Recent Applications of Solid Phase Microextraction Coupled to Liquid Chromatography

Abstract

Solid phase microextraction (SPME) is one of the most popular sample preparation methods which can be applied to organic compounds allowing the simultaneous extraction and pre-concentration of analytes from the sample matrix. It is based on the partitioning of the analyte between the extracting phase, generally immobilized on a fiber substrate, and the matrix (water, air, etc.), and has numerous advantages such as rapidity, simplicity, low cost, ease of use and automation, and absence of toxic solvents. Fiber SPME has been widely used in combination with various analytical instrumentation even if most of the work has been done coupling the extraction technique with gas and liquid chromatography (GC and LC). This manuscript presents an overview of the recent works (from 2010 to date) of solid phase microextraction coupled to liquid chromatography (SPME-LC) relevant to analytical applications performed using commercially available fibers or lab-made fibers already developed in previous papers, and to improved instrumental systems and approaches.

1. Introduction

The demand for modern, sensitive, and accurate analytical methods for the determination of compounds at low concentration levels in complex matrices, such as biological, food, and environmental, is always increasing. The main steps of an analytical process are sampling, sample preparation, instrumental analysis, and data processing, and any of them may deeply affect the analytical performance. Today, analytical instrumentation has reached an advanced level of technology and performances unimaginable until a few years ago, that allow selective and sensitive analytes determination even in complex extracts. On the other hand, sample preparation is usually the major source of inaccuracy and imprecision on analysis and remains a critical and time-consuming component of the workflow. It is devoted to the transfer of analytes from sample matrices to simplified final extracts compatible with the analytical instrument to be employed and is certainly a prerequisite for the attainment of reliable results. Consequently, any improvement on chemical analysis greatly depends also on the technological advances of modern sample preparation techniques.

Solid phase microextraction (SPME) is an evolving technique for the extraction of organic compounds from gaseous, aqueous, and solid matrices. The principle of SPME involves the equilibration of the analytes between a polymeric phase, generally coated onto a fiber, and the sample matrix. It has numerous advantages over other sample preparation techniques such as liquid–liquid (LLE), solid-phase (SPE), and supercritical fluid extraction (SFE), i.e., rapidity, low cost, ease of use and automation, and ability to perform in situ measurements. Furthermore, no harmful solvents are needed.

Since its introduction [1], many variants of the SPME technique have evolved and it can be found in different geometries, in particular in-tube [2], hollow fiber [3], thin-film [4], coated-tip [5], and magnetic nanoparticles [6]. However, the fiber geometry, where a fiber is covered by a specific sorbent to extract target compounds from the sample matrix by direct immersion (DI-SPME) or from the headspace (HS-SPME) [7], remains the most widespread.

Fiber SPME has been widely used in combination with various analytical techniques: gas and liquid chromatography (GC, LC) [8,9,10,11,12], capillary electrophoresis (CE) [13], mass spectrometry (MS) [10,11,12], inductively coupled plasma MS (ICP-MS) [14], and ICP-optical emission spectrometry (ICP-OES) [15]. However, most applications have been performed with gas chromatography (GC) and, to a lesser extent, liquid chromatography (LC).

Of course, SPME-GC applications are limited to the analysis of volatile and thermally stable compounds, while SPME-LC can be used for the analysis of thermally labile compounds with poor volatility. Nevertheless, for many years, most efforts towards applications and practical implementations of SPME have privileged coupling with GC. The main reason for that is the different nature between the desorption steps used in LC and GC. In fact, solid phase microextraction devices are easily interfaced with GC injectors since the thermal desorption process follows the same steps used for standard GC injections. On the contrary, the SPME-LC coupling requires a slower liquid desorption process, due to slower diffusion kinetics. Desorption is generally performed off-line, in a vial, or on-line, in the chamber of a dedicated interface, using two different modes: in the flowing mobile phase (dynamic mode) or in an appropriate solvent mixture for a specific time before injection is performed (static mode). Furthermore, the on-line liquid desorption process must be carefully optimized to avoid the decrease of the chromatographic efficiency.

Despite the problems, SPME-LC has gained a good degree of popularity over the years, as demonstrated by dedicated articles that have extensively reviewed its state of the art in the past [8,9]. Back then, most of the reviewed works were focused on applications of the technique to the determination of various analytes in environmental, biological, and food matrices, as well as on partitioning studies, thanks to its peculiar non-exhaustive extractive features.

On the contrary, the many additional works published on the subject over the past decade have followed differentiated trends. Most of them have been focused on the search, discovery, and application of novel materials [16,17,18,19,20,21] to be used as extractive phases. In fact, a wide range of new sorbents has been developed and recently reviewed for both SPME-GC and LC applications, including carbon-based [22], metal organic frameworks [23], molecular imprinted polymers [24], ionic liquids [25,26,27], immunosorbents, sol-gel-based compounds [28,29], and nanomaterial-based [30], among others.

On the other hand, a substantial part of the fiber SPME-LC literature of the past 10 years has not yet been reviewed and is the object of the present work. The purpose of this review is therefore to provide an overview of the main recent applications performed with the traditional SPME-LC approach using the fiber geometry. The manuscript is deliberately focused on analytical applications mainly performed using commercially available fibers, in order to provide a collection of works immediately usable by professionals and/or a starting point for scientists for developing future applications. Some interesting, selected works dealing with the development of new instrumental systems and approaches are also reported. This work could represent a very useful tool for those interested in SPME-LC fast and practical applications.

2. Applications

2.1. Biological

2.1.1. Fluids and Tissues

Liquid chromatography can be considered as the technique of choice currently used for drugs and metabolites analysis in biological fluids and tissues. However, biological matrices, such as serum, plasma, and tissues, are complex mixtures incompatible with a direct injection into conventional LC supports and a sample preparation step that limits the number of interferents co-extracted with the analytes is always needed, especially when working with LC coupled to electrospray mass spectrometry (ESI-MS) to prevent ionization issues. The commonly used extraction techniques (SPE, LLE, ultrafiltration, dialysis) generally work off-line, therefore being complex and tedious, and a substantially time-limiting step in the analytical process. Furthermore, they often require protein precipitation procedures that can induce analyte losses due to the known phenomenon of co-precipitation. SPME-LC has proven to be a good fast and sensitive approach for the determination of semi- or non-volatile analytes in biological samples, also for its ability to determine free and/or total drug concentration, depending on the extraction conditions in the sample under investigation [31].

The liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination of the antifibrinolytic drug tranexamic acid in plasma samples extracted by SPME, using a commercial biocompatible C18 coating, was accomplished by Bojko et al. [32]. Good accuracy and precision were obtained, and the matrix effect was minimized making the sample compatible with ESI detection. A comparison between the new method and standard techniques of protein precipitation and ultrafiltration showed no significant differences among the performances of the approaches. The potential of the procedure was demonstrated by the successful analysis of clinical plasma samples, determining the analyte at concentration levels that exceeded the capability of current assays employing sample preparation using protein precipitation followed by LC/MS analysis. In another study, the SPME-LC-UV simultaneous determination of clenbuterol, salbutamol, and ractopamine residues using polydimethylsiloxane/divinylbenzene (PDMS/DVB) fibers and on-line desorption was reported by Dua et al. [33], and its application to real samples demonstrated by the analysis of pig samples (muscle and liver), while Gonzalez and co-workers proposed a one-step simple extraction (using two fibers, carboxen-polydimethylsiloxane (CAR/PDMS) and polyacrylate (PA)) of BTEX (benzene, toluene, ethylbenzene, and xylenes and its metabolites) from spiked tooth followed by SPME-HPLC or HS-SPME-GC/MS determination [34]. In particular, the SPME-LC procedure showed the typical advantages of SPME (simple, rapid, accurate, solvent-free), good precision, linearity, and sensitivity, even if limits of detection (LODs) were slightly lower than those obtained with alternative approaches due to the limitation of the UV detector. The method was successfully applied in human teeth as environmental biomarker of BTEX and metabolites.

SPME (biocompatible C18 fibers) coupled to LC-MS/MS (triple quadrupole, QqQ) was also used to determine selected antibiotics and their metabolites in human blood and tissue taken from afflicted areas [35]. Samples of 25 patients under treatment were successfully analyzed to estimate pharmacokinetic profiles. Interestingly, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), coupled off-line with LC, was also used for identification and quantification of the analytes, showing results comparable to those obtained with LC-MS/MS. Another pharmacokinetic study in plasma for methylphenidate, a stimulant for the human central nervous system, was successfully performed coupling SPME (PDMS fibers) and thermal desorption-electrospray ionization/mass spectrometry (TD-ESI/MS) [36]. The applicability of the approach was demonstrated by the validation of the relevant results by means of LC-MS/MS.

Post-mortem human blood and bone marrow aspirate samples have been the target of a rapid and simple method for the determination of 25 common psychotropic drugs of different classes by SPME and liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-QqTOF-MS) [37]. All the validation parameters were within the acceptable range for toxicological analyses. Scanning electron microscope images of used fibers showed some few networks (likely proteins) that did not affect the performances of the fiber itself despite the intensive use, indicating that it can be safely employed for multiple extractions.

2.1.2. In Vivo Sampling

Another crucial branch of biological applications is represented by in vivo sampling procedures. They should allow the preservation of the sample to ensure that the information eventually obtained are effectively representative of the situation of the living organism under investigation. Fiber SPME perfectly fits this requirement thanks to its syringe-like format that allows a faster and simpler sampling, compared to alternative methods (i.e., microdialysis or ultrafiltration), followed by retraction into the holder for storage, protection, and transportation of the extracted compounds.

Togunde et al. developed three original SPME-LC-MS/MS applications for the analysis of target pharmaceuticals (carbamazepine, diazepam, nordiazepam, and others) in fish samples. In the first study [38], a method for the determination of the bioconcentration of trace contaminants in fish bile was first validated in rainbow trout after laboratory exposures and then applied to fathead minnow caged upstream and downstream of a local wastewater treatment plant. The method performances in field were demonstrated by the detection of some analytes detected in fish bile at the downstream location. In the second one [39], the same approach was applied to the in vitro and in vivo study of kinetic desorption rate constants of the analytes in agarose gel model used to simulate tissue matrix and fish muscle (dorsal muscle), respectively, using a triple quadrupole-linear ion trap (Qtrap) mass spectrometer as LC detector. The third work [40] was focused on the development and evaluation of a new improved in vivo SPME device (PDMS coating), that enabled a simplified sampling procedure without the need of fish anesthetization, as demonstrated by the in vivo determination of the target drugs in the muscle tissue of wild river fishes.

In situ SPME (mixed-mode fibers, octadecyl and benzenesulfonic acid groups) with LC-MS/MS detection was proposed as a green in vivo method for the direct underwater sampling of a very wide range of different small compounds from corals, so that the organisms can remain in their natural habitat [41]. More than 280 species were identified, mostly eicosanoids, steroids, prenol lipids, fatty acids, glycerolipids, and glycerophospholipids, likely because of the sampling location, near tentacles, biological membranes of corals.

Three SPME (reverse phase amide fiber, RPA) calibration methods for in vivo application, i.e., on-fiber standardization, dominant pre-equilibrium desorption, and diffusion-based interface model were compared by Yeung et al. [42] using fenoterol as a drug model and LC-MS/MS detection. The methods were also validated against SPME equilibrium extraction external calibration and protein precipitation. The study demonstrates that the diffusion-based model was the most convenient for future in-vein blood SPME investigations, to sample the circulating blood of animals without the need to withdraw representative samples. In fact, the same research group subsequently developed, using the same RPA fiber, a new in vivo SPME-LC-MS/MS procedure [43] to determine the pharmacokinetics of fenoterol and methoxyfenoterol in rat blood using the diffusion-based calibration interface model. The results obtained were in good agreement with those obtained by a traditional approach of blood withdrawal followed by protein precipitation, indicating the utility of the proposed method.

A protocol for the in vivo determination of benzodiazepines and metabolites in beagles by SPME-LC-MS/MS was validated by Lord and co-workers [44], using a polypyrrole wire coating exposed to the bloodstream through a SPME probe directly inserted into a peripheral vein by means of a standard medical catheter. A similar approach was employed for the development of two more applications performed using C18 biocompatible extractive coating: a further pharmacokinetic study in mice [45] to determine free and total concentrations of carbamazepine and a derived active metabolite, and the in vivo monitoring of the concentration of eicosanoids [46], known lipid mediators involved in the development of inflammatory conditions, in the blood of rats subjected to induced inflammation. In both cases, the results obtained were comparable with those obtained with conventional sample pre-treatment methods or literature data, demonstrating once again that in vivo SPME represents a quite reliable technique. Roszkowska et al. [47] successfully developed a SPME procedure for the determination of the anticancer drug doxorubicin using homogenized lamb’s lungs as a surrogate matrix that was subsequently applied to the LC-MS/MS analysis of pig lung biopsies obtained via in vivo lung perfusion. A future perspective of this application can be the possibility to perform repeated non-invasive samplings in the organ continuously measuring the concentration levels of drugs, to eventually optimize a personal dosage for each patient.

2.1.3. Metabolomic

SPME, including the in vivo format, can be also a very suitable means for metabolomic studies. Metabolomics, which is defined as the comprehensive analysis of metabolites in a biological specimen, is currently one of the most powerful analytical approaches for studying the functional status of biological systems in numerous research fields. As metabolites are the end-products of anabolic and catabolic pathways within living organisms, their comprehensive profiling may considerably help to understand many molecular mechanisms. As a matter of fact, metabolomics is deeply influenced by the sample pre-treatment procedure; it is crucial to maintain the metabolic integrity of biological samples so that metabolomic profiles can really be the direct expression of the biochemical status of a given organism.

In vivo solid-phase microextraction was proposed by Vuckovic and co-workers [48] as a sampling method for metabolomics studies of living systems using liquid chromatography-mass spectrometry and mice as a model system. The results showed that it was possible to extract more than 1500 untargeted metabolites with different polarities using a single mixed-mode coated fiber. This sampling approach is potentially able to capture unique metabolites and detect changes in metabolic profiles associated with any kind of modification occurring in the organism under investigation. For instance, two recent different in vivo metabolomic studies were carried out in the muscle tissue of living trout by Roszkowska et al. [49,50] using SPME fibers with biocompatible coatings and LC coupled to high resolution mass spectrometry (HRMS). Lipidic profiles were obtained [49] and compared to those obtained with an ex vivo SPME study of samples stored for a prolonged period, to determine the influence of sample storage, and to those obtained by solid liquid extraction. The great potential of the new approach was evident, since it enabled the characterization of a variegated lipidome, even detecting compounds not determinable with ex vivo SPME or SLE. Furthermore, the changes in the metabolic profile associated to short- and long-term exposition to sublethal doses of benzo[a]pyrene (BaP) [50] were determined in the muscle tissue of living fish by SPME-LC-HRMS, focusing on interesting dose and time-dependent alterations observed in the exposome by case-control model. The results indicated that BaP exposition was able to decrease the number of detected compounds.

In vivo SPME, using a mixed-mode (C18 with benzenesulfonic acid) coating, coupled to LC-MS allowed the detection of more than 50 oxylipins, brain mediators involved in key processes, under normal physiological conditions in awake moving rats [51]. Using the same well established experimental set-up as in vivo microdialysis, the approach permitted to obtain detailed oxylipin profiles in real time, eliminating post-mortem changes randomly observed in oxylipin concentrations.

SPME-LC metabolomic studies were also performed ex vivo in biological fluids. A comprehensive study [52] reported the extraction of 36 different metabolites with a wide range of polarities in biological fluids by comparing the performances of more than 40 fiber coatings. Mixed-mode coatings, polar-enhanced polystyrene−divinylbenzene, and phenylboronic acid resulted to be the most suitable for the simultaneous extraction of the largest number of heterogeneous compounds and, consequently, for untargeted metabolomic profiling applications. The mixed-mode SPME coated fiber coupled to LC- HRMS was then employed to develop a fast method for the metabolomic profiling of human plasma. A comparison with ultrafiltration and solvent precipitation clearly showed the numerous advantages of the new approach in terms of metabolite coverage, method precision, and ESI ionization suppression.

A new SPME-LC-qTOF-MS method for the solventless extraction of human milk lipidome has been reported by Garwolinska et al. [53]. Almost 70 lipids were detected and identified through an automated search of a lipid database, with the goal to properly understand the health benefits of human milk and to improve the quality and composition of artificial formulas.

In a further work, SPME directly coupled to ESI-QqQ mass spectrometer provided similar ions profiles as LC-MS/MS for the screening of secondary metabolites in actinobacteria solid medium [54]. In this case, SPME was performed by means of nitinol wires coated using a slurry composed by hydrophilic–lipophilic balance (HLB) particles (5 μm) and polyacrylonitrile (PAN).

2.1.4. Partitioning Studies

Binding of chemicals to dissolved organic carbon, sediment, proteins, or other components may reduce the free concentration and thus the bioavailability or effectivity of the chemical. Therefore, knowledge of the extent of binding or partitioning of the chemical of interest is essential in several scientific disciplines and much research is focused on measuring partition coefficients or binding affinities. For highly bound compounds, the concentration of the free fraction is frequently low and difficult to detect. SPME is the perfect solution for these applications due to its ability to determine chemicals in aqueous matrices even at very low concentration and to extract only a minimum amount of ligand (negligible depletion), so that the free concentration in the solution will not change significantly, and the equilibrium between the bound and free form will remain undisturbed. Thus, when partitioning studies involving semi- or non-volatile molecules must be performed, SPME-LC coupling is a good alternative to more conventional approaches. For instance, a method for the determination of the free fraction of chlorpromazine in medium containing bovine serum albumin (BSA) by SPME with commercial polyacrylate fibers and LC-UV detection was developed [55] and used to measure the protein binding of chlorpromazine and to calculate the chlorpromazine-bovine serum albumin (BSA) affinity constant. A similar analytical challenge, i.e., the study of the binding of human serum albumin (HSA) with carbamazepine, was faced by Bojko et al. [56] by SPME coupled to LC–UV or LC-MS, and by spectroscopic fluorescence, and nuclear magnetic resonance (1H NMR). The multi-instrumental approach provided synergic information that allowed for the identification of high and low binding sites for carbamazepine, while SPME-LC-MS was used to determine in vivo the free concentrations of carbamazepine and to verify in vitro binding predictions at therapeutically relevant doses. Drugs-bovine serum albumin interactions were also the object of an SPME (C18 fibers) study [57] devoted to quantifying the protein binding of diclofenac, 2,4-dichlorophenoxyacetic acid (2,4-D), ibuprofen, naproxen, torasemide, warfarin, and genistein to BSA, phospholipid liposomes, fetal bovine serum (FBS), and cells.

2.2. Food

Food safety and quality control are great concerns for national and international regulatory committees and the scientific community. Therefore, food sample analysis constitutes one of the most important challenges in analytical chemistry and numerous scientists are highly focused on the enhancement of the speed and reliability of analytical processes, that should also be easy to perform, be cost-effective, require small amounts of solvents, provide a high selectivity, and be able to simultaneously detect multiple analytes. However, food-based matrices are complex and characterized by a wide range of chemical compositions that influence the performance of chemical analytical measurements, making sample preparation a crucial step before final analysis. SPME-LC represents a useful tool to face some of the analytical problems related to food.

A comparison of the performances of four procedures, including QuEChERS, SPE, pressurized liquid extraction (PLE), and SPME, for the extraction of different organophosphorus (OPPs) and carbamates (CMs) insecticides from honey samples before LC-MS/MS analysis was reported by Blasco and co-workers [58]. All the approaches were successfully able to recover the selected analytes and the results obtained applying the four extraction techniques to real honey samples were similar.

The five methods that will be described below have been developed using LC-UV as an analytical technique. This aspect could be considered by many as a liability due to the low selectivity and sensitivity of the technique, especially if compared to LC-MS. However, looking from a different perspective, it should be considered an added value, due to the low cost and reliability of a standard instrumentation that can be easily found in most laboratories. In fact, if a SPME-LC-UV analytical method can detect the target compounds at the concentration levels typically encountered in real matrices, even below the required maximum residue levels (MRLs), it could represent a very useful tool for cost-effective determinations. An SPME-LC-UV protocol (PDMS fiber) has been developed [59] and validated in terms of linearity, accuracy, precision, and sensitivity for the simultaneous determination of ten pesticides in mango fruits. The method was proposed as a fast screening prior to LC-MS analysis, even if it proved to be sensitive enough to detect some of the target compounds in mango samples at concentration levels below the Brazilian MRLs. A further SPME-LC-UV screening method for the simultaneous determination of six triazoles (diniconazole, fluquinconazole, flusilazole, myclobutanil, tebuconazole, and tetraconazole) in grape and apple fruit samples (peel, pulp, juice, and rain under the fruit trees) was fully validated by Bordagaray et al. [60]. The procedure was applied to real samples previously sprayed with commercial products containing the analytes and, eventually, myclobutanil and tebuconazole were found in the peel and rain, but not in pulp and juice samples. Aresta and co-workers successfully developed three different fast and simple SPME-LC-UV applications in various food matrices using commercially available fibers, on-line desorption, and LC isocratic conditions. The first work [61] reported the simultaneous determination of benzoic acid, salicylic acid, and some of its derivatives in fava beans, blueberries, kiwi, tangerines, lemons, oranges, and fruit juice (lemon and blueberry) samples, using a PDMS/DVB fiber, that was also the coating used in the development of another procedure for the simultaneous determination of major isoflavones in soy milks and blended rice/soy beverages [62]. In the latter study, the binding constants of isoflavones with serum albumin bovine were also defined to evaluate their bioavailability and to study the interaction with the main constituents in plants. A further method for the determination of free and total (after enzymatic deconjugation of piceid) concentration of trans-resveratrol in wines, spirits, and grape juices [63], was carried out using a polyacrylate fiber. The analyte was found in all the considered samples at different levels.

2.3. Environmental

To determine pollutants and/or the fate and impact of their potentially harmful degradation products in matrices of environmental interest, sensitive analytical methods to be applied to aqueous-based matrices that do not require the use of organic solvent are required. Furthermore, environmental samples are normally complex, and sample pre-treatment should be able to minimize interference arising from undesired interfering compounds. Considering that many contaminants are not suitable for GC analysis because of their physico-chemical properties, the SPME-LC approach represents an attractive choice to solve many of these analytical problems.

To allow the SPME extraction and simultaneous LC-MS/MS determination of 16 drugs having acid and basic properties, urban wastewaters samples were split in two aliquots that were adjusted at pH 3 and 11, respectively, and extracted by two different Carbowax/Templated Resin (CW-TPR) fibers [64]. The fibers were then consecutively transferred in the desorption chamber for desorption in static mode and subsequent analysis. This original “dual SPME” approach permitted the quantification of all the analytes in influent and effluent wastewater treatment plant samples by means of a single chromatographic run. Another environmental application was focused on the SPME extraction (CW-TPR fiber) and LC-UV determination of chloramphenicol in tap and sea water [65]. Since the introduction of drugs into municipal sewage-treatment plants is often due to human excretion, the method was also applied to the determination of the analyte to urine samples. The optimized procedure required a simple sample pre-treatment, isocratic elution, and provided enough sensitivity for the analyte determination in the considered samples.

An interesting study has considered for the first time the issues potentially caused in ESI-MS ionization by interferents extracted by the SPME fibers from complex matrices (environmental and biological) [66], finding significant enhancement or suppression of ionization in all the samples under investigation. Then, an isotopic internal standard method was developed and suggested as a tool to correct matrix effects in samples where its occurrence is unavoidable. A further work proposed a homemade interface to couple SPME (PDMS fiber) to LC-UV for the reliable and simultaneous determination of the antibacterial triclosan, triclocarban, and four transformation products [67] in river water samples. More recently, the performances of three fibers (acrylate, nylon, and polydimethylsiloxane) for the extraction of endocrine disruptors (EDCs), triclosan, bisphenol A, and 17α-ethinylestradiol, from seawater and for bioavailability studies were compared [68]. LC-MS/MS was used as analytical device, and acrylate fibers were demonstrated to be the most suitable for the analytes.

A fast SPME-LC-MS/MS method (about 35 min of pre-treatment time) for the determination of tetrabromobisphenol A and hexabromocyclododecane, two currently used brominated flame retardants (BFRs), was developed [69] using spiked natural waters and subsequently applied to the analysis of tap water, even if the analytes were not found in the real matrix. Finally, the ultra-trace determination of sudan dyes in wastewater has been very recently reported [70] using two different extraction protocols, namely SPME and on-line SPE, in combination with LC-UV. As far as the SPME approach is concerned, using a PDMS/DVB coating, it was able to detect sudan I and II at sub-µg/l levels while sudan III and IV were not detectable at these levels likely due to strong binding to dissolved organic matter.

2.4. Technical Improvements

Since the beginning, SPME has grown and evolved continuously, as demonstrated by the huge number of publications relating not only to its applications or to the search of new coatings and geometries, but also to experimental setups and novel coupling approaches. Many SPME-LC works have been recently published in the attempt to improve the performances of the whole SPME-LC analytical procedure.

Back to 2010, the idea to use electrochemistry as a concentration approach led to the development of a method to electrochemically assist SPME extraction of parathion in water [71]. A pencil lead electrode simultaneously played the role of electrode and fiber. The extraction occurred with the application of a suitable reductive potential to the electrode/fiber. A static/dynamic desorption step was subsequently performed in the commercial SPME-LC desorption chamber, followed by LC-APCI-MS/MS analysis at trace levels.

In the attempt to fasten the desorption process, a heated lab-made interface chamber was introduced by Alves et al. and adopted in the development of a SPME-LC protocol for the determination of anticonvulsants in human plasma samples [72]. The method was fully validated and demonstrated to be adequate for therapeutic drug monitoring.

Modifications on the SPME-LC interface and on the commercial PAN/C18 fiber assembly were also made by Chen et al. [73], to avoid fiber coating damage and solvent/mobile phase leaks. The interface original tubes, replaced to minimize extra column volume, together with the optimized desorption and elution conditions, produced a good chromatographic efficiency. The system was tested by analyzing polycyclic aromatic hydrocarbons (PAH), proving to be robust and reliable.

Analytes extracted by the SPME fibers from complex matrices were desorbed into solvent droplets in a digital microfluidics (DMF) device prior to LC-MS analysis in a further study [74] focused on the improvement of the desorption process. The brand new SPME-DMF approach was successfully applied to the sensitive determination of free steroids in urine. A relevant pre-concentration of the analytes was observed, due to the low volumes inherent to DMF, indicating the applicability of the device to the determination of different analytes in complex matrices at trace levels.

A microfluidic open interface was designed and used to directly couple biocompatible SPME to tandem mass spectrometry [75]. Using this approach, the authors have optimized a rapid method for the determination of the antifibrinolytic drug tranexamic acid in patients undergoing cardiac surgery and experiencing renal disfunction, demonstrating the ability of this interface to perform an ultrafast monitoring of its concentration during administration (30 s per sample, 15 min with sample preparation) and then correcting the dosage in real time. A cross validation against a more conventional technique, such as standard thin film SPME-LC-MS/MS, confirmed the reliability of the procedure.

Kayali-Sayadi et al. [76] proposed a modification of the commercial SPME-LC interface to perform both vibrations-assisted extraction and desorption, directly placing the fiber in the chamber usually dedicated to the desorption step. Extraction in dynamic mode was made by continuously supplying the sample matrix using a syringe, while a mixed static/dynamic mode was chosen for desorption. Selected PAH were determined in water samples at low concentration levels by LC with fluorescence detection and the results were found not significantly different from those obtained with the 550.1 EPA method.

3. Conclusions

SPME has undoubtedly become an established and ever-growing extraction technique. Its coupling with liquid chromatography has always been problematic due to the criticalities associated with the desorption process. Nevertheless, the SPME-LC applications to biological (

Table 1. Biological applications of SPME-LC.

Application	Analyte	Matrix	Coating	Desorption	Detector	LOQ	Ref
Fluids and tissues	tranexamic acid	human plasma	C18	off-line	MS/MS, QqQ	1.5 µg/mL	[32]
	β2-agonists	pig tissues	PDMS/DVB	on-line	UV	0.2–0.5 µg/L	[33]
	BTEX	human teeth	CAR-PDMS and PA	off-line	UV/DAD	-	[34]
	antibiotics	human blood/tissue	C18	off-line	MS/MS, QqQ	0.085–0.135 µg/mL	[35]
	methylphenidate	human plasma	PDMS	on-line thermal	direct TD-ESI/MS	below 0.2 ng/mL	[36]
	psychotropic drugs	human blood/bone marrow	C18	off-line	MS/MS, QqTOF	5.60–42.80 ng/mL	[37]
In vivo	psychotropic drugs	fishes bile	PDMS	off-line	MS/MS	-	[38]
	psychotropic drugs	trout muscle	PDMS	off-line	MS/MS, Qtrap	0.40–0.97 (ng/g)	[39]
	psychotropic drugs	trout muscle	PDMS	off-line	MS/MS	0.40 0.97 ng/g	[40]
	untargeted	coral	mixed-mode	off-line	MS/MS, Orbitrap	-	[41]
	fenoterol	for future in-vein studies	reverse phase amide	off-line	MS/MS, QqQ	-	[42]
	fenoterol and methoxyfenoterol	rats blood	reverse phase amide	off-line	MS/MS, QqQ	1 ng/mL	[43]
	benzodiazepines	beagles blood	polypyrrole wire	off-line	MS/MS	-	[44]
	carbamazepine and metabolite	mouse blood	C18	off-line	MS/MS, QqQ	1 ng/mL	[45]
	eicosanoids	rats blood	C18	off-line	MS/MS; QqQ	0.4–0.8 ng/mL	[46]
	doxorubicin	pig lung	mixed-mode	off-line	MS/MS, QqQ	2.5 µg/g	[47]
Metabolomic	untargeted	mouse blood	mixed-mode	off-line	MS	-	[48]
	lipidome	trout muscle	mixed-mode	off-line	HRMS, Orbitrap	-	[49]
	BaP induced metabolites	trout muscle	mixed-mode	off-line	HRMS	-	[50]
	oxilipins	rats brain	mixed-mode	off-line	HRMS	-	[51]
	untargeted	human plasma	mixed-mode	off-line	HRMS, Orbitrap	-	[52]
	lipidome	human milk	C18	off-line	MS/MS, QqTOF	-	[53]
	untargeted	actinobacteria	HLB-PAN	off-line	MS/MS, QqQ	-	[54]
Application	Analyte	Binding host	Coating	Desorption	Detector	LOQ	Ref
Partitioning	chlorpromazine	BSA	PA	off-line	UV	-	[55]
	carbamazepine	HSA	C18	off-line	UV or MS	-	[56]
	various drugs	BSA, FBS, phospholipid liposomes, cells	C18	off-line	MS/MS, Qtrap	-	[57]

), food, and environmental (

Table 2. Food and environmental applications of SPME-LC.

Application	Analyte	Matrix	Coating	Desorption	Detector	LOQ	Ref
Food	OPPs/CMs insecticides	honey	CW/TPR	on-line	MS/MS	(CCα) 1–55 ng/g	[58]
	pesticides	mango	PDMS	on-line	UV	2.0–10.0 µg/kg	[59]
	triazoles	grape/apple	PDMS/DVB	on-line	UV	(LOD) 0.08–0.3 mg/kg	[60]
	salicylates	fruits/vegetables	PDMS/DVB	on-line	UV	0.007–0.095 µg/mL	[61]
	isoflavones	soy milk and beverages	PDMS/DVB	on-line	UV	0.016–0166 µM	[62]
	trans-resveratrol	Wine/grape juices	PA	on-line	UV	1.6–3.7ng/mL	[63]
Environmental	pharmaceuticals	wastewater	CW/TPR	on-line	MS/MS, ion trap	0.005–0.05 ng/mL	[64]
	chloramphenicol	tap/sea water	CW-TPR	on-line	UV	0.3/0.7 ng/mL	[65]
	-	Environm./Biological	C18	off-line	MS/MS	-	[66]
	antibacterials	river	PDMS	on-line	UV	(MDL) 0.12–0.73 ng/mL	[67]
	EDCs	seawater	Acrylate	off-line	MS/MS, QqQ	-	[68]
	BFRs	tap water	PDMS/DVB	on-line	MS/MS, Qtrap	(LOD) 0.01−0.04 ng/mL	[69]
	sudan dyes	wastewater	PDMS/DVB	on-line	UV	0.2 and 0.5 µg/L	[70]

) samples produced in recent years with commercial or already described fibers, reported in this review, are quite considerable and, added to the numerous SPME-LC works dedicated to the development of new extractive materials that will likely bring to a fast increase of commercial fibers, make clear the impact that this coupling continues to have in the analytical chemical context.

As regards the desorption phase, several users and developers have turned to off-line desorption mode, which provides longer fiber lifetime due to the lower probability of damages in the interface and is still able to guarantee good results in terms of sensitivity. The disadvantage of this approach is a loss in terms of rapidity of analysis, which is not indispensable for many partitioning, in vivo, and/or metabolomics applications. The on-line desorption mode is also widely used, as demonstrated by several applications carried out with the commercial interface as well as by studies dedicated to its improvement.