Rapid and Simple Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) Method for Simultaneous Quantifications of Triazole Antifungals in Human Serum

Purpose To develop and validate a one-step, rapid and simple reversed-phase high-performance liquid chromatography ( HPLC)-based protocol for the simultaneous measurement of voriconazole (VCZ), posaconazole (POSA), itraconazole (ITC) in serum/plasma. Methods Calibration standards (CS) and quality control samples were prepared in drug-free serum by spiking with the triazoles at different concentrations. HPLC was performed with C 18 column, isocratic mobile phase after extraction with cold acetonitrile. The standardized method was tested in 2693 patients’ serum/plasma samples. Results Linearity of CS for ITC, VCZ and POSA was proportional to the nominal concentration (correlation coefficient > 0.999). Limit of detection (mg/L) for ITC, VCZ and POSA was 0.25, 0.25 and 0.125, respectively. The lower limit of quantification (mg/L) for ITC, VCZ and POSA was 0.5, 0.5 and 0.25, respectively. Precision and accuracy were in acceptable range with 100% average percentage recovery. No interferences from endogenous substances and other antimicrobial compounds were noted. In clinical samples, the therapeutic range achieved for VCZ was 39.9%. Whereas, 61.1% and 44% of samples with ITC and POSA, respectively, were in the sub-therapeutic range. Conclusion We developed a rapid and simple HPLC method to quantify common triazoles in a single chromatographic run allowing simultaneous measurement of different antifungals in a small volume of serum/plasma. Thus, therapeutic drug monitoring requests can be processed in one run without changing the protocol parameters, column or column conditioning thereby improving turnaround time.