Separation of p-Aminobenzoic Acid Derivatives

According to the literature, p-aminobenzoic acid (PAB) is excreted in the urine mostly as the free acid and its N-acetyl derivative; the remaining part is eliminated conjugated with glucuronic acid and as p-aminohippuric acid (PAH) (Ellinger & Hensel, 1914'; Hensel, 1914; Cohen & McGilvery, 1946; Salassa, Bollman & Dry, 1948). There is no agreement concerning the formation of PAH. Thus, Beyer, Mattis, Patch & Russo (1945) attach little importance to PAH formation, and according to Quick (1926, 1932 a, b) PAH formation does not even occur. The technical difficulties encountered on attempting simultaneous demonstration ofPAH and PAB probably explain these divergent results, as the methods at present in general use are based solely upon the reactions of the aromatic amino group. It is important for a number of reasons to have available a method permitting simultaneous determination ofPAH and PAB. Thus, Schreiner, Wesson & Anslow (1949) reported that one ofthe widely used PAH preparations was contaminated with 23% of PAB. Lundquist (1945) and Halmagyi & Kelemen (1949) reported that the renal clearance of PAB, provided that the level in the plasma is low and urine flow is high, yields values close to renal blood flow. The question arose whether these results were to be attributed to PAB itself or to its possible metabolic conversion to PAH. The methods used were in each case incapable of deciding this question. Furthermore, the simultaneous determination may be of some importance in investigation of the role ofPAB and p-aminosalicylic acid (PAS) in the metabolism of Mycobacterium tuberculosis (Landy, Larkum & Oswald, 1943; Bird, 1947; Lutz, 1949). Numerous other problems, which need not be enumerated here, could be attacked, using a method of this kind. Ekman (1948) chromatographed a number of primary aromatic amines on filter paper with various organic solvents, using diazotization and N-ethyl-l-naphthylamine hydrochloride for colouring. Tabone, Robert & Troestler (1948) and Tabone & Robert (1949) separated o-, mandp-aminobenzoic acids and kynurenine by paper chromatography using a method similar to our own, but found nbutanol inconvenient for their purpose. In what follows we describe the use of filter-paper chromatography for attacking problems such as those outlined above. METHODS