Determination of Busulfan in Human Plasma Using an ELISA Format

Abstract

High-dose busulfan is an important component of many bone marrow transplantation-preparative regimens. High busulfan plasma levels have been shown to increase the chance of venoocclusive disease and low levels are associated with recurrence of disease or graft rejection. Currently, busulfan levels are monitored by physical methods that are expensive and time consuming, resulting in relatively low overall use of busulfan testing for dose adjustment. Novel highly selective antibodies for busulfan have been generated and a microtiter plate immunoassay capable of quantifying busulfan levels in plasma has been developed. The assay was configured using a busulfan-horseradish peroxidase (HRP) conjugate as the reporter group and busulfan monoclonal antibodies. The assay requires 30 microL of plasma with no sample preparation. The immunoassay has a standard curve based on busulfan with a range of 75-2000 ng/mL. The time to first result is 30 minutes with up to 40 patient samples in duplicate; multiple plates can be run at once. The coefficient of variation (CV) on signal is <5% for an entire plate, and the 95% confidence interval for negative samples (n = 78) is below the lowest calibrator of 75 ng/mL. Cross-reactivity with the major inactive metabolites (tetrahydrothiophene, tetramethyl sulfone, and tetrahydrothiophene-3-ol-1,1-dioxide) was <0.1%. Results generated with clinical samples (n = 35 and n = 70) correlate well to gas chromatography-mass spectrometry (R = 0.976 and 0.985, respectively) with a slope of 1.05 +/- 0.05. This immunoassay method is suitable for determining levels of busulfan in human plasma. It offers the advantages of using a smaller sample size, does not require sample preparation, and is less labor intensive than other methods. The ability to make 240 determinations per hour enables effective and timely routine monitoring of busulfan levels in clinical practice.

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

“…GC-based methods and LC-fluorescence/UV techniques are time-consuming and laborious, requiring derivatization steps for preparation and a large quantity of plasma of up to 1 mL, along with a long analysis time of up to 30 min per run [10-13]. 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.…”

Therapeutic Drug Monitoring in Cancer Chemotherapy

“…ELISA formats have also recently been adapted to detect busulfan [81]. Newer LC and MS/MS methods to detect busulfan concentrations in oral fluids have recently been described [69].…”

An Automated Method for Supporting Busulfan Therapeutic Drug Monitoring

“…Blood samples were stored on ice until all samples were collected. Plasma was separated from the cells, and busulfan concentrations were measured by the ELISA of Courtney et al, 10 using reagents obtained from Saladax Biomedical. Patient samples from Q24 dosing were diluted 32 before analysis by using Roche c 111 or Olympus AU400e.…”