Determination of N-(Phosphonacetyl)-L-aspartic acid (PALA) in dog serum and urine by gas chromatography and selected lon monitoring.

Methods are described for quantitating N-(phosphonacetyl)L-aspartic acid (PALA), a new antitumor agent presently undergoing clinical evaluation. These methods were developed to measure this compound in serum by gas chromatography using the selectivity of a nitrogen-phosphorus detector and in serum and urine by the gas chromatographic/mass spectrometric technique of selected ion monitoring. PALA and the internal standard, N-(phosphonacetyl)-L-glutamic acid (HOMOPALA), were analyzed as their tetramethyl derivatives. Extraction from the biologic fluids was achieved by first precipitating the protein with acetone and then drying the serum with subsequent washes of acetone and diethyl ether. PALA and HOMOPALA were than extracted from the dried serum into an anhydrous HCL/methanol/tetrahydrofuran solution. After removing the solvent, the extract was reacted with diazomethane to form the tetramethyl derivatives. The feasibility of the method was demonstrated in the dog by comparing the total radioactivity with PALA equivalents determined in blood and urine samples following iv administration of PALA-14C. The range of the analysis was 20.0--200 microgram/ml for the urine samples and 0.5--15 microgram/ml for the serum samples.