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International Journal of Analytical Chemistry
Volume 2015, Article ID 576250, 8 pages
http://dx.doi.org/10.1155/2015/576250
Research Article

Development and Validation of Simple RP-HPLC Method for Intracellular Determination of Fluconazole Concentration and Its Application to the Study of Candida albicans Azole Resistance

Scientific Center of Drug and Medical Technology Expertise JSC, Ministry of Health of Armenia, Komitas 49/4, 0051 Yerevan, Armenia

Received 25 August 2015; Revised 21 October 2015; Accepted 4 November 2015

Academic Editor: Josef Havel

Copyright © 2015 Tigran K. Davtyan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Candida albicans (strains NCTC-885-653 and ATCC-10231) long-term cultivated in the presence of antifungal agent fluconazole (FLC) and classical microbiological methods for determination of minimal inhibitory concentration (MIC) were used in this study. A simple and sensitive method based on reverse-phase high-performance liquid chromatography (RP-HPLC) has been developed for the determination of FLC intracellular concentration in C. albicans using tinidazole as an internal standard. Following extraction with dichloromethane, the chromatographic separation was achieved on a Machery-Nagel EC250/2 Nucleodur-100-3 C18 column by gradient elution using the mobile phase consisting of (A) 0.01 M ammonium acetate buffer, pH = 5.00, and (B) acetonitrile. Different analytical performance parameters such as linearity, precision, accuracy, limit of quantification (LOQ), and robustness were determined according to US DHHS FDA and EMEA guidelines. The method was linear for FLC ranging from 100 to 10000 ng/mL. The intraday and interday precisions (relative standard deviation) were within 2.79 and 2.64%, respectively, and the accuracy (relative error) was less than 2.82%. The extraction recovery ranged from 79.3 to 85.5%. The reliable method was successfully applied to C. albicans azole-resistance study and it was shown that intracellular concentration of FLC correlated with a yeast drug susceptibility profile and MIC values.