Determination of specificity of endopeptidases by combined high-performance liquid chromatography and amino acid analysis

The specificity of three neutral endopeptidases toward several biologically active peptides was determined by combined high-performance liquid chromatography and amino acid analysis of the degradation products. Incubation mixtures were chromatographed on a reversed-phase column equilibrated with a mixture of acetonitrile and potassium phosphate buffer (0.05 M; pH 2.0). Reaction products were eluted with a linear gradient of acetonitrile and the absorbance of the effluent monitored at 210 nm. Fractions corresponding to discrete peaks were subjected to quantitative amino acid analysis. The peptide bond undergoing cleavage is readily assigned from the knowledge of the primary structure of the peptide and the amino acid composition of the reaction products.     

High-performance liquid chromatography of amino acid peptides and proteins

Summary The retention behaviour of seven globular proteins ranging in molecular weight from 12,000 to 69,000 was investigated using Mono-Q anion-exchange resin as the stationary phase and sodium chloride as the displacer salt. In particular the influence of changes in ionic strength and mobile phase pH on the isocratic retention properties was assessed. Several proteins were found to have significant retention when the pH of the mobile phase was below the reported pl values of the proteins. This behaviour results from the non-uniform charge distribution on the protein surface, which allows interaction with the charged stationary phase even though the protein net charge is equal to or greater than zero. The influence of pH and ionic strength on experimentally observed bandwidths was also investigated. The dependence of the effective reduced plate height on solute capacity factor was found to vary significantly with the mobile phase pH, a behaviour consistent with the interplay of complex multisite binding kinetics. These results provide a basis for further detailed investigations into the mechanism of interaction of proteins not only with charged surfaces associated with adsorptive chromatographic media but also with other macromolecules. For Part LXXXII, see ref. [27].     

Enantiomeric analysis of the common protein amino acids by liquid chromatography

Methods for resolving amino acids into their enantiomers are of importance in the preparation of peptides, drugs, food additives, and in other areas where optical purity is important. HPLC methods of separation are particularly useful.     

Post-column derivatization in liquid chromatography using immobilized enzyme reactors and amperometric detection

The advantages to be gained by conducting enzyme assays of carbohydrates in liquid chromatography are discussed. The enzymes are contained in immobilized enzyme reactors and used in the post-column mode. The product formed in the reactor is selectively detected amperometrically at a chemically modified electrode mounted in a flow-through detector. Selected examples are given to illustrate the advantages obtained.     

Comparison of liquid chromatography with fluorescence detection to liquid chromatography-mass spectrometry for amino acid analysis with derivatisation by 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate: Applications for analysis of amino acids in skin

The analysis of nineteen amino acids found in collagen was optimised using 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate (AQC) as a derivatisation reagent. The analysis and detection of nineteen AQC-amino acids using fluorescence and mass spectrometry were compared at different mobile phase pH’s and column temperatures. The pH range of the mobile phase was set between 2.7 and 6.0 and column temperatures, 15–60 °C. The majority of amino acids produced a mono-derivatised product with AQC, except cystine, lysine and hydroxylysine which were di-derivatised. Hydroxylysine’s retention time was affected most by changes in the pH, whilst hydroxyproline’s retention time was more affected by column temperature. Hydroxylysine was detected as two diastereomers which were completely resolved. The relative standard deviation of the retention times of AQC-amino acids was less than 1% and the limit of detection (LOD) and limit of quantitation (LOQ) were ranged from (0.05–0.23) µM and (0.07–0.76) µM on fluorescence and (0.02–0.10) µM and (0.06–0.33) µM on mass spectrometry respectively. This method was successfully applied for the quantitation of amino acids in different animal skins.     

Retention prediction of o-phthalaldehyde amino acid derivatives in reversed-phase liquid chromatography

Summary Retention prediction of o-phthalaldehyde amino acid derivatives in reversed-phase liquid chromatography has been investigated. The retention of all derivatives could be predicted within about 10% relative error under the appropriate separation conditions in both isocratic and gradient-elution modes.     

Retention prediction of phenylthiohydantoin amino acid derivatives in reversed-phase liquid chromatography

Summary The concept of retention prediction for the separation of phenylthiohydatoin-amino acid derivatives in isocratic reversed-phase liquid chromatography is described. A novel retention-solubility parameter, R, is defined, for the retention prediction strategy and the performance of this R value is evaluated by comparing measured and predicted retention data. Excellent agreement between these values were observed. It is concluded that the R value has a very high potential in describing the retention of phenylthiohydantopinamino acid derivatives withdifferent types of separation systems consisting of C-18, C-8 and phenethyl bonded stationary phases and various mobile phases.     

Comparison of different amino acid derivatives and analysis of rat brain microdialysates by liquid chromatography tandem mass spectrometry

The efficiencies of three derivatisation reagents that react with either the amine (9-fluorenylmethyl chloroformate (FMOC)) or the carboxylic acid group (butanol) of amino acid or with both types of functional groups (propyl chloroformate) were compared in the analysis of amino acids by liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS). Separation of 20 amino acids derivatised with these three reagents was studied on reversed-phase chromatography. Linearity, repeatability and limits of detection of the LC-ESI-MS/MS method were determined by analysing FMOC-, butanol- and propyl chloroformate-derivatised lysine, β-aminobutyric acid, threonine and glutamic acid. The limits of detection for the derivatised amino acids (7.5-75 fmol) were as much as 2-60 times lower than those of the corresponding underivatised molecules. The best linearity was observed for amino acids derivatised with propyl chloroformate or butanol (r² = 0.996-0.999, range = 100-8500 nmol L⁻¹). Propyl chloroformate was the best suited of the reagents tested for the analysis of amino acids with LC-MS/MS and was used for the analysis of amino acids in rat brain microdialysis samples.     

Determination of 27 dansyl amino acid derivatives in biological fluids by reversed-phase high-performance liquid chromatography

The concentrations of free amino acids in plasma and in ascitic liquid of mice with Ehrlich ascitic tumours were determined by reversed-phase high-performance liquid chromatography using pre-column derivatization with Dns chloride and UV detection at 254 nm. Sample preparation is simple, and the Dns derivatives are stable. Complete separation of 27 amino acids, including proline and cysteine, was achieved in 70 min with detection limits of less than 25 pmol. There was no interference from Dns-Cl, Dns-OH and Dns-NH2. Retention time reproducibility was better than 1%. The described method enables a rapid, economical and reproducible quantification of free amino acids in biological fluids.     

Investigation of liquid and gas chromatography techniques for separation of diastereomers of beta-(alpha-methylbenzyl) amino isobutyric acid

Cryptophycins are macrolides investigated as potential anticancer agents. These large cyclic molecules are generated via a convergent process, utilizing the coupling of several smaller fragments synthesized individually. During early synthetic development of the beta-amino acid fragment C, analytical methods are necessary for the characterization of products resulting from the various routes being studied. One route being evaluated produces (RR) and (RS) diastereomers of beta-(alpha-methylbenzyl) amino isobutyric acid as intermediates. To measure diastereomeric excess (%de), assay conditions using high-performance liquid chromatography (HPLC) and capillary gas chromatographic (GC) techniques are explored. Derivatization methods using trifluoroacetyl- and silyl-derivatives are investigated for use with capillary GC. The results of the GC investigations are found to be only partially successful. Ion-pair HPLC is determined to be the optimal technique, utilizing pentanesulfonic acid as the counter ion to the amine group of beta-(alpha-methylbenzyl) amino isobutyric acid.     

Application of amino acid analysis by high-performance liquid chromatography with phenyl isothiocyanate derivatization to the rapid determination of free amino acids in biological samples.

An amino acid analysis by reversed-phase high-performance liquid chromatography after precolumn derivatization with phenyl isothiocyanate was adapted to the determination of free amino acids in plasma or other biological fluids and in tissue homogenates. Preparation of samples included deproteinization by 3% sulphosalicylic acid, and careful removal under high vacuum of residual phenyl isothiocyanate after derivatization. A Waters Pico-Tag column (15 cm long) was used, immersed in a water-bath at 38 degrees C. In rat or human plasma, separation of 23 individual amino acids, plus the unresolved pair tryptophan and ornithine, was obtained within 13 min. Including the time for column washing and re-equilibration, samples could be chromatographed at 23-min intervals. Variability was tested for each amino acid by calculating the coefficients of variation of retention times (less than 1% in the average) and peak areas (less than 4% for both intra-day and inter-day determinations). The linearity for each standard amino acid was remarkable over the concentration range 3-50 nmol/ml. The mean recovery of amino acid standards added to plasma prior to derivatization was 97 +/- 0.8%, except for aspartate (82%) and glutamate (81%). This method is rapid (almost three samples per hour can be analysed, more than in any other reported technique), with satisfactory precision, sensitivity and reproducibility. Therefore, it is well suited for routine analysis of free amino acids in both clinical and research work.     

Post-column continuous-flow analysis combined with reversed-phase liquid chromatography and computer-aided photodiode-array detection for the characterization of phloroglucinols by second-derivative difference spectroscopy

Flow-injection analysis coupled with liquid chromatography (LC) and a photodiode-array detector was applied to the investigation of phloroglucinol derivatives (kosins) from Hagenia abyssinica. Because of the structural similarities of the kosins, it was not possible to distinguish between the individual components in the crude mixture by their spectra alone. Post-column pH changes with various reagents, added using a flow-injection system, induced a bathochromic shift of the UV absorption maxima and these were found to furnish additional qualitative information. Addition of 0.3 M potassium hydroxide induced a smaller red shift in the spectrum for α-kosin compared with the shifts for kosotoxin and protokosin. This was confirmed in the normalized difference spectra, ΔA(λ), computed for each kosin by subtracting the normalized spectrum (in eluent at pH 6) from the normalized pH-shifted spectrum (at pH 12). The small pH-induced difference in ΔA(λ) was enhanced in the second-derivative transformation of the difference spectrum. This novel technique for solute recognition permits closely similar pH-sensitive spectra to be discriminated and characterized.     

Analysis of anthranilic acid by liquid chromatography

Analytical methods for the assay of anthranilic acid and for determination of the impurities methyl anthranilate, anthranoylanthranilic acid and 3- and 4-aminobenzoic acid are described. A Microbondapak C18 column is used for both the assay and the impurity determination. The assay is based on isocratic development with a mobile phase of 35:65 v v methanol/pH-3 phosphate buffer, with benzoic acid as internal standard. The impurities are separated by gradient elution. The standard deviation of the assay method is about 1% and the limit of detection for the impurities is about 0.01%.