New Stability Indicating Method for the Simultaneous Determination of Impurities Present in Candesartan Cilexetil and Hydrochlorothiazide Tablets by Ultra Performance Liquid Chromatography with Photo Diode Array Detector

© Authors. Terms and conditions of Creative Commons Attribution 4.0 International (CC BY 4.0) apply. Correspondence: M. V. V. N. Murali Krishna, APL Research Centre (A Division of Aurobindo Pharma Limited), Survey No. 313, Bachupally, Quthubullapur Mandal, Ranga reddy District, Hyderabad-500072, Telangana, India. murali_mantri@yahoo.com New Stability Indicating Method for the Simultaneous Determination of Impurities Present in Candesartan Cilexetil and Hydrochlorothiazide Tablets by Ultra Performance Liquid Chromatography with Photo Diode Array Detector


INTRODUCTION
Candesartan cilexetil (CAN) is a drug used for treating high blood pressure (hypertension) [1].It belongs to the class of drugs called angiotensin receptor blockers (ARBs).Angiotensin, formed in the blood by the action of angiotensin converting enzyme (ACE), is a powerful chemical that attaches to angiotensin receptors found in many tissues but primarily on smooth muscle cells surrounding blood vessels.Angiotensin's attachment to the receptors causes the muscle cells to contract and the blood vessels to narrow (vasoconstrict) which leads to an increase in blood pressure [2].CAN is classified as an angiotensin II receptor type 1 antagonist which is widely used in treatment of diseases like hypertension, heart failure, myocardial infarction and diabetic nephropathy.CAN causes reduction in blood pressure and is used in treatment of hypertension.It is also used in the treatment of congestive heart failure and given as prophylaxis to reduce the severity and duration of migraine.CAN is also available in a combination formulation with a low dose thiazide diuretic, invariably hydrochlorothiazide, to achieve an additive antihypertensive effect [3].
Hydrochlorothiazide (HCT) is a thiazide diuretic that helps prevent the body from absorbing too much salt, which can cause fluid retention.HCT treats fluid retention (edema) in people with congestive heart failure, cirrhosis of the liver, or kidney disorders, or edema caused by taking steroids or estrogen.This medication is also used to treat high blood pressure (hypertension) [4].
CAN and HCT tablets are a fixed dose combination (FDC) product with the brand name of Atacand HCT tablets in US market.The available three strengths are 16 mg/12.5mg, 32 mg/12.5 mg and 32 mg/25 mg.The combination of CAN and HCT is an effective treatment for patients with hypertension.The combination of these two agents showed an excellent adverse event profile.This combination, usually at doses of 16 and 12.5 mg respectively, which has been shown to be more effective in lowering blood pressure than either agent alone and to be capable of reducing blood pressure to a similar or greater extent than the combination of HCT with an angiotensin-converting enzyme (ACE) inhibitor or another angiotensin II antagonist, without loss of the placebo-like tolerability that angiotensin II antagonist have when used alone [5][6][7][8].CAN is chemically described as (±)-1-Hydroxyethyl 2-ethoxy-1-[p-(o-1H-tetrazol-5ylphenyl) benzyl]-7-benzimidazole carboxylate, cyclohexyl carbonate (ester).It is a white to off-white powder.It is practically insoluble in water and sparingly soluble in methanol.Candesartan cilexetil is a racemic mixture containing one chiral center at the cyclohexyloxycarbonyloxy ethyl ester group.The empirical formula is C33H34N6O6 and Molecular weight is 610.67.The chemical name of HCT is 6 It is a white, or practically white, crystalline powder which is slightly soluble in water, but freely soluble in sodium hydroxide solution.The empirical formula of HCT is C7H8ClN3O4S2 and its molecular weight is 297.74Chemical structures were illustrated in Figure 1.
From the study of literature, it was found that, various analytical methods like UV, HPLC, and UPLC methods are available for determination of CAN and its impurities in its individual dosage forms [9][10][11][12][13][14]. HPLC method for determining HCT in pharmaceutical dosage forms was reported [15].Different analytical methods using HPLC, HPTLC and UPLC are mentioned for simultaneous determination of CAN and HCT in its combined pharmaceutical dosage forms and in biological samples [16][17][18][19][20][21][22][23] and also HPLC method reported for determination of impurities in CAN and HCT in combined dosage form [24].However, there are no UPLC methods available for the simultaneous determination of CAN, HCT impurities in their fixed dosage forms.Also, drug product of this FDC is official in United States Pharmacopeia.However, the related substances method given by HPLC with a long run time of 70 minutes for monitoring seven impurities.However, the developed method is by UPLC technique for monitoring 13 impurities with a short run time of 26 minutes.Since UPLC offers better selectivity, sensitivity, fast analysis, eco-friendly due to less solvent consumption, RP-UPLC equipment was selected for the simultaneous determination of CAN and HCT impurities in the combination drug product.
Total thirteen specified impurities are present for both CAN and HCT out of which, nine impurities (including two pairs of isomers) related to CAN and four impurities are due to HCT.Structures were given in Figure 2 and Figure 3.
Present scenario in pharmaceutical industry requires fast analytical methods to deliver the products within stringent timelines to meet customer requirements.This can be accomplished by the development of shorter runtime chromatographic methods especially for the estimation of related substances which require longer runtimes to achieve desired separation.Also, these methods should be sensitive enough to detect low level impurities and sufficiently stability indicating for the assessment of shelf life stability for pharmaceutical dosage forms.Hence, it was proposed to develop the related substances method by UPLC to meet the specific targets.Forced degradation studies were conducted to check the stability indicating power of the developed method by producing a degradation profile similar to that what would be observed in a formal stability study under ICH conditions.Forced degradation studies are able to determine the intrinsic stability of drug substance in formulation and useful to establish degradation pathways of drug substances and drug products.These studies can solve stability related problems by differentiating degradation products that are related to drug product from those which are generated from placebo matrix present in the formulation.The present method was validated as per the requirements of United States Pharmacopeia (USP) and ICH [25][26][27][28][29][30][31].

Chemicals and reagents
CAN and HCT drug substances, impurities of CAN and HCT, Atacand HCT (Candesartan cilexetil /Hydrochlorothiazide) tablets were provided by Aurobindo Pharma limited.Acetonitrile of UPLC grade, Perchloric acid and Ortho phosphoric acid of AR grade were purchased from Merck chemicals.Ultrapure water is prepared by using Millipore Milli-Q plus water purification system.

Instrumentation
The experimental work was carried out on Waters-Acquity UPLC system with high pressure binary gradient pump, column oven, Photo diode array detector, Auto injector, Computer with Empower-2 software for data acquisition.The main drug components along with their impurities were separated using Acquity UPLC HSS T3, (100 mm x 2.1 mm id) with 1.8 m particle size column.

Summary of UPLC method optimization
The method is intended for the simultaneous determination of impurities present in both CAN and HCT in their fixed dose combination product.The critical step in the development is to separate both active ingredients along with their respective impurities with satisfactory resolution and at reduced runtime.The developed method should be sensitive to determine the impurities even at lower levels to ensure safety and efficacy of drug product.
From the structures, it was found that, both the drugs are containing functional groups of basic nature.Due to this fact, there will be interaction of these groups with acidic residual silanol groups left in the column.This effect is more prominent while using mobile phase at alkaline pH and leads to peak tailing.To avoid this, it is necessary to acidify the mobile phase to protonate the residual silanols and helps to improve peak symmetry.Also, HCT and its impurities HCT Imp-1 and HCT Imp-2 are highly polar in nature and do not have sufficient retention in the column with buffers such as phosphate and acetate.In order to retain these impurities, mobile phase should contain ion pair reagents such as sodium octane sulfonate.However, there are disadvantages with these long chain ion pair reagents under UPLC system.Also, they require longer equilibration times and not suitable for gradient elution.Considering these facts, trials were taken with 0.1% perchloric acid in water for mobile phase.Perchloric acid is a strong acid and acts as smaller ion pair reagent.It generates perchlorate ion in the mobile phase which will neutralize the positively charged basic polar analytes and helps to retain them.Hence the same was preferred for mobile phase buffer preparation.Acetonitrile was used as organic solvent which is a strong eluent and elutes non polar impurities of CAN.
HCT and Impurities are polar in nature.Impurities of CAN are non-polar in nature and some of the impurities are mid polar in nature.Hence, gradient elution mode was adopted with higher buffer composition for initial gradient steps followed by high percentage of acetonitrile for eluting highly non polar impurity namely CAN Imp-9.Initial flow rate of 0.5 mL.min -1 used to retain the polar impurities of HCT which gradually increased to 0.6 mL.min - 1 to elute late eluting non polar peaks of CAN.After taking different trials, the gradient program (Table 1) which can separate all the impurities with satisfactory resolution was finalized.To have symmetric peak shapes and to reduce the column back pressure, column oven temperature is fixed at 45°C.With these chromatographic parameters, resolution between critical pair of impurities and main drug components is found to be at satisfactory level of more than 1.5.Hence the same were adopted in the finalized methodology.Column selection also plays very important role in the separation of required components ranges from polar, mid polar to nonpolar.Among different columns tried, Acquity HSS T3 (100 mm x 2.1 mm), 1.8 m particle size column shows optimum separation between all the desired peaks.HSS T3 column contains tri functional C18 alkyl phase bonded which provides more retention of polar compounds and compatible to higher compositions of aqueous mobile phase.This T3 column is more effective than conventional end capped columns, and has shown better retention of polar impurities.Hence the same column was finalized for the entire study.
The placebo matrix in the drug product may interact with the impurities which lead to poor recovery of impurities.Considering this issue, diluent pH was kept at acidic side.Since drug components are soluble in organic solvents, a degassed mixture 0.1% ortho phosphoric acid in water and acetonitrile in the ratio of 50:50 v/v is fixed as diluent and found satisfactory solubility for impurities of CAN and HCT.
For better responses of impurities, concentrations of HCT and CAN were fixed at 250 µg/mL and 320 µg/mL respectively.Based on ICH limit for drug products, impurity solutions were prepared at a level of 0.4% for and 0.5% for HCT and CAN impurities respectively and injected along with drug components to check the retention times and relative retention times.(Table 2).
Spectral data obtained from Photo Diode Array detector shows that, majority of impurities of HCT and CAN exhibit wavelength maxima at about 220 nm (Figure 4 and Figure 5).Hence, the same wavelength of 220 nm has been chosen for quantification of impurities.For sufficient area counts, 4 µL injection volume has been chosen and found precise area counts for impurities as well as main drugs.

Preparation of standard solution
Initial Standard stock solution of HCT and CAN (0.4 mg/mL of HCT and 0.5 mg/mL of CAN) was prepared by dissolving in diluent.This stock solution was further diluted to obtain a concentration of 1.28 μg/mL for HCT and 1.6 μg/mL for CAN.All impurities were prepared by initially dissolving in an appropriate amount of acetonitrile, followed by using diluent at desired concentration levels for validation purpose.

System suitability criteria
The below system suitability criteria was adopted from standard solution.
1.The column efficiency as determined from CAN peak is not less than 25000 plate count 2. The Tailing factor for CAN and HCT peaks is not more than 2.0.3. RSD for peak areas of six injections of the standard solution is not more than 5.0%.System suitability results obtained in the finalized method are tabulated in (Table 3).

Reporting of unknown impurities for quantification purpose
a) Specified impurities of CAN and HCT will be reported using respective diluted standard solutions.b) Unspecified impurities of CAN and HCT will be identified from the degradation data of individual drug components of CAN and HCT with placebo reported accordingly.c) Other unspecified impurity for which is not matching with the above data shall be identified by spectra using Photo diode array detector and reported accordingly.d) Any other unspecified impurities which are unaccountable shall be reported using diluted standard of lower label claim component (i.e., HCT).

Analytical method validation
Atacand HCT (Candesartan cilexetil /Hydrochlorothiazide) tablets 32 mg/25 mg has been considered for the method validation of developed method.The validation parameters such as Specificity, Forced degradation, Precision, Ruggedness, Sensitivity (Limit of detection and Limit of Quantification), Linearity, Range, Accuracy, solution stability and Robustness were performed as per ICH general recommendation.

Specificity and Stress studies
The specificity of the related substances method was evaluated by injecting diluent, placebo used in sample matrix, standard and sample with thirteen potential impurities in presence of Atacand HCT tablets at a test concentration of 320 µg/mL for CAN and 250 µg/mL for HCT and its corresponding degradation products (Figure 6a to 6f).The stress conditions used for degradation study are Acid hydrolysis (1 N HCl / 5 mL/ 25°C / 30 minutes), Base hydrolysis (1 N NaOH / 5 mL / 25°C / Immediately), Oxidation (30% H2O2 /5 mL/ 85°C / 60 minutes), Thermal (105°C /48 hours), Humidity (95%RH / 48 Hours) and Photolytic (white fluorescent 1.2 million lux hours UV 200 watt hr/m 2 for 7 days).

Precision and Intermediate precision
The precision of the method was checked by injecting six individual preparations of CAN/HCT tablets spiked with 0.4% level for HCT-Imp-1,2,3 and 4; 0.5% for CAN-Imp-1, 2, 3, 4, 5&6 (two isomers), 7&8 (two isomers) and 9 against sample test concentration of 250 µg/mL and 320 µg/mL for HCT and CAN respectively.The percentage RSD for % w/w of each impurity is calculated.The intermediate precision (Ruggedness) of the method was

Sensitivity
For the establishment of Limit of detection (LOD) and Limit of Quantitation (LOQ) levels, a series of test solutions were prepared from 1 to 150% with respective impurity specification level by diluting the impurity stock solution to the required concentration.Linearity curves were drawn by plotting concentration versus area of the individual impurity.From these plots, LOD and LOQ were predicted from the formulae 3.3/S and 10/S respectively where  is the standard deviation of the response and S is the slope of the linearity curve.Precision was performed at predicted LOD and LOQ values and finalized the levels.

Linearity and Range
Linearity curves were plotted from the finalized LOQ level to 150% of the impurity specification level.The correlation coefficient, slope and Y-intercept of the Linearity curve are calculated for each impurity.

Accuracy
A known amount of the impurity stock solutions were spiked to the samples at LOQ concentration, 50%, 100% and 150% of the analyte concentration.The % w/w of recoveries for all the impurities was calculated.Each concentration level is prepared for triplicate preparation.

Solution Stability
In order to demonstrate the stability of both reference and sample solutions, these solutions were injected immediately after preparation and at periodical intervals by maintaining at room temperature or cooler temperature.

Robustness
To determine the robustness of the developed method, experimental conditions are deliberately changed and the impact of the variation on each impurity was evaluated.The flow rate of the mobile phase is 0.50 and 0.60 mL/min.To study the effect of flow rate ± 0.05 mL/min unit was changed i.e., 0.45 & 0.55 mL/min and 0.55 & 0.65 mL/min.The effect of column temperature (actual 45°C) is studied at 40°C and 50°C.For gradient programme variation, the composition of mobile phase-B was changed by ± 2% absolute.For wavelength variation, ± 5 nm was changed from the working wavelength i.e., 220 nm.

Specificity and Stress studies
Stress studies on Candesartan cilexetil and Hydrochlorothiazide tablets under different stress conditions suggested the following degradation behavior.(Figure 6g to 6l and Table 4a & Table 4b).

Acid stress
In Acid hydrolysis stress condition, CAN Imp-4 and HCT imp-1 were formed in significant levels.

Alkaline stress
In base hydrolysis stress condition, CAN Imp-1 and HCT imp-1 were formed in significant levels.

Oxidative stress
In Peroxide stress condition, CAN Imp-4 formed in low level.HCT not degraded in this condition.

Thermal stress
In thermal degradation, CAN Imp-5&6 impurity formed at higher levels.Significant levels of CAN Imp-7&8, CAN Imp-4 and CAN Imp-1were also generated.HCT significantly degraded to HCT Imp-1.

Humidity and Photolytic stress
There is no significant degradation observed for CAN and HCT.

Robustness
Close observation of analysis results for deliberately changed chromatographic conditions such as Flow rate, column temperature, wave length and change of organic component in gradient programme revealed that there is no significant change observed in the relative retention times of the main analyte and their corresponding impurities illustrating the robustness of the developed method.

CONCLUSION
The proposed RP-UPLC method enables the separation and simultaneous quantitative determination of specified and unspecified impurities of CAN and HCT in CAN/HCT tablets.The developed method is validated as per ICH requirements.The stress studies indicated that method is selective and stability indicating.UV detection at 220 nm was found to be suitable without any interference from excipients.All the calibration curves obtained were found to linear with values of correlation coefficients greater than 0.995.LOD and LOQ values are well below the specification limits.Recovery tests confirmed the accuracy of the method.The proposed RP-UPLC method is fast, precise, accurate, sensitive and efficient.

Figure 3 .
Figure 3. Structures for impurities of HCT Weighed and crushed not less than 10 tablets.Transferred an accurately weighed portion of sample powder, equivalent to about 32 mg of Candesartan cilexetil into a 100 mL clean, dry volumetric flask, added about 70 mL of diluent and sonicated for about 20 minutes, at room temperature (25°C), with intermediate shaking.Diluted to volume with diluent and mixed.Filtered the sample solution through a 0.22 μ filter (Millipore PVDF or Nylon mdi).

Figure
Figure 5. Spectra for CAN impurities

Figure 6 .
Typical chromatograms of diluent, placebo, standard solution, control sample

(e )
Impurity mixture (f) Sample spiked with impurities (g) Acid stress sample (h) Alkaline stress sample Figure 6.(continued) Typical chromatograms of impurity mixture, spiked, stress sample

Table 1 .
Gradient program

5 .
Spectra for CAN impurities

Table 3 .
Chromatographic system suitability data

Table 4a .
Summary of forced degradation study for CAN

Table 4b .
Summary of forced degradation study for HCT