Novel experimental design approach for development of high-performance liquid chromatography method with fluorescence detection for amlodipine and telmisartan in their dosage form

Abstract

This study endeavors to investigate and authenticate a proposed more sensitive and selective high-performance liquid chromatography with a fluorescence detector technique for the assessment of a binary formulation of Amlodipine (ADP) and Telmisartan (TMS). Both analytes were chromatographically separated by an optimized solvent system comprising acetonitrile and phosphate buffer at pH 4 (58:42, v/v), using Discovery^®C18 reverse phase high-performance liquid chromatography column (150 mm). The fluorescence detector excitation and emission wavelengths were determined to be 240 and 440 nm, respectively. The experiment followed Plackett–Burman’s design to optimize chemigraphic experimental variables. To assess the linearity of the method, calibration curves were generated using 12.5–200 μg/mL for ADP and 0.5–10 μg/mL for TMS, with good correlation coefficients of r = 0.9980 and 0.9970, respectively. In addition, the limits of detection for ADP and TMS were 1.04 and 0.15 µg/mL, respectively, while the corresponding limits of quantification were 3.14 and 0.44 µg/mL. The accuracy of the technique was confirmed by high percentage recovery with low relative standard deviation for both within-day and between-day precision. The analysis of the two medications in their prescribed form (tablet) was executed, and the retrieval and relative standard deviation were computed, followed by the application of a t-test. The results of the tablet analysis indicated that the overall accuracy (mean ± relative standard deviation) of the method was 99.78% ± 0.90% for ADP and 100.99% ± 0.07% for TMS. Hence, the proposed high-performance liquid chromatography method can be utilized for regular quality control of binary formulations consisting of ADP and TMS.

Keywords

amlodipine fluorescence detector high-performance liquid chromatography Plackett–Burman’s design telmisartan

Introduction

Due to societal progress and population growth, chronic illnesses are now one of the major global health issues,¹ with hypertension being the most common disease. Most hypertensive patients require a combination of at least two anti-hypertensive drugs to accomplish timely blood pressure management and avert early events. Several drugs are used to treat high blood pressure. The combination of Amlodipine (ADP) and Telmisartan (TMS) is a new pharmaceutical formulation of two blood pressure drugs in one tablet for adequate blood pressure control.²

Amlodipine (ADP, Figure 1(a)) is a third-generation dihydropyridine calcium channel blocker (CCB) utilized in the treatment of hypertension. The mechanism of action of CCB is the obstruction of extracellular calcium convergence by a voltage-gated L-type calcium channel, resulting in a reduction in the intracellular calcium concentration in vascular smooth muscle cells.^3,4 Telmisartan (TMS, Figure 1(b)), a specific angiotensin II (AII) type 1 (AT1) receptor inhibitor, is endorsed for the treatment of hypertension, either alone or in conjunction with other antihypertensive agents.⁵

Figure 1.

Chemical structures of Amlodipine (a) and Telmisartan (b).

Different high-performance liquid chromatography (HPLC) methods for the analysis of antihypertensive medication formulations in pharmaceutical preparations have been detailed in the past. The application of HPLC in pharmaceutical analysis was discussed in a survey article, and the application of HPLC within the pharmaceutical industry is significant because of the fast, accurate, and selective quantification of analytes.^6,7 A literature review demonstrated the feasibility of enhancing the environmental sustainability of reverse phase–HPLC (RP-HPLC) methods utilized in pharmaceutical analysis through the implementation of specific strategies.⁸ The RP-HPLC approach offers a significant advantage compared to the high-performance thin-layer chromatography (HPTLC) method in terms of sensitivity, resolution, and cost and it is compatible with a diverse array of detectors, these studies aimed to establish and validate the RP-HPLC method for the simultaneous analysis of chemical and pharmaceutical formulations.^9,10

There are many analytical techniques for the concurrent quantification of ADP and TMS in formulations, including UV spectrophotometry. Kondwaret al.¹¹ used a UV spectrophotometric technique to estimate ADP and TMS in bulk drug and dosage forms by multiwavelength analysis, while Sinha etal.¹² validated an HPLC-UV detector method for the analysis of ADP and TMS in their dosage form. A normal phase HPLC with UV detector methodology was applied by Deshmukh et al.¹³ to investigate ADP and TMS in dosage form. In addition, Marolia et al.¹⁴ developed an HPTLC method for the simultaneous estimation of ADP and TMS in their combined dosage form. Modroiuet al.¹⁵ developed a method for the analysis of ADP and TMS using capillary electrophoresis.

Achieving good analytical performance has been correlated with linearity, precision, reproducibility, and robustness. However, the reported HPLC methods have long analysis times, narrow linearity ranges, and low sensitivities due to the use of UV detectors. Furthermore, no HPLC method with a fluorescence detector has been reported for concurrent quantification of ADP and TMS in formulations.

In contrast to HPLC-UV, fluorescence detection offers significantly enhanced sensitivity and selectivity. Within fluorescence, photon absorption at a molecular level induces the emission of a photon with an extended wavelength. This disparity in wavelengths between absorption and emission contributes to heightened selectivity, and the fluorescence intensity is compared against a background of minimal light, consequently enhancing the signal-to-noise (S/N) ratio.¹⁶–¹⁸ The novelty of this study is attributed to the creation of a novel and extensively refined HPLC methodology integrated with fluorescence detection for the simultaneous analysis of ADP and TMS within their combined dosage forms. What distinguishes this work is the enhancement of the methodology, thereby ensuring superior sensitivity, selectivity, and efficiency in comparison to conventional methodologies. The purpose of this study is to examine a novel HPLC-RF method (calibration range, precision, limit of detection, limit of quantification, and sample frequency) in accordance with international guidelines. Moreover, proposed methods for the analysis of these medicinal products in combination dosage forms.

Materials and methods

Equipment and chromatographic conditions

High Performance liquid chromatography (SHIMADZU Prominence-i LC-2030) with fluorescence detector (RF 20 detector), two sample injectors (A 1.5 mL and B 1.5 mL), 440 bar LC-2030 pump low-pressure gradient mode, Heater/Cooler oven and LC-2030 Auto sampler and LC-2030 Auto purge was used for the analysis. Lab software solution consists of LC-2030 Controller was used to monitor the eluent. RP HPLC Discovery^®C18 column 150 mm × 4.6 mm × 5 µm particle size and a mobile phase consisting of acetonitrile and phosphate buffer at pH 4 (58:42, v/v/) used for separation of analytes at room temperature. The flow rate of mobile was adjusted to 1 mL/min in low-pressure mode, with 20 µL of sample injection. The fluorescence detector excitation and emission wavelengths were determined to be 240 and 440 nm, respectively.

Chemicals and reagents

ADP and TMS in combination TWYNSTA (claimed labeled amount 10 mg ADP and 80 mg TMS per tablet), was obtained from local pharmacies (manufacturer, Boehringer Ingelheim Pharma GmbH& Co. KG, Germany) All additional chemicals (analytical grade) and HPLC-grade methanol were utilized. ADP and TMS standards were provided from Sigma Aldrich company. The CAS No. for ADP is 111470-99-6, while the CAS No. for TMS is 144701-48-4. As for the solvents employed in the process, all of them were of HPLC grade. These solvents include water, methanol, acetonitrile, and disodium hydrogen phosphate.

Stock and standard solutions

To prepare stock solutions for ADP and TMS, 40 mg of TMS and 5 mg of ADP were weighed out. These weighed amounts were dissolved independently in methanol. Each drug was dissolved in a 100-mL volumetric flask. After preparing the stock solutions, advance dilutions were carried out to achieve a final concentration of 40 µg/mL for TMS and 5 µg/mL for ADP.

System suitability tests

Linearity and sensitivity

A series of solutions containing ADP and TMS were prepared in 25 mL volumetric flasks, and the original solutions were appropriately diluted with the mobile phase to achieve a concentration range of 12.5–200.0 μg/mL for ADP and 0.5–10.0 μg/mL for TMS. For each solution, six injections were performed at each level. The peak areas were graphed against the corresponding drug concentrations.²⁰ The limits of detection (LOD) and limits of quantification (LOQ) were determined using the equation (1)

LOQ or LOD = κ SDa / b

(1)

where κ = 10 for LOQ and 3.3 for LOD. SDa is the standard deviation of the intercept and b is the slope.²¹

Precision

The precision of the assay was evaluated by examining solutions with varying concentrations of ADP and TMS: ADP = 12.5, 100, and 200 μg/mL; TMS = 1, 5, and 10 μg/mL. Each of these solutions was subjected to analysis on five replicates on the same day (intraday). This allowed for the evaluation of precision within a day, often referred to as intraday precision. Furthermore, between-day (interday) precision was assessed by analyzing the same solutions three times over a 3-day period. This enabled the examination of variations in results across different days. Precision was quantified using percentage relative standard deviation (RSD%) values. RSD is a metric that assesses the variability in results and is obtained by dividing the standard deviation (SD)by the mean, expressed as a percentage.²⁰

Accuracy

The validity of the proposed approach was established by quantifying three concentrations of ADP (100, 50, and 25 μg/mL) and TMS (5, 2.5, and 1 μg/mL) using the technique of standard addition.^20,22 A measured quantity of a powdered tablet containing 10 mg of ADP and 80 mg of TMS was transferred into a 25-mL flask. Subsequently, the contents within the flask were subjected to sonication for 10 min. The solution then underwent dilution up to a specific volume of 25 mL and was filtered using a 0.45-μm nylon membrane filter. A volume of 0.05 mL of the filtered solution was supplemented with three corresponding solutions from the stock solution. The process of supplementation entailed adding a known quantity of the analytes of interest to the sample to create predetermined concentrations. This step allowed for the assessment of the accuracy of the method by comparing the anticipated concentration based on the added amount with the observed concentration. After supplementation, the solution was further diluted to a final volume of 10 mL. A volume of 0.05 mL from the resulting diluted solution was pipetted and transferred into a 1-mL vial. An equal volume of 12.4 mg/mL ADP standard was introduced, and the solution was brought up to the desired volume with a mobile phase. The resulting solution was subsequently subjected to analysis using the proposed analytical method. These operations, encompassing supplementation and analysis, were repeated three times for each quantity added.²⁰

Assay in tablets

Twenty tablets of the medication (TWYNSTA) were accurately weighed and finely powdered. The medication powder corresponding to 10 mg of ADP and 80 mg of TMS was transferred to a 25-mL volumetric flask for the purpose of analyzing the amount of ADP and TMS in the tablet samples. The contents within the flask were subjected to sonication for 10 min. This step was expected to facilitate the dissolution and homogenization of the tablet powder in the solvent, which in this case was methanol. After sonication, the solution was diluted with methanol until it reached the mark (25 mL). This dilution procedure was implemented to ensure that the concentration of the analytes, specifically ADP and TMS, fell within the detectable range of the analytical method. Part of the diluted solution, measuring 0.05 mL, was then transferred from the 25 mL volumetric flask to a 10-mL volumetric flask, where it was further diluted with methanol.

From the aforementioned solutions, 0.05 mL of the final solution was transferred to a 1-mL vessel. Subsequently, an equal quantity of ADP standard, possessing a concentration of 12.4 mg/mL, was introduced into this 1 mL vessel. The purpose of this standard was to validate the precision of the analysis. The solution was then supplemented to its maximum volume by adding the mobile phase. A volume of 20 μL from the previously prepared solutions was introduced into the column. The test was repeated six times, and during each repetition, the pellet corresponding to the tablet powder equivalent was weighed individually. This process of replication was undertaken to ensure the reliability and reproducibility of the assay results.²³

Results and discussion

Chromatography optimization

The optimization of chromatographic conditions was performed using the proposed method to achieve the most favorable peak resolution and shape for the analysis of ADP and TMS. Various mobile phases were examined, including methanol in phosphate buffer and acetonitrile in phosphate buffer. However, the results with methanol and phosphate buffers were deemed unsatisfactory as the peaks of ADP and TMS were broad. Attempts were made to use phosphate buffers with a pH ranging from 3.0 to 5.0, the peak shape of the drug exhibited sufficient symmetry only at a pH value of 4. Consequently, the optimal final mobile phase was determined to consist of acetonitrile and the phosphate buffer at pH 4 (in a ratio of 58:42, v/v). This selection allowed for the attainment of well-defined and resolved peaks, with average retention times of 2.1 and 3.55 min for ADP and TMS, respectively.

The chromatograms obtained from the solutions of the ADP and TMS tablets, as well as those of the dissolution medium, specifically the phosphate buffer at a pH of 4, exhibited a remarkable similarity to chromatograms obtained from the standard solutions containing equivalent concentrations. This confirms the adequate selectivity of the proposed method. ADP and TMS were monitored using RF detectors throughout the investigation. The optimal wavelengths for excitation and emission were determined to be 240 or 440 nm respectively.²⁴

Experimental design

Table 1²⁵ lists the variables that were typically examined in this study at two levels: low (−1) and high (+1), as well as their nominal values. In order to set the Plackett–Burman design for n = 8,²⁰ one dummy variable was introduced since six genuine factors were selected. The resolution factor (Rs) between ADP and TMS was chosen as the most crucial metric for further investigation. The relevant statistics are displayed in Table 2.

Table 1.

Variables employed for performing analyses in this study.

Variable		Lower value(–1)	Normal value(0)	Upper value(+1)
A	Flow rate (mL; Min-1)	0.80	1.00	1.20
B	Acetonitrile content (%)	50.00	58.00	60.00
C	pH of the buffer	3.50	4.00	4.50
D	Concentration of the buffer (M)	0.01	0.10	0.20
E	Excitation wavelength (nm)	228.00	240.00	255.00
F	Emission wavelength (nm)	400.00	440.00	448.00
G	Dummy 1	−1.00	0.00	1.00

Table 2.

Outline of the Plackett–Burman design, which is based on the A-G Variables described in Table 1. The design incorporates the resolution factor, denoted as Rs.

Run	A	B	C	D	E	F	G	Rs
1	1	1	1	1	1	1	1	3.02
2	1	1	–1	1	–1	1	–1	2.72
3	1	–1	–1	–1	–1	–1	1	2.05
4	1	–1	1	–1	1	–1	–1	2.36
5	–1	1	1–	–1	1	1	1	2.24
6	–1	1	–1	1	–1	–1	–1	2.85
7	–1	–1	1	1	–1	1	1	2.45
8	–1	–1	1	–1	1	–1	–1	2.65

System suitability

Standard solutions of ADP (100 µg/mL) and TMS (5 µg/mL) solutions were analyzed using the optimized chromatographic conditions in six replicates as a part of system suitability tests. The resolutions of the peaks and theoretical plate values were above 2 and 2000, respectively. The SDs calculated for retention time, resolution theoretical plate, and peak symmetry were within the acceptable ranges, as shown in Table 3. Figure 2 shows the representative chromatogram for ADP and TMS and the specific conditions and Figure 3 shows the chromatogram obtained by tablet formulation of ADP and TMS.

Table 3.

System suitability and validation parameters results.

HPLC Parameters	ADP	TMS
Retention time (min) ± SD	2.1 ± 0.01	3.5 ± 0.02
Resolution ± SD	—	3.05 ± 0.02
Capacity factor	1.40	2.40
Theoretical plate ± SD	7052 ± 54.60	5045 ± 52.10
Peak asymmetry	1.0 ± 0.05	0.95 ± 0.07
Linearity range (µg/mL)	12.50-200	0.50-10.00
Slope	1.26	56.72
Intercept	8.87	97.63
Correlation coefficient (R²)	0.998	0.998
LOD (µg/mL)	1.00	0.15
LOQ (µg/mL)	3.10	0.44

Figure 2.

Representative chromatogram for 100 and 5 µg/mL ADP and TMS, respectively (Mobile phase Acetonitrile and 0.1 M Disodium hydrogen phosphate buffer (pH 4) 58:42, flow rate 1, excitation and emission wavelengths are 240 and 440 nm, respectively).

Figure 3.

Chromatogram obtained by tablet formulation of ADP and TMS (Mobile phase Acetonitrile and 0.1 M Disodium hydrogen phosphate buffer (pH 4) 58:42, flow rate 1, excitation and emission wavelengths are 240 and 440 nm, respectively).

Method validation

The International Council for Harmonization (ICH) guidelines for method validation was followed for validation of the suggested method.²⁶

Linearity

The LOD and LOQ were determined using the equation (1)²¹ according to the ICH guidelines.²⁶

A total of six calibrators were employed for TMS (0.5, 1, 2.5, 3.5, 5, and 10 µg/mL) and an equal number of calibrators were utilized for ADP (12.5, 25, 50, 75, 100, and 200 µg/mL). The regression parameters were determined in accordance with the method of least squares. Consequently, ADP and TMS demonstrated satisfactory linearity, as indicated by their respective correlation coefficients of r = 0.998 and 0.997.²⁷ The slope values obtained for ADP and TMS also revealed a significant level of sensitivity, with values of 1.3 and 34.3, respectively, and SDs of 0.03 and 0.89, respectively. The minimal SD values, in comparison to the slope values for both drugs, signify a minimal degree of uncertainty in the regression analysis. This minimal uncertainty can be attributed to the influence of indeterminate errors on the slopes.²⁷

Regarding calibrations, the y-intercept values were +8.87 and +85.94, respectively. Although the calibration for ADP yielded a positive intercept, the modest outcome suggested a low level of interference with the response or a low degree of saturation of the responses at high concentrations. Similarly, the positive y-intercept for TMS, which is of considerable magnitude, lends support to the assumption of acceptable sensitivity. In addition, the SDs of the y-intercepts for ADP and TMS were 2.78 and 4.38, respectively. The minimal SDs in relation to the intercepts of both medications suggested a minimal level of uncertainty in the calibration. Both ADP and TMS displayed standard errors of the estimate, or alternatively, SDs of 4.29 and 6.94, respectively, indicating data points closer to the calibration curve. On the contrary, the determination of the LOD indicated that the values for ADP and TMS were established at 1.04 and 0.15 µg/mL, respectively, while the respective LOQ were 3.14 and 0.44 µg/mL. It is worth noting that the LOD and LOQ values for TMS were lower than those for ADP. Nevertheless, both medications possess active constituents and are found in elevated concentrations; thus, all these values were considered satisfactory for the analysis of both drugs in their pharmaceutical formulations.

Precision

The precision of the assay was evaluated according to ICH guidelines,²⁶ by examining solutions with varying concentrations of ADP and TMS: ADP = 12.5, 100, and 200 μg/mL; TMS = 1, 5, and 10 μg/mL. The data obtained from the precision experiments are presented in Table 4. The RSDs revealed that the interday precision of ADP ranged from 1.5% for the lowest concentration to 0.8% for the highest concentration. TMS, by contrast, exhibited corresponding values ranging from 0.8% to 1.0%. In terms of the intraday accuracy of ADP, the RSDs were determined to be 2.9% for the lowest concentration and 1.5% for the highest concentration. For TMS, the corresponding values ranged from 0.7% to 0.8%. Further analysis was conducted to evaluate intraday precision.

Table 4.

Precision of the method for standard solutions for ADP and TMS.

Drug (μg/mL)	Intraday precision (N = 5)		Interday precision (N = 3)
Drug (μg/mL)	Recovery mean ± SD	RSD%	Recovery mean ± SD	RSD%
ADP
12.5	98.5 ± 1.14	2.89	98.33 ± 0.58	1.47
100	100.0 ± 0.82	1.22	101.90 ± 1.15	1.62
200	101.7 ± 2.07	1.45	99.05 ± 1.15	0.83
TMS
1	99.84 ± 0.84	0.67	100.00 ± 1.00	0.79
5	99.85 ± 0.96	0.55	100.19 ± 0.58	0.32
10	99.24 ± 2.10	0.80	99.23 ± 2.65	1.03

The RSD values demonstrate a high level of precision of the proposed method.

Accuracy

The validity of the proposed approach was established by quantifying three concentrations of ADP (100, 50, and 25 µg/mL) and TMS (5, 2.5, and 1.0 µg/mL) using the technique of standard addition.^20,23 The method’s accuracy, as shown in Table 5, was demonstrated by examining treated samples of ADP and TMS according to ICH guidlines.²⁶ Accordingly, the recovery of the active components from the substrate was obtained, and the proposed analytical method illustrated adequate accuracy.

Table 5.

The method's accuracy in the fortified samples of ADP and TMS.

Drug	Expected concentration (μg/mL)	Measured concentration (μg/mL)	Recovery%
ADP	100.00	100.10	100.10
	50.00	50.10	100.20
	25.00	25.10	100.40
TMS	5.00	4.90	98.00
	2.50	2.40	96.00
	1.00	0.98	98.00

Assay in tablets

This assay was conducted to analyze the amount of ADP and TMS in the tablet samples. The test was repeated six times according to ICH guidelines.²⁶ Table 6 showcases the statistical analysis conducted on the outcomes derived from the examination of ADP and TMS levels in the tablets. The tablets exhibited a total recovery of 99.78% ± 0.34% and 100.99% ± 0.07% for ADP and TMS, respectively. This confirmed the consistency of the results. A Student t-test of the samples assuming equal variances did not demonstrate statistically significant differences between the content of the tablets and the reported content, as the statistical t-value was less than the critical one for both drugs, and their p-value was more than 0.9. Furthermore, the estimated outcome was derived by calculating the 95% confidence interval and verifying whether the declared amount fell within that range. According to ICH guidelines,²⁶ the reported levels aligned with the confidence intervals, indicating the satisfactory accuracy of the tablet determination method.

Table 6.

Statistical assessment of the results achieved for the analysis of ADP and TMS in tablets.

Assay	Declared Amount (μg/mL)	RecoveryMean ± SD%	RSD	95% Confidence interval	t-test
Tablets	Declared Amount (μg/mL)	RecoveryMean ± SD%	RSD	95% Confidence interval	t	p
ADP	12.70	99.80 ± 0.34	2.70	99.00-100.60	0.03	0.97
TMS	0.80	100.90 ± 0.07	8.70	100.90 -101.10	0.10	0.90

t critical value = 2.78²³

Comparison of the reported RP-HPLC methods for analysis of ADP and TMS with the method under study

A comparison was made between the proposed HPLC-RF method and previous HPLC-UV methods for the determination of ADP and TMS. Table 7 illustrates the contrast between the novel HPLC technique utilizing an RF detector, as employed in the present study, and the HPLC method outlined in prior research, all of which relied on the UV detector.

Table 7.

Comparison between the reported HPLC methods and the employed RF-HPLC method.

Drug	Mobile phase	Column	Detector	Retention time	Linearity	LOD and LOQ	RecoveryMean ± SD%	Reference
ADP	Acetonitrile, 0.05 M Sodium dihydrogen phosphate buffer (60:40), pH 6.0	C-18 column 150 mm × 4.6 mm., 5 μm	UV detector	4 min	5–30 μg/mL	50.0 ng/mL, 140.0 ng/mL	99.86 ± 1.18	Suárez¹⁴
TMS		C-18 column 150 mm × 4.6 mm., 5 μm	UV detector	8.2 min	10–60 μg/mL	2 µg/mL, 4.0 µg/mL	100.87 ± 1.18	Suárez¹⁴
ADP	Acetonitrile: Methanol: Triethylamine buffer, pH 5.0	C18 (250 mm × 4.6 mm, 5 μm)	UV detector	4 min	50.0–60.0 µg/mL	0.7 µg/mL, 2.2 µg/mL	98.85 ± 0.69	Chabukswar et al.²⁰
TMS	Acetonitrile: Methanol: Triethylamine buffer, pH 5.0	C18 (250 mm × 4.6 mm, 5 μm)	UV detector	8.2 min	320.0–480.0 µg/mL	0.04, 0.1 µg/mL	101.39 ± 0.39	Chabukswar et al.²⁰
ADP	Methanol: potassium dihydrogen Phosphate buffer at pH 4.5 (75:25 v/v)	C18 column (250 × 4.6 mm ID)	UV detector	3.1 min	2.0–20.0 μg/mL	-	99.83 ± 0.42	Chabukswar et al.²⁰
TMS		C18 column (250 × 4.6 mm ID)	UV detector	5.8 min	16.0–160.0 μg/mL	-	99.80 ± 0.42	Chabukswar et al.²⁰
ADP	Acetonitrile and disodium hydrogen phosphate buffer pH 4 (58:42, v/v/)	C18 column (150 mm × 4.6 mm × 5 µm)	RF 20 detector	2.1 min	12.5–200.0 μg/mL	1.0 μg/mL, 3.1 μg/mL	99.80 ± 0.3	This study
TMS		C18 column (150 mm × 4.6 mm × 5 µm)	RF 20 detector	3.55 min	0.5–10.0 μg/mL	0.15 μg/mL, 0.44 μg/mL	100.90 ± 0.07	This study

From Table 7, it can be observed that the results of this study in which RF detector was applied show a shorter retention time for ADP and TMS compared to the other reported methods in which UV detector was used.^14,20,20 The proposed method demonstrates a greater level of sensitivity, as indicated. Moreover, the process of this study was linear in a larger range for ADP and TMS compared to the other reported methods.^14,20,20 In addition, the proposed method shows a favorable level of accuracy when compared to prior methods,^14,20,20 highlighting a significant level of recovery with minimal standard deviation in both ADP and TMS.

Conclusion

A novel, reliable, and validated HPLC-fluorescence detection method was established for the concurrent determination of ADP and TMS. Under the conditions that were optimized, a baseline separation of the two analytes was achieved in less than 4 min. The suggested approach exhibited accuracy, within-day and between-day precision and linearity. Furthermore, the optimized method was effectively utilized to determine the analytes in the co-formulated tablets. This indicates that the tablets exhibited a satisfactory level of accuracy overall (mean ± RSD). The Student t-test did not demonstrate statistically significant differences between the content of the tablets and the reported content. It can be affirmed that this methodology adheres to all designated standards and is appropriate for analytical objectives as well as for quantifying ADP and TMS in mixtures of different pharmaceutical compounds and can be efficiently employed in the realm of quality control.

Footnotes

Author contributions

Conceptualization, A.Y.A. and R.E.E.; methodology M.S.E. and A.Y.A.; software, A.Y.A.; validation, R.E.E., M.A. and A.Y.A.; formal analysis, M.S.E.; investigation, A.Y.A.; resources, M.S.E.; data curation, A.Y.A. and M.S.E.; writing—original draft preparation, A.Y.A.; writing—review and editing, M.A. and A.A.E.; visualization, A.A.E.; supervision, A.Y.A. project administration, A.Y.A.; funding acquisition, M.S.E. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The data is available upon request.

Declaration of conflicting interests

The author declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was funded by Deanship of Scientific Research, King Faisal University, under Ambitious Researcher, grant No. 250602.

Ethical approval

This manuscript does not contain any studies with human or animal participants.

Consent to participate

The authors confirm that all authors participate in the research work of this manuscript.

Consent for publication

The authors confirm that we give our full consent for publishing this manuscript.

ORCID iDs

Amel Y Ahmed

Abdalla A Elbashir

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