Simultaneous determination of pentoxifylline, metabolites M1 (lisofylline), M4 and M5, and caffeine in plasma and dried blood spots for pharmacokinetic studies in preterm infants and neonates

Advances in bioanalytical methods are facilitating micro-volume and dried blood spot (DBS) analysis of drugs in biological matrices for pharmacokinetic studies in children and neonates. We sought to develop a UPLC–MS/MS assay for simultaneous measurement of caffeine, pentoxifylline (PTX) and three metabolites of PTX in both plasma and DBS. Caffeine, PTX, the metabolites M1 (lisofylline), M4 and M5, and the internal standards (caffeine-d₉ and PTX-d₆) were separated using a Waters Aquity T3 UPLC C₁₈ column and gradient mobile phase (water-methanol-formic acid). Retention times for caffeine, M5, M4, PTX and M1 were 1.6, 1.7, 1.9, 2.0 and 2.1 min, respectively, with a run time of 5 min. The precision (≤10%) and accuracy (≤15%) across the concentration range 0.1–50 mg/L for caffeine, PTX and the three metabolites in plasma and DBS were within accepted limits, as were the limits of quantification (100 μg/L for caffeine and 10 μg/L for PTX, M1, M4 and M5). Caffeine, PTX and the metabolites were stable in DBS for >34 days at room and refrigerated temperatures. Plasma and DBS samples were obtained from 24 preterm infants recruited into a clinical pharmacokinetic study of PTX. Paired analysis indicated that DBS concentrations were 9% lower than concurrent plasma concentrations for caffeine, 7% lower for PTX (consistent with the blood:plasma ratio) and 13% lower for M1 (lisofylline). The validated UPLC–MS/MS method is suitable for micro-volume plasma and DBS analysis of caffeine, PTX and its metabolites for pharmacokinetic studies in paediatric patients.