Quantitative Analysis of Mixtures of Metal–Carbonyl Complexes by Fourier-Transform Infrared Spectroscopy: Application to the Simultaneous Double Immunoassay of Antiepileptic Drugs by the Nonisotopic Carbonyl Metalloimmunoassay Method

Abstract

The feasibility of a double immunoassay of haptens by the nonisotopic carbonyl metalloimmunoassay (CMIA) method is demonstrated. Three different pairings of antiepileptic medications from the groups carbamazepine, diphenylhydantoin, and phenobarbital (for each of which a mono-CMIA is already available) were assayed by double CMIA. The assay method employs as tracers metal–carbonyl complexes that give very strong signals in the range of 1850–2200 cm−1in the infrared spectrum, permitting quantitative analysis by Fourier-transform infrared spectroscopy. The fact that the signals are individually assignable and of comparable intensity permits quantitative analysis of mixtures of two tracers. The analysis may proceed in one of two ways: in the simpler case, there is no peak overlap with the two tracers and the quantitative analysis can be performed by simply measuring the absorbance of characteristic peaks of the two tracers. In the second case, in which there is partial or total overlap of peaks, a stepwise calculation provides rapid quantification of the two tracers. These findings allowed us to perform the double CMIA of two antiepileptics in which experimental conditions and time of analysis were strictly identical to those for mono-CMIA. We show here that there is a very good correlation between the results obtained in mono- and double-immunoassay by the CMIA method (correlation coefficient >0.990).