Forensic Science International 217 (2012) 207–215

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Forensic Science International

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Target screening and confirmation of 35 licit and illicit drugs and metabolites
in hair by LC–MSMS
Elena Lendoiro a, Óscar Quintela b, Ana de Castro a,c, Angelines Cruz a, Manuel López-Rivadulla a,
Marta Concheiro a,*
a
Servicio de Toxicologı´a Forense, Instituto de Ciencias Forenses, Universidad de Santiago de Compostela, Spain
b
Instituto Nacional de Toxicologı´a y Ciencias Forenses, Madrid, Spain
c
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: A liquid chromatography–tandem mass spectrometry (LC–MSMS) target screening in 50 mg hair was
Received 21 July 2011 developed and fully validated for 35 analytes (D9-tetrahidrocannabinol (THC), morphine, 6-
Received in revised form 11 October 2011 acetylmorphine, codeine, methadone, fentanyl, amphetamine, methamphetamine, 3,4-methylenediox-
Accepted 5 November 2011
yamphetamine, 3,4-methylenedioxymethamphetamine, benzoylecgonine, cocaine, lysergic acid diethy-
Available online 30 November 2011
lamide, ketamine, scopolamine, alprazolam, bromazepam, clonazepam, diazepam, flunitrazepam, 7-
aminoflunitrazepam, lorazepam, lormetazepam, nordiazepam, oxazepam, tetrazepam, triazolam,
Keywords:
zolpidem, zopiclone, amitriptyline, citalopram, clomipramine, fluoxetine, paroxetine and venlafaxine).
Hair
Hair decontamination was performed with dichloromethane, and incubation in 2 mL of acetonitrile at
Screening
LC–MSMS 50 8C overnight. Extraction procedure was performed in 2 steps, first liquid–liquid extraction,
THC 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.

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
Toxicologı́a Forense, Instituto de Ciencias Forenses, Universidad de Santiago de
Compostela, C/San Francisco s/n, 15782 Santiago de Compostela (A Coruña), Spain.
Tel.: +34 881812446; fax: +34 981580336.
E-mail addresses: marta.concheiro@gmail.com, marta.concheiro@usc.es
(M. Concheiro).
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 physico-
chemical properties (lipophilicity, melanine affinity) [2]. Some hair
analysis disadvantages are external contamination (particularly
important for smoked drugs), low concentrations of some com-
pounds and metabolites, and limited amount of sample supplied for
testing. Hair analysis of illicit drugs and medicines (benzodiaze-
pines, 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, post-
mortem 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

0379-0738/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.forsciint.2011.11.006
208 E. Lendoiro et al. / Forensic Science International 217 (2012) 207–215

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, multi-
analyte 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 non-
volatile compounds [2]; whereas, in LC–MS methods derivatiza-
tion 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-9-
tetrahidrocannabinol (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, anti-
depressants 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 recommenda-
tions 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-monoacetylmor-
phine (6-AM), cocaine, lysergic acid diethylamide (LSD), 7-aminoflunitrazepam (7-
AF), 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, MA-
d5, 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, 2-
propanol, 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-sur-
Lausanne, 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) Retention time (min)

Morphine 286.0 > 201.2 40 25 2.0

286.0 > 229.2 40 25
Morphine-d6 292.3 > 201.5 40 24 2.0
Codeine 300.1 > 215.2 40 25 4.2
300.1 > 243.2 40 25
A 136.0 > 90.6 20 15 5.0
136.0 > 119.0 20 9
A-d5 141.2 > 124.1 15 9 4.9
MDA 180.0 > 163.0 18 10 5.8
180.0 > 104.8 18 20
Scopolamine 304.4 > 138.2 34 24 5.6
304.4 > 103.0 34 41
MA 150.0 > 90.6 24 17 5.9
150.0 > 119.0 24 11
MA-d5 155.1 > 120.9 22 11 5.8
6-AM 328.0 > 165.1 45 40 6.1
328.0 > 211.1 45 25
6-AM-d6 334.5 > 165.3 34 35 6.1
MDMA 194.0 > 163.0 20 12 6.5
194.0 > 104.8 20 25
MDMA-d5 199.1 > 165.0 22 12 6.4
Ketamine 238.3 > 125.0 25 25 8.2
238.3 > 179.3 25 17
Ketamine-d4 242.4 > 129.1 24 28 8.1
 E. Lendoiro et al. / Forensic Science International 217 (2012) 207–215 209

Table 1 (Continued )

Compound MRM Cone voltage (V) Collision energy (eV) Retention time (min)

BE 290.1 > 168.2 30 18 8.5

290.1 > 104.9 30 30
BE-d3 293.2 > 171.2 30 20 8.5
Zopiclone 389.2 > 245.2 20 13 10.0
389.2 > 217.2 20 37
Zopiclone-d4 393.4 > 245.2 20 13 10.0
7-AF 284.4 > 135.1 40 27 10.7
284.4 > 227.2 40 27
Cocaine 304.2 > 182.2 30 20 10.8
304.2 > 81.8 30 30
Cocaine-d3 307.1 > 185.2 30 20 10.8
Zolpidem 308.3 > 235.4 45 41 11.2
308.3 > 92.0 45 53
LSD 324.1 > 223.2 35 25 11.4
324.1 > 281.2 35 20
LSD-d3 327.2 > 226.3 35 25 11.4
Venlafaxine 278.1 > 57.8 25 18 11.5
278.1 > 260.3 25 12
Fentanyl 337.2 > 188.3 30 22 13.1
337.2 > 105.0 30 36
Fentanyl-d5 342.2 > 188.3 30 24 13.0
Bromazepam 316.2 > 182.3 38 30 13.7
316.2 > 209.3 38 23
Citalopram 325.1 > 109.1 35 26 14.1
325.1 > 260.1 35 20
Paroxetine 330.0 > 69.7 35 30 16.3
330.0 > 192.3 35 20
Paroxetine-d6 336.1 > 76.0 35 30 16.2
Amitriptyline 278.1 > 90.8 30 24 18.8
278.1 > 233.3 30 18
Oxazepam 287.2 > 103.9 25 39 18.9
287.2 > 76.9 25 59
Oxazepam-d5 292.2 > 246.23 30 21 18.7
Methadone 310.2 > 265.3 20 15 19.2
310.2 > 104.9 20 25
Methadone-d3 313.3 > 268.4 31 15 19.1
Lorazepam 321.1 > 275.2 25 21 20.4
321.1 > 303.2 25 17
Alprazolam 309.2 > 281.2 40 27 20.4
309.2 > 205.3 40 41
Alprazolam-d5 314.2 > 286.3 40 27 20.3
Fluoxetine 310.1 > 43.7 22 12 20.7
310.1 > 148.2 22 8
Fluoxetine-d6 316.1 > 43.7 25 12 20.6
Clonazepam 316.1 > 270.2 40 35 20.8
316.1 > 214.3 40 25
Nordiazepam 271.2 > 140.1 30 29 21.1
271.2 > 165.1 30 29
Triazolam 343.1 > 308.3 25 29 21.1
343.1 > 315.2 25 31
Clomipramine 315.0 > 85.9 22 20 21.5
315.0 > 57.8 22 32
Flunitrazepam 314.1 > 268.3 40 27 22.0
314.1 > 239.3 40 37
Flunitrazepam-d7 321.2 > 275.4 35 27 21.8
Tetrazepam 289.3 > 225.5 48 30 22.1
289.3 > 197.4 48 33
Lormetazepam 335.1 > 289.3 25 15 22.7
335.1 > 317.2 25 15
Diazepam 285.2 > 154.2 20 31 23.1
285.2 > 193.3 20 31
Diazepam-d5 290.2 > 154.2 40 27 23.0
THC 315.2 > 193.2 30 25 26.8
315.2 > 135.1 30 25
THC-d3 318.2 > 196.2 35 25 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, flunitra-
zepam, lormetazepam and diazepam; 10–10,000 pg/mg for zopiclone, venlafaxine,
amitriptyline, triazolam, fluoxetine and alprazolam; 20–20,000 pg/mg for mor-
phine, 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
210 E. Lendoiro et al. / Forensic Science International 217 (2012) 207–215

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 diaze-
pam), 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, flunitra-
zepam, lormetazepam and diazepam), 1500 (for zopiclone, venlafaxine, amitripty-
line, 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:2-
propanol (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, extrac-
tion 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 chromatogra-
phy, 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 (<LOD), the method was considered
selective.
Imprecision and analytical recovery were determined at low, medium and high
QC levels, with 5 replicates on 4 different days (n = 20). Imprecision, expressed as
coefficient of variation (CV) of the measured values, was expected to be less than
15%. Krouwer and Rabinowitz guidelines [14] were followed for calculation of
pooled intra-day, inter-day and total imprecision. The analytical recovery was
evaluated as the percentage of the target concentration (n = 20), with an acceptance
criterion of 85–115%.
Extraction efficiency, process efficiency and matrix effect were determined at 2
concentration levels (LOQ and 10LOQ). Extraction efficiency was calculated

Table 2
Limit of detection (LOD), calibration range and linearity results for the 35 compounds analyzed.

Compound LOD (pg/mg) Calibration range (pg/mg) Intercept  SD (n = 4) Slope  SD (n = 4) r2  SD (n = 4)

Morphine 5 20–20,000 0.0853  0.0331 0.0159  7.6E 4 0.9949  0.0021

Codeine 2 20–20,000 0.2006  0.1872 0.0091  7.9E 4 0.9894  0.0041
A 2 20–20,000 0.0261  0.0295 0.0014  2.3E 4 0.9907  0.0021
MDA 2 20–20,000 0.0210  0.0197 0.0010  8.9E 5 0.9885  0.0034
Scopolamine 2 5–2000 0.0002  0.0004 0.0007  6.9E 5 0.9912  0.0036
MA 2 20–20,000 0.0226  0.0228 0.0023  1.5E 4 0.9943  0.0022
6-AM 2 20–20,000 0.0438  0.0139 0.0119  8.6E 4 0.9954  0.0012
MDMA 2 20–20,000 0.0199  0.0051 0.0024  1.0E 4 0.9980  0.0015
Ketamine 2 5–2000 0.0115  0.0043 0.0073  3.4E 4 0.9891  0.0039
BE 10 20–20,000 0.0428  0.0202 0.0021  4.6E 5 0.9937  0.0031
Zopiclone 5 10–10,000 0.0137  0.0012 0.0038  1.5E 4 0.9885  0.0013
7-AF 2 10–5000 0.0012  0.0012 0.0007  1.4E 4 0.9955  0.0032
Cocaine 2 20–20,000 0.0123  0.0057 0.0017  4.6E 5 0.9948  0.0025
Zolpidem 2 5–5000 0.0003  0.0003 0.0019  2.0E 4 0.9901  0.0031
LSD 0.2 0.5–2000 0.0074  0.0008 0.0112  2.5E 4 0.9983  0.0012
Venlafaxine 2 10–10,000 0.0003  0.0003 0.0004  7.7E 5 0.9896  0.0033
Fentanyl 2 5–5000 0.0037  0.0009 0.0049  1.2E 4 0.9907  0.0018
Bromazepam 5 20–20,000 0.0001  0.0002 2.3E 5  4.7E 6 0.9867  0.0045
Citalopram 2 20–10,000 0.0061  0.0034 0.0007  8.3E 5 0.9868  0.0010
Paroxetine 2 5–5000 0.0051  0.0043 0.0047  1.6E 4 0.9892  0.0027
Amitriptyline 5 10–5000 0.0035  0.0037 0.0016  2.3E 4 0.9866  0.0093
Oxazepam 10 20–20,000 0.0076  0.0039 0.0011  6.4E 5 0.9898  0.0024
Methadone 2 20–20,000 0.0036  0.0016 0.0008  4.0E 5 0.9941  0.0024
Lorazepam 2 5–5000 0.0001  0.0001 0.0001  1.8E 5 0.9877  0.0029
Alprazolam 5 10–10,000 0.0038  0.0023 0.0009  5.2E 5 0.9935  0.0028
Fluoxetine 2 10–10,000 0.0094  0.0074 0.0009  4.0E 5 0.9872  0.0021
Clonazepam 10 20–10,000 2.0E 5  0.0001 1.1E 5  1.0E 6 0.9890  0.0018
Nordiazepam 2 5–5000 0.0049  0.0020 0.0036  5.6E 4 0.9901  0.0050
Triazolam 5 10–10,000 0.0031  0.0012 0.0007  1.8E 4 0.9886  0.0025
Clomipramine 2 5–2000 0.0896  0.0316 0.0491  5.2E 3 0.9850  0.0030
Flunitrazepam 2 5–5000 0.0076  0.0021 0.0057  2.5E 4 0.9863  0.0042
Tetrazepam 10 20–10,000 0.0021  0.0010 0.0003  3.9E 5 0.9898  0.0041
Lormetazepam 2 5–5000 0.0205  0.0123 0.0052  3.4E 4 0.9973  0.0018
Diazepam 2 5–5000 0.0065  0.0026 0.0030  1.0E 4 0.9922  0.0015
THC 50 100–20,000 0.0398  0.0060 0.0005  1.5E 5 0.9976  0.0011

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) 207–215 211

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,
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, tria-
zolam, 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 Pooled intra-day imprecision Inter-day imprecision Total imprecision Analytical recovery
(n = 20, CV) (n = 20, CV) (n = 20, CV) (n = 20, % target)

Low Med High Low Med High Low Med High Low Med High

Morphine 7.6 3.7 4.0 6.3 2.7 4.9 9.8 4.6 6.3 94.4 105.2 100.7
Codeine 6.1 3.9 3.5 6.6 6.2 6.2 9.0 7.3 7.1 86.0 102.2 96.4
A 5.6 4.5 2.7 11.8 9.1 5.5 13.1 10.2 6.1 98.3 105.6 99.7
MDA 14.6 2.6 4.0 15.1 2.3 7.1 21.0 3.5 8.1 98.7 109.2 97.6
Scopolamine 7.8 6.8 5.5 5.6 7.1 6.0 9.6 9.8 8.2 104.3 94.2 94.0
MA 6.5 1.4 1.1 13.9 5.6 3.7 15.3 5.8 3.9 101.2 107.0 101.7
6-AM 6.0 2.6 2.3 4.0 2.6 5.1 7.2 3.6 5.6 95.6 100.1 98.4
MDMA 15.9 2.2 1.7 6.8 0 2.2 17.3 0 2.7 91.1 106.9 102.6
Ketamine 5.3 2.1 1.7 9.2 2.4 1.7 10.6 3.2 2.4 90.9 98.2 87.2
BE 11.9 15.7 9.1 10.2 0 0 15.7 0 0 98.6 102.8 98.8
Zopiclone 8.2 2.7 2.3 3.3 2.3 2.6 8.8 3.5 3.5 92.5 98.1 94.0
7-AF 7.8 9.6 23.5 6.7 10.3 21.2 10.3 14.1 31.6 108.0 83.6 72.8
Cocaine 4.6 2.5 3.8 8.6 0 0 9.8 0 0 104.5 108.2 103.6
Zolpidem 7.7 2.5 3.0 4.0 2.9 1.4 8.7 3.8 3.3 95.1 88.8 87.9
LSD 19.2 3.3 2.1 0 3.7 2.9 0 5.0 3.6 104.2 92.7 102.2
Venlafaxine 12.8 7.7 5.3 8.5 3.2 6.6 15.4 8.4 8.4 97.4 91.4 102.2
Fentanyl 7.9 2.2 1.6 0 1.8 0.4 0 2.9 1.7 98.3 97.4 89.4
Bromazepam 21.2 7.4 8.4 0 7.8 6.5 0 10.8 10.6 111.3 96.5 100.5
Citalopram 16.0 5.2 7.1 0 5.4 5.8 0 7.5 9.2 104.1 107.8 96.7
Paroxetine 25.0 1.9 5.2 7.4 0.7 2.0 26.1 2.0 5.6 107.7 89.9 89.9
Amitriptyline 15.0 5.3 7.0 6.5 1.5 9.6 16.3 5.5 11.9 109.6 105.5 98.9
Oxazepam 11.1 3.2 2.0 0 1.6 3.1 0 3.6 3.7 96.8 109.0 101.8
Methadone 4.7 4.7 5.8 8.7 2.7 0 9.9 5.4 0 105.7 102.1 104.7
Lorazepam 6.3 8.4 10.6 3.0 2.9 7.7 7.0 8.9 13.1 109.9 93.9 90.6
Alprazolam 10.6 7.2 6.1 0 4.1 2.3 0 8.2 6.5 101.2 102.3 99.8
Fluoxetine 15.6 10.6 4.6 3.0 2.9 6.2 15.9 11.0 7.8 98.5 100.2 104.0
Clonazepam 6.3 9.6 7.9 7.4 5.3 9.5 9.7 11.0 12.3 111.9 97.7 95.3
Nordiazepam 16.8 6.8 10.3 5.4 0 0 17.7 0 0 106.6 106.6 95.4
Triazolam 7.6 9.5 7.5 6.7 0 0 10.1 0 0 94.8 102.6 96.2
Clomipramine 10.6 6.5 6.8 3.0 4.0 0 11.0 7.6 0 94.2 100.0 88.1
Flunitrazepam 8.8 3.0 3.2 8.6 6.3 9.9 12.3 6.9 10.4 94.5 103.0 95.4
Tetrazepam 7.9 4.1 5.1 1.1 3.0 8.2 8.0 5.1 9.6 103.9 109.8 101.3
Lormetazepam 9.8 6.6 5.8 9.2 6.9 4.6 13.5 9.6 7.4 95.3 100.6 100.9
Diazepam 16.5 2.3 5.6 0 2.7 1.9 0 3.5 5.9 106.4 95.8 91.5
THC 9.6 6.4 5.5 0 0 0.6 0 0 5.5 91.5 102.1 103.1

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).
212 E. Lendoiro et al. / Forensic Science International 217 (2012) 207–215

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.

Analyte EF % PE % ME % (CV, %)

LOQ 10LOQ LOQ 10LOQ LOQ 10LOQ

Morphine 8.5 10.4 5.7 5.8 32.9 (7.6) 44.2 (7.2)

Codeine 44.5 51.9 33.6 29.7 24.6 (10.0) 42.7 (7.8)
A 41.0 42.4 61.8 51.2 50.6 (21.3) 20.7 (8.9)
MDA 54.7 51.0 46.8 41.4 14.5 (12.4) 18.8 (9.5)
Scopolamine 31.7 39.8 28.0 26.8 11.6 (8.0) 32.7 (7.3)
MA 63.0 62.4 80.9 77.3 54.6 (27.4) 19.0 (11.5)
6-AM 62.1 74.0 37.3 37.3 39.9 (14.5) 49.6 (15.4)
MDMA 69.2 58.7 60.6 51.6 7.4 (18.0) 12.0 (10.8)
Ketamine 57.9 60.1 60.4 55.8 4.5 (8.8) 7.1 (11.8)
BE 4.6 4.1 4.5 3.2 1.8 (6.2) 22.0 (7.9)
Zopiclone 28.1 29.8 18.1 14.0 35.6 (14.4) 53.1 (13.4)
7-AF 10.1 7.4 3.7 2.5 63.5 (34.5) 66.8 (19.7)
Cocaine 85.6 50.9 38.0 27.8 33.6 (16.6) 43.0 (11.6)
Zolpidem 36.5 34.1 22.2 16.5 40.6 (19.4) 46.0 (11.6)
LSD 40.8 27.9 12.7 8.7 67.3 (13.1) 64.4 (14.2)
Venlafaxine 45.9 49.5 27.0 21.8 41.3 (14.3) 53.8 (14.3)
Fentanyl 39.5 35.9 14.4 11.7 61.8 (18.6) 58.4 (11.9)
Bromazepam 35.6 33.6 103.9 149.5 192.3 (19.3) 372.6 (14.0)
Citalopram 33.2 36.3 12.7 13.1 61.7 (18.0) 55.8 (9.9)
Paroxetine 38.9 49.6 12.0 10.4 69.1 (19.5) 76.4 (15.3)
Amitriptyline 31.9 61.7 19.8 17.2 55.9 (14.0) 67.6 (13.3)
Oxazepam 59.0 83.7 43.1 36.5 27.0 (10.9) 56.5 (7.4)
Methadone 70.8 49.2 16.5 25.6 53.7 (21.7) 58.9 (8.5)
Lorazepam 65.5 79.3 68.1 54.1 29.8 (15.9) 38.5 (10.6)
Alprazolam 26.4 28.8 19.2 13.5 20.1 (19.5) 47.5 (11.8)
Fluoxetine 39.9 52.0 17.2 17.3 56.8 (19.9) 63.4 (10.7)
Clonazepam 52.9 67.2 27.2 21.1 48.6 (20.4) 68.5 (16.3)
Nordiazepam 51.4 62.3 11.6 8.6 77.5 (19.5) 86.2 (20.6)
Triazolam 28.4 32.2 19.1 14.0 35.2 (20.3) 51.0 (8.0)
Clomipramine 39.3 64.9 9.2 10.3 76.5 (18.2) 82.2 (14.0)
Flunitrazepam 48.1 65.6 25.6 27.1 46.9 (19.6) 57.4 (13.6)
Tetrazepam 31.6 35.1 9.4 8.1 68.5 (17.6) 75.1 (16.5)
Lormetazepam 54.5 62.6 30.6 22.0 43.9 (18.4) 63.8 (12.9)
Diazepam 41.2 44.5 13.3 9.8 55.1 (13.0) 80.2 (15.4)
THC 26.8 27.8 123.3 207.7 483.6 (18.2) 647.1 (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 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13

Morphine 3869.6
Codeine 881.4 18.3 17.6
6-AM 18265.1 121.6 51.2 279.5
Methadone 44.0 206.3 604.0 4436.0 514.3
Fentanyl 7.7 416.0
Cocaine 535.0 88.1 307.9 >20,000 467.9 117.4 424.0 6103.5 137.0 >20,000
BE 509.0 85.4 326.0 19443.6 467.5 120.7 432.7 6101.0 134.6 >20,000
A 533.9 106.8 107.0
MDMA 95.5 31.3
THC 143.2
Ketamine 612.1
Alprazolam 170.9 59.2
Bromazepam 47.1 23.3
Diazepam 17.0 7.9 42.6 33.5
Flunitrazepam 8.7
Lorazepam 39.4
Nordiazepam 53.2 9.9 70.3
Tetrazepam 124.1
Zolpidem 113.2 12.8
Zopiclone 32.0
Amitriptiline 39.6
Citalopram 5027.7 6534.0 >10,000 >10,000
Fluoxetine 85.8
Paroxetine 95.2 162.6

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) 207–215 213

zepam and triazolam, which % loss was up to 54.8% at low, medium both substances were detected in hair. Positive results are shown
and high QC concentrations; paroxetine, alprazolam, tetrazepam in Table 5. Confirmation was performed by sample reinjection,
and THC at low concentrations (% loss < 35.2%); and scopolamine monitoring 2 transitions per compound. All results were con-
and clonazepam at medium and high concentrations (% firmed, except one false positive to bromazepam. The three washes
loss < 29.1%). of each specimen were analyzed and total removal of external
contamination was confirmed in all cases. Fig. 1 shows a
3.2. Real cases analysis chromatogram of a real specimen positive to A, MDMA, BE,
cocaine, ketamine and THC.
Seventeen hair specimens from forensic cases were analyzed as
proof of the method. Most of the cases were from patients 4. Discussion
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). A target screening method in hair for simultaneous identifica-
Specimens 14, 15, 16 and 17 were negative, and the other tion and quantification of drugs of abuse (THC, opiates, amphe-
specimens showed low concentrations of drugs of abuse. Positive tamines, cocaine, LSD, ketamine, and scopolamine) and medicines
results to benzodiazepines and antidepressants corresponded to (benzodiazepines, antidepressants, and hypnotics), all of them
medical prescriptions. Specimens 1, 5 and 12 were from frequently present in forensic cases, was developed by LC–MSMS
individuals suspected to consume drugs of abuse, and specimen and fully validated. One transition per compound was monitored
13 was a medical case, where drug monitoring was requested. In in MRM mode. In order to confirm positive results, a second
this case, the patient was prescribed citalopram and fentanyl, and injection monitoring 2 transitions per compound was performed.

100916_19 Smooth(Mn,1x2) F2:MRM of 3 channels,ES+ 100916_19 Smooth(Mn,1x2) F3:MRM of 8 channels,ES+

Pelo 215_2010_segmento 2 136 > 90.6 Pelo 215_2010_segmento 2 194 > 163
Anfetamina;4.69;29293.2;135734 1.373e+005 MDMA;6.26;28645.4;146426 1.472e+005
100 100

% %

0 min 0 min

100916_19 Smooth(Mn,1x2) F2:MRM of 3 channels,ES+ 100916_19 Smooth(Mn,1x2) F3:MRM of 8 channels,ES+

Pelo 215_2010_segmento 2 141.2 > 124.1 Pelo 215_2010_segmento 2 199.1 > 165
Anfetamina-d5;4.65;171621.8;824972 8.298e+005 MDMA-d5;6.21;966923.1;4897422 4.908e+006
100 100

% %

0 min 0 min
3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00

100916_19 Smooth(Mn,1x2) F4:MRM of 6 channels,ES+ 100916_19 Smooth(Mn,1x2) F5:MRM of 6 channels,ES+

Pelo 215_2010_segmento 2 290.1 > 168.2 Pelo 215_2010_segmento 2 304.2 > 182.2
BE;8.24;4275.2;21657 2.184e+004 Cocaina 9.275e+005
100 100
10.71
126851.9
925952

% %

0 min 0 min

100916_19 Smooth(Mn,1x2) F4:MRM of 6 channels,ES+ 100916_19 Smooth(Mn,2x2) F5:MRM of 6 channels,ES+

Pelo 215_2010_segmento 2 293.2 > 171.2 Pelo 215_2010_segmento 2 307.1 > 185.2
BE-d3;8.24;37633.7;188497 1.900e+005 Cocaina-d3 9.083e+006
100 100
10.71
1341157.3
9069586

% %

0 min 0 min
7.50 8.00 8.50 9.00 9.50 10.00 10.25 10.50 10.75 11.00 11.25 11.50 11.75 12.00 12.25 12.50

100916_19 Smooth(Mn,1x2) F9:MRM of 3 channels,ES+

100916_19 Smooth(Mn,1x2) F4:MRM of 6 channels,ES+
Pelo 215_2010_segmento 2 315.2 > 193.2
Pelo 215_2010_segmento 2 238.3 > 125
THC 9.412e+002
Ketamina;7.94;59364.2;297722 2.988e+005 100
100 26.80
111.4
704
25.38 25.90
25.03
%
%
27.11 27.39 27.70 28.84
28.03

0 min
0 min

100916_19 Smooth(Mn,1x2) F9:MRM of 3 channels,ES+

100916_19 Smooth(Mn,1x2) F4:MRM of 6 channels,ES+
Pelo 215_2010_segmento 2 318.2 > 196.2
Pelo 215_2010_segmento 2 242.4 > 129.1
THC-d3 5.131e+003
Ketamina-d4 1.079e+006 100
100 26.78
7.83
661.1
221741.3
4959
1076957

%
%

0 min
0 min
25.50 26.00 26.50 27.00 27.50 28.00 28.50
7.50 8.00 8.50 9.00 9.50 10.00

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).
214 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 validat-
ed 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 per-
formed. 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 antipsy-
chotic 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, paroxe-
tine 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 proce-
dures. 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 (Consellerı́a
de Innovación e Industria, Xunta de Galicia), project number
INCITE08PXIB208090PR and INCITE09228166PR.
References
[1] Society of Hair Testing, Recommendations for hair testing in forensic cases,
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