Forens Sci Med Pathol (2007) 3:119–123 DOI 10.1007/s12024-007-0008-3
ORIGINAL PAPER
Individual variations of amitriptyline biotransformation examined in scalp hair samples Detlef Thieme Æ Dagmar Schmid Æ Hans Sachs
Accepted: 22 December 2006 / Published online: 6 September 2007 Ó Humana Press Inc. 2007
Abstract The identification of amitriptyline, nortriptyline and its hydroxy-metabolites including a subsequent measurement of concentration profiles in hair samples was carried out to evaluate the administration history of amitriptyline. Analyses were carried out using liquid chromatography—tandem mass spectrometry, which permits simultaneous identification of all relevant substances in hair at low target concentrations (limit of detection better than 0.5 pg/mg). Standard hair sample preparation was applied for the estimation of average substance concentration in a hair bundle, while segmentation of individual hairs was utilised to examine accurate concentration profiles. Replication of analyses demonstrated a good reproducibility of individual hair concentration profiles, which proved to coincide for all relevant compounds. Significant variations of metabolite ratios (e.g. nortriptyline to amitriptyline and E10- to Z10-hydroxynortriptyline) between individuals suggest a correlation between hair concentration and metabolic phenotype. Different concentration ratios of certain metabolites in hair are highly correlated, indicating a systematic association between demethylation and stereo-specificity of hydroxylation. The trans isomers The data were presented at the Hair Testing Meeting, Vadstena, Sweden, May 2006. We hereby state that we do not have a significant financial interest or other relationship with any product manufacturer or provider of services discussed in this article. D. Thieme (&) D. Schmid Institute of Forensic Medicine, Frauenlobstr. 7a, 80337 Munich, Germany e-mail: [email protected] D. Thieme H. Sachs Forensic Toxicological Centre, Bayerstr. 53, 80335 Munich, Germany
of hydroxy-metabolites become significantly more prevalent with increasing degree of demethylation of amitriptyline or hydroxylation of amitriptyline. Keywords Antidepressants Amitriptyline Liquid chromatography Mass spectrometry Hair analysis Forensic toxicology
Introduction Many of the classical tricyclic antidepressants undergo a complex biotransformation, which is typically influenced by large individual variations, depending on parameters like sex, age, ethnicity and administration of other pharmaceutical substances. Therefore, therapeutic dosages should be adjusted and controlled individually, making a clinical routine application rather complex. Amitriptyline in particular was the subject of recent studies to investigate the influence of individual variations in metabolism and the resulting response to substance administration [1–3]. Typical side effects (dizziness, somnolence, unconsciousness, swollen tongue, speech disorders, circulatory failure, diarrhoea, constipation) are mainly correlated with serum concentrations of the active metabolite nortriptyline (Fig. 1), while the parent compound as well as active metabolites are not thought to be critical [4, 5]. The case of an illegal administration of amitriptyline to a group of children (aiming to the intended sedation) was retrospectively investigated and hair analysis was the only available option due to a time span between last administration and sample collection of at least 3 weeks. No reliable data on the amounts and duration of amitriptyline administration were available and systematic and qualified exploration of side effects was not carried out. Hair anal-
120
Forens Sci Med Pathol (2007) 3:119–123
Liquid chromatography – Electrospray – Mass spectrometry
N- Demethylation
N N
OH
NH
E( Z)10- H ydro xyl atio n
OH
NH
Fig. 1 Amitriptyline is mainly biotransformed by N-demethylation and hydroxylation in position 10, resulting in mainly trans (E) and partially cis (Z) isomers. Other metabolic reactions (e.g. glucuronidation, N-oxidation) can be ignored in hair analysis due to their low incorporation rate
ysis was carried out in 23 infants (age 2–6 years) to study additional details like administration interval and individual metabolite response.
Methods and materials
All analyses were carried out using an API 4000 mass spectrometer (Applied Biosystems), in electrospray ionisation mode (Turbo-Ion-Spray source) coupled to an Agilent 1100 LC system (binary pump and autosampler). Reference material of amitriptyline, nortriptyline (both Cerilliant), E10-hydroxynortriptyline and Z10-hydroxynortriptyline (both Sigma) was used for optimisation of the ionisation and fragmentation conditions while MS parameters of hydroxyamitriptyline were obtained from investigation of extracts of biological material. The relevant substance specific fragmentation reactions and corresponding values of declustering potential (DP) and collision energy (CE) are listed in Table 1. Both isomers of corresponding hydroxy-metabolites are mass spectrometrically identical and need to be separated chromatographically, which was achieved using a Zorbax Eclipse RP 18 column (4.6 * 75 mm, 3.5 lm particle size). The mobile phases consist of a 2 mM ammonium acetate buffer (Baker) in a mixture of water (Merck) and acetonitrile (Baker) at a composition of (90/10, v/v, buffer A) or (10/90, v/v, buffer B). The solvent gradient starts at a composition of 100%A (0–1 min) continuously changing to 100%B (isocratic from 6.5 to 7.5 min) at a constant flow of 0.7 ml/min. To accelerate the chromatography for profiling purposes, isochratic (100%B) chromatography on Synergy Polar-RP narrow bore column (Phenomenex, 75 * 2.0 mm, 4 lm particle size) was utilised (Fig. 2). Respective injection volumes were 10 ll.
Sample preparation There are two relevant protocols for preparation of hair samples either for routine analyses of entire hair bundles (representing the total time frame) or for profiling small segments of individual hairs [6]. The standard procedure for drug testing consists of decontamination of the intact hair strands by 5 min agitation in a gas-tight tube with 5 ml petroleum benzene (boiling range to 40°C, Merck), drying and cutting of 50 mg hair into pieces of 1–2 mm length. After adding 100 ng of the internal standard (MPPH, 5-(4methylphenyl)-5-phenyl hydantoine, Serva), the hair powder was extracted in 3 ml of methanol (Merck) (3 h ultrasonification at 55°C). Individual hairs were typically segmented into 5 mm pieces after fixation on an adhesive tape. Resulting hair concentrations were expressed as amount per cm [pg/cm] due to the unknown total mass of these segments. The resulting hair segments were transferred into vials, containing 5 ng of the internal standard (MPPH) in 30 ll of the mobile phase. The vials were directly analysed by LCMS after 3 h of ultrasonification at 55°C.
Results and discussion Significance of concentration profiles The concentration profiles of amitriptyline and nortriptyline were examined in single hairs leading to high Table 1 Ionisation and fragmentation parameters of the relevant compounds
Amitriptyline Nortriptyline
Declustering potential [V]
Precursor ion [Da]
Product ion [Da]
Collision energy [eV]
61
278
117
35
191
49
56
264
117 233
29 21
Hydroxyamitriptyline 71
294
215
53
Hydroxynortriptyline 71
280
216
35
215
49
216
33
Forens Sci Med Pathol (2007) 3:119–123
320
Max. 320.0 cps.
XIC of +MRM: 264.0/233.0 and 264.0/117.0 amu
Intensity, cps
Fig. 2 Comparison of multiple reaction monitoring data of nortriptyline, corresponding to two adjacent segments of a single hair (0.5 cm segment length). The data refer to the concentration profile as labelled by arrows in Fig. 4, comparing an almost blank sample close to the hair root (dashed) with the following positive segment (solid arrow)
121
0
1.0
XIC of +MRM: 264.0/233.0and 264.0/117.0 amu
2.0 Max. 1.8e4 cps.
1.22
Intensity, cps
1.8e4
Time, min
1.0
reproducibility within individuals and characteristic interindividual concentration variations (Figs. 3, 4). The primary identification of amitriptyline and nortriptyline in the total hair strands demonstrated that nortriptyline concentrations were significantly higher than corresponding amounts of amitriptyline. This distinction to typical metabolite ratios determined in blood is likely due to the predominant incorporation of basic compounds (e.g. nortriptyline) into hair. After the apparent termination of substance administration, the hair concentration drops to zero within a time equivalent to the length of a few millimetres. This suggests that no relevant diffusion of incorporated amitriptyline and nortriptyline towards the hair root occurs. The drug history of these substances, in particular the time of termination can accurately be determined, the main limitation of accuracy seems to be the
Time, min
2.0
uncertainty of the hair growth dynamics. The concentration profiles of corresponding metabolites are coincident. There is no indication for any long-term variation of metabolic ratios within one subject. Inter-individual variations of metabolite ratios Variations between the subjects are evaluated based on the average metabolite ratios obtained from analyses of hair bundles. There are significant inter-individual variation of metabolite ratios with respect to several parameters. The stability of metabolite ratios (NT/AT) over a hair profile (Fig. 3) was confirmed by examination and statistical evaluation of concentrations along a single hair [6]. The concentration of nortriptyline, relative to amitriptyline ranges from 0.5 to 4.8 that reproducibly and significantly
122
Forens Sci Med Pathol (2007) 3:119–123 200
Concentration [normalized]
100 AT NT E10-OHNT Z10-OHNT E10-OHAT Z10-OHAT
80
60
160
120
40
80
20
40
0
0 3
8
13
Distance from root [cm]
Fig. 3 Concentration profiles of amitriptyline and its metabolites along the hair strand are in good coincidence. Analyte concentrations are normalised to 100%, all hydroxy-metabolites refer to the secondary axis. The metabolite ratios within an individual are rather constant during an administration cycle
40
Ami-T Nor-T
35
Conc [pg/cm]
30 25 20 15 10
excluded. Another variable parameter is the ratio of the isomers of hydroxy-metabolites. 10-hydroxylation of both amitriptyline as well as nortriptyline yields two isomeric reaction products, the trans (E-) and cis (Z-) conformations, which are supposed to be synthesised by different enzymes. The ratio of E- to Z-hydroxynortriptyline concentrations in hair (ranging from 1.6 to 10.3) represents a measure of stereo-selectivity and efficiency of hydroxylation. Both reactions (i.e. demethylation and hydroxylation) seem to be significantly correlated. The stereo-selectivity of nortriptyline hydroxylation (E-/ Z- hydroxynortriptyline) depends markedly on the amount of hydroxylation (total hydroxynortriptyline/nortriptyline, Fig. 5). This is in good accordance to the expectations, because the most potent enzyme for nortriptyline hydroxylation (CYP2D6) is known to catalyse the synthesis of the E10-nortriptyline [4, 7, 8]. The lack of this enzyme should cause a reduction of both, amount and specificity of hydroxylation. Moreover, an increase of demethylation efficiency is associated with an elevation of stereo selectivity of the hydroxylation (Fig. 6), which is in contrast to the conventional theory. In general, it is assumed that a reduced efficiency of one metabolic pathway (e.g. N-demethylation) will induce alternative metabolic reaction (hydroxylation of amitriptyline) resulting in a higher amount and specificity of E10-hydroxyamitriptyline [9].
5 0 0
5
10
15
20
25
30
Distance from root [cm] 90 Ami-T
80
Nor-T
12.00
60
10.00
50 40 30
(E/Z)-OHNT
Conc [pg/cm]
70
20 10 0 0
5
10
15
20
25
30
8.00
6.00 4.00
Distance from root [cm]
2.00
Fig. 4 There are significant variations of metabolic ratio (e.g. amitriptyline over nortriptyline) in between subjects, which are reproducible over a long period of administration. Samples tagged by arrows refer to the chromatograms shown in Fig. 2
demonstrates the different potential of amitriptyline N-demethylation between the 23 subjects (Fig. 6) and suggests different metabolic phenotypes, because a co-administration of nortriptyline can be certainly
0.00 0.00
0.20
0.40
0.60
0.80
1.00
OH-NT/NT
Fig. 5 The relative amount of hydroxylation of nortriptyline (i.e. the ratio of total hydroxynortriptyline/nortriptyline, OH-NT/NT) influences the stereo-selectivity of hydroxylation considerably (r = 0.704, P = 0.001). An increasing amount of hydroxylation of nortriptyline is associated with an enhanced stereo-specificity of the reaction in favour of the trans conformation
Forens Sci Med Pathol (2007) 3:119–123
123
6.00
E/Z O H A T
5.00
2.
4.00 3.00 2.00
3.
1.00 0.00 0.00
1.00
2.00
3.00
4.00
5.00
6.00
metabolites often contribute significantly to these properties. Metabolism is mainly governed by enzymes which are characterised by a large inter-individual variation. Therefore, the biological response of substances administered in identical doses may well result in a different individual biological response. Identification of typical metabolite ratios (e.g. nortriptyline/amitriptyline) in hair is introduced as a powerful probe to examine individual variations of individual metabolic phenotypes.
NT/AT
Fig. 6 The relative amount of demethylation (i.e. the ratio nortriptyline/amitriptyline, NT/AT) and the specificity of hydroxylation (ratio of trans- to cis isomers, E/Z-OHAT) of amitriptyline, as quantified in hair samples of 23 individuals, are significantly correlated (r = 0.697, P = 0.001)
Conclusions Several advantages of hair analysis could be demonstrated by examination of a case of an illegal amitriptyline administration to infants. A long record of retrospective drug incorporation (which covered several years in many cases) was available. Detailed information on the course of substance administration (including its termination) could be collected based on a segmental hair analysis. Large inter-individual variations of relevant metabolite ratios seem to be correlated with the individual metabolic phenotype. Hair testing appears to be a powerful tool for optional examination of illegal substance administration, long-term compliance testing and evaluation of individual biotransformation phenotype. The fact that hair analysis is dealing with parent compounds rather than metabolites seems to be beneficial for consideration of certain metabolic reactions, because concentration of the biological active compounds are not superimposed by inactive metabolites (glucuronides).
Educational message 1.
The biological activity (incl. side effects) of drugs are mainly influenced by their biotransformation and
References 1. Breyer-Pfaff U. The metabolic fate of amitriptyline, nortriptyline and amitriptylinoxide in man. Drug Metab Rev 2004; 36(3– 4):723–46. 2. Bertilsson L, Dahl ML, Dalen P. Al-Shurbaji a molecular genetics of CYP2D6: clinical relevance with focus on psychotropic drugs. Br J Clin Pharmacol 2002; 53(2):111–22. 3. Lane RM, Baker GB. Chirality and drugs used in psychiatry: nice to know or need to know? Cell Mol Neurobiol 1999; 19(3):355– 72. 4. Nordin C, Bertilsson L. Active hydroxymetabolites of antidepressants. Emphasis on E-10-hydroxy-nortriptyline. Clin Pharmacokinet 1995; 28(1):26–40. 5. Steimer W, Zopf K, von Amelunxen S, Pfeiffer H, Bachofer J, Popp J, Messner B, Kissling W, Leucht S. Amitriptyline or not, that is the question: pharmacogenetic testing of CYP2D6 and CYP2C19 identifies patients with low or high risk for side effects in amitriptyline therapy. Clin Chem 2005; 51(2):376–85. 6. Thieme, D.; Sachs H. Examination of a long-term clozapine administration by high resolution segmental hair analysis. Forensic Sci Int. 2006; [Epub ahead of print]. 7. Dahl ML, Bertilsson L, Nordin C. Steady-state plasma levels of nortriptyline and its 10-hydroxy metabolite: relationship to the CYP2D6 genotype. Psychopharmacology (Berl) 1996; 123(4):315–9. 8. van der Weide J, van Baalen-Benedek EH, Kootstra-Ros JE. Metabolic ratios of psychotropics as indication of cytochrome P450 2D6/2C19 genotype. Ther Drug Monit 2005; 27:478–83. 9. Shimoda K, Someya T, Yokono A, Morita S, Hirokane G, Takahashi S, Okawa M. The impact of CYP2C19 and CYP2D6 genotypes on metabolism of amitriptyline in Japanese psychiatric patients. J Clin Psychopharmacol 2002; 22(4):371–8.
ORIGINAL PAPER
Individual variations of amitriptyline biotransformation examined in scalp hair samples Detlef Thieme Æ Dagmar Schmid Æ Hans Sachs
Accepted: 22 December 2006 / Published online: 6 September 2007 Ó Humana Press Inc. 2007
Abstract The identification of amitriptyline, nortriptyline and its hydroxy-metabolites including a subsequent measurement of concentration profiles in hair samples was carried out to evaluate the administration history of amitriptyline. Analyses were carried out using liquid chromatography—tandem mass spectrometry, which permits simultaneous identification of all relevant substances in hair at low target concentrations (limit of detection better than 0.5 pg/mg). Standard hair sample preparation was applied for the estimation of average substance concentration in a hair bundle, while segmentation of individual hairs was utilised to examine accurate concentration profiles. Replication of analyses demonstrated a good reproducibility of individual hair concentration profiles, which proved to coincide for all relevant compounds. Significant variations of metabolite ratios (e.g. nortriptyline to amitriptyline and E10- to Z10-hydroxynortriptyline) between individuals suggest a correlation between hair concentration and metabolic phenotype. Different concentration ratios of certain metabolites in hair are highly correlated, indicating a systematic association between demethylation and stereo-specificity of hydroxylation. The trans isomers The data were presented at the Hair Testing Meeting, Vadstena, Sweden, May 2006. We hereby state that we do not have a significant financial interest or other relationship with any product manufacturer or provider of services discussed in this article. D. Thieme (&) D. Schmid Institute of Forensic Medicine, Frauenlobstr. 7a, 80337 Munich, Germany e-mail: [email protected] D. Thieme H. Sachs Forensic Toxicological Centre, Bayerstr. 53, 80335 Munich, Germany
of hydroxy-metabolites become significantly more prevalent with increasing degree of demethylation of amitriptyline or hydroxylation of amitriptyline. Keywords Antidepressants Amitriptyline Liquid chromatography Mass spectrometry Hair analysis Forensic toxicology
Introduction Many of the classical tricyclic antidepressants undergo a complex biotransformation, which is typically influenced by large individual variations, depending on parameters like sex, age, ethnicity and administration of other pharmaceutical substances. Therefore, therapeutic dosages should be adjusted and controlled individually, making a clinical routine application rather complex. Amitriptyline in particular was the subject of recent studies to investigate the influence of individual variations in metabolism and the resulting response to substance administration [1–3]. Typical side effects (dizziness, somnolence, unconsciousness, swollen tongue, speech disorders, circulatory failure, diarrhoea, constipation) are mainly correlated with serum concentrations of the active metabolite nortriptyline (Fig. 1), while the parent compound as well as active metabolites are not thought to be critical [4, 5]. The case of an illegal administration of amitriptyline to a group of children (aiming to the intended sedation) was retrospectively investigated and hair analysis was the only available option due to a time span between last administration and sample collection of at least 3 weeks. No reliable data on the amounts and duration of amitriptyline administration were available and systematic and qualified exploration of side effects was not carried out. Hair anal-
120
Forens Sci Med Pathol (2007) 3:119–123
Liquid chromatography – Electrospray – Mass spectrometry
N- Demethylation
N N
OH
NH
E( Z)10- H ydro xyl atio n
OH
NH
Fig. 1 Amitriptyline is mainly biotransformed by N-demethylation and hydroxylation in position 10, resulting in mainly trans (E) and partially cis (Z) isomers. Other metabolic reactions (e.g. glucuronidation, N-oxidation) can be ignored in hair analysis due to their low incorporation rate
ysis was carried out in 23 infants (age 2–6 years) to study additional details like administration interval and individual metabolite response.
Methods and materials
All analyses were carried out using an API 4000 mass spectrometer (Applied Biosystems), in electrospray ionisation mode (Turbo-Ion-Spray source) coupled to an Agilent 1100 LC system (binary pump and autosampler). Reference material of amitriptyline, nortriptyline (both Cerilliant), E10-hydroxynortriptyline and Z10-hydroxynortriptyline (both Sigma) was used for optimisation of the ionisation and fragmentation conditions while MS parameters of hydroxyamitriptyline were obtained from investigation of extracts of biological material. The relevant substance specific fragmentation reactions and corresponding values of declustering potential (DP) and collision energy (CE) are listed in Table 1. Both isomers of corresponding hydroxy-metabolites are mass spectrometrically identical and need to be separated chromatographically, which was achieved using a Zorbax Eclipse RP 18 column (4.6 * 75 mm, 3.5 lm particle size). The mobile phases consist of a 2 mM ammonium acetate buffer (Baker) in a mixture of water (Merck) and acetonitrile (Baker) at a composition of (90/10, v/v, buffer A) or (10/90, v/v, buffer B). The solvent gradient starts at a composition of 100%A (0–1 min) continuously changing to 100%B (isocratic from 6.5 to 7.5 min) at a constant flow of 0.7 ml/min. To accelerate the chromatography for profiling purposes, isochratic (100%B) chromatography on Synergy Polar-RP narrow bore column (Phenomenex, 75 * 2.0 mm, 4 lm particle size) was utilised (Fig. 2). Respective injection volumes were 10 ll.
Sample preparation There are two relevant protocols for preparation of hair samples either for routine analyses of entire hair bundles (representing the total time frame) or for profiling small segments of individual hairs [6]. The standard procedure for drug testing consists of decontamination of the intact hair strands by 5 min agitation in a gas-tight tube with 5 ml petroleum benzene (boiling range to 40°C, Merck), drying and cutting of 50 mg hair into pieces of 1–2 mm length. After adding 100 ng of the internal standard (MPPH, 5-(4methylphenyl)-5-phenyl hydantoine, Serva), the hair powder was extracted in 3 ml of methanol (Merck) (3 h ultrasonification at 55°C). Individual hairs were typically segmented into 5 mm pieces after fixation on an adhesive tape. Resulting hair concentrations were expressed as amount per cm [pg/cm] due to the unknown total mass of these segments. The resulting hair segments were transferred into vials, containing 5 ng of the internal standard (MPPH) in 30 ll of the mobile phase. The vials were directly analysed by LCMS after 3 h of ultrasonification at 55°C.
Results and discussion Significance of concentration profiles The concentration profiles of amitriptyline and nortriptyline were examined in single hairs leading to high Table 1 Ionisation and fragmentation parameters of the relevant compounds
Amitriptyline Nortriptyline
Declustering potential [V]
Precursor ion [Da]
Product ion [Da]
Collision energy [eV]
61
278
117
35
191
49
56
264
117 233
29 21
Hydroxyamitriptyline 71
294
215
53
Hydroxynortriptyline 71
280
216
35
215
49
216
33
Forens Sci Med Pathol (2007) 3:119–123
320
Max. 320.0 cps.
XIC of +MRM: 264.0/233.0 and 264.0/117.0 amu
Intensity, cps
Fig. 2 Comparison of multiple reaction monitoring data of nortriptyline, corresponding to two adjacent segments of a single hair (0.5 cm segment length). The data refer to the concentration profile as labelled by arrows in Fig. 4, comparing an almost blank sample close to the hair root (dashed) with the following positive segment (solid arrow)
121
0
1.0
XIC of +MRM: 264.0/233.0and 264.0/117.0 amu
2.0 Max. 1.8e4 cps.
1.22
Intensity, cps
1.8e4
Time, min
1.0
reproducibility within individuals and characteristic interindividual concentration variations (Figs. 3, 4). The primary identification of amitriptyline and nortriptyline in the total hair strands demonstrated that nortriptyline concentrations were significantly higher than corresponding amounts of amitriptyline. This distinction to typical metabolite ratios determined in blood is likely due to the predominant incorporation of basic compounds (e.g. nortriptyline) into hair. After the apparent termination of substance administration, the hair concentration drops to zero within a time equivalent to the length of a few millimetres. This suggests that no relevant diffusion of incorporated amitriptyline and nortriptyline towards the hair root occurs. The drug history of these substances, in particular the time of termination can accurately be determined, the main limitation of accuracy seems to be the
Time, min
2.0
uncertainty of the hair growth dynamics. The concentration profiles of corresponding metabolites are coincident. There is no indication for any long-term variation of metabolic ratios within one subject. Inter-individual variations of metabolite ratios Variations between the subjects are evaluated based on the average metabolite ratios obtained from analyses of hair bundles. There are significant inter-individual variation of metabolite ratios with respect to several parameters. The stability of metabolite ratios (NT/AT) over a hair profile (Fig. 3) was confirmed by examination and statistical evaluation of concentrations along a single hair [6]. The concentration of nortriptyline, relative to amitriptyline ranges from 0.5 to 4.8 that reproducibly and significantly
122
Forens Sci Med Pathol (2007) 3:119–123 200
Concentration [normalized]
100 AT NT E10-OHNT Z10-OHNT E10-OHAT Z10-OHAT
80
60
160
120
40
80
20
40
0
0 3
8
13
Distance from root [cm]
Fig. 3 Concentration profiles of amitriptyline and its metabolites along the hair strand are in good coincidence. Analyte concentrations are normalised to 100%, all hydroxy-metabolites refer to the secondary axis. The metabolite ratios within an individual are rather constant during an administration cycle
40
Ami-T Nor-T
35
Conc [pg/cm]
30 25 20 15 10
excluded. Another variable parameter is the ratio of the isomers of hydroxy-metabolites. 10-hydroxylation of both amitriptyline as well as nortriptyline yields two isomeric reaction products, the trans (E-) and cis (Z-) conformations, which are supposed to be synthesised by different enzymes. The ratio of E- to Z-hydroxynortriptyline concentrations in hair (ranging from 1.6 to 10.3) represents a measure of stereo-selectivity and efficiency of hydroxylation. Both reactions (i.e. demethylation and hydroxylation) seem to be significantly correlated. The stereo-selectivity of nortriptyline hydroxylation (E-/ Z- hydroxynortriptyline) depends markedly on the amount of hydroxylation (total hydroxynortriptyline/nortriptyline, Fig. 5). This is in good accordance to the expectations, because the most potent enzyme for nortriptyline hydroxylation (CYP2D6) is known to catalyse the synthesis of the E10-nortriptyline [4, 7, 8]. The lack of this enzyme should cause a reduction of both, amount and specificity of hydroxylation. Moreover, an increase of demethylation efficiency is associated with an elevation of stereo selectivity of the hydroxylation (Fig. 6), which is in contrast to the conventional theory. In general, it is assumed that a reduced efficiency of one metabolic pathway (e.g. N-demethylation) will induce alternative metabolic reaction (hydroxylation of amitriptyline) resulting in a higher amount and specificity of E10-hydroxyamitriptyline [9].
5 0 0
5
10
15
20
25
30
Distance from root [cm] 90 Ami-T
80
Nor-T
12.00
60
10.00
50 40 30
(E/Z)-OHNT
Conc [pg/cm]
70
20 10 0 0
5
10
15
20
25
30
8.00
6.00 4.00
Distance from root [cm]
2.00
Fig. 4 There are significant variations of metabolic ratio (e.g. amitriptyline over nortriptyline) in between subjects, which are reproducible over a long period of administration. Samples tagged by arrows refer to the chromatograms shown in Fig. 2
demonstrates the different potential of amitriptyline N-demethylation between the 23 subjects (Fig. 6) and suggests different metabolic phenotypes, because a co-administration of nortriptyline can be certainly
0.00 0.00
0.20
0.40
0.60
0.80
1.00
OH-NT/NT
Fig. 5 The relative amount of hydroxylation of nortriptyline (i.e. the ratio of total hydroxynortriptyline/nortriptyline, OH-NT/NT) influences the stereo-selectivity of hydroxylation considerably (r = 0.704, P = 0.001). An increasing amount of hydroxylation of nortriptyline is associated with an enhanced stereo-specificity of the reaction in favour of the trans conformation
Forens Sci Med Pathol (2007) 3:119–123
123
6.00
E/Z O H A T
5.00
2.
4.00 3.00 2.00
3.
1.00 0.00 0.00
1.00
2.00
3.00
4.00
5.00
6.00
metabolites often contribute significantly to these properties. Metabolism is mainly governed by enzymes which are characterised by a large inter-individual variation. Therefore, the biological response of substances administered in identical doses may well result in a different individual biological response. Identification of typical metabolite ratios (e.g. nortriptyline/amitriptyline) in hair is introduced as a powerful probe to examine individual variations of individual metabolic phenotypes.
NT/AT
Fig. 6 The relative amount of demethylation (i.e. the ratio nortriptyline/amitriptyline, NT/AT) and the specificity of hydroxylation (ratio of trans- to cis isomers, E/Z-OHAT) of amitriptyline, as quantified in hair samples of 23 individuals, are significantly correlated (r = 0.697, P = 0.001)
Conclusions Several advantages of hair analysis could be demonstrated by examination of a case of an illegal amitriptyline administration to infants. A long record of retrospective drug incorporation (which covered several years in many cases) was available. Detailed information on the course of substance administration (including its termination) could be collected based on a segmental hair analysis. Large inter-individual variations of relevant metabolite ratios seem to be correlated with the individual metabolic phenotype. Hair testing appears to be a powerful tool for optional examination of illegal substance administration, long-term compliance testing and evaluation of individual biotransformation phenotype. The fact that hair analysis is dealing with parent compounds rather than metabolites seems to be beneficial for consideration of certain metabolic reactions, because concentration of the biological active compounds are not superimposed by inactive metabolites (glucuronides).
Educational message 1.
The biological activity (incl. side effects) of drugs are mainly influenced by their biotransformation and
References 1. Breyer-Pfaff U. The metabolic fate of amitriptyline, nortriptyline and amitriptylinoxide in man. Drug Metab Rev 2004; 36(3– 4):723–46. 2. Bertilsson L, Dahl ML, Dalen P. Al-Shurbaji a molecular genetics of CYP2D6: clinical relevance with focus on psychotropic drugs. Br J Clin Pharmacol 2002; 53(2):111–22. 3. Lane RM, Baker GB. Chirality and drugs used in psychiatry: nice to know or need to know? Cell Mol Neurobiol 1999; 19(3):355– 72. 4. Nordin C, Bertilsson L. Active hydroxymetabolites of antidepressants. Emphasis on E-10-hydroxy-nortriptyline. Clin Pharmacokinet 1995; 28(1):26–40. 5. Steimer W, Zopf K, von Amelunxen S, Pfeiffer H, Bachofer J, Popp J, Messner B, Kissling W, Leucht S. Amitriptyline or not, that is the question: pharmacogenetic testing of CYP2D6 and CYP2C19 identifies patients with low or high risk for side effects in amitriptyline therapy. Clin Chem 2005; 51(2):376–85. 6. Thieme, D.; Sachs H. Examination of a long-term clozapine administration by high resolution segmental hair analysis. Forensic Sci Int. 2006; [Epub ahead of print]. 7. Dahl ML, Bertilsson L, Nordin C. Steady-state plasma levels of nortriptyline and its 10-hydroxy metabolite: relationship to the CYP2D6 genotype. Psychopharmacology (Berl) 1996; 123(4):315–9. 8. van der Weide J, van Baalen-Benedek EH, Kootstra-Ros JE. Metabolic ratios of psychotropics as indication of cytochrome P450 2D6/2C19 genotype. Ther Drug Monit 2005; 27:478–83. 9. Shimoda K, Someya T, Yokono A, Morita S, Hirokane G, Takahashi S, Okawa M. The impact of CYP2C19 and CYP2D6 genotypes on metabolism of amitriptyline in Japanese psychiatric patients. J Clin Psychopharmacol 2002; 22(4):371–8.
