Stability is defined as the extent to which a pharmaceutical
product retains, within specified limits and throughout its storage period and
use, i.e. its shelf life, the same properties and characteristics that are
possessed at the time of its manufacture.
Microbial stability of formulations is defined as the
formulation which does not have any microbiological attack and meets the
standard for lack of contamination or sterility.
Microbial contamination in formulation mainly occurs from
water (Pseudomonas, Xanthomonas, etc.), air (Penicillium, Aspergillus, Bacillus
species, Yeasts, etc.) and raw materials (Micrococci).
Stability is used to determine:
• Quality of a drug substance or drug products.
• Shelf life of drug products.
• Recommended storage conditions.
Hence, stability testing is recommended because chemical
degradation sometimes leads to a lower concentration of drugs in the dosage form, and
also, a toxic product is formed due to the degradation of active ingredients.
In another way,
Stability is the capacity of a drug product to remain within the established specifications to ensure its identity, strength, quality, and purity.
There are three types of stability studies:
(a) Long-term study (Minimum 1 year and maximum 5 years):
Studies carried out at 25°C ± 2°C / 60% RH ± 5% RH. For 1 year every 3 months,
for 2 years every 6 months and for 3 years every year once.
(b) Intermediate (Minimum 6 months and maximum 12
months): Studies carried out at 30°C ± 2°C / 65% RH ± 5% RH.
(c) Accelerated (6 Months): Studies carried out at
40°C ± 2°C/75% RH ± 5% RH.
Types: Various types of stability are listed in the Flowchart.
Flowchart: Types of stability
Stability evaluation for various formulations:
1. Tablets: Colour, odour, assay, degradation,
dissolution, moisture content, hardness, friability.
2. Capsules: Appearance, brittleness, colour, odour,
content uniformity, assay, degradation product, dissolution, moisture and
microbial content.
3. Emulsions: Appearance, phase separation, colour,
odour, assay, pH, viscosity, microbial assay, preservative content, mean size
of globules, distribution of dispersed globules.
4. Oral solutions and suspensions: Mean size and
distribution of particles, for suspensions, dispersibility, rheological
properties, etc.
5. Oral powders for reconstitution: Moisture,
Reconstitution time.
6. Inhalations and nasal aerosols: Appearances
(content, container, valve, its components), dose content uniformity, labelled
number of medication actuations per container meeting, aerodynamic particle
size distribution, water content, microscopic evaluation, Leak rate, Microbial
limit.
7. Topical formulations (ointments, cream, lotions, gel,
paste, solutions, nonmetered aerosols): Clarity, colour, odour, pH,
resuspendability (for lotions), consistency, viscosity, preservatives,
antioxidant content, microbial limit, sterility testing, weight loss.
8. Ophthalmic preparations (cream, ointments, solutions,
suspensions): Sterility, particulate matters, non-metered aerosols,
delivery rate, microbial limit, spray patterns, water content, particle size.
9. Suppositories: Softening range, dissolution at
37°C, microbial limits.
10. Small and large volume parenterals: Particulate
matters, pH, sterility, endotoxins, pyrogens.
11. Transdermal patches: In-vitro release rate,
leakage, microbial limits, sterility test, peel and adhesive force, drug
release rate.
12. Freeze drying products: Appearance, reconstituted
products, assay, degradation products, pH, water content, rate of solution.
Microbial Stability Test: Microbial contaminants
usually originate from two different sources during production and filling, and the
use of the cosmetics by the consumers (by the consumer’s hands and body). It is
necessary to carry out routine microbiological analysis of each batch of the
finished product. The main potential pathogens in cosmetics are Pseudomonas
aeruginosa, Candida albicans, and Staphylococcus aureus. These pathogens must
not be detectable more than 0.1 g or 0.1 ml in a cosmetic product. Microbial
contamination in some cosmetic products is given in the table below.
Table: Microbial contamination in cosmetic products
|
Products |
Microorganisms |
CFU/g |
|
Skin cream |
Enterobacter
gergoviae |
120000 |
|
Eye cream |
Enterobacter
gergoviae |
290000 |
|
Herbal tooth
powder |
E. faecium,
K. pneumonia |
540000 |
|
Hair shampoo |
P. aeruginosa |
570000 |
|
Sun lotion |
E. cloacae,
E. faecium, Escherichia spp |
8000000 |
Some tests are as follows:
1. Screening test: It is also known as the plate count or
dip slide method. This method is used to detect aerobic bacteria in an aqueous
sample. The dip slide is coated on both sides with a solid agar gel medium. A
small quantity of TTC (2,3,5-triphenyl tetrazolium chloride) is added to detect
aerobic bacteria in the sample. The slide is dipped into the aqueous solution
for 10 seconds, and excess liquid is drained off from the slide. Then it is
incubated at 35-37°C for 18-48 hours. The colour that appeared is compared with
the calibration chart. Aerobic bacteria species grow on this medium and are
detected by their ability to reduce TTC dye to a red-coloured formazan dye.
Developing bacterial colonies alter the TTC dye and appear as red spots.
2. Quantitative test: Quantitative tests determine
the actual count level of bacteria, moulds and yeasts in cosmetic products.
This method is used for the isolation of microorganisms from cosmetic products, including direct colony counts and enrichment culturing.
Microbial stability studies are also carried out in
Pharmaceutical products. Raw materials play a vital role in the product
formulation. Pharmaceutical raw material is defined as a substance that is used
in the manufacturing of pharmaceutical products. During the manufacture of
pharmaceuticals and cosmetics, untreated raw materials are contaminated with
microorganisms beyond acceptable limits. Hence, it is necessary to control
microbiological contamination from raw materials under GMP regulations in the
pharmaceutical industry. These microorganisms from plants (e.g., species of
Erwinia, Pseudomonas, Lactobacillus, Bacillus or Streptococcus), such as gum
acacia, tragacanth, agar, powdered rhubarb, and starches, contain bacteria,
which cause disease. Water plays a major role in product formulation because
water is a good medium for microbial growth. The water activity (Aw)
of pharmaceutical and cosmetic products is the measure of free water in the
formulation. The amount of water that is in free form is available to
microorganisms for survival. The measurement of Aw in pharmaceutical
and cosmetic products predicts the type and number of microorganisms
responsible for product degradation and maintains chemical stability.
Water activity is determined by the dew point method and
using an electric hygrometer (which measures relative humidity).
Antimicrobial Effectiveness Test: This test is used
for estimating preservatives in a product. This test is mainly used during the development
of formulation and stability studies. In this test, a product is inoculated
with a controlled quantity of specific microorganisms. The test then compares
the level of microorganisms found on a control sample versus the test sample
over 28 days. Test organisms are used to challenge the preservative system in a
product. The suitable media are Soyabean Casein digest or Sabouraud Dextrose
Agar used for the test. The bacteria (likely, Escherichia coli, Pseudomonas
aeruginosa, and Staphylococcus aureus), yeast (Candida albicans) and mould
(Aspergillus niger) are used for this test. A product is inoculated or
contaminated with several organisms between 1 × 105 (100,000) to 1 ×
106 (1,000,000) colony-forming units (CFU) per ml of product. At
various intervals, the product is tested to determine its ability to control
reproduction or destroy the microorganisms.
Tests and standards are incubated at 32.5 ± 2.5 oC
and 22.5 ± 2.5 oC for bacteria and yeast, respectively, and the
concentration of the microorganisms in each of the standardised inocula is
determined by the plate count method.
Pharmaceutical Products: For testing purposes, the
USP has divided test articles into four separate categories:
Category 1 – Injections, other parenterals including
emulsions, sterile nasal products made with aqueous bases or vehicles.
Category 2 – Topically used products made with aqueous bases
or vehicles, non-sterile nasal products, and emulsions, including those applied
to mucous membranes.
Category 3 – Oral products other than antacids made with
aqueous bases or vehicles.
Category 4 – Antacids made with an aqueous base.