Isolation of a 2:1 hydrochlorothiazide-formaldehyde adduct impurity in hydrochlorothiazide drug substance by preparative chromatography and characterization by electrospray ionization LC-MS.

Hydrochlorothiazide drug substance (19 lots) from five different manufacturers and four different countries of origin (USA, Italy, Hungary, and Croatia) were analyzed for the presence of impurities using a gradient elution chromatographic system, with acetonitrile-water as the mobile phase. Two known impurities of hydrochlorothiazide, 4-amino-6-chloro-1,3-benzenedisulfonamide and chlorothiazide, were separated, as well as a late-eluting, unknown, recurring impurity. The unknown impurity was isolated by preparative liquid chromatography followed by preparative thin-layer chromatography. It was characterized by electrospray ionization LC-MS as a 2:1 hydrochlorothiazide-formaldehyde adduct of the parent drug substance. The adduct is believed to form through the double condensation reaction of hydrochlorothiazide with excess formaldehyde during the parent compound's synthesis. The concentration of this impurity ranged from 0.02 to 1.1% (area%), and was above the 0.1% USP Other Impurities threshold in 16 of the 19 lots examined.