Development of a rapid and specific assay for detection of busulfan in human plasma by high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry

Abstract

A sensitive and specific assay for detection of busulfan in human plasma was developed. The assay is based on rapid isolation of busulfan by liquid-liquid extraction with ethyl acetate, and detection by high-performance liquid chromatography with electrospray ionization and tandem mass spectrometry. 1,6-Bis(methanesulfonyloxy)hexane, a synthesized analogue of busulfan, was used as the internal standard (IS). The acquisition was performed in the multiple reaction monitoring mode; busulfan and the IS were detected with no interferences from plasma matrix. The method was linear over the range 5-2500 ng mL(-1), with r2 > 0.99 and a run time of only 3.5 min. The intra- and inter-assay precisions were in the ranges 2.1-11.9% and 3.2-10.1%, respectively, and the intra- and inter-assay accuracies were 92.2-107.6% and 94.7-104.1%, respectively. The absolute recoveries were 82.0% (20 ng mL(-1)), 90.6% (1000 ng mL(-1)) and 80.0% (2000 ng mL(-1)) for busulfan, and 89.1% for the IS (1000 ng mL(-1)). The limits of detection and quantification were 2 and 5 ng mL(-1), respectively. The validated method was successfully applied to analyze plasma samples obtained from six adults receiving doses of 1 mg kg(-1) in a conditioning regimen prior to bone marrow transplantation. A marked intra-patient variation in busulfan concentrations during the steady state was observed, which limits the application of pharmacokinetic modeling and suggests that continuous therapeutic monitoring is necessary for adequate individualized dosing. In this regard, the present assay brings important advantages relative to other methods described in the literature, i.e., it is highly specific and simple to perform, with a rapid chromatographic run time (3.5 min), and the whole procedure can be completed in 4-5 h, which would permit dose corrections after the third dose allowing earlier and better dosing adjustments towards the target level of busulfan.

Novel pharmacokinetic behavior of intravenous busulfan in children with thalassemia undergoing hematopoietic stem cell transplantation: a prospective evaluation of pharmacokinetic and pharmacodynamic profile with therapeutic drug monitoring

“…27 Bu concentration was determined using a linear 7-point calibration curve ranging from 39 to 2500 ng/mL. [ 2 H 8 ]-Bu (Eurisotop) was used as an internal standard.…”

Quantification of Human Plasma-Busulfan Concentration by Liquid Chromatography-Tandem Mass Spectrometry

“…In comparison with other known LC-MS/MS methods [17, 18, 27], a run time of 10 min required for chromatographic analysis is somewhat longer than the 3-4 min run time in previously described methods. However, considering the time needed for sample preparation, the total elapsed time is nearly as much as or slightly shorter than previous methods, as the present method does not require complex extraction procedures or evaporation processes.…”

“…The recently developed ELISA-based automated method shows similar precision, required time, and sample volume compared with previously described methods, while having the risk of cross-reactivity and lot-to-lot variability of reagents [14]. In contrast, the LC-MS/MS provides the highest sensitivity and specificity, minimizes the preparation steps, and requires the smallest sample volume along with remarkably short analysis time [17, 18, 27]. …”

“…Gas chromatography (GC) coupled with electron capture detector [10] or mass spectrometry (MS) [11], liquid chromatography (LC) coupled with UV detectors [12] or fluorescence detectors [13] have been introduced, and an ELISA-based automated method was developed recently [14]. Among the existing methods, LC coupled with MS [15, 16] or with tandem MS (MS/MS) [17, 18] offers a high level of sensitivity and requires a small sample volume, which is beneficial in a pediatric setting. Moreover, other major advantages include the possibility to eliminate complex derivatization procedures and the requirement of only a 10-min run.…”

Determination of content uniformity of busulfan capsules by liquid chromatography–mass spectrometry

“…Different methods for quantification of busulfan in plasma samples have been published, applying either HPLC or gas chromatography–mass spectrometry (MS) 5–9. Following the introduction of liquid chromatography (LC)–MS systems into routine clinical chemistry laboratories, several LC–MS methods for the determination of busulfan were established 10–12. We therefore adapted an LC–MS method established in our laboratory for quantification of plasma levels of busulfan to the specific requirements of quantification of the content uniformity of high dose busulfan capsules.…”