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Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by cognitive decline and memory loss. Recent studies have suggested a potential role for steroid synthesis in AD pathology. This study investigated the co-localization of steroidogenic enzymes in neuronal cells, changes in enzyme expression in an AD mouse model, and steroid expressions in human AD samples. Additionally, we conducted a steroidomic metabolomics analysis and evaluated the effects of dehydroepiandrosterone sulfate (DHEAS) treatment in an AD mouse model. Immunofluorescence analysis revealed significant co-localization of cytochrome P450 family 17 subfamily A member 1 (CYP17A1) and steroidogenic acute regulatory protein (StAR) proteins with α-synuclein in presynaptic neurons, suggesting active steroid synthesis in these cells. Conversely, such co-localization was absent in astrocytes. In the AD mouse model, a marked decrease in the expression of steroidogenic enzymes (Cyp11a1, Cyp17a1, Star) was observed, especially in areas with amyloid beta plaque accumulation. Human AD and MS brain tissues showed similar reductions in StAR and CYP17A1 expressions. Steroidomic analysis indicated a downregulation of key steroids in the serum of AD patients. DHEAS treatment in AD mice resulted in improved cognitive function and reduced Aβ accumulation. Our findings indicate a neuron-specific pathway for steroid synthesis, potentially playing a crucial role in AD pathology. The reduction in steroidogenic enzymes and key steroids in AD models and human samples suggests that impaired steroid synthesis is a feature of neurodegenerative diseases. The therapeutic potential of targeting steroid synthesis pathways, as indicated by the positive effects of DHEAS treatment, warrants further investigation., Competing Interests: Declaration of Competing Interest Authors have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Drug abuse is a severe social problem worldwide. Particularly, the issue of new psychoactive substances (NPSs) have increasingly emerged. NPSs are structural or functional analogs of traditional illicit drugs, such as cocaine, cannabis, and amphetamine; these molecules provide the same or more severe neurological effects. Usually, immunoassays are utilized in the preliminary screening method. However, NPSs have poor detectability in commercially available immunoassay kits. Meanwhile, various chromatography combined with the mass spectrometry platform have been developed to quantify NPSs. Still, a significant amount of time and resources are required during these procedures. Therefore, we established a rapid analytical platform for NPSs employing paper-loaded direct analysis in real time triple quadrupole mass spectrometry (pDART-QqQ-MS). We implemented this platform for the semiquantitative analysis of forensic drug tests in urine. This platform significantly shrinks the analytical time of a single sample within 30 s and requires a low volume of the specimen. The platform can detect 21 NPSs in urine mixtures at a lower limit of qualification of concentration ranging from 20 to 75 nanograms per milliliter (ng mL -1 ) and is lower than the cutoff value of currently available immune-based devices for detecting multiple drugs (1000 ng mL -1 ). Urine samples from drug addicts have been collected to verify the platform's effectiveness. By combining efficiency and accuracy, our platform offers a promising solution for addressing the challenges posed by NPSs in drug abuse detection.

Methcathinone, a psychoactive substance with stimulant properties, has raised concerns in recent years due to its presence in urine screenings, even among individuals with no history of drug abuse. To prevent misjudgment, this work aims to explore the source of methcathinone in urine. A total of 58 urine samples tested positive for methcathinone in the National Taiwan University Hospital cohort, with 27 linked to illicit drug use and 31 from individuals with no drug use history. Co-occurrence analysis revealed a strong association between methcathinone and over-the-counter cold medications containing pseudoephedrine or ephedrine. In an in vivo experiment, participants who consumed pseudoephedrine-containing drugs showed the presence of methcathinone in their urine, suggesting a connection between these substances. Additionally, tests on pharmaceutical products containing pseudoephedrine detected small amounts of methcathinone as impurities. The findings suggest that the presence of methcathinone in nonillicit drug users may be attributed to impurities in over-the-counter pseudoephedrine-containing medications. This raises concerns about potential misinterpretations of drug screening results and underscores the need for more comprehensive criteria for assessing drug use. This study contributes to our understanding of the origin of methcathinone in urine, which has implications for legal justice and drug screening practices., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Virus-induced changes in host lipid metabolism are an important but poorly understood aspect of viral pathogenesis. By combining nontargeted lipidomics analyses of infected cells and purified extracellular quasi-enveloped virions with high-throughput RNA sequencing and genetic depletion studies, we show that hepatitis A virus, an hepatotropic picornavirus, broadly manipulates the host cell lipid environment, enhancing synthesis of ceramides and other sphingolipids and transcriptionally activating acyl-coenzyme A synthetases and fatty acid elongases to import and activate long-chain fatty acids for entry into the fatty acid elongation cycle. Phospholipids with very-long-chain acyl tails (>C22) are essential for genome replication, whereas increases in sphingolipids support assembly and release of quasi-enveloped virions wrapped in membranes highly enriched for sphingomyelin and very-long-chain ceramides. Our data provide insight into how a pathogenic virus alters lipid flux in infected hepatocytes and demonstrate a distinction between lipid species required for viral RNA synthesis versus nonlytic quasi-enveloped virus release.

Adrenal venous sampling (AVS) is the gold standard for identifying curable unilateral aldosterone excess in primary aldosteronism (PA). Studies have demonstrated the value of steroid profiling through liquid chromatography-tandem mass spectrometry (LC-MS/MS) in AVS interpretation. First, the performance of LC-MS/MS and immunoassay in assessing selectivity and lateralization was compared. Second, the utility of the proportion of individual steroids in adrenal veins in subtyping PA was analyzed. We enrolled 75 consecutive patients with PA who underwent AVS between 2020 and 2021. Fifteen adrenal steroids were analyzed in peripheral and adrenal veins through LC-MS/MS before and after adrenocorticotropic hormone (ACTH) stimulation. Through selectivity index that was based on cortisol and alternative steroids, LC-MS/MS rescued 45% and 66% of failed cases judged by immunoassay in unstimulated and stimulated AVS, respectively. LC-MS/MS identified more unilateral diseases than did immunoassay (76% vs. 45%, P < 0.05) and provided adrenalectomy opportunities to 69% of patients judged through immunoassay to have bilateral disease. The secretion ratios (individual steroid concentration/total steroid concentration) of aldosterone, 18-oxocortisol, and 18-hydroxycortisol were novel indicators for identifying unilateral PA. The 18-oxocortisol secretion ratio of ≥0.785‰ (sensitivity/specificity: 0.90/0.77) at pre-ACTH and aldosterone secretion ratio of ≤0.637‰ (sensitivity/specificity: 0.88/0.85) at post-ACTH enabled optimal accuracy for predicting ipsilateral and contralateral disease, respectively, in robust unilateral PA. LC-MS/MS improved the success rate of AVS and identified more unilateral diseases than immunoassay. The secretion ratios of steroids can be used to discriminate the broad PA spectrum., (© 2023. The Author(s).)

Motivation: Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used in metabolomics studies, while HILIC LC-MS is particularly suited for polar metabolites. Determining an optimized mobile phase and developing a proper liquid chromatography method tend to be laborious, time-consuming and empirical., Results: We developed a containerized web tool providing a workflow to quickly determine the optimized mobile phase by batch-evaluating chromatography peaks for metabolomics LC-MS studies. A mass chromatographic quality value, an asymmetric factor, and the local maximum intensity of the extracted ion chromatogram were calculated to determine the number of peaks and peak retention time. The optimal mobile phase can be quickly determined by selecting the mobile phase that produces the largest number of resolved peaks. Moreover, the workflow enables one to automatically process the repeats by evaluating chromatography peaks and determining the retention time of large standards. This workflow was validated with 20 chemical standards and successfully constructed a reference library of 571 metabolites for the HILIC LC-MS platform., Availability and Implementation: MetaMOPE is freely available at https://metamope.cmdm.tw. Source code and installation instructions are available on GitHub: https://github.com/CMDM-Lab/MetaMOPE., Supplementary Information: Supplementary data are available at Bioinformatics Advances online., (© The Author(s) 2023. Published by Oxford University Press.)

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are commonly used to treat advanced non-small cell lung cancer (NSCLC). A rapid and reliable method for measuring plasma and cerebrospinal fluid (CSF) concentrations of EGFR-TKIs is needed for therapeutic drug monitoring. By using UHPLC‒MS/MS with multiple reaction monitoring mode, we developed a method for rapidly determining the plasma and CSF concentrations of gefitinib, erlotinib, afatinib, and osimertinib. Protein precipitation was employed to remove protein interference for plasma and CSF matrix. The LC‒MS/MS assay was validated to be satisfactory in terms of linearity, precision, and accuracy. This method was successfully applied to measure plasma (n = 44) and CSF (n = 6) concentrations of EGFR-TKIs in NSCLC patients. The chromatographic separation was achieved by a Hypersil Gold aQ column within 3 min. The median plasma concentrations were 325.76, 1981.50, 42.62, 40.27, and 340.92 ng/ml for gefitinib erlotinib, afatinib 30 mg/day, afatinib 40 mg/day, and osimertinib, respectively. The CSF penetration rates were 2.15% for the patients receiving erlotinib therapy, 0.59% for afatinib, 0.08-1.12% for osimertinib 80 mg/day, and 2.18% for those receiving osimertinib 160 mg/day. This assay helps to predict the effectiveness and toxicities of EGFR-TKIs in the pursuit of precision medicine for lung cancer patients., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The intestinal barrier, a complex structure consisting of multiple layers of defense barriers, blocks the transfer of intestinal and foreign bacteria and their metabolites into the internal environment of the human body. Intestinal permeability can be used to evaluate the integrity of the intestinal barrier. Increased intestinal permeability has been observed in patients with depressive disorder. Some studies have reported an interaction between depressive disorder and intestinal barrier. Herein, we reviewed reported findings on the mechanisms of how systematic low-grade inflammation, vagal nerve dysfunction, and hypothalamic-pituitary-adrenal axis dysfunction cause changes in intestinal permeability in patients with depressive disorder and the pathogenic mechanism of how bacterial translocation caused by damaged intestinal barrier leads to depressive disorder. In addition, the potential mechanisms of how antidepressants improve intestinal permeability and how probiotics improve depressive disorder have been discussed., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Sciences).)

Determination of urine organic acids (UOAs) is essential to understand the disease progress of inborn errors of metabolism (IEM) and often relies on GC-MS analysis. However, the efficiency of analytical reports is sometimes restricted by data processing due to labor-intensive work if no proper tool is employed. Herein, we present a simple and rapid workflow with an R-based script for automated data processing (AutoDP) of GC-MS raw files to quantitatively analyze essential UOAs. AutoDP features automatic quality checks, compound identification and confirmation with specific fragment ions, retention time correction from analytical batches, and visualization of abnormal UOAs with age-matched references on chromatograms. Compared with manual processing, AutoDP greatly reduces analytical time and increases the number of identifications. Speeding up data processing is expected to shorten the waiting time for clinical diagnosis, which could greatly benefit clinicians and patients with IEM. In addition, with quantitative results obtained from AutoDP, it would be more feasible to perform retrospective analysis of specific UOAs in IEM and could provide new perspectives for studying IEM., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The application of dried blood spots (DBS) has gradually increased in different fields because of its several advantages. The hematocrit (Hct) effect is one major analytical challenge that may affect the quantification accuracy of DBS samples and should be investigated when developing a novel DBS method. However, previous studies usually overlooked the Hct-related distribution bias when evaluating the Hct effect. This study aimed to propose an effective DBS preparation protocol for the comprehensive evaluation of the Hct effect. We selected voriconazole and posaconazole as the demonstration drugs. Fifteen microliters of the blood samples were spotted on DBS cards followed by whole spot extraction. An LC-MS/MS method was first developed to quantify voriconazole and posaconazole in DBS samples. The quantitation accuracy for both azole drugs was within 93.5%-111.7%, except for the accuracies of posaconazole at the LLOQ, which were less than 119%. The intra- and interday precision were below 11%. The validated LC-MS/MS method was used to develop the DBS preparation protocol for evaluating the Hct effect. Three critical parameters that may affect the observed Hct effect were investigated. The results showed that using the solid-state of the target analytes, spiking the target analytes before preparing different Hct levels, and allowing enough equilibrium time after spiking target analytes can provide a more holistic Hct effect evaluation. The validity of the proposed new protocol was verified by conversion factors obtained from 71 paired DBS and plasma samples. Conversion factors calculated by clinical samples were consistent with the Hct effect evaluated by manually prepared DBS samples. This new DBS preparation protocol eliminated the common pitfalls in studying the Hct effect and offered a comprehensive strategy to assess the Hct effect for further DBS studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Antemortem specimens are sometimes the sole sources available for forensic investigation, and samples collected in nonideal ways are inevitably employed to achieve toxicological analysis. It is essential to assess the effects of blood collection tubes on the recoveries of emerging synthetic cathinones (SC) to estimate actual drug concentrations, and no such systematic investigations have been previously carried out. Seventy-one SC with various LogP values were employed to examine commonly used blood collection tubes, including plasma tubes, serum tubes and gel-containing tubes in recoveries which determined by a reliable LC-MS/MS method. Significantly poor recoveries for hydrophobic SC were obtained using serum separating tubes (SST). Notably, the suppressed recoveries in SST can be reversed by adding anticoagulants. Adding a procoagulant to a plasma separating tube (PST) considerably reduced recoveries, which indicated that clotting processes in the presence of polymeric gels contributed to poor recoveries of these hydrophobic drugs. In this study, we find that clotting formation in the presence of polymeric gels could significantly affect the determination of hydrophobic drugs. However, in real-world scenarios, nonideal collection methods are inevitably employed for antemortem specimens. Thus, it is important to rigorously interpret forensic toxicological results, especially for susceptible species., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Immunoglobulin A nephropathy (IgAN) is a highly prevalent autoimmune renal disease. Human IgA1 with galactose deficiency in the hinge region (HR) has been identified as an autoantigen for this disease. Therefore, analyzing IgA1 HR glycoforms in biofluids is important for biomarker discovery. Herein, an analytical method that includes one-pot sample preparation with unbiased plasma IgA purification, dual internal standard addition, and sensitive ultra-high-performance liquid chromatography-triple quadrupole tandem mass spectroscopy (UHPLC-QqQ-MS/MS) was developed. Targeted O-glycopeptides detection was performed in pooled plasma with the validation of theoretical retention times, enzymatic treatment outcomes, product ion scans, and signal repeatability. A total of 42 IgA1 O-glycopeptides with N-acetylgalactosamines, galactoses, and sialic acids were determined from 8 µL of plasma. The newly developed method was applied to plasma samples from 16 non-IgAN controls and 19 IgAN patients. Comparing the 42 targets, 16 IgA1 HR O-glycopeptides were statistically different between the two groups (p<0.05). Decreased sialylation was identified in the IgA1 hinge region of IgAN patients, which was also correlated with the estimated glomerular filtration rate (eGFR). The developed method is sensitive and precise and can be used to identify plasma biomarkers for IgA nephropathy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Trastuzumab is a standard molecular targeted therapy for human epidermal growth factor receptor 2(HER2) -positive breast cancer, which can significantly improve the survival of patients with this molecular subtype of breast cancer. However, the clinical problem of onset or secondary resistance to trastuzumab has limited its efficacy. Therefore, it is very important to explore the mechanism of trastuzumab resistance and formulate countermeasures. Our study described the underlying molecular mechanism of trastuzumab resistance including ERBB2 mutations and nuclear localization, transcriptional and post-translational alterations of ERBB2 , over-activation of bypass signaling pathways activation and so on. Then summarize the potential emerging predicting biomarkers and therapeutic strategies for trastuzumab resistance, in order to provide research direction for reversing trastuzumab resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Zheng, Jia, Liu, Xiao, Chen, Liang and Lu.)

Background: Synthetic cathinones (SC) are popular new psychoactive substances that produce sympathomimetic toxicity. Meth/amphetamine and SC have similar chemical structures and pharmacological effects. We aimed to compare the clinical characteristics between meth/amphetamine and SC users presenting to the emergency department (ED)., Methods: This retrospective observational cohort study included patients who presented to six EDs from May 2017 to April 2021 with symptoms that related to recreational drug use and whose urine toxicology tests were positive only for meth/amphetamine or SC through liquid chromatography-tandem mass spectrometry., Results: There were 379 patients who tested positive only for meth/amphetamine (MA group), and 87 patients tested positive only for SC (SC groups). Patients in the MA group were older than those in the SC group (median (IQR); MA: 37.0 (30-43.7), SC: 25.0 (21.0-32.7), p  < 0.001). There were no significant between-group differences in the sex distribution and initial chief complaints. Compared with the MA group, the SC group had more cases of tachycardia (≥ 135/min; MA: 29 (8.2%), SC:16 (19.0%), p  = 0.0031) and hyperthermia (≥ 38 °C; MA: 31 (8.2%), SC:18 (20.7%), p  = 0.001). Besides, the SC group had significantly higher levels of creatinine kinase (CK, IU/L; MA: 263 (115-601), SC: 497 (206-9216), p  = 0.008) as well as a higher risk of rhabdomyolysis (CK > 1000; MA:32 (8.4%), SC: 16 (18.4%), p  = 0.006) and severe rhabdomyolysis (CK > 10,000; MA:10 (2.6%), SC:10 (11.5%), p  = 001). Multivariable logistic regression analyses indicated SC group in comparison with the MA group (adjusted odds ratio: 2.732, 95% confidence interval: 1. 250-5.972, p  = 0.012) was an association with the risk of rhabdomyolysis., Conclusion: Our findings demonstrate that tachycardia, hyperthermia, and rhabdomyolysis were more common among cathinone users than among meth/amphetamine users presented to EDs.

Background: The pathogenesis of sarcopenia is complex and has not been well explored. Identifying biomarkers is a promising strategy for exploring the mechanism of sarcopenia. This study aimed to identify potential biomarkers of sarcopenia through a metabolomic analysis of plasma metabolites in elderly subjects (≥65 years of age) vs. younger adults (<65 years of age)., Methods: Of the 168 candidates in the Comprehensive Geriatric Assessment and Frailty Study of Elderly Outpatients, 24 elderly subjects (≥65 years of age) with sarcopenia were age and sex matched with 24 elderly subjects without sarcopenia. In addition, 24 younger adults were recruited for comparison. Muscle strength, gait speed, and metabolic and inflammatory parameters, including plasma tumour necrosis factor-α, C-reactive protein, irisin, and growth differentiation factor 15 (GDF-15) levels were assessed. Metabolomic analysis was carried out using the plasma metabolites., Results: Seventy-two participants were enrolled, including 10 (41.6%) men and 14 (58.3%) women in both groups of elderly subjects. The median ages of elderly subjects with and without sarcopenia were 82 (range: 67-88) and 81.5 (range: 67-87) years, respectively. Among the 242 plasma metabolic peaks analysed among these three groups, traumatic acid was considered as a sarcopenia-related metabolite. The plasma traumatic acid signal intensity level was significantly higher in elderly subjects with sarcopenia than in elderly subjects without sarcopenia [591.5 (inter-quartile range, IQR: 491.5-664.5) vs. 430.0 (IQR: 261.0-599.5), P = 0.0063]. The plasma concentrations of traumatic acid were 15.8 (IQR: 11.5-21.7), 21.1 (IQR: 16.0-25.8), and 24.3 (IQR: 18.0-29.5) ppb in younger adults [age range: 23-37 years, 12 (50%) men], elderly subjects without sarcopenia, and elderly subjects with sarcopenia, respectively, thereby depicting an increasing tendency (P for trend = 0.034). This pattern was similar to that of GDF-15, a recognized sarcopenia-related factor. Plasma traumatic acid concentrations were also positively correlated with the presence of hypertension (r = 0.25, P = 0.034), glucose AC (r = 0.34, P = 0.0035), creatinine (r = 0.40, P = 0.0006), and GDF-15 levels (r = 0.25, P = 0.0376), but negatively correlated with the Modification of Diet in Renal Disease-simplify-glomerular filtration rate (r = -0.50, P < 0.0001). Similarly, plasma GDF-15 concentrations were associated with these factors., Conclusions: Traumatic acid might represent a potential plasma biomarker of sarcopenia. However, further studies are needed to validate the results and investigate the underlying mechanisms., (© 2021 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Emerging new psychoactive substances (NPS) poses a great risk to public health. Analyzing these large numbers of NPS and other associated substances often relies on liquid chromatography coupled to triple quadrupole mass spectrometry (LC-QqQ-MS) with multiple-reaction monitoring (MRM) mode. However, the differentiation of critical pairs, coeluted isobaric and/or isomeric species, is one of the challenges for this analytical platform. MRM transitions with poor selectivity can jeopardize accurate quantification and lead to biased interpretation. Herein, we refined a novel workflow for developing an MRM-based method with in-house CriticalPairFinder and TransitionFinder tools for the effective identification of unique and selective MRM transitions. Transitions selected by TransitionFinder showed much better accuracies than those selected only by fragment abundance in some mixtures of critical pairs. Using the proposed analytical strategy, a method that can simultaneously determine 219 NPS and 65 other substances across a variety of NPS classes in urine samples was developed, validated and applied to analyze clinical urine samples. This automated workflow is anticipated to facilitate method development for analyzing complex analytes while considering selectivity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Alcohol metabolism in the liver simultaneously generates toxic metabolites and disrupts redox balance, but the regulatory mechanisms have not been fully elucidated. The study aimed to characterize the role of PPARα in alcohol detoxification. Hepatic PPARα and catalase levels were examined in patients with severe alcoholic hepatitis. Mouse studies were conducted to determine the effect of PPARα reactivation by Wy14,643 on alcoholic hepatotoxicity and how catalase is involved in mediating such effects. Cell culture study was conducted to determine the effect of hydrogen peroxide on cellular NAD levels. We found that the protein levels of PPARα and catalase were significantly reduced in the livers of patients with severe alcoholic hepatitis. PPARα reactivation by Wy14,643 effectively reversed alcohol-induced liver damage in mice. Global and targeted metabolites analysis revealed a fundamental role of PPARα in regulating the tryptophan-NAD pathway. Notably, PPARα activation completely switched alcohol metabolism from the CYP2E1 pathway to the catalase pathway along with accelerated alcohol clearance. Catalase knockout mice were incompetent in alcohol metabolism and hydrogen peroxide clearance and were more susceptible to alcohol-induced liver injury. Hydrogen peroxide-treated hepatocytes had a reduced size of cellular NAD pool. These data demonstrate a key role of PPARα in regulating hepatic alcohol detoxification. Catalase-mediated hydrogen peroxide removal represents an underlying mechanism of how PPARα preserves the NAD pool. The study provides a new angle of view about the PPARα-catalase pathway in combating alcohol toxicity., (Published by Elsevier Inc.)

Glioblastoma multiforme (GBM) is the most fatal cancer among brain tumors, and the standard treatment of GBM patients is surgical tumor resection followed by radiotherapy and temozolomide (TMZ) chemotherapy. However, tumors always recur due to the developing drug resistance. It has been shown that neurosteroids, including dehydroepiandrosterone and 17β-estradiol, are synthesized in TMZ-resistant GBM tumors. Therefore, we sought to explore the possible role of 17β-estradiol in the development of drug resistance in GBM. Bioinformatics analysis revealed that aromatase/cytochrome P450 19A1 expression was gradually increased in the development from normal, astrocytoma to GBM. The level of 17β-estradiol was significantly increased in TMZ-resistant cells characterized by ultra performance liquid chromatography-tandem mass spectrometry. Furthermore, 17β-estradiol attenuated TMZ-induced cell death and reduced reactive oxygen species production by mitochondria. In addition, 17β-estradiol attenuated oxidative stress by increasing the expression of superoxide dismutase 1/2, catalase, and nuclear factor erythroid 2-related factor (NRF) 2. We found that NRF2 expression was essential for the induction of drug resistance by 17β-estradiol through the reduction of oxidative stress in GBM., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Recently, there has been significant interest in the influences of the human gut microbiota on many diseases, such as cardiovascular disease (CVD) and metabolic disorders. Trimethylamine N-oxide (TMAO) is one of the most frequently discussed gut-derived metabolites. Dried blood spot (DBS) sampling has been regarded as an attractive alternative sampling strategy for clinical studies and offers many advantages. For DBS sample processing, whole-spot analysis could minimize hematocrit-related bias, but it requires blood volume calibration. This study developed a method combining matrix-induced ion suppression (MIIS) with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to estimate blood volume and quantify TMAO and its precursors and derivatives, including choline, carnitine and acetylcarnitine, in DBSs. The MIIS method used an ion suppression indicator (ISI) to measure the extent of ion suppression caused by the blood matrix, which was related to the blood volume. The results showed that the volume estimation accuracy of the MIIS method was within 91.7-109.7%. The combined MIIS and LC-MS/MS method for quantifying TMAO, choline, carnitine and acetylcarnitine was validated in terms of linearity, precision and accuracy. The quantification accuracy was within 91.2-113.2% (with LLOQ <119%), and the imprecision was below 8.0% for all analytes. A stability study showed that the analytes in DBSs were stable at all evaluated temperatures for at least 30 days. The validated method was applied to quantify DBS samples (n = 56). Successful application of the new method demonstrated the potential of this method for real-world DBS samples and to facilitate our understanding of the gut microbiota in human health., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Oxylipins are an important class of bioactive lipids derived from polyunsaturated fatty acids. They can be both pro- and anti-inflammatory and function as important mediators in various pathological conditions. However, comprehensive analysis of oxylipins still remains a challenge because of their low abundance in plasma and the dominance of structurally similar isomeric species. Herein, we describe a simple and rapid method to comprehensively analyze oxylipins in blood plasma, which utilizes solid-phase extraction in 96-well format for efficient sample cleanup. Separation and detection of more than 130 oxylipins is accomplished by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring in negative-ion mode. The absolute concentrations of oxylipins in human plasma are determined using the calibration curves constructed from internal standards. Detailed methods and precautions are presented for a successful adoption of this method in analytical laboratory.

The glycerol phosphate pathway produces more than 90% of the liver triacylglycerol (TAG). LysoPA, an intermediate in this pathway, is produced by glycerol-3-phosphate acyltransferase. Glycerophosphodiester phosphodiesterase domain containing 3 (GDPD3), whose gene was recently cloned, contains lysophospholipase D activity, which produces LysoPA from lysophospholipids. Whether human GDPD3 plays a role in hepatic TAG homeostasis is unknown. We hypothesized that human GDPD3 increases LysoPA production and availability in the glycerol phosphate pathway, promoting TAG biosynthesis. To test our hypothesis, we infected C57BL/6J mice with adeno-associated virus encoding a hepatocyte-specific albumin promoter that drives GFP (control) or FLAG-tagged human GDPD3 overexpression and fed the mice chow or a Western diet to induce hepatosteatosis. Hepatic human GDPD3 overexpression induced LysoPA production and increased FA uptake and incorporation into TAG in mouse hepatocytes and livers, ultimately exacerbating Western diet-induced liver steatosis. Our results also showed that individuals with hepatic steatosis have increased GDPD3 mRNA levels compared with individuals without steatosis. Collectively, these findings indicate that upregulation of GDPD3 expression may play a key role in hepatic TAG accumulation and may represent a molecular target for managing hepatic steatosis., (Copyright © 2020 Key et al.)

Free fatty acid (FFA) and acylcarnitine (AcCar) are key elements of energy metabolism. Dysregulated levels of FFA and AcCar are associated with genetic defects and other metabolic disorders. Due to differences in the physicochemical properties of these two classes of compounds, it is challenging to quantify FFA and AcCar in human plasma using a single method. In this work, we developed a chemical isotope labeling (CIL)-based liquid chromatography-multiple reaction monitoring (LC-MRM) method to simultaneously quantify FFA and AcCar. Dansylhydrazine (DnsHz) was used to label the carboxylic acid moiety on FFA and AcCar. This resulted in the formation of a permanently charged ammonium ion for facile ionization in positive ionization mode and higher hydrophobicity for enhanced retention of short-chain analogs on reversed-phase LC columns and enabled absolute quantification by using heavy labeled DnsHz analogs as internal standards. Labeling conditions including the concentration and freshness of cross-linker, reaction time, and temperature were optimized. This method can successfully quantify all short-, medium- and long-chain FFAs and AcCars with greatly enhanced sensitivity. Using this method, 25 FFAs and 13 AcCars can be absolutely quantified and validated in human plasma samples within 12 min. Simultaneous quantification of FFA and AcCar enabled by this CIL-based LC-MRM method facilitates the investigation of fatty acid metabolism and has potential in clinical applications.

Various lipidomics studies have revealed the potential of using phospholipids as disease biomarkers for conditions such as Alzheimer's disease, cancer, and sepsis. Establishing accurate quantification methods for targeted phospholipid analysis is important for making these potential markers more clinically relevant. Although a stable isotope labelled-internal standard method can provide good quantification accuracy for endogenous metabolite quantification, there are limited isotope labelled phosphatidylcholines (PCs) commercially available. For this reason, this study proposed a postcolumn infused-internal standard (PCI-IS) method for the accurate quantification of PCs. To demonstrate the quantification accuracy of the PCI-IS method combined with the matrix normalization factor (MNF), 2 LPCs and 6 PCs have been quantified in the human plasma specimens, and the results showed that the PCI-IS combined with MNF method can provide quantification results as accurate as those of the standard addition method (SAM) but without the need for the labor-intensive SAM procedure. We additionally applied the PCI-IS method for improving the PC profiling accuracy, and the results indicated that the biased estimation of the PC composition caused by the MEs can be resolved by PCI-IS correction. Finally, the method was applied to investigate drug resistance in lung cancer cells. Decreased levels of PCs in drug resistant cells disclose the potential role of PCs in drug resistance. We anticipate that the PCI-IS strategy could help quantitative lipidomics move forward and further contribute to various clinical and biomedical studies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Dysregulated lipid species are linked to various disease pathologies and implicated as potential biomarkers for type 1 diabetes (T1D). However, it is challenging to comprehensively profile the blood specimen lipidome with full structural details of every lipid molecule. The commonly used reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS)-based lipidomics approach is powerful for the separation of individual lipid species, but lipids belonging to different classes may still co-elute and result in ion suppression and misidentification of lipids. Using offline mixed-mode and RPLC-based two-dimensional separations coupled with MS/MS, a comprehensive lipidomic profiling was performed on human sera pooled from healthy and T1D subjects. The elution order of lipid molecular species on RPLC showed good correlations to the total number of carbons in fatty acyl chains and total number of double bonds. This observation together with fatty acyl methyl ester analysis was used to enhance the confidence of identified lipid species. The final T1D serum lipid library database contains 753 lipid molecular species with accurate mass and RPLC retention time uniquely annotated for each of the species. This comprehensive human serum lipid library can serve as a database for high-throughput RPLC-MS-based lipidomic analysis of blood samples related to T1D and other childhood diseases. Graphical abstract.

Amadori compound modified lipids are the result of nonenzymatic glycation and play an important role in several physiological and pathological processes. However, glycation of phosphatidylethanolamine (PE), the most abundant amine-containing lipid in blood plasma, is underexplored and so far only a few glycated PEs have been reported. Herein, we report comprehensive profiling of Amadori-PE and -LysoPE species in human plasma. Using synthetic standards, we first optimized the enrichment procedure for extracting Amadori-PE/LysoPE from plasma. On the basis of the characteristic neutral losses of 303 Da in positive and 162 Da in negative ionization mode, we then applied neural loss scanning-liquid chromatography tandem mass spectrometry (LC-NLS-MS) to identify potentially glycated PE and LysoPE, which was followed by targeted product ion scanning (LC-PIS-MS) to confidently confirm the fatty acyl substitutions of the modified lipids. A total of 20 Amadori-LysoPE and 62 Amadori-PE species, including diacyl, plasmanyl, and plasmenyl, were identified. Among them, the concentrations of 12 Amarodi-LysoPE and 54 Amadori-PE were also quantified in native human plasma, using stable isotope labeled Amadori lipids as internal standards.

Introduction: Oxylipins are bioactive oxidation products derived from n-6 and n-3 polyunsaturated fatty acids (PUFAs) in the linoleic acid and α-linolenic desaturation pathways., Purpose: This study determined if carbohydrate intake during prolonged and intensive cycling countered post-exercise increases in n-6 and n-3 PUFA-derived oxylipins., Methods: The research design utilized a randomized, crossover, counterbalanced approach with cyclists (N = 20, overnight fasted state, 7:00 am start) who engaged in four 75-km time trials while ingesting two types of bananas (Cavendish, Mini-yellow), a 6% sugar beverage, and water only. Carbohydrate intake was set at 0.2 g/kg every 15 minutes, and blood samples were collected pre-exercise and 0 h-, 0.75 h-,1.5 h-, 3 h-, 4.5 h-, 21 h-, 45 h-post-exercise. Oxylipins were measured with a targeted liquid chromatography-multiple reaction monitoring mass spectrometric method., Results: Significant time effects and substantial fold-increases (immediately post-exercise/pre-exercise) were measured for plasma levels of arachidonic acid (ARA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and 43 of 45 oxylipins. Significant interaction effects (4 trials x 8 time points) were found for plasma ARA (P<0.001) and DHA (P<0.001), but not EPA (P = 0.255), with higher post-exercise values found in the water trial compared to the carbohydrate trials. Significant interaction effects were also measured for 12 of 45 oxylipins. The data supported a strong exercise-induced increase in plasma levels of these oxylipins during the water trial, with carbohydrate ingestion (both bananas types and the sugar beverage) attenuating oxylipin increases, especially those (9 of 12) generated from the cytochrome P-450 (CYP) enzyme system. These trials differences were especially apparent within the first three hours of recovery from the 75-km cycling bout., Conclusions: Prolonged and intensive exercise evoked a transient but robust increase in plasma levels of oxylipins, with a significant attenuation effect linked to acute carbohydrate ingestion for 28% of these, especially those generated through the CYP enzyme system., Trial Registration: ClinicalTrials.gov, U.S. National Institutes of Health, NCT02994628., Competing Interests: We have the following interests. This study was funded by Dole Foods. Dr. Nicholas Gillitt works as a scientist in the Dole Nutrition Research Laboratory. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Objectives: Recent metabolomic studies of sepsis showed that increased circulatory acylcarnitines were associated with worse survival. However, it is unknown whether plasma carnitine and acylcarnitines can reflect the severity of sepsis, and the role of specific acylcarnitines in prognostic assessment need further confirmation. This study aimed to clarify these questions., Design: Prospective multicenter cohort studies with derivation and validation cohort design., Setting: ICUs at two medical centers and three regional hospitals in Taiwan., Patients: Patients with sepsis and acute organ dysfunction were enrolled. Recruitment of the derivation (n = 90) and validation cohorts (n = 120) occurred from October 2010 through March 2012 and January 2013 through November 2014, respectively., Interventions: Plasma samples were collected immediately after admission, and the levels of carnitine and acylcarnitines were measured by ultra-high performance liquid chromatography-mass spectrometry., Measurements and Main Results: In the derivation cohort, increased plasma levels of short- and medium-chain acylcarnitines were significantly associated with hepatobiliary dysfunction, renal dysfunction, thrombocytopenia, and hyperlactatemia. However, acetylcarnitine is the only acylcarnitine significantly correlating with various plasma cytokine concentrations and also associated with blood culture positivity and 28-day mortality risk. The association between plasma acetylcarnitine and multiple organ dysfunction severity, blood culture positivity, and 28-day mortality, was confirmed in the validation cohort. Patients with high plasma acetylcarnitine (≥ 6,000 ng/mL) had significantly increased 28-day mortality compared with those with plasma acetylcarnitine less than 6,000 ng/mL (52.6% vs 13.9%; hazard ratio, 5.293; 95% CI, 2.340-11.975; p < 0.001 by Cox proportional hazard model)., Conclusions: We confirm that plasma acetylcarnitine can reflect the severity of organ dysfunction, inflammation, and infection in sepsis and can serve as a prognostic biomarker for mortality prediction.

Oxylipins, a subclass of lipid mediators, are metabolites of various polyunsaturated fatty acids with crucial functions in regulation of systemic inflammation. Elucidation of their roles in pathological conditions requires accurate quantification of their levels in biological samples. We refined an ultra-performance liquid chromatography-multiple reaction monitoring-mass spectrometry (UPLC-MRM-MS)-based workflow for comprehensive and specific quantification of 131 endogenous oxylipins in human plasma, in which we optimized LC mobile phase additives, column, and gradient conditions. We employed heatmap-assisted strategy to identify unique transitions to improve the assay selectivity and optimized solid phase extraction procedures to achieve better analyte recovery. The method was validated according to FDA guidelines. Overall, 94.4% and 95.7% of analytes at tested concentrations were within acceptable accuracy (80-120%) and precision (CV < 15%), respectively. Good linearity for most analytes was obtained with R 2  > 0.99. The method was also validated using a standard reference material-SRM 1950 frozen human plasma to demonstrate inter-lab compatibility. Graphical abstract ᅟ.

Tryptophan (TRP) and its catabolites have attracted a lot of attention because of their clinical significance to human health. Recently, microbiome-gut-brain axis was found to have links to many diseases based on the imbalance of TRP catabolism. By using ultra-high performance liquid chromatography coupled to electrospray ionization triple quadrupole mass spectrometry, we present a rapid, robust and comprehensive method to determine 31 TRP catabolites covering three major pathways - kynurenic, serotonergic and bacterial degradation - within 5 min. Polarity switching was employed to analyze catabolites in both ionization modes simultaneously for greatly improved analytical throughput. The intra-day and inter-day precision were 0.5-15.8% and 1.5-16.7%, respectively. Accuracy was between 75.8 and 126.9%. The developed method was applied to study the tissue level of TRP catabolites in the liver, ileum, ileal contents, brain and plasma samples from 8 mice, and clear differences in the distribution of TRP catabolites were observed in different tissues. Ratios of key catabolites to TRP were used to evaluate the activities of specific enzyme and pathway in respective tissues. This method has potential in high throughput analysis of TRP catabolites in biological matrices, which can facilitate understanding the influence of TRP catabolites on microbiome-gut-brain axis and on human health., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Glutamine, glutamate and glutathione are key modulators of excessive oxidative stress in tumor cells. In this study, we developed a rapid and accurate HILIC-MS/MS method to simultaneously determine concentrations of cellular glutamine, glutamate and glutathione. A bared silica HILIC column was employed to analyze these polar metabolites. The LC-MS parameters were optimized to achieve high sensitivity and selectivity. The analysis can be completed within 4 min under optimal conditions. The method was validated in terms of accuracy, precision, and linearity. Intra-day (n = 9) precision was within 2.68-6.24% among QCs. Inter-day precision (n = 3) was below 12.4%. The method accuracy was evaluated by the recovery test, and the accuracy for three analytes were between 91.6 and 110%. The developed method was applied to study antioxidant function of GLS2 in non-small cell lung cancer cells. Changes in concentrations of glutamine, glutamate and glutathione revealed that the overexpression of GLS2 could effectively decrease oxidative stress. In summary, this study developed a rapid HILIC-MS/MS method for quantification of GLS2-related metabolites that could facilitate elucidation of the role of GLS2 in tumor development., (Copyright © 2017. Published by Elsevier Inc.)

Cellular lipidomic studies have been favored approaches in many biomedical research areas. To provide fair comparisons of the studied cells, it is essential to perform normalization of the determined concentration before lipidomic analysis. This study proposed a cellular lipidomic normalization method by measuring the phosphatidylcholine (PC) and sphingomyelin (SM) contents in cell extracts. To provide efficient analysis of PC and SM in cell extracts, flow injection analysis-electrospray ionization-tandem mass spectrometry (FIA-ESI-MS/MS) with a precursor ion scan (PIS) of m/z 184 was used, and the parameters affecting the performance of the method were optimized. Good linearity could be observed between the cell extract dilution factor and the reciprocal of the total ion chromatogram (TIC) area in the PIS of m/z 184 within the dilution range of 1- to 16-fold (R 2  = 0.998). The calibration curve could be used for concentration adjustment of the unknown concentration of a cell extract. The intraday and intermediate precisions were below 10%. The accuracy ranged from 93.0% to 105.6%. The performance of the new normalization method was evaluated using different numbers of HCT-116 cells. Sphingosine, ceramide (d18:1/18:0), SM (d18:1/18:0) and PC (16:1/18:0) were selected as the representative test lipid species, and the results showed that the peak areas of each lipid species obtained from different cell numbers were within a 20% variation after normalization. Finally, the PIS of 184 normalization method was applied to study ischemia-induced neuron injury using oxygen and glucose deprivation (OGD) on primary neuronal cultured cells. Our results showed that the PIS of 184 normalization method is an efficient and effective approach for concentration normalization in cellular lipidomic studies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

An increased rate of drug abuse is a major social problem worldwide. The dried blood spot (DBS) sampling technique offers many advantages over using urine or whole blood sampling techniques. This study developed a simple and efficient ultra-high-performance liquid chromatography-ion booster-quadrupole time-of-flight mass spectrometry (UHPLC-IB-QTOF-MS) method for the analysis of abused drugs and their metabolites using DBS. Fifty-seven compounds covering the most commonly abused drugs, including amphetamines, opioids, cocaine, benzodiazepines, barbiturates, and many other new and emerging abused drugs, were selected as the target analytes of this study. An 80% acetonitrile solvent with a 5-min extraction by Geno grinder was used for sample extraction. A Poroshell column was used to provide efficient separation, and under optimal conditions, the analytical times were 15 and 5min in positive and negative ionization modes, respectively. Ionization parameters of both electrospray ionization source and ion booster (IB) source containing an extra heated zone were optimized to achieve the best ionization efficiency of the investigated abused drugs. In spite of their structural diversity, most of the abused drugs showed an enhanced mass response with the high temperature ionization from an extra heated zone of IB source. Compared to electrospray ionization, the ion booster (IB) greatly improved the detection sensitivity for 86% of the analytes by 1.5-14-fold and allowed the developed method to detect trace amounts of compounds on the DBS cards. The validation results showed that the coefficients of variation of intra-day and inter-day precision in terms of the signal intensity were lower than 19.65%. The extraction recovery of all analytes was between 67.21 and 115.14%. The limits of detection of all analytes were between 0.2 and 35.7ngmL -1 . The stability study indicated that 7% of compounds showed poor stability (below 50%) on the DBS cards after 6 months of storage at room temperature and -80°C. The reported method provides a new direction for abused drug screening using DBS., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The confident identification and in-depth profiling of molecular lipid species remain to be a challenge in lipidomics analysis. In this work, an off-line two-dimensional mixed-mode and reversed-phase liquid chromatography (RPLC) method combined with high-field quadrupole orbitrap mass spectrometer (Q Exactive HF) was developed to profile lipids from complex biological samples. In the first dimension, 22 different lipid classes were separated on a monolithic silica column with elution order from neutral to polar lipids. A total of 13 fractions were collected and run on a RPLC C30 column in the second dimension for further separation of the lipid molecular species based on their hydrophobicity, with the elution order being determined by both the length and degree of unsaturation in the fatty-acyl chain. The method was applied to analyze lipids extracted from rat plasma and rat liver. Fatty acid methyl ester analysis by gas chromatography-mass spectrometry was used to identify the fatty acyls from total lipid extracts, which provided a more confident identification of the lipid species present in these samples. More than 800 lipids were identified in each sample and their molecular structures were confidentially confirmed using tandem mass spectrometry (MS/MS). The number of lipid molecular species identified in both rat plasma and rat liver by this off-line two-dimensional method is approximately twice of that by one-dimensional RPLC-MS/MS employing a C30 column. This off-line two-dimensional mixed-mode LC-RPLC-MS/MS method is a promising technique for comprehensive lipid profiling in complex biological matrices., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Quantitative metabolomics has become much more important in clinical research in recent years. Individual differences in matrix effects (MEs) and the injection order effect are two major factors that reduce the quantification accuracy in liquid chromatography-electrospray ionization-mass spectrometry-based (LC-ESI-MS) metabolomics studies. This study proposed a postcolumn infused-internal standard (PCI-IS) combined with a matrix normalization factor (MNF) strategy to improve the analytical accuracy of quantitative metabolomics. The PCI-IS combined with the MNF method was applied for a targeted metabolomics study of amino acids (AAs). D8-Phenylalanine was used as the PCI-IS, and it was postcolumn-infused into the ESI interface for calibration purposes. The MNF was used to bridge the AA response in a standard solution with the plasma samples. The MEs caused signal changes that were corrected by dividing the AA signal intensities by the PCI-IS intensities after adjustment with the MNF. After the method validation, we evaluated the method applicability for breast cancer research using 100 plasma samples. The quantification results revealed that the 11 tested AAs exhibit an accuracy between 88.2 and 110.7%. The principal component analysis score plot revealed that the injection order effect can be successfully removed, and most of the within-group variation of the tested AAs decreased after the PCI-IS correction. Finally, targeted metabolomics studies on the AAs showed that tryptophan was expressed more in malignant patients than in the benign group. We anticipate that a similar approach can be applied to other endogenous metabolites to facilitate quantitative metabolomics studies.

Two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC/TOF-MS) is superior for chromatographic separation and provides great sensitivity for complex biological fluid analysis in metabolomics. However, GC×GC/TOF-MS data processing is currently limited to vendor software and typically requires several preprocessing steps. In this work, we implement a web-based platform, which we call GC 2 MS, to facilitate the application of recent advances in GC×GC/TOF-MS, especially for metabolomics studies. The core processing workflow of GC 2 MS consists of blob/peak detection, baseline correction, and blob alignment. GC 2 MS treats GC×GC/TOF-MS data as pictures and clusters the pixels as blobs according to the brightness of each pixel to generate a blob table. GC 2 MS then aligns the blobs of two GC×GC/TOF-MS data sets according to their distance and similarity. The blob distance and similarity are the Euclidean distance of the first and second retention times of two blobs and the Pearson's correlation coefficient of the two mass spectra, respectively. GC 2 MS also directly corrects the raw data baseline. The analytical performance of GC 2 MS was evaluated using GC×GC/TOF-MS data sets of Angelica sinensis compounds acquired under different experimental conditions and of human plasma samples. The results show that GC 2 MS is an easy-to-use tool for detecting peaks and correcting baselines, and GC 2 MS is able to align GC×GC/TOF-MS data sets acquired under different experimental conditions. GC 2 MS is freely accessible at http://gc2ms.web.cmdm.tw .

Dried blood spots (DBSs) have had a long history in disease screening in newborns but have gained attention in recent years in the medical care of adults because of the growing importance of personalized medicine. DBSs have several advantages, such as easy transportation, cost-effectiveness, and minimally invasive biological sampling. There are two strategies to process DBS samples. One method takes a fixed diameter of subsample, and another requires the extraction of the whole spot. The whole-spot extraction method is less affected by hematocrit-caused errors, but it requires calibration of the blood volume. We propose a novel strategy using a postcolumn infused-internal standard (PCI-IS) method with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) for estimating and correcting blood volume variations on the DBS cards. By using PCI-IS to measure the extent of ion suppression in the first ion suppression zone in the chromatogram, the blood volume on the DBS cards can be calculated and further calibrated. We used reference blood samples with different volumes (5 to 25 μL) to construct a calibration curve between the blood volume and the extent of ion suppression. The calibration curve was used to estimate the blood volume on the DBS cards collected from 6 volunteers, with 5 designated volumes from each volunteer. The estimation accuracy of the PCI-IS method was between 74.5% and 120.3%. The validated PCI-IS method was used to estimate and calibrate blood volume variation and also to quantify the voriconazole concentration for 26 patients undergoing voriconazole therapy. A high correlation was found for the quantification results between the DBS samples and the conventionally used plasma samples (r = 0.97). The PCI-IS method was demonstrated to be a simple and accurate method for estimating and calibrating the blood volume variation on DBS cards, which greatly facilitates using the DBS method for therapeutic drug monitoring (TDM) for improving the efficacy and safety of drug therapy.

Studies of the cell metabolome greatly improve our understanding of cell biology. Currently, most cellular metabolomics studies control only cell numbers or protein content without adjusting the total metabolite concentration, mainly because of the lack of an effective concentration normalization method for cell metabolites. This study proposed a matrix-induced ion suppression (MIIS) method to measure the total amount of cellular metabolites by utilizing flow injection analysis coupled with electrospray ionization mass spectrometry (FIA-ESI-MS).We used series dilutions of HL-60 cell extracts to establish the relationship between cellular metabolite concentration and the degree of ion suppression of the ion suppression indicator, and a good correlation was obtained between 2- and 12-fold dilutions of cell extracts (R(2) = 0.999). Two lung cancer cells, CL1-0 and CL1-5, were selected as the model cell lines to evaluate the efficacy of the MIIS method and the importance of metabolite concentration normalization. Through MIIS analysis, CL1-0 cells were found to contain metabolites at a concentration 2.1 times higher than in CL1-5, and the metastatic properties of CL1-5 could only be observed after 2.1-fold dilution of CL1-0 before metabolomic analysis. Our results demonstrated that the MIIS method is an effective approach for metabolite concentration normalization and that controlling metabolite concentrations can improve data integrity in cellular metabolomics studies.

Normalizing the total urine concentration is important for minimizing bias in urinary metabolomics analysis comparisons. In this study, we report a matrix-induced ion suppression (MIIS)-based method to normalize concentration using flow injection analysis coupled with electrospray ionization mass spectrometry (FIA-ESI-MS). An ion suppression indicator (ISI) was spiked into urine samples, and the intensity of the extracted ion chromatogram (EIC) for ISI in a urine matrix was subtracted by the EIC for a blank solution and used to calculate the extent to which the signal was reduced by the urine matrix. A series dilution of pooled urine samples was used to correlate the urine concentration and level of ion suppression for ISI. A regression equation was used to estimate the relative concentration of unknown urine samples. The MIIS method was validated for linearity, precision and accuracy. We obtained a good correlation using a quadratic regression model for 1- to 32-fold urine dilutions (R(2)=0.998). The reproducibility (n=4) and intermediate precision (n=3) were below 5% RSD, and the accuracy ranged from 97.15% to 102.10%. The established method was used to estimate the relative concentrations of 16 urine samples, and the results were compared with commonly used normalization methods. Pearson's correlation test was used to demonstrate that the MIIS method correlated highly with the creatinine and osmolarity methods; the correlation coefficients were 0.93 and 0.99, respectively. We successfully applied this method to a urinary metabolomics study on breast cancer. This study demonstrated the MIIS method is simple, accurate and can contribute to data integrity in urinary metabolomics studies., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objectives: Aspiration of the lower airways due to foreign body is rare in adults. This study aimed to determine the outcome of patients who received flexible bronchoscopy with different modalities for foreign body removal in the lower airways., Patients and Methods: Between January 2003 and January 2014, 94 patients diagnosed with foreign body in the lower airways underwent flexible bronchoscopy with different modalities, which included forceps, loop, basket, knife, electromagnet, and cryotherapy. The clinical presentation, foreign body location and characteristics, and applications of flexible bronchoscopy were analyzed., Results: Forty (43%) patients had acute aspiration, which developed within one week of foreign body entry and 54 (57%) had chronic aspiration. The most common foreign bodies were teeth or bone. More patients with chronic aspiration than those with acute aspiration were referred from the out-patient clinic (48% vs. 28%), but more patients with acute aspiration were referred from the emergency room (35% vs. 6%) and intensive care unit (18% vs. 2%). Flexible bronchoscopy with different modalities was used to remove the foreign bodies (85/94, 90%). Electromagnet or cryotherapy was used in nine patients to eliminate the surrounding granulation tissue before foreign body removal. In the nine patients with failed flexible bronchoscopy, eight underwent rigid bronchoscopy instead and one had right lower lung lobectomy for lung abscess., Conclusions: Flexible bronchoscopy with multiple modalities is effective for diagnosing and removing foreign bodies in the lower respiratory airways in adults, with a high success rate (90%) and no difference between acute and chronic aspirations.

Quantification of endogenous metabolites has enabled the discovery of biomarkers for diagnosis and provided for an understanding of disease etiology. The standard addition and stable isotope labeled-internal standard (SIL-IS) methods are currently the most widely used approaches to quantifying endogenous metabolites, but both have some limitations for clinical measurement. In this study, we developed a new approach for endogenous metabolite quantification by the postcolumn infused-internal standard (PCI-IS) method combined with the matrix normalization factor (MNF) method. MNF was used to correct the difference in MEs between standard solution and biofluids, and PCI-IS additionally tailored the correction of the MEs for individual samples. Androstenedione and testosterone were selected as test articles to verify this new approach to quantifying metabolites in plasma. The repeatability (n=4 runs) and intermediate precision (n=3 days) in terms of the peak area of androstenedione and testosterone at all tested concentrations were all less than 11% relative standard deviation (RSD). The accuracy test revealed that the recoveries were between 95.72% and 113.46%. The concentrations of androstenedione and testosterone in fifty plasma samples obtained from healthy volunteers were quantified by the PCI-IS combined with the MNF method, and the quantification results were compared with the results of the SIL-IS method. The Pearson correlation test showed that the correlation coefficient was 0.98 for both androstenedione and testosterone. We demonstrated that the PCI-IS combined with the MNF method is an effective and accurate method for quantifying endogenous metabolites., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: As fatty acids play an important role in biological regulation, the profiling of fatty acid expression has been used to discover various disease markers and to understand disease mechanisms. This study developed an effective and accurate comparative fatty acid analysis method using differential labeling to speed up the metabolic profiling of fatty acids., Methods: Fatty acids were derivatized with unlabeled (D0) or deuterated (D3) methanol, followed by GC-MS analysis. The comparative fatty acid analysis method was validated using a series of samples with different ratios of D0/D3-labeled fatty acid standards and with mouse liver extracts., Results: Using a lipopolysaccharide (LPS)-treated mouse model, we found that the fatty acid profiles after LPS treatment were similar between the conventional single-sample analysis approach and the proposed comparative approach, with a Pearson's correlation coefficient of approximately 0.96. We applied the comparative method to investigate voriconazole-induced hepatotoxicity and revealed the toxicity mechanism as well as the potential of using fatty acids as toxicity markers., Conclusions: In conclusion, the comparative fatty acid profiling technique was determined to be fast and accurate and allowed the discovery of potential fatty acid biomarkers in a more economical and efficient manner., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) has become one of the most widely used methods in pharmaceutical laboratories. Although LC-ESI-MS provides high sensitivity and high specificity for quantifying target analytes in complicated biofluids, the associated severe matrix effects (MEs) generally result in large quantification errors. Here, we propose a novel strategy for correcting MEs in various biofluids using a postcolumn infused-internal standard (PCI-IS) method in combination with matrix normalization factors (MNFs). We used the MNFs to normalize the encountered MEs in various biofluids to the MEs encountered in standard solutions. The use of a postcolumn infused-internal standard also corrects the MEs for individual samples. When using the PCI-IS method in combination with MNFs, the calibration curve generated from standard solutions can be applied to quantify the target analytes in various biofluids. We applied this new approach to quantify etoposide and etoposide catechol in plasma and CSF. The accuracy of the test results showed that over 93% of the data have quantification errors less than 20% and that 99% of the data have quantification errors less than 30%. The successful application of this method to evaluate real clinical samples revealed that our proposed MNFs in combination with the PCI-IS method largely simplifies the entire method development and validation processes, saves a great deal of time and cost without sacrificing quantification accuracy, and provides a simple means of quantifying target analytes in various biofluids. This method will be particularly useful in fields in which the same target analytes need to be quantified in various types of matrices, including bioanalysis, forensic toxicology, environmental studies, and food safety control., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Matrix effects (MEs) are a major problem affecting the quantitative accuracy of liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) when analyzing complicated samples. While analyzing urine specimens, the wide diversity of endogenous materials and different urine concentrations may result in inaccurate quantification. In this study, we propose a postcolumn-infused internal standard (PCI-IS) strategy for universal correction of MEs in urine specimens. MEs can be effectively corrected by dividing the target analyte signal intensity by the PCI-IS intensity. To evaluate the performance of PCI-IS, we used 6 benzodiazepine (BZD) drugs in 5 different concentrations of urine matrixes as a test model. The divergence of the BZD drug signal responses in 5 different urine matrixes was expressed using their respective coefficients of variation (CV) to evaluate the efficiency of using PCI-IS in correcting matrix effects. The CV of the BZD drug signal intensities in these 5 different concentrations of the urine matrixes were reduced from 10 to 30% to less than 10% when the PCI-IS correction method was employed. When the PCI-IS method was used to correct the 6 BZDs in 25 real human urine samples, over 90% of the test results exhibited quantification errors of less than 20%, and all of the test results had quantification errors of less than 30%. These results demonstrate that the PCI-IS method can resolve the problem of inaccurate quantification that arises from the diversity of urine specimens. The PCI-IS method is particularly useful for clinical analysis or forensic toxicology to improve the quantification accuracy in an economical way., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Antibiotic-resistant bacterial infection is one of the most serious clinical problems worldwide. Vancomycin, teicoplanin, daptomycin, and colistin are glycopeptide and lipopeptide antibiotics that are frequently used to treat multidrug-resistant bacterial infections. Therapeutic drug monitoring is recommended to ensure both safety and efficacy and to improve clinical outcomes. This study developed a fast, simple, and sensitive ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of the concentrations of these four drugs in human plasma. The sample preparation process includes a simple protein denaturation step using acetonitrile, followed by an 11-fold dilution with 0.1% formic acid. Eight target peaks for the four drugs can be analyzed within 3 min using a Kinetex™ 2.6 μm C18 column. The mass spectrometry parameters were optimized, and two transitions for each target peak were used for multiple reaction monitoring, which provided high sensitivity and specificity. The UHPLC-MS/MS method was validated over clinical concentration ranges. The intra-day and inter-day precisions for the ratio of the peak area of each analyte to the peak area of the internal standard were all below 12.7 and 14.7% relative standard deviations, respectively. The accuracy at low, medium, and high concentrations of the eight target peaks was between 89.3 and 110.7%. The standard curves for the analytes were linear and had coefficients of determination higher than 0.997. The limits of detection were all below 70 ng mL(-1). The use of this method to analyze patient plasma samples confirmed that it is effective for the therapeutic drug monitoring of these four drugs and can be used to improve the therapeutic efficacy and safety of treatment with antibiotics., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This study developed CE and ultra-high-pressure LC (UHPLC) methods coupled with UV detectors to characterize the metabolomic profiles of different rhubarb species. The optimal CE conditions used a BGE with 15 mM sodium tetraborate, 15 mM sodium dihydrogen phosphate monohydrate, 30 mM sodium deoxycholate, and 30% ACN v/v at pH 8.3. The optimal UHPLC conditions used a mobile phase composed of 0.05% phosphate buffer and ACN with gradient elution. The gradient profile increased linearly from 10 to 21% ACN within the first 25 min, then increased to 33% ACN for the next 10 min. It took another 5 min to reach the 65% ACN, then for the next 5 min, it stayed unchanged. Sixteen samples of Rheum officinale and Rheum tanguticum collected from various locations were analyzed by CE and UHPLC methods. The metabolite profiles of CE were aligned and baseline corrected before chemometric analysis. Metabolomic signatures of rhubarb species from CE and UHPLC were clustered using principle component analysis and distance-based redundancy analysis; the clusters were not only able to discriminate different species but also different cultivation regions. Similarity measurements were performed by calculating the correlation coefficient of each sample with the authentic samples. Hybrid rhizome was clearly identified through similarity measurement of UHPLC metabolite profile and later confirmed by gene sequencing. The present study demonstrated that CE and UHPLC are efficient and effective tools to identify and authenticate herbs even coupled with simple detectors., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

CYP2D6 (cytochrome P450 2D6) is one of the most important enzymes involved in drug metabolism, and CYP2D6 gene variants may cause toxic effects of therapeutic drugs or treatment failure. In this research, a rapid and simple method for genotyping the most common mutant alleles in the Asian population (CYP2D6*1/*1, CYP2D6*1/*10, CYP2D6*10/*10, CYP2D6*1/*5, CYP2D6*5/*10, and CYP2D6*5/*5) was developed by allele-specific polymerase chain reaction (AS-PCR) combined with capillary electrophoresis (CE). We designed a second mismatch nucleotide next to the single nucleotide polymorphism (SNP) site in allele-specific primers to increase the difference in PCR amplification. Besides, we established simulation equations to predict the CYP2D6 genotypes by analyzing the DNA patterns in the CE chromatograms. The multiplex PCR combined with CE method was applied to test 50 patients, and all of the test results were compared with the DNA sequencing method, long-PCR method and real-time PCR method. The correlation of the analytical results between the proposed method and other methods were higher than 90%, and the proposed method is superior to other methods for being able to simultaneous detection of SNPs and copy number variations (CNV). Furthermore, we compared the plasma concentration of aripiprazole (a CYP2D6 substrate) and its major metabolites with the genotype of 25 patients. The results demonstrate the proposed genotyping method is effective for estimating the activity of the CYP2D6 enzyme and shows potential for application in personalized medicine. Similar approach can be applied to simultaneous detection of SNPs and CNVs of other genes., (Copyright © 2012 Elsevier B.V. All rights reserved.)