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目的建立同时测定给予丹红注射液主要组分后脑缺血再灌注损伤大鼠血浆中丹参素、原儿茶酸、香草酸、丹酚酸B和羟基红花黄色素A(HYSA)质量浓度的HPLC-DAD方法,为其药动学研究提供准确可靠的方法。方法制备大鼠大脑中动脉栓塞(MCAO)模型,iv丹红注射液主要有效成分丹参素、原儿茶醛、丹酚酸B和HYSA后,收集血样,采用Eclipse XDB-C18(150 mm×4.6 mm,5μm)色谱柱,以乙腈-0.4%磷酸水溶液为流动相进行梯度洗脱,检测波长280 nm用于检测丹参素、原儿茶酸、香草酸和丹酚酸B,403 nm用于检测HYSA,柱温为30℃,内标法定量。结果原儿茶醛在MCAO大鼠体内迅速被代谢,3 min即可检测到其代谢产物原儿茶酸和香草酸。丹参素、原儿茶酸、香草酸、丹酚酸B和HYSA的进样浓度分别在0.35~140 mg/L(R2=0.999 8)、0.15~60 mg/L(R2=0.999 0)、0.05~20 mg/L(R2=0.998 8)、0.25~100 mg/L(R2=0.999 6)、0.075~30 mg/L(R2=0.998 5)呈良好线性关系,平均回收率均在80%~120%,日内、日间RSD均小于10%,稳定性符合体内药物分析要求。大鼠iv给予丹红注射液主要有效成分后丹参素、原儿茶酸、香草酸、丹酚酸B和HYSA的主要药动学参数:AUC0~∞分别为(1143.862±230.840)、(427.024±59.293)、(135.785±47.631)、(418.631±66.242)、(288.788±87.809)mg·min/L,t1/2z分别为(71.496±29.067)、(82.379±26.279)、(40.331±6.006)、(125.164±59.709)、(177.577±112.836)min。结论所建立的HPLC-DAD分析方法专属性强,各成分分离度好,分析时长适宜,操作简单快速,可用于MCAO大鼠体内丹参素、原儿茶酸、香草酸、丹酚酸B、HYSA的同时测定及复方丹红注射液的药动学研究。
OBJECTIVE To establish a method for the simultaneous determination of Danshensu, protocatechuic acid, vanillic acid, salvianolic acid B and hydroxysafflor yellow A (HYSA) in plasma of rats with cerebral ischemia-reperfusion injury after the administration of Danhong injection HPLC-DAD method provides an accurate and reliable method for its pharmacokinetic study. Methods The middle cerebral artery occlusion (MCAO) model of rats was established. Blood samples were collected after the main effective components of Danshen Injection (Danshensu, protocatechuic aldehyde, salvianolic acid B and HYSA) were collected. The blood samples were collected using Eclipse XDB-C18 mm, 5μm) column with a mobile phase of acetonitrile-0.4% phosphoric acid as mobile phase. The detection wavelength was 280 nm for detection of Danshensu, protocatechuic acid, vanillic acid and salvianolic acid B at 403 nm for detection HYSA, column temperature is 30 ℃, internal standard method. Results Protocatechuic aldehyde was rapidly metabolized in MCAO rats and its metabolites protocatechuic acid and vanillic acid were detected in 3 minutes. Danshensu, protocatechuic acid, vanillic acid, salvianolic acid B and HYSA were in the range of 0.35-140 mg / L (R2 = 0.999 8), 0.15-60 mg / L (R2 = 0.998 8), 0.25-100 mg / L (R2 = 0.999 6) and 0.075-30 mg / L (R2 = 0.998 5). The average recoveries were in the range of 80% 120%, day and day RSD were less than 10%, stability in line with the requirements of in vivo drug analysis. The main pharmacokinetic parameters of Danshensu, protocatechuic acid, vanillic acid, salvianolic acid B and HYSA after iv administration of Danhong injection were (1143.862 ± 230.840) and (427.024 ± 59.293, 135.785 ± 47.631, 418.631 ± 66.242, 288.788 ± 87.809 mg · min / L and t1 / 2z were (71.496 ± 29.067), (82.379 ± 26.279), (40.331 ± 6.006) and 125.164 ± 59.709), (177.577 ± 112.836) min. Conclusion The established HPLC-DAD method has the advantages of high specificity, good resolution, long analysis time and simple and quick operation. It can be used for the determination of Danshensu, protocatechuic acid, vanillic acid, salvianolic acid B, HYSA Simultaneous Determination and Pharmacokinetics of Compound Danhong Injection.