高效液相色谱法测定葡萄酒中溶菌酶的含量

Determination of lysozyme in wines by high performance liquid chromatography

目的建立高效液相色谱法检测葡萄酒中溶菌酶的含量。方法葡萄酒样品经5%盐酸溶液酸化后,采用流动相A乙腈∶三氟乙酸∶水（50：0.2：49.8,V：V：V）和流动相B乙腈∶三氟乙酸∶水（1：0.2：98.8,V：V：V）进行梯度洗脱,经反相苯基柱分离后,用高效液相色谱仪荧光检测器或紫外检测器进行测定,通过保留时间定性,外标法定量,并优化流动相的配比、梯度洗脱程序和样品的酸化条件。结果本方法在50、200、500和750 mg/L添加水平下的回收率为93.5%-99.2%,相对标准偏差为1.62-5.74%（n=6）,方法定量限为50 mg/L。结论本方法具有分离效能高、重现性好、准确可靠,灵敏度高等优点,测定结果与酶联免疫法结果一致。该方法可以满足葡萄酒中溶菌酶的检测需求。

Objective To establish a method for the determination of lysozyme content in wines by high performance liquid chromatography（HPLC）. Methods Wine samples were acidified by 5% hydrochloric acid solution. The gradient elution solvents were composed of the mobile phase A acetonitrile： trifluoroacetic acid： water（50：0.2：49.8, V：V：V） and the mobile phase B acetonitrile： trifluoroacetic acid： water（1：0.2：98.8, V：V：V）. The samples were separated through a column of reverse phase with phenyl functional groups and determined by HPLC with fluorescence detector（FLD） or ultraviolet detector（UV）. The results were qualitative by retention time and quantified by external standard method. Meanwhile, the ratio of mobile phase, gradient elution program and acidification conditions were optimized. Results The recoveries of lysozyme spiked at the levels of 50, 200, 500 and 750 mg/L were ranged from 93.5% to 99.2% with the relative standard deviations（RSDs） of 1.62%-5.74%（n=6）, and the limit of quantitation（LOQ） of lysozyme was 50 mg/L. Conclusion The method has high efficiency on separation and good reproducibility with the measured results confirming to the results of enzyme-linked immunosorbent assay（ELISA）, which is also accurate, reliable and sensitive, and can meet the requirements for the detection of lysozyme in wines.