Reversed-phase High-performance Liquid Chromatography Analysis Research Articles

Lipolysis in reduced sodium Kefalograviera cheese made by partial replacement of NaCl with KCl

Abstract Kefalograviera cheeses (five trials) of different sodium content were made from split lots of curd by varying the salting processes, i.e. brine — and dry — salting, with NaCl (control) or a mixture of NaCl/KCl (3:1 or 1:1, w/w basis). The extent and characteristics of proteolysis in the cheeses were monitored during aging by Kjeldahl determination of soluble nitrogen fractions (water-soluble nitrogen [WSN], trichloroacetic acid [TCA]-SN, phosphotungstic acid [PTA]-SN), the cadmium-ninhydrin method for the determination of total free amino acids (FAA), urea-polyacrylamide gel electrophoresis of cheese proteins, followed by densitometric analysis of the αs1- and β-casein fractions, reverse-phase high-performance liquid chromatography (HPLC) analysis of the water-soluble extracts of cheeses, and ion-exchange HPLC analysis of FAA. The results showed that proteolysis was similar in control and experimental cheeses at all sampling ages, indicating that the partial substitution of NaCl by KCl in the manufacture of Kefalograviera cheese did not significantly influence the extent and characteristics of proteolysis during cheese aging.

Specificity Assay of Serine Proteinases by Reverse-Phase High-Performance Liquid Chromatography Analysis of Competing Oligopeptide Substrate Library

In this paper we present an HPLC method developed for quick activity and specificity analysis of serine proteinases. The method applies a carefully designed peptide library in which the individual components differ only at the potential cleavage site for enzymes. The library has seven members representing seven different cleavage sites and it offers substrates for both trypsin and chymotrypsin-like enzymes. The individual peptide substrates compete for the proteinase during the enzymatic reaction. The reaction is monitored by RP-HPLC separation of the components. We describe the systematic design of the competitive peptide substrate library and the test of the system with eight different serine proteinases. The specificity profiles of the investigated enzymes as determined by the new method were essentially identical to the ones reported in the literature, verifying the ability of the system to characterize substrate specificity. The tests also demonstrated that the system could detect even subtle specificity differences of two isoforms of an enzyme. In addition to recording qualitative specificity profiles, data provided by the system can be analyzed quantitatively, yielding specificity constant values. This method can be a useful tool for quick analysis of uncharacterized gene products as well as new forms of enzymes generated by protein engineering.

Supercritical Carbon Dioxide Extraction of High-Value Substances from Soybean Oil Deodorizer Distillate

This work attempted to recover high-value substances such as free fatty acids, tocopherols, sterols, and squalene by investigating a supercritical fluid CO 2 (SF CO 2 ) distillation-extraction processing of soybean oil deodorizer distillate. Experimental conditions varied between 50 and 90 °C, at 24.1 and 31.0 MPa. Reverse-phase high-performance liquid chromatography analysis was performed to analyze 14 components in the extracts after SF CO 2 treatment. Experimental results indicated that when tocopherols were extracted at 31.0 MPa, 90 (top) to 70 (bottom) °C, and 1000-L STP CO 2 usage, their recovery reached 83.6% and the average value of the concentration factor was 1.38. Furthermore, the concentration factor of tocopherols grew to 1.70 when the extracts collected in the top exceeded 400-L STP CO 2 . The concentration factors of fatty acids, squalene, and stereols were 1.37, 1.26, and 0.60, respectively. This investigation also found that the percentage of total fatty acids decreased, while polyunsaturated fatty acids increased, with an increase in the CO 2 volume given the same extraction condition.

Heme O is present in Paracoccus denitrificans cells and accumulates under anoxic growth.

In spite of the published claims to the contrary, Paracoccus denitrificans was shown to contain a heme derivative, virtually indistinguishable from the Escherichia coli heme O on the basis of the reverse-phase high-performance liquid chromatography and MALDI-TOF mass spectrometry analyses. Aeration of the anoxically grown culture resulted in the disappearance of a significant portion of this compound with concomitant build-up of heme A.

Heme O is present in Paracoccus denitrificans cells and accumulates under anoxic growth

In spite of the published claims to the contrary, Paracoccus denitrificans was shown to contain a heme derivative, virtually indistinguishable from the Escherichia coli heme O on the basis of the reverse-phase high-performance liquid chromatography and MALDI-TOF mass spectrometry analyses. Aeration of the anoxically grown culture resulted in the disappearance of a significant portion of this compound with concomitant build-up of heme A.

Anthocyanin inheritance and instability in purple basil (Ocimum basilicum L.).

The instability of the purple pigments (anthocyanins) in purple basil varieties (Ocimum basilicum L.) limits their use as ornamental plants and as a potential anthocyanin source. Several self-pollinated generations of all purple plants were unsuccessful in stabilizing anthocyanin expression. In this study we investigated the inheritance and stability patterns of leaf traits using the Purple Ruffles variety. The results from the complete diallele crosses indicated anthocyanin expression in vegetative tissue is controlled by two dominant genes and ruffled leaf texture is controlled by a single recessive gene. Genes controlling leaf margin and leaf base structures were tightly linked to leaf texture. Essential oil production and oil constituents in leaves did not change as a result of the reversion in color. Color stability in cuttings was affected by the environment and the location where cuttings were taken. An accumulation of secondary metabolites (apigenin, genistein, and kaempferol) in green-reverted sectors on purple leaves was detected using reverse-phase high-performance liquid chromatography (HPLC) analysis; this suggested a potential block in the anthocyanin pathway. We hypothesize the reversion mutation is occurring in an anthocyanin regulatory gene.

Reversed-phase high-performance liquid chromatographic analysis of atenolol enantiomers in rat hepatic microsome after chiral derivatization with 2,3,4,6-tetra- O-acetyl-β- d-glycopyranosyl isothiocyanate

A reversed-phase high-performance liquid chromatographic method for the determination of the enantiomers of atenolol in rat hepatic microsome has been developed. Racemic atenolol was extracted from alkalinized rat hepatic microsome by ethyl acetate. The organic layer was dried with anhydrous sodium sulfate and evaporated using a gentle stream of air. Atenolol racemic compound was derivatized with 2,3,4,6-tetra- O-acetyl-β- d-glycopyranosyl isothiocyanate at 35°C for 30 min to form diastereomers. After removal of excess solvent, the diastereomers were dissolved in phosphate buffer (pH 4.6)–acetonitrile (50:30). The diastereomers were separated on a Shimadzu CLC-C18 column (10 μm particle size, 10 cm×0.46 cm I.D.) with a mobile phase of phosphate buffer–methanol–acetonitrile (50:20:30, v/v) at a flow-rate of 0.5 ml/min. A UV–VIS detector was operated at 254 nm. For each enantiomer, the limit of detection was 0.055 μg/ml (signal-to-noise ratio 3) and the limit of quantification (signal-to-noise ratio 10) was 0.145 μg/ml (RSD <10%). In the range 0.145–20 μg/ml, intra-day coefficients of variation were 1.0–7.0% and inter-day coefficients of variation were 0.4–16.5% for each enantiomer. The assay was applied to determine the concentrations of atenolol enantiomers in rat hepatic microsome as a function of time after incubation of racemic atenolol.

Characterization of commercial soybean products by conventional and perfusion reversed-phase high-performance liquid chromatography and multivariate analysis

Conventional and perfusion reversed-phase high-performance liquid chromatography are used to characterize commercial soybean products for human consumption. For this purpose, previously optimized methods of conventional and perfusion chromatography applied to the separation of soybean proteins are employed. Sixty different samples corresponding to 26 different trademarks of soybean products [soybean protein isolate, soybean flour, textured soybean, soybean milks (liquid and powdered), and soybean infant formulas] are analyzed. Characterization of soybean products is carried out on the basis of their protein profiles obtained by both chromatographic methods. Data obtained are processed using multivariate methods such as principal components and discriminant analysis. Perfusion chromatography enables a further and faster characterization of commercial soybean products than conventional chromatography, of great value in the quality control of this kind of product.

Glycosylation of the capsid proteins of cowpea mosaic virus: a reinvestigation shows the absence of sugar residues.

The previously reported (Partridge et al., Nature 247, 391-392, 1974 ) glycosylation of the capsid proteins of cowpea mosaic virus (CPMV) has been reinvestigated. In initial studies, a preparation of purified CPMV particles was hydrolysed with HCl and amino acids and sugars were derivatized with o-phthalaldehyde (OPA). No glucosamine or galactosamine, amino sugars previously reported to occur in significant quantities in CPMV capsids, could be detected by reverse-phase high-performance liquid chromatography (RP-HPLC) of the derivatized hydrolysates. A complete analysis of all sugars potentially present was carried out by hydrolysing a sample of purified CPMV capsid proteins and derivatizing the sugars with 1-phenyl-3-methyl-5-pyrazolone. RP-HPLC analysis demonstrated that the capsids do not contain significant quantities of any sugar. The results show that, contrary to the previous report, the coat proteins of CPMV are not glycosylated.

Comparison of in-line connected diode array and electrochemical detectors in the high-performance liquid chromatographic analysis of coenzymes Q(9) and Q(10) in food materials.

In-line connected electrochemical (EC) and diode array (DAD) detectors were compared in the reversed-phase high-performance liquid chromatographic (RP-HPLC) analysis of coenzymes Q(9) and Q(10) in some food materials (beef steak, beef heart, Baltic herring fillet, and rye flour). Coenzymes Q(9) and Q(10) were extracted from the samples using a 5:1 n-hexane-ethanol mixture. Coefficient of variation (CV%) of quadruplicate or quintuplicate determined samples for coenzymes Q(9) and Q(10) was <10 by both EC detector and DAD. Responses of the detection systems were linear in the range evaluated, 10-200 ng/injection, and had correlation coefficients exceeding 0.999. Recoveries of added coenzymes Q(9) and Q(10) varied 73-105% for DAD and 74-103% for EC detector, respectively. Detection limits for coenzymes Q(9) and Q(10) using the DAD system were 4 and 6 ng/injection, respectively, and 0.2 and 0.3 ng/injection by EC detection. Results derived from the two detection systems were generally similar. However, although EC detector was 20-fold more sensitive, the selectivity was, in some cases, poorer than that of DAD.

Analysis of major ovine milk proteins by reversed-phase high-performance liquid chromatography and flow injection analysis with electrospray ionization mass spectrometry

Ovine milk proteins were analyzed both by coupling HPLC and electrospray ionization mass spectrometry (ESI-MS) and by flow injection analysis and ESI-MS detection after separation and collection of fractions from gel permeation chromatography. These methods resolved the four ovine caseins and whey proteins and made it possible to study the complexity of these proteins associated with genetic polymorphism, post-translational changes (phosphorylation and glycosylation) and the presence of multiple forms of proteins. The experimental molecular masses of ewe milk proteins were: 19 373 for κ-casein 3P; 25 616 for α s2-casein 10P; 23 411 for α s1-casein C-8P; 23 750 for β-casein 5P; 18 170 and 18 148 for β-lactoglobulins A and B; 14 152 for α-lactalbumin A and 66 322 for serum albumin.

Excretion pattern investigation of urinary normal and modified nucleosides of breast cancer patients by RP-HPLC and factor analysis method.

Modified nucleosides, formed post-transcriptionally in RNA by a number of modification enzymes, are excreted in abnormal levels in the urine of patients with malignant tumors. To test their usefulness as tumor markers, and to compare them with the conventional tumor markers, a reversed-phase high-performance liquid chromatographic (RP-HPLC) method and a factor analysis method have been used to study the excretion pattern of nucleosides of breast cancer patients. A clear cut differentiation of the breast cancer group and the healthy individuals in two clusters without overlapping was obtained.

Metallothioneins in Human Kidneys and Associated Tumors

Human kidneys and their associated tumors (nonneoplastic kidney tissues from patients with a transitional cell carcinoma or an adenocarcinoma and the adenocarcinomas themselves) were evaluated for their Zn, Cd, and Cu contents as well as for their metallothionein (MT) level. The total Cd content was correlated with the MT content, and both values were significantly decreased in the adenocarcinomas in comparison with the other tissues. After extraction and separation by anion-exchange chromatography, MT-0 was identified in the nonneoplastic tissues from both the adenocarcinomas as well as the transitional cell carcinomas. Since until now MT-0 protein was only found in human fetal liver and in Zn-stimulated human monocytes, a possible role for this isoform as an oncofetal marker is hypothesized. Separation of the isoforms of MT by reversed-phase high-performance liquid chromatography and sequence analysis showed besides MT-1e and MT-1l the isoform-MT-1g, which is not expressed in the healthy kidney, and MT-1k, an isoform which is not yet demonstrated in renal tissues. We conclude that the expression profile of the MT isoforms in the kidney changes due to the presence of a tumor.

Reversal of MDR1-associated resistance to topotecan by PAK-200S, a new dihydropyridine analogue, in human cancer cell lines.

Recent data suggest that expression of the membrane P170-glycoprotein (P-gp) may confer resistance to the topoisomerase- I-interactive agent topotecan. The present study describes the cellular effects of a new dihydropyridine analogue, PAK-200S, on P-gp-mediated resistance to topotecan in human breast and ovarian tumour cells. PAK-200S at a non-cytotoxic concentration of 2.0 μM completely reversed resistance to topotecan in P-gp-expressing MCF-7/adr (breast) and A2780/Dx5 (ovarian) tumour cells, respectively, with no effects on parental cells. Cellular pharmacokinetic studies by reversed-phase high-performance liquid chromatography analysis showed significantly lower cellular drug concentrations of the pharmacologically active closed-ring lactone of topotecan in multidrug-resistant cells than in parental cells. PAK-200S was effective in restoring the cellular lactone concentrations of topotecan in resistant MCF-7/adr cells to levels comparable to those obtained in parental cells. Furthermore, exposure of MCF-7/adr cells to topotecan in the presence of PAK-200S significantly increased the induction of protein-linked DNA breaks. PAK-200S did not alter nuclear topoisomerase I-mediated ex vivo pBR322 DNA plasmid unwinding activity and topoisomerase-I protein expression. These results suggest that reversal of P-gp-mediated resistance to topotecan by PAK-200S was related to the restoration of cellular drug concentrations of the active lactone form of topotecan rather than a direct effect on topoisomerase-I function. © 1999 Cancer Research Campaign

Sample cleanup and reversed-phase high-performance liquid chromatographic analysis of polar aromatic compounds in groundwater samples from a former gas plant

A method for the analysis of the polar aromatic compounds 1 H-quinolin-4-one (Q), 10 H-acridin-9-one (A), 5 H-phenanthridin-6-one (P) and 9 H-fluoren-9-one (F) in aqueous solutions has been developed. The method comprises steps for sample preparation (solid-phase extraction, cleanup) and analytical determination by means of reversed-phase high-performance liquid chromatography (RP-HPLC). For the cleanup step the suitability of two different sorbents (alternative A: silica gel, alternative B: LiChrolut EN) was investigated. Alternative B depicted several advantages, in particular higher sorbent capacity, faster and less complicated handling, higher recovery and better reproducibility. For Q, A and P, reproducibility of all method steps is better than 13%, with recovery rates ranging from 76% to 105% ( n=3). Alternative B was applied to groundwater samples from a former gas plant. The analytes A and P could be detected at concentrations in the μg/l range.

Identification of an urotensin I-like peptide in the pituitary of the lungfish Protopterus annectens: immunocytochemical localization and biochemical characterization

In the present study we have investigated the localization and biochemical characteristics of urotensin I (UI)-like and urotensin II (UII)-like immunoreactive peptides in the central nervous system (CNS) and pituitary of the lungfish, Protopterus annectens, by using antisera raised against UI from the white sucker Catostomus commersoni and against UII from the goby Gillichythys mirabilis. UI-like immunoreactive material was found within the melanotrope cells of the intermediate lobe of the pituitary. By contrast, no UI-immunoreactive structures were found in the brain. No UII-like peptides structurally similar to goby UII were found in the brain and pituitary of P. annectens. The UI-immunoreactive material localized in the pituitary was characterized by combining reversed-phase high-performance liquid chromatography (HPLC) analysis and radioimmunological detection. The UI-like immunoreactivity contained in a pituitary extract eluted as a single peak with a retention time intermediate between those of sucker UI and rat corticotropin-releasing factor (CRF). Control tests on adjacent sections of pituitary showed that the UI antiserum cross-reacted with the frog skin peptide sauvagine, but lungfish UI did not co-elute with synthetic sauvagine on HPLC. On the contrary, no cross-reaction was observed between the UI antiserum and CRF or α-melanocyte-stimulating hormone (α-MSH). The occurrence of an UI-like peptide in the intermediate lobe of the pituitary of P. annectens suggests that, in lungfish, this peptide may act as a classic pituitary hormone or may be involved in the control of melanotrope cell secretion.

Simple and sensitive high-performance liquid chromatographic method for the determination of an everninomycin, SCH 27899, in rat plasma

A simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of SCH 27899, an everninomycin antibiotic, in rat plasma. The method involved plasma protein precipation with acetonitrile, followed by reversed-phase HPLC analysis using a polymeric column and a mobile phase containing acetonitrile and ammonium phosphate, pH 7.8. The linear relationship between detector response and concentration was demonstrated with a correlation coefficient of larger than 0.996 at concentrations ranging from 0.2 to 100 μg/ml. The results showed that the HPLC method was accurate (bias ≤6%) and precise (coefficient of variation, C.V.≤6%). The limit of quantitation was 0.2 μg/ml with a C.V. of 2.6% and bias of 5%. SCH 27899 was stable in rat plasma at −20°C for at least 40 days. The HPLC method has been utilized for the determination of SCH 27899 in plasma samples from rats following single intravenous administration (3 mg/kg).

Subcellular Accumulation of β-Carotene and Retinoids in Growth-Inhibited NCI-H69 Small Cell Lung Cancer Cells

Delivery of β-carotene in tetrahydrofuran slowed the growth of NCI-H69 small cell lung cancer cells. Analysis of cells and cellular fractions revealed that β-carotene-treated cells accumulated β-carotene as well as some polar metabolites, primarily in the crude nuclei. Cells were grown at 1 × 105 cells/ml and treated with 20 μM β-carotene. Growth monitoring up to 15 days indicated an inverse relationship between the duration of β-carotene treatment and the rate of cell growth. Reverse-phase high-performance liquid chromatography analysis of treated cells showed the presence of β-carotene, retinoic acid, retinol, and retinal, with β-carotene accounting for the major material recovered. When cellular fractions were analyzed for β-carotene, it was found to be located primarily in the crude nuclei. These results demonstrate that treatment of small cell lung cancer cells with β-carotene results in a reduced growth of the cells. Further investigation is required to show a direct effect of β-carotene or its intracellular polar metabolites on these cells. Accumulation of β-carotene in the nucleus suggests a need for evaluating the nuclear role for β-carotene

Estimation of the microcystin content in cyanobacterial field samples from German lakes using the colorimetric protein-phosphatase inhibition assay and RP-HPLC

The protein-phosphatase (PP-1) inhibition potency of microcystin was employed to estimate the microcystin content in cyanobacterial field samples from three German eutrophic lakes collected mainly in summer 1996. The microcystin content of lyophilized samples extracted with aqueous methanol was determined on the basis of the colorimetric PP-1 inhibition assay with p-nitrophenyl phosphate as substrate. With the test system described, an IC 50 of 1 ng/mL reaction volume (∼0.6 nM PP-1) was determined for microcystin-LR standard, corresponding to 20 ng microcystin-LR per milliliter sample volume. The microcystin content of field samples estimated on the basis of the PP-1 inhibition assay was compared to that obtained by reversed phase high performance liquid chromatography (RP-HPLC), and a good correlation was observed (r = 0.9202, P < 0.0001). The mean difference in microcystin content based on PP-1 inhibition was determined to be 27.9 ± 22.9% (n = 50) related to measurements based on RP-HPLC analysis. There was no observation of false positive or false negative results in this study. The results confirm the suitability of the PP-1 inhibition assay with regard to a preliminary screening of cyanobacterial field samples for microcystin.

Hydrolysis of pork muscle sarcoplasmic proteins by lactobacillus curvatus and lactobacillus sake.

Lactobacillus curvatus CECT 904 and Lactobacillus sake CECT 4808 were selected on the basis of their proteolytic activities against synthetic substrates. Further, the effects of whole cells, cell extracts, and a combination of both enzymatic sources on muscle sarcoplasmic proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography analyses. Strains of both species displayed proteinase activities on five sarcoplasmic proteins. The inoculation of whole cells caused a degradation of peptides, whereas the addition of cell extracts resulted in the generation of both hydrophilic and hydrophobic peptides. This phenomenon was remarkably more pronounced when L. curvatus was involved. Whole cells also consumed a great amount of free amino acids, while the addition of intracellular enzymes contributed to their generation. L. sake accounted for a greater release of free amino acids. In general, cell viability and also proteolytic events were promoted when cell suspensions were provided with cell extracts as an extra source of enzymes.