A Novel Stability Indicating Method for Determination of Major Alkaloid in Black Pepper by RP-HPLC in Different Pharmaceutical Dosage Forms

Background: Piperine is the major alkaloid found in the fruits of Black pepper. Recent studies revealed the antiurolithiatic effect of piperine. So, an attempt was made to develop an analytical method for the assay of Piperine in the formulated dosage forms. Objectives: The present study was done with the aim of developing a simple, accurate, precise and sensitive RP-HPLC method for estimation of Piperine in different dosage forms. Materials and Methods: Some trials were performed during method development using different solvents, mobile phase compositions and flow rate for the estimation of piperine in the dosage form. The developed optimized method was validated as per ICH guidelines and was employed to estimate the amount of piperine in the given dosage form. Results: The optimized chromatographic conditions were achieved using BDS C8 column with mobile phase having of water: Acetonitrile in 50: 50 ratio at 1.0ml/min flow rate. Detection was observed at 247nm using PDA detector. The retention time obtained for piperine peak was found to be 2.4 min. Conclusion: The analytical method which was developed for estimation of piperine is simple, rapid, economic, specific, precise, stable and can be successfully employed for its estimation in syrup and tablet dosage forms.


Instrumentation
Development and validation of a method for the assay were performed on HPLC (Waters 2690), PDA detector with empowering 2 software. Hypersil BDS C 8 (150mm x 4.6mm x 5.0mm) reverse phase column was used for chromatographic separation.

Methods
Different trials were performed during method development using different columns, buffers, mobile phase compositions for the estimation of piperine in tablet and syrup dosage form. As piperine is a photosensitive drug, ambered coloured glassware was used.
Preparation of Tablet formulation [14] As Piperine is slightly soluble in water, a solid dispersion was formulated by solvent evaporation method to enhance its solubility using HP β Cyclodextrin as a polymer in 1:1 ratio with methanol as solvent. Piperine tablet was prepared by direct compression by taking the equivalent weight of Piperine and suitable excipients. All the ingredients were passed through # 60 mesh sieve separately. Piperine and Microcrystalline cellulose were mixed thoroughly by adding small quantities each and properly blended to get a homogenous mixture. Then other excipients like sodium starch glycolate (SSG), talc, magnesium stearate were mixed in geometrical series and allowed to pass through a coarse sieve (#44 mesh) and the tablets were obtained by compressing in the hydraulic press. Preparation of Syrup formulation [15] The sugar base for the syrup was prepared by mixing 85g of sucrose in 45g of water and the mixture heated to its boiling point. After straining the liquid, the volume made up to 100ml with distilled water. The preservatives methyl paraben and propyl paraben were dissolved in a small volume of boiled and cooled water and then it was added to the sugar base. The required amount of Piperine was dissolved in propylene glycol which was heated to 45-50°C and then sorbitol, glycerin was added to it. Finally, sodium saccharin was added and thoroughly mixed. pH was adjusted between 5.5-6.5 with citric acid, if necessary. Then volume was made up to 25ml with boiled and cooled water.
Mobile phase preparation 0.1% OPA buffer was prepared by adding 1ml OPA in a 1000ml of volumetric flask followed by adding 100 ml milli-Q water and mixed thoroughly. Finally, the volume was made up to mark with milli-Q water and it was degassed. Buffer and acetonitrile (50:50%v/v) were employed as mobile phase.

Preparation of Diluent
Equal volumes of water and ACN were mixed and used as diluent for preparing solutions.

Preparation of Standard stock solutions
Piperine of 39mg was accurately weighed and transferred into a volumetric flask of 100ml volume followed by the addition of diluents to half of its volume. The mixture was sonicated for 10 min. Then it was made up to volume with diluent and marked as Standard stock solution (390µg/ml of Piperine)

Preparation of working Standards
Different working standards are prepared by diluting Piperine standard stock solution with diluents.

Preparation of working Standard (100% solution)
From standard stock solution, 1ml was transferred to 10ml volumetric flask and made up to mark with diluent. (39µg/ml of Piperine).

Preparation of Sample stock solutions
For tablets assay, twenty tablets of piperine were weighed accurately and pulverized. An amount of tablet powder equivalent to 39mg of piperine was weighed and transferred into 100ml volumetric flask. 50 ml of the prepared diluent was transferred and sonicated for 25 min. The solution was made up to volume with diluent and filtered through 0.45µ filter (390 µg/ml of Piperine).
For syrup formulation, the sample stock solution was prepared by taking an amount of syrup equivalent to 39mg into a 100 ml volumetric flask, followed by adding 50ml of diluents. The mixture was sonicated for 25 min and the volume was made up with diluents. The solution was filtered by passing through 0.45µ filter (390 µg/ml of Piperine).

Solution stability
The stability of the solution was studied by injecting the standard solution into the instrument immediately after preparation (0 hr) and again injected after 24 hr storage at room temperature. The peaks obtained were compared to understand their stability.

Validation System Suitability Parameters
The system suitability parameters of the optimized method were assessed by taking standard solutions of Piperine (39ppm) and were injected six times. The system suitability parameters like USP plate count, resolution and peak tailing were determined.
The % RSD of peak areas of six standard injections results should be NMT 2%.

Specificity
It was done for checking interference in the optimized method if any. There should not be any interfering peaks in blank and placebo at the retention times of these drugs in this method.

Linearity
Linearity was studied by preparing 25, 50, 75, 100, 125 and 150% solutions from the standard stock solution of Piperine. Calibration curve was plotted by taking concentration on X-axis and peak area on Y-axis. Linearity was determined by the least square regression method. The slope value (m) and correlation coefficient (R 2 ) were calculated.

Precision (Repeatability)
Six piperine standard solutions of the same concentration were injected into the instrument and peak areas were interpreted to obtain peak areas. The concentrations and % RSD were estimated. The % RSD should be NMT 2%.

Accuracy
The accuracy of the method was studied by spiking a known amount of standard to the sample solution (50, 100 and 150%) and the % recovery was determined by using the optimized method. The percent difference between the expected and measured concentrations was determined and represented as % RSD. The % recovery and % RSD of the assay should be ± 100% and NMT 2% respectively.

Robustness
Robustness was studied by causing small variations in chromatographic conditions like flow rate, mobile phase ratio, pH, column, column temperature. The solution obtained was diluted to get (39ppm) solution. From this solution, 10 µl were injected and the obtained chromatograms were evaluated to assess its stability.
Acid and Alkali degradation Studies 1 ml of standard s tock s solution Piperine was taken separately in two different volumetric flasks (A, B). 1 ml of 2N Hydrochloric acid, 1ml of 2 N sodium hydroxide was added to A and B flask respectively. The above solutions were refluxed at 60°C for 30min. The obtained solutions were diluted to obtain (39ppm) solution and 10 µl solutions were injected into the system and the chromatograms were recorded to assess the stability of t h e sample.

Dry Heat Degradation Studies
Piperine standard stock solution was kept in an oven at 105°C for 6 hr. 39ppm solution was prepared by diluting 1ml of obtained solution and 10µl volumes were injected into the system. The chromatograms were recorded to evaluate the Piperine stability.

Water Degradation Studies (Hydrolysis)
Stress testing was studied under neutral conditions by refluxing the drug in water for 6 hr at a temperature of 60°C. From the obtained solution, 1ml was taken and diluted to 39ppm solution and 10µl volumes were injected. The chromatograms were recorded to assess the stability.

Optimization of mobile phase
The method development for assay of piperine was done by using Altima C 18 column with mobile phase phosphate buffer (pH 3.5) and Acetonitrile in 40: 60 ratio but the peak obtained showed more retention time with USP pate count below the acceptance criteria. In the next trial BDS C8 column was used so that less retention time was obtained but USP plate count is not acceptable and the peak shape is not good. The optimized method with the following chromatographic conditions showed better results having less retention time, acceptable USP plate count with good peak shape and the chromatogram as shown in Figure 2.

LOD and LOQ
LOD is defined as the least concentration of analyte that gives response accurately but need not be quantified exactly. LOQ is the least concentration of analyte that gives the accurate response and can be quantified exactly. These LOD and LOQ were calculated using the following formulae Where σ is the standard deviation of response S is slope of the calibration curve

Assay Methodology
Assay of the marketed formulation was carried out by diluting the sample stock solution of tablet and syrup formulations to a suitable concentration and injected into the HPLC system. Peak areas were interpreted for the obtained peaks and the percent assay was calculated. Degradation studies [16] Forced degradation studies are performed to estimate the stability of the drug which in turn affects its purity, safety and potency. So, degradation studies are important to understand the stability of molecules under different stress conditions. The degradation limit defined by the regulatory agencies for validation of chromatographic assays is 5-20%.
Oxidation 20% hydrogen peroxide (H 2 O 2 ) of 1 ml was added to 1 ml of standard stock solution of Piperine and the mixture was kept at 60°C for 30 min.

Robustness
Robustness was studied by changing flow rate (0.8-1.2 ml/min), mobile phase ratio (55:45 and 45: 55), column temperature (28, 32°C). These changes didn't show much influence on R T and peak area. The % RSD for the obtained results was found to be within the limits and was shown in Table 5.

LOD and LOQ
LOD and LOQ of piperine for the developed method were found to be 0.076 and 0.230 µg/ml respectively.

Assay
The developed method was successfully employed for the assay of piperine in tablet formulation. The % assay of piperine for tablet and

Solution stability
The stability of the prepared standard solution was checked for stability up to 24 hr and the solution was found to be stable.

Method validation System suitability parameters
The optimized method showed USP plate count of more than 2000 with tailing factor less than 1.5. The % RSD for peak area and retention time was found be less than 2 which indicate that the developed method was suitable for the estimation of Piperine. The results of the System suitability study was given in Table 1.

Specificity
Blank and placebo were injected separately into HPLC. The chromatograms obtained didn't show any peaks at the Rt of piperine.

Linearity
A standard curve was plotted by taking 25, 50, 75, 100, 125 and 150% solutions from the standard stock solution of piperine. The correlation coefficient (R 2 ) for the linearity plot was found to 0.999 ( Figure 3) which confirms that the chosen concentration range of piperine follows linearity. The results of linearity were given in Table 2.

Precision (Repeatability)
The repeatability of the method was studied by interpreting peak areas of the samples injected in different days (Table 3). % RSD was calculated and found to be 0.16 which confirms that the developed method is precise.

Accuracy
The % recovery was calculated for the 50, 100, 150% spiked solutions and the average % recovery was found to be 98.97. The % RSD was found to be 0.73 which indicates that the accuracy of the method was within the acceptable range. The recovery results were shown in Table 4.

CONCLUSION
In the present world, a rapid, economic and stable analytical method was developed for the assay of Piperine in syrup and tablet dosage forms by RP-HPLC. All the validation parameters were tested according to the ICH guidelines and the results fall within acceptable limits. The developed method was found to be specific for the analyte of interest in presence of excipients. As the retention time is short, the analyst can analyze more number of samples in less duration. So, the proposed method can be successfully employed for routine analysis of Piperine in pharmaceutical dosage forms.
syrup formulation was found to be 95.216 and 95.158 respectively and were shown in Figure 4 and Figure 5.

Degradation studies
In order to assess the intrinsic stability of the piperine in the dosage form forced degradation studies were performed. The stressed sample did not show any interfering peaks of degradation product which indicates that the developed method is stable and the results are shown in Table 6.

DISCUSSION
A stable RP-HPLC method was developed for assay of piperine in tablet dosage form and the same method was successfully employed for assay of piperine in syrup. The chromatographic conditions like BDS C8 column with mobile phase composition of 0.1% OPA buffer: Acetonitrile in 50:50 ratio at 1.0 ml/min flow rate, using PDA detector at λ of 247 nm eluted piperine at R T of 2.4 min. The % assay of piperine for tablet and syrup formulation was found to be 95.216 and 95.158 respectively. For forced degradation studies peak purity angle should be less than the peak purity threshold according to ICH guidelines, [17] and the same was obtained in the stability studies which indicates that the developed method is stable.