Development of a fast extraction method and optimization of liquid chromatography–mass spectrometry for the analysis of phenolic compounds in lentil seed coats
Introduction
Phenolic compounds (also known as polyphenols) make up a large group of secondary metabolites characterized by the presence of an 
OH group and an aromatic ring [1]. The basic C6-C3-C6 (A-, C-, and B-ring) structure (Fig. 1) is typically observed, with different phenolic sub-classes being dependent upon further hydroxylation, methylation, or other modifications [2]. In human nutrition, phenolic compounds act as a “double-edged sword”, exerting both adverse effects and health benefits [3]. Several publications report health benefits of phenolic compounds, for example in protection against cancer [3], [4] and cardiovascular diseases [3]. Conversely, adverse effects described in the literature include inhibition of non-haem iron [5], [6] and induction of pro-oxidative stress and H2O2 production when polyphenols are present in high amounts [7], [8]. These contrasting effects highlight the importance of analyzing these compounds in food resources.
Lentil (Lens culinaris Medik.) is a good source of protein, carbohydrates, dietary fibre components, minerals, vitamins, and secondary metabolites that include phenolic compounds [9]. Simple phenolic compounds [10], phenolic acids [9], [11], [12], [13], flavan-3-ols and proanthocyanidins [11], [12], [14], [15], [16], [17], [18], anthocyanidins [19], [20], flavonols [9], [10], [12], [14], [16], [17], [18], [21], stilbenes [14], flavones [9], [12], [14], [20], and flavanones [21] are the major sub-classes of phenolic compounds found in lentil seeds. Phenolic compounds are much more diverse in the lentil seed coat than in the cotyledon and mostly consist of oligomers and polymers of proanthocyanidins [14]. Quantifying phenolic compound concentrations in lentils is needed to assess the potential for long-term development of breeding crops with improved quantity and quality of such compounds. Any suitable analytical method must be able to simultaneously quantify different sub-classes of phenolic compounds and do so as efficiently as possible due to the need to analyze large numbers of samples.
Several methods are used to analyze phenolic compounds. Liquid chromatography–mass spectrometry (LC–MS) methods are well-suited because of their selectivity and sensitivity. When coupled with ultraviolet detection (LC–UV–MS), these methods offer a fast solution to determine unknown phenolic compounds [2]. A critical step in these methods is sample preparation. Although several methods have been reported for extraction of phenolic compounds in lentil seeds, no systematic comparisons among these methods have been made to determine the differences among them or relative advantages or disadvantages of each. Extraction time is another important parameter as long extraction times (for example [11], [14], [19]) make analytical methods very time consuming and not readily applicable to the analysis of phenolic compounds in a large number of lentil genotypes. If extraction efficiencies are similar, the method that can be accomplished in the shortest time would be the preferred approach.
Previous experiments involving lentil seeds have used chromatographic gradients to separate phenolic compounds in a time range of 70 to 120 min [11], [14], [17], [21]. These separations were typically done using columns with large particles (e.g., 5 μm); in particular, C18 columns are commonly used [10], [11], [12], [13], [14], [15], [16], [17], [19], [20], [21]. However, the implementation of newer column technologies (e.g., core–shell) with smaller particle sizes (e.g., 2.6 μm) can improve the separation efficiency and thereby allow for a shorter analysis time [22]. As the column is a critical parameter in LC–MS experiments, an investigation of the performance of core–shell columns for the analysis with phenolic compounds in lentil is warranted; to our knowledge, no such work has been previously reported.
In addition, optimizing the gradient and the organic modifier [23] can contribute to improvements in LC–MS methods. We employed H2O: acetonitrile (ACN): formic acid (FA) to affect separation, while making changes to the amount of acid and organic solvent (separately) to improve the resolution of peaks.
We optimized phenolic compound extraction for speed and efficiency by examining both solvent type and extraction duration for several sub-classes of phenolic compounds in lentil seed coats. We also examined three different types of columns, the amount of organic modifier, and the gradient of the organic phase for their ability to achieve fast separation and sufficient resolution of peaks for different types of isomeric phenolic compounds.
Ultimately, the optimized method was applied to three different seed coat colors of lentil to demonstrate similarities and differences in the phenolic compound profiles. The results indicate that the time efficient procedure we developed can be successfully applied to the extraction and analysis of phenolic compounds for phytochemical screening of various lentil genotypes in breeding programs.
Section snippets
Plant material
Seeds from three lentil genotypes (CDC Maxim, 946a-46, Indianhead) were obtained from the Crop Development Centre at the University of Saskatchewan (Saskatoon, Canada). The seeds were decorticated using an abrasive mill (SATAKE Engineering Co. Ltd., no. 554046 Japan) and the seed coats separated using sieves and a column blower. To analyze the lentil seed coat, ∼50 mg (for each replicate) was weighed into micro centrifuge tubes. The tubes were covered, put in a −80 °C freezer for 1 h, and then
Optimizing compounds for use as standards in an LC–MS method
The MS allows for quantification of expected phenolic compounds, and UV detection will help to identify unexpected phenolic compounds. Because phenolic compounds absorb UV radiation between 250 and 600 nm, we can look for peaks in that UV range that are not associated with a corresponding MS peak and investigate further. For this study, quantification of expected phenolic compounds was done with a triple quadrupole mass spectrometer using MRM as described in Section 2.2. For all phenolic
Conclusions
A systematic approach was used to develop and optimize an LC–MS method for the analysis of phenolic compound composition in lentil seed coats. The optimum MS conditions (CV and CE), molecular and fragment ions, Rt, and UV wavelength were determined for 18 different phenolic standards using LC–MS with UV detection. The use of long duration maceration was found to be unnecessary for extracting phenolic compounds from lentil seed coats; 1 h maceration times provided similar results to those
Acknowledgements
The authors acknowledge financial assistance from the NSERC Industrial Research Chair Program and Saskatchewan Pulse Growers as well as additional support provided by the National Research Council of Canada, the University of Saskatchewan, and the Pulse Research Crew at the Crop Development Centre, U of S.
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