Amino acid analysis by reverse-phase high-performance liquid chromatography: precolumn derivatization with phenylisothiocyanate

Methods for the quantitative derivatization of amino acids with phenylisothiocyanate and for the separation and quantitation of the resulting phenylthiocarbamyl derivatives by reverse-phase high-performance liquid chromatography are described. Phenylthiocarbamylation of amino acids proceeds smoothly in 5 to 10 min at room temperature. Coupling solvents, reagent, and some byproducts are removed by rotary evaporation under high vacuum, and the phenylthiocarbamyl derivatives are dissolved in 0.05 M ammonium acetate, pH 6.8, for injection onto the octyl or octadecylsilyl reverse-phase column. Columns are equilibrated with the same solvent and the effluent stream is monitored continuously at 254 nm for detection of the amino acid derivatives. Elution of all of the phenylthiocarbamyl amino acids is achieved in about 30 min utilizing gradients of increasing concentrations of ammonium acetate and acetonitrile or methanol. This approach to amino acid analysis offers select advantages, both with respect to methods which employ reverse-phase separation of prederivatized samples and to the classical ion-exchange procedure. All amino acids, including proline, are converted quantitatively to phenylthiocarbamyl compounds and these are stable enough to eliminate any need for in-line derivatization. Furthermore, results comparable in sensitivity and precision to those obtained by state-of-the-art ion-exchange analyzers may be generated with equipment that need not be dedicated to a single application.     

Separation of phycobiliprotein subunits by reverse-phase high-pressure liquid chromatography

Baseline separation of subunits of diverse phycobiliproteins was achieved by a reverse-phase HPLC gradient method with a C4 large-pore column and a solvent system consisting of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in 2:1 (v/v) acetonitrile:isopropanol. The procedure was successfully applied to cyanobacterial allophycocyanin and C-phycocyanins, an unusual phycocyanin from a marine cyanobacterium, red algal B- and R-phycoerythrins, and a cryptomonad phycoerythrin. The subunit sizes in these proteins range from about 7.5 to 30 kDa. Sample recovery was in excess of 85% in all cases. On-line spectroscopic analysis with a multiple diode array detector allowed determination of the type and number of bilins carried by each subunit.     

Amino acid analysis of collagen hydrolysates by reverse-phase high-performance liquid chromatography of 9-fluorenylmethyl chloroformate derivatives

A recently described procedure for amino acid analyses has been modified and adapted for use in quantitating the unique mixture of products commonly found in hydrolysates of the collagens. The method involves precolumn derivatization of hydrolysates with 9-fluorenylmethyl chloroformate (FMOC-CL), chromatographic separation of the derivatives and excess reagent on a reverse-phase column, and quantitation based on the fluorescent properties of the derivatives. The method takes advantage of the ease with which stable derivatives are formed with the FMOC reagent. Using a ternary gradient system, a complete amino acid analysis with good resolution of all components can be performed within 35 min. The sensitivity of the method is comparable to levels attained by other derivatives and the fluorescence response of each derivative is linear over the total range of 1-800 pmol. Given these parameters, the method allows complete amino acid analyses to be performed on 100 ng of collagen corresponding to a single picomole of a collagen chain (Mr 100,000).     

Rapid separation of acetylated oligosaccharides by reverse-phase high-pressure liquid chromatography.

Peracetylated saccharides were separated by chromatography on a reverse-phase support, eluting with mixtures of acetonitrile-water. Gradient elution for 2.5 h gave significant separations of all linear glucose oligomers containing up to 35 sugar residues. With isocratic elution retention was exponentially related to molecular mass and only slightly affected by linkage or anomeric configuration. The presence of glucosamine in various saccharides markedly reduced their retention.     

Analysis of Neisseria gonorrhoeae peptidoglycan by reverse-phase, high-pressure liquid chromatography

The muramidase digest of peptidoglycan from Neisseria gonorrhoeae was isolated and analyzed by the use of a reverse-phase, high-pressure liquid chromatography system. As was found previously in the case of Escherichia coli, gonococci peptidoglycan is also composed of a greater number of muropeptides than can be resolved with thin-layer chromatography systems. Preliminary classification of the muropeptide components into subclasses based on O-acetyl modification and degree of cross-linkage was achieved. Examination of a penicillin-susceptible strain and a highly resistant strain with two penicillin-binding protein alterations synthesized distinctly different peptidoglycan structures, as revealed by this technique.     

The analysis of nanogram levels of free amino acids by reverse-phase high-pressure liquid chromatography.

A simple method and apparatus are described for the efficient recovery of proteins from sodium dodecyl sulfate-polyacrylamide gel systems after electrophoretic resolution. This procedure provides for high yields of proteins which are free of sodium dodecyl sulfate and in certain cases, exhibit significant levels of biological activity.     

Analysis by reverse-phase high-pressure liquid chromatography of phenylisothiocyanate-derivatized 1-aminocyclopropane-1-carboxylic acid in apple extracts

A rapid and sensitive method for the determination of 1-aminocyclopropane-1-carboxylic acid (ACC) in apple tissues is described. This method is based on the derivatization of ACC with phenylisothiocyanate, and the subsequent separation and quantification of the resulting phenylthiocarbamyl-ACC by reverse-phase high-pressure liquid chromatography. Phenylthiocarbamylation of ACC (and other amino acids) in apple extracts is complete within 20 min at room temperature. After removing solvents and reagent, the phenylthiocarbamyl derivatives are separated on an octadecyl reverse-phase column, eluted with a mixture of acetonitrile and sodium acetate buffer at pH 4.6, and monitored with a uv detector set at 254 nm. An analysis of apple extract can thus be achieved in 23 min and detect quantities as low as 1 pmol. Assays have been done to compare the efficiency of this method with that of a method using an ion-exchange amino acid analyzer and with that of Lizada and Yang's method [(1979), Anal. Biochem. 100, 140-145]. The latter method proved to yield markedly less accurate results than the other two, but the derivatization-HPLC method was preferred because of simplicity of operation and a better separation of ACC.     

Resolution of some glycopeptides of hen ovalbumin by reverse-phase high-pressure liquid chromatography

Reverse-phase high-pressure liquid chromatography has been shown to be useful in fractionation of oligosaccharides containing N-acetyl amino sugars on the basis of stereochemical differences. Data in this paper show this same stereochemical selectivity can be obtained for glycopeptides. Using dilute aqueous phosphate for elution, three major high-mannose glycopeptides from ovalbumin can be separated on the analytical scale in a few minutes and the same method can also be used for preparation of the glycopeptides on the milligram scale. High-field proton NMR spectroscopy is used to identify two of the compounds as the mannose-5 and -6 glycopeptides (E3 and D3) and the third as the mannose-6 glycopeptide substituted by two N-acetyl glucosamine (GlcNAc) residues (C3B). The NMR spectra and analytical chromatograms of the glycopeptides show high purity and freedom from contaminants previously identified in ovalbumin glycopeptides.     

Fractionation of oligosaccharides containing N-acetyl amino sugars by reverse-phase high-pressure liquid chromatography

High-pressure liquid chromatography (hplc) of N-acetylglucosamine (GlcNAc) and galactosamine (GalNAc) containing carbohydrates was performed on several reverse-phase silica columns. Nanomolar level detection was accomplished using far uv-absorbance monitoring. Baseline separations of the α and β anomers of GlcNAc, chitobiose, chitotriose, and chitotetraose were observed with water elution of the reverse-phase column. With the addition of up to 3% acetonitrile to the eluting solvent, similar resolution of chitin oligomers up to a chain length of seven was observed. Anomerization of the residue could be followed by isolation of either anomeric peak with subsequent rechromatography. Reduction of chitotriose with borohydride yielded a single sharp peak with a retention volume similar to that of the reducing trisaccharide. Semipreparative reverse-phase hplc allowed for the separation and identification by ¹³C NMR of the GlcNAc-α-1→6 GlcNAc disaccharide from the β-1→6 isomer. Methyl glycosides of GalNAc and GlcNAc were shown to have retention times much longer than the free sugar.     

The behaviour of peptides on reverse-phase supports during high-pressure liquid chromatography.

High-pressure ('performance') liquid chromatography has been used to investigate the reverse-phase chromatographic behaviour of peptides, ranging in length from 2 to 65 amino acid residues, which have originated from primary-sequence determinations or solution/solid-phase syntheses. By using a pyridine/formate-pyridine/acetate/propan-1-ol buffer system, as previously described [Hughes, Winterhalter & Wilson (1979) FEBS Lett. 108, 81-86], the influence of various experimental parameters were examined. (a) Peptide retention was observed to be temperature-independent between 25 and 55 degrees C. (b) The dependence of chromatographic retention on pH decreases with increasing peptide hydrophobicity. (c) Chromatographic results from C8- and C18-chain-length, as well as from 5 micrometers- and 10 micrometers-particle-size, supports were comparable. (d) The hydrophobic strength of the organic solvent in the mobile phase was observed to decrease: propan-1-ol approximately equal to propan-2-ol greater than acetonitrile much greater than methanol. (e) When gradient rates (% of buffer B/unit time) were systematically decreased, peptide retention decreased in a hyperbolic manner. Comparisons of the peptides chromatographed with respect to their measured retention properties and calculated hydrophobicities were performed by computer analysis. Deviation of peptide chromatographic behaviour was observed to be essentially independent of hydrophobicity, chain length and charge. On the basis of the measured retention properties of the chromatographed peptides, hydrophobic constants for the various amino acid side chains were determined and compared with similar constants available from the literature.     

Determination of plasma oxalic acid by high-pressure liquid chromatography

A new specific and sensitive method for determination of oxalic acid in plasma by High Performance Liquid Chromatography (HPLC) is described. The plasma sample is deproteinized by ultrafiltration. The oxalic acid in the ultrafiltrate is purified by precipitation with CaCl2, new dilution of calcium oxalate precipitate, oxalic acid extraction with diethyl-ether and total dryness of the sample. The losses of oxalic acid during this process are evaluated by the addition of oxalic acid (U-14C) before the precipitation step. The dried samples are redissolved in mobile phase (o-H3PO4, 0.05 M) and injected into a HPLC chromatograph, with reversed phase column (Lichrosorb RP-8, Merck). Oxalate peak is detected spectrophotometrically at 220 nm with a retention time of 3.20 minutes. The method shows a mean recovery value of 82.11, with an intra-run and between-run CV values of 2.54 and 6.95 respectively. The oxalic acid measured in plasma by this method is 291 +/- 89 micrograms/100 ml plasma ultrafiltrate, in 16 normal subjects.     

The analysis of acyl-coenzyme A derivatives by reverse-phase high-performance liquid chromatography

A method for determining tissue levels of Coenzyme A and various short-chain-length acyl-CoA derivatives using high-performance liquid chromatography is presented. Separation of the various compounds was accomplished using a reverse-phase Spherisorb ODS II, 5-microns C18 column. Mobile-phase solvents were (a) potassium phosphate, 220 mM; thiodiglycol (2,2-thiodiethanol), 0.05% (v/v), pH 4.0 and (b) methanol, 98%; chloroform; 2% (v/v). The various acyl-CoA derivatives were detected by monitoring the column effluent at 254 nm. Nearly baseline separation was obtained for a standard mixture of free CoASH, methylmalonyl-CoA, beta-hydroxy-beta-methylglutaryl-CoA, succinyl-CoA, acetoacetyl-CoA, acetyl-CoA, propionyl-CoA, isobutyryl-CoA, beta-methyl-crotonyl-CoA, and isovaleryl-CoA. CoA derivative profiles were determined in neutralized perchloric acid extracts of perfused rat hearts and livers and of isolated rat liver mitochondria to demonstrate the utility of this method for assessing the levels of CoA derivatives in biological samples.     

Analysis of demethylaminoazobenzene-thiohydantoins of amino acid by high-pressure liquid chromatography

Dimethylaminoazobenzene-thiohydantoins of amino acid can be quantitatively analyzed by high-pressure liquid chromatography at picomole level. As little as 5 to 10 pmol of dimethylaminoazobenzene-thiohydantoins of amino acid can easily be detected in the visible region (436 nm) against a stable baseline. Three amino acid pairs, namely glutamine and threonine, methionine and proline, and leucine and isoleucine, have not yet been separated. This new technique provides a sensitive and efficient tool for measuring the recovery of amino terminal amino acids using the dimethylaminoazobenzene-isothiocyanate method and the repetitive yield of sequence determination using the dimethylaminoazobenzene-isothiocyanate phenylisothiocyanate double-coupling method.     

Separation of amino acid phenylthiohydantoin derivatives by high-pressure liquid chromatography

Separation of the phenylthiohydantoin (PTH) derivatives of all 20 common amino acids is accomplished in approximately 11 min with excellent resolution by using high-pressure liquid chromatography. The chromatography is achieved at 50 degrees C on an Altex reversed-phase PTH-C18 column in an ammonium acetate-buffered acetonitrile, pH 4.5, mobile phase. Simple isocratic and linear gradient steps are used. Retention times for the various PTH-amino acids are very reproducible. Because the baseline is flat and free of background noise, PTH-amino acids can be detected in the low picomole range. The simplicity of this chromatographic system allows it to be easily automated.     

Determination of tissue UDP-glucuronic acid levels by high-pressure liquid chromatography

A new and sensitive method to measure UDP-glucuronic acid extracted from as little as 25 mg wet weight tissue has been developed. This procedure employs high-pressure liquid chromatography and liquid scintillation spectrophotometry to measure p-[¹⁴C]nitrophenylglucuronide generated enzymatically from p-[¹⁴C]nitrophenol and UDP-glucuronic acid. The reaction was catalyzed by UDP-glucuronyltransferase obtained from rat liver microsomes. The tissue levels of UDP-glucuronic acid assayed were 2 to 20 μmol/100 g wet wt, which are well below the levels detectable by the widely used spectrophotometric method.     

Desalting of nucleotides by reverse-phase high-performance liquid chromatography

Chromatography of AMP, NAD⁺, or NADH on a reverse-phase C₁₈ Porasil B column rapidly removes ammonium formate or potassium phosphate from 90% of the nucleotide. Earlier reports showed these salts could not be separated from nucleotides by conventional desalting using gel filtration.     

Determination of uric acid in biological fluids by high-pressure liquid chromatography

A high-pressure liquid chromatographic assay for uric acid in biological fluids has been developed. Blood uric acid can be analyzed in as little as 20 μl of plasma. The mean and range of plasma uric acid concentrations in healthy adults determined by high-pressure liquid chromatography were similar to these obtained by enzymatic analysis. One of the advantages of the present method is that naturally occurring metabolites in biological fluids or drugs do not interfere with the analysis. Data are presented for blood and urine specimens obtained from mice fed a known uricase inhibitor, potassium oxonate. Comparisons are made between the present method and methods previously employed for uric acid determination.