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Asian Journal of Biochemical and Pharmaceutical Research Issue 2 (Vol. 2) 2012 ISSN: 2231-2560

CODEN (USA): AJBPAD

Research Article

69

Asian Journal of Biochemical and Pharmaceutical Research

Importance, Polarographic Behaviour and Determination of Riboflavinnnn

Imrana Siddiqui

Assistant professor, Govt Arts and commerce college Sagar.

Received: 07 April 2012; Revised: 21 April 2012; Accepted: 06 May. 2012

Abstract: vitamin B2 is an easily absorbed micronutrient with a key role in maintaining health in humans and other animals. vitamin B2 is required for a wide variety of cellular processes. It plays a key role in energy metabolism, and for the metabolism of fats, ketone bodies, carbohydrates, and proteins. Riboflavin is best known visually as the vitamin which imparts the orange color to solid B-vitamin preparations, the yellow color to vitamin supplement solutions, and the unusual fluorescent yellow color to the urine of persons who supplement with high-dose B-complex preparations. . Riboflavin produces a well defined wave in a wide range of pH from 1.8 to 8.1 when determined polarographycally This wave was found to be directly proportional to its concentration in the solution. As such vitamin B2 has been determined in various pharmaceutical formulations by polarographic method. The results have been discussed in this paper, which were found to be in good agreement with those reported by the manufacturer. The method may thus be recommended for quality control purpose. Keywords-Riboflavin, vitamin B2, polarography, Direct current polarography, Differential pulse, polarogtraphy, pharmaceutical formulation.

INTRODUCTION:

A vitamin is an organic compound required as a nutrient in tiny amounts by an organism [1].

Vitamins are classified by their biological and chemical activity. Some have hormone-like functions as

regulators of mineral metabolism (e.g., vitamin D), or regulators of cell and tissue growth and

differentiation (e.g., some forms of vitamin A). Others function as antioxidants (e.g., vitamin E and

sometimes vitamin C) [2]. The largest number of vitamins (e.g., B complex vitamins) function as

precursors for enzyme cofactors, that help enzymes in their work as catalysts in metabolism. Vitamins

are either water-soluble or fat-soluble. Water-soluble vitamins dissolve easily in water and, in general,

are readily excreted from the body, to the degree that urinary output is a strong predictor of vitamin

consumption [3]. Because they are not readily stored, consistent daily intake is important [4]. The B-

complex vitamins are actually a group of eight vitamins, which include:

• thiamine (B1)

• riboflavin (B2)

• niacin (B3)

• pantothenic acid (B5)

• pyridoxine (B6)

Asian Journal of Biochemical and Pharmaceutical Research Issue 2 (Vol. 2) 2012

70

• cyanocobalamin (B12)

• folic acid

• biotin

Riboflavin,

7,8-dimethyl-10-((2R,3R,4S)-2,3,4,5- tetrahydroxypentyl) benzo [g] pteridine- 2,4 (3H,10H)-

dione also known as vitamin B2 or additive E101,[5] is an easily absorbed micronutrient with a key

role in maintaining health in humans and other animals. It is the central component of the cofactors

FAD and FMN, and is therefore required by all flavoproteins. As such, vitamin B2 is required for a

wide variety of cellular processes. It plays a key role in energy metabolism, and for the metabolism of

fats, ketone bodies, carbohydrates, and proteins.

Milk, cheese, leafy green vegetables, liver, kidneys, legumes, tomatoes, yeast, mushrooms, and

almonds [6] are good sources of vitamin B2, but exposure to light destroys riboflavin. The name

"riboflavin" comes from "ribose" (the sugar which forms part of its structure, which in turn is a

transposition of arabinose [7] ) and "flavin", the ring-moiety which imparts the yellow color to the

oxidized molecule (from Latin flavus, "yellow"). The reduced form, which occurs in metabolism, is

colorless. Riboflavin is best known visually as the vitamin which imparts the orange color to solid B-

vitamin preparations, the yellow color to vitamin supplement solutions, and the unusual fluorescent

yellow color to the urine of persons who supplement with high-dose B-complex preparations (no other

vitamin imparts any color to urine).

Riboflavin is yellow or yellow-orange in color and in addition to being used as a food coloring,

it is also used to fortify some foods. It is used in baby foods, breakfast cereals, pastas, sauces,

processed cheese, fruit drinks, vitamin-enriched milk products, and some energy drinks. Wheat bran,

eggs, meat, milk, and cheese are important sources in diets containing these foods. Cereals grains

contain relatively low concentrations of flavins, but are important sources in those parts of the world

where cereals constitute the staple diet. [8-9] Egg white and egg yolk contain specialized riboflavin-

binding proteins, which are required for storage of free riboflavin in the egg for use by the developing


71

embryo. Riboflavin is generally stable during the heat processing and normal cooking of foods if light

is excluded. The alkaline conditions in which riboflavin is unstable are rarely encountered in

foodstuffs. Riboflavin degradation in milk can occur slowly in dark during storage in the refrigerator

[10]. The latest (1998) RDA recommendations for vitamin B2 are similar to the 1989 RDA, which for

adults, suggested a minimum intake of 1.2 mg for persons whose caloric intake may be > 2,000 Kcal

[11].

Riboflavin is continuously excreted in the urine of healthy individuals [12], making deficiency

relatively common when dietary intake is insufficient. However, riboflavin deficiency is always

accompanied by deficiency of other vitamins. [12] In humans, signs and symptoms of riboflavin

deficiency (ariboflavinosis) include cracked and red lips, inflammation of the lining of mouth and

tongue, mouth ulcers, cracks at the corners of the mouth (angular cheilitis), and a sore throat. A

deficiency may also cause dry and scaling skin, fluid in the mucous membranes, and iron-deficiency

anemia. The eyes may also become bloodshot, itchy, watery and sensitive to bright light.

Riboflavin has been used in several clinical and therapeutic situations. For over 30 years,

riboflavin supplements have been used as part of the phototherapy treatment of neonatal jaundice. The

light used to irradiate the infants breaks down not only bilirubin, the toxin causing the jaundice, but

also the naturally occurring riboflavin within the infant's blood, so extra supplementation is necessary.

High dose riboflavin appears to be useful alone or along with beta-blockers in the prevention of

migraine [13-14].

Riboflavin has also been used as a muscle pain reliever. Riboflavin in combination with UV

light has been shown to be effective in reducing the ability of harmful pathogens found in blood

products to cause disease [15-17].The recommended dietary allowance of vitamin has been given in

table 3.

EXPERIMENTAL:

Instrumentation: All the polarograms /voltammograms were recorded on an Elico (India) DC

Polarograph, model CL-357 and Elico Pulse Polarograph, model CL 90. Systronics Digital pH meter

335 was used for pH measurements.The polarizable micro electrode, a slowly growing drop of

mercury issuing, under a head of 60 cm of mercury column from a resistance glass capillary (0.05 mm

in bore diameter and 7 cm long) in small, uniform drops was used as indicator electrode.

A saturated calomal electrode was used as a reference electrode.


72

The positive electrode was the counter electrode which provide for potentiostatic control of the DME.

A platinum wire electrode has been used for this purpose. Doubly distilled mercury is usually

recommended for polarographic work.

Solutions : All the chemicals used were of Anala R/BDH grade. Doubly distilled water was used to

prepare the solutions. pH adjustments were made using dilute solutions of HCl, NaOH, NH4OH or

acetic acid or buffers where even necessary.

Determination of Vitamin B2 (Riboflavin) :

0.01M solution of Riboflavin was prepared by dissolving a requisite quantity of compound in 5ml of

0.1M NaOH. The solution was then made up to 100ml with distilled water. The Britton Robinson

buffer (mixture of 0.04M phosphoric acid, 0.04M acetic acid and 0.04 M boric acid) was prepared by

mixing 20 ml of 0.2M phosphoric acid, 20 ml of 0.2M boric acid and 20ml of 0.2M acetic acid in a

measuring flask. Total volume of the solution was made up to 100 ml with distilled water. pH

adjustments were made with 0.2 N NaOH as described in the literature.

A known concentration of vitamin B2 was taken in polarographic cell containing 10 ml of BR

buffer. 0.2 N NaOH was added to the test solution to adjust the pH at 1.8. Volume of the analyte was

made up to 100 ml with distilled water. The test solution was deacrated by bubbling hydrogen gas

through the solution before recording the polarogram for ten minutes. Polarogram was recorded

keeping the initial emf set to -0.00V.

The polarograms thus obtained were not useful in the determination of the compound in

pharmaceutical formulations. At this pH the wave of riboflavin was suppressed by other compounds

present in the formulation. The polarogram of riboflavin was therefore recorded at pH 6.5 with initial

emf set to -0.50V. The wave produced at this pH was though a distorted one, yet it’s height was found

to be proportional to the concentration of riboflavin in the capsule powder.

Calibration curve was obtained by taking varying concentration of riboflavin. Polarograms

were recorded with in the same supporting electrolyte under identical experimental conditions at pH

6.5. The pharmaceutical formulation in which the study was carried out, was a multivitamin dry filled

capsule purchased from a medical shop. 20 capsules were weighed, opened and their contents were


73

emptied. The capsule shells were cleaned of adhering powder with absorbent cotton on the end of a

tooth pick. The shells were weighed and this weight was subtracted from that of the filled capsules.

This gave the weight of contents of the capsule used. The contents were mixed thoroughly and a

convenient aliquot was taken for analysis.

For the determination of vitamin B2, the aliquot was dissolved in 10ml of 0.1N NaOH. The

volume was made upto 50ml with distilled water. The solution was stirred vigourously. The

insoluble matter was filtred off. Clear filtrate was used to prepare the analyte. 10 ml of this solution

was transferred to polarographic cell containing 10 ml of BR buffer. The volume of the test solution

was made up to 100 ml with distilled water. The pH of the analyte was adjusted to 6.5 and the

polarograms were recorded under the same conditions as described earlier. Special care was taken to

prevent undue exposure of the sample to light.The concentration of the vitamin in capsule powder was

determined with the help of calibration curve.

RESULTS AND DISCUSSION:

The reduction wave for riboflavin occurs at relatively positive potential, so that it can be used

for its determination in mixtures with the other vitamins of B group, such as thaimin, nicotinic acid

and folic acid. Its determination is not effected by pantothenic acid and pyridoxine. It produces a well

defined curve in a wide range of pH from 1.8 to 8.1 (Fig.1) but because of change in riboflavin in

alkaline medium into lumiflavin, a pH of 6.5 was used for analytical purposes.

During the study of reduction of riboflavin in acid medium (eg at pH 1.8), a prewave was

always observed before the normal reduction wave. The polarogram consist of two waves, a small

prewave of constant height which was independent of the concentration, followed by a normal

reduction wave (i.e. the second wave). The diffusion current corresponding to the second wave is

proportional to the concentration of riboflavin in the solution. The half wave potential of the prewave

was more positive than the true oxidation potential of the system. On the other hand, the E1/2 of the

second wave corresponds closely to the oxidation potential of the thermodynamically reversible

riboflavin system.


74

The occurrence of the prewave at more positive potential may be attributed to an adsorption of

the reduced from of riboflavin on the surface of the DME. As a result of this adsorption, the activity

of the reduced form is considerably less than when it is in true solution. The difference in E1/2

between the prewave and the normal one corresponds to the energy involved in the adsorption of the

reduced form. At pH 1.8 the prewave was observed with E1/2 -0.13 V and the second wave was

observed with -0.34 V vs SCE (Fig. 2). Both the waves were found to be reversible reduction waves.

The number of electrons involved for the first wave was found to be one and for the second wave to be

two. The log plot slopes for both the waves show the reactions to be reversible (Fig. 3 and Fig. 4).

The most probable mode of reduction may be as follows -

Riboflavin readily takes up two hydrogen atoms. It is revesibly reduced, to dihydro compound

called leuco - riboflavin. The leuco-compound is easily oxidized to riboflavin. By reduction of

riboflavin to the leuco compound, three intermediate compounds results, which consist of molecular

forms of reduced and unreduced molecules . Verdoflavin consist of riboflavin and mono hydro

riboflavin, chloroflavin is a quinhydrone of riboflavin and leucoriboflavin and rhodoflavin

hydrochloride contains the hydrochlorides of leuco-riboflavin and mono hydro-riboflavin.

C ON H

C ON

N

N

C ONH

C ON

N

NH

C ONH

C ON

N

NH

+

Riboflavin (Yellow) Verdoflavin (green)

CONH

CON

N

NH

+

HCl

CONH

CON

N

NH

HCl

N CONH

CONH

N

Chloroflavin (green) Rhodoflavin (carmine red)

C ONH

C ON

N

N H

H

Leucoflavin (colourless)

Reduction of Riboflavin


75

Since the quinhydrone is the only form that exists in dilute solution, it is assumed that only this form

occurs under physiological conditions.These waves, obtained in acidic medium were not used for the

analytical purpose because the capsule powder don’t produce any wave at this pH. At this pH, the

wave of riboflovin may be supressed due to the presence of other compounds in the capsule powder.

The analytical studies were therefore done at pH 6.5.

At pH 6.5 riboflavin produced a well defined reversible two electron reduction wave/peak with

E1/2 = -1.1 V and Ep= -1.2. The height of the wave was found to be proportional to the vitamin

concentration in the solution. The polarograms of vitamin B2 at pH 6.5 are shown in Fig 5 and Fig 6.

The determination of vitamin B2 content of the multivitamin capsule was carried on by calibration

method and standard addition method. The polarograms of vitamin B2 in pharmaceutical formulation

are depicted in Fig. 7 and Fig. 8. The concentration of Riboflavin was found to be 9.92 mg per 240 mg

of capsule powder. The concentration of Riboflavin was found to be 9.92 mg per 240 mg of capsule

powder. The results have been subjected to statistical analysis which revealed high accuracy and

precision. The statistical analysis of results have been shown in table. 1. The recovery was more than

99% in each case. The values of Relative Mean Deviation, Standard Deviation and Coefficient of

Variance which were less than 0.02, 0.1 and 3.3, respectively speaks the reliability of the results. The

final analysis results on the multivitamin capsule for its vitamin B2 content have been shown in table

2. The table shows that the results are in good agreement with that reported by the manufacturer.

Table 1 Results* on multivitamin capsule for its vitamin B2 content (mg/240 mg) of capsule

powder.

Vitamin Parameter DCP DPP

Added Found Added Found

B2 Amount - 9.92 - 10.80

10.8 20.72 10.8 21.40

% R 99.8 99.0

RMD 0.001 0.005

SD 0.094 0.1

% CV 0.94 0.92 *Average of four determinations. Table 2 Final analysis results on multivitamin capsule for its vitamin B2 content.

Compound Reported by the Found

Manufacturer DCP DPP

Vitamin B2 10.0 9.92 10.80


76

Table 3 Recommended dietary allowances

Recommended by the Food and Nutrition Board, National Research Council

Group Riboflavin (B2)

(mg)

Man (70 kg)

Sedentary 2.2

Moderately active 2.7

Very active 3.3

Woman (56kg)

Sedentary 1.8

Moderately active 2.2

Very active 2.7

Pregnancy

(latter half) 2.5

Lactation 3.0

Children upto 12 years.

Under 1 year 0.6

1-3 years 0.9

4-6 years 1.2

7-9 years 1.5

10-12 years 1.8

Children over 12 years

Girls 13-15 yrs. 2.0

16-20 yrs 1.8

Boys, 13-15 yrs 2.4

16-20 yrs 3.0


77


78


79


80


81

References-

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2. Bender, David A. (2003). Nutritional biochemistry of the vitamins. Cambridge, U.K.:

Cambridge University Press.

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Pauling Institute at Oregon State University.

7. onlinelibrary.wiley.com/doi/10.1002/sce.3730420523/pdf

8. Food Standards Agency, McCance and Widowhood’s The Composition of Foods, 6th summary

Ed, Royal Society of Chemistry, Cambridge, 2002

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PP.132-37

12. Brody, Tom (1999). Nutritional Biochemistry. San Diego: Academic Press.

13. Sándor PS, Afra J, Ambrosini A, Schoenen J. Headache. 2000 Jan; 40(1):30-5.

14. Schoenen J, Jacquy J, Lenaerts, M. Neurology. 1998 Feb; 50(2):466-70.

15. Ruane PH, et al Transfusion 2004; 44: 877-885

16. Goodrich RP, et al Transfusion and Apheresis Science 2006; 35 (1): 5-17.

17.Goodrich RP, et.al,Chapter 5 Photochemistry and Photobiology, Vol. 6, 2006, Royal Society

of Chemistry; Cambridge, United Kingdom. E Silva and AM Edwards, editors.

*Correspondence Author: Imrana Siddiqui, Assistant professor, Govt Arts and commerce college Sagar.

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