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Chiral analysis of methadone and its main metabolite EDDP in hair: Incorporation depending on hair colour and metabolizer status


Fisichella, M; Steuer, Andrea E; Kraemer, Thomas; Baumgartner, Markus R (2014). Chiral analysis of methadone and its main metabolite EDDP in hair: Incorporation depending on hair colour and metabolizer status. Toxicologie Analytique et Clinique, 26(2):S12.

Abstract

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, 100×4, 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<blonde<brown<black). These factors range from 1 (white hair) up to > 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.

Abstract

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, 100×4, 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<blonde<brown<black). These factors range from 1 (white hair) up to > 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.

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Additional indexing

Item Type:Journal Article, not refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Legal Medicine
Dewey Decimal Classification:340 Law
610 Medicine & health
Language:English
Date:2014
Deposited On:29 Jul 2014 10:36
Last Modified:05 Apr 2016 17:59
Publisher:Elsevier Masson
ISSN:2352-0078
Publisher DOI:https://doi.org/10.1016/S2352-0078(14)70026-5

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