p-Toluene-sulfonic acid, cholesterol determination, and explosions.

HAVE BEEN USING the method of Pearson, Stern, and McGavack (1) for the determination of cholesterol for some time. It has proved to be a convenient and accurate procedure and the reaction had always gone smoothly. One day recently, however, the tubes were set up for the determination as usual, using a freshly prepared solution of p-toluenesulfonic acid. Solutions made from the same bottle of solid acidt had previously been used with smooth results. However, on this occasion, a minute or two after the serum, acetic acid, p-toluenesulfonic acid solution, and acetic anhydride were measured into the tubes and not mixed, a series of explosions occurred and most of the contents was blown out of the tubes. The serum blank and its zero reference tube, not containing acetic anhydride, did not blow up. A fresh solution of p-toluenesulfonic acid was prepared from the same solid used above, with a repeat performance of the explosions. A new solution made from a different batch of solid2 gave smooth results. Unfortunately the offending p-toluenesulfonic acid was discarded before it could be analyzed to determine the change which caused the trouble. On cursory examination the bottle of solid reagent had appeared somewhat moist, particularly on the inside of the screwcap. This note is not intended to discourage use of the Pearson, Stern, and McGavack method, which is still our method of choice for determining total cholesterol, but merely to advise caution in keeping away from above the tubes after the reagents are added, when a new solution of p-toluenesulfonic acid is first used. It might be well to set the rack of tubes under a shelf or to cover them with a board immediately after the components are pipeted in, when an unproved solution is used.