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Development and validation of a sensitive and specific HPLC assay of cladribine for pharmacokinetics studies in rats.
J Pharm Pharm Sci. 2007; 10(2):231-6.JP

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.

Authors+Show Affiliations

Pharmacokinetics and Metabolism Laboratory, College of Pharmacy and Department of Medicine, Faculty of Health Professions, Dalhousie University, Halifax, Nova Scotia, Canada. Pollen.Yeung@Dal.Ca <Yeung@Dal.Ca>No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Evaluation Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

17706181

Citation

Yeung, Pollen K F., et al. "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, vol. 10, no. 2, 2007, pp. 231-6.
Yeung PK, Ferguson C, Jarrar A, et al. Development and validation of a sensitive and specific HPLC assay of cladribine for pharmacokinetics studies in rats. J Pharm Pharm Sci. 2007;10(2):231-6.
Yeung, P. K., Ferguson, C., Jarrar, A., King, B., & Li, M. L. (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), 231-6.
Yeung PK, et al. Development and Validation of a Sensitive and Specific HPLC Assay of Cladribine for Pharmacokinetics Studies in Rats. J Pharm Pharm Sci. 2007;10(2):231-6. PubMed PMID: 17706181.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Development and validation of a sensitive and specific HPLC assay of cladribine for pharmacokinetics studies in rats. AU - Yeung,Pollen K F, AU - Ferguson,Carrie, AU - Jarrar,Ameer, AU - King,Brian, AU - Li,Mary L, PY - 2007/8/21/pubmed PY - 2007/10/5/medline PY - 2007/8/21/entrez SP - 231 EP - 6 JF - Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences, Societe canadienne des sciences pharmaceutiques JO - J Pharm Pharm Sci VL - 10 IS - 2 N2 - 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. SN - 1482-1826 UR - https://www.unboundmedicine.com/medline/citation/17706181/Development_and_validation_of_a_sensitive_and_specific_HPLC_assay_of_cladribine_for_pharmacokinetics_studies_in_rats_ L2 - http://www.ualberta.ca/~csps/JPPS10_2/jourcont10_2.htm DB - PRIME DP - Unbound Medicine ER -