Determination of Rofecoxib (MK-0966), a cyclooxygenase-2 inhibitor, in human plasma by high-performance liquid chromatography with tandem mass spectrometric detection

doi:10.1016/S0378-4347(99)00565-4

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 748, Issue 1, 1 October 2000, Pages 31-39

https://doi.org/10.1016/S0378-4347(99)00565-4 Get rights and content

Abstract

A method for the determination of 4-(4-methanesulfonylphenyl)-3-phenyl-5H-furan-2-one (Rofecoxib, Vioxx, MK-0966, I) a cyclooxygenase-2 inhibitor, in human plasma has been developed. The method is based on high-performance liquid chromatography (HPLC) with atmospheric pressure chemical ionization tandem mass spectrometric (APCI-MS–MS) detection in negative ionization mode using a heated nebulizer interface. Drug and internal standard (II) were isolated from basified plasma using liquid–liquid extraction. The organic extracts were dried, reconstituted in mobile phase and injected into the HPLC-MS–MS system. Compounds I and II were chromatographed on a narrow bore (100 mm×3.0 mm) C₁₈ analytical column, with mobile phase consisting of acetonitrile:water (1:1, v/v) at a flow-rate of 0.4 ml/min. The MS–MS detection was performed on a PE-Sciex API III Plus tandem mass spectrometer operated in selected reaction monitoring mode. The parent→product ion combinations of m/z 313→257 and 327→271 were used to quantify I and II, respectively, after chromatographic separation of the analytes. The assay was validated in the concentration range of 0.1 to 100 ng/ml of plasma. The precision of the assay (expressed as coefficient of variation) was less than 10% at all concentrations within the standard curve range, with adequate assay accuracy. The effect of HPLC mobile phase components on the ionization efficiency and sensitivity of detection in the positive and negative ionization modes, and the detailed description of all necessary steps involved in the assay for I in plasma are presented.

Introduction

Reduction of prostaglandin synthesis by direct inhibition of cyclooxygenase (COX, prostaglandin-endoperoxide synthase) is believed to be the mechanism of nonsteroidal antiinflammatory drugs [1]. There are two forms of COX, namely, COX-1 and COX-2. COX-1 is expressed in tissues and is involved in the physiological functions of prostaglandins, while COX-2 is inducible and is expressed in inflammatory cells and in kidney [2], [3], [4]. Hence, specific inhibitors of COX-2 are potential antiinflammatory agents which will not produce the toxicity associated with the inhibition of COX-1 derived prostaglandins. Compound I (4-(4-methanesulfonylphenyl)-3-phenyl-5H-furan-2-one, MK-0966) has been developed as a specific COX-2 inhibitor for treating acute pain and chronic inflammatory disorders without gastric side effects associated with the use of COX-1 inhibitors.

Concentrations of I in biological fluids from the majority of clinical pharmacokinetic studies with I were determined using highly sensitive high-performance liquid chromatographic (HPLC) methods based on postcolumn photochemical conversion of I to a substituted phenanthrene analog followed by fluorescence detection [5]. The selectivity, sensitivity and speed of analysis of this HPLC-fluorescence (FLU) method were comparable or even better than many typical HPLC-MS–MS assays. However, it was of interest to develop a HPLC-MS–MS method for I to serve as a back-up method for analyses of human plasma samples in cases when confirmatory data were needed. In addition, the availability of the HPLC-MS–MS method for unlabeled I could form a basis for the future development of simultaneous assays for determination of unlabeled and stable isotope labeled analogs of I that may be utilized in a number of mechanistic and bioavailability studies with I. Atmospheric pressure chemical ionization (APCI) HPLC-MS and HPLC-MS–MS techniques have proven to be of great use for both the identification and quantification of drugs and metabolites in biological fluids at very low concentrations. Some recent representative examples from our laboratories are listed in Refs. [6], [7], [8], [9], [10], [11]. In APCI, ionization occurs by proton/or electron transfer reactions and is mediated by the gas phase acidities/or basicities of the reagent ions and the compounds of interest. The effect of mobile phase components on sensitivity in APCI HPLC-MS has been recognized [12] and extensively investigated by Schaefer and Dixon [13]. Decrease in ionization efficiencies of I and internal standard (II) due to the presence of modifiers in the mobile phase was observed during the HPLC-MS–MS method development for these compounds. The proper choice of mobile phase and the type of ionization mode (positive or negative ionization modes) were found to be critical to the successful development of a sensitive assay for I and will be discussed in detail in this paper.

Section snippets

Materials and reagents

Rofecoxib (MK-0966, Vioxx, I) and internal standard (I.S., II) were synthesized at Merck (Rahway, NJ, USA). Ammonium acetate was purchased from Sigma (St. Louis, MO, USA). Formic acid was purchased from Aldrich (Milwaukee, WI, USA). All other chemicals were obtained from Fisher (Fair Lawn, NJ, USA). Control human plasma was obtained from Biological Specialties (Lansdale, PA, USA)

Instrumentation

The HPLC system consisted of a Perkin Elmer Biocompatible Binary pump 250, a WISP 715 Autoinjector (Waters-Millipore,

Effect of mobile phase modifiers on the ionization efficiency and sensitivity of detection

The Sciex mass spectrometer provides two ways for interfacing HPLC with MS: heated pneumatic nebulization and ISP or turbo ISP (TISP). Although ISP or TISP are widely used in quantitative analyses, the technique has some limitations, namely, a limited dynamic range [14], [15] and a higher probability of exhibiting a matrix effect [15], [16], [17], [18]. APCI using heated pneumatic nebulization has been widely used in quantitative analysis and is becoming even more popular because of its

Acknowledgements

The authors would like to thank Dr. Siu-Long Yao, clinical monitor, and Dr. Russell Dixon, clinical investigator of the clinical program from which the biological fluid samples were made available for analyses.

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Determination of Rofecoxib (MK-0966), a cyclooxygenase-2 inhibitor, in human plasma by high-performance liquid chromatography with tandem mass spectrometric detection

Abstract

Introduction

Section snippets

Materials and reagents

Instrumentation

Effect of mobile phase modifiers on the ionization efficiency and sensitivity of detection

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

J. Chromatogr. B.

J. Chromatogr. B.

J. Chromatogr. B.

J. Chromatogr. B.

J. Chromatogr. A

J. Pharm. Sci.

J. Am. Soc. Mass Spectrom.