A sensitive and rapid ultra HPLC-MS/MS method for the simultaneous detection of clopidogrel and its derivatized active thiol metabolite in human plasma

Abstract

A sensitive, selective, and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (uHPLC-MS/MS) was developed for the simultaneous quantification of clopidogrel (Plavix(®)) and its derivatized active metabolite (CAMD) in human plasma. Derivatization of the active metabolite in blood with 2-bromo-3'-methoxy acetophenone (MPB) immediately after collection ensured metabolite stability during sample handling and storage. Following addition of ticlopidine as an internal standard and simple protein precipitation, the analytes were separated on a Waters Acquity UPLC™ sub-2 μm-C(18) column via gradient elution before detection on a triple-quadrupole MS with multiple-reaction-monitoring via electrospray ionization. The method was validated across the clinically relevant concentration range of 0.01-50 ng/mL for parent clopidogrel and 0.1-150 ng/mL (r(2)=0.99) for CAMD, with a fast run time of 1.5 min to support pharmacokinetic studies using 75, 150, or 300 mg oral doses of clopidogrel. The analytical method measured concentrations of clopidogrel and CAMD with accuracy (%DEV) <±12% and precision (%CV) of <±6%. The method was successfully applied to measure the plasma concentrations of clopidogrel and CAMD in three subjects administered single oral doses of 75, 150, and 300 mg clopidogrel. It was further demonstrated that the derivatizing agent (MPB) does not affect clopidogrel levels, thus from one aliquot of blood drawn clinically, this method can simultaneously quantify both clopidogrel and CAMD with sensitivity in the picogram per mL range.

Figure 1. Formation of MPB-Derivatized Clopidogrel Active Metabolite

Clopidogrel is oxidized to the inactive intermediate 2-oxo-clopidogrel, before subsequent further oxidation to the free thiol-containing active metabolite. Due to its reactivity, the active metabolite is added to 2-bromo-3’-methoxyphenone (MPB) to derivatize the thiol to provide stability for more accurate quantification.

Figure 2. Chromatogram of A) blank plasma extract, B) mid quality control sample, and C) clinical sample

Panel A represents a blank plasma extract, Panel B represents a mid quality control sample (MQC), and Panel C represents a clinical sample. There are three separate LC-MS/MS chromatographic tracings within each panel resulting from three simultaneous multiple reaction monitoring (MRM) transitions. The bottom pane represents the internal standard ticlopidine (m/z 264>154); the middle pane represents CAMD (m/z 504>155); and the top pane represents clopidogrel (m/z 322>212). The racemic CAMD reference standard produces a broad peak (B), due to the presence of both stereoisomers. Clinical samples demonstrate the stereoselective metabolism, preferentially forming one stereoisomer as evident by the relatively sharper peak shape.

Figure 2. Chromatogram of A) blank plasma extract, B) mid quality control sample, and C) clinical sample

Panel A represents a blank plasma extract, Panel B represents a mid quality control sample (MQC), and Panel C represents a clinical sample. There are three separate LC-MS/MS chromatographic tracings within each panel resulting from three simultaneous multiple reaction monitoring (MRM) transitions. The bottom pane represents the internal standard ticlopidine (m/z 264>154); the middle pane represents CAMD (m/z 504>155); and the top pane represents clopidogrel (m/z 322>212). The racemic CAMD reference standard produces a broad peak (B), due to the presence of both stereoisomers. Clinical samples demonstrate the stereoselective metabolism, preferentially forming one stereoisomer as evident by the relatively sharper peak shape.

Figure 2. Chromatogram of A) blank plasma extract, B) mid quality control sample, and C) clinical sample

Panel A represents a blank plasma extract, Panel B represents a mid quality control sample (MQC), and Panel C represents a clinical sample. There are three separate LC-MS/MS chromatographic tracings within each panel resulting from three simultaneous multiple reaction monitoring (MRM) transitions. The bottom pane represents the internal standard ticlopidine (m/z 264>154); the middle pane represents CAMD (m/z 504>155); and the top pane represents clopidogrel (m/z 322>212). The racemic CAMD reference standard produces a broad peak (B), due to the presence of both stereoisomers. Clinical samples demonstrate the stereoselective metabolism, preferentially forming one stereoisomer as evident by the relatively sharper peak shape.

Figure 3. Clinical Concentration-Time Curves over 3 dose levels for A) Clopidogrel, and B) Clopidogrel Active Metabolite-Derivatized (CAMD)

Three subjects were administered clopidogrel orally at three dose levels: 75 mg, 150 mg, and 300 mg. Two separate aliquots of blood were drawn from the subjects at the following time points: pre-dose, 0.25, 0.50, 1, 2, and 4 hours post dose. The first aliquot of blood was drawn into an EDTA tube for analysis of parent clopidogrel. The second aliquot of blood was drawn into an EDTA tube pretreated with 30 µL of 500 mM of MPB to immediately derivatize the clopidogrel active metabolite for accurate analyses.