Development and validation of a sensitive and specific hplc assay of cladribine for pharmacokinetics

Purpose: To develop and validate a sensitive and specific HPLC assay for cladribine (CdA) in plasma for pharmacokinetic studies in rats. Methods: CdA and the internal standard AZT were purchased from Sigma-Aldrich Chem. The HPLC system consisted of a Shimadzu LC-9A pump, a 3 μm, 250 x 2.0 mm I.D. high speed C18 column (Jupiter), preceded by a 5 μm 4 x 4 mm I.D. C18 guard column (Licrocart), an Agilent Model 1050 UV-VIS detector and a 3395 Integrator. The mobile phase was made up of 0.01M KH2PO4 (pH 5): methanol: acetonitrile (90:5:5). The system was operated at ambient temperature with a flow rate of 0.3 mL/min, and UV wavelength at 265 nm, and an operating pressure of ~ 1.56 kpsi. Extraction of cladribine and AZT from plasma was achieved by solid phase extraction using 100 mg/mL C18 SPE columns (Extra-sep®). The assay was validated for sensitivity, precision, specificity and application for pharmacokinetic study in rats. Results: Under these conditions, the average retention times of CdA and AZT were 13.5 and 21 min, respectively, and recoveries were between 80 – 95%. Standard curve constructed from plasma standards was linear from 0.1 ug/mL to 1 ug/mL with regression coefficient (r) 0.99 or greater. Sensitivity assessed by oncolumn injection was < 1 ng. Using a 50-uL plasma sample size, the mean intra-assay variations at 0.1 ug/mL were 7%, and inter-assay variations over a period of 3 months for 5 separate batches were less than 20 %. The assay was used to study a single dose pharmacokinetic study of CdA in rats after a 2 mg/kg subcutaneous injection. Conclusion: The described HPLC assay has adequate sensitivity and specificity to study pharmacokinetics of CdA in rats, and could be adapted also to clinical pharmacokinetic studies.