Determination of 2,4-Dichlorophenoxyacetic acid (2,4-D) in rat serum for pharmacokinetic studies with a simple HPLC method

Abstract

2,4-Dichlorophenoxyacetic acid (2,4-D) is a chlorophenoxy herbicide used worldwide. We describe a high-performance liquid chromatography (HPLC) method with UV detection for the determination of 2,4-D in female and male rat serum. This allows to observe the change of serum 2,4-D concentration in rats with time and its pharmacokinetics characteristics with a simple, rapid, optimized and validated method. The serum samples are pretreated and introduced into the HPLC system. The analytes are separated in a XDB-C18 column with a mobile phase of acetonitrile (solvent A) and 0.02 M ammonium acetate (containing 0.1% formic acid) (solvent B) using a gradient elution at a flow rate of 1.0 mL/min. The wavelength for UV detection was set at 230 nm. Calibration curve for 2,4-D was constructed over a range of 0.1-400 mg/L. The method was successfully applied to study the pharmacokinetics of 2,4-D in rats in this study. After oral administration of 300 mg/kg and 60 mg/kg 2,4-D, the mean Cmax values were 601.9 and 218.4 mg/L, the AUC0→∞ values were 23,722 and 4,127 mg×h/L and the clearance (Cl) were 1.10 and 0.02 L/(h×kg), respectively. The developed method was found to be specific, precise, reproducible and rapid.

Fig 1. Chromatogram of 2,4-D obtained during analysis of rat serum samples.

Representative chromatograms of (A) blank rat serum, (B) serum spiked with 2,4-D, and (C) serum sample obtained 15 min after oral administration 2,4-D.

Fig 2. Concentration-time curve of 96% 2,4-D in rat serum.

Mean serum concentration-time profile of 96% 2,4-D after oral administration of 300 mg/kg bw (A) and 60 mg/kg bw (B) to rats (n = 16). Values at 168 h were below the limit of quantification. (OBS means observed value; PRED means predicted value).