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Microextractions in forensic toxicology: The potential role of ionic liquids
Accepted Manuscript Microextractions In Forensic Toxicology: The Potential Role Of Ionic Liquids Marieke De Boeck, Wim Dehaen, Jan Tytgat, Eva Cuypers PII:
S0165-9936(18)30355-8
DOI:
https://doi.org/10.1016/j.trac.2018.11.036
Reference:
TRAC 15338
To appear in:
Trends in Analytical Chemistry
Received Date: 6 August 2018 Revised Date:
21 November 2018
Accepted Date: 22 November 2018
Please cite this article as: M. De Boeck, W. Dehaen, J. Tytgat, E. Cuypers, Microextractions In Forensic Toxicology: The Potential Role Of Ionic Liquids, Trends in Analytical Chemistry, https://doi.org/10.1016/ j.trac.2018.11.036. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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MICROEXTRACTIONS IN FORENSIC TOXICOLOGY: THE POTENTIAL ROLE OF IONIC LIQUIDS Authors
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Marieke De Boecka, Wim Dehaenb, Jan Tytgata, Eva Cuypersa Toxicology and Pharmacology, Department of Pharmaceutical and Pharmacological
Sciences, University of Leuven (KU Leuven), Campus Gasthuisberg, O&N II, P.O. Box 922, Herestraat 49, 3000 Leuven, Belgium
Molecular Design and Synthesis, Department of Chemistry, University of Leuven (KU
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b
ORCID:
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Leuven), Campus Arenberg, P.O. Box 2404, Celestijnenlaan 200F, 3001 Leuven, Belgium
Marieke De Boeck
0000-0001-7910-7465
Wim Dehaen
0000-0002-9597-0629
Jan Tytgat
Corresponding author
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Prof. Dr. Eva Cuypers
0000-0002-6411-2461
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Eva Cuypers
Toxicology and Pharmacology
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Department of Pharmaceutical and Pharmacological Sciences University of Leuven (KU Leuven) Campus Gasthuisberg, O&N II Herestraat 49 – P.O. Box 922 3000 Leuven BELGIUM Tel: + 32 16 32 34 03 Fax: + 32 16 32 34 05 Email: [email protected]
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MICROEXTRACTIONS IN FORENSIC TOXICOLOGY:
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THE POTENTIAL ROLE OF IONIC LIQUIDS Abstract
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In forensic toxicology, scientists are faced with complex samples and trace concentrations of low-dose
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legal and illegal drugs. To enable accurate and sensitive analysis, a thorough sample preparation step
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is crucial. In the context of green chemistry, sample preparation techniques are rapidly evolving.
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Microextractions are introduced to avoid the use of large solvent volumes and obtain high analyte
8
enrichment. Another trend is the search for alternative solvents to replace toxic and volatile organic
9
solvents. Ionic liquids (ILs) - liquid salts - are proposed as promising alternatives, thanks to their low
10
volatility, low flammability, good thermal and chemical stability. Moreover, the IL structure can be
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altered by combining different cations and anions, resulting in ILs with optimized physicochemical
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properties for the extraction of a specific analyte. This review describes the current state of
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microextraction techniques that apply ILs for the extraction of forensically relevant drugs in biological
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samples.
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15 Keywords
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Analytical toxicology; Forensic toxicology; Ionic liquids; Microextractions; Biological samples;
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Sorbent; Solvent
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Abbreviations
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EC2MIm: 1-ethoxyethyl-3-methylimidazolium; EMIm: 1-ethyl-3-methylimidazolium; BF4: tetra-
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fluoroborate; BMIm: 1-butyl-3-methylimidazolium; Br: bromide; C2OHMIm: 1-hydroxyethyl-3-
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methylimidazolium; Cl: chloride; DAD: diode array detector; DLLME: dispersive liquid-liquid
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microextraction; dLPME: dynamic liquid-phase microextraction; ESI: electrospray ionization; GC:
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gas chromatography; HF-LPME: hollow fiber liquid-phase microextraction; HMIm: 1-hexyl-3-
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methylimidazolium; IL: ionic liquid; ATPS: aqueous two-phase system; ISFME: in situ solvent
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formation microextraction; LC: liquid chromatography; LOD: limit of detection; LPME: liquid-phase
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microextraction; ME: matrix effect; MEPS: microextraction by packed sorbent; MS: mass
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spectrometry; NaCl: sodium chloride; OH: hydroxide; OMIm: 1-octyl-3-methylimidazolium; PF6:
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hexafluorophosphate; PMIm: 1-pentyl-3-methylimidazolium; RE: recovery; RTIL: room-temperature
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ionic liquid; SDME: single drop microextraction; SPME: solid-phase microextraction; SBSE: stir bar
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sorptive extraction; Tf2N: bis(trifluoromethylsulfonyl)imide; TiO2: titanium dioxide; TSIL: task-
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specific ionic liquid; UA-IL-SE-ME: ultrasound-assisted ionic liquid surfactant-based emulsification
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microextraction
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1. Introduction In forensic toxicology, biological samples are analyzed to determine the presence of xenobiotics and
3
their metabolites in the human body. Both qualitative and quantitative results are obtained, interpreted
4
and finally reported in judicial proceedings. To obtain accurate results, appropriate analytical methods
5
are developed and validated for a specific forensic application [1]. They typically consist of an initial
6
sample preparation step, followed by the analysis of the final extract using an appropriate analytical
7
instrument. Sample preparation is considered the most time-consuming and error-inducing step in the
8
bioanalytical process. However, it remains indispensable, as it delivers compatible samples with the
9
detection instrument and reduces matrix interferences, such as endogenous compounds (e.g. proteins,
10
lipids). Furthermore, sample preparation can lead to analyte enrichment and increased method
11
sensitivity [2,3].
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Forensic toxicology is highly demanding in terms of sample preparation. It requires sensitive and
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multi-analyte procedures, since in many cases, only a limited amount of sample is available and the
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substances of interest are often taken in combination and at low doses. Moreover, complex sample
16
matrices may pose additional challenges for accurate analysis. For routine toxicological examination,
17
often peripheral blood, urine and gastric fluid are analyzed, however; hair and oral fluid are also
18
collected, due to extended drug surveillance windows and ease of on-site sampling, respectively [1].
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Generally, biological samples contain a wide range of endogenous and exogenous substances that can
20
hamper analyte quantification and identification. For instance, blood proteins or phospholipids are
21
abundantly present and can result in viscous samples that are difficult to handle, or they can hamper
22
analyte ionization using atmospheric pressure ionization sources [4]. Even more challenging is the
23
analysis of postmortem samples. For instance, postmortem blood is often putrefied, clotted,
24
hemolyzed, contaminated with tissue fluid and analytes undergo redistribution processes. All these
25
factors make it almost impossible to accurately determine drug concentrations and interpret the
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obtained results [5].
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To date, a broad diversity of sample preparation techniques are applied, using different extraction
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solvents and sorbents. Nevertheless, there is a persistent need for efficient and cost-effective methods
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that take into consideration environmental aspects [2,3,6]. Microextractions were introduced in 1990,
31
by Arthur and Pawliszyn, as a solution for tedious procedures with high solvent consumption [7].
32
They are considered green alternatives for well-known and established sample preparation techniques,
33
such as liquid-liquid extraction (LLE) and solid-phase extraction (SPE). Microextractions are
34
characterized by the consumption of small solvent volumes (µL range) and are in some cases even
35
solvent free. This leads to a reduction of hazardous volatile organic solvent (VOS) consumption, a
36
reduction of waste and high enrichment of the analyte. Additionally, microextractions generally
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consist of simplified protocols that lower the chance of manual errors [8].
2 Next to the microextraction trend, the possibility of using alternative extraction solvents has been
4
studied. Ionic liquids (ILs) were introduced in analytical chemistry to lower the environmental and
5
toxic burden of VOSs, but most importantly, to increase the current solvent functionality range [9]. To
6
date, only 600 VOSs are available, while more than one billion possible ILs can be created [9]. ILs
7
differ from conventional molecular solvents as they are purely composed of ions. However, they
8
cannot be categorized as conventional salts due to the presence of organic ions and their liquid state
9
below 100°C [10]. Some ILs even melt at/below room temperature, these are referred to as room-
10
temperature ionic liquids (RTILs) [11]. Thanks to their low volatility and low flammability, ILs hold
11
great promise as extraction solvents in bioanalytical research. Additionally, their easy tunable nature
12
allows to create a multitude of ILs with a wide variety of physicochemical properties. With relatively
13
easy chemical processes (e.g. metathesis or acid-base neutralization), certain functionalities can be
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incorporated into their chemical structure [12]. This is probably the most useful advantage of ILs,
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since it allows the design of TSILs with improved applicability for a specific task [13].
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This review focusses on the use of ILs in forensic toxicology, more specifically, as solvents in
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microextraction procedures. First, an introduction is provided on the properties and advantages of the
19
novel IL solvents. Second, principles of the most relevant solid-phase and liquid-phase
20
microextraction techniques are outlined. Third, an overview is provided of research performed on IL-
21
based microextractions for the analysis of forensically relevant substances in biological samples.
22
Finally, a conclusion is formulated, combined with a critical look at the future of ILs in forensic
23
toxicology.
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In the following section, conventional organic solvents and ionic liquids are discussed, with the
27
emphasis on the potential of ILs and their physicochemical properties.
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2.1. Conventional solvents
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A solvent is characterized by its liquid state and ability to dissolve or extract other substances, without
31
changing their chemical appearance [14]. They are extensively consumed in chemical and
32
pharmaceutical industry, for example in synthesis processes [15]. Also in sample preparation, solvent
33
extractions are frequently performed using organic solvents [16]. Traditionally, VOSs (e.g.
34
chloroform, diethyl ether, hexane, methanol) are used, as they can be easily obtained at low cost.
35
However, care should be taken when working with VOSs, due to their health and environmental
36
hazards [17]. People are mainly exposed to VOSs via inhalation. Acute effects can be headache, 3
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Long-term exposure cases report the carcinogenic impact of some organic solvents. Among
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youngsters, VOSs are even abused as legal highs [14]. Furthermore, VOSs are easily released in the
4
environment and aquatic systems, owing to their high volatility, thereby potentially severely harming
5
essential microorganisms. Additionally, with the use of VOSs comes the risk for ignition and
6
explosion [18]. It is clear that conventional VOSs need to be replaced in solvent extraction processes,
7
as they largely affect the safety and “greenness” of such procedures. To date, several strategies have
8
been proposed: the use of organic solvents with improved biodegradability and safety, organic
9
solvents that are produced with renewable energy, supercritical fluids and the use of solvents that are
10
less emitted into the environment, thanks to their negligible vapor pressures [17]. With respect to this
11
last category, ILs were introduced as credible alternatives [10].
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2.2. Ionic liquids 2.2.1.
History
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High temperature molten salts are considered the foundation of current IL chemistry. They are defined
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by melting points above 100°C [19]. Initially, these molten salts were used as electrolytes, however,
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they were considered hard to work with and a great deal of energy was needed to maintain their liquid
18
state. As a logical consequence, scientists started looking for salts with lower melting points. The first
19
low melting salt (i.e. IL) was synthesized in 1914 by Paul Walden, namely, ethylammonium nitrate. It
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took until the end of World War II for ILs to finally break through. The class of imidazolium- and
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pyridinium-based chloroaluminates formed the first-generation ILs and triggered genuine attention of
22
electrochemists. However, the wide applicability of these chloroaluminates was hampered, due to their
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water-reactive nature. In 1992, Wilkes and Zaworotko introduced ILs that consisted of a 1-ethyl-3-
24
methylimidazolium cation, combined with different anions. These were called second-generation ILs
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and were air- and water-stable compounds [10,20]. Currently, IL research focusses on developing less
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toxic and more renewable ILs, e.g. using renewable resources such as sugars and amino acids (third-
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generation ILs) [10]. Also, the task-specific design of ILs (task-specific ILs (TSILs)) is being
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extensively explored, thanks to the easily tunable physiochemical characteristics of ILs [13].
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2.2.2.
Physicochemical properties
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ILs are low temperature melting salts. Their low melting points can be attributed to small lattice
31
energies, as a result of bulky and asymmetric ions with large conformational flexibilities [21]. Figure
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1. shows the physical appearance of a conventional salt (sodium chloride; NaCl) and a commonly used
33
RTIL (1-butyl-3-methylimidazolium hexafluorophosphate; BMIm PF6) at room temperature. In
34
conventional salts, strong Coulombic interactions form compact lattice structures and result in solid
35
state salts, while ILs have larger cations and/or anions, leading to a lower charge density and less
36
pronounced Coulombic interaction [22]. Next to low melting points, ILs display negligible vapor 4
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the environment. Furthermore, IL viscosities differ from typical organic solvents. In general, ILs have
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viscosities ranging from 10 to 40,000 mPa⋅s, while organic solvent viscosities are much lower, ranging
4
up to 100 mPa⋅s. For instance, alkyl chain length is directly proportional to viscosity [22,23]. IL
5
densities are typically higher than those of organic solvents. This can be related to heavy anions that
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are often incorporated in the IL structure (e.g. bis(trifluoromethylsulfonyl)imide (Tf2N),
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hexafluorophosphate (PF6)) [22,23]. Extra IL advantages are their high thermal stability and low
8
flammability. Moreover, ILs have the ability to solvate a wide range of molecules [22,23]. The
9
solvation strength or polarity of ILs is often expressed by means of three Kamlet-Taft solvation
10
parameters: hydrogen bond acidity (α), hydrogen bond basicity (β) and polarizability/dipolarity (π*)
11 12 13
[24].
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There is an almost infinite number of possible anion-cation combinations that can be formed.
15
Furthermore, ILs can be tuned to obtain desirable physicochemical properties, as certain
16
functionalities can be incorporated into their structure, for instance, additional aromatic rings,
17
hydroxyl groups, etc. This is probably the most useful advantage of ILs, since it allows the design of
18
TSILs with improved applicability for a specific task [13]. Which ions are typically used in IL
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systems? As a cation, imidazolium is the most documented and sold IL class. Furthermore,
20
ammonium, pyridinium, pyrrolidinium and phosphonium are also frequently applied [22,25]. As an
21
anion, tetrafluoroborate (BF4), PF6, Tf2N, tosylate, acetate and halides are used [22,25]. Figure 2.
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shows their chemical structures. Most ILs are synthesized using straightforward techniques; either by
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metathesis of a cation-halide salt and metal-anion salt or by acid-base neutralization [12].
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Overall, the mentioned physicochemical properties largely depend on the selected cation and anion.
24 2.2.3.
Applications
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ILs are applied in different scientific fields: analytical chemistry [26–28], electrochemistry [25,29],
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organic chemistry [25,30], material science [31] and even in medicine [32–34]. Relevant to this review
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is their use in analytical chemistry, more specifically, their application as extraction solvents and
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sorbents. In this case, high specificity and affinity for the analyte is desired. These properties can be
30
optimized by altering the chemical structure of the IL solvent, thereby creating designer solvents.
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Furthermore,
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desorption/ionization (MALDI) of both proteins and small organic molecules. Their non-crystalline
33
characteristics allow more homogenous spots to be sampled with improved sensitivity [26]. Other
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popular IL applications in analytical chemistry are their use as organic modifiers in mobile phases to
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optimize separations in liquid chromatography (LC) and as modifiers of capillary column coatings in
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both gas chromatography (GC) and capillary electrophoresis (CE) [27].
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based systems have been reported. BASF has been a pioneer when it comes to the implementation of
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ILs. In 2002, they developed the BASILTM process in which 1-methylimidazole is used to scavenge
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unwanted acids. As a result, an IL is formed that can be easily separated from the reaction mixture
5
[25,35]. Overall, IL popularity is gradually increasing, as can be deduced from the large amount of IL
6
patent submissions. With different types of ILs entering the market, a larger diversity of
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physicochemical properties is offered that greatly exceeds the potential of only 600 VOSs that are
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available today [9].
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Toxicity and environmental impact
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Throughout the years, ILs have been presented as greener alternatives for VOSs. ILs are considered to
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be less toxic and have a negligible impact on the environment. However, this statement is solely based
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on their negligible volatilities that limit their release into the atmosphere. They can be spilled into the
14
environment by disposal into aquatic systems, followed by their adsorption onto soil [36]. This can
15
have negative consequences, since studies have demonstrated IL toxicity toward bacterial and even
16
mammalian cells. Results show that second-generation ILs are not as harmful as initially presumed.
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Their toxicity is directly linked to their chemical structure and lipophilicity. ILs with high lipid
18
solubility are more likely to interfere with cell membranes and result in cell leakage or even cell death
19
[36–38]. A clear structure-toxicity relationship has been reported. For example, long alkyl side chains
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and fluorine-containing anions should be avoided. Likewise, biodegradability can be directly linked to
21
the IL’s structural features [36–38]. These features can be easily tuned to obtain safer and greener
22
solvents, e.g. cholinium and amino acid-based ions. Moreover, renewable resources, such as sugars,
23
are increasingly used to establish a greener image [36,37]. To date, the active removal of ILs from
24
industrial wastewater mainly relies on adsorption and oxidation techniques [36]. In order to reduce IL
25
waste, researchers have reported recycle processes to enable their reuse, such as extraction with
26
supercritical CO2 and distillation processes [39]. In case reuse is not feasible, ILs are disposed as
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organic waste and degraded via oxidation. Several publications report the adequate degradation of ILs
28
by advanced oxidation processes consisting of UV treatment in the presence of oxidative catalysts
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[40].
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3. Sorbent-based microextractions
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The following section discusses three established sorbent-based microextraction techniques: solid-
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phase microextraction (SPME), stir bar sorptive extraction (SBSE) and microextraction by packed
34
sorbent (MEPS). Additionally, the application of ILs as promising sorbents and their implementation
35
in forensic toxicology are presented.
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3.1. General extraction principles The introduction of solid-phase microextractions (SPMEs) in 1990, symbolizes the beginning of a
3
new era in sample clean-up. SPME is a solventless technique that uses a syringe, on which a coated
4
fused silica fiber is mounted. This fiber can be lowered into a liquid sample (direct-immersion) or
5
headspace to obtain analyte adsorption. The analyte can be eluted from the fiber with a solvent or it
6
can be directly introduced into a GC device [41–43]. Stir bar sorptive extraction (SBSE) is another
7
popular solventless microextraction technique. It uses a magnetic stir bar that is coated with a layer of
8
sorbent. The bar is introduced into a liquid sample and the stirring process effectuates analyte
9
adsorption onto the sorbent surface. Next to liquid samples, SBSE can also be applied for headspace
10
extractions [8,43]. Microextraction by packed sorbent (MEPS) is a more recently introduced (2004)
11
technique for sample clean-up. The sorbent is packed as a small plug inside of a syringe. By pulling
12
the plunger up- and downwards, the plug can be conditioned, loaded, washed and eluted. MEPS can be
13
defined as a small-scale variant of conventional SPE techniques [8,43,44]. Figure 3. gives a schematic
14
overview of the sorbent-based procedures. Advantages and disadvantages are listed in Table 1.
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3.2. Ionic liquids as sorbents
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In recent years, ILs have been incorporated as solid supports for sorbent-based extraction techniques.
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Especially in SPME and SBSE applications, the possibility of implementing ILs is increasingly
20
studied. Their unique physicochemical properties can be exploited to obtain attractive solid supports
21
with improved selectivity. Appropriate ILs should have good thermal stability, high viscosity and high
22
affinity for the analyte. The first two properties are required to avoid decomposition or leakage of the
23
IL due to high GC injector operating temperatures. In both cases, the nature of the anion and cation
24
should be carefully considered, as is described by Tien D. Ho et al. [45].
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The preparation of the IL-based SPME fibers can be done in various ways. A first approach is to
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immobilize the IL onto a conventional silica-based fiber, by impregnation. This results in a physically
28
bonded phase. A disadvantage of physical bonding is the lower reusability of the fiber, compared to a
29
chemically bonded variant. The latter is reported for both silica and polymer supports [45,46]. Next to
30
the implementation of ILs as such, researchers have studied their polymerization in order to create
31
supports with higher viscosities and mechanical stability, referred to as polymeric ionic liquids (PILs).
32
In 2008, Zhao et al., described the first SPME application to use PILs for the extraction of esters. In
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this study, imidazolium-based polymers were prepared and coated onto a fused silica fiber by dipping.
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Next to viscosity and stability benefits, the tested PILs were found to give equal or superior recoveries,
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compared to commercial coatings [47]. Overall, the discussed SPME developments are promising and
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boost further research of task-specific IL/PIL coatings.
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2
developed for the extraction of NSAIDs from water, urine and milk, using a stir bar that was
3
chemically modified with 1-allylimidazolium tetrafluoroborate [48]. More recent research focusses on
4
the implementation of magnetic ionic liquids (MILs) to improve SBSE yields. The novel techniques
5
was developed by Chisvert et al. and is termed stir bar dispersive liquid microextraction (SBDLME).
6
The extraction mechanism is based on the magnetic attractive forces between the MIL and the
7
magnetic stir bar. The MIL is attracted toward the stir bar, however, during intense stirring, the MIL is
8
dispersed into the sample in order to create a high contact surface for good analyte transfer. After
9
extraction, the stirring process is ended and the analytes can be thermally desorbed from the stir bar
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[49].
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Table 2 summarizes publications that use ILs as sorbents in forensically relevant SPME procedures.
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So far, three SPME-based techniques have been reported for the analysis of benzodiazepines,
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psychostimulants and pesticides.
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For the extraction of amphetamine and methamphetamine from urine samples, He et al. described a
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headspace SPME method in which a silicone elastomer was chemically linked onto a fused silica fiber
19
[50]. Prior to cross-linkage, the elastomer was impregnated with a 1-ethoxyethyl-3-methylimidazolium
20
bis(trifluoromethylsulfonyl)imide (EC2MIm Tf2N) solution. The actual SPME extraction was
21
performed for 30 min, followed by the thermal desorption of the analytes and GC-MS analysis. The
22
authors reported no stability issues with the physically coated IL layer during desorption (220°C), with
23
a durability of more than 100 extractions. Validation results showed good repeatability and limits of
24
detection (LODs) ranging from 0.1 – 0.5 ng/mL. It should be noted that in real case urine samples,
25
amphetamine and methamphetamine are expected to be present at higher concentrations than 1 ng/mL,
26
more specifically, a urinary cutoff concentration of abuse of 200 ng/mL for amphetamine by
27
confirmatory test was set by the U.S. Substance Abuse and Mental Health Services Administration. To
28
validate the usability of the developed IL-SPME method in forensic practice, spiked urine samples
29
were analyzed (n=3) at four concentration levels (100 – 1000 ng/mL) and RSD values lower than
30
5.0% were obtained [50].
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Ebrahimi et al. developed a sol-gel based HF-SPME procedure for the extraction of pesticides from
33
hair samples [51]. The sol-gel sorbent was prepared based on 1-butyl-3-methylimidazolium hydroxide
34
(BMIM OH) mediated nanoparticles. Six µL of sol-gel sorbent was loaded into a hollow
35
polypropylene fiber, resulting in a disposable HF-SPME system. The hair samples were extracted
36
using methanol (5 h, 55°C) and the disposable device was immersed in the extract for 40 min, under
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continuous stirring. LC-DAD analysis was performed on the methanolic extract of the sample fiber.
2
Here, the IL was not only used as an extraction solvent, it rather served as a porogenous agent in order
3
to obtain a porous sol-gel system with an increased contact surface for efficient analyte extraction. The
4
developed method obtained LOD values lower than 0.08 ng/mL and recoveries ranging from 80 – 94%
5
for real case human hair samples [51]. The authors did not report the LOD values relating to the
6
analyte concentration in hair.
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Es’haghi et al. developed a similar hollow fiber solid-liquid-phase microextraction (HF-SLPME)
9
procedure for the extraction of four benzodiazepines (alprazolam, clonazepam, diazepam, lorazepam)
10
from urine and hair matrices [52]. In this study, titanium dioxide (TiO2) was studied as a potential
11
sorbent. The TiO2 extraction capacity was extended by enlarging its contact surface, thanks to the IL-
12
mediated synthesis of TiO2 nanoparticles. In a next step, 1-pentyl-3-methylimidazolium bromide was
13
coated onto the nanoparticles, from which a sol-gel system was prepared. The sol-gel was loaded into
14
a hollow fiber and could be used for extraction by immersing the device into the sample during 45
15
min. Methanol was used for desorption, followed by LC-UV analysis. The procedure obtained LOD
16
values lower than 0.10 ng/mg for the extraction of benzodiazepines from hair samples and relative
17
recoveries were within 93 – 103%. The analysis of urine samples gave relative recoveries within 45 –
18
106% [52].
19 4. Solvent-based microextractions
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The following section discusses three established solvent-based microextraction techniques, also
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termed liquid-phase microextraction (LPME): single drop microextraction (SDME), hollow fiber
23
liquid phase microextraction (HF-LPME) and dispersive liquid-liquid microextraction (DLLME).
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Additionally, the application of ILs as promising solvents and their implementation in forensic
25
toxicology
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4.1. General extraction principles
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Liquid microextractions are defined by the use of small solvent volumes; i.e. smaller than 100 µL[53].
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They were introduced in 1996, with the development of the single drop microextraction (SDME)
30
technique. SDME uses a single microdrop (< 10 µL) of extraction solvent that is positioned at the end
31
of a syringe needle. This drop is lowered into a liquid sample or headspace. After analyte extraction,
32
the drop is withdrawn into the syringe and introduced into an analytical measuring device. SDME has
33
received much attention, however, the instability of the drop has shown to be a major issue
34
[8,41,53,54]. Three years later, hollow fiber liquid phase microextraction (HF-LPME) was
35
developed to address the problem. In this method, a low volume (10 - 20 µL) of solvent is loaded
36
inside of a polymeric hollow fiber. In this way, a membrane is created that supports the solvent and 9
ACCEPTED MANUSCRIPT forms an additional barrier for large unwanted biomolecules. Finally, the acceptor phase is collected
2
and analyzed [8,41,53,54]. In 2004, dispersive liquid-liquid microextraction (DLLME) was
3
introduced. DLLME is characterized by simple and straightforward procedures, without the need for
4
fibers. A sample is injected with a combination of disperser and extraction solvent, resulting in a
5
turbid solution that consists of finely dispersed solvent microdroplets in the sample. This high contact
6
surface enables efficient extraction. Finally, both phases are separated and the extraction solvent is
7
analyzed. Thanks to easy protocols and high extraction yields, DLLME is currently the most
8
frequently used solvent-based ME technique [8,41,53,54]. Figure 4 gives a schematic overview of the
9
solvent-based procedures. Advantages and disadvantages of the procedures are listed in Table 3. 4.2. Ionic liquids as solvents
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As solvents in LPME, a variety of ILs can be used, depending on the application and their
13
physicochemical properties. For the extraction of aqueous (biological) samples, a water-immiscible
14
solvent is needed with the ability of forming two distinct phases. Hydrophobic ILs have been applied
15
in this respect and offer the additional advantage of a high solvating capacity for small organic
16
molecules. Furthermore, in both SDME and HF-LPME applications, the low IL volatility avoids
17
evaporation of the microdroplet or acceptor phase during preparation, respectively. This results in
18
techniques with improved repeatability. Additionally, the high surface tension of the IL permits the
19
formation of larger microdroplets in SDME and, therefore, higher extraction yields can be obtained
20
[55]. The most mature application domain of ILs as extraction solvents is probably DLLME. IL-
21
DLLME has the advantage of using solvents with low volatility and flammability, combined with
22
efficient DLLME protocols that generate high extraction yields [8,26,56,57]. The first IL-DLLME
23
procedure was described in 2008 by Zhou et al. and Baghdadi and Shemirani for the extraction of
24
pyrethroid pesticides and mercury, respectively [58,59]. To date, IL-DLLME is applied for the
25
extraction of a wide range of metal ions [60], small organic compounds, such as pharmaceuticals (e.g.
26
tetracycline drugs [61], sulfonamides [62], non-steroidal anti-inflammatory drugs [63] and
27
antihypertensives [64]) and biomolecules, such as DNA [65].
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Conventional IL-DLLME is characterized by the use of an organic disperser solvent to obtain a fine
30
dispersion. Conventional DLLME is therefore a three-phase system: sample + extraction solvent +
31
disperser solvent. However, the use of an organic disperser solvent is not in line with green chemistry
32
principles. Besides, it can increase analyte solubility in the aqueous sample, which results in lower
33
extraction yields [41]. In this regard, several alternatives have been introduced to avoid the use of a
34
disperser solvent; so-called two-phase IL-DLLME techniques [57]. A first alternative for the use of
35
a disperser solvent is mechanical agitation. Two major examples are ultrasonic-assisted IL-DLLME
36
(UA-IL-DLLME) and vortex-assisted IL-DLLME (VA-IL-DLLME). Also, the use of a rotary mixer
37
or a syringe to rapidly draw and inject the extraction solvent, have been described. Other approaches 10
ACCEPTED MANUSCRIPT 1
are the use of a microwave or temperature differences to obtain a fine dispersion [8,26,56]. In the
2
case of temperature-controlled IL-DLLME, ILs are used with an upper or lower critical solution
3
temperature; meaning that the IL is soluble in the aqueous sample above or below respective
4
temperatures. This creates a “dispersion” on a molecular level [66].
5 IL-DLLME applications require ILs with specific properties. Conventionally, hydrophobic ILs are
7
used, as they form a separate layer with aqueous samples. ILs with low viscosities are preferred, since
8
highly viscous ILs may hamper mass transfer during extraction [67,68]. Furthermore, ILs should have
9
a higher density than water, as this allows the IL to be more easily collected from conical-bottom
10
tubes, compared to floating low density solvents (e.g. phosphonium-based ILs) [60]. In general, 1-
11
alkyl-3-methylimidazolium hexafluorophosphate is the most frequently used IL class in extraction
12
applications, thanks to its relatively low cost.
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Next to conventional hydrophobic ILs, hydrophilic ones are also used. Two important applications are
15
in situ solvent formation microextraction (ISFME) and IL-based aqueous two-phase system (IL-
16
ATPS). ISFME consists in adding an anion-exchange reagent to the sample – hydrophilic IL mixture.
17
This result in the formation of a hydrophobic IL that can be separated from the aqueous sample [69].
18
IL-ATPS is based on the salting-out principle, where a hydrophilic IL is separated from the aqueous
19
sample by adding kosmotropic salts [70].
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4.3. Applications in forensic toxicology
Table 4 summarizes publications that use ILs as solvents in forensically relevant microextraction
23
procedures. So far, IL-based LPME has been studied for different classes of drugs: antipsychotics,
24
antidepressants, benzodiazepines, psychostimulants, opioids, and cannabinoids.
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Antipsychotics
27
As for the group of antipsychotics, Cruz-Vera et al. developed an IL-based dynamic liquid-phase
28
microextraction (IL-dLPME) for the determination of seven phenothiazine drugs (chlorpromazine,
29
fluphenazine, levomepromazine, prochlorperazine, promethazine, thioridazine, trifluoperazine) in
30
urine [71]. This dynamic approach involves the constant flow of the sample through a high-density IL
31
plug, present at the bottom of a Pasteur pipette. This technique generally results in higher enrichment
32
factors compared to a static approach. The selected extraction solvent, 1-butyl-3-methylimidazolium
33
hexafluorophosphate, was able to recover 72 – 98% of the analytes, with LOD values between 21 – 60
34
ng/mL [71].
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The extraction of clozapine and its metabolites (norclozapine and clozapine-N-oxide) from urine and
11
ACCEPTED MANUSCRIPT serum samples was studied by Breadmore and involved a vortex-assisted ionic liquid-based liquid-
2
phase microextraction (VA-IL-LPME) technique [72]. The extraction solvent, 1-ethyl-3-
3
methylimidazolium bis(trifluoromethylsulfonyl)imide, was added to the untreated biological samples,
4
followed by a 30-sec vortex step and centrifugation to separate both phases. In the offline approach,
5
the lower IL phase is collected with a pipette and transferred into a sample vial for capillary
6
electrophoresis (CE) analysis, while in the online approach, extraction and centrifugation is performed
7
directly in the sample vial, which can be subsequently placed in the CE autosampler for analysis. In
8
order to enable sufficient CE separation in the presence of the IL salt, a background electrolyte cation
9
with lower ion mobility than EMIm was selected, at high concentration. Validation results showed
10
analyte recoveries near 90% in urine and 85% in serum, except for clozapine-N-oxide (20%, serum).
11
LOD values were all lower than 13 ng/mL, except for clozapine-N-oxide (55 ng/mL, serum) [72].
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Zare et al. studied the extraction of an antipsychotic (perphenazine) and antidepressant (doxepin) drug
14
from urine, using an ultrasound-assisted ionic liquid surfactant-emulsified microextraction (UA-IL-
15
SE-ME) procedure [73]. The use of a surfactant (sodium dodecyl sulfate) and ultrasound irradiation
16
avoided the use of a disperser solvent and resulted in the optimal contact between extraction solvent
17
(1-hexyl-3-methylimidazolium hexafluorophosphate) and analytes. The final extraction procedure took
18
up to 20 min, recoveries of 89 – 98% and LOD values of 0.1 – 1 ng/mL were obtained [73].
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Recently, De Boeck et al. developed an IL-DLLME procedure for a broader group of antidepressants:
22
agomelatine, amitriptyline, bupropion, clomipramine, dosulepin, doxepin, duloxetine, escitalopram,
23
fluoxetine, imipramine, maprotiline, mianserin, mirtazapine, nortriptyline, paroxetine, reboxetine,
24
trazodone and venlafaxine [74]. The method used 1-butyl-3-methylimidazolium hexafluorophosphate
25
(BMIm PF6) as the extraction solvent, which was added directly to the whole blood sample, followed
26
by a 5-min rotary mixing step. The final extract was diluted 1/10 (v/v) in methanol prior to LC-
27
MS/MS analysis, in order to lower the viscosity of the IL and enable accurate LC sample injection.
28
Furthermore, the authors demonstrated that diluting the final extract lowered ion suppression by
29
limiting the injected amount of non-volatile IL and by decreasing IL viscosity. However, the optimal
30
dilution factor should be selected as a reasonable balance between ion suppression diminution and
31
sensitivity loss. Validation results showed that LOD values ranged from 0.8 to 2 ng/mL (35 ng/mL for
32
trazodone) and recoveries were within 53 – 133% (80 – 115% for the majority of analytes). It should
33
be noted that ion suppression was observed with ME values ranging from 62 – 123%, however, ME
34
results were found to be repeatable and could be taken into account [74].
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Benzodiazepines
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De Boeck et al. developed a similar multi-analyte IL-DLLME procedure for the extraction of 17 12
ACCEPTED MANUSCRIPT benzodiazepines
(7-aminoflunitrazepam,
alprazolam,
bromazepam,
clobazam,
clonazepam,
2
clotiazepam, diazepam, estazolam, ethyl loflazepate, etizolam, flurazepam, lormetazepam, midazolam,
3
oxazepam, prazepam, temazepam, triazolam) and 2 Z-drugs (zolpidem, zopiclone) in whole blood
4
samples [75]. The same extraction solvent, BMIm PF6, gave the best extraction efficiencies.
5
Moreover, a clear trend was observed for both antidepressant and benzodiazepine extraction; an
6
increase of the alkyl side chain length on the IL imidazolium cation resulted in lower extraction
7
efficiencies. This could be attributed to the increase in viscosity and/or decrease of polarity.
8
Recoveries were within 24.7 – 127.2% and LOD values ranged from 0.003 – 5 ng/mL. Since LC-ESI-
9
MS/MS was used for the analysis of the final extracts, again ion suppression was observed due to co-
10
elution of the IL and analytes [75]. An additional study by De Boeck et al. compared a set of 11 ILs
11
for their benzodiazepine extraction potential [76]. It was concluded that low-viscosity ILs are required
12
for IL-DLLME procedures, to enable efficient mass transfer during the extraction, to avoid
13
electrospray ionization issues and obtain data with good repeatability. Furthermore, sp2 hybridization
14
of the IL cation is desirable in order to effectuate π-stacking interactions with the 1,4-benzodiazepine
15
skeleton. Moreover, avoiding long alkyl chains and arene substitution of the cation was advised.
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16 Psychostimulants
18
Ephedrine and ketamine extraction from urine was studied by Liu et al [77]. The method consisted of a
19
normal dispersive liquid-liquid microextraction that uses BMIm PF6 as the extraction solvent, in
20
combination with acetonitrile as the disperser solvent. Prior to extraction, urine samples were treated
21
with a protein precipitation step (1:1 methanol) and a filtration step (4000 rpm, 5 min). The extraction
22
consisted of an IL-mediated extraction step, followed by an acidic back-extraction and CE-UV
23
analysis, which took about 5 minutes in total. The method gave recoveries of 79 – 90% from urine
24
samples and LOD values of 210 ng/mL for ephedrine and 390 ng/mL for ketamine [77].
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Wang et al. developed a simple IL-DLLME procedure for the extraction of methamphetamine in urine
27
samples, using 1-octyl-3-methylimidazolium hexafluorophosphate (OMIm PF6) and a disperser
28
solvent (methanol) [78]. The actual extraction only took a few seconds by rapidly injecting the IL
29
solvent into the samples. After centrifugation, the lower phase was collected for LC-UV analysis. The
30
LOD of methamphetamine was 1.7 ng/mL and recoveries ranged up to 82% [78].
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The extraction of nicotine from plasma and urine, using an IL-based HF-LPME procedure, was
33
studied by Lin and Yan [79]. To select the ideal extraction solvent, three commonly used VOSs were
34
compared to a hydrophobic IL; BMIm PF6. The authors found that the IL gave the highest extraction
35
efficiencies and six µL of the solvent was placed in a polypropylene hollow fiber (0.2 µm pore size).
36
The fiber was submerged in the sample for 20 min, followed by the collection of the IL extractant and
37
LC-UV analysis. The LOD of nicotine was found to be 50 ng/mL and recoveries ranged between 94 – 13
ACCEPTED MANUSCRIPT 1
98% for real case biological samples [79].
2 Opioids
4
A new and fast sample treatment strategy for the separation of opium alkaloids from an aqueous
5
Pericarpium Papaveris extract was developed by Li et al. [80]. An IL-ATPS procedure was used to
6
extract polar analytes (codeine, papaverine) from the solid matrix. As the hydrophilic IL, 1-butyl-3-
7
methylimidazolium chloride (BMIm Cl) was selected. The IL was dissolved in the plant extract,
8
followed by the addition of an alkaline K2HPO4 salt. This was thoroughly mixed and a two-phase
9
system (IL-rich and salt-rich phase) was formed within 2 minutes due to sufficient concentration of
10
K2HPO4 salt that initiated a salting-out process. Finally, the IL-rich phase was collected for analysis,
11
using LC-UV. Overall, recoveries ranged from 90 – 100% and 99 – 102% for codeine and papaverine,
12
respectively. LOD values were lower than 30 ng/mL. It should be noted LOD values were calculated
13
based on aqueous samples spiked with codeine and papaverine standard. No real plant matrices were
14
included, except for accuracy verification. Here, the IL-ATPS procedure gave similar results,
15
compared to a LLE method for the extraction of both opium alkaloids from plant samples (n=3).
16
Overall, the developed method showed to be fast (approx. 5 min) and did not require the use of
17
volatile organic solvents [80].
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The decontamination of hair samples using IL solvents was studied by Restolho et al. A contactless
20
procedure was developed for the extraction of morphine and its main active metabolite, 6-
21
monoacetylmorphine (6-MAM), from the hair surface, using a hydrophilic IL extraction solvent [81].
22
The procedure required a customized extraction flask with two separated compartments; one that holds
23
the fragmented hair sample and one that holds the extraction solvent; IL. The closed flask was placed
24
in a GC oven and heated at 120°C for 16 h. The temperature was selected as the optimal balance
25
between avoiding decomposition of IL and hair and promoting the evaporation of the studied analyte.
26
The evaporated analyte was then ‘trapped’ in the IL compartment. The method was validated by
27
comparison to a reference method, based on external contamination of blank hair samples (n = 12).
28
For both methods, drug incorporation was observed resulting in false positive results. However, this
29
was countered by applying the wash criterion. Overall, the authors state that for most hair samples the
30
difference between both the developed and reference method was less than ± 12%. It should be noted
31
that IL-based hair washes were not analyzed for the developed method, only drug concentrations
32
found in the hair digest itself were reported. This is a shortcoming of the method, as according to the
33
Society of Hair Testing, it is recommended to store the hair washes for later analysis [82]. In a follow-
34
up study, Restolho et al. compared a set of six ILs based on their morphine/6-MAM extraction ability
35
in order to better understand the specific IL-analyte interactions [83]. From the selected ILs, it was
36
found that 1-hydroxyethyl-3-methylimidazolium tetrafluoroborate (C2OHMIm BF4) gave the highest
37
extraction efficiencies, which was attributed to the formation of hydrogen bonds between the analyte
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ACCEPTED MANUSCRIPT 1
and the IL cation with high hydrogen bond donor potential thanks to the presence of a hydroxyl group.
2
Decontamination efficiencies were near 30% and 70% for morphine and 6-MAM, respectively, at 100
3
°C during 24 h. The presence of water seemed to enhance extraction efficiencies, values up to 80%
4
were obtained.
5 Cannabinoids
7
So far, one publication has demonstrated the use of ILs for the extraction of ∆9-tetrahrydrocannabinol
8
(THC) [84]. Similar to the contactless decontamination of morphine from hair, Restolho et al.
9
described a procedure for THC decontamination. The final method consisted of a 13-h heating step
10
(100°C) of hair in the presence of a hydrophilic IL in a specialized glass flask. Fifty-three ILs were
11
screened for their extraction efficiency, among which the best results were obtained using C2OHMIm
12
BF4. The authors state that the simple protocol and the ability of decontaminating several samples
13
simultaneously are the main benefits of contactless decontamination compared to conventional
14
methods [84]. Analogous to the morphine decontamination procedure by Restolho et al. [81], it should
15
be noted that the IL-based hair wash was not analyzed, only drug concentrations found in the hair
16
digest itself were reported. This is a shortcoming, as according to the Society of Hair Testing, it is
17
recommended to store hair washes for later analysis [82].
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18 5. Conclusion and outlook
19
Sample preparation remains a crucial aspect of analytical forensic toxicology and should be
21
continuously explored and improved. The introduction of ILs as extraction solvents in microextraction
22
procedures has shown to be beneficial on many levels. First, ILs generally have a low flammability
23
and negligible vapor pressures, which results in safer extractions with reduced air pollution. Second,
24
the chemical structure of ILs can be easily modified in order to obtain solvents with desired
25
physiochemical properties and increased affinity toward the analyte. Third, the estimated number of
26
possible ILs that can be created is 1018, which offers a broad functionality range for distinct extraction
27
applications. Over the last decade, IL-based analytical research in forensic toxicology has expanded
28
significantly, with the focus on complex biological matrices (urine, serum, plasma, whole blood, hair)
29
and
30
psychostimulants, opioids, pesticides and THC). The majority of the discussed research articles
31
developed an IL-based LPME technique, since LPME protocols are generally faster and simpler when
32
compared to sorbent-based alternatives. The selected LPME procedures had optimal extraction times
33
ranging from a few seconds to 20 minutes, while sorbent-based techniques showed extraction times of
34
at least 30 min. Despite extended extraction times, sorbent-based microextractions seems to obtain
35
slightly higher recoveries. This was seen for the extraction of methamphetamine from urine samples,
36
where the IL-SPME procedure obtained 10% higher extraction yields compared to the IL-DLLME
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forensically
relevant
substances
(antipsychotics,
15
antidepressants,
benzodiazepines,
ACCEPTED MANUSCRIPT alternative [50,78]. Taking a closer look at IL-LPME research, it was observed that IL-DLLME was
2
the most frequently applied technique for drug extraction purposes, using a high-density and
3
hydrophobic IL. Overall, imidazolium-based cations are dominating the sample preparation field, as
4
they are easy to obtain at relatively low cost. Furthermore, it was reported that the aromatic properties
5
of the imidazolium cation are crucial for benzodiazepine binding and thus its efficient extraction. As
6
anions, PF6 and Tf2N are often used in combination with an imidazolium cation to obtain hydrophobic
7
solvents. While BF4 and chloride anions generally result in hydrophilic solvents that are used for the
8
extraction of polar substances, such as codeine and morphine. Next to the choice of an appropriate
9
extraction solvent, the appropriate analytical instrument should be considered. Most research groups
10
analyzed their final extracts using LC, coupled to UV or DAD instruments. However, in forensic
11
toxicology, UV and DAD are no longer golden standard techniques, due to limited selectivity, limited
12
sensitivity and thus problematic compound identification. Therefore, mass spectrometry is generally
13
considered first choice. De Boeck et al. tested eleven commercially available ILs (e.g. ammonium,
14
imidazolium, pyridinium and pyrrolidinium cations, combined with hexafluorophosphate or
15
bis(trifluoromethylsulfonyl)imide) and extracts were analyzed using LC-ESI-MS/MS [76]. The
16
authors concluded that the IL solvents all had a tendency to stick to reversed phased LC columns and
17
therefore partially co-eluted with the analytes, resulting in a significant impact on analyte ionization
18
and sensitivity loss. To obtain optimal sensitivities, one should carefully select the IL extraction
19
solvent based on its chromatographic behavior. In general, it can be concluded that for the optimal
20
analysis of polar (e.g. amphetamines, opioids) and apolar compounds (e.g. antidepressants,
21
benzodiazepines), co-elution with IL should be avoided by increasing analyte retention in terms of
22
selecting an appropriate column and gradient, under the assumption that the IL remains minimally or
23
unretained. Furthermore, De Boeck et al. suggested avoiding ILs with high viscosity (> 300 mPa⋅s) as
24
they tend to form a broader chromatographic front and severely suppress ionization [76]. Another
25
specific challenge coupled to high IL viscosity is the reduced accuracy and repeatability of the LC
26
sample injection step. In this respect, GC seems be a more promising technique. Nevertheless, GC-MS
27
applications are rarely documented for the analysis of IL-based extracts. In this review, only He et al.
28
described a headspace SPME method for amphetamine and methamphetamine extraction [50].
29
Generally, GC-MS is avoided due to the non-volatile nature of ILs. In the rare case GC-MS is used,
30
SDME or HF-LPME should be considered, as these extraction techniques only produce small volumes
31
of IL, limiting the contamination of the GC liner. Additionally, the injector temperature should not
32
exceed the IL’s melting point or decomposition temperature to avoid contamination [41].
33
To effect further implementation of ILs in forensic toxicological sample preparation, researchers need
34
to focus on developing IL-based microextraction procedures for the quantification of a larger set of
35
drugs, since targeted multi-analyte methods save costs and time. Moreover, the applicability of ILs in
36
untargeted toxicological screening procedures has so far not been investigated. A universal IL
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ACCEPTED MANUSCRIPT extraction solvent should be selected that has affinity for a wide range of analyte polarities. From the
2
discussed procedures, it was observed that imidazolium-based ILs show a broad applicability and
3
could be tested for potential toxicological screening purposes. However, attention should be paid to
4
the extraction of polar drugs and metabolites, as these will probably require a specific extraction
5
approach as for instance was presented by Li et al. [80]. Furthermore, the implementation of a serial-
6
coupled LC system, described by Loos et al. [85], could improve separation of IL and both polar/non-
7
polar analytes by combining a hydrophilic interaction chromatography (HILIC) and reversed-phase
8
liquid chromatography (RPLC) column, which provides an orthogonal separation mechanism and
9
avoids co-elution and thus analyte suppression. Finally, in forensics, postmortem samples form a
10
significant portion of the analyzed matrices. Here, the question remains whether microextractions are
11
able to deal with these clotted and putrefied liquids.
12
Despite all advantages, the absolute number of IL-based extraction applications is still low.
13
Economical restrictions are probably the major cause. Since ILs are still produced on small scale from
14
pricey raw materials, this results in solvents that are typically 2 - 100 times more expensive than VOSs
15
[29,86]. With an increasing number of IL-based applications, its market share will expand and
16
production scale-up will automatically result in lower costs. Next to IL cost, toxicity and
17
biodegradability issues are even more important. Mostly, fluorinated anions are studied, due to their
18
attractive features for solvent extractions; they generally result in ILs with high hydrophobicity and
19
higher density than water. However, fluorine-containing anions have been reported to lower EC50
20
values of bacteria and can undergo hydrolysis with the formation of hydrofluoric acid [36,87].
21
Luckily, IL structural features can be easily modified in such a way to positively influence IL
22
biodegradability and IL toxicity. Moreover, the introduction of renewable ions based on amino acids,
23
choline or sugars form a possible solution [88]. When designing green ILs, physicochemical properties
24
should be carefully monitored to obtain a final compound that is usable for solvent- or sorbent-based
25
extraction applications. Furthermore, it still remains difficult to select the ideal IL extraction solvent
26
from an almost infinite number of potential anion-cation combinations. To ensure further
27
implementation of ILs in sample preparation, a rational IL design approach is needed. This would
28
allow rational combining of the desired anion and cation, resulting in an IL with the required
29
physicochemical properties for a certain application. Progress has been made with the launch of a US
30
National Institute of Standards and Technology (NIST) database that provides researchers with
31
relevant publications on IL structure and associated physicochemical properties. However, knowledge
32
should be extended by screening sets of ILs for a specific extraction application and defining crucial
33
IL-solute binding interactions in order to elucidate IL-based extraction mechanisms.
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Acknowledgements 17
ACCEPTED MANUSCRIPT This research did not receive any specific grant from funding agencies in the public, commercial, or
2
not-for-profit sectors.
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ACCEPTED MANUSCRIPT References
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Figure 2. Chemical structures of typical ionic liquid cations and anions.
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Figure 1. Sodium chloride (NaCl) and 1-butyl-3-methylimidazolium hexafluorophosphate (BMIm PF6). 1: NaCl; 2: BMIm PF6; a: physical appearance at room temperature; b: simplified overview of chemical appearance (blue/black/white = cation, orange = anion).
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Figure 3. Schematic overview of sorbent-based microextraction procedures. SPME: solid-phase microextraction; SBSE: stir bar sorptive extraction; MEPS: microextraction by packed sorbent.
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Figure 4. Schematic overview of solvent-based microextraction procedures. SDME: single drop microextraction; HF-LPME: hollow fiber liquid phase microextraction; DLLME: dispersive liquid-liquid microextraction.
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Figure captions
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Table 1.
2
Advantages and disadvantages of sorbent-based microextractions Advantages Simple Solvent free Small sample size Portable on field sampling Online applications Molecularly imprinted polymer Reusable
-
Low operating temperature Breakage of fiber (fused silica) Chemical instability Swelling of fiber High cost* Low sensitivity Carryover with reuse?
SBSE (°1999)
-
Simple Solvent free High sensitivity High extraction capacity High robustness Molecularly imprinted polymer Reusable
-
Lack of appropriate coatings High cost* High extraction times Carryover with reuse?
MEPS (°2004)
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Fast (1-4 min) Simple Molecularly imprinted polymer Reusable
-
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SPME (°1990)
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Requires solvent (small volume) Syringe blockage High cost* Carryover with reuse?
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SPME: solid-phase microextraction; SBSE: stir bar sorptive extraction; MEPS: microextraction by packed sorbent; *if not reused; ° year of first introduction. [8,41–44,54]
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Disadvantages
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Table 2.
Pesticides (6) Benzodiazepines (4)
Sample amount
Extraction technique
Ionic liquid
Urine
4 mL
IL-SPME
EC2MIm Tf2N
Water Hair River water Urine Hair
5 mL 50 mg 10 mL 10 mL 50 mg
IL-HF-SPME
BMIm OH
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Amphetamine Methamphetamine
Sample
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Analyte
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Applications of ILs as sorbents for the extraction of forensically relevant drugs.
IL-HF-SLPME
PMIm Br@TiO2
Recovery (%)
LOD (ng/mL)
89 – 103 94 – 114 86 – 99 82 – 94 95 – 102 45 – 106 93 – 103
0.5 0.1 0.004 – 0.095 0.003 – 0.08 0.08 – 0.5
Detection
Ref.
GC-MS
[50]
LC-DAD
[51]
LC-UV
[52]
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IL-SPME: ionic liquid-based solid-phase microextraction; IL-HF-SPME: ionic liquid-based hollow fiber solid-phase microextraction; IL-HF-SLPME: ionic liquid-based hollow fiber solidliquid-phase microextraction; BMIm: 1-butyl-3-methylimidazolium; EC2Mim: 1-ethoxyethyl-3-methylimidazolium; PMIm: 1-pentyl-3-methylimidazolium; @TiO2: coated onto titanium dioxide nanoparticles; Br: bromide; OH: hydroxide; Tf2N: bis(trifluoromethylsulfonyl)imide.
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Simple Enhanced selectivity (pores) Molecularly imprinted polymer Reusable
Instability of drop Fluctuation of drop volume Poor reproducibility Low contact surface Low extraction capacity Cost of fiber Long extraction times Fiber pore blockage Air bubbles near pores Carryover with reuse? Use of disperser solvent Difficult automation
- Fast (5 min) - Simple - High recovery
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DLLME (°2006)
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HF-LPME (°1999)
- Simple - Low cost - High enrichment factor
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SDME (°1996)
Disadvantages
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SDME: single drop microextraction; HF-LPME: hollow fiber liquid phase microextraction; DLLME: dispersive liquid-liquid microextraction. °: year of first introduction. [8,41,43,53,54]
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Table 4.
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Applications of Ils as solvents for the extraction of forensically relevant drugs.
Analyte
Sample
Sample amount
Phenothiazines (7)
Urine Urine Serum
10 mL 0.2 mL 0.2 mL
Urine
6 mL
UA-IL-SE-ME
Whole blood
1 mL
IL-DLLME
BMIm PF6
Whole blood
1 mL
IL-DLLME
BMIm PF6
Urine
0.5 mL
IL-DLLME
BMIm PF6
Urine Urine Plasma Pericarpium Papaveris extract
10 mL
IL-DLLME
OMIm PF6
Nicotine Codeine Papaverine Morphine 6-Monoacetylmorphine ∆9-Tetrahydrocannabinol
25 mL 1000 mg
Hair
20 mg
Hair
20 mg
53 – 133
0.8 – 35
25 – 127 59 – 98 86 – 90 79 – 82 80 – 82 98 94 90 – 100 99 – 102
0.003 – 5 0.3 – 0.4 210 390 2
C2OHMIm BF4 C2OHMIm BF4
EMIm Tf2N
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VA-IL-LPME
HMIm PF6
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Benzodiazepines (17) Z-drugs (2) Ephedrine Ketamine Methamphetamine
21 – 60 3 – 11 7 – 55 0.1 1
BMIm PF6
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Antidepressants (18)
72 – 98 87 – 93 20 – 85 93 – 98 89 – 96
IL-dLPME
IL-HF-LPME
BMIm PF6
IL-ATPS
BMIm Cl
Contactless decontamination Contactless decontamination
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Doxepin Perphenazine
LOD (ng/mL)
Ionic liquid
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Clozapine (+ metabol.)
Recovery (%)
Extraction technique
Detection
Ref.
LC-UV
[71]
CE-DAD
[72]
LC-UV
[73]
LC-MS/MS
[74]
LC-MS/MS
[75,76]
CE-UV
[77]
LC-UV
[78]
50
LC-UV
[79]
30 20
LC-UV
[80]
NA
NA
GC-MS
[81,83]
NA
NA
GC-MS
[84]
IL-dLPME: ionic liquid-based dynamic liquid-phase microextraction; VA-IL-LPME: vortex-assisted ionic liquid-based liquid-phase microextraction; UA-IL-SE-ME: ultrasound-assisted ionic liquid surfactant-based emulsification microextraction; IL-DLLME: ionic liquid-based dispersive liquid-liquid microextraction; IL-ATPS: ionic liquid-based aqueous two-phase system; IL-HFLPME: ionic liquid-based hollow fiber liquid-phase microextraction; EMIm: 1-ethyl-3-methylimidazolium; BMIm: 1-butyl-3-methylimidazolium; HMIm: 1-hexyl-3-methylimidazolium; OMIm: 1-octyl-3-methylimidazolium; C2OHMIm: 1-hydroxyethyl-3-methylimidazolium; BF4: tetrafluoroborate; Cl: chloride; PF6: hexafluorophosphate; Tf2N: bis(trifluoromethyl-sulfonyl)imide; NA: not applicable.
29
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ACCEPTED MANUSCRIPT Highlights An overview of general ionic liquid physicochemical features is provided.
•
Benefits of using ionic liquids as extraction sorbents or solvents are discussed.
•
Ionic liquid-based forensic toxicological analytical methods are reviewed.
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An outlook on the applicability of ionic liquids in forensic toxicology is given.
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Challenges associated with ionic liquid-based extractions are discussed.
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S0165-9936(18)30355-8
DOI:
https://doi.org/10.1016/j.trac.2018.11.036
Reference:
TRAC 15338
To appear in:
Trends in Analytical Chemistry
Received Date: 6 August 2018 Revised Date:
21 November 2018
Accepted Date: 22 November 2018
Please cite this article as: M. De Boeck, W. Dehaen, J. Tytgat, E. Cuypers, Microextractions In Forensic Toxicology: The Potential Role Of Ionic Liquids, Trends in Analytical Chemistry, https://doi.org/10.1016/ j.trac.2018.11.036. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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MICROEXTRACTIONS IN FORENSIC TOXICOLOGY: THE POTENTIAL ROLE OF IONIC LIQUIDS Authors
a
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Marieke De Boecka, Wim Dehaenb, Jan Tytgata, Eva Cuypersa Toxicology and Pharmacology, Department of Pharmaceutical and Pharmacological
Sciences, University of Leuven (KU Leuven), Campus Gasthuisberg, O&N II, P.O. Box 922, Herestraat 49, 3000 Leuven, Belgium
Molecular Design and Synthesis, Department of Chemistry, University of Leuven (KU
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b
ORCID:
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Leuven), Campus Arenberg, P.O. Box 2404, Celestijnenlaan 200F, 3001 Leuven, Belgium
Marieke De Boeck
0000-0001-7910-7465
Wim Dehaen
0000-0002-9597-0629
Jan Tytgat
Corresponding author
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Prof. Dr. Eva Cuypers
0000-0002-6411-2461
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Eva Cuypers
Toxicology and Pharmacology
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Department of Pharmaceutical and Pharmacological Sciences University of Leuven (KU Leuven) Campus Gasthuisberg, O&N II Herestraat 49 – P.O. Box 922 3000 Leuven BELGIUM Tel: + 32 16 32 34 03 Fax: + 32 16 32 34 05 Email: [email protected]
ACCEPTED MANUSCRIPT 1
MICROEXTRACTIONS IN FORENSIC TOXICOLOGY:
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THE POTENTIAL ROLE OF IONIC LIQUIDS Abstract
4
In forensic toxicology, scientists are faced with complex samples and trace concentrations of low-dose
5
legal and illegal drugs. To enable accurate and sensitive analysis, a thorough sample preparation step
6
is crucial. In the context of green chemistry, sample preparation techniques are rapidly evolving.
7
Microextractions are introduced to avoid the use of large solvent volumes and obtain high analyte
8
enrichment. Another trend is the search for alternative solvents to replace toxic and volatile organic
9
solvents. Ionic liquids (ILs) - liquid salts - are proposed as promising alternatives, thanks to their low
10
volatility, low flammability, good thermal and chemical stability. Moreover, the IL structure can be
11
altered by combining different cations and anions, resulting in ILs with optimized physicochemical
12
properties for the extraction of a specific analyte. This review describes the current state of
13
microextraction techniques that apply ILs for the extraction of forensically relevant drugs in biological
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samples.
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15 Keywords
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Analytical toxicology; Forensic toxicology; Ionic liquids; Microextractions; Biological samples;
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Sorbent; Solvent
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Abbreviations
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EC2MIm: 1-ethoxyethyl-3-methylimidazolium; EMIm: 1-ethyl-3-methylimidazolium; BF4: tetra-
22
fluoroborate; BMIm: 1-butyl-3-methylimidazolium; Br: bromide; C2OHMIm: 1-hydroxyethyl-3-
23
methylimidazolium; Cl: chloride; DAD: diode array detector; DLLME: dispersive liquid-liquid
24
microextraction; dLPME: dynamic liquid-phase microextraction; ESI: electrospray ionization; GC:
25
gas chromatography; HF-LPME: hollow fiber liquid-phase microextraction; HMIm: 1-hexyl-3-
26
methylimidazolium; IL: ionic liquid; ATPS: aqueous two-phase system; ISFME: in situ solvent
27
formation microextraction; LC: liquid chromatography; LOD: limit of detection; LPME: liquid-phase
28
microextraction; ME: matrix effect; MEPS: microextraction by packed sorbent; MS: mass
29
spectrometry; NaCl: sodium chloride; OH: hydroxide; OMIm: 1-octyl-3-methylimidazolium; PF6:
30
hexafluorophosphate; PMIm: 1-pentyl-3-methylimidazolium; RE: recovery; RTIL: room-temperature
31
ionic liquid; SDME: single drop microextraction; SPME: solid-phase microextraction; SBSE: stir bar
32
sorptive extraction; Tf2N: bis(trifluoromethylsulfonyl)imide; TiO2: titanium dioxide; TSIL: task-
33
specific ionic liquid; UA-IL-SE-ME: ultrasound-assisted ionic liquid surfactant-based emulsification
34
microextraction
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1. Introduction In forensic toxicology, biological samples are analyzed to determine the presence of xenobiotics and
3
their metabolites in the human body. Both qualitative and quantitative results are obtained, interpreted
4
and finally reported in judicial proceedings. To obtain accurate results, appropriate analytical methods
5
are developed and validated for a specific forensic application [1]. They typically consist of an initial
6
sample preparation step, followed by the analysis of the final extract using an appropriate analytical
7
instrument. Sample preparation is considered the most time-consuming and error-inducing step in the
8
bioanalytical process. However, it remains indispensable, as it delivers compatible samples with the
9
detection instrument and reduces matrix interferences, such as endogenous compounds (e.g. proteins,
10
lipids). Furthermore, sample preparation can lead to analyte enrichment and increased method
11
sensitivity [2,3].
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Forensic toxicology is highly demanding in terms of sample preparation. It requires sensitive and
14
multi-analyte procedures, since in many cases, only a limited amount of sample is available and the
15
substances of interest are often taken in combination and at low doses. Moreover, complex sample
16
matrices may pose additional challenges for accurate analysis. For routine toxicological examination,
17
often peripheral blood, urine and gastric fluid are analyzed, however; hair and oral fluid are also
18
collected, due to extended drug surveillance windows and ease of on-site sampling, respectively [1].
19
Generally, biological samples contain a wide range of endogenous and exogenous substances that can
20
hamper analyte quantification and identification. For instance, blood proteins or phospholipids are
21
abundantly present and can result in viscous samples that are difficult to handle, or they can hamper
22
analyte ionization using atmospheric pressure ionization sources [4]. Even more challenging is the
23
analysis of postmortem samples. For instance, postmortem blood is often putrefied, clotted,
24
hemolyzed, contaminated with tissue fluid and analytes undergo redistribution processes. All these
25
factors make it almost impossible to accurately determine drug concentrations and interpret the
26
obtained results [5].
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To date, a broad diversity of sample preparation techniques are applied, using different extraction
29
solvents and sorbents. Nevertheless, there is a persistent need for efficient and cost-effective methods
30
that take into consideration environmental aspects [2,3,6]. Microextractions were introduced in 1990,
31
by Arthur and Pawliszyn, as a solution for tedious procedures with high solvent consumption [7].
32
They are considered green alternatives for well-known and established sample preparation techniques,
33
such as liquid-liquid extraction (LLE) and solid-phase extraction (SPE). Microextractions are
34
characterized by the consumption of small solvent volumes (µL range) and are in some cases even
35
solvent free. This leads to a reduction of hazardous volatile organic solvent (VOS) consumption, a
36
reduction of waste and high enrichment of the analyte. Additionally, microextractions generally
2
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consist of simplified protocols that lower the chance of manual errors [8].
2 Next to the microextraction trend, the possibility of using alternative extraction solvents has been
4
studied. Ionic liquids (ILs) were introduced in analytical chemistry to lower the environmental and
5
toxic burden of VOSs, but most importantly, to increase the current solvent functionality range [9]. To
6
date, only 600 VOSs are available, while more than one billion possible ILs can be created [9]. ILs
7
differ from conventional molecular solvents as they are purely composed of ions. However, they
8
cannot be categorized as conventional salts due to the presence of organic ions and their liquid state
9
below 100°C [10]. Some ILs even melt at/below room temperature, these are referred to as room-
10
temperature ionic liquids (RTILs) [11]. Thanks to their low volatility and low flammability, ILs hold
11
great promise as extraction solvents in bioanalytical research. Additionally, their easy tunable nature
12
allows to create a multitude of ILs with a wide variety of physicochemical properties. With relatively
13
easy chemical processes (e.g. metathesis or acid-base neutralization), certain functionalities can be
14
incorporated into their chemical structure [12]. This is probably the most useful advantage of ILs,
15
since it allows the design of TSILs with improved applicability for a specific task [13].
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This review focusses on the use of ILs in forensic toxicology, more specifically, as solvents in
18
microextraction procedures. First, an introduction is provided on the properties and advantages of the
19
novel IL solvents. Second, principles of the most relevant solid-phase and liquid-phase
20
microextraction techniques are outlined. Third, an overview is provided of research performed on IL-
21
based microextractions for the analysis of forensically relevant substances in biological samples.
22
Finally, a conclusion is formulated, combined with a critical look at the future of ILs in forensic
23
toxicology.
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In the following section, conventional organic solvents and ionic liquids are discussed, with the
27
emphasis on the potential of ILs and their physicochemical properties.
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2.1. Conventional solvents
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A solvent is characterized by its liquid state and ability to dissolve or extract other substances, without
31
changing their chemical appearance [14]. They are extensively consumed in chemical and
32
pharmaceutical industry, for example in synthesis processes [15]. Also in sample preparation, solvent
33
extractions are frequently performed using organic solvents [16]. Traditionally, VOSs (e.g.
34
chloroform, diethyl ether, hexane, methanol) are used, as they can be easily obtained at low cost.
35
However, care should be taken when working with VOSs, due to their health and environmental
36
hazards [17]. People are mainly exposed to VOSs via inhalation. Acute effects can be headache, 3
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2
Long-term exposure cases report the carcinogenic impact of some organic solvents. Among
3
youngsters, VOSs are even abused as legal highs [14]. Furthermore, VOSs are easily released in the
4
environment and aquatic systems, owing to their high volatility, thereby potentially severely harming
5
essential microorganisms. Additionally, with the use of VOSs comes the risk for ignition and
6
explosion [18]. It is clear that conventional VOSs need to be replaced in solvent extraction processes,
7
as they largely affect the safety and “greenness” of such procedures. To date, several strategies have
8
been proposed: the use of organic solvents with improved biodegradability and safety, organic
9
solvents that are produced with renewable energy, supercritical fluids and the use of solvents that are
10
less emitted into the environment, thanks to their negligible vapor pressures [17]. With respect to this
11
last category, ILs were introduced as credible alternatives [10].
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2.2. Ionic liquids 2.2.1.
History
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High temperature molten salts are considered the foundation of current IL chemistry. They are defined
16
by melting points above 100°C [19]. Initially, these molten salts were used as electrolytes, however,
17
they were considered hard to work with and a great deal of energy was needed to maintain their liquid
18
state. As a logical consequence, scientists started looking for salts with lower melting points. The first
19
low melting salt (i.e. IL) was synthesized in 1914 by Paul Walden, namely, ethylammonium nitrate. It
20
took until the end of World War II for ILs to finally break through. The class of imidazolium- and
21
pyridinium-based chloroaluminates formed the first-generation ILs and triggered genuine attention of
22
electrochemists. However, the wide applicability of these chloroaluminates was hampered, due to their
23
water-reactive nature. In 1992, Wilkes and Zaworotko introduced ILs that consisted of a 1-ethyl-3-
24
methylimidazolium cation, combined with different anions. These were called second-generation ILs
25
and were air- and water-stable compounds [10,20]. Currently, IL research focusses on developing less
26
toxic and more renewable ILs, e.g. using renewable resources such as sugars and amino acids (third-
27
generation ILs) [10]. Also, the task-specific design of ILs (task-specific ILs (TSILs)) is being
28
extensively explored, thanks to the easily tunable physiochemical characteristics of ILs [13].
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2.2.2.
Physicochemical properties
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ILs are low temperature melting salts. Their low melting points can be attributed to small lattice
31
energies, as a result of bulky and asymmetric ions with large conformational flexibilities [21]. Figure
32
1. shows the physical appearance of a conventional salt (sodium chloride; NaCl) and a commonly used
33
RTIL (1-butyl-3-methylimidazolium hexafluorophosphate; BMIm PF6) at room temperature. In
34
conventional salts, strong Coulombic interactions form compact lattice structures and result in solid
35
state salts, while ILs have larger cations and/or anions, leading to a lower charge density and less
36
pronounced Coulombic interaction [22]. Next to low melting points, ILs display negligible vapor 4
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2
the environment. Furthermore, IL viscosities differ from typical organic solvents. In general, ILs have
3
viscosities ranging from 10 to 40,000 mPa⋅s, while organic solvent viscosities are much lower, ranging
4
up to 100 mPa⋅s. For instance, alkyl chain length is directly proportional to viscosity [22,23]. IL
5
densities are typically higher than those of organic solvents. This can be related to heavy anions that
6
are often incorporated in the IL structure (e.g. bis(trifluoromethylsulfonyl)imide (Tf2N),
7
hexafluorophosphate (PF6)) [22,23]. Extra IL advantages are their high thermal stability and low
8
flammability. Moreover, ILs have the ability to solvate a wide range of molecules [22,23]. The
9
solvation strength or polarity of ILs is often expressed by means of three Kamlet-Taft solvation
10
parameters: hydrogen bond acidity (α), hydrogen bond basicity (β) and polarizability/dipolarity (π*)
11 12 13
[24].
14
There is an almost infinite number of possible anion-cation combinations that can be formed.
15
Furthermore, ILs can be tuned to obtain desirable physicochemical properties, as certain
16
functionalities can be incorporated into their structure, for instance, additional aromatic rings,
17
hydroxyl groups, etc. This is probably the most useful advantage of ILs, since it allows the design of
18
TSILs with improved applicability for a specific task [13]. Which ions are typically used in IL
19
systems? As a cation, imidazolium is the most documented and sold IL class. Furthermore,
20
ammonium, pyridinium, pyrrolidinium and phosphonium are also frequently applied [22,25]. As an
21
anion, tetrafluoroborate (BF4), PF6, Tf2N, tosylate, acetate and halides are used [22,25]. Figure 2.
22
shows their chemical structures. Most ILs are synthesized using straightforward techniques; either by
23
metathesis of a cation-halide salt and metal-anion salt or by acid-base neutralization [12].
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Overall, the mentioned physicochemical properties largely depend on the selected cation and anion.
24 2.2.3.
Applications
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ILs are applied in different scientific fields: analytical chemistry [26–28], electrochemistry [25,29],
27
organic chemistry [25,30], material science [31] and even in medicine [32–34]. Relevant to this review
28
is their use in analytical chemistry, more specifically, their application as extraction solvents and
29
sorbents. In this case, high specificity and affinity for the analyte is desired. These properties can be
30
optimized by altering the chemical structure of the IL solvent, thereby creating designer solvents.
31
Furthermore,
32
desorption/ionization (MALDI) of both proteins and small organic molecules. Their non-crystalline
33
characteristics allow more homogenous spots to be sampled with improved sensitivity [26]. Other
34
popular IL applications in analytical chemistry are their use as organic modifiers in mobile phases to
35
optimize separations in liquid chromatography (LC) and as modifiers of capillary column coatings in
36
both gas chromatography (GC) and capillary electrophoresis (CE) [27].
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as
promising
37 5
liquid
matrices
for
matrix-assisted
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ACCEPTED MANUSCRIPT The mentioned IL applications do not simply stay within academic barriers; several industrial IL-
2
based systems have been reported. BASF has been a pioneer when it comes to the implementation of
3
ILs. In 2002, they developed the BASILTM process in which 1-methylimidazole is used to scavenge
4
unwanted acids. As a result, an IL is formed that can be easily separated from the reaction mixture
5
[25,35]. Overall, IL popularity is gradually increasing, as can be deduced from the large amount of IL
6
patent submissions. With different types of ILs entering the market, a larger diversity of
7
physicochemical properties is offered that greatly exceeds the potential of only 600 VOSs that are
8
available today [9].
9 2.2.4.
10
Toxicity and environmental impact
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Throughout the years, ILs have been presented as greener alternatives for VOSs. ILs are considered to
12
be less toxic and have a negligible impact on the environment. However, this statement is solely based
13
on their negligible volatilities that limit their release into the atmosphere. They can be spilled into the
14
environment by disposal into aquatic systems, followed by their adsorption onto soil [36]. This can
15
have negative consequences, since studies have demonstrated IL toxicity toward bacterial and even
16
mammalian cells. Results show that second-generation ILs are not as harmful as initially presumed.
17
Their toxicity is directly linked to their chemical structure and lipophilicity. ILs with high lipid
18
solubility are more likely to interfere with cell membranes and result in cell leakage or even cell death
19
[36–38]. A clear structure-toxicity relationship has been reported. For example, long alkyl side chains
20
and fluorine-containing anions should be avoided. Likewise, biodegradability can be directly linked to
21
the IL’s structural features [36–38]. These features can be easily tuned to obtain safer and greener
22
solvents, e.g. cholinium and amino acid-based ions. Moreover, renewable resources, such as sugars,
23
are increasingly used to establish a greener image [36,37]. To date, the active removal of ILs from
24
industrial wastewater mainly relies on adsorption and oxidation techniques [36]. In order to reduce IL
25
waste, researchers have reported recycle processes to enable their reuse, such as extraction with
26
supercritical CO2 and distillation processes [39]. In case reuse is not feasible, ILs are disposed as
27
organic waste and degraded via oxidation. Several publications report the adequate degradation of ILs
28
by advanced oxidation processes consisting of UV treatment in the presence of oxidative catalysts
29
[40].
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3. Sorbent-based microextractions
32
The following section discusses three established sorbent-based microextraction techniques: solid-
33
phase microextraction (SPME), stir bar sorptive extraction (SBSE) and microextraction by packed
34
sorbent (MEPS). Additionally, the application of ILs as promising sorbents and their implementation
35
in forensic toxicology are presented.
36
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3.1. General extraction principles The introduction of solid-phase microextractions (SPMEs) in 1990, symbolizes the beginning of a
3
new era in sample clean-up. SPME is a solventless technique that uses a syringe, on which a coated
4
fused silica fiber is mounted. This fiber can be lowered into a liquid sample (direct-immersion) or
5
headspace to obtain analyte adsorption. The analyte can be eluted from the fiber with a solvent or it
6
can be directly introduced into a GC device [41–43]. Stir bar sorptive extraction (SBSE) is another
7
popular solventless microextraction technique. It uses a magnetic stir bar that is coated with a layer of
8
sorbent. The bar is introduced into a liquid sample and the stirring process effectuates analyte
9
adsorption onto the sorbent surface. Next to liquid samples, SBSE can also be applied for headspace
10
extractions [8,43]. Microextraction by packed sorbent (MEPS) is a more recently introduced (2004)
11
technique for sample clean-up. The sorbent is packed as a small plug inside of a syringe. By pulling
12
the plunger up- and downwards, the plug can be conditioned, loaded, washed and eluted. MEPS can be
13
defined as a small-scale variant of conventional SPE techniques [8,43,44]. Figure 3. gives a schematic
14
overview of the sorbent-based procedures. Advantages and disadvantages are listed in Table 1.
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In recent years, ILs have been incorporated as solid supports for sorbent-based extraction techniques.
19
Especially in SPME and SBSE applications, the possibility of implementing ILs is increasingly
20
studied. Their unique physicochemical properties can be exploited to obtain attractive solid supports
21
with improved selectivity. Appropriate ILs should have good thermal stability, high viscosity and high
22
affinity for the analyte. The first two properties are required to avoid decomposition or leakage of the
23
IL due to high GC injector operating temperatures. In both cases, the nature of the anion and cation
24
should be carefully considered, as is described by Tien D. Ho et al. [45].
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The preparation of the IL-based SPME fibers can be done in various ways. A first approach is to
27
immobilize the IL onto a conventional silica-based fiber, by impregnation. This results in a physically
28
bonded phase. A disadvantage of physical bonding is the lower reusability of the fiber, compared to a
29
chemically bonded variant. The latter is reported for both silica and polymer supports [45,46]. Next to
30
the implementation of ILs as such, researchers have studied their polymerization in order to create
31
supports with higher viscosities and mechanical stability, referred to as polymeric ionic liquids (PILs).
32
In 2008, Zhao et al., described the first SPME application to use PILs for the extraction of esters. In
33
this study, imidazolium-based polymers were prepared and coated onto a fused silica fiber by dipping.
34
Next to viscosity and stability benefits, the tested PILs were found to give equal or superior recoveries,
35
compared to commercial coatings [47]. Overall, the discussed SPME developments are promising and
36
boost further research of task-specific IL/PIL coatings.
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2
developed for the extraction of NSAIDs from water, urine and milk, using a stir bar that was
3
chemically modified with 1-allylimidazolium tetrafluoroborate [48]. More recent research focusses on
4
the implementation of magnetic ionic liquids (MILs) to improve SBSE yields. The novel techniques
5
was developed by Chisvert et al. and is termed stir bar dispersive liquid microextraction (SBDLME).
6
The extraction mechanism is based on the magnetic attractive forces between the MIL and the
7
magnetic stir bar. The MIL is attracted toward the stir bar, however, during intense stirring, the MIL is
8
dispersed into the sample in order to create a high contact surface for good analyte transfer. After
9
extraction, the stirring process is ended and the analytes can be thermally desorbed from the stir bar
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[49].
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Table 2 summarizes publications that use ILs as sorbents in forensically relevant SPME procedures.
14
So far, three SPME-based techniques have been reported for the analysis of benzodiazepines,
15
psychostimulants and pesticides.
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For the extraction of amphetamine and methamphetamine from urine samples, He et al. described a
18
headspace SPME method in which a silicone elastomer was chemically linked onto a fused silica fiber
19
[50]. Prior to cross-linkage, the elastomer was impregnated with a 1-ethoxyethyl-3-methylimidazolium
20
bis(trifluoromethylsulfonyl)imide (EC2MIm Tf2N) solution. The actual SPME extraction was
21
performed for 30 min, followed by the thermal desorption of the analytes and GC-MS analysis. The
22
authors reported no stability issues with the physically coated IL layer during desorption (220°C), with
23
a durability of more than 100 extractions. Validation results showed good repeatability and limits of
24
detection (LODs) ranging from 0.1 – 0.5 ng/mL. It should be noted that in real case urine samples,
25
amphetamine and methamphetamine are expected to be present at higher concentrations than 1 ng/mL,
26
more specifically, a urinary cutoff concentration of abuse of 200 ng/mL for amphetamine by
27
confirmatory test was set by the U.S. Substance Abuse and Mental Health Services Administration. To
28
validate the usability of the developed IL-SPME method in forensic practice, spiked urine samples
29
were analyzed (n=3) at four concentration levels (100 – 1000 ng/mL) and RSD values lower than
30
5.0% were obtained [50].
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Ebrahimi et al. developed a sol-gel based HF-SPME procedure for the extraction of pesticides from
33
hair samples [51]. The sol-gel sorbent was prepared based on 1-butyl-3-methylimidazolium hydroxide
34
(BMIM OH) mediated nanoparticles. Six µL of sol-gel sorbent was loaded into a hollow
35
polypropylene fiber, resulting in a disposable HF-SPME system. The hair samples were extracted
36
using methanol (5 h, 55°C) and the disposable device was immersed in the extract for 40 min, under
8
ACCEPTED MANUSCRIPT 1
continuous stirring. LC-DAD analysis was performed on the methanolic extract of the sample fiber.
2
Here, the IL was not only used as an extraction solvent, it rather served as a porogenous agent in order
3
to obtain a porous sol-gel system with an increased contact surface for efficient analyte extraction. The
4
developed method obtained LOD values lower than 0.08 ng/mL and recoveries ranging from 80 – 94%
5
for real case human hair samples [51]. The authors did not report the LOD values relating to the
6
analyte concentration in hair.
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Es’haghi et al. developed a similar hollow fiber solid-liquid-phase microextraction (HF-SLPME)
9
procedure for the extraction of four benzodiazepines (alprazolam, clonazepam, diazepam, lorazepam)
10
from urine and hair matrices [52]. In this study, titanium dioxide (TiO2) was studied as a potential
11
sorbent. The TiO2 extraction capacity was extended by enlarging its contact surface, thanks to the IL-
12
mediated synthesis of TiO2 nanoparticles. In a next step, 1-pentyl-3-methylimidazolium bromide was
13
coated onto the nanoparticles, from which a sol-gel system was prepared. The sol-gel was loaded into
14
a hollow fiber and could be used for extraction by immersing the device into the sample during 45
15
min. Methanol was used for desorption, followed by LC-UV analysis. The procedure obtained LOD
16
values lower than 0.10 ng/mg for the extraction of benzodiazepines from hair samples and relative
17
recoveries were within 93 – 103%. The analysis of urine samples gave relative recoveries within 45 –
18
106% [52].
19 4. Solvent-based microextractions
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The following section discusses three established solvent-based microextraction techniques, also
22
termed liquid-phase microextraction (LPME): single drop microextraction (SDME), hollow fiber
23
liquid phase microextraction (HF-LPME) and dispersive liquid-liquid microextraction (DLLME).
24
Additionally, the application of ILs as promising solvents and their implementation in forensic
25
toxicology
27
are
presented.
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28
Liquid microextractions are defined by the use of small solvent volumes; i.e. smaller than 100 µL[53].
29
They were introduced in 1996, with the development of the single drop microextraction (SDME)
30
technique. SDME uses a single microdrop (< 10 µL) of extraction solvent that is positioned at the end
31
of a syringe needle. This drop is lowered into a liquid sample or headspace. After analyte extraction,
32
the drop is withdrawn into the syringe and introduced into an analytical measuring device. SDME has
33
received much attention, however, the instability of the drop has shown to be a major issue
34
[8,41,53,54]. Three years later, hollow fiber liquid phase microextraction (HF-LPME) was
35
developed to address the problem. In this method, a low volume (10 - 20 µL) of solvent is loaded
36
inside of a polymeric hollow fiber. In this way, a membrane is created that supports the solvent and 9
ACCEPTED MANUSCRIPT forms an additional barrier for large unwanted biomolecules. Finally, the acceptor phase is collected
2
and analyzed [8,41,53,54]. In 2004, dispersive liquid-liquid microextraction (DLLME) was
3
introduced. DLLME is characterized by simple and straightforward procedures, without the need for
4
fibers. A sample is injected with a combination of disperser and extraction solvent, resulting in a
5
turbid solution that consists of finely dispersed solvent microdroplets in the sample. This high contact
6
surface enables efficient extraction. Finally, both phases are separated and the extraction solvent is
7
analyzed. Thanks to easy protocols and high extraction yields, DLLME is currently the most
8
frequently used solvent-based ME technique [8,41,53,54]. Figure 4 gives a schematic overview of the
9
solvent-based procedures. Advantages and disadvantages of the procedures are listed in Table 3. 4.2. Ionic liquids as solvents
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As solvents in LPME, a variety of ILs can be used, depending on the application and their
13
physicochemical properties. For the extraction of aqueous (biological) samples, a water-immiscible
14
solvent is needed with the ability of forming two distinct phases. Hydrophobic ILs have been applied
15
in this respect and offer the additional advantage of a high solvating capacity for small organic
16
molecules. Furthermore, in both SDME and HF-LPME applications, the low IL volatility avoids
17
evaporation of the microdroplet or acceptor phase during preparation, respectively. This results in
18
techniques with improved repeatability. Additionally, the high surface tension of the IL permits the
19
formation of larger microdroplets in SDME and, therefore, higher extraction yields can be obtained
20
[55]. The most mature application domain of ILs as extraction solvents is probably DLLME. IL-
21
DLLME has the advantage of using solvents with low volatility and flammability, combined with
22
efficient DLLME protocols that generate high extraction yields [8,26,56,57]. The first IL-DLLME
23
procedure was described in 2008 by Zhou et al. and Baghdadi and Shemirani for the extraction of
24
pyrethroid pesticides and mercury, respectively [58,59]. To date, IL-DLLME is applied for the
25
extraction of a wide range of metal ions [60], small organic compounds, such as pharmaceuticals (e.g.
26
tetracycline drugs [61], sulfonamides [62], non-steroidal anti-inflammatory drugs [63] and
27
antihypertensives [64]) and biomolecules, such as DNA [65].
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Conventional IL-DLLME is characterized by the use of an organic disperser solvent to obtain a fine
30
dispersion. Conventional DLLME is therefore a three-phase system: sample + extraction solvent +
31
disperser solvent. However, the use of an organic disperser solvent is not in line with green chemistry
32
principles. Besides, it can increase analyte solubility in the aqueous sample, which results in lower
33
extraction yields [41]. In this regard, several alternatives have been introduced to avoid the use of a
34
disperser solvent; so-called two-phase IL-DLLME techniques [57]. A first alternative for the use of
35
a disperser solvent is mechanical agitation. Two major examples are ultrasonic-assisted IL-DLLME
36
(UA-IL-DLLME) and vortex-assisted IL-DLLME (VA-IL-DLLME). Also, the use of a rotary mixer
37
or a syringe to rapidly draw and inject the extraction solvent, have been described. Other approaches 10
ACCEPTED MANUSCRIPT 1
are the use of a microwave or temperature differences to obtain a fine dispersion [8,26,56]. In the
2
case of temperature-controlled IL-DLLME, ILs are used with an upper or lower critical solution
3
temperature; meaning that the IL is soluble in the aqueous sample above or below respective
4
temperatures. This creates a “dispersion” on a molecular level [66].
5 IL-DLLME applications require ILs with specific properties. Conventionally, hydrophobic ILs are
7
used, as they form a separate layer with aqueous samples. ILs with low viscosities are preferred, since
8
highly viscous ILs may hamper mass transfer during extraction [67,68]. Furthermore, ILs should have
9
a higher density than water, as this allows the IL to be more easily collected from conical-bottom
10
tubes, compared to floating low density solvents (e.g. phosphonium-based ILs) [60]. In general, 1-
11
alkyl-3-methylimidazolium hexafluorophosphate is the most frequently used IL class in extraction
12
applications, thanks to its relatively low cost.
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Next to conventional hydrophobic ILs, hydrophilic ones are also used. Two important applications are
15
in situ solvent formation microextraction (ISFME) and IL-based aqueous two-phase system (IL-
16
ATPS). ISFME consists in adding an anion-exchange reagent to the sample – hydrophilic IL mixture.
17
This result in the formation of a hydrophobic IL that can be separated from the aqueous sample [69].
18
IL-ATPS is based on the salting-out principle, where a hydrophilic IL is separated from the aqueous
19
sample by adding kosmotropic salts [70].
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4.3. Applications in forensic toxicology
Table 4 summarizes publications that use ILs as solvents in forensically relevant microextraction
23
procedures. So far, IL-based LPME has been studied for different classes of drugs: antipsychotics,
24
antidepressants, benzodiazepines, psychostimulants, opioids, and cannabinoids.
25
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Antipsychotics
27
As for the group of antipsychotics, Cruz-Vera et al. developed an IL-based dynamic liquid-phase
28
microextraction (IL-dLPME) for the determination of seven phenothiazine drugs (chlorpromazine,
29
fluphenazine, levomepromazine, prochlorperazine, promethazine, thioridazine, trifluoperazine) in
30
urine [71]. This dynamic approach involves the constant flow of the sample through a high-density IL
31
plug, present at the bottom of a Pasteur pipette. This technique generally results in higher enrichment
32
factors compared to a static approach. The selected extraction solvent, 1-butyl-3-methylimidazolium
33
hexafluorophosphate, was able to recover 72 – 98% of the analytes, with LOD values between 21 – 60
34
ng/mL [71].
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The extraction of clozapine and its metabolites (norclozapine and clozapine-N-oxide) from urine and
11
ACCEPTED MANUSCRIPT serum samples was studied by Breadmore and involved a vortex-assisted ionic liquid-based liquid-
2
phase microextraction (VA-IL-LPME) technique [72]. The extraction solvent, 1-ethyl-3-
3
methylimidazolium bis(trifluoromethylsulfonyl)imide, was added to the untreated biological samples,
4
followed by a 30-sec vortex step and centrifugation to separate both phases. In the offline approach,
5
the lower IL phase is collected with a pipette and transferred into a sample vial for capillary
6
electrophoresis (CE) analysis, while in the online approach, extraction and centrifugation is performed
7
directly in the sample vial, which can be subsequently placed in the CE autosampler for analysis. In
8
order to enable sufficient CE separation in the presence of the IL salt, a background electrolyte cation
9
with lower ion mobility than EMIm was selected, at high concentration. Validation results showed
10
analyte recoveries near 90% in urine and 85% in serum, except for clozapine-N-oxide (20%, serum).
11
LOD values were all lower than 13 ng/mL, except for clozapine-N-oxide (55 ng/mL, serum) [72].
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Zare et al. studied the extraction of an antipsychotic (perphenazine) and antidepressant (doxepin) drug
14
from urine, using an ultrasound-assisted ionic liquid surfactant-emulsified microextraction (UA-IL-
15
SE-ME) procedure [73]. The use of a surfactant (sodium dodecyl sulfate) and ultrasound irradiation
16
avoided the use of a disperser solvent and resulted in the optimal contact between extraction solvent
17
(1-hexyl-3-methylimidazolium hexafluorophosphate) and analytes. The final extraction procedure took
18
up to 20 min, recoveries of 89 – 98% and LOD values of 0.1 – 1 ng/mL were obtained [73].
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19 Antidepressants
21
Recently, De Boeck et al. developed an IL-DLLME procedure for a broader group of antidepressants:
22
agomelatine, amitriptyline, bupropion, clomipramine, dosulepin, doxepin, duloxetine, escitalopram,
23
fluoxetine, imipramine, maprotiline, mianserin, mirtazapine, nortriptyline, paroxetine, reboxetine,
24
trazodone and venlafaxine [74]. The method used 1-butyl-3-methylimidazolium hexafluorophosphate
25
(BMIm PF6) as the extraction solvent, which was added directly to the whole blood sample, followed
26
by a 5-min rotary mixing step. The final extract was diluted 1/10 (v/v) in methanol prior to LC-
27
MS/MS analysis, in order to lower the viscosity of the IL and enable accurate LC sample injection.
28
Furthermore, the authors demonstrated that diluting the final extract lowered ion suppression by
29
limiting the injected amount of non-volatile IL and by decreasing IL viscosity. However, the optimal
30
dilution factor should be selected as a reasonable balance between ion suppression diminution and
31
sensitivity loss. Validation results showed that LOD values ranged from 0.8 to 2 ng/mL (35 ng/mL for
32
trazodone) and recoveries were within 53 – 133% (80 – 115% for the majority of analytes). It should
33
be noted that ion suppression was observed with ME values ranging from 62 – 123%, however, ME
34
results were found to be repeatable and could be taken into account [74].
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Benzodiazepines
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De Boeck et al. developed a similar multi-analyte IL-DLLME procedure for the extraction of 17 12
ACCEPTED MANUSCRIPT benzodiazepines
(7-aminoflunitrazepam,
alprazolam,
bromazepam,
clobazam,
clonazepam,
2
clotiazepam, diazepam, estazolam, ethyl loflazepate, etizolam, flurazepam, lormetazepam, midazolam,
3
oxazepam, prazepam, temazepam, triazolam) and 2 Z-drugs (zolpidem, zopiclone) in whole blood
4
samples [75]. The same extraction solvent, BMIm PF6, gave the best extraction efficiencies.
5
Moreover, a clear trend was observed for both antidepressant and benzodiazepine extraction; an
6
increase of the alkyl side chain length on the IL imidazolium cation resulted in lower extraction
7
efficiencies. This could be attributed to the increase in viscosity and/or decrease of polarity.
8
Recoveries were within 24.7 – 127.2% and LOD values ranged from 0.003 – 5 ng/mL. Since LC-ESI-
9
MS/MS was used for the analysis of the final extracts, again ion suppression was observed due to co-
10
elution of the IL and analytes [75]. An additional study by De Boeck et al. compared a set of 11 ILs
11
for their benzodiazepine extraction potential [76]. It was concluded that low-viscosity ILs are required
12
for IL-DLLME procedures, to enable efficient mass transfer during the extraction, to avoid
13
electrospray ionization issues and obtain data with good repeatability. Furthermore, sp2 hybridization
14
of the IL cation is desirable in order to effectuate π-stacking interactions with the 1,4-benzodiazepine
15
skeleton. Moreover, avoiding long alkyl chains and arene substitution of the cation was advised.
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16 Psychostimulants
18
Ephedrine and ketamine extraction from urine was studied by Liu et al [77]. The method consisted of a
19
normal dispersive liquid-liquid microextraction that uses BMIm PF6 as the extraction solvent, in
20
combination with acetonitrile as the disperser solvent. Prior to extraction, urine samples were treated
21
with a protein precipitation step (1:1 methanol) and a filtration step (4000 rpm, 5 min). The extraction
22
consisted of an IL-mediated extraction step, followed by an acidic back-extraction and CE-UV
23
analysis, which took about 5 minutes in total. The method gave recoveries of 79 – 90% from urine
24
samples and LOD values of 210 ng/mL for ephedrine and 390 ng/mL for ketamine [77].
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Wang et al. developed a simple IL-DLLME procedure for the extraction of methamphetamine in urine
27
samples, using 1-octyl-3-methylimidazolium hexafluorophosphate (OMIm PF6) and a disperser
28
solvent (methanol) [78]. The actual extraction only took a few seconds by rapidly injecting the IL
29
solvent into the samples. After centrifugation, the lower phase was collected for LC-UV analysis. The
30
LOD of methamphetamine was 1.7 ng/mL and recoveries ranged up to 82% [78].
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The extraction of nicotine from plasma and urine, using an IL-based HF-LPME procedure, was
33
studied by Lin and Yan [79]. To select the ideal extraction solvent, three commonly used VOSs were
34
compared to a hydrophobic IL; BMIm PF6. The authors found that the IL gave the highest extraction
35
efficiencies and six µL of the solvent was placed in a polypropylene hollow fiber (0.2 µm pore size).
36
The fiber was submerged in the sample for 20 min, followed by the collection of the IL extractant and
37
LC-UV analysis. The LOD of nicotine was found to be 50 ng/mL and recoveries ranged between 94 – 13
ACCEPTED MANUSCRIPT 1
98% for real case biological samples [79].
2 Opioids
4
A new and fast sample treatment strategy for the separation of opium alkaloids from an aqueous
5
Pericarpium Papaveris extract was developed by Li et al. [80]. An IL-ATPS procedure was used to
6
extract polar analytes (codeine, papaverine) from the solid matrix. As the hydrophilic IL, 1-butyl-3-
7
methylimidazolium chloride (BMIm Cl) was selected. The IL was dissolved in the plant extract,
8
followed by the addition of an alkaline K2HPO4 salt. This was thoroughly mixed and a two-phase
9
system (IL-rich and salt-rich phase) was formed within 2 minutes due to sufficient concentration of
10
K2HPO4 salt that initiated a salting-out process. Finally, the IL-rich phase was collected for analysis,
11
using LC-UV. Overall, recoveries ranged from 90 – 100% and 99 – 102% for codeine and papaverine,
12
respectively. LOD values were lower than 30 ng/mL. It should be noted LOD values were calculated
13
based on aqueous samples spiked with codeine and papaverine standard. No real plant matrices were
14
included, except for accuracy verification. Here, the IL-ATPS procedure gave similar results,
15
compared to a LLE method for the extraction of both opium alkaloids from plant samples (n=3).
16
Overall, the developed method showed to be fast (approx. 5 min) and did not require the use of
17
volatile organic solvents [80].
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The decontamination of hair samples using IL solvents was studied by Restolho et al. A contactless
20
procedure was developed for the extraction of morphine and its main active metabolite, 6-
21
monoacetylmorphine (6-MAM), from the hair surface, using a hydrophilic IL extraction solvent [81].
22
The procedure required a customized extraction flask with two separated compartments; one that holds
23
the fragmented hair sample and one that holds the extraction solvent; IL. The closed flask was placed
24
in a GC oven and heated at 120°C for 16 h. The temperature was selected as the optimal balance
25
between avoiding decomposition of IL and hair and promoting the evaporation of the studied analyte.
26
The evaporated analyte was then ‘trapped’ in the IL compartment. The method was validated by
27
comparison to a reference method, based on external contamination of blank hair samples (n = 12).
28
For both methods, drug incorporation was observed resulting in false positive results. However, this
29
was countered by applying the wash criterion. Overall, the authors state that for most hair samples the
30
difference between both the developed and reference method was less than ± 12%. It should be noted
31
that IL-based hair washes were not analyzed for the developed method, only drug concentrations
32
found in the hair digest itself were reported. This is a shortcoming of the method, as according to the
33
Society of Hair Testing, it is recommended to store the hair washes for later analysis [82]. In a follow-
34
up study, Restolho et al. compared a set of six ILs based on their morphine/6-MAM extraction ability
35
in order to better understand the specific IL-analyte interactions [83]. From the selected ILs, it was
36
found that 1-hydroxyethyl-3-methylimidazolium tetrafluoroborate (C2OHMIm BF4) gave the highest
37
extraction efficiencies, which was attributed to the formation of hydrogen bonds between the analyte
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ACCEPTED MANUSCRIPT 1
and the IL cation with high hydrogen bond donor potential thanks to the presence of a hydroxyl group.
2
Decontamination efficiencies were near 30% and 70% for morphine and 6-MAM, respectively, at 100
3
°C during 24 h. The presence of water seemed to enhance extraction efficiencies, values up to 80%
4
were obtained.
5 Cannabinoids
7
So far, one publication has demonstrated the use of ILs for the extraction of ∆9-tetrahrydrocannabinol
8
(THC) [84]. Similar to the contactless decontamination of morphine from hair, Restolho et al.
9
described a procedure for THC decontamination. The final method consisted of a 13-h heating step
10
(100°C) of hair in the presence of a hydrophilic IL in a specialized glass flask. Fifty-three ILs were
11
screened for their extraction efficiency, among which the best results were obtained using C2OHMIm
12
BF4. The authors state that the simple protocol and the ability of decontaminating several samples
13
simultaneously are the main benefits of contactless decontamination compared to conventional
14
methods [84]. Analogous to the morphine decontamination procedure by Restolho et al. [81], it should
15
be noted that the IL-based hair wash was not analyzed, only drug concentrations found in the hair
16
digest itself were reported. This is a shortcoming, as according to the Society of Hair Testing, it is
17
recommended to store hair washes for later analysis [82].
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18 5. Conclusion and outlook
19
Sample preparation remains a crucial aspect of analytical forensic toxicology and should be
21
continuously explored and improved. The introduction of ILs as extraction solvents in microextraction
22
procedures has shown to be beneficial on many levels. First, ILs generally have a low flammability
23
and negligible vapor pressures, which results in safer extractions with reduced air pollution. Second,
24
the chemical structure of ILs can be easily modified in order to obtain solvents with desired
25
physiochemical properties and increased affinity toward the analyte. Third, the estimated number of
26
possible ILs that can be created is 1018, which offers a broad functionality range for distinct extraction
27
applications. Over the last decade, IL-based analytical research in forensic toxicology has expanded
28
significantly, with the focus on complex biological matrices (urine, serum, plasma, whole blood, hair)
29
and
30
psychostimulants, opioids, pesticides and THC). The majority of the discussed research articles
31
developed an IL-based LPME technique, since LPME protocols are generally faster and simpler when
32
compared to sorbent-based alternatives. The selected LPME procedures had optimal extraction times
33
ranging from a few seconds to 20 minutes, while sorbent-based techniques showed extraction times of
34
at least 30 min. Despite extended extraction times, sorbent-based microextractions seems to obtain
35
slightly higher recoveries. This was seen for the extraction of methamphetamine from urine samples,
36
where the IL-SPME procedure obtained 10% higher extraction yields compared to the IL-DLLME
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forensically
relevant
substances
(antipsychotics,
15
antidepressants,
benzodiazepines,
ACCEPTED MANUSCRIPT alternative [50,78]. Taking a closer look at IL-LPME research, it was observed that IL-DLLME was
2
the most frequently applied technique for drug extraction purposes, using a high-density and
3
hydrophobic IL. Overall, imidazolium-based cations are dominating the sample preparation field, as
4
they are easy to obtain at relatively low cost. Furthermore, it was reported that the aromatic properties
5
of the imidazolium cation are crucial for benzodiazepine binding and thus its efficient extraction. As
6
anions, PF6 and Tf2N are often used in combination with an imidazolium cation to obtain hydrophobic
7
solvents. While BF4 and chloride anions generally result in hydrophilic solvents that are used for the
8
extraction of polar substances, such as codeine and morphine. Next to the choice of an appropriate
9
extraction solvent, the appropriate analytical instrument should be considered. Most research groups
10
analyzed their final extracts using LC, coupled to UV or DAD instruments. However, in forensic
11
toxicology, UV and DAD are no longer golden standard techniques, due to limited selectivity, limited
12
sensitivity and thus problematic compound identification. Therefore, mass spectrometry is generally
13
considered first choice. De Boeck et al. tested eleven commercially available ILs (e.g. ammonium,
14
imidazolium, pyridinium and pyrrolidinium cations, combined with hexafluorophosphate or
15
bis(trifluoromethylsulfonyl)imide) and extracts were analyzed using LC-ESI-MS/MS [76]. The
16
authors concluded that the IL solvents all had a tendency to stick to reversed phased LC columns and
17
therefore partially co-eluted with the analytes, resulting in a significant impact on analyte ionization
18
and sensitivity loss. To obtain optimal sensitivities, one should carefully select the IL extraction
19
solvent based on its chromatographic behavior. In general, it can be concluded that for the optimal
20
analysis of polar (e.g. amphetamines, opioids) and apolar compounds (e.g. antidepressants,
21
benzodiazepines), co-elution with IL should be avoided by increasing analyte retention in terms of
22
selecting an appropriate column and gradient, under the assumption that the IL remains minimally or
23
unretained. Furthermore, De Boeck et al. suggested avoiding ILs with high viscosity (> 300 mPa⋅s) as
24
they tend to form a broader chromatographic front and severely suppress ionization [76]. Another
25
specific challenge coupled to high IL viscosity is the reduced accuracy and repeatability of the LC
26
sample injection step. In this respect, GC seems be a more promising technique. Nevertheless, GC-MS
27
applications are rarely documented for the analysis of IL-based extracts. In this review, only He et al.
28
described a headspace SPME method for amphetamine and methamphetamine extraction [50].
29
Generally, GC-MS is avoided due to the non-volatile nature of ILs. In the rare case GC-MS is used,
30
SDME or HF-LPME should be considered, as these extraction techniques only produce small volumes
31
of IL, limiting the contamination of the GC liner. Additionally, the injector temperature should not
32
exceed the IL’s melting point or decomposition temperature to avoid contamination [41].
33
To effect further implementation of ILs in forensic toxicological sample preparation, researchers need
34
to focus on developing IL-based microextraction procedures for the quantification of a larger set of
35
drugs, since targeted multi-analyte methods save costs and time. Moreover, the applicability of ILs in
36
untargeted toxicological screening procedures has so far not been investigated. A universal IL
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ACCEPTED MANUSCRIPT extraction solvent should be selected that has affinity for a wide range of analyte polarities. From the
2
discussed procedures, it was observed that imidazolium-based ILs show a broad applicability and
3
could be tested for potential toxicological screening purposes. However, attention should be paid to
4
the extraction of polar drugs and metabolites, as these will probably require a specific extraction
5
approach as for instance was presented by Li et al. [80]. Furthermore, the implementation of a serial-
6
coupled LC system, described by Loos et al. [85], could improve separation of IL and both polar/non-
7
polar analytes by combining a hydrophilic interaction chromatography (HILIC) and reversed-phase
8
liquid chromatography (RPLC) column, which provides an orthogonal separation mechanism and
9
avoids co-elution and thus analyte suppression. Finally, in forensics, postmortem samples form a
10
significant portion of the analyzed matrices. Here, the question remains whether microextractions are
11
able to deal with these clotted and putrefied liquids.
12
Despite all advantages, the absolute number of IL-based extraction applications is still low.
13
Economical restrictions are probably the major cause. Since ILs are still produced on small scale from
14
pricey raw materials, this results in solvents that are typically 2 - 100 times more expensive than VOSs
15
[29,86]. With an increasing number of IL-based applications, its market share will expand and
16
production scale-up will automatically result in lower costs. Next to IL cost, toxicity and
17
biodegradability issues are even more important. Mostly, fluorinated anions are studied, due to their
18
attractive features for solvent extractions; they generally result in ILs with high hydrophobicity and
19
higher density than water. However, fluorine-containing anions have been reported to lower EC50
20
values of bacteria and can undergo hydrolysis with the formation of hydrofluoric acid [36,87].
21
Luckily, IL structural features can be easily modified in such a way to positively influence IL
22
biodegradability and IL toxicity. Moreover, the introduction of renewable ions based on amino acids,
23
choline or sugars form a possible solution [88]. When designing green ILs, physicochemical properties
24
should be carefully monitored to obtain a final compound that is usable for solvent- or sorbent-based
25
extraction applications. Furthermore, it still remains difficult to select the ideal IL extraction solvent
26
from an almost infinite number of potential anion-cation combinations. To ensure further
27
implementation of ILs in sample preparation, a rational IL design approach is needed. This would
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allow rational combining of the desired anion and cation, resulting in an IL with the required
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physicochemical properties for a certain application. Progress has been made with the launch of a US
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National Institute of Standards and Technology (NIST) database that provides researchers with
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relevant publications on IL structure and associated physicochemical properties. However, knowledge
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should be extended by screening sets of ILs for a specific extraction application and defining crucial
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IL-solute binding interactions in order to elucidate IL-based extraction mechanisms.
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ACCEPTED MANUSCRIPT This research did not receive any specific grant from funding agencies in the public, commercial, or
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not-for-profit sectors.
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Figure 2. Chemical structures of typical ionic liquid cations and anions.
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Figure 1. Sodium chloride (NaCl) and 1-butyl-3-methylimidazolium hexafluorophosphate (BMIm PF6). 1: NaCl; 2: BMIm PF6; a: physical appearance at room temperature; b: simplified overview of chemical appearance (blue/black/white = cation, orange = anion).
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Figure 3. Schematic overview of sorbent-based microextraction procedures. SPME: solid-phase microextraction; SBSE: stir bar sorptive extraction; MEPS: microextraction by packed sorbent.
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Figure 4. Schematic overview of solvent-based microextraction procedures. SDME: single drop microextraction; HF-LPME: hollow fiber liquid phase microextraction; DLLME: dispersive liquid-liquid microextraction.
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Table 1.
2
Advantages and disadvantages of sorbent-based microextractions Advantages Simple Solvent free Small sample size Portable on field sampling Online applications Molecularly imprinted polymer Reusable
-
Low operating temperature Breakage of fiber (fused silica) Chemical instability Swelling of fiber High cost* Low sensitivity Carryover with reuse?
SBSE (°1999)
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Simple Solvent free High sensitivity High extraction capacity High robustness Molecularly imprinted polymer Reusable
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Lack of appropriate coatings High cost* High extraction times Carryover with reuse?
MEPS (°2004)
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Fast (1-4 min) Simple Molecularly imprinted polymer Reusable
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SPME (°1990)
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Requires solvent (small volume) Syringe blockage High cost* Carryover with reuse?
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SPME: solid-phase microextraction; SBSE: stir bar sorptive extraction; MEPS: microextraction by packed sorbent; *if not reused; ° year of first introduction. [8,41–44,54]
AC C
3 4 5 6
Disadvantages
26
ACCEPTED MANUSCRIPT
Table 2.
Pesticides (6) Benzodiazepines (4)
Sample amount
Extraction technique
Ionic liquid
Urine
4 mL
IL-SPME
EC2MIm Tf2N
Water Hair River water Urine Hair
5 mL 50 mg 10 mL 10 mL 50 mg
IL-HF-SPME
BMIm OH
SC
Amphetamine Methamphetamine
Sample
M AN U
Analyte
RI PT
Applications of ILs as sorbents for the extraction of forensically relevant drugs.
IL-HF-SLPME
PMIm Br@TiO2
Recovery (%)
LOD (ng/mL)
89 – 103 94 – 114 86 – 99 82 – 94 95 – 102 45 – 106 93 – 103
0.5 0.1 0.004 – 0.095 0.003 – 0.08 0.08 – 0.5
Detection
Ref.
GC-MS
[50]
LC-DAD
[51]
LC-UV
[52]
AC C
EP
TE D
IL-SPME: ionic liquid-based solid-phase microextraction; IL-HF-SPME: ionic liquid-based hollow fiber solid-phase microextraction; IL-HF-SLPME: ionic liquid-based hollow fiber solidliquid-phase microextraction; BMIm: 1-butyl-3-methylimidazolium; EC2Mim: 1-ethoxyethyl-3-methylimidazolium; PMIm: 1-pentyl-3-methylimidazolium; @TiO2: coated onto titanium dioxide nanoparticles; Br: bromide; OH: hydroxide; Tf2N: bis(trifluoromethylsulfonyl)imide.
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ACCEPTED MANUSCRIPT Table 3. Advantages and disadvantages of solvent-based microextractions Advantages
Simple Enhanced selectivity (pores) Molecularly imprinted polymer Reusable
Instability of drop Fluctuation of drop volume Poor reproducibility Low contact surface Low extraction capacity Cost of fiber Long extraction times Fiber pore blockage Air bubbles near pores Carryover with reuse? Use of disperser solvent Difficult automation
- Fast (5 min) - Simple - High recovery
M AN U
DLLME (°2006)
-
-
RI PT
HF-LPME (°1999)
- Simple - Low cost - High enrichment factor
SC
SDME (°1996)
Disadvantages
AC C
EP
TE D
SDME: single drop microextraction; HF-LPME: hollow fiber liquid phase microextraction; DLLME: dispersive liquid-liquid microextraction. °: year of first introduction. [8,41,43,53,54]
28
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Table 4.
RI PT
Applications of Ils as solvents for the extraction of forensically relevant drugs.
Analyte
Sample
Sample amount
Phenothiazines (7)
Urine Urine Serum
10 mL 0.2 mL 0.2 mL
Urine
6 mL
UA-IL-SE-ME
Whole blood
1 mL
IL-DLLME
BMIm PF6
Whole blood
1 mL
IL-DLLME
BMIm PF6
Urine
0.5 mL
IL-DLLME
BMIm PF6
Urine Urine Plasma Pericarpium Papaveris extract
10 mL
IL-DLLME
OMIm PF6
Nicotine Codeine Papaverine Morphine 6-Monoacetylmorphine ∆9-Tetrahydrocannabinol
25 mL 1000 mg
Hair
20 mg
Hair
20 mg
53 – 133
0.8 – 35
25 – 127 59 – 98 86 – 90 79 – 82 80 – 82 98 94 90 – 100 99 – 102
0.003 – 5 0.3 – 0.4 210 390 2
C2OHMIm BF4 C2OHMIm BF4
EMIm Tf2N
SC
VA-IL-LPME
HMIm PF6
M AN U
Benzodiazepines (17) Z-drugs (2) Ephedrine Ketamine Methamphetamine
21 – 60 3 – 11 7 – 55 0.1 1
BMIm PF6
TE D
Antidepressants (18)
72 – 98 87 – 93 20 – 85 93 – 98 89 – 96
IL-dLPME
IL-HF-LPME
BMIm PF6
IL-ATPS
BMIm Cl
Contactless decontamination Contactless decontamination
EP
Doxepin Perphenazine
LOD (ng/mL)
Ionic liquid
AC C
Clozapine (+ metabol.)
Recovery (%)
Extraction technique
Detection
Ref.
LC-UV
[71]
CE-DAD
[72]
LC-UV
[73]
LC-MS/MS
[74]
LC-MS/MS
[75,76]
CE-UV
[77]
LC-UV
[78]
50
LC-UV
[79]
30 20
LC-UV
[80]
NA
NA
GC-MS
[81,83]
NA
NA
GC-MS
[84]
IL-dLPME: ionic liquid-based dynamic liquid-phase microextraction; VA-IL-LPME: vortex-assisted ionic liquid-based liquid-phase microextraction; UA-IL-SE-ME: ultrasound-assisted ionic liquid surfactant-based emulsification microextraction; IL-DLLME: ionic liquid-based dispersive liquid-liquid microextraction; IL-ATPS: ionic liquid-based aqueous two-phase system; IL-HFLPME: ionic liquid-based hollow fiber liquid-phase microextraction; EMIm: 1-ethyl-3-methylimidazolium; BMIm: 1-butyl-3-methylimidazolium; HMIm: 1-hexyl-3-methylimidazolium; OMIm: 1-octyl-3-methylimidazolium; C2OHMIm: 1-hydroxyethyl-3-methylimidazolium; BF4: tetrafluoroborate; Cl: chloride; PF6: hexafluorophosphate; Tf2N: bis(trifluoromethyl-sulfonyl)imide; NA: not applicable.
29
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Highlights An overview of general ionic liquid physicochemical features is provided.
•
Benefits of using ionic liquids as extraction sorbents or solvents are discussed.
•
Ionic liquid-based forensic toxicological analytical methods are reviewed.
•
An outlook on the applicability of ionic liquids in forensic toxicology is given.
•
Challenges associated with ionic liquid-based extractions are discussed.
AC C
EP
TE D
M AN U
SC
RI PT
•