Sample preparation consisted of a protein precipitation step performed by adding 15 ¦ÌL of trifluoroacetic acid to 100 ¦ÌL of serum/plasma. Following vortexing and centrifugation, an aliquot of the supernatant was injected onto a polymeric PLRP-S column (250 mm ¡Á 4.6 mm i.d., dp: 8 ¦Ìm, 100 ?), maintained at 30 ¡ãC. The mobile phase consisted of water (A) and methanol (B) and a gradient elution (flow-rate: 1.0 mL min?, 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 ¦Ìg mL?) and endo-iohexol (0.62-93.0 ¦Ìg mL?). 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 ¦Ìg mL? for exo-iohexol and 0.62 and 0.20 ¦Ìg mL? 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.