Studies On Preparation Of Tulathromycin And Dicyclanil Marker Residue Reference Materials

Tulathromycin is a new broad-spectrum antibiotic of macrolides for animals.The antibacterial activity is more superior to Tylosin and Tilmicosin,which is characterized by antibacterial medicines for animals,less used amount,good bioavailability,long half life and low residue.So it is widely used in animal production at home and abroad.The(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10,13-tetrahydroxy-3,5,8,10,12,14-hexamethyl-11-[[3,4,6-trideoxy-3-(dimethylamino)-?-D-xylohexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one(TUM)is the marker residue of Tulathromycin,and provides the MRL in liver?kidney and fat is 3000,3000,100 pg/kg in cattle and pigs.Dicyclanil as a new type of outside parasite growth regulators is widely used in veterinary clinic,which is characterized by less toxicity,environment protective and high efficiency.Under JECFA and EMEA set the marker residue of Dicyclanil is 2,4,6-triamino-5-pyrimidine-carbonitrile.The MRLs of sheep target tissues were 400,400,200 and 50?g/kg for liver,kidney,muscle and fat.The reference material of TUM and 2,4,6-triamino-5-pyrimidinecarbonitrile is not commercialized,so bring inconvenience to residue detection and metabolism study of Tulathromycin and Dicyclanil.The paper mainly studies the synthesis of TUM and 2,4,6-triamino-5-pyrimidinecarbonitrile,preparing the high candidate reference materials,measurement of the purity and content,determination the homogeneity and stability,certified the value and total uncertainty analysis.This provides the material for the two drugs in pharmacology,toxicology and residue monitoring.It is good for the drugs pharmacokinetics research and supplying references for the synthesis of the metabolites Macrolides and Amino pyrimidines drugs.This study also provides references to preparation the purity reference material.1 Synthesis and purification of TUM and 2,4,6-triamino-5-pyrimidine-carbonitrileTUM was synthesized from Erythromycin A(E)-oxime.This reaction involves Beckmann rearrangement reaction,reduction reaction and hydrolytic reaction.The yield of the product under optimum conditions was up to 77.8%.The structural of the TUM was characterized by IR spectra,UV spectra,1HNMR,13CNMR and ESI-MS.The candidate reference material of TUM was prepared by acetone and ethyl acetate for several times.The purity of candidate reference material was detected by full spectrum scan of high performance liquid chromatogram(HPLC).The purity of TUM was above 99.5%by area normalization method.2,4,6-triamino-5-pyrimidinecarbonitrile was synthesized from Sodium dicyanamide and Malonitrile.This reaction involves nucleophilic addition,cyclization reactions and nucleophilic substitution.The yield of the product under optimum conditions was up to 81.0%.The structural of the 2,4,6-triamino-5-pyrimidinecarbonitrile was characterized by IR spectra,UV spectra,1HNMR,13CNMR and ESI-MS.The candidate reference material of 2,4,6-triamino-5-pyrimidinecarbonitrile was prepared by recrystallization from DMSO and methanol for several times.The purity of candidate reference material was detected by full spectrum scan of high performance liquid chromatogram(HPLC).The purity of 2,4,6-triamino-5-pyrimidinecarbonitrile was above 99.5%by area normalization method.2 Homogeneity study of TUM and 2,4,6-triamino-5-pyrimidinecarbonitrileThe TUM and 2,4,6-triamino-5-pyrimidinecarbonitrile reference materials was packed into 50 bottles,selected 16 bottles randomly,determined the purity by HPLC area normalization method,using F test method to analysis of measurement results,the results show that TUM and 2,4,6-triamino-5-pyrimidinecarbonitrile are uniform.3 Stability study of TUM and 2,4,6-triamino-5-pyrimidinecarbonitrileThe stability influence factors experiments of TUM and 2,4,6-triamino-5pyrimidine-carbonitrile include high temperature test(60?,respectively),high humidity test(90%±5%.25?),light test(25?,45001x±5001x).The results show that TUM and 2,4,6-triamino-5-pyrimidinecarbonitrile have deliquescence,unstable in light and high temperature.So TUM and 2,4,6-triamino-5-pyrimidinecarbonitrile preferably store in dark,cool,dry,airproof conditions.The stability test of TUM include short term stability test for 4 weeks(40?)and long term stability test for 9 months(4?),use T test inspection,there was no significant difference at the 95%confidence level.The results show that TUM has good stability.2,4,6-triamino-5-pyrimidinecarbonitrile in short term stability test for 4 weeks(40 ?)and long term stability test for 6 months(4?),were no significant difference at the 95%confidence level.The 2,4,6-triamino-5-pyrimidine-carbonitrile has good stability.4 Certified value and total uncertainty of TUM and 2,4,6-triamino-5-pyrimidinecarbonitrileThe certified values are based on results of determinations by two analytical techniques,HPLC-DAD and differential scanning calorimetry.The certified value for TUM was assigned as 98.47%with an expanded uncertainty of 0.78%.The certified value for 2,4,6-triamino-5-pyrimidinecarbonitrile was assigned as 98.56%with an expanded uncertainty of 0.76%.This paper was first preparation of TUM and 2,4,6-triamino-5-pyrimidine-carbonitrile reference materials,determined the physicochemical properties,determined the homogeneity and stability fixed the storage and transport conditions and certified the value of two reference materials.This study provides the material foundation for the two drugs in pharmacology,toxicology and residue monitoring.