Avanthi Institute of Pharmaceutical Sciences, Hyderabad.
A novel, rapid, and precise stability-indicating ultra-performance liquid chromatography (UPLC) method was developed and validated for the simultaneous estimation of chlorthalidone, telmisartan, and cilnidipine in combined pharmaceutical dosage forms. Chromatographic separation was achieved on a C18 column (250 × 4.6 mm, 5 µm) using a mobile phase of 0.1% orthophosphoric acid buffer and acetonitrile (57:43, v/v) at a flow rate of 1.2 mL/min, with detection at 238 nm. The retention times were 2.57, 3.10, and 3.92 minutes for chlorthalidone, telmisartan, and cilnidipine, respectively, with a total run time of 8 minutes. The method demonstrated excellent linearity (r² > 0.99) across the tested concentration ranges, with mean recovery values close to 100% and %RSD values below 2%, confirming accuracy and precision. Specificity studies showed no interference from excipients, impurities, or degradation products. Forced degradation under acidic, alkaline, oxidative, thermal, photolytic, and neutral conditions confirmed the stability-indicating capability of the method. Limits of detection (LOD) and quantification (LOQ) were found to be within acceptable ranges, reflecting high sensitivity. Assay of marketed formulations indicated drug content within pharmacopoeial limits, demonstrating the applicability of the method for routine quality control. Overall, the developed UPLC method is simple, rapid, and reliable for simultaneous quantification of chlorthalidone, telmisartan, and cilnidipine, and can be applied effectively in quality control and stability testing of pharmaceutical products.
Hypertension remains one of the leading risk factors for cardiovascular morbidity and mortality globally, frequently necessitating combination therapy to achieve optimal blood pressure control. Three agents of interest are chlorthalidone, a long-acting thiazide diuretic; telmisartan, an angiotensin II receptor blocker; and cilnidipine, a dihydropyridine calcium-channel blocker with unique dual L- and N-type channel activity. The combination of these drugs offers complementary mechanisms—chlorthalidone reducing blood volume, telmisartan blocking vasoconstrictive angiotensin II effects, and cilnidipine relaxing vascular smooth muscle—yielding greater antihypertensive efficacy and favorable cardiovascular and renal protection. Analytical quality assurance of such fixed-dose combinations is essential. Regulatory guidelines (e.g. ICH Q2(R1)) mandate that analytical methods be validated—covering specificity, linearity, accuracy, precision, sensitivity, robustness—and, where relevant, stability-indicating to detect potential degradation products under stress (acidic, basic, oxidative, thermal, photolytic conditions). Existing literature has reported RP-HPLC and UV spectroscopic methods for simultaneous quantification of chlorthalidone, telmisartan, and cilnidipine in bulk and tablet forms. However, many of these methods exhibit limitations in run-time, sensitivity, solvent usage, or in-depth forced degradation studies. Ultra Performance Liquid Chromatography (UPLC) addresses several of these limitations by enabling higher chromatographic resolution, much shorter run times, and lower solvent consumption. Incorporating a validated stability-indicating UPLC method for the simultaneous determination of chlorthalidone, telmisartan, and cilnidipine would therefore serve both pharmaceutical quality control and regulatory compliance. The present study aims to develop and validate such a method for combined dosage forms, including comprehensive forced degradation profiling and system suitability, to ensure reliable assay in the presence of degradation products.
MATERIALS & METHODS
Chemicals and Reagents
Fig-1. Chemical Structures of drugs
Pharmaceutical-grade standards of Chlorthalidone (CHT), Telmisartan (TEL), and Cilnidipine (CIL) were obtained from certified suppliers. Marketed fixed-dose combination tablets containing the three drugs were purchased from a local pharmacy. HPLC-grade acetonitrile, methanol, and water were used as solvents. Orthophosphoric acid and hydrogen peroxide (analytical grade) were procured for buffer preparation and degradation studies. All solutions were filtered through a 0.45 µm nylon membrane filter prior to analysis.
Instrumentation
Chromatographic separation was carried out on a Waters Acquity UPLC system equipped with a photodiode array (PDA) detector. Data acquisition and processing were performed using Empower software. A Kromasil C18 column (50 mm × 2.1 mm, 1.7 µm particle size) was employed for method development. Additional instruments included a pH meter, ultrasonicator, and analytical balance with sensitivity ±0.1 mg.
Chromatographic Conditions
The optimized mobile phase consisted of 0.1% orthophosphoric acid buffer and acetonitrile (57:43, v/v), delivered at a flow rate of 0.3 mL/min. The injection volume was 5 µL, and detection was performed at 230 nm. Column temperature was maintained at 30 °C. The run time per analysis was approximately 4 minutes.
Preparation of Standard Solutions
Accurately weighed 10 mg each of CHT, TEL, and CIL were transferred into separate 10 mL volumetric flasks, dissolved in methanol, and diluted with mobile phase to obtain 1 mg/mL stock solutions. Working standards were prepared by suitable dilution to cover the calibration range of 2–50 µg/mL.
Sample Preparation
For tablet analysis, twenty tablets were weighed and powdered. A quantity equivalent to one tablet was transferred into a 100 mL volumetric flask, dissolved in methanol with sonication for 10 min, and filtered. Aliquots were diluted with mobile phase to obtain concentrations within the calibration range.
Forced Degradation Studies
Stress testing was performed as per ICH Q1A(R2) guidelines under acidic, alkaline, oxidative, thermal, and photolytic conditions. Samples were neutralized where necessary and analyzed to evaluate the stability-indicating capacity of the method.
Method Validation
The method was validated in accordance with ICH Q2(R1) for specificity, linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), robustness, and system suitability. Each parameter was assessed using replicate injections and statistical analysis.
RESULTS AND DISCUSSION
Method Development
The UPLC method was systematically optimized through six trials, involving adjustments in mobile phase composition, column dimensions, and operational parameters. The finalized method provided sharp and symmetrical peaks for chlorthalidone (CLTD), telmisartan (TELM), and cilnidipine (CILN). The optimized chromatographic conditions are summarized in Table 1, and the final chromatogram is shown in Fig. 1. The retention times for CLTD, TELM, and CILN were 2.57, 3.10, and 3.92 minutes, respectively, indicating efficient separation.
Fig: 1. Optimized method (Trail-6)
Table 1: Optimized Conditions
|
Parameter |
Optimized Condition |
|
Column |
C18, 250 × 4.6 mm, 5?µm |
|
MP |
0.1% OPA buffer : Acetonitrile (57:43 v/v) |
|
FR |
1.2 mL/min |
|
Detector λ |
238 nm |
|
Column Temperature |
30°C |
|
Injection Volume |
10 µL |
|
Run Time |
8 minutes |
|
Diluent |
Methanol, made up with Water:ACN (50:50) |
|
Retention Time (CLTD) |
2.57 min |
|
Retention Time (TELM) |
3.10 min |
|
Retention Time (CILN) |
3.92 min |
Method Validation:
The UPLC demonstrated excellent specificity, sensitivity, reproducibility, and consistency of chlorthalidone, telmisartan, cilnidipine combined forms.
System Suitability
System suitability testing confirmed the robustness of the developed method. Six replicate injections of a standard mixture demonstrated consistent retention times, sharp peaks, and satisfactory resolution. The theoretical plates exceeded 2000, peak tailing factors were <2, and %RSD values remained below 2% for all analytes. These findings confirm high reproducibility and compliance with ICH guidelines (Table 2).
Table 2: System Suitability
|
Parameter |
CLTD |
TELM |
CILN |
Acceptance Criteria |
|
RT (min) |
2.58 ± 0.02 |
3.11 ± 0.03 |
3.93 ± 0.02 |
Consistent, no drift |
|
Theoretical Plates |
4200 ± 150 |
4500 ± 130 |
7100 ± 180 |
> 2000 |
|
Tailing Factor |
1.1 ± 0.05 |
1.3 ± 0.04 |
1.2 ± 0.03 |
≤ 2 |
|
%RSD of Peak Area |
1.10% |
1.00% |
0.90% |
≤ 2% |
This data confirms that the UPLC system is suitably optimized with high precision and chromatographic performance consistent with regulatory standards.
Specificity
Specificity was confirmed by injecting blank, placebo, and stressed samples. The developed UPLC method successfully resolved the three drugs from each other and from degradation products without interference (Fig. 3). This highlights the method’s ability to selectively quantify the analytes in combined dosage forms.
Fig: 3. Specificity overlay chromatogram
Linearity
Linearity studies demonstrated an excellent correlation between peak area and drug concentration across the tested ranges (3–20 µg/mL for CLTD, 20–120 µg/mL for TELM, and 5–30 µg/mL for CILN). Regression analysis yielded correlation coefficients (r²) above 0.99 for all three drugs (Table 4), confirming suitability for quantitative analysis.
Table 4. Linearity Data
|
S.No |
Pipetted Volume (mL) |
Concentration (µg/mL) CLTD |
Concentration (µg/mL) TELM |
Concentration (µg/mL) CILN |
Linearity Level (%) |
|
1 |
0.25 |
3.25 |
20 |
5 |
25 |
|
2 |
0.5 |
6.5 |
40 |
10 |
50 |
|
3 |
0.75 |
9.75 |
60 |
15 |
75 |
|
4 |
1 |
13 |
80 |
20 |
100 |
|
5 |
1.25 |
16.25 |
100 |
25 |
125 |
|
6 |
1.5 |
19.5 |
120 |
30 |
150 |
|
S.No |
Pipetted Volume (mL) |
CLTD Conc. (µg/mL) |
CLTD Peak Area |
TELM Conc. (µg/mL) |
TELM Peak Area |
CILN Conc. (µg/mL) |
CILN Peak Area |
|
1 |
0.25 |
3.3 |
1,17,500 |
20 |
7,20,000 |
5 |
1,11,200 |
|
2 |
0.5 |
6.6 |
2,28,000 |
40 |
1,38,000 |
10 |
1,98,000 |
|
3 |
0.75 |
9.8 |
3,18,000 |
60 |
2,10,000 |
15 |
2,90,000 |
|
4 |
1 |
13.2 |
4,35,000 |
80 |
2,75,000 |
20 |
3,90,000 |
|
5 |
1.25 |
16.5 |
5,32,500 |
100 |
3,35,000 |
25 |
4,75,000 |
|
6 |
1.5 |
19.8 |
6,35,000 |
120 |
3,95,000 |
30 |
5,70,000 |
|
Drug |
Slope |
Correlation Coefficient (r²) |
|
Chlorthalidone |
32,100 |
0.991 |
|
Telmisartan |
32,850 |
0.99 |
|
Cilnidipine |
18,600 |
0.99 |
Accuracy
Recovery studies performed at 80%, 100%, and 120% levels confirmed the method’s accuracy. The mean recoveries were 100.10% for CLTD, 100.20% for TELM, and 100.13% for CILN, with %RSD values below 1.5% (Table 5 & 6). These results demonstrate high reliability for routine estimation.
Table 5. Accuracy Data
|
Drug |
Mean % Recovery |
%RSD |
|
CLTD |
100.10% |
1.30% |
|
TELM |
100.20% |
1.20% |
|
CILN |
100.10% |
1.40% |
Table 6: Results of Recovery
|
Drug |
Conc. Level (%) |
Amount Added (µg/mL) |
Std (µg/mL) |
Amount Found (µg/mL) |
% Recovery |
|
CLTD |
50 |
6.5 |
13 |
6.59 |
101.49 |
|
100 |
13 |
13 |
13.12 |
100.96 |
|
|
150 |
19.5 |
13 |
19.12 |
98.09 |
|
|
Mean % Recovery |
100.09 |
||||
|
%RSD |
1.32 |
||||
|
TELM |
50 |
40 |
80 |
40.61 |
101.54 |
|
100 |
80 |
80 |
81.54 |
101.93 |
|
|
150 |
120 |
80 |
118.05 |
98.38 |
|
|
Mean % Recovery |
100.18 |
||||
|
%RSD |
1.16 |
||||
|
CILN |
50 |
10 |
20 |
10.06 |
100.66 |
|
100 |
20 |
20 |
20.07 |
100.35 |
|
|
150 |
30 |
20 |
30.27 |
100.91 |
|
|
Mean % Recovery |
100.13 |
||||
|
%RSD |
1.4 |
||||
Precision
Intra-day and inter-day precision studies confirmed the method’s reproducibility. Method precision values were 1.2% for CLTD, 1.2% for TELM, and 0.9% for CILN. Intermediate precision showed %RSD values below 2% for all analytes (Table 7). These findings confirm consistent performance across different days and analysts.
Table.7: Repeatability & Intermediate precision
|
S.No |
Repeatability |
Intermediate Precision |
||||
|
CLTD Area (Intraday) |
TELM Area (Intraday) |
CILN Area (Intraday) |
CLTD Area (Interday) |
TELM Area (Interday) |
CILN Area (Interday) |
|
|
1 |
4,28,000 |
26,78,000 |
3,82,500 |
4,15,000 |
26,68,000 |
3,82,000 |
|
2 |
4,28,500 |
27,43,500 |
3,89,500 |
4,22,000 |
27,20,000 |
3,88,000 |
|
3 |
4,40,500 |
27,13,000 |
3,87,500 |
4,14,000 |
26,58,000 |
3,80,500 |
|
4 |
4,27,500 |
26,70,000 |
3,82,000 |
4,20,000 |
26,95,000 |
3,82,000 |
|
5 |
4,26,000 |
27,48,000 |
3,82,000 |
4,33,000 |
26,83,000 |
3,82,000 |
|
6 |
4,32,000 |
27,17,000 |
3,90,000 |
4,22,000 |
27,35,000 |
3,90,500 |
|
Avg |
4,30,000 |
27,12,000 |
3,85,000 |
4,20,000 |
26,92,000 |
3,83,000 |
|
% RSD |
1.30% |
1.30% |
0.90% |
1.80% |
1.20% |
1.10% |
Robustness
The method remained unaffected by small variations in flow rate, mobile phase ratio, and column temperature. No significant differences in retention time, peak symmetry, or %RSD were observed under modified conditions (Table 8). This indicates the robustness of the developed UPLC method.
Table 8: Robustness studies
|
Parameter |
Condition |
Drug |
Peak Area |
Retention Time (RT) (min) |
%RSD RT |
|
Flow Rate |
Optimized 1.2 mL/min |
CLTD |
7,48,941 |
2.58 |
1.5 |
|
Altered 1.1 mL/min |
CLTD |
6,56,517 |
3.94 |
1.8 |
|
|
Optimized 1.2 mL/min |
TELM |
48,69,203 |
2.99 |
0.3 |
|
|
Altered 1.1 mL/min |
TELM |
5,90,419 |
2.87 |
0.2 |
|
|
Optimized 1.2 mL/min |
CILN |
8,42,365 |
3.74 |
1.2 |
|
|
Altered 1.1 mL/min |
CILN |
9,13,918 |
2.53 |
0.8 |
|
|
Mobile Phase Ratio |
Optimized 57:43 |
CLTD |
5,22,591 |
2.57 |
1.2 |
|
Altered 52:38 |
CLTD |
4,67,907 |
3.91 |
1.4 |
|
|
Optimized 57:43 |
TELM |
32,99,325 |
3.1 |
0.5 |
|
|
Altered 52:38 |
TELM |
14,16,617 |
3.79 |
1.5 |
|
|
Optimized 57:43 |
CILN |
15,32,303 |
2.56 |
0.4 |
|
|
Altered 52:38 |
CILN |
96,04,089 |
2.88 |
0.8 |
|
|
Column Temperature |
Optimized 30°C |
CLTD |
7,26,410 |
2.55 |
0.2 |
|
Altered 25°C |
CLTD |
6,61,393 |
3.82 |
1 |
|
|
Optimized 30°C |
TELM |
46,68,240 |
2.92 |
0.3 |
|
|
Altered 25°C |
TELM |
8,38,444 |
3.71 |
0.2 |
|
|
Optimized 30°C |
CILN |
9,03,335 |
2.53 |
0.5 |
|
|
Altered 25°C |
CILN |
58,55,092 |
2.85 |
0.2 |
Stability of Sample Solution
Sample and standard solutions were found stable at room temperature for 24 hours. Chromatograms at 0 and 24 hours showed negligible changes in retention times and peak areas, confirming solution stability.
LOD and LOQ
The LOD and LOQ values indicate high sensitivity of the method. The LOD was 0.035 µg/mL for CLTD, 0.85 µg/mL for TELM, and 0.14 µg/mL for CILN. The LOQ values were 0.11, 2.45, and 0.42 µg/mL, respectively (Table 9).
Table9. LOD and LOQ
|
Drug |
LOD |
LOQ |
|
CLTD |
0.035 |
0.11 |
|
TELM |
0.85 |
2.45 |
|
CILN |
0.14 |
0.42 |
Assay of Marketed Formulation (MF)
The assay of the marketed formulation showed average recoveries of 99.80% for CLTD, 99.90% for TELM, and 100.20% for CILN, with %RSD values below 2% (Table 10). These results confirm accuracy and suitability of the method for routine quality control.
Table 10. Assay of MF
|
S.No |
% Assay of CLTD |
% Assay of TELM |
% Assay of CILN |
|
1 |
99.2 |
98.7 |
99.2 |
|
2 |
99.3 |
101 |
101 |
|
3 |
102 |
99.8 |
100.3 |
|
4 |
99 |
98.5 |
99.7 |
|
5 |
98.7 |
101.5 |
99.8 |
|
6 |
100.5 |
100.3 |
101.3 |
|
Avg |
99.80% |
99.90% |
100.20% |
|
% RSD |
1.30% |
1.20% |
1.00% |
Fig.40. Assay of Marketed Formulation
Forced Degradation Studies
Forced degradation under acidic, alkaline, and oxidative conditions revealed notable degradation, whereas thermal, photolytic, and neutral conditions showed minimal impact. Peak purity tests confirmed separation of degradation products from parent compounds. Mass balance values remained within 98–102%, ensuring stability-indicating capacity (Table 11).
Table 11: Mass Balance Data for Forced Degradation
|
Degradation Condition |
Assay (%) CLTD |
% Degraded CLTD |
Mass Balance (%) CLTD |
Assay (%) TELM |
% Degraded TELM |
Mass Balance (%) TELM |
Assay (%) CILN |
% Degraded CILN |
Mass Balance (%) CILN |
|
Control |
100 |
– |
100 |
100 |
– |
100 |
100 |
– |
100 |
|
Acid |
97.6 |
2.48 |
100.1 |
96.7 |
3.29 |
99.9 |
96.1 |
3.92 |
100 |
|
Alkali |
96.3 |
3.65 |
99.9 |
97.1 |
2.94 |
100 |
97.8 |
2.21 |
100 |
|
Oxidation |
97.2 |
2.71 |
99.9 |
96.6 |
3.36 |
99.9 |
98.5 |
1.56 |
100.1 |
|
Thermal |
99.5 |
0.47 |
100 |
99.7 |
0.33 |
100 |
99.1 |
0.96 |
100.1 |
|
UV |
99.6 |
0.46 |
100.1 |
99.8 |
0.02 |
99.8 |
99.2 |
0.76 |
99.9 |
|
Water (Neutral) |
99.3 |
0.7 |
100 |
99.6 |
0.39 |
99.9 |
99.1 |
0.92 |
100 |
CONCLUSION
A simple, robust, and stability-indicating UPLC method was successfully developed and validated for the simultaneous estimation of chlorthalidone, telmisartan, and cilnidipine in combined pharmaceutical dosage forms. The optimized chromatographic conditions provided sharp and well-resolved peaks with satisfactory system suitability parameters. Validation studies confirmed the method’s accuracy, precision, linearity, specificity, robustness, and sensitivity in compliance with ICH guidelines. Forced degradation studies further established the method’s ability to distinguish the active drugs from their degradation products, confirming its stability-indicating nature. The assay results of marketed formulations were within acceptable limits, demonstrating the method’s applicability in routine quality control and regulatory analysis. Overall, this validated UPLC method offers a reliable analytical tool for simultaneous determination of these three antihypertensive drugs in bulk and combined dosage forms.
Summary
|
Parameter |
Chlorthalidone (CLTD) |
Telmisartan (TELM) |
Cilnidipine (CILN) |
|
Max Absorbance Wavelength |
238 nm |
238 nm |
238 nm |
|
Retention Time (min) |
2.57 |
3.11 |
3.92 |
|
LOD (µg/mL) |
0.03 |
0.8 |
0.13 |
|
LOQ (µg/mL) |
0.1 |
2.42 |
0.4 |
|
Linearity Range (µg/mL) |
3.25 – 19.5 |
20 – 120 |
5 – 30 |
|
Correlation Coefficient (r²) |
0.999 |
0.9993 |
0.9992 |
|
% Assay in Formulation |
99.60% |
99.89% |
99.90% |
|
% Recovery |
100.09% |
100.18% |
100.13% |
|
% RSD (intra-day precision) |
1.20% |
1.20% |
0.90% |
|
% RSD (inter-day precision) |
1.70% |
1.10% |
1.00% |
|
% Drug Degraded (Forced Degradation - Acid) |
2.51% |
3.26% |
3.96% |
|
% Drug Degraded (Forced Degradation - Alkali) |
3.67% |
2.96% |
2.18% |
|
% Drug Degraded (Forced Degradation - Oxidation) |
2.68% |
3.38% |
1.54% |
|
% Drug Degraded (Forced Degradation - Thermal) |
0.45% |
0.35% |
0.98% |
|
% Drug Degraded (Forced Degradation - UV) |
0.44% |
0.01% |
0.74% |
|
% Drug Degraded (Forced Degradation - Neutral) |
0.72% |
0.37% |
0.90% |
|
Robustness |
Stable over variations |
||
|
Solution Stability |
Stable up to 24 Hr |
||
REFERENCES
Vankayalapati Sai Sravani*, Gopi Swapna, Development and Validation of a Stability Indicating UPLC Method for Simultaneous Determination of Chlorthalidone, Telmisartan and Cilnidipine in Pharmaceutical Combined Dosage Forms, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 10, 1826-1839 https://doi.org/10.5281/zenodo.17375018
10.5281/zenodo.17375018
