Steroids in porcine follicular fluid: analysis by HPLC, capillary CG and capillary CG/MS after purification on SEP-PAK C18 and ion exchange chromatography

Steroids in porcine follicular fluid have been concentrated by reverse phase chromatography in SEP-PAK C18 and purified further on the cation exchanger SP-Sephadex C-25. Fractionation into unconjugated neutral and phenolic steroids, glucuronides and sulfates was carried out on triethylaminohydroxypropyl Sephadex LH-20 (TEAP-LH-20). The unconjugated neutral fraction was analysed by high pressure liquid chromatography (HPLC) on a C18 radial cartridge 5 mm I.D.; 10 mu, or on a C18 5 mu RESOLVE column, and by capillary gas chromatography (GC) on a 12 M OV-1 cross linked fused silica column. Testosterone, progesterone and androstenedione were the major steroids detected by HPLC monitored at 254 nm, although 17- hydroxy-, 20 alpha-dihydro- and 20 beta-dihydroprogesterone were also present. Pregnenolone, pregnanediol, dehydroepiandrosterone, 17-hydroxypregnenolone and androsterone were detected by capillary CG as their 0-methyloxime trimethylsilyether derivatives. Further confirmation of structure was provided by complete mass spectral data or by selective ion monitoring (SIM).     

Fractionation of fecal neutral steroids by high performance liquid chromatography

Fecal neutral steroids were fractionated by high performance liquid chromatography (HPLC) into three major fractions: 5 beta-H, 3-keto steroids; 5 beta-H, 3 beta-hydroxy steroids; and 5 alpha-H and delta 5-3 beta-hydroxy steroids. This separation was achieved in about 10 minutes, with greater than 97% recovery of standards in each fraction. Gas-liquid chromatographic quantitation of fecal steroids fractionated by either HPLC or thin-layer chromatography gave nearly identical results. A method using both C18 reverse phase and silica HPLC to purify radiolabeled sterols is also described.     

Chromophoric determination of putrescine, spermidine and spermine with dabsyl chloride by high-performance liquid chromatography and thin-layer chromatography

A fast and sensitive method for the determination of putrescine, spermidine, spermine and ammonia by high-performance liquid chromatography (HPLC) with dabsyl chloride is described. These compounds are converted to their chromophoric dabsyl derivatives and are separated by a normal-phase chromatographic column (μPorasil, 10 μm) with 2% acetone in chloroform as isocratic mobile phase. The sensitivity of the method is 20 pmoles. The present method was shown to be a straightforward procedure for estimating polyamines in various rat tissues.The chromophoric derivatives of polyamines are also well separated by thin-layer chromatography (TLC) on silica gel, and the combination of the HPLC and TLC procedures provides a reliable method for qualitative and quantitative analysis of polyamines.     

Oxalate-silica gel thin-layer system for free 2-hydroxy fatty acids and for fatty acyl coenzyme A

The 2-hydroxy fatty acids tend to yield streaks in thin-layer chromatography on silica gel plates. If potassium oxalate is included with binder-free silica gel, good spots are obtained. Similar difficulties are found in paper chromatography of the fatty acid derivatives of coenzyme A, especially with long-chain acids. The same thin-layer system gives good spots with these compounds.     

Synthesis of lipids from acetate by human preputial and abdominal skin in vitro

Lipogenesis in vitro from acetate-1-(14)C was studied in human preputial skin and abdominal skin. Radioactive lipids were separated by column chromatography on Florisil and by thin-layer chromatography on silica gel. Radioactivity was incorporated chiefly into the triglyceride, sterol, and polar lipid fractions, while lesser amounts of (14)C were found in the hydrocarbon, wax, diglyceride, monoglyceride, and fatty acid fractions; labeling of steryl esters was minimal. On thin-layer chromatography, the radioactive polar lipids had mobilities similar to lysolecithin, phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidic acid. The radioactive fatty acids of the different lipid fractions were separated by gas-liquid chromatography. The major (14)C-labeled acids were 16:0 and 18:0. Radioactivity was also detected in acids 14:0, 15:0, 16:1, 18:1, 18:2, 20:0, 20:1, 22:0, 24:0, 24:1, and 26:0. No radioactivity could be detected in arachidonic acid, although this fatty acid comprises 9% of the chromatographed fatty acids. The pattern of incorporated (14)C was different from the percentage mass composition of the fatty acids. Skin is therefore active in the biosynthesis of a wider variety of lipids than previously demonstrated.     

Separation and quantification of prostaglandins E1 and E2 as their panacyl derivatives using reverse phase high pressure liquid chromatography

Separation and quantification of prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2) were achieved using reverse phase high performance liquid chromatography (HPLC). Panacyl bromide (p-(9-anthroyloxy)phenacyl bromide) (PAB) derivatives of PGE2 and PGE1 were prepared. Reverse phase HPLC using a linear gradient of 56% to 80% acetonitrile in water containing 0.10% acetic acid gave baseline resolution of the two derivatives. A 3 um diameter particle, C18 column provided good resolution and reproducible recoveries. Human synovial tissue cells were incubated with the precursor fatty acids for PGE1 or PGE2 and stimulated with a crude Interleukin 1 (IL-1) preparation. Cells grown in the presence of dihomogammalinolenic acid (DGLA), the precursor for PGE1, made significantly more PGE1 than cells grown in control medium or in the presence of arachidonic acid, precursor for PGE2. PGE2 synthesis was reduced when DGLA was added to cells (resting or IL-1-stimulated).     

Purification of fatty acid methyl esters by high-performance liquid chromatography

The determination by gas chromatography (GC) of fatty acid methyl esters (FAMEs) prepared from complex biological samples is subject to interference from cholesterol. During sample injection on the GC system of FAMEs prepared from tissues that contain cholesterol, we observed a major contaminant that co-eluted with docosahexaenoic acid (DHA, 22:6n-3). To address this problem, FAMEs were purified on an amino-phase high-performance liquid chromatography (HPLC) column using a hexane–isopropanol gradient. The HPLC retention times for both the FAME fraction and cholesterol were stable and reproducible when the amino column was used for sample purification. The purified extracts were analyzed by GC without artifacts or impurity peaks after 50 analytical runs. The method described here will be useful for measurement of 22:6n-3 and other fatty acids important for studies of nutrition or pathology.     

Monitoring the location profile of fluorophores in phosphatidylcholine bilayers by the use or paramagnetic quenching.

Spin probes differing in the position of their paramagnetic centre are used to quench the fluorescence of pyrene derivatives and chlorophylls incorporated into dimyristoyl phosphatidylcholine membranes. Pyrene butyric acid and pyrene decanoic acid with known orientation relative to the membrane surface are investigated. The quenching efficiency of fatty acid spin probes is dependent on the position of the nitroxide radical group in the fatty acid chain. Using this short range interaction we developed a spectroscopic method to chlorophyll-containing vesicles, we were able to characterize the orientation of the porphyrin ring within the membrane. Moreover, the chlorophyll fluorescence is also quenched by a water-soluble spin label. Therefore the porphyrin ring appears to be orientated in the polar head group region of the lipid layer, but not to be protruding out into the water phase. This conclusion is confirmed by the use of pyrene derivatives. Fluorescence quenching by a water-soluble spin label within the lipid matrix is observed even in the rigid state of the membrane. Fluorescence lifetime measurements suggest the existence of two different quenching mechanisms: (1) a static quenching occurring below the lipid phase transition temperature, and (2) an additional dynamic quenching taking place in the fluid state of the lipid bilayer.     

Isolation and characterization of gangliosides from pig lymphocytes

Two major gangliosides from pig spleen lymphocytes, accounting for 57% of the total lipid-bound sialic acids, were isolated and purified to homogeneity by column chromatography on DEAE-Sephadex and silica gel. They were identified as GM3 (II3Neu5GcLacCer), and GD3 (II3(Neu5Gc)2LacCer), by thin-layer chromatography in comparison with standards and by analysis of the constituent sugars. The major fatty acids of these gangliosides were stearic acid and myristic acid, respectively. In addition to these gangliosides, GD2 and bands comigrating on thin-layer chromatography with authentic GM2, GM1, GD1a and GD1b were found. These compounds also occur in pig peripheral blood lymphocytes, where, however, GD3 represents about 70% of the total lipid-bound sialic acid.     

High pressure liquid chromatography methods for separation of omega- and (omega-1)-hydroxy fatty acids: their applications to microsomal fatty acid omega-oxidation

Fatty acids (C12-C18) and their omega- and (omega-1)-hydroxy derivatives, when converted to p-bromophenacyl (PBP) esters, can be completely separated from one another by high pressure liquid chromatography (HPLC) on a silicic acid column using 0.5% (v/v) isopropanol in n-hexane. In this system, fatty acid PBP esters are eluted at the solvent front, whereas the retention times of the omega- and (omega-1)-hydroxy derivatives are 14-20 and 24-29 min, respectively. The PBP esters can also be separated by reverse phase HPLC on a muBondapak C18 column, a method which has been developed by Fan et al. (Fan, L. L., Masters, B. S. S., and Prough, R. A. (1976) Anal. Biochem. 71, 265-272) for separation of methyl esters of fatty acids and their omega- and (omega-1)-hydroxy derivatives. In the latter method, however, the retention times of omega- and (omega-1)-hydroxy derivatives are only about 2 min apart and an increase in the solvent polarity is needed for elution of the esters of unmodified fatty acids. Fatty acid PBP esters, however, can be obtained as independent peaks which are not disturbed by the solvent front. An application of the former method to measure fatty acid omega oxidation by liver microsomes and by a reconstituted monooxygenase system containing purified cytochrome P-450 is described.     

Chromatographic behavior of oxygenated derivatives of cholesterol

Oxygenated derivatives of cholesterol have important functions in many biochemical processes. These oxysterols are difficult to study because of their low physiological concentrations, the facile formation of cholesterol autoxidation artifacts, and lack of information on their chromatographic behavior. Focusing on metabolites and autoxidation products of cholesterol, we have documented the chromatographic mobilities of 35 oxysterols under a variety of conditions: eight solvent systems for thin-layer chromatography on silica gel, several mobile phases for reversed-phase high-performance liquid chromatography (HPLC), and two types of stationary phase for capillary gas chromatography (GC) using trimethylsilyl derivatives. Notable differences in selectivity could be obtained by modifying the stationary or mobile phases. Separations of oxysterol pairs isomeric at side-chain carbons or C-7 were achieved on normal-phase, reversed-phase, chiral, or silver-ion HPLC columns. Chromatographic behavior is also described for side-chain hexadeuterated and heptafluorinated oxysterols, which are useful as standards in isotope dilution analyses and autoxidation studies, respectively. The overall results are relevant to many problems of oxysterol analysis, including the initial separation of oxysterols from cholesterol, determination of highly polar and nonpolar oxysterols, separation of isomeric pairs, selection of derivatization conditions for GC analysis, and quantitation of the extent of cholesterol autoxidation.     

HPLC analysis of geometrical isomers of lutein epoxide isolated from dandelion (Taraxacum officinale F. Weber ex Wiggers)

Lutein epoxide has been isolated from petals of dandelion (Taraxacum officinale F. Weber ex Wiggers) by thin-layer chromatography (TLC) on silica to be used for the accurate identification of this carotenoid in other sources. The extract was analyzed by high-performance liquid chromatography (HPLC) using a C(30) column, as a result of which six geometrical isomers were separated. The identification of these isomers was performed on the basis of their UV/vis spectroscopic features in the mobile phase. In quantitative terms, it was observed that all-E-lutein epoxide was the major carotenoid and that there were also high amounts of the (9Z)- and (9'Z)-isomers, although the latter may be an artifact.     

Presence of fatty acid esters of pregnenolone in follicular fluid from women undergoing follicle stimulation

In the following investigation the presence of lipoidal pregnenolone derivatives in the preovulatory follicular fluid obtained from women undergoing in vitro fertilization was established. Concentrations of lipoidal pregnenolone proved to be at least twofold greater than those of the unconjugated counterpart. Indirect identification of these lipoidal pregnenolone derivatives was achieved by comparing the C-18 column, thin-layer silica gel (TLC), high performance liquid chromatography (HPLC), and gas chromatography/mass spectrometry (GC/MS) chromatographic properties of the endogenous lipoidal pregnenolone derivatives in follicular fluid with those of synthetic acyl pregnenolone esters. Lipoidal pregnenolone derivatives recovered after HPLC subfractionation were treated with alkali to hydrolyze the acyl group thus liberating nonconjugated pregnenolone. Concentrations of this steroid were then measured using radioimmunoassay upon which analysis of HPCL and gas chromatograms permitted the calculation of the individual pregnenolone ester contributions within the samples. Five lipoidal pregnenolone derivatives constituted more than 90% of the total lipoidal pregnenolone concentration observed, these derivatives being: pregnenolone oleate (30.7%), linoleate (20.7%), palmitate (20.1%), linolenate (14.8%), and palmitoleate (7.1%).     

Induction of tryptophan oxygenase and tyrosine aminotransferase by metabolites of hydrocortisone

Metabolites of hydrocortisone were isolated from rat liver on a preparative scale, fractionated by column chromatography on Sephadex Lh-20 and silica gel and tested for biological activity. Apart from the well known neutral metabolites, steroid glucuronides and sulfates, we obtained metabolite fractions containing non-conjugated steroidal carboxy acids and acid metabolites of unknown structure. One of these fractions induced tyrosine aminotransferase (EC 2.6.1.5) in adrenalectomized female rats but not tryptophan oxygenase (EC 1.13.11.11), whereas another one mainly increased activity of tryptophan oxygenase. The doses necessary to significantly induce both enzymes were much lower in case of these metabolites than in the case of hydrocortisone itself. The active fractions eluting from silica gel column were analyzed by thin-layer chromatography in two different solvent systems. Absence of hydrocortisone in these fractions could be clearly demonstrated. Furthermore, the active fractions eluting from the silica gel column were characterized by treatment with an extract from Helix pomatia and/or diazomethane and subsequent analysis by thin-layer chromatography. We conclude, considering the biological activity of some synthesized derivatives of hydrocortisone, that the biologically active components are acid metabolites of hydrocortisone which are not identical to any of the known metabolites.     

Analysis of dimeric species derived from the reaction of phosphatidylethanolamine with dimethylsuberimidate

Phosphatidylethanolamines of various fatty acyl species in vortexed lipid dispersions were reacted with dimethylsuberimidate to produce dimeric products. The yield was 34% at pH 10 and 2% at pH 7. The crosslinked phosphatidylethanolamine species were separated from minor products and the reactants by extraction and two-dimensional thin-layer chromatography on silica gel G, gel filtration on lipophilic Sephadex, or C18-reversed phase high-performance liquid chromatography (HPLC). Reversed phase HPLC was also used to resolve the dimers into individual molecular species. Analysis of the dimers revealed the extinction coefficients at 205 nm to be higher than those of the reactant phosphatidylethanolamines. Proton nuclear magnetic resonance spectroscopy and chemical analysis implied that the dimers contain an amidine group.     

Characterization of Alkylgalactolipids from Calf Brain by High Performance Liquid Chromatography

Minor nonpolar galactolipids were isolated from the total lipids of calf brain stem by column chromatography and were separated by preparative thin-layer chromatography into four groups. The material recovered from the bottom band of the thin-layer chromatography consisted of monogalactosyl diglyceride and its 1-0-alkyl isomer, alkylgalactolipid, present in a molar ratio of 11 :9. After perbenzoylation. they were separated by preparative thin-layer chromatography and characterized. The fatty acid compositions of these lipids were similar to each other and to those of the ester-linked fatty acids of cerebroside esters. The major alkyl group of alkylgalactolipid was palmityl, and the other, minor components were oleyl. myristyl, and stearyl ethers. Perbenzoylated derivatives of these lipids were further separated by reverse-phase high performance liquid chromatography. The chromatograms from these two lipids were similar; however, most of the peaks were still mixtures of homologs containing different fatty acids or an alkyl group.     

高效液相色谱法测定毛豆中吡虫啉残留量

采用高效液相色谱法测定毛豆中吡虫啉农药残留。试样用二氯甲烷超声波提取,中性氧化铝柱净化,石油醚去除脂类杂质,以0.2%冰乙酸水溶液-乙腈(70:30,体积比)为流动相,配备Agilent TC-c18柱、高效液相色谱紫外检测器检测(HPLC-UV),外标法定量。实验表明,毛豆样品中吡虫啉添加回收率为84.0%~102.8%,相对标准偏差(n=6)为6.8%~10.9%,样品中吡虫啉最低检测浓度为0.003 mg/kg。     

High-performance liquid chromatographic analysis of acylated lipids containing pyrene fatty acids

A high-performance liquid chromatographic method for the separation and quantification of acylated lipids containing pyrene fatty acids is described. The method is adapted from a procedure originally developed for the analysis of tissue lipids (Christie, W. W. (1985) J. Lipid Res. 26, 507-512). Pyrenyl lipid analogs ranging in polarity from cholesteryl ester to lysophosphatidylcholine are completely resolved on a silica column in 50 min by gradient elution with a ternary solvent system. Furthermore, pyrene-labeled triglycerides are resolved according to the number of pyrene fatty acid residues incorporated. Pyrenyl lipids are detected at levels of 10(-13) mol by high-sensitivity fluorescence detection. Accurate quantification of pyrenyl lipids is obtained by correcting peak areas for mobile-phase quenching effects. The close correspondence between chromatograms obtained for the separation of labeled lipids extracted from Hep-G2 cells incubated with either 12-(1-pyrenyl)dodecanoic acid (fluorescence detection) or [1-14C]oleic acid (radioactivity detection) indicates that this HPLC method is equally suitable for analysis of native lipids.     

Diacylglycerols interfere in normal phase HPLC analysis of lipoxygenase products of docosahexaenoic or arachidonic acids

A methodological problem with the normal phase high performance liquid chromatography (HPLC) of hydroxylated products of docosahexaenoic and arachidonic acids is described. Diacylglycerols present in lipid extracts of rat retina co-elute with monohydroxy derivatives of docosahexaenoic or arachidonic acid, when samples are applied to uPorosil columns and eluted with hexane/isopropanol/acetic acid. Analysis of fatty acid composition of diacylglycerols which were acetone-extracted from the incubation medium showed a profile similar to diacylglycerols extracted from the tissue by hexane/isopropanol, although acetone extraction resulted in extremely variable recovery of diacylglycerols. This co-elution of diacylglycerols with monohydroxy polyunsaturated fatty acids can lead to a significant error in estimation of lipoxygenation activity by conversion of radiolabeled precursors, because the incorporation of fatty acids into diacylglycerols is very active in many tissues. An alternative extraction method and reverse phase HPLC procedures that result in the complete separation of hydroxy fatty acids and diacylglycerols are described.     

Induction of tryptophan oxygenase and tyrosine aminotransferase by metabolites of hydrocortisone

Metabolites of hydrocortisone were isolated from rat liver on a preparative scale, fractionated by column chromatography on Sephadex LH-20 and silica gel and tested for biological activity. Apart from the well known neutral metabolites, steroid glucuronides and sulfates, we obtained metabolite fractions containing non-conjugated steroidal carboxy acids and acid metabolites of unknown structure. One of these fractions induced tyrosine aminotransferase (EC 2.6.1.5) in adrenalectomized female rats but not trptophan oxygenase (EC 1.13.11.11), whereas another one mainly increased activity of tryptophan oxygenase. The doses necessary to significantly induce both enzymes were much lower in case of these metabolites than in the case of hydrocortisone itself. The active fractions eluting from silica gel column were analyzed by thin-layer chromatography in two different solvent systems. Absence of hydrocortisone in these fractions could be clearly demonstrated. Furthermore, the active fractions eluting from the silica gel column were characterized by treatment with an extract from Helix pomatia and/or diazomethane and subsequent analysis by thin-layer chromatography. We conclude, considering the biological activity of some synthesized derivatives of hydrocortisone, that the biologically active components are acid metabolites of hydrocortison which are not identical to any of the known metabolites.