Kinetic characteristics of native gamma-glutamylcysteine ligase in the aging housefly, Musca domestica L

The catalytic activity of gamma-glutamylcysteine ligase (gamma-GCL; EC 6.3.2.2) was compared between relatively young (4-day-old) and old (19-day-old) houseflies (Musca domestica) in order to understand the mechanism of putative deterioration of glutathione homeostasis during the aging process. Hanes-Woolf analyses ([S]/v vs [S]) indicated that gamma-GCL had significantly higher affinities for its substrates in the young than in the old flies. The K(m) values in the young and old flies were, respectively, for glutamate 0.6 and 5.5 mM; for cysteine 0.3 and 4.6 mM; and for ATP 1.2 and 2.9 mM. Furthermore, young but not old flies exhibited substrate-dependent inhibition of gamma-GCL activity at >5 mM cysteine indicating a loss of metabolic regulation during aging. The age-associated differences in the affinity of native gamma-GCL towards its substrates suggest that de novo synthesis of glutathione would be relatively less efficient in the old houseflies.     

Intact glucosinolate analysis in plant extracts by programmed cone voltage electrospray LC/MS: performance and comparison with LC/MS/MS methods

We present a comprehensive, sensitive, and highly specific negative ion electrospray LC/MS method for identifying all structural classes of glucosinolates in crude plant extracts. The technique is based on the observation of simultaneous maxima in the abundances of the m/z 96 and 97 ions, generated by programmed cone voltage fragmentation, in the mass chromatogram. The abundance ratios lie in the range 1:2-1:4 ([m/z 96]/[m/z 97]). Examination of the corresponding full-scan mass spectra allows individual glucosinolates of all structural classes to be identified rapidly and with confidence. The use of linearly programmed cone voltage fragmentation enhances characteristic fragment ions without compromising the abundance of the analytically important [M - H]- ion and its associated (and analytically useful) sulfur isotope peaks. Detection limits are in the low nanogram range for full-scan, programmed cone voltage spectra. Comparison of the technique with LC/MS/MS methods (product ion, precursor ion, and constant neutral loss scans) has shown that the sensitivity and selectivity of the programmed cone voltage method is superior. Data obtained on a variety of plant extracts confirmed that the methodology was robust and reliable.     

GC/MS and LC/MS Phytochemical Analysis of Vigna unguiculata L. Walp Pod

Vigna unguiculata (L. Walp) or Cowpea pod methanolic extracts phytochemical analysis, total phenolic content (TPC), and secondary metabolite profiling were determined using gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC/MS) analysis. GC/MS analysis revealed twenty compounds in the extract, while LC/MS analysis identified twenty-four compounds. GC/MS chromatogram analysis suggested the presence of opioid α-N-Normethadol a major constituent found in methanolic extract and fatty acid esters carotenoid is found second major constituent. LC/MS chromatogram and the mass spectral analysis demonstrated the presence of flavonoids, carotenoids, and alkaloids as major phytochemicals. We investigated the antibacterial, anti-fungal, and anti-oxidant activity of pod methanolic extract. The extract was found equally effective against E. coli, S. pyogenes, and P. aeruginosa with MIC 100 μg/mL similar to the standard Ampicillin (MIC 100 μg/mL). C. albicans were found to be most susceptible to Vign unguiculata pods methanolic extract with a MIC of 250 μg/mL. The pod extract showed significant DPPH scavenging activity (IC₅₀=78.38±0.15) which suggests its antioxidant potential.     

Accelerating high quality bioanalytical LC/MS/MS assays using fused-core columns

High quality, ultra-fast bioanalytical LC/MS/MS methods were developed using short columns packed with fused-core particles and high (1.0–3.0 mL/min) flow rates. For more than two years, at flow rates up to 3.0 mL/min, using 0.33 min non-ballistic gradients, these methods were shown to provide comparable or better performance than slower assays for accuracy, precision, sensitivity, specificity, and ruggedness, and met all criteria required by the bioanalytical regulatory guidance.     

无土栽培番红花的LC/MS分析

利用高效液相色谱质谱联用(LC/MS)法,分析比较了有土栽培和无土栽培番红花(Crocus sativusL.)药材的高效液相色谱指纹图谱,发现两者有一致的指纹图谱,并利用质谱作检测器(MSD)从无土栽培番红花药材中检测到了西红花苷-Ⅰ[crocin-Ⅰ,C44H64O24,分子质量(M.M.)976]、西红花苷-Ⅱ(crocin-Ⅱ,C38H54O19,M.M.814)、西红花苷-Ⅲ(crocin-Ⅲ,C32H44O14,M.M.652)、苦藏花素(picrocrocin,C16H26O7,M.M.330)、苦藏红花酸(picrocro-cinic acid,C16H26O8,M.M.346)、双葡萄糖基莰非醇(di-glucosyl-kaempferol,C27H30O16,M.M.610)以及1个西红花苷-Ⅱ的异构体(crocin-Ⅱs isomer,C38H54O19,M.M.814)。从化学成分角度,说明无土栽培技术可以用于无公害番红花药材的生产,在缺乏对照品的情况下,LC/MS法可作为检测番红花药材质量的有效方法。     

Biological monitoring of bisphenol A with HLPC/FLD and LC/MS/MS assays

Biological monitoring is a necessary process for risk assessment of endocrine disrupting chemicals (EDCs), particularly, bisphenol A (BPA), in breast milk, because its human risks are not clear yet, and infants, who feed on breast milk, are highly susceptible for EDCs. Concerning biological monitoring of BPA, the HPLC/FLD has been widely used before the LC/MS/MS. However, there was no report, which simultaneously evaluated the two methods in real analyses. Therefore, we analyzed BPA with LC/MS/MS and HPLC/FLD in human breast milk and conducted comparison of two methods in analyzed BPA levels. After establishing optimal condition, e.g. linearity, recovery, reproducibility and free BPA system, we analyzed BPA levels in human breast milk samples (N = 100). The LOQs were similar in the two methods, i.e. 1.8 and 1.3 ng/mL for the HPLC/FLD and LC/MS/MS assays, respectively. There were strong associations between total BPA levels with the two methods (R² = 0.40, p < 0.01), however, only 11% of them were analyzed as similar levels with 15% CVs. In addition, the detection range of BPA was broader in the HPLC method than the LC/MS/MS method. However, the BPA levels in the HPLC/FLD analysis were lower than those in the LC/MS/MS analysis (p < 0.01). Thus, the differences in BPA levels between the two methods may come from mainly over-estimation with the LC/MS/MS method in low BPA samples and some of poor resolution with the HPLC/FLD in high BPA samples.     

Relationship Between Phenolic Content Determined by LC/MS/MS and Antioxidant Capacity and Enzyme Inhibition of Cyclotrichium niveum L.

Cyclotrichium niveum (Boiss.) Manden & Scheng belonging to the Lamiaceae family, which is an endemic species in the eastern Anatolian region of Turkey, has an important place in terms of ethno-botany. The phytochemical composition of the plant, inhibition of acetylcholinesterase (AChE) (which hydrolyzes the neurotransmitter acetylcholine), inhibition of paraoxonase for antiatherosclerotic activity (hPON 1) (which detoxifies organophosphates), and antioxidant capacity were all investigated in this study. Phytochemical content was determined by LC/MS/MS, and enzyme inhibition and antioxidant capacity studies were determined by spectrophotometer. Antioxidant capacity of C. niveum extracts (methanol, hexane, and water) was determined by applying ABTS⋅⁺, DPPH⋅, FRAP, and CUPRAC methods. Both the water and the methanol extracts of the C. niveum exhibited significant inhibition on the AChE (IC₅₀ value for methanol and water extract 0.114±0.14 mg/mL (R2:0.997) and 0.178±0.12 mg/mL (R²: 0.994), respectively). In contrast, the methanol and water extracts of the C. niveum did not exhibit the inhibition effect on hPON 1. The highest activity for ABTS⋅⁺ was 66.53 % in the water extract, and DPPH⋅ was 55.03 % in the methanol extract. In the metal-reducing power assay, the absorbance was 0.168±0.04 for FRAP water extract and 0.621±0.01 for CUPRAC methanol extract. According to LC/MS/MS analyses, hydroxybenzoic acid, salicylic acid, syringic acid, acetohydroxamic acid and luteolin determined in the plant extract. As a consequence, C. niveum which has antioxidant, anti-atherogenic and anti-neurodegenerative properties has the potential to be used as a natural medication instead of synthetic drugs used in Alzheimer's patients.     

Determination of nicotine and cotinine in human serum by means of LC/MS

As part of a joint clinical research project to study the effects of nicotine on the brain, a HPLC electrospray ionisation mass spectrometry method with a solid-phase extraction sample preparation was developed for the quantitative determination of nicotine and cotinine in human serum in volunteers. The measured concentrations of nicotine and cotinine were used as control for smoking behaviour. A X-Bridge-column from Waters, and a SSQ 7000 single quadropole mass spectrometer with a TSP liquid chromatographic system were used. The method includes a simple and robust sample preparation and this assay has been shown to be of a sufficient sensitivity for this application. The limits of quantification were 5 and 2 ng/ml for cotinine and nicotine, respectively. A simultaneous study was conducted to measure nicotine receptor availability and the vigilance in the same group of volunteers.     

Membrane glycoproteomics of fetal lung fibroblasts using LC/MS

Some aberrant N‐glycosylations are being used as tumor markers, and glycoproteomics is expected to provide novel diagnosis markers and targets of drug developments. However, one has trouble in mass spectrometric glycoproteomics of membrane fraction because of lower intensity of glycopeptides in the existence of surfactants. Previously, we developed a glycopeptide enrichment method by acetone precipitation, and it was successfully applied to human serum glycoproteomics. In this study, we confirmed that this method is useful to remove the surfactants and applicable to membrane glycoproteomics. The glycoproteomic approach to the human fetal lung fibroblasts membrane fraction resulted in the identification of over 272 glycoforms on 63 sites of the 44 glycoproteins. According to the existing databases, the structural features on 41 sites are previously unreported. The most frequently occurring forms at N‐glycosylation site were high‐mannose type containing nine mannose residues (M9) and monosialo‐fucosylated biantennary oligosaccharides. Several unexpected N‐glycans, such as fucosylated complex‐type and fucosylated high‐mannose and/or fucosylated pauci‐mannose types were found in ER and lysosome proteins. Our method provides new insights into transport, biosynthesis, and degradation of glycoproteins.     

Method for therapeutic drug monitoring of azole antifungal drugs in human serum using LC/MS/MS

Fungal infections occur in immunocompromised patients. Azole antifungal agents are used for the prophylaxis and treatment of these infections. The interest in therapeutic drug monitoring azole agents has increased over the last few years. Inter- and intra-patient variability of pharmacokinetics, drug–drug interactions, serum concentration related toxicity and success of therapy has stressed the need of frequently therapeutic drug monitoring of the drugs, belonging to the group of azoles. Therefore a simple, rapid and flexible method of analysis is required. This method is based on the precipitation of proteins in human serum with LC/MS/MS detection. Validation was performed according to the guidelines for bioanalytical method validation of the food and drug administration agency. The four most used azole drugs can be detected in human serum within the clinical relevant serum levels with good accuracy and reproducibility at the limit of quantification. Intra- and inter-day validation demonstrated good accuracy and reproducibility. A rapid, sensitive and flexible LC/MS/MS method has been developed and validated to measure voriconazole (VRZ), fluconazole (FLZ), itraconazole (ITZ) and posaconazole (PSZ) in human serum. This new method is suitable for clinical pharmacokinetic studies and routine monitoring in daily practice.     

Achiral-chiral LC/LC-MS/MS coupling for determination of chiral discrimination effects in phenprocoumon metabolism

Many physiological processes show a high degree of stereoselectivity, including the metabolism of xenobiotics as catalyzed by cytochrome P450 enzymes. An analysis of these chiral discrimination effects in drug metabolism is essential for an in-depth understanding of metabolic pathways that differ between enantiomers of a given chiral drug or metabolite thereof. Achiral chromatographic separation and structural identification followed by chiral analysis of metabolites from blood specimens usually requires a time-consuming multistage analytical technique. In an effort to optimize such a complicated analytical scheme, a novel two-dimensional online achiral-chiral liquid chromatography-tandem mass spectrometry (LC/LC-MS/MS) coupling method was developed by using a peak parking technique in combination with a makeup flow system. Metabolites were separated in the first dimension using a C18 reversed-phase system. A makeup eluent of water/methanol (95/5) was split into the flow before storing the metabolites separately on chiral cartridges. Subsequently, the metabolite enantiomers were eluted backward onto the analytical chiral column and separated, and the ratio of enantiomers was determined. The method was successfully validated with respect to limit of detection, linearity, intra- and interday accuracy, and precision. In the course of a human volunteer study investigating the influence of CYP (cytochrome) 2C9 genetic polymorphism on phenprocoumon (PPC) metabolism, we used this new two-dimensional online analytical technique for the analysis of PPC metabolites in plasma. The enantiomeric forms of 4'-, 6-, and 7-hydroxy-PPC metabolites as well as two novel metabolites were identified, and the ratio of the enantiomers was calculated. We found that the enantiomeric ratio for the different metabolites in the plasma sample of each measured individual differs markedly from a nearly 100% chiral discrimination for the two new putative metabolites. This new analytical coupling method possesses general utility in the analysis of chiral discrimination effects, particularly as it relates to pharmacokinetics and dynamics, a scientific field that is rapidly becoming an area of concern and interest.     

Quantitative Detection of 15 Serum Bile Acid Metabolic Products by LC/MS/MS in the Diagnosis of Primary Biliary Cholangitis

To determine 15 bile acid metabolic products in human serum by liquid chromatography-tandem mass spectrometry (LC/MS/MS) and value their diagnostic outcome in primary biliary cholangitis (PBC). Serum from 20 healthy controls and 26 patients with PBC were collected and went LC/MS/MS analysis of 15 bile acid metabolic products. The test results were analyzed by bile acid metabolomics, and the potential biomarkers were screened and their diagnostic performance was judged by statistical methods such as principal component and partial least squares discriminant analysis and area under curve (AUC). 8 differential metabolites can be screened out: Deoxycholic acid (DCA), Glycine deoxycholic acid (GDCA), Lithocholic acid (LCA), Glycine ursodeoxycholic acid (GUDCA), Taurolithocholic acid (TLCA), Tauroursodeoxycholic acid (TUDCA), Taurodeoxycholic acid (TDCA), Glycine chenodeoxycholic acid (GCDCA). The performance of the biomarkers was evaluated by the AUC, specificity and sensitivity. In conclusion, DCA, GDCA, LCA, GUDCA, TLCA, TUDCA, TDCA and GCDCA were identified as eight potential biomarkers to distinguish between healthy people and PBC patients by multivariate statistical analysis, which provided reliable experimental basis for clinical practice.     

Identifying new PCR targets for pathogenic bacteria using top-down LC/MS protein discovery.

We have investigated the use of a top-down liquid chromatography/mass spectrometric (LC/MS) approach for the identification of specific protein biomarkers useful for differentiation of closely related strains of bacteria. The sequence information derived from the protein biomarker was then used to develop specific polymerase chain reaction primers useful for rapid identification of the strains. Shiga-toxigenic Escherichia coli (STEC) strains were used for this evaluation. The expressed protein profiles of two closely related serotype 0157:H7 strains, the predominant strain implicated in illness worldwide, and the nonpathogenic E. coli K-12 strain were compared with each other in an attempt to identify new protein markers that could be used to distinguish the 0157:H7 strains from each other and from the E. coli K-12 strain. Sequencing of a single protein unique to one of the 0157:H7 strains identified it as a cytolethal distending toxin, a potential virulence marker. The protein sequence information enabled the derivation of genetic sequence information for this toxin, thus allowing the development of specific polymerase chain reaction primers for its detection. In addition, the top-down LC/MS technique was able to identify other unique biomarkers and differentiate nearly identical 0157:H7 strains, which exhibited identical phenotypic, serologic, and genetic traits. The results of these studies demonstrate that this approach can be expanded to other serotypes of interest and provide a rational approach to identifying new molecular targets for detection.     

Quantification of dinor, dihydro metabolites of F2-isoprostanes in urine by liquid chromatography/tandem mass spectrometry

The F2-isoprostanes (F2-IsoP) are a series of prostaglandin (PG)-F2-like compounds that are produced by free-radical-mediated oxidation of arachidonic acid. One F2-IsoP with potent biological activity is 15-F2t-IsoP and increased levels of 15-F(2t)-IsoP have been measured in several diseases. The major urinary metabolite of 15-F2t-IsoP (8-iso-PGF(2alpha)) is 2,3-dinor-5,6-dihydro-15-F2t-IsoP (15-F2t-IsoP-M). Previously, we developed a stable isotope dilution gas chromatography/negative chemical ionization/mass spectrometry (MS) assay for 15-F2t-IsoP-M, which, while highly sensitive, required time-consuming derivatization and thin-layer chromatography purification. We now report the development of a more rapid high-performance liquid chromatography method coupled to electrospray ionization-tandem mass spectrometry (LC/MS/MS) to analyze all of the dinor,dihydro metabolites of the F2-IsoP isomers (F2-IsoP-M). The precision of this assay was +/-5.0% and the accuracy 80%. The assay remained linear over a range of 1-100 ng injected onto the LC column. Levels of F2-IsoP-M determined by the LC/MS/MS assay method significantly correlated with levels of 15-F2t-IsoP-M determined by the GC/MS assay (R = 0.77y = 67.2x-0.5). The levels of F2-IsoP-M detected in spot urines from 40 normal subjects were 38.1+/-19.1 ng/mg creatinine (mean+/-SD). This method provides an accurate and rapid assay to assess oxidative status in vivo.     

Enantioselective analysis of citalopram and demethylcitalopram in human and rat plasma by chiral LC-MS/MS: application to pharmacokinetics

Citalopram (CITA) is available as a racemic mixture and as a pure enantiomer. Its antidepressive action is related to the (+)-(S)-CITA and to the metabolite (+)-(S)-demethylcitalopram (DCITA). In the present investigation, a method for the analysis of CITA and DCITA enantiomers in human and rat plasma was developed and applied to the study of pharmacokinetics. Plasma samples (1 ml) were extracted at pH 9.0 with toluene:isoamyl alcohol (9:1, v/v). The CITA and DCITA enantiomers were analyzed by LC-MS/MS on a Chiralcel OD-R column. Recovery was higher than 70% for both enantiomers. The quantification limit was 0.1 ng/ml, and linearity was observed up to 500 ng/ml plasma for each CITA and DCITA enantiomer. The method was applied to the study of the kinetic disposition of CITA administered in a single oral dose of 20 mg to a healthy volunteer and in a single dose of 20 mg/kg (by gavage) to Wistar rats (n = 6 for each time). The results showed a higher proportion of the (-)-(R)-CITA in human and rat plasma, with S/R AUC ratios for CITA of 0.28 and 0.44, respectively. S/R AUC ratios of DCITA were 0.48 for rats and 1.04 for the healthy volunteer.     

Bioanalytical method for the simultaneous determination of d- and l-serine in human plasma by LC/MS/MS

d-Serine is an endogenous modulator of N-methyl-d-aspartate (NMDA) receptors. Plasma concentrations of d-serine and the ratio of d-serine to total serine may be used as clinically-translatable biomarkers in NMDA receptor-related disease. We developed a highly sensitive and specific method using high performance liquid chromatography tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of the d- and l-isomers of serine in human plasma. Since d- and l-serine are endogenous components, phosphate buffered saline was used as the surrogate matrix. d- and l-serine in human plasma and PBS were treated by cationic exchange solid phase extraction. d-Serine (m/z 106.1 > 60.0), l-serine (m/z 106.1 > 60.1) and dl-serine-d₃ (m/z 109.1 > 63.0) were detected using a multiple reaction monitoring. The enantiomer separation of d- and l-serine was successfully achieved without any derivatization step using tandemly-arranged and ice-cold CROWNPAK CR-I(+) columns with an isocratic mobile phase comprised of 0.3% trifluoroacetic acid in 10% acetonitrile. The standard curves were linear throughout the calibration range with 0.01–10 μg/mL (d-serine) and 0.1–100 μg/mL (l-serine), respectively. Intra-day and inter-day precision and accuracy of the quality control samples were within relative standard deviations of less than 15%. The endogenous concentrations of d- and l-serine in human plasma were 0.124–0.199 and 7.97–13.1 μg/mL, respectively.     

Development of an LC/MS/MS Method for Simultaneous Detection of 11 Polyphenols in Rat Plasma and Its Pharmacokinetic Application after Oral Administration of Coreopsis tinctoria Extract

Eleven polyphenols, classified as flavonoid glycosides, flavonoid aglycones, and phenolic acids, are important bioactive components in the capitula of Coreopsis tinctoria (CCT). Nevertheless, their full pharmacokinetic profiles have not been demonstrated simultaneously. Therefore, a liquid chromatography – tandem mass spectrometry (LC/MS/MS) method was developed in the present work and used it to study the pharmacokinetics of these 11 compounds. We performed LC/MS/MS with a gradient mobile phase composed of water containing 0.1 % formic acid and acetonitrile containing 0.1 % formic acid on a Proshell 120 SB C18 column (2.1 mm×100 mm, 2.7 μm). We achieved a good chromatographic peak shape, resolution, and mass signal response, and multiple reaction monitoring facilitated the simultaneous detection of 11 analytes. In addition, we validated the selectivity, correlation coefficient, precision, extraction recovery, matrix effects, and stability of the LC/MS/MS method to be acceptable for 11 analytes in rat plasma. Subsequently, rats were orally administered with 50 % ethanol eluent of CCT (ECCT). Nine of 11 polyphenols were absorbed quickly (except for QCD and TCA), and their plasma levels peaked within 40 min. The exposure and C_max values of flavonoid glycosides and phenolic acids were lower than those of flavonoid aglycones. This is the first report to demonstrate the pharmacokinetics of 11 polyphenols in ECCT, which may play an important role in future studies of the bioactive components of ECCT and their bioactive mechanisms.     

自旋捕捉结合液相色谱/电子自旋共振/质谱联用技术用于检测多不饱和脂肪酸过氧化过程中产生的自由基(英文)

ω-6和ω-3类多不饱和脂肪酸是两种人体所需的重要营养物质。人体内的很多生理病理过程均涉及到这些多不饱和脂肪酸,以及它们在环氧合酶(cyclooxygenase,COX)和脂氧合酶(lipoxygenase,LOX)催化下产生的过氧化代谢物。环氧合酶和脂氧合酶催化的多不饱和脂肪酸的过氧化是复杂的生化过程,会产生一系列的自由基产物。这些自由基产物又会与蛋白质、DNA和RNA结合,从而导致很多生理功能的改变。然而一直以来,缺乏合适的分析方法来有效分离和鉴定这些自由基产物,限制了人们对环氧合酶和脂氧合酶,以及多不饱和脂肪酸的过氧化在生理作用方面的研究。直到最近,才出现了对COX/LOX催化产生的活泼自由基定性和定量分析的报道。这里将对一种可以用来鉴定体外脂类过氧化产生的自由基产物的自旋捕捉-LC/ESR/MS联用技术的发展与改进过程进行综述。这种新颖的LC/ESR/MS联用技术首次使得直接检测多不饱和脂肪酸代谢产生的自由基成为可能,这对自由基的生理学作用研究是一个重大突破,为人们在多不饱和脂肪酸的生理作用以及环氧合酶和脂氧合酶催化的脂质过氧化方面的研究带来了极大便利。     

Determination of glutamate-cysteine ligase (gamma-glutamylcysteine synthetase) activity by high-performance liquid chromatography and electrochemical detection

The tripeptide glutathione (gamma-glutamylcysteinylglycine; GSH) is the predominant low molecular mass thiol in cells. The function of GSH is of considerable interest, with the molecule being implicated in numerous cellular processes in addition to being a major cellular antioxidant. The enzyme glutamate-cysteine ligase (GCL) is the rate-limiting step in GSH synthesis. The GCL assay described here is based on high-performance liquid chromatography and exploits the electrochemically active nature of gamma-glutamylcysteine (gamma-GC), the product of GCL activity. This method allows for the direct detection of gamma-GC rather than relying on derivatization of the molecule or linked assays. The sensitivity of the assay is sufficient to allow for the measurement of GCL activity in cultured cells. The specific activity of GCL in rat primary culture astrocytes was 9.7 +/- 1.7 nmol gamma-GC synthesized/min/mg protein.