Development and validation of a dried blood spot assay for the quantification of ribavirin using liquid chromatography coupled to mass spectrometry

•Measurement of RBV in dried blood spot to 0.05μg/mL.•DBS storage was stable at room temperature for up to 140 days.•Analysis was accurate and precise for hematocrit in the normal range.•Punch location and spot volume (10–60μL) did not affect measurement.•DBS can be used for pharmacokinetic/dynamic analyses in place of plasma. Efficient, inexpensive and sensitive assays for the measurement of drugs are of interest for pharmacokinetic and pharmacodynamics (PK–PD) analysis. Dried blood spots (DBS) are a unique bioanaltyical matrix with the potential to fulfill this interest for the measurement of numerous analytes. Here we describe the development and validation of a reversed-phase high performance liquid chromatographic (LC), tandem mass spectrometry (MS/MS) assay for the determination of ribavirin (RBV) in DBS. A 3mm punch from spotted and dried whole blood was extracted in methanol utilizing isotopically labeled internal standard for LC–MS/MS analysis. Validation was performed over a range of 0.05μg/mL to 10.0μg/mL and the method was shown to be precise (coefficient of variation ≤15%) and accurate (within ±15% of control). These acceptance criteria were met for hematocrit ranges of 20–54%, for center versus edge punches and for spot volumes from 10 to 60μL. RBV was stable for up to 140 days at room temperature and −20°C as well as for three freeze/thaw cycles. Correlation of RBV in DBS versus in plasma yielded r2≥0.98 demonstrating that DBS can be used as an alternative to plasma for PK–PD studies in human subjects.