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1. PURPOSE/SCOPEC

This method is described for the determination of niacin (nicotinic acid) in foods by colorimetric method. Results are expressed as milligram per 100g of food.

2. SAFETY

Cyanogen bromide is extremely poisonous. All operations involving this reagent should be carried out in an efficient fume cupboard. Do not breathe any vapour, and if solution comes in contact with skin, wash immediately with water.

3. REFERENCE

3.1 Deutsch MJ (1984). Vitamins and other nutrients. In Official Methods of Analysis of the Association of Official Analytical Chemists. 14th Edition (Williams S, ed.), AOAC, Virginia; pp. 841-842.

3.2 Association of Vitamin Chemists, Inc. (1966). Methods of Vitamin Assay.

3rd Edition, Interscience Publishers, New York;pp. 169-172.

4. DEFINITION

Niacin, and the amide of niacin, niacinamide, is widely distributed in foods.

5. PRINCIPLE

Niacin, and the amide of niacin, niacinamide, are usually bound to other chemical compounds and must therefore be freed by hydrolysis with strong acid or alkali or by enzymatic treatment prior to analysis by chemical methods.

Hence niacin derivatives such as coenzymes and niacinamide are converted into free niacin, which is being estimated by this procedure. To eliminate the interference of biologically inactive materials that may be present, purification procedures have to be carried out and blank determinations are necessary during colour development. Niacin in the extract is reacted with cyanogen bromide to give a pyridinium compound. The latter undergoes rearrangement yielding derivatives that couple with aromatic amines to produce coloured compounds. Under proper conditions the density of the colour produced is proportional to the niacin present and may be measured in a colorimeter.

It is to be noted that niacin and niacinamide are both stable in the dry form and in aqueous solutions and are unaffected by light and pH.

6. REAGENTS

6.1 Dilute ammonium hydroxide solution Dilute 5 ml ammonia to 250 ml with water 6.2 1N sodium hydroxide

6.3 10 N sodium hydroxide

6.4 Dilute hydrochloric acid

Dilute concentrated hydrochloric acid with water in the ratio of 1:5 6.5 9N sulphuric acid

6.6 Cyanogen bromide solution, 10%

Warm 80 ml water to 40oC in a large flask and add 10 g cyanogen bromide (CNBr). Shake until dissolved, cool, and dilute to 100 ml. Store in refrigerator.

6.7 Sulphanilic acid solution, 10%

Add ammonia drop by drop to mixture of 20 g sulphanilic acid (C6H7NO3S) and 120 ml water until dissolved. Adjust pH to 4.5 with a solution of a mixture of hydrochloric and water (1+1), using pH meter. Dilute to 200 ml.

Solution should be almost colourless.

6.8 Ammonium sulphate [(NH4)2SO4)]

6.9 Niacin standard solutions

a. Stock solution: dissolve 50 mg pure niacin standard in 25% alcohol to make 500 ml. Store in refrigerator. Concentration of solution is 100 μg per ml.

b. Working solution: dilute 6 ml stock solution to 100 ml with water.

Concentration of solution is 6 μg per ml.

7. APPARATUS

7.1 Analytical balance 7.2 Spectrophotometer 7.3 pH-meter

7.4 Glassware: test tubes, volumetric flasks, pipettes, beakers, cylinder, etc.

8. PROCEDURE

8.1 Preparation of extract

1) Weigh 5-20 g of the sample (containing about 0.5-1.0 mg niacin) into a beaker.

2) Add 20 ml 1N sulphuric acid and 20 ml water, mix, and heat 1 hour over a boiling water-bath.

3) Cool and adjust pH to 4.5 with 10 N sodium hydroxide and 9N H2SO4 .

4) Dilute to 100 ml (V1) with water and filter.

5) Add 17 g ammonium sulphate into 50 ml (V2) of the filtered extract in a stopper measuring cylinder.

6) Dilute to 100 ml (V3) with water and shake vigorously.

7) Filter, mix well, and use 1.0 ml (V4) for colour development. This solution should contain approximately 3 μg niacin per ml.

8) Pipette 50 ml of the working standard solution (6 μg per ml) into 17 g ammonium sulphate in a 100 ml stoppered measuring cylinder.

9) Dilute to volume with water and filter.

10) Use 1.0 ml of this standard solution, containing 3.0 μg niacin, for colour development.

8.2 Colour development

Prepare tubes and add reagents in the order as outlined in the following table. Detail instructions for the addition of these solutions and reagents are given below the table and should be strictly followed.

Prepare separate sample blank for each sample being analysed.

Standard

Blank Standard Test Blank Test

Standard solution (ml) 1.0 1.0 - -

Test solution (ml) - - 1.0 1.0

Water (ml) 5.0 - 5.0 -

Dilute NH4OH (ml) 0.5 0.5 0.5 0.5

10% sulphanilic acid (ml) 2.0 - 2.0 -

CNBr solution (ml) - 5.0 - 5.0

10% sulphanilic acid (ml) - 2.0 - 2.0

Dilute HCl (ml) 0.5 - 0.5 -

Water (ml) - 0.5 - 0.5

Caution: add sulphanilic acid and CNBr solutions in fume cupboard from burette or pipettes filled by mechanical suction.

Note : After adding the test, standard solution and water to the respective tubes, the subsequent reagents must be added to a single tube and measure the color before proceeding to the next tube.

Starting with the standard blank:

1. Swirl tube to impart a rotary motion in liquid immediately.

2. Add dilute ammonium hydroxide, swirl again.

3. Add sulphanilic acid solution, and swirl.

4. Immediately add 0.5 ml dilute hydrochloric acid [and mix again.

5. Place in colorimeter with wavelength adjusted to 450 nm (or any specific wavelength between 430 and 450 nm)

6. Adjust instrument to 0 absorbance within about 30 seconds after addition of sulphanilic acid solution.

Treat standard solution in the same way as for the standard blank with respect to addition of dilute ammonium hydroxide.

1. Immediately swirl tube, add cyanogen bromide solution and swirl again.

2. At 30 seconds after addition of cyanogen bromide solution, swirl tube, add sulphanilic acid solution and swirl again.

3. Immediately add 0.5 ml water, mix again and stopper.

4. Read absorbance of standard solution at maximum, with instrument set at 0 absorbance for standard blank, as performed above.

Colour reaches maximum in about l.5 minutes after addition of sulphanilic acid solution, remains at peak for about 2 minutes, and then fades slowly.

5. With test blank set at 0 absorbance, determine absorbance of test solution in a similarly manner.

Niacin content is proportional to absorbance if standard and test solutions are of approximately the same concentrations.

9. CALCULATION, UNIT OF EXPRESSION AND TEST REPORT

mg niacin per 100 g sample = test O.D. x C x V3 x V1 x 100 x 1

std O.D. V4 V2 W 1000

where,

C = μg niacin standard in reaction tube (3.0 μg) W = weight, in g, of food taken for analysis V1 = }

V2 = } see explanation given in procedure V3 = }

V4 = }

10. ACCEPTANCE OF RESULTS 11. METHOD VALIDATION 12. APPENDIX