4-Methylhippuric acid

A high-performance liquid chromatographic assay for the simultaneous analysis of hippuric and methylhippuric acids in urine is described. Compounds are first extracted from acidified urines after addition of o-methylbenzoylalanine (internal standard), using methyl-t-butyl ether. The organic phase is evaporated under nitrogen flow and the residue dissolved in the mobile phase which consisted of 91% potassium phosphate buffer (12.0mM, pH: 2.0), 4.5% methanol, and 4.5% tetrahydrofuran. The method described allows complete separation of meta- and para-methylhippuric acids in less than 20 min using a stainless steel column packed with octadecyl-dimethysilyl silica. The authors used this method to determine the urinary excretion of hippuric and methylhippuric acids in rabbits following exposure to a mixture of toluene and isomers of xylene. Data indicated that under the actual experimental conditions of exposure o-xylene is excreted to a lesser extent as a methylhippuric acid than the meta- and para-xylenes.

Hippuric acid (HA) and 4-methylhippuric acid (4-MHA) are metabolites as well as biological indicators for toluene and xylenes, respectively, and their determination in urine samples is very important, in order to monitor the occupational exposition to these solvents, ensuring a safe working environment. Thus, this paper describes the synthesis and characterization of a probe impregnated with molecularly imprinted polymers (MIPs) for the solid-phase extraction of HA and 4-MHA directly from untreated urine samples followed by micellar electrokinetic chromatography (MEKC) analyses. The MIP probe selectivity was compared to the non-imprinted polymer probe. The MEKC separations were carried out in 50 mmol/L sodium tetraborate pH 10.0/0.5 mmol/L cetyltrimethylammonium bromide aqueous solution, with a constant voltage of -15 kV. The system variables were optimized to provide ideal conditions for the extraction and desorption of the analytes, as well as for the MEKC analyses. The method was linear from 0.5 to 5.0 g/L for both analytes, with correlation coefficients > 0.994. Precisions and accuracies, expressed as relative standard deviation and relative error, were < 20.0 and within -15.4 to 16.6%, respectively, in accordance with the United States Food and Drug Administration recommendation. The MIP probe has proven to be simple, cheap, resistant, and synthetically reproducible, being successfully used to analyze both HA and 4-MHA from real samples.

4-Methylhippuric acid {systematic name: 2-[(4-methylbenzoyl)amino]ethanoic acid}, a p-xylene excreted metabolite with a backbone containing three rotatable bonds (R-bonds), is likely to produce more than one stable molecular structure in the solid state. In this work, we prepared polymorph I by slow solvent evaporation (plates with Z' = 1) and polymorph II by mechanical grinding (plates with Z' = 2). Potential energy surface (PES) analysis, rotating the molecule about the C-C-N-C torsion angle, shows four conformational energy basins. The second basin, with torsion angles near -73°, agree with the conformations adopted by polymorph I and molecules A of polymorph II, and the third basin at 57° matched molecules B of polymorph II. The energy barrier between these basins is 27.5 kJ mol-1. Superposition of the molecules of polymorphs I and II rendered a maximum r.m.s. deviation of 0.398 Å. Polymorphs I and II are therefore true conformational polymorphs. The crystal packing of polymorph I consists of C(5) chains linked by N-H...O interactions along the a axis and C(7) chains linked by O-H...O interactions along the b axis. In polymorph II, two molecules (A with A or B with B) are connected by two acid-amide O-H...O interactions rendering R22(14) centrosymmetric dimers. These dimers alternate to pile up along the b axis linked by N-H...O interactions. A Hirshfeld surface analysis localized weaker noncovalent interactions, C-H...O and C-H...π, with contact distances close to the sum of the van der Waals radii. Electron density at a local level using the Quantum Theory of Atoms in Molecules (QTAIM) and the Electron Localization Function (ELF), or a semi-local level using noncovalent interactions, was used to rank interactions. Strong closed shell interactions in classical O-H...O and N-H...O hydrogen bonds have electron density highly localized on bond critical points. Weaker delocalized electron density is seen around the p-methylphenyl rings associated with dispersive C-H...π and H...H interactions.