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Chemicals and reagents
EtG (ref: EGL-332-10) and its deuterated analogue EtG-D5 (ref: EGL-780-10), used as an internal standard (IS), were obtained from Lipomed (Souffelweyersheim, France). Methanol (MeOH, ref: 20837.320) and formic acid (99-100%, ref: 20318-297) were obtained from VWR Prolabo (Fontenay-sous-Bois, France). Ammonium hydroxide solution (25%, ref: 1.05432.1000) and hexane (ref: HEO2212500) were purchased from Merck (Chibret, France) and Scharlau (France), respectively. The derivatization agent, pentafluoropropionic anhydride (PFPA 99%, ref: 206-604-2), was obtained from Sigma Aldrich (Saint-Quentin Fallavier, France). All chemicals were of the highest analytical grade. Solid phase extraction (SPE) Oasis® MAX cartridges (3 mL, 60 mg, ref: 86000368) and a SPE tank system working under vacuum were supplied by Waters (Saint-Quentin en Yvelines, France).
Samples preparation
Blank urine and serum samples were collected from five volunteers (who are not alcohol consumers or had stopped drinking alcohol for at least one week), here referred to as alcohol abstainers, and were analysed for the presence of EtG before the validation phase. Citrated tubes were used for blood sampling, whereas clean and dry containers were used for urine samples. Blood samples were centrifuged immediately to separate the serum. No additional preservative was used during the sampling. All samples were stored at – 20°C in order to maintain a good stability along the validation time [23].
Urine sample preparation
Appropriate volumes of standard EtG solutions were added to 1 mL of blank urine, resulting in final concentrations of 10, 100, 1000, 5000, 8000 and 10000 ng of EtG per mL of urine. Then, 25 µL of each urine sample was diluted by the addition of 975 µL of distilled water in the presence of 25 μL of EtG-D5 solution (1000 ng/mL in MeOH). The final mixture was vortexed and transferred onto an Oasis® MAX SPE cartridge.
Serum sample preparation
Calibration samples were prepared by adding suitable amounts of the EtG standard solutions to 0.5 mL of blank serum, resulting in final concentrations of 5, 10, 50, 100, 500 and 1000 ng/mL. 50 μL of EtG-D5 solution (1000 ng/mL in MeOH) was added to each sample. Then, these samples were applied for clean-up and extraction to an (Oasis® MAX) cartridge.
Extraction procedure and derivatization
The prepared samples were applied to an Oasis® MAX cartridge conditioned with 1 mL of MeOH and 1 mL of deionized water. Special care was taken to ensure that the columns did not dry out between the conditioning steps. To prevent the column from drying-out, which could reduce the extraction yield, once the conditioning has started, we maintained water in the SPE column by replacing water that drained through the column. The cartridge was then washed with 1 mL of ammonium hydroxide (NH4OH, 2%) and, secondly, with 1 mL of MeOH. A strong vacuum was applied for 5 min to remove all residual liquid. EtG was eluted from the cartridge using 1 mL of a methanol/formic acid (98:2, v/v) solution. The eluate was evaporated to dryness under a stream of nitrogen using a heated metal block at 70°C. The residue was derivatized with 100 µL of pentafluoropropionic anhydride (PFPA) which had been previously shown to be the best agent for EtG derivatization with good stability up to 1 h of incubation at room temperature, as well as at 60°C [16]. The tubes were tightly closed, mixed by vortexing (10 s), heated for 30 min at 70°C, then dried under N 2 and, finally, the residue was reconstituted in 50 µL of hexane. One µL of extract was injected into the GC-MS/MS system.
Instruments and GC-MS/MS conditions
Identification and quantification of EtG were performed in a GC-MS/MS system, which consists of a gas chromatograph (7890A series, Agilent, Massy, France) equipped with an automatic injector (7683B series, Agilent), coupled with a tandem mass spectrometer (Quattro MicroTM GC MICROMASS®, Waters). Chromatographic separation was achieved with a fused silica capillary column AT5-ms (ref: 15807, Alltech, Templemars, France) (30 m × 0.25 mm. × 0.25 µm).
The carrier gas was helium with a constant flow of 1 mL/min. One µL was injected in splitless mode at an injection temperature of 250°C. The initial oven temperature of 60°C was kept for 2 min, increased first at 35°C/min to 250°C, and then kept at this temperature for 8.43 min. The transfer line was held at 270°C. Retention times were of 6.53 min for EtG and 6.52 min for EtG-D5. Samples were ionized by NCI with methane, as the reagent gas, at a pressure of 0.2 mTorr. The ion source temperature was kept at 100°C.
Data acquisition and MS control were performed using the software Mass-Lynx version 4.1 (Waters). The GC-MS/MS was performed in multiple reaction monitoring (MRM) mode. The precursor ions m/z 496 and 347 for EtG and m/z 501 for EtG-D5 were selected in the first quadrupole. These precursor ions were chosen for further fragmentation according to their selectivity and abundance in the mass spectra. The resulting product ions m/z 119 and 163 for EtG and m/z 163 for EtG-D5 were selected in the second quadrupole after collision (in the collision cell) with argon, as the collision gas, at a pressure of 5 mTorr. The collision energy.
was maintained at γ0, 10 and 8 eV for the transitions m/z 496→119, γ47→16γ and 496→16γ, respectively, and 10 eV for the transition m/z 501→16γ. Transition m/z 496→16γ.
was retained for the EtG quantification, whereas transitions m/z γ47→16γ and m/z 496→119
were used for the EtG identification. The electron multiplier was set up at 550 V.
Table of contents :
Abstract
Résumé
INTRODUCTION
Part I: Biomarkers of alcohol consumption
1. What is an ideal biomarker of alcohol consumption?
2. Indirect biomarkers of alcohol consumption
2.1. Gamma–glutamyltransferase
β.β. Aminotransferases
2.3. Mean corpuscular volume
2.4. Carbohydrate–deficient transferrin
2.5. GGT–CDT combinations
3. Direct biomarkers of alcohol consumption
3.2. Fatty acid ethyl esters
3.3. Phosphatidylethanol
3.4. Ethylglucuronide & Ethylsulfate
4. Other biomarkers of alcohol consumption
4.1. Acetaldehyde & acetaldehyde-protein adducts
4.2. 5-hydroxytryptophol
4.3. Total serum sialic acid
4.4. Plasma sialic acid index of apolipoprotein J
4.5. Beta-hexosaminidase
4.6. Other markers
Part II: Ethylglucuronide and research methodology
1. Generality
2. Interest of ethylglucuronide …
2.1. Characteristics
2.2. Stability
2.3. Detection window
2.4. Cut-off values
3. Context of EtG determination: clinical & forensic applications
3.1. In clinical settings
3.2. In forensic settings
4. Analytical methods for measurement of EtG
5. Interpretation, precautions, and confounding factors
6. UDP-glucuronosyltransferases & interindividual variability
6.1. The glucuronidation reaction
6.1. Genetic polymorphisms of UDP-glucuronosyltransferases
7. In vitro phenotyping of drug-metabolizing UGT enzymes
8. Quantitative in vitro–in vivo extrapolation
8.1. Determination of kinetic parameters
8.β. Interpretation and prediction of metabolic clearance
9. Prediction of Drug-Drug Interactions
9.1. Enzyme inhibition
9.2. Enzyme induction
OBJECTIVES & CONTEXT
PERSONAL WORK & RESEARCH FINDINGS
GENERAL DISCUSSION
PERSPECTIVES & CONCLUSION
BIBLIOGRAPHY
