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

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.