One-pot determination of amino acids in drugs by pre-column derivatization with phenyl isothiocyanate

Objectives. To develop a new method to determine amino acids in drugs by means of reverse-phase high-performance chromatography (RP HPLC) with pre-column derivatization using phenyl isothiocyanate (PITC) and one-pot sample preparation.

Methods. The initial standard solutions of amino acids were prepared by weighing, followed by dissolution in water. Working solutions were then prepared: standard, test, and blank, by dilution in 20 mM hydrochloric acid. Further sample preparation was carried out in Safe-lock polypropylene tubes (Eppendorf) in a reaction buff containing mobile phase A, acetonitrile, and triethylamine in a ratio of 85 : 10 : 5, labeled with a 5% PITC solution in acetonitrile. After thorough mixing for 3–5 min on a vortex, the tubes were kept in a solid-state thermostat with a thermally insulating lid for 2 h. The samples were then cooled for 10 min, centrifuged for 1 min at 13000 rpm, the supernatant was transferred into vials, and the mixture of amino acids was separated by RP HPLC using hydrophobic silica gel with grafted C18 groups as a stationary phase. The quantitative determination of amino acid derivatives was carried out using a diode array detector.

Results. A new method for the separation and determination of amino acids in medicinal preparations was developed and validated. Simple one-pot sample preparation using available reagents and equipment enabled studies to be carried out without using commercial kits, for example, the AccQ·Tag Ultra Derivatization Kit, USA. Using the analysis of mixtures of histidine and glycine as an example, it was shown that when using two mobile phases, an acceptable separation of amino acid derivatives in a gradient mode can be achieved for 20 min at a flow rate of 1.0 mL/min. The samples prepared according to the new method demonstrated a high level of stability in use and storage. A composition of mobile phases A and B consisting of 10 mM acetate buffer pH 3.5 and 80% acetonitrile solution was proposed. Validation of the method hereby developed in the analysis of the drug Innonafactor®, containing glycine and histidine as excipients, demonstrated high convergence of the results of the quantitative determination of these amino acids.

Conclusions. The new method to determine amino acids in medicinal preparations by RP HPLC with PITC pre-column derivatization has a wide range of applications, has a number of advantages when compared to imported commercial kits for the determination of amino acids. These include: lower cost of reagents and materials, high accuracy and repeatability. Thus, it can be recommended for use in quality control laboratories of pharmaceutical enterprises.

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