Elsevier

Journal of Chromatography B

Volume 845, Issue 1, 1 January 2007, Pages 169-173
Journal of Chromatography B

Short communication
Determination of metolazone in human blood by liquid chromatography with electrospray ionization tandem mass spectrometry

https://doi.org/10.1016/j.jchromb.2006.07.033Get rights and content

Abstract

A rapid, sensitive and accurate liquid chromatographic–tandem mass spectrometric method is described for the determination of metolazone in human blood. Metolazone was extracted from blood using ethyl acetate and separated on a C18 column interfaced with a triple quadrupole tandem mass spectrometer. The mobile phase consisting of a mixture of acetonitrile, 10 mmol/l ammonium acetate and formic acid (60:40:0.1, v/v/v) was delivered at a flow rate of 0.5 ml/min. Electrospray ionization (ESI) source was operated in positive ion mode. Selected reaction monitoring (SRM) mode using the transitions of m/z 366  m/z 259 and m/z 321  m/z 275 were used to quantify metolazone and the lorazepam (internal standard), respectively. The linearity was obtained over the concentration range of 0.5–500 ng/ml for metolazone and the lower limit of quantitation (LLOQ) was 0.5 ng/ml. For each level of QC samples, inter- and intra-run precision was less than 8.07 and 3.56% (relative standard deviation (RSD)), respectively, and the bias was within ±4.0%. This method was successfully applied to the pharmacokinetic study of metolazone formulation after oral administration to humans.

Introduction

Metolazone (Fig. 1), (7-chloro-1,2,3,4-tetrahydro-2-methyl-4-oxo-3-o-tolyl-6-quinazoline-sulfonamide), is a diuretic agent. It has been clinically used for the treatment of hypertension and edema [1], [2]. For the pharmacokinetic study of metolazone formulation in humans, an analytical method with simplicity and high sensitivity was required in our laboratory. Various methods have been reported for the determination of metolazone in biological samples, which involved high-performance liquid chromatographic method [3], [4], liquid chromatography with fluorescence detection [5] and liquid chromatography–tandem mass spectrometry (LC–MS–MS) [6]. Taking into consideration the low levels of metolazone in human blood, LC–MS–MS method is the first choice for our purpose. But the published LC–MS–MS method [6] addressed the qualitative analysis of a metolazone metabolite, not the quantitative determination of metolazone. This paper describes a simple, specific and highly sensitive LC–MS–MS method with an electrospray ionization (ESI) source in selected reaction monitoring (SRM) mode for the determination of metolazone in human blood. The described method was validated in terms of matrix effect, selectivity, sensitivity, linearity, accuracy, precision and stability of analyte in human blood, and successfully applied to the pharmacokinetic studies of metolazone formulation in humans.

Section snippets

Chemicals and reagents

Metolazone (purity, 99.0%) and tablets (2.5 mg, batch no. 20051124) were supplied by Tianjin Institute of Pharmaceutical Research (Tianjin, China). Lorazepam (purity, 99.5%, internal standard) was also supplied by Tianjin Institute of Pharmaceutical Research. HPLC-grade acetonitrile and formic acid were purchased from Tianjin Concord Tech Reagent Company (Tianjin, China). Ammonium acetate, analytical-grade, was purchased from Tianjin Chemical Reagent Co. Ltd. (Tianjin, China). Water was purified

Method development

Liquid–liquid extraction (LLE) was used for the sample preparation in this work. LLE can be helpful in producing a clean sample and avoiding the introduction of non-volatile materials onto the column and MS system. Clean samples are essential for minimizing ion suppression and matrix effect in LC–MS–MS analyses. Two organic solvents, ethyl acetate and chloroform were evaluated. Finally, ethyl acetate was found to be optimal, which can produce a clean chromatogram for a blank blood sample and

Conclusions

A rapid, sensitive and accurate liquid chromatography with electrospray ionization tandem mass spectrometry was developed for the determination of metolazone in human blood. The method was successfully applied to the pharmacokinetic studies of metolazone formulation in humans.

Acknowledgement

This work was supported by the National High Technology Program (863 program) of China (grant number 2003AA2Z347D).

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