Simultaneous estimation of ibuprofen, ca ﬀ eine, and paracetamol in commercial products using a green reverse-phase HPTLC method

: A fast, sensitive, and green reverse-phase “ high-performance thin-layer chromatography ” approach for the simultaneous estimation of ibuprofen (IBF), ca ﬀ eine (CAF), and paracetamol (PCM) in marketed formulations was established and veri ﬁ ed in this study. The binary combination of acetone and water (80:20 v/v) was used as the green eluent system. The current method ’ s greenness was predicted using four di ﬀ erent approaches, namely National Environmental Method Index, Analytical Eco-Score (89), ChlorTox (1.08 g), and the Analytical GREENness (83) approaches, which demonstrated an outstanding greener pro ﬁ le. The present approach was linear in the range of 25 – 800 ng·band − 1 for the simultaneous estimation of IBF, CAF, and PCM. In addition, the current method was accurate (% recoveries = 100 ± 2), precise (%CV < 2%), robust (%CV < 2), sensitive (LOD = 1.13 – 2.71ng·band − 1 and LOQ = 3.39 – 8.10ng·band − 1 ), and green. The amount of IBF, CAF, and PCM in commercial tablets was determined to be 99.51%, 98.25%, and 100.64%, respectively. The present method for the simultaneous determination of IBF, CAF, and PCM in marketed tablets is supported by these data. The ﬁ ndings of this study suggested that the current approach may be consistently applied to analyze IBF, CAF, and PCM in marketed tablets.


Introduction
Ibuprofen (IBF) is a commonly used anti-inflammatory medicine (Figure 1a) [1].It is advised to use it to treat mild to moderate pain and inflammation, including that caused by dysmenorrhea, migraines, dental pain, postoperative pain, and muscle and joint condition [1,2].It works by inhibiting the enzyme cyclooxygenase-2 (COX-2) [1].Caffeine (CAF) is a pseudo-alkaloidal medicine, which is also used to treat various kind of pain (Figure 1b) [3,4].The most popular anti-inflammatory and antipyretic medication, particularly for pediatric and geriatric patients, is paracetamol (PCM) (Figure 1c) [5,6].It is marketed and offered in a variety of dosage forms [6].The combination of IBF, CAF, and PCM is commonly used to treat several pains and inflammatory conditions [7].These drugs such as IBF, CAF, and PCM are violable in various commercially available multicomponent formulations.It is therefore required to standardize IBF, CAF, and PCM in qualitative and quantitative terms in marketed multicomponent products.
Various spectrometry [29][30][31], absorption-based spectrometry [7], and derivative spectrometry approaches [32,33] have been established for the simultaneous estimation of IBF, CAF, and PCM in bulk forms and multicomponent preparations.Numerous HPLC methods have also been developed and validated for the simultaneous estimation of IBF, CAF, and PCM in bulk forms and multicomponent pharmaceutical products [31,34,35].Capillary electrophoresis [34] and chemometry [36] approaches have also been suggested for the simultaneous estimation of IBF, CAF, and PCM in bulk forms and multicomponent pharmaceutical preparations.The simultaneous quantification of IBF, CAF, and PCM in mixed dosage forms has also been done using an HPTLC approach [37].Furthermore, our research group has produced an environmentally friendly HPTLC methodology for the quantification of CAF in commercially available energy drinks and dosage forms [38].Green normalphase and reverse-phase HPTLC methods have also been reported for the simultaneous estimation of CAF and PCM in commercial formulations by our research team [39].The simultaneous estimation of IBF, CAF, and PCM in commercial formulations, however, has not been reported using the green HPTLC techniques.
Various analytical techniques were recommended in published articles on the simultaneous estimation of CAF and PCM or IBF, CAF, and PCM.However, none of the reported analytical methods approximated the greenness scale.Furthermore, the simultaneous estimation of IBF, CAF, and PCM has not employed green HPTLC techniques.One of the twelve guiding principles of "green analytical chemistry (GAC)" is the use of substitute environmentally safe solvents to decrease the detrimental effects of toxic/ hazardous eluents on the environment [40].The application of green solvents has increased exponentially during the past few decades, according to a literature search [41][42][43].Several qualitative and quantitative techniques have been described in the literature to evaluate the greenness profiles of analytical methodologies.These include the Analytical GREENness (AGREE), Red, Green, and Blue, ChlorTox, Analytical Eco-Score (AES), Green Analytical Procedure Index, and the National Environmental Method Index (NEMI) [44][45][46][47][48][49][50].In the present study, four different tools, namely, NEMI, AES, ChlorTox, and AGREE approaches, were used to gauge the greener profile of the present methodology [44,45,49,50].Compared to traditional liquid chromatographic techniques, the green HPTLC method has a number of benefits, including low solvent consumption, short analysis times, nondestructive mode of detection, simplicity of use, minimal pretreatment, sensitivity, efficiency, simultaneous analysis of multiple samples, non-toxicity, and environmental friendliness [51][52][53][54][55][56].Based on the aforementioned data and observations, the current method seeks to create and establish a fast, sensitive, and green reverse-phase HPTLC method for the simultaneous estimation of IBF, CAF, and PCM in commercially available tablets.The proposed method for the simultaneous estimation of IBF, CAF, and PCM was verified using "The International Council for Harmonization (ICH)-Q2-R1" guidelines [57].

Materials
The reference standards of IBF, CAF, and PCM were obtained from "Sigma Aldrich (St. Louis, MO, USA)."Liquid chromatography-grade acetone was obtained from "E-Merck (Darmstadt, Germany)."The Milli-Q device was used to obtain the purified water.The marketed multicomponent tablets (containing 200 mg of IBF, 40 mg of CAF, and 325 mg of PCM) were bought at the neighborhood pharmacy in "Riyadh, Saudi Arabia."The other chemicals and solvents used were of the analytical variety.

Instrumentation and chromatographic conditions
The "HPTLC CAMAG TLC system (CAMAG, Muttenz, Switzerland)" was utilized for the simultaneous estimation of IBF, CAF, and PCM in the bulk forms and procured formulations.The samples were applied as 6 mm bands using the "CAMAG Automatic TLC Sampler 4 (ATS4) Sample Applicator (CAMAG, Geneva, Switzerland)."The "CAMAG microliter Syringe (Hamilton, Bonaduz, Switzerland)" was loaded into the sample applicator.The TLC plates were "glass plates (plate size: 10 × 20 cm 2 ) pre-coated with reverse-phase silica gel (particle size: 5 µm) 60F254S plates," which were used as the stationary phase.The binary combination of acetone-water (80:20 v/v) was used as the green eluent system.For the concurrent measurement of IBF, CAF, and PCM, the application rates were set at 150 nL

Calibration plots and quality control (QC) solutions for IBF, CAF, and PCM
Separate batches of IBF, CAF, and PCM stock solutions were made by dissolving the necessary quantities of each medication in the appropriate amount of the green eluent system.The resultant stock solution of each medication included 100 µg•mL −1 of active ingredient.IBF, CAF, and PCM concentrations in the 25-800 ng•band −1 range were created by diluting varying volumes of the stock solutions using the green eluent system.The peak area of each concentration of IBF, CAF, and PCM was recorded after 20 µL of each concentration of IBF, CAF, and PCM were placed on TLC plates for the current approach.IBF, CAF, and PCM calibration plots were produced by graphing the concentrations of IBF, CAF, and PCM versus the measured peak response in six replications (n = 6).In order to assess numerous validation criteria, three distinct QC solutions were produced.

Sample preparations for the simultaneous estimation of IBF, CAF, and PCM in procured tablets
Twenty commercial tablets were measured, and the mean weight for the concurrent determination of IBF, CAF, and PCM in procured tablets was computed.Each commercial tablet included 200 mg of IBF, 40 mg of CAF, and 325 mg of PCM.The commercial tablets were powdered after being roughly crushed.An amount of fine powder, equal to the mean weight of a tablet, was dispersed in 100 mL of the green eluent system.For the current procedure, 1 mL of this commercial tablet solution was diluted once more using 10 mL of the proposed solvent system.In order to remove any insoluble excipients, the produced solutions for procured formulations were sonicated for approximately 10 minutes and filtered.The obtained samples were used to assess IBF, CAF, and PCM concurrently in commercially available tablets by the existing methodology.

Validation studies
Using the ICH-Q2-R1 guidelines, the current method for the simultaneous estimation of IBF, CAF, and PCM was validated for numerous parameters [57].Plotting the concentrations of IBF, CAF, and PCM against the measured peak area allowed for the establishment of their linear ranges.
To assess the parameters for the proposed approach's system suitability for the simultaneous estimation of IBF, CAF, and PCM, "retardation factor (R f ), asymmetry factor (As), theoretical plates number per meter (N•m −1 ), and resolution factor (Rs)" computation was used.For the current method, the "R f , As, N•m −1 , and Rs" values were derived by their reported equations [43,58].
Using the spiking/standard addition methodology, the accuracy of the present method for the concurrent determination of IBF, CAF, and PCM was assessed as % recoveries [57].An additional 50%, 100%, and 150% of the IBF, CAF, and PCM solutions were spiked into the previously measured IBF, CAF, and PCM solutions (100 ng•band −1 ) in order to establish low-QC (LQC) solutions of IBF, CAF, and PCM of 150 ng•band −1 , moderate-QC (MQC) levels of 200 ng•band −1 , and high-QC (HQC) levels of 250 ng•band −1 .The above IBF, CAF, and PCM QC solutions were reanalyzed to measure the accuracy.The % recovery was computed at each concentration level of IBF, CAF, and PCM.Six replicates (n = 6) were utilized to measure the accuracy.
Intra/inter-assay precision was evaluated for the existing method for the simultaneous estimation of IBF, CAF, and PCM.Examining intra-assay variation for these three substances was made possible by quantifying freshly generated IBF, CAF, and PCM solutions at previously described QC levels on the same day (n = 6).Examination of freshly prepared solutions was done at previously described QC levels on three consecutive days (n = 6) as part of the evaluation of interassay variation for IBF, CAF, and PCM solutions for the current technique.
The robustness of IBF, CAF, and PCM was evaluated for the current method by introducing some, purposeful alterations to the composition of the green eluent system.After switching between acetone-water (82:18 v/v) and acetone-water (78:22 v/v) as the green eluent systems for IBF, CAF, and PCM, the alterations in peak area and R f data were observed (n = 6).
By utilizing a "standard deviation" approach, the sensitivity of the current method for the concurrent determination of IBF, CAF, and PCM was measured as "limit of detection (LOD) and limit of quantification (LOQ)."IBF, CAF, and PCM "LOD and LOQ" were computed using Eqs. 1 and 2 (n = 6) [57]: where S is the slope of the calibration curve for IBF, CAF, and PCM, and σ is the standard deviation of the intercept.
To assess the specificity of the current approach for the simultaneous estimation of IBF, CAF, and PCM, the R f data and 3D spectrum of IBF, CAF, and PCM in marketed tablets were contrasted to that of standards IBF, CAF, and PCM.

Application of current approach in the concurrent determination of IBF, CAF, and PCM in procured formulations
Under the same experimental settings as the simultaneous estimation of standards IBF, CAF, and PCM, the chromatographic responses of the processed samples of procured formulations were determined on TLC plates for the current approach (n = 3).The calibration curves for IBF, CAF, and PCM were used to approximate the amounts of IBF, CAF, and PCM in procured formulations for the present methodology.

Greenness assessment
Four different approaches, namely, NEMI, AES, ChlorTox, and AGREE, were used to assess the greenness profile of the current method for the simultaneous estimation of IBF, CAF, and PCM [44,45,49,50].NEMI is used to obtain preliminary judgment based on persistent, bioaccumulative, and toxic (PBT), hazardous, corrosive, and waste [44].
According to the NEMI method, four quarter circles are drawn and each quarter is colored green or left blank indicating the PBT, hazardous, corrosive, and waste [44].AES is a semi-quantitative approach, which considers all steps of analytical procedures, instruments, and waste.Analysis of compounds with no or minimal use of reagents, low energy consumption, and no waste is expected to be an ideal analysis with 100 points.If any of these parameters are deviated, penalty points are assigned, and total penalty points are subtracted from 100 [45].
The ChlorTox technique states that the ChlorTox score is determined using Eq. 3 [49]: where m sub is the mass of the substance of interest (acetone in the present study) needed for a single analysis, CH sub is the chemical risks of the acetone, and CH CHCl3 is the chemical hazard of standard chloroform.With the aid of the safety data sheet from "Sigma Aldrich (St. Louis, MO, USA)," the values of CH sub and CH CHCl3 were determined using the weighted hazards number (WHN) model [49].Using the WHN model and safety data sheet from "Sigma Aldrich (St. Louis, MO, USA)," CH sub value was determined using Eq.4: where, N cat1 , N cat2 , N cat3 , and N cat4 are the number of toxicities under the categories 1, 2, 3, and 4, respectively.For substance (acetone), N cat1 = 0, N cat2 = 2, N cat3 = 1, and N cat4 = 0 were taken from the safety data sheet of "Sigma Aldrich (St. Louis, MO, USA)."The AGREE-metric technique was used to evaluate the AGREE score for the current method for the simultaneous estimation of IBF, CAF, and PCM [50].This method assigns 0-1 scores to each of the twelve GAC components, after which the average score is determined.The "AGREE: The Analytical Greenness Calculator (version 0.5, Gdansk University of Technology, Gdansk, Poland, 2020)" was used to gauge the AGREE scores in the range from 0.0 to 1.0 for the current procedure.
3 Results and discussions

Method development
A variety of acetone-to-water ratios were studied as green eluent systems, including acetone-water ratios of 50:50, 60:40, 70:30, 80:20, and 90:10 v/v.The development of every green eluent system put to the test took place in saturated chambers (Figure 2).The green eluent systems such as acetone-water (50:50 v/v), acetone-water (60:40 v/v), acetone-water (70:30 v/v), and acetone-water (90:10 v/v) displayed unsatisfactory chromatography signals of IBF, CAF, and PCM with unreliable As for IBF (As > 1.20), CAF (As > 1.25), and PCM (As > 1.30).It was observed when the green eluent system acetone-water (80:20 v/v) was examined that this green eluent system displayed well-separated and unbroken chromatography signals of IBF at R f = 0.55 ± 0.01, CAF at R f = 0.67 ± 0.02, and of PCM at R f = 0.85 ± 0.02 (Figure 3a).Additionally, As values of 1.08, 1.12, and 1.05, respectively, were projected for IBF, CAF, and PCM, all of which are extremely reliable values.As a consequence, it was agreed that acetone-water (80:20 v/v) would be the final eluent system for the simultaneous estimation of IBF, CAF, and PCM in commercial tablets using the existing method.The densitometric recording of the IBF, CAF, and PCM spectral bands revealed that the strongest response was at 260 nm in wavelength.The entire simultaneous estimation of the IBF, CAF, and PCM therefore occurred at 260 nm.

Validation studies
The ICH-Q2-R1 criteria were utilized to measure a range of factors for the simultaneous estimation of IBF, CAF, and PCM [57].Table 1 shows the outcomes of the linearity evaluation of the IBF, CAF, and PCM calibration plots performed using the current method.The 25-800 ng•band −1 range of the IBF, CAF, and PCM calibration curves were linear.All drugs showed linearity over the 25-800 ng•band −1 range.The linearity was not maintained beyond 25-800 ng•band −1 range.As a result, it was the same for all drugs.IBF, CAF, and PCM's correlation coefficient (R 2 ) were predicted to be 0.9982, 0.9950, and 0.9949, respectively.Regression coefficients (R) for IBF, CAF, and PCM were calculated as 0.9991, 0.9974, and 0.9974,  Simultaneous estimation of ibuprofen, caffeine, and paracetamol  5 respectively.For IBF, CAF, and PCM, the R 2 and R data were considerably significant (p < 0.05).These outcomes suggested a significant correlation between IBF, CAF, and PCM concentration and measured responses.The linear range for IBF, CAF, and PCM for a reported HPTLC method was found to be 300-1,100 ng•band −1 [37].The reported linear range for IBF, CAF, and PCM was much inferior to the current method [37].All these results showed the reliability of the current method for the simultaneous estimation of IBF, CAF, and PCM.Table 2 mentions system appropriateness criteria for the current technique.The "R f , As, N•m −1 , and Rs" for the current approach were computed to be adequate for the concurrent determination of IBF, CAF, and PCM.
The accuracy of the current method was assessed as the % recovery for the simultaneous estimation of the IBF, CAF, and PCM.Table 3 contains the accuracy measurement outcomes for the current methodology.The % recoveries of IBF, CAF, and PCM at three distinct QC solutions were determined to be 98.90-101.24,99.12-100.82,and 98.24-101.99,respectively, using the current approach.The % recovery for IBF, CAF, and PCM for a reported HPTLC method was found to be 99.31-99.99,99.14-99.96,and 99.66-101.25,respectively [37].The reported % recovery for IBF, CAF, and PCM was identical to the current method [37].All these outcomes showed that the current method was accurate for the concurrent determination of IBF, CAF, and PCM.
The precision of the current method was evaluated as intra/inter-assay precision and reported as a percentage of coefficient of variation (%CV) for the simultaneous determination of IBF, CAF, and PCM.The results of the simultaneous estimation of IBF, CAF, and PCM utilizing the current technique are shown in Table 4 for intra-and inter-day precisions.IBF, CAF, and PCM %CVs were calculated to be 0.88-0.92%,0.92-1.08%,and 0.91-1.08%,respectively, for the intra-assay precision.The %CVs of IBF, CAF, and PCM for inter-day variation were measured to be 0.95-1.03%,0.95-1.13%,and 0.98-1.13%,respectively.The %CVs for IBF, CAF, and PCM for a reported HPTLC method were found to be 1.16-1.50,1.29-1.64,and 1.21-1.62,respectively [37].The reported %CVs for IBF, CAF, and PCM were identical to the current method [37].All of these outcomes showed that the current method was precise for the simultaneous estimation of IBF, CAF, and PCM.
By making minor, deliberate changes to the suggested solvent system, the robustness of the current method for the simultaneous determination of IBF, CAF, and PCM was evaluated.The results of the robustness research carried out with the current methodology are shown in Table 5.
It was established that the %CVs for IBF, CAF, and PCM were 0.87-0.97,0.93-0.99,and 0.95-1.02,respectively.It was discovered that the IBF, CAF, and PCM R f values were 0.54-0.56,0.66-0.68,and 0.84-0.86,respectively.The sensitivity of the current approach for the simultaneous determination of IBF, CAF, and PCM was assessed as "LOD and LOQ."The computed values of "LOD and LOQ" for IBF, CAF, and PCM are included in Table 1.For the present methodology, the "LOD and LOQ" for IBF were calculated to be 1.13 ± 0.03 and 3.39 ± 0.09 ng•band −1 , respectively.The "LOD and LOQ" for CAF were determined using the current method to be 2.29 ± 0.05 and 6.87 ± 0.15 ng•band −1 , respectively.The "LOD and LOQ" for PCM were determined using the current method to be 2.71 ± 0.06 and 8.10 ± 0.18 ng•band −1 , respectively.Using a reported HPTLC method, the "LOD and LOQ" for IBF were found to be 430 and 1,304 ng•mL −1 , respectively [37].Using a reported HPTLC method, the "LOD and LOQ" for CAF were found to be 36 and 109 ng•mL −1 , respectively [37].Using a reported HPTLC method, the "LOD and LOQ" for PCM were found to be 3 and 10 ng•mL −1 , respectively [37].The reported "LOD and LOQ" for IBF, CAF, and PCM were much inferior to those of the current method [37].Thus, compared to the previously disclosed HPTLC method, the proposed approach was significantly more sensitive for the simultaneous estimation of IBF, CAF, and PCM [37].All of these findings indicated that the current method was highly sensitive for the simultaneous estimation of IBF, CAF, and PCM.
By contrasting the R f data and 3D spectrum of IBF, CAF, and PCM in marketed tablets with that of standards IBF, CAF, and PCM, the specificity of the current method for the simultaneous determination of IBF, CAF, and PCM was evaluated.The results are shown in Figure 4.
At a wavelength of 260 nm, IBF, CAF, and PCM in standards and marketed tablets showed the greatest chromatographic response.The specificity of the present methodology for the concurrent determination of IBF, CAF, and PCM was revealed by the similar 3D spectrum, R f values, and wavelengths found in standards and marketed tablets.Overall, all validation parameters, including system suitability parameters, were acceptable for the concurrent determination of IBF, CAF, and PCM.For the simultaneous estimation of IBF, CAF, and PCM in their commercially available tablets, the proposed technique was used as an alternative to traditional liquid chromatography procedures.By contrasting the TLC signals at R f = 0.55 ± 0.01 for IBF, R f = 0.67 ± 0.02 for CAF, and R f = 0.85 ± 0.02 for PCM to those of standards IBF, CAF, and PCM utilizing the current methodology, the chromatograms of IBF, CAF, and PCM from procured tablets were recognized.
Figure 3b shows the chromatograms of IBF, CAF, and PCM found in marketed tablets.These peaks were identical to those of the standards for IBF, CAF, and PCM.Using the current method, the amount of IBF, CAF, and PCM in commercial tablets was determined to be 99.51 ± 1.38%, 98.25 ± 1.32%, and 100.64 ± 1.46%, respectively.These results suggested the applicability of the current methodology for the simultaneous estimation of IBF, CAF, and PCM in procured tablets.

Greenness assessment
Numerous quantitative and qualitative approaches are established for the greenness assessment of pharmaceutical assays [44][45][46][47][48][49][50].In the present work, four different approaches, namely NEMI, AES, ChlorTox, and AGREE approaches were used to assess the greenness of the   current approach [44,45,49,50].NEMI is used to obtain the preliminary assessment.According to the NEMI method, four quarter circles are drawn and each quarter is colored green or left blank indicating the following criteria [44]: PBT, corrosive, hazardous, and waste.Figure 5 shows the representative diagram for the NEMI of the current method.As all of the reagents employed are neither toxic, PBT, or corrosive and produce minimal waste, the current procedure produced four circles that were green.AES is a good semi-quantitative approach, which considers all the steps of the analytical procedures, instruments, and waste.The results of AES scores with penalty points for the current approach in comparison with the reported HPTLC approach are included in Table 6.The AES value of greater than 75 indicated an excellent greenness, the value of less than 75 but greater than 50 indicated adequate greenness, and the value of less than 50 indicated inadequate greenness [45].The AES score of the present method was derived to be 89, indicating an excellent greenness profile.The AES score for the reported HPTLC approach was derived to be 67 [37].The AES score of the present approach was much superior to the reported HPTLC method, indicating the excellent greenness profile of the present approach compared to the reported HPTLC method [37].
Table 7 includes the findings of the individual greener solvent ChlorTox scores and the overall ChlorTox for the suggested technique in comparison with the reported HPTLC approach.The total ChlorTox value for the suggested approach was anticipated to be 1.08 g, indicating that it was both relatively safe and environmentally friendly [49].The total ChlorTox value for the reported HPTLC approach was calculated to be 1.81 g [37].The total ChlorTox value of the present approach was much superior to the reported HPTLC method, indicating the excellent greenness and safety profile of the present approach compared to the reported HPTLC method [37].
The AGREE approach is the most widely used quantitative approach for greenness assessment as it consumes all 12 GAC principles [50].Figure 6 displays the overall AGREE score for the current methodology.The AGREE score of greater than 0.75 indicated excellent greenness, the AGREE score of less than 0.75 but greater than 0.50 indicated adequate greenness, and AGREE score of less than 0.50 indicated inadequate greenness [50].The current methodology projected that the overall AGREE score would be 0.83.The AGREE results again demonstrated the current method's excellent green features.Overall, the results of all greenness approaches indicated the excellent greener profile of the current method for the simultaneous estimation of IBF, CAF, and PCM in commercial products.

Conclusions
There are no green analytical methods in the literature for the simultaneous determination of IBF, CAF, and PCM.In this study, a fast, sensitive, and green HPTLC methodology was designed and validated for the simultaneous determination of IBF, CAF, and PCM in their commercially available products.For the simultaneous determination of IBF, CAF, and PCM, the current method is linear, accurate, precise, robust, highly sensitive, and green.The current method was successfully utilized to determine IBF, CAF, and PCM contents in their commercial tablets.The results of NEMI, AES, ChlorTox, and AGREE assessment showed an excellent greenness characteristic of the current method for the simultaneous determination of IBF, CAF, and PCM.The current method for the simultaneous determination of IBF, CAF, and PCM has been found more linear and highly sensitive than the previously reported HPTLC method.All of these findings suggested that the current method can be regularly used for the simultaneous determination of IBF, CAF, and PCM in their commercial products.

Figure 2 :
Figure 2: The representative TLC image for standard IBF, CAF, PCM, and marketed tablets established utilizing acetone-water (80:20 v/v) as the green eluent system for the current method.

Figure 5 :
Figure 5: NEMI Evaluation of greenness for the current methodology.

Figure 6 :
Figure 6: AGREE scale for the present methodology.

Table 1 :
Results of linearity assessment for the simultaneous estimation of IBF, CAF, and PCM utilizing the proposed methodology (mean ± SD; n = 6)

Table 2 :
The system suitability criteria for IBF, CAF, and PCM for the proposed methodology (mean ± SD; n = 3)

Table 3 :
Accuracy results of IBF, CAF, and PCM for the proposed technology (mean ± SD; n = 6)

Table 4 :
Assessment of intra/inter-day precision of IBF, CAF, and PCM for the current method (mean ± SD; n = 6)

Table 5 :
Robustness assessment results of IBF, CAF, and PCM for the current method (mean ± SD; n = 6)

Table 6 :
Analytical eco-score (AES) and penalty point assessment for the greenness of the current method and comparison with reported HPTLC method

Table 7 :
Results of ChlorTox scores for the proposed method in comparison with the reported HPTLC method in terms of the relative hazards with respect to chloroform (CH sub /CH CHCl3 ) and the mass of individual reagents used for single analysis (m sub )