中药及保健食品中PDE_5抑制剂检测的再研究

Restudy on PDE_5 inhibitors in traditional Chinese medicine and health food

目的：尝试优化和建立可同时快速检测多种PDE5抑制剂类非法添加化学成分的方法。方法：分别采用TLC法、HPLC-PDA-MS联用及UPLC技术。TLC法：使用硅胶GF254薄层板，以三氯甲烷-乙酸乙酯-甲醇-水（40：40：15：12）下层溶液为展开剂，254 nm和366 nm下观察。HPLC-PDA-MS法：采用Inertsil ODS-3（4.6 mm×250 mm，5 μm）色谱柱，以0.01 mol·L^-1乙酸铵（A）-乙腈（B）为流动相，梯度洗脱（0～17 min，36%B；17～18 min，36%B→42%B；18～27 min，42%B；27～28 min，42%B→52%B；28～43 min，52%B；43～44 min，52%B→80%B），流速1 mL·min^-1，检测波长229 nm，UV光谱扫描（200～400 nm）;正离子模式，氮气为雾化和干燥气体，毛细管电压3 kV，锥孔电压60 V，气体流量80 L·h-1，离子源温度120℃，去溶剂温度200℃，去溶剂气体流量350 L·h-1。UPLC法：采用Waters BEH柱（2.1 mm×100 mm，1.8 μm）色谱柱，以甲醇（A）-水（B）为流动相，梯度洗脱（0～3 min，90%B→65%B；3～10 min，65%B→50%B；10～13 min，50%B→25%B；13～16 min，25%B；16～20 min，25%B→90%B），流速0.4 mL·min^-1，检测波长229 nm，UV光谱扫描（200～400 nm）。结果：在TLC检测中添加了366 nm作为检测波长，提高了检测的灵敏度与准确度;优化HPLC-PDA-MS法，获得了更好的8个PDE5抑制剂色谱峰的分离度;建立了快速准确的UPLC法，可在13 min内实现6个PDE5抑制剂的快速分离与检测。结论：该法可用于中药及保健食品中非法添加PDE5抑制剂类化合物的检测，具有高效、简便、专属性强等特点。

Objective： To develop a method for detecting PDE5 inhibitors illegally added in traditional Chinese medicine and health food. Methods： TLC,HPLC-PDA-MS and UPLC were used in the experiment with two-phase system and gradient elution.TLC separation was performed on the silica gel GF254 with the lower layer of chloroform-ethyl acetate-methanol-water（40： 40： 15： 12）as the developing solvent,the detection wavelength was 254 nm and 366 nm;HPLC-PDA-MS method was carried out on an Inertsil ODS-3 column（4.6 mm×250 mm,5 μm）with mobile phase at a flow rate of 1 mL·min^-1 and 229 nm as the detection wavelength.The mobile phase was 0.01 mol· L^-1 ammonium acetate（A）- acetonitrile（B）and the ratios of B were 36% at 0-17 min,36%→42% at 17-18 min,42% at 18-27 min,42%→52% at 27-28 min,52% at 28-43 min,52%→80% at 43-44 min.The UV spectral scan range was from 200 nm to 400 nm.A positive electrospray ion mode was used with nitrogen as the desolvation gas and dry gas,the voltages of capillary was 3 kV,the voltages of cone was 60 V and the gas flow of cone was 80 L·h-1,the temperature of ion source was 120℃,the temperature of desolvation was 200℃ and the gas flow of desolvation was 350 L·h-1;UPLC method was performed on a Waters BEH column（2.1 mm×100 mm,1.8 μm）with mobile phase at a flow rate of 0.4 mL·min^-1 and 229 nm as the detection wavelength.The mobile phase was 0.01 mol·L^-1 methanol（A）- water（B）and the ratios of B were 90%→65% at 0-3 min,65%→50% at 3-10 min,50%→25% at 10-13 min,25% at 13-16 min,25%→90% at 16-20 min.The UV spectral scan range was from 200 nm to 400 nm. Results： The sensitivity and accuracy of TLC analysis were improved when 366 nm was added as the detection wavelength.The HPLC-PDA-MS method was optimized and the resolutions of eight PDE5 inhibitors were better.Six PCE5 inhibitors were completely separated in 13 minutes with UPLC. Conclusion： The method is high efficient,convenient,and specific,which can be used for detection of PDE5 inhibitors illegally added in traditional Chinese medicine and health food.