Determination of para-aminohippuric acid (PAH) in human plasma and urine by liquid chromatography–tandem mass spectrometry

In this manuscript, we present a simple and reliable method for the quantitation of para-aminohippuric acid (PAH; 2-(4-aminobenzamido)acetic acid) in human plasma and urine using liquid chromatography coupled to electrospray ionisation low-energy collision-induced dissociation tandem mass spectrometry (HPLC–ESI-CID-MS/MS) analysis (negative ion mode) via multiple reaction monitoring (MRM). Sample preparation involved protein precipitation of plasma samples with acetonitrile and subsequent dilution of urine samples with the mobile phase. The internal standard (IS) adopted was para-aminosalicylic acid (PAS; 4-amino-2-hydroxybenzoic acid). Chromatographic separation was achieved on a Cosmosil HILIC column using an isocratic mobile phase consisting of ammonium acetate buffer (20 mM) and acetonitrile (45:55, v/v) at a flow rate of 200 μl/min. The transactions monitored were m/z 192.9 → 149.1 for PAH and m/z 152.1 → 108.1 for IS. Linear calibration curves were generated over the PAH concentration range of 0.2–100 mg/L in human plasma and urine. The method was validated for selectivity, accuracy, precision, recovery and stability according to USFDA criteria, and has been successfully applied to a pharmacokinetic study in healthy volunteers administered an intravenous dose of 440 mg PAH.