一种高效快速的HPLC-QQQ-MS方法用于测定药用植物中托烷类生物碱含量

摘要

本研究建立了一种高效液相色谱串联三重四极杆质谱法（HPLC-QQQ-MS），用于鉴定和定量分析唐古特山莨菪（Anisodus tanguticus）中的托烷类生物碱（莨菪碱、山莨菪碱、东莨菪碱）。该方法快速、准确且可靠，适用于药用植物的高通量分析。

摘要

Anisodus tanguticus 是一种重要的藏药植物。其托烷类生物碱（TAs），如莨菪碱、山莨菪碱和东莨菪碱，具有显著的镇痛和镇静药理活性，使该物种成为提取这些生物活性成分的重要天然资源。然而，A. tanguticus 的化学成分极为复杂，包含大量结构尚未明确的化合物，因此对其活性成分进行系统的鉴定与结构表征尤为重要。尽管高效液相色谱法（HPLC）已广泛用于该植物提取物的分离，但即使借助光谱数据库比对，许多未知组分仍难以准确鉴定。

本研究建立了一种基于高效液相色谱串联三重四极杆质谱法（HPLC-QQQ-MS）的分析方法，用于测定莨菪烷类生物碱 A. tanguticus该方法包含一系列系统性步骤，包括标准化的样品制备、质谱参数设置、液相色谱预平衡、方法建立、质谱数据采集、多级质谱（MSⁿ）扫描以及人工数据分析。利用该策略，我们成功鉴定出三种具有代表性的托烷类生物碱 A. tanguticus：莨菪碱、山莨菪碱和东莨菪碱。此外，该研究验证了所建立的HPLC-QQQ-MS方法的精确性，证实该方法快速、灵敏、可行且准确，适用于托烷类生物碱（TAs）的定量测定 A. tanguticus 已建立的方法具有良好的通用性和可靠性，适用于莨菪烷类生物碱的高通量鉴定与定量分析 A. tanguticus 及其他药用植物。

引言

中药的治疗效果在很大程度上取决于其产生的次生代谢产物，例如生物碱、黄酮类、萜类化合物以及其他活性成分^1,2,3。因此，对这些复杂混合物中特定目标代谢物进行精确且灵敏的定性与定量分析，是确保中药质量、阐明其疗效物质基础并推动现代研究的关键步骤³。然而，中药成分的多样性和复杂性，加之许多关键活性成分的浓度极低，为实现高效且准确的检测带来了巨大挑战。

Anisodus tanguticus (Maxim.) Pascher 是莨菪烷类生物碱（TAs）的重要植物来源⁴，以其富含具有药理活性的莨菪烷型化合物而著称⁵。其主要特征性生物碱包括山莨菪碱、东莨菪碱和莨菪碱，这些化合物具有共同的莨菪烷母核，但在功能基团取代上存在差异（图1），从而表现出不同的药理学特性^6,7,8。尽管其结构相似性给色谱分离带来困难，但也为比较植物化学与代谢研究提供了有价值的模型。近年来，已有研究采用代谢组学与网络药理学方法对 A. tanguticus 的化学成分及其地理变异进行了探讨^9,10。目前针对 A. tanguticus 代谢物的分析方法主要依赖高效液相色谱法（HPLC）¹¹。然而，A. tanguticus 幼苗阶段的莨菪烷类生物碱含量可低至0.01%（万分之一），而传统的HPLC方法常受限于分辨率、灵敏度和分析通量不足。这些局限性阻碍了结构类似生物碱同系物的完全分离，也影响了痕量成分的检测，从而降低了分析准确性，并限制了对更深入科研应用的支持。近年来，高效液相色谱串联质谱技术的进步为解决这些分析瓶颈提供了有力工具。特别是高效液相色谱-三重四极杆质谱法（HPLC-QQQ-MS），在色谱分辨率、分离效率和灵敏度方面均显著提升^12,13,14。该技术已成为中药中次生代谢产物研究的先进平台，能够在复杂的生物基质中高效、准确地定性和定量痕量目标分析物^11,15,16,17。因此，它为 A. tanguticus 等药用植物的全面化学轮廓分析与质量评价提供了坚实的技术基础。本研究针对 A. tanguticus 中莨菪烷类生物碱检测的长期挑战，重点解决痕量成分的准确定性与定量问题。通过系统优化色谱与质谱条件，我们建立并验证了一种新型的HPLC-QQQ-MS方法，用于多种主要莨菪烷类生物碱的 simultaneous 测定。该优化方法显著提高了灵敏度、选择性和方法稳健性，从而克服了传统基于质谱的检测方法中常见的局限性——如灵敏度不足、基质效应和共洗脱干扰——这些问题在痕量生物碱化合物分析中尤为突出。

方案

1. Sample preparation

1. Preparation of standard solutions
   1. Accurately weigh 10.0 mg each of hyoscyamine, anisodamine hydrobromide, and scopolamine hydrobromide reference standards using a one-ten-thousandth analytical balance, and place them into separate 10 mL volumetric flasks, respectively.
   2. Add chromatographic-grade methanol to the marked line, and thoroughly dissolve the standards using ultrasonic treatment to prepare single standard stock solutions with a concentration of 1.0 mg/mL.
   3. Transfer the stock solutions to brown reagent bottles, store them at 4 °C in the dark.
      NOTE: The alkaloids used are highly toxic. Take safety precautions when handling the standard samples. All solutions were prepared freshly prior to analysis and stored at 4 °C in the dark to avoid degradation.
2. Preparation of mixed working solutions
   1. Pipette 100 µL of each aforementioned stock solution into a 10 mL volumetric flask, and dilute to the marked line with chromatographic-grade methanol to prepare a mixed standard intermediate solution of 10 µg/mL.
   2. According to the linear range requirements, further dilute the intermediate solution stepwise with methanol to obtain a series of mixed working solutions.
   3. Take 50 µL, 200 µL, and 500 µL, respectively. Obtain concentrations of 0.5 µg/mL, 2 µg/mL, 5 µg/mL, and 10 µg/mL. Prepare fresh before use.
   4. Transfer 100 µL of the 10 µg/mL intermediate solution into a 1 mL volumetric flask to prepare a 1 µg/mL dilute solution.
   5. Then, transfer 100 µL of the 1 µg/mL solution into another 1 mL volumetric flask and dilute it to the volume with methanol.
3. Sample pretreatment
   1. Carry out freeze-drying of fresh materials of Sophora flavescens and subsequently pulverize into a homogeneous powder using a high-speed grinder.
   2. Sieve the powder through a 0.25 mm standard test sieve. Accurately weigh 20 mg of this powder, transfer it into a 100 mL volumetric flask, and add chromatographic-grade methanol to nearly the marked line.
   3. Perform ultrasonic extraction at room temperature for 30 min. After extraction, take out the volumetric flask, allow it to cool to 25 °C at room temperature, replenish chromatographic-grade methanol to the 100 mL marked line, and vortex mix for 2 min.
   4. Pipette 1 mL of the above extract into a 100 mL volumetric flask, and dilute to the marked line with methanol (100-fold dilution). After shaking well, it serves as the mother solution for HPLC injection (Figure 2).
      NOTE: When operating the high-speed grinding machine, it is essential to wear protective goggles and a dust mask; control the grinding amount and duration to prevent overheating.
4. Sample filtration and storage
   1. Prepare a 1 mL disposable needle-free syringe (chromatography-specialized, non-adsorptive) and a 0.22 µm organic phase microporous membrane.
   2. Aspirate 1.2 mL of the mother solution for injection, and push slowly for filtration.
   3. Collect the subsequent filtrate into a 2 mL sample vial with an inner insert, and seal tightly with a cap.
      NOTE: The frequency of the ultrasonic cleaner is fixed at 40 kHz. Avoid loosening of the volumetric flask cap during extraction to prevent solvent volatilization. Adjust the dilution factor according to the sample peak response in the pre-experiment to ensure the target peak area falls within the linear range of the standard curve. All samples must be immediately stored in a refrigerator at 4 °C.

2. HPLC prerun, method establishment, and MS acquisition

1. Preparation of mobile phases
   1. Prepare mobile phase A: Measure 1000 mL of ultrapure water, add 1.0 mL of chromatographic-grade formic acid (volume fraction: 0.1% v/v), and mix uniformly using a magnetic stirrer for 10 min.
   2. Prepare mobile phase B: Use chromatographic-grade methanol directly without additional treatment.
   3. Pour mobile phases A and B into ultrasonic degassing devices, respectively, and perform ultrasonic degassing at 40 kHz for 15 min.
   4. Immediately transfer the degassed mobile phases to solvent bottles after degassing, seal them, and connect them to the corresponding A and B solvent lines of the HPLC system.
      NOTE: All solvents are of chromatographic grade and must be filtered through a 0.45 µm organic phase microporous membrane before use. Monitor the system pressure regularly. If the pressure fluctuation exceeds ±±5%, check for potential column clogging or air bubbles in the pipeline.
2. Setting of HPLC conditions
   1. Use a C₁₈ column (2.1 mm × 100 mm, 2.7 µm) for chromatographic separation. Equilibrate the column with methanol:water = 80:20 (v/v) for 30 min before use, with a flow rate of 0.3 mL/min during equilibration. Set the column oven temperature to 30 °C, and start preheating 10 min in advance.
   2. Optimize a gradient elution program as follows: 0–10.0 min, 5% to 10% B; 10.0–15.0 min, 10% to 35% B; 15.0–20.0 min, 35% to 60% B; 20.0–25.0 min, 60% to 95% B. Set the flow rate to 0.60 mL/min, the injection volume to 2 µL, and detection wavelength to 210 nm.
3. Mass spectrometric conditions
   1. Perform mass spectrometric (MS) detection using a triple quadrupole (QQQ) mass spectrometer equipped with an electrospray ionization (ESI) source operating in positive ionization mode (ESI+).
   2. Set the operation parameters as follows: nebulizer pressure: 15 psi; capillary voltage: 4000 V; gas temperature: 300 °C; and gas flow: 11 L/min.
   3. Systematically optimize the MS/MS parameters for each analyte.
      1. First, identify precursor ions by directly infusing individual standard solutions in MS2 SCAN mode.
      2. Optimize the Fragmentor voltage for each precursor ion to maximum signal intensity using MS2 SIM mode.
      3. Subsequently, employ the Product Ion Scan mode to determine the characteristic product ions. Systematically optimize the Collision Energy (CE) for the precursor ion to maximize the product-ion signal intensity.
      4. Select the most intense product ion for quantification, and use the second-most-intense ion for qualitative confirmation.
         NOTE: Identify precursor ions via direct infusion of individual standard solutions in MS2 SCAN mode. Ensure standard solution concentration is appropriate to avoid signal saturation. Calibrate the instrument regularly to avoid parameter drift.
4. Method validation
   1. Specificity
      1. Inject blank solvent (methanol), mixed standard working solution, and sample extract separately, and compare the chromatograms.
      2. Ensure the following requirements are met: No interfering peaks in the blank solvent; the retention time of target components in the sample is consistent with that of the standard (deviation ≤ ±±0.1 min); the peak area ratio of quantifier ion to qualifier ion conforms to that of the standard (deviation ≤ ±±10%).
   2. Linearity
      1. Inject the series of mixed working solutions prepared in step 1.1 (0.1, 0.5, 2, 5, 10 µg/mL) sequentially under the aforementioned HPLC-MS conditions, with 3 injections per concentration.
      2. Plot the standard curve with the concentration (x, µg/mL) of each component as the abscissa and the average peak area (y) of the quantifier ion as the ordinate. Perform linear regression analysis, requiring a correlation coefficient (R²) ≥ 0.995.
   3. Precision and accuracy
      1. Intraday precision (repeatability): Analyze 6 replicates of quality control (QC) samples at three concentration levels (2 µg/mL, 5 µg/mL, and 10 µg/mL) within one day, and calculate the relative standard deviation (RSD%).
      2. Interday precision (intermediate precision): Independently prepare QC samples from the same batch of dried plant raw material and analyze them by different operators on three consecutive days. and calculate RSD%. Ensure RSD% ≤ 10% for both.
      3. Evaluate accuracy using a spike-recovery test, and express as a recovery rate (%). Ensure the average recovery rate of low, medium, and high concentrations ranges from 85% to 115%, with RSD% ≤ 5%.
         NOTE: The linear range must cover the expected concentration range of target components in actual samples (including low, medium, and high levels) to reflect practical applicability. All validation experiments were performed in triplicate, and the results were expressed as the average value.

3. Data acquisition

1. Perform data acquisition in multiple reaction monitoring (MRM) mode. Refer to Table 1 for the optimized MRM parameters-including precursor ion, product ion transitions, fragmentor voltage, and collision energy (CE)-for each analyte.
2. Calculate the dwell time to ensure an adequate number of data points across each chromatographic peak for each transition while maintaining an appropriate cycle time.

4. Data analysis

1. Launch the qualitative analysis software, click File > Import Data, and import all collected data files (.d format).
2. Extraction of extracted ion chromatograms (EICs): In the Quantitation module, select MRM Transition, input the precursor ion-product ion pairs of each component, and extract the corresponding EICs.
3. Peak validation and integration: Check the peak symmetry and retention time consistency (deviation ≤ ±±0.1 min compared with the standard) of the target peaks in each EIC.
4. The software performs automatic integration by default; if baseline drift or interfering peaks exist, manually adjust the integration parameters (integration start and end points) to ensure accurate integration.
5. Calculate the concentration of each component in unknown samples using the standard curve. Click Report > Generate Report, select Excel format to export the report. The report includes the compound name, retention time, peak area, concentration, RSD%, etc.

结果

HPLC-QQQ-MS 分离与鉴定萜类生物碱
由标准混合物分析获得的典型 MRM 色谱图及一个 A. tanguticus 根提取物被展示于 图3 和 图4在优化的HPLC条件下，三种目标托烷生物碱——莨菪碱（hyoscyamine）、东莨菪碱（anisodamine）和山莨菪碱（scopolamine）均在20分钟内实现有效分离，表明所建立方法具有高效率。山莨菪碱与东莨菪碱之间的分离度（Rs）为8.67，东莨菪碱与莨菪碱之间的分离度达到11.58。所有分离度数值均显著高于基线分离标准（1.5），表明无色谱干扰，证实该方法适用于准确定量分析。所有化合物均呈现尖锐且对称的峰形，显示出优异的色谱性能及极低的基质干扰。每种托烷生物碱（TA）的鉴定均基于两个标准：（1）保留时间与对照标准品一致（±0.1 min以内）；（2）至少存在两个特异性的MRM跃迁（定量离子和定性离子）。

讨论

随着人们对植物次生代谢物的兴趣日益增长，生物碱检测的分析技术受到了广泛关注。尽管用于分析托烷类生物碱（TAs）的基础色谱体系已经建立¹⁸，但近年来的新进展——例如毛细管电泳（CE）的应用、GC–MS 和 HPLC–MS 仪器性能的提升，以及对旋花素（convolvine）和类似托品酮毒素等新型结构化合物的鉴定——凸显了对当前这些生物碱色谱分析方法进行系统综述的必要性^{11,19,20,21,22}。TAs 的分析需求因应用场景而异：在植物组织分析中（如植物育种、生物碱生物合成及提取研究），分析方法必须具备高选择性和高灵敏度，以区分结构相似的次生代谢物并检测痕量成分^23,

材料

本文使用的材料清单

姓名	公司	目录编号	评论
安捷伦 MassHunter 软件	Agilent Technologies
安捷伦 ZORBAX SB-C18 色谱柱	Agilent Technologies	880975-902	C18 反相色谱柱，4.6 × 250 mm，5 μm
氢溴酸山莨菪碱	成都格利普生物科技有限公司	101-31-5	HPLC>98%
甲酸	Sigma-Aldrich	5.33002
冷冻干燥机	EYELA，日本东京	FDU-2110
高速万能粉碎机	天津市泰斯特仪器有限公司	6010210100	FW-100，24,000 rpm，100 g 容量，80 目
莨菪碱	成都格利普生物科技有限公司	55449-49-5	HPLC>98%
甲醇	赛默飞世尔科技	022909.K2
0.22 μm 微孔滤膜	Millipore	SLGV033RB
Milli-Q Direct 8 纯水系统	MilliporeSigma，美国马萨诸塞州伯灵顿	ZRQSVP0JP
氢溴酸东莨菪碱	成都格利普生物科技有限公司	114-49-8	HPLC>98%