Development and validation of spectroscopic simultaneous equation method for simultaneous estimation of Azelnidipine and Valsartan in synthetic mixture

A simple, specific, accurate and precise spectrophotometric method was developed and validated for simultaneous estimation of Azelnidipine and Valsartan in Synthetic Mixture. The wavelength of estimation for Azelnidipine was 240.00 nm and for Valsartan was 250.00 nm. Beer’s law is obeyed in the concentration range of 2-10μg/ml and 16-80μg/ml and correlation coefficient of 0.9999 and 0.9993 for Azelnidipine and Valsartan respectively. The % recovery for Azelnidipine and Valsartan were found to be 101.2 % - 101.9 % and 101.8 - 101.9 % respectively. Intraday precision of Azelnidipine and Valsartan were found to be 0.06 – 0.24 and 0.08 – 0.27 % RSD and Interday precision were found to be 0.14 – 0.33 and 0.11 – 0.37 % RSD respectively. The proposed method was also evaluated by the Assay of Synthetic mixture containing Azelnidipine and Valsartan. The % Assay was found to be 100.50 ± 0.002 for Azelnidipine and 100.87 ± 0.001 for Valsartan. Validation of proposed methods was carried out according to ICH Q2R1Guidelines. The proposed methods were found accurate and reproducible for routine analysis of both the drugs in synthetic mixture.


Introduction
Hypertension is defined as a sustained diastolic blood pressure greater than 90 mm Hg accompanied by an elevated systolic blood pressure (>140 mm Hg).(1) Hypertension results from increased peripheral vascular smooth muscle tone, which leads to increased arteriolar resistance and reduced capacitance of the venous system.Elevated blood pressure is an extremely common disorder.Although many of these individuals have no symptoms, chronic hypertension either systolic or diastolic can lead to congestive heart failure, myocardial infarction, renal damage, and cerebrovascular accidents.
Method for producing pharmaceutical preparation containing calcium antagonist/ angiotensin II receptor antagonist, Patent No. WO201405807A1; French, Japanese, 2014 Hence, there is a scope to develop analytical methods for Azelnidipine and Valsartan in combination.
Literature review reveals that, various analytical methods have been reported for the estimation of Azelnidipine and Valsartan in pharmaceutical formulation and bulk drug include UV spectrophotometric, High-performance liquid chromatography method (HPLC), Stability indicating RP-HPLC method, HPTLC method, TLC method, LC/MS/MS method and UPLC method in individual and/or in combination of other drug.
Literature review shows that, there is no reported method available for simultaneous estimation of both the drugs in combination.Therefore, it is thought of interest to developed simple, accurate, precise and rapid methods for simultaneous estimation of Azelnidipine and Valsartan in combination.

Reagents and material
All the Reagents and Solvents used were of AR or HPLC grades.

Standard solution of Azelnidipine (AZL)
 Preparation of stock solution of AZL An accurately weighed quantity of AZL (10 mg) was transferred to a separate 100 ml volumetric flask and dissolved and diluted to the mark with methanol to obtain standard solution having concentration of AZL (100μg/ml).

Standard solution of Valsartan (VAL)
 Preparation of standard stock solution of VAL An accurately weighed quantity of VAL (10 mg) was transferred to a separate 100 ml volumetric flask and dissolved and diluted to the mark with methanol to obtain standard solution having concentration of VAL (100μg/ml).

Preparation of Standard Mixture Solution (AZL+ VAL):
0.2 ml of working standard stock solution of AZL (100μg/ml) and 1.6 ml of standard Stock solution of VAL (100μg/ml) were pipetted out into 10ml volumetric flask and volume was adjusted to the mark with methanol to get 2μg/ml of AZL and 16μg/ml of VAL.

Preparation of Test Solution
The preparation of synthetic mixture.
 Azelnidipine: 10 mg  Valsartan: 80 mg  Crosscamellose Sodium: 10 mg  HydroxyPropyl Cellulose: 10 mg  Hydrated Silicone Dioxide: 10 mg  Macrogel (PEG) 6000: 30 mg All the excipients were mixed in 100 ml volumetric flask containing 25 ml of Methanol and Sonicated for 15min.make up the volume with Methanol.The solution was filtered through Whatman filter paper No. 42. (AZL100μg/ml and VAL 800μg/ml) Finally, the solution had concentration 100μg/ml for AZL and 800μg/ml for VAL from that pipette out 2ml in 100ml volumetric flask and make up to the mark with Methanol.

Simultaneous equation method
To determine wavelength for measurement, standard spectra Azelnidipine and Valsartan was scanned between 200-400 nm against Methanol.The method was based on the measurement of absorbance of Azelnidipine and Valsartan at 240.00nm and 250.00nm respectively.This method obeyed Beer's law in the concentration range of 2-10μg/ml for Azelnidipine and 16-80μg/ml for Valsartan. Where

Calibration Curve for Azelnidipine
This series consisted of five concentrations of standard AZL solution ranging from 2 to 10μg/ml.The solutions were prepared by pipetting out Standard AZL stock solution (100μg/ml).Then pipetting out (0.2ml, 0.4ml, 0.6ml, 0.8ml, and 1.0ml) was transferred into a series of 10 ml volumetric flask and volume was adjusted up to mark with methanol.A zero-order spectrum of the resulting solution was recorded, measured the absorbance at 240.00 nm against a reagent blank solution (methanol).

Calibration Curve for Valsartan
This series consisted of five concentrations of standard VAL solution ranging from 16 to 80μg/ml.The solutions were prepared by pipetting out Standard VAL stock solution (1.6ml, 3.2ml, 4.8ml, 6.4ml, and 8.0ml) was transferred into a series of 10 ml volumetric flask and volume was adjusted up to mark with methanol.A zero-order spectrum of the resulting solution was recorded, measured the absorbance at 250.00 nm against a reagent blank solution (methanol).

Linearity and range
The linearity response was determined by analyzing 5 independent levels of concentrations in the range of 2 to 10μg/ml for Azelnidipine and 16 to 80μg/ml for Valsartan.(n=6).

Intraday Precision
The precision of the developed method was assessed by analysing samples of the same batch in nine determinations with three Standard solutions containing concentrations 2, 4, 6μg/ml for AZL and 16,32,48 for VAL and three replicate (n=3) each on same day.Zero order absorbance (D1) was measured at 240.00nm for AZL and 250.00nm for VAL.The %RSD value of the results corresponding to the absorbance was expressed for intra-day precision.

Interday Precision
The precision of the developed method was assessed by analysing samples of the same batch in nine determinations with three Standard solutions containing concentrations 2, 4, 6μg/ml for AZL and 16,32,48 for VAL in triplicate (n=3) per day for consecutive 3 days for inter-day precision.Zero order absorbance (D1) was measured at 240.00 nm for AZL and 250.00nm for VAL.The %RSD value of the results corresponding to the absorbance was expressed for inter-day precision.

Accuracy
It was determined by calculating the recovery of AZL and VAL by standard addition method.

LOD and LOQ
The Limit of detection and quantitation of the developed method was assessed by analysing 10 replicates of standard solutions containing concentrations 2μg/ml for AZL and 16μg/ml for VAL.

LOD
The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.
LOD was calculated out by using following formula: 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.

LOQ
The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.LOQ was calculated out by using following formula: DL = 10 σ /S

Robustness & ruggedness
Robustness and Ruggedness of the method was determined by subjecting the method to slight change in the method condition, individually, the:  Change in Wavelength (240±0.2nmand 250±0.2nm) Change in instrument (UV-Vis Spectrophotometer model 1800 and 2450), Three replicates were made for the concentration (2, 4, 6μg/ml of AZL and 16, 32, 48μg/ml of VAL) with different stock solution preparation and the recording of absorbance were done on both the UV-Vis spectrophotometer.

Assay by UV spectrophotometric method
Composition of synthetic mixture  Azelnidipine:10 mg  Valsartan: 80 mg  Crosscamellose Sodium: 10 mg  HydroxyPropyl Cellulose: 10 mg  Hydrated Silicone Dioxide: 10 mg  Macrogel (PEG) 6000: 30 mg All the excipients were mixed in 100 ml volumetric flask and Sonicate for 15min.make up the volume with Methanol.The solution was filtered through Whatman filter paper No. 42.
Finally, the solution had concentration 100μg/ml for AZL and 800μg/ml for MET.From that pipette out 0.2 ml in 100 ml volumetric flask and volume was made up to mark with Methanol to obtain final solution containing 2 µg/ml of AZL and 16 µg/ml of VAL.A zero-order spectrum of the resulting solution was recorded and measure the absorbance at 240.00 nm and 250.00 nm were noted for estimation of AZL and VAL, respectively.The concentrations of AZL and VAL in formulation were determined using the corresponding calibration graph.

Results and discussion
The methods were validated with respect ICH Q2R1 guidelines.

Linearity and range
The linearity response was determined by analyzing 5 independent levels of calibration curve in the range of 2-10μg/ml for Azelnidipine and 16-80μg/ml for Valsartan.(n=6).

Intraday precision
% RSD was found to 0.06 to 0.24 % for Azelnidipine and 0.08 to 0.27 % for Valsartan.

Conclusion
A simple, specific, accurate and precise spectrophotometric method was developed and validated for simultaneous estimation of Azelnidipine and Valsartan in Synthetic Mixture.The wavelength of estimation for Azelnidipine was 240.00 nm and for Valsartan was 250.00 nm.Beer's law is obeyed in the concentration range of 2-10μg/ml and 16-80μg/ml and correlation coefficient of 0.9999 and 0.9993 for Azelnidipine and Valsartan respectively.The % recovery for Azelnidipine and Valsartan were found to be 101.2% -101.9 % and 101.8 -101.9 % respectively.Intraday precision of Azelnidipine and Valsartan were found to be 0.06 -0.24 and 0.08 -0.27 % RSD and Interday precision were found to be 0.14 -0.33 and 0.11 -0.37 % RSD respectively.The proposed method was also evaluated by the Assay of Synthetic mixture containing Azelnidipine and Valsartan.The % Assay was found to be 100.50± 0.002 for Azelnidipine and 100.87 ± 0.001 for Valsartan.Validation of proposed methods was carried out according to ICH Q2R1Guidelines.

Figure 2 Figure 3
Figure 2 Calibration curve for AZL at 240.00nm

Figure 4
Figure 4 Calibration curve for AZL and VAL in combination

Table 1
Lists of Instrument and Apparatus

Table 2
Working Standard API

Table 3
Spectrophotometric conditions for Spectroscopic Method

Table 5
Average of absorptivity at 240nm and 250nm

Table 6
Average of absorptivity at 240nm and 250nm

Table 7
Intraday precision data for estimation of AZL and VAL *(n=3)

Table 8
Interday precision data for estimation of AZL and VAL *(n=3) Accuracy of the method was confirmed by recovery study from Synthetic mixture at three levels.The % recovery was found within 101.2% to 101.9% and 101.8 to 101.9% for Azelnidipine and Valsartan respectively.

Table 11
LOD and LOQ data of AZL and VAL *(n=10)