Reversed-phase High-performance Chromatography Research Articles

Distribution of Bound Hydroxycinnamic Acids and Their Glycosyl Esters in Barley (Hordeum vulgare L.) Air-Classified Flour: Comparative Study between Reversed Phase-High Performance Chromatography−Mass Spectrometry (RP-HPLC/MS) and Spectrophotometric Analysis

The level of bound hydroxycinnamic acid was determined by spectrophotometry (as total hydroxycinnamic compounds and free-radical-scavenging activity) and reversed-phase high-performance chromatography (RP-HPLC) coupled to mass spectrometry (MS) in barley flours (whole meals and air-classified fractions: coarse fraction and fine fraction). Hydroxycinnamic acids and their derivatives were the main bound phenols in barley flours. A total of 12 different hydroxycinnamic acids were identified and quantified by HPLC/diode array detector (DAD)-MS within 90 min. Ferulic acid (as a simple and glycosylated derivative) was the main phenolic acid in barley flours, representing 89-93% of total hydroxycinnamic acids. The amount of total hydroxycinnamic acid in air-classified coarse fraction was 2 and 3 times higher than those of whole meal and the air-classified fine fraction, respectively. Similarly, the coarse fraction showed higher antioxidant activity (650.03 micromol of TEAC/100 g of flour) compared to whole meal and the fine fraction (388.78 and 320.27 micromol of TEAC/100 g of flour, respectively).

Study of the migration of benzophenone from printed paperboard packages to cakes through different plastic films

Benzophenone is one of the most commonly used photoinitiators, which are mostly used in UV-cured inks for which drying times are much shorter than for conventional solvent or water-based coatings. Benzophenone levels were determined in different packaging materials used for cakes, to evaluate the safety of printed paperboard intended for food contact. For this, a simple extraction method and a rapid specific reverse-phase high performance chromatography method with UV detection were developed. Benzophenone was quantified in different samples by a rapid specific reverse-phase high performance chromatography method after extraction with acetonitrile at 70 °C for 24 h. A novel method for testing benzophenone diffusion towards plastics was also developed and different experiments were conducted in order to measure diffusion coefficients at 70 °C for 2 days and at 40 °C for 10 days. Polypropylene plastics sleeves were found not to be an effective barrier against benzophenone diffusion (3,800 μg/g of benzhophenone was found after 48 h at 70 °C in cakes protected with this film) whereas, the corresponding value in cakes wrapped with a multilayer film of PP/EVOH/PP was reduced to 1,400 μg/g, and the concentration of benzophenone in cakes wrapped with a multilayer film of PET/SiOx/PE was negligible.

Effect of Biotic Elicitors on Enrichment of Antioxidant Properties and Induced Isoflavones in Soybean

The antioxidant properties of methanolic extracts from soybean obtained with germination, wounding, and application of biotic elicitors were evaluated. Also, the relationship between observed antioxidant properties and compositional changes in isoflavone content was determined. The 2 biotic elicitors used in this study were the food-grade fungus Aspergillus sojae and A. sojae cell wall extract. Isoflavone content was determined by C(18) reverse phase high-performance chromatography coupled with a photodiode array detector. Antioxidant activities of the extracts were measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and beta-carotene cooxidation in a linoleate system. Higher antioxidant activities were observed in wounded and elicitor-treated extracts when compared with nonwounded control extracts. In addition, the phenolic content was higher in extracts from wounded and elicitor-treated soybean. Germination for 3 d slightly decreased total isoflavone content (-4.3%); however, wounding increased total isoflavone content (25.8%). The soybean extracts from seeds treated with A. sojae biotic elicitors had the highest total isoflavone contents (9.8 to 11.6 mg/g extract) and displayed the highest antioxidant activities in both the DPPH and beta-carotene assays. Also identified in the wounded and elicitor-treated extracts were the induced isoflavones glyceollins that contributed to the higher isoflavone contents observed.

99mTc(CO)3-DTMA bombesin conjugates having high affinity for the GRP receptor

Targeted diagnosis of specific human cancer types continues to be of significant interest in nuclear medicine. 99mTc is ideally suited as a diagnostic radiometal for in vivo tumor targeting due to its ideal physical characteristics and diverse labeling chemistries in numerous oxidation states. In this study, we report a synthetic approach toward design of a new tridentate amine ligand for the organometallic aqua-ion [99mTc(H2O)3(CO)3]+. The new chelating ligand framework, 2-(N,N'-Bis(tert-butoxycarbonyl)diethylenetriamine) acetic acid (DTMA), was synthesized from a diethylenetriamine precursor and fully characterized by mass spectrometry and nuclear magnetic resonance spectroscopy (1H and 13C). DTMA was conjugated to H2N-(X)-BBN(7-14)NH2, where X=an amino acid or aliphatic pharmacokinetic modifier and BBN=bombesin peptide, by means of solid phase peptide synthesis. DTMA-(X)-BBN(7-14)NH2 conjugates were purified by reversed-phase high-performance chromatography and characterized by electrospray-ionization mass spectrometry. The new conjugates were radiolabeled with [99mTc(H2O)3(CO)3]+ produced via Isolink radiolabeling kits to produce [99mTc(CO)3-DTMA-(X)-BBN(7-14)NH2]. Radiolabeled conjugates were purified by reversed-phase high-performance chromatography. Effective receptor binding behavior was evaluated in vitro and in vivo. [99mTc(CO)3-DTMA-(X)-BBN(7-14)NH2] conjugates displayed very high affinity for the gastrin releasing peptide receptor in vitro and in vivo. Therefore, these conjugates hold some propensity to be investigated as molecular imaging agents that specifically target human cancers uniquely expressing the gastrin releasing peptide receptor subtypes.

Variation in the monocyte proteome.

We have determined the variability of the monocyte proteome and identified those proteins that demonstrate the greatest variation in the general control population. Monocytes were isolated from 18 healthy (9 male and 9 female) donors ages 18-50 and with no known genetic or blood disorder. A combination of Ficoll-Paque PLUS density centrifugation of cells found in the buffy coat and positive selection with monoclonal antibodies against CD14, coupled to magnetic beads, led to >98% purity of monocytes. A 100,000 g microsomal membrane fraction or 100,000 g supernatant fraction from a control subject was compared to the equivalent fractions from a distinct control subject by two-dimensional differential gel electrophoresis (2D DIGE). Those protein spots that demonstrated Cy3-/Cy5- ratios greater than 2.5-fold in at least one experiment were selected for further statistical analysis. We determined variability for 31 cytosolic and 12 membrane protein spots. Proteins have been identified for 27 of the cytosolic protein spots and 9 of the microsomal membrane protein spots by in-gel digestion with trypsin followed by reverse-phase high-performance liquid chromatography in line with tandem mass spectrometry. We identified 24 distinct monocyte proteins that demonstrated the greatest variability in this general control population. The proteins demonstrating the greatest variance in the cytosolic fraction were enolase-1 and WD (tryptophan-aspartate) repeat-containing protein 1, and in the membrane fraction they were lamin B1 and L-plastin. This study demonstrates the importance of considering variance in the control population when performing future protein profiling comparisons of monocytes derived from disease versus control populations.

Analysis of European and North American Maize Inbred and Hybrid Lines by Monolithic and Perfusion Reversed-Phase High-Performance Chromatography and Multivariate Analysis

Monolithic and perfusion RP-HPLC methods have been employed for the separation of maize proteins from several European and North American inbred and hybrid lines in analysis times close to 4 min for the perfusion column and close to 8 min for the monolithic column. A study of the repeatability of the protein extraction conditions and of the perfusion and monolithic RP-HPLC methods was performed, indicating low values of variance for the relative peak areas and for the retention times. On the other hand, a low inter-kernel variability of these chromatographic parameters was also found, confirming the possibility of a variety identification and classification of maize inbred and hybrid lines by using a RP-HPLC analysis of the maize proteins. A multivariate analysis of the chromatographic data was carried out in order to characterize and identify the inbred and hybrid maize lines. Cluster analysis of the data showed how far or closely related were the maize lines. Principal component analysis showed that protein chromatographic data had enough information to distinguish between the different groups of maize lines. Finally, a linear discriminant analysis enabled the correct classification of the inbred and hybrid lines according to their geographical origin showing the best percentage of cases correctly classified for monolithic RP-HPLC.

Combining Fluorescence Detection and Mass Spectrometric Analysis for Comprehensive and Quantitative Analysis of Redox-Sensitive Cysteines in Native Membrane Proteins

Monobromobimane (MBB) is a lipophilic reagent that selectively modifies free cysteine residues in proteins. Because of its lipophilic character, MBB is capable of labeling cysteine residues in membrane proteins under native conditions. Reaction of MBB with the sulfhydryl groups of free cysteines leads to formation of highly fluorescent derivatives. Here we describe a procedure for the detection and relative quantitation of MBB-labeled cysteines using fluorescence and mass spectrometric analyses, which allow determination of free cysteine content and unambiguous identification of MBB-modified cysteine residues. We have applied this approach to the analysis of the free and redox-sensitive cysteine residues of a large membrane protein, the sarcoplasmic reticulum Ca2+ release channel with a molecular mass of 2.2 million Da. Labeling was performed under physiologic conditions where the channel complex is in its native environment and is functionally active. The purified MBB-labeled channel complex was enzymatically digested, and the resulting peptides were separated by reversed-phase high-performance chromatography. MBB-labeled peptides were detected by fluorescence and identified by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Under MALDI conditions, partial photolytic fragmentation of the MBB-peptide bound occurred, thus allowing convenient screening for the MBB-modified peptides in the MS spectrum by detection of the specific mass increment of 190.07 Da for MBB-modified cysteine residues. Modification of the peptides was further confirmed by tandem mass spectrometric analysis, utilizing sequencing information and the presence of the specific immonium ion for the MBB-modified cysteine residues at m/z 266.6. Quantitative information was obtained by comparison of both fluorescence and MS signal intensities of MBB-modified peptides. Combination of fluorescence with MS detection and analysis of MBB-labeled peptides supported by a customized software program provides a convenient method for identifying and quantifying redox-sensitive cysteines in membrane proteins of native biological systems. Identification of one redox-sensitive cysteine (2327) in the native membrane-bound sarcoplasmic reticulum Ca2+ release channel is described.

Features of the separation of pesticides of different classes by reversed-phase high-performance chromatography

The effect of the concentration and the nature of an organic modifier, the composition of a buffer solution, and the pH of the mobile phase on the efficiency and selectivity of the separation of some pesticides (difenoconazole, propiconazole, penconazole, fludioxonil, thiram, 2,4-dichlorophenoxyacetic acid, linuron, lenacil, benomyl, acetamiprid, chlorsulfoxime, rimsulfuron, chlorsulfuron, and dicamba) on a Diaspher C16 silica gel were examined. The dependence of the retention on the hydrophobicity of these pesticides were compared. The possibility of the group separation and determination of pesticides with the use of the mobile phase acetonitrile-phosphate buffer solution (0.01 M, pH 4.2) (40: 60) was demonstrated.

Erratum to “Urinary high performance reverse phase chromatography cortisol and cortisone analyses before and at the end of a race in elite cyclists”

Erratum to “Urinary high performance reverse phase chromatography cortisol and cortisone analyses before and at the end of a race in elite cyclists”

Heat shock induces the synthesis of the inflammatory mediator leukotriene B4 in human pulp cells

To measure the synthesis of leukotriene B4 (LTB4) in cultures of human dental pulp cells induced by heat shock. Primary pulp cells (PC) and dental pulp stem cells (DPSC) were cultivated under appropriate conditions. For the characterization of PC the expression of dentine sialophosphoprotein (DSPP) was evaluated by reverse transcription-polymerase chain reaction. Thermal stimulation of cell cultures was performed at temperatures of 37, 38, 39, 40, 42 and 45 degrees C for stimulation times of 5 and 30 s. LTB4 was quantified by reversed-phase high-performance chromatography and differences between the LTB4 concentrations of controls and heat stimulated cells were analysed with Friedman analysis of variances by ranks and multiple comparisons (P < 0.05). Both cell cultures expressed DSPP under the conditions of the present experiment. The analysis revealed significantly enhanced LTB4 synthesis following thermal stimulations at 38, 39, 40, 42 and 45 degrees C compared with unstimulated controls for both PC and DPSC. The present study demonstrated the capability of pulp cells to synthesize the arachidonic acid mediator LTB4 in response to heat shock. LTB4 has the capacity to induce inflammatory reactions and to sensitise afferent nociceptive nerve endings. LTB4 synthesis is induced by minor temperature changes, which are relevant for various clinical situations.

A novel “reverse screening” to identify refolding additives for activin-A

A general approach for refolding recombinant proteins from inclusion bodies (IBs) is to screen conditions, that facilitate a conversion of unfolded to folded structure and minimize a conversion of unfolded to misfolded and aggregated structures. In this simplified model, such conditions may be those that stabilize the native protein and/or reduce aggregation. In this paper, a novel screening approach, termed reverse screening, was developed using a native activin. Activin-A, a member of transforming growth factor β superfamily, is a homodimeric protein with nine disulfide bonds. We examined partial unfolding process of native activin-A dissolved in a buffer containing moderate concentrations of denaturant and reducing reagent (i.e., 1.5 M urea and 0.2 mM dithiothreitol). The recovery of the protein was followed by reverse-phase high performance chromatography analysis. Without additives, activin-A showed about 60% loss of the protein due to aggregation after 12-h incubation in the above condition. We then tested various additives for their effects on the recovery after partial unfolding. One of these additives, sodium taurodeoxycholate (TDCA), greatly increased recovery and suppressed aggregation of the protein. These additives were then tested for refolding activin-A from IBs. TDCA among others is proved to be a highly effective refolding additive. These results strongly suggest that reverse screening using native proteins, if available, may be another approach to discovering effective refolding additives.

Urinary high performance reverse phase chromatography cortisol and cortisone analyses before and at the end of a race in elite cyclists

A functional and basic method for the quantitative analysis of urine cortisol (F) and cortisone (E) using a Solid-Phase Extraction column and HPLC with ultraviolet detection is here described and validated to analyse urine samples. Urine specimens were analysed to study F and E relation and ratio in athletes and healthy sedentary subjects. The F and E concentrations in random urine specimens were significantly higher in the post exercise versus pre exercise condition in cyclists (F: 136+/-93 nmol/l versus 67+/-50 nmol/l (p<0.001); E: 797+/-400 nmol/l versus 408+/-252 nmol/l (p<0.001)). The F/E ratio was 0.18+/-0.11 versus 0.16+/-0.07, respectively, and a significant difference was only demonstrated comparing sedentary (0.11+/-0.07) and cyclist individuals at rest (p<0.05).

Determination of epimedin C in rat plasma by reversed-phase high-performance chromatography after oral administration of Herba Epimedii extract

A simple high-performance liquid chromatographic (HPLC) method has been developed for the determination of epimedin C in rat plasma and applied to a pharmacokinetic study in rats after administration of Herba Epimedii extract. After addition of carbamazepine as an internal standard plasma samples were extracted with ethyl acetate. HPLC analysis of the extracts was performed on a Hypersil ODS2 analytical column using acetonitrile −0.4% acetic acid (25:75, v/v) as the mobile phase. The UV detector was set at 260 nm. The standard curve was linear over the range 0.05–4.0 μg/mL. The lower limit of quantification was 0.05 μg/mL. The HPLC method developed could be easily applied to the determination and pharmacokinetic study of epimedin C in rat plasma after giving the animals Herba Epimedii extract.

Application of urinary nucleosides in the diagnosis and surgical monitoring of colorectal cancer

To evaluate the value of urinary normal and modified nucleosides in diagnosis and surgical monitoring of colorectal cancer (CRC). Between October 2002 and July 2003, 52 consecutive patients with pathological confirmed CRC were included in this study. Spontaneous urine samples were collected 1 d before and 8 d after surgery and 14 kinds of urinary nucleosides in the samples were determined by reversed-phase high-performance liquid chromatography (RP-HPLC) method. Another 62 healthy volunteers were also enrolled as controls. The routine clinical tumor markers, including serum CEA, CA199, CA125 and AFP levels of CRC patients were evaluated by electrochemical-luminescence immunoassay simultaneously. The mean levels of pseudouridine (Pseu), adenosine (A), cytidine (C), 1-methyladenosine (m1A), 1-methylinosine (m1I), 3-methyluridine + 5-methyluridine (mU), 2,2-methylguanosine (m22G), inosine (I), 1-methylguanosine (m1G), N4-acetylcytidine (ac4C), N6-methyladenosine (m6A) among 14 kinds of determined urinary nucleosides in CRC group were much higher than those of controls (P < 0.05). Based on principal component analysis, 76.9% of CRC patients were correctly identified, which was much higher than that of CEA (38.5%), CA199 (40.4%), CA125 (15.4%), and AFP (17.3%) (P < 0.01). ROC curve analysis of m1G, and Pseu showed good sensitivity-specificity profiles to CRC. Two classification equations, Y(normal) = -3.009 + 0.0272 x Pseu + 4.918 x m1G and Y(CRC) = -8.057 + 0.0667 x Pseu + 8.258 x m1G, were established by Bayes stepwise discriminate analysis for predicting carcinogenesis of CRC. The elevated levels of Pseu, C, U (uridine), m1A, m1I, m1G, ac4C, A, m22G dramatically decreased after curative resection of 40 cases of CRC. And our data also showed that the preoperative levels of Pseu, m1G, m1A and m22G were positively related with tumor size and the preoperative levels of m1A, m22G and ac4C were positively related with Duke's staging of CRC (P < 0.05). Normal and modified urinary nucleosides may become additional tumor markers which are feasible in the clinical setting and will prove helpful in the diagnosis, management and follow-up of CRC, and Pseu and m1G may be more promising for clinical application.

High-performance reverse phase chromatography with fluorescence detection assay for characterization and quantification of pneumococcal polysaccharides

A methods using high-performance reverse phase (RP) chromatography with fluorescence detection, has been developed to determine the composition and identity of Streptococcus pneumoniae capsular polysaccharide used in formulating conjugate vaccine for prevention of pneumococcal infection. For the monosaccharide composition, the polysaccharides were subjected to hydrofluoric acid (HF) hydrolysis followed by trifluoroacetic acid (TFA). After acid hydrolysis, the released monosaccharides were re- N-acetylated and labeled with 2-aminobenzamide (2AB) by reductive amination reaction. High-performance RP chromatography was performed on C18 TSKODS 120T column. Nuclear magnetic resonance was used to confirm chemical structure and purity of pneumococcal capsular polysaccharides.

Chirality determination of unusual amino acids using precolumn derivatization and liquid chromatography–electrospray ionization mass spectrometry

Unusual amino acids such as β-methoxytyrosine (β-MeOTyr), allo-threonine ( allo-Thr) and allo-isoleucine ( allo-Ile) were derivatized with N-α-(2,4-dinitro-5-fluorophenyl)- l-alaninamide (FDAA), 2,3,4,6-tetra- O-acetyl-β- d-glucopyranosyl isothiocyanate (GITC), ( S)- N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester ( S-NIFE), or o-phthalaldehyde/isobutyryl- l-cysteine (OPA-IBLC), and then separated via reversed-phase high-performance chromatography followed by UV and electrospray ionization mass spectrometry detection. FDAA generally showed the highest enantioselectivity but the lowest sensitivity among the chiral derivatizing agents (CDAs) investigated. The detection limit of FDAA-derivatized amino acids was in the low picomolar range. Although the enantioselectivity of FDAA derivatives was generally quite high, its selectivity among β-MeOTyr isomers was poor. The best separation of β-MeOTyr stereoisomers was achieved with S-NIFE. Due to the complex relationships between the investigated CDAs, stereochemical analyses using a combination of two or more of the CDAs gave the most reliable results for a given separation problem. In general, the methods described are selective and reliable, and are being applied to the analysis of unusual amino acids as they occur in marine peptides.

Screening for peptides from fish and cheese inhibitory to prolyl endopeptidase.

Peptides from hydrolysates of fish proteins and from cheeses were analysed for inhibition of prolyl endopeptidase (PE) isolated from porcine muscle. Muscles of cod, salmon, and trout were homogenised and incubated at pH 4.0 with pepsin and then at pH 7.5 with trypsin to obtain fish protein hydrolysates. Homogenates were incubated without exogenous enzymes at pH 4.0 and 7.5 to obtain fish protein autolysates. Water-soluble extracts from "rakfisk" (a Norwegian fermented/autolysed trout muscle dish) and water-soluble extracts from Cheddar, Norvegia, Jarlsberg, and Blue cheese were also prepared. Peptides in the supernatants obtained after heat-treatment of fish hydrolysates, autolysates and water-soluble extracts of rakfisk and cheeses at 95 degrees C for 15 min were analysed for inhibition of PE. Inhibition was also measured in peptide fractions separated by reversed-phase high-performance chromatography and by gel permeation chromatography. The peptide fractions from fish hydrolysates, fish autolysates, and water-soluble extracts of cheeses inhibited PE in hydrolysing Z-Gly-Pro-amidomethylcoumarin. Inhibition by peptides from rakfisk was negligible. Pepsin + trypsin hydrolysates from the three fish species contained PE inhibitory peptides with a broad range of apparent hydrophobicity and apparent molecular mass. Autolysates from muscles of the 3 fish species contained narrow peptide peaks of different molecular mass and different apparent hydrophobicity with strong PE inhibitory activity. The content of hydrophilic inhibitory peptides was lower in cheeses than in pepsin + trypsin hydrolysates of fish muscle.

Determination of spinosin in rat plasma by reversed-phase high-performance chromatography after oral administration of Suanzaoren decoction

A sensitive, simple, and accurate method for determination of spinosin in rat plasma with sulfamethoxazole (SMZ) as internal standard was developed using RP-HPLC with UV detection. Sample preparations were carried out by protein precipitation with acetonitrile, followed by the evaporation of the acetonitrile to dryness. The resultant residue was then reconstituted in mobile phase and injected onto a Hypersil C 18 (200×4.6 mm I.D., 5 μm) analytical column. The mobile phase consisted of acetonitrile–water (15:85, v/v) with 1% glacial acetic acid. The assay was shown to be linear over the range of 18.07–903.5 ng/ml ( R 2=0.995). Mean recovery was determined as 93.6%. Within- and between-day precisions were ≤8.9% RSD. The limit of quantitation was 18.07 ng/ml. The HPLC method developed has been applied to determine the pharmacokinetics of spinosin in rat plasma after having taken Suanzaoren decoction.

INFLUENCE OF PHOSPHORYLATION ON PROTEOLYTIC CLEAVAGE

ABSTRACT Phosphorylation of an amino acid residue close to the peptide bond, which is a target of a specific proteolytic enzyme, seems to cause inhibition of such a cleavage. This finding may have a number of important consequences. The effect of phosphorylation of a model peptide on its proteolytic cleavage by trypsin was studied. The velocity of peptide bond cleavage of nonphosphorylated synthetic peptide was compared with cleavage velocity of the same bond of synthetic peptide phosphorylated on serine residue located in closed proximity of the specifically cleaved peptide bond. It was shown that the enzymatic cleavage was inhibited by phosphorylation of mentioned amino acid residue. Reversed-phase high-performance chromatography (RP-HPLC) was used for the fast and reliable analysis of reaction mixtures. Influence of the addition of alkaline phosphatase on proteolytical cleavage of casein was also investigated. RP-HPLC peptide maps of β-casein cleaved by trypsin in the presence and absence of alkaline phosphatase were compared. It was proven that the action of acid phosphatase affects the composition of the resulting proteolytic digest.

Site-directed mutagenesis of exon 5 of p53: purification, analysis, and validation of amplicons for DHPLC.

Reversed-phase ion-pair chromatography is used in its three modes of operation--(1) non-denaturing, (2) partially denaturing, (3) fully denaturing - to isolate and characterize five site-directed mutant amplicons of exon 5 of the p53 gene (GenBank X54156). The mutant fragments were prepared using a simplified mutagenesis procedure without cloning and isolated in high purity under non-denaturing conditions. Then, under partially denaturing conditions, heteroduplices formed by hybridization of each of the five mutants with wild-type were all detected by denaturing reversed-phase high-performance chromatography (DHPLC). The fidelity of the PCR step was also assessed. Finally, the homogeneity of each mutant was confirmed by minisequencing under fully denaturing conditions. The versatility and facility of the method for DNA manipulations was exploited to demonstrate a strategy for validating amplicons for mutation detection by DHPLC.