二维高效液相色谱-三重四极杆/复合线性离子阱质谱联用法快速测定鸡肉和鸡蛋中利巴韦林总残留量

Rapid Determination of Total Residues of Ribavirin in Chicken and Egg by Two-Dimensional High Performance Liquid Chromatography-Triple Quadrupole/Linear Ion Trap Mass Spectrometry

建立了二维超高效液相色谱-三重四极杆/复合线性离子阱质谱联用方法快速测定鸡肉、鸡蛋中利巴韦林总残留量。样品在0.10mol/L乙酸铵缓冲液(pH 4.8)中,于37℃下经酸性磷酸酯酶酶解和提取,提取液经超滤后直接进样分析。样品先经Zorbax SB-Aq色谱柱(3.0mm×150mm×1.8μm)分离,将分离出含利巴韦林的流分切割至捕集柱中捕集,然后再将捕集柱切换至第2维色谱流路中,于Hypercarb PGC色谱柱(2.1mm×150mm×3μm)分离,利用电喷雾正离子模式多离子监测触发的增强子离子扫描方式(MRM-IDA-EPI)检测,稳定同位素内标法定量。结果表明:鸡肉、鸡蛋中利巴韦林的平均加标回收率为87.5%~97.7%,相对标准偏差在2.8%~8.3%之间(n=6),方法的检出限(S/N=3)为0.2μg/kg。本法操作简单、灵敏度高、选择性好,已成功应用于食品安全风险检测。

Ribavirin is antiviral agent for the treatments of influenza A virus infections. Due to the potential resistance for human beings, ribavirin has been banned as antiviral agent during poultry farming in many countries. Nevertheless, this antiviral drug may be still illegally used for the treatment of avian influenza in poultry farming. Therefore, it is necessary to develop a sensitive method for the analysis of ribavirin in chicken muscle tissues and eggs. A simple and sensitive method for the determination of total residues of ribavirin in chicken muscle tissues and egg by heart-cutting two-dimensional high performance liquid chromatography-triple quadrupole/linear ion trap mass spectrometry was developed. The main factors influencing the purification and separation efficiency including methods of sample pretreatment, column types of purification and separation, compositions of mobile phases, times of valve switch, and instrumental conditions of mass spectrometry were optimized. During analysis of two types of matrixes （chicken and egg）, the matrix effects of ribavirin under these conditions were evaluated. Under the optimal conditions, the matrix effects of ribavirin in chicken and eggs were 48.9% and 74.2%, respectively. The optimal extraction conditions were as follows： 2.00 g sample was extracted with 10 mL of 0.10 mol/L ammonium acetate buffer （pH 4.8） under enzymatically hydrolyzed for 2 h with acid phosphatase at 37 ℃, and then the extraction was centrifuged for 5 min at speed of 12 000 r/min and the supernatant was ultrafiltrated. The first dimension separation of ribavirin was carried out on a Zorbax SB-Aq column （3.0 mm×150 mm×1.8 μm） with elution with 0.2% formic acid aqueous solution. The flow rate was 0.400 mL/min , and the retention time of ribavirin was 2.32 min. During chromatographic ran for 2.10 to 2.50 min, the fraction containing ribavirin was switched into a Hypercarb PGC guard column （4.6 mm×10 mm×5 μm）. After the trap column retained completely the ribavirin, it was switched into the stream of 2 nd dimension chromatographic system, the ribavirin was separated on a Hypercarb PGC column （2.1 mm×150 mm× 3 μm ） with gradient elution of acetonitrile-aqueous solution containing 0.1% formic acid, and detected by positive electrospray ionization mass spectrometry in the multiple reaction monitoring-information-dependent acquisition-enhanced product ion （MRM-IDA-EPI） scanning mode, and quantified by stable isotope internal standard method. The correlation coefficient of linear calibration cuve of ribavirin is better than 0.999 at the corresponding concentration range of 0.1-100 μg/L. The average recoveries are 87.5%-97.7% for ribavirin in chicken and eggs with relative standard deviations of 2.8%-8.3% （ n =6）. The limit of detection and quantification of ribavirin are 0.2 and 0.7 μg/kg, respectively. The method is simple, sensitive and selective, and has been successfully applied to the total residue determination of ribavirin in chicken and egg samples.