Rapid and sensitive quantification of pemetrexed in human plasma by LC-MS/MS using a stable isotope-Labeled internal standard for therapeutic drug monitoring.

OBJECTIVE To develop and validate a reliable LC-MS/MS method for quantifying pemetrexed in human plasma to investigate exposure-toxicity relationships.

METHODS Separation utilized an Agilent ZORBAX Eclipse Plus C18 column with a gradient of acidified water and acetonitrile over 5 min. Detection was by triple quadrupole MS with positive electrospray ionization, monitoring qualifier MRM transitions m/z 428.20 → 163 (pemetrexed) and 433.20 → 163 (internal standard, pemetrexed-13C5), and quantifier transitions m/z 428.20 → 280.90 and 433.20 → 280.90 . The method was applied to therapeutic drug monitoring in a renal-impaired patient.

RESULTS The method was linear from 10 to 1000 ng/mL (R2 = 0.9992). Accuracy ranged from 103.67% to 108.83%, with precision (RSD) <15%. The extraction recovery ranged from 94.35% to 99.19%, while matrix effects were between 102.84% and 110.22%. Pemetrexed exhibited satisfactory stability under all tested conditions, meeting bioanalytical method validation requirements. Clinical application revealed delayed elimination of pemetrexed in the patient, suggesting a threshold-based relationship between exposure and toxicity. Notably, plasma concentrations remained above the proposed toxicity threshold (0.11 mg/L) for 11 days post-chemotherapy, correlating with the development of grade IV bone marrow suppression.

CONCLUSION A rapid, sensitive, and specific LC-MS/MS method for determining pemetrexed in human plasma was successfully developed and validated. This method is suitable for therapeutic drug monitoring (TDM). The clinical application suggests that pemetrexed-induced toxicity is likely driven by exposure exceeding a critical threshold. For patients with renal dysfunction or those on medications affecting its clearance, TDM using this method could be a valuable tool for individualized dose adjustment, thereby improving treatment safety and efficacy.