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

P. M. Krämer, Q. X. Li, B. D. Hammock

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18 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)1275-1287
Number of pages13
JournalJournal of AOAC International
Issue number5
StatePublished - Sep 1994

ASJC Scopus subject areas

  • Food Science
  • Analytical Chemistry


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