Impurity Testing

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IMPURITY TESTING Presented by , Guided by, MR.HEM G. SHAH Dr. Vipul M. Vaghela M.Pharm Sem.-II(Q.A) Associate Professor Enrollment No:- 03 Department of p’ceutical Chemistry ARCP Department of Quality Assurance A.R. College Of Pharmacy & G.H. Patel Institute of Pharmacy

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content Introduction Definition Objective ICH guideline (Qualification of impurity) Sources of impurities ICH limits Classification of impurities To be continue…… 2

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Analytical Method Development Reference standard method Spectroscopic method Separation method Isolation method Characterization method Purposeful degradation study Drug substance degradation study Drug product degradation study 3

Introduction: 

Introduction The control of pharmaceutical impurities is currently a critical issue to the pharmaceutical industry . The International Conference on Harmonization (ICH) has formulated a workable guideline regarding the control of impurities. There are various types and origins of impurities in relation to ICH guidelines and, degradation routes. 4

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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. 5

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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 a designed approach and guidance for isolating and identifying process-related impurities and degrada-tion products using mass spectrometry, Nuclear Magnetic Resonance (NMR), high-performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy ( FTIR), and tandem mass spectrometry for pharmaceutical substances. 6

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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 . 7

Definition: 

Definition Impurity: Unwanted chemicals that remains with the active pharmaceutical ingredients (API), develop during formulation and upon aging of API/drug products. Impurity Profile Description of the identified and unidentified impurities present in a typical batch of API (Active Pharmaceutical Ingredient) produced by a specific controlled production process. 8

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Objective Various regulatory authorities and Health Agency are emphasizing on the purity requirements and the identification of impurities API. Qualification of the impurities is the process of acquiring and evaluating data that establishes biological safety of an individual impurity; thus, revealing the need and scope of impurity profiling of drugs in pharmaceutical research. 9

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Maximum daily dose Reporting T hreshold Identification T hreshold Qualification threshold ≤2g/day 0.05% 0.10% or 1.0mg per day intake (whichever is lower) 0.15% or 1.0mg per day intake (whichever is lower) >2g/day 0.03% 0.05% 0.05% 10 ICH LIMITS FOR IMPURITIES

ICH limits: 

ICH limits Threshold for impurity in new drug substance (Q3A(R1)) Reporting threshold : A limit above which an impurity should be analytically reported . Identification threshold : A limit above which an impurity should be structurally identified. Qualification threshold : A limit above which an impurity should be toxicologically qualified. 11

ICH guidelines : 

ICH guidelines Codes Title Q 3 A (R1 ) Dated 25 October 2006 Impurities in new drug substances Q 3 B (R2 ) Dated 2 June 2006 Impurities in new drug product Q 3 C (R3 ) Dated 17 July 1997 Impurities: Guidelines for residual solvents 12

Classification of impurity: 

Classification of impurity 1. Impurities associated in with APIs Organic impurities Inorganic impurities Residual solvent 2. Impurities forms during formulation Method related Environmental related Dosage form related 3. Formation of impurities on aging Mutual interaction amongst ingredients Functional group related typical degradation 13

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Impurity associated with API Organic Impurity Starting material 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 some one manufacturer or be an intermediate for another. By product 14

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Degradation product 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 ß-lactam ring as well as that of an amino group in the C6/C7 side chain plays a critical role in their degradation. 15

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Trans- phenylcyclopropylamine cis - phenylcyclopropylamine 16 Isomeric impurity Trans (+) or (-) phenylcyclopropylamine active form of the sympathominetic drug and is official in USP. The USP prescribed a paper chromatography method for detection of the geometric impurity cis - phenyl cyclopropylamine .

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Enantiomeric impurities: - There are many drugs in which only single enantiomer is active. In such cases the inactive enantiomer is considered as an impurity, e. g. In pilocarpine only Dextro form is active, here levo form is considered as an impurity. 17

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B . Inorganic impurity Reagent , ligands, and catalysts : The chances of having this impurities are rare. However , in some processes, these could create a problem unless the manufacturers take proper care during production. Heavy metal : T he main sources of heavy metals are water used in process and the reactor , where acidification or acid hydrolysis takes place. These impurities can easily avoided using demineralised water. 18

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C . Residual Solvent Residual solvents are defined as the organic volatile chemicals that are used or produced in the manufacture of drug substances or excipients or in the preparation of the drug products. The solvents are not completely removed. No therapeutic benefit. All the solvent should be removed to the extent possible to meet product specification or other quality based requirements 19

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There are several nonspecific methods available that can determine the total amount of solvent(s) in a sample . Loss on drying (LOD) determines the amount of volatile components that are released from a sample under specific temperature and/or vacuum conditions . Thermal gravimetric analysis ( TGA) measures the loss of volatile components from a sample over a temperature gradient . The advantage of these methods is that they give an estimate of the volatile component content of a sample relatively quickly. The disadvantages of these methods are that they do not speciate and cannot account for volatile components that are trapped in the lattice structure of the compound. 20

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To determine the identity and quantity of residual solvents in a sample, a separation of the residual solvents from the matrix and from each other must occur. Gas chromatography (GC) is well suited for this type of separation since GC relies on sample volatilization for its separation mechanism . Most solvents used in manufacturing processes have low boiling points ( < 200 ◦C), so they are easily volatilized and can be separated using the proper chromatographic and instrument conditions. 21

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Classification of Residual solvent Solvents are evaluated for their possible risk to human health and placed in to one of three classes as follows: Class 1 solvents : solvent to be avoided. Class 2 solvents : solvents to be limited. Class 3 solvents : solvents with law toxic potential. Other solvents (“class 4”) : no adequate toxicological data. 24

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Class 1 Solvent : Solvent to be avoided Solvents in class 1 should not be employed in the manufacturing of drug substance because of their unacceptable toxicity or their deleterious environmental effect. However , if we want to use it in order to produce a drug product with significant therapeutic advance, their level should be restricted as shown ; 25

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Solvent Concentration in ppm concern Benzene 2 Carcinogen Carbontetrachloride 4 Toxic and environmental hazard 1,2 Dichloroethan 5 Toxic 26

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Class 2 Solvent : Solvent To Be Limited Solvent in the following table should be limited in pharmaceutical product because of their inherent toxicity. Nongenotoxic animal carcinogens or possible causative agent other irreversible toxicity such as neurotoxicity or teratogenicity . 27 Solvent Permitted daily exposure (mg/day) Concentration limit (ppm) Acetonitrile 4.1 4100 Chlorobenzene 3.6 3600

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Class 3 Solvent : Solvent with low toxic potential Regarded as less toxic and of lower risk to human health. No long term toxicity or carcinogenicity studies. less toxic in acute or short term studies and negative in Genotoxicity studies. E.g - acetic acid - acetone - anisole - 2-propanol - methyl acetate - ethyl ether. 28

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Other solvents (“class 4”): no adequate toxicological data The following solvent may also be of interest to manufacture of Drug substances or drug product. E,g : - Isooctane - petroleum ether - methyl isopropyl ketone - trichloroacetic acid . 29

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2 . Impurity Forms during formulation Method Related known impurity 1-(2, 6-dichlorophenyl) indoline-2 one is formed in the production of parenteral dosage form of diclofenac sodium if it is terminally sterilized by autoclave. 30

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Environmental Related Exposures to adverse temperatures- Vitamins as drug substances are very heat sensitive and degradation frequently leads to loss of potency in vitamin products, especially in liquid formulation. Light: Methyl ergometrine injection is unstable under tropical condition such as light. 50% of marketed sample was found to be low level of active ingredient and did not comply with the BP/USP limit of 90% to 110% of stated content. The injection of ergometrine showed almost complete degradation when kept 42 hours in direct sunlight. 31

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Humidity; For hygroscopic product, humidity is considered detrimental to both bulk powder and formulated solid dosage forms. E.g Aspirin and ranitidine . Dosage form related Flucinonide topical solution USP , 0.05% in 60 ml bottles, was recalled in US because of impurities leading to sub potency. In general , liquid dosage form are very much susceptible to both degradation and microbiological contamination. In this regard , water content , pH of solution, mutual interaction of ingredient and the primary container are critical factor. 32

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3. Formation of impurities on aging Mutual interaction among the ingredients The presence of nicotinamide in a formulation containing four vitamin ( nicotinamide , pyridoxine, riboflavin, thiamine ) can cause degradation of thiamine to sub standard level within a five year shelf life of vitamin B complex injection. Functional group related impurities Ester hydrolysis : common phenomenon to ester type of drug e.g. aspirin to salicylic acid and acetic acid, benzocaine , cefotataxime 33

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Hydrolysis - Hydrolysis is a common phenomenon for the ester type of drugs, especially in liquid dos-age forms. Examples include benzylpenicillin , barbitol , chloramphenicol, chlordiazepoxide , lincomycin , and oxazepam . R 1 COOR 2 + H 2 O R 1 –COOH + R 2 – OH Oxidative degradation - Oxidation is well known chemical degradation pathways for pharmaceutical. Oxidation mechanism for drug substances depend on the chemical structure of drug & the presence of reactive O 2 species or other oxidant. 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 ac-ids), heterocyclic aromatic rings, nitroso and nitrite derivatives, and aldehydes ( eg , flavorings) are all susceptible to oxidative degradation. 34

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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. 35 Decarboxylation - Some dissolved carboxylic acids, such as p- aminosalicylic acid, lose carbon dioxide from the carboxyl group when heated. Decarboxylation also occurred in the case of photoreaction of rufloxacin .

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Packaging materials : Impurities result also from 25 packaging materials i.e., containers and closures . For most drugs the reactive species for impurities consists of; Water – hydrolysis of active ingredient. Small electrophiles – Aldehydes and carboxylic acid derivatives . Peroxides – oxidize some drugs. Metals – catalyze oxidation of drugs and their degradation pathway. Extractable or leachables – Emerge from glass, rubber stoppers and plastic materials, in which oxides like NO , SiO CaO , MgO are major components leached or extracted from glass . 36

Analytical Method Development: 

Analytical Method Development The impurities can be identified predominantly by following methods; Reference standard method Spectroscopic method Separation method Isolation method Characterization method 37

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1. Reference standard method The key objective of this is to provide clarity to the overall life cycle, qualification and governance of reference standards used in development and control of new drugs. Reference standards serve as the basis of evaluation of both process and product performance and are the benchmarks for assessment of drug safety for patient consumption. These standards are needed ,not only for the active ingredients in dosage forms but also for impurities, degradation products, starting materials, process intermediates, and excipients 38

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2. Spectroscopic methods The UV, IR, MS, NMR and Raman spectroscopic methods are routinely being used for characterizing impurities. 3. Separation method The Capillary electrophoresis (CE), Chiral Separations, Gas Chromatography (GC), Supercritical Fluid Chromatography (SFC), TLC, HPTLC, HPLC are regularly being used for separation of impurities and degradation products. 39

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4. Isolation methods Generally, chromatographic and non-chromatographic techniques are used for isolation of impurities prior its characterization. In loratidine , impurity found was ofloratidine . A list of methods that can be used for isolation of impurities is given below. • Solid-phase extraction methods • Liquid-liquid extraction methods • Accelerated solvent extraction methods • Supercritical fluid extraction • TLC, GC, HPLC, HPTLC, Capillary electrophoresis , 40

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5. Characterization methods Highly sophisticated instrumentation, such as MS attached to a GC or HPLC, are inevitable tools in the identification of impurities. For characterization of impurities, different techniques are used; which are as follows; 5.1. NMR Provide information regarding the specific bonding structure and stereochemistry of molecules of pharmaceutical interest has made it a powerful analytical instrument for structural elucidation. Unfortunately, NMR has traditionally been used as a less sensitive method compared to other analytical techniques. Conventional sample requirements for NMR are on the order of 10 mg, as compared with MS, which requires less than 1 mg 41

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5.2. MS Advantage over the NMR, that directly connect separation techniques with Mass Spectrometers have afforded new opportunities for monitoring, characterizing, and quantification of impurity. If single method fails to provide the necessary selectivity, orthogonal coupling of chromatographic techniques such as HPLC-TLC and HPLC-CE, but hopefully only as a development tool rather than a tool for routine QC use. Hyphenated Methods: • LC-MS-MS • HPLC-DAD-MS • HPLC-DAD-NMR-MS • GC-MS • LC-MS 42

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An example of reverse-phase LC-MS analysis in gradient elution with two distinct soft ionization techniques is the Atmospheric pressure ionization with electrospray source (API-ESI) and the chemical ionization of d- allethrine . The popularity of LC-MS-MS systems for complex mixture analysis of thermally labile and biologically relevant molecules is largely attributed to the “soft” nature of atmospheric pressure chemical ionization (APCI), and atmospheric pressure ionization (APPI), and such other techniques are almost routinely used. In GC-MS of methamphetamine and in LC-MS of risperidone , and cetrizine tablets are found to be perfectly suitable for initial characterization of the impurities . 43

HPLC-DAD-MS: 

HPLC-DAD-MS 44

Degradation studies: 

Degradation studies Degradation product : According to ICH Guidelines on impurities in new drug product, a degradation product is defined as a chemical change in the drug molecule brought about over time and/or by action of e.g. light, temperature , pH or water or by reaction with an excipient and/or the immediate container/closure system. 45

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Objective : To determine the intrinsic stability of the molecule by establishing degradation pathways To identify the likely degradation products and to validate the stability indicating power of the analytical procedures used. to provide data on forced decomposition products and decomposition mechanisms . 46

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1. Drug substance degradation study The ICH guidelines specifically state: Stress testing is likely to be carried out on a single batch of material and to include the effect of temperatures in 10 ◦ C increments above the accelerated temperature test condition, humidity where appropriate , oxidation and photolysis on the drug substance plus its susceptibility to hydrolysis across a wide range of pH values when in solution or suspension . 47

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A) Acid B ase stress testing Acid/base stress testing is performed to force the degradation of a drug substance to its primary degradation products by exposure to acidic and basic conditions over time. To initiate studies, a preliminary solubility screen of the drug substance is performed. Solubility of at least 1 mg/mL in 1 N acidic and 1 N basic conditions is recommended. Acid/base reactions should be initiated at room temperature in the absence of light and heat . If no degradation is observed at room temperature, then the temperature can be increased. 48

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If the 24-h time point shows 10–20% degradation and the primary degradants are understood, there is no need to continue the reaction out to the 1-week point If degradation is not achieved, additional hydrolysis experiments should be performed at no more than 70 ◦C for a 1-week total reaction time. Guidance on Acid/Base Experimental Setup Samples : Drug substance + acid (1 N HCl ) Drug substance + base (1 N NaOH ) Drug substance “as is” Acid control (1 N HCl ) Base control (1 N NaOH ) Kinetic points : 0–1 week 49

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B) Thermal and Thermal/Humidity Stress Testing 10◦C increase in temperature results in a doubling of the reaction rate and a decrease in the reaction time . Using this rule of thumb, 1 year at 30 ◦C is equivalent to 3 weeks at 70 ◦C. As a result, the recommended study length for samples to predict a 2-year room temperature shelf life is 6 weeks at 70 ◦C. it is important to determine early kinetic points at 70 ◦C to get an understanding of the primary degradants . 50

Arrhenius expression: 

Arrhenius expression Based on this estimate, a 10◦C increase in temp. results in a doubling of the reaction rate and a decrease in the reaction time by a factor of 2. Temperature Length of storage 30 °C 1 year 40 °C 6 months 50 °C 12 weeks 60 °C 6 weeks 70 °C 3 weeks 80 °C 11 days Guidance for Thermal/Humidity Experimental Setup Samples: 70 ◦C/30% RH (ambient humidity) 70 ◦C/75% RH Time points: 0–6 weeks

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C). Oxidation Mechanism Initiation RH R∙ + (H∙) 2. Propagation R∙ + O 2 RO 2 ∙ X= free radical inhibiter RO 2 ∙ + RH ROOH + R∙ RH= drug substance 3. Hydroperoxide decomposition ROOH RO∙ + ∙OH 4 Termination RO 2 ∙ + RO 2 ∙ Inactive product RO 2 ∙ + X Inactive Product 52

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Our pressurized oxidation approach is run with radical initiators to accelerate oxidation. key predictive samples with 10–20% degradation are typically generated within 10 days using the addition of 1–10 mole % radical initiator. Additionally, the system is heated to accelerate degradation . The temperature depends on the free radical initiator selected . Another variable is the reaction solvent. Oxygen solubility depends on the solvent used in an oxidation reaction. 53

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Kinetic points are taken along the reaction pathway and quenched using 1–10 mole% antioxidant. Guidance for Oxidative Degradation Experimental Setup Samples: Oxygen with initiator Oxygen without initiator Argon with initiator Thermal control Initiator and antioxidant without drug substance Time points : 1-10 days 54

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D). Photostability Purposeful degradation is used to evaluate the overall photosensitivity of the material to elucidated degradation pathway stress testing can be performed using the Suntest chamber , the total time needed to perform a photostability study can be decreased from 1 month to approximately 5 days. Solid drug substances should be spread across the container to give a thickness of typically not more than 3 millimeters . The Rayonet RPR-200 reactor is positioned horizontally to get maximum stirring and homogeneous light coverage. 55

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For stress testing using a Rayonet RPR-200 reactor , vials to be used should be filled with sample, sealed, and then placed along the length of the vial to measure the sample depth. The depth of the material in the vial should not be greater than 3 mm. If the depth of the material is greater than 3 mm, a vial with a larger diameter should be used. Samples should rotate continuously within the chamber to obtain uniform exposure. Light measurements should be taken with an actinometer . Actinometry , the calculation of the number of molecules reacted per photon absorbed, is performed with either a physical device or a chemical system. 56

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57 Rayonet RPR-200 reactor

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E). Ultraviolet Exposure Guidance for Ultraviolet Degradation Experimental Set Up Samples: Ultraviolet Thermal foil-wrapped control Time points: 5× and 10× ICH * Note : ICH ultraviolet conditions = 200 watt h/m2. F). Fluorescence Exposure Guidance for Fluorescence Degradation Experimental Set Up Samples: Fluorescence Thermal foil-wrapped control Time points: 5× and 10× ICH * Note : ICH fluorescent conditions = 1.2 × 106 lux hrs 58

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2). Drug Product Degradation Studies Drug product degradation cannot be predicted from the stability studies of the drug substance in the solid state or solution. The excipients can also react with the drug substance or catalyze degradation reactions. Purposeful degradation studies are performed to determine the physical and chemical compatibility of the drug substance with excipients. It is critical to run the proper controls: the drug substance, drug product, and placebo for the purpose of complete understanding of the degradation pathway. 59

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In the chromatographic screening of degradation samples, it is extremely useful to use the same methods for drug substance and drug product to allow easier understanding of chromatographic differences. For a solid drug product, key experiments are thermal, humidity, photostability and oxidation , if applicable. The most common type of interaction in solid dosage forms is between water and the drug substance. Hence, thermal and thermal/humidity challenges are critical. For solution formulations, key experiments are thermal, acid/base hydrolysis, oxidation and photostability . 60

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Additionally, effects of actual drug product storage containers should be built into the solution drug product studies since the container is considered to be a part of the formulation. Oxygen permeation is also more significant in plastic than in glass containers; hence, the container should be considered in the drug product design as well. All excipient chemical reactions should be incorporated into the experimental design. For example, drugs that contain primary and secondary amines functionality undergo Maillard reactions with lactose and other reducing carbohydrates such as glucose and maltose under pharmaceutically “reasonable conditions.” This reaction should be considered during formulation development. Alternative excipients such as mannitol , sucrose, and trahalose , which are not subject to the Maillard reaction, should be used in place of lactose in such cases. 61

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References: 

References S.Ahuja & S.Scypinski , Morden Pharmaceutical Analysis, Volume 3, 95-111 http://www.pharmainfo.net/reviews/impurity-profile-active-pharmaceutical-ingredient-review http://www.ijperonline.com/july_sep_2010/301-310.pdf http://www.aapspharmscitech.org Eurasian journal of analytical chemistry;volume-2;2007. 66

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