Zhou Jian-Liang, Li Ping, Li Hui-Jun, Jiang Yan, Ren Mei-Ting, Liu Ying
Department of Pharmacognosy, China Pharmaceutical University, No. 24, Tongjia Lane, Nanjing 210009, China.
J Chromatogr A. 2008 Jan 4;1177(1):126-37. doi: 10.1016/j.chroma.2007.11.030. Epub 2007 Nov 17.
Steroidal alkaloids are naturally occurring nitrogen-containing compounds in many edible or medicinal plants, such as potato, tomato, Fritillaria and American hellebore, which possess a variety of toxicological and pharmacological effects on humans. The aim of this study is to explore the potential of liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) method in the determination of these important alkaloids in plant matrices. The application of this method has been proven through 26 naturally occurring steroidal alkaloids in Fritillaria species. Accurate mass measurements within 4 ppm error were obtained for all the alkaloids detected out of various plant matrices, which allowed an unequivocal identification of the target steroidal alkaloids. The bunching factor for mass spectrometer, an important parameter significantly affecting the precision and accuracy of quantitative method, was firstly optimized in this work and satisfactory precision and linearity were achieved by the optimization of that parameter. The ranges of RSD values of intra-day and inter-day variability for all alkaloids were decreased remarkably from 41.8-159% and 13.2-140% to 0.32-7.98% and 2.37-16.1%, respectively, when the value of bunching factor was optimized from 1 to 3. Linearity of response more than two orders of magnitude was also demonstrated (regression coefficient >0.99). The LC/TOF-MS detection method offered improvements to the sensitivity, compared with previously applied LC (or GC) methods, with limits of detection down to 0.0014-0.0335 microg/ml. The results in this paper illustrate the robustness and applicability of LC/TOF-MS for steroidal alkaloids analysis in plant samples. In addition, relative quantitative determination of steroidal alkaloid with one popular analyte verticinone which is commercially available was also investigated in order to break through the choke point of lack of standards in phytochemical analysis. The accuracies of relative quantitative method for steroidal alkaloids determinations with verticinone were 90.6-110.0% (average 98.5%) suggesting that it is feasible to quantify steroidal alkaloids by the proposed relative quantitative determination method within acceptable errors.
甾体生物碱是许多可食用或药用植物中天然存在的含氮化合物，如马铃薯、番茄、贝母和嚏根草，它们对人体具有多种毒理学和药理学作用。本研究的目的是探索液相色谱/电喷雾电离飞行时间质谱（LC/ESI-TOF-MS）方法在测定植物基质中这些重要生物碱方面的潜力。通过对贝母属植物中的26种天然甾体生物碱的研究，证明了该方法的应用。从各种植物基质中检测出的所有生物碱均获得了误差在4 ppm以内的精确质量测量结果，这使得能够明确鉴定目标甾体生物碱。本研究首次优化了对定量方法的精密度和准确性有显著影响的重要参数——质谱仪的聚集因子，并通过该参数的优化实现了令人满意的精密度和线性。当聚集因子的值从1优化到3时，所有生物碱日内和日间变异的相对标准偏差（RSD）值范围分别从41.8 - 159%和13.2 - 140%显著降低至0.32 - 7.98%和2.37 - 16.1%。还证明了响应线性超过两个数量级（回归系数>0.99）。与先前应用的液相色谱（或气相色谱）方法相比，LC/TOF-MS检测方法提高了灵敏度，检测限低至0.0014 - 0.0335 μg/ml。本文的结果说明了LC/TOF-MS在植物样品甾体生物碱分析中的稳健性和适用性。此外，还研究了以一种市售的常见分析物浙贝乙素对甾体生物碱进行相对定量测定，以突破植物化学分析中缺乏标准品的瓶颈。用浙贝乙素进行甾体生物碱相对定量测定方法的准确度为90.6 - 110.0%（平均98.5%），表明在所提出的相对定量测定方法中，在可接受误差范围内对甾体生物碱进行定量是可行的。
