Z Lebensm Unters Forsch (1995) 200:235-237 Zeitschrift far Lebensmittet- Untersuchur N und-Forschuag 9 Springer-Verlag 1995 Short communication Fast and sensitive determination of furosine Thomas Henle, Giinther Zehetner, Henning Klostermeyer Lehrstuhl far Milchwissenschaft, Technische Universit~t Mfinchen, V6ttinger Strasse 45, D-85350 Freising-Weihenstephan, Germany Received July 25, 1994 Methode zur sehnellen und empfindlichen Bestimmung yon Furosin Zusammenfassung. Mittels isokratischer Ionenpaar-RP- HPLC gelang die empfindliche Bestimmung von Furosin in S~urehydrolysaten yon Lebensmitteln innerhalb von 5 Minuten Laufzeit und mit einer Nachweisgrenze yon unter 1,5 mg pro kg Protein. Die Bildung yon Furosin w~hrend der Hydrolyse von Lebensmittelproben erwies sich zwi- schen Konzentrationen von 4 und 8 mol/L als linear vonder Salzs~urekonzentration abh~ingig. Abstract. Sensitive determination of furosine in acid hydrolysates of foods was achieved by isocratic ion-pair reversed-phase HPLC and direct UV-detection within a run time of 5 minutes and levels lower than 1.5 nag per kg of protein. The formation of furosine during hydrolysis of food samples with hydrochloric acid of varying concentra- tion was studied. Furosine formation increased linearly with acid concentration (4 to 8 tool/L). l'-pyridyl)-L-norleucine] and lysine [6-9] during acid hydrolysis of foods. Quantification of furosine generally was achieved by ion-exchange chromatography with post- column ninhydrin derivatisation [10-13] and RP-HPLC with direct UV-detection [14-17]. For all methods, the use of a furosine standard [18] proved to be essential for accurate quantification. Further questions still remain concerning the amount of furosine formed during acid hydrolysis. Although already known that an increase in the concentration of the acid used leads to higher furosine values [19], published results widely differed in the conditions applied for hydrolysis, thus disabeling a comparison of the reported furosine values. This makes also the fixation of critical values rather difficult. We describe a simplified isocratic ion-pair RP-HPLC method for a fast and sensitive determination of furosine in foods. In addition, the formation of furosine during hydro- lysis with hydrochloric acid of varying concentrations was investigated. Introduction Furosine [1] is widely used as a measure for protein-bound Amadori products and thus for the extent of the Maillard reaction in food and biological samples [2]. Especially for milk based foods, furosine values are rather good indicators for the classification of heat treatments, ranging from below pasteurisation up to sterilisation [3, 4]. Furosine indicates also the use of reconstituted milk in cheesemak- ing. For "pasta filata" type cheeses, a threshold value of 12 mg furosine per 100 g protein is fixed [5]. Furosine [N e- (furoylmethyl)-L-lysine] is formed out of Amadori com- pounds beside pyridosine [s-(6'-methyl-3'-hydroxy-4'-oxo- Correspondence to: T. Henle Materials and methods Chemicals. Hydrochloric acid was from J. T. Baker (Deventer, The Netherlands). Ethanol (HPLC-grade) and pentane sulphonic acid were from Laborbedarf Mtfller (Fridolfing, Germany). Furosine was pre- pared according to [18]. All other chemicals were from Merck (Darmstadt, Germany). - Food samples were obtained from local retail stores. - In the standard procedure, samples (corresponding to 40 mg of protein) were hydrolysed in stoppered test tubes in the presence of 10 ml of 8 N HC1 for 23 h at I10~ After cooling (ice bath), aliquots of 1 ml were dried in vacuo and dissolved in 500 gl of 0.2 moll1 sodium citrate, pH 2.20. - Amino acid analysis (samples of 10 to 160 ml) was performed as described in [13] and [18]. Ion-pair RP-HPLC. This was performed on a 625 solvent delivery system with a variable wavelength detector (Waters, Milford, USA), using a stainless steel column (125 x 4.6 ram) and a pre-column (4 x 4.6 mm, both from Knauer, Heidelberg, Germany), each filled with Superspher RP18-100, 4 gm (Merck, Darmstadt, Germany). The columns were isocratically eluted at ambient temperature. The mobile phase consisted of a solution of 7.5 mmol pentane sulphonic acid in