Metabolic signatures and risk of sarcopenia in suburb-dwelling older individuals by LC-MS-based untargeted metabonomics.

Background: Untargeted metabonomics has provided new insight into the pathogenesis of sarcopenia. In this study, we explored plasma metabolic signatures linked to a heightened risk of sarcopenia in a cohort study by LC-MS-based untargeted metabonomics. Methods: In this nested case-control study from the Adult Physical Fitness and Health Cohort Study (APFHCS), we collected blood plasma samples from 30 new-onset sarcopenia subjects (mean age 73.2 ± 5.6 years) and 30 healthy controls (mean age 74.2 ± 4.6 years) matched by age, sex, BMI, lifestyle, and comorbidities. An untargeted metabolomics methodology was employed to discern the metabolomic profile alterations present in individuals exhibiting newly diagnosed sarcopenia. Results: In comparing individuals with new-onset sarcopenia to normal controls, a comprehensive analysis using liquid chromatography-mass spectrometry (LC-MS) identified a total of 62 metabolites, predominantly comprising lipids, lipid-like molecules, organic acids, and derivatives. Receiver operating characteristic (ROC) curve analysis indicated that the three metabolites hypoxanthine (AUC=0.819, 95% CI=0.711-0.927), L-2-amino-3-oxobutanoic acid (AUC=0.733, 95% CI=0.598-0.868) and PC(14:0/20:2(11Z,14Z)) (AUC= 0.717, 95% CI=0.587-0.846) had the highest areas under the curve. Then, these significant metabolites were observed to be notably enriched in four distinct metabolic pathways, namely, "purine metabolism"; "parathyroid hormone synthesis, secretion and action"; "choline metabolism in cancer"; and "tuberculosis". Conclusion: The current investigation elucidates the metabolic perturbations observed in individuals diagnosed with sarcopenia. The identified metabolites hold promise as potential biomarkers, offering avenues for exploring the underlying pathological mechanisms associated with sarcopenia.

Comparative Analysis of Protein Quantification by the SomaScan Assay versus Orthogonal Methods in Urine from People with Diabetic Kidney Disease.

To our knowledge, calibration curves or other validations for thousands of SomaScan aptamers are not publicly available. Moreover, the abundance of urine proteins obtained from these assays is not routinely validated with orthogonal methods (OMs). We report an in-depth comparison of SomaScan readout for 23 proteins in urine samples from patients with diabetic kidney disease (n = 118) vs OMs, including liquid chromatography-targeted mass spectrometry (LC-MS), ELISA, and nephelometry. Pearson correlation between urine abundance of the 23 proteins from SomaScan 3.2 vs OMs ranged from -0.58 to 0.86, with a median (interquartile ratio, [IQR]) of 0.49 (0.18, 0.53). In multivariable linear regression, the SomaScan readout for 6 of the 23 examined proteins (26%) was most strongly associated with the OM-derived abundance of the same (target) protein. For 3 of 23 (13%), the SomaScan and OM-derived abundance of each protein were significantly associated, but the SomaScan readout was more strongly associated with OM-derived abundance of one or more "off-target" proteins. For the remaining 14 proteins (61%), the SomaScan readouts were not significantly associated with the OM-derived abundance of the targeted proteins. In 6 of the latest group, the SomaScan readout was not associated with urine abundance of any of the 23 quantified proteins. To sum, over half of the SomaScan results could not be confirmed by independent orthogonal methods.

[Applications of chromatography in glycomics].

Glycomics, an emerging "omics" technology that was developed after genomics and proteomics, is a discipline that studies the composition, structure, and functions of glycomes in cells, tissues, and organisms. Glycomics plays key roles in understanding the laws of major life activities, disease prevention and treatment, and drug quality control and development. At present, the structural analysis of glycans relies mainly on mass spectrometry. However, glycans have low abundance in biological samples. In addition, factors such as variable monosaccharide compositions, differences in glycosidic bond positions and modes, diverse branching structures, contribute to the complexity of the compositions and structures of glycans, posing great challenges to glycomics research. Liquid chromatography can effectively remove matrix interferences and enhance glycan separation to improve the mass spectrometric response of glycans. Thus, liquid chromatography and liquid chromatography coupled with mass spectrometry are important technical tools that have been actively applied to solve these problems; these technologies play indispensable roles in glycomics research. Different studies have highlighted similarities and differences in the applications of various types of liquid chromatography, which also reflects the versatility and flexibility of this technology. In this review, we first discuss the enrichment methods for glycans and their applications in glycomics research from the perspective of chromatographic separation mechanisms. We then compare the advantages and disadvantages of these methods. Some glycan-enrichment modes include affinity, hydrophilic interactions, size exclusion, and porous graphitized carbon adsorption. A number of newly developed materials exhibit excellent glycan-enrichment ability. We enumerate the separation mechanisms of reversed-phase high performance liquid chromatography (RP-HPLC), high performance anion-exchange chromatography (HPAEC), hydrophilic interaction chromatography (HILIC), and porous graphitic carbon (PGC) chromatography in the separation and analysis of glycans, and describe the applications of these methods in the separation of glycans, glycoconjugates, and glyco-derivatives. Among these methods, HILIC and PGC chromatography are the most widely used, whereas HPAEC and RP-HPLC are less commonly used. The HILIC and RP-HPLC modes are often used for the separation of derived glycans. The ionization efficiency and detectability of glycans are significantly improved after derivatization. However, the derivatization process is relatively cumbersome, and byproducts inevitably affect the accuracy and completeness of the detection results. HPAEC and PGC chromatography exhibit good separation effects on nonderivative glycans, but issues related to the detection integrity of low-abundance glycans owing to their poor detection effect continue to persist. Therefore, the appropriate analytical method for a specific sample or target analyte or mutual verification must be selected. Finally, we highlight the research progress in various chromatographic methods coupled with mass spectrometry for glycomics analysis. Significant progress has been made in glycomics research in recent years owing to advancements in the development of chromatographic separation techniques. However, several significant challenges remain. As the development of novel separation materials and methods continues, chromatographic techniques may be expected to play a critical role in future glycomics research.

[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.

[Establishment of collection methods and laboratory testing methods for active pharmaceutical ingredient in the workplace air].

Objective: To establish collection methods and laboratory testing methods for qualitative and quantitative analysis of 9 typical active pharmaceutical ingredient in the workplace air. Methods: In December 2021, a mixed solution of nine analytes was prepared and then dispersed in aerosol state to simulate sampling. Glass fiber filter membrane was selected as air collector and collected active pharmaceutical ingredient in the air at a rate of 2.0 L/min for 15 minutes. Then, the obtained filter membrane samples were eluted with 25%ACN/75%MeOH. Finally, the eluent was qualitatively and quantitatively analyzed with liquid chromatography-triple quadrupole mass spectrometer. Results: This method could effectively collect active pharmaceutical ingredient in the air, with an average sampling efficiency of more than 98.5%. The linear correlation coefficient r was greater than 0.9990. The lower limit of quantification for each analyte ranged from 0.6~500.0 ng/ml, and the average recovery rate ranged from 97.6%~102.5%. Conclusion: This method could simultaneously collect 9 active pharmaceutical ingredient in the workplace air, and could provide accurate qualitative and quantitative analysis in subsequent laboratory tests.

Metabolomics-based search for lung cancer markers among patients with different smoking status.

Tobacco smoking is the main etiological factor of lung cancer (LC), which can also cause metabolome disruption. This study aimed to investigate whether the observed metabolic shift in LC patients was also associated with their smoking status. Untargeted metabolomics profiling was applied for the initial screening of changes in serum metabolic profile between LC and chronic obstructive pulmonary disease (COPD) patients, selected as a non-cancer group. Differences in metabolite profiles between current and former smokers were also tested. Then, targeted metabolomics methods were applied to verify and validate the proposed LC biomarkers. For untargeted metabolomics, a single extraction-dual separation workflow was applied. The samples were analyzed using a liquid chromatograph-high resolution quadrupole time-of-flight mass spectrometer. Next, the selected metabolites were quantified using liquid chromatography-triple-quadrupole mass spectrometry. The acquired data confirmed that patients' stratification based on smoking status impacted the discriminating ability of the identified LC marker candidates. Analyzing a validation set of samples enabled us to determine if the putative LC markers were truly robust. It demonstrated significant differences in the case of four metabolites: allantoin, glutamic acid, succinic acid, and sphingosine-1-phosphate. Our research showed that studying the influence of strong environmental factors, such as tobacco smoking, should be considered in cancer marker research since it reduces the risk of false positives and improves understanding of the metabolite shifts in cancer patients.

Illuminating the dark space of neutral glycosphingolipidome by selective enrichment and profiling at multi-structural levels.

Glycosphingolipids (GSLs) are essential components of cell membranes, particularly enriched in the nervous system. Altered molecular distributions of GSLs are increasingly associated with human diseases, emphasizing the significance of lipidomic profiling. Traditional GSL analysis methods are hampered by matrix effect from phospholipids and the difficulty in distinguishing structural isomers. Herein, we introduce a highly sensitive workflow that harnesses magnetic TiO2 nanoparticle-based selective enrichment, charge-tagging Paternò-Büchi reaction, and liquid chromatography-tandem mass spectrometry. This approach enables mapping over 300 distinct GSLs in brain tissues by defining sugar types, long chain bases, N-acyl chains, and the locations of desaturation and hydroxylation. Relative quantitation of GSLs across multiple structural levels provides evidence of dysregulated gene and protein expressions of FA2H and CerS2 in human glioma tissue. Based on the structural features of GSLs, our method accurately differentiates human glioma with/without isocitrate dehydrogenase genetic mutation, and normal brain tissue.

Immunohistochemical Diagnosis of Amyloid Typing: Utility and Limitations as Determined by Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Although immunohistochemical techniques and proteomic analysis are widely used for typing diagnosis of amyloidosis, the diagnostic utility of immunohistochemical evaluation is not well understood. METHODS: We used immunohistochemical techniques to characterize staining patterns of in-house rabbit polyclonal anti-κ, anti-λ, anti-transthyretin antibodies, and commercial anti-amyloid A and anti-ß2-microglobulin antibodies in 40 autopsy cases. RESULTS: In thirty cases (75%), the subtype was determined by using the criterion that amyloid is strongly and diffusely positive for one antibody while negative for other antibodies. We then performed proteomic analysis of all 40 cases. In 39 cases, we identified only one amyloid protein and confirmed the immunohistochemically determined subtypes of the abovementioned 30 cases. In seven other cases, we could retrospectively determine subtypes with immunohistochemistry by using information from proteomic analysis, which increased the immunohistochemistry diagnosis rate to 92.5% (37/40). In one case, we identified double subtypes, both immunohistochemically and with proteomic analysis. In the remaining three cases, proteomic analysis was essential for typing diagnosis. CONCLUSIONS: The present findings suggest that combined immunohistochemistry and proteomic analysis is more useful than immunohistochemistry alone. Our findings highlight the importance of carefully interpreting immunohistochemistry for anti-TTR and light chain and offer insights that can guide amyloid typing through immunohistochemistry.

Mass spectrometry-based metabolomics study of nicotine exposure in THP-1 monocytes.

The tobacco alkaloid nicotine is known for its activation of neuronal nicotinic acetylcholine receptors. Nicotine is consumed in different ways such as through conventional smoking, e-cigarettes, snuff or nicotine pouches. The use of snuff has been associated with several adverse health effects, such as inflammatory reactions of the oral mucosa and oral cavity cancer. We performed a metabolomic analysis of nicotine-exposed THP-1 human monocytes. Cells were exposed to 5 mM of the alkaloid for up to 4 h, and cell extracts and medium subjected to untargeted liquid chromatography high-resolution mass spectrometry. Raw data processing revealed 17 nicotine biotransformation products. Among these, cotinine and nornicotine were identified as the two major cellular biotransformation products. The application of multi- and univariate statistical analyses resulted in the annotation, up to a certain level of identification, of 12 compounds in the cell extracts and 13 compounds in the medium that were altered by nicotine exposure. Of these, four were verified as methylthioadenosine, cytosine, uric acid, and L-glutamate. Methylthioadenosine levels were affected in both cells and the medium, while cytosine, uric acid, and L-glutamate levels were affected in the medium only. The effects of smoking on the pathways involving these metabolites have been previously demonstrated in humans. Most of the other discriminating compounds, which were merely tentatively or not fully identified, were amino acids or amino acid derivatives. In conclusion, our preliminary data suggest that some of the potentially adverse effects related to smoking may also be expected when nicotine is consumed via snuff or nicotine pouches.

Investigating the etiology of Haff disease: Optimization and validation of a sensitive LC-MS/MS method for palytoxins analysis in directly associated freshwater and marine food samples from Brazil.

Haff disease typically develops after eating contaminated marine or freshwater species, especially fish. Despite still having an unknown etiology, recent reports have suggested its possible correlation with palytoxins. Therefore, the present work aimed to optimize and perform a validation of a sensitive method using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the analysis of palytoxin and some of its analogs, with the main purpose of investigating their presence in marine and freshwater food samples associated with Haff disease in Brazil. The method optimization was performed using a central composite rotatable design and fish samples fortified with the palytoxin standard. Then, the optimized method was validated for different food matrices, including freshwater and marine fish, mollusks, and crustaceans. The sample preparation involved a solid-liquid extraction using methanol and water, solid-phase extraction using Strata-X cartridges, and on-column palytoxin oxidation. The detection of the main oxidized fragments (amino and amide aldehydes) was achieved by LC-MS/MS with electrospray ionization in positive mode, using a C18 column, as well as acetonitrile and water as mobile phases, both acidified with 0.1 % of formic acid. After optimization and validation, the etiological investigation involved the analysis of 16 Brazilian Haff disease-related food samples (in natura and leftover meals) from 2022. The method was demonstrated to be appropriate for quantitative analysis of freshwater and marine species. So far, it has proven to be one of the most sensitive methods related to palytoxin detection (LOD 10 µg/kg), being able to work in a range that includes the provisional ingestion limit (30 µg/kg). Regarding the Haff disease-related samples analysis, there is a strong indication of palytoxin contamination since the amino aldehyde (common fragment for all palytoxins) was detected in 15 of the 16 samples. Selected results were confirmed using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS).

Broad-spectrum and sensitive screening of more than 1000 compounds in equine urine using liquid chromatography/high-resolution mass spectrometry.

RATIONALE: To uphold the integrity of horseracing and equestrian sports, it is critical for an equine doping control laboratory to develop a comprehensive screening method to cover a wide range of target substances at the required detection levels in equine urine. METHODS: The procedure involved the enzymatic hydrolysis of 3 mL urine samples followed by solid-phase extraction using HF Bond Elut C18 cartridge. The resulting extracts were then separated on a C18 reversed-phase column and analyzed using liquid chromatography/high-resolution mass spectrometry (LC/HRMS) in both electrospray ionization positive and negative modes in two separate injections. The analytical data were obtained in full scan and product ion scan (PIS) modes in an 11 min LC run. RESULTS: The method can detect 1011 compounds (in both positive and negative ion modes). Over 95% of the target compounds have limits of detections (LODs) ≤10 ng/mL, and more than 50% of the LODs are ≤0.5 ng/mL. The lowest LOD can reach down to 0.01 ng/mL. The applicability of the method was demonstrated by the successful detection of prohibited substances in overseas and domestic equine urine samples. CONCLUSIONS: We have successfully developed a regular screening method for equine urine samples that can detect more than 1000 compounds at sub-ppb levels in both positive and negative ion modes with full scan and PIS using LC/HRMS. Furthermore, this method can theoretically be expanded to accommodate an unlimited number of prohibited substances in full-scan mode.

A Novel Peptidome Technology for the Diagnosis of Mild Cognitive Impairment and Alzheimer's Disease by Selected Reaction Monitoring.

Background: With the aging of populations worldwide, Alzheimer's disease (AD) has become a concern due to its high prevalence and the continued lack of established treatments. Early diagnosis is required as a preventive intervention to modify the disease's progression. In our previous study, we performed peptidomic analysis of serum samples obtained from AD patients and age-matched healthy subjects to seek peptide biomarker candidates for AD by using BLOTCHIP-MS analysis, and identified four peptides as AD biomarker candidates. Objective: The objective was to validate the serum biomarker peptides to distinguish mild cognitive impairment (MCI) and AD in comparison to cognitively healthy controls using a new peptidome technology, the Dementia Risk Test. Methods: We enrolled 195 subjects with normal cognitive function (NC; n = 70), MCI (n = 55), and AD (n = 70), The concentrations of cognitive impairment marker peptides (Fibrinogen α chain (FAC), Fibrinogen ß chain (FBC), Plasma protease C1 inhibitor (PPC1I), α2-HS-glycoprotein (AHSG)) were quantified by using a selected reaction monitoring assay based on liquid chromatography-MS/MS. Results: The present study confirmed that three peptides, FAC, FBC, and PPC1I, were significantly upregulated during the onset of AD. This three-peptide set was both highly sensitive in determining AD (sensitivity: 85.7%, specificity: 95.7%, AUC: 0.900) and useful in distinguishing MCI (sensitivity: 61.8%, specificity: 98.6%, AUC: 0.824) from NC. Conclusions: In this validation study, we confirmed the high diagnostic potential of the three peptides identified in our previous study as candidate serum biomarkers for AD. The Dementia Risk Test may be a powerful tool for detecting AD-related pathological changes.

Analysis of Metabolic Changes in Endogenous Metabolites and Diagnostic Biomarkers for Various Diseases Using Liquid Chromatography and Mass Spectrometry.

Analysis of endogenous metabolites in various diseases is useful for searching diagnostic biomarkers and elucidating the molecular mechanisms of pathophysiology. The author and collaborators have developed some LC/tandem mass spectrometry (LC/MS/MS) methods for metabolites and applied them to disease-related samples. First, we identified urinary conjugated cholesterol metabolites and serum N-palmitoyl-O-phosphocholine serine as useful biomarkers for Niemann-Pick disease type C (NPC). For the purpose of intraoperative diagnosis of glioma patients, we developed the LC/MS/MS analysis methods for 2-hydroxyglutaric acid or cystine and found that they could be good differential biomarkers. For renal cell carcinoma, we searched for various biomarkers for early diagnosis, malignancy evaluation and recurrence prediction by global metabolome analysis and targeted LC/MS/MS analysis. In pathological analysis, we developed a simultaneous LC/MS/MS analysis method for 13 steroid hormones and applied it to NPC cells, we found 6 types of reductions in NPC model cells. For non-alcoholic steatohepatitis (NASH), model mice were prepared with special diet and plasma bile acids were measured, and as a result, hydrophilic bile acids were significantly increased. In addition, we developed an LC/MS/MS method for 17 sterols and analyzed liver cholesterol metabolites and found a decrease in phytosterols and cholesterol synthetic markers and an increase in non-enzymatic oxidative sterols in the pre-onset stage of NASH. We will continue to challenge themselves to add value to clinical practice based on cutting-edge analytical chemistry methodology.

Improving analytical performance for pesticides and mycotoxins determination in Brazilian table olives: one extraction and one analysis.

This paper describes an extensive study in which a multiclass QuEChERS based approach was optimized for determination of 150 pesticides and 7 mycotoxins in table olives. Three versions of QuEChERS were evaluated and compared (unbuffered, citrate and acetate buffering). A combination of EMR-Lipid cartridges and liquid nitrogen or freezer freezing out were tested for clean-up of the oily olive extracts. Analysis of the extracts were performed by LC-MS/MS triple quadrupole. The best results were achieved using acetate QuEChERS with liquid nitrogen for clean-up. For validation, organic olives were ground and spiked at 4 concentrations with pesticides and mycotoxins (n = 5). The linearity of the calibration curves was assessed by analyzing calibration standards of 7 concentrations which were prepared separately in acetonitrile and in blank olive extract (n = 5). The validation study demonstrated that the calculated r2 was ≥0.99 for 144 pesticides and 6 mycotoxins, when the calibration curves were prepared in matrix extract, showing satisfactory linearity. Matrix effects were within the range of ±20% for only 46 pesticides and one mycotoxin. Then, to ensure reliable quantification, calibration standards had to be matrix-matched. In accuracy experiments 138 pesticides and 6 mycotoxins presented recoveries from 70 to 120% and RSD ≤ 20% for at least 2 of the 4 spike concentrations evaluated, being successfully validated. The integrated QuEChERS and LC-MS/MS method meet MRL for 11 of the 21 pesticides regulated for olives in Brazil and for 132 pesticides which are regulated in the EU law. Eleven commercial table olive samples were analyzed and 4 of them tested positive for pesticides. All the positive samples violate the Brazilian law and one sample violates also the European law.

Evaluation of environmental antibiotic contamination by surface wipe sampling in a large care centre.

OBJECTIVES: Exposure of healthcare workers to antibiotics may cause adverse health effects. Results of environmental contamination with antibiotics, obtained by taking surface wipe samples, can be used as an indicator for potential exposure to these sensitizing drugs. The objective was to describe the results of repeated measurements of contamination with antibiotics on multiple surfaces in hospital wards. Standardized needle and syringe preparation techniques and cleaning procedures were used. METHODS: The preparation table and the floor around the waste bin in six wards were sampled and analysed for contamination with the antibiotics amoxicillin, benzylpenicillin, cefotaxime, ceftriaxone, flucloxacillin, meropenem, piperacillin and vancomycin. Sampling was performed in four trials during 8 months. Depending on the outcome of the trials, the cleaning procedure was adapted. Liquid chromatography with tandem mass spectrometry was used for the analysis of the drugs. RESULTS: During the four trials, contamination with all eight antibiotics was omnipresent on all preparation tables and floors in the six wards. The highest contamination was found for amoxicillin (1291 ng/cm2). Changing the cleaning procedure did not reduce the level of contamination. CONCLUSIONS: Surface contamination with the antibiotics was widespread and most probably caused by spillage during the preparation in combination with an ineffective cleaning procedure. Strategies should be developed and implemented by institutions for safe handling of antibiotics to reduce environmental contamination and potential exposure of healthcare workers to these sensitizing drugs.

Hyphenation of Liquid Chromatography and Trapped Ion Mobility - Mass Spectrometry for Characterization of Isomeric Phosphatidylethanolamines with Focus on N-Acylated Species.

In prior research, hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry (HILIC-MS/MS) has demonstrated applicability for characterizing regioisomers in lipidomics studies, including phosphatidylglycerols (PG) and bis(monoacyl)glycerophosphates (BMP). However, there are other lipid regioisomers, such as phosphatidylethanolamines (PE) and lyso-N-acyl-PE (LNAPE), that have not been studied as extensively. Therefore, hyphenated mass spectrometric methods are needed to investigate PE and LNAPE regioisomers individually. The asymmetric structure of LNAPE favors isomeric species, which can result in coelution and chimeric MS/MS spectra. One way to address the challenge of chimeric MS/MS spectra is through mobility-resolved fragmentation using trapped ion mobility spectrometry (TIMS). Therefore, we developed a multidimensional HILIC-TIMS-MS/MS approach for the structural characterization of isomeric phosphatidylethanolamines in both negative and positive ionization modes. The study revealed the complementary fragmentation pattern and ion mobility behavior of LNAPE in both ionization modes, which was confirmed by a self-synthesized LNAPE standard. With this knowledge, a distinction of regioisomeric PE and LNAPE was achieved in human plasma samples. Furthermore, regioisomeric LNAPE species containing at least one unsaturated fatty acid were noted to exhibit a change in collision cross-section in positive ionization mode, leading to a lipid characterization with respect to fatty acyl positional level. Similar mobility behavior was also observed for the biological LNAPE precursor N-acyl-PE (NAPE). Application of this approach to plasma and cereal samples demonstrated its effectiveness in regioisomeric LNAPE and NAPE species' elucidation.

Double Cyclization Tandem Mass for Identification and Quantification of Phosphatidylcholines Using Isobaric Six-Plex Capillary nLC-MS/MS.

Multiplexing of phosphatidylcholine analysis is hindered by a lack of appropriate derivatization. Presented here is a tagging scheme that uses a quaternary amine tag and targets the hydroxy group of the phosphate, which switches the net charge from neutral to +2. Quantitative yields were achieved from >99% reaction completion derived by dimethoxymethyl morpholinium (DMTMM) activation. Fragmentation of phosphatidylcholines (PCs) and lysophosphatidylcholines (LPCs) releases two trimethylamines and the acyl chains through neutral loss and generates a unique double cyclization constant mass reporter. Selective incorporation of isotopes onto the tag produces a six-plex set of isobaric reagents. For equivalent six-plex-labeled samples, <14% RSD was achieved, followed by a dynamic range of 1:10 without signal compression. Quantification of PCs/LPCs in human hepatic cancer cells was conducted as six-plex using data-dependent analysis tandem MS. We report a six-plex qualitative and quantitative isobaric tagging strategy expanding the limits of analyzing PCs/LPCs.

Organophosphate esters in human serum: a relatively simple and efficient liquid chromatography-mass spectrometry method.

Organophosphate esters (OPEs) are widely used as flame retardants and plasticizers, which are of growing concern due to their endocrine-disrupting effects, developmental toxicity, and potential carcinogenicity. However, data on human exposure to OPEs is still scarce. In this study, a relatively simple and efficient method with less serum consumption for the detection of OPEs in human serum was developed and validated. Nine OPEs in 200 µL of human serum were extracted by an acetonitrile-formic acid system and analyzed using ultra-high-performance liquid chromatography-quadrupole tandem time-of-flight high-resolution mass spectrometry. Several experiments were conducted to optimize the chromatographic and mass spectrometric conditions as well as sample preparation to obtain a more sensitive and efficient analytical protocol. The proposed method was examined in terms of its linearity, accuracy, precision, detection limit, and matrix effect. The matrix-spiked recoveries of the target OPEs ranged from 83.3% to 111.1%, with relative standard deviations between 2.7% and 16.6%. The detection limits were within (0.002 to 0.029) ng mL-1, while the quantification limits were within (0.007 to 0.098) ng mL-1. The internal standard-corrected matrix effects varied from 82.7% to 113.9%. Finally, the method was applied to detect OPEs in actual human serum samples. All nine OPEs were detected in 269 serum samples to varying degrees, with the average concentrations ranging from (0.08 to 1.77) ng mL-1. After validation, the method was found to be simple in pretreatment, high in sensitivity, good in practicality, and suitable for exposure evaluation of OPEs in populations.

Alterations in plasma short-chain fatty acids in preadolescence children: The Hokkaido study.

The purpose of this study is to explore the plasma short-chain fatty acid (SCFA) concentrations in 9-12-year-old Japanese children collected in the Hokkaido study, focusing on how factors such as age, sex, and body mass index (BMI) correlate with these levels. The Hokkaido Study on Children's Health is an ongoing longitudinal study since 2002, encompassing 20,926 pregnant women in Hokkaido Prefecture, Japan, between 2003 and 2012. We contacted 1881 children aged 9-12 born between April 2006 and January 2010, and 342 non-fasting plasma samples (boys = 181, girls = 161) were obtained from this cohort, alongside assessments of their height and weight. Plasma SCFA concentrations were determined using N,N-dimethylethylenediamine derivatization method coupled with liquid chromatography-mass spectrometry. Ethyl acetate was used to extract SCFAs from plasma, and the recovery ranged from 83 % to 108 %. Our findings indicate that acetic acid had the highest concentration across all age groups and sexes. The concentrations of butyric acid, valeric acid, and hexanoic acid increased with age, peaking in 12-year-old children. Conversely, the level of 4-hydroxy valeric acid showed a decreasing trend with increasing age groups. This study also explored the correlation between BMI and SCFA concentrations, comparatively higher level of propionic acid was observed in the overweight group. The results obtained in this study enhance our understanding of the role of SCFAs in the growth and development of children and provide a foundation for future nutritional intervention and health promotion strategies.

Toxicologic analysis of 35 drugs in post mortem human blood samples with focus on antihypertensive and antiarrhythmic drugs.

Antiarrhythmic and antihypertensive drugs are frequently encountered in post mortem analysis, and the question may arise as to whether they were administered in therapeutic doses, and if they were taken in accidental, intentional, or suicidal overdose scenarios. Therefore, a novel analytical method was developed and validated for the quantification of 35 drugs with toxicological relevance, including antihypertensive and antiarrhythmic drugs (ajmaline, amlodipine, amiodarone, atenolol, bisoprolol, carvedilol, clonidine, desethylamiodarone, diltiazem, donepezil, doxazosin, dronedarone, esmolol, flecainide, lercanidipine, lidocaine, metoprolol, nebivolol, nimodipine, pindolol, prajmaline, propafenone, propranolol, sotalol, urapidil, and verapamil), as well as other medications commonly found in combination (sildenafil, tadalafil, atorvastatin, clopidogrel, dapoxetine, memantine, pentoxifylline, rivastigmine, and ivabradine). The method enables simultaneous identification and quantification in blood samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Validation exhibited excellent linearity across the concentration range for all analytes. Precision and accuracy were within acceptable limits, with bias and relative standard deviation (RSD) values consistently below 9 % and 10 %, respectively. Selectivity and specificity assessments confirmed the absence of any interference from contaminants or co-extracted drugs. The method demonstrated very high sensitivity, with limits of detection (LOD) as low as 0.01 ng/ml and limits of quantification (LOQ) as low as 0.04 ng/ml. Extraction recovery exceeded 57.5 % for all analytes except atenolol, and matrix effects were <17 % for all analytes except pindolol. Processed sample stability evaluations revealed consistent results with acceptable deviations for all analytes. In addition, the method was specifically tested for the use in post mortem analysis. The applicability of our method was demonstrated by the analysis of two authentic human autopsy blood samples.