超高效液相色谱-四极杆-飞行时间高分辨质谱用于乳液类化妆品中9种抗过敏违禁药物的筛查和定量分析

Screening and quantitative analysis of nine illicit antiallergics in emulsion cosmetics by ultra-high performance liquid chromatography-quadrupole-time-of-flight high-resolution mass spectrometry

建立了超高效液相色谱-四极杆-飞行时间高分辨质谱(UPLC-Q-TOF-HRMS)快速筛查和定量检测乳液类化妆品中9种抗过敏违禁药物的方法。乳液样品经乙腈涡旋和超声提取,采用PRiME HLB固相萃取小柱净化,以0.1%甲酸水-乙腈为流动相在XBridge C18色谱柱上进行梯度洗脱和分离,采用电喷雾电离源,在正离子模式下采用全景式数据非依赖性扫描模式(SWATH)进行数据采集,结合保留时间、母离子精确质量数、同位素峰及二级子离子碎片进行快速定性筛查,采用特征子离子碎片的峰面积进行外标法定量。实验比较了不同仪器条件和前处理条件对乳液类样品中9种抗过敏物质测定的影响,并对比了不同级别离子定量对基质效应的影响。结果表明,较其他净化柱而言,PRiME HLB固相萃取小柱具有更好的净化效果,能降低杂质对目标峰的干扰,同时保证目标物的回收;在优化的条件下,9种抗过敏药物在5~100μg/L质量浓度范围内线性良好,相关系数(r2)均大于0.99;9种目标物的定量限在0.05~0.10 mg/kg之间;在3个添加水平(0.10、0.20和0.60 mg/kg)下,其回收率在65.3%~107%之间,相对标准偏差(RSD,n=6)均小于20%;基质效应实验结果表明,采用二级子离子定量能够降低基质效应,提高定量准确度。与文献方法相比,该法操作简便,前处理耗时更短,其高精度质量数和二级指纹图谱比对技术能保证检测结果的准确度和灵敏度,可实现乳液类化妆品中抗过敏违禁药物的快速筛查和准确定量。

A rapid and accurate analysis method based on ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight high-resolution mass spectrometry(UPLC-Q-TOF-HRMS)was developed to screen and determine nine antiallergy drugs in emulsion cosmetics.First,a standard library of the target compounds was established.The library contained the TOF-MS information and secondary MS information such as retention time,ion addition mode,mass error,isotope distribution,mass-to-charge ratio of the parent ion,and fragment ion distribution.According to the European Union regulation(SANTE/11945/2015),the standard for the qualitative determination by HRMS was determined;that is,each compound was confirmed by two ions with a mass error below 5%,and the abundance ratio of the two ions was less than 30%.Second,the instrument conditions and sample pretreatment conditions for the determination of different compounds were optimized,and the influence of different levels of quantitative ions on the matrix effect was compared.The following observations were made:(1)the addition of 0.1%formic acid to the water phase improved the response of the chromatographic peaks;(2)among the various solvent amounts tested(4,5,6,8 mL acetonitrile and 4,5,6,8 mL methanol),4 mL acetonitrile showed the best extraction efficiency;(3)PRiME HLB had a better purification effect than the other two purification columns(C18 and HLB solid-phase extraction cartridges),thus reducing the interference of impurities and ensuring good recovery of the target compounds;(4)the use of two pairs of secondary product ion quantification could significantly reduce the matrix effect of anti-allergic compounds and improve the quantification accuracy.Finally,based on the above findings,the experimental procedure was established.The lotion samples were first ultrasonically extracted with acetonitrile and purified on the PRiME HLB column.Chromatographic separation was performed on a Waters XBridge C18 column with gradient elution using 0.1%(v/v)formic acid in water and acetonitrile.Finally,the sequential window acquisition of all theoretical mass spectra(SWATH),which shows obvious advantages in continuous and high-throughput acquisition,was selected for MS data acquisition.The retention time,mass accuracy,isotope distribution,and fragment ion matching ratio were used for fast qualitative screening,while the peak areas of characteristic product ions were used for precise quantification.All the calibration curves showed good linearity(r2>0.99)within the tested ranges(5-100μg/L)under the optimum conditions.The limits of quantification(LOQs)were in the range of 0.05-0.10 mg/kg.The recoveries were in the range of 65.3%-107%at three spiked levels(0.10,0.20,and 0.60 mg/kg),with relative standard deviations(RSDs,n=6)below 20%.Compared with the existing ion exchange column methods,the proposed“one-step”purification method based on PRiME HLB is simpler and more rapid,where the extraction solution is filtered directly after allowing it to pass through the column,without any subsequent washing and elution procedures.In addition,the LOQs of this method are lower than those of other LC-MS/MS methods,indicating that the proposed method has higher sensitivity.The application of SWATH data acquisition makes it possible to achieve quantification with two pairs of product ions,thus reducing the matrix effect and ensuring accuracy of the quantitative results.Therefore,the proposed method is less time-consuming and operationally convenient,and it can be used for the rapid screening and accurate quantification of antiallergics in lotion samples.