Development and validation of an ultra-high performance liquid chromatography–tandem mass spectrometry method for the simultaneous determination of iohexol, p-aminohippuric acid and creatinine in porcine and broiler chicken plasma

Abstract

In order to study the renal function, in terms of glomerular filtration and effective renal plasma flow, in broiler chickens and pigs, an ultra-high performance liquid chromatography–tandem mass spectrometry method for the simultaneous determination of iohexol, p-aminohippuric acid (PAH) and exogenously administered creatinine in plasma was developed and validated. Sample preparation consisted of a deproteinization step using methanol for porcine plasma and an Ostro™ Protein Precipitation & Phospholipid Removal Plate was used for broiler chicken plasma. Chromatographic separation was achieved on a Hypersil Gold aQ column using 0.1% formic acid in water and 0.1% formic acid in methanol as mobile phases. The total run time was limited to 10 min. Matrix-matched calibration curves for iohexol and PAH were prepared and good linearity (r ≥ 0.9973; gof ≤ 6.17%) was achieved over the concentration range tested (0.25–90 μg/mL). Limits of quantification were 0.25 μg/mL for iohexol and PAH. Water was used as surrogate matrix for analysis of creatinine in plasma. This surrogate calibration curve showed good linearity over the concentration range tested (0.25–90 μg/mL) (r ≥ 0.9979; gof ≤ 5.66%). For creatinine, the relative lower limit of quantification was 201.03 ± 49.20% and 60.14 ± 7.64% for chicken and porcine plasma, respectively. The results for within-day and between-day precision and accuracy fell within the specified ranges.This straightforward, cost-effective and rapid method, determining iohexol, PAH and creatinine within one single chromatographic run, has been successfully used for the analysis in porcine and broiler chicken plasma samples in order to determine the renal function of these species.