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.
Trang 1Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/chroma
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 treat- ments 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 restricted- access 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 prepara- tion, 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 quantifi- cation 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 ( http://creativecommons.org/licenses/by-nc-nd/4.0/)
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
in-volving 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,
cus-tody of minors, drive license regranting, etc.) and criminal
inves-tigations (e.g postmortem toxicology, drug-facilitated assault, etc.)
[3–5] Additional advantages of hair for drugs of abuse
detec-tion include non-invasive and simple sample collection, stability of
drugs at room temperature for long periods, and difficulty for
sam-ple 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: a42caasn@uco.es (N Caballero-Casero)
LC-MS/MS), although given the polar character of most drugs, LC-MS/MS is gradually replacing GC–MS in both screening and confir-mation 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 am-phetamines, cocaine and opioid drugs the highest groups con-sumed 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 con-centrations 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
https://doi.org/10.1016/j.chroma.2022.463100
0021-9673/© 2022 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )
Trang 2the other hand, some drugs can undergo hydrolysis in alkaline or
acidic environments [9] So long, complex and cumbersome
sam-ple 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
ver-tex posterior, where hair has a more uniform growth rate; less
in-fluenced by age and sex-related factors [9] Also, particular
atten-tion 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
sol-vents, 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
or-ganic solvents (e.g methanol, acetonitrile, solvent mixtures, etc.)
[4] Hair digestion damages proteins and helps the release of
ana-lytes but it requires the use of elevated temperatures and
incuba-tion periods between 16 and 20 h [7] On the other hand,
diges-tion at extreme pH values causes degradation of some drugs (e.g.
heroin and cocaine are hydrolyzed in alkaline conditions, while
6-acetylmorphine may originate morphine in an acidic environment)
[7] Extraction with organic solvents is simpler and primarily
car-ried 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
acetoni-trile/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
tech-niques, such as microwave-assisted extraction [17] or
pressurized-liquid 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
re-quired, even though it extends analysis time and adds complexity
and cost to sample treatment.
Supramolecular solvents (SUPRAS), nanostructured liquids
ob-tained 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
po-larity 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
per-fluorinated compounds from natural waters, log P from 0.4 to 11.6,
[22] ) On the other hand, they can be tailored to provide
matrix-independent 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
multi-class drugs of abuse in human hair by LC-ESI-MS/MS For this
pur-pose, 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 mi-croenvironments 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 μ L−1) As a result, solutes can be ex-tracted 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 pro-cesses [20]
The SUPRAS approach here proposed was tested for the extrac-tion of multiclass abuse drugs (i.e opioids, cocaine, amphetamines and their metabolites) in human hair Table 1 shows the chem-ical 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 Al-though 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
All chemicals were utilized according to supplier recommen-dations Solvents used for chromatographic separation were LC grade 1-Hexanol and acetonitrile were purchased from VWR-Prolabo (Bois, France) Tetrahydrofuran (THF) and formic acid were supplied by Panreac (Barcelona, Spain) Ammonium formate and dichloromethane (DCM) were got from Fluka (India) Indi-vidual 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), co-caine (COC, 0.25 mg mL−1), cocaethylene (COE, 1 mg mL−1), ec-gonine 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 μ g mL−1) were pre-pared in acetonitrile and stored at −20 °C Intermediate solutions
of drug mixtures and their working solutions were prepared by ap-propriate dilution in acetonitrile and acetonitrile/ ammonium for-mate buffer (95:05, v/v) respectively, and stored at −20 °C until use.
Trang 3Table 1
Chemical structures and relevant parameters for the selected abuse drugs
A Basic Magmix magnetic stirrer from Ovan (Barcelona, Spain)
and a digitally regulated centrifuge Mixtasel equipped with an
an-gle 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
(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 (SB-HCONC/1 and SBH130D/3, Stuart, France) was used for the evap-oration of SUPRAS extracts Samples pulverization was performed
by a mixer mill MM-301 from Restch (Asturias, Spain).
Trang 4Fig 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
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
equilib-rium solution (21.4 mL) volumes obtained were enough to treat 71
hair samples Fig 1 A depicts the general SUPRAS production
pro-cess.
Drug-free hair samples used for method optimization were
ob-tained from three healthy volunteers having no consumption
his-tory of drugs of abuse Sampling was carried out under the data
protection and management of biological samples policy
estab-lished by the ethical committee of the University of Córdoba
Con-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 ob-tained from the Society of Toxicological and Forensic Chemistry using a control material produced within the proficiency test DHF 2/12 organized by Arvecon GmbH.
In order to remove external contamination (e.g hair care prod-ucts, sweat, sebum, potential contaminants from the environment), the hair sample was washed first with ultrapure water, by gen-tle 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 pulver-ized for 4 min (2 cycles of 2 min) at a vibrational frequency of 28
s−1.
Trang 5Table 2
MS parameters applied for the quantification of the selected drugs of abuse
Drug class Analyte Precursor Ion ( m/z ) aProduct Ions ( m/z ) bDP (V) cCE (V) dT R (min)
a Quantitation transition in bold
b DP: Declustering potential
c CE: collision energy
d T R : time retention
Approximately 25 mg of pulverized hair was transferred to a
2 mL-microtube containing three glass-small balls (3 mm
diame-ter) and it was moistened with 300 μ L of the equilibrium
solu-tion (section 2.3) Extraction of drug/metabolites was carried out
by adding 100 μ L 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 μ L of the SUPRAS extract were
evaporated to dryness under a nitrogen stream at 60 °C, and the
target drugs were redissolved in 75 μ L of reconstitution solution
(acetonitrile:ammonium formate buffer, 95:5 v/v) Aliquots of 20
μL were analyzed by liquid chromatography-tandem mass
spec-trometry Fig 1 B shows a general scheme of the extraction
pro-cedure.
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
Anal-ysis MassHunter The stationary phase was a Gemini 110 C18
col-umn (4.6 mm x 150 mm, 5 μ m) The mobile phase consisted of
ammonium formate buffer (2 mM; pH 3.57; solvent A) and
ace-tonitrile/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
ap-proximately 3 min.
Mass-spectrometry conditions for drug detection were
opti-mized by direct infusion of 1 μ g mL−1 of individual drug
stan-dards 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
in-tense peaks The first abundant fragment was used as quantifier
ion while the second served as qualifier ion The positive
electro-spray ionization mode (ESI + ) was used for all of the target
com-pounds with the following settings: source gas temperature 350 °C,
capillary voltages of 60 0 0 V, nebulizer gas pressure of 50 psi
Com-pound specific MS/MS parameters for each compound are shown
in Table 2 The dwell time was 100 ms.
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 lev-els up to a maximum of 500 ng mL−1 Signal variability was cor-rected with the signal of the IS The correlation between peak ar-eas 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 sig-nal in sample extracts with signals obtained from standards.%SSE parameter may be referred to as absolute matrix effect: percent-ages higher than 115 indicate ion enhancement, while percent-ages lower than 85 are indicative of ion suppression [27] For that purpose, six aliquots of a pooled hair sample (25 mg) were sub-jected to the extraction method (section 2.6) After SUPRAS ex-tracts evaporation, residue reconstitution was performed with 300
μL of acetonitrile:ammonium formate buffer (section 2.6) contain-ing 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 sol-vent was completely evaporated Then, the six samples were sub-jected to the whole analytical procedure (section 2.6 and 2.7)
Trang 6Pre-cision of the method was evaluated in terms of repeatability and
within-laboratory reproducibility For this purpose, six hair
sam-ples, fortified as above described for the recovery study, were
an-alyzed for three consecutive days ( n = 18) The repeatability was
calculated as the square root of the average value of the
intra-day variances obtained and, the within-laboratory reproducibility
as the square root of the mean intra-day variance plus the
inter-day variance.
3 Results and discussion
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
fixa-tion 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
ef-ficient extraction of drugs of abuse and their metabolites (without
digestion or incubation stages) and complete removal of matrix
in-terferences Among available SUPRAS, the ones synthesized from
ternary mixtures of 1-hexanol, THF and water were selected
be-cause 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 1 A) In addition to
the general characteristics of SUPRAS, already specified in
Intro-duction, 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 ,
23-25 ].
The capability of the SUPRAS-based sample treatment workflow
( Fig 1 B) 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
complexa-tion 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,
evapo-ration 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
ma-trix 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
con-stant amount of hexanol (10%) After samples extract evaporation,
reconstitution of the residue was performed with 300 μL of
ace-tonitrile:ammonium formate buffer 95:5 v/v (section 2.6)
contain-ing the target analytes at 1 ng mL−1 (5 ng mL−1 IS) ( Fig 1 B).
Table 3 shows the%SSE values obtained for the extractions
per-formed with SUPRAS synthesized at different THF:water volume
ratios The concentration of both water and THF in the SUPRAS
in-creases as the THF/water ratio in the synthesis solution does, while
the amount of hexanol incorporated into the SUPRAS keeps
con-stant So, the synthesized SUPRAS becomes progressively more
di-luted in hexanol [25] According to these results, determinations
were interference-free (i.e.% SSE in the interval 85–115% [27] ) for
Table 3
Values of signal suppression and enhancement (%SSE), along with their corre- sponding 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
Drug THF:water (v/v,%)
MDEA 133 ± 14 132 ± 11 113 ± 8 124 ± 10 128 ± 12 MDMA 110 ± 22 85 ± 14 112 ± 3 69 ± 2 110 ± 12 COC 118 ± 5 110 ± 17 104 ± 5 102 ± 12 102 ± 19
EME 119 ± 8 114 ± 8 96 ± 5 111.7 ± 0.1 85 ± 10
6-AM 127 ± 7 98 ± 127 92 ± 6 119 ± 13 110 ± 17 COD 128 ± 16 119 ± 11 91 ± 6 115 ± 18 121 ± 16 MOR 122 ± 20 117 ± 16 107 ± 1 115 ± 18 106 ± 20 MET 116 ± 12 115 ± 6 112 ± 3 115 ± 11 102 ± 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 be-tween the SUPRAS phase and the equilibrium solution was clearly visualized (schematic in Fig 1 B) SUPPRAS synthesized in other THF:water ratio gave some matrix effects, particularly in the de-termination of MDEA ( Table 3 ), however, except for SUPRAS syn-thesized 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 sam-ple (25 mg) was fortified with 8 pg mg−1 of drugs of abuse by adding a proper volume ( < 20μL) of a working solution contain-ing their mixture in acetonitrile The sample was stood at room temperature until acetonitrile was evaporated to dryness The ex-traction was performed with different volumes of SUPRAS (60, 70,
80, 90 and 100 μL) and keeping constant the volume of equilib-rium 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 ab-solute recoveries obtained, along with the respective standard de-viations ( n = 3), for the different volumes of SUPRAS assessed The highest extraction yield was achieved with 100 μ L of SUPRAS and the recoveries obtained (87–102%) were within the admissible in-terval (85–115% [27] ) Thus 4:1 SUPRAS:sample ratio was selected
as optimal.
The optimal volume of the reconstitution solution [(acetoni-trile/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 recon-stitution 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 μ L was selected as optimal.
Finally, the extraction time was optimized As it can be ob-served in Table 4 , absolute recoveries near 100% for all the target drugs were obtained at 15 min of vortex-shaking The short time
Trang 7Table 4
Absolute recoveries (%) and standard deviations (%) obtained for the selected drugs of abuse in human hair extracted under different experimental conditions
Analyte
Recovey ± SD a(%)
Volume of SUPRAS b(μL) Volume of reconstitution solution c(μL) Extraction time d(min)
AP 81 ±5 88 ±9 92 ±7 86 ±3 101 ±4 74 ±6 87 ±9 94 ±5 88 ±3 63 ±7 89 ±8 100 ±5 104 ±7 107 ±5 111 ±2
MDA 82 ± 5 87 ±3 91 ±4 92 ±3 94 ±1 73 ±7 89 ±3 101 ±4 101 ±3 57 ±6 71 ±7 89 ±5 97 ±7 101 ±5 108 ±8
MDMA 72 ±6 75 ±2 83 ±5 91 ±4 99 ±2 67 ±5 85 ±7 92 ±4 103 ±3 64 ±9 84 ±8 90 ±6 91 ±8 99 ±4 103 ±7
6-AM 66 ±8 78 ±3 79 ±5 82 ±2 88 ±4 56 ±7 86 ±4 101 ±2 100 ±2 58 ±6 87 ±4 92 ±7 95 ±7 102 ±6 101 ±8 COD 79 ±3 82 ±6 90 ±4 98 ±5 102 ±3 65 ±2 84 ±7 97 ±5 99 ±2 49 ±5 69 ±3 91 ±5 95 ±7 101 ±4 107 ±6 MOR 59 ±5 67 ±3 83 ±1 85 ±4 87 ±2 67 ±9 79 ±1 87 ±3 91 ±2 67 ±9 87 ±6 96 ±4 102 ±3 103 ±5 103 ±2
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 ±SD
a (mL ng −1 ) Intercept ±SD a(ng mL −1 ) r b
MQL c (pg mg −1 ) Drug cutoff
d (pg mg −1 ) Recovery ± SD a(%) Repeatability(%)
Within-laboratory reproducibility (%)
a SD: standard deviation ( n = 3)
b r: correlation coefficient
c MQL: method quantitation limit
d recommended cutoff concentrations by the Society of Hair Testing
needed to reach a high extraction efficiency with SUPRAS is
usu-ally related to the high number of binding sites, the multiple types
of interactions and the high surface area that SUPRASs offer.
Linearity was kept in the ranges specified in Table 5 ,
be-ing 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
be-low 1.1 pg mg−1 for all drugs/metabolites investigated These
val-ues were far below the cutoff concentrations for these drugs in
hu-man hair recommended by the Society of Hair Testing [13]
Table 6 shows some features of the LC-MS/MS methods
re-ported 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
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 simi-lar 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 compo-nents were evaluated through the SSE parameter The obtained values of SSE ranged from 93 to 102% for all the analyzed sam-ples 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 re-ported methods, the use of SPE usually decreased matrix inter-ferences [ 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 con-sistent with the EMA guidelines [27] that establishes an acceptable
Trang 8Table 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) Sample treatment Organic solvent (mL) Treatment of sample extract Chemical group/ number of analytes Matrix effect Recovery (%) / MQL (pg mg −1 ) Ref
20 Extraction with methanol at 45 °C for 2 h
in ultrasonic bath 0.4 Dilution: water:sample
extract 1:3 (v/v)
Opiates, amphetamines, marijuana, cocaine and heroin/14
Signal suppression: < 16.7% n.a / 25 −250 [28]
50 Extraction with phosphate buffer (pH = 5)
at 45 °C for 18 h in a shaking water bath
Supernatant transferred to SPE cartridges
7 Evaporation Opiates, cocaine, and
amphetamines/28
Signal suppression: < 20% 80–120 / 50 [29]
50 Extraction with methanol at 50 °C for 3 h
in ultrasonic bath 3 Evaporation Narcotic drugs, opioids, antidepressants,
antipsychotics, benzodiazepines / > 100
50 Extraction with methanol overnight in
ultrasonic bath
44–60%
> 50 / 10 [31]
10 Extraction with methanol at 38 °C for
15 h
2 Evaporation Amphetamines/2 Signal suppression: < 21% 83–95/ 0.5–1 [32]
enhancement: < 265%
79.4–104.3 / 5–500
[33]
10 Extraction with methanol for 16 h at
room temperature
1 Evaporation Propofol/1 Signal suppression: < 19% 90.8–99.9 / 5 [34]
10 Extraction with methanol and HCl (5 M)
for 16 h Redissolved extracts in
phosphate buffer and transferred to SPE
cartridges
10 Evaporation Erectile dysfunction
drugs with high abuse risk/9
Signal suppression/enhancement:
< 80%
34–100 / 1000-
2500
[35]
10 Extraction with methanol/acetonitrile/
ammonium formate (pH = 5.3) in a bead
mill homogenizer for 10 min
20 Extraction with HCl (1 M) at 45 °C for
16 h Then, vortexed with phosphate
buffer (pH 6) Supernatant transferred to
SPE cartridges
9 Evaporation Synthetic cathinones/16 Signal suppression: < 17% 88–100 / 1–5 [37]
10 Extraction with methanol/TFA (85:15,
v/v) and microwave (700 W) for 3 min
Then, addition of methanol/HCl
(99:1, v/v)
0.4 Derivatization
+ Evaporation
Amphetamines and opioids/8
Signal suppression/enhancement:
< 9%
n.a / 45–125 [38]
20 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
suppression/enhancement:
< 20%
50 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
suppression/enhancement:
< 9%
85–109 / 100 [24]
25 SUPRAS extraction in a vortex-shaker for
15 min at room temperature
0.1 Evaporation Amphetamines, opioids,
cocaine, and their metabolites/13
Signal suppression/enhancement:
< 7%
93–107 / 0.5–1.1
This pa- per 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
aTarget value(ng mg −1 )
bControl range(ng mg −1 )
cConfidence range(ng mg −1 )
dExperimental concentration (ng mg −1 ) ± SD c
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
Trang 9Fig 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
stan-dard 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 ).
Due to the complexity of diffusion and fixation mechanisms of drug compounds into hair fiber, the strategy of fortifying hair sam-ples 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 con-centrations of the target drugs in the control material are given in Table 7 All the drug/metabolite concentrations found, except three
Trang 10of them, were within the confidence range established in the
profi-ciency 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
devia-tions), 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
deter-mination 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
mate-rial proved the suitability of SUPRAS for the simultaneous
extrac-tion and clean-up in the determination of drugs of abuse in human
hair.
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 ],
ul-trasounds [ 28 , 31 ] or microwaves [38] , that confirming our
work-ing hypothesis Thus, the high recoveries obtained (93–70%) in
a short extraction time (15 min), at room temperature and
us-ing tiny volumes of SUPRAS (100 μL), prove that the SUPRAS
ef-ficiently releases the drugs from the hair matrix, and no
diges-tion either incubation processes were needed The process is
cost-effective and eco-friendly and it is within the reach of any
labora-tory The synthesis only requires a mixture of ingredients (section
2.3) and the whole process is carried out with standard
conven-tional 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
rec-ommended by the Society of Hair Testing.
Declaration of Competing Interest
The authors declare that they have no known competing
finan-cial 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
Caballero-Casero acknowledges her post-doctoral contract from the
Andalu-sian Government (Ref Doc_00289).
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