Introduction

Bacterial contamination of several pharmaceuticals and agri-food products due to consumer use may result in the presence of mixed and harmful microbial flora in the product (Wilson, Kuehne, Hall, & Ahearn, 1971). Preservatives are used in a wide field of application to maintain the quality of products. They are used to deal with the chemical, physical and microbiological problems and the constraints of the development of several formulations.

They can be used in foods, beverages, pharmaceuticals, and personal care products. There are three main classes of preservatives used, antioxidants, antimicrobials, and chelating agents (Sznitowska, Janicki, Dabrowska, & Gajewska, 2002), including natural compounds. Antimicrobials are added primarily to pharmaceutical products to maintain sterility and to extend shelf life. Some of which act on yeasts, molds and bacteria, while others specifically target certain classes of bacteria.

The choice of preservatives is limited, and generally depends on the effectiveness against the appearance of molds and the growth of yeast. In this stability study, methylparabens and propylparabens, commonly used as antimicrobial preservatives in pharmaceutical formulations because of their broad antimicrobial spectrum with good stability and non-volatility, were analyzed under different stress degradation conditions in order to evaluate their degree of effectiveness. (Kashid, Singh, & Singh, 2011).

The parabens commonly employed in pharmaceutical formulation are the methyl parabens (PBM) and the propyl parabens (PBP), which are methyl and propyl esters of p-hydroxybenzoic acid (Figure 1). Acceptable daily intake methyl was established for adults at 10 mg / kg / day (Soni, Carabin, & Burdock, 2005). But no acceptable daily intake has been attributed to propyl parabens. PBM and PBP are generally used in combination because they possess synergistic activity, however an overuse of these preservatives can cause allergic reactions (Klöcker et al., 2004).

According to a thorough literature review there are several stability studies of several drugs preserved with parabens (Beasley, Shaw, Zhao, & Reed, 2005; Karbane et al., 2015) and stability studies of parabens in human body compartments (organs or body fluids) (Harville, Voorman, & Prusakiewicz, 2007; Ye, Wong, Jia, Needham, & Calafat, 2009) but there are no studies that focuses on parabens themselves.

The objective of this study is to make a stability test of the prabenes under different pH and temperature conditions and to see the resulting behavior while taking inspiration from the ICH QA1 guidelines, which suggests conducting stress tests to establish the inherent link between the stability characteristics of the molecule such as the pathways of degradation leading to identification of degradation products and therefore support the adequacy of the proposed analytical procedures.

Materials and Methods

Method

The method is based on a test of stability of parabens under different conditions of pH and temperature and others (light, additive in the middle). The solutions of PBM and PMP are prepared at a concentration of 1%, in a solution composed of mixing water and methanol (80%, 20% respectively) to facilitate the dissolution of parabens. In the first time we started with four stability tests; the PBP and PBM were incubated in different solutions at different pH, Temperatures, and light conditions,

• Four buffer solutions of pH 4,6,8 and 10 under light at 6-8 ° C.

• Four buffer solutions of pH 4,6,8 and 10 under light at 25 ° C.

• Four buffer solutions of pH 4,6,8 and 10 Without light at 25 ° C.

• Four pH buffer solutions 4,6,8 and 10 Without light at 40 ° C.

Secondly, the medium was changed and PBM and PBP were incubated in saturated sucrose solution (artificial syrup), to create a medium similar of a syrup (simulation mode).

The PBP and PBM were incubated in sucrose solution with,

• Four buffer solutions of pH 4,6,8 and 10 Without light at 25 ° C.

• Four pH buffer solutions 4,6,8 and 10 Without light at 40 ° C.

All the solutions are prepared under the condition of keeping the concentration of 1% and the assays were analyzed using high performance liquid chromatography (HPLC) method. The HPLC method was used for the assay, which is validated method for the parabens assay developed by (Karbane et al., 2015).

Materials and Reagents

HPLC Waters Alliance was used and the method used is a validated method for the parabens assay. The system was equipped with a quaternary pump, a 2695 automatic injector, a temperature controlled column compartment, an in-line degasser, and a 2998 diode array detector. Data analysis and reporting were performed by the Empower pro 2 chromatography software.

The products used were of analytical quality and came from commercial sources. Reagents and solvents (HPLC grade) used to prepare the mobile phase or diluent for HPLC analyzes were purchased from MERCK. the purified water used is filtered on a nitrocellulose membrane with a porosity of 0.45 μm (Sartorium medium). Methylparabens , propylparabens, sucrose, were obtained from Moroccan pharmaceutical companies.

Statistical analysis

The statistical analysis of the data is carried out by SAS JMP software

Results and Discussion

After incubation of parabens solutions under different temperature and pH conditions with changes in media (with sucrose and no sucrose), the results show many variations in parabens content in solutions (Table 2; Table 1). Figure 3; Figure 2 made by the JMP software show the results of our study (for tests in solutions without sucrose).

For medium without sucrose: there was a decrease in parabens levels over time and especially in the solutions with the extreme values of temperature and pH (pH 4 and 10 at 40 ° C). The decreases obtained for PBM and PBP were 5 to 10% after 60 days of incubation at 25 ° C, and 15 to 20% after 60 days of incubation at 40 ° C, with a strong decrease with pH 4 and 10.

For medium with sucrose: there was a decrease in parabens levels over time and especially in the solutions with extreme values of temperature and pH (pH 4 and 10 at 40 ° C).

The decreases obtained for PBM and PBP were 13 to 19% after 60 days of incubation at 25 ° C, and 23 to 35% after 60 days of incubation at 40 ° C, we observed a strong decrease with pH 4 and 10, and the appearance of a new peak that is identified as phenol peak according to the handbook of pharmaceutical excipients. For the two mediums used, the media incubated at pH 4 and 10 at a temperature of 40 ° C. underwent a strong decrease in parabens levels.

The stability tests of parabens themselves are of paramount importance given the integration of these products in several pharmaceutical formulations. The resuts of this study provided evidence on how the quality of parabens varies with time as a result of various environmental factors, such as Temperature, pH, light, and the nature of the solution thus providing an image on the conditions of conservation and determining the stability of these products under stress.

The results remain very satisfactory seen the information they provide. However, there are studies that are interested in the stability of parabens in drugs or body fluids, which may be a small comparison with our results.

In the literature, most studies on the development of stability indication tests involve forced decomposition studies under one or two conditions of the drug preserved with parabens and the search for the main degradation products (Karbane et al., 2015; Patil, Wankhede, & Chaudhari, 2013).

Our results show a direct influence of temperature and pH on the level of parabens studied, while the influence of light remains negligible. The increase in temperature gives a degree of immunity of parabens levels, especially with the extreme values of pH (pH 4 and pH 10). The diminution obtained at 40 ° C with pH 4 and 10 is greater than the one obtained at 40 ° C with pH 6 and 8, which explains the influence of pH variation on the stability of parabens.

The obtained parabens level values under light and those in darkness are almost the same, which eliminates the theory of the influence of light on parabens. The influence of sucrose is indirect via the appearance of bacteria which is responsible for the appearance of the p-hydroxybenzoic acid peak due to degradation as shown by the study of (Karbane et al., 2015). According to this research, the degradation of parabens could occur in two stages; the first relates to the enzymatic hydrolysis of the ester bond leading to the production of p-hydroxybenzoic acid and methyl alcohol; the second is an aerobic decarboxylation by a bacterial enzyme. A recent study confirms the biodegradation of parabens and bacteria responsible for this biodegradation (Lu, Li, Tu, & Yang, 2018).

According to the handbook of pharmaceutical excipients, the simultaneous presence of the two preservatives, prevent the decarboxylation of carboxylic acid (p-hydroxybenzoic acid) into phenol and prolong the stability period of the syrups after opening. These results are compliant with the observations described. Indeed, the authors of this document specify that the use of a mixture of parabens, namely MP and PP, in pharmaceutical formulations leads to a more efficient preservation (Sheskey, Cook, Walter, & Cable, 2017).

In our study it was reached that the first stage (appearance of the carboxylic acid), and the appearance of phenol is not detected, despite the presence of sugar that promotes bacterial degradation. This can be justified by different explanations: first, we have to work under good aseptic conditions (bacteriologically clean media and flasks), and the products should not be exposed to the air. There were no other substances that often exist in products that contain parabens (including the syrups) that can promote bacterial degradation and the appearance of phenol. For the parameter of moisture it was neglected because we are studying the stability of parabens in solutions, so moisture is not evocable.

In another context, parabens constitute a real danger for human health via the existence of traces in wastewater and even in natural water sources (rivers ....) (Kasprzyk-Hordern, Dinsdale, & Guwy, 2008; Kimura et al., 2014; Ramaswamy, Shanmugam, Velu, Rengarajan, & Larsson, 2011) our study gives a conclusion on the degradation of parabens and the resistances of these against the different proposed conditions, which require stronger means of detoxification by degradation, to combat this resistance (Gomes et al., 2017).

Conclusions

This study is one of the first studies of forced decomposition of parabens carried out under the various conditions suggested. The results give an idea of the stability profile of the tested parabens and propose a model of the conditions of conservation and use of these products in different domains and under different conditions.

Funding Support

None.

Conflict of Interest

None.