复方龙胆碳酸氢钠片中非法成分土大黄苷定性筛查与定量测定方法的建立

目的:建立复方龙胆碳酸氢钠片组方药材大黄伪品中指标性成分土大黄苷的定性筛查与定量测定方法。方法:从药品流通领域抽样获取来自国内8家药品生产企业(编号:A^H)的45批复方龙胆碳酸氢钠片,采用薄层色谱法(TLC)初步定性鉴别样品中的土大黄苷;采用高效液相色谱法(HPLC)对土大黄苷进行含量测定;采用超高效液相色谱-串联质谱法(UPLC-MS/MS)进一步对检出的土大黄苷成分进行结构确证。结果:TLC法检测结果显示,来自D企业的10批样品在紫外光灯(365nm波长)可见土大黄苷的亮蓝色荧光斑点。HPLC法方法学考察结果显示,土大黄苷质量浓度在0.884~88.4μg/mL范围内与峰面积呈良好的线性关系(r=0.9999);进样量检出限、定量限分别为0.7072、3.536ng;精密度、重复性、稳定性试验RSD均小于1%;平均加样回收率为96.55%(RSD=0.53%,n=6)。D企业产10批样品中土大黄苷含量为0.7324~2.8908mg/g。UPLC-MS/MS法检测结果显示,D企业产样品与土大黄苷对照品均有质荷比(m/z)419.0的准分子离子峰和m/z257.1、241.2的碎片离子峰。结论:建立的TLC法定性初筛、HPLC法定量测定与UPLC-MS/MS法结构确证相结合的检测方法操作简便、灵敏、可靠,可用于复方龙胆碳酸氢钠片中土大黄苷的定性筛查和定量测定。45批次抽检样品中,有来自1个生产企业的10批样品中检出土大黄苷成分,提示该企业在生产复方龙胆碳酸氢钠片时存在以伪品大黄药材替代正品的现象。

Method Establishment for Qualitative Screening and Quantitative Determination of Illegal Composition Rhaponiticin in Compound Gentian and Sodium Bicarbonate Tablets

OBJECTIVE: To establish a method for qualitative screening and quantitative determination of indicative composition rhaponiticin from counterfeit Rheum palmatum in Compound gentian and sodium bicarbonate tablets. METHODS: Totally 45 batches of Compound gentian and sodium bicarbonate tablets were collected from 8 domestic pharmaceutical manufacurers(No. A-H)in the field of drug distribution. TLC method was used to identify rhaponiticin in the samples primarily. The content of rhaponiticin was determined by HPLC,and then UPLC-MS/MS method was used to confirm the structure of rhaponiticin. RESULTS:TLC results showed that bright blue fluorescent spots of rhaponiticin could be seen in 10 batches of samples from manufacturer D at 365 nm wavelength of ultraviolet lamp. Results of HPLC methodology investigation showed that the linear range of rhaponiticin was 0.884-88.4 μg/mL(r=0.999 9);the detection limit and quantitative limit were 0.707 2,3.536 ng;RSDs of precision,reproducibility and stability tests were all lower than 1%;average recovery was 96.55%(RSD=0.53%, n=6). The contents of rhaponiticin in 10 batches of samples from manufacturer D were 0.732 4-2.890 8 mg/g. Results of UPLC-MS/MS method showed that quasimolecular ions with m/z of 419.0 and fragment ions with m/z 257.1 and 241.2 were found in both samples from manufacturer D and rhaponiticin control. CONCLUSIONS:TLC for primary screening,HPLC for content determination and UPLC-MS/MS for structure confirmation is simple,sensitive and reliable,and can be used for qualitative screening and quantitative determination of rhaponiticin in Compound gentian and sodium bicarbonate tablets. Among 45 batches of samples tested,rhaponiticin is detected in 10 batches of samples from one manufacturer,suggesting that the manufacturer substitute fake R. palmatum for genuine ones in the production of Compound gentian sodium bicarbonate tablets.