Study On The Analytical Methods Of22Benzodiazepines In Chinese Patent Drugs、Health-care Foods And Foodstuffs Of Animal Origin

BZDs is derivative by1,4-benzodiazepine, there are more than20species used in the clinic, because of its sedative、hypnotic、anxiolytic、anticonvulsant and muscle relaxant, illegal manufacturers often add it inChinese patent drugs or health-care foods to enhance its efficacy; orindividual livestock breeders add it in the feed as the animal growthpromoter to reduce animal nutrients that support the consumption ofphysical fitness to weight gain, or it be used in animal transport andslaughter process to reduce the animal body response to noxious stimulationor to reduce mortality and prevent fleshy changes. However, BZDs canproduce dizziness、drowsiness、fatigue、coma、respiratory depression andother side effects, it easily lead to tolerance、dependence and addiction forlong time. Therefore, Regulations for Implementation of The DrugAdministration Law of The People’s Republic of China、The Food HygieneLaw of the Peoples Republic of China clearly forbid chemical compoundadding in Chinese patent drugs or health-care foods. The Ministry ofAgriculture No.176announcement prohibite adding benzodiazepine in animal feed and drinking water. Therefore, it is important to establishe themethod for Chinese patent drugs、 health-care foods and foodstuffs ofanimal origin.This study established a sensitive, accurate, fast and practicaldetermination method to simultaneously detect22BZDs in Chinesepatent drugs、health-care foods、spork、milk. The full text is composed ofthe three parts.Part I Simultaneous Determination of22Benzodiazepines inChinese Patent Drugs and Health-Care Foods by LC-MS/MS0bjective: To develop a LC-MS/MS method for the determination of22benzodiazepines in Chinese patent drugs and health-care foods samples.Methods: Compounds were extracted with ether-dichloromethane(8:2,V/V) in pH11borax buffer solution, the extract was concentrated todry, dissolved in acetonitrile:water (2:8,v/v). The analytes were separatedon an Shimadzu VP-ODS（150mm×2.0mm，5.0μm）, eluted by gradientwith mobile phase of acetonitrile and2mmol/L ammonium acetic(include0.2％formic acid). The flow rate was0.3mL/min, the column temperaturewas40℃and the injection volume was30L. Conditions of MS/MS:electrospray voltage:5.5KV; atomizing gas pressure:0.448MPa; aircurtain air pressure:0.310MPa; auxiliary gas flow rate:0.207MPa;collision gas flow rate:0.069MPa; ion source temperature:650℃;collision chamber entrance voltage:10V, collision chamber outlet voltage: 12V. The analytes detected by positive electrospray ionization tandemmass spectrometry in the multiple reaction monitoring(MRM) mode, andquantified by isotope internal standard method. Results: There were goodlinear correlations between the peak areas and concentration of the22benzodiazepines, the linear rang was0.5～50.0μg/L and correlationcoefficients were above0.9990. The limits of detection (LOD) of these22benzodiazepines were in the range of0.02～0.21μg/kg, limits ofquantification (LOQ) were in the range of0.06～0.68μg/kg. The averagerecoveries of benzodiazepines were72.2%～116.5%, at spiked levels of1.0、2.0and5.0μg/kg, the relative standard deviations (RSDs) of themethod was between2.4%~12.2%. Conclusions:This method is highaccuracy, high sensitivityand good repeatability, it can be used to determinethe content of22BZDs in Chinese patent drugs and health-care foodssamples.Part II Simultaneous Determination of22Benzodiazepines inpork by LC-MS/MS0bjective: To develop a LC-MS/MS method for the determination of22benzodiazepines in pork sample. Methods: The sample was hydrolysisedby enzyme in pH5.2ammonium acetate buffer, followed by adjustment ofpH value more than9.5with ammonium hydroxide．The benzodiazepinesresidues were extracted by aceticether-isopropol(5:1,v/v), defatted byhexane and cleaned up by MCX solid phase extraction column．The analytes were detected by LC-MS/MS that established in the first part.Results: LOD of the22BZDs were in the range of0.01～0.13μg/kg，LOQ were in the range of0.04～0.45μg/kg. The average recoveries ofBZDs were76.0%～107.2%, at spiked levels of1.0,2.0and5.0μg/kg, therelative standard deviation (RSD)of the method was between2.3%～9.1%.Conclusions: This method is high accuracy, high sensitivity and goodrepeatability, it can be used to determine the content of22BZDs in in porksample.Part III Simultaneous determination of22benzodiazepines inmilk by LC-MS/MS0bjective: To develop a LC-MS/MS method for the determination of22benzodiazepines in milk sample. Methods: The sample was hydrolysisedby enzyme in pH5.2ammonium acetate buffer, the proteins wereprecipitated by saturated solution of lead acetate, followed by adjustment ofpH value more than9.5with ammonium hydroxide．The benzodiazepinesresidues were extracted by aceticether-isopropol(5:1,v/v), defatted byhexane and cleaned up by MCX solid phase extraction column．Theanalytes were detected by LC-MS/MS that established in the first part.Results: LOD of the22benzodiazepines were in the range of0.01～0.11μg/kg，LOQ were in the range of0.04～0.37μg/kg. The average recoveriesof benzodiazepines were70.9%～108.1%, at spiked levels of1.0、2.0and5.0μg/kg, the relative standard deviation (RSD) of the method was between1.7%～10.9%. Conclusions: This method is high accuracy, highsensitivityand good repeatability, it can be used to determine the content of22BZDs in milk sample.