Evaluation /Quality Control / assessment of crude drug

 according to WHO guidelines 

(Part-V)

 (Analytical & Biological Evaluation) 
(S.Y. B. Pharm Unit -IV ) Unit -I


(T.Y. B. Pharm Sem -VI ) -Unit - IV


(Final Yr. B. Pharm) Section -II

As per PCI Syllabus 

METHODS OF DRUG EVALUATION

 The evaluation of a drug is done by following methods. 

1. Organoleptic evaluation

2. Morphological evaluation

3. Microscopic evaluation

4. Physical evaluation

5. Chemical evaluation

6. Analytical evaluation
7. Biological evaluation

Analytical Evaluation


A) Chromatographic techniques

 i. TLC-Thin layer chromatography
ii. HPTLC-High performance thin layer chromatography
iii HPLC-High performance / pressure liquid chromatography
iv. GLC-Gas chromatography /CC-column chromatography
Vi. Gel permeation chromatography
Vii Affinity chromatography

Chromatography:

Pharmacopoeias are increasingly insisting to employ TLC as a mean for evaluating quality & purity. The Rf value obtained by TLC is used as an aid for identity.
Rate of flow (Rf) =     Distance moved by the solute
                          ----------------------------
                        Distance moved by the solvent front    


The qualitative extracts of crude drug are prepared & are compared with standard or marker solution of known constituents. Rf value varies from 0.0 -1.0,
So Rf value some time converted into hRf value by multiplying Rf  with 100 to obtain range from 0.0- 100 & used for better characterization.

The spots obtained are visualized either by observing in UV light or by using spray reagents like I% vanillin-sulfuric acid for steroids & terpenoids, dragendroff reagent for alkaloids etc.

TLC method is rapid & also gives information about the chief constituents of plant drug so enables in assessment of quality of the drug, so TLC is widely used adopted. Further more TLC also provide drug fingerprint, if any adulterants or substituent present they can be detected. So TLC is used to monitor the identity & purity of the drug.

High performance thin chromatography (HPTLC)
It is one of the very useful methods for the qualitative and quantitative analysis of plant extracts. It is the advanced form of TLC with shorter time and precise results.

HPLC: 
HPLC assay method is also mentioned in the monographs to estimate the amount of markers present in the drug. In this method standard of known concentration is prepared & injected into HPLC system to get peak area, which is proportional to its concentration. Similarly the peak areas of the sample because of the presence of the active constituents are taken which is proportional to the amount of constituents present in the sample. 

Then the amount of active constituents present in the sample can be calculated by comparing its peak areas in the both standard & sample, dilutions are also taken into account.

e.g. HPLC method of estimation of vasicine in Adhatoda vasika, Andrographolides in Andrographis paniculata, Phyllanthin & Hypophyllanthin in Pamarus. 

Gas liquid chromatography (GLC)

GLC is the most selective and versatile form of gas chromatography. Commonly it is used in the assay and analysis of starting materials and drug substances, quantification of drug substances in formulations and assay of impurities and solvents in the drug substances.

Gas chromatography is also employed for the analysis of volatile oil & fatty  acids.


Column chromatography


Basically it is a liquid chromatography in which mobile phase in the form of liquid passes over the stationery phase packed in a column. The column is made of either glass or metal. It is the oldest method and most commonly practiced method for the isolation of pure compounds.



Note: Chemical tests are usually performed to identify unorganised crude drugs.

HPTLC is the advanced form of TLC and HPLC is the advanced form of column chromatography.


B) Spectrophptometric methods


i. UV Ultra violet / visible spectroscopy
ii. IR-Infra Red spectroscopy
iii. Fluorescence analysis
iv. NMR-nuclear magnetic resonance spectroscopy
v. MS-Mass spectroscopy
vi. X-ray diffraction
vii. RIA-radio immune-assay


BIOLOGICAL EVALUATION

It is employed when the drug cannot be evaluated satisfactorily by Chemical and physical methods.
In this method, the response produced by the test drug on a living system is compared with that of the stranded preparation.

Such an activity is represented in units as International Units (I.U.) The dose is termed as International units (IU).

Example
 Digitalis 1 IU=76mg of standard
 Vit-A 1IU=O.344

SIGNIFICANCE
i. The method is generally used when standardization is not done satisfactory by chemical or physical methods.

 ii. When the quantities of the drug / sample are very less, then the drugs are evaluated by biological methods.
 iii These methods are performed on living animals, isolating living organ and tissue, animal preparation, and micro-organism (Bioassay).


Bitterness value
Medicinal plant materials that have a strong bitter taste ("bitters") are employed therapeutically, mostly as appetizing agents. Their bitterness stimulates secretion in then gastrointestinal tract, especially of gastric juice.

The bitter properties of plant material are determined by comparing the threshold bitter concentration of an extract of the materials with that of a dilute solution of quinine hydrochloride.

The bitterness value is expressed in units equivalent to the bitterness of a solution containing lgm of quinine hydrochloride in 2000 ml water.
 Bitterness value calculated in units per g using the following formula:

 2000 X c/ a X b 8

Where,
a= the concentration of the stock test solution (ST) (mg/ ml), atjo oil:
b= the volume of test solution ST (in ml) 1n the tube with the threshold bitter Concentration.
C = the volume of quinine hydrochloride R(in mg) in the tube with the threshold bitter conc.

Note : Bitterness value is evaluated by tasting the drug and Comparing its bitterness with the standard.

Haemolytic activity:

The most characteristic property of saponins is their ability to cause haemolysis; when added to a suspension of blood, saponin: produce changes in erythrocyte membranes, causing haemoglobin to diffuse into the surrounding medium.

The haemolytic activity of plant materials, or a preparation containing saponins, is determined by comparison with that of a reference material, saponin R, which has a haemolytic activity of 1000 units per gram. 

The haemolytic activity of the medicinal plant is calculated using the following formula:

1000 x a/b 

Where,
1000 = the defined haemolytic activity of saponin R in relation to ox blood,
a = quantity of saponin R that produces total haemolysis (g)
b = quantity of plant material that produces total haemolysis (g).

Determination of pesticide residues

WHO and FAQ (Food and Agricultural Organization) set limits of pesticides, which are usually present in the herbs. These pesticides are mixed with herbs during the time of cultivation.

Pesticides like DDT, BHC, toxaphene, cause serious side-effects in human beings if crude drugs are mixed with these agents.

The average pesticidal residue should not exceed more than 1%.
The average pesticidal residual limit (ARL) in mg of pesticide per kg of plant material can be calculated on the basis of the maximum acceptable daily intake of the pesticide for humans (ADI), as, recommended by WHO, & the mean daily intake (MDl) of the medicinal plant material. 

Determination of arsenic and other heavy metals

Contamination of medicinal plant materials with arsenic and heavy metals can be attributed to many causes including environmental pollution and traces of pesticides.

The contents of lead and cadmium may be determined by inverse voltametry or by atomic emission spectrophotometry.

The following maximum amounts in dried plant materials, which are based on the ADI values, are proposed. lead, 10 mg/ kg, cadmium, 0.3 mg / kg. 

Determination of microorganisms
Usually medicinal plants containing bacteria and molds are coming from soil and atmosphere. Analysis of the limits of E. coli and molds clearly throws light towards the harvesting and production practices. The substance known as afflatoxins will produce serious side-effects if consumed along with the crude drugs.

Aflatoxins content
Aflatoxins are naturally occuring mycotoxin produced mainly by Asperglllus flavus and Aspergillus parasiticus.

The presence of aflatoxins can be determined b standard aflatoxins Bl, B2, Gl, G2 mixtures.

IP method: NMT 2 µg/kg of aflatoxins B1 and Total aflatoxins 4 µg/kg.

USP method: NMT 5ppb of aflatoxins Bl & Total aflatoxins 20ppb. 

Radioactive contamination

Microbial growth in herbals is usually avoided by irradiation. This process may sterilize the plant material but radioactivity hazard should be taken into account.
The range of radionuclides that may be released into the environment as a result of nuclear accident might include long-lived and short-lived fission products, actinides, and activation products.

The nature & intensity of radionuclides released may differ markedly & depend on the source (reactor, reprocessing plant, fuel fabrication plant, isotope production unit, etc).