Development and validation of a UPLC–ELSD method for fast simultaneous determination of five bile acid derivatives in Calculus Bovis and its medicinal preparations

Abstract

A reverse phase ultra-performance liquid chromatography coupled with evaporative light scattering detection (UPLC–ELSD) method was successfully established for simultaneous determination of five bile acid derivatives in Calculus Bovis and its medicinal preparations using a Waters Acquity UPLC T3 C₁₈ column (2.1 × 50 mm, 1.8 μm) with gradient elution of 0.2% aqueous formic acid and acetonitrile. The influence of liquid flow-rate and column temperature, gas flow-rate and drift tube temperature on resolution was investigated to get the optimized parameters and conditions. This method was validated according to the regulatory guidelines with respect to precision, accuracy and linearity. Under optimum conditions, the five analytes were baseline separated in 11 min with detection and quantification limits ranged in 3.0–6.0 ng and 6.0–18.0 ng, respectively, which provided about a twofold reduction in analysis time and good efficiency and sensitivity by comparing a conventional high performance liquid chromatography (HPLC) method.

Introduction

Calculus Bovis (C. Bovis) (Niuhuang in Chinese), the dry gallstone of Bos taurus domesticus Gmelin, a type of rare Chinese medicinal material known as cow bezoar, has long been used in oriental preparations and is officially listed in Chinese Pharmacopoeia containing two species, natural C. Bovis and artificial C. Bovis (China Pharmacopoeia Committee, 2005). This important medicinal material has been recognized for centuries in traditional medicine for its multiple pharmacological actions including sedation, relieving fever, diminishing inflammation, normalizing function of the gallbladder, immunoregularity, anti-cancer and anti-hyperspasmia, etc. (Cai et al., 2003, Dai et al., 2009, Li et al., 1995, Liang et al., 2006, Tai et al., 2006). The major active components of C. Bovis are bile acids (BAs) and their derivatives, namely cholic acid (CA), deoxycholic acid (DCA), hyodeoxycholic acid (HDCA), chenodeoxycholic acid (CDCA), taurocholate sodium (TCANa) and others (Nambara et al., 1988, Wei et al., 2009, Zhang et al., 2009), which are often used as the chemical markers for quality control of this medicinal material. Because of limited source of natural C. Bovis, artificial C. Bovis is always applied as a substitute in the medicinal preparations. The contents of BAs and their derivatives in these artificial C. Bovis medical materials with various sources and species are largely different, resulting in the different internal qualities of these artificial products. So, it is necessary to establish suitable method for accurately and fast determining the contents of the major active components (BAs and their derivatives) for controlling the quality of these substitutes.

Sample preparation, chromatographic and detection methods for the quantitative analysis of the BAs and their derivatives in C. Bovis materials have been reported in several papers (Alnoutia et al., 2008, Hua et al., 2006, Wei et al., 2009, Yang et al., 2003, Zhou et al., 2007). However, the absence of a chromophore in these bile acids hampers their detection using a UV detector. The use of refractive index (RI) detector has limitation from its low sensitivity and non-applicability of gradient elution. And the instrument of mass spectrometry (MS) detector is more expensive and sophisticated technique is required to use this instrument routinely in industrial fields. So, using these methods, a derivatization process of the samples is usually needed, making the results of determinations more uncertain than they are when this process is omitted. Evaporative light scattering detector (ELSD), as a universal detector with high sensitivity of detection, does not require a chromophore in the analyte and can be used in the underivatized case. Recently, a few reports on the quantitative analysis of BAs using high performance liquid chromatography–evaporative light scattering detection (HPLC–ELSD) have appeared (Feng et al., 2000, Sebök et al., 2008, Wei et al., 2009). Unfortunately, the HPLC–ELSD method suffers from either long analysis time of more than 40 min or expensive equipment or much solvent consumption. With the objective of reducing analysis time and maintaining good efficiency, there has been substantial focus on high-speed chromatographic separations. The commercially available technique of ultra-performance liquid chromatography (UPLC) has been proven to be one of the most promising developments in the area of fast chromatographic separation (Chen et al., 2008, Plumb et al., 2006). This UPLC technique offers the possibility of significantly increased efficiency of the chromatographic separation through the utilization of columns packed with smaller diameters particles (1.8 μm) that can withstand high backpressures compared to the conventional HPLC instrumentation. It has the advantages of the fast analysis, high peak capacity, good sensitivity and low consumption of samples and has been applied for the pharmaceutical, toxicological and biochemical analysis (Bruce et al., 2009, Kong et al., 2009b, Kong et al., 2009a, Paliakov et al., 2009). The combination of UPLC and ELSD could better make up some disadvantages and inconveniences in HPLC with ELSD or other detectors for the determination of the main active components in C. Bovis raw material and its medicinal preparations.

So, in this present study, the ELSD detector was successfully connected to the UPLC instrument; then the main parameters of this combinatorial system were optimized for fast and simultaneously determining the contents of five bile acid derivatives in the raw material, i.e. natural and artificial C. Bovis and its three medicinal preparations.