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.
Zhang
Shimin
S
Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, Pennsylvania 15261, USA.
Mada
Sripal Reddy
SR
Torch
Marilyn
M
Goyal
Rakesh K
RK
Venkataramanan
Raman
R
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