Forensic Science International 234 (2014) 132–138
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Development and validation of a single LC–MS/MS assay following SPE for simultaneous hair analysis of amphetamines, opiates, cocaine and metabolites L. Imbert a,*, S. Dulaurent a, M. Mercerolle a,1, J. Morichon a, G. Lachaˆtre a,b, J.-M. Gaulier a a b
Department of Pharmacology and Toxicology, University Hospital, 2, Avenue Martin-Luther-King, 87042 Limoges Cedex, France Laboratory of Toxicology, Faculty of Pharmacy, 2 rue Docteur Raymond Marcland, 87000 Limoges, France
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 August 2013 Received in revised form 28 October 2013 Accepted 6 November 2013 Available online 15 November 2013
The two major challenges in hair analysis are the limited amount of samples usually available and the low targeted concentrations. To overcome these limitations, a liquid chromatography–electrospraytandem mass spectrometry method (LC–ESI-MS/MS) allowing the simultaneous analysis of 17 amphetamines (amphetamine, BDB, m-CPP, dexfenfluramine, DOB, DOM, ephedrine, MBDB, MDA, MDEA, MDMA, methamphetamine, methylphenidate, 4-MTA, norephedrine, norfenfluramine and PMA), 5 opiates (morphine, codeine, heroin, ethylmorphine, and 6AM), cocaine and 5 metabolites [ecgonine methyl ester (EME), benzoylecgonine (BZE), anhydroecgonine methyl ester (AME), cocaethylene, and norcocaine] has been developed. The validation procedure included linearity, intra-day and inter-day variability and accuracy for 5 days (5 replicates at 3 concentration levels). Proficiency studies were used to check the accuracy of the method. As a result, all amphetamines, opiates and cocaine derivatives were satisfactory identified by 2 MRM transitions in 15 min. Calibration curves were performed by a quadratic 1/X weighted regression. The calibration model fits from 0.05 to 10 ng/mg. The limits of detection (LODs) range between 0.005 and 0.030 ng/mg. Precision has been checked by intra-day and inter-day RSD, and associated relative bias, which were lower than 25% for the limits of quantifications (LOQs) and lower than 20% for the other levels tested. This method was routinely applied to hair samples: two positive results of adult drug addicts are presented. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Forensic toxicology Hair Cocaine Opiates Amphetamines Tandem mass spectrometry
1. Introduction The hair matrix allows the detection of analytes in a broader time-window than other usual biological matrices, such as blood or urine [1,2]. It also has the advantage of being easily stored at room temperature, in a properly ventilated room. Hair analysis is being used more and more, to follow drug or medication intake for several months prior to sampling. Thus, hair analysis of drugs, and more specifically illicit drugs, is a useful technique that allows to answer several clinical as well as forensic issues, such as the detection of abuse of drugs [3,4], gestational exposure to drugs, the following of drug abstinence, the research for the cause of drug
* Corresponding author. Tel.: +33 5 55 05 61 40; fax: +33 5 55 05 61 62. E-mail addresses: [email protected], [email protected] (L. Imbert), [email protected] (S. Dulaurent), [email protected] (M. Mercerolle), [email protected] (J. Morichon), [email protected] (G. Lachaˆtre), [email protected] (J.-M. Gaulier). 1 Present address: Shimadzu France, Le Luzard II – Bat. A, Bd Salvador Allende, Noisiel – 77448 Marne la Valle´e Cedex 2, France. 0379-0738/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2013.11.004
withdrawal syndrome [5–8], suspension of driving licences [9–11], or the research for the causes of death [12–16]. Concerning more specifically cocaine, amphetamines and opiates drugs, several analytical methods have been described and use either gas chromatography coupled to mass spectrometry (GC–MS) [17– 19], gas chromatography coupled to tandem mass spectrometry (GC–MS/MS) [4], liquid chromatography coupled to ultraviolet detector (UV) [20], liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) using electrospray ionization (ESI) [21–23] or atmospheric pressure chemical ionization (APCI) [3], and LC coupled with a time of flight (TOF) mass spectrometer [24]. These methods allow the detection of at most 22 molecules among cocaine and metabolites, opiates and amphetamines, using at least 10 mg up to 50 mg of hair sample. Particularly among amphetamines and related drugs, only amphetamine, methamphetamine, methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA) and N-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine (MBDB) are detected at the most. The reported limits of quantifications (LOQ) were between 0.015 and 0.31 ng/mg. Immunoassays have
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also been developed, and allow the analysis of hair within a short time period (around 2 h), using the same amount of samples as GC or LC methods [25,26]. These techniques display a lower specificity than mass spectrometric methods, and only detect a few molecules among cocaine, opiates, and amphetamines. However, the two major problems in hair analysis are the limited amount of samples available and the low targeted concentrations: LC–MS/MS methods allow the analysis of such challenging samples. We therefore present a LC–ESI-MS/MS method allowing the simultaneous analysis of 17 amphetamines or related compounds, 4 opiates, cocaine and 5 metabolites. This method allows in particular the detection of several designer drugs among amphetamines, like 1-(3-chlorophenyl)-piperazine (mCPP), 2,5-dimethoxy-4-bromoamphetamine (DOB) or 2,5dimethoxy-4-methylamphetamine (DOM). Finally, two forensic cases for which this method has been used are described. 2. Materials and methods 2.1. Materials All solvents were of analytical grade. Methanol was purchased from Carlo Erba Reagents (Val de Reuil, France); dichloromethane, propan-2-ol, hydrochloric acid, potassium dihydrogenophosphate and ammonium hydroxide came from VWR (Fontenay-sous-bois, France). Amphetamine, 1-(3,4-methylenedioxyphenyl)-2-butanamine (BDB), ephedrine, methamphetamine, methylphenidate, Nmethyl-1-(1,3-benzodioxol-5-yl)-2-butanamine (MBDB), methylenedioxyamphetamine (MDA), methylenedioxyethylamphetamine (MDEA), methylenedioxymethamphetamine (MDMA), pmethoxyamphetamine (PMA), morphine, codeine, 6-monoacetylmorphine (6AM), cocaine, ecgonine methyl ester (EME), benzoylecgonine (BZE), anhydroecgonine methyl ester (AME), cocaethylene (CocaEt), norcocaine, and the following deuterated internal standards (I.S.): amphetamine-D5, methamphetamineD5, methylenedioxyamphetamine-D5 (MDA-D5), methylenedioxy-methamphetamine-D5 (MDMA-D5), methylenedioxyethylamphetamine-D5 (MDEA-D5), ephedrine-D3, morphine-D3, codeine-D3, 6-monoacetylmorphine-D3 (6AM D3), heroin-D9, cocaine-D3, benzoylecgonine-D3 (BZE-D3), ecgoninemethylester-D3 (EME-D3) and cocaethylene-D3 (CocaEt-D3) were purchased from LGC Promochem (Molsheim, France). 2,5-Dimethoxy4-bromoamphetamine (DOB), 2,5-dimethoxy-4-methylamphetamine (DOM) and ethylmorphine, 1-(3-chlorophenyl)-piperazine (m-CPP) and norephedrine, were all obtained from Sigma–Aldrich (Saint-Quentin Fallavier, France). Dexfenfluramine and norfenfluramine were kindly provided by Laboratoires Servier (Orle´ans, France) and 4-methylthioamphetamine (4-MTA) was kindly provided by the Belgian Public Health Scientific Institute. Mixed cation exchange mode (OASIS MCX 3 cc, 60 mg) solid phase extraction (SPE) cartridges were purchased from Waters (Saint Quentin en Yvelines, France). Drug-free hair samples were collected from volunteers among the laboratory staff, and the absence of any drugs was previously verified in these samples using this method. 2.2. Standard solutions 4-MTA, dexfenfluramine, norfenfluramine, norephedrine, DOB, DOM, m-CPP and ethylmorphine powders were separately dissolved in methanol to obtain stock solutions at 1 g/L. Others drugs of abuse standards and all deuterated standards (IS) are presented as 1 g/L and 100 mg/L solutions, respectively, in methanol or acetonitrile (except heroin-D9 solution at 1 g/L). Two pools of working solutions were prepared: one containing amphetamines and deuterated equivalents at 1 mg/L in 0.2 N
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hydrochloride solution, and the other containing opiates, cocaine and derivatives at 0.1 mg/L in methanol. The appropriate dilutions of amphetamines and opiates, cocaine and derivatives solutions were performed twice in order to prepare control working solutions used to obtain internal quality controls. These standard solutions were stored at +4 8C for 3 months. 2.3. Hair sample decontamination and extraction procedure Hair strands were decontaminated by a gentle mixing of samples in two glass tubes containing 10 mL water for 2 min each, followed by two 1-min baths in 10 mL of dichloromethane. Hair samples were dried between the different washing steps using sheets of absorbent paper. To 50 mg of a washed and finely cut (1– 2 mm length, using perfectly decontaminated scissors) hair sample introduced in a 15 mL glass tube, 20 mL of the two IS solutions at 2 mg/L were added. Calibration standards at 0, 0.05, 0.10, 0.50, 1.0, 5.0 and 10.0 ng/mg were prepared by spiking 50 mg of drug-free human hair with 20 mL of both IS solutions prepared at 2 mg/L, and with the appropriate volumes of the different working solution mixtures. Internal quality controls at 0.2 and 2 ng/mg were prepared in the same conditions using appropriate control working solutions. After the addition of 3 mL of pH 5.0 phosphate buffer (0.1 N), samples were incubated for 18 h at about 45 8C in a OLS 200 orbital shaking water bath (Grant Instruments1). After incubation, hair samples were vortex-mixed for ten seconds and centrifuged at 3000 rpm for 5 min. Then supernatants were loaded on SPE cartridges (MCX1, Oasis1, Waters) previously conditioned with 2 mL methanol and 3 mL pH 5.0 phosphate buffer (0.1 N). Cartridges were washed successively by 2 mL of deionised water and by 1.5 mL of 0.1 N hydrochloride solution, before a drying step of 5 min, and were finally washed by 2 mL methanol. Solutes were then eluted by 3 mL of a dichloromethane/propan-2-ol (80:20, v:v) mixture and collected in 10 mL conic glass tubes. Eluates were evaporated under nitrogen flux at room temperature and reconstituted with 50 mL of a (5:95, v:v) mixture of acetonitrile and formate buffer (2 mM, pH 3.0). These extracts were introduced in 200 mL vials for injection and 10 mL were injected into the LC– ESI-MS/MS system. 2.3.1. Liquid chromatography–tandem mass spectrometry The chromatographic system consisted of a Series 200LC microflow rate, high-pressure gradient pumping system and a Series 200 Auto-sampler (Perkin-Elmer Instruments, Les Ulis, France) including a Rheodyne Model 7725 injection valve equipped with a 20 mL external loop. Chromatographic separation was performed on an Atlantis1 T3 (150 2.1-mm I.D., 3 mm) column (Waters, SaintQuentin en Yvelines, France). The mobile phase consisted of (A) ammonium formate 2 mM, pH 3.0, and (B) acetonitrile/A (90:10, v/ v), and a flow-rate of 200 mL/min was used. Separation started with 5% B for 3 min, then B increased at 20% for 1 min, followed by an increase to 40% for 10 min, and to 90% for 2 min maintained for 4 min. Initial conditions were achieved within 1 min and maintained for 3 min before the next injection. The total run time was 24 min. Detection was carried out with an API 2000 MS/MS System (AB SCIEX, Concord, Canada) equipped with a TurboIonSpray ionization source and controlled by the Analyst1 software. The Multiple Reaction Monitoring (MRM) transitions followed for each analyte and retention times are presented in Table 1. The TurboIonSpray settings were optimized in positive ionization mode, by infusing at 5 mL/min a 1 mg/L solution of the 41 compounds (17 amphetamines, 5 opiates, 6 cocaine and derivatives and 13 equivalent deuterated internal standards) prepared in a mixture of acetonitrile and formate buffer (2 mM, pH 3.0) (30:70, v:v). The optimal
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Table 1 Mass transitions, relative abundances, retention times and relative retention times of amphetamines, opiates, cocaine, derivatives and associated IS (compounds are regrouped with their corresponding IS in bold type, from which relative retention time is calculated). Analytes
Mass transition 1 (m/z > m/z)
Mass transition 2 (m/z > m/z)
Relative abundance (M2/M1)
Retention time (min)
Relative retention time (min)
Norephedrine Ephedrine-D3 Ephedrine Amphetamine-D5 Amphetamine Methamphetamine-D5 Methamphetamine MDA-D5 MDA PMA MDMA-D5 MDMA MDEA-D5 MDEA BDB MBDB 4-MTA Methylphenidate m-CCP DOM Norfenfluramine DOB Dexfenfluramine AME EME-D3 EME Cocaine-D3 Cocaine Norcocaine Cocaethylene-D3 Cocaethylene BZE-D3 BZE Codeine-D3 Codeine Ethylmorphine Morphine-D3 Morphine 6AM-D3 6AM Heroin-D9 Heroin
152.1 > 134.2 169.4 > 151.1 166.3 > 148.2 141.0 > 124.1 136.1 > 119.0 155.1 > 121.1 150.1 > 119.3 185.3 > 168.2 180.2 > 163.0 166.2 > 121.1 198.8 > 165.1 194.4 > 163.4 213.2 > 163.1 208.2 > 163.0 195.3 > 136.3 208.3 > 135.1 182.2 > 165.0 234.2 > 84.1 197.1 > 154.0 210.1 > 193.2 203.9 > 187.0 274.1 > 257.1 231.8 > 159.0 182.2 > 91.0 203.1 > 185.2 200.2 > 182.2 307.1 > 185.2 304.2 > 182.2 290.2 > 168.1 321.1 > 199.2 318.2 > 196.2 293.1 > 171.2 290.2 > 168.1 303.1 > 152.1 300.1 > 152.0 314.2 > 152.0 289.1 > 152.1 286.1 > 152.2 331.1 > 165.1 328.1 > 165.1 379.2 > 212.1 370.2 > 165.2
152.1 > 117.2 – 166.3 > 91.2 – 136.1 > 91.0 – 150.1 > 91.1 – 180.2 > 105.1 166.2 > 91.0 – 194.4 > 135.2 – 208.2 > 105.1 195.3 > 178.0 208.3 > 177.3 182.2 > 137.0 234.2 > 115.0 197.1 > 118.2 210.1 > 178.3 203.9 > 159.0 274.1 > 229.1 231.8 > 108.9 182.2 > 117.9 – 200.2 > 82.0 – 304.2 > 77.0 290.2 > 136.1 – 318.2 > 82.1 – 290.2 > 105.0 – 300.1 > 165.0 314.21 > 128.1 – 286.1 > 128.1 – 328.1 > 211.1 – 370.2 > 152.2
37% – 6% – 206% – 320% – 37% 41% – 55% – 45% 62% 29% 47% 1.1% 118% 31% 33% 40% 45% 55% – 40% – 25% 65% – 24% – 57% – 62% 82% – 52% – 76% – 102%
7.02 7.26 7.29 7.87 7.92 8.34 8.40 8.23 8.28 8.43 8.71 8.76 9.45 9.50 9.58 10.10 10.80 10.80 11.20 11.60 12.20 12.50 13.70 6.69 2.42 2.44 11.90 12.10 12.1 13.50 13.50 9.61 9.62 7.32 7.33 8.72 6.73 6.74 8.07 8.09 10.63 10.70
0.967 – 1.004
settings of the source were the following: ion spray voltage at 5500 V, curtain gas, ion source gas 1 and gas 2 at 50, 50 and 60 units, respectively. The declustering potential (DP) was optimized for each compound as shown in Table 2. 2.4. Validation procedure Quantitation of amphetamines, opiates, cocaine and derivatives was performed by the internal standard method. A six point calibration curve was made for all the substances by quadratic in 1/ X weighting regression analysis of the ratio of the peak area of analyte to the peak area of its associated deuterated internal standard. Calibration curves were prepared daily, by spiking finely cut (fragments < 2 mm) drug-free hair samples with internal standard solutions and corresponding analytical working solution mixtures to obtain calibration concentrations of 0, 0.05, 0.10, 0.50, 1.0, 5.0 and 10.0 ng/mg for each analyte, as described in the ‘‘Hair sample decontamination and extraction procedure’’ section. Linearity was assessed by analyzing five replicates for each calibration level on different days. For 5 days, validation samples at 0.05, 0.5, 5.0 and 10 ng/mg amphetamines, opiates, cocaine and metabolites were prepared in duplicate for the inter-assay study (5 series) and in 5
1.006 – 1.007 – 1.006 1.024 – 1.005 – 1.005 1.014 1.069 1.143 1.143 1.185 1.227 1.291 1.323 1.450 2.764 – 1.008 1.000 1.017 – 1.000 – 1.001 – 1.001 1.191 – 1.001 – 1.002 – 1.006
replicates (a different concentration per day) for the intra-assay study. Relative standard deviation (RSD) and bias were calculated for inter- and intra-assay studies. For acceptance, both parameters must simultaneously be lower than 25% for the LOQ and lower than 20% for the other concentrations in both studies. The limit of detection (LOD) was defined as the lowest concentration giving a response of at least three times the average of the baseline noise (S/ N > 3, as determined by Analyst1 software), and the LOQ was defined as the lowest concentration that could be measured with an intra-assay precision RSD and relative bias less than 20%. Carry over was evaluated, by analysing a sample of mobile phase subsequently to the highest calibration level (n = 5). The absence of any signal on the corresponding chromatogram was verified. Ion suppression phenomenon was studied following the experimental system previously proposed by Antignac et al. [27]. Briefly, a standard solution containing the compounds of interest (at 100 mg/L) was continuously and directly infused into the mass spectrometer interface. A simultaneous LC Flow containing either a pure mobile phase or a blank biological extract (hair from 10 non drugs consumers were sampled) was introduced through a tee. Evolution of the signal of the transitions at the retention times of the corresponding compounds of interest was studied to evaluate presence and intensity of ion suppression.
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Table 2 Optimized mass spectrometric parameters. IS are in bold type. Analytes
Q1 mass (amu)
Q3 mass (amu)
DP (V)
FP (V)
EP (V)
CEP (V)
CE (V)
CXP (V)
Norephedrine
152.1 152.1 169.4 166.3 166.3 141.0 136.1 136.1 155.1 150.1 150.1 185.3 180.2 180.2 166.2 166.2 198.8 194.4 194.4 213.2 208.2 208.2 195.3 195.3 208.3 208.3 182.2 182.2 234.2 234.2 197.1 197.1 210.1 210.1 203.9 203.9 274.1 274.1 231.8 231.8 182.2 182.2 203.1 200.2 200.2 290.2 290.2 307.1 304.2 304.2 321.1 318.2 318.2 293.1 290.2 290.2 303.1 300.1 300.1 314.2 314.2 289.1 286.1 286.1 331.1 328.1 328.1 379.2 370.2 370.2
134.2 117.2 151.1 148.2 91.2 124.1 119.0 91.0 121.1 119.3 91.1 168.2 163.0 105.1 121.1 91.0 165.1 163.4 135.2 163.1 163.0 105.1 136.3 178.0 135.1 177.3 165.0 137.0 84.1 115.0 154.0 118.2 193.2 178.3 187.0 159.0 257.1 229.1 159.0 108.9 91.0 117.9 185.2 182.2 82.0 168.1 136.1 185.2 182.2 77.0 199.2 196.2 82.1 171.2 168.1 105.0 152.1 152.0 165.0 152.0 128.1 152.1 152.2 128.1 165.1 165.1 211.1 212.1 165.2 152.2
31 31 41 21 11 6 16 16 11 16 16 6 11 11 21 26 21 16 16 31 1 1 16 31 31 31 26 21 26 36 31 31 21 21 46 51 31 31 36 41 66 66 26 51 51 36 36 26 26 26 31 41 41 31 46 46 51 71 71 66 66 61 71 71 61 71 71 81 56 56
360 360 120 360 360 330 360 360 360 360 360 320 360 360 330 330 310 360 360 310 360 360 360 360 190 190 290 280 370 370 360 330 370 370 240 250 370 370 350 370 50 50 370 360 360 370 370 370 370 370 370 370 370 350 370 370 370 350 350 370 370 370 350 350 370 350 350 140 360 360
6.5 6.5 5.5 6 9.5 8.5 8 7.5 8 8 7.5 4.5 10.5 10.5 12 8 8.5 10 10.5 10.5 5.5 6.5 11 6.5 7 7 7 7 8 8.5 9.5 10.5 10.5 10.5 11.5 11.5 10 10 11 10.5 9.5 9.5 10 12 12 9.5 9.5 10 9.5 9.5 8.5 10 10 9 10 10 12 10.5 10.5 12 12 10.5 10.5 10.5 11.5 12 12 8 11.5 11.5
8 8 10 6 52 10 8 8 8 10 8 10 10 10 10 10 10 12 10 10 10 8 10 14.5 12 12 8 10 12 14 10 10 12 12 12 10 14 14 12 12 12 12 12 12 12 12 12 14 14 14 14 14 14 14 12 12 14 14 14 14 14 14 14 14 14 14 14 16 14 14
15 25 17 17 39 13 13 21 13 15 25 15 13 27 25 39 17 15 29 19 17 35 23 11 25 15 11 27 29 63 27 45 15 27 13 25 15 23 33 57 31 31 23 25 35 21 33 27 25 79 25 27 47 25 27 43 89 81 51 81 87 75 81 79 51 53 35 43 71 109
2 2 2 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 2 0 0 2 0 2 2 2 0 0 0 0 2 0 2 2 4 4 0 0 0 2 2 2 0 2 2 2 2 0 4 2 0 2 2 0 0 0 0 0 0 0 0 0 2 2 2 2 0 0
Ephedrine-D3 Ephedrine Amphetamine-D5 Amphetamine Methamphetamine-D5 Methamphetamine MDA-D5 MDA PMA MDMA-D5 MDMA MDEA-D5 MDEA BDB MBDB 4-MTA Methylphenidate m-CCP DOM Norfenfluramine DOB Dexfenfluramine AME EME-D3 EME Norcocaine Cocaine-D3 Cocaine Cocaethylene-D3 Cocaethylene BZE-D3 BZE Codeine-D3 Codeine Ethylmorphine Morphine-D3 Morphine 6AM-D3 6AM Heroin-D9 Heroin
Q mass, parent ion m/z; Q mass, daughter ion m/z; DP, declustering potential (orifice plate); FP, focusing potential (focusing ring); EP, entrance potential (Q0 less); CEP, cell entrance potential; CE, collision cell potential; CXP, cell exit potential.
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3. Results and discussion All 28 compounds were analyzed within 15 min. It is of note that heroin can be detected in hair which is rarely the case in blood and urine. Authors have reported the presence of heroin in hair addicts in spite of fast degradation (less than 5 min) in 6AM in blood [28]. Total ion current (TIC) Chromatograms obtained for a drug-free hair extract and for the LOQ (i.e. 0.05 ng/mg hair) are presented in Fig. 1(A) and (B), respectively. Results obtained for the repeatability and intermediate precision are presented in Fig. 2 (A) and (B), respectively. RSD obtained for each compound, at all four concentrations evaluated (0.05, 0.5, 5 and 10 ng/mg), were below the required values of 25% for the LOQ and 20% for other values. A Bias below 20% was also obtained for all the compounds. The LODs and LOQs determined for each compound are presented in Table 3. Values between 0.005 and 0.030 ng/mg hair were achieved for the LOD, and a value of 0.05 ng/mg for the LOQ, corresponding to the lowest calibration level, was the same for all the compounds. The latter is in accordance with the values recommended in the consensus of the Society of Hair Testing (SoHT) on hair testing in forensic cases [29], which require a LOQ of at most 0.2 ng/mg for amphetamines and opiates, 0.5 ng/mg for cocaine and 0.05 ng/mg for other cocaine derivatives. The fit of the quadratic, 1/X weighted regression was evaluated by computing the correlation coefficient r, over five replicates for each calibration level. Values over 0.993 were obtained for all 28 compounds, thus the calibration model was considered acceptable. Finally, no ion suppression phenomenons were highlighted at the retention time of all compounds, for each MRM transition. Beside quantifying opiates and cocaine, the present method allows to detect routinely a broader range of amphetamines and related compounds (i.e. ephedrine and norephedrine, BDB, 4-MTA,
Fig. 2. (A) Repeatability and (B) intermediate precision (% RSD) assessed for each compound, at 0.05, 0.5, 5 and 10 ng/mg.
Fig. 1. (A) Chromatogram (TIC) of a drug-free hair extract and (B) chromatogram (TIC) of a drug-free hair sample spiked with 0.05 ng/mg of each compound. (a) EME; (b) AME, morphine; (c) norephedrine; (d) ephedrine and codeine; (e) 6AM and amphetamine; (f) MDA, methamphetamine and PMA; (g) MDMA and ethylmorphine; (h) BDB and MDEA; (i) MBDB and BZE; (j) 4-MTA and methylphenidate; (k) heroin; (l) m-CPP; (m) DOM, cocaine; (n) norcocaine; (o) norfenfluramine, DOB; (p) cocaethylene; and (q) dexfenfluramine.
m-CPP, DOM, DOB, methylphenidate, norfenfluramine and dexfenfluramine) than previously reported methods, that only focus on amphetamine, methamphetamine, MDA, MDMA, MDEA and MBDB at most [3,4,17–24]. This analytical method has now been validated, and has been used in forensic cases involving possible drug use. 3.1. Case 1 A man suspected of taking drugs and being a drug dealer was arrested by the police. Urine and hair samples were gathered in
L. Imbert et al. / Forensic Science International 234 (2014) 132–138 Table 3 Limits of detection and quantification evaluated for each compound. Compound
LOD (ng/mg)
LOQ (ng/mg)
Norephedrine Ephedrine Amphetamine Methamphetamine MDA PMA MDMA MDEA BDB MBDB 4-MTA Methylphenidate m-CPP DOM Norfenfluramine DOB Dexfenfluramine AME EME Cocaine Norcocaine Cocaethylene BZE Codeine Ethylmorphine Morphine 6AM Heroin
0.010 0.030 0.010 0.005 0.010 0.010 0.005 0.005 0.030 0.005 0.020 0.020 0.010 0.010 0.010 0.020 0.005 0.020 0.010 0.005 0.010 0.005 0.005 0.010 0.005 0.020 0.005 0.010
0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050
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codeine is usually co-extracted with morphine from raw opium and then acetylated into acetylcodeine. The acetylcodeine contained in heroin would be metabolized to codeine just like heroin is metabolized to 6AM. These results reflect most probably regular heroin consumption for approximately 7 months before sampling, which could explain the behaviour of the patient. Owing to the toxicological results in urine, the hypothesis of an overdose might not be plausible, and further blood analysis would have been required to investigate the cause of death. 4. Conclusion The present work described an analytical method for the detection of 28 opiates, cocaine and derivatives, and amphetamines. This method has also been evaluated and found to be successful in 4 external quality controls proposed by the German Society of Toxicological and Forensic Chemistry (GTFCh) and 3 by the SoHT in 2012 and the beginning of 2013. After successfully passing the validation process, this method has been routinely used for forensic cases, two of which have been described. Unfortunately new illicit drugs, particularly among amphetamines (i.e. cathinone derivatives, N-(1-phenylcyclohexyl)-2-ethoxyethanamine derivatives, etc.), which are regularly marketed by drug dealers, would not be detected by the current method. It is therefore necessary to constantly modify such a method, as is done in the doping control field, in order to be able to assess these latest drugs and to limit false-negative results as much as possible. References
order to verify if this man had taken any illicit drugs for several months before being arrested. A 4 cm hair sample was collected, and segmented as follows prior to analysis: 0 ! 2 cm and 2 ! 4 cm from the hair scalp. Methamphetamine and amphetamine were detected at 0.63 ng/mg and 0.06 ng/mg respectively in the 0 ! 2 segment, and only methamphetamine was detected at 0.24 ng/mg in the 2 ! 4 segment. These results favour a consumption of methamphetamine for approximately 4 months before the sampling, from which amphetamine would be a metabolite in this case. The increase of methamphetamine, associated to the detection of amphetamine in the first segment, may indicate that the suspect took more of this drug during the 2 months before sampling. However, the lower values obtained in the second segment could also be due to a wash-out of the compounds with the use of capillary cosmetics. The suspect was finally found guilty as these concentrations result from consumption without doubt. 3.2. Case 2 A man hospitalized in an intensive care unit (ICU) due to a loss of consciousness and a cardio-respiratory arrest died 2 weeks after his admission. Relatives reported that he had fainted several times in the weeks leading up to the incident, and a packet of brown powder was found in his belongings on his admission to the hospital. Traces of heroin and cocaine were detected in urine sampled on admission to the ICU. A 7 cm hair strand was then sampled during the autopsy in order to further investigate the cause of death. The sample was segmented and analyzed as follows: 0 ! 2 cm, 2 ! 4 cm and 4 ! 7 cm. 6AM, morphine and codeine were detected in all segments. Concentrations were more or less constant throughout the whole sample, with 6AM between 0.49 and 0.58 ng/mg, morphine between 0.22 and 0.30 ng/mg and codeine below the LOQ. No cocaine and metabolites were detected. In this case, 6AM and morphine may originate from the metabolism of heroin, and codeine would be detected in low amounts as it is a product extracted from opium during the production of heroin. Indeed, during the semi-synthesis of heroin,
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Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Development and validation of a single LC–MS/MS assay following SPE for simultaneous hair analysis of amphetamines, opiates, cocaine and metabolites L. Imbert a,*, S. Dulaurent a, M. Mercerolle a,1, J. Morichon a, G. Lachaˆtre a,b, J.-M. Gaulier a a b
Department of Pharmacology and Toxicology, University Hospital, 2, Avenue Martin-Luther-King, 87042 Limoges Cedex, France Laboratory of Toxicology, Faculty of Pharmacy, 2 rue Docteur Raymond Marcland, 87000 Limoges, France
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 August 2013 Received in revised form 28 October 2013 Accepted 6 November 2013 Available online 15 November 2013
The two major challenges in hair analysis are the limited amount of samples usually available and the low targeted concentrations. To overcome these limitations, a liquid chromatography–electrospraytandem mass spectrometry method (LC–ESI-MS/MS) allowing the simultaneous analysis of 17 amphetamines (amphetamine, BDB, m-CPP, dexfenfluramine, DOB, DOM, ephedrine, MBDB, MDA, MDEA, MDMA, methamphetamine, methylphenidate, 4-MTA, norephedrine, norfenfluramine and PMA), 5 opiates (morphine, codeine, heroin, ethylmorphine, and 6AM), cocaine and 5 metabolites [ecgonine methyl ester (EME), benzoylecgonine (BZE), anhydroecgonine methyl ester (AME), cocaethylene, and norcocaine] has been developed. The validation procedure included linearity, intra-day and inter-day variability and accuracy for 5 days (5 replicates at 3 concentration levels). Proficiency studies were used to check the accuracy of the method. As a result, all amphetamines, opiates and cocaine derivatives were satisfactory identified by 2 MRM transitions in 15 min. Calibration curves were performed by a quadratic 1/X weighted regression. The calibration model fits from 0.05 to 10 ng/mg. The limits of detection (LODs) range between 0.005 and 0.030 ng/mg. Precision has been checked by intra-day and inter-day RSD, and associated relative bias, which were lower than 25% for the limits of quantifications (LOQs) and lower than 20% for the other levels tested. This method was routinely applied to hair samples: two positive results of adult drug addicts are presented. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Forensic toxicology Hair Cocaine Opiates Amphetamines Tandem mass spectrometry
1. Introduction The hair matrix allows the detection of analytes in a broader time-window than other usual biological matrices, such as blood or urine [1,2]. It also has the advantage of being easily stored at room temperature, in a properly ventilated room. Hair analysis is being used more and more, to follow drug or medication intake for several months prior to sampling. Thus, hair analysis of drugs, and more specifically illicit drugs, is a useful technique that allows to answer several clinical as well as forensic issues, such as the detection of abuse of drugs [3,4], gestational exposure to drugs, the following of drug abstinence, the research for the cause of drug
* Corresponding author. Tel.: +33 5 55 05 61 40; fax: +33 5 55 05 61 62. E-mail addresses: [email protected], [email protected] (L. Imbert), [email protected] (S. Dulaurent), [email protected] (M. Mercerolle), [email protected] (J. Morichon), [email protected] (G. Lachaˆtre), [email protected] (J.-M. Gaulier). 1 Present address: Shimadzu France, Le Luzard II – Bat. A, Bd Salvador Allende, Noisiel – 77448 Marne la Valle´e Cedex 2, France. 0379-0738/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2013.11.004
withdrawal syndrome [5–8], suspension of driving licences [9–11], or the research for the causes of death [12–16]. Concerning more specifically cocaine, amphetamines and opiates drugs, several analytical methods have been described and use either gas chromatography coupled to mass spectrometry (GC–MS) [17– 19], gas chromatography coupled to tandem mass spectrometry (GC–MS/MS) [4], liquid chromatography coupled to ultraviolet detector (UV) [20], liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) using electrospray ionization (ESI) [21–23] or atmospheric pressure chemical ionization (APCI) [3], and LC coupled with a time of flight (TOF) mass spectrometer [24]. These methods allow the detection of at most 22 molecules among cocaine and metabolites, opiates and amphetamines, using at least 10 mg up to 50 mg of hair sample. Particularly among amphetamines and related drugs, only amphetamine, methamphetamine, methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA) and N-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine (MBDB) are detected at the most. The reported limits of quantifications (LOQ) were between 0.015 and 0.31 ng/mg. Immunoassays have
L. Imbert et al. / Forensic Science International 234 (2014) 132–138
also been developed, and allow the analysis of hair within a short time period (around 2 h), using the same amount of samples as GC or LC methods [25,26]. These techniques display a lower specificity than mass spectrometric methods, and only detect a few molecules among cocaine, opiates, and amphetamines. However, the two major problems in hair analysis are the limited amount of samples available and the low targeted concentrations: LC–MS/MS methods allow the analysis of such challenging samples. We therefore present a LC–ESI-MS/MS method allowing the simultaneous analysis of 17 amphetamines or related compounds, 4 opiates, cocaine and 5 metabolites. This method allows in particular the detection of several designer drugs among amphetamines, like 1-(3-chlorophenyl)-piperazine (mCPP), 2,5-dimethoxy-4-bromoamphetamine (DOB) or 2,5dimethoxy-4-methylamphetamine (DOM). Finally, two forensic cases for which this method has been used are described. 2. Materials and methods 2.1. Materials All solvents were of analytical grade. Methanol was purchased from Carlo Erba Reagents (Val de Reuil, France); dichloromethane, propan-2-ol, hydrochloric acid, potassium dihydrogenophosphate and ammonium hydroxide came from VWR (Fontenay-sous-bois, France). Amphetamine, 1-(3,4-methylenedioxyphenyl)-2-butanamine (BDB), ephedrine, methamphetamine, methylphenidate, Nmethyl-1-(1,3-benzodioxol-5-yl)-2-butanamine (MBDB), methylenedioxyamphetamine (MDA), methylenedioxyethylamphetamine (MDEA), methylenedioxymethamphetamine (MDMA), pmethoxyamphetamine (PMA), morphine, codeine, 6-monoacetylmorphine (6AM), cocaine, ecgonine methyl ester (EME), benzoylecgonine (BZE), anhydroecgonine methyl ester (AME), cocaethylene (CocaEt), norcocaine, and the following deuterated internal standards (I.S.): amphetamine-D5, methamphetamineD5, methylenedioxyamphetamine-D5 (MDA-D5), methylenedioxy-methamphetamine-D5 (MDMA-D5), methylenedioxyethylamphetamine-D5 (MDEA-D5), ephedrine-D3, morphine-D3, codeine-D3, 6-monoacetylmorphine-D3 (6AM D3), heroin-D9, cocaine-D3, benzoylecgonine-D3 (BZE-D3), ecgoninemethylester-D3 (EME-D3) and cocaethylene-D3 (CocaEt-D3) were purchased from LGC Promochem (Molsheim, France). 2,5-Dimethoxy4-bromoamphetamine (DOB), 2,5-dimethoxy-4-methylamphetamine (DOM) and ethylmorphine, 1-(3-chlorophenyl)-piperazine (m-CPP) and norephedrine, were all obtained from Sigma–Aldrich (Saint-Quentin Fallavier, France). Dexfenfluramine and norfenfluramine were kindly provided by Laboratoires Servier (Orle´ans, France) and 4-methylthioamphetamine (4-MTA) was kindly provided by the Belgian Public Health Scientific Institute. Mixed cation exchange mode (OASIS MCX 3 cc, 60 mg) solid phase extraction (SPE) cartridges were purchased from Waters (Saint Quentin en Yvelines, France). Drug-free hair samples were collected from volunteers among the laboratory staff, and the absence of any drugs was previously verified in these samples using this method. 2.2. Standard solutions 4-MTA, dexfenfluramine, norfenfluramine, norephedrine, DOB, DOM, m-CPP and ethylmorphine powders were separately dissolved in methanol to obtain stock solutions at 1 g/L. Others drugs of abuse standards and all deuterated standards (IS) are presented as 1 g/L and 100 mg/L solutions, respectively, in methanol or acetonitrile (except heroin-D9 solution at 1 g/L). Two pools of working solutions were prepared: one containing amphetamines and deuterated equivalents at 1 mg/L in 0.2 N
133
hydrochloride solution, and the other containing opiates, cocaine and derivatives at 0.1 mg/L in methanol. The appropriate dilutions of amphetamines and opiates, cocaine and derivatives solutions were performed twice in order to prepare control working solutions used to obtain internal quality controls. These standard solutions were stored at +4 8C for 3 months. 2.3. Hair sample decontamination and extraction procedure Hair strands were decontaminated by a gentle mixing of samples in two glass tubes containing 10 mL water for 2 min each, followed by two 1-min baths in 10 mL of dichloromethane. Hair samples were dried between the different washing steps using sheets of absorbent paper. To 50 mg of a washed and finely cut (1– 2 mm length, using perfectly decontaminated scissors) hair sample introduced in a 15 mL glass tube, 20 mL of the two IS solutions at 2 mg/L were added. Calibration standards at 0, 0.05, 0.10, 0.50, 1.0, 5.0 and 10.0 ng/mg were prepared by spiking 50 mg of drug-free human hair with 20 mL of both IS solutions prepared at 2 mg/L, and with the appropriate volumes of the different working solution mixtures. Internal quality controls at 0.2 and 2 ng/mg were prepared in the same conditions using appropriate control working solutions. After the addition of 3 mL of pH 5.0 phosphate buffer (0.1 N), samples were incubated for 18 h at about 45 8C in a OLS 200 orbital shaking water bath (Grant Instruments1). After incubation, hair samples were vortex-mixed for ten seconds and centrifuged at 3000 rpm for 5 min. Then supernatants were loaded on SPE cartridges (MCX1, Oasis1, Waters) previously conditioned with 2 mL methanol and 3 mL pH 5.0 phosphate buffer (0.1 N). Cartridges were washed successively by 2 mL of deionised water and by 1.5 mL of 0.1 N hydrochloride solution, before a drying step of 5 min, and were finally washed by 2 mL methanol. Solutes were then eluted by 3 mL of a dichloromethane/propan-2-ol (80:20, v:v) mixture and collected in 10 mL conic glass tubes. Eluates were evaporated under nitrogen flux at room temperature and reconstituted with 50 mL of a (5:95, v:v) mixture of acetonitrile and formate buffer (2 mM, pH 3.0). These extracts were introduced in 200 mL vials for injection and 10 mL were injected into the LC– ESI-MS/MS system. 2.3.1. Liquid chromatography–tandem mass spectrometry The chromatographic system consisted of a Series 200LC microflow rate, high-pressure gradient pumping system and a Series 200 Auto-sampler (Perkin-Elmer Instruments, Les Ulis, France) including a Rheodyne Model 7725 injection valve equipped with a 20 mL external loop. Chromatographic separation was performed on an Atlantis1 T3 (150 2.1-mm I.D., 3 mm) column (Waters, SaintQuentin en Yvelines, France). The mobile phase consisted of (A) ammonium formate 2 mM, pH 3.0, and (B) acetonitrile/A (90:10, v/ v), and a flow-rate of 200 mL/min was used. Separation started with 5% B for 3 min, then B increased at 20% for 1 min, followed by an increase to 40% for 10 min, and to 90% for 2 min maintained for 4 min. Initial conditions were achieved within 1 min and maintained for 3 min before the next injection. The total run time was 24 min. Detection was carried out with an API 2000 MS/MS System (AB SCIEX, Concord, Canada) equipped with a TurboIonSpray ionization source and controlled by the Analyst1 software. The Multiple Reaction Monitoring (MRM) transitions followed for each analyte and retention times are presented in Table 1. The TurboIonSpray settings were optimized in positive ionization mode, by infusing at 5 mL/min a 1 mg/L solution of the 41 compounds (17 amphetamines, 5 opiates, 6 cocaine and derivatives and 13 equivalent deuterated internal standards) prepared in a mixture of acetonitrile and formate buffer (2 mM, pH 3.0) (30:70, v:v). The optimal
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Table 1 Mass transitions, relative abundances, retention times and relative retention times of amphetamines, opiates, cocaine, derivatives and associated IS (compounds are regrouped with their corresponding IS in bold type, from which relative retention time is calculated). Analytes
Mass transition 1 (m/z > m/z)
Mass transition 2 (m/z > m/z)
Relative abundance (M2/M1)
Retention time (min)
Relative retention time (min)
Norephedrine Ephedrine-D3 Ephedrine Amphetamine-D5 Amphetamine Methamphetamine-D5 Methamphetamine MDA-D5 MDA PMA MDMA-D5 MDMA MDEA-D5 MDEA BDB MBDB 4-MTA Methylphenidate m-CCP DOM Norfenfluramine DOB Dexfenfluramine AME EME-D3 EME Cocaine-D3 Cocaine Norcocaine Cocaethylene-D3 Cocaethylene BZE-D3 BZE Codeine-D3 Codeine Ethylmorphine Morphine-D3 Morphine 6AM-D3 6AM Heroin-D9 Heroin
152.1 > 134.2 169.4 > 151.1 166.3 > 148.2 141.0 > 124.1 136.1 > 119.0 155.1 > 121.1 150.1 > 119.3 185.3 > 168.2 180.2 > 163.0 166.2 > 121.1 198.8 > 165.1 194.4 > 163.4 213.2 > 163.1 208.2 > 163.0 195.3 > 136.3 208.3 > 135.1 182.2 > 165.0 234.2 > 84.1 197.1 > 154.0 210.1 > 193.2 203.9 > 187.0 274.1 > 257.1 231.8 > 159.0 182.2 > 91.0 203.1 > 185.2 200.2 > 182.2 307.1 > 185.2 304.2 > 182.2 290.2 > 168.1 321.1 > 199.2 318.2 > 196.2 293.1 > 171.2 290.2 > 168.1 303.1 > 152.1 300.1 > 152.0 314.2 > 152.0 289.1 > 152.1 286.1 > 152.2 331.1 > 165.1 328.1 > 165.1 379.2 > 212.1 370.2 > 165.2
152.1 > 117.2 – 166.3 > 91.2 – 136.1 > 91.0 – 150.1 > 91.1 – 180.2 > 105.1 166.2 > 91.0 – 194.4 > 135.2 – 208.2 > 105.1 195.3 > 178.0 208.3 > 177.3 182.2 > 137.0 234.2 > 115.0 197.1 > 118.2 210.1 > 178.3 203.9 > 159.0 274.1 > 229.1 231.8 > 108.9 182.2 > 117.9 – 200.2 > 82.0 – 304.2 > 77.0 290.2 > 136.1 – 318.2 > 82.1 – 290.2 > 105.0 – 300.1 > 165.0 314.21 > 128.1 – 286.1 > 128.1 – 328.1 > 211.1 – 370.2 > 152.2
37% – 6% – 206% – 320% – 37% 41% – 55% – 45% 62% 29% 47% 1.1% 118% 31% 33% 40% 45% 55% – 40% – 25% 65% – 24% – 57% – 62% 82% – 52% – 76% – 102%
7.02 7.26 7.29 7.87 7.92 8.34 8.40 8.23 8.28 8.43 8.71 8.76 9.45 9.50 9.58 10.10 10.80 10.80 11.20 11.60 12.20 12.50 13.70 6.69 2.42 2.44 11.90 12.10 12.1 13.50 13.50 9.61 9.62 7.32 7.33 8.72 6.73 6.74 8.07 8.09 10.63 10.70
0.967 – 1.004
settings of the source were the following: ion spray voltage at 5500 V, curtain gas, ion source gas 1 and gas 2 at 50, 50 and 60 units, respectively. The declustering potential (DP) was optimized for each compound as shown in Table 2. 2.4. Validation procedure Quantitation of amphetamines, opiates, cocaine and derivatives was performed by the internal standard method. A six point calibration curve was made for all the substances by quadratic in 1/ X weighting regression analysis of the ratio of the peak area of analyte to the peak area of its associated deuterated internal standard. Calibration curves were prepared daily, by spiking finely cut (fragments < 2 mm) drug-free hair samples with internal standard solutions and corresponding analytical working solution mixtures to obtain calibration concentrations of 0, 0.05, 0.10, 0.50, 1.0, 5.0 and 10.0 ng/mg for each analyte, as described in the ‘‘Hair sample decontamination and extraction procedure’’ section. Linearity was assessed by analyzing five replicates for each calibration level on different days. For 5 days, validation samples at 0.05, 0.5, 5.0 and 10 ng/mg amphetamines, opiates, cocaine and metabolites were prepared in duplicate for the inter-assay study (5 series) and in 5
1.006 – 1.007 – 1.006 1.024 – 1.005 – 1.005 1.014 1.069 1.143 1.143 1.185 1.227 1.291 1.323 1.450 2.764 – 1.008 1.000 1.017 – 1.000 – 1.001 – 1.001 1.191 – 1.001 – 1.002 – 1.006
replicates (a different concentration per day) for the intra-assay study. Relative standard deviation (RSD) and bias were calculated for inter- and intra-assay studies. For acceptance, both parameters must simultaneously be lower than 25% for the LOQ and lower than 20% for the other concentrations in both studies. The limit of detection (LOD) was defined as the lowest concentration giving a response of at least three times the average of the baseline noise (S/ N > 3, as determined by Analyst1 software), and the LOQ was defined as the lowest concentration that could be measured with an intra-assay precision RSD and relative bias less than 20%. Carry over was evaluated, by analysing a sample of mobile phase subsequently to the highest calibration level (n = 5). The absence of any signal on the corresponding chromatogram was verified. Ion suppression phenomenon was studied following the experimental system previously proposed by Antignac et al. [27]. Briefly, a standard solution containing the compounds of interest (at 100 mg/L) was continuously and directly infused into the mass spectrometer interface. A simultaneous LC Flow containing either a pure mobile phase or a blank biological extract (hair from 10 non drugs consumers were sampled) was introduced through a tee. Evolution of the signal of the transitions at the retention times of the corresponding compounds of interest was studied to evaluate presence and intensity of ion suppression.
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Table 2 Optimized mass spectrometric parameters. IS are in bold type. Analytes
Q1 mass (amu)
Q3 mass (amu)
DP (V)
FP (V)
EP (V)
CEP (V)
CE (V)
CXP (V)
Norephedrine
152.1 152.1 169.4 166.3 166.3 141.0 136.1 136.1 155.1 150.1 150.1 185.3 180.2 180.2 166.2 166.2 198.8 194.4 194.4 213.2 208.2 208.2 195.3 195.3 208.3 208.3 182.2 182.2 234.2 234.2 197.1 197.1 210.1 210.1 203.9 203.9 274.1 274.1 231.8 231.8 182.2 182.2 203.1 200.2 200.2 290.2 290.2 307.1 304.2 304.2 321.1 318.2 318.2 293.1 290.2 290.2 303.1 300.1 300.1 314.2 314.2 289.1 286.1 286.1 331.1 328.1 328.1 379.2 370.2 370.2
134.2 117.2 151.1 148.2 91.2 124.1 119.0 91.0 121.1 119.3 91.1 168.2 163.0 105.1 121.1 91.0 165.1 163.4 135.2 163.1 163.0 105.1 136.3 178.0 135.1 177.3 165.0 137.0 84.1 115.0 154.0 118.2 193.2 178.3 187.0 159.0 257.1 229.1 159.0 108.9 91.0 117.9 185.2 182.2 82.0 168.1 136.1 185.2 182.2 77.0 199.2 196.2 82.1 171.2 168.1 105.0 152.1 152.0 165.0 152.0 128.1 152.1 152.2 128.1 165.1 165.1 211.1 212.1 165.2 152.2
31 31 41 21 11 6 16 16 11 16 16 6 11 11 21 26 21 16 16 31 1 1 16 31 31 31 26 21 26 36 31 31 21 21 46 51 31 31 36 41 66 66 26 51 51 36 36 26 26 26 31 41 41 31 46 46 51 71 71 66 66 61 71 71 61 71 71 81 56 56
360 360 120 360 360 330 360 360 360 360 360 320 360 360 330 330 310 360 360 310 360 360 360 360 190 190 290 280 370 370 360 330 370 370 240 250 370 370 350 370 50 50 370 360 360 370 370 370 370 370 370 370 370 350 370 370 370 350 350 370 370 370 350 350 370 350 350 140 360 360
6.5 6.5 5.5 6 9.5 8.5 8 7.5 8 8 7.5 4.5 10.5 10.5 12 8 8.5 10 10.5 10.5 5.5 6.5 11 6.5 7 7 7 7 8 8.5 9.5 10.5 10.5 10.5 11.5 11.5 10 10 11 10.5 9.5 9.5 10 12 12 9.5 9.5 10 9.5 9.5 8.5 10 10 9 10 10 12 10.5 10.5 12 12 10.5 10.5 10.5 11.5 12 12 8 11.5 11.5
8 8 10 6 52 10 8 8 8 10 8 10 10 10 10 10 10 12 10 10 10 8 10 14.5 12 12 8 10 12 14 10 10 12 12 12 10 14 14 12 12 12 12 12 12 12 12 12 14 14 14 14 14 14 14 12 12 14 14 14 14 14 14 14 14 14 14 14 16 14 14
15 25 17 17 39 13 13 21 13 15 25 15 13 27 25 39 17 15 29 19 17 35 23 11 25 15 11 27 29 63 27 45 15 27 13 25 15 23 33 57 31 31 23 25 35 21 33 27 25 79 25 27 47 25 27 43 89 81 51 81 87 75 81 79 51 53 35 43 71 109
2 2 2 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 2 0 0 2 0 2 2 2 0 0 0 0 2 0 2 2 4 4 0 0 0 2 2 2 0 2 2 2 2 0 4 2 0 2 2 0 0 0 0 0 0 0 0 0 2 2 2 2 0 0
Ephedrine-D3 Ephedrine Amphetamine-D5 Amphetamine Methamphetamine-D5 Methamphetamine MDA-D5 MDA PMA MDMA-D5 MDMA MDEA-D5 MDEA BDB MBDB 4-MTA Methylphenidate m-CCP DOM Norfenfluramine DOB Dexfenfluramine AME EME-D3 EME Norcocaine Cocaine-D3 Cocaine Cocaethylene-D3 Cocaethylene BZE-D3 BZE Codeine-D3 Codeine Ethylmorphine Morphine-D3 Morphine 6AM-D3 6AM Heroin-D9 Heroin
Q mass, parent ion m/z; Q mass, daughter ion m/z; DP, declustering potential (orifice plate); FP, focusing potential (focusing ring); EP, entrance potential (Q0 less); CEP, cell entrance potential; CE, collision cell potential; CXP, cell exit potential.
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3. Results and discussion All 28 compounds were analyzed within 15 min. It is of note that heroin can be detected in hair which is rarely the case in blood and urine. Authors have reported the presence of heroin in hair addicts in spite of fast degradation (less than 5 min) in 6AM in blood [28]. Total ion current (TIC) Chromatograms obtained for a drug-free hair extract and for the LOQ (i.e. 0.05 ng/mg hair) are presented in Fig. 1(A) and (B), respectively. Results obtained for the repeatability and intermediate precision are presented in Fig. 2 (A) and (B), respectively. RSD obtained for each compound, at all four concentrations evaluated (0.05, 0.5, 5 and 10 ng/mg), were below the required values of 25% for the LOQ and 20% for other values. A Bias below 20% was also obtained for all the compounds. The LODs and LOQs determined for each compound are presented in Table 3. Values between 0.005 and 0.030 ng/mg hair were achieved for the LOD, and a value of 0.05 ng/mg for the LOQ, corresponding to the lowest calibration level, was the same for all the compounds. The latter is in accordance with the values recommended in the consensus of the Society of Hair Testing (SoHT) on hair testing in forensic cases [29], which require a LOQ of at most 0.2 ng/mg for amphetamines and opiates, 0.5 ng/mg for cocaine and 0.05 ng/mg for other cocaine derivatives. The fit of the quadratic, 1/X weighted regression was evaluated by computing the correlation coefficient r, over five replicates for each calibration level. Values over 0.993 were obtained for all 28 compounds, thus the calibration model was considered acceptable. Finally, no ion suppression phenomenons were highlighted at the retention time of all compounds, for each MRM transition. Beside quantifying opiates and cocaine, the present method allows to detect routinely a broader range of amphetamines and related compounds (i.e. ephedrine and norephedrine, BDB, 4-MTA,
Fig. 2. (A) Repeatability and (B) intermediate precision (% RSD) assessed for each compound, at 0.05, 0.5, 5 and 10 ng/mg.
Fig. 1. (A) Chromatogram (TIC) of a drug-free hair extract and (B) chromatogram (TIC) of a drug-free hair sample spiked with 0.05 ng/mg of each compound. (a) EME; (b) AME, morphine; (c) norephedrine; (d) ephedrine and codeine; (e) 6AM and amphetamine; (f) MDA, methamphetamine and PMA; (g) MDMA and ethylmorphine; (h) BDB and MDEA; (i) MBDB and BZE; (j) 4-MTA and methylphenidate; (k) heroin; (l) m-CPP; (m) DOM, cocaine; (n) norcocaine; (o) norfenfluramine, DOB; (p) cocaethylene; and (q) dexfenfluramine.
m-CPP, DOM, DOB, methylphenidate, norfenfluramine and dexfenfluramine) than previously reported methods, that only focus on amphetamine, methamphetamine, MDA, MDMA, MDEA and MBDB at most [3,4,17–24]. This analytical method has now been validated, and has been used in forensic cases involving possible drug use. 3.1. Case 1 A man suspected of taking drugs and being a drug dealer was arrested by the police. Urine and hair samples were gathered in
L. Imbert et al. / Forensic Science International 234 (2014) 132–138 Table 3 Limits of detection and quantification evaluated for each compound. Compound
LOD (ng/mg)
LOQ (ng/mg)
Norephedrine Ephedrine Amphetamine Methamphetamine MDA PMA MDMA MDEA BDB MBDB 4-MTA Methylphenidate m-CPP DOM Norfenfluramine DOB Dexfenfluramine AME EME Cocaine Norcocaine Cocaethylene BZE Codeine Ethylmorphine Morphine 6AM Heroin
0.010 0.030 0.010 0.005 0.010 0.010 0.005 0.005 0.030 0.005 0.020 0.020 0.010 0.010 0.010 0.020 0.005 0.020 0.010 0.005 0.010 0.005 0.005 0.010 0.005 0.020 0.005 0.010
0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.050
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codeine is usually co-extracted with morphine from raw opium and then acetylated into acetylcodeine. The acetylcodeine contained in heroin would be metabolized to codeine just like heroin is metabolized to 6AM. These results reflect most probably regular heroin consumption for approximately 7 months before sampling, which could explain the behaviour of the patient. Owing to the toxicological results in urine, the hypothesis of an overdose might not be plausible, and further blood analysis would have been required to investigate the cause of death. 4. Conclusion The present work described an analytical method for the detection of 28 opiates, cocaine and derivatives, and amphetamines. This method has also been evaluated and found to be successful in 4 external quality controls proposed by the German Society of Toxicological and Forensic Chemistry (GTFCh) and 3 by the SoHT in 2012 and the beginning of 2013. After successfully passing the validation process, this method has been routinely used for forensic cases, two of which have been described. Unfortunately new illicit drugs, particularly among amphetamines (i.e. cathinone derivatives, N-(1-phenylcyclohexyl)-2-ethoxyethanamine derivatives, etc.), which are regularly marketed by drug dealers, would not be detected by the current method. It is therefore necessary to constantly modify such a method, as is done in the doping control field, in order to be able to assess these latest drugs and to limit false-negative results as much as possible. References
order to verify if this man had taken any illicit drugs for several months before being arrested. A 4 cm hair sample was collected, and segmented as follows prior to analysis: 0 ! 2 cm and 2 ! 4 cm from the hair scalp. Methamphetamine and amphetamine were detected at 0.63 ng/mg and 0.06 ng/mg respectively in the 0 ! 2 segment, and only methamphetamine was detected at 0.24 ng/mg in the 2 ! 4 segment. These results favour a consumption of methamphetamine for approximately 4 months before the sampling, from which amphetamine would be a metabolite in this case. The increase of methamphetamine, associated to the detection of amphetamine in the first segment, may indicate that the suspect took more of this drug during the 2 months before sampling. However, the lower values obtained in the second segment could also be due to a wash-out of the compounds with the use of capillary cosmetics. The suspect was finally found guilty as these concentrations result from consumption without doubt. 3.2. Case 2 A man hospitalized in an intensive care unit (ICU) due to a loss of consciousness and a cardio-respiratory arrest died 2 weeks after his admission. Relatives reported that he had fainted several times in the weeks leading up to the incident, and a packet of brown powder was found in his belongings on his admission to the hospital. Traces of heroin and cocaine were detected in urine sampled on admission to the ICU. A 7 cm hair strand was then sampled during the autopsy in order to further investigate the cause of death. The sample was segmented and analyzed as follows: 0 ! 2 cm, 2 ! 4 cm and 4 ! 7 cm. 6AM, morphine and codeine were detected in all segments. Concentrations were more or less constant throughout the whole sample, with 6AM between 0.49 and 0.58 ng/mg, morphine between 0.22 and 0.30 ng/mg and codeine below the LOQ. No cocaine and metabolites were detected. In this case, 6AM and morphine may originate from the metabolism of heroin, and codeine would be detected in low amounts as it is a product extracted from opium during the production of heroin. Indeed, during the semi-synthesis of heroin,
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