Corresponding author: Zlatina Kokanova-Nedialkova ( zlatina.kokanova@pharmfac.mu-sofia.bg ) Academic editor: Plamen Peikov
© 2021 Zlatina Kokanova-Nedialkova, Paraskev Nedialkov.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Kokanova-Nedialkova Z, Nedialkov P (2021) Validated UHPLC-HRMS method for simultaneous quantification of flavonoid contents in the aerial parts of Chenopodium bonus-henricus L. (wild spinach). Pharmacia 68(3): 597-601. https://doi.org/10.3897/pharmacia.68.e69781
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A UHPLC-HRMS method for simultaneous quantification of flavonoid contents in the aerial parts of Chenopodium bonus-henricus L. was developed and validated. The amount of 12 detected flavonoids was calculated relative to external standard hyperoside. The calibration curve of hyperoside showed very good linear regressions and the correlation coefficient was R2 > 0.9979. The limits of detection and quantitation limits were 0.39 ng/mL and 1.17 ng/mL, respectively. The UHPLC-HRMS method showed acceptable accuracy. At three different concentrations the recoveries of hyperoside ranging from 99.63% to 100.70% with RSD from 1.58% to 2.31%. The intra-day and inter-day precision were determined by analyzing the retention times and recovery of the external standard. The glycosides of spinacetin and patulenin (1) were the predominant compounds in the wild spinach which contents ranging from 1.79 to 4.41 mg g-1 D.W., calculated as hyperoside. The total amount of flavonoids was found to be 15.12 mg g-1 D.W.
Amaranthaceae, Chenopodium bonus-henricus, flavonoids, quantification, UHPLC-HRMS
The genus Chenopodium (Amaranthaceae) numbers a wide range of species (more than 200) and is native to all the continents with exception of Antarctica as well as in some distant archipelagoes (such as Juan Fernandez, New Zealand, and Hawaii) (
Chenopodium bonus-henricus
L. is widespread in Europe, Western Asia, and North America. The leaves and flowering tops of Good King Henry (C. bonus-henricus L.) are used as a vegetable similar to spinach in some European traditional cuisines. In Italy, Spain, and England it is used in soups or stews and roughly in salads. In Turkey, it is known as “yabanı ispanak” (wild spinach). Canadians have also cultivated the plant as a daily vegetable. The shoots and flower clusters are eaten like asparagus and broccoli, respectively (
UHPLC-HRMS analysis was performed using a Thermo Scientific Dionex Ultimate 3000 RSLC (Germering, Germany) consisting of 6-channel degasser SRD-3600, high-pressure gradient pump HPG-3400RS, autosampler WPS-3000TRS, and column compartment TCC-3000RS coupled to Thermo Scientific Q Exactive Plus (Bremen, Germany) mass spectrometer. All the reagents used were of analytical grade. Hyperoside (≥ 97%, HPLC) was purchased from Sigma-Aldrich (Taufkirchen, Germany).
The aerial parts of Chenopodium bonus-henricus L. were collected from Beglica, Western Rhodopes, Bulgaria in June 2019. The plant was identified by P. Nedialkov and a voucher specimen from the plant population (No. SOM-Co-169849) was deposited at the National Herbarium, Bulgarian Academy of Sciences, Sofia, Bulgaria.
The aerial parts of C. bonus-henricus L. were dried in а shade, and the powdered plant material (200.08 mg) was extracted with 80 vol. % MeOH (60 mL) by ultrasonic-assisted extraction for 30 minutes. The MeOH extract was diluted to 100 mL 80 vol. % MeOH. The resulting solution was filtered, and the first 10 mL were removed. An aliquot (10 mL) of this solution was evaporated to dryness, then dissolved in water, and further purified by solid-phase extraction over RP18. The sorbent was first washed with H2O, then eluted with 80 vol. % MeOH (12 × 500 µL) in a 10.0 mL volumetric flask and diluted to the nominal volume with the same solvent (solution A). Subsequently, 1 mL of solution A was diluted to 10 mL 80 vol. % MeOH (solution B). Solution B was used for LC-MS quantification of flavonoid contents.
UHPLC separations were performed on a Kromasil Eternity XT C18 column (AkzoNobel, Sweden) (2.1×100 mm, 1.8 μm) equipped with precolumn SecurityGuard ULTRA UHPLC EVO C18 (Phenomenex, USA) at 40 °C. Each chromatographic run was carried out with a binary mobile phase consisting of water containing 0.1% (v/v) formic acid (A) and acetonitrile also with 0.1% (v/v) formic acid (B). A gradient program was used as follows: 0–0.5 min, 10% B; 0.5–1 min, 10–12% B; 1–7 min, 12–14% B; 7–13 min, 14–17% B; 13–16 min, 17–28% B, 16–17.50 min, 28–95% B, 17.50–19.50 min, 95% B. The flow rate was 0.3 mL.min−1 and the sample injection volume was 2 µL.
Operating conditions for the HESI source used in a positive ionization mode were as follows: +3.5 kV spray voltage, 320 °C capillary and probe heater temperature, sheath gas flow rate 36 a.u., auxiliary gas flow 11 a.u., spare gas flow 1 a.u. (a.u. refer to arbitrary values set by the Exactive Tune software) and S-Lens RF level 50.00. Nitrogen was used for sample nebulization as well as collision gas in the HCD cell. The All Ion Fragmentation (AIF) mode was used as an MS experiment where the resolution, automatic gain control (AGC) target, maximum inject time (IT) and mass range were 70000 (at m/z 200), 3e6, 200 ms, and m/z 200–1200, respectively. The product ions at m/z 303.0499 (for hyperoside), 317.0656 (for 3, 4, 6, 7, 10 and 12), 333.0605 (for 1, 2 and 9) and 347.0761 (for 5, 8 and 11) with 5.0 ppm isolation window were used as quantifiers. Xcalibur software ver. 4.0 was used for data acquisition and processing.
The quantification of flavonoids was carried out using the external standard method. The amount of 12 previously detected flavonoids (
The limit of detection (LOD) of an analytical procedure is the lowest analytical concentration at which an analyte(s) could be detected qualitatively. Typically, peak heights are two or three times the noise level. The LODs were calculated according to the expression 3.3 σ/S, where σ was the standard deviation of the response and S the slope of the calibration curve. The quantitation limit (LOQ) is also the lowest concentration at that level analyte can be quantitated with acceptable precision, requiring peak heights 10 to 20 times higher than the baseline noise. The LOQs were established from the expression 10 σ/S (
Accuracy is the closeness of the analytical results obtained by the analyses to the true values and is usually presented as a percent of nominal (
The precision of an analytical method is the amount of variation in the results obtained from multiple analyses of the homogeneous samples. Intra‐day precision (repeatability) defines the precision obtained using the same operating conditions over a designated short period (typically ≤1 day). Inter‐day precision (intermediate precision) defines the precision obtained using the same operating conditions, typically within the same laboratory, over a designated period (typically ≥1 day) (
Ultra-high performance liquid chromatography – high-resolution mass spectrometry (UHPLC-HRMS) was used to detect the flavonoids in the aerial parts of C. bonus-henricus (Fig.
The efficiency of the extraction procedure was tested by using different solvents (80 vol. % MeOH, 70 vol. % MeOH and 50 vol. % MeOH) and techniques (ultrasonic-assisted and magnetic stirring extractions). The results showed that the highest extraction efficiency was achieved using ultrasonic-assisted extraction with 80% MeOH. The excellent selectivity of solid-phase extraction over RP18 for the preparation of MeOH extract helped to improve the LC-MS analysis.
The chromatographic conditions were optimized to achieve effective separation, symmetric peak shape, and short run time. Two chromatographic columns, Kromasil Eternity XT C18 column (2.1×100 mm, 1.8 μm) and Phenomenex Kinetex EVO C18 (100×2.1 mm, 1.7 μm) were pretested, and the best separation efficiency was obtained with the Kromasil Eternity XT C18 column. Acetonitrile was selected as the mobile phase due to its improved separation, and reduced column backpressure compared with methanol. The addition of formic acid in the mobile phase improved the peak shape, sensitivity, and retention time of individual flavonoids, which was consistent with the previous report (
Quantitative determination of flavonoid contents in the aerial parts of C. bonus-henricus L. was performed by the method of the external standard. The amount of 12 previously detected flavonoids (
The calibration curve of hyperoside was linear over the concentration range from 12.588 to 402.8 ng/mL and showed very good linear regression. The correlation coefficient was R2 = 0.9979. The method showed that LOD and LOQ were 0.39 ng/mL and 1.17 ng/mL, respectively (Table
The accuracy of hyperoside was checked at three different concentrations (40.08, 80.16, and 160.32 ng/mL). The external standard showed overall recoveries ranging from 99.63% to 100.70% with RSD from 1.58% to 2.31% (Table
The precision of the retention times was determined by analyzing the calibration sample during a single day and on three different days, respectively. The RSDs of retention times of hyperoside were 0.07 for intra‐day and 0.09 for inter‐day precision assays. Also, the external standard showed recoveries at 98.04% (for intra‐day precision assay) and 99.23% (for inter‐day precision assay) with RSDs at 2.38% and 1.32% respectively (Tables
The results (Table
External standard | Linear range (ng/mL) | Regression equation | R2 | LOD (ng/mL) | LOQ (ng/mL) |
---|---|---|---|---|---|
Hyperoside | 12.588–402.8 | Y = -391191+110556*X | 0.9979 | 0.39 | 1.17 |
External standard | Added (ng/mL) | Founda (ng/mL) | Recoverya (%) | RSD (%) |
---|---|---|---|---|
Hyperoside | 40.08 | 40.36 ± 0.82 | 100.70 ± 2.05 | 2.03 |
80.16 | 80.47 ± 1.86 | 100.39 ± 2.32 | 2.31 | |
160.32 | 159.73 ± 2.53 | 99.63 ± 1.58 | 1.58 |
Evaluation of intra‐day (repeatability) and inter‐day (intermediate precision) precision of the UHPLC-HRMS method applied on hyperoside.
Precision type | RT ± SD (min) | RSD (%) | Recovery ± SD (%) | RSD (%) |
---|---|---|---|---|
Intra‐day | 10.50 ± 0.008 | 0.07 | 98.04 ± 2.34 | 2.38 |
Inter‐day | 10.49 ± 0.009 | 0.09 | 99.23 ± 1.31 | 1.32 |
No. | tR (min) | Flavonoids | mg g-1 D.W. ± SD |
1 | 7.00 | patuletin-3-O-[β-apiofuranosyl (1→2)]-β-glucopyranosyl (1→6)-β-glucopyranoside | 1.79 ± 0.0007 |
2 | 8.39 | patuletin-3-O-gentiobioside | 0.82 ± 0.0036 |
3 | 9.13 | isorhamnetin-Hex-Hex-Pent | 0.32 ± 0.0006 |
4 | 9.32 | 6-methoxykaempferol-3-O-[β-apiofuranosyl (1→2)]-β-glucopyranosyl (1→6)-β-glucopyranoside | 0.74 ± 0.0010 |
5 | 10.20 | spinacetin-3-O-[β-apiofuranosyl (1→2)]-β-glucopyranosyl (1→6)-β-glucopyranoside | 4.41 ± 0.0075 |
6 | 11.63 | 6-methoxykaempferol-3-O-gentiobioside | 0.34 ± 0.0035 |
7 | 11.82 | isorhamnetin-Hex-Hex | 0.20 ± 0.0017 |
8 | 12.89 | spinacetin-3-O-gentiobioside | 3.19 ± 0.0104 |
9 | 15.66 | patuletin-3-O-(5˝΄-О-Е-feruloyl)-β-D-apiofuranosyl(1→2) [β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside | 0.70 ± 0.0022 |
10 | 16.16 | isorhamnetin-Hex-Hex-Pent-FA | 0.20 ± 0.0016 |
11 | 16.17 | spinacetin-3-O-(5˝΄-О-Е-feruloyl)-β-D-apiofuranosyl (1→2) [β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside | 2.07 ± 0.0031 |
12 | 16.32 | 6-methoxykaempferol-3-O-(5˝΄-О-Е-feruloyl)-β-D-apiofuranosyl (1→2) [β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside | 0.34 ± 0.0036 |
A novel UHPLC-HRMS method for simultaneous quantification of flavonoid contents from the aerial parts of the wild spinach (Chenopodium bonus-henricus L.) was developed. The optimized method was validated for specificity, the limit of detection and quantitation limit, linearity, accuracy, and precision. The glycosides of spinacetin (5, 8, and 11), and patulenin (1) were the predominant compounds in the aerial parts of C. bonus-henricus L. The results show that the proposed method can be easily used to quantify the total content of flavonoids in plant substances to control their quality, as well as to facilitate the isolation of potentially phytochemically interesting molecules.