Biocompatible Solid-phase Microextraction Research Articles

Portable Miniature Mass Spectrometry for Enhanced On-Site Detection of Analytes in Complex Samples by Integrating Solid-Phase Microextraction and Nano-Electrospray Ionization.

On-site mass spectrometry (MS) analysis plays a crucial role in timely understanding chemical compositions of field samples but presents a challenge to miniaturization, portability, and sensitivity. In this work, a portable MS approach was developed by integrating biocompatible solid-phase microextraction (SPME) and a nano-electrospray ionization (nESI) emitter into a kit to couple miniature MS (mMS). The SPME fiber was used for on-site extractive sampling of analytes from complex liquid samples and living organisms and was then inserted into an nESI emitter for on-site MS analysis via the facile kit. The limit of detection was found to be at the pg/mL level for various compounds tested. Acceptable relative standard deviation (RSD) values (5.5-7.6%, n = 6) were obtained for direct measurement of analytes in complex matrixes; acceptable linear responses (0.1-50 ng/mL) and matrix effects (76.0-82.6%) were also found. Enhanced detection of compounds of interest in various real samples, such as food samples, human fluids, environmental water, and living organisms, was unambiguously demonstrated. Our experimental data showed that SPME-nESI-mMS is a promising tool for on-site analysis of various complex samples in significant applications including but not limited to food safety, environmental monitoring, forensic investigation, and bioanalysis.

Chemical bonding of cross-linked glutaraldehyde/chitosan on the surface of a titanium wire to prepare a robust biocompatible SPME fiber for analysis of phytohormones in plants

A robust biocompatible solid-phase microextraction (SPME) fiber, so-called Ti/APTS/GA/CS, was prepared by chemical bonding of cross-linked glutaraldehyde-chitosan to the surface of a titanium wire using APTS. The fiber was applied for sampling of phytohormones in plant tissues, followed by HPLC-UV analysis. The structure and morphology of the fiber coating was investigated by FT-IR, SEM, EDX, XRD, and TGA techniques. A Box-Behnken design was implemented to optimize the experimental variables. The calibration graphs were linear over a wide linear range (0.5–200 μg L−1) with LODs over the range of 0.01–0.06 μg L−1. The intra-day and inter-day precisions were found to be 1.3–6.3% and 4.3–7.3%, respectively. The matrix effect values ranged from 86.5% to 111.7%, indicating that the complex sample matrices had an insignificant effect on the determination of phytohormones. The fiber was successfully employed for the direct-immersion SPME (DI-SPME-HPLC) analysis of the phytohormones in cucumber, tomato, date palm, and calendula samples.

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.

Mapping the metabolic responses to oxaliplatin-based chemotherapy with in vivo spatiotemporal metabolomics

Adjuvant chemotherapy improves the survival outlook for patients undergoing operations for lung metastases caused by colorectal cancer (CRC). However, a multidisciplinary approach that evaluates several factors related to patient and tumor characteristics is necessary for managing chemotherapy treatment in metastatic CRC patients with lung disease, as such factors dictate the timing and drug regimen, which may affect treatment response and prognosis. In this study, we explore the potential of spatial metabolomics for evaluating metabolic phenotypes and therapy outcomes during the local delivery of the anticancer drug, oxaliplatin, to the lung. 12 male Yorkshire pigs underwent a 3 h left lung in vivo lung perfusion (IVLP) with various doses of oxaliplatin (7.5, 10, 20, 40, and 80 mg/L), which were administered to the perfusion circuit reservoir as a bolus. Biocompatible solid-phase microextraction (SPME) microprobes were combined with global metabolite profiling to obtain spatiotemporal information about the activity of the drug, determine toxic doses that exceed therapeutic efficacy, and conduct a mechanistic exploration of associated lung injury. Mild and subclinical lung injury was observed at 40 mg/L of oxaliplatin, and significant compromise of the hemodynamic lung function was found at 80 mg/L. This result was associated with massive alterations in metabolic patterns of lung tissue and perfusate, resulting in a total of 139 discriminant compounds. Uncontrolled inflammatory response, abnormalities in energy metabolism, and mitochondrial dysfunction next to accelerated kynurenine and aldosterone production were recognized as distinct features of dysregulated metabolipidome. Spatial pharmacometabolomics may be a promising tool for identifying pathological responses to chemotherapy.

Determination of amphetamines, opioids, cocaine metabolites and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol in urine using biocompatible solid-phase microextraction and ultra-performance LC-MS/MS.

Background: Workplace drug testing primarily relies on urine analysis, targeting multiple compounds with varying physicochemical characteristics. Biocompatible solid-phase microextraction (BioSPME) is a miniaturized solid-phase extraction technique that enables the simultaneous extraction and preconcentration of analytes directly from the biological matrix. Methods: The BioSPME procedure consisted of the sequential extraction of 50-μl urine samples using LC Tips C18 in basic and acidic pH, followed by desorption with methanol and n-hexane, respectively. The extracts were analyzed by ultra-performance LC-MS/MS. Results: Intra-day precision was 1.2-8.6% and inter-day precision was 1.8-14.2%. Accuracy was 96.8-107.4%. The extraction yields were 62.8-109.4%. The matrix effects were -3.98% to 1%. Conclusion: BioSPME shows promise as an alternative method for preparing urine samples prior to drug measurement by ultra-performance LC-MS/MS.

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.

Quantitative determination of pesticides in human plasma using bio-SPME-LC-MS/MS: a robust tool to assess occupational exposure to pesticides.

Analysis of biofluids, such as plasma, can be used to investigate occupational pesticide exposure in the agricultural industry. Considering the chemical complexity and variability of plasma samples, any protocol for pesticide analysis should achieve efficient sample cleanup to minimize matrix effects and enhance method sensitivity through analyte pre-concentration. In this work, a high-throughput method was developed for analysis of 79 pesticides, commonly used in agricultural practices, in human plasma, using biocompatible solid-phase microextraction (SPME) coupled to liquid chromatography-tandem mass spectrometry. An SPME method was developed using abiocompatible hydrophilic-lipophilic balance/polyacrylonitrile (HLB/PAN) extraction phase and demonstrated negligible matrix effects. The performance of the developed SPME method was compared to a QuEChERS-Quick, Easy, Cheap, Effective, Rugged, and Safe- method, the most common sample preparation and cleanup approach for pesticide analysis in complex matrices. Comparable accuracy and precision were achieved for both methods, with accuracy values within 70-120% and relative standard deviation < 15%. Overall, the developed SPME and QuEChERS methods extracted 79 out of 82 monitored pesticides in human plasma. The SPME protocol demonstrated higher sensitivity than the QuEChERS method and a drastic reduction of matrix effects.

The metabolic fate of oxaliplatin in the biological milieu investigated during in vivo lung perfusion using a unique miniaturized sampling approach based on solid-phase microextraction coupled with liquid chromatography-mass spectrometry.

Adjuvant chemotherapy after pulmonary metastasectomy for colorectal cancer may reduce recurrence and improve survival rates; however, the benefits of this treatment are limited by the significant side effects that accompany it. The development of a novel in vivo lung perfusion (IVLP) platform would permit the localized delivery of high doses of chemotherapeutic drugs to target residual micrometastatic disease. Nonetheless, it is critical to continuously monitor the levels of such drugs during IVLP administration, as lung injury can occur if tissue concentrations are not maintained within the therapeutic window. This paper presents a simple chemical-biopsy approach based on sampling with a small nitinol wire coated with a sorbent of biocompatible morphology and evaluates its applicability for the near-real-time in vivo determination of oxaliplatin (OxPt) in a 72-h porcine IVLP survival model. To this end, the pigs underwent a 3-h left lung IVLP with 3 doses of the tested drug (5, 7.5, and 40 mg/L), which were administered to the perfusion circuit reservoir as a bolus after a full perfusion flow had been established. Along with OxPt levels, the biocompatible solid-phase microextraction (SPME) probes were employed to profile other low-molecular-weight compounds to provide spatial and temporal information about the toxicity of chemotherapy or lung injury. The resultant measurements revealed a rather heterogeneous distribution of OxPt (over the course of IVLP) in the two sampled sections of the lung. In most cases, the OxPt concentration in the lung tissue peaked during the second hour of IVLP, with this trend being more evident in the upper section. In turn, OxPt in supernatant samples represented ∼25% of the entire drug after the first hour of perfusion, which may be attributable to the binding of OxPt to albumin, its sequestration into erythrocytes, or its rapid nonenzymatic biotransformation. Additionally, the Bio-SPME probes also facilitated the extraction of various endogenous molecules for the purpose of screening biochemical pathways affected during IVLP (i.e., lipid and amino acid metabolism, steroidogenesis, or purine metabolism). Overall, the results of this study demonstrate that the minimally invasive SPME-based sampling approach presented in this work can serve as (pre)clinical and precise bedside medical tool.

Dynamic Metabolic Changes During Prolonged Ex Situ Heart Perfusion Are Associated With Myocardial Functional Decline.

Ex situ heart perfusion (ESHP) was developed to preserve and evaluate donated hearts in a perfused beating state. However, myocardial function declines during ESHP, which limits the duration of perfusion and the potential to expand the donor pool. In this research, we combine a novel, minimally-invasive sampling approach with comparative global metabolite profiling to evaluate changes in the metabolomic patterns associated with declines in myocardial function during ESHP. Biocompatible solid-phase microextraction (SPME) microprobes serving as chemical biopsy were used to sample heart tissue and perfusate in a translational porcine ESHP model and a small cohort of clinical cases. In addition, six core-needle biopsies of the left ventricular wall were collected to compare the performance of our SPME sampling method against that of traditional tissue-collection. Our state-of-the-art metabolomics platform allowed us to identify a large number of significantly altered metabolites and lipid species that presented comparable profile of alterations to conventional biopsies. However, significant discrepancies in the pool of identified analytes using two sampling methods (SPME vs. biopsy) were also identified concerning mainly compounds susceptible to dynamic biotransformation and most likely being a result of low-invasive nature of SPME. Overall, our results revealed striking metabolic alterations during prolonged 8h-ESHP associated with uncontrolled inflammation not counterbalanced by resolution, endothelial injury, accelerated mitochondrial oxidative stress, the disruption of mitochondrial bioenergetics, and the accumulation of harmful lipid species. In conclusion, the combination of perfusion parameters and metabolomics can uncover various mechanisms of organ injury and recovery, which can help differentiate between donor hearts that are transplantable from those that should be discarded.

Biocompatible SPME coupled to GC/MS for analysis of xenobiotics in blood plasma

This work proposes a new method for biomonitoring studies focused on the screening and quantification of xenobiotics in blood-derived samples. The performance of a polydimethylsiloxane/divinylbenzene/polydimethylsiloxane (PDMS/DVB/PDMS) biocompatible extraction phase was investigated for extraction of pesticides and pharmaceuticals from plasma samples via direct immersion solid-phase microextraction (SPME) prior to gas chromatography-mass spectrometry. Under the optimum extraction settings, which included an attentive optimization of the fiber rinsing conditions, the microextraction device was able to endure 100 consecutive extractions from undiluted and diluted plasma with an overall reproducibility up to 28% for all the analytes tested, except chlorpyrifos-methyl. Optimized conditions were used to validate a quantitative method using matrix-matched calibration with isotopically labeled internal standard correction. Accuracy and precision values obtained for analysis of bovine plasma were within 96–132% and 0.05–5.82% respectively. LLOQs for all the analytes were at 1 µg L−1 and LDR ranged within 1–100 µg L−1. The applicability of this method to plasma from different species (human, rat, rabbit) was also investigated. This work represents the first step toward broader use of the biocompatible PDMS/DVB/PDMS extraction phases for analysis of multiclass xenobiotics in plasma and other complex biofluids.

In Vivo Contaminant Monitoring and Metabolomic Profiling in Plants Exposed to Carbamates via a Novel Microextraction Fiber.

In this study, a biocompatible solid-phase microextraction (SPME) fiber with high-coverage capture capacity based on a nitrogen-rich porous polyaminal was developed. The fiber was used to track the bioaccumulation and elimination of carbamates (isoprocarb, carbofuran, and carbaryl) and their metabolites (o-cumenol, carbofuran phenol, and 1-naphthalenol) in living Chinese cabbage plants (Brassica campestris L. ssp. chinensis Makino (var. communis Tsen et Lee)). A case-and-control model was applied in the hydroponically cultured plants, with the exposed plant groups contaminated under three carbamates at 5 μg mL-1. Both bio-enrichment and elimination of carbamates and their metabolites in living plants appeared to be very fast with half-lives at ∼0.39-0.79 and ∼0.56-0.69 days, respectively. Statistical differences in the endogenous plant metabolome occurred on day 3 of carbamate exposure. In the exposed group, the plant metabolic alterations were not reversed after 5 days of contaminant-free growth, although most contaminates had been eliminated. Compared with prior nutriological and toxicological studies, >50 compounds were first identified as endogenous metabolites in cabbage plants. The contents of the glucosinolate-related metabolites demonstrated significant time-dependent dysregulations that the fold changes of these key metabolites decreased from 0.78-1.07 to 0.28-0.82 during carbamate exposure. To summarize, in vivo SPME provided new and important information regarding exogenous carbamate contamination and related metabolic dysregulation in plants.

Fluorometer for Screening of Doxorubicin in Perfusate Solution and Tissue with Solid-Phase Microextraction Chemical Biopsy Sampling.

The recent development of an in vivo solid-phase microextraction (SPME) method capable of analyzing drugs and metabolic products in biofluids and living tissues holds great promise. The standard in vivo SPME protocol based on mass spectrometry is a very powerful analytical approach, but it is not practical for on-site analysis in many cases. In this paper, we present a fluorescence-based SPME method and a prototype of a portable fluorometer that is capable of quickly quantifying concentrations of the anticancer drug, doxorubicin (DOX). The instrument uses thin coated, biocompatible SPME fibers, which we have previously presented as a chemical biopsy tool for use during in vivo lung perfusion (IVLP) procedures within a hospital setting. In this research, we test SPME fibers with C8-SCX, C18, and HLB coatings with our fluorometer. The mixed-mode C8-SCX fibers showed the best sensitivity of the three and were therefore used to examine DOX extraction from perfusate solution and a homogenized lamb lung tissue. The maximum concentration of free active sites in the C8-SCX fiber and the adsorption equilibrium constant were determined to be (9.1 ± 0.3) × 10-7 mol m-2 and 420 ± 30 m3 mol-1, respectively. Finally, the detection limits for DOX extracted from buffer, perfusate, and lung tissue were 40, 100, and 3700 μg L-1, respectively.

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.

Biocompatible solid-phase microextraction coupled to liquid chromatography triple quadrupole mass spectrometry analysis for the determination of phthalates in marine invertebrate

This paper describes the development and validation of a new procedure for the determination of phthalates in marine invertebrates, based on biocompatible solid-phase microextraction (BioSPME) followed by liquid chromatography coupled to mass spectrometry. The importance of this application relies on the current use of marine organisms as bioindicators for microplastic contamination through the detection of phthalates in their tissues. Challenges originate from the availability and/or possible restriction on the use of the biological materials, the need for user- friendly procedures for simplifying and speeding up operations in the marine environments, and the ubiquitous presence of phthalates in the laboratory environments that may cause background contamination. BioSPME served as an effective solution to all these issues, owing to the extreme minimization of the sample manipulation. Sampling operations were limited to the direct transfer of small amounts of the biological materials (150 mg) inside glass vials capped with aluminium lids; extractions were carried out by ultrasonication in acetone followed by dilution in ultrapure water and BioSPME clean-up; Electrospray (ESI) LC–MS/MS was employed for the final analytical determinations on the purified extracts. Tests were carried out on samples belonging to three different phyla, namely Cnidaria, Porifera, and Mollusca obtained from Maldivian coral reef environments and used for a current microplastic contamination biomonitoring project. Overall, very good sensitivity and repeatability, with negligible back contamination of the blanks were observed. The limits of detections were between 0.2 and 2.1 ng/g and precision, calculated as relative standard deviation (RSD), was below 14% for all the tested phthalates.

Investigation into the Analysis of Fentanyl in Postmortem Blood using Biocompatible Solid-Phase Microextraction

Over recent years, the abuse of fentanyl and other opioids has become a slow motion mass disaster in the United States resulting in an increased number of drug-related deaths. Various postmortem biological samples are collected by forensic pathologists during autopsy and then sent to a toxicology laboratory to be analyzed for the presence of various compounds such as fentanyl. This process can be time consuming and results in a backlog, potentially hindering an investigation. A possible solution is Biocompatible Solid-Phase MicroExtraction (BioSPME) fibers. These fibers can be directly inserted into a biological matrix and absorb drug compounds without the interference of macromolecules that may be present allowing for a faster analysis time. An initial method has been developed to analyze fentanyl in postmortem blood using BioSPME followed by GC-MS and LC-MS-MS analysis. BioSPME fibers were conditioned, washed, directly inserted into postmortem blood, washed, filtered, desorbed into solution, dried down and reconstituted. The extracted samples were screened by GC-MS and subsequently analyzed by LC-MS-MS. GC-MS was performed using splitless injection on a Rxi-5Sil MS column (30.0 m × 0.25 mm, 0.25 μm) in the SIM mode. Samples were confirmed using an AB SCIEX™ 3200 QTRAP® triple quadrupole mass spectrometer with ElectroSpray Ionization (ESI) source in the positive ion mode. Liquid Chromatography was performed on a Shimadzu® LC system using an Ascentis® Express Biphenyl column (50 mm × 2.1 mm, 2.7 μm) with the weak mobile phase of 0.1%(v/v) formic acid in water and the strong mobile phase of 0.1%(v/v) formic acid in acetonitrile for an analysis time of seven min per sample. This method was developed using bovine blood and then applied to 43 postmortem blood samples provided by the Lehigh County Coroner’s Office (Allentown, PA, USA).

Understanding the Method and Results from the Analysis of Fentanyl in Postmortem Blood Using Biocompatible Solid-Phase Microextraction

The use of the Biocompatible Solid-Phase Microextraction (BioSPME) fiber in Grant et al. original work was to create an analysis method that is faster and simpler in identifying fentanyl in postmortem blood than current analysis methods. While the study was able to demonstrate that fentanyl was able to be absorbed onto the fiber and analyzed through the use of GC-MS and LC-MS-MS, the data reported was beyond the sensitivity for both instruments. The purpose of this communication is to provide more information regarding the methodology and sensitivity for both instruments and to reinterpret the data when comparing to the results from Health Network Laboratories. Based on the reinterpreted data, this current method was consistent with the results from Health Network Laboratories in identifying fentanyl in postmortem blood concentration equal to or greater than 21.4 ng/mL.

Analysis of endocannabinoids in plasma samples by biocompatible solid-phase microextraction devices coupled to mass spectrometry

Anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) represent two of the most important endocannabinoids (ECs) investigated in neurobiology as therapeutic targets for several mental disorders. However, the determination of these ECs in biological matrices remains a challenging task because of the low concentrations, low stability and high protein-bound (LogP ∼ 6). This work describes innovative analytical methods based on biocompatible SPME (Bio-SPME), SPME-UHPLC-MS/MS and Bio-SPME-Nano-ESI-MS/MS, to determine AEA and 2-AG in human plasma samples. The direct coupling of Bio-SPME with nano-ESI-MS/MS can be considered an alternative tool for faster analysis.Different Bio-SPME fibers based on silica and polymeric coating (i.e. C18, C30, and HLB) were evaluated. Different desorption solvents based on combinations of methanol, acetonitrile, and isopropanol were also evaluated for efficient elution with minimum carry-over. Given the high protein binding analytes and the fact that SPME extracts the free-concentration of the analytes, the plasma samples were modified with additives such as guanidine hydrochloride (Gu-HCl), trifluoroacetic acid, and acetonitrile. This study was carried out by experimental design to achieve complete protein denaturation and the release of target analytes. The maximum extraction efficiency was obtained under the following conditions: HLB coated fibers (10 mm length, 20 μm coating thickness), matrix modified (300 μL of plasma) with 50 μL of Gu-HCL 1 mol L−1, 75 μL of ACN and 75 μL of water, and desorption with methanol/isopropanol solution (50:50, v/v). Both methods were validated based on current international guidelines and can be applied for monitoring of concentrations of endocannabinoids in plasma samples.SPME-UHPLC-MS/MS method presented lower LOQ values than SPME-nanoESI-MS/MS. The additional separation (chromatographic column) favored the detectability of LC-MS/MS method. However, the SPME-nano-ESI-MS/MS decrease the total analysis time, due to significant reductions in desorption and detection times.

Coupling Paternò-Büchi Reaction with Surface-Coated Probe Nanoelectrospray Ionization Mass Spectrometry for In Vivo and Microscale Profiling of Lipid C═C Location Isomers in Complex Biological Tissues.

Lipids are important structural components of biological systems, and lipid C═C locations play important roles in their biophysical and biochemical properties. Rapid, in vivo, in situ, and microscale lipidomics investigation (including precise identification of lipid C═C locations and isomers) of biological specimen has great potential for clinical diagnosis, biological studies, and biomarker discovery. Here we report a novel lipidomics methodology by coupling Paternò-Büchi (PB) reaction with surface-coated probe nanoelectrospray ionization mass spectrometry (SCP-nanoESI-MS) for in vivo, in situ, and microscale analysis of lipid species and C═C location isomers in complex biological tissues. The proposed SCP-PB-nanoESI-MS method was performed by application of a biocompatible solid-phase microextraction (SPME) probe for in vivo, in situ, and microscale sampling and extraction of lipids from biological tissues, and then some spray solvent containing PB reagent was applied to desorb lipid species enriched on SPME probe within a nanospray tip. Subsequently, ultraviolet irradiation was employed to initiate PB reaction for unsaturated lipids within the nanospray tip. After that, a high voltage was applied on the SPME probe to induce nanoESI for MS analysis under ambient and open-air conditions, and collision-induced dissociation was performed to the PB reaction product ions for determination of lipid C═C locations and isomers. By using our proposed SCP-BP-nanoESI-MS method, microscale investigation of lipid compositions and C═C location isomers for lipid droplet of Perilla seed and human intestinal tissue were successfully achieved, and in vivo analysis of lipid species and C═C locations for zebrafish was accomplished.

Application of solid-phase microextraction in current biomedical research.

Extraction of endogenous compounds and drugs and their corresponding metabolites from complex matrices, such as biofluids and solid tissues, requires adequate analytical approach facilitating qualitative and quantitative analysis. To this end, solid-phase microextraction has been introduced as modern technology that is capable of efficient and high-throughput extraction of compounds due to its ability to amalgamate sampling, extraction, and pre-concentration steps, while requiring minimal use of organic solvents. The ability of solid-phase microextraction to enable analyses on small-volume biological samples and growing availability of biocompatible solid-phase microextraction coatings make it a highly useful technology for variety of applications. For example, solid-phase microextraction is particularly useful for identifying biomarkers in metabolomics studies, and it can be successfully applied in pharmaceutical and toxicological studies requiring the fast and sensitive determination of drug levels, especially those that are present at low levels in biological matrices such as plasma, urine, saliva, and hair. Moreover, solid-phase microextraction can be directly applied in in vivo studies because this extraction technique is non-exhaustive and its biocompatible probes offer minimal invasiveness to the analyzed system. In this article, we review recent progress in well-established solid-phase microextraction technique for in vitro and in vivo analyses of various metabolites and drugs in clinical, pharmaceutical, and toxicological applications.

Open Port Probe Sampling Interface for the Direct Coupling of Biocompatible Solid-Phase Microextraction to Atmospheric Pressure Ionization Mass Spectrometry.

In recent years, the direct coupling of solid phase microextraction (SPME) and mass spectrometry (MS) has shown its great potential to improve limits of quantitation, accelerate analysis throughput, and diminish potential matrix effects when compared to direct injection to MS. In this study, we introduce the open port probe (OPP) as a robust interface to couple biocompatible SPME (Bio-SPME) fibers to MS systems for direct electrospray ionization. The presented design consisted of minimal alterations to the front-end of the instrument and provided better sensitivity, simplicity, speed, wider compound coverage, and high-throughput in comparison to the LC-MS based approach. Quantitative determination of clenbuterol, fentanyl, and buprenorphine was successfully achieved in human urine. Despite the use of short extraction/desorption times (5 min/5 s), limits of quantitation below the minimum required performance levels (MRPL) set by the world antidoping agency (WADA) were obtained with good accuracy (≥90%) and linearity (R2 > 0.99) over the range evaluated for all analytes using sample volumes of 300 μL. In-line technologies such as multiple reaction monitoring with multistage fragmentation (MRM3) and differential mobility spectrometry (DMS) were used to enhance the selectivity of the method without compromising analysis speed. On the basis of calculations, once coupled to high throughput, this method can potentially yield preparation times as low as 15 s per sample based on the 96-well plate format. Our results demonstrated that Bio-SPME-OPP-MS efficiently integrates sampling/sample cleanup and atmospheric pressure ionization, making it an advantageous configuration for several bioanalytical applications, including doping in sports, in vivo tissue sampling, and therapeutic drug monitoring.