波长切换HPLC法同时测定水蛭蛴螬化瘀片中10个活性成分的含量

Simultaneous determination of 10 active components in Shuizhi Qicao Huayu tablets by HPLC with wavelength switching

目的:建立多波长切换HPLC法同时测定水蛭蛴螬化瘀片中丹参素钠、丹酚酸B、黄芩素、汉黄芩素、黄芩苷、汉黄芩苷、异鼠李素-3-O-新橙皮苷、甘草酸铵、延胡索乙素、芍药苷10个成分的含量。方法:采用岛津Insertsustain C_(18)柱(250 mm×4.6 mm,5μm),以0.2%醋酸水溶液-甲醇-乙腈为流动相梯度洗脱,DAD检测器,检测波长为280 nm(0~20 min,33~36 min,38.8~42 min检测丹参素钠、延胡索乙素、黄芩苷),230 nm(20~29 min,检测芍药苷),254 nm(29~33 min,检测异鼠李素-3-O-新橙皮苷),252 nm(62~70 min检测甘草酸铵),286 nm(36~38.8 min检测丹酚酸B),274 nm(42~48 min,70~80 min检测汉黄芩苷、汉黄芩素),276 nm(48~62 min,检测黄芩素),流速1.0 mL·min^(-1),柱温为25℃。结果:通过1次进样,同时测定了上述10个成分的含量,丹参素钠、丹酚酸B、黄芩素、汉黄芩素、黄芩苷、汉黄芩苷、异鼠李素-3-O-新橙皮苷、甘草酸铵、延胡索乙素、芍药苷的进样质量浓度分别在6.32~158μg·mL^(-1)(r=0.999 8)、53.4~1 336μg·mL^(-1)(r=0.999 9)、1.51~37.8μg·mL^(-1)(r=0.999 9)、0.888~22.2μg·mL^(-1)(r=0.999 9)、18.6~465μg·mL^(-1)(r=0.999 9)、7.92~198μg·mL^(-1)(r=0.999 9)、2.14~53.6μg·mL^(-1)(r=0.999 8)、3.30~82.6μg·mL^(-1)(r=1.000 0)、1.12~28.0μg·mL^(-1)(r=0.999 9)、38.5~962μg·mL^(-1)(r=0.999 9)与峰面积呈良好线性关系;平均加样回收率(n=6)分别为97.0%(RSD=1.6%)、99.1%(RSD=1.4%)、99.0%(RSD=1.4%)、98.1%(RSD=1.6%)、99.2%(RSD=0.98%)、100.2%(RSD=1.8%)、98.7%(RSD=2.6%)、100.4%(RSD=1.7%)、100.2%(RSD=3.0%)、100.5%(RSD=2.2%);3批中试样品中上述10个成分含量测定结果依次为2.112~2.119 mg·g^(-1)、15.642~15.648 mg·g^(-1)、1.558~1.568 mg·g^(-1)、0.553~0.568 mg·g^(-1)、16.515~16.527 mg·g^(-1)、4.412~4.423 mg·g^(-1)、0.554~0.569 mg·g^(-1)、3.353~3.368 mg·g^(-1)、0.192~0.201 mg·g^(-1)、8.749~8.761 mg·g^(-1)。结论:该方法可用于水蛭蛴螬化瘀片的质量控制,为其质量标准的建立奠定了基础。

Objective： To establish a method of simultaneous determination of sodium Danshensu, salvianolic acid B, baicalein, wogonin, baicalin, wogonoside, isorhamnetin-3-O-neohespeidoside, ammonium glycyrrhizinate, tetrahydropalmatine and paeoniflorin in Shuizhi Qicao Huayu tablets by HPLC with wavelength switching. Methods： SHIMADZU Insertsustain C18 column （250 mm× 4.6 mm, 5 μm ）was chosen, and gradient elution was conducted with 0.2% acetic acid-methanol-acetonitrile as the mobile phase. The detection wavelength was set at 280 nm （ 0-20 min, 33-36 min, 38.8-42 rain for salvianic acid A sodium, tetrahydropalmatine, and baicalin ）, at 230 nm （ 20-29 rain for paeoniflorin ）, at 254 nm （ 29-33 rain for isorhamnetin-3-O- neohespeidoside ）, at 252 nm （ 62-70 min for ammonium glycyrrhizinate ）, at 286 nm （ 36-38.8 min for salvianolic acid B ）, at 274 nm （ 42-48 rain, 70-80 rain for wogonin and wogonoside ） and at 276 nm （ 48- 62 min for baicalein ）. The flow rate was 1.0 mL＂ rain-1 and the column temperature was 25 ~C. Results： The linear ranges of sodium Danshensu, salvianolic acid B, baicalein, wogonin, baicalin, wogonoside, isorhamnetin-3- O-neohespeidoside, monoammonium glycyrrhizinate, tetrahydropalmatine and paeoniflorin were 6.32-158 μg· mL-1 （ r=0.999 8 ）, 53.4-1 336 μg· mL-1 （ r=0.999 9 ）, 1.51-37.8 μg· mL-1 （ r=0.999 9 ）, 0.888-22.2 μg· mL-1 （ r=0.999 9 ）, 18.6-465 μg· mL-J （ r=l.000 0 ）, 7.92-198 μg· mL-1 （ r=0.999 9 ）, 2.14-53.6 μg· mL-1 （ r=0.999 8 ）, 3.30-82.6 μg· mL-1 （ r=1.000 0 ）, 1.12-28.0 μg· mL-1 （ r=0.999 9 ） and 38.5-962 μg· mL-1 （ r=0.999 9 ）, respectively. The average recoveries （ n=6 ） were 97.0% （ RSD=l.6% ）, 99.1% （ RSD=1.4% ）, 99.0% （ RSD=1.4% ）, 98.1% （ RSD=1.6% ）, 99.2% （ RSD=0.98% ）, 100.2% （ RSD=1.8% ）, 98.7% （ RSD=2.6% ）, 100.4% （ RSD=1.7% ）, 100.2% （ RSD=3.0% ） and 100.5% （ RSD=2.2% ）, respectively. The content ranges of three batches of pilot samples were 2.112-2.119 mg· g-1, 15.642-15.648 mg·g-1, 1.558-1.568 mg· g-Z, 0.553- 0.568 mg· g-1 16.515-16.527 mg· g-1 4.412-4.423 mg·g-l, 0.554-0.569 mg·g-1 3.353-3.368 mg·g-1 0.192-0.201 mg·g-1 and 8.749-8.761 mg· g-1, respectively. Conclusion： The method could be used for the quality control of Shuizhi Qicao Huayu tablets, and offered basis for the establishment of the standard.