Julien Déglon

Background: Dried blood spots (DBS) sampling has gained popularity in the bioanalytical community as an alternative to conventional plasma sampling, as it provides numerous benefits in terms of sample collection and logistics. The aim of this work was to show that these advantages can be coupled with a simple and cost-effective sample pretreatment, with subsequent rapid LC–MS/MS analysis for quantitation of 15 benzodiazepines, six metabolites and three Z-drugs. For this purpose, a simplified offline procedure was developed that consisted of letting a 5-µl DBS infuse directly into 100 µl of MeOH, in a conventional LC vial. Results: The parameters related to the DBS pretreatment, such as extraction time or internal standard addition, were investigated and optimized, demonstrating that passive infusion in a regular LC vial was sufficient to quantitatively extract the analytes of interest. The method was validated according to international criteria in the therapeutic concentration ranges of the selected comp...

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 μL 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 (–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 μL of whole blood. Copyright © 2012 John Wiley & Sons, Ltd.

Because of the emergence of dried blood spots (DBS) as an attractive alternative to conventional venous plasma sampling in many pharmaceutical companies and clinical laboratories, different analytical approaches have been developed to enable automated handling of DBS samples without any pretreatment. Associated with selective and sensitive MS–MS detection, these procedures give good results in the rapid identification and quantification of drugs (generally less than 3 min total run time), which is desirable because of the high throughput requirements of analytical laboratories. The objective of this review is to describe the analytical concepts of current direct DBS techniques and to present their advantages and disadvantages, with particular focus on automation capacity and commercial availability. Finally, an overview of the different biomedical applications in which these concepts could be of major interest will be presented.

This work presents a strategy for the evaluation of differences in plasma phospholipid content between atherosclerotic and healthy mice from micro volumes (2 microL) spotted on filter paper. Dried plasma spots (DPS) were directly desorbed into a triple quadrupole linear ion trap mass spectrometer using a homemade prototype, ensuring high-throughput analysis of dried spots without any sample pretreatment. Multiple positive and negative neutral loss and precursor ion scans were simultaneously acquired in a single loop, allowing oriented fingerprinting until 2700 potential species including phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and sphingomyelin (SM) classes. The phospholipidic variations between 15 healthy and 15 atherosclerotic mice were evaluated using t tests, matrix reduction and merging, and principal component analysis (PCA) as a chemometric statistical approach. The discriminating ions in PCA analysis were qualitatively identified in an information dependent acquisition (IDA) manner using enhanced resolution and enhanced product ion scans. PCA demonstrates a clear clustering between healthy and diseased mice. Regarding the most relevant variables identified, this procedure has confirmed the role of SM and PS classes in atherosclerosis and has established potential biomarkers shown to be significantly up- or down-regulated with the disease. The results presented in this work demonstrate the sample processing and analysis potential of the developed strategy to evaluate new biomarkers and the state of a disease.

An assay for the simultaneous analysis of pharmaceutical compounds and their metabolites from micro‐whole blood samples (i.e. 5 μL) was developed using an on‐line dried blood spot (on‐line DBS) device coupled with hydrophilic interaction/reversed‐phase (HILIC/RP) LC/MS/MS. Filter paper is directly integrated to the LC device using a homemade inox desorption cell. Without any sample pretreatment, analytes are desorbed from the paper towards an automated system of valves linking a zwitterionic‐HILIC column to an RP C18 column. In the same run, the polar fraction is separated by the zwitterionic‐HILIC column while the non‐polar fraction is eluted on the RP C18. Both fractions are detected by IT‐MS operating in full scan mode for the survey scan and in product ion mode for the dependant scan using an ESI source. The procedure was evaluated by the simultaneous qualitative analysis of four probes and their relative phase I and II metabolites spiked in whole blood. In addition, the method was successfully applied to the in vivo monitoring of buprenorphine metabolism after the administration of an intraperitoneal injection of 30 mg/kg on adult female Wistar rat.