A novel UHPLC method for quantitative analysis of five anthraquinone derivatives in Asphodeline plant extracts

A novel UHPLC method for quantitative analysis of five anthraquinone derivatives in Asphodeline plant extracts Irina Lazarova1, Silviya Ruseva2, Emiliya Cherneva1, Alexandrina Mateeva3, Iva Valkova1, Emilio Mateev3 1 Department of Chemistry, Faculty of Pharmacy, Medical University of Sofia, Sofia, Bulgaria 2 Department of Medical Chemistry and Biochemistry, Medical Faculty, Medical University of Sofia, Sofia, Bulgaria 3 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Medical University of Sofia, Sofia, Bulgaria Corresponding author: Irina Lazarova (lazaro[email protected]u-sofia.bg) Received 30 October 2025♦ Accepted 1 December 2025♦ Published 10 December 2025 Citation: Lazarova I, Ruseva S, Cherneva E, Mateeva A, Valkova I, Mateev E (2025) A novel UHPLC method for quantitative analysis of five anthraquinone derivatives in Asphodeline plant extracts. Pharmacia 72: 1–6. https://doi.org/10.3897/pharmacia.72.e176577 Abstract A reverse-phase ultra-high-performance liquid chromatography (UHPLC) method was established in this study for the simultaneous determination of five anthraquinones in extracts of Asphodeline species. A gradient program was used for chromatographic separation of the components under investigation. The analytes were separated on a Waters Acquity BEH C18 column with a flow rate of 0.3 mL/min. The mobile phase, consisting of acetonitrile and water containing 0.1% (v/v) formic acid, was used to elute the components under study. The compounds were detected at 435 nm. The method was validated according to ICH guidelines with respect to specificity, precision, linearity, and robustness. The proposed method was found to be convenient, reliable, and accurate for the quantitative determination of the described anthraquinones in Asphodeline species. Keywords Asphodeline, anthraquinones, UHPLC method, validation Introduction The genus Asphodeline Rchb. (Asphodelaceae) includes about seventeen perennial, grass-like flowering species distributed across southeastern Europe, the eastern Mediterranean, Krym, the northern and Transcaucasus regions, western Asia, and northern Africa. Members of the Asphodeline genus have been traditionally used in folk medicine and culinary practices (Locatelli et al. 2018). The plants of the genus Asphodeline Rchb. are renowned for containing bioactive anthraquinones with antitumor, antioxidant, antifungal, antibacterial, antiviral, purgative, and other health-beneficial properties (Wang et al. 2021; Mantareva et al. 2024). However, some recent articles describing the toxicity of plants containing anthraquinones and their derivatives, especially side liver effects, have been published (Hudson et al. 2018; Yang et al. 2018). In an earlier investigation, we described the dual biological potential of the dry root extract of Asphodeline lutea (L.) Rchb. (AsLuE). The extract exhibited mild hepatotoxic effects and moderate pro-oxidant activity, likely linked to its anthraquinone constituents. Our findings indicated that AsLuE contained a higher total level of anthraquinone derivatives Copyright Lazarova I et al. 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. Pharmacia 72: 1–6 DOI 10.3897/pharmacia.72.e176577 Research Article Lazarova I et al.: A UHPLC method for analysis of five anthraquinones in Asphodeline plant extracts2 than those typically found in vegetables commonly consumed in the human diet (Mueller et al. 1999). The in vivo analysis of AsLuE also displayed some toxic effects in Wistar rats related to increased enzyme levels of ALT, ALP, and malondialdehyde quantity and decreased GSH levels without affecting the activity of the antioxidant enzymes GPx, GR, and GST (Lazarova et al. 2016). Conversely, the antioxidant and hepatoprotective properties of AsLuE were also observed in both in vitro and in vivo models of CCl4-induced liver damage, where the extract restored all evaluated parameters to normal levels. In addition, AsLuE maintained the reduced cytochrome P450 content and EMND activity while having no effect on AH activity (Lazarova et al. 2016). These hepatoprotective and antioxidant effects can be explained by the high amount of caffeic acid and flavonoids as well as by metabolic interaction, prohibiting the production of CCl4 toxic metabolites (Kondeva-Burdina et al. 2018). There is conflicting evidence in the literature regarding the benefits and potential risks of using wild edible plants, food products, and supplements containing hydroxyanthracene derivatives (HADs) such as anthraquinones. Studies on the pharmacological properties and safety of HADs in different plant species are extremely lacking (Xu et al. 2022). Additional toxicological studies and development of reliable methods for the analysis of target compounds in plant extracts and marketed products are needed (Ekor 2013). Monitoring the main components, especially anthraquinones, in Asphodeline species is essential for assessing the safety of consumption of this edible plant. Therefore, the development of a simple, accurate, and reliable analytical method is crucial for its quality control. Materials and methods Chemicals and reagents The compounds aloe-emodin, rhein, emodin, chrysophanol, and physcion (with HPLC purity ≥ 98) were sourced from LGC Standards (Teddington, UK). Formic acid and acetonitrile were obtained from Merck (Darmstadt, Germany). Ultrapure water was produced using a Milli-Q Millipore Water Purification System (Bedford, MA, USA). All other chemicals used were of the highest purity available within the laboratory. Plant material and sample preparation The Asphodeline lutea roots were gathered near the town of Pernik, Bulgaria (42°29'35.15"N, 23°8'7.25"E). The Asphodeline taurica roots were collected from Slavyanka Mts (41°24'37.86"N, 23°39'31.15"E). The voucher specimens were preserved at the National Herbarium of the Institute of Biodiversity and Ecosystem Research at the Bulgarian Academy of Sciences. The samples were prepared according to the procedure described by Lazarova et al. (2016) with modifications. Finally, 5 µL of the sample were injected into the UHPLC system. Preparation of standard solutions A solution of each of the five standards was initially prepared at a concentration of 1 mg/mL in methanol. Working mixtures at concentrations of 1, 12.5, 25, 50, and 100 µg/mL were generated by dilution of a mixed stock solution at 1 mg/mL. Calibration curves were constructed by analyzing freshly prepared standard solutions in triplicate and using a weighted linear least squares regression equation. The concentrations were determined by interpolating the analyte peak areas against the calibration curve. All quantifications were carried out by measuring absorbance at 435 nm. UHPLC study of anthraquinones An anthraquinone quantification was conducted using a Dionex UltiMate 3000 RSLC UHPLC system (Thermo Scientific, Waltham, MA, USA). Chromatographic separation was achieved through gradient elution at a flow rate of 0.3 mL/min on a Waters Acquity BEH C18 column (50 mm × 2.1 mm internal diameter, 1.7 μm particle size). The mobile phase, composed of acetonitrile and water with 0.1% (v/v) formic acid, facilitated the elution of the analytes. The gradient conditions were set as follows: 0 min/45% B, 1 min/45% B, 8 min/63% B, 9 min/90% B, 13 min/90% B, 13.1 min/45% B, 15 min/45% B (see Table 1). A 5 µL injection volume was used, and the column temperature was maintained at 30 °C. Analyte detection was performed at 435 nm, and data processing was carried out with Chromeleon 7.2 software. Validation of the method Validation of the developed method was performed according to the recommendations of the International Conference on Harmonisation (ICH) guideline (ICH 2005). Because the effectiveness of extraction was outside the scope of this study, the recovery was not evaluated. Statistical analysis The statistical analyses were performed using SigmaPlot 12.0 (Systat Software, USA) and Excel software. All measurements were made in triplicate, and the data were presented as mean ± standard errors. Table 1. The mobile phase gradient. Time (min) A (%) B (%) 0 55 45 1 55 45 8 37 63 9 10 90 13 10 90 13,1 55 45 15 55 45 Pharmacia 72: 1–6 3 Results and discussion Optimization of chromatographic conditions Chromatographic parameters were optimized to improve peak shape, enhance sensitivity, and reduce analysis time for the simultaneous measurement of five anthraquinones. Various mobile phase compositions were tested to achieve optimal detection, sensitivity, and separation efficiency. Different combinations of methanol–water and acetonitrile–water systems, supplemented with varying concentrations of additives such as formic acid and phosphoric acid, were evaluated. A 0.1% formic acid in acetonitrile–0.1% formic acid in water system was found to produce the best chromatographic separation. Subsequently, the gradient elution approach was preferred and optimized. Optimal separation was accomplished using a Waters Acquity BEH C18 column (50 mm x 2.1 mm i.d., 1.7 μm) with a gradient program: 0–1 min, 45% B; 1–8 min, 45–63% B; 8–9 min, 63–90% B; 9–13 min, 90% B; 13–13.1 min, 90–45% B; 13.1–15 min, 45% B. The post-run time was 4 min at 45% B to re-equilibrate the column. The flow rate of 0.3 mL/min and the column temperature of 30 °C were preferred. Detection for quantitative analysis was performed at 435 nm. Figure of merits • Specificity The specificity was demonstrated by running a blank solution, standard, and sample (Fig. 1). The column efficiency and peak symmetry for the standard compounds were assessed in accordance with ICH guidelines. Figure 1. A chromatogram of the blank sample, standard mixed solution, Asphodeline lutea sample, and Asphodeline taurica sample. Lazarova I et al.: A UHPLC method for analysis of five anthraquinones in Asphodeline plant extracts4 • Linearity Linearity was assessed by analyzing five known concentrations of the mixed standards solution ranging from 1 to 100 µg/mL, with each concentration injected six times. Calibration curves for each anthraquinone were generated by plotting the peak areas against their respective concentrations (Fig. 2). The correlation coefficients, calculated using linear regression, were all greater than 0.99 (Table 2), indicating excellent linearity. • LOD and LOQ The limit of quantification (LOQ) represents the smallest concentration of an analyte in a sample that can be measured with acceptable accuracy and precision. In contrast, the limit of detection (LOD) is the lowest amount of analyte in a sample that can be detected but not necessarily quantitatively defined. The LOD and LOQ were calculated using the following equations after injection of standard solutions at low concentrations: LOQ = 10SD/Sl and LOD = 3SD/Sl, where SD is the standard deviation of the response and Sl is the slope of the calibration curve. • Precision Method precision was evaluated by assessing repeatability as well as inter-day and intra-day precision to ensure consistent performance. The intraday and interday precision tests were performed with a mixed standard solution of 50 μg/mL in six replicates. The inter-day precision was analyzed by different analysts. Peak area was evaluated, and the results for precision were determined as the percentage relative standard deviation (% RSD) (Table 3). • Robustness The robustness of the UHPLC method was evaluated based on the optimized conditions, considering slight and acceptable variations in key parameters to ensure consistent performance. The estimated parameters were mobile phase composition (0.1%, 0.4%, and 0.7% formic acid) and wavelength (430 nm, 435 nm, and 440 nm). Retention times and peak areas were verified for ruggedness by injecting the standard mixture at 50 μg mL-1 (Table 4). Table 2. The linear equations, correlation coefficients, LODs, and LOQs for anthraquinones under investigation. Analyte Linearity (µg/ml) Regression equation (y = ax + b) Correlation coefficient (R2) LOD (µg/ml) LOQ (µg/ml) Aloe-emodin 1–100 y = 0.2347x + 0.0869 0.9981 0.03 0.10 Rhein 1–100 y = 0.1420x + 0.1113 0.9975 0.06 0.17 Emodin 1–100 y = 0.2457x – 0.1954 0.9975 0.07 0.22 Chrysophanol 1–100 y = 0.5964x – 0.5090 0.9974 0.05 0.15 Physcion 1–100 y = 0.0634x – 0.0267 0.9980 0.08 0.25 Table 3. Repeatability and reproducibility of the analytical method. Analyte Repeatability (n = 6) Reproducibility (n = 6) Mean (μg/ml) RSD (%) Mean (μg/ml) RSD (%) Aloe-emodin 51.11 0.27 46.77 3.03 Rhein 49.89 0.27 46.44 4.01 Emodin 48.96 0.44 44.54 2.67 Chrysophanol 48.34 0.43 43.65 5.34 Physcion 48.99 0.29 45.98 5.11 Figure 2. Standard calibration curves of aloe-emodin, rhein, emodin, chrysophanol, and physcion in the range of 1 to 100 µg/mL. Pharmacia 72: 1–6 5 Table 4. Robustness of the analytical method. Compound Retention times ± SD Peaks area ± SD 0.1% FA 0.4% FA 0.7% FA 0.1% FA 0.4% FA 0.7% FA 430 nm 435 nm 440 nm Aloe-emodin 3.54 ± 0.03 3.54 ± 0.02 3.49 ± 0.02 9.5628 ± 0.05 9.0370 ± 0.10 9.5716 ± 0.11 9.1164 ± 0.19 9.5628 ± 0.05 8.6743 ± 0.07 Rhein 4.92 ± 0.03 4.91 ± 0.03 4.83 ± 0.02 6.1067 ± 0.07 5.8301 ± 0.18 6.3071 ± 0.10 6.0492 ± 0.07 6.1067 ± 0.07 5.7530 ± 0.05 Emodin 9.72 ± 0.02 9.73 ± 0.02 9.66 ± 0.00 10.3355 ± 0.17 10.7673 ± 0.02 11.1029 ± 0.18 10.7574 ± 0.09 10.3355 ± 0.17 10.8256 ± 0.17 Chrysophanol 9.97 ± 0.02 9.98 ± 0.01 9.89 ± 0.01 19.1168 ± 0.19 18.6537 ± 0.08 18.8842 ± 0.18 18.4507 ± 0.22 19.1168 ± 0.19 17.5724 ± 0.19 Physcion 11.78 ± 0.01 11.78 ± 0.01 11.73 ± 0.00 1.7798 ± 0.04 1.8194 ± 0.03 1.8106 ± 0.01 1.7093 ± 0.15 1.7798 ± 0.04 1.7380 ± 0.16 Table 5. The anthraquinone content in Asphodeline lutea and Asphodeline taurica root extracts. Analyte Asphodeline lutea Asphodeline taurica Content µg/g (n = 3) Content µg/g (n = 3) Aloe-emodin 22.13 ± 0.22 27.63 ± 0.31 Rhein 22.73 ± 0.27 21.21 ± 0.18 Emodin Not detected Not detected Chrysophanol 37.90 ± 0.20 38.26 ± 0.11 Physcion Not detected 3.76 ± 0.12 Two samples of Asphodeline lutea and Asphodeline taurica roots were analyzed using the method developed above. The content of each of the five anthraquinones in the analyzed samples was calculated according to the dry material (Table 5). Conclusion A reverse-phase ultra-performance liquid chromatography (UHPLC) technique was successfully developed and thoroughly validated for the simultaneous quantification of five key anthraquinones – aloe-emodin, rhein, emodin, chrysophanol, and physcion – in different species of Asphodeline. The method proved to be straightforward, dependable, precise, and efficient, significantly reducing the time required for analysis while maintaining high accuracy. Given its robustness and sensitivity, this UHPLC method is well suited for routine monitoring and quality control assessments of plant materials and their derived products containing the targeted anthraquinones, thereby ensuring both their consistency and safety for use. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statements The authors declared that no clinical trials were used in the present study. The authors declared that no experiments on humans or human tissues were performed for the present study. The authors declared that no informed consent was obtained from the humans, donors or donors’ representatives participating in the study. The authors declared that no experiments on animals were performed for the present study. The authors declared that no commercially available immortalised human and animal cell lines were used in the present study. Use of AI No use of AI was reported. Funding This work was supported by Grant D-142/29.05.2024 from the Medical Science Council at the Medical University– Sofia, Bulgaria. Author contributions All authors have contributed equally. Author ORCIDs Irina Lazarova https://orcid.org/0000-0002-8966-545X Silviya Ruseva https://orcid.org/0009-0005-1078-3787 Emiliya Cherneva https://orcid.org/0000-0002-1097-1850 Alexandrina Mateeva https://orcid.org/0000-0003-1905-7130 Iva Valkova https://orcid.org/0000-0002-6221-5002 Emilio Mateev https://orcid.org/0000-0002-5885-7213 Data availability All of the data that support the findings of this study are available in the main text. References Ekor M (2013) The growing use of herbal medicines: Issues relating to adverse reactions and challenges in monitoring safety. Frontiers in Pharmacology 4: е177. https://doi.org/10.3389/fphar.2013.00177 Hudson A, Lopez E, Almalki A J, Roe A L, Calderón A I (2018) A review of the toxicity of compounds found in herbal dietary supplements. 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