Application of central composite design and structural analysis to the optimization of HPLC method for simultaneous analysis of hydrochlorothiazide,amlodipine besylate and valsartan

Abstract

Central composite experimental design was used for fast, simple, and accurate high-performance liquid chromatography (HPLC) determination of hydrochlorothiazide, amlodipine and valsartan in combined dosage forms. This method avoids the disadvantages of the traditional analytical approach, which is time-consuming, involves a large number of runs, and does not allow establishing the multiple interacting parameters. On the basis of preliminary experiments and physicochemical characteristic of analyzed substances, three independent variables (methanol content, pH of the mobile phase, and column temperature) were selected as input, while as dependent variables, six responses (retention time of hydrochlorothiazide, retention time of amlodipine, retention time of valsartan, asymmetry of hydrochlorothiazide peak, asymmetry of amlodipine peak, and asymmetry of valsartan peak) were chosen. Face centered central composite design enables an estimation of factors which have the most importance. After optimizing experimental conditions, a separation was conducted on a Zorbax C₈ (150 mm×4.6 mm, 5 μm) column with a mobile phase consisting of methanol-acetonitrile-acetate buffer 40:20:40 (v/v/v), pH adjusted to 3.5 with acetic acid, flow rate of 1 mL/min and column temperature of 40 ^degC. The method was successfully applied to the simultaneous separations of these active drug compounds in their commercial dosage forms.

Keywords

HPLC central composite design hydrochlorothiazide amlodipine valsartan

1 Introduction

Recent guidelines for the treatment of hypertension have focused on the need for multiple medications to get most patients to goal blood pressure (BP). Two to three different classes of antihypertensive agents are frequently required, increasing the risk of poor compliance with therapy. Hence, the guidelines have recommended starting with combination therapy in patients with BP that is over 20 mm Hg systolic or 10 mm Hg diastolic above goal. The latest advance in treatment regimen has been the development of triple-therapy combinations of an angiotensin receptor blocker, amlodipine and hydrochlorothiazide [1, 2]. Hydrochlorothiazide [HCT, 6-chloro-1,1-dioxo-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide, Fig. 1 (a)] is a diuretic drug of the thiazide class, frequently used for treatment of hypertension and congestive heart failure. It reduces blood volume by acting on the kidneys to reduce sodium resorption in the distal convulated tubule, resulting in a decrease of blood pressure. Amlodipine [AML, (RS)-3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate, Fig. 1 (b)] is a long-acting calcium channel blocker which belongs to dihydropyridine (DHP) class, used as an antihypertensive and in the treatment of angina pectoris. Amlodipine acts by relaxing the smooth muscle in the arterial wall, decreasing total peripheral resistance and hence reducing blood pressure. Valsartan [VAL, (S)-3-methyl-2-(N-{[2’-(2H-1,2,3,4-tetrazol-5-yl)biphenyl-4-yl]methyl}pentanamido) butanoic acid, Fig. 1 (c)], is an angiotensin II receptor (AT1) antagonist. Angiotensin II receptor antagonists represent a relatively new pharmacological class [3] which acts mainly by selective blockade of AT1 receptors and reduces the effects of angiotensin II. By blocking the action of angiotensin, valsartan dilates blood vessels and reduces blood pressure.

Fig.1

Structural formulae of HCT (a), AML (b) and VAL (c).

There are many manuscripts focusing on the determination of HCT, AML and VAL only or in combination with other drugs [4 –21]. Several published papers describe the simultaneous determination of binary combinations HCT, AML and VAL [22 –25]. Galande et al. [26] describe estimation of all three drugs in combination by UV Spectrophotometry. The literature revealed only one paper dealing with the determination of HCT, AML and VAL by HPLC in human plasma [27]. Neither of the already published methods reports HPLC method for simultaneously determination all three components in a solid dosage form.

Central composite design (CCD) and response surface methodology (RSM) have been applied to obtain precise, simple and rapid HPLC method. Face centered CCD enables an estimation of factors which have the most importance. RSM is generally employed in order to provide a description of the response pattern in the region of the studied observations and to assist in finding the region where the optimal response occurs [28 –33]. CCD requires a minimum number of experiments to be performed and has been widely used for fitting a second-order model. The independent variables including methanol content, pH of mobile phase and column temperature were defined as factors and retention times and asymmetries peaks of HCT, AML and VAL (t_R _HCT , t_R _AML , t_R_VAL , As _HCT, As _AML and As _VAL) were used separately as the responses in the CCD.

A market tablet formulation (ExforgeHCT, Novartis GmbH, Wien, Austria) containing 25 mg HCT, 10 mg AML and 160 mg VAL was analyzed. The significant feature of this combination lies in the fact that hydrochlorthiazide and amlodipine are present in minute amounts compared to valsartan, which makes an analysis more complicated and tedious. In the present paper, a fast, simple and accurate HPLC method has been proposed without the tedious extraction procedure.

2 Experimental

2.1 Chemicals and reagents

The hydrochlorothiazide, amlodipine and valsartan standards (HCT = 101.12%, AML = 100.97%, VAL = 100.13%), ExforgeHCT tablets (Noavartis GmbH, Wien, Austria), consisting of 25 mg HCT, 10 mg AML and 160 mg VAL, were used. All solvents: methanol, acetonitrile and sodium-acetate (purchased by Chromosol, Sigma-Aldrich, Munich, Germany), and acetic acid (Fluka, Eindhoven, The Nederlands) were of a grade suitable for high-performance liquid chromatography analysis.

2.2 Preparation of standard solutions

Stock solutions were prepared by dissolving standard substances in mobile phase to obtain concentrations of 0.25 mg/mL for HCT, 0.1 mg/mL for AML and 1.6 mg/mL for VAL. Solutions for method optimization were prepared by dissolving stock solutions in mobile phase in order to obtain a concentration of 0.025 mg/mL for HCT, 0.01 mg/mL for AML and 0.16 mg/mL for VAL. Standard solutions for linearity testing for the calibration curves were a series of ten solutions prepared in the concentration range from 0.0125 to 0.125 mg/mL for HCT, 0.005 to 0.05 mg/mL for AML and 0.08 to 0.8 mg/mL for VAL. Solutions for accuracy testing containing placebo were prepared as the laboratory mixture of solutions of HCT, AML and VAL in the ratio related to the investigated tablets. For quantitative analysis, three solutions corresponding to 80, 100 and 120% of the label amount were prepared. To estimate precision, three series (0.025, 0.05, and 0.075 mg/mL for HCT; 0.01, 0.02, and 0.03 mg/mL for AML, 0.08, 0.16, and 0.24 mg/mL for VAL) were prepared with ten solutions for each of the concentrations.

2.3 Sample solutions

A tablet mass corresponds to 25 mg HCT, 10 mg AML and 160 mg VAL (ten tablets were accurately weighed and ground to fine powder) was dissolved in a 100-mL volumetric flask with mobile phase, placed into an ultrasonic bath, and filtrated. Filtrate (5 mL) was diluted with mobile phase to 50 mL.

2.4 Apparatus and software

The HPLC analyses were done by using a Shimadzu chromatographic system, consisting of a Shimadzu HPLC system (Shimadzu Technologies, Kyoto, Japan), a pump (LC-20AD VP), UV-Visible detector (SPD-20AV VP), column oven (CTO-20AC VP), column Zorbax C₈ (150×4.6 mm, 5 μm), degasser system (DGU-20AS) and injector with a 10 μL sample loop. Experimental design, structural analysis, statistical analysis and calculation of desirability function were performed by using Marvin Sketch 5.8.2 (Chem Axon Ltd., Somerville, MA, USA, and Budapest, Hungary) and Design-Expert 8.0.7.1 (Stat-Ease Inc.).

2.5 Procedure

Separations were performed on a Zorbax C₈ column (150 mm×4.6 mm, 5 μm particle size) with detection at 254 nm. Mobile phases were prepared according to the experimental plan given in Table 1. The resulting mobile phases were degassed and vacuum filtered through a 0.45-μm membranes filter (Alltech Associates, Lokeren, Belgium).

Table 1
CCD design and results of experiments

Factors A B C Results

Run % Methanol pH T (C) t _RHCT t _RAML t _RVAL As_HCT As_AML As_VAL

1 30 2.5 30 1.71 5.05 14.6 1.3 1.05 0.91

2 50 2.5 30 1.56 1.89 3.11 1.55 1.27 1.11

3 30 4.5 30 1.78 8.69 18.3 1.25 0.97 0.93

4 50 4.5 30 1.56 2.52 2.4 1.44 1.15 1.15

5 30 2.5 50 1.63 3.75 9.87 1.25 1.01 0.82

6 50 2.5 50 1.57 1.69 5.68 1.4 1.3 1.1

7 30 4.5 50 1.67 5.98 11.87 1.31 0.87 0.84

8 50 4.5 50 1.52 2.2 2.45 1.34 1.18 1.08

9 30 3.5 40 1.73 3.79 15.32 1.19 1.19 1.17

10 50 3.5 40 1.56 1.83 4.21 1.4 1.26 1.22

11 40 2.5 40 1.6 3.13 6.8 1.31 1.16 0.9

12 40 4.5 40 1.57 3.6 3.46 1.4 1.16 1.19

13 40 3.5 30 1.65 4.16 6.96 1.23 1.09 1.24

14 40 3.5 50 1.59 3.23 4.72 1.25 1.07 1.14

15 40 3.5 40 1.62 3.16 6.02 1.27 1.07 1.39

16 40 3.5 40 1.62 3.75 5.58 1.24 1.06 1.21

17 40 3.5 40 1.61 3.14 6.93 1.24 1.02 1.37

18 40 3.5 40 1.62 2.84 6.02 1.24 1.12 1.36

19 40 3.5 40 1.62 3.75 6.92 1.14 1.13 1.23

20 40 3.5 40 1.59 3.23 6.02 1.25 1.11 1.19

Factors	A	B	C	Results
1	30	2.5	30	1.71	5.05	14.6	1.3	1.05	0.91
2	50	2.5	30	1.56	1.89	3.11	1.55	1.27	1.11
3	30	4.5	30	1.78	8.69	18.3	1.25	0.97	0.93
4	50	4.5	30	1.56	2.52	2.4	1.44	1.15	1.15
5	30	2.5	50	1.63	3.75	9.87	1.25	1.01	0.82
6	50	2.5	50	1.57	1.69	5.68	1.4	1.3	1.1
7	30	4.5	50	1.67	5.98	11.87	1.31	0.87	0.84
8	50	4.5	50	1.52	2.2	2.45	1.34	1.18	1.08
9	30	3.5	40	1.73	3.79	15.32	1.19	1.19	1.17
10	50	3.5	40	1.56	1.83	4.21	1.4	1.26	1.22
11	40	2.5	40	1.6	3.13	6.8	1.31	1.16	0.9
12	40	4.5	40	1.57	3.6	3.46	1.4	1.16	1.19
13	40	3.5	30	1.65	4.16	6.96	1.23	1.09	1.24
14	40	3.5	50	1.59	3.23	4.72	1.25	1.07	1.14
15	40	3.5	40	1.62	3.16	6.02	1.27	1.07	1.39
16	40	3.5	40	1.62	3.75	5.58	1.24	1.06	1.21
17	40	3.5	40	1.61	3.14	6.93	1.24	1.02	1.37
18	40	3.5	40	1.62	2.84	6.02	1.24	1.12	1.36
19	40	3.5	40	1.62	3.75	6.92	1.14	1.13	1.23
20	40	3.5	40	1.59	3.23	6.02	1.25	1.11	1.19

3 Results and discussion

3.1 Structural analysis

The presence of several functional groups in the molecular structures, such as biphenyl, imidazole, dihydropyridine, and benzene (Fig. 1), makes a reversed-phased (RP)-HPLC method with SPDA detection suitable for the determination. Since the RP-HPLC method is based on use of a polar mobile phase, a complete description of the ionization profile of the examined substances was used for the evaluation of retention behavior and also for the separation.

The degree of ionization of the drug strongly affects solubility and retention. Additionally, the knowledge of dissociation constant of ionisable compounds at different pH values and the solvent composition is also significant to determine the optimal separation conditions in reversed phase liquid chromatography (RP-LC).

As it can be seen, HCT has three pKa values (9.09, sulfonamide group; 9.83 cyclic sulfonamide; 11.31, secondary amine), AML has two pKa values of nitrogen based groups (4.16, dihydropyrimidine nitrogen; 9.45, amine nitrogen) and VAL has two pKa values (4.37, carboxyl group; 7.40, tetrazole nitrogen) (Fig. 2). Considering the chemical structures and pKa values, it is possible to assume ionized structures of HCT, AML and VAL. Having in mind the acid-base properties of HCT, AML and VAL, the pH interval from 2.5 to 4.5 was chosen for further investigation. At the pH values from 2.5 to 4.5 molecules are present in its ionizing or non-ionizing various forms. Major dominant species of HCT and VAL at pH 2.5 and 3.5 is non-ionized structure form, while two nitrogens in AML are ionized (Fig. 3).

Fig.2

pKa values of functional groups of HCT, AML and VAL.

Fig.3

Major dominant molecular species at pH 2.5 and pH 3.5.

At maximum pH value 4.5 HCT has still been in non ionizing form, AML has only protonated amine nitrogen, and VAL carboxyl group is ionized (Fig. 4).

Fig.4

Major dominant molecular species at pH 4.5.

To predict the order of peak appearance on chromatogram, logP was analyzed for HCT (logP = –0.58), AML (logP = 1.64) and VAL (logP = 5.27). Several papers report the effect of logP on the retention time of substance in RP-LC. Papers report that an increase of logP leads to an increase of logt_R [34, 35].

3.2 Optimization of HPLC Conditions

The objective of the optimization was to perform a screening of the factors that could potentially influence chromatographic retention, thus the independent variables were defined during the preliminary study. Some chromatographic parameters, such as flow rate were excluded as its influence can usually be predicted by common chromatographic theory knowledge. The factors generally selected to optimize the chromatographic separation of ionisable compounds are pH and the content of organic solvent of the mobile phase. The variations of these parameters induce a variation of the degree of ionization and thus affect chromatographic behavior. In addition, column temperature affects retention behavior, thus three factors-independent variables were selected as inputs: methanol content, pH of the mobile phase and column temperature. CCD has been used for fitting a second-order model. Since a good separation is characterized by good resolution, run time is very important (from a practical point of view) and since the shape of the peak is determined by its asymmetry, eight responses were chosen as dependent variables: retention time of hydrochlorothiazide (t_RHCT), retention time of amlodipine (t_RAML), retention time of valsartan (t_RVAL), asymmetry of the hydrochlorothiazide peak (As_HCT), asymmetry of the amlodipine peak (As_AML), asymmetry of the valsartan peak (As_VAL) and resolution (Rs_AML and Rs_VAL). Retention parameters were measured using Class VP software with the USP computing option selected.

On the basis of structures, physicochemical characteristics of these three molecules and composition of mobile and stationary phase it is possible to assume the following order of retention: HCT, AML and VAL. The retention of a substance is a function of the volume fraction of the organic modifier in the mobile phase. Taking into account the variation of the retention factors of compounds with polarity of the mobile phase, a range of methanol concentrations from 30% to 50% was selected for investigation.

The analysis of HCT, AML and VAL was started on a non-polar stationary phase (Zorbax C8 column, 150 mm×4.6 mm, 5 μm particle size) with the mobile phase consisting of methanol-acetonitrile- acetate buffer (pH of mobile phase was adjusted to 4.0 with acetic acid). The column temperature was set at 25 °C and the flow rate at 1 mL/min. Separation was acceptable with methanol-acetonitrile-acetate buffer mixtures ranging from 30:20:50 to 50:20:30 (v/v/v), but peak shape and run time needed to be improved (Fig. 5).

Fig.5

Chromatogram before optimization.

In order to evaluate the effect of the most important factor, a face centered central composite design (CCD) which includes six replicates at the zero level was chosen. A central composite design runs needed 20 runs to complete a whole design. As dependent variables, eight responses were chosen: retention time of hydrochlorothiazide (t_RHCT), retention time of amlodipine (t_RAML), retention time of valsartan (t_RVAL), asymmetry of the hydrochlorothiazide peak (As_HCT), asymmetry of the amlodipine peak (As_AML), asymmetry of the valsartan peak (As_VAL) and resolution (Rs_AML and Rs_VAL).

Since the peak resolution of AML and VAL was greater than 2.0 on preliminary chromatogram in all further runs this parameter has been excluded (all runs meet the criteria for peak resolution (Rs > 1.5) and do not need further optimization of this dependent variable). Retention time of non-retained peak (t₀) was determined by injection of methanol and the k value of HCT was calculated. The obtained result was greater than 1 (not shown in chromatogram). At t₀ mobile phase has a small response and cannot be seen on chromatograms. The matrix of experiments and results obtained as an average value of three runs are presented in Table 1.

Appropriate calculations were done with Design-Expert 8.0.7.1 software (Stat-Ease Inc. Minneapolis, MN, USA). A quadratic interaction model was suggested for the relationship between input and output except for As_AML where linear model was suggested. Quadratic model interaction is presented by Equation (1):

$\begin{matrix} y = b_{0} + b_{1} A + b_{2} B + b_{3} C + b_{12} AB + b_{13} AC + b_{23} BC + b_{11} A^{2} \\ + b_{22} B^{2} + b_{33} C^{2} \end{matrix}$ (1) where b ₀ is the intercept, b _i (b ₁, b ₂, b ₃), bi _j (b ₁₂, b ₁₃, b ₂₃) and b _ijk represent the regression coefficients, and A, B, and C represent independent variables. The calculated coefficient and model of polynomial regression is presented in Table 2.

Table 2

The estimates of the coefficients for CCD regression models

Coefficients	t _RHCT	t _RAML	t _RVAL	As_HCT	As_AML	As_VAL
b₀	1.64	3.21	6.21	1.24	1.11	1.27
b₁	–0.075	–1.71	–5.21	0.083	0.11	0.099
b₂	–	0.75	–0.16	–	–0.046	0.035
b₃	–0.028	–0.55	–1.08	–0.022	–0.01	–0.036
b₁₂	–0.02	–0.59	–1.2	–0.022	–	–
b₁₃	–0.02	0.44	1.72	–0.032	–	0.013
b₂₃	–	–0.19	–0.53	0.02	–	–
b₁₁	0.032	–0.24	3.62	0.038	–	–0.044
b₂₂	–0.028	0.32	–1.01	0.098	–	–0.19
b₃₃	–	0.65	–0.3	–0.017	–	–0.049

CCD provides a precise estimation of the experimental errors and the measure of the adequacy of the models (lack of fit). The results were analyzed by the analysis of variance (ANOVA) method, and the results are presented in Table 3.

Table 3

Statistical parameters of model obtained by ANOVA

	_SSmodels	SS_models/df	F	p	Lack of fit	r²	r²_adj
t _RHCT	0.076	0.0084	45.25	0.0001	0.3263	0.976	0.9545
t _RAML	45.26	5.0289	16.12	0.0001	0.0893	0.9355	0.8775
t _RVAL	365.55	40.62	53.05	0.0001	0.0758	0.9795	0.9610
As_HCT	0.15	0.017	7.2	0.0024	0.3928	0.8662	0.7459
As_AML	0.14	0.047	10.28	0.0005	0.1081	0.6585	0.5945
As_VAL	0.45	0.05	5.28	0.0079	0.3865	0.8262	0.6698

The lack-of-fit test was determined by performing a Fischer F test. The high value of F with a very low probability (only model terms with corresponding p-value less than 0.05 are significant at 95% confidence level) implies that there was no evidence of the models lack of fit (lack of fit value is not significant) and the models could be accepted as an adequate representation of the data. In addition, the values of r ² and adjusted r ² taking into account the degrees of freedom indicated that the regression model fits the data well. The data collected from the performed CCD design led to the following conclusions: it was noticed that methanol contents in the mobile phase has the largest influence on retention time of hydrochlorothiazide (t_RHCT), amlodipine (t_RAML) and retention time of valsartan (t_RVAL). This effect has a negative sign, which means that the increase of methanol contents leads to a decrease in retention time. Influence of methanol on asymmetries of peaks has positive sign, which implies that increasing of methanol content lead to increase asymmetry of peaks. These results indicate that change of pH does not significantly affect the retention time and peak asymmetry of HCT, while on the retention time and asymmetry of AML and VAL has varying impact (positive sign for t_RAML and As_VAL and a negative sign for t_RVAL and As_AML). Effect of temperature is presented by a negative sign, which implies that increasing in temperature shortens retention time of valsartan and amlodipine and reduced asymmetry of peaks.

Since the selected responses were not affected in the same manner an additional optimization procedure was needed. In order to get the best chromatographic performance, a multicriterion methodology was employed by means of Derringer’s desirability function [28, 36]. It is based on constructing desirable ranges for each response (individual desirable function, di) and establishing an overall desirability function (the Derringer desirability function). The Derringer’s desirability function is defined as the geometric mean of individual desirability functions and can be expressed by Equation (2) as $D = {(d^{p 1} 1 \times d^{p 2} 2 \times \dots d^{pn} n)}^{n^{- 1}}$ (2) where n is the number of responses, and pⁿ is the weight of the responses. In Equation (2) pi is the relative importance assigned to the response i.

The relative importance pi is a comparative scale for weighting each of the resulting d_i in the overall desirabi-lity product and it varies from the least important (pi = 1) to the most important (pi = 5). It is noteworthy that the outcome of the overall desirability D depends on the pi value that offers users flexibility in the definition of desirability functions.

The weight of the response is the relative importance of each individual functions d_i and may range from 0.1 to 10. With a weight of 1, d_i varies in a linear way. In this study, weights equal to 1 were selected. Individual desirability functions range from 0 (undesired response) to 1 (a fully desired response). D value close to 1 means that the combination of different criteria was globally optimal (Figs. 6 and 7). If any of the responses or factors fall outside their desirability range, the overall function becomes zero. The goals of multi-criteria optimization for each response in this paper are presented in Table 4.

Fig.6

Graphical representation of the constraints accepted fot the determination of global desirabilty and obtained optimal conditions (Solution 1).

Fig.7

Graphical representation of the constraints accepted fot the determination of global desirabilty and obtained optimal conditions (Solution 6).

Table 4

Criteria for multivariate optimization of the individual responses

	Goal	Lower Limit	Upper Limit	Importance
% Methanol	is in range	30	50	3
pH	is in range	2.5	4.5	3
T(C)	is in range	30	50	3
t _RHCT	is in range	1.6	1.78	3
t _RAML	is in range	2.5	5	3
t _RVAL	is in range	6	8	3
As_HCT	is in range	1.14	1.3	3
As_AML	is in range	0.9	1.3	3
As_VAL	is in range	0.9	1.3	3

Software has released 42 solutions that meet the set criteria with resulting desirability (D = 1.000). Review of all the solutions, it was observed that two of the 42 are those that belong to the already existing points of the experiments (Table 5).

Table 5

Two solutions that meet criteria for analysis

Solutions	% Methanol	pH	T (°C)	t _RHCT	t _RAML	t _RVAL	As_HCT	As_AML	As_VAL
Solution 1	40	3.5	30	1.64	4.16	6.96	1.24	1.12	1.25
Solution 6	40	3.5	40	1.61	3.20	6.20	1.24	1.12	1.27

These two solutions are chosen for solutions on the basis of which should be validated method for analysis of samples (Figs. 8 and 9).

Fig.8

Overlay plot highlighted with yellow shows solutions obtained by optimization keeping the temperature at 30^°C.

Fig.9

Overlay plot highlighted with yellow shows solutions obtained by optimization keeping the temperature at 40^°C.

Figure 10 and Fig. 11 shows graphically and numerically the standard errors of design, depending on the pH and methanol mobile phase. Comparing numerical values of standard errors of design (0.344 and 0.701) it can be concluded that the temperature should be set at 40 ^°C. The representative chromatogram taken under these conditions are represented in Figs. 12 and 13.

Fig.10

Relation of standard error of design to methanol content and pH, temperature is set as constant value at 30^°C.

Fig.11

Relation of standard error of design to methanol content and pH, temperature is set as constant value at 40^°C.

Fig.12

Chromatogram after optimization keeping temperature at 30^°C.

Fig.13

Chromatogram after optimization keeping temperature at 40^°C.

3.3 Validation of analytical method

After setting the optimal conditions, the proposed method was validated [37]. Interfering peaks were not detected at the retention time of HCT, AML and VAL, indicating good selectivity of the method. Linear dependence of the peak areas versus concentration was determined for the proposed ranges. Parameters of the linear regression equations were calculated and are presented in Table 6. The statistical significance of the intercept was tested using Student’s t-test. The limit of detection (LOD) and limit of quantification (LOQ) were calculated as LOD = 3σ/S and LOQ = 10σ/S, where σ is the standard deviation of the response and S the intercept determined from the corresponding calibration curve.

Table 6
Statistical parameters for individual calibration curves

Parameter HCT AML VAL

Conc.(mg/mL) 0.0125–0.125 0.005–0.05 0.08–0.8

Slope 0.285 0.036 0.347

Intercept 0.014 0.002 0.028

r² 0.9926 0.9944 0.9977

LOD 0.0035 0.0019 0.0086

LOQ 0.0117 0.0066 0.0287

Parameter	HCT	AML	VAL
Conc.(mg/mL)	0.0125–0.125	0.005–0.05	0.08–0.8
Slope	0.285	0.036	0.347
Intercept	0.014	0.002	0.028
r²	0.9926	0.9944	0.9977
LOD	0.0035	0.0019	0.0086
LOQ	0.0117	0.0066	0.0287

The precision of the procedure was assessed by analyzing nine solutions containing known quantities of the investigated compounds. Low values of relative standard deviation for repeatability, RSD < 2.5%, and high recovery (Table 7) indicate very good precision of the proposed method.

Table 7

Precision of the RP-HPLC method

Sample	Injected (mg/mL)	Found (mg/mL)	RSD (%)
Hydrochlorothiazide	0.025	0.0253	0.83
	0.05	0.0521	0.93
	0.075	0.0767	0.92
Amlodipine	0.01	0.0104	1.68
	0.02	0.0214	0.97
	0.03	0.0314	0.68
Valsartan	0.08	0.0821	0.86
	0.16	0.1641	0.91
	0.24	0.2452	0.96

The applicability of the proposed method was examined by analyzing commercially available ExforgeHCT tablets.

4 Conclusions

The experimental design methodology was used for simultaneous HPLC determination of hydrochlorothiazide, amlodipine and valsartan in combined dosage forms. The significant feature of these combinations lies in the fact that hydrochlorothiazide and amlodipine are present in minute amounts compared to valsartan which makes for a more complicated and tedious analysis. The chemometric approach for optimization of chromatographic separation of hydrochlorothiazide, amlodipine and valsartan has been demonstrated. Central composite design and response surface methodology is generally employed in order to provide a description of the response pattern in the region of the studied observations and to assist in finding the region where the optimal response occurs. Central composite design requires a minimum number of experiments to be performed and has been widely used for fitting a second-order model. Since there was a mix of linear responses with different targets, Derringer’s desirability function was applied. After defining a global desirability according to the accepted constraints, optimal chromatographic conditions were established.

The proposed HPLC method was validated according to ICH guidelines. From the study of validation parameters, it was observed that the method is specific, accurate, precise, reproducible and is not time-consuming (run time is less than ten minutes). Since there was no interference from other components present in the dosage forms, complicated procedures for extraction were not required. The results obtained in this study corroborate that the proposed HPLC method can be used for routine quantitative analyses of the investigated compounds in a mixture or for their individual determination in pharmaceutical dosage forms.

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Application of central composite design and structural analysis to the optimization of HPLC method for simultaneous analysis of hydrochlorothiazide,amlodipine besylate and valsartan

Abstract

Keywords

1 Introduction

2.1 Chemicals and reagents

2.2 Preparation of standard solutions

2.3 Sample solutions

2.4 Apparatus and software

2.5 Procedure

3.1 Structural analysis

Table 6 Statistical parameters for individual calibration curves Parameter HCT AML VAL Conc.(mg/mL) 0.0125–0.125 0.005–0.05 0.08–0.8 Slope 0.285 0.036 0.347 Intercept 0.014 0.002 0.028 r2 0.9926 0.9944 0.9977 LOD 0.0035 0.0019 0.0086 LOQ 0.0117 0.0066 0.0287

References

Table 6
Statistical parameters for individual calibration curves

Parameter HCT AML VAL

Conc.(mg/mL) 0.0125–0.125 0.005–0.05 0.08–0.8

Slope 0.285 0.036 0.347

Intercept 0.014 0.002 0.028

r² 0.9926 0.9944 0.9977

LOD 0.0035 0.0019 0.0086

LOQ 0.0117 0.0066 0.0287