A novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously quantify phytosterols (brassicasterol, campesterol, stigmasterol and β-sitosterol) and tocopherols (alpha, beta, gamma and delta) entrapped in the lipid bilayer of a liposomal formulation. Apart from liposomes (a pharmaceutical product), the developed method was able to quantify target analytes in agricultural products, thus showing wide applications. Atmospheric pressure chemical ionization (APCI) was employed due to the enhanced ionization of phytosterols and tocopherols in comparison to electrospray ionization. Unlike published work, the chromatographic conditions were modified to simplify the analytical approach. For the first time, a simple isocratic elution (acetonitrile:methanol 99:1 v/v) was utilized for the separation of four phytosterols and four tocopherols in a single run. A substantially better baseline separation of phytosterols were obtained in comparison to reported methods by using poroshell C18 column. The method has a total run time of 7 min, which is the shortest run time among all reported quantitative methods for the simultaneous determination of four phytosterols and four tocopherols. Calibration curves for all phytosterols were linear in the range of 0.05-10 μg/mL. In the case of tocopherols, alpha tocopherol showed linear response in the range of 0.25-10 μg/mL. However, gamma and delta tocopherols exhibited quadratic relationship in the same concentration range (0.25-10 μg/mL). Validation parameters met the International Conference on Harmonization (ICH) guidelines in terms of selectivity, accuracy, precision, repeatability, sensitivity, matrix effects, dilution integrity and stability. The method was, for the first time, successfully applied for the quantifying phytosterols and tocopherols entrapped inside liposomes. An interesting chromatographic phenomenon was observed during sample analysis. Alpha tocopherol (entrapped in the liposomal lipid bilayer) was found to elute at two retention times, 2.53 min and 3.60 min. Such dual separation was not observed in calibration standards and quality controls. It was concluded that the chiral recognition ability of liposomes made up of phosphatidylcholine separated the enantiomers of alpha tocopherol, giving rise to two peaks at two different retention time. To sum, the reported novel LC-MS/MS method addresses three major analytical shortcomings, namely i)longer run time, ii)complex gradient elution and iii)poor baseline separation of phytosterols and tocopherols.

中文翻译：

---

使用经过验证的大气压化学电离-液相色谱-串联质谱法同时定量脂质体制剂中的植物甾醇和生育酚。

开发并验证了一种新颖的液相色谱串联质谱（LC-MS / MS）方法，以同时定量检测脂质双层中截留的植物甾醇（巴西甾醇，菜油甾醇，豆甾醇和β-谷甾醇）和生育酚（α，β，γ和δ）脂质体制剂。除了脂质体（药物产品）以外，开发的方法还能够定量农产品中的目标分析物，因此显示了广泛的应用。使用大气压化学电离（APCI）是由于与电喷雾电离相比，植物甾醇和生育酚的电离增强。与已发表的论文不同，对色谱条件进行了修改以简化分析方法。第一次，简单的等度洗脱（乙腈：甲醇99：1 v / v）可在一次运行中分离出四种植物甾醇和四种生育酚。与报告的方法相比，使用Poroshell C18色谱柱可获得基本更好的植物甾醇基线分离。该方法的总运行时间为7分钟，在所有同时测定四种植物甾醇和四种生育酚的定量方法中，这是最短的运行时间。所有植物甾醇的校准曲线在0.05-10μg/ mL范围内呈线性关系。就生育酚而言，α生育酚在0.25-10μg/ mL范围内表现出线性响应。但是，γ和δ生育酚在相同浓度范围（0.25-10μg/ mL）中表现出二次关系。验证参数在选择性，准确性，精密度，可重复性，灵敏度，基质效应，稀释完整性和稳定性。该方法首次成功地用于定量包裹在脂质体内的植物甾醇和生育酚。在样品分析过程中观察到一个有趣的色谱现象。发现α-生育酚（包裹在脂质体脂质双层中）在两个保留时间（2.53分钟和3.60分钟）洗脱。在校准标准品和质量控制中未观察到这种双重分离。结论是，由磷脂酰胆碱组成的脂质体的手性识别能力分离了α-生育酚的对映异构体，在两个不同的保留时间出现两个峰。总而言之，报告的新颖LC-MS / MS方法解决了三个主要的分析缺陷，即i）运行时间长，