背景:Kratom是一种草药物质,属于新的精神活性物质。它含有精神活性吲哚生物碱mitragynine和7-羟基mitragynine。在低剂量下,它们充当精神兴奋剂,在较高剂量时,它们介导类阿片样作用。kratom滥用的增加需要开发分析方法,以准确,可靠地识别和量化生物样品中的精神活性生物碱。因此,有效的发展,精确,和可靠的绿色分析方法,易于在实践中实施是非常重要的。毛细管区带电泳与串联质谱(CZE-MS/MS)的在线组合似乎是一个有前途的解决方案。
结果:我们提出了一种基于毛细管区带电泳-串联质谱(CZE-MS/MS)方法的新型绿色方法,该方法具有在线动态pH连接样品预处理功能,以鉴定和测定尿液样品中的mitragynine和7-羟基mitragynine。在由100mM甲酸(pH2.39)组成的背景电解质中进行分离。通过在样品之前注射12.5%NH4OH的短塞来确保动态pH连接。在最优条件下,对所开发的方法进行了验证,参数如线性(R2>0.99),精度(2.2-8.7%),样品的准确性(89.2-102.5%)或稳定性(86.6-114.7%)符合FDA定义的指南标准(%RSD和%RE值,±15%以内).引入基于动态pH连接的简单毛细管内预浓缩策略可以显着改善分析信号强度以及该方法的适用性。应用所提出的方法,如检测限值所示,实现了高灵敏度,mitragynine和7-羟基mitragynine分别为0.5ngmL-1和2ngmL-1,分别。所提出的方法的绿色性得到了AGREE指标的证实(得分为0.63)。使用盲法尿液模型样品成功验证了所开发方法的应用潜力。
结论:首次引入了完全验证的用于kratom生物碱测定的CZE-MS/MS方法。所提出的新方法是常规使用的色谱技术的更便宜,更生态的替代方法,其绿色性评估以及与先前发布的液相色谱(LC)方法的比较清楚地证实了这一点。毛细管内样品预处理(动态pH值连接)已被证明是生物分析中有效且快速的工具,最小化预处理步骤的数量和样品的操作。此外,记录与通过LC方法获得的那些相当的LOD值。预计在不久的将来将这种方法应用于毒理学环境的潜力很大。
BACKGROUND: Kratom is a herbal substance belonging to the group of new psychoactive substances. It contains psychoactive indole alkaloids mitragynine and 7-hydroxymitragynine. At low doses, they act as psychostimulants and at higher doses they mediate an opioid-like effect. The increasing misuse of kratom requires the development of analytical methods that will accurately and reliably identify and quantify its psychoactive alkaloids in biological samples. Therefore, the development of effective, precise, and reliable green analytical methods that are easy to implement in practice is of great importance. On-line combination of capillary zone electrophoresis with tandem mass spectrometry (CZE-MS/MS) seems to be a promising solution.
RESULTS: We present a novel green approach based on capillary zone electrophoresis - tandem mass spectrometry (CZE-MS/MS) method with on-line dynamic pH junction sample pretreatment to identify and determine mitragynine and 7-hydroxymitragynine in urine samples. The separation was performed in a background electrolyte composed of 100 mM formic acid (pH 2.39). The dynamic pH junction was ensured by injection of a short plug of 12.5 % NH4OH before the sample. Under optimal conditions, the developed method was validated and parameters such as linearity (r2 > 0.99), precision (2.2-8.7 %), accuracy (89.2-102.5 %) or stability of the sample (86.6-114.7 %) met the defined FDA guideline criteria (%RSD and %RE values where within ±15 %). Introduction of a simple in-capillary preconcentration strategy based on dynamic pH junction enabled significant improvement in analytical signal intensity and also the applicability of the method. Applying the presented approach, high sensitivity was achieved as indicated by limit of detection values, which were 0.5 ng mL-1 and 2 ng mL-1 for mitragynine and 7-hydroxymitragynine, respectively. Greenness of the proposed approach was confirmed by the AGREE metrics (score 0.63). The application potential of the developed method was successfully verified using blinded urine model samples.
CONCLUSIONS: For the first time a fully validated CZE-MS/MS method for kratom alkaloids determination was introduced. The presented novel method is a cheaper and more ecological alternative to conventionally used chromatographic techniques what was clearly confirmed by its greenness evaluation and comparison with previously published liquid chromatography (LC) approaches. In-capillary sample pretreatment (dynamic pH junction) has been demonstrated to be an effective and fast tool in bioanalysis, minimizing the number of pretreatment steps and the manipulation with the sample. Moreover, LOD values comparable to those obtained by LC methods were recorded. High potential for the implementation of this approach into the toxicology environment in the near future is expected.