Reversed-phase High-performance Liquid Chromatography Analysis Research Articles

Rapid and sensitive high-performance liquid chromatographic method for the analysis of tryptophan, tyrosine and phenylalanine in biological samples

In order to overcome problems related to the determination of free tryptophan in biological fluids using conventional methods, we have developed an accurate and reliable procedure based on a specific pretreatment of samples followed by a very rapid and sensitive reversed-phase high-performance liquid chromatographic analysis. The pretreatment consists of adding to the sample of a very low amount of 3 M phosphate buffer to maintain pH in the physiological range followed by ultrafiltration. The precision, reproducibility and sensitivity of our method were also evaluated. The recovery of each amino acid was greater than 92%. The use of a microbore column allows the detection of up to 0.2 pmol/μl of amino acid. The method has been applied to the analysis of samples obtained from 25 normal and 10 phenylketonuric subjects.

Peptide maps at picomolar levels obtained by reversed-phase high-performance liquid chromatography and pre-column derivatization with phenyl isothiocyanate: Microsequencing of phenylthiocarbamyl peptides

A new reversed-phase high-performance liquid chromatography approach to the production of analytical peptide maps by pre-column derivatization using phenylisothiocyanate is described. Tryptic peptide digests were derivatized with phenyl isothiocyanate to form the phenylthiocarbamyl peptides followed by reversed-phase high-performance liquid chromatographic analysis. The phenylthiocarbamyl peptides were separated by reversed-phase high-performance liquid chromatography with the conventional gradient elution system of water-acetonitrile containing trifluoroacetic acid. The sensitivity of detection of these peptide derivatives was within the range 5–10 pmol with a constant baseline at 254–260 nm. The isolated phenylthiocarbamyl peptides can be subjected to automatic Edman degradation. The effectiveness of this method was exemplified by microsequencing of phenylthiocarbamyl peptides isolated from tryptic digests of three different proteins: α-lactalbumin, β-lactoglobulin and a λ light-chain immunoglobulin.

Reversed-phase high-performance liquid chromatographic analysis of atenolol enantiomers in plasma after chiral derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate

A sensitive high-performance liquid chromatographic method for the determination of the enantiomers of atenolol in rat plasma has been developed. Racemic atenolol and practolol (internal standard) were extracted from alkalinized plasma (pH 12) into dichloromethane containing 3% (v/v) heptafluoro-1-butanol, and the organic layer was evaporated. The samples were derivatized with (+)-1-(9-fluorenyl)ethyl chloroformate at pH 8.5 for 30 min. After removal of excess reagent, the diastereomers were extracted into dichloromethane. The diastereomers were separated on a Microspher C 18 column (3 μm) with a mobile phase of acetonitrile—sodium acetate buffer (0.01 M, pH 7) (50:50, v/v) at a flow-rate of 0.8 ml/min. Fluorescence detection (λ ex = 227 nm, λ em = 310 nm) was used. When 100 μl of plasma were used, the quantitation limit was 10 ng/ml for the atenolol enantiomers. The assay was applied to measure concentrations of atenolol enantiomers in plasma after intravenous administration of racemic atenolol to rats.

Rapid high-performance liquid chromatographic assay for total homocysteine levels in human serum

A rapid, isocratic high-performance liquid chromatographic (HPLC) method is described for the determination of total homocysteine levels in human serum. Prior to reversed-phase HPLC analysis, the serum thiols were derivatized with SBD-F (ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate), a thiol-specific fluorogenic probe which is commercially available. Retention of SBD-homocysteine was sensitive to pH, and a mobile phase pH of 2.1 ensured baseline separation of serum thiols within 6 min. The method is simple, sensitive, reproducible (between-run coefficient of variation of 6.6%) and very suitable for routine determination of serum homocysteine levels in a clinical pathology laboratory.

Sandwich-Enzyme Immunoassays for Endothelin Family Peptides

Sandwich-enzyme immunoassays (sandwich-EIAs) for endothelin-1 (ET-1), endothelin-3 (ET-3), big ET-1, big endothelin-2 (big ET-2), and big endothelin-3 (big ET-3) have been established using two antibodies directed against the N-terminal and C-terminal portions of each ET and big ET. These sandwich-EIAs are sensitive enough to detect 0.1-0.4 pg/well of each ET or big ET with cross-reactivity of less than 1% with other ETs and big ETs (except in the case of the EIA for ET-1, which detects immunoreactivity of ET-2 as well as ET-1). Plasma levels of immunoreactive (ir-) ET-1, big ET-1, and ET-3 in normal humans were approximately 2.0, 5.1, and 0.9 pg/ml, respectively, when corrected for recovery efficiency. In reverse-phase high-performance liquid chromatography (HPLC) analysis, immunoreactivities detected by these sandwich-EIAs were eluted at the positions of authentic ET-1, big ET-1, and ET-3, indicating that plasma levels of ET-1, big ET-1, and ET-3 were precisely measured by our methods. Levels of ir-big ET-2 and ir-big ET-3 were also detected in normal human plasma; however, further studies are needed to determine their precise values. The establishment of reliable sandwich-EIAs and the measurement of specific ETs and big ETs in normal human plasma will contribute to clarifying the biological and clinical significance of ET family peptides.

Separation and Determination of Sulfonate Type Anionic Surfactants Including 2-Sulfonatofatty Acid Methyl Ester by Methyl Ester Derivatization and HPLC Analysis

A method for the separation and determination of the components of a sulfonate type anionic surfactant mixture including 2-sulfonatofatty acid methyl ester (α-SF) was developed. It is quite difficult to isolate linear alkylbenzenesulfonate (LAS), α-olefin sulfonate (AOS), alkane sulfonate (SAS) and α-SF from mixture of these compounds. However, when converted to their sulfonic acid methyl ester derivatives, separation is possible by high performance liquid chromatography (HPLC). The methyl sulfonate mixture was separated into α-SF, AOS, and (LAS and SAS) by normal phase HPLC, and the content of each was determined from the peak area. The eluents were collected into flasks and subject to reversed phase HPLC analysis of alkyl homologue distribution. The ratio of LAS to SAS was determined by means of NMR spectroscopy. Mixtures of α-SF, LAS, AOS, and SAS with definite composition were analyzed by this method. The results for alkyl homologue distribution agreed well with those for raw materials of fatty acid such as methyl ester, linear alkylbenzene, α-olefin or n-paraffin.

Fibrinogen Baltimore I: Polymerization Defect Associated With a γ292Gly→Val (GGC→GTC) Mutation

Fibrinogen Baltimore I is one of the very first congenital abnormal fibrinogens reported over several decades ago; however, the molecular defect of this dysfibrinogen has eluded identification. In fact, several reports misidentified the functional defect of Baltimore I, which has impaired fibrin monomer polymerization. Reversed-phase high-performance liquid chromatography analysis of lysyl endopeptidase digest of the purified Baltimore I gamma-chain showed an abnormal peptide not found in the co-existing normal gamma-chain of this heterozygote. Amino acid sequencing of this peptide indicated that gamma-chain Gly292 is replaced by valine. This observation was confirmed, and the genetic defect was determined by direct nucleotide sequencing of a polymerase chain reaction product containing codon gamma 292, which is mutated: GGC----GTC. The molecular defect of Fibrinogen Baltimore I lies in a region of the gamma-chain required for fibrin polymerization, suggesting that the integrity of gamma Gly292 is critical for fibrin assembly.

Protein HMG‐17 is hyper‐expressed in rat glucagonoma

High-mobility-group protein 17 (HMG-17) was identified by reversed-phase high-performance liquid chromatography analysis as a major component in acidic extracts of transplantable rat glucagonoma tissue but not in insulinoma tissue of similar origin. The peptide was purified in a single step and the entire sequence of 89 amino acids was determined. Rat HMG-17 has a molecular mass of 9238 Da and shows strong similarity to human, bovine (94.4%) and chicken (88.8%) HMG-17. Six of the seven residues which vary among the mammalian sequences are located within a short segment (positions 64-83) present in the acidic, non-DNA-binding C-terminal part of HMG-17. This region shows least similarity to the otherwise related proteins HMG-14 and H6 (a trout HMG protein). Interestingly, four of the six variable positions are Asp in rat HMG-17 which results in an overall net increase in the negative charge of the C-terminal region. The nature of selective hyper-expression of HMG-17 in glucagon but not in insulin-producing tumor tissue remains to be clarified.

Analysis of guarana seeds II. Studies on the composition of the tannin fraction

The content of the total tannins, proanthocyanidins and prototannins in the seeds of guarana, Paullinia cupana var. sorbilis HKB (Sapindaceae) was studied. A total tannin content of 12.1% dry matter was found in air dried samples. Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis resulted in 6.0% (+)-catechin and 3.8% (−)-epicatechin on an average. Hydrolysable tannins were not detectable. The results for one of two analyzed ground samples from the Brazilian market were substantially lowered. It is concluded that this product was adulterated with another no tannin containing material.

Expression of alcohol-soluble endosperm proteins in maize single and double mutants

Many maize (Zea mays L.) mutant genes exist. Some affect protein content or composition, while others modify carbohydrates or kernel phenotype. In doublemutant lines, two mutant genes are present. We know little about interactions of such genes, however. We therefore examined a normal maize inbred, B37, 10 near-isogenic single mutants and 46 double mutants to analyze quantitative effects on alcohol-soluble endosperm proteins. Proteins were extracted with 70% ethanol0.5% sodium acetate-5% mercaptoethanol, and fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC). Early peaks were alcohol-soluble glutelin (ASG) subunits, while late peaks contained zein. Results were quantified and statistically analyzed. In many double mutants, protein compositions differed significantly from averages of compositions of corresponding single mutants. For example, a high-methionine, water-insoluble ASG is absent when the opaque-2 (o2) gene combines with shrunken-1 (sh1) or surgary-1 (su1). Another water-insoluble ASG nearly doubled when floury-2 (fl2) andsu1 combined. A high-proline, high-histidine, water-soluble ASG nearly doubled in combinations offl2 witho2,su1 and sugary-2 (su2). Zein was about half its expected value wheno2 combined with amylose-extender (ae), floury-1 (fl1), soft-starch (h),sh1 andsu1. Thus, rapid protein extraction and quantitative RP-HPLC showed major new epistatic and synergistic effects of several mutant genes on protein composition. Unexpectedly, these effects often involve genes that primarily affect starch composition or kernel phenotype. Alcohol-soluble proteins often vary in amount, as ino2 lines. They also differ in nutritional value. Thus, RP-HPLC analysis of these proteins can identify nutritionally superior genotypes, and may help explain the basis of such quality.

Chemical composition of lipids, especially triacylglycerol, in grape seeds.

Total lipids were extracted from five varieties of grape seeds and systematically analyzed for their chemical compositions. The yields of the total lipids were 10–16%, and triacylglycerol (TG) usually amounted to c. 90% of the whole. From a reversed-phase high-performance liquid Chromatographic analysis, the major molecular species of TG were shown to be trilinolein (40%), oleoyldilinolein (21%) and palmitoyldilinolein (18%). The component fatty acids were asymmetrically distributed at C-1 and C-3 of the TG molecule. Palmitic acid was exclusively located at the C-1 position, although unsaturated fatty acids, especially linoleic acid, were predominant at the C-1 position, as at the C-2 and C-3 positions. Compared with TG, higher proportions of palmitic and linolenic acids were generally observed in thirteen other lipid classes isolated from grape seeds, although the fatty acid compositions of the diacylglycerol and free fatty acids were roughly identical with that of TG. As component sterols, sitosterol, c...

Comparison between ethanol and methanol for the reversed-phase high-performance liquid chromatographic analysis of leukotriene B 4 and its metabolites in cell incubation supernatants

Comparison between ethanol and methanol for the reversed-phase high-performance liquid chromatographic analysis of leukotriene B 4 and its metabolites in cell incubation supernatants

Autoxidation of polyunsaturated triacylglycerols. III. Synthetic triacylglycerols containing linoleate and linolenate

AbstractFour triacylglycerols containing linoleate (L) and linolenate (Ln) in specific positions were synthesized to determine the effect of fatty acid position on their relative rates and products of autoxidation. Analyses by reversedphase high performance liquid chromatography (HPLC) showed that autoxidation of L‐ and Ln‐containing triacylglycerols form monohydroperoxides and hydroperoxy epidioxides as the main products. The peroxyl radicals of internal 12‐ and 13‐mono‐hydroperoxides of Ln triacylglycerol components cyclized rapidly and their relative triacylglycerol position had no influence on their rates of cyclization. A good linear relation was obtained between total HPLC peak areas (detected at 235 nm) of the main oxidation products and peroxide values. Reversed phase HPLC analyses thus provide a useful method to estimate oxidation of polyunsaturated triacylglycerols. The ratios of Ln to L mono‐hydroperoxides were twice the ratios of Ln to L in the triacylglycerol substrates. Ln triacylglycerol components, therefore, oxidized twice as much as the L components. At 40°C, LnLnL oxidized slightly faster than LnLLn with respective induction periods of 45 and 47 hr. LLnL oxidized faster than LLLn with respective induction periods of 56 and 60 hr. Dilinolenoyl‐linoleoylglycerols are, therefore, slightly less stable to oxidation when Ln is in the 1,2‐ than the 1,3‐triacylglycerol position. Dilinoleoyl‐linolenoylglycerols are less stable when L is in the 1,3‐ than the 1,2‐triacylglycerol position.

High-performance liquid chromatography of insulin : Accessibility and flexibility

Current ideas suggest that a conformational change in the insulin monomer may play an important part in its interaction with the insulin receptor. An investigation is reported in which analytical reversed-phase high-performance liquid chromatography of insulin analogues was used to investigate the solution conformation of the insulin monomer. The results are interpreted in terms of elution coefficients modified by the calculated surface accessibilities of individual residues. The results suggest a partial unfolding of the insulin monomer under the experimental conditions used, which is consistent with current ideas on the biologically active conformation of insulin.

High-performance liquid chromatographic analysis of bile pigments as their native tetrapyrroles and as their dipyrrolic azosulfanilate derivatives

A reversed-phase high-performance liquid chromatographic (HPLC) analysis of bile pigments is described that provides baseline separation of the major bilirubin conjugates found in bile. The advantage of the technique is that the bile pigments can be analyzed directly as their native tetrapyrroles without prior solvent extractions or derivatization. The use of ammonium acetate in place of sodium salts permits preparative isolation and lyophilization of the pigments for mass spectroscopy. The derivatization of the pigments as their dipyrrolic azosulfanilates with subsequent HPLC analysis demonstrates baseline separation of the endo- and exovinyl azodipyrroles and allows identification of that half of the tetrapyrrole which contains the conjugate in the instances of monoglycosides.

Secretion of islet amyloid polypeptide in response to glucose

The content of isolet amyloid polypeptide (IAPP) in isolated rat pancreatic islets was determined by a radioimmunoassay. Reverse-phase high-performance liquid chromatography analysis revealed that a main peak of IAPP immunoreactivity in the extracts from the islets corresponded to a synthetic rat IAPP. Secretion of IAPP from the cells is regulated by the extracellular glucose concentration. Thus, IAPP may be a novel regulator for glucose homeostasis and changes in the secretion perhaps relate to insular amyloid deposits and impaired glucose tolerance in type 2 diabetes mellitus.

Kinetics of tryptic hydrolysis as a probe of the structure of human plasma apolipoprotein A-II

As a model system to understand apolipoprotein structure-function and their relationships to proteolytic events, the kinetics of tryptic hydrolysis of apolipoprotein A-II (apo A-II) was investigated in solution and in association with phospholipid. The rates of appearance and identities of specific peptides were determined by reversed-phase high-performance liquid chromatography and amino acid analysis, respectively. For the kinetics of hydrolysis of apo A-II in solution, the carboxyl-terminal peptides of residues 55–77 and 56–77 appeared first, followed by peptides of residues 4–23, 29–39, 40–44 and 45–54, which appeared at nearly identical rates. The kinetics of hydrolysis of apo A-II associated with 1,2-dimyristoyl- sn-glycero-3-phosphocholine showed several differences. First, a 100-fold larger amount of trypsin was needed to obtain a similar rate of product formation; second, a new peptide appeared, eluting earlier than apo A-II but having a similar amino acid composition; and third, the relative rates of appearance of peptides were different. The secondary structure surrounding the bonds susceptible to trypsin cleavage was determined by several predictive algorithms. The lysine amino acid bonds were found to be in regions defined by a high helical amphipathic moment. The reduced susceptibility to tryptic hydrolysis of apo-II associated with phospholipid appears to be due to a higher free energy of stabilization of protein secondary structure. As a consequence, the lysine amino acid bonds are in folded regions of the protein where they are conformationally inaccessible to enzymatic hydrolysis. By use of structure-prediction methods, it is possible to designate which regions of apolipoproteins may be important in proteolysis.

Catechol estrogen formation in MCF-7 cell culture and effects of bromoestrogen inhibitors

Agonist and antagonist activities have been reported for several catechol estrogens given exogenously. Since the metabolic clearance rate for catechol estrogens in the body is very rapid, catechol estrogens produced at other tissues will have minimal effect on breast tissue. Information of the extent of catechol estrogen formation within cells is critical in assessing the overall importance of these estrogen metabolites. Investigations of the conversion of estrogens to catechol estrogens were performed in the MCF-7 human mammary carcinoma cell culture system. Reverse-phase high-performance liquid chromatography (HPLC) analysis demonstrated that very little metabolism of estradiol occurs after 48 h, with only small amounts of estrone, 2-hydroxyestradiol, 2-hydroxyestrone, and estriol being observed. The total amount of 2-hydroxyestrogen products formed from 1 μM estradiol was 406.2 pmol (SD = 60.9) per 3 × 10 7 cells in 48 h. Similar results were obtained using the simpler radiometric assay for estrogen 2-hydroxylase, which measures the release of 3H 2O from [2- 3H]estradiol. The effects of inhibitors of estrogen 2-hydroxylase were also examined in MCF-7 cells. 2-Bromoestradiol, 4-bromoestradiol, and 2,4-dibromoestradiol effectively block estrogen 2-hydroxylase in a dose-dependent manner in MCF-7 cultures, with ED 50 of approximately 1 μM for each inhibitor. Furthermore, these bromoestrogens bind poorly to estrogen receptors in MCF-7 cells and do not alter cell growth. Thus, in MCF-7 mammary cell cultures, metabolism of estradiol occurs to only a minor degree, and it is unlikely that the levels of catechol estrogens would reach physiologically relevant concentrations in the intact breast cancer cells. In addition, the bromoestrogens are effective inhibitors of estrogen 2-hydroxylase in cell culture systems, bind poorly to the estrogen receptor, and do not exhibit estrogenic effects at concentrations up to 1 μM.

Analysis of parathyroid hormone in bovine parathyroid cysts.

Parathyroid cysts were found in 2-5% of beef steers and heifers while collecting parathyroids at a local abattoir. The cyst fluid contained parathyroid hormone (PTH), averaging 15 ng/ml of fluid. Reversed-phase high-performance liquid chromatographic (HPLC) analysis of the fluid using an acetonitrile-trifluoroacetic acid solvent system, followed by carboxyl-terminal specific PTH radioimmunoassay of the eluant fractions, revealed two major peaks of PTH immunoreactivity. The first eluted at approximately 25% acetonitrile, a position similar to the commercially available human carboxyl terminal fragment of PTH-(39-84). The second peak, at approximately 37% acetonitrile, corresponded to the elution position for bovine PTH-(1-84). The cyst fluid was tested for enzyme activity by incubation of 125I-labeled bovine PTH with cyst fluid for 30 minutes at 37 degrees C, which resulted in no fragmentation of the labeled peptide. Immunohistochemistry of the cells lining the parathyroid cyst indicated that they contained PTH.

Characterization of α-MSH-related peptides released from rat hypothalamic neurons in vitro

Reverse-phase high-performance liquid chromatography analysis, coupled with a sensitive radioimmunoassay for α-melanocyte-stimulating hormone (α-MSH), was used to characterize the α-MSH-related peptides stored in the rat hypothalamus or released from perifused hypothalamic slices. Four peaks of α-MSH-like immunoreactivity (α-MSH-LI) co-eluting with synthetic des- Nα-acetyl α-MSH, α-MSH and their respective sulfoxide derivatives were resolved and quantified. In hypothalamic extract, deacetyl α-MSH which was the predominant peptide represented 94.4% of total α-MSH-LI content, while the relative amount of α-MSH was only 5.6%. Analysis of α-MSH-related peptides contained in effluent perifusates showed that deacetyl α-MSH and its oxidized form were the major peptides released from neurons in basal conditions or under KCl-induced depolarization (50 mM KCl for 75 min). However, the proportion of acetylated peptide was 3–4 times higher in the perifusion medium than in hypothalamic extracts. Our data indicate that acetylation of des- Nα-acetyl α-MSH may occur during the process of exocytosis. Since acetylation of α-MSH markedly increases the behavioural potency of the peptide, these results suggest that regulation of the acetyltransferase activity could be a key mechanism to modulate the bioactivity of α-MSH-related peptides in the brain.