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O18: Chiral analysis of methadone and its main metabolite EDDP in hair: Incorporation depending on hair colour and metabolizer status
S12
Wednesday 11 June 2004 / Toxicologie Analytique & Clinique (2014) 26, S4-S14
displayed significantly higher MCV (median 94.0 vs 92.1 fL; p=0.001), GGT (28 vs 19 UI/L; p<0.0005) and % CDT (1.4% vs 1.1%; p<0.0005), but not AST and ALT, than EtG-negative subjects. Interestingly, up to 6 out of 12 (50%) only % CDT-positive subjects had EtG<15 pg/mg, whereas up to 27 out of 46 (59%) only EtG-positive subjects had % CDT<1.1%. Conclusion. – Large variability exists between % CDT and EtG in detecting chronic alcohol consumption. To identify alcohol abuse in a forensic context, such as traffic medicine, the combination of different alcohol biomarkers and appropriate medical history is mandatory for the correct interpretation of individual cases.
SESSION 4: ANALYTICAL APPROACH O18
Chiral analysis of methadone and its main metabolite EDDP in hair: Incorporation depending on hair colour and metabolizer status M. Fisichella (1), A. E. Steuer (2), T. Kraemer (2), M. R. Baumgartner (2) (1) Institute of legal medicine, catholic university of sacred heart, Rome, Italy; (2) Zurich institute of forensic medicine, department of forensic pharmacology and toxicology, university of Zurich, Zurich, Switzerland. Introduction. – Methadone (MTD) is an analgesic compound used for treatment of severe pain or opioid addiction. MTD is therapeutically administered as racemic mixture although opioid activity is attributed to the R-enantiomer. In some countries the pure R-enantiomer is also used. Three cytochrome p450 (CYP) enzymes are involved in MTD metabolism, namely 2B6, 2C19, and 3A4 with different stereo selectivity and two of them (2B6, 2C19) being polymorphically expressed. Incorporation of a drug and its metabolite into hair matrix might be a mirror mimicking genetic polymorphism. The aim was to establish an enantioselective quantification of MTD and EDDP and to adopt this method for non-pigmented and pigmented hair samples of different colours. Methods. – After washing, the hair samples were cut into snippets and extracted in a single step with ultrasonication. These extracts were analysed without any purification. MTD and EDDP enantiomers were determined in hair samples by HPLC (CHIRALPAK AGP column, 100x4, 5um) coupled to a tandem mass spectrometer (ABSciex QTrap 3200, 2 MRMs) using deuterated standards of MTD and EDDP, respectively. The method was validated and applied to hair samples collected during driving licence assessments of 152 participants of a long term MTD maintenance program (16% women, 84% men). Unpigmented hair samples were obtained by manual separation of grey hair strands (n = 40) providing the possibility of a direct comparison of the incorporation rate of MTD and EDDP into unpigmented hair versus that of pigmented hair of different colours (blond, brown, dark brown/black). The incorporation rate was defined by the clearance index (CI) calculated as ratio of the self-declared monthly dose to the MTD concentration. Results and Discussion. – The ratio of MTD concentration in pigmented to MTD concentration in not-pigmented hair (mean value 5.5) was smaller than that for EDDP (8.5), indicating that the incorporation of EDDP is more affected by pigmentation. However, these ratios exhibited only slight enantioselectivity. For the pigmented samples (n=159), the metabolic ratio EDDP/MTD was always lower for the R-enantiomers (0.06 ± 0.025) than for the S-form (0.13 ± 0.061). This distribution pattern of these ratio values might be indicative for the phenotype of the subject. CI was calculated to be the lowest for participants with black hair and the highest for participants with non-pigmented hair (white hair); the pigmentationdependent incorporation factors ranged from 1 (non-pigmented) up to > 10 (black hair).
Conclusion. – A chiral LC-MS/MS method for the quantification of methadone and EDDP in hair was established and validated. Incorporation factors for MTD were depending on the hair colour (white 10 for black hair samples; the clearance index shows an inverse order. The metabolic ratio for S-enantiomers was higher than for the R-enantiomers, indicating a stereo-selective biotransformation (CYP2B6) of MTD. O19
Identification and quantification of THC-COOH glucuronide in hair by ultra-performance liquid chromatography tandem mass spectrometry S. Pichini (1), S. Martello (2), M. Gottardi (3), E. Marchei (1), M. Pellegrini (1), F. Svaizer (3), A. Lotti (3), R. Pacifici (1), M. Chiarotti (2) (1) Department of therapeutic research and medicines evaluation, istituto superiore di sanitá, Rome, Italy; (2) Università cattolica del sacro cuore, policlinico Agostino-Gemelli, Rome, Italy; (3) Laboratorio di sanità pubblica (LSP), azienda provinciale servizi sanitari, Trento, Italy. Introduction. – Disclosing chronic use of cannabis has always been a difficult task due to the fact that hair testing of THC alone is not conclusive of use and hair testing of THC-COOH require specific and sensitive instrumentation and laboratory skill, not available for many analytical laboratories. We hypothesized the presence of THC-COOH glucuronide in hair as possible alternative biomarker of repeated consumption of cannabis products. Methods. – We developed and validated an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to identify and quantify THC-COOH glucuronide in hair after applying one hour digestion of 25 mg keratin matrix with M3 (Comedical Spa, Italy) reagent at 100 °C, using deuterated THC-COOH glucuronide as internal standard. An amount of 10 μl was injected in the ULPC-MS/MS system. Chromatographic separation was carried out on a Acquity UPLC HSS C18 column (2,1 mm x 150 mm, 1.8 μm) using a linear gradient elution with two solvents:0.1% formic acid in acetonitrile (solvent A) and 5 mM ammonium formate pH 3 (solvent B). The flow rate was kept constant at 0.40 mL/min during the analysis. The separated analytes were detected with a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode via positive electrospray ionization (ESI). The applied ESI conditions were the following: capillary voltage 1,3 kV, desolvation temperature 600 °C, source temperature 150 °C, cone gas flow rate 20 L/h, desolvation gas flow rate 1000 L/h and collision gas flow rate 0.13 mL/min, cone voltage of 65 V. Following transitions were considered: 521.2→345.03 (Collision energy:16), 521.2 →327.07 (Collision energy: 22),521.2 → 299.09 (Collision energy: 32), with the first one used for quantification. Results. – Linear calibration curves were obtained for THC-CCOH glucuronide with correlation coefficients (r2) of 0.99 and a LOQ of 0.1 pg/mg hair. Analytical recovery was between 70.9 and 100.7%. Intra and inter-assay imprecision and inaccuracy were always lower than 10%. No additional peaks due to endogenous substances which could have interfered with the detection of the analytes under investigation were observed in drug-free hair samples. No psychoactive drugs other than the compounds under investigation interfered with the assay. Blank hair samples injected after the highest point of the calibration curve did not present any traces of carryover. The matrix effect in quality control samples ranged from 80 to 101%. Preliminary analysis on 9 different hair samples of consumers disclosed the presence of THC-CCOH glucuronide in the range of 0.30-1.22 pg/mg with by median value of 0.58 pg/mg hair. Conclusions. – Simple extraction, identification and quantification of THC-COOH glucuronide in hair by UPLC-MS/MS was developed, validated and tested for its feasibility in clinical samples and provided a good start to consider THC-CCOH glucuronide as alternative hair biomarker of repeated cannabis consumption.
Wednesday 11 June 2004 / Toxicologie Analytique & Clinique (2014) 26, S4-S14
displayed significantly higher MCV (median 94.0 vs 92.1 fL; p=0.001), GGT (28 vs 19 UI/L; p<0.0005) and % CDT (1.4% vs 1.1%; p<0.0005), but not AST and ALT, than EtG-negative subjects. Interestingly, up to 6 out of 12 (50%) only % CDT-positive subjects had EtG<15 pg/mg, whereas up to 27 out of 46 (59%) only EtG-positive subjects had % CDT<1.1%. Conclusion. – Large variability exists between % CDT and EtG in detecting chronic alcohol consumption. To identify alcohol abuse in a forensic context, such as traffic medicine, the combination of different alcohol biomarkers and appropriate medical history is mandatory for the correct interpretation of individual cases.
SESSION 4: ANALYTICAL APPROACH O18
Chiral analysis of methadone and its main metabolite EDDP in hair: Incorporation depending on hair colour and metabolizer status M. Fisichella (1), A. E. Steuer (2), T. Kraemer (2), M. R. Baumgartner (2) (1) Institute of legal medicine, catholic university of sacred heart, Rome, Italy; (2) Zurich institute of forensic medicine, department of forensic pharmacology and toxicology, university of Zurich, Zurich, Switzerland. Introduction. – Methadone (MTD) is an analgesic compound used for treatment of severe pain or opioid addiction. MTD is therapeutically administered as racemic mixture although opioid activity is attributed to the R-enantiomer. In some countries the pure R-enantiomer is also used. Three cytochrome p450 (CYP) enzymes are involved in MTD metabolism, namely 2B6, 2C19, and 3A4 with different stereo selectivity and two of them (2B6, 2C19) being polymorphically expressed. Incorporation of a drug and its metabolite into hair matrix might be a mirror mimicking genetic polymorphism. The aim was to establish an enantioselective quantification of MTD and EDDP and to adopt this method for non-pigmented and pigmented hair samples of different colours. Methods. – After washing, the hair samples were cut into snippets and extracted in a single step with ultrasonication. These extracts were analysed without any purification. MTD and EDDP enantiomers were determined in hair samples by HPLC (CHIRALPAK AGP column, 100x4, 5um) coupled to a tandem mass spectrometer (ABSciex QTrap 3200, 2 MRMs) using deuterated standards of MTD and EDDP, respectively. The method was validated and applied to hair samples collected during driving licence assessments of 152 participants of a long term MTD maintenance program (16% women, 84% men). Unpigmented hair samples were obtained by manual separation of grey hair strands (n = 40) providing the possibility of a direct comparison of the incorporation rate of MTD and EDDP into unpigmented hair versus that of pigmented hair of different colours (blond, brown, dark brown/black). The incorporation rate was defined by the clearance index (CI) calculated as ratio of the self-declared monthly dose to the MTD concentration. Results and Discussion. – The ratio of MTD concentration in pigmented to MTD concentration in not-pigmented hair (mean value 5.5) was smaller than that for EDDP (8.5), indicating that the incorporation of EDDP is more affected by pigmentation. However, these ratios exhibited only slight enantioselectivity. For the pigmented samples (n=159), the metabolic ratio EDDP/MTD was always lower for the R-enantiomers (0.06 ± 0.025) than for the S-form (0.13 ± 0.061). This distribution pattern of these ratio values might be indicative for the phenotype of the subject. CI was calculated to be the lowest for participants with black hair and the highest for participants with non-pigmented hair (white hair); the pigmentationdependent incorporation factors ranged from 1 (non-pigmented) up to > 10 (black hair).
Conclusion. – A chiral LC-MS/MS method for the quantification of methadone and EDDP in hair was established and validated. Incorporation factors for MTD were depending on the hair colour (white
Identification and quantification of THC-COOH glucuronide in hair by ultra-performance liquid chromatography tandem mass spectrometry S. Pichini (1), S. Martello (2), M. Gottardi (3), E. Marchei (1), M. Pellegrini (1), F. Svaizer (3), A. Lotti (3), R. Pacifici (1), M. Chiarotti (2) (1) Department of therapeutic research and medicines evaluation, istituto superiore di sanitá, Rome, Italy; (2) Università cattolica del sacro cuore, policlinico Agostino-Gemelli, Rome, Italy; (3) Laboratorio di sanità pubblica (LSP), azienda provinciale servizi sanitari, Trento, Italy. Introduction. – Disclosing chronic use of cannabis has always been a difficult task due to the fact that hair testing of THC alone is not conclusive of use and hair testing of THC-COOH require specific and sensitive instrumentation and laboratory skill, not available for many analytical laboratories. We hypothesized the presence of THC-COOH glucuronide in hair as possible alternative biomarker of repeated consumption of cannabis products. Methods. – We developed and validated an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to identify and quantify THC-COOH glucuronide in hair after applying one hour digestion of 25 mg keratin matrix with M3 (Comedical Spa, Italy) reagent at 100 °C, using deuterated THC-COOH glucuronide as internal standard. An amount of 10 μl was injected in the ULPC-MS/MS system. Chromatographic separation was carried out on a Acquity UPLC HSS C18 column (2,1 mm x 150 mm, 1.8 μm) using a linear gradient elution with two solvents:0.1% formic acid in acetonitrile (solvent A) and 5 mM ammonium formate pH 3 (solvent B). The flow rate was kept constant at 0.40 mL/min during the analysis. The separated analytes were detected with a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode via positive electrospray ionization (ESI). The applied ESI conditions were the following: capillary voltage 1,3 kV, desolvation temperature 600 °C, source temperature 150 °C, cone gas flow rate 20 L/h, desolvation gas flow rate 1000 L/h and collision gas flow rate 0.13 mL/min, cone voltage of 65 V. Following transitions were considered: 521.2→345.03 (Collision energy:16), 521.2 →327.07 (Collision energy: 22),521.2 → 299.09 (Collision energy: 32), with the first one used for quantification. Results. – Linear calibration curves were obtained for THC-CCOH glucuronide with correlation coefficients (r2) of 0.99 and a LOQ of 0.1 pg/mg hair. Analytical recovery was between 70.9 and 100.7%. Intra and inter-assay imprecision and inaccuracy were always lower than 10%. No additional peaks due to endogenous substances which could have interfered with the detection of the analytes under investigation were observed in drug-free hair samples. No psychoactive drugs other than the compounds under investigation interfered with the assay. Blank hair samples injected after the highest point of the calibration curve did not present any traces of carryover. The matrix effect in quality control samples ranged from 80 to 101%. Preliminary analysis on 9 different hair samples of consumers disclosed the presence of THC-CCOH glucuronide in the range of 0.30-1.22 pg/mg with by median value of 0.58 pg/mg hair. Conclusions. – Simple extraction, identification and quantification of THC-COOH glucuronide in hair by UPLC-MS/MS was developed, validated and tested for its feasibility in clinical samples and provided a good start to consider THC-CCOH glucuronide as alternative hair biomarker of repeated cannabis consumption.