Abstract
SummaryBackgroundBusulfan (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.MethodsA 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.ResultsThe 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% CI: 0.97 to 1.01) and –6.82 intercept (95% CI: –15.23 to 3.53). Residuals analysis against the 2.5th–97.5th percentiles range showed four samples with significant bias individually.ConclusionsThe 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.
Highlights
Hematopoietic stem cell transplantation (HSCT) represents the only therapeutic approach to grant blood transfusion-free survival to patients affected by severe thalassemia [1]
We report a robust CSTbased LC-MS/MS Bu assay with on-line solid phase extraction (SPE), developed to speed up the therapeutic drug monitoring (TDM) of Bu in pediatric patients undergoing a conditioning regimen for HSCT
The retention time measured by repeated injections was 1.58±0.003 minutes for Bu and 1.58± 0.005 minutes for D8-Bu, with a 4 minutes total run time
Summary
Hematopoietic stem cell transplantation (HSCT) represents the only therapeutic approach to grant blood transfusion-free survival to patients affected by severe thalassemia [1]. Some antibody-based methods are available for dosing Bu [6, 7] In spite of this fact, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) represents the widespread choice to perform its TDM on several different matrices [8,9,10,11]. Superiority of LC-MS/MS in immunometric assays relies on its higher specificity, the extractive steps like liquid-liquid extraction (LLE) or protein precipitation (PP) if required cannot be automated [12]. In this regard, solid phase extraction (SPE) represents a valuable technique suitable to achieve an automated extraction of samples within the chromatographic apparatus. The on-line SPE through CST allows a reduced time analysis and increased reproducibility, limiting to the minimum the manual intervention in sample cleanup
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