QbD-driven RP-HPLC method for the simultaneous analysis of dihydropyridines calcium channel blockers in pharmaceuticals

Abstract

A rapid, sensitive, and selective reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the simultaneous determination of five dihydropyridines calcium channel blockers, namely: amlodipine (AML), nifedipine (NIF), lercanidipine (LER), nimodipine (NIM) and nitrendipine (NIT) using Quality by Design approaches (QbD). The chromatographic separation was achieved on Luna C8 (100 × 4.6 mm 3 μm) column using an isocratic mobile phase consisting of acetonitrile—methanol − 0.7% triethylamine pH 3.06 (30-35-35) adjusted with ortho phosphoric acid at a flow rate of 1 mL/min with UV detection at 237 nm. The optimized method demonstrated good chromatographic resolution with average retention times of 2.93, 3.98, 4.98, 6.32 and 7.75 min for AML, NIF, LER, NIT and NIM, respectively. The method was validated according to ICH guidelines, showing linearity in the concentration range of 10–50 µg/mL with correlation coefficients (r²) ≥ 0.9989 for all analytes. The method demonstrated high trueness (99.11–100.09%) and precision (RSD < 1.1%). The validated method was successfully applied for the determination of these drugs in their pharmaceutical formulations, with recovery values ranging from 99.57 to 100.07% and without significant interference from excipients. Greenness and practicality evaluations detected using AGREE, MoGAPI, complex MoGAPI, AGSA, CaFRI, BAGI and CACI tools which were, indicating the method’s environmental friendliness and excellent practical applicability for routine pharmaceutical quality control analysis.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13065-025-01661-5.

Introduction

The incidence of hypertonic disease is increasing significantly every year. This disease is accompanied by high mortality among people of working age. Often, due to a stable increase in blood pressure, the risk of disability and cerebrovascular disorders increases. Some of the drugs used to treat this pathology belong to calcium channel blockers in particularly dihydropyridine derivatives. Such drugs as amlodipine (AML), nifedipine (NIF), lercanidipine (LER), nimodipine (NIM) and nitrendipine (NIT) are used both for monotherapy and in combination therapy in the treatment of hypertonic disease and other diseases of the cardiovascular system [1–6]. The main challenges in HPLC of calcium channel blockers are their prone and tendency for appearing as skewing peaks in form of peak tailing, as a results of their dihydropyridine structure which generate secondary interactions with free Si–OH groups of the silica particles in stationary phase matrixes. Thereafter, the main nominator for all these five mentioned molecules of API with dihydropyridine-based structure, use significant quantity of strongest competitor for residual matrix silanol groups, the very strong base triethylamine, or search for most expensive best end-capped and base-deactivated RP-silica sorbent, especially newer hybrid and synthetic based. Quality by design (QbD) approaches are introduced and interpreted in implementing the principles of «green chemistry», reducing the number of chemical reagents and solvents used, that will not only help preserve the environment but also reduce health risks for laboratory personnel. HPLC and redox titration methods have been presented in United State Pharmacopeia (USP) and European Pharmacopeia (Eur.Ph.) monographs for the assay of various calcium channel blockers in bulk and dosage form [7, 8]. Liquid chromatography is widely used for drug analysis due to its high sensitivity, reproducibility, and selectivity. Several HPLC methods for the determination of AML [9–13], NIM [14–16], NIF [17–19], LER [20–27], NIT [28–31] in mono or in combination in pharmaceutical dosage forms are presented in scientific literature. However, these methods often present a significant risk of environmental pollution and danger to laboratory workers due to the use of toxic solvents and reagents. In addition, in some chromatographic methods, the retention is very long, which has a negative impact on the load and service life of the equipment. In scientific literature no methods for the simultaneous chromatographic determination of five calcium channel blockers. The development of such a method is extremely difficult because all the analytes to be determined have a similar structure and are derivatives of 1,4-dihydropyridine. Therefore, considering new approaches to the components of mobile phases and optimization of chromatographic conditions we propose a new, environmentally friendly and simple approach for the simultaneous determination of five calcium channel blockers.

The aim of our work is the development of simple, rapid and selective RP HPLC method for the simultaneous determination of dihydropyridines calcium channel blockers (AML, LER, NIF, NIM, NIT) in pharmaceutical formulations and model mixture using QbD approaches for the optimization and creation of new environmentally safe analytical methods, reducing the pharmaceutical cost of products due to the rational use of materials and reducing the load on equipment. By employing QbD based presentations, calculations and conclusions, our work will contribute to new other targeted HPLC method development intentions, by assuming our experiments for peaks retention movement behaviors from the changes of experimental conditions, and their impacts on system suitability parameters interpreted by peak capacity, shape, symmetry and resolutions. The value and scientific contribution of our work become valuable by the facts of employment and testing different reversed phase types C18, C8 and phenyl based stationary phases and inter-manufacturer specifications and separation manifestations of tested columns with chromatographic experimental variables. For example, the two different octadecylsilane or C18-based columns, like Luna(2)C18 and InertClone C18 ODS, have very different features, specified characteristic’s, like carbon load in percentages, active surfaces in m²/g, level of end-capped residual silanol Si-OH groups, type of alkyl-bonding reaction process and success, different level of elimination of residual metal cations in silica particles with base-deactivation step, recommended pH working range as resistance to degradation from solvent acidity, all parameters are available in manufacturers issued and available columns specifications.

Experimental

Instrumentation

HPLC analysis was performed using Shimadzu LC-2050 C with diode array detector (DAD). Chromatographic data acquisition and post run analysis integration were performed using LabSolutions software. A digital pH meter (Mettler-Toledo, model LE438, Switzerland) was used for pH measurements. An ultrasonic bath (Elmasonic Easy 40 H, Germany) was used for degassing the mobile phase and dissolving the samples. RADWAG AS 200/C precision analytical balances were used for accurate weighing.

Used chromatographic columns: Luna C8 (150 × 4.6 mm 3 μm), Luna C18 (100 × 4.6 mm 3 μm), InertClone ODS (150 × 4.6 mm 3.5 μm) purchased from Phenomenex; Zorbax SB C8 (150 × 4.6 mm 3.5 μm) and Zorbax SB Phenyl (150 × 4.6 mm 3.5 μm) purchased from Agilent Technologies.

Reagents and standards

AML, NIF, NIM, LER and NIT (purity ≥ 98% (HPLC)) were supplied from Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA).

Amlodipine besylate 10 mg tablets («Amlodipine» Farmak), Nitrendipine 10 mg tablets («Nitresan» PRO. MED. CS Praha a. s.), Nifedipine 10 mg tablets («Nifedipine» Lekhim), Nimodipine 30 mg tablets («Nimodipine» PJSC SIC “Borshchahivskiy CPP”), Lercanidipine 10 mg tablets («Lercamen» Berlin-Chemie. Menarini) were purchased from local drugstore.

Methanol (MeOH), acetonitrile (ACN) and water (HPLC grade, ≥ 99.9% purity) were acquired from Honeywell, Riedel-de Haen (Germany). Phosphoric acid 88% (for pH adjustment) was obtained from Honeywell Fluka. Triethylamine (TEA), potassium dihydrogen phosphate, trifluoroacetic acid (TFA) (HPLC purity) were supplied from Scharlab S.L.(Spain). A filter regenerated cellulose (RC) Membrane (pore size 0.45 μm, Phoenix, Germany) was used to filter the tested solutions. All chemicals utilized in the experiment were analytical grade.

Standard solutions

Preparation of stock standard solutions

Stock standard solutions of AML, NIF, NIM, NIT, LER (1000 µg/mL) were prepared separately dissolving 25 mg of each drug into 25 mL volumetric flasks, each containing 15 mL of MeOH. The contents of each flask were sonicated to ensure complete dissolution. Then the volume was completed with MeOH. A volume of 1 mL was transferred from each flask into a 10 mL volumetric one to prepare a standard solution mixture of the five analytes.

Preparation of working standard solutions

To prepare a working standard solution (100 µg/mL) 1 mL of each stock solution (1000 µg/mL) was diluted with MeOH into a 10 mL flask.

Chromatographic conditions

Isocratic chromatographic separation was performed on a Luna C8 column (150 mm × 4.6 mm; 3 μm particle size) maintained at 30 ± 2 °C. The mobile phase consisted of ACN, MeOH and 0.7% TEA with pH adjusted to 3.06 using phosphoric acid in the ratio of 30:35:35% v/v. The mobile phase was degassed ultrasonically prior to use. The flow rate 1.0 mL/min, the injection volume was 3 µL. UV detection was performed at 237 nm with a total run time of only 7.60 min. Baseline was adjusted by equilibrating the column with the mobile phase for 30 min before starting the analysis.

Procedures

Working standard solutions AML, NIF, NIM, NIT, LER (each 100 µg/mL) were transferred into a series of 10 mL volumetric flasks. All flasks were made up to the mark with water to produce final concentrations of 10, 20, 30, 40, and 50 µg/mL for each analyte. The studies were performed under optimized chromatographic conditions 2.4. Calibration curves were constructed in the coordinates of the dependence of peak areas and the analyte concentration in µg/mL. Regression equations were calculated for each analyte.

Application to tablet analysis

Twenty tablets were thoroughly crushed and weighed. The tablet weight equivalent to 25 mg of AML, LAC, NIF, NIM and NIT was weighed and extracted with 15 ml of MeOH, sonication was done and then the volume was made up to the mark in a 25 mL flask. The resulting solution was filtered using Whatman No. 42 filter paper and a filter. A working solution was prepared by diluting 2.50 mL of the resulting filtrate with water in a 25 mL flask to a concentration of 100 µg/mL.

Results and discussion

Preliminary method development

The primary objective of this work was to develop a simple, rapid and selective RP HPLC method for the simultaneous determination of dihydropyridines calcium channel blockers (AML, LER, NIF, NIM, NIT) in pharmaceutical formulations and model mixture. The investigated analytes have a similar chemical structure, making their separation a challenging task. pKa values are as follows for AML 8.6–9.1, NIF—5.41, LER—6.83, NIM—5.41, NIT—2.79. While LogP values are as follows for AML 3, NIF—3.41, LER—6.4, NIM—3.05–4.05, NIT—2.57 [32]. These five analytes are not present in a single dosage form. Their simultaneous determination will demonstrate not only the possibility of their detection in mono-preparations but also in biological fluids, wastewater, and rinses from equipment. The most important task was to obtain excellent separations of these five analytes with optimal chromatographic time. However, before applying QbD, it was necessary to select optimal chromatographic conditions. During previous experimental studies, various alkyl and phenyl bonded chromatographic columns such as Luna C8 (150 × 4.6 mm 3 μm), Luna C18 (100 × 4.6 mm 3 μm), Zorbax SB C8 (150 × 4.6 mm 3.5 μm), InertClone ODS (150 × 4.6 mm 3.5 μm), and Zorbax SB Phenyl (150 × 4.6 mm 3.5 μm), as well as approaches to mobile phase components, were tested. Columns with different bonded phases, C18 and phenyl, with lower end-capped and less base-deactivated silica matrixes usually manifest lower peak symmetries, shapes, and quite distinct relative retentions. These only confirms the facts that from qualitative aspects of higher potential for separation of all presented components in samples, more different interactions between analytes and matrixes is highly favorable however that increases the possibilities most similar compounds to be separated/ The modern type based silica RP columns with less, present silanol groups and higher presence of metal cations on stationary phases has less different type of interactions and thereafter little lower possibilities to select all present components. It can be concluded that better end-capped and base-deactivated matrixes like Phenomenex Luna (2) C18, have less possibility to separate small peak of contaminant or degradant.

To determine the optimal detection wavelength, UV spectra of five analytes were recorded (Fig. 1). AML, LER, NIM, NIT demonstrated a prominent absorption maxima at approximately 237 nm, while NIF showed strong absorption at 278 nm. After evaluating several wavelengths, 237 nm was selected as the optimum detection wavelength as it provided adequate sensitivity for all five analytes with minimal interference from mobile phase components. Figures S1–S7 depicts approaches using TFA, potassium dihydrogen phosphate buffer, and TEA in various ratios with organic components in the mobile phase on a Luna C8 column. The best resolution, peak shapes, and performance parameters of the chromatographic system were achieved using 0.7% TEA in the mobile phase at pH 2.7. As can be seen from the chromatograms on Figs. S1–S7, not only the concentration of TEA was critical in the separation of analytes however also the ratio of organic components (MeOH and ACN). When increasing ACN to 35%, unsatisfactory separation between NIF and LER was observed. Further reducing ACN in the mobile phase to 30% allowed for a chromatogram with satisfactory chromatographic system parameters. Therefore, in subsequent studies, we used a mobile phase composition of: ACN-MeOH-0.7% TEA pH 3.06 (30-35-35). To optimize the parameters, we studied the flow rate, column temperature, pH, and injection volume (flow rate—1 ml/min, temperature—30 °C, pH-3.06, injection volume—3 µL). As seen from the obtained results, it was not enough to simply specify the type of column in the methodology, as results vary significantly even within the same type of column, especially when it comes to the separation of multicomponent mixtures. Preliminary studies (before applying QbD) are presented in Table S1. The choice of injection volume is presented in Table S2. To select the injection volume, an analysis was performed with 3, 4, and 5 µL injections. Since the suitability parameters of the chromatographic system were satisfactory at all injection volumes, we proposed an approach with the smallest sample selection. Volume of 3 µL was experimentally chosen. The developed technique has high sensitivity and is relevant for the determination of BCC in equipment washouts, wastewater, and small research objects.

Fig. 1.

UV absorption spectra AML, LER, NIF, NIM, NIT in the wavelength range of 210–400 nm

Method development and optimization

For the RP-HPLC method, various critical method parameters (CMPs) which are crucial for method development were identified as per ICH Q9 guideline [33]. The risk assessment for each input variable (Xn) was assessed as low (L), medium (M), high (H) with respect to each method’s responses (Yn). The result of the risk assessment is shown in Table S3. Based on risk assessment, three high-potential risk factors were identified: X1 (MeOH (%V/V)), X2 (pH of the mobile phase), and X3 (TEA (%V/V)). These factors were found to significantly affect Critical Analytical Attributes (CAAs) like NTP (number of the theoretical plates (Y1), tailing factor (TF) (Y2), resolution (Rs) (Y3) and retention time (Rt) (Y4). The independent factors and their respective levels, chosen for the study, are summarized in Table 1. For the optimization study, Box-Behnken design (BBD) was chosen, which included three factors and a total of 26 experiments (Table 2). In our research we have five analysts, which complicated the statistical processing of the experiment. However, we show only QbD NIM as last eluted analyte. The Rt of NIM varied from 4.96 to 7.68 min, Rs—from 4.004 to 6.333, TF—1.186–1.243, NTP—13011–16609. The data analysis was performed by selecting the linear model for main and interaction effects. For statistical analysis, the free trial version of Minitab was utilized to evaluate the significance of models, terms, and their interactions via ANOVA. The model generates regression equation for each CAA parameter.

Table 1.

Chromatographic factors and level for central composite design

Independent factors	Units	Low level (− 1)	Medium level (0)	Hight level (+ 1)
X1: MeOH (%V/V)	%	30	32.5	35
X2: pH	NO.	2.7	2.9	3.1
X3: TEA (%V/V)	%	30	32.5	35

Table 2.

Central composite design optimization layout of NIM

StdOrder	RunOrder	PtType	Blocks	X1 MeOH	X2 pH	X3 TEA	Y1 NTP	Y2 TF	Y3 Rs	Y4 Rt
1	1	2	1	30	2.7	32.5	14,743	1.216	4.990	5.932
2	2	2	1	35	2.7	32.5	14,767	1.214	5.192	6.254
3	3	2	1	30	3.1	32.5	14,533	1.217	4.917	5.866
4	4	2	1	35	3.1	32.5	14,591	1.215	5.123	6.189
5	5	2	1	30	2.9	30	13,046	1.242	4.006	4.963
6	6	2	1	35	2.9	30	13,071	1.241	4.162	5.18
7	7	2	1	30	2.9	35	16,301	1.186	6.066	7.214
8	8	2	1	35	2.9	35	16,393	1.194	6.330	7.684
9	9	2	1	32.5	2.7	30	13,109	1.241	4.107	5.078
10	10	2	1	32.5	3.1	30	13,030	1.24	4.088	5.069
11	11	2	1	32.5	2.7	35	16,425	1.187	6.221	7.465
12	12	2	1	32.5	3.1	35	16,609	1.202	6.251	7.502
13	13	0	1	32.5	2.9	32.5	14,796	1.221	5.089	6.084
14	14	2	1	30	2.7	32.5	14,662	1.214	4.972	5.928
15	15	2	1	35	2.7	32.5	14,835	1.212	5.200	6.25
16	16	2	1	30	3.1	32.5	14,597	1.216	4.922	5.871
17	17	2	1	35	3.1	32.5	14,570	1.218	5.125	6.203
18	18	2	1	30	2.9	30	13,025	1.243	4.004	4.962
19	19	2	1	35	2.9	30	13,116	1.239	4.165	5.18
20	20	2	1	30	2.9	35	16,326	1.195	6.071	7.203
21	21	2	1	35	2.9	35	16,439	1.195	6.333	7.682
22	22	2	1	32.5	2.7	30	13,011	1.241	4.075	5.071
23	23	2	1	32.5	3.1	30	13,053	1.243	4.074	5.058
24	24	2	1	32.5	2.7	35	16,435	1.194	6.207	7.448
25	25	2	1	32.5	3.1	35	16,481	1.197	6.263	7.501
26	26	0	1	32.5	2.9	32.5	14,836	1.219	5.092	6.08

Effect of variables on NTP

The impact of MeOH and TEA, pH of mobile phase on NTP is displayed in Fig. 2. According to Pareto chart (Fig. S8), the greatest influence on NTP had factor X3 (TEA (%V/V)). With an increase in the amount of TEA, NTP rises. Amount of MeOH and pH did not significantly affect NTP.

Fig. 2.

Surface, countour, interation plots and main effects Plots of NTP

Effect of variables on TF

The impact of MeOH and TEA, pH of mobile phase on TF is displayed in Fig. 3. According to Pareto chart (Fig. S9), the greatest influence on TF had factor X3 (TEA (%V/V)). With an increase in the amount of TEA, TF decreases.

Fig. 3.

Surface, countour, interation plots and main effects Plots of TF

Effect of variables on Rs

The impact of MeOH and TEA, pH of mobile phase on Rs is displayed in Fig. 4. According to Pareto chart (Fig. S10), the greatest influence on Rs had factors X3 (TEA (%V/V)) and X1 (MeOH (%V/V)). With an increase in the amount of TEA, Rs rise. With an increase in the amount of MeOH, Rs slowly rose. pH did not significantly affect Rs.

Fig. 4.

Surface, countour, interation plots and main effects Plots of Rs

Effect of variables on Rt

The impact of MeOH and TEA, pH of mobile phase on Rt is displayed in Fig. 5. According to Pareto chart (Fig. S11), the greatest influence on Rt had factors X3 (TEA (%V/V)) and X1 (MeOH (%V/V)). With an increase in the amount of TEA, Rt rise. With an increase in the amount of MeOH, Rt slowly rises. pH did not significantly affect Rt.

Fig. 5.

Surface, countour, interation plots and main effects Plots of Rt

Optimized method conditions

Numerical optimization was attempted to achieve the intended goals of Rt, Res, TF, NTP (Fig. 6). The ideal chromatographic conditions were a mobile phase ratio of ACN-MeOH-0.7% TEA pH 3.06 (30-35-35) adjusted with ortho phosphoric acid, flow rate of 1.0 mL/min, column temperature − 30 °C on a Luna C8 (150 × 4.6 mm 3 μm) column with PDA detection at 237 nm. HPLC chromatogram of five dihydropyridines calcium channel blockers (AML, LER, NIF, NIM, NIT) in model mixture on optimized chromatographic condition is shown in Fig. 7. The optimized method demonstrated good chromatographic resolution with retention times of 2.93, 3.98, 4.98, 6.32 and 7.75 min for AML, NIF, LER, NIT and NIM, respectively. Peak purity indexes: AML—0.374149, NIF 0.089436, LER—0.499402, NIT—0.020103, NIM—0.048646. This method was identified based on acceptable system suitability test parameters and method applicability (Table 3). The other method variables such as detection wavelength, column and its temperature, were controlled and which are of low risk.

Fig. 6.

Interaction plots illustrating optimal analytical design space region

Fig. 7.

HPLC Chromatogram of AML (0.1 mg/mL), LER (0.1 mg/mL), NIF (0.1 mg/mL), NIM (0.1 mg/mL), NIT (0.1 mg/mL) in their mixture using ACN-MeOH-0.7% TEA pH 3.06 (30-35-35) adjusted with ortho phosphoric acid, flow rate of 1.0 mL/min, column temperature − 30 °C on a Luna C8 (150 × 4.6 mm 3 μm) column with PDA detection at 237 nm

Table 3.

System suitability results for the determination of AML, LER, NIF, NIM, NIT by the proposed HPLC method

Parameters	AML	NIF	LER	NIT	NIM	Reference value
Retention time (tR), min	2.93	3.97	4.98	6.23	7.75	–
Retention factor (K´)	1.15	2.11	2.89	3.95	5.06	1–10
Theoretical Plates (N)	8236	9468	3629	6722	4047	> 2000
Tailing factor (T)	1.38	1.4	1.24	1.2	1.17	< 2
Resolution factor (Rs)	6.46		5.48	6.7	6.4	> 2

Method validation

Validation of the proposed method was performed following ICH guidelines, assessing its linearity, specificity, trueness, precision, and robustness [34].

Linearity and range

Linearity was assessed using five levels of each of the investigated drugs (namely AML, LER, NIF, NIM, NIT), where concentration varied in the range of 10–50 µg/mL (Fig. 8).

Fig. 8.

Calibration curves showing the relationship between peak area and concentration (10–50 µg/mL) for the five calcium channel blockers

Table 4 summarizes the respective regression equations, correlation coefficients, concentration ranges, slopes, and intercepts. All five compounds displayed strong linear relationships within the range of 10–50 µg/mL, with correlation coefficients (r²) exceeding 0.9989, reflecting the method’s good linearity. Furthermore, the high r² values alongside low intercepts confirm the method’s linearity across the studied range.

Table 4.

Regression and validation parameters of the proposed HPLC method

Parameters	AML	LER	NIF	NIT	NIM
Wavelength (nm)	237
Linearity range (µg/mL)	10–50
- Regression Equation	y* = b x** + a	y* = b x** + a	y* = b x** + a	y* = b x** + a	y* = b x** + a
Slope (b)	5158.82	8794	15,459	3472.6	3189.21
Intercept (a)	7800.40	8189.5	− 11,424	13,890	17,091
Coefficient of determination(r2)	0.9995	0.9997	0.9989	1.000	0.9997
Trueness (%R)***	99.11 ± 0.67	99.49 ± 0.85	100.09 ± 0.32	99.39 ± 0.60	100.08 ± 0.11
Precision (%RSD)****
Repeatability	0.605	0.912	0.784	0.736	0.915
Intermediate precision	0.695	0.694	1.017	0.894	0.789

* Peak area of AML, LER, NIF, NIM, NIT

** Concentration of AML, LER, NIF, NIM, NIT in µg/mL

***Average of nine determinations (triplicate determination of three concentration level)

****%RSD of %Recovery of nine determinations (triplicate determination of three concentration level)

The low limits of detection (LOD) and quantification (LOQ) for all five drugs were determined using the slope of the calibration graph and the standard deviation of the response. Calculations were performed using the following formulas LOD = 3.3σ/S and LOQ = 10σ/S, where σ denotes the standard deviation of the intercept and S stands for the slope of the calibration graph. Table 4 shows that for the proposed method the LOD values fell within the range of 0.393–2.340 µg/mL, while the LOQ values were in the range of 1.191–7.080 µg/mL, thereby demonstrating the high sensitivity of the latter for all five drugs.

Trueness and precision

The method’s trueness was evaluated by testing laboratory-prepared mixes with known concentrations of the five pharmaceuticals at three different concentrations (10, 30, and 50 µg/mL) in triplicate. The percentage recoveries varied from 99.11 to 100.08%, with minimal standard deviations (Table 4), indicating that the approach is very accurate.

To assess repeatability, three distinct drug concentrations (10, 30, and 50 µg/mL) were tested in triplicate within 1 day. RSD values were lower than 1.0% for each tested drug (Table 4), showing high repeatability. To determine intermediate precision, three distinct drug levels (10, 30 and 50 µg/mL) were analyzed in triplicate over 3 days. Table 4 shows that each drug’s RSD values were below 1.1%, demonstrating the method’s excellent intermediate precision.

Specificity

The method’s specificity was assessed by evaluating synthetic mixtures comprising the five drugs in various proportions and comparing them to separate standard solutions. There was no interference between the examined drugs, and all peaks were resolved clearly, having resolution factors better than 2.0 for neighboring peaks. Furthermore, the specificity was established by using the conventional addition procedure on drug formulations. Pre-evaluated pharmaceutical formulations were mixed with known quantities of pure standards before being analyzed. Table 5 shows that percentage recoveries varied between 99.29 and 100.57%, demonstrating that excipients in drug formulations weren’t interfering with the determination of active pharmaceutical ingredients.

Table 5.

Quantitative determination of AML, LER, NIF, NIM, NIT in its pharmaceutical dosage form by HPLC method

Drug	Pharmaceutical taken (µg/mL)	Pharmaceutical found* (µg/mL)	Pure added (µg/mL)	Pure found ** (µg/mL)	%Recovery
AML	10	10.05	10	10.07	100.73
			20	20.08	100.42
			30	30.15	100.52
Mean					100.56
LER	10	9.97	10	9.94	99.42
			20	19.90	99.53
			30	29.67	98.93
Mean					99.29
NIF	10	10.03	10	10.08	100.82
			20	20.09	100.45
			30	30.12	100.43
Mean					100.57
NIM	10	9.96	10	10.06	100.59
			20	19.80	99.00
			30	29.87	99.57
Mean					99.72
NIT	30	30.19	30	30.07	100.23
			40	40.11	100.28
			50	50.84	101.68
Mean					100.73

* Average of five experiments

** Average of three experiments

Robustness

The method’s robustness was tested by applying little modifications to the chromatographic conditions, which included the flow rate (± 0.2 mL/min) and the temperature of the column (± 2 °C). The results, which are presented in Table 6, reveal that these minor alterations had no substantial effect on Tf as well as on Rt, proving the robustness of the proposed method.

Table 6.

Robustness results for the determination of AML, LER, NIF, NIM, NIT by the proposed HPLC method

Parameters		AML	NIF	LER	NIT	NIM		AML	NIF	LER	NIT	NIM
Flow rate (ml/min.)
0.8	Tailing factor (T)	1.37	1.41	1.23	1.17	1.15	Resolution (Rs)	–	6.82	6.03	6.86	6.67
1		1.38	1.40	1.24	1.2	1.17		–	6.46	5.48	6.70	6.37
1.2		1.35	1.25	1.39	1.16	1.2		–	6.08	5.04	6.25	5.99
Temperature ( °C )
28	Tailing factor (T)	1.35	1.35	1.23	1.22	1.18	Retention time (Rt)	2.99	4.04	5.15	6.49	7.99
30		1.38	1.40	1.24	1.2	1.17		2.93	3.98	4.98	6.32	7.75
32		1.39	1.45	1.24	1.22	1.18		2.90	3.93	4.92	6.2	7.56

Greenness assessment

AGREE assessment

The Analytical GREEnness (AGREE) instrument was used to assess the developed method’s greenness [35]. A high degree of greenness was indicated by the method’s AGREE result of 0.78 out of 1 (Fig. 9). The AGREE result was positively impacted by the low flow rate, the isocratic method of elution, the use of MeOH, ACN, and TEA rather than more hazardous solvents, and the lack of sample derivatization. The HPLC system’s energy consumption and the usage of organic solvent in the mobile phase were the main factors that determined of the greenness result. These are the intrinsic shortcomings of HPLC methods, though, and have been lowered by reducing the procedure to accomplish quick analysis while using a manageable amount of solvent.

Fig. 9.

A AGREE metric assessment of the developed RP-HPLC method (overall greenness score of 0.78); B MoGAPI assessment (overall score of 79)

MoGAPI and Complex MoGAPI assessments

We assessed the developed method’s applicability and feasibility using the Modified Green Analytic Procedure Index (MoGAPI) [36] and Complex MoGAPI [37]. With a MoGAPI result of 79 out of 100, the approach demonstrated great practical value for everyday analysis (Fig. 9). The method’s merits across several laboratory practicality parameters are reflected in this high result. The method uses straightforward equipment that is easily found in the majority of analytical labs to offer quantitative and qualitative examination of five drugs. Including time for sample preparation, the analysis performance is effective, processing 5–7 samples in an hour. Additionally, the approach is more accessible and economical because it utilizes widely used, readily accessible chemicals. Typical HPLC equipment’s semi-automated process of analysis minimizes human interaction while providing flexibility. Due to the low sample requirements, precious pharmaceutical components are preserved. These useful benefits, as indicated by the high MoGAPI result, show that the proposed approach is not only very feasible to use in pharmaceutical quality control labs with standard equipment and resources but also ecologically benign. With a Complex MoGAPI result of 79 out of 100, the approach demonstrated excellent green analysis (Fig. 10).

Fig. 10.

A Complex MoGAPI metric assessment of the developed RP-HPLC method (overall greenness score of 79); B AGSA assessment (overall score of 80.56)

AGSA, CaFRI, BAGI and CACI assessments

Analytical Green Star Area (AGSA) as a new tool to assess greenness of analytical methods was used in our work [38]. The AGSA metric delivers a robust framework for evaluating the greenness of analytical methods, proving quantitative and visual understanding of their environmental impact. A high degree of greenness was indicated by the method’s AGSA result of 80.56 (Fig. 11). Carbon Footprint Reduction Index (CaFRI) [39] is a new tool to assess greenness of analytical methods which focused on estimating greenhouse gas emissions [39]. The total CaFRI score of our proposed HPLC method was 80 (Fig. 11). Two important metrics such as Blue Applicability Grade Index (BAGI) (overall score of 70) [40] and Click Analytical Chemistry Index (CACI) (overall score of 80) [41] also were used to evaluate the method’s applicability.

Fig. 11.

A CaFRI metric assessment of the developed RP-HPLC method (overall greenness score of 80); B BAGI assessment (overall score of 70); C CACI assessment (overall score of 80)

Application to pharmaceutical formulations

The validated method was successfully applied to the determination of AML, LER, NIF, NIM, NIT in their respective pharmaceutical formulations. With RSD values less than 1.38% and good recovery percentages that vary between 99.57 and 100.07%, the obtained results, which are shown in Table 7, demonstrate the method’s trueness and precision for drug formulation testing. These findings demonstrate that the formulations’ typical excipients had no effect on the active components’ ability to be quantified. The results were statistically compared to those obtained from earlier published methods for different drugs using the student’s t-test as well as F-test at the confidence level of 95% in order to further validate the applicability of the method. There was no discernible difference in accuracy and precision between the developed approach and the published methods, as the computed t and F values were lower than the tabular values (Table 7). These results demonstrate that the developed method, which offers the benefit of simultaneous analysis with reduced time and resource consumption compared to separate methods, is appropriate for everyday quality control testing involving these five dihydropyridine calcium channel blockers in their various pharmaceutical dosage forms.

Table 7.

Determination of AML, LER, NIF, NIM, NIT in its pharmaceutical dosage form by the proposed HPLC method and the reported methods

Parameters	Proposed HPLC method					Reported methods
	AML	NIF	LER	NIT	NIM	AML	NIF	LER	NIT	NIM
Mean	10.05	100.07	100.30	99.57	99.84	100.8	99.80	99.77	99.87	100.01
%RSD	0.839	1.056	0.709	1.111	1.380	0.710	0.873	0.894	0.594	0.893
Number of measurements	5	5	5	5	5	5	5	5	5	5
Student’s t-test	0.784 (2.306)	0.736 (2.306)	1.710 (2.306)	1.338 (2.306)	1.363 (2.306)
F-value	1.649 (6.388)	1.794 (6.388)	1.395 (6.388)	1.584 (6.388)	1.723 (6.388)

Conclusion and future directions

This study presents the first validated RP-HPLC method for the simultaneous determination of five dihydropyridines calcium channel blockers: AML, LER, NIF, NIM, NIT. The developed method offers several significant advantages, including simplicity, sensitivity, selectivity, and rapid analysis time. The chromatographic separation was achieved on Luna C8 (150 × 4.6 mm 3 μm) column using a mobile phase of ACN-MetOH-0.7% TEA pH 3.06 with UV detection at 237 nm. All five compounds were well-separated with retention times under 8 min, making the method suitable for high-throughput analysis.

Following ICH criteria, the approach was thoroughly validated and showed high linearity (r² >0.9989) for all compounds in the concentration ranging from 10 to 50 µg/mL, good trueness (99.11–100.09%) and precision (RSD < 1.1%). With outstanding recovery results, the approach was effectively used to determine these APIs in their pharmaceutical forms, demonstrating that excipient interference was not present.

The procedure is both very practical for routine analysis (MoGAPI result: 79) and ecologically friendly (AGREE result: 0.78), according to environmental and practical evaluations conducted utilizing the AGREE and MoGAPI tools. The method’s greenness profile was enhanced by the use of less hazardous solvents, the isocratic method of elution, and the lack of intricate sample preparation procedures. The method’s outstanding applicability with regard to resource usage, sample productivity, and equipment needs is reflected in its very good MoGAPI result.

In general new and old metrics (AGREE, MoGAPI, complex MoGAPI, AGSA, CaFRI, BAGI and CACI) were used to evaluate the greenness of analytical methods.

It’s interesting that this work suggests several options for further investigation. In order to facilitate pharmacokinetic research and therapeutic medication monitoring of these drugs, the technique could first be expanded to biological samples, including urine and blood plasma. Further matrix impact validation and possibly adjusted sample preparation methods would be needed for this. Secondly, the technique may be modified for monitoring these substances’ environmental effects in sewage, which would help determine how they affect the environment.

Thirdly, it would be beneficial to create a stability-indicating version of this approach that includes induced degradation tests for determining shelf-life and maintaining quality over an extended period of storage. Furthermore, the developed approach can be used to analyze the effects of modifications on formulations during the introduction of new excipient manufacturers, modifications to manufacturing technology, product composition, and other stages in the lifespan of finished dosage forms of dihydropyridine calcium channel antagonists.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

RP-HPLC

Reversed-phase high-performance liquid chromatography

AML

Amlodipine

NIF

Nifedipine

LER

Lercanidipine

NIT

Nitrendipine

NIM

Nimodipine

QbD

Quality by design

USP

United State Pharmacopeia

Eur. Ph.

European Pharmacopeia

QC

Quality control

API

Active pharmaceutical ingredient

MeOH

Methanol

CAN

Acetonitrile

TEA

Triethylamine

RC

Regenerated cellulose

CAAs

Critical analytical attributes

NTP

Number of the theoretical plates

Tf

Tailing factor

Rs

Resolution

Rt

Retention time

LOD

Limits of detection

LOQ

Limits of quantification

Author contributions

Mariana Horyn: Methodology and writing the original draft, Validation and reviewing, Mariana Horyn, Marjan Piponski, Liubomyr Kryskiw, Mamdouh R. Rezk, Nadiya Zarivna, Dmytro Korobko***,*** Liliya Logoyda: reviewing and publishing editing, Liliya Logoyda: supervision.

Funding

This research was not received any funds.

Data availability

Data is provided within the manuscript or supplementary information files.

Declarations

Ethics approval and consent to participate

Guidelines according to BMC Chemistry.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.