Efficient extraction and sensitive HPLC-MS/MS quantification of selected ecdysteroids in plants

Abstract

Ecdysones, analogs of the insect molting hormone ecdysone, have been found in many plant species. Plants usually contain one or two dominant ecdysones, in addition to a few structurally related compounds in smaller amounts. The most well-known plant ecdysone is 20-hydroxyecdysone (20E). Phytoecdysones have long been recognized for their multiple potential health benefits in mammals including humans. There are thus high hopes that these compounds and the raw materials in which they dominate may be successfully used in phytotherapy. Species from the Chenopodiaceae family, e.g., spinach (Spinacia oleracea L.), show a high content of phytoecdysones. However, our knowledge of ecdysones in plants is limited as only 20E has been studied in-depth, whereas other important ecdysones have not been systematically examined. This study aimed to develop a new analytical approach for the simultaneous determination of four ecdysones (muristerone A, makisterone A, 20E, and ponasterone A) in plants using high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). Several extraction processes were evaluated and optimized and the best performing method, involving solid-liquid extraction (SLE) (a mixture of methanol:ethanol:water (30:25:45; v/v/v)) and solid-phase extraction (SPE) (C18), was validated and used to analyze different plant samples (e.g., spinach, arugula, and lamb's lettuce). 20E (0.52–428 µg/g) was found in higher concentration than makisterone A (2.18–67.2 µg/g), while ponasterone A and muristerone A were not detected in any plant. The spinach samples contained the highest levels of these compounds. Moreover, the impact of culinary practices (cooking and freezing) on ecdysone stability was evaluated. In general, the compounds were stable during cooking (reductions < 10%). The freezing of samples affected the content of ecdysteroids in plants more negatively (reductions of 46% for 20E and 94% for MaA). The results demonstrated that the newly developed analytical method was effective, sensitive, and selective for the determination of the selected ecdysones (20E and makisterone A) in plants. Moreover, as far as we know, the content of makisterone A was reported for the first time in spinach.