Minimal Matrix Research Articles

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.

Simulating Compaction and Cementation of Clay Grain Coated Sands in a Modern Marginal Marine Sedimentary System

Reservoir quality prediction in deeply buried reservoirs represents a complex challenge to geoscientists. In sandstones, reservoir quality is determined by the extent of compaction and cementation during burial. During compaction, porosity is lost through the rearrangement and fracture of rigid grains and the deformation of ductile grains. During cementation, porosity is predominantly lost through the growth of quartz cement, although carbonate and clay mineral growth can be locally important. The degree of quartz cementation is influenced by the surface area of quartz available for overgrowth nucleation and thermal history. Clay grain coats can significantly reduce the surface area of quartz available for overgrowth nucleation, preventing extensive cementation. Using a coupled-effect compaction and cementation model, we have forward-modelled porosity evolution of surface sediments from the modern Ravenglass Estuary under different maximum burial conditions, between 2000 and 5000 m depth, to aid the understanding of reservoir quality distribution in a marginal marine setting. Seven sand-dominated sub-depositional environments were subject to five burial models to assess porosity-preservation in sedimentary facies. Under relatively shallow burial conditions (&lt;3000 m), modelled porosity is highest (34 to 36%) in medium to coarse-grained outer-estuary sediments due to moderate sorting and minimal fine-grained matrix material. Fine-grained tidal flat sediments (mixed flats) experience a higher degree of porosity loss due to elevated matrix volumes (20 to 31%). Sediments subjected to deep burial (&gt;4000 m) experience a significant reduction in porosity due to extensive quartz cementation. Porosity is reduced to 1% in outer estuary sediments that lack grain-coating clays. However, in tidal flat sediments with continuous clay grain coats, porosity values of up to 30% are maintained due to quartz cement inhibition. The modelling approach powerfully emphasises the value of collecting quantitative data from modern analogue sedimentary environments to reveal how optimum reservoir quality is not always in the coarsest or cleanest clastic sediments.

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 influence of pulse cell wall structure and cellular protein matrix on the in vitro digestion kinetics of starch: A dual encapsulation mechanism

The intrinsic characteristics and extrinsic processing of whole-pulse food modulate the starch digestion rate and extent. This study investigated the dual encapsulation mechanism of cell wall structure and protein matrix on the in vitro digestion properties of intracellular starch, using an isolated whole-pulse food model of intact pea cotyledon cells subjected to alkaline buffer and enzymatic treatments. Results showed that intact cells with the maximum protein matrix content (18.9 %) exhibited the lowest peak temperature (71.4 °C, uncooked and 58.1 °C, cooked), enthalpy change (3.4 J/g, uncooked and 2.0 J/g, cooked), relative crystallinity (11.6 %), and starch digestion rate (0.0248 min−1) and extent (11.9 %) compared to alkaline buffer and enzymatic treatments. Even after enzymatic treatment, cells with minimal protein matrix content (1.8 %) exhibited a starch digestion rate (0.0387 min−1) and extent (39.7 %), which were still lower than those of isolated starch (0.0480 min−1 and 56.8 %). These findings indicate that the protein matrix and cell walls act as a dual encapsulation system to slow starch hydrolysis. This provides a theoretical basis and technical guidance for developing low-glycemic whole-pulse foods.

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.

Deccan volcanism's influence on Chicxulub impact signatures at the continental Cretaceous-Paleogene boundary in the Sichuan Basin, China

The Cretaceous-Paleogene (K-Pg) extinction is linked to two primary causes: the Chicxulub asteroid impact in Mexico and the Deccan Traps eruptions in India. While substantial geological evidence supporting both events has been found worldwide, to understand their potential interactions, it is crucial to study sites that preserve records of both events simultaneously. The terrestrial K-Pg boundary located in the Sichuan Basin, China (∼15,500 km from the Chicxulub Crater and ∼5,600 km from the Deccan Traps correspond to 66 Ma), preserves evidence of both the Chicxulub impact and the Deccan Traps eruptions, along with the effects of subsequent events on the earlier ones. It offers a new perspective on the K-Pg boundary. Detailed petrological examination and geochemical analyses, including platinum-group element (PGE) and black carbon concentrations, were conducted across a 2 m K-Pg boundary section. The K-Pg boundary layer (∼2.5 cm thick) is composed of very fine-grained, brick-red sandstone and exhibits significant enrichments in Ir (9.050 ppb) and Os (10.72 ppb), along with elevated black carbon (1.10 ‰), strongly indicating contributions from the impact. Intriguingly, another PGE anomaly was detected approximately 50 cm below the K-Pg boundary. The strata surrounding the K-Pg boundary display evidence of alteration by acidic and HSˉ-rich fluids. These fluids likely caused the observed downward migration of PGE within the boundary, resulting in two distinct PGE anomalies - one at the K-Pg boundary itself and another approximately 50 cm below. These fluids resulted in the overlying sandstones exhibiting minimal matrix content and bleached the underlying brick-red sandstones. Volcanic gas emissions from the Deccan Traps after the impact are a potential source for these fluids. Our findings support the hypothesis that the most voluminous Deccan eruptions had occurred close to, but not necessarily immediately after, the Chicxulub impact, potentially lasting 100–131 ka. The hiatus after the impact weakens the possibility of a direct causal link between the Deccan eruptions and the impact.

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

Carbon-coated Fe3O4 derived from metal-organic frameworks on reduced graphene as electrochemical sensor platform for Hg(II) determination

Mercury, a well-known neurotoxicant, mainly occurs as Hg0, Hg2+ or Hg (CH3)2 in the environment. Therefore, there is an urgent need to develop new analytical methods to rapidly screen for mercury in natural water. In this work, rGO-MIL-101(Fe) was fabricated using reduced graphene oxide (rGO) and MIL-101 by one spot solvothermal method, and then was pyrolyzed at 500 °C to yield carbon-coated Fe3O4 dispersed on rGO (hereafter rGO-Fe@C). The rGO-Fe@C was characterized by SEM, TEM, TGA, XRD, XPS and Raman spectroscopic methods for its morphological features, particulate size & gradations, element redox states. An electrochemical sensing interface was constructed for Hg2+ determination using rGO-Fe@C modified glassy carbon electrode(rGO-Fe@C/GCE) by square wave anode stripping voltammetry (SWASV). The rGO-Fe@C/GCE exhibits high sensitivity for Hg2+ determination with a sensitivity of 34.47 μA/μM and a limit detection of 4.5 nM. The sensitive mechanism of Hg2+ in the electrochemical interface was investigated by XPS and DFT calculations. The presence of common cations as Cd2+, Cu2+ and Pb2+ in comparable molarities does not interfere with Hg2+ detection. Moreover, the electrode sensor possesses good stability and reproducibility for repetitive analysis of samples. The rGO-Fe@C/GCE sensor can be used in Hg2+ detection with minimal matrix interference by ions ubiquitous in natural water.

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.

Novel Admissibility Criteria and Multiple Simulations for Descriptor Fractional Order Systems with Minimal LMI Variables

In this paper, we first present multiple numerical simulations of the anti-symmetric matrix in the stability criteria for fractional order systems (FOSs). Subsequently, this paper is devoted to the study of the admissibility criteria for descriptor fractional order systems (DFOSs) whose order belongs to (0, 2). The admissibility criteria are provided for DFOSs without eigenvalues on the boundary axes. In addition, a unified admissibility criterion for DFOSs involving the minimal linear matrix inequality (LMI) variable is provided. The results of this paper are all based on LMIs. Finally, numerical examples were provided to validate the accuracy and effectiveness of the conclusions.

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.

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.

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.

An Optimized and Validated QuEChERS-Based Method for the Determination of PCBs in Edible Aquatic Species

AbstractIn the present research, a quick, easy, cheap, effective, rugged, and safe (QuEChERS) method was optimized and validated for the determination of 14 selected PCB congeners in mussel (Mytilus galloprovincialis) and fish samples (Sparus aurata). The optimization included the selection of different QuEChERS procedures, extraction solvent, sample weight, and suitable sorbents for the clean-up step to achieve high sensitivity and minimal matrix interferences simultaneously. The identification and quantification of the selected PCBs were carried out using GC–MS. The method was validated providing in all cases excellent linearity (r2 above 0.99). Recoveries were estimated at three different fortification levels (10, 50, and 100 ng g−1) and ranged from 72.5 to 109.5% and 72.1 to 88.4% for mussel and fish samples, respectively. In addition, for both matrices, the LOQs ranged from 1 to 2.5 ng g−1. The matrix effect was in all cases &lt; 29%, while the expanded uncertainty U%, which was estimated at all the fortification levels, was found below 53% in all cases. Eventually, the optimized and validated method was applied to mussel and fish samples acquired from aquacultures in NW Greece, revealing the absence of the selected congeners in all cases.

Screening Non-neutralizing Anti-idiotype Antibodies Against a Drug Candidate for Total Pharmacokinetic and Target Engagement Assay.

Non-neutralizing anti-idiotype antibodies against a therapeutic monoclonal antibody (mAb) play a crucial role in the creation of total pharmacokinetic (PK) assays and total target engagement (TE) assays during both pre-clinical and clinical development. The development of these anti-idiotype antibodies is challenging. In this study, we utilized a hybridoma platform to produce a variety of anti-idiotype antibodies against GSK2857914, a humanized IgG1 anti-BCMA monoclonal antibody. The candidate clones were evaluated using surface plasmon resonance (SPR) and bio-layer interferometry (BLI) for binding affinity, binding profiling, matrix interference, and antibody pairing determination. We discovered that three anti-idiotype antibodies did not prevent BCMA from binding to GSK2857914. All three candidates demonstrated high binding affinities. One of the three exhibited minimal matrix inference and could pair with the other two candidates. Additionally, one of the three clones was biotinylated as a capture reagent for the total PK assay, and another was labeled with ruthenium as a detection reagent for both the total PK assay and total TE assay. The assay results clearly show that these reagents are genuine non-neutralizing anti-idiotypic antibodies and are suitable for total PK and TE assay development. Based on this and similar studies, we conclude that the hybridoma platform has a high success rate for generating non-neutralizing anti-idiotype antibodies. Our methodology for developing and characterizing non-neutralizing anti-idiotype antibodies to therapeutic antibodies can be generally applied to any antibody-based drug candidate's total PK and total TE assay development.

On area-minimizing Pfaffian varieties

There are two significant families of minimal real matrix varieties: determinantal varieties and skew-symmetric determinantal varieties, the later ones are also known as Pfaffian varieties. In 1999, Kerckhove and Lawlor [26] proved that determinantal varieties are area-minimizing except for two families. In this paper we prove that all Pfaffian varieties are area-minimizing with the exception of Pfaffian hypersurfaces.