Substances Assayed After Conversion - Gravimetric Analysis

SUBSTANCES ASSAYED AFTER CONVERSION

There are certain pharmaceutical substances that can be assayed gravimetrically after their suitable conversion to free acid, or free base, or free compound or corresponding derivatives (or substitution products). All these typical cases shall be discussed briefly with their appropriate examples in the following sections.

1. Substances Assayed after Conversion to Free Acid

A few official pharmaceutical substances may be assayed gravimetrically by affecting separation, purification, and weighing an organic medicinal compound without causing any permanent change in composition. It is an usual practice that before extraction of the organic medicinal compound, the sample of the crushed tablets is carefully washed with petroleum benzene to get rid of undesirable components, for instance : lubricants and binders that would be extracted along with the organic medicinal compound by such solvents as ether or chloroform which is employed subsequently.

In case, the organic medicinal compound is acidic in nature e.g., amobarbital in sodium amobarbital tablets, it is first and foremost extracted with an aqueous solution of an acid or base to cause separation from the neutral substance which might be present. The resulting aqueous solution of the salt of the respective organic medicinal compound is subsequently made acidic and the liberated organic acid (amobarbital) is finally extracted with ether or chloroform.

Interestingly, in a situation where either magnesium stearate or stearic acid forms a component in the formulation, the organic medicinal compound which is acidic (amobarbital) cannot be extracted with NaOH solution for obvious reason that sodium stearate shall also be extracted along with the salt of the organic acid. Therefore, instead a saturated solution of Ba(OH)₂ is employed thereby the insoluble precipitate of barium stearate may be discarded by filtration.

1.1.  Phenobarbitone Sodium

Materials Required : Phenobarbitone sodium : 0.5 g ; hydrochloric acid (2 M) : (dissolve 17.0 ml (^~  11.5 N) in 100 ml DW) : 5.0 ml ; ether : 13.5 ml ; absolute ethanol : 2.0 ml.

Procedure : Weigh accurately 0.5 g phenobarbitone sodium and dissolve in 15 ml of DW. Add to it 5 ml of 2 M hydrochloric acid and extract with 50 ml of ether and then with successive 25 ml quantities of ether until complete extraction is affected. Wash the combined extracts with two 5 ml quantities of DW and wash the combined aqueous extracts with 10 ml quantities of ether. Add the ether to the main ethereal extract, evaporate to low bulk, add 2 ml of absolute ethanol, evaporate to dryness and dry the residue to constant weight at 105°C. Each g of residue is equivalent to C₁₂H₁₁N₂NaO₃.

Calculations :

1.2.  Cognate Assays

There are certain pharmaceutical substances that may be assayed after their conversion to the respec-tive free acids as shown in Table 10.3.

2. Substances Assayed after Conversion to Free Base

In a specific instance where the organic medicinal substance is basic in nature e.g., papaverine in papaverine hydrochloride, it is primarily treated with an aqueous solution of a base and subsequently the liberated organic base is extracted with either chloroform or ether.

A typical example is described below :

2.1. Papaverine Hydrochloride Tablets

Materials Required : Sodium hydroxide (2 M) (dissolve 8.0 g of NaOH pellets in 100 ml of CO₂ free DW : 50 ml ; chloroform : 100 ml ; absolute ethanol : 5 ml.

Calculations :

Procedure : Weigh 20 tablets and crush them in a pestle mortar and find out the average weight of a single tablet. Accurately weigh 0.5 g equivalent of papaverine hydrochloride and dissolve in 15 ml of DW. Add to it 15 ml of 2 M sodium hydroxide and extract with 50 ml of chloroform and then with successive 25 ml quantities of chloroform until complete extraction is affected. Wash the combined extracts with two 5 ml quantities of DW and wash the combined aqueous extract with two 10 ml quantities of chloroform. Add the chloroform to the main chloroform extract, evaporate to a small volume, add 2 ml of absolute ethanol, evaporate to dryness and dry the residue to constant weight at 105°C.

Each g of the residue is equivalent to 1.105 g of C₂₀H₂₁NO₄ . HCl.

2.2.  Amodiaquine Hydrochloride

Materials Required : Amodiaquine hydrochloride : 0.3 g ; dilute ammonia solution (42.5 ml of strong ammonia solution to 100 ml in water) ; NO. 4 sintered glass crucible.

Theory : Amodiaquine hydrochloride possesses two moles of inherent water of crystallization, and hence the precentage base is calculated with reference to the substance dried over P₂O₅ at a pressure not exceeding 5 mm of Hg. Usually, the assay is performed on one portion of the sample and the drying on a separate portion altogether.

The underlying principle of the method is based upon the precipitation of amodiaquine base that is generated as a precipitate when the salt is decomposed in aqueous medium with dilute ammonia.

Procedure : Weigh accurately 0.3 g of previously dried amodiaquine hydrochloride into a 100 ml beaker provided with a stirring rod and watch glass cover. Dissolve it in 50 ml of DW and dilute ammonia solution with constant gentle stirring until the solution is just alkaline (to litmus paper). Allow the contents of the flask to stand for 30 minutes and then quantitatively filter through a NO. 4 sintered glass-crucible previously dried to a constant weight at 105°C. Wash the precipitate several times with DW, until the washings do not give a positive test for chloride (test with standard AgNO₃ Solution). Dry the residue to a constant weight at 105°C. Each gram of residue is equivalent to 1.306 g of C₂₀H₂₂ON₃Cl, 2HCl, 2H₂O.

2.2.  Cognate Assays

A few other pharmaceutical substances are also determined after conversion to free bases as recorded in Table : 10.4.

3. Substances Assayed After Conversion to Free Compound

In certain specific cases either the pure pharmaceutical substance or dosage forms are quantitatively converted to free compound. This conversion to free compound is quantitative and hence forms the basis of gravimetric analysis. A few typical examples belonging to this category are, namely : progesterone suspension sterile, progesterone tablets, sodium lauryl sulphate, mephobarbital tablets and sorbitan monooleate.

3.1. Mephobarbital Tablets

Materials Required : Mephobarbital : 300 mg ; hexane : 100 ml ; chloroform : 150 ml ; alcohol (95% v/v) : l0 ml.

Procedure : Weigh and finely powder not less than 20 mephobarbital tablets. Transfer an accurately weighed portion of the powder equivalent to about 300 mg of mephobarbital to an extraction thimble. Extract with 15 ml of solvent hexane, allow the thimble to drain, transfer to a continuous extraction apparatus pro-vided with a tared flask, and extract the mephobarbital with chloroform for 2 hours. Evaporate the chloroform on a steam bath with the aid of a current of air, cool, dissolve the residue in about 10 ml of alcohol, evaporate, dry the residue at 105°C for 1 hour, cool and weigh.

The weight of the residue represents the weight C_l3H₁₄N₂O₃ in the portion of the tablets taken.

4. Substances Assayed after Conversion to Derivatives or Substitution Products

In pharmaceutical drug analysis a host of organic pharmaceutical substances are invariably converted quantitatively to their corresponding derivatives by virtue of interactions with certain functional entities, namely : aldehyde, ketone, amino, carboxyl, phenolic, hydroxyl etc. However, in some cases it may be feasible to obtain uniform substitution products of organic pharmaceutical substances quantitatively, for instance : tetraido derivative of phenolphthalein is obtained from the phenolphthalein tablets. It is important to mention here that the number of organic pharmaceutical substances which may be analysed by this method is limited because of two vital reasons, they are :

(a) the reversible nature of reactions, and

(b) the formation of products of side reactions simultaneously.

4.1. Benzylpenicillin(Syn : Benzylpenicillin Sodium or Potassium Salt)

Materials Required : Benzylpenicillin sodium (say) : 0.12 g ; amyl acetate (previously saturated with 1-ethylpiperidinium benzylpencillin at room temperature, cooled in ice and filtered) : 5.0 ml ; phosphoric acid (20% v/v) : 0.5 ml ; anhydrous sodium sulphate (freshly ignited and powdered) : 0.5 g ; dry acetone (previously saturated with 1-ethylpiperidinium benzylpenicillin at room temperature cooled in ice and filtered) : 3.0 ml ; 1-ethylpiperidine amyl acetate solution (prepared from l-ethyl piperidine, 1 .0 ml, and amyl acetate, 8.0 ml, saturated at room temperature with 1-ethylpiperidinium benzylpenicillin, cooled in ice and filtered) : 1.5 ml ; dry acetone in amyl acetate (1 : 1) previously saturated with 1-ethylpiperidinium benzylpenicillin : 2.0 ml ; solvent ether : 4.0 ml.

Theory : Benzylpenicillin (sodium or potassium salt) may be assayed gravimetrically by quantitative conversion to the 1-ethylpiperidinium benzylpenicillin derivative. The ultimate precipitation is caused by l-ethyl piperidine after the respective sodium or potassium salt of benzylpencillin has been duly converted with phosphoric acid to the corresponding penicillanic acid (i.e. parent acid) and the latter finally extracted with amyl alcohol. The reactions may be expressed as follows :

Procedure : Weigh accurately 0.12 g of benzyl penicillin sodium, dissolve in 5 ml of ice-cold DW in a flask and cool in an ice-bath. Add to it 5.0 ml of amyl acetate followed by 0.5 ml of ice-cold H₃PO₄, stopper, shake the contents immediately for 15 seconds, and centrifuge for 30 seconds. Remove the aqueous layer as completely as possible with the help of a pipette. Add 0.5 g anhydrous Na₂SO₄, stir the contents vigorously and cool in an ice-bath for 5 minutes. Centrifuge for about 30 seconds and again cool in ice-bath for 5 minutes. Pipette 3.0 ml of the supernatant liquid into a tared centrifuge tube. Add to it 3.0 ml of ice-cold acetone and 1.5 ml of 1-ethylpiperidine amyl acetate solution, stir, stopper the tube and cool in ice-bath for 2 hours. Now, centrifuge for 1 minute, break the surface with the help of a pointed glass rod, so that all crystalline particles are covered by liquid, and again centrifuge for 1 minute. Decant off the supernatant liquid, wash the precipitate with 2 ml of ice-cold dry acetone in amyl acetate (1 : 1) and again centrifuge for 1.5 minutes. Decant the supernatant liquid, wash twice with 2.0 ml portion of solvent ether, centrifuging for 1.5 minutes and decanting each time. Dry to constant weight under vacuum at room temperature. Each gram of residue is equivalent to 0.8300 g of C₁₆H₁₇N₂NaO₄S.

4.2.  Cholesterol

Materials Required : Cholesterol : 0.1 g ; ethanol (90% v/v) : 12.0 ml ; digitonin solution (0.5% w/v in 90% v/v ethanol) : 40.0 ml ; ethanol (90% v/v) : 100 ml ; acetone ; carbon tetrachloride.

Theory : The assay of cholesterol is solely based on the fact that practically all 3 β-hydroxysterols e.g., cholesterol, readily produces an insoluble molecular addition complex with pure digitonin (1 : 1)—a steroidal saponin isolated from either Digitalis purpurea or Digitalis lanata. The complex thus obtained is crystalline in nature, fairly stable and possesses very low solubilities.

The complexation of cholesterol and digitonin may be expressed as follows :

Procedure : Weigh accurately about 0.1 g of cholesterol into a 100 ml flask and dissolve it in 12.0 ml ethanol. Insert the stopper and allow to stand at room temperature (25 ± 2°C) for 12 hours, filter through a Gooch crucible, and wash with 5.0 ml of ethanol. Mix the washings to the filtrate and add to it 40.0 ml solution of digitonin and make it warm to 60°C to ensure that the complexation is almost complete. Filter the precipitate of the resulting complex through a prepared Gooch crucible, previously dried to constant weight at 105°C. Wash the precipitate with ethanol followed by acetone and carbon tetrachloride, allow to drain as completely as possible, and dry to a constant weight at 105°C. Each g of the residue is equivalent to 0.2390 g of cholesterol.

Note : All solutions must be ice-cold.

4.3. Thiamine Hydrochloride

Materials Required : Thiamine hydrochloride : 0.5 g ; hydrochloric acid ( ~  11.5 N) : 2.0 ml ; silicotungstic acid solution (10% w/v in water) : 4.0 ml ; NO : 4-sintered glass-crucible ; dilute hydrochloric acid (1 part HCl + 19 parts H₂O) : 50 ml.

Theory : The gravimetric assay of thiamine hydrochloride is based upon the precipitation of it as thiamine silicotungstate with silicotungstic acid in a slightly acidic medium. It has been observed that the precipitating reagent is a complex silicate SiO₂, 12 WO₂, n H₂O having somewhat variable composition with regard to the degree of hydration. For a reasonably precise and accurate determination the precipitating reagent must contain <| 1.85% SiO₂ and <| 85% WO₃. Interestingly, the thiamine silicotungstate complex possesses more or less a constant composition.

The precipitation of insoluble thiamine silicotungstate may be designated by the following reaction :

Procedure : Weigh accurately 0.05 g of thiamine hydrochloride, previously dried at 105°C, and dissolve it in 50 ml DW in a 250 ml beaker having a stirring rod and watch glass cover. Add to it 2.0 ml of hydrochloric acid, heat to boiling and then add 4.0 ml of silicotungstic acid solution as rapidly as possible. Now, boil the solution gently for 2 minutes and quickly filter through a NO. 4 sintered-glass crucible, previously dried to a constant weight at 105°C. Wash the residue with a boiling mixture of HCl and H₂O (1 : 19) about 40 ml, then with DW 10.0 ml and ultimately with two portions of 5 ml each of acetone. Finally dry the residue to constant weight at 105°C. Each g of thiamine silicotungstate residue is equivalent to 0.1938 g of C₁₂H_l7ON₄SCl, HCl.

Precautions :

(a) An excess of HCl is a must so as to produce a readily filterable precipitate,

(b) In case the sample is pure enough, the rate of addition of silicotungstic acid has little influence on the result, but on the contrary if the sample has significant impurity it may afford poor results,

(c) To achieve complete complexation boiling must be done for more than 2 minutes, otherwise it would yield low results, and

(d) A 50-ml wash-liquid is quite ideal, further washings (volume) may offer poor results.

4.4. Histamine Acid Phosphate (C₅H₉N₃, 2H₃PO₄)

Materials Required : Histamine : 0.15 g ; nitranilic acid solution (3.5% w/v in 95% ethanol) : 10.0 ml ; ethanol (95%) : 30.0 ml ; sintered-glass crucible (NO : 3) ; ether : 10.0 ml.

Theory : The gravimetric assay of histamine acid phosphate is based upon the formation of insoluble histamine-nitranilic acid complex as depicted in the following equation :

Procedure : Weigh accurately about 0.15 g of histamine acid phosphate into a 250 ml beaker provided with a stirring rod and watch glass cover. Add to it 10.0 ml of DW to dissolve the sample. Now, add 10.0 ml of nitranilic acid solution, stir and allow to stand for 15 ininutes. Pour in 10.0 ml of ethanol, keep it in an ice-bath for 3 hours and filter through a No. 3 sintered-glass crucible, previously dried to a constant weight at 130°C. Transfer the precipitate quantitatively and wash it thoroughly with four quantities each of 5.0 ml of ethanol and ultimately with 10.0 ml of ether. Dry to constant weight at 130°C. Simultaneously, determine the loss in weight on drying a separate portion of the sample at 105°C. Each gram of the histamine-nitranilic acid complex is equivalent to 0.8998 g of C₅H₉N₃, 2 H₃PO₄.

4.5.  Proguanil Hydrochloride

Materials Required : Proguanil hydrochloride : 0.6 g ; ammoniacal cupric chloride solution (dissolve 22.5 g of copper (II) chloride in 200 ml of DW and mix with 100 ml of 13.5 M ammonia) ; NO. 4 sintered-glass crucible ; mixture of dilute solution of ammonia and DW (1 : 5).

Theory : Gravimetric analysis of proguanil hydrochloride involves the precipitation of the proguanil-cupric complex that results on the addition of ammoniacal cupric chloride solution to a solution of proguanil hydrochloride. The reaction can be expressed by the following equation :

Procedure : Weigh accurately 0.6 g of proguanil hydrochloride into a 250 ml beaker fitted with a stirring rod and watch-glass cover. Add to it 50.0 ml of DW and heat gently to dissolve the sample. Chill the solution below 10°C in an ice-bath and then add ammoniacal-cupric-chloride solution with continuous stirring till the resulting solution attains a permanent deep-colour. Allow the solution to stand for 90 minutes to complete the complexation and then filter through a No. 4 sintered glass crucible previously dried to constant weight at 130°C. Transfer the precipitate quantitatively into the crucible, wash first with a mixture of dilute solution of ammonia and DW (1 : 5) adequately followed by cold water until the washings are practically colourless thereby showing the complete absence of soluble copper salts. Dry the precipitate to a constant weight at 130°C. Simultaneously, find out the loss in weight on drying with a separate portion of the sample at 105°C and incorporate this in the calculation. Each gram of proguanil-cupric-complex is equivalent to 1.0199 g of C₁₁H_l6N₅Cl, HCl.

4.6. Benzethonium Chloride

Theory : In general, quaternary nitrogen containing compounds like—choline chloride, acetylpyridinium chloride, benzethonium chloride, and bethanechol chloride readily form insoluble salts quantitatively with tetraphenyl boron and this puts forward the basis for the gravimetric assay of the above cited pharmaceutical substances.

The various reactions involved may be summarized and expressed as follows :

Eq. (a) shows that the quaternary salt gets quantitatively precipitated by sodium tetraphenyl boron as the complexing agent. Eq. (b) depicts that quaternary compounds shall readily react with certain anionic dye, such as bromophenol blue, to yield a blue, chloroform-soluble complex.

Eq. (c) finally illustrates that the blue-coloured complex shall react quantitatively with sodium tetraphenyl boron to give an insoluble compound.

Therefore, we have :

Materials Required : Benzethonium chloride : 0.15 g ; Chloroform : 50 ml ; bromophenol blue solution (Dissolve with heating 0.2 g of bromophenol blue in 3 ml of 0.1 M NaOH and 10 ml of ethanol (96%). Allow to cool and dilute to 100 ml with ethanol 96%] : 50 ml ; sodium tetraphenyl borate solution (1% w/v in chloroform) : 50 ml ; sintered-glass crucible No : 4.

Procedure : Weigh accurately about 0.15 g of benzethonium chloride sample into a 250-ml beaker placed on a magnetic-stirrer and watch-glass cover. Add to it 25 ml of chloroform and warm gently to dissolve. Cool to ambient temperature and add suffcient bromophenol blue solution gradually till the solution yields a blue Chloroform-soluble complex. Now, add sodium tetraphenyl borate solution in small lots at intervals with constant stirring until the complete precipitation of insoluble benzethonium tetraphenyl borate complex takes place. Allow the solution to stand for 60 minutes to complete the complexation and subsequently filter through a No. 4 sintered-glass crucible previously dried to constant weight at 130°C. Transfer the precipitate quantitatively into the crucible and wash the precipitate with cold chloroform. Dry the precipitate to a constant weight at 110°C. Each gram of benzethonium tetraphenyl borate complex is equivalent to 0.6117 g of C₂₇H_4lO₂NCl.

4.7.  Cognate Assays

Quite a few official pharmaceutical substances and their respective dosage forms can be assayed gravimetrically after conversion to their corresponding derivatives or substitution products. Table 10.5 records some examples from official compendia.