RP-HPLC Method Development and Validation for Simultaneous Estimation of Amlodipine, Telmisartan and Chlorthalidone in Tablet Dosage Form

Hypertension is a chronic condition often managed by multi-drug regimens. The fixed-dose combination of Chlorthalidone (thiazide-like diuretic), Telmisartan (Angiotensin II Receptor Blocker), and Amlodipine (Calcium Channel Blocker) provides synergistic control of blood pressure through complementary mechanisms of action.^1,2Chlorthalidone is a long-acting thiazide-like diuretic that has demonstrated superior cardiovascular outcomes in clinical trials.³ Telmisartan is an angiotensin II receptor antagonist with metabolic benefits beyond blood pressure reduction.⁴ Amlodipine besylate is a dihydropyridine calcium channel blocker with proven efficacy in hypertension management.⁵

Figure 1: Chemical Structures Of (A) Chlorthalidone, (B) Telmisartan, And (C) Amlodipine

While monographs for dual combinations exist in official compendia (e.g., Telmisartan and Amlodipine in USP),⁶ there is currently no official pharmacopoeial method for this triple combination. A literature survey revealed several HPLC methods for related combinations,^7,10 but existing methods for triple combinations often employ complex ion-pairing buffers, require long run times (>15 minutes), or use expensive gradient elution systems.^11,12 The objective of this study was to develop a rapid, simple, and reliable isocratic RP-HPLC method using an Agilent Zorbax SB-C18 column and a simple TEA-phosphate buffer system for routine Quality Control (QC) analysis. The method was validated according to ICH Q2(R1) guidelines¹³ for linearity, accuracy, precision, specificity, robustness, and sensitivity.

MATERIALS AND METHODS

Chemicals and Reagents

Pharmaceutically pure reference standards of Chlorthalidone (purity 99.8%), Telmisartan (purity 99.6%), and Amlodipine besylate (purity 99.5%) were obtained as gift samples from pharmaceutical companies. Commercial tablets (each containing Telmisartan 40 mg, Chlorthalidone 12.5 mg, and Amlodipine besylate equivalent to 5 mg Amlodipine) were purchased from a local pharmacy. HPLC grade Acetonitrile (Merck, Germany), Orthophosphoric Acid (OPA, Merck), and Triethylamine (TEA, Sigma-Aldrich) were used. Water was purified using a Milli-Q system (Millipore, USA).

Instrumentation

The analysis was performed on a ChromeIn HPLC system equipped with a quaternary solvent delivery pump (LC-10AT VP), manual Rheodyne injector with a 10 µL loop, and a UV-Visible detector (SPD-20A). Data acquisition and processing were performed using Empower 2 software (Waters Corporation).

Chromatographic Conditions

Based on preliminary optimization studies and literature review,^14,15 the following chromatographic conditions were established:

• Column: Agilent Zorbax SB-C18 (250 × 4.6 mm, 5 µm particle size)
• Mobile Phase: 0.1% Triethylamine in Water (pH adjusted to 3.0 with Orthophosphoric Acid): Acetonitrile (65:35 % v/v)
• Diluent: Mobile Phase
• Flow Rate: 1.0 mL/min
• Column Temperature: Ambient (25 ± 2°C)
• Detection: 222 nm
• Injection Volume: 10 µL
• Run Time: 10 minutes

Wavelength Selection

The standard solutions of all three drugs were scanned individually and in combination in the UV range of 200–400 nm using a UV-Visible spectrophotometer (Shimadzu UV-1800). An isosbestic point (wavelength of overlapping absorbance) was identified at 222 nm, where all three drugs showed significant absorbance, ensuring adequate sensitivity for simultaneous quantification.

Preparation of Solutions

Standard Stock Solutions: Accurately weighed quantities of Chlorthalidone (12.5 mg), Telmisartan (40 mg), and Amlodipine (5 mg) were transferred to a 100 mL volumetric flask. Approximately 70 mL of Mobile Phase was added, the solution was sonicated for 10 minutes to ensure complete dissolution, and the volume was made up to the mark with Mobile Phase. This provided concentrations of 125 µg/mL (Chlorthalidone), 400 µg/mL (Telmisartan), and 50 µg/mL (Amlodipine).

Working Standard Solution: The stock solution was diluted with Mobile Phase to obtain working standard concentrations of 12.5 µg/mL (Chlorthalidone), 40 µg/mL (Telmisartan), and 10 µg/mL (Amlodipine), representing 100% of the label claim.

Sample Preparation (Tablet Assay): Twenty tablets were weighed accurately, and the average weight per tablet was calculated. The tablets were crushed to a fine, homogeneous powder using a mortar and pestle. An accurately weighed portion of powdered sample equivalent to one average tablet (nominally containing 40 mg Telmisartan, 12.5 mg Chlorthalidone, and 5 mg Amlodipine) was transferred to a 100 mL volumetric flask. Approximately 70 mL of Mobile Phase was added, and the mixture was sonicated for 20 minutes with intermittent shaking to ensure complete extraction. The solution was cooled to room temperature, the volume was made up to the mark with Mobile Phase, and the solution was filtered through a 0.45 µm Nylon syringe filter (Millipore). The first 5 mL of filtrate was discarded. Further dilutions were made with Mobile Phase to achieve the target working concentrations matching the standard solution.

Ethical Matter

This research work involves the analysis of pharmaceutical dosage forms (in vitro) and does not involve any experiments on animals or human subjects. Therefore, ethical committee approval was not required.

RESULTS

Method Optimization

The primary objective was to achieve baseline separation of all three analytes within a short run time while maintaining good peak shape and system suitability parameters. Several parameters were systematically optimized.

Column Selection: The Agilent Zorbax SB-C18 (250 × 4.6 mm, 5 µm) column was selected based on its superior stability at low pH and excellent peak shape for basic compounds.¹⁴ Initial trials with a standard C18 column (without stabilized bonding) showed significant peak tailing for Amlodipine (Tailing Factor > 2.5), likely due to residual silanol interactions.

Mobile Phase Optimization: Basic compounds like Amlodipine are prone to peak tailing due to interaction with unbonded silanol groups on the stationary phase.^14,15 The addition of 0.1% Triethylamine (TEA) to the aqueous component of the mobile phase acted as a silanol masking agent, effectively suppressing these interactions and reducing the Tailing Factor (T) for Amlodipine from 2.4 (without TEA) to 1.18 (with TEA). The pH was adjusted to 3.0 with orthophosphoric acid to ensure ionization suppression and improve peak symmetry.¹⁴

Various ratios of Buffer: Acetonitrile were tested (70:30, 65:35, 60:40). The optimized ratio of 65:35 (v/v) provided optimal resolution between all peaks, with a Resolution (Rs) of 5.8 between Chlorthalidone and Telmisartan, and Rs of 3.2 between Telmisartan and Amlodipine, while maintaining a total run time under 10 minutes.

Figure 2: Specificity Study Showing (A) Blank Mobile Phase Chromatogram And (B) Standard Solution Chromatogram

System Suitability

System suitability testing is an integral part of chromatographic analysis and ensures that the resolution and reproducibility of the chromatographic system are adequate for the analysis to be performed.^13,14 Six replicate injections of the working standard solution were performed, and the system suitability parameters were calculated. The method met all acceptance criteria as per ICH guidelines,¹³ including Resolution (Rs) and Tailing Factor (T).

Table 1: System Suitability Data (N=6)

The high number of theoretical plates (N > 3700) indicates excellent column efficiency. Tailing factors below 1.25 for all peaks indicate symmetric peak shapes, which are essential for accurate integration.¹⁴ Resolution values greater than 3.0 confirm complete baseline separation between adjacent peaks.

Figure 3: Typical RP-HPLC Chromatogram Of Chlorthalidone (Rt = 4.33 Min), Telmisartan (Rt = 6.06 Min), And Amlodipine (Rt = 7.60 Min) Under Optimized Conditions

Linearity

Linearity is the ability of an analytical method to obtain test results that are directly proportional to the concentration of analyte in samples within a given range.¹³ Linearity was established at five concentration levels spanning 50% to 150% of the target concentration (representing typical ranges encountered during assay and content uniformity testing). The calibration curves were constructed by plotting peak area (y) against concentration (x) for each analyte.

Table 2: Linearity Data

Figure 4: Calibration Curves Of (A) Chlorthalidone, (B) Telmisartan, And (C) Amlodipine Showing Linear Response

Note: Small positive intercepts are attributed to minor instrumental baseline offset and do not affect quantification accuracy, as evidenced by recovery data.

The correlation coefficients (R² > 0.9998) for all three analytes demonstrate excellent linearity over the tested concentration ranges, meeting ICH acceptance criteria (R² ≥ 0.999).13

Sensitivity (LOD and LOQ)

The Limit of Detection (LOD) represents the lowest concentration of an analyte that can be detected but not necessarily quantified. The Limit of Quantitation (LOQ) is the lowest concentration that can be quantified with acceptable precision and accuracy.¹³ LOD and LOQ were calculated using the standard deviation of the response (σ) and the slope (S) of the calibration curve based on the formulae LOD = 3.3σ/S and LOQ = 10σ/S.

Table 3: Limit Of Detection (LOD) And Limit Of Quantitation (LOQ)

The low LOD and LOQ values indicate that the method is sufficiently sensitive for quality control applications, including the detection of low-level impurities or degradation products.

Precision

Precision is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings.¹³ Two types of precision were evaluated. Method Precision (Repeatability): Six independent sample preparations from the same homogeneous batch were analyzed on the same day under identical conditions by the same analyst. The % RSD of the assay results was calculated as a measure of method precision. Intermediate Precision (Ruggedness): The analysis was repeated on a different day by a different analyst using a different HPLC instrument from the same manufacturer. This evaluates the method's robustness to variations in analytical conditions.¹³

Table 4: Precision And Intermediate Precision Study

The % RSD values well below 2.0% for both repeatability and intermediate precision confirm that the method is precise and rugged, meeting ICH acceptance criteria (% RSD ≤ 2.0%).13

Accuracy (Recovery)

Accuracy is the closeness of agreement between the value found and the value that is accepted as the conventional true value or an accepted reference value.¹³ Accuracy was determined by the standard addition method (spiking). Known amounts of standard drugs were added to pre-analyzed sample solutions at three concentration levels (50%, 100%, and 150% of the label claim), and the percentage recovery was calculated.

Table 5: Accuracy (Recovery Studies)

*Average of 3 determinations at each level.

The mean recovery values ranging from 99.5% to 99.7% with low standard deviations demonstrate excellent accuracy. The results fall well within the ICH acceptance range of 98.0–102.0% for pharmaceutical assays,¹³ confirming that the method is free from systematic errors.

Analysis of Marketed Formulation (Assay)

The validated method was successfully applied to the simultaneous quantitative determination of Chlorthalidone, Telmisartan, and Amlodipine in commercially available fixed-dose combination tablets. Six replicate determinations were performed.

Table 6: Assay of Commercial Tablet Formulation