Optimization and validation of an ion-pair RP-HPLC-UV method for the determination of total free iodine in rabbit plasma: application to a pharmacokinetic study.

An ion-pair reverse-phase high performance liquid chromatographic method with UV-vis detection has been developed for the determination of total free iodine in rabbit plasma after vaginal administration of povidone-iodine (PVP-I). Sample preparation was done by protein precipitation with acetonitrile in 96-well format and aspirin was used as the internal standard. The 100 microL sodium thiosulfate solution (5 g L(-1)) was added to 100 microL plasma sample before protein precipitation, to convert the total free iodine in plasma to iodide (I(-)). Separation was performed on a C(18) column (200 x 4.6 mm i.d., 5 microm). The mobile phase consisting of a mixture of water phase (containing 10 mmol L(-1) 18-crown-6 ether, 5 mmol L(-1) octylamine and 5 mmol L(-1) sodium dihydrogen phosphate, pH adjusted to 6.0 with phosphoric acid) and acetonitrile in the ratio 70:30 (v/v) was delivered isocraticly at a flow rate of 1.0 mL min(-1). The method was sensitive with a lower limit of quantification of 0.005 microg mL(-1), with good linearity (r(2) > 0.9990) over the linear range of 0.005-2 microg mL(-1). All the validation data, such as linearity, accuracy and precision, were within the required limits. The method was successfully applied to study the pharmacokinetic of PVP-I in rabbits after vaginal administration.