Journal of Chromatography A 1673 (2022) 463100

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Supramolecular solvent-based sample treatment workflow for
determination of multi-class drugs of abuse in hair by liquid
chromatography-tandem mass spectrometry
Noelia Caballero-Casero∗, Gedifew Nigatu Beza, Soledad Rubio
Department of Analytical Chemistry, Institute of Fine Chemistry and Nanochemistry, Universidad de Córdoba, Marie Curie Annex Building, Campus de
Rabanales, Córdoba 14071, Spain

a r t i c l e

i n f o

Article history:
Received 25 January 2022
Revised 9 April 2022
Accepted 28 April 2022
Available online 2 May 2022
Keywords:
Hair analysis
Drugs of abuse
Supramolecular solvent
Microextraction
Liquid chromatography/tandem mass
spectrometry

a b s t r a c t
Hair is becoming a main matrix for forensic drug analyses due to its large detection window compared
to traditional matrices (i.e. urine & blood) and the possibility of establishing the temporal pattern of drug
consumption. However, the extremely time- and solvent-consuming nature of conventional sample treatments render it difficult for routine use of hair analysis in forensics. In this paper, this drawback was
intended to be addressed by the use of hexanol-based supramolecular solvents (SUPRAS) with restrictedaccess properties. The aim was to develop a fast and interference-free sample treatment workflow for the
determination of opioids, cocaine, amphetamines and their metabolites in human hair. The main variables
affecting the extraction were optimized and the method was validated following the European Medical
Agency guideline. Major advantages of the proposed method were the straightforward sample preparation, which combines a high extraction yield (93–107%) and matrix effect removal (93–102%SSE) in a
single step, the high sample throughput, and the reduced volume of organic solvent required (100 μL
of SUPRAS per sample), which makes sample treatment cost-effective and eco-friendly. Method quantification limits were lower enough for all the target drugs (0.5–1.1 pg mg−1 ) to allow their quantitation
in human hair routine analyses. The method was successfully applied to the determination of drugs of
abuse in a human hair control sample.
© 2022 The Authors. Published by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license
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1. Introduction
The large detection window of drugs of abuse in hair (weeks to
years) compared to conventional matrices (hours to days in urine
and blood) has rendered hair highly valuable in forensic cases involving drug-facilitated crimes [1]. Analysis of hair segments from
the hair root allows determine drug consumption pattern [2], and
provides data for judicial decisions (e.g. firearms licenses, custody of minors, drive license regranting, etc.) and criminal investigations (e.g. postmortem toxicology, drug-facilitated assault, etc.)
[3–5]. Additional advantages of hair for drugs of abuse detection include non-invasive and simple sample collection, stability of
drugs at room temperature for long periods, and difficulty for sample adulteration [6].
Hair toxicological analysis is commonly carried out by both gas
and liquid chromatography coupled to mass spectrometry (GC–MS,
∗

Corresponding author.
E-mail address: (N. Caballero-Casero).


LC-MS/MS), although given the polar character of most drugs, LCMS/MS is gradually replacing GC–MS in both screening and confirmation methods [5]. Hair is a complex matrix mainly consisting of
proteins (65–95%) and lipids (1–9%) [7]. On the other hand, drugs
of interest for being analyzed in hair include a wide variety of both
parent substances and their metabolites, which range broadly in
polarity. Thus, sample preparation continues as the most important
challenge in hair toxicological analysis, and particular attention has
been paid to this critical step in order to tackle the different issues
involved [5,7-9].
According to the 2021 report of the European Monitoring
centre for Drugs and Drug Addiction, drug trafficking seems to
have adapted rapidly to pandemic-related restrictions, being amphetamines, cocaine and opioid drugs the highest groups consumed by the European population [10]. The content of drugs
of abuse in hair usually ranges from a few to several hundreds
of picograms per milligram [11,12], so recommended cutoff concentrations by the Society of Hair Testing (SoHT) for analysis of
these drugs in hair are in the range of 50–500 pg mg−1 [13]. On

/>0021-9673/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license ( />

N. Caballero-Casero, G.N. Beza and S. Rubio

Journal of Chromatography A 1673 (2022) 463100

the other hand, some drugs can undergo hydrolysis in alkaline or
acidic environments [9]. So long, complex and cumbersome sample treatments are required prior to drug determination in order
to address all these issues [5,7-9].
Typically, sample treatment for drug testing in hair includes
sample collection, hair segmentation (if needed [14]), washing
of hair samples to remove any possible external contamination,
grinding, extraction of drugs and their metabolites, clean-up of hair
extracts, and drug preconcentration [5,7-9]. In general, the most
recommended sampling site is the back of the head, in the vertex posterior, where hair has a more uniform growth rate; less influenced by age and sex-related factors [9]. Also, particular attention has been paid to removing the external contamination of hair

with exogenous substances deposited from the environment [15].
Although both protic and aprotic solvents have been used for this
purpose, the last ones are recommended because, unlike protic solvents, they do not swell the hair and should ideally remove only
the analytes on the surface [13]. Drug/metabolite extraction and
hair matrix cleanup are by far the longest and the most complex
steps of sample treatment and although considerable progress has
been made in the last ten years, a number of significant challenges
still remain [5,7-9].
Release of drugs/metabolites from hair is commonly achieved
by digestion (acid, basic or enzymatic) or solubilization in organic solvents (e.g. methanol, acetonitrile, solvent mixtures, etc.)
[4]. Hair digestion damages proteins and helps the release of analytes but it requires the use of elevated temperatures and incubation periods between 16 and 20 h [7]. On the other hand, digestion at extreme pH values causes degradation of some drugs (e.g.
heroin and cocaine are hydrolyzed in alkaline conditions, while 6acetylmorphine may originate morphine in an acidic environment)
[7]. Extraction with organic solvents is simpler and primarily carried out with methanol at temperatures in the range of 30–60 °C
for 5–18 h [5,7-9]. Methanol penetrates into the hair, leading to
swelling and solubilization of neutral and lipophilic compounds.
Extraction with acetonitrile is less efficient because hair swelling
occurs to a lesser extent. However, extraction yields of acetonitrile/water, or two-steps extractions involving methanol in the first
step and methanol/acetonitrile/formate buffer, methanol/formate
buffer or methanol/hydrochloric acid in the second step are more
efficient compared to methanol [16]. In order to reduce extraction
time, attention has been paid to the use of assisted extraction techniques, such as microwave-assisted extraction [17] or pressurizedliquid extraction [18,19]. In general, hair extracts contain matrix
components that can cause signal enhancement or suppression
when electrospray ionization LC-ESI-MS/MS is used [5,7-9]. Thus,
in order to prevent potential matrix effects a cleanup step is required, even though it extends analysis time and adds complexity
and cost to sample treatment.
Supramolecular solvents (SUPRAS), nanostructured liquids obtained by self-assembly and coacervation of amphiphiles, have
proved valuable in developing innovative sample treatments that
are not affordable by conventional organic solvents [20]. Thus, they
are able to efficiently extract multiclass substances in a wide polarity range from liquid samples (e.g. 92 substances from urine in
human sport drug testing, log P from −2.4 to 9.2 [21] or 15 perfluorinated compounds from natural waters, log P from 0.4 to 11.6,

[22]). On the other hand, they can be tailored to provide matrixindependent methods in LC-ESI-MS/MS (e.g. determination of 21
bisphenols in canned beverages, urine, serum, canned food and
dust [23] or 5 amphetamines in oral fluid, urine, serum, sweat, hair
and fingernails [24]).
In this research, we tried to develop a SUPRAS-based sample
treatment workflow for simplifying the determination of multiclass drugs of abuse in human hair by LC-ESI-MS/MS. For this purpose, we selected hexanol-based SUPRAS [25], which consists of

inverted hexagonal aggregates of the amphiphile, where the polar
groups surround aqueous cavities and the hydrocarbon chains are
dispersed in tetrahydrofuran (THF). Our working hypothesis was
that this SUPRAS could greatly increase extraction efficiency for
multiclass drugs of abuse in hair, erasing digestion and incubation
steps. This hypothesis is supported by the fact that alcohol-based
SUPRAS should have the ability to penetrate hair capillaries since
proteins (the main component of hair) are easily denatured by
THF and flocculated by complexation with the amphiphile [26]. On
the other hand, hexanol-based SUPRAS have different polarity microenvironments where targeted drugs/metabolites spanning wide
polarity ranges can be solubilized through mixed mechanisms (e.g.
hydrogen bonding, dipole-dipole, ionic, etc. in the polar region and
dispersion, π -π , etc. in the nonpolar region). Likewise, they offer
multiple binding sites owing to the huge concentration of hexanol
in the SUPRAS (0.09–0.5 mg μL−1 ). As a result, solutes can be extracted at low SUPRAS/hair ratios. Additionally, SUPRAS are formed
by individual droplets in the nm-μm range, which provide a large
surface area and enable fast solute mass transfer in extraction processes [20].
The SUPRAS approach here proposed was tested for the extraction of multiclass abuse drugs (i.e. opioids, cocaine, amphetamines
and their metabolites) in human hair. Table 1 shows the chemical structure of the selected drugs/metabolites along with some
physicochemical parameters. Illicit drugs in a wide polarity range
(log P from −0.59 to 3.93) were investigated. The sample treatment
procedure was optimized and the method was in-house validated
and applied to the analysis of a human hair reference material. Although SUPRAS have been previously used for our research group

for the extraction of 5 amphetamines from hair, in the framework
of research intended to develop a matrix-independent method for
these drugs, not attempts were made to avoid hair digestion and
incubation [24].
2. Materials and methods
2.1. Chemicals
All chemicals were utilized according to supplier recommendations. Solvents used for chromatographic separation were LC
grade. 1-Hexanol and acetonitrile were purchased from VWRProlabo (Bois, France). Tetrahydrofuran (THF) and formic acid
were supplied by Panreac (Barcelona, Spain). Ammonium formate
and dichloromethane (DCM) were got from Fluka (India). Individual standard solutions and isotopically internal standards (IS)
were all obtained from Sigma-Aldrich (Barcelona, Spain). They
were: amphetamine (AP, 1 mg mL−1 ), methamphetamine (MA,
0.25 mg mL−1 ), 3,4-methylenedioxyamphetamine (MDA, 1 mg
mL−1 ), 3,4-methylenedioxymethamphetamine (MDEA, 1 mg mL−1 ),
3,4-methylenedioxyethylamphetamine (MDMA, 1 mg mL−1 ), cocaine (COC, 0.25 mg mL−1 ), cocaethylene (COE, 1 mg mL−1 ), ecgonine methyl ester (EME, 1 mg mL−1 ), benzoylecgonine (BZE,
1 mg mL−1 ), codeine (COD, 1 mg mL−1 ), 6-acetylmorphine (6-AM,
1 mg mL−1 ), morphine (MOR, 1 mg mL−1 ), methadone (MET, 1 mg
mL−1 ), methamphetamine-d14 (MA-d14, 0.1 mg mL−1 ), cocaine-d3
(COC-d3, 0.1 mg mL−1 ), 6-acetylmorphine-d6 (6-AM-d6, 0.1 mg
mL−1 ), benzoylecgonine-d3 (BZE-d3, 0.1 mg mL−1 ), methadone-d3
(MET-D3, 0.1 mg mL−1 ). Ultra-high-quality water was produced
in a Milli-Q water purification system (Millipore-Sigma, Madrid,
Spain).
Stock solutions for individual drugs (25 μg mL−1 ) were prepared in acetonitrile and stored at −20 °C. Intermediate solutions
of drug mixtures and their working solutions were prepared by appropriate dilution in acetonitrile and acetonitrile/ ammonium formate buffer (95:05, v/v) respectively, and stored at −20 °C until
use.
2


N. Caballero-Casero, G.N. Beza and S. Rubio


Journal of Chromatography A 1673 (2022) 463100

Table 1
Chemical structures and relevant parameters for the selected abuse drugs.
Drug class

Drug

Chemical structure

Log Ko/w

pKa

Acceptor / donor hydrogen bonds

1.76

10.13

1/1

Methamphetamine (MA)

2.07

9.87

1/1


3,4-methylenedioxyamphetamine (MDA)

1.43

10.01

1/1

3,4-methylenedioxyethylamphetamine (MDEA)

2.33

10.34

3/1

3,4-methylenedioxymethamphetamine (MDMA)

1.86

10.14

3/1

Cocaine (COC)

2.30

8.61


5/0

Cocaethylene (COE)

2.64

8.77

5/0

Ecgonine methyl ester (EME)

−0.21

9.04

4/1

Benzoylecgonine (BZE)

−0.59

9.54

5/1

Codeine (COD)

1.19


8.21

4/1

Morphine (MOR)

0.89

8.21

4/2

6-Monoacetylmorphine (6-AM)

1.31

9.08

5/1

Methadone (MET)

3.93

8.94

2/1

AmphetaminesAmphetamine (AP)


Cocaine

Opioids

2.2. Apparatus

(Schwabach, Germany) with an attachment for 10 tubes, and a
high-speed brushless centrifuge MPW-350R with 36 × 2.2/1.5 ml
angle rotor from MPW Med- Instruments (Warschaw, Poland) were
used for sample extraction. A sample evaporator/concentrator (SBHCONC/1 and SBH130D/3, Stuart, France) was used for the evaporation of SUPRAS extracts. Samples pulverization was performed
by a mixer mill MM-301 from Restch (Asturias, Spain).

A Basic Magmix magnetic stirrer from Ovan (Barcelona, Spain)
and a digitally regulated centrifuge Mixtasel equipped with an angle rotor 4 × 100 mL obtained from JP-Selecta (Abrera, Spain) were
used for SUPRAS production. Two mL-microtubes Safe-Lock from
Eppendorf Ibérica (Madrid, Spain), a Reax Heidolph vortex mixer

3


N. Caballero-Casero, G.N. Beza and S. Rubio

Journal of Chromatography A 1673 (2022) 463100

Fig. 1. A) Schematic illustration for the production and structure of SUPRAS and B) for the simultaneous SUPRAS-RAM-based microextraction and interferences removal in
the quantification of illicit drugs in human hair by LC-MS-MS.

2.3. Supramolecular solvent production


sequently, all volunteers were duly informed about the process,
their rights and other considerations. Hair samples were collected
from the vertex posterior region of the head and cut as close to
the scalp as possible, following the recommendations of SoHT [13].
Samples were stored in aluminum foil at room temperature until
analysis. A hair sample for the method proficiency test was obtained from the Society of Toxicological and Forensic Chemistry
using a control material produced within the proficiency test DHF
2/12 organized by Arvecon GmbH.

1-Hexanol (3 mL) was dissolved in THF (9 mL) in a centrifuge
tube, whereupon water (18 mL) was added as the coacervating
agent. The SUPRAS formed instantaneously and the mixture was
centrifuged at 2400 g for 30 min to facilitate its separation from
the bulk (equilibrium) solution. The SUPRAS, standing at the top
of the mixture solution, was collected with a syringe, transferred
to a hermetically closed vial, and stored at room temperature until
use. The equilibrium solution was also stored and used as wetting
agent of hair during extraction. The SUPRAS (8.6 mL) and equilibrium solution (21.4 mL) volumes obtained were enough to treat 71
hair samples. Fig. 1A depicts the general SUPRAS production process.

2.5. Hair decontamination and milling
In order to remove external contamination (e.g. hair care products, sweat, sebum, potential contaminants from the environment),
the hair sample was washed first with ultrapure water, by gentle mixing for 2 min, followed by immersion in dichloromethane
for 1 min. Excess solvent from the hair sample was absorbed by
clean cellulose paper, and then the hair was immersed again in
dichloromethane. Samples were air-dried and subsequently pulverized for 4 min (2 cycles of 2 min) at a vibrational frequency of 28
s−1 .

2.4. Hair samples
Drug-free hair samples used for method optimization were obtained from three healthy volunteers having no consumption history of drugs of abuse. Sampling was carried out under the data

protection and management of biological samples policy established by the ethical committee of the University of Córdoba. Con-

4


N. Caballero-Casero, G.N. Beza and S. Rubio

Journal of Chromatography A 1673 (2022) 463100

Table 2
MS parameters applied for the quantification of the selected drugs of abuse.
Drug class

Analyte

Precursor Ion (m/z)

a

Amphetamines

AP
MA
MA-d14
MDA
MDEA
MDMA
COC
COC-d3
COE

EME
BZE
BZE-d3
6-AM
6-AM-d6
COD
MOR
Methadone
Methadone-d3

136.0
150.0
164.0
180.0
208.0
194.0
304.0
307.0
318.0
200.0
290.0
293.0
328.0
334.0
300.0
286.0
310.0
313.0

91.165.1

91.165.1
98.2130.1
163.0105.1
163.0105.1
163.0105.1
182.1150.0
185.0153.0
196.1150.0
182.082.1
168.0105.0
171.0105.0
165.0210.9
165.0211.0
165.0201.0
165.0201.0
265.0223.0
268.0226.0

Cocaine

Opiates

a
b
c
d

Product Ions (m/z)

b


DP (V)

95
95
95
95
95
95
100
130
120
100
110
121
144
144
148
154
105
100

c

CE (V)

1840
1840
2210
1022

1026
1026
1822
1826
1826
1426
1531
1830
3927
4026
4022
4026
1018
1018

d

TR (min)

12.77
12.80
12.80
13.27
12.86
12.83
26.67
26.67
27.55
7.90
27.83

27.83
12.75
12.75
12.72
8.14
29.39
29.39

Quantitation transition in bold.
DP: Declustering potential.
CE: collision energy.
TR : time retention.

2.6. SUPRAS-based extraction and cleanup

pound specific MS/MS parameters for each compound are shown
in Table 2. The dwell time was 100 ms.

Approximately 25 mg of pulverized hair was transferred to a
2 mL-microtube containing three glass-small balls (3 mm diameter) and it was moistened with 300 μL of the equilibrium solution (section 2.3). Extraction of drug/metabolites was carried out
by adding 100 μL of SUPRAS and vortex-shaking the mixture at
2700 rpm for 15 min. After that, the mixture was centrifuged at
14.160 x g for 10 min. Finally, 50 μL of the SUPRAS extract were
evaporated to dryness under a nitrogen stream at 60 °C, and the
target drugs were redissolved in 75 μL of reconstitution solution
(acetonitrile:ammonium formate buffer, 95:5 v/v). Aliquots of 20
μL were analyzed by liquid chromatography-tandem mass spectrometry. Fig. 1B shows a general scheme of the extraction procedure.

2.8. Method validation
Validation of the proposed method was in accordance with

the guidelines set by the european medical agency (EMA) [27]. A
pooled human hair sample from three independent hair samples
(see Section 2.4.) was used for method validation.
Calibration curves (n = 8) were plotted by running series
of standard solutions in acetonitrile/ammonium formate buffer
(95:05, v/v) containing the analytes at different concentration levels up to a maximum of 500 ng mL−1 . Signal variability was corrected with the signal of the IS. The correlation between peak areas and concentration of drugs of abuse was determined by linear
regression. Method detection and quantification limits (MDLs and
MQLs) were estimated as three and ten times the average standard
deviation obtained for the determination of six blank hair samples
subjected to the whole proposed method.
Matrix effects were calculated through the percentage of signal
suppression or enhancement (%SSE), which compares analytes signal in sample extracts with signals obtained from standards.%SSE
parameter may be referred to as absolute matrix effect: percentages higher than 115 indicate ion enhancement, while percentages lower than 85 are indicative of ion suppression [27]. For that
purpose, six aliquots of a pooled hair sample (25 mg) were subjected to the extraction method (section 2.6). After SUPRAS extracts evaporation, residue reconstitution was performed with 300
μL of acetonitrile:ammonium formate buffer (section 2.6) containing the mixture of target analytes at 1 ng mL−1 (5 ng mL−1 IS).
Then, the extracts were analyzed by LC-MS/MS analysis (section
2.7.).
Since a reference or certified hair material for all the target
drugs of this study was not available, the accuracy of the method
was assessed by calculating the recovery obtained in the analysis
of six aliquots (25 mg) of a pooled hair sample fortified with the
analytes. Samples were fortified with 8 pg mg−1 of drugs of abuse
and 40 pg mg−1 of IS by adding the proper volume (<20μL) of
working solutions containing a mixture of analytes in acetonitrile.
The sample was stood at room temperature until the organic solvent was completely evaporated. Then, the six samples were subjected to the whole analytical procedure (section 2.6 and 2.7). Pre-

2.7. Liquid chromatography-mass spectrometry analysis
All analyses were carried out in an Agilent Technologies 1200
series LC coupled to a 6420 triple quadrupole mass spectrometer
equipped with an electrospray ionization source (ESI) (Waldbronn,

Germany). The software used for the data processing was the Analysis MassHunter. The stationary phase was a Gemini 110 C18 column (4.6 mm x 150 mm, 5 μm). The mobile phase consisted of
ammonium formate buffer (2 mM; pH 3.57; solvent A) and acetonitrile/solvent A (90:10 v/v; solvent B), at a flow rate of 0.2 mL
min−1 . The elution program started with 10% of B for 5 min, after
B increased to 25% for 5 min, followed by an increase to 50% for
7 min, to 80% for 7 min, and to 100% for 6 min, keeping constant
these conditions for 5 min. Reconditioning of the column took approximately 3 min.
Mass-spectrometry conditions for drug detection were optimized by direct infusion of 1 μg mL−1 of individual drug standards prepared in a (95:5 v/v) mixture of acetonitrile:ammonium
formate/formic acid buffer (pH 3.57, 2 mM). The selection criteria
were set to provide the four most abundant product ions for the
target analytes and to set the final method with the two most intense peaks. The first abundant fragment was used as quantifier
ion while the second served as qualifier ion. The positive electrospray ionization mode (ESI+) was used for all of the target compounds with the following settings: source gas temperature 350 °C,
capillary voltages of 60 0 0 V, nebulizer gas pressure of 50 psi. Com5


N. Caballero-Casero, G.N. Beza and S. Rubio

Journal of Chromatography A 1673 (2022) 463100
Table 3
Values of signal suppression and enhancement (%SSE), along with their corresponding standard deviations, for drugs of abuse in human hair as a function of
the THF:water volume ratios used in the synthesis of the SUPRAS.

cision of the method was evaluated in terms of repeatability and
within-laboratory reproducibility. For this purpose, six hair samples, fortified as above described for the recovery study, were analyzed for three consecutive days (n = 18). The repeatability was
calculated as the square root of the average value of the intraday variances obtained and, the within-laboratory reproducibility
as the square root of the mean intra-day variance plus the interday variance.

Drug

AP
MA

MDA
MDEA
MDMA
COC
COE
EME
BZE
6-AM
COD
MOR
MET

3. Results and discussion
3.1. Optimization of the SUPRAS-based microextraction of
drugs/metabolites from human hair
Drugs are incorporated via bloodstream or sebum secretions
into a keratinized matrix. The analytes are fixed into the matrix
and diffusion mechanisms are not well described [5]. This fixation is a crucial factor in the development and validation of an
analytical methodology to determine drugs in hair. The extraction
method must be selective and no promote hydrolysis, oxidation
nor trans-esterification processes that are usually observed for this
type of analytes [7–9]. For that reason, two objectives were set to
be achieved by the SUPRAS-based sample treatment; direct and efficient extraction of drugs of abuse and their metabolites (without
digestion or incubation stages) and complete removal of matrix interferences. Among available SUPRAS, the ones synthesized from
ternary mixtures of 1-hexanol, THF and water were selected because they have the characteristics to meet the objectives set [25].
These SUPRAS consist of inverted hexagonal aggregates, produced
by the addition of water at solutions of hexanol in THF, where the
alcohol groups of the amphiphile surround aqueous cavities and
the THF solvates their hydrocarbon chains (Fig. 1A). In addition to
the general characteristics of SUPRAS, already specified in Introduction, hexanol-based SUPRAS are environment-responsive. This

means that both the chemical composition of SUPRAS and the size
of the aqueous cavities of the hexagonal aggregates can be tailored
by controlling the THF:water ratio in the synthesis solution, which
gives a number of opportunities for improving selectivity [20,2325].
The capability of the SUPRAS-based sample treatment workflow
(Fig. 1B) to remove matrix interferences present in hair (mainly
proteins and lipids) was investigated. Proteins are expected to be
denatured in the presence of THF and flocculate by complexation with the amphiphile (i.e. hexanol) [26]. On the other hand,
lipids are supposed to be efficiently extracted from hair by the
formation of mixed lipid-hexanol aggregates. In this case, evaporation of SUPRAS components after extraction will give a residue
of lipids from which the analytes can be redissolved. This strategy
has proved successful in the reduction of phospholipid-based matrix effects in the determination of bisphenol A in urine [25].
The influence of SUPRAS composition on method selectivity
was investigated by extracting a pooled hair sample (25 mg) with
SUPRAS synthesized in different THF/water volume ratios (10/90;
20/80; 30/70; 40/60; and 50/50 (%), v/v), while maintaining a constant amount of hexanol (10%). After samples extract evaporation,
reconstitution of the residue was performed with 300 μL of acetonitrile:ammonium formate buffer 95:5 v/v (section 2.6) containing the target analytes at 1 ng mL−1 (5 ng mL−1 IS) (Fig. 1B).
Table 3 shows the%SSE values obtained for the extractions performed with SUPRAS synthesized at different THF:water volume
ratios. The concentration of both water and THF in the SUPRAS increases as the THF/water ratio in the synthesis solution does, while
the amount of hexanol incorporated into the SUPRAS keeps constant. So, the synthesized SUPRAS becomes progressively more diluted in hexanol [25]. According to these results, determinations
were interference-free (i.e.% SSE in the interval 85–115% [27]) for

THF:water (v/v,%)
10:90

20:80

30:70

40:60


50:50

84 ± 9
98 ± 20
93 ± 27
133 ± 14
110 ± 22
118 ± 5
79 ± 20
119 ± 8
81 ± 2
127 ± 7
128 ± 16
122 ± 20
116 ± 12

87 ± 8
85 ± 13
83 ± 5
132 ± 11
85 ± 14
110 ± 17
89 ± 4
114 ± 8
85 ± 4
98 ± 127
119 ± 11
117 ± 16
115 ± 6


90 ± 3
100 ± 5
89 ± 12
113 ± 8
112 ± 3
104 ± 5
96 ± 4
96 ± 5
90 ± 5
92 ± 6
91 ± 6
107 ± 1
112 ± 3

85 ± 11
54 ± 4
88 ± 17
124 ± 10
69 ± 2
102 ± 12
109 ± 8
111.7 ± 0.1
101 ± 11
119 ± 13
115 ± 18
115 ± 18
115 ± 11

81 ± 10

55 ± 7
104 ± 9
128 ± 12
110 ± 12
102 ± 19
115 ± 14
85 ± 10
101 ± 4
110 ± 17
121 ± 16
106 ± 20
102 ± 1

n = 3; SUPRAS synthesized with 10% hexanol. Final extracts fortified with 1 ng
mL−1 of target drugs and 5 ng mL−1 IS.

SUPRAS synthesized in a 30:70%, v/v THF:water mixture. In other
words, both proteins and lipids were efficiently removed by the
strategy proposed. Flocculation of proteins as a white layer between the SUPRAS phase and the equilibrium solution was clearly
visualized (schematic in Fig. 1B). SUPPRAS synthesized in other
THF:water ratio gave some matrix effects, particularly in the determination of MDEA (Table 3), however, except for SUPRAS synthesized in the lowest percentage of THF (i.e.10%), only 3–4 drugs
were out of the recommended SSE values. A mixture of THF:water
of 30:70%, v/v, was selected as optimal for SUPRAS synthesis, in
order to achieve an interference-free determination of the selected
drugs of abuse and their metabolites.
Once SUPRAS composition was selected, extraction yield was
investigated as a function of SUPRAS/sample ratio, volume of
the reconstitution solution (acetonitrile:ammonium formate buffer,
95:5 v/v) and extraction time. For this purpose, a pooled hair sample (25 mg) was fortified with 8 pg mg−1 of drugs of abuse by
adding a proper volume (<20μL) of a working solution containing their mixture in acetonitrile. The sample was stood at room

temperature until acetonitrile was evaporated to dryness. The extraction was performed with different volumes of SUPRAS (60, 70,
80, 90 and 100 μL) and keeping constant the volume of equilibrium solution used for sample wetting (i.e. 300 μL). The sample
was subjected to the whole analytical process described in section
2.6, and the analysis was made in triplicate. Table 4 shows the absolute recoveries obtained, along with the respective standard deviations (n = 3), for the different volumes of SUPRAS assessed. The
highest extraction yield was achieved with 100 μL of SUPRAS and
the recoveries obtained (87–102%) were within the admissible interval (85–115% [27]). Thus 4:1 SUPRAS:sample ratio was selected
as optimal.
The optimal volume of the reconstitution solution [(acetonitrile/ammonium formate buffer (2 mM; pH = 3.57, 90:10 v/v)]
used for drug solubilization after SUPRAS extract evaporation was
investigated based on absolute recoveries. A pooled hair sample
(25 mg), fortified with 8 pg mg−1 of drugs of abuse was subjected
to the whole sample treatment (see section 2.6) but analytes were
solubilized in different volumes (25, 50, 75 and 100 μL) of reconstitution solution. As it is shown in Table 4, almost quantitative
recoveries were obtained for aliquots of 75 and 100 μL. In order
to get as minimal method quantitation limits (MQL) as possible, a
volume of 75 μL was selected as optimal.
Finally, the extraction time was optimized. As it can be observed in Table 4, absolute recoveries near 100% for all the target
drugs were obtained at 15 min of vortex-shaking. The short time
6


N. Caballero-Casero, G.N. Beza and S. Rubio

Journal of Chromatography A 1673 (2022) 463100

Table 4
Absolute recoveries (%) and standard deviations (%) obtained for the selected drugs of abuse in human hair extracted under different experimental conditions.
Recovey ± SDa (%)
Analyte


AP
MA
MDA
MDEA
MDMA
COC
COE
EME
BZE
6-AM
COD
MOR
MET

Volume of SUPRASb (μL)

Volume of reconstitution solutionc (μL)

Extraction timed (min)

60

70

80

90

100


25

50

75

100

0

1

5

10

15

30

81±5
73±2
82 ± 5
69±1
72±6
60±7
80±4
65±2
80±5
66±8

79±3
59±5
77±2

88±9
83±5
87±3
77±5
75±2
68±4
85±2
71±6
84±3
78±3
82±6
67±3
84±5

92±7
87±3
91±4
80±3
83±5
76±2
86±5
77±4
86±1
79±5
90±4
83±1

86±3

86±3
90±4
92±3
83±4
91±4
84±1
88±7
80±1
94±3
82±2
98±5
85±4
92±4

101±4
91±2
94±1
89±3
99±2
89±3
95±3
87±2
98±1
88±4
102±3
87±2
97±4


74±6
66±4
73±7
54±3
67±5
61±4
74±5
52±7
71±8
56±7
65±2
67±9
59±4

87±9
82±5
89±3
71±6
85±7
80±5
87±2
68±5
83±7
86±4
84±7
79±1
83±3

94±5
93±1

101±4
88±2
92±4
89±3
98±3
88±5
91±7
101±2
97±5
87±3
85±2

88±3
95±1
101±3
90±1
103±3
98±2
97±2
88±1
97±5
100±2
99±2
91±2
94±2

63±7
57±6
57±6
54±9

64±9
57±8
59±8
49±5
54±8
58±6
49±5
67±9
64±7

89±8
90±6
71±7
82±7
84±8
85±6
83±6
84±7
83±6
87±4
69±3
87±6
85±8

100±5
92±3
89±5
93±4
90±6
86±9

89±7
90±4
89±7
92±7
91±5
96±4
89±6

104±7
94±6
97±7
92±8
91±8
92±7
96±5
93±5
92±5
95±7
95±7
102±3
92±3

107±5
99±2
101±5
98±3
99±4
97±6
98±4
101±2

98±3
102±6
101±4
103±5
93±5

111±2
102±7
108±8
102±8
103±7
96±8
101±4
98±6
99±7
101±8
107±6
103±2
98±5

a
SD: Standard deviation (n = 3); Fortification level: 8 pg mg−1 of target compounds; SUPRAS synthesis conditions: 30% THF, 70% water; Volume of equilibrium solution:
300 μL.
b
Volume of reconstitution solution: 100 μL; Extraction time: 20 min.
c
SUPRAS volume: 100 μL; Extraction time: 20 min.
d
SUPRAS volume: 100 μL; Volume of reconstitution solution: 75 μL.


Table 5
Analytical figures of merit of the proposed method.

Drug

Linear range
(ng mL−1 )

Slope ±SDa
(mL ng−1 )

Intercept ±
SDa (ng mL−1 )

rb

MQLc
(pg mg−1 )

Drug cutoffd
(pg mg−1 )

Recovery
± SDa (%)

Within-laboratory
Repeatability(%) reproducibility (%)

AP
MA

MDA
MDEA
MDMA
COC
COE
EME
BZE
6-AM
COD
MOR
MET

0.02–500
0.05–500
0.04–500
0.06–500
0.13–500
0.11–500
0.04–500
0.06–500
0.13–500
0.16–500
0.07–500
0.15–500
0.03–500

0.114±0.001
0.283±0.002
0.0453±0.0004
0.304±0.003

0.163±0.002
0.182±0.002
0.150±0.002
1.486±0.003
0.228±0.002
0.188±0.002
0.099±0.001
0.107±0.002
0.136±0.002

0.056±0.009
0.03±0.01
0.021±0.003
0.02±0.02
0.09±0.01
0.43±0.02
0.34±0.01
0.065±0.006
0.55±0.02
0.14±0.01
0.118±0.008
0.21±0.02
0.05±0.02

0.9996
0.9999
0.9997
0.9997
0.9997
0.9996

0.9996
0.9999
0.9996
0.9997
0.9996
0.9988
0.9995

0.5
0.6
0.7
0.7
0.9
1.0
0.8
0.9
1.1
1.1
0.7
1.0
0.9

200
200
200
200
200
500
50
50

50
200
200
200
200

107±5
99±2
101±5
98±3
99±4
97±6
98±4
101±2
98±3
102±6
101±4
103±5
93±5

2
1
1
2
1
3
2
1
1
1

1
2
1

a
b
c
d

7
8
8
6
7
9
7
9
7
6
8
7
7

SD: standard deviation (n = 3).
r: correlation coefficient.
MQL: method quantitation limit.
recommended cutoff concentrations by the Society of Hair Testing.

method here proposed (0.5–1.1 pg mg−1 ) were much lower than
those reported by Cardoso et al. (25–250 pg mL−1 ) [28] for similar classes of compounds and number of analytes. In general, the

MQLs obtained for drugs of abuse with the method here developed
were much lower or had the same magnitude as those obtained
with the methods previously reported, some of which used a much
higher volume of organic solvent (e.g. references 29, 30, 37, 39 in
table 6).
Potential interferences in analytes signal from matrix components were evaluated through the SSE parameter. The obtained
values of SSE ranged from 93 to 102% for all the analyzed samples and so were in agreement with the recommendations of EMA
guidelines [27]. Therefore, no interference from matrix components
was expected to affect the determination of drugs of abuse in
human hair by the SUPRAS method. Regarding the previous reported methods, the use of SPE usually decreased matrix interferences [29,37], although it was not always effective [35]. LLE
was also used for interference removal in the analysis of hair
[39].
Recoveries for the drugs of abuse in six aliquots of a spiked
pooled hair sample were in the range 93–107%, which was consistent with the EMA guidelines [27] that establishes an acceptable

needed to reach a high extraction efficiency with SUPRAS is usually related to the high number of binding sites, the multiple types
of interactions and the high surface area that SUPRASs offer.
3.2. Analytical method validation
Linearity was kept in the ranges specified in Table 5, being 500 ng mL−1 the maximum concentration tested for all the
drugs/metabolites investigated. These ranges varied from 0.02 to
50 0 to 0.16–50 0 ng mL−1 and their correlation coefficients were
above 0.998. As Table 5 shows, MQL values were equal to or below 1.1 pg mg−1 for all drugs/metabolites investigated. These values were far below the cutoff concentrations for these drugs in human hair recommended by the Society of Hair Testing [13].
Table 6 shows some features of the LC-MS/MS methods reported in the last decade for the determination of drugs of abuse
in hair [24, 28-39]. Regarding method sensitivity, huge differences
in MQL values have been reported for methods involving drugs of
abuse of the same class. For instance, both Accioni et al. [24] and
Jang et al. [32] reported methods for amphetamines, but MQLs
were different by two orders of magnitude (100 pg mg−1 and 0.5
pg mg−1 , respectively). On the other hand, MQLs obtained with the


7


N. Caballero-Casero, G.N. Beza and S. Rubio

Journal of Chromatography A 1673 (2022) 463100

Table 6
Representative analytical methods for the determination of drugs of abuse in human hair by LC-MS/MS reported in the last decade.
Sample
size (mg)

Organic
solvent (mL)

Sample treatment

20

Extraction with methanol at 45 °C for 2 h 0.4
in ultrasonic bath

50

Extraction with phosphate buffer (pH=5) 7
at 45 °C for 18 h in a shaking water bath.
Supernatant transferred to SPE cartridges
Extraction with methanol at 50 °C for 3 h 3
in ultrasonic bath


50

50
10
10
10
10

10

20

10

20

50

25

Treatment of
sample extract

Chemical group/
number of analytes

Matrix effect

Recovery (%) /
MQL (pg mg−1 )


Dilution:
water:sample
extract 1:3
(v/v)
Evaporation

Ref.

Opiates, amphetamines,
marijuana, cocaine and
heroin/14

Signal suppression: <16.7%

n.a. / 25 −250

[28]

Opiates, cocaine, and
amphetamines/28

Signal suppression: < 20%

80–120 / 50

[29]

Evaporation


n.a.

n.a. / 10

[30]

Signal enhancement:
44–60%
Signal suppression: <21%

>50 / 10

[31]

83–95/ 0.5–1

[32]

79.4–104.3 /
5–500
90.8–99.9 / 5

[33]

34–100 / 10002500

[35]

Extraction with methanol overnight in
ultrasonic bath

Extraction with methanol at 38 °C for
15 h
Extraction with methanol for 16 h

1

Evaporation

Narcotic drugs, opioids,
antidepressants,
antipsychotics,
benzodiazepines / > 100
Propofol/2

2

Evaporation

Amphetamines/2

2

Evaporation

Opioids/18

Extraction with methanol for 16 h at
room temperature
Extraction with methanol and HCl (5 M)
for 16 h. Redissolved extracts in

phosphate buffer and transferred to SPE
cartridges
Extraction with methanol/acetonitrile/
ammonium formate (pH=5.3) in a bead
mill homogenizer for 10 min
Extraction with HCl (1 M) at 45 °C for
16 h. Then, vortexed with phosphate
buffer (pH 6). Supernatant transferred to
SPE cartridges
Extraction with methanol/TFA (85:15,
v/v) and microwave (700 W) for 3 min.
Then, addition of methanol/HCl
(99:1, v/v)
Extraction with borate buffer (pH=9.5 at
40 °C overnight. Then, extraction with
ether/dichloromethane/hexane/
isoamylic alcohol (50/30/20/0.5, v/v) in a
shaker for 15 min.
Extraction with sodium hydroxide (1 M)
at 80 °C for 1 h. Then SUPRAS extraction
in a vortex-shaker for 10 min at room
temperature.
SUPRAS extraction in a vortex-shaker for
15 min at room temperature

1

Evaporation

Propofol/1


Signal suppression/
enhancement:<265%
Signal suppression: <19%

10

Evaporation

Erectile dysfunction
drugs with high abuse
risk/9

Signal
suppression/enhancement:
<80%

0.5

–

Benzodiazepines/2

Signal suppression: 13–54% 86–95 / 10

[36]

9

Evaporation


Synthetic cathinones/16

Signal suppression: <17%

88–100 / 1–5

[37]

0.4

Derivatization
+ Evaporation

Amphetamines and
opioids/8

Signal
suppression/enhancement:
<9%

n.a. / 45–125

[38]

5

Evaporation

Tizanidine/1


Signal
suppression/enhancement:
<20%

80 / 1

[39]

0.9

–

Amphetamines/5

Signal
suppression/enhancement:
<9%

85–109 / 100

[24]

0.1

Evaporation

Amphetamines, opioids,
cocaine, and their
metabolites/13


Signal
suppression/enhancement:
<7%

93–107 /
0.5–1.1

This
paper

[34]

SLE: solid-liquid extraction; SPE: solid phase extraction; LLE: liquid-liquid extraction; MAE: microwave-assisted extraction; SUPRAS: supramolecular solvent extraction; MQL:
method quantitation limits; n.a.: not available.
Table 7
Obtained results for the analysis of a human hair control material obtained from the Society of Toxicological and Forensic Chemistry using a control material produced
within the proficiency test DHF 2/12 organized by Arvecon GmbH.

Analyte

a
Target
value(ng mg−1 )

b

Drug class

Control

range(ng mg−1 )

c
Confidence
range(ng mg−1 )

d
Experimental concentration
(ng mg−1 ) ± SDc

Amphetamines

AP
MA
MDA
MDEA
MDMA
COC
COE
EME
BZE
6-AM
COD
MOR
MET

1.170
0.767
0.428
0.589

1.740
2.990
N.I.
N.I.
2.840
1.160
N.I.
0.826
1.800

0.790–1.550
0.511–1.023
0.272–0.584
0.383–0.795
1.220–2.260
2.170–3.810
N.I.
N.I.
2.060–3.620
0.780–1.540
N.I.
0.552–1.100
1.260–2.340

1.080–1.260
0.736–0.798
0.389–0.467
0.547–0.631
1.610–1.870
2.820–3.160

N.I.
N.I.
2.670–3.010
1.080–1.240
N.I.
0.552–1.100
1.650–1.950

1.07±0.05
0.76±0.03
0.43±0.04
0.59±0.08
1.75±0.07
3.1 ± 0.2
N.I.
N.I.
3.54±0.05
0.79±0.12
N.I.
0.991±0.004
1.8 ± 0.7

Cocaine

Opiates

a
The target values of hair control material were determined within the proficiency test DHF 2 / 12 - drugs in hair of the GTFCh (Society of Toxicological and Forensic
Chemistry) under the organizational management of ARVECON GmbH.
b

The control ranges were determined using the standard deviation according to Horwitz. They were determined by the target value and two standard deviations (mean
± 2SDHorwitz).
c
The confidence interval indicates the range in which the target value is located with a significance level of 99%.
d
Standard deviation (n = 3).N.I. Drug not include in the hair control material.

8


N. Caballero-Casero, G.N. Beza and S. Rubio

Journal of Chromatography A 1673 (2022) 463100

Fig. 2. Obtained extracted chromatograms for the analysis of a control material of drugs of abuse in human hair. The highest and lowest intense peaks correspond to
quantitative and qualitative ions, respectively.

recovery interval of 85–115%. Table 5 shows the obtained recovery
value for each drug compound. Regarding the recoveries obtained
by previously reported methods (table 6), they were not available
for some of them (e.g. in references 28, 30, 38) and in other cases
were below the interval recommended by EMA guidelines (e.g. in
references 31, 33, 35).
Method precision was evaluated in terms of repeatability and
within-laboratory reproducibility and expressed as relative standard deviation (RSD). Precision was acceptable if RSD was equal
to or below 15%. The repeatability and reproducibility were in the
ranges 1–3% and 6–9%, respectively (Table 5).

3.3. Analysis of control human hair material
Due to the complexity of diffusion and fixation mechanisms of

drug compounds into hair fiber, the strategy of fortifying hair samples with the target drugs might be not representative of a realistic
scenario. For this reason, in order to prove the applicability of the
proposed SUPRAS-based extraction method under real conditions,
it has been applied to the analysis of a control material of drugs
of abuse in human hair (from LGC Standards, Spain). Found concentrations of the target drugs in the control material are given in
Table 7. All the drug/metabolite concentrations found, except three

9


N. Caballero-Casero, G.N. Beza and S. Rubio

Journal of Chromatography A 1673 (2022) 463100

of them, were within the confidence range established in the proficiency test DHF 2/12 organized by Arvecon GmbH (i.e. the range in
which the target value is located with a significance level of 99%).
The rest of the drug concentrations found (i.e. 6-AM, BZE and AP)
were within the control range (target value ± two standard deviations), where the significance level is around 95%. So, the method
proved to be suitable for the extraction and quantification of the
drugs accomplishing the analytical features required for the determination of drugs of abuse in hair for forensic or medical purposes.
Fig. 2 shows a typical chromatogram of the analysis of the control
material. The results obtained for the analysis of the control material proved the suitability of SUPRAS for the simultaneous extraction and clean-up in the determination of drugs of abuse in human
hair.

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4. Conclusions
In the sample treatment workflow here developed, we have
proved that both extraction of opioids, cocaine, amphetamines and
their metabolites and removal of major matrix components can
be efficiently achieved in hair samples by using an hexanol-based
SUPRAS. Compared to the LC-MS/MS methods reported in the last
decade for this purpose (table 6), the method here proposed is
faster and drugs/metabolites are released from the hair matrix
without the need for high temperatures [24, 28-30, 32,37], ultrasounds [28,31] or microwaves [38], that confirming our working hypothesis. Thus, the high recoveries obtained (93–70%) in
a short extraction time (15 min), at room temperature and using tiny volumes of SUPRAS (100 μL), prove that the SUPRAS efficiently releases the drugs from the hair matrix, and no digestion either incubation processes were needed. The process is costeffective and eco-friendly and it is within the reach of any laboratory. The synthesis only requires a mixture of ingredients (section
2.3) and the whole process is carried out with standard conventional equipment. Apart from the excellent recoveries obtained and
the efficient removal of interferences, another valuable asset of the
method is the low method quantification methods achieved, which
allows drug determination far below the cutoff concentrations recommended by the Society of Hair Testing.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to
influence the work reported in this paper.
Acknowledgments
This work was supported by the Andalusian Department of
Knowledge, Innovation and University (P18-RT-2654). Dr CaballeroCasero acknowledges her post-doctoral contract from the Andalusian Government (Ref. Doc_00289).
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