ABSTRACT
High-performance liquid chromatographic (HPLC) and capillary electrophoretic (CE) methods have been developed for the separation of 22 related peptides (potential and real impurities) from a new immunostimulant tetrapeptide derivative (thymocartin, Arg-Lys-Asp-Val) being under clinical examination. The described methods are suitable for the purity control of the bulk drug material. In the course of the reversed-phase ion-pair HPLC separations C-18 columns (Hypersil BDS and Ultrasphere IP) were used. In the case of using sodium hexanesulphonate as the ion-pairing reagent, the eluent contained phosphate buffer (pH = 3) and 32% v/v methanol, while in another method a gradient system with a sodium perchlorate solution (pH = 2 set by perchloric acid) and acetonitrile was applied. The optimum separation in the CE investigations was achieved at pH = 3 using triethylammonium phosphate buffer and 10% v/v acetonitrile as the organic modifier.
Subject(s)
Adjuvants, Immunologic/isolation & purification , Oligopeptides/isolation & purification , Peptide Fragments/isolation & purification , Thymopoietins/isolation & purification , Amino Acid Sequence , Chromatography, High Pressure Liquid/methods , Electrophoresis, Capillary/methods , Structure-Activity RelationshipABSTRACT
The following methods are described for the analytical investigation of pipecuronium bromide. 1. HPLC method. Of the several systems tried for the separation and quantification of impurities and degradation products the best results were obtained using silica as the stationary phase and 43:43:14 mixture of methanol, acetonitrile and concentrated aqueous ammonia containing 0.1 mole/l each of ammonium chloride and ammonium carbonate as the eluent. The validation of this method is presented. The above described aggressive eluent can be successfully replaced by an ion-pairing system using silica as the stationary phase and 96:4 mixture of acetonitrile and water containing 0.1 mole/l sodium perchlorate as the eluent. 2. Thin-layer chromatography. TLC systems are described for the separation and densitometric quantification of the impurities and degradation products of pipecuronium bromide. 3. Spectrophotometry. Two methods are described. The ester groups of the molecule can be determined by the iron(III)-hydroxamate method while for the ion-pair extraction of the quaternary ammonium steroid picric acid or bromthymol blue are used as the reagents. 4. Titrimetry. In addition to the titration with acetous perchloric acid for the assay of the bulk material a microtitration method is described for the determination of pipecuronium bromide in individual lyophylized ampoules (potentiometric titration with 0.1 M silver nitrate).
