High-performance Liquid Chromatography-electrospray Ionization-mass Spectrometry Research Articles

Disposition of sanguinarine in the rat

Sanguinarine is an alkaloid with known antibiotic and anti-inflammatory activity and its pharmacokinetics have been studied in the rat after a single oral dose (10 mg kg−1 body weight). Alkaloid determination in the plasma and liver was carried out by high-performance liquid chromatography–electrospray ionization mass spectrometry (HPLC/ESI-MS). The pharmacokinetic parameters (tmax, cmax, AUC0→t and AUC0→∞) were determined for sanguinarine and dihydrosanguinarine, the major components detected in plasma. The first step in sanguinarine metabolism in the rat was the reduction of the iminium bond resulting in formation of the less toxic dihydrosanguinarine. Both compounds were completely eliminated from the plasma and liver after 24 h and not detected in urine. After a single oral dose of 3H-sanguinarine, more than 42% of the ingested radioactivity was present in gastrointestinal tract. Benz[c]acridine, up to date the only sanguinarine metabolite referred to in the literature, was not detected in the plasma, liver or urine.

Analysis of chemical and metabolic components in traditional Chinese medicinal combined prescription containing Radix Salvia miltiorrhiza and Radix Panax notoginseng by LC‐ESI‐MS methods

High-performance liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) methods were developed for the analysis of chemical and metabolic components in traditional Chinese medicinal combined prescription containing Radix Salvia miltiorrhiza and Radix Panax notoginseng (commonly known as Fufang Danshen prescription, FDP). The HPLC experiments used a reversed-phase Zorbax C(18) column with the column temperature at 30 degrees C and a binary mobile phase system consisting of aqueous formic acid (0.1%, v/v) and acetonitrile using a gradient elution at the flow rate of 1.0 mL/min. The ESI-MS was operated with a single-quadrupole mass spectrometer in both negative and positive ion modes. 36 major chromatographic peaks of FDP, including 14 saponins, 13 phenolic acids and nine diterpenoid quinones were characterized by their MS spectra and in comparison with some of the reference standards. In addition, after oral administration of extraction of FDP, the rat's plasma, urine and feces were also analyzed; 53 metabolic components including 30 original components and 23 transformative components of FDP were detected, and possible metabolic pathways of some components in FDP were given. The analysis of chemical and metabolic components in FDP by HPLC-MS methods could be a useful means of identifying the multi-components of FDP and to hint at their possible metabolic mechanism of action in the body.

Inhibition of phosphatidylserine biosynthesis in developing rat brain by maternal exposure to ethanol

Phosphatidylserine (PtdSer), major acidic phospholipids in neuronal membranes, participate in important cell signaling processes. The PtdSer in brain is highly enriched with docosahexaenoic acid (DHA; 22:6n-3), and the DHA status or ethanol exposure has been shown to influence the PtdSer level. This study shows that ethanol exposure during prenatal and developmental period significantly attenuates microsomal PtdSer biosynthetic activities and reduces PtdSer, particularly 18:0, 22:6-PtdSer, in developing rat brain cortices. Brain microsomes were incubated with deuterium labeled exogenous substrates in vitro and the products formed were detected by reversed phase HPLC-electrospray ionization mass spectrometry (ESI-MS). These in vitro bioassays showed that 1-stearoyl-2-docosahexaenoyl (18:0, 22:6) species is the best substrate for PtdSer synthesis from both phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn). The PtdSer biosynthetic activity of brain, especially for 18:0, 22:6-PtdSer production, was hampered significantly by maternal exposure to ethanol. PtdSer levels were consistently reduced significantly in brain cortices of the pups from ethanol-exposed dams, due mainly to the depletion of 18:0, 22:6-PtdSer. The mRNA expression of PtdSer synthase 1 (PSS1) and PtdSer synthase 2 (PSS2) was not reduced by ethanol. Similarly, the PSS1 enzyme level did not change after ethanol exposure but PSS2 could not be probed with the antibody available currently. Degradation of PtdSer by mitochondrial PtdSer decarboxylation was not enhanced but also inhibited. Taken together, attenuated PtdSer biosynthetic activities are largely responsible for the PtdSer reduction observed in developing rat brains after maternal exposure to ethanol.

Comparative Analysis of Ligusticum chuanxiong and Related Umbelliferous Medicinal Plants by High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry

A highly precise and accurate method, based on high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS), was developed for the qualitative and quantitative comparison of the main constituents in the rhizome of Ligusticum chuanxiong (LC) and three related umbelliferous medicinal plants. A comprehensive validation of the developed method was conducted, and the method was highly sensitive, reproducible and accurate. The unique properties of the present method were validated by analyzing 20 related herbal samples including 5 LC samples, 5 Cnidium officinale samples (CO), 5 Angelica sinensis samples (AS) and 5 Angelica acutiloba samples (AA). Twelve compounds including phenolic constituents, alkylphthalides and phthalide dimers were identified by online ESI-MS and by comparison with literature data and standard compounds, and six of them were quantified by HPLC-DAD simultaneously. The results demonstrated that identical compound types were identified as the main constituents of LC, CO, AS and AA herbs. The results also support the alternative application of these medicinal plants in Chinese and Japanese folk medicines. In the present study, it was found that the variation in the abundance of senkyunolide A was significant in these related herbs; it is therefore feasible to choose senkyunolide A as a characteristic compound for quality evaluation and chemical authentication of these herbs.

To improve the quantification sensitivity of large molecular weight compounds – with ginsenosides as example

High cone voltage was used to improve the quantification sensitivity of large molecular weight compounds in high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC/ESI-MS), with ginsenosides as example. Investigations on the effect of cone voltage showed that within a voltage range of 30-130 V, for all the ginsenosides tested, i.e., Rb(1), Rb(2), Rc, Rd, Re, R(f) and R(g1), an increase in the applied cone voltage can significantly increase the sensitivity of the method. The maximum sensitivity in the determination decreases with the decreasing molecular weight of the ginsenosides in the order of Rb(1) > Rb(2) > Rc > Re > Rd > R(g1) > R(f). At the high cone voltage of 130 V, both molecular weight and structural information was obtained from a single mass spectrum. It can also be used for isomer differentiation and determination of O-glycosidic linkages in ginsenosides. Linear relationships between the peak area response and concentration were observed in the range of 50-2 x 10(5) ng/mL, with the correlation coefficients >0.99. The limits of detection reached down to pg for ginsenosides. The method was successfully applied to the determination of ginsenosides in commercial ginseng samples.

Catalytic Reduction of 1,1,1-Trichloro-2,2,2-trifluoroethane (CFC-113a) by Cobalt(I) Salen Electrogenerated at Vitreous Carbon Cathodes in Dimethylformamide

Catalytic reduction of 1,1,1-trichloro-2,2,2-trifluoroethane (CFC-113a or Freon 113a) by cobalt(I) salen electrogenerated at a carbon cathode in dimethylformamide (DMF) containing tetramethylammonium tetrafluoroborate has been investigated with the aid of cyclic voltammetry, controlled-potential electrolysis, gas chromatography–mass spectrometry, and high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS). Cyclic voltammetry reveals that CFC-113a and two of its degradation products, 2,2-dichloro-1,1,1-trifluoroethane (HCFC-123) and 2-chloro-1,1,1-trifluoroethane (HCFC-133a), can all undergo catalytic reduction by cobalt(I) salen. Controlled-potential (bulk) electrolyses of cobalt(II) salen in the presence of CFC-113a lead to the production of HCFC-123, HCFC-133a, 2-chloro-1,1-difluoroethene (HCFC-1122), and 1,1-difluoroethene (HFC-1132a). HPLC-ESI-MS has been employed to demonstrate that, during the bulk catalytic reduction of CFC-113a, the salen ligand of the catalyst is modified through addition of a or moiety to an imino bond. On the basis of documented knowledge about the electrochemistry of cobalt-containing complexes, together with the results of our cyclic voltammetry and bulk electrolysis experiments, a mechanistic scheme is proposed for the cobalt(I) salen-catalyzed reduction of CFC-113a.

Simultaneously determination of five ginsenosides in rabbit plasma using solid-phase extraction and HPLC/MS technique after intravenous administration of ‘SHENMAI’ injection

In this study, a sensitive and reliable analytical method for the simultaneous determination of five ginsenosides ( R g1, R f, R e, R d and R b1) in rabbit plasma was developed by high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS). Chromatographic separation was carried out on a Zorbax SB-C 18 Column (150 mm × 2.1 mm i.d., 5.0 μm particle size) with a simple linear gradient elution. The detection was conducted on a single quadrupole mass spectrometer by selected ion monitoring mode via electrospray ionization source. Good linearity over the investigated concentration ranges was observed with the values of R 2 higher than 0.991 for all the analytes. Limits of detection of the analytes varied from 0.25 ng/ml to 1.45 ng/ml, and the average recoveries, examined at three concentration levels, ranged from 90.6% to 106.9%. The validated method was successfully applied to the determination of the ginsenosides in the rabbit plasma after intravenous administration of ‘SHENMAI’ injection.

Rapid measurement of deuterium-labeled long-chain fatty acids in plasma by HPLC-ESI-MS

Imbalanced fatty acid metabolism contributes significantly to the increased incidence of metabolic disorders. Isotope-labeled fatty acids (2H, 13C) provide efficient means to trace fatty acid metabolism in vivo. This study reports a new and rapid method for the quantification of deuterium-labeled fatty acids in plasma by HPLC-MS. The sample preparation protocol developed required only hydrolysis, neutralization, and quenching steps followed by high-performance liquid chromatography-electrospray ionization-mass spectrometry analysis in negative ion mode using single ion monitoring. Deuterium-labeled stearic acid (d7-C18:0) was synthesized to reduce matrix interference observed with d5 analog, which improved the limit of detection (LOD) significantly, depending on the products analyzed. Linearity > 0.999 between the LOD (100 nM) and 30 microM, accuracy > 90%, precision > 88%, and adequate recovery in the dynamic range were obtained for d7-C18:0 and d7-oleic acid (C18:1). Upon oral dosing of d7-C18:0 in rats, the parent compound and its desaturation and beta-oxidation products, d7-C18:1 and d7-C16:0, were circulating with a maximal concentration ranging from 0.6 to 2.2 microM, with significant levels of d7-fatty acids detected for up to 72 h.

Analysis of glycolysis products of polyurethane fiber waste with diethylene glycol

Diethylene glycol and dibutyl tin dilaurate were used to degrade polyurethane fiber waste in this paper. The glycolysis products were separated into two phases including white solid and brown liquid by a freezing process. The chemical structure and thermal property of the purified solid product were characterized by Fourier transform infrared (FTIR) spectrometer,1H nuclear magnetic resonance (1H NMR) and differential scanning calorimetry (DSC). The solid product was mainly polytetrahydrofuran. The analysis of the liquid product was carried out by FTIR and high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS). The liquid phase included various components such as aromatic amine, diethylene glycol-propylene diamine compound and so on. The glycolysis mechanism of polyurethane fiber waste is also discussed in this paper.

Simultaneous stereoselective analysis of venlafaxine and O-desmethylvenlafaxine enantiomers in human plasma by HPLC-ESI/MS using a vancomycin chiral column

A high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI/MS) method for simultaneous stereoselective analysis of venlafaxine (VEN) and its major metabolite O-desmethylvenlafaxine (ODV) enantiomers in human plasma has been developed and validated. Chiral chromatography is performed on the CHRIOBIOTIC V ™ (5 μm, 250 mm × 4.6 mm) column with mobile phase constituted of 30 mmol/l ammonium acetate–methanol (15:85, pH 6.0) at a flow rate of 1.0 ml/min and a postcolumn splitting ratio of 3:1. The compounds were ionized in the electrospray ionization (ESI) ion source of the mass spectrometer and detected using the selected ion recording (SIR) mode. Calibration curves obtained from spiked plasma were linear in the range of 5.0–400 ng/ml for S-(+)-VEN and R-(−)-VEN, 4.0–280 ng/ml for S-(+)-ODV and R-(−)-ODV, respectively, with linear correlation coefficient all above 0.999. The average extraction recoveries for all the four analytes were above 76%. The methodology recoveries were higher than 92%. The limit of detection were 1.0 ng/ml for S-(+)-VEN and R-(−)-VEN, 1.5 ng/ml for S-(+)-ODV and R-(−)-ODV, respectively. The intra- and inter-day variation coefficients were less than 9%.

Determination of palonosetron in human plasma by liquid chromatography–electrospray ionization-mass spectrometry

A high performance liquid chromatography–electrospray ionization-mass spectrometry (HPLC–ESI-MS) method for the determination of palonosetron (PALO) in human plasma using naloxone as the internal standard (IS) was established. After adjustment to a weakly basic pH with saturated sodium bicarbonate, plasma samples were extracted with ethyl acetate and separated on a Hanbon Lichrospher 5-C 18 column with a mobile phase of 40 mM ammonium acetate buffer solution containing 0.04% formic acid–methanol (46:54, v/v). PALO was determined with electrospray ionization-mass spectrometry (ESI-MS). HPLC–ESI-MS was performed in the selected-ion monitoring (SIM) mode using target ions at [M + H] + m/ z 297.2 for PALO and [M + H] + m/ z 328.2 for the IS. Calibration curve was linear over the range of 0.02124–10.62 ng/ml. The lower limit of quantification (LLOQ) was 0.02124 ng/ml. The intra- and inter-run variability values were all less than 10.4%. The method has been successfully applied to determine the plasma concentration of PALO in healthy Chinese volunteers.

Detection of Saponins in Extracts from the Rhizomes of Paris Species and Prepared Chinese Medicines by High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry

A highly sensitive and specific method, based on high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS), was developed for simultaneous detection of eleven steroidal saponins and one phytoecdysone in extracts from the rhizomes of thirteen PARIS species and four prepared Chinese medicines (PCMs). The HPLC experiments were performed by means of a reversed-phase C18 column (4.6 mm x 150 mm, 5 microm) and mobile phase system consisting of 0.1 % aqueous formic acid and acetonitrile under gradient elution conditions. The most intensive electrospray ionization signals were found in the negative ion spectra due to HCOO- adducts. The limits of detection (LODs) for the saponins were lower than 40 ng/mL in selected ion monitoring (SIM) mode. The identification of the saponins in the extracts of PARIS species and PCMs was confirmed using their retention times and mass spectral comparisons to standard compounds. The validated method was successfully applied for simultaneous detection of twelve standard compounds in the analytes so that it provided a new means for quality evaluation of Rhizoma Paridis and Chinese multiherb remedies that contain Rhizoma Paridis. The results showed that most species of PARIS contain steroidal saponins, which provided evidence for expanding the botanical origin of Rhizoma Paridis.

The pharmacokinetics of orally administered fudosteine in healthy Chinese volunteers

The pharmacokinetics of fudosteine in healthy Chinese volunteers was investigated for the first time after single- and multiple-dose administration. Five male and five female volunteers were enrolled in this study. Each subject received 400 mg fudosteine capsules (the therapeutic dose) on day 1 after overnight fasting for the single-dose study and three times daily oral administration (400 mg) for 5 consecutive days until the sixth morning for the multiple-dose study. Serial blood samples were collected at specified time intervals up to 16 hours following the first and last doses of fudosteine. Plasma harvested from the blood was separated and analyzed for fudosteine levels by a validated high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC/ESI/MS) method employing percolumn derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl). Noncompartmental analysis was used for the calculation of the total area under the plasma concentration-time curve (AUC) from time zero to time infinity and the terminal half-life (t1/2) of fudosteine. The pharmacokinetic parameters for single- and multiple-dose administration were estimated as follows: Cmax amounted to 10.13+/-4.39 microg/mL and 11.75+/-6.51 microg/mL, tmax to 0.69+/-0.36 h and 0.53+/-0.12 h and t1/2 to 2.33+/-0.63 h and 2.40+/-0.37 h, respectively. No significant differences were found between single- and multiple-dose oral administration, although gender differences were observed.

Development of a protocol for the semi‐synthesis and purification of betaxanthins

A method for the analytical and semi-preparative chromatographic purification of betaxanthins is described together with an improved procedure for the semi-synthesis of these compounds from betalamic acid. Standard conditions for obtaining preparative amounts of betaxanthins free of the precursor amino acids are provided. Following this procedure, 14 pure betaxanthins were obtained with yields of up to 100%. A simple reversed-phase HPLC protocol for pigment identification and quantification is also provided. Calibration for betaxanthins is reported for the first time using the synthesised and purified pigments as standards. Structures were confirmed by UV-vis spectroscopy, HPLC retention times and electrospray ionization mass spectrometry. Betaxanthins can be obtained pure, and in sufficient amounts for further studies, which opens up new perspectives in the research and applications of these pigments.

High-Performance Liquid Chromatographic-Mass Spectrometric Determination of Methamphetamine and Amphetamine Enantiomers, Desmethylselegiline and Selegiline, in Hair Samples of Long-Term Methamphetamine Abusers or Selegiline Users

We devised a highly sensitive method for simultaneously determining methamphetamine (MA) and amphetamine (AP) enantiomers, desmethylselegiline (DMSG) and selegiline (SG), in human hair using a derivatization technique and high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS). MA and AP enantiomers and DMSG were effectively converted to trifluoroacetic acid (TFA) derivatives, and the sensitivity of MA and DMSG increased five times over compared with that of free bases. The TFA derivatives of each compound were stable within one week in a stock solution of methanol or for 24 h in the HPLC mobile phase (mixture of methanol and ammonium formate buffer). Each compound was well separated, and calibration curves were linear in the concentration range 0.04-40 ng/mg for MA enantiomers, SG and DMSG, and 0.2-40 ng/mg for AP enantiomers. The accuracy and precision of the method were evaluated, and relative standard deviations were within 7%. Our method was successfully applied to hair samples obtained from long-term MA abusers and SG users. (+)-MA and (+)-AP were detected from three MA abusers at concentrations of 0.79-20.85 and 0.04-3.30 ng/mg, respectively. On the other hand, (-)-MA, (-)-AP, DMSG, and SG were detected in three SG users at concentrations of 2.48-9.05, 0.72-3.10, 0.12-0.59, and 0-0.04 ng/mg, respectively. Based on our obtained data, discrimination of MA abusers from SG users was considered to be possible by comparing optical isomers of MA and AP, the existence of DMSG and/or SG, and the concentration ratio of AP to MA in hair samples.

Comparative assessment of distribution of blackcurrant anthocyanins in rabbit and rat ocular tissues

Anthocyanins (ACs) are phenolic compounds that are distributed widely in fruits and vegetables. Although consumption of these compounds has been shown to improve visual function, the distribution of ACs in ocular tissue has not been examined in detail. The aim of this study was therefore to evaluate the ocular distribution of blackcurrant anthocyanins (BCAs) in rats and rabbits after oral, intravenous (i.v.) and intraperitoneal (i.p.) administration. Identification and quantification of ACs were carried out using high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) and high-performance liquid chromatography (HPLC) with UV-visible detection, respectively. BCAs were identified in the plasma and whole eye after oral and i.p. administration in rats. No other peaks were detected in either plasma or ocular tissues after administration when the absorbance of the eluate was monitored at 520 nm. This finding indicates that intact forms of ACs were present in rats after administration of BCA. In rats given i.p. administration, the concentration of total ACs in the whole eye and some ocular tissues was higher than that measured in plasma. These results suggested that ACs detected in the ocular tissues were not due to residual blood. Following i.v. administration in rabbits, four ACs were identified in the plasma and several ocular tissues including the aqueous humor, cornea, sclera, choroid, ciliary body, iris and retina. A small amount of ACs was also detected in the vitreous and lens. In conclusion, this study demonstrated that BCAs were absorbed and distributed in ocular tissues as intact forms. Our data show clearly that intact forms of BCAs pass thorough the blood–aqueous barrier and blood–retinal barrier in both rats and rabbits.

Cross-Linking of the Human DNA Repair Protein O6-Alkylguanine DNA Alkyltransferase to DNA in the Presence of 1,2,3,4-Diepoxybutane

1,2,3,4-Diepoxybutane (DEB) is a key carcinogenic metabolite of the important industrial chemical 1,3-butadiene. DEB is a bifunctional alkylating agent capable of reacting with DNA and proteins. Initial DNA alkylation by DEB produces N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-guanine monoadducts, which can react with another nucleophilic site to form cross-linked adducts. A recent report revealed a strong correlation between cellular expression of the DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) and the cytotoxic and mutagenic activity of DEB, suggesting that DEB induces AGT-DNA cross-links (Valadez, J. G., et al. (2004) Activation of bis-electrophiles to mutagenic conjugates by human O6-alkylguanine-DNA alkyltransferase. Chem. Res. Toxicol. 17, 972-982). The purpose of our study was to analyze the formation and structures of DEB-induced AGT-DNA conjugates and to identify specific amino acid residues within the protein involved in cross-linking. DNA-protein cross-link formation was detected by SDS-PAGE when 32P-labeled double-stranded oligodeoxynucleotides were exposed to DEB in the presence of either wild-type hAGT or a C145A hAGT mutant. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of hAGT that had been treated with N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-deoxyguanosine (dG monoepoxide) revealed the ability of the protein to form either one or two butanediol-dG cross-links, corresponding to mass shifts of +353 and +706 Da, respectively. HPLC-ESI+ -MS/MS sequencing of the tryptic peptides obtained from dG monoepoxide-treated protein indicated that the two cross-linking sites were the alkyl acceptor site, Cys145, and a neighboring active site residue, Cys150. The same two amino acid residues of hAGT became covalently cross-linked to DNA following DEB treatment. Modification of Cys145 was further confirmed by HPLC-ESI+ -MS/MS analysis of dG monoepoxide-treated synthetic peptide GNPVPILIPCHR which represents the active site tryptic fragment of hAGT (C = Cys145). The replacement of the catalytic cysteine residue with alanine in the C145A hAGT mutant abolished DEB-induced cross-linking at this site, while the formation of conjugates via neighboring Cys150 was retained. The exact chemical structure of the cross-linked lesion was established as 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol by HPLC-ESI+ -MS/MS analysis of the amino acids resulting from the total digestion of modified proteins analyzed in parallel with an authentic standard. AGT-DNA cross-linking is a likely mechanism of DEB-mediated cytotoxicity in cells expressing this important repair protein.

Bilirubin degradation by uncoupled cytochrome P450. Comparison with a chemical oxidation system and characterization of the products by high‐performance liquid chromatography/electrospray ionization mass spectrometry

Bilirubin is a protective antioxidant; however, when its conjugation and excretion are impaired, as in neonatal and hereditary jaundice, bilirubin accumulates and may cause severe neurotoxicity. Degradation of bilirubin takes place (a) on interaction with oxidative free radicals and (b) when cytochrome P450 (CYP) enzymes are uncoupled by polyhalogenated substrate analogues. The products of pathways (a) and (b) above have now been characterized by high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and the mechanisms of fragmentation in part clarified. Oxidation of bilirubin by uncoupled CYP1A5 and by a Fe-EDTA/H2O2 system produced both biliverdin and an identical profile of dipyrrolic fragments, as detected by positive ESI-MS. A similar profile of oxidation products was found from mesobilirubin, all showing the expected increase in mass, thus providing direct evidence for fragmentation at the central methene bridge of the tetrapyrroles. Two degradation products, also detected by negative ESI-MS, were characterized as dipyrroles retaining the central bridge carbon, with one or two oxygen atom(s) bound (probably as the aldehyde and hydroperoxide derivatives). Ions compatible with propentdyopents and bilifuscins were also detected, but here the assignment was less certain. It is concluded that the first step in the oxidation of bilirubin may be hydrogen abstraction at the central methene bridge. This is followed either by loss of another hydrogen to give biliverdin, or by oxygen binding and fragmentation. Fe-EDTA/H2O2 and uncoupled CYP(Fe=O) may both initiate the reaction, the latter in an attempt to reduce the ferryl oxygen to water. These studies shed light on the CYP uncoupling mechanism and are of potential significance for the therapy of severe jaundice.

Analyses of Stemona alkaloids in Stemona tuberosa by liquid chromatography/tandem mass spectrometry

Alkaloid profiles in Stemona tuberosa were found to be highly variable. Six Stemona alkaloids isolated from the plant were subjected to on-line high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and tandem mass spectrometry (MS/MS) analyses. Their fragmentation patterns and products were useful for their characterization. The LC/MS fingerprints of these alkaloids, though variable among samples, could provide an overall characterization of the authenticity and quality of this species and help to differentiate it from S. japonica and S. sessilifolia, as all three species are recognized as genuine sources of the herb Radix Stemonae in the Pharmacopoeia of the People's Republic of China.

Assay of ochratoxin A in grape by high-pressure liquid chromatography coupled on line with an ESI–mass spectrometry

In this paper, we propose a method for detection of ochratoxin A (OTA) in grapes by using nano-reversed-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry (nano-RP-HPLC-ESI-MS). The method is rapid, highly sensitive and reproducible. OTA is extracted preferably from the entire acinus, rather than must; using chloroform at long incubation time period, lyophilized, resolubilized in acetonitrile (AcCN) and injected onto a reversed phase capillary or analytical column. Capillary columns are the method of choice because it requires a reduced amount of injected sample and consequently the chloroform necessary for OTA extraction, which is a toxic agent. This method gives a detection limit of femtog/ml, without resorting to an immunoaffinity clean-up or concentration, which makes it by far superior to any other method reported. Moreover, by using MS as a detection method it is possible, in the case of a complex matrix, to measure its molecular mass and to confirm the presence of OTA by MS-MS, which cannot be done by fluorescent detection. The method has a high sample extraction throughput (24/h) and has adequate precision (between batch C.V. <8%) and sensitivity (limit of detection (LOD)=1 pg/g; limits of quantification (LOQ)=2 pg/g) for OTA measured.