经典名方两地汤基准样品的质量标准建立

Establishment of Quality Standard for Benchmark Samples of Liangditang

目的:建立经典名方两地汤基准样品的质量标准。方法:采用超高效液相色谱-四极杆飞行时间质谱法(UPLCQTOF-MS),根据分子离子峰和碎片离子峰信息,结合质谱裂解规律对两地汤的化学成分进行定性分析,流动相甲醇(A)-0.05%磷酸水溶液(B)(0~10 min,5%~23.5%A;10~20 min,23.5%A;20~58 min,23.5%~63%A;58~60 min,63%~90%A),流速0.8 mL·min^(-1),检测波长254 nm;电喷雾离子源(ESI),正离子模式,检测范围m/z 100~1700。通过与单味药及对照品进行对比指认,确定关键质量属性及来源;采用高效液相色谱法(HPLC)对多批次饮片-基准样品进行量质传递分析,建立两地汤的指标成分含量测定及特征图谱检测方法,通过对15批基准样品的测定,确定指标成分含量范围及特征图谱共有峰;采用薄层色谱法(TLC)对处方中的5味药进行鉴别;采用烘干法对基准样品进行水分和干膏得率测定。结果:两地汤基准样品中共鉴定出27个化合物,其中9个与对照品比对后确认,分别为梓醇、哈巴苷、没食子酸、芍药内酯苷、芍药苷、毛蕊花糖苷、安格洛苷C、肉桂酸、哈巴俄苷。建立了基准样品的特征图谱检测方法,确定13个共有峰,特征峰主要来源于酒地黄、玄参和酒白芍,15批基准样品的特征图谱与对照特征图谱的相似度均>0.9;建立了芍药苷、哈巴俄苷、L-羟脯氨酸、甘氨酸4个指标成分的含量测定方法,15批基准样品的测定结果均在对应均值的±30%范围内,且饮片-基准样品的转移率稳定可控;建立了采用2种供试品溶液对处方5味药(阿胶除外)进行鉴别的TLC,检测结果显示,该方法专属性良好;15批基准样品的平均干膏得率48.06%,平均水分5.58%,分别在各自均值的±10%和±30%范围内。结论:建立的两地汤基准样品的质量标准为基准样品与对照特征图谱的相似度>0.9,芍药苷、哈巴俄苷、L-羟脯氨酸、甘氨酸的含量标准分别为217~403、24~46、634~1178、1253~2328 mg/剂,干膏得率43.0%~53.0%,水分4.0%~7.0%,5味药在设定的TLC检测条件下,对照药材和两地汤基准样品存在对应特征斑点。该质量标准稳定可靠,填补了经典名方两地汤质量控制方面的空白,可为两地汤颗粒制剂质量标准的建立提供参考依据。

Objective:To establish the quality standard of Liangditang benchmark samples.Method:Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-QTOF-MS)was used to qualitatively analyze the chemical composition of Liangditang on the basis of molecular and fragment ion peak information with cracking law.The mobile phase was methanol(A)-0.05%phosphate aqueous solution(B)for gradient elution(0-10 min,5%-23.5%A;10-20 min,23.5%A;20-58 min,23.5%-63%A;58-60 min,63%-90%A),the flow rate was 0.8 mL·min^(-1),and the detection wavelength was 254 nm.Electrospray ionization was employed under positive ion mode,the detection range was m/z 100-1700.Key quality attributes and sources were determined by comparing with single medicine and reference substances.Through mass transfer analysis of multiple batches from decoction pieces to benchmark samples,high performance liquid chromatography(HPLC)for determining the contents of index components and HPLC detection of characteristic maps were established.Through the determination of 15 batches of benchmark samples,the content range of the index components and the common peaks of the characteristic map were determined.Thin layer chromatography(TLC)was applied to the identification of 5 medicines in the formula.Moisture and dry extract yield of the benchmark samples were determined by drying method.Result:A total of 27 compounds were inferred from the benchmark samples of Liangditang,among which 9 compounds were confirmed by comparison with the control,including catalpol,harpagide,gallic acid,albiflorin,paeoniflorin,verbascoside,angoroside C,cinnamic acid and harpagoside.A method for determining the characteristic maps of the benchmark samples were established and 13 peaks were assigned,and the characteristic peaks were mainly derived from wine-processed products of Rehmanniae Radix,Scrophulariae Radix and wine-processed products of Paeoniae Radix Alba.The similarity between the characteristic map of 15 batches of benchmark samples and the control characteristic map was>0.9.Methods for the determination of paeoniflorin,harpagoside,L-hydroxyproline and glycine were established,and the contents of these four components in 15 batches of benchmark samples were within±30%of the corresponding mean value,and the transfer rate of decoction pieces to the benchmark samples was stable and controllable.TLC was established to identify 5 prescription drugs(except Ejiao)with two kinds of test solutions,and the results showed that the method had good specificity.The average dry extract yield was48.06%,and the average moisture was 5.58%,which were within the range of±10%and±30%of their mean values,respectively.Conclusion:The quality standard of Liangditang benchmark samples was as follows:the similarity between the benchmark samples and the control characteristic map is>0.9,the contents of paeoniflorin,harpagoside,L-hydroxyproline and glycine are 217-403,24-46,634-1178,1253-2328 mg per dose,the dry extract yield is 43.0%-53.0%,the moisture is 4.0%-7.0%,under the set detection conditions,the benchmark samples have corresponding characteristic spots by comparing with the control herbs of 5 medicines.This quality standard is stable and reliable,which fills the gap in the quality control of Liangditang,and can provide a reference for the establishment of the quality standard of Liangditang granules.