ANALYSIS OF VITAMIN B SERIES

PRESENTED BY-
MINI JHA
MPHARM ANALYSIS
INTRODUCTION TO VITAMINS:
 Vital amines/growth factors/accessory factors.
 Any group of organic compounds required in
small amount to perform specific biological
functions for normal maintenance of optimum
growth and health of organism.
 Classification:
 F a t soluble vitamins: vit-A, vit-D, vit-E, vit-K.
 Water soluble vitamins: vit-C, B-complex, vit-H.
 *THIAMINE*
 Chemistry: NH3+ S
H3C N CH2CH O
2
H

.22 Cl-
Contains pyridine a nd thiazole ring structures.
N N
 Functions: + CH3

 Prosthetic group in decarboxylation. a n d transketolase reactions.

 Deficiency disorders: Beri beri.

 Properties:
1) White crystalline powder with slight characteristic odour.
2) It is hygroscopic.
3) Soluble in water, glycerol, alcohol. Practically insoluble in ether, benzene,
hexane, a nd chloroform
 Chemical, biological a n d microbiological methods are available for the analysis of
thiamine.
◦ The bio-assay is based on prevention a nd cure of polyneuritis a nd weight gain
in animals. Microbiological methods using Phycomyces blakesleeanus a nd
Lactobacillus ermenti are available. A yeast fermentation method is also
available.
 FLOURIMETRIC METHOD (Thiochrome)
1. Thiamine is quantitatively isolated from the foods, biologicals,
and pharmaceuticals usually by boiling with dilute acids and
treatment with enzyme preparations containing phosphatases
which will free thiamine from its n atu ral complexes.
2. Protein substances must be digested with proteolytic enzyme like
papain.
3. Purification of extract is done by passing through zeolite, a n
inorganic ion exchanger an d thiamine is retained in the zeolite.
4. Add acidified potassium chloride to elute thiamine.
5. Further it is oxidized with alkaline potassium ferricyanide to
produce thiachrome. This compound is having blue fluorescence.

H3C N NH3+ S CH22CCH-2l O H

H3C N N S CH2CH2O H
K3 Fe (C N)6
N N
CH3 N N
+ CH3
 SILICO TUNGST IC ACID METHOD (Gravimetry):
{H4Si(W3O10)4 nH 2 O }

Prepared by One mole of Silicic acid for every twelve moles of

Sodium Tungstate.
 Thiamine in tablets and solutions may be determined by
precipitation with silicotungstic acid.
 The sample is dissolved in acidified water an d heated to boiling.

 The precipitate is filtered washed with acid and water finally

with acetone. Then its weighed to constant weight.

Each gram is equivalent to 0.1936 g of thiamine hydrochloride.

 COLORIMETRIC METHOD:
 Method 1: with p-Amino acetophenone:

C O C H3
H3C N NH3 S CH2 CH2 O H
complex
N N +
CH3 520 nm
 The extraction is done as gNivNenHin thiochrome method.
diazotised p-amino acetophenone

 Thiamine couples with diazotized p-Amino Acetophenone

which has a n absorption maximum a t 520nm.

 This method can be used to determine thiamine in presence

of phosphorylated thiamine an d is useful in urine analysis.

 This method is not recommended for assay of materials rich

in protein content an d low in thiamine.

 Results of this method agrees with the biological method.

 Method 2: using 6-Aminothymol.
OH
H3C N NH3 S CH2 CH2 O H CH (CH 3)2

N N + color complex
CH3 N NH
H3C

Acolor reaction between thiamine and diazotized 6-

Amino thymol is seen. This method is simple fast and
no interference is seen between the degraded thiamine
and 6-Aminothymol.
 Miscellaneous Methods:

 Method 3: Non-aqueous titration:

 Thiamine HCl can be titrated with perchloric acid in glacial acetic acid solution, if a n
excess of mercuric acetate is added. Both the nitrogen are titrated. p-
Naphthol-benzein and quinaldine red are suitable indicators.

Method 4: Argentometric Method:.

 Total chlorine in thiamine HCl can be determined by dissolving in acidified water
(HNO 3 ) added with excess of silver nitrate. The precipitate is filtered and washed.
The filtrate is then titrated with 0.1N Ammonium Thiocyanate.
1ml of 0.1N AgNO3 is equivalent to 0.003546 g of chlorine.

Method 5:
 Chloride as hydrochloride is determined by titration with 0.1 N sodium hydroxide to
pH7 using bromothymol blue as indicator.
Each ml of 0.1 N NaOH is equivalent to 0.003546 g of chlorine

Method 6
 The nitrate in thiamine mononitrate is determined by precipitation with nitron
(1,4-Diphenyl-3-phenylamino-1,2,4-triazolium hydroxide inner salt)
from a n acidified solution.
 Nitron is a compound C20H16N4 used in the qualitative and quantitative determination
of nitric acid with which it forms a n insoluble nitrate
 * NIACIN (NICOTINIC ACID)*
COOH
Chemistry:
◦ It’s a heterocyclic. 3-pyridine carboxylic acid.
Functions: N
◦ It functions as oxidising co-enzymes of many dehydrogenases
Deffiency disorders:
◦ Pellagra, characterised by dementia, dermatitis a nd diarrhoea.

PROPERTIES:
1. Solubility: Soluble in boiling water a nd in boiling ethanol (95%). Sparingly
soluble in water. Very slightly soluble in chloroform. Practically insoluble in
ether. It dissolves in dilute solutions of alkali hydroxides a nd carbonates.
2. Non-hygroscopic a nd stable in air.
3. It has absorption maximum in UV a t 262nm.
4. Identification test:
Thiamine is dissolved in water, a nd neutralized to litmus paper with 0.1M
sodium hydroxide, add 3 ml of copper sulphate solution; a blue precipitate is
 ACID BASE TITRATION:
COOH
COONa
+ NaOH
H2 O
N +
N

 Nicotinic acid can be titrated with NaOH using

phenolphthalein solution as indicator.

 COLORIMETRIC METHOD U S I N G CYANOGEN

BROMIDE:
SO3H
COOH
+ + CNBr cleavage of CN bond of pyridine by CNBr red color measured spectroscopically
N

NH2

 This is based on a color reaction of pyridine an d

unsubstituted derivatives. Cyanogen bromide breaks one
carbon-nitrogen linkage an d produces a color compound upon
addition of amine or ammonia.
 DETERMINATION OF NICOTINAMIDE
 ACID-BASE TITRATION:
◦ Nicotinamide when boiled with sodium hydroxide solution, it
releases the nitrogen of the amido group in the form of ammonia,
which can be collected in sulphuric acid and determined by
titration.

CONH2
COOH
alkaline hydrolysis
N
N
+ NH3
Nicotinic acid
 The liberated ammonia collected in sulphuric acid and
determined by titration with NaOH.
2NH 3 + H2 SO4 ----------- (NH4)2
(NH4)2 + 2 NaOH----- Na2SO4 + 2 H2O + 2NH 3
 End- point being determined using phenolphthalein
indicator.
 RIBOFLAVIN (VITAMIN B2)
 It is also called as lactoflavin.
 Properties: CH 2OH
 Yellow to orange-yellow, crystalline powder with slight. Odour HO C H
 Deffiency:
HO C H
 S o lu bility :
HO C
 Very slightly soluble in water; more soluble in saline solution t h a n H
CH 2
in water; practically insoluble in chloroform, in ethanol (95%)
H 3C N N O
an d in ether. N
H3 C N
 Aqueous solutions exhibit a n intense yellow color-green fluorescen ce a t pH6. O
 Specific rotation is Between –115o and –135o, determined in a 0.5% w/v solution in
carbonate-free 0.05M sodium hydroxide .
 Riboflavin shows absorption maxima a t 224, 267, 373, 445, an d 475nm.
 Irradiation either with uv or visible light, of alkaline solutions, produces luminoflavin,
irradiation of acid or alkaline solutions produces luminochrome, a blue fluorescent
substance.
 Reducing agents such as, sodium hydrosulphite, reduce riboflavin to a dihydro
compound, leucoflavin which is not fluorescent. But this is reversible and leucoflavin is
readily oxidized back to riboflavin by atmospheric oxygen.


 METHOD 1: FLUORIMETRIC METHOD

 There are three methods are in use in

fluorimetric determination of riboflavin;
 Direct determination

 Direct additive determination

 Adsorptive additive determination

 FLUORIMETRIC METHOD: DIRECT
DETERMINATION:

 This method is employed for the mixtures which are free of interfering pigments
or substances and contain relatively high concentration of riboflavin.
 An appropriate quantity is weighed and add with boiling distilled water and
shaken for few minutes, if required, boil the solution. It is then centrifuged.
 S a m p l e solution: a suitable aliquot of clear liquid is diluted appropriately to
yield concentration of 0.2mcg/ml.
 B l a n k solution: add a few granules of sodium hydrosulphite to the aliquot of
sample solution. This shows indication of purity of solution.
 M e a s u re m e n t a n d calculation: Readings must be taken as rapidly as possible.
 Mcg of r ibofla vin = A– C * dilution factor
B–C wt of sample (g.)
 A  reading of unknown concentration.
 C  unknown blank
 B  reading of stan d ard
 D  reading of stan dard blank.
 FLUORIMETRIC METHOD: DIRECT ADDITIVE
METHOD
 The interference of other substances can be avoided by this method. In this method the
addition of the known quantity of riboflavin to the assay solution is used to compensate
for interfering substance which may absorb the incident or fluorescent light.
 Sample treatment: samples of natural origin should be subjected for enzymatic
hydrolysis to digest starchy substances and to “free” any “bound” riboflavin.
 Standard solution I: 40mcg/ml of USP reference standard in 20% of ethanol.
 Standard solution II: dilute solution I such that it contains 1.6mcg of riboflavinin
distilled water.
 Measurement and calculation:
 Mcg of riboflavin per gram= A – (1.07C) * 1.6 * 1 dilution factor
B- (0.94A) 16 wt of sample (g)
 A  fluorescence of sample solution
 B  fluorescence of standard II
 C  blank solution ( obtained by adding HYDROGEN SULPHITE to standard solution IIand
this should be repeated until successive additions shows no deflections)
 1.07 and 0.94  constants due to change in volume of measurement.


 USPmethod: in this an additional purification step is
involved by treating with potassium permanganate
and this produces oxidation of interfering
substances. Excess permanganate is removed by
treating with hydrogen peroxide.

Applications:
 This method can be used with vitamin mixtures of
wafers, flour enrichment mixtures and simple
pharmaceutical preparations, provided they are
substantially free from coloring or fluorescencing
matter.
FLUORIMETRIC METHOD: ADSORPTIVE ADDITIVE
DETERMINATION:
 In this method most of the interfering substances are eliminated by an
adsorption step and those not eliminated are compensated by addition of a
known quantity of riboflavin to the assay solution. . this method can be
applied to universally to all samples.

 Extraction: the extraction procedure is same as the direct additive

determination.

 Adsorption and elution:

 An aliquot of sample solution or suitable dilution of the sample extract is

measured accurately and passed through an adsorption column. The column
is prepared by using fluorosil. After elution the column is washed with hot
distilled water and the excess water is drained using vacuum.
 The riboflavin is being retained in the column is then eluted using hot acetic
acid-pyridine eluent is collected and mixed well.
FLUORIMETRIC METHOD: ADSORPTIVE
ADDITIVE DETERMINATION:

 Standard solution I: 40mcg/ml of usp reference standard in 20% of

ethanol.
 Standard solution III: dilute solution I such that it contains 1.6mcg of
riboflavinin in acetic acid pyridine mixture.


 Measurement and calculation:

Mcg of riboflavin per gram= A – (1.07C) * 1.6 * 1 dilution factor
B- (0.94A) 16 wt of sample (g)
A  fluorescence of sample solution
B  fluorescence of standard III
C  blank solution ( obtained by adding hydrogen sulphite to standard
solution II and this should be repeated until successive additions shows no
deflections)
1.07 and 0. 94  constants due to change in volume of measurement.
METHOD 2: SPECTROPHOTOMETRIC METHOD (IP 1996):

 Procedure:

 Carry out the procedure in subdued light.

 Weigh accurately about 65 mg and transfer to an amber-glass 500-
ml volumetric flask, suspend in 5 ml of water, ensuring that it is
completely wetted.
 Dissolve in 5 ml of 2M sodium hydroxide. As soon as dissolution is
complete add 100 ml of water and 2.5 ml of glacial acetic acid and
dilute to 500.0 ml with water.
 To 20.0 ml of this solution add 3.5 ml of a 1.4% w/v solution of
sodium acetate and dilute to 200.0 ml with water.
 Measure the absorbance of the resulting solution at the maximum at
about 444 nm.
 Calculate the content of C17H20N4O6taking 328 as the value of A(1%, 1

cm) at the maximum at about 444 nm.

 METHOD 2:
 Riboflavin has a characteristic absorption spectrum in water with a
maximum at 267nm. This is the basis of the method. This method is
based on the assumption that extraction with chloroform will remove
impurities from an aqueous solution.
 Procedure:
 Carry out in subdued light.
 Weighed about 20mg of riboflavin transferred to a 1000ml volumetric
flask, dilute the solution and add few drops of 1N NaOH.
 Shake gently until the solution is complete, then add few drops of 5N
acetic acid and dilute upto the mark.
 A 20ml of aliquot of solution is taken and shaken with 25ml of chloroform
for 1min. separate the chloroform layer and discard. The extraction is
repeated twice.
 METHOD 2 CONTD…

 The absorbance of clear aqueous layer is

determined at 267nm.with water as a
reference.
 Repeat the procedure with reference
sample.
% Riboflavin= 100 (As/Ar) . (Wr/Ws)
 Ws and A s  weight in mg and absorbance
of sample respectively.
 Wr and A r  corresponding values of
standard and reference sample of riboflavin.
REFERENCES:
 Pharmaceutical analysis by Takeru Higuchi et.al. page no. 649-707.
 Bentley and Drivers textbook of pharmaceutical chemistry. 8thedition.
 Vogel’s textbook of quantitative chemical analysis.
 IP-1996.
 Instrumental methods of chemical analysis by G R Chatwal and Sham K
Anand. Enlarged edition. 2005. p no. 2.413-2.414
 Guyton textbook of medical physiology. 11thedition.
 Biochemistry by Leninger 4thedition 2005.
 Biochemistry By Jeremy M Berg, John L Tymoczko And Lubert Stryler. 5th
Edition
Thank You