Simultaneous determination of busulfan, fludarabine, phenytoin, and posaconazole in plasma from patients undergoing hematopoietic stem cell transplantation

A highly efficient, rapid, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of busulfan, fludarabine, phenytoin, and posaconazole in plasma samples from pediatric patients. This method was designed to optimize sample preservation while maintaining accuracy and reliability. By incorporating in-source collision-induced dissociation, the linear range of detection was fine-tuned, particularly for phenytoin, which exhibited an excessive response. Each of the four drugs presents distinct physical and chemical properties, making their simultaneous analysis a complex task that was successfully addressed in this study.

The procedure required only 20 μL of plasma, minimizing sample volume requirements, which is particularly advantageous in pediatric applications. A straightforward protein precipitation technique was employed for sample preparation, eliminating the need for complex extraction procedures. Chromatographic separation was performed using a reversed-phase C18 column (50 × 2.1 mm, 2.6 μm) under gradient elution conditions. The mobile phase consisted of water with 0.1% formic acid and 2 mM ammonium acetate, along with acetonitrile containing 0.1% formic acid. With an injection volume of 2 μL, the total run time was limited to 3.6 minutes, ensuring a fast and efficient analytical process.

Mass spectrometric detection was carried out on a Triple Quad™ 4500MD tandem mass spectrometer equipped with an electrospray ionization (ESI) source operating in positive ion mode. The use of in-source collision-induced dissociation played a crucial role in adjusting the linear range of phenytoin, optimizing its quantification. The method demonstrated excellent linearity across the calibration ranges: 20–2560 ng/mL for busulfan, 10–1280 ng/mL for fludarabine, 0.4–51.2 μg/mL for phenytoin, and 0.1–12.8 μg/mL for posaconazole, with correlation coefficients (r²) exceeding 0.997 for all analytes.

Further validation, conducted in accordance with European Medicines Agency guidelines, confirmed the method’s exceptional accuracy, ranging from 90.5% to 106.7%, and precision, which remained within 2.0%–13.0%. Additionally, its robustness was tested against atypical matrices, including hemolytic and hyperlipidemic plasma, demonstrating its applicability in diverse clinical scenarios. The developed method was effectively implemented for therapeutic drug monitoring (TDM) in pediatric patients undergoing hematopoietic stem cell transplantation. By ensuring precise and reliable quantification of these critical drugs, this method supports optimized dosing strategies and improved patient outcomes.