Preparative High Pressure Liquid Chromatography Research Articles

A dipyrrole derivative from Aloe vera inhibits an anti-diabetic drug target Dipeptidyl Peptidase (DPP)-IV in vitro

Aloe vera, a succulent herb, has a long history of use in traditional medicine, including diabetes. Earlier studies from our laboratory demonstrated that the Aloe vera extract has the ability to inhibit the diabetic drug target dipeptidyl peptidase (DPP) IV in vitro. This current study focuses on the isolation of small water soluble active molecule(s) involved in DPP-IV inhibition from Aloe vera extract, and further to characterize its structure and to elucidate the mode of inhibition of the DPP-IV enzyme. Aloe vera gel ethanolic extract was subjected to preparative reverse-phase high-pressure liquid chromatography (RP-HPLC), LH-20 Sephadex gel filtration chromatography, followed by analytical RP-HPLC, to isolate the active molecule involved in DPP-IV inhibition. Based on the spectroscopic studies, the structure of the isolated DPP-IV inhibitor was predicted to be 3, 6-dioxo-3, 3a, 6, 6 a-tetrahydropyrrolo [3, 4-c] pyrrole-1, 4-dicarboxamide with the chemical formula C8H6N4O4, having the molecular weight of 225.175 Da. This molecule inhibited the DPP-IV enzyme in a noncompetitive manner with an IC50 value of 8.59 ± 2.61 µM, with a Ki of 4.7 ± 0.038 µM. Thus, the mechanism of DPP-IV inhibition and the inhibitory constants were determined. The results of our studies suggested that the inhibition of the DPP-IV enzyme as one of the pathways by which the Aloe vera extract may restore the pancreatic islets cell mass in diabetic animal model.

Taste Modulating Peptides from Overfermented Cocoa Beans.

Activity-guided fractionation of an aqueous extract of overfermented cocoa beans, which were recently found to be a rich source of previously unknown taste enhancing substances, revealed the presence of a series of taste modulating short peptides. Fractionation was achieved by means of sequential solvent extraction, medium pressure liquid chromatography, as well as preparative high pressure liquid chromatography, and the taste modulating activity was determined by means of matrix assisted taste dilution analysis. By means of ultra performance liquid chromatography-time-of-flight-mass spectrometry (UPLC-ToF-MS) screening, liquid chromatography-MS/MS methods, and customized syntheses, numerous short peptides could be identified in the taste modulating fractions. Sensory experiments of the target peptides showed umami enhancing and salt taste enhancing properties as well as kokumi effects when applied in a savory taste matrix. Evaluation of the taste threshold concentrations in model broth demonstrated a high taste modulating potential of 11 out of 13 identified peptides. Lowest threshold concentrations were determined for the salt taste enhancing tripeptide pEEE (55 μmol/L) and the kokumi active tripeptide VPA (90 μmol/L). Furthermore, a large number of dipeptides, carrying either a prolyl- or pyro-glutamyl moiety were located in the aqueous extract, exhibiting taste modulating properties and revealing a pH dependency of the taste modulating effect of the savory taste matrix. Additionally, synergistic effects of a mixture of five umami enhancing pyro-glutamyl dipeptides in the model matrix were demonstrated.

Efficient Synthesis of Norbuprenorphines Coupled with Enkephalins and Investigation of Their Permeability.

An efficient approach for the synthesis of norbuprenorphin derivatives through coupling of enkephalins and norbuprenorphine intermediates is described. Norbuprenorphine derivative was synthesized from thebaine and then, its reaction with succinic acid and phthalic acid was also studied. Meanwhile, the synthesis of enkephalins was done using solid phase peptide synthesis approach. Furthermore, after cleavage of the peptide from the surface of the resin, the coupling of enkephalins with norbuprenorphine derivative was done using TBTU as a coupling reagent then the derivatives were purified using preparative high-pressure liquid chromatography and their structures were confirmed using high-resolution mass spectrometry data. Later, their permeability across membranes was investigated. After PAMPA studies, it was found that the permeability of all norbuprenorphin-enkephalin derivatives was increased; however, succinic and phthalic acid derivatives showed higher permeability than norbuprenorphine-Leu-enkephalin.

H-Bonded Duplexes based on a Phenylacetylene Backbone.

Complementary phenylacetylene oligomers equipped with phenol and phosphine oxide recognition sites form stable multiply H-bonded duplexes in toluene solution. Oligomers were prepared by Sonogashira coupling of diiodobenzene and bis-acetylene building blocks in the presence of monoacetylene chain terminators. The product mixtures were separated by reverse phase preparative high-pressure liquid chromatography to give a series of pure oligomers up to seven recognition units in length. Duplex formation between length complementary homo-oligomers was demonstrated by 31P NMR denaturation experiments using dimethyl sulfoxide as a competing H-bond acceptor. The denaturation experiments were used to determine the association constants for duplex formation, which increase by nearly 2 orders of magnitude for every phenol-phosphine oxide base-pair added. These experiments show that the phenylacetylene backbone supports formation of extended duplexes with multiple cooperative intermolecular H-bonding interactions, and together with previous studies on the mixed sequence phenylacetylene 2-mer, suggest that this supramolecular architecture is a promising candidate for the development of synthetic information molecules that parallel the properties of nucleic acids.

Isolation and characterization of a tadalafil analogue, N-cyclopentyl nortadalafil in health supplement

A tadalafil analogue was detected for the first time during the screening of a health supplement for undeclared sexual enhancement drugs. The compound had been isolated and purified by preparative high-pressure liquid chromatography (HPLC). Its chemical structure was elucidated using high-resolution mass spectrometry (HRMS), electrospray ionization tandem mass spectrometry (ESI-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. The compound had a protonated molecular ion at m/z 444 with a chemical formula of C26H25N3O4. The data obtained from the MS analysis of the compound suggested that the N-methyl group on the piperazinedione moiety of tadalafil was substituted with a –C5H9 group. Analysis using NMR was performed and the –C5H9 group was characterized as a cyclopentyl moiety. The analogue was named N-cyclopentyl nortadalafil.

A new approach to δ15N compound‐specific amino acid trophic position measurements: preparative high pressure liquid chromatography technique for purifying underivatized amino acids for stable isotope analysis

Compound specific isotope analysis of amino acids (CSI‐AA) has emerged as an important new method for investigating trophic dynamics in both aquatic and terrestrial systems. Multiple studies have shown that δ15N values of glutamic acid (Glu) and phenylalanine (Phe) can be coupled to provide precise estimates of trophic position (TP), while simultaneously decoupling baseline δ15N values from the effects of trophic transfer. However, the current standard gas chromatography‐combustion‐isotope ratio mass spectrometry (GC‐C‐IRMS) approach is limited by high expense, limited availability, and relatively low precision. We present a new method for making TP estimates in biological samples by CSI‐AA (TPCSIA), based on a high‐pressure liquid chromatography (HPLC) purification of underivatized amino acids, followed by offline elemental analysis‐isotope ratio mass spectrometry (EA‐IRMS). We compare results from our new HPLC/EA‐IRMS method versus GC‐C‐IRMS in both standard and natural materials. Nitrogen isotopic values of purified Glu and Phe standards were identical within error for both methods. In five widely different marine organisms, the δ15N values of Glu and Phe were also indistinguishable within error between the two approaches; however, the δ15N values produced by the HPLC/EA‐IRMS approach had higher average precision (average SD = 0.3 ± 0.2 ‰) than the GC‐C‐IRMS measurements (average SD = 0.45 ± 0.15 ‰). The resulting TPCSIA estimates were statistically indistinguishable (t < 1.2, df = 6, P > 0.3) between the two methods for all organisms examined. Our HPLC/EA‐IRMS method may therefore allow significant expansion of TPCSIA applications, requiring only commonly available instrumentation to produce high precision TPCSIA values.

Anticancer Constituents and Cytotoxic Activity of Methanol-Water Extract of <em>Polygonum Bistorta</em> L.

This study was specifically designed to identify anticancer constituents in methanol-water extract of Polygonum bistorta L. and evaluate its cytotoxicity. For this purpose methanol-water (40:60 v/v) extract was subjected to conventional preparative high pressure liquid chromatography and 13 fractions were obtained. Constituents of obtained fractions were separated and identified with the help of GC-MS and LC-DAD-ESI-MS. Anticancer phenolic compounds such as gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, syringic acid, catechol, 4-methyl catechol, syringol and pyrogallol and fatty acids such as linoleic acid, myristic acid and palmitic acid were separated from different fractions. Fractions were evaluated for their cytotoxic activity on a rarely studied human hepatocellular carcinoma cell line (HCCLM3). 11 fractions showed good to strong cytotoxicity in a range of 200 µg/mL-800 µg/mL, whereas 2 fractions did not show any activity even at 800 µg/mL and no anticancer constituent was detected from them. 50 percent growth inhibition (GI50) values for five most active fractions were calculated and results were in a range of 86.5 (±3) µg/mL-126.8 (±3) µg/mL. 3 out of these 5 most active fractions were found to contain phenolic content in them whereas all other fractions containing phenolic content did possess cytotoxic activity that may suggest the importance of phenolic constituents in anticancer activity. Moreover, the results also showed a definite dose dependent relationship between amount of fractions and cytotoxic activity.

PREPARATIVE ISOLATION AND PURIFICATION OF CHEMICAL CONSTITUENTS OF BELAMCANDA BY MPLC, HSCCC, AND PREP-HPLC

Combined with medium-pressure liquid chromatography (MPLC) and preparative high-pressure liquid chromatography (Prep-HPLC), high-speed countercurrent chromatography (HSCCC) was successfully applied for separation and purification of isoflavonoids from the extract of belamcanda. HSCCC separation was performed on a two-phase solvent system composed of methyl tert-butyl ether-ethyl acetate-n-butyl alcohol-acetonitrile-0.1% aqueous trifluoroacetic acid at a volume radio of 1:2:1:1:5. Semi-purified peak fractions from HSCCC separation were further purified by Prep-HPLC. Nine well-separated fractions were analyzed by HPLC-UV absorption spectrometry to determine their purities and characterized with ESI-MSn. Except for peaks I and VII (unknown), seven compounds were identified as apocynin (peak II), mangiferin (peak III), 7-O-methylmangiferin (peak IV), hispidulin (peak V), 3′-hydroxyltectoridin (peak VI), iristectorin B (peak VIII), and isoiridin (peak IX).

Biotransformation of Flurbiprofen by Cunninghamella Species

The biotransformation of the fluorinated anti-inflammatory drug flurbiprofen was investigated in Cunninghamella spp. Mono- and dihydroxylated metabolites were detected using gas chromatography-mass spectrometry and fluorine-19 nuclear magnetic resonance spectroscopy, and the major metabolite 4'-hydroxyflurbiprofen was isolated by preparative high-pressure liquid chromatography (HPLC). Cunninghamella elegans DSM 1908 and C. blakesleeana DSM 1906 also produced a phase II (conjugated) metabolite, which was identified as the sulfated drug via deconjugation experiments.

Chemical and toxicological characterization of an unresolved complex mixture‐rich biodegraded crude oil

Chemical and toxicological characterization of unresolved complex mixtures in the water-soluble fraction of an artificially weathered Norwegian Sea crude oil was determined by a combination of chemical analysis and toxicity testing in fish in vitro bioassays. The water-soluble fraction of the crude oil was separated into 14 increasingly polar fractions by preparative high-pressure liquid chromatography. The in vitro toxicity (7-ethoxyresorufin O-deethylase activity, estrogenicity, and metabolic inhibition) of these fractions was characterized in a primary culture of liver cells (hepatocytes) from rainbow trout (Oncorhynchus mykiss). The main contributor to toxicity was one of the most polar fractions, accounting gravimetrically for more than 70% of the organic material in the water-soluble fraction and dominated by an unresolved complex mixture. Chemical analysis by gas chromatography-mass spectrometry and comprehensive two-dimensional gas chromatography-time of flight-mass spectrometry identified a large number of cyclic and aromatic sulfoxide compounds and low amounts of benzothiophenes (<0.1% of total mass) in this fraction. Commonly monitored toxic components of crude oil (e.g., naphthalenes, polycyclic aromatic hydrocarbons, and alkylated phenols) eluted in less polar fractions, characterized by somewhat lower toxicity. Normalization of in vitro responses to the mass in each fraction demonstrated a more even distribution of toxicity, indicating that toxicity in the individual fractions was related to the amount of material present. Although polar and nonpolar compounds contribute additively to crude oil toxicity, the water-soluble fraction was dominated by polar compounds because of their high aqueous solubility and the high oil-water loading. Under these conditions, the polar unresolved complex mixture-rich fraction might account for a large portion of crude oil toxicity because of its high abundance in the water-soluble fraction.

Nitration of Solvent-exposed Tyrosine 74 on Cytochrome c Triggers Heme Iron-Methionine 80 Bond Disruption: NUCLEAR MAGNETIC RESONANCE AND OPTICAL SPECTROSCOPY STUDIES

Cytochrome c, a mitochondrial electron transfer protein containing a hexacoordinated heme, is involved in other physiologically relevant events, such as the triggering of apoptosis, and the activation of a peroxidatic activity. The latter occurs secondary to interactions with cardiolipin and/or post-translational modifications, including tyrosine nitration by peroxynitrite and other nitric oxide-derived oxidants. The gain of peroxidatic activity in nitrated cytochrome c has been related to a heme site transition in the physiological pH region, which normally occurs at alkaline pH in the native protein. Herein, we report a spectroscopic characterization of two nitrated variants of horse heart cytochrome c by using optical spectroscopy studies and NMR. Highly pure nitrated cytochrome c species modified at solvent-exposed Tyr-74 or Tyr-97 were generated after treatment with a flux of peroxynitrite, separated, purified by preparative high pressure liquid chromatography, and characterized by mass spectrometry-based peptide mapping. It is shown that nitration of Tyr-74 elicits an early alkaline transition with a pKa = 7.2, resulting in the displacement of the sixth and axial iron ligand Met-80 and replacement by a weaker Lys ligand to yield an alternative low spin conformation. Based on the study of site-specific Tyr to Phe mutants in the four conserved Tyr residues, we also show that this transition is not due to deprotonation of nitro-Tyr-74, but instead we propose a destabilizing steric effect of the nitro group in the mobile Omega-loop of cytochrome c, which is transmitted to the iron center via the nearby Tyr-67. The key role of Tyr-67 in promoting the transition through interactions with Met-80 was further substantiated in the Y67F mutant. These results therefore provide new insights into how a remote post-translational modification in cytochrome c such as tyrosine nitration triggers profound structural changes in the heme ligation and microenvironment and impacts in protein function.

Trishomocubane Amino Acid as a β‐turn scaffold

The synthesis and X-ray structure of two diasteriomeric heptapeptides [Ac-Ala-Ala-Ala-(R/S)-Cage-Ala-Ala-Ala-NH2] with a trishomocubane amino acid as a beta-turn scaffold are reported. The amino acid was synthesized as a racemate and two diastereomeric peptides were obtained. The two peptides were separated by preparative high-pressure liquid chromatography and crystals suitable for X-ray analysis were grown for both diasteriomeric peptides. In general, both the peptides satisfy the criteria for beta-turn conformations. Five of the six Ala residues of both cage peptide crystals satisfy the criteria for 3(10)-helix characteristics and the cage amino acid residue satisfied the alpha-helix classification. These experimental results confirm previous theoretical studies in our laboratory which predicted that the cage moiety would be a strong/active beta-turn inducer.

Dynamic Developmental Elaboration of N-Linked Glycan Complexity in the Drosophila melanogaster Embryo

The structural diversity of glycoprotein N-linked oligosaccharides is determined by the expression and regulation of glycosyltransferase activities and by the availability of the appropriate acceptor/donor substrates. Cells in different tissues and in different developmental stages utilize these control points to manifest unique glycan expression patterns in response to their surroundings. The activity of a Toll-like receptor, called Tollo/Toll-8, induces a pattern of incompletely defined, but neural specific, glycan expression in the Drosophila embryo. Understanding the full extent of the changes in glycan expression that result from altered Tollo/Toll-8 signaling requires characterization of the complete N-linked glycan profile of both wild-type and mutant embryos. N-Linked glycans harvested from wild-type or mutant embryos were subjected to direct structural analysis by analytic and preparative high pressure liquid chromatography, by multidimensional mass spectrometry, and by exoglycosidase digestion, revealing a predominance of high mannose and paucimannose glycans. Di-, mono-, and nonfucosylated forms of hybrid, complex biantennary, and triantennary glycans account for 12% of the total wild-type glycan profile. Two sialylated glycans bearing N-acetylneuraminic acid were detected, the first direct demonstration of this modification in Drosophila. Glycan profiles change during normal development consistent with increasing alpha-mannosidase II and core fucosyl-transferase enzyme activities, and with decreasing activity of the Fused lobes processing hexosaminidase. In tollo/toll-8 mutants, a dramatic, expected loss of difucosylated glycans is accompanied by unexpected decreases in monofucosylated and nonfucosylated hybrid glycans and increases in some nonfucosylated paucimannose and biantennary glycans. Therefore, tollo/toll-8 signaling influences flux through several processing steps that affect the maturation of N-linked glycans.

Studies related to the origin of C18 neutral steroids isolated from extracts of urine from the male horse: The identification of urinary 19-oic acids and their decarboxylation to produce estr-4-en-17β-ol-3-one (19-nortestosterone) and estr-4-ene-3,17-dione (19-norandrost-4-ene-3,17-dione) during sample processing

For almost two decades we have known that enzymatic hydrolysis of “normal” urine samples from the entire male horse using Escherichia coli (E. coli) followed by solvolysis (ethyl acetate:methanol:sulphuric acid) results in the detection of significant amounts of estr-4-ene-3,17-dione (19-norandrost-4-ene-3,17-dione) along with estr-4-en-17β-ol-3-one (19-nortestosterone, nandrolone) in extracts of the hydrolysed urine and that both steroids are isolated from the solvolysis fraction. This solvolysis process is targeted at the steroid sulphates. Also we have shown that 19-norandrost-4-ene-3,17-dione and 19-nortestosterone are isolated from testicular tissue extracts.Subsequently, evidence was obtained that 19-nortestosterone detected in extracts of “normal” urine from male horses may not be derived from the 17β-sulphate conjugate. However, following administration of 19-nortestosterone based proprietary anabolic steroids to all horses (males, females and castrates), the urinary 19-nortestosterone arising from the administration is excreted primarily as the 17β-sulphate conjugate. Thus, if the 19-nortestosterone-17β-sulphate conjugate arises only following administration this has interesting implications for drug surveillance programmes to control administration of 19-nortestosterone based anabolic preparations to male horses.These results have led us to consider that the precursors to 19-nortestosterone and 19-norandrost-4-ene-3,17-dione, present in the urine prior to the hydrolysis steps, have the same basic structure except for the functionality at the 17-position. We have used preparative high pressure liquid chromatography (LC) and LC fractionation to separate these precursors from the high amounts of oestrogenic sulphates present in “normal” urine from the entire male horse. Purified fractions have then been studied by liquid chromatography–mass spectrometry (LC–MS) and gas chromatography–mass spectrometry (GC–MS) to identify the precursors.

A novel approach to the synthesis of EGF‐like domains: a method for the one‐pot regioselective formation of the three disulfide bonds of a human blood coagulation Factor VII EGF‐1 analogue

The study of EGF-like domains is of great general interest in protein science because of their participation in a multitude of protein-protein interactions. A common structural feature of EGF-like modules is the presence of three disulfide bonds, the regioselective formation of which still remains a challenge to peptide chemists. We report here on a method for the one-pot regioselective synthesis of an analogue of the EGF-1 domain of human coagulation Factor VII (residues 45-83) comprising an Asn57beta-Asp substitution. The cysteine protecting groups trityl, t-butyl and acetamidomethyl were chosen for the three disulfide bond pairings. All three disulfide bridges were prepared directly from the crude starting product, obviating the need for the timely and costly purification of the intermediate folded products. The fully folded product was purified by preparative high-pressure liquid chromatography prior to evaluation of its biological activity in an assay to detect inhibition of FVII/TF complex formation. In addition circular dichroism spectroscopy was employed to elucidate the main structural similarities between this peptide analogue and the native human Factor VII EGF-1 domain.

Formation, isolation, spectroscopic properties, and calculated properties of some isomers of C(60)H(36).

Isomers of C(60)H(36) and He@C(60)H(36) have been synthesized by the Birch or dihydroanthracene reduction of C(60) and isolated by preparative high-pressure liquid chromatography. (3)He, (13)C, and (1)H NMR spectroscopic properties were then determined. A comparison of experimental chemical shifts against those computed using density functional theory (B3LYP) with polarized triple- and double-zeta basis sets for He and C,H, respectively, allowed provisional assignment of structure for several isomers to be made. Theoretical calculations have also been carried out to identify low-energy structures. The transfer hydrogenation method using dihydroanthracene gives a major C(60)H(36) isomer and a minor C(60)H(36) isomer with C(3) symmetry as determined by the (13)C NMR spectrum of C(60)H(36) and the (3)He NMR spectrum of the corresponding sample of (3)He@C(60)H(36). In view of the HPLC retention times and the (3)He chemical shifts observed for the Birch and dihydroanthracene reduction products, the two isomers generated by the latter procedure can be only minor isomers of the Birch reduction. A significant energy barrier apparently exists in the dihydroanthracene reduction of C(60) for the conversion of the C(3) and C(1) symmetry isomers of C(60)H(36) to the T symmetry isomer previously predicted by many calculations to be among the most stable C(60)H(36) isomers. Many of the (1)H NMR signals exhibited by C(60)H(36) (and C(60)H(18), previously reported) are unusually deshielded compared to "ordinary" organic compounds, presumably because the unusual structures of C(60)H(36) and C(60)H(18) result in chemical shift tensors with one or more unusual principal values. Calculations clearly show a relationship between exceptionally deshielded protons beta to a benzene ring in C(60)H(18) and C(60)H(36) and relatively long C-C bonds associated with these protons. The additional information obtained from 1D and 2D (1)H NMR spectra obtained at ultrahigh field strengths (up to 900 MHz) will serve as a critical test of chemical shifts to be obtained from future calculations on different C(60)H(36) isomers.

The influence of automobile exhausts on mutagenicity of soils: contamination with, fractionation, separation, and preliminary identification of mutagens in the Salmonella/reversion assay and effects of solvent fractions on the sister-chromatid exchanges in human lymphocyte cultures and in the in vivo mouse bone marrow micronucleus assay

To test the assumption that automobile exhausts contribute to soil mutagenicity, two soils with low levels of mutagenic activities were exposed to traffic exhausts at a heavily charged junction of German motorways (Autobahnen) for 3, 7, 10, 13, 17, 21, and 26 weeks. Indeed, in the presence of a metabolic activation system from rat liver (S9), an average increase of 8 and 9 (4 and 12) revertants per gram per week was found in Salmonella typhimurium TA 98 (TA 100). In the absence of S9, meaningful measurements were impossible on account of a concurrent dose dependent increase of toxicity. No correlation between the increase of mutagenicity and the contents of polycyclic aromatic hydrocarbons (PAH) could be detected. In another series, soils sampled at the roadside and at distances of 10 and 50 m of five roads near Mainz expressed 10–20-fold higher mutagenicity (revertants per gram) under identical test conditions as compared with the average of agricultural soils. Toxic effects, however, again confounded the results and no correlation between the distance from roads and the levels of mutagenicity could be demonstrated. Subsequently, Soxhlet-extraction with the solvent sequence dichloromethane, acetone, and toluene/diethylketone was found to be an optimum procedure for soils at roadsides. The mass balance of solvent fractionation of such soils revealed that <2% each belonged to organic acids and bases, ∼4% to fractions designed polar neutrals, ∼8% to polar aromatics, ∼7% to dichloromethane solubles, and ∼79% to cylohexane solubles, among them ∼63% acetone soluble compounds. The major part of mutagenicity (55–65%) was present in the fraction of polar aromatics, followed by polar neutrals and the acetone subfraction of cyclohexane solubles (∼10% each) summarizing the results obtained with S. typhimurium TA 98, TA 98NR, YG 1021, YG 1024, TA 100, YG 1026, and YG 1029 with and without addition of S9. The modified tester strains, either deficient in nitroreductase (TA 98NR) or overproducing nitroreductase (YG 1021, 1026) or O-acetyl-transferase (YG 1024, 1026), indicated a major contribution of nitroarenes to soil mutagenicity. With respect to mutagenic PAH, high pressure liquid chromatography (HPLC) revealed that >90% of dibenz[a,h]anthracene (4.18 mg/kg soil), benzo[a]pyrene (1.96 mg), benzofluoranthenes (0.14 mg), and benz[a]anthracene (0.18 mg) were present in the acetone subfraction of cyclohexane solubles. Concentrations and mutagenic activities, however, did not correlate. Additional preparative and analytical HPLC of the solvent fractions of polar neutrals and polar aromatics, resulted in the tentative identification of 2-nitrofluorene. Analysis of the vertical profile of soil revealed an increase of mutagenicity per gram from the surface to a maximum at 5–15 cm depth and a subsequent decrease with very little activity remaining deeper than 35 cm. In human lymphocyte cultures, the fraction of polar aromatics, 0.01–0.3 μg/ml, induced 11.27±4.76–20.70±6.19 sister-chromatid exchanges (SCE) per cell in the absence of S9 (solvent control: 10.16±4.83 SCE per cell) and 12.77±6.53–17.87±4.93 SCE per cell in the presence of S9 (solvent control: 8.37±3.92 SCE per cell). However, no activities could be detected in the fractions of polar neutrals and non-polar neutrals. Again, negative results were obtained in the in vivo mouse bone marrow micronucleus assay at 2000 mg/kg p.o. with all fractions.

Preparative HPLC with ultrastable-Y zeolite for compound-specific carbon isotopic analyses

Preparative high pressure liquid chromatography on US-Y zeolite shape-selective molecular sieve was studied for carbon isotopic fractionation effects. We tested a standard mixture [17β, 21β(H)-hopane, 5α-cholestane] and complex natural hydrocarbon mixtures dominated by tetra- and pentacyclic triterpenoids extracted from Oxford Clay shales. We confirmed that steroids and hopanoids were separated on the basis of stereochemical configuration while isotopic analysis of eluents indicated that shape-selective chromatography did not result in isotopic fractionation. US-Y zeolite chromatography can be used to simplify hydrocarbon mixtures and prepare well resolved mixtures of molecular fossils for compound-specific isotopic analyses (CSIA).

Cuticular waxes of the holm (Quercus ilex l. subsp.ballota (desf.) samp.) and cork (Q. suber l.) oaks

The epicuticular leaf waxes of young leaves of the Holm oak (Quercus ilex L. ssp. ballota (Desf.) Samp.) and the cork oak (Q suber L.) were extracted in chloroform and separated into their constituent wax classes by preparative thin layer and high pressure liquid chromatography. The wax coverage was about 125 μg/cm2 in Q. suber and about 71 μg/cm2 in Q. ilex. The main classes encountered (by weight) were odd chain n-alkanes (9% in Q. ilex, and 4–27% in Q. suber) and even chain amphiphilic compounds. The major classes of amphiphilic compounds were n-alkan-1-ols (20–27% in Q. ilex, and 18–50% in Q. suber), n-alkanals (<3% in Q. ilex, and up to 25% in Q. suber), n-alkanoic acids (11–12% in Q. ilex, and <5% in Q. suber) and n-alkyl esters (50–56% in Q. ilex, and 25–45% in Q. suber). The major components of each class, identified by gas chromatography coupled to mass spectrometry were C29 for the n-alkanes (54–58% in Q. ilex, and 47–57% in Q. suber), C24 for the n-alkan-1-ols (78–87% in Q. ilex, and 77–93% in Q. suber), C30 for the n-alkanals (up to 57% in Q. ilex, and up to 77% in Q. suber), C22 and C24 for the n-alkanoic acids in Q. ilex (39–62%) and C30 in Q. suber (49%). The n-alkyl esters were composed mainly from C22 and C24 n-alkanoic acids (∼38%) and n-alkan-1-ols (43–54%) in Q. ilex, whereas in Q. suber these esters were composed mainly from C22 and C24 n-alkanoic acids (44–52%) and C20 and C22 n-alkan-1-ols (32–66%). In addition to the above compounds, trace amounts (<5%) of C28 and C30 n-alkenes and the triterpenone, friedelin, were encountered in Q. suber and both plant waxes contained small amounts of the triterpenols α- and β-amyrin (5–9% of the total alkanol content). The variability observed in the chemical composition of the leaf waxes were attributable to seasonal causes and/or leaf age. Copyright © 1999 John Wiley & Sons, Ltd.

Characterization of monomeric and oligomeric flavan-3-ols from unripe almond fruits

The flavan-3-ol composition of unripe sweet almond fruits (Prunus amygdalus) was analysed. Isolation of compounds was performed on a methanol extract of the fruit by gel chromatography on Sephadex LH-20 and then by preparative high pressure liquid chromatography. Identification of compounds was carried out by different kinds of hydrolyses. Two monomers, (+)-catechin and (−)-epicatechin, and 15 oligomeric procyanidins, (six dimers, seven trimers and two tetramers) were identified. Higher procyanidins present in the extract were estimated to have an average degree of polymerization of five by using a method based on hydrolysis in the presence of phloroglucinol. A tentative analysis was also carried out by liquid chromatography-mass spectrometry (LC-MS) in order to explore the potential of this recently introduced technique for the analysis of proanthocyanidins. The results obtained permitted the confirmation of the identifications made by hydrolysis techniques and also the detection and pre-identification of other minor compounds as two possible A-type procyanidins. The possibility of resolving peaks of co-eluting compounds was shown as another interesting aspect offered by the LC-MS technique. © 1998 John Wiley & Sons, Ltd.