Use Of Liquid Chromatography-mass Spectrometry Research Articles

Germplasm Resources and Metabolite Marker Screening of High-Flavonoid Tartary Buckwheat (Fagopyrum tataricum).

Tartary buckwheat is an annual minor cereal crop with a variety of secondary metabolites, endowing it with a high nutritional and medicinal value. Flavonoids constitute the primary compounds of Tartary buckwheat. Recently, metabolomics, as an adjunct breeding method, has been increasingly employed in crop research. This study explores the correlation between the total flavonoid content (TFC) and antioxidant capacity in 167 Tartary buckwheat varieties. Ten Tartary buckwheat varieties with significant differences in flavonoid content and antioxidant capacity were selected by cluster analysis. With the use of liquid chromatography-mass spectrometry, 58 flavonoid compounds were identified, namely, 42 flavonols, 10 flavanols, 3 flavanones, 1 isoflavone, 1 anthocyanidin, and 1 proanthocyanidin. Different samples were clearly separated by employing principal component analysis and partial least-squares discriminant analysis. Eight differential flavonoid compounds were further selected through volcano plots and variable importance in projection. Differential metabolites were highly correlated with TFC and antioxidant capacity. Finally, metabolic markers of kaempferol-3-O-hexoside, kaempferol-7-O-glucoside, and naringenin-O-hexoside were determined by the random forest model. The findings provide a basis for the selection and identification of Tartary buckwheat varieties with high flavonoid content and strong antioxidant activity.

LC-MS Approaches for Oxysterols in Various Biosamples.

Oxysterols are involved in a plethora of biological processes, including a wide variety of diseases. Therefore, monitoring oxysterols is important for obtaining a deeper understanding of their biological roles and utilizing them as, for example, biomarkers. However, oxysterols can be challenging compounds to study, as they can be very similar in chemical structure but still have distinct biological roles. In addition, oxysterols may be difficult to detect, even with advanced analytical instrumentation. We here focus on the use of liquid chromatography-mass spectrometry (LC-MS) for the analysis of oxysterols, with an additional focus on the steps needed to prepare oxysterols for LC-MS. Steps can include chemical modification of the oxysterols for improving LC-MS sensitivity and adding chemicals that can reveal if the oxysterol levels have been perturbed during preparation. We then round off with descriptions and applications of various sample preparations for different biological matrices, from blood to cells, and biosamples with emerging attention, for example, exosomes and organoids. Taken together, oxysterol analysis is highly compatible with a wide variety of biosamples, allowing for a deeper understanding of these challenging analytes.

Current State of Fluid Lipid Biomarkers for Personalized Diagnostics and Therapeutics in Schizophrenia Spectrum Disorders and Related Psychoses: A Narrative Review.

Schizophrenia spectrum disorders (SSD) are traditionally diagnosed and categorized through clinical assessment, owing to their complex heterogeneity and an insufficient understanding of their underlying pathology. However, disease progression and accurate clinical diagnosis become problematic when differentiating shared aspects amongst mental health conditions. Hence, there is a need for widely accessible biomarkers to identify and track the neurobiological and pathophysiological development of mental health conditions, including SSD. High-throughput omics applications involving the use of liquid chromatography-mass spectrometry (LC-MS) are driving a surge in biological data generation, providing systems-level insight into physiological and pathogenic conditions. Lipidomics is an emerging subset of metabolomics, largely underexplored amongst the omics systems. Lipid profiles in the brain are highly enriched with well-established functions, including maintenance, support, and signal transduction of neuronal signaling pathways, making them a prospective and exciting source of biological material for neuropsychiatric research. Importantly, changes in the lipid composition of the brain appear to extend into the periphery, as there is evidence that circulating lipid alterations correlate with alterations of psychiatric condition(s). The relative accessibility of fluid lipids offers a unique source to acquire a lipidomic “footprint” of molecular changes, which may support reliable diagnostics even at early disease stages, prediction of treatment response and monitoring of treatment success (theranostics). Here, we summarize the latest fluid lipidomics discoveries in SSD-related research, examining the latest strategies to integrate information into multi-systems overviews that generate new perspectives of SSD-related psychosis identification, development, and treatment.

Method development for the quantitative determination of captopril from Caco-2 cell monolayers by using LC-MS/MS

Aim. Caco-2 cells are a human colon epithelial cancer cell line used as a model of human intestinal absorption of drugs and other compounds. Although compounds were used in the original Caco-2 cells monolayer assays, compounds have been replaced in most laboratories by the use of liquid chromatography-mass spectrometry (LC-MS) and LC-tandem mass spectrometry (LC-MS/MS). Mass spectrometry not only eliminates the need for compounds, but permits the simultaneous measurement of multiple compounds. The measurement of multiple compounds per assay reduces the number of incubations that need to be carried out, thereby increasing the throughput of the experiments. Furthermore, LC-MS and LC-MS-MS add another dimension to Caco-2 assays by facilitating the investigation of the metabolism of compounds by Caco-2 cells. A simple, rapid LC-MS/MS method has been developed for determination of captopril from confluent Caco-2 monolayers and from aqueous solution. Materials and methods. Chromatography was achieved on Discovery C18, 50 × 2.1 mm, 5 μm column. Samples were chromatographed in a gradient mode (eluent A (acetonitrile – water – formic acid, 5 : 95 : 0.1 v/v), eluent B (acetonitrile – formic acid, 100 : 0.1 v/v)). The initial content of the eluent B is 0%, which increases linearly by 1.0 min to 100% and to 1.01 min returns to the initial 0%. The mobile phase was delivered at a flow rate of 0.4 mL/min into the mass spectrometer ESI chamber. The sample volume was 5 μl. Results. Under these conditions, captopril was eluted at 1.42 min. A linear response function was established at 2 – 200 ng/mL. The regression equation for the analysis was y =0.0187x+0.000248 with coefficient of correction (r2) = 0.9993. According to the Caco-2 test results, captopril showed low permeability. It should be noted that the recovery value is 103.20%. The within-run coefficients of variation ranged between 0.321% and 0.541%. The within-run percentages of nominal concentrations ranged between 99.13% and 101.12%. The between-run coefficients of variation ranged between 0.314% and 0.663%. The between-run percentages of nominal concentrations ranged between 99.17% and 101.03%.The assay values on both the occasions (intra- and inter-day) were found to be within the accepted limits. Conclusion. From results of analysis, it can be concluded that developed method is simple and rapid for determination of captopril from confluent Caco-2 monolayers and from aqueous solution. Acquired results demonstrate that proposed strategy can be effortlessly and advantageously applied for examination of captopril from Caco-2 cell monolayers.

Recommendations for the Use of Liquid Chromatography-Mass Spectrometry in the Clinical Laboratory: Part I. Implementation and Management

Recommendations for the Use of Liquid Chromatography-Mass Spectrometry in the Clinical Laboratory: Part I. Implementation and Management

Quantitative analysis of γ-glutamylpeptides by liquid chromatography-mass spectrometry and application for γ-glutamyltransferase assays

γ-Glutamylpeptides are largely produced via the action of γ-glutamylcysteine synthetase or γ-glutamyltransferase (GGT). GGT transfers the γ-glutamyl moiety from glutathione (GSH) and other γ-glutamyl compounds to amino acids, peptides, or water. A conventional GGT assay employs a synthetic donor substrate, which facilitates monitoring cleavage activity by means of colorimetric analyses but provides no information on the resulting γ-glutamylpeptides. In this study, we report on the use of liquid chromatography-mass spectrometry (LC-MS) to quantitatively measure the levels of 21 γ-glutamylpeptides including GSH and 45 amino acids, including Cys. Authentic compounds consisting of 17 chemically synthesized and commercially available 4 γ-glutamylpeptides were adopted as references. We applied this method to the characterization of γ-glutamylpeptides in blood plasma and livers of mice that had been treated with an overdose of acetaminophen. The established LC-MS-based assay was found to be useful for characterizing the γ-glutamylation reaction under in vivo and in vitro conditions and was clearly helpful for understanding the physiological significance of the production of γ-glutamylpeptides.

Efficient and irreversible antibody-cysteine bioconjugation using carbonylacrylic reagents.

There is considerable interest in the development of chemical methods for the precise, site-selective modification of antibodies for therapeutic applications. In this protocol, we describe a strategy for the irreversible and selective modification of cysteine residues on antibodies, using functionalized carbonylacrylic reagents. This protocol is based on a thiol-Michael-type addition of native or engineered cysteine residues to carbonylacrylic reagents equipped with functional compounds such as cytotoxic drugs. This approach is a robust alternative to the conventional maleimide technique; the reaction is irreversible and uses synthetically accessible reagents. Complete conversion to the conjugates, with improved quality and homogeneity, is often achieved using a minimal excess (typically between 5 and 10 equiv.) of the carbonylacrylic reagent. Potential applications of this method cover a broad scope of cysteine-tagged antibodies in various formats (full-length IgGs, nanobodies) for the site-selective incorporation of cytotoxic drugs without loss of antigen-binding affinity. Both the synthesis of the carbonylacrylic reagent armed with a synthetic molecule of interest and the subsequent preparation of the chemically defined, homogeneous antibody conjugate can be achieved within 48 h and can be easily performed by nonspecialists. Importantly, the conjugates formed are stable in human plasma. The use of liquid chromatography-mass spectrometry (LC-MS) analysis is recommended for monitoring the progression of the bioconjugation reactions on protein and antibody substrates with accurate resolution.

Metabolic fingerprinting of carp and rainbow trout seminal plasma

Rainbow trout and common carp are commercially important aquaculture fish species. To the best of our knowledge, the global composition of fish seminal plasma (SP) metabolites has never been studied. The aim of this study was to identify metabolites in carp and rainbow trout SP and to compare their relative abundances through the use of liquid chromatography-mass spectrometry (LC-MS)-based metabolic fingerprinting. Different solvents and sample/solvent ratios were tested for metabolite extraction. The largest number of metabolic features and the highest signal reproducibility were achieved using a 1:1 sample/acetone ratio for extraction. Accurate masses were searched against metabolite databases, and the identities of 48 and 40 metabolites for rainbow trout and carp SP, respectively, were confirmed using LC-MS/MS or through the standards. Most of the identified metabolites have not been previously described in fish SP. Several metabolites (amino acids, acylcarnitines, vitamins) were found in the SP of both species. However, differences between carp and rainbow trout SP metabolic fingerprints were observed. Certain metabolites appeared to be species-specific, e.g., propionic acid for carp and adipic and picolinic acids, homocitrulline and niacin for rainbow trout. Functional analysis revealed the involvement of SP metabolites in free radical scavenging, lipid and energy metabolism, molecular transport, cell death and survival, and inflammatory response. The identification of a high number of carp and rainbow trout seminal plasma metabolites provides new opportunities to develop novel biomarkers for sperm quality, optimise artificial reproduction and develop efficient cryopreservation procedures for these important aquaculture species.

The penetration and distribution of topical atropine in animal ocular tissues.

To conduct a multi-tissue investigation on the penetration and distribution of topical atropine in myopia treatment, and determine if atropine is detectable in the untreated contralateral eye after uniocular instillation. Nine mature New Zealand white rabbits were evenly divided into three groups. Each group was killed at 5, 24 and 72hr, respectively, following uniocular instillation of 0.05ml of 1% atropine. Tissues were sampled after enucleation: conjunctiva, sclera, cornea, iris, ciliary body, lens, retina, aqueous, and vitreous humors. The assay for atropine was performed using liquid chromatography-mass spectrometry (LC-MS), and molecular tissue distribution was illustrated using matrix-assisted laser desorption ionization-imaging mass spectrometry (MALDI-IMS) via an independent experiment on murine eyes. At 5hr, the highest (mean±SEM) concentration of atropine was detected in the conjunctiva (19.05±5.57ng/mg, p<0.05) with a concentration gradient established anteriorly to posteriorly, as supported by MALDI-IMS. At 24hr, preferential binding of atropine to posterior ocular tissues occurred, demonstrating a reversal of the initial concentration gradient. Atropine has good ocular bioavailability with concentrations of two magnitudes higher than its binding affinity in most tissues at 3days. Crossing-over of atropine to the untreated eye occurred within 5hr post-administration. Both transcorneal and transconjunctival-scleral routes are key in atropine absorption. Posterior ocular tissues could be important sites of action by atropine in myopic reduction. In uniocular atropine trials, cross-over effects on the placebo eye should be adjusted to enhance results reliability. Combining the use of LC-MS and MALDI-IMS can be a viable approach in the study of the ocular pharmacokinetics of atropine.

An improved method for fast and selective separation of carotenoids by LC-MS.

Carotenoids are a large class of compounds that are biosynthesized by condensation of isoprene units in plants, fungi, bacteria, and some animals. They are characteristically highly conjugated through double bonds, which lead to many isomers as well susceptibility to oxidation and other chemical modifications. Carotenoids are important because of their potent antioxidant activity and are the pigments responsible for color in a wide variety of foods. Human consumption is correlated to many health benefits including prevention of cancer, cardiovascular disease, and age-related disease. Extreme hydrophobicity, poor stability, and low concentration in biological samples make these compounds difficult to analyze and difficult to develop analytical methods for aimed towards identification and quantification. Examples in the literature frequently report the use of exotic stationary phases, solvents, and additives, such as ethyl acetate, dichloromethane, and methyl tert-butyl ether that are incompatible with liquid chromatography mass spectrometry (LC-MS). In order to address these issues, we implemented the use of LC-MS friendly conditions using a low-hydrophobicity cyano-propyl column (Agilent Zorbax SB-CN). We successfully differentiated between isomeric carotenoids by optimizing two gradient methods and using a mixture of 11 standards and LC-MS in positive ionization mode. Three complex biological samples from strawberry leaf, chicken feed supplement, and the photosynthetic bacterium Chloroflexus aurantiacus were analyzed and several carotenoids were resolved in these diverse backgrounds. Our results show this methodology is a significant improvement over other alternatives for analyzing carotenoids because of its ease of use, rapid analysis time, high selectivity, and, most importantly, its compatibility with typical LC-MS conditions.

Agricultural potential of rhizospheric Bacillus subtilis strains exhibiting varied efficiency of surfactin production

Six Bacillus subtilis strains collected from broad-leaved trees rhizosphere and exhibiting an ability of surfactin production were investigated as potent plant biofertilizers with biocontrol activity. Lipopeptide (LP) biosurfactants were extracted by a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) procedure proposed as a quick and effective method of isolating LP biosurfactants. Matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF) was applied to detect the lipopeptides. Predominant peaks at m/z 1016, 1030, 1044, 1058 and 1072 were putatively assigned as surfactin Na adducts possess a Glu-Leu-Leu-Val-Asp-Leu–Leu peptide and the β-OH fatty acid with a different length (C13-C16) whereas m/z 1016 was C13 surfactin Na adduct with Val in position 7 on the peptide moiety. Four other ions m/z 1051, 1065, 1079 and 1093 were recognized as iturin A Na adducts. Five fengycin homologues with Na adducts (m/z 1471, 1485, 1499, 1513 and 1527) were also determined. The quantitative analyses of the compounds were performed with the use of liquid chromatography-mass spectrometry (LC–MS/MS). Strains IM 7, IM 16 and IM 17 cultivated in Luria-Bertani medium for 48h produced surfactin alone at the concentrations below 10mgl−1. In contrast, strains KP7, IM 13 and IM 14 produced surfactin much more efficiently. Surfactin concentrations in 72-h-old cultures reached 100.3, 55.3 and 124.4mgl−1, respectively and were accompanied by the production of iturin in concentrations 5.7, 142.5 and 2.3mgl−1, respectively. Additionally, in Cooper’s medium, strain KP7 increased twofold the production of surfactin and iturin. Only surfactin overproducers (KP7, IM 13 and IM 14) exhibited swarming motility and formed biofilms, limited the growth of Botrytis cinerea and Aspergillus flavus strains, produced cellulases and inulinases and enhanced cucumber seed germination in vitro. All examined surfactin overproducers produced siderophores and, additionally, KP7 and IM 14 solubilized phosphates. Among all examined surfactin overproducers only IM 13 during the growth in the presence of F. oxysporum mycelium (in dual liquid culture) increased about twofold surfactin and iturin secretion and almost completely suppressed fungal biomass growth. The obtained results revealed that the studied surfactin overproducers were equipped with various plant growth-promoting (PGP) traits allowing them to promote plant development in a diverse manner.

Glycyrrhizin and glycyrrhetinic acid inhibits alpha-naphthyl isothiocyanate-induced liver injury and bile acid cycle disruption.

Alpha-naphthyl isothiocyanate (ANIT) is a common hepatotoxicant experimentally used to reproduce the pathologies of drug-induced liver injury in humans, but the mechanism of its toxicity remains unclear. To determine the metabolic alterations following ANIT exposure, metabolomic analyses was performed by use of liquid chromatography-mass spectrometry. Partial least squares discriminant analysis (PLS-DA) of liver, serum, bile, ileum, and cecum of vehicle- and ANIT-treated mice revealed significant alterations of individual bile acids, including increased tauroursodeoxycholic acid, taurohydrodeoxycholic acid, taurochenodeoxycholic acid, and taurodeoxycholic acid, and decreased ω-, β- and tauro-α/β- murideoxycholic acid, cholic acid, and taurocholic acid in the ANIT-treated groups. In accordance with these changes, ANIT treatment altered the expression of mRNAs encoded by genes responsible for the metabolism and transport of bile acids and cholesterol. Pre-treatment of glycyrrhizin (GL) and glycyrrhetinic acid (GA) prevented ANIT-induced liver damage and reversed the alteration of bile acid metabolites and Cyp7a1, Npc1l1, Mttp, and Acat2 mRNAs encoding bile acid transport and metabolism proteins. These results suggested that GL/GA could prevent drug-induced liver injury and ensuing disruption of bile acid metabolism in humans.

Stretch-Activated Paracrine/Autocrine Signalling in the Heart: A Role for Prostaglandins?

It is well known that stretch increases the hearts contractility, although the cellular mechanisms that produce the stretch response are not fully understood. Recently, using a unique bioassay system, we showed unequivocally that stretching the left ventricle of isolated perfused rat hearts released paracrine/autocrine factors [[1]Ward M.-L. Shen X. Greenwood D.R. Use of liquid chromatography-mass spectrometry (LC-MS) to detect substances of nanomolar concentration in the coronary effluent of isolated perfused hearts.Prog Biophys Mol Biol. 2014 Jul 24; Google Scholar]. Preliminary analysis of our data using a metabolomics approach suggested these factors were prostanoids, constituting a set of signalling pathways not previously associated with regulation of the heart. Quantitative and statistical analyses using differential LCMS/MS of the coronary effluent collected from un-stretched and stretched hearts identified prostaglandins F2α and E2 as significantly different between stretched and un-stretched samples (P < 0.01). Exogenous PGF2α and PGE2 applied to isolated trabeculae from rat hearts elicited a positively inotropic response from PGF2α, but not PGE2. PGF2ɑ, over a concentration range of 1 nM - 1 μM, increased both the Ca2+ transients and isometric stress in trabeculae, reaching steady-state after 10 - 15 min, without altering the time course of Ca2+ transient decay. The positive inotropic effect of PGF2α was mediated through a protein kinase C (PKC) signalling pathway that involved activation of the sarcolemmal Na+/H+ exchanger. These data suggest PGF2α is important in maintaining homeostasis during volume loading in healthy hearts. However, under pathophysiological conditions, sustained release of PGF2α might be important in that activation of detrimental hypertrophic pathways.

Use of LC-MS and GC-MS Methods to Measure Emerging Contaminants Pharmaceutical and Personal Care Products (PPCPs) in Fish

Aquatic ecosystems are continuously contaminated by manufactured pharmaceutical and personal care products (PPCPs). Non-regulated, multi-purpose PPCP contaminants enter aquatic systems through sewage/ wastewater treatment plants after consumption and use by humans and animals. These micro-pollutants receive increased attention worldwidesince significant levels of contamination have been found in various environmental compartments and organisms. Highly sophisticated equipment such as liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) are reliable ways to determine PPCPs at sub-ppb levels from air, waters, sediments, effluents, aquatic organisms and human body fluids. Although the consequences of these pollutants are gradually becoming visible, their potential impacts on aquatic ecosystems and organisms are poorly known. Some studies have suggested that PPCPs are persistent and have bioaccumulation potential leading to ecological effects and abnormalities in fish. Other findings illustrate that PPCPs can impair swimming behavior in fathead minnow (Pimephales promelas), interfere with thyroid axis in the zebra fish (Danio rerio), or form adducts with hemoglobin and/or suitable protein breakdown PPCPs products. Thus, this review focuses on PPCPs emerging contaminants concern with regards to sources, occurrences, analytical methods, fate and biological transformation.

Replacement of “Amide Chemistry” with “Click Chemistry”: Current Trend in Proteomics

In the past decades of proteomics research, amide chemistry played a vital and indispensable role. Presence of amino and carboxylic groups in proteins, availability of respective amide/carboxyl and amine labeled ligands and mild reaction environment for corresponding amide chemistry have fulfilled the requirement of advanced proteomics research. We have used the amide chemistry for enantioseparation of biologically important amino acids in recent years [1-3]. But in recent years amide chemistry has lost its importance to quite an extent due to its one drawback: on account of ample presence of amino and carboxylic groups it has become kind of “non-selective” reaction. The use of liquid chromatography-mass spectrometry and its variations in proteomics research [4,5] has widen the gap of conventional amide chemistry protocol and current click-chemistry protocol.

1409The Use of Liquid Chromatography-Mass Spectrometry (LC-MS) for the Diagnosis of Community-Acquired Pneumonia

1409The Use of Liquid Chromatography-Mass Spectrometry (LC-MS) for the Diagnosis of Community-Acquired Pneumonia

Use of liquid chromatography-mass spectrometry (LC-MS) to detect substances of nanomolar concentration in the coronary effluent of isolated perfused hearts

When the heart is stretched there is an abrupt increase in force that coincides with the stretch that is then followed by a second phase that develops slowly over a period of minutes, known as the “slow force response”, or “SFR”. The SFR is accompanied by a slow increase in the Ca2+ transients, but the underlying cellular mechanisms are still debated. We therefore devised a simple bioassay system to investigate whether stretch released autocrine/paracrine factors that could explain the stretch-dependent Ca2+ influx during the SFR. Our analysis was based around a metabolomics approach using tandem mass spectrometry whereby secreted compounds were analysed by liquid chromatography mass spectrometry (LC-MS/MS) at high resolution and accurate mass.Our bioassay consisted of two isolated cardiac preparations in series. A sustained stretch was applied to an “upstream” Langendorff-perfused heart, whilst monitoring the performance of an un-stretched, “downstream” trabecula perfused with coronary effluent collected from the “upstream” heart. Switching from “un-stretched” to “stretched” coronary effluent increased the amplitude of the Ca2+ transients (340/380) fura-2 ratio) in the “downstream” trabeculae from 0.87 ± 0.04 to 0.94 ± 0.05 (p < 0.05, n = 4), and active stress (mN mm−2) from 15.4 ± 1.5 to 19.2 ± 1.9 (p < 0.01, n = 13), confirming the stretch-dependent release of one or more positively inotropic factor(s). To identify the factor, coronary effluent samples were concentrated by Solid Phase Extraction (SPE) treatment, lyophilised, then analysed using a Thermo Finnigan LTQ FT-ICR hybrid mass spectrometer. Differential profile data was obtained initially followed by targeting specific compounds at elevated levels in the stretched coronary effluent samples. Quantitative and statistical analyses of the differential LC-MS/MS results were then carried out using SIEVE 2.1, statistical software capable of performing differential analysis of peptides and small molecules. A number of substances were identified that were different between stretched and unstretched effluents (p < 0.01).

Glycan analysis: scope and limitations of different techniques—a case for integrated use of LC-MS(/MS) and NMR techniques

The structure of glycans from glycoproteins is highly relevant for their function. We tightly integrate liquid chromatography-mass spectrometry (LC-MS), MS/MS, and nuclear magnetic resonance (NMR) data to achieve a complete characterization of even isobaric glycans differing in only one linkage position or in the substitution in one branch. As example, we analyzed ten desialylated underivatized glycans from bovine fibrinogen. The molecules were separated on a PGC column, and LC-MS data allowed an assignment of the compositions of the glycans. MS/MS data of the same glycans allowed elucidation of sequence and to some extent of branching and linkage. All MS/MS fragmentation methods led to multiple dissociations, resulting in several cases in ambiguous data. The MS/MS data were interpreted both by scientists and automatically by software, and the differential results are compared. Additional data from a tight integration of LC-MS and NMR data resulted in a complete structural characterization of the glycans. The acquisition of simple 1D (1)H NMR data led--in combination with LC-MS and MS/MS data--to an unambiguous assignment of the isobaric glycans. Compounds that were not separated in the chromatography could easily be assigned structurally by applying the 3D cross-correlation (3DCC) technology to arrive at NMR spectra of the pure components-without actually separating them. By applying LC-MS, MS/MS, 1D (1)H NMR, and 3DCC together, one can assign glycan structures from glycoconjugates with high confidence affording only 200 pmol of glycan material.

Muscle fiber typing by identification of the myosin heavy chain using LCMS

Identifying skeletal muscle fiber types is valuable to the ongoing research efforts to understand the relationships between structure and function, particularly in humans. While existing methods of fiber typing (gel electrophoresis, histochemistry, etc.) are effective in identifying fiber types, liquid chromatography‐mass spectrometry (LCMS) has the potential for also monitoring many other proteins or metabolites simultaneously. A new method is being developed using the technology of LCMS to match peptide sequences with myosin heavy chain isoforms. Samples of chicken were digested by the enzyme trypsin, followed by analysis using reversed phase LCMS. Detected peptides were mapped to their respective myosin chains based on a theoretical digest list. Using single ion monitoring (SIM), the relative amounts of each peptide were quantified by numerical integration. A blind sample was analyzed and correctly identified. The research seemed to suggest that the use of LCMS for muscle fiber peptide quantification could be a viable alternative to currently used methods. Future research plans are for the study of human muscle samples.

Pharmacokinetics of gabapentin in cats

To determine the pharmacokinetics of gabapentin in cats after IV and oral administration. 6 healthy female adult domestic shorthair cats. Gabapentin was administered IV (4 mg/kg) or orally (10 mg/kg) in a crossover randomized design. Blood samples were obtained immediately before gabapentin administration and at various times up to 960 minutes after IV administration or up to 1,440 minutes after oral administration. Blood samples were immediately transferred to tubes that contained EDTA and were centrifuged at 4 degrees C. Plasma was harvested and stored at -20 degrees C until analysis. Plasma concentrations of gabapentin were determined by use of liquid chromatography-mass spectrometry. Gabapentin concentration-time data were fit to compartment models. A 3-compartment model with elimination from the central compartment best described the disposition of gabapentin administered IV to cats, but a 1-compartment model best described the disposition of gabapentin administered orally to cats. After IV administration, the mean +/- SEM apparent volume of the central compartment, apparent volume of distribution at steady state, and clearance and the harmonic mean +/- jackknife pseudo-SD for terminal half-life were 90.4 +/- 11.3 mL/kg, 650 +/- 14 mL/kg, 3 +/- 0.2 mL/min/kg, and 170 +/- 21 minutes, respectively. Mean +/- SD systemic availability and harmonic mean +/- jackknife pseudo-SD terminal half-life after oral administration were 88.7 +/- 11.1% and 177 +/- 25 minutes, respectively. The disposition of gabapentin in cats was characterized by a small volume of distribution and a low clearance.