Development of an assay by liquid chromatography coupled to tandem mass spectrometry for thiopurines drugs in whole blood and application for their Therapeutic Drug Monitoring

Diplôme Master Development of an assay by liquid chromatography coupled to tandem mass spectrometry for thiopurines drugs in whole blood and application for their Therapeutic Drug Monitoring eng 2020-12-11 author Nguyen Nathalie Than-Mai-Vy 875067 director Decosterd Laurent 826211 Context: Thiopurines drugs are cytotoxic and immunosuppressive drugs, which are mainly used in acute lymphocytic leukemia, as well in various disorders of immune regulation and in inflammatory bowel disease. Currently, these thiopurines analyses are outsourced at the Laboratory of the University Hospital of Zurich (Universität Spital Zurich, USZ). However, the turnaround time (TAT) is slow (about twelve days according to the electronic hospital system). Clinicians have therefore requested to have this service available on site (i.e. at the CHUV) for a faster TAT. This work will help at improving the clinical follow-up of patients receiving thiopurine drugs. Objective: The first aim of this project is to develop and implement an in-house method for the quantification of thiopurines by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The two metabolites quantified are the 6-Thioguanine nucleotide (6-TGN) and the 6-Methyl-mercaptopurine (6-MMP). This step also includes all the different optimizations of the performance of the method in terms of recovery of the analytes, and to improve laboratory and clinical practicality. Extensive characterization of the preanalytic information (i.e. sample stability on various storage conditions) is also realized for hospital needs. Once all these steps are completed, a cross-validation between CHUV and USZ, to which the analyses have been so far outsourced, is carried-out to verify the strength of the agreement between both laboratories. In parallel, a comparison between the concentrations of the analytes determined in washed red blood cells and whole blood is performed. Finally, the information required for the therapeutic drug monitoring of Thiopurines has been reviewed and compiled in a “Thiopurines TDM information sheet”. The review of the current state of knowledge on TDM of thiopurines will help at interpreting thiopurines metabolites levels and if necessary, to adjust drug dosage accordingly so that 6-TGN and 6-MMP blood concentrations would lie in patients within the recommended therapeutic ranges. Methods: Extraction procedure for thiopurines is based on the landmark method established by Dervieux et al.: the main steps are the addition of DTT and perchloric acid in blood sample, followed by an incubation at 100°C for 60 minutes in a dry block heater. Such hydrolysis cleaves the 6-TGN to 6-TG and transforms the 6- MMP to its derivative (6-MMPD) which is the species that is actually quantified. Sensitivity and selectivity optimization also include improvement of LC and MS parameters. Several parameters are tested for extraction optimization, like DTT concentration, duration of hydrolysis, etc. Then, this analytical method is validated throughout a series of bioanalytical assays recommended by the FDA and EMEA authority. A Bland-Altman test (statistic test) is used for the cross-validation between the results obtained with the assay established at CHUV in comparison to those obtained by USZ. Results: These analytical optimizations allowed a fair chromatographic separation and resolution of analytes, while having a short analysis time (5 minutes). The trueness of the method was excellent when compared to reference external quality controls (i.e. bias lower than ±15%). Stability studies have demonstrated that the 10ml collecting tube of heparin supplemented with DTT prepared by USZ -that has been used so far- can be conveniently replaced by standard 2.6 ml EDTA blood tube, the latter being stable at least 72 hours at RT. This modification improves the convenience for both patients and health care personal. A good correlation with USZ results could be observed (slope of 0.94 for 6-TG (R=0,966) and 0,92 for 6-MMP (R=0,964) respectively). We have observed comparable thiopurines levels either determined in washed erythrocytes or whole blood. The validated range of the assay is comprised between 5 to 2’000 ng/ml for 6-TGN, and 50 to 20’000 ng/ml for 6-MMP. All analyzed patients samples were found to lie within these intervals. Conclusion: A LC-MS/MS method for quantifying simultaneously the two main metabolites of thiopurines drugs (6-thioguanine nucleotide and 6-methyl-mercaptopurine) in whole blood has been developed, optimized, and validated. A comprehensive optimization of the assay provides several improvements in terms of performance of the method (sensitivity, selectivity, trueness, etc.), and for the clinical perspective and patient management. A cross-validation with the laboratory of Zurich has been carried-out to implement this assay as a routine service at the University Hospital Center in Lausanne (CHUV). Whole blood can be directly used to quantify the target analytes because their concentration in plasma is insignificant. Finally, the “Thiopurines TDM information sheet” that has been prepared provides helpful information for the routine interpretation of 6-TGN and 6-MMP levels. Dewey 615 Master en Pharmacie 31 Section des sciences pharmaceutiques 17 false eabec74c-6640-4b0b-a491-d77db6a10453 Master thesis Restricted 210112200919_nguyena7__TPR.pdf 9787671 application/pdf restrictedAccess Faculté des sciences / Section des sciences pharmaceutiques

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Development of an assay by liquid chromatography coupled to tandem mass spectrometry for thiopurines drugs in whole blood and application for their Therapeutic Drug Monitoring				2020	90	0

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Nguyen, Nathalie Than-Mai-Vy