Chapter-XI-XV - Lecture notes 10 PDF

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SPECIAL METHODS USED IN PHARMACEUTICAL ANALYSES (FINAL LECTURE)Quantitative analyses of crude drugs – establish purity or to determine the amount of therapeutically active constituents present for the purpose of standardization. SPECIAL METHODS: CHEMICAL METHOD – ash, moisture, crude fiber, extracti...

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SPECIAL METHODS USED IN PHARMACEUTICAL ANALYSES (FINAL LECTURE) Quantitative analyses of crude drugs – establish purity or to determine the amount of therapeutically active constituents present for the purpose of standardization. SPECIAL METHODS: 1. CHEMICAL METHOD – ash, moisture, crude fiber, extractives from different solvents, estimation of alkaloidal content, etc. 2. BIOLOGICAL METHOD -affect drugs such as microbes, animals, or animal tissues are measured. Official standard for drugs or preparations include biological assays or tests for such medicinal agents as adrenal cortex, corticotropin, digitalis, heparin sodium, insulin, oxytocin, parathyroid and vasopressin. -Microbiological assays are required for antibiotics, liver, vitamin B12, and other related drugs. Tests are required for the toxicity and potency of serological preparations such as vaccines, serums, toxoids and antitoxins. CHAPTER XI. ASH AND WATER DETERMINATIONS I. ASH CONTENT – residue remaining after incineration, representing the inorganic salts naturally occurring in the drug and adhering to it, or it may also include inorganic matter added for the purpose of adulteration. - A basis for judging the identity and cleanliness of a drug and gives information relative to its adulteration with inorganic matter - A measure of the amount of inorganic matter present as impurity - Ash content standards have been established for a number of the official drugs, they set a maximum limit on the total ash or on the acid insoluble ash permitted. **TOTAL ASH – is the total residue remaining after incineration **ACID INSOLUBLE ASH – part of the total ash which is insoluble in diluted HCl. - Diluted HCl dissolves calcium carbonate, alkali chlorides leaving the silica which is derived from the soil adhering to the drug. Factor Affecting Ash determination: Temperature – careful control of temperature is the most important analytical factor to regulate - When using Electric furnace for ignitions, the following temperature equivalents should be considered a. Very dull red heat – 500 to 550.C b. Dull red heat – 550 to 700.C c. Bright red heat – 800 to 1000.C d. Yellow red heat – 1000 to 1200.C e. White heat – 1200 to 1600.C Example: Determination of the Total acid insoluble ash Content of Acacia Different sources of ash of a drug: Carbonates, phosphates, sulfates, chlorides, oxides of Calcium, Magnesium, Potassium, Sodium, Aluminum, Iron and other metallic elements **RESIDUE ON IGNITION – the allowable limit of residue that may remain on the drug - Residue of expensive chemicals are determined by using small amounts and the ash yielded be negligible.

Negligible – defined as the quantity not exceeding 500 microgram - Residue on ignition limit is given in the USP XIX and NF XIV monographs Example: 1 to 2 grams of the following drugs i. Acetazolamide – URL (Upper Residue Limit) 0.1% ii. Aspirin – URL 0.05% **LOSS ON IGNITION – define as the percentage of test material which is volatilized and driven off under the conditions specified. Limitations on the amount of volatile matter to be lost when heated are in the USP Example: Calamine (2.0), Magnesium Sulfate (40.0 to 52.0), Titanium dioxide (0.5) B. WATER CONTENT – many drugs official in the USP and NF contain varying quantities of water either as water of crystallization (hydrates) or as water in the adsorbed form. - USP and NF specify certain water content limits in the drug monographs Importance of water content: i. crude drugs – percent active constituent must be calculated on the basis of moisture free drug ii. scientific work – variable quantities of moisture would adversely affect the calculated results iii. affect stability/degradation of crude drugs iv. important in making solutions of different concentrations v. determination of the dosage of substances used as medicaments Methods of water content determination: 1. Gravimetric for drugs containing no constituents other than water, volatile at 105.C 2. Gravimetric for drugs containing ether soluble constituents, volatile at 105.C 3. Azeotropic distillation (toluene) - AKA Xylene method in the USP and Moisture method by toluene in the NF. It was developed by the US Forestry service - specified for the determination of moisture content of many vegetable drugs containing 2% or more moisture 4. Titrimetric – Karl Fischer 5. Dewpoint 6. Electrolytic hydrometric 7. Drugs which contain no constituents volatile at 105.C, other than water, 10 grams of the drug is accurately weighed and dried in an oven at 105.C for 5 hours. After 5 hours the samples is weighed and dried again at 1 hour intervals until the loss in weight is NMT 0.25% in 1 hour of drying. The weight loss of the drug represents the moisture present in the sample. 8. Drug contains matter, other than water, volatile at 105.C, the volatile ether soluble extractive be determined and the weight of the latter subtracted from the weight lost by the drug upon drying, the difference is the moisture content of the drug. Example: Determination of the water content of Acacia Determination of the moisture content of digitalis by the Toluene distillation method CHAPTER XII. EXTRACTIVE AND CRUDE FIBER CONTENT

I. EXTRACTIVE – yield to a solvent, gives an approximate measure of the amount of a certain constituent or group of related constituents the drug contains - Solvent used will not dissolve quantities of substances other than those sought in the extraction. - For efficient and complete extraction, the solvent should be divided into a number of relatively small proportions and used successively. Each portion should be brought into intimate contact with all the parts of the sample and then be removed before addition of the next portion. - The use of fresh portions of solvent not saturated with extractive favors the rapid and thorough removal of the solute with the minimum quantity of solvent - It is convenient and economical to be able to use the same portion of solvent repeatedly. This is accomplished by the Soxhlet (apparatus used for extraction with volatile solvents), which separates the solvent from solute after the extraction, the separated solvent being employed in a second extraction. Procedure on how to use the Soxhlet apparatus: i. Solvent in a weighed flask A, heated over a hot plate or water bath (absence of flame since solvent is flammable) ii. Weighed sample of drug in a porous paper extraction thimble, fitted in the extraction tube B iii. reflux condenser C is attached to the top of Extraction tube B iv. solvent vapor pass upward from flask A to the side of the tube D of the extractor to the condenser v. the condensate drips into the thimble until a sufficient amount has accumulated to raise the level of liquid in the extraction tube to the top of the siphon tube E. vi. the solution that has been standing in contact with the drug is the discharged into the flask where the solvent is vaporized again. vii. solvent is passes through this cycle repeatedly, solute is collected in the flask viii. heat should be adjusted so that the solvent passes through the cycle about once every 10 minutes ix. top of thimble should be above the level of the top of the siphon side tube x. solvent must not be volatilized more rapidly than it will filter through the porous thimble, or it will overflow and carry insoluble matter over mechanically xi. after the extraction is complete, the flask is removed, the solvent is driven off until the flask and its contents are of constant weight xii. percentage of extractive in the drug is calculated from the weight of the residue TYPES OF EXTRACTIVES: 1. Volatile and Non-volatile Ether soluble Extractive - total ether soluble volatile extractive is applied to drugs which contain volatile oils - total ether soluble non-volatile extractive is applied to drugs having active constituents associated with volatile matter. Example: Determination of the nonvolatile ether soluble content of Cocoa 2. Alcohol soluble extractive - alcohol is a good solvent for resinous matter - employed to determine the amount of resin in drugs Example: Assay of Benzoin

3. Diluted Alcohol Extractive a. Macerate about 2 grams of the prepared drug, accurately weighed, in about 70 ml of diluted alcohol in suitable flask b. Shake the mixture during 8 hours at 30 minutes intervals and then allow it to stand for 16 hours without shaking. c. Filter and wash the flask and residue with small portions of diluted alcohol, passing the washings through the filter, until the filtrate measures 100 ml. d. Evaporate a 50 ml aliquot portion of this filtrate to dryness in a suitable tared dish on a water bath and dry the constant weight at 110.C. e. Calculate the percentage of this extractive from the weight of drug taken. 4. Water soluble Extractive and Water insoluble residue - applied to drugs of which one or more of the most important constituents are soluble in water. Example: Assay of Aloe 5. Solvent Hexane soluble Extractive - hexane is a good solvent for fats and fatty acids - the hexane extractive is an index to the amount of fat present and serves as a check on the amount of seeds contained in colocynth pulp, since the seeds contain a large amount of fatty matter II. CRUDE FIBER - Residue, consisting of cellulose that remains undissolved after successive treatment with boiling acid and alkali. - Important in the examination of certain drugs and particular spices, since the most commonly used adulterants consist of waste or refuse material derived from the drugs or spices themselves or from other food products. - Cellulose is the outer cellular layer or protective coating which contains a larger proportion of lignified tissues and consequently, more crude fiber than the official part. Example: Determination of the crude fiber content of Cocoa REFERENCE: JENKIN’S QUANTITATIVE PHARMACEUTICAL CHEMISTRY CHAPTER XIII. CONSTANTS OF FATS, FATTY OILS, WAXES, BALSAMS, RESINS CONSTANTS – number of physical and chemical properties or values of fatty substances, waxes, resins - When taken in conjunction with color, odor, taste and special identity tests for the given substance and for the common adulterants are the basis upon which purity and quality of these substances are judged. I. ACID VALUE – AKA Acid number, acidity index - define as the number of milligrams (mg) of KOH necessary to neutralize the free acid in 1 grams of oil, fat, wax, resin, balsam or similar organic substance of complex composition. - gives the amount of KOH expressed in tenths of 1% required to neutralize the free acids in a substance. - maybe expressed as the number of ml of 0.1 N NaOH required to neutralize 10 grams of the substance. - official lists of acid value on USP/NF

Method: Titrate a weighed sample containing an alcoholic or alcohol-ether solution with a standard base (either 0.5N, 0.1N, 0.02N NaOH) using phenolphthalein as indicator. ** Balsams and resins – use mixed solvent of alcohol and ether **Solid fats and waxes – melted on a water bath and titrated while hot NOTE: Fresh or recently prepared fatty substances contain little or no free acids. Upon aging, the acid value increases slowly at first and more rapidly later, especially if the substance is not well protected from the simultaneous action of light and air If acid value exceeded indicate that the substances have undergone hydrolytic decomposition in their preparation or purification or during the period of storage. High acid value is NOT necessarily an indication of rancidity since rancidity is a result of the action of air or possible bacteria on liberated fatty acids. Example of Acid value limits: Tolu – NLT 112 and NMT 168 (less than 112 Tolu is of low quality, more than 168 Tolu is adulterated) Example of Assay: Determination of the acid value of Rosin COMPUTATION ON ACID VALUE Sample problem: Find the acid value (mg/g) of Rosin sample weighing 1.100 grams which required 28 ml of 0.1100 N NaOH to bring about the end point. Given: Volume of NaOH – 28 ml Normality of NaOH – 0.1100 N Milliequivalent weight of KOH – 56.11mg/meq MW K = 39.10, O=16, H-1.01 Weight of sample = 1.100 grams Equation: AV = VNaOH x NNaOH x mg/meqKOH Weight of sample AV = 28 x 0.1100 N x 56.11 mg/meq 1.100 grams AV = 157.1 mg of KOH/ g of sample II. SAPONIFICATION VALUE – AKA saponification number, Koettsdorfer number (from originator) - defined as the number of milligrams of KOH required to neutralize the free acids and saponify the esters contained in 1 gram of fat, fatty or volatile oils, wax, resin, balsam or other substance of similar composition - serve to aid in the detection of the presence of the glycerides of acids containing less than 16 or more than 18 Carbon atoms, since the value of this constant is inversely proportional to the mean molecular weights of the acids present. - it may also indicate adulteration of the sample with unsaponifiable matter such as mineral oil Example of Assay: Determination of the Saponification value of Cottonseed oil COMPUTATION ON SAPONIFICATION VALUE: Sample problem: Find the SV (mg/g) of cottonseed oil if a 1.532 gram sample refluxed with 25 ml of about 0.5N alcoholic KOH, required 15.70 ml of 0.5100 N HCl for the residual titration. The blank was

run using the same value of 0.5N alcoholic KOH and required 26 ml of 0.51100 N HCl to bring about the end point. Equation: SV = (VHCl blank - VHCl residual) x N HCl x mg/meq KOH Weight of sample = (26 ml – 15.70 ml) x 0.5100 N x 56.11 mg/meq 1.532 grams SV = 192 mg KOH/g of sample III. ESTER VALUE – AKA ester number - defined as the number of milligrams of KOH to saponify the esters in 1 gram of fatty or volatile oil, fat, wax, balsam, resin or similar substance. - important in the analysis of yellow and white wax, since it indicate the presence of adulterant which is the paraffin, or the presence of rosin and stearic acid in the wax NOTE: If the sample DO NOT contain free acid: Ester value is equal to Saponification value If the sample contains free acids: Ester value = SV – Acid value Sample problem: 1. If a sample of beeswax is found to have an acid value of 20.4 mg/g and saponification value of 89.8 mg/g, what is the Ester value of the sample? Solution: EV = SV – AV = 89.8 – 20.4 = 69.4 2. If a sample of white beeswax has an acid value of 18.45 mg/g and an ester value of 74 mg/g, what is the Saponification value of the sample? Solution: EV = SV – AV, 74 = SV – 18.45, SV = 74 + 18.45 = 92.45 IV. UNSAPONIFIABLE MATTER – defined as the substances present in oils and fats that are not saponified by basic/alkali hydroxides but are soluble in ordinary fat solvents. *oils and fats = saponified = remain small amount of residue Residue : Physosterol – vegetable oils and fats Cholesterol – animal oils and fats Unsaponifiable subtances – purpose of adulteration *Normal unsaponifiable matter present in most oils and fats dissolves in the alcoholic soap solution *Large amount of unsaponifiable matter may separate out and float on the surface of the mixture. Example: linseed oil – addition of petroleum oils increases the unsaponifiable matter content of linseed oil. Corn oil – NMT 1.5% Cod liver oil – NMT 1.3 % V. IODINE VALUE – AKA Iodine number - Defined as the number of grams of iodine absorbed under specified conditions by 100g of oil, fat, wax or other substances. - Quantitative measure of the proportion of unsaturated fatty acids, both free and combined as esters, that have the property of absorbing iodine. - Indicate whether the fats and oils are pure or admixtures Example:

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  

Drying oils – linseed oil, fish oils (cod liver oil) - High iodine numbers of above 120 since they contain large proportion of unsaturated fatty acids Non drying oils – olive oil, almond oil - Low iodine numbers below 100 Semi drying oils – cottonseed oil, sesame oil - Intermediate Between 100 to 120 Animal fats – less than 90

Conclusion: iodine number does not only serve as identification of known oils but also to indicate which class an unknown fat or oil belongs. NOTE: Relationship between saponification and iodine value – means of detecting adulteration Example: olive oil adulterated with cotton seed oil – saponification does not change but the iodine number increases. Castor oil adulterated with olive oil – iodine does not change but the saponification increases. Example of Assay: Determination of the Iodine value of Olive oil Methods of determining iodine number: A. Hubl B. Hanus C. Wijs – method used in USP which required Iodochloride TS COMPUTATION ON IODINE VALUE: Sample problem: Determine the Iodine value of a sample of Olive oil weighing 0.2100 grams if 24.15 ml and 12 ml of 0.1100 N Sodium thiosulfate solutions are required for the blank and residual titrations? Equation: Iodine value = (Vblank – Vresidual) x N Na2s2O4 x mg/meq x 100 Weight of sample MW Iodine = 126.90 = 1269/1 X 1000 = 0.1269 g/meq Solution: IV = (24.15 ml – 12 ml) x 0.1100 N x 0.1269 g/meq x 100 = 80.8 grams of Iodine consumed by 100 g 0.2100 grams of oil VI. HYDROXYL VALUE – AKA Hydroxyl number - Defined as the number of milligrams of potassium hydroxide equivalent to the hydroxyl content of 1 gram of the substance. - Indicate the identity and purity of fatty substances possessing alcoholic hydroxyl groups. - Adulteration is present if the Hydroxyl value is inversely proportional to the molecular weight - Low hydroxyl value, there’s an adulteration with high molecular weight alcohols. Formula: ml of 1N NaOH x 56.11 Wt of sample VII. ACETYL VALUE OF FATTY ACIDS – corresponds closely to hydroxyl value of fatty alcohols

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Number of milligrams of potassium hydroxide required to neutralize the acetic acid obtained by the saponification of 1 gram of acetylated fatty acids. Significant in identity and purity Acetyl value is found by acetylating hydroxyl fatty acids and determining the saponification value of the product. From the saponification value and the acid value of the original fatty acid, the acetyl value is then calculated.

FORMULA: A

=

S-F 1- (0.00075S) Where : A = acetyl value S = SAPONIFICATION value of acetylated fatty acid F = acid value of original fatty acids 0.00075 = number of grams of acetyl group corresponding to 1 mg of KOH.

VIII. WATER AND SEDIMENT IN FATTY OILS - Fatty oils particularly of animal origin frequently contain some moisture and non fatty tissue residues - Official limit for moisture and sediment have been fixed by the USP for oils of this type. Example: cod liver oil – NMT 0.5% by volume of water and sediment - Makes use of pear shaped centrifuge tubes with different scale division

REFERENCE: Jenkins’s Quantitative Pharmaceutical Chemistry CHAPTER XIV. ASSAY OF VOLATILE OILS VOLATILE OILS - AKA as ethereal oils, essential oils, essences - Complex products composed of mixtures of widely variant chemical characteristics OFFICIAL VOLATILE OILS BASED ON CHEMICAL COMPONENTS: 1. Hydrocarbons Two types: a. acyclic series (heptane and myrcene) b. Isocyclic series (pinene, camphene, limonene, bornylene, fenchene, dipentene, sylvestrene, phellandrene) 2. Alcohols – free alcohols and alcohols combined with acids as esters Ex. Linalool, geraniol, citronellol, terpineol, borneol, menthol, santalol 3. Aldehydes Ex. Benzaldehyde, cinnamic aldehyde, salicyl aldehyde, citral and citronellal 4. Ketones Ex. Camphor, carvone, fencone, thujone, menthone 5.Phenol Ex. anethol, eugenol, carvacrol, safrol, chavicol, thymol

6. Acids Two types: a. Free acids Ex. Acetic, propionic, butyric, valeric, benzoic, cinnamic, hydrocyanic b. combination with alcohols Ex. Linaly acetate, bornyl acetate, menthyl acetate 7.

Sulfur compounds – allyl isothiocyante (mustard oil)

PURPOSE OF ANALYSIS OF VOLATILE OILS: 1) Determine purity – by quantitative measurement of certain physical characteristics 2) Test for adulterants – by qualitative tests for the various substances present METHODS OF GENERAL APPLICATION: I. SPECIFIC GRAVITY – use of Westphal balance and pycnometer (latter is the most accurate and official method) Ratio of the weight of the volume of oil to that of an equal volume of pure water at 25 0C Spec.gravity of volatile oils ranges from 0.84 to 1.2 Spec. gravity is not constants because of t...