A novel liquid chromatography–mass spectrometry (LC-MS) method was developed and validated for quantification of topiramate (TPM) and its metabolites 10-hydroxy topiramate (10-OH-TPM), 9-hydroxy topiramate (9-OH-TPM), and 4,5-O-desisopropylidene topiramate (4,5-diol-TPM) in plasma and urine. The method uses 0.5 mL of plasma or 1 mL of urine that is extracted with diethyl ether and analyzed by LC-MS. Positive ion mode detection enables tandem mass spectrometric (MS/MS) identification of the aforementioned four compounds. Calibration curves of TPM, 4,5-diol-TPM, 9-OH-TPM, and 10-OH-TPM in plasma and urine were prepared and validated over the concentration range of 0.625 to 40 μg/mL using TPM-d12 as an internal standard. Calibration curves were linear over this concentration range for TPM and its metabolites. Accuracy and precision ranged in urine from 83% to 114% and 4% to 13% (%CV), respectively, and in plasma from 82% to 108% and 6% to 13%, respectively. The applicability of the assay was evaluated by analyzing plasma samples from a healthy subject who received a single oral dose of TPM (200 mg) and urine samples from 11 patients with epilepsy treated with TPM (daily dose between 100 to 600 mg) alone or with other antiepileptic drugs. Only TPM was detected and quantified in the plasma samples, and its concentration ranged between 0.7 and 4.3 μg/mL. The concentrations of TPM and 10-OH TPM were quantifiable in all urine samples and ranged from 20 to 300 μg/mL for TPM and from 1 to 50 μg/mL for 10-OH-TPM. The metabolites 4,5-diol-TPM and 9-OH-TPM were also detected in all urine samples, but their concentrations were quantifiable only in 4 patients. An unidentified peak in the chromatograms obtained from patients' urine was attributed to 2,3-O-desisopropylidene topiramate (2,3-diol-TPM). Due to a lack of reference material of 2,3-diol TPM and the similar MS/MS spectrum with 4,5-diol-TPM, the calibration curves of 4,5-diol-TPM were used for the quantification of its isomer 2,3-diol-TPM. Based on these determinations, the apparent 2,3-diol-TPM-to-TPM concentration ratio in patients' urine ranged from 0.05 to 0.51 and the 10-OH-TPM-to-TPM ratio ranged from 0.02 to 0.17. In conclusion, a novel LC-MS method for the assay of TPM and four of its metabolites in plasma and urine was developed. Its utilization for analysis of urine samples from patients with epilepsy showed that the method was suitable for analysis of TPM and its metabolites in clinical samples. Two quantitatively significant TPM metabolites (10-OH-TPM and 2,3-diol-TPM) and two quantitatively minor metabolites (9-OH-TPM and 4,5-diol-TPM) were detected and quantified in urine samples from patients with epilepsy.
Topiramate (TPM;Fig. 1) is a new antiepileptic drug (AED) that has been approved worldwide in more than 100 countries as adjunctive therapy for refractory partial onset seizures. Additional indications approved in many countries include adjunctive therapy for primary generalized tonic–clonic seizures and Lennox-Gastaut syndrome and monotherapy for refractory partial onset seizures and newly diagnosed epilepsy in adults (1–4). It is currently being evaluated for its effect in various neurologic and psychiatric disorders.
TPM is characterized by linear pharmacokinetics over the clinically used dose range, a low oral clearance (22–36 mL/min) that in monotherapy is predominantly through renal excretion (renal clearance, 10–20 mL/min), and a long half-life (19–25 hours), which is decreased by coadministration of enzyme-inducing AEDs such as phenytoin (PHT) and carbamazepine (CBZ) (4–9). The absolute bioavailability or oral availability of TPM is 81% to 95% and is not affected by food (6). The clinically relevant TPM plasma concentration range has been found to extend up to 33 μg/L (4). Although TPM is not extensively metabolized when administered as monotherapy, with a fraction metabolized (fm) ∼20% (4,10), its metabolism to various inactive metabolites (Fig. 1) is induced during polytherapy with CBZ and PHT, and its fm increases appreciably (5,8,9,10). During concomitant treatment with CBZ or PHT, TPM oral clearance increases twofold, and its half-life decreases by approximately 50%. These changes may require TPM dose adjustment when PHT or CBZ therapy is added or discontinued (5,8,9). Such an increase in total body clearance implies that the formation clearance(s) of metabolite(s) through inducible pathways (cytochrome P450-mediated oxidation and glucuronide conjugation) may be increased by as much as 10-fold. Nevertheless, there is no information in the literature on quantitative contributions of individual metabolic pathways of TPM.
This lack of information may be due to lack of: 1) synthesized TPM metabolites that can be used as reference material (10,11), and 2) a sensitive and specific assay for the quantification of TPM metabolites in urine or plasma. Consequently, the aims of the current study were to (1) develop a sensitive and specific liquid chromatography–mass spectrometry (LC-MS) method for quantification of TPM and its metabolites in body fluids, and (2) apply this assay to the quantification of TPM and its metabolites in the plasma or urine of healthy subjects and patients with epilepsy.
*Department of Pharmaceutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel; †National Residue Control Laboratory, Kimron Veterinary Institute, Beit Dagan, Israel; ‡School of Pharmacy, University of Washington, Seattle, Washington; §Clinical Pharmacology Unit, Department of Internal Medicine and Therapeutics, University of Pavia, Pavia, Italy; ∥Johnson & Johnson Pharmaceutical Research and Development, L.L.C., Raritan, New Jersey and Spring House, Pennsylvania; #David R. Bloom Center for Pharmacy, Hebrew University of Jerusalem, Jerusalem, Israel
Received December 9, 2002; accepted January 17, 2003.
Supported by an unrestricted grant from Johnson & Johnson Pharmaceutical Research & Development, L.L.C., Raritan, New Jersey, and partially supported by an FAR 2001-2002 grant from The University of Pavia Fondi di Ateneo per la Ricerca.
This work is abstracted from the PhD thesis of Ms. Malka Britzi in partial fulfillment of the PhD degree requirements for The Hebrew University of Jerusalem.
Address correspondence and reprint requests to Professor Meir Bialer, Department of Pharmaceutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, P.O. Box 12065, Ein Karem, Jerusalem 91120, Israel; E-mail: email@example.com