The hair samples of six opiate addicts, who died after heroin overdose, were investigated by gas chromatography-mass spectrometry after extraction with 10 ...
Journal of Analytical Toxicology, Vol. 19, July/August 1995
Solvent Optimization for the Direct Extraction of Opiates from Hair Samples Michael Rothe and Fritz Pragst* Institute of Forensic Medicine, Humboldt University, Hannoversche Strasse 6, D- 10115 Berlin, Germany
Abstract The hair samples of six opiate addicts, who died after heroin overdose, were investigated by gas chromatography-mass spectrometry after extraction with 10 solvents differing in polarity and hydrophilicity in an ultrasonic bath. Morphine, 6monoacetylmorphine (MAM), and codeine were detected in all cases, and heroin was detected in four cases. With toluene, which is hydrophobic, almost no extraction occurred, and with the nonprotic solvents dioxane, acetonitrile, acetone, and dimethyl sulfoxide, only a relatively low extraction rate was found. The yield increased in the series of alcohols from n-propanol to isopropanol to ethanol to methanol. Water proved to have almost the same extraction capability as methanol. Using equal conditions, the extraction rate of the opiates decreased in the following order: heroin > MAM > morphine = codeine. Addition of 1% acetic acid or 1% triethylamine to methanol led to a decrease in the heroin yield and an increase in the morphine yield. The results are discussed in terms of the binding between the hair matrix and the drug, the penetration of the solvent into the hair, and the solubility of the drug in the solvent.
Introduction The analytical investigation of hair samples from opiate addicts and fatalities after heroin overdose is used to an increasing extent to prove long-term abuse, to characterize the abuse intensity, and to elucidate the addiction history (1-24). Although other analytical methods can be used, gas chromatography with mass spectrometry (GC-MS) is the most important technique for this purpose. Sample preparation in the past has first involved the destruction of the hair matrix by heating in NaOH (2-8,12,16,17), followed by radioimmunoassay or by liquid-liquid or solid-phase extraction and derivatization for GC-MS. Using these methods, only morphine and codeine were detected. In other laboratories, the drugs were extracted from the hair with HCI (13,19,20), or the hair matrix was enzymatically digested (10,20). The primary heroin *Author to whom correspondence should be addressed
236
metabolite, 6-monoacetylmorphine (MAM),could be detected along with morphine after enzymatic sample preparation, as described by Raff et al. (10), or in the HCI extract used by Nakahara et al. (19). If hydrolytic conditions are avoidedduring the sample preparation, unchanged heroin is also found in a surprisingly high concentration, as was described by Goldberger et al. (18), who simply cut the hair into small pieces and extracted it by stirring in methanol at 37~ for 18 h. The identification of MAM and heroin after direct extraction of hair was confirmed by Kauert and co-workers (21,22) and also reported by Ahrens et al. (23) and Sachs (24). In order to optimize this direct extraction of hair samples for GC-MS analysis of opiates, we investigated the capability of 10 solvents differing in polarity and hydrophilicity.
Materials and Methods Hair samples Samples of hair with their roots were chosen from eight fatalities whose case histories revealed a drug addiction and whose body fluids and tissues proved an opiate overdose upon analytical investigation. Solvents All solvents except water (i.e., toluene, dioxane, acetonitrile, acetone, dimethyl sulfoxide, isopropanol, n-propanol, ethanol, and methanol) were purchased from Merck (Darmstadt, Germany). The solvents were of analytical-grade quality and were used without further purification.
Extractionprocedureand derivatization Each hair sample (0.5 g) was precleaned by treatment with approximately 20 mL acetone for 5 min in an ultrasonic bath, was rinsed further with acetone, and was then dried between filter papers. The sample was then cut in pieces of 1-3 mm in length and thoroughly mixed. From 10 to 15 mg of this sample pool and I mL of the solvent were tightly enclosed in a 5-mL polyethylene vessel and were treated in the ultrasonic bath
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Journal of Analytical Toxicology, Vol. 19, July/August 1995
(35 kilocycles/s, 120 W) for 2 h. During this time, the bath temperature increased to 45-50~ After this, the extract solution was separated, and 200 ng each codeine-d3and morphined3 dissolvedin 2 pL n-propanol was added by a 10-uL Hamilton syringe. The solvent was removed by evaporation, and the residue was derivatized by a 30-rain treatment at 80~ with 50 pL trifluoroaceticanhydride in 200 uL acetonitrile as similarly describedby Kintz et al. (25). It was then evaporatedagain, and the residuewas dissolved in 50 uL ethyl acetate for the GC-MS analysis. There is a possibility that the roots may have had traces of tissue adhering to them; however, the effect of this on the results of the extraction procedure and on the results of the analysis can be neglected because their part of the sample weight is estimated to be far less than 1%.
several times until no further drug was found in the extract. Figure 2 shows the increase of the extracted amount with the number of extractions for the four compounds in case 4. This was also representative of the methanol extraction used for the other cases. The extraction rate decreased in the order heroin > MAM> morphine = codeine. The concentrations are given in Table I. Heroin was found in four cases. The concentration of MAM was significantly higher than that of morphine in all cases. For the investigation of the extraction efficiency of the different solvents, only the hair samples of cases 1-6 proved to be suitable. Samples 7 and 8 were not involved because the concentrations were too small for unambiguous results. The six samples were extracted only once for 2 h as already described. The yields of this single extraction in relation to the total concentrations given in Table I are shown in Table II. Figure 3 illustrates the differencebetween the solvents used to extract heroin, MAM,and morphine in case 4. With toluene, which is nonpolar and hydrophobic, the yield was only between 0 and 7%. In addition, the cyclic ether, dioxane, which gave a yield between 0 and 11%, was quite ineffective.Using the
GC-MS analysis
For the quantitative determination of opiates, we used the same routine GC-MS method as used in the investigation of blood and urine samples. The Hewlett-Packard GC-MS device consisted of an HP 5890 series II gas chromatograph, an HP 5971 mass selective detector, and an HP 7673 autosampler. A 25-m HP-1 capillary Abundance MAM column (100% methyl silicone) was used _ Morphlne-d for the chromatographic separation with 80O"1 the following temperature program: hold at 120~ for 2 min, then ramp to 280~ at 4oo-I 20~ then hold at 280~ for 10 rain. Heroin The following fragment ions were chosen L 367 000" I ~ for the determination of opiates in the seMorphine lected ion monitoring mode: morphine, m/z 600 J i Co([eine 364, 477; morphine-d3, m/z 367, 480; ~ = _ 395 codeine,m/z 282,395; codeine-d3,m/z 285, . . . . 364 398; MAM, m/z 364, 423; and heroin, m/z 200~ ---~ 327, 369. Each sample was measured twice. 12.00 14.00 8.OO 10.00 Time (mln) This method was characterized by a reFigure 1. Selected ion chromatograms of heroin, 6-monoacetylmorphine (MAM), morphine, and producibilityof +5% at a morphine solution codeine after a single methanol extraction of the hair sample in case 3. concentration of 200 ng/mL (corresponding to a hair concentration of approximately0.5 ng/mg), a limit of detection of 10 ng/mL (approximately0.02 ng/mg hair), and a limit of quantitation of 30 ng/mL (approximately 100 0.06 ng/mg hair). The GC-MS response was linear to the opiate concentration between 8O 30 ng/mL and 20 IJg/mL (approximately 0.075-50 ng/mg hair). An example of the selected ion monitoring chromatograms is shown in Figure 1. -0- Morphine 4O
.e-Codeine ..*-Heroin
Results and Discussion To determine the concentration of the opiates, the hair samples of all eight cases were exhaustively extracted with methanol by repeating the extraction procedure described above using the same sample
9Q-
0 0
I 1
I 2
I 3
J 4
5
6
MAM [
L
7
8
Number of extraction steps Figure 2. Cumulative extraction yields of heroin, 6-monoacetylmorphine (MAM), morphine, and codeine for case 4 as a function of the number of 2-h ultrasonic treatment steps in methanol.
237
Journal of Analytical Toxicology, Vol. 19, July/August 1995
polar aprotic solvents acetone, acetonitrile, and dimethyl sulfoxide, a medium extraction yield was observed. Because of the very small extraction yields, the error of the analytical measurement rendered a comparison difficult; however, it is obvious that with these five aprotic solvents heroin was preferentially extracted. Within the series of alcohols, the yield increased from propanol to methanol. There was no significant difference between n-propanol and isopropanol. As already described for methanol, the extractability also decreased for the other alcohols in the order heroin > MAM > morphine = codeine. A relatively high yield was also determined with water. For cases 3-5, the same order was seen as with methanol. To examine the influence of acidic and basic conditions, 1% acetic acid and 1% triethylamine were added to methanol. The effect for case 4 is shown in Figure 4: The yield of heroin was diminished and that of morphine was increased, and this effect was more evident in the presence of the amine. The concentration of MAM increased in some cases and decreased in other
Table I. Concentrations of Heroin, 6-Monoacetylmorphine (MAM), Morphine, and Codeine in the Hair Samples of Eight Fatalities after Heroin Overdose Determined by GC-MS after Repeated Extraction with Methanol Age Heroin Case (gender) (ng/mg)
MAM (ng/mg)
Morphine Codeine (ng/mg) (ng/mg)
1
40 (m)
2.5
12.0
5.8
1.4
2
35 (m)
2.7
3.6
2.1
0.6
3
33 (f)
3.0
4.3
1.6
1.1
4
31 (m)
1.3
3.8
0.9
0.6
5
22 (m)
0.0
1.2
0.6
0.5
6
55 (m)
0.0
0.8
0.5
0.4
7
19 (m)
0.0
0.2
0.1
0.0
8
23 (m)
0.0
0.1
0.1
0.1
Table II. Extraction Yields of Opiates from Six Hair Samples Obtained after 2 Hours of Treatment with Different Solvents in an Ultrasonic Bath* Dimethyl Methanol, Methanol, Toluene Dioxane Acetonitrile Acetone sulfoxide Isopropanol n-Propanol Ethanol Methanol Water acidict basic* Case Compound (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) Heroin MAMw Morphine Codeine
7 0 1 2
10 1 1 4
18 3 4 6
15 4 3 8
23 8 9 13
13 5 3 6
15 6 4 7
30 24 13 21
45 51 24 31
26 20 7 16
17 49 46 31
5 66 51 46
Heroin MAM Morphine Codeine
0 3 4 0
11 4 3 5
11 5 3 5
2 4 5 7
6 33 21 25
22 3 4 6
20 6 5 7
37 19 15 15
59 50 38 40
17 47 42 61
3 28 62 36
0 19 93 78
Heroin MAM Morphine Codeine
6 2 0 0
10 6 5 0
6 7 7 7
40 9 11 12
31 32 18 15
5 6 6 7
11 10 9 9
34 26 15 16
76 69 42 46
71 70 54 86
27 38 38 47
0 42 83 55
Heroin MAM Morphine Codeine
7 5 4 5
7 7 5 5
15 6 5 8
12 13 9 6
13 6 7 8
17 8 7 12
21 10 11 16
40 31 25 14
69 60 43 21
62 57 43 20
29 55 67 25
11 46 71 22
Heroin MAM Morphine Codeine
. 0 0 0
8 2 7
. 9 7 6
6 9 10
. 5 5 7
. 13 10 11
. 21 27 23
53 51 40
51 53 20
38 71 33
38 73 38
Heroin MAM Morphine Codeine
. 0 0 0
0 0 0
15 12 13
. 18 19 21
51 50 57
27 70 0
51 53 26
15 95 37
.
. 4 6 7 .
. 16 20 9
* Yields in relation to the total concentrations given in Table I. t 1% acetic acid in methanol. =~ 1% triethylamine in methanol. w 6-Monoacetylmorphine.
238
.
. 18 22 27
. 13 15 18
. 12 14 11
Journal of Analytical Toxicology, Voi. 19, July/August 1995
also explain the increase of the extraction rate in this order. 80 It is known that hair can uptake between 15 and 25% water, which is assumed to be 70 placed between the peptide hydrogen bonds 60 "o (26). Also, alcohols penetrate into the hair 50 keratin to a decreasing degree from g 40 methanol to ethanol to propanol (26). These 30 swelling properties should improve the dif20 fusion of the drugs from the binding site to the hair surface the higher the content of 10 the solvent is in the hair. For this reason the 0 extraction rate should decrease in the series ~_. %, % % % . % ,'~-e~, ~'e~ >o/% from water to methanol to higher alcohols. % o, % % %,,o,,+@ .,o ,,o On the other hand, the solubility of the drugs in water is less than in methanol. Figure 3. Extraction yields of heroin, 6-monoacetylmorphine (MAM), and morphine for case 4 after a Therefore, no significant increase of the exsingle 2-h ultrasonic extraction step in different solvents. The yields are in relation to the total contraction rate is observedwhen water is used centrations given in Table I. instead of methanol. It can be concluded from the results that cases. Obviously, hydrolysis of heroin to MAM and partial methanol is the most suitable solvent for the direct extraction hydrolysis of MAMto morphine occurred under these condiof opiates from human hair because it sufficiently penetrates tions. Becauseno water was added, its content in the solvent or into the matrix, it is able to displace the drug from its binding in hair should be quite sufficient for this reaction. sites, it sufficientlydissolves the drugs, and it does not promote In contrast with liquid-liquid extraction of body fluids, the hydrolysis of heroin and MAM. which is determined by a partition equilibrium, the recoveryof drugs from hair should be regarded from a kinetic point of view. Efficiencymainly depends upon extraction rate, and no equilibrium should be attained during the limited extraction 80 time. Therefore, it should be affected to a high extent by the special properties of the hair (e.g., diameter, intactness of the 60 cuticle, alterations due to treatment during the lifetime of the ~ 4o deceased), and no agreement of the results between different Q. cases can be expected. Nevertheless, the effect of the solvent ~9 20 and the opiate structure on the extraction rate is clearly U.I 0 demonstrated. ~ 2"0 2'0 The results can be interpreted in terms of the type and ~ strength of the intermolecular bonds between the hair proteins [~Heroin and the drug, as well as in terms of the capabilityof the solvent IIMAM to penetrate into the hair, to loosen the hydrogen bonds IIMorphine between the protein chains, and to displace the drug from its Figure 4. Effect of 1% acetic acid (AcOH) and triethylamine (TEA) on the position. In addition, the solubility of the drug in the solvent extraction yields of heroin, 6-monoacetylmorphine (MAM), and morphine may have an effect on the results. in case 4 after a single 2-h ultrasonic extraction step with methanol in rePresently,experimental results about the binding of drugs to lation to the total concentrations given in Table I. the hair matrix are not available. With regard to a possible analogy between the binding of drugs to the hair matrix and the binding to plasma protein, ionic and hydrogen bonds and / / hydrophobic interactions must be taken into account. Because R-HC HN \ \ c _ _ o . . . . . H-O c water and methanol extract the opiates significantly faster HN O~C than the higher alcohols or the aprotic solvents, it can be conCH-. H .... O cluded that the hydrogen bridges to the OH groups of mor/ / \ / o~ , / ~ / O=C / N" C--CHs phine and MAM,as well as the ionic interaction between the \NH ~CH 3 0// / /C=O opiate nitrogen atom protonated at neutral pH and the anionic R - HC\ HN\ protein side groups, play a predominant role. This is schemat/ C = O ...... H - - O / CH-R HN On-C ically illustrated in Figure 5. Water and protic solvents are \ \ particularly suitable for the displacement of opiates from this binding. Because the OH groups are successivelyacetylated in Figure 5. Simplified schematic model of the binding of morphine to hair the series morphine < MAM < heroin, this assumption can protein. 239
Journal of Analytical Toxicology, Vol. 19, July/August 1995
Acknowledgments These investigations were supported by the Deutsche Forschungsgemeinschaft. Furthermore, we thank Dr. H. Sachs (Institute of Forensic Medicine, University of Ulm) for practical advice and useful discussions.
14. 15. 16. 17.
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