Three dimensions of chromatographic separation, using split-flow two-dimensional liquid chromatography (SF-2D-LC) with two parallel second dimensions, LC × 2LC, combined with quadruple parallel mass spectrometry (LC3MS4) is demonstrated for analysis of NIST SRM 1849a adult/infant formula. The first dimension, 1D, was a conventional non-aqueous reversed-phase (NARP) HPLC separation using two C18 columns in series, followed by detection using an ultraviolet (UV) detector, a fluorescence detector (FLD), with flow then split to a corona charged aerosol detector (CAD), and then dual parallel mass spectrometry (MS), conducted in atmospheric pressure photoionization (APPI) and electrospray ionization (ESI) modes. The first second dimension, 2D(1), UHPLC was conducted on a 50.0 mm C30 column using a NARP-UHPLC parallel gradient for separation of short-chain triacylglycerols (TAGs) from long-chain TAGs, with detection by UV and ESI-MS. The second dimension, 2D(2), UHPLC was conducted using a 100.0 mm C30 column with a NARP-UHPLC parallel gradient for improved separation of TAG isomers, with detection by UV, an evaporative light scattering detector, and high-resolution, accurate-mass (HRAM) ESI-MS. Transferred eluent dilution was used to refocus peaks and keep them sharp during elution in both 2Ds. The separation space in the 2D(2) was optimized using multi-cycle (aka, "constructive wraparound") elution, which employed flow rate programming. In the 1D, calibration lines for quantification of fat-soluble vitamins were constructed. A lipidomics approach to TAG identification and quantification by HRAM-ESI-MS was applied to the 2D(2). These experiments can be represented: LC1MS2 × (LC1MS1 + LC1MS1) = LC3MS4, or three-dimensional liquid chromatography with quadruple parallel mass spectrometry.
- MeSH
- Adult MeSH
- Spectrometry, Mass, Electrospray Ionization * MeSH
- Humans MeSH
- Infant Formula * MeSH
- Triglycerides MeSH
- Chromatography, High Pressure Liquid MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Publication type
- Journal Article MeSH
The present study explores pharmacological potential and phytochemicals profiling of Picrorhiza kurroa extracts against mammalian cancer cell lines and pathogenic microbes. Bioactive extracts from roots of Picrorhiza kurroa were recovered in the methanol, 50% aqueous dichloromethane (50 : 50 v/v) and n-hexane. Antimicrobial activity of the bioactive extracts was assessed against selected strains of bacteria and pathogenic fungi. Aqueous dichloromethane extract showed highest zone of growth inhibition (39.06 ± 1.0 mm) towards Staphylococcus aureus bacteria while methanolic extract showed the lowest inhibition (6.3 ± 4.1 mm) to Escherichia coli bacteria. The tested extracts such as methanol and aqueous dichloromethane exhibited higher inhibition antifungal activity against Aspergillus flavus compared to Fusarium oxysporum. As far as cytotoxicity (MTT assay) of the tested extracts is concerned, n-hexane and aqueous dichloromethane extracts were found to be very active against all cancer cell lines (breast cancer MCF7, MDA-MB-231, SKBR3 and ovarian cancer SKOV3). A preliminary phytochemicals profiling was performed in extracts using GC-MS. Several fractions of active extract were separated with HPLC and analyzed using High Resolution Atmospheric Pressure Chemical Ionization Mass Spectrometry (HR-APCI-MS). Two purified compounds (Dihydromikanolide and 1,3-Dicyclohexyl-4-(cyclohexylimino)-2-(cyclohexylethylamino)-3,4-dihydro-1,3-diazetium) were further evaluated for their anticancer activity against ovarian cancer cell line. Our findings depict that all the tested extracts showed considerable anticancer potential through cell viability assays. The purified compound 1 - Dihydromikanolide from methanolic extract was found to be active against ovarian cancer cells and can be explored as a promising nutra-pharmaceutical candidate against ovarian cancer. However, further studies exploring the molecular pathways and in vivo testing are required.
- MeSH
- Anti-Infective Agents MeSH
- Anticarcinogenic Agents MeSH
- Plants, Medicinal MeSH
- Humans MeSH
- Tumor Cells, Cultured drug effects MeSH
- Ovarian Neoplasms * drug therapy MeSH
- Neoplasms drug therapy MeSH
- Picrorhiza * MeSH
- Antineoplastic Agents MeSH
- Plant Extracts MeSH
- Research MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
Arrayan and peumo fruits are commonly used in the traditional medicine of Chile. In this study, the concentration of the extracts halving the bacterial viability and biofilms formation and disruption of the drug-sensitive and drug-resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa was determined. The chemical composition of extracts was analyzed by high-resolution liquid chromatography coupled with mass spectrometry (U-HPLC/MS). The arrayan extract (Inhibitory concentration IC50 0.35 ± 0.01 mg/mL) was more effective than peumo extract (IC50 0.53 ± 0.02 mg/mL) in the inhibition of S. aureus planktonic cells. Similarly, the arrayan extract was more effective in inhibiting the adhesion (S. aureus IC50 0.23 ± 0.02 mg/mL, P. aeruginosa IC50 0.29 ± 0.02 mg/mL) than peumo extracts (S. aureus IC50 0.47 ± 0.03 mg/mL, P. aeruginosa IC50 0.35 ± 0.01 mg/mL). Both extracts inhibited quorum sensing in a concentration-dependent manner, and the most significant was the autoinducer-2 type communication inhibition by arrayan extract. Both extracts also disrupted preformed biofilm of P. aeruginosa (arrayan IC50 0.56 ± 0.04 mg/mL, peumo IC50 0.59 ± 0.04 mg/mL). However, neither arrayan nor peumo extracts disrupted S. aureus mature biofilm. U-HPLC/MS showed that both fruit extracts mainly possessed quercetin compounds; the peumo fruit extract also contained phenolic acids and phenylpropanoids. Our results suggested that both extracts could be used as natural antimicrobials for some skin and nosocomial infections.
- Publication type
- Journal Article MeSH
Triacylglycerols (TAGs) containing less common fatty acids (FAs) were isolated from the seeds of three plants (Santalum album, Crepis foetida, and Leucas aspera). These FAs had allenic (laballenic acid, Lb) and acetylenic (crepenynic, C; ximenynic acids, Xi) bonds. TAGs were analyzed on reversed-phase and chiral columns. High-resolution tandem mass spectrometry identified TAGs by positive electrospray ionization (ESI+). Twenty-two molecular species of TAGs isolated from the seed oil of Santalum album were separated by RP-HPLC and chiral HPLC methods and identified by positive electrospray ionization tandem MS detection (ESI+-MS). Two major enantiomers, i.e., sn-OOLb and sn-LLLb (O represents oleic acid; and L represents linoleic acid), were synthesized from the appropriate phosphatidylcholines. This allowed the identification of enantiomers after separation by chiral chromatography by tandem mass spectrometry. Similarly, TAGs from the seeds of Crepis foetida, and Leucas aspera were analyzed by reversed-phase chromatography and identified by mass spectrometry. Four enantiomers (sn-OOC, sn-LLC, sn-OOXi, and sn-LLXi) were synthesized. A total of six and three enantiomers of TAGs containing crepenynic and ximenynic acids, respectively, were identified by chiral column analysis. The retention times of TAGs containing allenic and acetylenic bonds were always greater on the reversed-phase column than TAGs with the same number of carbon atoms and the same unsaturation (e.g., LLL versus LLLb). From the chiral column, the regioisomers and enantiomers were eluted in the order of symmetric-asymmetric-asymmetric (i.e., sn-OCO, sn-COO, and sn-OOC). Through tandem mass spectrometry, we were able to identify and distinguish regioisomer [DAG]+-type ions, i.e., [MNH4NH3RCOOH]+, that can be considered diagnostic. Unfortunately, enantiomers and TAGs with the same numbers of carbon atoms and the same unsaturation levels have identical mass spectra, such as LLL and LLLb.
- MeSH
- Alkynes analysis chemistry MeSH
- Chromatography, Liquid MeSH
- Chromatography, Reverse-Phase MeSH
- Phosphatidylcholines chemistry MeSH
- Spectrometry, Mass, Electrospray Ionization MeSH
- Linoleic Acid analysis MeSH
- Oleic Acids analysis MeSH
- Fatty Acids analysis chemistry MeSH
- Seeds chemistry MeSH
- Stereoisomerism MeSH
- Tandem Mass Spectrometry * MeSH
- Triglycerides analysis chemistry isolation & purification MeSH
- Chromatography, High Pressure Liquid * MeSH
- Publication type
- Journal Article MeSH
Streptomyces are among the most promising genera in terms of production ability to biosynthesize a variety of bioactive secondary metabolites with pharmaceutical interest. Coinciding with the increase in genomic sequencing of these bacteria, mining of their genomes for biosynthetic gene clusters (BGCs) has become a routine component of natural product discovery. Herein, we describe the isolation and characterization of a Streptomyces tendae VITAKN with quorum sensing inhibitory (QSI) activity that was isolated from southern coastal part of India. The nearly complete genome consists of 8,621,231bp with a GC content of 72.2%. Sequence similarity networks of the BGCs detected from this strain against the Minimum Information about a Biosynthetic Gene Cluster (MIBiG) database and 3365 BGCs predicted by antiSMASH analysis of publicly available complete Streptomyces genomes were generated through the BiG-SCAPE-CORASON platform to evaluate its biosynthetic novelty. Crude extract analysis using high-performance liquid chromatography connected to high resolution tandem mass spectrometry (HPLC-HRMS/MS) and dereplication through the Global Natural Product Social Molecular Networking (GNPS) online workflow resulted in the identification of cyclic dipeptides (2, 5-diketopiperazines, DKPs) in the extract, which are known to possess QSI activity. Our results highlight the potential of genome mining coupled with LC-HRMS/MS and in silico tools (GNPS) as a valid approach for the discovery of novel QSI lead compounds. This study also provides the biosynthetic diversity of BGCs and an assessment of the predicted chemical space yet to be discovered.
- Publication type
- Journal Article MeSH
Deoxynivalenol (DON) is one of the most common mycotoxins produced by field fungi (especially Fusarium). Contamination of livestock feed is a significant risk factor, especially for pigs that are highly susceptible to the toxic effects of deoxynivalenol. In this study, validated ultra-high performance liquid chromatography (U-HPLC) combined with a HR-Orbitrap-MS analysis method is described for the identification and quantitative determination of the mycotoxin compounds (DON and deepoxy-deoxynivalenol (DOM-1)) in pig colostrum (milk) and serum. Pre-treatment of the samples involved a deproteinisation step with methanol followed by a purification step by solid phase extraction (HLB cartridges). The chromatographic separation was performed on a C18 column with 1.7 μm-particle size using a water-methanol mobile phase. Detection of analytes was achieved on the tandem hybrid mass spectrometer Q Exactive, with a heated electrospray ionisation probe measured in positive mode (H-ESI+). For the confirmation of identification, a mass spectrometer was utilized in the full scan mode with resolving power (PR) = 140,000 (FWHM) and for quantification analysis, it was utilized in the parallel reaction monitoring mode (PRM). The method has been fully validated according to the requirements of Commission Decision 2002/657/EC for confirmatory analyses, plus the addition of a mass accuracy (MA) parameter. For the confirmation of the presence of these analytes in pig colostrum and serum, matching of the retention time with mass accuracy for the precursor ion from MS and product ions from MS/MS was used. A deuterium isotopically labelled internal standard and a matrix-matched calibration curve were employed for quantification. The linear range of quantification was 0.5-20 μg L-1 and the correlation coefficient (R2) was >0.999 for all calibrations. The limit of detection for DON and DOM-1 in colostrum was 0.48 μg L-1 and 0.54 μg L-1, respectively, and in serum 0.24 μg L-1 and 0.36 μg L-1, respectively. The limit of quantification for DON and DOM-1 in colostrum was 0.80 μg L-1 and 0.89 μg L-1, respectively, and in serum 0.39 μg L-1 and 0.60 μg L-1, respectively. The method was successfully evaluated using the obtained samples of pig colostrum and serum.
- MeSH
- Chromatography, Liquid methods MeSH
- Colostrum chemistry MeSH
- Food Contamination analysis MeSH
- Animal Feed MeSH
- Limit of Detection MeSH
- Linear Models MeSH
- Swine MeSH
- Reproducibility of Results MeSH
- Tandem Mass Spectrometry methods MeSH
- Pregnancy MeSH
- Trichothecenes analysis MeSH
- Animals MeSH
- Check Tag
- Pregnancy MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Lipid-like compounds containing a dimethylarsinoyl group, i.e. Me2As(O)-, have been identified by liquid chromatography/inductively coupled plasma mass spectrometry (LC/ICP-MS) and non-aqueous reversed-phase high-performance liquid chromatography (positive and/or negative high-resolution tandem electrospray ionization mass spectrometry (NARP-HPLC/HR-ESI+(-)-MS/MS) from three strains of green algae of the genus Coccomyxa (Trebouxiophyceae, Chlorophyta). The algae were cultivated in a medium containing 10 g arsenic/L, i.e. 133.5 mmol/L of Na2HAsO4.7H2O. After extraction by methyl-tert-butyl ether (MTBE), total lipids were analyzed by ICP-MS or ESI-MS without any further separation or fractionation. A total of 39 molecular species of arsenic triacylglycerols (AsTAG), 15 arsenic phosphatidylcholines (AsPC), 8 arsenic phosphatidylethanolamines (AsPE), 6 arsenic phosphatidylinositols (AsPI), 2 arsenic phosphatidylglycerols (AsPG) and 5 unknown lipids (probably ceramides) were identified. The structures of all molecular species were confirmed by tandem MS. Dry matter of the individual strains contained different amounts of total arsenolipids, i.e. C. elongata CCALA 427 (0.32 mg/g), C. onubensis (1.48 mg/g), C. elongata S3 (2.13 mg/g). On the other hand, there were only slight differences between strains in the relative abundances of individual molecular species. Possible biosynthesis of long-chain lipids with the end group Me2As(O) has also been suggested.
Vernix caseosa, the waxy substance that coats the skin of newborn babies, has an extremely complex lipid composition. We have explored these lipids and identified nonhydroxylated 1-O-acylceramides (1-O-ENSs) as a new class of lipids in vernix caseosa. These ceramides mostly contain saturated C11-C38 ester-linked (1-O) acyls, saturated C12-C39 amide-linked acyls, and C16-C24 sphingoid bases. Because their fatty acyl chains are frequently branched, numerous molecular species were separable and detectable by HPLC/MS: we found more than 2,300 molecular species, 972 of which were structurally characterized. The most abundant 1-O-ENSs contained straight-chain and branched fatty acyls with 20, 22, 24, or 26 carbons in the 1-O position, 24 or 26 carbons in the N position, and sphingosine. The 1-O-ENSs were isolated using multistep TLC and HPLC and they accounted for 1% of the total lipid extract. The molecular species of 1-O-ENSs were separated on a C18 HPLC column using an acetonitrile/propan-2-ol gradient and detected by APCI-MS, and the structures were elucidated by high-resolution and tandem MS. Medium-polarity 1-O-ENSs likely contribute to the cohesiveness and to the waterproofing and moisturizing properties of vernix caseosa.
- MeSH
- Ceramides metabolism MeSH
- Spectrometry, Mass, Electrospray Ionization MeSH
- Mass Spectrometry MeSH
- Skin metabolism MeSH
- Humans MeSH
- Lipids blood MeSH
- Magnetic Resonance Spectroscopy MeSH
- Infant, Newborn MeSH
- Gas Chromatography-Mass Spectrometry MeSH
- Vernix Caseosa metabolism MeSH
- Chromatography, High Pressure Liquid MeSH
- Check Tag
- Humans MeSH
- Infant, Newborn MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Cholesteryl esters of ω-(O-acyl)-hydroxy FAs (Chl-ωOAHFAs) were identified for the first time in vernix caseosa and characterized using chromatography and MS. Chl-ωOAHFAs were isolated using adsorption chromatography on silica gel and magnesium hydroxide. Their general structure was established using high-resolution and tandem MS of intact lipids, and products of their transesterification and derivatizations. Individual molecular species were characterized using nonaqueous reversed-phase HPLC coupled to atmospheric pressure chemical ionization. The analytes were detected as protonated molecules, and their structures were elucidated in the negative ion mode using controlled thermal decomposition and data-dependent fragmentation. About three hundred molecular species of Chl-ωOAHFAs were identified in this way. The most abundant Chl-ωOAHFAs contained 32:1 ω-hydroxy FA (ω-HFA) and 14:0, 15:0, 16:0, 16:1, and 18:1 FAs. The double bond in the 32:1 ω-HFA was in then-7 andn-9 positions. Chl-ωOAHFAs are estimated to account for approximately 1-2% of vernix caseosa lipids.
- MeSH
- Cholesterol Esters metabolism MeSH
- Humans MeSH
- Fatty Acids chemistry metabolism MeSH
- Infant, Newborn MeSH
- Vernix Caseosa metabolism MeSH
- Check Tag
- Humans MeSH
- Infant, Newborn MeSH
- Publication type
- Journal Article MeSH
This study describes the identification of very long chain polyunsaturated fatty acids (VLCPUFAs) in three strains of dinoflagellates (Amphidinium carterae, Cystodinium sp., and Peridinium aciculiferum). The strains were cultivated and their lipidomic profiles were obtained by high resolution mass spectrometry with the aid of positive and negative electrospray ionization (ESI) mode by Orbitrap apparatus. Hydrophilic interaction liquid chromatography (HILIC/ESI) was used to separate major lipid classes of the three genera of dinoflagellates by neutral loss scan showing the ion [M + H-28:8](+), where 28:8 was octacosaoctaenoic acid, and by precursor ion scanning of ions at m/z 407, which was an ion corresponding to the structure of acyl of 28:8 acid (C27H39COO(-)). Based on these analyzes, it was found that out of more than a dozen lipid classes present in the total lipids, only two classes of neutral lipids, i.e. major triacylglycerols and minor diacylglycerols contain VLCPUFAs. In polar lipids, VLCPUFAs were identified only in phosphatidic acid (PA) and phosphatidyl choline (PC) or in their lyso-forms (LPA and LPC). Further analysis of individual lipid classes by reversed-phase high-performance liquid chromatography (RP-HPLC) showed the presence of triacylglycerols (TAGs) containing VLCPUFAs, i.e. molecular species of the sn-28:7/28:8/28:8, sn-26:7/28:7/28:8, or sn-26:7/28:8/28:8 types. These TAGs are the longest and most unsaturated TAGs isolated from a natural source that have yet been synthesized. In the case of PA and PC, tandem MS identified sn-28:8/16:0-PA and sn-28:8/16:0-PC and the corresponding lyso-forms (28:8-LPC and 28:8-LPA). All these results indicate that TAGs containing VLCPUFAs are biosynthesized in dinoflagellates in the same manner as in higher eukaryotic organisms, which means that the PA, after conversion to DAG, serves as a precursor in the biosynthesis of other phospholipids, e.g. PC, and, after further acylation, also of TAG.
- MeSH
- Quinolines MeSH
- Chromatography, Liquid methods MeSH
- Chromatography, Reverse-Phase MeSH
- Dinoflagellida chemistry MeSH
- Phosphatidylcholines analysis MeSH
- Phospholipids MeSH
- Hydrophobic and Hydrophilic Interactions MeSH
- Morphinans MeSH
- Fatty Acids, Unsaturated chemistry isolation & purification MeSH
- Publication type
- Journal Article MeSH
