Multivitamin analysis methods

Recent multivitamin analytical methods, including thiamine analysis recent development of high quality instrumental methods for thiamine analysis when a food sample is subjected to the multivitamin assay. The application of the methods to pharmaceutical materials is indicated. 

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. 

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 Colorimetric, fluorometric, and microbiological methods have often been replaced by HPLC analysis.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. 

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. 

Both WSV and FSV methods were rapid, with extraction and analysis completed in an honr. A single tablet was used to minimize sample volume and solvent nse. This is more acceptable for OTC prodncts. Further details and data were published elsewhere. Table 4 shows assay data on precision and recovery and Figure 7 shows HPLC chromatograms of WSV in standard solution and in multivitamin extracts. 

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. 

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). 

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). 

Thiamine Analysis by Multivitamin Methods with Potential to Improve Performance in the Future... 

Particular attention has been paid in recent years for thiamine analysis with multivitamin methods with potential to improve performance in the future. These methods include up-do-date techniques of liquid chromatography and electrochemical methods based mainly on the voltammetric methods of analysis. 

The most common detector for HPLC and UHPLC is a diode array deteetor (DAD). Although there is an earlier report of determination of pantothenic add using UV (210 nm in multivitamin pharmaceutical preparations (Hudson and Allen 1984), most HPLC-DAD methods used for the analysis of WSVs do not include pantothenic acid as part of the analysis (Kirchmeier and Upton 1978 Kwok et al. 1981 Li 2002 Markopoulou et al. 2002 Walker et al. 1981 Wills et al. 1977) as the interferences may undermine the quality of the quantitation. 

Other widely used detectors for HPLC include refractive index (RI), fluorescence and evaporative light-scattering (ELS). The use of Rl and ELS detectors for pantothenic acid analysis in multivitamin dietary supplements has not been reported. The main reason is that the two detectors are not selective and thus cannot resolve pantothenic acid from other components existing in a multivitamin dietary supplement. Although fluorescence detection can be highly selective depending on the application, pantothenic acid does not have fluorescence excitation and emission and so fluorescence detection cannot be used for pantothenic acid analysis unless derivatization methods are applied (Pakin et al. 2004 Takahashi et al. 2009). Derivatization adds more complexity to analytical method and should not be used unless neeessary. For deteetion and quantitation of pantothenic add in multivitamin dietary supplements with HPLC/UHPLC, a highly selective detector such as MS should be the instrument of choice. 

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. 

Using a single LC-MS/MS method to quantify all WSVs at such different levels of concentrations is very difficult. However, quantitation of all the WSVs except cyanocobalamin in a single chromatographic run has been achieved (Chen and Wolf 2007 Chen et al. 2009). Compromises have to be made to accommodate WSVs at different concentration levels. For multi WSV analysis using LC-MS or LC-MS/MS in multivitamin dietary supplements, the instrument may need to be intentionally tuned to lower the sensitivity for pantothenic acid to accommodate higher concentrations if folic acid and biotin need to be analysed together (Chen and Wolf 2007). 

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. 

Overpressure derivatization (OPD) is a relatively new method in which an absorbent polymeric pad prewetted with detection reagent is pressed onto the TLC plate. OPD was found to give more reproducible results when compared to spraying of ninhydrin reagent in the quantitative analysis of tryptophan on a cellulose layer using densitometric scanning. Use of OPD for a variety of other detection reagents and application to the analysis of a multivitamin preparation was described by Postaire et al. (1990). 

Nicotinamide is present in pork, beef, chicken, and fish. 2-Py and 4-Py are the major metabolites of nicotinic acid and nicotinamide excreted in urine. The contents of 2-Py and 4-Py in blood are below the limit of detection (33). The HPLC methods for analysis of nicotinamide and the related compounds are summarized in Table 4. Shibata et al. (34) reported the simultaneous measurement of nicotinamide, 2-Py, and 4-Py. This method is commonly applicable not only to urine and pharmaceutical preparations but also biological materials and foods. Chromatograms of a reference mixture of isonicotinamide (used as an internal standard), nicotinamide, 2-Py, and 4-Py and of extracts of rat urine, human urine, rat liver, and of the extract of multivitamin preparations are shown in Figure 10. The detection limits for nicotinamide, 2-Py, and 4-Py were 4 pmol (552 pg), 10 pmol (1220 pg), and 2 pmol (304 pg), respectively, at a signal-to-noise ratio of 5 1. Daily urinary excretion of nicotinamide, 2-Py and 4-Py in rats, mice, guinea pigs, hamsters and humans is given in Table 5. 

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. 

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. 

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. 

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