A simplified method for busulfan monitoring using dried blood spot in combination with liquid chromatography/tandem mass spectrometry

Abstract

RATIONALE: Busulfan (Bu) is an important component of the myeloablative conditioning regimen prior to hematopoietic stem cell transplantation (HSCT) especially in children. Intravenously administered Bu exhibits a therapeutic window phenomenon requiring therapeutic drug monitoring. Analytical methods developed for Bu routine monitoring were aimed at using low volumes of biological fluids and development of simple procedures to facilitate the dosage adjustment. In this report, we describe a simple, rapid method for Bu measurement using dried blood spots (DBS) from only 5 mL of whole blood. METHODS: Bu extracted from DBS with methanol was measured by high-performance liquid chromatography with electrospray ionization and tandem mass spectrometry in multiple reaction monitoring mode using D8-Bu as an internal standard. The method was in-house validated evaluating trueness, repeatability, within-laboratory reproducibility, specificity and the lower limit of quantification (LLOQ). RESULTS: The method was linear in the calibration range of 100-2000 ng mL -1 (r 2 >0.99) encompassing the therapeutic concentrations of Bu. A good trueness (<14%), precision (<10%), and recovery (100%) were observed during validation of the method with quality controls of 300, 600 and 1400 ng mL -1. The LLOQ was determined as 50 ng mL -1 and no matrix or carryover effects were observed. The validated method was applied to measure Bu levels in four children receiving infusion of Bu prior to HSCT. A good correlation was observed between the Bu levels measured by DBS and dried plasma spot (DPS) (r 2 = 0.96) and between DPS and the GC/MS method (r 2 = 0.92). Bu was found to be stable in DBS up to 6 h at room temperature and for 24 h at 4 C. CONCLUSIONS: The new DBS method facilitates earlier dosage adjustment during Bu therapy by its specific and simple procedure using 5 mL of whole blood.

Applying Dried Blood Spot Sampling with LCMS Quantification in The Clinical Development Phase of Tasquinimod

“…To date, the DBS methodology is not covered by international guidelines for bioanalytical validation and is, therefore, seldom applied in clinical studies, even though several successful applications have been described [12,[22][23]. Procedures that demand small volumes of blood could also be of interest in pediatric clinical studies as well as in preclinical studies.…”

“…The drawbacks in DBS sampling that have drawn attention recently is its susceptibility to sample inhomogeneity and variations in hematocrit [7,8], which might compromise the accuracy of the method. However, recent developments in which the entire DBS sample is analyzed rather than a subsample have been reported, resolving this issue [9][10][11][12]. Another drawback is when comparison of quantitative data from a method based on DBS with methods using conventional plasma sampling is needed (i.e., when switching sampling approach in an ongoing drug development process).…”

Procedures and Practices for The Validation of Bioanalytical Methods Using Dried Blood Spots: A Review

“…Subsequently, a fixed diameter subpunch (usually 3-8 mm) is taken from the DBS and extraction solvent containing the internal standard is added to the punched sample. However, in four papers the use of the whole spot instead of a fixed diameter subpunch is described; for three assays venous blood is obtained and subsequently spotted onto the card with an accurate pipette [51,68,72], one assay uses a precision capillary to transfer blood from a fingerprick to the card [44]. With this approach, the spotting volume and spot homo geneity will not be an issue.…”

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

“…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 (8–11). Superiority of LC-MS/MS in immunometric assays relies on its higher specificity, although the extractive steps like liquid-liquid extraction (LLE) or protein precipitation (PP) if required cannot be automated (12).…”