Integration of liquid chromatography with immunoassay: an approach combining the strengths of both methods

The integration of liquid chromatography (LC) with immunochemical detection combines the superior separation power of LC and the sensitivity and specificity of immunoassays. This approach is shown with 3 LC systems (Perkin-Elmer, C18 RP, 4.6 mm; Varian, C18 RP, 1 mm microbore; Michrom, C18 RP, 1 mm microbore) integrated with an enzyme-linked immunosorbent assay (ELISA) selective for five 4-nitrophenols. The nitrophenols were separated with the 3 LC systems with isocratic runs of 15 to 20 min. Microbore LC separation showed a 10-20 times reduction in solvent amount compared to conventional separation. LC-immunoassay was about 8- to 10-fold more sensitive compared with LC with UV detection. Integrated LC-immuno-assay proved to be a very selective method when 2-methylphenol was injected with an equimolar mixture of 2-amino-4-nitrophenol and 3-methyl-4-nitrophenol; 2-methylphenol does not cross-react with the serum used. Only 2 peaks could be seen in the detection, even when 2-methylphenol was present in very high amounts (3000 pmol). Further, the ELISA-LC detection proved to be selective and sensitive for complex matrixes. 2-Amino-4-nitrophenol was clearly identified in spiked extracts of soil and plant, even when a very small amount (2.4 ng) was injected. Although LC-immunoassay is more labor intensive than LC with UV detection, it offers great advantages in multiresidue analysis and is generally applicable for peak confirmation.