Assessment of ∆9-THC and ∆9-THCCOOH Bias, Precision, and Ionization Suppression/Enhancement between Solid Tissue Homogenate and Supernatant by LC-MS/MS.

LC-MS-MS assays are frequently utilized for screening and confirmatory purposes in the forensic toxicology laboratory. While these techniques are excellent for the targeted identification and quantitation of a wide variety of drug classes, validation and determining fit-for-purpose is a requirement for each method. In the United States, ANSI/ASB Standard 036 currently serves as a primary resource in forensic toxicology method validation, and mandates that laboratories evaluate critical performance characteristics to help ensure the production of forensically defensible results. Due to the variability of specimen quality frequently encountered in the discipline of postmortem toxicology, the [Author Information Removed] Office of the Chief Medical Examiner Forensic Toxicology Laboratory routinely analyzes solid tissue specimens as part of the medicolegal death investigation process and evaluates liver as a representative solid tissue matrix during method validation. Authentic postmortem specimens (e.g., liver, kidney, skeletal muscle, and spleen) were used to investigate the effects of analyzing solid tissue homogenate versus solid tissue supernatant on bias, precision, and ionization suppression/enhancement of ∆9-THC and ∆9-THCCOOH. Bias was <20% for Δ9-THC and ∆9-THCCOOH in liver homogenate and supernatant with a single exception of the low QC concentration for Δ9-THC in liver homogenate (-29%). Within-run and between-run CV was <20% for Δ9-THC and ∆9-THCCOOH in liver homogenate and supernatant. Δ9-THC and Δ9-THC-d3 exhibited significant ion suppression in both liver homogenate and supernatant, while ∆9-THCCOOH and ∆9-THCCOOH-d3 showed both ion suppression and enhancement in these matrices. Noticeable quantitative differences were observed in authentic postmortem solid tissue homogenate and supernatant specimens despite evaluating from identical tissue samplings. A brief discussion of the results is presented using a validated LC-MS-MS method for the confirmation and quantitation of ∆9-THC and ∆9-THCCOOH in postmortem casework.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of polymixin B1, B2, ile-B1, E1, and E2 in human plasma and its clinical pharmacokinetic application.

Polymyxin B (PB) and Polymyxin E (PE, also called colistin) are used as the last treatment resort for multidrug-resistant Gram-negative bacterial infections. The nephrotoxicity and neurotoxicity of polymyxins limit their clinical use, and guidelines recommend therapeutic drug monitoring (TDM) to optimize efficacy and reduce toxicity. However, there are limited analytical methods available for the determination of PB and PE. This study aimed to develop a simple and robust liquid chromatography with tandem mass spectrometry (LC-MS/MS) analytical method for determining the main compounds of PB and PE, namely PB1, PB2, ile-PB1, PE1, and PE2, in human plasma and to investigate of their pharmacokinetics in critically ill patients with the use of PB and PE, respectively. Plasma PB1, PB2, ile-PB1, PE1, and PE2 were chromatographically separated on a Welch LP-C18 column and detected using electrospray ionization mode coupled with multiple reaction monitoring. The calibration curve showed acceptable linearity over 20-10,000 ng/mL for PB1, PE1, and PE2 and 10-5000 ng/mL for PB2 and ile-PB1 in the plasma, respectively. After validation following approved guidelines, this method was successfully applied for PB and PE pharmacokinetic analysis and TDM in critically ill patients. Additionally, the composition of PB1, PB2, ile-PB1, PE1, and PE2 remains unchanged from 0 to 12 h after entering the patient's body.

Analysis of total microcystins by Lemieux oxidation and liquid chromatography-mass spectrometry in fish and mussels tissues: Optimization and comparison of protocols.

Microcystins (MCs) can be detected in various matrices in two forms: a freely extractable fraction and a total (free and covalently protein-bound) fraction. Although the majority of MCs analyses are limited to the free fraction, they do not allow the analysis of all MCs variants or protein-bound forms. Other methods, known as total MCs analysis methods, enable simultaneous analysis of all MCs variants, as well as bound forms, which may be a major form of toxin accumulation in organisms. Among these techniques, the chemical oxidation method (e.g. Lemieux) allows the detection of total forms of MC (and nodularins) by oxidizing the common part to all MC and nodularins, and analyzing the resultant MMPB product (2-methyl-3-methoxy-4-phenylbutyric acid). However, the execution of this method in the context of health monitoring is challenging due to the variability of the protocols, the recoveries obtained with these protocols, and the important matrix effects associated with the method. The objectives of this study were i) to optimize an existing protocol of chemical oxidation "Lemieux1" on fresh fish fillet matrices, ii) to compare two existing protocols ("Lemieux1" and "Lemieux2"), and iii) apply Lemieux oxidation to fish fillets and livers naturally contaminated with MCs-producing cyanobacteria and to freshwater mussels contaminated with MCs in laboratories. Optimization of the "Lemieux1" protocol, in particular in the oxidation and SPE (solid phase extraction) steps improved the method's yields on the fresh fish fillet matrix (from <5 % to around 40 %). Moreover, several quantification methods have been compared through various calibration techniques (solvent calibration curve, matrix-matched calibration curve, oxidized MC-LR calibration curve and also by testing the addition of d3-MMPB as an internal standard). Comparison with the "Lemieux2" protocol showed the best results on the same matrix, with yields of around 65 %. MMPB was analyzed using this "Lemieux 2" protocol, in livers of carps sampled during an episode of cyanobacteria proliferation, at concentrations ranging from 17.9 to 27.5 µg/kg MMPB and at concentrations ranging from 50 to 2890 µg/kg MMPB in freshwater mussels laboratory contaminated to MCs.

Comparative proteomic analysis of saliva from chewing tobacco users and oral cancer patients reveals shared biomarkers: A case control observational study.

OBJECTIVE: The aim of this study was to compare the salivary proteomic profile of smokeless tobacco users with that of non-users and oral cancer patients using Liquid Chromatography-Mass Spectrometry/ Mass Spectrometry (LC-MS/MS). METHODS: Saliva samples from 65 participants were collected in three groups: control (25 participants), smokeless tobacco users (25 participants), and oral cancer (15 participants). RESULTS: The analysis revealed 343 protein groups with significantly altered abundance in the saliva samples (P < 0.05). Among these, 43 out of 51 dysregulated proteins in the smokeless tobacco group were also dysregulated in the oral cancer group. Notably, Apolipoprotein A1 (ApoA1) and Pon1 were found to be significantly increased in both smokeless tobacco users and oral cancer patients (p < 0.05). Furthermore, six out of the 20 most significantly altered proteins were mitochondrial proteins, and all of these were decreased relative to controls in both smokeless tobacco users and cancer samples. CONCLUSION: The proteomic profile of users of chewing (smokeless) tobacco (SLT) shows substantial overlap in the altered pathways and dysregulated proteins with those altered in oral cancer samples, suggesting that SLT use induces a shift toward an oncogenic state. Specifically indicated pathways included blood microparticles, platelet α-granules and protease inhibitors as well as indicators of oxidative stress and exogenous compound processing. What differentiates oral cancer samples from SLT users is enrichment of alterations related to cytoskeletal organisation and tissue remodelling. CLINICAL SIGNIFICANCE: The findings emphasize the importance of salivary proteomic profiles because changes in certain proteins may be indicators for early oral cancer identification and risk assessment in smokeless tobacco users.

Development and validation of a novel 7α-hydroxy-4-cholesten-3-one (C4) liquid chromatography tandem mass spectrometry method and its utility to assess pre-analytical stability.

OBJECTIVES: 7α-Hydroxy-4-cholesten-3-one (C4) is the common intermediary of both primary bile acids. C4 is recommended by the British Society of Gastroenterology for the investigation of bile acid diarrhoea (BAD) in patients with chronic diarrhoea. This project aimed to develop and validate an assay to quantitate C4 in serum and assess the stability of C4 in unseparated blood. METHODS: Accuracy was underpinned by calibrating to quantitative nuclear magnetic resonance analysis. C4 was analysed in a 96-well plate format with a deuterated C4 internal standard and liquid-liquid extraction. Validation followed the 2018 Food and Drug Administration guidelines. To assess C4 stability, healthy volunteers (n=12) donated 8 fasted samples each. Samples were incubated at 20 °C for up to 72 h and retrieved, centrifuged, aliquoted and frozen for storage at different time points prior to C4 analysis. RESULTS: The C4 method demonstrated excellent analytical performance and passed all validation criteria. The method was found to be accurate, precise, free from matrix effects and interference. After 72 h of delayed sample separation, C4 concentration gradually declined by up to 14 % from baseline. However, the change was not significant for up to 12 h. CONCLUSIONS: We present a robust method of analysing serum C4, offering a convenient alternative to 75SeHCAT for BAD investigation. C4 was found to decline in unseparated blood over time; however, after 12 h the mean change was <5 % from baseline. Our results suggest C4 is suitable for collection from both primary and secondary care prior to gastroenterology referral.

Phytochemical profile and antioxidant capacity of the endemic species Bellevalia sasonii Fidan.

The study investigated total phenolic-flavonoid content, antioxidant activity, and phytochemical compounds across various parts (bulb, stem, leaf, and flower) of the endemic Bellevalia sasonii, commonly known as hyacinth, belonging to the Asparagaceae family. Phenolic content was highest in bulb extracts (117.28 µg GAE) and lowest in stems (45.11 µg GAE). Conversely, leaf extracts exhibited the highest flavonoid content (79.44 µg QEs), while stems showed the lowest (22.77 µg QEs). When the antioxidant activities were compared, by DPPH method leaf = flower > bulb > stem; in ABTS and CUPRAC methods bulb > flower > leaf > stem, respectively. Considering the results in general, it was revealed that bulbs and flowers displayed higher activity, while stem exhibited lower activity compared to other parts. The phytochemical analysis identified 53 active substances, with 27 absent in any extract and 15 detected across all extracts. The distribution of phytochemicals varied among parts, with bulbs, stems, flowers, and leaves also different numbers. The LC-MS/MS analysis revealed prominent metabolites including fumaric acid in leaves, caffeic acid in bulbs, and cosmosiin and quinic acid in flowers. This study provides foundational insights into B. sasonii, an important endemic plant in Türkiye, laying the groundwork for future research on its medicinal and ecological roles.

A sensitive, expandable AQC-based LC-MS/MS method to measure amino metabolites and sphingolipids in cell and serum samples.

Sphingolipids are a major lipid species found in all eukaryotes. Among structurally complex and diversified lipids, sphingoid bases have been heavily linked to various metabolic diseases. However, most current LC-MS-based methods lack the sensitivity to detect low-abundant sphingoid bases. The 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization reagent, which efficiently forms covalent bonds with amino groups, has been widely used for amino acid detection. Nevertheless, the commonly used reverse-phase HPLC method for amino acid analysis is not suitable for amphipathic sphingolipids. To address this issue, we report a robust reverse-phase HPLC-MS/MS method capable of separating and detecting hydrophilic amino acids and sphingoid bases in a single run with high sensitivity. This method is also inclusive of other amino metabolites with an expandable target list. We tested this method under various conditions and samples, demonstrating its high reproducibility and sensitivity. Using this approach, we systematically analyzed human serum samples from healthy individuals, dyslipidemia, and type II diabetes mellitus (T2DM) patients, respectively. Two sphingolipids and five amino acids were identified with significant differences between the control and T2DM groups, highlighting the potential of this method in clinical studies.

Potential (co-)contamination of dairy milk with AFM1 and MC-LR and their synergistic interaction in inducing mitochondrial dysfunction in HepG2 cells.

Several toxic metabolites, such as aflatoxin M1 (AFM1), are known to contaminate dairy milk. However, as mentioned in an external EFSA report, there is a knowledge gap regarding the carry-over of certain emerging toxins such as microcystin-LR (MC-LR). Therefore, this work aimed to develop an LC-MS/MS method for MC-LR quantification in dairy milk. Also, the method included AFM1 as a common fungal metabolite and applied to analyze 113 dairy milk samples collected directly after the end of the summer peak. Both toxins were below their LODs, keeping the question on MC-LR carry-over still unanswered. Moreover, an in silico analysis, using a 3D molecular modeling was performed, pointing to a possible interaction between MC-LR and milk proteins, especially ß-lactoglobulin. Since AFM1 and MC-LR are hepatotoxic, their interaction in inducing mitochondrial dysfunction in HepG2 cells was investigated at low (subcytotoxic) concentrations. Live cell imaging-based assays showed an inhibition in cell viability, without involvement of caspase-3/7, and a hyperpolarization in the mitochondrial membrane potential after the exposure to a mixture of 100 ng mL-1 AFM1 and 1000 ng mL-1 MC-LR for 48h. Extracellular flux analysis revealed inhibitions of several key parameters of mitochondrial function (basal respiration, ATP-linked respiration, and spare respiratory capacity).

Screening and identification of reactive metabolic compounds of Cortex Periplocae based on glutathione capture-mass spectrometry.

As a traditional Chinese medicine (TCM), Cortex Periplocae (CP) has a wide range of pharmacological effects, as well as toxic side effects. The main toxic components of it are cardiac glycosides, which tend to cause cardiotoxicity. Currently, it has also been reported in studies to cause hepatotoxicity, but it is not clear whether the hepatotoxicity is related to the toxicity caused by the reactive metabolites. This study aims to investigate the target components of CP that generate reactive metabolic toxicity. The fluorescent probe method was used to detect glutathione (GSH)-trapped reactive metabolites in a co-incubation system of CP extract with rat liver microsomes. Identification of GSH conjugates was performed by LC-MS/MS and that of the possible precursor components that produce reactive metabolites was conducted by UPLC-Q-TOF/MS. Cell viability assays were performed on HepG2 and L02 cells to determine the cytotoxicity of the target components. The findings of our study demonstrate that the extract derived from CP has the ability to generate metabolites that exhaust the intracellular GSH levels, resulting in the formation of GSH conjugates and subsequent cytotoxic effects. Through the utilization of the UPLC-Q-TOF/MS technique, we were able to accurately determine the molecular weight of the precursor compound in CP to be 355.1023. The primary evidence to determining the GSH conjugetes relies on the appearance of characteristic product ions resulting from central neutral loss (CNL) scanning of 129 Da and product scanning of m/z 660 in the positive MS/MS spectrum. Through analysis, it was ultimately ascertained that the presence of chlorogenic acid (CGA) and its isomers, namely neochlorogenic acid (NCGA) and cryptochlorogenic acid (CCGA), could lead to the production of GSH conjugates, resulting in cytotoxicity at elevated levels. Taking these findings into consideration, the underlying cause for the potential hepatotoxicity of CP was initially determined.

Comprehensive phytochemical analysis of Salvia hispanica L. callus extracts using LC-MS/MS.

In this research, the study utilized the root, leaf, and petiole parts of in vitro grown Salvia hispanica plants as explants. Following UV-C treatment applied to developing callus, methanol extracts were obtained and analyzed using liquid chromatography-mass spectrometry (LC/MS) to investigate their anticancer properties. First, the seeds of S. hispanica were soaked in commercial bleach for 6 min to ensure surface sterilization. The most effective antimicrobial activity on Gram-negative bacteria, with a zone diameter (11 ± 0.82 mm), was noticed in callus extracts obtained from the petiole explant in the second protocol against Klebsiella pneumoniae EMCS bacteria. Anticancer activities on SH-SY5Y human neuroblastoma cells were investigated by using 1000, 500, 250, 125, 62.5, 31.25, 15.62, and 78.12 µg/mL doses of the extracts, and the most effective cytotoxic activity was determined at the 1000 µg/mL dose of the extracts obtained from both protocols. The extracts were determined to inhibit hCAI, hCAII, AChE, and BChE enzymes. The content of 53 different phytochemical components of the extracts was analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Rosmarinic acid, quinic acid, and caffeic acid were found in the highest concentration. The comprehensive LC-MS/MS analysis of S. hispanica extracts revealed a diverse array of phytochemical compounds, highlighting its potential for therapeutic applications.

Screening the quality of legal and illegal dietary supplements by LC-MS/MS.

Dietary supplements are widely consumed. However, the lack of mandatory testing results in limited data on their quality, particularly in Eastern Europe. In this study, 21 legally registered and 9 illegal supplements, seized from an underground facility by the Polish Police, were examined. Contaminants were screened by utilising high-performance liquid chromatography coupled with untargeted mass spectrometry. The analysis identified 32 contaminants in the 30 dietary supplements examined. Untargeted analysis revealed a concerning issue: the intentional adulteration of both legal and illegal supplements with pharmacologically active substances that are prohibited in this category of products. This study indicated that many dietary supplements are of low quality due to deliberate adulteration or inadequate manufacturing conditions. The presence of unregistered or unapproved substances in these supplements poses serious health risks. Strong legal regulations are essential to address this issue effectively.

Determination of Normal Range of Acylcarnitine in Neonatal Dried Blood Spots using LC-MS/MS.

Background: Acylcarnitine is one of the crucial markers of fatty acid metabolism, and examination of their level in infants can reveal several Inherited Metabolic Disorders (IDM) or Inborn errors of Metabolism (IEM). Because of the great importance of hereditary, metabolic, and other inherited disorders early diagnosis before the appearance of clinical symptoms, this study was carried out to establish a reference range for carnitine analytes and to identify acylcarnitine profiles in normal weight neonatal dried blood spots (DBS) specimens. Methods: By using liquid chromatography tandem mass spectrometry (LC-MS/MS) for neonatal screening and eventually the examination and analysis of LC-MS/MS results, 34 acylcarnitine derivatives were identified. Results: The normal range for acylcarnitine analytes with carbon numbers ranging from zero to 18, both main and the branched ones, were ultimately measured. Afterward, they were compared with the results of some other diagnostic laboratories to be verified. Conclusions: This study differed from the other findings, which could be due to diversity in population and work methods. However, the reference range of most acylcarnitine derivatives in Tehran closely aligned with this study's findings.

A multianalyte LC-MS/MS method for accurate quantification of Nitrosamines in Olmesartan tablets.

This research summaries the development, optimization and validation of liquid chromatography tandem mass spectrometric (LC-MS/MS) method for concurrent measurement of seven nitrosamines viz; NDMA, NDEA, NDIPA, NDPA, NEIPA, NMPA & NMBA in Olmesartan tablet. Controlling these nitrosamines at trace levels is imperative for ensuring the safety of drug substances and products for consumption. Various regulatory authorities stress the significance of utilizing highly sensitive analytical methods to precisely measure nitrosamines at trace levels. The method applied effective chromatographic separation and optimized parameters for mass spectrometric detection. Detection was carried out using APCI positive ion mode. Chromatographic separation was achieved using a Thermo Accucore PFP column (150 mm x 4.6 mm, 2.6 µ), with a simple gradient elution of mobile phase consisting of 0.1 % formic acid in water (mobile phase A) and methanol (mobile phase B). The total run time was 20 min, with a flow rate of 0.800 mL/min. The method was validated according to the International Council on Harmonisation (ICH Q2 (R2)) guidelines. The established method demonstrated excellent linearity (R2> 0.99) and sensitivity for all the nitrosamines. Detection and quantification limits were sufficiently low for trace nitrosamine levels having good S/N ratio. The method showed good accuracy in Olmesartan tablet samples, with recoveries ranges between 80 % to 120 %. The new analytical approach has exceptional repeatability and reliability, making it possible to precisely quantify the levels of seven nitrosamines in Olmesartan medoxomil tablets in a single analytical run.

Forensic toxicological studies of acute glufosinate poisoning: A case series.

Glufosinate is a widely and increasingly used non-selective, broad-spectrum herbicide. Although cases of glufosinate poisoning are frequently reported, they are rarely documented in forensic case reports, particularly in fatal instances. The present study examined six cases of glufosinate poisoning, including a fatal case involving a 25-year-old female found deceased by the roadside, with an empty 1000 mL bottle labeled "glufosinate" by her side. Biological specimens such as plasma or cardiac blood, gastric contents, and liver tissues were collected for quantitative analysis of glufosinate levels using LC-MS/MS. In five cases of acute glufosinate poisoning, glufosinate plasma concentrations ranged from 0.62 to 3.92 µg/mL. In the fatal case, the concentrations of glufosinate in cardiac blood, gastric contents, and liver tissues were 8.41 µg/mL, 31.25 µg/mL, and 66.1 µg/g, respectively. The pathological autopsy concluded that the cause of death was acute cardio-respiratory failure due to glufosinate poisoning, characterized by multi-organ congestion without specific pathological findings. The toxicological data provided in this study aim to serve as a critical reference for future clinical treatment and forensic validation of glufosinate poisoning-related deaths.

Enhanced analysis of equine plasma for the presence of recombinant human erythropoietin - Implementation of an improved workflow.

An improved screening workflow and a robust capillary flow LC-MS confirmatory method for the detection of recombinant human erythropoietin (rHuEPO) has been implemented to increase the sensitivity of rHuEPO detection and to reduce the number of suspect samples committed to confirmatory testing. The influence of repeated dosing of epoetin-ß on the detection window of rHuEPO in equine plasma was assessed using the optimised method. Samples were initially assessed using an economical R&D Human EPO Duo-Set ELISA Development System. Samples indicating a result greater than the batch baseline were analysed using the complementary R&D Human EPO Quantikine IVD ELISA kit. All samples recording an abnormal screening result were subjected to confirmatory analysis. Confirmation of rHuEPO in plasma (≥2.5 ml) in the range of 4-13 mIU/ml (n = 6) was achieved using immunoaffinity enrichment, tryptic digestion, and capillary flow LC-MS/MS. Four horses were administered a single dose of epoetin-ß (10,000 IU) via the subcutaneous and intravenous routes, on two occasions, seven days apart. The excretion profile was rapid with epoetin-ß detection times of 48 to 72 h following each administration, with no appreciable difference observed between the two routes of administration. This workflow has been shown as an effective anti-doping strategy related to rHuEPO misuse and supports the use of out-of-competition testing of horses in the 2 to 3-day period prior to race-day.

Validation of analytical methods for newly regulated veterinary drug residues in livestock and fishery products with maximum residue limits in Republic of Korea.

Veterinary drugs play a crucial role in the treatment of various animal diseases. However, their residues, stemming from issues, such as withdrawal period lapses, overuse, or abuse, can jeopardize food safety and human health. This study addresses recent regulations in Korea concerning specific veterinary drugs (anacolin, ephedrine, menichlopholan, piperonyl butoxide, and etisazole HCl) and their ongoing discussions. This study aimed to validate two pre-developed methods for quantifying residues in livestock and fishery products using QuEChERS and liquid chromatography-tandem mass spectrometry. Both methods exhibited excellent linearity, recoveries (70.3-119%), and coefficient of variations (1.3-28%), along with low limits of detection and quantification (0.3-4 ng/g and 1-12 ng/g). This study is significant for its contribution to the detection of veterinary drugs in livestock and fishery products, given the limited research available on the methods for analyzing these substances.

Identification of chemoresistance targets in doxorubicin-resistant lung adenocarcinoma cells using LC-MS/MS-based proteomics.

Acquired chemoresistance remains a significant challenge in the clinics as most of the treated cancers eventually emerge as hard-to-treat phenotypes. Therefore, identifying chemoresistance targets is highly warranted to manage the disease better. In this study, we employed a label-free LC-MS/MS-based quantitative proteomics analysis to identify potential targets and signaling pathways underlying acquired chemoresistance in a sub-cell line (A549DR) derived from the parental lung adenocarcinoma cells (A549) treated with gradually increasing doses of doxorubicin (DOX). Our proteomics analysis identified 146 upregulated and 129 downregulated targets in A549DR cells. The KEGG pathway and Gene ontology (GO) analysis of differentially expressed upregulated and downregulated proteins showed that most abundant upregulated pathways were related to metabolic pathways, cellular senescence, cell cycle, and p53 signaling. Meanwhile, the downregulated pathways were related to spliceosome, nucleotide metabolism, DNA replication, nucleotide excision repair, and nuclear-cytoplasmic transport. Further, STRING analysis of upregulated biological processes showed a protein-protein interaction (PPI) between CDK1, AKT2, SRC, STAT1, HDAC1, FDXR, FDX1, NPC1, ALDH2, GPx1, CDK4, and B2M, proteins. The identified proteins in this study might be the potential therapeutic targets for mitigating DOX resistance.

Antioxidant and Anti-Inflammatory Capacities of Three Dry Bean Varieties after Cooking and In Vitro Gastrointestinal Digestion.

The present study delved into the chemical composition, antioxidant, and anti-inflammatory properties of three dry edible beans: Black (BL), Great Northern (GN), and Pinto (PN). The beans were soaked, cooked, and subjected to in vitro gastrointestinal (GI) digestion. BL bean exhibited significantly higher gastric (42%) and intestinal (8%) digestion rates. Comparative assessment of soluble GI-digested fractions (<3 kDa) revealed that the GN bean exhibited the highest abundance of dipeptides (P < 0.05). The BL bean fraction displayed a 4-fold increase in tripeptides (P < 0.05). Both BL and PN bean fractions are high in essential free amino acids, flavonols, and derivatives of hydroxybenzoic acid when compared to the GN bean. All the beans exhibited the ability to mitigate TNF-α-induced pro-inflammatory signaling; however, the BL bean fraction was the most effective at lowering AAPH-induced oxidative stress in HT-29 cells, followed by the GN bean (P < 0.05). In contrast, a low antioxidant effect was observed with PN beans.

Detection and quantification of selected cannabinoids in oral fluid samples by protein precipitation and LC-MS/MS.

Cannabis is the most widely consumed illicit drug worldwide. As consumption rates increase, partially due to the decriminalization of its use for medicinal and recreational purposes, analytical methods for monitoring different cannabinoids in several biological matrices have been developed. Herein, a simple and fast extraction procedure to extract natural cannabinoids from oral fluid (OF) samples was developed and fully validated according to the ANSI/ASB 2019 Standard Practices for Method Validation in Forensic Toxicology. Using only 0.2 mL of neat OF, the analytes [Δ9-tetrahidrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), cannabinol (CBN) and cannabidiol (CBD)] were extracted by protein precipitation with a mixture of methanol:acetonitrile (80:20, v/v); the extracts were centrifuged, evaporated to dryness and reconstituted in 100 µL of methanol. Analysis was performed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The developed methodology produced linear results for all compounds, with working ranges of 0.1-50 ng/mL for THC, 0.5-50 ng/mL for THC-OH, CBN and CBD, and 0.05-1 ng/mL for THC-COOH. Ion suppression was observed for THC, CBN and CBD, which did not impair sensitivity considering the low limits of quantification (LOQs) and limits of detection (LODs) obtained (which varied between 0.05 and 0.5 ng/mL). The extraction procedure produced great recoveries, and the compounds were stable. No interferences were found, and the method proved to be extremely fast, selective, precise, and accurate for use in routine analysis. The method was successfully applied to authentic samples.

Validation of a method for the determination of Aderamastat (FP-025) in K2EDTA human plasma by LC-MS/MS.

Aderamastat (FP-025) is a small molecule, selective matrix metalloproteinase (MMP)-12 inhibitor, under development for respiratory conditions which may include chronic inflammatory airway diseases and pulmonary fibrosis. To support evaluation of the pharmacokinetic parameters of Aderamastat in humans, we developed and validated a high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analytical method for the quantification of Aderamastat in human plasma. This assay was validated in compliance with the Food and Drug Administration (FDA) Good Laboratory Practice Regulations (GLP) and European Medicines Agency (EMA) guidelines. K2EDTA human plasma samples were spiked with internal standard, processed by liquid-liquid extraction, and analyzed using reversed-phase HPLC with Turbo Ion Spray® MS/MS detection. Separation was done using a chromatographic gradient on 5 µm C6-Phenyl 110 Å, 50*2 mm analytical column at a temperature of 35 °C. The LC-MS/MS bioanalytical method, developed by QPS Taiwan to determine the concentration of Aderamastat in K2EDTA human plasma, was successfully validated with respect to linearity, sensitivity, accuracy, precision, dilution, selectivity, hemolyzed plasma, lipemic plasma, batch size, recovery, matrix effect, and carry-over. These data indicate that the method for determination of Aderamastat concentrations in human K2EDTA plasma can be used in pharmacokinetics studies and subsequent clinical trials with Aderamastat. Authors declare that, this novel data is not published and not under consideration for publication by another journal than this journal. All data will be made available on request.