HPLC Peak Research Articles

Improved method for the determination of triacylglycerols in olive oils by high performance liquid chromatography

The analysis of triacylglycerols has great importance as quality control and origin determination aid in olive oils analytical methodologies. New improvements in the analysis of triacylglycerols by high-performance liquid chromatography in olive oils are developed in order to increase the separation between LLL/OLLn and OLL/OOLn critical pairs used for detection of seed oils in olive oils. Elution with acetone/acetonitrile (55:45) using gradient temperature and isothermal elution with propionitrile were investigated, in comparison with the European Union official method. The best resolution was achieved using propionitrile at 20 oC. Although the HPLC profile was similar using propionitrile and acetone/acetonitrile, differences in minor triacylglycerols contributing to each HPLC peak were encountered. The precision of the method was good.

In vitro steroidogenesis of the gonads of the protogynous Pacific wrasse, Haliochoeres trimaculatus

Testicular and ovarian fragments of the protogynous Pacific wrasse Haliochoeres trimaculatus were incubated in vitro with [3H]pregnenolone ([3H]P5), [3H]17‐hydroxyprogesterone ([3H]17OHP4), non‐radioactive (nr) 17β‐oestradiol (nrE2) or nrP5 to identify the major gonadal steroidogenic pathways and steroid products in females and in the two male variants of this species, the terminal phase (TP) and initial phase (IP) males. Both testis and ovarian tissues exhibited 7 hydroxylase activity resulting in the formation of 7α‐hydroxypregnenolone (7OHP5) from [3H]P5, and many HPLC peaks were identified as products of testicular (c. 29) and ovarian (c. 23) steroidogenesis, and only c. 50% of these metabolites co‐eluted with authentic reference standards; only very small amounts of conjugated steroid were synthesized from any of the precursors. [3H]P5 was converted by testis mainly to 7αOHP5, and two unknown steroids, whereas [3H]17OHP4 metabolism gave rise to [3H]17,20β‐dihydroxy‐4‐pregnen‐3‐one (DHP), 11‐ketotestosterone (11KT), and two unknown steroids. For ovarian tissues, [3H]17OHP4 and [3H]P5 were metabolized to form E2, oestrone (E1), androstenedione (A4), 20α‐ and 20β‐dihydroprogesterone (20αDHP and 20βDHP), 7αOHP5 (from [3H]P5) and a major unknown. The HPLC steroid profiles for testis incubations for IP and TP males were similar, however, the steroidogenic response of the testis of TP males to human chorionic gonadotrophin, in vitro (determined by hormone assay), was significantly higher than that of IP males.

Characterization of the purity and stability of commercially available dichlorotriazine chromophores used in nonlinear optical materials

Nonlinear optical (NLO) materials consisting of organic chromophores have unique optical properties due to the polar molecular structure and noncentrosymmetric ordering of the chromophores. Chromophores with dichlorotriazine moieties can be incorporated with a consistent polar ordering into layer-by-layer films via covalent reaction with a nucleophilic polymeric species. While the manufacture of optical materials traditionally requires highly pure reagents, commercially available azo dyes originally manufactured for textile industry use are usually impure. The purities of three commercially available chromophores, Reactive Red 2, Reactive Orange 4, and Reactive Brown 23, that are being used in NLO materials research were determined by HPLC and found to be highly variable. MALDI-TOF MS analysis of collected HPLC peaks was used to confirm the identity of the chromophores and impurities that resulted from basic hydrolysis. Additionally, since noncentrosymmetric deposition of these chromophores into NLO films occurs at basic pH and hydrolyzed chromophores are not reactive, the stability of the chromophores at pH 10.5 was determined. Base hydrolysis experiments also revealed whether other impurities in the dyes were reactive. Because the layer-by-layer method of making NLO films relies on covalent reaction, the presence of non-reactive impurities may not be a critical issue. However, reactive impurities can take up space and inhibit incorporation of the primary chromophore in the film, and if their dipole moment orientation is different, they may cancel out the molecular ordering of the primary chromophore and further decrease NLO performance.

Determination of thiobarbituric acid adduct of malondialdehyde using on-line microdialysis coupled with high-performance liquid chromatography.

An on-line analytical system for the continuous monitoring of malondialdehyde (MDA) was developed. This method involves the use of microdialysis perfusion, on-line derivatization and on-line HPLC analysis. This method gave a linear response for MDA concentrations and HPLC peak areas in the range from 0.051 microM to 2.43 microM. The intra-day (RSD = 1.6-10.5%) and inter-day (RSD = 1.1-9.3%) precisions were acceptable. The average in vitro probe recovery of MDA standard was 18.4 +/- 1.0%. The detection limit was 0.03 microM, corresponding to 0.6 pmol for an injection volume of 20 microl. This method was used for in vitro peroxidation investigations, which provided evidence for elevated MDA levels following the incubation of metal ions to a linoleic acid solution.

Radiolabeling morpholinos with 90Y, 111In, 188Re and 99mTc

This laboratory is investigating morpholinos (MORF), a DNA analogue, for radiopharmaceutical applications. While we routinely radiolabel with 99mTc, we have now labeled MORFs with 111In, 188Re and 90Y in anticipation of therapeutic studies. Methods: A 25 mer MORF with a primary amine on the 3′ equivalent end attached via a 10 member linker was conjugated with an isothiocyanate backbone derivative of DOTA (for labeling with 111In and 90Y) and with NHS-MAG 3 (for labeling with 188Re and 99mTc). The in vitro stability of labeled MORFs were investigated and biodistribution was carried out in normal mice. Results: As evident by size exclusion HPLC, ITLC and Sep-Pak analysis, all four radiolabeled MORFs were successfully radiolabeled. In each case, the labeled MORFs showed one sharp peak in HPLC that shifted completely to earlier retention times following addition of a polymer conjugated with the complementary MORF. In saline at room temperature and in 37°C serum, the radioactivity profile of 111In, 188Re and 99mTc was unchanged over 48 h while over the same period, the 90Y profile showed a pronounced lower molecular weight peak which did not shift and was shown to be most probably due to 90Y-DOTA resulting from radiolysis. In addition, the recovery of 188Re on HPLC decreased as samples aged probably due to oxidation to perrhenate which was retained by the HPLC column. The biodistributions at 1, 3 and 6 h in normal mice showed no important differences among all four labels with the exception that levels of radioactivity in stomach and thyroid were higher in the case of 188Re due to in vivo oxidation of the radiolabel to perrhenate. Conclusions: When radiolabeled with DOTA, 90Y-labeled MORF showed increased instabilities relative to that of 111In and when radiolabeled with MAG 3, 188Re showed in vitro and in vivo instabilities compared to 99mTc, but all labels were still largely intact after 48 h in saline or serum. Possibly because of the rapid clearance of MORFs, no important differences in biodistribution among 90Y, 111In and 99mTc labels were evident in normal mice. These strategies for labeling MORF with 90Y and 188Re therefore appear to be suitable for therapeutic applications although both show some evidence of instabilities.

Further studies on the structure of polysaccharides from the lichen Flavoparmelia caperata (L.) Hale.

A water-soluble polysaccharide called PC-2 was previously isolated from Flavoparmelia caperata (L.) Hale and assigned to alpha(1-3)(1-4)glucan. However, PC-2 separated into three components, PC-2A, PC-2B, and PC-2C (a single peak in HPLC, respectively) on further purification. Methylation analysis and (1)H- and (13)C-NMR spectroscopic studies suggested that PC-2A is composed of repeating units of [alpha-D-Glc1-3](3), : [alpha-D-Glc1-4](2), while PC-2B and PC-2C are partly branched galactoglucomannans consisting of (1-3)- and (1-4)-linked alpha-D-glucopyranosyl units as the main chain. In addition we confirmed that the polysaccharide fraction PB-2 from the lichen of the same genus, Flavoparmelia baltimorensis (Gyelink & Foriss) Hale, is identical to PC-2 based on the chemical and spectroscopic data.

Characterisation and partial purification of a white mustard kairomone that attracts the beet cyst nematode, Heterodera schachtii

AbstractA kairomone from white mustard, Sinapis alba , that attracts infective juveniles (J2) of the beet cyst nematode, Heterodera schachtii, was extracted from root exudates and enriched by rotary evaporation. The kairomone-containing root exudate fractions were then repeatedly enriched and separated on a combination of octyl and octadecyl columns, leaving a single UV-absorbing HPLC peak. Glucose and fructose were identified by a subsequent HPLC separation in the ion moderated partition chromatography mode. The kairomone appeared in the fructose-containing fraction. As fructose did not cause nematode attraction, the fructose fraction contained at least one further substance. HPLC separations showed that the kairomone shares several properties with fructose and that it is relatively more polar than those compounds giving an UV signal. The repeated reversed-phase and ion moderated partition chromatography separations of single kairomone-containing fractions indicate that the kairomone is composed of one single substance which could not be identified with the methods applied. Gas chromatographic and mass spectrometric analyses revealed the presence of glucose, fructose, myo-inositol, sucrose and xylofuranose/ribofuranose in S. alba root exudates.

Evidence that glycoprotein 96 (B2), a stress protein, functions as a Th2-specific costimulatory molecule.

After the engagement of Ag receptor, most of the Th cells for their optimal activation require a second (costimulatory) signal provided by the APCs. We demonstrate the isolation and characterization of a 99- to 105-kDa protein (B2), from LPS-activated B cell surface, and its function as a Th2-specific costimulatory molecule. Appearance of B2 as a single entity on two-dimensional gel electrophoresis and as a distinct peak in reverse-phase HPLC ascertains the fact that B2 is homogeneous in preparation. Electron microscopy as well as competitive binding studies reveal that (125)I-labeled B2 specifically binds anti-CD3-activated T cell surface and also competes with its unlabeled form. Internal amino acid sequences of B2 are found to be identical with stress protein gp96. The identity of B2 as gp96 is also revealed by immunological characterization and by confocal microscopic colocalization studies of B2 and gp96 on LPS-activated B cells. Confocal imaging studies also demonstrate that gp96 can be induced on B cell surface without association of MHC molecules. Furthermore, the novel role of gp96 in Th cell proliferation skewing its differentiation toward Th2 phenotype has also been established. Ab-mediated blocking of gp96-induced signaling not only abrogates in vitro proliferation of CD4(+) T cells, but also diminishes the secretion of Th2-specific cytokines. Notably, the expression of CD91 (receptor of gp96/B2) is up-regulated on anti-CD3-activated Th cells and also found to be present on Th1 and Th2 subsets.

Molecular species of acylglycerols incorporating radiolabeled fatty acids from castor (Ricinus communis L.) microsomal incubations.

Sixty-one molecular species of triacylglycerols (TAG) and diacylglycerols produced from castor microsomal incubations incorporating six different (14)C-labeled fatty acids have been identified and quantified. The preference for incorporation into TAG was in the order ricinoleate > oleate > linoleate > linolenate > stearate > palmitate. Ricinoleate was the major fatty acid incorporated, whereas stearate, linolenate, and palmitate were incorporated at low levels. Twenty-one molecular species of acylglycerols (HPLC peaks) in castor oil have also been assigned. The levels of TAG in castor oil are RRR (triricinolein) >> RR-TAG >> R-TAG > no R-TAG. The levels of the molecular species within the groups of RR-TAG, RL-TAG, and LL-TAG individually are ricinoleate > linoleate > oleate > linolenate, stearate, and palmitate. The results of the labeled fatty acid incorporation are consistent with ricinoleate being preferentially driven into TAG and oleate being converted to ricinoleate in castor oil biosynthesis.

Intersexual variations in the venom of the Brazilian ‘armed’ spider Phoneutria nigriventer (Keyserling, 1891)

Venom from male and female specimens of the medically most important Brazilian Ctenidae spider Phoneutria nigriventer (Keyserling, 1891) has been compared. Males showed a slightly higher venom yield (386 μg) than equal sized females (296 μg), while adult females showed nearly a three times higher venom yield (1079 μg). High-pressure liquid-chromatography analyses revealed differences in the venom composition between males and females. A single peak in HPLC (peak 11) was only detected in venom from females carrying egg-sacs, and sodium dodecyl sulfate–gel electrophoresis showed a series of high molecular weight proteins only in the male venom pool. The median lethal dose (LD 50) in mice for female venom was 0.63 μg kg −1 (95% confidence interval [0.54; 0.71] and 0.61 [0.56; 0.73] μg kg −1) for females with egg-sacs, when compared to the male venom which showed a LD 50 of 1.57 [1.46; 1.88] μg kg −1. The venom of both sexes was also tested in insects using a termite bioassay with doses of 2, 3, 4, and 5 μg per termite. No effect was detected for the lowest dose of female venom, whereas all the other venom doses from both sexes caused a decreased paralysis time and death of the termites. Comparing the venom of both sexes, it was observed that female venom provoked a faster reaction than male venom. The results indicate that males and females of P. nigriventer have differing venom composition which lead to different effects in biological assays.

Identification of protein-derived tyrosyl radical in the reaction of cytochrome c and hydrogen peroxide: characterization by ESR spin-trapping, HPLC and MS.

The reaction of cytochrome c and H(2)O(2) is known to form a protein-centred radical that can be detected with the spin trap 2-methyl-2-nitrosopropane (MNP). To characterize the MNP/tyrosyl adduct structure that had previously been determined incorrectly [Barr, Gunther, Deterding, Tomer and Mason (1996) J. Biol. Chem. 271, 15498-15503], we eliminated unreasonable structure models by ESR studies with a series of (13)C-labelled tyrosines, and photochemically synthesized an authentic MNP/tyrosyl adduct that has its trapping site on the C-3 position of the tyrosine phenyl ring. The observation of the identical ESR spectra for this radical adduct from the UV irradiation of 3-iodo-tyrosine and the adduct from the cytochrome c reaction demonstrated that the radical trapping site of MNP/tyrosyl is located on the equivalent C-3/C-5 positions instead of the C-1 position, as was proposed by Barr et al. In an on-line HPLC/ESR system, an identical retention time (17.7 min) was observed for the ESR-active HPLC peak of the MNP/tyrosyl adduct from the following three reactions: (i) the tyrosine oxidation via horseradish peroxidase/H(2)O(2); (ii) UV irradiation of 3-iodo-tyrosine and (iii) the reaction of cytochrome c with H(2)O(2). This result demonstrated that the radical adducts of all three reactions are most probably the same. The mass spectrometric analysis of the HPLC fractions from reactions (i) and (ii) showed an ion at m/z 267 attributed to the MNP/tyrosyl adduct. We conclude that the cytochrome c-derived tyrosyl radical was trapped by MNP, leading to a persistent radical adduct at the C-3/C-5 positions of the tyrosine phenyl ring.

Identification of protein-derived tyrosyl radical in the reaction of cytochrome c and hydrogen peroxide: characterization by ESR spin-trapping, HPLC and MS

The reaction of cytochrome c and H2O2 is known to form a protein-centred radical that can be detected with the spin trap 2-methyl-2-nitrosopropane (MNP). To characterize the MNP/tyrosyl adduct structure that had previously been determined incorrectly [Barr, Gunther, Deterding, Tomer and Mason (1996) J. Biol. Chem. 271, 15498–15503], we eliminated unreasonable structure models by ESR studies with a series of 13C-labelled tyrosines, and photochemically synthesized an authentic MNP/tyrosyl adduct that has its trapping site on the C-3 position of the tyrosine phenyl ring. The observation of the identical ESR spectra for this radical adduct from the UV irradiation of 3-iodo-tyrosine and the adduct from the cytochrome c reaction demonstrated that the radical trapping site of MNP/tyrosyl is located on the equivalent C-3/C-5 positions instead of the C-1 position, as was proposed by Barr et al. In an on-line HPLC/ESR system, an identical retention time (17.7min) was observed for the ESR-active HPLC peak of the MNP/tyrosyl adduct from the following three reactions: (i) the tyrosine oxidation via horseradish peroxidase/H2O2; (ii) UV irradiation of 3-iodo-tyrosine and (iii) the reaction of cytochrome c with H2O2. This result demonstrated that the radical adducts of all three reactions are most probably the same. The mass spectrometric analysis of the HPLC fractions from reactions (i) and (ii) showed an ion at m/z 267 attributed to the MNP/tyrosyl adduct. We conclude that the cytochrome c-derived tyrosyl radical was trapped by MNP, leading to a persistent radical adduct at the C-3/C-5 positions of the tyrosine phenyl ring.

Development of a system to evaluate compound identity, purity, and concentration in a single experiment and its application in quality assessment of combinatorial libraries and screening hits.

The development and use of a new assay system for the simultaneous determination of identity, purity, and concentration of sample components from combinatorial libraries produced by parallel synthesis are described. The system makes use of high-performance liquid chromatography with UV/vis photodiode array (PDA), evaporative light scattering (ELSD), chemiluminescent nitrogen (CLND), and time-of-flight mass spectrometer (TOFMS) detectors (HPLC-PDA-ELSD-CLND-TOFMS). Although these detectors have previously been utilized separately for the analysis of combinatorial chemistry libraries, the use of TOFMS along with CLND provides a synergistic combination enabling target and side-product structures to be identified and their concentrations and purities determined in a single experiment from a solution containing microgram levels of material. The CLND was found to give a linear response based on the number of moles of nitrogen present. Therefore, if the number of nitrogens per molecule is known, the concentration of each nitrogen-containing sample component may be determined utilizing an unrelated co-injected standard. A molecular formula for an impurity may often be calculated from the exact mass determined by the TOFMS and knowledge of the chemistry involved. Thus, if the sample components contain nitrogen, the concentration of every identified HPLC peak may be determined even in the absence of primary standards. This combination of detectors enabled the characterization of both target compounds and byproducts in combinatorial libraries, allowing the optimization of library synthetic procedures. This system was also used to survey the quality of libraries, enabling the selection of the best libraries for screening. This method also facilitated the characterization of samples from combinatorial libraries found as hits in high-throughput screening to establish the potency of the leads based on their actual concentration. In addition, concentrations and potencies of impurities were determined after identification of their structures, utilizing exact mass data, determination of charge states, and knowledge of the synthetic chemistry.

Stabilization and improvement of catalytic activity of a low molar mass cellobiase by cellobiase-sucrase aggregation in the culture filtrate of Termitomyces clypeatus.

The extracellular cellobiase (EC 3.2.1.21) of Termitomyces clypeatus separated in two protein fractions when culture filtrate or ammonium sulfate precipitated proteins were chromatographed on BioGel P-200 column. During purification of cellobiase (CBS) from the lower molar mass (LMM) protein fraction, the enzyme behaved like a low molecular weight multimeric protein. The purified enzyme gave a single 56 kDa band in SDS-PAGE but ladderlike bands (14, 28, 42, and 56 kDa) on denaturation by reducing-SDS and urea. The protein, however, dissociated on dilution and protomeric (14 kDa) and multimeric forms (28 and 60 kDa) were eluted separately during HPGPLC. Specific activity of CBS gradually decreased as the molar mass of the enzyme was lowered in different eluted peaks. Protein present in all CBS pool fractions had the same amino acid composition and all displayed the same, single protein peak in reverse-phase HPLC and 56 kDa band in SDS-PAGE. Thus, T. clypeatus CBS was a multimeric 14 kDa protein that was optimally active as a tetramer. CBS purified from the higher molar mass fraction (HMM) as a SDS-PAGE homogeneous 110-kDa protein did not dissociate on dilution or by SDS-urea. The purified protein was a protein aggregate as CBS consistently contained 20 +/- 5% sucrase (SUC) Units in the preparation. The aggregate resolved during reverse-phase chromatography on a C(4) column, and an additional protein peak other than CBS was detected. The aggregated CBS had a higher temperature optimum and was more stable toward thermal and chemical denaturations than SUC-free CBS. Increase of stability and catalytic activity of CBS by aggregation with SUC was much higher than those by the multimerization of CBS itself. All of these observations for the first time suggested that the heterologous protein-protein aggregation, observed for a long time for fungal enzymes, might have a significant role in modulating physicochemical properties of the extracellular enzyme.

Identification of selenocompounds in yeast by electrospray quadrupole-time of flight mass spectrometry

The performance of a quadrupole-time of flight (Q-TOF) tandem mass spectrometer was evaluated for the identification of low-molecular weight selenocompounds in an aqueous yeast extract. The accuracy of mass measurements, evaluated with selenomethionine, selenoethionine and selenocystine standards was found to be between 0.005 and 0.01% (0.01–0.02 u). The low molecular weight fraction was obtained by collecting selenocompounds eluted in the total volume of a G-15 size-exclusion column. The fraction gave three peaks in anion-exchange HPLC of which the identity was investigated by ES-Q-TOFMS/MS. Altogether, eight clusters with the isotopic pattern corresponding to the presence of Se were observed. They were further investigated by collision induced dissociation TOFMS leading to the demonstration of the presence of a series of new selenocompounds, all characterized by the presence of an adenosyl functional group.

Evaluation of capillary liquid chromatography–electrospray ionization ion mobility spectrometry with mass spectrometry detection

Due to the proteomics revolution, multi-dimensional separation and detection instruments are required to evaluate many peptides and proteins in single samples. In this study, electrospray ionization (ESI) ion mobility spectrometry (IMS) was evaluated as an additional separation after HPLC separations. Common HPLC mobile phase compositions (solvents, acid modifiers, and buffers) were assessed for the effect on ESI-IMS response. Up to 5 m M sodium phosphate, a non-volatile buffer, was able to be electrosprayed into the IMS without degradation of the instrumental performance. Due to the rapid separation times of IMS, multiple IMS spectra were obtained within a single HPLC peak. A five-peptide mixture was separated in a capillary HPLC column under isocratic conditions within 3 min. Coelution of two peaks due to non-optimal HPLC conditions occurred and these two peaks could not be distinguished by HPLC with UV detection. In contrast, the single ion mobility chromatograms provided separation of each peptide as well as providing a second degree of analyte identification (HPLC retention time and IMS mobility). Furthermore, IMS–MS analysis of the five peptides and comparison with HPLC retention times showed that each peptide had a unique retention time–ion mobility–mass to charge value. This work showed that IMS could be employed for direct separation and detection of HPLC eluents and also could be combined with HPLC–MS for three unique dimensions of separation.

Anti‐elastase and anti‐hyaluronidase of phenolic substance from Areca catechu as a new anti‐ageing agent

We have previously screened 150 medicinal plants for the inhibition of elastase and found significant inhibitory effects of the extracts of Areca catechu L. on the ageing and inflammation of skin tissues. To isolate and identify the compounds having biological activity, they were further purified by each fraction of solvents, silica gel column chromatography, preparative TLC and reversed-phase HPLC. The peak in HPLC, which coincided with the inhibitory activity against elastase, was identified as a phenolic substance by using various colorimetric methods, UV and IR. IC(50) values of this phenolic substance were 26.9 mug mL(-1) for porcine pancreatic elastase (PPE) and 60.8 mug mL(-1) for human neutrophil elastase (HNE). This phenolic substance showed more potent activity than that of reference compounds, oleanolic acid (76.5 mug mL(-1) for PPE, 219.2 mug mL(-1) for HNE) and ursolic acid (31.0 mug mL(-1) for PPE, 118.6 mug mL(-1) for HNE). According to the Lineweaver-Burk plots, the inhibition against both PPE and HNE by this phenolic substance was competitive inhibition with the substrate. The phenolic substance from A. catechu effectively inhibited hyaluronidase activity (IC(50) : 210 mug mL(-1) ). These results suggest that the phenolic substance purified from A. catechu has an anti-ageing effect by protecting connective tissue proteins.

Speciation of selenoamino acids, selenonium ions and inorganic selenium by ion exchange HPLC with mass spectrometric detection and its application to yeast and algae

Cation and anion exchange HPLC were used to separate a mixture of 12 selenium species comprising selenoamino acids, selenonium ions and inorganic selenium. The cationic species were separated from each other and from the co-injected anions using a cation exchange column with gradient elution by aqueous pyridinium formate at pH⊕∼3 as the mobile phase. The anionic species were separated using an anion exchange column with isocratic elution by an aqueous salicylate–TRIS mobile phase at pH 8.5. The separated selenium species were detected as 80Se by ICP-dynamic reaction cell (DRC)-MS. The analytical methods were applied to the analysis of yeast and algae enriched in selenium. The yeast was treated with β-glucosidase followed by a protease mixture for dissolution of the cell walls and selenium-containing peptides, respectively. The second to largest HPLC peak after that corresponding to selenomethionine was ascribed to selenomethionine-Se-oxide (SeOMet) by retention time matching with a SeOMet standard, which was characterised by HPLC-electrospray (ES)-MS. Se-methylselenocysteine was detected based on co-chromatography with the standard substance spiked to the yeast hydrolysate. A trichloroacetic acid extract of Chlorella algae contained dimethylselenonium propionate (DMSeP), which was verified by HPLC-ES-MS. Se-allylselenocysteine and selenoethionine was detected at the low ng g−1 concentration level based on co-chromatography with the standard substances spiked to the algal extract.

Recognition and strand displacement of DNA oligonucleotides by peptide nucleic acids (PNAs): High-performance ion-exchange chromatographic analysis

Peptide nucleic acids (PNAs) are oligonucleotide mimics containing a pseudopeptide chain, which are able to bind complementary DNA tracts with high affinity and selectivity. Two mixed-sequence PNA undecamers ( 1 and 2) were synthesized and their double-stranded adducts with the complementary oligonucleotides ( 3 and 4) were revealed by the appearance of the corresponding peak in anion-exchange HPLC. A DEAE column was used and elution was performed with aqueous Tris buffer (pH 8) and an ionic strength gradient (0–0.5 M NaCl). The same effect was not observed with non-complementary oligonucleotides. The stability of the PNA–DNA adducts under the conditions used in the chromatographic system was studied as a function of temperature. Furthermore, in competition experiments double-stranded oligonucleotides were challenged by a PNA complementary to one strand: the formation of the PNA–DNA hybrid and the displacement of the non-complementary strand were observed with high specificity. The results suggest a possible use of ion-exchange HPLC for studying PNA–DNA interactions, and indicate the efficiency of PNA probes in the chromatographic analysis of DNA.

Identification of new diketopiperazines in roasted coffee

This paper reports on the identification of new diketopiperazines (DKPs) in roasted coffee. The roasted coffee was extracted with hot water and CHCl3 to achieve both a cleanup and a concentration of the DKPs. To get rid of the huge caffeine peak in both HPLC and GC analysis, this extract was cleaned up again by ion-exchange chromatography on Dowex 50×8 followed by gel chromatography on Sephadex G10. The identification was accomplished by HPLC-ESI-MS, HPLC-ESI-MS/MS, GC-EI-MS and by comparison with synthesised reference compounds. In roasted coffee the following DKPs were identified: cyclo(pro-gly), cyclo(pro-ala), cyclo(phe-val), cyclo(phe-leu), and cyclo(phe-ile). Except for cyclo(pro-gly), where only one isomer can be formed, each DKP was present in both possible isomeric forms.