【作者】 杜小燕；

【导师】 蒋金书； 朱蓓蕾；

【作者基本信息】 中国农业大学 ， 预防兽医学， 2004， 博士

【摘要】 复方丁氨丙磷注射液（商品名Catosal）是一种有机磷酸化合物和维生素组成的复方注射液，其主要有效成分为丁氨丙磷和维生素B₁₂，二者结合可起到提高能量代谢和增强免疫力的作用。作为代谢促进剂和强壮剂用于各种家畜，其主要的适应症为：由营养不良、管理不善和疾病引起的家畜代谢紊乱和免疫力下降，幼畜发育不良、营养失调；同时还用于治疗母畜不孕、过度使役和疲劳以及轻瘫和肌强直时钙、镁治疗的辅助剂。为了在我国自主开发该产品，本课题组研究合成了其主要成分丁氨丙磷，并在此基础上研制出两种制剂，命名为复方丁氨丙磷注射液和溶液剂，本文主要进行复方丁氨丙磷注射液的药学和药代动力学研究。 本研究建立了电位滴定和紫外衍生化HPLC两种丁氨丙磷原料药的含量测定方法。用0.1mol／L的氢氧化四丁基氨对丁氨丙磷进行电位滴定，该法的日内变异系数在0.8％～2.3％之间，日间变异系数2.2％，精密度和重复性良好，简便易行。本研究成功地对丁氨丙磷进行了丹磺酰化衍生，对衍生产物进行了HPLC、TLC和MS鉴定，对衍生条件进行优化。结果表明，对丁氨丙磷成功地进行了丹磺酰化反应，在浓度为0.2mg／mL～2.0mg／mL之间，用pH值9.80的缓冲液、乙睛溶解Dns-Cl、反应温度为50℃、反应60min的条件下，衍生产物峰高和丁氨丙磷的浓度呈线性相关，线性方程为Y=16026X+180.89，R²=0.9978，可用于定量检测。本研究还建立了丁氨丙磷原料药的TLC杂质检查方法。 选用德国拜耳公司的注射液配方，进行适当调整后研制成复方丁氨丙磷注射液，建立了测定注射液主要成分丁氨丙膦的HPLC-RID方法和另一有效成分维生素B₁₂的HPLC含量测定方法。用加温加湿和光加速试验对注射液的稳定性进行研究，结果表明，所研制的注射液对温湿比较稳定，六个月加温加湿试验末的存留率分别为丁氨丙磷96.58％和维生素B₁₂91.16％；对强光不稳定，需避光保存。 本试验进行了马血清中丁氨丙磷的SPE净化、LC-MS检测方法的研究。以甲醇作为血清中丁氨丙磷的提取溶剂和蛋白沉淀剂，提取液经减压旋转蒸发后加入1.5mL缓冲液（pH3.5）。对多种SPE柱进行筛选后，选用C18固相萃取柱进行净化。洗脱液经减压旋转蒸发和吹干后，加水溶解进行LC-MS选择离子检测。检测条件如下：LC-MS系统中LC的测定条件：色谱柱为Symmetry（?）C18（3.5μm，2.1×150mm），流动相为甲醇：醋酸水溶液（pH值3.0）／10:90，等度洗脱；流速：0.2mL／min；柱温为20℃；进样量20uL。以ESI+电离模式下的选择离子检测m／z114进行定量。在浓度0.02～10.0ug／mL浓度范围内，丁氨丙磷浓度和峰面积呈良好的线性关系（R²=0.999），五倍噪音的检测限为10ng／mL。添加水平0.1～10ug／mL浓度范围内，马血清样品中丁氨丙磷的添加回收率为94.51％～114.23％，变异系数C．V．在4.028％～8.23％之间。 本试验以马为试验对象，用自行建立的LC-MS方法进行了复方丁氨丙磷注射液的药代动力学研究。共用6匹马分两个剂量组进行试验（每组三匹马），剂量分别为5mg／kg bw和2.5mg／kg bw。静脉注射后，分别在1、6、12、30min和1、2、4、8、16、24、32h时间点采血，分离血清，LC-MS测定血清中的丁氨丙磷含量并用3P97药代动力学程序进行处理。结果表明，血清中丁氨丙磷的浓度变化符合权重1／C²的二室模型。平均最高浓度分别为高剂量组87.811ug／mL更多还原

【Abstract】 Compound butafosfan injection (trade name: Catosal) is an aqueous solution. Both of butafosfan and vitamin B12 are the main ingredients of the compound. Butafosfan is an organic phosphorus compound and is active in stimulating metabolism and modulating the immune system. The preparation has been used as metabolic stimulant and tonic in a wide variety of species. The indications are metabolic disorder through malnutrition or faulty husbandry, developmental and nutritional disorder in young animals, metaphylaxis of sterility and puerperal diseases, tetany and pareses as adjunct to calcium and magnesium therapy and as effctive tonic in cases of over-exertion and exhaustion. In order to develop this drug in our country, our research group had synthesized butafosfan and developed two preparations. In our studies, the injection, one of the two preparations and its pharmacokinetics has been studied.For the determination of butafosfan, electronic diluting method and HPLC assay after derivatization with dansyl chloride (Dns-Cl) were developed. The butafosfan was diluted by tetrabutylammoniumoidide (0.1 mol/L). Electronic diluting method was simple and convenient and the C.V.% of intra-day and inter-day was 0.8-2.3% and 2.2%, respectively. The Dns-Cl derivatized butafosfan was identified with HPLC, TLC and MS and the reaction conditions were optimized. The results show that butafosfan was derivatied by Dns-Cl successfully and at pH9.8, the reaction between butafosfan and Dns-Cl acetonitrile solution can be finished after 60 min.The products can be seperated on an inertsil ODS column, acetonitrile-water (1.6:l,v/v) as mobile phase with ultraviolet detection at 254 nm. The peak area of derivative and concentration of butafosfan has good linear (r=0.9988) between 0.2mg/mL~2.0mg/mL. A TLC assay was used in this study to identify impurity in butafosfan.A kind of compound butafosfan injection was prepared based on the formula of the Bayer company after certain changes. For the determination of both injectable butafosfan and vitamin B12, HPLC-RID and HPLC-UV assay were developed, respectively. Thermal stability and the light stability test were undergone. At the end of the thermal stability test, the remains of butafosfan and vitamin B12 were 96.58%and 91.16%, respectively.The experimental results for stability study of the compound showed that our injection was do stable to heat and humidity during the testing period of 6 months, but unstable to strong light. The injection should be stored avoiding the direct sunshine.A specific and rapid LC-MS method to quantitate butafosfan concentration in horse serum was developed. Methanol was applied to extract butafosfan from lmL aliquots of serum and deposit proteins. The extraction was cleaned-up by selected C18 SPE column after evaporation and added 1.5mL buffer (pH3.5). The elute was dried and dissolved with water and injected onto LC column, which was eluted using a methanol/acetic acid gradient (pH3.0) over 10 min. Butafosfan was detected by a mass spectrometer operated in the positive single ion monitoring mode using ESI as an ionization process at m/z 114. The assay is linear between 0.02 and 10ug/mL and the determination limit of the assay was 0.01ug/mL calculated to be five times the baseline noise. The recoveries of the analyte in serum at the fortified level of 0.1, 0.5, 2.0 and 10 ug/mL ranged from 94.51%~ 114.23%. The coefficients of variation are less than 8.23%.The pharmacokinetics of the compound butafosfan injection administrated intravenously at dose rate of 5 mg/kg bw and 2.5 mg/kg bw to 6 horses was studied (3 horses per dose). Serum samples were taken at 1,6,12,30 min and 1,2,4,8,16,24,32h and determined by the LC-MS assay. The butafosfan concentrations in serum were calculated by the software 3P97. The results suggested that the kinetics of the injection was fitted to two compartments. Serum levels of butafosfan declined rapidly from the mean peak levels of 87.811ug/mL for high dose group and 10.224 ug/mL for the low dose group at the first sampling time (1更多还原