Preservation of pharmaceutical products using antimicrobial agents, evaluation of microbial stability of formulations. preservative efficacy test

1. Preservation of
Pharmaceutical Products
By.S.D.Mankar
Assistant Professor
Department of Pharmaceutcs
Pravara Rural College Of Pharmacy,Pravaranagar

2. Definition:-
The agent which may reduce the risk of contamination or
and to kill any contaminants.
The correct approach to preservation has as its foundation in
important principles.
The first of these is that the addition of a preservatives to a
product must not be done to mask any deficiencies in the mfg.
procedures & second is that the preservative should be an
integral part of the formulation, chosen to afford protection in
particular environment.

3. Single preservative is not suitable for preservation of all
pharmaceutical formulations.
The selection of preservative must be made on an individual basis,
using published information, & microbiological studies.
Combination of two or more preservatives are used to extent the
range and spectrum of preservation.

4. NEED:-
To protect our drug from microbial attack.
To enhance activity and efficacy of drug.
 To increase shelf life of our product.
 To stabilize our product

5. Ideal Preservative:-
Compatible with drug components
Effective
Stable
Non toxic and non reactive
Cost effective
Highly soluble
Tasteless & odourless

6. FACTORS AFFECTING PRESERVATIVES EFFICACY:-
1.Interaction with formulation
 2.Properties of the preservation
3.Effects of containers
4.Type of microorganisms
5.Influence of pH

7.  1. Interaction with formulation:-
 Hydrocollids such as methylcellulose, alignates, tragacanth can interacts with
preservatives and diminish their activity.
 Many emulgents are used in pharmaceutical preparations to produce elegant
applications. Interaction may occur between preservatives and emulsified oil
phase and with emulgent molecules.
 Nature of oil, oil water ratio, type of concentration of emulgent, influence the
concentration of preservatives needed to protect the system.
 Many tablet additives cause problems in tablet preservations due to their
interaction with added preservatives.

8. 2. Properties of the preservation:-
 The distribution of preservative must be homogeneous and more
solubility in the bulk phase is preferable in multi phase system.
Some chemicals such as chlorobutol may hydrolyse on storage if
pH is unfavourable.
Preservatives may react with substances leached from the
and lose its antimicrobial activity.

9. 3. Effects of containers :-
 Formulations packed in glass containers can be expected to retain
their preservative content if closure is airtight.
 Preservatives may penetrate through the plastic container and
interacts with it.
 Rubber also reacts with many preservatives but is still used for
closures.
Containers or closures may cause contamination of pathogens.
Screw–capped containers and corks are the common source of
spores.

10. 4. Type of microorganisms :-
 Plants products may contain pathogenic microorganisms from the
soil. E.g Clostridium species, Bacillus anthracis.
These soil microorganisms can cause spoilage of pharmaceutical
products.
 Many products prepared from animal sources may contain
pathogens like Salmonella typhi.
 Spores of tetanus and gas gangrene have been isolated from
geletin.

11. 5. Influence of pH :-
Adjustment of the pH of solution may affect the chemical
stability and the activity of the preservative.
The majority of preservatives are less dependent upon pH,
although cationic active quaternary ammonium
are more active at high pH values.

12. EVALUATION OF PRESERVATIVES:
Preservative Efficacy Test (PET)
First appeared in USP in 18th revision, 1 st sept. 1970.
• Applied to the formulated medicine in its final container to determine
whether it is adequately protected from microbial spoilage.
 Tests/standards apply only to the product in original, unopened
containers.
 This test is done to determine the efficacy of preservative against
microorganism.

13. Involve challenging a product with a defined number of colony
forming units (CFU) of a variety of test microorganisms (bacteria,
yeasts and fungi).
Then monitoring the kill /survival rate at defined time intervals up to
28- days. It is examined by the duplicate plate count method
(CFU/ml = number of colonies per ml plated /Total dilution factor).
 Multiple dosages forms- Parenterals, otics, nasal, oral, topical and
ophthalmic products made with aqueous bases or vehicles.

14. Medium:-
Soyabean casein digest agar medium (SCDM) & Sabouraud -
Dextrose medium.
Test organisms :- Choice of test microorganisms:
The intension is to use M.O. which are likely to arise in the raw
materials used in the product, occur in the mfg. environment &
represent a particular health hazard if they grew in the product.

15. Preservatives should be active against as wide as a range of
microorganisms as possible hence the choice should be of both Gram+ve
and Gram-ve bacteria, yeast and moulds in IP Test.
Test organism that are recommended by all of the pharmacopoeias
includes:
Staphylococcus aureus ATCC 6538
Pseudomonas aeruginosa ATCC 9027
E. coli ATCC 8739
Fungi/mould, Aspergillus Brasiliensis ATCC 16404
Yeast, Candida albicans ATCC 10231

16. Preparation of inoculum :-
Fresh stock culture of each test M.O.are subcultured on the surface of
soyabean casein digest agar medium.
Incubate the bacterial culture at 30 -35 ˚C for 18-24 hrs & incubate the
culture of candida albicans & aspergillus niger to 20-25 ˚C for 48 hrs &
07 days using sterile saline solution.
After this incubation period, microbial culture is harvested by washing
with sterile saline(0.9% w/v) to obtain a microbial count of about 10 8
CFU/ mL (collect it in a sterile container and treat this as Stock solution
test solution)

17. Procedure:-
Inoculate each original product container with one of the std.
microbial suspension using ratio eq. to 0.1 ml of inoculum
suspension to 20 ml of product & mix.
The final conc. Should between 1 X 10 5 & 1 X 10 6 m.o./ml of
product.
 Determine the no. of viable m.o. by the plate count method in
inoculum suspension and from these calculate the initial conc. Of
m.o./ml of product examined.

18. Incubate the inoculated containers or tubes at 20-25 ˚C.
Determine the viable count by the plate count method at 7, 14, 21 & 28
days to inoculation.
Interpretation of results:-
The preservative is effective in the product examined if
1. for parenteral, ophthalmic, sterile nasal & otic preparation:-
A) the conc. Of viable bacteria are NMT 10 % of intial conc. By the 7th day
& NMT 0.1 % of the intial conc. At 14 th day.
B) the conc. Of viable yeast & molds remain at or below the initial conc.
During the 7,14 & 28 days of initial count.

19. 2 for topical preparation made with aq.base, non sterile nasal
preparation & emulsion.
A) the conc. Of viable bacteria is NMT 1% of the initial conc at 14
days and there is further decrease in count at 28 days.
B) there is no increase in yeast & mould count at 14 & 28 day from
the initial count.
3) For oral preparation:-
A) the conc. Of viable bacteria is NMT 10% of the initial conc at 14
days and there is further decrease in count at 28 days.

20. B) there is no increase in yeast & mould count at 14 & 28 day from
the initial count.
