高效液相色谱法测定畜禽肉中8种生物胺含量国家标准方法的改进

Improvement of National Standard Method for Determination of 8 Biogenic Amines in Livestock and Poultry Meat by High Performance Liquid Chromatography

针对国家标准方法GB 5009.208-2016操作繁琐,检测时间较长(约6 h)等问题,改进了提取方法和衍生方法,并应用于畜禽肉中色胺、尸胺、β-苯乙胺、酪胺、亚精胺、腐胺、组胺和精胺等8种生物胺含量的测定。取绞碎样品10 g,加入50 g·L^(-1)三氯乙酸溶液20 mL、正己烷10 mL及1 000 mg·L^(-1) 1,7-二氨基庚烷内标溶液1.0 mL,混匀后涡旋15 min,超声提取15 min,离心5 min。取0.5 mL上清液置于10 mL比色管中,依次加入2 mol·L^(-1)氢氧化钠溶液100μL、饱和碳酸氢钠溶液300μL、10 g·L^(-1)丹磺酰氯衍生溶液2.0 mL,振荡混匀,于60℃反应15 min。加入100μL氨水,于60℃保温15 min,以终止衍生反应。加入乙腈稀释至5 mL,用0.22μm滤膜过滤,滤液注入高效液相色谱仪,在Phenomenex Kinetex色谱柱上以不同体积比的甲醇-水体系进行梯度洗脱,并用二极管阵列检测器检测。结果显示:样品可在1.5 h内完成检测,8种生物胺的质量浓度均在5.00~250 mg·L^(-1)内与其对应的峰面积与内标物峰面积的比值呈线性关系,检出限(3S/N)为4~21μg·kg^(-1);对实际样品进行加标回收试验和日内、日间精密度试验,回收率为80.4%~95.6%,日内(n=6)、日间(n=5)精密度试验测定值的相对标准偏差分别为1.9%~5.1%和2.3%~6.2%;方法用于20批畜禽肉的分析,除组胺外,其他7种生物胺均有不同程度检出。

In view of the cumbersome operation and the long detection time(about 6 h)of the national standard method of GB 5009.208-2016,extraction and derivatization methods were improved,and the proposed method was applied to the determination of 8 biogenic amines in livestock and poultry meat,including tryptamine,cadaverine,β-phenethylamine,tyramine,spermidine,putrescine,histamine and spermine.The minced sample(10 g)was taken and mixed with 20 mL of 50 g·L^(-1) trichloroacetic acid solution,10 mL of n-hexane and 1.0 mL of 1000 mg·L^(-1)1,7-diaminoheptane internal standard solution by vortex for 15 min.After ultrasonic extraction for 15 min,and centrifugation for 5 min,0.5 mL of the supernatant was taken and placed into 10 mL-colorimetric tube.100μL of 2 mol·L^(-1) sodium hydroxide solution,300μL of saturated sodium bicarbonate solution,and 2.0 mL of 10 g·L^(-1) dansyl chloride derivative solution were added subsequently.The mixed solution was mixed by shaking,reacted at 60℃for 15 min,and 100μL of ammonia water was added for reaction at 60℃for 15 min to stop the derivatization reaction.The solution obtained was diluted to 5 mL with acetonitrile,and passed through a 0.22μm filter membrane.The filtrate was introduced into high performance liquid chromatograph,separated on Phenomenex Kinetex column with methanol-water system at different volume ratios for gradient elution,and analyzed with diode array detector.It was shown that the sample could be analyzed within 1.5 h,and the mass concentrations of the 8 biogenic amines were all linearly related with ratios of the peak areas between the target and the internal standard within 5.00-250 mg·L^(-1),with detection limits(3 S/N)of 4-21μg·kg^(-1).Tests for spiked recovery and intra-day and inter-day precision were made on the actual samples,giving recoveries in the range of 80.4%-95.6%,and RSDs of the determined values were 1.9%-5.1%(for intra-day precision test,n=6)and 2.3%-6.2%(for inter-day precision test,n=5),respectively.The proposed method was used for the analysis of 20 batches of livestock and poultry meat.Except for histamine,the other 7 biogenic amines were detected to varying degrees.