Yongxin Zhu, Hwa Jiang, Rita Hilt, Phil Wong, Candice B. Kissinger and Peter T. Kissinger
Bioanalytical Systems, Inc., 2701 Kent Avenue, West Lafayette, IN 47906
Carbamazepine (CBZ) (5-H-dibenz[b,f]azepine-5-carboxamide) is an anticomvulsant used in clinical practice as first-line treatment for generalised tonic-clonic and partial seizures . Over the last two decades, thirty-three metabolites of CBZ have been isolated and identified in the urine from patients on an oral dose . Of these metabolites, carbamazepine 10,11-epoxide (CBZ-E) is the most important one from a clinical point of view. CBZ-E is pharmacologically as active as the parent compound in experimental animals. Figure 1 shows the structures of CBZ, CBZ-E and D -carbamazepine. A fast, sensitive and specific LC/MS/MS method for the simultaneous determination of CBZ and its metabolite CBZ-E in rat plasma is described. After administration of CBZ, blood samples were periodically collected from awake, freely moving animals by a BASi Culex ® automated blood sampler. The PK parameters of CBZ and CBZ-E were evaluated. This study has demonstrated that BASi Culex ® automated blood sampling system provided a powerful tool for unattended pharmacokinetic and pharmacodynamic studies involving low stress freely moving rats.
F1 Structures of carbamazepine and related compounds
Male Sprague-Dawley rats weighting 300-400 g were used for the study. For automatic sampling, rats were implanted with a jugular vein catheter (CX-2010, BAS,West Lafayette, IN, USA) and/or femoral vein catheter (CX-2020, BAS). After surgery, the rats were installed in the Raturn™ BASi and allowed to recover for one day with free access to food and water. The rats were fasted overnight before dosing orally with CBZ at a dose of 5 mg/kg. A volume of 0.25 mL blood sample was withdrawn from the jugular vein into a vial, containing heparine and kept in a refrigerated fraction collector according to a preset schedule in BASi Culex ® BASi automated blood sampling system. For manual sampling, rats were restrained in a restraint box and the blood samples were collected via the tail vein. Figures 2 and 3 illustrate the automated blood sampling and manual sampling system. The analytical method consists of a liquid-liquid extraction procedure and electrospray LC/MS/MS analysis The samples were then analyzed off-line by reverse phase liquid chromatography electrospray tandem mass spectrometry. The LC/MS/MS system was equipped with a BASi PM-80 pump coupled to a Finnigan LCQ Deca ion trap mass spectrometer (ThermoQuest, San Jose, CA, USA). The mass spectrometer was operated in ESI positive ion mode. The column was a Supecol Discovery C8, 5 mm, 150 x 2.1 mm with. A mobile phase containing 0.5% acetic acid and 30% acetonitrile at a flow-rate of 0.4 mL/min. D - Carbamazepine (D10-CBZ) is used as the internal standard for all compounds.
F2 BASi Culex ® Automated Blood Sampling System
F3 Manual sampling system
T1 Positive product ion mass parameters of CBZ, CBZ-E and D10-CBZ
Quantitation was conducted using the selected reaction monitoring (SRM) mode. Table 1 summarizes the product ion spectra of the two analytes and D -carbamazepine. The best sensitivities and minimum interferences were achieved by monitoring the transitions stated in Table 1. The chromatographic separation was achieved within 5 min using a C8 (150 x 2.1 mm) 5 mm column with a mobile phase containing 0.5% acetic acid and 30% acetonitrile at a flow- rate of 0.4 mL/min. Figures 4, 5 and 6 show typical chromatograms of an extracted drug-free rat plasma blank, plasma spiked with all analytes at 5 ng/mL (LLOQ) and plasma spiked with all analytes at 500 ng/mL, respectively.
F4 Representative chromatogram of extracted blank rat plasma
Representative chromatogram of two analytes from an extracted rat plasma
LLOQ (5ng/mL) sample
Representative chromatogram of two analytes from an extracted rat plasma
No endogenous rat plasma components were observed at the retention times corresponding to all two analytes and D10-cabamazepine (internal standard). The lower limit of quantitation (LLOQ) is 5 ng/mL for each analyte, based on 0.1 mL aliquots of rat plasma. The extraction recovery of analytes from rat plasma was over 87%. Linearity is observed over the range of 5-2000 ng/mL. The intra- and inter-day accuracy and precision values for QC samples are present in Table 2.
T2 Accuracy and precision for CBZ and CBZ-E from rat plasma
The precision values (coefficient of variation) at the three concentrations in the intraassay study varied between 2.6 and 5.2% for CBZ and 4.9 and 9.5% for CBZ-E while that of inter-assay study varied between 4.0 and 4.7% for CBZ and 8.7 and 9.6% for CBZ-E. The accuracy (relative error) values for all three concentrations deviated less than 4.7% FOR CBZ and 5.2% for CBZ-E from the corresponding nominal concentrations. This method has been used for the pharmacokinetic study of CBZ and CBZ-E.
Culex® automated blood sampler is designed to collect whole blood, urine and faces over a long period of time from awake, freely-moving animals like rats, dogs and primates as shown in Fig. 2 for a BASi Culex ® system. Each unit can house four rats. The BASi Culex ® system collects blood in sealed, refrigerated vials, which are transferable to a 96-well plate for automated sample preparation. It provides simultaneously automated serial blood sampling, behavior monitoring and microdialysis from a rodent metabolism cage for PK, PD and CNS effect studies .
After giving a single oral dose of 5 mg/kg of CBZ, the rat was sampled by an automated blood sampling system or by traditional manual draws. The plasma was collected at specific time points for the determination of the analytes. The plasma concentration-time plot of CBZ and CBZ-E for both automatic and manual sampling is shown in Figure 7.
The plasma concentration-time profiles of CBZ and CBZ-E in rat following a single 5 mg/kg oral administration
by BASi Culex ® and manual sampling
It has been recognized that stress of conventional sampling from awake rats drastically influences rodent physiology (blood pressure, blood flow) and biochemistry (stress home release, metabolism, even protein expression) . Automated blood sampling permits awake cannulated animals to be sampled stress free (without restraint and human handling). The latter situation provides the most consistent and meaningful data for PK studies. Table 3 shows PK parameters of CBZ and its epoxide metabolite in plasma from the same rat using two different blood sampling methods. It can be seen clearly that when PK data are obtained from an animal under stress, the normal parameters of Cmax, Tmax, AUC and CL can all vary very substantially.
T3 PK parameters of carbamazepine and its epoxide metabolite in plasma from the same rat using two different sampling methods
LC/MS/MS with automated blood sampling system has been proven to be powerful for the pharmacokinetic study. The current studies demonstrate the significant difference in PK results between automated blood sampling and manual withdraw, with more consistent and meaningful data for the automated blood sampling system and the predicted stress influences on the traditional manual approach.
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