Contrasting the validation parameters and greenness of normal-phase and reverse-phase stability-indicating HPTLC methods for lemborexant analysis

2.1 Materials

“Beijing Mesochem Technology (Beijing, China)” provided pure LMB (purity: 99 % by HPLC). “E-Merck (Darmstadt, Germany)” provided the HPLC grades ethanol (EtOH) and acetone (Ace). The ACS grade of petroleum ether (PE) was obtained from “Sigma Aldrich (St. Louis, MO, USA)”. The “Milli-Q device (Lyon, France)” was used to obtain the water (H₂O) of HPLC-grade. Commercial pharmaceutical tablets of LMB, each containing 10 mg of LMB, were purchased at a Mumbai, Indian pharmacy. The glass-coated 60 NP-18F254S and 60 RP-18F254S plates (plate size: 10 × 20 cm) pre-coated with silica gel (particle size: 5 μm) were obtained from “E-Merck (Darmstadt, Germany)”. The remaining materials were of AR grade.

2.2 Instrumentation and analytical settings

The “HPTLC system (Muttenz, Switzerland)” was used to analyze LMB in marketed pharmaceutical tablets. The solutions were applied in the form of 6 mm bands utilizing an “Automatic TLC Sampler 4 (ATS4) Sample Applicator (CAMAG, Geneva, Switzerland)”. The sample applicator was loaded to the “Microliter Syringe (Hamilton, Bonaduz, Switzerland)”. 150 nL/s was used as the application rate for LMB measurement. The glass-coated plates were developed using a linear ascending mode at a distance of 8 cm in an “automated developing chamber 2 (ADC2) (CAMAG, Muttenz, Switzerland)”. The development chamber was filled with the vapors of the corresponding developing system for 30 min at 22 °C. Under densitometry mode, LMB was detected at a wavelength of 199 nm using a UV detector. The slit size and scan speed were set to 4 × 0.45 mm² and 20 mm/s, respectively. For every measurement, there were three or six replicates used. The software that was utilized was “WinCAT's (version 1.4.3.6336, CAMAG, Muttenz, Switzerland)”.

Both procedures employed same equipment and analytical conditions. The developing systems and stationary phases were the primary differences between the NP and RP procedures. In the NP procedure, the optimal developing system was Ace/PE (40:60 v/v), while in the RP procedure, the optimal developing system was EtOH/H₂O (85:15 v/v). The stationary phase was “glass-coated plates (plate size: 10 x 20 cm) pre-coated with silica gel (particle size: 5 μm) 60 NP-18F254S plates” in NP procedure. The stationary phase was “glass-coated plates (plate size: 10 x 20 cm) pre-coated with silica gel (particle size: 5 μm) 60 RP-18F254S plates” in RP procedure.

2.3 LMB calibration curves

The precisely measured 10 mg of LMB was dissolved into 100 mL volumes of Ace/PE (40:60 v/v) for NP procedure and EtOH/H₂O (85:15 v/v) for RP procedure to form a stock solution with a concentration of 100 μg/mL of LMB (working standards for NP and RP procedures). For the NP procedure, LMB concentrations in the range of 50–500 ng/band were further produced by diluting the variable amount of LMB stock solution with Ace/PE (40:60 v/v). Conversely, the RP procedure was used to produce LMB concentrations in the 20–1000 ng/band range. This involved dilution of the different LMB stock solution volumes using EtOH/H₂O (85:15 v/v) (ICH, 2023). 10 μL of each LMB concentration were added to NP- and RP-TLC plates, respectively, for the NP and RP processes. For each LMB concentration, the peak response was determined using both techniques. LMB calibration curves were created by plotting the measured spot area vs. the LMB concentrations using six replicates (n = 6).

2.4 Sample preparation for the measurement of LMB in commercial tablets

Twenty-five tablets containing 10 mg of LMB each were randomly ingested in order to measure the amount of LMB in commercial tablets. Next, the average weight was calculated. The fine powder was obtained by crushing and triturating the tablets. The fine powder, containing a total of 10 mg of LMB, was dispersed using 10 mL of the corresponding developing system. The resultant mixes were sonicated for 15 min and filtered via 0.45 μm membrane filter (Foudah et al., 2021). The obtained solution was diluted using the respective developing system to obtain the solution in the LMB concentration of 300 ng/band for both procedures. The 10 μL samples from both solutions were injected for the determination of LMB in commercial tablets using both procedures.

2.5 Validation assessment

Both LMB measurement techniques were validated for multiple validation criteria in accordance with ICH-Q2-R2 guidelines (ICH, 2023). By plotting the recorded peak response against the LMB concentrations, LMB linearity was evaluated. Six repetitions (n = 6) were run separately to evaluate the linearity of the NP procedure of LMB analysis in the 50–500 ng/band range. Six replicates (n = 6) of the RP procedure were run separately to evaluate the LMB linearity over the 20–1000 ng/band range.

The “retardation factor (R_f), asymmetry factor (As), and number of theoretical plates per meter (N/m)” were computed to determine the parameters for the system appropriateness for both LMB measurement methods. Using their published formulae (Bele and Khale, 2011; Malinowska et al., 2013), the “R_f, As, and N/m” for both LMB measurement procedures were computed.

The accuracy of the two LMB measuring techniques was calculated utilizing spiking technology/standard addition strategy, expressed as % recoveries (ICH, 2023). For the NP procedure, additional 50, 100, and 150 % LMB solution was spiked into the pre-analyzed LMB solution (100 ng/band) to achieve low-quality control (LQC) levels of 150 ng/band, middle-quality control (MQC) levels of 200 ng/band, and high-quality control (HQC) levels of 250 ng/band of LMB. To attain LQC, MQC, and HQC levels of 300 ng/band, 400 ng/band, and 500 ng/band of LMB by the RP procedure, additional 50, 100, and 150 % LMB solution was mixed with the 200 ng/band pre-analyzed LMB solution. The pre-analyzed solution of LMB (100 ng/band) was first prepared freshly. Then, spiking was performed. The total volume after spiking was 1.0 mL for each QC sample. An investigation was conducted on three distinct LMB QC solutions in order to evaluate the accuracy of both approaches. For both approaches, six replications (n = 6) were run separately to calculate the % recovery at every level of QC.

The intra- and inter-assay precision for LMB of the NP- and RP-HPTLC techniques was evaluated. Using six replicates of freshly made LMB solutions at LQC, MQC, and HQC on the same day for both procedures (n = 6), the intra-assay precision for LMB was assessed. Using six replicates (n = 6) of newly generated LMB solutions at the same QC samples spaced out over three days, LMB inter-day precision was evaluated for each strategy.

A number of deliberate modifications can be made to the relevant developing system's content in order to evaluate the robustness of LMB for both approaches. The standard Ace/PE (40:60 v/v) developing system for LMB was changed to Ace/PE (42:58 v/v) and Ace/PE (38:62 v/v) for the NP-HPTLC experiment. Six replications (n = 6) were run separately to record the variations in peak response and R_f. The developing system EtOH/H₂O (85:15 v/v) for the RP-HPTLC method was changed to EtOH/H₂O (87:13 v/v) and EtOH/H₂O (83:17 v/v), and six replications (n = 6) were used to record the uncertainties in spot area and R_f (ICH, 2023).

Using a standard deviation methodology, the sensitivity of both techniques for LMB was assessed in terms of “limit of detection (LOD) and limit of quantification (LOQ)”. A blank sample (one devoid of LMB) was injected six times for each of the two methods, and the sample's standard deviation was computed. The published equations for both approaches were utilized to calculate LMB “LOD and LOQ” through six replications (n = 6) (ICH, 2023).

The R_f values, UV-absorption spectra, and 3D spectrum of LMB in commercially available pharmaceutical tablets were compared to that of pure LMB in order to assess the specificity and peak purity of both procedures for LMB (ICH, 2023).

2.6 Forced-degradation investigations

Under acidic, alkaline, oxidative, and thermal stress conditions, the forced-degradation investigations were carried out for both approaches (Alam et al., 2021b; Foudah et al., 2021). LMB is not a photosensitive drug and hence photostability study was not performed in this work. The LMB in the concentration of 400 ng/band for both techniques was subjected to 24 h of thermal stress conditions in a hot air oven at 55 °C, 1 M HCl (acid), 1 M NaOH (alkaline), and 30 % v/v H₂O₂ (oxidative). The acid and base stress solutions were not neutralized before chromatographic developments. The solutions were diluted with corresponding developing systems. For these investigations, the comprehensive protocols as described in our most recent article (Foudah et al., 2021) were adhered to. Under the previously stated stress conditions, LMB chromatograms were acquired for both approaches and examined for degradation products.

2.7 Application of NP- and RP-HPTLC methods in the measurement of LMB in commercial tablets

NP and RP techniques were used to capture the peak responses for LMB in three replicates (n = 3) using commercial tablet solutions on NP- and RP-TLC plates, respectively. The LMB calibration plot for both procedures was used to calculate the LMB content of the commercial tablet.

2.8 Greenness assessment

Four different methodologies were used to analyze the greenness profile of both LMB determination techniques: NEMI (Keith et al., 2005), AES (Galuszka et al., 2012), ChlorTox (Nowak et al., 2023), and AGREE (Pena-Pereira et al., 2020). To obtain the initial evaluation based on persistent, bioaccumulative, and toxic (PBT), hazardous, corrosive, and waste materials, NEMI is employed (Keith et al., 2005). AES is a semi-quantitative method that takes into account all of the analytical steps, waste, and tools. A perfect analysis with 100 points is anticipated for the solvents/reagents that need little to no reagent use, low energy, and no waste. If any of these requirements are not fulfilled, penalty points are given and subtracted from the final score of 100 (Galuszka et al., 2012). The ChlorTox scale is determined using equation (1) (Nowak et al., 2023) in accordance with the ChlorTox scale technique.

where CH_CHCl3 is the chemical hazard of standard CHCl₃, CH_sub is the chemical risks of the substance of interest, and m_sub is the mass of the substance of interest required for a single analysis. The safety data sheet from “Sigma Aldrich (St. Louis, MO, USA)” was utilized to help with the weighted hazards number (WHN) model computation of the values of CH_sub and CH_CHCl3 (Nowak et al., 2023). The AGREE-metric technique was used to create the AGREE scale for both approaches to the LMB analysis (Pena-Pereira et al., 2020). The “AGREE: The Analytical Greenness Calculator (version 0.5, Gdansk University of Technology, Gdansk, Poland, 2020)” was used to calculate the AGREE scales for both approaches. The values, which were determined by 12 distinct GAC principles, varied from 0.0 to 1.0.

3.1 Method development and optimization

Table 1 shows a summary of the different developing systems and measured chromatographi responses for each technique. Chamber saturation conditions were utilized in the development of the TLC plates for both procedures and representative TLC images are presented in Fig. 1.

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Fig. 1

The representative TLC images for standard lemborexant (LMB), marketed tablets, and forced-degradation samples derived using (A) NP- and (B) RP-HPTLC techniques.

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As the developing systems for the LMB analysis by the NP procedure, a variety of Ace/PE combinations between 40 and 90 % Ace were investigated. Table 1 shows the combinations of various developing systems and measured chromatographic responses. The developing system Ace/PE (40:60 v/v) produced a well-eluted and sharp chromatographic signal for LMB at R_f = 0.44 ± 0.01 (Fig. 2A), according to the results. However, there was some fronting in LMB peak, which could be possible due to the poor sample solubility. Furthermore, it was observed that the As value of 1.10 ± 0.04 for LMB is suitable for LMB assessment. As a result, Ace/PE (40:60 v/v) was selected as the optimal developing system for the NP-HPTLC method's LMB assessment.

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Fig. 2

Representative spectrodensitograms of standard LMB obtained by (A) NP- and (B) RP-HPTLC methods.

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As the developing systems for the LMB analysis by the RP procedure, a variety of EtOH/H₂O combinations between 35 and 95 % EtOH were investigated. Table 1 summarizes the combinations of the RP-HPTLC method's several chromatographic parameters and developing systems. The results showed that an intact and well-resolved LMB chromatographic signal at R_f = 0.69 ± 0.02 was obtained with the EtOH/H₂O (85:15 v/v) mixture (Fig. 2B). Additionally, there was no fronting in LMB peak, which could be possible due to the excellent sample solubility. Furthermore, a projection of 1.04 ± 0.02 for LMB's As value was made, which was reliable for LMB assessment. Because of this, the EtOH/H₂O (85:15 v/v) was selected as the optimal greener developing system for the LMB assessment using the RP-HPTLC method. When spectrodensitometry mode was used to evaluate the LMB spectral bands in the range of 190–400 nm, the maximum TLC response was observed at 199 nm. Thus, the whole LMB analysis was performed at 199 nm.

3.2 Validation studies

The ICH-Q2-R2 procedures were utilized to calculate the different LMB validation parameters (ICH, 2023). Table 2 displays the statistical data for the linear regression analysis of LMB calibration plots performed with both techniques. Between 50 and 500 ng/band, the LMB calibration curve for the NP method was linear. The linearity for NP procedure was not maintained beyond the 500 ng/band concentration. However, between 20 and 1000 ng/band, the LMB calibration curve for the RP process was linear. Therefore, different calibration ranges for NP and RP procedures were based on the linearity results. The correlation coefficient (R) and coefficient of determination (R²) for LMB were 0.9987 and 0.9976 for the NP-HPTLC procedure, respectively. For the RP-HPTLC procedure, LMB's R² and R were, respectively, 0.9979 and 0.9989. In addition, the standard deviations and standard error values of slope and calibration curves were also too low compared to their mean values for both methods. These findings showed a robust correlation between the LMB concentrations and the measured peak areas. These results all showed that the two LMB measurement techniques were linear. However, the RP procedure was more linear than the NP procedure.

Table 1 shows a results of the system suitability parameters for each technique. For the NP procedure, the “R_f, As, and N/m” for LMB analysis were found to be 0.44 ± 0.01, 1.10 ± 0.04, and 4517 ± 4.41, respectively. However, for the RP procedure, the “R_f, As, and N/m” for LMB analysis were measured to be 0.69 ± 0.02, 1.04 ± 0.02, and 5164 ± 4.21, respectively. The recorded system suitability parameters for both methods were reliable and acceptable for LMB analysis.

Both LMB measurement procedures were evaluated for accuracy in terms of % recovery. Table S1 indicates the accuracy measurement results for both procedures. For the NP-HPTLC procedure, the recoveries of LMB at three distinct QC samples were found to be 96.34–103.72 %. Using the RP-HPTLC procedure, the recoveries of LMB at three distinct QC samples were examined and found to be 98.24–101.57 %. These outcomes demonstrated the accuracy of both methods for assessing LMB. On the other hand, when it came to quantifying LMB, the RP-HPTLC method outperformed the NP-HPTLC method.

The data are given as a percentage of the coefficient of variation (%CV), and the LMB was determined by evaluating the intra- and inter-assay precision of both procedures. Table S2 displays the intra-assay and inter-assay precisions for the two LMB measurement methods. The intra-assay CVs of LMB for NP procedure varied from 2.53 to 3.05 %. The NP procedure's LMB inter-assay CVs varied from 2.64 to 3.17 %. The intra-assay CVs of LMB for RP procedure varied from 0.90 to 0.95 %. The range of the LMB CVs for inter-assay in RP procedure was 0.87 to 1.00 %. These measurements demonstrated the precision of both LMB measurement procedures. For measuring LMB, however, the RP procedure proved more precise than NP procedure.

In order to evaluate the robustness of both LMB measurement procedures, intentional planned modifications were made to the developing systems. Table S3 displays the results of the robustness evaluation for both procedures. The CVs for LMB using the NP-HPTLC technique were 2.98–3.24 %. The LMB R_f values for the NP-HPTLC technique were found to be 0.42–0.46. The LMB CVs for the RP-HPTLC procedure varied from 0.90 to 0.95 %. The RP-procedure's LMB R_f values were found to range from 0.68 to 0.70. These measurements demonstrated the robustness of both procedures for LMB measurement. In terms of LMB measurements, however, the RP-HPTLC procedure fared better than the NP-HPTLC procedure.

Both LMB measurement methods' sensitivity was assessed in terms of “LOD and LOQ”. Table 2 lists the calculated values of “LOD and LOQ” for LMB for both procedures. The “LOD and LOQ” for LMB for NP procedure were calculated to be 1.50 ± 0.12 and 4.50 ± 0.36 ng/band, respectively. The “LOD and LOQ” for LMB for RP procedure were computed to be 0.92 ± 0.03 and 2.76 ± 0.09 ng/band, respectively. These results demonstrated that both procedures were highly-sensitive for LMB assessment. However, compared to NP procedure, the RP procedure was more sensitive in measuring LMB.

By contrasting the R_f values, superimposed UV spectrum, and 3D spectrum of LMB in marketed pharmaceutical tablets with those of pure LMB, we were able to evaluate the specificity and peak purity of the proposed method for LMB assessment. The superimposed UV spectrum of marketed pharmaceutical tablets' LMB and pure LMB are shown in Fig. 3. Fig. S2 indicates the 3D spectrum of marketed pharmaceutical tablets and pure LMB. By contrasting the spectrum at the peak start (S), peak apex (M), and peak end (E) positions of the spot, the peak purities of standard LMB and LMB in marketed pharmaceutical tablets were evaluated (El-Kimary et al., 2014; El-Kimary and Ragab, 2015). Standard LMB and marketed pharmaceutical tablets' computed values of r (S,M) and r (M,E) were found to be more than 0.99, demonstrating the homogeneity of the peaks (El-Kimary et al., 2018; Shakeel et al., 2024). The maximum chromatographic response was observed for LMB in standard and commercial tablets at a wavelength of 199 nm. The usage of the same UV spectrum, 3D spectrum, R_f data, and wavelengths found in standard and marketed pharmaceutical tablets demonstrated the specificity of the current methods for LMB measurement. Based on these results, a simple spectrophotometric technique can also be used for the measurement of LMB with no overlap from any matrix components. However, simple spectrophotometric techniques are not enough sensitive compared to separations techniques such as HPTLC and HPLC. Therefore, the separation technique, HPTLC was used for LMB analysis in this study.

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Fig. 3

UV spectrum of standard LMB and marketed tablets, superimposed.

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3.3 Comparison of current HPTLC method’s validation parameters with reported HPLC techniques

Various validation parameters, including linear range, accuracy, precision, LOD, and LOQ, were compared between the present HPTLC methods and reported HPLC techniques for the analysis of LMB. Table 3 includes the comparison evaluation results. Two reported HPLC techniques have been shown to have linear ranges of 2–12 µg/mL (Muralikrishna et al., 2020) and 10–70 µg/mL (Kamble et al., 2022), respectively. The linear ranges of present NP- and RP-HPTLC approaches were determined to be 50–500 and 20–1000 ng/band, respectively. It has been determined that the present RP-HPTLC approach of LMB analysis is superior to both reported HPLC techniques and present NP-HPTLC method (Muralikrishna et al., 2020; Kamble et al., 2022). Both reported HPLC techniques from the literature were found to have accuracy (100 ± 2 %) and precision (<2 %) levels within the ICH-Q2-R2 guidelines' suggested range, indicating a similarity to the current RP-HPTLC approach (Muralikrishna et al., 2020; Kamble et al., 2022). However, the accuracy and precision of present NP-HPTLC approach was deviated from the prescribed limit of ICH-Q2-R2 guidelines, and hence found to be inferior to the present RP-HPTLC and reported HPLC techniques of LMB analysis (Muralikrishna et al., 2020; Kamble et al., 2022). According to reports, the LOD for two reported HPLC techniques is 0.27 µg/mL (Muralikrishna et al., 2020) and 0.54 µg/mL (Kamble et al., 2022), respectively. Nonetheless, it has been discovered that the LOQ for two reported HPLC techniques is 0.67 µg/mL (Muralikrishna et al., 2020) and 1.60 µg/mL (Kamble et al., 2022), respectively. The present RP-HPTLC method's LOD (0.92 ng/band) and LOQ (2.76 ng/band) of LMB were much lower than reported HPLC techniques and present NP-HPTLC method (Muralikrishna et al., 2020; Kamble et al., 2022). Therefore, it is found that the current RP-HPLC method is significantly more sensitive than reported HPLC techniques. All things considered, it has been found that the present RP-HPTLC analytical strategy is more linear and highly-sensitive than reported HPLC techniques and present NP-HPTLC method (Muralikrishna et al., 2020; Kamble et al., 2022).

3.4 Forced-degradation evaluation

Under varied stress conditions, the forced-degradation of the NP and RP procedures was evaluated. Fig. 4 and Table 4 present the results obtained from the NP-HPTLC procedure. At different stress settings, the LMB peak was clearly separated (Fig. 4). Under the conditions of acid degradation (Fig. 4A), base degradation (Fig. 4B), and thermal degradation (Fig. 4D), LMB was maintained at 100.00 %, and no signs of LMB degradation were found. Consequently, in conditions of acid, base, and thermal breakdown, LMB was highly stable. Under the conditions of oxidative degradation (Fig. 4C), 62.53 % of LMB was degraded and 37.47 % was remained intact. Consequently, in conditions of oxidative degradation, LMB was highly unstable. The compounds detected under oxidative degradation (peaks 2, 3, and 4 in Fig. 4C) showed separation at R_f = 0.54, 0.60, and 0.73, in that order. During acid, base, oxidative, and thermal-degradation settings, the LMB R_f value was remained constant (R_f = 0.44).

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Fig. 4

Chromatograms of LMB recorded under (A) acid, (B) base, (C) oxidative, and (D) thermal degradations of LMB by NP-HPTLC method.

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Table 5 and Fig. 5 indicate the results of the RP-HPTLC procedure. At various stress levels, the LMB peak exhibited good separation as well (Fig. 5). Under the conditions of acid degradation (Fig. 5A), base degradation (Fig. 5B), and thermal degradation (Fig. 5D), LMB was maintained at 100.00 %, and no signs of LMB degradation were found. Consequently, in conditions of acid, base, and thermal breakdown, LMB was highly stable. During acid, base, and thermal-degradation settings, the LMB R_f value was remained constant (R_f = 0.69). 50.39 % of the LMB had degraded after the application of oxidative stress, leaving 49.61 % of it intact (Table 5 and Fig. 5C). The LMB R_f value was slightly shifted (R_f = 0.68) during the oxidative-degradation settings. At R_f = 0.76 and 0.82, respectively, the oxidative-degradation compounds (peaks 2 and 3 in Fig. 5C) were separated. Consequently, in conditions of oxidative breakdown, LMB was extremely unstable. Both procedures were used to record the highest decomposition of LMB during oxidative-degradation settings. The purpose of present method development was to established stability-indicating HPTLC methods for the determination of LMB. Stability-indicating methods are those methods which are able to detect the target drug in the presence of its degradation products and hence can be applicable to stability evaluation of the target drug. The identification of degradation products was beyond the scope of the present work. Therefore, the identification of degradation products was not performed in this work. These findings showed that in the presence of its breakdown products, LMB might be detected by both NP- and RP-HPTLC techniques. The stability-indicating characteristics of both processes were indicated by these results. Both approaches were stability-indicating for LMB detection.

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Fig. 5

Chromatograms of LMB recorded under (A) acid, (B) base, (C) oxidative, and (D) thermal degradations of LMB by RP-HPTLC method.

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3.5 Application of NP- and RP-HPTLC methods in LMB estimation in commercial tablets

The green HPTLC method has several advantages over traditional LC procedures: it uses less solvent, takes less time to analyze samples, is nondestructive, requires little preparation, easy to use, can analyze multiple samples at once, non-toxic, and environmentally friendly (Alam et al., 2020; Alam et al., 2021b; Foudah et al., 2021; Shakeel et al., 2024). In spite of several merits of HPTLC techniques, they offer some demerits such as lack of standardization and reproducibility, which could influence the accuracy and precision of the results (El-Kimary et al., 2018; Alqarni et al., 2023; Shakeel et al., 2024). Both procedures were applied in order to determine the LMB in marketed pharmaceutical tablets. The chromatogram of LMB from pharmaceutical tablets was assessed by using the NP-HPTLC method to compare the single TLC band at R_f = 0.44 ± 0.01 for LMB with standard LMB. The chromatographic peak of LMB in pharmaceutical tablets, as determined by the NP-HPTLC method, matched the peak of pure LMB. Using the RP-HPTLC technique, the single TLC band at R_f = 0.69 ± 0.02 for LMB was compared with standard LMB to analyze the chromatogram of pharmaceuticals tablets. Using the RP-HPTLC method, the chromatographic peak of LMB in pharmaceutical tablets was likewise comparable to that of pure LMB. Additionally, neither of the two procedures revealed any additional signals related to the tablet contents in the commercial tablets, suggesting that there was no interaction between LMB and the tablet ingredients. The amount of LMB in pharmaceutical tablets was established using the LMB calibration plot for both processes. Using the NP procedure, the amount of LMB in pharmaceutical tablets was found to be 89.24 ± 1.32 %. Using the RP procedure, the amount of LMB in pharmaceutical tablets was found to be 98.79 % ± 1.36 %. The amount of LMB in commercial tablets has been determined in the magnitude of 100 ± 2 % by Muralikrishna et al., (2020). However, the amount of LMB in commercial tablets was determined to be 96.59 ± 1.34 % by Kamble et al., (2022). Using the Student's t-test and the variance ratio F-test, the outcomes of the current LMB analysis methods in commercial tablets were compared with documented HPTLC techniques (Muralikrishna et al., 2020; Kamble et al., 2022). There were no discernible differences in the accuracy and precision of the examined methods, according to the acquired t and F values of the current RP-HPLC method and one of the reported HPLC procedures, which did not exceed their theoretical values (Muralikrishna et al., 2020). There were noticeable differences in the accuracy and precision of the compared methods, as evidenced by the obtained t and F values of the current RP-HPLC method, NP-HPLC method, and another reported HPLC approaches, which greatly exceeded their theoretical values (Kamble et al., 2020). These findings showed that the current RP-HPTLC method is better than the NP-HPTLC method and the HPLC method published by Kamble et al., (2022), and comparable to the HPLC method reported by Muralikrishna et al., (2020). These findings demonstrated that the RP procedure worked better than the NP procedure for the assessment of LMB.

3.6 Greenness evaluation

A number of greenness tools such as NEMI, EAT, AMVI, AES, GAPI, AMGS, RGB, AGREE, and ChlorTox can be used to assess the greenness of developed analytical procedures. The greenness tools have been used to determine the greenness of the wide range of analytical procedures such as HPLC, LC-MS, LC-MS/MS, UPLC-MS/MS, HPTLC etc. (Obaydo and Sakur, 2019; Alam et al., 2021a; Kannaiah et al., 2023a, 2023b; Saleh et al., 2024a, 2024b). In the current work, four different approaches—NEMI (Keith et al., 2005), AES (Galuszka et al., 2012), ChlorTox (Nowak et al., 2023), and AGREE (Pena-Pereira et al., 2020)—were used to assess the greenness of both procedures. The typical pictograms for the NEMI of both procedures are displayed in Fig. S3. Just two of the circles for the NP-HPTLC procedure were green (Fig. S3A). However, the RP-HPTLC procedure yields all four circles green (Fig. S3B), reflecting the method's greenness, because all of the chemicals used are neither toxic, PBT, or corrosive and produce little waste. The RP-HPTLC procedure fared better than the NP-HPTLC procedure based on the NEMI results.

The results of AES scales with penalty points for both procedures are displayed in Table 6. An AES rating of more than 75 suggested excellent greenness, a scale of less than 75 but more than 50 indicated appropriate greenness, and a scale of less than 50 indicated inadequate greenness (Galuszka et al., 2012). The AES scale for the NP-HPTLC procedure was found to be 65. On the other hand, the AES scale of the RP-HPTLC procedure was found to be 93. The RP-HPTLC procedure fared better than the NP-HPTLC procedure, according to the AES results. Additionally, we calculated the AES scales of two HPLC methods found in the literature and compared them to the NP- and RP-HPTLC procedures used currently for LMB assessment (Table 6). AES scales for two literature HPLC methods were derived to be 47 and 71, respectively (Muralikrishna et al., 2020; Kamble et al., 2022). Based on AES scales, it was found that both previously published HPLC methods for LMB determination were significantly inferior to the current RP-HPTLC procedure (Muralikrishna et al., 2020; Kamble et al., 2022). According to AES scales, one of the documented HPLC methods for LMB determination was judged to be better than the current NP-HPTLC procedure (Muralikrishna et al., 2020).

Table 7 displays the total ChlorTox and individual solvent ChlorTox scale results for both procedures in relation to reported HPLC methods. The current NP-HPTLC method's estimated total ChlorTox scale was 2.42 g, indicating that it was hazardous and unsafe (Nowak et al., 2023). On the other hand, the current RP-HPTLC method's computed total ChlorTox scale was 0.88 g, indicating that it was environmentally benign and safe (Nowak et al., 2023). Additionally, we calculated the ChlorTox scales for two literature HPLC methods and compared them to the NP- and RP-HPTLC methods used currently for LMB assessment (Table 7). It was found that the ChlorTox scales for two literature HPLC methods were 1.78 g and 5.81 g, respectively (Muralikrishna et al., 2020; Kamble et al., 2022). Based on ChlorTox scales, it was inferred that both literature HPLC methods for LMB detection were significantly less effective than the present RP-HPTLC procedure (Muralikrishna et al., 2020; Kamble et al., 2022). On the basis of ChlorTox scales, one of the documented HPLC methods for LMB detection was discovered to be better than the current NP-HPTLC procedure (Muralikrishna et al., 2020).

The most popular quantitative approach for assessing greenness is the AGREE methodology (Pena-Pereira et al., 2020), which considers all 12 GAC criteria (Galuszka et al., 2013). The overall AGREE scale for both approaches is shown in Fig. 6. The total AGREE scale was predicted by the current NP-HPTLC approach to be 0.59 (Fig. 6A). Nonetheless, the total AGREE scale of 0.89 was estimated by the current RP-HPTLC procedure (Fig. 6B). The AGREE results once more showed that, in terms of the AGREE scale, the present RP-HPTLC procedure performed better than the NP-HPTLC procedure. The quantitative greenness parameters such as AES, ChlorTox, and AGREE for NP and RP procedures were compared using the Student’s t-test and the variance ratio F-test. There were noticeable differences between the compared approaches, as evidenced by the calculated t and F values of the RP and NP procedures, which greatly exceeded their theoretical values. These findings indicated that there was a statistically significant difference between the NP and RP procedures. Overall greenness approaches compared to literature HPLC methods show that the current RP-HPTLC procedure for LMB assessment in commercial tablets has an excellent greener profile.

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Fig. 6

AGREE calculator-derived representative images for AGREE scales for (A) NP-HPTLC and (B) RP-HPTLC procedures.

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