A novel ion-pairing chromatographic method for the simultaneous determination of both nicarbazin components in feed additives: Chemometric tools for improving the optimization and validation

DE ZAN, MERCEDES; TEGLIA, CARLA; ROBLES, JC; GOICOECHEA, H C

TALANTA

ELSEVIER SCIENCE BV

Año: 2011 vol. 85 p. 142 - 150

0039-9140

The development, optimization and validation of an ion-pairing high performance liquid chromatographymethod for the simultaneous determination of both nicarbazin (NIC) components: 4,4-dinitrocarbanilide(DNC) and 2-hydroxy-4,6-dimethylpyrimidine (HDP) in bulk materials and feed additives are described.An experimental design was used for the optimization of the chromatographic system. Four variables,including mobile phase composition and oven temperature, were analyzed through a central compositedesign exploring their contribution to analyte separation. Five responses: peak resolutions, HDP capacityfactor, HDP tailing and analysis time, were modelled by using the response surface methodologyand were optimized simultaneously by implementing the desirability function. The optimum conditionsresulted in a mobile phase consisting of 10.0mmolL−1 of 1-heptanesulfonate, 20.0mmolL−1 ofsodium acetate, pH= 3.30 buffer and acetonitrile in a gradient system at a flow rate of 1.00mLmin−1. Columnwas an INERSTIL ODS-3 (4.6mm×150mm, 5mparticle size) at 40.0 ◦C. Detection was performedat 300nm by a diode array detector. The validation results of the method indicated a high selectivityand good precision characteristics, with RSD less than 1.0% for both components, both in intra andinter-assay precision studies. Linearity was proved for a range of 32.0–50.0gmL−1 of NIC in samplesolution. The recovery, studied at three different fortification levels, varied from 98.0 to 101.4 for HDPand from 99.1 to 100.2 for DNC. The applicability of the method was demonstrated by determining DNCand HDP content in raw materials and commercial formulations used for coccidiosis prevention. Assaysresults on real samples showed that considerable differences in molecular ratio DNC:HDP exist amongthem.