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`==========:-------------------------------- www.japtr.org
`
`REVIEW ARTICLE
`
`Recent trends in the impurity profile of pharmaceuticals
`
`Kavita Pilaniya,
`Harish K. Chandrawanshi',
`Urmila Pilaniya',
`Pooja Manchandani2,
`Pratishtha Ja.in.3 , Nitin Singh3
`
`Department of Pharmaceutics ,
`Shivdan Singh Institute of Technology
`and Management, Aligarh, U.P.,
`'V.N.S. Institute of Pharmacy, Bhopal,
`2SGSITS, Indore, ' Oriental College of
`Pharmacy, Bhopal, M.P., India
`
`DOI: 10.4103/0110-5558. 72422
`
`J. Adv. Phann. Tech. Res.
`
`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 (APls). The va rious
`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 APls 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.
`
`Key words: Characterization, chromatography, identification, impurities, NMR,
`mass spectrometry
`
`INTRODUCTION
`
`The impurities in drug products can be attributed not only to
`the drug substance or inert ingredients u sed for formulating
`a drug product; but they can also be brought into the drug
`product through the formulation process or by contact with
`packaging of the various impurities that can be found in
`drug products.
`
`"Any component of the drug product tha t is not the chemical
`entity defined as the drug substance or an excipient in the
`drug product." (ICH Q6A: Specifications)_lll I tis important to
`give greater consideration to these detrimental impurities.
`In general, most of these impurities are small molecules.
`This is especially true in solid dosage forms where the
`limited mobility restricts the reactivity of larger molecules.
`For most drugs, the r eactive species consist of water
`
`Address for correspondence
`Dr. Harish K Chandrawanshi, Department of Pharmaceutics,
`Saraswati Nagar, Vidisha - 464 001, M.P., India.
`E-mail: hchandrawanshi@gmail.com
`
`(which can hydrolyze some drugs or effect the dosage
`form performance), small electrophiles (e.g., aldehyde
`and carboxylic acid derivatives), peroxides (which can
`oxide some drugs), an d m etals (which can catalyze
`oxida tion and o ther drug d egradation p a thways).
`Additionally, some impurites can cause toxicological
`problems. The presen ce of these unwanted chemicals,
`even in small amounts, may influence the efficacy and
`safety of the pharmaceutical products. Impurity profiling
`(i.e., the identity as well as the quantity of impurity in the
`pharmaceuticals), is now receiving critical attention from
`regulatory authorities. Th e different pharmacopoeias
`such as BP (British pharmacopoeias), USP (United States
`pharmacopoeias), IP (Indian pharmacopoeias), and so on,
`are slowly incorporating limits to the allowable levels of
`impurities present in active pharmaceutical ingredients
`(AP!s) or formulations. The large number of compounds
`under investigation in drug discovery presents a significant
`analy tical challenge for the detection, quantitation,
`and c harac teriza tion of the compoun ds alone .1'1
`Here, in Figure 1, we have summarized all classes of
`impurities.
`
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`Pilaniya, et al. : Recent t rends in impurity profile
`
`SOURCES OF IMPURITY IN MEDICINES
`
`According to the International Conference on Harmon.iza tion
`(!CH) guidelines, impuri ties associated with APis are
`classified in different ways.
`
`Organic Impurities
`Organic impurities are the most common impurities found
`in every AP! unless pro per care is taken in every step
`involved, throughout the multi-step synthesis. Although
`the end products are always washed with solvents, there
`is always a chance that the residual unreacted starting
`materials remain, unless the manufacturers are very careful
`about the impurities. In a paracetamol bulk, there is a lim it
`test for p-aminophenol, which could be a starting material
`for one manufacturer or be an intermediate for others
`[Figure 2].
`
`Oxidative degradation
`Hydrocortisone, methotrexate, adinazolam, hydroxyl group
`directly bonded to an aromatic ring (e.g., phenol derivatives
`such as catecholamines and morphine), conjugated dienes,
`heterocyclic aromatic rings, nitroso and nitrite derivatives,
`and aldehydes (e.g., flavones) are all susceptible to oxidative
`degradation.
`
`Decarboxi;lation
`Some dissolved carboxylic acids, such as p-aminosalicylic
`acid, lose carbon dioxide from the carboxyl group w hen
`heated, in the case of photoreaction of rufloxacin.131
`
`Hydrolysis
`Hydrolysis is a common phenomenon for the ester type
`of drugs, especially in liquid dosage forms. Examples
`include ben zyl penicillin, barbitol, chloramphenicol,
`chlord iazepoxide, lincomycin, ethyl paraben,1•1 and
`cefpodoxime proxetiJ.151 Moreover, the hydrolysis scheme
`of benzocain has been depicted in Figure 3.
`
`PHOTOLYTIC CLEAVAGE
`
`Pharmaceutical prod ucts are exposed to light while being
`manufactured as a solid or solution, and then they are
`packaged. Most compounds will degrade as solutions
`when exposed to high energy UV exposure (Ergometrine,161
`Nifedipine,171 riboflavin, and phenothiazines are very labile to
`photCrOxidation. ). Fluoroquinolones antibiotics are also found
`to be susceptible to photolytic cleavage.1•1 In ciprofloxacin
`eye d rops, the photocleavage reaction produces the
`ethylenediamine analog of ciprofloxacin.191
`
`HeaYy mct:1.b or
`other residual
`mtub:
`
`O.tbcbasssof
`lnorv,uric
`11npun1ie:1
`
`Other materials
`c,1 -, filtaa1cb,
`
`,....,.,.,
`
`Figure I: Flow chart depicting various kinds of impurities
`
`OH t 0 -COCH, ~
`¢ ¢"'
`
`+
`
`dacetylated
`Paraceta11a
`as bi-produc
`NHCOCH,
`
`✓,;.
`
`NHCOCH3
`Paracetamol
`
`OH
`
`( : ) (CH,CO)2O
`
`Y acetyla:ion
`
`NH2
`p-Aminophenol
`
`Figure 2: Production of paracetamol from intermediate. p-Amin(cid:173)
`o phenol
`
`O~OEI
`
`0~
`~c
`
`+ EtOH
`
`NH2
`
`-EtOH
`¢ C-0 ¢
`=
`NH2 w
`o~0 ~oH 9 0
`¢ FGI ¢ FGI
`= -H2
`
`o~c~OH
`
`FGI
`
`NH2
`
`Pd,C
`
`N02
`
`KUn04
`
`HN03
`N02 H2SQJ
`
`Figure 3: Ester hydro lysis ofbenzocaine
`
`Enantiomeric Impurities
`However, the pharmacokinetic profiles of levofloxacin
`The single enantiomeric form of a chiral drug is now
`(S-isomeric form) and ofloxacin (R-isomeric form) are
`considered as an improved chemical entity that may offer a
`comparable, suggesting the lack of advantages of a single
`better pharmacological profile and an increased therapeutic
`isomer in this regard. For the manufacturers of a single
`index, with a more favorable adverse reaction profile.
`enantiomeric drug (eutomer), the undesirable stereoisomers
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`Pilaniya, et al.: Recent trends in impurity profile
`
`in drug control are considered in the same manner as other
`organic impurities.11°1
`
`Inorganic Impurities
`Inorganic impurities may also be d erived from th e
`manufacturing processes used for bulk drugs. They are
`normally known and identified, and include the following:
`
`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.
`
`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 acidification or acid hydrolysis takes place.
`These impurities of heavy metals can easily be avoided
`using demineralized water and glass-lined reactors.
`
`Other materials (e.g., filter aids, charcoal etc.)
`The filters or filtering aids such as centrifuge bags are
`routinely used in the bulk drugs manufacturing plants and
`in many cases, activated carbon is also used. The regular
`monitoring of fibers and black particles in the bulk drugs
`is essential to avoid these contaminations.
`
`In-Process Production Impurities
`Crystallization related impurities
`Impurity can be any substance other than the material
`being crystallized. Therefore, even the solvent from which
`the crystals are grown can be considered as an impurity.
`When impurities are added specifically to produce a desired
`morphological effect they are referred to as additives. The
`presence of impurities or additives in a crystallization
`system can have a radical effect on cr ystal growth,
`nucleation, and agglomeration, asn also on the uptake of
`foreign ions in the crystal structure.1111
`
`Stereochemistrt; related impurities
`It is of paramount importance to look for stereochemistry
`related compounds; that is, those compounds that
`h ave a similar chemical structure, but different spatial
`orientation. These compounds can be considered as
`impurities in the APis. The single enantiorneric form of a
`chiral drug is now considered as an improved chemical
`entity that may offer a better pharmacological profile and
`an increased therapeutic index, with a more favorable
`
`adverse reaction profile, for example, the pharmacokinetic
`profile of levofloxacin (S-isomeric form) and ofloxacin
`(R-isomeric form) are comparable, other examples are
`levofloxacin (S-ofloxacin), esomeprazole (S-omeprazole),
`and lavalbuterol (R-albuterol).11°1
`
`Solvents remain after processing
`Residual solvents are organic volatile chemicals used
`during the manufacturing process or generated during
`the production. Some solvents that are known to cause
`toxicity should be avoided in the production of bulk
`drugs.1121 Depending on the possible risk to human health,
`residual solvents are divided into three classes [Table l].
`
`Sy11thetic i11termediates and by-products
`Impurities in pharmaceutical compounds or a new chemical
`entity (NCE) can originate during the synthetic process,
`from raw materials, intermediates, and / or by-products.
`Impurity profiling of tablets by GC-MS and MOMA (3,
`4-Methylene dioxy methamphetamine) samples produced
`impurities in the intermediates via the reductive amination
`route.1131
`
`Impurities generated during storage
`A number of impurities can originate during storage or
`shipment of drug products. It is essential to carry out
`stability studies to predict, evalua te, and ensure drug
`product safety.1141
`
`Metal impurities
`Metal acts as an impurity in the APis and excepients. Metals
`can be divided into three classes, as mentioned in Table 2.1151
`
`Leachables I Extractables
`Regulatory, safety, and scientific considerations in evaluating
`extractables and leachables is important, along with strategy
`studies, for analytical identification, quantification, and
`monitoring.11•1
`
`ICH Guidelines
`We have summarized various classes based on impurities
`according to the ICH guideline in Table 3.
`
`ICH Limits for Impurities
`According to the ICH guidelines on impurities in new drug
`products, identification of impurities below 0.1 % level is not
`considered to be necessary, unless potential impurities are
`expected to be unusually potent or toxic. According to the
`
`Table 1: Classification of solvents on the basis of their limit in parts per million (ppm)
`Category
`Name of the solvent/limits
`Unit/specification
`Class I
`Benzene (2 ppm), carbon tetrachloride (4 ppm), methylene chloride
`More than this should be avoided
`(600 ppm), methanol (3000 ppm, pyridine (200 ppm), toluene (890 ppm)
`N, Ndimethylformamide (880 ppm), acetonitrile (41 O ppm)
`Acetic acid, ethanol, acetone (50 mg)
`
`Class II
`Class Ill
`
`More than this should be avoided
`Have permitted daily exposure of 50 mg o r
`less per day, as per the ICH guidelines
`
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`Pilaniya, et al.: Recent trends in impurity profile
`
`Table 2: Classification of metals on the basis of
`their safety concern
`Category
`Class I (metals of significant
`safety concern)
`
`Examples
`Ir (iridinium), Pt (platinum), Rh
`(rhubedium), Mo (molibidnum).
`V (vanadium), Cr (chromium),
`and Ni (nickel)
`Class II (metals with low safety Cu (copper) and Mn
`concern)
`(manganese)
`Class Ill (metals with minimal
`Fe (iron) and Zn (zinc)
`safety concern)
`
`!CH, the maximum daily dose qualification threshold to be
`considered is as follows; <2 g / day, 0.1 % or 1 mg per day
`intake (whichever is lower) >2 g / day, 0.05%.
`i) Organic Impurities
`Each specific identified impurity
`- Each specific unidentified impurity at or above 0.1 %
`-Any unspecific impurity, with a limit of not more than
`0.1%
`- Total impurities
`ii) Residual solvents
`iii) Inorganic impuritiesl 171
`
`Isolation Methods
`It is often necessary to isolate impurities. However, if
`instrumental meth ods arc used, isolation of impurities
`is avoided, as it directly characterizes the impurities.
`Generally, chromatographic and non-chromatographic
`techniques are used for the isolation of impurities prior to
`its characterization. The term 'chromatographic reactor'
`refers to the use of an analytical-scale column, servin g
`both as a flow-through reactor and a separation medium
`for the reactant(s) and product(s). High-performance
`liquid chromatography (HPLC) and the chromatographic
`reactor approach, with solution-phase hydrolysis kinetics
`can be used for an aprepitant (EmendTM) prodrug and
`fosaprepitant dimeglumine.1"1 In loratidine, the impurity
`found was oflora tid ine;l191 other examples include
`celecoxib1201 and amikacin.12'1
`
`T he structure of impurities - unknown degradation
`products in drug substances and drug products - must,
`according to the current FDA a nd EMEA guidelines,
`elucidate if they exceed a level of g reater than 0.1 %.
`Analytical Services provide the latest analytical techniques
`for structure elucidation (e.g., high-field NMR, LC-MSMS,
`GC-MS, and MALDI-TOF) as well as for preparative
`isola tion of unknown impurities (e.g., semi and fully
`preparative HPLC). Software tools for the prediction of
`spectra support the study of our experts.
`
`Table 3: Classification of a guideline on the
`basis of impurities
`Section
`Impurities
`Q3A(R2)
`Impurities in new drug substances
`Q3B(R2)
`Impurities in new drug products
`Q3C(R4)
`Impurities: Guideline for residual
`solvents
`Impurities: Guideline for residual
`solvents (Maintenance)
`PDE for tetrahydrofuran (in Q3C(R3))
`PDE for N-methylpyrrolidone
`(in Q3C(R3))
`
`Q3C(M)
`
`Q3C(M)
`
`Sub-section
`Q3A(R)
`Q3B(R)
`Q3C
`
`such as incomplete ph ase separation, .less-than-qu antitative
`recoveries, use of expensive, breakable specialty glassware,
`and disposal of large quantities of organic solvents. SPE
`is more efficient than liquid -
`liquid extraction, y ields
`qu antitative extractions that are easy to perform, is rapid,
`and can be automated. Solvent u se and laboratory time
`are reduced. SPE is used very often to prepare liquid
`samples and extract semi-volatile or nonvolatile analytes,
`and can also be used w ith solids that are pre-extracted into
`solvents. SPE products are excellent for sample extraction,
`concentration, and cleanup. They are available in a wide
`variety of chemistries, adsorbents, and sizes. Selecting the
`most suitable product for each application and sam ple is
`important.
`
`Liquid - Liquid Extraction Methods
`Liquid - liquid extraction, also known as solvent extraction
`and partitioning, is a method to separate com pounds based
`on their relative solubilities in two d ifferent immiscible
`liquids, usually water and an organic solvent. It is an
`extraction of a substance from one liquid phase into
`another liquid phase. Liquid - liquid extraction is a basic
`technique in chemical laboratories, where it is performed
`using a separating funnel. This type of process is commonly
`performed after a chemical reaction as part of the workup.
`
`3os I
`
`Accelerated Solvent Extraction Methods
`Accelerated Solvent Extraction (ASE) is a better technique
`for th e extraction of solid and semisolid sample matrices,
`using common solvents, at elevated temperatures and
`p ressures. ASE systems are available in the entry level ASE
`150 system and the fully automated ASE 350. Extractions
`that normally take hours can be done in minutes using
`ASE w ith pH hardened pathways, using Dioniumni
`components. Compared to techniques such as Soxhlet and
`sonication, ASE generates results in a fraction of the time.
`The many steps involved in sample preparation can now be
`automated with the ASE flow-through technology. Filtration
`Solid-Phase Extraction Methods
`and clean up of solid samples can be achieved as part of
`the solvent extraction process in a single step. ASE offers
`Solid phase extraction (SPE) is an increasingly useful sample
`a lower cost per sam ple than other techniques, reducing
`preparation technique . With SPE, many of the problems
`solvent consumption by up to 90%.
`associated with liquid - liquid extraction can be prevented,
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`Pilaniya, et al.: Recent trends in impurity profile
`
`Supercritical Fluid Extraction
`Supercritical Fluid Extraction (SFE) is the process of
`separating one component (the extractant) from another (the
`matrix), using supercritical fluids as the extracting solvent.
`Extraction is usually from a solid matrix, but can also be
`from liquids. SFE can be used as a sam ple preparation step
`for analytical purposes, or on a larger scale to either strip
`unwanted material from a product (e.g., decaffeination) or
`collect a desired product (e.g., essential oils). Carbon dioxide
`(CO2) is the most used supercritical fluid, sometimes
`modified by co-solvents su ch as ethanol or methanol.
`Extraction conditions for supercritical CO2 are above the
`critical temperature of 31 °C and critical pressure of 72 bar.
`Addition of modifiers may slightly alter this.
`
`Column Chromatography
`Column chromatography in chemistry is a method used
`to purify individual chemical compounds from mixtures
`of compounds. It is often used for preparative applications
`on scales from micrograms to kilograms. The classical
`preparative chromatography column is a glass tube with a
`diameter of 50 mm and a height of 50 cm to 1 m with a tap
`at the bottom. Two methods are generally used to prepare a
`column; the dry method and the wet method. The individual
`components are retained by the stationary phase differently
`and separate from each other while they are running at
`different speeds through the column with the eluent. At
`the end of the column they elute one at a time. During the
`entire chromatography process the eluent is collected in a
`series of fractions. The composition of the eluent flow can
`be monitored and each fraction is analyzed for dissolved
`compounds, for example, by analytical chromatography,
`UV absorption or fluorescence. Colored compounds (or
`fluorescent compounds, with the aid of an UV lamp) can
`be seen through the glass wall as moving bands.
`
`Flash Chromatography
`Distillation, re-crystallization, and extraction are al l
`important techniques for the purification of organic
`compounds. However, the technique used most commonly
`in modern organic research is 'flash' chromatography.
`In traditional column chromatography the sample to be
`purified is placed on top of a column containing some solid
`support, often silica gel. The rest of the column is then filled
`with a solvent (or a mixture of solvents), which then runs
`through the solid support under the force of gravity. The
`various components to be separated travel through the
`column at different rates and are then collected separately as
`they emerge from the bottom of the column. Unfortunately,
`the rate at which the solvent percolates through the column
`is slow. In flash chromatography, however, air pressure
`is used to speed up the flow of the solvent, dramatically
`decreasing the time needed to purify the sample.1221
`
`Thin Layer Chromatography
`Thin layer chromatography (TLC) is a chromatography
`I 306
`
`technique used to separate mi xtures. Thin la yer
`chromatography is performed on a sheet of glass, plastic
`or aluminum foil, which is coated with a thin layer of
`adsorbent material, usually silica gel, aluminium oxide or
`cellulose. This layer of adsorbent is known as the stationary
`phase.
`
`After the sample has been applied on the plate, a solvent
`or solvent mixture (known as the mobile phase) is drawn
`up the plate via capillary action. As different analytes
`ascend the TLC plate at different rates, separation is
`achieved.
`
`Thin layer chromatography finds many applications to
`determine the components that are contained in plants.
`It is also used for monitoring organic reactions and
`analyzing ceramides and fatty acids; for the detection of
`pesticides or insecticides in food and water; for analyzing
`the dye composition of fibers in forensics and identifying
`compounds present in a given substance, and for assaying
`the radi ochemical purity of radiopharmaceuticals
`[Figure 4]. A number of enhancements can be made to
`the original method, to automate the different steps, to
`increase the resolution achieved with TLC, and to allow
`more accurate quantization. This method is referred to as
`HPTLC or 'high performance TLC'.
`
`Gas Chromatography
`Gas-liquid chromatography (GLC) o r simply gas
`chromatography (GC), is a common type of chromatography
`used in analytical chemistry for separating and analyzing
`compounds that can be vaporized without decomposition.
`Typical uses of CC include testing the purity of a particular
`substance or separating the different com ponents of a
`mixture (the relative amounts of such components can
`also be determined). In some situations, CC may help in
`identifying a compound. In preparative chromatography,
`GC can be used to prepare pure com pounds from a
`mixture.1231
`
`Figure 4: Separation of different chemical constituents by TLC
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`High Performance Liquid Chromatography
`High performance liquid chromatography ( or high pressure
`liquid chromatography, HPLC) is a form of column
`chromatography u sed frequently in biochemistry and
`analytical chemistry, to separate, identify, and quantify
`compounds, based on their idiosyncratic polarities and
`interactions with the column's stationary phase. HPLC
`utilizes d ifferent types of stationary phases (typically,
`hydrophobic saturated carbon chains), a pump that moves
`the mobile phase(s) and analy te through the column,
`and a detector that provides a characteristic retention
`time for the analyte. The d etector may also provide other
`characteristic information (i.e., UV / Vis s pectroscopic
`data for the analyte if so equipped). Analyte retention
`time varies depending on the strength of its interactions
`with the stationary phase, the ratio/ composition of the
`solvent(s) used, and the flow rate of the mobile phase.1241
`
`Supercritical Fluid Chromatography
`Supercritical Fluid Chromatograph y (SFC) is a form
`of normal phase chromatography that is u sed for the
`analysis and purification of low-to-moderate molecular
`weight, thermally labile molecules. It can also be used
`for the separation of chiral compounds. Its pr inciples are
`similar to those of HPLC, however SFC typically utilizes
`carbon dioxide as the mobile phase; therefore, the entire
`chromatographic flow path must be pressurized.
`
`Capillary Electrophoresis
`Capillary electrophoresis (CE), also known as capillary
`zone electrophoresis (CZE), can be used to separate ionic
`species by their charge and friction al forces and mass. In
`traditional electrophoresis, electrically charged analytes
`move in a conductive liquid medium under the influence
`of an electric field. Introduced in the 1960s, the tech nique
`of capillary electrophoresis (CE) was designed to separate
`species based on their size, to charge ratio in the interior
`of a small capillary filled with an electrolyte.
`
`is a method that combines the features of gas - liquid
`chromatograph y and mass s pectrom etry, to identify
`different substances within a test sample. Applications
`of GC - MS include drug detection, fi re in vestigation,
`environmental analysis, explosives investigation, and
`identification of unknown samples. Additionally, it can
`identify trace elements in materials that were previously
`thought to have disintegrated beyond identification. The
`GC - MS has been widely heralded as a 'gold standard'
`for forensic substance identification because it is used
`to perform a specific test. A specific test positively
`identifies the actual presence of a particular substance
`in a g iven sample. A non-specific test merely indicates
`that a substance falls into a category of substances.
`Although a non-specific test could statistically suggest the
`identity of the substance, this could lead to false positive
`identification.
`
`Gravimetric Analysis
`Gravimetric analysis descr ibes a set of method s in
`analytical chemistry for the quantitative determination
`of an analyte based on the mass of a solid. A simple
`example is the measurement of solids suspended in a
`water sample: A known volume of water is filtered, and
`the collected solids are weighed. In most cases, the analyte
`m ust first be converted to a solid by precipitation, with an
`appropriate reagent. The precipitate can then be collected
`by filtration, washed, dried to remove traces of moisture
`from the solution, and weighed. The amount of analyte in
`the original sample can then be calculated from the mass
`of the precipitate and its chemical com position.
`
`UV Spectrometry
`Ultraviolet (UV) spectroscopy is a p h ysical technique
`of the optical spectroscopy that uses light in the visible,
`ultraviolet, and near infrared ranges. The Beer-Lambert
`law states that the absorbance of a solution is d irectly
`proportional to the concentration of the absorbing species
`in the solution and the path length. Thus, for a fixed path
`length, UV / VIS spectroscopy can be used to determine
`the concentra tion of the absorber in a solution. It is
`necessary to know how rapid ly the absorbance changes
`with concentration.
`
`Electron Paramagnetic Resonance
`Electron paramagnetic resonance (EPR) or electron spin
`resonance (ESR) spectroscopy is a technique for studying
`the chemical species that have one or more unpaired
`electrons, such as organic and inorganic free radicals or
`inorganic complexes possessing a transition metal ion. The
`basic physical concepts of EPR are analogous to those of
`NMR, but it is electron spins that are excited here instead
`of spins of the atomic nuclei. As the most stable molecules
`have all their electrons paired, the EPR technique is less
`widely used than NMR. However, this limita tion to the
`paramagnetic species also means that the EPR technique
`is one of great specificity, as ordinary chemical solvents
`and matrices do not give rise to EPR spectra.
`
`Infrared Spectroscopy
`Infrared spectroscopy is the su bset of spectroscopy that
`d ea ls with the infrared region of the electromagnetic
`spectrum. It covers a range of techniqu es, the most
`common being a form of absorption spectroscopy. As with
`all spectroscopic techniques, it can be used to identify
`compoun ds and inves tigate sample compositions. A
`common laboratory instrument that uses this technique
`is an infrared spectrophotometer. The infrared portion
`of the electromagnetic spectrum is usually divided into
`Gas Chromatography - Mass Spectroscopy
`three regions; the near-, mid- and far-infrared, named
`Gas chromatography - mass spectrometry (GC - MS)
`according to their relation to the visible spectrum. TI1e
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`Pilaniya, et al. : Recent trends in impurity profile
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`far-infrared, approximately 400 - 10 cm-1 (1000 - 30 µm),
`lying adjacent to the microwave region, has low energy
`and may be used for rotational spectroscopy. The mid(cid:173)
`infrared, approximately 4000-400 cm-1 (30-2.5 µm), may
`be used to study the fundamental vibrations and associated
`rotational-vibrational structure.
`
`Fluorescence Spectroscopy
`Fluorescence spectroscopy is called as fluorometry
`or spectrofluorometry. It is a type of electromagnetic
`spectroscopy, which analyzes the fluorescence from a
`sample. It involves using a beam oflight, usually ultraviolet
`light, which excites the electrons in the molecules of certain
`compounds and causes them to emit light of a lower energy,
`typically, but not necessarily, visible light. A complementary
`technique is absorption spectroscopy. Devices that measure
`fluorescence are called fluorometers or fluorimeters.
`
`Characterization Method
`H ig hly sophisticated instrumentation, such as MS
`attached to a GC or HPLC, are inevitable tools in the
`identification of minor components (drugs, impurities,
`degradation products, metabolites) in various matrices.
`For characterization of impurities, different techniques
`are used; which are as follows;
`
`Mass spectrometn;
`Mass spectrometers are used in the industry and academia
`for both routine and research purposes. The fol lowing list
`is just a brief summary of the major mass spectrometric
`applications:
`Biotechnology: Is the analysis of proteins, peptides, and
`oligonucleotides.
`Pharmaceuticals: Deal with drug discovery, combinatorial
`chemistry, pharmacokinetics, and drug metabolism.
`Clinical: Deals with neonatal screening, hemoglobin
`analysis, and drug testing.
`En v i ronmen ta l: Dea ls with po lycycl ic aroma tic
`hydrocarbons (PAHs), Polychlorina ted biphenyls (PCBs),
`water quality, and food contamination.
`Geological: Deals with the oil composition.
`
`The instruments include: Ionization source, for example,
`electrospray ionization (ES!) and matrix-assisted laser
`desorption ionization (MALDI); Analyzer mass to charge
`(m/z), for example, quadruple a nd magnet; FT-ICR
`d etector, for example, the photomultiplier micro-channel
`plate electron multiplier.
`
`NMR spectroscopy
`Nuclear Magnetic Resonance (NMR) spectroscopy is a
`powerful and theoretically complex analytical tool. In NMR,
`the chemical environment of the specific nuclei is deduced
`from the information obtained about the nuclei.
`
`Nuclear m agnetic resonance (NMR) is a property that
`magnetic nuclei have in a magnetic field and the applied
`electromagnetic (EM) pulse or pulses, which cause the
`nuclei to absorb energy from the EM pulse and radiate
`this energy back out. The ene