A high-performance liquid chromatographic method for the sensitive determination of 2,2'-[(2-aminoethyl)imino]diethanol bis(butylcarbamate) (I) and its metabolites in human serum has been developed. The method was based on a pre-column derivatization with o-phthalaldehyde. The derivatives were stabilized at least for 24 h at 4 degrees C by using N-acetyl-L-cysteine as a thiol and by eliminating the excess o-phthalaldehyde in the reaction mixture by solvent extraction and the addition of an ammonium salt after the reaction. The recoveries and reproducibilities in human serum spiked with I and its two metabolites were satisfactory, and the responses were linear over a wide range of analyte concentrations. The detection limits of I and its metabolites, II and III, in serum were 0.5, 4 and 2 ng/ml, respectively, at a signal-to-noise ratio of 5. The method was satisfactorily applied to the clinical study of I.
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