title: Development and validation of a dried blood spot assay for the quantification of ribavirin using liquid chromatography coupled to mass spectrometry.
abstract: 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 r(2)≥0.98 demonstrating that DBS can be used as an alternative to plasma for PK-PD studies in human subjects.
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical Sciences, University of Colorado Denver, Anschutz Medical Campus, 12850 East Montview Blvd, C-238 V20, Aurora, CO 80045, USA.
authors.forename: Leah C
authors.initials: LC
authors.lastname: Jimmerson
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical Sciences, University of Colorado Denver, Anschutz Medical Campus, 12850 East Montview Blvd, C-238 V20, Aurora, CO 80045, USA.
authors.forename: Jia-Hua
authors.initials: JH
authors.lastname: Zheng
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical Sciences, University of Colorado Denver, Anschutz Medical Campus, 12850 East Montview Blvd, C-238 V20, Aurora, CO 80045, USA.
authors.forename: Lane R
authors.initials: LR
authors.lastname: Bushman
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical Sciences, University of Colorado Denver, Anschutz Medical Campus, 12850 East Montview Blvd, C-238 V20, Aurora, CO 80045, USA.
authors.forename: Christine E
authors.initials: CE
authors.lastname: MacBrayne
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical Sciences, University of Colorado Denver, Anschutz Medical Campus, 12850 East Montview Blvd, C-238 V20, Aurora, CO 80045, USA.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Anderson
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical Sciences, University of Colorado Denver, Anschutz Medical Campus, 12850 East Montview Blvd, C-238 V20, Aurora, CO 80045, USA. Electronic address: jennifer.kiser@ucdenver.edu.
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Kiser
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D059788:Dried Blood Spot Testing / Q000379:methods*; D004355:Drug Stability; D006801:Humans; D016014:Linear Models; D015203:Reproducibility of Results; D012254:Ribavirin / Q000097:blood* / Q000737:chemistry; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D012254:Ribavirin
keywords: ALQ; Analytical method; DBS; Dried blood spot; HCT; Hepatitis C; LC–MS/MS; LLOQ; Nucleoside analog; PK–PD; QC; RBV; RT; Ribavirin; STD; UPH2O; above limit of quantitation; dried blood spots; hematocrit; liquid chromatography tandem–mass spectrometry; lower limit of quantitation; pharmacokinetics/dynamics; quality control; ribavirin; room temperature; standard; ultra-pure water