About the lab
International working group dedicated to pharmaceutical analysis and bioanalysis, expertise in drug metabolism, GC-MS, LC-MS, SFC-MS, steroid synthesis, drug-drug interactions, anti-doping research
Featured projects (4)
method developement and validation for different analytes and different matrices using chromatography coupled to (tandem) mass spectrometry
Featured research (20)
Differences in metabolic profiles can link to functional changes of immune cells in disease conditions. Here, we detail a protocol for the detection and quantitation of 19 metabolites in one analytical run. We provide the parameters for chromatographic separation and mass spectrometric analysis of isotopically labeled and unlabeled metabolites. We include steps for incubation and sample preparation of PBMCs and monocytes. This protocol overcomes the chromatographic challenges caused by the chelating properties of some metabolites.
Propranolol is a competitive non-selective beta-receptor antagonist that is available on the market as a racemic mixture. In the present study, glucuronidation of propranolol and its equipotent phase I metabolite 4-hydroxypropranolol by all 19 members of the human UGT1 and UGT2 families was monitored. UGT1A7, UGT1A9, UGT1A10 and UGT2A1 were found to glucuronidate propranolol, with UGT1A7, UGT1A9 and UGT2A1 mainly acting on (S)-propranolol, while UGT1A10 displays the opposite stereoselectivity. UGT1A7, UGT1A9 and UGT2A1 were also found to glucuronidate 4-hydroxypropranolol. In contrast to propranolol, 4-hydroxypropranolol was found to be glucuronidated by UGT1A8 but not by UGT1A10. Additional biotransformations with 4-methoxypropanolol demonstrated different regioselectivities of these UGTs with respect to the aliphatic and aromatic hydroxy groups of the substrate. Modeling and molecular docking studies were performed to explain the stereoselective glucuronidation of the substrates under study.
Terbutaline is mainly metabolized by sulfoconjugation stereoselectively, favoring its (S)-(+) enantiomer. Reported chiral separations of Terbutaline enantiomers were achieved by various chromatographic methods. However, the simultaneous enantioseparation of Terbutaline and the monosulfate conjugate metabolites was never reported. This study aims at shedding light on the influential factors and interactions leading to successful enantioseparation of Terbutaline and its monosulfate conjugate pairs by Supercritical Fluid Chromatography (SFC) for the first time within a Quality by Design framework using Design of Experiments. The effect of molarity of mobile phase additive, mobile phase flow rate, column temperature and back pressure were evaluated. Compared to previous reports, the response surface interestingly revealed the favorability of high temperature and high flow rate up to 2.25 ml/min for resolution of the two pairs of enantiomers on polysaccharide chiral stationary phase CHIRALPAK IC. In addition, a switch in the elution order of Terbutaline and the sulfate conjugate peak pairs was observed upon elimination of the mobile phase additive where the sulfate conjugate underwent intra-molecular ionic interactions and the change in elution order was only due to TER behavior. The multifactorial interactions would not have been detected with the common one-factor-at-a-time approach during method development, demonstrating the superiority and importance of the Analytical Quality by Design frame in enantioseparation.
Rationale: This work demonstrated the high potential of combining high-resolution mass spectrometry with chemometric tools, using metabolomics as a guided tool for anti-doping analysis. The administration of 7-keto-DHEA was studied as a proof-of-concept of the effectiveness of the combination of knowledge-based and machine-learning approaches to differentiate the changes due to the athletic activities from those due to the recourse to doping substances and methods. Methods: Urine samples were collected from 5 healthy volunteers before and after an oral administration by identifying three-time intervals. Raw data were acquired by injecting less than one microliter of derivatized samples into an Agilent Technologies 8890 Gas Chromatograph coupled to an Agilent Technologies 7250 Accurate-Mass Quadrupole Time-of-Flight, by using a low energy electron ionization source, and then they were preprocessed to align peak retention times with the same accurate mass. The resulting data table was subjected to multivariate analysis. Results: Multivariate analysis showed a high similarity between the samples belonging to the same collection interval and a clear separation between the different excretion intervals. The discrimination between blank and long excretion groups may suggest the presence of long excretion markers, which are particularly significant in anti-doping analysis. Furthermore, matching the most significant features with some of the metabolites reported in the literature data demonstrated the rationality of the proposed metabolomics-based approach. Conclusions: The application of metabolomics tools as an investigation strategy could reduce the time and resources required to identify and characterize intake markers maximizing the information that can be extracted from the data and extending the research field by avoiding a priori bias. Therefore, metabolic fingerprinting of prohibited substance intakes could be an appropriate analytical approach to reduce the risk of false-positive/negative results, aiding in the interpretation of "abnormal" profiles and discrimination of pseudo-endogenous steroid intake in the anti-doping field.
Dehydrochloromethyltestosterone (DHCMT) is an anabolic-androgenic steroid that was developed by Jenapharm in the 1960s and was marketed as Oral Turinabol ® . It is prohibited in sports at all times; nevertheless, there are several findings by anti-doping laboratories every year. New long-term metabolites have been proposed in 2011/12, which resulted in adverse analytical findings in retests of the Olympic games of 2008 and 2012. However, no controlled administration trial monitoring these long-term metabolites was reported until now. In this study, DHCMT (5 mg, p.o.) was administered to five healthy male volunteers and their urine samples were collected for a total of 60 days. The unconjugated and the glucuronidated fraction were analyzed separately by gas chromatography coupled to tandem mass spectrometry. The formation of the described long-term metabolites was verified, and their excretion monitored in detail. Due to interindividual differences there were several varieties in the excretion profiles among the volunteers. The metabolite M3, which has a fully reduced A-ring and modified D-ring structure, was identified by comparison with reference material as 4α-chloro-17β-hydroxymethyl-17α-methyl-18-nor-5α-androstan-13-en-3α-ol. It was found to be suitable as long-term marker for the intake of DHCMT in four of the volunteers. In one of the volunteers, it was detectable for 45 days after single oral dose administration. However, in two of the volunteers M5 (already published as long-term metabolite in the 1990s) showed longer detection windows. In one volunteer M3 was undetectable but another metabolite, M2, was found as the longest detectable metabolite. The last sample clearly identified as positive was collected between 9.9 and 44.9 days. Furthermore, the metabolite epiM4 (partially reduced A-ring and a modified D-ring structure which is epimerized in position 17 compared to M3) was identified in the urine of all volunteers with the help of chemically synthesized reference as 4-chloro-17α-hydroxymethyl-17β-methyl-18-nor-androsta-4,13-dien-3β-ol. It may serve as additional confirmatory metabolite. It is highly recommended to screen for all known metabolites in both fractions, glucuronidated and unconjugated, to improve identification of cheating athletes. This study also offers some deeper insights into the metabolism of DHCMT and of 17α-methyl steroids in general.