Abstract
Due to the frequent use of paracetamol formulations, it is useful to develop an analytical technique for the determination of intact paracetamol in presence of other drugs and excipients or the degradation products. In this study, a simple, isocratic, fast, specific, accurate and precise stability-indicating high performance liquid chromatography (HPLC) method has been developed and validated for simultaneous quantitative determination of paracetamol (PCM) and potassium sorbate (PS) in oral liquid formulations. The chromatographic separation was achieved on Zorbax SB C18 column (150 × 4.6 mm, 5 µm) with Zorbax SB C18 precolumn (12.5 × 4.6 mm, 5 µm) using distilled water pH 2 with ortho-phosphoric acid and acetonitrile (70:30, v/v) as a mobile phase, and UV detection at 235 nm. The temperature of the column was kept constant at 25 °C. The method was validated according to International Conference on harmonization (ICH) guidelines. The method demonstrated excellent linearity, with a correlation coefficient of 0.9996 and 0.9998 for PCM and PS, respectively, over the concentration ranges of 10–600 μg mL−1 (PCM) and 6–500 μg mL−1 (PS). The retention time was found to be 1.98 and 4.86 min for PCM and PS, respectively. Oral liquid formulation samples were subjected to various stress conditions (acidic and alkaline hydrolysis, as well as oxidative, heat and photolytic degradation) for the purpose of forced degradation study. The major degradation of paracetamol was achieved in acidic and basic stress conditions, while thermal and photolytic degradation generally had the least influence. On the other hand, potassium sorbate was highly susceptible to photolytic degradation. It was also shown that the formulation has strong influence on stability of tested compounds. Forced degradation studies demonstrated the stability-indicating power of the method and can be used to assess the stability of paracetamol and potassium sorbate in oral liquid formulations.
Introduction
Paracetamol or acetaminophen (PCM) (Fig. 1) is para-aminophenol derivative (N-(4-hydroxyphenyl) acetamide) which exhibits analgesic and antipyretic effects [1]. It is used in pain therapy of varying intensity, alone in acute pain, or more often in combination with non-steroidal anti-inflammatory drugs (NSAIDs) [2]. Also, it can be combined with opioid analgesics in the treatment of pain in oncology patients [3]. Additionally, paracetamol is indicated for use in pregnant women, children, patients with gastric ulcer, i.e. in all conditions where the use of NSAIDs is contraindicated, which makes it one of the most commonly used analgesics and antipyretics [4]. Paracetamol can be administered orally, rectally and intravenously [5]. Liquid formulations in the form of suspensions and solutions are most often used in the pediatric population and in addition to the active substance, they also contain preservatives [6, 7]. The usage of preservatives is essential in the formulations with high content of water to prevent microbiological contamination during the shelf life [6–8]. Among the most commonly used preservatives in liquid drug formulations are potassium sorbate (Fig. 2), sodium benzoate and parabens [8–11]. Oral liquid formulations intended for the use in pediatric population which readily available in the Serbian market contain potassium sorbate or parabens. Comparing the risks and suitability of these preservatives, potassium sorbate shows the best risk-benefit ratio, but it exhibits the greatest activity at pH values lower than 6 [9, 12]. In general, potassium sorbate (PS) is used at concentrations of 0.1–0.2% in oral and topical formulations [12].
Chemical structure of paracetamol
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01215
Chemical structure of potassium sorbate
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01215
Forced degradation studies (stress studies) are carried out with the aim of causing intentional degradation of the drug in order to obtain information about the possible degradation pathways of the investigated substance, its degradation products, then to determine the intrinsic stability of the investigated molecule, as well as for the development and validation of analytical methods for stability monitoring [13, 14]. Acidic and basic hydrolysis, oxidation, thermal and photolytic degradation are the most common stress factors whose influence is examined in forced degradation studies. However, the regulatory guidelines are not very precise regarding the conditions (pH, temperature, oxidizing agent) under which these tests are performed, which leaves room for analysts to adapt the conditions to the specific active substance and the formulation. For the validation of stability-indicating HPLC methods, it is preferable that the degradation of the active substance is in the range of 5–20%. It is considered that this level of degradation is sufficient in order to predict the degradation products that could realistically be formed during long-term stability studies, and anything beyond that would only complicate the analysis because it would potentially lead to the formation of secondary degradation products [15].
Due to the frequent use of paracetamol formulations, it is very important to have an analytical technique for the determination of intact paracetamol in presence of other drugs and excipients and the degradation products. In previously published studies, paracetamol in combination with other active compounds (e.g. caffeine, ibuprofen, tramadol…) has been determined usually using HPLC methods with C18 stationary phase, water or buffer combined with methanol or acetonitrile as mobile phase, and UV detection [16–21]. These methods were usually applied to quality control of solid dosage forms. However, as authors are aware only few papers [22–24] reported the application of a HPLC method for simultaneous separation of paracetamol and preservatives (mainly sodium benzoate and/or parabens) in pharmaceutical formulations, especially in oral liquid formulations. Also, forced degradation of these type of preparations, especially syrups containing potassium sorbate, has not been studied. Bearing in mind that in Serbian pharmacies most of the oral liquid formulations with paracetamol contain potassium sorbate as preservative, the aim of this paper was to develop and validate a simple stability-indicating HPLC method for simultaneous determination of PCT and PS in oral liquid formulations for quality control applications. Additional goal was to compare the stability of different PCT liquid formulations.
Experimental
Chemicals and reagents
The following substances and solvents were used during the experimental work: analytical grade 35% hydrochloric acid (HCl), sodium hydroxide granules (NaOH) and 3% hydrogen peroxide (H2O2) (Lach-Ner, Czech Republic), analytical grade 85% ortho-phosphoric acid (Poch, Poland), distilled water produced by AC-L8 water distiller (JP Selecta, Spain), HPLC-grade acetonitrile (J.T. Baker, USA), paracetamol and potassium sorbate standard substances of purity higher than 99% (Sigma-Aldrich, USA).
Samples
Four commercially available paracetamol oral liquid formulations were purchased from a local pharmacy in Novi Sad, Serbia. The detailed composition of the tested samples is listed in Table 1.
Description and composition of the tested samples
| Sample | Declared paracetamol content | Formulation type | Excipients |
| P | 120 mg 5 mL−1 | Oral suspension | Malic acid, azorubine (E122), xanthan gum, liquid maltitol, natural-synthetic strawberry flavor, sorbitol, sorbitol 70% w/v crystallizing solution, anhydrous citric acid, sodium paraben mixture: sodium methyl parahydroxybezoate, sodium ethyl parahydroxybezoate, sodium propyl parahydroxybezoate, purified water |
| PG | 120 mg 5 mL−1 | Syrup | Methyl parahydroxybenzoate (E218), propylene glycol, dispersible cellulose, xanthan gum, sucrose, non-crystallizing liquid sorbitol (E420), glycerol, color Allura Red AC Cl16035 (E123), cherry flavor, purified water |
| F | 120 mg 5 mL−1 | Syrup | Propylene glycol, sucrose, methylcellulose 15 cP, hydroxyethylcellulose 5500 cP, ethanol 96%, potassium sorbate, saccharin sodium dihydrate, citric acid monohydrate, cherry flavor, purified water |
| E | 150 mg 5 mL−1 | Oral solution | Macrogol 6000, sucrose solution, saccharin sodium, potassium sorbate, anhydrous citric acid, vanilla caramel flavor (contains propylene glycol (E1520) and caramel color (E150d)), purified water |
Apparatus and instruments
Analytical balance (Kern, Germany), magnetic stirrer (Velp Scientifica, Italy), pH meter (InoLab, Germany) and NU-8 KL UV lamp (Konrad Benda, Germany) were used for the experiments. The HPLC system (Agilent 1100 Series liquid chromatograph, USA) equipped with a binary pump, an inline degasser and a UV/DAD detector was used for chromatographic separation.
HPLC analysis
Chromatographic separation was performed on a Zorbax SB C18 column (150 × 4.6 mm, 5 µm) with Zorbax SB C18 precolumn (12.5 × 4.6 mm, 5 µm). The mobile phase used was distilled water with ortho-phosphoric acid (pH 2) and acetonitrile (70:30, v/v), under isocratic elution mode. The solvent system had a constant flow rate of 1.0 mL min−1. The injection volume was 10 µL and the temperature was kept constant (25 °C). Detection wavelength was set at 235 nm and a run time was 10 min.
Method validation
The proposed HPLC method was validated as per ICH guidelines [25] and it involved demonstration of validation characteristics such as selectivity, linearity, range, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ) and robustness.
Preparation of standard stock solutions for validation
To prepare standard stock solutions, 49.5 mg of paracetamol or 25.3 mg of potassium sorbate standard substance was accurately weighted and separately transferred into 50-mL volumetric flasks. The flasks were then completed to volume with distilled water to give stock solutions with PCM and PS concentrations of 990 and 506 μg mL−1, respectively. The required concentration levels were prepared by serial dilutions to obtain working calibration standard solutions of PCM and PS.
Preparation of sample solutions for validation
Sample solutions were prepared in 100 mL volumetric flasks by diluting 1 mL of liquid formulation with distilled water.
Selectivity
Method selectivity was investigated by analyzing peak purity and resolution between obtained PCM and PS peaks.
Linearity and range
The linearity of method was studied by preparing and analyzing nine and ten different concentrations of calibration standard solutions, over a range of 10–594 and 6–506 μg mL−1 for PCM and PS, respectively. Calibration curves were obtained by plotting chromatogram's peak areas against concentrations. Each concentration was analyzed twice. The least-squares linear regression method was used to determine correlation coefficient, slope of the regression line, and y-intercept.
Accuracy
Accuracy was determined by spiking of real samples at three different concentration levels (50, 200 and 400 μg mL−1 for PCM and 25, 100 and 200 μg mL−1 for PS). Each analysis was performed in triplicate. The amount of PCT and PS was calculated from the earlier made calibration curves and the percentage recovery was determined and used as an indicator for accuracy.
Precision
The precision of the proposed method was assessed by intra-day and inter-day repeatability evaluation. Intra-day precision was determined by repeated injections of independently prepared standard solutions on three concentration levels within the same day while inter-day reproducibility was evaluated by analysis of a standard solution on three different days. For both intra- and inter-day evaluations, analysis was performed in triplicate. Three concentration levels were 50, 200 and 400 μg mL−1 for PCM and 25, 100 and 200 μg mL−1 for PS. As a measure of precision, % RSD was calculated.
Limit of detection (LOD) and limit of quantification (LOQ)
The LODs and LOQs of PCM and PS for the proposed method were determined as concentrations of analyte at signal-to-noise ratios (S/N) of 3 and 10, respectively.
Robustness
Robustness of the method was assessed by analyzing the effects of deliberate changes in the experimental settings. Sample solutions were prepared and analyzed using the established conditions and by varying the chromatographic conditions as follows: flow rate (±0.1 mL min−1), detection wavelength (±2 nm) and column oven temperature (±5 °C). One factor at a time was changed to estimate the effect.
Paracetamol content determination
Paracetamol content was evaluated by validated HPLC method in control (unstressed) samples. In order to prepare control sample solutions 1 mL of a sample was diluted in a 100-mL volumetric flask with distilled water. Triplicate measurements were performed.
Forced degradation study
A validated HPLC method was developed to study the degradation behavior of paracetamol and preservative potassium sorbate in liquid oral formulations under acidic, alkali, oxidation, thermal and photolytic degradation. Percentage loss of paracetamol and potassium sorbate in stress exposed samples was calculated in comparison to control samples.
Preparation of standard solutions for stress degradation studies
The stress studies' standard stock solutions were prepared by accurately weighing and dissolving PCM and PS standard substances in volumetric flasks by distilled water. Working calibration standard solutions were prepared by serial dilutions of stock solutions to obtain required concentration ranges (0.1–0.35 mg mL−1 and 0.0055–0.067 mg mL−1 μg mL−1 for PCM and PS, respectively). The regression equations for standard calibration curves were y = 35357.44x – 125.68 for PCM and y = 17789.08x + 10.78 for PS.
Preparation of test solutions for test degradation studies
Samples (liquid oral formulations) were exposed to stress conditions undiluted. After test degradation studies, 1 mL of each sample was transferred to a 100-mL volumetric flask and diluted up to the mark with distilled water to obtain sample solutions. As control solutions 100-fold diluted samples (distilled water was used as diluting solvent) which were not exposed to stress conditions were used.
Acid- and base-induced degradation
Acidic and alkaline hydrolysis were performed by adding 1 mL of 1M HCl or 1M NaOH to 1 mL of sample, respectively, and prepared mixtures were left for 24 h at room temperature. After acidic and alkali degradation, the solutions were neutralized with 1M NaOH and 1M HCl, respectively. Finally, sample solutions and control solutions were prepared as previously described, filtered and injected into the HPLC system. Triplicate measurements were performed.
Oxidative degradation
A volume of 1 mL of 3% H2O2 was added to 1 mL of sample and the mixture was stored for 24 h at room temperature in order to evaluate oxidative degradation of PCM and PS in oral liquid formulations. After oxidative degradation study, sample solutions and control solutions were prepared as previously described, filtered and triplicate HPLC measurements were performed.
Thermal degradation
The effect of elevated temperature on the stability of the tested components (PCM and PS) was analyzed heating of 1 mL of sample at 70 °C in a water bath for 2 h. Afterwards, sample solutions and control solutions were prepared as previously described, filtered and injected into the HPLC system. The analysis was performed in triplicate.
Photolytic degradation
A volume of 1 mL of sample was exposed to indoor indirect daylight and UV light at wavelengths of 254 and 366 nm for 24 and 48 h. Finally, sample solutions and control solutions were prepared as previously described, filtered and injected into the HPLC system. Triplicate measurements were performed.
Results and discussion
Method validation
Selectivity
Potential interfering substances were not observed. Spectral peak purity for PCM and PS was confirmed using UV/DAD detection. Also, resolution value between PCM and PS was 18.94 ± 0.15 min (Table 2). Therefore, method was found to be selective.
Results of the proposed method validation
| Parameter | Paracetamol | Potassium sorbate |
| Selectivity | ||
| Resolution (±SD) | – | 18.94 (0.15) |
| Linear regression | ||
| Regression equation | y = 33.2966x + 88.6291 | y = 29.58499x + 27.75085 |
| Slope | 33.2966 | 29.58499 |
| y-intercept | 88.6291 | 27.75085 |
| R2 | 0.999586176 | 0.99978409 |
| Linearity range (µg mL−1) | 10–600 | 6–500 |
| Accuracy | ||
| Average recovery (%) | 98–101 | 99–101 |
| Precision | ||
| Average intra-day precision, RSD (%) | 1.52 | 1.37 |
| Average inter-day precision, RSD (%) | 1.34 | 1.22 |
| Limit of detection and limit of quantification | ||
| LOD (µg mL−1) | 2 | 1 |
| LOQ (µg mL−1) | 10 | 6 |
| Robustness | ||
| % RSD of Area | 0.030–0.191 | 0.251–0.770 |
| % RSD of Rt | 0.054–1.027 | 0.022–1.064 |
RSD – relative standard deviation, LOD – limit of detection, LOQ – limit of quantification.
Calibration curve, linearity and range
The standard calibration curves for PCM and PS were obtained by plotting peak areas against corresponding concentrations. The analytical data for the PCM and PS calibration curves are summarized in Table 2. The results obtained showed that the method is linear for both PCM and PS in the range of 10–600 and 6–500 μg mL−1, respectively, with their correlation coefficients (R2) > 0.9995, indicating excellent linearity for both analytes [26].
Accuracy
For both analytes accuracy was calculated as % recovery by spiking of real samples at three different concentration levels. The accuracy results presented in Table 2 for active substance and preservative indicated satisfactory recovery (98–102%) [26].
Precision
The precision of the proposed method was determined based on intra- and inter-day precisions and the results are displayed in Table 2. The requirement for intra-day and inter-day precision is RSD ≤2% [26] and therefore, precision of the method showed good agreement with the requirement.
Limit of detection and limit of quantification
The results of LOD and LOQ for PCM and PS (Table 2) indicated the method's suitability for the assay of both analytes over a wide concentration range.
Robustness
There were no significant differences between the results obtained with the deliberate modifications of the method parameters and those from the original chromatographic conditions. The degree of reproducibility of the results obtained as a result of small variations in the method parameters indicated that the developed method shows a high level of robustness (Tables 2 and 3).
Results of robustness for paracetamol (PCM) and potassium sorbate (PS)
| Compound | Changed parameters | Mean Peak Area ±SD | % RSD of Area | Mean Rt ± SD | % RSD of Rt | |
| PCM | Flow rate (mL min−1) | 0.9 | 3054.0 ± 2.85 | 0.093 | 2.140 ± 0.001 | 0.054 |
| 1 | 2778.8 ± 5.15 | 0.185 | 1.949 ± 0.020 | 1.027 | ||
| 1.1 | 2542.7 ± 1.87 | 0.073 | 1.749 ± 0.004 | 0.231 | ||
| Change in wavelength (nm) | 233 | 2558.8 ± 3.74 | 0.146 | 1.949 ± 0.019 | 1.001 | |
| 235 | 2778.8 ± 5.15 | 0.185 | 1.949 ± 0.020 | 1.027 | ||
| 237 | 2987.1 ± 5.69 | 0.191 | 1.949 ± 0.019 | 1.001 | ||
| Column oven temperature (°C) | 20 | 2779.8 ± 0.85 | 0.030 | 1.932 ± 0.004 | 0.196 | |
| 25 | 2778.8 ± 5.15 | 0.185 | 1.949 ± 0.020 | 1.027 | ||
| 30 | 2775.4 ± 1.46 | 0.050 | 1.905 ± 0.006 | 0.328 | ||
| PS | Flow rate (mL min−1) | 0.9 | 1268.7 ± 9.78 | 0.770 | 5.127 ± 0.003 | 0.070 |
| 1 | 1149.8 ± 2.88 | 0.251 | 4.623 ± 0.001 | 0.022 | ||
| 1.1 | 1065.6 ± 2.79 | 0.262 | 4.212 ± 0.004 | 0.099 | ||
| Change in wavelength (nm) | 233 | 981.3 ± 4.94 | 0.503 | 4.624 ± 0.001 | 0.033 | |
| 235 | 1149.8 ± 2.88 | 0.251 | 4.623 ± 0.001 | 0.022 | ||
| 237 | 1341.6 ± 8.39 | 0.620 | 4.623 ± 0.001 | 0.033 | ||
| Column oven temperature (°C) | 20 | 1166.9 ± 6.74 | 0.578 | 4.716 ± 0.029 | 0.608 | |
| 25 | 1149.8 ± 2.88 | 0.251 | 4.623 ± 0.001 | 0.022 | ||
| 30 | 1159.6 ± 3.83 | 0.330 | 4.531 ± 0.048 | 1.064 | ||
Paracetamol content in samples
One of the integral parts of the quality control of drugs represents the control of drug substance content. Most often, a deviation from the declared active substance content of ±5% or ±10% is allowed [27]. In a total of four samples declared content of paracetamol was 120 mg 5 mL−1 (samples P, PG and F) while in sample E it was declared 150 mg 5 mL−1, as stated in Table 1. Table 4 shows the percentage deviation of determined paracetamol content in relation to the declared one. In all examined samples, the content of paracetamol was in accordance with the declaration, i.e., the content of drug substance is within 95%–105% in relation to the declared content.
Paracetamol content in relation to the declared content (mg 5 mL−1)
| Samples | Declared content (mg 5 mL−1) | Determined content (mg 5 mL−1) | % of paracetamol content |
| P (mg 5 mL−1) | 120 | 123.25 | 102.71 |
| PG (mg 5 mL−1) | 120 | 124.05 | 103.37 |
| F (mg 5 mL−1) | 120 | 121.15 | 100.96 |
| E (mg 5 mL−1) | 150 | 147.9 | 98.6 |
Stress degradation study
Intentional degradation was attempted at stress conditions of acidic (using 1 M HCl), basic degradation (using 1 M NaOH), oxidative degradation (using 3% H2O2), photolytic degradation (UV and daylight degradation during 24/48 h) and thermal degradation (heated at 70° C) in order to evaluate the ability of the proposed method to separate paracetamol and potassium sorbate as well as their degradation products. In all cases of stress conditions, the retention times for PCM and PS remained unaltered, compared to control solution (1.98 ± 0.003 and 4.88 ± 0.023 min for paracetamol and potassium sorbate, respectively). The analytes were quantified and the degradation products were separated, which indicated the high specificity of the proposed method as well as its stability-indicating power.
Table 5 shows the concentrations of the active component (paracetamol) and analyzed preservative (potassium sorbate) in the tested liquid formulations for oral use. The data are presented at the starting point, i.e., before exposure to any factor (control), and after exposure to each of the stress factors. It was observed that the content of PS in the control of sample E is almost three times higher than in sample F (Table 5).
The content of paracetamol and potassium sorbate (mg mL−1) in the samples before (control) and after the effect of the stress factor
| Sample | Substance | Control | Acidic (1 M HCl 24 h) | Alkaline (1 M NaOH 24 h) | Oxidative (3% H2O2 24 h) | Thermal (70 °C 2 h) | Photolytic | |
| 24 h | 48 h | |||||||
| Concentration (mg mL−1) | ||||||||
| P | PCM | 24.65 | 21.78 | 20.59 | 22.22 | 21.21 | 24.22 | 23.87 |
| PG | PCM | 24.81 | 21.68 | 21.04 | 21.48 | 24.62 | 24.37 | 24.31 |
| F | PCM | 24.23 | 20.34 | 21.92 | 22.69 | 23.47 | 24.23 | 24.09 |
| PS | 1.50 | 1.30 | 1.38 | 1.29 | 1.28 | 1.14 | 0.92 | |
| E | PCM | 29.58 | 29.15 | 28.64 | 29.44 | 29.58 | 29.56 | 29.58 |
| PS | 4.10 | 3.91 | 4.05 | 3.75 | 3.82 | 2.20 | 1.58 | |
The extent of paracetamol and potassium sorbate degradation under stress conditions was also calculated in terms of percentage loss in comparison to unstressed samples. The results for acidic, alkaline, oxidative, thermal and photolytic degradation of paracetamol and potassium sorbate are presented in Figs 3 and 4, respectively. Also, chromatograms of standard solution and the sample E before and after photolytic degradation (after 24 and 48 h) are presented in Fig. 5.
The percentage loss of paracetamol in the analyzed samples P, PG, F and G after exposure to different stress factors
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01215
The percentage loss of potassium sorbate in samples after exposure to different stress factors
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01215
Chromatogram of standard solution containing PCM and PS (A), chromatograms of the sample E before stress degradation (B), after 24 h of photolytic degradation (C) and after 48 h of photolytic degradation (D) chromatogram of the solvent (E)
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01215
Based on the obtained results, it was noticed that alkaline and acidic hydrolysis led to the greatest average degradation of paracetamol in the samples (11.1% and 10.4%, respectively) and the same pattern was observed in most individual samples. The greatest individual degradation was noticed in sample P after exposure to alkaline conditions (percentage loss after 24 h was 16.47%). Acidic conditions caused the greatest paracetamol degradation in sample F (16.06%), where this factor caused the highest degradation compared to other stress factors. The similar results were obtained in certain previous studies. Namely, Khan et al. (2013) investigated the stability of paracetamol and piroxicam in a combined tablet form and observed that more extreme conditions (e.g., 5 M HCl under storage conditions of 40 °C/75% RH for 48 h) led to paracetamol degradation of up to 62.4% [28]. Similarly, the most pronounced degradation of PCM in a commercial tablet form of PCM and tramadol was achieved under acidic conditions (0.1 N HCl for 24 h) of 32.82% [20]. On the other hand, thermal and photolytic conditions generally result in only modest degradation of PCM. Namely, in this study, the loss of paracetamol achieved by thermal and photolytic degradation for all samples was below 5%, except sample P, whose exposure to high temperature led to almost 14% loss of PCM. Similarly, thermal and photolytic stress conditions applied by Khan et al. (2013) did not result in a desired degradation rate of PCM (between 5 and 20%) when samples are exposed to 40 °C/75% RH or sunlight [28]. Also, Kanthale et al. (2020) noticed that thermal degradation (80 °C during 1 h) and photolytic degradation of paracetamol tablet formulation resulted in a loss of substance less than 5%, without formation of degradant peak, which is in accordance to our results [21]. In the same paper, a significant degradation of PCM was achieved in the oxidative condition (3% H2O2) with the recovery of only 70.46% and without the appearance of degradant peaks [21]. In our work, exposure of the samples to 3% H2O2 for 24 h resulted in a decrease in PCM concentration between 0.47 and 13.42%. It should be noted that in a study of Jahan et al. (2014) an excessive paracetamol standard substance degradation (53.4%) was obtained after exposure to 10% H2O2, which justifies the use of milder conditions for oxidative degradation (e.g., 3% H2O2) [19]. As a result, the desired degree of degradation (5–20 %) was achieved in three out of four analyzed samples in this work. Similar to our results, only a low degradation of PCM was observed in oxidative (3% H2O2, 12 h), photolytic (UV degradation, 12 h) and thermal (dry heat at 100 °C) degradation studies of PCM and tramadol commercial medicinal tablets [20]. Moreover, a study of Jahan et al. (2014) confirmed that the reducing agent had no major effect on the degradation of paracetamol [19]. In our paper, under all conditions peaks of degradation products were not detected at 235 nm and the peak purities were confirmed (Fig. 5). Based on the above results, it was confirmed that paracetamol is the most susceptible to acid- or base-catalyzed hydrolysis reactions, as well as to oxidation under certain conditions. According to the literature, under the conditions of acidic and alkaline media, paracetamol undergoes hydrolysis forming para-aminophenol (chemically 4-aminophenol or 4-hydroxyaniline) as a major degradation product [29–30]. However, in several studies including acidic or alkaline treatment of paracetamol preparations, one or two unknown degradation peaks were observed [19–21]. Additionally, in one of these studies, no degradation products were detected after alkaline hydrolysis [21]. Furthermore, in a study focused on determination of paracetamol degradation process in alkaline conditions, it was observed that one or more intermediates are formed during the production of p-aminophenol and it was concluded that mechanism is not as simple as the first order reaction, from reactant to product [30]. Also, in our study the degradation of paracetamol did not exceed 20% after exposure to any stress factor, which confirms that stress conditions are adequately chosen.
Potassium sorbate was detected in two out of four paracetamol oral liquid formulations found on the Serbian market, as stated on the declarations. Regarding this preservative, a completely different degradation pattern was observed after exposure to stress conditions compared to the drug substance (Fig. 4). Significant degradation of PS in both samples was observed after exposure to photolytic stress conditions, high temperature, oxidizing agent and hydrochloric acid, while the lowest loss of PS was noticed after exposure of the samples to 1 M NaOH for 24 h. This result can be explained by the fact that sorbic acid is unstable in aqueous solution and undergoes an autoxidation reaction. The oxidizing agent acts on the double bond of sorbic acid, causing the formation of peroxide, its degradation and polymerization, which results in the formation of various carbonyl compounds as final degradation products [31]. Autooxidation is greatly influenced by the pH value. Namely, at higher pH values, this reaction is slower since ionized molecules are less susceptible to this type of degradation compared to non-ionized molecules. Also, hydrochloric acid has a pronounced catalytic activity because it reacts with the hydroxyl group of sorbic acid. Also, as the temperature increases, the stability of sorbic acid decreases and its degradation is pronounced [31]. One very notable finding is that the highest loss of PS in both samples was achieved during photolytic degradation (more than 20% after both 24 and 48 h). Un unknown degradation product of potassium sorbate was formed during photolytic degradation in both samples (peaks observed at around 4.6 min). Furthermore, photolytic degradation was more pronounced in sample E compared to sample F, while all other stress factors led to more extensive degradation of PS in sample F. Namely, the concentration of potassium sorbate in sample E decreased between 1.41 and 8.65% after exposure to alkaline, acidic, oxidative and high-temperature conditions, while the loss of preservative in sample F was between 7.95 and 14.51% under these conditions.
If the stability of different samples is observed, it can be seen that in general sample E is the most stable formulation, i.e. it was shown that paracetamol degraded the least (less than 4%) under the influence of different stress factors in this sample (Fig. 3). Additionally, a lower percentage of loss of potassium sorbate generally was observed in sample E, compared to formulation F (except during photolytic degradation). On the other hand, samples P and PG showed, on average, the highest percentage of active component loss under the influence of applied stress factors, so it could be said that they are the least stable formulations. Taking this into account, it can be seen that the specific formulation of liquid preparations of paracetamol for oral administration, i.e. all the excipients present, have a significant influence on the stability of the active component itself.
Conclusion
The method developed for the quantitative analysis of the active substance paracetamol and the preservative potassium sorbate in oral liquid formulations is fast, accurate, precise, selective and reproducible. The method was validated and satisfactory results were obtained. Forced degradation studies demonstrated the stability-indicating power of the method and can be used to assess the stability of paracetamol and potassium sorbate in oral liquid formulations. The major degradation of paracetamol was achieved in acidic and basic stress conditions, while thermal and photolytic degradation generally had the least influence. On the other hand, potassium sorbate was highly susceptible to photolytic degradation. It was also shown that the formulation and excipients have strong influence on stability of tested compounds. The method could be conveniently used for assay of paracetamol-containing liquid oral dosage forms with potassium sorbate as preservative in a quality control laboratory.
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