Glucosinolates and amines in Reseda media

The content of glucosinolates and amines in green parts of Reseda media has been investigated. Benzylglucosinolate, 2-phenethylglucosinolate, and m-hydroxybenzylglucosinolate occur in appreciable amounts accompanied by minor amounts of other glucosinolates, benzylamine and m-hydroxybenzylamine. Isolation and identification of these compounds was made using ion-exchange chromatography, high voltage electrophoresis, GC, MS, and ¹³C-NMR spectroscopy. The glucosinolates were transformed into corresponding nitriles and isothiocyanates by thioglucoside glucohydrolase-catalysed hydrolysis and to the corresponding carboxylic acids by acid-catalysed hydrolysis. The content of glucosinolates and amines in leaves and inflorescences of R. media has been determined by UV-spectroscopy and GC.     

A liquid chromatography-electrospray ionization tandem mass spectrometric assay for quantitation of the histone deacetylase inhibitor, vorinostat (suberoylanilide hydroxamicacid, SAHA), and its metabolites in human serum

Vorinostat (suberoylanilide hydroxamic acid, SAHA) is undergoing evaluation as an antineoplastic agent. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for quantitating vorinostat and its major metabolites, vorinostat glucuronide and 4-anilino-4-oxobutanoic acid, in human serum. The assay uses: deuterated internal standards; acetonitrile protein precipitation; a BDS Hypersil C18 (3 microm, 100 mm x 3 mm) column; a gradient mobile phase of 0.5% acetic acid in acetonitrile and water; and electrospray positive-mode ionization with selected reaction monitoring (SRM) detection. The lower limit of quantitation was 3.0 ng/ml for each analyte. The assay is being employed in at least 12 clinical studies of vorinostat-containing regimens.     

Rapid and sensitive liquid chromatography-tandem mass spectrometry: assay development, validation and application to a human pharmacokinetic study

Rasagiline is a highly potent, selective and irreversible second-generation monoamine oxidase inhibitor with selectivity for type B of the enzyme (MAO-B). The present studies aimed at developing and validating a rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for determination of rasagiline in human plasma and urine. LC-MS/MS analysis was carried out on a Finnigan LC-TSQ Quantum mass spectrometer using positive ion electrospray ionization (ESI(+)) and selected reaction monitoring (SRM). The assay for rasagiline was linear over the range of 0.01-40 ng/mL in plasma and 0.025-40 ng/mL in urine. It took 5.5 min to analyze a sample. The average recoveries in plasma and urine samples were both >85%. The RSD of precision and bias of accuracy were less than 15% and 10%, respectively, of their nominal values based on the intra- and inter-day analysis. The developed method was proved to be suitable for use in clinical pharmacokinetic study after single oral administration of 0.5, 1 and 2 mg rasagiline mesylate tablets in healthy Chinese volunteers.     

The quantitative analysis of heroin, methadone and their metabolites and the simultaneous detection of cocaine, acetylcodeine and their metabolites in human plasma by high-performance liquid chromatography coupled with tandem mass spectrometry

For a pharmacokinetic-pharmacodynamic study in opioid tolerant patients, who were treated with heroin in combination with methadone, a liquid chromatographic assay with tandem mass spectrometry detection (LC-MS/MS) was developed for the simultaneous determination of heroin, methadone, heroin metabolites 6-monoacetylmorphine, morphine, and morphine-6 and 3-glucuronide and methadone metabolite EMDP. To detect any abuse of substances besides the prescribed opioids the assay was extended with the detection of cocaine, its metabolites benzoylecgonine and norcocaine and illicit heroin adulterants acetylcodeine and codeine. Heroin-d6, morphine-d3, morphine-3-glucuronide-d3 and methadone-d9 were used as internal standards. The sample pre-treatment consisted of solid phase extraction using mixed mode sorbent columns (MCX Oasis). Chromatographic separation was performed at 25 degrees C on a reversed phase Zorbax column with a gradient mobile phase consisting of ammonium formate (pH 4.0) and acetonitrile. The run time was 15 min. MS with relatively mild electrospray ionisation under atmospheric pressure was applied. The triple quadrupole MS was operating in the positive ion mode and multiple reaction monitoring (MRM) was used for drug quantification. The method was validated over a concentration range of 5-500 ng/mL for all analytes. The total recovery of heroin varied between 86 and 96% and of the heroin metabolites between 76 and 101%. Intra-assay and inter-assay accuracy and precision of all analytes were always within the designated limits (< or =20% at lower limit of quantification (LLQ) and < or =15% for other samples). This specific and sensitive assay was successfully applied in pharmacokinetic studies with medically prescribed heroin and toxicological cases.     

Quantitative analysis of a synthetic peptide, NR58-3.14.3, in serum by LC-MS with inclusion of a diastereomer as internal standard

A method for quantifying an intramolecularly linked all-d-amino acid peptide, NR58-3.14.3, in rat serum by LC-MS using selected ion monitoring with inclusion of a diastereomer as internal standard was developed. The reproducible quantitation of multiply charged compounds by LC-MS using single ion or selective reaction monitoring is often a challenge as the intensity ratio of the ions in a series of different charge states can vary. Good precision was obtained in the selected ion monitoring mode by integrating the summed ion currents of the singly, doubly, and triply charged molecular ions. Since stable isotope analogs are costly and integration of residual unlabeled material can be of concern, a diastereomer of NR58-3.14.3, NR58-3.14.5, was used as internal standard. The diastereomers were indistinguishable by electrospray MS, but fully separated by reversed-phase LC. Consequently, interference due to isotopic impurities or coelution was not encountered. The calibration plot was linear throughout a concentration range of 0.2 to 200.0 microg/ml (r(2) = 0.9996). Intraday precision of the standards analyzed was less than 12% RSD over the calibration range and the accuracy within +/-11% RE. Serum pharmacokinetics were in good agreement with the pharmacokinetic profiles of small, ionic, and polar molecules.     

A sample extraction and chromatographic strategy for increasing LC/MS detection coverage of the erythrocyte metabolome

Reproducible and comprehensive sample extraction and detection of metabolites with a broad range of physico-chemical properties from biological matrices can be a highly challenging process. A single LC/MS separation method was developed for a 2.1mmx100mm, 1.8mum ZORBAX SB-Aq column that was used to separate human erythrocyte metabolites extracted under sample extraction solvent conditions where the pH was neutral or had been adjusted to either, pH 2, 6 or 9. Internal standards were included and evaluated for tracking sample extraction efficiency. Through the combination of electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) techniques in both positive (+) and negative (-) ion modes, a total of 2370 features (compounds and associated compound related components: isotopes, adducts and dimers) were detected across all pHs. Broader coverage of the detected metabolome was achieved by observing that (1) performing extractions at pH 2 and 9, leads to a combined 92% increase in detected features over pH 7 alone; and (2) including APCI in the analysis results in a 34% increase in detected features, across all pHs, than the total number detected by ESI. A significant dependency of extraction solvent pH on the recovery of heme and other compounds was observed in erythrocytes and underscores the need for a comprehensive sample extraction strategy and LC/MS analysis in metabolomics profiling experiments.     

LC-MS metabolic profiling of Arabidopsis thaliana plant leaves and cell cultures: optimization of pre-LC-MS procedure parameters

This study treats the optimization of methods for homogenizing Arabidopsis thaliana plant leaves as well as cell cultures, and extracting their metabolites for metabolomics analysis by conventional liquid chromatography electrospray ionization mass spectrometry (LC-ESI/MS). Absolute recovery, process efficiency and procedure repeatability have been compared between different pre-LC-MS homogenization/extraction procedures through the use of samples fortified before extraction with a range of representative metabolites. Hereby, the magnitude of the matrix effect observed in the ensuing LC-MS based metabolomics analysis was evaluated. Based on relative recovery and repeatability of key metabolites, comprehensiveness of extraction (number of m/z-retention time pairs) and clean-up potential of the approach (minimum matrix effects), the most appropriate sample pre-treatment was adopted. It combines liquid nitrogen homogenization for plant leaves with thermomixer based extraction using MeOH/H(2)O 80/20. As such, an efficient and highly reproducible LC-MS plant metabolomics set-up is achieved, as illustrated by the obtained results for both LC-MS (8.88%+/-5.16 versus 7.05%+/-4.45) and technical variability (12.53%+/-11.21 versus 9.31%+/-6.65) data in a comparative investigation of A. thaliana plant leaves and cell cultures, respectively.     

Detection of new exemestane metabolites by liquid chromatography interfaced to electrospray-tandem mass spectrometry

Exemestane is an irreversible aromatase inhibitor used for anticancer therapy. Unfortunately, this drug is also misused in sports to avoid some adverse effects caused by steroids administration. For this reason exemestane has been included in World Anti-Doping Agency prohibited list. Usually, doping control laboratories monitor prohibited substances through their metabolites, because parent compounds are readily metabolized. Thus metabolism studies of these substances are very important. Metabolism of exemestane in humans is not clearly reported and this drug is detected indirectly through analysis of its only known metabolite: 17β-hydroxyexemestane using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and gas chromatography coupled to mass spectrometry (GC-MS). This drug is extensively metabolized to several unknown oxidized metabolites. For this purpose LC-MS/MS has been used to propose new urinary exemestane metabolites, mainly oxidized in C6-exomethylene and simultaneously reduced in 17-keto group. Urine samples from four volunteers obtained after administration of a 25mg dose of exemestane were analyzed separately by LC-MS/MS. Urine samples of each volunteer were hydrolyzed followed by liquid-liquid extraction and injected into a LC-MS/MS system. Three unreported metabolites were detected in all urine samples by LC-MS/MS. The postulated structures of the detected metabolites were based on molecular formulae composition obtained through high accuracy mass determination by liquid chromatography coupled to hybrid quadrupole-time of flight mass spectrometry (LC-QTOF MS) (all mass errors below 2ppm), electrospray (ESI) product ion spectra and chromatographic behavior.     

Identification of the major metabolites of resveratrol in rat urine by HPLC-MS/MS

To identify the major metabolites of resveratrol in rat, rat urine samples were pretreated by using solid-phase extraction technique (SPE) with polyamide cartridges. And a LC-MS/MS method with electrospray ionisation (ESI), negative ion mode and collision induced dissociation (CID), was used to elucidate the structures of the major metabolites of resveratrol. According to the results of our experiment, we found that the main metabolites of resveratrol were resveratrol monoglucuronide (M1), dihydroresveratrol monosulfate (M2), resveratrol monosulfate (M3) and dihydroresveratrol (M4).     

Determination of esomeprazole and its two main metabolites in human, rat and dog plasma by liquid chromatography with tandem mass spectrometry

A LC-MS/MS method was developed for quantitative determination of esomeprazole, and its two main metabolites 5-hydroxyesomeprazole and omeprazole sulphone in 25 microL human, rat or dog plasma. The analytes and their internal standards were extracted from plasma into methyl tert-butyl ether - dichloromethane (3:2, v/v). After evaporation and reconstitution of the organic extract the analytes were separated on a reversed-phase LC column and measured by atmospheric-pressure positive ionisation MS. The linearity range was 20-20,000 nmol/L for esomeprazole and omeprazole sulphone, and 20-4000 nmol/L for 5-hydroxyesomeprazole. The extraction recoveries ranged between 80 and 105%. The intra- and inter-day imprecision were less than 9.5% with accuracy between 97.7% and 100.1% for all analytes.     

Glucuronidation, oxidative metabolism, and bioactivation of enterolactone in rhesus monkeys

Enterolactone (ENL) and enterodiol (END) are mammalian lignans derived from the plant lignans matairesionol (MAT), secoisolariciresinol (SECO), and other dietary precursors. ENL was found to undergo extensive glucuronidation with rhesus liver microsomes to form O-glucuronides at both phenolic hydroxy groups. In addition to glucuronidation, ENL was found to be a good substrate for oxidative metabolism. The major products had a m/z of 313 or 295 by LC-MS analysis in negative ion mode and were determined to be products of monohydroxylation of ENL. The m/z 295 products were the result of a dehydration of the m/z 313 in the MS source. Addition of N-acetylcysteine (NAC) to the NADPH incubations resulted in a decrease of at least 2 major monohydroxylated products and the formation of a major and several minor new products with a m/z of 474. The major adduct was isolated, purified for NMR, and confirmed to be the NAC adduct of the ENL catechol. Incubations of ENL with liver microsomes containing UDPGA, NADPH, and NAC resulted in the formation of ENL-glucuronides; no NAC adducts were detected by LC-MS. Incubations of ENL with human and rhesus hepatocytes resulted in several metabolites. The major metabolites in hepatocytes were the glucuronic acid conjugates; minor amounts of the sulfate conjugate(s) and monohydroxylated products were also detected by LC-MS. Glutathione or other thiol adducts were not detected in hepatocytes. Conclusion. The high efficiency and specificity for the glucuronidation of ENL decrease its potential toxicity via CYP450 bioactivation.     

The antimicrobial effects of glucosinolates and their respective enzymatic hydrolysis products on bacteria isolated from the human intestinal tract

Aims:  The aim of the study was to evaluate the in vitro antibacterial activity of glucosinolates and their enzymatic hydrolysis product against bacteria isolated from the human intestinal tract.
Methods and results:  Using a disc diffusion bioassay, different doses of intact glucosinolates and their corresponding hydrolysis products were tested. There were clear structure–activity and concentration differences with respect to the in vitro growth inhibition effects as well as differences in the sensitivities of the individual bacteria. The most effective glucosinolate hydrolysis products were the isothiocyanates; sulforaphane and benzyl isothiocyanate were the best inhibitors of growth. Indole-3-carbinol had some inhibitory effects against the Gram-positive bacteria but had no effect, even at the highest dose, against the Gram-negative bacteria. Indole-3-acetonitrile had some inhibitory activity against the Gram-negative bacteria. Glucosinolates, nitriles and amines were ineffective at all the doses used.
Conclusions:  Glucosinolate hydrolysis products and specifically the isothiocyanates SFN and BITC have significant antimicrobial activity against Gram-positive and Gram-negative bacteria, and might be useful in controlling human pathogens through the diet.
Significance and Impact of the Study:  This the first major in vitro study demonstrating the potential of these natural dietary chemicals as an alternative to, or in combination with, current antibiotic-based therapies for treating infectious diseases.     

Quantification of plant surface metabolites by matrix‐assisted laser desorption–ionization mass spectrometry imaging: glucosinolates on Arabidopsis thaliana leaves

The localization of metabolites on plant surfaces has been problematic because of the limitations of current methodologies. Attempts to localize glucosinolates, the sulfur‐rich defense compounds of the order Brassicales, on leaf surfaces have given many contradictory results depending on the method employed. Here we developed a matrix‐assisted laser desorption–ionization (MALDI) mass spectrometry protocol to detect surface glucosinolates on Arabidopsis thaliana leaves by applying the MALDI matrix through sublimation. Quantification was accomplished by spotting glucosinolate standards directly on the leaf surface. The A. thaliana leaf surface was found to contain approximately 15 nmol of total glucosinolate per leaf with about 50 pmol mm^?2 on abaxial (bottom) surfaces and 15–30 times less on adaxial (top) surfaces. Of the major compounds detected, 4‐methylsulfinylbutylglucosinolate, indol‐3‐ylmethylglucosinolate, and 8‐methylsulfinyloctylglucosinolate were also major components of the leaf interior, but the second most abundant glucosinolate on the surface, 4‐methylthiobutylglucosinolate, was only a trace component of the interior. Distribution on the surface was relatively uniform in contrast to the interior, where glucosinolates were distributed more abundantly in the midrib and periphery than the rest of the leaf. These results were confirmed by two other mass spectrometry‐based techniques, laser ablation electrospray ionization and liquid extraction surface analysis. The concentrations of glucosinolates on A. thaliana leaf surfaces were found to be sufficient to attract the specialist feeding lepidopterans Plutella xylostella and Pieris rapae for oviposition. The methods employed here should be easily applied to other plant species and metabolites.     

Simultaneous determination of sulforaphane and its major metabolites from biological matrices with liquid chromatography-tandem mass spectroscopy

A simple, sensitive and specific LC-MS/MS method for the simultaneous determination of sulforaphane (SFN) and its major metabolites, the glutathione (SFN-GSH) and N-acetyl cysteine conjugates (SFN-NAC) from biological matrices was developed and validated. The assay procedure involved solid-phase extratcion of all three analytes from rat intestinal perfusate using C2 extraction cartridges, whereas from rat plasma, metabolites were extracted by solid-phase extraction and SFN was extracted by liquid-liquid extraction with ethyl acetate. Chromatographic separation of SFN, SFN-GSH and SFN-NAC was achieved on a C8 reverse phase column with a mobile phase gradient (Mobile Phase A: 10mM ammonium acetate buffer, pH: 4.5 and Mobile Phase B: acetonitrile with 0.1% formic acid) at a flow rate of 0.3 mL/min. The Finnigan LCQ LC-MS/MS was operated under the selective reaction monitoring mode using the electrospray ionization technique in positive mode. The nominal retention times for SFN-GSH, SFN-NAC and SFN were 8.4, 11.0, and 28.2 min,, respectively. The method was linear for SFN and its metabolites with correlation coefficients >0.998 for all analytes. The limit of quantification was 0.01-0.1 microm depending on analyte and matrix, whereas the mean recoveries from spiked plasma and perfusate samples were approximately 90%. The method was further validated according to U.S. Food and Drug Administration guidance in terms of accuracy and precision. Stability of compounds was established in a battery of stability studies, i.e., bench top, auto-sampler and long-term storage stability as well as freeze/thaw cycles. The utility of the assay was confirmed by the analysis of intestinal perfusate and plasma samples from single-pass intestinal perfusion studies with mesenteric vein cannulation in rats.     

Linear and accurate quantitation of proenkephalin-derived peptides by isotopic labeling with internal standards and mass spectrometry

Proenkephalin (PE) represents the precursor protein of the active peptide neurotransmitter enkephalin. Quantitative analysis of peptides and proteins is an objective of mass spectrometry-based studies of biological systems and will be important for studying the proteolytic conversion of proproteins to active enkephalin and neuropeptides. The goal of this study was to define and optimize quantitation of different amounts of tryptic peptides derived from PE using light (H4, 4 hydrogens) and heavy (D4, 4 deuteriums) succinic anhydride for isotopic labeling of peptides analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Comparisons were made between PE-derived peptides with and without internal standards. Importantly, incorporation of internal standards of known amounts of heavy isotope-labeled tryptic peptides of PE provided linear calibration plots with accurate quantitation. In contrast, comparison of light and heavy isotope-labeled peptides without internal standards produced variable and inaccurate nonlinear isotopic ratio comparisons of PE-derived peptides. These results demonstrate that use of internal standards composed of a defined amount(s) of standard peptides (PE-derived tryptic peptides) is necessary for high-quality linear quantitation of peptides by isotopic labeling and MS/MS.     

Role of camalexin, indole glucosinolates, and side chain modification of glucosinolate-derived isothiocyanates in defense of Arabidopsis against Sclerotinia sclerotiorum

Plant secondary metabolites are known to facilitate interactions with a variety of beneficial and detrimental organisms, yet the contribution of specific metabolites to interactions with fungal pathogens is poorly understood. Here we show that, with respect to aliphatic glucosinolate‐derived isothiocyanates, toxicity against the pathogenic ascomycete Sclerotinia sclerotiorum depends on side chain structure. Genes associated with the formation of the secondary metabolites camalexin and glucosinolate were induced in Arabidopsis thaliana leaves challenged with the necrotrophic pathogen S. sclerotiorum. Unlike S. sclerotiorum, the closely related ascomycete Botrytis cinerea was not identified to induce genes associated with aliphatic glucosinolate biosynthesis in pathogen‐challenged leaves. Mutant plant lines deficient in camalexin, indole, or aliphatic glucosinolate biosynthesis were hypersusceptible to S. sclerotiorum, among them the myb28 mutant, which has a regulatory defect resulting in decreased production of long‐chained aliphatic glucosinolates. The antimicrobial activity of aliphatic glucosinolate‐derived isothiocyanates was dependent on side chain elongation and modification, with 8‐methylsulfinyloctyl isothiocyanate being most toxic to S. sclerotiorum. This information is important for microbial associations with cruciferous host plants and for metabolic engineering of pathogen defenses in cruciferous plants that produce short‐chained aliphatic glucosinolates.     

Quantitation of paclitaxel and its two major metabolites using a liquid chromatography-electrospray ionization tandem mass spectrometry

A sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the determination of paclitaxel (Taxol) and its two major metabolites in human plasma has been developed. Samples were prepared after liquid-liquid extraction and analyzed on a C(18) column interfaced with a Q-Trap tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile-water (0.05% formic acid) (65:35) at the flow rate of 0.25 mL/min. The analytes and internal standard docetaxel were both detected by use of multiple reaction monitoring mode. The method was linear in the concentration range of 0.5-500.0 ng/mL for paclitaxel, 6α-hydroxypaclitaxel and p-3'-hydroxypaclitaxel, respectively. The lower limit of quantification (LLOQ) was 0.5 ng/mL for paclitaxel, 6α-hydroxypaclitaxel and p-3'-hydroxypaclitaxel, respectively. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 8.18%. The accuracy determined at three concentrations was within ±10.8% in terms of relative error. The total run time was 7.0 min. This assay offers advantages in terms of expediency, and suitability for the analysis of paclitaxel and its metabolites in various biological fluids.     

Direct detection and quantification of 19-norandrosterone sulfate in human urine by liquid chromatography-linear ion trap mass spectrometry

19-Norandrosterone sulfate (19-NAS) is the sulfoconjugated form of 19-norandrosterone (19-NA), the major metabolite of the steroid nandrolone. A sensitive and accurate liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay was developed for the direct measurement of 19-NAS in human urine samples. The method involved a quaternary amine SPE protocol and subsequently injection of the extract onto an analytical column (Uptisphere ODB, 150 mm x 3.0 mm, 5 microm) for chromatographic separation and mass spectrometry detection in negative electrospray ionisation mode. The sulfoconjugate of 19-NA was identified in urine by comparison of mass spectra and retention time with a reference substance. The limit of detection (LOD) and lowest limit of quantification (LLOQ) of 19-NAS were of 40 pg/mL and 200 pg/mL, respectively. For a nominal concentration of 2 ng/mL, recovery (94%), intra-day precision (2.7%), intra-assay precision (6.6%) and inter-assay precision (14.3%) were determined. Finally, this analytical method was applied for quantifying the concentration of 19-NAS in doping samples, using calibration curves (0.2-20 ng/mL) and the standard-addition method. The results show the feasibility of applying this LC-MS/MS assay as a complementary tool to detect misuse of nandrolone or nandrolone precursors.     

Quantitative profiling of nucleotides and related phosphate-containing metabolites in cultured mammalian cells by liquid chromatography tandem electrospray mass spectrometry

A method has been developed for the quantitative profiling of over twenty nucleotides and related phosphorylated species using ion-pair reversed-phase liquid chromatography hyphenated to negative ion tandem electrospray mass spectrometry. The influence of mobile phase pH and ion-pairing agent concentration were assessed to optimise separation and peak shapes. Full quantitative analysis was obtained for the nucleotides by reference to structurally related calibration standards. The developed method was applied to profile changes in nucleotides and related compounds in monolayer cultured Chinese hamster ovary (CHO) cells expressing the beta(2) adrenoceptor when exposed to pharmacological stimuli. These experiments demonstrate the potential of the LC-MS/MS method to detect changes in nucleotide drug targets as well as the simultaneous monitoring of levels of other nucleotides.