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
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-...
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| Format: | Article |
| Language: | English |
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Wiley
2015-01-01
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| Series: | International Journal of Analytical Chemistry |
| Online Access: | http://dx.doi.org/10.1155/2015/576250 |
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| author | Tigran K. Davtyan Levon A. Melikyan Nune A. Nikoyan Hripsime P. Aleksanyan Nairi G. Grigoryan |
| author_facet | Tigran K. Davtyan Levon A. Melikyan Nune A. Nikoyan Hripsime P. Aleksanyan Nairi G. Grigoryan |
| author_sort | Tigran K. Davtyan |
| collection | DOAJ |
| description | 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 (r=0.9999) 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. |
| format | Article |
| id | doaj-art-ddd5af2a5094408591acc420e6dde34a |
| institution | Kabale University |
| issn | 1687-8760 1687-8779 |
| language | English |
| publishDate | 2015-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | International Journal of Analytical Chemistry |
| spelling | doaj-art-ddd5af2a5094408591acc420e6dde34a2025-08-20T03:26:29ZengWileyInternational Journal of Analytical Chemistry1687-87601687-87792015-01-01201510.1155/2015/576250576250Development and Validation of Simple RP-HPLC Method for Intracellular Determination of Fluconazole Concentration and Its Application to the Study of Candida albicans Azole ResistanceTigran K. Davtyan0Levon A. Melikyan1Nune A. Nikoyan2Hripsime P. Aleksanyan3Nairi G. Grigoryan4Scientific Center of Drug and Medical Technology Expertise JSC, Ministry of Health of Armenia, Komitas 49/4, 0051 Yerevan, ArmeniaScientific Center of Drug and Medical Technology Expertise JSC, Ministry of Health of Armenia, Komitas 49/4, 0051 Yerevan, ArmeniaScientific Center of Drug and Medical Technology Expertise JSC, Ministry of Health of Armenia, Komitas 49/4, 0051 Yerevan, ArmeniaScientific Center of Drug and Medical Technology Expertise JSC, Ministry of Health of Armenia, Komitas 49/4, 0051 Yerevan, ArmeniaScientific Center of Drug and Medical Technology Expertise JSC, Ministry of Health of Armenia, Komitas 49/4, 0051 Yerevan, ArmeniaCandida 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 (r=0.9999) 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.http://dx.doi.org/10.1155/2015/576250 |
| spellingShingle | Tigran K. Davtyan Levon A. Melikyan Nune A. Nikoyan Hripsime P. Aleksanyan Nairi G. Grigoryan 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 International Journal of Analytical Chemistry |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| url | http://dx.doi.org/10.1155/2015/576250 |
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