Determination of potato carboxypeptidase inhibitor in African Green Monkey plasma using 96-well SPE and LC-MS/MS

Potato carboxypeptidase inhibitor (CPI), a peptide with multiple isoforms (MW > 4000 Da) was determined from African Green Monkey plasma using a PE Sciex API-3000 LC-MS/MS in the positive ionization mode with the turbo ionspray interface (450 °C). Samples were prepared using an Oasis MCX 96-well solid phase extraction plate and chromatographed on an Allure C18 HPLC Column (50 mm × 1.0 mm, 5 μm) using gradient elution. Upon analysis of the extracts using LC-MS/MS, the concentration of CPI was calculated using a single MS/MS transition (m/z 830.5 → 221.0) that was reflective of the mass concentration (μg/mL) of main the CPI isoforms present in plasma from monkeys after they were given an intravenous dose of CPI. The assay was linear for CPI over concentrations of 0.05-10 μg/mL when extracting 200-μL aliquots of African Green Monkey plasma. The assay was applied to the determination of CPI in African Green Monkey plasma samples in two separate analytical runs (correlation of standard curves, r1 = 0.9991 and r2 = 0.9953). Quality control (QC) samples were run at 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 μg/mL for each assay. Average ranges (n = 12) for accuracy and precision for all concentrations of QCs during the two runs were 92.0-102.0% of expected potency and 10.4-21.8% (coefficient of variations), respectively.