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Target screening and confirmation of 35 licit and illicit drugs and metabolites in hair by LC–MSMS
Forensic Science International 217 (2012) 207–215
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Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Target screening and confirmation of 35 licit and illicit drugs and metabolites in hair by LC–MSMS ´ scar Quintela b, Ana de Castro a,c, Angelines Cruz a, Manuel Lo´pez-Rivadulla a, Elena Lendoiro a, O a, Marta Concheiro * a b c
Servicio de Toxicologı´a Forense, Instituto de Ciencias Forenses, Universidad de Santiago de Compostela, Spain Instituto Nacional de Toxicologı´a y Ciencias Forenses, Madrid, Spain Departamento de I+D. Cienytech S.L., Santiago de Compostela, Spain
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 July 2011 Received in revised form 11 October 2011 Accepted 5 November 2011 Available online 30 November 2011
A liquid chromatography–tandem mass spectrometry (LC–MSMS) target screening in 50 mg hair was developed and fully validated for 35 analytes (D9-tetrahidrocannabinol (THC), morphine, 6acetylmorphine, codeine, methadone, fentanyl, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, benzoylecgonine, cocaine, lysergic acid diethylamide, ketamine, scopolamine, alprazolam, bromazepam, clonazepam, diazepam, flunitrazepam, 7aminoflunitrazepam, lorazepam, lormetazepam, nordiazepam, oxazepam, tetrazepam, triazolam, zolpidem, zopiclone, amitriptyline, citalopram, clomipramine, fluoxetine, paroxetine and venlafaxine). Hair decontamination was performed with dichloromethane, and incubation in 2 mL of acetonitrile at 50 8C overnight. Extraction procedure was performed in 2 steps, first liquid–liquid extraction, hexane:ethyl acetate (55:45, v:v) at pH 9, followed by solid-phase extraction (Strata-X cartridges). Chromatographic separation was performed in AtlantisT3 (2.1 mm 100 mm, 3 mm) column, acetonitrile and ammonium formate pH 3 as mobile phase, and 32 min total run time. One transition per analyte was monitored in MRM mode. To confirm a positive result, a second injection monitoring 2 transitions was performed. The method was specific (no endogenous interferences, n = 9); LOD was 0.2– 50 pg/mg and LOQ 0.5–100 pg/mg; linearity ranged from 0.5–100 to 2000–20,000 pg/mg; imprecision <15%; analytical recovery 85–115%; extraction efficiency 4.1–85.6%; and process efficiency 2.5–207.7%; 27 analytes showed ion suppression (up to 86.2%), 4 ion enhancement (up to 647.1%), and 4 no matrix effect; compounds showed good stability 24–48 h in autosampler. The method was applied to 17 forensic cases. In conclusion, a sensitive and specific target screening of 35 analytes in 50 mg hair, including drugs of abuse (THC, cocaine, opiates, amphetamines) and medicines (benzodiazepines, antidepressants) was developed and validated, achieving lower cut-offs than Society of Hair Testing recommendations. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Hair Screening LC–MSMS THC
1. Introduction Hair analysis is becoming a routine practice in forensic toxicology laboratories. This alternative matrix offers several advantages, highlighting its large window of detection (months), non-invasive collection, and easy storage and transport conditions (envelop at room temperature). In addition, it is possible to perform segmental analysis, which allows the determination of the historic pattern of drug use if the sample is cut as close as possible to scalp on vertex
* Corresponding author at: Investigadora Parga Pondal (IPP), Servicio de Toxicologı´a Forense, Instituto de Ciencias Forenses, Universidad de Santiago de ˜ a), Spain. Compostela, C/San Francisco s/n, 15782 Santiago de Compostela (A Corun Tel.: +34 881812446; fax: +34 981580336. E-mail addresses: [email protected], [email protected] (M. Concheiro). 0379-0738/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2011.11.006
posterior region [1]. Drugs are mainly incorporated into hair by passive diffusion from blood, but also from sweat and sebaceous glands. The incorporation mechanism is not yet well understood, although it depends on melanine hair content and analyte’s physicochemical properties (lipophilicity, melanine affinity) [2]. Some hair analysis disadvantages are external contamination (particularly important for smoked drugs), low concentrations of some compounds and metabolites, and limited amount of sample supplied for testing. Hair analysis of illicit drugs and medicines (benzodiazepines, hypnotics, antidepressants) is currently employed in a wide range of situations, such as workplace drug testing, driving ability probation, doping control, chronic drug abuse intoxication, postmortem toxicology, therapy compliance control, and drug facilitated sexual assault cases. According to SAMHSA [3], most admissions to substance abuse treatment reported the use of multiple substances (multiple drug
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use), being cannabis the most common illicit drug, followed by cocaine, opiates and other drugs. In Europe, the combined use of different kind of licit and/or illicit drugs also was a common fact, and also cannabis showed the highest prevalence [4]. Owing to this polydrug consumption tendency, and to the small amount of hair specimen usually available for analysis, multianalyte methods are recommended, saving time, costs and amount of specimen required. Several multianalyte procedures for drugs of abuse and/or medicines analysis in hair have been developed by GC–MS [5–7] and LC–MS [8–13]. GC–MS methods require different derivatization procedures for analysis of thermolabile and nonvolatile compounds [2]; whereas, in LC–MS methods derivatization procedures are avoided, and determination of multiple groups of compounds can be performed in a single method. Among these multianalyte methods, only Kronstrand et al. [10] included D-9tetrahidrocannabinol (THC) in the same extraction procedure along with other drugs; however, THC was analyzed separately by GC–MS, and the other drugs by LC–MSMS. For the first time, a target multianalyte screening method by LC–MSMS in hair including THC is presented. The method was developed and validated for the simultaneous identification and quantification of 35 licit and illicit drugs and metabolites, including THC, opiates and opioids, amphetamines, cocaine and its main metabolite, LSD, ketamine, scopolamine, benzodiazepines, antidepressants and hypnotics, and it was applied to the analysis of real specimens. The method was specific and sensitive, achieving at least the Society of Hair Testing (SoHT) cut-off recommendations for opiates, amphetamines, cocaine and THC. 2. Materials and methods 2.1. Chemical and reagents Codeine, methadone, morphine, fentanyl, benzoylecgonine (BE), ketamine, THC, amphetamine (A), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), alprazolam, clonazepam, diazepam, flunitrazepam, lormetazepam, nordiazepam, oxazepam, triazolam, zolpidem, amitriptyline, clomipramine, fluoxetine, paroxetine, 6-monoacetylmorphine (6-AM), cocaine, lysergic acid diethylamide (LSD), 7-aminoflunitrazepam (7AF), lorazepam and zopiclone standards at 1 mg/mL in methanol or acetonitrile were supplied by Cerilliant (Round Rock, TX, USA). The rest of compounds were purchased as a solid form; bromazepam and scopolamine were supplied by Sigma– Aldrich (St. Louis, MO, USA); citalopram by H. Lundbeck A/S (Copenhagen,
Denmark); tetrazepam by Cerriliant (Round Rock, TX, USA) and venlafaxine by European Pharmacopoeia (Strasbourg, France). The internal standards (IStd) methadone-d3, morphine-d6, fentanyl-d5, BE-d3, ketamine-d4, THC-d3, A-d5, MAd5, MDMA-d5, alprazolam-d5, diazepam-d5, flunitrazepam-d7, oxazepam-d5, fluoxetine-d6, paroxetine-d6, 6-AM-d6, cocaine-d3, LSD-d3 and zopiclone-d4 at 100 mg/mL in methanol or acetonitrile were obtained from Cerilliant (Round Rock, TX, USA). Acetonitrile, methanol and ammonium hydroxide in a 25% solution were provided by Panreac Quimica S.A.U. (Barcelona, Spain). Dichloromethane, 2propanol, ethyl acetate and formic acid were supplied by Scharlau (Sentmenat, Spain). Hexane was provided by Merck (Darmstadt, Germany). All solvents were analytical grade. Ammonium formate was from Fluka Chemie (Bachs, Switzerland). Purified water was obtained in the laboratory using a Milli-Q system (Le Mont-surLausanne, Switzerland). Stata-X cartridges (3 mL, 60 mg) were from Phenomenex (Torrance, CA, USA). 2.2. Instrumentation The HPLC system was a Waters Alliance 2795 Separation Module with a Waters Alliance series column heater/cooler (Waters Corp., Milford, USA). An Atlantis T3 column (2.1 mm 100 mm, 3 mm) (Waters Corp., Milford, USA) was used for separation at 30 8C. The chromatographic separation was performed in gradient mode, and the mobile phase was ammonium formate 2 mM with 0.1% formic acid (pH 3) (A) and acetonitrile (B). The gradient program was as follows: 0–1 min 10% B; 1–3 min from 10% to 15% B; 3–5.2 min 15% B; 5.2–7 min from 15% to 25% B; 7–9 min from 25% to 30% B; 9–16 min 30% B; 16–19 min from 30% to 45% B; 19–21 min from 45% to 90% B; 21–26 min 90% B; 26–26.3 min return to initial conditions; and 26.3– 32 min column re-equilibration. A divert valve was set to direct the LC flow to the mass spectrometer from 1 to 29 min and to waste the remaining time. A Quattro MicroTM API ESCI triple quadrupole (Waters Corp., Milford, USA) was used for analyses. The instrument was operated in electrospray in positive mode (ESI+) to produce protonated molecules of the analytes under the following optimized settings: capillary voltage 3.0 kV; source block temperature 150 8C; desolvation gas (nitrogen) temperature 450 8C; desolvation gas flow rate 550 L/h; and cone gas (nitrogen) flow rate at 45 L/h. Data were acquired in MRM (multiple reaction monitoring) mode. Transitions, cone voltage and collision energy were optimized by infusion of each individual analyte (10 mg/mL in methanol) at 20 mL/min. One transition per compound was monitored for the initial screening. If compound confirmation was required, a second injection was performed monitoring two transitions per compound. Table 1 shows MRM transitions, cone voltage, collision energy and retention time (RT) for each analyte and the corresponding IStd. Data acquisition was controlled using MassLynx 4.0 software and processed with QuanLynx 4.0 software (Waters Corp., Milford, USA).
2.3. Calibrators and quality controls preparation Working solutions of the 35 compounds were prepared in methanol at 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5 and 10 mg/mL. Calibration curves (0.5–2000 pg/mg for LSD; 5–5000 pg/mg for scopolamine, ketamine, 7-AF,
Table 1 MRM transitions, cone voltage, collision energy and retention time for all the analytes and internal standards. The transition used in the screening quantification purposes is underlined. Compound
MRM
Cone voltage (V)
Collision energy (eV)
Morphine
286.0 > 201.2 286.0 > 229.2 292.3 > 201.5 300.1 > 215.2 300.1 > 243.2 136.0 > 90.6 136.0 > 119.0 141.2 > 124.1 180.0 > 163.0 180.0 > 104.8 304.4 > 138.2 304.4 > 103.0 150.0 > 90.6 150.0 > 119.0 155.1 > 120.9 328.0 > 165.1 328.0 > 211.1 334.5 > 165.3 194.0 > 163.0 194.0 > 104.8 199.1 > 165.0 238.3 > 125.0 238.3 > 179.3 242.4 > 129.1
40 40 40 40 40 20 20 15 18 18 34 34 24 24 22 45 45 34 20 20 22 25 25 24
25 25 24 25 25 15 9 9 10 20 24 41 17 11 11 40 25 35 12 25 12 25 17 28
Morphine-d6 Codeine A A-d5 MDA Scopolamine MA MA-d5 6-AM 6-AM-d6 MDMA MDMA-d5 Ketamine Ketamine-d4
Retention time (min) 2.0 2.0 4.2 5.0 4.9 5.8 5.6 5.9 5.8 6.1 6.1 6.5 6.4 8.2 8.1
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Table 1 (Continued ) Compound
MRM
Cone voltage (V)
Collision energy (eV)
BE
290.1 > 168.2 290.1 > 104.9 293.2 > 171.2 389.2 > 245.2 389.2 > 217.2 393.4 > 245.2 284.4 > 135.1 284.4 > 227.2 304.2 > 182.2 304.2 > 81.8 307.1 > 185.2 308.3 > 235.4 308.3 > 92.0 324.1 > 223.2 324.1 > 281.2 327.2 > 226.3 278.1 > 57.8 278.1 > 260.3 337.2 > 188.3 337.2 > 105.0 342.2 > 188.3 316.2 > 182.3 316.2 > 209.3 325.1 > 109.1 325.1 > 260.1 330.0 > 69.7 330.0 > 192.3 336.1 > 76.0 278.1 > 90.8 278.1 > 233.3 287.2 > 103.9 287.2 > 76.9 292.2 > 246.23 310.2 > 265.3 310.2 > 104.9 313.3 > 268.4 321.1 > 275.2 321.1 > 303.2 309.2 > 281.2 309.2 > 205.3 314.2 > 286.3 310.1 > 43.7 310.1 > 148.2 316.1 > 43.7 316.1 > 270.2 316.1 > 214.3 271.2 > 140.1 271.2 > 165.1 343.1 > 308.3 343.1 > 315.2 315.0 > 85.9 315.0 > 57.8 314.1 > 268.3 314.1 > 239.3 321.2 > 275.4 289.3 > 225.5 289.3 > 197.4 335.1 > 289.3 335.1 > 317.2 285.2 > 154.2 285.2 > 193.3 290.2 > 154.2 315.2 > 193.2 315.2 > 135.1 318.2 > 196.2
30 30 30 20 20 20 40 40 30 30 30 45 45 35 35 35 25 25 30 30 30 38 38 35 35 35 35 35 30 30 25 25 30 20 20 31 25 25 40 40 40 22 22 25 40 40 30 30 25 25 22 22 40 40 35 48 48 25 25 20 20 40 30 30 35
18 30 20 13 37 13 27 27 20 30 20 41 53 25 20 25 18 12 22 36 24 30 23 26 20 30 20 30 24 18 39 59 21 15 25 15 21 17 27 41 27 12 8 12 35 25 29 29 29 31 20 32 27 37 27 30 33 15 15 31 31 27 25 25 25
BE-d3 Zopiclone Zopiclone-d4 7-AF Cocaine Cocaine-d3 Zolpidem LSD LSD-d3 Venlafaxine Fentanyl Fentanyl-d5 Bromazepam Citalopram Paroxetine Paroxetine-d6 Amitriptyline Oxazepam Oxazepam-d5 Methadone Methadone-d3 Lorazepam Alprazolam Alprazolam-d5 Fluoxetine Fluoxetine-d6 Clonazepam Nordiazepam Triazolam Clomipramine Flunitrazepam Flunitrazepam-d7 Tetrazepam Lormetazepam Diazepam Diazepam-d5 THC THC-d3
Retention time (min) 8.5 8.5 10.0 10.0 10.7 10.8 10.8 11.2 11.4 11.4 11.5 13.1 13.0 13.7 14.1 16.3 16.2 18.8 18.9 18.7 19.2 19.1 20.4 20.4 20.3 20.7 20.6 20.8 21.1 21.1 21.5 22.0 21.8 22.1 22.7 23.1 23.0 26.8 26.8
Amphetamine (A), 3,4-methylenedioxyamphetamine (MDA), methamphetamine (MA), 6-acetylmorphine (6-AM), 3,4-methylenedioxymethamphetamine (MDMA), benzoylecgonine (BE), 7-aminoflunitrazepam (7-AF), lysergic acid diethylamide (LSD), D-9-tetrahidrocannabinol (THC).
zolpidem, fentanyl, paroxetine, clomipramine, nordiazepam, lorazepam, flunitrazepam, lormetazepam and diazepam; 10–10,000 pg/mg for zopiclone, venlafaxine, amitriptyline, triazolam, fluoxetine and alprazolam; 20–20,000 pg/mg for morphine, 6-AM, codeine, A, MA, MDA, MDMA, BE, cocaine, bromazepam, citalopram, methadone, tetrazepam, oxazepam and clonazepam; and 100–20,000 pg/mg for THC), were prepared by adding 25, 50 or 100 mL of the corresponding working solution, and 50 mL IStd solution at 0.5 (methadone-d3, morphine-d6, fentanyl-d5, BE-d3, ketamine-d4, A-d5, MA-d5, MDMA-d5, alprazolam-d5, diazepam-d5, fluni-
trazepam-d7, oxazepam-d5, fluoxetine-d6, paroxetine-d6, 6-AM-d6, cocaine-d3, and zopiclone-d4), 2 (THC-d3,) and 0.1 ng/mL (LSD-d3) in methanol to 50 mg hair sample. Low QC working solutions at 0.001, 0.0025, 0.005, 0.01, and 0.1 mg/mL, medium QC working solutions at 0.01, 0.025, 0.05, and 0.1 mg/mL, and high QC working solutions at 0.25, 0.5, 1 and 10 mg/mL, depending on the analyte, were prepared in methanol from different stock solutions than those used for calibrators. QC samples were prepared adding 25, 30, 40, 50, 75, 100 or 150 mL of the corresponding
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working solution and 50 mL IStd solution to 50 mg blank hair sample. Low QC was 0.5 (for LSD), 5 (for scopolamine, ketamine, 7-AF, zolpidem, fentanyl, paroxetine, clomipramine, nordiazepam, lorazepam, flunitrazepam, lormetazepam and diazepam), 10 (for zopiclone, venlafaxine, amitriptyline, triazolam, fluoxetine and alprazolam), 20 (for morphine, 6-AM, codeine, A, MA, MDA, MDMA, BE, cocaine, bromazepam, citalopram, methadone, tetrazepam, oxazepam and clonazepam), and 100 pg/mg (for THC); medium QC was 10 (for LSD), 37.5 (for scopolamine, ketamine, 7-AF, zolpidem, fentanyl, paroxetine, clomipramine, nordiazepam, lorazepam, flunitrazepam, lormetazepam and diazepam), 75 (for zopiclone, venlafaxine, amitriptyline, triazolam, fluoxetine and alprazolam), 150 (for morphine, 6-AM, codeine, A, MA, MDA, MDMA, BE, cocaine, bromazepam, citalopram, methadone, tetrazepam, oxazepam and clonazepam), and 300 pg/mg (for THC); and high QC was 800 (for LSD), 750 (for scopolamine, ketamine, 7-AF, zolpidem, fentanyl, paroxetine, clomipramine, nordiazepam, lorazepam, flunitrazepam, lormetazepam and diazepam), 1500 (for zopiclone, venlafaxine, amitriptyline, triazolam, fluoxetine and alprazolam), 3000 (for morphine, 6-AM, codeine, A, MA, MDA, MDMA, BE, cocaine, bromazepam, citalopram, methadone, tetrazepam, oxazepam and clonazepam), and 6000 pg/mg (for THC). 2.4. Hair decontamination, incubation and extraction Hair samples were decontaminated with 3 consecutive 2 mL dichloromethane washes, for 2 min each. The 3 wash solvents were collected and analyzed to confirm total elimination of external contamination. The wash solvent was dried under nitrogen at 35 8C, reconstituted in 100 mL of initial mobile phase and 45 mL was injected into LC–MSMS. 50 mg of decontaminated hair was dried at 70 8C 40 min, and pulverized with a ball-mill (Precellys, Montigny le Bretonneux, France). The powder was incubated with 2 mL acetonitrile for 12 h at 50 8C in a bath, after the addition of 50 mL IStd solution. Samples were centrifuged at 4000 rpm for 10 min at 4 8C. Supernatants were evaporated to dryness under nitrogen at 35 8C, and reconstituted in 200 mL methanol (to improve THC recovery) and 2 mL borate buffer (pH 9). Sample extraction was performed in 2 steps, first a liquid–liquid extraction (LLE) followed by a solid phase extraction (SPE). LLE was performed adding 4 mL of hexane:ethyl acetate (55:45, v:v). Samples were shaken for 15 min in a rotor, centrifuged (4000 rpm, 10 min, 4 8C), and the organic phases were collected and evaporated to dryness under nitrogen at 35 8C. The dried supernatants were
reconstituted again in 200 mL methanol and 2 mL borate buffer (pH 9), and submitted to SPE with Strata-X cartridges (Phenomenex, Torrance, CA, USA). After cartridges conditioning with 2 mL methanol and 2 mL water, sample was loaded. Cleanup was accomplished by sequential washes with 2 mL 5% methanol in water and 2 mL water:methanol:ammonium hydroxide (75:24.5:0.5, v:v). Cartridges were dried for 10 min under vacuum before elution with 2 mL dichloromethane:2propanol (75:25, v:v). Eluates were evaporated to dryness with nitrogen at 35 8C, reconstituted in 100 mL initial mobile phase, and 45 mL were injected into LC– MSMS. 2.5. Validation The method was fully validated, including linearity, limit of detection (LOD), limit of quantification (LOQ), selectivity, imprecision, analytical recovery, extraction efficiency, process efficiency, matrix effect, and autosampler stability. Linearity was determined by least-squares regression with 1/x or 1/x2 weighting. Acceptable linearity was achieved if coefficient of determination (r2) was at least 0.98, and calibrator residual was 20% at the LOQ and 15% at the other concentration levels. The LOD was defined as the lower concentration with acceptable chromatography, the presence of all transitions with signal-to-noise ratio of at least 3, and retention time within 0.2 min of the average retention time of calibrators. The LOQ was the lowest concentration that met the LOD criteria, and a signal-to-noise of at least 10, imprecision lower than 20%, and analytical recovery between 80% and 120%. Interferences from endogenous matrix components were evaluated by the analysis of 9 different blank hair samples from healthy non-drug-consuming volunteers. If analytes were not detected (
Table 2 Limit of detection (LOD), calibration range and linearity results for the 35 compounds analyzed. Compound
LOD (pg/mg)
Calibration range (pg/mg)
Morphine Codeine A MDA Scopolamine MA 6-AM MDMA Ketamine BE Zopiclone 7-AF Cocaine Zolpidem LSD Venlafaxine Fentanyl Bromazepam Citalopram Paroxetine Amitriptyline Oxazepam Methadone Lorazepam Alprazolam Fluoxetine Clonazepam Nordiazepam Triazolam Clomipramine Flunitrazepam Tetrazepam Lormetazepam Diazepam THC
5 2 2 2 2 2 2 2 2 10 5 2 2 2 0.2 2 2 5 2 2 5 10 2 2 5 2 10 2 5 2 2 10 2 2 50
20–20,000 20–20,000 20–20,000 20–20,000 5–2000 20–20,000 20–20,000 20–20,000 5–2000 20–20,000 10–10,000 10–5000 20–20,000 5–5000 0.5–2000 10–10,000 5–5000 20–20,000 20–10,000 5–5000 10–5000 20–20,000 20–20,000 5–5000 10–10,000 10–10,000 20–10,000 5–5000 10–10,000 5–2000 5–5000 20–10,000 5–5000 5–5000 100–20,000
Intercept SD (n = 4) 0.0853 0.0331 0.2006 0.1872 0.0261 0.0295 0.0210 0.0197 0.0002 0.0004 0.0226 0.0228 0.0438 0.0139 0.0199 0.0051 0.0115 0.0043 0.0428 0.0202 0.0137 0.0012 0.0012 0.0012 0.0123 0.0057 0.0003 0.0003 0.0074 0.0008 0.0003 0.0003 0.0037 0.0009 0.0001 0.0002 0.0061 0.0034 0.0051 0.0043 0.0035 0.0037 0.0076 0.0039 0.0036 0.0016 0.0001 0.0001 0.0038 0.0023 0.0094 0.0074 2.0E 5 0.0001 0.0049 0.0020 0.0031 0.0012 0.0896 0.0316 0.0076 0.0021 0.0021 0.0010 0.0205 0.0123 0.0065 0.0026 0.0398 0.0060
Slope SD (n = 4)
r2 SD (n = 4)
0.0159 7.6E 0.0091 7.9E 0.0014 2.3E 0.0010 8.9E 0.0007 6.9E 0.0023 1.5E 0.0119 8.6E 0.0024 1.0E 0.0073 3.4E 0.0021 4.6E 0.0038 1.5E 0.0007 1.4E 0.0017 4.6E 0.0019 2.0E 0.0112 2.5E 0.0004 7.7E 0.0049 1.2E 2.3E 5 4.7E 0.0007 8.3E 0.0047 1.6E 0.0016 2.3E 0.0011 6.4E 0.0008 4.0E 0.0001 1.8E 0.0009 5.2E 0.0009 4.0E 1.1E 5 1.0E 0.0036 5.6E 0.0007 1.8E 0.0491 5.2E 0.0057 2.5E 0.0003 3.9E 0.0052 3.4E 0.0030 1.0E 0.0005 1.5E
0.9949 0.0021 0.9894 0.0041 0.9907 0.0021 0.9885 0.0034 0.9912 0.0036 0.9943 0.0022 0.9954 0.0012 0.9980 0.0015 0.9891 0.0039 0.9937 0.0031 0.9885 0.0013 0.9955 0.0032 0.9948 0.0025 0.9901 0.0031 0.9983 0.0012 0.9896 0.0033 0.9907 0.0018 0.9867 0.0045 0.9868 0.0010 0.9892 0.0027 0.9866 0.0093 0.9898 0.0024 0.9941 0.0024 0.9877 0.0029 0.9935 0.0028 0.9872 0.0021 0.9890 0.0018 0.9901 0.0050 0.9886 0.0025 0.9850 0.0030 0.9863 0.0042 0.9898 0.0041 0.9973 0.0018 0.9922 0.0015 0.9976 0.0011
4 4 4 5 5 4 4 4 4 5 4 4 5 4 4 5 4 6 5 4 4 5 5 5 5 5 6 4 4 3 4 5 4 4 5
Amphetamine (A), 3,4-methylenedioxyamphetamine (MDA), methamphetamine (MA), 6-acetylmorphine (6-AM), 3,4-methylenedioxymethamphetamine (MDMA), benzoylecgonine (BE), 7-aminoflunitrazepam (7-AF), lysergic acid diethylamide (LSD), D-9-tetrahidrocannabinol (THC).
E. Lendoiro et al. / Forensic Science International 217 (2012) 207–215 comparing average peak areas of blank hair specimens fortified prior to extraction (n = 9) with those obtained in specimens fortified after extraction (n = 9) at the same concentration. Process efficiency was determined comparing average peak areas of blank hair specimens fortified prior to extraction (n = 9), with peak areas of samples at the same nominal concentrations prepared in initial mobile phase (neats). Matrix effect was assessed by comparing analyte peak areas in 9 different blank extracted hair samples fortified after extraction, with analyte peak areas of neats. Matrix effect was calculated as follows: (100 mean peak area of fortified hair after extraction/mean peak area of neats) 100. Autosampler analyte stability was assayed at LOQ, medium and high QC levels after 24 and 48 h at 10 8C. Analyte stability was calculated comparing percentage of mean concentration after storage in the autosampler for 24 or 48 h (n = 5), versus mean concentration of fresh prepared QCs (n = 5). In order to demonstrate the applicability of the method, 17 real hair specimens were analyzed according to the previously described method. Hair specimens were collected from head vertex posterior region, indicating with a string the hair root. Specimens were stored at room temperature in an envelope until analysis. 2.6. Identification criteria Identification criteria included RT within 0.2 min of average calibrator RT, presence of 2 transitions, and relative ion intensities (% of base peak) within 20%, of calibrator relative ion intensity if it was >50%; 25% if it was 20–50%; 30% if it was 10–20%; and 50% if it was 10% [15].
3. Results 3.1. Validation Linearity of analyte-to-IStd peak area ratio versus theoretical concentration was verified in hair. All compounds calibration curves fitted with 1/x2-weighted linear regression, except MDMA,
211
7-AF, LSD, lormetazepam and THC, which fitted with 1/x-weighting factor. The curvature tested on a set of 4 calibration curves yielded determination coefficients (r2) above 0.98, with residuals within 20% at LOQs and 15% at other calibrator concentrations for all compounds. LODs ranged between 0.2 and 50 pg/mg, and LOQ was between 0.5 and 100 pg/mg, depending on the analyte. These results are summarized in Table 2. In order to guarantee the correct RT for each analyte, a mobile phase was injected every 2 injections. No interferences with any extractable endogenous compounds in hair (n = 9) were detected. Imprecision and analytical recovery were satisfactory at all tested concentrations (Table 3), except for paroxetine imprecision at low QC (26.1%), and for 7-AF imprecision and analytical recovery at high QC (31.6% and 72.8%). Extraction efficiencies ranged from 4.1% to 85.6%, and process efficiency 2.5–207.7%. Matrix effect was evaluated for all analytes; 27 analytes (morphine, codeine, 6-AM, scopolamine, zopiclone, 7-AF, cocaine, zolpidem, venlafaxine, LSD, fentanyl, citalopram, paroxetine, amitriptyline, methadone, triazolam, fluoxetine, tretrazepam, clomipramine, nordiazepam, oxazepam, alprazolam, clonazepam, lorazepam, flunitrazepam, lormetazepam and diazepam) showed ion suppression up to 86.2%. Four analytes showed ion enhancement; A and MA up to 54.6%, and extremely high for bromazepam and THC with results up to 647.1%. And 4 compounds have no matrix effect (MDMA, MDA, ketamine and BE). These data are shown in Table 4. All analytes were stable for 24 h in autosampler at 10 8C (% loss < 18.0%). The majority of analytes also were stable for 48 h under the same conditions (% loss < 18.5%), except 7-AF, nordia-
Table 3 Pooled intra-day, inter-day and total imprecision, and analytical recovery for the 35 analytes. Analyte
Morphine Codeine A MDA Scopolamine MA 6-AM MDMA Ketamine BE Zopiclone 7-AF Cocaine Zolpidem LSD Venlafaxine Fentanyl Bromazepam Citalopram Paroxetine Amitriptyline Oxazepam Methadone Lorazepam Alprazolam Fluoxetine Clonazepam Nordiazepam Triazolam Clomipramine Flunitrazepam Tetrazepam Lormetazepam Diazepam THC
Pooled intra-day imprecision (n = 20, CV)
Inter-day imprecision (n = 20, CV)
Total imprecision (n = 20, CV)
Analytical recovery (n = 20, % target)
Low
Med
High
Low
Med
High
Low
Med
High
Low
Med
High
7.6 6.1 5.6 14.6 7.8 6.5 6.0 15.9 5.3 11.9 8.2 7.8 4.6 7.7 19.2 12.8 7.9 21.2 16.0 25.0 15.0 11.1 4.7 6.3 10.6 15.6 6.3 16.8 7.6 10.6 8.8 7.9 9.8 16.5 9.6
3.7 3.9 4.5 2.6 6.8 1.4 2.6 2.2 2.1 15.7 2.7 9.6 2.5 2.5 3.3 7.7 2.2 7.4 5.2 1.9 5.3 3.2 4.7 8.4 7.2 10.6 9.6 6.8 9.5 6.5 3.0 4.1 6.6 2.3 6.4
4.0 3.5 2.7 4.0 5.5 1.1 2.3 1.7 1.7 9.1 2.3 23.5 3.8 3.0 2.1 5.3 1.6 8.4 7.1 5.2 7.0 2.0 5.8 10.6 6.1 4.6 7.9 10.3 7.5 6.8 3.2 5.1 5.8 5.6 5.5
6.3 6.6 11.8 15.1 5.6 13.9 4.0 6.8 9.2 10.2 3.3 6.7 8.6 4.0 0 8.5 0 0 0 7.4 6.5 0 8.7 3.0 0 3.0 7.4 5.4 6.7 3.0 8.6 1.1 9.2 0 0
2.7 6.2 9.1 2.3 7.1 5.6 2.6 0 2.4 0 2.3 10.3 0 2.9 3.7 3.2 1.8 7.8 5.4 0.7 1.5 1.6 2.7 2.9 4.1 2.9 5.3 0 0 4.0 6.3 3.0 6.9 2.7 0
4.9 6.2 5.5 7.1 6.0 3.7 5.1 2.2 1.7 0 2.6 21.2 0 1.4 2.9 6.6 0.4 6.5 5.8 2.0 9.6 3.1 0 7.7 2.3 6.2 9.5 0 0 0 9.9 8.2 4.6 1.9 0.6
9.8 9.0 13.1 21.0 9.6 15.3 7.2 17.3 10.6 15.7 8.8 10.3 9.8 8.7 0 15.4 0 0 0 26.1 16.3 0 9.9 7.0 0 15.9 9.7 17.7 10.1 11.0 12.3 8.0 13.5 0 0
4.6 7.3 10.2 3.5 9.8 5.8 3.6 0 3.2 0 3.5 14.1 0 3.8 5.0 8.4 2.9 10.8 7.5 2.0 5.5 3.6 5.4 8.9 8.2 11.0 11.0 0 0 7.6 6.9 5.1 9.6 3.5 0
6.3 7.1 6.1 8.1 8.2 3.9 5.6 2.7 2.4 0 3.5 31.6 0 3.3 3.6 8.4 1.7 10.6 9.2 5.6 11.9 3.7 0 13.1 6.5 7.8 12.3 0 0 0 10.4 9.6 7.4 5.9 5.5
94.4 86.0 98.3 98.7 104.3 101.2 95.6 91.1 90.9 98.6 92.5 108.0 104.5 95.1 104.2 97.4 98.3 111.3 104.1 107.7 109.6 96.8 105.7 109.9 101.2 98.5 111.9 106.6 94.8 94.2 94.5 103.9 95.3 106.4 91.5
105.2 102.2 105.6 109.2 94.2 107.0 100.1 106.9 98.2 102.8 98.1 83.6 108.2 88.8 92.7 91.4 97.4 96.5 107.8 89.9 105.5 109.0 102.1 93.9 102.3 100.2 97.7 106.6 102.6 100.0 103.0 109.8 100.6 95.8 102.1
100.7 96.4 99.7 97.6 94.0 101.7 98.4 102.6 87.2 98.8 94.0 72.8 103.6 87.9 102.2 102.2 89.4 100.5 96.7 89.9 98.9 101.8 104.7 90.6 99.8 104.0 95.3 95.4 96.2 88.1 95.4 101.3 100.9 91.5 103.1
Amphetamine (A), 3,4-methylenedioxyamphetamine (MDA), methamphetamine (MA), 6-acetylmorphine (6-AM), 3,4-methylenedioxymethamphetamine (MDMA), benzoylecgonine (BE), 7-aminoflunitrazepam (7-AF), lysergic acid diethylamide (LSD), D-9-tetrahidrocannabinol (THC).
E. Lendoiro et al. / Forensic Science International 217 (2012) 207–215
212
Table 4 Extraction efficiency (EF) (n = 9), process efficiency (PE) (n = 9) and matrix effect (ME) (n = 9), and coefficient of variation (CV) of ME for the 35 analytes. EF %
Analyte
Morphine Codeine A MDA Scopolamine MA 6-AM MDMA Ketamine BE Zopiclone 7-AF Cocaine Zolpidem LSD Venlafaxine Fentanyl Bromazepam Citalopram Paroxetine Amitriptyline Oxazepam Methadone Lorazepam Alprazolam Fluoxetine Clonazepam Nordiazepam Triazolam Clomipramine Flunitrazepam Tetrazepam Lormetazepam Diazepam THC
PE %
ME % (CV, %)
LOQ
10LOQ
LOQ
10LOQ
LOQ
8.5 44.5 41.0 54.7 31.7 63.0 62.1 69.2 57.9 4.6 28.1 10.1 85.6 36.5 40.8 45.9 39.5 35.6 33.2 38.9 31.9 59.0 70.8 65.5 26.4 39.9 52.9 51.4 28.4 39.3 48.1 31.6 54.5 41.2 26.8
10.4 51.9 42.4 51.0 39.8 62.4 74.0 58.7 60.1 4.1 29.8 7.4 50.9 34.1 27.9 49.5 35.9 33.6 36.3 49.6 61.7 83.7 49.2 79.3 28.8 52.0 67.2 62.3 32.2 64.9 65.6 35.1 62.6 44.5 27.8
5.7 33.6 61.8 46.8 28.0 80.9 37.3 60.6 60.4 4.5 18.1 3.7 38.0 22.2 12.7 27.0 14.4 103.9 12.7 12.0 19.8 43.1 16.5 68.1 19.2 17.2 27.2 11.6 19.1 9.2 25.6 9.4 30.6 13.3 123.3
5.8 29.7 51.2 41.4 26.8 77.3 37.3 51.6 55.8 3.2 14.0 2.5 27.8 16.5 8.7 21.8 11.7 149.5 13.1 10.4 17.2 36.5 25.6 54.1 13.5 17.3 21.1 8.6 14.0 10.3 27.1 8.1 22.0 9.8 207.7
32.9 24.6 50.6 14.5 11.6 54.6 39.9 7.4 4.5 1.8 35.6 63.5 33.6 40.6 67.3 41.3 61.8 192.3 61.7 69.1 55.9 27.0 53.7 29.8 20.1 56.8 48.6 77.5 35.2 76.5 46.9 68.5 43.9 55.1 483.6
10LOQ (7.6) (10.0) (21.3) (12.4) (8.0) (27.4) (14.5) (18.0) (8.8) (6.2) (14.4) (34.5) (16.6) (19.4) (13.1) (14.3) (18.6) (19.3) (18.0) (19.5) (14.0) (10.9) (21.7) (15.9) (19.5) (19.9) (20.4) (19.5) (20.3) (18.2) (19.6) (17.6) (18.4) (13.0) (18.2)
44.2 42.7 20.7 18.8 32.7 19.0 49.6 12.0 7.1 22.0 53.1 66.8 43.0 46.0 64.4 53.8 58.4 372.6 55.8 76.4 67.6 56.5 58.9 38.5 47.5 63.4 68.5 86.2 51.0 82.2 57.4 75.1 63.8 80.2 647.1
(7.2) (7.8) (8.9) (9.5) (7.3) (11.5) (15.4) (10.8) (11.8) (7.9) (13.4) (19.7) (11.6) (11.6) (14.2) (14.3) (11.9) (14.0) (9.9) (15.3) (13.3) (7.4) (8.5) (10.6) (11.8) (10.7) (16.3) (20.6) (8.0) (14.0) (13.6) (16.5) (12.9) (15.4) (47.6)
Amphetamine (A), 3,4-methylenedioxyamphetamine (MDA), methamphetamine (MA), 6-acetylmorphine (6-AM), 3,4-methylenedioxymethamphetamine (MDMA), benzoylecgonine (BE), 7-aminoflunitrazepam (7-AF), lysergic acid diethylamide (LSD), D-9-tetrahidrocannabinol (THC).
Table 5 Analytes’ concentrations (pg/mg) detected in the 13 positive real hair specimens. Analyte
Hair specimen H1
Morphine Codeine 6-AM Methadone Fentanyl Cocaine BE A MDMA THC Ketamine Alprazolam Bromazepam Diazepam Flunitrazepam Lorazepam Nordiazepam Tetrazepam Zolpidem Zopiclone Amitriptiline Citalopram Fluoxetine Paroxetine
H2
H3
H4
88.1 85.4
47.1 17.0 8.7
H6
3869.6 881.4 18265.1 206.3 7.7 >20,000 19443.6 106.8 31.3
44.0 535.0 509.0 533.9 95.5 143.2 612.1
H5
307.9 326.0
H7
18.3 121.6 604.0
H8
H9 17.6 51.2 4436.0
279.5 514.3
424.0 432.7
6103.5 6101.0
H10
H11
H12
H13
416.0 467.9 467.5
117.4 120.7
170.9
59.2
7.9
42.6
9.9 124.1 113.2
70.3
137.0 134.6
>20,000 >20,000 107.0
23.3 33.5
39.4 53.2
12.8 32.0 39.6
5027.7
6534.0 95.2
>10,000 85.8
>10,000 162.6
Amphetamine (A), 6-acetylmorphine (6-AM), 3,4-methylenedioxymethamphetamine (MDMA), benzoylecgonine (BE), D-9-tetrahidrocannabinol (THC).
E. Lendoiro et al. / Forensic Science International 217 (2012) 207–215
zepam and triazolam, which % loss was up to 54.8% at low, medium and high QC concentrations; paroxetine, alprazolam, tetrazepam and THC at low concentrations (% loss < 35.2%); and scopolamine and clonazepam at medium and high concentrations (% loss < 29.1%). 3.2. Real cases analysis Seventeen hair specimens from forensic cases were analyzed as proof of the method. Most of the cases were from patients following withdrawal treatment for at least 2 months previous hair collection (specimens 2, 3, 4, 6, 7, 8, 9, 10, 11, 14, 15, 16 and 17). Specimens 14, 15, 16 and 17 were negative, and the other specimens showed low concentrations of drugs of abuse. Positive results to benzodiazepines and antidepressants corresponded to medical prescriptions. Specimens 1, 5 and 12 were from individuals suspected to consume drugs of abuse, and specimen 13 was a medical case, where drug monitoring was requested. In this case, the patient was prescribed citalopram and fentanyl, and 100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2
F2:MRM of 3 channels,ES+ 136 > 90.6 1.373e+005
both substances were detected in hair. Positive results are shown in Table 5. Confirmation was performed by sample reinjection, monitoring 2 transitions per compound. All results were confirmed, except one false positive to bromazepam. The three washes of each specimen were analyzed and total removal of external contamination was confirmed in all cases. Fig. 1 shows a chromatogram of a real specimen positive to A, MDMA, BE, cocaine, ketamine and THC. 4. Discussion A target screening method in hair for simultaneous identification and quantification of drugs of abuse (THC, opiates, amphetamines, cocaine, LSD, ketamine, and scopolamine) and medicines (benzodiazepines, antidepressants, and hypnotics), all of them frequently present in forensic cases, was developed by LC–MSMS and fully validated. One transition per compound was monitored in MRM mode. In order to confirm positive results, a second injection monitoring 2 transitions per compound was performed. 100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2
Anfetamina;4.69;29293.2;135734
100
213
MDMA;6.26;28645.4;146426
F3:MRM of 8 channels,ES+ 194 > 163 1.472e+005
MDMA-d5;6.21;966923.1;4897422
F3:MRM of 8 channels,ES+ 199.1 > 165 4.908e+006
100
%
%
0
min
100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2
F2:MRM of 3 channels,ES+ 141.2 > 124.1 8.298e+005 Anfetamina-d5;4.65;171621.8;824972
100
0
min
100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2 100
%
%
0
min 3.40
3.60
3.80
4.00
4.20
4.40
4.60
4.80
100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2 100
min 5.00
F4:MRM of 6 channels,ES+ 290.1 > 168.2 2.184e+004
BE;8.24;4275.2;21657
0
5.00
5.20
5.40
5.60
5.80
6.00
6.20
6.40
6.60
100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2 Cocaina 100 10.71 126851.9 925952
%
6.80
7.00
F5:MRM of 6 channels,ES+ 304.2 > 182.2 9.275e+005
%
0
min
100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2
F4:MRM of 6 channels,ES+ 293.2 > 171.2 1.900e+005
BE-d3;8.24;37633.7;188497
100
0
min
100916_19 Smooth(Mn,2x2) Pelo 215_2010_segmento 2 Cocaina-d3 10.71 1341157.3 9069586
100
%
F5:MRM of 6 channels,ES+ 307.1 > 185.2 9.083e+006
%
0
min 7.50
8.00
8.50
9.00
9.50
100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2 Ketamina;7.94;59364.2;297722 100
0
10.00
min 10.25
F4:MRM of 6 channels,ES+ 238.3 > 125 2.988e+005
10.75
11.00
11.25
11.50
0
min
100
F4:MRM of 6 channels,ES+ 242.4 > 129.1 1.079e+006
Ketamina-d4 7.83 221741.3 1076957
12.00
100
25.03
25.38
12.25
12.50
F9:MRM of 3 channels,ES+ 315.2 > 193.2 9.412e+002
THC 26.80 111.4 704 25.90 27.11
100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2
11.75
100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2
%
%
10.50
27.39
27.70
28.84
28.03
0
min
100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2
F9:MRM of 3 channels,ES+ 318.2 > 196.2 5.131e+003
THC-d3 26.78 661.1 4959
100
%
%
0
min 7.50
8.00
8.50
9.00
9.50
10.00
0
min 25.50
26.00
26.50
27.00
27.50
28.00
28.50
Fig. 1. Chromatogram of a real positive specimen to amphetamine, A (533.9 pg/mg); 3,4-methylenedioxymethamphetamine, MDMA (95.5 pg/mg); benzoylecgonine, BE (509.0 pg/mg); cocaine (535.0 pg/mg); ketamine (612.1 pg/mg); and D-9-tetrahidrocannabinol, THC (143.2 pg/mg).
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E. Lendoiro et al. / Forensic Science International 217 (2012) 207–215
For incubation, different buffers in basic and acidic conditions, and organic solvents (methanol, acetonitrile) were assayed. Basic conditions hydrolyzed several compounds (cocaine, BE, 6AM, for example), and acidic conditions did not extract THC. Methanol incubation yielded good recoveries for all compounds; however, the obtained extracts after LLE and SPE, were not clean enough, and the chromatography was affected (broad peaks, RT shift). Finally, acetonitrile allowed a good recovery for all compounds and clean extracts. THC is the most frequently encountered illicit drug worldwide and drug users are usually poly-drug consumers [4]; however, previously published screening methods for hair analysis did not include this compound in the same analysis along with other drugs of abuse and medicines [8–13,16]. Although Kronstrand et al. [17] analyzed 21 licit and illicit drugs, including THC in the same extraction procedure, two different detection techniques (THC by GC–MS and the rest of the compounds by LC–MS/MS) were employed. LC–MS hair screening methods including a larger number of compounds, even more than 800 analytes [16,18], have been previously published. However, these methods were only validated for analytes’ identification, but not for quantification purposes, and they did not include THC. In the present method, all analytes were extracted using the same procedure and analyzed in the same LC–MSMS run. If further confirmation was required, a second injection monitoring two transitions per compound was performed. Laloup et al. [11] also employed a first injection with the most prominent precursor-product transition for quantification, and confirmation was performed monitoring 2 transitions in a second injection; however, only benzodiazepines and hypnotics were determined in this method. Miller et al. [12] used 3 separate injections to determine cocaine, opiates and metabolites in the first injection, amphetamines in the second injection, and diazepam and metabolites in the third injection. Method’s LOQs were at least those recommended by the SoHT [1] for opiates (200 pg/mg), cocaine and its main metabolite, BE (500 pg/mg and 50 pg/mg, respectively), amphetamines (200 pg/mg) and THC (100 pg/mg). Ketamine (and its main metabolite) was determined by Harun et al. [19] with a LOD of 100 pg/mg and a LOQ of 180 pg/mg, both of them are higher values than those achieved in the present method. For the licit drugs, the limits are consistent with previous publications. Agius and Kintz [20] recommended a cut-off of 50 pg/mg in workplace for bromazepam, nordiazepam, oxazepam, lorazepam, alprazolam, diazepam and flunitrazepam, higher than those achieved in this method. For fentanyl, Kronstrand et al. [17] obtained a LOD of 3 pg/ mg and a LOQ of 8 pg/mg, similar to the limits of this work. Nielsen et al. [13] determined antidepressants, benzodiazepines and their metabolites, and hypnotics (zolpidem and zopiclone) with a LOD of 10 pg/mg and a LOQ of 50 pg/mg. Shen et al. [7] performed a GC– MS-CI method for the detection of antidepressants and antipsychotic with a LOD of 100 pg/mg and LOQ of 200 pg/mg. In all these studies, LODs and LOQs were higher than the limits obtained in the present work. Lower LODs (from 0.5 to 5 pg/mg) for 16 benzodiazepines and hypnotics were obtained in a LC–MSMS method developed by Villain et al. [21] to monitor single dose exposure. With regard to method validation, low extraction efficiency (<50%) was observed for 18 compounds (morphine, fentanyl, BE, A, THC, LSD, scopolamine, 7-AF, alprazolam, bromazepam, diazepam, triazolam, tetrazepam, zolpidem, zopiclone, citalopram, paroxetine and velafaxine). These extraction efficiencies were lower than those described in previously published methods [8–13,16,22]; however, good sensitivity was achieved, with LOQ ranging from 0.5 to 100 pg/mg depending on the compound. Although 2 extraction procedures were performed (LLE and SPE), 31 out of 35 analytes
showed matrix effect; 27 showed ion suppression, ranging from 24.6% to 86.2%, and 4 ion enhancement from 20.7% to 647.1%. In all cases % CV was below 20%, except for A, MA, 7-AF, methadone and THC, with values up to 47.6%. Nevertheless, the use of the corresponding deuterated analogs for these analytes as internal standard compensated for these effects. No significant losses were observed after 24 h storage in the autosampler and, therefore, positive specimens could be reinjected for confirmation purposes. Some analytes (scopolamine, alprazolam, 7-AF, clonazepam, nordiazepam, triazolam, tetrazepam, paroxetine and THC) showed 48 h autosampler losses up to 54.8% and, therefore, positive results of these analytes should be confirmed within 24 h to guarantee a good quantification. Seventeen hair specimens were analyzed to demonstrate method applicability. Several groups of drugs were identified and confirmed in the majority of these specimens, proving the polydrug use pattern and the usefulness of multianalyte procedures. The concentrations found were in accordance with previous published methods [8–12,19,21]. Most of the positive results were confirmed when 2 transitions were monitored in a second injection. A false positive was observed for bromazepam in one specimen. Although RT was correct, the qualifier transition monitored in the second injection was not detected and, therefore, the identification criteria could not be fulfilled. In conclusion, a selective and sensitive LC–MSMS method for the simultaneous identification and quantification of 35 licit and illicit drugs and metabolites in 50 mg of hair was developed and fully validated. The drugs analyzed were THC, opiates, cocaine, amphetamines, hallucinogens, benzodiazepines, antidepressants, and hypnotics. Confirmation of positive results was performed by reinjection of the sample, monitoring 2 transitions per compound. LOQ’s were at least those proposed by the SoHT for opiates, cocaine, amphetamines and THC. Acknowledgment We gratefully acknowledge the support by INCITE (Consellerı´a de Innovacio´n e Industria, Xunta de Galicia), project number INCITE08PXIB208090PR and INCITE09228166PR. References [1] Society of Hair Testing, Recommendations for hair testing in forensic cases, Forensic Sci. Int. 145 (2004) 83–84. [2] F. Pragst, M.A. Balikova, State of the art in hair analysis for detection of drug and alcohol abuse, Clin. Chim. Acta 370 (2006) 17–49. [3] Substance Abuse and Mental Health Services Administration (SAMHSA), The DASIS report, polydrug admissions: 2002, http://www.oas.samhsa.gov/2k5/polydrugTX/polydrugTX.pdf (Accessed May 2011). [4] United Nations, United Nations office on drugs and crime 2010, World Drug Report 2010, http://www.unodc.org/documents/wdr/WDR_2010/World_Drug_Report_2010_lo-res.pdf (Accessed May 2011). [5] R. Cordero, S. Paterson, Simultaneous quantification of opiates, amphetamines, cocaine and metabolites and diazepam and metabolite in a single hair sample using GC-MS, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 850 (2007) 423– 431. [6] G. Merola, S. Gentili, F. Tagliaro, T. Macchia, Determination of different recreational drugs in hair by HS-SPME and GC/MS, Anal. Bioanal. Chem. 397 (2010) 2987–2995. [7] M. Shen, P. Xiang, H. Wu, B. Shen, Z. Huang, Detection of antidepressant and antipsychotic drugs in human hair, Forensic Sci. Int. 126 (2002) 153–161. [8] F. Bucelli, A. Fratini, P. Bavazzano, N. Comodo, Quantification of drugs of abuse and some stimulants in hair samples by liquid chromatography-electrospray ionization ion trap mass spectrometry, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 877 (2009) 3931–3936. [9] S. Hegstad, H.Z. Khiabani, L. Kristoffersen, N. Kunoe, P.P. Lobmaier, A.S. Christophersen, Drug screening of hair by liquid chromatography-tandem mass spectrometry, J. Anal. Toxicol. 32 (2008) 364–372. [10] R. Kronstrand, I. Nystrom, J. Strandberg, H. Druid, Screening for drugs of abuse in hair with ion spray LC-MS-MS, Forensic Sci. Int. 145 (2004) 183–190. [11] M. Laloup, M. Ramirez Fernandez Mdel, G. De Boeck, M. Wood, V. Maes, N. Samyn, Validation of a liquid chromatography-tandem mass spectrometry method for
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[12]
[13]
[14] [15] [16]
[17]
the simultaneous determination of 26 benzodiazepines and metabolites, zolpidem and zopiclone, in blood, urine, and hair, J. Anal. Toxicol. 29 (2005) 616–626. E.I. Miller, F.M. Wylie, J.S. Oliver, Simultaneous detection and quantification of amphetamines, diazepam and its metabolites, cocaine and its metabolites, and opiates in hair by LC-ESI-MS-MS using a single extraction method, J. Anal. Toxicol. 32 (2008) 457–469. M.K. Nielsen, S.S. Johansen, P.W. Dalsgaard, K. Linnet, Simultaneous screening and quantification of 52 common pharmaceuticals and drugs of abuse in hair using UPLC-TOF-MS, Forensic Sci. Int. 196 (2010) 85–92. J.S. Krouwer, R. Rabinowitz, How to improve estimates of imprecision, Clin. Chem. 30 (1984) 290–292. European Union Decision 2002/657/EC (17/8/2002), Off. J. Eur. Commun. 221 (2002) 8–36. A. Pelander, J. Ristimaa, I. Rasanen, E. Vuori, I. Ojanpera, Screening for basic drugs in hair of drug addicts by liquid chromatography/time-of-flight mass spectrometry, Ther. Drug Monit. 30 (2008) 717–724. R. Kronstrand, I. Nystrom, M. Forsman, K. Kall, Hair analysis for drugs in driver’s license regranting. A Swedish pilot study, Forensic Sci. Int. 196 (2010) 55–58.
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Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Target screening and confirmation of 35 licit and illicit drugs and metabolites in hair by LC–MSMS ´ scar Quintela b, Ana de Castro a,c, Angelines Cruz a, Manuel Lo´pez-Rivadulla a, Elena Lendoiro a, O a, Marta Concheiro * a b c
Servicio de Toxicologı´a Forense, Instituto de Ciencias Forenses, Universidad de Santiago de Compostela, Spain Instituto Nacional de Toxicologı´a y Ciencias Forenses, Madrid, Spain Departamento de I+D. Cienytech S.L., Santiago de Compostela, Spain
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 July 2011 Received in revised form 11 October 2011 Accepted 5 November 2011 Available online 30 November 2011
A liquid chromatography–tandem mass spectrometry (LC–MSMS) target screening in 50 mg hair was developed and fully validated for 35 analytes (D9-tetrahidrocannabinol (THC), morphine, 6acetylmorphine, codeine, methadone, fentanyl, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, benzoylecgonine, cocaine, lysergic acid diethylamide, ketamine, scopolamine, alprazolam, bromazepam, clonazepam, diazepam, flunitrazepam, 7aminoflunitrazepam, lorazepam, lormetazepam, nordiazepam, oxazepam, tetrazepam, triazolam, zolpidem, zopiclone, amitriptyline, citalopram, clomipramine, fluoxetine, paroxetine and venlafaxine). Hair decontamination was performed with dichloromethane, and incubation in 2 mL of acetonitrile at 50 8C overnight. Extraction procedure was performed in 2 steps, first liquid–liquid extraction, hexane:ethyl acetate (55:45, v:v) at pH 9, followed by solid-phase extraction (Strata-X cartridges). Chromatographic separation was performed in AtlantisT3 (2.1 mm 100 mm, 3 mm) column, acetonitrile and ammonium formate pH 3 as mobile phase, and 32 min total run time. One transition per analyte was monitored in MRM mode. To confirm a positive result, a second injection monitoring 2 transitions was performed. The method was specific (no endogenous interferences, n = 9); LOD was 0.2– 50 pg/mg and LOQ 0.5–100 pg/mg; linearity ranged from 0.5–100 to 2000–20,000 pg/mg; imprecision <15%; analytical recovery 85–115%; extraction efficiency 4.1–85.6%; and process efficiency 2.5–207.7%; 27 analytes showed ion suppression (up to 86.2%), 4 ion enhancement (up to 647.1%), and 4 no matrix effect; compounds showed good stability 24–48 h in autosampler. The method was applied to 17 forensic cases. In conclusion, a sensitive and specific target screening of 35 analytes in 50 mg hair, including drugs of abuse (THC, cocaine, opiates, amphetamines) and medicines (benzodiazepines, antidepressants) was developed and validated, achieving lower cut-offs than Society of Hair Testing recommendations. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Hair Screening LC–MSMS THC
1. Introduction Hair analysis is becoming a routine practice in forensic toxicology laboratories. This alternative matrix offers several advantages, highlighting its large window of detection (months), non-invasive collection, and easy storage and transport conditions (envelop at room temperature). In addition, it is possible to perform segmental analysis, which allows the determination of the historic pattern of drug use if the sample is cut as close as possible to scalp on vertex
* Corresponding author at: Investigadora Parga Pondal (IPP), Servicio de Toxicologı´a Forense, Instituto de Ciencias Forenses, Universidad de Santiago de ˜ a), Spain. Compostela, C/San Francisco s/n, 15782 Santiago de Compostela (A Corun Tel.: +34 881812446; fax: +34 981580336. E-mail addresses: [email protected], [email protected] (M. Concheiro). 0379-0738/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2011.11.006
posterior region [1]. Drugs are mainly incorporated into hair by passive diffusion from blood, but also from sweat and sebaceous glands. The incorporation mechanism is not yet well understood, although it depends on melanine hair content and analyte’s physicochemical properties (lipophilicity, melanine affinity) [2]. Some hair analysis disadvantages are external contamination (particularly important for smoked drugs), low concentrations of some compounds and metabolites, and limited amount of sample supplied for testing. Hair analysis of illicit drugs and medicines (benzodiazepines, hypnotics, antidepressants) is currently employed in a wide range of situations, such as workplace drug testing, driving ability probation, doping control, chronic drug abuse intoxication, postmortem toxicology, therapy compliance control, and drug facilitated sexual assault cases. According to SAMHSA [3], most admissions to substance abuse treatment reported the use of multiple substances (multiple drug
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use), being cannabis the most common illicit drug, followed by cocaine, opiates and other drugs. In Europe, the combined use of different kind of licit and/or illicit drugs also was a common fact, and also cannabis showed the highest prevalence [4]. Owing to this polydrug consumption tendency, and to the small amount of hair specimen usually available for analysis, multianalyte methods are recommended, saving time, costs and amount of specimen required. Several multianalyte procedures for drugs of abuse and/or medicines analysis in hair have been developed by GC–MS [5–7] and LC–MS [8–13]. GC–MS methods require different derivatization procedures for analysis of thermolabile and nonvolatile compounds [2]; whereas, in LC–MS methods derivatization procedures are avoided, and determination of multiple groups of compounds can be performed in a single method. Among these multianalyte methods, only Kronstrand et al. [10] included D-9tetrahidrocannabinol (THC) in the same extraction procedure along with other drugs; however, THC was analyzed separately by GC–MS, and the other drugs by LC–MSMS. For the first time, a target multianalyte screening method by LC–MSMS in hair including THC is presented. The method was developed and validated for the simultaneous identification and quantification of 35 licit and illicit drugs and metabolites, including THC, opiates and opioids, amphetamines, cocaine and its main metabolite, LSD, ketamine, scopolamine, benzodiazepines, antidepressants and hypnotics, and it was applied to the analysis of real specimens. The method was specific and sensitive, achieving at least the Society of Hair Testing (SoHT) cut-off recommendations for opiates, amphetamines, cocaine and THC. 2. Materials and methods 2.1. Chemical and reagents Codeine, methadone, morphine, fentanyl, benzoylecgonine (BE), ketamine, THC, amphetamine (A), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), alprazolam, clonazepam, diazepam, flunitrazepam, lormetazepam, nordiazepam, oxazepam, triazolam, zolpidem, amitriptyline, clomipramine, fluoxetine, paroxetine, 6-monoacetylmorphine (6-AM), cocaine, lysergic acid diethylamide (LSD), 7-aminoflunitrazepam (7AF), lorazepam and zopiclone standards at 1 mg/mL in methanol or acetonitrile were supplied by Cerilliant (Round Rock, TX, USA). The rest of compounds were purchased as a solid form; bromazepam and scopolamine were supplied by Sigma– Aldrich (St. Louis, MO, USA); citalopram by H. Lundbeck A/S (Copenhagen,
Denmark); tetrazepam by Cerriliant (Round Rock, TX, USA) and venlafaxine by European Pharmacopoeia (Strasbourg, France). The internal standards (IStd) methadone-d3, morphine-d6, fentanyl-d5, BE-d3, ketamine-d4, THC-d3, A-d5, MAd5, MDMA-d5, alprazolam-d5, diazepam-d5, flunitrazepam-d7, oxazepam-d5, fluoxetine-d6, paroxetine-d6, 6-AM-d6, cocaine-d3, LSD-d3 and zopiclone-d4 at 100 mg/mL in methanol or acetonitrile were obtained from Cerilliant (Round Rock, TX, USA). Acetonitrile, methanol and ammonium hydroxide in a 25% solution were provided by Panreac Quimica S.A.U. (Barcelona, Spain). Dichloromethane, 2propanol, ethyl acetate and formic acid were supplied by Scharlau (Sentmenat, Spain). Hexane was provided by Merck (Darmstadt, Germany). All solvents were analytical grade. Ammonium formate was from Fluka Chemie (Bachs, Switzerland). Purified water was obtained in the laboratory using a Milli-Q system (Le Mont-surLausanne, Switzerland). Stata-X cartridges (3 mL, 60 mg) were from Phenomenex (Torrance, CA, USA). 2.2. Instrumentation The HPLC system was a Waters Alliance 2795 Separation Module with a Waters Alliance series column heater/cooler (Waters Corp., Milford, USA). An Atlantis T3 column (2.1 mm 100 mm, 3 mm) (Waters Corp., Milford, USA) was used for separation at 30 8C. The chromatographic separation was performed in gradient mode, and the mobile phase was ammonium formate 2 mM with 0.1% formic acid (pH 3) (A) and acetonitrile (B). The gradient program was as follows: 0–1 min 10% B; 1–3 min from 10% to 15% B; 3–5.2 min 15% B; 5.2–7 min from 15% to 25% B; 7–9 min from 25% to 30% B; 9–16 min 30% B; 16–19 min from 30% to 45% B; 19–21 min from 45% to 90% B; 21–26 min 90% B; 26–26.3 min return to initial conditions; and 26.3– 32 min column re-equilibration. A divert valve was set to direct the LC flow to the mass spectrometer from 1 to 29 min and to waste the remaining time. A Quattro MicroTM API ESCI triple quadrupole (Waters Corp., Milford, USA) was used for analyses. The instrument was operated in electrospray in positive mode (ESI+) to produce protonated molecules of the analytes under the following optimized settings: capillary voltage 3.0 kV; source block temperature 150 8C; desolvation gas (nitrogen) temperature 450 8C; desolvation gas flow rate 550 L/h; and cone gas (nitrogen) flow rate at 45 L/h. Data were acquired in MRM (multiple reaction monitoring) mode. Transitions, cone voltage and collision energy were optimized by infusion of each individual analyte (10 mg/mL in methanol) at 20 mL/min. One transition per compound was monitored for the initial screening. If compound confirmation was required, a second injection was performed monitoring two transitions per compound. Table 1 shows MRM transitions, cone voltage, collision energy and retention time (RT) for each analyte and the corresponding IStd. Data acquisition was controlled using MassLynx 4.0 software and processed with QuanLynx 4.0 software (Waters Corp., Milford, USA).
2.3. Calibrators and quality controls preparation Working solutions of the 35 compounds were prepared in methanol at 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5 and 10 mg/mL. Calibration curves (0.5–2000 pg/mg for LSD; 5–5000 pg/mg for scopolamine, ketamine, 7-AF,
Table 1 MRM transitions, cone voltage, collision energy and retention time for all the analytes and internal standards. The transition used in the screening quantification purposes is underlined. Compound
MRM
Cone voltage (V)
Collision energy (eV)
Morphine
286.0 > 201.2 286.0 > 229.2 292.3 > 201.5 300.1 > 215.2 300.1 > 243.2 136.0 > 90.6 136.0 > 119.0 141.2 > 124.1 180.0 > 163.0 180.0 > 104.8 304.4 > 138.2 304.4 > 103.0 150.0 > 90.6 150.0 > 119.0 155.1 > 120.9 328.0 > 165.1 328.0 > 211.1 334.5 > 165.3 194.0 > 163.0 194.0 > 104.8 199.1 > 165.0 238.3 > 125.0 238.3 > 179.3 242.4 > 129.1
40 40 40 40 40 20 20 15 18 18 34 34 24 24 22 45 45 34 20 20 22 25 25 24
25 25 24 25 25 15 9 9 10 20 24 41 17 11 11 40 25 35 12 25 12 25 17 28
Morphine-d6 Codeine A A-d5 MDA Scopolamine MA MA-d5 6-AM 6-AM-d6 MDMA MDMA-d5 Ketamine Ketamine-d4
Retention time (min) 2.0 2.0 4.2 5.0 4.9 5.8 5.6 5.9 5.8 6.1 6.1 6.5 6.4 8.2 8.1
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Table 1 (Continued ) Compound
MRM
Cone voltage (V)
Collision energy (eV)
BE
290.1 > 168.2 290.1 > 104.9 293.2 > 171.2 389.2 > 245.2 389.2 > 217.2 393.4 > 245.2 284.4 > 135.1 284.4 > 227.2 304.2 > 182.2 304.2 > 81.8 307.1 > 185.2 308.3 > 235.4 308.3 > 92.0 324.1 > 223.2 324.1 > 281.2 327.2 > 226.3 278.1 > 57.8 278.1 > 260.3 337.2 > 188.3 337.2 > 105.0 342.2 > 188.3 316.2 > 182.3 316.2 > 209.3 325.1 > 109.1 325.1 > 260.1 330.0 > 69.7 330.0 > 192.3 336.1 > 76.0 278.1 > 90.8 278.1 > 233.3 287.2 > 103.9 287.2 > 76.9 292.2 > 246.23 310.2 > 265.3 310.2 > 104.9 313.3 > 268.4 321.1 > 275.2 321.1 > 303.2 309.2 > 281.2 309.2 > 205.3 314.2 > 286.3 310.1 > 43.7 310.1 > 148.2 316.1 > 43.7 316.1 > 270.2 316.1 > 214.3 271.2 > 140.1 271.2 > 165.1 343.1 > 308.3 343.1 > 315.2 315.0 > 85.9 315.0 > 57.8 314.1 > 268.3 314.1 > 239.3 321.2 > 275.4 289.3 > 225.5 289.3 > 197.4 335.1 > 289.3 335.1 > 317.2 285.2 > 154.2 285.2 > 193.3 290.2 > 154.2 315.2 > 193.2 315.2 > 135.1 318.2 > 196.2
30 30 30 20 20 20 40 40 30 30 30 45 45 35 35 35 25 25 30 30 30 38 38 35 35 35 35 35 30 30 25 25 30 20 20 31 25 25 40 40 40 22 22 25 40 40 30 30 25 25 22 22 40 40 35 48 48 25 25 20 20 40 30 30 35
18 30 20 13 37 13 27 27 20 30 20 41 53 25 20 25 18 12 22 36 24 30 23 26 20 30 20 30 24 18 39 59 21 15 25 15 21 17 27 41 27 12 8 12 35 25 29 29 29 31 20 32 27 37 27 30 33 15 15 31 31 27 25 25 25
BE-d3 Zopiclone Zopiclone-d4 7-AF Cocaine Cocaine-d3 Zolpidem LSD LSD-d3 Venlafaxine Fentanyl Fentanyl-d5 Bromazepam Citalopram Paroxetine Paroxetine-d6 Amitriptyline Oxazepam Oxazepam-d5 Methadone Methadone-d3 Lorazepam Alprazolam Alprazolam-d5 Fluoxetine Fluoxetine-d6 Clonazepam Nordiazepam Triazolam Clomipramine Flunitrazepam Flunitrazepam-d7 Tetrazepam Lormetazepam Diazepam Diazepam-d5 THC THC-d3
Retention time (min) 8.5 8.5 10.0 10.0 10.7 10.8 10.8 11.2 11.4 11.4 11.5 13.1 13.0 13.7 14.1 16.3 16.2 18.8 18.9 18.7 19.2 19.1 20.4 20.4 20.3 20.7 20.6 20.8 21.1 21.1 21.5 22.0 21.8 22.1 22.7 23.1 23.0 26.8 26.8
Amphetamine (A), 3,4-methylenedioxyamphetamine (MDA), methamphetamine (MA), 6-acetylmorphine (6-AM), 3,4-methylenedioxymethamphetamine (MDMA), benzoylecgonine (BE), 7-aminoflunitrazepam (7-AF), lysergic acid diethylamide (LSD), D-9-tetrahidrocannabinol (THC).
zolpidem, fentanyl, paroxetine, clomipramine, nordiazepam, lorazepam, flunitrazepam, lormetazepam and diazepam; 10–10,000 pg/mg for zopiclone, venlafaxine, amitriptyline, triazolam, fluoxetine and alprazolam; 20–20,000 pg/mg for morphine, 6-AM, codeine, A, MA, MDA, MDMA, BE, cocaine, bromazepam, citalopram, methadone, tetrazepam, oxazepam and clonazepam; and 100–20,000 pg/mg for THC), were prepared by adding 25, 50 or 100 mL of the corresponding working solution, and 50 mL IStd solution at 0.5 (methadone-d3, morphine-d6, fentanyl-d5, BE-d3, ketamine-d4, A-d5, MA-d5, MDMA-d5, alprazolam-d5, diazepam-d5, fluni-
trazepam-d7, oxazepam-d5, fluoxetine-d6, paroxetine-d6, 6-AM-d6, cocaine-d3, and zopiclone-d4), 2 (THC-d3,) and 0.1 ng/mL (LSD-d3) in methanol to 50 mg hair sample. Low QC working solutions at 0.001, 0.0025, 0.005, 0.01, and 0.1 mg/mL, medium QC working solutions at 0.01, 0.025, 0.05, and 0.1 mg/mL, and high QC working solutions at 0.25, 0.5, 1 and 10 mg/mL, depending on the analyte, were prepared in methanol from different stock solutions than those used for calibrators. QC samples were prepared adding 25, 30, 40, 50, 75, 100 or 150 mL of the corresponding
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working solution and 50 mL IStd solution to 50 mg blank hair sample. Low QC was 0.5 (for LSD), 5 (for scopolamine, ketamine, 7-AF, zolpidem, fentanyl, paroxetine, clomipramine, nordiazepam, lorazepam, flunitrazepam, lormetazepam and diazepam), 10 (for zopiclone, venlafaxine, amitriptyline, triazolam, fluoxetine and alprazolam), 20 (for morphine, 6-AM, codeine, A, MA, MDA, MDMA, BE, cocaine, bromazepam, citalopram, methadone, tetrazepam, oxazepam and clonazepam), and 100 pg/mg (for THC); medium QC was 10 (for LSD), 37.5 (for scopolamine, ketamine, 7-AF, zolpidem, fentanyl, paroxetine, clomipramine, nordiazepam, lorazepam, flunitrazepam, lormetazepam and diazepam), 75 (for zopiclone, venlafaxine, amitriptyline, triazolam, fluoxetine and alprazolam), 150 (for morphine, 6-AM, codeine, A, MA, MDA, MDMA, BE, cocaine, bromazepam, citalopram, methadone, tetrazepam, oxazepam and clonazepam), and 300 pg/mg (for THC); and high QC was 800 (for LSD), 750 (for scopolamine, ketamine, 7-AF, zolpidem, fentanyl, paroxetine, clomipramine, nordiazepam, lorazepam, flunitrazepam, lormetazepam and diazepam), 1500 (for zopiclone, venlafaxine, amitriptyline, triazolam, fluoxetine and alprazolam), 3000 (for morphine, 6-AM, codeine, A, MA, MDA, MDMA, BE, cocaine, bromazepam, citalopram, methadone, tetrazepam, oxazepam and clonazepam), and 6000 pg/mg (for THC). 2.4. Hair decontamination, incubation and extraction Hair samples were decontaminated with 3 consecutive 2 mL dichloromethane washes, for 2 min each. The 3 wash solvents were collected and analyzed to confirm total elimination of external contamination. The wash solvent was dried under nitrogen at 35 8C, reconstituted in 100 mL of initial mobile phase and 45 mL was injected into LC–MSMS. 50 mg of decontaminated hair was dried at 70 8C 40 min, and pulverized with a ball-mill (Precellys, Montigny le Bretonneux, France). The powder was incubated with 2 mL acetonitrile for 12 h at 50 8C in a bath, after the addition of 50 mL IStd solution. Samples were centrifuged at 4000 rpm for 10 min at 4 8C. Supernatants were evaporated to dryness under nitrogen at 35 8C, and reconstituted in 200 mL methanol (to improve THC recovery) and 2 mL borate buffer (pH 9). Sample extraction was performed in 2 steps, first a liquid–liquid extraction (LLE) followed by a solid phase extraction (SPE). LLE was performed adding 4 mL of hexane:ethyl acetate (55:45, v:v). Samples were shaken for 15 min in a rotor, centrifuged (4000 rpm, 10 min, 4 8C), and the organic phases were collected and evaporated to dryness under nitrogen at 35 8C. The dried supernatants were
reconstituted again in 200 mL methanol and 2 mL borate buffer (pH 9), and submitted to SPE with Strata-X cartridges (Phenomenex, Torrance, CA, USA). After cartridges conditioning with 2 mL methanol and 2 mL water, sample was loaded. Cleanup was accomplished by sequential washes with 2 mL 5% methanol in water and 2 mL water:methanol:ammonium hydroxide (75:24.5:0.5, v:v). Cartridges were dried for 10 min under vacuum before elution with 2 mL dichloromethane:2propanol (75:25, v:v). Eluates were evaporated to dryness with nitrogen at 35 8C, reconstituted in 100 mL initial mobile phase, and 45 mL were injected into LC– MSMS. 2.5. Validation The method was fully validated, including linearity, limit of detection (LOD), limit of quantification (LOQ), selectivity, imprecision, analytical recovery, extraction efficiency, process efficiency, matrix effect, and autosampler stability. Linearity was determined by least-squares regression with 1/x or 1/x2 weighting. Acceptable linearity was achieved if coefficient of determination (r2) was at least 0.98, and calibrator residual was 20% at the LOQ and 15% at the other concentration levels. The LOD was defined as the lower concentration with acceptable chromatography, the presence of all transitions with signal-to-noise ratio of at least 3, and retention time within 0.2 min of the average retention time of calibrators. The LOQ was the lowest concentration that met the LOD criteria, and a signal-to-noise of at least 10, imprecision lower than 20%, and analytical recovery between 80% and 120%. Interferences from endogenous matrix components were evaluated by the analysis of 9 different blank hair samples from healthy non-drug-consuming volunteers. If analytes were not detected (
Table 2 Limit of detection (LOD), calibration range and linearity results for the 35 compounds analyzed. Compound
LOD (pg/mg)
Calibration range (pg/mg)
Morphine Codeine A MDA Scopolamine MA 6-AM MDMA Ketamine BE Zopiclone 7-AF Cocaine Zolpidem LSD Venlafaxine Fentanyl Bromazepam Citalopram Paroxetine Amitriptyline Oxazepam Methadone Lorazepam Alprazolam Fluoxetine Clonazepam Nordiazepam Triazolam Clomipramine Flunitrazepam Tetrazepam Lormetazepam Diazepam THC
5 2 2 2 2 2 2 2 2 10 5 2 2 2 0.2 2 2 5 2 2 5 10 2 2 5 2 10 2 5 2 2 10 2 2 50
20–20,000 20–20,000 20–20,000 20–20,000 5–2000 20–20,000 20–20,000 20–20,000 5–2000 20–20,000 10–10,000 10–5000 20–20,000 5–5000 0.5–2000 10–10,000 5–5000 20–20,000 20–10,000 5–5000 10–5000 20–20,000 20–20,000 5–5000 10–10,000 10–10,000 20–10,000 5–5000 10–10,000 5–2000 5–5000 20–10,000 5–5000 5–5000 100–20,000
Intercept SD (n = 4) 0.0853 0.0331 0.2006 0.1872 0.0261 0.0295 0.0210 0.0197 0.0002 0.0004 0.0226 0.0228 0.0438 0.0139 0.0199 0.0051 0.0115 0.0043 0.0428 0.0202 0.0137 0.0012 0.0012 0.0012 0.0123 0.0057 0.0003 0.0003 0.0074 0.0008 0.0003 0.0003 0.0037 0.0009 0.0001 0.0002 0.0061 0.0034 0.0051 0.0043 0.0035 0.0037 0.0076 0.0039 0.0036 0.0016 0.0001 0.0001 0.0038 0.0023 0.0094 0.0074 2.0E 5 0.0001 0.0049 0.0020 0.0031 0.0012 0.0896 0.0316 0.0076 0.0021 0.0021 0.0010 0.0205 0.0123 0.0065 0.0026 0.0398 0.0060
Slope SD (n = 4)
r2 SD (n = 4)
0.0159 7.6E 0.0091 7.9E 0.0014 2.3E 0.0010 8.9E 0.0007 6.9E 0.0023 1.5E 0.0119 8.6E 0.0024 1.0E 0.0073 3.4E 0.0021 4.6E 0.0038 1.5E 0.0007 1.4E 0.0017 4.6E 0.0019 2.0E 0.0112 2.5E 0.0004 7.7E 0.0049 1.2E 2.3E 5 4.7E 0.0007 8.3E 0.0047 1.6E 0.0016 2.3E 0.0011 6.4E 0.0008 4.0E 0.0001 1.8E 0.0009 5.2E 0.0009 4.0E 1.1E 5 1.0E 0.0036 5.6E 0.0007 1.8E 0.0491 5.2E 0.0057 2.5E 0.0003 3.9E 0.0052 3.4E 0.0030 1.0E 0.0005 1.5E
0.9949 0.0021 0.9894 0.0041 0.9907 0.0021 0.9885 0.0034 0.9912 0.0036 0.9943 0.0022 0.9954 0.0012 0.9980 0.0015 0.9891 0.0039 0.9937 0.0031 0.9885 0.0013 0.9955 0.0032 0.9948 0.0025 0.9901 0.0031 0.9983 0.0012 0.9896 0.0033 0.9907 0.0018 0.9867 0.0045 0.9868 0.0010 0.9892 0.0027 0.9866 0.0093 0.9898 0.0024 0.9941 0.0024 0.9877 0.0029 0.9935 0.0028 0.9872 0.0021 0.9890 0.0018 0.9901 0.0050 0.9886 0.0025 0.9850 0.0030 0.9863 0.0042 0.9898 0.0041 0.9973 0.0018 0.9922 0.0015 0.9976 0.0011
4 4 4 5 5 4 4 4 4 5 4 4 5 4 4 5 4 6 5 4 4 5 5 5 5 5 6 4 4 3 4 5 4 4 5
Amphetamine (A), 3,4-methylenedioxyamphetamine (MDA), methamphetamine (MA), 6-acetylmorphine (6-AM), 3,4-methylenedioxymethamphetamine (MDMA), benzoylecgonine (BE), 7-aminoflunitrazepam (7-AF), lysergic acid diethylamide (LSD), D-9-tetrahidrocannabinol (THC).
E. Lendoiro et al. / Forensic Science International 217 (2012) 207–215 comparing average peak areas of blank hair specimens fortified prior to extraction (n = 9) with those obtained in specimens fortified after extraction (n = 9) at the same concentration. Process efficiency was determined comparing average peak areas of blank hair specimens fortified prior to extraction (n = 9), with peak areas of samples at the same nominal concentrations prepared in initial mobile phase (neats). Matrix effect was assessed by comparing analyte peak areas in 9 different blank extracted hair samples fortified after extraction, with analyte peak areas of neats. Matrix effect was calculated as follows: (100 mean peak area of fortified hair after extraction/mean peak area of neats) 100. Autosampler analyte stability was assayed at LOQ, medium and high QC levels after 24 and 48 h at 10 8C. Analyte stability was calculated comparing percentage of mean concentration after storage in the autosampler for 24 or 48 h (n = 5), versus mean concentration of fresh prepared QCs (n = 5). In order to demonstrate the applicability of the method, 17 real hair specimens were analyzed according to the previously described method. Hair specimens were collected from head vertex posterior region, indicating with a string the hair root. Specimens were stored at room temperature in an envelope until analysis. 2.6. Identification criteria Identification criteria included RT within 0.2 min of average calibrator RT, presence of 2 transitions, and relative ion intensities (% of base peak) within 20%, of calibrator relative ion intensity if it was >50%; 25% if it was 20–50%; 30% if it was 10–20%; and 50% if it was 10% [15].
3. Results 3.1. Validation Linearity of analyte-to-IStd peak area ratio versus theoretical concentration was verified in hair. All compounds calibration curves fitted with 1/x2-weighted linear regression, except MDMA,
211
7-AF, LSD, lormetazepam and THC, which fitted with 1/x-weighting factor. The curvature tested on a set of 4 calibration curves yielded determination coefficients (r2) above 0.98, with residuals within 20% at LOQs and 15% at other calibrator concentrations for all compounds. LODs ranged between 0.2 and 50 pg/mg, and LOQ was between 0.5 and 100 pg/mg, depending on the analyte. These results are summarized in Table 2. In order to guarantee the correct RT for each analyte, a mobile phase was injected every 2 injections. No interferences with any extractable endogenous compounds in hair (n = 9) were detected. Imprecision and analytical recovery were satisfactory at all tested concentrations (Table 3), except for paroxetine imprecision at low QC (26.1%), and for 7-AF imprecision and analytical recovery at high QC (31.6% and 72.8%). Extraction efficiencies ranged from 4.1% to 85.6%, and process efficiency 2.5–207.7%. Matrix effect was evaluated for all analytes; 27 analytes (morphine, codeine, 6-AM, scopolamine, zopiclone, 7-AF, cocaine, zolpidem, venlafaxine, LSD, fentanyl, citalopram, paroxetine, amitriptyline, methadone, triazolam, fluoxetine, tretrazepam, clomipramine, nordiazepam, oxazepam, alprazolam, clonazepam, lorazepam, flunitrazepam, lormetazepam and diazepam) showed ion suppression up to 86.2%. Four analytes showed ion enhancement; A and MA up to 54.6%, and extremely high for bromazepam and THC with results up to 647.1%. And 4 compounds have no matrix effect (MDMA, MDA, ketamine and BE). These data are shown in Table 4. All analytes were stable for 24 h in autosampler at 10 8C (% loss < 18.0%). The majority of analytes also were stable for 48 h under the same conditions (% loss < 18.5%), except 7-AF, nordia-
Table 3 Pooled intra-day, inter-day and total imprecision, and analytical recovery for the 35 analytes. Analyte
Morphine Codeine A MDA Scopolamine MA 6-AM MDMA Ketamine BE Zopiclone 7-AF Cocaine Zolpidem LSD Venlafaxine Fentanyl Bromazepam Citalopram Paroxetine Amitriptyline Oxazepam Methadone Lorazepam Alprazolam Fluoxetine Clonazepam Nordiazepam Triazolam Clomipramine Flunitrazepam Tetrazepam Lormetazepam Diazepam THC
Pooled intra-day imprecision (n = 20, CV)
Inter-day imprecision (n = 20, CV)
Total imprecision (n = 20, CV)
Analytical recovery (n = 20, % target)
Low
Med
High
Low
Med
High
Low
Med
High
Low
Med
High
7.6 6.1 5.6 14.6 7.8 6.5 6.0 15.9 5.3 11.9 8.2 7.8 4.6 7.7 19.2 12.8 7.9 21.2 16.0 25.0 15.0 11.1 4.7 6.3 10.6 15.6 6.3 16.8 7.6 10.6 8.8 7.9 9.8 16.5 9.6
3.7 3.9 4.5 2.6 6.8 1.4 2.6 2.2 2.1 15.7 2.7 9.6 2.5 2.5 3.3 7.7 2.2 7.4 5.2 1.9 5.3 3.2 4.7 8.4 7.2 10.6 9.6 6.8 9.5 6.5 3.0 4.1 6.6 2.3 6.4
4.0 3.5 2.7 4.0 5.5 1.1 2.3 1.7 1.7 9.1 2.3 23.5 3.8 3.0 2.1 5.3 1.6 8.4 7.1 5.2 7.0 2.0 5.8 10.6 6.1 4.6 7.9 10.3 7.5 6.8 3.2 5.1 5.8 5.6 5.5
6.3 6.6 11.8 15.1 5.6 13.9 4.0 6.8 9.2 10.2 3.3 6.7 8.6 4.0 0 8.5 0 0 0 7.4 6.5 0 8.7 3.0 0 3.0 7.4 5.4 6.7 3.0 8.6 1.1 9.2 0 0
2.7 6.2 9.1 2.3 7.1 5.6 2.6 0 2.4 0 2.3 10.3 0 2.9 3.7 3.2 1.8 7.8 5.4 0.7 1.5 1.6 2.7 2.9 4.1 2.9 5.3 0 0 4.0 6.3 3.0 6.9 2.7 0
4.9 6.2 5.5 7.1 6.0 3.7 5.1 2.2 1.7 0 2.6 21.2 0 1.4 2.9 6.6 0.4 6.5 5.8 2.0 9.6 3.1 0 7.7 2.3 6.2 9.5 0 0 0 9.9 8.2 4.6 1.9 0.6
9.8 9.0 13.1 21.0 9.6 15.3 7.2 17.3 10.6 15.7 8.8 10.3 9.8 8.7 0 15.4 0 0 0 26.1 16.3 0 9.9 7.0 0 15.9 9.7 17.7 10.1 11.0 12.3 8.0 13.5 0 0
4.6 7.3 10.2 3.5 9.8 5.8 3.6 0 3.2 0 3.5 14.1 0 3.8 5.0 8.4 2.9 10.8 7.5 2.0 5.5 3.6 5.4 8.9 8.2 11.0 11.0 0 0 7.6 6.9 5.1 9.6 3.5 0
6.3 7.1 6.1 8.1 8.2 3.9 5.6 2.7 2.4 0 3.5 31.6 0 3.3 3.6 8.4 1.7 10.6 9.2 5.6 11.9 3.7 0 13.1 6.5 7.8 12.3 0 0 0 10.4 9.6 7.4 5.9 5.5
94.4 86.0 98.3 98.7 104.3 101.2 95.6 91.1 90.9 98.6 92.5 108.0 104.5 95.1 104.2 97.4 98.3 111.3 104.1 107.7 109.6 96.8 105.7 109.9 101.2 98.5 111.9 106.6 94.8 94.2 94.5 103.9 95.3 106.4 91.5
105.2 102.2 105.6 109.2 94.2 107.0 100.1 106.9 98.2 102.8 98.1 83.6 108.2 88.8 92.7 91.4 97.4 96.5 107.8 89.9 105.5 109.0 102.1 93.9 102.3 100.2 97.7 106.6 102.6 100.0 103.0 109.8 100.6 95.8 102.1
100.7 96.4 99.7 97.6 94.0 101.7 98.4 102.6 87.2 98.8 94.0 72.8 103.6 87.9 102.2 102.2 89.4 100.5 96.7 89.9 98.9 101.8 104.7 90.6 99.8 104.0 95.3 95.4 96.2 88.1 95.4 101.3 100.9 91.5 103.1
Amphetamine (A), 3,4-methylenedioxyamphetamine (MDA), methamphetamine (MA), 6-acetylmorphine (6-AM), 3,4-methylenedioxymethamphetamine (MDMA), benzoylecgonine (BE), 7-aminoflunitrazepam (7-AF), lysergic acid diethylamide (LSD), D-9-tetrahidrocannabinol (THC).
E. Lendoiro et al. / Forensic Science International 217 (2012) 207–215
212
Table 4 Extraction efficiency (EF) (n = 9), process efficiency (PE) (n = 9) and matrix effect (ME) (n = 9), and coefficient of variation (CV) of ME for the 35 analytes. EF %
Analyte
Morphine Codeine A MDA Scopolamine MA 6-AM MDMA Ketamine BE Zopiclone 7-AF Cocaine Zolpidem LSD Venlafaxine Fentanyl Bromazepam Citalopram Paroxetine Amitriptyline Oxazepam Methadone Lorazepam Alprazolam Fluoxetine Clonazepam Nordiazepam Triazolam Clomipramine Flunitrazepam Tetrazepam Lormetazepam Diazepam THC
PE %
ME % (CV, %)
LOQ
10LOQ
LOQ
10LOQ
LOQ
8.5 44.5 41.0 54.7 31.7 63.0 62.1 69.2 57.9 4.6 28.1 10.1 85.6 36.5 40.8 45.9 39.5 35.6 33.2 38.9 31.9 59.0 70.8 65.5 26.4 39.9 52.9 51.4 28.4 39.3 48.1 31.6 54.5 41.2 26.8
10.4 51.9 42.4 51.0 39.8 62.4 74.0 58.7 60.1 4.1 29.8 7.4 50.9 34.1 27.9 49.5 35.9 33.6 36.3 49.6 61.7 83.7 49.2 79.3 28.8 52.0 67.2 62.3 32.2 64.9 65.6 35.1 62.6 44.5 27.8
5.7 33.6 61.8 46.8 28.0 80.9 37.3 60.6 60.4 4.5 18.1 3.7 38.0 22.2 12.7 27.0 14.4 103.9 12.7 12.0 19.8 43.1 16.5 68.1 19.2 17.2 27.2 11.6 19.1 9.2 25.6 9.4 30.6 13.3 123.3
5.8 29.7 51.2 41.4 26.8 77.3 37.3 51.6 55.8 3.2 14.0 2.5 27.8 16.5 8.7 21.8 11.7 149.5 13.1 10.4 17.2 36.5 25.6 54.1 13.5 17.3 21.1 8.6 14.0 10.3 27.1 8.1 22.0 9.8 207.7
32.9 24.6 50.6 14.5 11.6 54.6 39.9 7.4 4.5 1.8 35.6 63.5 33.6 40.6 67.3 41.3 61.8 192.3 61.7 69.1 55.9 27.0 53.7 29.8 20.1 56.8 48.6 77.5 35.2 76.5 46.9 68.5 43.9 55.1 483.6
10LOQ (7.6) (10.0) (21.3) (12.4) (8.0) (27.4) (14.5) (18.0) (8.8) (6.2) (14.4) (34.5) (16.6) (19.4) (13.1) (14.3) (18.6) (19.3) (18.0) (19.5) (14.0) (10.9) (21.7) (15.9) (19.5) (19.9) (20.4) (19.5) (20.3) (18.2) (19.6) (17.6) (18.4) (13.0) (18.2)
44.2 42.7 20.7 18.8 32.7 19.0 49.6 12.0 7.1 22.0 53.1 66.8 43.0 46.0 64.4 53.8 58.4 372.6 55.8 76.4 67.6 56.5 58.9 38.5 47.5 63.4 68.5 86.2 51.0 82.2 57.4 75.1 63.8 80.2 647.1
(7.2) (7.8) (8.9) (9.5) (7.3) (11.5) (15.4) (10.8) (11.8) (7.9) (13.4) (19.7) (11.6) (11.6) (14.2) (14.3) (11.9) (14.0) (9.9) (15.3) (13.3) (7.4) (8.5) (10.6) (11.8) (10.7) (16.3) (20.6) (8.0) (14.0) (13.6) (16.5) (12.9) (15.4) (47.6)
Amphetamine (A), 3,4-methylenedioxyamphetamine (MDA), methamphetamine (MA), 6-acetylmorphine (6-AM), 3,4-methylenedioxymethamphetamine (MDMA), benzoylecgonine (BE), 7-aminoflunitrazepam (7-AF), lysergic acid diethylamide (LSD), D-9-tetrahidrocannabinol (THC).
Table 5 Analytes’ concentrations (pg/mg) detected in the 13 positive real hair specimens. Analyte
Hair specimen H1
Morphine Codeine 6-AM Methadone Fentanyl Cocaine BE A MDMA THC Ketamine Alprazolam Bromazepam Diazepam Flunitrazepam Lorazepam Nordiazepam Tetrazepam Zolpidem Zopiclone Amitriptiline Citalopram Fluoxetine Paroxetine
H2
H3
H4
88.1 85.4
47.1 17.0 8.7
H6
3869.6 881.4 18265.1 206.3 7.7 >20,000 19443.6 106.8 31.3
44.0 535.0 509.0 533.9 95.5 143.2 612.1
H5
307.9 326.0
H7
18.3 121.6 604.0
H8
H9 17.6 51.2 4436.0
279.5 514.3
424.0 432.7
6103.5 6101.0
H10
H11
H12
H13
416.0 467.9 467.5
117.4 120.7
170.9
59.2
7.9
42.6
9.9 124.1 113.2
70.3
137.0 134.6
>20,000 >20,000 107.0
23.3 33.5
39.4 53.2
12.8 32.0 39.6
5027.7
6534.0 95.2
>10,000 85.8
>10,000 162.6
Amphetamine (A), 6-acetylmorphine (6-AM), 3,4-methylenedioxymethamphetamine (MDMA), benzoylecgonine (BE), D-9-tetrahidrocannabinol (THC).
E. Lendoiro et al. / Forensic Science International 217 (2012) 207–215
zepam and triazolam, which % loss was up to 54.8% at low, medium and high QC concentrations; paroxetine, alprazolam, tetrazepam and THC at low concentrations (% loss < 35.2%); and scopolamine and clonazepam at medium and high concentrations (% loss < 29.1%). 3.2. Real cases analysis Seventeen hair specimens from forensic cases were analyzed as proof of the method. Most of the cases were from patients following withdrawal treatment for at least 2 months previous hair collection (specimens 2, 3, 4, 6, 7, 8, 9, 10, 11, 14, 15, 16 and 17). Specimens 14, 15, 16 and 17 were negative, and the other specimens showed low concentrations of drugs of abuse. Positive results to benzodiazepines and antidepressants corresponded to medical prescriptions. Specimens 1, 5 and 12 were from individuals suspected to consume drugs of abuse, and specimen 13 was a medical case, where drug monitoring was requested. In this case, the patient was prescribed citalopram and fentanyl, and 100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2
F2:MRM of 3 channels,ES+ 136 > 90.6 1.373e+005
both substances were detected in hair. Positive results are shown in Table 5. Confirmation was performed by sample reinjection, monitoring 2 transitions per compound. All results were confirmed, except one false positive to bromazepam. The three washes of each specimen were analyzed and total removal of external contamination was confirmed in all cases. Fig. 1 shows a chromatogram of a real specimen positive to A, MDMA, BE, cocaine, ketamine and THC. 4. Discussion A target screening method in hair for simultaneous identification and quantification of drugs of abuse (THC, opiates, amphetamines, cocaine, LSD, ketamine, and scopolamine) and medicines (benzodiazepines, antidepressants, and hypnotics), all of them frequently present in forensic cases, was developed by LC–MSMS and fully validated. One transition per compound was monitored in MRM mode. In order to confirm positive results, a second injection monitoring 2 transitions per compound was performed. 100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2
Anfetamina;4.69;29293.2;135734
100
213
MDMA;6.26;28645.4;146426
F3:MRM of 8 channels,ES+ 194 > 163 1.472e+005
MDMA-d5;6.21;966923.1;4897422
F3:MRM of 8 channels,ES+ 199.1 > 165 4.908e+006
100
%
%
0
min
100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2
F2:MRM of 3 channels,ES+ 141.2 > 124.1 8.298e+005 Anfetamina-d5;4.65;171621.8;824972
100
0
min
100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2 100
%
%
0
min 3.40
3.60
3.80
4.00
4.20
4.40
4.60
4.80
100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2 100
min 5.00
F4:MRM of 6 channels,ES+ 290.1 > 168.2 2.184e+004
BE;8.24;4275.2;21657
0
5.00
5.20
5.40
5.60
5.80
6.00
6.20
6.40
6.60
100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2 Cocaina 100 10.71 126851.9 925952
%
6.80
7.00
F5:MRM of 6 channels,ES+ 304.2 > 182.2 9.275e+005
%
0
min
100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2
F4:MRM of 6 channels,ES+ 293.2 > 171.2 1.900e+005
BE-d3;8.24;37633.7;188497
100
0
min
100916_19 Smooth(Mn,2x2) Pelo 215_2010_segmento 2 Cocaina-d3 10.71 1341157.3 9069586
100
%
F5:MRM of 6 channels,ES+ 307.1 > 185.2 9.083e+006
%
0
min 7.50
8.00
8.50
9.00
9.50
100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2 Ketamina;7.94;59364.2;297722 100
0
10.00
min 10.25
F4:MRM of 6 channels,ES+ 238.3 > 125 2.988e+005
10.75
11.00
11.25
11.50
0
min
100
F4:MRM of 6 channels,ES+ 242.4 > 129.1 1.079e+006
Ketamina-d4 7.83 221741.3 1076957
12.00
100
25.03
25.38
12.25
12.50
F9:MRM of 3 channels,ES+ 315.2 > 193.2 9.412e+002
THC 26.80 111.4 704 25.90 27.11
100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2
11.75
100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2
%
%
10.50
27.39
27.70
28.84
28.03
0
min
100916_19 Smooth(Mn,1x2) Pelo 215_2010_segmento 2
F9:MRM of 3 channels,ES+ 318.2 > 196.2 5.131e+003
THC-d3 26.78 661.1 4959
100
%
%
0
min 7.50
8.00
8.50
9.00
9.50
10.00
0
min 25.50
26.00
26.50
27.00
27.50
28.00
28.50
Fig. 1. Chromatogram of a real positive specimen to amphetamine, A (533.9 pg/mg); 3,4-methylenedioxymethamphetamine, MDMA (95.5 pg/mg); benzoylecgonine, BE (509.0 pg/mg); cocaine (535.0 pg/mg); ketamine (612.1 pg/mg); and D-9-tetrahidrocannabinol, THC (143.2 pg/mg).
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For incubation, different buffers in basic and acidic conditions, and organic solvents (methanol, acetonitrile) were assayed. Basic conditions hydrolyzed several compounds (cocaine, BE, 6AM, for example), and acidic conditions did not extract THC. Methanol incubation yielded good recoveries for all compounds; however, the obtained extracts after LLE and SPE, were not clean enough, and the chromatography was affected (broad peaks, RT shift). Finally, acetonitrile allowed a good recovery for all compounds and clean extracts. THC is the most frequently encountered illicit drug worldwide and drug users are usually poly-drug consumers [4]; however, previously published screening methods for hair analysis did not include this compound in the same analysis along with other drugs of abuse and medicines [8–13,16]. Although Kronstrand et al. [17] analyzed 21 licit and illicit drugs, including THC in the same extraction procedure, two different detection techniques (THC by GC–MS and the rest of the compounds by LC–MS/MS) were employed. LC–MS hair screening methods including a larger number of compounds, even more than 800 analytes [16,18], have been previously published. However, these methods were only validated for analytes’ identification, but not for quantification purposes, and they did not include THC. In the present method, all analytes were extracted using the same procedure and analyzed in the same LC–MSMS run. If further confirmation was required, a second injection monitoring two transitions per compound was performed. Laloup et al. [11] also employed a first injection with the most prominent precursor-product transition for quantification, and confirmation was performed monitoring 2 transitions in a second injection; however, only benzodiazepines and hypnotics were determined in this method. Miller et al. [12] used 3 separate injections to determine cocaine, opiates and metabolites in the first injection, amphetamines in the second injection, and diazepam and metabolites in the third injection. Method’s LOQs were at least those recommended by the SoHT [1] for opiates (200 pg/mg), cocaine and its main metabolite, BE (500 pg/mg and 50 pg/mg, respectively), amphetamines (200 pg/mg) and THC (100 pg/mg). Ketamine (and its main metabolite) was determined by Harun et al. [19] with a LOD of 100 pg/mg and a LOQ of 180 pg/mg, both of them are higher values than those achieved in the present method. For the licit drugs, the limits are consistent with previous publications. Agius and Kintz [20] recommended a cut-off of 50 pg/mg in workplace for bromazepam, nordiazepam, oxazepam, lorazepam, alprazolam, diazepam and flunitrazepam, higher than those achieved in this method. For fentanyl, Kronstrand et al. [17] obtained a LOD of 3 pg/ mg and a LOQ of 8 pg/mg, similar to the limits of this work. Nielsen et al. [13] determined antidepressants, benzodiazepines and their metabolites, and hypnotics (zolpidem and zopiclone) with a LOD of 10 pg/mg and a LOQ of 50 pg/mg. Shen et al. [7] performed a GC– MS-CI method for the detection of antidepressants and antipsychotic with a LOD of 100 pg/mg and LOQ of 200 pg/mg. In all these studies, LODs and LOQs were higher than the limits obtained in the present work. Lower LODs (from 0.5 to 5 pg/mg) for 16 benzodiazepines and hypnotics were obtained in a LC–MSMS method developed by Villain et al. [21] to monitor single dose exposure. With regard to method validation, low extraction efficiency (<50%) was observed for 18 compounds (morphine, fentanyl, BE, A, THC, LSD, scopolamine, 7-AF, alprazolam, bromazepam, diazepam, triazolam, tetrazepam, zolpidem, zopiclone, citalopram, paroxetine and velafaxine). These extraction efficiencies were lower than those described in previously published methods [8–13,16,22]; however, good sensitivity was achieved, with LOQ ranging from 0.5 to 100 pg/mg depending on the compound. Although 2 extraction procedures were performed (LLE and SPE), 31 out of 35 analytes
showed matrix effect; 27 showed ion suppression, ranging from 24.6% to 86.2%, and 4 ion enhancement from 20.7% to 647.1%. In all cases % CV was below 20%, except for A, MA, 7-AF, methadone and THC, with values up to 47.6%. Nevertheless, the use of the corresponding deuterated analogs for these analytes as internal standard compensated for these effects. No significant losses were observed after 24 h storage in the autosampler and, therefore, positive specimens could be reinjected for confirmation purposes. Some analytes (scopolamine, alprazolam, 7-AF, clonazepam, nordiazepam, triazolam, tetrazepam, paroxetine and THC) showed 48 h autosampler losses up to 54.8% and, therefore, positive results of these analytes should be confirmed within 24 h to guarantee a good quantification. Seventeen hair specimens were analyzed to demonstrate method applicability. Several groups of drugs were identified and confirmed in the majority of these specimens, proving the polydrug use pattern and the usefulness of multianalyte procedures. The concentrations found were in accordance with previous published methods [8–12,19,21]. Most of the positive results were confirmed when 2 transitions were monitored in a second injection. A false positive was observed for bromazepam in one specimen. Although RT was correct, the qualifier transition monitored in the second injection was not detected and, therefore, the identification criteria could not be fulfilled. In conclusion, a selective and sensitive LC–MSMS method for the simultaneous identification and quantification of 35 licit and illicit drugs and metabolites in 50 mg of hair was developed and fully validated. The drugs analyzed were THC, opiates, cocaine, amphetamines, hallucinogens, benzodiazepines, antidepressants, and hypnotics. Confirmation of positive results was performed by reinjection of the sample, monitoring 2 transitions per compound. LOQ’s were at least those proposed by the SoHT for opiates, cocaine, amphetamines and THC. Acknowledgment We gratefully acknowledge the support by INCITE (Consellerı´a de Innovacio´n e Industria, Xunta de Galicia), project number INCITE08PXIB208090PR and INCITE09228166PR. References [1] Society of Hair Testing, Recommendations for hair testing in forensic cases, Forensic Sci. Int. 145 (2004) 83–84. [2] F. Pragst, M.A. Balikova, State of the art in hair analysis for detection of drug and alcohol abuse, Clin. Chim. Acta 370 (2006) 17–49. [3] Substance Abuse and Mental Health Services Administration (SAMHSA), The DASIS report, polydrug admissions: 2002, http://www.oas.samhsa.gov/2k5/polydrugTX/polydrugTX.pdf (Accessed May 2011). [4] United Nations, United Nations office on drugs and crime 2010, World Drug Report 2010, http://www.unodc.org/documents/wdr/WDR_2010/World_Drug_Report_2010_lo-res.pdf (Accessed May 2011). [5] R. Cordero, S. Paterson, Simultaneous quantification of opiates, amphetamines, cocaine and metabolites and diazepam and metabolite in a single hair sample using GC-MS, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 850 (2007) 423– 431. [6] G. Merola, S. Gentili, F. Tagliaro, T. Macchia, Determination of different recreational drugs in hair by HS-SPME and GC/MS, Anal. Bioanal. Chem. 397 (2010) 2987–2995. [7] M. Shen, P. Xiang, H. Wu, B. Shen, Z. Huang, Detection of antidepressant and antipsychotic drugs in human hair, Forensic Sci. Int. 126 (2002) 153–161. [8] F. Bucelli, A. Fratini, P. Bavazzano, N. Comodo, Quantification of drugs of abuse and some stimulants in hair samples by liquid chromatography-electrospray ionization ion trap mass spectrometry, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 877 (2009) 3931–3936. [9] S. Hegstad, H.Z. Khiabani, L. Kristoffersen, N. Kunoe, P.P. Lobmaier, A.S. Christophersen, Drug screening of hair by liquid chromatography-tandem mass spectrometry, J. Anal. Toxicol. 32 (2008) 364–372. [10] R. Kronstrand, I. Nystrom, J. Strandberg, H. Druid, Screening for drugs of abuse in hair with ion spray LC-MS-MS, Forensic Sci. Int. 145 (2004) 183–190. [11] M. Laloup, M. Ramirez Fernandez Mdel, G. De Boeck, M. Wood, V. Maes, N. Samyn, Validation of a liquid chromatography-tandem mass spectrometry method for
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