Vitamin analytical techniques

In addition to being necessary for all forms of life, biopolymers, especially enzymes (proteins), have found commercial applications in various analytical techniques (see Automated instrumentation, clinical chemistry Automated instrumentation, hemtatology Biopolymers, analytical techniques Biosensors Immunoassay) in synthetic processes (see Enzyme applications, industrial Enzyme applications in organic synthesis) and in prescribed therapies (see Enzyme applications, THERAPEUTICS IMMUNOTHERAPEUTIC AGENTS Vitamins). Other naturally occurring biopolymers having significant commercial importance are the cellulose (qv) derivatives, eg, cotton (qv) and wood (qv), which are complex polysaccharides. 

It is essential for a successful assay that the vitamins be quantitatively extracted from the food matrix in a form that can be accurately measured by the particular HPLC technique to be used. An effective extraction procedure serves to homogenize and concentrate the sample, isolate the vitamin analyte from its association with protein, eliminate as far as possible known interfering substances, and destroy any indigenous enzyme activity. The vitamin-rich fraction thus obtained may require some form of cleanup before the vitamins can be measured, particularly when measuring the trace amounts of naturally occurring vitamins D and K. 

Factors to consider in selecting a suitable extraction procedure are (a) the analytical information required, (b) the nature of the food matrix, (c) the form in which the vitamin occurs naturally or is added, (d) the nature and relative amounts of potentially interfering substances, (e) the stability of the vitamin in heat and extremes of pH, and (f) the selectivity and specificity of the analytical technique to be used. 

The first HPLC methods for quantitating vitamin B12 in foods are beginning to appear (Tables 21 and 22). The detection problems are being addressed by coupling HPLC with other analytical techniques. Further work is needed for routine analysis of these vitamers by HPLC. 

The increasing interest in a-tocopherol as the principal lipid-soluble antioxidant in mammalian cells creates a need for rapid, reproducible and accurate methods for measurement of the vitamin in body fluids, cultured cells, whole tissues and, in some instances, in food products. Early methods identified a number of difficulties that arise in achieving this analytical objective and an understanding of the possible problems that may arise is fundamental to the successful application of the chosen analytical methods. The early methods were summarized in three categories (Bunnell, 1971) which cover the three stages of analysis that may still be found to be necessary with the availability of modem analytical techniques. These stages are ... 

Although modern analytical techniques have considerable precision and sensitivity, food composition tables carmot be considered to give more than an approximation to vitamin intake. Apart from the problems of biological availability (Section 1.1.2), there is considerable variation in the vitamin content of different samples of the same food, depending on differences between varieties, differences in growing conditions (even of the same variety), losses in storage, and losses in food preparation. 

It is a truism that nothing in the universe is free from trace elements. As analytical techniques have improved with the development of ever more sophisticated measuring devices, the impact of trace and ultratrace concentrations of elements has been revealed much more clearly over the last two decades or so. We know from nutritional studies the importance of ultratrace concentrations of several elements including, for example, cobalt in cyanocobalamine (vitamin B12), where the daily requirement for this vitamin is only 1 pg/day for an adult. 

In this chapter, pharmaceutical and health-care products, such as prescription drugs, generic drugs, OTC products, animal health products, dietary supplements (vitamins and herbal drugs), and biotechnology-derived products, are discussed in relationship to the format of preformulation reports. Topics of the preformulation study are discussed in detail. Models for some of the reports are provided in the hope that the pharmaceutical development team will devise an individual report format based on particular needs and resources. Analytical techniques useful for preformulation and regulatory conformity or requirements relative to product registration processes are also enumerated. 

Thiamine, a soluble vitamin, is an essential nutrient for humans and is important in carbohydrate metabolism, maintaining normal neural activity and preventing beriberi. Various analytical techniques have been reported for the determination of thiamine in pure form, in pharmaceutical preparations, or in biological fluids. Spectrophotometric methods suffer from poor sensitivity (mg/L detection limit). Spectrofluorometric methods usually involve the conversion of thiamine to thiochrome.2 High performance liquid chromatography requires a post-column derivatization step3 and instrumentation for electrophoresis-based methods is expensive.4... 

When your blood is drawn, for example, the doctors use techniques borrowed from analytical chemistry to determine levels of several analytes in your body, including cholesterol, vitamins, glucose, and white blood cells. Analysis of tissue samples and other biological fluids is also based on analytical techniques. Other examples of medically related applications include tests for illegal drugs and steroids, glucose sensors used by diabetic patients, or to check for toxic substances in your body if you are exposed to hazardous chemicals. 

Supercritical fluid extraction (SEE) has a very good field of application in cosmetics analysis as separation treatment before different analytical techniques (e.g., waxes, UV filters, preservatives, or vitamins have been extracted from different cosmetic matrices). 

Research into new analytical techniques for foodstuffs continues, striving for greater accuracy, sensitivity or simplicity, for more rapid methods, for simultaneous multielement analysis, etc. Chromatographic techniques, e.g., LC, GLC, GC-MS, have led to great improvements in the levels of accuracy, sensitivity, and detection that can be achieved for many analytes including carbohydrates, certain vitamins, chemical residues, and additives. Work is still required, for instance, in the area of vitamin analysis in order to provide standard techniques that are applicable to all food types and that would enable concurrent multi-vitamin analysis to take place. Many of the microbiological assays currently used for vitamin determination involve long incubation times and more rapid techniques are needed. 

Research on bioavailability of vitamins is a modern field of biochemistry with a strong need for non-biological and biological analytical techniques to promote the vmderstanding of the complicated subject of bioavailability. 

A number of cyclic polyols also are known to occur (Fig. 15.13). myo-Inositol (62), widespread in animals, is a growth factor in yeasts and a group B vitamin. This compound, one of nine possible stereoisomers, is widespread in plants (six other isomers also are naturally occurring). When plant cells are incubated with radiolabeled myo-inositol, a large portion of the label appears in the pentose and uronic acid residues of the cell wall (Karr, 1976 Loewus and Loewus, 1980). myo-Inositol serves as a precursor for o-glucuronic acid (3). Furthermore, when glucose and other monosaccharides are incorporated into cell walls, myo-inositol appears to be an intermediate (Karr, 1976 Loewus and Loewus, 1980). Analytical techniques for the study of cyclitols and sugar alcohols (polyols) have been reviewed (Beck and Hopf, 1990 Loewus, 1990). 

In 2002, Indyk and co-workers evaluated vitamin B12 in a range of foods such as milks, infant formulas, meat and liver using SPR technology with performance parameters including a quantitation range of 0.08-2.4 ng/mL with recoveries of 89-106% (Indyk et al. 2002). The analytical technique was biosensor based utilizing bimolecular interaction. 

Various CL-based analyses of vitamin B12 are listed in Tables 27.2 and 27.3 for pharmaceutical and food samples. Compared with conventional analytical techniques, such as microbiological assay, high-performance liquid chromatography (HPLC), atomic absorption spectroscopy (AAS), radioisotope isotope assay, and fluorimetric detection, the current proposed CL method was more sensitive with a detection limit of 5 pg/mL. 

---