柱前衍生-反相高效液相色谱法测定仔猪结肠内容物生物胺含量

DETERMINATION OF BIOAMINES IN COLONIC CONTENTS OF PIGLETS BY PRE-COLUMN DERIVATIZATION RP-HPLC

目的建立高效液相色谱结合紫外检测器（HPLC—VWD）方法测定仔猪结肠内容物中生物胺的含量。方法以0．4mol／L的高氯酸溶液-20℃过夜处理仔猪结肠内容物，离心后取上清液，用丹磺酰氯衍生，以Zorbax SB—C18柱为分析柱，乙腈和0．02mol／L的乙酸铵水溶液为流动相，流速1．0ml／min进行梯度洗脱，结合保留时间和紫外光谱进行定性分析，定量检测波长254nm，并对样品前处理过程进行优化。结果色胺、苯乙胺、腐胺、尸胺、组胺、1，7-庚二胺、酪胺、亚精胺和精胺分别在0．29～220．00、0．33～250．00、0．20～508．00、0．32～246．00、0．32～240．00、0．28～352．00、0．19～228．00、0．32～396．00、0．25～306．00的浓度（μg／m1）范围内线性关系良好，相关系数均达到0．999，检测限（RSN=3）为O．07～0．19μg／ml，样品的加样回收率为76．74％～115．31％，相对标准偏差均低于4．8％。结论该方法简单、快速、结果准确可靠。

Objective A method based on high-performance liquid chromatography （HPLC） combined with visible wavelength detector （HPLC/VWD） was developed to determine the bioamines in colonic contents of piglets. Methods The colonic contents of piglets were treated overnight with 0.4 mol/L perchloric acid at -20℃, and the supernatant obtained by centrifugation was derived with dansyl chloride. The separation of the derivatives was performed on a Zorbax SB-C18 column by gradient elution using acetonitrile and 0.02 mol/L ammonium acetate at a flow rate of 1.0ml/min. The qualitative analysis was performed by using retention time and UV spectrum. The conditions for sample pre-treatment were investigated and optimized. Results There was good linearity （r2〉0.999） between concentrations （μg/ml） of tryptamine, phenylethylamine, putrescine, cadaverine, histamine, 1, 7-heptyldiamine, tyramine, spermidine and spermine and their peak areas over the range of 0.29-220.00, 0.33-250.00, 0.20-508.00, 0.32-246.00, 0.32-240.00, 0.28-352.00, 0.19-228.00, 0.32-396.00 and 0.25-306.00 respectively. The detection limits （RSN=3） ranged from 0.07 to 0.19 μg/ml. The recoveries of spotted samples ranged from 76.74 % to 115.31% with relative standard derivations less than 4.8 %. Conclusion The method of pre-column derivatization RP-HPLC for determination of biomanines in colonic contents of piglets is accurate, rapid and sensitive.