A rapid hydrolysis method and DABS-Cl derivatization for complete amino acid analysis of octreotide acetate by reversed phase HPLC

Abstract

Octreotide as a synthetic cyclic octapeptide is a somatostatin analog with longer half-life and more selectivity for inhibition of the growth hormone. The acetate salt of octreotide is currently used for medical treatment of somatostatin-related disorders such as endocrine and carcinoid tumors, acromegaly, and gigantism. Octreotide contains both cysteine and tryptophan residues which make the hydrolysis part of its amino acid analysis procedure very challenging. The current paper introduces a fast and additive-free method which preserves tryptophan and cysteine residues during the hydrolysis. Using only 6 M HCl, this hydrolysis process is completed in 30 min at 150 °C. This fast hydrolysis method followed by pre-column derivatization of the released amino acids with 4-N,N-dimethylaminoazobenzene-4'-sulfonyl chloride (DABS-Cl) which takes only 20 min, makes it possible to do the complete amino acid analysis of an octreotide sample in a few hours. The highly stable-colored DABS-Cl derivatives can be detected in 436 nm in a reversed phase chromatographic system, which eliminates spectral interferences to a great extent. The amino acid analysis of octreotide acetate including hydrolysis, derivatization, and reversed phase HPLC determination was validated according to International Conference of Harmonization (ICH) guidelines.

Evaluation of the Hydrolysis Specificity of an Aminopeptidase from Bacillus licheniformis SWJS33 Using Synthetic Peptides and Soybean Protein Isolate

“…DABS-Cl Derivatization for FAA Analysis of Peptide Hydrolysates by Reversed Phase HPLC. The FAA from single peptide hydrolysates were determined by DABS-Cl derivatization based on Akhlaghi 20 with some modifications. Briefly, 200 μL sample and 200 μL derivatization solution (7.5 mM DABS-Cl, dissolved by acetonitrile) were added in a 2 mL vial.…”

Rapid and Accurate Screening of Lysine-Producing Edible Mushrooms via the Homocitrate Synthase Gene as a Universal Molecular Marker

“…Reagent interference, instability of derivatives, long preparation time, and difficulties in derivatization to specific amino acids are the disadvantages of an ordinary HPLC method . Therefore, reversed-phase high-performance liquid chromatography (HPLC) and high-throughput UPLC–MS/MS were established for the detection of amino acids with adequate separation and high sensitivity. , The use of LC–MS/MS eliminates the derivatization step and allows for overlapping amino acid retention times, thereby shortening the analysis time . However, no method has been developed to quantify amino acids rapidly and accurately with molecular biological techniques in edible mushrooms.…”

Development of specificl-methionine sensors by FRET-based protein engineering

“…3,4 Owing to their biochemical importance, much effort has been made for the analysis of AAs. [5][6][7][8][9][10][11][12][13][14][15] The most common analytical method is liquid chromatography coupled with chemical modication of AAs or mass spectrometry. [5][6][7][8] Alternatively, chemical and protein sensors based on UV/vis or uorescence spectroscopy have been developed for AA analysis.…”

“…[5][6][7][8][9][10][11][12][13][14][15] The most common analytical method is liquid chromatography coupled with chemical modication of AAs or mass spectrometry. [5][6][7][8] Alternatively, chemical and protein sensors based on UV/vis or uorescence spectroscopy have been developed for AA analysis. [9][10][11][12][13][14][15][16] Recently, array-based sensing has been developed for analyzing AAs, in which a series of sensors are used in combination with chemometric techniques such as principal components analysis (PCA) and linear discriminant analysis (LDA).…”