Department of Drug Analysis, University of Belgrade - Faculty of Pharmacy, Vojvode Stepe 450, Belgrade, Serbia.
Antidoping Agency of Serbia, Belgrade, Serbia; Sport Medicine Association of Serbia, Belgrade, Serbia.
J Pharm Biomed Anal. 2017 Nov 30;146:48-52. doi: 10.1016/j.jpba.2017.08.017. Epub 2017 Aug 15.
From 1st January 2017 higenamine was added on the WADA (World Anti-doping Agency) Prohibited list under S3 group beta-2 agonists as at all times banned substance for the athletes. The main origine of higenamine (or norcoclaurine) are different plants including Nandina domestica, Aconitum carmichaelii, Asarum heterotropioides, Galium divaricatum, Annona squamosa, Nelumbo nucifera etc. Higenamine main use is related to weight loss and it could be found (un)labeled in different dietary supplements. The objective of this study was development of sensitive and reliable UHPLC/MS/MS method for determination of higenamine in various dietary supplement samples. In order to obtain high method sensitivity, hydrophilic interaction liquid chromatography (HILIC) mode was applied. Separation was carried out on UHPLC Acquity BEH HILIC analytical column (2.1mm×100mm, 1.7μm particle size). Mobile phase consisted of 0.1% formic acid in water and acetonitrile, respectively, was mixed in ratio of 30:70, v/v. Flow rate was set at 0.2mLmin. Quercetin was used as an internal standard. ESI (+) source ionization mode using multi reaction monitoring (MRM) mode was utilized and three ion transitions of higenamine were followed 272.08→107.01, 272.08→161.07 and 272.08→77.08. Developed method was fully validated and applied for identification and quantification of higenamine in different dietary supplements. According to the results, the most of investigated supplements were free of higenamine, and on the other hand, presence of higenamine was confirmed in some samples while it was not declared on the label.
自 2017 年 1 月 1 日起， higenamine 被世界反兴奋剂机构（WADA）列入 S3 组β-2 激动剂的禁用清单，成为运动员始终被禁止使用的物质。 higenamine（或诺卡枯林）的主要来源是包括南天竹、乌头、细辛、筋骨草、番荔枝、莲等不同植物。 higenamine 的主要用途与减肥有关，它可以在不同的膳食补充剂中被发现（未）标注。本研究的目的是开发一种灵敏可靠的 UHPLC/MS/MS 方法，用于测定各种膳食补充剂样品中的 higenamine。为了获得高方法灵敏度，采用亲水相互作用液相色谱（HILIC）模式。分离在 UHPLC Acquity BEH HILIC 分析柱（2.1mm×100mm，1.7μm 粒径）上进行。流动相由 0.1%甲酸水和乙腈分别组成，按 30:70，v/v 混合。流速设定为 0.2mLmin。槲皮素被用作内标。采用电喷雾（ESI）（+）源离子化模式，采用多反应监测（MRM）模式，跟踪 higenamine 的三个离子跃迁 272.08→107.01、272.08→161.07 和 272.08→77.08。开发的方法进行了全面验证，并应用于不同膳食补充剂中 higenamine 的鉴定和定量。根据结果，大多数被调查的补充剂中不含 higenamine，而另一方面，在一些样品中证实存在 higenamine，而标签上并未声明。
