J Pharm Educ Res Vol. 3, Issue No.

1, June 2012

Current trends in forced degradation study for pharmaceutical product development

Ranjit Singh*and Zia ur Rehman
PCTE Institute of Pharmacy, near Baddowal Cantt. Ludhiana, India *E-mail: ranjeet@pcte.edu.in

Received : 21 May, 2012; Accepted: 15 June, 2012 Abstract

Forced degradation is a powerful tool used routinely in pharmaceutical development in order to develop stabilityindicating methods that lead to quality stability data and to understand the degradation pathways of the drug substances and drug products. This article describes the mechanism of formation and characterization of generated impurities during force degradation studies in pharmaceuticals. Force degradation studies ensure appropriate stability of final pharmaceutical products in very early stages of pharmaceutical development. Keywords: Forced degradationstudy, Degradation related impurities, Active pharmaceutical ingredient, International conference on harmonization.

Introduction Forced degradation (FD)study is a process in which the natural degradation rate of a pharmaceutical product is increased by the application of an additional stress. FD studies (i) help to identify reactions that cause degradation of pharmaceutical product, ii) are part of the development strategyand an integral component of validating analytical methods that indicate stability and detect impurities which are formed during manufacture, storage, or use and their properties are different from the desired product with respect to activity, efficacy and safety1 and iii) are designed to generate product-related variants and develop analytical methods to determine the degradation products formed during accelerated and longterm stability studies. Any significant degradation product should be evaluated for characterization and quantization for its potential hazard 2,3. Impurities in pharmaceuticals are the unwanted chemicals that can develop during synthesis or with aging of active pharmaceutical ingredient (API). Presence of impurity even in small quantity may influence the efficacy and safety of pharmaceutical products4,5. Hence, there is increasing interest in impurities present in APIs. Recently, impurity profile has become essential components of the drug development process6. Various regulatory authorities such as International Conference on Harmonization (ICH) and Food and Drug Administration (FDA) recommend the reporting of impurities present
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beyond or allowable limits7-9. The British Pharmacopeia (BP) and United State Pharmacopeia (USP) have also mentioned allowable limits of impurities present in the APIs and formulation. The regulatory authorities have also developed guidelines regarding stress testing on drug substances and drug products to predict the degradation products and degradation pathways which can help in establishing intrinsic stability of the drug substance as well as developing stability indicating assay method (SIAM). There is a significant increase in demand for the impurityreference standard by both the regulatory authorities and pharmaceutical companies10. Metal impurities have been historically monitored in drug articles intended for use by humans and animals. Drug articles include active pharmaceutical ingredients (API), excipients, drug products and dietary supplements along with their ingredients. Some heavy metals, such as arsenic, mercury, cadmium and lead are toxic at very low levels and should be monitored to ensure consumer safety. Sources of metal impurities may include those that are added to the process (such as catalysts), those derived from the process (such as leachables from equipment and contact surfaces), or undetected contaminants from starting materials or reagents11. Rationale for Reporting of Impurity Recently not only purity profile but also impurity profile has become essential as per various regulatory requirements. An impurity is any chemical (organic or

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inorganic) present in a drug substance other than itself or in a drug product and ingredient. It may originate during synthesis of drug substance or formed due to degradation of drug substance or reaction between drug substance and excipients. Hence impurity may be either process related impurity (PRI) which includes starting materials, reaction intermediates, chemical reagents, ligands and catalysts, by-product of synthetic route and residual solvents or degradation related impurity (DRI) which is formed due to hydrolysis, oxidation or photolysis of drug. Any impurity that is required to be identified is present at a level greater than threshold. The purpose of impurity identification and quantification is to provide information on how the quality of a drug substance or drug product varies under the influence of degradation related factors such as temperature, humidity and light and process related factors such as pH, solvents, or reagents which are used in synthetic process. Information on impurity profiling of drug substance is an essential component of chemistry, manufacturing and control (CMC) part of dossier12,13. The ICH guidelines Q3A (R2) require identification of each impurity with respect to both chemistry and safety perspectives. The chemistry perspectives include classification and identification of impurities, report generation, listing of impurities in specification and a brief discussion of analytical procedures while the safety perspectives include specific guidance for qualifying those impurities that were not present or were present at substantially lower levels in batch of a new drug substance and used in safety and clinical studies. These ICH guidelines classify the impurities into three categories 1. Organic impurities 2. Inorganic impurities 3. Residual solvents The organic impurities may arise during the manufacturing process or during the storage of the drug substance and these may be identified or unidentified, volatile or non volatile. The various kind of organic impurities include starting material or intermediate, by products, degradation products, and reagents, ligands, catalysis. The inorganic impurities are normally known and identified which may arise during the manufacturing processes. These are generally detected and quantified

using pharmacopoeial or other appropriate procedures. It mainly includes heavy metals, reagents, ligands, catalysts and other materials like inorganic salts, filters aids, charcoal etc. The residual solvents are organic volatile chemicals used during the manufacturing process or generated during the production of the API. It is very difficult to remove all these solvents completely by the work-up process, however efforts should be taken to maintain the level to meet the safety data. The use of toxic solvents should be avoided in the production of bulk drugs. The ICH guidelines Q3C(R2) guidelines for residual solvents have classified residual solvents into three classes on the bases of their possible risk to human health. Class I solvents like benzene (2 ppm limit) and carbon tetrachloride (4 ppm limit) are to be avoided. Class II include the most commonly used solvents such as methylene chloride (600 ppm), methanol (3000 ppm), pyridine (200 ppm), toluene (890 ppm), N, N dimethylformamide (880 ppm) and acetonitrile (410 ppm). Class III solvents which are least harmful include (acetic acid, acetone, isopropyl alcohol, butanol, ethanol and ethyl acetate) have permitted daily exposure of 50 mg or less per day9. The ICH guidelines Q1A(R2) (2003) elaborate on stability testing of APIs and drug products in order to determine storage conditions, retest period, maximum expiring dating period of drug products, correct packaging to protect the product and transport conditions14. These guidelines recommend the conduct of long term stability studies (25C 2C/60% RH 5% or 30C 2C/65% RH) for 12 months, intermediate stability studies (30C 2C/65% RH 5% RH) for 6 months and accelerated stability study ( 40C 2C/75% RH 5% RH) for minimum 6 months duration. The purpose of stability testing is to provide evidence on how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors such as temperature, humidity and light and other is to establish a retest period for the drug substance or a shelf life for the drug product and recommended storage conditions15,16. Origin of Degradation product/Degradation Related Impurities (DRIs) The main cause of appearance of impurities in drug substance or product is due to its degradation. The chemical instability of the drug substance under
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the conditions of heat, humidity, solvent, pH, and light encountered during manufacture, isolation, purication, drying, storage, transportation, and/or formulation is main cause of its degradation. It is governed by inherent chemical stability of the drug substance. The major routes of degradation of any drug substance include hydrolysis, oxidation, heat and photolysis. The stress testing helps in generation all possible degradation products that may form under different conditions12. 1. Hydrolytic condition Hydrolysis is one of the most common degradation chemical reactions over wide range of pH. Hydrolysis is a solvolytic process in which drug reacts with water to yield breakdown products of different chemical compositions. Water either as a solvent or as moisture in the air comes in contact with pharmaceutical dosage forms is responsible for degradation most of the drugs. For example, aspirin combines with water and hydrolyzed to salicylic acid and acetic acid12,17. Hydrolytic study under acidic and basic condition involves catalyzation of ionisable functional groups present in the molecule. HCl

and NaOH are employed for generating acidic and basic stress samples, respectively18. The hydrolytic degradation of a new drug in acidic and alkaline condition can be studied by refluxing the drug in 0.1 N HCl / 0.1 N NaOH. If reasonable degradation is seen, testing can be stopped at this point. However in case no degradation is seen under these conditions the drug should be refluxed in acid/alkali of higher strength and for longer duration of time (Fig 1). Alternatively if total degradation is seen after subjecting the drugs to initial condition, acid/alkali strength can be decreased with decrease in reaction temperature19. In general temperature and pH are the major determinant in stability of the drug prone to hydrolytic decomposition. Hydrolysis of most of the drugs is dependent upon the relative concentration of hydronium and hydroxyl ions such as (i) Anastrozole, significantly degraded in basic conditions as compared to acidic conditions and two new degradation products were formed under basic pH, ii) Doxofylline, a bronchodilator drug that show degradation more in acidic condition. Hence pH at which each drug is optimaly stable can be determined20,21.

Fig 1: Flow chart for performing hydrolytic degradation 56

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2. Oxidative condition Many drug substances undergo autoxidation i.e., oxidation under normal storage condition and involving ground state elemental oxygen. Therefore it is an important degradation pathway of many drugs. Autoxidation is a free radical reaction that requires free radical initiator to begin the chain reaction. Hydrogen peroxide, metal ions, or trace level of impurities in a drug substance act as initiators for autoxidation12. Hydrogen peroxide is very common oxidant to produce oxidative degradation products which may arise as minor impurities during long term stability studies. It can be used in the concentration range of 3-30% at a temperature not exceeding 40 C for 2-8 days. The oxidative stress testing is initially carried out in 3% H2O2 at room temperature for 6 hr and it can be increased/ decreased to achieve sufficient degradation. The time can also be increased up to 24 hr with 30% or decreased up to 30 min with 1% of H2O222 (Fig 2). The mechanism of oxidative degradation of drug substance involves an electron transfer mechanism to form reactive anions and cations. Amines, sulphides and phenols are susceptible to electron transfer oxidation to give N-oxides, hydroxylamine, sulphones and sulphoxide21. The functional group with labile hydrogen like benzylic

carbon, allylic carbon, and tertiary carbon or positions with respect to hetro atom is susceptible to oxidation to form hydroperoxides, hydroxide or ketone23,24. For example Rapamycin an immunosuppressant drug used in drug-eluting stents reacts with oxygen to form a complex mixture of monomeric and oligomeric products. Formation of the majority of the rapamycin degradation products could be rationalized with free radical-mediated autoxidation reactions involving alkene and alcohol sites25. 3 Thermal condition In general, rate of a reaction increase with increase in temperature. Hence, the drugs are susceptible to degradation at higher temperature. Many APIs are sensitive to heat or tropical temperatures. For example, vitamins, peptides, etc. Thermal degradation involves different reactions like pyrolysis, hydrolysis, decarboxylation, isomerization, rearrangement and polymerization. Effect of temperature on thermal degradation of a substance is studied through Arrhenius equation: K= Ae-Ea/RT where k is specific reaction rate, A is frequency factor, Ea is energy of activation, R is gas constant(1.987 cal/ deg mole) and T is absolute temperature12,24,26.

Fig 2: Flow chart for performing oxidative degradation 57

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Thermal degradation study is carried out at 40C to 80C. The most widely accepted temperature is 70C at low and high humidity for 1-2 months. High temperature (>80C) may not produce predictive degradation pathway9,27. The use of high-temperatures in predictive degradation studies assumes that the drug molecule will follow the same pathway of decomposition at all temperatures. This assumption may not hold true for all drug molecules, and therefore great care must be taken in using the extreme temperatures easily accessible in a sealed-vessel microwave experiment for predictive degradation studies28. 4. Photolytic condition Exposure of drug molecules may produce photolytic degraded products. The rate of photodegradtion depends upon the intensity of incident light and quantity of light absorbed by the drug molecule. Photolytic degradation is carried out by exposing the drug substance (in solid as well as in the solution form) or drug product to a combination of visible and UV light. The most commonly accepted wavelength of light is in the range of 300-800 nm to cause the photolytic degradation29,30. The photolytic degradation can occur through nonoxidative or oxidative photolytic reaction. The nonoxidative photolytic reaction include isomerization, dimerization, cyclization, rearrangements, decarboxylation and hemolytic cleavage of X-C hetero bonds, N-alkyl bond

(dealkylation and deamination), SO2-C bonds etc and while oxidative photolytic reaction occur through either singlet oxygen(1O2) or triplet oxygen(3O2) mechanism. The singlet oxygen reacts with the unsaturated bonds, such as alkenes, dienes, polynuclear aromatic hydrocarbon to form photoxidative degradation products whereas triplet oxygen react with free radical of the drug molecule, which than react with a triplet oxygen molecule to form peroxide. Hence, light can also act as a catalyst to oxidation reactions31. For example photodegradtion of Barnidipin involves probable formation of singlet oxygen that can modify or destroy tissues causing significant damages and loss of therapeutic activity. Hence, the characterization of photodegradtion process involves the study of the transient species and the interaction between precursor and products, is a crucial way to understand the potential photo-toxicity of a drug and determining it32. The overall illumination showed not be less than 1.2 million lux hours and an integrated near ultraviolet energy of not less than 200 W-h/m2. However, illumination is decreased or increased to achieve sufficient degradation. The maximum illumination recommended is 6 million lux h33 (Fig 3). Identification of Degradation Products Detection of the unknown impurity is the first step in impurity identification. The unknown impurity that are observed during the analysis, pharmaceutical

Fig 3: Flow chart for performing photolytic degradation 58

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development, stress studies and formal stability studies of the drug substances and drug product by using various chromatographic techniques like Reversed Phase High Performance Liquid Chromatography (RP-HPLC), Thin Layer Chromatography (TLC), Gas Chromatography (GC), Capillary Electrophoresis (CE) , Capillary Electrophoresis Chromatography (CEC) and Super critical Fluid Chromatography (SFC). The RP-HPLC is most widely used analytical tool for separation and quantifying the impurities and it is most frequently coupled with UV detector12. The behavior of the functional group present in the drug molecules proved helpful in the determination of the nature and origin DRI of the possible degradation. The simplest situation for the DRI identification is when there is a reference impurity standard available. If the DRIs have the resolution factor (Rf) or retention time (RT) is same as the reference impurity than the characterization of DRI is not required but it is denoted as the reference impurity standard. If the resolution factor (Rf) or retention time (RT) of DRI and reference standard does not match , than DRI is isolated from the sample matrix by crystallization, selective solvent-solvent extraction, preparative TLC, HPTLC (High Performance Thin Layer Chromatography) OR HPLC and structural information of DRI can be obtained from IR (Infrared) Spectroscopy, NMR (Nuclear Magnetic Resonance) and Mass spectra12,34. An excellent combination of hyphenated chromatographic and spectroscopic technique such as HPLC-DAD (High Performance Liquid ChromatographyPhotodiode Array ultraviolet Detector), LC-MS (Liquid Chromatography-Mass Spectrometry), LC-NMR (Liquid Chromatography-Nuclear Magnetic Resonance) and GCMS (Gas Chromatography-Mass Spectrometry) are used when DRI cannot be isolated in pure form. HPLC-DAD and LC-MS are used to compare the RRT (relative retention time), UV spectra, mass spectra (MS/MS or MSN)12. 1. Role of HPLC-DAD HPLC-DAD provides diagnostic information about the structure of impurity, but HPLC-DAD is not as informative as LC-MS and LC-NMR. The photodiode array ultraviolet detector (DAD) has proven to be an important tool in identication of compounds as it allows on-line acquisition of their UV spectra. One of the strengths of HPLC-DAD is that oxidation does not
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alter to some extent the chromophores of a drug/impurity, so the drug can be easily identified. If the UV spectrum of an unknown impurity is identical to that of the drug substance, it is likely that the impurity has the same chromophores as that of the drug substances. On other hand, if the impurity has a different UV spectrum from the drug substances, it is an indication that chromophores have been changed in the impurity. In some cases, the UV spectrum can also be used to differentiate target impurity peaks from other components in the sample12. 2. Role of LC-MS LC-MShas become primary tools for identificationof low-level pharmaceutical impuritiesin drug substances and drug products. Because of its superior sensitivity, selectivity and speed, it allows identification of trace components in complex mixtures directly with no prior preparative purification or fractionation. More importantly, automation such as data-dependant multi-stage MS analysis (MS)n allows comprehensive structural information of multiple impurity peaks to be collected on-the-fly during chromatographic separation despite their structural similarity to the main component35. MS detectors are the main part of the LC-MS, where analytes in the eluent are detected. The use of MS detectors as characterization tool depends upon response per unit weight of the analyte which is ultimately affected by ionization mode and ionization efficiency of the molecule. The selection of the ionization mode is depending upon polarity, molecular mass and thermal stability of the analyte and ionization chamber environment. Electrospray Ionization (ESI), Atmospheric Pressure Photo Ionization (APPI) and Atmospheric Pressure Chemical Ionization (APCI) are the most commonly used ionization technique for LC-MS analysis of pharmaceutical active ingredients36. ESI can be used for the molecules with low polarity to ionic compounds, while APCI is better for weak to medium polar compounds. APPI is better technique than ESI and APCI for non polar compounds. For most of the compounds, ESI and APCI are almost interchangeable. ESI can be performed in positive or negative ion mode. In the positive ion mode, the mass spectrum usually featured a protonated molecule [M+H]+ and in many cases along with adduct ions such as [M+Na]+ , [M+K]+ , [M+NH4]+ or [M+solvent or modifier]+. These adduct ions serve as conformation of a protonated molecule because of

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distinctive mass difference between the adduct ions and the protonated ions facilitating the determination of correct molecular mass of the unidentified impurity. Adduct ions are particularly useful when the signal is low and there are interference from the background or other components in the sample. In +ESI mode, the molecules containing functional group like NH2 , -OH , -SH etc form [M+H]+ ions and in ESI mode, the molecule with COOH group or other acidic group form [M-H]- ions37,38. APPI is recent technique which uses a krypton lamp which emits photons of energies 10.0 and 10.6 eV which is sufficient for ionization of most analytes but lower than ionization potential of common reverse phase solvents such as water (12.62eV) and methanol (10.58eV). The background noise in APPI spectrum is significantly reduced than in ESI and APCI spectrum38,39. Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization (AP-MALDI) is the technique which is used to ionize heavy molecules by mechanism similar to MALDI but at atmospheric pressure. This technique has been used in the biopolymers analysis40. Desorption Electrospray Ionization (DESI) is a new technique for analysis of the surfaces. In this technique charged droplets from an electrospray are directed on to sample surface and cause desorption ionization of the analyte on the surface and cause desorption ionization of the analyte on the surface41. The main advantage of this technique is that it gives quick analysis of analyte in the tablets and impurity spots on TLC plates42,43. The liquid samples such as blood, urine or pharmaceutical solutions can be deposited on a neutral surfaces (e.g., Teflon) or PMMA (ploymethylmethacrylate) and then analyzed using DESI44. Direct Analysis in Real Time (DART) is useful for the direct analysis of the molecules from the surface similarly to DESI. In DART the sample expose to a stream of electronically and vibronically excited gas such as helium or nitrogen, which cause the ionization of the analyte. It is used to analyze gas as well as liquid samples and works similarly to DESI for solid surface analysis45. The DART and DESI as compare to ESI are simple, fast, no need of sample preparation and allow direct determination analyte in tablets. But these techniques shows disadvantage that these are not directly coupled to HPLC, although automated sampling from TLC plates can be done33,46.
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Matrix-Assisted Laser Desorption/Ionization (MALDI) mainly used for large biomolecules or biopolymers such as proteins or peptides. In MALDI the sample in a matrix is dispersed on a surface, and desorbed and ionized by the energy of the laser beam. Matrix selection is critical and depends upon the wavelength of laser beam and nature of the sample. The combination of the pulsed laser beam and time-of-flight mass spectrometer is particularly effective47. 3. Role of Tandem Mass spectrometry (Tandem MS) or MS/MS The tandem mass spectrometry is widely used technique during these days to analyze particular component in the mixture. The tandem mass spectrometer consists of a mass spectrometer followed by a field-free collision chamber, followed by second mass spectrometer. The sample is sorted in MS1, which is fragmented in the collision chamber and these fragments are further sorted in the MS2. In other words, the specific ions (parent ions) are separated in the first mass spectrometer (MS1) and passed into the collision chamber where daughter ions are formed and these ions passed into the second mass spectrometer (MS2) and finally these daughter ions are analyzed (Fig 4).

Fig 4: Tandem Mass Spectrometer arrangement

Collision-induced dissociation (CID) is the commonly used dissociation method in which the neutral gas such as helium, argon, nitrogen etc at high collide with the parent ions and give the daughter ions48. Tandem instrument is divided into two types that are tandem-in-time and tandem-in-space. In case of tandemin-time type, different phases of MS/MS are carried out in same analyzer, but in separated time. The trapping instrument falls in this category and they facilitate conducting the MSn experiments. The trapping instrument fail to provide the information regarding scanning of the parent ion as well as the neutral ion. In case of tandemin-space, each stage of MS/MS needs distinct analyzer and normally beam type analyzer suits49,50. The triple

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quadrupole (QpQ) is a typical example, in which three quadrupole exist in linear series. The first is a analyzer, the second act as CID while third is an ion trap respectively. The QpQ can be couple either with TOF or the ion trap to give variety of tandem mass spectrometers51. 4. Role of LC-NMR LC-NMR is an indispensable tool for low level impurity identification. The combination of NMR with HPLC resembles the idea of other techniques such as HPLC-DAD or HPLC-MS. LC-NMR is used when LCMS data is not sufficient to elucidate the structure of DRI. The sensitivity of LC-NMR is low as compare to LC-MS, but this technique has complementary selectivity and very accurately defines an unknown molecular structure. Stop flow and loop collections approaches improve the sensitivity in LC-NMR by increasing the observation time by tracing impurity peak of interest. The main problem of HPLC method compatibility with LC-NMR lies in the interference from the protons of mobile phase components and this can be overcome by replacing Protic solvents such as methanol and water with deutrated solvents to avoid solvent suppression. In LC-NMR mineral acid buffers are preferred over organic buffer or modifier as in LC-MS52,53. The availability of cryogenically cooled low-volume probes and C/N data from indirect proton detection using polarization transfer techniques has radically increased the amount of information available from a small amount of analyte in the NMR coils. An SPE (Solid Phase Extraction) cartridge trapping interface between the LC column and the NMR now allows on-line concentration and purification of very low-level minor components into a low-volume flow cell for high quality NMR spectral data54. 5. Role of LC-NMR-MS Though NMR is arguably the most versatile analytical platform for complex mixture analysis, it is rarely possible to solve the structure of a novel compound/ impurity by NMR alone. Specifically, interfacing liquid chromatography with parallel NMR and mass spectrometry (LCNMRMS) gives comprehensive structural data on identification of impurities. Common functional groups such as carboxylic acid, phenol and amino groups are NMR-silent in many solvents because of proton deuterium exchange. Nitro groups and sulfate conjugates do not contain protons and although these functional groups
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are not directly detectable in proton NMR spectra, they can be readily detected by mass spectrometry (MS). Conversely, MS data might give molecular weight, fragmentation and molecular formulae that are insufficient to unambiguously assign the molecular structure of an unknown compound. In the most difficult cases closely eluting isobars and isomers are indistinguishable by LC MS55. Parallel on-line NMR and MS detection efficiently provides complementary data and minimizes ambiguities between LCMS and LCNMR systems. These integrated LCNMRMS systems are highly versatile56. 6. Role of GC-MS HPLC based technique such as HPLC- DAD and HPLC-MS can analyze at least 80% of the organic compounds and GC based technique such as GC-FID (Gas Chromatography-Flame Ionization Detector) and GS-MS play major role in analysis of the remaining 20% of the organic compounds57-59. These compounds are either not suitable for HPLC analysis (e.g, volatile compounds) or not suitable for ionization by API techniques (e.g nonpolar compounds. Electron Ionization (EI) is a better technique for non polar compounds. EI is hard ionization technique, therefore an EI spectrum can contain many fragment ions because of this inherent feature it is usually not necessary to acquire MS/MS data with EI. On other hand, extensive fragmentation in EI often results in the absence of the molecular ion, which adds the difficulty in the determination of molecular weight of the unknown impurity. Therefore Chemical Ionization (CI) and other soft ionization techniques have been developed12. The identification and quantification of the residual solvents and other organic volatile impurities is done using GC-FID or GC-MS methods. But the importance of GC-MS in the identification of pharmaceutical impurity has decreased due to routine availability of API based LC-MS60.

Conclusions
Forced degradation studies are used to facilitate the development of analytical methodology, to gain a better understanding of active pharmaceutical ingredient (API) and drug product (DP) stability and to provide information about degradation pathways and degradation products. The impurity profiling of the pharmaceuticals is of increasing importance as drug safety receives more and more attention from the public and from the media. Future directions in pharmaceutical DRI profiling will involve

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improvements in both the technique-oriented and the chemistry-guided approaches. As technologies continue to progress rapidly, significant improvements in analytical techniques will lead to much faster DRI profiling and greatly reducing the risk of missing the important DRIs. Multi-dimensional approaches such as coupled analytical separator with multiple detectors are likely to play leading role in pharmaceutical development and impurity profiling. The chemistry-guided approaches will also advance as a result of faster structure-elucidation technologies, improved computational tools and better procedures for stress-testing and mechanistic investigations.

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