First Identification of the Hepatotoxic Microcystins in the Serum of a Chronically Exposed Human Population Together with Indication of Hepatocellular Damage

Hepatotoxic microcystins (MCs) are the most commonly reported cyanotoxins in eutrophic freshwaters. In 1996, human intoxications by MCs caused deaths of 76 patients at Caruaru dialysis centers in Brazil. So far, there have been no direct evidences of MC occurrence in human tissue in consequence of exposure to MC. In this study, we improved cleanup procedures for detecting MCs in serum sample using liquid chromatographymass spectrometry, and confirmed for the first time the presence of MCs in serum samples (average 0.39 ng/ml, which amounts to ca. 1/87 of the concentrations found in tissue samples of the Caruaru victims) of fishermen at Lake Chaohu. Daily intake by the fishermen was estimated to be in the range of 2.2-3.9 mu g MC-LReq, whereas the provisional World Health Organization tolerable daily intake (TDI) for daily lifetime exposure is 0.04 mu g/kg or 2-3 mu g per person. Moreover, statistical analysis showed closer positive relationships between MC serum concentrations and concentrations of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase than between the MC concentrations and other biochemical indicators. Thus, the data raise the question whether extended exposure in the range of the TDI or up to a factor of 10 above it may already lead to indication of liver damage. The results also demonstrate a risk of health effects from chronic exposure to MCs at least for populations with high levels of exposure, like these fishermen.

Determinations of MC-LR and [Dha(7)] MC-LR Concentrations and Physicochemical Properties by Liquid Chromatography-Tandem Mass Spectrometry

A liquid chromatography electrospray mass spectrometry (LC/ESI/MS) method working in multiple reactions monitoring mode for the determination of trace amounts of microcystin variants (MC-LR and [Dha(7)] MC-LR) in waters was developed. The limit of quantification was 0.05 mu g/L and the limit of detection was 0.015 mu g/L for MC-LR and [Dha(7)] MC-LR, respectively. Recoveries for MCs were in the range of 68%-81%. MC-LR and [Dha(7)] MC-LR were chemically stable with similar physiochemical behavior.

Simultaneous determination of microcystin-LR and its glutathione conjugate in fish tissues by liquid chromatography-tandem mass spectrometry

A sensitive and selective liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantitative determination of microcystin-LR (MC-LR) and its glutathione conjugate (MC-LR-GSH) in fish tissues. The analytes were extracted from fish liver and kidney using 0.01 M EDTA-Na-2-5% acetic acid, followed by a solid-phase extraction (SPE) on Oasis HLB and silica cartridges. High-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry, operating in selected reaction monitoring (SRM) mode, was used to quantify MC-LR and its glutathione conjugate in fish liver and kidney. Recoveries of analytes were assessed at three concentrations (0.2, 1.0, and 5 mu g g(-1) dry weight [DW]) and ranged from 91 to 103% for MC-LR, and from 65.0 to 75.7% for MC-LR-GSH. The assay was linear within the range from 0.02 to 5.0 mu g g(-1) DW, with a limit of quantification (LOQ) of 0.02 mu g g(-1) DW. The limit of detection (LOD) of the method was 0.007 mu g g(-1) DW in both fish liver and kidney. The overall precision was determined on three different days. The values for within- and between-day precision in liver and kidney were within 15%. This method was applied to the identification and quantification of MC-LR and its glutathione conjugate in liver and kidney of fish with acute exposure of MC-LR. (c) 2007 Elsevier B.V. All rights reserved.

Determination of peptides and amino acids from wool and beer with sensitive fluorescent reagent 2-(9-carbazole)-ethyl chloroformate by reverse phase high-performance liquid chromotography and liquid chromotography mass spectrometry

A new method for the sensitive determination of amino acids and peptides using the tagging reagent 2-(9-carbazole)-ethyl chloroformate (CEOC) with fluorescence (FL) detection has been developed. Identification of derivatives was carried out by liquid chromotography mass spectrometry. The chromophore in the 2-(9-fluorenyl)-ethyl chloroformate (FMOC) reagent was replaced by carbazole, which resulted in a sensitive fluorescence lerivatizing agent CEOC. CEOC can easily and quickly label peptides and amino acids. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. Studies on derivatization demonstrate excellent derivative yields over the pH range 8.8-10.0. Maximal yields close to 100% are observed with three- to fourfold molar reagent excess. Derivatives exhibit strong fluorescence and allow direct injection of the reaction mixture with no significant disturbance from the major fluorescent reagent degradation by-products, such as 2(9-carbazole)-ethanol and bis-(2-(9-carbazole)-ethyl) carbonate. In addition, the detection responses for CEOC derivatives are compared to those obtained with FMOC. The ratios AC(CEOC)/AC(FMOC) = 1.00-1.82 for fluorescence (FL) response and AC'(CEOC)/AC'(FMOC) = 1.00-1.21 for ultraviolet (UV) response are observed (here, AC and AC' are, respectively, FL and UV F response). Separation of the derivatized peptides and amino acids has been optimized on a Hypersil BDS C18 column. Excellent linear responses are observed. This method was used successfully to analyze protein hydrolysates from wool and from direct-derivatized beer. (C) 2003 Elsevier Science (USA). All rights reserved.

Screening and Structural Characterization of alpha-Glucosidase Inhibitors from Hawthorn Leaf Flavonoids Extract by Ultrafiltration LC-DAD-MSn and SORI-CID FTICR MS

In vitro a-glucosidase inhibition assays and ultrafiltration liquid chromatography with photodiode array detection coupled to electrospray ionization tandem mass spectrometry (ultrafiltration LC-DAD-ESI-MSn) were combined to screen a-glucosidase inhibitors from hawthorn leaf flavonoids extract (HLFE). As a result, four compounds were identified as alpha-glucosidase inhibitors in the HLFE, and their structures were confirmed to be quercetin-3-O-rha-(1-4)-glc-rha and C-glycosylflavones (vitexin-2 ''-O-glucoside, vitexin-2 ''-O-rhamnoside and vitexin) by high-resolution sustained off resonance irradiation collision-induced dissociation (SORI-CID) data obtained by Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS).

Identification of Phenylethanoid Glycosides in Plant Extract of Plantago asiatica L. by Liquid Chromatography-Electrospray Ionization Mass Spectrometry

The present work describes a liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method for rapid identification of phenylethanoid glycosides in plant extract from Plantago asiatica L. By using a binary mobile phase system consisting of 0.2% acetic acid and acetonitrile under gradient conditions, a good separation was achieved on a reversed-phase C-18 column. The [M-H](-) ions, the molecular weights, and the fragment ions of phenylethanoid glycosides were obtained in the negative ion mode using LC-ESI-MS. The identification of the phenylethanoid glycosides (peaks 1-3) in the extract of P. asiatica L. was based on matching their retention time, the detection of molecular ions, and the fragment ions obtained by collision-induced dissociation (CID) experiments with those of the authentic standards and data reported in the literature.

Analysis of Saponins from Leaves of Aralia elata by Liquid Chromatography and Multi-stage Tandem Mass Spectrometry

Four saponins were isolated from the leaves of Aralia elata, and established using NMR and other spectroscopic methods, as well as data reported in the literature. Three Aralia saponins from the leaves of Aralia elata sharing the same structures as those isolated from the root bark suggested that the leaves would be a good substitute for the root bark of Aralia elata. These four Aralia saponins were then extensively investigated using complementarily positive and negative electrospray ionization multistage tandem mass spectrometry (ESI-MSn). Two isomers of saponins with different sugar linkages were then successfully differentiated by positive ESI-MSn and verified with different retention times and the collision-induced dissociation (CID) spectra by LC-MS. A simple and effective LC-MS method was thus developed for the rapid identification and screening of these saponins in plant extracts from leaves of Aralia elata.

Determination of tetrodotoxin by liquid chromatography coupled with electrospray ion-trap mass spectrometry

Two methods for tetrodotoxin analysis using liquid chromatography coupled with electrospray iontrap mass spectrometry (LC-ESI-MS) have been established with C,, reversed phase column and hydrophilic interaction liquid chromatography (HILIC) column, respectively. Sensitivity and reproducibility of the methods were compared. The method using C-18 column in selected ion monitoring (SIM) mode had a detection limit (S/N = 3) of 120 pg, and a good linearity of the calibration curve was obtained for tetrodotoxin (r = 0. 9992). High reproducibility of the method was observed, with a relative standard deviation (RSD) below 10%. The method using HILIC column in SIM mode and selected reaction monitoring (SRM) mode had detection limits (S/N = 3) of 15 and 3.75 pg, respectively. Good linearity of the calibration curves was obtained for tetrodotoxin (r = 0. 9996 and 0. 9998 in SIM and SRM mode, respectively). T he reproducibility was high in SIM mode but relatively poor in SRM mode. Based on the results, the method using HILIC column in SIM mode was suggested for the analysis of tetrodotoxin with LC-MS system.

Development of a sensitive fluorescent derivatization reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) and its application for determination of amino acids from seeds and bryophyte plants using high-performance liquid chromatography with fluorescence detection and identification with electrospray ionization mass spectrometry

A pre-column derivatization method for the sensitive determination of amino acids and peptides using the tagging reagent 1,2-benzo-3,4dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS). The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent was replaced by 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEOC. BCEOC can easily and quickly label peptides and amino acids. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z (M + H)(+) under electrospray ionization (ESI) positive-ion mode with an exception being Tyr detected at negative mode. The collision-induced dissociation of protonated molecular ion formed a product at m/z 246.2 corresponding to the cleavage of C-O bond of BCEOC molecule. Studies on derivatization demonstrate excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% are observed with a 3-4-fold molar reagent excess. Derivatives exhibit strong fluorescence and extracted detzvatization solution with n-hexane/ethyl acetate (10:1, v/v) allows for the direct injection with no significant interference from the major fluorescent reagent degradation by-products, such as 1,2-benzo-3,4-dihydrocarbazole-9-ethanol (BDC-OH) (a major by-product), mono- 1,2-benzo-3,4-dihydrocarbazole-9-ethyl carbonate (BCEOC-OH) and bis-(1,2-benzo-3,4-dihydrocarbazole-9-ethyl) carbonate (BCEOC)(2). In addition, the detection responses for BCEOC derivatives are compared to those obtained with previously synthesized 2-(9-carbazole)-ethyl chloroformate (CEOC) in our laboratory. The ratios AC(BCEOC)/AC(CEOC) = 2.05-6.51 for fluorescence responses are observed (here, AC is relative fluorescence response). Separation of the derivatized peptides and amino acids had been optimized on Hypersil BDS C-18 column. Detection limits were calculated from 1.0 pmol injection at a signal-to-noise ratio of 3, and were 6.3 (Lys)-177.6 (His) fmol. The mean interday accuracy ranged from 92 to 106% for fluorescence detection with mean %CV < 7.5. The mean interday precision for all standards was < 10% of the expected concentration. Excellent linear responses were observed with coefficients of > 0.9999. Good compositional data could be obtained from the analysis of derivatized protein hydrolysates containing as little as 50.5 ng of sample. Therefore, the facile BCEOC derivatization coupled with mass spectrometry allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids and peptides from biological and natural environmental samples. (c) 2005 Elsevier B.V. All rights reserved.

Integration of ion-exchange chromatography fractionation with reversed-phase liquid chromatography-atmospheric pressure chemical ionization mass spectrometer and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for isolation and identification of compounds in Psoralea corylifolia

An approach for the separation and identification of components in a traditional Chinese medicine Psoralea corylifolia was developed. Ion-exchange chromatography (IEC) was applied for the fractionation of P corylifolia extract, and then followed by concentration of all the fractions with rotary vacuum evaporator. Each of the enriched fractions was then further separated on an ODS column with detection of UV absorbance and atmospheric pressure chemical ionization mass spectrometer (APCI/MS), respectively, and also analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) with matrix of oxidized carbon nanotubes. Totally more than 188 components in P. corylifolia extract were detected with this integrated approach, and 12 of them were preliminary identified according to their UV spectra and mass spectra performed by APCI/MS and MALDI-TOF/MS. The obtained analytical results not only demonstrated the powerful resolution of integration IEC fractionation with reversed-phase liquid chromatography (RPLC)-APCI/MS and MALDI-TOF/MS for analysis of compounds in a complex sample, but also exhibited the superiority of APCI/MS and MALDI-TOF/MS for identification of low-mass compounds, such as for study of traditional Chinese medicines (TCMs) and metabolome. (c) 2005 Published by Elsevier B.V.

High-performance liquid chromatography-tandem mass spectrometry for identification of isoflavones and description of the biotransformation of kudzu root

High-performance liquid chromatography-tandem mass spectrometry has been used to identify isoflavone aglycones and glycosides in kudzu root. Fourteen isoflavones were detected. Among these, six were identified by comparison with authentic standards. Tentative identifications of the other isoflavones are based on UV spectra, mass spectra of protonated and deprotonated molecules, and MS-MS data. Several are reported for the first time in kudzu root. The bioactivity and bioavailability of isoflavone aglycones are usually greater than those of their glycosides. To improve the bioavailability of kudzu root isoflavones, crude beta-glycosidases prepared from microbes were used to hydrolyze the isoflavone glycosides. Several MS modes are combined not only to identify the isoflavones in kudzu root, but also to describe the biotransformation of kudzu root isoflavone glycosides. It is also proved that crude beta-glycosidases have high selectivity toward the O-glycosides of isoflavones.

Analysis of carbohydrates in a tibetan medicine using new labeling reagent, 1-(2-naphthyl)-3-methyl-5-pyrazolone, by HPLC with DAD detection and ESI-MS identification

A new labeling reagent, 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP), coupling with liquid chromatography (LC) with electrospray ionization mass spectrometry (ESI-MS) for the detection of carbohydrates from a famous Tibetan medicine is reported. Carbohydrates were derivatized to their bis-NMP-labeled derivatives. The method, in conjunction with a gradient elution, offered a baseline resolution of carbohydrate derivatives on a reversed phase Hypersil ODS-2 column. The carbohydrates such as mannose, galacturonic acid, glucuronic acid, rhamnose, glucose, galactose, xylose, arabinose, and fucose could be successfully detected by UV and ESI-MS. Derivatives showed intense protonated molecular ion at m/z [M+H]+ in positive ion mode. The mass to charge ratios of characteristic fragment ions at m/z 473.0 could be used for the accurately qualitative identification of carbohydrates; this characteristic fragment ion was from the cleavage of C2-C3 bond in the carbohydrate chain giving the specific fragment ions at m/z [MH-CmH2m+1Om-H2O](+) for pentose, hexose, and glyceraldehydes, and at m/z [MH-CmH2m-1Om+1-H2O](+) for alduronic acids, such as galacturonic acid and glucuronic acid (m=n-2, n is carbon atom number of carbohydrate). Compared with the traditional 1-phenyl-3-methyl-5-pyrazolone (PMP) reagent, currently synthesized NMP show the advantage of higher sensitivity to carbohydrate compounds with UV and ESI-MS detection.

LC determination of amino acids in rat plasma with fluorescence detection: Application to exercise physiology

An LC method for the determination of 20 amino acids (AAs), using 1,2-Benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) as fluorescent labeling reagent, has been validated and applied for the analysis of AAs in rat plasma at three different states concerning exercise physiology. Identification of AA derivatives was carried out by LC-MS with electrospray ion (ESI), and the MS-MS cleavage mode of the representative tyrosine (Tyr) derivative was analyzed. Gradient elution on a Hypersil BDS C-18 column gave good separation of the derivatives. Excellent linear responses were observed and good compositional data could be obtained from as little as 50-200 mu L of plasma samples. The contents of 20 AAs in rat plasma of three groups (24 rats, group A: quiet state, group B: at exercising exhaust, group C: 12 h after exercising exhaust) exhibited evident difference corresponding to the physiological states. Facile BCEOC derivatization coupled with LC-FLD-ESI-MS analysis allowed the development of a highly sensitive method for the quantitative analysis of trace level of AAs from plasma or other biochemical samples.

2-(2-Phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA) and its application for determination of amines by high performance liquid chromatography with fluorescence detection and identification with mass spectroscopy/atmospheric pressure chemical ionization

A simple, sensitive, and mild method for the determination of amino compounds based on a condensation reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC-HCI) as the dehydrant with fluorescence detection has been developed. Amines were derivatized to their acidamides with labeling reagent 2-(2-phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA). Studies on derivatization conditions indicated that the coupling reaction proceeded rapidly and smoothly in the presence of a base catalyst in acetonitrile to give the corresponding sensitively fluorescent derivatives with an excitation maximum at lambda(ex) 260nm and an emission maximum at lambda(em) 380nm. The labeled derivatives exhibited high stability and were enough to be efficiently analyzed by high-performance liquid chromatography. Identification of derivatives was carried out by online post-column mass spectrometry (LC/APCI-MS/MS) and showed an intense protonated molecular ion corresponding m/z [MH](+) under APCI in positive-ion mode. At the same time, the fluorescence properties of derivatives in various solvents or at different temperature were investigated. The method, in conjunction with a gradient elution, offered a baseline resolution of the common amine derivatives on a reversed-phase Eclipse XDB-C-8 column. LC separation for the derivatized amines showed good reproducibility with acetonitrile-water as mobile phase. Detection limits calculated from 0.78 pmol injection, at a signal-to-noise ratio of 3, were 3.1-18.2 fmol. The mean intra- and inter-assay precision for all amine levels were < 3.85% and 2.11%, respectively. Excellent linear responses were observed with coefficients of > 0.9996. The established method for the determination of aliphatic amines from real wastewater and biological samples was satisfactory. (c) 2006 Elsevier B.V. All rights reserved.

Pharmacokinetics of sifuvirtide, a novel anti-HIV-1 peptide, in monkeys and its inhibitory concentration in vitro

Aim: To study the pharmacokinetics of sifuvirtide, a novel anti-human immunodeficiency virus (HIV) peptide, in monkeys and to compare the inhibitory concentrations of sifuvirtide and enfuvirtide on HIV-1-infected-cell fusion. Methods: Monkeys received 1.2 mg/kg iv or sc of sifuvirtide. An on-line solid-phase extraction procedure combined with liquid chromatography tandem mass spectrometry (SPELC/MS/MS) was established and applied to determine the concentration of sifuvirtide in monkey plasma. A four-I-127 iodinated peptide was used as an internal standard. Fifty percent inhibitory concentration (IC50) of sifuvirtide on cell fusion was determined by co-cultivation assay. Results: The assay was validated with good precision and accuracy. The calibration curve for sifuvirtide in plasma was linear over a range of 4.88-5000 mu g/L, with correlation coefficients above 0.9923. After iv or sc administration, the observed peak concentrations of sifuvirtide were 10626 +/- 2886 mu g/L and 528 +/- 191 mu g/L, and the terminal elimination half-lives (T,12) were 6.3 +/- 0.9 h and 5.5 +/- 1.0 h, respectively. After sc, T-max was 0.25-2 h, and the absolute bioavailability was 49% +/- 13%. Sifuvirtide inhibited the syncytium formation between HIV-1 chronically infected cells and uninfected cells with an IC50 of 0.33 mu g/L. Conclusion: An on-line SPE-LC/MS/MS approach was established for peptide pharmacokinetic studies. Sifuvirtide was rapidly absorbed subcutaneously into the blood circulation. The T-1/2 of sifuvirtide was remarkably longer than that of its analog, enfuvirtide, reported in healthy monkeys and it conferred a long-term plasma concentration level which was higher than its IC50 in vitro.