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Cited by 22Phytochemical Studies and BioactivitiesAnalytical Methods in PharmaceuticalsPesticide Residue Analysis and Safety

Efficient Extraction and Sensitive HPLC-MS/MS Quantification of Selected Ecdysteroids in Plants

Open access
Fang, Xiangyu, Szołtysik, Rafał, Tang, Ji, Bajkacz, Sylwia·2022·Journal of Food Composition and Analysis
DOI 10.1016/j.jfca.2022.104580
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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.

AI snapshot

Written from the paper’s full text.

TL;DR

A new SLE-SPE followed by HPLC-MS/MS method was developed and validated for simultaneous determination of muristerone A, makisterone A, 20-hydroxyecdysone, and ponasterone A in plants. In the analyzed samples, 20E and MaA were detected, with spinach containing the highest levels, while MuA and PoA were not detected.

Key findings

  • The validated method showed good linearity (R² > 0.9924), intra-day precision 0.87–5.41% RSD, inter-day precision 1.78–8.73% RSD, recovery 75.2–98.7% (SD ≤ 5.72%), and negligible matrix effects (1.36–6.47%).
  • 20E ranged from 0.52 to 428 µg/g dry mass and MaA from 2.18 to 67.2 µg/g dry mass; MuA and PoA were not detected in any plant. Spinach samples contained the highest levels, with 20E between 79.6 and 428 µg/g and MaA up to 67.2 µg/g.
  • Cooking affected 20E only slightly in two spinach samples (3–10% decrease; one sample increased 2%), but MaA decreased by 1.5%, 67%, and 82% across three groups. Freezing reduced 20E by 46% and MaA by 94%.
  • The paper reports the first detection of MaA in spinach, as far as the authors know.

Methods

  • Extraction was optimized using a simplex-centroid mixture design for solvent composition and a central composite design for mixing speed, extraction time, and solid/liquid ratio. The final SLE conditions were 200 mg plant sample, 15 mL methanol:ethanol:water (30:25:45, v/v/v), 2 h extraction, and 700 rpm mixing, followed by C18 SPE clean-up.
  • HPLC-MS/MS used a Poroshell 120 SB-C18 column with 0.1% formic acid in water and acetonitrile, positive ESI, and scheduled MRM with two transitions per compound.
  • Plant samples included spinach, arugula, lamb’s lettuce, curly kale, green lettuce, black kale, and mixed salad, purchased in Poland; fresh, cooked, and frozen spinach were also analyzed. Validation covered calibration linearity, LOD/LOQ, intra- and inter-day precision and accuracy, recovery, and matrix effect.

Limitations

  • Stated: the authors note there is no literature information on MaA content in spinach or the other studied plants, and no literature on the effect of processing on ecdysteroid content in spinach; they also suggest small differences after cooking may be due to measurement error.
  • Evident: MuA and PoA were not detected in any plant, so the study provides no plant-content or processing-stability data for those two analytes.
  • Evident: the paper has no formal limitations section, and the plant sample set is limited to selected vegetables bought in local Polish markets and supermarkets.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.jfca.2022.104580

Cites

30 of 30 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 6 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Most of the selected solvents were capable of at least partially extracting ecdysteroids from plant samples.

    “The results showed that most of the selected solvents were capable of at least partially extracting ecdysteroids from plant samples.”
    Quote in the paperSubstantial claim
  • Findingown data

    All selected compounds produced stronger and more reproducible ions in positive ion mode.

    “It was noted that all the selected compounds produced stronger and more reproducible ions in positive ion mode.”
    Quote in the paperSubstantial claim
  • Findingown data

    All responses, for both mixtures 1 and 2, increased with methanol proportion.

    “All responses, for both mixtures 1 and 2, increased with methanol proportion; these results show that methanol is an adequate solvent to extract these compounds from plant samples.”
    Quote in the paperSubstantial claim
  • Findingown data

    Analytical recoveries for C18, Oasis HLB, Bond Elut C18 LO, and Bond Elut PPL were within 97-107%.

    “Analytical recoveries were within the following ranges: 99.0 -105% for C18, 97.0 -103% for Oasis HLB, 98.0 -103 for Bond Elut C18 LO, and 102 -107% for Bond Elut PPL.”
    Quote in the paperSubstantial claim
  • Findingown data

    The extract corresponding to the tertiary mixture presented a higher value than predicted by the model (93.2% for 20E, 84.0% for MaA).

    “In regard to the multiresponse optimization (Fig. 4), it was verified that the extract corresponding to the tertiary mixture presented a higher value than the value predicted by the model (93.2% for 20E, 84.0% for MaA), indicating the efficiency of the mixture in extracting these phytocompounds.”
    Quote in the paperSubstantial claim
  • May combine two claims. 29
  • Classified as probably not a substantial claim. 2

  • May combine two claims.

    Findingown data

    The C18 sorbent was selected for further research because it gave the best removal of interferents from the matrix and the lowest matrix effect.

    “Finally, the C18 sorbent was selected for further research due to it giving the best removal of interferents from the matrix and also the lowest matrix effect.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    For all four ecdysteroids, the R2 values of the calibration curves were higher than 0.9924, showing good linearity.

    “For all four ecdysteroids, the R 2 values of the calibration curves were higher than 0.9924, which showed good linearity (Table 4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Freezing of samples reduced 20E content by 46% and MaA content by 94%.

    “The freezing of samples affected the content of ecdysteroids in plants more negatively (reductions of 46% for 20E and 94% for MaA).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Extraction efficiency was good, with recoveries in the range 75.2-98.7% and SD ≤ 5.72%.

    “The extraction efficiency of all the analytes was good, with acceptable and appropriate recoveries in the range of 75.2-98.7%with SD≤ 5.72% (Table 4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Extraction efficiency was higher after use of mixture 2 compared to mixture 1, by 46% for 20E and by 15% for MaA.

    “The extraction efficiency was higher after the use of mixture 2 compared to mixture 1, by 46% for 20E and by 15% for MaA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Pure ethyl acetate extracted no analytes, and acetone extracted only a small amount.

    “The exceptions were pure ethyl acetate (no responses for all analytes, mixture 1, assay 6) and acetone (a small amount of extracted analytes, mixture 1, assay 5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both intra- and inter-day measurements showed excellent concordance.

    “Both intra-and inter-day measurements showed excellent concordance.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The highest extraction efficiency and best removal of interferences was obtained using the C18 column (200 mg, 3 mL).

    “The highest extraction efficiency and the best removal of interferences was obtained using the C18 column (200 mg, 3 mL), so this column was selected for the clean-up procedure.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The ANOVA model for extraction yield had R2 and adjusted R2 of 0.9888 and 0.9721 for 20E and 0.9295 and 0.8239 for MaA.

    “The ANOVA results of the model (Table S1) indicated a good performance with R 2 and R 2adjusted of 0.9888 and 0.9721 for 20E and 0.9295 and 0.8239 for MaA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    Extraction yield improved with an increase in time to 120 min, but a further increase to 240 min did not significantly augment the content of analytes in the extract.

    “The extraction yields improved with an increase in time to 120 min, but a further increase to 240 min did not significantly augment the content of analytes in the extract.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    20E was found in higher concentration than makisterone A in plant samples, with 20E ranging from 0.52 to 428 μg/g and MaA from 2.18 to 67.2 μg/g.

    “20E (0.52-428 µg/g) was found in higher concentration than makisterone A (2.18-67.2µg/g), while ponasterone A and muristerone A were not detected in any plant.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Accuracy ranged from -5.78% to 4.89% for intra-day and from -8.14% to 6.39% for inter-day measurements.

    “The accuracy ranged from -5.78-4.89%and from -8.14-6.39%for intraand inter-day measurements, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    For mixture 1, best results were obtained for methanol as extraction solvent; for mixture 2, most effective was methanol:ethanol:water (1:1:1).

    “In conclusion, for mixture 1 the best results were obtained for methanol as extraction solvent, but for mixture 2 the most effective for isolation of ecdysteroids from plants was the methanol:ethanol:water (1:1:1; v/v/v) mixture.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    There was no significant difference in degree of separation between methanol-water and acetonitrile-water, but peaks were sharper and more symmetrical with acetonitrile-water mobile phase.

    “There was no significant difference in the degree of separation, but the peaks was sharper and more symmetrical with the acetonitrile-water mobile phase, so this mixture was selected as the mobile phase.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The method was linear in the concentration range 50-1000 ng/g for 20E and MaA, 200-1000 ng/g for MuA, and 100-1000 ng/g for PoA.

    “The linearity of the method was established in the concentration range 50-1000 ng/g for 20E and MaA, 200-1000 ng/g for MuA, and 100-1000 ng/g for PoA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The best results were obtained with a simple, linear gradient with the first analyte eluting after 1.3 min and the last before 3.0 min.

    “The best results were obtained with a simple, linear gradient with the first analyte eluting after 1.3 min and the last before 3.0 min (Fig. 2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Maximum yields of ecdysones were obtained with ternary solvent mixture 2 (assay 4), followed by methanol:water (assay 3) and ethanol:water (assay 7).

    “The maximum yields of ecdysones were obtained with ternary solvent mixture 2 (assay 4), which were followed by yields from the methanol:water (assay 3) and ethanol:water (assay 7) binary mixtures.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Fresh spinach contained the highest amount of ecdysones, 0.042% of dry mass, while the other studied plants contained below 0.008% of dry mass.

    “According to our analysis, fresh spinach contained the highest amount of ecdysones, 0.042% of dry mass, while in the other studied plants, the content of ecdysones was below 0.008% of dry mass.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The content of ecdysone in the different plants studied was very variable, and the quantitative ecdysone profiles of the studied plants were significantly different.

    “As shown in Table 5, the content of ecdysone in the different plants was very variable.”
    • Also stated in: § 3.4. Analysis of plant extract samples · hedged
      “As a summary of the analysis of studied plants, it may be conducted that their quantitative ecdysone profiles are significantly different.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The analyzed plant samples had 20E content ranging from 0.52 μg/g in arugula to 428 μg/g in spinach and MaA content ranging from 2.18 μg/g in spinach (leaves after cooking) to 67.2 μg/g in spinach (fresh leaves).

    “The analyzed plant samples had an 20E and MaA content ranging from 0.52 µg/g in arugula to 428 µg/g in spinach and from 2.18 µg/g in spinach (leaves after cooking) to 67.2 µg/g in spinach (fresh leaves), respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The content of individual ecdysones depended on the plant type and the area of cultivation.

    “Furthermore, our research on plant samples showed that the content of individual ecdysones depended on the plant type and the area of cultivation, which could be practically used in the verification of prohealth properties.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Among three spinach samples, 20E content declined after cooking in two samples (3-10%) and increased in one sample (2%), with differences very slight and possibly due to measurement error.

    “Among the three spinach samples, the content of 20E in two of them declined after cooking (3-10%), and one (spinach 3) increased instead (2%); however, the differences were very slight and may be due to measurement error.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Most methods available in the literature concern determination of 20E, and there are no accurate data on content of other ecdysones in plant tissues.

    “Most of the methods available in the literature concern the determination of 20E, and there are no accurate data on the content of other ecdysones in plant tissues (Glazowska et al., 2018).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Glazowska et al., 2018 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Each sample was analyzed in triplicate.

    “Each sample was analyzed in triplicate.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Because the Bond Elut AccuCAT mixed-mode SCX/SAX sorbent is effective for acidic or basic compounds, ecdysone recoveries with it were relatively low.

    “Such a mixture is effective for the extraction of acidic or basic compounds, so the recoveries of ecdysones were relatively low.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Mixed-mode SCX/SAX sorbents are effective for the extraction of acidic or basic compounds.

  • May combine two claims.

    Interpretationown data

    The adjusted R2 values of 0.97 and 0.82 imply that 97% and 82% of the variation in extraction yield is attributed to the selected independent variables.

    “The model R 2 -adjusted values of 0.97 and 0.82 imply that 97% and 82% of the variations associated with extraction yield are attributed to the selected independent variables (extraction time, temperature, and solid/ liquid ratio).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Both non-polar sorbents and sorbents with hydrophilic-hydrophobic properties are suitable for ecdysteroid extraction.

    “Therefore, both non-polar sorbents and those with hydrophilic-hydrophobic properties are suitable for their extraction.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Ecdysteroids are intermediate polar compounds with logP 2.01 for MaA and 1.98 for 20E.

  • May combine two claims.

    Interpretationown data

    The newly developed analytical method was effective, sensitive, and selective for determination of 20E and MaA in plants.

    “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.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Arugula used as a blank sample contained no ecdysones.

  • May combine two claims.

    Interpretationreasoning

    With the advantages of time-efficiency and detection limits as low as 50 ng/g sample, the method shows promise for many other plants and large sample sets.

    “With the advantages of time-efficiency and detection limits as low as 50 ng/g sample, this method shows promise for many other plants and large sample sets.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    Grucza et al. (2022) reported lower 20E content in spinach (10 μg/100 g), but that amount was given for 100 g fresh spinach, not for dry mass as in this work.

    “Grucza et al. (2022) reported lower content of 20E in spinach sample (10 µg/100 g), however the amount of 20E was given for 100 g of fresh spinach, not for dry mass, as in this work.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Grucza et al. (2022) source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    Metabolic reactions within the plants may also explain the ecdysteroid decrease due to the process.

    “Metabolic reactions (e.g.attachment of hydroxyl groups, detachment of hydroxyl groups, oxidation, epimerization) within the plants may also explain the ecdysteroids decrease due to the process.”
    Quote in the paperNot a substantial claim
31 of 31

  • The paper relies on this without support.

    Arugula used as a blank sample contained no ecdysones.

    “The arugula was used as a blank sample because its leaves are similar to spinach, but contained no ecdysones.”

  • The paper relies on this without support.

    The procedures described so far mainly used a non-selective spectrophotometric detector.

    “Moreover, the procedures described so far mainly used a non-selective spectrophotometric detector.”

  • The paper relies on this without support.

    The literature lacks information on the influence of processing (cooking or freezing) on the content of ecdysteroids in spinach samples.

    “The literature also lacks information on the influence of processing (cooking or freezing) on the content of ecdysteroids in spinach samples.”

  • The paper relies on this without support.

    Spinach contains high levels of phytoecdysones, particularly 20E.

    “Species from the Chenopodiaceae family, e.g., spinach (Spinacia oleracea L.), show a high content of phytoecdysones.”
    “Spinach is rich in 20-ecdysone (20E), which acts as an antifeedant against herbivorous insects and is a natural ecdysone that plays an important role in the normal physiological metabolism of arthropods.”

  • The paper relies on this without support.

    Ecdysteroids are intermediate polar compounds with logP 2.01 for MaA and 1.98 for 20E.

    “Based on the logP values, ecdysteroids can be considered as intermediate polar compounds (logP=2.01 for MaA, logP=1.98 for 20E).”

  • The paper relies on this without support.

    Mixed-mode SCX/SAX sorbents are effective for the extraction of acidic or basic compounds.

    “Such a mixture is effective for the extraction of acidic or basic compounds, so the recoveries of ecdysones were relatively low.”
6 of 6

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • The reported content of 20-hydroxyecdysone in spinach leaves ranges from 17.1 to 885 μg/g.

    “The reported content of 20HE in spinach leaves ranges from 17.1 to 885 µg/g [158,159].”

    From Ecdysterone and Turkesterone—Compounds with Prominent Potential in Sport and Healthy Nutrition (2024) · cites this paper as [159]

    Section 3.4 reports 20E in the spinach samples at 79.6–428 µg/g, not 17.1–885 µg/g. Table 5 (listed under Conclusions; page unavailable) gives the individual measurements in µg/g dry mass and contains neither claimed endpoint. (LLM, conf=0.88)

    Checked by a reasoning model — may be wrong.

    Experimental — accuracy not yet measured

  • The finding that none of the prepared plant extracts contained PA is consistent with the study by Fang et al. in 2022, which reported that various types of spinach did not contain PA.

    “This finding is consistent with the study by Fang et al. in 2022, which reported that various types of spinach did not contain PA [4].”

    From Phytoecdysteroids: Quantification in Selected Plant Species and Evaluation of Some Effects on Gastric Smooth Muscles (2024) · cites this paper as [4]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Abstract says cooking reductions were below 10%, but MaA fell much more

    Major

    The abstract characterizes the compounds as stable during cooking, with reductions below 10%. The results report MaA reductions of 67% and 82% in two of the three heated spinach samples.

  • Chosen extraction-solvent composition differs from the stated procedure

    Major

    In methanol:ethanol:water order, the mixture described as chosen for continued study is 25:20:45, not the 30:25:45 mixture specified for the SLE procedure. The two passages give different compositions for the selected extractant.

  • Arugula is called analyte-free but is reported to contain 20E

    Major

    The calibration procedure describes arugula as a blank containing no ecdysones, whereas Table 5 reports 0.52 μg/g of 20E in arugula. The text does not establish that the arugula used for calibration was the same sample, but the unqualified claim that arugula leaves contained no ecdysones conflicts with the reported result.

  • The stated ranking of binary extraction solvents reverses Table 2

    Minor

    For mixture 2, Table 2 gives ethanol:water (assay 7) higher responses than methanol:water (assay 3) for both 20E (0.99 versus 0.87) and MaA (0.88 versus 0.78). The text ranks assay 3 ahead of assay 7.

4 of 4

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. Rybin, 2007, Application of high-performance liquid chromatography for simultaneous identification of integristerone а, 20-hydroxyecdysone, ecdysone and 2-deoxy-20-hydroxyecdysone, Nat. Prod. Commun., 2, 1101

Cited by

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Related

Authors
Fang, X., Szołtysik, R., Tang, J., & Bajkacz, S.
Affiliations
  • Ningbo University (CN)
  • Silesian University of Technology (PL)
Corresponding author
Bajkacz, Sylwia
ORCID
Bajkacz, Sylwia
Venue
Journal of Food Composition and Analysis
Publisher
Elsevier BV
Published
2022-07
Volume
110
Pages
104580
ISSN
0889-1575 (ISSN-L)
DOI
10.1016/j.jfca.2022.104580
OpenAlex
W4223960660
Cited by
20
References
30
Citations by year
2026: 3 · 2025: 5 · 2024: 10 · 2023: 2
FWCI
1.39
Type
journal article
Language
en
Open access
Licence
Locations
Journal of Food Composition and Analysis (publishedVersion, cc-by)
Funders
Silesian University of Technology (04/010/RGJ20/0122)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Phytochemical Studies and Bioactivities
  • Physical Sciences › Chemistry › Analytical Chemistry › Analytical Methods in Pharmaceuticals
  • Life Sciences › Agricultural and Biological Sciences › Food Science › Pesticide Residue Analysis and Safety
Keywords
ecdysteroids, phytoecdysone, 20-hydroxyecdysone, makisterone A, ponasterone A, spinach, Eruca sativa, lamb's lettuce, solid-phase extraction, solid-liquid extraction, herbal medicine

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