分散固相萃取-液相色谱-串联质谱法测定不同品种柑橘果实不同部位中3种柠檬苦素类似物的含量

Determination of 3 Limonin Analogues in Different Parts of Citrus Fruits of Different Varieties by Liquid Chromatography-Tandem Mass Spectrometry with Dispersed Solid Phase Extraction

提出了分散固相萃取-液相色谱-串联质谱法同时测定4个品种柑橘果实的4个部位(油胞层、白皮层、囊衣、果肉)中柠檬苦素、诺米林、黄柏酮含量的方法。取5.0 g均质后的样品,加入5 mL水,涡旋混匀后加入20 mL体积比5∶5的丙酮-乙腈混合溶液,混匀后超声提取10 min,离心5 min。用3 mL水活化2 g EMR-Lipid吸附剂,加至上清液中,涡旋30 s,加入5 g质量比1∶4的氯化钠-硫酸镁混合物和15 mg石墨化碳黑,迅速摇匀后离心1 min。吸取全部上清液,于40℃氮吹至近干,残渣用10.0 mL体积比9∶1的0.1%(体积分数,下同)甲酸溶液和乙腈的混合溶液复溶,过0.22μm滤膜,滤液按照优化的仪器工作条件测定。进行色谱分析时,以CAPCELL PAK ADME HR色谱柱作固定相,以不同体积比的0.1%甲酸溶液和乙腈的混合溶液作流动相进行梯度洗脱分离;进行质谱分析时,以电喷雾离子源正离子(ESI+)和多反应监测(MRM)模式检测。结果显示:3种柠檬苦素类似物的质量浓度在0.10~10.0 mg·L^(-1)内和对应的峰面积呈线性关系,检出限(3S/N)均为0.01 mg·kg^(-1);按标准加入法进行回收试验,回收率为83.1%~90.5%,测定值的相对标准偏差(n=6)为2.6%~5.6%;方法用于30个实际样品的分析,柠檬苦素类似物在不同品种柑橘果实及其不同部位中的含量分布差异显著,柚类品种中柠檬苦素类似物总测定值较高且主要分布在囊衣部位,其他品种主要分布在白皮层和油包层。

The method of liquid chromatography-tandem mass spectrometry with dispersion solid phase extraction was proposed to simultaneously determine limonin,normillin,and phellosterone in 4 parts(oil cell layer,white layer,capsule,and pulp)of citrus fruits of different varieties.The 5.0 g of homogenized sample was taken,and 5 mL of water was added.The mixture was mixed well by vortex,and 20 mL of mixed solution composed of acetone and acetonitrile at volume ratio of 5∶5 was added.After mixing,ultrasonic extraction was made for 10 min,and centrifugation was made for 5 min.The 2 g of EMR-Lipid adsorbent was activated by 3 mL of water,which was added into the above supernatant.The mixture was vortexed for 30 s,ang 5 g of sodium chloride and magnesium sulfate at mass ratio of 1∶4 and 15 mg of graphitized carbon black were added.After quickly shaking well,the mixture was centrifuged for 1 min,and all the supernatant was collected,and blown to near dryness by nitrogen at 40℃.The residue was dissolved in 10.0 mL of mixed solution consisted of 0.1%(volume fraction,the same below)formic acid solution and acetonitrile at volume ratio of 9∶1,and the solution was passed through a 0.22μm filter membrane.The filtrate was determined according to the optimized instrument working conditions.In chromatographic analysis,CAPCELL PAK ADME HR chromatographic column was used as the stationary phase,and mixed solutions composed of 0.1%formic acid solution and acetonitrile at different volume ratios were used as the mobile phase for gradient elution separation.In MS analysis,the modes of electric spray ion source positive ion(ESI~+)and multiple reaction monitoring(MRM)were used for detection.It was shown that linear relationships between values of the mass concentration and peak area of 3 limonin analogues were kept in the range of 0.10-10.0 mg·L^(-1),with detection limits(3S/N)of 0.01 mg·kg^(-1).Test for recovery was made according to the standard addition method,giving recoveries in the range of 83.1%-90.5%,and RSDs(n=6)of the determined values ranged from 2.6%to 5.6%.The proposed method was used for the analysis of 30 actual samples.The distribution of limonin analogues in different parts of citrus fruits of different varieties showed significant differences.Among pummelo varieties,the total determined values of limonin analogues were higher and mainly distributed in the capsule part,while other varieties were mainly distributed in the white and oil layers.