METHODS OF ANALYSIS OF DIURETICS

INTRODUCTION TO FUROSEMIDE Structure:

HOOC NH CH2 O2SH 2N Cl O

Molecular formula Molecular weight Appearance Co lour Odor Taste Solubility

: : : : : :

C12H11ClN2O5S 330.77g/mol crystalline powder slightly yellow odorless tasteless

: soluble in water, chloroform, ether, acetone, methanol, dimethyl formamide and in alkali hydroxides : high ceiling efficacy diuretic

Category

METHODS OF ANALYSIS:
1. Titrimetric method 2. Column chromatography 3. Thin layer chromatography 4. Gas chromatography 5. High performance liquid chromatography 6. Ultraviolet spectroscopy 7. Colorimetric method 8. Infrared spectroscopy

TITRIMETRIC METHOD:
Principle: Acid-base titration Reaction:
COOH NH O

H2NO 2S

NaOH

COONa NH O

H2NO 2S

Procedure:
0.5g of furosemide dissolve

40ml Dimethyl formamide bromothymol blue

Titrated with 0.1N NaOH End point is blue colour. Each ml of 0.1NaOH = 0.03308g of C12H11ClN2O5S

COLUMN CHROMATOGRAPHY:
Principle Column Adsorbent : Column adsorption chromatography : Stainless-steel column(15cm x 4.5cm) : Lichrosorb RP-18 or RP-8

Mobile phase : Various mobile phases

Sample Detection : 20mcl of urine : U.V at 243nm

THIN LAYER CHROMATOGRAPHY:

Principle Adsorbent Solvent Standard : Adsorption : Kiesulghur 60 : Chloroform: ethyl acetate: formic acid(14:6:1) : 0.2 to 10mg of furosemide and its metabolite

Sample

: 0.5ml of plasma with 1ml methanol is centrifuged, a 50l of the aliquot of the supernatant liquid is used.
: 3hours

Drying time

Spraying reagent : 10% citric acid in aqueous ethanediol(1:1)

HPLC:
Sl Stationary No phase
1. Bondapack C18 corasil (1.8x2mm) Bondapack C18 column (30x4mm) Lichrosorb RP8 (5)

Mobile phase

Flow rate Detection

Retention time
3min

0.02M KCl-HCl 0.9ml/min U.V at buffer pH 2 280nm 10mM (NH4)2HPO4 in 25% methanol 2ml/min U.V at 254nm

2.

3min

3.

Methanol1ml/min 0.02M, phosphate buffer of pH 3.0(1:1) Methanol: water: acetic acid (40:57:3)

Flourimetric detection

3min

4.

Column packed with ODS(5)

1.45ml/mi Flourimetric n detection

3min

ULTRAVIOLET SPECTROSCOPY:
Sample : Lasix (furosemide preparation) Solvent : Water Absorbance maxima : 228nm E1%1cm : 945

COLORIMETRIC METHOD:
Furosemide produces blue colour when it reacts with butylamine,carbonyl chloride and acetic acid in a medium of anhydrous methanol.

Colour is measured at 570nm.

It can also be detected by mixing a solution of p-dimethyl aminocinnamaldehyde in 65%H2SO4, containing FeCl3, diluting with ethanol and measuring the absorbance at 530nm.

INFRARED SPECTROSCOPY:
Frequency(cm-1) 3350-3400 1671 1576 1322 582 Type of vibration NH C=O NH -S=O Cl Assignment C-NH -COOH -NH2 -SO2 C-Cl

GAS CHROMATOGRAPHY:
Principle Sample : : Partition Trimethyl derivative of furosemide

Carrier gas :
Column

Nitrogen

: Silanisedglass column(1.8x2mm) packed with 3% of JXR (supelco) on Gas-ChromQ(100-120mesh)

Temperature : 245oC
Detector :
63Ni

electron-capture detector

Internal standard : Trimethyl furosemide

INTRODUCTION TO SPIRONOLACTONE Structure :

O O

SCOCH

Molecular formula : C24H32O4S Molecular weight : 416.59 g/mol Appearance : Crystalline powder

Colour
Odor

: Yellowish-white
: Faint mercaptan

METHODS OF ANALYSIS:
1. Thin layer chromatography 2. Ultraviolet spectroscopy 3. Colorimetric method 4. Infrared spectroscopy

5. Flourimetric analysis

THIN LAYER CHROMATOGRAPHY:

Example 1: Solvent system : Ethyl acetate(100%) Adsorbent : Silica gel GF(Woelm) Detection : Short wave U.V, : Spraying with 50%H2SO4, heat to 80oC for 10 minutes, observe under long U.V, : Spray with phosphomolybdic acid. Rf value : 0.53

Example 2: Solvent system : Benzene:Ethyl acetate:Methanol(73:25:2) Adsorbent : Silica gel G Detection : Spray with phosphomolybdic acid and heat at 80oC for 10 minutes Rf value : 0.67

INFRARED SPECTROSCOPY:
Wave number (cm-1) 1775 1670-1690 1620 Assignment 5 -membered,lactone carbonyl 3- ketone,7-thioester carbonyl 4,5-double bond

ULTRAVIOLET SPECTYROSCOPY:

COLORIMETRIC METHOD:
Reaction of spironolactone with methanolic hydroxylamine hydrochloride and ferric perchlorate yields a red ferric hydroxamate complex having an absorbance at about 515nm. Colour is stable upto 2hrs. Isonicotinic acid hydrazide may be reacted with spironolactone in methanolic solution,yielding a soluble yellow product having an absorbance at 575nm.

FLUORIMETRIC ANALYSIS:
Spironolactone

Dethioacetylated under mild acid Or alkaline conditions

4,6-dienone,canrenone

In 62% sulphuric acid

Fluorescent trienone (excitation maximum at 483nm and an emission maximum at 525nm)

AMILORIDE
Structure :

.HCl.2H2O

Mol formula : C6H8ClN7O.HCl.2H2O Mol weight : 302.12g/ml Description : Yellow to greenish yellow crystalline powder which is odorless. Solubility : Freely soluble in Dimethylsulfoxide, sparingly soluble in water, very slightly in ethanol. Brand name : MIDAMOR

METHODS OF ANALYSIS :
Non aqueous titration Thin layer chromatography UV spectroscopy HPLC IR spectroscopy

1. Non aqueous titration : Principle : Substances which are either too weakly basic or too weakly acidic to give sharp end point in aqueous solutions are determined by non-aqueous method. Procedure : Dissolve 450mg of sample in 100ml of glacial acetic acid Add 10ml of mercuric acetate. Add 15ml of dioxane. Titrate with 0.1N perchloric acid using crystal violet as indicator until a blue color. Blank determination is done to make necessary corrections. Each ml 0.1N perchloric acid = 26.61gm of Amiloride hydrochloride.

HClO

ClO 4

CH 3COOH HClO

CH 3COO

4 + CH 3COOH + CH 3COO + CH 3COOH 2

CH 3COOH

+ 2

ClO 4

2 CH 3COOH

O Cl N

NH2 Cl .HCl.2H 2O N

NH2
+

CN CNH 2

CN C NH

CH3COOH 2

H2N

NH2

H2N

NH2

Amiloride HCl

1. Thin layer chromatography : Principle :

Main principle is adsorption. Adsorbent acts as stationary phase, Mobile phase selection depend on elutropic series based upon polarity of solvents.

Amiloride hydrochloride is analyzed by Normal phase TLC on silica gel by employing two solvent system ;

Solvent system

Sample spot Detection of spot

Rf value

Sensitivity

AMILORIDE HYDRO CHLORIDE

1) 10% npropanol in chloroform.

5l of 1% aqueous soln of drug.

0.01%

2) 3N aqueous 1l of 0.1% ammonium aqueous hydroxide + soln of tetrahydrofura drug. n (4 : 30)

By UV at 254nm or 360nm.

0.7 0.1 to 0.5%

UV spectroscopy :
Principle : Any molecule has either , and or a combination of these

electrons. Bonding ( and ) & non-bonding () electrons abosorb the characteristic radiation and undergoes transition from ground state to excited state. By this characterization absorption peaks are obtained. There by the nature of electrons present and hence the molecular structure can be elucidated.

Procedure : UV spectrum of amiloride can be determined in a solution

of 0.01N aqueous hydrochloric acid. Absorption maxima is approximately at 212nm, 285nm and 362nm . Molar absorptivity of 1% drug solution in a 1cm cell is 642, 555 and 617 for the wavelengths of 212nm, 285nm and 362nm respectively.

4. High Performance Liquid Chromatography :

Principle : Amiloride is determined by Rp-HPLC method Where stationary phase is non-polar and mobile phase is polar.
Procedure : Column : C-2 ( 300mm X 4.6 mm i.d) Adsorbent : Silica Gel G Particle size of adsorbent : 10m Temp : Ambient Mobile Phase : 85% aqueous 0.01Msodium hexane sulphonate in acetonotrile Flow rate : 2ml/min Retention time : 2mins Detection : By UV at 280nm.

5. IR spectroscopy : Principle :
For any molecule to absorb IR radiation it should posses, Dipole moment Applied frequency of IR must be equal to natural frequency of radiation. IR spectra is nothing but a fingerprint of a molecule.

Procedure :
1) IR spectrum in a KBr pellet was obtained using a Fourier-transform (FT IR ) spectrometer. 2) IR spectrum obtained in a mineral oil called mull ( nujol )

IR spectral assignments :
Frequency ( cm-1 )
3250 -3500 3150 1680 1640 1600 1240 770

Assignments
N-H stretch ( NH2) N-H stretch (NH) C=O stretch H2 deformation mode H2 deformation mode N- (C6H6) stretch C-H out of plane mode

REFERENCES :
Text book of Pharmacology by P.D Seth. Pg 292 303. Analytical Profiles of Drug Substances by Klaus Florey, vol 15, Pg 1 30. Clinical Pharmacology by P.N.Bennett and M.J.Brown. 9th edition, Pg 529 547. Modern Pharmacology by Charles.R.Craig and Robert.E.Stitzel,3rd edition, pg 270 270. The Pharmacological basis of Therapeutics by Goodmann and Gilman, 8th edition, vol 1, pg 708 730.