Determination of 1-phenyl-3-methyl-5-pyrazolone-labeled carbohydrates by liquid chromatography and micellar electrokinetic chromatography

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Abstract

In this paper, the method for the derivatization of carbohydrates with 1-phenyl-3-methyl-5-pyrazolone (PMP) was simplified. One-third of the derivatization time was saved. Five monosaccharide derivatives have been well separated by MEKC and HPLC under optimized conditions. Good reproducibility could be obtained with relative standard deviation (RSD) values of the migration times within 5.0 and 2.3%, respectively. Furthermore, the developed methods have been successfully applied to the analysis of carbohydrates in Aloe powder and food. These methods are quite useful for routine analysis of monosaccharides and oligosaccharides in real samples.

Introduction

The important roles of carbohydrates in biological processes have been increasingly recognized. A considerable amount of research has already been carried out in recent years in this area. Since carbohydrates encompass a number of homologues having very similar structures and many of them exist concurrently in real-life samples, carbohydrate analysis inevitably requires high-resolution separation techniques. However, these compounds generally have low intrinsic UV spectral activity. Therefore the derivatization of carbohydrates is indispensable to obtain highly sensitive detection [1]. In practical analyses, carbohydrates are usually derived with either UV [2], [3] or fluorescent tags [4], [5]. The introduction of some labels also endows carbohydrates with ionic properties, and changes their hydrophilicity, so that different separation modes can be adopted to analyze them.

The reagent 1-phenyl-3-methyl-5-pyrazolone (PMP) is one of the popular labels that can react with reducing carbohydrates under mild conditions, requiring no acid catalyst and causing no desialylation and isomerization. PMP yields strong UV absorbance at 245 nm [6]. This derivatization method was first developed for the analysis of carbohydrates by high-performance liquid chromatography (HPLC) [7], [8], [9], [10], [11], and later successfully applied to capillary electrophoresis (CE) [12], [13], [14], [15], [16], [17], [18], [19], [20]. With high efficiency, high speed, low sample requirement and low solvent cost, CE has proved to be an excellent method for the analysis of carbohydrates with capillary zone electrophoresis (CZE) [14], micellar electrokinetic chromatography (MEKC) [15], [16], [17], [18], capillary gel electrophoresis (CGE) [19] and ion-exchange electrokinetic chromatography (IXEKC) [20]. PMP derivatization increases the hydrophobicity of carbohydrates, therefore HPLC and MEKC are quite suitable for analyzing PMP derivatives.

In this study, five monosaccharide derivatives have been prepared using a simplified PMP reaction method. These derivatives have been well separated by MEKC and HPLC under optimized conditions. Furthermore, the developed methods have been successfully applied to the analysis of carbohydrates in Aloe powder and food.

Section snippets

Instrumentation

Capillary electrophoresis experiments were carried out on a Beckman P/ACE 5010 instrument equipped with a UV detector set at 254 nm (Beckman, Fullerton, CA, USA). Fused-silica capillaries with 50 μm I.D. and 375 μm O.D. were purchased from Yongnian Optical Fiber Factory (Hebei, China). Data acquisition and processing were carried out with Beckman System Gold software.

The HPLC apparatus was composed of a P200II liquid chromatography pump system, a Rheodyne 7125 injector with a 20-μl loop, a

Simplification of PMP derivatization method

In our method, drying of the aqueous layer after extraction with chloroform (as described by [6], [7]) was omitted. This drying step was used to remove residual derivatization reagents and impurities produced in the derivatization procedure. Although this step could minimize the interference of PMP to separation and detection and prolong the lifespan of columns, it complicated the derivatization procedure and resulted in the loss of the derivatives.

Ma et al. [8] did not employ any drying steps

Conclusion

The procedure of derivatization of carbohydrates by PMP is simple, rapid and reproducible, and proved to be suitable for analyses by HPLC and MEKC. Several real samples, such as Aloe powder and its hydrolysates, candy, and beer were analyzed by both HPLC and MEKC. The methods developed in this context are quite useful for routine analysis of monosaccharides and oligosaccharides in real-life samples.

Acknowledgements

The authors are grateful for the financial support from the National Natural Science Foundation of China (No. 990401).

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