Pharmacokinetic Analysis

The concentration of phenobarbital was measured in plasma by liquid chromatography tandem-mass spectrometry (LC-MS/MS) using negative electrospray ionization. Briefly, phenobarbital working solutions were prepared by dilution of the 1 mg/mL stock solution (d5 phenobarbital, Cerilliant, Round Rock, Texas, USA) with methanol to concentrations of 0.01, 0.1, 1, and 10 ng/μL. Plasma calibrators were prepared by dilution of the working standard solutions with drug free plasma to concentrations ranging from 0.2 to 30,000 ng/mL. Calibration curves and negative control samples were prepared fresh for each quantitative assay. In addition, quality control samples (plasma fortified with analyte at three concentrations within the standard curve) were included with each sample set as an additional check of accuracy. The response for phenobarbital was linear and produced correlation coefficients (R²) of ≥ 0.99. The precision and accuracy of the assay were determined by assaying quality control samples in replicates (n = 6) for phenobarbital. Accuracy was reported as percent nominal concentration and precision was reported as percent relative standard deviation. Accuracy for phenobarbital determination was 102% for 0.6 ng/mL, 98% for 40 ng/mL, and 95% for 400 ng/mL. Precision was 5% for 0.6 ng/mL, 2% for 40 ng/mL, and 4% for 400 ng/mL. The technique was optimized to provide a limit of quantitation of 0.2 ng/mL and a limit of detection of approximately 0.05 ng/mL for phenobarbital.