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

Date

2007

Authors

Yeung, P. K.
Ferguson, C.
Jarrar, A.
King, B.
Li, M. L.

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Abstract

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 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. 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 (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. 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.

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Yeung, P. K., C. Ferguson, A. Jarrar, B. King, et al. 2007. "Development and validation of a sensitive and specific HPLC assay of cladribine for pharmacokinetics studies in rats." Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences, Societe canadienne des sciences pharmaceutiques 10(2): 231-236.

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