Highly sensitive quantification of pemetrexed in human plasma using UPLC‐MS/MS to support microdosing studies

Pemetrexed is an antifolate drug approved for the treatment of non‐small‐cell lung cancer and mesothelioma. Assessing pemetrexed pharmacokinetics after administration of a microdose (100 μg) may facilitate drug–drug interaction and dose individualization studies with cytotoxic drugs, without causing harm to patients. Therefore, a highly sensitive bioanalytical assay is required. A reversed‐phase ultra‐high performance liquid chromatography method was developed to determine pemetrexed concentrations in human ethylenediaminetetraacetic acid–plasma after microdosing. [(13)C(5)]‐Pemetrexed was used as the internal standard. The sample preparation involved solid‐phase extraction from plasma. Detection was performed using MS/MS in a total run time of 9.5 min. The assay was validated over the concentration range of 0.0250–25.0 μg/L pemetrexed. The average accuracies for the assay in plasma were 96.5 and 96.5%, and the within‐day and between‐day precision in coefficients of variations was <8.8%. Extraction recovery was 59 ± 1 and 55 ± 5% for pemetrexed and its internal standard. Processed plasma samples were stable for 2 days in a cooled autosampler at 10°C. The assay was successfully applied in a pharmacokinetic curve, which was obtained as a part of an ongoing clinical microdosing study.