Stability-Indicating RP-HPLC Method Development and Validation with Forced Degradation Studies of Echinops echinatus Marker Compounds

Medicinal plants have served as an important source of therapeutic agents for centuries and continue to play a significant role in modern healthcare systems. The growing global demand for herbal medicines has highlighted the need for scientifically validated analytical methods to ensure their quality, safety, efficacy, and consistency. Unlike synthetic drugs, herbal materials contain complex mixtures of phytoconstituents whose chemical composition may vary depending on geographical origin, harvesting conditions, processing, and storage. Therefore, the standardization of herbal products through the identification and quantification of characteristic marker compounds has become an essential component of quality control.2,3,4

Echinops echinatus Roxb., belonging to the family Asteraceae, is a medicinal plant widely distributed in India and other parts of Asia. It has been traditionally used in the treatment of inflammation, fever, microbial infections, reproductive disorders, liver ailments, and various other health conditions. Pharmacological investigations have demonstrated that the plant possesses diverse biological activities, including anti-inflammatory, antioxidant, antimicrobial, hepatoprotective, analgesic, and immunomodulatory effects. These therapeutic properties are primarily attributed to the presence of bioactive phytochemicals such as flavonoids, phenolic compounds, alkaloids, triterpenoids, and other secondary metabolites. The quantitative determination of selected marker compounds is therefore important for establishing the identity and quality of E. echinatus and its formulations.

Reverse-phase high-performance liquid chromatography (RP-HPLC) is one of the most widely employed analytical techniques for the qualitative and quantitative analysis of phytochemicals because of its high sensitivity, selectivity, reproducibility, and suitability for complex herbal matrices. The development of a stability-indicating RP-HPLC method is particularly valuable, as it enables the separation of marker compounds from their degradation products and potential impurities, thereby ensuring reliable analysis throughout the product's shelf life. Such methods are essential for routine quality control, stability assessment, formulation development, and regulatory compliance.5,6

Method validation is a critical step in analytical method development and is performed to demonstrate that the method consistently produces reliable and reproducible results for its intended purpose. According to the International Council for Harmonisation (ICH) guidelines, analytical methods should be evaluated for parameters such as specificity, linearity, accuracy, precision, robustness, sensitivity, and solution stability. In addition, forced degradation studies conducted under acidic, alkaline, oxidative, thermal, photolytic, and hydrolytic stress conditions provide valuable information regarding the intrinsic stability of the analytes and establish the stability-indicating capability of the developed method.7,8

The objective of the present study was to develop and validate a stability-indicating RP-HPLC method for the analysis of selected marker compounds of Echinops echinatus and to evaluate their degradation behavior under various forced degradation conditions in accordance with ICH guidelines.

MATERIALS AND METHODS

The whole plant of Echinops echinatus was authenticated and shade-dried before being powdered for analysis. HPLC-grade methanol, acetonitrile, water, orthophosphoric acid, hydrochloric acid, sodium hydroxide, and hydrogen peroxide (Merck KGaA, Darmstadt, Germany) were used throughout the study. The reference standard of the selected marker compound was obtained from a certified commercial supplier.

Chromatographic analysis was performed using an Agilent 1260 Infinity II RP-HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a C18 column (250 mm × 4.6 mm, 5 µm). Standard and sample solutions were prepared in HPLC-grade methanol, filtered through a 0.45 µm membrane filter, and analyzed under optimized chromatographic conditions.9,10,11

The developed RP-HPLC method was validated according to ICH Q2(R2) guidelines for specificity, linearity, accuracy, precision, robustness, limit of detection (LOD), and limit of quantification (LOQ). Forced degradation studies were carried out under acidic, alkaline, oxidative, thermal, photolytic, and neutral hydrolytic conditions to evaluate the stability-indicating capability of the method.

As the study involved only plant material and analytical experiments, ethical approval was not required. All experiments were performed in triplicate, and the results were expressed as mean ± standard deviation (SD). Statistical analysis was carried out using GraphPad Prism version 10.0, with statistical significance considered at p < 0.05.12,13

A stability-indicating RP-HPLC method was developed for the analysis of the selected marker compounds of Echinops echinatus. Standard and sample solutions were prepared in HPLC-grade methanol and filtered through a 0.45 µm membrane filter before analysis. Chromatographic conditions, including the mobile phase composition, flow rate, detection wavelength, and column temperature, were optimized to achieve satisfactory separation and peak resolution.

The optimized method was validated according to ICH Q2(R2) guidelines by evaluating system suitability, specificity, linearity, accuracy, precision, robustness, limit of detection (LOD), and limit of quantification (LOQ).14,15

Forced degradation studies were performed by exposing the marker compounds and plant extract to acidic, alkaline, oxidative, thermal, photolytic, and neutral hydrolytic stress conditions. The stressed samples were analyzed using the developed RP-HPLC method to assess degradation behavior and to confirm that the method could effectively separate the marker compounds from their degradation products.16

RESULTS

Extraction of Flower of Echinopus echinatus The flowers of plant shade dried were powdered and extracted with various solvent. The appearance and percent yield of extracts are reported as

  follows:

 

 

 

 

Fig No :1 TLC Identification Around 0.35–0.50 Rf value was noted and it is in the acceptable range

 

 

Fig No 2- UV-Visible Spectroscopy Dissolve sample in methanol and get clear λmax (approx.) at 268 nm and it show exact same for std

 

 

Fig No 3-Development and validation of RP-HPLC method-

 

 

Fig No 4 Chromatogram of Trial no.1a (Methanol: Water with Composition 70:30v/v)

 

 

Fig No 5 Chromatogram of Trial 1b (Methanol: Water with composition 65:35v/v)

 

 

Fig No 6-Chromatogram of Trial no.1c (Methanol: Water with proportion 60:40v/v)

 

 

Fig No 7-Chromatogram of Trial no.2a (Methanol: OPA at pH 2.51 with ratio 65: 35v/v

 

 

Fig No 8 - Chromatogram of Trial no.2b (Methanol: OPA at pH 2.51 with ratio 60:40v/v)

 

 

Fig No 9- Chromatogram of Trial no.2c (Methanol: OPA at pH 3 with ratio 70:30v/v)

 

 

Fig No 10-Chromatogram obtained in Optimized mobile phase (Methanol: Phosphate buffer at pH 3.4 with 45:55v/v proportion with flow rate 1ml/min)

 

 

Fig No.-11 Concentration  Apigenin 7-O-Glucoside

CONCLUSION

In this present work RP-HPLC method development and validation as per regulatory requirements along with Stability Indicating Assay Methods is discussed.

HPLC method is available for simultaneous determination of Apigenin 7-O-glucoside in various plant like Chameli, Night Jasmine and various other plants but , No RP-HPLC method is available for simultaneous determination of Apigenin 7-O-glucoside from the flower part of echinopus echinatus roxob plant Similarly no studies have been carried out on Stability Indicating Assay methods for simultaneous determination of Apigenin 7-O-glucoside. This constituent is used as Anti-inflammatory, antioxidant, and anti-cancer agents. Hence this combination is selected for simultaneous estimation using HPLC method and Stability indicating assay method. HPLC remains the method of choice because it gives more sensitive results along with greater accuracy. It can be used both for qualitative as well as quantitative analysis. Isocratic method involves increase in solvent strength with time. This method is chosen since a sample containing compounds of a wide range of polarities can be separated by a isocratic elution in a shorter time period without a loss of resolution in the earlier peaks or excessive broadening of later peaks. Force degradation studies on Apigenin 7-O-glucoside were carried at different stress conditions like acid, base, oxidation, thermal, Photolysis degradation, to check the stability of the drugs. The percentage degradation of Apigenin 7-O-glucoside is more in hydrogen peroxide mediated oxidative degradation and less in thermal hydrolysis. The Apigenin 7-O-glucosid is degraded more in acid hydrolysis and less in photolytic condition. The stability indicating nature and specificity of the method was demonstrated as no degradation products from different stress conditions affected the detection and quantification of Apigenin 7-O-glucosid. The method was found to be stability indicating from the forced degradation studies which was found to be within limit and thus can be used for the simultaneous estimation of Apigenin 7-O-glucoside . The RP-HPLC method was validated and found to be specific, linear, accurate, precise and reproducible. Thus it is concluded that the developed method can be thus successfully applied to analyze various formulations containing Apigenin 7-O-glucoside

AKNOWLEGEMENT

The  authors  are  grateful  to  the  DJPS  College  of  Pharmacy,  Pathri  for providing research facilities.

FUNDING

Nil

AUTHORS CONTRIBUTIONS

All the authors have contributed equally

CONFLICTS OF INTERESTS

Declare none

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