Multiple Reaction Monitoring Research Articles

Integration of deep neutral network modeling and LC-MS-based pseudo-targeted metabolomics to discriminate easily confused ginseng species

Metabolomics covers a wide range of applications in life sciences, biomedicine, and phytology. Data acquisition (to achieve high coverage and efficiency) and analysis (to pursue good classification) are two key segments involved in metabolomics workflows. Various chemometric approaches utilizing either pattern recognition or machine learning have been employed to separate different groups. However, insufficient feature extraction, inappropriate feature selection, overfitting, or underfitting lead to an insufficient capacity to discriminate plants that are often easily confused. Using two ginseng varieties, namely Panax japonicus and P. japonicus var. major, containing the similar ginsenosides, we integrated pseudo-targeted metabolomics and deep neural network (DNN) modeling to achieve accurate species differentiation. A pseudo-targeted metabolomics approach was optimized through data acquisition mode, ion pairs generation, comparison between multiple reaction monitoring (MRM) and scheduled MRM, and chromatographic elution gradient. In total, 1980 ion pairs were monitored within 23 min, allowing for the most comprehensive ginseng metabolome analysis. The established DNN model demonstrated excellent classification performance (in terms of accuracy, precision, recall, F1 score, area under the curve, and receiver operating characteristic) using the entire metabolome data and feature-selection dataset, exhibiting superior advantages over random forest (RF), support vector machine (SVM), extreme gradient boosting (XGBoost), and multilayer perceptron (MLP). Moreover, DNNs were advantageous for automated feature learning, nonlinear modeling, adaptability, and generalization. This study confirmed practicality of the established strategy for efficient metabolomics data analysis and reliable classification performance even when using small-volume samples. This established approach holds promise for plant metabolomics and is not limited to ginseng.

Simultaneous determination of 61 hormones in water by solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry

Concerns over the emergence of steroid hormones as pollutants in water have grown. Steroid hormone compounds present challenges in the simultaneous detection of total residual hormones owing to their analogous structures and diverse types. In this study, we established a rapid and high-throughput continuous online method based on solid phase extraction (SPE) coupled with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the simultaneous determination of 61 hormone components, including 48 glucocorticoids, 1 mineralocorticoid, 4 androgens, and 8 progesterones, in water. Various SPE columns were investigated to assess their extraction efficiency for enriching and purifying target compounds in a large sample volume (1 L). An HC-C18 SPE column was selected because of its superior performance. Acetonitrile was used as a washing solution during SPE to ensure that the majority of the tested substances achieved recoveries exceeding 70% and effectively avoid interferences from water-soluble components. Various C8 and C18 columns were tested, and the optimal HPLC conditions for hormone retention were established. We systematically evaluated different UPLC columns and mobile phases, including methanol-water and acetonitrile-water systems with 0.1% formic acid added to the aqueous phase. The optimized UPLC conditions were as follows: BEH C18 column (100 mm×2.1 mm, 1.7 μm); column temperature, 40 ℃; flow rate, 0.3 mL/min; injection volume, 5 μL; mobile phase A: 0.1% formic acid aqueous phase; mobile phase B: acetonitrile. Gradient elution was performed as follows: 0-0.5 min, 30%B; 0.5-15.0 min, 30%B-75%B; 15.0-18.0 min, 75%B-98%B; 18.0-19.0 min, 98%B; 19.0-19.1 min, 98%B-30%B; 19.1-20.0 min, 30%B. Both positive- and negative-ion modes were explored in the UPLC-MS/MS experiment to obtain the full scan of the parent ions, and positive mode was finally selected for electrospray ionization (ESI). Two product ions exhibiting strong signals and minimal interference were selected for quantitative and qualitative ion analyses, using an external standard method for quantification. MS/MS was performed in positive-ion (ESI+) mode with multiple reaction monitoring (MRM) scanning. The MS/MS parameters were as follows: atomizing gas pressure, 379 kPa; curtain air pressure, 241 kPa; spray voltage, 5500 V; desolvation temperature, 550 ℃; collision exit voltage (CXP), 13 V; intake voltage (EP), 10 V; and residence time of each ion pair, 0.5 ms. Other instrument settings, such as the collision energy and declustering voltage, were also optimized. The 61 hormones exhibited excellent linear relationships within their corresponding concentration ranges, with correlation coefficients greater than 0.99. The method detection limits (MDLs) were in the range of 0.05-1.50 ng/L. The average recoveries of the 61 hormones across three spiked levels ranged from 62.3% to 125.2%, with relative standard deviations (RSDs, n=6) of 1.1%-10.5%. Using this method, we successfully detected 10 hormone components (cortisone, fluticasone propionate, ciclesonide, betamethasone dipropionate, clobetasone butyrate, diflucortolone valerate, halobetasol propionate, isoflupredone, difluprednate, and hydroxyprogesterone caproate) in various surface water and groundwater samples collected from the Taihu Basin region. The SPE-UPLC-MS/MS method presented in this paper is simple, highly sensitivity, and exceptionally accurate. Thus, it exhibits promising potential for tracing targeted hormone residues in water and will be of great value in monitoring and ensuring water safety. Finally, a regional analysis was conducted on the hormone levels in water, and suggestions were made for the targeted treatment of hormone residues in future sewage treatment processes.

Circulating Exosomes Studied by Label-free Proteomics Analysis Reveal Transition Signatures from Diabetes Mellitus to Diabetic Kidney Disease

Background: Diabetic kidney disease (DKD) is a common microvascular complication of diabetic mellitus (DM). At present, the early diagnosis of DKD mainly depends on microalbuminuria, which is prone to be affected by confounding factors such as urinary tract infections. Methods: To identify the more stable early diagnosis markers, the whole proteome in the circulating exosomes from controls, DM patients, and DKD patients was quantified by label-free proteomics analysis and then validated with parallel reaction monitoring. Results: Three hundred ninety-one quantitative proteins were detected, and the expression trends of 7 proteins in the validation phase were consistent with that in the discovery phase. The expression level assessment results revealed that the expression of EFEMP1 and ApoA4 in the DKD group was significantly higher than those in DM and controls. Correlation analysis showed that EFEMP1 and APOA4 were positively correlated with urinary microalbumin and urinary albumin creatinine ratio and had excellent diagnostic values for distinguishing DKD from DM and controls. Conclusions: ApoA4 and EFEMP1 could serve as the early diagnosis markers of DKD. These findings provide a possibility for the development of a clinical diagnostic index that can efficiently distinguish DKD from DM in the near future.

Comprehensive Proteomic Analysis Reveals Distinct Features and a Diagnostic Biomarker Panel for Early Pregnancy Loss in Histological Subtypes

Early pregnancy loss (EPL) is a common event in human reproduction and is classified into histological subtypes such as hydropic abortion (HA) and hydatidiform moles (HMs), including complete hydatidiform moles (CHMs) and partial hydatidiform moles (PHMs). However, accurate diagnosis and improved patient management remain challenging due to high rates of misdiagnosis and diverse prognostic risks. Therefore, diagnostic biomarkers for EPL are urgently needed. Our study aimed to identify biomarkers for EPL through comprehensive proteomic analysis. Ten CHMs, six PHMs, ten HAs and ten normal control (NC) products of conception (POC) were used to obtain a proteomic portrait. Parallel reaction monitoring (PRM)-targeted proteomic and regression analyses were used to verify and select the diagnostic signatures. Finally, 14 proteins were selected and a panel of diagnostic classifiers (DLK1, SPTB/COL21A1, and SAR1A) was built to represent the CHM, PHM, and NC groups (auROC=0.900, 0.804/0.885, and 0.991, respectively). This high diagnostic power was further validated in another independent cohort (n = 148) by immunohistochemistry (IHC) (n = 120) and western blot (WB) analyses (n = 28). The protein SPTB was selected for further biological behaviour experiments in vitro. Our data suggest that SPTB maintains trophoblast cell proliferation, angiogenesis, cell motility and the cytoskeleton network. This study provides a comprehensive proteomic portrait and identifies potential diagnostic biomarkers. These findings enhance our understanding of EPL pathogenesis and offer novel targets for diagnosis and therapeutic interventions.

Determination of eight neonicotinoid pesticides in wastewater by solid phase extraction combined with liquid chromatography-tandem mass spectrometry

Neonicotinoid pesticides are a relatively new class of pesticides that have garnered significant attention owing to their potential ecological risks to nontarget organisms. A method combining solid phase extraction with liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) was developed for the rapid and accurate detection of eight neonicotinoid pesticides (dinotefuran, E-nitenpyram, thiamethoxam, clothianidin, imidacloprid, imidaclothiz, acetamiprid, and thiacloprid) in wastewater. The chromatographic mobile phase and MS parameters were selected, and a single-factor method was used to determine the optimal column type, extraction volume, sample loading speed, and pH for SPE. The optimal parameters were as follows: column type, HLB column (500 mg/6 mL); sample extraction volume, 500 mL; sample loading speed, 10 mL/min; and sample pH, 6-8. The matrix effects of the wastewater samples were reduced by optimizing the chromatographic gradient-elution program, examining the dilution factor of the samples, and using the isotope internal standard calibration method. Prior to analysis, the wastewater samples were diluted 5-fold with ultrapure water for pretreatment. Subsequently, 2 mmol/L ammonium acetate aqueous solution containing 0.1% (v/v) formic acid and methanol was used as mobile phases for gradient elution on a ZORBAX Eclipse Plus C18 column (100 mm×2.1 mm, 1.8 μm). The samples were quantified using positive-ion multiple reaction monitoring (MRM) mode for 10 min. Imidacloprid-d4 was used as the isotope internal standard. The SPE process was further optimized by applying response surface methodology to select the type and mass of rinsing and elution solvents. The optimal pretreatment of the SPE column included rinsing with 10% methanol aqueous solution and elution with methanol-acetonitrile (1∶1, v/v) mixture (7 mL). The eight neonicotinoid pesticides showed satisfactory linearity within the relevant range, with linear correlation coefficients (r) all greater than 0.9990. The method detection limits (MDLs) ranged from 0.2 to 1.2 ng/L, and the method quantification limits (MQLs) ranged from 0.8 to 4.8 ng/L. The average recoveries of the eight neonicotinoid pesticides were in the range of 82.6%-94.2% at three spiked levels, with relative standard deviations (RSDs) ranging from 3.9% to 9.4%. Finally, the optimized method was successfully applied to analyze wastewater samples collected from four sewage treatment plants. The results indicated that the eight neonicotinoid pesticides could be generally detected at concentrations ranging from not detected (ND) to 256 ng/L. The developed method has a low MDL and high accuracy, rendering it a suitable choice for the trace detection of the eight neonicotinoid pesticides in wastewater when compared with other similar methods. The proposed method can be utilized to monitor the environmental impact and assess the potential risks of neonicotinoid pesticides in wastewater, thus promoting the protection of nontarget organisms and the sustainable use of these pesticides in agriculture.

Application of a Novel UPLC-MS/MS Method for Analysis of Rivaroxaban Concentrations in Dried Blood Spot and Plasma Samples Collected from Patients with Venous Thrombosis.

Despite a higher safety profile compared to vitamin K antagonists, rivaroxaban therapy is still connected with multiple adverse effects, such as a high risk of bleeding. Thus, therapeutic drug monitoring (TDM) of rivaroxaban concentrations is suggested. An alternative to plasma samples can be dried blood spots (DBS), which minimize the cost of sample storage and transport. In this study, we developed a UPLC-MS/MS method for the analysis of rivaroxaban in DBS and plasma samples. Chromatographic separation was achieved on a Zorbax Eclipse Plus C18 column (2.1 × 100 mm; 3.5 µm, Agilent Technologies Inc., Santa Clara, CA, USA) with a mobile phase consisting of water and acetonitrile, both containing 0.1% formic acid. The analytes were detected using a positive ionization mode by multiple reaction monitoring. We validated the method according to ICH guidelines. The precision and accuracy were satisfactory. Extraction recovery was approximately 57% and 66% for DBS and plasma samples, respectively. A high correlation between rivaroxaban concentrations in plasma and DBS samples collected from patients was confirmed with Deming regression. The suitability of both sampling techniques for the rivaroxaban TDM was also verified by Bland-Altman plots based on DBS-predicted and observed plasma concentrations. In addition, we found a significant relationship between rivaroxaban concentrations and coagulation parameters, including prothrombin time (PT) and international normalized ratio (INR).

Determination of Site-Specific Phosphorylation Occupancy Using Targeted Mass Spectrometry Reveals the Regulation of Human Apical Bile Acid Transporter, ASBT.

The human apical bile acid transporter (hASBT, SLC10A2) reabsorbs bile acids in the distal ileum, facilitating their recycling to the liver and resecretion. Its activity has been implicated in various disease states, including Crohn's disease, hypercholesterolemia, cholestasis, and type-2 diabetes. Post-translational modifications such as N-glycosylation, ubiquitination, and S-acylation regulate ASBT function by controlling its translocation and stability. However, the precise role of phosphorylation and its relationship with activity remains unknown. Here, we employed parallel reaction monitoring targeted mass spectrometry to investigate ASBT phosphorylation in the presence of various kinase inhibitors and activators. Our study ascertains phosphorylation at multiple sites (Thr330, Ser334, and Ser335), with Ser335 being the predominant phosphosite. We further demonstrate the critical involvement of PKC in regulating ASBT activity by phosphorylation at Ser335. Importantly, we establish a proportional relationship between the phosphorylation level of Ser335 and ASBT bile acid uptake activity. Collectively, our findings shed light on the molecular mechanisms underlying phosphorylation-mediated regulation of ASBT.

LC-MS based strategy for chemical profiling and quantification of dispensing granules of Ginkgo biloba seeds

Ginkgo biloba seeds have been used as a traditional Chinese medicine for hundreds of years to treat diseases such as cervicitis, cough, asthma and other lung diseases. As a novel form, the dispensing granules (GSDG) of Ginkgo biloba seeds have been widely employed in clinic. However, its chemical profiling is not yet clear, which has restricted in-depth research in many fields.In this study, a high performance liquid chromatography coupled with quardrupole time-of-flight mass spectrometry method was used for the component characteration with the help of accurate molecular weights, fragmentation pathways, reported data, literatures and even some reference standards. Furthermore, in multiple-reaction monitoring mode, a high performance liquid chromatography coupled with quadrupole linear ion trap mass spectrometry method was developed and applied for simultaneous determination of the bioactive phytochemicals.As a result, a total of 56 components in GSDG were identified including 12 amino acids, 9 organic acids, 6 nucleosides and nucleobases, 6 flavonoids, 5 vitamins, 5 terpenoid lactones, 4 carbohydrates and 9 other compounds As for quantitative analysis, glutamic acid, asparatic acid, histidine, ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, eucomic acid, N-(N-glucopyranosyl)-indoleacetylaspartate and N-(N-glucopyranosyl)-indoleacetylglutamate were selected as the analytes for quanlity marker of GSDG. After necessary validation tests, the developed quantitative method was successfully put into use for 10 batches of GSDG. In all batches, N-(N-glucopyranosyl)-indoleacetylaspartate was the richest phytochemical with the amount of 17.3–25.7 mg/g while ginkgolide J (0.0197–0.0335 mg/g) was determined to be the poorest.The study is supposed to exhibit a comprehensive chemical profiling and to provide some strong basis for preparation technology, quality control and even for action mechanism of GSDG, this novel form of Chinese medicine.

Serum N-Glycan Changes in Rats Chronically Exposed to Glyphosate-Based Herbicides.

Glyphosate, the active ingredient in many herbicides, has been widely used in agriculture since the 1970s. Despite initial beliefs in its safety for humans and animals due to the absence of the shikimate pathway, recent studies have raised concerns about its potential health effects. This study aimed to identify glycomic changes indicative of glyphosate-induced toxicity. Specifically, the study focused on profiling N-glycosylation, a protein post-translational modification increasingly recognized for its involvement in various disorders, including neurological conditions. A comprehensive analysis of rat serum N-glycomics following chronic exposure to glyphosate-based herbicides (GBH) was conducted using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results revealed significant changes in the N-glycan profile, particularly in sialylated and sialofucosylated N-glycans. The analysis of N-glycans across gender subgroups provided insights into gender-specific responses to GBH exposure, with the male rats exhibiting a higher susceptibility to these N-glycan changes compared to females. The validation of significantly altered N-glycans using parallel reaction monitoring (PRM) confirmed their expression patterns. This study provides novel insights into the impact of chronic GBH exposure on serum N-glycan composition, with implications for assessing glyphosate toxicity and its potential neurological implications.

Fast and reliable quantification of aldosterone, cortisol and cortisone via LC-MS/MS to study 11β-hydroxysteroid dehydrogenase activities in primary cell cultures

Cell culture experiments can support characterization of enzymatic activities in healthy and tumorous human tissues. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) enables simultaneous measurement of several steroids from a single sample, facilitating analysis of molecular pathways involved in steroid biosynthesis. We developed a reliable but fast method for quantification of cortisol, cortisone and aldosterone in cell culture supernatant. Validation, including investigation of matrix-matched calibration, was performed for two different cell types. Utility of the method was demonstrated in the study of 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) activity under conditions of glucocorticoid and mineralocorticoid excess in different cell types. Aldosterone, cortisol and cortisone were extracted by liquid-liquid extraction (LLE) with methyl tert-butyl ether from 1 mL of cell culture supernatant. Steroids were separated on a Kinetex biphenyl column (50 ×2.1 mm, 2.6 µm) with gradient elution of water and methanol containing 2 mM ammonium format and analysed in multiple reaction monitoring mode after positive electrospray ionization. Application of the method included cell culture experiments with two different primary cell types, human coronary artery smooth muscle cells (HCSMC) and human coronary artery endothelial cells (EC). Cells were treated with different concentrations of cortisol, aldosterone and mifepristone, a glucocorticoid receptor antagonist and quantitative PCR was performed. The method exhibits high precision (CV ≤ 6 %) and accuracy (deviation from nominal concentration ≤ 6 %) for concentrations above the limit of quantification (LoQ) which is 0.11, 0.56 and 0.69 nmol/L for aldosterone, cortisone and cortisol, respectively. Calibration curves did not differ when prepared in media or solvent. The method enabled us to confirm activity of HSD11B2 and concentration dependent conversion of cortisol to cortisone in HCSMC (median conversion ratio at 140 nM cortisol = 1.46 %). In contrast we did not observe any HSD11B2 activity in EC. Neither addition of high aldosterone, nor addition of 1 µM mifepristone had impact on glucocorticoid concentrations. Quantitative PCR revealed expression of HSD11B1 and HSD11B2 in HCSMC but not in EC. We present a fast and reliable method for quantification of cortisol, cortisone and aldosterone in cell culture supernatants. The method enabled us to study HSD11B2 activity in two different cell types and will support future experiments investigating mechanisms of target organ damage in conditions of glucocorticoid and mineralocorticoid excess.

Qualitative and Quantitative Analysis of Phytochemicals in Sayeok-Tang via UPLC-Q-Orbitrap-MS and UPLC-TQ-MS/MS.

Sayeok-tang (SYT) is a traditional herbal formula comprising three medicinal herbs: Glycyrrhiza uralensis, Zingiber officinale, and Aconitum carmichaeli. Several studies have employed liquid chromatography-mass spectrometry (LC-MS) to qualitatively analyze the components and metabolites of SYT in vitro and in vivo; however, studies on quantitative analysis of SYT, which is important for quality control, are absent or limited to only a few components. In this study, ultrahigh-performance liquid chromatography coupled with quadrupole (UPLC-Q)-Orbitrap-MS was used to screen the phytochemicals of SYT, revealing a total of 42 compounds. Among them, 24 compounds were simultaneously quantified within 20 min via UPLC-TQ-MS/MS in the multiple reaction monitoring mode. The developed analytical method was validated for its linearity (r2 ≥ 0.9992), precision (0.36-2.96%), accuracy (-6.52-4.64%), and recovery (94.39-119.07%) for all analytes, exhibiting acceptable results. The validated method was applied in the analysis of SYT extracts, and the 24 compounds were quantified in the range of 0.004-6.882 mg/g (CV ≤ 3.746%). Among them, liquiritin apioside (6.870-6.933 mg/g), glycyrrhizic acid (5.418-5.540 mg/g), and liquiritin (1.303-1.331 mg/g) from G. uralensis were identified as the relatively abundant compounds. The presented validated analytical method is highly promising for the comprehensive quality control of SYT, offering fast, highly sensitive, and reliable analysis.

Novel Technique for Simultaneous Ethylene Glycol and Its Metabolites Determination in Human Whole Blood and Urine Samples Using GC-QqQ-MS/MS.

Toxicological analyses often necessitate the identification of compounds belonging to diverse functional groups. For GC-MS analyses, derivatization of compounds belonging to different functional groups can pose a challenge and requires the development of comprehensive methods of analysis. One example could be ethylene glycol, whose widespread use is related to possible unintentional or suicidal intoxications. This fact clearly indicates the need to develop sensitive methods for the determination of ethylene glycol and its metabolites in biological material, as only such complex analysis allows for proper toxicological expertise. A simultaneous GC-QqQ-MS/MS method for the determination of ethylene glycol together with its metabolites, glyoxal and glycolic acid, as well as the detection of glyoxylic acid and oxalic acid, was developed and fully validated. A novel approach for simultaneous derivatization of substances from different groups (alcohols, aldehydes, and carboxylic acids) was established. Sample preparation included the addition of three internal standards (BHB-d4, ethylene glycol-d4 and methylglyoxal), precipitation with acetonitrile and subsequent derivatization with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA), as well as pentafluorophenylhydrazine (PFPH). Detection was carried out with the use of triple quadrupole mass spectrometer. The ionization method was electron impact, and quantitative analysis was carried out in multiple reaction monitoring mode. The lower limit of quantification was 1 μg/mL, 0.1 μg/mL, and 500 μg/mL for ethylene glycol, glyoxal, and glycolic acid, respectively. The presented method was applied in three authentic postmortem cases of ethylene glycol intoxication.

Non-human peptides revealed in blood reflect the composition of intestinal microbiota

BackgroundThe previously underestimated effects of commensal gut microbiota on the human body are increasingly being investigated using omics. The discovery of active molecules of interaction between the microbiota and the host may be an important step towards elucidating the mechanisms of symbiosis.ResultsHere, we show that in the bloodstream of healthy people, there are over 900 peptides that are fragments of proteins from microorganisms which naturally inhabit human biotopes, including the intestinal microbiota. Absolute quantitation by multiple reaction monitoring has confirmed the presence of bacterial peptides in the blood plasma and serum in the range of approximately 0.1 nM to 1 μM. The abundance of microbiota peptides reaches its maximum about 5 h after a meal. Most of the peptides correlate with the bacterial composition of the small intestine and are likely obtained by hydrolysis of membrane proteins with trypsin, chymotrypsin and pepsin – the main proteases of the gastrointestinal tract. The peptides have physicochemical properties that likely allow them to selectively pass the intestinal mucosal barrier and resist fibrinolysis.ConclusionsThe proposed approach to the identification of microbiota peptides in the blood, after additional validation, may be useful for determining the microbiota composition of hard-to-reach intestinal areas and monitoring the permeability of the intestinal mucosal barrier.

Extraction and Quantification of Phenolic Compounds in Maize.

Plants accumulate hundreds of thousands of specialized metabolites that participate in their interactions with the environment. Among these compounds, phenolics represent a large class, and they play important physiological roles, such as providing a first barrier against pathogens, cues to pollinators, and radiation protection. Maize is one of the most important crops worldwide for food, animal feed, and biofuels, and it has the potential to accumulate different phenolics in vegetative tissues as well as in seeds. Recent studies have identified a large number of phenolic compounds-with a diversity of chemical decorations-in different maize tissues, but these likely represent just a fraction of the metabolic diversity of maize. In this protocol, we describe a specific method for the extraction and quantification of maize phenolic compounds by ultra-high-pressure liquid chromatography-tandem multiple reaction monitoring mass spectrometry (UHPLC-MRM-MS/MS) analysis. We provide detailed instructions for the extraction of phenolics using acidic methanol, and for the quantification of 33 different compounds in maize stems, including flavonoids, phenolic acids, and lignin precursors.

Determination of three ephedrine psychoactive substances in sewage using solid-phase extraction-ultra-performance liquid chromatography-tandem mass spectrometry.

In recent years, ephedrine psychoactive substances have attracted much attention due to their prevalence in water bodies and potential threat to aquatic ecosystems. Psychoactive substances have been considered as a new type of environmental pollutant due to their unpredictable potential risks to the behavior and nervous system of non-target organisms. A rapid, sensitive, selective, and robust method for the quantification of three ephedrine psychoactive substances in sewage is needed. An ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of three ephedrine psychoactive substances in water. The optimal processing conditions were determined by optimizing the chromatography-mass spectrometry and solid-phase extraction (SPE) conditions (e.g., the SPE column, sample pH, washing, and elution), and the treatment conditions were determined; this was achieved via positive ion scanning in multiple reaction monitoring mode. Poly-Sery MCX was selected as the extraction column, with samples loaded at pH 3. And 4-mL solution of 2% formic acid (FA) aqueous solution was used as the eluent; the target compounds were eluted with 5 mL of 5% NH4OH in acetonitrile (ACN) solution. The best results were obtained when the residue was resolubulization in ACN after nitrogen evaporation. The developed UPLC-MS/MS showed a good linear relationship in the range of 0-50.00 μg/L, with determination coefficients (R2) greater than 0.9990. The detection limit and quantitation limit were 0.05-0.10 and 0.20-0.50 μg/L, respectively. Recovery rates of the target compounds in blank sewage at three different concentrations ranged from 92.37% to 106.31%, with relative standard deviations (RSDs) of 0.77%-4.83% (n = 7). This method has been successfully applied to the analysis of surface water and domestic sewage, and the samples were processed stably, indicating that the method is practical for the determination of ephedrine psychoactive drugs in water bodies.

Improving the Sensitivity of Protein Quantification by Immunoaffinity Liquid Chromatography─Triple Quadrupole Mass Spectrometry Using an Iterative Transition Summing Technique.

The desire to reach ever-diminishing lower limits of quantification (LLOQ) to probe changes in low abundance protein targets has led to enormous progress in sample preparation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrumentation. To maximize signal and reduce noise, many approaches have been employed, including specific immunoaffinity (IA) enrichment and reducing the LC flow to the nanoflow (nLC) level; however, additional sensitivity gains may still be required. Recently, a technique termed "echo summing" has been described for small-molecular-weight analytes on a triple quadrupole (QqQ) MS where multiple iterations of the same, single selected reaction monitoring (SRM) transition are collected, summed, and integrated, yielding significant analyte dependent signal-to-noise (S/N) improvements. Herein, the direct applicability of echo summing to protein quantification by sequential IA combined with nLC-MS/MS (IA-nLC-MS/MS) is described for a beta nerve growth factor (NGF) and a soluble asialoglycoprotein receptor (sASGPR) assay from human serum. Five iterations of echo summing outperformed traditional collection in relative average accuracy (-1.5 ± 7.7 vs -41.7 ± 10.7% bias) and precision (7.8 vs 18.4% coefficient of variation (CV)) of the low-end quality control (QC) sample (N = 4) for NGF and improved functional sensitivity of serially diluted serum QC samples (N = 5 each population) approximately 2-fold (1.96 and 2.00-fold) for two peptides of sASGPR. Echo summing also extended the minimum quantifiable QC level for sASGPR 4-fold lower. Similar gains are believed to be achievable for most protein IA-nLC-MS/MS assays.

Determination of Taurine in Infant Formula and Adult/Pediatric Nutritionals by Ultra High-Performance Liquid Chromatography with Multiple Reaction Monitoring-Mass Spectrometry: Single-Laboratory Validation, First Action 2022.08.

Taurine (2-aminoethanesulfonic acid) is an essential β-amino acid, which is one of the most abundant intracellular amino acid component in humans. The level of free taurine in human milk is higher than in bovine milk. Since taurine is considered important for the development of a newborn, fortification of infant nutrition with taurine is common. This publication describes a rapid method for the determination of taurine in infant formula and adult/pediatric nutritionals. This publication demonstrates the suitability and applicability of the method to determine taurine content in infant formula and adult/pediatric nutritionals. Test portions are deproteinized by acid in an aqueous environment prior to analysis. The level of taurine is analyzed by hydrophilic interaction chromatography (HILIC) coupled with multiple reaction monitoring MS (MRM-MS). Quantitation is accomplished using a stable isotope-labeled internal standard (SILS) for taurine. The accuracy of the method was validated using standard reference materials and spike recovery studies. The average recoveries ranged between 92% and 105%. The relative standard deviation repeatability (RSDr) and relative standard deviation intermediate precision (RSDiR) were in the range of 0.7-4.6% (RSDr) and 1.3-6.7% (RSDiR). The limit of quantitation (LOQ) was below the requirement of 0.5 mg/100 g. The single-laboratory validation (SLV) of the method for determination of taurine content in infant formula and adult/pediatric nutritionals demonstrates that it meets the requirements per AOAC INTERNATIONAL Standard Method Performance Requirements (SMPR) 2014.013. A fast method to selectively determine the content of taurine in infant formula and adult/pediatric nutritionals was evaluated and found to be fit for purpose for routine product compliance testing. Based on the results of this SLV, the method was approved by the AOAC Stakeholder Program on Infant Formula and Adult Nutritionals (SPIFAN) Expert Review Panel for SPIFAN Nutrient Methods for First Action Official MethodsSM status.

Development and Validation of a Novel Isotope Dilution-Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry Method for Serum C-Peptide.

Mass spectrometry (MS) methods exhibit higher accuracy and comparability in measuring serum C-peptide concentrations than immunoassays. We developed and validated a novel isotope dilution-ultraperformance liquid chromatography-tandem MS (ID-UPLC-MS/MS) assay to measure serum C-peptide concentrations. Sample pretreatment involved solid-phase extraction, ion-exchange solid-phase extraction, and derivatization with 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (Cayman Chemical, Ann Arbor, Michigan, USA). We used an ExionLC UPLC system (Sciex, Framingham, MA, USA) and a Sciex Triple Quad 6500+ MS/MS system (Sciex) for electrospray ionization in positive-ion mode with multiple charge states of [M+3H]3+ and multiple reaction monitoring transitions. The total run time was 50 mins, and the flow rate was 0.20 mL/min. We evaluated the precision, trueness, linearity, lower limit of quantitation (LLOQ), carryover, and matrix effects. Method comparison with electrochemiluminescence immunoassay (ECLIA) was performed in 138 clinical specimens. The intra- and inter-run precision coefficients of variation were <5% and the bias values for trueness were <4%, which were all acceptable. The verified linear interval was 0.050-15 ng/mL, and the LLOQ was 0.050 ng/mL. No significant carryover or matrix effects were observed. The correlation between this ID-UPLC-MS/MS method and ECLIA was good (R=0.995, slope=1.564); however, the ECLIA showed a positive bias (51.8%). The developed ID-UPLC-MS/MS assay shows acceptable performance in measuring serum C-peptide concentrations. This will be useful in situations requiring accurate measurement of serum C-peptide in clinical laboratories.

Parallel and High Throughput Reaction Monitoring with Computer Vision.

We report the development and applications of a computer vision based reaction monitoring method for parallel and high throughput experimentation (HTE). Whereas previous efforts reported methods to extract bulk kinetics of one reaction from one video, this new approach enables one video to capture bulk kinetics of multiple reactions running in parallel. Case studies, in and beyond well-plate high throughput settings, are described. Analysis of parallel dye-quenching hydroxylations, DMAP-catalysed esterification, solid-liquid sedimentation dynamics, metal catalyst degradation, and biologically-relevant sugar-mediated nitro reduction reactions have each provided insight into the scope and limitations of camera-enabled high throughput kinetics as a means of widening known analytical bottlenecks in HTE for reaction discovery, mechanistic understanding, and optimisation. It is envisaged that the nature of the multi-reaction time-resolved datasets made available by this analytical approach will later serve a broad range of downstream efforts in machine learning approaches to exploring chemical space.

Validated UPLC-MS/MS method for quantification of melatonin receptor agonists and dual orexin receptor antagonists in human plasma and breast milk: Application to quantify suvorexant and lemborexant in clinical samples

Pharmaceutical care is important for mental health during the perinatal period, which is often characterized by insomnia. In recent years, prescriptions of melatonin receptor agonists (MRAs) and dual orexin receptor antagonists (DORAs) for insomnia have increased; however, their use during the perinatal period has scarcely been reported. In the present study, we developed a UPLC-MS/MS method for the quantification of ramelteon, its metabolite M-II, suvorexant, and lemborexant in human plasma and breast milk to accumulate information on the safety and transfer of MRAs and DORAs into breast milk. Samples of MRAs (ramelteon and M-II) in plasma and breast milk were prepared using liquid–liquid extraction (LLE) with ethyl acetate. For DORAs (suvorexant and lemborexant), LLE with ethyl acetate was applied to plasma samples. For breast milk samples, significant ion suppression was observed for LLE with ethyl acetate. Solid-phase extraction (SPE) cartridges capable of removing phospholipids improved the matrix effects. Finally, protein precipitation with methanol and an SPE cartridge, InertSep® Phospholipid Remover, were selected for breast milk sample preparation. An ACQUITY UPLC BEH C18 column was used for analyte separation. MRAs and DORAs were eluted using isocratic and gradient elution, respectively, and analyzed using electrospray ionization in the positive mode with multiple reaction monitoring. The range of calibration curve for MRAs and DORAs was 0.1–25 and 0.5–50 ng/ml, respectively. Both the plasma and breast milk samples exhibited good linearity over this range. The method was validated by evaluating its accuracy and precision, matrix effect, recovery, carry-over, stability, and dilution integrity. The validated method was successfully applied to clinical samples donated by breastfeeding women and the milk/plasma (M/P) ratio and relative infant dose (RID) of lemborexant (one case) and suvorexant (two cases) were estimated. The M/P ratio of lemborexant was <1, and the RID was 1.05 %. The M/P ratio of suvorexant was <0.1, and RID was 0.11–0.20 %. This method will be useful for future studies evaluating the safety of these drugs during breastfeeding.