Derivatization-independent cholesterol analysis in crude lipid extracts by liquid chromatography/mass spectrometry: applications to a rabbit model for atherosclerosis

Direct measurement of various sterols in crude lipid extracts in a single experiment from limited biological samples is challenging. Current mass spectrometry (MS) based approaches usually require chemical derivatization before subjecting to MS analysis. Here, we present a derivatization-independent method for analyzing various sterols, including cholesterol and its congeners, using liquid chromatography and atmospheric pressure chemical ionization mass spectrometry. Based on the specific tandem mass spectrometry pattern of cholesterol, multiple reaction monitoring (MRM) transitions were used to quantify free cholesterol and its fatty acyl esters. Several cholesterol oxidation products could also be measured using the upfront liquid chromatography separation and specific MRM transitions. The method was validated alongside established enzymatic assays in measuring total cholesterol. As a proof of concept, we analyzed plasma sterols in rabbits administrated with a high cholesterol diet (HCD) which is a classical atherosclerotic model. Free cholesterol, cholesterol esters, 7-hydroxycholesterol, and 7-ketocholesterol were elevated in plasma of rabbits on HCD. This method could also serve as an excellent tool for quantitative analysis of other sterols such as ergosterol and sitosterol in other organisms beside mammalian. In Saccharomyces cerevisiae, our results indicated dramatic increases of the ratio of ergosterol esters to free ergosterol in both yeh2Δ and tgl1Δ cells, which are consistent with the function of the respective enzymes.     

Profiling of grape monoterpene glycosides (aroma precursors) by ultra‐high performance‐liquid chromatography‐high resolution mass spectrometry (UHPLC/QTOF)

A ‘suspect screening analysis’ method for grape metabolomics by ultra‐high performance‐liquid chromatography (UHPLC) and high‐resolution quadrupole‐time of flight (QTOF) mass spectrometry was recently developed. This method was applied to study grape monoterpene glycosides, the main grape aroma precursors. Since standard compounds were not available, they were tentatively identified by overlapping various analytical approaches, in agreement with the indications recommended in mass spectrometry (MS)‐based metabolomics. Accurate mass and isotopic pattern, MS/MS fragmentation, correlation between fragments observed and putative structures and between liquid chromatography coupled with mass spectrometry (LC/MS) and gas chromatography/mass spectrometry signals were studied. Seventeen monoterpene glycosides were identified without performing the hydrolytic artifacts commonly used to study these compounds which may affect sample profile. This is the first time that a detailed study of these aroma precursors has been carried out by direct LC/MS analysis. Copyright © 2014 John Wiley & Sons, Ltd.     

Sterols of scallop. I. Application of hydrophobic sephadex derivatives to the resolution of a complex mixture of marine sterols.

Column chromatography on a hydroxyalkoxypropyl derivative of Sephadex LH-20 and on Anasil B has been applied to the resolution of complex marine sterol mixture in combination with argentation thin-layer chromatography and gas chromatography. This approach permits isolation in quantity of individual sterols from a complex mixture and separation of sterol mixtures that were not resolved without the modified Sephadex step. Seventeen sterols were detected in the scallop Placopecten magellanicus. 24-Methyl-cholesterol, 24-ethyl-cholesterol, 24-methyl-22-dehydrocholesterol and 24-ethyl-22-dehydrocholesterol, i.e. sterols whose configuration at C-24 had not been definitively established, were isolated in sufficient quantities for further study by nuclear magnetic resonance spectroscopy.     

Determination of ergosterol as an indicator of fungal biomass in various samples using non-discriminating flash pyrolysis

Ergosterol is the major sterol constituent of most fungi. Since it is present in negligible amounts in higher plants, it can be used as a chemical marker for the presence of fungal contamination. A number of different ergosterol assays have been developed for the quantification of fungi in various samples. The paper presents the development of a new method for ergosterol detection based on the combination of non-discriminating flash pyrolysis with gas chromatography/mass spectrometry (Py-GC/MS). The design of the non-discriminating Py-GC/MS systems assures efficient transfer of high-molecular-weight pyrolysis products to the GC column for separation, followed by analyte detection by MS. The method was tested on different types of samples, including baker's yeast (Saccharomyces cerevisiae), moldy bread, indoor dust, and a leaf infected with powdery mildew. Ergosterol was detected in all these samples at levels ranging from approximately 4mg/g for the baker's yeast to approximately 6mug/g for household dust. The main benefits of non-discriminating pyrolysis over other techniques include elimination of the need for sample preparation, small sample size required and short analysis time.     

皱瘤海鞘的化学成分研究

从中国广东惠州大亚湾海域采集的皱瘤海鞘的甲醇-氯仿提取物中分离出混合甾醇和神经酰胺两类化合物,混合甾醇经波谱分析和GC/MS联机分析,发现基主要由9种甾醇组成,含量约为甲醇-氯仿提取物的20%。通过波谱分析（如IR,^1HNMR,^13CNMR（DEPT）、^1H-^1H COSY、RCT、FABMS）和GC/MS分析证明神经酰胺结构是由4个同系物组成,含量为提取物的0.1%。同时也初步探讨了共生的皱瘤海鞘与冠瘤海鞘化学成分差异的原因。     

Explanation through density functional theory of the unanticipated loss of CO2 and differences in mass fragmentation profiles of ritonavir and its rCYP3A4‐mediated metabolites

In the present study, the metabolism of ritonavir was explored in the presence of rCYP3A4 using a well‐established strategy involving liquid chromatography–mass spectrometry (LC–MS) tools. A total of six metabolites were formed, of which two were new, not reported earlier as CYP3A4‐mediated metabolites. During LC–MS studies, ritonavir was found to fragment through six principal pathways, many of which involved neutral loss of CO₂, as indicated through 44‐Da difference between masses of the precursors and the product ions. This was unusual as the drug and the precursors were devoid of a terminal carboxylic acid group. Apart from the neutral loss of CO₂, marked differences were also observed among the fragmentation pathways of the drug and its metabolites having intact N‐methyl moiety as compared to those lacking N‐methyl moiety. These unusual fragmentation behaviours were successfully explained through energy distribution profiles by application of the density functional theory. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of fluorotelomer alcohols by liquid chromatography/tandem mass spectrometry in water

Fluorotelomer alcohols (FTOHs) are important polyfluorinated raw materials that belong to the general category of perfluoroalkyl substances (PFAS). PFAS, including perfluoroalkyl carboxylates (PFCAs) and perfluoroalkyl sulfonates, have recently attracted considerable attention because they are persistent and found globally in the environment. FTOHs are precursors that may degrade in the environment to PFCAs. The development of analytical methods for determination FTOHs in environmental samples is necessary to determine the environmental presence of FTOHs. This work presents the development and validation of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of FTOHs (6-2, 8-2, 10-2) in aqueous samples. Chromatographic conditions were optimized in order to obtain focused FTOH chromatographic peaks. The mobile phase and mass spectrometric conditions were optimized to enable formation of deprotonated FTOH molecules in the negative ion electrospray mode. Two extraction methods were investigated using acetonitrile and methyl tert-butyl ether (MTBE). These methods were validated for a range of environmental water samples fortified with FTOHs at three different levels. Both extraction methods resulted in recoveries from 70 to 120%. Detection limits of FTOHs were estimated to be approximately 0.09 ng/mL for LC/MS/MS detection. An LC/MS method was also developed for FTOH determination with an estimated 1.2 ng/mL limit of detection. Various sample storage scenarios were investigated. It was determined that the aqueous samples of FTOHs are best preserved by storing them frozen in sealed vials with aluminum foil lined septa.     

Design and Synthesis of New Bile AcidSterol Conjugates Linked via 1,2,3‐Triazole Ring

A new steroid conjugates have been obtained from bile acids and sterol derivatives using ‘click chemistry’. Intermolecular 1,3‐dipolar cycloaddition of the propargyl ester of bile acids (lithocholic, deoxycholic, and cholic acid) and azide derivatives of sterols (ergosterol and cholesterol) gave a new bile acid? sterol conjugates linked with a 1,2,3‐triazole ring. The structures of all products were confirmed by spectroscopic (¹H‐ and ¹³C‐NMR, and FT‐IR) analyses, mass spectrometry (ESI‐MS), and in silico biological activity evaluation methods (PASS), as well as PM5 semiempirical methods.     

Development of supercritical fluid chromatography coupled with mass spectrometry method for characterization of a nonionic surfactant and comparison with liquid chromatography coupled with mass spectrometry method

The supercritical fluid chromatography coupled with mass spectrometry (SFC‐MS) method and liquid chromatography coupled with mass spectrometry (LC‐MS) method were developed for the separation and characterization of poly (ethylene oxide) methyl glucose sesquistearate (PEO‐Glu‐sesquistearate). The products of PEO‐Glu‐sesquistearate are composed of complex oligomers. The relationship between molecular structure of these oligomers and chromatographic retention behavior in both SFC and LC were discussed and compared. As compared with LC, hydrophobic moieties of compounds favor the fast elution in SFC. The different series can be better separated by LC, while the homologues compounds in same series can be better separated by SFC, and SFC‐MS provided more comprehensive structural information. Different series such as PEO‐distearate, PEO‐stearate, PEO, PEO‐Glu‐tetrastearate, PEO‐Glu‐tristearate, PEO‐Glu‐distearate, PEO‐Glu‐stearate, and PEO‐Glu were identified by MS/MS.     

Profiling of hydroxycinnamic acid amides in Arabidopsis thaliana pollen by tandem mass spectrometry

Phenylpropanoid polyamine conjugates are widespread in plant species. Their presence has been established in seeds, flower buds, and pollen grains. A biosynthetic pathway proposed for hydroxycinnamoyl spermidine conjugates has been suggested for the model plant Arabidopsis thaliana with a central acyl transfer reaction performed by a BAHD-like hydroxycinnamoyl transferase. A detailed liquid chromatography (LC)–electrospray ionization–mass spectrometry- and tandem-mass-spectrometry (MS/MS)-based survey of wild-type and spermidine hydroxycinnamoyl transferase (SHT) mutants identified more than 30 different bis- and tris-substituted spermidine conjugates, five of which were glycosylated, in the methanol-soluble fraction of the pollen exine. On the basis of characterized fragmentation patterns, a high-throughput LC–MS/MS method for highly sensitive HCAA relative quantification (targeted profiling) was developed. Only minor qualitative and quantitative differences in the pattern of bis-acyl spermidine conjugates in the SHT mutant compared to wild-type plants provide strong evidence for the presence of multiple BAHD-like acyl transferases and suggest a much more complex array of enzymatic steps in the biosynthesis of these conjugates than previously anticipated.     

Liquid chromatography/multi-stage mass spectrometry of bisphenol A and its halogenated derivatives

We report a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for analyzing bisphenol A (BPA) and its halogenated derivatives. Since only tetrachlorobisphenol A and tetrabromobisphenol A (TBBPA) are commercially available, mono-, di- and trichlorobisphenol A were synthesized and purified in order to be used as analytical standards. This family of compounds was studied using electrospray ionization and an ion trap mass analyzer in order to characterize the new compounds and to propose fragmentation pathways. Multi-stage mass spectrometry was used to confirm the genealogical relationship between the ions. Some product ions were traced from MS/MS to MS(4) and the labelled compounds BPA-d(16) and TBBPA-(13)C(12) were used to assign some product ion structures. In general, the deprotonated molecule [M--H](-) loses a methyl and/or a halogen group during both MS/MS and MS(3), while the neutral loss of CO was also observed in MS(3) spectra. We selected the most intense and characteristic MS/MS transitions for LC/MS/MS analysis. LC separation was performed in a reversed-phase column; methanol/water (no additives) was used as the mobile phase in gradient elution mode; and BPA-d(16) was chosen as the internal standard. Solid-phase extraction (SPE) was used to pre-concentrate and to clean up water samples. The SPE LC/MS/MS method allows BPA and its halogenated derivatives to be detected at a few parts-per-billion (ppb) in surface water.     

Analysis of free and esterified sterols in fats and oils by flash chromatography, gas chromatography and electrospray tandem mass spectrometry.

A method for the analysis of free and esterified sterols has been developed. Fat or oil samples were separated on solid-phase extraction silica gel columns into a sterol ester fraction, a fraction of triacylglycerols, and a free sterol fraction containing partial acylglycerols and residual triacylglycerols. Sterol esters and acylglycerols of the free sterol fraction were transesterified to methyl esters. The fatty acid methyl esters from sterol ester fraction and the free sterols from sterol ester fraction and free sterol fraction were determined by GLC. Precursor ion electrospray MS-MS of sterol fragment ions of sterol ester fractions were recorded and used for determination of sterol ester proportions in butterfat and vegetable oil samples.     

Separation and purification of ergosterol and stigmasterol in Anoectochilus roxburghii (wall) Lindl by high-speed counter-current chromatography

Ergosterol and stigmasterol are the most common phytosterols in the traditional Chinese medicine. They are two major sterol compounds in Anoectochilus roxburghii (wall) Lindl (A. roxburghii) and have been proved to have many important biological activities. A method by using high-speed counter-current chromatography (HSCCC) has been successfully developed for separation and purification of ergosterol and stigmasterol in A. roxburghii simultaneously in this paper. The optimum conditions used in this method were as follows: The two-phase solvent system consisted of n-hexane-ethylacetate-butanol-methanol-water (3.5:0.3:0.5:2.5:0.3, v/v); the rotation speed was 900 rpm; the flow rate of the lower phase was 1.5 mL/min. About 36.5 mg of ergosterol and 43.6 mg of stigmasterol were obtained from 100 g of A. roxburghii. The purity of ergosterol and stigmasterol was examined to be 92.0 and 95.5%, respectively, by using HPLC. The chemical structures of these components were identified by UV spectra, FT-IR, MS, (1) H-NMR and (13) C-NMR. The results demonstrated that high-speed counter-current chromatography was a feasible method to separate and purify ergosterol and stigmasterol from the herb. This separation and purification method was more effective than many other conventional techniques.     

N-(1-piperidinepropionyl)amphotericin B methyl ester (PAME)--a new derivative of the antifungal antibiotic amphotericin B: searching for the mechanism of its reduced toxicity

N-(1-piperidinepropionyl)amphotericin B methyl ester (in short, PAME), a low-toxicity amphotericin B derivative, has been investigated in Langmuir monolayers at the air/water interface alone and in mixtures with cellular membrane sterols (a mammalian sterol, cholesterol, and a fungal sterol, ergosterol) and a model phospholipid (DPPC). The analysis of the strength of interaction between PAME and both sterols as well as DPPC was based, on surface pressure measurements and analysis of the isothermal compressibility (C(s)(-1)), the mean area per molecule (A(12)), the excess free energy of mixing (DeltaG(Exc)) and the total free energy of mixing (DeltaG(M)). It has been found that the interactions between PAME and sterols are attractive; however, their strength is significantly weaker for mixtures of PAME with cholesterol than with ergosterol. This casts light on the improved selectivity of PAME toward fungal cells. The strongest interactions, found for PAME/DPPC mixtures, proved an important role of DPPC in the mechanism of reduced toxicity of PAME as compared to amphotericin B. Due to stable complex formation between PAME and DPPC the antibiotic is immobilized with DPPC molecules, which reduces the concentration of free antibiotic, which is capable of interacting with membrane sterols.     

Antifungal Activities of cis-trans Citral Isomers against Trichophyton rubrum with ERG6 as a Potential Target

Trichophyton rubrum causes ringworm worldwide. Citral (CIT), extracted from Pectis plants, is a monoterpene and naturally composed of geometric isomers neral (cis-citral) and geranial (trans-citral). CIT has promising antifungal activities and ergosterol biosynthesis inhibition effects against several pathogenic fungi. However, no study has focused on neral and geranial against T. rubrum, which hinders the clinical application of CIT. This study aimed to compare antifungal activities of neral and geranial and preliminarily elucidate their ergosterol biosynthesis inhibition mechanism against T. rubrum. Herein, the disc diffusion assays, cellular leakage measurement, flow cytometry, SEM/TEM observation, sterol quantification, and sterol pattern change analyses were employed. The results showed geranial exhibited larger inhibition zones (p < 0.01 or 0.05), higher cellular leakage rates (p < 0.01), increased conidia with damaged membranes (p < 0.01) within 24 h, more distinct shriveled mycelium in SEM, prominent cellular material leakage, membrane damage, and morphological changes in TEM. Furthermore, geranial possessed more promising ergosterol biosynthesis inhibition effects than neral, and both induced the synthesis of 7-Dehydrodesmosterol and Cholesta-5,7,22,24-tetraen-3β-ol, which represented marker sterols when ERG6 was affected. These results suggest geranial is more potent than neral against T. rubrum, and both inhibit ergosterol biosynthesis by affecting ERG6.     

Active site mapping of 2-deoxy-scyllo-inosose synthase, the key starter enzyme for the biosynthesis of 2-deoxystreptamine. Mechanism-based inhibition and identification of lysine-141 as the entrapped nucleophile

A key enzyme in the biosynthesis of clinically important aminoglycoside antibiotics including neomycin, kanamycin, gentamicin, etc. is 2-deoxy-scyllo-inosose synthase (DOIS), which catalyzes the carbocycle formation from d-glucose-6-phosphate to 2-deoxy-scyllo-inosose (DOI). To clarify its precise reaction mechanism and crucial amino acid residues in the active site, we took advantage of a mechanism-based inhibitor carbaglucose-6-phosphate (pseudo-dl-glucose, C-6-P) with anticipation of its conversion to a reactive alpha,beta-unsaturated carbonyl intermediate. It turned out that C-6-P clearly showed time- and concentration-dependent inhibition against DOIS, and the molecular mass of the resulting modified-DOIS with C-6-P was 160 mass units larger than that of native DOIS. Thus, the expected alpha,beta-unsaturated intermediate appeared to trap a specific nucleophilic group in the active site through the Michael-type 1,4-addition. The covalently modified amino acid residue was determined to be Lys-141 by means of enzymatic digestion and subsequent LC/MS and LC/MS/MS of the digest. Also discussed are the role of Lys-141 in the substrate recognition and the reaction pathway and comparison with evolutionary related dehydroquinate synthase.     

identification of the sterols of the yeasts Saccharomyces cerevisiae and Candida guilliermondii by the mass-spectrometric method

The sterol compositions of mutants of the yeasts ofSaccharomyces cerevisiae andCandida guilliermondii have been characterized by the methods of GLC, TLC, UV spectroscopy, and chromato-mass spectrometry. The possibility has been shown of identifying yeast sterols on the basis of a comparison of the intensities of the peaks of the fragmentary ions having a common nature for all sterols (m/z200). A change in the sterol composition in mutant yeasts as compared with the initial strain has been detected which is obviously connected with the blockage of the routes for the biosynthesis of ergosterol in the yeast cell.Lensovet Leningrad Technological Institute. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 196–201, March–April, 1986.     

Identification of beta-sitosterol, stigmasterol and ergosterin in A. roxburghii using supercritical fluid extraction followed by liquid chromatography/atmospheric pressure chemical ionization ion trap mass spectrometry

beta-Sitosterol and stigmasterol are the most common phytosterols in traditional Chinese medicine. They have been proved to have many important bioactivities. To the best of our knowledge, this is the first report that beta-sitosterol, stigmasterol and ergosterol coexisting in A. roxburghii herbs can be simultaneously extracted by a supercritical fluid extraction (SFE) procedure; then a simple high-performance liquid chromatography/atmospheric pressure chemical ionization ion trap mass spectrometry (HPLC/APCI/MS) method was developed for simultaneous identification and determination of these three compounds. The ion trap MS/MS detector was equipped with an atmospheric pressure chemical ionization source operating in the positive ion mode, APCI(+). The linear responses were obtained in the concentration range of 0.50-150 microg/mL (r = 0.9999) for ergosterol, 5-400 microg/mL (r = 0.9999) for stigmasterol, and 10-2000 microg/mL (r = 0.9998) for beta-sitosterol. An orthogonal L(9) (3(3)) test design was employed for optimization of the SFE process. Under the optimized conditions, i.e. pressure of 25 mPa, temperature of 45 degrees C and ethanol as modifier, the concentrations of sterols in the extract were found to be 2.89% (g/g) for beta-sitosterol, 3.56% (g/g) for stigmasterol and 2.96% (g/g) for ergosterin. The SFE method was also compared with a previously developed Soxhlet extraction. The SFE method produced higher yields of sterols than that of the Soxhlet extraction. The proposed method has been successfully used for identification and quantitation of beta-sitosterol, stigmasterol and ergosterin in a real A. roxburghii sample.     

Simultaneous determination of the urinary metabolites of benzene, toluene, xylene and styrene using high-performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry

A simple and rapid method using reversed-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of the urinary metabolites of benzene, toluene, xylene and styrene in human urine specimens and standard solutions is described. A hybrid quadrupole/time-of-flight (QqTOF) mass spectrometer was compared for the determination of metabolite of aromatic solvents in urine samples. The metabolites selected were: trans,trans-muconic acid, hippuric acid, o-, m- and p-methylhippuric acid and phenylglyoxylic acid. The compounds were well separated from each other on narrow-bore 1-mm i.d. reversed-phase LC C-18 columns. Average recoveries for loading 100 microL of urine samples varied from 88-110% and the quantification limits were less than 30 ng/mL for each analyte (3 ng/mL for trans,trans-muconic acid). The qualitative information obtained (mass accuracy, resolution and full-scan spectra) with the QqTOF mass spectrometer allows a secure identification of analytes in biological matrices.     

Polyoxygenated ergostane-type sterols from the liquid culture of Ganoderma applanatum

A new polyoxygenated ergostane-type sterol, 3β,5α,6β,8β,14α-pentahydroxy-(22E,24R)-ergost-22-en-7-one (1), has been isolated from the liquid culture of the basidiomycete Ganoderma applanatum together with four known sterols, 3β,5α,9α-trihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (2), ergosterol peroxide (3), 6-dehydrocerevisterol (4) and cerevisterol (5). Two of these sterols (2, 4) are reported to have been isolated from this species for the first time. The structures of these compounds were determined by chemical and spectroscopic analyses, including 1D- and 2D-NMR, as well as by comparison of their spectroscopic data with those reported in the literature.