HPLC Fingerprint Analysis and Multiple Ingredients Determination of Sanse Powder Gel Paste
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摘要: 目的 建立三色散凝胶贴膏剂的HPLC指纹图谱，同时对其多种成分进行含量测定，为其质量控制提供依据。方法 采用Hypersil Gold C18色谱柱(250 mm×4.6 mm，5 μm)，流动相为乙腈-0.1%磷酸水溶液，梯度洗脱，流速为1.0 mL/min，柱温为30 ℃，检测波长为240 nm，进样量为20 μL，建立三色散凝胶贴膏剂的HPLC指纹图谱，运用《中药色谱指纹图谱相似度评价系统》2012A版对其进行相似度评价；采用外标法，利用混合对照品测定12个成分的含量。结果 在选定的色谱条件下建立了三色散凝胶贴膏剂的HPLC指纹图谱，共标定16个共有峰，与对照品比对出12个成分，10批三色散凝胶贴膏剂的相似度在0.943~0.994之间；12个成分在一定浓度范围内均成良好的线性，相关系数均大于0.999 1，平均加样回收率在95.35%~99.43%之间，RSD均小于3.0%，12个成分在10个批次中的含量基本一致。结论 建立的三色散凝胶贴膏剂指纹图谱及含量测定方法，准确可靠，重复性好，可为进一步完善三色散凝胶贴膏剂的质量控制提供参考。Abstract: OBJECTIVE To develop a HPLC method for the figerprint analysis and multiple ingredients determination of Sanse powder gel paste and provide a basis for the quality control.METHODS HPLC analysis was performed on a Hypersil Gold C18 column (250 mm×4.6 mm, 5 μm) with a gradient mobile phase of acetonitrile-0.1% phosphoric acid at a fow rate of 1.0 mL/min. The detection wavelength was set at 240 nm and the column temperature was set at 30 ℃. The sample volume was 20 μL, and the HPLC fingerprints of Sanse powder gel paste were established. Similarity evaluation of 10 batches of Sanse powder gel paste were evaluated by using "The similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine" (2012A Version). The content of the 12 components were determined by the external standard method and mixed control solution.RESULTS The HPLC fingerprints of Sanse powder gel paste were established under the selected chromatographic conditions, and a total of 16 common peaks were calibrated. 12 components were identified by standard comparison, and the similarity of the 10 batches of Sanse powder gel paste ranged from 0.943 to 0.994. The 12 components were all showed good linearity in a certain concentration range with the correlation coefficients greater than 0.999 1. The average recoveries were between 95.35% and 99.43% with the RSD less than 3%. The contents of 12 substances in 10 batches of preparations were basically the same.CONCLUSION The HPLC fingerprinting method and assay are reproducible, stable and reliable for the quality control of Sanse powder gel paste.
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Key words:
- Sanse powder gel paste /
- HPLC /
- fingerprint /
- content determination
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图 1 混合对照品溶液的HPLC色谱图
1.马钱苷酸；2.士的宁；4.升麻素苷；6.甘草苷；7.紫花前胡苷；8.杯苋甾酮；9.蔓荆子黄素；10.甘草酸；11.欧前胡素；12.羌活醇；13.异欧前胡素；16.丹参酮ⅡA；λ＝240 nm
图 2 供试品溶液的HPLC色谱图
1.马钱苷酸；2.士的宁；4.升麻素苷；6.甘草苷；7.紫花前胡苷；8.杯苋甾酮；9.蔓荆子黄素；10.甘草酸；11.欧前胡素；12.羌活醇；13.异欧前胡素；16.丹参酮ⅡA；λ＝240 nm
表 1 梯度洗脱程序
时间/min 乙腈/% 0.1%磷酸水溶液/% 0 5 95 65 18 82 120 38 62 150 45 55 200 51 49 205 95 5 210 5 95 220 5 95 
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表 2 10批三色散凝胶贴膏剂的相似度
批号 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 R S1 1 0.973 0.931 0.969 0.998 0.975 0.960 0.942 0.952 0.977 0.994 S2 0.973 1 0.837 0.971 0.983 0.916 0.878 0.904 0.867 0.933 0.947 S3 0.931 0.837 1 0.835 0.911 0.960 0.990 0.846 0.983 0.943 0.959 S4 0.969 0.971 0.835 1 0.971 0.932 0.891 0.972 0.892 0.913 0.952 S5 0.998 0.983 0.911 0.971 1 0.960 0.941 0.932 0.931 0.973 0.986 S6 0.975 0.916 0.960 0.932 0.960 1 0.987 0.941 0.988 0.949 0.990 S7 0.960 0.878 0.990 0.891 0.941 0.987 1 0.906 0.998 0.956 0.983 S8 0.942 0.904 0.846 0.972 0.932 0.941 0.906 1 0.919 0.894 0.943 S9 0.952 0.867 0.983 0.892 0.931 0.988 0.998 0.919 1 0.945 0.978 S10 0.977 0.933 0.943 0.913 0.973 0.949 0.956 0.894 0.945 1 0.980 R 0.994 0.947 0.959 0.952 0.986 0.990 0.983 0.943 0.978 0.980 1
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表 3 各成分的线性方程及其浓度范围
成分 线性范围/(μg·mL-1) 线性方程 r 马钱苷酸 14.56~182.06 Y=2.739×107X+2.057 4×105 0.999 4 士的宁 6.80~85.03 Y=6.883×106X+9.091 5×104 0.999 5 升麻素苷 5.71~71.38 Y=3.201×107X+6.349 2×104 0.999 1 甘草苷 0.48~6.11 Y=2.415×107X-2.697 0×103 0.999 1 紫花前胡苷 9.72~121.60 Y=9.509×106X+2.791 4×104 0.999 1 杯苋甾酮 0.22~2.86 Y=2.340×107X-0.032 9×104 0.999 5 蔓荆子黄素 2.05~25.66 Y=4.520×107X+1.683 0×104 0.999 5 甘草酸 7.69~96.22 Y=1.208×107X+2.337 3×104 0.999 1 欧前胡素 2.71~33.91 Y=6.622×107X+3.618 3×104 0.999 3 羌活醇 1.20~15.01 Y=4.799×107X+3.069 0×103 0.999 4 异欧前胡素 5.59~69.99 Y=4.865×107X+3.696 2×104 0.999 9 丹参酮ⅡA 11.06~138.33 Y=3.312×107X+2.134 1×104 0.999 2
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表 4 12个成分的方法学考察结果(%)
成分 精密度RSD 重复性RSD 稳定性RSD 回收率 均值 RSD 马钱苷酸 1.8 1.5 1.6 95.35 2.6 士的宁 1.6 2.5 2.2 98.33 1.4 升麻素苷 2.1 2.6 2.1 97.42 1.4 甘草苷 2.5 2.7 2.1 99.33 1.8 紫花前胡苷 1.7 1.8 2.2 95.47 1.7 杯苋甾酮 2.3 2.6 1.8 95.57 2.7 蔓荆子黄素 2.1 2.4 2.7 99.43 1.2 甘草酸 1.8 1.6 2.6 96.62 2.1 欧前胡素 2.3 1.7 2.5 99.04 1.7 羌活醇 2.4 1.4 1.9 95.49 2.3 异欧前胡素 1.5 1.3 1.5 96.69 2.3 丹参酮ⅡA 2.3 2.5 1.2 98.23 2.2
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表 5 10批三色散凝胶贴膏剂含量测定结果(mg·g-1)
样品 马钱苷酸 士的宁 升麻素苷 甘草苷 紫花前胡苷 杯苋甾酮 蔓荆子黄素 甘草酸 欧前胡素 羌活醇 异欧前胡素 丹参酮ⅡA S1 0.717 0.429 0.137 0.059 8 0.183 0.018 0 0.094 0 0.202 0.092 7 0.148 0.099 5 0.539 S2 0.684 0.457 0.165 0.064 9 0.195 0.023 5 0.081 7 0.184 0.078 2 0.157 0.104 0 0.583 S3 0.582 0.355 0.141 0.050 2 0.178 0.020 1 0.087 8 0.203 0.083 4 0.154 0.082 9 0.496 S4 0.793 0.462 0.200 0.061 1 0.181 0.017 4 0.091 5 0.229 0.094 8 0.174 0.090 5 0.456 S5 0.786 0.458 0.135 0.056 1 0.179 0.021 3 0.090 0 0.236 0.084 3 0.154 0.100 0 0.480 S6 0.735 0.433 0.142 0.065 8 0.203 0.020 9 0.078 5 0.264 0.083 8 0.194 0.109 0 0.559 S7 0.671 0.374 0.160 0.048 1 0.166 0.021 9 0.085 7 0.264 0.108 0 0.150 0.080 0 0.593 S8 0.614 0.409 0.156 0.042 3 0.174 0.019 6 0.076 4 0.215 0.086 0 0.134 0.089 3 0.549 S9 0.588 0.396 0.138 0.040 2 0.178 0.023 7 0.088 7 0.256 0.102 0 0.144 0.091 6 0.553 S10 0.685 0.422 0.211 0.051 5 0.184 0.023 5 0.098 5 0.260 0.090 1 0.144 0.096 8 0.582
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