Development and validation of a sensitive and specific HPLC assay of cladribine for pharmacokinetics studies in rats

To develop and validate a sensitive and specific HPLC assay for cladribine (CdA) in plasma for pharmacokinetic studies in rats.

CdA and the internal standard AZT were purchased from Sigma-Aldrich Chem. The HPLC system consisted of a Shimadzu LC-9A pump, a 3 im, 250 x 2.0 mm I.D. high speed C18 column (Jupitertrade mark), preceded by a 5 im 4 4 mm I.D. C18 guard column (Licrocarttrade mark), 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 ca. 1.56 kpsi. Extraction of cladribine and AZT from plasma was achieved by solid phase extraction using 100 mg/mL C18 SPE columns Extra-septrade mark). The assay was validated for sensitivity, precision, specificity and application for pharmacokinetic study in rats.

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 (r2) 0.99 or greater. Sensitivity assessed by on column injection was < 1 ng. Using a 50-uL plasma sample size, the mean intra assay variations 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.

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.