Alkaloidal Assay:

-Alkaloidal substances are organic nitrogenous chemical compounds. -Difficult to extract from the drug. -Present in relatively small quantities. -In many cases, easily destroyed by improper handling. -The proportion of alkaloids in plant material & preparations(extracts, tincture) can be quatitatively determined. -The process usually involves the separation of the alkaloids from non-basic materials.

Following general properties of alkaloids are made use of:

i)Usually sparingly soluble in water but freely soluble in non-polar organic solvents, like ether, chloroform, benzene, or light petroleums.

ii)Combine with acids to form salts which are freely soluble in water but only sparingly soluble in the above organic solvents. iii)Alkaloids are liberated from their salts by alkalis.

Separation of non-alkaloidal matters:

-In addition to active principles, crude drugs & galenical preparation contains large quantities of extraneous vegetable matters.

-With the above mentioned 3 properties of alkaloids, following non-alkaloidal substances are removed.

- Total alkaloid is usually calculated as the main constituent(proximate assay)

For separation & estimation of alkaloids:

1)Extraction of the drug(Preparation of gelenicals) 2)Separation and purification of the alkaloids. 3)Determination of alkaloids (Gravimetry, titrimetry(volumetric)

1)Extraction of the drug(Preparation of gelenicals)

a)The drug substance is reduced to proper fineness by grinding(30-60mesh). -Moisten with the solvent , allowed standing for penetration of the tissues. b)Made alkaline by moistening with a small amount of ammonia. -Kept standing for -1hour for maceration.

c)Alkaloid is by automatic continuous extraction apparatus or pharmacopoeial percolation.

-In continuous extraction are two types: Liquid/solid & liquid/liquid -For the dry sample(liquid/solid) Soxhlets a & b

-The sample is held in a paper thimble or in tube A. -For liquid/liquid: c) for the solvent heavier than water, d) for the solvent lighter than water.

-Continuous extraction calls for small volume of solvents, tedious evaporation of large volumes of solvents are avoided.

-Its a rapid extraction process.Disadvantage is that for some alkaloids can not withstand heat. -If alkaloids are heat sensitive or yields undue(un- suitable) amount of non-alkaloidal extractive, use of cold solvent in an ordinary percolator is an alternative. Requires large

quantity of solvents.

2)Separation and purification of the alkaloids.

A)Liquid extract is diluted with water, -Acidified to convert the alkaloids to their water- soluble salts. -Solution is extracted shaking with the chloroform. -Repeatedly extracted with chloroform for several times. -Extracts are combined.

Nux Vomica:

-Contains brucine & strychnine as principal alkaloids. -Assay of strychnine is to be carried out. -Formerly, brucine used to be converted to an alkali soluble red nitro compd by nitrous & nitric acid. -The red compound is separated from strychnine.

-Nowadays assay of Strychnine is carried out by differential absorption method without physically separating them. Cognate determination: Nux vomica liquid extract & tincture.

Belldonnna leaf: Determination of alkaloidal content as Atropine -Moderately course powder is weighed & transferred into a soxhlet extraction thimble. -The thimble is placed in the extractor. -The powder is moisten with a mixture of concentrated ammonum hydroxide, ethanol & ether. -Allowed for maceration for overnight & extract the drug with ether for 3Hrs. -The etherial alkaloid is transferred into a separating funnel & extracted with sulphuric acid. -The combined acid extract is basify by ammonia solution . -The alkaloid is extracted into chloroform. -The combined extract is evaporated to dryness on a steam bath. -The residue is dissolved into few ml of chloroform and sulphuric acid V.S. is added. -The solution is heated to dry off chloroform. -The excess acid the back titrated with sodium hydroxide V.S. using methyl red as indicator.

-Alkaloid content of the belladonna leaf is calculated as(C17H23NO3). 1000ml 0.5M H2SO4 = 289.4g C17H23NO3