ABSTRACT
Ornipressin is a peptide drug which is usually assayed by a test on live rats. In order to reduce the animal experiments an alternative method was developed which uses gradient high-performance liquid chromatography (HPLC) on reversed-phase. The HPLC method was validated and shown to be selective and precise. Correlation studies were performed on samples of different dosage strengths and on thermally degraded samples, showing good correlation with the results obtained by the biological assay. The HPLC method was applied on various batches of ornipressin in bulk and in pharmaceutical preparations. HPLC is a rapid and inexpensive method which can replace the animal assay. A new quality control concept is proposed which uses HPLC for the analysis of ornipressin in bulk and in pharmaceutical preparations. With this concept animal testing can be reduced by 90%.
Subject(s)
Animal Testing Alternatives , Ornipressin/analysis , Amino Acid Sequence , Animals , Biological Assay , Chromatography, High Pressure Liquid , Molecular Sequence Data , Ornipressin/pharmacology , Peptides/analysis , Peptides/pharmacology , Quality Control , Reference StandardsABSTRACT
A high-performance liquid chromatographic (HPLC) method is described for the assay of salmon calcitonin. The method uses a 5-microns octadecasilyl silica column (100 x 4.6 mm) at 50 degrees C and an initial mobile phase (flow rate 1 ml min-1) comprising 35% of B (1 M tetramethylammonium hydroxide-water-acetonitrile, 8:392:600) and 65% of A (1 M tetramethylammonium hydroxide-water-acetonitrile, 20:880:100) with linear gradient elution over 21 min to a final mobile phase of 57% B; solutions A and B are adjusted to pH 2.5 with phosphoric acid. Detection was by UV spectrophotometry at 210 nm. The method has been shown to be selective, precise and rapid and, in a collaborative study to give excellent correlation with the results obtained by using the biological assay method of the European Pharmacopoeia. The method, which has been applied successfully to the assay of different batches of salmon calcitonin in bulk drug and in formulated products, is recommended for adoption as the pharmacopoeial assay method.
Subject(s)
Calcitonin/analysis , Amino Acid Sequence , Animals , Biological Assay , Chickens , Chromatography, High Pressure Liquid , Eels , Humans , Molecular Sequence Data , Reference Standards , Species Specificity , SwineABSTRACT
Optimized C18 reversed-phase systems for oxytocin, desamino-oxytocin, lysine-vasopressin, ornithine-vasopressin and felypressin with gradient elution are discussed, focussing on precision, selectivity and ruggedness of the methods. Data from collaborative studies are presented, demonstrating the equivalence of high-performance liquid chromatography (HPLC) assays to bioassays. The findings suggest that HPLC is an excellent alternative to the time-consuming and less reliable animal testing.
