An LC-MS Assay with Isocratic Separation and On-Line Solid Phase Extraction to Improve the Routine Therapeutic Drug Monitoring of Busulfan in Plasma

Background: Busulfan (Bu) requires therapeutic drug monitoring (TDM) in subjects undergoing a conditioning regimen for hematopoietic stem cell transplantation (HSCT). To speed up the procedure and increase reproducibility, we improved our routine LC-MS/MS assay using the on-line solid-phase extraction (SPE) of samples. Methods: A protein precipitation (PP) step was performed before the on-line SPE of Bu from 200 μL of plasma spiked with octa-deuterated Bu (D8-Bu) as the internal standard. Bias was assessed with respect to our routine LC-MS/MS Bu assay with off-line extraction using the Passing-Bablok robust regression. Root cause of bias for individual samples was assessed by analyzing the regression residuals. Results: The method was linear in the range 37.75-2,416 ng/mL (r2>0.999), with 19.74 ng/mL LLOQ and 10.5% CV at 20 ng/mL. Precision and accuracy were both within ±5%, and neither appreciable matrix nor carryover effects were observed. The Passing-Bablok regression analysis returned a 0.99 slope (95% Cl: 0.97 to 1.01) and -6.82 intercept (95% Cl: -15.23 to 3.53). Residuals analysis against the 2.5th-97.5th percentiles range showed four samples with significant bias individually. Conclusions: The method presented can be successfully employed for the routine analysis of Bu in plasmatic samples, and can replace the LC-MS/MS method with off-line extraction without any statistically significant overall bias. In this regard, samples with individual significant bias were reasonably produced by preanalytical issues which had no relation with the conversion to the on-line SPE extraction.