Quantitative determination of exo- and endo-iohexol in canine and feline samples using high performance liquid chromatography with ultraviolet detection

Abstract

A sensitive and specific high performance liquid chromatography–ultraviolet detection (HPLC–UV) method for quantitative determination of exo- and endo-iohexol in cat and dog serum/plasma is presented. Sample preparation consisted of a protein precipitation step performed by adding 15 μL of trifluoroacetic acid to 100 μL of serum/plasma. Following vortexing and centrifugation, an aliquot of the supernatant was injected onto a polymeric PLRP-S column (250 mm × 4.6 mm i.d., dp: 8 μm, 100 Å), maintained at 30 °C. The mobile phase consisted of water (A) and methanol (B) and a gradient elution (flow-rate: 1.0 mL min −1, total run-time: 21 min). The UV detector was set at a wavelength of 254 nm. Matrix-matched calibration graphs were prepared for both exo- (0.44–657 μg mL −1) and endo-iohexol (0.62–93.0 μg mL −1). Correlation and goodness-of-fit coefficients were between 0.9985–0.9999 and 4.44–9.87%, respectively. Limits of quantification and detection were 0.44 and 0.15 μg mL −1 for exo-iohexol and 0.62 and 0.20 μg mL −1 for endo-iohexol, respectively. Results for within-day and between-day precision and accuracy fell within the ranges specified. The reported method is simple and cost-effective. It has been successfully used for the analysis of exo- and endo-iohexol in serum/plasma samples of cats and dogs as part of pharmacokinetic studies with iohexol in order to determine plasma clearance of exo- and endo-iohexol. This indicates the usefulness of the developed method for application in the field of veterinary clinical practice and research.