Fast determination of intact glucosinolates in broccoli leaf by pressurized liquid extraction and ultra high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry

- PLE was employed to extract intact-GLSs in broccoli leaves for the first time.
- The PLE extraction was fully optimized by means of an experimental design.
- The 15 intact-GLSs were separated in less than 8 min by UHPLC-MS/MS.
- Different intact-GLS contents were found in broccoli leaves from 3 cultivars.

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 15 min; meanwhile, the average analyte recoveries were between 85% and 96% in all cases. Chromatography was performed on a UHPLC BEH Shield RP18 1.7 μm 110 Å (2.1 × 100 mm) 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 0.3 mL/min, resulted in baseline-separated peaks and a run time of 13 min. 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 26 μg/g, wide linear ranges from LOQ to 2500 μg/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 1136 μg/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.