Proteomic Analysis of Human Saliva via Solid-Phase Microextraction Coupled with Liquid Chromatography-Mass Spectrometry.

Proteomics of human saliva samples was achieved for the first time via biocompatible solid-phase microextraction (bio-SPME) devices. Upon introduction of a porogen to a conventional C18 coating, porous C18/polyacrylonitrile (PAN) SPME blades were able to extract peptides up to 3.0 kDa and more peptides than commercial SPME blades. Following Trypsin digestion, salivary proteomic analysis was achieved via SPME-LC-MS/MS. Seven endogenous proteins were consistently identified in all saliva samples via bio-SPME. Taking advantage of this strategy, untargeted peptidomics was applied for the comparison of saliva samples between healthy and SARS-CoV-2 positive individuals. The results showed clear peptidomic differences between the viral and healthy saliva samples. This proof-of-concept study demonstrates the potential of bio-SPME-LC-MS/MS for peptidomics and proteomics in biomedical applications.

High-Throughput and Rapid Screening of Drugs of Abuse in Saliva by Multi-Segment Injection Using Solid-Phase Microextraction-Automated Microfluidic Open Interface-Mass Spectrometry.

There is great demand for analytical methods capable of providing high-throughput and rapid screening, especially for anti-doping and clinical point-of-care applications. In this work, automated microfluidic open interface-mass spectrometry (MOI-MS) was used for coupling with high-throughput, automated solid-phase microextraction (SPME) to achieve this objective. The design of the MOI-MS interface provides a continuous and stable electrospray fluid flow to the MS without introducing any bubble, a feature that we exploit to introduce the concept of multi-segment injection for the determination of multiple samples in a single MS run. By eliminating the need to start a new MS run between sample assays, the developed approach provides significantly simplified protocols controlled by programmed software and increased reproducibility. Furthermore, the biocompatible SPME device, which utilizes coating consisting of hydrophilic-lipophilic balanced particles embedded in a polyacrylonitrile (PAN) binder, can be directly used for biological sample analysis, as the PAN acts as both a binder and a matrix-compatible barrier, thus enabling the enrichment of small molecules while eliminating interferences associated with the presence of interfering macromolecules. The above design was employed to develop a fast, quantitative method capable of analyzing drugs of abuse in saliva samples in as little as 75 s per sample. The findings indicate that the developed method provides good analytical performance, with limits of detection ranging between 0.05 and 5 ng/mL for analysis of 16 drugs of abuse, good calibration linear correlation coefficients (R2 ≥ 0.9957), accuracy between 81 and 120%, and excellent precision (RSD% < 13%). Finally, a proof-of-concept experiment was performed to demonstrate the method's suitability for real-time analysis in anti-doping applications.

The Effect of Sorbent Particles in a Binder on the Mass Transfer Kinetics in Separation Media: In Silico Study and Experimental Verification.

Selecting the optimal binder and the sorbent affinity for selected compounds can cause the composite to behave either as an efficient extraction coating, as a permeable membrane, or as an impermeable barrier. If the compound partitions onto the sorbent with high preference, it becomes stationary and the composite behaves as an impermeable barrier, while appropriately optimized affinity will result in effective permeation. To understand this phenomenon, we utilize solid-phase microextraction to characterize the mass transfer attributes of different separation composites. Our results indicate that for strong sorbents, the extraction rate is primarily controlled by the diffusion in the extraction phase rather than the sample matrix, even if it is relatively thin. Low analyte diffusion is caused by the retarding force generated by the partitioning of analytes into the sorbent, as migration through the composite is driven by the unbound form of the compound in the binder. One of the main contributions of this work is that an understanding of the extraction composite parameters that control mass transfer during extraction enables better optimization of binder/sorbent extraction phase composition for a given application. Another contribution of this work shows how a heterogeneous coating model can be simplified into a homogeneous coating model. The developed models enable an enhanced understanding of mass transfer kinetics, and they provide insight into how to optimize the extraction phase parameters for a given method involving sorbent particles in polymeric media, including membranes and paints, in addition to extraction coatings.

Polymer Ligand-Sensitized Lanthanide Metal-Organic Frameworks for an On-Site Analysis of a Radionuclide.

Herein, a new strategy to increase the sensitivity of a lanthanide metal-organic framework (Ln-MOF) to UO22+ was proposed by using polymeric ligands. By utilizing [Tb(1,3,5-benzenetrisbenzoate)]n (Tb-TBT) MOF as the host, preloaded 2-vinyl terephthalic acid (VTP) was polymerized in situ, which produced a novel fluorescent composite denoted as PVTP⊂Tb-TBT. Benefiting from the coordination of PVTP to the Tb nodes, the polymeric chains performed both as molecular scaffolds that improved the water stability of the framework and as additional antennae that sensitized the photoluminescence of the Tb nodes. More importantly, the detection sensitivity and selectivity of PVTP⊂Tb-TBT to UO22+ were much improved compared to those of Tb-TBT. Detailed characterizations indicated that the incorporation of PVTP efficiently enriched UO22+ in the probe, which promoted the energy dissipation to UO22+. Besides, UO22+ was also supposed to release PVTP from PVTP⊂Tb-TBT and, thus, exposed the open metal sites to water molecules, which interrupted the sensitization effect of PVTP and induced a nonradiative energy dissipation. A limit of detection (LOD) as low as 0.75 nm was recorded by suspending the PVTP⊂Tb-TBT probe in a water sample, far below the limit in drinking water set by the United States Environmental Protection Agency (130 nm). Furthermore, a remotely controlled sampling and an on-site analysis of real water samples were realized by facilely loading PVTP⊂Tb-TBT on thin films (TFs). The LOD for UO22+ was 2.5 nm by using the TFs. This study reports a new strategy for boosting the sensitivity and selectivity of Ln-MOF to monitor UO22+ and expands the application of the strategy to an on-site analysis.

Development of a Biocompatible Solid Phase Microextraction Thin Film Coating for the Sampling and Enrichment of Peptides.

While currently available methods for peptide sample preparation are mostly suitable for ex situ analysis via exhaustive extraction techniques, these techniques do not allow for in situ extraction of peptides from biological samples, such as blood or plasma collected from patients for routine clinical applications. Biocompatible solid phase microextraction (Bio-SPME) has shown great potential in metabolomics for in situ extraction of metabolites including labile compounds from biological matrices in a biocompatible and non-exhaustive fashion, thus facilitating even in vivo sampling. However, the amounts of peptides extracted by such Bio-SPME chemical biopsy tools are deemed too low for quantification when porous polyacrylonitrile (PAN)-based biocompatible thin film sorbent coatings are used, since such materials have been commonly applied as means to restrict access of high molecular weight compounds such as proteins. Aiming to improve peptide extraction by the SPME sorbent while still preventing protein adsorption, thin films with nanoscale irregularities and mesopores were prepared by inclusion of the porogen lithium perchlorate in the slurries of the coatings. The novel thin film coating method significantly improved extraction of a range of angiotensins known to possess important roles in blood pressure regulation and electrolyte balance. Model low abundance peptides covering a wide range of hydrophobicities were successfully extracted from physiological buffers and human plasma using the increased porosity coating, while the SPME protocol on the tryptic digestion of a protein supported that enzymes were excluded during peptide extraction. Surface rheological analysis, which displayed mesopores on the C18/PAN coatings, confirmed that the porosity of the coating facilitated the mass transport of peptides through the PAN layer, thus enabling extraction of high amounts of peptides by the new C18/PAN coating.

Systematic Evaluation of Different Coating Chemistries Used in Thin-Film Microextraction.

A systematic evaluation of eight different coatings made of solid phase extraction (SPE) and carbon-based sorbents immobilized with polyacrylonitrile in the thin-film microextraction (TFME) format using LC-MS/MS was described. The investigated coatings included graphene, graphene oxide, multi-walled carbon nanotubes (MWCNTs), carboxylated MWCNTs, as carbon-based coatings, and polystyrene-divinylbenzene (PS-DVB), octadecyl-silica particles (C18), hydrophilic-hydrophobic balance particles (HLB) and phenyl-boronic acid modified particles (PBA), as SPE-based coatings. A total of 24 compounds of diverse moieties and of a wide range of polarities (log P from -2.99 to 6.98) were selected as probes. The investigated coatings were characterized based on their extraction performance toward the selected probes at different pH values and at optimized desorption conditions. In the case of SPE-based coatings, PS-DVB and HLB exhibited a balanced extraction for compounds within a wide range of polarities, and C18 showed superior extraction recoveries for non-polar analytes. Carbon-based coatings showed high affinity for non-polar compounds given that their main driving force for extraction is hydrophobic interactions. Interestingly, among the studied carbon-based coatings, graphene oxide showed the best extraction capabilities toward polar compounds owing to its oxygen-containing groups. Overall, this work provided important insights about the extraction mechanisms and properties of the investigated coatings, facilitating the coating selection when developing new TFME applications.

Single-Use Poly(etheretherketone) Solid-Phase Microextraction-Transmission Mode Devices for Rapid Screening and Quantitation of Drugs of Abuse in Oral Fluid and Urine via Direct Analysis in Real-Time Tandem Mass Spectrometry.

The analysis of oral fluid (OF) and urine samples to detect drug consumption has garnered considerable attention as alternative biomatrices. Efficient implementation of microextraction and ambient ionization technologies for rapid detection of target compounds in such biomatrices creates a need for biocompatible devices which can be implemented for in vivo sampling and easily interfaced with mass spectrometry (MS) analyzers. This study introduces a novel solid-phase microextraction-transmission mode (SPME-TM) device made of poly(etheretherketone) (PEEK) mesh that can rapidly detect prohibited substances in biofluids via direct analysis in real-time tandem MS (DART-MS/MS). PEEK mesh was selected due to its biocompatibility, excellent resistance to various organic solvents, and its ability to withstand relatively high temperatures (≤350 °C). The meshes were coated with hydrophilic-lipophilic-balance particle-poly(acrylonitrile) (HLB-PAN) slurry. The robustness of the coated meshes was tested by performing rapid vortex agitation (≥3200 rpm) in LC/MS-grade solvents and by exposing them to the DART source jet stream at typical operational temperatures (∼250-350 °C). PEEK SPME-TM devices proved to be robust and were therefore used to perform ex vivo analysis of drugs of abuse spiked in urine and OF samples. Excellent results were obtained for all analytes under study; furthermore, the tests yielded satisfactory limits of quantitation (median, ∼0.5 ng mL-1), linearity (≥0.99), and accuracy (80-120%) over the evaluated range (0.5-200 ng mL-1). This research highlights plastic SPME-TM's potential usefulness as a method for rapidly screening for prohibited substances in on-site/in vivo scenarios, such as roadside or workplace drug testing, antidoping controls, and pain management programs.

Development of a Biocompatible In-Tube Solid-Phase Microextraction Device: A Sensitive Approach for Direct Analysis of Single Drops of Complex Matrixes.

The aim of the current study is to develop a sensitive solid-phase microextraction (SPME) device for direct and rapid analysis of untreated complex matrixes (i.e., single drop of the samples, V ≤ 2 µL). A thin layer of a biocompatible nanostructured polypyrrole (PPy) was electrochemically deposited inside a medical grade spinal needle, minimizing the matrix effect. Microsampling was facilitated by loading the sample inside the in-tube SPME device (withdraw of sample via plunger), where extraction was performed under static conditions. Two strategies were used for analysis of the compounds including offline desorption and running the extract to the liquid chromatograph-tandem mass spectrometer (LC-MS/MS) or direct coupling of the in-tube SPME device to the MS. Given the high surface-area-to-volume ratio of the coating, a short equilibrium time (i.e., t ≤ 2 min) was obtained. The whole analytical procedure (i.e., extraction, rinsing, desorption, and LC-MS/MS analysis) was performed within 10 min by LC-MS/MS, and 3 min by in-tube-MS/MS. Possible matrix effects for the prepared device were evaluated in whole blood samples at three levels of concentration, and encouraging results were achieved in the range of 83-120%. The obtained results, no matrix effect, are attributed to the smooth surface and small pore size of the biocompatible PPy coating, which was prepared in the presence of cetyltrimethylammonium bromide (CTAB) surfactant. The in-tube SPME device was shown to be very sensitive, with high total recoveries obtained for all compounds in phosphate-buffered saline (PBS) and urine samples owing to the large volume and capacity of the coating. Subnanogram per milliliter levels of detection were achieved for urine samples, and low nanogram per milliliter levels were found in whole blood samples for all studied compounds with a high protein binding index. Rapid analysis of whole blood samples was achieved without need of any pretreatment or manipulation of sample, revealing the developed in-tube SPME device as an ideal probe for forensic application, drug monitoring, and point-of care-diagnosis.

Biocompatible Solid-Phase Microextraction Nanoelectrospray Ionization: An Unexploited Tool in Bioanalysis.

In recent years, different geometrical configurations of solid-phase microextraction (SPME) have been directly coupled to mass spectrometry, resulting in benefits such as diminishing matrix effects, improvement of detection limits, and considerable enhancement of analysis throughput. Although SPME fibers have been used for years, their potential for quantitative analysis when directly combined with mass spectrometry has not been explored to its full extent. In this study, we present the direct coupling of biocompatible SPME (Bio-SPME) fibers to mass spectrometry via nanoelectrospray ionization (nano-ESI) emitters as a powerful tool for fast quantitative analysis of target analytes in biofluids. Total sample preparation time does not exceed 2 min, and by selecting an appropriate fiber length and sample vessel, sample volumes ranging between 10 and 1500 µL can be used. Despite the short extraction time of the technique, limits of detection in the subnanogram per milliliter with good accuracy (≥90%) and linearity (R(2) > 0.999) were attained for all the studied probes in phosphate-buffered saline (PBS), urine, and whole blood. Given that Bio-SPME-nano-ESI efficiently integrates sampling with analyte extraction/enrichment, sample cleanup (including elimination of matrix effects in the form of particles), and ionization, our results demonstrated that it is an advantageous configuration for bioanalytical applications such as therapeutic drug monitoring, doping in sports, and pharmacological studies in various matrixes.

Solid phase microextraction devices prepared on plastic support as potential single-use samplers for bioanalytical applications.

This study presents new thin-film solid phase microextraction (SPME) devices prepared on plastic as potential single-use samplers for bioanalysis. Polybutylene terephthalate (PBT) was selected as a support due to its well-known chemical resistance, low cost, and suitability as a material for different medical grade components. The herein proposed samplers were prepared by applying a hydrophilic-lipophilic balanced (HLB)-polyacrylonitrile (PAN) coating on rounded and flat PBT pieces previously sanded with regular sandpaper. SPME devices prepared on PBT were evaluated in terms of robustness, chemical stability, and possible interferences upon exposure to different solvents and matrixes. Rewarding results were found when these samplers were employed for the quantitative analysis of multiple doping substances in common biological matrixes such as urine, plasma, and whole blood. Finally, the proposed thin-film SPME devices made on a PBT were evaluated by conducting multiple extractions from whole blood and plasma using the Concept 96 system. Results showed that more than 20 extractions from plasma and whole blood can be performed without observed decreases in coating performance or peeling of the extraction phase from the plastic surface. These findings demonstrate the robustness of PAN-based coatings applied on such polymeric substrate and open up the possibility of introducing new alternatives and cost-effective materials as support to manufacture SPME biocompatible devices for a wide range of applications, particularly in the clinical field.

Headspace versus direct immersion solid phase microextraction in complex matrixes: investigation of analyte behavior in multicomponent mixtures.

This work aims to investigate the behavior of analytes in complex mixtures and matrixes with the use of solid-phase microextraction (SPME). Various factors that influence analyte uptake such as coating chemistry, extraction mode, the physicochemical properties of analytes, and matrix complexity were considered. At first, an aqueous system containing analytes bearing different hydrophobicities, molecular weights, and chemical functionalities was investigated by using commercially available liquid and solid porous coatings. The differences in the mass transfer mechanisms resulted in a more pronounced occurrence of coating saturation in headspace mode. Contrariwise, direct immersion extraction minimizes the occurrence of artifacts related to coating saturation and provides enhanced extraction of polar compounds. In addition, matrix-compatible PDMS-modified solid coatings, characterized by a new morphology that avoids coating fouling, were compared to their nonmodified analogues. The obtained results indicate that PDMS-modified coatings reduce artifacts associated with coating saturation, even in headspace mode. This factor, coupled to their matrix compatibility, make the use of direct SPME very practical as a quantification approach and the best choice for metabolomics studies where wide coverage is intended. To further understand the influence on analyte uptake on a system where additional interactions occur due to matrix components, ex vivo and in vivo sampling conditions were simulated using a starch matrix model, with the aim of mimicking plant-derived materials. Our results corroborate the fact that matrix handling can affect analyte/matrix equilibria, with consequent release of high concentrations of previously bound hydrophobic compounds, potentially leading to coating saturation. Direct immersion SPME limited the occurrence of the artifacts, which confirms the suitability of SPME for in vivo applications. These findings shed light into the implementation of in vivo SPME strategies in quantitative metabolomics studies of complex plant-based systems.

Sequential thin film microextraction and overcoated thin film microextraction devices for characterization of sparkling wine aroma profiles and partitioning equilibria.

Solid Phase Microextraction (SPME) is a commonly used, robust method for characterization of aroma profiles in food matrices. However, challenges such as saturation, swelling, and competition can occur when sampling such complex matrices, resulting in decreased accuracy in the quantitation of polar compounds. In this study, sequential thin film micro-extraction (TFME) was employed to study the aroma profile of sparkling wine, with a focus to evaluate the displacement of polar analytes at extraction times longer than their corresponding equilibrium time. This investigation also describes advancements in the production of TFME devices, specifically the overcoating of hydrophilic-lipophilic balance/polydimethylsiloxane (HLB/PDMS) thin films to increase their matrix compatibility. Sequential thin film micro-extraction and overcoated HLB/PDMS thin films were evaluated for characterization of sparkling wine samples. The results were encouraging, showing that these advancements can decrease competition phenomena and increase the calibration linearity range compared to traditional micro-extraction approaches more commonly used for the characterization of such samples. In addition, multiphase equilibria investigation involving micellar systems enabled by the microextraction technology provides better understanding between wine aroma and its composition.

Reusable solid-phase microextraction coating for direct immersion whole-blood analysis and extracted blood spot sampling coupled with liquid chromatography-tandem mass spectrometry and direct analysis in real time-tandem mass spectrometry.

Three different biocompatible polymers were tested and evaluated in order to improve the whole-blood biocompatibility of previously developed C18-polyacrylonitrile (C18-PAN) thin-film solid-phase microextraction (SPME) coating. Among all methods of modification, UV-dried thin PAN-over C18-PAN provided the best results. This coating presented reusable properties and reproducible extraction efficiency for at least 30 direct extractions of diazepam from whole blood [relative standard deviation (RSD) = 12% using external calibration and 4% using isotope dilution calibration]. The amount of absolute recovery for direct immersion analysis and based on the free concentration of diazepam in blood matrix was about 4.8% (desorption efficiency = 98%). The limit of quantitation (LOQ) for the developed solid-phase microextraction liquid chromatography-tandem mass spectrometry (SPME-LC-MS/MS) method for direct whole-blood analysis was 0.5 ng/mL. The optimized modification of the coating was then used for an extracted blood spot (EBS) sampling approach, a new sampling method which is introduced to address the limitations of dried blood spot sampling. EBS was evaluated using LC-MS/MS and direct analysis in real time (DART)-MS/MS, where, for a 5 µL blood spot, LOQs of 0.2 and 1 µg/mL, respectively, were achieved for extraction of diazepam.

Systematic evaluation of solid-phase microextraction coatings for untargeted metabolomic profiling of biological fluids by liquid chromatography-mass spectrometry.

In this study, we propose for the first time the use of solid-phase microextraction (SPME) in combination with liquid chromatography-mass spectrometry for untargeted metabolomic profiling of biological fluids. To achieve this goal, we first systematically evaluated 42 different SPME coatings for the extraction of 36 metabolites from different chemical classes and of widely varying polarities (log P range of -7.9 to 7.4) in order to identify SPME coatings which are the most suitable for metabolomic studies and to improve the extraction of polar metabolites over the existing commercial SPME devices. Three types of SPME coatings (mixed-mode coatings, polar-enhanced polystyrene-divinylbenzene, and phenylboronic acid) performed the best for simultaneous extraction of both hydrophilic and hydrophobic metabolites at physiological conditions, thus making them suitable for untargeted metabolomic profiling applications. A rapid and simple SPME method was then developed with single-use biocompatible mixed-mode coating for the metabolomic profiling of human plasma in combination with liquid chromatography-high-resolution mass spectrometry on a benchtop Orbitrap system. This optimized SPME method was evaluated versus ultrafiltration and solvent precipitation in terms of metabolite coverage and method precision. SPME detected 1592-3320 features versus 2082-3245 features detected by solvent precipitation methods and 2093-2686 detected for ultrafiltration using the same pooled human plasma sample. Method precision of SPME ranged between 11% and 18% (expressed as median relative standard deviation (RSD) of n = 7 replicates) versus 8-19% for solvent precipitation and 20-22% for ultrafiltration. The results demonstrate that the proposed SPME methodology reduces ionization suppression, provides free concentration information for hydrophobic analytes which are not detected by ultrafiltration methods, and can improve metabolite coverage over existing methodologies.

Optimization of the coating procedure for a high-throughput 96-blade solid phase microextraction system coupled with LC-MS/MS for analysis of complex samples.

Biocompatible C18-polyacrylonitrile (PAN) coating was used as the extraction phase for an automated 96-blade solid phase microextraction (SPME) system with thin-film geometry. Three different methods of coating preparation (dipping, brush painting, and spraying) were evaluated; the spraying method was optimum in terms of its stability and reusability. The high-throughput sample preparation was achieved by using a robotic autosampler that enabled simultaneous preparation of 96 samples in 96-well-plate format. The increased volume of the extraction phase of the C18-PAN thin film coating resulted in significant enhancement in the extraction recovery when compared with that of the C18-PAN rod fibers. Various factors, such as reusability, reproducibility, pH stability, and reliability of the coating were evaluated. The results showed that the C18-PAN 96-blade SPME coating presented good extraction recovery, long-term reusability, good reproducibility, and biocompatibility. The limits of detection and quantitation were in the ranges of 0.1-0.3 and 0.5-1 ng/mL for all four analytes.

Fully integrated PDMS/SU-8/quartz microfluidic chip with a novel macroporous poly dimethylsiloxane (PDMS) membrane for isoelectric focusing of proteins using whole-channel imaging detection.

A fully integrated polydimethylsiloxane (PDMS)/modified PDMS membrane/SU-8/quartz hybrid chip was developed for protein separation using isoelectric focusing (IEF) mechanism coupled with whole-channel imaging detection (WCID) method. This microfluidic chip integrates three components into one single chip: (i) modified PDMS membranes for separating electrolytes in the reservoirs from the sample in the microchannel and thus reducing pressure disturbance, (ii) SU-8 optical slit to block UV light (below 300 nm) outside the channel aiming to increase detection sensitivity, and (iii) injection and discharge capillaries for continuous operation. Integration of all these components on a single chip is challenging because it requires fabrication techniques for perfect bonding between different materials and is prone to leakage and blockage. This study has addressed all the challenges and presented a fully integrated chip, which is more robust with higher sensitivity than the previously developed IEF chips. This chip was tested by performing protein and pI marker separation. The separation results obtained in this chip were compared with that obtained in commercial cartridges. Side-by-side comparison validated the developed chip and fabrication techniques.

Direct immersion thin film solid phase microextraction of polychlorinated n-alkanes in cod liver oil.

A thin film-solid phase microextraction (TF-SPME) method was developed to test for 5 individual polychlorinated n-alkanes (PCAs) from commercial cod liver oil samples. This was accomplished by preparing a novel aluminum supported, hydrophilic-lipophilic balance/polydimethylsiloxane (HLB/PDMS) TF-SPME device that enabled direct immersion extraction from fish oil. Matrix-matched calibration gave a linear range from 0.075 µg/g to 0.75 µg/g with method limits of quantitation (MLOQ) ranging from 0.07 µg/g to 0.217 µg/g in oil. Standard addition calibration was performed using other fish oils demonstrating comparable slope to the external calibration. As a proof of concept, four fish oil brands were tested for contaminants; 1,1,1,3-tetrachlorodecane, 1,2,9,10-tetrachlorodecane, 1,2,13,14-tetrachlorotetradecane, and 1,1,1,3,14,15-hexachloropentadecane were detected above the MLOQ but below the range provided by the Stockholm Convention. This method provides an effective approach for cleanup and preconcentration of PCAs from oily matrices using inexpensive, and reusable microextraction devices that limit environmental impact of the sample preparation protocol.

Direct-immersion SPME in soy milk for pesticide analysis at trace levels by means of a matrix-compatible coating.

This study demonstrates a newly developed PDMS/DVB/PDMS fiber's suitability for the determination of pesticides in soy milk via direct-immersion solid-phase microextraction (SPME) combined with gas chromatography-mass spectrometry, eliminating the need for extensive sample pre-treatment procedures. Fouling accumulation on the coating surface was further minimized by implementing rapid and effective pre- and post-desorption cleaning steps. Under optimum conditions, the fiber was used to perform over 120 extractions while maintaining RSD values of less than 24.5% for 10 extracted pesticides. By comparison, the RSD values ranged from 8.4% to 42.8% over 80 extractions using a commercial PDMS/DVB fiber. The optimized conditions were used to fully validate a quantitative method for the targeted analytes by matrix-matched calibration and isotopically labeled internal standard correction. Significantly, the proposed method was able to achieve limits of quantitation (1-2.5 µg/kg) for the targeted analytes that were below the Maximum Residue Levels mandated for soy-based products. Accuracy, intra- and inter-day repeatability were also satisfactory. The proposed PDMS/DVB/PDMS fiber dramatically improved repeatability and suitability for direct-immersion SPME in soy milk, and represents a good alternative to other extraction methods for high-throughput quantitative analysis of pesticide residues in soy-based products.

Mechanism of interactions between organophosphorus insecticides and human serum albumin: Solid-phase microextraction, thermodynamics and computational approach.

Organophosphates insecticides (OPs) are one of the major environmental pollutants and their interaction with human serum albumin (HSA) has been shown to have significant effects on their bioavailability which is related to toxicokinetics and toxicodynamics in human body. In this research, solid-phase microextraction methods were developed to analyse the free concentrations of three OPs (chlorpyrifos, parathion-methyl and malathion) in buffered HSA solution and that provide a useful method for the determination of binding affinity constants (Ka), binding forces and binding location. Polydimethylsiloxane fibers were selected for analysing the free concentrations of OPs, with an external calibration approach. Good linearities conducted in PBS solution were observed in the range of 0.0025-1.7 µmol L-1 (R2 = 0.9975) for chlorpyrifos, 1.0-27 µmol L-1 (R2 = 0.9974) for parathion-methyl, and 0.5-70 µmol L-1 (R2 = 0.9973)for malathion, respectively. The LODs for instrument response were 1 ng, 5 ng and 10 ng for chlorpyrifos, parathion-methyl and malathion, respectively. The Ka values for chlorpyrifos, parathion-methyl and malathion showed that they were positively correlated with hydrophobicity and negatively correlated with temperature. The OP binding sites on HSA were confirmed by site marker competition test and further proven by computational approaches. The recognition region of parathion-methyl was situated within residues 199-292 in subdomain IIA. Malathion bonded to residues 404-558 in subdomain IIIA. The mode of action between HSA-parathion-methyl and HSA-malathion is found to involve mainly by H-bonds, π-π stacking and hydrophobic effects. These results clearly demonstrate the noncovalent binding of OPs with HSA and provide new insight into solid-phase microextraction, thermodynamics and computational approaches.

Development and validation of an improved, thin film solid phase microextraction based, standard gas generating vial for the repeatable generation of gaseous standards.

This work presents the development and validation of novel thin film solid phase micro extraction (TF-SPME) based standard gas generating vials suitable for repeatable generation of gaseous standards for GC-MS analysis and quality control. The vials were developed using carbon mesh membranes loaded with pure polydimethylsiloxane (PDMS), divinylbenzene (DVB/PDMS), hydrophilic-lipophilic balance (HLB/PDMS), and carboxen (Car/PDMS) sorbents that were then spiked with modified McReynolds standards including benzene, 2-pentanone, 1-nitropropane, pyridine, 1-pentanol, octane, dodecane, and hexadecane. Sorbent strength was determined to follow the aforementioned order, with pure PDMS presenting the weakest sorption capabilities and Car/PDMS the strongest. While the weaker, pure PDMS based gas generating vials transferred an instrument-overloading amount of McReynolds probes to the 1.1 mm DVB/PDMS SPME arrows used for extraction, vials prepared using Car/PDMS TF-SPME as a sorbent failed to provide consistently detectable amounts of analytes less volatile than 1-nitropropane. The DVB/PDMS and HLB/PDMS based vials were found to maintain optimal sorption capabilities for the tested analytes, providing a sorption strength strong enough to not exhibit any depletion in 10 replicate runs, while still delivering a consistent amount of all the regular McReynolds components. Moreover, with intra-vial%RSDs of 5% or less for all analytes tested, these HLB and DVB vials were found to deliver very good repeatability. After purposely submitting vials to 200 accelerated depletion extractions (1.1 mm DVB/PDMS arrow at 55 °C for 3 min), vials prepared with DVB/PDMS were found to deplete by 33%, 38%, 34%, 33%, 40%, and 33% while vials prepared with HLB/PDMS were found to deplete by 21%, 16%, 12%, 31%, 16% and 0% for benzene, 2-pentanone, 1-nitropropane, pyridine, 1-pentanol, and octane, respectively. When user typical extractions conditions were used instead (50/30 µm DVB/Car/PDMS SPME fiber at 35 °C for 1 min), no depletion could be observed from the HLB/PDMS based vial while%RSDs ranged from 1.1-3.0% after the 300 extraction/desorption cycles. Finally, in efforts to demonstrate its real world applicability, the DVB/PDMS vial was used to evaluate the inter-fiber repeatability of commercial DVB/PDMS SPME arrows, with results demonstrating that arrows from a single package were statistically similar (ANOVA at 95% confidence).