Vitamins multivitamin methods

Russell (44) and Ball (45) summarized multivitamin methods for foods that determined various combinations of thiamine, riboflavin, niacin, vitamin B6, folacin, and biotin. 

Pharmaceuticals. In commerce, ascorbic acid is produced exclusively by synthesis (98). Because of its rather pure nature and high concentrations in vitamin-multivitamin tablets, analysis by conventional or sophisticated procedures can be performed easily. The USP provides a reference standard of L-ascorbic acid for assay purposes. The methods used can be chosen from the many discussed above. The method officially approved by the Association of Official Analytical Chemists is the micro-fluorometric procedure developed by Deutsch and Weeks (44). 

The recently elaborated multivitamin methods for B vitamins assay using LC with MS/MS detection, micellar LC, LC-DAD/MS, LC/ESI-MS or LC-IDMS systems offer better sensitivity and selectivity, and can be potentially used for thiamine determination in fortified foods and infant formulas. These methods have potential to improve performance in the future. 

HPLC analyses of multivitamin preparations are today widely accepted to ensure quality control. Colorimetric, fluorometric, and microbiological methods have often been replaced by HPLC analysis. The main advantage of HPLC analysis is its versatility and flexibility published vitamin analysis methods are usually easily adapted to suit different requirements of the pharmaceutical industry. 

The key step of a multivitamin method is the development of a simultaneous and quantitative extraction procedure. The intra- and intergroup heterogeneity of water-soluble vitamins makes it difficult to realize this goal. The application of an acid treatment, to hydrolyze the bound forms, can be used for simultaneous extraction of Bi, B2, B3, Bg, Bs,... 

Preferably, high pressure Hquid chromatography (hplc) is used to separate the active pre- and cis-isomers of vitamin D from other isomers and allows their analysis by comparison with the chromatograph of a sample of pure reference i j -vitainin D, which is equiUbrated to a mixture of pre- and cis-isomers (82,84,85). This method is more sensitive and provides information on isomer distribution as well as the active pre- and cis-isomer content of a vitamin D sample. It is appHcable to most forms of vitamin D, including the more dilute formulations, ie, multivitamin preparations containing at least 1 lU/g (AOAC Methods 979.24 980.26 981.17 982.29 985.27) (82). The practical problem of isolation of the vitamin material from interfering and extraneous components is the limiting factor in the assay of low level formulations. 

A large number of methods have been developed for analysis of water-soluble vitamins simultaneously in pharmaceutical products (like multivitamin tablet supplements). In fact, for these products no particular sample preparations are required and the high concentrations simplify the detection, enabling the use of UV [636]. The use of MS is also reported [637]. As well, Moreno and Salvado [638] reports also the use of a unique SPE cartridge (C18) for separating fat-soluble and water-soluble vitamins, which are, then analyzed using different chromatographic systems. 

HPLC is the method of choice for multivitamin determinations. Tables 25-27 summarize simultaneous HPLC determinations of multiple B vitamins in foods, published from 1992 to 1997. 

DeVries et al. (67) reported the summary of studies by a number of collaborating laboraties for the HPLC assay of vitamin D in multivitamin preparations. Saponification and a reverse-phase Merck LiChrosorb RP-8 column were used for sample cleanup. The analytical column was a Partisil , 5 nm column (Whatman, Clifton, N.J.) with hexane-amyl alcohol (99.65 0.35%) as the mobile phase. The cleanup procedure although a deparature from the usual analytical methods, was incorporated to ensure predictable, interference-free vitamin D assays (D2 and D3 co-elute). 

Intravenous vitamins and trace elements should be initiated on the first day of therapy and continued as a daily component of the PN solution. Children under age 11 should receive a vitamin product formulated for pediatric patients. Two multivitamin dosing schemas have been suggested for infants and children. One method recommends 2 mL/kg per day for infants weighing less than 2.5 kg and 5 mL... 

Multivitamin + Calcium Syrup (1 RDA of Vitamins/20 ml) 3. Chemical stability (20-25 °C HPLC methods)... 

The determination of vitamin in multivitamin preparations can be hampered by extraction difficulties but the use of sodium diethylene penta-acetic acid facihtates recoveries in excess of 90% (Walker et al., 1981). Subsequently, reversed phase ion-pair chromatography can be carried out using a mobile phase of 0.001 M hexanesulphonic acid in 1% acetic acid-methanol (75 25). Using this method, thiamine, niacinamide, riboflavin and pyridoxine can be quantitated. 

Figure 14.1 presents chromatograms of vitamins analysed in fruit drinks. The detection limit for thiamine hydrochloride deteetion was 9.2 ng/ml, whereas the limits for pyridoxine and cyanoeobalamin were 2.7 and 0.08 ng/ml, respectively. The proposed separation and detection procedure was applied sueeess-fully for quantitative evaluation of the studied B vitamins in pharmaeeutieal preparations and dietary supplements, and for routine control of multivitamin enriched foods. Based on those sueeessful results, we have developed also a method for analysis of vitamins Bg, B12 and Bi in seafood produets (Lebiedzinska et al. 2007). 

Vidovic, S., Stojanovic, B., Veljkovic, J., and Prazic-Arsic, L., 2008. Simultaneous determination of some water-soluble vitamins and preservatives in multivitamin syrup by validated stability-indicating high-performance liquid chromatography method. Journal of Chromatography A. 1202 155-162. 

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. 

Chen, P., Atkinson, R., and Wolf, W.R., 2009. Single-laboratory validation of a high-performance liquid chromatographic-diode array detector-fluorescence detector/mass spectrometric method for simultaneous determination of water-soluble vitamins in multivitamin dietary tablets. Journal of AOAC... 

Markopoulou, C.K., Kagkadis, K.A., and Koundourellis J.E., 2002. An optimized method for the simultaneous determination of vitamins Bl, B6, B12 in multivitamin tablets by high performance hquid chromatography. Journal of Pharmaceutical and Biomedical Analysis. 30 1403 1410. 

Folic acid can be determined in methods designed for multivitamin analysis either from multivitamin preparations (90-93) or from fortified samples such as infant milk (94). In these methods trichloroacetic acid extraction of liquid and powdered infant milk followed by ion pair chromatography with reversed-phase Cig column was applied. Satisfactory separation was achieved with octanesulfonic acid (5 mM) with triethylamine (0.5%) in methanol-water (15 85 v v) at pH 3.6 (Fig. 6). UV detection and wavelength switching were used for six vitamins giving sensitivity of 1 ng folic acid per injection (282 nm for folic acid). Total run time of this isogratic separation was 55 min. This approach has not been reported for nonfortified samples. 

A simultaneous detection of all water-soluble vitamins in multivitamin solution by only one TLC analysis was described by Postaire et al. in 1991 (31). The method used HPTLC plates with silica gel as stationary phase and n-butanol/ pyridine/water (50 35 15, v/v) as mobile phase. The quantitation was carried out by photodensitometric detection without derivatization (B, B2, Bg, C, folic acid, nicotinamide) or after spraying ninhydrin reagent (calcium panthothenate) or 4-dimethylaminocinnamaldehyde (B12, biotin). The use of an over-pressured layer chromatograph improved the resolution of the HPTLC plates. Good reproducibility, satisfactory standard deviations, and recoveries were obtained for all the vitamins. 

Riboflavin is an ingredient of many pharmaceutical multivitamin preparations. Methods have been described for the determination of riboflavin and other, water-soluble vitamins (Bj, B, B12, niacin, niacinamide, folic acid, ascorbic acid). For... 

The simultaneous determination of the B vitamins, thiamine, riboflavin, pyridoxal, pyridoxine, and pyridoxamine in a pharmaceutical product using CZE was described by Huopalahti and Sunell (90). Hydrochlorid acid was used for the extraction of the vitamins from the multivitamin-multimineral tablet. The applied potential was 6.0 kV, and a 75-p.m fused-silica capillary tubing was used. The electrolyte used was a 20-mM sodium phosphate buffer pH 9.0. A clear separation of standards as well as of the pharmaceutical sample is shown in Figure 14. This method appears to be a fast and simple technique for the simultaneous determination of water-soluble vitamins in pharmaceutical products, where the... 

Nuttall and Bush (102) described a TLC chromatographic method for the analysis of multivitamin preparations. After extraction of fat-soluble vitamins, water-soluble vitamins and water-soluble materials were separated in three TLC systems. Biotin was resolved with acetone-acetic acid-benzene-methanol (1 1 14 4) as solvent and visualized by spraying o-toluidine-potassium iodide. Standards can be included if quantitative results are required. However, the reproducibility of the technique has not been tested. Groningsson and Jansson (105) worked out a TLC method for the determination of biotin in the presence of other water-soluble vitamins. After dissolution of the lyophilized preparation and addition of the internal standard (2-imidazolidone), the sample was applied on a silicagel plate and eluted with chloroform-methanol-formic acid (70 40 2). Biotin was visualized by spraying with p-DACA and determined in situ by reflectance measurements. The sensitivity of the method could be increased by spraying with paraffin after the coloring procedure. LFnder these conditions the detection limit was 10 ng. 

Pantothenic acid/calcium pantothenate in pharmaceutical products and vitamin premixes was also analyzed using low-wavelength ultraviolet (UV) detection (64,66). The vitamin was extracted from tablets or powdered premixes with 0.005 M NaH2P04 buffer (pH 4.5) and separated from other water-soluble vitamins on an aminopropyl-bonded silica column (LiChrosorb NH2) eluted with an acetonitrile-0.005 MNaH2P04 buffer (pH 4.5) (87 13, v/v) and detected at 210 nm. Quantitative recoveries (>95%) and relative standard deviations 0.79% to 2.2% were obtained for multivitamin tablets, vitamin premixes, fortified yeasts, and raw materials. The limit of sensitivity was approximately 1 mg/g sample. The results were compared with those obtained by the standard microbiological procedure. Low levels of calcium pantothenate (<3 mg per tablet) were more precisely analyzed by the HPLC procedure than by the microbiological method. 

Gennaro (51) proposed a method for the separation of water-soluble vitamins by means of the ion interaction reagent using octylamine < -phosphate or octylamine salicylate buffer (at pH 6.4) as the interaction reagent and the mobile phase at a flow rate of 1 mL/min, and a 2.5-p.m Spherisorb ODS C18 column (250 X 4.6 mm) as the stationary phase. The column effluent was monitored at 210 nm. Retention times of pantothenic acid obtained with octylamine t)-phos-phate and octylamine salicylate were 64.0 and 9.8 min. The method was used for the determination of pantothenic acid in a model mixture of water-soluble vitamins and also in a commercial multivitamin isotonic salt dietetic drink (Fig. 9). 

Pantothenic acid, its salts, and panthenol as such are not volatile enough for direct gas-liquid chromatography (GLC). However, it is possible to use this chromatographic technique after derivatization of the polar hydroxyl and carboxyl groups of the vitamin (23,38,72,73,75-79). The majority of the developed methods are, however, applicable only to relatively pure and simple samples such as multivitamin preparations, and certain biological samples, such as urine (75-77). Only a few methods are suitable for the determination of the vitamin in complex matrices such as foods. An overview of methods was given by Velisek et al. (5). 

The UV—diode array [132,134—136,139,140] and fluorescence detection [141,142] have been used to develop multivitamin LC methods, which, however, remain limited to a few analytes responding to the same detection system and extracted quantitatively with the same procedure. Moreover, by means of a UV detector, other difficulties are represented by the absence of a strong chromophoric group in some vitamins (pantothenic acid and biotin), which absorb with modest sensitivity in the low UV region only, where the selectivity is scarce (absorption of interfering compoimds). 

Amin (2001) reported a colorimetric method for vitamin E in pure form and multivitamin capsules based on the reduction of tetrazolium blue to formazan derivative by vitamin E in alkaline medium. The reaction mixture was incubated at 90 2 C for 10 min and the absorbance of the reaction product was monitored at 526 nm with relative standard deviation of 0.7%-1.5%, a limit of detection 0.012 mg/E and sample throughput of approximately 6/h. The reduction of tetrazolium blue to formazan derivative requires 3 h at room temperature and the color developed was stable for 3 h. EDTA was added to sample solution for masking any metal ions during analysis. 

Hossu et al. (2009) presented spectrofluorimetric methods for the determination of fat-soluble vitamin E in multivitamin pharmaceutical products. In method I, M-hexane was used as solvent for a-tocopherol assay, while in method II, ethanol was used as a carrier between the aqueous solution and the n-hexane fluorescent solution of a-tocopherol. At 290/306 nm excitation and emission wavelengths, method I was linear for a-tocopherol over the range 1-100 p,g/mL R = 0.97687), having a limit of detection 1 p,g/mL and a limit of quantification 2 p,g/mL, and method II was linear over the range 2-50 p,g/mL R = 0.9709) with a limit of detection 0.68 J.g/mL and a limit of quantification 2.27 j,g/mL. 

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