Metabolism Study Of Ribavirin In Laying Hens By Using Ultra High Performance Liquid Chromatography-tandem Mass Spectrometry

China has a big consumption on poultry meat and eggs,but veterinary drugs residue or pathogen contamination often threaten the safety of poultry products due to lack of management control,epidemic diseases and so on.In the event of "fast-grown chicken",ribavirin and amantadine,antiviral drugs developed for human,are abused in foodborneanimal,which lead to a big concern of consumer and revealed that veterinary drugs residue made a big strike on food safety.When we face the objective fact of ribavirin abusing,little data can be accessed because of lacking research on metabolism of ribavirin in poultry.To support regulation of food safety and facilitate food quality,this study established an ultra high performance liquid chromatography-tandem mass spectrometryfor simultaneous determination of ribavirin and its metabolites,1H-1,2,4-triazole-3-carboxamide（TCONH₂）and 1-P-D-ribofuranosyl triazole-3-carboxylic acid（RTCOOH）in laying hens plasma.Pharmacokinetics and metabolism of ribavirin in laying hens plasma were studied for the first time based on the UPLC-MS/MS method,which provides theoretical and technical support for ribavirin residue monitoring of poultry.1.A validated method for determination of ribavirin and its metabolites in hens plasmaAfter precipitation of proteins in laying hens plasma by adding methanol,the pretreated sample was separated on a ZORBAX SB-Aq column（3.0 mm×100mm,1.8μm）with a gradient elution program by using 0.1%formic acid water solution and methanol as mobile phase.The target analytes were measured by using electrospray ionization-mass（?）spectrometry under positive MRM mode with ¹³C₅-labelled-ribavirin as internal standard.The result indicate that the linearity of the calibration curve ranging from 5ng/mL to 5000ng/mLfor ribavirin andTCONH₂,and from 50ng/mL to 5000ng/mL for RTCOOH were all very well.The correlation coefficients were 0.9947,0.9992 and 0.9976,respectively.The average recovery of ribavirin,TCONH₂ and RTCOOH were 84.1%～108.2%with intra-assay relative standard deviations（RSDs）of 1.6%～13.4%（n=6）and inter-assay RSDs of 3.8%～16.9%（n=12）.The limits of detection（LOD）for ribavirin,TCONH₂ and RTCOOH in chicken plasma were2μg/kg,2μg/kg andl10μg/kg,respectively.The limits of quantificationfor ribavirin,TCONH₂ and RTCOOH in chicken plasma were 5μg/kg,5μg/kg,20μg/kg,respectively.Theseparation of ribavirin,TCONH₂ and RTCOOH fromendogenous isobaric compounds using MRM for the first time.Moreover,the method with merits of rapid analysis,good repeatability and wide linearity range could be a powerful tool forthe study of ribavirin pharmacokinetics and metabolism in chicken plasma.2.Pharmacokinetics and metabolism of ribavirin in laying hens plasma.ElevenBeijing Red Hens were fed with 30 mg/kg BW ribavirin,theblood sampleswere collected from a vein under the wing at different time points.Ribavirin in the plasma sample was measured using an UPLC-MS/MS method and the data was analysed by WinNonlin 6.3software with the non-compartmental model.Ribavirin concentration increased rapidly after administration,then decreased gradually with elimination processing.The main parameters of ribavirin pharmacokinetics in hens plasma are as follows:the area under the curve of drug(AUC_0-t)is（10658.18±2627.348）h·ng/mL;elimination half-lives(T_1/2β)of plasma ribavirin is（2.9461 ±0.9010）h;peaking time after dosing the drug to chickens(T_max)is（1.5909±0.7355）h;the maximal plasma concentration(C_max)is（2796.54± 1120）ng/mL.The results suggest that the pharmacokinetic characters of ribavirin in hens are quick absorption,broad distribution and rapidelimination.Plasma concentration of TCONH₂ and RTCOOH went up rapidly and down gradually from original to time determined,showing a stable generating and elimination.The major metabolite of ribavirin in laying hens plasma is RTCOOH,and the proportion of RTCOOH in both the metabolites and ribavirin decreased slowly but steady in 75%.3.Metabolism of ribavirin in liver microsomesLiver samples were collected from three blank Beijing Red Henswith proper pretreatment.Those liver were homogenized and separated with differential centrifugation.The sediment of lowerlayer were liver microsomes.Ribavirin level and laying hens liver microsomal protein concentration were changed to obtain optimal condition for drug metabolism.The results indicate that,there is no reaction between ribavirin and laying hens liver microsomes.Neither the incubation time nor ribavirin concentration,even liver microsomal protein concentration do affect the metabolism of ribavirin by laying hens liver microsomes.The LC-IT-TOF analysis of incubation samples showed the same outcome,which means ribavirin is not asubstrate for CYP 450.