Development and Validation of Ultra-Performance Liquid Chromatography (UPLC) Method for Simultaneous Quantification of Hydrochlorothiazide, Amlodipine Besylate, and Valsartan in Marketed Fixed-Dose Combination Tablet

: Fixed-dose combination therapy is considered a practical approach in the treatment of various diseases, as it can simultaneously target different mechanisms of action that achieve the required therapeutic efficacy through a synergistic effect. A combination of hydrochlorothiazide (HTZ), amlodipine (AMD), and valsartan (VLS) has been created for the treatment of hypertension. Therefore, the aim of this study was to develop an optimized UPLC method for the simultaneous quantification of this combination. A DoE at a level of 32 was used to investigate the effects of column temperature (20, 30, and 40 ◦ C) and formic acid concentration (0.05, 0.15, and 0.25%) on the retention time of each active pharmaceutical ingredient (API), the peak area, and the peak symmetry, as well as the resolution between HTZ-AMD and AMD-VLS peaks. The optimized analytical method was validated and used to extract the three APIs from the marketed product. The optimized analytical condition with a column temperature of 27.86 ◦ C and a formic acid concentration of 0.172% showed good separation of the three APIs in 1.62 ± 0.006, 3.59 ± 0.002, and 3.94 ± 0.002 min for HTZ, AMD, and VST, respectively. The developed method was linear with the LOQ for a HTC, AMD, and VST of 0.028, 0.038, and 0.101 ppm, respectively. Moreover, the developed assay was sustainable and robust, with an RSD % of less than 2%. The application of this method in the extraction of HTZ, AMD, and VST from the Exforge ® marketed product showed good separation with a measurable drug content of 23.5 ± 0.7, 9.68 ± 0.1, and 165.2 ± 5.2 mg compared to the label claims of 25/10/160 for HTZ, AMD, and VST, respectively.


Introduction
Fixed-dose combination therapy is considered a practical approach during the treatment of various medical conditions [1].A combination of diverse therapeutic molecules effectively targets different pathways simultaneously that achieve the required therapeutic efficacy through a synergistic effect.This leads to a reduction in the required dose of each drug and, thus, to a reduction in the expected side effects as well [2,3].Furthermore, this approach increases patient compliance with the therapeutic regimen and reduces the chance of medication errors owing to the reduction in the total number of pills administered [4].
Various combinations of antihypertensive drugs have been invented and put on the market to achieve optimal clinical outcomes [5].Among them, a combination of hydrochlorothiazide (HTZ), amlodipine (AMD), and valsartan (VLS) was invented for the treatment of elevated blood pressure [6,7].In addition, it was found that this triple therapy was able to reduce cardiovascular complications and fatal outcomes for patients diagnosed with hypertension [8].
2 of 15 HTZ inhibits the sodium chloride symporter located in the distal tubule of the kidneys.The predicted decrease in blood volume is caused by a reduction in salt and water reabsorption.This leads to a significant reduction in blood pressure [9,10].In addition, HTZ has been found to reduce cardiac remodeling in patients diagnosed with heart failure [11].
AMD is one of the calcium channel blockers that act in the myocardium and vascular smooth muscle.This leads to a remarkable reduction in cardiac output or peripheral vascular resistance [12].It was also found that the administration of AMD has a protective effect on the heart and kidneys.It is therefore suitable for the treatment of patients diagnosed with chronic kidney disease and diabetes mellitus [13,14].
VLS exerts its antihypertensive effect by inhibiting the binding of the potent vasoconstrictor angiotensin II to its receptor [15].In addition, it reduces the salt and water retention effect by inhibiting the activation of the aldosterone system.This leads to a significant reduction in cardiac load by reducing blood volume and peripheral back pressure [16].As a result, VLS has a beneficial effect on patients with heart problems, especially heart failure and ventricular hypertrophy.This is due to the reduction in stress exerted on the heart muscle [17,18].The simultaneous administration of this combination therefore not only lowers blood pressure but also protects patients from serious complications.
Different methods have been used for the simultaneous quantification of the therapeutic molecules contained in this pharmaceutical formulation.The UPLC technique has been widely used for this purpose due to its superiority in terms of its short separation time and excellent efficiency and sensitivity [19,20].The accurate quantification of drug molecules ensures the efficacy and safety of the pharmaceutical formulation [21].
A high level of solvent consumption in the development of the UPLC method is expected in the traditional optimization method [22].This is due to the unpredictable results that arise when the independent variables are changed randomly.Therefore, Design of Experiments (DoE) using Statgraphics Centurion program Version 17.2.02software has recently been used to select the optimal conditions for the separation of drugs based on a prediction of the interaction of the factors [23].
The present study aims to develop an optimized UPLC method for the simultaneous quantification of amlodipine, valsartan, and hydrochlorothiazide in marketed tablet form.This method will help ensure patient safety and therapeutic efficacy in the treatment of hypertension.

Experimental Design (DoE)
A 3 2 full factorial experimental design was used to investigate and optimize the impacts of the independent factors on the simultaneous analysis of the three compounds (HTZ, AMD, and VLS).The temperature of the analytical column (A) and the percentage of formic acid in the aqueous phase (B) are the two independent parameters that were investigated.These parameters were estimated in terms of their effects on the following analytical characteristics (responses) for each compound: retention time, peak area, peak symmetry, and the resolution between the HTZ-AMD and AMD-VLS peaks.This is shown in Table 1.A statistical analysis of the obtained data was achieved by using the Statgraphics Centurion program version 17.2.02software.According to the statistical design, nine (9) analytical runs were created based on different column temperatures and flow rates.The analytical design for the simultaneous analysis of the three compounds (HTZ, AMD, and VLS) was created with a susceptible UPLC system (Ultimate 3000 ® binary solvent manager) using an Acquity ® UHPLC BEH C18 1.8 µm (2.1 mm × 50 mm) column connected to an automatic sampler and a photodiode array (PDA) detector.A 50 ppm working solution of all APIs in methanol was prepared, and the analytical determination of the three APIs was achieved simultaneously by isocratic reverse-phase elution at a flow rate of 0.2 mL/min using the following mobile phase composition: water containing different concentrations of 90% formic acid: 10 ACN for 1 min, followed by a change in mobile phase composition to water containing different concentrations of 10% formic acid: 90 ACN for a further 4 min.The mobile phases were run at different column temperatures together with varying concentrations of formic acid, as described in the Design of Experiments (DoE).It is worth mentioning that the analytical determination of HTZ, AMD, and VLS was achieved simultaneously (at the same time), but each compound was determined at its corresponding wavelength channel (271 nm, 237 nm, and 237 nm, respectively) and retention time.

Method Validation
To define the suitability of the developed method to analyze the three different APIs simultaneously, a validation procedure according to the ICH was performed.This included the determination of its linearity, accuracy, and precision and how robust it is [24].Linearity A calibration curve was constructed for each API, and this was repeated in triplicate.The calibration was carried out in a range of 2-50 ppm.The data were plotted, and based on the results of the slope and standard deviation, the LOD and LOQ were calculated [25,26].

Accuracy and Precision
The % recovery was calculated, and this reflects the accuracy of the desired method.The precision was determined by measuring the intra-day (same day) and inter-day (between days) variability.Three selected concentrations (2, 5, 20 ppm) for HTZ, AMD, and VLS were used to perform the test.The RSD % was calculated, and it should not be more than 2% [27].

Robustness
The sustainability and robustness of the developed assay against changes in the analytical conditions were determined by changing the flow rate (0.4 mL/min ± 0.2).The effect of the wavelength on the robustness of the analytical method was determined for the three drugs as follows: HTZ (271 ± 2 nm), AMD (237 ± 2 nm), and VLS (237 ± 2 nm).

Application of Developed UPLC Method
The actual content of drugs within the marketed tablet (EXFORGE HCT) was determined as follows: The tablet was crushed in the mortar using a pestle until a uniform powder was obtained.After that, the ground powder was transferred to a 50 mL volumetric flask, and the volume was completed with methanol to dissolve the three APIs.The mixture was subjected to sonication for 15 min to ensure the complete extraction of drugs in a methanolic solution.The obtained solution was subjected to centrifugation for 5 min at 14,000 rpm to precipitate undissolved tablet components.The actual concentration of drugs in the supernatant was estimated following an appropriate dilution.This mean value was derived from the three individual measurements to determine the drug concentration.As for the symmetry of the HTZ peak, the Pareto standardized chart in Figure 1 shows that the individual parameters (column temperature and formic acid concentration) and the quadratic and interactive parameters did not significantly affect the drug peak.The column temperature showed an agonistic effect on the peak symmetry.In contrast, the formic acid and the interaction between column temperature and formic acid concentration had an antagonistic effect on the reaction.Figure 2 shows that an increase in the column temperature increased the value of the peak symmetry, while an increase in formic acid showed the opposite effect.Table 2 shows that the values for the HTZ peak asymmetry ranged from 1.22 to 1.64.The values for the peak symmetry at the lowest column temperatures were close to this range, regardless of the effects of the formic acid concentration in the aqueous mobile phase.
Also, the individual effects and their quadratic and interactive parameters had no significant impact on the resolution between HTZ and the adjacent drug (AMD), as shown in the Pareto chart in Figure 1.The formic acid content in the mobile phase and the interactive effect had an agonistic effect on the HTZ peak resolution, while the effect of the column temperature on the response was feeble, as shown in Figure 2. Table 2 also shows that the resolution range between the HTZ and AMD peaks is 11.21-16.70,which can be considered a reasonable value to avoid peak overlap [28].In terms of the HTZ peak retention time, the results showed that the column temperature and formic acid concentration had significant antagonistic effects (p values of 0.0007 and 0.02, respectively) on the HTZ peak retention time, while the quadratic effect Processes 2024, 12, 1259 5 of 15 of the column temperature (AA) showed significant antagonism (p = 0.003).In addition, the individual main effects (Figure 2) showed that an increase in column temperature was associated with an aggressive shortening of the peak retention time, while an increase in the formic acid content in the aqueous mobile phase resulted in a noticeable reduction in the retention time of HTZ.In addition, Table 2 shows that the shortest retention time (0.98 min) was observed when the highest column temperature (40 • C) was used with the highest formic acid content in the aqueous phase (0.25%), while using the lowest column temperature (20

AMD Analysis
The UPLC analytical chromatogram showed that the AMD peak appeared at a retention time after that of the HTZ peak (around 3.6 min).It is clear from the Pareto chart Concerning the peak area of HTZ, all the quadratic and interactive parameters studied had insignificant impacts, as shown in the standardized Pareto chart in Figure 1.The main effects in Figure 2 show that increasing the column temperature led to a decrease in the drug's peak area in the chromatogram, while changing the formic acid concentration had no significant effect.Moreover, Table 2 shows that all peak area values were close to each other in the 5.78-6.26mAu/min range.
As for the symmetry of the HTZ peak, the Pareto standardized chart in Figure 1 shows that the individual parameters (column temperature and formic acid concentration) and the quadratic and interactive parameters did not significantly affect the drug peak.The column temperature showed an agonistic effect on the peak symmetry.In contrast, the formic acid and the interaction between column temperature and formic acid concentration had an antagonistic effect on the reaction.Figure 2 shows that an increase in the column temperature increased the value of the peak symmetry, while an increase in formic acid showed the opposite effect.Table 2 shows that the values for the HTZ peak asymmetry ranged from 1.22 to 1.64.The values for the peak symmetry at the lowest column temperatures were close to this range, regardless of the effects of the formic acid concentration in the aqueous mobile phase.Also, the individual effects and their quadratic and interactive parameters had no significant impact on the resolution between HTZ and the adjacent drug (AMD), as shown in the Pareto chart in Figure 1.The formic acid content in the mobile phase and the interactive effect had an agonistic effect on the HTZ peak resolution, while the effect of the column temperature on the response was feeble, as shown in Figure 2. Table 2 also shows that the resolution range between the HTZ and AMD peaks is 11.21-16.70,which can be considered a reasonable value to avoid peak overlap [28].

AMD Analysis
The UPLC analytical chromatogram showed that the AMD peak appeared at a retention time after that of the HTZ peak (around 3.6 min).It is clear from the Pareto chart (Figure 3) that the AMD peak retention time was antagonistically and significantly affected by the column temperature (p = 0.0175).In contrast, the response was affected agonistically and significantly by the formic acid concentration in the mobile phase (p = 0.05).These findings are clearly illustrated in the main effects (Figure 4).Raising the column temperature shortened the drug peak retention time, while increasing the formic acid level in the mobile phase caused retention time prolongation.Table 2 shows that the retention values of all the tested runs were close (in the range of 3.57-3.62min), which might be due to the opposing effects of the column temperature and the formic acid concentration on the responses.
and their quadratic as well as interactive actions (Figure 3).The main effects in Figure 4 clarified that there was anopposing action between the peak symmetry-enhancing effect of the column temperature and the reducing effect of the formic acid level.Moreover, the values of AMD peak symmetry were in the range of 1.15-1.20,which could be considered an ideal range [22].
The resolution between the adjacent AMD and VLS peaks was not significantly impacted by the individual effects, nor were their quadratic and interactive parameters.As shown in the Pareto chart in Figure 3, the interactive and formic acid effects showed antagonistic actions on the response.In contrast, other parameters (A, AA, and BB) showed agonistic actions on such responses.The slight effect on the AMD peak resolution of the column temperature and the augmenting effect of the formic acid concentration are revealed in Figure 4, showing the individual effects.All the values of the AMD peak resolutions obtained for the nine runs tested were very close, in a range of 3.36-3.82,as shown in Table 2.

VLS Analysis
The impacts of the tested independent analytical factors (column temperature, A, formic acid concentration, B, and their quadratic effects and interactive effects) on the retention time of the VLS peak are presented in the standardized Pareto chart in Figure 5.The The peak area of AMD was insignificantly affected by the individual effects and their quadratic and interactive actions, as shown in Figure 3.However, the column temperature and the formic acid concentration exerted an antagonistic effect on the response, while the quadratic effect of formic acid (BB) and the interactive effect showed antagonism.Figure 4, which illustrates the main effects, shows that raising the column temperature caused a reduction in the drug peak area, while increasing the formic acid concentration in the mobile phase first decreased the peak area, followed by an increasing peak area at higher formic acid levels.The AMD peak area values in the UPLC chromatograms were 2.76-3.20 mAU/min, as depicted in Table 2.
The AMD peak symmetry was insignificantly influenced by the individual effects and their quadratic as well as interactive actions (Figure 3).The main effects in Figure 4 clarified that there was anopposing action between the peak symmetry-enhancing effect of the column temperature and the reducing effect of the formic acid level.Moreover, the values of AMD peak symmetry were in the range of 1.15-1.20,which could be considered an ideal range [22].
The resolution between the adjacent AMD and VLS peaks was not significantly impacted by the individual effects, nor were their quadratic and interactive parameters.As shown in the Pareto chart in Figure 3, the interactive and formic acid effects showed antagonistic actions on the response.In contrast, other parameters (A, AA, and BB) showed agonistic actions on such responses.The slight effect on the AMD peak resolution of the column temperature and the augmenting effect of the formic acid concentration are revealed in Figure 4, showing the individual effects.All the values of the AMD peak resolutions obtained for the nine runs tested were very close, in a range of 3.36-3.82,as shown in Table 2.

VLS Analysis
The impacts of the tested independent analytical factors (column temperature, A, formic acid concentration, B, and their quadratic effects and interactive effects) on the retention time of the VLS peak are presented in the standardized Pareto chart in Figure 5.The column temperature had a highly significant antagonistic effect on the drug's peak retention time (p value was 0.001), while the effects of the other tested independent factors were not significant.As depicted in Figure 6, increasing the column temperature led to a significant and pronounced shortening of the drug's peak retention time, while the effect of formic acid was shallow.Furthermore, the retention times for the VLS peak in all nine runs ranged from 3.8 to 3.95 min, which seems closely similar, as shown in Table 2.
Regarding the effects of the independent analytical parameters on the VLS peak area, the column temperature and its quadratic action showed antagonistic impacts, while the formic acid concentration and its antagonistic effect had an agonistic effect on the response; however, all these effects were insignificant (p values were higher than 0.05), as illustrated in the Pareto chart (Figure 5). Figure 5, showing the main effects on the drug peak area, reveals that the column temperature exerted a reducing effect on the response at low values, followed by an increasing peak area upon raising the column temperature, while faint effects at all formic acid levels on the response were detected.Table 2 supports these insignificant effects on the independent factors by clarifying that all the peak area values were found to be close (2.793.62 mAu/min).
Similarly, the symmetry of the VLS peak was insignificantly agonized by the individual independent parameters and their quadratic effects, while the interactive effect had an insignificantly antagonistic action on the response (Figure 5). of the VLS peak symmetry was very close and in an acceptable range between 1.10 and 1.25, as shown in Table 2.   of the VLS peak symmetry was very close and in an acceptable range between 1.10 and 1.25, as shown in Table 2.The main effect plot for the drug's peak symmetry (Figure 6) showed the indistinct action of the column temperature at all formic acid levels on the responses detected, while the formic acid concentration showed a symmetryenhancing effect.In addition, the range of the VLS peak symmetry was very close and in an acceptable range between 1.10 and 1.25, as shown in Table 2.

Optimization of UPLC Analytical Parameters for Simultaneous Separation of HTZ, AMD, and VLS
The optimization of the analytical independent factors (column temperature, A, and formic acid concentration in the mobile phase, B) for the concurrent analysis of the three tested APIs was carried out based on the previously described statistical analysis of all the studied responses.The optimization procedures were generated according to the following analytical desirability features: the minimization of the peak retention time, the maximization of the peaks' areas, and making the peaks' asymmetry values fall in the range of 0.9-1.4 for all the APIs.The resolution between the HTZ and AMD peaks and between the AMD and VLS peaks was designed to be minimized in the actual analytical range.According to the previously mentioned desirable analytical conditions, the statistical software proposed using a column temperature of 27.86 • C and a formic acid concentration in the mobile phase of 0.172% as the analytically optimized settings.
UPLC chromatograms of the three APIs based on the optimized simultaneous analytical conditions are presented in Figure 7, and the optimum analytical conditions for the separation of the HTZ, AMD, and VLS chromatograms comparing the observed values with the predicted values are tabulated in Table 3.The results of the observed values indicated that the HTZ peak was detected at 1.62 ± 0.006 min, and its measured area was 5.92 ± 0.007 mAU/min, with a 1.34 asymmetry factor.The resolution value between the HTZ and AMD peaks was 11.74 ± 0.02.The AMD peak was detected based on the optimum settings at 3.59 ± 0.002 min, and its area was 2.87 ± 0.0.004mAU/min, with a symmetry factor of 1.17 ± 0.02.The resolution factor between the AMD and VLS peaks was 3.96 ± 0.015.Moreover, the last peak in the analytical chromatogram of VLS was separated at 3.94 ± 0.002 min, with an area of 3.28 ± 0.006 mAU/min, and the asymmetry of this peak was 1.2 ± 0.02.The observed analytical data were found to be close to the predicted values.5.92 ± 0.007 mAU/min, with a 1.34 asymmetry factor.The resolution value between HTZ and AMD peaks was 11.74 ± 0.02.The AMD peak was detected based on the mum settings at 3.59 ± 0.002 min, and its area was 2.87 ± 0.0.004mAU/min, with a s metry factor of 1.17 ± 0.02.The resolution factor between the AMD and VLS peaks 3.96 ± 0.015.Moreover, the last peak in the analytical chromatogram of VLS was separ at 3.94 ± 0.002 min, with an area of 3.28 ± 0.006 mAU/min, and the asymmetry of this was 1.2 ± 0.02.The observed analytical data were found to be close to the predicted va

Linearity
A calibration curve was constructed by plotting the peak area, as the instrument's response, against the concentration (Figure 8).The regression coefficient values (r 2 ) indicate a linear correlation between the peak area and the concentration.These were 0.999 for HTZ, 0.9988 for AMD, and 0.997 for VLS (Table 4).A calibration curve was constructed by plotting the peak area, as the instrument's response, against the concentration (Figure 8).The regression coefficient values (r 2 ) indicate a linear correlation between the peak area and the concentration.These were 0.999 for HTZ, 0.9988 for AMD, and 0.997 for VLS (Table 4).
The LOD and LOQ were calculated based on the slope obtained from the calibration curve, and they are shown in Table 4.The LOQ for HTZ, AMD, and VLS was 0.028, 0.038, and 0.101 ppm, respectively.The LOD and LOQ were calculated based on the slope obtained from the calibration curve, and they are shown in Table 4.The LOQ for HTZ, AMD, and VLS was 0.028, 0.038, and 0.101 ppm, respectively.

Accuracy and Precision
The accuracy of the developed analytical assay was determined by measuring the percentage recovery of the APIs at different concentrations of 2, 5, and 20 ppm (Table 5).For HTZ, the recovered concentration ranged from 94 to 110%, while for AMD, it ranged from 93 to 108%, and for VLS, it ranged between 95 and 115%, which all met the requirement.The precision of the developed assay was estimated using intra-day and inter-day precision analyses (Table 5).Good precision is always predicted by the % RSD.A % RSD of less than 2 indicates that the developed assay has good precision.The calculated % RSD for all the components is less than 2, which indicates that the developed assay is precise.

Robustness
The developed assay's sustainability against changes in the absorbance wavelength and flow rate was determined in terms of peak area, retention time, resolution, and symmetry.The data were expressed as RSD%.Table 6 shows that the RSD% for the effects of the flow rate and wavelength changes was less than 2%, indicating the assay's robustness.

Analysis of Drugs in EXFORGE HCT Tablet
The developed method was successfully used to estimate the HTZ, AMD, and VLS concentrations in the marketed tablet.The measured concentration of the drugs within the marketed formulation was 23.5 ± 0.7, 9.68 ± 0.1, and 165.2 ± 5.2 mg, respectively.These results demonstrate the accuracy of the developed method, with a percentage of drug recovery between 93.79 and 103.26%.This indicates that the developed UPLC method is a reliable tool for drug analysis.Figure 9 shows the HTZ, AMD, and VLS chromatograms extracted from Exforge ® tablets.The chromatograms show the applicability of the developed method for separating the tablet components.
the marketed formulation was 23.5 ± 0.7, 9.68 ± 0.1, and 165.2 ± 5.2 mg, respectively.These results demonstrate the accuracy of the developed method, with a percentage of drug recovery between 93.79 and 103.26%.This indicates that the developed UPLC method is a reliable tool for drug analysis.Figure 9 shows the HTZ, AMD, and VLS chromatograms extracted from Exforge ® tablets.The chromatograms show the applicability of the developed method for separating the tablet components.

Conclusions
A production line for a dosage form that contains a combination therapy is now adopted by many pharmaceutical companies.Therefore, developing an analytical analysis that analyzes the combined ingredients simultaneously is crucial to determining the accurate quantification of the drugs and maintaining patients' safety.The developed UPLC was sensitive, easy, accurate, and precise in determining the concentrations of HTZ, AMD, and VST in bulk and pharmaceutical dosage forms.

1 .
Effect of Analytical Conditions on the Simultaneous Separation of the Three APIs 3.1.1.HTZ Analysis The standardized Pareto chart for the effect of individual parameters (column temperature, A, and formic acid concentration, B) and their quadratic and interactive effect (AB) on the analytical attributes of HTZ are displayed in Figure 1.Processes 2024, 12, x FOR PEER REVIEW 5 of 15

Figure 1 .
Figure 1.Pareto standardized chart for the effects of independent analytical parameters on the responses of the HTZ peak.The vertical line is the significant reference line.

Figure 1 .
Figure 1.Pareto standardized chart for the effects of independent analytical parameters on the responses of the HTZ peak.The vertical line is the significant reference line.

Figure 2 .
Figure 2. Main individual effects of independent analytical parameters on the responses of the HTZ peak.

Figure 2 .
Figure 2. Main individual effects of independent analytical parameters on the responses of the HTZ peak.

Figure 3 .
Figure 3. Pareto standardized chart for the effects of independent analytical parameters on the responses of the AMD peak.The vertical line is the significant reference line.

Figure 3 .
Figure 3. Pareto standardized chart for the effects of independent analytical parameters on the responses of the AMD peak.The vertical line is the significant reference line.

Figure 4 .
Figure 4. Main individual effects of independent analytical parameters on the responses of the AMD peak.

Figure 4 .
Figure 4. Main individual effects of independent analytical parameters on the responses of the AMD peak.

Figure 5 .
Figure 5. Pareto standardized chart for the effects of independent analytical parameters on the responses of the VLS peak.The vertical line is the significant reference line

Figure 6 .
Figure 6.Main individual effects of independent analytical parameters on the responses of the VLS peak.

Figure 5 .
Figure 5. Pareto standardized chart for the effects of independent analytical parameters on the responses of the VLS peak.The vertical line is the significant reference line.

Figure 5 .
Figure 5. Pareto standardized chart for the effects of independent analytical parameters on the responses of the VLS peak.The vertical line is the significant reference line

Figure 6 .
Figure 6.Main individual effects of independent analytical parameters on the responses of the VLS peak.

Figure 6 .
Figure 6.Main individual effects of independent analytical parameters on the responses of the VLS peak.

Figure 7 .Table 3 .Figure 7 .
Figure 7. UPLC chromatograms of HTZ, AMD, and VLS obtained from the optimized analy method.Table3.The optimized analytical settings for the simultaneous UPLC analysis of HTZ, AMD VLS comparing the predicted and observed analytical values.

Figure 9 .
Figure 9. UPLC chromatograms of HTZ, AMD, and VLS extracted from Exforge ® tablets and analyzed according to the optimized analytical method.

Table 1 .
Variables in 3 2 full factorial design of analytical procedures for the separation of HTZ, AMD, and VLS.

Table 2 .
Results for the simultaneous determination of HTZ, AMD, and VLS.

Table 2 .
Results for the simultaneous determination of HTZ, AMD, and VLS.

Table 3 .
The optimized analytical settings for the simultaneous UPLC analysis of HTZ, AMD, and VLS comparing the predicted and observed analytical values.

Table 5 .
Accuracy and precision results in terms of recovered concentration and RSD %.

Table 6 .
Robustness results (RSD) against flow rate and wavelength changes.