High-performance liquid chromatography coupled to positive ion mode thermospray ionization mass spectrometry has been used to characterize triprolidine and its known metabolite hydroxymethyltriprolidine. The method allows direct analysis of these compounds in complex biological matrices. Analysis of an extract from a microbial biotransformation experiment with triprolidine was performed and the results included two baseline-resolved chromatographic peaks. The [M + H]+ ion obtained from the mass spectrum of the first peak was consistent with that of hydroxymethyltriprolidine, and the mass spectrum of the second peak corresponded to triprolidine. Under the described conditions, the minimum detectable level of triprolidine without filament enhancement was estimated at about 10 micrograms injected on column.