Multiple Reaction Monitoring Research Articles

Development and Validation of a Sensitive Liquid Chromatography-Tandem Mass Spectrometry Method for Therapeutic Drug Monitoring of Ceftolozane and Tazobactam in Human Plasma Microsamples.

Ceftolozane/tazobactam (C-T) is a novel beta-lactam/beta-lactamase inhibitor combination approved for the treatment of various infections caused by difficult-to-treat Pseudomonas aeruginosa. In critically ill patients, C-T may exhibit significant pharmacokinetic variability, both between individuals and within individuals, warranting therapeutic drug monitoring for clinical purposes. We aim to develop and validate a novel and sensitive analytical method for concurrently determining C and T in human plasma microsamples (3μL). The method was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with positive electrospray ionization and multiple reaction monitoring (MRM) detection modes, employing specific mass transitions for both drugs. Sample preparation was simple, and the chromatographic run lasted only 4 minutes. Validation was conducted according to European Medicines Agency (EMA) guidelines, encompassing specificity, sensitivity, linearity, precision, accuracy, matrix effect, extraction recovery, limit of quantification, and drug stability. The validated method was applied to measure C and T in 32 plasma samples collected from critically ill patients with multidrug-resistant, gram-negative, bacterial infections. The method ensured accurate (BIAS% 2.1-9.6 for C and -2.2 to 15.2 for T) and precise intraday CV% for C: 6.7-5.5; for T: 1.3-8.9; interday CV% for C 6.0-10.8; for T 4.1-10.2) measurements of C-T over a wide concentration range (0.2-200.0 mg/L for C and 0.1-100.0 mg/L for T). Overall, the recovery at quality control concentration levels was high for both C and T (mean values: 90-91 for C and 89-92 for T). Analyte stability was satisfactory in both human plasma and extracts under various storage conditions. The clinical applicability of the assay was confirmed by the reliably quantifying C and T in clinical plasma samples. The developed and validated LC-MS/MS method is sensitive and suitable for monitoring C and T in human plasma microsamples.

New Sweet-Tasting Gypenosides from "Jiaogulan" (Gynostemma pentaphyllum) and Their Interactions with the Homology Model of Sweet Taste Receptors.

Gynostemma pentaphyllum has been used as an herbal tea, vegetable, and dietary supplement for hundreds of years in East Asia. The sweet variety, grown in large areas in Fujian Province, China, is an essential source of "Jiaogulan" herbal tea. However, its sweet components are unknown. To investigate the sweet constituents of Fujian "Jiaogulan" and discover new natural high-potency sweeteners, phytochemical and sensory evaluations were combined to obtain 15 saponins, of which 11 (1-11) were sweet-tasting, including 2 new ones with sweetness intensities 20-200 times higher than that of sucrose, and four (12-15) were bitter-tasting. Their structures were elucidated using spectroscopic methods (NMR, MS, IR, UV), hydrolysis, and comparison with literature data. The contents of the 15 saponins were quantitatively analyzed using UPLC-MS/MS in multiple reaction monitoring mode. The contents of 1 and 2 sweet-tasting gypenosides were 9.913 ± 1.735 and 35.852 ± 1.739 mg/kg, respectively. The content of the sweetest compound (6) was 124.969 ± 0.961 mg/kg. Additionally, compound 4 was the most abundant sweet component (422.530 ± 3.702 mg/kg). Furthermore, molecular docking results suggested interactions of sweet saponins with sweet taste receptors. In general, this study revealed the material basis of the Fujian "Jiaogulan" taste.

A Validated Method for the Simultaneous Determination of Oxytocin and Cortisol in Human Saliva

Oxytocin and cortisol (OXY and CORT) are hormones related to stress, cognitive, and social behaviors. Their detection is relevant to epidemiological studies aimed at investigating the effects of stressor factors on human life. The aim of this study was to develop and validate an assay for the measurement of OXY and CORT in saliva samples using liquid chromatography/tandem mass spectrometry (LC-MS/MS) in the presence of deuterated analogs. A 500 mL aliquot of oral fluid, obtained by the centrifugation of a chewed swab, was purified by solid-phase extraction. Analytes were then separated using C18 reversed-phase chromatography, subjected to positive electrospray ionization, and then quantified using a triple-quadrupole mass detector in multiple-reaction monitoring mode. The limits of quantification and the linear dynamic ranges were 2.0 × 10−3 and 0.5 nmol/L, and up to 1.0 × 10−1 and 20 nmol/L for OXY and CORT, respectively. Inter- and intra-run precision, expressed as relative standard deviation, was <7%, and accuracy was within 93–104% of the theoretical concentrations. The evaluation of matrix effects showed that the use of internal standards controlled sources of bias. The high sensitivity of the method allowed the quantification of OXY and CORT in the salivary samples of both adults and children: levels of CORT ranged from 0.6 to 18.5 nmol/L, while OXY levels were two orders of magnitude lower (from 1.7 × 10−3 to 1.1 × 10−2 nmol/L). To our knowledge, this is the first method that can analyze, in the same chromatographic run, both hormones in saliva samples.

Systematic Proteome Profiling of Maternal Plasma for Development of Preeclampsia Biomarkers

Preeclampsia (PE) is a hypertensive disorder of pregnancy with various clinical symptoms. However, traditional markers for the disease including high blood pressure and proteinuria are poor indicators of the related adverse outcomes. Here, we performed systematic proteome profiling of plasma samples obtained from pregnant women with PE to identify clinically effective diagnostic biomarkers. Proteome profiling was performed using TMT-based liquid chromatography-mass spectrometry (LC-MS/MS) followed by subsequent verification by multiple reaction monitoring (MRM) analysis on normal and PE maternal plasma samples. Functional annotations of differentially expressed proteins (DEPs) in PE were predicted using bioinformatic tools. The diagnostic accuracies of the biomarkers for PE were estimated according to the area under the receiver-operating characteristics curve (AUC). A total of 1307 proteins were identified, and 870 proteins of them were quantified from plasma samples. Significant differences were evident in 138 DEPs, including 71 upregulated DEPs and 67 downregulated DEPs in the PE group, compared with those in the control group. Upregulated proteins were significantly associated with biological processes including platelet degranulation, proteolysis, lipoprotein metabolism, and cholesterol efflux. Biological processes including blood coagulation and acute-phase response were enriched for down-regulated proteins. Of these, 40 proteins were subsequently validated in an independent cohort of 26 PE patients and 29 healthy controls. APOM, LCN2, and QSOX1 showed high diagnostic accuracies for PE detection (AUC >0.9 and p<0.001, for all) as validated by MRM and ELISA. Our data demonstrate that three plasma biomarkers, identified by systematic proteomic profiling, present a possibility for the assessment of PE, independent of the clinical characteristics of pregnant women.

Development of a Fit-For-Purpose Multi-Marker Panel for Early Diagnosis of Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) suffers from a lack of an effective diagnostic method, which hampers improvement in patient survival. Carbohydrate antigen 19-9 (CA19-9) is the only FDA-approved blood biomarker for PDAC, yet its clinical utility is limited due to suboptimal performance. Liquid chromatography-mass spectrometry (LC-MS) has emerged as a burgeoning technology in clinical proteomics for the discovery, verification, and validation of novel biomarkers. A plethora of protein biomarker candidates for PDAC have been identified using LC-MS, yet few has successfully transitioned into clinical practice. This translational standstill is owed partly to insufficient considerations of practical needs and perspectives of clinical implementation during biomarker development pipelines, such as demonstrating the analytical robustness of proposed biomarkers which is critical for transitioning from research-grade to clinical-grade assays. Moreover, the throughput and cost-effectiveness of proposed assays ought to be considered concomitantly from the early phases of the biomarker pipelines for enhancing widespread adoption in clinical settings. Here, we developed a fit-for-purpose multi-marker panel for PDAC diagnosis by consolidating analytically robust biomarkers as well as employing a relatively simple LC-MS protocol. In the discovery phase, we comprehensively surveyed putative PDAC biomarkers from both in-house data and prior studies. In the verification phase, we developed a multiple-reaction monitoring (MRM)-MS-based proteomic assay using surrogate peptides that passed stringent analytical validation tests. We adopted a high-throughput protocol including a short gradient (<10min) and simple sample preparation (no depletion or enrichment steps). Additionally, we developed our assay using serum samples, which are usually the preferred biospecimen in clinical settings. We developed predictive models based on our final panel of 12 protein biomarkers combined with CA19-9, which showed improved diagnostic performance compared to using CA19-9 alone in discriminating PDAC from non-PDAC controls including healthy individuals and patients with benign pancreatic diseases. A large-scale clinical validation is underway to demonstrate the clinical validity of our novel panel.

Simultaneous determination of 21 perfluorinated and polyfluoroalkyl substances in plant oils by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry

Edible plant oils are a key component of the daily human diet, and the quality and safety of plant oils are related to human health. Perfluorinated and polyfluoroalkyl substances (PFASs) are pollutants that can contaminate plant oil through the processing of raw materials or exposure to materials containing these substances. Thus, establishing a sensitive and accurate analytical method for the determination of PFASs is critical for ensuring the safety of plant oils. In this study, a method based on acetonitrile extraction and solid phase extraction purification combined with ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS/MS) was developed for the simultaneous determination of 21 PFASs, including perfluorocarboxylic acids, perfluoroalkyl sulfonic acids, and fluorotelomer sulfonic acids, in edible plant oils. The chromatographic conditions and MS parameters were optimized, and the influences of the extraction solvents and purification method were systematically studied. Plant oil samples were directly extracted with acetonitrile and purified using a weak anion-exchange (WAX) column. The 21 target PFASs were separated on a reversed-phase C18 chromatographic column and detected using a triple quadrupole mass spectrometer with an electrospray ionization source. The mass spectrometer was operated in negative-ion mode. The target compounds were analyzed in multiple reaction monitoring (MRM) mode and quantified using an internal standard method. The results demonstrated that the severe interference observed during the detection of PFASs in the co-extracted substances was completely eliminated after the extraction mixture was purified using a WAX column. The 21 target PFASs showed good linearity in their corresponding ranges, with correlation coefficients greater than 0.995. The limits of detection (LODs) and limits of quantification (LOQs) of the method were in the range of 0.004-0.015 and 0.015-0.050 μg/kg, respectively. The recoveries ranged from 95.6% to 115.8%, with relative standard deviations (RSDs) in the range of 0.3%-10.9% (n=9). The established method is characterized by simple sample pretreatment, good sensitivity, high immunity to interferences, and good stability, rendering it suitable for the rapid analysis and accurate determination of typical PFASs in edible plant oils.

Determination of Cotinine, 3'-Hydroxycotinine and Nicotine 1'-Oxide in Urine of Passive and Active Young Smokers by LC-Orbitrap-MS/MS Technique.

Tobacco smoke is probably the most significant factor conducing to toxic xenobiotics exposure to humans. The aim of the study was to develop a rapid and sensitive method for the determination of selected nicotine metabolites in urine of tobacco smokers and passive smokers. The method for removing protein and extracting the metabolites involved the centrifugation of urine with acetonitrile. Cotinine, trans-3'-hydroxycotinine, and (2'S)-nicotine 1'-oxide in the supernatant were determined using the LC-Orbitrap-MS/MS technique, with the selected ion monitoring (SIM) and parallel reaction monitoring (PRM) modes used. The recovery of these analytes added to the urine samples ranged from 72% to 101%. Repeatability and reproducibility were less than 3.1% and 10.1%, respectively. The study was carried out among medical students. The group was selected as representatives of young people and who as future physicians should be more aware of the effects of nicotine use. Concentration levels of cotinine and trans-3'-hydroxycotinine determined in ng/mL in the urine of cigarette smokers were 70- and 58-fold higher, respectively, compared to passive smokers. Higher concentrations were recorded in the urine of those passively exposed to tobacco smoke than in non-smokers, confirming that passive exposure to tobacco smoke is not harmless to the human body. However, no significant differences were observed in the concentration of (1'S,2'S)-nicotine 1'-oxide in the samples of individuals from various groups.

TWO-DIMENSIONAL ION CHROMATOGRAPHY TANDEM-MASS SPECTROMETRIC (2D-IC-MS/MS) METHOD FOR THE ANALYSIS OF PHOSPHORUS COMPOUNDS IN SOIL

Investigations into soil organic phosphorus (Po) dynamics are instrumental in understanding the transformations and processes responsible for ecosystem productivity. However, quantitative analysis of Po in a soil environment is extremely challenging due to low target analyte concentrations and matrix interferences with chromatography and analysis. Consequently, a two-dimensional ion chromatography-tandem mass spectrometric (2D-IC-MS/MS) method was developed to estimate soil Po concentrations. The first dimension diverted early eluting anions to waste while preconcentrating P compounds in a trap column, followed by chromatographic separation and detection in the second dimension. Detection was done using a mass spectrometer, and quantification was performed using the multiple reaction monitoring scan (MRM) method. The linear range of the studied P compounds, mostly nucleotides, was 0.05 – 50 ng/mL. Most P compounds were detected and quantified in calcareous subsoil samples in the concentration ranges 0.70 – 51.78 ng/g. The developed method achieved chromatographic separation that allowed unambiguous identification of isobars/isomers and isotopologues contributing to interferences in MS detection. However, improvements to the extraction method and post-clean-up procedures are required due to the complexity of soil extract composition, extreme matrix effect and/or loss of analyte during preconcentration. The method is ideal for simultaneously analyzing P compounds from environmental samples to elucidate key components of the soil P dynamics.

Bacteria-photocatalyst biohybrid system for sustainable ammonium production

Although the Haber–Bosch process supports the growth of modern agriculture with abundant ammonia and fertilizer production, substantial energy consumption and enormous greenhouse emissions demand an alternative and sustainable approach. Here, we report a novel approach that combines the non-photosynthetic bacterium Shewanella oneidensis MR-1 (S. oneidensis MR-1) with cadmium sulfide nanoparticles (CdS NPs) to enable the photosynthesis of ammonium (NH4+) from nitrate (NO3−) using photoexcited electrons as donors. The NO3− reduction efficiency reached almost 100%, with an NH4+ production selectivity of over 90%. The maximum instantaneous quantum efficiency was 3.01% under light irradiation. The reverse metal-reducing (Mtr) pathway is responsible for the transfer of photoexcited electrons to intracellular compartments. Parallel reaction monitoring analysis illustrated that NO3− to NH4+ was produced via the dissimilatory nitrate reduction to ammonium (DNRA) pathway in S. oneidensis MR-1. This study provides a facile strategy for light-driven ambient NH4+ synthesis and solar-to-chemical conversion.

UPLC-MS/MS-Based Target Screening of 90 Phosphodiesterase Type 5 Inhibitors in 5 Dietary Supplements.

The aim of individuals consuming health supplements is to attain a robust state through nutritional regulation. However, some unscrupulous manufacturers, motivated by profit, fraudulently incorporate drugs or unauthorized components with therapeutic effects into the product for instant product performance enhancement. The long-term use of these products may inadvertently inflict harm on human health and fail to promote nutritive healthcare. The illegal inclusion of these substances is prevalent in kidney-tonifying and sexuality-enhancing products. Developing effective analytical methods to identify these products and screen for illegal added ingredients can effectively prevent such products from reaching and remaining on the market. A target screening method for the detection and quantification of 90 phosphodiesterase type 5 inhibitors (PDE-5is) in 5 kinds of health products was developed and validated. The type of dietary supplements varied from tablets, capsules, and protein powder to wine and beverages. Sample preparation was completed with a one-step liquid phase extraction. The screening process of 90 PDE-5is was done efficiently within 25 min by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) using the dynamic multiple reaction monitoring (dMRM) technique. The LODs of 90 PDE-5is were detected at levels ranging from 25 to 85 ng/g or ng/mL. This novel targeting methodology was effective and can be applied to routine market supervision. Among 286 batches of samples, 8 batches were found to be positive. Three kinds of PDE-5is were first detected in healthy products. The screening method demonstrated herein will be a promising and powerful tool for rapid screening of PDE-5is.

Simultaneous quantification of baloxavir marboxil and its active metabolite in human plasma using UHPLC-MS/MS: Application to a human pharmacokinetic study with different anticoagulants

Baloxavir marboxil (BXM) is a cap-dependent nucleic acid endonuclease inhibitor, which exerts its antiviral effects after being metabolized to its active form baloxavir acid (BXA). Ethylenediamine tetra-acetic acid (EDTA) and heparin are the two most used anticoagulants in clinical blood sample collection to estimate drug levels in plasma. However, compared to heparin plasma, there is a lack of clinical pharmacokinetic data of BXA using EDTA anticoagulant tubes for blood collection. In the present study, an efficient, rapid, and sensitive ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for simultaneous quantification of BXM and its active metabolite BXA in human plasma with its isotopic baloxavir-d5 (BXA-d5) as internal standard (IS). Plasma samples (50 μL) were undergone using acetonitrile containing 0.1 % formic acid a precipitant. Chromatographic separation was achieved by a Waters XBridge®C8 (2.1 mm × 50 mm, 2.5 µm) column. The gradient mobile phase was 0.1 % formic acid in water (A, pH 2.8) and 0.1 % formic acid in acetonitrile (B) and delivered at a flow rate of 0.6 mL/min for 4.5 min. BXM and BXA were monitored using a positive electrospray triple quadrupole mass spectrometer (TRIPLE QUAD™ 6500+) via multiple reaction monitoring mode. The mass-to-charge ratios (m/z) were 572.2→247.0, 484.2→247.0 and 489.2→252.0 for BXM, BXA, and BXA-d5 (IS). Calibration curves exhibited excellent linearity in the range of 0.1–10 ng/mL for BXM (r2 > 0.996), and 0.3–300 ng/mL for BXA (r2 > 0.998). Within-run and between-run precisions in coefficients of variations were less than 11.62 % for BXM and less than 7.47 % for BXA, and accuracies in relative error were determined to be within −7.78 % to 5.70 % for BXM and −6.67 % to 8.56 % for BXA. Extraction recovery efficiency was 92.76 % for BXM, 95.32 % for BXA, and 99.26 % for BXA-d5, respectively. The matrix effect of BXM and BXA was in line with the requirements, where the relative deviation of the accuracy was less than 6.67 % and the precision was less than 6.69 %. The validated efficient and simple UHPLC-MS/MS method was successfully used in the pharmacokinetic study of BXM and BXA in healthy human volunteers with K2EDTA and heparin tubes for blood collection. EDTA might compete with BXA for chelating metal ions and thereby decrease the plasma ratio in whole blood, leading to approximately 50 % lower measurement of pharmacokinetic parameters as compared with those obtained from heparin plasma anticoagulant tubes.

Quantitative Analysis of Glutathione and Carnosine Adducts with 4-Hydroxy-2-nonenal in Muscle in a hSOD1G93A ALS Rat Model.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the dysfunction and death of motor neurons through multifactorial mechanisms that remain unclear. ALS has been recognized as a multisystemic disease, and the potential role of skeletal muscle in disease progression has been investigated. Reactive aldehydes formed as secondary lipid peroxidation products in the redox processes react with biomolecules, such as DNA, proteins, and amino acids, resulting in cytotoxic effects. 4-Hydroxy-2-nonenal (HNE) levels are elevated in the spinal cord motor neurons of ALS patients, and HNE-modified proteins have been identified in the spinal cord tissue of an ALS transgenic mice model, suggesting that reactive aldehydes can contribute to motor neuron degeneration in ALS. One biological pathway of aldehyde detoxification involves conjugation with glutathione (GSH) or carnosine (Car). Here, the detection and quantification of Car, GSH, GSSG (glutathione disulfide), and the corresponding adducts with HNE, Car-HNE, and GS-HNE, were performed in muscle and liver tissues of a hSOD1G93A ALS rat model by reverse-phase high-performance liquid chromatography coupled to electrospray ion trap tandem mass spectrometry in the selected reaction monitoring mode. A significant increase in the levels of GS-HNE and Car-HNE was observed in the muscle tissue of the end-stage ALS animals. Therefore, analyzing variations in the levels of these adducts in ALS animal tissue is crucial from a toxicological perspective and can contribute to the development of new therapeutic strategies.

Development and validation of an LC-MSMS method for the quantitation of pacritinib; application of kinetics in rabbits

BackgroundAccurate and selective LC/ESI-MSMS method development and validation for the quantitation of pacritinib is the primary goal of this study to perform kinetic studies in the healthy rabbit. MethodsChromatographic resolution was accomplished with a hypersil/ODS (50 mm × 4.6 mm, 3 μ) analytical C18 column and a mobile phase composition of 0.1% formic acid and ACN in the proportion of 25:75 with a 0.6 ml/min flow of the mobile phasic system from the analytical column. The method was employed by monitoring the established ionic transitions of m/z-473.25/98.09 for Pacritinib and 506.18/57.12 for the internal standard (Amprenavir) in multiple reaction monitoring. ResultsThe calibration plot regression line was y = 0.0002× + 0.007, with a correction coefficient (r2) of 0.9989. The CV outcomes for the matrix effect at low-QC and high-QC levels were 4.79% and 4.91%, respectively. The percentage average recoveries for Pacritinib in High-QC (12.70 μg/ml), MQC (8.50 μg/ml), and Low-QC (1.19 μg/ml) were 95.87%, 103.64%, and 94.32%, respectively. The obtained values were found between 2.98 and 5.07% for the QC (1.19, 8.50, and 12.70 μg/ml) samples. The established procedure was subjected to kinetics study of Pacritinib after oral administration in rabbits. Cmax, Tmax, and T1/2, of the Pacritinib tablets were 247.25 ± 3.32 ng/ml, 6.0 ± 0.03 h, and 12.24 ± 0.53 h, respectively. AUC0-∞ infinity for Pacritinib tablets was 1691.74 ± 3.67 ng h/ml. ConclusionAfter oral administration of Pacritinib to healthy rabbits, pharmacokinetic characteristics were presented, and the established technique was effectively verified.

Bridging Targeted (Zeno MRM-HR) and Untargeted (SWATH) LC-HRMS in a Single Run for Sensitive Exposomics.

Traditionally, chemical exposure has been assessed by low-resolution mass spectrometry via targeted approaches due to the typically extremely low concentration of such compounds in biological samples. Nevertheless, untargeted approaches are now becoming a promising tool for a broader investigation of the exposome, covering additional compounds, their biotransformation products, and possible metabolic alterations (metabolomics). However, despite broad compound coverage, untargeted metabolomics still underperforms in ultratrace biomonitoring analysis. To overcome these analytical limitations, we present the development of the first combined targeted/untargeted LC-MS method, merging MRM-HR and SWATH experiments in one analytical run, making use of Zeno technology for improved sensitivity. Multiple reaction monitoring transitions were optimized for 135 highly diverse toxicants including mycotoxins, plasticizers, PFAS, personal care products ingredients, and industrial side products as well as potentially beneficial xenobiotics such as phytohormones. As a proof of concept, standard reference materials of human plasma (SRM 1950) and serum (SRM 1958) were analyzed with both Zeno MRM-HR + SWATH and SWATH-only methodologies. Results demonstrated a significant increase in sensitivity represented by the detection of lower concentration levels in spiked SRM materials (mean value: 2.2 and 3 times lower concentrations for SRMs 1950 and 1958, respectively). Overall, the detection frequency was increased by 68% (19 to 32 positive detections) in the MRM-HR + SWATH mode compared to the SWATH-only. This work presents a promising avenue for addressing the outstanding key challenge in the small-molecule omics field: finding a balance between high sensitivity and broad chemical coverage. It was demonstrated for exposomic applications but might be transferred to lipidomics and metabolomics workflows.

Mercaptans in malodorants break disulfide bridges in human serum albumin and form adducts suitable as biomarkers of exposure in vitro.

Malodorants comprise notoriously smelling mercaptans and might be applied for crowd control. Because exposure to malodorants may lead to irritation of the respiratory system, choking, and coma, bioanalytical verification of poisoning might be required in a medical and forensic context. We herein present the detection and identification of novel biomarkers of exposure to ethyl mercaptan, n-butyl mercaptan, tert-butyl mercaptan, and iso-amyl mercaptan. These alkyl thiol compounds were found to form disulfide adducts in human serum albumin (HSA) in plasma in vitro with the only non-disulfide-bridged Cys34 residue and with other residues being part of the disulfide-bridged pattern in HSA. After proteinase K-catalyzed proteolysis, adducts of all mercaptans were detected simultaneously as the tripeptide Cys34*ProPhe and the dipeptides Cys369*Tyr, ValCys316*, and Cysx*Ala (x denominates either Positions 91, 200, 253, 361, and/or 448) by a sensitive micro-liquid chromatography-electrospray ionization tandem mass spectrometry (μLC-ESI MS/MS) method working in the scheduled multiple reaction monitoring (sMRM) mode. Time- and concentration-dependent adduct formations while exposure and proteolysis were investigated and the suitability of adducts as biomarkers of exposure was elaborated. Adducts at Cys34 showed the lowest limits of identification (LOIs, 6nM to 1.2μM mercaptan in plasma) and superior stability in plasma at 37°C. Therefore, Cys34*ProPhe appears as the most promising target to prove exposure to mercaptans at least in vitro.

Small RNA-regulated expression of efflux pump affects tigecycline resistance and heteroresistance in clinical isolates of Klebsiella pneumoniae

Tigecycline and the newly Food and Drug Administration-approved tetracyclines, including eravacycline and omadacycline, are regarded as last-resort treatments for multidrug-resistant Enterobacterales. However, tigecycline resistance in Klebsiella pneumoniae has increased, especially the underlying mechanism of heteroresistance is unclear. This study aimed to elucidate the mechanisms underlying tigecycline resistance and heteroresistance in clinical K. pneumoniae isolates. A total of 153 clinical K. pneumoniae isolates were collected, and identified 15 tigecycline-resistant and three tigecycline-heteroresistant isolates using broth microdilution and population analysis profile methods, respectively. Total RNAs from K. pneumoniae ATCC13883 and the laboratory-induced tigecycline-resistant strain were extracted and sequenced on an Illumina platform. Differentially expressed genes and regulatory small RNAs (sRNAs) were analyzed and validated in clinical isolates of K. pneumoniae using quantitative real-time PCR. RNA sequencing results showed that mdtABC efflux pump genes were significantly upregulated in the tigecycline-resistant strains. Overexpression of mdtABC was observed in a clinical K. pneumoniae isolate, which increased tigecycline minimum inhibitory concentrations (MICs) and was involved in tigecycline heteroresistance. Sequencing analysis of sRNA demonstrated that candidate sRNA-120 directly interacted with the mdtABC operon and was downregulated in tigecycline-resistant strains. We generated an sRNA-120 deletion mutation strain and a complemented strain of K. pneumoniae. The sRNA-120 deletion strain displayed increased mRNA levels of mdtA, mdtB, and mdtC and an increase in MICs of tigecycline. The complemented strain of sRNA-120 restored the mRNA levels of these genes and the susceptibility to tigecycline. RNA antisense purification and parallel reaction monitoring mass spectrometry were performed to verify the interactions between sRNA-120 and mdtABC. Collectively, our study highlights that the post-transcriptional repression of mdtABC through sRNA-120 may provide an additional layer of efflux pump gene expression control, which is important for resistance and heteroresistance in clinical K. pneumoniae isolates.

Rapid assay development for low input targeted proteomics using a versatile linear ion trap.

Advances in proteomics and mass spectrometry enable the study of limited cell populations, where high-mass accuracy instruments are typically required. While triple quadrupoles offer fast and sensitive low-mass accuracy measurements, these instruments are effectively restricted to targeted proteomics. Linear ion traps (LITs) offer a versatile, cost-effective alternative capable of both targeted and global proteomics. Here, we describe a workflow using a new hybrid quadrupole-LIT instrument that rapidly develops targeted proteomics assays from global data-independent acquisition (DIA) measurements without needing high-mass accuracy. Using an automated software approach for scheduling parallel reaction monitoring assays (PRM), we show consistent quantification across three orders of magnitude in a matched-matrix background. We demonstrate measuring low-level proteins such as transcription factors and cytokines with quantitative linearity below two orders of magnitude in a 1 ng background proteome without requiring stable isotope-labeled standards. From a 1 ng sample, we found clear consistency between proteins in subsets of CD4 + and CD8 + T cells measured using high dimensional flow cytometry and LIT-based proteomics. Based on these results, we believe hybrid quadrupole-LIT instruments represent an economical solution to democratizing mass spectrometry in a wide variety of laboratory settings.

Phytochemical characterization and quality evaluation of stirred goat milk yoghurt with added fruit and vegetable puree during storage

PurposeGoat milk yoghurt differs from cow milk yoghurt in that it has a different casein composition and content, which presents several technical challenges, including consistency with an appropriate flavor.Design/methodology/approachIn this study, the antioxidant potential and phytochemical profiling of the fruits (pineapple and papaya) and vegetable (carrot) extracts was evaluated and the effect of their purees on the quality and stability of stirred goat milk yoghurt (GMY) were investigated. The qualities of stirred GMY with carrot (CrY), pineapple (PaY) and papaya (PpY) purees were assessed against the product without puree (CY).FindingsThe carrot puree had the highest moisture, ash contents and pH value. The carrot extract had the highest DPPH radical scavenging activity, while the pineapple extract had the highest total phenolic value (1.59 µg GAE/g) and flavonoids content (0.203 µg CE/g). The scanning of all the puree extracts in GC-MS indicated that 5-hydroxymethylfurfural was a major component. The phytochemical quantification of the extracts through multiple reaction monitoring (MRM) against 16 compounds showed the presence of sinapic acid, cinnamic acid, pthalic acid, ferulic acid, 4-OH-benzoic acid, 3-OH-benzoic acid, p-coumaric acid, caffeic acid and vanillic acid in different quantities. The addition of purees and storage period had a significant (p < 0.05) effect on the moisture, pH, titratable acidity, syneresis, viscosity, color values and sensory properties of the products. In all the samples after 15 days of storage, Streptococcus thermophilus and Lactobacillus bulgaricus counts remained above the recommended level of 106CFU/g. Stirred GMY sample produced with pineapple puree showed a higher syneresis and viscosity, but the CrY sample demonstrated the highest antioxidant activity. The developed formulations remained stable with minimum changes in quality and sensory attribute during refrigerated storage for 10 days.Originality/valueThis study suggests that addition of fruit and vegetable improve the viscosity and sensory perception of the product with minimal use of synthetic flavor and preservatives.

IS-PRM-Based Peptide Targeting Informed by Long-Read Sequencing for Alternative Proteome Detection.

Alternative splicing is a major contributor of transcriptomic complexity, but the extent to which transcript isoforms are translated into stable, functional protein isoforms is unclear. Furthermore, detection of relatively scarce isoform-specific peptides is challenging, with many protein isoforms remaining uncharted due to technical limitations. Recently, a family of advanced targeted MS strategies, termed internal standard parallel reaction monitoring (IS-PRM), have demonstrated multiplexed, sensitive detection of predefined peptides of interest. Such approaches have not yet been used to confirm existence of novel peptides. Here, we present a targeted proteogenomic approach that leverages sample-matched long-read RNA sequencing (lrRNA-seq) data to predict potential protein isoforms with prior transcript evidence. Predicted tryptic isoform-specific peptides, which are specific to individual gene product isoforms, serve as "triggers" and "targets" in the IS-PRM method, Tomahto. Using the model human stem cell line WTC11, LR RNaseq data were generated and used to inform the generation of synthetic standards for 192 isoform-specific peptides (114 isoforms from 55 genes). These synthetic "trigger" peptides were labeled with super heavy tandem mass tags (TMT) and spiked into TMT-labeled WTC11 tryptic digest, predicted to contain corresponding endogenous "target" peptides. Compared to DDA mode, Tomahto increased detectability of isoforms by 3.6-fold, resulting in the identification of five previously unannotated isoforms. Our method detected protein isoform expression for 43 out of 55 genes corresponding to 54 resolved isoforms. This lrRNA-seq-informed Tomahto targeted approach is a new modality for generating protein-level evidence of alternative isoforms─a critical first step in designing functional studies and eventually clinical assays.

Safety and human health: The landscape of an effective UPLC-MS/MS method for the identification and quantification of N-Nitroso Hydrochlorothiazide impurity in Hydrochlorothiazide

N-nitrosamines (NSAs) and Nitrosamine Drug Substance Related Impurities (NDSRIs) are class of compounds that contains the nitroso functional group. These compounds are carcinogens and mutagens for both humans and animals. As a result, all global regulatory agenesis are requiring risk assessments of NSAs in each pharmaceutical product. The present research aims to provide a single pre-packaged mass spectrometry method parameter set, which includes Ultra performance liquid chromatography and a triple quadrupole mass spectrometer (UPLC-MS-MS) configuration technique for qualifying and quantifying NNHCT in HCT. The analysis was performed on a YMC ODS-A liquid chromatography column with an oven temperature of 35 °C. In this method, mobile phase was prepared by mixing 10 mM ammonium formate buffer and acetonitrile was mixed in the ratio of 60:40 (v/v) with an isocratic program and a flow rate of 0.8 mL/min. The mass detector was used along with electrospray ionization (ESI) Negative mode with multiple reaction monitoring (MRM). The method has been validated in accordance with the current regulatory guidelines and shown the ability to detect NNHCT with a LOQ of 0.01 ppm and LOD of 0.005 ppm.