Clinical Chemistry, Vol 35, 1342-1347, Copyright © 1989 by American Association for Clinical Chemistry

HPLC measurement of chlorophenoxy herbicides, bromoxynil, and ioxynil, in biological specimens to aid diagnosis of acute poisoning

RJ Flanagan and M Ruprah
Poisons Unit, Guy's Hospital, London, U.K.

A simple high-performance liquid-chromatographic assay for eight chlorophenoxy (2,4-D and related compounds) and two benzonitrile (bromoxynil and ioxynil) herbicides has been developed to aid in the diagnosis of acute poisoning. Sample (whole blood, plasma/serum, urine, or tissue homogenate) or standard (100 microL) is vortex-mixed (ca. 5 s) with 20 microL of internal standard solution [1.00 g/L 2,4,5-TP in 0.02 mol/L Tris buffer, pH 9.6:methanol (1 + 1)]. Dilute (0.2 mL/L) hydrochloric acid in methanol, 200 microL, is added and the mixture is again vortex-mixed (30 s). After centrifugation (9950 X g, 2 min) a 10- 20 microL portion of the supernate is analyzed on a 250 X 5 mm (i.d.) Spherisorb S5 Phenyl column, with aqueous potassium dihydrogen orthophosphate (50 mmol/L, pH 3.5) and acetonitrile (3 to 1 by vol) at a flow-rate of 1.8 mL/min as eluent. The method is capable of resolving the chlorophenoxy/benzonitrile mixtures (2,4-D/MCPP, 2,4-D/DCPP, 2,4- D/ioxynil, 2,4-D/MCPP/DCPP, 2,4-D/2,4,5-T, and MCPP/ioxynil) encountered in the U.K. The limit of detection (at 240 nm) is 20 mg/L (10 mg/L for bromoxynil and ioxynil). Intra-assay and interassay CVs were less than 5% and less than 8%, respectively, for all analytes. Plasma:whole blood distribution ratios ranged from ca. 1.7 for 2,4-DB to ca. 2.0 for 2,4-D, emphasizing that results of whole-blood measurements must be multiplied by a factor of ca. 2 for comparison with plasma/serum data.