Stability Indicating Rp-Hplc Method Development And Validation For The Simultaneous Determination Of Propranolol Hydrochloride And Ascorbic Acid In Tablet Dosage Form

Figure 1: Structure of Propranolol and Ascorbic acid

MATERIALS AND METHODS

Chemicals and Reagents

Propranolol hydrochloride and Ascorbic acid were kindly provided as gift samples by Ipca Laboratories, Mumbai.Tablets of (Ascorbic acid 0.25mg, Propranolol hydrochloride 20 mg) were purchased from local market. Water LiChrosolv® (Merck Specialties Pvt. Ltd., Mumbai.), Methanol LiChrosolv® (Merck Specialties Pvt. Ltd., Mumbai.), Acetonitrile LiChrosolv® (Merck Specialties Pvt. Ltd., Mumbai.), Di potassium hydrogen phosphate anhydrous AR (Merck Specialties Pvt. Ltd., Mumbai.), Potassium dihydrogen orthophosphate AR (S.D. Fine-chem. Ltd., Mumbai).

Instruments

The chromatographic separation was carried out on a Younglin 9000 HPLC system. Chromatographic system equipped with Quaternary Gradient HPLC system (SP930D), fitted with UV-VIS Detector. Phenomenex C18 (150 x 4.6mm, 5μm) column as stationary phase with Phenomenex Security Guard Cartridges C18, 4x3mm. The mobile phase was degassed by sonication using ultrasonic bath (Microclean-103). Shimadzu UV Spectrophotometer 1800 was used for wavelength selection. The standard substances were weighed on, electronic balance Shimadzu, Japan AY220. Vigo Melting point apparatus was used.

Preparation of Standard Stock Solution

Standard Stock Solution of PRO

Weighed exactly 10 mg of standard PRO and transferred it to a 10 ml volumetric flask, then dissolved it in methanol LiChrosolv®.To achieve the desired concentration, the volume was adjusted to the mark using the same solvent. of 1000μg/ml of PRO. The "Std Stock PRO" (800 μg/ml) was prepared by diluting 8 ml of the solution to 10 ml with the same solvent.

Standard Stock Solution of Vit C

Weigh 10 mg of standard Vitamin C, transfer it to a 10ml volumetric flask, and dissolve it using methanol (LiChrosolv®).The solution was diluted to the mark with the same solvent to obtain a 1000 μg/ml Vitamin C concentration.To obtain the ‘Std Stock Vit C’ (10 μg/ml), 1 ml of the initial solution was diluted to 10 ml with the same solvent to reach a concentration of 100 μg/ml; subsequently, 1 ml of this intermediate solution was diluted again to 10 ml.

Combined Standard Stock Solution of PRO and Vit C

5ml of ‘Std Stock Vit C’ (10μg/ml) and 5ml of ‘Std Stock PRO’ (800μg/ml) mixed to get conc. of 5μg/ml of Vit C and 400μg/ml of PRO. The solution was labeled as ‘Std Stock MIX’.

Selection of Analytical Wavelength

To investigate the appropriate wavelength for simultaneous determination of Vit C and PRO, individual solutions in the mobile phase were scanned in the range of 200-300nm.

Chromatographic Condition

Analytical Column: Phenomenex C18 column (150 mm × 4.6 mm, 5 μm)

Mobile Phase: Methanol Phosphate Buffer pH7 (70:30)

Flow Rate: 1ml/min

Column temperature: Ambient

Injection Volume: 20 μl

Detection Wavelength: 221nm

Preparation of phosphate buffer pH7

Accurately weighed 0.136gm of potassium dihydrogen phosphate and 0.174gm of dipotasium hydrogen phosphate transferred in the 100ml volumetric flask and dissolved in HPLC water, then volume was made up to the mark with HPLC water.

Preparation of Mobile phase

Various combination of mobile phase including water, methanol, acetonitrile, phosphate buffer were tried finally Methanol and phosphate buffer of pH7 in the ratio (70:30) was selected for analysis of Vit C and PRO.

Identification of Separated Peak of the Drugs

For identification of peak of the drugs; the standard solutions of Vit C (10μg/ml) and PRO (10μg/ml) were injected separately into HPLC system and retention time were matched with retention time of mixture.

Method Validation

The method was validated according to ICH guidelines with respect to specificity, accuracy, precision, linearity, robustness, ruggedness, system suitability.

Specificity

The chromatogram of standard solution of mixture of Vit C and PRO was compared with formulation to observe the interference of excipient.

Linearity

Into a series of 10 mL volumetric flasks, transfer "Std Stock MIX" (Vit C 5 μg/mL & PRO 400 μg/mL) aliquots of 1.6, 1.8, 2.0, 2.2, and 2.4 mL.

The volume was made up to the mark with mobile phase to obtain the conc. of 0.8, 0.9, 1, 1.1 and 1.2μg/ml of Vit C and 64, 72,80,88 and 96 μg/ml of PRO.

After filtration through a syringe filter, 20 μL of each solution was injected into the HPLC system, and chromatograms were recorded for 10 minutes. Peak areas were recorded for all the peaks. Calibration curves of Vit C and PRO were constructed by plotting the peak area v/s conc. The correlation coefficient (r2) of least square linear regression for Vit C and PRO was calculated.

Range

The analytical range was determined by plotting the calibration curve and using the interval between its lowest and highest levels.

Accuracy

From the ‘Sample Stock 1’ solution 1 ml was transferred to four different 10ml volumetric flasks separately along with 0, 0.8, 1, 1.2 ml from the ‘Std Stock MIX’ (Vit C - 5μg/ml and PRO -400μg/ml) solution. The volume was brought to the mark with the mobile phase. All the solutions were filtered through syringe filter and injected into the HPLC system and their chromatograms were recorded under the same chromatographic conditions after getting a stable baseline. Peak areas were recorded for all the peaks and percent recoveries were calculated.

Precision

Evaluated the precision of the analytical method by assessing its repeatability and intermediate precision.

Repeatability

Repeatability was determined by analyzing the standard solution of Vit C (1μg/ml) and PRO (80μg/ml) six times and %RSD calculated

Intermediate Precision Intra-day Precision

Intra-day precision was determined by analyzing the standard solution of Vit C (1μg/ml) and PRO (80μg/ml) at 9.00am and 6.00pm on same day following the procedure of repeatability.

Robustness

Combined standard solution of Vit C (1μg/ml) and PRO (80μg/ml) was prepared and analyzed at different flow rates (0.9, 1.0, 1.1 ml/min) separately.

System Suitability

Sample solutions containing Vitamin C (1μg/ml)and PRO (80μg/ml)  were prepared and analyzed six times.. Chromatograms were studied for different parameters such as tailing factor, resolution and theoretical plates to evaluating whether they stay within the recommended limits.

Assay of Tablet Formulation

Twenty tablets (containing 0.25mg of Vitamin C and 20mg of Propranolol hydrochloride each) were weighed and finely powdered. An accurately weighed portion of the powder equivalent to 0.25mg of Vitamin C and 20mg of Propranolol hydrochloride was then transferred to a 50mL volumetric flask. Add 5 mL of methanol, sonicate the mixture for 10 minutes, dilute it to volume with methanol, and filter it through Whatman No. 41 filter paper. (‘Sample Stock1’). 2ml of resulting solution diluted upto10ml with mobile phase to obtain 1μg/ml Vit C and 80μg/ml PRO(‘Sample Stock2’).

Sample Stock2 was filtered through syringe filter and injected into HPLC system. The amount of Vit C and PRO present in the tablets were calculated using calibration curve equations, y= 142x+6.2 and y= 130.91x+1058.4 respectively.

Forced degradation study

Forced degradation study performed on Vit C and PRO both standard sample and tablet formulation.

Preparation of standard stock solution of Vit C (API)

20mg of standard Vit C was weighed and transferred to a 20ml volumetric flask then dissolved in the methanol LiChrosolv®. The volume was made up to the mark with methanol to obtain conc. of 1000μg/ml of Vit C (Sample stock A).1 ml of above solution dilute up to 10 ml with methanol resulting concentration 100μ/ml. Dilute 4 mL of this solution to 10 mL with mobile phase to obtain a final concentration of 40 μg/mL.solution filtered through syringe filter and injected in HPLC. This is chromatograph of fresh sample.

Preparation of standard stock solution of PRO (API)

20mg of standard PRO was weighed and transferred to a 20ml volumetric flask then dissolved in the methanol LiChrosolv®. The volume was made up to the mark with methanol to obtain conc. of 1000μg/ml of PRO (Sample stock A).1 ml of above solution dilute up to 10 ml with methanol resulting concentration 100μ/ml. Dilute 4 mL of this solution to 10 mL with mobile phase to obtain a final concentration of 40 μg/mL.solution filtered through syringe filter and injected in HPLC. (Chromatograph of fresh sample as a reference)

To conduct forced degradation study on API, standard stock solution was prepared using above procedure. These solutions subjected to acid hydrolysis i.e. refluxing with 0.1 N HCL at 700C. The mixture was neutralized by using 0.1N NAOH. Alkaline hydrolysis i.e. refluxing with 0.1N NAOH 700C. The mixture was neutralized by using 0.1N HCL. Oxidation using 5% H2O2, heating at 700C for 24 hrs. Photolysis degradation using UV Cabinet for 24 hrs. Thermal degradation using hot air oven at 700C, 24hrs.

Stress study on tablet formulation was performed by exposing tablets to oxidative, thermal, photolytic conditions. Stressed samples analyzed by validated HPLC method. Stressed samples were analyzed for peak purity via chromatograms.

RESULT AND DISCUSSION

Wavelength selection

Vit C (10μg/ml) and PRO (10μg/ml) in mobile phase were scanned separately in range of 200-400 nm.

λ max of Vit C 221nm and PRO 290nm.based on spectral characteristic and overlain spectra 221 selected as wavelength for analysis.

Figure 2: Overlain spectra of Vitamine C and PRO between 200- 400nm in mobile phase

Linearity and Range

Graphs were plotted concentration in μg/ml on X axis verses area on Y axis and the correlation coefficients were determined. The method was found to be linear in concentration range of 0.8-1.2μg/ml for Vit C and 64-96 μg/ml for PRO.

Accuracy

This technique involves the addition of standard drug solution to preanalyzed sample solution at 80%, 100%, and 120%. The % recoveries were 100-105% (Vit C), 104- 109% (PRO).

Table 1: Result of Accuracy for RP-HPLC Method (n=3)

Precision

Table 2: Result of Repeatability and intermediate precision Study for Vit C and PRO

Robustness

Table 3: Result of Robustness Study: Variation in Flow Rate (ml/min)

Assay

The amounts of Vit C and PRO in marketed formulation were 107% and 101% respectively

System Suitability Testing

Table 4: Results of System Suitability Parameters

Table 5: Forced Degradition Study of Vitamine C and PRO