Method 1: Extraction Method (The method currently employed in my country; it yields lower purity but offers lower production costs.)
Extraction of Oxytocin Solution: Take 100 g of dried posterior pituitary powder and 30 g of quartz powder, place them into a ball mill, and add distilled water. Perform the extraction four times using this same procedure. The volumes of distilled water added for each step are as follows: 1.4 L for the first two extractions, and 1.3 L for the subsequent two. Each extraction is carried out for 45 minutes, followed by centrifugation to collect the liquid; the remaining residue is then subjected to further extraction. The four resulting extracts are combined to yield the oxytocin solution. In summary: Dried Posterior Pituitary Powder [Water, Ball Mill] → Oxytocin Extract → Chromatographic Separation.
Take 1,500 g of pre-treated synthetic zeolite, add 20 L of a 0.25% acetic acid solution, and-after stirring-pour the mixture into an ion-exchange column. Once the acetic acid solution level has dropped to a point 2–3 cm above the surface of the zeolite bed, immediately introduce the oxytocin extract. Maintain an appropriate flow rate and collect the resulting white, turbid eluate (this fraction primarily contains oxytocin; since oxytocin has an isoelectric point [pI] of 7.7-whereas vasopressin has a pI of 10.9-vasopressin readily forms positive ions and is thus adsorbed onto the column). As soon as the liquid level of the extract drops to the surface of the zeolite bed, immediately add distilled water and continue collecting the turbid eluate until the flow ceases. Adjust the pH of the collected turbid eluate to 3.5 using glacial acetic acid. Rapidly heat the solution in a water bath to 95°C, maintain this temperature for 3 minutes, then rapidly cool the solution and refrigerate it overnight. In summary: Oxytocin Extract [HAc, Synthetic Zeolite Column] → Turbid Eluate [HAc, Heating] → Separated Solution → Adsorption & Elution. On the following day, filter the refrigerated solution. To the filtrate, add a 10% bentonite slurry while stirring (adding 3 mL of slurry per 100 mL of filtrate). Continue stirring for 1 hour to facilitate adsorption, then centrifuge the mixture. Test the supernatant liquid using a sulfosalicylic acid reagent solution; the absence of any precipitate indicates that the adsorption process is complete. If the adsorption is incomplete, repeat the bentonite treatment process; subsequently, combine the bentonite precipitates obtained from both adsorption steps. The bentonite is eluted four times using 1% acetic acid (with volumes of 1.6 L, 1.4 L, 1.2 L, and 0.8 L, respectively). For each eluate, *tert*-butyltrichloride is added once the solution is heated to 80°C; the temperature is then raised to 95°C, rapidly cooled to 25°C, and either filtered or centrifuged. The four resulting filtrates are combined to determine the potency and vasopressin limit. The process flow is as follows: Refrigerated Solution [Filtration] → Filtrate [Bentonite Slurry] → Adsorbate [1% HAc] → Eluate. Preparation of Oxytocin Injection:
The assayed oxytocin solution, having met quality standards, is diluted to a concentration of 5 U/mL or 10 U/mL. It is then filtered through a No. 4 or No. 5 sintered glass funnel, filled into ampoules, and sterilized using flowing steam at 100°C for 30 minutes to yield the final product. The process flow is as follows: Oxytocin Solution (Assay Approved) [Filtration, Filling, Sterilization] → Oxytocin Injection.
Regeneration of Synthetic Zeolite: The synthetic zeolite is washed twice with distilled water. Then, 1.5 L of water (calculated based on 500 g of zeolite) and 150 g of sodium chloride are added, and the mixture is stirred for 2 hours to wash away and remove vasopressin. The zeolite is then washed with distilled water until no chloride ions remain. An appropriate amount of water is added, and the pH is adjusted to 9 using sodium hydroxide; the mixture is stirred for 30 minutes, after which the supernatant is decanted. The zeolite is washed with distilled water, the pH is adjusted to 5.5–6 using sulfuric acid, and it is washed again with water until nearly neutral. It is then filtered to remove excess water and dried at 105°C for future use. Before use, an appropriate amount of water is added, and the pH is adjusted using glacial acetic acid while stirring to allow for soaking. Preparation of 10% Bentonite Slurry: Bentonite and water are mixed at a mass-to-volume ratio of 1:10. The mixture is ground for approximately 1 hour, during which time acetic acid is added to maintain a stable pH of 3.5. The resulting slurry is then stored in a refrigerator for future use. Method 2: Chemical Synthesis. First, the heptapeptide amide (segments 3–9) is synthesized starting from benzyloxycarbonyl-leucine *p*-nitrophenyl ester; next, benzyloxycarbonyl-S-benzylcysteine-tyrosine azide (segments 1–2) is synthesized; and in the third step, oxytocin is synthesized.