Novel Stability Indicating RP-HPLC Method for The Estimation of Pinaverium Bromide in Tablet Formulation: Assay Development and Validation

A selective RP-HPLC method has been developed for the estimation of pinaverium bromide (PB) in tablet dosage form. A Phenomenox C18 (250 x 4.6 mm i.d., particle size 5 μm) column with ammonium acetate buffer (pH 3.0) and acetonitrile (20:80) was employed as the mobile phase. The flow rate of 1 mL/min was utilized employing 214 nm as detector range where the drug eluted at 5.067 min. The method was validated further for linearity, accuracy, precision, and robustness according to the ICH guidelines. Forced degradation studies were performed under neutral, acidic, alkaline conditions along with thermal, photolysis and oxidative stress conditions, where the drug exhibited degradation and various peaks were monitored. The method was found to be linear over the range concentration range of 12.575.0 μg/mL (r values of 0.999), accurate (recovery of about 100.22% with % RSD of < 2%), highly precise (% RSD of < 2% in both intra-day and inter-day study), and robust enough to deliver accurate results when the chromatographic conditions were altered deliberately. No interference of additives/excipients was encountered while developing this method. Highest degradation was observed under oxidative stress condition. This method will help in the quantitative determination of PB and its degraded products.


INTRODUCTION
Pinaverium bromide (PB) chemically, 4-(2-bromo-4,5-dimethoxybenzyl)-4-(2-[2- (6,6dimethylbicyclo[3.1.1]heptan-2-yl)ethoxy]ethyl)morpholin-4-iumbromide, is an antispasmodic used mainly for gastrointestinal (GI) disorders (Figure 1).It relaxes GI structures primarily by inhibiting Ca 2+ influx through potential-dependent channels of surface membranes of smooth muscle cells [1].The literature revealed that little efforts were put in developing an accurate and simple method for estimating PB either in pharmaceutical formulations or in biological fluids.Balaji et al. developed a method for estimation of PB in bulk drug using C8 column with 30:70 v/v of water: acetonitrile (ACN), where Rt was found to be 4.84 min [2].In another research, elution of PB was performed using phosphate buffer (pH 6.5): methanol in a C18 column with Rt of 1.52 min [3].An assay was developed recently for estimation of PB in urine utilizing a coated wire silver electrode having a detection limit of 6.3 × 10 -7 mol/L in the pH range of 3-11 [4].Similarly, PB has been determined using liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) [5], by gas chromatographic mass spectrometry (GC-MS) in human plasma [6], and by high throughput ultra-performance liquid chromatography (UPLC) [7].Under diverse environments, a change in the physicochemical and biological properties of the PB results gradually.In order to maintain quality aspects of PB, study of degradation profile is of prime importance in determination of the degraded content.Therefore, a stability indicating analytical method will detect the small and deliberate changes apart from the determination of analyte content in tablets.Since, there were no significant results reported on degradation profile for specific quantification of degradation product using RP-HPLC C18 column.This leads to the development of a stability indicating reverse phase chromatographic assay method for the analysis of PB.

Chemicals and reagents
PB was obtained as a generous gift from Zim Laboratories Ltd., Nagpur, India.Eldicet ® , Solvay Pharmaceutical Ltd. a fixed dose marketed combination containing 50 mg of PB was purchased from Pharmacy in Nagpur, India.The weight of each tablet was found to be approximately 182 mg and assay results revealed 99.83% drug content in the tablet.HPLC grade reagents were procured from Merck Chemicals Ltd., India.Double distilled water (Millipore) was used during the study and was suitably filtered through 0.45 μm nylon membrane filter.

Instrumentation
The constitution of HPLC system involves Shimadzu LC-2010 CHT (Japan) system with in-built SPD 20-AD UV-Vis detector.The resolution was carried out on Phenomenox C18 (250 x 4.6 mm i.d., 5 µm) column.UV-spectrophotometric analysis was carried out using double-beam Shimadzu Ultraviolet-Visible Spectrophotometer (Kyoto, Japan) model UV-1800 equipped with a pair of 1 cm path length quartz cells.Weighing was carried out by Shimadzu balance (Kyoto, Japan) model AUW220D.The pH was measured using Contech ® digital pH meter.Photostability chamber (SVI equipments, Germany), and sonicator (Transonic Digital S) were employed for study.

Preparation of stock solution
50 mg of PB reference standard was transferred into volumetric flask and diluted with 50 mL ACN was added.The content was sonicated for 10 min, cooled to the room temperature; volume was made up to 100 mL with diluents and filtered suitably with 0.45 μm filter.

Preparation of standard solution
The standard solution was prepared by taking 5 mL of above stock solution of PB and diluted with 50 mL diluents.The content was further sonicated, cooled to the room temperature and filtered through 0.45 μm filter.

Preparation of buffer
The buffer was prepared by dissolving 154 mg ammonium acetate in 1000 mL of water containing 5 mL of formic acid.The pH was adjusted to 3.0 with triethylamine and the resulting solution was filtered through 0.45 µm filter and degassed before use.

Preparation of blank
Blank was prepared by taking 5 mL of ACN in 50 mL volumetric flask and the volume was made up to mark with the mobile phase and filtered through 0.45 µm filter.

Preparation of sample solutions of tablet
Tablets of PB were accurately weighed; their mean weight was determined, and grounded to fine powder in a glass mortar.Powder content equivalent to 50 mg of PB was weighed suitably, transferred in 100 mL volumetric flask.70 mL of diluent was added, sonicated for 30 minutes with intermittent shaking and volume was made upto 100 mL to produce 500 μg/mL concentration (Sample-A).The content was centrifuged at 3500 rpm for 5 minute in stoppered centrifuge tube and 5 mL of supernatant solution was pipetted out in 50 mL volumetric flask and volume was made upto the mark to produce 50 μg/mL (Sample-B).

Detection of wavelength
The standard solution of PB was scanned in UV-Vis spectrophotometer over the range of 400-200 nm and the λmax was found to be 214 nm.

Chromatographic conditions
For chromatographic separation, Phenomenex C18 column (250 mm × 4.5 mm i.d., 5 μm) was utilized as stationary phase.The selection of mobile phase was critical and after a number of trials with solvent mixtures, parameters like separation, symmetry of peak, peak purity index, and theoretical plate covered were considered for selecting the best mobile phase.This reverse phased chromatography was resumed using C18 stationary phase through 0.002 M ammonium acetate buffer (pH 3.0 adjusted using triethylamine) with ACN in ratio of 20:80 after final selection.

Force degradation studies
The forced degradation studies were performed by hydrolyzing PB under acidic and basic conditions to determine primary degradation products.PB was also subjected to UV degradation, oxidative and thermal stress conditions.Under hydrolytic reactions, the functional groups like ester and amide undergo hydrolysis, leading to formation of degraded components.The efficacy of the method in resolving and determining the degraded analytes from the sample from the drug was studied.

Alkaline degradation
PB was forced degraded in alkaline condition by reacting with 2N NaOH solution.20 tablets were crushed in a glass mortar and drug equivalent to 50 mg was taken in a 100 mL volumetric flask.To the above content, 50 mL of ACN was added, sonicated for 15 min and volume was made up to 100 mL.The sample solution was further stirred for 30 min using magnetic stirrer, and centrifuged at 3000 rpm for 5 min.5 mL of the resulting solution was pipette out in 50 mL volumetric flask and equal quantity of 2N NaOH was added with occasional shaking.The content was kept on boiling water bath for 1 hour, cooled to room temperature, neutralized with 5 mL of 2N HCl, sufficient mobile phase was added to make up the volume up and filtered through the 0.45 µm pore size nylon membrane and 20 μL sample was injected.

Acidic degradation
PB underwent degradation in acidic environment by reacting with 2N HCl solution.Tablets were crushed and PB equivalent to 50 mg was taken in 100 mL volumetric flask.50 mL of ACN, the diluent was added to the above content, stirred with a magnetic stirrer, sonicated for 15 min to promote complete dissolution and volume was made up to 100 mL with ACN.The sample solution was duly centrifuged at 3000 rpm for 5 min and 5 mL of the resulting solution was pipette out in 50 mL volumetric flask and equal quantity of 2N HCl was added with intermittent shaking.The content was boiled for an hour, cooled, neutralized with 5 mL of 2N NaOH, and sufficient mobile phase to produce 50 mL.The content was further filtered through the 0.45 µm pore size nylon membrane and 20 μL sample was injected.

Oxidative degradation
Oxidative degradation results by the interaction between drug and molecular oxygen which resumes the process of auto-oxidation and drug substance gets converted into several small fragments.For the analysis, PB equivalent to 50 mg was taken in volumetric flask, dissolved in 5 mL of H2O2, boiled for 60 min and placed at room temperature for commencing degradation.Further 50 mL diluent was added, stirred, sonicated and the volume was made up to 100 mL.The above content was centrifuged, filtered, 20 μL sample was injected into the HPLC system and obtained chromatogram was studied.

Thermal degradation
Thermolysis at high temperature (70-100°C) leads to arising in several degradation products in the solid state.Drugs begin to degrade via varied mechanisms which results in the formation of degradation products.The study includes PB equivalent to 50 mg of tablet mixture, produced by crushing 20 tablets.The drug material was exposed to heat at 80±1°C for 1 hour and analyzed for stability under thermal stress.The content was transferred to a volumetric flask, duly diluted with acetonitrile, stirred for 30 min using magnetic stirrer, sonicated for 15 min and further diluent (ACN) was added to produce 100 mL.The solution was centrifuged at 3000 rpm for 5 min, filtered using 0.45 µm membrane and drug solution (20 μL) was injected for analysis.

Photolytic degradation
UV-degradation study involves exposure of UV radiations to the drug where the molecule absorbs these radiations causing absorption band overlapping and ultimately produces excitation of valence electron.For this study, 50 mg equivalent of PB was taken from tablet mixture and placed in the UV chamber for 72 hr at 254 nm and degradation products were analyzed.After exposure, the content was transferred suitably in a volumetric flask, 50 mL ACN was added.The solution was stirred for 30 min, sonicated and the volume was made up to 100 mL.The sample was further centrifuged at 3000 rpm for 5 min, filtered through 0.45 µm nylon membrane and 20 μL sample was analyzed.

Method validation
Validation of analytical methods, in general, has been extensively covered in the ICH guidelines Q2A and Q2B, in the FDA guidance and by USP.The validation has been carried out as per ICH guidelines Q2A and Q2B.

Linearity and range
The linearity of the proposed method was determined by taking six concentrations ranging from 25-150% of the analyte concentrations in PB tablet.Solutions were prepared using acetonitrile as diluents and equivalent volume of solutions was injected under the given chromatographic condition.The obtained average area corresponding to the concentration of standard drugs was plotted and the linearity was expressed in regression coefficient value (r 2 ).

Accuracy
The accuracy represents the proximity between the standard reference and the observed value during analysis.The accuracy was determined as % recovery of the standard spiked to previously analyzed test sample of PB and expressed as % recovery ± (% confidence interval) with % relative error.The recovery was calculated based on the actual and estimated concentrations by spiking equivalent drug substance in placebo at three different concentrations (50%, 100%, and 150%) of the standard drug.

Precision
The precision is the closeness of results obtained from analysis of the same sample repetitively.Precision studies involve inter-day and intra-day variability.Relative standard deviation (RSD) values are marked indicator of precision of any analytical method and values <2% are acceptable.Intra-day studies involve injecting six times, three concentrations (50%, 75%, and 150%) of standard solution of PB in a single day.In contrast, the inter-day study involves repeating the above protocol in three consecutive days in similar manner.

Robustness
Robustness expresses the ability of a system to resist tiny but purposeful changes without disturbing the analysis of analytes.The chromatographic conditions were purposely changed (changing the flow rate by ±0.2 mL/min and wavelength by ±2 nm) keeping the other chromatographic conditions constant and chromatogram for PB was evaluated.

System suitability parameters
System suitability parameters signify the vital component which validates the ability of reproducibility of an analytical system.The study involves injecting the standard solution of PB five times repetitively and retention time, peak area, tailing factor and theoretical plates of the peaks were calculated.

Method development and optimization of chromatographic conditions
Earlier experiments have demonstrated the use of C8 (250 × 4.5 mm i.d., 5 μm) and C18 column (250 × 4.5 mm i.d., 1.7 μm) for resolution of PB, hence in this study, Phenomenex C18 column (250 × 4.5 mm i.d., 5 μm) was utilized.For the achievement of the highest degree of separation, the mobile phase composition was thoroughly studied.A lot of trials were taken using number of binary eluants like methanol, water, acetonitrile, and various buffer at pH 3.0-6.0and mobile phase was chosen on the basis of peak purity index, peak symmetry, and theoretical plate.In addition, pH played a decisive role in the resolution of analytes as an alteration in pH modifies the retention by 2 units of pKa.Although, a low pH is beneficial in minimizing the peak tailing along with enhancement in system robustness, however, pH >7 often results in complete dissolution of column silica.Therefore, it is judicious to regulate the pH of mobile phase 2.0 units higher or lower the pKa to assure unionization of the analyte.Initially, water: methanol (30:70) showed the appearance of multiple peaks with visible tailing (Figure 2a); followed by a trial with water: ACN (60:40) displayed several distorted peaks with poor resolution (Figure 2b).Subsequently, a trial with phosphate buffer (pH 4.5): ACN (50:50) resulted in very high distortion and tailing (Figure 2c).When phosphate buffer (pH 5.0): methanol (30:70) was employed, a marked improvement in the resolution was observed (Figure 2d).This provided a clue that elution with a buffer solution and methanol in ratios will lead to the reduction in tailing and the sharp peak will be achieved.Serendipitously, Ammonium acetate buffer (pH 3.0): Methanol (20:80) was employed which exhibited sharp peak, good theoretical plate, and peak purity index, therefore, it was chosen for the analytical purpose (Figure 2e).The experiment was carried out employing Phenomenex C18 column maintained at 25°C with mobile phase optimized 10 min runtime in isocratic mode at a flowrate of 1 mL/min keeping detector at 214 nm.The retention time of PIM was found to be 5.067 min.

Linearity and range
The graph was found to be linear over the range 12.5-75 μg/mL with linear regression equation of y = 56859x -27881 (Figure 3).The regression coefficient value was observed to be 0.999, which indicates an acceptable degree of linearity (Table 1).

Accuracy
The recovery data for accuracy studies are depicted in Table 2.The recovery was estimated using calibration curve, where the slope and Y-intercept of the plotted graph served as a tool to calculate appropriately the % recovery.The % RSD values for the proposed method was found to be within the acceptance limit of ±2%, i.e. 0.73, 0.30 and 0.39 at three different concentrations, which indicated a good accuracy of the developed method.

Precision
The method was found to be highly precise over the range of 50-150% of the standard drug.The % RSD values were found to be less than 2% in both the cases.In intra-day variability, the % RSD was found to be in range 0.16-0.47%,and in inter-day variability, the % RSD range was 0.19-0.46%.This concluded that the variability was found to be minimal and the method is precise enough to determine the drug.The intra-and inter-day variability or precision data are given in Table 3.

Robustness
The slight and deliberate change in the chromatographic conditions did not hamper analysis of PB by the proposed method.However, minor changes were observed, which is conclusive enough to prove the robustness of the analytical method.The actual peak observed at 5.067 min (under a flow rate of 1.0 mL/min) gets shifted to 4.976 min when the flow rate was changed to 1.2 mL/min.An intentional change in detecting wavelength from 214 nm to 216 nm, leads to increasing retention of PB at 5.108 min.Therefore, the robustness of this method is sufficient to detect the PB content even at minute alteration in the chromatographic environment.

System suitability parameters
The system suitability parameters reflect that the proposed method demonstrate the proficiency as per the minimum requirements of monographs of United States Pharmacopoeia (USP) [8].The system expressed a mean of 4192 theoretical plates (minimum pharmacopoeial limit of 2000) which reflect high column efficacy with better resolution.The peak area was calculated to be 2837025 averagely with % RSD of 1.127 (i.e. less than 2%), indicating high column efficacy and reproducibility.The tailing factor (TF) was found to 1.207 which revealed that the peak exhibit symmetry.The value of unity is indicative of the fact that the asymmetric factor (AF) is equal to 1, therefore no tailing will be observed till it exceeds and a value of TF less than 2 describes the shape of an ideal Gaussian peak.An ideal Gaussian peak has TF always less than 2, AF equal to 1, and exhibit symmetry.A nearly identical retention time of 5.067 min was detected in every case implying that the system exhibits good reproducibility.Altogether, the newer method possesses the attributes of robustness, reproducibility, accuracy and is of immense interest for applications in drug analysis.The system suitability parameters are represented in Table 4.

Forced degradation studies
The forced degradation study revealed that PB underwent immense degradation under acidic, basic and oxidative conditions, as compared to the UV and thermal stress, where no degradant peaks were observed (Figure 4).Furthermore, it may possibly be assumed that the stability of PB is dependent on temperature, pH, concentration of buffer, and environment impact.Under neutral environment, no hydrolysis of PB took place over the period of 4 hr.PB in presence of 0.01 M NaOH at 80°C displayed complete degradation with a single peak at 6.169 min in the chromatogram.No drug peak was observed indicating a total change in drug content.The possible cause for maximum degradation in the alkaline media involved mechanistic abstraction of the proton by the base (OH) radical.The alpha proton is weakly acidic and the resulting bond between the proton and the carbon is relatively strong, and a strong base can cleave the proton away from the carbon atom.In disparity, under acidic hydrolysis using 0.1M HCl at 80°C, peaks of degraded products at were observed at 2.562 and 6.078 min in the chromatogram.The peak area demonstrated that 51.62% of drug degradation occurred when the drug was kept under lower pH.Similarly, in the oxidative degradation study, PB underwent dreadful degradation exhibiting degradation peaks at 2.214, 2.588, and 6.178 min, respectively, with no appearance of parent peak.In both thermal degradation and UV exposure, PB chromatogram displayed no significant degradation and only a characteristic peak at 5.070 min was observed, which may be the drug peak itself (5.067 min).On comparing acidic and oxidative degradation chromatograms, few similarities were observed.In 2.5 min, a small peak was observed in both the conditions with different intensity; more pronounced under the influence of H2O2.The forced degradation study results are described in Table 5.In the present investigation, RP-HPLC method was developed for the estimation of PB in tablet formulation where the drug was eluted in isocratic mode with ammonium acetate buffer (pH 3.0): acetonitrile in the ratio of 20:80 at a flow rate of 1 mL/min using Phenomenox C18 column.No interference of degradation product and diluents was encountered in this method.With a robustness value in terms of pH, wavelength and flow rate, the newer method possesses all necessary attributes to be utilized for regular quality control analysis of PB in pharmaceutical dosage form.The previous methods have demonstrated a shorter retention time of 1-3 min and this method has an optimum retention time of 5.067 min.For pharmaceutical analyses in industrial scale, HPLC method is favored in several cases owing to the accurate, precise, and reproducible results and flexibility in routine quality control analysis of PB in pharmaceutical dosage form.Furthermore, this method with suitable modification(s) may be utilized in the estimation of drugs of similar chemical skeleton like PB, several active pharmaceutical ingredients (APIs), non-pharmaceutical products, miscellaneous products, etc.The forced degradation studies performed under various conditions as per ICH Guidelines highlighted that PB underwent the highest degradation under oxidative stress with the formation of three different products followed by acidic and alkaline degradations.Therefore, the method is highly reliable in determining the content of degraded products in the formulation.

Table 1 .
Linearity levels solutions of PB

Table 2 .
Recovery for accuracy studies of PB

Table 3 .
Precision data of inter-and intra-day variability of PB

Table 4 .
System suitability parameters

Table 5 .
Forced degradation studies