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 quater-nary pyridinium NBP derivative was extracted into ethyl acetate and read on a Beckman DU spectrophotometer utilizing microcells. A LKYLATJN(; AGENTS are used extensively in cancer chemotherapy. Bio-logic actions and clinical effects of these (lrugS have 1)eeli intensively in-vestigated. Serious toxicity is a comistant threat. The development of newer techiuics of administration, such as intraluminal, infusion, and perfusion, present further deniamids for a sensitive, rapid, afl(i reliable determinatiomi of these drugs in biologic fluids. The niost sensitive meth-0d! that has appeared imi the literature is the fluorimetric estimation de-veloped by Mellctt and Woods (1. 2). This procedure, how-ever, is very tedious alid, ill the extraction step, also presents a serious prol)lem due to the presence of protein colloid iii plasma. Furthermore, the required 10 ml. of whole blood (3 mI. of plasma) for each estiniation is- a large quantity to be taken from the animal or human for serial specimens. A colorimetric procedure for the estimation of alkylating agents in water amid nomuaqueous solvents with y-(4-miitrobenzyl) pyridine (NBP) has been established by Epstein (3). This method was later adapted by Klatt et al. (4). They succeeded in detecting 0.05 M of plienylalanin