Direct Analysis in Real Time-Mass Spectrometry Measurement of Antifungals in Human Serum: A Proof-of-Concept Study

Abstract

INTRODUCTION: Direct analysis in real time (DART) is a soft ionization technique that uses a heated gas such as helium, argon, or nitrogen to generate a cascade of ionized atmospheric molecules that subsequently ionize analytes in biological samples. OBJECTIVES: In this proof-of-concept study evaluating selected antifungals in human serum, 5-fluorocytosine (5FC), itraconazole (ITR), hydroxyitraconazole (OH-ITR), isavuconazole (ISV), ketoconazole (KETO), fluconazole (FLU), and posaconazole (POSA) were assessed as DART candidates using a Bruker EVOQ^® DART-TQ+. METHODS: Human serum samples were extracted using protein precipitation or liquid–liquid extraction. Quantification was performed by DART tandem mass spectrometry (DART-MS/MS) with internal standards. Precision and linearity across individual analytical measurement ranges (AMRs) were evaluated with rapid sample acquisition (0.24 min linear scans), and the results were compared with those obtained using validated LC-MS/MS methods. RESULTS: Linearity and precision were acceptable for all evaluated compounds. Good agreement with LC-MS/MS was observed for 5FC, ITR, OH-ITR, and FLU, with slopes ranging from 0.875 to 1.112 and correlation coefficients (r) ≥ 0.949. POSA extraction by protein precipitation resulted in high bias; however, liquid–liquid extraction or the use of an alternative ion transition reduced bias to <7%. DART replicates demonstrated coefficients of variation <15%. All samples met ion-ratio tolerance criteria of ±50%. CONCLUSION: This proof-of-concept study demonstrates the feasibility of quantitative antifungal measurement in human serum using DART-MS/MS with simple sample preparation. A moderate linear association with bias for POSA was likely attributable to matrix effects. More selective sample preparation may improve DART accuracy. Overall, DART-MS/MS shows promise as a rapid, high-throughput alternative to conventional LC-MS/MS.