Elsevier

Journal of Chromatography B

Volume 877, Issue 24, 15 August 2009, Pages 2537-2544
Journal of Chromatography B

Novel molecularly imprinted polymers for the selective extraction and determination of metoclopramide in human serum and urine samples using high-performance liquid chromatography

https://doi.org/10.1016/j.jchromb.2009.06.030Get rights and content

Abstract

This work was performed in order to study the possibilities in using molecularly imprinted polymers (MIPs) as sorbent material in solid-phase extraction (MISPE) for the sample clean-up technique for the determination of metoclopramide (MCP) in biological fluids. The effective factors influencing the bulk polymerization have been studied. Molecular recognition properties, binding capability and selectivity of the molecularly imprinted polymers (MIPs) were evaluated and the results revealed the obtained MIPs have high affinity for MCP in aqueous environment. The optimal conditions for solid-phase extraction (SPE) consisted of conditioning with 1 mL of methanol and 1 mL of deionized water at neutral pH, loading with 1 mL of the water sample (50 μg L−1) at pH 8.5, washing using 1 mL of acetone and elution with 2× 1 mL methanol/acetic acid (10/1, v/v). After optimization of SPE procedure, the MIP was then directly used to selectively extract the target drug from human serum and urine with an extraction recovery of more than 90%. Chromatograms of the eluate solutions show an efficient clean-up, which supports the potential of MISPE for clean-up of trace amounts of MCP from serum and urine. The limits of detection of MCP in human serum and urine were 3 and 1.2 μg L−1, respectively.

Introduction

Metoclopramide (MCP), 4-amino-5-chloro-2-methoxy-N-(2-diethylamino-ethyl), benzamide is a dopamine-receptor antagonist active on gastrointestinal motility. It is used as an anti-emetic in the treatment of some forms of nausea and vomiting and to increase gastrointestinal motility. It is also used at much higher doses for the prevention of cancer chemotherapy-induced emesis [1]. Both the British Pharmacopoeia (BP) and the United States Pharmacopoeia (USP) recommend a nonaqueous acid–base titration with potentiometric detection of the end-point for the evaluation of the raw material of metoclopramide; for its dosage forms, the BP describes spectrophotometric methods while the USP recommends high-performance liquid chromatography (HPLC) methods [2]. The drug has been quantified using different methods such as chemiluminescence [3], potentiometry [4], stripping voltammetry [5], liquid chromatography (LC) [6], gas chromatography–mass spectrometry (GC–MS) [7], gas chromatography-electron capture detection [8], high-performance capillary electrophoresis (HPCE) [9] and UV–vis spectrophotometry [10]. High-performance liquid chromatography (HPLC) has become a powerful tool for the analysis of pharmaceutical products and has been applied to the determination of the MCP in biological fluids [11], [12], [13], [14]. HPLC methods sometimes requiring specific solvent including ion-pairs and evaporation of organic solvents or combined with a liquid-liquid extraction (LLE) step that is time-consuming and hindered the degree of automation [15], [16]. A HPLC method for quantification of MCP was already introduced by Vergin and coworker [17] with a detection limit of 8 μg L−1. The procedure, however, requires an Extrelut® column, large solvent volumes of chloroform (70 mL), and two evaporation steps for dryness of organic eluate which are not possible for routine use in monitoring plasma MCP levels in a clinical laboratory. Further, Guang-yue et al. [18] published an article about the quantification of MCP in human plasma with HPLC coupled to tandem mass spectrometry. Although, the obtained detection limit (0.5 μg L−1) is lower than the limit of detection presented in this work, but the method plasma samples were prepared by LLE using ethyl ether and then separated using the Alltima C18 column which is costly and have an expensive sample clean-up. In this work, our novelty in introducing a simple clean-up technique based on molecularly imprinted solid-phase extraction can be highlighted. On the other hand, none of the available methods was ideal, and an improved method for selective extraction and determination of MCP was necessary. Analysis of pharmaceutical compounds in biological matrices such as serum requires sample pretreatment to clean-up the sample before the chromatographic separation that can be commonly achieved by solid-phase extraction (SPE). SPE is the most popular of clean-up techniques due to factors such as convenience, cost, time saving and simplicity. Compared to LLE, SPE can reduce the time required, especially for automated methods, can handle small samples, and consumes small amount of solvent [19]. As a result, SPE is the most accepted sample pretreatment method today [20]. However, classical SPE sorbents, used for the treatment of serum samples, mainly cause non-specific hydrophobic interactions that lead to the co-extraction of interfering compounds often preventing an easy and reliable quantification of the analyte. A relatively new development in the area of SPE is the use of molecularly imprinted polymers (MIPs) for the sample clean-up [21], [22], [23], [24]. The use of molecularly imprinted polymers (MIPs) to clean-up and preconcentration the drugs of interest are becoming popular in high-performance liquid chromatography, and many applications have been reported [25], [26], [27].

The procedure for synthesizing an MIP is based on the chemical polymerization of a functional monomer and a cross-linking agent in the presence of a molecule used as a template. Removal of the template yields a functional polymeric matrix with recognition sites complementary in functionality and shape to the print molecule structure and consequently allows its selective uptake. MIPs are often referred to as ‘artificial antibodies’. Unlike antibodies, MIPs are stable to extremes of pH, organic solvents and temperature which allows for more flexibility in the analytical methods [21], [28]. The use of MIPs for SPE can involve various modes, including conventional SPE where the MIP is packed into columns or cartridges [29], [30] and batch mode SPE where the MIP is incubated with the sample [31]. Another major advantage of MIP-based SPE, related to the high selectivity of the sorbent, is the achievement of an efficient sample clean-up. The application of MIP technology to solid-phase extraction (MISPE) has been used for both biological and environmental samples and has been reviewed [32], [33]. Study on the mechanism of binding specificity of metoclopramide-imprinted polymers is the only work on MCP-based MIPs that was reported by Xu et al. [34]. The study described that the imprinted polymers were prepared with EDMA as the cross-linker and MAA as functional monomer. The molar ratio of cross-linker, functional monomer and template was 20:4:1 that is different from this work. The study reveals that the proton NMR studies can provide useful information for us to understand the mechanism of molecular recognition and a more directed choice of MIP ingredients, which in turn should help us to shorten the process of making MIPs.

Recently, we described the use of MIPs as new sensing material in potentiometric detection of hydroxyzine [35], cetirizine [36] and SPE of verapamil [37]. The purpose of this paper is to develop a novel, fast and accurate method for the performance evaluation of MCP-based MIPs as selective SPE sorbents for efficient sample clean-up and followed determination of MCP from complex matrices by high-performance liquid chromatography. This method allows the sensitive, simple and inexpensive extraction and detection of MCP in human serum and urine samples at low concentration levels using MIPs-based sorbent material.

Section snippets

Reagents

Methacrylic acid (MAA) from Merck (Darmstadt, Germany) was distilled in vacuum prior to use in order to remove the stabilizers. Ethylene glycol dimethacrylate (EGDMA) and 2,2′-azobis isobutyronitrile (AIBN) from Merck (Darmstadt, Germany), were of reagent grade and were used without any further purification. The phosphate buffer solutions with a pH value of 8.5 were prepared in deionized water and were used. All solvents used in chromatography analyses were HPLC grade and supplied by Merck.

Characterization

The IR spectra of NIP, the unleached and leached MIPs displayed similar characteristic peaks, indicating the similarity in the backbone structure of the different polymers. The IR spectra of the unleached and leached imprinted poly(MAA-co-EGDMA) are shown in Fig. 2. As a result of the hydrogen binding with the –COOH group of MAA, the Cdouble bondO stretching, the O–H stretching and the bending vibrations at 1722, 3473 and 1394 cm−1 in the leached MIP materials were shifted to 1710, 3457 and 1388 cm−1 in the

Conclusions

In this paper, a polymer imprinted for MCP has been synthesized via a non-covalent molecular imprinting approach. The MIP particles as new sorbents in SPE were successfully investigated for the clean-up of human serum and urine samples with an optimized procedure.

A SPE-HPLC method based on MIP has been developed for the extraction of MCP from aqueous solutions. This efficient method allowed cleaner extracts to be obtained and interfering peaks arising from the complicated biologic samples to be

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