The development and validation of assay methods for the quantitative determination of drugs and their metabolites in biological specimens: clarithromycin, carbamazepine and carbamazepine-10, 11-epoxide
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Date
2003-05
Authors
Van Rooyen, Gert Frederick
Journal Title
Journal ISSN
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Publisher
University of the Free State
Abstract
The projects described in this dissertation use cutting edge technology to quantitate various drugs in plasma. Highly
sensitive assay methods for the quantification of clarithromycin, carbamazepine and its major metabolite,
carbamazepine-l0, Il-epoxide in human plasma were developed and validated. The application of these methods to
analyse samples generated during pharmacokinetic studies is also discussed where up to 230 samples were analysed per
day. The methods were sensitive enough to quantitate the analytes for at least 5 half-lives of the drug after an oral dose
to human volunteers.
A sensitive method for the determination of clarithromycin, a macrolide antibiotic, USing high-performance liquid
chromatographic separation with tandem mass spectrometry, and the method development thereof, are described in this
dissertation. Samples were prepared using liquid-liquid extraction and were separated on a CI8 column with a mobile
phase consisting of acetonitrile, methanol and acetic acid. Detection was performed by a mass speetrometer in the
multiple reaction monitoring (MRM) mode, using TurboIonSpray ionisation. The mean recovery of clarithromycin was
87.3%, with a lower limit of quantification of 2.95 ng/ml when using 300f!1 plasma. This high-throughput method was
used to quantify 230 samples per day, and is sufficiently sensitive to be employed in pharmacokinetic studies.
Carbamazepine, an anticonvulsant, and its major metabolite, carbamazepine-10, l l-epĆ³xide, were quantified using
tandem liquid chromatography-mass spectrometry, with electrospray ionisation. Both these analytes were extracted
from 500f!1 of human plasma, using a liquid-liquid extraction method that showed recoveries greater than 95% for both
these analytes. Concentrations as low as 0.722 ng/ml for carbamazepine and 5.15 ng/ml for carbamazepine-10, 11-
epoxide could be quantified by using this method.
These are the first LC-MSIMS assay methods described for the quantification of carbamazepine and its metabolite,
carbamazepine-10, ll-epoxideas well as clarithromycin and their applications to pharmacokinetic studies.
Description
Keywords
Method development, Validation, Bioequivalence, High-performance liquid, Chromatography, Plasma, Mass spectrometry, Carbamazepine, 11-epoxide, Clarithromycin, Carbarnazepine-10, Clarithromycin, Macrolide antibiotics, Biological assay, Drugs -- Analysis, Dissertation (M.Med.Sc. (Pharmacology))--University of the Free State, 2003