Barbara Bojko

SPME – Quo vadis?

Solid phase microextraction (SPME) has experienced rapid development and growth in number of application areas since its inception over 20 years ago. It has had a major impact on sampling and sample preparation practices in chemical analysis, bioanalysis, food and environmental sciences. A significant impact is expected in clinical analysis as well as pharmaceutical and medical sciences in the near future. In this review, recent developments of SPME and related technologies are discussed including an in-vial standard gas system for calibration of SPME in high throughput mode; a thin film geometry with high extraction efficiency SPME for gas chromatography (GC) and liquid chromatography (LC) analyses; and couplings of SPME with portable instruments permitting on-site measurements. Also, the latest advances in the preparation of sorbents applicable for direct extraction from complex biological matrices as well as applications of these extraction phases in food analysis and biomedical studies such as therapeutic drug monitoring and pharmacokinetics are described. Finally, recent trends in metabolomics analysis and examples of clinical monitoring of biomarkers with SPME are reviewed.

Solid-Phase Microextraction: A Complementary In Vivo Sampling Method to Microdialysis†

Given the intricate organization of the brain, tissue sampling for chemical profiling studies have always been a challenging task. It is often exceptionally difficult to obtain homogeneous samples for in vitro/ex vivo experiments without altering or losing valuable information. The obvious approach has been to develop in vivo analytical methods that may cause minimal perturbation to this complex chemical network so as to improve overall reliability of acquired information. Methods such as biosensors and microdialysis (MD) are among sampling methods applied to in vivo brain chemical profiling studies despite their unique challenges. MD is a well-established in vivo analytical sampling method used over the years for monitoring often low-molecular-weight hydrophilic compounds from the interstitial space. The successful application of the method to neuroscience, especially monitoring of neurotransmitters, led to its expansion to a wider range of analytes, including drugs, metabolites, and peptides. A major challenge, however, associated with MD is its difficulty in sampling hydrophobic compounds. Hydrophobic compounds are often highly protein-bound and bind to the MD probe and tubing, thereby affecting relative recovery. The addition of modifiers, such as bovine serum albumin, glycerol in water, or cyclodextrin, is among the approaches that have been used to prevent hydrophobic interactions and to improve relative recoveries. But these techniques often may complicate the pharmacology of the neurological analytes, as the additives are known to interact with the tissue surrounding the probe. Thus, in typical global metabolomics studies, for example, the composition of a measured metabolome can be significantly affected by the analytical procedure, leaving the analysts with results which likely do not adequately reflect accurate composition of the metabolome during sampling. In effect, it will compromise the already challenging efforts in diagnosis, prognostics, and searching for potential biomarkers for therapeutic purposes. Herein, we demonstrate a novel application of solid-phase microextraction (SPME) for in vivo sampling for brain study. For the first time an application of in vivo SPME as a complementary method to MD for braintissue bioanalysis has been presented. Our technique was first validated against MD in targeted analysis of selected neurotransmitters. Their complementary nature was subsequently shown in global profiling of the brain metabolome. From the profiling study, SPME detected groups of lipids such as gangliosides, fatty acids, and lysophospholipids, which are of particular interest in relation to neurodegenerative diseases. SPME derives its selectivity from the extracting sorbent type. Thus, SPME provides the needed flexibility to analysts to tailor investigations to specific biologically hydrophilic/ hydrophobic compounds. For a global study of the metabolome, however, the sorbent choice is one of low selectivity; that is, the sorbent chemical property must enhance simultaneous extraction of hydrophilic and hydrophobic biochemical species. A unique advantage of the new biocompatible in vivo SPME probe is, it prevents extraction of proteins and other bio-interferences owing to the small pore size of the coating and the adhesive biocompatibility, and thus minimizes matrix effect significantly. Furthermore, the in vivo characteristics guarantees enriched chemical information for tissue bioanalysis over other methods when coupled to analytical techniques. Herein, we introduce in vivo SPME and MD coupled to liquid chromatography mass spectrometry (LC-MS) to study the chemical components of the brain extracellular fluid in freely moving rat. Briefly, the approach involved surgically implanting the two probes (SPME and MD), respectively, into the left and right hemispheres of the striata of freely moving rats for continuous chemical monitoring over a period of time. The SPME probe is a simple device placed in a commercial MD guide cannula without extended tubes to an external pumping system contrary to what is typical for the MD probe. Samples collected for both MD and SPME were subjected to a reversed reverse-phase chromatographic separation on a pentafluorophenyl stationary phase in a positive-mode mass spectrometry analysis. Knowing that SPME sorbent can extract relatively wider range of analytes, including hydrophobic biomolecules contrary to MD, initial investigations focused on the effectiveness of the sampling technique for monitoring small polar endogenous compounds in a targeted metabolomics study. Subsequently, we simultaneously monitored the effect of single dose (10 mgkg ) fluoxetine on multiple neurotransmitters: dopamine (DA), serotonin (5-HT), gamma aminobutyric acid (GABA), and glutamic acid (GA) using both SPME and MD. The purpose was to evaluate the ability of SPME to monitor at each time point changes in multiple neurochemicals with a single probe similar to MD. The [*] E. Cudjoe, Dr. B. Bojko, Prof. J. Pawliszyn Department of Chemistry, University of Waterloo 200 University Avenue, Waterloo, N2L 3G1 (Canada) E-mail: janusz@uwaterloo.ca Homepage: http://spme.uwaterloo.ca

Quantitative structure–retention relationships models for prediction of high performance liquid chromatography retention time of small molecules: Endogenous metabolites and banned compounds

Quantitative structure-retention relationship (QSRR) is a technique capable of improving the identification of analytes by predicting their retention time on a liquid chromatography column (LC) and/or their properties. This approach is particularly useful when LC is coupled with a high-resolution mass spectrometry (HRMS) platform. The main aim of the present study was to develop and describe appropriate QSRR models that provide usable predictive capability, allowing false positive identification to be removed during the interpretation of metabolomics data, while additionally increasing confidence of experimental results in doping control area. For this purpose, a dataset consisting of 146 drugs, metabolites and banned compounds from World Anti-Doping Agency (WADA) lists, was used. A QSRR study was carried out separately on high quality retention data determined by reversed-phase (RP-LC-HRMS) and hydrophilic interaction chromatography (HILIC-LC-HRMS) systems, employing a single protocol for each system. Multiple linear regression (MLR) was applied to construct the linear QSRR models based on a variety of theoretical molecular descriptors. The regression equations included a set of three descriptors for each model: ALogP, BELe6, R2p and ALogP(2), FDI, BLTA96, were used in the analysis of reversed-phase and HILIC column models, respectively. Statistically significant QSRR models (squared correlation coefficient for model fitting, R(2)=0.95 for RP and R(2)=0.84 for HILIC) indicate a strong correlation between retention time and the molecular descriptors. An evaluation of the best correlation models, performed by validation of each model using three tests (leave-one-out, leave-many-out, external tests), demonstrated the reliability of the models. This paper provides a practical and effective method for analytical chemists working with LC/HRMS platforms to improve predictive confidence of studies that seek to identify small molecules.

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.

Pharmacokinetics of tranexamic acid in patients undergoing cardiac surgery with use of cardiopulmonary bypass

We conducted a study to assess pharmacokinetics of high‐dose tranexamic acid for 24 h after administration of the drug in patients undergoing cardiac surgery with cardiopulmonary bypass. High‐dose tranexamic acid involved a bolus of 30 mg.kg−1 infused over 15 min followed by a 16 mg.kg−1.h−1 infusion until chest closure with a 2 mg.kg−1 load within the pump prime. Tranexamic acid followed first‐order kinetics best described using a two‐compartment model, with a total body clearance that approximated the glomerular filtration rate. Mean plasma tranexamic acid concentrations during the intra‐operative period and in the first 6 postoperative hours were consistently higher than the suggested threshold to achieve 100% inhibition and 80% inhibition of tissue plasminogen activator. With recent studies implicating high‐dose tranexamic acid as a possible aetiology of postoperative seizures following cardiac surgery, the minimum effective yet safe dose of tranexamic acid in high‐risk cardiac surgery needs to be refined.

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.

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.

High throughput quantification of prohibited substances in plasma using thin film solid phase microextraction

Simple, fast and efficient sample preparation approaches that allow high-throughput isolation of various compounds from complex matrices are highly desired in bioanalysis. Particularly sought are methods that can, without sacrificing time, easily remove matrix interferences capable of inducing ionization suppression/enhancement, or causing detrimental effects in instrumental performance. In this work, an automated high-throughput sample preparation method using thin film solid phase microextraction (SPME) for the analysis of multiple prohibited substances in plasma is proposed. A biocompatible SPME extraction phase made of hydrophilic-lipophilic balance particles immobilized with polyacrylonitrile (PAN) demonstrated satisfactory extraction capabilities for 25 compounds of a wide range of polarities (logP from -2 to 6.8). Due to the well-known biocompatible characteristics of PAN-based SPME coatings, minimum sample handling was required. Experimental conditions for pre-conditioning, extraction, wash and desorption were carefully optimized for the proposed method. By taking full advantage of the 96 thin film handling capability of the automated system, a preparation time of approximately 1.5min per sample can be achieved. Satisfactory results in terms of absolute matrix effects were found for the majority of the studied analytes, given that 24 out of 25 compounds exhibited values in the range of 100 and 120%. The method was validated in terms of linearity (R(2)>0.99), inter and intra-day accuracy (85-130%) and precision (<20%) and limits of quantitation (0.25-10ngmL(-1) for most compounds).

Determination of tranexamic acid concentration by solid phase microextraction and liquid chromatography–tandem mass spectrometry: First step to in vivo analysis

A solid phase microextraction (SPME) method followed by LC-MS/MS analysis was developed to determine the concentration of tranexamic acid (TA) in plasma. The use of a new biocompatible C18 coating allowed the direct extraction from complex biological samples without prior sample preparation; no matrix effect was observed. The results revealed that SPME was suitable for the analysis of polar drugs such as TA; such an application was previously inaccessible because of the limited availability of SPME coatings that can extract polar molecules. The proposed method was validated according to the bioanalytical method validation guidelines. LOD and LLOQ were 0.5 and 1.5 μg/ml, respectively. The recovery for the method was 0.19%, and the accuracy and precision of the method were <9 and <11%, respectively, with the exception of LLOQ, where the values were <16 and <13%, respectively. The performance of the proposed method was also compared against that of the standard techniques of protein precipitation and ultrafiltration. A statistical analysis indicated a clinically significant agreement among all assays. Another advantage of SPME over conventional techniques was the easy automation and feasibility of in vivo analysis; this advantage makes it possible to use the proposed method for an on-site analysis in clinical practice.

Analysis of human saliva metabolome by direct immersion solid-phase microextraction LC and benchtop orbitrap MS

BACKGROUND Saliva samples collected from one 58 year old male and one 35 year old female during 7 days of fasting were analyzed by direct immersion of both C18 and mixed-mode biocompatible solid-phase microextraction fibers, in combination with a LC-MS method using a benchtop orbitrap instrument in both positive and negative ionization modes, in order to evaluate the difference in terms of metabolite coverage. RESULTS The mixed-mode coating provided better results, with the simultaneous extraction of a higher number of both hydrophilic and hydrophobic metabolites. The ability of detected features to distinguish differences between the individuals and changes in saliva metabolome induced by diet was also demonstrated. CONCLUSION Saliva may be useful for medical diagnostics as it is non-invasive. The use of biocompatible solid-phase microextraction fibers can play an important role as an alternative sample preparation method for untargeted LC-MS metabolomics studies on human saliva because it provides simultaneous extraction of metabolites with a wide range of polarity, thus allowing the detection of changes in metabolic pathways with unsupervised statistical analyses.