Abstracts

Rationale: The Bioequivalence in Epileptic Patients (BEEP) study at the University of Maryland School of Pharmacy has been initiated to address neurologists' and epileptic patients' concerns about the bioequivalence of generic lamotrigine to Lamictal. This bioequivalence study requires the quantification of lamotrigine in human plasma, including in the presence of other drugs, as this study employs epileptic patients rather than healthy volunteers. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the bioanalytical method of choice for quantification of analytes in complex matrices. An essential aspect of method validation is the assessment of the influence of concomitant drugs and plasma components due to the possibility of ion suppression/enhancement. The objective was to develop and validate a LC-MS/MS method to measure the concentration of lamotrigine in patient plasma. A range of analytical conditions, as well as a backflush step to reduce matrix effects, were examined. Methods: Lamotrigine was extracted from plasma through protein precipitation with acetonitrile. The supernatant of each sample was diluted in water before injection. Lamotrigine-13C3-d3, a stable isotope-labeled compound, was used as an internal standard. Samples were run utilizing a two pump system in a LC-MS/MS. The method was validated for accuracy, precision, inter- and intraday coefficient of variation, specificity, limit of detection (LOD), limit of quantitation (LOQ), linearity, range, and instrument precision. Specificity considered interference of other anti-epileptic drugs (AEDs), including levetiracetam, topiramate, phenytoin, and carbamazepine. Freeze-thaw, dilution, and sample stability tests were also performed. All calculations were based on the peak area response ratio of lamotrigine to lamotrigine-13C3-d3. Results: A backflush step reduced matrix effects. A final method was validated for a linear range of 8 to 10,000 ng/mL of lamotrigine (r2 = 0.9999). Tests were ran at various concentration levels, including LOQ at 8 ng/mL, 2.5 x LOQ at 20 ng/mL, low quality control (QC) sample at 50 ng/mL, medium QC at 500 ng/mL, and high QC at 5000 ng/mL. LOD was determined to be 1 ng/mL, with interday and intraday results less than 10.0% and 10.4%, respectively (range: 20-5000 ng/mL). The percent recovery varied from 96.6 to 109.3% (range: 20-5000 ng/mL), with no interference peak from plasma or other AEDs found. The freeze-thaw and dilution results were also similar to the standard curve. The only elevated stability result was the short-term temperature test, with peak areas increased by 7.0 to 19.5% (range: 50-5000 ng/mL). Conclusions: An LC-MS/MS method was developed and fully validated to measure lamotrigine concentration in patient plasma. A backflush step allowed for consistent results and reduced matrix effects, affording a simple precipitation and dilution procedure to determine lamotrigine in patient plasma. The methods will be applied in the BEEP study to compare brand versus generic lamotrigine.