Mass Spectrometry Method Research Articles

Detection of Pharmaceutical Contamination in Amphipods of Lake Baikal by the HPLC-MS Method.

Pollution by active ingredients is one of the most significant and widespread forms of pollution on Earth. Medicines can have a negative impact on ecosystems, and contamination can have unpredictable consequences. An urgent and unexplored task is to study the Lake Baikal ecosystem and its organisms for the presence of trace concentrations of active pharmaceutical ingredients. Our study aimed to conduct a qualitative analysis of active pharmaceutical ingredients, and quantitative analysis of ibuprofen in endemic amphipods of Lake Baikal, using methods of high-performance liquid chromatography and mass spectrometry (HPLC-MS). Acetylsalicylic acid (aspirin), ibuprofen, acetaminophen, azithromycin, dimetridazole, metronidazole, amikacin, spiramycin, and some tetracycline antibiotics were detected in the studied littoral amphipods. We also detected different annual loads of active pharmaceutical ingredients on amphipods. Using the multiple reaction monitoring (MRM) mode mentioned in GOST International Technical Standards, we detected molecules, fragmented as amikacin, chlortetracycline, doxycycline, oxytetracycline, dimetridazole, metronidazole and spiramycin. Thus, we first revealed that invertebrates of Lake Baikal can uptake pharmaceutical contaminants in the environment.

Enabling high-sensitivity live single-cell mass spectrometry using an integrated electrical lysis and nano electrospray ionization interface

BackgroundLive single-cell metabolomic studies encounter inherent difficulties attributed to the limited sample volume, minimal compound quantity, and insufficient sensitivity in the Mass Spectrometry (MS) method used to obtain single-cell data. However, understanding cellular heterogeneity, functional diversity, and metabolic processes within individual cells is essential. Exploring how individual cells respond to stimuli, including drugs, environmental changes, or signaling molecules, offers insights into biology, oncology, and drug discovery. Efficient release of cell contents (lysis) is vital for accurate metabolite detection at the single-cell level. Despite this, traditional approaches in live single cell metabolomics methods do not emphasize efficient lysis to prevent sample dilution. Instead, current live single cell metabolomics methods use direct infusion to introduce the cell into the mass spectrometry without prior chromatographic separation or a lysis step, which adversely affects sensitivity and metabolic coverage. ResultsTo address this, we developed an integrated single-cell electrical lysis and nano spray (SCEL-nS) platform coupled to an Orbitrap MS capable of efficiently lysing a single cell after being sampled with specially manufactured micropipettes. Lysis efficiency was validated by comparing live cell stain fluorescent intensities of intact and electrically lysed cells through microscopy imaging. The SCEL-nS platform successfully induced the breakdown of a single cell, significantly reducing the live cell stain's fluorescent intensity indicating cell membrane breakdown. Additionally, SCEL-nS was validated by measuring single cells spiked with the anti-cancer drug tamoxifen by MS. SCEL-nS use resulted in statistically significant increase in the peak measured by the method compared to the traditional non-lysis method. SignificanceOverall, our results demonstrate that the newly incorporated SCEL-nS platform achieved higher sensitivities compared to traditional live single cell analysis methods.

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.

Time-resolved interactome profiling deconvolutes secretory protein quality control dynamics.

Many cellular processes are governed by protein-protein interactions that require tight spatial and temporal regulation. Accordingly, it is necessary to understand the dynamics of these interactions to fully comprehend and elucidate cellular processes and pathological disease states. To map de novo protein-protein interactions with time resolution at an organelle-wide scale, we developed a quantitative mass spectrometry method, time-resolved interactome profiling (TRIP). We apply TRIP to elucidate aberrant protein interaction dynamics that lead to the protein misfolding disease congenital hypothyroidism. We deconvolute altered temporal interactions of the thyroid hormone precursor thyroglobulin with pathways implicated in hypothyroidism pathophysiology, such as Hsp70-/90-assisted folding, disulfide/redox processing, and N-glycosylation. Functional siRNA screening identified VCP and TEX264 as key protein degradation components whose inhibition selectively rescues mutant prohormone secretion. Ultimately, our results provide novel insight into the temporal coordination of protein homeostasis, and our TRIP method should find broad applications in investigating protein-folding diseases and cellular processes.

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.

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. 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. 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, C4concentration gradually declined by up to 14 % from baseline. However, the change was not significant for up to 12 h. 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.

Sample-Specific MS/MS Methods in High-Throughput Mass Spectrometry.

The drug discovery process increasingly relies on high-throughput sample analysis to accelerate the identification of viable drug candidates. Recently, chromatographic-free high-throughput mass spectrometry (HT-MS) technologies have emerged, significantly increasing sample readout speed and enabling the analysis of large sample sets. These HT-MS platforms continuously acquire data from various samples into a single data file, presenting challenges in applying distinctive data acquisition methods to specific samples. This study introduces a novel approach that integrates real-time sample loading status to activate sample-specific MS/MS data acquisition methods on the high-throughput acoustic ejection mass spectrometry platform. Effective method switching and high signal reproducibility were demonstrated across different data acquisition window durations in multiple reaction monitoring (MRM), high-resolution MRM (MRMHR), and information-dependent acquisition modes. This advancement provides a user-friendly and robust solution to the method-setting challenges of HT-MS, expanding the implementation of HT-MS platforms in drug discovery and other high-throughput analytical applications.

Eco-friendly one-step egg white gel preparation for sensitive detection of 13 trichothecenes in oats using UHPLC-MS/MS.

Oat products have gained widespread recognition as a health food due to their rich and balanced nutritional profile and convenience. However, the unique matrix composition of oats, which differs significantly from other cereals, presents specific challenges for mycotoxin analysis. This study presents an ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method enhanced with an innovative egg white gel pretreatment for the simultaneous analysis of 13 regulated and unregulated trichothecenes in oats. The method demonstrated excellent performance with high accuracy (> 87.5%), repeatability (< 5.7%), and reproducibility (< 8.1%). Analysis of 100 commercial oat products revealed a concerning detection rate (78%) for at least one of the 11 trichothecenes investigated. Notably, deoxynivalenol, exceeding the standard limit in 2% of samples, exhibited the highest detection rate (62%). Additionally, concerning co-occurrence patterns and positive correlations were observed, highlighting potential synergistic effects. The first-time detection of unregulated mycotoxins (T-2 triol, 4,15-diacetoxyscirpenol, 15-acetoxyscirpenol, and neosolaniol) underscores the need for comprehensive monitoring. This method, while developed for oats, shows potential for broader application to other cereals, though further investigation and confirmation are necessary. These findings suggest a potentially underestimated risk of trichothecenes in oats, necessitating continuous monitoring to ensure consumer safety.

Thermal instability of 1-butyl-3-methylimidazolium chloride ionic liquid

The evaporation and thermal decomposition (thermolysis) of 1-butyl-3-methylimidazolium chloride ionic liquid, [BuMIm][Cl](l), were studied in the temperature range of 373–463 K by thermogravimetry–mass spectrometry (TG–MS) and Knudsen cell mass spectrometry (KCMS) methods. The saturated vapor pressures and the enthalpy of vaporization of [BuMIm][Cl](l) were determined within 373–398 K, where the thermolysis of this ionic liquid occurs via the SN2 mechanism at a low rate. With increasing temperature, the thermolysis becomes more extensive, which is accompanied by the C2-methylation of [BuMIm][Cl](l). The resulting ion pairs [BuMMIm+][Cl-] are thermally unstable and decomposed A decrease in the vapor pressure of the ion pairs [BuMIm+][Cl-] at a constant temperature takes place due to side reactions, resulting in the suppression of the evaporation. Complex ionic compounds were found in the thermolysis/evaporation residues using atmospheric pressure chemical ionization (APCI) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) methods. The priority and possible pathways of reactions involving ion pairs and the thermolysis products were established by quantum chemical calculations.

An ultra-fast green ultra-high-performance liquid chromatography-tandem mass spectrometry method for estimating the in vitro metabolic stability of zotizalkib in human liver microsomes.

Zotizalkib (ZTK, TPX-0131) is a fourth-generation highly effective inhibitor of wild-type anaplastic lymphoma kinase (ALK) and ALK-resistant mutations that can penetrate the central nervous system. It exhibited greater potency compared to all five officially approved ALK inhibitors. The aim of this study was to develop a rapid, accurate, eco-friendly, and highly sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for measuring the concentration of ZTK in human liver microsomes (HLMs). The validation aspects of the current UHPLC-MS/MS methodology in the HLMs were conducted in accordance with the bioanalytical method validation standards specified by the US Food and Drug Administration. ZTK and encorafenib were separated using an Agilent C8 column (Eclipse Plus) and an isocratic mobile phase. The calibration curve for the developed ZTK exhibited a linear relationship within the concentration range of 1-3000ng/mL. The results from the Analytical Green-ness Metric Approach program (0.76) suggested that the created method demonstrated a significant degree of environmental sustainability. The in vitro half-life (t1/2) and intrinsic clearance (Clint) of ZTK were determined to be 15.79min and 51.35mL/min/kg, respectively that suggests the ZTK exhibits characteristics similar to those of a medication with a high extraction ratio. These approaches are crucial for the progress of novel pharmaceutical development, especially in improving metabolic stability.

Integration of serum pharmacochemistry with network pharmacology to reveal the potential mechanism of Yangqing Chenfei formula for the treatment of silicosis.

To explore the mechanisms of Yangqing Chenfei formula (, YCF) in the treatment of silicosis through a comprehensive strategy consisting of serum pharmacochemistry, network pharmacology analysis, and in vitro validation. An ultrahigh-performance liquid chroma-tography-tandem mass spectrometry method was used to confirm the active components in YCF-medicated serum. Then, we obtained targets for active components and genes for silicosis from multiple databases. Furthermore, a protein-protein interaction network was constructed, and Kyoto Encyclopedia of Genes and Genomes pathway and biological process analyses were conducted to elucidate the mechanisms of YCF for the treatment of silicosis. Finally, we validated the important components and mechanisms in vitro. Altogether, 19 active components were identified from rat serum after YCF administration. We identified 724 targets for 19 components, which were mainly related to inflammation [phosphatidy linositol 3 kinase/protein kinase B, forkhead box O, hypoxia inducible factor, and T-cell receptor signaling pathway, nitric oxide biosynthetic process], fibrotic processes [vascular endothelial growth factor signaling pathway, extracellular signal regulated kinase (ERK) 1 and ERK2 cascade, smooth muscle cell proliferation], and apoptosis (negative regulation of apoptotic process). In addition, 218 genes for silicosis were identified and were mainly associated with the inflammatory response and immune process [cytokine?cytokine receptor interaction, tumor necrosis factor alpha (TNF-α), toll-like receptor, and nucleotide binding oligomerization domain-like receptor signaling pathway]. Taking an intersection of active component targets and silicosis genes, we obtained 61 common genes that were mainly related to the inflammatory response and apoptosis, such as the phosphatidylinositol-3-kinase/protein kinase B signaling pathway, mitogen activated protein kinases signaling pathway, TNF signaling pathway, toll-like receptor signaling pathway, biosynthesis of nitric oxide, and apoptotic process. In the herb-component-gene-pathway network, paeoniflorin, rutin and nobiletin targeted the most genes. In vitro, paeoniflorin, rutin and nobiletin decreased the mRNA levels of inflammatory factors [interleukin (IL)-6, TNF-α, and IL-1β], suppressed p-AKT and cleaved caspase-3, and increased B cell lymphoma (Bcl)-2 protein expression in silica-induced macrophages in a concentration-dependent manner. YCF could significantly relieve the inflammatory response of silicosis via suppression of the AKT/Bcl-2/Caspase-3 pathway.

LC-MS analysis of chiral amino acids in human urine reveals D-amino acids as potential biomarkers for colorectal cancer

Colorectal cancer (CRC) is a common malignant tumor in the gastrointestinal tract. Changes in amino acid metabolites have been implicated in tumorigenesis and disease progression. Biomarkers on the basis of chiral amino acids, especially D-amino acids, have not been established for early diagnosis of CRC. Quantification of chiral amino acids, especially very low concentrations of endogenous D-amino acids, is technically challenging. We report here the quantification of L- and D-amino acids in urine samples collected from 115 CRC patients and 155 healthy volunteers, using an improved method. The method of chiral labeling, liquid chromatography, and tandem mass spectrometry enabled separation and detection of 28 amino acids (14 L-amino acids, 13 D-amino acids and Gly). Orthogonal partial least squares discriminant analysis identified 14 targeted variables among these chiral amino acids that distinguished the CRC from the healthy controls. Binary logistic regression analysis revealed that D-α-aminobutyric acid (D-AABA), L-alanine (L-Ala), D-alanine (D-Ala), D-glutamine (D-Gln) and D-serine (D-Ser) could be potential biomarkers for CRC. A receiver operating characteristic curve analysis of combined multi-variables contributed to an area under the curve (AUC) of 0.995 with 98.3 % sensitivity and 96.8 % specificity. A model constructed with D-AABA, D-Ala, D-Gln, and D-Ser achieved an AUC of 0.988, indicating important contributions of D-amino acids to the association with CRC. Further analysis also demonstrated that the metabolic aberration was associated with age and the development of CRC, D-methionine (D-Met) was decreased in CRC patients with age over 50, and D/L-Gln in patients at stage IV was higher than patients at stage I. This study provides the signature of D-amino acids in urine samples and offers a promising strategy for developing non-invasive diagnosis of CRC.

Therapeutic drug monitoring of free perampanel concentrations in practice: A practical analytical technique based on centrifugal ultrafiltration sample separation

ObjectivesThe centrifugal ultrafiltration–high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was established to determine the free perampanel (PER) concentration in children with epilepsy. MethodsFree PER concentration was obtained using centrifugal ultrafiltration devices. The internal standard was PER-D5. The method was investigated for selectivity, carryover, lower limit of quantification, calibration curve, accuracy, precision, matrix effects, recovery, and stability. The Spearman's correlation coefficient was used to evaluate the correlation between the free and total PER concentrations. A nonparametric test was used to estimate the effects of PER along with other antiepileptic drugs on the total and free PER concentrations. ResultsThe free PER concentration was positively correlated with the total PER concentration in the 57 plasma samples (r = 0.793 > 0, P < 0.001). Additionally, the free PER concentrations were significantly (P < 0.05) increased in valproic acid (VPA) co-therapy (9.87 ± 5.83) compared with non-VPA co-therapy (5.03 ± 4.57). ConclusionsThe proposed method is efficient, sensitive, and suitable for detecting free PER concentrations in children with epilepsy. Simultaneously, the free PER concentration response to clinical outcomes in children with epilepsy was more clinically significant, particularly when combined with VPA.

Targeted proteomic approach for quantification of collagen type I and type III in formalin-fixed paraffin-embedded tissue

Collagen is the most abundant protein in mammals and a major structural component of the extracellular matrix (ECM). Changes to ECM composition occur as a result of numerous physiological and pathophysiological causes, and a common means to evaluate these changes is the collagen 3 (Col3) to collagen 1 (Col1) ratio. Current methods to measure the Col3/1 ratio suffer from a lack of specificity and often under- or over-estimate collagen composition and quantity. This manuscript presents a targeted liquid chromatography tandem mass spectrometry (LC–MS/MS) method for quantification of Col3 and Col1 in FFPE tissues. Using surrogate peptides to generate calibration curves, Col3 and Col1 are readily quantified in FFPE tissue sections with high accuracy and precision. The method is applied to several tissue types from both human and reindeer sources, demonstrating its generalizability. In addition, the targeted LC–MS/MS method permits quantitation of the hydroxyprolinated form of Col3, which has significant implications for understanding not only the quantity of Col3 in tissue, but also understanding of the pathophysiology underlying many causes of ECM changes. This manuscript presents a straightforward, accurate, precise, and generalizable method for quantifying the Col3/1 ratio in a variety of tissue types and organisms.

LC-HRMS analysis of phospholipids bearing oxylipins

BackgroundSeveral oxylipins including hydroxy- and epoxy-polyunsaturated fatty acids act as lipid mediators. In biological samples they can be present as non-esterified form, but the major part occurs esterified in phospholipids (PL) or other lipids. Esterified oxylipins are quantified indirectly after alkaline hydrolysis as non-esterified oxylipins. However, in this indirect analysis the information in which lipid class oxylipins are bound is lost. In this work, an untargeted liquid chromatography high-resolution mass spectrometry (LC-HRMS) method for the direct analysis of PL bearing oxylipins was developped. ResultsOptimized reversed phase LC separation achieved a sufficient separation of isobaric and isomeric PL from different lipid classes bearing oxylipin positional isomers. Individual PL species bearing oxylipins were identified based on retention time, precursor ion and characteristic product ions. The bound oxylipin could be characterized based on product ions resulting from the α-cleavage occurring at the hydroxy/epoxy group. Phospholipid sn-1/sn-2 isomers were identified based on the neutral loss of the fatty acyl in the sn-2 position. A total of 422 individual oxPL species from 7 different lipid classes i.e., PI, PS, PC, PE, PC-P, PC-O, and PE-P were detected in human serum and cells. This method enabled to determine in which PL class supplemented oxylipins are incorporated in HEK293 cells: 20:4;15OH, 20:4;14Ep, and 20:5;14Ep were mostly bound to PI. 20:4;8Ep and 20:5;8Ep were esterified to PC and PE while other oxylipins were mainly found in PC. SignificanceThe developed LC-HRMS method enables the comprehensive detection as well as the semi-quantification of isobaric and isomeric PL species bearing oxylipins. With this method, we show that the position of the oxidation has a great impact and directs the incorporation of oxylipins into the different PL classes in human cells.

Determination of sodium cyclamate in Baijiu by pre-column derivatization-high performance liquid chromatography with fluorometric detection

Sodium cyclamate in Baijiu is a key item in the China National Food Safety Supervision and Inspection Plan. A simple, economical, sensitive, and reliable method is urgently needed for routine analysis and internal quality control. A method based on high performance liquid chromatography with fluorescence detection (HPLC-FLD) was developed for the determination of sodium cyclamate in Baijiu by o-phthalaldehyde derivatization. First, the sodium cyclamate in the sample solution was converted into amino compounds using the desulfurization reaction under acidic conditions. Next, 400 g/L sodium hydroxide solution was added to the sample solution for neutralization. The amino compounds in the sample solution were then derivatized with o-phthalaldehyde to produce indole-substituted derivatives that are capable of producing fluorescence signals. Separation was carried out on a C18 column (250 mm×4.6 mm, 5 μm) in isocratic elution mode using a mobile phase consisting of acetonitrile and phosphate buffer. Finally, the eluate was monitored using a fluorescence detector, and an external standard method was used for quantification. A good linear relationship was obtained in the range of 0.1-2.0 mg/L, with correlation coefficients greater than 0.999. The average recoveries of sodium cyclamate spiked at levels of 0.1-1.0 mg/kg in Baijiu samples ranged from 90.7% to 100.9%, with relative standard deviations (RSDs) of 3.5%-5.6% (n=6). The limits of detection and quantification were 0.03 and 0.10 mg/kg, respectively. Nine Baijiu samples collected from the market were tested, and the results demonstrated that the contents of sodium cyclamate detected by the developed method were consistent with those obtained using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method described in GB 5009.97-2016 (the third method). The proposed method is economical, sensitive, specific, and accurate; thus, it provides a basic approach for the determination of sodium cyclamate in Baijiu samples and has great potential for routine analysis in foodstuffs.

Evaluation of Cannabis Product Mislabeling: The development of a unified cannabinoid LC-MS/MS method to analyze e-liquids and edible products

The 2018 Agricultural Improvement Act federally legalized the cultivation of hemp and manufacture and sale of cannabidiol (CBD). The overproduction of CBD led to a significant drop in price. To maintain profitability, manufacturers began producing synthetic and semi-synthetic cannabinoids, marketing them as “legal” and “hemp-derived” due to their synthesis from CBD. The cannabis industry is adaptable, and laboratories have struggled to keep up with the rapid emergence of new compounds. This has resulted in products with unlabeled or mislabeled cannabinoids, causing unexpected adverse events. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the separation and quantitation of 21 cannabinoids. The method was applied in the analysis of 55 commercially available products. Results demonstrate significant issues with quality assurance and labeling in the unregulated cannabis market. Many products either did not contain the cannabinoids listed on their labels or contained cannabinoids that were not listed. When cannabinoids were present, their concentrations were often incorrect, with some products showing high concentrations that could pose a potential health risk. Moreover, safety, dosing, and other pharmacological data of these newly proliferated cannabinoids are lacking. Reports of adverse events are increasing to poison controls centers and emergency departments following cannabis product use. Often, the extract cannabinoids involved are not determined. It is critical to develop more comprehensive testing methodologies to determine the content of cannabis products and to implement stronger quality assurance measures and regulations to protect the public from low-quality and potentially dangerous products.

Forensic differentiation of blue pen inks using time-of-flight secondary ion mass spectrometry and multivariate statistical methods

This study investigates the use of time-of-flight secondary ion mass spectrometry (ToF-SIMS) combined with multivariate statistical analysis for the identification of blue pen strokes in document forgery cases. Strokes of six commercially available blue pens were analysed using ToF-SIMS, and the data were interpreted using principal component analysis (PCA) and multivariate curve resolution (MCR). PCA effectively reduced the dimensionality of the complex ToF-SIMS data, enabling clear differentiation of the blue pen inks based on the obtained PCA scores. The first three principal components accounted for most of the variance, with PCA score plots showing distinct clusters of samples corresponding to the spectra of each pen. PCA analysis of the large-area ToF-SIMS images further revealed some characteristic spatial patterns and variations across the pen strokes; however, this analysis was less useful for the identification process. On the other hand, MCR provided a more chemically intuitive analysis by extracting pure component spectra from the complex mixtures. Each MCR factor was individually associated with a specific pen, allowing for exact identification of the pen stroke made. MCR score images displayed clear correlations with individual pen strokes, and the associated loadings identified signals at particular m/z values that were characteristic of each pen. This study shows that an efficient method for non-destructive forensic analysis of blue pen strokes can be achieved by combining ToF-SIMS with PCA and MCR. This analytical method provides reliable identification, which is crucial for forgery detection without requiring any sample pretreatment, i.e. the samples (pen strokes) were used just as they were made on paper.

Evaluation of pharmacokinetic herb-drug interaction of diabecon and losartan by UHPLC-MS/MS

The diabecon is an ayurvedic herbal formulation that contains a mixture of herbs traditionally used as antidiabetic which is reported in the ayurvedic pharmacopeia of India and Indian Materia medica. The diabetic population has a common co-morbidity of hypertension for which losartan drug is commonly used for the treatment of hypertension. However, there is a lack of research on the pharmacokinetics interaction between diabecon and losartan. This research aims to investigate the influence of diabecon on the pharmacokinetics of losartan drugs in rats by establishing a highly sensitive ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UHPLC-MS/MS) method. The method was validated according to the USFDA guidelines and was applied for the pharmacokinetic study. The lowest concentration of losartan detection in rat plasma was found to be 1 ng/mL and the accuracy and precision were within the linear range (1–1500 ng/mL). The results revealed that after 28 days of dosing diabecon, it has altered the pharmacokinetic parameters like area under the curve (AUC0-t), drug clearance (Cl/F), and rate of elimination (Ke) of losartan, which may happen due to enzyme induction. Although there was a change in the pharmacokinetic parameters of losartan when administered in the presence of diabecon, it was found to be nonsignificant in rats (p > 0.05). According to the best of our knowledge, this is the first article that discusses the pharmacokinetic herb-drug interaction between diabecon and losartan.

Data acquisition approaches for single cell proteomics.

Single-cell proteomics (SCP) aims to characterize the proteome of individual cells, providing insights into complex biological systems. It reveals subtle differences in distinct cellular populations that bulk proteome analysis may overlook, which is essential for understanding disease mechanisms and developing targeted therapies. Mass spectrometry (MS) methods in SCP allow the identification and quantification of thousands of proteins from individual cells. Two major challenges in SCP are the limited material in single-cell samples necessitating highly sensitive analytical techniques and the efficient processing of samples, as each biological sample requires thousands of single cell measurements. This review discusses MS advancements to mitigate these challenges using data-dependent acquisition (DDA) and data-independent acquisition (DIA). Additionally, we examine the use of short liquid chromatography gradients and sample multiplexing methods that increase the sample throughput and scalability of SCP experiments. We believe these methods will pave the way for improving our understanding of cellular heterogeneity and its implications for systems biology.

Real-time platelet P2Y12 receptor occupancy as a promising pharmacodynamics biomarker for bridging the gap between PK/PD of clopidogrel therapy

Clopidogrel effectively inhibits platelet aggregation in response to ADP by irreversibly binding to the platelet P2Y12 receptor through its active metabolite. However, the observed discrepancies between the pharmacokinetics (PK) and pharmacodynamics (PD) of clopidogrel present substantial challenges in individualizing of antiplatelet therapy. To address these challenges, a robust liquid chromatography–tandem mass spectrometry method has been developed to facilitate the real-time assessment of platelet P2Y12 receptor occupancy. This method has been validated in animal models, providing a reliable link between individual PK profiles and PD effects. Target receptor occupancy offers a comprehensive overview of interindividual variations in clopidogrel metabolism, regulation of P2Y12 receptor expression, and platelet turnover. Moreover, it directly correlates with the inhibitory effect on platelet aggregation. The levels of platelet P2Y12 occupancy accurately reflect the extent of clinical factors influencing the PD of clopidogrel, including dosage, drug–drug interactions (DDI), and type 2 diabetes mellitus (T2DM). As a normalized metric, platelet P2Y12 occupancy not only serves potential as a diagnostic tool for personalized clopidogrel therapy but also aids in elucidating the role of the P2Y12 signaling pathway in cases of abnormal on-treatment platelet reactivity.