Pesticide analysis:
For the purposes of the Pharmacopoeia, a pesticide is any substance or mixture of substances intended for preventing, destroying or controlling any pest, unwanted species of plants or animals causing harm during or otherwise interfering with the production, processing storage, transport or marketing of vegetable drugs. The item includes substances intended for use as growth-regulators, defoliants or desiccants and any substance applied to crops either before or after harvest to protect the commodity from deterioration during storage and transport.

Reagents. All reagents and solvents are free from any contaminants, especially pesticides, that might interfere with the analysis. It is often necessary to use special quality solvents or, if this is not possible, solvents that have recently been re-distilled in an apparatus made entirely of glass. In any case, suitable blank tests must be carried out.

Apparatus. Clean the apparatus and especially glassware to ensure that they are free from pesticides, for example, soak for at least 16 h in a solution of phosphate-free detergent, rinse with large quantities of distilled water R and wash with acetone and hexane or heptane.

Quanitative analysis of Organophosphorus insecticides.
Examine by gas chromatography, using carbophenothion R as internal standard. It may be necessary to use a second internal standard to identify possible interference with the peak corresponding to carbophenothion.

Test solution. Concentrate solution B in a current of helium for chromatography R almost to dryness and dilute to 100 μl with toluene R.

Reference solution. Prepare at least three solutions in toluene R containing the insecticides to be determined and carbophenothion at concentrations suitable for plotting a calibration curve.

The chromatographic procedure may be carried out using :
– a fused-silica column 30 m long and 0.32 mm in internal diameter the internal wall of which is covered with a layer 0.25 μm thick of poly (dimethyl) siloxane R.
– hydrogen for chromatography R as the carrier gas. Other gases such as helium for chromatography R or nitrogen for chromatography R may also be used provided the chromatography is suitably validated.
– a phosphorus-nitrogen flame-ionisation detector or a atomic emission spectrometry detector. Maintaining the temperature of the column at 80ºC for 1 min, then raising it at a rate of 30ºC/min to 150ºC, maintaining at 150ºC for 3 min, then raising the temperature at a rate of 4ºC/min to 280ºC and maintaining at this temperature for 1 min and maintaining the temperature of the injector port at 250ºC and that of the detector at 275ºC. Inject the chosen volume of each solution. When the chromatograms are recorded.

Quanitative analysis of Organochlorine and pyrethroid insecticides:
Examine by gas chromatography, using carbophenothion as the internal standard.It may be necessary to use a second internal standard to identify possible interferencewith the peak corresponding to carbophenothion.

Microbial content determination:

The tests for sterility are intended for detecting the presence of viable forms of micro-organisms in or on pharmacopoeial preparations. The tests must be carried out under conditions designed to avoid accidental contamination of the product during the test.

The working conditions in which the tests are performed should be monitored regularly by sampling the air and surfaces of the working area and by carrying out control tests, The tests are based upon the principle that if micro-organisms are placed in a medium which provides nutritive material and water, and kept at a favourable temperature, the organisms will grow and their presence can be indicated by a turbidity in the originally clear medium. The tests for sterility are designed to reveal the presence of micro-organisms in the samples used in the tests; interpretation of results is based on the assumption that the contents of every container in the batch, had they been tested, would also have complied with the the tests. Since every container cannot be tested, a sufficient number of containers should be examined to give a suitable degree of confidence in the results of the tests.

Culture Media:
Fluid thioglycollate medium – For use with clear fluid products

Alternative thioglycollate medium – For use with turbnid and viscid products and for devices having tubes with small lumina.

Test Procedures:
The tests can be carried out using Method A, Membrane Filtration or Method B, Direct Inoculation. Method A is to be preferred where the substance being examined is (a) an oil, (b) an ointment that can be put into solution, (c) a non-bacteriostatic solid not readily soluble in the culture medium and (d) a soluble powder or a liquid that possesses inherent bacteriostatic and fungistatic properties.

Method A, Memberane filteration test
Prepare each membrane by aseptically transferring a small quantity (sufficient to moisten the membrane) of fluid A on to the membrane and filtering it. For each medium to be used, transfer aseptically into two separate membrane. Alternatively, transfer aseptically the combined quantities of the preparation being examined prescribed in the two media onto one membrane. Draw the liquid rapidly through the filter with the aid of vacuum. If the solution being examined has antimicrobial properties, wash the membrane (s) by filtering through it (them) not less than three successive quantities, each of approximately 100 ml, of sterile fluid A. The quantities of fluid used should be sufficient to allow growth of a small inoculum of organisms (approximately 50) sensitive to the antimicrobial substance in the presence of the residual inhibitory material on the membrane. After filtration, aseptically remove the membrane (s) from the holder, cut the membrane in half, if only one is used, immerse the membrane, or one-half of the membrane, in 100 ml of soyabean-casein digest medium and incubate at 20º to 25º for not less than 7 days. Similarly, immerse the other membrane, or other half of the membrane, in 100 ml of fluid thioglycollate medium and incubate at 30º to 35º for not less than 7 days.

Diluting fluids
a. Fluid A: Dissolve 1 g of peptic digest of animal tissue (such as bacteriological peptone) or its equivalent in water to make 1 litre, filter or centrifuge to clarify, adjust to pH 7.1±0.2, dispense into flasks in 100-ml quantities and sterilize at 121º for 20 minutes.
b. Fluid B: If the test sample contains lecithin or oil, use fluid A to each litre of which has been added 1 ml of polysorbate 80, adjust to pH 7.1±0.2, dispense into flasks and sterilize at 121º for 20 minutes.

Note – A sterile fluid shall not have antibacterial or antifungal properties if it is to be considered suitable for dissolving, diluting or rinsing a preparation being examined for sterility.

Method B: Direct Inoculation:

Method of test:
Remove the liquid from the test containers with a sterile pipette or with a sterile syringe or a needle. Aseptically transfer the specified volume of the material from each container to a vessel of the culture medium. Mix the liquid with the medium but do not aerate excessively. Incubate the inoculated media for not less than 14 days, unless otherwise specified in the monograph, at 30º to 35º in the case of fluid thioglycollate medium and at 20º to 25º in the case of soyabean-casein digest medium. When the material being examined renders the medium turbid so that the presence or absence of microbial growth cannot be determined readily by visual examination, transfer suitable portions of the medium to fresh vessels of the same medium between the third and seventh days after the test is started. Continue incubation of the transfer vessels for not less than 7 additional days after the transfer and for a total of not less than 14 days.

Observation and Interpretation of Results:
At intervals during the incubation period, and at its conclusion, examine the media for macroscopic evidence of microbial growth. If no evidence of growth is found, the preparation being examined passes the tests for sterility. If evidence of microbial growth is found, reserve the containers showing this and, unless it is demonstrated by any other means that their presence is due to causes unrelated to the preparation being examined and hence that the tests for sterility are invalid and may therefore be recommenced, perform a retest using the same number of samples, volumes to be tested and the media as in the original test. If no evidence of microbial growth is then found, the preparation being examined passes the tests for sterility. If evidence of microbial growth is found, isolate and identify the organisms. If they are not readily distinguishable from those growing in the containers reserved in the first test, the preparation being examined fails the tests for sterility. If they are readily distinguishable from those growing in the containers reserved in the first test, perform a second retest using twice the number of samples. If no evidence of microbial growth is found in the second retest, the preparation being examined passes the tests for sterility. If evidence of growth of any micro-organisms is found in the second retest the preparation being examined fails the tests for sterility.

Result and Conclusion:
Involved Evaluation and Standardization techniques for crude drugs, mono or Polyherbal Frormulation. They involved the macroscopic techniques, microscopic techniques, physical evaluation and biological evaluation. They also involved the Quantitative analysis of Organophosphorus insecticides, Organochlorine and pyrethroid insecticides, microbial content determination.

Summary report :
The given  test are applied for quality, safety and efficacy built up in the product. And these tests are most important for pharmaceutical industry who manufacture from the crude drug.


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