超高效液相色谱检测黄樟素方法研究

Determination of Safrole by Ultra Performance Liquid Chromatography

本文建立了检测黄樟素的超高效液相色谱定量分析方法。将黄樟素样本以甲醇稀释，1500 r/min振荡10 min或超声3 min，以0.22μm滤膜过滤后供仪器分析，对流动相梯度程序进行了考察和优化。采用岛津ODS-SP型（250 mm×4.6 mm,5μm）色谱柱，以高纯水（A）-乙腈（B）体系作为流动相，梯度程序为60%B（0.01 min）~66.5%B（13 min）~95%B（13.01 min）~95%B（20 min），流速1 mL/min，柱温40℃，进样量5μL，主定量波长210 nm，辅定量波长235 nm。实验结果表明，随着有机相初始浓度的升高，黄樟素色谱峰的理论塔板数减小、峰高增加、保留时间缩短；随着梯度陡度的增加，黄樟素色谱峰的理论塔板数和峰高均增加、保留时间缩短。经过方法优化，所测黄樟素样品色谱行为良好，在2~200μg/mL范围内线性良好，外标工作曲线为Y=1833.39X＋5675.32，相关系数高于0.9999。以信噪比S/N＞3计算，检测限为0.1μg/mL。日内精密度≤1.26%，日间精密度≤2.08%。加标回收率在98.21%~102.18%之间，RSD为0.35%~0.96%。在室温放置96 h、-20℃放置15 d以及在室温和-20℃反复冻融3次的条件下均可保持稳定。该方法快速、简便、高效、可靠。

To establish a quantitative method for determination of safrole by ultra performance liquid chromatography （UPLC）, safrole samples were diluted by methanol, oscillated with the speed of 1500r/min for 10 minutes or ultrasound-assisted oscillated for 3 minutes, then ifltered the solution with 0.22 μm membrane and drew 1.5 mL for automatic injection. The separation was optimized and performed on a Shimadzu ODS-SP column （250 mm×4.6 mm, 5 μm）. The mobile phase was pure water （A）-acetonitrile （B） and the mode of elution was gradient. The initial condition was 60%B, and the gradient steepness of organic phase was 0.5%/min between 0~13 min, then the organic phase’s content was raised to 95% immediately and held on for 7 minutes. The complete gradient elution procedure was 60%B （0.01 min）~66.5% B （13 min）~95%B （13.01 min）~95% B （20 min）. Flow rate was set as 1 mL/min, column temperature was set as 40℃ and injection volume was set as 5 μL. The primary quantitative wavelength of UV detector was set as 210 nm and the auxiliary one was set as 235 nm. When the organic phase’s initial content increased, theoretical plate number and retention time decreased, but peak height increased. When the gradient steepness increased, theoretical plate number and peak height increased, but retention time decreased. Using the optimized method, good linearity was achieved over the range of 2~200 μg/mL. External standard working curve Y=1833.39X＋5675.32, r2〉0.9999. Limit of detection was 0.1 μg/mL under the condition of S/N〉3. The precision was good with the intra-day RSDs less than 1.26% and inter-day RSDs less than 2.08%. The accuracy was also satisifed. The recovery rates were in the range of 98.21% to 102.18%, with RSDs between 0.35% and 0.96%. The test samples showed good stability under room temperature, freeze condition and three cycles of freeze-thaw condition. The RSDs of peak area of 6 samples placed at room temperature for 3, 6, 12, 24, 48, 96 hours were between 0.35% and 1.02%. The RSDs of 3 samples placed in refrigerator at -20℃ for 5, 10, 15 days were between 0.58% and 1.67%. The RSDs of 3 samples which were frozen and thawed three times were between 0.26% and 0.93%. The method was proved to be good when applied to case samples. For example, once we received an unknown sample seized by police, and then used the extraction and chromatography methods to analyze the sample, the major analyte’s retention time was consistent with safrole standard and the quantitative result was 7.08%. This method was rapid, simple, effective and suited for quantitative determination of safrole.