Development of a background electrolyte for the determination of inorganic cations in high ionic strength samples by capillary electrophoresis with indirect UV-absorption detection.

In this study, a background electrolyte capable to separate and quantify inorganic cations in high ionic strength samples by UV-absorption indirect detection was designed. In this regard, the four most abundant monovalent and divalent cations in earth crust (K+, Na+, Ca+2, Mg+2) were selected as model compounds. A group of small carboxylic acids and, several toluidines and pyridines were evaluated as mild strength complexing agents and chromophoric probes, respectively. The optimized background electrolyte was composed of 200 mM 2,4,6-trimethylpyridine as the chromophoric probe, 250 mM lactic acid as the weak complexing agent and pH buffering reagent (adjusted to pH 4.5), and 5% v/v methanol as organic solvent modifier. Based on a minimum number of components, it provided outstanding separation performance in less than 4 min in a wide linear dynamic range (10 - 2500 µg·mL-1). Performances were contrasted against a reference method based on conductometric detection. Furthermore, studies of separation efficiency and peak shape were carried out at different analyte concentrations in high electric conductivity solutions. The herein developed method demonstrated exceptional features in terms of limits of detection (~10 µg·mL-1), resolution, speed of analysis, sensitivity and peak capacity in high electric conductivity samples. Moreover, the method was successfully applied to high ionic strength samples such as rock digest, sea water, soy sauce and isotonic drinks.

Solid phase microextraction chemical biopsy tool for monitoring of doxorubicin residue during in vivo lung chemo-perfusion.

Development of a novel in vivo lung perfusion (IVLP) procedure allows localized delivery of high-dose doxorubicin (DOX) for targeting residual micrometastatic disease in the lungs. However, DOX delivery via IVLP requires careful monitoring of drug level to ensure tissue concentrations of this agent remain in the therapeutic window. A small dimension nitinol wire coated with a sorbent of biocompatible morphology (Bio-SPME) has been clinically evaluated for in vivo lung tissue extraction and determination of DOX and its key metabolites. The in vivo Bio-SPME-IVLP experiments were performed on pig model over various (150 and 225 mg/m2) drug doses, and during human clinical trial. Two patients with metastatic osteosarcoma were treated with a single 5 and 7 µg/mL (respectively) dose of DOX during a 3-h IVLP. In both pig and human cases, DOX tissue levels presented similar trends during IVLP. Human lung tissue concentrations of drug ranged between 15 and 293 µg/g over the course of the IVLP procedure. In addition to DOX levels, Bio-SPME followed by liquid chromatography-mass spectrometry analysis generated 64 metabolic features during endogenous metabolite screening, providing information about lung status during drug administration. Real-time monitoring of DOX levels in the lungs can be performed effectively throughout the IVLP procedure by in vivo Bio-SPME chemical biopsy approach. Bio-SPME also extracted various endogenous molecules, thus providing a real-time snapshot of the physiology of the cells, which might assist in the tailoring of personalized treatment strategy.

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.

Solid phase microextraction chemical biopsy tool for monitoring of doxorubicin residue during in vivo lung chemo-perfusion / 药物分析学报

Development of a novel in vivo lung perfusion(IVLP)procedure allows localized delivery of high-dose doxorubicin(DOX)for targeting residual micrometastatic disease in the lungs.However,DOX delivery via IVLP requires careful monitoring of drug level to ensure tissue concentrations of this agent remain in the therapeutic window.A small dimension nitinol wire coated with a sorbent of biocompatible morphology(Bio-SPME)has been clinically evaluated for in vivo lung tissue extraction and determina-tion of DOX and its key metabolites.The in vivo Bio-SPME-IVLP experiments were performed on pig model over various(150 and 225 mg/m2)drug doses,and during human clinical trial.Two patients with metastatic osteosarcoma were treated with a single 5 and 7 μg/mL(respectively)dose of DOX during a 3-h IVLP.In both pig and human cases,DOX tissue levels presented similar trends during IVLP.Human lung tissue concentrations of drug ranged between 15 and 293 μg/g over the course of the IVLP procedure.In addition to DOX levels,Bio-SPME followed by liquid chromatography-mass spectrometry analysis generated 64 metabolic features during endogenous metabolite screening,providing information about lung status during drug administration.Real-time monitoring of DOX levels in the lungs can be per-formed effectively throughout the IVLP procedure by in vivo Bio-SPME chemical biopsy approach.Bio-SPME also extracted various endogenous molecules,thus providing a real-time snapshot of the physi-ology of the cells,which might assist in the tailoring of personalized treatment strategy.

Photophysics and photochemistry of carminic acid and related natural pigments.

Carminic acid (CA) and other related compounds have been widely used as dyes in cultural heritage, cosmetics and the food industry. Therefore, the study of their properties upon photoexcitation is particularly important. In this work, the photophysical and photochemical properties of CA, carminic lake and other related pigments in aqueous solutions are revisited. Novel quantitative information regarding the fate of the photoexcited states is provided including the efficiency of reactive oxygen species (ROS) photosensitized production (i.e., singlet oxygen and hydrogen peroxide) as well as the efficiency of nonradiative deactivation pathways. Laser-induced optoacoustic spectroscopy (LIOAS) data revealed that for all the investigated compounds, almost all the absorbed energy is released as prompt heat to the media. This is in agreement with the fact that other deactivation pathways, including fluorescence (ΦF ∼ 10-3-10-5), photochemical degradation (ΦR ∼ 10-4) and/or photosensitized ROS formation (ΦH2O2 < 10-5 and ΦΔ âˆ¼ 0), are negligible or null. In addition, a comprehensive investigation of the photodegradation of CA and lake is herein reported. The influence of different experimental parameters such as irradiation wavelength and oxygen partial pressure was evaluated. UV-vis absorption and fluorescence emission spectroscopy in combination with chemometric data analysis were used to elucidate the relevant aspects of the photodegradation mechanism involved and the spectroscopic features of the photoproducts generated. In aqueous media, CA follows an O2-dependent photochemical degradation when subject to elapsed photoexcitation in the UVB, UVA and visible regions. The photoproduct profile depends on the excitation wavelength giving rise to quite distinctive spectroscopic profiles. With respect to lake, our data suggest that upon photoexcitation, this pigment releases a CA-like chromophore that follows a similar fate to CA.

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.

A multi-analytical approach for the characterization of modern white paints used for Argentine concrete art paintings during 1940-1960.

Modern art has challenged many aspects of the analytical approaches that are typically used for traditional paint characterization and ageing studies. The paint industry has changed significantly throughout the twentieth century, frequently altering its manufacturing techniques in order to achieve paints with improved appearance, application and performance for a range of diverse household, industrial and artistic uses. This has led to the appearance and use of a multitude of new binding media, pigments and additives, most of which require new analytical methods for their identification. Concrete art is the name given to a significant art movement that took place in Argentina (and other nearby countries) during the 1940s and 1950s, at the exact same time as a flourishing paint industry was utilizing many of these new products and diversifying formulas. This paper reports on some initial findings from a long-term study to develop and apply analytical methodologies on paint samples from a number of Concrete artworks, that will help to better understand art history and advance the conservation field by shedding light on these artist's painting techniques, and the ageing behavior of their materials. Specifically, samples of white paints manufactured by local paint companies in Argentina from that time period were purchased and studied with a multi-analytical approach, which will serve as a reference collection for the field. The analytical techniques used were X-ray diffraction (XRD), scanning electron microscopy with energy dispersive X-ray analysis (SEM-EDX) and gas chromatography coupled with mass spectrometry (GC/MS) with previous derivatization. Artworks samples were studied with a similar multi-analytical approach, and utilized micro-Raman spectroscopy instead of XRD, for its non-destructive application. A wide range of possible compounds was identified due to the complementarity use of analytical techniques, representing a significant first step in Argentinian modern art research.

Improving separation optimization in capillary electrophoresis by using a general quality criterion.

In this paper we extend the use of the quality criterion t' to optimize separations in capillary electrophoresis (CE). The theoretical parameter t' takes into account not only the relative separation between a given pair of compounds but also their separation from the neutral species migrating with the electroosmotic flow (EOF). Furthermore, it can be composed for complex mixtures as a global multicriterium optimization function T', for a rapid, simple and reliable selection of optimized separation conditions by mathematical maximization. Here, we demonstrate the applicability of T' using as a variable the electrophoretic mobility (me) for the optimization of pH in the separation of a mixture of amyloid beta (Aß) peptide fragments. In addition, it is shown the versatility of T' using other variables related to me, which do not require experimental measurements. This is the case with ionizable compounds as the Aß peptide fragments, whose charge-to-mass ratios can be calculated if accurate pKa values are available in the literature. The excellent performance of T' for Aß peptide fragments is further validated optimizing the pH for the separation of mixtures of harmala alkaloids (HAlks) and quinolone antibiotics.

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.

Measurement of Free Drug Concentration from Biological Tissue by Solid-Phase Microextraction: In Silico and Experimental Study.

In this article, the use of an SPME technique is reported for the first time for direct measurement of free drug concentration in solid tissue. In our investigations, we considered doxorubicin (DOX) spiked in homogenized tissue matrix at transient and equilibrium extraction conditions, with subsequent assessment of obtained experimental results by an in silico approach using mathematical models developed in COMSOL Multyphysics. In silico studies were performed on the basis of transported diluted species (tds) and reaction engineering (re) modules from COMSOL Multiphysics, using the same conditions as those used to attain experimental results. To determine the apparent binding affinity of DOX to the tissue matrix which contains multiple binding species, the experimentally determined binding affinity of DOX with human serum albumin (HSA) was considered to simplify the mathematical calculations. Here, the value of the binding affinity was considered for a single binding site and adjusted by fitting the experimental results with the mathematical model. Bovine lung tissue homogenate was selected as a surrogate matrix, and a biocompatible C-8 commercial SPME fiber was used for extraction of DOX. In total, four mathematical models were herein developed to describe the mass transfer kinetics of solid coatings: in agar gel at static conditions, in PBS solution with agitated conditions, extraction in PBS solution in the presence of an HSA binding matrix, and static extraction in homogenized lung tissue. For all conditions, simulated results were in good agreement with experimental results. The developed mathematical model allows for measurements of free drug concentrations inside the tissue matrix and facilitates calculations of local depletion of DOX by a solid SPME coating. Results of the investigations indicate that local depletion of the free form of DOX, even at the kinetic stage, is negligible for tissue extraction, as the release of the heavily bound analyte (over 99% binding to tissue matrix) is very rapid, thus easily compensating for the loss of the drug to the SPME coating. This indicates that the dissociation rate constant of DOX from lung tissue components is very rapid; therefore, the mass transfer of drug to the fiber coating via free from is very efficient. Our results also indicate that thin coating SPME fibers provide a good way to measure drug distribution after dosing, as extractions via thin coating SPME fibers do not affect the free concentration of the drug, which is responsible for drug distribution in tissue.

Solid phase microextraction coupled to mass spectrometry via a microfluidic open interface for rapid therapeutic drug monitoring.

Tranexamic acid (TXA) is an antifibrinolytic used during cardiac surgery that presents high inter-patient variability. High plasma concentrations have been associated with post-operative seizures. Due to the difficulties with maintaining acceptable concentrations of TXA during surgery, implementation of a point-of-care strategy for testing TXA plasma concentration would allow for close monitoring of its concentration during administration. This would facilitate timely corrections to the dosing schedule, and in effect tailor treatment for individual patient needs. In this work, a method for the rapid monitoring of TXA from plasma samples was subsequently carried out via biocompatible solid-phase microextraction (Bio-SPME) coupled directly to tandem mass spectrometry via a microfluidic open interface (MOI). MOI operates under the concept of a flow-isolated desorption volume and was designed with aims to directly hyphenate Bio-SPME to different detection and ionization systems. In addition, it allows the desorption of Bio-SPME fibers in small volumes while it concurrently continues feeding the ESI with a constant flow to minimize cross-talking and instabilities. The methodology was used to monitor six patients with varying degrees of renal dysfunction, at different time points during cardiac surgery. MOI proves to be a reliable and feasible tool for rapid therapeutic drug monitoring. Affording total times of analysis as low as 30 seconds per sample in its high throughput mode configuration while the single sample turn-around time was 15 minutes, including sample preparation. In addition, cross-validation against a standard thin film solid phase microextraction using liquid chromatography coupled to tandem mass spectrometry (TFME-LC-MS/MS) method was performed. Bland-Altman analysis was used to cross-validate the results obtained by the two methods. Data analysis demonstrated that 92% of the compared data pairs (n = 63) were distributed within the acceptable range. The data was also validated by the Passing Bablok regression, demonstrating good statistical agreement between these two methods. Finally, the currently presented method offers comparable results to the conventional liquid chromatography with acceptable RSDs, while only necessitating a fraction of the time. In this way, TXA concentration in plasma can be monitored in a close to real time throughput during surgery.

Direct Coupling of Dispersive Extractions with Magnetic Particles to Mass Spectrometry via Microfluidic Open Interface.

Microextraction coupled to mass spectrometry (MS) has great potential in analytical chemistry laboratories operating in a variety of fields. Indeed, microextraction methods directly coupled to MS can be of large value given that they can provide not only analyte extraction and enrichment but also effective sample cleanup. In recent years, the practicality in handling high active surface area, versatility, and environmentally friendly features of magnetic dispersive microextraction technologies has contributed to an explosion in the number of methods and technologies reported in the literature for a wide range of applications. However, to the best of our knowledge, no technology to date has been capable of efficiently merging these two rising concepts in a simple and integrated analytical workflow. In this context, the microfluidic open interface is presented for the direct coupling of dispersive magnetic extraction to mass spectrometry. This technology operates under the concept of a flow-isolated desorption volume, which generates a stagnant droplet open to ambient conditions while continuously feeding the ionization source with solvent by means of the self-aspiration process intrinsic of the electrospray ionization (ESI) interface. To improve the efficiency of the final analytical workflow, a novel dispersive magnetic micro- and nanoparticle extraction protocol for biofluid droplets was developed. The final methodology entailed the dispersion of a small amount of magnetic particles (20-70 µg) in a droplet of biofluid (≤40 µL) for extraction, followed by a particle collection step using a homemade 3D-printed holder containing an embedded rare-earth magnet. In the final step, the holder is set on top of the microfluidic open interface (MOI) for desorption in the isolated droplet. Switching the valve transfers the desorbed analytes to the ESI source in less than 5 s. As proof of concept, the completely new setup was applied to the determination of prohibited substances from phosphate-buffered saline (PBS) and human urine using Fe2O3 magnetic nanoparticles (50 nm) functionalized with C18. The limits of quantitation (LOQs) obtained were in the low-ppb range in all cases, and acceptable precision (≤20%) and accuracy (80-120%) were attained. Also, taking advantage of the fast extraction kinetics provided by the radial diffusion associated with small particles, we employed the methodology for the selective extraction of phosphopeptides from 40 µL of tryptic ß-casein digest using 70 µg of magnetic Ti-IMAC microparticles. To conclude, the technology and methodology herein presented provided excellent capabilities comparable to those of other solid-phase microextraction (SPME-MS) approaches while dramatically minimizing the amount of sample and sorbent required per analysis, as well as affording significantly fast extraction times due to the enhanced kinetics of extraction.

Effect of Transport Parameters and Device Geometry on Extraction Kinetics and Efficiency in Direct Immersion Solid-phase Microextraction.

An alternative strategy to increase mass transfer entails geometry optimization of the extraction systems including design of solid-phase microextraction (SPME) probes. In this work, a computational model was employed to elucidate practical aspects such as efficiency and kinetics of extraction by employing several new geometries. Extraction of a model analyte at static conditions with the configurations, such as thin-film, fiber, coated tip, and nanoparticles, was numerically simulated to obtain an in-depth understanding of the advantages and limitations of each geometry in microextraction and exhaustive modes. The attained results associated with the equilibration time dependency on shape were in good agreement with previously reported experimental observations. They demonstrate that the mass-transfer is highly dependent on the size and shape of the coatings and increases with a decrease in size of the devices particularly rapidly below 10 µm caused by radial diffusion effect. Nevertheless, extractions performed using octadecyl-functionalized magnetic nanoparticles demonstrated that higher enrichment factors are achievable with the use of a fewer number of particles in comparison to factors achieved via exhaustive extraction, where a larger number of particles must be employed, confirming theoretical predictions. The conclusions reached are valid for any extraction method. The results obtained herein are very useful toward the design and optimization of future extraction technologies and approaches.

Equilibrium ex vivo calibration of homogenized tissue for in vivo SPME quantitation of doxorubicin in lung tissue.

The fast and sensitive determination of concentrations of anticancer drugs in specific organs can improve the efficacy of chemotherapy and minimize its adverse effects. In this paper, ex vivo solid-phase microextraction (SPME) coupled to LC-MS/MS as a method for rapidly quantitating doxorubicin (DOX) in lung tissue was optimized. Furthermore, the theoretical and practical challenges related to the real-time monitoring of DOX levels in the lung tissue of a living organism (in vivo SPME) are presented. In addition, several parameters for ex vivo/in vivo SPME studies, such as extraction efficiency of autoclaved fibers, intact/homogenized tissue differences, critical tissue amount, and the absence of an internal standard are thoroughly examined. To both accurately quantify DOX in solid tissue and minimize the error related to the lack of an internal standard, a calibration method at equilibrium conditions was chosen. In optimized ex vivo SPME conditions, the targeted compound was extracted by directly introducing a 15 mm (45 µm thickness) mixed-mode fiber into 15 g of homogenized tissue for 20 min, followed by a desorption step in an optimal solvent mixture. The detection limit for DOX was 2.5 µg g-1 of tissue. The optimized ex vivo SPME method was successfully applied for the analysis of DOX in real pig lung biopsies, providing an averaged accuracy and precision of 103.2% and 12.3%, respectively. Additionally, a comparison between SPME and solid-liquid extraction revealed good agreement. The results presented herein demonstrate that the developed SPME method radically simplifies the sample preparation step and eliminates the need for tissue biopsies. These results suggest that SPME can accurately quantify DOX in different tissue compartments and can be potentially useful for monitoring and adjusting drug dosages during chemotherapy in order to achieve effective and safe concentrations of doxorubicin.

Development of a Microfluidic Open Interface with Flow Isolated Desorption Volume for the Direct Coupling of SPME Devices to Mass Spectrometry.

Technologies that efficiently integrate the sampling and sample preparation steps with direct introduction to mass spectrometry (MS), providing simple and sensitive analytical workflows as well as capabilities for automation, can generate a great impact in a vast variety of fields, such as in clinical, environmental, and food-science applications. In this study, a novel approach that facilitates direct coupling of Bio-SPME devices to MS using a microfluidic design is presented. This technology, named microfluidic open interface (MOI), which operates under the concept of flow-isolated desorption volume, consists of an open-to-ambient desorption chamber (V ≤ 7 µL) connected to an ionization source. Subsequently, compounds of interest are transported to the ionization source by means of the self-aspiration process intrinsic of these interfaces. Thus, any ionization technology that provides a reliable and constant suction, such as electrospray ionization (ESI), atmospheric-pressure chemical ionization (APCI), or inductively coupled plasma ionization (ICP), can be hyphenated to MOI. Using this setup, the desorption chamber is used to release target compounds from the coating, while the isolation of the flow enables the ionization source to be continuously fed with solvent, all without the necessity of employment of additional valves. As a proof of concept, the design was applied to an ESI-MS/MS system for experimental validation. Furthermore, numerical simulations were undertaken to provide a detailed understanding of the fluid flow pattern inside the interface, then used to optimize the system for better efficiency. The analytical workflow of the developed Bio-SPME-MOI-MS setup consists of the direct immersion of SPME fibers into the matrix to extract/enrich analytes of interest within a short period of time, followed by a rinsing step with water to remove potentially adhering proteins, salts, and/or other interfering compounds. Next, the fiber is inserted into the MOI for desorption of compounds of interest. Finally, the volume contained in the chamber is drained and moved toward the electrospray needle for ionization and direct introduction to MS. Aiming to validate the technology, the fast determination of selected immunosuppressive drugs (e.g., tacrolimus, cyclosporine, sirolimus, and everolimus) from 100 µL of whole blood was assessed. Limits of quantitation in the subppb range were obtained for all studied compounds. Good linearity (r2 ≥ 0.99) and excellent precision, with (8%) and without (14%) internal standard correction, were attained.

Coated blade spray: shifting the paradigm of direct sample introduction to MS.

Coated blade spray (CBS) is a solid-phase microextraction-based technology that can be directly coupled to MS to enable the rapid qualitative and quantitative analysis of complex matrices. The goal of this mini review is to concisely introduce CBS's operational fundamentals and to consider how it correlates/contrasts with existing direct-to-MS technologies suitable for bioanalytical applications. In addition, we provide a fair comparison of CBS to other existing solid-phase microextraction-to-MS approaches, as well as an overview of recent CBS applications/strategies that have been developed to analyze diverse compounds present in biofluids.

Rapid determination of immunosuppressive drug concentrations in whole blood by coated blade spray-tandem mass spectrometry (CBS-MS/MS).

Coated Blade Spray (CBS) is a technology that efficiently integrates sample preparation and direct coupling to mass spectrometry (MS) on a single device. In this article, we present CBS-tandem mass spectrometry (CBS-MS/MS) as a novel tool for the rapid and simultaneous determination of four commonly used immunosuppressive drugs (ISDs) in whole blood: tacrolimus (TAC) and cyclosporine-A (CycA), which are calcineurin inhibitors; and sirolimus (SIR) and everolimus (EVR), which are both mTOR (mechanistic target of rapamycin) inhibitors. Given that CBS extracts via free concentration, analytes that are largely bound to plasma proteins or red blood cells provide considerably lower extraction recovery rates. Therefore, we defy the solventless philosophy of SPME-based techniques, like CBS, by performing the analyte-enrichment step via direct immersion in a solvent-modified matrix. The assay was linear within the evaluated range of concentrations (between 1 and 100 ng/mL for EVR/SIR/TAC and 10-1000 ng/mL for CycA), and the limits of quantification were determined to be 10 ng/mL for CycA and 1 ng/mL for EVR/SIR/TAC. Good accuracy (87-119%) and linearity (r2 ≥ 0.99) were attained over the evaluated range for all ISDs. Interassay imprecision (CV) determined from incurred sample reanalysis was ≤10% for all ISDs. Our method was validated using Liquichek™ whole blood immunosuppressant quality control (QC) standards purchased from Bio-Rad. Concentrations determined by CBS-MS/MS were inside the range specified by Bio-Rad and within 15% of the expected mean value for all ISDs at all QC levels. Furthermore, the effect of different hematocrit levels (20, 45, and 70%) in the entire calibration range was carefully studied. No statistical differences (RSD ≤ 7%) in the calibration curve slopes of ISDs in blood were observed. CBS offers a simpler workflow than that of traditional methods; it eliminates the need for chromatographic separation and provides a clean extract that allows for long-term MS instrumental operation with minimal maintenance. Additionally, because CBS integrates all analytical steps into one device, it eliminates the risk of instrumental carry-over and can be used as a low-cost disposable device for sample preparation and analysis. Fully-automated sample preparation simplifies the method and allows for total analysis times as short as 3 min with turn-around times of less than 90 min.

Quantitative analysis of biofluid spots by coated blade spray mass spectrometry, a new approach to rapid screening.

This study demonstrates the quantitative capabilities of coated blade spray (CBS) mass spectrometry (MS) for the concomitant analysis of multiple target substances in biofluid spots. In CBS-MS the analytes present in a given sample are first isolated and enriched in the thin coating of the CBS device. After a quick rinsing of the blade surface, as to remove remaining matrix, the analytes are quickly desorbed with the help of a solvent and then directly electrosprayed into the MS analyzer. Diverse pain management drugs, controlled substances, and therapeutic medications were successfully determined using only 10 µL of biofluid, with limits of quantitation in the low/sub ng·mL-1 level attained within 7 minutes.

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, ß-2 agonists, diuretics, stimulants, narcotics, and ß-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 CBS's 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.

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

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 6µg/mL and limited efficacy at concentrations below 2µg/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 (1min) of the target analyte from 300µL of human plasma using conventional laboratory vessels (e.g. vial, 96-well plate). Alternatively, the second strategy consists of a 2min extraction from a plasma droplet (10µL) 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 (0.1-50µg/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.