Manufacturing Process
Step 1). A 100 L quantity of urine is adjusted by the addition of acid
(hydrochloric acid is preferred but not essential) to a pH of 4 and extracted
with a suitable solvent such as n-butyl alcohol, benzol, chloroform or ether in
a continuous extraction apparatus. By using the countercurrent principle we
find that this volume of urine may readily be extracted during one day's time
and the active fraction transferred completely to a 4 L volume of butyl alcohol.
This alcoholic solution is chilled and filtered from salts and other insoluble
matter.
Step 2). The butyl alcohol extract is distilled to dryness in vacuum and the
brown tarry residue (300 to 600 g). Residue is extracted with benzol using
successive volumes of 1.5, 1.0, and 0.75 L of hot benzol, which treatment
dissolves the active principle.
Step 3). The benzol solution is then chilled, poured from the insoluble matter
and distilled using a vacuum to complete removal of benzol. The residue from
the distillation is treated with e 200 mL of butyl alcohol to which solution or
suspension 4 L of petroleum ether (boiling point 60-80°C) are added. The
resultant solution and suspension then extracted five to eight times with 800
mL of water to each portion of which sufficient 10% NaOH is added to
maintain a reaction alkaline to phenolphthalein. In this manner the hormone
is transferred to the alkaline aqueous solution. This solution is chilled to 2°C
for a day and poured from tarry material which separates. Subsequent
purification of the hormone is based upon the fact that it possesses a
sufficient acidic property so that it can be removed from certain organic
solvents by means of alkali, and that it can be removed in turn from alkaline solutions by successive extractions with organic solvents.
Step 4). The slightly alkaline aqueous solution is extracted five times with
successive (800-1200 mL) portions of ether (peroxide-free). This combined
ether extract is then distilled and the active residue treated first with 80 mL
butyl alcohol and then with 1500 mL of petroleum ether as in Step 3.
Step 5). The petroleum ether solution is then extracted 4 to 6 times with 300
mL portions of dilute NaOH solution and filtered. The alkaline filtrate is then
extracted six times with 400 mL portions of sulfuric ether, thus again
transferring the hormone to ether solution. Up to this stage usually 60-75% of
the total activity is accounted for. For example in a typical experiment the
original crude material contained 300.000 units and the assay of the ether
solution obtained at the end of Step 5, assayed fully 200.000 units. In the
subsequent steps, however, a considerable amount of scattering of the active
material occurs and hence all by-products are worked back into the process.
Step 6). The ether solution is distilled to dryness and yields a yellowish oil.
The oil is leached with 200-240 mL of cold 0.2 N NaOH solution, repeating the
extraction 4 or 5 times, and combining and filtering the alkaline extract. This
aqueous alkaline solution is then extracted with six successive portions of
sulfuric ether using about 300 mL of peroxide-free ether extract.
Step 7). The ether solution resulting from Step 6 is distilled and the residue
crystallized from 25% aqueous ethyl alcohol or from 25% aqueous acetone.
As an alternative method of procedure, the following may be substituted for
Steps 4 to 7 inclusive of the above process. After distilling the benzol, the
tarry mass may be stirred directly with 2000 mL of hot 0.3 N NaOH with a
mechanical stirrer. The suspension is chilled and the supernatant Liquid
poured or siphoned off. Repetition of the extraction two or three times is
advisable. The alkaline aqueous solution is then extracted five or six times
with 400 mL portions of sulfuric ether, thus transferring the hormone to ether
solution. After distillation of the ether the residue is steam distilled as long as
a distillate other than water is obtained. The condensed water is removed by
vacuum distillation and the small amount of dark tarry residue leached 5
times with 50 mL of hot 0.3 N NaOH. This solution is filtered and the filtrate
extracted with sulfuric ether (100 mL, 6 times). The ether solution is distilled
and the residue leached with cold 0.3 N NaOH using 20 mL five times. This
alkaline solution is filtered and extracted with 50 mL of sulfuric ether five
times. Upon distillation of the ether and solution of the residue in a small
quantity of hot ethyl alcohol, the hormone separates in semi-crystalline balls
which may be filtered off. A further quantity is obtained by adding 3 volumes
of water to the alcoholic solution. It may be recrystallized from 25% aqueous
ethyl alcohol or from 25% aqueous acetone or from any of the following:
chloroform, benzol, ethyl acetate, ethyl ether or petroleum ether. The final
product consists of colorless crystals which, when crystallized from dilute
alcohol, possess a distinct rhomboid outline. The crystals melt at 242-243°C
(248-249°C corrected) with some decomposition.
Hormone was used with piperazine (1:1).
