LC/MS) And Liquid Chromatography/tandem Mass Spectrometry Research Articles

Stability-Indicating Liquid Chromatography Method for Rifaximin and LC-MS Characterization of its Potential Degradants

Rifaximin (RFX) is a structural analog of rifampin that has been shown to be a gastrointestinal-selective antibiotic with broad-spectrum antibacterial properties, a pronounced safety profile, and limited drug interactions. Crohn’s disease, diarrhea, hepatic encephalopathy, irritable bowel syndrome, and traveler’s diarrhea caused by non-invasive diarrheagenic Escherichia coli, can all be treated with rifaximin. FDA has designated RFX as an orphan drug for the treatment of hepatic encephalopathy. There is a substantial need for analytical quality monitoring of Rifaximin to ensure the safety and efficacy of treatment. To generate degradants, forced degradation study of the drug was carried out in acid, alkali, oxidative, thermal, and photolytic stress conditions. The optimum separation of the drug, and its degradants was achieved on an Inertsil ODS-3V C18 column (250 X 4.6 mm X 5 μm) under gradient elution conditions using, 10 mM potassium dihydrogen orthophosphate (pH 5.0 ± 0.05) as mobile phase A, and acetonitrile as mobile phase B, at a flow rate of 1.0 mL/min. The run time was 41 minutes and the detection wavelength was 258 nm. The method was validated in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human use Guidelines. The drug was stable in neutral hydrolysis, photo and dry heat degradation conditions. The percentage degradation observed during acid, alkali hydrolysis and oxidative stress conditions were 70.46, 15.11 and 24.18, respectively. The degradants were investigated by LC-MS which indicated the presence of four (m/z 784.2, 744.5, 784.3, 753.8), three (m/z 744.5, 784.3, 753.8) and five (m/z 772.4, 838.4, 744.5, 753.8, and 801.9) degradants in acid, alkali and oxidative stress conditions, respectively. The structures of degradants were elucidated using Liquid chromatography–mass spectrometry (LC-MS) and Liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses.

Catabolism of the Lipofuscin Cycloretinal by MsP1.

Age-related macular degeneration (AMD) is a debilitating eye disease that tends to affect people over the age of 55. Lipofuscins are autofluorescent, toxic byproducts of the visual cycle thought to contribute toward the progression of the disease. Targeting the accumulation of lipofuscin through catabolism may serve as a method for the early treatment of AMD. Thus, an enzymatic approach capable of degrading lipofuscin, cycloretinal (all-trans retinal dimer), was examined. A peroxidase from the organism Marasmius scorodonius (MsP1) has shown capability of degrading this toxic metabolite into nontoxic byproducts. A catalytic triad within MsP1 (D228, H365, and R388) was identified through multiple-sequence alignment and homology modeling and confirmed by kinetic analysis. MsP1-associated cleavage products were detected by gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and liquid chromatography-tandem mass spectrometry (LC-MSMS). MsP1 degradation byproducts of cycloretinal show reduced cytotoxicity within cell culture (ARPE-19), demonstrating its potential as a gene therapeutic to alleviate the buildup of lipofuscin within AMD.

Simultaneous Determination of Multiple Active Components from Bushen Pills and Application in a Pharmacokinetic Study in Rats.

Bushen Pills (BSPs), as a traditional Chinese medicine (TCM), is widely used in clinic to enrich Yang, nourish Yin, stem essence, and strengthen kidneys. Two chromatographic methods, liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), were applied to analyze the multiple active components of BSPs in dosage form for quality evaluation and in rat plasma for pharmacokinetics study, respectively. Three active constituents of BSPs, including paeoniflorin (PF), berberine hydrochloride (BBR), and schizandrin (SCH), were simultaneously determined by the established LC-MS method with electrospray ionization (ESI) in positive selected ion monitoring (SIM) mode at m/z 503.1, 336.0, and 455.2. The contents of PF, BBR, and SCH were (6.112 ± 0.166) mg/g, (335.1 ± 14.95) μg/g, and (5.867 ± 0.136) μg/g in BSPs. On this basis, PF and BBR were selected as targeted analytes for the pharmacokinetic study of BSPs in rats. Memantine hydrochloride was used as an internal standard (IS), and the plasma samples were processed by liquid-liquid extraction with ethyl acetate. All the analytes were separated on a C18 reversed phase column, eluted with a mobile phase consisting of acetonitrile-formic acid (0.01%) (25 : 75, v/v), and detected by ESI in the selected ion mode with multiple reaction monitoring (MRM). The target fragment ions were m/z 525.3 ⟶ 449.5 for PF, 336.2 ⟶ 320.2 for BBR, and 180.1 ⟶ 163.1 for IS. The linear ranges of PF and BBR were 5–500 ng/mL and 0.1–20 ng/mL with good linearity (r2 > 0.99). No obvious matrix effect was observed, and acceptable accuracy, precision, recovery, and stability were obtained. The proposed method has been successfully applied to the pharmacokinetic study of BSPs in rats after a single dose.

3-Nitrotyrosine quantification methods: Current concepts and future challenges.

Measurement of 3-nitrotyrosine (3-NT) in biological samples can be used as a biomarker of nitrosative stress, since it is very stable and suitable for analysis. Increased 3-NT levels in biological samples have been associated with several physiological and pathological conditions. Different methods have been described for the detection and quantification of this molecule, such as (i) immunological methods; (ii) liquid chromatography, namely high-pressure liquid chromatography (HPLC)-based methods that use ultraviolet-visible (UV/VIS) absorption, electrochemical (ECD) and diode array (DAD) detection, liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS); (iii) gas chromatography, such as gas chromatography-mass spectrometry (GC-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS). A literature review on nitrosative stress, protein nitration, as well as 3-NT quantification methods was carried out. This review covers the different methods for analysis of 3-NT that have been developed during the last years as well as the latest advances in this field. Overall, all methods present positive and negative aspects, although it is clear that chromatography-based methods present good sensitivity and specificity. Regarding this, GC-based methods exhibit the highest sensibility in the quantification of 3-NT, although it requires a prior time consuming derivatization step. Conversely, HPLC does not require such derivatization step, despite being not as accurate as GC. It becomes clear that all the methods described during this literature review, although accurate for 3-NT quantification, need to be improved regarding both sensitivity and specificity. Moreover, optimization of the protocols that have been described is clearly needed.

Structural Basis for dsRNA Recognition, Filament Formation, and Antiviral Signal Activation by MDA5

MDA5, a viral double-stranded RNA (dsRNA) receptor, shares sequence similarity and signaling pathways with RIG-I yet plays essential functions in antiviral immunity through distinct specificity for viral RNA. Revealing the molecular basis for the functional divergence, we report here the crystal structure ofMDA5 bound to dsRNA, which shows how, using the same domain architecture, MDA5 recognizes the internal duplex structure, whereas RIG-I recognizes the terminus of dsRNA. We further show that MDA5 uses direct protein-protein contacts to stack along dsRNA in a head-to-tail arrangement, and that the signaling domain (tandem CARD), which decorates the outside of the core MDA5 filament, also has an intrinsic propensity to oligomerize into an elongated structure that activates the signaling adaptor, MAVS. These data support a model in which MDA5 uses long dsRNA as a signaling platform to cooperatively assemble the core filament, which in turn promotes stochastic assembly of the tandem CARD oligomers for signaling.

Identification of new minor metabolites of penicillin G in human serum by multiple‐stage tandem mass spectrometry

Liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were applied to characterize drug metabolites. Although these two methods have overcome the identification and structural characterization of metabolites analysis, they remain time-consuming processes. In this study, a novel multiple-stage tandem mass spectrometric method (MS(n) ) was evaluated for identification and characterization of new minor metabolism profiling of penicillin G, one of the β-lactam antibiotics, in human serum. Seven minor metabolites including five phase I metabolites and two phase II metabolites of penicillin G were identified by using data-dependent LC/MS(n) screening in one chromatographic run. The accuracy masses of seven identified metabolites of penicillin G were also confirmed by mass spectral calibration software (MassWorks™). The proposed data-dependent LC/MS(n) method is a powerful tool to provide large amounts of the necessary structural information to characterize minor metabolite in metabolism profiling.

Study on the quality control of cell therapy products: Determination of N-glycolylneuraminic acid incorporated into human cells by nano-flow liquid chromatography/Fourier transformation ion cyclotron mass spectrometry

N-Glycolylneuraminic acid (NeuGc), an acidic nine-carbon sugar, is produced in several animals, such as cattle and mice. Since human cells cannot synthesize NeuGc, it is considered to be immunogenic in humans. Recently, NeuGc contamination was reported in human embryonic stem cells cultured with xenogeneic serum and cells, suggesting that possibly NeuGc may harm the efficacy and safety of cell therapy products. Sialic acids have been determined by derivatization with 1,2-diamino-4,5-methylenedioxybenzene (DMB) followed by liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS); however, the limited availability of cell therapy products requires more sensitive and specific methods for the quality test. Here we studied the use of nano-flow liquid chromatography/Fourier transformation ion cyclotron resonance mass spectrometry (nanoLC/FTMS) and nanoLC/MS/MS for NeuGc-specific determination at a low femtomole level. Using our method, we found NeuGc contamination of the human cell line (HL-60RG cells) cultured with human serum. Our method needs only 2.5 × 10 3 cells for one injection and would be applicable to the determination of NeuGc in cell therapy products.

Occurrence of Estrogenic Compounds in and Removal by a Swine Farm Waste Treatment Plant

The total estrogenic activity of the wastewater from a swine farm in Japan was quantitatively characterized, and the compounds responsible for the estrogenic activity were identified and quantified. The wastewater treatment process consisted of a series of an up-flow anaerobic sludge blanket (UASB) and a trickling filter. Samples were collected at each treatment step, and the total estrogenic activity was determined by use of an in vitro gene expression assay (MVLN; MCF-7 human breast cancer cell stably transfected with the pVit-tk-LUC receptor plasmid). Individual estrogenic compounds were identified and quantified using liquid chromatography-mass spectrometry (LC/MS) and liquid chromatography-tandem mass spectrometry (LC/ MS/MS). To further identify the compounds contributing to the estrogenic activity in the wastewater, the sample extracts were fractionated into 12 fractions (fractions 1-12) by HPLC. The rate of removal of estrogenic activity between the effluent and the influent was greater than 97%. The trickling filter removed the majority of the estrogenic activity. The removal rates of specific estrogenic compounds ranged from 44 to 99%. Estrogenic activity was detected mainly in the fractions containing estrone (El), 17beta-estradiol (betaE2), 17alpha-estradiol (alpha E2), estriol (E3), bisphenol A (alphaPA), and equol (EQ0). The ratios of betaE2-EQc (betaE2 equivalents derived from chemical analysis) to betaE2-EQB (betaE2 equivalent derived from bioassay) in the 12 fractions collectively were contributed by El (17-30%), betaE2 (23-30%), acE2 (<1%), E3 (1-2%), BPA (<1%), and EQO (2-3%) in the influent and El (16-37%), PE2 (<1-7%), alphaE2 (<1%), E3 (<1-3%), BPA (<1%), and EQO (<1%) in the effluent. The compounds responsible for most of the estrogenic activity measured in the bioassay were natural estrogens such as El and betaE2.

Development of a new standard reference material: SRM 1955 (homocysteine and folate in human serum)

Total homocysteine (tHCY) and folate are interrelated biomarkers for arteriosclerosis and coronary heart disease. Although many different methods for both tHCY and folate are clinically available, the intermethod and interlaboratory results are often poor, resulting in the need for a matrix reference material and reference methods. The National Institute of Standards and Technology (NIST) has developed isotope dilution liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/ tandem mass spectrometry (LC/MS/MS) methods for determination of tHCY and several folate forms including 5-methyltetrahydrofolic acid (5MT) and folic acid (FA). Additionally, a method for simultaneous measurement of tHCY, 5MT, and FA has been developed and validated. In collaboration with the Centers for Disease Control and Prevention (CDC), mass spectrometric methods and methods used in clinical laboratories have been applied to characterize a new Standard Reference Material (SRM), SRM 1955, "Homocysteine and Folate in Human Serum," containing low, medium, and high levels of tHCY and 5MT. Additionally, FA, 5-formyltetrahydrofolic acid (5FT), vitamin B12, and total folate values are provided. Use of the new SRM should improve clinical measurements and will permit traceability to internationally recognized certified reference materials, as described by European Directive 98/79/EC on in vitro diagnostic medical devices.

HUMAN METABOLISM OF THE PROTEASOME INHIBITOR BORTEZOMIB: IDENTIFICATION OF CIRCULATING METABOLITES

Bortezomib [N-(2,3-pyrazine)carbonyl-L-phenylalanine-L-leucine boronic acid] is a potent first-in-class dipeptidyl boronic acid proteasome inhibitor that was approved in May 2003 in the United States for the treatment of patients with relapsed multiple myeloma where the disease is refractory to conventional lines of therapy. Bortezomib binds the proteasome via the boronic acid moiety, and therefore, the presence of this moiety is necessary to achieve proteasome inhibition. Metabolites in plasma obtained from patients receiving a single intravenous dose of bortezomib were identified and characterized by liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). Metabolite standards that were synthesized and characterized by LC/MS/MS and high field nuclear magnetic resonance spectroscopy (NMR) were used to confirm metabolite structures. The principal biotransformation pathway observed was oxidative deboronation, most notably to a pair of diastereomeric carbinolamide metabolites. Further metabolism of the leucine and phenylalanine moieties produced tertiary hydroxylated metabolites and a metabolite hydroxylated at the benzylic position, respectively. Conversion of the carbinolamides to the corresponding amide and carboxylic acid was also observed. Human liver microsomes adequately modeled the in vivo metabolism of bortezomib, as the principal circulating metabolites were observed in vitro. Using cDNA-expressed cytochrome P450 isoenzymes, it was determined that several isoforms contributed to the metabolism of bortezomib, including CYP3A4, CYP2C19, CYP1A2, CYP2D6, and CYP2C9. The development of bortezomib has provided an opportunity to describe the metabolism of a novel boronic acid pharmacophore.

Liquid Chromatography-Mass Spectrometric and Liquid Chromatography-Tandem Mass Spectrometric Determination of Hallucinogenic Indoles Psilocin and Psilocybin in “Magic Mushroom” Samples

Accurate and sensitive analytical methods for psilocin (PC) and psilocybin (PB), tryptamine-type hallucinogens contained in "magic mushrooms," were investigated using liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). The chromatographic separation on an ODS column and mass spectral information gave complete discrimination between PC and PB without derivatization. The mass spectrometric detection had a high sensitivity, and the tandem mass spectrometric detection provided more specificity and accuracy, as well as high sensitivity. The detection limits ranged from 1 to 25 pg by LC-MS in the selected ion monitoring mode, and the intra- and inter-day coefficients of variation were estimated to be 4.21-5.93% by LC-MS-MS in the selected reaction monitoring mode. By applying the present LC-MS-MS technique to four real samples, the contents of PC and PB were found to vary over a wide range (0.60-1.4 and 0.18-3.8 mg/g dry wt. for PC and PB, respectively) between samples.

Development and Evaluation of a Candidate Reference Method for the Determination of Total Cortisol in Human Serum Using Isotope Dilution Liquid Chromatography/Mass Spectrometry and Liquid Chromatography/Tandem Mass Spectrometry

Cortisol is an important diagnostic marker for the production of steroid hormones, and accurate measurements of serum cortisol are necessary for proper diagnosis of adrenal function. A candidate reference method involving isotope dilution coupled with liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed and critically evaluated. An isotopically labeled internal standard, cortisol-d(3), was added to serum, followed by equilibration and solid-phase and ethyl acetate extractions to prepare samples for liquid chromatography/mass spectrometry electrospray ionization (LC/MS-ESI) and liquid chromatography/tandem mass spectrometry electrospray ionization (LC/MS/MS-ESI) analyses. (M + H)(+) ions at m/z 363 and 366 for cortisol and its labeled internal standard were monitored for LC/MS. The transitions of (M + H)(+) --> [(M + H)(+) - 2H(2)O] at m/z 363 --> 327 and 366 --> 330 were monitored for LC/MS/MS. The accuracy of the measurement was evaluated by a comparison of results of this candidate reference method on lyophilized human serum reference materials for cortisol [Certified Reference Materials 192 and 193] with the certified values determined by gas chromatography/mass spectrometry reference methods and by a recovery study for the added cortisol. The results of this method for total cortisol agreed with the certified values within 1.1%. The recovery of the added cortisol ranged from 99.8% to 101.0%. This method was applied to the determination of cortisol in samples of frozen serum pools. Excellent precision was obtained with within-set CVs of 0.3%-1.5% and between-set CVs of 0.04%-0.4% for both LC/MS and LC/MS/MS analyses. The correlation coefficients of all linear regression lines ranged from 0.998 to 1.000. The detection limits (at a signal-to-noise ratio of approximately 3-5) were 10 and 15 pg for LC/MS and LC/MS/MS, respectively. This method, which demonstrates good accuracy and precision, and is free from interferences from structural analogues, qualifies as a candidate reference method and can be used as an alternative reference method to provide an accuracy base to which the routine methods can be compared.

Identification of oxidative degradates of the TRIS salt of a 5,6,7,8-tetrahydro-1,8-naphthyridine derivative by LC/MS/MS and NMR spectroscopy—interactions between the active pharmaceutical ingredient and its counterion

The Tris(hydroxymethyl)aminomethane (TRIS) salt of a substituted 5,6,7,8-tetrahydro-1,8-naphthyridine compound (I) in a mannitol-based formulation was stressed at various conditions. Liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses of the stressed samples revealed that oxidation and dimerization were the primary degradation pathways for this compound. 1H- and 13C-nuclear magnetic resonance (NMR) spectroscopy were used to characterize the isolated dimers. The aromatized degradate, N-oxide, amide, and three dimeric products were all confirmed by either LC/MS using authentic standards or NMR spectroscopy. In general, the aromatized product was always the primary degradate produced under all stress conditions. When stressed at 80 °C, the TRIS counterion also underwent thermal degradation to yield formaldehyde in situ which reacted with the parent compound to form a unique methylene-bridged dimeric product and an N-formyl degradate. A minor condensation product between the compound I and the TRIS counterion was also detected in the 80 °C stressed samples. Under 40 °C/75% RH stress conditions, TRIS derived degradates were insignificant, while dimers formed by compound I became predominant. In addition, two hydroxylated products (7-OH and 5-OH) were also detected. Mechanisms for the formation of the oxidative and dimeric degradates were proposed.

Determination of estrogens and progestogens by mass spectrometric techniques (GC/MS, LC/MS and LC/MS/MS).

Steroid sex hormones and related synthetic compounds have been shown to provoke alarming estrogenic effects in aquatic organisms, such as feminization, at very low concentrations (ng/L or pg/L). In this work, different chromatographic techniques, namely, gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS), are discussed for the analysis of estrogens, both free and conjugated, and progestogens, and the sensitivities achieved with the various techniques are inter-compared. GC/MS analyses are usually carried out after derivatization of the analytes with bis(trimethylsilyl)trifluoroacetamide (BSTFA). For LC/MS and LC/MS/MS analyses, different instruments, ionization techniques (electrospray (ESI) and atmospheric pressure chemical ionization (APCI)), ionization modes (negative ion (NI) and positive ion (PI)) and monitoring modes (selected ion monitoring (SIM) and selected reaction monitoring (SRM)) are generally employed. Based on sensitivity and selectivity, LC/ESI-MS/MS is generally the method of choice for determination of estrogens in the NI mode and of progestogens in the PI mode (instrumental detection limits (IDLs) 0.1-10 ng/mL). IDLs achieved by LC/ESI-MS in the SIM mode and by LC/ESI-MS/MS in the SRM mode were, in general, comparable, although the selectivity of the latter is significantly higher and essential to avoid false positive determinations in the analysis of real samples. Conclusions and future perspectives are outlined.

Mycosporine-glutamicol-glucoside: a natural UV-absorbing secondary metabolite of rock-inhabiting microcolonial fungi.

Microcolonial ascomycetes are known to inhabit bare rock surfaces in cold and hot deserts and thus are habitually exposed to high levels of solar radiation. Several of these stress-tolerant fungal isolates, cultivated in the laboratory under daylight illumination, were studied for the presence of effective UV-radiation protection substances. Liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses allowed for efficient separation and structure clarification of two mycosporines. It was demonstrated that both mycosporine-glutamicol-glucoside and mycosporine-glutaminol-glucoside are natural and constitutive secondary metabolites of microcolonial fungi. The function and relation of these substances in the fungal cell are discussed.

Characterization of dehydroascorbic acid solutions by liquid chromatography/mass spectrometry.

The composition of a commercial dehydroascorbic acid (DA) solution at pH 2 was investigated by liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) to establish the nature of its different forms and its decomposition products. In freshly prepared solutions, dimeric forms of DA and the hydrated bicyclic hemiketal of DA are the species mainly present in solution. In the presence of light, the initial dimeric species disappears over time to give other dehydrated dimers some of which decompose to the monomer. The comparison of these data with similar data obtained for ascorbic acid (AA) solutions under the same experimental conditions revealed that, in the presence of light, the aging of such AA solutions gives rise to only the hemiketal form of DA, and that no dimeric species of DA were formed. The presence of the hemiketal form of DA was not revealed by analysis of the same AA solutions using the conventional LC/UV technique. The natural form of DA from the oxidation of AA is the hydrated bicyclic form.

Detection of oxidative species for 4-phenoxyphenol derivatives during the electrospray ionization process.

Analyses by flow injection as well as liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were performed with four 4-phenoxyphenol derivatives. When ambient temperature nitrogen gas was used to facilitate solvent evaporation, [M + H]+, [M + NH4]+, and [2M + NH4]+ ions were observed as the major ions. As the nitrogen gas temperature increased from ambient to 250 and 450 degrees C, [M]+*, [M - 1]+ and [M + 15]+ ions were the predominant ions. Heat-induced oxidation was found to be the primary source for the formation of oxidative species. Aqueous solvents were found to be essential for the formation of the [M + 15]+ ions. The [M]+* and [M + 15]+ ions were further characterized by tandem mass spectrometry. Based on the MS/MS data, it was proposed that the [M + 15]+ ions were the in-source generated 1,2-quinone ions.

Structure elucidation of three isomeric metabolites of SYN-2836, a novel antifungal agent, in dogs via liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry methodologies.

This paper presents liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) approaches for the rapid characterization of three urinary isomeric metabolites and their two precursor metabolites of SYN-2836, a novel antifungal agent, in dogs administered multiple oral doses of the agent (30 mg kg(-1) day(-1)). A collection of correlative data regarding the SYN-2836 metabolites was obtained by LC/MS and LC/MS/MS performed under complementary conditions such as the columns (C(18) vs cyano type), the mobile phase systems (acetonitrile-water-formic acid vs acetonitrile-water-ammonium acetate) and the electrospray ionization modes (positive vs negative). Metabolite identification was accomplished based on not only the LC/MS/MS data (product ion spectra) but also the LC/MS data indicating chromatographic behaviors of the metabolites. SYN-2836 and SYN-2869, an analog of the former, showed almost the same metabolic pathways following the same multiple-dose administration of the individual agents to the dogs. Therefore, correlation analysis in product ion spectra between corresponding metabolites of SYN-2836 and SYN-2869, and also in metabolic pathways between the two agents, was strategically used to facilitate the identification of the SYN-2836 (and SYN-2869 if necessary) metabolites. For the reason that various elucidation strategies were used complementarily, the chemical structures of the metabolites were unambiguously attained and the isomeric metabolites were explicitly differentiated without the use of other analytical methods. The methodologies used in this study may be applicable to metabolite screening of several structurally related agents simultaneously, promoting lead finding and optimization of drug candidates using a metabolism-based approach.

Analysis of DNA Adduct,S-[2-(N7-Guanyl)ethyl]glutathione, by Liquid Chromatography/Mass Spectrometry and Liquid Chromatography/Tandem Mass Spectrometry

Sensitive and specific isotope dilution liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods were developed for the detection and quantitative analysis ofS-{2-(N7-guanyl)ethyl}glutathione as a DNA adduct formed upon exposure of animals to carcinogenic 1,2-dihaloethanes. Separation and analysis were performed using microbore HPLC coupled in-line to an electrospray ionization triple quadrupole mass spectrometer.S-{2-(N7-guanyl)[2H4]ethyl} glutathione was synthesized and used as internal standard. These methods provide structural confirmation of the adduct as well as quantitative analysis with the accuracy and precision necessary to measure biologically relevant levels in small tissue sample sizes (< 1 g). The sample detection limits inin vivotissue extracts were 100 pg and 5 pg on-column for LC/MS and LC/MS/MS methods, respectively. Selected-ion monitoring mode was used to monitor the product ions of the doubly charged molecular ion. The application of these methods was demonstrated by measuring the DNA adduct levels in rat and fish samples after exposure to 1,2-dihaloethanes. The method has application in studies of DNA adduct formation as a biological marker of exposure to carcinogens and for environmental monitoring of 1,2-dihaloethanes.

Characterization of the primary structure of H-protein from Pisum sativum and location of a lipoic acid residue by combined liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry.

A purified extract of H-protein, a subunit of the glycine cleavage complex of the pea leaf mitochondria, was investigated by liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS), using both continuous flow fast atom bombardment (CF-FAB) and electrospray ionization (ESI) mass spectrometry. Determination of the molecular weight of the entire protein, a 14 kDa subunit of the glycine decarboxylase complex, was achieved by ESI mass spectrometry and revealed covalent binding of the protein to the stabilizing agent beta-mercapto-ethanol. On-line LC/MS analysis of peptides arising from the endoproteinase Glu-C digestion of the H-protein was achieved using capillary columns (0.25 mm i.d.), and permitted confirmation of the previously reported sequence deduced from cDNA cloning experiments. The detailed interpretation of data extracted from these LC/MS experiments facilitated identification of peptides containing modified amino acid residues. In particular the identification of a lipoic acid cofactor, a rather unusual modified lysine residue which interacts with different active sites in the enzyme complex, was achieved using both LC/CF-FAB-MS and LC/ESI-MS. The exact location of this modified lysine residue was determined by obtaining fragment spectra of multiply protonated precursor ions of selected peptides, using on-line LC/MS/MS techniques.