New Method for Determination of Alkylating Agents in Biologic Fluids.

An accurate, reproducible colorimetric method for determining minute amounts of alkylating agents in biologic fluids has been developed. Utilizing this method, we have been able to detect less than 0.1 g. of nitrogen mustard (HN2), or Thio-TEPA in 1 ml. of plasma, lymph, urine, or tissue homogenate. The biologic fluid was deproteinized with zinc sulfate, barium hydroxide, and methanol; the supernatant was used to alkylate y-(4-nitrobenzyl) pyridine (NBP) under optimum conditions. The color of the quaternary pyridinium NBP derivative was extracted into ethyl acetate and read on a Beckman DU spectrophotometer utilizing microcells.