Analytical Method Development of Antihypertensive Drugs in Bulk and Tablet Dosage Form

METHOD DEVELOPMENT AND OPTIMIZATION OF METHOD

Preparation of Buffer solution

1ml of formic acid was taken into a clean in 1000ml beaker and dissolved in HPLC grade water and the volume was adjusted to 1000ml. The resulting solution was sonicated for 15min. Then the solution was filtered through 0.45µl nylon filter.

Preparation of mobile phase

The mobile phase was prepared by taking 25 volumes of buffer and 75 volumes of methanol mixed well and sonicated for 15 min. Then the solution was filtered through 0.45µl nylon filter.

Diluent : Water: Acetonitrile (50:50% v/v).

Preparation of standard solution

A 48.5 µg of pure Valsartan and 51.5 µg of Sacubitril were weighed and transferred to 50 ml of volumetric flask and dissolved in diluent. The flask was shaken and volume was made up to mark with diluent to give a primary stock solution. From the above solution 1ml of solution is pipette out into a 10 ml volumetric flask and volume was made up to mark with water to give a solution containing 97µg/ml of valsartan and 103 µg/ml sacubitril.

Preparation of sample solution

Accurately weighed twenty tablets were ground to obtain fine powder equivalent to 970µg of valsartan and 1030µg of sacubitril sample were weighed and transferred to 10 ml of volumetric flask and dissolved in diluent. The flask volume was made up to mark with diluent and was shaken to    give a solution containing 97µg/ml of valsartan and 103 µg/ml of sacubitril.

Determination of Working Wavelength (λ max)

10 mg of the Valsartan and Sacubitril standard drug is taken in a 10 ml volumetric flask and dissolved in Diluent and volume made up to the mark, from this solution 0.1ml is pipette into 10 ml Volumetric flask and made up to the mark with the water to give a concentration of 10µg/ml. The above prepared solution is scanned in UV between 200-400 nm using water as blank. The λ max was found to be 267nm.

 After several initial trails with mixtures of methanol, water, ACN and buffer in various combinations and proportions, a trail with a mobile phase mixture of 0.1%v/v Formic acid in water: Methanol (25:75%v/v). The flow rate was 0.8ml/ minute brought sharp peaks. The chromatogram was shown in Results & Discussion.

Trial-1 Chromatographic Conditions

• Buffer : 0.01 M Ammonium acetate in Water
• Mobile Phase : Buffer: Methanol (40:60% v/v)
• Column : Inertsil ODS 3V column (150*4.6mm, 5µm)
• Flow Rate : 0.8ml/min
• Temperature : Ambient
• Injection Volume : 10µl
• Detector : 267nm
• Diluents : Water: Acetonitrile (50:50)
• Run time : 10 Min

Trial-2 Chromatographic Conditions

• Buffer : 0.1% Formic acid in Water
• Mobile Phase : Buffer: Acetonitrile (50:50%v/v)
• Column : X-Terra-RP18 column (150*4.6mm, 5µm)
• Flow Rate : 0.8ml/min
• Temperature      : Ambient
• Injection Volume : 10µl
• Detector : 267nm
• Diluent : Water: Acetonitrile (50:50%v/v)
• Run time : 10 Min

Trial-3 Chromatographic Conditions

• Buffer : 0.1% Formic acid in water
• Mobile Phase : Buffer: Methanol (25:75%v/v)
• Column : X- Terra RP-18column (150*4.6mm, 5µm)
• Flow Rate : 0.8ml/min
• Temperature : Ambient
• Injection Volume : 10µl
• Detector : 267nm
• Diluent : Water: Acetonitrile (50:50%v/v)
• Run time : 10 Min

Optimization Method for Simultaneous Estimation of Sacubitril and Valsartan in Bulk and Tablet Dosage Form by RP-HPLC

Chromatographic Conditions

• Buffer : 0.1% Formic acid in water
• Mobile Phase : Buffer: Methanol (25:75%v/v)
• Column : X- Terra RP-18column (150*4.6mm, 5µm)
• Flow Rate : 0.8ml/min
• Temperature : Ambient
• Injection Volume : 10µl
• Detector : 267nm
• Diluent : Water: Acetonitrile (50:50%v/v)
• Run time : 10 Min

ANALYTICAL METHOD VALIDATION

Specificity

Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically, these might include impurities, degradants, matrix, etc.

Linearity

Linearity was studied by analyzing five standard solutions covering the range of 50% - 150% for valsartan and 50%- 150% of sacubitril. From the primary stock solution 0.5ml, 0.75ml, 1.0ml, 1.25ml, 1.5 ml of aliquots are pipette into 10 ml volumetric flasks and made up to the mark with the water to give a concentrations of 48.5 µg /mL,  72.75µg/mL,  97µg/mL,  121.25µg/mL and 145.5µg/mL of Valsartan and 51.5g/mL, 77.25µg/mL, 103µg/mL, 128.75µg/mL and 154.5µg/mL Sacubitril.

Accuracy

The accuracy of the method was determined by calculating the recoveries of Valsartan and Sacubitril by analyzing solutions containing approximately 50%, 100% and 150% of the working strength of valsartan and sacubitril.

Method Precision (Repeatability)

The  precision  of  an  analytical  procedure  expresses  the  closeness  of  agreement  (degree   of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.  Precision may be considered at three levels: repeatability, intermediate precision and reproducibility.

Precision should be investigated using homogeneous, authentic samples.  However, if it is not possible to obtain a homogeneous sample it may be investigated using artificially prepared samples or a sample solution.

The precision of the method was checked by repeated preparation (n=6) of 97µg/ml of valsartan and 103µg/ml sacubitril without changing the parameter of the proposed chromatographic method and measured the peak areas and retention times.

Detection Limit

Based on the Standard Deviation of the Response and the Slope

The detection limit (DL) may be expressed as: DL = 3.3 σ/ S where σ = the standard deviation of the response S = the slope of the calibration curve The slope S may be estimated from the calibration curve of the analyte. The estimate of σ may be carried out in a variety of ways.

Quantification Limit

Based on the Standard Deviation of the Response and the Slope

The quantification limit (QL) may be expressed as: QL = 10 σ/ S where σ = the standard deviation of the response S = the slope of the calibration curve. The slope S may be estimated from the calibration curve of the analyte. The estimate of σ may be carried out in a variety of ways.

Robustness

Robustness is the measure of a method remain unaffected by small, deliberate changes in method parameters like flow rate and detection wavelength on assay of the analyte of interest. Here the detection wavelength varied ±2nm and flow rate was varied ±0.2 ml/min.

Ruggedness (Intermediate Precision)

The Method is said to be rugged when the results produced after analysis performed by another analyst are similar to the precision data of prior or another analyst performed. The ruggedness of the method was studied by analyzing the sample and standard preparations by two analysts.

System Suitability

The system suitability parameters such as US tailing factor, US theoretical Plates and resolution was achieved by  injecting  the  prepared  solution  five  times  individually  into  the  chromatographic system separately.

RESULTS AND DISCUSSIONS

METHOD DEVELOPMENT

Determination of Working Wavelength (λ max)

Fig No: 1 UV spectrum of standard Valsartan

Fig No: 2 UV spectrum of standard Sacubitril

OPTIMIZED CHROMATOGRAPHIC CONDITIONS FOR SIMULTANEOUS ESTIMATION OF SACUBITRIL AND VALSARTAN IN BULK AND TABLET DOSAGE FORM BY RP-HPLC

Chromatographic Conditions

• Buffer: 0.1% Formic acid in water
• Mobile Phase : Buffer: Methanol (25:75%v/v)
• Column Flow : X- Terra RP-18column (150*4.6mm,5µm)
• Rate : 0.8ml/in
• Temperature: Ambient
• Injection Volume : 10µl
• Detector: 267nm
• Diluent: Water: Acetonitrile (50:50%v/v)
• Run time: 10 Min

Specificity

The chromatograms of standard and sample are identical with nearly same retention time. No interference due to placebo and sample at the retention time of analyte which shows that the method was specific. The results were given in the Table No 1

Table No: 1 Specificity data for Sacubitril and Valsartan

Result:

Chromatograms explain that retention time for standard, sample and commercial product of Valsartan and Sacubitril are same. This proves that, recipients have no effect on the analytical method. On the other hand, blank peak did not overlap drug peak. So, the method is highly selective.

Linearity

Calibration curve with concentration verses peak areas was plotted by injecting the prepared solutions (Fig No: 3 & 4) and the obtained data were subjected to regression analysis using the least squares method and the data was represented in the Table No: 2 & 3

Fig No: 3 Linearity (calibration) curve of Sacubitril

Fig No: 4 Linearity (calibration) curve of Valsartan

Table No: 2 Linearity data of sacubitril

Table No: 3 Linearity data of valsartan

Result

A linear relationship between peak areas versus concentrations was observed for Valsartan and Sacubitril in the range of 50% to 150% of nominal concentration. Correlation coefficient was 0.9997 and 0.9997 for both valsartan and sacubitril which prove that the method is linear in the range of 50% to 150%.

Table No: 4 Recovery data of Sacubitril and Valsartan

Results of accuracy study are presented in the above table. All the results indicate that the method is highly accurate.

Method Precision (Repeatability)

Table No: 5 Summary of peak areas for method precision of sacubitril and Valsartan

Results of variability were summarized in the above Table No: 5 Percentage Relative standard deviation (%RSD) was found to be less than 2.0% which proves that method is precise.

Detection Limit

Table No: 6 LOD Values of valsartan and sacubitril

Quantification Limit

Table No: 7 LOQ Values of valsartan and sacubitril

Robustness

Table No: 8 Results of robustness of Sacubitril and Valsartan

(Where SAC: Sacubitril; VAL: Valsartan)

Result

The results of Robustness of the present method had shown  that changes made in the flow and wavelength did not produce significant changes in analytical results which were presented  in    the above table. As the changes are not significant, we can say that the method is robust.

Ruggedness (Intermediate Precision)

Table No: 9 Results of Ruggedness of Sacubitril and Valsartan

Result

The %RSD assay values between two analysts was calculated, this indicates the method was rugged.

Table No: 10 System suitability data of Sacubitril and Valsartan

Table No: 11 Standard Results of Sacubitril and Valsartan