A simple and highly sensitive high-performance liquid chromatography assay is proposed to test salivary diamines (putrescine and cadaverine). Derivation was carried out with the orthophthalic aldehyde 2-mercaptoethanol. A rapid purification procedure for derivatives on the cartridges packed with 10 mg of hypercrosslinked polystyrene (Purosep-200) was first developed. Separation was made on a Chromolith (Merck), 100 x 4.6 mm in size, with monolithic reverse-phase silica gel (RP-18e) in the isocratic mode with ultraviolet (UV) detection at 230 nm. The eluent contained 55% acetonitrile and 45% 0.01 M phosphate buffer pH 6.8, added by 1% of isopropanol; flow rate was 1400 pl/min; pressure was 28 bars. Complete separation of diamine derivatives lasted at least 5 min. The sensitivity of the assay with UV detection (230 nm) was about 0.1 ngfor diamines and about 0.5 ng for the internal standard (IS) at a signal/noise ratio of 3.0, which enabled diamines to be determined in I pl (0.001 ml) of saliva. The simplicity, reproducibility, and high sensitivity of the assay along with the feasibility of its application on standard chromatographic equipment (an isocratic pump and an UV detector) make it suitable for routine clinical application.
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