TuP-05



THE MEASUREMENT OF CAPTOPRIL IN HUMAN PLASMA USING GC-MS WITH SELECTED ION MONITORING AND ITS APPLICATION TO SINGLE DOSE PHARMACOKINETIC STUDIES

ME Franklin, RS Addison, PV Baker and WD Hooper

Department of Medicine, University of Queensland, Royal Brisbane Hospital, Brisbane, QLD, 4029



A robust assay for the highly specific ACE-inhibitor, captopril, was required for the analysis of large batches of plasma samples from volunteers in a bioequivalence study. Captopril is unstable in blood and plasma and requires the addition of a suitable fixative immediately upon sample collection. The addition of the water soluble fixative, N-ethylmaleimide (NEM) to form stable N-ethylsuccinimide analogues has been used for GC methods, prior to esterification of the carboxylic acid moiety by methylation, pentafluorobenzylation or hexafluoroisopropylation. However, the final esterified derivatives generally result in more than one peak making quantitation difficult. Ito et al.1 developed a GC-MS assay using derivatisation of captopril to its bis-pentafluorobenzyl (PFB) derivative where the NEM moiety is displaced, leading to a single chromatographic peak. We have modified this assay to use powdered NEM in the collection tubes, more rapid extraction, a commercially available internal standard and a capillary GC column.

Plasma samples (1 mL) containing internal standard (thiosalicylic acid, 1 µg) were acidified and extracted with dichloromethane (5 mL). The drug was then back extracted into 5% NaHCO3 (2 mL) followed by reacidification and reextraction into dichloromethane (2 mL). This extract was evaporated to dryness and the residue derivatised by the addition of pentafluorobenzylbromide (2% in acetone, 100 µL) in the presence of base. Following incubation at 60°C for 1 hour, the derivatising reagents were evaporated and the product dissolved in chloroform (200 µL). The derivatives were resolved on a 25 m length of 0. 2 mm ID HP-5 capillary column within 10 min following on-column injection. The HP 5971A mass spectrometer was tuned using the maximum sensitivity option and two fragment ions were monitored for both the captopril derivative (m/z 294, m/z 396) and the thiosalicylic acid derivative (m/z 333), m/z 514). The assay was linear from 10 to 5000 ng/mL with a captopril recovery of 77%. Assay precision at mean values of 45.9, 187 and 980 ng/mL were 4.0, 2.9 and 3.5 % respectively while accuracy was 8.2, 6.5 and 3.1 % across the assay range. Analysis of plasma samples from 20 volunteers following administration of 100 mg of captopril provided the following pharmacokinetic data (mean ± sd): Cmax, 1470 ± 467 ng/mL; AUC 0-inf., 1736 ± 481 ng/mL.h; Tmax 0.73 h; t1/2, 1.58 ± 0.41 h. We conclude that this assay is precise, accurate and robust and suitable for high throughput analysis of large batches of plasma samples such as those encountered in bioequivalence studies.

  1. Ito T, Matsuki Y, Kurihara H and Nambara T. (1 987) J Chromatography, 417,79 - 87.