Journal of Analytical Toxicology, Vol. 16, July/August 1992
Hair Analysisfor Drugs of Abuse. III. Movementand Stability of Methoxyphenamine(As a Model Compound of Methamphetamine)along Hair Shaft with Hair Growth Y u j i N a k a h a r a * , M o c h i h i k o S h i m a m i n e , and K a z u n o r i T a k a h a s h i
National Institute of Hygienic Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo, Japan
Abstract This paper describes the movement of methoxyphenamlne (MOP, a model compound of methamphetamlne) along the hair shaft at the rate of hair growth and the stability of drugs in hair for several months. Five healthy subjects (3 males and 2 females) took 50 mg of methoxyphenamlne orally once a day for 7 days. Scalp hairs from the posterior vertex were collected every 2 weeks or every 8 weeks after the first dosage. The hairs were cut into 1-cm sections and extracted with methanol-5N HCl (20:1). MOP In the extract was determined by gas chromatography/mass spectrometry (GC/MS) with tetradeuterlum labeled MOP as an internal standard. The drug moved along hair shaft at the rate of 2.8-3.2 mm/week, according to hair growth, without diffusion. When drug bands were extrapolated according to the sections In which drug was detected, the bands were approximately 5 mm wide, equivalent to 1.7-2.4 periods of 7-day hair growth. In the case of identical doses, the drug level was highest In the root side and lowest in the distal side. In our data, we found that the drug level in hair had decreased approximately 50% five months later. The ratio of drug levels in corresponding sections correlated well to the ratio of doses, except where the hair shafts had been damaged or the drugs had decomposed.
according to hair growth without diffusion, are drugs stable in hair for long periods, is the width of the drug bands the same as the length of hair growth for the period of drug use, do drug levels in hair correlate to the amount of drug used, is the excretion of drugs into hair affected by their physical properties, and how should sampling, pretreatment, and analysis be done to achieve accurate results? To address these problems, we first studied the movement and stability of drugs excreted (or incorporated) into human hair. Methoxyphenamine (bronchodilator, MOP), a compound structurally related to methamphetamine, was used as a model because previously reported studies on its hair analysis could be utilized (6, 7). The determination of MOP in human hair was carfled out by gas chromatography/mass spectrometry (GC/MS) with homemade deuterium-labeled MOP as an internal standard. This paper describes the movement of MOP along the hair shaft according to hair growth and the stability of drugs in hair for several months.
Experimental Subjects
Introduction
In recent years, several studies on hair analysis for drug use history have been reported. Baumgartner et al. reported that sectional analysis of hair could serve as a valuable tool in the determination of drug abuse with opiates (1) and phencyclidine (2). Uematsu et al. (3-5) reported that human scalp hair could serve as a useful tool for monitoring individual dosage history over several months of haloperidol use. We have also reported (6,7) that the period of drug use corresponds approximately to the drug distribution along the hair shaft. These reports describe the advantages of hair analysis compared to analysis of other body specimens. However, for the practical use of hair analysis in forensic toxicology laboratories there are several problems that must be addressed. For example, do drugs excreted in hair move "Authorto whom correspondenceshould be addressed.
Three healthy male (YN, KT HS) and two healthy female (KN, AI) volunteer subjects (28-52 years) took 250 mg of methoxyphenamine preparation (20% powder, 0.8-1.1 mg/kg) orally once a day for seven days during weeks I, 8, and 20 after the first dosage. All subjects washed their scalp hair with shampoo every two or three days. On the day the hair sample was collected, hair was washed, dried, and brushed, and approximately 50 hairs from the posterior vertex region were pulled out from the root. The root side of hair samples was lightly fixed, wrapped in aluminum foil, and stored in a refrigerator. The hair samples of YN were collected every 2 weeks from week 4 to week 30 after the first dosage. The other samples were collected on the first day of weeks 9, 17, 26, and 30 after the first dosage. In order to compare the dosage with the drug level in hair, YN took an additional 20, 40, and 60 mg of methoxyphenamine orally for 7 days during weeks 1, 5, and 9, respectively. On the first day of the 12th week hair samples were collected and drug levels in each 6-mm section were determined.
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253
Joumsl of Analytical Toxicology, Vol. 16, July/August 1992
methylamine gas into acetaldehyde-d4 in ether), the reaction mixture was refluxed for 1 h. After cooling, 200 mL of 0.75M sulfuric acid (H2504) was added to the reaction mixture to decompose excess reagent. The acid layer was separated, made basic with 6M sodium hydroxide (NaOH), and extracted three times with 200 mL of chloroform (CHC13). The combined CHCI 3 extract was treated with a few drops of concentrated hydrochloric acid (HC1)-ethanol (1:9) and evaporated to dryness. The solid residues were recrystallized from ethanol under the following conditions: MOP-d4/HCI; mp 130~ Ultraviolet spectrum; ;%ax = 271 nm (H20), high resolution mass measurement [isobutane/Cl]; MH + at m/z 184.156 (Ctt H]4NOD4 = 184.159); El-MS (m/z) of TFA-MOP-d4:M+ (279), M+-69 (210), M+-91 (158 = base peak), M*-157 (122). Preparation of hair samples. In order to align the roots of 40-50 hairs, the samples were pasted onto adhesive paper (a commercially available carpet cleaner [20 cm x 15 cm]). Scissors were then used to cut 1-cm sections, beginning with the root. Each section was soaked for 10-20 min in 20 mL of 0.1% SDS in a beaker, and the hair was peeled from the paper. After removal from the paper, the hair was washed three times with 10 mL of 0.1% SDS and distilled water, respectively, under ultra-
Materials
Methoxyphenamine preparation (20% powder) was purchased from Nihon Shinyaku Co., Ltd. (Kyoto, Japan). Synthesis of 2,3,3,3-tetradeuterium methoxyphenamine. After 60 mL of o-methoxybenzyl magnesium chloride in ether (obtained by refluxing o-methoxybenzyl chloride [ 13.1 g] and magnesium [2.75 g] for 1 h in anhydrous ether) was added to 20 mL of N-methylethyleneimide-d4 in ether (obtained by introducing T a b l e I. P r e c i s i o n f o r D e t e r m i n a t i o n Methoxyphenamine In H a i r *
of
C0ncentratl0n (ng/mg) Added
Measured (means + SD)
CV (%)
10 5 1
10.24 + 0,21 5.12 + 0,30 0.99 + 0.05
2,05 5,86 5.05
"No. of analyses = 10
Scan
"=
199
(5.451
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MOP-d4 Figure 2. Selected ion monitoring of the derivatized methoxyphenamine and O-desmethyl metabolite extracted from (A) hair and (B) internal standard at m/z 154 and 158.
Figure 1. El mass spectra and major fragments of (A) TFA-methoxyphenamine and (B) TFA-methoxyphenamine-d4.
T a b l e II. C o n c e n t r a t i o n s
r
380
Ma=;s;/Ch ar~a
MOP
amu.
i 180881
}"
_LL ~
148
Ion
e
(ng/mg)
of Methoxyphenamine
in E a c h ( 1 - c m ) H a i r S e c t i o n Section
Week
1
2
4 6 8 10 12
10.1
10.3 4.7
. 8.5 5.1 3.6
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-
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7
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-
9.9
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1.9
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4.5 3.2
4.8 5.9
"Hair sampleswere collectedon the first day of the week from subject YN, who had taken the drug for seven daysduringweeks 1, 8, and 20.
254
1.7 3.8
J o u m a l of Analytical Toxicology, Vol. 16, July/August 1992
and warmed at 55~ for 20 min. The reaction solution was evaporated with a nitrogen stream, and the residue was redissolved in 100 laL of ethylacetate. One ILL of the ethylacetate solution was automatically injected into a gas chromatograph. The E1 mass spectra of TFA-MOP and TFA-MOP-d4 are shown in Figure 1. Calibration curves, linearity, and reproducibility. The calibration curve for the measurement of MOP was constructed by the analysis of extracted and derivatized samples of 10 mg of control hair, to which the standard solutions of MOP had been added (1-100 ng/mg of hair), together with MOP-d4 (IS), as mentioned above. The calibration curve for MOP (y = 1.01x + 0.0025) in the hair was linear over the concentration range 1-100 ng/mg of hair, with correlation coefficients r = 0.9999. Values less than 1 ng/mg of hair were cut off. Precision of the method was determined using hair of healthy volunteers spiked with MOP at three concentrations with respect to a calibration curve (10 samples were analyzed for each concentration). The coefficients of variations (CVs) for MOP were 2.05, 5.86, and 5.05 at 10, 5, and 1 ng/mg, respectively.
sonication for 1 min, as described in our previous paper (6). Samples were dried with a paper towel and placed in a desiccator under reduced pressure. Analytical method. Each section of hair (4-8 mg) was precisely weighed and extracted with 2 mL of MeOH/5M HCI (20:1) for 1 h under ultrasonication. The solution was kept at room temperature overnight. After I00 laL of the IS methanol solution containing MOP-d4 at 3 I.tg/mL was added into the extracted solution and the hair was filtered off, the filtrate was evaporated under a stream of nitrogen. The residue was dissolved in 200 laL of trifluoroacetic anhydride-ethylacetate (1 : 1)
YN o
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Figure 3. Schematic diagram of drug movement along hair shaft from 4th to 30th week after dosages of methoxyphenamine at weeks 1, 8, and 20 (Subject: YN).
Table
III. C o n c e n t r a t i o n s
(ng/mg)
of Methoxyphenemlne
KT Section
8wk
16wk
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In Each
(1-cm)
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Section
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1.2 5.1
Discussion
Sampling. It is reported (8) that the growth rate of human scalp hair is ordinarily 0.35--0.4 mm/day (10.5-1.2 cm/month). However, growth rate is also dependent to a certain extent on anatomical region, race, sex, and age. Moreover, hair follicles exist in three phases, the anagen (growing), catagen (intermediate), and talogen (resting) phases. On the scalp of an adult, approximately 15% of the follicles are in a resting stage while the remaining 85% are in their growing stage. Scalp hair is divided into five regions, as follows: frontal, temporal, anterior vertex, posterior vertex, and occipital. In humans, hair is best collected from the area at the back of the head called the "posterior vertex" (9). In this area, the number of follicles in the growing phase is more constant, there are more long terminal hairs, and the hairs are less subject to age and sex influences. We also have found (7) that the variation of drug levels in the vertex hair of several methamphetamine abusers was minimal when compared with the other regions (frontal, temporal, occipital). In this study, therefore, hair samples were collected from the posterior vertex after removing as much of the resting phase of hair as possible by brushing. Analytical method. The determination of MOP in hair was carried out by the stable isotope dilution GC/MS method using deuterium-labeled MOP (MOP-d4) as an internal standard. The base peak of TFA derivatives of MOP and MOP-d4 are m/z 154
I
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8wk
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AI
25wk
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16wk
25wk
29wk
5.9 . -
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"Hair samples were collected on a first day of the week from subject YN, who had taken the drug for seven days during weeks 1, 8 and 20
255
Journal of Analytical Toxicology, Vol. 16, July/August 1992
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Fioure 4. Schematic diagrams of drug movement along hair shaft from 8th to 29th week after the dosages of methoxyphenamine on week 1, 8, and 20. (A) subject KT, (B) subject HS, (C) subject KN, (D) subject AI.
256
Figure 5. Stability of methoxyphenamine in hair. All subjects took the same oral dosage of methoxyphenamine(50 mg x 7). Only drug bands entirely in one (1-cm) section were selected; drug bands divided into two sections were omitted.
and 158, respectively, so that selected ion monitoring (SIM) of their ions was used for the determination. Figure 2 shows the typical SIM chromatogram of TFA derivatives of the extract from hair. One of the metabolites, O-desmethyl MOP, was detected together with unchanged MOR Movement of drug along the hair shaft after drug use. For the elucidation of movement of drug along the hair shaft, five healthy subjects (3 males and 2 females) took 250 mg of MOP powder orally (20% powder) once a day for 7 days during weeks l, 8, and 20. The hair samples of YN (male) were collected on the first day of every 2 weeks from week 4 to week 30 after the first dosage. The samples of KT (male), HS (male), AI (female), and KN (female) were collected on the first day of weeks 8, 16, 25, and 29 alter the first dosage. The results of the sectional analyses of YN are shown in Table 11. MOP was detected in the hair samples three weeks after the first dosage at a concentration of 10.1 ng/mg in the first 1-cm section. This drug band moved from the first to the eighth section according to hair growth, as shown in Figure 3. In a similar manner, the MOPs of the dosages administered during weeks 8 and 20 also moved from the root to the tip. The movement of the drug bands along hair shafts was also observed in the other subjects (KT, HS, KN, AI) without exception, as shown in Table III and Figure 4. When drug bands were extrapolated in Figures 3 and 4, according to the sections in which drugs were detected, their width was found to be approximately 5-6 mm, which is equivalent to 2.4-1.7 times of seven days' hair growth. When MOP is taken orally, the concentration of drug in blood reaches a maximum after 30 min and then rapidly decreases. Within 24 h, 85% is excreted in urine (10). Therefore, MOP is not considered to be present in blood several days after the last administration. Though the mechanism by which drugs are incorporated into hair is not well understood, we have considered that the clearance of drugs from hair matrix cells takes more days, while drugs in the hair matrix cell are gradually incorporated into the hair shaft with tailing. This would explain why the drug bands for 7-day doses are approximately twice as long as the length of 7-days' hair growth. It was also found that the drug bands moved to the hair tip at the rate of 2.8-3.2 mm/week. Stability of drug in hair. Figure 5 shows the decay of the drug
Journal of Analytical Toxicology, Vol. 16, July/August 1992
Table IV. Relationship between Dosage and Drug Level In Each Section Ratios** Section 1 2 3 4 5 6
Lengthfrom root(mm) 0- 6 6-12 12-18 18-24 24-30 30-36
Dosage* (mg)
Level detected (ng/mg)
60
4.9 tracer 2.6 tracer 1.4
40 20
drug level
dosage
3.5
3
1.9
2
1
1
* Amount of methoxyphenamineas a free base. *" The ratiosbased on level detectedand dosage in section5. t Lessthan 1 ng/mg.
levels according to the forward movement of drug bands. Only drug bands contained within the 1-cm sections were selected; drug bands spread across two sections were omitted because of the comparison between the equivalents to 7 doses in each 1-cm section. In our data, it is found that the drug level in hair decreased roughly 50% 5 to 6 months later. We found that the drug levels in the root-side sections were higher than those in the tip-side sections. It is well known that the hair shaft is gradually damaged by environmental influences according to hair growth. Therefore, it is assumed that some drugs in the hair cortex may escape from damaged hair by washing. Furthermore, drugs in hair are assumed to gradually decompose over long periods of time. Correlation between the drug level in hair and the dosage. YN took 20, 40, and 60 mg of methoxyphenamine orally for 7 days during the first, fifth, and ninth week, respectively. On the first day of the 12th week after the first dosage, hair samples were collected and the drug levels in 6-mm sections were determined, as shown in Table IV. When it was assumed that the rate of movement of drug bands would be 2.8 mm/week and the width of the drug band for 7-day doses was 5--6 mm, we figured that the drug bands corresponding to 20-, 40-, and 60-mg doses were in the 5th, 3rd, and 1st sections, respectively. The drug concentrations in the 5th, 3rd, and Ist sections were 1.4, 2.6, and 4.9 ng/mg, respectively. The ratio of the drug concentrations in the 5th, 3rd, and 1st sections was 1:1.9:3.5. This ratio closely corresponds to the ratio of doses (1:2:3). Because this result was obtained from the hair less than 3 cm from the root, it may indicate that the drug level in each hair section nearly corresponds to dose, assuming that the hair shaft has not yet been damaged and that the drugs in hair have not yet decomposed. Comparison between our results and the other reports. Baumgartner et al. reported that the pattern of past drug use of opiates (1), cocaine (11), and phencyclidine (2) can be obtained by sectional hair analysis. Uematsu et al. also reported that a history of individual dosage can be deduced from the distribution of drug level along the length of the hair and that the drug level in a section corresponds to dosage by the sectional analysis of scalp hair of patients that received haloperidol. On the other hand, Puschel et al. (12) concluded in their study that no correlation existed between administered doses of opiates and their concentrations in hair and that the approximate period of drug use in man could be detected by sectioning the hair, though the transport of drugs along the hair may be considerably influenced by various factors concerning hair, The authors have already demonstrated that the distribution of drug along the hair shaft determined by the sectional hair analysis of scalp hairs corresponds to the drug use histories of 11 metham-
phetamine abusers. However, for the elucidation of the movement of the drug along the hair shaft and the correlation between dosage and drug level, it was necessary to perform drug administration under control. Therefore, we undertook an experiment in which five volunteers took the correct dose of methoxyphenamine as a model compound of methamphetamine under control. Samples were collected from the posterior vertex after removing as much of the resting-phase hair as possible.
Conclusion From our present study the following conclusions can be summarized: (a) The drug excreted into hair has moved along hair shaft at the rate of 2.8-3.2 mm/week, according to hair growth, without diffusion. (b) When drug bands were extrapolated according to the sections in which drug was detected, the drug bands were approximately 5 mm wide, which was equivalent to 2.4--1.7 times the length of 7-day hair growth. (c) In case of the same dosages, the drug level in the root side was highest and in the tip side lowest. In our data, it is found that the drug level in hair decreased approximately 50% 5 months later. (d) The ratio of drug levels in the corresponding sections nearly correlates to the ratio of dosages, only in the case that hair shafts are not yet damaged and drugs in hair are not yet decomposed.
References 1. AM. Baumgartner, P.F. Jones, W.A. Baumgartner, and C.T. Black. Radioimmunoassay of hair for determining opiate-abuse histories. J. Nucl. Med. 20:748-52 (1979). 2. A.M. Baumgartner, P.F. Jones, and C.T. Black. Detection of phencyclidine in hair. J. Forensic ScL 26:576-81 (1981). 3. T. Uematsu, R. Sato, K. Suzuki, S. Yamaguchi, and M. Nakashima. Human scalp hair as evidence of individual dosage history of halopeddol: Method and retrospective study. Eur. J. Clin. PharmacoL 37:239-44 (1989). 4. R. Sato, T. Uematsu, S. Yamaguchi, and M. Nakashima. Human scalp hair as evidence of individual dosage history of haloperidol: Prospective Study. Ther. Drug Monitor. 11:686-91 (1989). 5. T. Uematsu, R. Sato, O. Fujimori, and M. Nakashima. Human scalp hair as evidence of individual dosage history of haloperidol: A possible linkage of haloperidol excretion into hair with hair pigment. Arch. DermatoL Res. 282:120-25 (1990). 6. Y. Nakahara, K. Takahashi, M. Shimamine, and Y. Takeda. Hair analysis for drug abuse: I. Determination of methamphetamine and amphetamine in hair by stable isotope dilution gas chromatography/mass spectrometry method. J. Forensic Sci. 36:70-78 (1991). 7. Y. Nakahara, K. Takahashi, Y. Takeda, K. Konuma, S. Fukui, and T. Tokui. Hair analysis for drug abuse, part II. Hair analysis for monitoring of methamphetamine abuse by stable isotope dilution gas chromatography/mass spectrometry. Forensic Sci. Int. 48:243-54 (1990). 8. M. Saitoh, M. Uzuka, M. Sakamoto, and T. Kobori. In Advances in Biology of Skin. W. Montagna and R.L. Dobson, Eds., VoL 5, Pergamon, New York, 1967. 9. M.R. Herkey and G.L. Henderson. In Advances in Analytical Toxicology, Vol. II, R.C. Baselt, Ed., Year Book Medical, 1989, pp. 298-329. 10. In Drugs In Japan, Japan Pharmaceutical Information Center, Yakugyo Jiho Co., Tokyo, Japan, 1985, p. 1029. 11. W.A. Baumgartner, C.T. Black, P.F. Jones, and W.H. Blahd. Radioimmunoassay of cocaine in hair: Concise communication. J. Nucl. Med. 23: 790-92 (1982). 12. K. Puschel, P. Thomasch, and W. Arnold. Opiate level in hair. Forensic Sci. Int. 21:181-86 (1983). Manuscript received August 6, 1991 ; revision received October 29, 1991.
257
Hair Analysisfor Drugs of Abuse. III. Movementand Stability of Methoxyphenamine(As a Model Compound of Methamphetamine)along Hair Shaft with Hair Growth Y u j i N a k a h a r a * , M o c h i h i k o S h i m a m i n e , and K a z u n o r i T a k a h a s h i
National Institute of Hygienic Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo, Japan
Abstract This paper describes the movement of methoxyphenamlne (MOP, a model compound of methamphetamlne) along the hair shaft at the rate of hair growth and the stability of drugs in hair for several months. Five healthy subjects (3 males and 2 females) took 50 mg of methoxyphenamlne orally once a day for 7 days. Scalp hairs from the posterior vertex were collected every 2 weeks or every 8 weeks after the first dosage. The hairs were cut into 1-cm sections and extracted with methanol-5N HCl (20:1). MOP In the extract was determined by gas chromatography/mass spectrometry (GC/MS) with tetradeuterlum labeled MOP as an internal standard. The drug moved along hair shaft at the rate of 2.8-3.2 mm/week, according to hair growth, without diffusion. When drug bands were extrapolated according to the sections In which drug was detected, the bands were approximately 5 mm wide, equivalent to 1.7-2.4 periods of 7-day hair growth. In the case of identical doses, the drug level was highest In the root side and lowest in the distal side. In our data, we found that the drug level in hair had decreased approximately 50% five months later. The ratio of drug levels in corresponding sections correlated well to the ratio of doses, except where the hair shafts had been damaged or the drugs had decomposed.
according to hair growth without diffusion, are drugs stable in hair for long periods, is the width of the drug bands the same as the length of hair growth for the period of drug use, do drug levels in hair correlate to the amount of drug used, is the excretion of drugs into hair affected by their physical properties, and how should sampling, pretreatment, and analysis be done to achieve accurate results? To address these problems, we first studied the movement and stability of drugs excreted (or incorporated) into human hair. Methoxyphenamine (bronchodilator, MOP), a compound structurally related to methamphetamine, was used as a model because previously reported studies on its hair analysis could be utilized (6, 7). The determination of MOP in human hair was carfled out by gas chromatography/mass spectrometry (GC/MS) with homemade deuterium-labeled MOP as an internal standard. This paper describes the movement of MOP along the hair shaft according to hair growth and the stability of drugs in hair for several months.
Experimental Subjects
Introduction
In recent years, several studies on hair analysis for drug use history have been reported. Baumgartner et al. reported that sectional analysis of hair could serve as a valuable tool in the determination of drug abuse with opiates (1) and phencyclidine (2). Uematsu et al. (3-5) reported that human scalp hair could serve as a useful tool for monitoring individual dosage history over several months of haloperidol use. We have also reported (6,7) that the period of drug use corresponds approximately to the drug distribution along the hair shaft. These reports describe the advantages of hair analysis compared to analysis of other body specimens. However, for the practical use of hair analysis in forensic toxicology laboratories there are several problems that must be addressed. For example, do drugs excreted in hair move "Authorto whom correspondenceshould be addressed.
Three healthy male (YN, KT HS) and two healthy female (KN, AI) volunteer subjects (28-52 years) took 250 mg of methoxyphenamine preparation (20% powder, 0.8-1.1 mg/kg) orally once a day for seven days during weeks I, 8, and 20 after the first dosage. All subjects washed their scalp hair with shampoo every two or three days. On the day the hair sample was collected, hair was washed, dried, and brushed, and approximately 50 hairs from the posterior vertex region were pulled out from the root. The root side of hair samples was lightly fixed, wrapped in aluminum foil, and stored in a refrigerator. The hair samples of YN were collected every 2 weeks from week 4 to week 30 after the first dosage. The other samples were collected on the first day of weeks 9, 17, 26, and 30 after the first dosage. In order to compare the dosage with the drug level in hair, YN took an additional 20, 40, and 60 mg of methoxyphenamine orally for 7 days during weeks 1, 5, and 9, respectively. On the first day of the 12th week hair samples were collected and drug levels in each 6-mm section were determined.
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253
Joumsl of Analytical Toxicology, Vol. 16, July/August 1992
methylamine gas into acetaldehyde-d4 in ether), the reaction mixture was refluxed for 1 h. After cooling, 200 mL of 0.75M sulfuric acid (H2504) was added to the reaction mixture to decompose excess reagent. The acid layer was separated, made basic with 6M sodium hydroxide (NaOH), and extracted three times with 200 mL of chloroform (CHC13). The combined CHCI 3 extract was treated with a few drops of concentrated hydrochloric acid (HC1)-ethanol (1:9) and evaporated to dryness. The solid residues were recrystallized from ethanol under the following conditions: MOP-d4/HCI; mp 130~ Ultraviolet spectrum; ;%ax = 271 nm (H20), high resolution mass measurement [isobutane/Cl]; MH + at m/z 184.156 (Ctt H]4NOD4 = 184.159); El-MS (m/z) of TFA-MOP-d4:M+ (279), M+-69 (210), M+-91 (158 = base peak), M*-157 (122). Preparation of hair samples. In order to align the roots of 40-50 hairs, the samples were pasted onto adhesive paper (a commercially available carpet cleaner [20 cm x 15 cm]). Scissors were then used to cut 1-cm sections, beginning with the root. Each section was soaked for 10-20 min in 20 mL of 0.1% SDS in a beaker, and the hair was peeled from the paper. After removal from the paper, the hair was washed three times with 10 mL of 0.1% SDS and distilled water, respectively, under ultra-
Materials
Methoxyphenamine preparation (20% powder) was purchased from Nihon Shinyaku Co., Ltd. (Kyoto, Japan). Synthesis of 2,3,3,3-tetradeuterium methoxyphenamine. After 60 mL of o-methoxybenzyl magnesium chloride in ether (obtained by refluxing o-methoxybenzyl chloride [ 13.1 g] and magnesium [2.75 g] for 1 h in anhydrous ether) was added to 20 mL of N-methylethyleneimide-d4 in ether (obtained by introducing T a b l e I. P r e c i s i o n f o r D e t e r m i n a t i o n Methoxyphenamine In H a i r *
of
C0ncentratl0n (ng/mg) Added
Measured (means + SD)
CV (%)
10 5 1
10.24 + 0,21 5.12 + 0,30 0.99 + 0.05
2,05 5,86 5.05
"No. of analyses = 10
Scan
"=
199
(5.451
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MOP-d4 Figure 2. Selected ion monitoring of the derivatized methoxyphenamine and O-desmethyl metabolite extracted from (A) hair and (B) internal standard at m/z 154 and 158.
Figure 1. El mass spectra and major fragments of (A) TFA-methoxyphenamine and (B) TFA-methoxyphenamine-d4.
T a b l e II. C o n c e n t r a t i o n s
r
380
Ma=;s;/Ch ar~a
MOP
amu.
i 180881
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148
Ion
e
(ng/mg)
of Methoxyphenamine
in E a c h ( 1 - c m ) H a i r S e c t i o n Section
Week
1
2
4 6 8 10 12
10.1
10.3 4.7
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1.9
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4.5 3.2
4.8 5.9
"Hair sampleswere collectedon the first day of the week from subject YN, who had taken the drug for seven daysduringweeks 1, 8, and 20.
254
1.7 3.8
J o u m a l of Analytical Toxicology, Vol. 16, July/August 1992
and warmed at 55~ for 20 min. The reaction solution was evaporated with a nitrogen stream, and the residue was redissolved in 100 laL of ethylacetate. One ILL of the ethylacetate solution was automatically injected into a gas chromatograph. The E1 mass spectra of TFA-MOP and TFA-MOP-d4 are shown in Figure 1. Calibration curves, linearity, and reproducibility. The calibration curve for the measurement of MOP was constructed by the analysis of extracted and derivatized samples of 10 mg of control hair, to which the standard solutions of MOP had been added (1-100 ng/mg of hair), together with MOP-d4 (IS), as mentioned above. The calibration curve for MOP (y = 1.01x + 0.0025) in the hair was linear over the concentration range 1-100 ng/mg of hair, with correlation coefficients r = 0.9999. Values less than 1 ng/mg of hair were cut off. Precision of the method was determined using hair of healthy volunteers spiked with MOP at three concentrations with respect to a calibration curve (10 samples were analyzed for each concentration). The coefficients of variations (CVs) for MOP were 2.05, 5.86, and 5.05 at 10, 5, and 1 ng/mg, respectively.
sonication for 1 min, as described in our previous paper (6). Samples were dried with a paper towel and placed in a desiccator under reduced pressure. Analytical method. Each section of hair (4-8 mg) was precisely weighed and extracted with 2 mL of MeOH/5M HCI (20:1) for 1 h under ultrasonication. The solution was kept at room temperature overnight. After I00 laL of the IS methanol solution containing MOP-d4 at 3 I.tg/mL was added into the extracted solution and the hair was filtered off, the filtrate was evaporated under a stream of nitrogen. The residue was dissolved in 200 laL of trifluoroacetic anhydride-ethylacetate (1 : 1)
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Figure 3. Schematic diagram of drug movement along hair shaft from 4th to 30th week after dosages of methoxyphenamine at weeks 1, 8, and 20 (Subject: YN).
Table
III. C o n c e n t r a t i o n s
(ng/mg)
of Methoxyphenemlne
KT Section
8wk
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Discussion
Sampling. It is reported (8) that the growth rate of human scalp hair is ordinarily 0.35--0.4 mm/day (10.5-1.2 cm/month). However, growth rate is also dependent to a certain extent on anatomical region, race, sex, and age. Moreover, hair follicles exist in three phases, the anagen (growing), catagen (intermediate), and talogen (resting) phases. On the scalp of an adult, approximately 15% of the follicles are in a resting stage while the remaining 85% are in their growing stage. Scalp hair is divided into five regions, as follows: frontal, temporal, anterior vertex, posterior vertex, and occipital. In humans, hair is best collected from the area at the back of the head called the "posterior vertex" (9). In this area, the number of follicles in the growing phase is more constant, there are more long terminal hairs, and the hairs are less subject to age and sex influences. We also have found (7) that the variation of drug levels in the vertex hair of several methamphetamine abusers was minimal when compared with the other regions (frontal, temporal, occipital). In this study, therefore, hair samples were collected from the posterior vertex after removing as much of the resting phase of hair as possible by brushing. Analytical method. The determination of MOP in hair was carried out by the stable isotope dilution GC/MS method using deuterium-labeled MOP (MOP-d4) as an internal standard. The base peak of TFA derivatives of MOP and MOP-d4 are m/z 154
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25wk
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"Hair samples were collected on a first day of the week from subject YN, who had taken the drug for seven days during weeks 1, 8 and 20
255
Journal of Analytical Toxicology, Vol. 16, July/August 1992
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Fioure 4. Schematic diagrams of drug movement along hair shaft from 8th to 29th week after the dosages of methoxyphenamine on week 1, 8, and 20. (A) subject KT, (B) subject HS, (C) subject KN, (D) subject AI.
256
Figure 5. Stability of methoxyphenamine in hair. All subjects took the same oral dosage of methoxyphenamine(50 mg x 7). Only drug bands entirely in one (1-cm) section were selected; drug bands divided into two sections were omitted.
and 158, respectively, so that selected ion monitoring (SIM) of their ions was used for the determination. Figure 2 shows the typical SIM chromatogram of TFA derivatives of the extract from hair. One of the metabolites, O-desmethyl MOP, was detected together with unchanged MOR Movement of drug along the hair shaft after drug use. For the elucidation of movement of drug along the hair shaft, five healthy subjects (3 males and 2 females) took 250 mg of MOP powder orally (20% powder) once a day for 7 days during weeks l, 8, and 20. The hair samples of YN (male) were collected on the first day of every 2 weeks from week 4 to week 30 after the first dosage. The samples of KT (male), HS (male), AI (female), and KN (female) were collected on the first day of weeks 8, 16, 25, and 29 alter the first dosage. The results of the sectional analyses of YN are shown in Table 11. MOP was detected in the hair samples three weeks after the first dosage at a concentration of 10.1 ng/mg in the first 1-cm section. This drug band moved from the first to the eighth section according to hair growth, as shown in Figure 3. In a similar manner, the MOPs of the dosages administered during weeks 8 and 20 also moved from the root to the tip. The movement of the drug bands along hair shafts was also observed in the other subjects (KT, HS, KN, AI) without exception, as shown in Table III and Figure 4. When drug bands were extrapolated in Figures 3 and 4, according to the sections in which drugs were detected, their width was found to be approximately 5-6 mm, which is equivalent to 2.4-1.7 times of seven days' hair growth. When MOP is taken orally, the concentration of drug in blood reaches a maximum after 30 min and then rapidly decreases. Within 24 h, 85% is excreted in urine (10). Therefore, MOP is not considered to be present in blood several days after the last administration. Though the mechanism by which drugs are incorporated into hair is not well understood, we have considered that the clearance of drugs from hair matrix cells takes more days, while drugs in the hair matrix cell are gradually incorporated into the hair shaft with tailing. This would explain why the drug bands for 7-day doses are approximately twice as long as the length of 7-days' hair growth. It was also found that the drug bands moved to the hair tip at the rate of 2.8-3.2 mm/week. Stability of drug in hair. Figure 5 shows the decay of the drug
Journal of Analytical Toxicology, Vol. 16, July/August 1992
Table IV. Relationship between Dosage and Drug Level In Each Section Ratios** Section 1 2 3 4 5 6
Lengthfrom root(mm) 0- 6 6-12 12-18 18-24 24-30 30-36
Dosage* (mg)
Level detected (ng/mg)
60
4.9 tracer 2.6 tracer 1.4
40 20
drug level
dosage
3.5
3
1.9
2
1
1
* Amount of methoxyphenamineas a free base. *" The ratiosbased on level detectedand dosage in section5. t Lessthan 1 ng/mg.
levels according to the forward movement of drug bands. Only drug bands contained within the 1-cm sections were selected; drug bands spread across two sections were omitted because of the comparison between the equivalents to 7 doses in each 1-cm section. In our data, it is found that the drug level in hair decreased roughly 50% 5 to 6 months later. We found that the drug levels in the root-side sections were higher than those in the tip-side sections. It is well known that the hair shaft is gradually damaged by environmental influences according to hair growth. Therefore, it is assumed that some drugs in the hair cortex may escape from damaged hair by washing. Furthermore, drugs in hair are assumed to gradually decompose over long periods of time. Correlation between the drug level in hair and the dosage. YN took 20, 40, and 60 mg of methoxyphenamine orally for 7 days during the first, fifth, and ninth week, respectively. On the first day of the 12th week after the first dosage, hair samples were collected and the drug levels in 6-mm sections were determined, as shown in Table IV. When it was assumed that the rate of movement of drug bands would be 2.8 mm/week and the width of the drug band for 7-day doses was 5--6 mm, we figured that the drug bands corresponding to 20-, 40-, and 60-mg doses were in the 5th, 3rd, and 1st sections, respectively. The drug concentrations in the 5th, 3rd, and Ist sections were 1.4, 2.6, and 4.9 ng/mg, respectively. The ratio of the drug concentrations in the 5th, 3rd, and 1st sections was 1:1.9:3.5. This ratio closely corresponds to the ratio of doses (1:2:3). Because this result was obtained from the hair less than 3 cm from the root, it may indicate that the drug level in each hair section nearly corresponds to dose, assuming that the hair shaft has not yet been damaged and that the drugs in hair have not yet decomposed. Comparison between our results and the other reports. Baumgartner et al. reported that the pattern of past drug use of opiates (1), cocaine (11), and phencyclidine (2) can be obtained by sectional hair analysis. Uematsu et al. also reported that a history of individual dosage can be deduced from the distribution of drug level along the length of the hair and that the drug level in a section corresponds to dosage by the sectional analysis of scalp hair of patients that received haloperidol. On the other hand, Puschel et al. (12) concluded in their study that no correlation existed between administered doses of opiates and their concentrations in hair and that the approximate period of drug use in man could be detected by sectioning the hair, though the transport of drugs along the hair may be considerably influenced by various factors concerning hair, The authors have already demonstrated that the distribution of drug along the hair shaft determined by the sectional hair analysis of scalp hairs corresponds to the drug use histories of 11 metham-
phetamine abusers. However, for the elucidation of the movement of the drug along the hair shaft and the correlation between dosage and drug level, it was necessary to perform drug administration under control. Therefore, we undertook an experiment in which five volunteers took the correct dose of methoxyphenamine as a model compound of methamphetamine under control. Samples were collected from the posterior vertex after removing as much of the resting-phase hair as possible.
Conclusion From our present study the following conclusions can be summarized: (a) The drug excreted into hair has moved along hair shaft at the rate of 2.8-3.2 mm/week, according to hair growth, without diffusion. (b) When drug bands were extrapolated according to the sections in which drug was detected, the drug bands were approximately 5 mm wide, which was equivalent to 2.4--1.7 times the length of 7-day hair growth. (c) In case of the same dosages, the drug level in the root side was highest and in the tip side lowest. In our data, it is found that the drug level in hair decreased approximately 50% 5 months later. (d) The ratio of drug levels in the corresponding sections nearly correlates to the ratio of dosages, only in the case that hair shafts are not yet damaged and drugs in hair are not yet decomposed.
References 1. AM. Baumgartner, P.F. Jones, W.A. Baumgartner, and C.T. Black. Radioimmunoassay of hair for determining opiate-abuse histories. J. Nucl. Med. 20:748-52 (1979). 2. A.M. Baumgartner, P.F. Jones, and C.T. Black. Detection of phencyclidine in hair. J. Forensic ScL 26:576-81 (1981). 3. T. Uematsu, R. Sato, K. Suzuki, S. Yamaguchi, and M. Nakashima. Human scalp hair as evidence of individual dosage history of halopeddol: Method and retrospective study. Eur. J. Clin. PharmacoL 37:239-44 (1989). 4. R. Sato, T. Uematsu, S. Yamaguchi, and M. Nakashima. Human scalp hair as evidence of individual dosage history of haloperidol: Prospective Study. Ther. Drug Monitor. 11:686-91 (1989). 5. T. Uematsu, R. Sato, O. Fujimori, and M. Nakashima. Human scalp hair as evidence of individual dosage history of haloperidol: A possible linkage of haloperidol excretion into hair with hair pigment. Arch. DermatoL Res. 282:120-25 (1990). 6. Y. Nakahara, K. Takahashi, M. Shimamine, and Y. Takeda. Hair analysis for drug abuse: I. Determination of methamphetamine and amphetamine in hair by stable isotope dilution gas chromatography/mass spectrometry method. J. Forensic Sci. 36:70-78 (1991). 7. Y. Nakahara, K. Takahashi, Y. Takeda, K. Konuma, S. Fukui, and T. Tokui. Hair analysis for drug abuse, part II. Hair analysis for monitoring of methamphetamine abuse by stable isotope dilution gas chromatography/mass spectrometry. Forensic Sci. Int. 48:243-54 (1990). 8. M. Saitoh, M. Uzuka, M. Sakamoto, and T. Kobori. In Advances in Biology of Skin. W. Montagna and R.L. Dobson, Eds., VoL 5, Pergamon, New York, 1967. 9. M.R. Herkey and G.L. Henderson. In Advances in Analytical Toxicology, Vol. II, R.C. Baselt, Ed., Year Book Medical, 1989, pp. 298-329. 10. In Drugs In Japan, Japan Pharmaceutical Information Center, Yakugyo Jiho Co., Tokyo, Japan, 1985, p. 1029. 11. W.A. Baumgartner, C.T. Black, P.F. Jones, and W.H. Blahd. Radioimmunoassay of cocaine in hair: Concise communication. J. Nucl. Med. 23: 790-92 (1982). 12. K. Puschel, P. Thomasch, and W. Arnold. Opiate level in hair. Forensic Sci. Int. 21:181-86 (1983). Manuscript received August 6, 1991 ; revision received October 29, 1991.
257
