Development and Clinical Validation of a Simultaneous Ultra‐High‐Performance Liquid Chromatography Tandem Mass Spectrometry Assay for Three Triazole Antifungal Agents and Two Metabolites Enabling Precision Therapeutic Drug Monitoring in Chinese Patients With Hematologic Malignancies

ABSTRACT Triazole antifungals are clinically established agents for the prophylaxis and treatment of invasive fungal diseases in patients with hematologic malignancies. To address the requirements of clinical therapeutic drug monitoring, we developed and validated a novel ultra‐high‐performance liquid chromatography tandem mass spectrometry method for the simultaneous quantification of three triazole antifungals and their metabolites in human plasma: itraconazole (ICZ), hydroxy‐itraconazole (ICZ‐OH), voriconazole (VCZ), voriconazole N‐oxide (VCZ N‐oxide), and posaconazole. The five target analytes were successfully separated using a BEH C18 column (2.1 × 50 mm, 1.7 µm) maintained at 40°C, with gradient elution employing mobile phase A (5.0 mM ammonium acetate in water containing 0.1% formic acid) and mobile phase B (100% acetonitrile [ACN]) at a constant flow rate of 0.4 mL/min. A positive ion pattern was chosen for quantification under multiple reaction monitoring. Following the addition of 10 µL of internal standard, 50 µL of plasma underwent protein precipitation with ACN, and the resulting supernatant was diluted for subsequent analysis. Method validation followed Food and Drug Administration guidelines and Chinese Pharmacopoeia regulations, demonstrating acceptable accuracy, precision, matrix effects, recovery, and stability. The calibration curves exhibited excellent linearity over the range of 0.1–10 µg/mL for all five analytes, with correlation coefficients (r 2 ) ≥ 0.9962, while the lower limit of quantification and limit of detection were established at 0.1 and 0.03 µg/mL, respectively. The intra‐ and inter‐day coefficients of variation were below 8.7% at all concentration levels, and the accuracy was 90.0%–113.0%. This methodology was successfully applied for TDM of three triazole antifungals and their metabolites in 150 patients with hematologic malignancies. Therefore, the method demonstrates good analytical performance, establishing its reliability for clinical TDM of triazole antifungals.