高效液相色谱-串联质谱法测定蜂蜜中的5种双稠吡咯啶类生物碱

Determination of five pyrrolizidine alkaloids in honeybyliquid chromatography-tandem mass spectrometry

建立了强阳离子固相萃取-高效液相色谱-串联质谱法用于测定蜂蜜中野百合碱、克氏千里光宁、倒千里光碱、千里光菲啉和千里光宁等5种双稠吡咯啶类生物碱.蜂蜜样品用0.1 mol/L盐酸溶解,强阳离子交换固相萃取柱富集净化后,高效液相色谱-串联质谱进行定性和定量.以Phenomenex C18柱（100 mm×4.6 mm,2.6 μm）为分析柱,乙腈和0.1％（体积分数）甲酸-5 mmol/L乙酸铵水溶液为流动相进行梯度洗脱,在电喷雾正离子多反应监测模式下进行检测.结果表明,5种双稠吡咯啶类生物碱在1 ～ 100 μg/L范围内线性关系良好,线性相关系数均大于0.99.在1、20和50 μg/kg加标水平下,11种不同种类蜂蜜中的5种双稠吡咯啶类生物碱的平均回收率为73.1％～107.1％,相对标准偏差（RSD）为4.1％～17.0％,方法检定量限可达到1.0 μg/kg.该方法准确灵敏,可适用于不同种类进出口蜂蜜中双稠吡咯啶类生物碱的监控分析.利用该方法对来自中国8个省及自治区的洋槐蜜、葵花蜜、棉花蜜、油菜蜜、荆条蜜、枣花蜜、荞麦蜜和来自新西兰、西班牙、澳大利亚等国家的进口蜂蜜进行了筛查.结果发现,野百合碱、克氏千里光宁和倒千里光碱均未检出,而千里光菲啉和千里光宁在大多数蜂蜜中均能检出,千里光菲啉含量在11.0 ～31.1 μg/kg范围内,千里光宁含量在8.3 ～ 29.1 μg/kg范围内.

A liquid chromatography-tandem mass spectrometry （LC-MS/MS） method was developed for the determination of five pyrrolizidine alkaloids （PAs） （monocrotaline,senkirkine,retrorsine,seneciphylline and senecionine） in honey.The honey samples were dissolved in 0.1 mol/L hydrochloric acid solution and a strong-cation exchange column was used to purify and concentrate the target analytes.The separation of the analytes was carried out on a Phenomenex C18 column（100 mm ×4.6 mm,2.6 μm）using the mobile phases of acetonitrile and 5 mmol/L ammonium acetate-0.1% （volume percentage） formic acid aqueous solution with gradient elution.The separated compounds were detected in multiple reaction monitoring （MRM） mode via positive electrospray ionization （ESI ＋）.The calibration curves were of good linearity in the range of 1-100 μg/L （r 〉0.99）.The limit of quantification of the method was 1.0 μg/kg.The average recoveries were between 73.1% to 107.1% at three spiked levels （1,20 and 50 μg/kg） with the relative standard deviations（RSDs） in the range of 4.1% to 17.0%.The proposed method was applied to different kinds of honey from China,New Zealand,Spain and Australia.The samples included rape,vitex,sunflower,cotton,tilia tree,date,acacia,buckwheat,manuka and eucalyptus honey.Monocrotaline,senkirkine and retrorsine were not detected in the collected honey samples.However,seneciphylline and senecionine were found in most of the honey samples.The concentrations of seneciphylline and senecionine were 11.0-31.1 μg/kg and 8.3-29.1 μg/kg,respectively.