1. PHCH 402: Analytical Quality Control
05: Limit tests (10 hrs)

2. OUTLINE Presence of impurities in pharmaceuticals and their sources.
Limit tests and factors considered in their design, negative and comparison tests.
Classification of limit tests: limits of soluble and insoluble matter; moisture; volatile matter; residual solvents; nonvolatile matter. Residue on ignition, loss on ignition, loss on drying, ash values, limit tests for metallic and non-metallic impurities, other specific limit tests

3. Is there any difference between IMPURITY and CONTAMINANT ?

4. Definition of Impurities:
Impurities in a pharmaceutical product may be defined as unwanted chemicals in the product that are not the active pharmaceutical ingredient (API) itself (or the excipients used to manufacture it), or which develop during formulation or upon aging of both API and formulation.
They are unwanted chemicals that remain within the formulation or API in small amounts and which can influence quality, safety and efficacy (QSE), thereby causing serious health hazard.

5. In general….
Impurities are undesirable elements or substances that occur commonly or naturally in a substance, thereby lowering its quality or value. Depending on its quantity, the impurity may or may not make the substance unfit for its intended use.
On the contrary, a contaminant is an external agent that is (or gets) added to something and usually renders it unfit for its intended use.

6. Sources of Impurities
Major sources of impurities may be classified broadly into two:
i) Synthesis related impurities
Raw materials employed
the manufacturing process
solvent
reaction vessel
stability of the final product which can be predicted from its chemistry i.e. degradation

7. Sources of Impurities….
ii) Formulation related impurities
Physical contamination
Improper storage conditions
Atmospheric contaminant
Microbial contamination
Particulate contamination
Filth

8. Types of Impurities
According to the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), impurities can be classified broadly into:
a) Organic impurities
b) Inorganic impurities and
c) Residual Solvents

9. I. Synthesis related impurities:
Organic Impurities:
The composition of impurities allows one to draw conclusions regarding the manufacturing of the products and its adulteration. Majority of the impurities are characteristics of the synthetic route of the manufacturing process.
Since there are several possibilities of synthesizing a
drug, it is possible that the same product of different sources may give rise to different impurities

10. Synthesis related impurities:
In the case of drugs prepared by multi-step synthesis, the number and the variety of structures of organic impurities are almost unlimited and highly dependent on the route and reaction conditions of the syntheses and several other factors such as the purity of the starting material, method of isolation, purification, conditions of storage etc.

11. 1. Starting Materials and Intermediates:
Starting materials and intermediates are the chemical building blocks used to construct the final form of a drug substance molecule. Unreacted starting materials and intermediates, particularly those involved in the last a few steps of the synthesis, can potentially survive the synthetic and purification process and appear in the final product as impurities
For example, in the synthesis of paracetamol, p-aminophenol could be a starting material or an intermediate and may appear in the final product. There is a limit test for it in bulk paracetamol:

12. Production of paracetamol from intermediate p-amino phenol:

13. i. Impurities in the Starting Materials:
Impurities present in the starting materials could be present in the final product, or follow the same reaction pathways as the starting material itself, and the reaction products could carry over to the final product as process impurities.
Knowledge of the impurities in starting materials helps to identify related impurities in the final product, and to understand the formation mechanisms of these related process impurities e.g

14. Effect of trace impurity in starting material:
Tolperisone trace impurity
Starting material trace impurity

15. ii. Degradation products during manufacturing
During manufacturing of bulk drugs degradation of end products results in the formation of impurities. For example Hydrochlorothiazide has a known degradation pathway through which it degrades to the starting material as 1,3- disulfonamide in its synthesis:

16. Dihydrochlorothiazide degradation:
Dihydrochloro thiazide degradation product

17. iii. By – products:
In synthetic organic chemistry, getting a single end – product with 100% yield is seldom. There is always a chance of having by-products.
can be formed through variety of side reactions, such as incomplete reaction, over-reaction, isomerization,
dimerization, rearrangement or unwanted reactions
between starting materials or intermediate with chemical reagents or catalysts . For example, in the case of paracetamol bulk production, diacetylated paracetamol may form as a by-product :

18. Formation of diacetylated paracetamol as a by-product:

19. iv. Organic Reagents, Ligands and Catalysts:
Chemical reagents, ligands, and catalysts used in the synthesis of a drug substance can be carried over to the final products as trace level impurities. For example, carbonic acid chloromethyl tetrahydro-pyran-4-yl ester (CCMTHP), which is used as an alkylating agent in the synthesis of a beta-lactam drug substance, was observed in the final product as an impurity
Pyridine, a catalyst used in the course of synthesis of mazipredone, reacts with an intermediate to form a pyridinium impurity

20. v. Organic Impurities originating from reaction solvents:
Impurities in the solvents can also be source of impurities.
For e.g. 2-hydroxytetrahydrofuran is an impurity in tetrahydrofuran, which is often used as the solvent of Grignard reagents
furan: 2-hydroxytetrahydrofuran:

21. B: Inorganic impurities
Inorganic impurities derive from the manufacturing process for the bulk drug, and excipients. They include the following:
1. Reagents, ligands, and catalysts
The chances of having these impurities are rare: however, in some processes, these could create a problem unless the manufacturers take proper care during production.

22. 2. Heavy metals
The main sources of heavy metals are: a) the water used in the processes and the reactors (if stainless steel reactors are used), where acidification or acid hydrolysis takes place. These heavy metal impurities can easily be avoided using demineralized water and glass-lined reactors.

23. b) Excipients: Generally, excipients may contain high levels of heavy metals such as arsenic, bismuth, cadmium, chromium, copper, iron, lead, mercury, nickel and sodium.
Sometimes they might present in the product during processing or may leach from packing material. For example, excipients such as hydrogenated oils and fats, which are produced using metal catalysts, are found to contain high concentrations of metals (platinum and palladium). This may be due to leaching from process equipment or storage container.

24. 3. Other materials (e.g., filter aids, charcoal etc.)
filters or filtering aids such as centrifuge bags are routinely used in the bulk drugs manufacturing plants and in many cases, activated carbon is also used. The regular monitoring of fibers and black particles in the bulk drugs is essential to avoid these contaminations.

25. C. Residual solvents
Residual solvents are volatile organic chemicals used during the manufacturing process or generated during production
Residual solvents are potentially undesirable substances.
either modify the properties of certain compounds or may be hazardous to human health.

26. Also affect physicochemical properties of the bulk drug substances such as crystallinity, which in turn may affect the dissolution properties, odor and colour changes in finished products
In addition, solvents used in synthesis may contain a number of impurities which can react with chemicals used in the synthesis to produce impurities

27. Water: Most commonly used solvent
Not considered as an impurity most of the time
However moisture content can be very important after packaging as moisture content may be sufficient to cause hydrolysis
Drug products may also be affected by water from environment
Hydrolysis due to presence of water causes chemical instability problems
Water can be present even in non-aqueous formulations in enough quantities to cause degradation
It can also be a major source of microbial contamination

28. II. FORMULATION RELATED IMPURITIES
DOSAGE FORM RELATED (EXCIPIENTS):
APIs are formulated with excipients (pharmaceutical aids) into solutions, tablets, capsules, semi-solids, aerosols and Novel Drug Delivery Systems.
During formulation, excipients are added to API to render the product elegant. They can be sometimes heterogeneous mixtures.
i. Excipients can be a source impurities and microbial contamination
ii. drug – excipient incompatibility may lead to undesirable products which can affect the therapeutic efficacy of the product eg: See table below

29. Effect of Pharmaceutical Aids on Stability of Active Ingredients
Pharamaceutical aid
Effect
Kanamycin
Honey, sugar syrup
Loss of activity at room
temperature (RT)
Cholecalciferol
2%polyoxy ethylene ester
surfactant, polysorbate
Change in pH resulted, degradation of active ingredient
Tetracyclines
Calcium or magnesium or metal ions
Complexation
Thiomersal
Bromine, chloride, iodide
Form different soluble halides of
cationic mercury compounds.
Adrenaline
Boric acid, povidone
Stabilization
Tryptophan
Sodium pyrosulfite, oxygen
Discoloration, precipitation.

30. In general…
Liquid dosage forms may undergo both degradation as well as microbial contamination
Water content, pH of formulation, compatibility of cations and anions, mutual interaction of ingredients and the primary container are the critical factors

31. B. METHOD RELATED IMPURITY
Eg. In production of parenteral dosage form of diclofenac Na, 1-(2,6 dichlorophenyl) indoline-2 –one is formed as an impurity when it is terminally sterilised by autoclaving. The formation of this derivative and NaOH occurs due to intramolecular cyclic reaction of diclofenac Na by autoclave conditions (1230C).

32. Diclofenac Na Indolinone derivative

33. C. Environmental related impurity
1. Temperature: Especially during formulation of vitamins and antibiotics, extreme care should be exercised to prevent them from degradation because these classes of compounds are heat liable. When subjected to extreme temperature, loss of potency takes place (for instance drying under heat)

34. 2. Light - UV light:
Light is one of the means by which the formulation degrades because of photolytic reaction. Exposure to light is known to be deleterious on a number of pharmaceutical compounds. For eg. Ergometrine injection has been reported to be unstable under tropical conditions of light and heat. Some other drugs that are affected are:

35. Drugs Affected By Light or Catalyst

36. 3. Humidity
Humidity is one of the important key factors incase of hygroscopic compounds. It is detrimental to both bulk powder and formulated solid dosage form. The classic examples are ranitidine and aspirin

37. D. Impurities on Aging (storage and transport)
Mutual interaction amongst ingredients
Because of the labile nature of vitamins, they undergo degradation in different dosage forms, especially liquid
degradation of vitamins such as folic acid, thiamine and cyanocobalamines does not yield toxic impurities but they lose their potency well below compendial specifications
An example of mutual interaction : presence of nicotinamide in formulation containing four vitamins (nicotinamide, pyridoxine, riboflavin and thiamine) causes the degradation of thiamine to a substandard level within a one year shelf life of vitamin–B complex injection .

38. 2. Instability of the Product
Chemical instability
Impurities can also arise during storage because of chemical instability of the pharmaceutical substance. Many pharmaceutically important substances undergo chemical decomposition when storage conditions are inadequate.
often catalyzed by light, traces of acid or alkali, traces of metallic impurities, air oxidation, carbon dioxide and water vapours (humidity)
The nature of the decomposition can easily be predicted from the knowledge of chemical properties of the substance(s)
All such decompositions can be minimized or avoided by using proper storage procedures and conditions
The photosensitive substances should be protected from light by storing them in darkened glass or metal containers thereby inhibiting photochemical decomposition.
Materials susceptible to oxidation by air or attack by moisture should be stored in sealed containers
if necessary the air from the containers can be displaced by an inert gas such as Nitrogen. Oxidation can also be prevented by adding suitable antioxidants

39. Types of degradation
Oxidation
Drugs which are prone to oxidation are those that contain OH groups directly bonded to aromatic rings eg. catechols, conjugated-dienes, heterocyclic aromatic rings, nitroso and nitrite derivatives e.g. Hydrocortisone, methotrexate, adinazolam, catecholamine, (Vitamin–A) etc.
In pharmaceuticals, the most common form of oxidative decomposition is auto oxidation through a free radical chain process commonly catalysed by metals.
For example, auto-oxidation of ascorbic acid studies reveals that cupric ion oxidises ascorbic acid rapidly to dehydroascorbic acid and potassium cyanide. As a result, there is a cleavage of chain due to the formation of copper complexes.

40. ii. Hydrolysis
A reaction in which water is the reactant causing precipitation.
Most well-known examples of such reactions in pharmaceutical compounds are esters and amides
Many drugs are derivatives of carboxylic acids or contain functional groups based on that moiety example esters, amides, lactones, lactams, imides and carbamates, others which are susceptible to acid-base hydrolysis include aspirin, atropine, chloramphenicol, barbiturates, chlordiazepoxide, oxazepam and lincomycin etc.

41. iii. Decarboxylation
Some of the carboxylic acids such as p-amino salicylic acid have shown loss of carbon dioxide from carboxyl group when heated
For instance, photo reaction of rufloxacin tablet enteric coated with cellulose acetate phthalate (CAP) and sub-coating with calcium carbonate cause hydrolysis of CAP liberating acetic acid, which on reacting with calcium carbonate produced carbon dioxide, a by-product that blew off the cap from the bottle after cap was loosened .

42. iv. Photolysis
Photolytic cleavage on aging includes examples of pharmaceutical drugs or products that are prone to degradation on exposure to UV-light
During manufacturing process as solid or solution, packaging or on storage, drugs like ergometrine, nifedipine, nitroprusside, riboflavin and phenothiazines are liable to photo oxidation
involves generation of free radical intermediate, which will degrade the products
For example, exposure of ciprofloxacin eye drop 0.3% to UV light induces photolysis thereby resulting in the formation of ethylene di-amine analogue of ciprofloxacin

43. b. Physical instability
Pharmaceuticals may undergo changes in physical properties during storage. There can be changes in crystal size and shape, sedimentation, agglomeration and caking of the suspended particles.
These physical changes are not always avoidable and may result in significant changes in the physical appearance, pharmaceutical and therapeutic effects of the product.
Particle size and consequently surface area is a critical factor in determining the bioavailability of the low solubility drug such as griseofulvin.
Physical changes such as sedimentation and caking in case of multidose suspension may constitute hazard leading to the possibility of under dosage and later to overdosage of the drugs.
Similarly increase in the globule size of the injectable emulsions on storage may lead to fat embolism.

44. E) Packaging material
Impurities result also from packaging materials i.e., containers and closures
For most drugs the reactive species for impurities consists of:
Water – hydrolysis of active ingredient.
Small electrophiles – Aldehydes and carboxylic acid derivatives.
Peroxides – oxidize some drugs.
Metals – catalyze oxidation of drugs and their degradation pathway.
Extractable or leachables – Emerge from glass, rubber stoppers and plastic materials, in which oxides like NO, SiO, CaO, MgO are major components leached or extracted from glass.
Some examples of synthetic materials include styrene from polystyrene, diethylhexylpthalate (DEHP) plasticizer in PVC, dioctyltin iso octyl mercaptoacetate stabilizer for PVC, zinc stearate stabilizer in PVC and polypropylene, bisphenol A from plastics etc.

45. Enantiomeric Impurities:
The majority of therapeutic chiral drugs used as pure enantiomers are natural products. The high level of enantioselectivity of their biosyntheses excludes the possibility of the presence of enantiomeric impurities
In the case of synthetic chiral drugs, the racemate of which is usually marketed, if the pure enantiomer is administered, the antipode is considered to be an impurity. The reason for its presence can be either
a) the incomplete enantioselectivity of the syntheses or b) incomplete resolution of the enantiomers of the racemate
Although the ICH guideline excludes enantiomeric impurities, pharmacopoeias consider them as ordinary impurities

46. A single enantiomeric form of chiral drug is now considered as an improved chemical entity that may offer a better pharmacological profile and an increased therapeutic index with a more favourable adverse reaction profile than the racemic mixture; and a lower dose
However, the pharmacokinetic profile of levofloxacin (S- Isomeric form) and ofloxacin (R-isomeric form) are comparable, suggesting the lack of advantages of single isomer in this regard
Typical examples of drugs containing enantiomeric impurities:
a) Dexchlorophenarmine maleate (R enantiomer impurity allowed NMT 0.5%)
b) Timolol maleate (R enantiomer impurity allowed NMT 1%)
c) Clopidogrel sulphate (R enantiomer impurity allowed NMT 1%)
In general, an individual API may contain all of the above-mentioned types of organic impurities at levels varying from negligible to significant

47. Pharmacopoeial Norms for the Enantiomeric Impurities:
Many medicinal substances that contain one or more chiral centres and that are already in the market have been made available for pharmaceutical use as racemic mixtures with little known about the biological activities of the separate isomers and this is reflected in the monograph in the pharmacopoeia.
Nevertheless, with increasing concern by regulatory authorities for substances to be made available as single isomers, tests for enantiomeric composition will become more common
As a result the following recommendations have been made by the BP 2001:

48. Chemical definition in monographs
1) -In the case of substances containing a single chiral centre, the descriptor ‘(RS)’ should be included at the appropriate position in the chemical definition of the substances to indicate a racemic mixture. 2) - For substances containing multiple chiral centres and comprising mixture of all possible stereomers the term ‘all-rec’ should be used, for example Iso-aminile. In those few substances existing as diastereomeric mixtures, that is where in one or more centres the stereochemistry is explicit but in other centres it is not, each centre is defined either as the specific (R)- or (S) – configuration , or as racemic (RS)-, respectively.

49. In future…
Tests:
1) - when a monograph describes an enantiomer, it will include both a test for specific optical rotation under identification and a test using methods such as chiral chromatography, to control enantiomeric purity.
2) - When only the racemic mixture is available, the monograph for the racemic mixture will simply specify a test for angle of rotation.

50. LIMIT TESTS

51. OUTLINE
Limit tests and factors considered in their design, negative and comparison tests.
Classification of limit tests: limits of soluble and insoluble matter; moisture; volatile matter; residual solvents; nonvolatile matter. Residue on ignition, loss on ignition, loss on drying, ash values, limit tests for metallic and non-metallic impurities, other specific limit tests

52. Limit Tests
Limit tests are quantitative or semi-quantitative tests designed to identify and control small amount of impurities that are most likely to be present in a pharmaceutical substance i.e. they assume no gross contamination, that GMP has been followed.
They involve simple comparisons of opalescence, turbidity or colour produced in test with that of fixed standards.
Since the amount of any single impurity present in an official substance is usually small, the normal visible-reaction-response to any test for that impurity is also quite small.
it is therefore necessary and important to design the individual test in such a manner as to avoid possible errors in the hands of various analysts. It may be achieved by taking into consideration the following factors:

53. Factors considered in the design of Limit Tests:
GENERAL FACTORS:
(a) Specificity of the Tests :
A test employed as a limit test should imply some sort of selective reaction with the trace impurity. (However, it has been observed that a less specific test which limits a number of possible impurities rather instantly has a positive edge over the highly specific tests which limits only one impurity.
Example : Contamination with Pb2+ and other heavy metal impurities in Alum: these are precipitated by thioacetamide as their respective sulphides at pH 3.5.

54. (b) Sensitivity : The extent of sensitivity stipulated in a limit test varies widely as per the standard laid down by a pharmacopoeia. The sensitivity is governed by a number of variable factors having a common objective to yield reproducible results, for instance : (i) Gravimetric Analysis : The precipitation is guided by the concentration of the solute and of the precipitating reagent, reaction time, reaction temperature and the nature and amount of other substance(s) present in solution. (ii) Colour Tests : The production of visible and distinct colouration may be achieved by ascertaining the requisite quantities of reagents and reactants, time period and above all the stability of the colour produced.

55. (c) Personal Errors : personal errors must be avoided as far as possible such as: (i) Physical Impairment : A person suffering from colour blindness may not be in a position to assess colour-changes precisely ; or if he uses bifocals he may not take the burette readings accurately. (ii) Learning-Curve Syndrome : An analyst must practise a new assay method employing ‘known’ samples before making an attempt to tackle an unknown sample, thereby minimising the scope of personal errors.

56. SPECIFIC FACTORS
For Known Impurities:
For known impurities, several aspects are taken into account in designing the test. These include the nature of the impurity, its toxicity and the levels likely to be found in routine production. Analytical considerations such as the response factor for the impurity and practical issues such as availability of the impurity as a reference material or reagent also influence the test design

57. If a major and/or toxic impurity in a material is known to have a significantly different response (more than ±20%) from that of the substance being examined in the conditions of the test, the preferred manner of limiting this impurity is to use a reference substance of the impurity.
If this is not possible, a reference solution of the substance being examined containing a known amount of the impurity may be used. When neither of these approaches is possible, a dilution of the solution of the substance being examined may be used as a reference solution. This approach is also commonly used in tests where an impurity that is known (but not named within the test) has a response within ±20% of that of the substance being examined.

58. The response factor (k) is a relative term, being the response of equal weights of one substance relative to that of another in the conditions described in the test.
In the context of a related substances test where a response factor is quoted for an impurity, unless otherwise stated, this is the expected response for that impurity in relation to a response of unity for the substance being examined. The way in which a response factor is to be used in any subsequent calculation is stated in the monograph.
Response factors of less than 0.2 or more than 5 are not used. If the difference between the response of an impurity and that of the substance being examined is outside these limits, a different method of determination, such as a different detection wavelength (λ) or a different method of visualisation, is used.

59. For Unknown Impurities:
Unknown impurities may be limited by reference to a dilution of the solution of the substance being examined used as a reference solution together with an open design of statement limiting 'any' or 'any other' secondary peak or spot. Such a reference solution may be used in addition to those containing named impurities (any other secondary peak/spot) or, in some simple tests, control of unknown and known (but unnamed) impurities may be exerted by means of a comparison between the sample solution and a dilution of this solution (any secondary peak/spot).

60. For Formulated Preparations
Many monographs for formulated preparations in the British Pharmacopoeia also include tests for impurities. In general, wherever possible a test for impurities based on that in the monograph for the active ingredient is included with any necessary modification.
Wider limits and/or additional controls may be required for impurities arising on manufacture or storage of the dosage form.
Tests for impurities in monographs for formulated preparation are used to control not only degradation products but also by-products of the synthetic route used for manufacture of the active ingredient. It has been argued, for example in the ICH guideline Q3B, that by-products of synthesis have been controlled already during examination of the substance before formulation and that further testing for these impurities is unnecessary. Clearly it would be repetitious and wasteful of resources for tests, often complex in nature, to be repeated routinely simply to demonstrate acceptably low levels of impurities that could arise only during synthesis (as opposed to degradation) of the active ingredient.

61. However, this information is available only to those who know the detailed attributes of the active raw material that has been used. For an analyst who has access only to the dosage form, the profile of synthesis-related impurities offers one means of establishing whether or not the dosage form has been prepared from an active ingredient of pharmacopoeial quality. It is for this reason that such tests are included in British Pharmacopoeia monographs for formulated preparations

62. Examples:
i) Acetylsalicylic acid: Limit tests for salicylic acid (degradation product) are specified in both the active ingredient and the tablets
aspirin salicylic acid

63. ii) Chloramphenicol:
degradation product
Limit test for the degradation product is specified only for the capsules

64. iii) for metronidazole:
limit test for the degradation product is only specified for the tablets but not the active ingredient:
Metronidazole methyl-5-nitroimidazole

65. Limit tests involve either “negative” or “comparison” tests:
Negative test: refers to absence of spot
Comparison test: comparison with the response due to a known concentration/quantity of reference impurity

66. Limit Tests for Related Substances
It is usual to include a test for related substances in a monograph for a medicinal substance. These may be manufacturing impurities (intermediates or by-products) or degradation products or both.
When preparation of a monograph is initiated the manufacturer is asked to provide information concerning:
a) the nature of such impurities,
b) the reason for their presence,
c) the amounts that may be encountered in material prepared under conditions of good pharmaceutical manufacturing practice
d) the manner in which proportions may vary on storage,
e) an indication of the toxicity of any impurities in relation to that of the substance itself.
This is called an IMPURITY PROFILE which involves detection of the impurities using chromatography - either TLC, HPLC or GC (MS)

67. Where there is only one manufacturer of a substance, pharmacopoeia limits are set in the knowledge that the level of impurities in production batches of the substance will have been accepted by the registration authority based on the toxicity studies and clinical trials carried out before the granting of a licence.
Such studies and trials will have been carried out on material with an impurity profile that is qualitatively and quantitatively similar to that of subsequent production batches.

68. Any subsequent changes to the manufacturing process by the original manufacturer or the introduction of material from another manufacturer utilising a different route of synthesis will be subject to the need to demonstrate essential similarity or to provide equivalent data to the relevant registration authority.
In some cases a change in production or source may give rise to impurities that are not adequately controlled by the published pharmacopoeial monograph. Appropriate revision of the monograph will be carried out provided that the pharmacopoeial authority is notified of the need and that it is supplied with the relevant information

69. VARIOUS TYPES OF TESTS FOR QUANTITATIVE DETERMINATIONS
In actual practice, it has been observed that different official compendia describe a number of detailed types of tests with a view to obtain a constant and regular check that might be possible to maintain the desired degree of optimum purity both in the pure pharmaceutical substances and the respective dosage-forms made therefrom.

70. A number of such tests include
Limits of soluble matters
Limits of insoluble matters
Limit of moisture, volatile matter and residual solvent
Limit of non-volatile matter, residue on ignition and loss of ignition
Limit of ash values e.g acid insoluble ash, water soluble ash, water soluble extractives and sulphated ash
Limit test for acid radical impurities
Limit test for metallic impurities etc.

71. Limits of soluble matter
In order to detect the presence of some very specific impurities normally present in the official substances, the limits of soluble impurities have been laid down in different pharmacopoeias.
It is applied to limit soluble impurities which are completely insoluble in a particular solvent .
Some typical examples include
Water soluble barium salts are highly toxic and therefore, strictly excluded from barium sulphate used for X-ray work.
Limit of matter soluble in dilute HCl is applied to both light and heavy kaolin by refluxing with dilute HCl, filtering and evaporating the filtrate. NMT 0.5%

72. Limits of insoluble matter
The tests for clarity of solution offer a means of limiting insoluble impurities in an official preparation.
Clarity of solutions for injection is important to ensure complete freedom from particulate matter.
A special requirement for all injections and water for injection is that the solution must be free from insoluble matter on viewing against a black background in upward, horizontal and inverted position.
Another example of limit test of insoluble matter is the control of phenylbarbituric acid in phenobarbitone by the requirement that 1 g shall be completely soluble in 5 ml of boiling ethanol (90 %) within 3 minutes.

73. Limits of Moisture, Volatile Matter and Residual Solvents
A good number of pharmaceutical substances usually absorb moisture on storage thereby causing deterioration. Such an anomaly can be safely restricted and limited by imposing an essential requirement for the loss in weight (Loss on Drying) when the pharmaceutical chemical is subjected to drying under specified conditions.
The quantum of heat that may be applied to the substance varies widely as per the following norms :
(a) Nature of the substance
(b) Decomposition characteristics of the substance.
Various official compendia recommended different temperatures and duration of drying either at atmospheric or reduced pressure (vacuum). A few typical examples are stated below :

75. There are four types of hydrates which may be observed amongst the pharmaceutical chemicals, namely :
Inorganic Salt Hydrates e.g., Magnesium Sulphate (MgSO4.7H2O) ; Sodium Sulphate (Na2SO4.10 H2O).
Salts of Inorganic Cations and Organic Acids e.g., Calcium Lactate, Ferrous Gluconate.
Organic Hyrates e.g., Caffeine Hydrate, Theophylline Hydrate.
Organic Substances e.g., Acacia, Hydroxymethyl Cellulose.

77. Aquametry:It refers to the determination of water content titrimetrically with Karl Fischer Reagent (KFR). This technique has been used exclusively for the determination of water content in a number of pharmaceutical substances listed below:

78. Since the introduction of Gas-Liquid-Chromatography (GLC) as an essential analytical tool, it has been judiciously exploited as an useful alternative to KFR for not only determining water content in pharmaceutical chemicals but also limiting specific volatile substances present in them: eg:
For Determination of Water Content for Gonadorelin : (Limit NMT : 7.0 % w/v)
For Limiting Specific Volatile Substance for Orciprenaline Sulphate : (Limit of Water and Methanol : 6.0% w/w)

79. Limits of Non-Volatile Matter
Pharmaceutical chemicals belonging to the domain of inorganic as well as organic substances contain readily volatile matter for which the various official compendia prescribe limits of non-volatile matter. It is pertinent to mention here that the Pharmacopoeia usually makes a clear distinction between substances that are readily volatile and substances that are volatile upon strong ignition, for instance :
(a) Readily Volatile : e.g., Organic Substances—alcohol (95% v/v), isopropyl alcohol, chloroform, halothane, anaesthetic ether, chlorocresol and trichloroethylene ; and Inorganic substances—ammonia solution, hydrogen peroxide solution, water for injection.
(b) Volatile Upon Strong Ignition : e.g., hydrous wool fat (lanolin).

80. Limits of Residue on Ignition
the limits of residue on ignition are basically applicable to the following two categories of pharmaceutical substances, namely :
(a) Those which are completely volatile when ignited e.g., Hg.
(b) Those which undergo total decomposition thereby leaving a residue with a definite composition e.g., calamine—a basic zinc carbonate that gives rise to ZnO as the residue.
According to BP, 68.0 to 74.0% when ignited at a temperature not lower than 900°C until, after further ignition, two successive weighings do not differ, by more than 0.2% of the weight of the residue.

81. Limits of Loss on Ignition

82. Limits on Ash Value
The ash values usually represent the inorganic residue present in official herbal drugs and pharmaceutical substances. These values are categorized into four heads, namely :
(a) Ash Value (Total Ash),
(b) Acid-Insoluble Ash,
(c) Sulphated Ash, and
(d) Water-Soluble Ash.

83. Ash Value (Total Ash)
Ash value normally designates the presence of inorganic salts e.g., calcium oxalate found naturally in the drug, as well as inorganic matter derived from external sources. The official ash values are of prime importance in examination of the purity of powdered drugs as enumerated below : (i) To detect and check adulteration with exhausted drugs e.g., ginger. (ii) To detect and check absence of other parts of the plant e.g., cardamom fruit. (iii) To detect and check adulteration with material containing either starch or stone cells that would modify the ash values. (iv) To ensure the absence of an abnormal proportion of extraneous mineral matter incorporated accidentally or due to follow up treatment or due to modus operandi at the time of collection e.g., soil, floor sweepings and sand.

84. The most common procedure recommended for crude drugs is described below :
Procedure : Incinerate 2 to 3 g of the ground drug in a tared platinum or silica dish at a temperature not exceeding 450°C until free from carbon. Cool and weigh. If a carbon-free ash cannot be obtained in this way, exhaust the charred mass with hot water (DW), collect the residue on an ashless filter paper, incinerate the residue and filter paper, add the filtrate, evaporate to dryness and ignite at a temperature not exceeding 450°C.
Calculate the percentage of ash with reference to the air-dried drug.

86. Acid-Insoluble Ash
The method described above for ‘total ash’ present in crude drugs containing calcium oxalate has certain serious anomalies, namely : • Offers variable results upon ashing based on the conditions of ignition. • Does not detect soil present in the drug efficaciously. • The limits of excess of soil in the drug are not quite definite. Hence, the treatment of the ‘total ash’ with acid virtually leaves silica exclusively and thus comparatively forms a better test to detect and limit excess of soil in the drug than does the ash. The common procedure usually adopted for the determination of ‘acid insoluble ash’ is given below :

87. Procedure : Place the ash, as described earlier, in a crucible, add 15 ml DW and l0 ml hydrochloric acid (~–11.5 N), cover with a watch-glass, boil for 10 minutes and allow to cool. Collect the insoluble matter on an ashless filtre paper, wash with hot DW until the filtrate is neutral, dry, ignite to dull redness, allow to cool in a desiccator and weigh. Repeat until the difference between two successive weighings is not more than l mg.
Calculate the percentage of acid-insoluble ash with reference to the air-dried drug.

88. A few typical examples are listed below :

89. Sulphated Ash
The estimation of ‘sulphated ash’ is broadly employed in the case of :
(a) Unorganized drugs e.g., colophony, podophyllum resin, wool alcohols, wool fat and hydrous wool fat.
(b) Pharmaceutical substances containing inorganic impurities e.g.,
Natural Origin : Spray-dried acacia, Frangula Bark, Activated Charcoal
Organic Substances : Cephalexin, Lignocaine hydrochloride, Griseofulvin, Diazoxide, Medazapam, Saccharin.
Inorganic Substances : Ammonium chloride, Hydroxy urea.

90. Procedure : Heat a silica or platimum crucible to redness for 30 minutes, allow to cool in a desiccator and weigh. Place a suitable quantity of the substance being examined, accurately weighed in the crucible, add 2 ml of 1 M sulphuric acid and heat, first on a waterbath and then cautiously over a flame to about 600°C. Continue heating until all black particles have disappeared and then allow to cool. Add a few drops of 1 M sulphuric acid, heat to ignition as before and allow to cool. Add a few drops of a 16% solution of ammonium carbonate, evaporate to dryness and cautiously ignite. Cool, weigh, ignite for 15 minutes and repeat the procedure to constant weight.

92. Water-Soluble Ash
Water-soluble ash is specifically useful in detecting such samples which have been extracted with water.
A detailed procedure as per the official compendium is enumerated below :
Procedure : The ash as described earlier, is boiled for 5 minutes with 25 ml DW, collect the insoluble matter in a sintered-glass crucible or on an ashless filter paper, wash with hot DW and ignite for 15 minutes at a temperature not exceeding 450°. Subtract the weight of the residue thus obtained from the weight of the ash.The difference in weight represents the water-soluble ash. Now, calculate the percentage of water-soluble ash with reference to the air-dried drug.
A typical example of an official drug is that of ‘Ginger’, the water-soluble ash of which is found to be not more than 6.0%.

93. Limit test for acid radical impurities
Limit test for metallic impurities
(REFER TO NOTES)