Development and Validation of HPLC Method for Estimation of Pharmaceutical Drug and its Stability Studies in Simulated Biological Fluid: Comparative Study

 

Sushil D. Patil*, Tejashri Dugaje*, Dr. Sanjay J. Kshirsagar

MET’s Institute of Pharmacy, MET League of Colleges, Bhujbal Knowledge City, Adgaon, Nashik,

 Savitribai Phule Pune University, Maharashtra, India

 

ABSTRACT:

A simple and precise high performance liquid chromatography method for determination of candesartan cilexetil in their pharmaceutical formulation was developed and validated. The separation was carried out on Cosmosil C18 (250mm X 4.6, 5µ) RP-HPLC column using an 0.8ml/min as flow rate with methanol: water (80:20 v/v) mobile phase. Quantification was performed with a UV detector at 214nm, solubility and stability was determined individually in simulated biological fluids at various pH was investigated. The calibration curves of candesartan cilexetil were linear in the range of 10-50 µg/ml (R²=0.999). The developed method was applied to pharmaceutical formulation successfully with no interfering peaks. The percentage recovery was 99.71-100.29%. It was observed that solubility of candesartan cilexetil was increased in all invitro medium, in 1.2 and slightly 7.4 pH and the stability of candesartan cilexetil was stable in pH 1.2 or degraded in pH 7.4 for 24 hours.

 

 

 

INTRODUCTION:

Candesartan cilexetil is a pro-drug which shows ester hydrolysis in GIT where it transforms in to its active form Candesartan. Candesartan Cilexetil is a selective angiotensin II type 1 receptor blocker .This drug is used in the treatment of hypertension, heart failure, myocardial infarction. Oral tablet of Candesartan Cilexetil are available containing various doses (4mg to 32mg) alone or in combination with hydrochlorothiazide and amlodipine for the treatment of hypertension and diabetes. Analytical method is an important process for the identification and quantification of bulk drugs. For precise results there must be a validated method for the analysis of bulk drugs and finished dosage forms.

 

The validated method should be precise, accurate and reproducible .The estimation of CC is done by Potentiometric titration as per USP35-NF30, HPLC [1 -12], HPTLC [10] method is reported in literature. UV estimation also reported. [11] To investigate the Comparative study of sample in different biological fluid. Development of HPLC method used for estimation of candesartan cilexetil stability in given biological fluid and done comparative study with invitro interactions studies. The developed HPLC method will be applied for estimation of Candesartan Cilexetil. The method is validated according to the ICH guidelines. Invivo pharmacological studies were recommended, which can help with the invitro study in improvement of clinical efficacy of the drug. Thus we have developed and validated the analysis technique for the estimation of CC by HPLC which is simple, sensitive, accurate, precise, and reproducible. Stability study helps to know how much drug are stable in the simulated biological fluids given conditions. [13-15] The results of these studies are the basis for developing appropriate manufacturing process, formulation, suitable dosage forms, invivo studies.

 

 

Fig 1: Structure of candesartan cilexetil

 

 

MATERIAL AND EQUIPMENTS:

Drug sample:

Candesartan cilexetil was kindly supplied as gift samples by Glenmark pharmaceuticals Ltd. Mumbai. The procured drug standards were standardized by measurement of physical properties like Melting point, Infrared spectrum, UV absorption spectrum and comparing data with literature work. 

 

Chemical and Reagents:

Solvent Methanol used for chromatographic analysis was of HPLC grade purchased from S.D. Fine Chemicals, Mumbai. The water used for HPLC was double distilled assembly (BOROSIL) and passed through a 0.45 µm filter. All other chemicals and solvents were of AR grade and purchased from India Pvt. Ltd., Mumbai, India.  

 

INSTRUMENTATIONS:

For method development Weighing balance (Wenser High Precision Balance, (PGB 100), Ultra sonicator bath (Wenser Ultra Sonicator, WUC-4L), HPLC Binary Gradient System (HPLC 3000 series), with Detector UV-3000-M, Column: cosmosilC18 (250mm˟4.6 ID, Particle size: 5µm). A Shimadzu UV-Visible spectrophotometer (UV-1800, Shimadzu corporation, Kyoto Japan) was used for all absorbance measurement with the 1 cm paired quartz cell was used for the measurement of UV absorbance in the range of 400-200 nm.

 

EXPERIMENTAL WORK:

Method development Study on the chromatographic conditions of candesartan cilexetil:

An accurately weighed quantity of 10mg candesartan cilexetil was transferred to 10ml volumetric flask to form 1000µg/mL stock solution.Dissolved with sufficient quantity of methanol and volume was made up to the mark with same mobile phase and sonicated for 15min.From the resulting solution 0.3 ml was transferred to 10ml volumetric flask and the volume was made up to the mark with different ratios of mobile phases that are to be tried.The resulting  30ug/ml of solution was subjected to chromatographic analyses using mobile phases of different strengths with chromatographic conditions mention below and observed in fig 2.

 

Fig 2: Representative chromatogram of Candesartan in methanol: water (80:20%v/v) 0.8ml 254nm.

 

Preparation of mobile phase and diluent:

The mobile phase was prepared by mixing 200ml double distilled water and 800ml of methanol. The solution was degassed in ultrasonic water bath for 5 minutes and filtered through 0.45um filter under vacuum. The same mobile phase used as diluent.

 

Preparation of standard stock solution:

An accurate weighed quantity 10mg of the drug candesartan cilexetil was transferred in  three different 10ml volumetric flasks, dissolved with sufficient quantity of diluent (mobile phase) and volume was made up to the mark with diluent. This gave 1000 µg/ml standard stock solution for candesartan.

 

Method validation:

HPLC method was validated as per ICH guidelines for method validation. The performance parameters like linearity, precision, and accuracy were evaluated.[17-19]

 

Linearity

This was studied by diluting standard stock solution of Candesartan cilexetil 10-50 µg/ml concentrations. Calibration curve with concentrations verses absorbance were plotted at their respective wavelengths and the obtained data was subjected to regression analysis using the least square method. The standard curve of drug showed in Fig 3.

 

Preparation of calibration curve standard for candesartan cilexetil:

From the above standard stock solution 1000ug/ml of candesartan cilexetil aliquots five concentrations of 10, 20, 30, 40,50µg/ml was prepared in mobile phase.

 

Fig 3: Calibration /linearity curve of Candesartan Cilexetil

 

Table no 1: Linearity data for candesartan cilexetil

Sr. No	Concentration in µg/mL	Area
1	10	1177767
2	20	2210406
3	30	3263889
4	40	4175636
5	50	5197233
Equation	y = 100042+203738
R2 value	0.999
slope	100042

 

 

Calibration runs and regression analysis:

These calibration standard solutions were analyse using the under mentioned chromatographic conditions:

Analytical column: cosmosil C18 (250mm× 4.6mm, partical size 5um), Injection volume: 20ul, Flow rate: 0.8ml/min, Mobile phase: Methanol: water (80:20% v/v) at 254nm.

 

Accuracy and percentage recovery:

For accuracy studies were performed in triplicate way by standard addition method at 50%, 100%, 150% of label claimed respectively. In pre analyzed samples, the known amount of standard drugs were added and subjected to chromatographic analysis. From these we calculated %Recovery value which in limit as recommended limit. Also, we were studied triplicate concentration levels at 10,30,50 µg/ml which obtained %RSD value within limit; shows good result as recommended in ICH guideline. (Table- 2, 3)

 

Table 2: Accuracy study of the candesartan cilexetil

Sr No	Conc in µg/mL	%	Area	Mean	SD	%SD	Mean
1	10		1177767
2	10	80	1185638	1183373	4884.999	0.412803
3	10		1186715
4	30		3263889
5	30	100	3252430	3261348	7958.212	0.244016	0.292682
6	30		3267726
7	50		5197233
8	50	120	5182416	5194897	11492.46	0.221226
9	50		5205042

 

 

Table 3: Accuracy and percentage recovery study

Sr. No.	Conc. of tablet in µg/ml (A)	% level	Amount added of standard (B)	Area	Mean	Amount found (A+B)	% Recovery	Mean
1	20	50	10	3263889		30.5888	101.9622
2	20	100	20	4175636	4212253	39.7023	99.25576	100.3486
3	20	150	30	5197233		49.9139	99.82797
					SD	967192
					%RSD	0.229614

 

Table no 4: Precision Interday study of candesartan cilexetil

Sr. No		Conc. in ppm	Area	Mean	SD	%RSD
1	DAY1	30	3263889
2		30	3252430	3261348	7958.212	0.244016
3		30	3267726
1	DAY2	30	3258357
2		30	3257778	3261456	5875.613	0.180153
3		30	3268232
					Mean	0.212085

 

 

Table no5: Precision Intraday study of candesartan cilexetil

Sr. No		Conc. in ppm	Area	Mean	SD	%SD
1	morning	30	3263889
2		30	3252430	3261348	7958.212	0.244016
3		30	3267726
1	evening	30	3271350
2		30	3256402	3261385	8630.232	0.264619
3		30	3256402
					Mean	0.254317

 

Precision:

Precision is the degree of agreement among individual test results when the technique is applied repeatedly to numerous samplings. It was determined by studying repeatability, intra-day and inter-day precision of method. For intraday, the analysis was carried out at different intervals on the same day and for inter day, the analysis was carried on different days. (Table- 4, 5)

 

Detection Limit:

Based on the Standard Deviation (SD) of the response and the slope of calibration curve, the detection limit (DL) was calculated as 

 

DL=3.3 σ/s

Where, σ=the S.D. of the y-intercepts of regression lines

           S= the slope of the calibration curve

 

LOD=3.3X 8111.89/ 100042 = 0.26758 µg/ml 

The detection of limit found to be 0.26758 µg/ml.The slope 'S' may be estimated from the calibration curve and SD was used should be calculated from the y-intercepts of regression line in calibration curve.

 

Quantitation Limit:

Based on SD of the response and the slope of calibration curve, the quantitation limit (QL) was calculated as,

 

QL=10σ/S  

Where, σ=the SD of the y-intercept of regression lines

     S=the slope of the calibration curve

LOQ =10X8111.89/ 100042=0.810848 µg/ml

The slope 'S' may be estimated from the calibration curve and SD was used should be calculated from the y-intercepts of regression line in calibration curve.

 

Robustness study:

It was measured by small but measured variation in method parameter like change in physical parameters like, flow rate and wavelength.

    -change in wavelength (±2nm)

    -change in flow rate (± 0.20ml/min)

 

Analysis of tablet:

The tablet weight is 200mg which contains 8mg candesartan cilexetil as marketed tablet actasin 8mg, (candesartan cilexetil, ACTINSAR 8mg)10 tablet crushed and then further process carried. Accurately weighed 10mg then transfer to 10ml volumetric flask, dissolved in methanol and finally volume was made with methanol (1000 µg/ml). This solution was filtered through 0.45u filter. From this filtrate 0.3mL solution was pipette out and diluted with mobile phase to get final concentration of 30 µg/ml of candesartan cilexetil. The resulting solution was subjected to chromatographic analysis by RP-HPLC.

 

Table no 6: Assay of candesartan cilexetil

Sr. no	Composition 30 µg/ml	Area of standard	Area of sample	% Assay
1.	API sample	5192617	3263889	62.85634
2.	Tablet formulation	5192617	3246860	62.52839

 

Degradation or Stability Comparative Study:

Preparation of simulated biological fluids:

Preparation of simulated gastric fluid pH 1.2:

Dissolve 2 g of sodium chloride and 3.2g of pepsin in water. Add 80 mL 1M hydrochloric acid and diluted to 1000 mL with water. (As per IP) [17]

 

Preparation of simulated blood fluid pH 7.4:

This pH fluid was prepared by dissolving 0.6 gm. of potassium dihydrogen o-phosphate, 6.4 g of disodium hydrogen o-phosphate and 5.85 g of sodium chloride in necessary deionized water to produce 1000 mL and the pH was adjusted.[15]

 

Procedure for stability comparative study:

Prepared 1000 µg/ml solutions with addition of 10 ml simulated blood fluids (pH 7.4) in 10mg drug in 10ml volumetric flask .From this solution aliquot 0.5 ml in 10ml volumetric flask and make up volume upto mark with methanol. This was further 20µl solution was injected in column which obtained result in different time intervals as 0, 6, 12, 24 hrs. The following table shows the percent degradation or stability of candesartan cilexetil in different time interval.

 

 

 

 

Fig No.2. Representative chromatogram of degradation of Candesartan  cilexetil 50 µg/ml treated with simulated gastric fluid at 1.2pH at 0, 6, 12, 24hrs, in Methanol: Water (80:20%v/v) ,  0.8ml/min,  254nm.

 

Table 7: Degradation observed at pH 1.2 (Gastric artificial fluid)

Sr. No	Degradation in hrs.	Area of Standard	Area of degraded Sample	Degraded upto %	Actual % degradation
1	0	5197233	4950624	95.25499434	4.74500566
2	6	5197233	2186127	42.06328637	57.93671363
3	12	5197233	1677218	32.2713644	67.7286356
4	24	5197233	1484098	28.555541	71.444459

 

Fig No.3. Representative chromatogram of Candesartan cilexetil  50µg/ml treated with simulated blood at 7.4pH at 0,6 hrs,  Methanol: Water (80:20%v/v), 0.8ml/min,  254nm (Sample Volume: 20µl, Pressure: 10-11MPa, Run time: 11.25min) where at 6 hrs it totally degraded.

Table 8: Degradation observed in pH 7.4

Sr. No.	Degradation in hrs.	Area of standard	Area of degraded sample	Degraded upto %	Actual % degradation
1	0	5197233	5042820	97.02893828	2.971061717
2	6	5197233	663681	12.76989121	87.23010879

 

Table 9: Result obtained of method development and validation:

Sr. No.	Parameter	Result
1	λmax	254nm
2	Slope	100042
3	Regression value	0.0999
4	Linearity and range (ug/ml)	10-50 ug/ml
5	Accuracy (% recovery)	100.3486
6	Precision (% RSD) Interday precision (n=3) Intraday precision(n=3)	RSD<2% 0.212085 0.254317
7 8	Limit of Detection Limit of Quantitation	0.26758 μg/ml 0.810848 μg/ml

 

CONCLUSION:

A specific, precise, accurate, rapid and reliable HPLC method has been developed and validated for the analysis of Candesartan cilexetil. The method is appropriate analysis can be used in quality control department in pharmaceutical industries for the analysis of bulk, intermediate and pharmaceutical dosage form of Candesartan Cilexetil. By this compare study in different simulated fluid (invitro study) study in different time interval; it is observed that the given drug is more stable in the gastric fluid pH than blood pH. More precise result obtained by HPLC method.

 

ACKNOWLEDGMENT:

The authors are thankful to the management and trustees of Mumbai Educational Trust’s Bhujbal Knowledge City, Nashik, for providing necessary chemicals and analytical facilities and to Raptakos, Brett and CO. Ltd, Thane, India. Gift sample.

 

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Received on 02.06.2019            Accepted on 25.06.2019           

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