Archives of

Arch Toxicol (1992) 66:669-674

Toxicologr 9 Springer-Verlag 1992

Hair analysis for drugs of abuse IV. Determination of total morphine and confirmation of 6-acetylmorphine in monkey and human hair by GC/MS Yuji Nakahara 1, Kazunori Takahashi 1, Mochihiko Shimamine 1, and Atsushi Saitoh 2 J National Institute of Hygienic Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo, 158, Japan 2Kanagawa Prefectural Center of Psychiatry Serigaya Hospital, 2-3-1, Serigaya, Konan-ku, Yokohama, 233, Japan Received 13 May 1992/Accepted 21 July 1992

Abstract. The reliable analytical method for total morphine in hair was established by GC/MS-SIM. The calibration curve for morphine in hair showed linear over 0.5-100 ng/mg hair. Though the limit of detection was 0.1 ng/mg hair with an S/N >3 of the base ion(m/z 429) for morphine, the limit of confirmation by detection of three major ions was 0.5 ng/mg. The hydrolytic extraction of the morphine analogs in hair with 10% HC1 for 1 h at 100~ gave quantitative recovery of morphine. The reproducibility of recovery of morphine spiked to the control hair was 2.9-7.3% in a concentration range between 2 and 50 ng/mg hair. The three monkeys were administered once a day with morphine at 10 mg/kg and heroin at 2.5 mg/kg, respectively, for 10 days and their back hair newly grown for 10 weeks was cut for analysis. The levels of total morphine in monkey hair intoxicated with morphine and heroin were 3.4 and 5.2 ng/mg, respectively. Taking their doses into account, it is concluded that the morphine level in hair from monkeys administered with heroin was 6 times higher than that with morphine. In hair from monkeys and humans intoxicated with heroin, 6-acetylmorphine was detected at the level of 0.7-7.2 ng/mg as a major component in hair together with morphine and no heroin. Drug concentrations of sectional hair shaft cut 2 cm each from the root side were compared with the self-reported drug histories of three cases. The results of sectional analysis of heroin abuser's hair suggested that the relationship between the distribution of morphine along hair shaft and the drug use history showed a good correlation, though the accumulation of heroin metabolites in body could result from chronic use of heroin.

Introduction Hair analysis for drugs of abuse is a newly developing technique that offers the confirmation of past drug use and the monitoring of long drug history. In recent years, a number of the studies on the detection of drugs of abuse in human hair have been reported, especially on the detection of opiates. The most common method for hair analysis of opiates was hitherto immunoassay (Baumgartner et al. 1979; Valente et al. 1981; Puschel et al. 1983; Franceschin et al. 1987; Tagliaro et al. 1987, 1988; Sachs et al. 1989), whereas only a few gas chromatography/mass spectrometry (GC/MS) methods for it have been reported (Cone 1990; Goldberger et al. 1991). In forensic toxicology, the use of GC/MS has usually been required for confirmation of drugs. Therefore, the appropriate pretreatment and the precise analytical method should be studied for hair analysis of drugs of abuse using GC/MS method. We have already reported on hair analysis for methamphetamine and amphetamine by stable isotope dilution GC/MS (Nakahara et al. 1990, 1991). In our previous paper (Nakahara et al. 1992a, b), we showed that the drugs excreted into hair steadily moved forward along the hair shaft according to hair growth without diffusion, and the incorporation rate of drugs into hair strongly depended on their physical characters. The present study describes the extraction procedures and analytical method for measuring the levels of total morphine and detecting 6-acetylmorphine(6AM) in hair. The second purpose is to investigate if there is a correlation between the distribution of total morphine along hair shaft and heroin abuse history.

Key words: Hair analysis - Heroin - Morphine - 6Acetylmorphine - GC/MS

Materials and methods

Correspondence to: Y. Nakahara

Chemicals. Morphine hydrochloride (HCI) was purchased from Takeda Pharmaceutical Co. (Osaka, Japan). 6-Acetylmorphine(6AM) and normorphine were synthesized from heroin by the method of Fehn and Megges (1985) and the modified von Braun method (Niwaguchi et al. 1976), respectively. Paraformaldehyde-d2 (99.8 atom % D) and formic acid-d2 (95% in D20, 98.4 atom % D) were purchased from MSD isotope Co. Ldt. (Montreal, Canada). All other chemicals were of reagent grade.

670 Table 1. The levels of 6-acetylmorphine and morphine in monkey and human hair intoxicated with heroin Subjects

6-Acetylmorphine (ng/mg)

Morphine (ng/mg)

Heroin (ng/mg)

Monkey No. 30 No. 32 No. 33

0.8+_0.3 0.7 +_0.2 1.6+_0.4

0.4* +_0.2 0.3* +_0.1 0.5 _+0.2

ND ND ND

Human ST-2 ST-5 ST-12

6.3+_0.8 7.2_+ 1.0 1.9+-0.3

2.8 +_0.3 1.6 +-0.2 0.5 +_0.1

ND ND ND

* Though these values are less than the limit of confirmation, they are shown for reference n=3

Synthesis of morphine-d3. To a solution of normorphine (245 mg) in methanol (8 ml) was added 176 mg paraformaldebyde-d2 and 0.6 ml formic acid-d2 (95% in 1320). The reaction mixture was refluxed under stirring for 20 h. After cooling, the reaction mixture was filtered and evaporated to dryness. The residue was dissolved in 10 ml water and the solution was washed with 10 ml tetrachloromethane, made alkaline with ammonia and extracted three times with 15 ml chloroform-isopropanol (9: 1). The combined organic layers were dried over anhydrous Na2SO4 and evaporated to dryness. The residue was redissolved in 1 ml methanol-con. HCI (9: 1) and the solvent was evaporated again. The solid was recrystallized from methanol-ethanol (1 : 2) to give colorless crystals of morphine-d3 HCI (210 mg, yield 81%).

Animal experiments. Before drug administration, the back hair of the monkeys was cut with an animal electric shaver (Daitoh Electric Machine Co., Tokyo, Japan) and used as a control monkey hair. The six crab eating monkeys (male, 3.3-5.2 kg, age: 3 - 5 years) were subcutaneously administered once a day with morphine HCI at 10 mg/kg and heroin at 2.5 mg/kg once a day for 10 days, respectively. Their back hair grown on the same area where it had been cut was collected on 10 weeks after the first administration.

Human hair sample. Hair samples were collected from the posterior vertex of the scalp by cutting approximately 2 mm from the scalp. The root sides of the hair samples obtained were bundled with a rubber band and the samples were wrapped in aluminum foil. Scalp hair samples of heroin abusers, ST-2, ST-5 and ST-12, were collected and their drug histories recorded by Dr. A. Saitoh of Kanagawa Psychiatry Serigaya Hospital. The heroin abuse histories of three abusers are described in discussion.

Preparation of the standard hair samples. To 10 mg of the control human hair finely cut in a 10 ml glass tube was added 0.1 ml of the methanol solutions of morphine (0.05-10 p.g/ml) at concentrations of 0.5-100 ng/mg hair. The hair sample was mixed occasionally with a vortex mixer while the solvent was naturally evaporated at room temperature. This sample obtained in the glass tube was used as a standard hair sample (positive control).

glass tube at 100*C for 1 h. After cooling, the hair was filtered off. The filtrate was extracted three times with 10 ml CHCI3-isoPrOH (3:1) under the ammonium alkaline condition. The extract combined was evaporated to dryness and then the residue, dissolved in 50 ~tl bis(trimethylsilyl)acetamide, was heated at 90 ~C for 20 min. A 1 [.tl aliquot of the reaction mixture was analyzed by GC/MS (Hewlett-Packard Model 5890IUMSD5971; Neutrabond-1 capillary column, 0.25 mm • 30 m, 0.25 I.tm thickness, temperature-programmed from 60*C (0.5 min hold) to 280~ (5 min hold) at 20~ in the selected ion monitoring (SIM) for TMS2-morphine and TMS2-morphined3 at m/z 429 and 432, respectively. Helium was used as the carrier gas with a head pressure of 9 psi.

Confirmation of 6-acetylmorphine in the hair of monkeys and abusers. Methanol (3 ml) was added to a 10 ml tube containing washed hair (-20 rag) and the tube sealed with parafilm was placed in the ultrasonication bath. The hair was extracted under ultrasonication for 14 h with cooling under 40 ~C. After ultrasonication, the methanol was removed and concentrated to about 0.2 ml at 40*C under a stream of nitrogen. A 2 ml aliquot of phosphate buffer (pH 5.5) was added into the concentrated solution and i~oured on a Bond Elut Certify column under mild suction at about 1 ml/min. After washing with water (2 ml), 0.1 M acetic acid (2 ml) and water (2 ml), the column was dried at maximum suction for 5 rain. After further rinsing with methanol (2 ml), the target drugs were eluted with 2 ml CHCl3-methanol - 28% NH4OH (20 : 80 : 2). The eluent was evaporated to dryness under a stream of nitrogen. The residue was derivatized as mentioned before for GC/MS analysis.

Results Calibration curves and selected ion monitoring L i n e a r c a l i b r a t i o n l i n e s w e r e o b t a i n e d w h e n t h e p e a k area ratio m / z 4 2 9 / 4 3 2 w a s p l o t t e d a g a i n s t concentrations ( 0 . 5 - 1 0 0 n g / m g ) o f m o r p h i n e HC1 in s p i k e d standard hairs. A t the c o n c e n t r a t i o n r a n g e o f 0 . 5 - 1 0 0 n g / m g , the r e g r e s s i o n e q u a t i o n w a s Y = 0.0431 X + 0 . 0 1 3 4 (correlat i o n c o e f f i c i e n t , r = 1.000). T h o u g h the d e u t e r a t e d comp o u n d (14.43 m i n ) e l u t e s s l i g h t l y e a r l i e r t h a n the unlabeled s p e c i e s (14.45 m i n ) , t h e o c c u r r e n c e o f m a s s f r a g m e n t s at m / z 4 2 9 a n d 4 3 2 w i t h v e r y c l o s e r e t e n t i o n t i m e s on this r e l a t i v e l y c l e a n c h r o m a t o g r a m s t r o n g l y s u p p o r t s the prese n c e o f m o r p h i n e , In a d d i t i o n , it is r e c o m m e n d e d for the c o n f i r m a t i o n o f m o r p h i n e that the p r i n c i p a l p e a k s (m/z 429, 287, 236) are c o n f i r m e d at the s a m e r e t e n t i o n times as s h o w n in Fig. 1. T h o u g h the l i m i t o f d e t e c t i o n w a s 0.1 n g / m g hair with an S / N >3 o f the b a s e p e a k i o n ( m / z 4 2 9 ) f o r m o r p h i n e , the l i m i t o f c o n f i r m a t i o n by d e t e c t i o n o f t h r e e m a j o r ions was 0.5 n g / m g . F i g u r e 2 s h o w s a t e n t a t i v e m a s s fragmentation p a t t e r n o f T M S 2 - m o r p h i n e and T M S - 6 - a c e t y l m o r p h i n e .

Recovery of morphine from hair sample Analytical method. For the exact arrangement of the length from the root of 4 0 - 5 0 hairs, the samples were evenly pasted on adhesive paper from the root side. Then every 2 cm - sections from the root side were cut by scissors. At 10 or 20 min after each section had been soaked in 0.1% SDS, hair tips were peeled off from the paper. On removal of the paper, the hair tips were washed three times with 10 ml 0.1% SDS and 10 ml distilled water, respectively, under ultrasonication for 1 min. After the hair had been dried, to each section of hair ( 4 - 8 mg) 100 Ixl of the IS aqueous solution were added, containing morphine-d3 HCI at 1 ktg/ml. The extraction was carried out with 2 ml 10% HC1 in a 10 ml stoppered

T h e r e c o v e r i e s o f m o r p h i n e f r o m h a i r s a m p l e s at concentrations o f 2 - 5 0 n g / m g w e r e m o r e t h a n 9 7 . 5 % and the c o e f f i c i e n t s o f v a r i a t i o n ( C V ) w e r e f r o m 2.38 to 7.30% in the six a n a l y s e s . C o m p l e t i o n o f h y d r o l y s i s o f a c e t y l m o r p h i n e in hair was s t u d i e d w i t h the h a i r s a m p l e s f o r t i f i e d w i t h h e r o i n (50 ng) a n d 6 - a c e t y l m o r p h i n e (50 n g ) in the s a m e e x t r a c t i o n condition as m e n t i o n e d a b o v e . T h e r e c o v e r i e s o f m o r p h i n e from

671 ~bundance

Ion 399.15: 1101003.D Ion 340.10:1101003.0 Ion 287.00:1101003.0 Ion 236.05: 1101003.D I0n 429,15: iiOtO03.D I0n 432.10:1101003.0

4500-

~bundance

Ion 399.15: 0701002.D Ion 340.10: 0701002.D Ion EB7.O0: 0701002.D

t1000~

[on 231~.05: 0701002.D Ion 429.15: 0701002.D

Ion 43;!.t0:

tO000 :

0701002.D

400090003500,

6AM

00003000.

7000-

2500

60005000.

2000 -~ 1500 -4 morphine

tO00 -~

4000,

morphlale

2000]

IJll 14291upper}

0001

6AM

A

]

500 -q

|

Illl 12361mlddle)I

J

J340(middlelJ

1000~

oi ' ' ' ' I rl=e -:44. Po 14.40

14.60

14.80

15.00

'

iime ->

'

14.40 '

I

.

.

.

.

I

9

14.60

9

'

9

I

.

.

.

14.80

.

I

'

'

'

15.00

Fig. 1. The SIM chromatogramsof the extracts from monkey (A) and human (B) hair intoxicatedwith heroin. Selected ions of m/z 429, 287,236 for TMS2morphine and m/z 399, 340, 287 for TMS-6AM were monitored,respectively

---]+

TMSO

c.3 ----- soL, Jc.3 m/z 429 TMS2-morphine

m/z 236

TMS~~ + ~

,i

-~)NCH3

m/z 340

RDA

TMSO 1 0 ~

rlVz429

---1+

"-1 +

C H 3 C O OL ~~CH3 m/z 399 TMS-6AM

hair sample by hydrolysis of heroin and 6-acetylmorphine were 96.4 and 99.7%, respectively.

Detection of 6AM in monkey and human hair intoxicated with heroin Detection of 6AM or heroin in biological samples would serve as an acceptable marker for the confirmation of heroin exposure. All our hair samples from three monkeys and three humans intoxicated with heroin contained 6AM at the level of 0.7-7.2 ng/mg as a major metabolite derived from heroin (Table 1). The SIM chromatograms of the extracts from monkey and human hair intoxicated with heroin are shown in Fig. 1.

%~.,/NCH3

m/z 287

Fig. 2. Tentative mass fragmentation pattern of TMS2-morphineand TMS-6acetylmorphine

Though the methanol extraction of monkey hair samples intoxicated with heroin showed the presence of 6-acetylmorphine (0.7-1.6 ng/mg) as well as morphine (0.3-0.8 ng/mg) in hair, the total amount of morphine was lower than that (4.2-6.4 ng/mg) following hydrolytic extraction.

Difference of morphine level in hair between administration of morphine and heroin The 10-weeks-old hair samples of three monkeys administered morphine HCI at 10 mg/kg/day and heroin at 2.5 mg/kg/day for 10 days, respectively, were used for this study. Morphine levels in three monkey hair samples administered with morphine were 2.52-3.99 ng/mg (mean

672 Table 2. The analytical results of monkey hair administered with morphine and heroin

ST-2 30

84

9

Drug administrated

Monkey number

Morphine (ng/mg)

SD

Morphine 10 mg/kg

No. 14 No. 15 No. 16

2.52 3.72 3.99

0.28 1.13 0.74

11.1 30.4 9.3

Av.

3.41

No. 30 No. 32 No. 33

6.40 4.17 5.06

0.73 1.01 0.73

11.4 24.2 14.4

Av.

5.21

///

CV (%)

/// 26

///

84

///

Heroin 2.5 mg/kg

15 84

6

Section Length from Morphine root side* (ng/mg)

Drug abuse history (heroin)

Timetable month, year

(cm) (1) (2)

0- 2 2- 4

13.0 8.3

(3) (4) (5) (6) (7) (8) (9)

4- 6 6- 8 8-10 10-12 12-14 14-16 16+

5.0 2.1 9.1 13.3 17.0 20.8 27.5

Everyday use 3, (max. 1 g/day) in Thailand and Nairobi . . . .Drug-free . . . . . . . . . . . .in. . . . 11, Japan 7, Every day use (max. 1 g/day) in Nairobi and New York 12,

1989

1988 1988

1987

* Hair sample was cut at approximately 2 mm from the scalp

Table 4. Sectional analysis of scalp hair and drug abuse history of ST-5 Hair Section Length from Morphine Drug abuse collection root side* (ng/mg) history (cm)

Second

First

(1) (2)

0- 2 2- 4

1.1

MS.

The reliable analytical method for total morphine in hair was established by GC/MS-SIM. The calibration curve for morphine in hair showed linear over ...
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