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Vitamin Analysis, USP, GMP

Updated 10/98

The Nutritional Supplement industry is becoming more and more closely regulated. This is apparent from the addition of the Nutritional Supplement monographs to USP 23, and the rapid appearance of additional methods in the USP 23 Supplements. Methods for oral solutions have also been added to those for capsules and tablets.

We have been determining minerals in supplements for several years, primarily by ICPMS. Over the last few years, we have also increased our capabilities for vitamin analysis. The methods for vitamins analysis in supplements are not always the same as those given in the monographs for the individual materials (for example, injectables). For supplements, many vitamins are determined using liquid chromatography or wet chemical methods. Ascorbic acid (vitamin C) and beta-carotene are determined using titration and visible spectrophotometry, respectively. The more common B vitamins, thiamine, pyridoxine, riboflavin, and niacin (or niacinamide) are determined using ion pair reverse phase LC in one analysis.

B Vitamins Analysis by HPLC

Other reverse-phase LC methods are used for vitamin E, folic acid, pantothenic acid (or calcium pantothenate), phytonadione, and cyanocobalamin (B-12). Normal phase LC methods are used for vitamins A and D. 

folic acid analysis by HPLC

Listed below are the vitamin analyses conducted at WCAS in either raw material, finished product, or in some cases natural products.

  • ascorbic acid
  • beta-carotene
  • biotin (H)
  • calcium pantothenate
  • cyanocobalamine (B12)
  • cholcalciferol (D)
  • ergocalciferol (D2)
  • folic acid
  • niacin
  • niacinamide (B3)
  • phytonadione (K1)
  • pyridoxyl-5-phosphate
  • pyridoxine HCl (B6)
  • riboflavin (B2)
  • thiamine HCl (B1)
  • vitamin E (acetate, alcohol, succinate)
  • vitamin A (acetate, palmitate)


Vitamins by USP Methods (from Newsletter 7-22-97)

Over the past few years, WCAS has added analysis for several vitamins to our capabilities. The bulk of this work has been on USP nutritional supplements, using the methods described for these supplements in USP 23. However, these methods cannot always be applied to other products. Beta-carotene, especially, is prone to problems.

USP specifications for nutritional supplements do not allow the presence of other carotenoids, such as lutein or zeaxanthin. However, many products on the market, such as herbal preparations, do include these types of compounds. Since they are chemically similar to beta-carotene, they co-extract with the beta-carotene when using the USP methods and interfere with the colorimetric determination. This yields results which are biased high, sometimes drastically. To overcome these problems, we use a modification to the USP method to accurately determine beta-carotene in non-USP formulations.

We have also identified problems with riboflavin analysis. The USP method to determine riboflavin in nutritional supplements is a liquid chromatography procedure. However, to analyze riboflavin raw material or injection, a fluorescence method is used which has presented problems. It requires a sample preparation step using potassium permanganate and hydrogen peroxide without specifying the concentration of these reagents. (Although the permanganate is listed as "1 in 25," this concentration is too strong for the color to be destroyed by the peroxide in the time given in the method. The peroxide concentration is not given at all.)

When we attempted this method, we encountered a problem with the fluorescence measurement which we traced back to the permanganate/peroxide step. When that step was eliminated, the method performed as expected. Because of these problems with the fluorescence method, we have explored using the liquid chromatography method to determine lower levels of riboflavin, such as those found in injectable preparations, and even cell culture media. Although the nutritional supplement method determines riboflavin at levels of 100 ppm in solution, we have found that we can accurately measure it at concentrations down to 0.1 ppm, using the same conditions. We recommend using these conditions to determine low levels of riboflavin. These conditions can also determine niacin (or niacinamide), pyridoxine, and thiamine in the same analysis. However, since detection is by UV absorbance at 280 nm, the method can be subject to interferences, especially in complex matrices such as culture or fermentation media.

What does all this mean to you? Determination of vitamins in formulations more complex than USP nutritional supplements requires some thought to ascertain the best analytical approach. If you have any questions about what methods to use in your products, feel free to give Mike Shelton, or Eric a call. 

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Formerly West Coast Analytical Service (WCAS) and Bodycote Testing Group