The present invention relates to a new HPLC method for the analysis of the drug substance tiotropium and related substances. In particular, the present invention relates to a new HPLC method for the analysis of the salt tiotropium bromide and related substances. In a first method the mobile phase comprises two or more liquids and the relative concentration of the liquids is varied to a predetermined gradient. In a second method the mobile phase comprises a phosphate salt, a dihydrogen phosphate salt, a phosphoric acid, or a mixture thereof. A third method comprises the detection and optional quantification of methyl-di-2-thienyl glycolate. The present invention also relates to tiotropium and associated pharmaceutical compositions from which samples have been analysed by the methods of the invention and/or which are substantially free of specific impurities.
