Minimize Matrix Effects Research Articles

Impedimetric immunosensor versus qPCR for Huanglongbing detection

Citriculture is facing challenges due to the spread of various diseases, among which the most threatening, with worldwide occurrence, is Huanglongbing (HLB), caused by the bacteria Candidatus Liberibacter spp., vectored by psyllids. In orchards, HLB-infected plants are identified by visual observation of symptoms. For laboratory diagnosis, the gold standard is the polymerase chain reaction (PCR), requiring expertise and specific equipment, and with high financial cost. In this work, a selective impedimetric immunosensor was developed for the detection and determination of Ca. L. asiaticus (CLas) in citrus samples. An anti-HLB antibody against the outer membrane protein (OMP) sequence of CLas was obtained and immobilized on previously synthesized and characterized magnetic nanoparticles. The immobilized antibody was presented to various citrus leaf sample extracts. After the affinity reaction with the antigen, a washing step was performed to minimize matrix effects. The affinity reaction was monitored by electrochemical impedance spectroscopy, using a glassy carbon working electrode containing a neodymium magnet. The developed device was able to distinguish HLB-positive samples from HLB-negative samples and those with other infections. The results obtained with the proposed methodology were in good agreement with quantitative PCR (qPCR).

Development and validation of an LC-MS/MS method for highly concentrated tacrolimus and cyclosporine samples prepared from pharmaceutical products to assess drug loss from feeding tubes

IntroductionTacrolimus and cyclosporine are common immunosuppressants utilized post-organ transplantation to manage allograft rejection. Both have narrow therapeutic indices and are frequently measured to support dose adjustments. Although nasogastric tubes are commonly used to provide nutritional support and serve as a route for immunosuppressant administration, they were never validated for such purposes. ObjectiveTo develop and validate a liquid chromatography – tandem mass spectrometry (LC-MS/MS) method for highly concentrated tacrolimus and cyclosporine samples prepared from pharmaceutical products to support the validation of feeding tube administration of these immunosuppressants. MethodsThe method involved stepwise dilutions with dimethyl sulfoxide before analysis using online sample preparation and LC-MS/MS. It was validated in a CLIA-certified clinical laboratory that measures immunosuppressants by LC-MS/MS and is designed to support clinical studies evaluating drug loss from feeding tubes. ResultsThe method was linear between 6.8 µg/mL and 75 µg/mL for tacrolimus, and between 0.9 mg/mL and 10 mg/mL for cyclosporine, with r2 > 0.99 and total precision <5 % at all QC levels. The method demonstrated good recovery using certified cyclosporine reference material, European pharmacopeia reference standard tacrolimus, and prepared pharmaceutical products. Minimal matrix effects were observed. ConclusionAn analytical method was developed and validated for in vitro studies with simulated administration of tacrolimus or cyclosporine to assess loss during drug administration using feeding tubes.

Development of an automated immunologic mass spectrometry (iMS) method to overcome matrix effect for quantification: Steroid hormones as the example

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) shows great promise in clinical application for its high specificity, high sensitivity and wide linear range for the determination of small molecules. However, its application in clinical laboratory is hampered by matrix effect of clinical samples which could greatly affect quantification accuracy and the difficulty to be automated for the traditional sample preparation procedures. Thus, new techniques which could achieve selective enrichment to minimize matrix effect and automatic sample preparation of mass spectrometry are needed. We developed an immunologic mass spectrometry (iMS) method to overcome matrix effect and its clinical application was demonstrated for automatic analysis of testosterone (T), progesterone (P) and estradiol (E2) in human serum simultaneously. Firstly, three monoclonal antibodies were coupled to magnetic beads for selective enrichment of target hormones from serum. The immunomagnetic beads were separated, washed and eluted automatically for LC-MS/MS analysis. Analytical performance of the iMS method was validated and compared with traditional LC-MS/MS and chemiluminescence immunoassay (CLIA). Hormone levels were measured for 160 pregnancy women at different gestational weeks. Results showed that target hormones could be selectively captured with absolute recoveries of 93.9%–110.8 %. Relative responses for high, medium and low concentrations of the hormones between serum and methanol solution were 98.0%–109.7 %, 92.2%–105.3 % and 91.7%–96.0 % for T, P and E2, respectively. Calibration curves prepared in methanol solution, BSA solution and blank serum showed good consistency for the iMS method. The automated iMS method could overcome matrix effect of LC-MS/MS and cross-reaction of CLIA. Matrix effect of the iMS method was negligible as high specificity of target hormone enrichment before LC-MS/MS analysis. Matrix-matched calibration standards were no longer necessary for accurate quantification, which was of great benefit for the clinical application of mass spetrometry.

A-043 A Fast, Novel and Analytically Sensitive Clinical Research Method for the Analysis of Free Testosterone using UPLC-MS/MS

Abstract Background The aim of this work was to develop an analytically sensitive, rapid clinical research method for the analysis of free testosterone in serum using a small sample volume. Laboratories have found this difficult, partly due to the inherent low concentrations (less than 3% of total testosterone is free), technical challenges and variability in results have been noted with equilibrium dialysis, which is considered the gold standard. Methods Testosterone certified reference material (Merck, UK) was used to create calibrators in 52.75 mM HEPES buffer adjusted to pH 7.4 to mimic the ionic environment of serum. Precision was evaluated using in-house QC materials prepared in serum (Golden West Biologicals, USA and BioIVT, UK). 200 µL serum was incubated at 37°C with 400 µL pH-adjusted 52.75 mM HEPES buffer for 2 hours, mixing at 800 r.p.m. with Rapid Equilibrium Analysis (RED) inserts and plates (Thermo Scientific™, UK). Testosterone-13C3 (Merck, UK) was spiked into resultant diasylate, then liquid-liquid extraction (LLE) was used to concentrate and clean the sample. The ACQUITY™ UPLC™ I-Class System with the Waters™ ACQUITY UPLC BEH C18, 1.7µm, 2.1 x 100 mm Column using a water/methanol/ammonium fluoride gradient and the Waters Xevo™ TQ Absolute Mass Spectrometer were used for quantification. An accelerated protocol for equilibrium dialysis, using a 2-hour incubation and fast mixing step was used (4 hours or longer is more typical historically) though it is essential to control the pH of the dialysis buffer and, particularly, the temperature of the chamber. Results No significant carryover or matrix effects were demonstrated with linearity from 0.5-650 pg/mL. Addition of high concentrations of endogenous substances (albumin, bilirubin, creatinine, cholesterol, triglycerides and uric acid) did not affect quantification. Analytical sensitivity investigations indicated the analytical sensitivity would allow precise quantification (≤20%CV and ≤15% bias) at 0.50 pg/mL. Coefficients of variation (CV) for total precision and repeatability on 5 analytical runs for low, mid and high QCs at approximately 2.79, 8.80 and 134 pg/mL respectively were all ≤ 8.4% (n=25). Accuracy was assessed by analyzing 45 male external quality assurance samples (NEQAS, UK) ranging in concentration from 50.1-268.6 pg/mL and the Bland-Altman agreement showed a small negative bias of -7.6% compared with the all-laboratory trimmed mean (ALTM), though excellent agreement overall. When LC-MS/MS determined concentrations were processed against Vermeulen equation determined concentrations (total testosterone and SHBG concentrations were provided by the scheme, a concentration of 45 g/L albumin was assumed), again good agreement was shown by the Bland-Altman bias of -7.7%. Conclusions A novel clinical research method using a fast equilibrium dialysis procedure with LLE and LC-MS/MS analysis with only 200 µL of serum has been demonstrated for clinical research use. Sub pg/mL analytical sensitivity, excellent linearity and precision, with minimal matrix effects and a strong agreement with an EQA scheme were established.

The pharmacokinetics and tissue distribution of curcumin following inhalation administration in rats-A comparative analysis with oral and intravenous routes.

A sensitive and simple method using ultra-liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed and validated to determine the concentration of curcumin in rat plasma and tissue samples. Emodin was selected as the internal standard (IS), and biological samples were pretreated with simple one-step acetonitrile precipitation. The calibration curves exhibited linearity within the range of 1-1000 ng/ml for both rat plasma and tissue samples. The accuracy and precision of intra-day as well as inter-day determinations ranged from 99.3% to 117.3% and from 98.2% to 105.1%, respectively. This method demonstrated excellent recovery rates ranging from 76.4% to 96.4% along with minimal matrix effect ranging from 86.5% to 99.6%. The effectiveness of this method was successfully demonstrated through its application in an in vivo pharmacokinetic and tissue distribution study after single administration via inhalation (100 mg/kg), oral gavage (100 mg/kg) and intravenous injection (2.5mg/kg) of curcumin in rats. The results revealed that inhalation significantly improved the bioavailability of curcumin, with most of the drug being deposited in the lung. These findings highlight inhalation as an effective route for targeted delivery of drugs directly into lung tissues, thus suggesting potential future applications for treating pulmonary diseases utilizing inhaled curcumin.

Quantification of Letrozole, Palbociclib, Ribociclib, Abemaciclib, and Metabolites in Volumetric Dried Blood Spots: Development and Validation of an LC-MS/MS Method for Therapeutic Drug Monitoring

Therapeutic drug monitoring (TDM) may be beneficial for cyclin-dependent kinase 4/6 inhibitors (CDK4/6is), such as palbociclib, ribociclib, and abemaciclib, due to established exposure–toxicity relationships and the potential for monitoring treatment adherence. Developing a method for quantifying CDK4/6is, abemaciclib metabolites (M2, M20), and letrozole in dried blood spots (DBS) could be useful to enhance the feasibility of TDM. Thus, an optimized LC-MS/MS method was developed using the HemaXis DB10 device for volumetric (10 µL) DBS collection. Chromatographic separation was achieved using a reversed-phase XBridge BEH C18 column. Detection was performed with a triple quadrupole mass spectrometer, utilizing ESI source switching between negative and positive ionization modes and multiple reaction monitoring acquisition. Analytical validation followed FDA, EMA, and IATDMCT guidelines, demonstrating high selectivity, adequate sensitivity (LLOQ S/N ≥ 30), and linearity (r ≥ 0.997). Accuracy and precision met acceptance criteria (between-run: accuracy 95–106%, CV ≤ 10.6%). Haematocrit independence was confirmed (22–55%),with high recovery rates (81–93%) and minimal matrix effects (ME 0.9–1.1%). The stability of analytes under home-sampling conditions was also verified. Clinical validation supports DBS-based TDM as feasible, with conversion models developed for estimating plasma concentrations (the reference for TDM target values) of letrozole, abemaciclib, and its metabolites. Preliminary data for palbociclib and ribociclib are also presented.

Spectrophotometric determination of bisphenol A in beverages using multi-dispersion calibration

Bisphenol A (BPA) is an endocrine disruptor that may be present in plastics and coatings of food and beverage packages. In this regard, the World Health Organization recommends the maximum migration of BPA to foodstuffs as < 0.6 mg kg−1. Analytical BPA determination procedures have been based on chromatographic techniques, which generate waste with organic solvents. Alternatively, proposed spectrophotometric procedures have often shown high detection limits for foodstuffs analysis, requiring pre-concentration prior to determination. The analysis of beverages is susceptible to interferences because of their complex formulation comprising dyes and sugars, for example, that hinder spectrophotometric determinations. Multi-signal calibrations are powerful tools used to minimize matrix effects, and recently, they have proven efficient when performed in flow analysis systems, denominated as multi-dispersion calibration (MDC). This work proposes a multi-pumping flow system for the spectrophotometric determination of BPA based on the reaction with sulfanilamide (used for the first time) after diazotization to yield a compound with an absorption maximum at 446 nm. After the optimization of the main parameters, a linear response was observed between 0.25 and 10 mg L−1. The detection limit (by external calibration), coefficient of variation (n = 20), and determination rate were estimated at 0.11 mg L−1, 4.0 %, and 33 h−1, respectively. The MDC was exploited to analyze beverage samples to minimize interferences, especially due to dyes and sucrose. The detection limit estimated for this method was 0.057 mg L−1. Recoveries from 84 to 114 % and the agreement of comparative analysis of the samples with the reference procedure (at the 95% confidence level) demonstrated the accuracy of the proposed procedure. Therefore, the developed alternative is reliable with proper sensitivity and minimum sample preparation for the spectrophotometric determination of BPA in beverages.

Micro-solid phase extraction with in-lab-packed columns for cost-efficient drug extraction from urine samples

This study introduces a miniaturised solid-phase extraction (µ-SPE) method coupled with ultra-high performance liquid chromatography and tandem mass spectrometry (UHPLC-MS/MS) for the simultaneous quantification of 23 compounds, including psychoactive drugs, narcotics and their metabolites, in urine samples. The µ-SPE method, using standard laboratory equipment such as a centrifuge, was developed and validated, offering a practical and economical alternative to more traditional SPE methods. The key extraction parameters: elution solvent composition, sample and elution volumes, and sorbent mass were optimized to enhance performance. The method demonstrated high sensitivity, with detection limits between 0.06 and 0.41 ng cm−3 and quantification limits from 0.19 to 1.24 ng cm−3. The precision was confirmed with an intra-day precision ranging from 0.2% to 7.5% and an inter-day precision from 1.2% to 13.4%. Recovery rates ranged from 84.2% to 109.4%, with minimal matrix effects observed for most analytes (|ME|< 10%). The relative error remained below 5%, indicating good accuracy. The method was also evaluated in terms of greenness, practical, and analytical aspects using the WAC model. Based on the validation results, it was concluded that the developed method could be a good confirmatory method for results obtained by immunochemical tests and could also be used in both clinical analyses and forensic toxicology.Graphical abstract

Streamlined integrated protein isoelectric focusing using microfluidic paper-based device

An innovative integrated paper-based microdevice was developed for protein separation by isoelectric focusing (IEF), allowing for robust design thanks to a 3D-printed holder integrating separation channel, reservoirs, and electrodes. To reach robustness and precision, the optimization focused on the holder geometry, the paper nature, the reservoir design, the IEF medium, and various focusing parameters. A well-established and stable pH gradient was obtained on a glass-fiber paper substrate with simple sponge reservoirs, and the integration of the electrodes in the holder led to a straightforward system. The separation medium composed of water/glycerol (85/15, v/v) allowed for reducing medium evaporation while being an efficient medium for most hydrophobic and hydrophilic proteins, compatible with mass spectrometry detection for further proteomics developments. To our knowledge, this is the first report of the use of glycerol solutions as a separation medium in a paper-based microdevice. Analytical performances regarding pH gradient generation, pI determination, separation efficiency, and resolution were estimated while varying the IEF experimental parameters. The overall process led to an efficient separation within 25 min. Then, this methodology was applied to a sample composed of saliva doped with proteins. A minimal matrix effect was evidenced, underscoring the practical viability of our platform. This low-cost, versatile and robust paper-based IEF microdevice opens the way to various applications, ranging from sample pre-treatment to integration in an overall proteomic-on-a-chip device.

Determination of pesticide residues in five different corn-based products using a single and simple solid–liquid extraction without clean-up steps followed by comprehensive two-dimensional liquid chromatography coupled to tandem mass spectrometry

Pesticide residues in food products are a critical issue concerning food safety. Recent studies have increasingly focused on developing selective, rapid, innovative, and environmentally friendly analytical methods for detecting pesticides in foodstuffs. This study presents a novel analytical method utilizing comprehensive two-dimensional liquid chromatography-tandem mass spectrometry for the simultaneous determination of one hundred and thirteen multiclass pesticides in corn products. The method simplifies the process with a single-step solid–liquid extraction and benefits from two-dimensional separation to enhance resolution and minimize matrix effects. The methodology was validated according to European Commission DG-SANTE (SANTE/11312/2021) and European Committee for Standardization (EN 15662, 2018) guidelines, obtaining acceptable analytical parameters. Limits of detection ranged from 0.2 µg/L to 30.2 µg/L and limits of quantification from 0.3 to 91.6 μg/L, all bellow regulatory limits. Accuracy, ranged from 60 to 120 % and precision, estimated by relative standard deviation, was consistently below 15.1 % in all cases. The applicability of the method was demonstrated by analyzing pesticides in five representative corn products (tortilla, corn flakes, corn cake, starch, and polenta). Sixteen pesticides were detected, all within the regulatory residue limits. Three metric tools were applied to assess the analytical process. Environmental sustainability was evaluated using the AGREE metric, while analytical performance and method applicability were assessed with the Red-Green-Blue and Blue Applicability Grade Index models, respectively, yielding promising scores (0.5, ≥ 0.55, and 0.65). These findings and the comparison to previous methods indicated that this approach represents a promising, alternative and user-friendly strategy for pesticide determination, offering effective productivity and reliable analytical performance.

Next generation microsampling towards sustainable forensic analysis: Volumetric DBS for cocaine and metabolites

This study introduces for the first time a reliable whole blood microsampling method for forensic analysis of cocaine and its metabolites using quantitative dried blood spot (qDBS) technology and UHPLC-MS analysis. This methodology offers accurate and less invasive sampling and aligns with the current trend towards sustainable and accessible analytical methods. Microsampling is subject-friendly, improves logistics, stability and efficiency, marking a shift towards modern forensic practices with wide-ranging application potential.The qDBS-UHPLC-MS method underwent comprehensive validation, confirming its linearity, sensitivity, precision, extraction efficiency and stability. UHPLC-MS achieved effective chromatographic separation and suitable sensitivity, with detection limits between 1.0 and 2.5 ng/mL, and quantitation limits from 2.5 to 7.5 ng/mL. Analytes showed over 85.1 % extraction yield and less than 7.5 % relative standard deviation in precision. Stability tests indicated superior analyte preservation in qDBS at room temperature versus refrigerated plasma, while minimal matrix effect highlighted the sample clean-up efficiency. Application to real samples yielded consistent quali-quantitative results between qDBS and plasma samples, confirming method suitability for forensic cocaine bioanalysis, thus addressing critical needs in drug testing and pharmaco-toxicological analysis.

Analysis of Globotriaosylceramide (Gb3) in Liquid Urine: A Straightforward Assay Using Tandem Mass Spectrometry.

Fabry disease (FD) is a lysosomal storage disorder caused by variants in the GLA gene encoding α-galactosidase A, an enzyme required for catabolism of globotriaosylceramide (Gb3). Accumulation of Gb3 in patients' cells, tissues, and biological fluids causes clinical manifestations including ventricular hypertrophy, renal insufficiency, and strokes. This protocol describes a methodology to analyze urinary Gb3 and creatinine. Samples are diluted with an internal standard solution containing Gb3(C17:0) and creatinine-D3, centrifuged, and directly analyzed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) using an 8.7-min method. Eight Gb3 isoforms [C16:0, C18:0, C20:0, C22:1, C22:0, C24:1, C24:0, and (C24:0)OH] are analyzed and the total is normalized to creatinine. Confirmation ions are monitored to detect potential interferences. The Gb3 limit of quantification is 0.023 µg/ml. Its interday coefficients of variation (3 concentrations measured) are ≤15.4%. This method minimizes matrix effects (≤6.5%) and prevents adsorption or precipitation of Gb3. Urine samples are stable (bias <15%) for 2 days at 21°C, 7 days at 4°C, and 4 freeze/thaw cycles, whereas prepared samples are stable for 5 days at 21°C, and 14 days at 4°C. The Gb3/creatinine age-related upper reference limits (mean + 2 standard deviations) are 29 mg/mol creatinine (<7 years) and 14 mg/mol creatinine (≥7 years). This simple, robust protocol has been fully validated (ISO 15189) and provides a valuable tool for diagnosis and monitoring of FD patients. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Analysis of urinary globotriaosylceramide (Gb3) and creatinine by UHPLC-MS/MS Support Protocol 1: Preparation of the urinary quality controls Support Protocol 2: Preparation of the urine matrix used for the Gb3 calibration curve Support Protocol 3: Preparation of the Gb3 calibrators Support Protocol 4: Preparation of the working solution containing the internal standards Support Protocol 5: Preparation of the creatinine calibrators Support Protocol 6: Preparation of the UHPLC solutions and mobile phases.

Rapid and Comprehensive Analysis of 41 Harmful Substances in Multi-Matrix Products by Gas Chromatography-Mass Spectrometry Using Matrix-Matching Calibration Strategy.

Harmful substances in consumer goods pose serious hazards to human health and the environment. However, due to the vast variety of consumer goods and the complexity of their substrates, it is difficult to simultaneously detect multiple harmful substances in different materials. This paper presents a method for the simultaneous determination of 41 harmful substances comprising 17 phthalates (PAEs), 8 organophosphate flame retardants (OPFRs), and 16 polycyclic aromatic hydrocarbons (PAHs) in five types of products using the matrix-matching calibration strategy. The method employs an efficient ultrasonic extraction procedure using a mixture of dichloromethane and methylbenzene, followed by dissolution-precipitation and analysis through gas chromatography-mass spectrometry. Compared with previous experiments, we established a universal pretreatment method suitable for multi-matrix materials to simultaneously determine multiple harmful substances. To evaluate the effects of the matrix on the experimental results, we compared neat standard solutions and matrix-matching standard solutions. The results demonstrated that all compounds were successfully separated within 30 min with excellent separation efficiency. Additionally, the linear relationships of all analytes showed strong correlation coefficients (R2) of at least 0.995, ranging from 0.02 mg/L to 20 mg/L. The average recoveries of the target compounds (spiked at three concentration levels) were between 73.6 and 124.1%, with a relative standard deviation (n = 6) varying from 1.2% to 9.9%. Finally, we tested 40 different materials from consumer products and detected 16 harmful substances in 31 samples. Overall, this method is simple and accurate, and it can be used to simultaneously determine multiple types of hazardous substances in multi-matrix materials by minimizing matrix effects, making it an invaluable tool for ensuring product safety and protecting public health.

Alkanediol–hexafluoroisopropanol amphiphilic supramolecular solvent: Fabrication, characterization, and application potential for doping control of hormones and metabolic modulators in human urine

A novel amphiphilic supramolecular solvent (SUPRAS) was developed by hexafluoroisopropanol (HFIP)-induced self-assembly of 1,2-hexanediol in an aqueous matrix. Its application potential in multiclass determination was assessed by extracting from urine 10 prohibited hormones and metabolic modulators (log P from 2.38 to 7.57) listed by the World Anti-Doping Agency (WADA). This SUPRAS is stably formed across a wide pH range (2–12) and is unaffected by salt concentrations (<10 % g mL−1 of NaCl), and it has a higher density than water. The dehydration and hydrogen bonding ability of HFIP, as well as hydrophobic interactions between alkanediols, dominates the formation of the SUPRAS. The nanostructure of the SUPRAS, composed of 1,2-hexanediol, HFIP, and a high content of water (>25 %, w/w), forms large spherical micellar aggregates (10–100 μm) that exhibit amphiphilic properties. A 1,2-hexanediol-HFIP SUPRAS-based liquid-phase microextraction (LPME) method for detection of selected prohibited drugs in urine coupled with liquid chromatography–tandem mass spectrometry (LC-MS/MS) was established, optimized, and validated. The method showed fast extraction time (30 s of vortex) and low solvent consumption (160 μL of organic solvent). Under the optimal conditions (7 % g mL−1 of 1,2-hexanediol, 10 % v/v of HFIP), the SUPRAS achieved high extraction efficiencies (>80 %) and an enrichment factor of 6.0, with minimal matrix effects for all analytes due to its high amphiphilicity and large surface area. The method demonstrated excellent linearity (R ≥0.9909), with method limits of detection ranging from 0.018 to 0.18 ng mL−1 and method limits of quantitation ranging from 0.060 to 0.60 ng mL−1, which are well below the minimum required performance levels set by authorities. Furthermore, the intra- and inter-day relative recoveries for spiked human urine samples varied in the ranges of 90.3–121 % and 88.1–119 %, respectively, with relative standard deviations less than 13 % and 19 %, respectively. This SUPRAS-based LPME method proves to be simple, highly efficient, and eco-friendly, and it has promising potential for multiclass determination in doping control.

Development of a highly efficient solubilization method for mass spectrometric analysis of phospholipids in living single cells.

Phospholipids are vital constituents of the cell membrane and aid in signal transduction. Phospholipid profiles vary distinctively with the cell type. Notably, specific phospholipid molecules are present in significantly higher or lower concentrations in cancer cells versus normal cells. In this study, live single-cell mass spectrometry (MS) was developed for analyzing phospholipids at the single-cell level. This method facilitates rapid molecular analysis of cells under microscopic observation. For nanoelectrospray ionization, phospholipids were extracted from single cells isolated in a glass capillary through a high-efficiency process. Cell-derived phosphatidylcholines were detected with high sensitivity when trehalose C14 was added as a solubilizing reagent. Trehalose C14 can solubilize cells at low concentrations owing to its low critical micelle concentration, and exerts minimal matrix effects (such as suppressing ionization and causing peak overlap) in the MS analysis of cellular molecules. Analyses of phospholipids in Raji and HEV0070 cells using the developed method revealed specific peaks of phosphatidylcholine and sphingomyelin in the respective cells. The developed technique not only affords phospholipid profiles at the single-cell level, but also holds promise for identifying biomarkers associated with various diseases, particularly cancer.

Optimization of a fast and sensitive method based on matrix solid-phase dispersion-LC-ms/ms for simultaneous determination of phthalates and bisphenols in mussel samples

Bisphenols and phthalates are wide classes of endocrine disrupting chemicals (EDCs) extensively used as additives in plastic products. In this study, a fast and reliable analytical method based on matrix solid-phase dispersion (MSPD) coupled with LC-MS/MS was developed and optimized for simultaneous determination of 8 bisphenols and 7 phthalates in raw mussel extract. The LC-MS/MS method was tested for linearity (R2), inter- and intra-day repeatability, limit of detection and quantification, both for matrix-free and matrix-matched solutions. The MSPD method was optimized in terms of ratio between sample and sorbent, and the type and quantity of the eluents in order to maximize the recoveries and to minimize matrix effects. The obtained recoveries (values between 75% and 113%), limits of detection (values between 0.048 and 0.36 µg kg−1), limits of quantification (values between 0.16 and 1.28 µg kg−1), repeatability (RSD% between 1.30% and 8.41%) and linearity (R2 > 0.998) were satisfactory and suitable for the determination of target micropollutants in food samples. In addition, the low solvent consumption and fast execution make this method ideal for routinely determinations of bisphenols and phthalates in mussels.

Simplified processing and rapid quantification of buprenorphine, norbuprenorphine, and their conjugated metabolites in human plasma using UPLC-MS/MS: Assessment of buprenorphine exposure during opioid use disorder treatment.

Opioid use disorder (OUD) is a chronic neurobehavioral ailment and is prevalent in pregnancy. OUD is commonly treated with methadone or buprenorphine (BUP). Pregnancy is known to alter the pharmacokinetics of drugs and may lead to changes in drug exposure and response. A simple, specific, and sensitive analytical method for measuring the parent drug and its metabolites is valuable for assessing the impact of pregnancy on drug exposure. A new liquid chromatography-tandem mass spectrometric method that utilized a simple protein precipitation procedure for sample preparation and four deuterated internal standards for quantification was developed and validated for BUP and its major metabolites (norbuprenorphine [NBUP], buprenorphine-glucuronide [BUP-G], and norbuprenorphine-glucuronide [NBUP-G]) in human plasma. The standard curve was linear over the concentration range of 0.05-100 ng/mL for BUP and NBUP, and 0.1-200 ng/mL for BUP-G and NBUP-G. Intra- and inter-day bias and precision were within ±15% of nominal values for all the analytes. Quality controls assessed at four levels showed high recovery consistently for all the analytes with minimal matrix effect. Adequate analyte stability was observed at various laboratory conditions tested. Overall, the developed method is simple, sensitive, accurate and reproducible, and was successfully applied for the quantification of BUP and its metabolites in plasma samples collected from pregnant women in a clinical study assessing BUP exposure during OUD treatment.

Comparative Pharmacokinetics Research of 13 Bioactive Components of Jieyu Pills in Control and Attention Deficit Hyperactivity Disorder Model Rats Based on UPLC-Orbitrap Fusion MS.

Jieyu Pills (JYPs), a Chinese medicine consisting of 10 herbal elements, have displayed promising clinical effectiveness and low by-effects in the treatment of depression. Prior investigations mostly focused on elucidating the mechanism and therapeutic efficacy of JYPs. In our earlier study, we provided an analysis of the chemical composition, serum pharmacochemistry, and concentrations of the main bioactive chemicals found in JYPs. However, our precise understanding of the pharmacokinetics and metabolism remained vague. This study involved a comprehensive and meticulous examination of the pharmacokinetics of 13 bioactive compounds in JYPs. Using UPLC-Orbitrap Fusion MS, we analyzed the metabolic characteristics and established the pharmacokinetic parameters in both control rats and model rats with attention deficit hyperactivity disorder (ADHD) following oral administration of the drug. Before analysis, plasma samples that were collected at different time intervals after the administration underwent methanol pre-treatment with Puerarin used as the internal standard (IS) solution. Subsequently, the sample was chromatographed on a C18 column employing gradient elution. The mobile phase consisted of methanol solution containing 0.1% formic acid in water. The electrospray ionization source (ESI) was utilized for ionization, whereas the scanning mode employed was selected ion monitoring (SIM). The UPLC-Orbitrap Fusion MS method was subjected to a comprehensive validation process to assess its performance. The method demonstrated excellent linearity (r ≥ 0.9944), precise measurements (RSD < 8.78%), accurate results (RE: -7.88% to 8.98%), and appropriate extraction recoveries (87.83-102.23%). Additionally, the method exhibited minimal matrix effects (87.58-101.08%) and satisfactory stability (RSD: 1.52-12.42%). These results demonstrated adherence to the criteria for evaluating and determining biological material. The 13 bioactive compounds exhibited unique pharmacokinetic patterns in vivo. In control rats, all bioactive compounds except Ferulic acid exhibited linear pharmacokinetics within the dose ranges. In the ADHD model, the absorption rate and amount of most of the components were both observed to have increased. Essentially, this work is an important reference for examining the metabolism of JYPs and providing guidelines for clinical therapy.

DEVELOPMENT AND VALIDATION OF AN LC-MS/MS METHOD FOR THE DETERMINATION OF TENOFOVIR AND EMTRICITABINE

Objective: Therapeutic drug monitoring of tenofovir and emtricitabine, two commonly used antiretroviral drugs, is important to maximize effectiveness while minimizing side effects. Materials: A Liquid Chromatography-Mass spectrometry (LC-MS/MS) method was developed to quantify tenofovir and emtricitabine in human plasma samples. The method involves a simple solid phase extraction procedure followed by liquid chromatography separation using a Penta Fluoro Phenyl (PFP) column with a Phenomenex C18 column and a mobile phase of ammonium formate, acetonitrile, and methanol, achieving separation in under 4 min. Results: The method showed good accuracy, low limits of quantification, adequate recovery, minimal matrix effects, and specificity. Analyte stability under multiple storage conditions was demonstrated. Conclusion: The validated LC-MS/MS method provides a reliable tool for therapeutic drug monitoring and pharmacokinetic studies of anti-Human Immuno-deficiency Virus (HIV) regimens. The assay can be applied to large populations, especially in resource-poor settings, to help individualize dosing and improve clinical outcomes while reducing toxicity.

Developing, validating, and comparing an analytical method to simultaneously detect z-drugs in urine samples using the QuEChERS approach with both liquid chromatography and gas chromatography-tandem mass spectrometry

Detecting z-drugs, a sedative-hypnotic medication, is also misused for criminal activities. Therefore, the analysis of urine samples is crucial for clinical and forensic purposes. We conducted a study where we developed, validated, and compared an analytical method for simultaneously detecting z-drugs in urine samples. Our approach uses the QuEChERS method for sample preparation, combined with liquid chromatography (LC) and gas chromatography (GC) coupled with tandem mass spectrometry (MS/MS). We optimized the QuEChERS method to effectively extract z-drugs from urine samples while minimizing matrix effects and achieving high recovery rates. After extraction, we split the samples into two parts for analysis using LC-MS/MS and GC–MS/MS. We validated our methods, and the results showed good linearity over a broad concentration range (1–200 ng/mL) for each z-drug. The limits of detection and quantification were within clinically relevant ranges, ensuring sensitivity for detecting z-drugs in urine samples. We compared the two chromatographic techniques by analyzing a set of urine samples spiked with known concentrations of z-drugs using both LC-MS/MS and GC–MS/MS methods and then applied to the real samples. The results were statistically analyzed to assess any significant differences in accuracy and precision above 95 %, and both methods offered reliable and consistent results with the samples as well. In conclusion, our analytical method coupled with both LC-MS/MS and GC–MS/MS using the QuEChERS approach provides a comprehensive and robust solution for the simultaneous detection of z-drugs in urine samples. The choice between the two chromatographic techniques can be based on the specific z-drugs of interest and the required analytical performance. This method holds promise for applications in clinical toxicology, forensic analysis, and monitoring z-drug usage.