To Develop and Validate an RP HPLC Method for Estimation of Marker Compound and Compare Quality of Different Commercial Brands of Sitopaladi Churn

Table no 1 RPHPLC Conditions

UV Analysis of Piperine in Commercial Brands

The UV spectrophotometric analysis of Piperine present in different commercial brands of Sitopaladi Churn was carried out to confirm the presence of the marker compound and to compare the spectral characteristics across formulations

Table no 2 Various Extraction

Procedure

Accurately weighed 500 mg of Sitopaladi Churn from each commercial brand (e.g., Dabur, Baidyanath, Zandu, Himalaya, Charak). Each sample was extracted separately with methanol (50 mL) in a conical flask. The mixtures were sonicated for 30 minutes to ensure complete extraction of phytoconstituents. The extracts were filtered using Whatman filter paper and further filtered through a 0.45 µm syringe filter. Suitable dilutions were prepared from each filtrate to obtain measurable absorbance. Each sample solution was scanned in the range of 200–400 nm using UV-Visible spectrophotometer against methanol as blank. The spectra obtained were compared with the standard Piperine spectrum.

Observation

 All three commercial brands showed absorption peaks in the UV region similar to that of standard Piperine. A characteristic absorption maximum was observed at approximately 343 nm, confirming the presence of Piperine. Slight variations in absorbance intensity were observed among different brands,

Method development by RP-HPLC and optimization-

Preparation of Mobile phase

Mobile phase was prepared by using double distilled water of pH 3-4.5 adjusted with (0.1%v/v) OPA and acetonitrile. The double distilled water was taken which is adjusted to pH3-4.5with Ortho-Phosphoric Acid and was filtered through 0.45 µm Nylon 6,6 membrane filter and was degassed by ultra sonicator. Acetonitrile was filtered through 0.45 µm Nylon 6,6 membrane filter and was degassed by ultrasonicator.

Preparation of stock solution

The stock solution of Piperine was prepared for RP-HPLC analysis to construct the calibration curve and for quantitative estimation in different commercial brands of Sitopaladi Churn.

Procedure

Accurately weighed 10 mg of Piperine standard using an analytical balance. Transferred the weighed standard into a 10 mL volumetric flask. Added approximately 5 mL of methanol and sonicated for 5–10 minutes to ensure complete dissolution of the compound. The volume was then made up to 10 mL with methanol to obtain a stock solution of 1000 µg/mL (1 mg/mL). The stock solution was stored in a refrigerator (2–8°C) when not in use to maintain stability

Preparation of sample solution of marketed formulation

The sample solution of Sitopaladi Churn from different commercial brands was prepared for quantitative analysis of Piperine by RP-HPLC.

Procedure

Accurately weigh 500 mg of Sitopaladi Churn from a selected commercial brand. Transfer the powdered sample into a 50 mL volumetric flask. Add approximately 25 mL of methanol to the flask. Sonicate the mixture for 30 minutes to extract Piperine completely. Allow the solution to cool (if any heat is generated during sonication) and dilute to the mark with methanol. Mix well to obtain a homogeneous solution. Filter the solution through a 0.45 µm syringe filter to remove insoluble particles before HPLC injection. Transfer the filtered solution to an HPLC vial for analysis.

Dilution for HPLC Analysis

If required, prepare a suitable dilution of the sample solution to match the concentration range of the calibration curve. Example: Take 1 mL of filtered sample solution and dilute to 10 mL with methanol to obtain a working sample solution.

Optimization of Developed method-

Optimization in HPLC is the process of finding a set of conditions that adequately separate and enable the quantification of analyte from the endogenous material with acceptable accuracy, precision, sensitivity, specificity, cost, ease and speed. Various trials were carried out by using different composition of mobile phase. Initially the study was started with 100 % acetonitrile followed by use of various other compositions. It was observed that peak of Piperine was too broad to get satisfactory retention time of compound. The concentration of organic phase was decreased. The optimum mobile phase composition was found to be containing acetonitrile: double distilled water pH adjusted with ortho-phosphoric acid (50: 50) at flow rate 1ml/ min. This optimum condition was selected so as to shift the retention time of drug at sufficient length of time. The run time is kept as 20 minute.

Validation of method-

Validation of Analytical Method may be defined as the process by which it is established, by laboratories studies, that the performance characteristics of the method meet the requirements for the intended analytical application. Typical validation parameters to be considered are as follows:

Linearity:Linearity of Piperine(S1 S2 and S3) was performed using standard solution in the range of 1-7 µg/ml of each drugs.

Acceptance criteria: Correlation coefficient (R2) should be not less than 0.999.

Range:

For range, data shall be considered from linearity and accuracy.

Acceptance criteria- Correlation coefficient (R2) should be not less than 0.999 and individual % recovery of at each level should be between 98 % - 102 %.

System Suitability-

System suitability testing is essential for the assurance of the quality performance of the chromatographic system. The system suitability parameters with respect to theoretical plates, capacity factor, resolution factor, tailing factor were calculated. In system suitability was evaluated by analyzing different concentration of 1-7 µg/ml of Piperine(S1 S2 and S3) Limit of detection (LOD) and Limit of Quantitation (LOQ)-The limit of detection (LOD) and limit of quantitation (LOQ) were determined by calculating the signal to noise (S/N) ratio of the LOD preparation and LOQ preparation. The Limit of detection (LOD) and Limit of Quantitation (LOQ) were evaluated from calibration curve of linearity data.

Acceptance criteria- A lower LOD and LOQ value indicates high sensitivity of the method.

Accuracy and Recovery-

To evaluate the recovery at each level, working standards of the drugs were added at three different concentrations (levels) 80 %, 100 % and 120 %, to the known amount of sample which was kept constant. The measurements were done in triplicates.

Acceptance criteria: Individual % recovery of Piperine(S1 S2 and S3)  at each level should be between 98 % - 102% and % RSD should not be more than 2.0 at each level.

Precision-

Precision of the method was verified by repeatability and intermediate precision studies. Repeatability studies were performed by analyses of 1 µg/ml of the sample for five times on the same day. Intraday and Interday precision of the method was checked by repeating the studies on within same day and at different days.

Acceptance criteria: % RSD should not be more than 2.0.

Ruggedness-

Ruggedness of the method was verified by analyzing samples of the same batch used for method   precision as per proposed method by different analyst. The SD and % RSD of Piperine(S1 S2 and S3) was determined.

Acceptance criteria: % RSD should not be more than 2.0

Robustness-

To determine the robustness of the method, experimental conditions were purposely altered. Two parameters selected were flow rate and mobile phase ratio. The flow rate of mobile phase was 1 ml/min. This was changed to 0.8 ml/min and 1.2 ml/min and effect was studied. The mobile phase ratio was 50:50. This was changed to 55:45 and 53:47 and effect was studied. When the effect of altering one set of conditions was tested, the other conditions were held constant at the optimum values.

Forced Degradation Study-

Stress studies were carried as per ICH guideline. Degradation studies were performed with standard & bulk drug. The following conditions were applied for forced degradation.

Acidic Condition-

The degradation study was carried out by taking 1mg of each Piperine(S1 S2 and S3) in small RBF & 0.5 ml acetonitrile was added to dissolve all drugs. Then add 1 ml of 0.1 N HCl to it. This solution was refluxed at 80 0 C for 1 hours. Then solution were withdrawn from RBF in 10 ml volumetric flask & kept at room temperature. It is then neutralized with 0.1 N NaOH. This solution was filtered by membrane filtre.1 ml of degraded sample was diluted with mobile phase to 10 ml in volumetric flask.

Basic condition-

The degradation study was carried out by taking 1mg of each Piperine(S1 S2 and S3) in small RBF and add 1 ml/ mg of 0.1 N NaOH in RBF . This solution was refluxed at 80 0 C for 1 hours. Then solution were withdrawn from RBF in 10 ml volumetric flask and kept at room temperature. It is then neutralized with 0.1 N HCl. This solution filtered by membrane filter. 1 ml of degraded sample was diluted with mobile phase to 10 ml in volumetric flask.

Neutral Condition

The degradation study was carried out by taking 1 mg of each Piperine(S1 S2 and S3) in 10 ml volumetric flask and adds 1ml/ mg double distilled water to it. This solution was sonicated for 5 min in order to dissolve the drug and kept at room temperature for 1 hour. This solution was filtered by membrane filter. 1 ml of degraded sample was diluted with mobile phase to 10 ml in volumetric flask.

Oxidative Condition-

1mg of each Piperine(S1 S2 and S3) was taken in RBF and add 6 % v/v H 2O2. Then solution was refluxed at 80 0 C for 1 hours. Then solution was withdrawn from RBF in 10 ml volumetric flask allowed to room temperature. 1 ml of degraded sample was diluted with mobile phase to 10 ml in volumetric flask.

Thermal Degradation-

For thermal stress study Piperine(S1 S2 and S3) solid drugs were kept in dry oven at 1050 C for 24 Hours. The 1 µg/ml solution was prepared with mobile phase and analyzed.

Photolytic Degradation-

Forced degradation was carried out under stress conditions of photolysis (UV light -254 nm).The solid powder of all drugs was kept in petri plate for 5 hours. The degraded sample was dissolved in mobile phase and then injected.

RESULTS AND DISCUSSION-

Selection of various brands sitophaladi churn

Select three brands for the comparative study according to their celling situation. The brands contains various ingredients but the main marker constituent is piperine

Table no.3 Name of commercial brand

Purchase Samples

All sample was purchased from Ahilya medical and retail shop from Pathri within expiry. 

Table no.4 Purchase Samples information

Preliminary Evaluation

A preliminary evaluation of Sitopaladi Churna brands should focus on classical formulation accuracy, ingredient quality, manufacturing standards, consumer reliability, and overall consistency. Dabur, Baidyanath, and Patanjali all market the classical Ayurvedic formulation, but they differ in processing quality, market reputation, and user perception.

Table no.5 Preliminary Evaluation of various brands of Sitiphaladi

Physicochemical Evaluation of various Brands of sitophaldi

Table no.6 Physicochemical Evaluation of various brands of Sitiphaladi

7.5       Separation of Piperine from Sitopaladi Churn

From literature review, we identified that Pippali (Piper longum) contains the major bioactivecompound: Piperine which is a major marker compound. Piperine was successfully separated and identified from Sitopaladi Churna by solvent extraction and chromatographic techniques.

Extraction of Crude Piperine Sitopaladi Churn and transfer to a conical flask.

Add 50 mL methanol (or methanol:water)

Fig no 1 Various Extraction

Extraction of Dabur Baidynath and Patanjali Sitophaladi churn

The residue contains Piperine along with other compounds after the extraction with methanol and water in sonicate for 30 min.

Isolation of crude drug

By using seprating funnel organic layer containing crude extract was seprated. Crude piperine extract was successfully separated from the aqueous extract using solvent partition method in a separating funnel.

Fig No 2- Sepration of Dabur Baidynath and Patanjali Sitophaladi churn

Fig No 3- Evaporation of solvent from piperine

Evaporate the solvent on a water bath or rotary evaporator. Crude yellowish piperine extract remains. Various brands like Dabur Baidynath Patanjali brands extract was done and isolation of piperine was done liquid piperine was dried on water bath

TLC Identification

Around 0.35–0.50 Rf value was noted and it is in the acceptable range

TLC Identification of Piperine in Different Brands of Sitopaladi Churna

Mobile Phase Hexane : Ethyl acetate (7:3) and Stationary Phase Silica Gel 60 F254 TLC Plate

TLC analysis of methanolic extracts of different marketed Sitopaladi Churna formulations showed spots corresponding to standard Piperine with comparable Rf values. This confirmed the presence of Piperine in all selected brands.

he TLC chromatogram demonstrated effective separation of Piperine using hexane:ethyl acetate (7:3) as the mobile phase. All marketed formulations showed spots at Rf values comparable to standard Piperine. Dabur and Baidyanath formulations exhibited intense compact spots, whereas Himalaya formulation showed relatively lower spot intensity, suggesting comparatively lower Piperine content.

    Strong spot → higher Piperine concentration

    Faint spot → lower Piperine concentration

    Similar Rf value → confirms identity of Piperine

TLC Results of Different Sitopaladi Churna Brands

Table no.7 TLC Results of Different Sitopaladi Churna Brands

Dabur Sitopaladi Churna containing piperine has show the similar Rf value as standard

Fig No 4- TLC of  piperine

Development and validation of RP-HPLC method-

The present work, carried out to develop and validate simple, selective, sensitive, rugged and reproducible method for quantitative determination of marker compound of sitophaladi churn as  Piperine. The analytical method based on RP-HPLC using UV detection was developed and validated for determination of drug as chemical constituent Piperine from various commercial brands like Badynath Dabur and Patanjali for estimation of quality of marker constituent.

Table no 8 Comparative Initial Trial Observation for Three Brands

Table no.9 Initial Trials of Piperine in Commercial Brands

Fig 5 Chromatogram of Trial no Acetonitrile : Water (50:50) Better peak symmetry and reduced retention time

Fig 6 Chromatogram of Trial no Methanol: Water (50:50) Broad peak, high retention time, poor separation

Fig 7 Chromatogram of Trial no Acetonitrile : Water (60:40) Sharp peak, good resolution, symmetrical chromatogram

Validation of Drugs Piperine-

Linearity and Range-Linearity and range of standard drugs Stock solution of Piperine was prepared as: Weigh 10 mg Piperine standard, Dissolve in  methanol Make volume up to 10 mL Stock concentration = 1000 µg/mL From stock solution, different dilutions were prepared 20 to 100 µg/ml concentration was prepared.

Table no.15 concentration of S1

Fig no. 8 Calibration curve of S1

Table no.9 concentration

Fig 10 Calibration curve of S2

Table no.17 concentration of S3

Fig no. 11 Calibration curve of S3

Determination of range of linearity for standard Piperine

Table No.18 Study of Linearity and Range

Determination of range of linearity for standard Piperine

Table No.19 Study of Linearity and Range

Fig no 12 HPLC chromatogram of standard piperine

At an optimal wavelength of 345 nm, the obtained HPLC chromatogram using 0.02 % standard piperine revealed a mean area of 5241042  at a mean retention time of 8.578 min shown in Fig. 2. The relative standard deviation of peak area and retention time 0.273 % and 0.064 % respectively

1)         System suitability parameter

From table  all the system suitability parameters are within the acceptable range indicating the suitability of the method for estimation of Piperine.

Table No.10 Study of System suitability parameter

1)         Limit of Detection & Limit of Quantification-

Lower LOD and LOQ value indicates high sensitivity of the method. The limit of detection (LOD) and limit of quantitation (LOQ) were determined by calculating the signal to noise (S/N) ratio of the LOD preparation and LOQ preparation. The Limit of detection (LOD) and Limit of Quantitation (LOQ) were evaluated from calibration curve of linearity data.

Table no 21 LOD and LOQ of Piperine in Different Commercial Brands of Sitopaladi Churna

The developed RP-HPLC method exhibited good sensitivity for estimation of Piperine in different commercial brands of Sitopaladi Churna. The LOD values ranged from 0.45–0.46 µg/mL, while LOQ values ranged from 1.35–1.41 µg/mL, indicating that the method is sufficiently sensitive for routine quality control analysis of marketed formulations.

1)         Accuracy and analyte Recovery -

Recovery studies were conducted at three levels i.e. 80%, 100% and 120% of target concentration. It was carried out by adding known amount of standard drug of sample Piperine.to the pre-analyzed. The mixed sample solutions were analyzed to obtained peak, retention time, and area under curve respectively. The concentration of Piperine was calculated from area under curve of peak. At each concentration three were performed and obtained were compared with standard results. The % RSD of accuracy study was found to be within specific limit and thus we can concluded that the developed method is suitable for assay and there is no interference of excipients. The recovery data from the study were reported in Table: The recovery of Piperine from Sitopaladi Churna formulations ranged between 98–102%, indicating good accuracy of the developed RP-HPLC method. The low %RSD values confirmed the reliability and reproducibility of the method.

Table No.11 Accuracy study of Piperine

2)         Precision-

Precision of the developed RP-HPLC method for estimation of Piperine in different commercial brands of Sitopaladi Churna such as Dabur, Patanjali, and Baidyanath was evaluated in terms of intra-day and inter-day precision. The study was performed by repeated analysis of sample solutions under the same chromatographic conditions and the results were expressed as %RSD of peak area. Method precision was demonstrated by preparing six test solutions at 100% concentration as per the test procedure & recording the chromatograms of six test solutions. The % RSD of six samples was calculated. Intermediate precision of the analytical method was determined by performing method precision on another day by different analysts under same experimental condition. The % RSD values were found to be within specified limit (<2%), which indicates that the developed method is precise. The results 20 µg/ml of standard drug solution (Piperine.) was repeated for five times and the peak area was determined and was found to be consistent.

Table No.12 Precision study

The %RSD values for both intra-day and inter-day precision studies were found to be less than 2%, indicating that the developed RP-HPLC method is precise, reproducible, and suitable for routine quantitative estimation of Piperine in different commercial brands of Sitopaladi Churna.

Intraday precision-

The % RSD for retention time and area was found to be 0.18 of Piperine., % RSD is not more than 2 and is found within the specified limit.

Table No.13 Interday Precision study

The %RSD values for both intra-day and inter-day precision studies were found to be less than 2%, indicating good precision and reproducibility of the developed RP-HPLC method for estimation of Piperine in commercial brands of Sitopaladi Churna.

Ruggedness-

The ruggedness study of the developed RP-HPLC method showed %RSD values below 2% for all commercial brands analyzed by different analysts. The results indicate that the method is rugged, reliable, and reproducible under normal operating conditions for estimation of Piperine in Sitopaladi Churna formulations. The ruggedness study demonstrated that the developed RP-HPLC method for estimation of     Piperine in different commercial brands of Sitopaladi Churna was reliable and reproducible under varied analytical conditions. The low %RSD values obtained from analyses performed by different analysts confirmed the consistency and rugged nature of the method. Hence, the method was found to be suitable for routine quality control analysis of Piperine in marketed Sitopaladi Churna formulations.

Table No.14 Result of Robustness

STABILITY INDICATING ASSAY METHODS-

1)         Acidic condition

Acidic degradation study of Piperine was performed to evaluate the stability of the marker compound under acidic conditions. The sample solution was treated with 0.1 N hydrochloric acid (HCl) and kept at room temperature for a specified period, followed by RP-HPLC analysis. Acidic Degradation Study of Piperine in Commercial Brands of Sitopaladi Churna

Table No.15 Result of acidic degradation

Fig. No. 13 Degradation of drugs by acidic conditions

2.Alkaline Condition-

Alkaline degradation study of Piperine was performed by treating the sample solution with 0.1 N sodium hydroxide (NaOH) and keeping it at room temperature for a specified time period, followed by analysis using the developed RP-HPLC method.

Table No.16Alkaline Degradation of Piperine in Commercial Brands of Sitopaladi Churna

The alkaline degradation study indicated that Piperine is comparatively more unstable under alkaline conditions than acidic conditions. Significant reduction in assay values was observed in all tested brands, suggesting formation of degradation products. The RP-HPLC method efficiently separated Piperine from its degradation peaks, confirming that the method is stability-indicating and suitable for routine quality control analysis

Fig No 14  Degradation of drugs by alkaline condition

Neutral condition –

Neutral degradation study of Piperine was carried out by keeping the sample solution in distilled water (neutral pH) at room temperature for a specified period, followed by analysis using the developed RP-HPLC method.

Neutral Degradation of Piperine in Commercial Brands of Sitopaladi Churna

Table No.17 Neutral Degradation of Piperine in Commercial Brands of Sitopaladi Churna

Fig. No. 15 Degradation of drugs by neutral condition

1)         Thermal condition

Thermal degraded samples wherever degradation possible from about 1% to 30%.Preferably, the following stress conditions are was performed by exposing solid drug at 105 °C for 24 hours. Thermal degradation study of Piperine was performed by exposing the sample solutions of different commercial brands of Sitopaladi Churna to elevated temperature (e.g., 60°C for a specified time period) in a hot air oven, followed by analysis using the developed RP-HPLC method.

Table No.18 Thermal  Degradation of Piperine in Commercial Brands of Sitopaladi Churna

Fig .No. 16 Degradation of drugs by thermal condition

1)         Photolytic condition

 Photolytic degradation study of Piperine was performed by exposing the sample solutions of different commercial brands of Sitopaladi Churna to UV light (as per ICH guidelines) for a specified period of time. The exposed samples were then analyzed using the developed RP-HPLC method.

Table No.19 Photolytic Degradation of Piperine in Commercial Brands of Sitopaladi Churna

Fig .No.17 Degradation of drugs by photolysis condition

The forced degradation study demonstrated that Piperine is most unstable under alkaline and oxidative conditions, followed by photolytic and acidic conditions, while it remained comparatively stable under thermal and neutral conditions. The RP-HPLC method successfully resolved the degradation products from the parent peak without interference, confirming that the method is specific, accurate, and stability-indicating. Hence, the developed method is suitable for routine quality control and stability studies of Sitopaladi Churna formulations.

Table No.20 Result of Stability indicating assay methods.

Comparative Quality Evaluation of Brands

the comparative evaluation of different commercial brands of Sitopaladi Churna showed variation in Piperine content and chromatographic response. Among all tested formulations, Patanjali Sitopaladi Churna exhibited comparatively higher Piperine content and peak response, indicating better standardization of the marker compound. Dabur and Baidyanath formulations showed comparable results with slight variations in content.All three brands complied with method validation parameters including precision, accuracy, linearity, LOD, and LOQ, confirming the reliability of the developed RP-HPLC method for routine quality control and comparative assessment of Sitopaladi Churna formulations.

Table No.21 Result of Comparative Quality Evaluation of Brands