BIOLOGICAL ASSAY OF ALKALOIDAL DRUGS

Assay: An assay is an investigative (analytic) procedure for qualitatively assessing

or quantitatively measuring the presence or amount or the functional activity of a
target entity, which can be a drug or biochemical substance or organic sample.

Alkaloids: are groups of naturally occurring chemical compounds that contain

mostly basic nitrogen atoms. This group also includes some related compounds
with neutral and even weakly acidic properties. Some synthetic compounds of
similar structure are also attributed to alkaloids. In addition to carbon, hydrogen
and nitrogen, alkaloids may also contain oxygen, sulfur and more rarely other
elements such as chlorine, bromine and phosphorus.

Common Alkaloidal Drugs: Atropine, Belladonna, Opium, Hyoscyamus

Ephedrine, Rauwolfia, Datura stramonium, Ipecachuana, Lobelia inflata.

Chemical Nature of Alkaloids:

 Alkaloids are insoluble or slightly soluble in water, soluble in organic
solvents which are immiscible with water.
 Salts of alkaloids are soluble in water.
 This character makes it possible to separate the alkaloid by partitioning.

Shaking Out Process: the process of assay of drugs by immiscible solvents is

known as the Shaking out process.
 It is carried out by treating the drug, or a conc. liq. extract of it, with a
solvent immiscible with water, in the presence of an excess of alkali which
liberates the alkaloid.
 The free alkaloid is dissolved by the immiscible solvent from which it is
subsequently removed by means of an excess of dil. aq. acid.
 The acids solutions are then extracted with an immiscible solvent
evaporated to obtain the alkaloid which is either weighed or determined
volumetrically.

SCHEME:
 Alkaloid salt (preparation) + Alkali  Alkaloid base (free) in immiscible
solvent (chloroform)
 Alkaloid base (free) in immiscible solvent (chloroform) + dil. acid  Acid
alkaloidal salt in aq. solution.
 Acid alkaloidal salt in aq. soln. + Alkali  Alkaloidal base (free) in
immiscible solvent (chloroform)
 Chloroform is evaporated giving free alkaloid.

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  Free alkaloid+ Excess dil.acid of known normality Alkaloid salt.
 The excess acid back titrated against standard alkali.
 The amount of alkaloids is calculated by multiplying the factor under
individual monograph with the volume of standard acid used.

Steps of Assay:
1. Preparation Of Drug
2. Weighing For Assay
3. Extraction Of Drugs
4. Purification Of Alkaloids
5. Washing Of Glassware
6. Determination Of Alkaloids

1. Preparation of drugs for assay:

 The drug should be pulverized to the required fineness.
 Care should be taken to avoid the loss of water during the pulverization.

2. Weighing for Assay:

 In weighing bulky, crude drugs for the assay, accuracy to within 10 mg for
quantities of 5 g and over is sufficient.
 Portions of soft extracts or ointments may be weighed on a tarred piece of
waxed paper and transferred into the vessel containing the solvent.
 In transferring weighed portions to a separator, thoroughly rinse the vessel
in which the material to be assayed was weighed, and add the rinsing to
the separator.

3. Extraction:
The alkaloid bearing drugs are extracted with one of the following methods;
1. Maceration
2. Percolation
3. Continuous extraction

Maceration:
1. Accurately weighed powdered drug is dipped in a specified amount of
solvent or mixture of solvents.
2. Then made it alkaline with ammonia TS and mix well.
3. Then allow macerating for 12-24 hours with occasional shaking.
4. After some time allow the solvent to settle.
5. Decant the solvent and aliquot of which are used for analysis.

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Percolation:
1. Place accurately weighed powdered drug in a suitable container, saturate
with suitable solvent for 5 min.
2. Make it alkaline with ammonia test solution, mix thoroughly and put in
percolator.
3. Allow the drug to macerate for 10-12hours.
4. Percolate it slowly until the percolate is alkaloid free.

Alkaloidal test: Determine the completeness of extraction of the alkaloid by

evaporating about 4 ml of the last percolate to dryness, dissolving the residue in
0.5 ml of approx. 0.5 N acid, and adding a drop of mercuric iodide TS (Valser ‘s
reagent) ; not more than a slight turbidity is produced. Treat the percolate as
directed for purification of alkaloids.

Continuous Extraction:
1. Place an accurately weighed portion of ground drug in an extraction
thimble.
2. Insert thimble into suitable extractor.
3. Moist the drug with specified solvent & mix by means of stirring rod.
4. Allow to stand for 5min.
5. Make the mixture alkaline with ammonia TS & mix thoroughly.
6. Allow the drug to macerate for 12hours.
7. Then pack the drug in thimble & cover with cotton & add sufficient
solvent & extract the drug.

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4. Purification of Alkaloids:
1. The alkaloidal soln. obtained by any of the extraction methods is usually
contaminated with other extractives which interfere with the quantitative
determination of the alkaloids.
2. For purification, remove the alkaloids from the immiscible solvent by the
shaking out with an acid, then make the acid soln. alkaline, usually with an
alkali OH and extract with an immiscible solvent.
3. The vol. and strength of the acid to be used are usually left to the
discretion of the operator. It is best, however, to keep the total vol. as
small as possible.
4. For the first extraction use at least 10 ml of approx. 1 N acid or sufficient to
make the mixture distinctly acid.
5. For succeeding extractions, use a dilution of 5 ml of the acid with 5 ml of
water.
6. In all assays, continue the extraction until 0.5 ml of the last acid washing
shows no more than a slight turbidity on the addition of a drop of
mercuric iodide TS.
7. The acid extracts, before proceeding with the next step, should be clear. If
not clear, filter or treat as follows:
8. Shake the combined acid extracts with one or more 10 ml portions of the
appropriate immiscible solvent, until the acid soln. is clear.
9. Then wash the immiscible solvent extracts with one or more 5 ml portions
of water acidified with HCl or hydro sulfuric acid, and add these washings
to the acid soln.
10. Then make the acid soln. alkaline, in most cases with ammonia TS , and
extract it with several successive portions of the appropriate immiscible
solvent.
11. Use a vol. of the latter in each operation not less than half that of the
water soln. and repeat the operation as long as any alkaloid is extracted by
the immiscible solvent.
12. The completeness of extraction can be tested by mercuric iodide TS.
13. With most alkaloids extract several times before testing.

5. Washing of Glassware:
1. Carefully wash the stems of separators and funnels and the lips of flasks,
separators and graduates from which solvents containing alkaloids have
been drawn or poured with some of the solvent to prevent loss and to
remove any of the alkaloids left by evaporation.
2. Add these washings to the other extractions containing the alkaloids.

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6. Determination of alkaloids:
1. Evaporate the alkaloidal solution to dryness by steam or with a current of
air.
2. Let the residue to get soft with 1ml of neutral ether or alcohol.
3. Add a volume of standard acid and warm gently to make complete
solution.
4. Add the volume of water to make 25ml.
5. Titrate the excess of acid by a standard alkali using methyl red as an
indicator.
6. If alkaloidal residue is to be weighed, dry it at 105 C to a constant weight.
7. If solvent is chloroform then remove the traces with few ml of neutral
ether or alcohol followed by evaporation.

Indicator: Unless otherwise specified in the individual monograph, use methyl

red TS as the indicator in volumetric solns.

Special treatment:

Use of adsorbent:
 For fluid extract & tinctures having alkaloidal drugs, it is necessary to
evaporate then to dryness.
 To facilitate evaporation & reduce loss, extract are added on an adsorbent
which is previously acid or alkali wash then made neutral by washing with
water and dried before use.

Emulsions:
 Avoid shaking of immiscible solvent with water.
 If emulsion form, at excess of either of the solvents, it will results in the
breakdown of emulsion.
 Emulsion can also be broken down by adding anhydrous sodium sulphate
or some acid.