HPLC Assay of Fluconazole and Its Application to Patients with Early Septic Shock

Abstract

This work describes a precise, simple, and accurate HPLC method for the determination of fluconazole in plasma samples with enough sensitivity to monitor patients suffering from early septic shock for a month. The separation was done using an HPLC system with UV absorbance detector (set at 262 nm). It was performed using a μ Bondapak C₁₈ column (3.9 × 150 mm²) packed with 10 µm spherical particles. The mobile phase consisted of acetonitrile (20%) in 0.05 M ammonium acetate containing 0.1% triethylamine; acetic acid was used to adjust the pH to 7. Sample run time was 15 min. Metoclopramide was used as the assay internal standard (IS). Using the chromatographic conditions described, fluconazole and metoclopramide were well resolved with mean retention times of 6.7 and 10.2 min, respectively. Linear response (r > 0.996) was observed over the range of 0.2–15 µg/mL of fluconazole. There was no significant difference (p > 0.05) between inter‐ and intra‐day studies for fluconazole. The mean relative standard deviation (RSD%) of the results of within‐day precision and accuracy of the drug was ≤7.6%, which confirmed the reproducibility of the assay method. The applicability of the assay was demonstrated in measuring fluconazole pharmacokinetics in human plasma after daily intravenous infusion of 200 mg fluconazole to patient with early septic shock. Re‐analysis of samples several weeks after the initial analysis showed no loss in fluconazole. Patient with early septic shock showed a mean t _1/2 of fluconazole of 12.3 ± 9 h after a single dose and 41.3 ± 20 h after multiple administrations.

Keywords:

• Fluconazole
• HPLC
• pharmacokinetics
• metoclopramide
• methods validations
• patients
• early septic shock

Acknowledgment

The first two authors are indebted to the Research Center at the University Center for Women Students, King Saud University, for financial support.