High-performance liquid chromatography of side-chain-protected amino acid phenylthiohydantoins

Eighteen side-chain-protected amino acids, routinely employed in solid-phase peptide synthesis, were derivatized to their phenylthiohydantoins (PTH) by one cycle of the Edman degradation. All of these side-chain-protected PTH amino acids elute, with almost-baseline resolution, in less than 18 min by high-performance liquid chromatography, utilizing a biphasic gradient of acetonitrile in 0.01 n sodium acetate, pH 4.5, or a linear gradient of 0 to 100% acetonitrile with the exception of the coelution of a O-benzyl-threonine and carbobenzoxy-lysine phenylthiohydantoin amino acids. The derivatized amino acids were subjected to reverse-phase chromatography on a Zorbax ODS column and monitored at 254 nm. None of the PTH amino acids coelute with side-chain-protected PTH amino acid counterparts, although PTH-tosyl-histidine undergoes deprotection to PTH-histidine in the Edman degradation. A protected decapeptide attached to a chloromethylated polystyrene resin was degraded on a solid-phase sequencer in 16 h. The PTH amino acids resulting from the automated Edman degradation on the decapeptide were fully resolved and quantified in less than 3 h demonstrating that automated high-performance liquid chromatography can keep pace with both the automated sequencer and synthesizer which requires minimally 2–3 h for attachment of each residue to the growing peptide chain.     

Assay of L-aromatic amino acid decarboxylase by high performance liquid chromatography

A method is presented for the assay, by high-performance liquid chromatography, of l-aromatic amino acid decarboxylase. The K_m value of the enzyme for 3,4-dihydroxyphenylalanine (dopa) was, by this procedure, 0.16 mm, in good agreement with previous reports. When α-methyldopa was used as substrate, evidence was obtained indicating the formation of, besides α-methyldopamine, 3,4-dihydroxyphenylacetone, presumably through an internal transamination process.     

Analysis of amino acid phenylthiohydantoins by high-performance liquid chromatography using gradient elution with ethanol

The use of gradient elution with acetate-buffered ethanol for high-performance liquid chromatographic analysis of phenylthiohydantoins (PTHs) is described. The system separates all commonly encountered PTH derivatives in a total analysis time of 20 min on columns of Spherisorb 5 S ODS (4.6 × 250 mm) packed in the laboratory at moderate expense. Experience with routine operation of the method, particularly with regard to column stability is discussed. Ethanol is considerably cheaper and less toxic than previously described solvent systems.     

Analysis of the amino acid indospicine in biological samples by high performance liquid chromatography

Indospicine is a hepatotoxic amino acid that accumulates in the meat of horses that consume the legume Indigofera linnaei. A method to determine indospicine concentration in biological samples using an amino acid analyser has been reported, but the analysis time is long and therefore not suited to the analysis of large numbers of samples. A rapid and reliable method was developed for the analysis of indospicine in horsemeat and serum using High Performance Liquid Chromatography. Horsemeat and serum were extracted with either water or 0.01 N hydrochloric acid, respectively, and deproteinized by ultrafiltration. Precolumn derivatization of samples with phenylisothiocyanate was followed by separation of indospicine from other amino acids on a Pico-Tag C 18 column and UV detection at 254 nm. The calibration curves for indospicine in horsemeat extract were linear over the concentration range 0.4 microg ml(-1) to 20 microg ml(-1), while for indospicine in serum, the linear range was from 0.17 microg ml(-1) to 16.67 microg ml(-1). The mean recovery of indospicine in horsemeat extract was 87.2 +/- 6.8% and in serum was 97.3 +/- 9.9%. Analysis time for indospicine in horsemeat samples was 31 min and in serum samples was 36 min.     

Simple,rapid, and highly efficient separation of amino acid phenylthiohydantoins by reversed-phase high-performance liquid chromatography

A rapid and efficient separation of amino acid phenylthiohydantoins by high-performance liquid chromatography has been accomplished by step-gradient elution with use of a mobile phase of acetate-buffered aqueous acetonitrile and an octadecylsilyl stationary phase. Typical analyses are completed in less than 12 min. Peak elution positions in this procedure are highly reproducible (with about 0.2% variance) and allow unambiguous identification of all residues. A procedure for the optimal positioning phenylthiohydantoin-Arg and -His is given. Molar extinction coefficients at 254 nm, which are particularly useful with common fixed wavelength detectors, are given for 25 amino acid phenylthiohydantoins.     

High-pressure liquid chromatographic analysis of amino acid phenylthiohydantoins: Comparison with other techniques

High-pressure liquid chromatography, utilizing reverse phase μ Bondapak C₁₈ columns and elution with increasing acetonitrile concentrations, has been used to resolve amino acid phenylthiohydantoins obtained from the automated Edman degradation of proteins. Assignment of identity to residues which are difficult to distinguish or identify conclusively by other conventional techniques is easily achieved by high-pressure liquid chromatographic techniques. The use of high-pressure liquid chromatography, in parallel with gas-liquid and polyamide thin-layer chromatography, allows unequivocal assignments of identity to amino acid phenylthiohydantoins obtained in protein sequencing. Single protein sequence determinations can be extended by 20 to 100% by the use of high-pressure liquid chromatography with rapid, accurate, and quantitative identifications of amino acid phenylthiohydantoins.     

A simple and inexpensive high pressure liquid chromatographic system as an adjunct to gas chromatography in identification of amino acid phenylthiohydantoins.

A simple and inexpensive system has been developed in our laboratory for the identification and quantitation of amino acid phenylthiohydantoins by high pressure liquid chromatography. Isocratic methodology has been employed, so that expensive complex gradient makers can be avoided. The method is rapid, and retention times of 10 min or less are obtained in all but one case. This paper presents the rationale and details of the method, and demonstrates a comparison between gas-liquid chromatograms and high pressure liquid chromatograms of selected residues from actual automated sequence analyses of both sperm whale myoglobin and egg white lysozyme. The method is intended to complement rather than replace gas-liquid chromatography, as it cannot completely resolve all 20 phenylthiohydantoins, but rather identifies easily those residues which are difficult or impossible by gas chromatography.     

Quantitative determination of amino acids in tissue cells by high performance liquid chromatography

Amino acids derivatized with o-phthaldialdehyde were separated by high performance liquid chromatography on a reverse phase C8 column with a ternary gradient. Multilevel calibration permits the analytical quantitation within the range of 50 to 3500 pMol of the individual amino acids in one run. The reproducibility of the analysis in consecutive runs is ± 3%. HeLa cells grown in suspension cultures were harvested by either centrifugation in the cold or by centrifugation through dibutylphthalate. Amino acids were extracted with methanol-water. Cells not exposed to aqueous media prior to extraction show an up to 3 fold higher level of amino acids in the intracellular pool.     

Determination of free amino acids in burley tobacco by high performance liquid chromatography

A reversed-phase high performance liquid chromatographic method was developed for determining free amino acids in burley tobacco. The test was done by OPA/3-mercaptopropionic acid as the pre-column derivatizing reagent. Chromatographic column was Elitte C¹⁸ column (4.6 mm × 250 mm i.d., 5 μm). Mobile phase A was 18 mol/l NaAc (pH7.2) including 0.002%(v/v) triethylamine and 0.3%(v/v) furanidine. Mobile phase B was 100 mol/l NaAc (pH7.2)–acetonitrile–methanol (v/v = 1:2:2). The column temperature was 40 °C and the flow rate was 1.0 ml/min. The fluorescence detector was used with 350 nm excitation wave length and 450 nm emission wave length. The average recoveries of the method ranged from 95.3–100.7% with the relative standard deviation of 2.32–9.24%. The method is simple, accurate and has good repeatability. The results of the determination of seventeen kinds of free amino acids in burley leaves were produced by the way of different ratios of cake fertilizer and inorganic fertilizer. The results show that Aspartic acid has the highest content however ratio of cake fertilizer and inorganic fertilizer. The contents of most of the free amino acids are increased and then gradually decreased with the increase in organic manure. The contents of most of the free amino acids are very close at 15:85% ratio and 30:70% ratio of cake fertilizer and inorganic fertilizer. The total amount of free amino acids is the highest at 30:70% ratio of cake fertilizer and inorganic fertilizer. Considering comprehensively, the quality of burley leaves is the best at 30:70% ratio of cake fertilizer and inorganic fertilizer.     

Nucleic acid analogs for high performance liquid chromatography

HPLC resins containing nucleic acid base derivatives were successfully prepared. These resins were found to give excellent complementary separation of nucleic acid base derivatives, nucleosides, nucleotides, and oligonucleotides. These resins may be useful for separation of components of nucleic acids and polynucleotides as a specific separation system, while ion-exchange and reverse-phase systems are non-specific separation systems.     

Measurement of muramic acid in marine sediments by high performance liquid chromatography

Bacterial biomass in marine sediments may be estimated from the amount of muramic acid present. A method for determining muramic acid by high performance liquid chromatography is described, which is simpler and faster than other methods. Muramic acid is released from sediment by acid hydrolysis, and assayed as an o-phthaldialdehyde derivative.     

Measurement of branched chain amino acids in blood plasma by high performance liquid chromatography

A simple and rapid high performance liquid chromatographic technique is described for the separation and quantitation of plasma branched chain amino acids. After addition of a norleucine internal standard, plasma samples are acidified with acetic acid, and amino acids are separated from proteins and other plasma components by passage of the acidified plasma through an ion exchange resin. The ammonium hydroxide eluate from the resin is dried, phenylisothiocyanate derivatives are prepared, and the amino acids are separated on a Waters reverse-phase "Pico-Tag" column with an ultraviolet detector set at 254 nm. In addition to the branched chain amino acids (leucine, valine, and isoleucine), aspartate, glutamate, serine, threonine, alanine, and methionine are quantitated with high precision and accuracy, as verified by quantitative recovery and comparison with an automatic amino acid analyzer. The advantages of the method are its simplicity, speed, stability of derivatives, high reproducibility, low per-sample cost, and the use of a simple fixed-wavelength ultraviolet detector.     

Complete phenylthiohydantoin amino acid analysis by high-performance liquid chromatography on ULTRASPHERE-octadecyltrimethyloxysllane

All 25 phenylthiohydantoin amino acids have been separated by high-performance liquid chromatography on ULTRASPHERE-octadecyltrimethyloxysilane employing an acetate buffer (pH 5), acetonitrile gradient. The selectivity of the basic and acidic residues can be controlled by manipulation of pH and ionic strength of the mobile phase to optimize resolution between peaks.     

Measurement of amino acid release from cultured cerebellar granule cells by an improved high performance liquid chromatography procedure

Endogenous amino acid release was examined in rat cerebellar primary cultures comprising more than 95% of glutamatergic granule cells. Eighteen amino acids were determined in the cell extracts and in the release fractions by high performance liquid chromatography, using precolumn derivatization witho-phthaldialdehyde and separation on a reverse-phase column using a multi-step gradient system of two solvents (0.1 M Na⁺acetate, pH 7.2/methanol: tetrahydrofuran, 97:3). The fluorimetric response was linear, at least in the range of 2–162 pmol, for all the amino acids analysed, with a detection limit of 1 pmole. We observed a good reproducibility in within-assay and between-assay coefficients of variation of the retention times and fluorescence yield. When cultured granule cells were exposed to the excitatory amino acid receptor agonist quisqualic acid (50 M), we observed a net increase in the release of glutamate (3 fold over the baseline) and a smaller increase in that of aspartate (2 fold) and taurine (1.6 fold). Other amino acids were not significantly affected. GABA levels were below detection limits, due to the minimal number of GABAergic neurons present in the cultures.     

Application of L-Amino acid in determination of Huperzine A by high performance liquid chromatography

A selective HPLC method for determination of Huperzine A in Huperzia serrata Extract has been developed and validated. Huperzine A was dissolved in 0.01 mol/L HCl, chromatographed on an Agilent Zorbax SB-C18 (150 mm x 4.6 mm i.d., 5 microm) column, with a mobile phase consisting of methanol-1mM L-Lysine water solution (50:50, v/v), and detected at 310 nm. The UV peak areas were used for quantitation of Huperzine A content. The correlation coefficient (R(2)) of the calibration was 0.9999 over the range of 1-25 microg/ml and intra- and interday precision over this range were not more than 2%. The method was successfully applied to characterize and determine the Huperzine A in Huperzia serrata Extract.     

Fluorometric analysis of pectenotoxin-2 in microalgal samples by high performance liquid chromatography

A rapid HPLC method with fluorescence detection of pectenotoxin-2 (PTX2), a polyether macrolide toxin, in microalgae is presented. A dienophile reagent, DMEQ-TAD, was used for precolumn fluorescence labeling. PTX2 could be quantitatively detected in the range 1-200 ng. This method confirmed the occurrence of PTX2 in net haul samples mostly composed of dinoflagellates Dinophysisspp. collected in the Adriatic Sea, Italy and Mutsu Bay, Japan.