方法开发报告：2012-APP-RLC-0182D-UHPLC分析苦荞麦中12个主要化学成分（LC-UV）(1为Quercetin-3-O-β-D-galactoside，2为Quercetin-3-O-β-D-glucoside, 3为Kaempferol-3-O-β-D-galactoside，4为N-trans-feruloyltyramine, 5为Kaempferol-3-O-β-D-glucoside, 6为Quercetin-3-O-α-L-rhamnoside, 7为Quercetin-3-O-[β-D-xyloxyl-(1→2)-α-L-rhamnoside]，8为1, 3, 6-tri-p-coumaroyl-6′-feruloyl sucrose, 9为3,6-di-p-coumaroyl-1, 6-di-feruloyl sucrose, 10为1, 6,6′-tri-feruloyl-3-p-coumaroyl sucrose，11为1, 3, 6,126′-tetra-feruloyl sucrose，12为rutin)苦荞麦又名乌麦、花麦、三角麦，是蓼科取苦荞麦药材粉末0.1000 g, 精密称定，置具塞圆底玻璃试管中，精密加入70% 乙醇2 mL ，密塞，称定重量。
室温浸泡30 min 后，超声处理（功率100W ，频率40 kHZ ）30min ，放冷，再称定重量，用70%乙醇补足减失的重量，摇匀，4500 rpm 离心10 min 。
用0.22μm 微孔滤膜过滤，作为供试品溶液。
3. 标准曲线和线性范围取系列浓度的标准曲线样品，从低浓度到高浓度连续进样，以被测物浓度（X ）为横坐标，被测物的峰面积之比（Y ）为纵坐标，按最小二乘法进行标准曲线的I C -10 0m m拟合，获得标准曲线线性回归方程和线性相关系数r2值。
结果见表1。
结果表明，12个化学成分在各自考察的浓度范围内，线性关系良好，线性相关系数r2均大于0.999。
表1 Calibration curves, LOD and LOQ data of investigated compounds by 2D-UHPLCAnalytes Calibration curves a r2Linearrange(μg/m l)LOD(μg/ml)LOQ(μg/ml)1. Quercetin-3-O-rutinoside y =0.1258x -0.1089 0.99971.02 -162.800.20 0.302. Quercetin-3-O-β-D-galactoside y =0.1582x -0.0800 0.99960.50 -80.800.15 0.203. Quercetin-3-O-β-D-glucoside y =0.1319x -0.1375 0.99951.03 -165.200.21 0.414. Kaempferol-3-O-β-D-galactoside y =0.1956x -0.0660 0.99980.52 -82.400.10 0.215. N-trans-feruloyltyramine y =0.5018x -0.2307 0.99960.52 -83.600.05 0.166. Kaempferol-3-O-β-D-glucoside y =0.1932x -0.1552 0.99961.01 -162.00.10 0.307. Quercetin-3-O-α-L-rhamnoside y =0.1700x -0.1644 0.99961.0 -160.00.20 0.408. Quercetin-3-O-[β-D-xyloxyl-(1→2)-α-L- rhamnoside]y =0.1288x -0.14290.99941.01 -161.200.20 0.409. 1, 3, 6-tri-p-coumaroyl-6′-feruloyl sucrosey =0.5425x -0.15720.99950.26 -42.200.05 0.1010. 3, 6-di-p-coumaroyl-1, 6-di-feruloyl sucrosey =0.5627x -0.16270.99950.25 -40.00.05 0.1011. 1, 6, 6′-tri-feruloyl-3-p-coumaroyl sucrosey =0.5642x -0.33070.99950.49 -78.800.05 0.1512. 1, 3, 6, 6′-tetra-feruloyl sucrose y =0.4061x -0.1296 0.99960.24 -39.200.07 0.12a y is the logarithmic value of peak area and x is the logarithmic value of the reference compound’s concentration (μg/ml).4.精密度和质控样品溶液稳定性通过测定日内及日间质控样品浓度变化来验证方法的精密度，测定低、中、高三种不同浓度的质控样品，将峰面积带入相应的标准曲线计算浓度，日内精密度实验中质控样品连续测定6次，日间精密度实验中质控样品每天测定1次，连续测定6天，测定浓度的RSD作为衡量精密度的指标。
结果表明（Table 2）所建立的分析方法日内精密度RSD小于3.3%，日间精密度RSD小于3.9%。
82.60 2.3 2.0 2.4 2.01.032.83.14.1 3.14. Kaempferol-3-O-β-D-galactoside5.15 3.3 3.0 2.9 3.041.20 2.5 3.8 2.8 3.81.04 0.3 0.9 0.6 0.95. N-trans-feruloyltyramine 5.22 2.8 3.7 2.8 3.741.80 2.6 3.9 2.8 3.92.02 1.9 1.3 1.3 1.36. Kaempferol-3-O-β-D-glucoside10.12 2.6 3.1 2.9 3.181.0 2.5 3.6 2.7 3.62.0 1.4 1.9 1.8 1.97. Quercetin-3-O-α-L-rhamnoside10.0 2.3 2.0 2.1 2.080.0 2.8 2.8 2.9 2.82.02 0.9 2.1 2.1 2.1 8. Quercetin-3-O-[β-D-xyloxyl-(1→10.083.0 0.9 2.5 0.9 2)-α-L- rhamnoside] 80.60 2.6 2.3 2.6 2.30.53 2.0 2.1 1.7 2.1 9. 1, 3, 6-tri-p-coumaroyl-6′-feruloyl 2.64 2.4 3.3 2.3 3.3 sucrose 21.10 2.5 3.0 2.6 3.00.50 2.3 2.9 2.3 2.9 10. 3, 6-di-p-coumaroyl-1, 6-di- 2.50 2.4 3.3 2.4 3.3 feruloyl sucrose 20.0 2.6 2.5 2.5 2.50.98 1.1 1.4 1.0 1.4 11. 1, 6, 6′-tri-feruloyl-3-p- 4.92 2.5 3.0 2.6 3.0Stability StabilityRSD (%) (n=6) RSD (%)(n=6)RSD (%) (n=6)1. Quercetin-3-O-β-D-galactoside 1.0 1.12.32. Quercetin-3-O-β-D-glucoside 1.8 2.3 1.03. Kaempferol-3-O-β-D-galactoside 2.5 2.7 3.24. N-trans-feruloyltyramine 1.2 1.9 0.95. Kaempferol-3-O-β-D-glucoside 1.8 2.7 0.86. Quercetin-3-O-α-L-rhamnoside 3.2 4.4 3.37.Quercetin-3-O-[β-D-xyloxyl-(1→2)-α-L-rhamnoside]1.1 3.02.0 8. 1, 3, 6-tri-p-coumaroyl-6′-feruloyl sucrose 1.3 2.0 1.49. 3, 6-di-p-coumaroyl-1, 6-di-feruloyl sucrose 1.1 1.8 0.810. 1, 6, 6′-tri-feruloyl-3-p-coumaroyl sucrose 1.4 1.7 0.911. 1, 3, 6, 6′-tetra-feruloyl sucrose 1.8 2.0 1.16.回收率称取苦荞麦茎粉末9份，称取量为样品测定时称样品量的二分之一，按各测定化合物含量的1:0.5（低）、1:1（中）、1:1.5（高）的比例加入各对照品贮备液，每个浓度制备3份，按样品前处理方法处理后，进行2D-UHPLC分析，带入标准曲线，计算回收率。
结果见表4，12个待测成分的回收率均在91.21%～107.76%37.39 18.22 54.92 96.21±0.03 3.5 6. Kaempferol-3-O-β-D-glucoside 37.39 36.45 71.46 93.47±0.03 3.237.39 54.68 93.01 101.72±0.01 0.634.47 18.0 52.17 100.51±0.05 4.9 7. Quercetin-3-O-α-L-rhamnoside 34.47 36.0 71.36 102.47±0.02 1.934.47 54.0 88.32 99.72±0.02 2.439.29 18.14 56.52 95.00±0.04 4.1 8. Quercetin-3-O-[β-D-xyloxyl-(1→39.29 36.27 76.35 102.18±0.02 2.02)-α- L-rhamnoside] 39.29 54.4 93.08 98.88±0.03 2.95.48 4.75 10.72 107.73±0.04 3.6 9. 1, 3, 6-tri-p-coumaroyl-6′-feruloyl 5.48 9.50 14.88 98.92±0.03 3.4sucrose 5.48 14.24 20.17 103.17±0.02 1.710.43 4.5 15.33 107.76±0.02 1.8 10. 3, 6-di-p-coumaroyl-1,6-di- 10.43 9.0 19.23 97.80±0.02 2.4feruloyl sucrose 10.43 13.5 23.82 99.17±0.03 3.218.96 8.86 27.81 101.22±0.03 3.3 11. 1, 6, 6′-tri-feruloyl-3-p- 18.96 17.73 37.12 102.41±0.02 1.6coumaroyl sucrose 18.96 26.6 44.79 97.12±0.01 1.011.23 4.41 15.87 105.11±0.04 3.612. 1, 3, 6, 6′-tetra-feruloyl sucrose 11.23 8.82 19.66 97.41±0.04 4.411.23 13.23 24.01 96.58±0.03 2.7 Recovery (%) = 100×(amount found – original amount)/ amount spiked.a The values were means ± S.D. of three experiments.7.8.苦荞麦茎2D-UHPLC色谱图图2 12个对照品色谱图LQ-root 0.08 - - - 0.09 - 0.12 - 0.56 0.56 0.44 0.32 LQ-stem 0.86 - 0.04 0.04 0.02 - 0.07 - 0.16 0.13 0.18 0.08 LQ-leaf 33.41 - 0.33 0.65 0.02 - 0.06 - 0.01 0.01 0.02 0.01 SZ-herb 3.10 0.30 0.51 - 0.03 0.02 0.05 - 0.02 0.15 0.05 0.04 SZ-root 0.23 0.56 0.35 - - - 0.29 - 0.09 1.19 0.22 0.12 SZ-stem 1.15 0.49 0.41 - 0.06 - 0.09 - 0.02 0.19 0.07 0.06 SZ-leaf 36.95 - 0.63 - - 0.16 0.09 - - 0.04 - - 09-herb 1.62 0.17 0.32 - 0.09 - - - 0.04 0.06 0.07 0.03 09-root 0.08 0.08 0.10 - 0.03 - - - 0.35 0.52 0.52 0.20 09-stem 0.57 0.29 0.46 - 0.12 - - - 0.06 0.09 0.10 0.04 09-leaf 8.07 - 0.25 - 0.07 - - - - - - - 08-herb 1.60 0.11 0.50 - - - - - - 0.02 0.03 0.0208-root 0.25 0.12 0.28 - - - - - 0.06 0.16 0.18 0.12 08-stem 1.49 0.18 0.44 - - - - - 0.01 0.04 0.04 0.04 08-leaf 2.98 0.07 0.17 - - - - - 0.01 0.03 0.03 0.03 1. Quercetin-3-O-rutinoside, 2.Quercetin-3-O-β-D-galactoside, 3.Quercetin-3-O-β-D- glucoside, 4.Kaempferol-3-O-β-D-galactoside,5.N-trans-feruloyltyramine, 6. Kaempferol-3-O-β-D-glucoside, 7. Quercetin-3-O-α-L-rhamnoside, 8. Quercetin-3-O-[β-D-xyloxyl-(1→2)-α-L-rhamnoside]9. 1, 3, 6-tri-p-coumaroyl-6′-feruloyl sucrose, 10. 3, 6-di-p-coumaroyl-1, 6-di-feruloyl sucrose, 11. 1, 6, 6′-tri-feruloyl-3-p-coumaroyl sucrose, 12. 1, 3, 6, 6′-tetra-feruloyl sucrose10.可行性建议本方法使用Acclaim HILIC-10 把苦荞麦全草部分分成两部分分别使用Acclaim PAII和Acclaim Phenyl-1分析柱进行二维分析，对于我们的目标化合物得到了理想的分离状态，对于其他干扰物也能达到很好的分离，对于苦荞麦全草的分析提供了有力的条件。