Optimization of HPTLC conditions

The experimental conditions for the HPTLC method such as mobile phase composition and wavelength of detection were optimized to provide accurate, precise, reproducible and compact flat bands for the three drug mixtures.

Solvent system

The mobile phase is regarded as the most important factor that controls the peak shape and resolution; therefore, different solvent systems with different proportions were investigated before reaching the optimum mobile phase. Greener systems were first tried such as methanol with a little proportion of ammonia (9.95:0.05, V/V), but poor resolution was observed for mixtures 1 and 3, while well-separated and well-defined peaks were obtained for mixture 2. A system consisting of ethanol‒chloroform in different proportions was tried, but overlapped and distorted peaks were obtained. Increasing the ratio of chloroform led to poor resolution, while increasing the ratio of ethanol resulted in more distortion of the peaks, so, there was no improvement by changing the ratio of these two solvents. Finally, sharp and symmetric peaks were obtained for the three mixtures using toluene‒ethyl acetate‒methanol‒25% ammonia (3.5:4.5:2:0.2, V/V). The distance traveled by the developed APX, EDX, RIV and ROS bands increased by adding very small volumes of ammonia, thus, the differences between the retardation factor (RF) values of the APX and ROS bands and the RIV and ROS bands increased to reach values of about 0.50 and 0.60, respectively; in addition, the incorporation of ammonia helped in decreasing the tailing effect.

Well-defined bands for the two drugs in each mixture were obtained when the chamber was saturated with the mobile phase at room temperature for at least 30 min. It was required to eliminate the edge effect and to avoid unequal solvent evaporation losses from the developing plate that could lead to behavior resulting in a lack of RF values reproducibility.

Scanning and detection wavelength

Different scanning wavelengths were investigated and 291 nm was chosen for the three mixtures, as it gave reasonable response with all of them. The optimum bandwidth chosen was 5 nm, taking into consideration the range of concentrations applied and number of tracks. All tracks were scanned efficiently at the same wavelength (291 nm) for the three mixtures.

The optimized chromatographic conditions gave compact spots for the cited drugs at the specific RF values which were found to be 0.65 ± 0.01 for APX, 0.20 ± 0.01 for EDX, 0.75 ± 0.02 for RIV and 0.10 ± 0.01 for ROS using method I and 0.40 ± 0.02 for EDX and 0.90 ± 0.02 for ROS using method II. Typical densitograms obtained from the analysis of the mentioned mixtures at the selected wavelength using the proposed methods are shown in Figs. 2, 3, 4, and 5.

Fig. 2

A typical TLC chromatogram of 0.3 μg band–1 (15 μg mL.–1) of rosuvastatin calcium (ROS) and apixaban (APX) in their mixture using 20-μL band volume and toluene‒ethyl acetate‒methanol‒25% ammonia (3.5:4.5:2:0.2, V/V) as the mobile phase (method I)

Fig. 3

A typical TLC chromatogram of 0.3 μg band–1 (15 μg mL.–1) of edoxaban tosylate (EDX) and rosuvastatin calcium (ROS) in their mixture using 20-μL band volume and toluene‒ethyl acetate‒methanol‒25% ammonia (3.5:4.5:2:0.2, V/V) as the mobile phase (method I)

Fig. 4

A typical TLC chromatogram of 0.3 μg band–1 (15 μg mL.–1) of rosuvastatin calcium (ROS) and rivaroxaban (RIV) in their mixture using 20-μL band volume and toluene‒ethyl acetate‒methanol‒25% ammonia (3.5:4.5:2:0.2, V/V) as the mobile phase (method I)

Fig. 5

A typical TLC chromatogram of 0.5 μg band–1 (25 μg mL.–1) of rosuvastatin calcium (ROS) and edoxaban tosylate (EDX) in their mixture using 20-μL band volume and methanol‒25% ammonia (9.95:0.05, V/V) as the mobile phase (method II)

Method validation

For the analysis of the mixtures in bulk and in dosage forms the methods were validated according to the ICH guidelines [29], while for the quantitation of the drugs spiked in human plasma, validation was done according to the FDA guidelines (2001): Guidance for Industry on Bioanalytical Method Validation [28].

Analysis in bulk form and tabletsLinearity

Linearity was evaluated by analyzing a series of different concentrations of each of APX and ROS (mixture 1) and RIV and ROS (mixture 3) by applying method I and mixture 2 (EDX and ROS) by applying both methods I and II. Under the experimental conditions previously described, the graphs obtained by plotting peak areas of the drugs versus concentrations in the ranges stated in Table 1 showed linear relationships. The slopes, intercepts and correlation coefficients obtained by the linear least-squares regression treatment of the results are also given. The smaller the standard error of the estimate (Sy/x) obtained, the closer the points are to the straight line. The high values of correlation coefficients and F indicate the good linearity of the calibration curves.

Table 1 Parameters of the regression equations for the determination of APX, EDX and RIV with ROS mixtures using the proposed HPTLC methods (methods I and II)Limit of detection and limit of quantitation

The limit of detection (LOD) is considered as the concentration which has a signal-to noise ratio of 3:1. For the limit of quantitation (LOQ), the ratio considered is 10:1 [30]. Using the proposed methods, LOD and LOQ for each compound were calculated and are presented in Table 1. These values were calculated using the signal-to-noise ratio method. Both LOD and LOQ values indicate that the proposed method showed low noise levels along with the high drugs responses which enable the quantitation and detection of low concentrations. The LOD and LOQ values ranged from 0.0022–0.0282 to 0.0074–0.094 μg band–1, respectively.

Accuracy and precision

Accuracies either with intra-day or inter-day precision were evaluated using three concentration levels (n = 3) within the same day or on three consecutive days, respectively. The percentage relative standard deviation (RSD%) and percentage relative error (Er%) did not exceed 2.0% proving the high repeatability and accuracy of the developed method for the estimation of the analytes in their bulk form (Tables S1‒S4).

Selectivity

Method selectivity was checked by analyzing the laboratory-prepared synthetic mixtures containing different ratios of drugs in each mixture, where good percentage recoveries were obtained indicating that they did not interfere with each other (Tables S5‒S7). In addition, the selectivity of each of the proposed methods was confirmed by the absence of interference from adjuvants during the application to the analysis of pharmaceutical preparations. Figures S8‒S11 present TLC densitograms of the prepared tablet extracts for the three mixtures recorded at 291 nm. These densitograms showed no interfering peaks from the added excipients thus confirming the specificity of the proposed HPTLC methods.

Robustness

The robustness of the proposed method was assessed by slightly varying some parameters such as the time of saturation (30 min ± 2 min) and the detection wavelength (± 2 nm). It was found that small deliberate variations in the above parameters had no significant influence on the determination of any of the drugs using the proposed method. The low values of RSD% of the peak areas along with nearly unchanged RF values obtained after introducing small deliberate changes in the method parameters indicated the robustness of the developed method.

Stability of solutions

The stability of the standard solutions was investigated over 4 h, where the RF values and the peak areas were unchanged throughout the analysis time. In addition, no extra peaks were observed in the densitograms, confirming the stability of the working solutions. Also, the methanolic stock solutions of the four drugs were found to be stable for at least one week when refrigerated at 4 °C.

Analysis in spiked human plasmaLinearity and LLOQ

Method I

For mixtures 1 and 3, the calibration curves of the drugs were constructed from a blank sample (plasma sample processed without IS) (Figs. 6a and 7a), a zero calibrator sample (plasma sample processed with IS) (Figs. 6b and 7b) and non-zero calibration standards encompassing the entire range including LLOQ (Figs. 6c and 7c). Linearity was assessed by the IS method. The calibration curves were linear and the data of regression analysis showed good linearity as the correlation coefficient values were higher than 0.998. Regression equations were Y =  − 0.13 + 0.60X and Y = 0.09 + 0.05X, for APX and ROS (mixture 1) and Y =  − 0.08 + 0.27X and Y = 0.06 + 0.13X, for RIV and ROS (mixture 3), respectively (Y is the peak area ratio of drug; X is the drug concentration in μg mL–1).

Fig. 6

Typical TLC chromatograms of 20-μLvolume of a blank human plasma, b zero calibrator of human plasma spiked with rivaroxaban (RIV) (IS) only, c lower limit of quantification (LLOQ) of apixaban (APX) and rosuvastatin calcium (ROS) with RIV (IS) in human plasma using method I

Fig. 7

Typical TLC chromatograms of 20-μLvolume of a blank human plasma, b zero calibrator of human plasma spiked with apixaban (APX) (IS) only, c lower limit of quantification (LLOQ) of rivaroxaban (RIV) and rosuvastatin calcium (ROS) with APX (IS) in human plasma using method I

Method 2

For mixture 2, the calibration curves of the drugs were constructed from a blank sample (plasma sample processed without IS) (Fig. 8a), a zero calibrator sample (plasma sample processed with IS) (Fig. 8b) and non-zero calibration standards encompassing the entire range including LLOQ (Fig. 8c). Linearity was assessed by the IS method. The calibration curves were linear and the data of regression analysis showed good linearity as the correlation coefficient values were higher than 0.998. Regression equations were: Y = 1.30 + 1.05X and Y = 0.30 + 0.13X, for EDX and ROS (mixture 2), respectively (Y is the peak area ratio of drug; X is the drug concentration in μg mL–1).

Fig. 8

Typical TLC chromatograms of 20-μLvolume a blank human plasma, b zero calibrator of human plasma spiked with apixaban (APX) (IS) only, c lower limit of quantification (LLOQ) of edoxaban tosylate (EDX) and rosuvastatin calcium (ROS) with APX (IS) in human plasma using method II

Accuracy and precision

The validation batch used consisted of one set of calibration standards and six replicates (n = 6) of quality control samples at four levels (LLOQ, LQC, MQC and HQC). Accuracy and precision were evaluated as described under analysis in bulk form and tablets (RSD%) ranging between 1.00–1.89% and 1.21–2.44% for APX and ROS, respectively (mixture 1); between 0.48–2.90% and 1.24–2.44% for EDX and ROS, respectively (mixture 2); and between 1.16–2.41% and 1.53–2.75% for RIV and ROS, respectively (mixture 3) (Table 2).

Table 2 Evaluation of the precision and accuracy of the proposed HPTLC methods for the determination of the three mixtures in spiked human plasmaSpecificity

The specificity of the proposed methods was evaluated by processing control plasma from six different packets. The plasma samples were spiked with lower limit of quantification (LLOQ) working solutions along with IS to confirm the lack of interference at their RF values. No interfering peaks from endogenous plasma compounds were observed in blank plasma at the RF of analytes and IS. Typical chromatograms for the blank plasma, and plasma spiked with the drugs forming each mixture along with the corresponding internal standard are shown in Figs. 6, 7, and 8.

Stability studies

Stability experiments were performed to evaluate the analyte stability in plasma samples (LQC and HQC, n = 6) under different sample analysis conditions. Long-term stability was evaluated after storage of the samples at − 70 °C for 45 days. Short-term stability was assessed after storage of spiked QC samples at ambient temperature for 6 h. Post-preparative stability was assessed after storage at 5 °C for 24 h. Freeze–thaw stability was assessed by analyzing spiked QC samples after five freeze–thaw cycles. For all the stability experiments of analytes and IS in control plasma, excellent % recovery (97.14–103.90%) and RSD% (less than 2.52%) values for mixture 1; % recovery (97.31–103.28%) and RSD% (less than 2.82%) values for mixture 2; and % recovery (97.44–103.89%) and RSD% (less than 2.51%) values for mixture 3 were obtained, thus indicating their stability under different conditions (Table 3).

Table 3 Stability tests of APX and ROS (mixture 1), EDX and ROS (mixture 2) and RIV and ROS (mixture 3) in spiked human plasma (n = 6)Application to the analysis of pharmaceutical preparations

Due to the unavailability of commercial tablets containing these binary mixtures, single component tablets of ROS with each of the NOACs were mixed in ratios simulating their appropriate doses, and extracted with methanol, then spotted on the TLC plates. The lack of foreign peaks in the chromatograms confirmed that inactive ingredients did not interfere in the analysis. Percentage recoveries were calculated for three independently prepared solutions each repeated three times and were found acceptable (Table S12). Owing to the absence of any published method for the determination of these binary mixtures, the standard addition technique was applied by spiking the tablets’ extracts with portions of the standard solution of each drug to obtain total concentrations within the linearity ranges. The obtained % recoveries, RSD% and Er (%) were satisfactory, thus proving the absence of interference from the tablets excipients.

Application to the analysis of the binary mixtures in human plasma

The suggested HPTLC methods were successfully applied to the analysis of the three binary mixtures in spiked human plasma; method I was used to analyze mixtures 1 and 3, while method II was used for mixture 2. The procedure adopted was based on simple protein precipitation with acetonitrile with no need for tedious extraction steps (Figs. 6, 7, 8).

Assessment of method greenness

Green and eco-friendly practices have been recently adopted in different analytical procedures such as using green sample pretreatment, using environmentally friendly solvents and reagents, consuming less energy and shortening analysis times.

Greenness assessment was done using two different recent methods. The Green Analytical Procedure Index [31] (GAPI), a widely cited green assessing method newly introduced in 2018 was used. The GAPI pectogram is represented by five major pentagrams divided into 15 zones where each pentagram represents a step in the analytical procedure. These zones are colored by three colors (green, yellow and red) used in the greenness assessment, where theses colors represents low, moderate and high impact on the environment, respectively. As shown in Table 4, GAPI pictogram shows only one red zone which represents the off-line sampling since the sites of production of the pharmaceutical preparations must be away from the laboratories where the HPTLC method was designed. Such a pictogram indicates that the method is a good green method for the analysis of these binary mixtures. Also, Analytical GREEnness Metric Approach [32] (AGREE), a new method introduced in 2020, was used. AGREE assessment is based on the twelve principles of Green Analytical Chemistry (GAC) and is represented in a clock-like graph composed of twelve sections representing the twelve GAC principles. Each section is assessed and represented in a color of green, yellow and red. The overall greenness performance of the twelve sections is written in the middle of the clock-like graph with a score within 0–1 and with a color. As shown in Table 4, the clock-like graph shows an overall AGREE score of 0.74 with green color indicating the low impact on the environment. Only one red was found representing the off-line sampling that occurs in the analytical quality control laboratories of pharmaceuticals.

Table 4 Assessment of the greenness of the proposed HPTLC methods using two different assessment methods