同时测定不同人参加工产品中20种人参皂苷的UPLC-PDA方法开发和验证（英文）
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摘要
建立一种简单、有效、精密和准确的超高效液相色谱方法评价不同人参加工产品的质量,同时快速测定20种人参皂苷Rg1、Re、Rf、20(S)-Rg2、20(R)-Rg2、Rb1、Rc、Ra1、Rb2、Rb3、Rd、Rk3、F2、20(S)-Rg3、20(R)-Rg3、Compound K(CK)、Rg5、20(S)-Rh2、20(R)-Rh2和protopanaxadiol(PPD)。采用二极管阵列检测器和ACQUITY UPLC BEH-C18(2.1 mm×50 mm,1.7μm)色谱柱,以乙腈-水为流动相,流速0.3 mL/min,柱温30℃,梯度洗脱。20种人参皂苷在31 min内可达到良好的分离,考察方法的线性范围、回收率、日内和日间精密度。在本方法条件下,线性关系良好,相关系数R2均大于0.998,日内相对标准偏差不大于4.65%,日间相对标准偏差不大于4.88%,回收率为85.71%～108.50%。方法检出限为0.81～3.10μg/m L,方法定量限为2.88～10.00μg/m L。本方法快速、可靠,已成功用于不同人参加工产品包括保鲜参、红参和白参中20种人参皂苷的分析检测,有效揭示不同人参加工产品中人参皂苷含量水平的显著变化,可用于鲜人参及其加工产品中活性化合物的分析和质量控制。
A cost effective, simple, precise and accurate ultra-high performance liquid chromatography(UPLC) method was established and validated for simultaneous determination of 20 ginsenosides in various processed ginseng products, including Rg1, Re, Rf, 20(S)-Rg2, 20(R)-Rg2, Rb1, Rc, Ra1, Rb2, Rb3, Rd, Rk3, F2, 20(S)-Rg3, 20(R)-Rg3, compound K(CK), Rg5, 20(S)-Rh2, 20(R)-Rh2 and protopanaxadiol(PPD). The developed method was carried out on a Waters ACQUITY UPLC BEH C18 column(2.1 mm × 50 mm, 1.7 μm) using a photodiode array detector(PDA). Good chromatographic separation was performed using a mixture of acetonitrile(B) and water(A) as the mobile phase with gradient elution at a flow rate of 0.3 mL/min and column temperature of 30 ℃ within 31 min. The method was well validated with respect to linearity(R2 > 0.998), precision(intra-day RSD ≤ 4.65% and inter-day RSD ≤ 4.88%) and accuracy(recovery rate between 85.71% and 108.50%), and the limit of detection(LOD) and limit of quantification(LOQ) were in the range of 0.81–3.10 μg/mL and 2.88–10.00 μg/mL, respectively. This method was rapid and reliable and was successfully used for the analysis of various processed ginseng products, including freshly stored ginseng, red ginseng and white ginseng and the results revealed significant variations in the ginsenoside levels. This method can be suitable for the analysis of active compounds and the quality control of fresh and processed ginseng.
A cost effective, simple, precise and accurate ultra-high performance liquid chromatography(UPLC) method was established and validated for simultaneous determination of 20 ginsenosides in various processed ginseng products, including Rg1, Re, Rf, 20(S)-Rg2, 20(R)-Rg2, Rb1, Rc, Ra1, Rb2, Rb3, Rd, Rk3, F2, 20(S)-Rg3, 20(R)-Rg3, compound K(CK), Rg5, 20(S)-Rh2, 20(R)-Rh2 and protopanaxadiol(PPD). The developed method was carried out on a Waters ACQUITY UPLC BEH C18 column(2.1 mm × 50 mm, 1.7 μm) using a photodiode array detector(PDA). Good chromatographic separation was performed using a mixture of acetonitrile(B) and water(A) as the mobile phase with gradient elution at a flow rate of 0.3 mL/min and column temperature of 30 ℃ within 31 min. The method was well validated with respect to linearity(R2 > 0.998), precision(intra-day RSD ≤ 4.65% and inter-day RSD ≤ 4.88%) and accuracy(recovery rate between 85.71% and 108.50%), and the limit of detection(LOD) and limit of quantification(LOQ) were in the range of 0.81–3.10 μg/mL and 2.88–10.00 μg/mL, respectively. This method was rapid and reliable and was successfully used for the analysis of various processed ginseng products, including freshly stored ginseng, red ginseng and white ginseng and the results revealed significant variations in the ginsenoside levels. This method can be suitable for the analysis of active compounds and the quality control of fresh and processed ginseng.
引文
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[11]SUN B S,GU L J,FANG Z M,et al.Simultaneous quantification of19 ginsenosides in black ginseng developed from Panax ginseng by HPLC-ELSD[J].Journal of Pharmaceutical and Biomedical Analysis,2009,50(1):15-22.DOI:10.1016/j.jpba.2009.03.025.
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[13]SHI Y,SUN C J,ZHENG B,et al.Simultaneous determination of nine ginsenosides in functional foods by high performance liquid chromatography with diode array detector detection[J].Food Chemistry,2010,123(4):1322-1327.DOI:10.1016/j.foodchem.2010.06.014.
[14]SHAN S M,LUO J G,HUANG F,et al.Chemical characteristics combined with bioactivity for comprehensive evaluation of Panax ginseng C.A.Meyer in different ages and seasons based on HPLC-DAD and chemometric methods[J].Journal of Pharmaceutical and Biomedical Analysis,2014,89:76-82.DOI:10.1016/j.jpba.2013.10.030.
[15]YANG W Z,YE M,QIAO X,et al.A strategy for efficient discovery of new natural compounds by integrating orthogonal column chromatography and liquid chromatography/mass spectrometry analysis:its application in Panax ginseng,Panax quinquefolium and Panax notoginseng to characterize 437 potential new ginsenosides[J].Analytica Chimica Acta,2012,739:56-66.DOI:10.1016/j.aca.2012.06.017.
[16]WANG H P,ZHANG Y B,YANG X W,et al.Rapid characterization of ginsenosides in the roots and rhizomes of Panax ginseng by UPLC-DAD-QTOF-MS/MS and simultaneous determination of 19ginsenosides by HPLC-ESI-MS[J].Journal of Ginseng Research,2016,40(4):382-394.DOI:10.1016/j.jgr.2015.12.001.
[17]XIE Y Y,LUO D,CHENG Y J,et al.Steaming-induced chemical transformations and holistic quality assessment of red ginseng derived from Panax ginseng by means of HPLC-ESI-MS/MSn based multicomponent quantification fingerprint[J].Journal of Agricultural and Food Chemistry,2012,60(33):8213-8224.DOI:10.1021/jf301116x.
[18]ZHANG Y C,PI Z F,LIU C M,et al.Analysis of low-polar ginsenosides in steamed Panax ginseng at high-temperature by HPLC-ESI-MS/MS[J].Chemical Research in Chinese Universities,2012,28(1):31-36.
[19]SHI Y,SUN C,ZHENG B,et al.Simultaneous determination of ten ginsenosides in American ginseng functional foods and ginseng raw plant materials by liquid chromatography tandem mass spectrometry[J].Food Analytical Methods,2013,6(1):112-122.DOI:10.1007/s12161-012-9406-6.
[20]LI L,LUO G A,LIANG Q L,et al.Rapid qualitative and quantitative analyses of Asian ginseng in adulterated American ginseng preparations by UPLC/Q-TOF-MS[J].Journal of Pharmaceutical and Biomedical Analysis,2010,52(1):66-72.DOI:10.1016/j.jpba.2009.12.017.
[21]YANG H,LEE D Y,KANG K B,et al.Identification of ginsenoside markers from dry purified extract of Panax ginseng by a dereplication approach and UPLC-QTOF/MS analysis[J].Journal of Pharmaceutical and Biomedical Analysis,2015,109:91-104.DOI:10.1016/j.jpba.2015.02.034.
[22]SUN T T,LIANG X L,ZHU H Y,et al.Rapid separation and identification of 31 major saponins in Shizhu ginseng by ultrahigh performance liquid chromatography-electron spray ionizationMS/MS[J].Journal of Ginseng Research,2016,40(3):220-228.DOI:10.1016/j.jgr.2015.07.008.
[23]NOVáKOVáL,MATYSOVáL,SOLICH P.Advantages of application of UPLC in pharmaceutical analysis[J].Talanta,2006,68(3):908-918.DOI:10.1016/j.talanta.2005.06.035.
[24]LIU Y,SONG X,YAN R,et al.Development and validation of a UPLC-DAD-MS method for characterization and quantification of alkaloids in Menispermi Rhizoma and its preparations[J].Journal of Food and Drug Analysis,2013,21(2):206-218.DOI:10.1016/j.jfda.2013.05.012.
[25]PARK H W,IN G,HAN S T,et al.Simultaneous determination of 30ginsenosides in Panax ginseng preparations using ultra performance liquid chromatography[J].Journal of Ginseng Research,2013,37(4):457-467.DOI:10.5142/jgr.2013.37.457.
[26]STAVRIANIDI A,STEKOLSHCHIKOVA E,POROTOVA A,et al.Combination of HPLC-MS and QAMS as a new analytical approach for determination of saponins in ginseng containing products[J].Journal of Pharmaceutical and Biomedical Analysis,2017,132:87-92.DOI:10.1016/j.jpba.2016.09.041.
[27]PAN J Y,CHENG Y Y,WANG Y,et al.A method of smiultaneous determination of 9 ginsenosides for identifying the quality of ginseng[J].Chinese Journal of Analytical Chemistry,2005,33(11):1565-1568.DOI:10.3321/j.issn:0253-3820.2005.11.012.
[28]IN G,AHN N G,BAE B S,et al.New method for simultaneous quantification of 12 ginsenosides in red ginseng powder and extract:in-house method validation[J].Journal of Ginseng Research,2012,36(2):205-210.DOI:10.5142/jgr.2012.36.2.205.
[29]LEE G J,SHIN B K,YU Y H,et al.Systematic development of a group quantification method using evaporative light scattering detector for relative quantification of ginsenosides in ginseng products[J].Journal of Pharmaceutical and Biomedical Analysis,2016,128:158-165.DOI:10.1016/j.jpba.2016.05.030.
[30]YANG Y W,MENG F S,GAO Y G,et al.Simultaneous determination of twenty ginsenosides in ginseng preparations by HPLC[J].Food Science,2016,37(22):131-135.DOI:10.7506/spkx1002-6630-201622019.
[31]LIU Z,RUAN C C,LIU T Z,et al.Simultaneous determination of 14kinds of ginsenosides in similar wild ginseng,fresh ginseng,white ginseng,and red ginseng by HPLC[J].Chinese Traditional and Herbal Drugs,2012,43(12):2431-2434.
[2]CHOI K T.Botanical characteristics,pharmacological effects and medicinal components of Korean Panax ginseng C.A.Meyer[J].Acta Pharmacologica Sinica,2008,29(9):1109-1118.DOI:10.1111/j.1745-7254.2008.00869.x.
[3]LEE C H,KIM J H.A review on the medicinal potentials of ginseng and ginsenosides on cardiovascular diseases[J].Journal of Ginseng Research,2014,38(3):161-166.DOI:10.1016/j.jgr.2014.03.001.
[4]GUI Y,RYU G H.Effects of extrusion cooking on physicochemical properties of white and red ginseng(powder)[J].Journal of Ginseng Research,2014,38(2):146-153.DOI:10.1016/j.jgr.2013.12.002.
[5]Standardization Administration of China.Grade quality of fresh-keeping ginseng:GB/T 22534-2008[S].Beijing:Chinese Specification Press,2008.
[6]CHRISTENSEN L P.Ginsenoside:chemistry,biosynthesis,analysis and potential health effects[J].Advances in Food and Nutrition Research,2008,55:1-99.DOI:10.1016/S1043-4526(08)00401-4.
[7]KIM Y J,CHOI W I,JEON B N,et al.Stereospecific effects of ginsenoside 20-Rg3 inhibits TGF-β1-induced epithelial-mesenchymal transition and suppresses lung cancer migration,invasion and anoikis resistanc[J].Toxicology,2014,322:23-33.DOI:10.1016/j.tox.2014.04.002.
[8]LI B,ZHAO J,WANG C Z,et al.Ginsenoside Rh2 induces apoptosis and paraptosis-like cell death in colorectal cancer cells through activation of p53[J].Cancer Letters,2011,301(2):185-192.DOI:10.1016/j.canlet.2010.11.015.
[9]CORBIT R M,FERREIRA J F S,EBBS S D,et al.Simplified extraction of ginsenosides from American ginseng(Panax quinquefolius L.)for high-performance liquid chromatographyultraviolet analysis[J].Journal of Agricultural and Food Chemistry,2005,53(26):9867-9873.DOI:10.1021/jf051504p.
[10]LEE S I,KWON H J,LEE Y M,et al.Simultaneous analysis method for polar and non-polar ginsenosides in red ginseng by reversed-phase HPLC-PAD[J].Journal of Pharmaceutical and Biomedical Analysis,2012,60:80-85.DOI:10.1016/j.jpba.2011.08.030.
[11]SUN B S,GU L J,FANG Z M,et al.Simultaneous quantification of19 ginsenosides in black ginseng developed from Panax ginseng by HPLC-ELSD[J].Journal of Pharmaceutical and Biomedical Analysis,2009,50(1):15-22.DOI:10.1016/j.jpba.2009.03.025.
[12]KIM S N,HA Y W,SHIN H,et al.Simultaneous quantification of14 ginsenosides in Panax ginseng C.A.Meyer(Korean red ginseng)by HPLC-ELSD and its application to quality control[J].Journal of Pharmaceutical and Biomedical Analysis,2007,45(1):164-170.DOI:10.1016/j.jpba.2007.05.001.
[13]SHI Y,SUN C J,ZHENG B,et al.Simultaneous determination of nine ginsenosides in functional foods by high performance liquid chromatography with diode array detector detection[J].Food Chemistry,2010,123(4):1322-1327.DOI:10.1016/j.foodchem.2010.06.014.
[14]SHAN S M,LUO J G,HUANG F,et al.Chemical characteristics combined with bioactivity for comprehensive evaluation of Panax ginseng C.A.Meyer in different ages and seasons based on HPLC-DAD and chemometric methods[J].Journal of Pharmaceutical and Biomedical Analysis,2014,89:76-82.DOI:10.1016/j.jpba.2013.10.030.
[15]YANG W Z,YE M,QIAO X,et al.A strategy for efficient discovery of new natural compounds by integrating orthogonal column chromatography and liquid chromatography/mass spectrometry analysis:its application in Panax ginseng,Panax quinquefolium and Panax notoginseng to characterize 437 potential new ginsenosides[J].Analytica Chimica Acta,2012,739:56-66.DOI:10.1016/j.aca.2012.06.017.
[16]WANG H P,ZHANG Y B,YANG X W,et al.Rapid characterization of ginsenosides in the roots and rhizomes of Panax ginseng by UPLC-DAD-QTOF-MS/MS and simultaneous determination of 19ginsenosides by HPLC-ESI-MS[J].Journal of Ginseng Research,2016,40(4):382-394.DOI:10.1016/j.jgr.2015.12.001.
[17]XIE Y Y,LUO D,CHENG Y J,et al.Steaming-induced chemical transformations and holistic quality assessment of red ginseng derived from Panax ginseng by means of HPLC-ESI-MS/MSn based multicomponent quantification fingerprint[J].Journal of Agricultural and Food Chemistry,2012,60(33):8213-8224.DOI:10.1021/jf301116x.
[18]ZHANG Y C,PI Z F,LIU C M,et al.Analysis of low-polar ginsenosides in steamed Panax ginseng at high-temperature by HPLC-ESI-MS/MS[J].Chemical Research in Chinese Universities,2012,28(1):31-36.
[19]SHI Y,SUN C,ZHENG B,et al.Simultaneous determination of ten ginsenosides in American ginseng functional foods and ginseng raw plant materials by liquid chromatography tandem mass spectrometry[J].Food Analytical Methods,2013,6(1):112-122.DOI:10.1007/s12161-012-9406-6.
[20]LI L,LUO G A,LIANG Q L,et al.Rapid qualitative and quantitative analyses of Asian ginseng in adulterated American ginseng preparations by UPLC/Q-TOF-MS[J].Journal of Pharmaceutical and Biomedical Analysis,2010,52(1):66-72.DOI:10.1016/j.jpba.2009.12.017.
[21]YANG H,LEE D Y,KANG K B,et al.Identification of ginsenoside markers from dry purified extract of Panax ginseng by a dereplication approach and UPLC-QTOF/MS analysis[J].Journal of Pharmaceutical and Biomedical Analysis,2015,109:91-104.DOI:10.1016/j.jpba.2015.02.034.
[22]SUN T T,LIANG X L,ZHU H Y,et al.Rapid separation and identification of 31 major saponins in Shizhu ginseng by ultrahigh performance liquid chromatography-electron spray ionizationMS/MS[J].Journal of Ginseng Research,2016,40(3):220-228.DOI:10.1016/j.jgr.2015.07.008.
[23]NOVáKOVáL,MATYSOVáL,SOLICH P.Advantages of application of UPLC in pharmaceutical analysis[J].Talanta,2006,68(3):908-918.DOI:10.1016/j.talanta.2005.06.035.
[24]LIU Y,SONG X,YAN R,et al.Development and validation of a UPLC-DAD-MS method for characterization and quantification of alkaloids in Menispermi Rhizoma and its preparations[J].Journal of Food and Drug Analysis,2013,21(2):206-218.DOI:10.1016/j.jfda.2013.05.012.
[25]PARK H W,IN G,HAN S T,et al.Simultaneous determination of 30ginsenosides in Panax ginseng preparations using ultra performance liquid chromatography[J].Journal of Ginseng Research,2013,37(4):457-467.DOI:10.5142/jgr.2013.37.457.
[26]STAVRIANIDI A,STEKOLSHCHIKOVA E,POROTOVA A,et al.Combination of HPLC-MS and QAMS as a new analytical approach for determination of saponins in ginseng containing products[J].Journal of Pharmaceutical and Biomedical Analysis,2017,132:87-92.DOI:10.1016/j.jpba.2016.09.041.
[27]PAN J Y,CHENG Y Y,WANG Y,et al.A method of smiultaneous determination of 9 ginsenosides for identifying the quality of ginseng[J].Chinese Journal of Analytical Chemistry,2005,33(11):1565-1568.DOI:10.3321/j.issn:0253-3820.2005.11.012.
[28]IN G,AHN N G,BAE B S,et al.New method for simultaneous quantification of 12 ginsenosides in red ginseng powder and extract:in-house method validation[J].Journal of Ginseng Research,2012,36(2):205-210.DOI:10.5142/jgr.2012.36.2.205.
[29]LEE G J,SHIN B K,YU Y H,et al.Systematic development of a group quantification method using evaporative light scattering detector for relative quantification of ginsenosides in ginseng products[J].Journal of Pharmaceutical and Biomedical Analysis,2016,128:158-165.DOI:10.1016/j.jpba.2016.05.030.
[30]YANG Y W,MENG F S,GAO Y G,et al.Simultaneous determination of twenty ginsenosides in ginseng preparations by HPLC[J].Food Science,2016,37(22):131-135.DOI:10.7506/spkx1002-6630-201622019.
[31]LIU Z,RUAN C C,LIU T Z,et al.Simultaneous determination of 14kinds of ginsenosides in similar wild ginseng,fresh ginseng,white ginseng,and red ginseng by HPLC[J].Chinese Traditional and Herbal Drugs,2012,43(12):2431-2434.