INTRODUCTION:

Candesartan chemically, 1-{[(cyclohexyloxy)carbonyl]oxy}ethyl 2-ethoxy-1-{[2'-(1H-1,2,3,4-tetrazol-5-yl)-[1,1'-biphenyl]-4-yl]methyl}-1H-1,3-benzodiazole-7-carboxylateis an angiotensin-receptor blocker (ARB) that may be used alone or with other agents to treat hypertension(Figure-1).

Hydrochlorthiazidechemically,
6-chloro-1,1-dioxo-3,4-dihydro-2H-1λ⁶,2,4-benzothiadiazine-7-sulfonamide (Figure-2) is thiazide diuretic often considered the prototypical member of this class. It reduces the reabsorption of electrolytes from the renal tubules. This results in increased excretion of water and electrolytes, including sodium, potassium, chloride, and magnesium.

It has been used in the treatment of several disorders including edema, hypertension, diabetes insipidus, and hypoparathyroidism. Based on literature, many HPLC methods have been reported for the individual determination of candesartan and hydrochlorthiazide in pharmaceutical dosage forms and biological samples¹. A few chromatographic^2-7and spectroscopic⁸ methods have been reported for the simultaneous determination of candesartan and hydrochlorthiazide in combined dosage forms. The present work is aimed to develop and validate simple, sensitive and more precise RP- HPLC method for simultaneous estimation of candesartan and hydrochlorthiazide in bulk and pharmaceutical tablet dosage form.

MATERIALS AND METHODS:

Equipment:

Separation was carried out by using Shimadzu LC20A system equipped with LC20AT pump. SPD 20A Prominence UV-Visible detector and the peak areas were integrated by using spinchrome software CFR. Analysis was carried out on Hypersil BDS C8 (250 mm x 4.6 mm, 5 µm) column.

Figure 1: Chemical structure of Candesartan

Figure 2: Chemical structure of Hydrochlorthiazide

Chemicals and reagents:

HPLC grade methanol, acetonitrile and analytical grade orthophosphoric acid, triethylamine were obtained from M/s. Rankem Chemicals Ltd, Mumbai, India. Milli-Q water dispensed through a 0.22 μ filter of the Milli-Q water purification system (Millipore, Merck KGaA, Darmstadt, Germany) was used throughout the study.

Preparation of solutions:

Preparation of Mobile phase:

Mobile phase was prepared by mixture ofacetonitrile and 0.02% triethyl amine in the ratio 40:60v/v was prepared by diluting 400 mL of acetonitrile and 600 mL of 0.02% triethyl amine in a 1L flask and its pH was adjusted to 5.5 with o-phosphoric acid. After mobile phase was filtered through 0.45 µ membrane filter and sonicated for 10 min to remove dissolved gases before transferring in to the reservoir.

Preparation of mixed standard solution of candesartan and hydrochlorthiazide:

About 100 mg of candesartan and 150 mg of hydrochlorthiazide were accurately weighed and transferred into a 50 mL clean dry volumetric flask containing 25 mL of the methanol.The solution was sonicated for 5 min and then volume was made up to the mark with a further quantity of the diluent to get a concentration of 1000 μg/mL of candesartan and 1500 μg/mL of hydrochlorthiazide(Stock solution). A mixed working standard solution was further prepared by diluting the above stock solution to obtain a concentration of 100 μg/mL of candesartan and 150 μg/mL of hydrochlorthiazide.

Preparation of the tablet solution:

Twenty tablets of the commercial sample of ‘Candesar-H’ were weighed and finely powdered. An accurately weighed portion of powdered sample equivalent to 50 mg of candesartan and 100 mg of hydrochlorthiazide was transferred into a 50 mL volumetric flask containing 30 mL of the diluent. The contents of the flask were sonicated for about 10 min for complete solubility of the drugs and the volume made up with a further quantity of the diluent. Then, this mixture was filtered through a 0.45 μ membrane filter. This filtrate was used for further analysis.

Chromatographic conditions:

A reverse phase column hypersil BDS C8 column (250 mm x 4.6 mm, 5 µm particle size), equilibrated with mobile phase (acetonitrile: triethylamine 0.02%in the ratio of 40:60 v/v) was used. Mobile phase flow rate was maintained at 1 mL/min and effluents were monitored at 262 nm. The sample was injected using 20 micro litre manual sample injector and run time was 10 min (Figure 3 and 4).

Figure 3: A typical chromatogram showing the separation of candesartan and hydrochlorthiazide standard

Figure 4: A typical chromatogram showing the separation of candesartan and hydrochlorthiazide sample

METHOD VALIDATION:

Linearity:

Linear calibrations plots of the proposed method were obtained over concentration ranges of 50-150μg/mL for candesartan, (50, 80, 90, 100, 110, 120, 150μg/mL) and 75-225μg/mL for hydrochlorthiazide (75, 120, 135, 150, 165, 180, 225μg/mL). Each solution was prepared in triplicate. Regression coefficient was found to be 0.998 and 0.999 for both the drugs (Figure 5 and 6). Standard curve had a reliable reproducible over the standard concentrations across the calibration range. All back calculated concentrations did not differ from the theoretical value as no single calibration standard point was dropped during the validation.

Figure 5: Calibration curve for candesartan

Figure 6: Calibration curve for hydrochlorthiazide

Accuracy:

The standard addition method was used to demonstrate the accuracy of the proposed method. For this purpose, known quantities of candesartan and hydrochlorthiazide were supplemented to the previously analysed sample solution and then experimental and true values compared. Three levels of solutions were made corresponding to 50, 100 and 150 % of nominal analytical concentration (150 µg/mLcandesartan and 200 µg/mL for hydrochlorthiazide). Standard preparation & sample preparation was injected into the HPLC and % RSD forcandesartan and hydrochlorthiazidepeaks in standard preparation was calculated and tabulated in Table 1. The mean recovery values of candesartan and hydrochlorthiazidewere found to be 99.92, 99.85 % respectively.

Table 1: Results of recovery experiments of candesartan and hydrochlorthiazide

Preanalysed amount (μg/mL)		Spiked amount (μg/mL)		% recovered
Candesartan	Hydrochlorthiazide	Candesartan	Hydrochlorthiazide	Candesartan	Hydrochlorthiazide
150	200	125	175	99.65	99
150	200	125	175	100.1	100.1
150	200	125	175	99.91	99.78
150	200	150	200	100.01	100.1
150	200	150	200	99.75	99.91
150	200	150	200	100.05	99.72
150	200	175	225	100.06	100.13
150	200	175	225	99.88	99.62
150	200	175	225	99.91	99.81
			MEAN	99.92	99.85
			SD	0.15	0.35
			%RSD	0.15	0.35

Table 2: Results of repeatability of candesartan and hydrochlorthiazide

S. No.	Candesartan			Hydrochlorthiazide
S. No.	Area	USP Plate Count	USP Tailing	Area	USP Plate Count	USP Tailing
1	1195864	5236	1.45	1665203.7	53079	1.47
2	1189541	5201	1.43	1656215.0	52023	1.47
3	1199545	5122	1.47	1645413.57	53137	1.49
4	1185641	5262	1.47	1658452	52016	1.46
5	1198546	5145	1.46	1674589.2	52860	1.48
6	1198754	5290	1.48	1655613	52354	1.47
MEAN	1194649			1659248
SD	5239.6			8993.3
% RSD	0.43			0.54

Intermediate precision:

Six replicate injections of the same dilution were analyzed on two different days by different analyst for verifying the variation in the precision. The % RSD of the results for candesartan and hydrochlorthiazide were found to be 1.31 and 0.77 respectively, which are within acceptable limit of ≤2. Hence, the method is reproducible on different days with different analyst. This indicates that the method is precise. The results are shown in the Table 3a and 3b.

Table 3a: Results of intermediate precision of candesartan

S. No.	Average area (n=9)	USP Plate Count	USP Tailing
Day 1	1184625	5266	1.46
Day 2	1162857	5145	1.41
Overall average	1173741
SD	15392
% RSD	1.31

Table 3b: Results of intermediate precision of hydrochlorthiazide

S. No.	Average area (n=6)	USP Plate Count	USP Tailing
Day 1	1647456	53112	1.48
Day 2	1629571	52921	1.45
Overall average	1638514
SD	12647
% RSD	0.77

Robustness:

Robustness is generally done by changing the parameters like flow rate, organic phase of the mobile phase and pH of mobile phase. The results are shown in the following data is given in the Table 4 and 5.

Table 4: Robustness study for candesartan

Condition

Tailing factor

Theoretical plates

% assay

Flow rate at 0.9 mL/min

Flow rate at 1.1 mL/min

1.47

1.48

5102

5269

99.7

98.5

Mobile phase:

· Acetonitrile (38%)

· Acetonitrile (42%)

1.48

1.46

5235

5132

99.5

98.7

pH of mobile phase:

· at 5.3

· at 5.7

1.46

1.48

5145

5290

100.1

100.3

Table 5: Robustness study for hydrochlorthiazide

Condition

Tailing factor

Theoretical plates

% assay

Flow rate at 0.9 mL/min

Flow rate at 1.1 mL/min

1.48

53095

52015

99.5

98.4

Mobile phase:

· Acetonitrile (38%)

· Acetonitrile (42%)

1.48

53024

52044

99.4

98.8

pH of mobile phase:

· at 5.3

· at 5.7

1.48

53078

52158

100.3

100.2

Limit of detection (LOD):

The LOD for this method was found to be 0.08 µg/mL and 0.13 µg/mL for candesartan and hydrochlorthiazide respectively.

Limit of quantitation (LOQ):

The LOQ for this method was found to be 0.26µg/mL and 0.42 µg/mL for candesartan and hydrochlorthiazide respectively.

Assay:

Twenty micro liters of standard and sample solutions were injected separately in to the chromatographic system and the peak areas for the analyte peaks were measured. The % content of each drug was calculated.

RESULTS AND DISCUSSION:

To develop a new RP-HPLC method, several mobile phase compositions were tried. A satisfactory separation with good peak symmetry was obtained with zodiac. In the present study, a new simple, precise and accurate HPLC method was developed and validated for the simultaneous estimation of candesartan and hydrochlorthiazidein tablet dosage forms. In this method, a Hydrosil BDS C8 (250 x 4.6 mm; 5 µm) column using mobile phase containing acetonitrile and triethylamine 0.02% (40:60 v/v) at a flow rate of 1 mL/min. Quantification was achieved with UV detection at 262 nm based on peak area. The retention time for candesartan and hydrochlorthiazidewere found to be 2.449 min and 4.895 respectively. The optimized method was validated as per ICH guidelines. The System suitability parameters observed by using this optimized conditions were reported. A linearity range of 50-150μg/mL with correlation coefficient 0.9998 was established for candesartanand 75-225µg/mL with correlation coefficient 0.9999 was established for hydrochlorthiazide. The precision of the proposed method was carried in terms of the repeatability and the % RSD values of candesartan was found to be 0.43 % and of hydrochlorthiazide. was found to be 0.54 % and reveal that the proposed method is precise. The LOD and LOQ values for candesartan were 0.08 and 0.13µg/mL respectively and for hydrochlorthiazide were found to be 0.26 and 0.42µg/mL. The study of robustness in the present method shows no significant changes either in the peak area or room temperature. The results of analysis of commercial formulation indicated that there is no interference due to common formulation excipients with the developed method. Therefore, the proposed method can be used for routine analysis of these two drugs in their combined pharmaceutical dosage form.

CONCLUSION:

The proposed method was found to be simple, precise, accurate and rapid for determination of candesartan and hydrochlorthiazide from pure and its dosage forms. The mobile phase is simple to prepare and economical. The sample recoveries in the formulation were in good agreement with their respective label claims and they suggested non-interference of formulation excipients in the estimation. Hence, this method can be easily and conveniently adopted for routine analysis of candesartan and hydrochlorthiazidein pure form and its dosage form and also can be used for dissolution or similar studies.

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Received on 15.07.2019 Accepted on 09.08.2019

Asian J. Pharm. Ana. 2019; 9(4):224-228.

DOI: 10.5958/2231-5675.2019.00038.3