Liquid Chromatography High-resolution Tandem Mass Research Articles

Proteomic Identification of Novel Plasma Biomarkers and Pathobiologic Pathways in Alcoholic Acute Pancreatitis.

Acute pancreatitis (AP) is a painful and potentially life-threatening disorder with the potential for therapeutic interventions. Biomarkers that characterize cases by severity and pathogenic mechanisms involved are not yet available but needed for the implementation of rational therapies. Here, we used shotgun proteomics to obtain information from plasma samples about local and systemic pathologies taking place during cases of alcoholic AP. Plasma was obtained at Kaunas University of Medicine Hospital (Lithuania) from 12 AP patients of alcohol related etiology (median age of 40) within 24 h of presentation, and 12 age-matched, healthy controls. Patients entered into the study had moderately severe AP with the following characteristics: mean blood lactate dehydrogenase level of 1127 mg/dl; median APACHEII score of 5.5 and mean IMRIE score of 3.5. For proteomic analysis, less-abundant proteins in plasma samples were enriched using Top 12 abundant protein depletion columns. Further processing was performed by a modified filter-assisted sample preparation combined with tandem mass tag labeling for quantitation. Samples were analyzed using an Orbitrap Elite mass spectrometer for high resolution liquid chromatography–tandem mass spectrometry (LC–MS/MS). Our analysis revealed 31 proteins that exhibited significant 1.5-fold or higher increases in the AP compared to control patients, and six that were significantly decreased. Gene ontology analysis indicated a strong correlation with exosomal origin in the elevated proteins, with 29/31 (93.5%) associated with this extracellularly-secreted compartment. Elevated proteins included established and proposed biomarkers of AP including C-reactive protein, LPS-binding protein, intercellular adhesion molecule-1, and von Willebrand factor, as well as several novel potential biomarkers. These results provide the methodology for proteomic analysis of plasma samples to discover novel biomarkers that characterize pancreatitis cases by pathogenic mechanism as well as disease activity at an early stage that is highly informative for routine clinical practice and clinical trials.

In vitro glucuronidation of designer benzodiazepines by human UDP-glucuronyltransferases.

Multiple new psychoactive substances (NPS) are released into the recreational drug market each year. One NPS drug class that has become more common in recent years is that of the benzodiazepines (designer benzodiazepines, DBZ). Several metabolism studies have been performed to improve their bioanalytical detection via the best target. These studies have shown the presence of parent glucuronides and, as polymorphisms have been noted for the catalyzing enzymes (UDP-glucuronyltransferases) responsible for glucuronide conjugation reactions, it is important to keep this in mind when interpreting DBZ cases in clinical and/or forensic toxicology. Therefore, the aim of this study was to determine the UDP-glucuronyltransferases (UGTs) responsible for parent compound conjugation of nine DBZ to facilitate interpretation of related cases. Clonazolam, deschloroetizolam, etizolam, flubromazolam, flunitrazolam, metizolam, nifoxipam, nitrazolam, and pyrazolam were incubated with pooled human liver microsomes (pHLM) or 13 different human UGTs. The samples were analyzed using liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). Glucuronide conjugates of flunitrazolam and nifoxipam were only detected in pHLM, suggesting that these reactions are performed by dimer complexes of several UGTs or complexes between UGTs and other metabolizing enzymes contained in pHLM. Nitrazolam or pyrazolam glucuronides were not detected. Glucuronidation of clonazolam, deschloroetizolam, etizolam, flubromazolam, and metizolam was catalyzed exclusively by UGT1A4. The conjugation of the majority of the DBZ was performed by the UGT isoform 1A4 for which polymorphisms have been described. This underlines the importance of taking glucuronidation polymorphism into consideration when interpreting intoxication cases.

Two-dimensional gel and shotgun proteomics approaches to distinguish fresh and frozen-thawed curled octopus (Eledone cirrhosa)

The substitution and sale of frozen-thawed fish labeled as fresh is a widespread, difficult to unmask commercial fraud and a potential risk for consumer health. Proteomics could help to identify markers for the rapid screening of food samples and the identification of frozen-thawed seafood. Using two-dimensional electrophoresis (2-DE) and high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS), we identified biomarkers that are able to discriminate between fresh and frozen-thawed tissue samples of curled octopus (Eledone cirrhosa). The 2-DE analysis showed a significant reduction in two protein spots (molecular weight of 45–50 kDa, isoelectric point of 6.5–7) identified as transgelin. At shotgun analysis, nine proteins resulted modulated and transgelin was confirmed as down-regulated, making it a potentially useful marker for differentiating between fresh and frozen-thawed fish product samples. Biological significanceThis work, based on two different proteomics approaches, investigated differentially expressed proteins in the tentacles of the curled octopus (E. cirrhosa) after freezing-thawing processes. We were able to characterize the proteome of the tentacles, increasing our knowledge on this species, and a common down-regulated protein was identified by 2-DE and shotgun analysis, a calponin-like protein called transgelin, suggesting a potential use as a marker to distinguish different states of conservation in this species.

Endocrine disruptors and neonatal anthropometry, NICHD Fetal Growth Studies - Singletons

BackgroundIntrauterine exposure to endocrine disrupting chemicals (EDCs) has been equivocally associated with birth weight, length and head circumference with limited attention to anthropometric endpoints such as umbilical circumference and limb lengths. ObjectiveTo explore 76 prenatal maternal plasma EDC concentrations in a healthy obstetric cohort and 7 neonatal anthropometric endpoints by maternal race/ethnicity. MethodsThe study cohort comprised 2106 (564 White, 549 Black, 590 Hispanic, 403 Asian) healthy pregnant women recruited from 12 U.S. clinical sites between 2009 and 2012 who were followed through delivery. Neonates underwent standardized anthropometric assessment (weight, length, head and umbilical circumference, and mid- upper arm and thigh length). Plasma EDC concentrations were quantified using high resolution gas chromatography-high resolution mass spectrometry and liquid chromatography-tandem mass spectrometry. EDCs were log-transformed and rescaled by their deviations (SD) when modeled relative to neonatal endpoints using linear regression adjusting for age, education, pre-pregnancy body mass index (BMI), serum cotinine, serum lipids for lipophilic chemicals, and a race/ethnicity interaction term; p-values had false discovery rate correction (<0.05). ResultsThe cohort comprised women aged 28 (SD = 5) years with normal BMIs (23.6 kg/m2, SD = 3). Maternal EDC concentrations varied by self-identified race/ethnicity and neonatal outcomes, though no specific EDC was consistently associated with neonatal anthropometric outcomes across racial/ethnic groups. For the overall cohort, perfluorooctanoic acid was negatively associated with birth length per SD increase in concentration (β = −0.23 cm; 95% CI −0.35, −0.10), while perfluorohexanesulfonic acid was negatively associated with umbilical circumference (β = −0.26 cm; 95% CI −0.40, −0.13), perfluorodecanoic acid with arm length (−0.09 cm; 95% CI −0.14, −0.04), and PCBs congeners 118/106 (−0.12 cm; 95% CI −0.20, −0.04) and 146/161 (−0.14 cm; 95% CI −0.23, −0.05) with thigh length, as were 7 other poly-and-perfluorinated alkyl substances (PFASs). ConclusionsAmong healthy pregnant women with low risk antenatal profiles and relatively low EDC concentrations, reductions in umbilical circumference and bone lengths may be a sensitive marker of intrauterine EDC exposure, particularly for PFAS.

Study of the in vitro and in vivo metabolism of 4-HO-MET

4-Hydroxy-N-methyl-N-ethyltryptamine (4-HO-MET) is a new psychoactive substance (NPS) of the chemical class of tryptamines. It shows structural similarities to the endogenous neurotransmitter serotonin, and is a serotonergic hallucinogen, affecting emotional, motoric, and cognitive functions. The knowledge about its biotransformation is mandatory to confirm the abuse of the substance by urine analysis in forensic cases. Therefore, phase I metabolites were generated by the use of the pooled human liver microsomes (pHLM) in vitro model and analyzed by high-performance liquid chromatography high-resolution tandem mass spectrometry with information-dependent acquisition (HPLC-IDA-HR-MS/MS). Furthermore, three authentic urine samples was analyzed and results were compared: 12 different in vitro and 4 in vivo metabolites were found. The predominant biotransformation steps observed in vitro were mono- or dihydroxylation of 4-HO-MET, besides demethylation, demethylation in combination with monohydroxylation, formation of a carboxylic acid, deethylation, and oxidative deamination. In vivo, monohydroxylation, and glucuronidation were detected. A metabolic pathway based on these results was proposed.For the analysis of urine samples in forensic cases, the N-oxide metabolite and the HO-alkyl metabolite are recommended as target compounds, besides the glucuronides of 4-HO-MET and the parent compound 4-HO-MET itself.

Abstract 3335: Characterizing the MAXH28R secretome in endometrial cancer

Abstract We have previously shown that the recurrent endometrial cancer MAX p.His28Arg mutation (MAXH28R) is associated with a proangiogenesis phenotype in both in vitro and xenograft models. The purpose of this study is to characterize the MAXH28R secretome to identify the proangiogenic and other paracrine factors contributing to MAXH28R associated tumorigenesis. Bottom-up high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) of secreted proteins coupled with microarray analysis of global gene expression was performed in AN3CA endometrial cancer cell lines that stably express the long isoform of MAX-wild type (MAXWT) or MAXH28R. Orthogonal validation of differentially expressed proteins was performed by Western blot (WB) and enzyme-linked immunosorbent assay (ELISA) using MAXWT- or MAXH28R-expressing AN3CA cell lines. Bioinformatic analysis of the proteins secreted into conditioned media (secretome) identified 114 proteins differentially expressed by MAXH28R compared to MAXWT-expressing AN3CA cells (P &amp;lt; 0.05). Differential expression of proteins was highly correlated with mRNA expression (Pearson r = 0.75, P &amp;lt; 0.001), indicating that a subset of the differentially expressed proteins are potential direct transcriptional targets of MAX. Secretome analysis revealed that Laminin-β1 expression in MAXH28R was reduced more than 100-fold compared to MAXWT (P &amp;lt; 0.001), while ALCAM (CD166) expression was increased 3.5-fold (P &amp;lt; 0.001). Decreased expression of TGFβ1 (5.9-fold decrease, P = 0.023) and TGFβ2 (3-fold decrease, P = 0.009) was seen for MAXH28R cells compared to MAXWT. Differential expression of Laminin-β1 and ALCAM was confirmed by WB analysis and TGFβ1 and TGFβ2 by ELISA. Our analyses revealed extensive changes in the MAXH28R secretome, as exemplified above. Reduced Laminin-β1 highlights alteration in the extracellular matrix with implications for cell adhesion and integrin signaling. Increased ALCAM expression indicates changes to cell-cell adhesion. Elevated levels of ALCAM in conditioned media are consistent with increased ALCAM shedding, which is a marker of more aggressive tumors in a variety of malignancies. Reduced TGFβ ligand levels suggest potential alterations in paracrine and autocrine TGFβ signaling. However, the impact on canonical and noncanonical TGFβ signaling in MAX mutant endometrial cancers remains unknown. ChIP-qPCR experiments are ongoing to assess MAXH28R binding at the promoters of differentially expressed genes. Biologic validation of MAXH28R effects on differential expression of secreted proteins is being performed in the Ishikawa and RL95-2 endometrial cancer cell lines that stably express MAXH28R and MAXWT. The effects of reduced Laminin-β1 on integrin signaling and anchorage independent growth will be reported, as will effects of altered TGFβ expression on canonical and noncanonical TGFβ signaling. Citation Format: Craig M. Rush, Miranda L. Gardner, Caroline E. Sapp, Michael A. Freitas, Paul J. Goodfellow. Characterizing the MAXH28R secretome in endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3335.

Degradation and residues of indoxacarb enantiomers in rice plants, rice hulls and brown rice using enriched S-indoxacarb formulation and enantiopure formulation.

A chiral liquid chromatography-tandem high resolution mass spectroscopic method was developed for the analysis of indoxacarb enantiomers in rice plants, rice hulls and brown rice. Chiral separation of two enantiomers was carried out on a Superchiral S-OD column maintained at 20°C and eluted with 0.3 mL/min methanol. Samples were extracted by acetonitrile solution with ultrasound and cleansed by dispersive solid-phase extraction of 50 mg of primary secondary amine and 50 mg of C18 . This method was successfully used to study the degradation and residues of two enantiomers with enriched S-indoxacarb (2.33S/1R) and pure S-indoxacarb in rice plants. The half-lives of R-indoxacarb and S-indoxacarb were 4.20-4.33 and 3.45-3.57 days in rice plants during the degradation of enriched S-indoxacarb in Guizhou and Hunan, respectively, whereas the half-lives of pure S-indoxacarb were 2.68 and 3.69 days in Guizhou and Hunan, respectively. The results indicated that preferential S-indoxacarb degradation occurred and that enantiomeric transformation was absent in the total experiment periods of pure S-indoxacarb in rice plants. The final residue concentrations of indoxacarb enantiomers in brown rice were significantly less than those in rice plants and rice hulls in the same rice field after applying indoxacarb SC and indoxacarb EC.

Non-target screening to trace ozonation transformation products in a wastewater treatment train including different post-treatments

Ozonation and subsequent post-treatments are increasingly implemented in wastewater treatment plants (WWTPs) for enhanced micropollutant abatement. While this technology is effective, micropollutant oxidation leads to the formation of ozonation transformation products (OTPs). Target and suspect screening provide information about known parent compounds and known OTPs, but for a more comprehensive picture, non-target screening is needed. Here, sampling was conducted at a full-scale WWTP to investigate OTP formation at four ozone doses (2, 3, 4, and 5 mg/L, ranging from 0.3 to 1.0 gO3/gDOC) and subsequent changes during five post-treatment steps (i.e., sand filter, fixed bed bioreactor, moving bed bioreactor, and two granular activated carbon (GAC) filters, relatively fresh and pre-loaded). Samples were measured with online solid-phase extraction coupled to liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) using electrospray ionization (ESI) in positive and negative modes. Existing non-target screening workflows were adapted to (1) examine the formation of potential OTPs at four ozone doses and (2) compare the removal of OTPs among five post-treatments. In (1), data processing included principal component analysis (PCA) and chemical knowledge on possible oxidation reactions to prioritize non-target features likely to be OTPs. Between 394 and 1328 unique potential OTPs were detected in positive ESI for the four ozone doses tested; between 12 and 324 unique potential OTPs were detected in negative ESI. At a specific ozone dose of 0.5 gO3/gDOC, 27 parent compounds were identified and were related to 69 non-target features selected as potential OTPs. Two OTPs were confirmed with reference standards (venlafaxine N-oxide and chlorothiazide); 34 other potential OTPs were in agreement with literature data and/or reaction mechanisms. In (2), hierarchical cluster analysis (HCA) was applied on profiles detected in positive ESI mode across the WWTP and revealed 11 relevant trends. OTP removal was compared among the five post-treatments and 54–83% of the non-target features that appeared after ozonation were removed, with the two GAC filters performing the best. Overall, these data analysis strategies for non-target screening provide a useful tool to understand the behavior of unknown features during ozonation and post-treatment and to prioritize certain non-targets for further identification.

A stable isotope dilution tandem mass spectrometry method of major kavalactones and its applications.

Kava is regaining its popularity with detailed characterizations warranted. We developed an ultraperformance liquid chromatography high-resolution tandem mass spectrometry (UPLC-MS/MS) method for major kavalactones (kavain, dihydrokavain, methysticin, dihydromethysticin and desmethoxyyangonin) with excellent selectivity and specificity. The method has been validated for different matrices following the Food and Drug Administration guidance of analytical procedures and methods validation. The scope of this method has been demonstrated by quantifying these kavalactones in two kava products, characterizing their tissue distribution and pharmacokinetics in mice, and detecting their presence in human urines and plasmas upon kava intake. As expected, the abundances of these kavalactones differed significantly in kava products. All of them exhibited a large volume of distribution with extensive tissue affinity and adequate mean residence time (MRT) in mice. This method also successfully quantified these kavalactones in human body fluids upon kava consumption at the recommended human dose. This UPLC-MS/MS method therefore can be used to characterize kava products and its pharmacokinetics in animals and in humans.

Trace analysis of multi-class phytohormones in Oryza sativa using different scan modes in high-resolution Orbitrap mass spectrometry: method validation, concentration levels, and screening in multiple accessions.

Phytohormones are signaling and regulating metabolites involved in numerous plant processes, including growth, development, and responses to stress. Currently, the focus is on the analysis of multiple phytohormones in order to characterize crosstalk and hormone signaling networks. In this paper, representative phytohormones of the major classes are simultaneously determined in rice tissues by a generic solid-liquid extraction, followed by liquid chromatography and electrospray ionization high-resolution tandem mass spectrometry using a Q-Exactive™ instrument. After a thorough optimization of the sample preparation, the analytical method was fully validated toward the ultra-trace quantification of six a priori selected plant hormones using three scan modes of the quadrupole-Orbitrap instrument: full-scan high-resolution mass spectrometry, targeted single ion monitoring (t-SIM), and t-SIM followed by data-dependent tandem mass spectrometry. Overall, a similar quantitative performance was noticed for the different scan modes. The analytical method was successfully applied to measure basal phytohormone levels in six different rice accessions, comprising Oryza sativa ssp. japonica, indica, and Oryza glaberrima. Hormone concentrations were higher in shoots than in roots or at least similar. Except for a lower level of salicylic acid in shoots of O. glaberrima versus O. sativa, no other differences in hormone levels could be noticed that were dependent of the (sub)species assignment of the analyzed accessions. Making use of the benefits of full-scan high-resolution mass spectrometry, a first post-run suspect screening was performed, suggesting - based on accurate mass measurements and isotopic patterns - the possible presence of about 50 additional plant hormones in the rice tissues. Graphical abstract ᅟ.

New pathways for the biodegradation of diethyl phthalate by Sphingobium yanoikuyae SHJ

Abstract Diethyl phthalate (DEP) is a representative of phthalate esters (PAEs) that are largely used as industrial additives and have become environmental contaminants and endocrine disruptors. A newly isolated bacterial strain Sphingobium yanoikuyae SHJ was found to be capable of degrading DEP very efficiently. Four intermediates during the DEP degradation process were identified to be ethyl methyl phthalate (EMP), monoethyl phthalate (MEP), monomethyl phthalate (MMP) and phthalic acid (PA) using high-performance liquid chromatography tandem high-resolution mass spectrometry (HPLC-HRMS). When there was limited oxygen under SSA conditions, the rates of the further degradation of MEP and PA were slower compared to that under aerobic conditions. Although all intermediates EMP, MEP and MMP eventually decreased or disappeared, the DEP degradation kinetics showed that EMP formed much faster than that of MEP, and MMP appeared after the decreasing of MEP. It is proposed that two DEP degradation pathways must be present in S. yanoikuyae SHJ. The first is DEP → MEP → PA through a sequential hydrolysis process. The second is DEP → EMP → MEP → MMP → PA through a combination of demethylation or trans-esterification with methanol and hydrolysis processes. It is concluded that S. yanoikuyae SHJ has a versatile capability of degrading DEP and will have a great application potential in bioremediation of PAEs-contaminated shallow aquifer.

Plasma proteomic analysis of systemic lupus erythematosus patients using liquid chromatography/tandem mass spectrometry with label-free quantification.

ContextSystemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease with unknown etiology.ObjectiveHuman plasma is comprised of over 10 orders of magnitude concentration of proteins and tissue leakages. The changes in the abundance of these proteins have played an important role in various human diseases. Therefore, the research objective of this study is to identify the significantly altered expression levels of plasma proteins from SLE patients compared with healthy controls using proteomic analysis. The plasma proteome profiles of both SLE patients and controls were compared.MethodsA total of 19 active SLE patients and 12 healthy controls plasma samples were analyzed using high-resolution electrospray ionization liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) followed by label-free quantification.ResultsA total of 19 proteins showed a significant level of expression in the comparative LC-ESI-MS/MS triplicate analysis; among these, 14 proteins had >1.5- to three-fold up-regulation and five had <0.2- to 0.6-fold down-regulation. Gene ontology and DAVID (Database Annotation Visualization, and Integrated Discovery) functional enrichment analysis revealed that these proteins are involved in several important biological processes including acute phase inflammatory responses, complement activation, hemostasis, and immune system regulation.ConclusionOur study identified a group of differentially expressed proteins in the plasma of SLE patients that are involved in the imbalance of the immune system and inflammatory responses. Therefore, these findings may have the potential to be used as prognostic/diagnostic markers for SLE disease assessment or disease monitoring.

Development and validation of an ultrahigh performance liquid chromatography-high resolution tandem mass spectrometry assay for nine toxic alkaloids from endophyte-infected pasture grasses in horse serum

Endophyte fungi (e.g. Epichloë ssp. and Neotyphodium ssp.) in symbiosis with pasture grasses (e.g. Festuca arundinacaea and Lolium perenne) can produce toxic alkaloids, which are suspected to be involved in equine diseases such as fescue toxicosis, ryegrass staggers, and equine fescue oedema. The aim of this study was, therefore, to develop and validate a quantification method for these and related alkaloids: ergocristine, ergocryptine, ergotamine, ergovaline, lolitrem B, lysergic acid, N-acetylloline, N-formylloline, peramine, and paxilline in horse serum.Horse serum samples (1.5mL) were worked up by solid-phase extraction (OASIS HLB). The extracts were analyzed by ultra high performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS). Chromatographic separation was achieved by gradient elution with ammonium formate buffer and acetonitrile on a RP18 column (100×2.1mm; 1.7μm). HRMS/MS detection was performed on a QExactive Focus instrument with heated positive electrospray ionization and operated in the parallel reaction monitoring (PRM) mode. Method validation included evaluation of selectivity, matrix effect, recovery, linearity, limit of quantification (LOQ), limit of detection (LOD), accuracy, and stability.With exception of lolitrem B solid phase extraction yielded high recoveries (73.6–104.6%) for all analytes. Chromatographic separation of all analytes was achieved with a run time of 25min. HRMS/MS allowed sensitive detection with LODs ranging from 0.05 to 0.5ng/mL and LOQs from 0.1 to 1.0ng/mL. Selectivity experiments showed no interferences from matrix or IS, but N-acetylloline and N-formylloline were found to be ubiquitous in horse serum. Newborn calf serum was therefore used as surrogate matrix for the validation study. Calibration ranges were analyte-dependent and in total covered concentrations from 0.1 to 50ng/mL. Lolitrem B and paxilline could be sensitively detected, but did not meet quantification requirements. For the other analytes, accuracy and precision were shown for 3 different concentrations (QC low, medium, high) with acceptable bias (−10, 5%–7.9%) and precision (CV 2.6%–12.5%). Matrix effects varied from 55.0% to 121% (RSD 7.8–18.5%) and were adequately compensated by IS. Matrix effects of N-acetylloline and N-formylloline could only be estimated in newborn calf serum (n=1) and ranged from 52.5–88.3%. All analytes were stable under autosampler conditions and over 3 freeze and thaw cycles. Applicability was proven by analyzing authentic horse serum samples (n=24).In conclusion, the presented method allows a sensitive detection of ergocrisitine, ergocryptine, ergotamine, ergovaline, lolitrem B, lysergic acid, N-acetylloline, N-formylloline, peramine, and paxilline in horse serum and reliable quantification of all but lolitrem B and paxilline.

Pharmacokinetics, Metabolism, and Biodistribution of a Liver‐Targeted siRNA Therapeutic in Preclinical Species

Almost twenty years ago, small interfering RNA (siRNA) was first demonstrated as a tool to silence gene expression in vitro. Today siRNA is part of an emerging drug class of RNA interference‐based therapeutics undergoing development against formerly undruggable targets. siRNA is a double‐stranded molecule typically 21–23 nucleotides in length. Upon uptake into the cell, the siRNA duplex is loaded into a multi‐protein RNA‐induced silencing complex (RISC). One strand, referred to as the sense strand, of the siRNA duplex is degraded while the remaining strand, the antisense strand, guides RISC to a target mRNA sequence for cleavage, subsequently decreasing the expression of the corresponding protein. The primary objective of this work was to determine an extensive pharmacokinetic profile of a liver‐targeting siRNA construct in preclinical species. To aid in measuring low levels of siRNA drug, an ultrasensitive hybridization‐ligation enzyme‐linked immunosorbent assay was developed utilizing locked nucleic acid probes for the quantitation of the sense and antisense strands across multiple biological matrices. Following a single subcutaneous injection of siRNA (0.1, 1 or 10 mg/kg) in non‐human primates (NHP), we observed peak serum concentrations of sense and antisense strands to occur as soon as 1–2 hours post‐dose from which circulating siRNA levels then declined, but were detected out to 7 days post‐dose. In addition to this work, we aimed to gain a better understanding of siRNA tissue distribution, specifically to the liver and kidneys, as well as the metabolism of the molecule that may occur in vivo potentially compromising drug efficacy. Interestingly, we observed a similar accumulation of both sense and antisense strands in the liver, whereas, the sense strand reached concentrations three orders of magnitude greater than the antisense strand in the kidneys, suggesting denaturation of the siRNA duplex. We also confirmed siRNA loading of RISC via immunoprecipitation from the liver homogenate and observed comparable loading levels of both sense and antisense strands. Further, these liver homogenates were then analyzed for metabolite formation using high‐resolution liquid chromatography‐tandem mass spectrometry. Metabolite profiling was performed on the antisense strand of the siRNA duplex as this is the pharmacologically active component of the molecule. We were excited to observe that the full‐length antisense strand was the predominant analyte detected (71.3% of total drug and metabolites) in liver homogenate followed by six metabolites of lesser abundance that resulted from single nucleotide cleaving at the 3′ end, n‐1 (21.5%), n‐2 (1%), n‐3 (0.6%), n‐4 (1.5%), n‐5 (2.8%) and n‐6 (1.5%). Concurrent to this work, efforts are underway to develop a fluorescent‐based in situ hybridization to investigate intra‐ and extra‐hepatic distribution of the siRNA construct at the cellular level. Through the described studies, we have successfully achieved a more complete understanding of the absorption, distribution, metabolism and excretion properties of an siRNA therapeutic in preclinical species which will inform clinical translation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential expressions of plasma proteins in systemic lupus erythematosus patients identified by proteomic analysis.

Systemic lupus erythematosus (SLE) is a chronic and complex autoimmune disease with a wide range of clinical manifestations that affects multiple organs and tissues. Therefore the differential expression of proteins in the serum/plasma have potential clinical applications when treating SLE. We have compared the plasma/serum protein expression patterns of nineteen active SLE patients with those of twelve age-matched and gender-matched healthy controls by proteomic analysis. To investigate the differentially expressed proteins among SLE and controls, a 2-dimensional gel electrophoresis coupled with high-resolution liquid chromatography tandem mass spectrometry was performed. To further understand the molecular and biological functions of the identified proteins, PANTHER and Gene Ontology (GO) analyses were employed. A total of 14 significantly expressed (p<0.05, p<0.01) proteins were identified, and of these nine were up-regulated and five down-regulated in the SLE patients. The functional enrichment analysis assigned the majority of the identified proteins including alpha 2 macroglobulin, complement C4, complement factor H, fibrinogen beta chain, and alpha-1-antitrypsin were part of the complement/coagulation cascade, which is an important pathway that plays a crucial role in SLE pathogenesis. In addition to these proteins the differential expressions of ceruloplasmin, transthyretin, and haptoglobin play a potential role in the renal system abnormalities of SLE. Therefore, the identified differentially expressed proteins are relevant to SLE patient's cohort. Most importantly the up-regulated proteins might be the potential candidates for renal system involvement in SLE disease pathogenesis. In order to confirm the diagnostic/therapeutic potential of the identified proteins, future validation studies are required.

Identification of Polygonum orientale constituents using high-performance liquid chromatography high-resolution tandem mass spectrometry.

Our primary focus in this research was to identify and characterize its bioactive compounds for potential therapeutic use. Twenty-seven metabolites of Polygonum orientale were identified using LC-QTOF tandem mass spectrometry. Interestingly, P. orientale extracts included several highly oxygenated flavonoids were isolated from P. orientale by column chromatography. 13C NMR data of highly oxygenated flavonoids (1-7) are reported here for the first time. In addition, nitric oxide, 1,1-diphenyl-2-picrylhydrazyl, and water-soluble tetrazolium salt assays were carried out on the isolated compounds to investigate their anti-inflammatory, anti-oxidant, and neuroprotective activities, respectively. Compounds 1, 2, 3, 5, 7, and 8 significantly attenuated lipopolysaccharide-stimulated NO production in BV2 cells without affecting cell viability. Compounds 9-12 exhibited significant antioxidant activity, while compounds 8, 9, and 12 exhibited protective effects against glutamate-induced neurotoxicity in HT22 cells. Our results indicate that P. orientale is a promising source of natural agents for the potential treatment of inflammation and neurodegenerative diseases.

Determination of polar organic micropollutants in surface and pore water by high-resolution sampling-direct injection-ultra high performance liquid chromatography-tandem mass spectrometry

Hyporheic zones (HZs) are dynamic and complex transition regions between rivers and aquifers which are thought to play an important role in the attenuation of environmental micropollutants. Non-steady state and small-scale hyporheic processes which affect micropollutants in the HZ are poorly characterized due to limitations in existing analytical methodologies. In this work we developed a method for high spatio-temporal resolution analysis of polar organic micropollutants (POMs) in hyporheic pore- and surface waters by combining (semi-) automatic low volume sampling techniques with direct-injection ultra-high performance liquid chromatography tandem mass spectrometry. The method is capable of quantifying 25 parent compounds and 18 transformation products (TPs) using only 0.4 mL of water and few preparation steps. Application of the method to both surface and pore water revealed significant (i.e. > an order of magnitude) differences in POM concentrations over small time and spatial scales (i.e. < a few hours and tens of cm, respectively). Guanylurea, a TP of the antidiabetic drug metformin was detected at unprecedentedly high concentrations. Collectively, this method is suitable for in situ characterization of POMs at high spatial and temporal resolution and with minimal disturbance of natural flow paths and infiltration of surface water.

Development and validation of an ultrahigh performance liquid chromatography-high resolution tandem mass spectrometry quantification method for hypoglycin A and methylene cyclopropyl acetic acid carnitine in horse serum in cases of atypical myopathy.

Atypical myopathy (AM) is a fatal disease in horses presumably caused by hypoglycine A (HGA) from ingested maple seeds and its active metabolite methylene cyclopropyl acetic acid (MCPA). The aim of this study was the development and validation of a rapid and simple assay for HGA and MCPA-carnitine in horse serum and its application to authentic samples. Identification and quantification were carried out by ultra high performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS) with full-scan/data-dependent MS/MS. Chromatographic separation was performed by isocratic elution on a hydrophilic interaction liquid chromatography (HILIC) column (100 x 2.1mm, 1.7μm). Serum samples (250μL) were worked up by protein precipitation. The method was validated according to international guidelines with respect to selectivity, linearity, accuracy, precision, matrix effects, and recovery. The calibration range was from 100 to 2000ng/mL for HGA and from 10 to 1000ng/mL for MCPA-carnitine. HGA and MCPA-carnitine showed acceptable accuracy and precision (bias -3.0% to 1.1%; RSD 9.2% to 12.4%). The limit of quantification (LOQ) was defined as the lowest calibrator and well below the lowest published serum concentrations in affected horses. Matrix effects ranged from -79% to +20% (RSD 4.2% to 14.4%), recoveries from 17.9% to 21.1% (RSD 2.3% to 10.8 %) for low and high quality control samples, respectively. Applicability was tested in 10 authentic AM cases. In all specimens, relevant amounts of HGA and MCPA-carnitine were found (570-2000ng/mL; ~8.5-150ng/mL, respectively). The developed assay allows reliable identification and quantification of HGA and MCPA-carnitine in horse serum and will be helpful to further study the association between HGA/MCPA and AM.

High-Resolution Liquid Chromatography Tandem Mass Spectrometry Enables Large Scale Molecular Characterization of Dissolved Organic Matter

Dissolved organic matter (DOM) is arguably one of the most complex exometabolomes on earth, and is comprised of thousands of compounds, that together contribute more than 600×1015 g carbon. This reservoir is primarily the product of interactions between the upper ocean’s microbial food web, yet abiotic processes that occur over millennia have also modified many of its molecules. The compounds within this reservoir play important roles in determining the rate and extent of element exchange between inorganic reservoirs and the marine biosphere, while also mediating microbe-microbe interactions. As such, there has been a widespread effort to characterize DOM using high-resolution analytical methods including nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). To date, molecular information in DOM has been primarily obtained through calculated molecular formulas from exact mass. This approach has the advantage of being non-targeted, accessing the inherent complexity of DOM. Molecular structures are however still elusive and the most commonly used instruments are costly. More recently, tandem mass spectrometry has been employed to more precisely identify DOM components through comparison to library mass spectra. Here we describe a data acquisition and analysis workflow that expands the repertoire of high-resolution analytical approaches available to access the complexity of DOM molecules that are amenable to electrospray ionization (ESI) MS. We couple liquid chromatographic separation, with tandem MS (LC-MS/MS) and a data analysis pipeline, that integrates peak extraction from extracted ion chromatograms (XIC), molecular formula calculation and molecular networking. This provides more precise structural characterization. Although only around 1% of detectable DOM compounds can be annotated through publicly available spectral libraries, community-wide participation in populating and annotating DOM datasets could rapidly increase the annotation rate and should be broadly encouraged. Our analysis also identifies shortcomings of the current data analysis workflow that need to be addressed by the community in the future. This work will lay the foundation for an integrative, non-targeted molecular analysis of DOM which, together with next generation sequencing, meta-proteomics and physical data, will pave the way to a more comprehensive understanding of the role of DOM in structuring marine ecosystems.

Application of high-performance liquid chromatography with charged aerosol detection (LC–CAD) for unified quantification of synthetic cannabinoids in herbal blends and comparison with quantitative NMR results

The analysis of products which contain synthetic cannabinoids (SCs) is very challenging due to their diversity and rapidly changing SC structures, variable herbal matrices and, above all, inaccessibility of reference standards. Therefore, the aim of this study was to develop a method which allows quantification of SCs’ contents in herbal blends without their reference standards. Identification of SCs was performed using liquid chromatography–high-resolution tandem mass spectrometry with a quadrupole time-of-flight analyser (LC–QTOF-MS/MS). A liquid chromatography–charged aerosol detection (LC–CAD) method with unified calibration for the quantification of SCs was developed and validated. Two available reference standards were used as universal standards. Quantitative analysis using a nuclear magnetic resonance spectroscopy method was also performed to externally validate the developed LC–CAD method. All peaks of SCs observed in LC–CAD chromatograms were identified by LC–QTOF-MS/MS. Validation data and results from a CAD response evaluation indicated that the elaborated quantitative method was sufficiently accurate for the determination of SCs belonging to various chemical families. The LC–CAD method turned out to be very flexible, because it was successfully applied for the analysis of 19 herbal products. In this study, methods which enable identification and quantification of currently known SCs as well as novel unknown derivatives without their reference standards were developed. These methods can be applied to the control of suspect SC products and may support the risk assessment of SC presence on the market. To our knowledge, this is the first trial to use LC–CAD for unified quantification of SCs without their reference standards.