Liquid Chromatography-electrospray Ionization-mass Spectrometry Analysis Research Articles

Bacterial responses to plant antimicrobials: the case of alkannin and shikonin derivatives.

Alkannin, shikonin and their derivatives (A/S) are secondary metabolites produced in the roots of certain plants of the Boraginaceae family such as Lithospermum erythrorhizon Siebold & Zucc. and Alkanna tinctoria (L.) Tausch. These naphthoquinones express anti-cancer, wound healing, and antimicrobial activities. To study the interactions between endophytic bacteria isolated from A. tinctoria and the antimicrobials A/S, endophytic bacteria known to be resistant to the compounds were screened for their effect on A/S in liquid medium. Thereafter, the strain Pseudomonas sp. R-72008, was selected and tested for its ability to modify A/S in nutrient medium and minimal medium with A/S as sole carbon source. Bacterial growth was recorded, and high performance liquid chromatography-diode array and ultra-high performance liquid chromatography-electrospray ionization-mass spectrometry analyses were performed to detect and quantify metabolites. In nutrient medium inoculated with R-72008, a decrease in the amount of A/S monomers initially present was observed and correlated with an increase of A/S oligomers. Moreover, a significant decrease of initial A/S monomers in minimal medium was correlated with bacterial growth, showing for the first time that a bacterial strain, Pseudomonas sp. R-72008, was able to use the naphthoquinones A/S as sole carbon source. This study opens new perspectives on the interactions between bacteria and plant antimicrobials.

Identification of Antifungal Compounds from Piper Plants Against Fusarium oxysporum: An Untargeted Metabolite Profiling-Based Approach

The phytopathogen Fusarium oxysporum produces considerable losses in economically important crops, making alternative control measures urgently required. Piper plants are widely distributed in tropical regions, and they are also known to produce metabolites with biological activity against infectious agents. As part of our continuous search for antifungals, 18 Piper-derived ethanolic extracts were evaluated by their in vitro effect on F oxysporum mycelial growth inhibition. The total content of phenol and flavonoid measurements and liquid chromatography-electrospray ionization-mass spectrometry analysis served as the chemical characterization of the investigated extracts. Piper pulchrum, Piper barcoense, and Piper tuberculatum exhibited the highest mycelial growth inhibition (>74%). The integration of chemical fingerprints and bioactivity datasets led to recognizing 4 bioactive candidates among extracts through single- Y orthogonal partial least squares regression and univariate statistics. These candidates were 2 amides (1,3), an alkyl lactone (2), and a prenylated benzoquinone (4), subsequently isolated and identified by nuclear magnetic resonance spectroscopy. These isolated compounds exhibited reasonable antifungal activity (IC50 < 50 µM). The findings indicated that the correlative association is advantageous for identifying bioactive metabolites within active extracts.

Effects of Fengycins and Iturins on Fusarium oxysporum f. sp. physali and Root Colonization by Bacillus velezensis Bs006 Protect Golden Berry Against Vascular Wilt.

Bacillus velezensis Bs006 has shown antagonistic activity on Fusarium oxysporum f. sp. physali and biocontrol activity against Fusarium wilt (FW) in golden berry (Physalis peruviana). We hypothesized that strain Bs006 has the ability to synthesize antimicrobial cyclic lipopeptides (CLPs) like other members of the same species. However, if so, the real effects of CLPs on F. oxysporum f. sp. physali and their potential as a biocontrol tool against Physalis-FW have not been elucidated. In this study the CLPs profile of Bs006 in liquid culture and antagonist-plant-pathogen interactions were characterized. Also, the potential effects of supernatant free of bacteria against F. oxysporum f. sp. physali and FW were explored and compared with the effects of pure CLPs. Ultraperformance liquid chromatography-electrospray ionization-mass spectrometry analysis revealed the capacity of Bs006 to synthesize homologous compounds of iturins, surfactins, and fengycins in liquid culture and on the inhibition zone against F. oxysporum f. sp. physali in dual confrontation tests. Bs006 supernatant reduced the germination and growth of F. oxysporum f. sp. physali and caused vacuolization, swelling, and lysis of F. oxysporum f. sp. physali cells in a concentration-dependent manner. Pure fengycins affected the development of F. oxysporum f. sp. physali from 11 mg/liter and iturins from 21 mg/liter. In a gnotobiotic system, Bs006 colonized the root surface of golden berry, inhibited the growth of F. oxysporum f. sp. physali, and produced CLPs. Individual application of Bs006 and supernatant protected the plants from F. oxysporum f. sp. physali infections by 37 to 53%, respectively. Meanwhile, fengycins reduced the disease progress by 39%. These results suggest further studies to select an optimum combination of Bs006 and supernatant or CLPs, which might be a good option as biofungicide against F. oxysporum f. sp. physali.

Multiple phosphorylated protein selective analysis via fluorous derivatization and liquid chromatography-electrospray ionization-mass spectrometry analysis

Post-translational modification of proteins is involved in protein function and higher-order structure. Among such modification, phosphorylation is an important intracellular signal transduction pathway. Many studies on phosphorylated protein analysis using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) have been developed. However, there are few reports on the analysis of highly phosphorylated proteins because of their handling difficulty. Hence, we developed an analytical method that converts multiple phosphate groups contained in the peptides into perfluoroalkyl groups for selective analysis using fluorous affinity. Here, tryptic digested β-casein fragment peptides [RELEELNVPGEIVE(pSer)L(pSer)(pSer)(pSer)EESITR and FQ(pSer)EEQQQTEDELQDK] were used as model phosphorylated peptides. 1H,1H,2H,2H-Perfluorooctanethiol (PFOT) and 2,2,2-trifluoroethanethiol (TFET) were used as derivatization reagents for mono-phosphorylated peptides and multi-phosphorylated peptides, respectively, to derivatize via β-elimination/Michael addition. The derivatives were analyzed by LC-ESI-MS. A fluorous LC column is typically used to selectively retain the fluorous-derivatized peptides, which are expected to be separated from contaminants and non-phosphorylated peptides. When this method was applied to β-casein, TFET- and PFOT-derivatized peptides were strongly retained in the fluorous LC column and clearly separated from non-phosphorylated peptides on the chromatogram. Therefore, the developed method enables quantification of mono- and multi-phosphorylated peptides and is suitable for application in proteomics.

Microwave-assisted aqueous two-phase extraction of phenolic compounds from Ribes nigrum L. and its antibacterial effect on foodborne pathogens

Microwave-assisted ammonium sulfate/ethanol aqueous two-phase system was used to extract the phenolic compounds from blackcurrant (Ribes nigrum L.) fruits. After purification with AB-8 macroporous resin, the phenolics (RMAP) was obtained with a purity of 80.50 ± 0.26%. The minimum inhibitory concentration (MIC) and inhibition zone tests showed that RMAP exhibited more potent antibacterial properties against Staphylococcus aureus than against Escherichia coli and Salmonella typhimurium. Additionally, high-performance liquid chromatography-electrospray ionization-mass spectrometry analysis confirmed RMAP was mainly composed of four phenolic compounds including kaempferol-3-O-rutinoside, rutin, cyanidin-3-O-glucoside (C3G) and hyperoside. MIC and time-kill curve assays indicated that C3G displayed the highest inhibitory activity on S. aureus among four compounds. Moreover, crystal violet and MTT assays showed that C3G exhibited obviously inhibitory effect on fresh biofilm formation of S. aureus. Transmission electron microscopy observation, alkaline phosphatase, lactate dehydrogenase, protein and DNA leakage assays verified that C3G damaged cell wall and membrane of S. aureus. On the other hand, UV absorption spectroscopy and DNA cleavage assay revealed that C3G was able to interact with DNA in the form of chimera, but had no direct effect on DNA cleavage. Whereas, DNA topoisomerase IV (DNA Topo IV) inhibition assay and molecular docking model manifested that C3G could combine with DNA Topo IV to affect its activity and further restrained the synthesis of DNA. These results indicated that the antibacterial mechanism of C3G against S. aureus was mainly related to its inhibiting biofilm formation, disrupting cell structure and resisting DNA Topo IV activity.

Degradation of iohexol by UV/chlorine process and formation of iodinated trihalomethanes during post-chlorination

Degradation kinetics of iohexol by UV/chlorine advanced oxidation process (AOP) and the formation of iodinated trihalomethanes (I-THMs) during post-chlorination were investigated in this study. Iohexol, a commonly detected iodinated contrast media in water, can be effectively removed during UV/chlorine process with pseudo-first-order reaction kinetics due to the combination of UV photolysis and oxidation of hydroxyl radicals. The second-order rate constant between iohexol and hydroxyl radicals was determined as 3.8×109M−1s−1 by competition kinetic experiment. Five intermediates were identified by ultra performance liquid chromatography–electrospray ionization-mass spectrometry analysis and degradation pathways of iohexol during UV/chlorine were proposed. Effects of chlorine dose, pH and bromide concentration on iohexol degradation and I-THM formation during post-chlorination were also studied. The results showed that iohexol degradation was accelerated with the increase of chlorine concentration as well as the decrease of pH and bromide concentration. On the other hand, I-THM formation from post-chlorination of UV/chlorine treated iohexol favored relatively low chlorine doses, high bromide concentrations at circumneutral conditions. Raw water experiments showed that I-THM formation after post-chlorination of UV/chlorine-treated iohexol was lower compared to that from UV irradiation, indicating that UV/chlorine is superior to UV alone in controlling I-THM formation.

Quantitative liquid chromatography–electrospray ionization-mass spectrometry analysis of amine-containing metabolites derivatized with cyanuric chloride and methylamine isotopologues

Electrospray ionization (ESI) is the most useful interface for mass spectrometry associated with liquid chromatography (LC). However, analyses of polar metabolites become a challenge because the high polarity impairs the separation of metabolites in reversed-phase liquid chromatography and ionization of metabolites by ESI. In this article, we have used cyanuric chloride to couple the amine-containing molecules with methylamine and found that both resolution on LC chromatogram and ionization by ESI are greatly improved. Derivatives would be obtained in 2-h coupling reactions, and then resolved by LC–ESI-MS in 15min for each sample. Most amino acids can be quantified with linear range from 1nM to 1μM and with an R2 above 0.979. Although reversed-phase chromatography is suitable for resolving the derivatives, phenyl columns with methanol elution provide optimal separation and signal intensity. Moreover, most structural isomers are well separated following cyanuric chloride and methylamine derivatization. Instead of synthesizing a stable isotope-labeled cyanuric chloride, we can take advantage of using commercially available methyl-d3-amine for a novel stable isotope-coded derivatization method. Each metabolite can be directly quantified by the peak intensity ratio of each derivative isotopologue pair in a single LC–MS analysis. The coupling reactions are relatively easy and accessible to most investigators to generate multiple stable isotope-labeled derivatives of amine-containing compounds for a differential metabolomic analysis.

Chemical Constituents of Gold‐red Apple and Their α‐Glucosidase Inhibitory Activities

Ten compounds were isolated and purified from the peels of gold-red apple (Malus domestica) for the 1st time. The identified compounds are 3β, 20β-dihydroxyursan-28-oic acid (1), 2α-hydroxyoleanolic acid (2), euscaphic acid (3), 3-O-p-coumaroyl tormentic acid (4), ursolic acid (5), 2α-hydroxyursolic acid (6), oleanolic acid (7), betulinic acid (8), linolic acid (9), and α-linolenic acid (10). Their structures were determined by interpreting their nuclear magnetic resonance and mass spectrometry (MS) spectra, and by comparison with literature data. Compound 1 is new, and compound 2 is herein reported for the 1st time for the genus Malus. α-Glucosidase inhibition assay revealed 6 of the triterpenoid isolates as remarkable α-glucosidase inhibitors, with betulinic acid showing the strongest inhibition (IC50 = 15.19 μM). Ultra-performance liquid chromatography-electrospray ionization MS analysis of the fruit peels, pomace, flesh, and juice revealed that the peels and pomace contained high levels of triterpenes, suggesting that wastes from the fruit juice industry could serve as rich sources of bioactive triterpenes.

Differential activation of sporamin expression in response to abiotic mechanical wounding and biotic herbivore attack in the sweet potato.

BackgroundPlants respond differently to mechanical wounding and herbivore attack, using distinct pathways for defense. The versatile sweet potato sporamin possesses multiple biological functions in response to stress. However, the regulation of sporamin gene expression that is activated upon mechanical damage or herbivore attack has not been well studied.ResultsBiochemical analysis revealed that different patterns of Reactive oxygen species (ROS) and antioxidant mechanism exist between mechanical wounding (MW) and herbivore attack (HA) in the sweet potato leaf. Using LC-ESI-MS (Liquid chromatography electrospray ionization mass spectrometry analysis), only the endogenous JA (jasmonic acid) level was found to increase dramatically after MW in a time-dependent manner, whereas both endogenous JA and SA (salicylic acid) increase in parallel after HA. Through yeast one-hybrid screening, two transcription factors IbNAC1 (no apical meristem (NAM), Arabidopsis transcription activation factor (ATAF), and cup-shaped cotyledon (CUC)) and IbWRKY1 were isolated, which interact with the sporamin promoter fragment of SWRE (sporamin wounding-responsive element) regulatory sequences. Exogenous application of MeJA (methyl jasmonate), SA and DIECA (diethyldithiocarbamic acid, JAs biosynthesis inhibitor) on sweet potato leaves was employed, and the results revealed that IbNAC1 mediated the expression of sporamin through a JA-dependent signaling pathway upon MW, whereas both IbNAC1 and IbWRKY1 coordinately regulated sporamin expression through JA- and SA-dependent pathways upon HA. Transcriptome analysis identified MYC2/4 and JAZ2/TIFY10A (jasmonate ZIM/tify-domain), the repressor and activator of JA and SA signaling among others, as the genes that play an intermediate role in the JA and SA pathways, and these results were further validated by qRT-PCR (quantitative real-time polymerase chain reaction).ConclusionThis work has improved our understanding of the differential regulatory mechanism of sporamin expression. Our study illustrates that sweet potato sporamin expression is differentially induced upon abiotic MW and biotic HA that involves IbNAC1 and IbWRKY1 and is dependent on the JA and SA signaling pathways. Thus, we established a model to address the plant-wounding response upon physical and biotic damage.

In Vitro and In Vivo Characterization of Plant Growth Promoting Bacillus Strains Isolated from Extreme Environments of Eastern Algeria

This report is to our knowledge the first to study plant growth promotion and biocontrol characteristics of Bacillus isolates from extreme environments of Eastern Algeria. Seven isolates of 14 (50 %) were screened for their ability to inhibit growth of some phytopathogenic fungi on PDA and some roots exudates. The bacteria identification based on 16S r-RNA and gyrase-A gene sequence analysis showed that 71 % of the screened isolates belonged to Bacillus amyloliquefaciens and the rest were closely related to B. atrophaeus and B. mojavensis. Most of them had high spore yields (22 × 10(8)-27 × 10(8) spores/ml). They produced protease and cellulase cell wall-degrading enzymes while the chitinase activity was only observed in the B. atrophaeus (6SEL). A wide variety of lipopeptides homologous was detected by liquid chromatography-electrospray ionization-mass spectrometry analysis. Interestingly, some additional peaks with new masses were characterized, which may correspond to new fengycin classes. The isolates produced siderophores and indole-3- acetic acid phytohormone. The greenhouse experiment using a naturally infested soil with Sclerotonia sclerotiorum showed that the B. atrophaeus (6SEL) significantly increased the size of the chickpea plants and reduced the stem rot disease (P < 0.05). These results suggest that these isolates may be used further as bio-inoculants to improve crop systems.

Effects of the Veterinary Pharmaceutical Salinomycin and its Formulation on the Plant Brassica rapa

Veterinary pharmaceuticals are emerging contaminants found throughout the environment, and their presence and effects are a matter of concern. The purpose of this study was to compare the phytotoxicity of salinomycin (pure compound = 96 %) and Sacox 120 (formulated product = 120 g salinomycin/kg) to the plant species Brassica rapa as well as to investigate salinomycin persistence in soil. Calculated EC/IC(50) values for salinomycin and Sacox 120 were 1.10 and 2.88 and 2.19 and 18.03 mg/kg, respectively, based on salinomycin concentration. For exposure of B. rapa to salinomycin, significant adverse effects were observed for growth end points at the greater concentrations. For the reproduction end point (i.e., number of buds), as well as root length and wet mass, significant differences were observed at the lower concentrations (stimulating growth) and adverse effects at the greater concentrations. This study confirmed that the toxic effects of Sacox 120 are attributable to the active ingredient salinomycin. Liquid chromatography-electrospray ionization-mass spectrometry analyses confirmed that exposure concentrations of salinomycin were 90 and 83 % of the nominal concentrations, respectively, in the soils amended with either pure or formulated product. At the end of the experiment, after 14 days, salinomycin concentrations for both tests (salinomycin and Sacox 120) decreased to 6.2 and 5.8 % of the nominal exposure concentrations, respectively. Detected salinomycin concentrations in plant shoots ranged from 3.47 to 41.0 ng/g dry shoot. This study shows the importance of using plants as tools to evaluate environmental risk and as a bridge to relate environment and human health risks.

Biodegradation and removal of 3,4-dichloroaniline by Chlorella pyrenoidosa based on liquid chromatography-electrospray ionization-mass spectrometry

3,4-Dichloroaniline (3,4-DCA), widely used in the synthesis of dyes, textile and herbicides, is toxic to living organisms. The purpose of this study was to investigate the capability of green algae in degrading and removing 3,4-DCA in water. An environmentally ubiquitous green alga Chlorella pyrenoidosa was isolated from fresh aquatic environment. Then unicellular alga was incubated with 3,4-DCA at a concentration of 4.6 μg/ mL in water. The residual concentration of 3,4-DCA in the medium and the metabolites were analyzed. A removal percentage of 78.4 % was obtained over a 7-day period. Two major metabolites with less toxicity were identified as 3,4-dichloroformanilide and 3,4-dichloroacetanilide from the liquid chromatography-electrospray ionization-mass spectrometry analysis. The application of microalga C. pyrenoidosa may have potential for removing the environmental pollutant in aquatic environment.

Isolation of phlorotannins from Eisenia bicyclis and their hepatoprotective effect against oxidative stress induced by tert-butyl hyperoxide.

Eisenia bicyclis (Kjellman) Setchell is a common brown alga that inhabits the middle Pacific coast around Korea and Japan. In this study, the ethanol extract and its serial solvent fractions were prepared from fresh E. bicyclis, and their hepatoprotective effects were investigated against hepatotoxicity in tert-butyl hyperoxide(t-BHP)-injured HepG2 cells. When these samples were used at a dose of 10-40 μg/mL⁻¹, they significantly protected the t-BHP-induced cell death in HepG2 cells. Among fractions, ethyl acetate fraction (EF) and n-butanol extract (BF) exhibited potent hepatoprotective activities (62.60% for EF and 64.86% for BF) in t-BHP-injured HepG2 cells at a concentration of 10 μg/mL⁻¹. To find the potential factors for this activity, the samples were characterized on total phenolics, chlorophylls, carotenoids, and radical scavenging activity. Among them, EF showed the highest content of total phenolics and the strongest antioxidant activity both in on- and offline assays. Five phlorotannin compounds, oligomers of phloroglucinol, were isolated chromatographically from this fraction and structurally identified by (1)H-NMR and liquid chromatography-electrospray ionization-mass spectrometry analyses as eckol(1), 6,6'-bieckol(2), 8,8'-bieckol(3), dieckol(4), and phlorofucofuroeckol A(5). Compound 5 among five purified compounds showed the strongest protective activity (45.54%) at a concentration of 10 μM. At the high dose (40 μM), the protective activities of three compounds (compound 2, 4, and 5) were higher than that of quercetin treated with 10 μM concentration. Therefore, we can speculate that they can be developed as potential candidates for natural hepatoprotective agents.

Enantiomeric Purity of Chiral Derivatizing Reagents for Enantioresolution

BioanalysisVol. 3, No. 18 EditorialEnantiomeric purity of chiral derivatizing reagents for enantioresolutionRavi BhushanRavi BhushanDepartment of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247667, India. Search for more papers by this authorEmail the corresponding author at rbushfcy@iitr.ernet.inPublished Online:26 Sep 2011https://doi.org/10.4155/bio.11.211AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail View articleKeywords: chiral derivatizing reagentsenantiomeric purityenantioresolutionHPLCracemic drugsBibliography1 Bhushan R, Martens J. Amino Acids: Chromatographic Separation and Enantioresolution. HNB Publishing, NY, USA, 32–35 (2010).Google Scholar2 Bhushan R, Brückner H. Use of Marfey’s reagent and analogs for chiral amino acid analysis: assessment and applications to natural products and biological systems. J. Chromatogr. 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Enantioresolution of five β-blockers by reversed-phase high-performance liquid chromatography using fifteen chiral derivatizing reagents having amino acids or their amides as chiral auxiliaries on cyanuric chloride platform. Biomed. Chromatogr. DOI: 10.1002/bmc.1653 (2011) (Epub ahead of print).Google ScholarFiguresReferencesRelatedDetailsCited BySynthesis and Chiral Separation of Some 4-thioflavones9 February 2021 | Journal of Chromatographic Science, Vol. 59, No. 9Derivatization procedures and their analytical performances for HPLC determination in bioanalysis24 November 2020 | Biomedical Chromatography, Vol. 35, No. 1Bioanalysis and enantioseparation of dl-carnitine in human plasma by the derivatization approachVinod Kumar Vashistha & Ravi Bhushan12 October 2015 | Bioanalysis, Vol. 7, No. 19Quantitative analysis of drugs in biological matrices by HPLC hyphenated to fluorescence detectionEmmanuelle Lipka & Claude Vaccher14 April 2015 | Bioanalysis, Vol. 7, No. 6Preparative Enantioseparation of ( RS )-Baclofen: Determination of Molecular Dissymmetry14 February 2015 | Chirality, Vol. 27, No. 4Quantitative liquid chromatography–electrospray ionization-mass spectrometry analysis of amine-containing metabolites derivatized with cyanuric chloride and methylamine isotopologuesJournal of Chromatography A, Vol. 1388Amino acids as chiral auxiliaries in cyanuric chloride-based chiral derivatizing agents for enantioseparation by liquid chromatography18 August 2014 | Biomedical Chromatography, Vol. 28, No. 11Enantioseparation of amino alcohols by reversed-phase high-performance liquid chromatography using cyanuric chloride-based chiral derivatizing reagents having amino acids and their amides as chiral auxiliariesActa Chromatographica, Vol. 26, No. 3Liquid chromatographic enantioseparation of ( RS )-mexiletine and ( RS )-fluoxetine using chiral derivatizing reagents synthesized with ( S )-naproxen moiety25 May 2014 | Biomedical Chromatography, Vol. 28, No. 6Indirect enantioseparation of selenomethionine by reversed-phase high-performance liquid chromatography using a newly synthesized chiral derivatizing reagent based on ( S )-naproxen moiety21 March 2013 | Biomedical Chromatography, Vol. 28, No. 1LC Enantioseparation of 30-Component Diastereomeric Mixture of Amino Acids and Detection of d-Isomers Using New Reagents with Amines as Chiral Auxiliaries in Cyanuric Chloride18 July 2013 | Chromatographia, Vol. 76, No. 17-18LC-MS Bioanalysis of Chiral Compounds30 August 2013Indirect enantioseparation of proteinogenic amino acids using naproxen-based chiral derivatizing reagent and HPLC7 December 2012 | Biomedical Chromatography, Vol. 27, No. 6Application of optically pure amines as chiral auxiliaries to develop trichloro- s -triazine-based new chiral derivatizing reagents for reversed-phase high-performance liquid chromatographic enantioseparation of dl -selenomethionine26 March 2013 | Biomedical Chromatography, Vol. 15 Vol. 3, No. 18 Follow us on social media for the latest updates Metrics Downloaded 175 times History Published online 26 September 2011 Published in print September 2011 Information© Future Science LtdKeywordschiral derivatizing reagentsenantiomeric purityenantioresolutionHPLCracemic drugsFinancial & competing interests disclosureThe author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. 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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.

Structural Characterization of Anti-HIV Drug Candidate PA-457 [3-O-(3′,3′-Dimethylsuccinyl)-betulinic acid] and Its Acyl Glucuronides in Rat Bile and Evaluation of in Vitro Stability in Human and Animal Liver Microsomes and Plasma

PA-457 [3-O-(3',3'-dimethylsuccinyl)-betulinic acid] represents a new class of anti-HIV drug candidates termed maturation inhibitors. After oral administration to rats, PA-457 was metabolized to several glucuronide conjugates and mainly eliminated into rat bile. Liquid chromatography-electrospray ionization-mass spectrometry analysis showed that the glucuronidation products of PA-457 were acyl glucuronides including one di-glucuronide, di-PA-457G, and two mono-glucuronides, referred to as mono-PA-457G (I) and mono-PA-457G (II), respectively. In-source fragmentation of MS spectra supported the conclusion that mono-PA-457G (I) was glucuronidated at the C-28 carboxyl of PA-457, whereas mono-PA-457G (II) was conjugated at the dimethylsuccinic acid side chain of the C-3 position. Quantification demonstrated that the predominant glucuronide of PA-457 in rat bile was mono-PA-457G (I) with lower amounts of mono-PA-457G (II) and di-PA-457G. In vitro stability indicated that the mono-acyl glucuronides of PA-457 were not degraded after incubation with 0.1 M phosphate buffer (pH 4, 7.4 and 9), plasma (human, rat, and mouse), and UDP-glucuronosyltransferase reaction media (without uridine 5'-diphosphoglucuronic acid) with microsomes (human, rat, and mouse liver microsomes), respectively, whereas the minor diglucuronide was unstable in rodent liver microsomes. All glucuronides of PA-457 could be hydrolyzed both by beta-glucuronidase and alkaline (1 M NaOH). Minor putative acyl migration products were slowly formed at pH 9, suggesting that the acyl glucuronides of PA-457 have relatively high in vitro stability.

Biosynthesis of chloro-beta-hydroxytyrosine, a nonproteinogenic amino acid of the peptidic backbone of glycopeptide antibiotics.

The role of the putative P450 monooxygenase OxyD and the chlorination time point in the biosynthesis of the glycopeptide antibiotic balhimycin produced by Amycolatopsis balhimycina were analyzed. The oxyD gene is located directly downstream of the bhp (perhydrolase) and bpsD (nonribosomal peptide synthetase D) genes, which are involved in the synthesis of the balhimycin building block beta-hydroxytyrosine (beta-HT). Reverse transcriptase experiments revealed that bhp, bpsD, and oxyD form an operon. oxyD was inactivated by an in-frame deletion, and the resulting mutant was unable to produce an active compound. Balhimycin production could be restored (i) by complementation with an oxyD gene, (ii) in cross-feeding studies using A. balhimycina JR1 (a null mutant with a block in the biosynthesis pathway of the building blocks hydroxy- and dihydroxyphenylglycine) as an excretor of the missing precursor, and (iii) by supplementation of beta-HT in the growth medium. These data demonstrated an essential role of OxyD in the formation pathway of this amino acid. Liquid chromatography-electrospray ionization-mass spectrometry analysis indicated the biosynthesis of completely chlorinated balhimycin by the oxyD mutant when culture filtrates were supplemented with nonchlorinated beta-HT. In contrast, supplementation with 3-chloro-beta-HT did not restore balhimycin production. These results indicated that the chlorination time point was later than the stage of free beta-HT, most likely during heptapeptide synthesis.