Ares Ana M, Bernal José, Nozal María J, Turner Charlotta, Plaza Merichel
I.U. CINQUIMA, Analytical Chemistry Group, University of Valladolid, 47011 Valladolid, Spain.
Department of Chemistry, Centre for Analysis and Synthesis, Lund University, P.O. Box 124, SE-22100 Lund, Sweden.
Food Res Int. 2015 Oct;76(Pt 3):498-505. doi: 10.1016/j.foodres.2015.06.037. Epub 2015 Jul 6.
In this study, we investigate for the first time the efficiency of an environmentally sustainable extraction technique (pressurized liquid extraction, PLE) in conjunction with a fast separation technique (ultra-high performance liquid chromatography, UHPLC) coupled to a selective mass spectrometry (MS) detector (quadrupole time-of-flight, qTOF) to extract, separate and quantify fifteen intact-glucosinolates (GLSs) in broccoli leaves. Firstly, we have developed and optimized by means of an experimental design an efficient extraction procedure based on PLE (using ethanol/water as a solvent), giving complete extraction within 15min; meanwhile, the average analyte recoveries were between 85% and 96% in all cases. Chromatography was performed on a UHPLC BEH Shield RP18 1.7μm 110Å (2.1×100mm) analytical column with a mobile phase composed by formic acid in water (0.5%, v/v) and formic acid in acetonitrile (0.5%, v/v) in gradient elution mode at 0.3mL/min, resulted in baseline-separated peaks and a run time of 13min. The method was fully validated in terms of selectivity, limits of detection (LOD) and quantification (LOQ), linearity, precision, and trueness; meanwhile a study of the matrix effect was also performed. A good selectivity, low LODs and LOQs, ranging from 2 to 26μg/g, wide linear ranges from LOQ to 2500μg/g, and satisfactory precision and trueness with relative standard deviation and relative error values lower than or equal to 9%, were obtained for the studied GLSs. Finally, the proposed method was successfully applied to the analysis of intact-GLSs in fifteen broccoli leaf samples from three different cultivars (Parthenon, Nubia, and Naxos). Nine intact-GLSs were detected in all the varieties, although in different concentrations, which ranged between 14 and 1136μg/g, depending on the broccoli cultivar. In addition, the highest total content of GLSs was found in broccoli leaf samples from Parthenon cultivar, being the Naxos cultivar the poorest in GLS content. This study demonstrates the efficiency of PLE as an environmentally sustainable alternative to extract intact-GLS from broccoli leaves, and that UHPLC-qTOF-MS allowed a rapid, selective and sensitive determination of intact-GLSs in this matrix.
在本研究中，我们首次研究了一种环境可持续的萃取技术（加压液体萃取，PLE）与快速分离技术（超高效液相色谱，UHPLC）以及选择性质谱（MS）检测器（四极杆飞行时间，qTOF）相结合，用于萃取、分离和定量西兰花叶片中15种完整的硫代葡萄糖苷（GLSs）的效率。首先，我们通过实验设计开发并优化了一种基于PLE（使用乙醇/水作为溶剂）的高效萃取程序，在15分钟内实现了完全萃取；同时，在所有情况下，分析物的平均回收率在85%至96%之间。色谱分析在UHPLC BEH Shield RP18 1.7μm 110Å（2.1×100mm）分析柱上进行，流动相由水相甲酸（0.5%，v/v）和乙腈相甲酸（0.5%，v/v）组成，采用梯度洗脱模式，流速为0.3mL/min，峰基线分离，运行时间为13分钟。该方法在选择性、检测限（LOD）和定量限（LOQ）、线性、精密度和准确性方面得到了充分验证；同时还进行了基质效应研究。对于所研究的硫代葡萄糖苷，获得了良好的选择性、低检测限和定量限（范围为2至26μg/g）、从定量限到2500μg/g的宽线性范围，以及令人满意的精密度和准确性，相对标准偏差和相对误差值低于或等于9%。最后，所提出的方法成功应用于来自三个不同品种（帕台农神庙、努比亚和纳克索斯）的15个西兰花叶片样品中完整硫代葡萄糖苷的分析。在所有品种中均检测到9种完整的硫代葡萄糖苷，尽管浓度不同，其范围在14至1136μg/g之间，具体取决于西兰花品种。此外，帕台农神庙品种的西兰花叶片样品中硫代葡萄糖苷的总含量最高，而纳克索斯品种的硫代葡萄糖苷含量最低。本研究证明了PLE作为从西兰花叶片中萃取完整硫代葡萄糖苷的一种环境可持续替代方法的效率，并且UHPLC-qTOF-MS能够快速、选择性和灵敏地测定该基质中的完整硫代葡萄糖苷。
