Coupling Miniaturized Stir Bar Sorptive Dispersive Microextraction to Needle-Based Electrospray Ionization Emitters for Mass Spectrometry: Determination of Tetrahydrocannabinol in Human Saliva as a Proof of Concept.

Direct coupling of sample preparation with mass spectrometry (MS) can speed up analysis, enabling faster decision-making. In such combinations, where the analysis time is mainly defined by the extraction procedure, magnetic dispersive solid-phase extraction emerges as a relevant technique because of its rapid workflow. The dispersion and retrieval of the magnetic sorbent are typically uncoupled stages, thus reducing the potential simplicity. Stir bar sorptive dispersive microextraction (SBSDME) is a novel technique that integrates both stages into a single device. Its miniaturization (mSBSDME) makes it more portable and compatible with low-availability samples. This article reports the direct combination of mSBSDME and MS using a needle-based electrospray ionization (NESI) emitter as the interface. This combination is applied to determine tetrahydrocannabinol in saliva samples, a relevant societal problem if the global consumption rates of cannabis are considered. The coupling requires only the transference of the magnet (containing the sorbent and the isolated analyte) from the mSBSDME to the hub of a hypodermic needle, where the online elution occurs. The application of 5 kV on the needle forms an electrospray on its tip, transferring the ionized analyte to the MS inlet. The excellent performance of mSBSDME-NESI-MS/MS relies on the sensitivity (limits of detection as low as 2.25 ng mL-1), the precision (relative standard deviation lower than 15%), and the accuracy (relative recoveries ranged from 87 to 127%) obtained. According to the results, the mSBSDME-NESI-MS/MS technique promises faster and more efficient chemical analysis in MS-based applications.

Sustainable beeswax modified cellulose paper for the determination of tricyclic antidepressants in biofluids.

This article describes the synthesis of sorptive phases for bioanalysis based on the modification of cellulose paper with natural beeswax as sorbent, resulting in a substrate completely renewable and sustainable. The preparation of the sorptive phases consisted of the dissolution of beeswax in hexane, followed by its drop-casting on cellulose paper and subsequent evaporation of the solvent. The beeswax modification of paper renders it hydrophobic, enabling the extraction of the target analytes, i.e., imipramine, desipramine, amitriptyline and trimipramine, via hydrophobic interactions. The main variables affecting the extraction performance were investigated (e.g., pH, ionic strength, extraction time, eluent composition, agitation speed). The analytical workflow combines a straightforward sampling, simultaneous extraction of 30 samples in 1 h, and the rapid (<2 min) determination of the analytes via direct infusion mass spectrometry. The method provided limits of detection in the range 2.0 and 3.2 µg L-1, and the precision, expressed as relative standard deviation, was better than 5.4 % and 8.5 % for intra and inter-day analyses, respectively. The accuracy, in terms of relative recovery, ranged from 90 % to 121 % using saliva as model biofluid.

Bioinspired composite packed in blunt needles, integrated microextraction and determination of oxycodone and naloxone in saliva by substrate spray mass spectrometry.

BACKGROUND: Opioids are effective painkillers used for medical purposes. Their prolonged ingestion can provoke some side effects (including overdose or constipation) that are minimized by using opioid antagonists (e.g., naloxone). The rapid determination of opioids and their antagonists in biosamples is essential for an effective medical treatment. The direct combination of sample preparation and mass spectrometry (MS) fits well in this scenario. It can speed up the analysis achieving a good selectivity, which relies on the sample preparation and MS, and sensitivity levels. RESULTS: This article presents a novel substrate-spray mass spectrometry interface based on a polydopamine-cotton (PDA-Cel) composite hosted inside the inner diameter of a 14-gauge blunt needle to determine oxycodone and naloxone in saliva samples. The needle is used as a microextraction device and a substrate for mass spectrometric analysis. The lack of sharpness of the 14-gauge (14G) blunt needles challenges the formation of the electrospray (ESI), and a commercial 10 µL pipette tip is proposed as a simple solution to this shortcoming. Under the optimum parameters, the proposed method was validated, obtaining limits of detection lower than 0.6 µg L-1, linear range up to 200 µg L-1, and linearity better than 0.9915. Relative standard deviation (RSD) and relative recoveries (RR) were studied at three different concentration levels (2, 40, and 200 µg L-1). RSD values were better than 20.7 %, and RR ranged from 90 to 114 %. Finally, a positive sample from a patient under medical treatment was analyzed. SIGNIFICANCE AND NOVELTY: 14G blunt needles have been demonstrated as effective extraction devices due to their low price (<0.15 € per extraction unit), their better safety (avoiding finger pricking), and their higher hosting capacity (up to 8 mg of sorbent). The conductivity of stainless steel permits their use as electrospray emitters, making their direct combination to MS easier. The large variety of fibrous sorbents makes this approach versatile enough to be adapted to other analytical problems.

Septic arthritis caused by Candida dubliniensis following arthroscopic surgery.

A 37-year-old immunocompetent man was admitted to the emergency department due to recurrent pain and oedema of his right knee. Two months earlier, he had undergone surgery to repair his meniscus. Arthroscopic joint lavage was performed and Candida dubliniensis was recovered in culture. The authors describe the first case of septic arthritis caused by Candida dubliniensis.

Casein kinase 1 controls components of a TORC2 signaling network in budding yeast.

Tor kinases play diverse and essential roles in control of nutrient signaling and cell growth. Tor kinases are assembled into two large multiprotein complexes referred to as Tor Complex 1 and Tor Complex 2 (TORC1 and TORC2). In budding yeast, TORC2 controls a signaling network that relays signals regarding carbon source that strongly influence growth rate and cell size. However, the mechanisms that control TORC2 signaling are poorly understood. Activation of TORC2 requires Mss4, a phosphoinositol kinase that initiates assembly of a multi-protein complex at the plasma membrane that recruits and activates downstream targets of TORC2. Localization of Mss4 to the plasma membrane is controlled by phosphorylation and previous work suggested that yeast homologs of casein kinase 1γ, referred to as Yck1 and Yck2, control phosphorylation of Mss4. Here, we generated a new analog-sensitive allele of YCK2 and used it to test whether Yck1/2 influence signaling in the TORC2 network. We found that multiple components of the TORC2 network are strongly influenced by Yck1/2 signaling.

Sorptive tape-spray tandem mass spectrometry using aluminum foil coated with mixed-mode microparticles.

Direct mass spectrometry (MS) is an exciting strategy in bioanalysis, enabling rapid decision-making in different scenarios. Its application is usually hindered by matrix effects and the typically low concentration of the target compounds in the biofluids. In this sense, combining a previous sample preparation step minimizes or removes these shortcomings. This article describes sorptive tape-spray tandem mass spectrometry (STS-MS/MS) based on mixed-mode particles as a strategy to combine sample preparation and MS analysis in a single device. The technique uses a sorptive tape (ST) consisting of mixed-mode polymeric microparticles (combining ionic exchange and hydrophobic interactions) coated over aluminum foil in a spatial controlled way. The tapes act as the sorptive phases to isolate the analytes from the sample matrix and substrates for STS-MS/MS. The performance of the technique has been evaluated by developing a method to determine codeine in saliva as proof of concept. The affordability of the STs elements allows the preparation of many individual phases at low cost so that several samples can be extracted simultaneously, thus increasing the sample throughput. The extraction variables were optimized following a multivariate approach. Working under the optimum conditions, the limit of detection was 0.3 µg L-1, while the intraday precision, calculated as relative standard deviation (RSD) at three concentration levels, was better than 9.4 %. The accuracy, expressed as relative recovery, was in the range of 78-98 %. The method was also applied to the analysis of real samples. Despite being a powerful strategy, the direct combination of microextraction to MS is not always affordable in all laboratories. For this reason, the STs were also combined with commercial liquid chromatography-MS working under the direct infusion mode to demonstrate the usefulness of the ST in classical extraction workflows.

Volumetric absorptive microsampling for the therapeutic drug monitoring of psychiatric patients treated with cariprazine.

Psychiatric disorders are usually treated with antipsychotic agents belonging to different pharmacological and chemical classes, the most recent ones collectively known as "third-generation antipsychotics", such as cariprazine, approved in 2015 for the treatment of patients affected by schizophrenia. For these patients, a frequent therapeutic drug monitoring (TDM) becomes essential to assess compliance and to optimise and personalise their therapy, also due to cariprazine interindividual variability and narrow therapeutic range. In this study, a bioanalytical method featuring miniaturised sampling and pretreatment was developed, based on volumetric absorptive microsampling (VAMS) for TDM of psychiatric patients under cariprazine treatment and compared to a reference method based on fluid plasma analysis. Minimally invasive whole blood VAMS was coupled to an original instrumental method based on ultra-high performance liquid chromatography hyphenated to mass spectrometry (UHPLC-MS). A feasible and streamlined, yet reliable VAMS pretreatment protocol was carefully optimised and the VAMS-UHPLC-MS methodology was validated with satisfactory results in terms of linearity (r2 > 0.9970 in the 1.5-100 ng/mL range), precision (%RSD < 11.7), extraction yield (> 90.0 %) and matrix effect (8.2 ≤ %RE ≤ 10.9). Finally, the microsampling approach coupled to UHPLC-MS was successfully applied to the TDM of psychiatric patients treated with cariprazine and compared with standard fluid plasma analysis, providing reliable quali-quantitative results, and proving to be readily applicable to the clinical practice in TDM programs as a useful alternative to cariprazine plasma analysis. This is the first report of a successful microsampling application, and in particular the first report of VAMS application, for the TDM of cariprazine.

Mixed-mode cationic exchange sorptive tapes combined with direct infusion mass spectrometry for determining opioids in saliva samples.

This article describes the synthesis of mixed-mode cationic exchange (MCX) tapes as sorptive phases in bioanalysis, and it faces the determination of methadone and tramadol in saliva as the model analytical problem. The tapes are synthesized using aluminum foil as substrate, which is subsequently covered with double-sided adhesive tape where the MCX particles (ca. 1.4 ± 0.2 mg) finally adhere. MCX particles allow the extraction of the analytes at the physiological pH, where both drugs are positively charged, minimizing the potential co-extraction of endogenous matrix compounds. The extraction conditions were studied considering the main variables (e.g. ionic strength, extraction time, sample dilution). Under the optimum conditions and using direct infusion mass spectrometry as the instrumental technique, detection limits as low as 3.3 µg·L-1 were obtained. The precision calculated at three different levels, and expressed as relative standard deviation, was better than 3.8%. The accuracy, expressed as relative recoveries, ranged from 83 to 113%. The method was finally applied to determine tramadol in saliva samples from patients under medical treatment. This approach opens the door to easily preparing sorptive tapes based on commercial (or ad-hoc synthesized) sorbent particles.

Impact of sphingolipids on protein membrane trafficking.

Membrane trafficking is essential to maintain the spatiotemporal control of protein and lipid distribution within membrane systems of eukaryotic cells. To achieve their functional destination proteins are sorted and transported into lipid carriers that construct the secretory and endocytic pathways. It is an emerging theme that lipid diversity might exist in part to ensure the homeostasis of these pathways. Sphingolipids, a chemical diverse type of lipids with special physicochemical characteristics have been implicated in the selective transport of proteins. In this review, we will discuss current knowledge about how sphingolipids modulate protein trafficking through the endomembrane systems to guarantee that proteins reach their functional destination and the proposed underlying mechanisms.

Deep eutectic solvent coated paper: Sustainable sorptive phase for sample preparation.

Paper-based sorptive phases have gained attention recently due to the low-cost and sustainable character of the cellulosic substrate. However, the sustainability of the resulting phase can be limited by type of coating used for analytes isolation. In this article, this limitation is overcome by using deep eutectic solvents (DES) as coating. To this aim, a Thymol-Vanillin DES is synthesized and deposited on pre-cut cellulose paper strips. The paper-supported DES is employed as sorptive phase for the isolation of selected triazine herbicides for environmental waters analysis. The isolated analytes are finally determined by gas chromatography-mass spectrometry using selected ion monitoring. The method is optimized according to the critical variables that potentially affect its analytical performance such as sample volume, extractant amount, extraction time and sample ionic strength. The method was characterized in terms of sensitivity, accuracy and precision and its applicability was evaluated for the analysis of real environmental water samples. Good linearity values (R2>0.995) were obtained for all the analytes. Limits of detection (LODs) ranged from 0.4 to 0.6 µg L-1 and the precision, expressed as relative standard deviation (RSD) was better than 14.7%. The relative recoveries, calculated in spiked well and river samples, were in the range 90-106%.

Nylon 6-cellulose composite hosted in a hypodermic needle: Biofluid extraction and analysis by ambient mass spectrometry in a single device.

This study proposes a hypodermic needle (HN) as a sorbent holder and an electrospray (ESI) emitter, thus combining extraction and analysis in a single device. A novel nylon 6-cellulose (N6-Cel) composite sorbent is proposed to extract methadone from oral fluid samples. The cellulosic substrate provides the composite with high porosity, permitting the flow-through of the sample, while the polyamide contributes to the extraction of the analyte. The low price of the devices (considering the holder and the sorbent) contributes to the affordability of the method, and their small size allows easy transportation, opening the door to on-site extractions. Under the optimum conditions, the analyte can be determined by high-resolution ambient ionization mass spectrometry at a limit of detection (LOD) as low as 0.3 µg/L and precision (expressed as relative standard deviation, RSD) better than 9.3%. The trueness, expressed as relative recovery (RR), ranged from 90% to 109%. As high-resolution mass spectrometers are not available in many laboratories, the method was also adapted to low-resolution spectrometers. In this sense, the direct infusion of the eluates in a triple quadrupole-mass spectrometry provided an LOD of 2.2 µg/L. The RSD was better than 5.3%, and the RR ranged from 96% to 121%.

Comparative Study with and without the Use of 3D Prototyping of an Unconventional Technique in the Surgical Planning of Revision of Total Hip Arthroplasty.

This article presents a comparison between two cases in which there was a need for revision of total hip arthroplasty due to aseptic acetabular failure. We used 3D prototyping in one of the cases to perform an unconventional technique of molding synthesis material before the procedure to evaluate the time saved in the transoperative period in complex cases.

Comparative Study with and without the Use of 3D Prototyping of an Unconventional Technique in the Surgical Planning of Revision of Total Hip Arthroplasty / Estudo comparativo com e sem o uso de prototipagem em 3D de uma técnica não convencional no planejamento cirúrgico de revisão de artroplastia total de quadril

Abstract This article presents a comparison between two cases in which there was a need for revision of total hip arthroplasty due to aseptic acetabular failure. We used 3D prototyping in one of the cases to perform an unconventional technique of molding synthesis material before the procedure to evaluate the time saved in the transoperative period in complex cases.

Resumo Este artigo apresenta uma comparação entre dois casos em que havia a necessidade de revisão de artroplastia total de quadril por falha acetabular asséptica. Utilizamos prototipagem em 3D em um dos casos para realizar uma técnica não convencional de moldagem de material de síntese antes do procedimento, com objetivo de avaliar o tempo economizado no transoperatório em casos complexos.

Radiographic Evaluation of Postoperative Alignment in Total Knee Arthroplasty.

Objective To demonstrate the greater accuracy of panoramic radiographs of the lower limbs (long) in relation to short radiographs of the knee in the measurement of the mechanical axis of the lower limb after total knee arthroplasty (TKA). Methods A retrospective study was conducted to evaluate the accuracy of long and short postoperative radiographic images of 70 patients submitted to TKA in our service. The images were analyzed at random, at different times, by three orthopedists. In all images, the mechanical axis of the limb, femur and tibia were traced and femorotibial angles (FTAs) were calculated. The intraclass correlation coefficient (ICC) was calculated to evaluate the agreement of the measurement of the inter- and intra-observer mechanical axis. Results It was observed that there was high intra and interobserver agreement when panoramic radiographs were used, with minimum intra and interobserver ICC of 0.89, equivalent to a very strong agreement. On short radiographs in the anteroposterior incidence (AP) of the knee, the ICC showed moderate agreement, obtaining a maximum value of 0.75. Conclusion There is a significant difference in accuracy for the measurement of the mechanical axis of the lower limb, comparing long and short radiographs of the lower limb. Thus, for the proper measurement of the mechanical axis of the lower limb, we suggest the performance of long radiography in the postoperative period of TKA.

Radiographic Evaluation of Postoperative Alignment in Total Knee Arthroplasty / Avaliação radiográfica do alinhamento pós-operatório na artroplastia total de joelho

Abstract Objective To demonstrate the greater accuracy of panoramic radiographs of the lower limbs (long) in relation to short radiographs of the knee in the measurement of the mechanical axis of the lower limb after total knee arthroplasty (TKA). Methods A retrospective study was conducted to evaluate the accuracy of long and short postoperative radiographic images of 70 patients submitted to TKA in our service. The images were analyzed at random, at different times, by three orthopedists. In all images, the mechanical axis of the limb, femur and tibia were traced and femorotibial angles (FTAs) were calculated. The intraclass correlation coefficient (ICC) was calculated to evaluate the agreement of the measurement of the inter- and intra-observer mechanical axis. Results It was observed that there was high intra and interobserver agreement when panoramic radiographs were used, with minimum intra and interobserver ICC of 0.89, equivalent to a very strong agreement. On short radiographs in the anteroposterior incidence (AP) of the knee, the ICC showed moderate agreement, obtaining a maximum value of 0.75. Conclusion There is a significant difference in accuracy for the measurement of the mechanical axis of the lower limb, comparing long and short radiographs of the lower limb. Thus, for the proper measurement of the mechanical axis of the lower limb, we suggest the performance of long radiography in the postoperative period of TKA.

Resumo Objetivo Demonstrar a maior acurácia das radiografias panorâmicas de membros inferiores (longas) em relação às radiografias curtas do joelho na medida do eixo mecânico do membro inferior após a artroplastia total de joelho (ATJ). Métodos Foi realizado um estudo retrospectivo para avaliar a acurácia de imagens radiográficas longas e curtas pós-operatórias de 70 pacientes submetidos à ATJ em nosso serviço. As imagens foram analisadas ao acaso, em momentos distintos, por três ortopedistas. Em todas as imagens, o eixo mecânico do membro, do fêmur e da tíbia foram traçado,s e os ângulos femorotibiais (AFTs) foram calculados. O coeficiente de correlação intraclasse (CCI) foi calculado para avaliar a concordância da medida do eixo mecânico inter e intraobservador. Resultados Observou-se que houve alta concordância intra e interobservador quando utilizamos radiografias panorâmicas, apresentando CCI mínimo intrae interobservador de 0,89, equivalente a uma concordância fortíssima. Já nas radiografias curtas na incidência anteroposterior (AP) do joelho, o CCI mostrou-se com concordância moderada, obtendo valor máximo de 0,75. Conclusão Existe uma diferença significativa na acurácia para a medida do eixo mecânico do membro inferior, comparando-se radiografias longas e curtas do membro inferior. Assim, para a adequada mensuração do eixo mecânico do membro inferior, sugerimos a realização de radiografia longa no pós-operatório de ATJ.

Polydopamine inner wall-coated hypodermic needle as microextraction device and electrospray emitter for the direct analysis of illicit drugs in oral fluid by ambient mass spectrometry.

In this article, polydopamine inner wall-coated hypodermic needles (PDA-HNs) are evaluated as both microextraction devices and electrospray ionization (ESI) emitters for determining selected illicit drugs (methamphetamine, cocaine, and methadone) in oral fluid samples. The PDA film, located in the inner wall of the needle, allows the extraction of the analytes at alkaline pH, where their hydrophobic character is promoted. The extracted analytes are finally eluted in a methanol/formic acid mixture that also acts as ESI solution. For this purpose, a dedicated interface based on the connection of a PEEK tube with the needle hub is proposed. This assembly allows delivering the ESI solution by the infusion syringe pump of the mass spectrometer, providing an efficient ESI on the tip of the needle. The double use of the PDA-HNs as microextraction devices and ESI emitters permits the determination of the target analytes with limits of detection and precision (expressed as relative standard deviation) values better than 2.4 µg/L and 17.6%, respectively. The accuracy was evaluated by analyzing independent spiked oral fluid samples, obtaining good results.

Potential of hydrophobic paper-based sorptive phase prepared by in-situ thermal imidization for the extraction of methadone from oral fluid samples.

Paper-based sorptive phases (PSPs) are functional planar materials with a demonstrated potential in analytical sample preparation. This article describes the synthesis of a polyimide coated paper by an in-situ imidization at a high temperature. Polyimides (PI) are synthesized in two subsequent steps where a hydrophilic polymer, in this case, poly(amic acid) (PAA), is formed as an intermediate product. PAA is finally transformed into hydrophobic PI by thermal curing at 180 °C. The synthesis of PI-paper takes advantage of this two-step procedure. In the first stage, a segment of filter paper is immersed into an aqueous PAA solution. After the solvent evaporation, the paper is heated at 180 °C for 1 h inducing the formation of the hydrophobic PI over the cellulose fibers. Infrared spectroscopy has been used to characterize the synthesized materials by defining a coverage factor F. The hydrophobicity of the materials has been studied using an aqueous methylene blue solution as a marker. To fully demonstrate the usefulness of the material in the sample preparation field, the extraction of methadone from oral fluid (OF) samples has been considered as a model analytical problem. The main variables affecting the synthesis (PAA concentration on the precursor solution and number of dips) and the extraction (elution and extraction times) have been fully evaluated. Working under the optimum conditions, a limit of quantification of 9 µg/L, intraday and interday precision better than 14.6%, and accuracy in the range of 87-108% were obtained.

Pre-cleaned bare wooden toothpicks for the determination of drugs in oral fluid by mass spectrometry.

This article deepens the potential of pre-cleaned bare wooden toothpicks (pb-WTs) for extracting drugs (antidepressants and acetaminophen) from oral fluid samples. The leaching of the intrinsic compounds from the wood matrix is identified as the main challenge for the final determination of the targets, even when a very selective instrumental technique, such as mass spectrometry, is employed. The pre-cleaning of the WTs is proposed for improving the analytical performance. The number of cleaning cycles depends on the injection mode (direct infusion or chromatography) into the mass spectrometer. The different variables affecting the extraction of selected antidepressant drugs were studied in detail, and the optimum procedure was validated using the two mentioned injection modes. The limits of detection were in the ranges 0.1-0.5 ng/mL and 0.1-0.3 ng/mL for direct infusion and liquid chromatography, respectively. The intra-day precision (expressed as relative standard deviation) was better than 12.1% and 8.6%, for direct infusion and liquid chromatography, respectively. Single-blind samples were used to study the applicability of the method. Finally, as a proof-of-concept, the potential of pb-WTs for in vivo sampling was outlined.