Analytical and Bioanalytical Method for Quantification of Pure Azilsartan, Not its Salts by RP-HPLC

 

Mukta D. Naykode¹, Durgacharan A. Bhagwat¹*, Swapnil D. Jadhav², Harinath N. More²

¹Department of Pharmaceutics, Bharati Vidyapeeth College of Pharmacy, Near Chitranagari, Kolhapur-416013, M. S., India

²Department of Pharmaceutical Chemistry, Bharati Vidyapeeth College of Pharmacy, Near Chitranagari, Kolhapur-416013, M. S., India

ABSTRACT:

A simple, specific and accurate reverse phase liquid chromatographic method was developed for determination of Azilsartan in its tablet dosage form as well as from biological fluid like plasma. The determination was carried out on ODS Hypersil C18 column (4.6 mm x 250 mm 5µm) column using a mobile phase of acetonitrile: water (pH 4) [60: 40 %v/v]. The flow rate was 1 mL/min with detection at 249 nm. The linearity response of the HPLC system for Azilsartan was obtained over the range 1-64 µg/mL. Ambroxol (25µg/mL) was used as internal standard. Retention time for Azilsartan and ambroxol were found to be 3.74 and 1.84 minutes respectively. The correlation co-efficient (R² value) for Azilsartan was found to be 0.992. The proposed method was successfully used for quantification of Azilsartan in Azilva tablets and plasma. The method was validated as per ICH Q2B (Analytical) and USFDA (Bio-analytical) guidelines. The results of analysis have been validated statistically and by recovery studies. The method is found useful for quantification of Azilsartan in marketed formulations as well as from biological fluids and can be applied for quantification of Azilsartan in preclinical and clinical studies.

 

 

 

1. INTRODUCTION:

Azilsartan is an angiotensin II receptor blocker (ARB) that lowers blood pressure by blocking the action of angiotensin II. It is indicated for the treatment of hypertension. Azilsartan is chemically designated as 2-Ethoxy-1-[{2'(-5-oxo−4, 5−dihydro-1, 2, 4-oxadiazol-3-yl) biphenyl-4-yl] methyl}-1H-benzo[d]imidazole-7-carboxylic acid (Figure1a). It is new Angiotensin II type 1 receptor blocker discovered in 2011.

 

But till date it is used in the form of prodrug Azilsartan  medoxomil (Figure 1b) which has been approved in both the United State and Europe. In 2012, the parent compound Azilsartan was approved in Japan for the treatment of hypertension.¹

 

Figure 1 Structure of a) Azilsartan  b) Azilsartan  medoximil

 

Literature survey revealed few methods for quantitative analysis of Azilsartan medoxomil in biological samples and in pharmaceutical formulations alone as well as in combination with other drugs.^2-13 these include RP-HPLC method with PDA detection in human plasma by solid-phase extraction procedure. Vekariya et al. developed a reversed-phase high-performance liquid chromatography (RP-HPLC)method for quantification of AZM in human plasma.² Gorla et al. developed an ultraviolet (UV) spectrophotometric method to determine the presence of AZM in bulk drug and formulations.³ Ebeid et al. carried out spectrophotometric and spectrofluorimetric studies to estimate Azilsartan  medoxomil potassium and chlorthalidone in combination pharmaceutical dosage forms.⁴ Naazneen and Sridevi proposed a stability indicating HPLC assay method for fixed dose combination of Azilsartan  medoxomil potassium and chlorthalidone.⁵ Recently, Ebeid et al. have developed a stability-indicating method for the combination of Azilsartan medoxomil and chlorthalidone with it ssubsequent application to degradation kinetics.⁶ However, no method has been reported for quantification of the pure Azilsartan  from various dosage form like tablets as well as from biological fluids hence it becomes necessary to develop simple, cost effective, accurate, sensitive and selective method for quantification of Azilsartan.

 

2. MATERIALS AND METHODS:

2.1    Materials:

2.1.1.         Reagents and Chemicals:

For HPLC work, double distilled water was prepared in the laboratory. Acetonitrile, Orthophosphoric acid (Loba Chemie Pvt. Ltd. Mumbai, India) of HPLC grade were used. Pure drug samples Azilsartan and marketed formulation Azilva 20mg were obtained as gift samples from DRDL, USV Private Ltd. Govandi, Mumbai. Ambroxol (internal standard) was provided by Noven Life Sciences Private Ltd. Hyderabad. The plasma has been obtained from blood by centrifugation (Shahu Blood Bank, Kolhapur, M.S., India).

 

2.1.2.         Instrumentation:

The separation Azilsartan was carried out on an isocratic JASCO RP-HPLC system using C18 column (250x4.6mm internal diameter, particle size 5μm).The pump used in this HPLC system was PU 2080 pump (Dual piston with gear driven pump). Twenty microlitre sample solutions were injected to chromatographic system using Rheodyne Injector. The UV detector used in this HPLC system was Czerny turners mount monochromater with deuterium lamp as light source. The chromatographic and the integrated data were recorded using Hercule 2000 (interface) computer system. Data processing was carried out using Borwin® Version 1.5software.

 

2.2.  Methods:

2.2.1.         Chromatographic Conditions:

The analysis was carried out by HPLC using acetonitrile: water (pH 4) [60:40 %v/v] as a mobile phase and ODS Hypersil C18 column  (4.6 mm x 250 mm 5µm) as a stationary phase at a flow rate of 1ml /minute in an isocratic elution mode. Before delivering the mobile phase in to the system, it was degassed and filtered through 0.20μm syringe filter. The injection volume was 20 μl and the detection was performed at 249 nm.

 

2.2.2.         Standard Stock Solution:

Standard Stock Solution containing Azilsartan and ambroxol was prepared by dissolving 10mg of drug in 25ml of mobile phase then final volume of solution was made up to 100ml with mobile phase to get stock solutions containing 1000 µg/ml.

 

2.2.3.         Preparation of internal standard solution:

Internal standard solution containing ambroxol was prepared by dissolving 10mg of drug in 25ml of mobile phase  then final volume of solution was made up to 100ml with mobile phase to get stock solutions containing 1000 µg/ml.

 

2.2.4.         Calibration Curves:

For drug, appropriate aliquots were pipette out from the standard stock solution into a series of 10 ml volumetric flasks, and to each flask 0.25 ml of ambroxol (Internal Standard) was added and then final volume of the solution was made up to 10mL with mobile phase to obtain solutions having concentration 1-64µg/mL. Then it filtered to 0.45µ membrane filter. A 20 µl of sample solution was injected into the chromatographic system using fixed volume loop injector. Chromatograms were recorded. A retention times for Azilsartan and ambroxol found to be 4.19and 2.03 min respectively. Peak areas were recorded for all the peaks and plotted against concentrations to obtain the standard calibration curves.

 

2.2.5.         Analysis of Tablet Formulation:

From the powder content of 20 tablets, an amount equivalent to 10 mg of Azilsartan was weighed and dissolved in 25mL of mobile phase and sonicated for 20minutes. The solution was filtered through 0.45 µ filter. In the flask, 2.5mL of ambroxol solution was added and then final volume of the solution was made up to 100 ml with mobile phase. Appropriate aliquots were taken and analyzed by proposed method using the procedure described earlier. The concentrations of Azilsartan present in the sample solutions was calculated by using equation generated from calibration curve of Azilsartan .

 

Figure 2 Chromatograph of Tablet Analysis

 

Table 1 Result of Tablet (Azilva) Analysis and Recovery Studies

Drug	Lable Claim (mg/tablet)	% Lable Claim Estimated Mean± SDb	Amount Added (mg)	% Recovery Estimated Mean a±SDb
		99.72±0.5108	12	99.37±1.1184
Azilsartan	20	99.59±0.5097	10	99.31±0.3292
		100.16±0.8767	08	98.37±0.7725

^aAverage of Three Determinations; ^bStandard Deviation

 

2.2.6.         Sample Preparation for Bio-Analysis:

Drug stock a solution was prepared (1000µg/mL) then add to drug-free plasma in volumes not exceeding 2% of the plasma volume separately. The plasma samples were stored in the freezer at -17ºC and allowed to thaw at room temperature before processing. The plasma samples were centrifuged at 4000 g for 10 min. An aliquot (1 mL) and acetonitrile (2mL) was pipette into a 10mL polypropylene tube. The mixture was vortex mixed briefly and after standing for 5 min at room temperature the mixture was centrifuged at 4000 g for 20 min. The supernatant was stored as standards stock solution of this drug in freezer for bio-analysis.

 

2.2.7.         Calibration Curves for Analysis of Drugs in Plasma:

For drug, appropriate aliquots stored in freezer was pipette out from standard stock solution into a 10mL volumetric flask and each flask  add 0.25 mL of internal standard solution of ambroxol and then final volume of the solutions was made up to 10mL with mobile phase to get solutions having concentration ranges of 1-64µg/mL. A 20µL of sample solution was injected into the chromatographic system using fixed volume loop injector. Chromatogram was recorded. Retention time for Azilsartan and ambroxol was found to be 4.19and 2.03 min respectively. The response factor was plotted against concentration to get the standard calibration curve.

 

Figure 3 Chromatograms (a) drug analysis in plasma (b) blank plasma analysis

 

2.2.8.         Analysis of Quality Control (QC) Sample:

The known amounts of stock solutions of azilsarrtan, stored in freezer were pipette out in 10mL volumetric flask. In a flask, 0.25 mL of ambroxol solution was added and then final volume of the solutions was made up to 10mL with mobile phase. A 20 µL of sample solution was injected into the chromatographic system using fixed volume loop injector. Chromatograms were recorded as shown Figure 4. The concentrations of drugs were calculated using calibration curve data of the individual drug. The results of the quality control sample are reported in Table 2.

 

Table 2 Results of Quality Control Sample Analysis and Extraction Recovery Studies

^aAverage of Five Determinations; ^bStandard Deviation; ^cAverage of Four Determinations

 

3.      RESULT AND DISCUSSION:

Till date, all developed method are useful for quantification of Azilsartan salts like Azilsartan  medoximil  but recently pure drug Azilsartan has been approved by FDA. Hence it becomes necessary to develop a new, simpler, accurate, reproducible, and sensitive HPLC method for the quantification of pure Azilsartan in marketed formulation and biological fluids.

 

3.1.  RP-HPLC for Tablet Analysis:

In the present communication, development and validation of chromatographic method was carried out for quantification of pure Azilsartan in marketed formulation. The optimum wavelength for detection of 249 nm was selected because drug was found to have good detector responses at this wavelength, confirmed by analyzing 10 µg/mL solutions of drug on an UV-Visible spectrophotometer. To find the appropriate HPLC conditions for separation of the examined drug, various reversed-phase columns, isocratic, and gradient mobile phase systems were tried. After repeated trials with varying ratios of acetonitrilel: water (60:40) was found to yield relatively better resolution of azilsatan, ambroxol.  The drugs were resolved on a ODS Hypersil C18 column (4.6 mm x 250 mm 5µm) column using a mobile phase of acetonitrile: water (pH 4) [60: 40 %v/v]. The flow rate of 1mL/min was found be satisfactory for analysis of drugs within short period of five min. Retention time for Azilsartan  and ambroxol was found to be 4.19and 2.03 min respectively.

 

3.2.  Validation of HPLC Method:

The developed method was validated as per ICH guidelines including the parameters specificity, linearity, precision, accuracy, Limit of detection, Limit of quantification and Robustness.^{14, 15}

 

3.2.1.         Accuracy (Recovery Study):

Recovery studies were performed by standard addition method at three levels, that is, 80%, 100%, and 120%. A known amount of standard Azilsartan  was added to pre-analyzed samples and they were subjected to proposed HPLC method. The RSD (%) for the tablet analysis was less than 2% indicating high degree of accuracy. Results of recovery studies are shown in Table 1.

 

3.2.2.         Precision:

Precision study was performed for three consecutive days at three times to find out intra-day and inter-day variations. The % relative standard deviation (RSD) for intra-day precision was 0.4458%, and for inter-day, precision was 0.7946%, for Azilsartan which is less than 2% and indicates a high degree of precision.

 

3.2.3.         Linearity and Range Study:

The response factors calculated were found to be proportional to concentrations of analytes over ranges tested as specified in Table 3. This study was carried out separately on three consecutive days. The calibration curves were fitted by method of least square. The results of linearity study by regression analysis are given in Table 3.

 

3.2.4.         Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The LOD and LOQ were separately determined based on the calibration curves data. The standard deviation of the y-intercepts and slope of the regression lines were used in calculating these values using the following formulae:

LOD = 3:3 x σ/S

LOQ = 10 x σ/S

 

Where, σ = standard deviation of the response

S = slope of the calibration curve

 

The LOD for Azilsartan found to be 0.012µg/mL. The LOQ for Azilsartan was found to be 0.029µg/mL respectively.

 

3.2.5.         System suitability:

The system suitability parameters like capacity factor (k’), resolution (Rs), retention time in minute, number of theoretical plates (N), and tailing factor (t) were determined which were found to be satisfactory [16]. The results are shown in Table 3.

 

3.2.6.         Specificity:

The specificity of the RP-HPLC method was determined by comparison of the chromatogram of mixed standards and sample solutions. The parameters like retention time, resolution (RS), and tailing factor (t) were calculated. Good correlation was found between the results of mixed standards and sample solutions.

 

Table 3 System Suitability Parameters and Linearity Study of RP-HPLC for tablet analysis

^aRelative Standard Deviation (n=6). C=Concentration in mg/mL; Y=Unit of Response Factor

 

3.2.7.         ROBUSTNESS:

The robustness study was carried out by making small changes in the optimized method parameters like ±0.1 changes in pH, ±2% change in mobile phase ratio, and ±0.1mL/min change in flow rate. There was no significant impact on percentage recoveries of drugs. The results of the robustness study indicated that the method is robust and is unaffected by small variations in the chromatographic conditions. The results of robustness study are reported in Table 4.

 

Table 4 Results of Robustness Study

^aOptimized Parameter for Developed Method; ^bMean of Three Readings; ^cStandard Deviation

 

3.2.8.         RUGGEDNESS:

The results obtained by two analysts were compared statistically by performing Student’s t test. As calculated t values for Azilsartan  (0.8548) was less than theoretical t values (2.776), there is no significant difference between results obtained by two analysts at 95% confidence level for 5 degrees of freedom.

 

3.3.  RP-HPLC for Bio-Analysis:

The proposed RP-HPLC method for simultaneous estimation of Azilsartan from marketed formulations was optimized further to estimate drug from plasma. In this method, blood obtained from blood bank was centrifuged to obtain plasma and stored in freezer. After addition of drugs in plasma, precipitation and separation of plasma proteins are carried out by adding acetonitrile and centrifugation of mixture respectively. To find the appropriate HPLC conditions for separation of the examined drugs, various reversed-phase columns, isocratic and gradient mobile phase systems were tried. After these trials, it was observed that all chromatographic conditions used for tablet analysis were most suitable for analysis of drug in plasma too. As precipitation with plasma: acetonitrile (1:2), followed by centrifugation is found to remove all but trace amounts of plasma proteins larger than 20 kDa while use of plasma: acetonitrile (1:3) does not produce any significant difference in sample preparation and subsequent analysis, the former option was chosen and was found to produce good results. Whenever the protein precipitation step is followed by centrifugation there appears to be no significant advantage of using 3 parts of acetonitrile over 2 parts. The adopted procedure for sample preparation eliminates interference from plasma proteins and yields reasonably accurate results with acceptable precision. All the drugs were resolved on a ODS Hypersil C18 column (4.6 mm x 250 mm 5µm) column using a mobile phase of acetonitrile: water (pH 4) (60:340% v/v) on isocratic system. The wavelength of 249 nm and flow rate of 1mL/min was selected. Retention time for was found to be 4.19 min.

 

3.3.1.         Validation of Bio-Analytical Method:

The proposed RP-HPLC method for bio-analysis was validated as per USFDA guidelines.¹⁷

 

3.3.1.1.             Selectivity:

Individual specificity, in relation to endogenous plasma components, was demonstrated by analysis of series of randomly selected drug-free plasma samples. Typical chromatograms obtained after analysis of drug-free plasma and plasma samples after addition of drugs are reported in Figure 3. The retention time for the investigated drug was found to be different than that of endogenous plasma components. Thus, it indicates selectivity of method for elution of drug in plasma.

 

3.3.1.2.             Accuracy Study:

The recovery study has been carried out by analyzing quality control samples, (five determinations per three concentrations) spiked with analyte. Results of accuracy study are given in Table 5.

 

3.3.1.3.             Extraction Recovery Studies:

The recovery represents the efficiency of an analytical method within the variation limit. The recovery in an assay is the detector response obtained from an amount of analyte added and recovered from the biological matrix. Recovery experiments performed in quadruplicate for analyte by comparing the analytical results for extracted samples at three concentrations (equivalent to LLOQ, MQC, and HQC) with three unextracted concentrations that represent 100 % recovery. Results of the extraction recovery for analytes are given in Table 2. MQC-medium quality control concentrations, 50% of largest concentrations of calibration curve HQC high quality control concentrations, 75–90% of largest concentrations of calibration curve % recovery=(Mean response of extracted samples=Mean response of un-extracted samples)x100.¹⁸

 

3.3.1.4.             Precision Study:

The precision study was carried out by analyzing the plasma samples of three concentrations specified in the inaccuracy study for three consecutive days at two different times. This analysis illustrates the intra-day and inters day precision study. The plasma samples were stored in between analysis in a freezer at -17°C. The results of the precision studies are reported in Table 5.

 

3.3.1.5.             Linearity and Range Study:

The response factors calculated were found to be proportional to concentrations of analyte over the ranges tested as specified in Table 5. This study was carried out separately on three consecutive days. The calibration curves were fitted by method of least square. The results of linearity study by regression analysis are given in Table 5.

 

Table 5 Results of Accuracy, Precision and linearity Studies of Bio-analysis

Precision	Time/Day	Amount of Pure Drug Added in mg	% Concentration Found (Meana±SDb)	Parameter	Azilsartan
Intra-Day	T1	10	90.28±1.4562	Range in ng/ml	1-64µg/mL
	T2	20	89.21±1.3432	Regression Equation	Y=A+B*C
	T3	30	91.25±2.4623	Slope(B)	8532
Inter-Day	D1	-	90.21±2.0762	Intercept (A)	2.329
	D2	-	90.45±1.2546	Correlation coefficient	0.9997
	D3	-	88.56±2.7548	-

T= Time; D= Day. ^aAverage of Fifteen Determinations (calculated at three levels 3x5);  ^bStandard Deviation C=Concentration in mg/mL; Y=Unit of Response Factor

 

3.3.1.6.          Lower Limit of Quantitation (LLOQ):

It is the lowest serum concentration of AT quantified with a coefficient of variation of less than 20%. The LLOQ value of Azilsartan  was found to be 0.012 µg/mL.

 

3.4.  Stability Study:

This study was carried out by performing analysis of stock solutions, unextracted plasma samples, and freshly prepared solutions at various atmospheric conditions and times as shown in the following headings as per guidelines as the mean of 6 readings with its standard deviation.¹⁴ Stock Solution Stability. To test the stock solution stability of Azilsartan and ambroxol (IS), five aliquots of standard stock solutions were left at -17°C for 3 days. Then, the concentrations were analyzed and compared with the fresh stock solution. The percentage recovery of Azilsartan and ambroxol (IS) were found to be 95.08±1.52, and 94.12±1.87, respectively. Freeze and Thaw Stability. The effect of freeze and thaw cycles on the stability of plasma samples containing analytes were determined by subjecting five aliquots of QC samples at low, middle, and high concentration unextracted quality control samples to four freeze–thaw cycles. After completion of every cycle, the samples were analyzed and the experimental concentrations were compared with the nominal values obtained by analyzing fresh samples. The accuracy values of three concentrations in two freeze–thaw cycles were calculated. The percentage recovery of Azilsartan was found to be 95.46±3.57. Short-Term Stability. Five aliquots of QC samples at low, mid, and high concentration unextracted QC samples were kept at ambient temperature (15°C) for 12 hr in order to determine the short-term stability of analytes in plasma. Then, the samples were processed and analyzed and the concentrations obtained were compared with the nominal values obtained by analyzing fresh samples. The percentage recovery of Azilsartan  was found to be 95.33± 4.01. Post-Preparation Stability. In order to estimate the stability of Azilsartan in the prepared samples, five aliquots of QC samples at low, mid, and high concentration were kept at 4°C for about 4 hr. Then, the sample was analyzed and the concentrations obtained were compared with the nominal values obtained by analyzing fresh samples. The percentage recovery of Azilsartan was found to be 95.73±2.91.

 

4.      CONCLUSION:

The developed RP-HPLC method for tablet analysis is accurate, precise, sensitive, robust, and selective and can be employed successfully for the quantification of Azilsartan in both bulk and formulations. The adequate retention of active component of the formulation and IS in a HPLC runtime of 5 min clearly indicates the utility of the developed method for rapid and accurate analysis of formulations containing this drug with acceptable precision. The method optimized for analysis of drug from plasma was found to be effective for determination of Azilsartan from plasma without any interference from the additives and endogenous substances. It is a simple and accurate procedure requiring inexpensive reagents that could be used for rapid and reliable clinical and pharmacokinetic studies including Azilsartan. The application of the developed method for analysis of Azilsartan drug from plasma has advantage of not only being rapid, accurate, and precise but also of having adequate sensitivity for quantification of this drug at low concentration in biological matrix.

 

5. ACKNOWLEDGEMENT:

Authors are very thankful to DRDL, USV Private Ltd. Mumbai for providing gift sample of Azilsartan. The authors are also thankful to the Management and Principal, Bharati Vidyapeeth College of Pharmacy, Kolhapur for provision of facilities for this research work.

 

6. CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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