CAJ | 학술논문

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。
본문건립료검측황장소적초고효액상색보정량분석방법。장황장소양본이갑순희석，1500 r/min진탕10 min혹초성3 min，이0.22μm려막과려후공의기분석，대류동상제도정서진행료고찰화우화。채용도진ODS-SP형（250 mm×4.6 mm,5μm）색보주，이고순수(A)-을정(B)체계작위류동상，제도정서위60%B(0.01 min)~66.5%B(13 min)~95%B(13.01 min)~95%B(20 min)，류속1 mL/min，주온40℃，진양량5μL，주정량파장210 nm，보정량파장235 nm。실험결과표명，수착유궤상초시농도적승고，황장소색보봉적이론탑판수감소、봉고증가、보류시간축단；수착제도두도적증가，황장소색보봉적이론탑판수화봉고균증가、보류시간축단。경과방법우화，소측황장소양품색보행위량호，재2~200μg/mL범위내선성량호，외표공작곡선위Y=1833.39X+5675.32，상관계수고우0.9999。이신조비S/N＞3계산，검측한위0.1μg/mL。일내정밀도≤1.26%，일간정밀도≤2.08%。가표회수솔재98.21%~102.18%지간，RSD위0.35%~0.96%。재실온방치96 h、-20℃방치15 d이급재실온화-20℃반복동융3차적조건하균가보지은정。해방법쾌속、간편、고효、가고。
ABSTRACT: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.