Determination of eleven components in rat plasma by UPLC-MS/MS and GC-MS for pharmacokinetic studies after oral administration of Citri Reticulatae Pericarpium extract.

Citri Reticulatae Pericarpium (CRP) is used as common health-care food and traditional Chinese medicine (TCM), which exerts pharmacological effects, such as anti-cardiovascular, anti-tumor, anti-oxidant, anti-inflammatory, anti-virus, hepatoprotective, blood pressure-lowering and neuroprotective. In this study, reliable, and sensitive ultra-high performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) methods were developed and validated for the determination of eleven active components in rat plasma after oral administration of the CRP extract. The results of this method exhibited that the specificity, linearity (r > 0.999), precision and accuracy (the coefficient of variation (CV) < 11.5 %), recovery (52.9-107.9 %), matrix effects (63.8-107.5 %), and stability (CV < 10.8 %) met all requirements for the quantitation of plasma samples. The pharmacokinetic results showed that the Tmax of flavone glycosides was less than 0.7 h, and that of polymethoxyflavones and volatile components were within 1-7 h. Meanwhile, the area-under-the-curve (AUC) and concentration maximum (Cmax) of hesperidin, nobiletin, tangeretin, and D-limonene were higher than those of the other components, suggesting that the plasma exposure levels of these constituents were higher in CRP. The present research lays a foundation for elucidating the therapeutic material basis and provides a reference for further scientific research and clinical application of CRP.

[Research progress on detection techniques for effective albumin concentration].

Hypoalbuminemia is one of the important clinical features of decompensated cirrhosis. As the disease progresses, not only does the total albumin concentration decrease, but so does the proportion of albumin that remains structurally and functionally intact. The structural and functional integrity of albumin is essential for its normal physiological role in the body. This led to the concept of "effective albumin concentration," which may be much lower than the total albumin concentration routinely measured clinically in patients with advanced cirrhosis. Liquid chromatography-tandem mass spectrometry, and electron paramagnetic resonance (EMR) are emerging technologies for effective albumin concentration detection, showing promising clinical application prospects, but research in patients with cirrhosis is still in the preliminary stage. Therefore, this article will comprehensively summarize the latest research on the aspects of effective albumin detection methods, liquid chromatography-tandem mass spectrometry, and electron paramagnetic resonance, as well as their applications.

Liquid chromatography-tandem mass spectrometry in clinical laboratory protein measurement.

Proteins are essential components of human cells and tissues, and they are commonly measured in clinical laboratories using immunoassays. However, these assays have certain limitations, such as non-specificity binding, insufficient selectivity, and interference of antibodies. More sensitive, accurate, and efficient technology is required to overcome these limitations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful analytical tool that provides high sensitivity and specificity, making it superior to traditional methods such as biochemical and immunoassays. While LC-MS/MS has been increasingly used for detecting small molecular analytes and steroid hormones in clinical practice recently, its application for protein or peptide analysis is still in its early stages. Established methods for quantifying proteins and peptides by LC-MS/MS are mainly focused on scientific research, and only a few proteins and peptides can be or have the potential to be detected and applied in clinical practice. Therefore, this article aims to review the clinical applications, advantages, and challenges of analyzing proteins and peptides using LC-MS/MS in clinical laboratories.

Wastewater-based epidemiology to monitor 68 NPS/conventional drug use in Taipei metropolitan area in Taiwan during and after COVID-19 pandemic.

Amidst far-reaching COVID-19 effects and social constraints, this study leveraged wastewater-based epidemiology to track 38 conventional drugs and 30 new psychoactive substances (NPS) in northern Taiwan. Analyzing daily samples from four Taipei wastewater plants between September 2021 and January 2024-encompassing club reopenings, holidays, Lunar New Year, an outbreak, and regular periods-thirty-one drugs were detected, including 5 NPS. Tramadol, zolpidem tartrate, CMA, and MDPV were newly detected in Taiwanese sewage with frequency of 1.4 %- 89.0 %. Conventional drug use typically increased post-pandemic, aside from benzodiazepines and methadone. Methamphetamine showed 100 % frequency, indicating ongoing daily consumption despite COVID-19 measures. Methamphetamine and morphine's consumption dipped then rose around club reopening, hinting at limited access. The consumption trend of methadone appeared to compensate for the use of morphine. Ketamine and NPS demonstrated similar patterns throughout the entire period. NPS as party drugs seemed influenced by an unstable supply chain and complexities in implementation. Benzodiazepines, commonly abused alongside synthetic cathinones in Taiwan exhibited an opposing trend to NPS while aligned with acetaminophen, suggesting elevated stress and anxiety levels during the pandemic. No significant differences were observed in drug consumption between weekdays and weekends, potentially indicating that COVID-19 measures blurred the traditional distinctions between these timeframes. ENVIRONMENTAL IMPLICATION: New psychoactive substances refer to chemically modified variants of controlled drugs designed to mimic the effects of the original drugs while evading modern detection methods, categorizing them as hazardous materials. The study presents a sewage monitoring project conducted from 2021 to 2024, collecting samples from four WWTPs to analyze NPS and conventional drug trends during and after the COVID-19 pandemic. The findings uncovered connections between drug consumption patterns and pandemic-related policies. In light of the persistent drug abuse and their environmental presence, the results bear critical importance for both environmental and public health. We provide a thorough assessment of these relationships and prioritize areas for future research.

Acaricidal activity of Erigeron acer L. root against Haemaphysalis longicornis and phytochemical profiling by liquid chromatography-tandem mass spectrometry.

The present study is focused on evaluating acaricidal activity and chemical compositions of Erigeron acer root, which was identified as a promising candidate among fifteen Mongolian plant extracts tested for acaricidal activity. The acaricidal effect was evaluated against Haemaphysalis longicornis, assessed for toxicity to normal human skin fibroblast, and analyzed for its chemical constituents. The acetone extract of E. acer root showed significant activity against H. longicornis, with a lethal concentration (LC50) of 5.31 mg/mL and low toxicity, evidenced by a cytotoxic concentration (CC50) of 267.00 µg/mL. Using liquid chromatography-tandem mass spectrometry and molecular networking, thirteen natural compounds were identified, including pyrrolidines, alkaloids, fatty acids, and flavonoids, highlighting the efficacy of E. acer root extract as an effective acaricide against H. longicornis and offering insights for developing new tick control solutions.

Simultaneous measurement of 17 endogenous steroid hormones in human serum by liquid chromatography-tandem mass spectrometry without derivatization.

Mass spectrometric-based steroidomics is a valuable analytical approach that gives a comprehensive understanding of the interlinked steroid biosynthetic pathways. Here, we describe a rapid and versatile liquid chromatography-tandem mass spectrometry (LC-MS/MS) method designed to accurately quantify endogenous steroids in human serum. Sample preparation involved liquid-liquid extraction with methyl tert-butyl ether (MTBE) from 180 µL serum. The targeted steroids for quantification included androgens: dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), dihydrotestosterone (DHT), 11-oxyandrogens: 11ß-hydroxy-androstenedione (11OHA4), 11-keto-androstenedione (11KA4), 11ß-hydroxy-testosterone (11OHT), 11-keto-testosterone (11KT), progestogens: 17α-hydroxy-progesterone (17OHP4), progesterone (P4), 11ß-hydroxy-progesterone (11OHP4), 11-keto-progesterone (11KP4), mineralocorticoids: aldosterone, corticosterone, and glucocorticoids: 11-deoxycortisol, cortisol, and cortisone. The lower limits of quantification (LLOQ) were 0.05 ng/mL for A4, T, 11KA4, P4, and cortisone, 0.1 ng/mL for DHT, 11OHA4, 11OHT, 11KT, 17OHP4, 11OHP4, 11KP4, corticosterone, aldosterone, 11-deoxycortisol, and cortisol, and 0.5 ng/mL for DHEA. Accuracy, precision, reproducibility, and recovery fell within acceptable limits for bioanalytical method validation. Using serum samples from 29 premenopausal women in different menstrual phases, we demonstrated the clinical utility of our method, which showed sufficient sensitivity to reliably quantify all targeted steroids at levels typically found in circulation, except for 11OHP4 and 11KP4.

Analytical characterization of anthocyanins using trapped ion mobility spectrometry-quadrupole time-of-flight tandem mass spectrometry.

Anthocyanin profiles of juices from blueberry (Vaccinium myrtillus L.) and different grape varieties (Vitis labrusca L. cv. Concord, Vitis vinifera L. cvs. Accent, Dunkelfelder, Dakapo, and GM 674-1) were characterized by ultra-high performance liquid chromatography (UHPLC) coupled to trapped ion mobility spectrometry-quadrupole time-of-flight tandem mass spectrometry (TIMS-QTOF-MS/MS). Ion mobility and collision cross section (CCS) values of over 50 structurally related anthocyanins based on delphinidin, cyanidin, petunidin, peonidin, and malvidin were determined. Relations between molecular mass, mobility values, and specific structural features were revealed. The mass-to-charge (m/z) ratio of the molecular ions (M+) was found to be the major factor influencing anthocyanin ion mobilities, but structural characteristics also contributed to their variability. We were able to differentiate positional and geometrical isomers and certain epimers by their respective mobility values. For instance, whereas 3-O-hexosides (i.e., 3-O-glucosides and 3-O-galactosides) were separated by TIMS, epimers of 3-O-pentosides assessed could not be distinguished.

Rapid determination of toxic and essential metal binding proteins in biological samples by size exclusion chromatography-inductively coupled plasma tandem mass spectrometry.

Metalloproteins binding with trace elements play a crucial role in biological processes and on the contrary, those binding with exogenous heavy metals have adverse effects. However, the methods for rapid, high sensitivity and simultaneous analysis of these metalloproteins are still lacking. In this study, a fast method for simultaneously determination of both essential and toxic metal-containing proteins was developed by coupling size exclusion chromatography (SEC) with inductively coupled plasma tandem mass spectrometry (ICP-MS/MS). After optimization of the separation and detection conditions, seven metalloproteins with different molecular weight (from 16.0 to 443.0 kDa) were successfully separated within 10 min and the proteins containing iron (Fe), copper (Cu), zinc (Zn), iodine (I) and lead (Pb) elements could be simultaneously detected with the use of oxygen as the collision gas in ICP-MS/MS. Accordingly, the linear relationship between log molecular weight and retention time was established to estimate the molecular weight of unknown proteins. Thus, the trace metal and toxic metal containing proteins could be detected in a single run with high sensitivity (detection limits in the range of 0.0020-2.5 µg/mL) and good repeatability (relative standard deviations lower than 4.5 %). This method was then successfully used to analyze metal (e.g., Pb, Zn, Cu and Fe) binding proteins in the blood of Pb-intoxicated patients, and the results showed a negative correlation between the contents of zinc and lead binding proteins, which was identified to contain hemoglobin subunit. In summary, this work provided a rapid and sensitive tool for screening metal containing proteins in large number of biological samples.

Temperature and pH-dependent stability of fentanyl analogs: Degradation pathways and potential biomarkers.

The collection, storage, and transport of samples prior to and during analysis is of utmost importance, especially for highly potent analogs that may not be present in high concentrations and are susceptible to pH or thermally mediated degradation. An accelerated stability study was performed on 17 fentanyl analogs (fentalogs) over a wide range of pH (2-10) and temperature (20-60°C) conditions over 24 h. Dilute aqueous systems were used to investigate temperature and pH-dependent kinetics using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Liquid chromatography-quadrupole/time-of-flight-mass spectrometry (LC-Q/TOF-MS) was used for structural elucidation of degradants. With the exception of remifentanil, all fentalogs evaluated were stable at pH 6 or lower. Fentalogs were generally unstable in strongly alkaline environments and at elevated temperatures. Remifentanil was the least stable drug and N-dealkylated fentalogs were the most stable. Fentanyl degraded to acetylfentanyl, norfentanyl, fentanyl N-oxide, and 1-phenethylpyridinium salt (1-PEP). A total of 26 unique breakdown products were observed for 15 of the fentanyl derivatives studied. Common degradation pathways involved N-dealkylation, oxidation of the piperidine nitrogen, and ß-elimination of N-phenylpropanamide followed by oxidation/dehydration of the piperidine ring. Ester and amide hydrolysis, demethylation at the propanamide, and O-demethylation were observed for selected fentalogs only. The potential for analyte loss should be considered during the pre-analytical phase (i.e., shipping and transport) where environmental conditions may not be controlled, as well as during the analysis itself.

Making MS Omics Data ML-Ready: SpeCollate Protocols.

The increasing complexity and volume of mass spectrometry (MS) data have presented new challenges and opportunities for proteomics data analysis and interpretation. In this chapter, we provide a comprehensive guide to transforming MS data for machine learning (ML) training, inference, and applications. The chapter is organized into three parts. The first part describes the data analysis needed for MS-based experiments and a general introduction to our deep learning model SpeCollate-which we will use throughout the chapter for illustration. The second part of the chapter explores the transformation of MS data for inference, providing a step-by-step guide for users to deduce peptides from their MS data. This section aims to bridge the gap between data acquisition and practical applications by detailing the necessary steps for data preparation and interpretation. In the final part, we present a demonstrative example of SpeCollate, a deep learning-based peptide database search engine that overcomes the problems of simplistic simulation of theoretical spectra and heuristic scoring functions for peptide-spectrum matches by generating joint embeddings for spectra and peptides. SpeCollate is a user-friendly tool with an intuitive command-line interface to perform the search, showcasing the effectiveness of the techniques and methodologies discussed in the earlier sections and highlighting the potential of machine learning in the context of mass spectrometry data analysis. By offering a comprehensive overview of data transformation, inference, and ML model applications for mass spectrometry, this chapter aims to empower researchers and practitioners in leveraging the power of machine learning to unlock novel insights and drive innovation in the field of mass spectrometry-based omics.

Demystifying PTM Identification Using MODplus: Best Practices and Pitfalls.

Tandem mass spectrometry (MS/MS) facilitates the rapid identification of posttranslational modifications (PTMs), which play a pivotal role in regulating numerous biological processes. This chapter explores recent advancements that expand the types of detectable PTMs and enhance the speed of the PTM searches. We also delve into computational challenges associated with searching for a multitude of PTMs simultaneously. The latter section introduces an automated procedure to identify an extensive range of PTMs using MODplus, a free PTM analysis software tool. We guide the reader through the preparation of the modification search, the determination of optional search parameters, the execution of the search, and the analysis of results, exemplified by a case study using specific MS/MS dataset.

The analysis of methylamphetamine and para-hydroxy-methylamphetamine in post-mortem hair samples using liquid chromatography-tandem mass spectrometry.

External contamination is a well-recognized limitation of hair analysis for drugs of abuse like methylamphetamine (MA), and there are no guidelines regarding the analysis of specific metabolites of MA to assist interpretation. We developed an analytical method to detect MA, amphetamine (AMP), and para-hydroxy-methylamphetamine (p-OH-MA) in hair and present their concentrations among a cohort of deceased persons positive for MA in blood (n = 63). Hair samples (≤ 3 cm) were washed with dichloromethane and water prior to extraction using a methanolic micro-pulverization. The reconstituted hair extracts were separated on a UCT Selectra® Aqueous C18 HPLC Column (100 × 2.1 mm, 3 µm) by gradient elution and detected using a Sciex Triple Quad 6500+ system. Validation was satisfactory, and the lower limits of quantitation were 0.01 ng/mg for MA and AMP and 0.001 ng/mg for p-OH-MA. The median hair concentrations of MA, AMP, and p-OH-MA were 13 ng/mg (range = 0.015-49; n = 51), 1.1 ng/mg (range = 0.018-44; n = 60), and 0.020 ng/mg (range = 0.0012-0.38, n = 62), respectively. These concentrations in hair were strongly positively correlated (r = .7202 to .8641, p < .001), suggesting similar modes of incorporation. Moreover, the wash/hair ratios were indicative of external contamination, especially among the soiled group of hair samples. Therefore, further studies are necessary to determine concentrations of p-OH-MA in living MA users and confirm if this metabolite constitutes a potential marker of MA consumption.

Development and evaluation of a candidate reference measurement procedure for detecting 17α-hydroxyprogesterone in dried blood spots using isotope dilution liquid chromatography tandem mass spectrometry.

17α-Hydroxyprogesterone (17α-OHP) quantification in dried blood spots (DBS) is essential for newborn screening for congenital adrenal hyperplasia (CAH), which is challenging due to its low physiological concentration. The high false-positive rates of immunoassays necessitate the development of more accurate methods. Liquid chromatography tandem mass spectrometry (LC-MS/MS) offers increased specificity and sensitivity, yet standardized procedures for 17α-OHP measurement are required for clinical application. A candidate reference measurement procedure (cRMP) using isotope dilution LC-MS/MS was developed for 17α-OHP quantification in DBS. By utilizing stable isotope-labeled D8-17α-OHP as an internal standard, the cRMP was optimized, covering sample preparation, calibration, and LC-MS/MS analysis. The method performance was validated across several parameters, including precision, accuracy, specificity, detection limits, and matrix effects. Clinical applicability was further assessed through the establishment of reference intervals for healthy newborns. The developed cRMP exhibited a linear range of 1.00 to 80.00 ng/mL for 17α-OHP, with detection and quantification limits of 0.14 ng/mL and 0.52 ng/mL, respectively. Inter- and intraday precision demonstrated coefficients of variation within 1.27 to 5.69%. The recovery rates and matrix effects were well within acceptable limits, ensuring method reliability. Clinical application showed distinct reference intervals for healthy newborns that were unaffected by sex but influenced by weight and gestational age. This method significantly enhances CAH diagnostic accuracy in newborns, providing a valuable tool for clinical laboratories and improving newborn screening program standardization and traceability.

Characterizing lipid constituents of B. moojeni snake venom: a comparative approach for chemical and biological investigations.

Snake venoms are complex mixtures majorly composed of proteins with well-studied biological effects. However, the exploration of non-protein components, especially lipids, remains limited despite their potential for discovering bioactive molecules. This study compares three liquid-liquid lipid extraction methods for both chemical and biological analyses of Bothrops moojeni snake venom. The methods evaluated include the Bligh and Dyer method (methanol, chloroform, water), considered standard; the Acunha method, a modification of the Bligh and Dyer protocol; and the Matyash method (MTBE/methanol/water), featuring an organic phase less dense than the aqueous phase. Lipidomic analysis using liquid chromatography with high-resolution mass spectrometry (LC-HRMS) system revealed comparable values of lipid constituents' peak intensity across different extraction methods. Our results show that all methods effectively extracted a similar quantity of lipid species, yielding approximately 17-18 subclasses per method. However, the Matyash and Acunha methods exhibited notably higher proportions of biologically active lipids compared to the Bligh and Dyer method, particularly in extracting lipid species crucial for cellular structure and function, such as sphingomyelins and phosphatidylinositol-phosphate. In conclusion, when selecting a lipid extraction method, it is essential to consider the study's objectives. For a biological approach, it is crucial to evaluate not only the total quantity of extracted lipids but also their quality and biological activity. The Matyash and Acunha methods show promise in this regard, potentially offering a superior option for extracting biologically active lipids compared to the Bligh and Dyer method.

Distinguishing Isomeric Cyclobutane Thymidine Dimers by Ion Mobility and Tandem Mass Spectrometry.

Irradiation of the major conformation of duplex DNA found in cells (B form) produces cyclobutane pyrimidine dimers (CPDs) from adjacent pyrimidines in a head-to-head orientation (syn) with the C5 substituents in a cis stereochemistry. These CPDs have crucial implications in skin cancer. Irradiation of G-quadruplexes and other non-B DNA conformations in vitro produces, however, CPDs between nonadjacent pyrimidines in nearby loops with syn and head-to-tail orientations (anti) with both cis and trans stereochemistry to yield a mixture of six possible isomers of the T=T dimer. This outcome is further complicated by formation of mixtures of nonadjacent CPDs of C=T, T=C, and C=C, and successful analysis depends on development of specific and sensitive methods. Toward meeting this need, we investigated whether ion mobility mass spectrometry (IMMS) and MS/MS can distinguish the cis,syn and trans,anti T=T CPDs. Ion mobility can afford baseline separation and give relative mobilities that are in accord with predicted cross sections. Complementing this ability to distinguish isomers is MS/MS collisional activation where fragmentation also distinguishes the two isomers and confirms conclusions drawn from ion mobility analysis. The observations offer early support that ion mobility and MS/MS can enable the distinction of DNA photoproduct isomers.

Cytotoxicity of Physalis minima Linn (Solanaceae) fruit against HCT116 and HT29 colorectal cancer cell lines.

The pantropical Physalis minima are traditionally used for the prevention and treatment of various illnesses, diseases, and cancers. While most earlier studies on the species have focused on the phytochemistry of the leaf and stem extracts, recent studies have indicated that its fruit may contain bioactive compounds of medical interest. In this study, we investigated the cytotoxicity of extracts from the fruit of P. minima against colorectal cancer cell lines and revealed its phytochemical profile via high-resolution tandem mass spectrometry analysis. Following a 24-h treatment with the fruit extract, cytoplasm shrinkage and nucleus condensation were observed in the colorectal cancer cell lines HCT116 and HT29, indicating the induction of programmed cell death. Phytochemically, 71 putative metabolites were identified. Some of these metabolites have been reported to inhibit cancers to varying degrees, further supporting the correlation of the putative metabolites with the cytotoxicity against colorectal cancer cells demonstrated in this study.

Basal release of 6-cyanodopamine from rat isolated vas deferens and its role on the tissue contractility.

6-Cyanodopamine is a novel catecholamine released from rabbit isolated heart. However, it is not known whether this catecholamine presents any biological activity. Here, it was evaluated whether 6-cyanodopamine (6-CYD) is released from rat vas deferens and its effect on this tissue contractility. Basal release of 6-CYD, 6-nitrodopamine (6-ND), 6-bromodopamine, 6-nitrodopa, and 6-nitroadrenaline from vas deferens were quantified by LC-MS/MS. Electric-field stimulation (EFS) and concentration-response curves to noradrenaline, adrenaline, and dopamine of the rat isolated epididymal vas deferens (RIEVD) were performed in the absence and presence of 6-CYD and /or 6-ND. Expression of tyrosine hydroxylase was assessed by immunohistochemistry. The rat isolated vas deferens released significant amounts of both 6-CYD and 6-ND. The voltage-gated sodium channel blocker tetrodotoxin had no effect on the release of 6-CYD, but it virtually abolished 6-ND release. 6-CYD alone exhibited a negligible RIEVD contractile activity; however, at 10 nM, 6-CYD significantly potentiated the noradrenaline- and EFS-induced RIEVD contractions, whereas at 10 and 100 nM, it also significantly potentiated the adrenaline- and dopamine-induced contractions. The potentiation of noradrenaline- and adrenaline-induced contractions by 6-CYD was unaffected by tetrodotoxin. Co-incubation of 6-CYD (100 pM) with 6-ND (10 pM) caused a significant leftward shift and increased the maximal contractile responses to noradrenaline, even in the presence of tetrodotoxin. Immunohistochemistry revealed the presence of tyrosine hydroxylase in both epithelial cell cytoplasm of the mucosae and nerve fibers of RIEVD. The identification of epithelium-derived 6-CYD and its remarkable synergism with catecholamines indicate that epithelial cells may regulate vas deferens smooth muscle contractility.

Determination of Flunixin and 5-Hydroxy Flunixin Residues in Livestock and Fishery Products Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Flunixin is a veterinary nonsteroidal anti-inflammatory agent whose residues have been investigated in their original form within tissues such as muscle and liver. However, flunixin remains in milk as a metabolite, and 5-hydroxy flunixin has been used as the primary marker for its surveillance. This study aimed to develop a quantitative method for detecting flunixin and 5-hydroxy flunixin in milk and to strengthen the monitoring system by applying to other livestock and fishery products. Two different methods were compared, and the target compounds were extracted from milk using an organic solvent, purified with C18, concentrated, and reconstituted using a methanol-based solvent. Following filtering, the final sample was analyzed using liquid chromatography- tandem mass spectrometry. Method 1 is environmentally friendly due to the low use of reagents and is based on a multi-residue, multi-class analysis method approved by the Ministry of Food and Drug Safety. The accuracy and precision of both methods were 84.6%-115% and 0.7%-9.3%, respectively. Owing to the low matrix effect in milk and its convenience, Method 1 was evaluated for other matrices (beef, chicken, egg, flatfish, and shrimp) and its recovery and coefficient of variation are sufficient according to the Codex criteria (CAC/GL 71-2009). The limits of detection and quantification were 2-8 and 5-27 µg/kg for flunixin and 2-10 and 6-33 µg/kg for 5-hydroxy flunixin, respectively. This study can be used as a monitoring method for a positive list system that regulates veterinary drug residues for all livestock and fisheries products.

Plastic Quantification and Polyethylene Overestimation in Agricultural Soil Using Large-Volume Pyrolysis and TD-GC-MS/MS.

Quantification of microplastics in soil is needed to understand their impact and fate in agricultural areas. Often, low sample volume and removal of organic matter (OM) limit representative quantification. We present a method which allows simultaneous quantification of microplastics in homogenized, large environmental samples (>1 g) and tested polyethylene (PE), polyethylene terephthalate (PET), and polystyrene (PS) (200-400 µm) overestimation by fresh and diagenetically altered OM in agricultural soils using a new combination of large-volume pyrolysis adsorption with thermal desorption-gas chromatography-tandem mass spectrometry (TD-GC-MS/MS). Characteristic MS/MS profiles for PE, PET, and PS were derived from plastic pyrolysis and allowed for a new mass separation of PET. Volume-defined standard particles (125 × 125 × 20 µm3) were developed with the respective weight (PE: 0.48 ± 0.12, PET: 0.50 ± 0.10, PS: 0.31 ± 0.08 µg), which can be spiked into solid samples. Diagenetically altered OM contained compounds that could be incorrectly identified as PE and suggest a mathematical correction to account for OM contribution. With a standard addition method, we quantified PS, PET, and PEcorrected in two agricultural soils. This provides a base to simultaneously quantify a variety of microplastics in many environmental matrices and agricultural soil.

[A highly sensitive approach for the analysis of tyrosine phosphoproteome in primary T cells].

Tyrosine phosphorylation, a common post-translational modification process for proteins, is involved in a variety of biological processes. However, the abundance of tyrosine-phosphorylated proteins is very low, making their identification by mass spectrometry (MS) is difficult; thus, milligrams of the starting material are often required for their enrichment. For example, tyrosine phosphorylation plays an important role in T cell signal transduction. However, the number of primary T cells derived from biological tissue samples is very small, and these cells are difficult to culture and expand; thus, the study of T cell signal transduction is usually carried out on immortalized cell lines, which can be greatly expanded. However, the data from immortalized cell lines cannot fully mimic the signal transduction processes observed in the real physiological state, and they usually lead to conclusions that are quite different from those of primary T cells. Therefore, a highly sensitive proteomic method was developed for studying tyrosine phosphorylation modification signals in primary T cells. To address the issue of the limited T cells numbers, a comprehensive protocol was first optimized for the isolation, activation, and expansion of primary T cells from mouse spleen. CD3+ primary T cells were successfully sorted; more than 91% of the T cells collected were well activated on day 2, and the number of T cells expanded to over 7-fold on day 4. Next, to address the low abundance of tyrosine-phosphorylated proteins, we used SH2-superbinder affinity enrichment and immobilized Ti4+affinity chromatography (Ti4+-IMAC) to enrich the tyrosine-phosphorylated polypeptides of primary T cells that were co-stimulated with anti-CD3 and anti-CD28. These polypeptides were resolved using nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). Finally, 282 tyrosine phosphorylation sites were successfully identified in 1 mg of protein, including many tyrosine phosphorylation sites on the immunoreceptor tyrosine-based activation motif (ITAM) in the intracellular region of the T cell receptor membrane protein CD3, as well as the phosphotyrosine sites of ZAP70, LAT, VAV1, and other proteins related to signal transduction under costimulatory conditions. In summary, to solve the technical problems of the limited number of primary cells, low abundance of tyrosine-phosphorylated proteins, and difficulty of detection by MS, we developed a comprehensive proteomic method for the in-depth analysis of tyrosine phosphorylation modification signals in primary T cells. This protocol may be applied to map signal transduction networks that are closely related to physiological states.