As the name suggests, vitamins are “vital amines” that play an important role in human growth and development. Since the past decade, vitamins have drawn attention because of their nutritional implications and the need to develop standardized quality-control procedures [Hurst 2002]. However, development of physicochemical methods for characterization is diffi cult because these compounds are diverse in nature, present in low quantities in complex matrices, such as functional foods and vegetables, mimicking of their activity by other compounds, and the special stability considerations. Most of the analytical methods described in literature focus on extraction, purifi cation, and detection [Hurst 2002]. The extraction procedure from the complex matrices may involve heat, acid or alkali or enzymes, followed by cleanup procedures, and then quantitative estimation by RP-HPLC coupled with UV, fl uorescence, or protein binding. The water-soluble B vitamins, such as thiamin, ribofl avin, and niacin, may be estimated by RP-HPLC coupled with fl uorescence or UV absorption. For thiamine, after extraction, it is convenient to convert it to fl uorescent thiocrome by oxidation and then do quantitative separation and identifi cation by RP-HPLC coupled with fl uorometry using an excitation wavelength of 360–365 nm and an emission wavelength of 460–480 nm [Hurst 2002]. For RP-HPLC, usually C8 or C18 columns are used with the mobile phase ranging from organic solvents and ion pairs to organic-aqueous buffer mixtures. It should be noted that ion exchange and normal HPLC purifi cation has also been reported for thiamine and other B vitamins. Cobalamin or vitamin B12 can be characterized by HPLC coupled with specifi c protein binding assays involving radio isotopes or enzymes. Its diversity and low propensity in food matrices make it an unsuitable candidate for UV detection; the same applies to biotin or folic acid [Hurst 2002].
REFERENCES
American Nutraceutical Association. 2008. Nutraceutical Information. http://www.ana-jana. org/nut_info_details.cfm?NutInfoID=4.
Analytical Solutions. 2008. Nutraceuticals. http://www.asi-rtp.com.
Dureja, S., D. Kaushik, and V. Kumar. 2003. Developments in nutraceuticals. Indian J.
Pharmacol. 35:363–372.
Hurst, W. J. 2002. Methods of Analysis for Functional Foods and Nutraceuticals. 2nd edition. Boca Raton, FL: CRC Press.
Krull, I. and M. Swartz. 2001. Striving to validate nutraceuticals. LCGC 19:1142–1149. Mehta, J., R. L. Chapman, W. G. Chambliss, M. A. Murray, and W. Obermeyer. 2007. Formulation
and Stability of Nutraceuticals. http://www.aapspharma ceutica.com/meetings/annualmeet/ am07/ProgramInfo/Prelim/program/pt_form.asp.
131
Development of Techniques for Analysis
of Nutraceuticals with Specific Reference
to Glucosamine and Coenzyme Q
10John Adams and Brian Lockwood
CONTENTS
Introduction ... 132 Techniques for Analysis of Glucosamine ... 133 Radiolabeling ... 133 HPLC Separation ... 134 Precolumn Derivatization Agents for UV/Fluorescent Visualization ... 134 Postcolumn Agents Used for Indirect Fluorescent Detection ... 136 HPLC Using Electrospray Ionization-MS Detection ... 136 The Use of HPLC with Alternative Detection Methods ... 137 Gas Chromatography ... 138 High-Performance Thin-Layer Chromatography ... 138 Capillary Electrophoresis Determination ... 138 Capillary Electrophoresis with Dansylation of Glucosamine under
Microwave Irradiation ... 139 Microchip Capillary Electrophoresis with Fluorescamine Labeling for
Anomeric Composition Determination ... 139 The Pros and Cons of Capillary Electrophoresis ... 140 Infrared Spectroscopy with Chemometrics ... 140 Techniques for Analysis of Coenzyme Q10 ... 141 HPLC ... 142 UV or Electrochemical Detection? ... 142 Column Switching HPLC ... 143 Mass Spectrometry Detection ... 143 Derivative Spectrophotometry ... 144
Voltammetric Determination ... 145 Square-Wave Voltammetry with Glassy Carbon Electrode ... 145 Mercury Hanging Electrode ... 145 Electron Paramagnetic Spectroelectrochemistry ... 145 Conclusions ... 146 References ... 146
INTRODUCTION
Over the past two decades, the public has gained an increasing interest in the role of diet, nutrition, and dietary supplements in the prevention and treatment of disease [Childs and Poryzees 1998]. This has resulted in a dramatic increase in the use of commercially available nutraceuticals and their foodstuffs [G. Lockwood 2007].
This widespread use of nutraceuticals has stimulated the development of a number of analytical techniques to monitor their identifi cation and levels in available raw materials, simple extracts, formulated products, biological fl uids, and a number of other matrices.
This review covers the wide range of techniques developed for the analysis of two of the most commonly studied nutraceuticals. There are a number of scientifi c reasons for the development of improved analytical methods, predominantly in the area of additional understanding of disease states and their treatment and particu- larly the modes of action of specifi c nutraceuticals. Other important reasons include prediction of disease states and compliance.
The analytical procedures used for identification and quantification of nutra- ceuticals are becoming increasingly sophisticated, reflecting current analytical advances. Manufacturers, independent researchers, and consumer organizations such as ConsumerLab.com often produce detailed information about levels of active constituents, in both the natural materials and formulated products. Clinical researchers are increasingly investigating biological fluids, in clini- cal trials and in vivo research in an attempt to determine the biological fate of nutraceuticals.
There are few published reviews on the analytical techniques used for nutra- ceuticals. There has been a limited overview of 26 examples published in 2007 [B. Lockwood 2007], chapters in two edited texts published in 2002 [Ho and Zheng 2002; Hurst 2002], but, for the majority, there are few available methods. Monographs, such as those (being) produced by the FDA and the Offi ce of Dietary Supplements of the National Institutes of Health of the United States, list techniques for sample preparation and extraction, chromatographic separation, detection meth- ods, and quantitation methods [B. Lockwood 2007]. In addition, these publications highlight the wide range of techniques increasingly being used for their identifi ca- tion and quantifi cation in both manufactured products and biological fl uids. HPLC, using a range of different detectors, has been used for the majority of nutraceuticals, but newer techniques are also becoming widespread.
The phytoestrogens from soy and fl ax, n-3 PUFAs and CLA, carotenoids, lycopene, zeaxanthin, astaxanthin, and lutein have been the subject of a recent edited book on the subject [Ho and Zheng 2002]. The other text by Ho and col- leagues reviews the techniques available for cocoa, chocolate, cranberry, and gug- gul (Commiphora wightii) constituents [Hurst 2002]. Two recent publications have reviewed the analysis of soy [Dentith and Lockwood 2008] and tea polyphenols [Kirrane and Lockwood 2008].
This chapter will focus on the techniques used for two important and widely used nutraceuticals whose analysis has not been reviewed previously, namely glu- cosamine and CoQ10. The chapter will also explain the development of techniques used in analysis of such nutraceuticals.
Glucosamine is widely used to improve joint health, specifi cally osteoarthritis and rheumatoid arthritis, and skin health. It is also widely used for joint problems in domestic animals. CoQ10 has been reported to have activities in cardiovascular health, cancer prevention, respiratory and skin diseases, and animal ailments.