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86 Vitamins Methods​

86-01.02 Vitamin A—Ultraviolet Absorption Method

This method measures vitamin A in dry vitamin mixes, beadlets, oils, and emulsions, at concentrations greater than 10,000 international units (IU)/g.

86-02.01 Vitamin A—Carr-Price Method

This method measures vitamin A in nonfat and instantized dry milks. Milk samples are saponified and extracted in hexane or hexane/ethyl ether. Vitamin A is reacted with antimony trichloride and read spectroscopically.

86-03.01 Vitamin A in Enriched Flour

This method measures vitamin A in enriched flour. Vitamin A reacts with chloroform saturated with antimony trichloride (Carr-Price reagent), resulting in a transient, blue-colored compound. Intensity of color is proportional to vitamin A content. The maximum intensity is at 620 nm. The method is not applicable for products containing carotene, as carotenoids also produce blue color with Carr-Price reagent. If carotenoids are present, they must be removed with the alumina column step. See Method 86-05.01.

86-05.01 Vitamin A and Carotene

This method measures vitamin A and carotene in enriched cereals and mixed feeds. Ground samples are saponified with potassium hydroxide and extracted with hexane. Aliquots of hexane, containing the vitamin, are then applied to chromatographic columns. Carotene and vitamin A are eluted with 4% acetone (in hexane) and 15% acetone (in hexane), respectively, and read separately by a spectophotometer.

86-06.01 Analysis of Vitamins A and E by High-Performance Liquid Chromatography

This primary method measures the presence and quantity of vitamin A as retinol whether it is present as retinol or as retinol esters (retinyl acetate and/or retinyl palmitate) and vitamin E as alpha-tocopherol (expressed as alpha-tocopherol acetate) whether it is present as alpha-tocopherol or as alpha-tocopherol esters (such as alpha-tocopherol acetate) in foods. This method is applicable to all foods. The detection/quantitation limits are 0.15 µg/g for retinol and 0.001 mg/g for alpha-tocopherol. This method covers the range of 0.15 µg/g to 100% vitamin A and 0.001 mg/g to 100% vitamin E. Standards and samples are saponified in basic ethanol-water solution, neutralized, and diluted, converting fats to fatty acids and retinol esters and tocopherol esters to retinol and tocopherol, respectively. Retinol and alpha-tocopherol are quantitated on separate high-performance liquid chromatography (HPLC) systems, using UV detection at 313 or 328 nm for retinol and fluorescence detection (excitation 290 nm, emission 330 nm) for alpha-tocopherol. Vitamin concentrations are calculated by comparison of the peak heights or peak areas of vitamins in samples with those of standards. Caution: see Note 1.

86-10.01 Ascorbic Acid—Indophenol-Xylene Extraction Method

This method is based on the quantitative decolorization of a blue dye (2,6-dichlorophenolindophenol) by ascorbic acid, or vitamin C. The excess (unreacted) dye is extracted with xylene and measured in a spectrophotometer. In the absence of interfering materials, the amount of ascorbic acid present in the sample is proportional to the amount of dye decolorized.

86-31.01 Vitamin B₆ Complex—Microbiological Method

The vitamin B6 group consists of pyridoxine, pyridoxal, pyridoxamine, and one or more unidentified labile factors. All members of the complex show comparable activity for Saccharomyces uvarum (American Type Culture Collection [ATCC] No. 9080) and the rat. Hence this procedure measures the biological vitamin B₆ activity rather than the concentration of pyridoxine or any single derivative.

86-40.01 Vitamin B₁₂—Microbiological Method

This method measures vitamin B₁₂ content in relation to the growth of Lactobacillus delbrueckii ssp. lactis in a culture medium. The resultant turbidity is measured spectroscopically.

86-45.01 Choline

Choline (2-hydroxy-N,N,N-trimethylethanaminium hydroxide) is chemically measured as choline reineckate. Samples are extracted in methanol. Upon evaporation of methanol, choline is precipitated as reineckate, washed, dried, and reconstituted in acetone. The acetone solution of choline reineckate is spectroscopically measured at 526 nm.

86-47.01 Total Folate in Cereal Products—Microbiological Assay Using Trienzyme Extraction

This is a microbiological method employing the organism Lactobacillus casei subsp. rhamnosus (ATCC no. 7469) to determine the amount of folate present in foods and in vitamin concentrates. This method is semiautomated through the use of automated dilution and turbidity reading instruments or, optionally, the 96-well microtiter plate and reader system. See Note 1. Samples, with water added, are autoclaved to break up particles, gelatinize starch, and denature proteins to enhance enzymatic attack and make folate more extractable. Folate (pteroylglutamic acid in various forms) occurs naturally in foods bound to glutamic acid residues of varying chain lengths. Most naturally occurring folates cannot be utilized by the assay organism. Folic acid (pteroylglutamic acid) is extracted from the sample using a triple enzyme system. A protease and an amylase are used to digest the food matrix and aid in the release of folates. Desiccated chicken pancreas conjugase is used to hydrolyze folylpolyglutamates to folyldiglutamates, which, along with folic acid, can be utilized by the assay organism. The freed folates are extracted and diluted with basal medium containing all required growth nutrients except folate, and the turbidity of the L. casei subsp. rhamnosus growth response for the samples is compared quantitatively to that of known standard solutions. The method is applicable to cereal grains and cereal grain foods containing added folate (folic acid) or naturally occurring folates with levels from 5 µg/100 g to 100% folate.

86-49.01 Niacin in Enrichment Concentrates

Niacin is determined in vitamin and mineral concentrates (used to enrich cereals) by means of a colorimetric chemical reaction. Diluted sample solutions react with buffered ammonia solution and cyanogen bromide. The intensity of the resulting yellow color is proportional to niacin content.

86-50.02 Niacin and Niacinamide in Cereal Products

Niacin (either nicotinamide or nicotinic acid) is determined chemically using a colorimetric technique. Ground sample is extracted in alkaline medium, clarified, and reacted with sulfanilic acid and cyanogen bromide to yield a yellow color. The method employs the principle of the Konig reaction, in which the pyridine group of the niacin molecule is cleaved with CNBr before reaction with chromogen (sulfanilic acid). This reaction occurs at cold temperatures. CNBr should be used with caution. This method is applicable to cereal products.

86-51.01 Niacin—Microbiological Method

To determine niacin by a microbiological method. Growth of Lactobacillus plantarum ATCC 8014 is directly proportional to niacin content in the sample. Microbiological growth and the resulting metabolites can be measured spectrophotometrically.

86-70.01 Riboflavin—Fluorometric Method

Riboflavin, or vitamin B₂, exhibits native fluorescence, a property used in its measurement. Ground sample is extracted in sulfuric acid; impurities are oxidized with permanganate; and the intensity of the extract's fluorescence is measured by a fluorometer. This method is applicable to whole-grain products, grits, meal, flaked and puffed cereals, farina, bread, etc.

86-72.01 Riboflavin—Microbiological Method

The method measures riboflavin by a microbiological technique. Microorganisms such as Lactobacillus casei reproduce in proportion to the vitamin content in a culture medium. The resultant turbidity or metabolic products can be measured spectroscopically. In this method, the acidity resulting from microbial growth is measured by titration with a base (NaOH) and related to riboflavin content.

86-80.01 Thiamine—Thiochrome Method

Thiamine, or vitamin B₁, in the sample is oxidized to thiochrome using alkaline potassium ferricyanide, after treatment of sample with starch- and protein-degrading enzymes. Thiochrome, which is fluorescent, is extracted in isobutanol for fluorimetric measurement. Highly colored samples or samples containing interfering impurities may require additional purification after extraction and before thiochrome formation. This method measures thiamine in bread and other grain-based products and in corn grits, cornmeal, puffed cereals, farina, and flour.

86-90.01 B-Vitamins in Vitamin Concentrates by HPLC

B-vitamins are dissolved in ionic-strength-adjusted aqueous acetic acid. They are separated by paired-ion high-performance liquid chromatography (HPLC) and quantitated by comparing their absorbance with absorbance of standard solutions at 285 nm. This method measures the presence and quantity of niacin, niacinamide, pyridoxine (vitamin B₆), and riboflavin (vitamin B₂) in vitamin concentrates containing a minimum of 0.1% of any one of the analytes.