| Cheese was known as"king of the dairy products"in the world, It was one of high nutritional value products. Dairy industry of our country had experienced the milk powder and liquid milk stage, and then entered the cheese stage. Biogenic amines would be formed during the maturation process of cheese. Biogenic amines of high concentration would not only affect food flavor, but also would do harm to human health, therefore, it was necessary to establish a rapid analysis method of biogenic amines in cheese.In this study, a simple, rapid, sensitive and high performance liquid chromatography method was established from pretreatment of sample, derivatization and detector. The results of this study were as follows:1. o-phthaldialdehyde (OPA) was used as derivatization agent, and conditions of pre-column derivatization of biogenic amine for HPLC method were determined. All samples were analysised by HPLC after derivatized for two minutes and extracted by ethyl acetate for six minutes. Conditions for chromatography were as follow: Hypersil ODS2 column (4.6 mm×250 mm,5μm)was used. Wavelength of diode array detector was 335 nm; Under Fluorescence detector with Ex 336 nm and Em 466 nm. Mobile phase: 0.02 M KH2PO4- methanol(30:70 ,V:V); Flowing rate: 0.8 mL·min-1; Injection volume: 10μL; column temperature kept at 35℃.2. Five biogenic amines were detected by HPLC instrument with a diode array detector system or a fluorescence detector system. Fluorescence detection was linear in.the range of concentration from 0.1 mg·L-1 to 10 mg·L-1; the detection limit for histamine and tyramine was 0.010 mg·L-1 , for tryptamine was 0.005 mg·L-1, and that for putrescine and cadaverine was 0.015 mg·L-1. Ultraviolet detection was linear in the range of concentration from 4 mg·L-1 and 40 mg·L-1. The detection limit for histamine was 2 mg·L-1, for tyramine and cadaverine was 0.8 mg·L-1, and that for tryptamine and putrescine was 1 mg·L-1.3.The cheese samples were extracted by trichloroacetic acid,ethanol and methanol respectively. The cheese samples were homogenate extraction twice, purified with N-hexane and derivatized by OPA. Afterwards, the samples were quantitatively analyzed by HPLC with external standard method.(1) Extraction with trichloroacetic acid (TCA): When added concentration was 2.5 mg·L-1, recovery ranged from 80.4% to 107.1% and relative standard deviation (RSD) were less than 7.8%, detected by FL detector. When added concentration was 5.0 mg·L-1, recovery ranged from 78.2% to 101.3% and relative standard deviation (RSD) were less than 6.1%, detected by FL detector; recovery ranged from 80.0% to 110.7% and relative standard deviation (RSD) less than 6.7%, detected by ultraviolet detector. When added concentration was10 mg·L-1, recovery ranged from 79.6% to 106.6% and relative standard deviation (RSD) were less than 6.0%, detected by FL detector; recovery ranged from 75.7% to 103.1% and relative standard deviation (RSD) less than 7.6%, detected by ultraviolet detector.(2) Extraction with ethanol: When added concentration was 5.0 mg·L-1, recovery ranged from 37.8% to 118.0% with relative standard deviation (RSD) between 5.1% and 21.2 %, detected by FL detector; recovery ranged from 23.5% to 115.2% with relative standard deviation (RSD)between 1.1 % and 15.2 %, detected by ultraviolet detector. When added concentration was 10.0 mg·L-1, recovery ranged from 39.5% to 150.9% with relative standard deviation (RSD) between 2.9% and 19.5 %, detected by FL detector; recovery ranged from 38.3% to 131.1% with relative standard deviation (RSD)between 1.6 % and 15.7 %, detected by ultraviolet detector.(3) Extraction with methanol: When added concentration was 5.0 mg·L-1, recovery ranged from 48.2% to 127.7% with relative standard deviation (RSD) between 5.4% and 28.0 %, detected by FL detector; recovery ranged from 63.2% to 113.9% with relative standard deviation (RSD)between 4.0 % and 26.4%, detected by ultraviolet detector. When added concentration was 10.0 mg·L-1, recovery ranged from 26.7% to 148.4% with relative standard deviation (RSD) between 7.3% and 27.2%, detected by FL detector; recovery ranged from 31.3% to 162.1% with relative standard deviation (RSD)between 5.5% and 27.9 %, detected by ultraviolet detector.Trichloroacetic acid extraction was a rapid and effective extraction method compared with the other two extraction methods. The pretreatment time was 30 minutes and the total analysis time was 100 minutes, the TCA extraction method was fit for detection of biogenic amines in cheese.
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