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

• Published in: Journal of pharmaceutical and biomedical analysis 2017-11, Vol.146, p.302-313
• Main Authors: Page-Sharp, Madhu, Strunk, Tobias, Salman, Sam, Hibbert, Julie, Patole, Sanjay K., Manning, Laurens, Batty, Kevin T.
• Format: Article
• Language: English
• Subjects: Caffeine; Caffeine - chemistry; Chromatography, High Pressure Liquid - methods; Dried Blood Spot Testing - methods; Dried blood spots; Humans; Infant, Newborn; Infant, Premature; LC–MS/MS; Neonates; Pentoxifylline; Pentoxifylline - analogs & derivatives; Pentoxifylline - chemistry; Plasma; Plasma - chemistry; Tandem Mass Spectrometry - methods
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2017.08.030

[Display omitted] •Validated UPLC–MS/MS assay for caffeine, pentoxifylline (PTX) and three metabolites of PTX.•LLOQ was 100μg/L for caffeine and 10μg/L for PTX and its metabolites in plasma and DBS.•In preterm infants, DBS concentrations for caffeine, PTX and M1 were 9%, 7% and 13% lower than plasma, respectively. 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 2.1min, respectively, with a run time of 5min. The precision (≤10%) and accuracy (≤15%) across the concentration range 0.1–50mg/L for caffeine, PTX and the three metabolites in plasma and DBS were within accepted limits, as were the limits of quantification (100μg/L for caffeine and 10μg/L for PTX, M1, M4 and M5). Caffeine, PTX and the metabolites were stable in DBS for >34days 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.