A New Strategy To Stabilize Oxytocin in Aqueous Solutions: II. Suppression of Cysteine-Mediated Intermolecular Reactions by a Combination of Divalent Metal Ions and Citrate

A series of studies have been conducted to develop a heat-stable liquid oxytocin formulation. Oxytocin degradation products have been identified including citrate adducts formed in a formulation with citrate buffer. In a more recent study we have found that divalent metal salts in combination with citrate buffer strongly stabilize oxytocin in aqueous solutions (Avanti, C.; et al. AAPS J. 2011, 13, 284–290). The aim of the present investigation was to identify various degradation products of oxytocin in citrate-buffered solution after thermal stress at a temperature of 70 °C for 5 days and the changes in degradation pattern in the presence of divalent metal ions. Degradation products of oxytocin in the citrate buffer formulation with and without divalent metal ions were analyzed using liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS). In the presence of divalent metal ions, almost all degradation products, in particular citrate adduct, tri- and tetrasulfides, and dimers, were greatly reduced in intensity. No significant difference in the stabilizing effect was found among the divalent metal ions Ca2+, Mg2+, and Zn2+. The suppressed degradation products all involve the cysteine residues. We therefore postulate that cysteine-mediated intermolecular reactions are suppressed by complex formation of the divalent metal ion and citrate with oxytocin, thereby inhibiting the formation of citrate adducts and reactions of the cysteine thiol group in oxytocin.