Study On Detection Method Of Glucocorticoid In Feed And Urine Sample

This paper studied the detection method of glucocorticoid such as Prednisone, Prednisolone, MethylPrednisone, Demxamethasone, Betamethasone, Beclometasone, Fludrocortisone acetate , Cortisone acetate and Hydrocortisone in feed and urine samples.Feed specimen was extracted by using methanol as extraction solvent and was purified via solid phase extraction technics in which black carbon column connected with asahipak had the best purification effect, peristaltic pump was used in continuous sample introduction, mass spectrum parameter were optimized at ESI- mode.The data showed that glucocorticoid drug had diverse ions peak such as [M+H]+,[M-H]-,[M+HCOO]-,among which [M+HCOO]- had the highest peak,then we chose [M+HCOO]- in the target compound as parent ion in collision induced dissociation.Fludrocortisone acetate and Cortisone acetate had characteristic fragment ions of [M-H]-and [M-H-HCOOCH3]-,but of which in other 7 glucocorticoids were [M-H]-and [M-H-CH2O]-.30% acetonitrile was used as sample introduction solvent. Accuracy test was did through adding in the blank feed 3 different concentration (low, medium and high concentration )of nine glucocorticoid drugs, each concentration did six parallel, repeated 3 times. Glucocorticoids of 5ng/g～20ng/g were added to blank feed and the recovery rate of the method were 51.1%～88.2%, the relative standard deviation in one batch and between batched were all less than 20%.This method is applicable to detect single or mixture of the nine kinds of glucocorticoid in compound feed, concentrated feed and premix compound feed. The minimum detection limit was 2μg/kg and the limit of quantitation was 5μg/kg.50μLβ- Hydrochloric acid glycoside enzyme glucuronic - aryl sulfatase was added to 5ml urine sample,then the enzymatic hydrolysis was carried out in ammonium acetate buffer solution under 37℃. Using ethyl acetate as extraction solvent, the extractive was purified through HLB connected with asahipak, this method had stable recovery and good reproducibility.Ethyl acetate– methanol( 60:40) was used as elution solution and acetonitrile - water (30:70) as sample introduction solvent.Accuracy test was did through adding in the blank feed 3 different concentration (low, medium and high concentration )of nine glucocorticoid drugs, each concentration did six parallel, repeated 3 times.Glucocorticoids of 1ng/ml～5ng/ml were added to blank urine sample and the recovery rate of the method were 55.0%～106.2%, the relative standard deviation in one batch and between batched were all less than 20%.This method is applicable to detect single or mixture of the nine kinds of glucocorticoid in annimal urine. The minimum detection limit was 2μg/kg and the limit of quantitation was 5μg/kg.This paper studied the detection method of nine kinds of glucocorticoid in feed and urine using liquid chromatographic - tandem mass spectrometry method, Defined the sphere of application, determined the sampling quantity, purification, extraction method, liquid chromatography and tandem mass spectrometry conditions. It showed that this method has good sensitivity,accuracy and precision,could meet the requirements of detection of this 9 kinds of glucocorticoid in feed and urine samples and could provide technology support for quality and safety monitoring in feed.