ae VE 2 ae. A \ PROIKCT BEPORBT ON “EFFECTS OF IMPURITIES IN PHARMACEUTICAL PRODUCTS” This Project Work submitted to Talcher (Auto.) College, Talcher on the partial fulfillment for the Bachelor Degree of Science (Chemistry Hons) SESSION: 2022-23 Lipsarani Pradhan Mr. Chaitanya Sahu Exam. Roll No: 2002ACHEMO15 H.O.D College Roll No: BS(P)20-015 Department of Chemistry 43 Final Year Se. (Chemistry Hons) Talcher (Auto.) College, Talcher DEPARTMENT OF CHEMISTRY TALCHER AUTONOMOUS COLLEGE, TALCHERDEPARTMENT OF CHEMISTRY TALCHER AUTONOMOUS COLLEGE, TALCHER ERTIFI This is to certified that the candidate Lipsarani Pradhan, bearing Exam Roll No: 2002ACHEMO015, student of +3 Final Year Science (Chemistry Hons.), for the session 2022-23 is a bonafide student of Talcher Autonomous College, Talcher. She has done the project report entitled “EFFECTS OF IMPURITIES IN PHARMACEUTICAL PRODUCTS?” in her original piece of work and to the best of my knowledge no other candidate has submitted the same paper for the award of Bachelor’s Degree in Science under Talcher Autonomous College, Talcher I wish her all success in future life Sign of H.O.D Sign of Guide Date: Date:Acknowledgment Words fail me to express my heartfelt thankfulness to Mr. Chaitanya Sahu, Head of the Department of Chemistry Talcher Autonomous College, Talcher for his precious guidance and supervision for the completion of my project work. I am very grateful to Teaching Staff Members of Chemistry Dept. for their valuable advice and kind help. I would like to convey my hearty thanks to my respected parents, friends, and Suryakanta Sahu for their constant inspiration and support which has elevated my spirit to complete my project work. Signature of StudentDECLARATION I do here by declare that the project work entitled, “EFFECTS OF IMPURITIES IN PHARMACEUTICAL PRODUCTS” submitted by the for the partial fulfillment of the Bachelor Degree of Science (Chemistry) Talcher Autonomous College, Talcher is my own and original work and has not been submitted earlier to Talcher Autonomous College, Talcher or to any other institution for the requirement for any degree. Lipsarani Pradhan Exam, Roll No: 2002ACHEM015 College Roll No: BS(P)20-015 +3 Final Year Sc, (Chemistry HonsCONTENTS Sl. No Topic ABSTRACT CHAPTER-1 INTRODUCTION EFFECTS OF IMPURIES IN PHARMACEUCALS CHAPTER-2 IMPURITY PROFILE: © Critical Factors Affecting the Quality of Bulk Drugs CHAPTER-3 CLASSIFICATION OF IMPURITIES ORIGIN AND OCCURRENCE OF TYPES OF IMPURITIES IDENTIFIED OR UNIDENTIFIED o Degradation products CHAPTER-4 IMPURITIES RELATED TO FORMULATION FORMATION OF IMPURITIES ON AGING CRITICAL FACTORS REGARDING BULK DRUGS’ QUALITY CHAPTER-S IMPURITY DETECTION, ISOLATION AND CHARACTERIZATION METHODS FOR IMPURITY DETECTION CHARACTERIZATION METHODS o COMET Instrumentation CHAPTER-6 CONCLUSION REFERENCESABSTRACT Various regulatory authorities such as the International Conference on Harmonization (ICH), the United States Food and Drug administration (FDA), and the Canadian Drug and Health Agency (CDHA) are emphasizing on the purity requirements and the identification of impurities in Active Pharmaceutical Ingredients (APIs). The various sources of impurity in pharmaceutical products are — reagents, heavy metals, ligands, catalysts, other materials like filter aids, charcoal, and the like, degraded end products obtained during \ after manufacturing of bulk drugs from hydrolysis, photolytic cleavage, oxidative degradation, decarboxylation, enantiomeric impurity, and so on. The different pharmacopoeias such as the British Pharmacopoeia, United State Pharmacopoeia, and Indian Pharmacopoeia are slowly incorporating limits to allowable levels of impurities present in APIs or formulations. Various methods are used to isolate and characterize impurities in pharmaceuticals, such as, capillary electrophoresis, electron paramagnetic resonance, gas-liquid chromatography, gravimetric analysis, high performance liquid chromatography, solid-phase extraction methods, liquid-liquid extraction method, Ultraviolet. Spectrometry, infrared spectroscopy, supercritical fluid extraction column chromatography, mass spectrometry, Nuclear magnetic resonance (NMR) spectroscopy, and RAMAN spectroscopy. Among all hyphenated techniques, the most exploited techniques for impurity profiling of drugs are Liquid Chromatography (LC)- Mass Spectroscopy (MS), LC-NMR, LC-NMR-MS, GC-MS, and LC-MS. This reveals the need and scope of impurity profiling of drugs in pharmaceutical research.INTRODUCTION Impurities in pharmaceuticals are the unwanted chemicals that remain with the active pharmaceutical ingredients (APIs), or develop during formulation, or upon aging of both API and formulated APIs to medicines. The presence of these unwanted chemicals even in small amounts may influence the efficacy and safety of the pharmaceutical products. Impurity profiling (ie, the identity as well as the quantity of impurity in the pharmaceuticals), is now getting receiving important critical attention from regulatory authorities. The different pharmacopoeias, such as the British Pharmacopoeia (BP) and the United States Pharmacopoeia (USP), are slowly incorporating limits to allowable levels of impurities present in the APIs or formulations. A number of recent articles have described designed approaches and guidance for isolating and identifying process-related impurities and degradation products using mass spectrometry, Nuclear Magnetic Resonance (NMR), high-performance liquid chromatography (HPLC), Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), and tandem mass spectrometry for pharmaceutical substances. In general, according to ICH guidelines on impurities in new drug products, identification of impurities below the 0.1% level is not considered to be necessary unless the potential impurities are expected to be unusually potent or toxic. In all cases, impurities should be qualified. If data are not available to qualify the proposed specification level of an impurity, studies to obtain such data may be needed (when the usual qualification threshold limits given below are exceeded). According to ICH, the maximum daily dose qualification threshold is considered as follows — < 2g/day 0.1 % or 1 mg per day intake (whichever is lower) > 2giday 0.05%.EFFECTS OF IMPURIES IN PHARMACEUCALS ieee eS eS) The substance that are used in pharmaceucal field must be clear so that they can be used safely. But it is very difficult to obtain an almost absolute pure substance. Different substance and chemicals have varying grade of purity. Effect of Impuries: We know that almost all pure substances are difficult to get, and some amount of impurity is invariably present in the material. So, the impuries that are present in the substances may have the following effects. "= Impuries may bring about conflict with other substances. Impuries may lower the shelf life of the substances. Impuries may cause complicaons during formulaons and use of the substances. Somemes Impuries alters the physical and chemical properes of the substances. Therapeuc effect can be decreased. Shows toxic impact aer a certain period. Injurious when present beyond certain limits. It may change odour, colour, taste of the substance. To prevent these impuries many tests such as limit test are carried out to lower the impuries to ensure the pharmaceucals safer. At Oceanic Pharmachem Pvt. 3Ltd. we focus on quality of pharmaceucal intermediates and fine chemicals by regular quality audits. Quality runs across all our departments, varying from inial stage development to document control and manufacturing to validaon. Irrespecve of purpose, our people are passionate regarding achieving manufacturing excellence. Oceanic Pharmachem Pvt. Ltd. is one of the foremost manufacturers and exporters of Advanced Pharmaceucal Drug Intermediate.IMPURITY PROFILE: A description of the identified and unidentified impurities present in a drug product. DEGRADATION PRODUCT: An impurity resulting from a chemical change in the drug substance brought about during manufacture and/or storage of the new drug product by the effect of, for example, light, temperature, pH, water, or by reaction with an excipient and/or the immediate container closure system. IMPURITY PROFILE Impurity profile describes the identified and unidentified impurities present in a new drug substance. The aim of which is determination of organic and inorganic impurities as well as residual solvents in bulk drugs and pharmaceutical formulations. It helps identifying the impurity present in pharmaceutical formulation by analytical technique or methods. Numbers of impurities present in formulation such by-products, degradation products, interaction products, intermediates, penultimate intermediates, related products, Transformation product. According to USP impurities have various sections which are Impurities in Official Articles, Ordinary Impurities, and Organic Volatile Impurities. Impurity profiling is the common name of a group of analytical and quantitative identification and structural elucidation activities According to ICH impurities occurred or produced by chemical syntheses which are organic mpurities (Process and Drug related) Inorganic Impurities, Residual Solvents.There are various sources of impurity like heavy metals, ligands, catalysts other materials like degraded end products obtained during or after manufacturing of bulk drug or products. The expert working group of the international conference on harmonization of technical requirements for registration of pharmaceuticals for human use commonly known ICH has “defined impurity is any compound of the medicinal product which is not the chemical entity defined as the active substance or as an excipient in the product”. Critical Factors Affecting the Quality of Bulk Drugs 1. Crystallization Washing the wet cake Drying Appropriate packagingAPTER-3 CLASSIFICATION OF IMPURITIES 1) DOSAGE FORM RELATED CLASSIFICATION i, Mutual interaction amongst Ingredients ii. Functional group- related typical Degradation SOURCES OF IMPURITIES IN MEDICINES Medicines are the formulated forms _ of active pharmaceutical ingredients. There are 2 types of impurities in medicines: (1) Impuritiesassociated with APIs (active Pharmaceutical ingredients) (2) Impurities that are created during formulation and or with aging or that are related to the formulated forms. 1) Impurities associated in with APIs According to ICH guidelines, impurities associated with APIs are classified into the following categories Organic impurities (Process and Drug- related) Inorganic impurities Residual Solvent Ester hydrolysis Hydrolysis Oxidative degradation Impurities coming from : Interaction between the primary packaging and drug —_ product At the time of contact between processing materials and storage bags, closure, filters, tubing material etc. Impurities also introduce during storage of compound Impurities also. come from label, ink, overwrap, cardboard, boxes ete. Impurities can be present at the synthesis of product (called genotoxic impurities) that is solvent, residues, catalysts, reaction product in synthesis, etcUSP CLASSIFICATION According to united state pharmacopoeia impurities are classified as 1. Impurities in official articles Ordinary impurities 2. 3. Organic volatile impurities ICH CLASSIFICATION 1. Organic impurities 2. Inorganic mpurities a Residual solvents ORIGIN AND OCCURRENCE OF TYPES OF IMPURITIES, ORGANIC IMPURITIES. a) Starting materials or intermediates b) _ By-products c) Reagents, ligands, and catalysts ENANTIOMERIC IMPURITIES, INORGANIC IMPURITIES a) Reagents, ligands, and catalysts b) — Heavy metals ce) Other materials (eg, filter aids, charcoal) 4) SOLVENT RESIDUES 1) ORGANIC IMPURITIES Organic impurities may arise during the manufacturing process and/or storage of the drug substance. They may beIDENTIFIED OR UNIDENTIFIED, Volatile or non-volatile, and include the following. a) Starting materials or intermediates- These are the most common impurities found in every API unless a proper care is taken in every step involved throughout the multi-step synthesis. Although the end products are always washed with solvents, there are always chances of having the residual unreacted starting materials may remain unless the manufacturers are very careful about the impurities. In paracetamol bulk, there is a limit test for p-aminophenol, which could be a starting material for manufacturer or an intermediate for ans other. b) By-products: In synthetic organic chemistry, getting a single end product with 100% yield is very rare; there is always a chance of having by-products. In the case of paracetamol bulk, acetylated paracetamol (Figure) may form as a by product OH OH 0-00CH, (CHC0},0 acetylated —_———) Paracetamol acetylation as by-product NH, NHCOCH, | NHCOCH, p-Aminophenol Peracetamol Figure Production of Paracetamol from intermediate, p-Aminophenol Degradation products. Impurities can also be formed by degradation of the end product during manufacturing of bulk drugs. However, degradation products resulting from storage or formulation to different dosage forms or aging are common impurities in the medicines. The degradation of penicillins and cephalosporins is a well- known example of degradation products. The presence of a 8-lactam ring as well as that of an a-amino group in the C6/C7 side chain plays a critical role in their degradationFigure . General Structures of (1) Penicillins and (ID) Cephalosporins Quality, purity, safety and efficacy of pharmaceuticals are the fundamental issues of importance in drug therapy. A greater emphasis is being paid today on assurance of the quality and safety of a drug by monitoring and controlling the impurities and degradation products. The control of pharmaceutical impurities, unwanted substances is a critical issue for the pharmaceutical industry since their presence in the active pharmaceutical constituents and its dosage forms diminish both drug quality and safety By the identification or characterization and quantification of the impurities and related substances, the risk of their contribution to the side effect profile of the drug therapy can be avoided or minimized. Moreover, the characterization of such trace to minor analytes in the final product is the inherent prerequisites in the wake of stringent requirements from the regulatory authorities. Hence, the development of new analytical methods became an essential task for investigators to determine the stress-related impurities along with their degradation pathway. A forced degradation study (stress testing) is the main tool used to predict stability problems, develop analytical methods, and identify degradation products or pathways. Since, the subject of stress testing has insufficiently addressed in the scientific literature, the measurement, and identification of impurities by today’s standards present significant challenges to the analytical chemist. The development of modern quantitative and qualitative methods driven by these challenges has provided increasing opportunity to detect, identify, and quantify organic and inorganic impurities in bulk drugs and pharmaceutical formulations, and therefore, the origin and safety potential, of such impurities be established.Recent efforts by the International Conference on Harmonization (ICH) have led to greater regulative scrutiny of impurities, constraining their isolation, characterization and toxicological qualification when present at threshold levels. Moreover, the toxicological profiles of the active drug substance as well as the adverse effects of the related impurities govern the safety of a drug product. It is important to determine the cytotoxic, genotoxic and mutagenic activity of impurities thereby establishing their structure-toxicity relationship using various assay __ procedures. Worldwide, characterization or impurity profiling of drug substances has increasingly viewed as a precious complement to routine investigative work, adding valuable, scientific information in support of their vulnerability to stress conditions and degradation pathways. Impurity profiling has definite techno-commercial value since the pharmaceutical industry is facing the challenge of impurities that diminish the quality, safety and efficacy of drug substances. Due to lack of adequate scientific literature, it is necessary to incorporate stringent tests to control the impurities . This fact is also evident from the requirements of the Federal Food, Drug and Cosmetics Act and from a large number of pharmacopeias that provide tests for the control of specific impurities. Forced degradation and impurity profiling is one of the key prerequisites for investigational new drug (IND) and new drug application (NDA) registration. hyphenated techniques with liquid chromatography has made this much simpler than the traditional time- consuming approach of quantification, isolation, followed by acquisition of the spectral (mass, UV, NMR and IR) and elemental (C,H, N) data c) Reagents, ligands, and catalysts These chemicals are less commonly found in APIs; however, in some cases they may pose a problem as impurities. In general, an individual API may contain all of the above- mentioned types of organic impurities at lev-els varying from negligible to significant. A detailed investigation of impurities in semi- synthetic penicillin was performed both by the manufacturers and different research groups. A review paper on penicillins and cephalosporins describes methods of isolation, detection, and quantification of degradation products, and antigenic polymeric by-products. Studies show the presence of traces of ampicillin polymers and hydrolyzed products in the API. It has also been found that the presence of certain chemicals such as triethyl- amine has a degradative effect on the product. Am- picillin trihydrate samples having triethylamine content of 3000 to 4000ppm awere found to be stable under lerated stability testing. However, the product showed appreciable degradation when triethylamine content became 7000 ppm. Recent pharmacopoeia[20] included the limit tests for the traces of impu-rities present in ampicillin and amoxycillin bulk raw materials. The residual solvents associated with these APIs have also been determined. ‘As the organic impurities are the most common product- as well as process- related impurities, it is the responsibility of both the manufacturers of APIs and the users (ie, formulators) to take care of these impurities according to ICH guidelines or compendia. In addition, for an optically active single isomer drug there could be enantiomeric impurities present in the API 2) ENANTIOMERIC IMPURITIES — The single enantiomeric form of a chiral drug is now considered as an im-proved chemical entity that may offer a better pharmacological profile and an increased therapeu-tic index with a more favorable adverse reaction profile.[22] However, the pharmacokinetic profile of levofloxacin (S- isomeric form) and oflox-acin (R- isomeric form) are comparable, suggesting the lack of advantages of single isomer in this re-gard.[23] In any case, cost benefits as well as the patients compliance need to be considered in se-lecting drugs. For the manufacturers of single enan- tiomeric drug (eutomer), the undesirable stereoi- somers in drug control are considered in the same manner as other organic impurities. The prominent single isomer drugs, which are being marketed, in-clude levofloxacin (S- ofloxacin), levalbuterol (R-albuterol), esomeprazole (S- omeprazole). 3) INORGANIC IMPURITIES: Inorganic impurities may also derive from the manufacturing processes used for bulk drugs. They are normally known and identified and include the following a) 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. b) Heavy metals - The main sources of heavy metals are the water used in the processes and the reactors (if stainless steel reactors are used), where acidifi- cation or acid hydrolysis takes place. These impuri- ties of heavy metals can easily be avoided using demineralized water and glass- lined reactors. c) Other materials (eg, filter aids, charcoal) - The filters or filtering aids such as centrifuge bags are routinely used in the bulk drugs manufacturing plants, and, 2tivated carbon is also used. The regular monitoring of fibers and n the bulk drugs is essential to avoid these contaminations. 4) SOLVENT RESIDUES: Residual solvents are organic volatile chemicals used during the manufacturing process or generated during the production. It is very difficult to remove these solvents completely by the work-up process; however, efforts should be taken to the extent pos-sible to meet the safety data. Some solvents that are known to cause toxicity should be avoided in the production of bulk drugs. Depending on the possi-ble risk to human health, residual solvents are di-vided into 3 classes. Solvents such as benzene (Class I, 2 ppm limit) and carbon tetrachloride (Class I, 4 ppm limit) are to be avoided. On the other hand, the most commonly used solvents such as methylene chloride (600 ppm), methanol (3000 ppm), pyridine (200 ppm), toluene (890 ppm), ,N- dimethylformamide (880 ppm), and acetonitrile (410 ppm) are of Cl IL. Class III solvents (acetic acid, acetone, isopropyl alcohol, butanol, ethanol, and ethyl acetate) have permitted daily exposures of 50 mg or less per day. In this regard, ICH guidelines for limits should be strictly followed.APTER-4 IMPURITIES RELATED TO FORMULATION Apart from bulk drug related impurities, the formulated form of API may contain impurities that form in various ways. IMPURITY FORMS DURING FORMULATION a) Method Related b) Environmental Related c) Dosage form Factors Related a) Method related A known impurity, 1-(2,6- diclorophenyl) indolin-2-one is formed in the production of a parenteral dosage form of diclofenac sodium if it is terminally sterilized by autoclave. Itwas the condition of the autoclave method (ie, 123+2°c) thatenforced the intramolecular cyclic reaction of diclofenac sodium forming the indolinone derivative and sodium forming the indolinone derivative and sodium hydroxide. The formation of this impurity has been found to depend on the initial pH of the formulation. The concentration of the impurity in the resultant product in the ampoule exceeds the limit of the raw material in the BP. b) Environmental related The primary environmental factors that can reduce stability include the __ following exposures i) Temperatures - There are many APIs that are labile to heat or tropical temperatures. For example, vitamins as drug substances are very heat - sensitive and degradation frequently leads to loss of potency in vitamin products, especially in liquid formulations. ii) —_Light-especially UV light -Several studies have reported that ergometrine as well as methyl ergometrine injection is unstable under tropical conditions such as light and heat, and a very low level of active ingredient was found in many field samples. In only 50% of the marketed 4samples of ergometrine injections tested did the level of active ingredient comply with the BP/USP limit of 90% to 110% of the stated content. The custom-made injection of ergometrine (0.2 mg/mL) showed almost complete degradation when kept 42 hours in direct sunlight. Humidity : For hygroscopic products, humidity is considered detrimentalto both bulk powder & formulated solid dosage forms. Aspirin and ranitidine are classical examples. ©) Dosage form factors Although the pharmaceutical companies perform Pre-formulation studies, including a stability study before marketing the dosage form products, sometimes. the factors that influence drug stability force the company to recall the product. Fluocinomide Topical Solution USP, 0.05%, (Teva Pharmaceuticals USA, Inc., Sellersville, Pennsylvania) in 60-mL bottles, was recalled in the United States because of degradation/impurities leading to sub- potency. In general, liquid dosage forms are very much sus-ceptible to both degradation and microbiological contamination. In this regard, water content, pH of the solution/suspension, compatibility of anions and cations, mutual interactions of ingredients, and the primary container are critical factors. Microbiological growth resulting from the growth of bacteria, fungi, and yeast in a humid and warm environment may result in oral liquid products that are unusable for human consumption. Microbial Contamination may occur during the shelf life and subsequent consumer-use of a multiple dose prod-uct due to inappropriate use of certain preservatives in the preparations, or because of the semipermeable nature of primary containers.FORMATION OF IMPURITIES ON AGING a, Mutual interaction amongst ingredients Most vitamins are very labile and on aging they pose a problem of instability in different dosage forms, especially in liquid dosage forms. Degradation of vitamins such as folie acid, pantothenic acid, cyanocobalamin, and thiamine do not give toxic impurities; however, potency of active ingredients drops below pharmacopoeial specifications. Because of mutual nteraction, the presence of nicotinamide in a formulation containing 4 vitamins (nicotinamide, pyridoxine, riboflavin, and thiamine) causes the degradation of thiamine to a sub-standard level within a lyear shelf-life of vitamin B complex injections. The marketed samples of vitamin B- complex injections were found to have a pH in the range of 2.8-4.0. The custom-made formulation in a simple distilled water vehicle and in a typical formulated vehicle that included disodium editate and benzyl aleohol_ was _also investigated and similar mutual interaction causing degradation was observed b.Functional group related typical degradation Ester hydrolysis -Examples included the follow-ing: Aspirin, benzocaine, cefotaxime, cocaine echothiophate, ethyl paraben, cefpodoxime proxetil. i) Hydrolysis- Hydrolysis is a common phenomenon for the ester type of drugs, especially in liquid dosage forms. Examples include benzylpenicillin, barbitol, chloramphenicol, chlordiazepoxide, lincomy- cin, and oxazepam. ii) Oxidative degradation - Hydrocortisone, meth-otrexate, adinazolam, hydroxyl group directly bonded to an aromatic ring (eg, phenol derivatives such as catecholamines and morphine), conjugated dienes (eg, vitamin A and unsaturated free fatty acids), heterocyclic aromatic rings, nitroso and nitrite derivatives, and aldehydes (eg, flavorings) are all susceptible to oxidative degradation. Photolytic cleavage - Pharmaceutical products are exposed to light while being manufactured as a solid or solution, packaged, held in pharmacy shops or hospitals pending use, or held by the consumer pending use. Ergometrine , nifedipine , nitroprusside, riboflavin, and phenothiazines are very labile to photo-oxidation. In susceptible compounds, photo-chemical energy creates free radical intermediates, which can perpetuate chain reactions. Most compounds will degrade when exposed to high UV energy. 16Fluoroquinolone antibacterials are found to be susceptible to photolytic cleavage. In ciprofloxacin eye drops preparation (0.3%), sunlight induces photocleavage reaction producing ethylene-diamine analog of ciprofloxacin. iv) Decarboxylation: Some dissolved carboxylic acids, such asp- minosalicylic acid, lose carbon dioxide from the carboxyl group when heated. Decarboxylation also occurred in the case of photoreaction of rufloxacin CRITICAL FACTORS REGARDING BULK DRUGS’ QUALITY During crystallization - The size of crystals some-times determines the quality, especially the stability, of bulk drugs. Largesize crystals can entrap a minute amount of chemicals from the mother liquor, which ultimately causes the degradation of the drug. Thus, the manufacturers of bulk drugs should take care to produce finer crystals while isolating the products. Washing the wet cake - Washing the wet cake or powder in the centrifuge should be thorough to re-move unwanted chemicals including residual solvents. Drying- Use of a vacuum dryer or a fluid-bed dryer is always preferable to a tray dryer in the pharmaceutical bulk drug industry. The high thermal efficiencies, reduction of drying time, and more uniform drying are helpful in drying sensitive drug substances. However, if a tray dryer is used then initial airflow at ambient conditions should be considered before exposing the materials to a relatively higher fixed temperature. Appropriate packaging Light sensitive Pharmaceutical Products need light protective packaging. An accelerated stability test-ing conducted using marketed ampoules of er- gometrine wrapped with either black carbon paper or aluminum foil produced negligible degradation against direct sunlight. Similar use of opaque containers for ciprofloxacin eye- drop preparation can protect the active ingredient from photodegra-dation to some extent compared with transparent containers. Use of vproduction method based on stability studies In finalizing the method of preparation, a detailed investigation, including stability studies, should be undertaken. In the case of parenteral preparations, aseptic filtration versus terminal autoclaving method for sterilization should be evaluated before finalizing the method. For diclofenac sodium injections, the aseptic filtration process has been recently recommended as the alternative to the autoclave method that produces impurity. Measures by pharmacopoeias It has been observed in the pharmacopoeias that there is an impurity limit shown in the specifications of certain raw materials but not given in the case of products made of those raw materials. Al-though the impurity limit on the drug substances is applicable to the drug products, it would be convenient for the users if the impurity limits were also mentioned in each dosage forms. The limits may vary from orals to injectables. Diclofenac sodium is such an example where an impurity limit is not mentioned in the case of injections. ICH recommendations can be incorporated in the pharmacopeias. In addition, a generalized monograph on the impurity issues can be added in the pharmacopoeiaCHAPTER-5 IMPURITY DETECTION, ISOLATION AND CHARACTERIZATION METHODS FOR IMPURITY DETECTION 1, Isolation 2. Characterization a. Classical Approach b. Modern Approach Isolation Isolation of impurity is necessary for accurate monitor, but it is only necessary if instrumental method is not available as it directly characterizes the impurity. Which method used for isolation and characterization of impurity depends upon the nature of the impurity that means structure of impurity, its physicochemical properties and availability. For isolation of impurity methods used are, Chromatographic and Non-Chromatographic. Preparative chromatography In reversed phase chromatography good isolation can be achieved, by using stationary non polar phase and polar mobile phase with polarity modifiers, pairing ions, buffers. Due to diverse nature of impurity, not only RP-HPLC , but following are also may be of good choice. Normal phase chromatography © Supercritical fluid chromatography * Gas chromatography © Capillary electrophoresis After isolation sample may contain artifacts from the isolation process, this constituents are referred as “chemical noise” (Artifacts:-something observed in a scientific investigation or experiment that is not naturally present but occurs as a result of the preparative or investigative procedure.)NOISE (Chemical Noise) Chemical Noise is a small signal which indicates presence of unwanted foreign compounds other than the required compound. Chemical noise is derived from chemical components in the matrix other than the target analyte, and always increases the absolute intensity of the signal recorded for the analyte by increasing the absolute level of the measured signal in the measurement window. The presence of chemical noise has a predictably deleterious effect on the signal to noise (S/N) ratio for the analyte signal. CHARACTERIZATION METHODS Characterization of Impurity has 2 Approaches 1) CLASSICAL APPROACH 2) MODERN APPROACH 1) CLASSICAL APPROACH A) Separation and Determination of Impurities with Known Structure B) Isolation of the Impurity and off-Line Structure Elucidation 2) MODERN APPROACH Using on-line hyphenated separation/spectroscopic methods 1) Classical Approach A) SEPARATION AND DETERMINATION OF IMPURITIES WITH KNOWN STRUCTURE T) HPLC IDTLCII) Electrophoretic and Related Methods Y Capillary Electrophoresis Y Electrophoresis-related Chromatographic Techniques TV) Methods not Requiring Separation Y Polarography & UV 1) HPLC: HPLC is often used with UV Chemometric methods used for separation optimisation but also for improving quantitative analysis Dimension 150-250 x 4.0-4.6 mm RP-stationary phase with particle size of 3.5-5.0 ym Rt in range of 10-20 min * Ultra-performance liquid chromatography (UPLC) uses short and narrow-bore columns with small particle size packing Exp Impurities of primaquine phosphate dimensions of the column were 50 x 2.1mm C18 silica packing and 1.7 pm separation was achieved within 2 minutes 11) TLC It has good ease of use, cost-effectiveness, good sensitivity, speed of separation capacity to analyze multiple samples simultaneously complementary technique to HPLC HPTLC method is suitable for quantitation of impurities of alprazolam Over pressured liquid chromatography (OPLC) a forced flow variant of HPTLC for determination of impurities of norethisterone by fluorimetric scanning after sulphuric acid spray.II) Electrophoresis & Related Methods * Capillary Electrophoresis + CBis primarily suitable separation and quantification of charged impurities example for use detection of ammonium ion + Useful for ammonium salts as impurities not contributed by residue of ignition and not detected by TLC or HPLC methods * Electrophoresis-related Chromatographic Techniques © micro emulsion electro kinetic chromatography (MEEKC) and capillary electro chromatography (CEC). Exp determination of impurities in ketorolac by MEEKC Determination of the extremely toxic N-methy]-4- phenyl- 1,2,3,6 tetrahydropyridine impurity in pethidine Performances of HPLC, CE and MEKC compared Shortest run time (less than 5 min) was achieved by CE. Best sensitivity by MEKC IV) Methods not Requiring Separation A) POLAROGRAPHIC AND UV METHODS, Electro analytical methods never played important role in drug analysis due to limited selectivity and sensitivity An example is the POLAROGRAPHIC AND UV METHODS Exp Determination of benzophenone impurity in phenytoin impurities and low concentration of phenytoin do not allow direct use of UV spectrophotometry in drug impurity profiling UV Derivative spectrophotometry is advantageous Used in drug degradation studies where the relative concentration of the degradants is in per cent range Exp Determination of omeprazole sulphone in omeprazole monitoring of the photo degradation of amlodipine to it pyridine degradation product B) ISOLATION OF THE IMPURITY AND OFF-LINE STRUCTURE ELUCIDATION The Techniques applied for isolating impurity and elucidating its structure offline also utilizes some hyphenated analytical approaches. But offline elucidation is not clear in results as the quantity of Impurity is very less.2) Modern Approach Using On-Line Hyphenated Separation/Spectroscopic Methods 1) LC-MS Very often the molecular mass of the impurities are obtainable by using the standard soft ionisation techniques Tandem Mass Spectrometer is available (HPLCMS/ MS) so, fragmentation of impurity can be used for the structure elucidation Group exchanged on-line hydrogen to deuterium by using D20 as HPLC mobile phase component powerful tool for identifying active hydrogen atoms (hydroxyl, thiolamine, acid groups) Useful for distinguishing between isomeric structures, difficult by product ion spectral data or accurate mass data Separation power and usefulness of HPLC-MS as a tool for rapid identification of several impurities with different polarities can be improved by using the “heart-cut” technique 2) HPLC-APCI-MS + (HPLC — atmospheric pressure chemical ionisation — MS)for the identification of impurities co-eluting with the main component (acids, bases and zwitterions) not detectable by UV- DAD detector. 3) COMET (Comprehensive Orthogonal method Evaluation Technology) (HP 1100 LC System] Equipped with vacuum degasser quaternary pump auto-sampler UV diode array detector HP-MSD mode ID mass spectrometer thermo stated column selector 0 six-position Cheminert valve is inserted on “D” line of quaternary pump to extend mobile phase selection Both column switcher and valve are connected through contact closures controlled electronically through the Chem- Station software.COMET Instrumentation Column Spidering fully automated Comprehensive Orthogonal Method Evaluation Technology (COMET) 4). GC-MS: Now a days mass spectroscopy connected with various hyphenated techniques like GC-MS, LC-MS,LCMS-MS HPLC- DAD-MS, HPLCDAD- NMR-MS, Tandem Mass spectroscopy and capillary electrophoresis-Mass spectroscopy,. To identify different substances within a test sample gas chromatography-mass spectrometry (GC- MS) method used, that combines the features of gas- liquid chromatography and mass spectroscopy. In this method gas chromatography separate volatile and semi- volatile compounds with great resolution. MS: can provide detailed structural information on most compounds such that they can be exactly identified, but it cannot readily separate them. Sample vaporized by injection into a heated system, eluted through a column by inert gaseous mobile phase and detected. The sample is transported through the column by the flow of an inert, gaseous mobile phase, the carrier gas. Flow is regulated. The carrier gas must be removed and GC peak components transferred to the MS ion source‘Advantages high-resolution separations highly selective sensitive detection wide ionisation capabilities Sample injector T regulated oven Mass spectrometer Column: detector packed or open tubular (capillary) Gas: He, No, Hy 5) LC-MS: LC/MS is a hyphenated technique, combining the separation power of HPLC, with the detection power of mass spectrometry. LC/MS became really popular with the introduction of the thermo spray interface and the particle beam interface. This is same as GC-MS but removal of liquid carrier from an HPLC eluent before samples are passed in to the MS source. For handle normal eluent flow rate 0.5-2.0ml min-1 which is not handled by MS pumping system moving belt inlet systems, jet separators and vacuum nebulizers are used. 6) TLC-MS Separated spots on the plate could be subjected to direct matrix assisted-laser- desorption ionization * Time-of-flight mass spectrometry (TLCMALDI TOF - MS)no need to remove the spots from plate * Only wetting spot with methanolrequired to transfer the analytic from inside the silica gel to the surface + thus enhancing the MALDI-TOF-MS signal 7) LC-NMR Majority of applications are from the fields of metabolite and natural product analyses. stopped flow HPLC-NMR technique (together with HPLC- MS) in identifying impurities in acarboseCHAPTER-6 CONCLUSIO! Identification of impurities is very important task during the synthesis of drug substances and manufacture of dosage forms. It can provide crucial data regarding the toxicity, safety, various limits of detection and limits of quantitation of several organic and inorganic impurities, usually accompany with APIs and finished products. ICH has outlined guidelines with regard to impurities but much more need to be required. There is strong requirement to have unified specifications/standards with regard to impurities.Books & Author Gorog. Sandor, Chemical and analytical characterization of related organic impurities in drugs. Anal. Bioanal.Chem, 2003; 377: 852 — 862. Sanjay B. Bari, Bharati R. Kadam, Yogini S. Jaiswal, et al. Impurity profile: Significance in Active Pharmaceutical Ingredient. Eurasian J. Anal. Chem, 2007; 2(1): 32-53. Jiben roy, Pharmaceutical Impurities - A mini Review. AAPS Pharm. sci tech, 2002; 3(2): 1-9. Janko Zmitek et.al. Sources of impurities — investigation of 4 — (2 — chlorophenyl ) — 3 — ethoxy carbonyl — 5 — methoxy carbonyl — 6 — methyl — J.Surya_Hrd 2000 _Handbook of Modern Pharmaceutical analysis. Academic press, 2001; 298. Poonam Kushwaha. The organic impurities in pharmaceuticals. Pharma info. net., 2008, vol.6(4). Website ceutical products+chemistry+project+pdf&oq=effects+of+impurities +in+pharmaceutical+products+chemistry+ > www.google.com