Development and validation of a dissolution test with reversed-phase high performance liquid chromatographic analysis for Candesartan cilexetil in tablet dosage forms

1 Introduction

Candesartancilexetil (CC)is a commercially available antihypertensive prodrug containing one chiral center at the cyclohexyloxycarbonyloxy ethyl ester group (Ross and Papademetriou, 2004) (Fig. 1). In the gastrointestinal (GI) tract CC is rapidly absorbed and completely bioactivated by ester hydrolysis at the ester link, converted to active candesartan (McClellan and Goa, 1998). An important clinical application of CC is an angiotensin receptor blocker with insurmountable binding properties to the angiotensin-1 receptor, long duration of action and improved efficacy (Joost et al., 2011). Furthermore, it is a white to off-white crystalline powder with a molecular mass of 61kDa. The solubility nature in benzyl alcohol is 0.3M, and is insoluble in water (<8 × 10^–8M). The partition coefficient (C_octanol/C_aqueous) at pH1.1, 6.9 and 8.9 is >1000 indicating high hydrophobicity character (ATACAND®). It has a pK_a value of 6.0 (Cagigal et al., 2001).

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Figure 1

Chemical structure of Candesartan cilexetil.

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Analytical method validation is a process to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Food and Drugs Administration (FDA) regulations such as Good Manufacturing Practice (GMP), Good Laboratories Practice (GLP), Good Clinical Practice (GCP) and quality standards such as International Organization for Standardization (ISO17025) require analytical methods to be validated before and during routine use (Physicians’ Desk Reference®, 2009). Method validation is defined as the process of proving through scientific studies that an analytical method is acceptable for its intended use. USP provides regulatory guidance for method validation (FDA, 2000) and recent guidelines for methods development and validation for new non-compendial test methods are provided by the FDA draft document (USP 25-NF 20, 2002).

Developing dissolution methods for poorly soluble compounds has been a consistent challenge for the pharmaceutical scientist. Because of inherently slow dissolution, poorly soluble compounds are good candidates for developing in vitro and in vivo correlations (IVIVCs) if intestinal permeability is high and drug dissolution is the controlling mechanism for the release of drug from the dosage form (ICH, 2000). Drug absorption from a dosage form after oral administration depends on the release of the drug from the pharmaceutical formulation, the dissolution and/or its solubilization under physiological conditions and the permeability across the gastrointestinal tract. Because of the critical nature of the first two of these steps in vitro dissolution may be relevant to the prediction of in vivo performance (Amidon et al., 1995; Emami, 2006).

Over the past decade several published literatures reported that there is no validated method for dissolution and moreover no official monograph was available for CC. Parameters to set up the dissolution test should be researched and defined for the drugs that do not possess official monographs (Emami, 2006). Importantly, the present work describes the development and validation of an accurate and reliable RP-HPLC method for the estimation of CC release in solid dosage form. The best dissolution conditions were used to evaluate the development and validation of a dissolution method and this method was used to evaluate the dissolution profile of three different batches of tablets.

2 Experimental

3 Method

4 Results and discussion

4.1

4.1 Optimization of dissolution test conditions

The accomplishment of dissolution profile is recommended as a support in the development and optimization of drug formulation as well as in the establishment of in vitro/in vivo correlation. When dissolution test is not defined or if the monograph is not available, comparison of drug dissolution profiles is recommended on three different dissolution mediums (pH1–7.5). In vitro dissolution was used to perform the release rate of drug products and to assure the quality of solid dosage forms by the pharmaceutical industry and regulatory agencies (ICH (Q2R1), 2007). The sink conditions are determined and expressed as a percentage of drug released. Purified water, 0.1N hydrochloric acid, acetate buffer (pH1.2–2.2) and phosphate buffer (pH4.5–7.2) were used as dissolution medium and selected on the basis of solubility and screening study. From the above study the phosphate buffer (pH7.2) provided highest drug release profile with greater stability, ensured excellent sink conditions and was selected as the best dissolution medium. The drug release profiles are shown in Fig. 2. Based on the solubility and screening study, phosphate buffer (pH7.2), was selected as the dissolution medium and USP type 2 rotating paddle apparatus at 50rpm as an instrument. In these conditions, typical acceptance criteria for the amount of drug dissolved were in the range of 65–83%. In the present study, the percentage of drug released for all three different products were >80% in 60 min (Fig. 3) and the suggested acceptance criteria can be 75 (Q) in 60 min. The stability test indicated that CC is stable in the dissolution medium at room temperature and at 37.0 ± 0.5°C for 24 and 2h, respectively. The results obtained from the initial and final response factors were within the acceptable range and not much difference between the stability and freshly prepared solutions.

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Figure 2

The cumulative release curves of Candesartan cilexetil from tablets at different medium.

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Figure 3

In vitro release profile (n = 6) of three batches of Candesartan cilexetil tablet.

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4.2

4.2 Analytical method validation

In the current study, RP-HPLC method was used to determine the percentage drug release. HPLC is used to separate, identify and determine the concentration of a specific component in a mixture, moreover that this method is very fast, reproducible and easy to operate (Pharmacopoeial Forum, 2004). The developed method was validated to meet the requirements for a global regulatory filing. The validation parameters such as precision, linearity, specificity, accuracy, limit of quantitation and ruggedness were carried out in accordance with ICH and US Pharmacopoeia guidelines.

4.2.1

4.2.1 Linearity

The linearity of CC response is evaluated from the range of 2.0–20.0mcg/mL and showed a good correlation coefficient (r²)=1.0. To validate linearity, the standard curve of CC was constructed by plotting concentration (mcg/mL) versus area response (mAU) which is shown in Fig. 4. The linear regression and slope were calculated and are shown in Tables 1–3.

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Figure 4

Linearity graph of candesartan cilexetil.

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Table 1 Linear regression of Candesartan cilexetil.

Concentration (mcg/mL)	Areaa (average)	Standard deviation	RSD
2	4,360,646.3	26,083	0.598
4	8,762,327.3	3492.8	0.04
8	17,528,692.7	7573.4	0.043
12	26,288,297.3	5351.3	0.02
16	35,063,016.3	19,091.7	0.054
20	43,809,040.7	4709.3	0.011

a Average of five determinations.

Table 2 Calculation of regression line.

X (concentration in μg/mL)	Y (area obtained in mAU)a	XY	X2	Y2
2	4,360,646.3	8,721,292.6	4	1.90152E+13
4	8,762,327.3	35,049,309.2	16	7.67784E+13
8	17,528,692.7	140,229,541.6	64	3.07255E+14
12	26,288,297.3	315,459,567.6	144	6.91075E+14
16	35,063,016.3	561,008,260.8	256	1.22942E+15
20	43,809,040.7	876,180,814.0	400	1.91923E+15

a Average of five determinations.

Table 3 Slope calculation.

X axis	Y axisa	Y intercept	Slope
2	4,360,646.3	9862	2,185,254.2
4	8,762,327.3	9862	2,193,047.3
8	17,528,692.7	9862	2,192,319.3
12	26,288,297.3	9862	2,191,513.3
16	35,063,016.3	9862	2,192,054.9
20	43,809,040.7	9862	2,190,945.1

a Average of five determinations.

4.2.2

4.2.2 Specificity

Specificity is carried out with placebo solution and compared with the standard preparation. The drug release of CC in the dissolution medium was measured at 215nm and the run time was extended up to 30 min. Two peaks were observed in the chromatogram, the major and minor peaks retention time was 2.2 and 1.3, respectively. The primary peak was due to polysorbate 80 and was confirmed by correlating with the blank peak. The main peak was well separated from the blank peak and the resolution between these two peaks was more than 2 (Fig. 5). There were no other additional peaks observed which indicate no interferences by excipients and thus demonstrating that the proposed method is specific for the analysis of CC.

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Figure 5

Standard chromatogram of Candesartan cilexetil.

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4.2.3

4.2.3 Precision

The precision of an analytical procedure expresses the closeness of the agreement (degree of scatter) between a series of measurements obtained from the multiple samples of the same homogeneous sample under the prescribed conditions. Repeatability is a measure of the precision under the same operating conditions over a short interval of time and it is also known as intra assay precision. A minimum six determinations at 100% of the standard concentration were tested to find out the average, standard deviation and related standard deviation, and all the calculated parameters were well within the prescribed limit. Intra-day precision and intermediate precision were done for ensuring the robustness of the method. The related standard deviation (RSD) of both the tests was well within the desirable limit of NMT 1.8% which is clearly indicated that the developed method is robust. Intraday and intermediate precision results are shown in Table 4.

Table 4 Precision, intermediate and intraday precision of Candesartan cilexetil.

Replicates	Chemist-I (area)a	Chemist-II (area)a	Column-I (area)a	Column-II (area)a
Area-1	13,773,622	13,765,439	13,773,622	13,975,643
Area-2	13,991,726	13,756,393	13,991,726	13,759,234
Area-3	13,874,337	13,756,394	13,874,337	13,759,320
Area-4	13,705,374	13,850,324	13,705,374	13,856,290
Area-5	13,553,809	13,786,439	13,553,809	13,822,233
Area-6	13,571,089	13,745,633	13,571,089	13,944,752
Average	13,744,993	13,776,770	13,744,993	13,852,912
Standard deviation	171,341.9	38,554	171,341.9	91,635
RSD	1.247	0.2798	1.247	0.6615

a Average of five determinations.

4.2.4

4.2.4 Accuracy

The accuracy of an analytical procedure is the closeness of agreement between the values that are accepted either as conventional true values or an accepted reference value. Accuracy is usually reported as percent recovery by an assay using the proposed analytical procedure of known amount of analyte added to the sample. The ICH also recommended assessing a minimum of three determinations over a minimum of three concentration levels covering the specified range. The common method of determining accuracy is to apply the analytical procedure to the drug substance and to be quantitated against the reference standard of known purity. The range for the accuracy limit should be within the linear range. Typical accuracy of the recovery of the drug substance in the mixture is expected to be about 98–102%. Values of accuracy of the recovery data beyond this range are to be investigated. The precision concentration was 10mcg/mL, hence the linearity range was selected from 7 to 14mcg/mL. The known concentration of (20%, 40%, 60%, 80% and 100%) were added to the standard preparation (7mcg/mL). The percentage recoveries obtained were considered under the acceptable range as per the ICH guidelines (Table 5).

Table 5 Accuracy of Candesartan cilexetil.

Solution	Quantity added (known %)	Areaa	Average area	Standard deviation	RSD	Recovery	Actual value	Accuracy
Soln-1	100 (7mcg/mL)	9,629,430	9,654,878	43,944.8	0.455	–	–	–
		9,629,583
		9,705,621
Soln-2	120 (8.4mcg/mL)	11,583,211	11,572,808	12,646.94	0.109	119.86	120	99.89
		11,558,731
		11,576,483
Soln-3	140 (9.8mcg/mL)	13,588,943	13,744,906	150,576.3	1.096	142.36	140	101.69
		13,756,329
		13,889,445
Soln-4	160 (11.2mcg/mL)	15,234,754	15,425,980	182,200.9	1.181	159.77	160	99.86
		15,597,564
		15,445,623
Soln-5	180 (12.6mcg/mL)	17,123,893	17,347,168	234,157.7	1.350	179.67	180	99.82
		17,326,740
		17,590,870
Soln-6	200 (14mcg/mL)	19,345,786	19,540,023	317,808.7	1.626	202.39	200	101.19
		19,367,501
		19,906,783

a Average of three determinations.

4.2.5

4.2.5 Limit of quantitation

Limit of quantitation (LOQ) is the lowest amount of analyte in a sample that can be determined with acceptable precision and accuracy under stated experimental conditions. The quantitation limit is expressed as the concentration of analyte in the sample. The standard deviation and related standard deviation for the limit of quantitation were 15,686.2 and 1.1067%, respectively, which was well within the desirable limit of not more than 2.0%. The lowest quantifiable concentration was 1mcg/mL and this parameter can be used for predicting the drug release in low dose formulation.

4.2.6

4.2.6 Ruggedness

Intraday and intermediate precision were determined by analyzing the solutions by two different analysts, using different instruments, using multiple lots of column, in two different labs and on different days. The percentage RSD obtained under different conditions was below 2%. Table 4 represents the intermediate and intraday precision.

5 Conclusion

The simple, sensitive and inexpensive isocratic RP-HPLC method was developed to determine the percentage drug release of CC tablets. The dissolution study showed that CC has good stability and the percentage drug released was satisfactory for all the evaluated batches from the formulation. The validation results show that the method is specific, accurate, linear, precise, rugged and robust. The run time is relatively short (5.0min) which enables rapid quantification of many samples in routine analysis. Therefore this method is proposed for the quality control studies of CC modified and conventional pharmaceutical dosage forms contributing to assure the therapeutic efficacy of the drug.