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 0.05 μg/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 (10–60 μL) 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 3 mm 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 0.05 μg/mL to 10.0 μg/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 60 μL. 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.