Trends in Analytical Chemistry 112 (2019) 135e146

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Trends in Analytical Chemistry

journal homepage: www.elsevier.com/locate/trac

A critical review of solid-phase microextraction applied in drugs of

abuse determinations and potential applications for targeted doping
testing
Krzysztof Gorynski*
Department of Pharmacodynamics and Molecular Pharmacology, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in

, Bydgoszcz, 85-067, Poland
Torun

a r t i c l e i n f o a b s t r a c t

Article history: Conventional extraction and microextraction techniques have been extensively implemented as sample
Available online 9 January 2019 preparation methods for bioanalysis. Both types of techniques are affected by their own advantages and
drawbacks. Conventional extractions usually afford better extraction efficiency as they are exhaustive
Keywords: in nature. Conversely, equilibrium-based microextraction techniques aim at either minimizing or
SPME, solid phase microextraction completely circumventing the use of solvents, also reducing the time and cost of analysis. Furthermore,
Illicit drugs
additional microextraction features such as the varied dimensions of extracting devices, miniaturization,
Doping control
high-throughput operation, and automated coupling with analytical instruments have enabled
Prohibited substances analysis
Drugs of abuse
the development of exemplar analytical tools for bioanalysis applications. This review offers a brief
Bioanalytical discussion concerning achievements, limitations, and future directions of solid phase microextraction
Sample preparation (SPME) with respect to determinations of drugs of abuse and anti-doping applications. In addition,
Mass spectrometry commercially available devices, automation, and several geometric configurations of SPME compatible
Direct coupling with various mass spectrometry interfaces are described in several examples.
© 2019 Elsevier B.V. All rights reserved.

1. Introduction requirements as well as analytical challenges posed by the

complexity of the matrices under study increase the need for
According the 2018 World Drug Report [1] published by The “cleaner” extracts, rendering the sample preparation step as one of
United Nations Office on Drugs and Crime in close cooperation with the most significant steps of the analysis workflow [2]. Briefly, the
the World Health Organization, the overall number of users of illicit sample preparation step must be sophisticated enough so as to
drugs has increased steadily since the late 1990s; today, over 275 address the demand for trace-level quantitation, new regulatory
million people are estimated to have used an illicit drug at least requirements, as well as the complicated nature of the sample
once in 2016 alone. Further, the global number of illicit drug users is matrices, which are incompatible with analytical instrumentation
projected to grow by 25% by 2050, directly impacting road safety for direct analysis. Taking into account the fact that a universal
and the status of public health. Given the complexity of biological sample preparation protocol suitable for all biological matrices
matrices, the wide spectrum of drug compounds that must be does not exist, research in bioanalysis has focused on innovative
monitored, the size of the population that must be addressed, as extraction techniques that can be combined with the various
well as complexities related to time of analysis, effective screening analytical tools available to date. An ideal sample preparation
of drug (illicit and doping) use poses a tough practical and bio- method should enable easy operation, be comprised of as few
analytical challenge to regulatory agencies and analysts worldwide. extraction steps as possible, yield reproducible recovery, and afford
Despite significant recent developments in analytical instrumen- sufficient sensitivity for the compounds under study. Further,
tation, sample preparation still plays a crucial role in the determi- such methods should also ideally enable automation of the analysis
nation of target analytes in biological matrices. High sensitivity workflow and high throughput applications, afford low cost of
analysis per sample, be environmentally friendly, comply with
safety regulations, and lastly, afford easy coupling to analytical
* Corresponding author. instrumentation. Several sample pretreatment processes for
E-mail address: [email protected]. determination of drugs at trace levels from complex matrices have

https://doi.org/10.1016/j.trac.2018.12.029
0165-9936/© 2019 Elsevier B.V. All rights reserved.
136 K. Gorynski / Trends in Analytical Chemistry 112 (2019) 135e146

been described in the literature [3,4]. Conventional extraction extraction curves. Generally, according to IUPAC Recommendations
techniques, such as solid-phase extraction (SPE) and liquid-liquid [14], microextraction is defined as an extraction technique in which
extraction (LLE), very often described as exhaustive techniques, the extraction phase (liquid or solid) has considerably smaller
have been traditionally used to separate analytes from ‘aqueous’ volume (typically <100 mL or <10 mg) than the sample (>1 mL).
matrices [5e7]. However, LLE is associated with several disadvan- Consequently, at equilibrium, the extracted fraction of the analyte
tages, such as its time-consuming and labor-intensive nature, in under study is frequently negligible (or non-exhaustive). New
addition to its consumption of large volumes of costly and envi- instrumental technologies have enabled analysis of compounds
ronmentally unfriendly organic solvents. Although SPE techniques present at low detection limits, in the picograms or below levels,
require less solvent use in comparison to LLE, and can be easily advancements which have subsequently enabled faster analysis
semi-automated and coupled with LC-MS, nevertheless such times in cases where equilibrium-regime extractions are not other-
techniques still require multiple analytical steps, which introduce wise necessary or practical. Suitable calibration methods have also
potential sources of error to the analysis. Combined with the cur- been developed for SPME to facilitate quantitative analysis in cases
rent analytical demand for fast, integrated analytical platforms for where the partition coefficients are unknown [15]. As considerations
drug screening, the above listed drawbacks of traditional tech- about the kinetics of extraction governing the principles of operation
niques have propelled the search for methods that simplify the of SPME, the influence of coating chemistries and geometries, or the
sample preparation workflow via methodologies distinct from the selection calibration strategies are beyond the scope of this short
available standard sample preparation techniques [2]. Rather than review, readers seeking further information on these topics are
providing a discussion on recent trends in exhaustive techniques, directed elsewhere [12,15e20].
the current review aims to present a critical review of recent de- SPME sampling can be performed via two modes of extraction:
velopments in solid phase microextraction (SPME) technology for headspace extraction (HS-SPME), and direct immersion extraction
analysis of specific and targeted drugs of abuse, as well as discuss (DI-SPME), whereas the essential diversity remains in the position
their potential applications in doping control. of the fibers. Succinctly, in HS, the SPME fiber is placed in the
Over the course of the last decade, SPME technology has headspace of the sample for extraction of compounds. This mode is
increasingly observed a rise in popularity as a sample preparation most suitable for extraction of volatile compounds and semi-
technique of choice in various fields, including bioanalysis, phar- volatile compounds [16]. However in latter, an appropriate deriv-
maceutics, toxicology, and drug monitoring [8e10]. Since its first atization process is required to improve extraction efficiency as a
introduction by Janusz Pawliszyn in 1990 [11], SPME has widely step that can be performed in situ prior to analysis, directly on
spread into analytical practice worldwide through the develop- SPME [21]. The advantages of HS-SPME in drug of abuse de-
ment and implementation of new SPME configurations, geome- terminations are (i) the prevention of direct contact of the fiber
tries, coatings, procedures, and protocols for a plethora of novel with the biological sample, consequently preventing contamina-
applications in various fields [12]. The attractiveness of SPME is tion of the coating; (ii) the possibility of analysis of solid samples
owed to the unique advantages it affords over traditional tech- (e.g. hair, tissue, skin), or volatile types of matrix (e.g. breath); (iii)
niques, such as operation simplicity, compatibility with different greatly suited for GC injection ports, where the thermal desorption
routine separation (GC, LC or CE) and detection techniques (MS, FID, process is carried out. Several methods employing HS-SPME were
UV), possibility of automation, ‘tailoring’ of its geometry to suit developed for simultaneous extraction drugs of abuse from com-
innumerous applications, as well as the commercial availability of plex biological matrices, including urine [22], breast milk [23],
multiple configurations and coating materials. In addition, SPME sweat [24] and oral fluid [25]. Conversely, the direct immersion (DI-
enables integration of several analytical workflow steps, such as SPME) approach is carried out by exposing the extraction phase
sampling (direct or headspace), preconcentration, extraction, and directly into the sample, facilitating direct extraction of analytes
sample introduction into various analytical instruments, thus from the matrix. This mode has been adapted more often to LC, as it
allowing for easy, quick, and accurate analysis. is based on solvent desorption of retained compounds from the
This review discusses applications of SPME toward drug of abuse surface of the extraction phase.
determinations for various biological matrices, critically examining The various possible configurations of SPME, including several
their role in improving the overall efficiency of the analytical pro- geometries, sorbents, and devices, have enabled the development
cess, from extraction to determination. Additionally, commercially of unique methods that can be adapted to difficult applications
available equipment, automation, and novel geometrical configu- limited by factors such as the physicochemical properties of the
rations of SPME compatible with different mass spectrometry analytes under study, the limited volume of the samples, the type
interfaces are also discussed in this review with respect to their and complexity of the matrix, necessary limits of detections, as well
applicability in the drug screening and monitoring fields. At the as high-throughput and/or automation needs. SPME may be per-
end, a short discussion is provided regarding the achievements, formed in a variety of arrangements [17], such as fiber SPME [11],
limitations, and future directions of SPME in these fields of study. in-tube SPME [26], stir-bar sorptive extraction [27], Thin-Film
SPME [28], in-tip SPME [29], or electromembrane-surrounded
2. Principles SPME [30]. Historically, fiber SPME [11] was the first configura-
tion to be introduced as a new strategy in sample preparation
In SPME, an extraction phase layered on a solid support is used as (1990), and it is still popularly used in many applications today [13];
extractant through direct or headspace exposure (Fig. 1) for a spec- for instance, of the 10 260 articles found in Scopus based on the
ified period of time to the sample matrix under study [13]. Here, the ‘solid-phase microextraction’ keyword, 6020 entries apply to ‘fiber’.
partitioning of analytes occurs between the two phases, namely the A few years later, Eisert and Pawliszyn [26] developed in-tube
extracting phase and the sample matrix, until equilibrium is estab- SPME (1997), a technique suitable for on-line coupling with HPLC,
lished, whereupon the transfer of analytes ceases to happen. While providing a unique alternative to the fiber configuration. In-tube
SPME is generally associated with equilibrium extraction, it is SPME uses a capillary column internally coated with an appro-
important to note that extraction does not necessarily need to be priate extractant to enable constant flow-through extraction, or
carried out until equilibrium is established; in cases where time of repeated draw/eject extraction cycles of samples. To address the
analysis is a priority, pre-equilibrium extraction can be carried out sensitivity limitation related to the volume of the SPME extraction
and analytes accurately quantified through the generation of phase, stir-bar sorptive extraction (1999) [27] and thin-film
 K. Gorynski / Trends in Analytical Chemistry 112 (2019) 135e146 137

Fig. 1. The principles of SPME. Figures modified based on Ref. [13].

microextraction [28] were introduced as alternative configurations and desorption conditions. Selection of fiber coating chemistry,
of SPME, with the latter offering a larger volume and area of geometry, and configuration is generally limited to commercially
extraction phase. In addition to these developments, technological available configurations and coatings, while mode of extraction is
innovations in high-throughput and automation also opened new limited to either headspace or direct immersion extraction. All
doors in SPME. In 2009, Xie et al. [29] combined automation with commercially available fiber coatings have been used to date in drug
the fiber format by using 96-well extraction plates, an off-line ro- analysis applications [8,12,17], including divinylbenzene/carboxen/
botic sample preparation station (Concept 96), and a pipette polydimethylsiloxane (DVB/CAR/PDMS), polydimethylsiloxane
tip-based SPME format. In 2013, Rezazedeh et al. [30] proposed the (PDMS), carboxen/polydimethylsiloxane (CAR/PDMS), poly-
electromembrane-surrounded SPME system as a novel approach dimethylsiloxane/divinylbenzene (PDMS/DVB), Carbowaxpolye-
for efficient extraction from complicated matrices. Jornet-Martínez thyleneglycol (PEG), polyacrylate (PA), and carbowax/template
et al. [31] most recently demonstrated for the first time, the reli- resin (CW/TPR) for GC applications, and C18 for LC-based investi-
ability of in-tube solid-phase microextraction (IT-SPME) coupled gation, as presently C18 is the only option on the market for
on-line to nanoliquid chromatography (nanoLC) for the analysis of LC-based SPME applications. Despite the advantages imparted by
complex matrices such as cannabis plant extracts to the detection commercially available coatings [17], such as (i) repeatability, (ii)
of traces of cannabis in different kind of items (plastic bags, office availability of reports describing inter-laboratory validations, (iii)
paper, aluminum foil, cotton cloths, and hand skin). different geometries, and (iv) biocompatibility, there may still be a
need to adapt new coatings to a specific application or particular
3. SPME e method development and crucial points in drug matrix. In this regard, home-made SPME devices tailored by using
determination protocols more selective extraction phases are very often applied. Detailed
information regarding the development of new support, geome-
A fundamental understanding of the theory and principles of tries, and main techniques widely used for preparation of SPME are
Solid Phase Microextraction [13] is indispensable for development documented in several publications [17,18,20,33].
of SPME methods for drug analysis, as a wide range of parameters As previously discussed, SPME drug analysis is either carried out
must be first optimized before a final protocol is established. in direct immersion mode for most compounds, or in headspace
Comprehensive information regarding the most important indi- mode for analysis of volatile drugs (e.g. amphetamines and volatile
vidual parameters in SPME method development, their corre- metabolites). Headspace SPME is employed wherever possible as a
sponding impact on the final results of analysis, as well as their means to avoid exposure of fibers to dirty matrices, although
rigorous control during optimization have all been thoroughly several biocompatible coatings are now available for direct
discussed in Handbook of Solid Phase Microextraction [13], as well extraction of drugs of abuse from complex biomatrices such as
as detailed step-by-step in Nature Protocol [32]. Fig. 2 illustrates a blood, urine, plasma, and saliva. In order to attain reproducible
typical SPME method development and optimization scheme. results that are not affected by variations in recoveries, the matrix
Parameters that require special attention during optimization of an must be adjusted prior to the extraction step by adjustment of the
SPME protocol are: fiber coating and extraction mode choice; pH or salt content of aqueous matrices (urine, blood, or saliva) [32].
time of extraction, adjustments to the matrix (pH and ionic An additional option to reduce variations in extraction recoveries,
strength) d unless an appropriate calibration technique is applied; particularly in cases where a protocol is followed for quantitative
138 K. Gorynski / Trends in Analytical Chemistry 112 (2019) 135e146

Fig. 2. Typical parameters considered in SPME method development and optimisation. Adopted from Ref. [13] with permission Elsevier 2011.

analysis, regards the employment of internal standards, or thermostated sample carousel to enable temperature control
alternatively, an SPME calibration technique [15,32]. (1998) [13]. A large number of developments and improvements to
commercially available autosamplers took place within the next
4. Automation, high-throughput and commercially available decade. The Combi-PAL autosampler was released in 1999 by CTC
equipment Analytics (Zwingen, Switzerland), allowing users to program many
parameters, including dilution, agitation, and extraction, while also
Automation is a desirable feature of a drug analysis method, supporting a derivatization step, online sample preparation, and
especially in pharmaceutical, toxicological, and doping-control fiber washing steps [13]. In turns improvements to the extraction
laboratories. Automation enables large reductions in time for and incubation processes by the use of both orbital agitation and a
both method development as well as during routine analysis, stirring mechanism were introduced by Gerstel GmbH (Mulheim
providing higher sample throughput and improving reproduc- an der Ruhr, Germany) in the form of the MultiPurpose Sampler
ibility. The first trial of automation of SPME was carried out in 1993 (MPS 2). Another interesting modification presented by the Thermo
on a Varian GC syringe autosampler, which matches very well with Fisher Scientific (Milan, Italy) company on their product, the TriPlus
SPME fibers [13]. Following the success of this investigation, both autosampler, concerns the addition of a rocking agitation mecha-
an SPME device and a GC autosampler were commercialized within nism instead of the orbital procedure enabled by Combi-PAL,
the year, by Supelco and Varian, respectively [13]. Automation at additionally providing the possibility of closing the agitator tray
that time meant that the extraction of the next sample was during extraction. High-throughput sample analysis was further
commenced whilst the previous one underwent GC analysis. Next improved by the option to connect samples to two separate GC
developments concerned device modification to allow vibration systems via a single TriPlus autosampler. Gerstel has also evaluated
of the fiber to agitate the sample (1996), and addition of a a wide range of additional options attractive to SPME technique
 K. Gorynski / Trends in Analytical Chemistry 112 (2019) 135e146 139

users, such as the automated tube exchange system (ATEX), ther- Solid Phase Microextraction [13] and explained step-by-step in
mal desorption system (TDS), automated dynamic headspace Nature Protocols [32,39].
(DHS), the twister thermal desorption unit (TDU), and the Multi-
Fiber Exchange (MFX). 5. Application
In 2008, high-throughput analysis, namely the handling of
multiple samples at once, was combined with SPME automation in Several SPME methods and protocols have been reported in the
the 96-well plate format [34] in the form of the commercial robotic literature, showing great potential as doping control applications
system Concept 96 (PAS Technology, Magdala, Germany). In the ten [35e37,42e44], as well as for drug of abuse screening and quan-
years since, large progress has been made in expanding the us- tification from biological matrices including urine, whole blood,
ability of the Concept 96 robotic system for a variety of applications plasma, saliva, hair, skin and breath [8,17,45,46]. A typical workflow
and SPME configurations, such as enabling applications using of bioanalytical processes applied in daily routine analysis in
multi-fiber SPME, in-tip SPME, and thin film SPME (TFME blades). doping control or industry laboratories involves several sequential
The Concept 96 system has also undergone a series of upgrades steps, such as planning of analysis, sampling (sample collection),
since its introduction (see Fig. 3), presently offering precise tem- sample preparation, LC or GC separation, detection, data evaluation,
perature control of each step (extraction, desorption), protection and lastly, generation of a report or decision. Taking into account
against solvent evaporation via use of a plate cover when the 96- that strict control of biological fluids is of great importance in such
well plate is in park position, and direct operation from a 10 inch applications (e.g. during sport competitions or in hospital/toxico-
palmtop offering a user-friendly interface. By coupling the thin-film logical laboratory determinations of presence of drugs of abuse),
configuration of SPME to the Concept 96, improvements in method large numbers of samples may need to be analyzed within a short
sensitivity can be attained by spreading a larger volume and larger time [2]. In this regard, high-throughput, simultaneous monitoring
surface area of the extraction phase, while simultaneously guar- of a wide variety of possible substances is thus a highly desirable
anteeing greater reproducibility of extracted analytes due to the feature of a given sample preparation method. Taking advantage of
simultaneous extract analysis capabilities of the Concept 96, which these same features enabled by SPME, Boyaci and co-workers [35]
improves control experimental parameters (e.g. extraction and developed a comprehensive protocol for analysis of doping
desorption time, temperature, stirring rate parameters) even when substances in urine with the use of C18 coated blades and an
extraction occurs in the pre-equilibrium regime. To date, several automated station (such as Concept 96 autosampler), followed by
methods employing TFME blades and Concept 96 were developed LC-MS analysis. The proposed protocol (summarized in Table 1)
for simultaneous extraction of varied drugs from complex biolog- allows for analysis of more than 100 drugs of varying physical and
ical matrices, including more than 100 drugs from urine [35], and chemical properties, and enables throughput speeds of less than
25 from blood and plasma [36,37]. These studies have had as 2 min per sample. Although the sample preparation step is typically
aim the development of methods for potential use in doping con- regarded as the most time-consuming and error-inducing step in
trol applications [35,36,38], drug-protein binding studies [39], and traditional analytical workflows, often introducing the majority of
high-throughput analysis of patient samples [40], and have sources of errors known to affect the accuracy and precision of the
included development of both targeted and untargeted analyses analytical outcome, the SPME technique seems to effortlessly
methods [8,12,41]. Detailed information regarding TFME blade overcome these drawbacks. Given that SPME protocols usually do
preparation has been published [33] and reviewed [17]. Further- not require any sample pretreatment (e.g. centrifugation or
more, important considerations to keep in mind when operating filtering), and also superior clean-up, which prevents matrix ef-
the Concept 96 robotic system are documented in the Handbook of fects, such methods can largely decrease the chances of errors as

Fig. 3. Evolution of the Concept 96 system since its invention in 2006 (top pictures courtesy of Dietmar Hein).
Table 1

140
Representative SPME applications toward multi-compounds prohibited substances analysis (potential in doping control) in variety of sample matrices.

Urine [35] Blood, urine, plasma [37] Plasma [36] Saliva [42] Urine, saliva [43] Urine, plasma [59] Blood, plasma [44]

No of 110 17 25 49 13 18 17
substances
Extraction C18 HLB-PAN HLB C18 HLB-PAN HLB-PAN HLB-PAN
phase
Sample 1200 ul 1080 ul 1080 ul 1200 ul 700ul urine 300 ul 10 ul
volume 300 ul saliva
Total time of 165/96 samples 140/96 samples 140/96 samples 165/96 samples 2/sample 15/96 samples 7/sample
sample
preparation
[min]
Format/ TFME Plastic support TFME TFME PEEK Mesh CBS-brush 96 CBS
support Stainless steel Polybutylene terephthalate Stainless steel Stainless steel Polybutylene terephthalate Stainless steel Stainless steel
Separation and LC-MS/MS LC-MS/MS LC-MS/MS LC-MS/MS DART-MS/MS CBS-MS/MS CBS-MS/MS
detection
method
Pros High-throughput Single-use samplers High-throughput High-throughput Fast on-site/in-vivo Rapid, direct coupling Only 10ul of sample

K. Gorynski / Trends in Analytical Chemistry 112 (2019) 135e146

Automated 96-concept Biocompatible device 1.5 min/sample Automated High-throughput Rapid
Sensitive 10 s/sample On-site
Photo of SPME
Device

Name of the 11-nor-9-carboxy-D-THC Amphetamine 17-a- Morphine-3B-glu 11-Nor-9-carboxy-THC Cocaine Methamphetamine Methamphetamine Methamphetamine
compounds 19norandrosterone trenbolone Morphine-6B-glu Morphine Cannabidiol Cannabinol Nordiazepam Fentanyl 3,4- Carbamazepine Carbamazepine
investigated 6acetylcodeine Benzoylecgonine Salbutamol Nikethamide Androstenedione Clostebol methylenedioxymethamphetamine Propranolol Clenbuterol Propranolol Clenbuterol
6acetylmorphine Bisoprolol Clenbuterol Codeine Benzoylecgonine Epitestosterone (MDMA) Heroin Phencyclidine (PCP) Diazepam Codeine Diazepam Codeine
Acebutolol Alprenolol Codeine Exemestane Amphetamine Fluoxymesterone Oxazepam Methadone Oxycodone Cocaine Sertraline Cocaine Sertraline
Aminoglutethimide GW501516 Prednisolone Furosemide Methandrostenolone Lorazepam Lysergic acid Citalopram Fentanyl Citalopram Fentanyl
Amphetamine Anastrozole Methamphetamine Methamphetamine Nandrolone Prostanozol dimethylamine (LSD) Diazepam Buprenorphine Morphine Buprenorphine
Androstenedione Atenolol Metoprolol Morphine Dexamethasone Testosterone 6- Methadone Salbutamol Morphine Methadone
Bambuterol Nikethamide Propanolol Trenbolone Strychnine Acetylcodeine Fentanyl Oxycodone Lorazepam Salbutamol Oxycodone
Bendroflumethiazide Salbutamol Stanozolol Metoprolol Exemestane Heroin Methadone Bisoprolol Stanozolol Lorazepam Bisoprolol
Benzphetamine Betaxolol Strychnine Toremifene Budesonide Clenbuterol Norfentanyl oxalate
Bisoprolol Boldenone THCCOOH-glu Stanozolol Acebutolol Alprenolol
Budesonide Bumetanide THCCOOH Bisoprolol Atenolol Betaxolol Esmolol
Buprenorphine Cannabidiol GW501516 Propanolol Labetalol Metoprolol
Cannabinol Canrenone Toremifene Nadolol Oxprenolol
Carvedilol Chlorthalidone Pindolol Propanolol Sotalol
Clenbuterol Clopamide Timolol Benzphetamine
Clostebol Cocaine Codeine Nikethamide
Dehydroepiandrosterone Phendimetrazine
Dexamethasone Selegiline Strychnine
Dihydrotestosterone Bambuterol Formoterol
Dimethylamphetamine Terbutaline Budesonide
Ephedrine Esmolol Fludrocortisone acetate
Etacrynic acid Exemestane Flunisolide Triamcinolone
Fenfluramine Fenoterol acetonide Triamcinolone
Fenproporex Fentanyl diactetate Clenbuterol
Finasteride Fludrocortisone Zilpaterol
Flumethasone Flunisolide Aminoglutethimide
Fluoxymesterone Exemestane Fulvestrant
Formestane Formoterol
Fulvestrant Furosemide
 GW501516
Hydromorphone
Indapamide Labetalol
Letrozole Mefenorex
Mesterolone
Metaproterenol Methadone
Methamphetamine
Methandriol
Methandrostenolone
Methyltestosterone
Metolazone Metoprolol
Modafinil Morphine
Nadolol Nandrolone
Nikethamide Norephedrine
Norfentanyl Oxandrolone
Oxprenolol Oxycodone
Oxymesterone
Oxymorphone Pentazocine
Pentetrazole Pethidine
Phendimetrazine Pindolol

K. Gorynski / Trends in Analytical Chemistry 112 (2019) 135e146

Piretanide Prednisolone
Prednisone Probenecid
Procaterol Propranolol
Prostanozol Raloxifene
Ritalin Salbutamol
Salmeterol Selegiline
Sotalol Stanozolol
Strychnine Tamoxifene
Terbutaline Testosterone
THC Timolol Torasemide
Toremifene Trenbolone
Triamcinolone
Triamcinolone acetonide
Triamcinolone diacetate
Zilpaterol

Abbreviations: C18 Octadecylsilane particles; HLB hydrophilic-lipophilic balance adsorptive particles; PAN polyacrylonitrile; TFME Thin Film MicroExtraction; PEEK poly(etheretherketone); CBS Coated Blade Spray; LC Liquid
Chromatography; MS/MS Tandem Mass Spectrometry; DART Direct Analysis in Real Time; SPME Solid Phase MicroExtraction.
Photo of SPME devices reprinted with permission from [35e37,42e44,59] American Chemical Society and Wiley and Elsevier.

141
142 K. Gorynski / Trends in Analytical Chemistry 112 (2019) 135e146

well as the time needed for analysis in comparison to traditional for over 49 prohibited drugs, using DI-TFME C18 operating on the
methods. While urine is considered a reasonably simple matrix Concept 96 robotic system. An additional approach was success-
when compared to other biological samples, and although it pro- fully employed using biocompatible commercial SPME C18 fibers
vides an easy and non-invasive sample collection process, identi- (Fig. 4; manuscript under preparation) coupled to LC-MS, the very
fication and quantification of prohibited substances and drugs of sensitive Shimadzu LC-MS/MS 8060 system for targeted analysis
abuse often proves to be difficult due to the varying salt content and high-resolution mass spectrometry QExactive Focus (Thermo)
(ionic strength) of urine, the dietary habits (pH) of the patient, as for untargeted analysis toward comparison two sampling (spitting
well as the various metabolite forms of drug excretion and the and Salivette) and sample preparation (SPME and Protein Precipi-
subsequent low concentrations of the corresponding drug. Taking tation) techniques. In addition, given that SPME integrates
into account the above difficulties, the SPME method would seem several sample preparation steps into one, thus simplifying the
to be an appropriate method for detection and quantification in this whole analytical workflow, while offering various geometries and
matrix due to its non-exhaustive extractive nature, which biocompatible probes, the SPME platform thus presents great po-
allows for single-analysis, simultaneous determination of various tential for in-vivo and/or on-site sampling and metabolomics
glucoronated conjugates of analytes, as well as polar or non-polar analysis of saliva matrix [41]. Simultaneous quantitative analysis of
drugs. SPME also optionally allows for removal of additional 46 prohibited drugs from saliva matrix based on C18 TFME and C18
error-prone deconjugation steps from the sample preparation SPME demonstrated great reproducibility, accuracy, and precision.
protocol. An interesting alternative procedure for fast urine sample Results on Fig. 4 (manuscript under preparation) from untargeted
analysis of cocaine and methadone presence was presented by approach coincide with observations obtained from targeted
Rodriguez-Lafuente [47] and co-workers. The developed micro- analysis (precision, accuracy, and reproducibility), while addition-
extraction protocol included TFME C18-PAN-coated mesh, 10 min ally demonstrating better performance than the traditional protein
extraction from 5 ul of urine sample, and direct introduction in the precipitation method. Recently, Vasiljevic et al. [43] developed an
LC-MS instrument, where the desorption step was coupled with SPME method comprised of a plastic mesh coated with hydrophilic-
direct ionization and introduction into the system. Improvements lipophilic balance particle polyacrylonitrile (HLB-BAN) for rapid
to SPME devices adapted to the front of the DART source were screening and quantification of drugs of abuse from saliva and urine
recently presented by Gomez-Rios and Pawliszyn [48]. As proof of via DART-MS/MS. The results obtained for the 13 selected drugs of
concept, experiments were based on 21 prohibited substances abuse, including limits of detection ca. 0.5 ng mL 1, linearity 0.99,
spiked on PBS at 20 ng mL 1, offering for most compounds limits of and accuracy of 80e120% within the 0.5e200 ng mL 1 range,
detection in the low pg mL 1 level in less than 3 min, with great proved its potential usefulness as a quick screening method suit-
reproducibility below 5%. Most recently, electrochemically co- able to on-site and in-vivo settings for use by the pharma industry
deposited solegel/Cu nanocomposites has been introduced as a and toxicological laboratories e.g. anti-doping controls, roadside, or
novel, simple and single-step technique for preparation of solid- workplace drug testing.
phase microextraction (SPME) coating applied to extraction of Less common matrices, such as hair and sweat/skin, may offer
drugs from biological matrices. Nanocomposite fiber was compared unique advantages over blood, urine, or saliva, including among
with previously reported techniques for determination of metha- them their comparatively easier and less invasive collection. Hair
done in urine samples presented good figures of merit even when can be applied for long-term monitoring of intake of drugs of abuse
combine with the HPLC-UV system (Table 2) showing the detection over time [51]. Given the nature of hair as a solid matrix, an HS-
limit about 0.2 ng mL 1 [49]. SPME-GC-MS platform presents itself as ideal for analysis of this
Reyes-Garces et al. [36] first developed a protocol towards high- matrix. In addition to affording advantages such as high purity of
throughput quantification of prohibited substances in plasma extract, this technique is able to detect compounds from small
(Table 1), which demonstrated acceptable extraction capabilities amounts of biological material. In this respect, a few studies of HS-
for 25 drugs using biocompatible hydrophilic-lipophilic balance SPME analysis of hair have been reported to date, mostly applied to
(HLB) TFME. As a continuation of this work, the authors proposed drug of abuse determinations (mainly cannabinoids) [52]. An
utilization of a stable plastic material (polybutyleneterepthalate) as example of such study entails work carried out by Moosmann et al.
a support for the HLB particles as a practical and inexpensive means [53]. In this work, the authors coupled SPME methods to different
to meet single-use, disposable device requirements [37]. It should types of separation and detection techniques (LC-MS, GC-MS, GC-
be stressed that the SPME technique additionally enables the FID), comparing all three sample preparation methods at the same
monitoring of both the free and bound fractions of compounds of time. Interestingly, based on results from analysis of hair samples
interest [39] in blood and plasma matrices without need of addi- from 41 children, 4 teenagers, and 34 drug-consuming parents,
tional steps during sample preparation. THCA-A (Delta-9-Tetrahydrocannabinolic acid A) was indicated as a
Saliva is considered as an alternative matrix for drug analysis marker that allows differentiation between external contamination
that can be obtained in a non-invasive manner without discrimi- and systemic uptake. In contrast, less frequently used DI-SPME
nation privacy during collection. Compared to urine, saliva samples analysis of hair has been reported by Gambelunghe et al. [54].
are more difficult to adulterate; as well, saliva offers a much lower Authors reported results obtained in 90 hair samples from addicts
risk of infection due to sample collection in comparison to blood, as using gas chromatography mass spectrometry techniques coupled
well as richer information regarding recent drug consumption [50]. with direct immersion solid-phase microextraction. All samples
Several protocols based on SPME have been developed to date for were analyzed toward presence of cocaine, benzoylecgonine, nor-
analysis of drugs of abuse in saliva [45], including applications for cocaine, cocaethylene, and tropococaine. Another interesting
measurement of five amphetamine-type stimulants using DI-SPME application of SPME regards its use towards analysis of volatiles
PDMS fibers and GC-MS analysis after derivatization, DI-SPME MIP from skin. In work by Jiang et al. [55], instead of exposing flexible
coatings applied for extraction of methamphetamine analysis PDMS sheets in direct contact with skin, a method involving a
without derivatization, and an automated on-line in-tube SPME for special sampling device for skin HS sampling was developed as a
hormone detection (testosterone, dehydroepiandrosterone) for means to avoid contamination of the GC-MS instrument by addi-
sensitive sample analysis. More comprehensive studies entailing tional adsorption of fatty acids and higher lipids present on the
doping control analysis were performed by Bessonneau et al. [42]. surface of skin. Another newer, less common, but nonetheless
The proposed protocol (Table 1) demonstrated good performance important application of SPME entails analysis of compounds in
Table 2
Representative Solid Phase Microextraction application for determination of different drug of abuse since 2016.

Analyte Abbreviation Matrix Method Phase material Analytical LOD LOQ Linear range Year of
method publication
Ref.

D9-tetrahydrocannabinol THC Skin IT-SPME TBR-5 column (95% polydimethyl nanoLC with 2 ng/mL 8 ng/mL 8e100 ng/mL 2018 [31]
siloxane, 5% polydiphenylsiloxane) UVeVis DAD
Cannabidiol CBD Skin IT-SPME TBR-5 column (95% polydimethyl nanoLC with 5 ng/mL 15 ng/mL 15e100 ng/mL 2018 [31]
siloxane, 5% polydiphenylsiloxane) UVeVis DAD
Cannabinol CBN Skin IT-SPME TBR-5 column (95% polydimethyl nanoLC with 5 ng/mL 15 ng/mL 15e100 ng/mL 2018 [31]
siloxane, 5% polydiphenylsiloxane) UVeVis DAD
±-Amphetamine AMP Urine HS-SPME Oxidized multiwalled carbon GC-MS 0.3 ng/mL 1 ng/mL 1e1000 ng/mL 2018 [22]
nanotubes
±-Methamphetamine MAMP Urine HS-SPME Oxidized multiwalled carbon GC-MS 0.2 ng/mL 0.7 ng/mL 0.5e1000 ng/mL 2018 [22]
nanotubes
3, 4-Methylenedioxyamphetamine MDA Urine HS-SPME Oxidized multiwalled carbon nanotubes GC-MS 1.3 ng/mL 4.3 ng/mL 4e1000 ng/mL 2018 [22]

K. Gorynski / Trends in Analytical Chemistry 112 (2019) 135e146

(±)-3, 4 Methylenedioxymethamphetamine MDMA Urine HS-SPME Oxidized multiwalled carbon nanotubes GC-MS 0.8 ng/mL 2.7 ng/mL 2e1000 ng/mL 2018 [22]
(±)-3,4- Methylenedioxy-N ethylamphetamine MDEA Urine HS-SPME Oxidized multiwalled carbon nanotubes GC-MS 0.7 ng/mL 2.4 ng/mL 2e1000 ng/mL 2018 [22]
Phentermine PTM Urine HS-SPME Oxidized multiwalled carbon nanotubes GC-MS 0.2 ng/mL 0.7 ng/mL 0.5e1000 ng/mL 2018 [22]
Methadone MDN Urine DI-SPME 3-(Trimethoxysilyl) propyl HPLC-UV/VIS 0.2 ng/mL 0.8 ng/mL 0.8e50 ng/mL 2017 [49]
methacrylate -based solegel
Cocaine COC hair DI-SPME polydimethylsiloxane GC-MS 0.01 ng/mg 0.02 ng/mg 0.02e40 ng/mg 2017 [54]
D9-tetrahydrocannabinol THC Breast milk HS-SPME PDMS GC-MS 10 ng/mL 20 ng/mL 20e200 ng/mL 2017 [23]
Cannabidiol CBD Breast milk HS-SPME PDMS GC-MS 10 ng/mL 20 ng/mL 20e200 ng/mL 2017 [23]
Cannabinol CBN Breast milk HS-SPME PDMS GC-MS 10 ng/mL 20 ng/mL 20e200 ng/mL 2017 [23]
Amphetamine AMP Sweat HS-SPME PDMS GC-MS 0.27 ng/pad 0.81 ng/pad 0.05e10 ng/mg 2016 [24]
Methamphetamine MAMP Sweat HS-SPME PDMS GC-MS 0.21 ng/pad 0.63 ng/pad 0.05e10 ng/mg 2016 [24]
3,4 methylenedioxyamphetamine MDA Sweat HS-SPME PDMS GC-MS 0.13 ng/pad 0.39 ng/pad 0.05e10 ng/mg 2016 [24]
Ketamine KET Sweat HS-SPME PDMS GC-MS 0.07 ng/pad 0.21 ng/pad 0.05e10 ng/mg 2016 [24]
3,4 methylenedioxymethamphetamine MDMA Sweat HS-SPME PDMS GC-MS 0.09 ng/pad 0.27 ng/pad 0.05e10 ng/mg 2016 [24]
3,4-methylenedioxy-N-ethylamphetamine MDEA Sweat HS-SPME PDMS GC-MS 0.15 ng/pad 0.35 ng/pad 0.05e10 ng/mg 2016 [24]
-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine MBDB Sweat HS-SPME PDMS GC-MS 0.12 ng/pad 0.36 ng/pad 0.05e10 ng/mg 2016 [24]
Methadon MDN Sweat HS-SPME PDMS GC-MS 0.08 ng/pad 0.24 ng/pad 0.05e10 ng/mg 2016 [24]
Cocaine COC Sweat HS-SPME PDMS GC-MS 0.07 ng/pad 0.21 ng/pad 0.05e10 ng/mg 2016 [24]
Cocaethylene COE Sweat HS-SPME PDMS GC-MS 0.10 ng/pad 0.30 ng/pad 0.05e10 ng/mg 2016 [24]
D9-Tetrahydrocannabinol THC Sweat HS-SPME PDMS GC-MS 0.09 ng/pad 0.27 ng/pad 0.01e10 ng/mg 2016 [24]
Amphetamine AMP Oral fluid HS-SPME PDMS GC-MS 0.37 ng/pad 1.11 ng/pad 0.25e5 ng/pad 2016 [25]
Methamphetamine MAMP Oral fluid HS-SPME PDMS GC-MS 0.68 ng/pad 2.04 ng/pad 0.25e5 ng/pad 2016 [25]
3,4 methylenedioxyamphetamine MDA Oral fluid HS-SPME PDMS GC-MS 0.60 ng/pad 1.80 ng/pad 0.25e5 ng/pad 2016 [25]
Ketamine KET Oral fluid HS-SPME PDMS GC-MS 0.73 ng/pad 2.19 ng/pad 0.25e5 ng/pad 2016 [25]
3,4 methylenedioxymethamphetamine MDMA Oral fluid HS-SPME PDMS GC-MS 0.34 ng/pad 1.02 ng/pad 0.25e5 ng/pad 2016 [25]
3,4-methylenedioxy-N-ethylamphetamine MDEA Oral fluid HS-SPME PDMS GC-MS 0.26 ng/pad 0.78 ng/pad 0.25e5 ng/pad 2016 [25]
-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine MBDB Oral fluid HS-SPME PDMS GC-MS 0.11 ng/pad 0.33 ng/pad 0.25e5 ng/pad 2016 [25]
Methadon MDN Oral fluid HS-SPME PDMS GC-MS 0.71 ng/pad 2.13 ng/pad 0.25e5 ng/pad 2016 [25]
Cocaine COC Oral fluid HS-SPME PDMS GC-MS 0.35 ng/pad 1.05 ng/pad 0.25e5 ng/pad 2016 [25]
Cocaethylene COE Oral fluid HS-SPME PDMS GC-MS 0.51 ng/pad 1.53 ng/pad 0.25e5 ng/pad 2016 [25]
D9-Tetrahydrocannabinol THC Oral fluid HS-SPME PDMS GC-MS 0.06 ng/pad 0.18 ng/pad 0.05e1 ng/pad 2016 [25]
Codeine COD Oral fluid HS-SPME PDMS GC-MS 0.06 ng/pad 0.18 ng/pad 0.25e5 ng/pad 2016 [25]
Morphine MOR Oral fluid HS-SPME PDMS GC-MS 0.43 ng/pad 1.29 ng/pad 0.25e5 ng/pad 2016 [25]
6-monoacetylmorphine 6-MAM Oral fluid HS-SPME PDMS GC-MS 0.39 ng/pad 1.17 ng/pad 0.25e5 ng/pad 2016 [25]

Abbreviations: IT In-Tube; HS Head Space; DI Direct Immersion; SPME Solid Phase MicroExtraction; TBR Poly(dimethyldiphenyl)siloxane; PDMS polydimethylsiloxane; LC Liquid Chromatography; UV Ultra Violet; VIS; DAD
Diode Array Detector; GC Gas Chromatography; MS Mass Spectrometry; LOD Limit of Detection; LOQ Limit of Quantification; ‘pad’ cotton (0.5  0.5 cm) the part of DrugWipe®5 device allowed the simultaneous detection of
Opiates, Cocaine, Amphetamines/Methamphetamines (Ecstasy) and Cannabis.

143
144 K. Gorynski / Trends in Analytical Chemistry 112 (2019) 135e146

Fig. 4. Simultaneous quantitative analysis of 46 prohibited drugs from saliva matrix based on C18 TFME coupled to LC-MS/MS (Shimadzu LC-MS 8060), on top; biocompatible
commercial SPME C18 fibers coupled to LC-HRMS, (Q Exactive Focus ThermoFisher) for untargeted analysis toward comparison two sampling (spitting and Salivette) and sample
preparation (SPME and PP) techniques, on bottom.

exhaled breath. SPME methods in head-space configurations using exhaled breath volatiles has mainly focused on disease diagnosis
mainly CAR/PDMS and DVB/CAR/PDMS have been broadly utilized and prognosis. Breath analysis is a non-invasive approach capable
to analyze several exhaled volatile organic compounds (VOCs) of targeting solitary volatile compounds that reflect concentrations
identified by GC-MS [45]. However, none of them have yet to be present in both the bloodstream and airways, thus providing
applied directly into doping control; to date, research in the field of information about both external exposures and endogenous

Fig. 5. Comparison between most relevant SPME-MS technologies developed recently (Abbreviations: OPP Open Port Probe; DBDI Dielectric Barrier Desorption Ionization; CBS
Coated Blade Spray; DART Direct Analysis in Real Time; nanoESI nano-electrospray ionization; SPME Solid Phase MicroExtraction; MS Mass Spectrometry; MS/MS Tandem Mass
Spectrometry; LOQ Limit of Quantification; S/N signal-to-noise ratio). Reprinted with permission [12].
 K. Gorynski / Trends in Analytical Chemistry 112 (2019) 135e146 145

metabolism. Recently, O. Beck et al. demonstrated detection of quick method optimization for the drugs and metabolites analysis
drugs of abuse in EBC carried out from patients following recovery by LC-MS (grant no. 2013/11/N/ST4/01017) attributed by the Na-
from intoxication [56]. Given this, VOCs testing is thus posed as a tional Science Centre, Poland.
potential rapid on-site control for clinical exercise health moni-
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