Reversed-phase high-performance liquid chromatography of fertirelin acetate and related compounds.

Separation of fertirelin acetate (FA) from process impurities, potential degradation products and related peptides including luteinizing hormone releasing hormone has been achieved by reversed-phase high-performance liquid chromatography (HPLC). A number of chromatographic conditions (column type, mobile phase composition, isocratic/gradient elution) and detection systems have been utilized to examine the bulk drug and formulation of FA. Examples of separations designed for potency and impurity determinations are described. Complete recovery of FA is obtained with an isocratic HPLC system. An external standard method is used to determine potency with a precision of less than 1% R.S.D. A gradient HPLC system is used to determine impurities with a precision of ca. 5-10% R.S.D. at the 1-2% impurity level. As little as ca. 0.1% (area%) of related peptides are detected at 214 nm.

Reversed-phase high-performance liquid chromatography peptide mapping of bovine somatotropin.

Reversed-phase high-performance liquid chromatography peptide mapping techniques have been used to examine the primary structure of bovine somatotropin (bSt) isolated from Escherichia coli modified by recombinant DNA techniques (rbSt) and from bovine pituitary (pbSt). Peptide fragments arising from tryptic digestion of bSt were separated on Baker wide pore C4 or C8 columns with linear gradients (acetonitrile-water with 0.1% trifluoroacetic acid). Major peaks eluted in a consistent order, but significant day-to-day variation in retention times was observed. Isolated peptide fragments were analyzed via acid hydrolysis followed by formation and separation of the phenylthiocarbamyl amino acids. These correspond to expected tryptic fragments.

Reversed-phase high-performance liquid chromatographic assay for the determination of potency and impurities in tazadolene succinate bulk drug and capsules.

A reversed-phase assay based on high-performance liquid chromatography with a water-acetonitrile-tetrahydrofuran (THF)-triethylamine (TEA)-perchloric acid (pH 2.5) mobile phase and a Zorbax C8 column has been validated for the determination of the purity of tazadolene succinate (I) [E-(+/-)-1-(2-benzylidenecyclohexyl)azetidine succinate, U-53996H] bulk drug, the potency of tazadolene succinate hard-filled capsule formulations and impurity levels in bulk drug. The system resolves E- and Z-isomers and other structurally related molecules. Retention of these compounds is mainly dependent on the amount of acetonitrile and THF in the mobile phase. An amine must be present in the mobile phase to bring about elution of I. The potency assay utilizes testosterone as internal standard. Potency assays exhibited relative standard deviations (R.S.D.) of less than 1%. Quantitative recovery from hard-filled capsules (HFC) is obtained by using a simple extraction procedure. Potential process impurities, potential degradation products, and formulation excipients are resolved. The assay is linear for tazadolene succinate concentrations equivalent to 50-150% of the assay concentration. Impurities can be quantitated to levels equivalent to about 0.1% by weight with R.S.D. less than 5%. The estimated limit of detection for I is about 2 ng for a 20 microliters injection.

Equilibrium denaturation of pituitary- and recombinant-derived bovine growth hormone.

Holladay and co-workers [Holladay, L. A., Hammonds, R. G., & Puett, D. (1974) Biochemistry 13, 1653-1661] reported the presence of an equilibrium intermediate in the guanidine hydrochloride (GdnHCl) induced denaturation of pituitary-derived bovine growth hormone (p-bGH). Since then, numerous reports have appeared demonstrating the inherent heterogeneity in p-bGH. In this report we show that a standard preparation of p-bGH can be separated into two components of almost equal abundance differing in molecular weight by approximately 1000. Each of these two components could give rise to different denaturation transitions which would be interpreted as evidence for equilibrium intermediates. We report here the equilibrium denaturation of bGH produced by Escherichia coli through recombinant DNA technology. The recombinant-derived bGH (r-bGH) is more homogeneous than that derived from pituitary sources and is greater than 95% a single polypeptide entity. Nevertheless, the GdnHCl-induced denaturation profiles of both recombinant bGH and pituitary bGH are very similar. The presence of equilibrium intermediates is verified by the asymmetry of the denaturation transition as measured by size-exclusion high-performance liquid chromatography and by noncoincidence of the denaturation transitions as observed by ultraviolet absorbance, fluorescence intensity, and circular dichroism. These findings conclusively show that the secondary structure of bovine growth hormone is more stable than the tertiary structure and is consistent with a framework model of protein folding.