HPLC-MS/MS method for the intracellular determination of ribavirin monophosphate and ribavirin triphosphate in CEM ss cells.
A sensitive and specific method using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for the determination of ribavirin monophosphate (RBV-MP) and ribavirin triphosphate (RBV-TP) in cells has been developed and validated. In this method, ribavirin phosphorylated metabolites were extracted and separated by anion exchange solid phase extraction (SPE). The RBV-MP and RBV-TP fractions were dephosphorylated using acid phosphatase and further purified by phenyl boronate SPE prior to HPLC-MS/MS analysis. (13)C(5)-uridine was added as internal standard to obtain better accuracy and precision of the analysis. The MS/MS detector was optimized at multiple reaction monitoring (MRM) using positive electrospray ionization to detect 245-->113 and 250-->133 transitions for ribavirin and internal standard, respectively. The calibration curve was linear over a concentration range of 0.01-10 microg/mL with a limit of quantitation of 0.01 microg/mL. Mean inter-assay accuracy and precision for RBV-MP and RBV-TP quality control samples at 0.03, 0.3 and 8 microg/mL were 5% and 10%, respectively. This method was successfully used for the in vitro determination of RBV-MP and RBV-TP in CEM(ss) cells cultured with RBV.
Meléndez
Margarita
M
Department of Chemistry, Río Piedras Campus, University of Puerto Rico, Río Piedras, Puerto Rico.
Rosario
Osvaldo
O
Zayas
Beatriz
B
Rodríguez
José F
JF
D000998:Antiviral Agents / Q000378:metabolism*; D002138:Calibration; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D005615:Freezing; D006801:Humans; D015394:Molecular Structure; D009711:Nucleotides / Q000032:analysis*; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D012254:Ribavirin / Q000378:metabolism*; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction / Q000379:methods; D012996:Solutions; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors
D000998:Antiviral Agents; D009711:Nucleotides; D012996:Solutions; C009174:ribavirin-5'-phosphate; D012254:Ribavirin; C037827:ribavirin 5'-triphosphate