Development of 2D Chiral Chromatography with Accelerator Mass Spectrometry for Quantification of ¹⁴ C-Labeled R - and S -Verapamil in Plasma

Abstract

Background: A microdose study was performed where 50 μg R/S- 14C-verapamil was dosed intravenously to human volunteers. In order to quantify the individual R- and S-enantiomers in human plasma a 2D chiral HPLC method with subsequent analysis by accelerator mass spectrometry was verified. Results: R/S-verapamil was separated on a C18 column and the isolated fraction was applied to a chiral column where the verapamil enantiomers were separated. Experimental recovery (�73 coefficient of variation {CV} = 16% and 66% CV = 21% for R- and S-verapamil, respectively) was accounted for by the use of internal standardization from the fluorescence response of nonlabeled R- and S-verapamil. The precision of the assay ranged from 4.1 to 15.9% CV and the limit of quantitation was 1.95-4.81 pg/ml for R-verapamil and 1.76-3.34 pg/ml for S-verapamil. Conclusion: This method was successfully applied to the analysis of R- and S-verapamil in human plasma. © 2010 Future Science Ltd

A Combined Accelerator Mass Spectrometry-Positron Emission Tomography Human Microdose Study with 14C- and 11C-Labelled Verapamil

“…The mass concentration of verapamil enantiomers in plasma were determined from the 14 C-content measured by AMS relative to the UV peak area (used as an internal standard). The accuracy of this assay, as previously reported [18] was assessed using quality control samples and the results were within 3% of the true value (13 and 2% coefficient of variation for ( R )- and ( S )-verapamil respectively). The limit of quantification of the assay was 1.95-4.81 pg/mL for ( R )-verapamil and 1.76-3.34 pg/mL for ( S )-verapamil.…”

“…For formulation, 0.05 mg of racemic verapamil (Isoptin®, 5-mg vials, Abbott, Vienna, 20 μL) was mixed with ( R / S )-[ 14 C]verapamil dissolved in ethanol (200 μL) and ( R )-[ 11 C]verapamil dissolved in physiological saline solution/ethanol (9/1, v/v, 3-6 mL) and the mixture filtered through a sterile Millex-GV filter (0.22 μm) (Millipore, Bedford, Mass, USA) into a sterile vial. Prior to iv injection, one aliquot (0.4 mL) was removed from each dose preparation for determining the concentrations of ( R )- and ( S )-verapamil by chiral high-performance liquid chromatography (HPLC) as described previously [18] and for determining the administered 14 C-amount by liquid scintillation counting. For iv injection, the final volume of each dose preparation was adjusted to 10 mL with physiological saline solution.…”

“…AMS analysis of ( R )- and ( S )-[ 14 C]verapamil was performed as described previously. [18] In brief, to each plasma sample an excess and known amount of unlabelled verapamil was added and the samples extracted into solvent. Aliquots of the extracts were injected onto a C18 HPLC system and verapamil collected as a discrete fraction.…”