Skip to main content
Download PDF
Download ePub

AQbD TLC-densitometric method approach along with green fingerprint and whiteness assessment for quantifying two combined antihypertensive agents and their impurities

BMC Chemistry volume 18, Article number: 15 (2024) Cite this article

Abstract

Preserving the environment, reducing the amount of waste resulting from chemical trials, and reducing the amount of energy consumed have currently become a pivotal global trend. An analytical quality by design (AQbD) based eco-friendly TLC-densitometric method was implemented for quantifying two antihypertensive agents, captopril (CPL) and hydrochlorothiazide (HCZ), along with their impurities; captopril disulphide (CDS), chlorothiazide (CTZ) and salamide (SMD). The analytical target profile (ATP) was first identified, followed by selecting the critical analytical attributes (CAAs), such as retardation factors and resolution between the separated peaks. Critical method parameters (CMPs) that may have a crucial influence on CAAs were identified and emanated through the quality risk assessment phase. A literature survey-based preliminary studies were performed, followed by optimization of the selected CMPs through a custom experimental design to attain the highest resolution with optimum retardation factors. Moreover, method robustness was also tested by testing the design space. Complete separation of the drugs and their impurities was achieved using ethyl acetate: glacial acetic acid (6: 0.6, v/v) as a developing system applied to a 12 cm length TLC plate at room temperature with UV scanning at 215 nm. Calibration graphs were found to be linear in the ranges of (0.70–6.00), (0.10–2.00), (0.20–1.00), (0.07–1.50) and (0.05–1.00) µg/band corresponding to CPL, HCZ, CDS, CTZ, and SMD, respectively. Four different green metric tools were used to evaluate the greenness profile of the proposed method, and results showed that it is greener than the reported HPLC method. Method whiteness assessment was also conducted. Moreover, the method performance was evaluated following the ICH guidelines, and the outcomes fell within the acceptable limits. The developed method could be approved for routine assay of the cited components in their pharmaceutical formulations and bulk powder without interference from the reported impurities. The issue of concern is saving money, especially in developing countries.

Highlights

  • A combination of hydrochlorothiazide and captopril is used in treating patients not adequately controlled with monotherapy.

  • The AQbD approach was used to develop the TLC- densitometric method to reduce the number of trials, solvent consumption, and waste production.

  • Increasing global attention for considering GAC principles in developing analytical methods; therefore, different green metric tools were implemented for the assessment of the greenness profile of the proposed and reported methods.

  • Integration between the AQbD and GAC principles was performed to develop a greener, more time- and money-saving method.

  • Whiteness assessment was conducted to evaluate method performance and perform a comparative study between the developed method and the reported ones.

Peer Review reports

Introduction

Captopril (CPL) is chemically known as ((2S)-1-[(2S)-2-Methyl-3-sulfanylpropanoyl]pyrrolidine-2-carboxylic acid) [1]. It acts as a competitive inhibitor of the angiotensin-converting enzyme, widely used to reduce elevated blood pressure [2]. Hydrochlorothiazide (HCZ) is chemically known as (6-Chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide) [1]. It is a thiazide diuretic commonly prescribed in treating hypertension and edema as it prevents the transport of sodium chloride in the distal convoluted tubule [3]. ACE inhibitors and HCZ are frequently used in combined products in patients whose blood pressure is not adequately controlled with monotherapy [3].

Among impurities of HCZ that are specified in British pharmacopeia (B.P) [1] are; Chlorothiazide (CTZ) (6-chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide) and salamide (SMD) (4-amino-6-chlorobenzene-1,3-disulfonamide). The maximum specified accepted limit for both CTZ and SMD is 0.50% [1]. On the other hand, the impurity of CPL is reported in B.P. to be captopril disulphide (CDS) with a maximum specified limit of 1.00% [1, 4]. CDS was designated to be (1,1¢-[disulfanediylbis[(2S)-2-methyl-1-oxopropane-3,1-diyl]] bis [(2S)-pyrrolidine-2- carboxylic] acid) and it is considered to be the oxidative product of CPL [4]. In pharmaceutical industries, it is crucial to investigate and quantify the supplied raw material in the presence of its specified impurities. The importance of their quantification has originated from their influential effect on human health [5, 6].

The literature survey reveals several reported methods regarding the analysis of HCZ and CPL either alone, in their binary mixture, in combination with other drugs, or with their impurities. The reported methods for their combined determination included UV-spectrophotometric [7], chemiluminescence and artificial neural network [8], HPLC [9, 10], and voltammetric methods of analysis [11, 12]. For their determination in the presence of other drugs, there are some methods such as HPLC [13], UPLC MS/MS [14], GC/MS [15], and voltammetric methods of analysis [16]. Only two reported methods were described for determining HCZ and CPL with their specified impurities including HPLC [17] and micellar electrokinetic chromatographic method [18].

It is worth noting that the studied impurities were subjected to ADME/TOX studies [19] in our previous work and were found to be hepatotoxic [20]. Several obstacles are raised from the similarity in chemical structure between the studied compounds and their impurities, which in consequence leads to the similarity in their physical and chemical properties, leading to difficulty in chromatographic separation, excessive number of trials, massive amounts of the consumed toxic solvents with a large amount of waste. Moreover, numerous factors control TLC-densitometric separation, such as developing system ratio, nature of stationary phase, scanning wavelength, saturation time, plate dimensions, temperature, etc. Usually, many trials and errors were performed to elicit critical process parameters (CPPs) and their influential effect on the responses of the method.

To face these dilemmas, reduce hazardous chemical waste, and preserve the environment, the attention of most analysts turned to merging and integrating the two concepts; the AQbD approach and green analytical chemistry (GAC) principles into one crucible that pours into one interest, namely the development of green analytical methods with the least number of experiments [21].

The principle of AQbD eliminates these difficulties and starts a new era in analyzing drugs in highly robust and accurate ways with the least number of experiments, which in consequence saves money [22,23,24,25].

On the other hand, green assessment of analytical methods is very important. It has been conducted by different methods including qualitative green metric tools (such as the National Environmental Method Index (NEMI) and Green Analytical Procedure Index (GAPI)), semi-quantitative tool (like Analytical eco-scale) and quantitative metric tools (such as AGREE). Additionally, an assessment of method whiteness was done by (RGB 12) [25,26,27].

The current work aims to develop a green, sensitive, and selective TLC-densitometric method for the separation and quantification of captopril (CPL) and hydrochlorothiazide (HCZ) and their impurities using a custom experimental design to decrease the number of trials, waste, and time with maximum efficient outcomes. Additionally, four green metric tools; (NEMI), (GAPI), Analytical eco-scale, and (AGREE) side by side with assessment of method whiteness using (RGB 12) were applied for green and whiteness assessment of the developed TLC-densitometric method with a comparative study of its profile with those of the reported ones [10, 17, 18].

Experimental

Instruments

  • A Linomat V applicator and a 100 μL syringe were used to apply the samples.

  • TLC Scanner III densitometer (CAMAG, Muttenz, Switzerland).

  • A UV lamp with a short wavelength of 254 nm (Vilber Lourmat, Marne La Vallee, Cedex, France) was used during the optimization of the implied TLC-densitometric method.

  • Digital balance (Sartorius, German).

  • Ultrasonicator Sonix TV SS-series (South Carolina, USA).

Software programs

  • JMP® software version 16.0.0 SAS institute Inc., Cary, North Carolina, USA software, copyright 2021 [28] was used for developing custom experimental design and data analysis for the developed TLC- densitometric method.

  • After scanning the developed plates, the outcome signals were measured using winCATS software (Version 3.15; CAMAG).

  • Software used for the application of the (AGREE) metric tool was downloaded following the link in the following article [29].

  • The pkCSM website was used for the prediction of pharmacokinetic parameters and toxicity profiles for molecules [19, 30].

Samples and chemicals

Pure samples

Captopril and hydrochlorothiazide pure samples were kindly supplied from (Pharco Pharmaceuticals Co. Alexandria, Egypt). Their purity was verified using the reported HPLC method [10] and the results were 99.83% and 99.91%, respectively. Chlorothiazide and salamide standards were purchased from (Sigma-Aldrich Chemie GmbH, Germany) and their purity was labeled 99.90% and 99.87%, respectively. Based on the literature survey, captopril disulphide was prepared from the supplied sample of captopril via oxidative degradation using an iodine solution [4].

Pharmaceutical formulation

Capozide® tablets 50/25 mg, its Batch No. was (J88C). They were manufactured by (Squipp—Smithkline Beecham) and are claimed to contain 50.00 mg CPL and 25.00 mg HCZ. Capozide® tablets under investigation were bought from the local market.

Preparation of captopril oxidative product (captopril disulphide, CDS)

The oxidative product of captopril; (CDS) was prepared by following the procedure developed by Moussa et al. [4]. Following the mentioned steps, a white precipitate was resulted [4]. The CPL oxidative product's structure was then confirmed by recording its molecular weight using mass spectrometric analysis.

Chemicals

Ethanol and ethyl acetate were of HPLC grade (Fischer, Loughborough, UK). The consumed glacial acetic acid, iodine, and sodium thiosulphate (for preparation of CDS) were of analytical grade (Piochem for Chemical Co., Giza, Egypt). Deionized water for injection (SEDICO Pharmaceuticals Co., 6th October City, Egypt) was also used.

Solutions

Standard solutions

Standard stock solutions for CPL, HCZ, CTZ, SMD, and CDS were prepared using ethanol at a concentration of (1000.00 µg mL−1) by weighing 0.01 gm of each pure powder in separate five 10 mL volumetric flasks. Final dilutions for the preparation of calibration and validation standards were prepared in 5.00 mL calibrated flasks by further diluting the corresponding stock solutions using ethanol as solvent.

Pharmaceutical formulation solution

Ten Capozide® tablets were precisely weighed and carefully grinded. Into a 5.00 mL volumetric flask, the weight of the grinded tablets containing amounts equivalent to 5 mg CPL and 2.5 mg HCZ was taken and then transferred cautiously to the flask. Ethanol of 3.00 mL was added and the prepared flask was ultra-sonicated for 20 min. the sonicated solution was filtered and the volume was completed to the mark using ethanol to prepare a sample solution containing (1000.00 µg mL−1) of CPL and (500.00 µg mL−1) of HCZ. Concentrations within the linear ranges of the constructed calibration curves were then prepared in ethanol and analyzed by the developed method. The standard addition technique was applied on three different levels to investigate the matrix effect on the performance of the proposed method.

Procedure

Experimental design

JMP® software version 16.0.0 SAS institute Inc., Cary, North Carolina, USA software, copyright 2021 [28] was used for establishing two levels of three factors custom experimental design with one center point. Preliminary univariate analyses based on a wide literature survey were used for the selection of the independent variables such as mobile phase composition, solvent ratio, plate length, and their critical ranges. Data was statistically analyzed, and the final optimization of the developed method was conducted via the desirability function. A surface plot was constructed to test the relationship between the selected factors and the desired responses. The selected responses included resolution between CTZ and HCZ, HCZ and SMD, and Rf of CDS. The resolution factors were desired to be (˃ 1.5), and the intended Rf of CDS was set to be (˃ 0.1) cm.

Chromatographic conditions

As a stationary phase, (20 × 12 cm) aluminum plates pre-coated with 0.25 mm Silica gel 60 F254 (Merck, Darmstadt, Germany) were used. Slit dimensions were optimized to be 6 × 0.45 mm, with a 20 mm/s scanning speed. Scanning mode was the absorbance mode.

Samples of the cited components were applied to TLC plates using a Camag Linomat V automatic applicator with a band width of 6 mm. The bands were spaced 5 mm apart and 15 mm from the bottom edge of the plate. Linear ascending development was carried out for the plates placed in a previously prepared chromatographic jar which was saturated for 30 min with the developing system composed of ethyl acetate: glacial acetic acid (6.00: 0.60, v/v) at room temperature. The separated bands were scanned at 215 nm.

Preparation of calibration standards

Accurate aliquots covered the concentrations range of (70–600 µg/mL), (10–200 µg/mL), (20–100 µg/mL), (7–150 µg/mL) and (5–100 µg/mL) corresponding to CPL, HCZ, CDS, CTZ and SMD, respectively, were accurately and separately transferred into 5 mL volumetric flask from their relative stock solutions (1000.00 µg mL−1). From each previously prepared solution, 10.00 μL was applied in triplicates to the TLC plates using a Camag Linomat V applicator. The plates were placed in a chromatographic jar saturated with the previously described developing system of ethyl acetate: glacial acetic acid (6.00: 0.60, v/v) using the linear ascending development to the solvent front. The separated bands were scanned at 215 nm and calibration curves were plotted using the recorded integrated peak areas against the corresponding concentrations as μg/mL and then the regression equations were computed.

Application of the method to pharmaceutical formulation

Using the previously prepared solution of Capozide® tablets mentioned under the pharmaceutical formulation solution section, a concentration equivalent to (100 µg/mL for CPL and 50 µg/mL for HCZ) was prepared, and then 10 μL was applied six times to the TLC plates. The previously mentioned chromatographic conditions were followed and peak areas were computed. The concentrations and recovery percent were calculated from linear regression equations previously calculated. For the application of the standard addition technique, samples were prepared by spiking the intended dosage form concentration by three different and separate concentrations of each drug. The prepared samples were analyzed and concentrations of the added pure drugs were obtained.

Results and discussion

Development and validation of stability indicating analytical methods for the determination of pharmaceutical components in the presence of their specified pharmacopeial impurities represents a great challenge. The importance of developing those methods originates from the direct effect of these impurities on human health because of their possible toxicity and side effects. The current study concerns merely quantifying the cited drugs; CPL and HCZ using a TLC-densitometric approach and their hepatotoxic impurities; CDS, CTZ, and SMD specified in British pharmacopoeia (B.P) [1]. It is worth mentioning that; CDS is also considered as the oxidative product of CPL and it was prepared in our laboratory following the procedure published by Moussa et al. [4]. Structural elucidation was conducted by using mass spectrometric analysis as depicted in Fig. 1. It was found that the molecular weight of the analyzed product is equal to 431, which agrees with the expected structural molecular weight.

Fig. 1
figure 1

Mass spectrum of precursor and product ion of captopril disulphide representing molecular weight (A), fragmentation and product ion (B) of captopril disulphide

Full size image

The cited mixture was quantified in the context of achieving goals of vital values such as minimization of the number of experiments, decreasing waste production, using low amounts of harmful solvents, and attaining optimum outcomes. To achieve this goal, AQbD was integrated with GAC principles to gain their great impact on the newly developed chromatographic method such as increasing method sensitivity, selectivity, and robustness with the lowest number of trials and environmental impact. AQbD is a multiple-step process that includes defining ATP and CAAs, quality risk assessment, experimental design for screening and optimization of CMPs, design space (DS) establishment, and method control strategy, all of are depicted in Additional file 1: Figure S1.

Defining ATP and CAAs

Defining the analytical target profile in definite terms to state the essential primary objectives and prospect outcomes is the first critical step in developing the analytical method using AQbD. The ATP for the current study is to develop a reliable, accurate, sensitive, precise, and eco-friendly TLC-densitometric method. ATP elements discussed for the developed TLC-densitometric method were summarized in Additional file 1: Table S1. The term CAAs could be translated to the measurable responses which were used as an indicator for method performance and aided in attaining the stated ATP. The most influential CAAs were retardation factors (Rf) and resolution (Rs) between the separated peaks.

Quality risk assessment

ICH Q9 [31] provided certain guidelines regarding risk assessment study considering it a pivotal part of QbD which has an impact on CAAs [24].

The risk assessment process identifies the various critical method factors influencing the ATP. Many tools were depicted for the risk assessment study, one of which is the cause and effect diagram which is also known as Ishikawa or fishbone diagram [24] Fig. 2.

Fig. 2
figure 2

A fishbone (cause and effect diagram) listing the most important critical method parameters (CMPs) regarding AQbD and green chemistry influencing the critical analytical attributes (CAAs) for developing a green TLC-densitometric method

Full size image

Brainstorming was conducted to compile and summarize all the conceivable factors influencing the CAAs via the Ishikawa diagram, highlighting the likely risky ones based on previous literature and experiments. Figure 2, represents the fishbone diagram concerning the developed TLC-densitometric analysis and also factors concerning GAC principles that will affect the CAAs [24, 32, 33]. Looking closely at Ishikawa Fig. 2, the effect of many parameters on CAAs was investigated. Considering GAC principles, ethanol was selected for sample preparation rather than other available solvents. Different stationary phases with different polarities were investigated, and preliminary studies concluded that the normal stationary phase was perfect for the separation of the cited components with satisfactory resolution. Additionally, the type of organic modifier and its ratio in the developing system were also chosen to be studied. Ethyl acetate as an organic modifier was selected rather than other organic solvents from the GAC point of view. Moreover, its ratio was critical for separation and hence it was consecutively optimized. Additionally, the effect of pH on the developing system was investigated, based on preliminary studies revealed that the acidic modifier was important for the separation of the studied components. Plate length optimization was also chosen as a significant factor to be studied. Finally, the type and ratio of organic and acidic modifiers along with the plate length were exposed to scanning studies for a closer look to figure out their impacts. On the other hand, the scanning wavelength was selected after running the UV-spectra for the five studied components and it was found that 215 nm was the best scanning wavelength regarding sensitivity. Other parameters such as temperature and saturation time (25 °C and 30 min, in order) were kept constant due to their insignificant effect on method performance.

Preliminary studies for critical method parameters (CMPs) screening and experimental design for their optimization

Two main steps are involved in the optimization of the developed TLC-densitometric method. The first one is performing preliminary experiments based on a literature survey to select factors that have the greatest impact on the selected responses. The second one is applying the AQbD principles to investigate the selected factors to obtain the optimum conditions to achieve the desired chromatographic separation.

Preliminary studies and experiments

Reviewing the mobile phase systems used in the previously published TLC-densitometric methods which were developed for the determination of either HCZ with other drugs [34,35,36,37,38,39,40], HCZ with its impurities [41,42,43], or CPL in combination with other drugs [44,45,46], it was found that, in most manuscripts, methanol was used in combination with either ethyl acetate or chloroform. Chloroform was excluded for its toxic effect. Trials were performed using a methanol: ethyl acetate mixture and it was observed that using methanol in any ratio resulted in the elution of all of the studied components with the developing system except for CDS, hence methanol was kept out. After that, preliminary experiments were done to test the effect of the amount of ethyl acetate on the mobile phase used. It is worth mentioning that, on increasing ethyl acetate amount (> 8 mL), bad resolution between HCZ and CTZ and retained CDS peak was observed. Upon decreasing the ethyl acetate amount (< 6 mL) no resolution between HCZ and SMD was noticed. Moreover, when testing the effect of ammonium hydroxide solution and glacial acetic acid, it was observed that ammonium hydroxide had no significant effect on the chromatographic separation. In contrast, glacial acetic acid improved the elution of CDS. Moreover, it enhanced the separation between CPL, CTZ, HCZ, and SMD. By moving on to studying the effect of the glacial acetic acid amount, it was found that its effect was remarkable only when it was used in a ratio between 0.5 to 0.7 mL. On the other hand, the effect of plate length on chromatographic resolution was tested and it significantly affected the separation between HCZ and SMD, HCZ and CTZ, and the retardation factor of CDS. Increasing the plate length (> 10 up to 12) enhanced the chromatographic resolution as well as the elution of CDS although it increased the analysis time.

Optimization design and desirability function

For attaining the optimum conditions, three CMPs (independent variables); ethyl acetate ratio, glacial acetic acid ratio, and plate length were found to affect significantly the selected CAAs (dependent variables); retardation factor (Rf) of CDS and resolution (Rs) between CTZ and HCZ and between HCZ and SMD. CMPs were subjected to multivariate custom design experimental analysis, studied, and optimized. Levels of each (CMP) were assigned depending on the previously mentioned preliminary studies to obtain the maximum desired values for the selected (CAAs).

In consequence, AQbD was performed at three different levels for ethyl acetate ratio (6.00, 7.00, and 8.00 mL/chromatographic development), glacial acetic acid levels were (0.50, 0.60, and 0.70 mL/chromatographic development) using plates length of (10, 11 and 12 cm). The custom design was established using JMP® software version 16.0.0 [28] and, consequently, sixteen experiments originated. In pursuit of measuring the curvature, the model design included three trials at the center point (the three independent variables are at zero values). Experiments were performed and the resulting responses were gathered, recorded, and fed into the model, Table 1, and then prediction models were constructed for each measured response. The models provided a coefficient for each main effect, interaction, and quadratic term, Fig. 3. The previously mentioned figure represented the nonlinear polynomial interactions, while, the coefficients measured the direction and strength of each impact on the related response. ANOVA (analysis of variance) was performed and measured the significance of each value on the responses and p-values were computed. P-values less than 0.05 indicated that the assigned factors affected the responses. From the results in Table 2, one can conclude that the ethyl acetate ratio had a significant effect on all the studied responses while the glacial acetic acid ratio affected significantly CTZ and HCZ separation. Additionally, plate length affected the Rf of CDS and resolution between HCZ and SMD remarkably.

Table 1 Design of experiments represents factors, their levels and responses for the proposed TLC-densitometric method
Full size table
Fig. 3
figure 3

Interaction plots represent the effect of the studied factors on the measured responses

Full size image
Table 2 The calculated coefficients of the prediction models and P-values obtained from the ANOVA test results
Full size table

Moreover, the ANOVA test was used to evaluate the model's statistical regression validity. Table 3, showed that the P-value of all responses was (< 0.05), indicating that the model was significant. The calculated R2 values for the three stated responses were almost unity indicating that the expected and the obtained responses were perfectly fitted, ensuring the high predictive ability of the model to new observations.

Table 3 Summary of fitting results for the predicted versus the found responses for the proposed method
Full size table

Moreover; the root mean square error of prediction (RMSEP) value is an indicator of the error between the predicted and the true values, the lower (RMSEP) values the better the prediction power. The calculated (RMSEP) was found to be at minimal values confirming the accuracy of the predicted model.

The response surface method as surface plots was additionally used to illustrate the effect of the CMPs (independent variables) on the selected CAAs (responses). It was given in Fig. 4 [25].

Fig. 4
figure 4

Surface plots for measured responses as (Rf of captopril disulphide, resolution between chlorothiazide and hydrochlorothiazide peaks and resolution between hydrochlorothiazide and salamide peaks)

Full size image

For the optimization of the multiple responses, the desirability function technique was the tool of choice, the response optimizer tool was used to attain the best separation parameters, Fig. 5 and the “maximum desirability” option was adopted [47]. The optimum conditions for separating the cited components was ethyl acetate: glacial acetic acid (6.00: 0.60, v/v) as a developing system applied to 12 cm plate length which in consequence led to acceptable and high resolution and retardation factor, as represented in Fig. 6.

Fig. 5
figure 5

Prediction profiler concerning the experimental design representing the optimum separation conditions

Full size image
Fig. 6
figure 6

Three and two dimensional (3D) and (2D) TLC-densitogram of mixture of captopril and hydrochlorothiazide and their impurities (chlorothiazide, salamide and captopril disulphide) using ethyl acetate: glacial acetic acid (60:6, v/v) as a developing system and scanning wavelength at 215 nm

Full size image

Design space (DS) establishment

Design space (DS) is a multidimensional configuration and interaction of input factors and process parameters that have been shown to assure quality [24]. It could be represented graphically as a multi-response surface plot in which each response’s contour plot is based on the factors being investigated, and factor interactions are represented in an overlapping manner [24]. Establishing design space is a crucial step to guarantee that minor changes in the method's parameters do not negatively impact the analysis's quality and maintain the resulting responses within the stated acceptable limits [25]. The design space for the developed experimental design is illustrated in Fig. 7. Looking at the red zone in the mentioned figure, it is clear that it is possible to move through a flexible range of changes in ethyl acetate ratio, glacial acetic acid ratio, and plate length between 6.00–6.25 mL, 0.58–0.65 mL, and 11–12 cm, respectively, to obtain the best results regarding retardation factor and resolution between the separated peaks.

Fig. 7
figure 7

The design space for the proposed experimental design for the three responses; retardation factor for captopril disulphide (A), resolution between hydrochlorothiazide and chlorothiazide (B) and resolution between hydrochlorothiazide and salamide (C)

Full size image

Method control strategy

System suitability parameters were calculated for the developed method to validate and assess the anticipated experimental conditions. All calculated parameters were found to be within the specified limits Table 4.

Table 4 System suitability parameters testing for determination of captopril and hydrochlorothiazide in presence of their impurities by the developed TLC-densitometric method
Full size table

Method validation results

To ensure the suitability of the method for routine analysis, validation parameters were calculated according to ICH guidelines [48], such as linearity, range, accuracy, specificity, selectivity, LOD, and LOQ and results were summarized in Additional file 1: Table S2.

Values of the resulting correlation coefficients confirmed that the developed method had good linearity within the tested ranges, Additional file 1: Figure S2. Moreover, the accuracy of the method was confirmed by the closeness of all obtained percent recovery to the reference value (100%). On the other hand, all the calculated (%RSD) values were small (< 2%) assuring the precision of the method either on repeatability or intermediate precision levels. Regarding method sensitivity, it was confirmed by low values of the calculated LOD and LOQ achieving the target of the proposed method. The high specificity of the proposed method was ensured by the well-defined and highly resolved peaks among the cited components. In addition to the absence of interference from encountered excipients or impurities Fig. 6.

Analysis of dosage form and statistical comparison

The proposed method was applied to pharmaceutical formulation (Capozide®) and obtained recovery percent results were found to be (101.96 and 101.55 for CPL and HCZ, respectively) revealing high method selectivity Fig. 6. Results conducted by the proposed method were statistically compared with the reported HPLC method [10]. It could be concluded that the resulting F and t values did not exceed the tabulated ones indicating that there was no fundamental difference between the two compared methods, results were listed in Table 5.

Table 5 Statistical comparison of the results obtained by the proposed TLC-densitometry method and the reported HPLC method for the analysis of Capozide ® tablet
Full size table

After that, the accuracy of the method was assured by the good results obtained on applying the standard addition technique Table 5, confirming no interference from pharmaceutical additives and ensuring method accuracy.

Greenness profile

Green chemistry is all about designing an eco-friendly process without a negative impact on the environment and human health [49]. It has gained increased global attention and consideration over the last few decades.

In this regard, numerous publications and reviews were published emphasizing how to develop more benign and eco-friendly methods. Evaluating the compatibility of the developed methods with the 12 green analytical chemistry (GAC) principles using several different tools has become important to ascertain the extent of the method's greenness and its influential effect on the environment [26, 50].

The first green metric tool used was the National Environmental Method Index (NEMI) [51]. It was represented in a circular form divided into four sections, each of them expressing one of the principles of GAC [51]. The greenness of the method and the extent of its impact on the environment, regarding the usage of toxic, hazardous, persistent, bio-accumulative, or corrosive solvents are expressed qualitatively [50]. Despite its advantages, it has many drawbacks, most of them originating from its inability to cover all GAC principles. Also, it gives general information about the greenness of the method, not detailed and in a qualitative way wasting more time searching official lists about chemicals used [50]. From the results depicted in Table 6, it could be concluded that the developed method was greener than the other comparable reported methods [10, 17, 18] with four green quadrants. This is due to the use of more green solvents with a minimum amount of consumed solvents and the least waste production.

Table 6 Green and whiteness assessment with comparison between the proposed and reported methods
Full size table

The second applied tool was the Green Analytical Procedure Index (GAPI). It is one of the semi-qualitative tools [52] that translates the twelve GAC principles into three aspects which are sample preparation, reagents and solvents, and finally, instruments assessment. From the depicted GAPI pictograms, in Table 6, it could be concluded that the developed method has the lowest number of red fields followed by the reported capillary electrophoresis [18] then the reported HPLC [10], and finally the reported HPLC [17] one. The previous results could be rationalized as the developed method used greener solvents compared to the other reported ones which depended on using less eco-friendly solvents than ethyl acetate such as (methanol, phosphoric acid, sodium hydroxide, etc.) [53]. It is worth mentioning that the reported HPLC [17] had the highest number of red fields as it consumed massive amounts of solvents leading to high amounts of the produced waste.

The third green metric tool is the analytical eco-scale [54]. This method is represented by one hundred points, from which the penalty points that are calculated for each step that does not apply one of the green chemistry principles are subtracted. Its most important advantages, in addition to its simplicity, lie in the fact that it calculates the hazard effect of each solvent separately, and also takes into consideration the amount of the produced waste and its treatment [26, 54]. From an eco-scale point of view; the developed TLC-densitometric method and the other reported ones [10, 18] were found to have eco-score (> 75) which indicated that these methods were excellent green methods Additional file 1: Table S3. It worth noticed that developed method had the highest eco-score = 85 followed by the reported capillary electrophoretic method [18]. Although the developed TLC-densitometric method produced a larger amount of waste compared to the reported capillary electrophoresis [18], the last depended upon using the less green solvents, n-butanol, and methanol with a higher number of pictograms compared to ethyl acetate, Additional file 1: Table S3.

Moreover, the principles of GAC have been summarized and expressed in a way that brings them all together and calculated quantitatively in one step through one program in the form of a watch calculator, this method is called AGREE [29]. Depending on how green the method is and its impact on the environment, each of the 12 GAC principles is evaluated and assigned a value between 0 and 1, with 1 being the highest possible score [26]. From the results listed in Table 6; the proposed TLC-densitometric method has the highest AGREE value followed by the reported methods [10, 17, 18] with values (0.74, 0.69, 0.59 and 0.49), respectively, Table 6. The proposed method had the advantage of analyzing many samples per run with the use of more ecological solvents than other reported methods [10, 17, 18]. However; the reported method [17] had the lowest AGREE value due to the high amount of waste production with a long run time that resulted in a lower number of analyzed samples per hour.

The urgent need for a closer look and a more holistic view of the method performance and quality was the catalyst for developing the white analytical chemistry (WAC) which was the extension of the GAC principles [27]. A white analytical method represents the adhesion between the method’s analytical, ecological, and practical traits [27]. The fourth used tool was RGB-12 (Red–Green–Blue-12) which indicated the method's sustainability by evaluating the method's whiteness percent [55, 56]. The percent of whiteness was listed in Table 6. Additionally, the details of scores for the coded colors were presented in Additional file 1: Figures S3 and S4. The proposed method had the highest whiteness percent (105.3%) followed by the reported capillary electrophoresis one [18] (101.5%) then the reported HPLC [10] (88.2%) and finally the reported HPLC [17] with whiteness score of (80.2%). Each coded color was then evaluated and explained individually. For the red color, the developed method had the highest score indicating the high sensitivity, validity, and well performance of the method. Moving to the green color, the reported capillary electrophoresis had the highest score as it consumed the least amount of solvents along with the use of an instrument that consumed low energy. Concerning the blue color, the developed method had the highest score due to the use of a cost-effective instrument compared to other expensive chromatographic tools, in addition to the higher number of samples analyzed per hour.

All results obtained by the four applied green metric tools are in the same context confirming the superiority of the developed method over the reported ones [10, 17, 18]. It could be deduced that the developed method had the lowest impact on the environment with the least number of trials and low amount of solvent consumption which highlights the prevalence of the proposed method over the reported ones [10, 17, 18], Table 6.

Conclusion

Currently, a highly sensitive quantification of active pharmaceutical ingredients encountered impurities in their specified pharmacopeial limit has become necessary, in anticipation of the expected severe serious effect on human health. Therefore, the utmost importance came to develop a method for separating impurities with high sensitivity from the drugs present in the mixture.

AQbD-based TLC-densitometric method with green fingerprint had been innovatively developed for the determination of two antihypertensive agents along with their impurities with a low number of trials and hence low solvent consumption and waste production. The developed method was successfully applied for quantitation of the drugs and their toxic impurities for the first time with high sensitivity and low analysis time. It was evaluated according to the ICH guidelines, and the results were in accordance with the accepted limits. Using four different green metric tools and a whiteness assessment tool has verified that the newly developed TLC-densitometric method is more sustainable than the other published methods. This method can be approved for routine analysis of the cited components in raw material and pharmaceutical formulation, especially in small laboratories that lack the instruments and facilities required for the highly expensive HPLC without affecting method performance or sensitivity.

Availability of data and materials

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request.

Abbreviations

AQbD:

Analytical quality by design

CPL:

Captopril

HCZ:

Hydrochlorothiazide

CDS:

Captopril disulphide

CTZ:

Chlorothiazide

SMD:

Salamide

ATP:

Analytical target profile

CAAs:

Critical analytical attributes

CMPs:

Critical method parameters

TLC:

Densitometric:thin layer chromatographic-densitometric

HPLC:

High Performance liquid chromatography

ICH:

International Conference on Harmonization

ACE inhibitors:

Angiotensin converting enzyme inhibitors

UPLC/MS/MS:

Ultra performance liquid chromatography/mass spectrometry

GC–MS:

Gas chromatography–mass spectrometry

ADME/TOX studies:

Absorption-distribution-metabolism-excretion/toxicological studies

GAC:

Green analytical chemistry

CPPs:

Critical Process Parameters

NEMI:

National Environmental Method Index

GAPI:

Green Analytical Procedure Index

AGREE:

Analytical GREEnness

RGB-12:

Red–Green–Blue-12

Rf:

Retardation Factor

Rs:

Resolution

B.P:

British Pharmacopeia

DS:

Design space

ANOVA:

Analysis of variance

RMSEP:

Root mean square error of prediction

LOD:

Limit of detection

LOQ:

Limit of quantitation

RSD:

Relative standard deviation

WAC:

White analytical chemistry

References

  1. Her Majesty’s Stationery Office; London: 2020 British Pharmacopoeia Commission - British Pharmacopoeia.

  2. Sweetman SC. Martindale, the complete drug reference. 36th ed. London: Pharmaceutical Press; 2009.

    Google Scholar 

  3. Harvey RA. Lippincott’s illustrated reviews pharmacology. 5th ed. Philadelphia: Lippincott Williams & Wilkins; 2012.

    Google Scholar 

  4. Moussa BA, Abadi AH, Abou Youssef H, Mahrouse MA. Development and validation of a stability indicating HPLC method for the simultaneous determination of captopril and indapamide. Anal Chem An Indian J. 2013;12(6):222–32.

    CAS  Google Scholar 

  5. Venkatesan P, Valliappan K. Impurity profiling: theory and practice. J Pharm Sci Res. 2014;6(7):254–9.

    Google Scholar 

  6. Roy J. Pharmaceutical impurities—a mini-review. AAPS PharmSciTech. 2002;3(2):1–8.

    Article  PubMed Central  Google Scholar 

  7. Rao KS, Panda M, Keshar NK, Yadav SK. Simultaneous estimation of captopril and hydrochlorothiazide in combined dosage forms. Chronicles Young Sci. 2012;3(1):37–41.

    Article  CAS  Google Scholar 

  8. Yao HC, Sun M, Yang XF, Zhang ZZ, Li H. Simultaneous determination of captopril and hydrochlorothiazide by time-resolved chemiluminescence with artificial neural network calibration. J Pharm Anal. 2011;1(1):32–8.

    Article  CAS  PubMed  Google Scholar 

  9. Huang T, He Z, Yang B, Shao L, Zheng X, Duan G. Simultaneous determination of captopril and hydrochlorothiazide in human plasma by reverse-phase HPLC from linear gradient elution. J Pharm Biomed Anal. 2006;41(2):644–8.

    Article  CAS  PubMed  Google Scholar 

  10. Ivanovic D, Medenica M, Malenovic A, Jancic B. Validation of the RP-HPLC method for analysis of hydrochlorothiazide and captopril in tablets. Accredit Qual Assur. 2004;9(1):76–81.

    Article  CAS  Google Scholar 

  11. Absalan G, Akhond M, Karimi R, Ramezani AM. Simultaneous determination of captopril and hydrochlorothiazide by using a carbon ionic liquid electrode modified with copper hydroxide nanoparticles. Microchim Acta. 2018;185(2):1–8.

    Article  CAS  Google Scholar 

  12. Gholivand MB, Khodadadian M. Simultaneous voltammetric determination of captopril and hydrochlorothiazide on a graphene/ferrocene composite carbon paste electrode. Electroanalysis. 2013;25(5):1263–70.

    Article  CAS  Google Scholar 

  13. Dawud ER, Shakya AK. HPLC-PDA analysis of ACE-inhibitors, hydrochlorothiazide and indapamide utilizing design of experiments. Arab J Chem. 2019;12(5):718–28.

    Article  CAS  Google Scholar 

  14. Mahrouse MA. Simultaneous ultraperformance liquid chromatography/tandem mass spectrometry determination of four antihypertensive drugs in human plasma using hydrophilic–lipophilic balanced reversed-phase sorbents sample preparation protocol. Biomed Chromatogr. 2018;32(12): e4362.

    Article  PubMed  Google Scholar 

  15. Gulyaev IV, RevelSkii AI. Analyssis of pharmaceutical substances by reaction gas chromatography/mass spectrometry. J Anal Chem. 2010;65(13):1341–6.

    Article  CAS  Google Scholar 

  16. Karimi-Maleh H, Ganjali MR, Norouzi P, Bananezhad A. Amplified nanostructure electrochemical sensor for simultaneous determination of captopril, acetaminophen, tyrosine and hydrochlorothiazide. Mater Sci Eng C. 2017;73:472–7.

    Article  CAS  Google Scholar 

  17. Kirschbaum J, Perlman S. Analysis of captopril and hydrochlorothiazide combination tablet formulations by liquid chromatography. J Pharm Sci. 1984;73(5):686–7.

    Article  CAS  PubMed  Google Scholar 

  18. Pasquini B, et al. Cyclodextrin- and solvent-modified micellar electrokinetic chromatography for the determination of captopril, hydrochlorothiazide and their impurities: a quality by design approach. Talanta. 2016;160:332–9.

    Article  CAS  PubMed  Google Scholar 

  19. Pires DEV, Blundell TL, Ascher DB. pkCSM: predicting small-molecule pharmacokinetic properties using graph-based signatures. J Med Chem. 2015;58(9):4066–72.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Nagieb HM, Abdelwahab NS, Abdelrahman MM, Zaazaa HE, Ghoniem NS. Greenness assessment of UPLC/MS/MS method for determination of two antihypertensive agents and their harmful impurities with ADME/TOX profile study. Sci Rep. 2023;13(1):19318.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Prajapati P, Ahir H, Prajapati B, Shah S. Chemometric-based AQbD and green chemistry approaches to chromatographic analysis of remogliflozin etabonate and vildagliptin. J AOAC Int. 2023;106(1):239–49.

    Article  Google Scholar 

  22. Prajapati P, Tailor P, Shahi A, Acharya A, Shah S. Application of chemometry and design of experiments to green HPTLC method for synchronous estimation of multiple FDCs of cilnidipine. J AOAC Int. 2022;105(5):1491–501.

    Article  PubMed  Google Scholar 

  23. Prajapati P, Patel A, Shah S. Simultaneous estimation of telmisartan, chlorthalidone, amlodipine besylate and atorvastatin by RP-HPLC method for synchronous assay of multiple FDC Products using analytical FMCEA-based AQbD approach. J Chromatogr Sci. 2023;61(2):160–71.

    Article  CAS  PubMed  Google Scholar 

  24. Hejmady S, Choudhury D, Pradhan R, Singhvi G, Dubey SK. Analytical quality by design for high-performance thin-layer chromatography method development. In: Handbook of analytical quality by design. London: Academic Press; 2021. p. 99–113.

    Chapter  Google Scholar 

  25. Fares MY, Hegazy MA, El-Sayed GM, Abdelrahman MM, Abdelwahab NS. Quality by design approach for green HPLC method development for simultaneous analysis of two thalassemia drugs in biological fluid with pharmacokinetic study. RSC Adv. 2022;12(22):13896–916.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Kannaiah KP, Sugumaran A, Chanduluru HK, Rathinam S. Environmental impact of greenness assessment tools in liquid chromatography—a review. Microchem J. 2021;170: 106685.

    Article  CAS  Google Scholar 

  27. Nowak PM, Wietecha-Posłuszny R, Pawliszyn J. White analytical chemistry: an approach to reconcile the principles of green analytical chemistry and functionality. TrAC Trends Anal Chem. 2021;138: 116223.

    Article  CAS  Google Scholar 

  28. JMP, Design of Experiments (DOE) with JMP®, JMP; 2020. https://www.jmp.com/en_us/home.html.

  29. Pena-Pereira F, Wojnowski W, Tobiszewski M. AGREE—analytical GREEnness metric approach and software. Anal Chem. 2020;92(14):10076–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. pkCSM properties. https://biosig.lab.uq.edu.au/pkcsm/prediction

  31. Elder D, Teasdale A. ICH Q9 quality risk management. ICH quality guidelines: an implementation guid; 2017. p. 579–610.

  32. Patil AS, Shirkhedkar AA. Application of quality by design in the development of HPTLC method for estimation of anagliptin in bulk and in-house tablets. Eurasian J Anal Chem. 2017;12(5):443–58.

    Article  CAS  Google Scholar 

  33. El-Koussi WM, Atia NN, Saleh GA, Hammam N. Innovative HPTLC method for simultaneous determination of ternary mixture of certain DMARDs in real samples of rheumatoid arthritis patients: an application of quality by design approach. J Chromatogr B. 2019;1124:135–45.

    Article  CAS  Google Scholar 

  34. Patel TR, Patel TB, Suhagia BN, Shah SA. HPTLC Method for simultaneous estimation of aliskiren, amlodipine and hydrochlorothiazide in synthetic mixture using quality by design approach. Liq Chromatogr Relat Technol. 2015;38(16):1546–54.

    Article  CAS  Google Scholar 

  35. Yadav SS, Rao JR. Simultaneous HPTLC analysis of bisoprolol fumarate and hydrochlorthiazide in pharmaceutical dosage form. Int J Pharm Pharm Sci. 2013;5(2):286–90.

    CAS  Google Scholar 

  36. Ahir KB, Patelia EM, Mehta FA. Simultaneous estimation of nebivolol hydrochloride and hydrochlorothiazide in tablets by TLC-densitometry. J Chromatogr Sep Tech. 2012;3(5):2.

    Article  Google Scholar 

  37. Sharma M, Kothari C, Sherikar O, Mehta P. Concurrent estimation of amlodipine besylate, hydrochlorothiazide and valsartan by RP-HPLC, HPTLC and UV-spectrophotometry. J Chromatogr Sci. 2014;52(1):27–35.

    Article  CAS  PubMed  Google Scholar 

  38. Youssef RM, Maher HM, Hassan EM, El-Kimary EI, Barary MA. Development and validation of HPTLC and spectrophotometric methods for simultaneous determination of candesartan cilexetil and hydrochlorothiazide in pharmaceutical preparation. Int J Appl Chem. 2010;6(2):233–46.

    Google Scholar 

  39. Santhana Lakshmi K, Lakshmi S. Simultaneous analysis of losartan potassium, amlodipine besylate, and hydrochlorothiazide in bulk and in tablets by high-performance thin layer chromatography with UV-absorption densitometry. J Anal Methods Chem. 2012;2012: 108281.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Kumbhar ST, Chougule GK, Tegeli VS, Gajeli GB, Thorat YS, Shivsharan US. A validated HPTLC method for simultaneous quantification of nebivolol and hydrochlorothiazide in bulk and tablet formulation. Int J Pharm Sci Drug Res. 2011;3(1):62–6.

    CAS  Google Scholar 

  41. Hegazy MA, Metwaly FH, Abdelkawy M, Abdelwahab NS. Validated chromatographic methods for determination of Hydrochlorothiazide and Spironolactone in pharmaceutical formulation in presence of impurities and degradants. J Chromatogr Sci. 2011;49(2):129–35.

    Article  CAS  PubMed  Google Scholar 

  42. Naguib IA, Abdelaleem EA, Zaazaa HE, Draz ME. Simultaneous determination of hydrochlorothiazide and benazepril hydrochloride or amiloride hydrochloride in presence of hydrochlorothiazide impurities: chlorothiazide and salamide by HPTLC method. J Chromatogr Sci. 2015;53(1):183–8.

    Article  CAS  PubMed  Google Scholar 

  43. Abou Kull ME, Naguib IA. Simultaneous determination of hydrochlorothiazide and its impurities (chlorothiazide and salamide) in a quaternary mixture with candesartan cilexetil by HPTLC method. Curr Pharm Anal. 2017;13(2):188–94.

    Article  CAS  Google Scholar 

  44. Czerwinska K, Wyszomirska E, Kaniewska T. Identification and determination of selected medicines reducing hypertension by densitometric and gas chromatographic methods. Acta Pol Pharm. 2001;58(5):331–8.

    CAS  PubMed  Google Scholar 

  45. Lin Ling B, Baeyens WRG, De Castillo B, Imai K, Moerloose PD, Stragier K. Determination of thiols of biological and pharmacological interest by high-performance thin-layer chromatography and fluorescence scanning densitometry. Pharm Biomed Anal. 1989;7(12):1663–70.

    Article  Google Scholar 

  46. Liliya L, Dmytro K, Olena S, Ihor B, Tamara K. Development of methodology for identification of captopril in medicines. Asian J Pharm. 2016;10(3):168–71.

    CAS  Google Scholar 

  47. Hussein OG, Ahmed DA, Rezk MR, Abdelkawy M, Rostom Y. Exquisite integration of quality-by-design and green analytical approaches for simultaneous determination of xylometazoline and antazoline in eye drops and rabbit aqueous humor, application to stability study. J Pharm Biomed Anal. 2023;235: 115598.

    Article  CAS  PubMed  Google Scholar 

  48. ICH, ICH Harmonised Tripartite Health. Validation of analytical procedures: text and methodology. Q2 (R1). ICH, ICH Harmonised Tripartite Health; 2005.

    Google Scholar 

  49. Paul AT, John WC. Green chemistry: theory and practice. Oxford: Oxford University Press; 2000.

    Google Scholar 

  50. Korany MA, Mahgoub H, Haggag RS, Ragab MAA, Elmallah OA. Green chemistry: analytical and chromatography. J Liq Chromatogr Relat Technol. 2017;40(16):839–52.

    Article  CAS  Google Scholar 

  51. Keith LH, Gron LU, Young JL. Green analytical methodologies. Chem Rev. 2007;107(6):2695–708.

    Article  CAS  PubMed  Google Scholar 

  52. Płotka-Wasylka J. A new tool for the evaluation of the analytical procedure: green analytical procedure index. Talanta. 2018;181:204–9.

    Article  PubMed  Google Scholar 

  53. list of N. codes and Standards, “NFPA Chemicals.” https://www.newenv.com/resources/nfpa-chemicals/. Accessed 19 Aug 2023.

  54. Gałuszka A, Konieczka P, Migaszewski ZM, Namiesnik J. Analytical Eco-Scale for assessing the greenness of analytical procedures. TrAC Trends Anal Chem. 2012;37:61–72.

    Article  Google Scholar 

  55. Prajapati P, Pulusu VS, Shah S. Principles of white analytical chemistry and design of experiments to development of stability-indicating chromatographic method for the simultaneous estimation of thiocolchicoside and lornoxicam. J AOAC Int. 2023;106(6):1654–65.

    Article  PubMed  Google Scholar 

  56. Prajapati P, Shahi A, Acharya A, Pulusu VS, Shah S. Implementation of white analytical chemistry-assisted analytical quality by design approach to green liquid chromatographic method for concomitant analysis of anti-hypertensive drugs in human plasma. J Chromatogr Sci. 2023. https://0-doi-org.brum.beds.ac.uk/10.1093/chromsci/bmad054.

    Article  PubMed  Google Scholar 

Download references

Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author information

Authors and Affiliations

  1. Pharmaceutical Chemistry, Faculty of Pharmacy, Nahda University [NUB], Beni-Suef, 62511, Egypt

    Hend M. Nagieb

  2. Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, 62514, Egypt

    Nada S. Abdelwahab & Maha M. Abdelrahman

  3. Analytical Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt

    Hala E. Zaazaa & Nermine S. Ghoniem

Authors
  1. Hend M. Nagieb
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nada S. Abdelwahab
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maha M. Abdelrahman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hala E. Zaazaa
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Nermine S. Ghoniem
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

HMN: Formal analysis, methodology and writing. NSA: Supervision, methodology, review & editing. MMA: Supervision, methodology, review & editing, HEZ: Supervision, methodology, review& editing and NSG: Supervision, methodology, review & editing. All authors approved the final manuscript.

Corresponding author

Correspondence to Nermine S. Ghoniem.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publications

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Figure S1.

Flow chart highlights the most important stages of AQbD step by step. Figure S2. Calibration graphs for captopril and hydrochlorothiazide and their impurities illustrating linear regression equation and correlation coefficient (r). Figure S3. Visualization and comparison of the evaluation results of the four model methods for determination of captopril and hydrochlorothiazide and their impurities according to the 12 principles of WAC, performed using the RGB 12 algorithm. Figure S4. Comparison of the main evaluation outcomes obtained from the RGB 12 analysis where the white dashed line indicates 100% - a full fitness for planned application. The values above 100 indicate additional capabilities exceeding current requirements. Table S1. Analytical target profile elements for TLC-densitometric method for determination of captopril and hydrochlorothiazide and their impurities. Table S2. Regression and validation parameters for the developed TLC – densitometry method for determination of captopril and hydrochlorothiazide in presence of their impurities. Table S3. Eco-scale scores of the developed and the reported method.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Nagieb, H.M., Abdelwahab, N.S., Abdelrahman, M.M. et al. AQbD TLC-densitometric method approach along with green fingerprint and whiteness assessment for quantifying two combined antihypertensive agents and their impurities. BMC Chemistry 18, 15 (2024). https://0-doi-org.brum.beds.ac.uk/10.1186/s13065-024-01125-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://0-doi-org.brum.beds.ac.uk/10.1186/s13065-024-01125-2

Keywords

BMC Chemistry

ISSN: 2661-801X

Contact us