Simultaneous determination of creatinine, iohexol and p-aminohippuric acid in animal plasma by ultra-high-performance liquid chromatography–tandem mass spectrometry Laura Dhondt1, Siska Croubels1, Pieter De Cock 2,3,4, Peter De Paepe3, Mathias Devreese1 1 Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium ; 2 Department of Pharmacy, Ghent University Hospital, Ghent, Belgium; 3 Heymans Institute of Pharmacology, Ghent University, Ghent, Belgium ; 4 Department of Paediatric Intensive Care, Ghent University Hospital, Ghent, Belgium Introduction The kidneys have a pivotal role in the excretion of various endogenous as exogenous substances, such as medicinal products. This excretory function can be significantly altered in case of illness. To measure the glomerular filtration rate, iohexol and exogenous administered creatinine clearance can be used, whereas para-aminohippuric acid (PAH) clearance reflects the effective renal plasma flow. To date, no methods have been reported for the simultaneous determination of creatinine, iohexol and PAH. The aim of this study was to develop and validate a sensitive and specific ultra-high-performance liquid chromatography–tandem mass spectrometry method for simultaneous determination of these renal markers in porcine and avian plasma. Quantification of these renal markers will be used to evaluate the renal function of piglets and various avian species.

Analyte and internal standard (IS) Material and Methods Sample preparation Instrumentation and analysis 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, following a gradient elution. Flow rate was set at 300 μL/min. The MS/MS instrument (Quattro Premier XE) operated in the positive electrospray ionization mode. Acquisition was performed in the selected reaction monitoring mode (Table 1). Table 1. Reaction monitoring transitions (m/z) of the compounds Analyte and internal standard (IS) Quantification ion Identification ion Creatinine 113.8 → 44.1 113.8 → 86.0 Creatinine-d3 (IS) 117.2 → 47.4 117.2 → 89.4 PAH 195.1 → 119.9 195.1 → 91.9 Para-aminobenzoic acid (PABA, IS) 137.8 → 77.2 137.8 → 65.1 Iohexol 822.0 → 804.0 822.0 → 731.2 Iohexol-d5 (IS) 827.0→ 809.0 827.0 → 736.1

Results & Discussion (Part 1) The method was in-house validated compliant with European and international guidelines1,2. Matrix-matched calibration graphs, for both avian and porcine plasma, were prepared for all compounds. Since creatinine is an endogenous substance, also a standard calibration curve and quality control samples (QCs) in water were extracted and analysed. Extracted plasma QCs were quantified using the standard calibration curve and corrected for the endogenous quantity of creatinine. Figure 1. LC-MS/MS chromatogram of porcine plasma spiked at 10 µg/mL with (1) creatinine, (3) iohexol and (5) PAH, and their respective internal standards (2) creatinine-d3, (4) iohexol-d5 and (6) PABA 1VICH GL49: Guidance for Industry: Studies to Evaluate the Metabolism and Residue Kinetics of Veterinary Drugs in Food-Producing Animals: Validation of Analytical Methods used in Residue Depletion Studies (February 2011) 22002/657/EC: Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results (notified under document number C(2002) 3044)

Results & Discussion (part 2) Table 2. Evaluation of validation parameters in porcine (P) and avian (A) plasma Iohexol PAH Creatinine Linearity Accuracy & precision Carry-over Limit of quantification (ng/mL) 250 2501 Limit of detection (ng/mL) 84 (P) 32 (A) 3.8 (P) 9.0 (A) 13.71 Calibration curves for all compounds were linear (1/x2 weighing) over a range from 0.25 - 90 µg/mL. Correlation coefficient (r) and goodness-of-fit (g) were used to evaluate linearity. The within- and between-run precision and accuracy were evaluated and the results fell within the ranges specified by the EC (2002/657/EC). Conclusion 1theoretical value, determined in water In conclusion, a fast, accurate and precise method was developed for the simultaneous quantitative determination of iohexol, PAH and creatinine in porcine as well as avian plasma. This method will be further used to assess the renal function in piglets and different avian species. Acknowledgment Contact Laura.Dhondt@Ugent.be vetftb.ugent.be safepedrug.eu This study was funded by the special research fund of Ghent University (BOF16/DOC/285).