1460 GÖKMEN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 5, 2009 FOOD COMPOSITION AND ADDITIVES
Determination of Furosine in Thermally Processed Foods by Hydrophilic Interaction Liquid Chromatography VURAL GÖKMEN and ARDA SERPEN Hacettepe University, Department of Food Engineering, 06800 Beytepe, Ankara, Turkey FRANCISCO J. MORALES Instituto del Frío, Consejo Superior de Investigaciones Científicas, José Antonio Novais 10, E-28040 Madrid, Spain
Furosine, a marker of the impairment of lysine residues in protein, is formed during acid hydrolysis of the Amadori compound generated at the early stage of the Maillard reaction in thermally treated foods. An analytical method is described for the determination of furosine in thermally processed foods. The method entails acid hydrolysis of food, SPE cleanup with a hydrophilic-lipophilic sorbent, and hydrophilic interaction LC separation. The main advantage of the method is the separation of furosine by means of hydrophilic interaction LC analysis, which simply avoids ion-pairing agents during the chromatography for a complete baseline separation, or avoids the use of a metal-free chromatographic device. In addition, by combining microwave hydrolysis with hydrophilic interaction LC, the complete determination of furosine in a food sample takes approximately 25–30 min. The LOD and the LOQ were 0.7 and 2.3 mg/kg, respectively, for furosine, based on S/Ns of 3 and 10, respectively. The recoveries ranged from 94.6 ± 3.1 to 98.6 ± 1.7% for spiking levels of 100–1000 mg/kg sample. The method is easy to use and cost-effective, and gave reproducible results for both within-day and day-to-day tests.
echnological processes applied to food can give rise to modifications in its composition. One of the most important modifications induced in food during heating is the Maillard reaction (MR), which involves amino acids and reducing carbohydrates, and is responsible for the brown color and the many organoleptic properties of foods appreciated by consumers (1). The loss of available lysine is the most significant consequence of this reaction (2). Furosine [e-N-(2-furoylmethyl)-L-lysine], which is found in acid hydrolysates of heated foods, has been considered as a suitable marker of the thermal process and food quality (3). It has been shown to be related to the formation of Amadori
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Received November 10, 2008. Accepted by SG February 18, 2009. Corresponding author’s e-mail:
[email protected]
compounds in the early stage of the MR by the condensation of reducing sugars with the e-amino group of protein-bound lysine (4). Although GC after derivatization of furosine to heptafluorobutyryl isobutyl esters and amino acids analysis has also been used for the determination of furosine, HPLC has been used traditionally. The chromatographic methods reported so far have been based mainly on ion-pair reversedphase LC using hydrophobic interaction columns (5–9). The aim of the study described in this paper was to develop an improved analytical method for the determination of furosine in thermally processed foods. The developed method entails hydrolysis of the sample (conventional or microwave) and SPE using an HLB sorbent cleanup before chromatographic analysis by hydrophilic interaction LC with diode-array detection (DAD). The results were compared with those obtained by classical ion-pair reversed-phase chromatography, as described by Delgado et al. (7) in a generic method. Experimental Chemicals and Consumables Formic acid, sodium heptanesulfonate, acetonitrile, and hydrochloric acid (HCl) were analytical grade and obtained from Merck (Darmstadt, Germany). Ultrapure water was used throughout the experiments (Milli-Q system; Millipore, Bedford, MA). Sep-Pak C18 (1 mL, 30 mg) and Oasis HLB (1 mL, 30 mg) were supplied by Waters (Milford, MA). The analytical column (Atlantis HILIC, 4.6 ´ 300 mm, 5 mm) was obtained from Waters. Furosine standard was purchased from Neosystem Laboratoire (Strasbourg, France). Sample Description Processed cereal and milk samples were obtained from the local market. The samples were comparatively analyzed by hydrophilic interaction chromatography or ion-pair reversed-phase chromatography methods. Two reference crisp bread samples (rye flour) identified as CB-HT927 and CB-LT927 were obtained from the intercomparison ring test for heat-induced markers analytical methods launched by COST-Action 927 in September 2005 (http://www.if.csic.es/ proyectos/cost927/index.htm).
GÖKMEN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 5, 2009 1461
Hydrophilic Interaction Chromatography Method
Figure 1. Chromatograms of furosine standard solution at a concentration of 1 mg/L, a cookie sample containing furosine at 667 mg/kg (834 mg/100 g protein), and an ultra-high temperature milk sample containing furosine at 214 mg/kg (669 mg/100 g protein). Asterisks show furosine peaks.
Thermally processed foods were hydrolyzed before chromatographic analysis. Solid samples were homogenized by using a grinder. The samples were hydrolyzed by both conventional and microwave procedures. Conventional hydrolysis.—A 0.5 g sample in a screw-cap glass tube was subjected to acid hydrolysis by adding 10 mL 8 N HCl. After the contents of the tube were purged with nitrogen for 2 min, the glass tube was capped and heated at 110°C for 23 h as described elsewhere (5). Microwave hydrolysis.—A 1.0 g sample was subjected to acid hydrolysis by adding 10 mL 8 N HCl in a screw-cap Teflon vessel. After the contents of the vessel were purged with nitrogen for 2 min, the vessel was capped and heated in a microwave oven. A closed-vessel microwave system [Model MDS-2000; power maximum, 1200 W (CEM Corp., Matthews, NC)] equipped with temperature control was used. (A feedback control signal to regulate microwave power output was used as the temperature control.) The oven was equipped with a turntable (3 rpm) and a mode stirrer, which distributed the microwave energy to prevent uneven heating. The hydrolyzed sample was filtered through a medium grade filter paper. The filtrate was diluted 100-fold with HPLC grade water; 1 mL was passed through an Oasis HLB or a Sep-Pak C18 cartridge to remove dark-colored interfering compounds before HPLC analysis. An Agilent Technologies (Waldbronn, Germany) 1100 HPLC system consisting of a quaternary pump, a DAD, and a temperature-controlled column oven was used. The chromatographic separation was performed on an Atlantis HILIC column by using 1% formic acid solution at a flow rate of 1.0 mL/min at 40°C. Chromatograms were obtained at a detection wavelength of 280 nm. The purity of the furosine
Figure 2. UV spectrum of pure furosine (solid line) and spectra corresponding to furosine in a cookie and in ultra-high termperature milk.
1462 GÖKMEN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 5, 2009
Figure 3. Effect of (a) hydrolysis time (T = 150°C) and (b) hydrolysis temperature (t = 10 min) on the resulting furosine concentration of a cookie sample (t = 10 min).
peak was confirmed by recording spectra in a wavelength range between 250 and 350 nm. The generic method described by Delgado et al. (7) was used as a reference. Chromatographic measurements were performed by using an ion-pair reversed-phase chromatography method. Results and Discussion The usual approach for the determination of furosine in acid hydrolysates of foods is to use reversed-phase LC with UV detection at 280 nm. Because furosine is relatively hydrophilic as a result of its amino acid lysine moiety, it is poorly retained by highly hydrophobic sorbents like C18. Its retention can be improved if a reversed-phase column is used, by introducing an ion-pairing agent into aqueous mobile phase in order to resolve interfering compounds. Sodium heptanesulfonate has been the choice as the ion-pairing agent for the determination of furosine (5, 7, 9, 10). In this study, hydrophilic interaction chromatography was successfully applied to the separation of furosine in acid hydrolysates of foods (processed cereals and milk) without a need for an ion-pairing agent. Furosine eluted at 6.1 min (6.102 ± 0.017 min, n = 10) with the use of 1% aqueous formic acid as the mobile phase at a flow rate of 1.0 mL/min at 40°C. The CV of the retention time of furosine was found to be
