Studies on neurosteroids. Part XIII. Characterization of catechol estrogens in rat brains using liquid chromatography-mass spectrometry-mass spectrometry

The existence of catechol estrogens in rat brains was clarified using liquid chromatography-atmospheric pressure chemical ionization-ion trap-mass spectrometry-mass spectrometry (LC-APCI-MS2). The catechol estrogens were extracted in the presence of ascorbic acid and then derivatized to acetates with acetic anhydride and pyridine. After a successive purification with silica gel mini-column chromatography, reversed-phase solid-phase extraction and preparative HPLC, the obtained fractions containing the catechol estrogen acetates were subjected to LC-APCI-MS2. 2-Hydroxyestrone, 2-hydroxyestradiol and their 4-hydroxy isomers were identified as acetates by comparison with authentic samples based on their chromatographic behavior and mass spectral data. The derivatization to acetate was useful for the treatment of labile catechol estrogens.     

Studies on neurosteroids X. Determination of pregnenolone and dehydroepiandrosterone in rat brains using gas chromatography-mass spectrometry-mass spectrometry

An assay method for pregnenolone and dehydroepiandrosterone in rat brains is developed using gas chromatography (GC)-electron ionization-mass spectrometry (MS)-MS. The extract of the rat brain homogenate containing deuterated internal standard with organic solvent is purified by silica gel minicolumn chromatography. The obtained fraction is derivatized into methyloxime, treated with dimethylisopropylsilylimidazole, and then subjected to GC-MS-MS. The method is applied to the determination of these steroids in the gray matter and olfactory bulbs of rat brains, which are divided into control and acute stressed specimens. Although pregnenolone in both regions of the rat brains increases more than three times after stress, dehydroepiandrosterone in both regions is not so clearly influenced by stress.     

Efficient new method for extraction and isolation of three flavonoids from Patrinia villosa Juss. by supercritical fluid extraction and high-speed counter-current chromatography

Supercritical fluid extraction (SFE) of orotinin, orotinin-5-methyl ether and licoagrochalcone B from Patrinia villosa was performed. The optimization of parameters including pressure, temperature, modifier and sample particle size on yield was carried out using an analytical-scale SFE system. The process was then scaled up by 100 times using a preparative SFE system under the optimized conditions of 25 MPa, 45 degrees C, a sample particle size 40-60 mesh and modified CO2 with 20% methanol. The yield of the preparative SFE was 2.82% (crude extract I) and the combined yield of orotinin, orotinin-5-methyl ether and licoagrochalcone B was 0.82 mg/g of dry sample mass. Then the crude extract I was re-dissolved in methanol and methanol soluble fraction (crude extract II, 0.17%) was obtained, which was successfully isolated and separated by a preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (5:6:6:6, v/v/v/v) by increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml/min after 3 h. The target compounds isolated and purified by HSCCC were analyzed by high performance liquid chromatography. The separation produced total of 38.2 mg of orotinin at 99.2% purity, 19.8 mg of orotinin-5-methyl ether at 98.5% purity and 21.5 mg of licoagrochalcone B at 97.6% purity from 400 mg of the crude extract in a one-step separation. The recoveries of orotinin, orotinin-5-methyl ether and licoagrochalcone B were 91.1, 91.6 and 90.3%, respectively, and the chemical structure identification was carried out by UV, IR, MS, 1H NMR and 13C NMR.     

Carbohydrate profiling of bacteria by gas chromatography-mass spectrometry and their trace detection in complex matrices by gas chromatography-tandem mass spectrometry.

Bacterial cellular polysaccharides are composed of a variety of sugar monomers. These sugars serve as chemical markers to identify specific species or genera or to determine their physiological status. Some of these markers can also be used for trace detection of bacteria or their constituents in complex clinical or environmental matrices. Analyses are performed, in our hands, employing hydrolysis followed by the alditol acetate derivatization procedure. Substantial improvements have been made to sample preparation including simplification and computer-controlled automation. For characterization of whole cell bacterial hydrolysates, sugars are analyzed by gas chromatography-mass spectrometry (GC-MS). Simple chromatograms are generated using selected ion monitoring (SIM). Using total ion GC-MS, sugars can be readily identified. In more complex clinical and environmental samples, markers for bacteria are present at sufficiently low concentrations that more advanced instrumentation, gas chromatography-tandem mass spectrometry (GC-MS-MS), is preferred for optimal analysis. Using multiple reaction monitoring, MS-MS is used (replacing more conventional SIM) to ignore extraneous chromatographic peaks. Triple quadrupole and ion trap GC-MS-MS instruments have both been used successfully. Absolute chemical identification of sugar markers at trace levels is achieved, using MS-MS, by the product spectrum.     

Optimization of an isotope dilution gas chromatography/mass spectrometry method for the detection of endogenous estrogen metabolites in urine samples

A gas chromatography/mass spectrometry (GC/MS) method for the simultaneous quantitation of ten estrogen metabolites in human urine was optimized. The method consists of initial enzymatic hydrolysis of the estrogen conjugates using beta-glucuronidase followed by solid-phase extraction (SPE) on Sep-pak C18 columns and further sample purification by ion-exchange chromatography on QAE-Sephadex cartridges in the acetate form. QAE-Sephadex cartridges in the borate form were used to separate estrogens into two fractions: one fraction containing estrogens lacking vicinal cis-hydroxyls (Fr 1) and another containing estrogens possessing vicinal cis-hydroxyls (catecholestrogens; Fr 2). Finally, following O-trimethylsilyl ether derivatization, the estrogens were analyzed by GC/MS in the selected ion monitoring mode. Estrogens were quantitated using deuterated internal standards, which were added to the samples at the initiation of the work-up procedures. After addition to estrogen-low male human samples the standards showed good chromatographic linear response and reproducibility. A reduction in the number of steps and improvements in the robustness of the work-up procedures were achieved. The modified method described is less complex, amenable to use with commercially available SPE columns and fulfils all the reliability criteria, resulting in highly specific and accurate results.     

Comparison of gas chromatographic-mass spectrometric methods for screening of chlorotriazine pesticides in soil.

The performance of a coupled technique resulting from the combination of gas chromatography with a selective mass spectrometric technique (tandem mass spectrometry) (GC-MS-MS) with collisionally activated dissociation (CAD) and multi-reaction monitoring (MRM) was compared with that of GC-low resolution MS (GC-LRMS) at a resolving power of 1000 and GC-high-resolution MS (GC-HRMS) at resolving powers of 5000 and 10,000 for the determination of atrazine, simazine, cyanazine, deethylatrazine and deisopropylatrazine in polluted soil samples. GC-MS-MS daughter ion spectra for the parent ions [M]+. and [M - CH3]+ were generated using collisionally activated dissociation and studied. Also, by optimizing the collision energy for maximum sensitivity a method for screening chlorotriazines by MRM was developed. Analyses of soil sample extracts showed that GC-MS-MS overcomes interferences from other chlorotriazines and interfering compounds that could not be removed by GC-HRMS or GC-LRMS at resolving powers of 10,000 and 1000, respectively. The limits of detection for GC-MS-MS and GC-HRMS at a signal-to-noise ratio of 10 ranged between 1 and 24 pg, with a mean relative standard deviation of 25-30%. Soil samples known to contain chlorotriazines and their degradation products were analysed by GC-MS-MS and the results obtained were compared with those given by GC-HRMS at resolving powers of 5000 and 10,000, with quantification differences of 25-30%.     

Selective detection of the tolyl cation among other [C7H7]+ isomers by ion/molecule reaction with dimethyl ether

The ion/molecule reaction of the tolyl cation with dimethyl ether has been investigated using triple quadrupole mass spectrometry. Three isomers with [C₇H₇]⁺ composition, the 3-tolyl, benzyl, and tropylium cations, were individually selected and reacted with dimethyl ether at a pressure of 1 mtorr in the second quadrupole (Q₂) collision cell. Only the tolyl ion reacted to yield a methoxylated product ion peak at m/z 122. This reaction product having m/z 122 is postulated to be identical in structure with the molecular ion of 3-methyl anisole, as supported by thermochemical data and the similarity of the collision induced dissociation (CID) daughter ion mass spectra of the product ion and the molecular ion of authentic 3-methyl anisole. The daughter ion mass spectra of the three [C₇H₇]⁺ isomers during CID, by using a triple quadrupole mass spectrometer, are nearly identical; on the other hand, the analytical approach based on the ion/molecule reaction with dimethyl ether clearly exhibits distinct gas-phase chemistry reflecting structural differences among the isomers. Sot     

Multiple-stage mass spectrometry analysis of bisphenol A diglycidyl ether, bisphenol F diglycidyl ether and their derivatives

The fragmentation of bisphenol A diglycidyl ether (BADGE), bisphenol F diglycidyl ether (BFDGE) and their derivatives was studied by electrospray ionization tandem mass spectrometry. Multiple-stage mass spectrometry and accurate mass measurements were combined to establish the fragmentation pathways. BADGEs and BFDGEs tend to form ammonium adducts under electrospray conditions which fragmented easily. The fragmentation of [M+NH(4)](+) for BADGEs started with the cleavage of the phenyl-alkyl bond, which was followed by the α-cleavage of the ether group to generate the characteristic product ions at m/z 135, [C(9)H(11)O](+), and m/z 107, [C(7)H(7)O](+). The fragmentation of the BFDGE isomer mixtures was studied by on-line reversed-phase liquid chromatography coupled to multiple-stage mass spectrometry (LC/MS(n)). Information obtained from product ion spectra for each BFDGE isomer and its comparison with the fragmentation pathway of BADGE allowed each isomer and the chromatographic elution order to be identified.     

Derivatization in Liquid Chromatograhy/Mass Spectrometric Analysis of Neurosteroids

 Liquid chromatography/mass spectrometry (LC/MS) is now considered to be the most promising analytical method for the determination of biological substances, especially nonvolatile or highly polar substances However, some compounds do not show enough sensitivity in LC/MS and soft ionization methods commonly used in LC/MS, such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), sometimes do not give satisfactory structural information This report presents an overview     

Derivatization in Liquid Chromatography/Mass Spectrometric Analysis of Neurosteroids

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Anthraquinones from Morinda officinalis roots enhance adipocyte differentiation in 3T3-L1 cells

To search for anti-diabetic and insulin-sensitising natural products, the effect on adipocyte differentiation was investigated by assessing fat accumulation in 3T3-L1 preadipocytes using Oil Red O staining. Fractionation and separation of n-hexane and CHCl? fractions of Morinda officinalis (Rubiaceae) using several chromatographic methods led to the isolation of three anthraquinones, 1,2-dimethoxyanthraquinone (1), alizarin-2-methyl ether (2) and rubiadin-1-methyl ether (3). Among them, alizarin-2-methyl ether (2) showed the strongest enhancing activity, followed by rubiadin-1-methyl ether (3) and 1,2-dimethoxyanthraquinone (1). At a concentration of 100?μM, alizarin-2-methyl ether (2) enhanced adipocyte differentiation by up to 131% (compared to insulin-treated cells). Thus, these compounds could be beneficial in the treatment of diabetes.     

Preparative Hydrophobic Interaction Chromatography of Proteins Using Ether Based Chemically Bonded Phases

Abstract

This paper examines the use of 15–20 micron wide-pore silica-based ether bonded phases for the preparative hydrophobic interaction chromatography of proteins. In particular, silyl ethers are immobilized on large particle silica in an analogous manner to previously developed ether bonded 5 um analytical supports. The preparative supports are reproducibly prepared and exhibit constant chromatographic retention for at least five months of continual use. Preparative columns can be operated for protein chromatography with peak shapes and capacity as predicted by the Snyder gradient elution model. Moreover, similar retention times are obtained relative to those on the 5 um analytical columns, enabling the direct transition and scale-up of separation. Gradient optimization is seen to directly parallel that performed on 5 um bonded ether analytical columns. Acceptable chromatographic resolution was obtained with sample capacity of >15 mg protein/ml column volume using a repetitive injection technique. A column clean-up strategy is examined for rapid and safe removal of contaminants. An illustrative example of use of the bonded ether preparative columns is made by application to soybean trypsin inhibitor purification. Initial results are presented on a column-switching method for the analytical monitoring of preparative separation.     

Preparative chromatographic separation of enantiomers.

The potential of the chromatographic separation of enantiomers on a preparative scale as a tool for the isolation of optically pure compounds is gaining increasing recognition. This review surveys the different chiral stationary phases (CSPs) used for preparative chromatography, emphasizing the advantages and drawbacks of each. The strategy to be followed for preparative separations is discussed and tables summarizing separations reported in the literature give an overview of practical applications. Cellulose triacetate has been used most frequently, probably because of its broad application range and its low production costs in comparison with more recently introduced CSPs. Nevertheless, the high efficiency of some of the novel CSPs is likely to contribute to the further development and expansion of the method.     

Glyconothio-O-lactones. Part III. Thermolysis of a 4,5-Dihydro-1,2,3- and a 2,5-Dihydro-1,3,4-thiadiazole

Addition of CH₂N₂ to 2,3:5,6-di-O-isopropylidene-1-thio-mannono-1,4-lactone ( 1 ) gave the 2,5-dihydro-1,3,4-thiadiazole 2 and the 4,5-dihydro-1,2,3-thiadiazole 3 . First-order kinetics were observed for the thermolysis of 3 (Scheme 3) at 80–110° in C₆D₅Cl solution and of 2 (Scheme 3) at 20–35° in CDC1₃, respectively. The 1,2,3-thiadiazole 3 led to mixtures of the thiirane 9 , the starting thionolactone 1 , the thiono-1,5-lactone 8 , and the enol ether 7 , while the isomeric 1,3,4-thiadiazole 2 led to mixtures of the anomeric thiiranes 9 and 12 , the O-hydrogen S,O,O-ortholactone α-D - 14 , the S-methyl thioester 15 , the S,S,O-ortholactone 13 , and the 2,3:5,6-di-Oisopropylidene-mannono-1,4-iactone ( 16 ). Pure products of the thermolysis were isolated by semipreparative supercritical fluid chromatography (SFC), whereas preparative HPLC led to partial or complete decomposition. Thus, the β-D -mannofuranosyl β-D -mannofuranoside 10 , contaminated by an unknown S species, was isolated by preparative HPLC of the crude product of thermolysis of 3 at 115–120° and partially transformed in CD₃OD solution into the symmetric di(α-D -mannofuranosyl) tetrasulfide 11 . Its structure was evidenced by X-ray analysis. Similarly, HPLC of the thermolysis product of 2 gave the enethiol 17 , the sulfide 19 , and the mercapto alcohol 18 as secondary products. Thermolysis of the thiirane 9 at 110–120° (Scheme 4) led to the anomeric thiirane 12 which was transformed into mixtures of the enethiol 17 and the enol ether 7. Addition of H₂O to 17 and 7 gave the corresponding hemiacetals 18 and 20. The mechanism of the thermolysis of the dihydrothiadiazoles 2 and 3 , and the thiiranes 9 and 12 is discussed.     

Application of ion chromatography to the determination of inorganic anions in foodstuffs.

A review on the applications of ion chromatography (IC) to the determination of inorganic anions in foodstuffs is presented. The anions were commonly determined in food, i.e., SO3(2-), NO3- and NO2-, and to a lesser extent Cl-, Br-, I-, SO4(2-), IO3-, BrO3- and phosphate, are considered. In comparison with standard methods for the determination of anions in food products, chromatographic methods are rapid, sensitive and precise. They also have the advantage of determining several ions simultaneously. The separation may be achieved by conventional IC, by ion interaction chromatography or by ion exclusion chromatography. IC has also been applied to the determination of Br, I, N and S in foods after oxidation or combustion of samples and conversion into anionic forms.     

Regiospecific analysis of neutral ether lipids by liquid chromatography/electrospray ionization/single quadrupole mass spectrometry: validation with synthetic compounds.

A reversed-phase high-performance liquid chromatography (HPLC) method with on-line electrospray ionization/collision-induced dissociation/mass spectrometry (ESI/CID/MS) is presented for the regiospecific analysis of synthetic reference compounds of neutral ether lipids. The reference compounds were characterized by chromatographic retention times, full mass spectra, and fragmentation patterns as an aid to clarify the regiospecificity of ether lipids from natural sources. The results clearly show that single quadrupole mass spectroscopic analysis may elucidate the regiospecific structure of neutral ether lipids. Ether lipid reference compounds were characterized by five to six major ions in the positive ion mode. The 1-O-alkyl-sn-glycerols were analyzed as the diacetoyl derivative, and showed the [M - acetoyl](+) ion as an important diagnostic ion. The diagnostic ions of directly analyzed 1-O-alkyl-2-acyl-sn-glycerols and 1-O-alkyl-3-acyl-sn-glycerols were the [M - alkyl](+), [M + H - H(2)O](+) and [M + H](+) ions. Regiospecific characterization of the fatty acid position was evident from the relative ion intensities, as the sn-2 species had relatively high [M + H](+) ion intensities compared with [M + H - H(2)O](+), whereas the reverse situation characterized the sn-3 species. Furthermore, corresponding sn-2 and sn-3 species were separated by the chromatographic system. However, loss of water was promoted as fatty acid unsaturation was raised, which may complicate interpretation of the mass spectra. The diagnostic ions of directly analyzed 1-O-alkyl-2,3-diacyl-sn-glycerols were the [M - alkyl](+), [M - sn-2-acyl](+) and [M - sn-3-acyl](+) ions. Regiospecific characterization of the fatty acid identity and position was evident from the relative ion intensities, as fragmentation of the sn-2 fatty acids was preferred to the sn-3 fatty acids; however, loss of fatty acids was also promoted by higher degrees of unsaturation. Therefore, both structural and positional effects of the fatty acids affect the spectra of the neutral ether lipids. Fragmentation patterns and optimal capillary exit voltages are suggested for each neutral ether lipid class. The present study demonstrates that reversed-phase HPLC and positive ion ESI/CID/MS provide direct and unambiguous information about the configuration and identity of molecular species in neutral 1-O-alkyl-sn-glycerol classes.     

PROFLAVIN-SENSITIZED PHOTOOXIDATION OF 3-METHYL INDOLE TO 2-METHYL-2-METHOXY-1, 4-BENZOXAZINE

Abstract— The dye-sensitized photochemical oxidation of 3-methyl indole was studied as a model for the photooxidation of the indole amino acid, tryptophan. The major product formed by proflavin-sensitized photooxidation is 2-methyl-2-methoxy-1,4-benzoxazine. The compound was isolated by preparative gas chromatography and identified by a combination of NMR, u.v., i.r., and mass spectroscopy on the photoproduct and its derivatives.     

Recent progress in the analysis of sugar monomers from complex matrices using chromatography in conjunction with mass spectrometry or stand-alone tandem mass spectrometry

Analysis of trace levels of carbohydrate monomers in complex matrices requires excellent discrimination of the peaks of interest from background noise. Minimizing contaminating peaks introduced during sample preparation and chromatography is extremely important. However, the exquisite selectivity of the mass spectrometer is essential as a chromatographic detector in this regard. Traditionally gas chromatography-mass spectrometry (GC-MS) has been the method of choice for trace analysis of derivatized carbohydrates. Recent improvements in commercial tandem mass spectrometers (MS-MS) are encouraging the use of GC-MS-MS for improved specificity in trace analysis. There has also been an explosion in applications of electrospray ionization (ESI) for sensitive introduction of polar molecules (including sugars) into the mass spectrometer. This has encouraged ongoing developments in high-performance liquid chromatography-mass spectrometry (LC-MS) and MS-MS of underivatized carbohydrates. This has the potential to dramatically simplify sample preparation. However, as yet LC-MS and MS-MS do not match the sensitivity of GC-MS or GC-MS-MS. Developments in analysis of sugar monomers from complex matrices using chromatography (GC/LC) in conjunction with mass spectrometry (MS, MS-MS) or stand-alone MS-MS are discussed.     

Bile acids. LIX. Purification of 5 alpha-anhydrocyprinol by preparative high-performance liquid chromatography

26,27-Oxido-5 alpha-cholestane-3 alpha,7 alpha,12 alpha-triol can be obtained in a homogeneous state in gram quantities by passing it through one PrepPak-500/Silica cartridge mounted in a Waters Assoc. preparative liquid chromatograph. The elution solvent was methanol-chloroform (1:14). The isolated material was analyzed for purity by several chromatographic means and by elemental analysis, and was finally characterized by the usual spectroscopic means. Gas-liquid chromatography of its trimethylsilyl ether indicated the formation of a tetrakis-trimethylsilyl-26-chloro derivative, in addition to the expected tris-trimethylsilylated substance. The structure of the former compound is deduced from the fragmentation and isotope abundance in its mass spectrum and from chemical principles.     

Endogenous alkaloids in man. XI. Analysis of glyoxylate-derived 1,3-thiazolidines by ion-pair-assisted reversed-phase chromatography.

Reversed-phase ion-pair chromatography, using cetyltrimethyl ammonium hydrogensulphate as detergent, has been applied to the analysis of the highly polar glyoxylate-derived 1,3-thiazolidines 1, 2 and 3. On the base of this high-performance liquid chromatographic separation of the diastereomeric compounds 1a/b and 3a/b was achieved. Removal of the hydrophobic couterion by precipitation as its insoluble iodide, followed by an extraction with chloroform, seems to be a promising first step to establish ion-pair-assisted chromatography as a preparative high-performance liquid chromatographic method for the isolation of polar compounds.