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.