Pharmacopoeias have played a crucial role in standardizing drug quality, safety, and efficacy. The history of pharmacopoeia dates back to ancient civilizations, with early records in Greek, Roman, and Arabic texts. Over time, national and international pharmacopoeias have established guidelines for pharmaceutical substances and preparations.

Pharmaceutical substances often contain impurities arising from raw materials, manufacturing processes, storage, or degradation. These impurities can be classified as organic, inorganic, and residual solvents, impacting drug quality and patient safety. Limit tests are essential quality control procedures designed to detect and quantify specific impurities within permissible limits.

The principles of limit tests for chloride, sulphate, iron, arsenic, lead, and heavy metals rely on precipitation, color formation, or complexation reactions that produce visible changes for comparison with standard solutions. Modified limit tests for chloride and sulphate involve variations in reagent concentration and reaction conditions to improve sensitivity and specificity. These tests ensure compliance with pharmacopoeial standards, maintaining drug purity and safety.

KEY WORDS: Pharmacopoeia, Impurities, limit test 1.1 INTRODUCTION The word Pharmacopoeia is derived from Greek words ‘pharmakon’ means a drug (both remedy and poison) and ‘poiein’ means to make or create. It is a legal and official book issued by recognized authorities usually appointed by Government of each country. It comprises list of pharmaceutical substances, formulae along with their description and standards.

This book

contains directions for the identification of samples and the preparation of compound medicines. The descriptions of individual drug/excipients or product is known as Monograph. 1.2 Importance

• To maintain the uniformity in standards as well as to control the adulterated medicines.
• It is important element to control the medicines licencing and inspection process.
• Play a significant role in generic drug manufacturing, contract research, production for export purpose.
• It is updated or amended time to time in the form of supplement or new edition where monographs of new drugs are added as well as outdated are deleted. 1.3 History and background Overwhelming empirical knowledge of mankind gained during centuries and constant effort to establish better health care possibilities have led to the creation of a list of origin, preparation and healing properties of medicines. The term Pharmacopoeia first appears as a distinct title in a work published in Basel, Switzerland in 1561 by Dr A. Foes, but does not appear to have come into general use until the beginning of the 17th century. Today’s pharmacopoeias focus mainly on assurance of quality of products by various tools of analytical

sciences. The aim to achieve a wide global harmonization of quality specifications for selected pharmaceutical products, excipients and dosage forms came with increased globalization and reciprocal collaboration. History of these approaches goes back to 1902–1925 when agreements established a ″Unified″ Pharmacopoeia.

In 1929 the "Brussels Agreement" stipulated the League of Nations to carry out related administrative functions. Eight years later, in 1937, the first meeting of the ″Technical Commission of Pharmaceutical Experts″ was held. An important date in the history of quality assurance of medicines is 1948, when the First World Health Assembly (WHA) approved the Expert Committee on Unification of Pharmacopoeias to continue this work.

One year later, the WHA renamed it the Expert Committee on International Pharmacopoeia . 1.4 List of Pharmacopoeias: a) Indian Pharmacopoeia b) British Pharmacopoeia c) United State Pharmacopoeia d) European Pharmacopoeia e) Chinese Pharmacopoeia f) Japanese Pharmacopoeia g) Ayurvedic Pharmacopoeia 1.4.1 Indian Pharmacopoeia

• First official Pharmacopoeia of India appeared in 1868 which was edited by Edward John Waring.
• In pre-independence days, British Pharmacopoeia was used in India.
• The colonial addendum of BP 1898 was published in 1900 appeared as Government of India edition in 1901.
• In 1946 Government of India issued one list known as “The Indian Pharmacopoeial list”
• Committee under chairmanship of Sir R. N. Chopra along with other nine members prepared “The Indian Pharmacopoeial list”
• It was prepared by Dept. of Health, Govt. of India, Delhi in 1946.
• In 1948 Government of India appointed an Indian Pharmacopoeia committee for preparing “Pharmacopoeia of India”. Tenure of this committee was five years.
• Indian Pharmacopoeia committee under chairmanship of Dr. B. N. Ghosh Published first edition of IP in 1955. First edition of IP was published in 1955 , followed by supplement in 1960. Second edition of IP was published in 1966 followed by supplement in 1975. Third edition of IP was published in 1985 with two volumes & nine appendices.
• 261 new monographs have been added.
• 450 monographs were deleted.
• Addendum I to IP was published in 1989 were 46 new monographs added and 126 amended.
• Addendum II was published in 1991 were 62 new monographs added and 110 amended. Fourth edition of IP was published in 1996 under the chairmanship of Dr. Nityanand.
• It has been made effective from 1st December 1996.
• It covered 1149 monographs and 123 appendices.
• It includes 294 new monographs & 110 monographs have been deleted.
• Addendum I have been made effective from 31st December 2000 were 42 new monographs have been added.
• Addendum II has been made effective from 30th June 2003 were 19 new monographs have been added.
• The veterinary supplement to IP 1996 contains 208 monographs & four appendices. Fifth edition of IP was published in 2007 & addendum to this edition was published in 2008.
• IP 2007 is presented in Three Volumes.
• Volume One contains general notices & general chapters.
• Volume Two & Three contains general monographs on drug substances, dosage forms & Pharmaceutical aids. Sixth edition of IP is published in 2010 .
• The 6th edition of the Indian Pharmacopoeia 2010 is published by the Indian Pharmacopoeia Commission (IPC) Ghaziabad in accordance with a plan and completed through the untiring efforts of its members, Secretariat and Laboratory over a period of about two years.
• It supersedes the 2007 edition but any monograph of the earlier edition that does not figure in this edition.
• This edition would be effective from 1st September, 2010.
• The Indian Pharmacopoeia 2010 is presented in three volumes.
• Volume I contain the Notices, Preface, the Structure of the IPC, Acknowledgements, Introduction, and the General Chapters.
• Volume II contains the General Notice, General Monographs on Dosage Forms and Monographs on drug substances, dosage forms and pharmaceutical aids (A to M).
• Volume III contains Monographs on drug substances, dosage forms and pharmaceutical aids (N to Z).
• Followed by Monographs on Vaccines and Immunosera for Human use, Herbs and Herbal products, Blood and blood- related products, Biotechnology products and Veterinary products.
• The scope of the Pharmacopoeia has been extended to include products of biotechnology, indigenous herbs and herbal products, veterinary vaccines and additional antiretroviral drugs and formulations, inclusive of commonly used fixed-dose combinations. Standards for new drugs and drugs used under National Health Programmes are added and the drugs as well as their formulations not in use now a day are omitted from this edition.
• The number of monographs of Excipients, Anticancer drugs, Herbal products and antiretroviral drugs has been increased in this edition.
• Monographs of Vaccines and Immunosera are also upgraded in view of development of latest technology in the field.
• A new chapter on Liposomal products and a monograph of Liposomal Amphotericin B injection is an added advantage in view of latest technology adopted for drug delivery.
• A chapter on NMR is incorporated in Appendices.
• The chapter on microbial contamination is also updated to a great extent to harmonise with prevailing international requirements. Seventh edition of IP published in 2014 .
• The seventh edition of the Indian Pharmacopoeia is published by the Indian Pharmacopoeia Commission (IPC) on behalf of the Government of India, Ministry of Health & Family Welfare.
• The Indian Pharmacopoeia 2014 is presented in four volumes. The scope of the Pharmacopoeia has been extended to include additional anticancer drugs & antiretroviral drugs and formulations, products of biotechnology, indigenous herbs and herbal products, veterinary vaccines.
• The IP 2014 incorporates 2550 monographs of drugs out of which 577 are new monographs consisting of APIs, excipients, dosage forms and herbal products etc.
• A list of 577 New Monographs not included in IP-2010 and its Addendum-2012 but added in this edition containing 313 New Monographs on drug substances, Dosage forms & Pharmaceutical aids (A to Z), 43 New Drugs Substances Monographs, 10 Antibiotic Monographs, 31 Herbal Monographs, 05 Vaccines & immunosera for human use, 06 Insulin Products, 07 Biotechnology Products etc. along with the 19 new General Chapters.
• 19 New Radiopharmaceutical Monographs & 1 General chapter is first time being included in this edition. Eighth edition of IP was published in 2018 with DVD.
• Four volumes
• 3060 monographs
• Allergen monograph incorporated
• Addendum in 2019 and 2021 Ninth edition of IP was published in 2022 .
• Four volumes
• 3152 monographs
• Phytopharmaceuticals monograph incorporated
• General chapters on elemental impurities and nitrosamines 1.4.2 British Pharmacopoeia First edition of BP was published in 1864 & consist of two sections: Part I: Materia Medica Part II: Preparation & compounds Second edition of BP was published in 1867 . Third edition of BP was published in 1885 . Fourth edition of BP was published in 1898 . Fifth edition of BP was published in 1914 . Sixth edition of BP was published in 1932 , in which diagnostic materials were added and standards for antitoxins as well as insulin were also included. Seventh edition of BP was published in 1948 , carrying the generic names of substances which are newly enlisted in the world of medicines. Several analytical

methods, tablet disintegration tests as well as sterilization techniques for parenteral preparations were expanded. Various novel monographs regarding the penicillins and sex hormones were also introduced. Eighth edition of BP was published in 1953 , where the Titles of drugs & preparations were in English instead of Latin and metric system.

It has been published annually. Ninth edition of BP was published in 1958 , having 160 newer monographs as well as tranquilising drugs and spectrophotometric analysis were enlisted. Tenth edition appeared in 1963 .

Thirteenth edition was published in 1980 . In BP 2007 monographs has been introduced for material specifically used in preparation of Traditional Chinese Medicines. Term “Prolonged release” has been replaced the term “Slow” and the term “Gastro-resistant” has been replaced with “Enteric coated” in number of monographs.

BP 2008 contains approximately 3100 monographs for substances, preparations and articles used in practice. It has been made effective from 1 st January 2008. BP 2007-2009 were given in 06 Volumes i.e.

Vol. I to Vol. VI.

• Volume I & II: Contains medicinal substances.
• Volume III: Contains formulated preparations, blood related products, immunological products, radiopharmaceutical preparations, surgical materials & homoeopathic preparations.
• Volume IV: Contains supplementary chapters, IR spectra etc.
• Volume V: Contains veterinary products.
• Volume VI: Contains CD ROM version. BP 2010 The Stationery Office, on behalf of BP Secretariat, part of the Medicines and Healthcare products Regulatory Agency (MHRA), has recently published the BP 2010. BP is the official collection of standards for UK medicinal products and pharmaceutical substances. Published annually, the BP contains monographs for pharmaceutical substances, formulated preparations and other articles used in the practice of medicine. The standards in the BP 2010 are legally effective in the UK from 1 January 2010. BP has been providing authoritative, official standards for pharmaceutical substances and medicinal products since 1864. It is used in almost 100 countries worldwide and remains an essential reference for any individual or organization working within pharmaceutical research and

development, manufacturing and testing across the globe. BP 2010 has 40 monographs for formulated preparations, including veterinary medicines and additional standards for widely used unlicensed formulations. All European Pharmacopoeia 6th edition material upto and including Supplement 6.5 is integrated into the text of the BP 2010.

BP supports regulatory work in the fields of herbal and complementary medicines by providing additional new and revised monographs for herbal medicinal products and for homeopathic stocks and mother tinctures. Print edition of BP 2010 comprises 4 volumes of BP 2010 and a single volume of BP (Veterinary) 2010. The BP 2013 package includes,

• 06 volume printed edition including the BP (Veterinary) 2013
• 41 new BP monographs
• 40 new European Pharmacopoeia monographs
• 619 amended monographs
• 6 new and 1 amended Infrared Reference Spectra BP 2013 The 2014 edition includes almost 3500 monographs which are legally enforced by the Human Medicines Regulations 2012. The BP 2014 package comprises 5 volumes of BP 2014 and a single volume of BP (Veterinary) 2014, along with a fully searchable CD ROM and online access to provide you with flexible resources. Legally effective from 1 January 2014, 40 new, 272 amended and 4 new BP (Vet) monographs. 03 new Supplementary Chapters and 01 new BP (Vet) Supplementary Chapter BP 2018
• 35 new monographs
• 185 amended BP monographs
• 04 new monographs for unlicensed formulations
• 04 new monographs for herbal medicines
• 06 new monographs for veterinary medicines 1.4.3 United State Pharmacopoeia
• First edition of United state Pharmacopeia was published on 15 th December 1820 in both Latin & English.
• From 1820 to 1942 it was published at Ten years intervals.
• From 1942 to 2000 it was published at Five years intervals.
• From 2002 it was published annually.
• First National Formulary of the united states appeared in 1888. USP21-NF16 have eight supplements.
• First appeared in January 1985 & last in November 1988. USP22-NF17 , 1990 is the third revision that consolidates USP & NF into a single volume.
• Electronic version of USP-NF on floppy disks was introduced in 1992. USP23-NF18 , was published in Mumbai as an Asian edition at the end of 1994 .
• USP23 has ten supplements.
• First supplement was published in January 1995 & Last in May 1999. USP24-NF19 , appeared from first January 2000. USP30-NF25 , appeared from May 2007.
• It contains Scientific standards for drugs, dietary substances, biological products & Excipients used in dosage forms.
• It contains 4,100 monographs and 200 general chapters.
• It has been printed in three volume set.
• Volume I contains general chapters & Volume II & III contains monographs.
• First supplement to USP30-NF25, appeared from August 2007 & second supplement from November 2007 which will be considered official from May 2008.
• From 2006, Spanish edition of USP is also being published.
• Current edition of USP 2014 is in process.
• United states pharmacopoeia 30 – national formulary 25 Highlights include: ✔ New heavier paper stock ✔ Complete table of contents and index in each volume ✔ Special 'Using the New USP-NF Print' tutorial CD ✔ Convenient slipcase for easy access and storage (English edition only). United states pharmacopoeia 31 - National Formulary 26 The USP-NF is a single-volume combination of two official compendia, the United States Pharmacopeia (USP) and the National Formulary (NF). Monographs for drug substances and preparations are featured in the USP, with monographs for dietary supplements and ingredients appearing in a separate section of the USP. Excipient monographs are included in the NF. United States Pharmacopoeia 32 - National Formulary 27 the USP 32-nf 27 contains:
• More than 4,200 monographs
• Includes over 200 general chapters, covering general tests and assays
• Displays helpful guides and charts that make it easy to find focus-specific information
• Includes information on emerging areas of science and medicine
• Helps ensure compliance with official standards
• Enables validation of test results against proven benchmarks
• Creates in-house standards for operating procedures and specifications
• Expedites new product development and approvals. United States Pharmacopoeia 33 - national formulary 28: the USP 33-NF 28 contains:
• More than 4,400 monographs
• Over 200 general chapters covering general tests and assays
• A new, easy-to-read format and monograph layout
• Helpful guides and charts that make it easy to find focus-specific information
• Ensures compliance with official standards
• Establishes in-house standard operating procedures and specifications
• Facilitates new product development and approval United States Pharmacopeia 34 - National Formulary 29: USP 34- NF 29 features more than 4,500 monographs for drug substances, dosage forms, excipients, biologics, dietary supplements, and other therapeutics. USP 34-NF 29 also offers harmonized material and more than 230 General Chapters with current guidelines for the full range of laboratory tests and established processes for validating methods. United States Pharmacopeia 35 - National Formulary 30 : The 'United States Pharmacopeia 35 - National Formulary 30' (USP-NF) is a combination of two official compendia: the 'United States Pharmacopeia (USP)' and the 'National Formulary (NF)' and is officially applicable from 1 May, 2012 to 30 April, 2013. 1.4.4 European Pharmacopeia European Pharmacopeia commission started working

since 1964 to prepare EP Editions

• 1st edition: published 1967
• 2nd edition: published 1980
• 3rd edition: published 1997
• 4th edition: published 2001, valid from 1 January 2002
• 5th edition: published 15 June 2004, valid from 1 January 2005
• 6th edition: published 16 July 2007, valid from 1 January 2008
• 7th edition: published June 2010, valid from 1 January 2011
• 8th edition: published June 2013, valid from 1 January 2014 Since its 5th edition, the pharmacopoeia is published in 2 volumes. Volume 1 contains general chapters and monographs (e.g. on dosage forms, methods of analysis, reagents), volume 2 contains all substance monographs. During runtime of current edition several supplements are published. Electronic versions are also available (CD-ROM, USB stick and online version). Eighth edition EP was published on June 2013.
• The European Pharmacopoeia defines requirements for the qualitative and quantitative composition of medicines, the tests to be carried out on medicines and on substances and materials used in their production.
• It covers active substances, excipients and preparations of chemical, animal, human or herbal origin, homoeopathic preparations and homoeopathic stocks, antibiotics, as well as dosage forms and containers.
• It also includes texts on biologicals, blood and plasma derivatives, vaccines and radiopharmaceutical preparations. The European Pharmacopoeia and its requirements are legally binding in the member states of the European Pharmacopoeia Convention and the European Union. 2. SOURCES AND TYPES OF IMPURITIES (4,5) 2.1 Definitions :
• Impurity : As per IP any component or drug substance for pharmaceutical use or a drug product not a chemical entity.
• Impure Chemical Compound : A compound is said to be impure if it is having foreign matter i.e. impurities.
• Pure Chemical Compound : A pure chemical compound refers to that compound which is having no foreign matter i.e. impurities. 2.2 Effect of impurities :
• Impurities having toxic effects may be injurious to health, if present above certain limits.
• Traces of impurities, may exert a cumulative toxic effect after a certain time.
• Impurities may lower the active strength of the substance.
• Impurity may decrease shelf life of substance.
• Impurity may cause incompatibility with other substances.
• Impurities may cause a physical or chemical change in the properties of the substance. So, making the substance medicinally useless.
• May cause change in color, odour and taste. 2.3 Steps of identification of impurities
• Check impurities: presence/absence
• Select the method to remove impurities
• Confirm for purity 2.4 Type of impurities a) Identified Impurity Definition: An impurity whose chemical structure has been characterized and confirmed using analytical techniques. Example: In the synthesis of paracetamol, p-aminophenol is an identified impurity because its structure is well known and characterized. b) Potential Impurity Definition: An impurity that is expected to form during the manufacturing process or storage but has not necessarily been observed in the final product. Example: In the synthesis of amoxicillin, penicilloic acid is a potential impurity because it can form due to hydrolysis during storage, even if not always detected. c) Unidentified Impurity Definition: An impurity that is detected in a drug substance or product but whose chemical structure is unknown. Example: In an

HPLC analysis of a drug formulation, an unknown peak at a different retention time from known impurities suggests the presence of an unidentified impurity. 2.5 Commonly found impurities Effect on aesthetic properties: Taste, odour, colour – easily notice Examples: Benzoic acid, Salicylic acid, Aspirin: Phenolic impurity- odour Toxic impurities: Metallic impurities - toxic even in trace amount Example: Arsenic, Lead, Mercury Harmless impurities: Beyond certain tolerance limit. They affect the efficacy and potency Example: Sodium impurities – harmful in the patient with restricted diet of sodium Due to improper storage condition: Modification on storage condition Example: NaCl – Moisture, tends to liquid Create incompatibility: Unable to live together Example: API + Excipients. Impurity present in any one ingredient create incompatibility Create technical and technical difficulty: Difficulty in manufacturing process Example: Mixing, granulation process 2.6 Sources of impurities The different sources of impurities in pharmaceuticals are listed below: 1) Raw material used in manufacture 2) Reagents used in manufacturing process 3) Method/ process used in manufacture or method of manufacturing 4) Chemical processes used in the manufacture 5) Atmospheric contamination during the manufacturing process 6) Intermediate products in the manufacturing process 7) Defects in the manufacturing process 8) Manufacturing hazards 9) Inadequate storage conditions 10) Decomposition of the product during storage 11)Accidental substitution or deliberate adulteration with spurious or useless materials 1.

Raw materials used in manufacture Impurities known to be associated with these chemicals may be carried through the manufacturing process and contaminate the final product. Example: Rock salt contains small amounts of Calcium sulphate and Magnesium chloride. Rock salt → CaSO 4 + MgCl 2 = NaCl prepared Thus, Sodium chloride prepared from this source will contain traces of Calcium and Magnesium compounds.

Impurities such as Arsenic, Lead and Heavy metals are present in raw materials and hence are found in substances. So, it is necessary to use pure chemicals and substances as raw materials for the manufacturing process. Example: Copper sulphate may be prepared by the action of sulphuric acid on copper turnings: Cu+ 2H 2 SO 4 _____________________ CuSO 4 + 2H 2 O + SO 2 > Copper turnings are known to have Iron and Arsenic as impurities.

If Large quantities of impurities are present in the raw material (e.g. Copper turnings), they may enter the final product. (CuSO 4 .5H 2 O) 2.

Reagents used in the manufacturing process: If reagents used in the manufacturing process are not completely removed by washing, these may find entry into the final products. Example : Ammoniated mercury may be prepared by adding a solution of Mercuric chloride to dilute ammonia solution. HgCl 2 + 2NH 4 OH → NH 2 HgCl + NH 4 Cl + 2H 2 O soluble soluble Ammoniated mercury soluble (ppt) The precipitate of Ammoniated mercury (Final Product) contains ammonium hydroxide.

Thus, this precipitate is washed with cold water to remove ammonium hydroxide. If it is not removed completely by washing with water, the final product may contain Ammonium hydroxide as impurity. 3. Method or the process used in the manufacture: Many drugs and chemicals (usually organic) are manufactured from different raw materials, by using different methods or processes.

• Some impurities are incorporated into the materials during the manufacturing process.
• The type and amount of impurity present in the drug/ chemical varies.
• In certain drugs, a multiple-step-synthesis procedure is used, which produces intermediate compounds.
• The purification of intermediates is also important, otherwise the impurities present in the intermediate will get incorporated in the final product.
• Usually side reactions occur during the synthesis.
• Impurities of the product side reactions also occur in the substances. This may introduce new impurities due to contamination by reagents and solvents at various stages of the process as described below: a) Reagents employed in the manufacturing process: • Soluble alkali in Calcium carbonate arises from sodium carbonate used in the process. • Calcium carbonate is obtained by interaction of a soluble calcium salt and a soluble carbonate and therefore the product will contain traces of soluble alkali, which the washing process has failed to remove. b) Reagents added to remove other impurities: Potassium bromide contains traces of Barium, which is added in the manufacturing process to remove excess of sulphate. c) Solvents: Water is the cheapest solvent available

and has been used wherever possible Tap Water: It has Ca +2 , Mg +2 , Na + , Cl - , SO -2 and CO -2 as impurities in small amounts Softened water : It is obtained by allowing the tap water to pass through the sodium form of Zeolite which removes divalent cations like Ca +2 and Mg +2 from tap water in exchange of sodium. So, softened water contains Na + , Cl - ions as impurity. De-mineralised water : It is obtained by passing tap water through columns packed with ion exchange resin.

The water obtained from this process is free from Ca +2 , Mg +2 , Na + , Cl - , SO -2 and CO -2 . Thus, the final product is free from these impurities. The water obtained from this source may still contain organic impurities and so final product contains organic impurities.

Distilled water : It is considered the best but it is very costly d) Action of solvents and reagents on reaction vessels: During manufacturing process, some of the solvents and reagent may undergo reaction with metals of react vessel and may dissolve these metals, which appear as impurities in the final product. Example: ✓ Iron is known to contain Arsenic impurity. ✓ The inorganic compounds manufactured in Iron vessel will contain Arsenic and Iron as impurities. ✓ Thus IP has prescribed limit test for Arsenic and Iron for most inorganic compounds. 4. Chemical process used in the manufacture: For the synthesis of drugs, many chemical reactions such as nitration, halogenation, oxidation, reduction, hydrolysis are involved.

• In these chemical processes, different chemicals are used.
• Tap water is generally used in the various processes and it is often having Cl - , Mg +2 , Ca +2 ions, which are generally found in the substance which is being manufactured 5. Atmospheric contamination during the manufacturing process In the industrial areas, the atmosphere is contaminated with dust particles and some gases like Hydrogen sulphide, Sulphur dioxide, and black smoke.
• During the manufacture or purification of the pharmaceutical products, these impurities enter the final products.
• There are many pharmaceutical products which when manufactured are contaminated with atmospheric CO 2 and water vapour. Example: NaOH absorbs atmospheric CO 2 . 2NaOH + CO 2 _____________________ Na 2 CO 3 + H 2 O > Due to this reaction, NaOH should not be kept open for a longer time during its manufacture. Therefore, IP has prescribed that Sodium hydroxide should not contain more than 3% of sodium carbonate. 6. Defects in the manufacturing process: In many manufacturing processes, there are defects like imperfect mixing, incompleteness, non-adherence to proper temperature, pressure, pH or reaction conditions, which may give chemical compounds with impurities in them. Example:
• Zinc oxide may be prepared by heating metallic zinc to bright redness in a current of air.
• The vapours of Zinc burn to form Zinc oxide which is collected as a fine white powder. But if there is less heat or air or both, zinc metal is not completely converted to zinc oxide.
• Thus, the final product, Zinc oxide may still contain metallic zinc as impurity.
• So, IP has prescribed a test for Zinc metal in zinc oxide. 7. Intermediate products in the manufacturing process: There are some intermediates which are produced during the manufacturing process. Sometimes these intermediates may be carried through to the final product as impurity. Example: Potassium iodide is prepared by reacting Iodine with Potassium hydroxide. 6KOH+ 3I 2 _____________________ 5KI + KIO 3 + 3H 2 O > The resulting solution is first evaporated and then heated with charcoal. KIO 3 + 3C _____________________ KI + 3CO > In this process if the intermediate product (KIO 3 ) is not completely converted into KI, then it may be carried through to the final product as an impurity. 8. Manufacturing hazards: a.

Particulate contamination

• The presence of unwanted particulate matter can arise due to dirt, dust, glass, porcelain or plastic fragments from sieves, granulating or tableting machines or from product containers.
• Ware and tare of equipment or improperly cleaned equipment may also cause particulate contamination.
• Clarity of solutions for injection is particularly important.
• Example: Metal particles which have been found in eye ointments packed in metal tubes. b. Process error:
• Gross errors arising from incomplete solution of a solute in a liquid preparation must be detected readily by the normal analytical control procedures.
• Minor errors arise if the manufacturing tolerance for the quantity of active ingredient in the product has been wide. c. Cross contamination:
• The handling of powders, granules, and tablets in large bulk creates air-borne dust, which leads to cross contamination of the product.
• So, face masks and special extraction equipment are used to protect operators from harmful effects of drugs.
• Example: penicillin preparation requires special handling during its manufacture. d. Microbial contamination • Parenteral preparations and ophthalmic preparations require special care against microbial contamination. • Many liquid preparations and creams are liable to bacterial and fungal contamination. • So, care should be taken • Example: Acacia, senna, tragacanth---→They should be controlled for Salmonellae e. Packaging error • Products of similar appearance such as tablets of same size, shape, colour packed in similar containers can constitute a potential source of danger. • Improper labelling or destruction of stock of unused labels also constitutes a major packaging hazard 9. Storage conditions: The chemical substances when prepared have to be stored in different types of containers depending upon: ✓ Nature of the material

✓ Batch size ✓ Quantity Many types of materials are used for storage purpose like plastic, polythene, iron vessels, stainless steel and aluminium. Leaching out effect: Alkalis stored in ordinary glass containers extract lead from it, which in found as impurity in the final product. Strong chemicals react with iron containers and extract Iron an impurity in final product.

Inadequate storage and their effects are as follows: a) Filth: Stored products may become contaminated with dust, bodies of insects, animal and insect excreta. b) Chemical instability: decomposition because of light, traces of acid or alkali, air oxidation, water vapour, CO 2 and traces of metallic ions. Example: light sensitive materials should be stored in amber coloured bottles. c) Reactions with container materials: Example: salicylic acid ointment must not be stored in metal tubes d) Physical changes: The occurrence of changes in the physical form of drug like change in crystal size can lead to change in efficiency of product. e) Temperature effect: Chemical and physical changes occur if materials are not stored at proper temperature. 10. Decomposition of the product during storage:

• Chemical decomposition, analysis or breakdown is the separation of a chemical compound into elements or simpler compounds. It is sometimes defined as the exact opposite of a chemical synthesis.
• Chemical decomposition is often an undesired chemical reaction.
• Some substances decompose on storing due to presence of air, light and oxygen. So, the final product is contaminated.
• Deliquescent substances, absorb water from the atmosphere and get liquefied.
• Decomposition products appear as impurities in the substances. 11. Accidental substitution or deliberate adulteration with spurious or useless materials: It is possible to avoid accidental substitution by storing the toxic substances together separately or in a locked cupboard. Many pharmaceutical chemicals are adulterated with cheaper substances. Example: The expensive potassium may be adulterated with sodium bromide. 2.7 LIMIT TEST (5-8) 2.7.1 TEST FOR PURITY: Pharmacopoeia prescribes the “Test for purity” for pharmaceutical substances to check their freedom from undesirable impurities. Pharmacopoeia will decide and fix the limit of tolerance for these impurities. For certain common impurities for which pharmacopoeia prescribes the test of purity are: ✔ Colour, odour, taste ✔ Physicochemical constants (Iodine value, saponification value, melting point, refractive index

etc.) ✔ Acidity, alkalinity, pH ✔ Humidity (Estimation of moisture) ✔ Cations and anions ✔ Ash ✔ Arsenic or lead ✔ Loss on drying ✔ Loss on ignition 2.7.2 LIMIT TEST Limit: an amount that is likely to be present Test: to examine, investigate Limit test is a semi quantitative analysis and design to identify and control small quantity of impurity which are present in the sample Importance of Limit test:

• To find amount of harmful impurities
• To find out avoidable/unavoidable amount of impurities Limit test is performed in Nessler Cylinder Specification: Capacity : 50 ml Total height: 150 mm Mark: up to 50 ml Wall thickness: 1 – 1.5 mm Base thickness : 1.5 – 3.0 mm Figure 1: Nessler Cylinder 2.7.3 Limit Test 1. Limit test of Chloride 2. Limit test of Sulphate 3. Limit test of Iron 4. Limit test of Lead 5. Limit test of Arsenic 6. Limit test of Heavy metals 7. Modified limit test of Chloride and Sulphate 1. LIMIT TEST OF CHLORIDE Requirements: Nessler cylinders, Glass rod, Stand, Dilute Nitric acid (10%), Silver nitrate (0.1 M), Sodium chloride, Sample, Dist. water Principle: It is based upon the chemical reaction between

silver nitrate and soluble chlorides in presence of dilute nitric acid to give opalescence of silver chloride. The opalescence produced is compared with the standard solution. Cl - + AgNO 3 _____________________ AgCl + NO 3 > Dilute HNO 3 Procedure: Take two 50 ml Nessler Cylinders.

Label one as “Test” and the other as ‘ Standard’ . Table 1: Procedure for the Limit Test of Chloride TEST STANDARD Dissolve specify quantity of substance as per monograph 10 ml Standard Chloride solution (25 ppm) Add 5 ml Distilled water Add 5 ml Distilled water Add 10 ml Dilute Nitric acid Add 10 ml Dilute Nitric acid Dilute up to 50 ml dist. water Dilute up to 50 ml dist. water Add 1 ml 0.1 M AgNO 3 Add 1 ml 0.1 M AgNO 3 Stir immediately with glass rod and allow to stand for 5 minutes Compare opalescence of test with standard Result:

• If the opalescence in the sample is less than the standard, it passes the test. If it is more than the standard, it fails the test. Significance:
• Dilute nitric acid used to solution make acidic. Important notes:
• Standard Sodium Chloride Solution (25 ppm): Dilute 5 volume of 0.0824 % w/v of NaCl to 100 volume of Dist. water
• Distilled water must be used otherwise chloride present in tap water: interfere with the result
• Different glass rod must be used.
• Silver nitrate: photosensitive, black spot will be produced if it contacts with skin. 2. LIMIT TEST OF SULPHATE Requirements: Nessler cylinders, Glass rod, Stand, Barium Chloride, Acetic acid, Ethanolic sulphate standard solution, Barium sulphate, Sample, Dist. water Principle: Reaction between Barium chloride and sulphate in presence of acetic acid gives opalescence of barium sulphate. The opalescence produced is compared with the standard solution. SO 4 -2 + BaCl2 _____________________ BaSO 4 + 2Cl - > CH 3 COOH Procedure: Take two 50 ml Nessler Cylinders. Label one as “Test” and the other as ‘ Standard’ . Table 2: Procedure for Limit Test of Sulphate TEST STANDARD Take 1 ml 25 % w/v BaCl 2 solution Take 1 ml 25 %

w/v BaCl 2 solution Add 1.5 ml ethanolic sulphate standard solution (10 ppm) mix and allow to stand for 1 min Add 1.5 ml ethanolic sulphate standard solution (10 ppm) mix and allow to stand for 1 min Add 15 ml sample solution (as per monograph) Add 15 ml Sulphate standard solution (10 ppm) 0.15 ml 5 M Acetic acid 0.15 ml 5 M Acetic acid Add sufficient dist. water up to 50 ml Add sufficient dist. water up to 50 ml Stir immediately with glass rod and allow to stand for 5 minutes Compare opalescence of test with standard Result:

• If the opalescence in the sample is less than the standard, it passes the test. If it is more than the standard, it fails the test. Significance:
• Acidity of solution controlled by acetic acid
• Ethanolic sulphate standard solution: to prevent super saturation and increase the sensitivity of reaction. Importance notes:
• Sulphate standard solution (10 ppm): 1 volume of 0.181 % w/v solution of potassium sulphate in100 ml Distilled water. ( Ethanolic sulphate standard solution , 10 ppm is prepared in ethanol (30 % v/v) instead of distilled water.
• Different glass rod must be used. 3. LIMIT TEST OF IRON Requirements: Nessler cylinders, Glass rod, Stand, Ferric ammonium sulphate solution, Thioglycolic acid, Ammonia solution, Citric acid Principle: Reaction between ferrous ion and thioglycolic acid in presence of citric acid in alkaline medium and formation of ferrous thioglycolate. The intensity of colour is compared with standard solution. Reaction: Figure 2: Principle Reaction for the Limit Test of Iron Procedure: Take two 50 ml Nessler Cylinders. Label one as “Test” and the other as ‘ Standard’ . Table 3: Procedure for Limit Test of Iron TEST STANDARD Prepare sample solution specified as per monograph Take 2 ml standard Iron solution (20 ppm) Add 2 ml 20 % w/v Citric acid Add

2 ml 20 % w/v Citric acid Add 0.1 ml Thioglycolic acid Add 0.1 ml Thioglycolic acid Make alkaline with ammonia solution Make alkaline with ammonia solution Dilute up to 50 ml with dist. water Dilute up to 50 ml with dist. water Stir immediately with glass rod and allow to stand for 5 minutes Compare colour intensity of test with standard Result: If the colour of the sample is less than the standard, it passes the test. If it is more than the standard, it fails the test. Significance:

• Citric acid is eliminating interference of other metal
• Thioglycolic acid is reducing agent.
• Ferrous thioglycolate gives colour in alkaline media only . Important note
• Ferric ammonium sulphate solution is used as standard (20 ppm)
• Used iron free citric acid
• Different glass rod must be used: affect on observation 4. LIMIT TEST OF LEAD Requirements: Separating funnel, Dithizone, lead acetate cotton plug, ammonium citrate, potassium cyanide, hydroxylamine hydrochloride Figure 3: Separating funnel Principle: Limit test of Lead (Pb) is based on the reaction between lead (impurity) and Diphenylthiocarbazone (Dithizone) in chloroform solution and alkaline medium to form Lead-dithizone complex, which is red in color. Dithizone is green in color in chloroform and lead-dithizone complex is violet in color, so the resulting color at the end of process is red. Dithizone in chloroform, extract lead from alkaline aqueous solution as lead dithizone complex (violet in color). In this experiment, ammonium citrate, potassium cyanide, hydroxylamine hydrochloride are used to extract and discard

and interfering metal ions (other than lead) at optimum pH in the form of complex. Reaction: Figure 4: Principle Reaction for the Limit Test of Lead Procedure: Take two separating funnels. Label one as “Test” and the other as ‘ Standard’ .

Table 4: Procedure for Limit Test of Lead TEST STANDARD A known quantity of sample as per monograph is transferred in separating funnel A standard lead solution (1 ppm) is prepared equivalent to the amount of lead permitted in the sample under examination. Add 6 ml of ammonium citrate solution Add 6 ml of ammonium citrate solution Add 2 ml of potassium cyanide and 2 ml of hydroxylamine hydrochloride Add 2 ml of potassium cyanide and 2 ml of hydroxylamine hydrochloride Add 2 ml of phenol red Add 2 ml of phenol red Make solution alkaline by adding strong ammonia solution Make solution alkaline by adding strong ammonia solution Extract with 5 ml of dithizone until it become green extract Extract with 5 ml of dithizone until it become green extract Combined dithizone extract are shaken for 30 mins with 30 ml nitric acid and dithizone layer is discarded (chloroform layer) Combined dithizone extract are shaken for 30 mins with 30 ml nitric acid and dithizone layer is discarded (chloroform layer) To the acid solution add 5 ml of standard dithizone solution. To the acid solution add 5 ml of standard dithizone solution Shake for 30 seconds, the color of chloroform layer is not more intense than that obtained in standard solution Result: The intensity of the color of complex will depend on the amount of lead in the solution.

The color produced in the sample solution should not be greater than standard solution. If color produces in sample solution is less than the standard solution, the sample will pass the limit test of lead and vice versa. Significance:

• Ammonium citrate, potassium cyanide, hydroxylamine hydrochloride is used to make pH optimum. So, interference and influence of other impurities have been eliminated.
• Phenol red is used as indicator to develop the color at the end of process.
• Uses of HNO 3 : Lead react with HNO 3 and formation of lead nitrate which is soluble in water. That’s why discard chloroform layer. Important note
• Lead standard solution (10 ppm)
• Dithizone solution prepared in Chloroform 5. LIMIT TEST OF ARSENIC Requirements: Sample, magnesium sulphate, potassium bromide, glass rod, filter paper, cotton, Arsenic test apparatus (gutzeit’s apparatus), dilute arsenic solution, stannated HCl, granulated Zinc, potassium Iodide, lead acetate solution, mercuric chloride, distilled water. Principle: The impurity of arsenic is present in trivalent (As 3+ ) and pentavalent (As 5+ ) form. Trivalent arsenic is concerted to arsenious acid and pentavalent arsenic is converted to arsenic acid. The arsenic acid is converted to arsenious acid. Figure 5: Principle Reaction for the Limit Test of Arsenic The amount of impurity present in the sample is first converted into arsenious acid by the action of reducing agents like potassium iodide, zinc, HCl, and stannous

chloride. Then further arsenic acid is then reduced to arsine by the action of hydrogen which is produced by the reaction between zinc and HCl. The arsine gas travels through a tube and finally reacts with mercuric chloride paper to produce a yellow stain.

The depth of the yellow stain depends on the amount of arsenic present in the sample compared with that of the standard stain produced from a known amount of arsenic. Reaction: H 3 AsO 4 _____________________ H 3 AsO 3 > Arsenic acid Reducing agent Arsenious acid Zn + 2HCl _____________________ ZnCl 2 + H 2 ( Nascent hydrogen) > H 3 AsO 3 + 6 [H] _____________________ AsH 3 + 3H 2 O > Arsenious acid Nascent hydrogen Arsine gas 2AsH 3 + HgCl2 _____________________ Hg (AsH 2 ) 2 + 2HCl > Arsine gas Mercurous arsenate (Yellow colour) Gutzeits test/ apparatus:

• The Gutzeit apparatus consists of a 120 ml wide-mouthed glass bottle with a mouth of 2.5cm diameter.
• The mouth of the bottle is closed with a rubber or glass stopper which passes a glass tube of 20cm long, having an external and internal diameter of 0.8cm and 0.65cm, respectively.
• Similar to the pipette’s lower end extremity, the glass tube is constricted to about 1mm in diameter. Approximately 15mm from the tip of the lower end is a lateral orifice which should be at least 3mm below the lower surface of the stopper.
• It must be noted that when the tube is in position in the stopper, the constricted end of the tube should be above the surface of the reaction liquid in the flask, and the hole in the side is 3mm below the bottom of the bung.
• The upper end of the glass tube is cut off square and has a flat surface at right angles to the axis of the tube.
• A second glass tube of the same internal diameter as that of the first and 30 mm long, with a similar flat surface, is placed in contact with the first tube (over the top position) and is held in position by two spiral springs or clips.
• Between the two flat surfaces of the glass tubes, a disc or a small square of mercuric chloride paper is placed, which should be large enough to cover the orifice of the tube (15 mm × 15 mm).
• A loosely packed cotton is inserted into the lower glass tube previously moistened with lead acetate solution. Figure 6: Gutzeits apparatus Procedure: Table 5: Procedure for the Limit Test of Arsenic TEST STANDARD A known quantity of sample as per monograph Add 1 ml standard solution of Arsenic (10 ppm) in 50 ml water. Add 10 ml of Stannous HCl acid. Add 10 ml of Stannous HCl acid. Add a further 1 gm of Potassium iodide + 10 gm of granulated zinc. Add a further 1 gm of Potassium iodide + 10 gm of granulated zinc. Immediately assemble the apparatus and immerse the flask in water bath at a temperature such a that uniform evolution of gas is maintained. Keep

the apparatus for 40 minutes Result: After 40 minutes, compare the mercuric chloride paper of both. the test has less stain as compared with the standard to pass the limit test. Significance: All the special reagents used in the limit test for arsenic are marked and distinguished by the letter “as T” which means they all should be arsenic-free and should themselves confirm to limit test for arsenic. Lateral orifice: To prevent condensed liquid produced by press of hydrogen developed in flask Stannous chloride is used for the complete evolution of arsine.

Zinc, potassium iodide and stannous hydrochloric acid are used as reducing agents. Arsenic is reduced to the arsine by the combined action of these reagents. Lead acetate-soaked cotton plug is used to trap any hydrogen sulfide that may be evolved along with arsenic.

Important notes: Arsenic standard solution (10 ppm) of Arsenic trioxide Mercuric chloride paper: Smooth white filter paper soaked in a saturated solution of mercuric chloride and pressed to removed extra solution and dried it 60 °C. Store in stopper bottle and in a dark place 6. LIMIT TEST OF HEAVY METALS Principle : Limit test of Heavy metals is based on the reaction of metallic impurities with Hydrogen sulphide (H 2 S) or Sodium sulphide in an acidic medium to produce metal sulphides which gives Brown color.

Here lead is used to make standard solution. Heavy metal like: Cobalt, tin, Manganese, Bismuth, Antimony, Silver, Arsenic, lead etc. Reaction: Method A and B Acidic medium Heavy metals + H 2 S _____________________ Sulphide of heavy metals > (brown coloration) Method C: Acidic medium Heavy metals + Na 2 S _____________________ Sulphide of heavy metals > (brown coloration) Method D: Heavy metals + Thioacetamide _____________________ Sulphide of heavy metals > (brown coloration) Procedure: Method A : For colourless substance, Method B : For coloured substance Method C : Colourless solution in NaOH, Method D : Reaction between heavy metal and thioacetamide Procedure: Method A and B Take two separating funnels.

Label one as “Test” and the other as ‘ Standard’ . Procedure for Method B It is similar to method A except in this case the coloured substance (Test sample) is given special treatment (sulphuric acid, ignition, nitric acid, ignition, HCl and finally digestion with water) to make it colourless before preparing its solution. Table 6: Procedure for Method A & B TEST STANDARD Place 25 ml of the solution of the test sample prepared according to monograph Place 2 ml of standard lead solution (0.02mg lead) and dilute with distilled water to 25 ml Adjust with dil.

Acetic acid or ammonia to a pH b/t 3 to 4 and dilute with water to 35 ml. Adjust with dil. Acetic acid or ammonia to a pH b/t 3 to 4 and dilute with water to 35 ml.

Add 10 ml of freshly prepared saturated solution of hydrogen sulphide Add 10 ml of freshly prepared saturated solution of hydrogen sulphide Make the volume to 50 ml with distilled water and mix. Make the volume to 50 ml with distilled water and mix. Allow to stand for 5 minutes Procedure: Method C and D Procedure for Method D To the cylinders containing test sample and standard solution add 2 ml of acetate buffer of pH 3.5 and mix/ to each of the cylinder add 1.2 ml thioacetamide reagent.

Allow to stand for two minutes and view downwards over a white surface and compare intensity of color in both. Table 7: Procedure for Method C & D TEST STANDARD Place 25 ml of the solution of the test sample prepared according monograph Place 2 ml of standard lead solution (0.02mg lead) and dilute with distilled water to 25 ml Add 5 ml of dilute sodium hydroxide solution Add 5 ml of dilute sodium hydroxide solution Add distilled water to make 50 ml and mix. Add distilled water to make 50 ml and mix.

Add 5 drops of sodium sulphide solution and mix. Add 5 drops of sodium sulphide solution and mix. Allow to stand for 5 minutes and compare the color Result: The turbidity/ color produce in sample solution should not be greater than standard solution.

If turbidity/color produces in sample solution is less than the standard solution, the sample will pass the limit test of heavy metals and vice versa. 7. Modified limit test of Chloride and sulphate i. Alkaline solution: Sample dissolve in acid and effervescence produce and free acid left behind in the solution This solution is used for test and follow the same as mentioned in limit test of chloride and sulphate. ii.

Insoluble substance: Sample is extract with water then filter. Filtrate is used for test and follow the same as mentioned in limit test of chloride and sulphate. iii. Salt of organic acid: Sample is first treated with acid.

Liberated free acid, which are insoluble in water and form precipitation. In this case, ppt. free acid if filtered and filtrate is subjected to limit test. iv. Coloured substance: Sample is ignited at high temp. and ash is subjected limit test of chloride and sulphate. v.

Deeply coloured substance: Sample is decolourised by boiling with ethanol. Then filter to remove ppt. of manganese dioxide and filtrate subject to limit test of chloride and sulphate. vi. Reducing substance: Sample is oxidized with oxidizing agent.

Then filter and filtrate is subjected to limit test of chloride and sulphate. REFERANCES: 1. Indian Pharmacopoeia Commission.

Indian Pharmacopoeia . Ghaziabad, India: Indian Pharmacopoeia Commission. 2. Medicines and Healthcare Products Regulatory Agency (MHRA).

British Pharmacopoeia . London, UK: The Stationery Office. 3. United States Pharmacopoeial Convention.

United States Pharmacopoeia . Rockville, MD: United States Pharmacopoeial Convention. 4. Chatwal, G.

R. (2015). Pharmaceutical chemistry – Inorganic (Vol. 1) Himalaya Publishing House 5.

Kar, A. (2005). Pharmaceutical drug analysis (2nd ed.).

New Age International (P) Ltd., Publishers. 6. Raval, H. G., Shah, D.

A., & Baldania, S. L. (2011).

Practicals in inorganic and analytical chemistry (1st ed.,) Nirav and Roopal Prakashan. 7. Mehta R.S., Vaghela V.M., Prajapati H.R. ( ).

Practicals on Pharmaceutical Analysis (ed.,) Mahajan Publishing House. 8. Chatwal, G. R.

(2015). Pharmaceutical chemistry – Inorganic (Vol. 1) Himalaya Publishing House