Journal of Analytical Toxicology,

2017;41:376–381 doi:
10.1093/jat/bkx020 Advance
Access Publication Date: 4
March 2017 Article

Article

Hair Testing for Drugs of Abuse and New Psychoactive Substances

in a High-Risk Population

Alberto Salomone1,*, Joseph J. Palamar2,3, Enrico Gerace1,

Daniele Di Corcia1, and Marco Vincenti1,4

1Centro Regionale Antidoping e di Tossicologia “A. Bertinaria”, Orbassano, Turin, Italy, 2Department of Population Health, New
York University Langone Medical Center, New York, USA, 3Center for Drug Use and
HIV Research, New York University Rory Meyers College of Nursing, New York, NY,
USA, and 4Dipartimento di Chimica, Università di Torino, Turin, Italy

*Author to whom correspondence should be addressed. Alberto Salomone, Centro

Regionale Antidoping “A. Bertinaria”, Regione Gonzole 10/1, 10043 Orbassano,
Torino, Italy. Email: [email protected]

Abstra
ct

Hundreds of new psychoactive substances (NPS) have emerged in the drug market
over the last decade. Few drug surveys in the USA, however, ask about use of NPS,
so prevalence and corre- lates of use are largely unknown. A large portion of NPS
use is unintentional or unknown as NPS are common adulterants in drugs like
ecstasy/Molly, and most NPS are rapidly eliminated from the body, limiting
efficacy of urine, blood and saliva testing. We utilized a novel method of exam- ining
prevalence of NPS use in a high-risk population utilizing hair-testing. Hair samples
from high-risk nightclub and dance music attendees were tested for 82 drugs and
metabolites (includ- ing NPS) using ultra-high performance liquid chromatography–
tandem mass spectrometry. Eighty samples collected from different parts of the body
were analyzed, 57 of which detected positive for at least one substance—either a
traditional or new drug. Among these, 26 samples tested posi- tive for at least one
NPS—the most commo`` n being butylone (25 samples). Other new drugs detected
include methylone, methoxetamine, 5/6-APB, α-PVP and 4-FA. Hair analysis proved
a powerful tool to gain objective biological drug-prevalence information, free from
possible biases of unintentional or unknown intake and untruthful reporting of use.
Such testing can be used actively or retrospectively to validate survey responses and
inform research on consumption pat- terns, including intentional and unknown use,
polydrug-use, occasional NPS intake and frequent or heavy use.

Introduction

Hundreds of new psychoactive substances (NPS) have emerged in the drug market in the last decade, taking
advantage of the delay occurring between their introduction into the market and their legal ban. NPS tend to
mimic the psychotropic effects of traditional drugs of abuse, but their acute and chronic toxicity, and side-effects
are largely unknown. This has led to funneling of resources worldwide to outline the phenomenon, identify the
molecules, describe effects,
interpret or update existing laws, enact new regulations, and develop appropriate and effective analytical
methods for their identi- fication in biological fluids and seized materials (1–9).
Unfortunately, little is known about the current diffusion of NPS among the general population or in
high-risk populations. Few sur- veys query NPS use and there is increasing evidence that much NPS use is
actually unknown or unintentional as they are common adul- terants in drugs like ecstasy/MDMA/Molly (10–
13). Most NPS are

also eliminated from the urine, blood and saliva of users within hours or days, which limits the ability of
toxicological confirmation. This is a serious concern, especially in cases of hospitalizations and deaths
resulting from intentional or unintentional use. A further challenge to detection of NPS in the biological
matrices commonly tested, especially urine, is posed by the extensive, yet insufficiently investigated, metabolic
transformation that these substances pos- sibly undergo once introduced in the human body. In practice, most
routine analyses do not presently include screening procedures for NPS, preventing clear knowledge of the
consumption of these new drugs in the population. As a consequence, a number of unresolved issues are raised,
including the number and variety of NPS present in different countries, frequency of their use and the social
features of users.

To circumvent the limitations of urine, blood and saliva testing, the detection of NPS in hair
samples was recently proposed as a practical means to provide preliminary information on the black
market penetration of NPS in specific territories and populations (14–18). The keratin matrix
incorporates the parent NPS consumed over extended time periods, providing access to a much wider
diag- nostic window than urine. This feature, combined with the analytical performances of the last
generation ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS-
MS) instruments (8), allows researchers to obtain significant information about past use, even a single
intake, of any targeted NPS, with older periods of use corresponding to the hair segments more distant
from the hair root (19, 20).

In the present study, we considered the specific population of electronic dance music (EDM) nightclubs
and festivals attendees in New York City (NYC), as a high-risk population particularly exposed to both
intentional and unintentional NPS intake (21, 22). Information on NPS prevalence in the US via biological
confirm- ation tends to be limited to drug confiscations (23, 24), but we tested hair analysis as a potential tool to
gain objective biological drug-prevalence information in the context of an epidemiology study, free from
possible biases of unintentional or unknown intake and untruthful reporting of use. Inclusion of both traditional
drugs and NPS in the screening allows us to distinguish different consump- tion patterns, including co-use,
occasional NPS intake and frequent or heavy use. To our knowledge, this is the first large epidemiology study
to examine biological hair results of NPS use in this high-risk scene in the USA. We believe these biological
results with retrospect- ive capability complement both surveys and analysis of biological fluids conducted in
EDM festivals population and can help encour- age future testing and inform prevention.

Experimental

Study protocol Six hundred and seventy-nine nightclub and festival attendees in NYC were surveyed
from July through September of 2015. Participants were surveyed outside of 10 different venues
(including two dance festivals) over 21 days. Participants were eligible if they identified as age 18–25 and
were about to attend the randomly selected party (21). After providing informed consent and taking a
survey, participants were asked if they were willing to donate a hair sample to be tested for “new drugs”
such as “bath salts”. If the par- ticipant agreed, the trained recruiter collected the sample by cutting a
small lock of hair (~100 hairs) from as close to the participant’s scalp as possible. Hair was cut with a
clean scissor (wiped with an
alcohol-wipe after each use), folded up in a piece of tin foil, and stored in a small envelope labeled with
the participant’s anonymous study ID number. In some cases, male participants volunteered to have the
recruiter to clip or buzz (with an electronic buzzer) body hair from the arm, chest or leg. Of the 679
participants surveyed, 80 (11.8%) provided a hair sample compatible with the quantity needed for the
analysis. This study was approved by the New York University Langone Medical Center Institutional
Review Board.
Hair analyses Hair samples were analyzed in their full length. The average length, calculated for the 80
analyzed samples, was 9.9 cm (median 9.0 cm). Assuming normal hair growth rate (25), the corresponding mean
time frame is ~1 cm = 1 month. A minimum quantity of 20 mg was needed to perform the analysis. The
collected specimens were tested using three previously published and validated methods using UHPLC–
MS-MS. One (26) was used to screen each sample for 11 common drugs of abuse or metabolites: morphine, 6-
acetylmorphine, codeine, amphetamine (AMP), methamphetamine, 3,4-methylene- dioxyamphetamine (MDA),
3,4-methylenedioxymethylamphetamine (MDMA), 3,4-methylenedioxyethylamphetamine (MDEA), cocaine,
benzoylecgonine and Δ9-tetrahydrocannabinol (THC). The second method (27) was addressed to detect the most
expected and com- mon NPS (26 substances)—namely stimulants (primarily synthetic cathinones) and
psychedelic substituted phenethylamines: mephe- drone, 3-methylmethcathinone, 4-methylethcathinone,
methylone, 4- fluoroamphetamine (4-FA), 3,4-methylenedioxypyrovalerone (MDPV), pentedrone,
ethcathinone, alpha-pyrrolidinovalerophenone (α-PVP; a.k.a.: “Flakka”), butylone, buphedrone, 25I-NBOMe,
25C- NBOMe, 25H-NBOMe, 25B-NBOMe, 2C-P, 2C-B, 1-(benzofur- an-5-yl)-N-methylpropan-2-amine (5-
MAPB), 5-(2-aminopropyl) benzofuran (5-APB)/6-(2-aminopropyl)benzofuran (6-APB), para-
methoxymethamphetamine, para-methoxyamphetamine, amfepra- mone, meta-chlorophenylpiperazine
(mCPP), and bupropione, plus five dissociative drugs, namely methoxetamine (MXE), phencyclid- ine (PCP),
4-methoxyphencyclidine (4-MeO-PCP), diphenidine and ketamine. The new designer drug mCPP can be
detected in bio- logical fluids as a metabolite of trazodone (28, 29); therefore, trazo- done was also included in
our method in order to discriminate between direct mCPP intake and biotransformation of trazodone. The limits
of detection of the analytical methods (26, 27) were set as the minimum criterion to identify the positive samples.
Hair samples were also screened for synthetic cannabinoids. Our existing method for synthetic cannabinoids
(15) was updated by introducing the fol- lowing compounds: MAM-2201, UR-144, XLR-11, AKB-48, STS-
135, PB-22, AB-PINACA, 5F-AB-PINACA, ADB-PINACA, AB- FUBINACA, ADBICA, JWH-302, AM-
2233, CB-13, JWH-016, JWH-098 and JWH-147. The total number of targeted analytes in the latter method
was 40. Altogether, each sample was screened for 82 drugs and metabolites.

Results

Eighty samples collected from different parts of the body were ana- lyzed, 57 of which detected positive for at
least one substance—either a traditional or new drug. Among these, 36 samples were head hair, 3 were leg hair
and in 18 cases the sampling site was not recorded. Among positive specimens, 40.4% (n = 23) were provided
by female participants. Donors mainly identified as White (n = 35, 61.4%), while other ethnic groups included
Hispanic (n = 12, 21.0%), Asian (n = 4,

377 Hair Testing for Drugs of Abuse and New Psychoactive Substances
7.0%) or other/mixed (n = 6, 10.6%). Among positive samples (n = 57), 31 tested positive for the exclusive
presence of traditional drugs, 2 were positive for NPS only, while in the remaining 24 cases of trad- itional drugs
were also positive for at least one NPS. No sample tested positive for synthetic cannabinoids. Among traditional
drugs, THC was detected in 35 samples (range 0.02−1.92 ng/mg, mean = 0.34 ng/ mg, median = 0.11ng/mg),
MDMA in 26 samples (range 0.08 −7.17 ng/mg, mean = 1.43 ng/mg, median = 0.68 ng/mg), AMP in 15
samples (range 0.04−1.68ng/mg, mean = 0.52ng/mg, median = 0.33 ng/mg), ketamine in 11 samples (range
0.21−11.1 ng/mg, mean = 1.90 ng/mg, median = 0.68ng/mg) and cocaine in 25 samples (range 0.10−33.5
ng/mg, mean = 4.13 ng/mg, median = 1.83 ng/mg). MDA, possibly arising from the metabolism of MDMA,
was measurable in 18 samples (range 0.01−0.70ng/mg, mean = 0.19ng/mg, median = 0.14 ng/mg). In all 25
samples that tested positive for cocaine, the ben- zoylecgonine metabolite was also detected (range 0.01−4.34
ng/mg, mean = 0.74 ng/mg, median = 0.38ng/mg). Two samples positive to AMP (0.04 and 0.52 ng/mg) were
also positive for methamphetamine at similar levels (0.01 and 0.34 ng/mg, respectively), suggesting that the
former drug was likely present as metabolite of the latter. One sporadic finding of MDEA at the concentration
of 0.34ng/mg was recorded. The remaining analytes considered in the analytical method have never been
identified in any of the analyzed samples.
Multiple positivity related to sole use of multiple traditional drugs was detected in 31 cases. Figure 1
presents the percentage of the sample positive for one drug, two, three and four drugs, for each class of traditional
compounds. More than 30% of participants who tested positive for MDMA or THC appear to have been inclined
to use only that single substance. On the other hand, the large majority of participants who used cocaine and
ketamine also tested positive for other substances. However, it is unknown whether multiple drugs were used
concomitantly or at different points in time.
As shown in Table I, 26 samples tested positive for at least one NPS. Among these, nine samples were
positive for two or more NPS. Positive specimens were almost equally distributed between males (n = 14;
53.8%) and females (n = 12; 46.2%). The most common NPS detected was butylone, which was found in 25
samples, often at high concentrations (range <7−4,900 pg/mg, mean = 440 pg/mg, median = 21pg/mg). Other
multiple detection included methylone (five cases; range <6−98pg/mg), MXE (four cases; range 3−19 pg/mg),
5/6-APB (one case; 82 pg/mg), α-PVP (one case; 6pg/mg) and 4-FA (one case; 29pg/mg). Multiple positive
findings that include at least one trad- itional drug of abuse and one NPS were detected in 24 cases. Eight samples
tested positive for five or more drugs. Traditional drugs detected in association with NPS were MDMA (17
cases), THC (15 cases), cocaine (15 cases), AMP (8 cases), ketamine (6 cases) and meth- amphetamine (1 case).
While butylone was detected in a wide range of concentrations, with nine samples above 100pg/mg, the
measured levels for the remaining NPS were interestingly below 100 pg of drug per milligrams of hair, either
suggesting sporadic exposure to these substances or low rate of incorporation into the keratin matrix.

Discussion
Our study shows that NPS were detected in a large number of NYC nightclub and dance festival attendees—a
high-risk population. Methylone, butylone and MXE were detected exclusively or more often concurrently in
25 out of 26 cases which tested positive for at least one NPS. Our previous work has already shown that many
nightclub and dance festival attendees reporting MDMA use tested positive for methylone and/or butylone, with
4 out of 10 self- reported MDMA/ecstasy/Molly users testing positive for these drugs after reporting no use of
synthetic cathinones or unknown pills or powders (13). NPS are often identified at festivals as being sold in

378 Salomone et al.

Figure 1. Percentage of the sample positive for one drug, two, three and four drugs, for each class
of traditional compounds. Positivity related to sole use of multiple traditional drugs was detected in
31 cases.
place of traditional drugs—primarily ecstasy/Molly. The fact that synthetic cathinones are often falsely
represented as a pure form of ecstasy to MDMA users has been verified in the past (30). Likewise,
ketamine is often replaced or adulterated with MXE. Therefore, it is not unlikely that most of our
positive findings would be unexpected by the users and can be attributed to unaware intake of NPS.
Synthetic cannabinoids were not detected in any of the tested hair samples. In the present study, it is
possible that the panel of tar- get analytes covered by our method did not fully reproduce the NYC
scenario at the time of sample collection. While it is possible that some newly introduced synthetic
cannabinoids were out of the range of the targeted substances, it should also be noted that differ- ent
groups of synthetic drugs are expected to be used by different social groups. Nightclub attendees and
partygoers are likely to use synthetic cathinones, not synthetic cannabinoids, whereas other social
groups (31) might resort to synthetic cannabinoids as cheap but more potent substitute of THC to
temporarily escape from per- sonal or social problems, such as unemployment, homelessness and/ or
incarceration (32).
It should be noted that prevalence of use of specific NPS— particularly synthetic cathinones—is
constantly shifting. According to national seizure data, for example, prevalence of confiscations of
synthetic cathinones fluctuated greatly between 2013 and 2015 (33). For example, methylone was the
most confiscated compound in 2013 (71.8%) and this decreased to 30.7% and 2.3% in 2014 and
379 Hair Testing for Drugs of Abuse and New Psychoactive Substances
Table I. Results from Samples which Tested Positive for at least One NPS (Concentrations
are Given in pg/mg)
Subject New psychoactive substances Traditional drugs of abuse
Case Sex Ethnic
group
Hair type
Length (cm)
Butylone Methylone MXE 5/6- APB
α-PVP
4-FA
AMP mAMP MDMA COC KET THC
1 F White Head 25 82 1,600 2 M White Leg 2.5 600 98 6 6,300 1,200 150 3 M White Leg 2 100 4 F
Hispanic Head 12 290 630 5,600 680 5 F White Head 12 <7 280 13,000 6 F Hispanic Head 25 17 8
1,040 1,800 2,050 240 7 F White Head 12 17 13 160 5,200 1,400 60 8 M White Head 12 <7 19 220
710 9,400 1,600 80 9 F Other/
mixed
Head 7 <7
10 M Hispanic Head 12 4,900 10 350 1,200 11 M White Head 8 <7 3 120 130 1,600 310 12 M White
Head 4.5 <7 320 13 M Other/
mixed
Head 23 <7 40 10 1,500 5,800 420 20
14 F Hispanic Head 23 <7 30 15 M Hispanic Head 8 25 150 40 16 F Asian n/a 12 23 850 17 M Other/
mixed
n/a 7 110 950 3,100 6,200 190
18 F Asian Head 23 <7 1,700 1,900 19 M Hispanic Head 12 120 540 540 2,600 90 20 F Hispanic Head
7 200 120 21 M Hispanic Head 11 <7 80 22 M White Head 4.5 21 29 710 1,100 23 F Hispanic Head
12 <7 <6 5 110 1,200 3,400 290 24 M White Head 6 300 <6 120 1,400 25 F Hispanic Head 10 190 940
26 M White Head 4 38 34,000 380 Total number of positive samples 25 5 4 1 1 1 8 1 17 15 6 15
2015, respectively. Various other synthetic cathinones (e.g., MDPV) rapidly decreased in prevalence
(or virtually disappeared) over the 3-year period. Therefore, it is possible that other/newer compounds
were used and not detected.
Conclusions
The present study demonstrates that hair testing offers a unique per- spective in the investigation of
drug consumption, provided that a large panel of target analytes is considered. The extended diagnostic
time-window covered by the keratin matrix (unlike urine, blood and oral fluid) allows retrospective
investigation of drug prevalence and diffusion of any targeted psychoactive substance among selected
populations. Furthermore, clues about occasional (even sporadic intake) vs. heavy NPS use are made
possible by quantitative results of hair testing. In particular, the present study highlighted the wide
occurrence of poly-use and likely unintentional intake of unknown NPS in the NYC area. The prevalent
diffusion of synthetic cathi- nones as recreational drugs, often used in combination with other more
traditional drugs, was clearly evident in the data, relative to young consumers attending EDM parties at
nightclubs and dance festivals. Butylone was the most frequently detected NPS, followed by methylone
and MXE.
In prospect, the limitations concerning the specific population under study will be surmounted by
investigating broader groups of young people. Similar occurrences are expected for social groups more
inclined to use synthetic cannabinoids, since the composition of “legal highs” sold in the black market
is typically variable, impli- cating that consumers do not have control over the quality of the product
they are using, potentially increasing risk of serious health threat. Large NPS screening panels using
hair testing and taking into account the timely fluctuation of the market situation can also be applied to
other individuals under periodic control (e.g., in work- place control or driving re-licensing) to check
how frequently NPS are used in substitution of traditional drugs to escape a positive outcome.
Funding

The sample collection in New York was funded by the Center for Drug Use and HIV Research (CDUHR—P30
DA011041). J.P. is funded by the National Institutes of Health (NIH) (K01DA-038800).