Analysis of Pantothenic Acid Vitamin

High-performance Liquid Chromatography Mass Spectrometry Analysis of Pantothenic Acid (Vitamin Bg) in Multivitamin Dietary Supplements... 

Velisakek et al. (1992) reviewed the use of TLC and paper chromatography for the analysis of pantothenic acid (vitamin B5). They presented considerable infor-... 

Chemical methods are mainly based on analysis of pantothenic acid hydrolysis products by specttophotometry or fluorometry. Although relatively rapid to perform, these methods lack the specificity and sensitivity needed to determine vitamin in foods or differentiate between d and l forms. Several spectrophotomet-ric determinations of pantothenic acid and its salts have been developed. They are based on the reaction of pantolactone with hydroxylamine and 2,7-naphtha-lenediol. Most published methods use the determination of P-alanine. Ninhydrin, 1,2-naphthoquinone, o-phthalaldehyde, acetylacetone, and some other reagents have been introduced for the spectrophotometric determination of pantothenates via p-alanine (5,46,47). 

Recently, HPLC-MS (electron impact, chemical ionization in positive-ion and negative-ion modes) has been used for the analysis of pantothenic acid in an artificial mixture with other water-soluble vitamins (62). 

Particle beam LC-MS was investigated by Careri et al. (62) for the analysis of pantothenic acid and 10 other water-soluble vitamins. A reversed-phase HPLC method making use of volatile buffers was set up for the simultaneous separation of this mixture of vitamins using narrow-bore columns. 

Appropriate GLC procedures have also been used for the analysis of pure vitamins, their optical isomers, pharmaceuticals, and, rarely, even foods. The main disadvantage of this group of chromatographic methods seems to be the tedious and relatively time-consuming sample preparation (derivatization and/or cleanup procedures) prior to the GLC analysis. Nevertheless, the newly developed and introduced GC-MS techniques show that GLC is a sufficiently sensitive tool for the trace analysis of pantothenic acid, its higher homolog and other related compounds such as acyl-CoA, even in biological samples (serum, brain, foodstuffs). 

Radioimmunoassay has been used for assay of pantothenic acid in physiological samples (sensitivity 250 nmol 1 ), and an enzyme-linked immunosorbent assay (ELISA) has been used for this vitamin in food analysis. 

Rychlik, M., 2003. Simultaneous analysis of folic acid and pantothenic acid in foods enriched with vitamins by stable isotope dilution assays. Analytica Chimica Acta. 495 133-141. 

Two approaches are currently used for the preparation of volatile derivatives of the vitamin. The first approach is mostly based on the conversion of pantothenic acid and/or panthenol to acetyl (72) or trimethylsilyl derivatives (73). A simpler and more convenient approach for most applications seems to be the procedure based on the hydrolysis of the vitamin in acidic medium and analysis of the hydrolysis products. Pantothenic acid, its salts, and coenzyme A as well as its analogs undergo acid hydrolysis with formation of P-alanine and pantolactone. Panthenol breaks down to 3-amino-l-propanol (P-alanol) and pantolactone. For instance, P-alanine can be analyzed as the corresponding jv-trifluoroacetyl methyl ester or AMrifluoroacetyl butyl ester. Pantolactone is sufficiently volatile to be amenable to direct GLC (23,76,77), but it can also be analyzed as the corresponding trimethylsilyl ether, trifluoroacetyl, or isopropylurethane derivative (5,78). 

Currently employed HPLC methods for pantothenic acid and/or pantothenates have been applied solely to pharmaceuticals and simple matrices such as fortified infant formulas, whereas assays of coenzyme A and its acyl analogs have also been successfully performed on animal tissues. In the last few years, chiral stationary phases have been developed for optical resolution of pantothenic acid and related compounds by HPLC, and also HPLC-MS has become a promising technique. However, the newly developed HPLC procedures still require increased sensitivity and selectivity to make them applicable for the analysis of the total vitamin content in complex matrices such as foods and feeds. 

In terms of amino acids bacterial protein is similar to fish protein. The yeast s protein is almost identical to soya protein fungal protein is lower than yeast protein. In addition, SCP is deficient in amino acids with a sulphur bridge, such as cystine, cysteine and methionine. SCP as a food may require supplements of cysteine and methionine whereas they have high levels of lysine vitamins and other amino acids. The vitamins of microorganisms are primarily of the B type. Vitamin B12 occurs mostly hi bacteria, whereas algae are usually rich in vitamin A. The most common vitamins in SCP are thiamine, riboflavin, niacin, pyridoxine, pantothenic acid, choline, folic acid, inositol, biotin, B12 and P-aminobenzoic acid. Table 14.4 shows the essential amino acid analysis of SCP compared with several sources of protein. 

Tanner, J.T., Barnett, S.A., and Mountford, M.K. 1993. Analysis of milk based infant formula. Phase V. Vitamins A and E, folic acid, and pantothenic acid Food and Dairy Administration-Infant Formula Council Collaborative study. J. AOAC Int. 76 399-401. 

J Velfsek, J Davfdek, T Davfdek. Pantothenic acid. In AP De Leenheer, WE Lambert, HJ Nelis, eds. Modem Chromatographic Analysis of Vitamins. 2nd ed. New York Marcel Dekker, 1992, pp 515-560. 

Several B vitamins, including folic acid, niacin, pyridoxine, and pantothenic acid, are routinely determined using microbiological assays, details of which can be found in the AOAC Official Methods of Analysis. Standard methods for thiamine determination using fluorimetric detection are also detailed in the AOAC methods in addition, LC techniques are now being used routinely for thiamine and other B vitamins, e.g., riboflavin. 

The method of choice for the determination of most vitamins is HPLC due to its high separation capability, its mild analytical conditions, and the possibility to use various specifically adapted detection methods, e.g., LTV, fluorescence, or MS detection. All fat-soluble vitamins and most water-soluble vitamins have chromophores suitable for UV detection. Separation of vitamers and stereoisomers can be achieved. If a higher sensitivity is required HPLC with fluorescence detection can be used, either directly (e.g., vitamins A and E) or after derivatization (e.g., thiamine). A further improvement in sensitivity and specificity has been achieved by introducing HPLC with mass spectrometric detection in vitamin analysis. Due to the structural information retrievable, e.g., molecular mass, fragmentation pattern, this is the method of choice for analysis of samples with complex mixtures or low vitamin concentrations. Examples for the use of HPLC-MS in vitamin analysis include the determination of 25-hydroxy-D3 and pantothenic acid. However, one drawback of mass spectrometry is the need for an isotopically labeled reference compound for reliable quantification. Due to the structural complexity of many vitamins, these reference compounds are often expensive and difficult to synthesize. An interesting unique application is the determination of vitamin B12 by HPLC-IPC-MS, which is possible due to its cobalt content. 

AOAC, 1996. Method 945.74. Pantothenic acid in vitamin preparations-microbiological methods. In Official Methods of Analysis of the Association of Official Analytical Chemists, 16th ed. Association of Official Analytical Chemists, Arlington VA, USA, pp. 45.2.05. 

Pure reference standards of B complex vitamins can be obtained from USP. Once a LC-MS method for pantothenic acid analysis from multivitamin dietary supplements is developed, the multi-element multivitamin dietary supplement Standard Reference Material 3280 (SRM 3280) from the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA) can be used for validation and quality control. 

Vitamin classification is initially based on fat solubility—i.e., A, D, E, and K— versus water solubility—i.e., C, folic acid, nicotinic acid, and nicotinamide (B3), B, B2, Bs, biotin, B,2, and pantothenic acid. DeLeenheer et al. (1985, 1992) edited two valuable treatises on modern chromatographic analysis of vitamins. Two tables have been compiled in this chapter with information on the chemical terms for vitamins and their derivatives, vitamin function, sources of vitamins and recent selected references containing TLC information. Table 19.1 summarizes pertinent information on the fat-soluble vitamins and Table 19.2 does the same for the water-soluble vitamins. 

A similar quantitative analysis of six water-soluble vitamins (B, B2, Bg, C, nicotinamide, and pantothenic acid) in a pharmaceutical formulation using CZE in uncoated fused silica capillaries with UV detection was described by Fotsing et al. (91). Eor the B-group vitamins, a good compromise among resolution, analysis time, and analyte stability was obtained by use of 50 mM borax buffer pH 8.5. A capillary wash with sodium hydroxide was necessary between successive runs to minimize absorption of excipients from the pharmaceutical formulation to the capillary surface, otherwise giving rise to a progressive decrease of the electro-osmotic flow. 

Capillary zone electrophoresis (CE) is a relatively new analytical method currently under investigation for use in research and control laboratories for the analysis of ionic forms of vitamins. Micellar electrokinetic capillary chromatography (MECC) is a modification of CE which allows the separation of both neutral and ionic forms using buffers with micellar additives. Both methods have been used to separate water-soluble vitamins but very rarely pantothenic acid, which has only been analyzed by this technique in model mixtures and pharmaceuticals. 

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