1. Introduction

2. Materials and Methods

3. Results and Discussion

3.1. Validation

The proposed method for the simultaneous determination of paracetamol, propyphenazone and caffeine in Saridon tablets has been fully validated. The validation results are presented in Tables 3-6 and Figures 1-8, Figures S1-S12, and described in the following subsections.

3.1.1. Selection of Chromatographic Conditions

According to the guidelines for validation of analytical methods [33,34] used in pharmaceutical analysis, specificity (selectivity) is an important parameter proving that an analytical procedure allows to determine the API presence in the tested pharmaceutical preparation along with related substances that may be their contaminants. In order to select the optimal mobile phase ensuring the separation of propyphenazone (PP), paracetamol (PA), caffeine (C), 4-chloroacetanilide (CA), 4-aminophenol (AF) and 4-nitrophenol (NF), 22 chromatographic conditions were tested (Table S1). The subject of the research were mobile phases consisting of selected solvents such as acetone, chloroform, ammonia, n-hexane, toluene, ethyl acetate, methanol, ethanol, acetic acid 80%), acetonitile, buffer at pH=5. Hałka-Grysińska et al. [30] determined the separation of propyphenazone (PP), paracetamol (PA) and caffeine (C) by RP-HPTLC on RP18W plates using the mobile phase acetonitrile + buffer pH=5.0 in a volume ratio of 22.5:77.5. However, these authors did not demonstrate the specificity of this method. The use of chromatographic conditions in this manuscript did not allow for separation of caffeine from 4-nitrophenol and 4-aminophenol from paracetamol (Figure S1). Further analyzes were performed on ordinary NP-TLC plates, i.e. aluminum foil precoated with silica gel 60F₂₅₄. Other selected mobile phases that have previously been used to analyze paracetamol and caffeine in simple and combined preparations were tested [12,38]. These mobile phases (acetone + chloroform + ammonia, 10 : 40: 0.5 and n-hexane + acetone + ammonia, 25 : 25: 0.5) failed to separate caffeine from 4-aminophenol and paracetamol from 4-nitrophenol (Figure S2), and paracetamol from 4-nitrophenol (Figure S3). The mobile phase chloroform + toluene + ethyl acetate + methanol + acetic acid 80%, 6:6:1:2:0.1 was previously used for the analysis of propyphenazone (PP), paracetamol (PA), caffeine (C), 4-nitrophenol (NF), and 4-aminophenol (AF) [24]. This mobile phase, however, fails to separate the caffeine from the 4-chloroacetanilide under these conditions (Figure S4). This mobile phase was modified in terms of changing the volume composition of individual components and the methanol was also replaced with ethanol. A total of 18 mobile phases were tested (Table S1). Studies have shown that the optimal mobile phase is: chloroform + toluene + ethyl acetate + ethanol + acetic acid (80%) in a volume ratio of 18: 18: 7.5: 5: 0.3. The densitogram of standard substances such as propyphenazone (PP), paracetamol (PA), caffeine (C), 4-chloroacetanilide (CA), 4-aminophenol (AF) and 4-nitrophenol (NF) is shown in Figure 1. Mobile phases: chloroform + toluene + ethyl acetate + methanol + acetic acid 80%, 6:6:2:2:0.1 and chloroform + toluene + ethyl acetate + ethanol + acetic acid 80%, 6:6:6:2:0.2 also allow for the separation of all tested substances. However, using the mobile phase of chloroform + toluene + ethyl acetate + ethanol + acetic acid (80%) in a volume ratio of 18: 18: 7.5: 5: 0.3, better separation of C from CA is obtained than using chloroform + toluene + ethyl acetate + methanol + acetic acid 80%, 6:6:2:2:0.1 (Figure S5) and better separation of C from PA than mobile phase chloroform + toluene + ethyl acetate + ethanol + acetic acid 80%, 6:6:6:2:0.2 (Figure S6).

Figure 1. Densitogram of a mixture of standard substances: propyphenazone (PP), paracetamol (PA), caffeine (C), 4-chloroacetanilide (CA), 4-aminophenol (AF), and 4-nitrophenol (NF) made in the range of 250-350 nm with a change in wavelength every 25 nm (different colours on the densitogram), using plates precoated with silica gel 60F₂₅₄ and phase mobile phase: chloroform + toluene + ethyl acetate + ethanol + acetic acid (80%) in a volume composition of 18: 18: 7.5: 5: 0.3.

Figure 1. Densitogram of a mixture of standard substances: propyphenazone (PP), paracetamol (PA), caffeine (C), 4-chloroacetanilide (CA), 4-aminophenol (AF), and 4-nitrophenol (NF) made in the range of 250-350 nm with a change in wavelength every 25 nm (different colours on the densitogram), using plates precoated with silica gel 60F₂₅₄ and phase mobile phase: chloroform + toluene + ethyl acetate + ethanol + acetic acid (80%) in a volume composition of 18: 18: 7.5: 5: 0.3.

It follows that the best mobile phase ensuring the separation of the reference substances propyphenazone (PP), paracetamol (PA) and caffeine (C) as well as 4-chloroacetanilide (CA), 4-aminophenol (AF) and 4-nitrophenol (NF) was mobile phase with the composition of chloroform + toluene + ethyl acetate + ethanol + acetic acid 80%, and the volume ratio 18:18:7.5:5:0.3. Using this mobile phase, the following R_f values were obtained: R_f(AF)= 0.20±0.05, R_f(PA)= 0.38±0.04, R_f(C)= 0.47±0.02, R_{f (CA)}=0.54±0.03, R_f(PP)=0.60±0.02, R_f(NF)=0.67±0.02. The resolution factor (R_S) calculated had the following values: R_S(AF/PA)= 3.00, R_S(PA/C)= 1.62, R_S(C/CA)= 2.14, R_{S(CA/ PP)}=1.23, R_S(PP/NF)=0.86. Spectrodensitometric analysis indicates that the maximum absorption of propyphenazone and caffeine occurs at 272 nm, and that of paracetamol at 248 nm.

The densitogram obtained from the Saridon drug extract for the optimal chromatographic conditions, shown in Figure 2, indicates that there are no additional chromatographic bands from the analyzed preparation. Only three chromatographic bands are observed on the densitogram, i.e. those corresponding to paracetamol, propyphenazone and caffeine. No matrix effect on the separation and determination of PA, PP, and C was observed.

Comparisons of the spectrodensitograms of propyphenazone (PP), paracetamol (PA) and caffeine (C) standards with the spectrodensitograms of these substances present in Saridon samples are presented in Figures S7, S8 and S9 respectively. The absorption maximum occurs at λ= 272 nm for PP and PA, as well as at λ= 248 nm for C. Therefore, the developed chromatographic conditions can also be successfully used to confirm the identity of paracetamol, propyphenazone and caffeine in their combined Saridon preparation.

The densitogram of propyphenazone (PP), paracetamol (PA) and caffeine (C) presented in Figure 2 confirms that the TLC technique combined with densitometry used for the quantitative determination of propyphenazone (PP), paracetamol (PA) and caffeine (C) in the pharmaceutical preparation Saridon is highly selective. The mean R_f of PA, C, and PP values ​​were 0.38, 0.47, and 0.60, respectively. The R_f values ​​of the propyphenazone (PP), paracetamol (PA) and caffeine (C) standards are consistent with the R_f for PP, PA and C determined in Saridon. What is more, spectrodensitograms of propyphenazone (PP), paracetamol (PA) and caffeine (C) standards are similar to PP, PA and C spectrodensitograms from the drug samples (Figures S7, S8 and S9).

Normal phase thin-layer chromatography (NP-TLC) with densitometry can be used for quantitative analysis of propyphenazone (PP), paracetamol (PA) and caffeine (C) in drugs, using the area values ​​of chromatographic bands for calculations. Qualitative identification of the above-mentioned compounds can be made on the basis of the values ​​of retardation coefficients and spectrodensitometric analysis.

3.1.2. Linearity and Range

To determine the range of linearity, the relationship between the area of ​​the densitometric band (Table 1, Figures S10A, S811A, S12A) and the concentration of propyphenazone (PP), paracetamol (PA) and caffeine (C) standards [µg/spot] was used. The linearity range for propyphenazone was found to be 0.8÷4.0 µg/spot, for paracetamol and caffeine 0.4÷4.0 µg/spot, as shown in Figures S10A, S11A and S12A, respectively. The differences between the real values ​​of the chromatographic band areas and the values ​​calculated using the appropriate calibration curves are shown in Figures S10B, S11B and S12B. Figures S10B, S11B and S12B indicate the correct choice of linearity ranges for paracetamol, propyphenazone and caffeine.

3.1.3. Precision

The measure of the precision of the developed method is the coefficient of variation (CV). Intra-day and inter-day precisions were determined by analyzing solutions of API samples with concentrations of 3.0; 2.0 and 1.0 µg per spot. The resulting chromatographic spots were evaluated by densitometric analysis and the CV was determined. The values ​​of the coefficients CV allow the method to be considered precise, because for propyphenazone they ranged from 0.68% to 1.10% and from 0.53% to 1.26%, for paracetamol from 0.35% to 1.56% and from 0 .79% to 1.54%, and for caffeine from 0.75% to 1.08% and from 0.82% to 1.06%, respectively for intraday and interday precisions (Table 1).

3.1.4. Accuracy

Measurement of the recovery allowed to assess the accuracy of the proposed NP-TLC method. The appropriate amount of the powdered tablet mass was supplemented with standard substances of propyphenazone, paracetamol and caffeine in the ratio of: 50%, 100% and 150% to their content in the weighed amount of the drug. The average recovery for propyphenazone was: 99.0%, 98.8% and 98.4%, for paracetamol: 100.2%, 100.0% and 99.6% and for caffeine: 99.7% , 100.2% and 99.2%, respectively for 50%, 100% and 150% of standard substances added to the Saridon samples (Table 1). The accuracy of the method is indicated by the low values ​​of the coefficient of variation, which was <3% for propyphenazone and paracetamol, and <2% for caffeine. The repeatability of the developed method, which is measured by recovery, is comparable, and in some cases better than the repeatability obtained by TLC and HPTLC techniques by other researchers for simple and combined pharmaceutical preparations containing paracetamol, propyphenazaone, caffeine [8,9,10,11,12,13,24,30,38.

3.1.5. Limit of Detection (LOD) and Limit of Quantification (LOQ) investigated APIs and Comparison with Literature Data

Based on the parameters of the special calibration curve obtained for paracetamol, propyphenazone and caffeine, respectively, the limit of detection (LOD) and the limit of quantification (LOQ) for PA, PP and C were estimated using the NPTLC method combined with densitometry. These calibration curves were obtained on the base of the analysis of three standard solutions containing paracetamol, propyphenazone, and caffeine with following concetrations: 0.20; 0.30 and 0.40 mg/5mL. An example densitogram of a mixture of PA, C, and PP standards with a concentration of each standard of 0.20 µg/spot is shown in Figure S13. The average values ​​of the limit of detection of the tested APIs were: 0.016, 0.032 and 0.054 µg/spot, respectively for PA, PP and C. The limit of quantification of tested APIs were: 0.048, 0.096 and 0.162 µg/spot for PA, PP and C, respectively (Table 1). Proposed method is characterized by low LOD and LOQ for the determination of PA, PP and C, which confirm sensitivity of the proposed method.

Table 2 compares the LOD and LOQ values ​​with the literature data for PA, PP and C. Comparing all LOD and LOQ values ​​presented in Table 2, it should be noted that particularly high LOD and LOQ values ​​for PA, C and PP were obtained by Ibrahim et al [24]. This may be due to, among others, from the fact that Ibrahim et al. [24] performed densitometric measurements at 220 nm, while the maximum absorption occurs at λ = 272 nm for PP and PA and at λ = 248 nm for C. The method developed in this workis more sensitive, with lower LOD and LOQ values ​​for PA, PP and C than previously described TLC methods for the simultaneous determination of PA, PP and C [24,30]. Better LOD and LOQ were obtained only when paracetamol and caffeine [9] or caffeine alone [39] were determined in pharmaceutical preparations. But these analyzes were carried out with more cost-intensive techniques, namely NP-HPTLC and RP-HPTLC.

3.1.6. Robustness

The rules for testing robustness were described in detail in reference publications [35,36,37].

Seven factors were changed, namely the activation temperature of the chromatographic plate, the time of tablet extraction, the time of saturation of the chromatographic chamber and slight changes in the volume of the mobile phase components, i.e. chloroform, toluene, ethyl acetate and ethanol (Table S2). These factors were tested on two levels in eight experiments (Table S2). Table 3 shows the results obtained for PA, PP, and C content (y_i) in Saridon tablets. The main effects (E) of the factors calculated from these results (yi) are also presented in Table 3. The calculated statistical data presented in Table 3 indicate that the changed analysis parameters have no impact on the analysis result, because the coefficient of variations is less than 2%. These results show that no factor has significant effect on the results. To evaluate whether the proposed NPTLC method combined with densitometry is robust, these results were evaluated by half-normal probability plotting of rank probabilities (p_i) as a function of the absolute values of the main effects. The effects of factors, and half-normal probability plot of effects for the determination of PA, PP, and C in Saridon tablets are presented in Figures S14, S15 and S16, respectively. The points of all factors lie near the straight line, which indicates that their effect is negligible (R²≥0.9424). Therefore, the presented NPTLC-densitometric method can be regarded as robust. The standard deviation of paracetamol, propyphenazone, and caffeine content (y_i) in Saridon tablets at seven parameters which have been changed in conducted experiment in order to check the robustness of applied method is placed of 1.5%, 1.2% and 1.8% for PA, PP, and C, respectively. The value of CV in percent (<2) indicates the reliability of proposed NPTLC-densitometric method during its normal use. The only criterion to fulfill the robustness of this method is the content of the acetic acid (80%) in the mobile phase must be constant.

3.2.. Quantification of APIs in Saridon Tablets by Proposed NPTLC-Densitometric Method and Comparison with Literature Method

Table 4 presents the results of the actual content of the tested paracetamol, propyphenazone and caffeine in Saridon tablets obtained by the developed NP-TLC method in combination with densitometry. Statistical parameters listed in Table 6 made it possible to evaluate the obtained results. According to Table 4, the actual APIs contents are 254.1 mg/tablet, 149.7 mg/tablet, and 50.4 mg/tablet for PA, PP and C, respectively. Compared to the values declared by the manufacturer of Saridon, the prestented method indicatess values of 101.6%, 99.8% and 100.8% o for PA, PP and C, respectively. These values ​​are in accordance with the pharmacopoeial guidelines, i.e. they should be in the range of 95-105% for paracetamol and caffeine and in the range of 90-110% for propyphenazone. All these results meet the recommendations of the Polish and USP pharmacopoeias [40,41]. The proposed method allows for the simultaneous determination of PA, PP, and C in the same sample.

To verify the results obtained by the proposed NPTLC-densitometric method (method A), comparison was made with a previous report using the method (method B). The method described by Hałka-Grysińska et al. [30] was used as an accurate method (method B). The comparison of the results obtained with both methods are presented in Table 4. The PA, PP and C contents in Saridon tablets obtained by both methods A and B were similar. The coefficients of variance were smaller than 3% in each case. High reproducibility and insignificant differences between the two compared methods were obtained at the 95% probability level for t-test and F-test of significance of 0.364<2.101 and 1.11<3.18; 1.756<2.101 and 2.04<3.18; and 0.923<2.101 and 2.94<3.18, respectively for paracetamol (PA), propyphenazone (PP) and caffeine (C). These results statistically confirmed the TLC-densitometric method is accurate and can be used as a substitute method. The calculated values ​​of the t and F tests indicate that the proposed in this work NP-TLC method is accurate.

4. Conclusions

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