Enantioselective determination of azelnidipine in human plasma using liquid chromatography–tandem mass spectrometry

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Abstract

A sensitive and simple method was developed for determination of the enantiomers of azelnidipine, (R)-(−)-azelnidipine and (S)-(+)-azelnidipine, in human plasma using chiral liquid chromatography with positive ion atmospheric pressure chemical ionization tandem mass spectrometry. Plasma samples spiked with stable isotope-labeled azelnidipine, [2H6]-azelnidipine, as an internal standard, were processed for analysis using a solid-phase extraction in a 96-well plate format. The azelnidipine enantiomers were separated on a chiral column containing α1-acid glycoprotein as a chiral selector under isocratic mobile phase conditions. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode, monitoring the transitions from m/z 583  167 for (R)-(−)-azelnidipine and (S)-(+)-azelnidipine, and from m/z 589  167 for [2H6]-azelnidipine. The standard curve was linear over the studied range (0.05–20 ng/mL), with r2 > 0.997 using weighted (1/x2) quadratic regression, and the chromatographic run time was 5.0 min/injection. The intra- and inter-assay precision (coefficient of variation), calculated from the assay data of the quality control samples, was 1.2–8.2% and 2.4–5.8% for (R)-(−)-azelnidipine and (S)-(+)-azelnidipine, respectively. The accuracy was 101.2–117.0% for (R)-(−)-azelnidipine and 100.0–107.0% for (S)-(+)-azelnidipine. The overall recoveries for (R)-(−)-azelnidipine and (S)-(+)-azelnidipine were 71.4–79.7% and 71.7–84.2%, respectively. The lower limit of quantification for both enantiomers was 0.05 ng/mL using 1.0 mL of plasma. All the analytes showed acceptable short-term, long-term, auto-sampler and stock solution stability. Furthermore, the method described above was used to separately measure the concentrations of the azelnidipine enantiomers in plasma samples collected from healthy subjects who had received a single oral dose of 16 mg of azelnidipine.

Introduction

Azelnidipine, (±)-(3)-(1-diphenylmethylazetidin-3-yl)-5-isopropyl-2-amino-1, 4-dihydro-6-methyl-4-(3-nitrophenyl)-3, 5–pyridinedicarboxylate, is a new dihydropyridine derivative with calcium channel antagonistic activity. Azelnidipine has two enantiomers due to an asymmetric carbon at the 4-position of the 1, 4-dihydropyridine ring (Fig. 1). In 2003, this drug was launched into the market as CALBLOCK ® in Japan. It is generally accepted that the (R)-(−) enantiomers of dihydropyridine calcium blockers possess intrinsic pharmacological activity [1], and it is therefore desirable to perform selective measurements of the enantiomers in their plasma concentrations. Previously, we have reported the plasma concentrations of azelnidipine and the metabolites following an oral administration of azelnidipine to healthy volunteers [2], while stereoselective measurement of the plasma levels of R-(−)- and S-(+)-enantiomers of azelnidipine has not yet been performed. Historically, numerous methods have been established for the quantitative determination of dihydropyridine calcium blockers as racemate in plasma, including high-performance liquid chromatography (HPLC) or gas chromatography coupled with mass spectrometry (HPLC or GC–MS) [3], [4], [5], [6], [7], [8], [9] or HPLC coupled with tandem mass spectrometry (LC–MS/MS) [10], and these were followed subsequently by the development of an assay of their enantiomers as in the case of amlodipine, felodipine and benidipine in human plasma, determined by LC–MS/MS using a chiral separation column [11], [12], [13].

In this study, we developed an ultrasenstive method by liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC/APCI–MS/MS) for determination of (R)-(−)-azelnidipine and (S)-(+)-azelnidipine in human plasma, using deutellium-labeled azelnidipine ([2H6]-azelnidipine) as an internal standard and a chiral column containing α1-acid glycoprotein as a stationary phase for chiral separation. Briefly, plasma samples were spiked with azelnidipine and [2H6]-azelnidipine, extracted by a solid-phase extraction using a 96-well plate format, and the plasma extracts were chromatographed on an HPLC system with the chiral column. Following separation, the (R)-(−)-azelnidipine, (S)-(+)-azelnidipine and the internal standard were selectively detected using APCI–MS/MS. The method has a lower limit of quantification (LLOQ) of 0.05 ng/mL for (R)-(−)-azelnidipine and (S)-(+)-azelnidipine, using a plasma sample volume of 1.0 mL. The LC/APCI–MS/MS method has a relatively short run time of 5 min/sample for high throughput. To the best of our knowledge, no quantitative LC/APCI–MS/MS methods suitable for the routine analysis of (R)-(−)- and (S)-(+)-isomers of azelnidipine have been reported yet.

This assay method was successfully applied to measurements of plasma concentrations of (R)-(−)-azelnidipine and (S)-(+)-azelnidipine after a single oral administration of 16 mg of azelnidipine (CALBLOCK® Tablets, 2 × 8 mg) to healthy male volunteers.

Section snippets

Standard substances, reagents and blank plasma

(R)-(−)-azelnidipine and (S)-(+)-azelnidipine (molecular weight 582.6), and the internal standard substance, [2H6]-azelnidipine (molecular weight 588.6), were synthesized by the Pharmaceutical Research Department of Ube Laboratory, Ube Industries Ltd., (Yamaguchi, Japan). Methanol of HPLC grade and formic acid, ammonium acetate and hydrochloric acid of reagent grade were obtained from Wako Pure Chemical Industries Co. Ltd., (Osaka, Japan). Oasis® HLB 96-well plates were from Waters Corporation

Separation of (R)-(−)-azelnidipine and (S)-(+)-azelnidipine by chiral column

In separating the enantiomers by HPLC equipped with a chiral column containing α1 acid glycoprotein, the proportion of the organic modifiers (generally methanol), the buffer concentration (generally ammonium acetate), and the buffer pH in the mobile phase strongly influence the retention time and enantioselectivity in HPLC, as well as the intensity in the subsequent mass spectrometric detection [15], [16]. Regarding the proportion of organic solvent in the mobile phase, we used 50% methanol in

Conclusion

An LC–MS/MS method for the quantification of (R)-(−)-azelnidipine and (S)-(+)-azelnidipine in human plasma was developed and fully validated according to the FDA guidance. The combination of techniques employed, including solid-phase extraction, use of stable isotope-labeled internal standard, and chiral LC and MS/MS, resulted in a robust, ultrasensitive, accurate and precise method for the determination of the azelnidipine enantiomers in human plasma at concentrations from 0.05 to 20 ng/mL. The

Acknowledgements

We would like to thank Dr. Toshihiko Ikeda of the Drug Metabolism and Pharmacokinetics Research Laboratories, Sankyo Co. Ltd. for his collaboration in this study.

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