A Novel RP-HPLC Analytical Method Development and Validation of Berberine In Bulk and Polyherbal Formulation

Bayye Kavitha Rani ⃰, Kedari Navya, Mrs. N.Ashritha

Department of Pharmaceutical Analysis, School of Pharmaceutical Sciences and Technologies, JNTUK Kakinada, Andhra Pradesh- 533003

*Correspondence: kavitabayye20@gmail.com,

DOI: https://doi.org/10.71431/IJRPAS.2025.4612          

INTRODUCTION

Berberine is a type of benzylisoquinoline alkaloid commonly present in the roots, rhizomes, and bark of plants belonging to the Berberidaceae, Ranunculaceae, and Rutaceae families. It exhibits a wide range of pharmacological activities including antimicrobial, anti-diabetic, anti-inflammatory, and cholesterol-lowering effects. Berberine is a naturally occurring compound found in several plants, including Berberis species (such as barberry, goldenseal, and Oregon grape). It belongs to a class of compounds known as alkaloids and has a bright yellow color, often used as a natural dye. Traditionally, berberine has been used in Chinese and Ayurvedic medicine for thousands of years to treat infections, gastrointestinal issues, and inflammation. In recent years, it has gained attention for its wide range of potential health benefits, particularly in managing blood sugar levels, cholesterol, and metabolic syndrome. Despite its promising effects, berberine should be used with caution, especially in combination with medications, as it can interact with enzymes in the liver that affect drug metabolism.

High-Performance Liquid Chromatography (HPLC) is a powerful and widely used analytical technique for the qualitative and quantitative analysis of various compounds, including natural alkaloids like berberine. Given berberine’s increasing importance in pharmaceutical and nutraceutical industries, developing accurate and reliable HPLC methods for its detection and quantification is essential.

Padmavathi S et al., RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR ESTIMATION OF BERBERINE IN BULK AND PHARMACEUTICAL DOSAGE FORMS 2019⁽³⁾. Berberine is a naturally occurring isoquinoline alkaloid and an important plant compound present in Berberis aristata, which belongs to the Berberidaceae family. In this study, a simple and easy-to-use RP-HPLC method was developed, making it suitable for the routine analysis and estimation of berberine.

Anubhuti Pasrija et al., A validated HPLC-UV technique was developed to precisely measure berberine content in the raw Daruharidra herb, and different Ayurvedic products available on the market 2010⁽⁴⁾. Berberis aristata is a plant species classified under the genus Berberis and belongs to the Berberidaceae family. This plant genus is commonly known as "berberry" in English and locally referred to as "Kashmol" or "Kinjosa." In this study, a new high-performance liquid chromatography (HPLC) method was developed and validated. The method is simple, sensitive, selective, and precise, and is suitable for analyzing berberine in raw plant material, herbal extracts, and Ayurvedic formulations.

Description of Berberine: (Figure 1: Chemical structure)

Figure 1 : Chemical structure

MATERIALS AND METHODOLOGY

Optimization of chromatographic conditions :

Proper selection of the chromatographic method depends upon the nature of the sample, its molecular weight and how easily it dissolves. The phytochemicals selected for the present study are polar in nature and hence either reverse phase or ion pair or ion exchange chromatography can be used. For the present study reverse phase HPLC method is considered to be more suitable because they are extremely specific, linear, precise, accurate, sensitive and rapid method.

Selection of detection wavelength for Berberine:

A 10 µg/ml solution of Berberine was prepared using a mixture of Acetonitrile and Water. This solution was scanned in the UV region of 200 - 400 nm and the UV spectrum was recorded ( Figure 2). From the spectra, detection wavelength 265 nm was selected.

Figure 2: UV Spectrum of Berberine

Diluent : used as a mobile phase.

Preparation of the mobile phase:

0.1% Orthophosphoric acid solution was prepared by taking 0.1ml make upto 100ml with HPLC water. The mobile phase used was a mixture of Acetonitrile and 0.1% Orthophosphoric acid in 60:40 v/v. The mobile phase was passed through a 0.45 µm membrane filter (Millipore) and then sonicated prior to use. Before injecting solutions, the column was run with the mobile phase for 30 minutes.

Preparation of standard stock and working solutions:

Standard stock solutions were prepared by transferring accurately weighed 100 mg of Berberine into separate 100 ml volumetric flask and the volume was made up with HPLC grade Acetonitrile. From this stock solution, standard solution (100ug/ml) was prepared by diluting 1ml up to 10ml with Acetonitrile and water in 1:1. Working Standard solution of Berberine (10 μg/ml) was prepared by further dissolving 1ml of the standard with the diluent (mobile phase).

RESULTS AND DISCUSSIONS

OPTIMIZED METHOD

Chromatographic conditions

Stationary phase                                  : Shiseido C18(250 x 4.6 mm;5µm)

Mobile Phase                                       : Acetonitrile: 0.1% Orthophosphoric acid (60:40 v/v)

Flow rate                                              : 1.0 ml/min

Column temperature                            : 25±2℃

Run time                                              : 6 min

Injection volume                                 : 20µL

Detection                                             : 265 nm

Figure 2 : Optimised chromatographic method

System Suitability:

The system suitability of the HPLC method was determined by making six replicate injections of 10µg/ml from freshly prepared standard solutions and analyzing each for their retention time, theoretical plates number(N) and tailing factor (T).

Table 1. System suitability studies

Specificity:

One of the significant features of HPLC is its ability to generate signals free from interferences. For this method, the active peak should be blank, sample and standard chromatograms. In order to prove that the method chosen was specific and selective the active peak of the drug and the sample peak were compared to the retention time against the blank and placebo chromatogram.  

Figure 4: Standard chromatogram of Berberine

Linearity:

The linearity was plotted within the range of 2,4,6,8, and 10 µg/ml for Berberine. The correlation coefficient (r²) greater than 0.99.

Figure 5 : Graph showing Linearity of Berberine

Precision:

The precision of the assay was measured by the percent coefficient of variation over the concentration range of low, middle and high-quality control samples of Berberine during the course of validation.

Accuracy:

The accuracy of the method was evaluated by determination of recovery of Berberine at three levels of concentrations 50,100,150 with three replicates.

LOD & LOQ:

LOD & LOQ prediction data was also considered from the linearity experiment and calculated the standard error as standard deviation from the linearity data.

                  LOD= ( standard deviation/slope)*3.3

                  LOQ= ( standard deviation/slope)*10.0

Figure 6: Typical chromatogram showing LOD

Figure 7 : Typical chromatogram showing LOQ

Robustness:

Robustness of the method was studied by injecting the standard solutions with slight variations in the optimized conditions namely, ± 2% in the ratio of Acetonitrile in the mobile phase, ± 0.1 ml of the flow rate.

SUMMARY OF VALIDATION REPORT

Table 2: Summary of validation report.

CONCLUSION

The developed HPLC method allows rapid and precise determinations of Berberine is to expand the optimization of the chromatographic conditions and develop a sensitive RP- HPLC method using Acetonitrile and 0.1% Orthophosphoric acid (60:40 v/v) as an ideal mobile phase. Since it gives a good resolution and peak shapes with perfect optimization. The flow rate at 1 ml/min was optimized.

The linearity and correlation coefficient (R²) of Berberine was found to be 2,4,6,8,10 µg/ml respectively and R² value is 0.996 respectively. The limit of detection for Berberine was found to be 0.1 ng/ml and the limit of quantification was found to be 1 ng/ml. The percentage recovery of Berberine was found to be 99.69% . Acceptance criteria according to ICH guidelines (98 to 102%). The isocratic elution technique developed for the determination of Berberine ideally suited for rapid and routine analysis. This method shows good reproducibility of the results. Hence this method was simple, sensitive and accurate. Robustness for the developed method was carried out and the results were observed to be within the limits.

The RP-HPLC method for analysis of Berberine was found to be accurate and precise. The proposed method was validated according to ICH guidelines like System suitability, Sensitivity, Linearity, Precision, Accuracy, Robustness. Satisfactory results were obtained when obtained values were correlated with standard values. The standard and sample preparation requires less time.

The reported methods in literature had limitations of more retention time, less sensitivity and more amount of organic phase. The present study was superior in terms of less retention time, low limit of detection and economical. So the developed method was said to be linear, accurate, precise , robust, sensitive. The developed and validated method was successfully applied for the estimation of Berberine in Polyherbal formulation.

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15.         Kamal, Y. T., M. Singh, E. T. Tamboli, R. Parveen, and S. Ahmad. "Quantitative analysis of berberine in Berberis aristata fruits and in a traditional anti-inflammatory unani formulation by use of a validated HPLC method." Acta Chromatographica 23, no. 1 (2011): 157-168.

16.         Pasrija, Anubhuti, Rahul Singh, and Chandra Kant Katiyar. "Validated HPLC-UV method for the determination of berberine in raw herb Daruharidra (Berberis aristata DC), its extract, and in commercially marketed ayurvedic dosage forms." International Journal of Ayurveda Research 1, no. 4 (2010): 243.

17.         Shigwan, Hemant, Arvind Saklani, P. D. Hamrapurkar, Tukaram Mane, and Priyanka Bhatt. "HPLC method development and validation for quantification of berberine from Berberis aristata and Berberis tinctoria." International Journal of Applied Science and Engineering 11, no. 2 (2013): 203-211.

18.         Jain, Shweta, Shalini Tripathi, and Pushpendra Kumar Tripathi. "Antiarthritic potential of berberine loaded invasomal gel." Phytomedicine Plus 2, no. 4 (2022): 100373.

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