Development and Validation of Hplc Method for Simultaneous Determination of Amlodipine, Valsartan, Hydrochlorothiazide in Dosage Form and Spiked Human Plasma

A simple, sensitive, and specific method was developed for simultaneous determination of Amlodipine besylate (AML), Valsartan (Vals) and Hydrochlorothiazide (HCT) by high performance liquid chromatography without previous separation. Satisfactory resolution was achieved using a RP-C18 chromatographic column, Phenomenex Kinetex (150 mm × 4.6 mm i.d) and a mobile phase consisting of acetonitrile-phosphate buffer (0.05 M) with pH 2.8 in the proportion of (40/60, v/v) at a flow rate 0.8 mL/min and the wavelength detection was 227 nm. The retention time for HCT, AML and VAls was 2.26, 3.16 and 11.19 min; respectively. The described method was linear over a range of 4-28 µg /ml, 5-40 µg/ml and 1-12 µg/ml for AML, Vals and HCT; respectively. The mean percent recoveries were 99.94%, 99.96% and 99.78% for AML, Vals and HCT; respectively. F-test and t-test at 95%confidence level were used to check the intermediate precision data obtained under different experimental setups. The method could be used for analysis of combined dose tablet formulation containing AML, Vals, HCT as well as spiked human plasma.

The aim of this work was the development of simple, sensitive and accurate analytical methods for the simultaneous determination of amlodipine besylate, valsartan and hydrochlorothiazide in mixtures without the need of prior separation step.The developed method was able to determine the content of the cited drugs in commercial tablets and spiked human plasma.
Egypt S.A.E.(Cairo, Egypt), Vals was kindly provided by Novartis Pharma S.A.E.(Cairo, Egypt) and HCT was kindly provided by Medical Union Pharmaceuticals, (Abu-sultan, Ismailia, Egypt).Exforge HCT (B.No.B8022) containing 10 mg AM-LO, 160 mg Vals and 12.5 mg HCT per tablet were obtained from Swaziland manufactured by Novartis Farmaceutica, Barbera, Spain for Novartis pharma AG, Basle Switzerland. Acetonitril, HPLC grade (Fisher Scientific, UK). Potassium dihydrogen phosphate and orthophosphoric acid were of HPLC grade (Merck, Darmstadt, Germany). Double distilled water was used in preparation of mobile phase.

Chromatographic Conditions
Chromatographic analysis was carried out at ambient temperature (22˚C -25˚C).The compounds were separated isocratically with a mobile phase consisting of acetonetrile-phosphate buffer (0.05 M) with pH 2.8 ± 0.2 in the proportion of (40/60, v/v) at a flow rate 0.8 mL/min with injection volume 20 µL.The effluent was monitored spectrophotometrically at wavelength 227 nm.The mobile phase was filtered by passing through a 0.45 µm membrane filter (Millipore, Bradford, MA), and then ultrasonicated for 15 min.

Preparation of Stock Solutions
Primary stock solutions of AML, Vals and HCT were prepared daily separately by dissolving 25 mg of each in 25 mL volumetric flasks (1.0 mg/mL) in mobile.Series of working solutions of AML, Vals and HCT were prepared by the appropriate dilution of the stock solutions with same solvent to reach the concentration ranges of 4 -28 µg/ml for AML, 5 -40 µg/ml for Vals and 1 -12 µg/ml for HCT.

Procedures
The standard solutions were prepared by dilution of the stock standard solution with mobile phase to reach a concentration range 4 -28 µg/mL for AML, 5 -40 µg /ml for Vals and 1 -12 µg/ml for HCT.Triplicate 20 μL injections were made for each concentration and chromatographed under the condition described above.The peak area of each concentration was plotted against the corresponding concentration to obtain the calibration graph and regression equation was computed.

Preparation of Pharmaceutical Dosage Sample
The contents of twenty tablets labeled to contain 10 mg of AML, 160 mg of Vals and 12.5 mg of HCT were individually weighed, mixed and finely powdered in a mortar.Amount of the powder equivalent to one tablet content was accurately weighed, transferred into 100 ml volumetric flask and diluted with mobile phase.The sample solution was then filtered using 0.45 μm filters (Millipore, Milford, MA).The procedures were completed as mentioned above and the concentration of AML and PER was obtained from corresponding regression equations.

Preparation of Spiked Serum Sample
To 1.0 ml plasma 100 µL of each standard solution was pipetted into 5 mL tapered bottom centrifuge tube, and the volume was made up by acetonitrile.The mixture was mixed briefly, standing for 5 min at room temperature, then vortex for 3 min, finally the mixture was centrifuged at 4000 r/min for 10 min.One ml of the clear supernatant was transferred to series of 10 ml volumetric flasks, completed to volume with the mobile phase, the final concentration of the spiked serum sample containing 4 µg/mL of AML, 5 µg/mL of vals and 1 µg/mL of HCT was obtained by further dilution with mobile phase.Finally, 20 µL of the clear supernatant was injected into the liquid chromatograph.

Calibration and Linearity
Calibration curves were constructed in the ranges of 4 -28, 5 -40 and 1 -12 µg/mL for AML, Vals and HCT, respectively, to encompass the expected concentrations in the measured samples.Triplicate 20 µL injections were made for each working standard solution.The peak area for each concentration was recorded and then plotted against the corresponding concentration to obtain the calibration graph.

Results and Discussion
In order to affect the simultaneous elution of more than one component under isocratic conditions, different chromatographic conditions (organic modifier, flow rate, and pH) have been investigated.Various stationary phases were used like C8 and C18 and phenyl column, poor and distorted peaks were observed with phenyl column while respectively.In triplicate run from which the linear regression equation was calculated.Correlation coefficient, C8 and C18 gave satisfactory resolution and free from tailing.Mobile phases containing methanol alone or acetonetrile alone were found to elute the compounds unresolved.Increasing the acetonitrile concentration to more than 65% of buffer led to inadequate separation.At a lower acetonitrile concentration (<40%), separation occurred, but with excessive tailing and increased retention time.To avoid multiple peaks of reversed phase columns, the pH must be controlled with buffers, for example potassium dihydrogen phosphate.
slope and intercept were listed in Table 2. Results indicate high sensitivity of the proposed procedures.According to ICH recommendation [35,36], the approach based on the S.D. of the response and the slope was used for determining the detection and quantitation limits.

Accuracy
Accuracy of the measurements was determined using the calibration standards of the three drugs, where mean percentage of 99.94, 99.96 and 99.78 for AML, Vals and HCT, respectively were obtained Table 3. Accuracy was also assessed by the recovery of added standard, five concentrations each in duplicate to know concentration of commercial tablets using the proposed HPLC procedures.
Our objective of the chromatographic method development was to achieve a peak tailing factor <2, retention time in between 2 and 12 min, along with good resolution.This objective was obtained using mobile phase consisting of acetonitrile-phosphate buffer (0.05 M) in the proportion of (40/60, v/v) with the pH adjusted to of 2.8 ± 0.2 with orthophosphoric acid.The mobile phase composition was optimized under the described conditions, the analyte peaks were well defined, resolved and free from tailing, the tailing factors were <2 for all peaks.The elution orders were HCT (t R 2.26 min), AML (t R 3.16 min) and Vals (t R 11.19) at a flow rate of 0.8 ml/min.The optimum wavelength for detection was 227 nm at which much better detector responses for the three drugs were obtained (Figure 1).According to U.S. Pharmacopeia (USP) [1], system suitability tests are an integral part of an LC method.System suitability tests are used to verify that the column efficiency (N), selectivity factor (resolution) and reproducibility of the chromatographic system are adequate for the analysis.System suitability tests were carried out on freshly prepared standard stock solutions of AML, Vals and HCT.The system was found to be suitable as shown in Table 1.

Precision
Injection repeatability: The RSD of drugs peak area in five triplicate injections of standard drug solution determined each day of 3 consecutive days.Intraday and Interday precision were assessed using three concentration and three replicates of each concentration.The calculated relative standard deviation values were found to be small   Hight equivalent theoretical plates (HETP) (cm) The smaller the value, the higher the column efficiency = L/N 0.004 0.005 0.003  below 2% indicating good repeatability and reliability of the proposed method.The results and their statistical analysis were summarized in Table 4.
The utility of the proposed method was verified by replicate estimations of the pharmaceutical preparation and results obtained are evaluated statistically Table 5.

Specificity
Retention time of the peak in the chromatogram of tablets and spiked human plasma was the same as that of standard drugs without Interference from excipients, additives or biological fluid components.Accordingly, specificity of the HPLC method was evaluated by its successful application to determine drugs in their tablets with mean recovery of 99.25% ± 0.73% for Exforge HCT ® tab.The results were summarized in Table 4.
A statistical comparison of the results obtained by the proposed method and the reported HPLC methods [37][38][39] is shown in Table 6.The values of the calculated t and F are less than the tabulated ones, which reveals that there is no significant difference with respect to accuracy and precision between the proposed and reported methods.Moreover, the proposed method was extended to analyze drugs in spiked human plasma (Figures 2 and 3) by simple extraction and de-proteination with acetonitrile, followed by centrifugation and the clear supernatant containing the drug was adjusted to volume by the mobile phase and analyzed directly by HPLC.Nearly non-destructive extraction was obtained as obvious from a mean % recovery of 98.19 -98.87 from plasma, Table 7.

Robustness and Ruggedness
For the evaluation of the method robustness, some parameters were interchanged such as pH, organic phase ratio of mobile phase and column oven temperature.The capacity remains unaffected by small deliberate variations.Method ruggedness was expressed as R.S.D. % of the same procedure applied by using two different instruments on different days.The results showed no statistical differences between different instruments suggesting that the developed methods were robust and HPLC method was rugged.

Application to Dosage
The proposed method was successfully applied for the simultaneous determination of three drugs in Exgorge * Average of three determinations Table 6.Determination of the studied drugs in their combined tablets using the proposed method compared to reference HPLC methods.

1 .
Linearity, Detection and Quantitation Limits Calibration curve representing the relation between the concentrations of drugs versus the peak area were constructed.Results show linear relationship in the range of

Figure 1 .
Figure 1.Representative chromatogram of amlodipine, valsartan and hydrochlorothiazide in pure form.

Figure 3 .
Figure 3. Representative chromatogram of amlodipine, valsartan and hydrochlorothiazide in spiked human plasma.