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Use of solid-phase microextraction (SPME) for the determination of methadone and EDDP in human hair by GC–MS
Forensic Science International 107 (2000) 225–232 www.elsevier.com / locate / forsciint
Use of solid-phase microextraction (SPME) for the determination of methadone and EDDP in human hair by GC–MS b ´ A.C.S. Lucas a , *, A.M. Bermejo b , M.J. Tabernero b , P. Fernandez , c S. Strano-Rossi a
b
Department of Clinical Analysis and Toxicology, University of Amazonas, Manaus, Brazil Department of Legal Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain c Institute of Legal Medicine, Catholic University of Sacred Heart, Rome, Italy Received 28 June 1999; received in revised form 4 August 1999; accepted 25 August 1999
Abstract Solid-phase microextraction (SPME) is a new extraction technique with many advantages: small sample volume, simplicity, quickness and solvent-free. It is mainly applied to environmental analysis, but is also useful for the extraction of drugs from biological samples. In this paper the use of SPME is proposed for the determination of methadone and its main metabolite EDDP in hair by GC–MS. The hair samples were washed, cut into 1-mm segments, and incubated with Pronase E for 12 h. A 100-mm polydimethylsiloxane (PDMS) film fibre was submerged for 30 min in a diluted solution of the hydrolysis liquid (1:4 with borax buffer) containing methadone-d 3 and EDDP-d 3 as internal standards. Once the microextraction was concluded the fibre was directly inserted into the CG injection port. Linearity was found for methadone and EDDP in the range studied, 1.0–50 ng / mg hair, with correlation coefficients higher than 0.99. Interassay relative standard deviation (R.S.D) was determined to be less than 13.30% for methadone and less than 8.94% for EDDP, at 3.0 and 30.0 ng / mg. Analytical recoveries were close to 100% for both compounds on spiked samples. The method was applied to the analysis of real hair samples from eight patients of a methadone maintenance programme. The concentration of methadone in hair ranged from 2.45 to 78.10 ng / mg, and for EDDP from 0.98 to 7.76 ng / mg of hair. 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Solid-phase microextraction (SPME); Methadone; EDDP; Hair; GC–MS
*Corresponding author. 0379-0738 / 00 / $ – see front matter 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S0379-0738( 99 )00165-6
226
A.C.S. Lucas et al. / Forensic Science International 107 (2000) 225 – 232
1. Introduction The analysis of drugs in hair is of particular interest because it can provide information about long-term use and is useful for many purposes including criminal investigations and monitoring the compliance on drug maintenance programmes. Immunological methods are normally used for the screening of drugs in hair, and their results must be confirmed by more sensitive and specific methods. Various methods have been applied to the determination of methadone in hair: radioimmunoassay [1,2], high-performance liquid chromatography [3], capillary electrophoresis [4], and gas chromatography–mass spectrometry (GC–MS) [5–7]. Confirmation analysis generally consists of a pretreatment of the sample, and hair hydrolysis followed by liquid–liquid or a solid-phase extraction. Solid-phase microextraction (SPME) is a new technique of extraction with many advantages: small sample volume, simplicity, quickness and solvent-free. It is mainly applied to environmental analysis, but is also useful for the extraction of drugs from biological samples [8]. Strano-Rossi and Chiarotti [9] demonstrated the suitability of SPME for the determination of methadone in hair samples. Recently, we proposed the use of SPME for the determination of methadone and its main metabolite (EDDP) in plasma, in a comparative study with the classical liquid–liquid extraction [10]. In the present work the use of SPME in the determination of methadone and EDDP in hair by GC–MS is described. The method was applied to the analysis of real hair samples from eight patients of a methadone maintenance programme (MMP).
2. Experimental.
2.1. Hair samples Drug-free black hair, taken from a laboratory volunteer, was used for preparation of standards and calibrators. Hair samples from eight patients of a MMP from the Autonomic Community of Galicia (Spain) were analyzed.
2.2. Chemicals and reagents Methadone hydrochloride and methadone-d 3 hydrochloride were obtained from Sigma (St. Louis, MO); 2-ethylene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) perchlorate and EDDP-d 3 perchlorate were obtained from Radian (Austin, TX). Methanol, borax and sodium chloride (analytical grade), Pronase E and 1,4-dithiothreitol (DTT) were obtained from Merck (Darmstadt, Germany). The SPME device equipped with a 100-mm polydimethylsiloxane film fibre supplied by Supelco (Bellefonte, PA) was used for the microextraction.
A.C.S. Lucas et al. / Forensic Science International 107 (2000) 225 – 232
227
2.3. Instrumentation GC–MS analysis was performed with a Hewlett Packard Model 6890 gas chromatograph (Hewlett-Packard, Avondale, PA), equipped with a HP-5 capillary column (12 m30.22 mm I.D., 0.33 mm film thickness of crosslinked 5% phenyl methyl silicone). The injection port (splitless, 2 min) was set at 2508C. The column temperature was initially held at 908C for 1 min, increased to 2008C at 308C / min, and remaining at 2008C for 5 min, then increased to 2908C at 308C / min. The carrier gas was helium at 1 ml / min. A HP 5973 mass selective detector in SIM mode coupled to GC was used for quantitative analysis. The electron impact of 70 eV was used for the ionization of the compounds. Ion currents at m /z (294), 295 and 223; (297) and 226; (277), 276 and 262; (280), 279 and 265, were monitored for methadone, methadone-d 3 , EDDP and EDDPd 3 , respectively.
2.4. Sample preparation and extraction procedure Hair samples were cut as close as possible to the scalp. To eliminate external contaminants, the samples were washed three times with distilled water and acetone, and then dried in a stream of warm air. The last wash was analysed to exclude external contamination. The hair samples were then cut into approximately 1-mm segments with surgical scissors and 0.5 ml of 6 mg / ml DTT (in Tris buffer at pH 7.2) were added to 50 mg of hair, after 2 h, 0.5 ml of 1 mg / ml Pronase E (in Tris buffer at pH 7.2), then incubated at 378C for 12 h for hydrolysis. To a 100-ml aliquot of the incubation medium 5 ml of a methanolic solution of the deuterated internal standards (methadone-d 3 and EDDP-d 3 at 0.01 mg / ml), 0.4 ml of borax buffer (pH 9.2) and 100 mg of sodium chloride was added and then mixed. The fibre (SPME) was submerged in this solution for 30 min to perform the microextraction, then directly inserted into the GC injection port and stripped at 2508C for 5 min.
2.5. Quantitation Standard calibration curves were obtained in triple runs with the described method using drug-free control hair spiked with methadone and EDDP to obtain the concentrations of 1, 3, 5, 10, 20, 30, 40 and 50 ng / mg hair for each compound. Quantitation was based on target peak area ratios of methadone (m /z 294) and EDDP (m /z 277) with their respective internal standards (m /z 297 and 280, respectively)
2.6. Limit of detection and limit of quantitation The limit of detection (LOD) and the limit of quantitation (LOQ) were determined by replicate analysis, as described above, of samples devoid of analytes (N511). LOD was defined as the mean value of analyte apparent concentration in the negative samples plus
228
A.C.S. Lucas et al. / Forensic Science International 107 (2000) 225 – 232
three times the standard deviation, and LOQ was defined as the same mean value plus ten times the standard deviation [11].
2.7. Reproducibility and analytical recovery The precision of the method for methadone and EDDP in hair was evaluated for eight replicate extractions of drug-free hair samples spiked with a 3.0 and 30.0 ng / mg concentration of each analyte and submitted to the described procedure. The analytical recovery for each analyte was expressed as the percentage over the theoretical samples concentration (3.0 and 30.0 ng / mg), for a confidence interval of 95%.
3. Results and discussion
3.1. Analytical method The gas chromatographic method used in this work was efficient to separate methadone and EDDP. The retention times (RT) were 7.21 min for EDDP-d 3 , 7.24 min for EDDP, 8.69 min for methadone-d 3 and 8.72 min for methadone, as can be seen in Fig. 1 that shows the extracted ion chromatogram from patient 6. No carry-over with the desorption time of 5 min at 2508C and no degradation of the fibre were observed after at least 90 runs. The standard calibration curves were obtained in a triple run. Simple linear regression analysis was performed and provided the calibration curve equations and the correlation coefficients (r) shown in Table 1. The assay was linear from 1.0 to 50.0 ng / mg of hair for methadone (r50.998) and for EDDP (r50.997). The precision of the methods for methadone and EDDP in hair was studied through the within-batch precision for eight replicate analysis at two concentration levels, 3.0 and 30.0 ng / mg, prepared with drug-free human hair, and submitted to the SPME procedure. The results (Table 2) are expressed as relative standard deviation (R.S.D.%), whose values were lower than 13.30% for methadone and lower than 8.94% for EDDP. Such results lead to a maximum error of 64.7% for methadone and 63.2% for EDDP, ´ assumed to be typical for analysis by SPME–GC according to Gorecki and Pawliszyn [12]. The results of analytical recovery, on spiked samples are also presented in the Table 2 expressed as the mean recovery obtained for the analytes, at 3.0 and 30.0 ng / mg, for a confidence interval of 95% (N58). As can be observed, adequate analytical recoveries were achieved, close to 100% for both analytes. The limit of detection (LOD) for methadone was 2.48 ng / mg and the limit of quantitation (LOQ) was 3.46 ng / mg. Despite the high LOD and LOQ values for methadone, due to the noise of the sample matrix, it was not enough to interfere with the range of concentrations found in the patient’s hair samples. The LOD for EDDP was 0.15 ng / mg and the LOQ was 0.36 ng / mg. These values are quite acceptable for the purposes of this method.
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Fig. 1. Extracted ion chromatograms from hair sample of Patient 6, in which 14.70 ng / mg of methadone and 3.37 ng / mg of EDDP were detected.
A.C.S. Lucas et al. / Forensic Science International 107 (2000) 225 – 232
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Table 1 Curve equations and correlation coefficients (r) for determination of methadone and EDDP in hair using SPME / GC–MS Compound
Curve equation
r
Methadone EDDP
y50.0333x20.0015 y50.0574x10.0467
0.998 0.997
Table 2 Precision (R.S.D. %) and analytical recoveries (%) for methadone and EDDP in hair, using SPME / GC–MS, at 3.0 and 30.0 ng / mg Compound
Concentration (ng / mg)
Analytical recoveries a (%)
R.S.D.b (%)
Methadone
3.0 30.0 3.0 30.0
102.50611.12 107.24610.24 102.7266.07 103.6367.47
13.30 12.25 7.26 8.94
EDDP a b
For a confidence interval of 95%. Relative standard deviation.
3.2. Analysis of MMP patient’ s hair samples The described method was used to determine methadone and EDDP in human hair from eight patients from a methadone maintenance programme from the Assistance Programme of the Autonomic Community of Galicia (Spain). As can be seen in Table 3, the hair concentrations of methadone in the samples ranged from 2.45 to 78.10 ng / mg and the EDDP concentrations ranged from 0 to 7.76 ng / mg. These values are higher than those found by other authors [2,5–7], possibly due to the higher doses of methadone that received our patients (55–100 mg / day) compared to the doses of other studies (25–80 mg / day). Nevertheless, the methadone / EDDP ratios ranged from 4.36 to 10.06 and are consistent with those previous studies. The correlation between methadone dose and hair concentration in our samples showed statistical significance (P,0.02). Goldberger et al. [7] found no correlation Table 3 Results of MMP a patient’s hair analysis, using SPME / GC–MS for determination of methadone and EDDP Patient
Age (years)
Hair colour
Methadone dose (mg / day)
Methadone (ng / mg)
EDDP (ng / mg)
MTD b / EDDP ratio
1 2 3 4 5 6 7 8
32 25 34 27 22 29 38 35
Black Brown Dark brown Black Dark brown Black Black Dark brown
55 100 70 80 80 60 60 70
23.83 78.10 7.88 25.12 31.31 14.70 2.45 7.34
4.19 7.76 1.36 2.97 5.07 3.37 – 0.98
5.69 10.06 5.79 8.46 6.18 4.36 – 7.49
a b
Methadone maintenance programme. Methadone
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231
between these parameters, and said that was probably due to the assay of hair trimmings rather than hair strands. Green et al. [13] found statistical differences between methadone concentrations in black and white rat hair. The effect of hair colour in the concentration of methadone was not evident in our samples.
4. Conclusion This work describes the application of solid-phase microextraction (SPME) for the determination of methadone and EDDP in hair by GC–MS. The method was successfully applied to the analysis of hair samples from patients of a methadone maintenance programme. The microextraction was performed directly on a small aliquot of the enzymatic hydrolysis liquid, commonly used for the screening techniques for drugs in hair, allowing the use of the remaining sample for confirmation analysis of other drugs, necessary for each positive screening results. Because the SPME needs small sample volume, and is a simple, rapid and solventfree procedure, it also made the analysis time (screening-confirmation) quicker for methadone and EDDP in hair.
References [1] S. Balabanova, H.U. Wolf, Methadone concentrations in human hair of the head, axillary and pubic hair, Z. Rechtsmed. 102 (1989) 293–296. [2] S. Balabanova, H.U. Wolf, Determination of methadone in human hair by radioimmunoassay, Z. Rechtsmed. 102 (1989) 1–4. [3] P. Kintz, H.P. Eser, A. Tracqui, M. Moeller, V. Cirimele, P. Mangin, Enantioselective separation of methadone and its main metabolite in human hair by liquid chromatography / ion trap–mass spectrometry, J. Forensic Sci. 42 (1997) 291–295. ¨ [4] M. Frost, H. Kohler, G. Blaschke, Enantioselective determination of methadone and its main metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in serum, urine and hair by capillary electrophoresis, Electrophoresis 18 (1997) 1026–1034. [5] M.R. Moeller, P. Fey, R. Wenning, Simultaneous determination of drugs of abuse (opiates, cocaine and amphetamine) in human hair by GC–MS and its application to a methadone treatment programme, Forensic Sci. Int. 63 (1993) 185–206. [6] D.G. Wilkins, P.R. Nagasawa, S.P. Gygi, R.L. Foltz, D.E. Rollins, Quantitative analysis of methadone and two major metabolites in hair by positive chemical ionization ion trap mass spectrometry, J. Anal. Toxicol. 20 (1996) 355–361. [7] B.A. Goldberger, A.G. Darraj, Y.H. Caplan, E.J. Cone, Detection of methadone, methadone metabolites, and other illicit drugs of abuse in hair of methadone treatment subjects, J. Anal. Toxicol. 22 (1998) 526–530. [8] L. Junting, C. Peng, O. Suzuki, Solid-phase microextraction (SPME) of drugs and poisons from biological samples, Forensic Sci. Int. 97 (1998) 93–100. [9] S. Strano-Rossi, M. Chiarotti, Solid-phase microextraction for cannabinoids analysis in hair and its possible application to other drugs, J. Anal. Toxicol. 23 (1999) 7–10.
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´ [10] A.M. Bermejo, R. Seara, A.C. dos Santos Lucas, M.J. Tabernero, P. Fernandez, R. Marsili, Use of solid-phase microextraction (SPME) for the determination of methadone and its main metabolite, EDDP, in plasma by gas chromatography–mass spectrometry, J. Anal. Toxicol. 23 (1999) 00, in press. [11] J.C. Miller, J.N. Miller, Statistics for Analytical Chemistry, Wiley, New York, 1993. ´ [12] T. Gorecki, J. Pawliszyn, Effect of sample volume on quantitative analysis by solid-phase microextraction. Part 1. Theoretical considerations, Analyst 122 (1997) 1079–1086. [13] S.J. Green, J.F. Wilson, The effect of hair color on the incorporation of methadone into hair in the rat, J. Anal. Toxicol. 20 (1996) 121–123.
Use of solid-phase microextraction (SPME) for the determination of methadone and EDDP in human hair by GC–MS b ´ A.C.S. Lucas a , *, A.M. Bermejo b , M.J. Tabernero b , P. Fernandez , c S. Strano-Rossi a
b
Department of Clinical Analysis and Toxicology, University of Amazonas, Manaus, Brazil Department of Legal Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain c Institute of Legal Medicine, Catholic University of Sacred Heart, Rome, Italy Received 28 June 1999; received in revised form 4 August 1999; accepted 25 August 1999
Abstract Solid-phase microextraction (SPME) is a new extraction technique with many advantages: small sample volume, simplicity, quickness and solvent-free. It is mainly applied to environmental analysis, but is also useful for the extraction of drugs from biological samples. In this paper the use of SPME is proposed for the determination of methadone and its main metabolite EDDP in hair by GC–MS. The hair samples were washed, cut into 1-mm segments, and incubated with Pronase E for 12 h. A 100-mm polydimethylsiloxane (PDMS) film fibre was submerged for 30 min in a diluted solution of the hydrolysis liquid (1:4 with borax buffer) containing methadone-d 3 and EDDP-d 3 as internal standards. Once the microextraction was concluded the fibre was directly inserted into the CG injection port. Linearity was found for methadone and EDDP in the range studied, 1.0–50 ng / mg hair, with correlation coefficients higher than 0.99. Interassay relative standard deviation (R.S.D) was determined to be less than 13.30% for methadone and less than 8.94% for EDDP, at 3.0 and 30.0 ng / mg. Analytical recoveries were close to 100% for both compounds on spiked samples. The method was applied to the analysis of real hair samples from eight patients of a methadone maintenance programme. The concentration of methadone in hair ranged from 2.45 to 78.10 ng / mg, and for EDDP from 0.98 to 7.76 ng / mg of hair. 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Solid-phase microextraction (SPME); Methadone; EDDP; Hair; GC–MS
*Corresponding author. 0379-0738 / 00 / $ – see front matter 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S0379-0738( 99 )00165-6
226
A.C.S. Lucas et al. / Forensic Science International 107 (2000) 225 – 232
1. Introduction The analysis of drugs in hair is of particular interest because it can provide information about long-term use and is useful for many purposes including criminal investigations and monitoring the compliance on drug maintenance programmes. Immunological methods are normally used for the screening of drugs in hair, and their results must be confirmed by more sensitive and specific methods. Various methods have been applied to the determination of methadone in hair: radioimmunoassay [1,2], high-performance liquid chromatography [3], capillary electrophoresis [4], and gas chromatography–mass spectrometry (GC–MS) [5–7]. Confirmation analysis generally consists of a pretreatment of the sample, and hair hydrolysis followed by liquid–liquid or a solid-phase extraction. Solid-phase microextraction (SPME) is a new technique of extraction with many advantages: small sample volume, simplicity, quickness and solvent-free. It is mainly applied to environmental analysis, but is also useful for the extraction of drugs from biological samples [8]. Strano-Rossi and Chiarotti [9] demonstrated the suitability of SPME for the determination of methadone in hair samples. Recently, we proposed the use of SPME for the determination of methadone and its main metabolite (EDDP) in plasma, in a comparative study with the classical liquid–liquid extraction [10]. In the present work the use of SPME in the determination of methadone and EDDP in hair by GC–MS is described. The method was applied to the analysis of real hair samples from eight patients of a methadone maintenance programme (MMP).
2. Experimental.
2.1. Hair samples Drug-free black hair, taken from a laboratory volunteer, was used for preparation of standards and calibrators. Hair samples from eight patients of a MMP from the Autonomic Community of Galicia (Spain) were analyzed.
2.2. Chemicals and reagents Methadone hydrochloride and methadone-d 3 hydrochloride were obtained from Sigma (St. Louis, MO); 2-ethylene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) perchlorate and EDDP-d 3 perchlorate were obtained from Radian (Austin, TX). Methanol, borax and sodium chloride (analytical grade), Pronase E and 1,4-dithiothreitol (DTT) were obtained from Merck (Darmstadt, Germany). The SPME device equipped with a 100-mm polydimethylsiloxane film fibre supplied by Supelco (Bellefonte, PA) was used for the microextraction.
A.C.S. Lucas et al. / Forensic Science International 107 (2000) 225 – 232
227
2.3. Instrumentation GC–MS analysis was performed with a Hewlett Packard Model 6890 gas chromatograph (Hewlett-Packard, Avondale, PA), equipped with a HP-5 capillary column (12 m30.22 mm I.D., 0.33 mm film thickness of crosslinked 5% phenyl methyl silicone). The injection port (splitless, 2 min) was set at 2508C. The column temperature was initially held at 908C for 1 min, increased to 2008C at 308C / min, and remaining at 2008C for 5 min, then increased to 2908C at 308C / min. The carrier gas was helium at 1 ml / min. A HP 5973 mass selective detector in SIM mode coupled to GC was used for quantitative analysis. The electron impact of 70 eV was used for the ionization of the compounds. Ion currents at m /z (294), 295 and 223; (297) and 226; (277), 276 and 262; (280), 279 and 265, were monitored for methadone, methadone-d 3 , EDDP and EDDPd 3 , respectively.
2.4. Sample preparation and extraction procedure Hair samples were cut as close as possible to the scalp. To eliminate external contaminants, the samples were washed three times with distilled water and acetone, and then dried in a stream of warm air. The last wash was analysed to exclude external contamination. The hair samples were then cut into approximately 1-mm segments with surgical scissors and 0.5 ml of 6 mg / ml DTT (in Tris buffer at pH 7.2) were added to 50 mg of hair, after 2 h, 0.5 ml of 1 mg / ml Pronase E (in Tris buffer at pH 7.2), then incubated at 378C for 12 h for hydrolysis. To a 100-ml aliquot of the incubation medium 5 ml of a methanolic solution of the deuterated internal standards (methadone-d 3 and EDDP-d 3 at 0.01 mg / ml), 0.4 ml of borax buffer (pH 9.2) and 100 mg of sodium chloride was added and then mixed. The fibre (SPME) was submerged in this solution for 30 min to perform the microextraction, then directly inserted into the GC injection port and stripped at 2508C for 5 min.
2.5. Quantitation Standard calibration curves were obtained in triple runs with the described method using drug-free control hair spiked with methadone and EDDP to obtain the concentrations of 1, 3, 5, 10, 20, 30, 40 and 50 ng / mg hair for each compound. Quantitation was based on target peak area ratios of methadone (m /z 294) and EDDP (m /z 277) with their respective internal standards (m /z 297 and 280, respectively)
2.6. Limit of detection and limit of quantitation The limit of detection (LOD) and the limit of quantitation (LOQ) were determined by replicate analysis, as described above, of samples devoid of analytes (N511). LOD was defined as the mean value of analyte apparent concentration in the negative samples plus
228
A.C.S. Lucas et al. / Forensic Science International 107 (2000) 225 – 232
three times the standard deviation, and LOQ was defined as the same mean value plus ten times the standard deviation [11].
2.7. Reproducibility and analytical recovery The precision of the method for methadone and EDDP in hair was evaluated for eight replicate extractions of drug-free hair samples spiked with a 3.0 and 30.0 ng / mg concentration of each analyte and submitted to the described procedure. The analytical recovery for each analyte was expressed as the percentage over the theoretical samples concentration (3.0 and 30.0 ng / mg), for a confidence interval of 95%.
3. Results and discussion
3.1. Analytical method The gas chromatographic method used in this work was efficient to separate methadone and EDDP. The retention times (RT) were 7.21 min for EDDP-d 3 , 7.24 min for EDDP, 8.69 min for methadone-d 3 and 8.72 min for methadone, as can be seen in Fig. 1 that shows the extracted ion chromatogram from patient 6. No carry-over with the desorption time of 5 min at 2508C and no degradation of the fibre were observed after at least 90 runs. The standard calibration curves were obtained in a triple run. Simple linear regression analysis was performed and provided the calibration curve equations and the correlation coefficients (r) shown in Table 1. The assay was linear from 1.0 to 50.0 ng / mg of hair for methadone (r50.998) and for EDDP (r50.997). The precision of the methods for methadone and EDDP in hair was studied through the within-batch precision for eight replicate analysis at two concentration levels, 3.0 and 30.0 ng / mg, prepared with drug-free human hair, and submitted to the SPME procedure. The results (Table 2) are expressed as relative standard deviation (R.S.D.%), whose values were lower than 13.30% for methadone and lower than 8.94% for EDDP. Such results lead to a maximum error of 64.7% for methadone and 63.2% for EDDP, ´ assumed to be typical for analysis by SPME–GC according to Gorecki and Pawliszyn [12]. The results of analytical recovery, on spiked samples are also presented in the Table 2 expressed as the mean recovery obtained for the analytes, at 3.0 and 30.0 ng / mg, for a confidence interval of 95% (N58). As can be observed, adequate analytical recoveries were achieved, close to 100% for both analytes. The limit of detection (LOD) for methadone was 2.48 ng / mg and the limit of quantitation (LOQ) was 3.46 ng / mg. Despite the high LOD and LOQ values for methadone, due to the noise of the sample matrix, it was not enough to interfere with the range of concentrations found in the patient’s hair samples. The LOD for EDDP was 0.15 ng / mg and the LOQ was 0.36 ng / mg. These values are quite acceptable for the purposes of this method.
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229
Fig. 1. Extracted ion chromatograms from hair sample of Patient 6, in which 14.70 ng / mg of methadone and 3.37 ng / mg of EDDP were detected.
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Table 1 Curve equations and correlation coefficients (r) for determination of methadone and EDDP in hair using SPME / GC–MS Compound
Curve equation
r
Methadone EDDP
y50.0333x20.0015 y50.0574x10.0467
0.998 0.997
Table 2 Precision (R.S.D. %) and analytical recoveries (%) for methadone and EDDP in hair, using SPME / GC–MS, at 3.0 and 30.0 ng / mg Compound
Concentration (ng / mg)
Analytical recoveries a (%)
R.S.D.b (%)
Methadone
3.0 30.0 3.0 30.0
102.50611.12 107.24610.24 102.7266.07 103.6367.47
13.30 12.25 7.26 8.94
EDDP a b
For a confidence interval of 95%. Relative standard deviation.
3.2. Analysis of MMP patient’ s hair samples The described method was used to determine methadone and EDDP in human hair from eight patients from a methadone maintenance programme from the Assistance Programme of the Autonomic Community of Galicia (Spain). As can be seen in Table 3, the hair concentrations of methadone in the samples ranged from 2.45 to 78.10 ng / mg and the EDDP concentrations ranged from 0 to 7.76 ng / mg. These values are higher than those found by other authors [2,5–7], possibly due to the higher doses of methadone that received our patients (55–100 mg / day) compared to the doses of other studies (25–80 mg / day). Nevertheless, the methadone / EDDP ratios ranged from 4.36 to 10.06 and are consistent with those previous studies. The correlation between methadone dose and hair concentration in our samples showed statistical significance (P,0.02). Goldberger et al. [7] found no correlation Table 3 Results of MMP a patient’s hair analysis, using SPME / GC–MS for determination of methadone and EDDP Patient
Age (years)
Hair colour
Methadone dose (mg / day)
Methadone (ng / mg)
EDDP (ng / mg)
MTD b / EDDP ratio
1 2 3 4 5 6 7 8
32 25 34 27 22 29 38 35
Black Brown Dark brown Black Dark brown Black Black Dark brown
55 100 70 80 80 60 60 70
23.83 78.10 7.88 25.12 31.31 14.70 2.45 7.34
4.19 7.76 1.36 2.97 5.07 3.37 – 0.98
5.69 10.06 5.79 8.46 6.18 4.36 – 7.49
a b
Methadone maintenance programme. Methadone
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231
between these parameters, and said that was probably due to the assay of hair trimmings rather than hair strands. Green et al. [13] found statistical differences between methadone concentrations in black and white rat hair. The effect of hair colour in the concentration of methadone was not evident in our samples.
4. Conclusion This work describes the application of solid-phase microextraction (SPME) for the determination of methadone and EDDP in hair by GC–MS. The method was successfully applied to the analysis of hair samples from patients of a methadone maintenance programme. The microextraction was performed directly on a small aliquot of the enzymatic hydrolysis liquid, commonly used for the screening techniques for drugs in hair, allowing the use of the remaining sample for confirmation analysis of other drugs, necessary for each positive screening results. Because the SPME needs small sample volume, and is a simple, rapid and solventfree procedure, it also made the analysis time (screening-confirmation) quicker for methadone and EDDP in hair.
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