针对肿瘤血管抑制剂DX1002的含量测定,构建一种基于新型纳米碳点材料(CDs)的特异性荧光定量方法.以柠檬酸和尿素为原料,热解法制得碳点(CACCDs),经TEM、IR、UV/Vis、Flu等方法对其进行结构表征.内滤光效应下碳点荧光可被DX1002特异性定量猝灭,CACCDs浓度为250?μg/mL时,于λex/λem=400/530处测定猝灭前后的荧光差值(ΔF).结果表明,DX1002浓度在2.5～75?μg/mL范围内与ΔF具良好线性关系(r2=0.9988),检出限为1.16?μg/mL,平均加标回收率为101.7％(RSD=2.08％),常见细胞阳离子、微量金属离子、糖类、氨基酸等潜在共存物质及有关物质对测定无干扰,DX1002含量测定的结果(101.3％±1.33％)与HPLC法测定结果(99.4％±1.19％)基本吻合(P>0.05),但较HPLC分析时间快约75倍.该方法快速、灵敏、特异性强,可为建立DX1002高通量特异性体内分析方法提供体外定量的依据.

该文建立大鼠血浆中DX1002的浓度的UPLC-MS/MS法,研究DX1002在大鼠体内的药代动力学特性及其生物利用度.DX1002分别经口给药和静脉给药100 mg/kg后,分别于设定的时间点采血,制备出的血浆通过预处理后采用UPLC-MS/MS法测定,色谱条件为UPLC C18为色谱柱(50 mm×2.10 mm,1.7μm),流量:0.5 mL/min,流动相A相为0.1％甲酸,B相为0.1％甲酸乙腈,采用梯度洗脱;质谱条件为ESI源的MRM检测模式;检测到的血药浓度采用Analyst软件计算药代动力学参数.结果DX1002在1.0～2000 ng/mL内线性良好(r2≥0.9979),精密度、准确度均满足测定需要.口服条件大鼠下平均Cmax分别为35597.0 ng/mL,平均AUC0～24 h为47291.5 h·ng/mL,平均t1/2为2.42 h,平均Tmax为0.235 h;经静脉给药后,雌雄大鼠的平均AUC0～24 h为174043.5 h·ng/mL,平均t1/2为4.26 h.同等剂量下大鼠口服给药的平均绝对生物利用度为27.29％.最后,所建方法是大鼠体内DX1002药代动力学研究的较好方法.

该文以有机弱酸正丁铵盐DX1002为对象,评价弱酸弱碱盐原料药的含量测定方法,优化出HPLC法为DX1002含量测定的最佳方法.根据DX1002的苯丙乙烯酸正丁铵盐的结构特点,分别针对本品的酸根部分、正丁铵离子部分和共轭体系部分等采用容量法(酸碱滴定和非水滴定)、紫外分光光谱法(UV)和高效液相色谱法(HPLC)进行含量测定,通过比较其精密度、回收率和含量结果,评价优选出最佳的方法.DX1002在4种方法分析中均具有良好的线性关系,重复性精密度RSD在0.063％～2.0％,平均加样回收率在99.4％～101.8％,平均含量99.3％～103.6％,其中非水滴定法和HPLC法的含量结果<100％,酸碱滴定法和光谱法的含量结果>100％.结果表明,4种定量方法没有显著性差异(P>0.05),但采用HPLC法可更好地克服供试样品中共存杂质的干扰,具有高专属、高灵敏和良好精密度等优点,建议可作为弱酸铵盐类化合物DX1002含量测定的较好方法.

To set up a systematic study, including the analysis on the physical and chemical features and the establishment of the methods for analysis of related substances, determination of content and the determination of residual solvent, on the CA4 acid-an important intermediates of Combretastatins, a tumor angiogenesis inhibitors-and provide the foundation for farther study on this kind of angiogenesis inhibitors. The chemical structure was confirmed by IR, UV, MS and NMR. Related substances was analyzed by HPLC and the chromatographic conditions were as follows: C18(150 mm×4.60 mm,5 μm); the mobile phase contained a mixture of 0.1% formic acid buffer (ammonia to pH 6.8), methanol and acetonitrile in a ratio of 75:22:3 (v/v/v); with the wavelongth of 220 nm; The HS-GC chromatographic conditions for determination of residual solvent ethanol were as follows: analyzing in PEG capillary column (30 m×0.53 mm, 1 μm)with column temperature at 80 ℃, the detector temperature of FID at 250℃ and injection port temperature at 200℃, the flow rate of carrier gas nitrogen was 4.0 mL/min with diversion ratio of 10:1. Related substances were identified by LC/MS, and the content of CA4 acid was determined by the volumetric analysis. CA4 acid was light yellow amorphous powder, and was almost insoluble in water.The structure of CA4 acid is (E)-3-[(3-hydroxy-4-methoxyphenyl)]-2-(3, 4, 5-trimethoxyphenyl)acrylic acid. The fifth and second of its' six related substances were trimethoxy phenylacetic acid and isovanillin, the starting materials of CA4 acid, while the sixth was the (Z)-CA4 acid, which transformed from CA4 acid mainly because of light influences. The content of residual solvent ethanol was less than 0.08%. As the carboxyl group in the molecule can react with an alkaline compound to form a soluble salt, CA4 acid is seen as the important intermediates of Combretastatins which could enhance the efficiency of inhibiting tumor blood vessels.

Zhang’s technique system organization (ZTSO) has been rapidly optimized and ion chromatography determination of n-butylamine content has been established. AcelaimTM TrinityTM P1 (3.0 mm ×100 mm,3μm) was adopted as chromatographic column. The leacheate was 18 mmol/L ammonium acetate(adjust pH 3.4 by using acetic acid)-acetonitrile(56∶44,υ∶υ),and the detector was conductivity detector. Under the condition of 37℃ column temperature and isocratic elution at a flow rate of 0.15 mL/min,the measurement of n-butylamine content could be done in 20 minutes. Show that there was a good linear relationship (r2=0.999 8) at range of the concentration of 10.01-500.5μg/mL,the limit of detection is 5.05μg/mL (S/N≥3),the limit of quantization is 10.01μg/mL (S/N≥10),the intra-day and the inter-day precision were at 0.33% and 0.90% respectively,n-butylamine solution was stable(RSD=0.54%) in 24 hours. The average content of n-butylamine raw materials in three batches are 99.79% (RSD=0.125%), the average n-butylamine content is 20.32%(RSD=4.09%) in three batches of tumor angiogenesis inhibitors XY03. When heat drying, the decrease rate of ammonium salt in XY03 was positively related to its solubility. ZTSO technology has good conditions for optimizing function. The method is rapid,simple and accurate, which is a better method for the assay of n-butylamine or containing the butyl ammonium of compounds.

The spectrums of 1H-NMR and 13C-NMR and others in combination with MS and IR were applied to analyze the chemical structure of tumor angiogenesis inhibitor DX-1002. Experimental results indicate that the chemical shifts of the 1H-NMR and 13C-NMR are consistent with the hydrogen atoms and carbon atoms of DX-1002. The spatial structure of molecules was determined on the basis of gCOSY,gHMQC and NOESY. And then the chemical structure of DX-1002 was confirmed via mass spectrum and infrared spectrum to be (E)-3-(3"-hydroxy-4"-methoxyl phenyl)-2-(3',4',5'-triethoxyphenyl) N-butyl acrylate ammonium salt.