Development and validation of a high-performance liquid chromatographic assay for the determination of fluconazole in human whole blood using solid phase extraction.
A sensitive and specific high-performance liquid chromatographic ultraviolet method for the determination of fluconazole in human whole blood has been developed and validated. Whole blood samples were processed by a solid phase extraction procedure using an Oasis HLB extraction cartridge before chromatography. Phenacetin was used as the internal standard. Chromatography was performed using Waters C18 Symmetry analytical column, 5 microm, 4.6 x 250 mm, using an isocratic elution with a mobile phase consisting of acetonitrile and water (36:64, v/v) at a flow rate of 0.8 mL/min. The retention times of fluconazole and phenacetin were 4.7 and 8.3 minutes, respectively, and the total run time was 10 minutes. Quantitative analysis was performed using a Waters UV-VIS detector at a wavelength of 210 nm. The assay was linear over the concentration range of 0.5 to 15 microg/mL for fluconazole. The extraction recoveries at concentrations of 2.5, 5, and 10 microg/mL were 105.9%, 98.4%, and 95%, respectively. The method can quantify 0.5 microg/mL fluconazole using 300 microL of whole blood. At concentrations of 2.5, 5, and 10 microg/mL, the intraday precision expressed as coefficient of variation was 3.47%, 8.81%, and 1.14% and the interday precision was 5.21%, 5.48%, and 7.18%, respectively. This method is simple, uses a low blood volume for analysis, and allows reproducible and accurate measurement of fluconazole in whole blood samples from pediatric patients.
Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, Pennsylvania 15261, USA.
Shimin
S
Zhang
Sripal Reddy
SR
Mada
Marilyn
M
Torch
Rakesh K
RK
Goyal
Raman
R
Venkataramanan
D000935:Antifungal Agents / Q000097:blood*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015725:Fluconazole / Q000097:blood*; D006801:Humans; D007165:Immunosuppression Therapy; D007202:Indicators and Reagents; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D012997:Solvents
D000935:Antifungal Agents; D007202:Indicators and Reagents; D012997:Solvents; D015725:Fluconazole