Rapid determination of tacrolimus and sirolimus in whole human blood by direct coupling of solid-phase microextraction to mass spectrometry via microfluidic open interface.

Immunosuppressive drugs are administered to decrease immune system activity (e.g. of patients undergoing solid organ transplant). Concentrations of immunosuppressive drugs (ISDs) in circulating blood must be closely monitored during the period of immunosuppression therapy due to adverse effects that take place when concentration levels fall outside of the very narrow therapeutic concentration range of these drugs. This study presents the rapid determination of four relevant immunosuppressive drugs (tacrolimus, sirolimus, everolimus, and cyclosporine A) in whole human blood by directly coupling solid-phase microextraction to mass spectrometry via the microfluidic open interface (Bio-SPME-MOI-MS/MS). The BioSPME-MOI-MS/MS method offers ≤ 10% imprecision of in-house prepared quality controls over a 10-day period, ≤ 10% imprecision of ClinCal® Recipe calibrators over a three-day period, and single total turnaround time of ∼ 60 min (4.5 min for high throughput). The limits of quantification were determined to be 0.8 ng mL-1 for tacrolimus, 0.7 ng mL-1 sirolimus, 1.0 ng mL-1 for everolimus, and 0.8 ng mL-1 for cyclosporine. The limits of detection were determined to be 0.3 ng mL-1 for tacrolimus, 0.2 ng mL-1 for sirolimus, 0.3 ng mL-1 for everolimus, and 0.3 ng mL-1 for cyclosporine A. The R2 values for all analytes were above 0.9992 with linear dynamic range from 1.0 mL-1 to 50.0 ng mL-1 for tacrolimus, sirolimus, and everolimus while from 2.5 ng mL-1 to 500.0 ng mL-1 for cyclosporine A. To further evaluate the performance of the present method, 95 residual whole blood samples of tacrolimus and sirolimus from patients undergoing immunosuppression therapy were used to compare the Bio-SPME-MOI-MS/MS method against a clinically validated reference method based on chemiluminescent microparticle immunoassay, showing acceptable results. Our results demonstrated that Bio-SPME-MOI-MS/MS can be considered as a suitable alternative to existing methods for the determination of immunosuppressive drugs in whole blood providing faster analysis, better selectivity and sensitivity, and a wider dynamic range than current existing approaches.

Assessment of solid phase microextraction as a sample preparation tool for untargeted analysis of brain tissue using liquid chromatography-mass spectrometry.

This work presents an evaluation of solid-phase microextraction (SPME) SPME in combination with liquid chromatography-high resolution mass spectrometry (LC-HRMS) as an analytical approach for untargeted brain analysis. The study included a characterization of the metabolite coverage provided by C18, mixed-mode (MM, with benzene sulfonic acid and C18 functionalities), and hydrophilic lipophilic balanced (HLB) particles as sorbents in SPME coatings after extraction from cow brain homogenate at static conditions. The effects of desorption solvent, extraction time, and chromatographic modes on the metabolite features detected were investigated. Method precision and absolute matrix effects were also assessed. Among the main findings of this work, it was observed that all three tested coating chemistries were able to provide comparable brain tissue information. HLB provided higher responses for polar metabolites; however, as these fibers were prepared in-house, higher inter-fiber relative standard deviations were also observed. C18 and HLB coatings offered similar responses with respect to lipid-related features, whereas MM and C18 provided the best results in terms of method precision. Our results also showed that the use of methanol is essential for effective desorption of non-polar metabolites. Using a reversed-phase chromatographic method, an average of 800 and 1200 brain metabolite features detected in positive and negative modes, respectively, met inter-fibre RSD values below 30% (n=4) after removal of fibre and solvent artefacts from the associated datasets. For features detected using a lipidomics method, a total of 900 and 1800 features detected using C18 fibers in positive and negative mode, respectively, met the same criteria. In terms of absolute matrix effects, the majority of the model metabolites tested showed values between 80 and 120%, which are within the acceptable range. Overall, the findings of this work lay the foundation for further optimization of parameters for SPME-LC-HRMS methods suitable for in vivo and ex vivo brain (and other tissue) untargeted studies, and support the applicability of this approach for non-destructive tissue metabolomics.

Evaluation of a coated blade spray-tandem mass spectrometry assay as a new tool for the determination of immunosuppressive drugs in whole blood.

Immunosuppressive drugs (ISDs) are primarily administered following solid organ transplant or for treatment of a variety of autoimmune conditions. Their principal function is to suppress the activity of the immune system; however, the levels must be carefully monitored due to adverse effects of over- or underadministration. A technology for rapid quantitative screening, named coated blade spray (CBS), was directly coupled to a triple quadrupole mass spectrometer (MS/MS) to measure the concentration of ISDs (i.e., cyclosporine A, tacrolimus, everolimus, sirolimus) in whole blood samples. We evaluated the stability of replicate measurements over a 10-day period (precision), assessed linearity and limit of quantification, and performed a method comparison against a validated clinical immunoassay (Abbott ARCHITECT). Total interday variation of less than 5% for all target compounds at three different concentrations was achieved. The sensitivity of the method was determined as 0.25, 1, 1, and 2.5 ng/mL for everolimus, sirolimus, tacrolimus, and cyclosporine A, respectively. The concentrations of three immunosuppressive drugs in 284 patient samples (i.e., ~ 95 samples of cyclosporine A, tacrolimus, or sirolimus) obtained using the CBS-MS/MS methodology were compared with concentrations previously quantified on an Abbott ARCHITECT immunoassay system. Our analysis demonstrated significant statistical similarities between both methods. The results demonstrate that CBS-MS/MS is a suitable alternative to conventional methodologies for monitoring of ISDs from whole blood in a clinical setting. Graphical abstract.

In Vivo Brain Sampling Using a Microextraction Probe Reveals Metabolic Changes in Rodents after Deep Brain Stimulation.

Brain metabolomics is an emerging field that complements the more traditional approaches of neuroscience. However, typical brain metabolomics workflows require that animals be sacrificed and tend to involve tedious sample preparation steps. Microdialysis, the standard technique to study brain metabolites in vivo, is encumbered by significant limitations in the analysis of hydrophobic metabolites, which are prone to adsorption losses on microdialysis equipment. An alternative sampling method suitable for in vivo brain studies is solid-phase microextraction (SPME). In SPME, a small probe coated with a biocompatible polymer is employed to extract/enrich analytes from biological matrices. In this work, we report the use of SPME and liquid chromatography-mass spectrometry for untargeted in vivo analysis of rodent's brains after deep brain stimulation (DBS). First, metabolite changes occurring in brain hippocampi after application of 3 h of DBS to the animals' prefrontal cortex were monitored with the proposed approach. As SPME allows for nonlethal sampling, the same group of animals was sampled again after 8 days of daily DBS therapy. After acute DBS, we detected changes in a broad range of metabolites, including the amino acid citrulline, which may reflect changes in nitric oxide production, as well as various phospho- and glycosphingolipids. Measurements conducted after chronic DBS showed a decrease in hippocampal corticosterone, indicating that DBS may have a regulatory effect in the hypothalamic-pituitary-adrenal axis. Our findings demonstrate the potential of in vivo SPME as a tool of scientific and clinical interest capable of revealing changes in a wide range of metabolites in brain tissue.

Rapid and Concomitant Analysis of Pharmaceuticals in Treated Wastewater by Coated Blade Spray Mass Spectrometry.

The widespread use of pharmaceuticals in both human and animal populations, and the resultant contamination of surface waters from the outflow of water treatment facilities is an issue of growing concern. This has raised the need for analytical methods that can both perform rapid sample analysis and overcome the limitations of conventional analysis procedures, such as multistep workflows and tedious procedures. Coated blade spray (CBS) is a solid-phase microextraction based technique that enables the direct-to-mass-spectrometry analysis of extracted compounds via the use of limited organic solvent to desorb analytes and perform electrospray ionization. This paper documents how CBS can be applied for the concomitant tandem mass spectrometric (MS/MS) analysis of nine pharmaceuticals in treated wastewater. The total analysis times of less than 11 min provided limits of detection lower than 50 ng L-1 for all target compounds in river water. The CBS methodology was then compared to a conventional solid-phase extraction technique for the analysis of the final effluent of six wastewater treatment facilities. The experimental results strongly suggest that CBS offers scientists a viable alternative method for analyzing water samples that is both rapid and relatively solvent-free.

Deposition of a Sorbent into a Recession on a Solid Support To Provide a New, Mechanically Robust Solid-Phase Microextraction Device.

To date, solid-phase microextraction (SPME) fibers used for in vivo bioanalysis can be too fragile and flexible, which limits suitability for direct tissue sampling. As a result, these devices often require a sheathing needle to prepuncture robust sample matrixes and protect the extraction phase from mechanical damage. To address this limitation, a new SPME device is herein presented which incorporates an extraction phase recessed into the body of a solid needle. This device requires no additional support or shielding during puncture events through protective tissue. The presented device was thoroughly tested, being fired at 90 m·s-1 through fish scales, forced through vial septa, and employed in a targeted study of polyunsaturated fatty acids in salmon where the protective outer skin was repetitively punctured during sampling. Finally, the recessed SPME device was applied to an on-site application for the tissue analysis of wild muskellunge. With this advancement, rapid, minimally invasive, and easily executed in vivo SPME is now possible opening the door to near endless sampling opportunities.

Solid Phase Microextraction On-Fiber Derivatization Using a Stable, Portable, and Reusable Pentafluorophenyl Hydrazine Standard Gas Generating Vial.

Solid phase microextraction (SPME) on-fiber derivatization methods have facilitated the achievement of lower detection limits and targeted analysis of various substances that exhibit poor chromatographic behavior, thermal instability, or high reactivity while limiting the use of organic solvents. However, previously developed on-fiber derivatization methods have been hindered by poor loading reproducibility and standard lifetime due to derivatization reagent reactivity. In addition, this reactivity often results in these reagents demonstrating toxic effects, complicating handling and standard formulation. To address this, a reusable standard gas generating vial containing pentafluorophenyl hydrazine (PFPH) has been developed. With this development, SPME fibers can now be reproducibly loaded with derivatization reagent, from an easy to use and safe platform. Validation of the vial using C4-C9 linear aldehyde standards as target analytes demonstrated intrabatch vial reproducibility (2% relative standard deviation (RSD), n = 4), along with PFPH headspace stability over a period of 11 weeks, facilitating reduced reagent consumption due to standard longevity. In addition, reproducibility of the derivatization reaction was observed over 1 week (RSD < 9%), and the linear concentration range was evaluated using headspace extractions from aqueous aldehyde solutions (R(2) > 0.996, 10-200 ppb v/v). Finally, the PFPH-generating vial was applied to the monitoring of volatile aldehydes generated during meat spoilage, as well as an on-site application where the free and total concentration of formaldehyde was determined in car exhaust using a portable GC/MS. To the best of our knowledge, the standard gas generating vial proposed in this work is the first documented device for the long-term storage of reusable headspace standards for a reactive, toxic, and otherwise unstable derivatization reagent standard.

Evaluation of a multi-fiber exchange solid-phase microextraction system and its application to on-site sampling.

Until recently, multiple solid-phase microextraction fibers could not be automatically desorbed in a single gas chromatographic sequence without manual intervention from an operator. This drawback had been a critical issue, particularly during the analysis of numerous on-site samples taken with various fiber assemblies. Recently, a Multi-Fiber Exchange system, designed to overcome this flaw found in other commercially available autosamplers, was released. In the current research, a critical evaluation of the Multi-Fiber Exchange system performance in terms of storage stability and long-term operation is presented. It was established in the course of our research that the Multi-Fiber Exchange system can operate continuously and precisely for multiple extraction/injection cycles. However, when the effect of residence time of commercial fibers on the Multi-Fiber Exchange tray was evaluated, results showed that among the evaluated fiber coatings, Carboxen/polydimethylsiloxane was the only coating capable of efficient storage on the tray for up to 24 h after field sampling without suffering significant loss of analytes (≤10% for benzene, toluene, ethylbenzene, o-xylene, decane, and limonene). Additionally, the system capability for high-throughput analysis was demonstrated by the unattended desorption of multiple fibers after on-site sampling of toluene, indoor air levels, in a polymer synthesis lab.

Development of a standard gas generating vial comprised of a silicon oil-polystyrene/divinylbenzene composite sorbent.

In this work, a highly reproducible standard gas generating vial is proposed. The vial is comprised of a silicon diffusion pump oil spiked with an appropriate calibration compound, such as modified McReynolds probes (benzene, 2-pentanone, pyridine, 1-nitropropane, 1-pentanol, and n-octane), and then mixed with polystyrene/divinylbenzene (PS/DVB) particles. The concentrations of these compounds in gaseous headspace were found to substantially decrease in comparison to previously developed hydrocarbon pump oil based vials; hence, the amount of standard loaded onto SPME fibers was at most, half that of the previous vial design. Depletion for all compounds after 208 successive extractions was shown to be less than 3.5%. Smaller quantities of standards being used resulted in a vial that depleted slower while remaining statistically repeatable over a wider number of runs. Indeed, it was found that depletion could be largely predicted by using a mass balance theoretical model. This behavior allowed a further increase in the number of loadings that could be performed repeatedly. At a 95% level of confidence, the ANOVA test demonstrated that the prepared vials were statistically identical, with no significant intra- or inter-batch differences. In addition, it was found that vials stored under different conditions (e.g. under light exposure, room temperature, and within a refrigerator) were stable over 10 weeks. Silicon based vials proved to be ideal for performing instrument quality control and loading of internal standards onto fibers, both of which are of great importance when performing on-site analysis using portable GC-MS instrumentation and high throughput determinations in laboratory.

Development of needle trap technology for on-site determinations: active and passive sampling.

This study presents a thorough evaluation of new prototypes of extended tip needle trap devices (NT), as well as their application to in situ sampling of biological emissions and active/passive on-site sampling of indoor air. A new NT prototype was constructed with a side hole above the sorbent and an extended tip that fits inside the restriction of the narrow neck liner to increase desorption efficiency. New prototype needles were initially packed with divinylbenzene particles at SGE Analytical Science for the purpose of studying biogenic emissions of pine trees. Prior to their final application, they were evaluated in terms of robustness after multiple use (n > 10), as well as amount extracted of volatile organic compounds (VOCs). An ANOVA test for all the probes showed that at a 95% level of confidence, there were not statistical differences observed among the 9 NTs tested. In addition, the needles were also packed in laboratory with synthesized highly cross-linked PDMS as a frit to immobilize carboxen (Car) particles for spot sampling. For passive sampling, the needles were packed with Car particles embedded in PDMS to simplify calculations in passive mode. The use of NTs as spot samplers, as well as a passive sampler under controlled conditions in the laboratoryyielded a relative standard deviation of less than 15%. Finally, a new, reusable and readily deployable penlike diffusive sampler for needle traps (PDS-NT) was built and tested. Application of the PDS-NT in combination with NT-spot sampling toward the analysis of indoor air in a polymer synthesis laboratory showed good agreement between both techniques for the analyte studied, yielding averages of 0.03 and 0.025 ng/mL of toluene, respectively.

Low invasive in vivo tissue sampling for monitoring biomarkers and drugs during surgery.

The techniques currently used for drug, metabolite, and biomarker determination are based on sample collection, and therefore they are not suitable for repeated analysis because of the high invasiveness. Here, we present a novel method of biochemical analysis directly in organ during operation without need of a separate sample collection step: solid-phase microextraction (SPME). The approach is based on flexible microprobe coated with biocompatible extraction phase that is inserted to the tissue with no damage or disturbance of the organ. The method was evaluated during lung and liver transplantations using normothermic ex vivo liver perfusion (NEVLP) and ex vivo lung perfusion (EVLP). The study demonstrated feasibility of the method to extract wide range of endogenous compounds and drugs. Statistical analysis allowed observing metabolic changes of lung during cold ischemic time, perfusion, and reperfusion. It was also demonstrated that the level of drugs and their metabolites can be monitored over time. Based on the methylprednisolone as a selected example, the impairment of enzymatic properties of liver was detected in the injured organs but not in healthy control. This finding was supported by changes in pathways of endogenous metabolites. The SPME probe was also used for analysis of perfusion fluid using stopcock connection. The evaluation of biochemical profile of perfusates demonstrated potential of the approach for monitoring organ function during ex vivo perfusion. The simplicity of the device makes it convenient to use by medical personnel. With the microprobe, different areas of the organ or various organs can be sampled simultaneously. The technology allows assessment of organ function by biochemical profiling, determination of potential biomarkers, and drug monitoring. The use of this method for preintervention analysis could enhance the decision-making process for the best possible personalized approach, whereas post-transplantation monitoring would be used for graft assessments and fast response in case of organ failure.

Development of a new in-vial standard gas system for calibrating solid-phase microextraction in high-throughput and on-site applications.

In this work, an innovative, reproducible, and reusable standard generator vial is presented. The standard generator vial consists of vacuum-pump oil doped with McReynolds probes (benzene, 2-pentanone, pyridine, nitropropane, 1-pentanol, and n-octane) mixed with a polystyrene-divinylbenzene resin without functional groups. The evaluation of this vial was fully automated on a multifiber exchanger system and the extraction/desorption cycle, together with the programmed GC-qMS analysis, did not exceed 13 min. The results showed that after 160 extraction/injections cycles of the vial the relative SDs were smaller than 4% for all the standards. A randomized block design was used to evaluate the inter and intravial repeatability, and at 95% level of confidence nonstatistical differences among vials were found. Because of its compacted granular appearance this vial is easy to transport, and it is an ideal calibration standard for bench and field instruments and devices.

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.