Study On Enrichment And Analysis Method Of Three Keto Aging Flavor Compounds In Beer

Beer becomes one of essential drink in human’s daily life because of its mellow taste and abundant nutrition. The flavor of beer has drawn great attention of general consumers as an important parameter of the quality of beer. During the process of production, storage and transportation, the flavor of beer changes continually and generates unpleasant smell gradually which is named as aging flavor. Different kinds of beer with different routes and rates produce different aging flavor compounds. Establishing a method for analysing and determining aging flavor compounds in beer, which can not only evaluate the aging extent of beer effectively, but also monitor the quality of beer, is very necessary.A high performance liquid chromatography (HPLC) method was established for the simultaneous determination of diacetyl,2,3-pentanedione and β-damascone in beer. The determination of diacetyl and2,3-pentanedione on ultraviolet detector was achieved through pre-column ultraviolet derivatization reaction. After optimizing the derivatization reaction conditions, o-phenylenediamine was used as derivatization reagent, whose concentration is twenty times of that of analytes; the temperature of water bath was40℃for2.5hours; the acidity of reaction medium was pH5.0. The HPLC analysis was carried out using a column Nova-Pak C18. A mixture of methanol (A) and ammonium acetate solution of30mmol/L (B) was acted as mobile phase at a flow-rate of0.9mL/min. A gradient elution style was used: firstly, A:B=50:50for three minutes, then changed to A:B=80:20linearly during the third to sixth minutes, remained for four minutes, and then changed to A:B=50:50linearly during the tenth to thirteenth minutes, balanced for two minutes. The detection program of wavelength changing with time was set up:detection wavelength of237nm for first six minutes, then227nm at the thirteenth to fifteenth minutes, and then237nm for the last two minutes. The automatically injection volumn was10μL and the column temperature was30℃. Under the optimal HPLC conditions, the simultaneous determination of the three targets was performed in15minutes.A dispersive liquid-liquid microextraction (DLLME) was established on the basis of improving the injection apparatus of HPLC to extract and enrich the analytes in beer effectively, meanwhile to reduce their detection limits. DLLME conditions were optimized.250μL dichloromethane and1.2mL isopropanol were selected as extractant and dispersing agent respectively. The pH of extraction system was6.0with0.3g NaCl added and the extraction time was ten minutes. The enrichment factors of diacetyl,2,3-pentanedione and β-damascone were45,63and121respectively under the optimal DLLME conditons.Under the optimal conditions, working curves of each compound were obtained with the linear correlation coefficients ranged from0.9996to0.9999and the linear ranges were greater than or equal to103. The detection limits of0.3～0.9ng/mL(S/N=3) were obtained with the relative standard deviations of2.4～4.4%and the average recoveries of86～97%obtained by spiking in samples.The results show that the DLLME-HPLC method which is convenient to operate with high sensitivity and enrichment factor, good accuracy, low analysis cost and friendly to environment can absolutely meet the requirements of simultaneous analysis and determination of the three main keto aging compounds in beer.