Journal of Chronzatogra~hy, 202 (1980) 317-318 Ekeviet Scientific zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Publishing Company, Amsterdam-Printed in The Netherlands CHROM. 13,245 Note zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Analysis of mimosine and 3_hydroxy4(1 H)-pyridone zyxwvutsrqponmlkjihgfedcbaZYXWV by high-pesformance liquid chromatography zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA B. TANGENDJAJA and R B. H. WLLS+ School of Food Te&wlogy. Universiiy of New So& Wales. P-0. Box I, Kenszkgton 2033 (Australia) (Received August l&h, 1980) Mimosine, fl-[N-(3-hydroxy4oxopyridyl)]-a-aminopropionic acid, is a non- protein amino acid that occurs in the tropical plants, Mimosa zyxwvutsrqponmlkjihgfe pm&a and more importantly, in Leucaena leucocephala. Its presence in Leucaena has prevented the widespread use of this legume for intensive animal feeding as mimosine induces the depilatory and other toxic effiits in ruminants and monogastric animals’*‘. 3-Hydroxy- 4(1H)-pyridone @HP), a metabolite of mimosine in both plants3 and animds4, has also been associated with the development of various abnormal growth or metabolic effects in ruminants5~6 . A range of methods has been developed for the analysis of mimosine and/or DHP utilising ion-exchange and paper chromatography’, gas chromatography, an amino acid analyserg, and calorimetry with an auto-analyser”. All these methods are unsatisfactory for use as a standard routine method as they are either specific for only mimosine or DHP, are not suitable for analysis of both plant and animal extracts, are tedious and time-co&uming, or are subject to variable losses during analysis. This paper describes a sensitive and simple method for the simultaneous analysis of mimosine and DHP in plant material and urine by high-performance liquid chroma- tography (HPLC). METHODS AND RE!WLTS HPLC analyses were performed on a PBondapak C1s column in a Waters liquid chromatograph (Model No. ALC/GPC 244) using a single wavelength UV (280 nm) absorbance detector. Rapid elution and good separation of mimosine and DHP in standard solutions was obtained using a solvent system of 0.2 % (w/v) ortho- phosphoric acid in double distilled water at a How-rate of 1 ml/min (Fig. la). mere was a linear response of both peak height and peak area to concentration of mimosine and DHP with the limits of detection being 1 ng mimosine and 2 ng DHP. Leaf samples of Leucaena were prepared for analysis by initially holding the leaf at 20°C for 24 h, to ensure the production of some DHP3, and then freeze dried. Dried leaf (25 mg) was ground in a mortar with 0.1 N hydrochloric acid (10 ml) to extract mimosine and DHP7, the mixture was then centrifuged for 10 min at 75OOg and the supernatant was Altered under nitrogen (60 p-s-i.) through a membrane ultrafilter. AnaIysis of the leaf extract (IO+ aliquot) showed that sharp resolution of mimosine and DHP was retained and there were no major components in the extract that interfered with the analysis (Fig. lb). OO2l-9673/ ISQ2.25 0 1980 EIsevie~ scientific J?ublish.ingCompany