Quantitative UHPLC–MS/MS Analysis of Gemcitabine and Its Active and Inactive Metabolites in Pig Plasma and Lung Tissue in Preparation of Locoregional Therapies for Non–Small Cell Lung Cancer

Gemcitabine (dFdC), an effective chemotherapeutic for non-small cell lung cancer (NSCLC), acts through its active metabolite, dFdCTP. Its current pharmacokinetics (PK) knowledge is based on intravenous infusion, which does not represent locoregional administration. In contrast, locoregional studies focus on dFdC, neglecting the role of dFdU (inactive) and dFdCTP, limiting our lung-specific PK insights. Existing dFdCTP assays require lengthy runs and complex protocols, reducing their translatability for large-scale studies. This study presents an ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method to quantify dFdC, dFdU, and dFdCTP in pig lung tissue and plasma, improving gemcitabine's PK assessment following both systemic and locoregional therapies. A high-throughput UHPLC-MS/MS method was developed and validated for the quantification of dFdC, dFdU, and dFdCTP in pig plasma and lung tissue. Protein precipitation was used, followed by chromatographic separation on a Luna Omega Polar C18 column with a 4-min run time. Linearity was established over the ranges: 0.5-400.0 μg/mL (plasma) and 0.5-400.0 μg/g (lung tissue). It demonstrated robust accuracy, precision, and selectivity with minimal carryover and negligible matrix effects. Stability was confirmed for 4 h at 4°C, 40°C, and on the autosampler. The method's applicability was verified in study samples treated with a 30-min intravenous gemcitabine (1.25 g/m2) infusion. This UHPLC-MS/MS assay overcomes previous methodological limitations by reducing run time and simplifying sample preparation. Its robustness to quantify dFdC, dFdU, and dFdCTP in pig plasma and lung tissue has been confirmed, providing a valuable tool for future PK studies with locoregional therapies for NSCLC.