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Volume 38 Issue 2
Apr.  2020
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SONG Weiguo, LU Yonghui, ZHANG Zhonghu, ZHANG Huimin. Simultaneously detect three illegal additive components in Yinhuang soft capsule by TLC-SERS[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(2): 166-169. doi: 10.3969/j.issn.1006-0111.201908092
Citation: SONG Weiguo, LU Yonghui, ZHANG Zhonghu, ZHANG Huimin. Simultaneously detect three illegal additive components in Yinhuang soft capsule by TLC-SERS[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(2): 166-169. doi: 10.3969/j.issn.1006-0111.201908092

Simultaneously detect three illegal additive components in Yinhuang soft capsule by TLC-SERS

doi: 10.3969/j.issn.1006-0111.201908092
  • Received Date: 2019-08-21
  • Rev Recd Date: 2020-01-27
  • Available Online: 2020-04-23
  • Publish Date: 2020-03-01
  •   Objective  To establish a simultaneous detection of diclofenac sodium, fenbufen and rotundine hydrochloride illegally added in Yinhuang soft capsules by TLC-SERS.  Methods  The samples to be measured were placed on the silica gel plate, and the thin layer was developed with petroleum ether-ethyl acetate (3:5). Location detection was carried out under 254 nm ultraviolet lamp. Nano-silver colloidal solution was sprayed at each spot of separation according to the optimum conditions, and then qualitative identification was done by TLC-SERS.  Results  A method for simultaneous determination of diclofenac sodium, fenbufen and rotundine hydrochloride illegally added in Yinhuang soft capsule by TLC-SERS was established. And the minimum detection limits of three chemical constituents were determined.  Conclusion  TLC-SERS was a rapid, accurate and sensitive method for the simultaneous and rapid detection of diclofenac sodium, fenbufen and rotundine hydrochloride illegally added into Yinhuang soft capsule.
  • [1] 蓝献泉, 黄义纯, 黄红雯, 等. 抗风湿类中成药及保健品中非法添加双氯芬酸钠的快速筛查方法研究[J]. 中南药学, 2014, 12(9):902-905. doi:  10.7539/j.issn.1672-2981.2014.09.020
    [2] 吴小红, 李焕德, 朱荣华, 等. HPLC-MS/MS法鉴别中药制剂中非法添加的多种解热镇痛类化学药物[J]. 中南药学, 2010, 8(10):724-729. doi:  10.3969/j.issn.1672-2981.2010.10.002
    [3] ZHANG J N, MA X Y, WANG Z P. Real-time and in situ monitoring of abrin induced cell apoptosis by using SERS spectroscopy[J]. Talanta,2019,195:8-16. doi:  10.1016/j.talanta.2018.11.015
    [4] LI D W, QU L L, ZHAI W L, et al. Facile on-site detection of substituted aromatic pollutants in water using thin layer chromatography combined with surface-enhanced Raman spectros- copy[J]. Environ Sci Technol,2011,45(9):4046-4052. doi:  10.1021/es104155r
    [5] 王磊, 陈梦云, 李晓, 等. TLC-SERS法快速分析抗精神失常类中药的化药成分[J]. 实验室研究与探索, 2016, 35(5):22-26. doi:  10.3969/j.issn.1006-7167.2016.05.006
    [6] 张彬彬, 史毅, 陈辉, 等. 薄层色谱-表面增强拉曼光谱法快速检测染色掺伪的西红花[J]. 药学实践杂志, 2017, 35(3):215-218. doi:  10.3969/j.issn.1006-0111.2017.03.006
    [7] 赵瑜, 尹利辉, 胡昌勤. 拉曼光谱法在快速筛查紫杉醇脂质体制剂中的应用[J]. 中国医药导报, 2018, 15(19):24-29.
    [8] 张中湖, 李军, 谢强胜, 等. TLC-SERS高通量筛查中成药与保健食品中添加的降糖化学物质[J]. 药物分析杂志, 2019, 39(3):467-476.
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Simultaneously detect three illegal additive components in Yinhuang soft capsule by TLC-SERS

doi: 10.3969/j.issn.1006-0111.201908092

Abstract:   Objective  To establish a simultaneous detection of diclofenac sodium, fenbufen and rotundine hydrochloride illegally added in Yinhuang soft capsules by TLC-SERS.  Methods  The samples to be measured were placed on the silica gel plate, and the thin layer was developed with petroleum ether-ethyl acetate (3:5). Location detection was carried out under 254 nm ultraviolet lamp. Nano-silver colloidal solution was sprayed at each spot of separation according to the optimum conditions, and then qualitative identification was done by TLC-SERS.  Results  A method for simultaneous determination of diclofenac sodium, fenbufen and rotundine hydrochloride illegally added in Yinhuang soft capsule by TLC-SERS was established. And the minimum detection limits of three chemical constituents were determined.  Conclusion  TLC-SERS was a rapid, accurate and sensitive method for the simultaneous and rapid detection of diclofenac sodium, fenbufen and rotundine hydrochloride illegally added into Yinhuang soft capsule.

SONG Weiguo, LU Yonghui, ZHANG Zhonghu, ZHANG Huimin. Simultaneously detect three illegal additive components in Yinhuang soft capsule by TLC-SERS[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(2): 166-169. doi: 10.3969/j.issn.1006-0111.201908092
Citation: SONG Weiguo, LU Yonghui, ZHANG Zhonghu, ZHANG Huimin. Simultaneously detect three illegal additive components in Yinhuang soft capsule by TLC-SERS[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(2): 166-169. doi: 10.3969/j.issn.1006-0111.201908092
  • 银黄软胶囊是一种清热、解毒、消炎作用的中成药。因中药起效慢、副作用少,一些不良商家为了寻求暴利,在中成药中添加西药成分,短时间内提高中成药的疗效,误导消费者以寻求高利润。因双氯芬酸钠有消炎镇痛作用、芬布芬有抗炎作用、盐酸罗通定有镇痛作用,就成了不良商家非法添加的成分之列,据报道有30%的抗风湿类健康产品中非法添加了双氯芬酸钠[1],多种抗风湿镇痛类中药制剂中非法添加双氯芬酸钠、芬布芬等此类化学药物[2]。因此,建立一种快速有效的检测方法,对可能非法添加了西药成分的中成药进行现场检测具有重要的意义。

    近几年,薄层色谱-表面增强拉曼光谱(TLC-SERS)联用技术广泛地应用于各种领域[3-4],尤其是在药物非法添加研究领域被逐渐认可[5-8]。本研究应用TLC-SERS联用技术,探索双氯芬酸钠、芬布芬、盐酸罗通定在中成药银黄软胶囊中非法添加的鉴别方法,以期为进一步的中成药快速检验提供实验依据。

    • 便携式拉曼光谱仪(BWS415,B&W Tek Inc., U.S.A),激光发射波长785 nm,分辨率5 cm–1;WFH-203B型三用紫外分析仪(上海精科实业有限公司);离心机(TG16-WS,上海卢湘仪离心机有限公司);KQ-250DB型数控超声波清洗器(昆山超声仪器有限公司);薄层板HSGF254(烟台市化学工业研究所)。

      双氯芬酸钠(批号:100334-200302,供含量测定用)、芬布芬(批号:100415-201102,纯度99.4%)、盐酸罗通定(批号:100222-200702,供鉴别用)均购自中国食品药品检定研究院。实验所用阴性样品由山东省食品药品检验所提供,银黄软胶囊(批号:359161004,石药集团欧意药业有限公司)。乙酸乙酯(国药集团化学试剂有限公司,批号:20150410)、石油醚(天津市富宇精细化工有限公司,批号:12589-2007);硝酸银(批号:20140320,国药集团化学试剂有限公司),柠檬酸钠(分析纯,批号:20120429,国药集团化学试剂有限公司);实验用水为蒸馏水。

    • 对照品溶液:精密称取对照品双氯芬酸钠、芬布芬、盐酸罗通定对照品2 mg,置EP管中,加入甲醇2 ml溶解,制成浓度为1 mg/ml的溶液,作为对照品溶液,冷藏保存,备用。

      模拟假阳性样品溶液:取中成药银黄软胶囊(经山东省食品药品检验院薄层色谱-质谱联用法检测,不含以上3种对照品,可作为阴性样品)一次剂量,置EP管中,并精密量取双氯芬酸钠、芬布芬、盐酸罗通定对照品溶液加入EP管中,与银黄软胶囊样品溶液混匀,甲醇溶解,振荡,超声(40 kHz,250 W)30 min,10 000 r/min离心10 min,收集上清液,微孔滤膜滤过,作为假阳性样品,冷藏保存,备用。

      阴性样品溶液:取中成药银黄软胶囊一次剂量,置EP管中,加入甲醇2 ml溶解,振荡,超声(40 kHz,250 W)30 min,10 000 r/min离心10 min,取上清液,微孔滤膜滤过,作为阴性样品,冷藏保存,备用。

    • 精密称取45 mg的硝酸银,加入少量去离子水使其溶解。溶解后移至250 ml的容量瓶中,用去离子水定容。用电磁搅拌加热器不断搅拌并将其加热至微沸,逐滴加入5 ml浓度为1 %柠檬酸钠溶液并继续加热,保持微沸60 min,继续搅拌至溶液冷却,得灰绿色银溶胶,避光保存[8]

    • 分别取1 mg/ml双氯芬酸钠、芬布芬、盐酸罗通定对照品溶液,以点样点胶比为变量,激光功率,积分时间为定值,检测出峰效果,筛选3种成分的最佳点样点胶比;同理筛选3种成分的最佳激光功率、积分时间。

    • 以对照品溶液与阴性样品溶液作为对照,将配制的添加不同浓度对照品的模拟阳性样品点在同一块硅胶薄层板上,按优选的最佳光谱条件设置点样点胶比、激光功率、积分时间,根据信噪比(S/N=3)评价出峰效果,确定各个对照品的检测限。

    • 将配置好的各种样品、对照品溶液置于薄曼联用仪内对应位置,高效硅胶薄层板置于仪器平台上,设置点样量与点样位置,由联用仪完成点样操作。取出点样后的薄层板,以选定的石油醚-乙酸乙酯(3:5)展开系统,室温下展开80 mm,取出晾干,放回联用仪,于内置254 nm紫外灯下检视定位以确定点胶位置,设置合适的点胶量、激光强度与积分时间,由联用仪完成点胶操作后,随即进行拉曼检测。

    • 对照品双氯芬酸钠、芬布芬、盐酸罗通定最佳出峰条件分别是:双氯芬酸钠点样点胶比2:3,激光功率30%,积分时间30 s;芬布芬点样点胶比2:2,激光功率40%,积分时间30 s;盐酸罗通定点样点胶比1:2,激光功率50%,积分时间30 s。拉曼图谱如图1所示。

    • 图2图3图4中可以看出,3种对照品在薄层色谱中分离程度高,双氯芬酸钠的比移植(Rf)值为0.75,芬布芬Rf值为0.54,盐酸罗通定Rf值为0.21。由“2.1”项可知,双氯芬酸钠、芬布芬、盐酸罗通定最佳点样点胶比分别为2:3、2:2、1:2(即点样量分别为2 μl、2 μl、1 μl,故银黄软胶囊中添加的双氯芬酸钠、芬布芬、盐酸罗通定最低检测限对应的点样沉积量分别为0.2 μg、0.2 μg、0.1 μg。

    • 图5薄层板中可以看出,同时添加了3种对照品的银黄软胶囊模拟的假阳性样品溶液4、5、6、7号中出现了与双氯芬酸钠、芬布芬、盐酸罗通定相同Rf值的斑点,可初步确定4、5、6、7中添加了双氯芬酸钠、芬布芬、盐酸罗通定。为进一步确证,对各斑点进行SERS检测,在拉曼仪数据处理系统中与相应对照品色谱斑点的SERS光谱进行比对,结果表明4、5、6、7号中对应斑点的SERS图谱与双氯芬酸钠、芬布芬、盐酸罗通定对照品的SERS图谱相同,见图5中SERS图谱。

    • 图6可知,双氯芬酸钠、芬布芬、盐酸罗通定3种成分的分子结构中均含有苯环、C—C、C=O基团,故3种成分的拉曼光谱特征峰有9个共有峰,在576、712、1 004、1 130、1 240、1 316、1 394、1 454、1 564 cm–1左右,同一共有峰的强度有所不同,取代基不同拉曼位移略有变化(见表1)。此外,由于3种成分的分子结构含有不同的基团,也有相应的差异峰,如740 cm–1为盐酸罗通定特有峰。共有峰、差异峰以及峰强度共同组成了区别于其他成分的特征峰群,这为TLC-SERS专属性鉴别提供了依据。

      成分特征峰
      12345678910111213
      双氯芬酸钠3905767128541 0041 1301 1621 2381 3161 3941 4541 570
      芬布芬3845767128541 0041 1301 2401 2761 3161 3941 4541 564
      盐酸罗通定3945767127401 0051 1321 1681 2401 2781 3201 3951 4561 564
    • 本研究通过反复试验,确定了TLC展开剂为石油醚-乙酸乙酯(3:5),得到了较好的展开效果。在展开的TLC薄层板上,3种对照品对应的斑点处,模拟假阳性样品与3种对照品有相同斑点;经过进一步的SERS检测,模拟假阳性样品的斑点拉曼光谱特征峰与对照品斑点拉曼光谱特征峰一致,且重复性良好,说明TLC-SERS联用技术简便、准确、可靠,可同时检测银黄软胶囊中非法添加的双氯芬酸钠、芬布芬、盐酸罗通定3种成分。

      本研究采用TLC-SERS联用技术所建立的检测方法,对中成药银黄软胶囊中非法添加西药双氯芬酸钠、芬布芬、盐酸罗通定3种成分的,能满足同时、快速检测的要求。该方法实用性强,对人员技术要求低,尤其适合现场快速检测。

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