‘Atomic Spectrophotometry: emission and Absorption pic emission spectrometry ign method 2.2.22) ‘principle emission isa process that occurs when Ae gnagnetc radiation is emited by excited atoms or j fponnic emission spectrometry the sample is subjected Bptertates high enough fo cause not only dissociation into $i bt soto cause significant amounts of cllsinal aon and ionisation ofthe sample atoms to take place ‘onthe atoms and fons are in the excited states, they ean {dayto lower states through thermal or radiative (emisien) formy taasitions and electromagnetic radiation is emitted. Sinemission spectrum of an element contains several more ‘ies tian the corresponding absorption spectrum, ‘sonic emission spectrometry isa technique for determining fpr comozntration ofan element in a sample by measuring theimensity of one of the emission lines of the atomic tapour of the element generated from the sample. on is earied out at the wavelength nding to this emission line. ‘only atomisation in flame is dealt with, od of inductively coupled plasma-atomic emission (ICP-AES) is described in a different general ary Sa wl SS __ Brean i Method ‘Use of plastic labware is recommended wherever possible: Operate an atomie emission spectrometer in accordance with the manufacturer's instructions a the prescribed wavelength Optimise the experimental conditions (Hume temperate, ‘bumer adjustment, use ofan ionic bulfer, concentration of solutions) for the specific element to be analysed and in respect ofthe sample matrix. Introduce a blank solution into the atomic generator and adjust the instrument reading 0 ero orto its blank valu. Introduce the most concentrated reference solution and adjust the sensitivity to obtain 8 suitable reading Its preferable to use concentrations which fall within the linea part ofthe calibration curve. If this is not possible, the calforation plots ray also be curved and are then t0 be sppled with appropriate calibration software. Determinations are made by comparison wit reference solutions with known concentrations ofthe element to be determined either bythe method of direct calibration (Method 1) or the method of standard additions (Method 1, Method I - Direct calibration For routine measurements 3 reference solutions of the clement to be determined and a blank are prepared and ‘examined, Prepare the solution of the substance to be examined (test solution) as prescribed in the monograph. Prepare not fewer than 3 reference solutions of the element to be determined, the concentrations of which span the expected value in the test solution. For assay purposes, optimal calibration levels are between 0.7 and 1.3 times the expected content of the ‘element to be determined othe limit prescribed in the ‘monograph, For purty determination, calibration levels are ‘between the Kimi of detection and 1.2 times the limit specified for the element to be determined. Any reagents ‘used in the preparation of the test solution are added to the teference solutions and to the blank solution at the same concentration. Tntroduce each of the solutions into the instrument using the same number of replicates for each solution, to obtain a steady reading Calculation Prepare a calibration curve from the mean of the readings obtained with the reference solutions by plotting the means as a function of concentration. Determine the concentration of the element in the test solution from the ceurve obtained. ‘Method Il - Standard additions ‘Ada to at least 3 siriar volumeric flasks equal volumes of ‘he solution of the substance to be examined (test solution) prepared as prescribed. Add to all but 1 ofthe flasks Frogrssvely larger volumes ofa reference solution etaining a known concentration of the clement ro be SRrermined to produce a series of solutions containing erally increasing concentrations ofthat element known to fre responses in the linear part ofthe curve, if a all ple, Die the contents of each fas ro volume with solvent Tyveduce each ofthe solutions into the instrument using the qo numberof replicates for each Solution, to obtain a steady reading. sd Came enn quan of he gph Cate ane-sqares fit, and desve fom ithe concentration UNE“ Jement to be determined in the test solution.SO —™ A172. Appendix II D a tangential stream of support gas "yatem consisting of 3 concentric al (the load coil) surrounds the top A mei is connected toa radio-Fequency (RF) "700-1500 W) is applied through a magnetic field coresponding tthe in most cases 27 MHz, 40 ME) when the support gas is made Validation of the method ‘Satisfactory performance of methods prescribed in. ‘monographs is verified at suitable time intervals. generator. Power berccind dan oscil Prepare and analyse not fewer than 4 reference solutions over the col and an me the calvation range and a blank solution Perform not fewer EQUENEY Nay forms the i {Spel eo ew a a eleieesnerescn oem produces seed electrons and ions. on the nea . from all measured data, The repression cure, the means the et cared paces (eons and os) a aaa etmmmas tacenstere ier ott caitexsan TAOS: Oe «cloned enna path, AS they mest aren treet eee forced 0 erin ow, eating takes place prodcing = the correlation coeficint is at least 0.99, ‘euisonal ionisation, The process occurs almost _~the residuals ofeach calibration level are randomly adeiomtously, and the plasma expands to it full tength cdiorrequency oscillation ofthe power ae eos eergm many an pan ‘the mean and relative standard deviation for the applied through the coil Von) peel ee Sle a a pars igri as be wi ee ‘ratio of the estimated standard deviation of the pighest calibration level is less than 0.5 oF esher seeding device is applied to the support gas flowing Fe cantons clitensos trough tbe toch vome clecuons re e-PP=d Sect i a Thoth support gas atoms. These clectrons are then caught up inthe led 3 magnetic field and accelerated. Adding energy to the Cleatons by the use ofa coil s known as inductive coupling ‘These high-energy clectrons in turn collide with other support-gas atoms, stripping off still more electrons. ‘The collisional ionisation of the support gas continues in a chain reaction, breaking down the gas into a physical plasma consisting of support-gas atoms, electrons and suppor-ss fons, The plasma is then sustained within the torch and load coil as radiofrequency energy is continually transfered tit ‘through the inductive coupling process, “The ICP appears as an intense, very bright, plume-shaped plasma, At the base the plasma is toroidal, and this is {eferred to asthe induction region (IR), i. the region in which the inductive energy transfer from the load coil to the plasma takes place. The sample is introduced through the induction region into the centre ofthe plasma Apparatus ‘The apparatus consists essentially ofthe following elements = sample-introduction system consisting ofa peristaltic ump divesing the soluion st constant fn ate 0 8 nnebuser ~ radio-frequency (RF) generator, plasma torch; —tansfer optics focussing th image of the plasma atthe eh for difficult matrices (alkalis, organics), whe ial Viewing gives more intens Lae 1 rea ity and better detection limits in wavelength dispersive devices consisting of diffraction ‘grtings, prisms, fer or a= dicen interferometers converting radiant energy int electrical enejos of certain elements or matrix scorers Goin jon of the sample and calibrat Piast by lion of the sample mar standards oF through applicat ese a ofintrference occasionally e Pega prcales cay inset ea oa are those elements that are lonised epich ifr example alkaline meas and alkaline cou ‘hat contain high concentrations of EIEs Gea fu pet cnt), SUPPrEssion or enhancement of vier gob is likely to occur. mt interference This may be due to other lines or sin background intensity. These lines may correspond 10 fepo served above 300 nm), OH bands due to the ition of water (at about 300 nm), NO bands due foie neraction ofthe plasma with the ambient air 200 nm and 300 nm), and other elements in the feng, especially those present at high concentrations Feinedrence falls ino 4 different categories: simple ‘uskzound shif, sloping background shit direct spectral tyedap, and complex background shift Martin interference This arises when pat of the ‘asion from an analyte is absorbed before it reaches the {Seow This effets observed particulary when the centration of a strongly emitting element is so high that fesoms or ions of that element that arc in the lower state of transition absorb significant amounts of the ‘emitted by the relevant excited species. This effect, determines the upper ead of the compounds, Viscosity and ion standards) can tatching, use the method of fitting Multiple emission-line ‘used to overcome problems ee ‘The peristaltic sually deliver Pumps used for ICP-AES usually deliver the Tae sample solutions at rate of 1 mLnin ores {the case of organic solvents being used the introduction ‘oxygen must be considered to avoid organic layers. CHOICE OF OPERATING CONDITIONS ‘The standard operating conditions prescribed by the ‘Manufacturer are to be followed. Usually different sets of Qferatng condone are we for aqueous sautons and fr anic solvents. Suitable operating parameters are t0 be property chosen: Bt — wavelength selection; — support-gas flow rates (outer; intermediate and inner tubes of the torch); — RF powers — viewing position (radial or axial); — pump speed; — conditions for the detector (gain/voltage for photomultiplier tube detectors, others for array detectors); — integration time (time set to measure the emission intensity at each wavelength) Control of instrument performance SYSTEM SUETABILITY ‘The following tests may be carried out with @ multi-clement control solution to ensure the adequate performance of the ICP-AES system: — energy transfer (generator, torch, plasma); measurement ‘of the ratio Mg II (280.270 nm)/Mg I (285.213 nm) may be used; sample transfer, by checking nebuliser efficiency and stability; — resolution (optical system), by measuring peak widths at hhalf height, for example As (189.042 nm), ‘Mn (257.610 nm), Cu (324.754 nm) oF Ba (455.403 nm); — analytical performance, by calculating detection limits of Selected elements over the wavelength range. Validation of the method Satisfactory performance of methods prescribed in monographs is verified at suitable time intervals. LINEARITY : Prepare and analyse not fewer than 4 reference solutions over the calibration range plus blank, Perform not fewer than 5 replicates. ‘The calbration curve i calculated by least-square regression trams all measured data of the calibration test. The regression voor the means, the measured data and the confidence Se of the ealoration curve are plotted. The operating method is valid when: = the correlation coefficient is atleast 0.99 — the residuals ofeach calibration level ae randomly distributed around the calibration curve standard deviation forthe Catculate the mean and relative standard devin fearest and for the highest calibration level ‘When the ratio ofthe estimated standard deviations of the Where and the highest calibration level is lest than 0.5 or tom than 2.0, a ore preci estimation of be cabraon Sea gay be obtained using weighed near represion. Both ta ad quads weigting fencions ae applied w the Tinea ind the mest appropdate weighting fonction o be ‘employed.A174 Appendix ID If the means compared to the calibration curve show a deviation from linearity, two-dimensional linear regression is used. accuRACY Verify the accuracy preferably by using a certified reference ‘material (CRM). Where this isnot possible, perform a test for recovery. Recovery For assay determinations a recovery of 90 per cent to 110 per cent isto be obtained. The testis not vali if recovery, for example for trace-element determination, is outsde of the range 80 per cent t0 120 per cent of the theoretical value. Recovery may be determined on a suitable reference solution (matrix solution) spiked with a known quantity of analyte (concentration range that is relevant to the samples to be determined). [REPEATABILITY ‘The repeatability isnot greater than 3 per cent for an assay and not greater than 5 per cent for an impurity test LIMIT OF QUANTIFICATION ‘Verify that the limit of quantification (for example, determined using the 10 « approach) is below the value to bbe measured. ‘Atomic absorption spectrometry (Ph. Bur, method 2.223) Goneral principle ‘Atomic absorption sa process that occurs when aground stare-atom absorbs electromagnetic radiation of a specific wavelength andi levated to an excited sate. The atoms in the ground state absorb energy at thei resonant frequency and the electromagnetic radiation i attenuated duc to fesonance absorption. The energy absorption is virtually © direc function of the numberof stoms presen. ‘This chapter provides general information and defines the procedures used in element determinations by atomic Absorption spectrometry, ether atomisaton by fame, by tlectroihermal vaporistion in a graphite furnace, by hydride generation or by cold vapour technique for mercury “Atomic absorption spectrometry isa techie fr determining the concentration of an clement in a sample by ‘measuring the absorption of eleeromagnetc radiation by the tromie vapour ofthe clement generated from the sample ‘The determination is cared outa the wavelength of one of the absorption {resonance} ines of the element concerned, “The amount of radiation sbsrbed is, according to the [Lambert Bee law, proportional to the element concentration. Apparatus: ‘This consists essentially of: =a source of radiation; =a sample itrodcton deviees —a sample atomiser ~ 4 monochromator or poyehromator =a detector, — a daraacquistion unit. “The apparatus is usually equipped with a background c on system. Hollow-cathode lamps and clectrodeless ps (EDL) are use as radiation source, 1 of such lamps consists of a spectrum showing, with half-width of about 0.002 nm of the smere are 3 sypes of sample atomisers Flame technique - 1 production accessory of a nebulisation system wit gas-flow regulation A flame ator and a bumer. Puce emperatures from about 2000 K to 3009 produce a range of tem rdsogen and acetylene; nde Pe ants The conf id to the gases used and the gs flow is ed, acidified water being th test and reference solutions, K. Fuel tig and mito fhe burner adap Scjustable,Sarples are nebuliss setrent of choice for Preparing sae of chk Fo be sea if precuons akan Organic str alvent doesnot interfere wit the stability of the flame IN lecuothermal somisation technique ‘An electrothermal atomiser is generally composed of « ? clectrie power sours. sraphit tube furnace Sietrhermal atomistin in a graphite tube farmace aoc he ene sample and retains the atomic vapour in delight path for an extended period. This improves the {eteton limit. Samples, liquid as well s solid, are ‘reduced drcey into the graphite cube furnace, which is tested ina programmed series of steps to dry the sample and tamove major matrix components by pyrolysis and to then ttomive all of the analyte, The furnace is cleaned using @ final temperature higher than the atomisaion temperature ‘The low ofan inert gas ring the pyrolysis step inthe graphite tbe furnace allows a beter periormance ofthe Subsequent atomisaton proces — Cold vapour and hyde technique “The atomic vapour may also be generated outside the spectrometer, This x notably the ease forthe cold-vapour method for mercury of for certain hydeie-forming elements Such as arsenic, anion, bismuth, selenium and tn For mercury, stoms are generated by chemical rection swith stannous chloride or sodium borohydride and the atomic vapour is swept by @ steam ofan inert gas into 2 ol 4quare cell mounted in the optical path of the insmume Harides thus generated are swept by an inert gas into a heated ell in which they are dissociated into atoms Interferences Chemical physical ionisation and spectra interie. encountered in atomic absorption measurement. imererence is compensated by addon of matrix moi of releasing agents or by using high temperature produced BY 4 nitrous oxide-acetlene flame; the wee of apecise ion bes or example, aan and eaesism) campers for ionisation interference; by dilution ofthe sample th the method of standard additions or by mae matin Physical ineerference due to high salt content or viscosity | sfiminated. Spectral interference results from the overlapping of resonance lines and can be avoided by using a difere resonance line. The use of Zeeman background corectio? also compensates for spect interfere : interference and interfeen from molecular absorption, especially when using the ‘ — fi : ||| e300 ct | —as008 ‘ i 200 mm long and with an external outs tanatnees eeiay {Pooved stopper though which the thermometer pases. bot as ofthe cup is placed about 15 mm from the tom of the ese, The le device immer in wih a capacity of about 1 its, led with water. ‘The bottom ofthe ete is placed about 25mm fom the bottom ofthe beaker The water vel reaches the oper pat teehee ‘Method Prepare the substance to be examined scoring tothe respons ofthe monograph Fl the cop 0 the st with the substance oe eamined. Remove the exes substance a the 2 end af he cup with espana. When Shea 4a hve nen aban pe tec ot 8 housing in sheath B un touches the suppor, Remove with a sprue the substance pune out by the thermometer ce the apparatus in the waterbath a described above Heat the waterbath and, when the temperature isa abot 10°C below the presumed drop point, ast the hesting fate o about 1°Clmin, Note the temperature atthe al of the fist drop. Cary out at east 3 determinations, each time vith afeesh sample of the substance. The difrence between the readings most not excend 3 °C. The mean of 3 readings isthe drop point ofthe substance. orn cece toe eee ee te eS Rope coin meat ae oe i ae aa eat aates ‘serene oe a Tapio ao cy. Te ig ag ce. an es 3 pe sana tin i eh ee See sey eg ct ae ee ear Pcie end ope Metin ee peers: feinto the sample cup according to the prescriptions of the 1 oe ng Fo a te a Ne te zcomet ae so st Co wi emeen nr essed, meget a ee Se beige i et eet ate soe eae i ee ea Brg te tease ft fh a imonren oben em 8 rea automatically.Acuphokle F beating element beating Mosk Galea ih source HL potosensor ange ‘Looe dee ccarige sembly temperature probe Figure 22.17.2, ~ Example of automated drop point apparatus Calibration Use the apparatus according 0 the ‘manufacturer's nsrutions and carryout the prescribed falaions and sytem performance test t regular intervals depending on the use ofthe apparass andthe substances 10 be examined, Benzpc acid and benzophenone ae usally ‘sed as cerfied reference materials. Other materials may be sed provided they show no polymorphism. Proced as follows or according to the manufacturer’ instructions. Prepare 3 sample cups foreach ofthe 2 cried reference ‘materials, Place the sample cups on a clean surface, Into tach sample cup, introduce a small quantity ofthe sample fnd pres it down with a rod (ameter about 4.5 mm), Cheek tat the opening is completely fled. Fil the sample up about half fill and compact dhe sample with rod (Giameter about 9 mm), Fill the sample cup completly and compact, adding more sample and compacting again if eceary, uni the sample cup is completely ful “Tempernture programme for benzoic acid start temperature = 118.0 C5 eating rate = 02 ‘Cmins fend temperature = 126.0 °C. Aer inserting the cup at 118 °C, a waiting time of 30 ss set before heating stars. “Temperature programme for benzophenone: start temperature = 44.0 Cheating rate = 0.2 Ciminy fend temperature = 56.0 °C. After inserting the cup at 44. 8 wing time of 30 is se before heating sat ‘Check the 3 single resus: the tests valid if the 3 results are within 03 °C ofthe mean valve Gatelate the comeced mean temperature (7): using the i a (Ds wing R-F temperature of 3 sample in“ 5 the difference in tempers Jean the point in the he 4 ‘between the samp! ee where the tempel block when ren the design of the automatic crature is measured; this wil epending UP yn instrument and is provi ided by the drop point the drop point (7) of the certified Taking to sce accuracy ofthe temperature scale is "Fp| is not greater than 0:3 °C. refer satisfactory if |7 Method 1V (Ph Bue method 2 Saermine he mel one determing he pa when determined by this method segs cap legen ‘external diameter of 1.4 mm to 1,5 mm and femal ameter of 1.0 mm to 1.2 mm. Tae ino each of capil tubes a suiient amount tp vabrance previously tte as described, to for in exchabe a enfoun about 10 min high and allow the 1 ita forte appropeat tine and atthe preserbed ‘np fing point (also referred 10 as sii ry tubes open at both ends, about 80 mm Unless otherwise prescribed, substances with a waxy consistency are carefully and completely melted on a ‘water-bath before introduction into the capillary tubes. Allow the tubes to stand at 2-8 °C for 2h. Attach one of the tubes to a thermometer graduated in (05 °C so that the substance is close to the bulb of th thermometer Introduce the thermometer with the at tube into a beaker so that the distance berween the bot the beaker and the lower part of the bull of the thermon is 1 cm, Fill the beaker with water to a depth of 5 em. of 1 °C/min. ‘The temperature at which the substance begins to rise inthe capillary tube is regarded as the melting point. Repeat the operation with the other 4 ca calculate the esl asthe mena of the 5 Method V (Ph. Ew method 2.2.16) ‘The instantaneous melting point expression: any tubes and dings. is calculated using the =a Shih te i temperature read under the conditions stated bel < Apparatus The apparatus consis a metal Bock ‘resistant to the subst Baia goek tn cay sch sre hr poled pine wpe: sutace, The bk sunny Bt eae its mass by means of a micro-adjustable 638 « block has a cylindrical cavity, wide enough t accomodate a thermometer, which should be nan wih the mera nen Which hold be mane ame position during eratus and the determination of Of the substance to be examined is parallel to the upper poised su jut 3-mm from it. The a eaibeeeA wion one apparatus isHeat the block at a suitably woe about 10 °C below the ot then adjust the heating rate to Said Particles of powdered and, DPropated as for the einity of the ‘S10 | at regular intervals on the block, cleaning te ee ears ace ene Sesabtnce ceases t0 melt instantaneously when it eens raat wth the metal jon ofthe apparatus The a using meting point reference | ne ofthe World Health Organisation ‘uheanees, Method VI (0h Bw. method 2.2.60) apparatus may be Substances such as ‘ischapter describes the measurement of melting point by ‘method using an instrumental method of teil Apparatus There are 2 modes of automatic observation mangement: mode A: by light transmission through the capillary tube Jaded with the sample; ‘mode B: by ight being reflected from the sample in the the capillary tube sits in a hollow of a metal cea 5 ich is heated electrically and control by a sor laced in another hollow of the metal ates slow and steady ‘or other appropriate Place the capillary tube inthe heating block withthe closed end down the owns, Start the temperature programme. When Substance stats melting, it changes its appearance in the ‘phils tube, As a result the temperature of the heating lock is recorded automatically following the signal changes fom the photosensor due to light transmission (mode A, Figure 2.2,60.-1), or following image processing (mode B, Figure 2.260. (Carry ou the rest on 2 otter samples and calculate dhe mean value ofthe 3 reals, Calibration The temperature sale of the apparatus is checked periodically by measuring the meting point of certified reference materials. Use capillary tubes having the Same dimensions as those used forthe determination of the ‘melting point (See Apparat). Prepare 3 capillary tubes for cach of at least 2 certified reference materials. Cary out the test and calculate the mean value of the 3 results foreach material System suitability In addition to the cabration, carry out a vetfction, before the measurements, using a suitable certified reference material whose melting point i close to that expected for the substance to be examined. Prepare 3 capillary tubes. Cary ou the test and ealelate the sean vaive of the 3 rer “The mean value is within the tolerance given on the cerifeate supplied with the ceried reference materi oO ‘Aidascopilarytube __D. temperature sensor Bese heating Nock photsensor ihe source Figure 2.2.60-1. - Mode A: transmissionA240 Appendix V B heating Bock light source transparent plate {isthe maximum temperature occuring ofa supercooled hau. apparatus (se Figure 22.181) consis yout 25 mm in diameter and 150 mm long, out 40 mm in diameter and Figure 2.2.18-1. ~ Apparatus for the determination of freezing point Dimensions in millimetres ‘Method Place in the inner tube sufficient quantity of the Figuid or previously melted substance to be examined, to cover the thermometer bulb and determine the approxima: freezing point by cooling rapidly. Place the inner tube in bath about 5 °C above the approximate freezing point until all but the last traces of crystals are melted. Fill the beaker ‘with water or a saturated solution of sodium chloride, at temperature about 5 °C lower than the expected freezing ‘point, insert the inner tube into the outer tube, ensuring that some seed crystals are present, and stir thoroughly until solidification takes place. Note the highest temperature observed during solidification, C. Determination of Distillation Range (Ph. Bar. method 2.2.11) ‘The distillation range is the temperature interval, corrected fra rene of 101 3 Pa (760 Tor), within vi bora specified faction ofa liquid, dis in the following conditions, a Apparatus The apparatus (see Figure 2.2.11.-1) consis ‘of a distillation flask (4), a straight tube condenset (B) ‘which ft on to the side arm of the flask and a plan-bend adaptor (C) attached to the end of the condenser. The lo" ‘end of the condenser may, alternatively, be bent to replae® the adaptor. A thermometer is inserted in the neck of flask so that the upper end of the mercury reservoir is 5 lower than the junction of the lower wall of the lateral tube ‘The thermometer is graduated at 0.2 °C intervals and the salecor age of bout 50°C. Dug determination, including its neck, is prove draughts by a suitable as seesieAppendix VD A241 ~ Apparatus for the determination of distillation range Dimensions in millimetres Place inthe flask (4) 50.0 mt ofthe me = oe eliid te, Determination of Boiling Point sting by (Ph Br, metiod 2.2.12) Pi is he comet repeat wich he ‘pour pressure of iid is equal to 1013 Ke Apparacis epee (211) wih te exception thatthe tbemaneet Range (the neck ofthe flank so that ce ower end of ie ise vd er wi the lwer endl of the nek of smear von flank and thatthe Sask placed on «Date of since by a ole 35 mm in diameter. vita Place in ve Sask (4) 20 mi. ofthe Hal 90 Method tafe piss of porous mate, Het the Sask caer oling x rapily achieved and record the $0 tat tee a wc gud rns om the seam no De hat +k(1013-4) ‘he corrected temperatures the SPoeved temperanae at barometric reste 2+ the Correction factor as shown in Table 2.2.11-b under Distillation Rang p= he baromeric pressure in lopascaly at he Se of the determination. ® k=A242. Appendix V E E. Determination of Refractive Index (Ph, Eur. mathod 2.2.6) ‘The refractive index of a medium with reference to air is ‘equal to the ratio ofthe sine ofthe angle of incidence of a ‘beam of light in air to the sine of the angle of reftaction of the refracted beam in the given medium, Unless otherwise prescribed, the refractive index is measured at 20 + 0.5 °C, with reference to the wavelength of the Daline of sodium (4 = 589.3 nm); the symbol is then nf Refractometers normally determine the critical angle. In such apparatus the essential paris a prism of known refractive {index in contact with the liquid to be examined. Calibrate the apparatus using cerified reference materi When white light is used, the refractometer is provided with 1 compensating system. The apparatus gives readings accurate to at least the third decimal place and is provided with a means of operation at the temperature presrbed. ‘The thermometer is graduated at intervals of 0.5 °C of less F. Determination of Optical Rotation and Specific Optical Rotation (Ph. Bur method 2.27) Optica rotation isthe propery displayed by chiral substances of rotating the plane of polarisation of polarised ight Optical rxtion is considered toe postive (+) for dentorotatory substances (ie those that rotate the plane of polarisation ina clockwise direction) and negauve (~) for Jaevorotatory substances ‘The specific optical rotation [ag isthe rotation, expressed ‘in radians (Fad), measured atthe temperature t and st che given by a 1m thickness of liquid oF = solution nih acl is, dese ies deci pag [O-mL-dm™'¢ 1 are understoo rr on actor from the Internationa Stem oh Tas come ter he allowing [aml = lal, x 0.1745 ceria case spectied inthe monograph the ang of In cern cn Pere at temperatures ote sn 20°C tn aber wavelengths “Te olaranter must be capable of ing readings he re acl anual checked by mean of tee seat plats, The near of the scale may be SESH by means f sucrose sluions Mashed Determine the zero ofthe poarincter an he Meet rotation of polarised ight a the wavelenath fhe Pec efuolum (2 389.3 nm at 20 403 C, unew Die preted, Measurements may be cared ot ihe tempers only where the monograph ine te ctoponareceecon tobe made tothe measured op ‘outon Deternine the 270 of the sppartun with the tbe “ved for liguds the aero determined with he tbe ‘topo for sls ied with the reseed sen Clete the specie opal oration using the following Somes For neat igus: B= Pa Tp» For substances in solution: ‘where is the concentration of the solution in grams pe lire Calculate the content cin grams per lire or the content c's per cent mim of a dissolved substance using the following formulae: 1000 I> 1 [ale + p20 angle of rotation in degrees read at 20 + 0.5°C Jength in decimetres of the polarimeter tube, density at 20 °C in grams per cubic centimete For the purposes of the Pharmacopoeia, density s eplaced by relative density (2.2 5). G. Determination of Weight per Millilitre, Density, Relative Density and Apparent Weight per millilitre (No Ph Eur, method) Ti weight pe mille ofa liquid isthe weigh in g of 1 liquid when weighed in air at 20°, unless otherwise specified in the monograph, The weight per mille is determined by dividing the we8t* 1 y dividing the wei in air, expressed in g, of the quantity of liquid that fills @ yenometer atthe specified temperature by the capaci: SpPressed in mL, of the pyenometer at the same remperst™ ‘The capacity of the pycnometer is ascertained from theAppendix V M_ A251 sir solutions in suitable chemically Ses, such 8 tyPe T glass bottles or plage ne tet Sone for aqueous solutions, °F Plastic containers, “astional points JOr Monographs of the ‘patish Pharmacopoeia [Garis lectodes and pH meters ofboth ose and digital type are described i the $56 1979 and 3145:1978,] in British Standards MM. Thermal Analysis (ph Br, method 2.2.34) ‘eral ants is group of techniques Seal ore Senet: temper The i common far thove which measure changes of ae or ‘ofa sample ofa substance. The ‘Pe sstnigue sued to determine the drench flow of heat (with reference to the temperature) evolved ot ‘teed by the rex spl compared with the reference Decte fntin of the temperature. Two pes of DSC spparatuses are avaiable, those using power ompenston to maintain a mull temperate diference beeween sample and reference and those that apy a Constant rate of heating and detect temperature fleas erence in heat flow between sample and reference. Apparatus ‘The spars forthe power compensation Consists ofa fumace containing a sample holder with a reference cell and atest cell. The apparatus for the eat fw SC consists ofa furnace containing a singe cell with a sample holder for the reference crucible and the test erucbe. ‘A temperatre-programming devie, thermal detector() and 8 recording sjstem which ean be connected to a computer ae atached. The measurements are caried out under a controlled atmosphere Figure 2234-1. ~ Thermogram Calibration of the apparatus Calibrate the apparatus for temperature and enthalpy change, using indium of high purity or anyother suitable certified material, according to the manufacture’ instrictons. A combination of 2 metals, eg indium and zine may be used to contol inary Operating procedure Weigh in a suitable crucible an appropriate quantity of the substance to be examined place itin the sample holder. Set the initial and final temperatures, land the heating rate according tothe operating conditions prescribed in the monograph. Begin the analysis and record the diferenial thermal analysis ‘cave, with the temperture or time on the abscissa (values increasing from lft o right) and the enengy change on the cndinte(opeify whether the change is endothermic or exothermic). ‘The temperature at which the phenomenon occurs (he onset temperature) corresponds tothe intersection (A) of the tertension of the basline with the tangent at the point of Greatest slope Gnflexion pois) ofthe curve (see Figure Fie dal), The end of the thermal phenomenon is indicated ‘by the peak ofthe curve “The enthalpy of the phenomenon is proportional to the area under the curve limited by the baseline; the proportionality fbotor is determined from the measurement of the heat of feaion ofa know substance (e. indium) under the same thermogram may be accompanied by the following a ye ed fat altran, sak Soe sod identficaon (cluding thermal history), container, ee pophere (identity, lw rate, pressure), drecion and rte Sfremperatare change, instrument and recorder sensititySe ere tt—C“i=‘:;S; APPLICATIONS Phase changes Determination of the temperature, heat capacity change and enthalpy of phase changes undergone by fa substance as a function of temperature solid - solid transition: allotropy - polymorphism lass transition desolvation amorphous-erystalline : melting sublimation freezing recrystallsation liquid - gas transition: evaporation Changes in chemical composition Measurement of heat ‘and temperatures of reaction under given experimental ‘conditions, o that, for example, the kinetics of decomposition oF of desolvation can be determined. Application to phase diagrams Establishment of phase diagrams for solid mixtures. The establishment of a phase diagram may be an important step in the preformulation and ‘optimisation of the freeze-drying process. Determination of purity The measurement of the heat of fusion and the melting point by DSC enables the ‘impurity content of a substance to be determined from a single thermal diagram, requiring the use of only a few ‘milligrams of sample with no need for repeated accurate ‘measurements of the true temperature. In theory, the melting of an entirely crystaline, pure ‘constant pressure is characterised by a heat of, ‘an infinitely narrow range, corresponding to tT. A broadening of this range isa sensitive Hence, samples of the same ry contents vary by a few tenths of a = mmole faction of the impurity i.e. the numberof Be ae crake impury divided by the ox amoliper of molectles in the liguid phase (or mo, hase) at temperaure 7 expressed in Kelis), Pe paint ofthe chemically PUe substance, n Ivins, kof iio ofthe subst in jo, gus constant for ideal gases, in joules kelvin” mole Hence, the determination of purty by DSC is fimited ote vrei of impurities forming a eutectic mixture with the dertipal campound and present at a mole fraction of es ven 2 per cent in the substance to be examined ‘This method cannot be applied to: — amorphous substancesy ~ Solvates or polymorphic compounds that are unstable ‘ithin the experimental temperature range, — impurities forming solid solutions with the principal substance, — impurities that are insoluble in the liquid phase or in the ‘melt of the principal substance. ‘During the heating of the substance to be examined, the impurity melts completely at the temperature of the eutectic mixture, Above this temperature, the solid phase contains ‘only the pure substance, As the temperarure increases progressively from the temperature of the eutectic mixture to the melting point of the pure substance, the mole faction of imparity in the liquid decreases constantly sine the guaniy of liquified pure substance increases constantly. For all temperatures above the eutectic point: te n=kxat ® F = molten fraction of the analysed sample, x} = mole fraction of the impurity in the analysed sample. ‘When the entire sample has melted, = 1 and x2 = x} equation (2) is combined with equation (1), the following equation is obtained: =ERTG ‘AH; ‘The value of the heat of fusion is obtained by integrating be peed obtained by integrating The melting point Ty of the pure substance is rapolated a ee ia ‘The slope a of the curve. obtajned after linearsatio® essary, coresponding to RI seq allows x 10 “The fraction x3, mal 4) multiplied by 100 gives the mole faction Der cet fr he wal exec inputting ‘Thermomicroscopy Phase changes may be visualised by thermomicrseoP # ethod which enables a sample subjected to a pros temperature change tobe examined, in polarised ih— “The apparatus consists of a yee microscope fitted aiarpoariset, a hot plate, a temperature Pai ding re roramane an a ont at n temperatures. A video camera andenies nay be added. and video y.osmolality - method 22:35) (isa practical means of giving an overall m: tion of the various solutes prevent in ecole, Ganotc pressure of the solution. ee reese approximation fo the osmolality &, of a gr ae Sn of given bn = umd fhe solute isnot ionised, » ~ 1; otherwise is dhe total ‘ofions already present or formed by solvolysis sabe of y solvolysis from ‘x = moldy ofthe solution, that is the number of moles of + salut er Kilogram of solvent {= neal osmotic coeficint which takes account ofthe {meractions between ions of opposite charge in the solution. I s dependent on the value of m. As the complexity of solutions increases, becomes dificult to measure. ‘The uit of osmolality is osmole per kilogram (osmol/kg), but submaltiple milosmole per Kilogram (rosmol kg) is ‘Table 2.2.35-1, ~ Reference solutions for osmometer eS ealtvation __ ass in ams of Ral Se Sor ‘9463, 00, 32383, 09264 0558 ae ene 0. Conductivity (Ph, Eur. method 2.2.38) ‘The current J (in amperes) flowing in a conductor is directiy proportional tothe applied electromotive force F (in volts) ‘and inversely proportional to the resistance R (in ohms) of ‘the conductor: E vs “The conductivity (formerly called specific conductance) of a solution (x) is, by definition, the reciprocal of resistivity (). Resistivity is defined as the quotient of the electric field and the density of the current. The resistance R (in ©) of = conductor of cross-section $ (in em’) and length (in em) is sven by the expression: Thus: LS corresponds to the ideal cell constant. “The unit of conductivity in che Intemational Sytem isthe siemens per metre (Sm). In practice, the electrical Conductivity ofa solution is expressed in siemens fer centimetre (S-em-") of in microsiemens per centimetre {GiSem). The unt of resistivity in the International System {fthe ohmmetre (Cem). The resistivity of «solution is fenerally expressed in ohim-ceatimetes (Q-em). Unless ‘cherwise preseribed, the reference temperature for the expression of conductivity or resistivity 25 °C. “The apparatus and operating procedure described below are Ticable to laboratory measurement of conductivity greater dan 10 yS-em-*, The measurement of conductivity of water js deal with in the relevant monographs. Apparatus ‘The apparatus used (conductivity meter or resistivity meter) the resistance of the column of liquid berween the ‘lecrodes ofthe immersed measuring device (conductivity EA), The apparatus is supplied with skerating current to saya the effec of electrode polarisation. Ics equipped with Srpinperarure probe and etemperatare compensation device.A254 Appendix V P ‘The conductivity cell contains 2 parallel platinum electrodes ‘coated with platinum black, each with a surface area S, and separated from the other by a distance Z, Bot are generally protected by a glass tube, Other types of cells may also be cused. Operating procedure DETERMINATION OF THE CELL CONSTANT ‘Choose a conductivity cell that is appropriate forthe [Properties and conductivity of the solution to be examines ‘The higher the expected conductivity, the higher the cell ‘constant that must be chosen (low p), Commonly used ‘conductivity cells have cell constants of the order of Oud em=*, 1 em™¥ and 10 em”. Use a certified reference ‘material, for example a solution of potassium chloride, thats appropriate for the measurement, The conductivity value of ‘the certified reference material, should be near the expected ‘conductivity value of the solution to be examined. Other certified reference materials may be used especially for cells ‘having a constant of 0.1 em". Rinse the cell several times ‘with dinilad eater R and at leat twice with the certified ‘reference material used for the determination ofthe cell ‘constant of the conductivity cell. Measure the resistance of ‘the conductivity cell using the certified reference material at (25 £ 1°C. The cell constant Kuz (in em”) depends on the we x[1-+0 (Tons) voy atthe dren temperature iy ofthe cried reference a en mate meneame performance of various operations in the monitor the sdicines. tae Sk standard solution is analysed at suitable interval, sees fei ance ote (a arenas a ee ee ere ae ee ee Secale ei veces ee eer ot ae ee ae ac eee cease ses soe ra oe ‘Use highly purified water complying with the following specifications: — conductivity: not greater than 1,0 S:cm~! at 25°C, total organic carbon: not greater than 0.1 mg/L. Depending on the type of apparatus used, the content of ‘heavy metals and copper may be critical. The manufscture’s instructions should be followed, Glassware preparation ‘Use glassware that has been scrupul i rupulously cleaned by a ‘method that will r¢move organic matter. Use TOC tater the final rinse of glassware, Standard solution Dissolve sucrose R, dred at 105° we R, dried at 105 °C for 3h in TOC water 10 obtain & solution containing 1.19 mg of sucrose pet lite (0.50 mg of carbon perlite. Test solution Using all due care to avoid con: tested in an airtight contin Examine the water with ming amination, collect water #0 % ner leaving minimal heap ‘minimum delay to reduce .75 mg of sls ‘Pet litre (0.50 mag of carbon per lt):water control 9 ad we ye TOA standard solution and the le control oo the TOC water cont, prepare sua patie che solutions nesded for eats ee si ofr calibration adjustments folowing fe a ers istrucions; ran the appre ao it Banks tn sce sit Sorte flowing solutions and record ther Ra (ws standard ston (7); sem by sbi the peteige respons ficiency wing te expression: 1 a8 that used to sostem suitability EG faa T a REF ‘pesytem is suitable if the response efiiency isnot les ¢hin85 percent and not more then 115 per cent ofthe ‘hese response. Procedure un he test solution and record the response (r,). The txt fxton complies withthe test ifr is not greater than F~r ‘The method can also be applied using online ‘agramentation that has been adequately calibrated and {Som to have acceptable system suitability, The location of don must be chosen to ensure thatthe responses af 2 z Appendix VQ A255 ‘True density ‘The true density of a substance is the ratio of the mass to the Talus ofthe unit cl, exclusive ofall woids that are not 2 damental part of the molecular packing arrangement. Its {an intrinsic property of the specified crystal structure of Substance, and hence should be independent of the method ‘of determination. The true density is determined by calculation. Its obeained using crystallographic data (volume and ‘composition ofthe unit cel) from, for example, X-ray slffraction data, ether on a singe crystal or by refinement of the crystaline structure from X-ray powder diffraction data. Particle density ‘The particle density takes into account both the true density and the inraparticulate porosity (Sealed andor experimentally non-accessible open pores). Thus, particle density depends on the value of the volume determined, ‘hich in tum depends on the method of measurement ‘The panicle density can be determined using one of the 2 following methods, ‘The gas pyenomeric density is determined by measuring the volume occupied by a known mass of powder, which i equivalent to the volume of gas displaced by the powder using a gas displacement pycnometer (2.9.23). In gs pycnometric density measurements, the volume determined excludes the volume occupied by open pores; however, it includes the volume occupied by sealed pores or pores inaccessible tothe gas. Due to the high diffusivity of hetium, ‘which isthe preferred choice of gas, most open pores are ‘accessible tothe gas. Therefore, the gas pycnometric density fof finely milled powder is generally not very different from the true density. Hence, this density is the best estimate of the tue density of an amorphous or partially cystalline sample and is therefore widely applicable for processed ‘pharmaceutical powder samples. "The mercury porsimeter density is also called gramalar density. ‘With this method the volume determined includes the volume occupied by sealed pores or pores inaccessible to ‘mercury; however it includes the volume only from open pores smaller than some size limit. This pore-size limit or ‘minimal access diameter depends on the maximal mercury fusion pressure applied during the measurement, and ‘under normal operating pressures the mercury does not penetrate the finest pores accessible to helium. Various {granular densities can be obtained from one sample since, for tach applied mercury intrusion pressure, a density can be determined that corresponds to the pore-size limit at that pressure. Bulk and tapped density “The bulk density ofa poder includes the contribution of inteparticlate void volume. Hence, the bulk density depends on both the density of power particles and the Spatial arrangement of particles inthe powder bed. ‘The bulk density of a powder is often very dificult to measure with good reproducibility since the slightest erurbance ofthe bed may result in a new density. Thus, it fs essential in reporting bulk density to specify how the determination wes made “The bulk density and the tapped density are determined as ‘mentioned in chapter 2.9.34. Bulk density and taped densA256 Appendix VI Appendix VI Qualitative Reactions and Tests (Ph. Bur. method 2.3.1) Acetates |A. Heat the substance to be examined with an equal quantity of oxalic acid R. Acid vapours with the characteristic odour of sestic acid are liberated, showing fan acid reaction (2.2.4) B. Dissolve about 30 mg of the substance to be examined fn 3 ml of ater R oF use 3 ml of the prescribed solution, Add successively 0.25 mL of danthanum mitrate solution R, 0.1 mi of 0.05 M iadine and 0.05 ml. of die ammonia R2, Heat carefully to boiling. Within few minutes a blue precipitate is formed or a dark blue colour develops. Acetyl Groups Ii a testtube sbout 180 mm long and 18 mi in extemal diameter, pace about 15 mg of the substance to be ‘examined, o the prescribed quantity, and 0.15 ml of phosphonic acid R, Close the tube witha stopper through ‘which pases a smal tes-tube about 100 mm long and 10 mn in extemal diameter containing ester Rto act a 2 condenset.On the outside ofthe smaller tube, hang @ drop of lanthanum rte son R. Except for substances Inydolyable only with dificult, place the apparatus in @ ‘water-bath for 5 min, then take out the smaller tube. Remove the drop and mix it with 0.05 mL of €.07 A iaine ona ie ‘Add at the edge 0.05 mL. of dilute ammonia R2. After 1 min ‘0 2'min, @ blue colour develops atthe junction ofthe neo ae colour intensifies and persists for a short time. For 05 hydrolysable only svith difficulty heat the mixture ae above. Alkaloids Dissolve a few miligrams of the substance to be examines, or the preseribed quantity, in 5 mL. of tater R, add dice Incochlre acid R until an acid reaction occurs (22-0, then 1 mL of passion iodobismuahate slution R. An orange or precipitate is formed immediately. eer Aluminium Salts Dissolve about 15 mg ofthe substance robe examined in 2 mL of tater R oF use 2 mL of the prescribed solution. ‘Add about 0.5 mL. of cite Indroclric acid R and about ex just beneath the surfce of @ mixture oft mp, seid and 0.05 ma. of met ed solution icator changes to yellow: On addition yy [00 g/L solution of sod flow precipitate is formed. that escap 0.1 M lydrochlor ‘The colour of the indi I mL of a freshly prepared 1 nite Ra ye nium Salts and Salts of Volatile Bases ae pout 20 mg of the substance tbe examined in Dis ator use 2 in. of the prescribed slun, 2m of et in sun hide lion R. On heig Ae occa eaceen w aaa pate alain reaction (2.2.9) Ammo ‘Antimony Compounds Dissolve with gentle heating about 10:mg of the substance tp be examined in a solution of 0.5 g of sodium porastom tartate Rin 10 ml. of tater R and allow to cool: to 2 mL of this solution, or to 2 mL of the prescribed solution, add Sodom sufde solution R dropwise an orange-red precipitate is formed which dissolves on addition of dilute sodium hychoxide solution B. Arsenic Compounds Heat 5 ml. of the prescribed solution on a water-bath with ‘an equal volume of hypophosphorous reagent R. A brown precipitate is formed. Barbiturates, Non-nitrogen Substituted Dissolve about 5 mg of the substance to be examined in 3 mL. of methanol R, add 0.1 ml. ofa solution containing 100 gL. of cobalt nitrate R and 100 giL. of calcium chide R ‘Mix and add, with shaking, 0.1 mL of dite sodium hydride solution R.A violet-blue colour and precipitate are formed Benzoates A. To 1 mil. of the preseribed solution add 0.5 mL of or chloride solution RI. A dull-yellow precipitate, soluble in ether R, is formed, B, Place 0.2 g of the substance to be examined, treated if ‘ecessary as prescribed, in a test-tube. Moisten with 0.2 mL t0 0.3 mL of sufi acid R, Gently warm the bottom of the tube. A white sublinate is deposited o0 the inner wall of the tube. Dissolve 0.5 g of the substance to be examined in 10 mL of waver R or use 10 mL. of the prescribed solution, Add 0.5 mL of hydrochloric acid R. ‘The precipitate obtained, after crystallisation from wart @219e120'Cw ia Bicarbonates ‘Ac Introduce into a test tu : ce no atest tbe 0.1 ofthe substance bing examined suspended in 2 mi. of rater or se 2 lof the precibed solution. Add 3 mi. of 2 ace ath se the tube immediately using a stopper ited wit © Bias ube bent at two right angles. The soluton of Stspension effervesces, Hear genly and cole the Sal of 84.73% wiy wh of Soro os Presptate is prociced which discles oP sition fa eee of heh ac. > esa solution of he substance being examined vi? Solution of magnesion sulfate no precipitate is OS sae ae Bowl» white presi A solution liberates carbon dioxide when boiled.ono sod Bismuth Compounds esl st mio wae Stcaceinnc an -R_Awhite or slightly yellow precipitate is m9 med which on addition of 0.05 ml. to 0.1 ml. of ion fide solution R turns brown ‘g. Toabout 45 mg of the substance to be examined 8 HaLtaiienin:eci Fores omeroe prescribed solution. Boil for 1 min. Allow to cool and fer necessary. To 5 mi ofthe solution obtained add ‘ofa 100 gL solution of thiwrea R.A yellowish colour or an orange precipitate is formed. of a 25 gi. solution of sodium fluoride R. Appendix VI_A257 C. To 5 mL of 0.4% wiv solution ofthe substance being, ‘examined add 0.2 mL. of a 2% wi solution of ‘ammoniion oxalate. A white precipitate is produced which is only sparingly soluble in 6x. acetic acd but is sobuble in Inydroclone acid Carbonates For monographs from the European Pharmacopociay use test A only. ‘A. Introduce into a test tube 0.1 g of the substance being examined suspended in 2 mL. of water or use 2 mL. of the prescribed solution, Add 3 ml. of 2st acetic acid, Close the tube immediately using a stopper fired with & ‘lass tube bent at rwo right angles. The solution or Suspension effervesces evolving 2 colourless and ‘dourlese gas. Heat gently and collet the gas in 5 ml. of (cla baru hydroxide. A white precipitate is produced which dissolves on addition of an excess of “1 Idrehloric acid B. Treata solution of the substance being examined with a sofution of magnesium sulfate. A white precipitate is ‘produced (distinction from bicarbonates)- Carbonates and Bicarbonates Traroduce into a tes-tube 0.1 g ofthe substance 10 De Caantined and suspend in 2 ml. of water Ror use 2 ral of the prescribed solution. Add 3 ml. of dilute acre acid R ‘Gore the tube immediately using 2 stopper fied with a eass tbe bent twice at right angles, The solution or the Jon becomes effervescent and gives offa colourless aT Edouress gas. Heat gently and collect che gas in 5 ml. of fariom hydroxide olution R. A white precipitate is formed that ‘Gasolves on addition of an excess of fndrochlrc aid RI. Chlorides ‘A. -Disoive in 2 mL of ear R a quantity of the substance Dobe examined equivalent to about 2 mg of chloride {erp or vie 2 mL ofthe prescribed solution. Acidity ith eae irc ocd R and add OA rl. of ser mtrate witin RE. Shake and allow to stand. A cordled, white Sprechiat is formed. Centrfage and wash the Precitate with three quanties, cach oft mil of aime R. Carry out this operation rapidly in subdued fighe dezarding de fact thatthe supernatant solution Pei not become perfectly clear. Suspend the precipitate Feo mal of water R and add 1.5 mL of ammonia R. ‘The precipitate dissolves easly with the posible ‘reception ofa few lage particles which dissolve slow'y 'B._ Introduce into a test-tube a quantity ofthe substance to Tareined equivalent to about 15 mg of chloride (CI) tr the presrbed quant. Add 0.2 g of potas Gebrmate Rand 1 al of sure acid R. Pace a fier aper sep impregnated with 0.1 mi. of aiphoncarbaside rap ron R over the opening ofthe test-tube, The paper tums violtred. The impregnated paper must not come ; from the European Pharmacopoeiay use only. Ih Disahein 5 ml of ater Ra quan ofthe sss Af beczamine gules 0 ou 50 ee ntti pst tion AO ot Sc ocid R an 1s. of potas permanente in Wm tl he clu of PEN Biren Add 05 mL a 100g solution of dion— DU - A258 Appendix VI = —— iopruside Rin dite sufi acid Rand 4 g of sulfaic ‘acid R. Make alkaline with concentrated ammonia R, added dropwise until all she sulfamic acid has dissolved. Addition of an excess of concentrated ammonia R produces a violet colour, ruming co violet-bive. B, To a neutral solution of the substance being examined ‘add a solution of calcium chloride; no precipitate is ‘produced. Boi the solution; a white precipitate is produced which is soluble in 6x acetic acid. Esters To about 30 mg of the substance to be examined or the ‘prescribed quantity add 0.5 mL of a 70 g/L solution of ‘nxdroxlamine hydrochloride R in methanol R and 0.5 ml. of 100 g/L solution of porasum hroside R in ethanol (96 per cont) R. Heat to boiling, cool, aidify with ditwe Jsdroclorie acid R and ada 0.2 ml. of fore chloe solion RI dikted ten times, A blush-ted or red colour is produced. Todides ‘A. Dissolve a quantity ofthe substance to be enanined ‘equivalent 10 about 4 mg of ide 1) in 2 ml of ‘eater Ror use 2 mL. of the preserved solution. Acidity with dae merc acid Rand add 0.4m. of ser nae solution RI. Shake and allow to stand. A cuted, pale- sellow precipitate is formed. Geneige and wash with ‘quantities, each of 1 ma of eater R. Cary out this ton rapidly in subdued light disregarding dhe fict ‘supernatant solution may not become perfec, the precipitate in 2 ml of eater 8 and 4 sion ofthe substance tobe Bees saat na orto 02 mL of he press solu, ad So ie irc aR 0. of possi "Grr Re Saks fra few seco ad alo vo | Sand The chorion lperin colored veto il Fe Iron and Iron Salts, A. Dissolve a quantity ofthe substance to be examined 0 about 10 mg of iron (Fe") in I al of ‘Rot use 1 mL ofthe prescribe solution “Add 1 ml of prs fernyanie soluion RA blue "precipitate is formed that does not dsolve on addition nt Torch acid R. BB. Dissolve a quantity ofthe substance to be examined NE eee au R. To 3 mL of this solution or wo 3 mL. of the dd 1 mL of dite hydrochloric of potas thiseyanate solution oloured red. Take two portions, each of . To one portion add 5 ml. of a of cher R. Shake and allow to yetates ratte: a quantity ofthe substance to be etamined Lead and Lead Compounds |. Dissolve 0.1 g ofthe substance to be examined in 1 ml of atc acid R or use 1 mL. of the prescribed solution. ‘Add 2 ml. of potassium chromate solution R. A yellow precipitate is formed that dissolves on addition of 2 mi, of strong sodium hydroxide solution R. B. Dissolve 50 meg of the substance to be examined in mil. of acetic acid R or use 1 mL of the prescribed solution, Add 10 mL. of mar R and 0.2 mL of pasion fadide solution R. A yellow precipitate is formed. Heat 1 boiling for 1 min to 2 min. The precipitate dissolves. Allow to cool. The precipitate is re-formed as glistening, yellow plates. Lignin ‘A. Lignin call walls are coloured bright red by soaking them ina 1% why solution of phoroglucinal in ethanol (90%) and adding 0.1 t0 0.2 ml of xdrochloric acid B Lignifid tissues are coloured yellow by aniline Inydrochloide solution. Magnesium and Magnesium Salts For monographs from the European Pharmacopocia wse tt A ‘A. Dissolve about 15 mg of the substance to be examined jn 2-mL of water R or use 2 mL. of the prescribed Solution. Add 1 mL of dite ammonia RI. A white respite i formed that disoles on addition of mL ‘of ammonizm chloride solution R. Add 1 ml. of ddim ‘iydrogen phosphate solution R. A white crystaline precipitate is formed, ‘To 0.5 mL- of « neutral or slightly acidic solution ofthe Sapte being examined add 0.2 mL of 0.1% w lion of titan yellow and 0.5 mL of 0.16 sodium Hhydroxide. bright red turbidity is produced which ‘radually sets to give a bright red precipitate we ‘and Mercury Compounds Place about 0.1 mL of a solut ce tobe aoe hn ution of the substanc ‘that becomes, B,_ Tothe presebed sluts bed Solution ad ne sodium horse until strongly alkain yellow ; alkane (2.24). A dense Precipitates formed (mercuric sal) Nitratesooo lll A260 Appendix VII —— Appendix VII Limit Tests (No Ph, Eur. method) Nessler Cylinders Where the use of Nesslor evinders is prescribed in atest of the Pharmacopocia, Nessler cylinders complying with the following requirements should be used. ‘esse cylinders comply with British Standard 612:1966 (Specification for Nessler cylinders). They are of clear glass with a nominal capacity of 50 mL; the overall height is about 15m, the external height to the 50-mL mark 11.0 to 124 em, the thickness of the wall 1.0 to 1.5 mm and the thickness of the base 1.0 to 3.0 mm. The external heights to the 50-ml. mark of cylinders used for a test must not differ bby more than 1 mm. ‘Tubes for Comparative Tests (Ph, Bur. mad 2.13) “Tubes used for comparative tests are matched tubes of colourless glass with a uniform internal diameter The base is ‘transparent and flat. A column of the liquid is examined down the vertical axis of ‘the tube against a white background, or if necessary, aginst ‘black background. The examination i carsed out in sifsed light. I's assumed that tubes with an internal diameter of 16 mim willbe used, Tulbes witha larger intemal diameter may be ‘used instead but the volume of liquid examined must then be {increased so that the depth of liquid in the tubes isnot less ‘than where the presribed volume of liquid and tubes 16 mm in intemal diameter are used. ‘Limit Test for Aluminium (Ph, Bur, method 24.17) ‘Place the prescribed solution in a separating funnel and shake ‘with 2 quantities, each of 20 mL, and then with one 10 ml. ‘quantity of 25 gL solution of iodroyauinaine R in chloroform R. Dilute the combined chloroform solutions to 50.0 ml. with chlorform R (test solution). ‘Prepare a standard in the same manner using the prescribed reference solution, ‘Prepare a blank in the same manner using the prescribed ae fess Measure the intensity of the fuorescense (2.2.21) of the tat ‘solution (J;), of the standard (J,) and of the blank (/3) using ‘an excitant beam at 392 nm and a secondary filter with a ‘transmission band centred on 518 nm or a monochromator ‘set to transmit at this wavelength. ‘The fluorescence (h~ 1) ofthe test solution isnot greater ees. }. Rowith 5 mL solution (1 ppm NHd Saya oe No piece of silver manganese paper R 5 mt square, Wetted witha low colour in the test solution is not the standard. Limit Test for Arsenic (Ph, Baer. method 2.4.2) ee ee et eee ee a com nei cee Position by two spiral springs. Into the lower tube insert ‘small plug of cotton and a rolled piece of lead acetate paper R ‘Weighing 50 mg to 60 mg. Between the flat surfaces of the ‘tubes place a disc or a small square of mercuric bronride paper R large enough to cover the orifice of the tube (ae es cyt ofa dea nie the prescribed volume to 25 mL. with ‘alin Be loo stand or 1 min and inosuce 34 inet ai mace re er, temperature such that a uniform evolution of ‘gas is maintained. Prepare a santana : Imhof ain in the same mannet sine 25 a andar oun 1 ppm As), dite © After not less than 2 bromide paper in sander, J the stain produced on the merc the testis mot more intense than that i | 5 mg of pasion ode Band add for 15 can eae R. Heat the misture ona wae ame nity shaking occasionally, Prepare a standart 110 pai 0.5 mL of arsenic standard solutionging on the wat er het ‘Mfgon not more intense than that in the standand ath, any colour in the test 30 Figure 2.4.2-1. - Apparatus for limit test A for arsenic Dimensions in millimetres Limit Test for Caleium (Ph Eu, method 2.43) AM soln wed fortis test shold be prepared with tiled ae R To02 mL of aloha calcium standard solution 100 ppm Ca) Ry add 1 mL. of ammonivom oxalate solution R. ‘Mier min, add a mixture of 1 mL of die ace acid R and 1S ofa solution containing the prescribed queniy of the ‘ubwance tobe examined and shake. Prepare a standard in ‘Besume manner using a mixture of 10 mL. of aqueous ‘aim sandard solution (10 ppm Ca) R, 1 ma of due aeric sad R and 5 mL of dsl water R. ‘fer 15 min, any opalescence in the test solution i not "or intense than chat in the standard Limit Test for Chlorides (Ph. Bur method 2.4.4) To 1st afte peeibed scuon a1 ma of die ie ‘cid Rand pour the mixture asa single addition into tes ‘Ube containing 1 mL. of silver mitrate solution R2. Prepare & ‘Statin the same manner using 10 lof cherie tered soluion (5 ppm CD R and 5 mal. of wer Str habs raat 2 Back stan Standing for 5 min protected from ligt a" {Plsesnce inthe est slution i nt more intense than that the Standard, Appendix VII A261 Limit Test for Fluorides (Ph, Bur. method 2.4.5) Figure 2.45.1. ~ Apparatus fr limit test for fluorides Dimensions in millimetres Introduce into the inner tube of the apparatus (see Figure 24.5.1) the prescribed quantity ofthe substance to be ‘examined, 0.1 g of acid-washed sand R and 20 mL. of a mixture of equal volumes of sufric acd R and tater R. Heat the jacket containing terachlorocthane R maintained atts boiling point (146°C), Heat the steam generator and distil, collecting the distillate in a 100 mL volumetric flask ontaining 0.3 mL of 0.1 M sodium hydroxide and 0.1 mL. of in solution R, Maintain a constant volume {20 mL) in the tube during dstilation and ensue that the istllate remains alkaline, adding 0.1 M sodium hydroide i necessary. Dilute the dstilate 1 100 mL with tater (test sofution). Prepare a standard in the same manner by Sistillaton, using 5 mL of fluoride standard slaion (10 ppm B) Rinsead ofthe substance t0 be examined. Ino «wo las-stoppered cylinders introduce 20 mL ofthe test Solution and 20 mL. ofthe standard and 5 mL. of D————————————— A262 Appendix VII [After 20 min, any blue colour in the test solution (originally red) isnot more intense than that in the standard, Limit Test for Heavy Metals (Ph. Eur, method 2.4.8) “The methods described below require the use of shioacetamide reagent R. As an alternative, sodium sufide solution RI (0.1 ml) is usualy suitable, Since tests prescribed in ‘monographs have been developed using thioaceramide reagent R, if sodium sulfde solution R1 is used instead, iis necessary to include also for methods A, B and Ha monitor solution, prepared from the quantity of the substance to be examined prescribed for the test, to which has been added the volume of lead standard solution prescribed for preparation ofthe reference solution The test invalid ifthe ‘monitor solution is not at least as intense as che reference solution. METHOD A Test solution 12 ra. ofthe prescribed aqueous solution of the substance to be examined. Reference solution (standard) A mixture of 10 mL of lead sandardsobion ppm PB) Rox lead standard solution 2 pom Pb) R, as prescribed, and 2 mL of the prescribed aqueous solution of the substance t0 be examine. Blank solution & mixture of 10 mL of water R and 2 mL. ‘of the preseribed aqueous solution ofthe substance 10 be examined, “To each solution, add 2 ml. of bufor solution pH 3.5 R ‘Mix andl add to 1.2 mi of shioaetamide reagent. ‘Mix immediately. Examine the solutions after 2 min ‘System suitability ‘The reference solution shows a slight ‘brown colour compared to the blank solution Result Any brown colour in the test solution is not more ingense dhan that i the reference solution Ifthe results dificult to judge, ter the solutions through a suitable membrane filter (nominal pore iz 0.45 yn). Cary ‘ut the filtration slowly and uniformly, applying moderate land constant pressure 1o the piston. Compare the spots on the filters obeined wih the diferent solution. METHOD B Test solution 12 mi ofthe prescribed solution ofthe ‘substance to be examined prepared using an organic solvent containing 2 minimum percentage of water (for example, ‘ioxan containing 15 percent of water or acetone coniaining 15 percent of water, Reference solution (standard) A mixture of 10 mL of dead standard solution (1 or 2 ppm Pb), as prescribed, and 2 mo the prescribed solution of the substance to be agents, Frome nd acd ‘solution (1 or 2 ppm Pb) by eiution of fad standard sation (pm ih res ed fe sb 0 ‘Amixture of 10 mL of the solvent used and 2 ml. of the METHOD ¢ : werent nt Teal soon Pio be omnes ck tan 2 of eon of mapesion sae Rin A a Eng aie so Het lt a ero 0 io ee pope bat eon oe en Be ee eee Mates de mie ge een oe ey ark jtoas om ee a eis ana Beer eae aaa al a Fy re nk ee ial ge coe act et ee Tete tate re ro toe tee 20 mt wth phere eee ten anand) repes w ccitd i Peer a aid pests otis otal oe Deen Fae ator oe vanance te Seo rn ofthe rola coated nde 2 Sor ninion Ree op pa acs te te en Be os cain we wie Saad eet sic Io ppm Po) R pcre fe tee cece tan io ome ote ee ees a fat re a en Flak elton Amis of 10 of wo Band? ae To 12 ml ofeach shin a2 of bf ok Bia Ris ant at slot oooate ae Gee eectcauiren tuiet ae Sur east eee ier eet kts kee Se solution. ‘i k as Tess hs Boh cain x pion ot ae ee Hie nuts dificle to judge, fter the solutions thot * ee txt fete ton and constant pressure to oo METHOD D. F the result isd suitable membran (nomial pore size 0.45 jm). C ad uniformly, applying mode the piston, Compare the so °F the differen solutions. Test solution tn asi Tn sca eruibe, mix thoroushly Memeo oR. ae wd ines wi fae te or grevish-thite mass i obtain Allow to cal mag Bat the mize remains cloud ition near fine las od and repeat te Tor abou 1 eSS8S28YFepeat the operation. Heat at 800 ©. Pees 1B. Take up the residue in 2 quanti «8% nd ater Ata go ages volumes of felon ee O.1 mL of phenipalin son R ammonia Rana pink clout 03\ ==-== Figure 24.8.1. Appendix VII A263 Membrane Prefiter fiter Membrane ~~~ Prefiter filter Prefitrtion Filtration ofthe ofthe solution solution after (Method &) ‘addition of the reagents (Method E) Apparatus for the test for heavy metals Dimensions in millimetres Conk ad lasil acre acd R until the solution is decolovised ‘od.add 05 mL in excess. Fiter if necessary and wash the fer Dilute to 20 ml. with aazer R. Reference solution (standard) Prepare as described for the est solution using the prescribed volume of ad stondand ‘alin (10pm PB) R instead of the substance to be feamied and drying in an oven at 100-105 °C. To 10 mL. he solution obtained add 2 ml. ofthe test solution Monitor solution Prepare as descrved for the test ‘ution, adding to the substance to be examined the volume ‘of ad standard solution (10 ppm Pb) R prescribed for persion ofthe reference solution and drying in an oven 4100-105 °C, To 10 ml. ofthe solution obtained add il. ofthe text solution. Blank solution A mixture of 10 mL. of water R and 2 ml. ofthe rest solution, To 12mL of each solution, add 2 mL of buf sion ‘PHS R. Mix and add to 1.2 mL of shioaceramide reagent B. Mix immediately, Examine the solutions after 2 min : System suitability the reference solution shows a slight brown colour compared to the blank solution, ~ the monitor solution is atleast as intense as the reference ‘sug, Result Any brown colour in the test solution tose han hatin he reference sluion * the result i dificult to judges filter the solutions through tbe membrane filter (nominal pore size 0.45 wm). Ca" oy Hatin slowly and uniformly, applying moder ‘constant pressure to the piston. Compare the spor O° ot more ‘Se fltes obtained with the different solutions. EB Tages the solution Dissolve quantity of 7 nce tobe cumined in 30 mio eur # oF Mrectibed volume. Reference solution (standard) Unie: prescribed, dilute the prescribed volum solution (1 ppm PB) Ro the same volume 3s solution. Prepare the filtration apparatus by adaptin 50 mL. syringe without its piston to a supp the plate, a membrane filter (nominal pore above ita prefter (Figure 2.4.8.-). ‘Transfer the test solution into the syringe piston in place and then apply an even the whole ofthe liquid has been fie support and removing the prefilter, check tha fier remains uncontaminated with i the ease replace it with another membra the operation under the same cond To the prefitrate or to the prescribed vo to 1.2 mL of shioace allow to stand for 10 min and again fi ‘but invering the order of the fil prefer (Figure 24.8.-1). The slowly and uniformly by applyn pressure to the piston of the syringe. A filtration, open the support, remove th dey using filter paper. In parallel, treat the reference solution the test solution. Result The colour of the spot obtained w solution is not more intense than that ob reference solution. METHOD F ‘Test solution Place the prescribed quantity the substance to be examined in 2 fong-necked combustion flask (a 300 ml. & if he reaction foams excessively). Clamp the fask at an Magle of 45" Ifthe substance to be examined isa slid, 2A264 Appendix VII sufficient volume of a mixture of 8 ml. of sure acid R dnd 10 ml of nitric acd R to moisten the substance thoroughly ifthe substance to be examined isa liquid add a few mililiues of a mixture of 8 mL. of sufuric acid R and 10 ml of mmc acid R. Warm gently until the reaction commences, allow the reaction to subside and add adeitional portions of the same acid mixture, heating after each Addition, until a total of 13 ml. ofthe acd mixture has been fadded, Increase the amount of heat and boil geny until the Solution darkens. Cool, add 2 ml. of ni acid R and heat ‘gan until the solution darkens. Continue the heating, followed by the addition of mime acid R until no further darkening occurs, then heat strongly until dense, white fumes are produced, Cool, cautiously add 5 mL of eater R, boil ently until dense, white fumes are produced and continue Fheating to reduce to 2-3 mL, Cool, cautiously add 5 mL. of toater Rand examine the colour ofthe solution, If the colour is yellow, cautiously add 1 ml. of srong ipdrogen peroxide Soliton R and again evaporate until dense, white fumes are produced and reduce to a volume of 2-3 mL. Ifthe solution {s sill yellow in colour, repeat the addition of 5 mL of ‘eater Rand 1 mL of song hycogen poroidesolucon R wos the solution is colourless. Cool, dilute cautiously with ‘eater R and rinse into « 50 mi. colour comparison tube, ensuring thatthe ttal volume does not exceed 25 ml. ‘Adjust the solution to pH 3.0-4.0, using short range pH indicator paper as extemal indicator, with concentrated ‘ammonia RI (dle ammonia RI may be used, if desired, 2s ‘the specified range is approached), dilute with teater R to 440 mL-and mix. Add 2 ml. of buforsolwion pH 3.5 R Mix and add 01.2 mi of thoacetamide reagent R. ‘Mix immediately. Dilute to 50 mL. with cater R and mix. ‘Reference solution (standard) Prepare at the same time and in the same manger asthe test solution, using the scribed volume of ad sandard soon C10 ppm) R ae ee tected tee jing to the substance to be examined the volume solution (10 ppm Pt) R prescribed for the of the reference Solution. of ead st Blank solution Prepare as described forthe tt solution, ‘omitting the substance to be examined. ‘Examine the solutions vericalyapninst a white background after 2 min. System suitability - the reference solution shows a brown colour compared to solution, aapea eres a ‘intense as the reference Resule Any inown colour in the test solution is not more ‘intense than that in the reference solution, ‘Ifthe results difficult to judge, filter the solutions through a filter (nominal pore size 0.45 im), Carry ‘out the filtration slowly and uniformly, applying moderate to the piston. Compare the spots on ‘with the diferent solutions. wing high-pressure digestion ves the safry ing instructions given by the manufacturer race the prescribed amount ofthe recat a ee se scans Menke, ad suecesitely 27 ma fs se eo marc ci abd 2.0 ma. f stone fg at 9 sing 0 magnetic sitet, AW the er et wh 2 reagent before ding te neo, to a dry high-pressure-resistant Tea sel civropamer oF sua) Reference solution (standard) Prepare a described fa Referens ion using the prescribed volume of lad sony the rv ppm PB) instead of the substance tobe examined. arfor aottion Prepare as prsceed for the tex ora andard solution (10 ppm Pb) R prescribed forte reparation ofthe reference solution Blank solution Prepare as described for the test solution, aenitng the substance to be examined ‘Close the vessels and place in a laboratory microwave oven Digest using a sequence of 2 separate suitable programmes Diuign the programmes in several steps in ordcr to contol the reaction, monitoring pressure, temperature oF enensy qTepcnding onthe ype of microwave oven available. After the first programme allow the digestion vessels to cool before opening. Add to each vessel 2.0 mL. of stone hyogen povoidesaluion R and digest using the second programme. ‘After dhe second programme allow the digestion vessels t0 ool before opening. If necessary to obtain a clear solution, repeat the addition of song hydrogen peroxide soluion R and the second digestion programme. Cool cite cautiously with water R and rinse int a fas ensuring thatthe total volume does not exceed 25 ml. ‘Using short-range pH indicator paper as extemal indicate, aust the solutions to pH 3.04.0 with concentrated ‘ammonia Ri (dae anmmonia RI may be used asthe specied range is approached). To avoid heating ofthe solutions use an ice-bath and a magnetic stirer. Dilute to 40 mL with tear Rand mix. Add 2 ral. of bur slion pH 3.5 R Mix and add to 1.2 mi. of thioacetamide reagent. Mix immediatly. Dilute to 50 mL with eater R, mix and allow to stand for 2 min, tthe sltos thr sate membrane fie (nomial pore size 0.45 jm). Carry ont the filtration shows ee ee oe the lon Compare he spas on thefts aban System suitabiity ~ the spot abiined with the reference solution shows @ own colour compared to the spot obtained with the SS i Sd — the spot obtained with the monitor station i atleast pettbe th aoe whe enc =" Revit The brow exour of the sot obtained wi O° ion is Not more intense than that of the spot obianed withthe efeence sluion, METHOD H Test solution Dissoh issolve the prescribed quantity ofthe substance to be examined in 2 quantity 3 ined in 20 ml. ofthe solvent oF Soh ‘mixture prescribed. Poh Reference solution Di ! itandard fon Dilute the prescribed volume of Sandard sation (10 ppm 8) Ro 20 en wih te S08 ‘mixture prescribed,Bes sisnircotamer peered: ~~ Fs? 8 eer nn DR paitereredecicin nape yee per oren Aix immediately and allow to stand for 2 min, Filter the ene erect omer ‘pe aiferent solutions. c System suitability The spot obtained with the reference ee ‘pnsined with the blank solution, a 5 Seelam ont Beg sts tthe ence tat a ot erence oe Limit Test for Tron (0% Ba, method 2.4.9) Disolve the prescribed quantity ofthe substance to be eamined in water R and dilute to 10 mL. with the same stent or use 10 mL. of the prescribed solution. Add 2 ml. £1200 gL solution of cin acid Rand 0.1 mL. of thebealic acid R. Mix, make alkaline with ammonia R and Geto 20 mL with wear R. Prepare a standard in the same runner, using 10 mL. of iron standard sluion (1 ppm Fe) . “After 5 min, any pink colour in the test solution is not more “intense than that in the standard. Limie Test for Lead in Sugars (Ph Ba, method 24.10) ‘Determine the lead by atomic absorption spectrometry (2.2.23, Method ID. os Dissolve 20.0 g of the substance to be 7 in a mixture of equal volumes of dite actc acid R water R and dilute to 100.0 mL. with the same mixture “Add 2,0 mL of ¢ clear 10 g/L solution of i idinedithio Rand 10.0 mL of met ‘io! hetane R and then shake for 30's protected from "right ight, Allow the layers to separate and use the methyl “iobuy ketone layer. | solutions. Prepare 3 reference solutions in the "spe manner as the test solution but adding 0.5 ml ‘of lead standard solution 20.0 g of the substance to Appendix VII A265 the solution of hydroxyquinoline in chloroform, shake for | min, allow to stand and separate. Use the lower layer for comparison, Prepare a standard in the same manner using & mixture of | mL, of magnesium standard solucion (10 70m Mg) Rand 9 mL. of water R ‘Any colour in the solution obtained from the substance to be examined is not more intense than that in the standard. Limit Test for Magnesium and Alkaline-earth. Metals (Ph, Bur. method 2.4.2) ‘To 200 mL of water R add 0.1 g of hyvromlamine Iydrociorde R, 10 mL. of ammonia chloride buffer slaton pH 10.0 R, mob of 0.1 M zine sulfate and about 15 mg of ‘mordant black 11 trituate R, Heat co about 40 °C. Titrate with 0.02 M sodium edetare until the violet colour changes to full blue. To the solution add the prescribed quantity of the substance to be examined dissolved in 100 ml. of water R oF ‘use the prescribed solution, Ifthe colour ofthe solution changes 10 violet, trate with 0.01 M sodium edetare until the full blue colour is agnin obuained. The volume of 0,01 1M sodium edetate used in the second titration does not exceed the presribed quantity Limit Test for Heavy Metals in Herbal Drugs and Fatty Oils (Ph, Ear, method 24.27) Examine by atomic absorption spectrometry (2.2.23) CAUTION: thon sing coed high-pressure digestion vessels and ico laboratory equipment, be famatar eth te sae and peracing instructions gin by the anufactre. Apparatus “The apparatus typically consists of the following — as digestion fis, polstetafluoroehylene flasks with a frolume of about 120 mi, fied with an aright closure, @ Calve to adjust the presure inside the container and a politetrafiuoroetislene tube to allow release of es, — a system to make flasks sich, using the same torsional force foreach of them, = a microwave oven, with 2 magnetron frequency of 2450 MHz, witha selectable ourput from 0 to {630 70 W in 1 percent increments, a programmable tigial computer, «polytctralluoroethyiene-coated ticrowave cavity witha variable speed exhaust fan, a Totatng turntable dive system and eshaust tubing .o vent fumes, — an atomie absorption spectrometer, equipped with holow- Cathode lamps as source of radiation and a deuterium lamp as background corrector; the sytem is ited with: (@) a graphite furnace as atomisation device for cadmium, copper fon, lead, nickel and zinc () an automated continuous-flow hydride vapour generation system for arsenic and mereuy Method Incase akernative appara is wed, am adjustment of he marae paramews may be neces (Clean all the glassware and laboratory equipment with a 10 gl solution of ioc acid R before we. ‘Test solution In digestion flask place the prescribed dani of the substance to be examined (about 0.50 of erred dra (1400) (2.9.12) of 050 g of faty oi, Be 6 mL of heavy metal-free mic acid Rand 4 mL of eae matalfreedochone acd R. Make the fask aright.A266 Appendix VII Place the digestion flasks in the microwave oven. Carty out the digestion in 3 steps according to the following programme, used for 7 flasks each containing the test Solution: 80 per cent power for 15 min, 100 per cent power for 5 min, 80 per cent power for 20 min. ‘At the end ofthe cycle allow the flasks to coo! in air and to teach add 4 mi. of heaeyy metal-fre sufi acd R. Repeat the digestion programme. After cooling in air, open each digestion flask and introduce the clear, colourless solution ‘obtained into a 50 mL. volumetric flask. Rinse each digestion flask with 2 quantities, each of 15 mL, of water R and collect the rinsings in the volumetric flask. Add 1.0 mL of a 10 a1 solution of magnesium mizaze R and 1.0 mL of a 100 g/L. solution of ammoniion dihydrogen phosphate R and dilute to 50.0 ml with eater R. Blank solution Mix 6 mL. of heawy metal-ee mii acid R ‘and 4 mL of heaey metai-ie hydrochloric acid Rin a digestion flask. Carry out the digestion in the same manner as for the tes solution. ‘CADMIUM, COPPER, IRON, LEAD, NICKEL AND ZINC ‘Measure the content of cadmium, copper, iron, lead, nickel ‘and zine by the standard additions method (2.2.23, _Mechod ID), using reference solutions of each heavy metal and the instrumental parameters described in Table 2.4.27.-1 ‘The absorbance value of the blank solution is automatically subtracted from the value obtained with the test solution. Table 24.271 Gt Ne Vecengh 88 ERS SS TR Se Siwidh M05 0502020505 Lampeurest, mA 6 75ST eation se aw 800 800 800 800 8m ‘epertare ‘Aumiston °C 1800 2300 2500 2500 2200 2000 ‘emperture — edo ott cores Nuwgesfow Wmin 3033333 ARSENIC AND MERCURY “Measure the content of arsenic and mercury in comparison ‘with the reference solutions of arsenic or mercury ata known concentration by direct calibration (2.2.23, Method J using an automated continuous-flow hydride vapour generation system, ‘The absorbance value of the blank solution is automatically ‘subtracted from the value obtained with the test solution, ARSENIC Sample solution To 19.0 mL. of the test solution or of ‘the blank solution as preseribed above, add 1 mL. of a 200 gL. solution of porassiom iodide R. Allow the test solution oe for about 50 min or at 70°C. for Jo jin Table 2.4.27.-2 may be ide wi me ‘or blank solution, as. ie rian —=—_—_-—lhl ;$wtC~O ‘peta reagent 515; sition OF hay meta re Iiydrochloric acid R. Reducing reagent in dilute heavy metal-free The instrumental parameters used. ‘A 10 g/L solution of stannous chords Inydrochloric acid R. in Table 2.4.27-2 may be Table 2.4.27-2 s,s sadn ep ee ee nee 1987s sit with = oz os knee ae rs nies noreaatam ati 282% bw Serta (GE ~ ants asia er ers toe Limit Test for Nickel in Polyols (Ph, Bur, method 2.4.15) Determine the nickel by atomic absorption spectrometry (2.2.23, Method 1D, Test solution Dissolve 20.0 g of the substance to be examined in a mixture of equal volumes of die aceic acd R and waver R and dilute to 100.0 ml. with the same mixture of solvents, Add 2.0 mL of a saturated solution of anmoniun Pyrralidinedthiacarbamate R (about 10 g/L) and 10.0 ml-of methyl isobusy! tone R and then shake for 30 s protected from bright light. Allow the layers to separate and use the ‘methyl isobutyl Ketone layer. Reference solutions, Prepare 3 reference solutions in the Same manner as the test solution but adding 0.5 ml, 10 mL and 1.5 mL respectively of mike! standard solution (20 ppm Ni) R in addition to the 20.0 ¢ of the substance t0 be examined, Set the zero of the instrument using meth! isobutyl ketone R eae as desenibed for preparation of the test sluion ‘omitting the substance to be examined, Measure the absorbance at 232.0 nm using a nickel hollow-cathode lam? 88 source of radiation and an air-acetylene flame. ‘The substance to/he examined contains not more Us pm of nickel, unless otherwise prescribed Limit Test for Phosphates Ph. Bur, method 24.11) To 100 mi of the sou ion prepared and, if necesars neutralised as prescribed add 4 mi of yi oy ‘Any colour in the in the stndang. Solution is mot more intense than tt Limit Test for Potassium. Ch. Ber maiad 24.12) ‘To 10 mL of the prescribed solution add 2 mi. of a fret S$— Appendix VIL A267 vpepae a standard in the same mann 2 inet using a mixture of 20 ppm K) Rand 5 mb. Pr pas standard solution oe 5 min, any opalescence Mose than that in the standard is not more Limit Test for Sulfates ‘pp Bur. tod 2.4.13) ns wed for this test must be prepared with did fait 3 mL of 250 g/L sation of barium chloride R to At aL of sulfate standard solution (10 ppm SO.) RI. Shake 4° ao to stand for 1 min, To 2.5 mL ofthis suspension sad 15 mL of the solution to be examined and 0.5 ml. of ‘ sit eacd R. Prepare a standard in the same manner using sete of sate standard soi (10 ppm S04) R instead of the solution to be examined. afer 5 mio, any opalescence inthe test solution isnot more pense than that in the standard,a 0 A » as Appendix VII F A271 appeal ts eka Seed § = sone le erode demoed eprinenay solutions whose concentrations dif ns difer by a factor ten and are situated within the range © = calibration curve is linear, * WT the F, Determination of Ethanol} ‘Use Method I ot, where appropriate, Method If unless “phenwise prescribed in the monograph, ; Method I (Ne Ph Eu method) (any ovt the method for gas chromatography, TIL, using the following solutions. Solution (1) contains 5.0% viv of absolute ethanol and 5.0% viv of prope Foal Gnteral standard). For solution (2) dilute a volume ot eparaton being examined with caer to contain feween 40 and 60% vv of eal, Prepress (3) in fhe sme manner a solution (2) but adding sufcient ofthe jniemal standard to produce a final concentration of 50% WW. ‘he chromatographic procedure may be cared out using a column (15m x 4 mm) packed with porous polymer beads (400 0 120:mesh) (Porapak Q and Chromosor® 101 are ‘siable) end maintained at 150° with both the inks port and the detector at 170°, the percentage content of ethanol ftom the aces of due to ethanol in the chromatograms obtained with Eur, method) dons in which, in accordance with the aubosty Industrial Methyated Spirit has the content of ethanol as described in solution (2) a volume of the diluted with rater to contain ‘of total ethanol and methanol. ‘of methanol in the following procedure described following solutions. Solution fand 0.25% vi of propa {@) dilute a volume of in grams of ethanol per 100g ofthe liquid. This is known a3 © “Percentage of ethanol by mass’ (per cent mim), Method A ‘Where preparations contain dissolved substances, the dissolved substances must be separated from the ethanol that 's to be determined by disilation. Where distillation would ist volatile substances other than ethanol and water, the ‘ppropriate precautions are stated in the monograph, ‘The relation between the density at 20 + 0.1 °C, the relative density (corrected to vacuum) and the ethanol content of a mixture of water and ethanol i given in the tables of the International Organisation for Legal Metrology (1972), Tterational Recommendation No. 22 Figure 2.9.10-1. ~ Apparatus for the determination of ethanol content Dimensions in millimetres ee pars Ge: Hes 20001) oe cre tak () etek a Seinen ed Fp rei cepa tc sree cma a lei ore pata le OF Oi casi fe po 100 at cn Te vlunci ft Srwetina no: ot nae (ee pac hea «coor sere om an, wc ik CD wees eT of Soe amy ane ss_& ‘A272. Appendix VIII F METHOD Pycnometer method/oscillating transducer density ‘meter method “Transfer 25.0 ml. of the preparation to be examined, measured at 20 + 0.1 °C, to the distillation flask Dilute with 100-150 ml. of diiled water R and add a few pieces of pumice. Attach the ditllation head and condenser. Distl and collect nor less than 90 ma. of distillate in 2 100 mi volumetric flask. Adjust the temperacure to 20 + 0.1 °C and dilute to 100.0 ml with dial sazor R at 20 £ 0.1 °C. Determine the relative density at 20 + 0.1 °C. ‘using a pycnometer or an oscillating transducer density ‘meter, ‘The values indicated in Table 2.9.10.-1, column 3, axe ‘multiplied by 4 to obtain the percentage of ethanol by volume (V/V) contained in the preparation. After calculation of the ethanol content using the table, round off the resul 10 1 decimal place ‘Table 29.101, - Relationship between density, relative density “and ethanol content hie eevee ‘iol ott (egw?) sate measured air ver coat 2 2120 se a ee MD 5 nee ni 840 9858 060 roe none 11s ia 9868 976 am ses 935 ey sis so 855 od as sro 688 4 srs esees ee a «9608 74 mie ‘ams 695 9.0 0s 656 05 sens en 0.0 ges 510 ns; 5 542 woo oe28 504 sous 983 467 woo ost08 430 ams a st x30 ss 358 ws 953 an 240 08 286 4s 969 251 50 968 216 55 osm 132 6.0 vos 1a 85 908 ia oon sas oxo ors sos 046 sre 686 an SEE a eT Hydrometer method Transfer 50.0 ml. ofthe Preparation to be examined, measured at 20 + 0.1 °C, 0 the dsitation fash, aed 200-300 ma. of diilat cate Rad Hee tan eed above nwo a volumetric ask unt 3 20 Oe a Bee lcd Adjust the emperatre ace % 250.0 mL. with dil water Rt Transfer the dstilate ro acyl Cylinder whose diameter sat st {im mde than the bulb of the hydrometer If the vlie PRRIEDY the strength by 5 tallow forthe dition dung— B 18 chromatography (2.2.26) Beonal standard solution Die. mit. of Sen trees eaceee ion is olution Dilute a volume of the prepara Maines coresponding to 1 g of ethanol 50.0 mh eh Ge Dilute 1.0 ml. of the solution to 20.0 mL with R To 2.0 ml of this solution add 1.0 ml. of the ssandard solution and dilute to 20.0 mL. with solution (a) Dilute 5.0 mL of eh sth eater R, Dilute 25.0 mL ofthe sonny swith water R. Dilute 1.0 mL of this solution we ‘water R. solution (b) Mix 0.5 mL of reference so ‘mal. of the internal standard solution and dilute (9200 mL with water R. erence solution (c) Mix 1.0 ml. of reference solution : Bpetieticeecirecs tckaion [le ofthe imemal standard scion and dite Dilute 1.0 mL of aniyaous ) mL with water R. Dilute 1.0 mL. of the Ses Vo Relative retention With reference to ethanol (Getention time = about 5.3 min): methanol = about 0.8; {propanol = about 1.6. ‘System suitability Reference solution (D; — resolution: minimum 5 between the peaks due to methanol and ethanol. Establish a calibration curve with the concentration of ethanol in reference solutions (b), (2), (4) and (a8 the albscissa and the mean rato of the peak area of ethancl to the Peak area ofthe internal standard in the comesponding chromatograms as the ordinate. Calculate the percentage content of ethanol in the preparation tobe examined. Method Gas chromatography (2.2.28). Internal standard solution to 100.0 mL with waer R. Test solution Dilute « volume of the preparation to be ‘examined corresponding to 1 g of ethanol to 50,0 mL. with seater R. To 1.0 ml. of this solution add 1.0 mL of the internal standard solution and dilute to 20.0 mL. with seater R. Reference solution (a) Dilute 1.0 mL. of ethanol RI vo 50.0 mL. with cater R. Reference solution (b) Dilute 1.0 mL. of anlyaiows ‘methanol Rto 100.0 ro with water R, Dilute 1.0 mL. of the solution to 20.0 mL. with eater R. Reference solution (c) Mix 1.0 ml. of the internal standard solution, 1.0 mL of reference solution (a) and 2.0 mL of reference solution (b) and dilute to 20.0 ml. with water R Column: — material used sic — sige: = 30 m, = 0.53 mms Dilute 1.0 mL. of propanol R — stationary phase: ‘pos(Cevanopropy) (phony) jidimetkysiloxane R (rm thickness 3 ym). Carrier gas helium for chromatography R. Flow rate 1 ml/min. Split ratio 1:50. Temperature: Sah Tementere ee ‘Column 0-16 0 16-99) 0465 99-188 69115 136-20 1 Injection port 200 Detector 200 Detection Flame ionisation. 1.0 jL-of the test solution and reference solution (c), atleast 3 times. ‘Elution order Methanol, ethanol, 1-propancl ‘Relative retention With reference to ethanol (fetention me ~ about 5.3 min): methanol Tepropanol = about 1.6, about 0.85A274 Appendix VIII G ‘System suitability Reference solution (©) “= resolution: minirmum 5 between the peaks due to methanol and ethanol Calculate the ethanol comtent in per cent V/V using the following expression: Ai xh xp Ax h xvi ea ofthe peak due to ethanol in the chromatogram ‘obtained with the test solution; ‘Ay, = area of the peak due to ethanol in the chromatogram obtained with reference solution (6); area of the peak due to the intemal standard in the chromatogram obtained with the test solution; area of the peak due to the intemal standard in the chromatogram obtained with reference solution ( Vi, = volume of the preparation to be examined in the test solution, in mililitres; p= percentage content of ethanol in ethanal RI. G. Determination of Methanol and Propan-2-ol (Ph Bur. method 2.9.11) Dilute 1.0 mL of propanol R R, Dilute 1.0 ml. of the solution to can 40465 99-138 +15 136-20 "5 Ineton post Detection Flame ionisation. Injection 1.0 ml. of the gaseous phase of the test solution tnd reference solution (C), at least 3 times. ‘Elution order Methanol, ethanol, 2-propanol, I-propano With reference to ethanol (retention time = about 5.3 min): methanol = about 0.8; 3 propanol = about 1.2; I-propanol = about 1.6 ‘System suitability Reference solution (6 — esohtion: mninimum 5 between the peaks due to methanol and ethanol. Calculate the methanol content in pet cent V/V using the following expression: Relative retention Ax Ia xp A xh x40 A, = area of the peak due to methanol in the chromatogram obtained with the test solution; ‘Ay = area of the peak due to methanol in the chromatogram obtained with reference solution (os J, = area of the peak due to the internal standard in the chromatogram obtained with the test solution; Jz = area of the peak due to the internal standard in the chromatogram obtained with reference solution (€) P= percentage content of methanol in anhydrous methanol R, Calculate the 2-propanol content in per cent V/V using the following expression: Asx h xp Ack hh x 40 Ay = ea of the peak due to 2-propanol in the chromatogram obtained with the test solution; Ay = area of the peak due to 2-propanol in the chromatogram obtained with reference solution (0s 1 = area of the peak due to the internal standard in the chromatogram obtained with the test solution; J = area of the peak due to the intemal standard inthe chromatogram obtained with reference solution (© P= percentage content of 2-propanol in 2-peparolR Method B Gas chromatography (2,2.28). Internal standard solution Dit propane 101000 mL with car NS LOE Test solution Mix 1.0 ml. of th \ mL ofthe intemal standard solution and 4.0 mL. of the preparation to be examined #™4 AEs 200 Wh ts= Appendix VII H_ A275 mL with eater R, Dilute 1.9 mL of finer, ME? 5 Ah aaawesaaerwapoaeres : tion (b) Dit ar Maa y Caen "SLO mL St tan Ri | _ 4, _ SMITA ne wi he et ohn ly = area o fue to 2-propanol in the , Bee eine See ore torso shun, 1.0 ml of teen ston (ea 4h = area of the peak due tothe internal standard inthe SoraL-ofreference solution (a) and dite to 20'9 chromatogram obtained with the test solution; Jip wer Re 4h = sre ofthe peak due wo the imal anda in he * chromatogram obtained with reference solution (€); ae P= percentage content of 2-propancl in 2-propanal R. |) Fier i= 30m, @ = 0.53 mm, rionary Phase: thine pbieamerens) (Phew) dmeosonane Rim H, Determination of Nitrogen ‘bickaess 3 umn). ‘Use Method T unless otherwise specified in the monograph Carrer gas hen for chromaxography R. Method 1 iow rate mLsmin, (Ph. Bur. method 2.59) ‘split ratio. 150. SEMI-MICRO METHOD Place a quantity ofthe substance tobe examined (m ) containing about 2 mg of nitrogen in a combustion flask, ada 4 gota powdered mixture of 100 g of dpoassion sulfate Ry 5 8 of copa nate R and 2.5 of slonion R, and three glass beads, Wash any adhering particles from the neck into the flask with 5 ml. of sufi acid, allowing it to run down the sides of the Bask, and mix the contents by roxation. Close the ‘mouth ofthe flask loosely, for example by means of a glass bulb witha shore stem, to avoid excessive loss of sulfuric acid, Heat gradually at fst, then increase the temperature tant there i vigorous boiling with condensation of sulfuric acid in the neck ofthe flask; precautions should be taken to prevent the upper part ofthe flask from becoming Covetheated. Comsinue the heating for 30 min, unless ‘otherwise prescribed. Cool, dissolve the solid material by cautiously adding tothe mixture 25 mL of rater R, cool again and place in & stcam-dstilaion apparatus. Add 30 mL. of sro scion hydroxide solution R and dist immediately by passing team through the mixture. Collect about 40 mL. of Aistilate in 20.0 ml. of 0.01 M Jydochoric acid and enough tear Rto cover th ip ofthe condenser Towards the end ofthe disllaton, lower the receiver so thatthe tip of the ‘condenser is above the surface of the acid. Take precautions to prevent any water on the outer surface of the condenser from reaching the contents of the receiver. Ttrate the distillate with 0.01 M sadium hydroxides using methyl red mixed solution Ras indicator (n, mL. of 0.0! Msoduon hydroxide. Repeat the test using about 50 mg of glucose R in place of the substance to be examined (2 mL of 0.01 M sodium yds) 0.01401 (na — ni) Content of nitrogen = S°MEN (2 =) per cont ‘Method II (Determination of Protein in Blood Products) (No Ph. Bur, method) For dried blood products prepare a solution of the preparation as directed in the monograph. To a volume expected to contain about 0.1 g of protein add sufficient saline solution to produce 20 mL. To 2 mL of the esulting solution, in a 75-mL. boiling tube, add 2 mL. of a Solution containing 75% wv of nimgen-free sulfuric acd, 4.5% wiv of porasi sulfate and 0.5% wiv of copper) sudan, enix and loosely stopper the tube. Heat gradually to polling, boil vigorously for 1.5 hours and cool Ifthe solution Pomme ar add 0.25 mL of hydrogen peroxide solutiona Appendix IX t Determination of Sulfateq Ash ffeMethod Lunlessothervise dizecod, ‘pethod I Ph, Ei. method) Hata plinum dish 0 redness or 10 minutes, low fadeiator and weigh. Unles otherwise sper eo orice rl nl een 6, moisten with sufrc acid, ignite genus, agnin mois sth seis acd and ignite at abou 800°. Coot, wagh Soir bmn cater ncaa an sss weishings do not differ by more than 05 me, Method IL (Ph Bur, method 2.4.14) fie asitable crucible (for example, silica, platinum, yocsan or quartz) at 600 + 50 °C for 30 min, allow to soon a desiccator over silica gel or other sulatle deccant tei, Place the prescribed teexamined inthe crucible and weigh. Mossten the sane to be examined with a smal amount of sue af R(osually 1m.) and heat gently at as fow 6 ‘enpeature as practicable until the sample is theroughiy tamed Alter cooing, moisten the residue with» small amount of ue acid R (usually 1 mL), heat genly unl sie fmes are no longer evolved and ignite at 600 2 50 °C antl the residue js completely incinerated. Ensure that flames ‘enot produced at any time during the procedure. Allow ste ceuible to cool ina desicestor over sic gel or other desiccant, weigh it again and calulae the percentage : ii While momentarily removin " 8 the stopper of the flask, 2 hed quantity of 50 to 100g of te substance being {ambos het gently and bl for 48 mince, Die ee the absorption tubes before ie mL of 0. wlio ere is equal wo 3203 mg of salar ee Peat the operation without the substance sxamined, The schon in the atnorpton tubes ema sea dioxide Method II (Ph. Bar. method 2.5.29) Introduce 150 mL of rater R into the flask (4) (see Figure 2.5.20-1) and pass carhon dios R through the whole ‘stem for 15 min at a rate of 100 mLmin, To 10 mL. of ddiuae hydrogen peroxide solution R add 0.15 mL. of a1 gL. solution of bromophenol blue R in alcohol (20 per cent VIV) R. ‘Add 0.1 M sodium hydroxide until a violecblue colour is ‘obtained, without exceeding the end-point. Place the solution Jn the testtube (D). Without interrupting the stream of carbon dioxide, remove the funnel (B) and introduce through the opening into the flask (4) 25.0 g ofthe substance to be ‘examined (mg) withthe aid of 100 ml. of eater R. ‘Add through the funnel 80 mL of dite hydrochloric acid R and boil for Ih. Open the tap of the funnel and stop the flow of carbon dioxide and also the heating and the cooling ‘water, Transfer the contents ofthe test-rube with the aid of a litle water R to a 200 mL. wide-necked, conical flask. Heat ‘on a water-bath for 15 min and allow to cool. Add 0.1 mL. of 41 pL solution of bromophenol blue Rin alcohol (20 percent WIV) Rand titrate with 0.1 M sodivon hydroxide until theA292 Appendix IX C colour changes from yellow to vilet-blue (Vj mL). Carry ost ‘blank titration (V; mL). Calculate the content of sulfur dioxide in parts per milion fiom the expression 2080 « (Y=) x molarity of the sodium hydroxide solution used as C. Determination of Water ‘Use Method IA unless otherwise directed Method I (Ph. Eur, method 2.5.12) “The semismicro determination of water based upon the quantitative reaction of water with sulfur dioxide and iodine ih a suitable anydrous medium in the presence of a base with sufficient bulfering capaci: Apparatus The apparatus consis of a tiraon vessel swith: — 2 identical platinum electrodes: the ines for introduction of solvent and strats amine for introduction of air via a desivant a sample inlet fitted with a stopper or, for liquids, a septum. Inlet systems for introduction of dey nitrogen oF for aspiration of solvents may also be fed. “The titration is cared out according othe instrument supplier’ instructions. Care s taken throughout zhe determination ro avoid exposure of reagents and solvents to umospheric moistuce, The end-point is determined using 2 identical indicator electrodes connected wo an electical Source that maintains between the electrodes either a Gonstant current or a constant voltage. Where cree sition ‘eed (method A), addon of tirant causes either Jecrease in voltage where constant current is maintained or fn increase in current where constant voltage is maintained, unt the end-point is reached. Instzuments with automatic end-point detection are commonly used Standardisation To the tivation vessl, add moiana R, Grid if necessary, othe solvent recommended bythe Supplier ofthe titrant, Where applicable for the apparatus ‘sed, eliminate residual water from the measurement cll o¢ any outa pre-itration, Intuce a suible amount of ‘water in an appropriate form (ater R ora certfed reference Imateril) and carry owt the tiation, string for the necessary time. The water equivalent isnot less than 80 percent of that indicated by the supplier. Standardie che titrant before the frst use and ot suitable imtervals thereafter ‘Unless otherwise prescribed, use Method IA. ‘Method IA. Introduce into the titration vessel methanol R or the solvent indicated in the monograph or recommended by the supplier ofthe vitrant. Where applicable forthe ‘apparatus used, eliminate residual water from the “measurement cell or carry out a pre-titration. Introduce the substance to be examined rapidly and carryout the titration, sting forthe necessary extraction time, TB. Introduce into the tiation vessel methane R indicated in the monograph or recommended plier of the titrant. Where applicable for the eliminate residual water from the ig onesie ti tn eo A eae pn a ee eee ee ess of about 1 mL. or the preserbe Solon, Add an accra qolune, AD ae ete ane iki, with sring. Titrate the exces of rege presribed ribed solvent, containing aq Pe pth Ror the pe {2ebsc aos a oe Sa pcre aocurcy ofthe determination Wh Siti be vere for each substance he ag proce, gen oh a tp, ne Te scanning 25°25 8 Of Wate suitabl ‘The water content ofthe subst deeenmined using the reagenvsolvent system chosen ‘Tharwafter, sequential known amounts of tater R are added in an appropriate form (atleast 5 additions) and the ‘cumulative water content determined after each addition, Calculate the percentage recovery (7) at each point using th following expression: Wa 10057 WW; = amount of water added, in milligrams, W, = amount of water found, in milligrams. Calculate the regression line ofthe cumulative water determined against the water added. Calculate the slope (6 the intercept with the y-axis (a) and the intercept of the | extrapolated calibration line with the x-axis (4). | Calculate the percentage mean recovery (7). Calculate percentage errors (e, and 02) sing the following expressions 4 = the y-axis intercept, in milligrams of water; the scaxis intercept, in milligrams of water, water content ofthe substance, in milligrams of water. ‘The reagenvsolvent system is consi —ai| and | sd 10 be acceptable if fare not greater than 2.5 per cents — bis between 0.975 and 1.025 (deviation + 2.5 per cet) — Fis between 97.5 per cent and 102.5 per cent Method II Determination of water by distillation (Ph. Fur, method 2.2.13) . ‘The apparatus (see Figure 2.2.13.-1) mre 2.2.13.-1) consists of a glass flask (A) connected by a tube (D) to a cylindrical tube (B) fed with a graduated receiving tube (E) and reflux condenset (©), The receiving tube (E) is graduated in 0.1 mL Fre sours of heat i preferably an electric heater ith ‘heostat control or an oil bath. The upper portion of the And the connecting tube maybe insane | Method Clean the rece - receiving tube and the condenser of apparatus, thoroughly rinse with water, and dry Introduce 200 mL of toluene R and about 2 mL of eater R into the dry flask. Distil for 2h, then allow to coo! for abo% 30 min and read the water volume to the nearest 0.05 ml- acs inthe flask a quantity ofthe substance, weighed Si fan accuracy of 1 per cent, expected to give about 2:mL:'® 3m. of water. ithe substance has a pasty consist Weigh it im a boat of metal foil, Add a few pieces of PO ‘material and heat the flask gently for 15 min, When the0 boil, dist at the rate of fail most of the water has distiled ave, ao a mes, ghen the water has al distiled over, snes he en: tube with lene R. Continue the Peso 5 me Now he oe Sel ener a Gli y Gp sate ch Pen err ee whe ad toluene have completely soe Seva and cles te cnet sen be is ‘neo lilies per log, using the formas peeea eee 7 ger ae ee x ‘he mass in grams ofthe substance to be a examine, Be mumber of mililiues of water obtained inthe —_Polbimety 95) ‘eet, _ enhionse eee oe Brat 70 on 2Atetylenecolsero a9 080 Campestrt ose ost competes! oa ose Stigmaser! on os? AF Campeste| oss ose 5,23 Sugmastacenl 095, os rea 096 096 Bssostew! 1 1 Stoxano! to 102 as Averaterl 103 Ls 5,24Stgmasadenl 109 108 srsigasteno!” us Lie ravers 138 116 Beta u u“ 1 Tis sterol may alo be ered o as AT stigmas in erature “The peak due to the intemal standard (beculin) must be clearly separated from the peaks due ro the sterols to be determined. For the chromatogram obtained with de test solution {identify the peaks and calculate the percentage content of teach sterol in the sterol fraction of the substance to be ‘examined using the following expression: A 3x10 ‘A = area of the peak due to the component to be determined ‘um of the areas of the peaks due to the components {ndicated in Table 2.4.23.-1 ; disregard the peak due to betulin 1 required in the monograph calculate the content of each xtra in milligrams per 100 grams of the substance to be texamined using the following expression: sA314 Appendix X Q Ax m! x 100 Alem A. = area of the peak due to the component to be determined; area of the peak due to betalin; ‘mass ofthe sample ofthe substance to be examined, in grams; im! = mass of beulin R added, in milligrams. Method B Preparation of the unsaponifiable matter Prepare the unseponifiable matter according to the method stated in the test for unsaponifiable mater of the monograph fn the substance to be examined. Falling this, prepare the ‘unsaponifiable matter according to the method described in chapter 2.5.7. Unsaponifable mater. After the final neutralisation step, evaporate the ethanol, then add 6 mL of ‘acetone R and evaporate the solvent. Dry the residue at 100-105 °C, It's not necessary to dry to constant mass, ‘Simultaneously prepare under the same conditions the ‘unsaponifiable matter of sunflower oil R. This will in particular serve to locate the sterol fraction 10 be collected ‘Separation of the sterol fraction (LC) Liquid chromatography (2.229). Test solution Take up the residue with 3 quantities, each ‘of 4 mL, of the solvent used during the preparation of the D ‘matter (generally eter R or lige petroleon R) 15 ml-tube, Evaporate to dryness under a Dissolve the residue in a volume of na solution with an vious pritne Rand 02 ML of © mi veering hn te mg 1 alu ool rfloroactamie R. Str the ty SO aC for 20min. AO 1 el the gud phase Reference soit te reference solution i the pre fasion ob Tot ands pine Rand 02 mk of EC we 0 cof cama R and ime of 1 Jn 0 brine efuoroaceanieR 99 a pe ead eat a 8D C108 20, Ang Stop due the uid phase. 10 oe of eletral coker! R) may aso Be sed, A andar ot ear wih the sterol faction of sunfover 9 atone or easton as described forthe tts, Columns — aera: sed sices — ise: | = 30 m, © = 0.25 mm; ~ stationary phase: poyfmedyi(95)phensi(S)siexane B (fim thickness 0.25 jm). Carrier gas helium for chromatography R. Flow rate 2.6 mL/min Dissolve the residue of the stro] ‘Split ratio 125. Tampeasie i foment Pimento ea tO pe 1-38 260 4 260280 we 20 eal 20 Detector 290 Detection Flame ionisation. Injection 1-3 yi. (depending onthe expected amount of ste inthe subwtance to be examined) dentfcation of peaks Use the chromatogram obained with the reference solution to identify the peaks duc to ampesteral sigmasterol, osteo] and A7-stigmasteo enti the peat du to the sterols inthe chromato bane wth the tex solution using the chromates shined with te reteence solaton and the race retentions wih reference to Pestoserol (main peak) sv Table 2.4.23.-1. ena ere System suitability Reference solution: ~ hon minim 4.0 beeen the pss © campesterol and stigmasterol, mapens Calculate the percentage content of each sterol in the sterol fraction of the substance to at to be examined using the following A $x 100 ‘A = ‘a8 ofthe peak due tothe component to be S = sum of the arcas OF the areas of the peaks due to the componenis indicated in Table'2.4.23.-1, except betulin= Appendix XIE A315 Appendix XI 4, Total Solids ipod Bi meth em oS fap to he reside btn vente prescribed amount ofthe preperaing Meee weight under the conditions specified beise’ sess ills fa-botiomed, flanged dish, about 7 beer about 25 mim deep, sade ota sable meal of high heat conductivity and low specie ven sch not affected by bong water Sure a ax escribed in Brdsh Standard 1742-1051 (Methods to thectemical analysis of condensed milk), us0D Pose quantity stated in the monograph in a tare iy, ‘qaporate at as low a temperature as possible until the urls emoved and heaton a Wwater-bath unt he resider isqarety dry. Transfer to an oven operating wince finand dry to constant weight at 105° unless einer sated inthe monograph. It may be necessary, for reeues of akygscopic nature, to use a dish provided with a wall fring cover and t0 ol ina desiceator. 81. Ethanol-soluble Extractive Bur, method) sate 5g ofthe air-dried drug, comely powdered, with Lf ean of the specified strength ina closed fask shaking fequentiy during the fist 6 hours and to sand for 18 hours. Fer rapidly taking a oss of ethanol, evapora Surface ofthe tiguid by fi rotating the ender abou a vertical any enSaste the volume occupied by the drug, including $3 adhering macage. Cary out 3 tests at the same time ‘he sweing inex is given by the mean ofthe 3 tests D. Foreign Matter Ph. Be. method 28.2) Hrrbal drugs should be fee from mould insets and other animal contamination. Foreign matter is material consisting of any or all of the following 1) Foreign ongans: matter | ‘coming from the source plant but not defined asthe drug, 2) Foi cemens: mater not coming from the source plant and either of vegetable or mineral origin Determination of Foreign Matter Weigh 100 g 0 500 g of the substance to be examined, or the minimum quantity prescribed in the monograph, and spread it out in a thin layer. Examine for foreign matter by {inspection with the unaided eye or by use ofa lens (6 x) Separate foreign mater and weigh it and calculate the Percentage present. E. Essential Oils in Herbal Drugs (Ph. Eur, method 2.8.12) ‘The determination of essential oils in herbal drugs is carried ‘out by steam disilation in a special apparatus in the conditions described below. The distillate is collected in the ‘graduated tube, using xylene to take up the essential il the aqueous phase is automatically returned to the distilation flask Apparatus The apparatus comprises the following pans (@) a suitable round-bottomed flask with a short, ground- lass neck having an internal diameter of about 29 mm atthe wide ends (®) a condenser assembly (See Figure 2.8.12.-1) that closely fis the fas, the different parts being Fused into one piece; the glass used has a low coefficient of expansion: =the stopper K’ is vented and the tube K has an orifice of diameter about 1 mm that coincides with the vent; the wide end of the tube K is of ground-alass and nas an incemal diameter of 10 rm; ‘a pear-shaped swelling, J, of 3 mL. capacity; =the tube JL is graduated in 0.01 mL; — the bulb-shaped swelling L has a capacity of about 2 mis = Misa three-way tap; — the junction B is ata level 20 mm higher than the uppermost graduation; (©) a suitable heating device, allowing a fine control; (@) a vertical support with a horizontal ring covered with insulating material._- Apparatus fr the determination of ols in herbal drugs cleaned apparatus. Carry out the o the nature of the drug to be examined. ‘volume of distilation liquid in the flask, ‘porcelain and atach the J wate R through the filing 8 at the level B. Remove the stopper K” prescribed quantity of fone R, using 3 Jat the bottom ofthe tube K. Replace the thatthe orifice coincides with the the flask to oiling and adjust the Figure 2.8.12-2 ace nt the fask he presided quant ofthe dy Inoue ite lato a desebed above fr thie a cont bed. Srp the Beating snd ar 1 aod atthe oe Pu coleted in Ge ested se rte ose prs 2. i oa tbe quant feel ln Toe ies en Calo the os 3 ml pe logam of . russ eaenl is 10 be wed for other ana Fa pe ements mae of ene and sen of Pa cred fol: remove the Spr K and Rfluce Of meatal gl soln of ttm sas Rand 05 mi of eater R Ler he mio id rca olan th bulsaped sling Ly PEN ofthe ieevay ap, allot stand for 5 min sa Bes ear oy oil fa acs he ke of ie MSM, Open heap enteclcvi ota th wat om uf i conesing ube iM, Wash the tbe with aero an hae ene nrodueed hough he fing ane! N. Tun the tap anecokve in ner0 root te murs of lene ed ere ol nan sop ak Intro F. Continuous Extraction of Drugs (No Ph Br, method) Wher the process of macatin or perl is sein Themen cary out te flowing procedures wih any teson indeed i he mons acanarion Pe the sold mater with the whole of the mens it 4 closed veel and alow to stand fer 7 da, shaking Oceana Stain, pss the mare and mix the gues brand. Clarify by subsidence or fleasen PERCOLATION Moin the solid materi with a suficint quay oe menseuum allow to stand ford hours a ellcned ese pack ina perslatr and ad sufcent o he enim to ttre te ters Whos the id B= to drop from the percolator, close the oud, a ufc the menstroum oleae layer above the dug an all © acerte or 24 hours, Allow pecan to proceed so ‘unl the percolate measures about tree quae te eguted Volume. Pres the mates ns tc eased ‘with the percolate and add sufficient of the menstruu ‘0 Produce the gue ohne Clan by seo Souris of pereolaing the drug with the solvent stated in monograph at a temperature approximately that of the Doda it of ote ao apparatus described below, or any similar pats ‘may be used, provided that it permits the uniform Continuous extraction ofa drug for the purpose of an 38Appendix XI H_ A317 | gon ofthe drug and the regular fo jround the percolator. Of # gus is shown in Fig. 11F-1, A ig an, vat ‘the wider prs abou ac aE of iff diameter of 4.8 t0 5 cms the lower end C it abort (gud hs an extra dimeer of abou ey 50g Bas tube OPEN at both ends, about 9 cm long sain esteral ameter of about 3.8 com ogee Alc el Fm a peace ther Maral, D sa glas coll which suppsea ne gee ke B and prevents it rom resting incontas wk ne dite pe A. The lower end C of the outer tube Ae Ret aikoe ground: ss joint to the datllation ese Ce yam, suitable quantity of the solvent has been placed. gto be extracted, previously moistened withthe ‘eCPor subjected t0 any preliminary treatment required, is inp the tnner tube B, which i supporead es ks Pe desrops inn we outer tube A put ot sashes Beet Gis vn of hs dg, te eae fetopostion andthe outer tbe connected by mens fsck of ground-slas oi with the tbe of Meadenser The fas is heated and the extraction oimued as directed. the vapour of RRO CTION WITH AN iMMUscIBLE Sou vENT nats meting a east three times with the solvent, add t0 erect ofthe next portion 1 to 2 ml. of 01 hydrochloric cid remove the organic solvent by evaporation, transfer the ‘aueous residue toa test tube and add 0.05 ml. of potas ‘etaiodemercurate solution or, for solanaceos alkaloid, 0.05 mal. of possum iodobiomuthate sbaion RA of, for metine, 0.05 ml of iodinated potassium iodide sion. ‘Not more than a very faint opalescence is produced, ‘CONTINUOUS EXTRACTION After percolating for at least 2 hours, collect 1 to 2 mi. of the effluent and carry out the procedure described under ‘Extraction with an aqueous or alcoholic liquid’ or “Extraction with an immiscible solvent’, as appropriate. H. Stomata Bi. mad 283) ‘here ae teed pe of soma (ne Fie 283-D sng byt fm ad range of sirodlag ee (1 The onomoget Grea ce) pte fm trond by a vayiag mune fsa no me fee icee ee eee (8) The antag ueqalcled) Ope the wa way pee feiely mul tn the Gs 3 dig (zoucad) ype: th vom (© Mhompared by? obi col wht ommon wall Segre ete eae ) ‘The para gural-eled) pe the som at on (0 Tah oe or more subiary cls paral the fang ans ofthe pore an gnc Stomatal Index Stomatal Index = 2 aoe the mero soma ingen ae of a ee rae of cpcmal cole (nude thom) a th same ae oe ror cach sample fe make 2 fever hn Fe cas and ite te met Figure 283.1—————————————————— A318 Appendix XIJ — era L. Pesticide Residues é 1p Use Method I untes otherwise directed in the monograph sansa Rf Das woe he ge Ceaition | For the Purp aman Method I Def gy etance oF mite of SUDAN eg a bstying or controling NY Pes, unwary Lae ects pean ee eI AT Pte r ot species ‘of plants of animals rah ‘during iio in species terfering with the production, Processing, Incinerate 2 to 3 g of the ground drug in tard platinum or silica dish ata temperature not exceeding 450° until frce from carbon, cool and weigh. Ifa carbon-free ash cannot be ‘obtained in this way, exhaust the charred mass with hot ‘ware, collec the residue on an ashless filter papes, incinerate the residue and filter paper, add the filtrate, evaporate t© dryness and ignite at «temperature not exceeding 450 Calculate the percentage of ash with reference to the air deed drug, FOR OTHER SUBSTANCES. Carry out the above method using I g, unless otherwise stated, Calculate the percentage of ash. ‘Method I (Ph. Ear, method 2.4.19) ‘Heat a silica or platinum crucible to redness for 30 min, allow to cool ina desiccator and weigh. Unless otherwise ‘prescribed, evenly distribute 1.00 g of the substance or the powdered herbal drug to be examined in the crucible. Dry at TOO C to 105 °C for 1 h and ignite to constant mass in a ‘muffle furnace at 600 °C + 25°C, allowing the crucible 0 ool in a desiccator after each ignition. Flames should not be produced at any time during the procedure. If after rc the ash sill contains black particles, take et or marketing of herbal drugs, The item sori eznec intended for use a owt repute, incades seein and any substance appl 0c, efi oes bares 0 protect the comm fry citer bof using storage and wanspor. Pesticide red deere ae are controled in herbal drops and et drug preparations pcs = cer Gales otherwise indicated inthe monograph Tema jrug o be examined at least complies with the lis ae arn Table 2813-1. The limits applying to iets that ae aot Usted in Table 28.13-1 ad whe ree is suspected for any reason comply with he ix ety cron referred to by Regulation (EC) No. 396205, including annexes and successive updates. Limits for vrealde that are not listed in Table 28,131 no in Bimtpean Union tests are calculated using the foloing expression: storages IFansP* ADI x M MDDyp x 100 ADI = acceptable daily intake, as published by FAO-WHO, in milligrams pet kilogram of body mass, M jody mass in Kilograms (60 ke), MDDyp = daily dose of the herbal drug, in kilograms ‘The limits for pesticides in herbal drug preparations a calculated using the following expressions APDER < 10; MRLupxDER I'DER > 10: ADI x M MDDap x 100 MRL = ‘maximum residue limit ofthe pesticide int herbal drug as given in Table 2813-1 ort EU tents or caleulated using the express ‘mentioned above; dtrugiextract ratio, ie, the ratio between the quantity of herbal drug used in the mame of herbal drug preparation and the quan 4 ppp Mit se0s preparation obaineds "ap = daily dose of the herbal drug preparatio® i lelograms, DER = ‘The competent authority may rity may grant total or partial exemption from he tet when the compete Ho fd quantity of the pesticides used, date ofeach Tm, uring culation and aftr the harvest) ofthe wea the batch is known and canbe checked precisely aso ‘od agricultural and collection practice (GACP) Sampling of herbal drugs Sampling is done acco? the general chapter 2.8.20 ‘an eee ‘Herbal drugs: samplingAppendix XIL A319 Table 281 ‘a Table 28:34 Longe ata ‘eae 6. ro as a ss TS ate inane dedi (64m of = a Tarn. Sees : a wel = iat ‘rnd, inorganic (akulated as bromide ion) o es i Pint od eae os sromophas-ethy 005 Parat Paieeericiratretil el oa seo fc o me Pentachloranisol of eg es el “f a ‘Permethrin and isomers (sum af) 7 aenvionhos Phosaone a Phosmet Ans Piperony! butoxide Pirimiphosethyt is Teeter mf ices ond Ristenchionata : nets, 1 Profenopbos oS Prothiophos bel Petes arse are cs ape sna Quinalphos _ eet area ae sen See nr = ie és “Tetradifon J Vinelozotin - Qualitative and quantitative analysis of pesticide residues ‘The analytical procedures used are validated (€«, according to Document N° SANCO110232/2006). Tn particular, they satisfy the following criteria: — the chosen method, especially the purification steps, is stable for the combination pesticide residuesubstance to ‘be examined, end not susceptible to interference from co-entactves _— natural occurrence of some constituents is considered in the interpretation of results (., disulfide from ‘Cruciferaceae); _— the concentration of test and reference solutions and the serting ofthe apparatus are such tht the responses used for quantification ofthe pesticide residues are within the ‘dynamic range of the detector. Test solutions containing residues at a level outside the dynamic range, nay be diluted within the ealibration range, provided that ‘the concentration of the matrix in the solution is adjusted jn the case where the calibration solutions must be matrix- ‘matched; — beryeen 70 per cent to 110 per cent of cach pesticide is ‘epeatabilty ofthe method: RSD is not greater than the ‘alues indicted in Table 2.8.13-2; — reproducibility ofthe method: RSD is not greater than the ~ fais indcated in Table 2.9.13-2.a A320 Appendix XI M Table 2.8:13-2 ‘Cencentraion age Repeatability (RSD) Reproetiity (RSD) Seal ‘percent (oer cen 1 al 9001-001 » o >oo1-01 » ” poner 6 » ot » 2» M. Tannins in Herbal Drugs (Ph Faw, method 2.8.19) ‘Carry out all the extraction and dition operations protected from light. ‘In the ease of a herbal drug ora dry extract, to the stated amount of the powdered drug (180) (2.9.12) or the extract in 0 ask add 150 mL of avr R. fn for 30 min. Cool under running water flask and collect the washings in 5 then dilute to 250.0 mL with tearer R. ctor tincture to 250.0 ml. with through a filter paper 125 mm in reagent R and 10.0 mL of 25.0 mL with a 290 g/L solution of 30 min measure the absorbance using warer R 2s the compensation d by hide powder To 10.0 ml. 10 of hie powder CRS an shake . Filter and ute 5.0 mL. ofthe fate mediately mg of ‘and dilute to 100.0 mL with the same ‘solution to 100.0 mL. with with 1.0 mL of CTT N. Bitterness Value sn method 2.8.15) ed, a liquid or an extract that still has a biter tan, Se th une paras is Sremess value of which is set at 200 000 Determination of the correction factor rina anmpring a eat 6 pens i recomend Ae pn cmt ae pe ee or india ference in ax teres To cae fr el member its neoary 1 determin an Mra foreach panel member SS coleton Diol 0.100 of enn dri R Se sete TOO wi the sane flee i at a of soln to 100.0 ta with wae He hace, esac $cc ot daioo by Tee eeeb cy atic woo atonal Br ocoutenen aie Terre ds ualoeia of eve ieee ee Dereon lows se lion wih te lowe tes Geese a evire caer, Tarr 100 mot Be pncen ar oe ac EPA to a See one de bck ecpajodog 90 0 Pte ota Fag eee gt eect AE va Roae Rie SE cr ARETD un we Gener chai ee ris ane oot ie eh ed ekintom a eens 1 = number of mililitres ofthe stock soation inthe ailuion of lowest concentration that is judged to be bitter. a Persons who are unable to taste any bittemess when wsing the reference solution prepared from 5.8 mi. of stock Solution have to be excluded from the panel Sample preparation If necessary, reduce the iple te der (710) ( : € sample to a powder (710) (2.82. To 1.0g of sample add 100 ml. of boiling rater R. Hest 00 water-bath for 30 min, stirring continuously. Allow to cool and cue to 100 ml. with water R. Shake vigorously and Aker ieardng the frst 2 ml ofthe ftmte. The State # Wels C-1 an has dui factor (DF) of 100 Pui have to be examined, 1 mi ofthe liquid is utd a suitable solvent to 100 mL. and designated C-1 Determination of the bitiemess value 10.0mL of C-1 is wi Too ant of Ct ete with water to 10.0 mL of C-2 is dituted wit 100mL: C-3 with carer R to (DF = 1000) (DF = 100%)with solution D prepare the fo meee lowing sequence of Gaon Dia) | 12 air Ral) the number of mili tte of solutiog seen diluted t0 10.0 mL. with water Regn D which, ‘ater R, stil has a bitter & taste Jate the bitterness value for cach tbe expression: ( Yxk Xx0. Re Sele rate tance ann Panel member from P. Dry Residue of Extracts (Ph, Ew: method 2.8.16) fa-bottomed dish about 50 mm in diameter and about ight, introduce rapidly 2.00 g or 2.0 mL of the xs Evaporate to dryness on a water-bath 100-105 °C for 3b. Allow to cool ina pentoxide R ot antyvous sca the result as a mess percentage or Appendix XIR A321 id at tees equal spl fren Astle ac ‘These methods arent designed for inclusion aay methods in monographs on those dus that pst, Asstolochic acs a secondary metabolites fer thee 4 more senate validated metho i egued to or greater than 2 ppm. It may be graphic data suguess the presence of Method A: screening test for aristolochic acids Thin layer chromatography (2.2.27) ‘Solvent mixture anhyrous formic acid R, arr Ry amethancl R (19:40 VIVIV). Test solution “To 1.0 g of the powdered herbal drug (710) (29.12) add 10.0 mi of the solvent mixture, sonicate for 10 min and centrifuge. Reference solution (a) Disperse an amount of aristalochia HRS corresponding t9 0.10 mg of aristolochic acid Tin 20.0 ml. of the solvent mixture, sonicae for 10 min and centrifuge Reference solution (b) Die 1.0 mil. of reference soletion (a) to 25.0 ma. with methanol R Plate TLC silica gel Fass plate R 2-10 uz) ‘Mobile phase anhyicus formic ac R, water Ry ethyl actateR, toluene R (33:30:60 VIVIVIV); use the upper layer. Application 20 iL as bands of § mm Development Over a path of 6 em. Drying In current of cold aie for 5 min, Detection Spray with 100 g/L. solution of stannous chloride Rin die hychochlorc acid R unt the plate is slightly ‘wet, heat at 100 °C for 1 min and examine in ultraviolet light a 365 am, System suitability ~ the chromatogram obtained with reference solution (a) shows 2 greenish-blue zones due to aristolochic acids 1 and Il berseon Rp = 0.35 and Ry = 0.55, which may not be completely separated —the chromatogram obssined with reference solution (6) shows at leat 1 of these zones (corresponding to 2 ppm of aristolochic acd D. Results In the chromatogram obtsined with the test ‘solution no zone is similar in position and fluorescence to ‘ny of the zones due to aristolochic acids in the ‘chromatogram obtained with reference solution (). [the chromatogram obtained with the test solution shows any zones similar in positon and fluorescence to any ofthe ‘ones due to aristolochic acids 1 and Il inthe chromatogram ‘obtained with reference solution (e), apply method B. ‘Method B: limit test for aristolochic acid 1 Liquid chromatography (2.2.29). Solvent mixture acetone R, water R (50:50 VV). Test solution Weigh 2.0 g of the powdered herbal drug G10) 2.9.12) ito-8 250 mL, brown, srew-cap bottle and $d 100.0 mL. ofthe solvent misture, Stir for 30 min at ibout 300 rimin and filter through a membrane filter {ominal pore siz 0.45 um). ‘solution (a) Dissolve the contents ofa vial of in the solvent mixture to obtain & anti pk yom of estloche 2 tncentation of 0.04 we ‘solution (b) Dissolve the contents ofa vial of cid foro sib ORS containing ar cack Land Tt) inthe solvent ture and di asa ah eh slvent me.A322 Appendix XI R Column sige: 1= 0.15 m, O = 2.1 mm; — nationary phase: octadecylsilica ge for chromatography R G5 um); = temperature: 40°C. Mobile phase — mobile phase A: wiftuoroacetc aid R, eater R (0.1:99.9 V1); — mobile phase B: wifuoroacerc aid R, acetonitrile R (0.1999 VY); Time Mobile phave Ail phase B in) (perce VM. (percent 1) 0-25) Sos 15-965 23-30 40 50 nen 04% 100-15 Flow rate 0.3 mL/min. Detection Spectrophotometer at 390 nm. Injection 25 iL. 5 sa ‘= resolution: minimum 3.0 between the peaks due t0 aristolochic acids 1 and II in the chromatogram obtained reference solution (b); ise ratio: minimum 10 for the peak due ro es Mile pve A rey est )_tor com To > ° 0 04m +9 Fw rate 04 mini, a ree on 20 ys inject reference solution () ews, the Inicio ee eeence vlwien (tc, then ‘teence shtion () tie Devcon Man detector a described below under Aor 8 pact gare temperate andthe detector Aas 5 bo comply with the stem subi exten A ensrap mass spectrometer equipped with n GeeSipy onan GSD ineace and MS" ana, ‘Set the mass spectrometer parameters for the MS? mode as ‘Sow Parent | soation width | Relive cose | Mote | twa) iw) energy per xat)_| sage 38 | MS | ocr a 2 Ms! 208 20 a = full scan of product ions: from m/z 80 to mis 370; — product ions to be monitored: miz 252, ‘iz 268 and m/z 281. System suitability — signal-to-noise rato: minimum 100 for the monitored ‘product fons in the chromatogram obtained with reference solution (a); — mani: imererence test; the average of the 2 ratios of ‘eference solution (b) is inside the + 40 per cent interval ‘of the average of the 2 ratios of reference solution (a) ‘otherwise itis necessary to adjust the detector settings Results Evaluate the average ratios (252/268 and 281/268) ‘of the relative intensity of the 3 product ions of aristalochic ‘acid Lin the test solution; evaluate the average of the 2 ratios ‘ofthe signals at the retention time of aristolochic acid I in Feference solution (a); ifthe average of the 2 ratios of the test solution is within the +t 40 per cent interval of the Average of the 2 ratios of reference solution (a), arstolochi acid Tis present in the test solution. B. Tuple-quadrupote mass spectrometer equipped with ESlinerice and MS" apace ‘Set the mass spectromet eters for the MS2 mode # = mass spectrometer parameters for the MS2 mos — prestinor ion: m/s 359 [M+ NH,)+; ‘= Product ions t0 be monitored: mi a eee tored: m/z 265, mis 281 and ‘System suitability Te Ra te: minimum 100 for the monitor oe biained with matrix imerference test: __ Teference solution (b) is inside the + ‘cent int _hthe average ofthe 2 ras of rene lo ‘otherwise itis necessary to adjust the detector 1 sages) Regis, Rett the werage ratios (265281 and 29628 relative intensity of the 3 product ions of arist \ Ne eee ea eaeAppendix XI $ A323 a shat eat, atten ine fae 2 est for Aristolochic Acids T and 11 x bal Drugs 5 (ay Bar Method) Aristolochic acids have been shoun to i aringeic. Extraordinary care shoud be in which they are used. jnbne with the prohibition ofthe use of Arisulochia species jnunlcnsed herbal medicines in the United Kingom, «est feabsence of aristolochic acids I and I in herbal drugs has texninciuded in the British Pharmacopoeia, The limit of dein has been shovin to be 0.00078 mgiml. (epposimately ppm of aristolochic acids I and ID). Its sdvsod thatthe limit of detection forthe system-in-use is deomined by the analyst, Aristolochic acids I and I are not caniaed to the genus Aritolochia, The acids are also sqpned as present in certain species of Asari be highly toxic taken in any Inject 10 ui. of sol lution (2) and allow the chromatography 10 proceed for 10 minutes. The tests not valid unless the i "solution factor between the peaks corresponding to aristolochic acid II (retention time about 6 minutes) and STeolocic acd retention tine about 7 minutes) i at least Inject solution (2) si times, The relative standard Aviation ofthe areas ofthe peaks is at most 1.5%. Inject 10 UL of solution (1) and allow the chromatography to proceed for 30 minutes. In the chromatogram obtained with solution (I) the peaks due to aristolochic acid I and aristolochic acid IT are absent. S. Determination of Mycotoxins in Herbal Drugs 1. Determination of Aflatoxin B, in Herbal Drugs (Ph. Eur. method 2.8.18) CAUTION: aflaoxins are very toxic and carcinogenic. Perform ‘manipulations under an extraction hood whenever posible. Take ‘particular precautions, such as use ofa glove box, whem toxins are tn dy form becauce oftheir electowaic properties and the tendency to disperse rough the caorkng areas. Decontamination procedures for laboratory castes of aflatoxins cere developed by the International Agency for Rescarch on Cancer (IARC). Aflatoxins are naturally occurring mycotoxins produced mainly by Asperglus flacus and Asprglus parasiticus. These fungi are common end widespread in nature and are most often found when cerain grains are grown under concitions of stress such as drought. The mould occurs in soil, decaying vegetation, hay, and grains undergoing microbial spoilage, and invades all types of organic substrates whenever and, ‘wherever the conditions are favourable for its growth. Favourable conditions include high moisture content and ‘high temperature. At least 13 diferent types of aflatoxin are produced in nature and most of these are known to be highly toxic and carcinogenic. Aflatoxin B, is considered the most toxic. Herbal drugs that are subject to contamination by aflatoxins are tested by a validated method. ‘Unless otherwise indicated in the monograph, herbal drugs contain not more than 2 pg/kg of aflatoxin B. "The competent authority may also require compliance with @ limit of 4 jgikg for the sum of aflatoxins By, Ba, Gy and Ga, ‘The method described below is cited as an example of a ‘method that has been showin to be suitable for devil's claw root ginger and senna pods. Its suitability for other herbal drugs must be demonstrated or another validated method used. Method Liquid chromatography (2.2.29, “Aflatoxin ore subject wo light degradation. Garry ou the ‘eermination protected from daylch by using UV-absonbng od on ends in combination exit subdued Fight, o curtain oF ‘ns combinaion ech adic light lorscent bes ae dacoprabe). Prov oflsosn-comaining solutions from dasligh. Rinse glstware before use with @10 percent V/V solution of tiie aid Rand then rinse carefully wit dsiledearer R tnt no more acid is present ‘Toot solution Use an immunoaffinity column containing ddoubodies against aflatoxin By with a capacity of not less ‘an 100 ng of aflacoxin B, and which gives a recovery of ‘ec les than 80 per cent when a solution of 5 ng of aflatoxin Br ina mature of 125 mL. of methanol Rand 87.5 mL of———ltrlrttttst— A324 Appendix XI S water R is passed through. Allow the immunoaffinity column to reach room temperature, To 5.00 g of the powdered drug (500) (2.9.12) add 100 mL. of a mixture of 30 volumes of seater R and 70 volumes of methanol R and extract by sonication for 30 min, Filter through folded filter paper. Pipette 10.0 mL. of the clear filtrate into a 150 mL. conical flask. Add 70 ml. of water R. Pass 40 ml. through the immunoaffinity column at a flow rate of 3 mL/min (not exceeding 5 ml/min). Wash the column with 2 volumes, each of 10 mL, of water R at a flow rate not exceeding 5 mL/min and dry by applying a slight vacuum for 5-10 s or bby passing air through the immunoaffinity column by means of a syringe for 10 s. Apply 0.5 ml. of methanol R to the column and allow to pass through by gravity. Collect the eluate in a 5 mf. volumetric flask. After 1 min, apply a 2© portion of 0.5 mL. of methanol R. After a further 1 min, apply a 3 portion of 0.5 mL of methanol R, Collect most of the applied elution solvent by pressing air through or applying vacuum to the column. Dilute to 5 mL with ‘eater R and shake well. Ifthe solution is clear it can be used directly for analysis. Otherwise, pass it through a disposable filter unit prior to injection, Use a disposable filter unit (eg. 0.45 jum pore size polytetrafluoroethylene filter) that has been shown not t0 cause loss of aflatoxin by retention. Aflatoxin B, primary stock solution Dissolve ‘aflaroxin By R in a mixture of 2 volumes of acetone R and 98 volumes of toluene R to give a 10 jig'mL. solution. ‘To determine the exact concentration of aflazaxin B, in the ‘tock solution, record the absorption curve (2.2.25) between 330 am and 370 nm in quart cells. Calculate the aflatoxin By mass concentration, in micrograms per mililitre, using the following expression: AxM x 100 ext A. = absorbance determined a the maximum of the “absorption curve; ‘M = molar mass of aflatoxin B, (312 gimol); ¢ = molar absorptivity of aflatoxin B, in the ‘toluene-acetonitrile mixture (1930 m’/mol); J = optical path length of the cell (1 cm). Aflatoxin B; secondary stock solution Prepare a secondary stock solution containing 100 ng/mL afiatoxin B, by diluting afiatoxin B, primary stock solution with a ‘mixture of 2 volumes of acetonitrile R and 98 volumes of toluene R. Wrap the flask tightly in aluminium foil and store. it below 4 °C. Before use, do not remove the aluminium foil ‘until the contents have reached room temperature. If the ‘solution has to be stored for a long period (for example, T month), weigh the flask and record the mass before and after each use of the solution. Aflatoxin B, standard solutions Place the volumes of aflatoxin secondary stock solution indicated in -18,-1 in separate 250 mL. volumetric flasks. Pass a of niogen through at room temperature unt the just evaporated. To cach flask, add 75 mL of 1 R, allow the aflaroxin By to dissolve and dilute to ith eer MERI 0. AO Be a Table 2818-1. ‘Standard solution Final concetra | {98 /mi) Volume of secondary sock ation) ‘bration curve Prepare the calibration curve using sanivalent to 1-8 uaikg of aflatoxin B, inthe herbal dug Ge the plot for linearity. If che content of aflatoxin By in the sample to be examined is ouside ofthe calibration fange, the test solution must be diluted to an alaoxin Content that is appropriate for the established calibration Column: — sice: = 0.25 m, © = 4.6 mm; — stationary phase: octadecylsilica ge for chromatography R 6 um). ‘Mobile phases — mobile phase A (for post-cokuran derivatisaton with photochemical reactor oF pyridinium bromide): acetonitrile R, methanol R, water R (2:3:6 VIVIV); ‘mobile phase B (for post-cokurin derivatisaton with clectrochemically derived bromine): add 0.12 g of potassium bromide R and 350 iL of die nic acid RY perlite of mobile phase A. Flow rate: 1 min, Detection: Fluorescence detector with a 360 nm exciton filer and a 420 nm cuvott emission fier, or equivalent Recommended settings for adjustable detectors are 365 am (excitation wavelength) and 435 nm (emission wavelength) Injection: 500 ul. Post-column derivatisation with pyridinium hydrobromide perbromide (PBPB): — puibeless pump; — Trpiece with zero dead volume; — polytetruoroethylene reaction tube, 1 = 0.45 m, ©=05 mm; — mobile phase As = post-column derivatsation reagent: dissolve 50 mg of Pyridinium hydrobromide berromide Rin 1000 mL. ‘f teater R (store protected from light and use within 4438 ~ flow rate of the derivatsaton reagent: 0.4 mini ae derivatisation with photochemical react” — reactor unit with one 254 nm low pressure mercury UV bulb (minimum 8 W), ~pahedspport las — knitted reactor coil: polytetrafluoroethylene tubing Kait™ sgh around the UV bulbs 5 ne 0.23 ‘nominal void volume 1.25 val; exposure time: 2 min; — mobile phase A.a Appendix XI S A325 ann derioatisation with e . Posted bromine (KOBRA). | “chemically FeonRA-el electrochemical cl that gon fom of bromine for derivatsaton ef agers a Tate fhenhanced fluorescence; available fom en euing Somers suppl; oe J peintion direct-curest supply in series KOBRA-cl providing a constant curene es roe os _ perafluoccethylene reaction tube, = seer tube, 0.12 m, motile phase B. itgwronier setorin Oy edanain G, sins Bi ; tion calculate the calibration curve y = ax +b (Gr astosn By concentration (gL) onthe x an te dal (S) on the yraxis. The aflatogn By concenscton tO ine measured solution sequal to 22 Cao the aflatoxin B, content ofthe herbal drug, n Sowa per gram, using the following expression aflatoxin Bay Vixvaxo meV a | = mass ofthe herbal drug taken for anal, in grams; -Nolume ofthe solvent used for extraction, in ‘and dilution, in miles; 'B, concentration of the test, Test solution Use an immunoaffinity column containing Antibodies aginst ocratorin A witha capacity of not less than 100 ng of ochratoxin A and which gies a recovery of not less than 70 percent. Allow the immunoafinty column to reach room temperature ‘To 2.00 g ofthe powdered drug (250) (2.9.12) add $0: 0f 30g solution of aun fyrogen carbonate R and ‘eruct by sonication for 30 min (change water of ultrasonic ‘bath afer 15 min). Cool 1 room temperature and de to 100.0 mi. (V,) wit the same solution. Centnfure ‘Mix thoroughly 5.0 mL. (V) ofthe clear supernatant with 30-ml buf seltion pH 74 Rand pass the whole soto through the inmunoafinity column ata los rate of 3 mLUmin (do not exceed 5 mL/min). Wash the column first with 10 mi bufersolaion pl 7.4 R then with 2 quantities, cach of 10 ml, of water Rata ow rate not exceeding 5 mlsmin and dry by applying a slight vacuum for 5-10 or ‘by pasing ar dough the inmuncafinty column by means ‘ofa syringe for 10s: Apply 0.5 mL of methanol Ro the column and alow to pass trough by grav Collect the chat in a4 nL glass via. After 30, apply 8 2 quantity of 0.5 mL. of methanol R and allow to pass through the column by gravity nto the same glas vial. Aer a further 30s, repeat witha 3 portion of 0.5 mL. of tmathanalR. Collect any solvent retained on the cohum by Dressing ir trough or applying vacuum to the column. Evaporate the combined eluates completly to dryness using 4 thermal block with a nitzogen blanket (40 °C). Dissolve the residue in 0.5 mL. (V2) of solution A. Ite solution is clese itcan be used directly for anal, Otherise, pas ic through 2 disposable filter unit port injection. Use a disposable fiter unit (eg 045 jm pore sze polytetrauoroctene fe) that hasbeen shown not 9 ease loss of ochratoxin A by renton. Ochratoxin A primary stock solution Dilute 1.0 mL of ehratxin A slon Ro 100.0 iL. with methanol Rand shake thoroughly Ockratoxin A secondary stock solution Ditte 10.0 ml. fochraoxin A primary stock solution to 100.0 ml. with amethanol R and shake thoroughly. ‘Ochratoxin A standard solutions Place the volumes of ‘chratorin A primary stock solution or ochratoxin A SEcondary stock solution indicted in Table 28.221 inxo ‘erate Hasks and make up to 50.0 mi with solution A. ‘Table 2822-1. ~ Ochratoxin A standard solutions “Tania slain Volume ofoehratoin A Final concentration Primary sick slton of ocracin Ain w ‘andard slate Se ee 7 0 ry 2 200 2s 3 1000 0 4 so 5 5 20 25 sso anid win Wome fornia A ial concentration Miockofachratnn Ala “elation GL) standard solton —s 0 sae 7 100, a SS ae Jon curve Prepare the calibration curve using cvosin A standard solutions 1 0 7, which cover a rsd aia 1 0.5-250 pag of ochratoxin A in the herbalA UPESIUSTARAIIY AMT BM SEMI IT A326 Appendix XI T 500-1000 ; Into subbatches, and the proceduer subd Ahoughitweres homogeneous bic, ons «20 SE Aertldrugin he tac asm 10.0 percent of te maybe ed asthe ine Prepare the bulk sampi i le by combining and thorough! Ising the samples taken fom ech a containers (See Table 2.8.20.-1) randomly seeAppendix XIU A327 der fo obtain the prescribed bulk sample “ at | Naa i 5 Soe me | 500 | [cri | Stes’ |e) Peat | onus. | tect | es | oe [aes] Sas “ ee peewee og | eee” (ren Se | eee ee eae | 250 w 5 55, 2 i, = : . 2000 80 10 3000 6 5 | 200 | a 3 000 3000 120, @ 3000 Ey 6 | 0 | 6 « ‘3000 “5000 200 6 5000 40 8 5000 | = 3000 | 10.000 400 a 8000 0 1 “8000 20 6 ‘e000 | [se 0 [| Bao | 0 | asm | _« «| 2s] tan Sa Minion weigiteftestsaaple the sieve must not be more than 10 per cent of the total ees lowes, sees ts ~ tsomple lesan 2508 | ken orfagmented drugs where 1256 | Bibeemas el the neces es ‘NOTE: quartering consists of placing the bulk sample, ‘mized, asa level and squareshaped heap and dividing it diagonally into 4 equal parts. 2 opposite quarters ‘are retained and carefully remixed. Tha process is repeated ‘as necessary until the required minimum mass is obtained ‘or the test sample. Test sample ‘Unless otherwise prescribed in the monograph, prepare the ‘st sample as follows. eed the siae ofthe bulk sample by quartet Kee ) or thod that produces @ a ipl making sure at ech resid porn emains representative of the whole, until the minimo™ eained quantity complies with the following conditions srandar seve o ae ned OD particle size in the monograph. If these conditions are met, the sample and residue are to be well mixed to form the test sample for anal, In those cases where these requirements are not met, the test sample for analysis is composed of the 2 pars measured separately. Therefore, the quantity required for each analysis is derived by weighing proportional quantities ofthe powder and the residue. NOTE: for determina the milled test sample is r-m m of microscopic cha ed ers, a portion of hrough a 0.355 mm scree U. Microscopic Examination of Herbal Drugs (Ph. Bue, method 2.8.23) ‘The microscopic examination of herbal drugs is cared out on the powdered drug (355) (29.12) unles etherise prescribed in the monograph, ioral hydrate solution Ris the most commonly prescribed reagent, However, certain Features are not visible or not easly Seen afer mounting in this reagent. In this case, other ‘Segent are use, for example, 250 percent VIV olin ofA328. App beard R, which makes it possible to visualise starch sranules, It may also be necessary to prescribe specifi reagents in a monograph, for example: lactic reagent R whish {is used to show the presence of various features, 10 per cent VV alcoholic solution of phiomlucinol R and hyrochloic acid R, which are used to identify the presence of lignin in cells oF tissues, ruthenitn red solution R, which is used to show the presence of mucilage in cells or giyert R used to show the presence of starch and inulin, Examination under polarised light (between crossed nico prisms) is used ro identify starch granules (black cross ‘phenomenon calcium oxalate crystal (refringence) ot Tigniied structures. MOUNTING IN CHLORAL HYDRATE SOLUTION Place 2-3 drops of chloral iydrae solution R on a glass microscope slide, Disperse a very small quantity ofthe powdered dug inthe liquid and cover the preparation with a ‘coverslip, Heat the preparation very gently to boiling on s hotplate or a micro gus burner, Maintain gentle boiling f shor time, Make sure that the quantity of mounting fit is sufficient. Ifnecessary, add more fluid using a tapered glass pipette. Allow to cool and then examine under a microscope: ‘Repeat the heating until the starch granules and the water soluble contents of the clls are no longer visible. Examine under a microscope. CChiloral hydrate tends to crystallite as long needles, To avoid this, proceed as follows ater heating, remove the cover sp to the preparation add 1 drop of a 10 per cent V/V mixture ‘of chloral hydrate solution R in sscerol Ry place a clean cover slip on the preparation; examine under a microscope. MOUNTING IN A 50 PER CENT 1/V SOLUTION ‘OF GLYCEROL. Place 2 drops of a 50 per cent V/V’ sotution of glcerl R on a ‘glass microscope slide. Disperse a very small quantity ofthe ‘powsdered drug in the liquid and cover the preparation with a ‘coverslip, Examine under a microscope. MOUNTING IN A 10 PER CENT V/V ALCOHOLIC SOLUTION OF PHLOROGLUCINOL AND HYDROCHLORIC ACID Place a very small quantity of the powdered drug on a glass ‘microscope slide. Add 1-2 drops of a 10 per cent V/V" ‘Alcoholic solution of phloroglucinolR. Mix and allow the solvent to evaporate almost completely. Add 1-2 drops of Invdrochloric acid R and cover the preparation with a cover slip. Examine immediately under a microscope. ‘The red colour indicates the presence of lignin. MOUNTING IN LACTIC REAGENT Place 2-3 drops of lactic reagent R on a glass microscope slide, Disperse a very small quantity ofthe powdered drug in the liquid and cover the preparation with a coverslip. Heat the preparation very gently to boiling. Maintain gente boiling fora short time, Make sure that the quantity of mounting ‘uid is sufficient. If necessary, add more fuid using a tapered ‘glass pipette Allow to cool and then examine under a ‘microscope. Lignifed structures stain bright yellow; ‘structures containing cellulose remain colourless. Starch ‘stain more or less violet; certain secretions (e.g, ‘resins, oleoresins) stain orange and cork stains ‘Kher about I minute, allow a drop of dined pow coverslip > R to be taken uP "under a microscope age stains viol etAppendix XII A, Disintegration 1, Disintegration Test for Tablets and Capsules! Bur. method 2.9.1) ee ‘his est is provided to determine wh. ether table intepate within the prescribed time when gk oct ime when placed in a atal conditions presented or the purposes of this test, disintegration does ropa dshuton of the ut or ncn or ont EY Gonsnent. Complete disintegration s defined oar inwhich any residue of the unit, except fragments of ilble coating or capsule shell, remaining oth seren of she rest apparatus or adhering to the lower surface of the dics, if used, is a soft mass having no palpably firm core {Be apparatus A for tablets and capsules that ae not geaer thn 8 mm long. For lager tablets or captor ace sopra B. Test A ~ Tablets and capsules of normal size “The apparatus consists of a basket rack bl 1 ie low-form beaker, 149 © 11 am in ‘having an inside diameter of 106 + 9 mm for flid, a thermostatic arrangement for heating n 35 °C and 39 °C, and a device for raising he basket in the immersion fluid at a constant ‘between 29 and 32 cycles per minute, of 55 + 2 mm. The volume of the fuid that at the highest point of the upward Femains at ast 15 mm el the descends to not less than 25 mm. selon the downward stroke. ofthe basketrack assembly time required forthe upward sock: for the downward stoke, and “a smooth anstin, rather “The basket-ic assembly . There is no appreciable rrngvement of the ai rom the vertical mbly The basket-rack assembly consists 19-mm thick; the tubes are held in a 2 plates, each 90 + 2 mm in diameter ii a i Appendix XII A A329 29.161 : os . hata Discs ‘The ue of dnc i permited only where specied or lowed. Each tbe provided witha ey dhe 8.5 £ 0.15 mm thick and 207 + 0.15 mm in dameter “The dis is made ofa suitable, transparent plastic mater having a specie gravy of 1.18-1.20,5 parallel 2 = 0.1 mm holes ented between the ends of the cinder. One of che hoe is centered on the cindal ax: The oe oles are centered 6 0.2 mm from the as on imaginary fins Perpendicular to the ani and parallel to each other. 4 idencaltapezoidaabape planes ae ct into the wall of the ofiader, nea perpendicular to the ends of the cvinde. ‘The trapeasdal shape is symmetrical parallel sides coincide withthe ends ofthe ends and are parallel to an imaginary Hie connecting the cannes of 2 adjacent holes 6 mum from the clndral ais. The parallel side ofthe trapezoid on te bottom ofthe ender has length of 1.6 = 0.1 mm and is boom edges le ata depth of 1.5 mm to 8 mm from the eyinder's deeuference “The parle side ofthe trapezoid on che top of the evinder has elengh of 9.4 © 0.2 mm ad scone cs at depth of 2.6 = 0.1 mm fom the cinde?'s crcumference. All surfaces ofthe dsc ae smooth Ifthe use of is is spect, ad a disc ro cach tube and operate the apparatus as decid under Procedure. The diss Conform to dhe dimensions shown in Figure 29.11 “The ase of automatic detection employing modifi dss is permited where the une of dc speed or allowed. Sich {dscs must comply withthe requirements of density and Alimension even in this chapter Procedure Place | dosage unit in cach of the 6 tubes of the basket and, it prescribed, add adie, Operate the appraising the speibed mim, mainained at 37 2 -Gyas the immersion fd, At the end of the Specie ine ft he basket from the uid and observe the dosage units all ofthe dowage units have diimerated completely. IF or 2 dosage ois fl to disintegrate, repeat the test on 12 addon dosage uns. The requremens of te tes are met if ao les than 16 ofthe 18 dosage units tested have dintezrated “Test B~ Large tablets and large capsules [Apparatus The main par of the apparaus (Figure 2.1-2) is rigid askeveack assembly supporting 5 eylndcal transparent tubes 775 + 2.5 mm long, 33.0 mm + 0.5 mm in intemal diameter, and with a wall chickness of 25 4 0.5 mn, Each tube i provided with a cslindrical dsc 314 + 0.13 mm in diameter and 15. 4 0.15 mm thick, made of transparent plastic with a relative density of 1.18-1,20. Each disc is pierced by 7 holes, tach 3.15 + 0.1 mm in diameter, 1 in the centre and the other 6 spaced equally of a circle of radius 4.2 mm from the Cente of the dsc, The tubes are held vertically by 2 separate hd superimposed rigid plastic plates 97 mm in diameter and ‘ mm thick, with 3 holes. The holes are equidistant from the ‘entre of the plate and equally spaced. Attched to the under Side ofthe lower plate isa piece of woven gauze made from Sains steal wire 0.63 + 0.03 mm in diameter and having mesh apertures of 2.0 + 0.2 mm. The plates are held rigidly ih postion and 77.5 mm apart by vertical metal rods atthe A metal ro abo fed othe cet ofthe oper plate to enable the assembly to be atached toa Pree device capable of ang and oven aanehly ata constant frequency of between 29 and SPegeles per minute, through a dixance of 35 = 2 mm.A330 Appendix XII A Baskot-rack ‘assembly Disc q a # view 5 Si rr 9520.15 | Figure 291-1, ~ Disintegration apparatus A Dimensions in millimetres tetris» fesse ben: ramumn the surface ofthe liquid. A suitable d ite levice maintains |__mpee ofthe guid a 35.39 °c design of the basket-rack assembly may be varied Drovided the specifications forthe tubes and wire mesh a25205 F 715425 Appendix XI A A331 97 25205 330205 | | | sraz09_| ee 4634045 Figure 29.1.2, - Disintegration apparatus B ‘Dimensions in millimetres er by usin Method ‘Test 6 tablets oF capsules & Ziaserack assemblies in parallel by repeating (De tMenlre Tn each ofthe 3 tubes, pace 1 tablet OF APSIC y gasped the assembly in the containing the specified liquid. Operate the apparatus fete reseed period, withdraw the assembly a4 Giaine the sate ofthe tablets or capsules, To Pass 150% 41.6 ofthe tablets or capsules must have since i wries and Disintegration Test for Supposito# Paces 2 Ph. Bu, method 2.9. The dtargtin otdctemins wine be SPT ° Pessares soften or disintegrate with the preseibed Hine ‘he lcd ini mem in the expen Senton described below. Disintegration is considered to be achieved when: a) dissolution is complete, {the components of the suppository or pessary have separated: melted faty substances collect on the surface fhe liquid, insoluble powders fall tothe bottom and Soluble components dissolve, depending on the type of preparation, the components may be distributed in one for more of these ways there is softening of the sample that may be accompanied by appreciable change of shape without Complete seperation of the components, the softening is Sic that the suppository or pessary no longer has a afd core offering resistance to pressure of a glass rod, 42) rupture of the gelatin shell of rectal or vaginal capsules ‘Gevurs allowing release of the contents, the perforated disc or if a residue ©) no residue remains on t renaing it consists only of a soft or fothy mass having1 solid cote offering resistance to pressure of a glass rod (vaginal tablets). Apparatus ‘The apparatus (Figure 2.9.2.1) consists ofa sleeve of glass or suitable transparent plastic, of appropriate thickness, 0 the interior of which is attached by means of three hooks a ‘metal device consisting of two perforated stainless metal dises ‘each containing 39 holes 4 mm in diameter; the diameter of the discs is similar to that of the interior ofthe sleeve; the discs are about 30 mm apart. The test i carried out using three such apparatuses each containing a single sample. ‘Each apparatus is placed in a beaker with a capacity of at least 4 L filled with water maintained at 36-37 °C, unless otherwise preseribed. The apparatuses may also be placed. fogether in a vessel with a capacity of atleast 12 L. ‘The beaker is fitted with a slow ster and a device that will hhold the cylinders vertically not less than 90 mm below the surface of the water and allow them to be inverted without ‘emerging from the wate. A332 Appendix XII B y ee the apparatus described above, arranged 50 28 tort og Pies i to ofthe Ui ae rl ape a he ae eee Some ee Se a ae Be a be KS. QW0 ‘Alas pate D. water vaginal tablet dish, beaker Cater surce Figure 2.9.2.2, Monographs of the British Pharmacopovia The flloving atonal pins apply to monographs ofthe British Pharmacopocia. : ee we AccRPrANE CRITERIA For moulded supostoris, disintegration oc in 0 ‘hun 30 mines for fabased suppor an inno 0 than 6 mines for watersohle vpposore los others justified and authored, For moulded pessaris,dntegration occur in ao 08 than 60 mites unl othervise sine and ators For etal capsules and vaginal aes and capes Asnerain occur in not more dn 30 nisses, B, Dissolution 1, Dissolution Test for Tablets aoe Dissolution Testor Solid Dosage Pores) (Ph, Eur. method 2,9,3) ‘This test is provided to determine compliance with the equitements for solid dosage forms adminis Grab. In this chapter, a dosage unit i defined as 1 tae“ 1 capsule or the amount specified.na. 4 a prARATUS: rus 1 (Basket apparatus) The Apfel: a veel, Which may be concen Hl efindsical basket (ning i OS 8 ie Sprenient size or heated by a suitable device saan con rs ae even faxsaining the temperature inside the yeast Se, Se ie weer siemens in which he seca gett lng eee ae pore fring the test is preferable. The vessel is cylindrical, with Gee Re encores P06 mm on Sect eds mS a ea te ne a eee woe ined that allows the shaft rotation speed to be selected and erro Ree. ee ree STS eee (00001 inch) thick may be used. ‘The dosage unit is placed. re a ee eee | SNe apenas Apparatus 2 (Paddle apparatus) Use the assembly from Apparatus 1, except that a paddle formed from a blade and shaft is used as the stirring element. The shaft is positioned ‘otha its exis is not more than 2 mm from the vertical axis of the vessel, at any point, and rotates smoothly without significant wobble that could affect the results. The vertical ‘enter line of the blade passes through the axis of the shaft $0 thatthe bottom of the blade flush with te bottom of the shaft, The paddle conforms to the specifications shown. in Figure 2.0.-2. The distance of 25 + 2 mm becween the bottom ofthe blade and the inside bottom of the wesc 8 ‘maintained during the test, The metallic or suitably inert, gd blade and shaft comprise a single entity. A suitable two-part detachable design may be used provided the sembly remains fimily engaged during the test. The pad’le ‘blade and shaft may be costed with a suitable costing, 0 as ‘to make them inert. The dosage unit is ‘allowed to sink to th bottom of the vessel before rotation of the Dads is i ‘aed. A small, loose piece of non-reactive matrah *eh Bot more han fe tran of wie el, maybe arash ‘dosage units that would otherwise float. AP ee, Avice is shown in Figure 2.9.3.3. Other validated Sn may be used ie Appendix XII B A333 6306s e040 104 Vento = 20205, 3 Retention sting wath tangs on BY cans "Sear pening w2s10 seen oo —[Etey a asi) [BEEN ft 39 EE 39 ee 44 eH 55 EH | 1 ¥ ‘ hearin oe | 1 1S in eed an: 022251 we ameter whe openig of 0360.44 mm. Aer Welling the res sy be shy ators. 2) Marin allable renaut at°A ie LO ‘heath prt seated on center ne 2 a rset outed Apparatus 1, Basket stirring element Dimensions in millimetres Figure 293. “Apparatus 3 (Reciprocating cylinder) The assembly consists of a set of cylindrical flat-bottomed glass vessels fa set of glass reciprocating cylinders; inet fittings (stainless sel type 316 or other suitable material) and screens that are ‘made of suitable nonsorbing and nonreactive material, that are designed to fit the tops and bottoms of the eeiprocating cylinders; a motor and drive assembly to Feciprocate the cylinders vertically inside the vesels, and if Feared, index the reciprocating cinders horizontally to 8 ‘Gierent row of vessels. The vessels are partially immersed in raitable water-bath of any convenient size that permits folding the temperature at 37 + 0.5 °C during the test No part ofthe assembly, including the environment in which fhe assembly is placed, contributes significant motion, tigtaton, oF ibradon beyond that due tothe smoot,A334 Appendix XII B xy WY, partis otated on centerline ais. Tolerances tus 8, Paddle stirring element oe ies ‘referable. The vessels are provided with 20 fn cap that remains in place forthe dustin of APonents conform to the dimensions shown iAc acidresistant wire clasp Appendix XII B A335 120202 Baca wie suport Figure 293.3. - Aternative sinker Dimensions in millimetres Apparatus 4 (Flow-through cell) The assembly consists ofa reservoir and a pump forthe dissolution medion tfow-through cell; a water-bath that maintains the Sscon metiam a 97 £03 °C, Use the spate The pump fores the dissolution medium upwards through the ow-through cell, The pump has a delivery range terween 240 mU/h and 960 mL, with standard flow rates f4 mini, 8 mL/min, and 16 mL/min. Ie must deliver a coostnt flow (E 5 per cent ofthe nominal flow rate), ‘ke ow profile is sinusoidal witha pulsation of 120 + 10 pulsesimin. A pump without pulsation may slo be tse Dissolution test procedures using the flow-through eel sms be characterised with respect ro rate and any pulsation. The flow-through cel (see Figures 2.9.35 and 2.9.36) of tansparent and inert material is mounted vertically with a fier system that prevents escape of uncisolved particles fom the top of the cell standard call diameters are 12 mm. and 22.6 mms the bottom cone is usually fled with small fs beads of about 1 mm diameter, with 1 bead of about mm positioned at the apex to protect the fluid entey tude; tibet holder (see Figures 2.9.3.-5 and 2.9.3-6 i available {or positioning of special dosage forms. The cells immersed “inavwaterbah, and the temperature is maintained "37 £05°C, F ‘The apparatus uses clamp mechanism and 2 O-rings for ‘be fixation of the cell assembly. The pump is separated from the dissolution unit in onder o shield the later eines ens ‘brations originating from the pump. The position of Dump mast not be on # level higher than the resevoi Has Tube connections are as sbott as possible, Use suitably inert ‘ubing, such as polytetrafiuoroethylene, with a 1.6 mm inne es ily of Apparatus suitability The determination of suiehth the apparatus to perform dissolution testing mus neh éonformance to the dimensions and tolerances of ‘te of medium (Apparatus 4)- Decmine the acceptable performance of ‘sembly periodically. Wines b the dissolution test PROCEDURE, Apparatus 1 and 2 Conventional-release solid dosage forms Procedure Place the stated volume ofthe dissolution ‘medium (+ 1 percent in the vessel ofthe specified apparatus. Assemble the apparatus, equilibrate the dissolution ‘medium w 37 + 0.5 °C, and remove the thermometer ‘The test may also be carried outwith the thermometer in place, provided tis shown that results equivalent to those ‘obtained without the thermometer are obtained, Place 1 dosage unit in the apparatus, taking care to exclude air bubbles from the surface ofthe dosage unit. Operate the apparatus atthe speciied rate, Within dhe time interval speciied or at each ofthe times stated, withdraw a specimen ffom a zone midway between the surface of the dissolution medium and the top ofthe rotating basket or Blade, not less than 1 cm from the vessel wall. Where multiple sampling times are specified, replace the aliquots withdrawn for analysis with equal volumes of fresh dissolution medium at 37 °C or, where it ean be shown that replacement ofthe ‘medium is not necessary, correct for the volume change in the calculation, Keep the vessel covered for the duration of the test and verfythe temperature of the medium at suitable times, Perform the analysis using a suitable assay method’ Repeat the test with additional dosage units. If automated equipment is sed for sampling or the apparatus is otherwise modified, verification that the ‘modified apparatus wil produce results equivalent ro those obtained with the apparatus described in this chapter, is Dissolution medium ‘used, The volume specified refers to measurements made bpetween 20°C and 25°C. Ifthe dissolution medium is a buffered solution, adjust the solution so that is pH is within table diechition medium is 10.05 units of the specified pH. Dissolved gases can cause ‘bubbles to form, which may change the results ofthe rest. In such cases, dissolved gases must be removed prior to testing Te cine are lee omy upon sampling wes. donna 1 be wuss. Use an inc flr ta trove the anabsic Tf mated of doen i 2 olla: hat the medion, ce string °C, manly fle under exci sing a fir having ely, 1 abou 4 “am a Spr of 05m oe thon rig, an cm ig ei psn for abou Sin. Or aliated deacon removal of sled gases may be wrell A336 Appendix XII B pee eee Time Where a single time specification is given, the rest ce may be concluded in « shorter period ifthe requirement for 2 —Aiteles minimum amount dissolved is met, Samples ae to be San ‘vithdrawm only a the stated times, within a tolerance oa 2pecount aS Prolonged-release solid dosage forms Ff \ Procedure Proved as described for conveniona-lease t ; dosage forms. ! Dissohution medium Proceed as describe for ‘coaventional-release dosage forms. | I Time “The testme points, generally 3, are expressed in - hours Delayed-release solid dosage forms 6821 Procedure Use Method A or Method B. Airholes ‘Method A COOH — Acid stage Place 750 mL. of 0.1 M foros acid in the vessel, and assemble the apparatus. Allow the medium to equilibrate to « temperature of 37 +: 0.5 °C, Place 1 dosage unit in the apparatus, cover the vessel and operate the apparatus atthe specified rate, After 2h of ‘operation in 0.1 M dhol acid, withdraw an aliquot ‘ofthe fluid and proceed immediately as directed under ‘Buffer stage, Perform an analysis ofthe aliquot using a suitable astay method. Patents poem pang oe the pH within 5 min, With the srr pr ef 50 mL. of a 0.20 M solution of miodion ate dodecahyavateR that has been equilibrated to f ‘if necessary, with 2 M hackle Rio a pH of 68 + 0.05. Glas reciprocsting cylinder = Mesh screen 47214 —— Giass vessel 10021 | | | | : Figure 293-4. Apparatus 3, glass vessel and reciproctt ‘ulinder Dimensions in miltimetres untess otherwise specifiedAppendix XII B A337 core SR teen os Figure 2.3.5, - Apparatus 4 large cll or tablets and capsules (top) tablet holder fr the large cell (ottom) sim mlietres unless otherzsespeciied Dimension’A338 Appendix 3 sr canbor 4 sowd02 w=045 ii at i nt Hh | id ora 02, ! Score for the holder he £ 20.82 0.05 ’ Sr a pan tT from a zone midway between the ium and the bottom of eh the analysis as directed, If necessary repet! test with additional dosage units. aliguot withdrawn for analysis with equal (of fresh dissolution medium at 37 °C or, where it lacement of the medium i aotaf Appendix XII B A339 a red a Coane the cll covered cation ee ett alpine ‘ te eaatOn ap for the ess Dae tere ouution medium Proceed as d peri re Sova ams tas Proceed described for conventionsieegs nt? a ander Appa 1 and 2, al release dosage fetagd-release dosage forms rere Droced 8 decribed for co Pomorns under Apparates 3,00 mmmahcese tiom medium Proceed as describe seein mre Sted tr es Time Proceed as described for prolonged-ceas arene peised-release dosage forms wre Proceed as described for delayed-reease fa tb Bde Apis In iene Shreve forthe acid stage media and the following row o aires fox the bllr stage medio and wing the wine of tedium specified (usually 300 mL). Tine Proceed as dzected for delayed-release dosage forms dr Apparatus Land 2. Apporatus 4 CCosventional-release dosage forms Procedure Place the glass beads into the cell specified Phor 1 dosage unit on top of the beads or, if specified, on wie care. Assemble the filter head and fx the pars together by means of a suitable clamping device. Introduce by the pump the dissolution medium warmed to 3105 °C through the bottom of te cell vo obtain the fiow rate specified and measured with an accuracy of S per cent. Collect the eluate by fractions at each of the ties stated. Perform the analysis as directed. Repeat the tse with additonal dosage writs. Dissolution medium Proceeds described for conventionabrelease dosage forms under Apparatus 1 and 2+ Time Proceed as described for conventionalreease dosaae forms under Apparatus 1 and 2. Prolonged-release dosage forms Procedure Proceed as described for conventional-rlease Aasage forms under Apparatus 4 Dissolution medium Proceed as described for Cenventinal-release dosage forms under Apparatus 4 Time Proceed as described for conventional-slease 4osaee forms under Apparatus 4. Jayed-release dosage firms under Apparatus 1 and 2, using the specified media Time Proceed as described for dlayes-elese

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