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Sample preparation with solid phase microextraction and exhaustive extraction approaches: Comparison for challenging cases.

In chemical analysis, sample preparation is frequently considered the bottleneck of the entire analytical method. The success of the final method strongly depends on understanding the entire process of analysis of a particular type of analyte in a sample, namely: the physicochemical properties of the analytes (solubility, volatility, polarity etc.), the environmental conditions, and the matrix components of the sample. Various sample preparation strategies have been developed based on exhaustive or non-exhaustive extraction of analytes from matrices. Undoubtedly, amongst all sample preparation approaches, liquid extraction, including liquid-liquid (LLE) and solid phase extraction (SPE), are the most well-known, widely used, and commonly accepted methods by many international organizations and accredited laboratories. Both methods are well documented and there are many well defined procedures, which make them, at first sight, the methods of choice. However, many challenging tasks, such as complex matrix applications, on-site and in vivo applications, and determination of matrix-bound and free concentrations of analytes, are not easily attainable with these classical approaches for sample preparation. In the last two decades, the introduction of solid phase microextraction (SPME) has brought significant progress in the sample preparation area by facilitating on-site and in vivo applications, time weighted average (TWA) and instantaneous concentration determinations. Recently introduced matrix compatible coatings for SPME facilitate direct extraction from complex matrices and fill the gap in direct sampling from challenging matrices. Following introduction of SPME, numerous other microextraction approaches evolved to address limitations of the above mentioned techniques. There is not a single method that can be considered as a universal solution for sample preparation. This review aims to show the main advantages and limitations of the above mentioned sample preparation approaches and the applicability and capability of each technique for challenging cases such as complex matrices, on-site applications and automation.

Introduction of solid-phase microextraction as a high-throughput sample preparation tool in laboratory analysis of prohibited substances.

A fully automated, high-throughput method based on thin-film solid-phase microextraction (SPME) and liquid chromatography-mass spectrometry was developed for simultaneous quantitative analysis of 110 doping compounds, selected from ten classes and varying in physical and chemical properties. Among four tested extraction phases, C18 blades were chosen, as they provided optimum recoveries and the lowest carryover effect. The SPME method was optimized in terms of extraction pH, ionic strength of the sample, washing solution, extraction and desorption times for analysis of urine samples. Chromatographic separation was obtained in reversed-phase model; for detection, two mass spectrometers were used: triple quadrupole and full scan orbitrap. These combinations allowed for selective analysis of targeted compounds, as well as a retrospective study for known and unknown compounds. The developed method was validated according to the Food and Drug Administration (FDA) criteria, taking into account Minimum Required Performance Level (MRPL) values required by the World Anti-Doping Agency (WADA). In addition to analysis of free substances, it was also shown that the proposed method is able to extract the glucuronated forms of the compounds. The developed assay offers fast and reliable analysis of various prohibited substances, an attractive alternative to the standard methods that are currently used in anti-doping laboratories.

Targeting Mitochondria with Avocatin B Induces Selective Leukemia Cell Death.

Treatment regimens for acute myeloid leukemia (AML) continue to offer weak clinical outcomes. Through a high-throughput cell-based screen, we identified avocatin B, a lipid derived from avocado fruit, as a novel compound with cytotoxic activity in AML. Avocatin B reduced human primary AML cell viability without effect on normal peripheral blood stem cells. Functional stem cell assays demonstrated selectivity toward AML progenitor and stem cells without effects on normal hematopoietic stem cells. Mechanistic investigations indicated that cytotoxicity relied on mitochondrial localization, as cells lacking functional mitochondria or CPT1, the enzyme that facilitates mitochondria lipid transport, were insensitive to avocatin B. Furthermore, avocatin B inhibited fatty acid oxidation and decreased NADPH levels, resulting in ROS-dependent leukemia cell death characterized by the release of mitochondrial proteins, apoptosis-inducing factor, and cytochrome c. This study reveals a novel strategy for selective leukemia cell eradication based on a specific difference in mitochondrial function.

Development of a Carbon Mesh Supported Thin Film Microextraction Membrane As a Means to Lower the Detection Limits of Benchtop and Portable GC/MS Instrumentation.

In this work, a durable and easy to handle thin film microextraction (TFME) device is reported. The membrane is comprised of poly(divinylbenzene) (DVB) resin particles suspended in a high-density polydimethylsiloxane (PDMS) glue, which is spread onto a carbon fiber mesh. The currently presented membrane was shown to exhibit a substantially lesser amount of siloxane bleed during thermal desorption, while providing a statistically similar extraction efficiency toward a broad spectrum of analytes varying in polarity when compared to an unsupported DVB/PDMS membrane of similar shape and size which was prepared with previously published methods. With the use of hand-portable GC-TMS instrumentation, membranes cut with dimensions 40 mm long by 4.85 mm wide and 40 ± 5 μm thick (per side) were shown to extract 21.2, 19.8, 18.5, 18,4, 26.8, and 23.7 times the amount of 2,4 dichlorophenol, 2,4,6 trichlorophenol, phorate D10, fonofos, chloropyrifos, and parathion, respectively, within 15 min from a 10 ppb aqueous solution as compared to a 65 μm DVB/PDMS solid phase microextraction (SPME) fiber. A portable high volume desorption module prototype was also evaluated and shown to be appropriate for the desorption of analytes with a volatility equal to or lesser than benzene when employed in conjunction with TFME membranes. Indeed, the coupling of these TFME devices to hand-portable gas chromatography toroidial ion trap mass spectrometry (GC-TMS) instrumentation was shown to push detection limits for these pesticides down to the hundreds of ppt levels, nearing that which can be achieved with benchtop instrumentation. Where these membranes can also be coupled to benchtop instrumentation it is reasonable to assume that detection limits could be pushed down even further. As a final proof of the concept, the first ever, entirely on-site TFME-GC-TMS analysis was performed at a construction impacted lake. Results had indicated the presence of contaminants such as toluene, ethylbenzene, xylene, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, and tris(1-chloro-2-propyl)phosphate, which stood out from other naturally occurring compounds detected.

Fast Quantitation of Target Analytes in Small Volumes of Complex Samples by Matrix‐Compatible Solid‐Phase Microextraction Devices

Herein we report the development of solid-phase microextraction (SPME) devices designed to perform fast extraction/enrichment of target analytes present in small volumes of complex matrices (i.e. V≤10 μL). Micro-sampling was performed with the use of etched metal tips coated with a thin layer of biocompatible nano-structured polypyrrole (PPy), or by using coated blade spray (CBS) devices. These devices can be coupled either to liquid chromatography (LC), or directly to mass spectrometry (MS) via dedicated interfaces. The reported results demonstrated that the whole analytical procedure can be carried out within a few minutes with high sensitivity and quantitation precision, and can be used to sample from various biological matrices such as blood, urine, or Allium cepa L single-cells.

In vivo solid phase microextraction sampling of human saliva for non-invasive and on-site monitoring.

On-site sample preparation is an analytical approach based on direct sampling from the system under investigation. It has the advantage of combining sampling and sample preparation into a single step, thus generally is fast, minimizes the potential sources of error and eliminates the risks for analytes instability. For such analysis solid phase microextraction in thin film geometry (TF-SPME) can provide robust and convenient in vivo sampling, offering in the same time faster analysis and higher extraction recovery (i.e., better sensitivity) due to large surface to volume ratio. In this study, TF-SPME in coated blade and membrane formats with a single extraction phase were used for in vivo and ex vivo saliva extraction and separation by LC and GC, respectively. Due to applicability for wide range of polarity of analytes as well as thermal and solvent stability during the desorption, hydrophilic lipophilic balanced particles (HLB) were chosen as extraction phase and used for fast (5 min) in vivo and ex vivo sampling. The results of metabolomic profiling of the saliva are indicating that even 5 min in vivo sampling using TF-SPME followed by GC and LC analyses provides complementary coverage of wide range of analytes with different physical and chemical properties. To demonstrate the applicability of the method for doping analyses, the SPME-LC-MS/MS method was validated for simultaneous quantification of 49 prohibited substances with limit of quantification (LOQ) ranging between 0.004 and 0.98 ng mL(-1). Moreover, the method was also validated and successfully applied for determination of endogenous steroids in saliva where the concentrations of the analytes are substantially low. The developed assay offers fast and reliable multiresidue analysis of saliva as an attractive alternative to the standard analysis methods.

Microextraction versus exhaustive extraction approaches for simultaneous analysis of compounds in wide range of polarity.

This article discusses comparison of microextraction versus exhaustive extraction approaches for simultaneous extraction of compounds in wide range of polarity at low and high volumes of sample by comparing solid phase extraction (SPE) and solid phase microextraction (SPME). Here, both systems are discussed theoretically and evaluated based on experimental data. Experimental comparisons were conducted in terms of extraction recovery, sensitivity, and selectivity for the extraction of doping agent compounds (logP: 0.14-4.98), using C18 as the extraction phase. The extraction recovery of both systems was studied at different volumes of phosphate buffered saline (PBS). The distribution constant of SPME in thin-film geometry (i.e., thin-film microextraction/TFME) as well as the retention factor and breakthrough volume for the SPE system were evaluated for the simultaneous extraction of polar and non-polar compounds. Using 1 mL of sample, the extraction recovery and sensitivity of the SPE system (100 mg sorbent) was comparable with that of TFME format of SPME (15 mg sorbent) for all analytes, with the exception of most polar compounds, due to the smaller amount of the extraction phase in SPME. Breakthrough in the SPE system was observed for more polar compounds in a 25 mL sample; however, this situation did not affect the quantitation of TFME, as this technique operates in equilibrium mode. Experimental values for breakthrough volume were in good match with the calculated theoretical values. Results indicate that the microextraction approach is more suitable for untargeted determinations, where the breakthrough volume cannot be determined prior to the experiment. In addition, when both methods are at optimum conditions, findings suggest that, despite the smaller volume of the extraction phase in TFME, the sensitivity of TFME can exceed that of SPE for samples where the target analytes vary substantially in polarity.

Advances in Solid Phase Microextraction and Perspective on Future Directions

Solid phase microextraction (SPME) is a versatile, non-exhaustive sample preparation tool that has been demonstrated to be well-suited for facile and effective analysis of a broad range of compounds in a plethora of studies. A growing number of reports describing diverse SPME workflows for novel investigations in a variety of fields, such as flavor and fragrance investigations, environmental studies, and diverse bioanalytical applications, among others, corroborate the applicability of this microextraction tool in the analytical sciences

Ultra-fast quantitation of voriconazole in human plasma by coated blade spray mass spectrometry

HIGHLIGHTSA Coated Blade Spray method for voriconazole determination from human plasma was developed.Analysis from from spots (10 &mgr;L) and small volumes (300 &mgr;L) of plasma were performed.Total analysis times obtained were between 120 and 180 s.The methodology was successfully validated for different plasma lots and patients.Relative matrix effects as low as 7% were achieved. ABSTRACT Voriconazole is a triazole broad‐spectrum antifungal medication often used to treat fungal infections caused by Aspergillus and Fusarium species. One of the main challenges associated with the implementation of this medication is its narrow therapeutic concentration range, demonstrating toxicity at concentrations above 6 &mgr;g/mL and limited efficacy at concentrations below 2 &mgr;g/mL. As a result, methodologies which permit the rapid and accurate quantitation of voriconazole in patients are highly desirable. In this work two different approaches based on coated blade spray directly coupled to mass spectrometry (CBS‐MS) are introduced; each enabling the quantitation of voriconazole in plasma samples with a simple and fast sample preparation and no chromatographic step. The first approach involves a rapid extraction (1 min) of the target analyte from 300 &mgr;L of human plasma using conventional laboratory vessels (e.g. vial, 96‐well plate). Alternatively, the second strategy consists of a 2 min extraction from a plasma droplet (10 &mgr;L) placed on the coated area of the blade. Both procedures were successfully validated and good linearity (R2 ≥ 0.998), accuracy (91–122%) and precision (<8%) were attained in the concentration range evaluated (0.1–50 &mgr;g/mL). Moreover, very good results in terms of relative matrix effects were obtained given that the slopes of the calibration curves constructed in five different plasma lots exhibited relative standard deviation (RSD) values below 7%. Herein we demonstrated that CBS‐MS is a technology suitable for the ultra‐fast determination of voriconazole in human plasma samples. Indeed, the proposed methodology can be easily used either for routine drug monitoring or for in vitro pharmacokinetic studies in applications where very small sample volumes are available and great temporal resolution is needed.

High-Throughput Screening and Quantitation of Target Compounds in Biofluids by Coated Blade Spray-Mass Spectrometry

Most contemporary methods of screening and quantitating controlled substances and therapeutic drugs in biofluids typically require laborious, time-consuming, and expensive analytical workflows. In recent years, our group has worked toward developing microextraction (μe)-mass spectrometry (MS) technologies that merge all of the tedious steps of the classical methods into a simple, efficient, and low-cost methodology. Unquestionably, the automation of these technologies allows for faster sample throughput, greater reproducibility, and radically reduced analysis times. Coated blade spray (CBS) is a μe technology engineered for extracting/enriching analytes of interest in complex matrices, and it can be directly coupled with MS instruments to achieve efficient screening and quantitative analysis. In this study, we introduced CBS as a technology that can be arranged to perform either rapid diagnostics (single vial) or the high-throughput (96-well plate) analysis of biofluids. Furthermore, we demonstrate that performing 96-CBS extractions at the same time allows the total analysis time to be reduced to less than 55 s per sample. Aiming to validate the versatility of CBS, substances comprising a broad range of molecular weights, moieties, protein binding, and polarities were selected. Thus, the high-throughput (HT)-CBS technology was used for the concomitant quantitation of 18 compounds (mixture of anabolics, β-2 agonists, diuretics, stimulants, narcotics, and β-blockers) spiked in human urine and plasma samples. Excellent precision (∼2.5%), accuracy (≥90%), and linearity (R2 ≥ 0.99) were attained for all the studied compounds, and the limits of quantitation (LOQs) were within the range of 0.1 to 10 ng·mL-1 for plasma and 0.25 to 10 ng·mL-1 for urine. The results reported in this paper confirm CBSs great potential for achieving subsixty-second analyses of target compounds in a broad range of fields such as those related to clinical diagnosis, food, the environment, and forensics.