RESUMO
BACKGROUND: Busulfan (BU) is an alkylating agent with a narrow therapeutic index and high intraindividual pharmacokinetic variability used in conditioning therapy for hematopoietic stem cell transplantation. Monitoring BU exposure during high-dose conditioning regimens is recommended and positively impacts outcomes. We aimed to develop, validate, and apply a ultra-high-performance liquid chromatography-mass spectrometry (MS)/MS assay to measure BU concentrations in oral fluid and dried plasma spots (DPS) as alternative matrices to plasma. METHODS: We prepared plasma and oral fluid samples by protein precipitation and DPS after liquid extraction. We analyzed extracts using an LC-MS/MS system with an Acquity HSS T3 column in the positive electrospray ionization mode. The method was validated and applied to 79 paired plasma and oral fluid samples from 7 patients on BU conditioning treatment. DPS were prepared by pipetting plasma onto Whatman 903 paper. The correlation between BU in plasma, oral fluid, and DPS samples was evaluated. RESULTS: Run time was 4.0 minutes. The assay was linear at 50-5000 ng mL-1 (r > 0.99), precise (1.9%-5.3% oral fluid and 1.8%-5.9% DPS), and accurate (98.1%-108.9% oral fluid and 93%-103.1% DPS). BU was stable in DPS at 23°C for 24 hours. BU levels in oral fluid (r = 0.927) and DPS (r = 0.982) were significantly correlated with plasma. Despite the good correlation, we found a wide variation between oral fluid and plasma levels. The area under curves (AUCs) calculated with oral fluid concentrations were 79.1%-167.1% of plasma AUCs. Bland-Altman plots found a better agreement for DPS, with AUCs estimated from corrected DPS levels at 83.1%-114.1% of plasma values. CONCLUSIONS: We developed and validated a simple and fast ultra-high-performance liquid chromatography-MS/MS assay to measure BU in oral fluid and DPS. The results do not support the use of oral fluid as a matrix for routine therapeutic drug monitoring of BU. The AUC estimated from BU measurements in DPS was comparable to that in plasma, supporting the use of DPS in BU therapeutic drug monitoring as an alternative matrix, with adequate short-term stability and logistic advantages.
