Antacids are agents that neutralize stomach acid containing at least one active acid neutralizing compound such as magnesium, aluminum or calcium salts. In general, liquid antacid suspensions are preferred to tablets or powders, since they are more rapidly acting and have a greater ability to neutralize gastric acid. Although the alkaline pH of liquid antacids is a critical factor in their acid neutralizing capacity and also controlling the microbial growth within the suspension, these formulations could still be susceptible to microbial contamination thus the need for an adequate preservative policy towards oral antacid suspensions should be recognized (Beveridge, 1998; Bloomfield, 1996). Under most circumstances, the ability to restrict microbial growth can be aided by addition of a preservative. The choice of a suitable preservative should be made as a function of pH and compatibility with other ingredients (Bloomfield and Sheppard, 1996). No approved preservative systems function optimally at pH higher than 8.5. The alkyl esters of parahydroxybenzoic acid especially methyl and propyl esters are still the most widely used preservatives in oral preparations because they offer the most efficacious options such as relative broad spectrum of activity, low toxicity and good stability (Morris and Leech, 1996; Soni et al., 2002, 2001), but they undergo hydrolysis in alkaline pH (Shija et al., 1992).
To achieve adequate preservative levels throughout the shelf life of a product
with an alkaline pH, we investigated the extent of methyl and propyl paraben
hydrolysis in magnesium hydroxide suspension using both real time (32 months
incubation at 25± 2°C with humidity of 60±5% RH) and also
accelerated (3 months incubation at 40±2°C with humidity of 70±5%
MATERIALS AND METHODS
All chemicals and culture media were obtained from Merck Co. Methyl paraben, propyl paraben and ethyl paraben were further purified by recrystallizing from ethanol-water admixture. P-hydroxy benzoic acid was recrystallized from water.
HPLC analysis: Methyl, propyl and p-hydroxy benzoic acid were determined
by HPLC method (Waters 600 pump, Waters 486 UV detector, Waters 746 integrator
and C18, 150χ 3.9 mm Novapack column). The injection volume was 25 μ
L. Detection was performed at 254 nm. The mobile phase was water and methanol
in 50:50 ratio with the flow rate of 1 mL min1. Ethyl paraben
was used as internal standard.
Chemical analysis: The stability study was evaluated using both accelerated
(40± 2°C, 70±5% RH) and real time (25±2°C, 60±5%
RH) conditions and amounts of methyl and propyl parabens and p-hydroxy benzoic
acid were estimated. The slope of regression line of concentration log versus
time represented the rate constant (k) of the decomposition (Gullory and Poust,
1996; Mechkovski, 1991).
Sample preparation: The test samples, magnesium hydroxide 8% suspensions,
were prepared by diluting a 30% magnesium hydroxide paste with distilled water.
Two kinds of samples were prepared, suspensions containing a mixture of methyl
and propyl paraben (1.2 mg mL1 methyl paraben and 0.6 mg
mL‾1 propyl paraben) and also preparations without any parabens.
Test organisms: Cultures of following microorganisms were used for the challenge test according to US Pharmacopeia 27 (10):
Staphylococcus aureus PTCC 1112 (ATCC 6538)
Pseudomonas aeruginosa PTCC 1074 (ATCC 9027)
Escherichia coli PTCC 1338 (ATCC 8739)
Aspergillus niger PTCC 5011 (ATCC 16404)
Candida albicans PTCC 5027 (ATCC 10231)
Antimicrobial preservative effectiveness test: The preservative effectiveness testing was carried out according to the US Pharmacopeia 27 guidelines (10).
The magnesium hydroxide suspensions with or without parabens were inoculated
separately with 105-106 cfu mL1 of
each challenge microorganism having cultured under the conditions described
in the US Pharmacopeia (10). Recovery of viable microorganisms was performed
on 7, 14 and 28 days of experiment by diluting serially 1 mL of the test product
into 0.9% sterile NaCl. Samples of 1 mL from each dilution were plated onto
Tryptone Soy Agar (TSA) media for bacteria and incubated at 30-35° C for
3 days. In term of the test fungi, Sabouraud Dextrose Agar (SDA) media was used
and the resulting plates were incubated at 20-25°C for 3-5 days. The number
of viable challenging organisms was determined and the results were interpreted
according to the US Pharmacopeia 27.
In use test: Six samples from each type of magnesium hydroxide suspension
(with or without parabens) were consumed by 6 individuals for a period of two
weeks. The test samples were maintained at room temperature and about 10 mL
suspension was drinked off directly from magnesium hydroxide bottle, three times
a day before each meal. The bioburden of magnesium hydroxide suspensions were
determined immediately after preparation and at 7 and 14 days during consumption.
Chemical analysis: The rate of methyl and propyl paraben hydrolysis
in accelerated and real time studies showed a pseudo first orders kinetic (Fig.
1 and 2). The rate of methyl paraben decomposition was
higher than propyl paraben in both accelerated and real time studies. By the
end of 12 weeks of accelerated study about 1 log decrease in methyl paraben
concentration was observed, while that for propyl paraben was about 0.3 log.
The hydrolysis rate of parabens in real time study was slower than accelerated
conditions, showing about 1 log decrease in methyl paraben and propyl paraben
concentrations after 48 weeks and more than 128 weeks, respectively. The pH
of suspensions reduced from 9.8 to 9.5 during 32 months of real time study.
Antimicrobial preservative effectiveness test: Both of magnesium
hydroxide formulations containing parabens and without parabens eradicated the
inoculated bacteria more than 4 log at 7 days. In both formulations the number
of A. niger and C. albicans at 7 days were at least 2 and 3 log
lower than the initial counts, respectively. The bacterial and fungal counts
at 14 and 28 days did not show any increase compared to those at 7 days counts
|| Antimicrobial effectiveness testing of magnesium hydroxide
suspensions with or without parabens
|| Microbial quality of magnesium hydroxide suspensions with
parabens (6 samples) or without parabens (6 samples) during in-use test
||Hydrolysis of methyl paraben and propyl paraben in accelerated
conditions of 40± 2° C, 70± 5% RH
||Hydrolysis of methyl paraben and propyl paraben in real time
conditions of 25± 2° C, 60± 5% RH
In use test: The results of in-use microbiological testing of magnesium
hydroxide suspensions containing parabens and without parabens at 7 and 14 days
were similar and total aerobic viable counts of lower than 100 cfu mL‾
1 were obtained for all 12 suspensions. Escherichia coli was
not detected in any samples (Table 2).
Sufficient preservation of liquid and semi liquid formulations in pharmaceuticals and cosmetics is essential for the efficacy and safety of these products. In this study the chemical stability of paraben esters in magnesium hydroxide suspension with initial pH of 9.8 was evaluated.
The methyl and propyl paraben hydrolysis showed a pseudo first order kinetic in both accelerated and real time conditions. While decomposition rate constant (k) in accelerated conditions was higher than real time, the rate of methyl paraben hydrolysis was about 3 times faster than propyl paraben in both conditions.
The methyl and propyl paraben concentrations were lower than the reported MIC
values of parabens after the first and the fifth months of real time study,
respectively (Table 3). It was supposed that suspensions containing
parabens will not be further preserved against microbial contamination after
first month of preparation. A reduction in pH values during 32 months of study
was related to benzoic acid production following parabens hydrolysis.
Preservative effectiveness testing results were similar in both suspensions
with or without parabens and conforms the US Pharmacopeias requirements for
oral antacids which requires no increase from the initial calculated counts
at 14 and 28 days with lower initial inoculation of 103-104
cfu mL1 of the test product at starting date (10). It was
concluded that alkaline pH of magnesium hydroxide suspension without parabens
could preserve the product against Pharmacopeias challenging microorganisms
and incorporation of parabens did not add any antimicrobial activity to the
Although the results of in-use testing was in accordance with the challenge test
results, indicating good microbiological stability of antacid suspensions with
high pH and without any parabens, there are some reports of their microbial contamination
(Vanhaecke et al., 1987). To achieve adequate preservative levels throughout
the shelf life of a product with an alkaline pH, higher initial levels of parabens,
due to their poor solubility in water, is practically impossible and usefulness.
Lowering the pH of the antacid suspensions to inhibit degradation of the parabens,
by addition of large amounts of buffers such as organic acids, will adversely
affect the acid neutralizing capacity of the antacid. Accordingly, there is still
a need for an effective preservative system in pH levels above 7 which inhibits
microbial contamination over the shelf life of liquid antacid preparations.
|| Methyl and propyl paraben concentrations and MIC values