HPLC Peak Research Articles

Production of tartrate and glycolate from the electrochemical reduction of glyoxylate

There has been considerable controversy about these reductions. Bewick and Greener [7, 8] claimed to have produced glycolic acid from CO2 on lead and malic acid from CO2 on mercury but they did not detect glyoxylic acid. Similar results were reported by Wolf and Rollin [9] but others were unable to repeat these results, or obtained different products [3]. A number of workers have studied the voltammetric reduction of glyoxylic acid [10, 11]. But there have been no direct preparative studies since Bauer [12] in 1931. Some preparative studies are presented here. It has been generally assumed that the electrochemical reduction of glyoxylate goes by a two electron process to give glycolate (perhaps explaining Bewick's results [7]). However another possibility exists: the formation of tartaric acid by a one-electron reduction followed by a dimerisation (pinacol formation). Further reduction of tartaric acid could lead to malic acid (as claimed by Bewick [8] and Wolf [9]). We observed an HPLC peak from the reduction products of the reduction of oxalic acid which could be attributable to malic acid [4].

Acid-catalyzed peptide bond hydrolysis of recombinant human interleukin 11.

Recombinant human interleukin 11 (rhIL-11) is a multispectrum cytokine that plays an important role in megakaryocytopoiesis and platelet production. Probing rhIL-11 chemical reactivity in aqueous solution is an important initial step in developing a dosage form for rhIL-11 clinical trials. This report documents rhIL-11 degradation kinetics at 50 degrees C in solutions adjusted to pH 3.0 to 9.5. Stressed samples were analyzed by reverse-phase HPLC and degradation product peaks were isolated for structural characterization. The results show maximal stability in the region pH 6.5 to 7.0. Degradation product identification shows that the major reaction pathway in acidic solution involves peptide cleavage at aspartate133-proline134. In alkaline solution, protein disappearance proceeds via nonspecific loss to container surfaces. Degradation products at alkaline pH have not been identified.

Use of polyethylene glycol and high-performance liquid chromatography for preparative separation of Aspergillus ficuum acid phosphatases

Proteins of Aspergillus ficuum culture filtrate were sequentially fractionated with 4, 9, 15, 19, 24, 30 and 36% polyethylene glycol (PEG) into seven acid phosphatases (APases) with 93% and 52% overall recoveries of activity and protein, respectively. Crude extract was also separated into seven APase peaks on a 30 cm × 2.5 cm I.D. anion-exchange column using 0.1 M Tris-HCl (pH 8.0) and a 0–0.4 M KCl gradient as the eluent, but their resolution was incomplete. However, when individual PEG precipitates were injected on to the column, each APase was eluted in a single, large peak resulting in 85% recovery and fifteen-fold purification of APase activity over the PEG precipitates. Use of PEG prior to HPLC separations also reduced the separation time to half and allowed a tenfold increase in sample load with complete resolution. The APases in PEG fractions and their corresponding HPLC peaks varied significantly in their kinetic parameters, including substrate specificity and pH optimum. The method developed is most beneficial for the isolation of these closely related APases from microbial or other sources for further molecular biology studies.

Isolation and characterization of a hemorrhagin from the venom of Calloselasma Rhodostoma (Malayan pit viper)

G. Ponnudurai, M. C. M. Chung and N.-H. Tan. Isolation and characterization of a hemorrhagin from the venom of Calloselasma rhodostoma (Malayan pit viper). Toxicon31, 997–1005, 1993.—The major hemorrhagin (termed rhodostoxin) of the venom of Calloselasma rhodostoma (Malayan pit viper) was purified to electrophoretic homogeneity by Sephadex G-200 gel filtration followed by high performance ion exchange chromatography. The purified hemorrhagin also yielded a single peak in reversed-phase HPLC. It had an isoelectric point of 5.3 and a mol. wt of 34,000. Rhodostoxin exhibited potent proteolytic, hemorrhagic and edema-inducing activities but was not lethal to mice at a dose of 6 μg/g (i.v.). Treatment of rhodostoxin with EDTA eliminated both the proteolytic and hemorrhagic activities completely. The N-terminal sequence of rhodostoxin was determined to be NHEIKRHVDIVVVXDSRFCTK.

Identification of compounds in an HPLC fraction from female extracts that elicit mating responses in male screwworm flies,Cochliomyia hominivorax

When hexane extracts of mature screwworm females were chromatographed on a silica gel column, mating stimulant activity was concentrated in a fraction that eluted with hexane-ether (94∶6, v/v). Separation of this fraction with HPLC (acetonitrile-acetone; 60∶40, isocratic) resulted in a chromatogram of some 20 peaks. Only peaks 4-11 elicited mating responses. Peaks 5-10 had most of the activity, with peak 8 producing the highest response. Sixteen compounds were characterized from peak 8 by gas chromatography-mass spectrometry: six unbranched secondary acetates (C31H62O2); seven previously unreported methyl-branched secondary acetates (C32H64O2); one unbranched ketone (C31H62O); and one methyl-branched ketone (C32H64O). The isomeric acetates were not completely resolved from each other by capillary gas chromatography (CGC) on methyl silicone columns. The sixteenth compound was an aldehyde (C30H60O) that was present only in occasional peak 8 preparations. These compounds and several derivatives were characterized by capillary gas chromatography-mass spectrometry (CGC-MS). The position of the acetate group was ascertained by conversion to a keto group or by replacement of the acetate with a methyl group. Pheromone activity was not observed in peaks trapped either from CGC or by recombination of the trapped CGC peaks from HPLC peak 8. This apparent loss of activity from CGC peaks or from TLC cannot currently be explained.

THE USE OF LIQUID CHROMATOGRAPHY‐MASS SPECTROMETRY TO MONITOR THE ALLOMERIZATION REACTIONS OF CHLOROPHYLL a and PHEOPHYTIN a:m IDENTIFICATION OF THE ALLOMERS OF PHEOPHYTIN a

Abstract— This paper reports a new method for monitoring the allomerization reactions of chlorophyll a and pheo‐phytin a. Complex mixtures are generated from illuminating pure compounds and monitored using both diode array high‐performance liquid chromatography (DAD‐HPLC) and liquid chromatography‐mass spectrometry (LC‐MS). LC‐MS allows molecular weight and fragment ion analysis of significant HPLC peaks. Five products of the degradation of chlorophyll a can be simultaneously detected in a mixture, namely the monohydroxy allomer, the methoxylactone allomer, pheophytin a and the two corresponding allomers of the pheophytin. It is demonstrated that more than one pathway must be involved in the in vitro photodegradation of chlorophyll a as shown by the simultaneous existence of several intermediates.

Enhanced Glutathione S-Transferase 7-7 Expression in Rat Hepatic Cytosol Following Treatment with Pyrrole

Rat hepatic GST 7.7 expression in cytosol and in the S-hexylglutathione-agarose affinity purified fraction of cytosol from saline- (control) and pyrrole-treated animals was examined using metabolic activity, SDS-PAGE, immunoblot, and HPLC analyses. Metabolic activity of hepatic cytosol from pyrrole-treated animals was assayed using the substrates 1 -chloro-2,4-dinitrobenzene, ethacrynic acid, and acrolein, and an ≍1.5-, 1.2-, and 1.3-fold increase, respectively, was monitored in the rate of GST-catalyzed substrate conjugation to reduced glutathione. SDS-PAGE and immunoblot analysis using Class P1 GST-specific lgG confirmed that the GST 7 subunit was expressed in hepatic cytosol and in the affinity purified fractions from pyrrole-treated rats. In contrast, the GST 7 subunit was below the limit of detection in hepatic cytosol of saline-treated animals. HPLC analysis demonstrated the presence of the GST 7 subunit in the affinity purified fraction from pyrrole-treated rat hepatic tissue and showed that the level of this subunit was increased ≍ 8-fold relative to the barely detectable level present in control tissue. N-terminal amino acid sequencing analysis confirmed the identity of the HPLC peak as GST 7-7. GST subunits 3, la, and lb were elevated ≍ 1.7-, 2.0-, and 2.4-fold, respectively, in response to pyrrole treatment. These data provide evidence that pyrrole treatment results in de novo expression of GST 7-7 in rat hepatic tissue and suggest that pyrrole-containing compounds potentially generated during disease and/or altered heme metabolism, or those ingested in food products, may alter GST expression in hepatic tissue.

A neutrophil chemotactic factor present in H. pylori but absent in H. mustelae.

This study was designed to compare the capabilities of Helicobacter pylori and Helicobacter mustelae to generate neutrophil chemotactic activity (NCA) in vitro. H. pylori and H. mustelae were grown in parallel cultures under identical conditions. The cultures were washed and transferred to saline solution for 3 hr to avoid detecting nonspecific chemotactic activity from culture media. Supernatants were subjected to size-exclusion HPLC. All peaks from HPLC were collected and assayed for NCA. Peaks having significant NCA were subjected to gel electrophoresis. H. pylori generated 85.9 +/- 1.7% NCA compared to only 41.6 +/- 2.5% for H. mustelae (P < 0.001). The HPLC peak containing the highest NCA from H. pylori revealed a band on gel electrophoresis at approximately 10.5 kDa. This band was not present on gels from H. mustelae. We conclude that H. pylori produces a neutrophil chemotactic factor lacking from H. mustelae. This offers an explanation for the histologic difference between gastritides caused by these organisms.

23] Enzymatic conversion of all-trans-β-carotene to retinal

Enzymatic conversion of all-trans-beta-carotene to retinal by a partially purified enzyme from rabbit, rat, and human neonatal intestinal mucosa has been demonstrated. The enzymatic product was characterized based on the following evidence. First, the product gave rise to its O-ethyl oxime by treatment with O-ethylhydroxylamine with an absorption maximum at 363 nm in ethanol characteristic of authentic retinal (O-ethyl) oxime. High-performance liquid chromatography of this derivative yielded a sharp peak with a retention time of 7.99 min, corresponding to the authentic compound. The enzyme blank and boiled enzyme blank failed to show any significant HPLC peaks corresponding to retinal (O-ethyl) oxime or retinal or retinol. Second, the mass spectrum of the O-ethyl oxime of the enzymatic product was identical to that of authentic retinal (O-ethyl) oxime (m/z 327, 45%; m+ and m/z 282, 100%, methoxy). Third, the 14C radioactivity persisted to constant specific activity even after repeated crystallization of the retinal (O-ethyl) oxime isolated from the enzyme reaction with purified beta-[14C]carotene. Fourth, the enzymatic product exhibited an absorption maximum at 370 nm in light petroleum characteristic of authentic retinal. Furthermore, it was reduced by horse liver alcohol dehydrogenase to retinol with an absorption maximum at 326 nm in light petroleum. This retinol was enzymatically esterified to retinyl palmitate by rat pancreatic esterase with a retention time of 10 min on HPLC, corresponding to authentic retinyl palmitate. Thus, the enzymatic product of beta-carotene cleavage by the partially purified intestinal enzyme has been unequivocally confirmed to be retinal. Similarly, enzymatic conversion of all-trans-beta-carotene to retinal by an intestinal mucosal enzyme from autopsy samples of human neonates has also been demonstrated. Based on the observed activities among intestinal samples from 12 premature infants, the BCC enzyme activity ranged from 3.3 to 1210 pmol/mg mucosal protein/hr. However, the observed activities in the human autopsy samples may be markedly underestimated, presumably because of marked loss of enzyme activity from the time of death to the time of assay. Therefore, the true activity of the enzyme can be assessed only after the extent of the loss of its activity on storage of the human samples can be accurately measured. Nonetheless, the demonstration of BCC enzyme activity in human neonates shows that beta-carotene may be an important source of vitamin A nutrition during gestation.

Chemotaxonomic analysis of Pappobolus (Asteraceae: Heliantheae)

Chemical constituents from capitate glandular trichomes were analyzed for 33 of the 38 species of Pappobolus. In total, 35 HPLC peaks were detected, 30 of which could be tentatively assigned to known compounds of the sesquiterpene lactone and benzofuran type. Compounds of the 1-keto-2,3-unsaturated-furanoheliangolide type (budleins) were present in all plants and quantitatively dominated the compound patterns. Several other heliangolides were also present. Xanthonolides were detected in about 50% of the species, and benzofurans from about two-thirds of them. Pappobolus is the only genus for which benzofurans have been demonstrated to occur in capitate glandular trichomes. Overall there was a high degree of chemical homogeneity, which is in accord with the suggestion that Pappobolus is monophyletic and of relatively recent origin. Pappobolus is distinct from other genera of the subtribe Helianthinae in the occurrence of xanthanolides and in the lack of germacrolides and eudesmanolides.

Tandem mass spectrometry: the competitive edge for pharmacology.

The notion of connecting two mass spectrometers to examine the chemistry of purified ion species was realized experimentally as early as 1954 (1), and ten years later instruments of this complexity had been constructed in five labora­ tories worldwide (2). The utility of collisions of fast-moving ionswith neutral target atoms to promote fragmentation was introduced by Jennings in 1968 (3). The analytical value of collisional activation and tandem mass spec­ trometry for components of mixtures received attention through the next decade (e. g. 4-6), and was demonstrated with a variety of instrumental configurations. The commercial availability around 1980 of tandem mass spectrometers based on three-sector analyzers and on three quadrupole an­ alyzers, and around 1985 of four-sector tandem instruments and of hybrids comprising two sectors and two quadrupole modules, rapidly increased the use of tandem (MSMS) techniques in analytical applications (7-14). Simply stated, these provide (a) a separation technique based on mass that removes chemical contamination (e. g. coelutants in HPLC peaks or matrix ions desorbed by fast atom bombardment) and separates components of mixtures, (b) reproducible and extensive fragmentation, and (c) increased selectivity through selected reaction monitoring. The selectivity and reliability provided by these powerful hyphenated instruments are compellingly relevant to prob­ lems in pharmacology. Applications of MSMS were included in about 100 papers in pharmacological journals in 1990.

Glycation of human lens proteins: Preferential glycation of αA subunits

Glycation of crystallins and high molecular weight (HMW) aggregates was followed during aging (16–85 years) and in diabetes (44 and 70 years old). Lens soluble and insoluble fractions were reduced with [ 3H]NaBH 4 and separated by molecular sieve HPLC. The protein content in each HPLC peak was measured by the Lowry method. The tritium incorporation, expressed as cpm mg −1 protein, was taken as a measure of early glycation and specific non-tryptophan fluorescence (E x: 370 nm; E m: 440 nm), expressed as relative fluorescence U mg −1 protein, was taken as a measure of advanced glycation. The youngest lenses analysed were 16 and 17 years old and these provided the baseline values. The results showed that during aging there was about a three-fold increase in tritium incorporation and fluorescence of α-crystallin, while the increases in β and γ were only two-fold from the levels seen in 16- and 17-year-old lenses. On the other hand, both the soluble and insoluble HMW aggregate fractions showed up to five-fold increase in tritium incorporation during aging. The fluorescence was about two-fold higher in the insoluble HMW aggregates as compared to the soluble HMW aggregates in 16- and 17-year-old lenses and both showed an increase of about three-fold during aging. Diabetes resulted in an approximately 10–50% increase in tritium incorporation and non-tryptophan fluorescence of various crystallins and HMW aggregates. Since α-crystallins showed a higher level of glycation than other crystallins, and soluble HMW aggregates appear to have consisted predominantly of α-crystallins, we further determined the level of glycation of α-crystallin subunits. Alpha-crystallins from 50–85-year-old lenses was used to separate the subunits by both reverse-phase HPLC (RP-HPLC) and gel electrophoresis. Determination of the protein bound radioactivity showed that αA was more glycated than αB. Furthermore, the RP-HPLC also separated a modified α peak, in addition to αA and αB, which presumably originated from HMW aggregates. The modified α had an even higher level of early glycation than αA. Interestingly, the fluorescence associated with the modified α was also several fold higher than αA and αB.

Identification of phenolic compounds and their relationships to in‐vitro digestibility of sorghum leaves from bird‐resistant and non‐bird‐resistant varieties

AbstractSoluble phenolics in leaf blades and sheaths from the crop residues of 24 sorghum varieties were studied. Apigenin, luteolin, apigenin and luteolin 7‐O‐glucosides, p‐coumaric acid, butin and apigeninidin were identified. This is the first report of butin in sorghum tissues. Derivatives of the following compounds were also detected but not characterised further: apigeninidin, luteolinidin, chalcone, flavanone and/or dihydroflavonol, cinnamic acid, apigenin and luteolin. The composition of phenolics was clearly different between leaf blades (LB) and sheaths (LS), and also between leaf sheaths of bird‐resistant (BR) and non‐bird‐resistant (NBR) varieties. Routes of flavonoid biosynthesis in BR and NBR varieties appear to diverge at the flavanone/dihydroflavonol level. Several negative correlations were found between HPLC peaks and in‐vitro digestibilities, ie true dry matter and neutral‐detergent fibre digestibility. These were highly significant with butin and significant with several luteolin derivatives but only with one apigenin derivative. Butin in turn was highly negatively correlated with colorimetric measurements of 3‐desoxyanthocyanidins. This may suggest that butin—rather than the 3‐desoxyanthocyanidins as previously reported—is implicated in reducing dry matter digestibility. Selecting varieties low in luteolin derivatives and butin and rich in apigenin should enhance the digestibilities of sorghum crop residues.

Phytoalexins, benzoxazinones, N-aroylanthranilates and N-aroylanilines, from Fusarium-infected carnation stems

Analysis of isolated HPLC peak fractions of extracts of cv Pallas carnation stems infected with Fusarium oxysporum f.sp. dianthi race 2 yielded a number of new 2-aryl- 1,3-benzoxazin-4 H-ones and dianthramides in addition to several known ones. At least one N-aroylaniline was also found to be a natural carnation phytoalexin. The presence of another phytoalexin, structurally unrelated to the dianthramides, was indicated.

?-Tocopherol alteration of soybean antiherbivory toTrichoplusia ni larvae

The antioxidant vitamin E, α-tocopherol, was tested as a candidate elicitor of alterable antiherbivory in soybean plants against cabbage looper larvae. Although a nonspecific antioxidant, vitamin E proved elicitory to the involved sulfhydryl-dependent receptor-energy transducer protein in soybean plasma membrane. Effects of α-tocopherol were dependent on dosage, time, and space in the plant. The observed elicited effects were all decreases in herbivory. The best negative phytochemical correlate of looper feeding was the percentage of increased total HPLC peak area of extractables from elicited as compared to nonelicited leaves. Some specific compounds, e.g., glyceollins, were quantitatively major components of the total profile of secondary metabolites.

Adenylate cyclase stimulating activity immunologically similar to parathyroid hormone-related peptide can be extracted from fetal rat long bones.

We observed that culture medium conditioned with fetal rat long bones contained peptides immunologically related to the parathyroid hormone-related peptide of malignancy (PTHrP) and stimulated cyclic AMP production in canine renal cortical membranes. Because the adenylate cyclase stimulating activity (CSA) of the medium increased when bone resorption was stimulated, it was suspected that these peptides were stored in the matrix and released during the resorption process. In this work, we extracted the noncollagenous proteins of fetal rat long bones and found that the extract contained significant amounts of CSA. The biologic activity of the extract was abolished after trypsin digestion and eluted at 24 and 37 kD on filtration HPLC. The CSA of bone extract and of both HPLC peaks could be inhibited by 3-34 and 7-34 parathyroid hormone analogs. It was not blocked by an antiserum directed against the N-terminal region of parathyroid hormone, but it was significantly inhibited after an overnight preincubation with an antiserum directed against the 1-11 fragment of PTHrP. One band migrating at 18 kD could be visualized after SDS-PAGE electrophoresis of bone extract and immunoblotting with the anti-PTHrP antiserum. We conclude that an adenylate cyclase stimulator immunologically similar to PTHrP is present in the matrix of fetal rat long bones. Adenylate cyclase stimulating peptides of lower molecular weight found in bone-conditioned medium could be active fragments formed by proteolysis during the resorption process.

Isolation of substance from sweet potato (Ipomoea batatas) periderm tissue that inhibits seed germination

Chromatographic procedures were used to isolate inhibitors of seed germination from sweet potato root periderm tissue. The inhibitory activity of all fractions was monitored using a proso millet seed germination bioassay. A single HPLC peak, representing approximately 1.2% of the periderm dry weight, accounted for most of the inhibitory activity. The active fraction was labile in methanolic solution. Further fractionation of this peak by HPLC methods was not successful. In vitro seed germination dose-response relationships were established for the peak. The various seed species exhibited an extremely wide range of sensitivity. The I50 values were 0.16, 0.013 and 0.011 mg/ml for redroot pigweed, velvetleaf, and proso millet, respectively. Tall morning glory was not inhibited by any concentration tested.

Synthesis of high specific activity 80mBr and 123I labeled 5‐halodeoxyuridines and other 80mBr compounds for the study of auger electron toxicity

AbstractConvenient preparations of 80mBr and 123I‐labeled 5‐halodeoxyuridines, required for basic investigations of Auger electron radiotoxicity, are described. These radioactive thymidine analogs were synthesized from deoxyuridine, radiohalide and N‐chlorosuccinimide in dilute sulfuric acid. Yields were 50–60% for 80mBr and 60–70% for 123I. Apparent specific radioactivities (based on UV absorption under the radioactive HPLC peaks) were 150–550 Ci/mmol and over 2000 Ci/mmol for 80mBr and 123I, respectively. 5‐[80mBr]bromouracil was produced in 89% yield when uracil was used. [80mBr]Bromoantipyrine was produced in higher yield and specific activity using peroxyacetic acid, rather than N‐chlorosuccinimide as oxidant (90% and 4,000 Ci/mmol versus 50% and 2000 Ci/mmol).

Synthesis of high specific activity (+)- and (-)-6-[18F]fluoronorepinephrine via the nucleophilic aromatic substitution reaction

The first example of a no-carrier-added 18F-labeled catecholamine, 6-[18F]fluoronorepinephrine (6-[18F]FNE), has been synthesized via nucleophilic aromatic substitution. The racemic mixture was resolved on a chiral HPLC column to obtain pure samples of (-)-6-[18F]FNE and (+)6-[18F]FNE. Radiochemical yields of 20% at the end of bombardment (EOB) for the racemic mixture (synthesis time 93 min), 6% for each enantiomer (synthesis time 128 min) with a specific activity of 2-5 Ci/mumol at EOB were obtained. Chiral HPLC peak assignment for the resolved enantiomers was achieved by using two independent methods: polarimetric determination and reaction with dopamine beta-hydroxylase. Positron emission tomography (PET) studies with racemic 6-[18F]FNE show high uptake and retention in the baboon heart. This work demonstrates that nucleophilic aromatic substitution by [18F]fluoride ion is applicable to systems having electron-rich aromatic rings, leading to high specific activity radiopharmaceuticals. Furthermore, the suitably protected dihydroxynitrobenzaldehyde 1 may serve as a useful synthetic precursor for the radiosynthesis of other complex 18F-labeled radiotracers.

Identity of a biliary metabolite formed from monocrotaline in isolated, perfused rat liver

A. R. Mattocks, S. Croswell, R. Jukes and R. J. Huxtable. Identity of a biliary metabolite formed from monocrotaline in isolated, perfused rat liver. Toxicon 29, 409–415, 1991.—A pneumotoxic pyrrolic metabolite, previously isolated from the bile when rat liver was perfused with the pyrrolizidine alkaloid, monocrotaline, has been identified as 7-glutathionyl-dehydroretronecine. The metabolite showed a TLC spot and HPLC peak corresponding with the latter compound, and a procedure for replacing the thioether group with an ethoxy group converted the metabolite to dehydroretronecine 7-ethyl ether, confirming that the glutathionyl moiety was attached to the 7-position of dehydroretronecine. The same metabolite was detected in bile from rat liver perfused with retrorsine, which is a diester alkaloid similar to monocrotaline, whereas it was not formed from heliotrine, an alkaloid lacking the 7-ester function.