Validation of HPLC-DAD method for analysis of paracetamol and potassium sorbate in liquid oral formulations and its application to forced degradation study (2024)

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 Table1.

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.

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.

Method validation

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 Table1. Table4 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.

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.

Table5 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 (Table5).

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.

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 etal. (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 etal. (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 etal. (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 etal. (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% H₂O_2, 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 etal. (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, underthe conditions of acidic and alkaline media, paracetamol undergoes hydrolysisforming para-aminophenol (chemically4-aminophenol or 4-hydroxyaniline) as a majordegradation product [29–30]. However, in several studiesincluding acidic or alkaline treatment of paracetamol preparations, one or two unknown degradation peaks wereobserved [19–21]. Additionally, in one of these studies, no degradation products were detected after alkaline hydrolysis[21]. Furthermore, in a study focused on determinationof paracetamol degradation process in alkaline conditions, it was observed that one or more intermediatesare formed during the production of p-aminophenoland it was concluded that mechanism is notas 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 insampleE 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.