d-Fagomine is an iminosugar first found in buckwheat (Fagopyrum esculentum Moench) which if used as a dietary supplement or functional food component may reduce the risks of developing insulin resistance, becoming overweight and suffering from an excess of potentially pathogenic bacteria. As d-fagomine may become increasingly important to the food industry, a reliable analytical method for its determination in natural plant sources and foodstuffs is desirable. We have devised a method to separate d-fagomine from its diastereomers 3-epi-fagomine and 3,4-di-epi-fagomine in a single run by cation exchange high-performance liquid chromatography (HPLC) with detection and quantification by mass spectrometry using electrospray ionisation and a simple quadrupole analyser (ESI-Q-MS). The method is validated and applied to the determination of d-fagomine in buckwheat groats (6.7-44 mg kg(-1)), leaves, bran and flour. We show that buckwheat contains 3,4-di-epi-fagomine (1.0-43 mg kg(-1)), which has not previously been reported in this source. The procedure is also applied to mulberry (Morus alba) leaves, which contain d-fagomine and 3-epi-fagomine as minor components. The new method provides a means for convenient and accurate determination of d-fagomine in plant samples and foodstuffs.