Abstract: Objective To establish HPLC methods for the determination of active components—vasopressin and oxytocin—in posterior pituitary and to investigate the correlation and equivalence between the newly developed methods and those specified in the pharmacopoeia. Methods For vasopressin, a C₁₈ column (4.6 mm×25 cm, 3.5 μm) was used. Mobile phase A consisted of 0.1 mol·L^-1 diammonium hydrogen phosphate (pH=6.0) while mobile phase B was composed of a 1∶1 mixture of acetonitrile and water. The flow rate was 1.0 mL·min^-1, detection wavelength 220 nm, and column temperature was 40°C. For oxytocin, a C₁₈ column (4.6 mm×25 cm, 5 μm) was adopted. Mobile phase A was 0.1 mol·L^-1 sodium dihydrogen phosphate solution (pH=4.5) while mobile phase B was a 1∶1 mixture of acetonitrile and water under the same conditions as vasopressin. Results Using either method, the resolution between the main peaks of vasopressin/oxytocin and adjacent impurity peaks met the required standard. A good linear relationship was observed between the concentration and chromatographic peak area: within the range of 0.21-13.33 IU·mL^-1 for vasopressin (r=0.999 9), and 0.26-21 IU·mL^-1 for oxytocin (r=0.999 9). The average recovery (n=9) was 100.3% and 99.8%, respectively. The RSD of repeatability precision was 0.09% and 0.44%, and that of reproducibility precision was 2.85% and 1.9%, respectively. Statistical analysis indicated good result equivalence and performance equivalence between the HPLC methods and the bioassay. Conclusion The established methods are user-friendly, accurate, reproducible, and equivalent to the bioassay, which can be used for determination of vasopressin and oxytocin contents in posterior pituitary.

Key words: Posterior Pituitary, Vasopressin, Oxytocin, Content Determination, Method Substitution, HPLC, Equivalence, Rats