list of publications: 1. prajesh prajapati, yadvendra k...

LIST OF PUBLICATIONS

Prajesh Prajapati: Institute of R & D, GFSU 21 of 121

LIST OF PUBLICATIONS:

1. Prajesh Prajapati, Yadvendra K. Agrawal, Analysis and impurity identification in pharmaceuticals, Reviews in Analytical Chemistry. 33 (2) (2014), 123–133. (IF=1.0)

2. Prajesh Prajapati, Y.K. Agrawal, SFC–MS/MS for identification and

simultaneous estimation of the isoniazid and pyrazinamide in its dosage form, The Journal of Supercritical Fluids, 95 (2014), 597-602 (IF=2.56)

List of communicated papers 1) SFC-MS/MS for identification and estimation of the Ethambutol in its dosage form

and human urine samples. (Journal of Chromatography B)

Rev Anal Chem 2014; 33(2): 123–133

Prajesh Prajapati and Yadvendra K. Agrawal*

Analysis and impurity identification in pharmaceuticals

Abstract: Impurity is not a much-liked word by pharma-ceutical and industry people, because they are concerned about quality. Here we discuss various impurities that might be present in API formulations. To fulfill our pur-pose we have compiled a variety of regulatory authorities’ guidelines (i.e., ICH, WHO, and pharmacopoeias), which serve in endlessly regulating the impurities by various means. As the impurity present in a drug can affect its quality and thus its efficiency, it is therefore crucial to know about impurities. The current article reveals the different terms, regulatory control, and basic techniques (e.g., HPLC, LC-MS, TLC) that will help novices to under-stand, identify, and quantitatively estimate impurities and that have the advantage of profiling. This article primarily focuses on identification and control of vari-ous impurities (i.e., organic, inorganic, and genotoxic). For any of the substances, quality is the prime objec-tive. Because impurities can alter quality, understand-ing the various impurities will help in producing quality products.

Keywords: analytical methods; genotoxic impurity; inor-ganic impurity; organic impurity; regulatory requirement in impurity profile.

DOI 10.1515/revac-2014-0001Received January 7, 2014; accepted March 19, 2014; previously published online May 7, 2014

IntroductionThe pharmaceutical world is dedicated to quality. Speak-ing from the customer’s perspective, quality means pleas-ant appearance with good packaging. But in the case of pharmaceutical industries, quality means providing

drug standards conforming to a variety of conditions and making profit from them. So, they should be aware of the various types of impurities and their regulation and control, which infer quality. Therefore, in this paper we have tried to summarize different types of impurities, along with their effects and limitations as given by the International Conference on Harmonization (ICH). ICH has given guidelines [ICH Q-3B (R2) 2006] for impurity in a drug, and according to ICH, it is a chemical entity, which is not defined as a drug per the Drugs and Cosmetic act and which has an impact on the purity of the active pharma-ceutical ingredient or drug substance.

Every pharmaceutical manufacturer defines impu-rity in its own words, making it difficult to find an exact definition of impurity. In the pharma world, impurity can be identified by various terms that we will see later. Drug substances or drug products are prepared with various solvents. Remaining solvents or residual solvents that might be present in the final product often are cited as organic volatile impurities (OVI) (ICH Q-3C [R4] 2009), and the impurities associated with the inactive pharmaceuti-cal ingredients used in formulation or as additives or adju-vants are rarely mentioned.

Bulk pharmaceutical chemicals (BPCs), can be obtained or synthesized from multiple sources and, therefore, it is very important that impurities in BCPs be carefully monitored and controlled. Recently British pharmacopoeia (BP), United State Pharmacopoeia (USP), and Indian pharmacopoeia (IP) started incorporating allowable limits of impurities present in drug substances or drug products (Kovaleski et al. 2007, Gad 2008). This article thoroughly reviews different impurities found in the pharmaceuticals by methods for isolation, extraction, and identity of possible impurities.

Impurity should be defined as identified impurity – an impurity available with information about the structural characterization, and unidentified impurity – an impu-rity that can be identified only with qualitative analytical values (e.g., peak area, retention time, etc.), for which structural information is not yet available.

Impurities present in new drug substances used in clinical and safety trials are covered under two aspects [ICH Q-3A (R2) 2006]. Chemistry aspects classify and iden-tify impurities, generate the report for different impurities,

*Corresponding author: Yadvendra K. Agrawal, Institute of Research and Development, Gujarat Forensic Sciences University, Sector-18 A, Nr. Police bhavan, Gandhinagar-382007, Gujarat, India, e-mail: [email protected] Prajapati: Institute of Research and Development, Gujarat Forensic Sciences University, Sector-18 A, Nr. Police bhavan, Gandhinagar-382007, Gujarat, India

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124 P. Prajapati and Y. K. Agrawal: Analytical methods impurity profile

list various impurities present in any substances, and give a brief discussion of analytical procedures for impurity detection. Safety aspects include those impurities that are present at a considerably lower amount or not present at all in a discovery of new drug substance.

Commonly used impurity termsA number of terms have been commonly used to describe an impurity or impurities (Francis et al. 1984, McNaught and Wilkinson 1997):

– Intermediate – Penultimate intermediate – By-product – Transformation product – Interaction product – Related product – Degradation product – Foreign substance – Toxic impurity – Concomitant component – Ordinary impurity – Organic volatile impurity (OVI).

Intermediate

The compounds formed in the process of synthesis for the desired product are called intermediates or reaction inter-mediates. They are defined as products that have under-gone a partial processing and are used as raw material in a successive productive step.

Penultimate intermediate

As the name suggests, this is the compound found in the synthesis chain before the production of the desired compound. Sometimes confusion arises when the desired material is a salt of a free base or acid. In our opinion, it is inappropriate to label the free base or acid as the penulti-mate intermediate if the drug substance is a salt.

By-product

The unintentional compounds that arise during the reac-tion are commonly called by-products. Not all by-products can be quantified easily; hence, they present a thorny

problem to the analytical chemist. A by-product can be useful and marketable or it can be considered waste.

Transformation product

This relates to an expected and non-expected product that may be formed in the reaction. Transformation products are very similar to by-products, except the term tends to connote that more is known about the reaction products than transformation products.

Interaction product

This term is slightly more comprehensive and more dif-ficult to evaluate than by-products and transformation products in that it considers interactions occurring among various chemicals involved in reaction.

Related product

As mentioned, impurity is a word that is not well liked. So a related product actually is similar to an impurity, but active pharmaceuticals use the term related prod-ucts instead, thus playing down the negativity frequently attached to the term impurity. These products can have similar chemical structure and might have standardized biological activity; however, this by itself does not provide any guarantee of effect.

Degradation product

The compounds produced due to decomposition of the material of interest or active ingredients often are referred to as degradation products.

Foreign substance

This is the material that may be present due to contamina-tion or adulteration, not as outcomes of synthesis.

Toxic impurity

Toxic impurities might affect the biological activity, even at very low concentrations. They require identification by qualitative or quantitative means.



P. Prajapati and Y. K. Agrawal: Analytical methods impurity profile 125

Concomitant component

Bulk pharmaceutical chemicals may contain concomitant components, which are geometric and optical isomers and antibiotics that are mixtures.

Ordinary impurity

An impurity having enough potency to have biologi-cal activity – even at trace level – is called an ordinary impurity.

Organic volatile impurity

A solvent that may remain in the drug substance should be considered as an organic volatile impurity (OVI).

Classification of impurities – Organic (process and drug related) – Inorganic – Residual solvents – Polymorphic – Enantiomeric.

Organic impurities

Organic impurities come into existence during the syn-thesis of the active and inactive materials. They may occur during manufacturing or during storage of the materials. These impurities can be deduced from deg-radation reactions and ongoing synthesis in active pharmaceutical entities and drug products. Impurities generated during the synthetic process are intermedi-ates, by-products, and reagents, as well as ligands and catalysts used in the chemical synthesis (Ahuja 1998, Qiu and Narwood 2007).

Starting materials and intermediates

These are the chemical compositions used to synthesize the desired constitute of a drug substance molecule. Start-ing materials and intermediates that are not reacted in the reaction, especially when the synthesis is about to complete, will remain in the final product as impurities

(Muehlen 1992, Gorog 2000, Gavin and Olsen 2006). One such example is 4-aminophenol, a starting mate-rial for synthesis of paracetamol bulk drug, which might be present in final product as an impurity having a toxic effect on the liver.

According to Dir. 2001/83/EC (EMEA 2012), for biologi-cal medicinal products, “Starting materials means any substance prevailed from the human or plant or micro-organisms or any alteration to the biological origins by means biotechnological cell constructs which will have tendency to formed drug product.” So measures for con-trolling sourcing of starting materials or intermediates must be strong.

An intermediate is a substance that is produced in the reaction vessel from the starting materials and which might undergo further chemical modification to provide the final product.

By-product

As mentioned earlier, the desired product is commonly called the main product, and product that is unwanted but might be useful is known as a by-product.

Degradation product

Degradation products are the compounds formed due to chemical changes in drug products during storage. Degra-dants may form because of chemical interactions with other compounds or due to contaminants present in the drug substances.

In certain cases, physical degradation occurs for a variety of reasons: change in the polymorphic state of the molecule, aggregation of proteinaceous material due to heat or residual solvents, absorption of water, loss of water, and others. A degradation product can be deter-mined by short- and long-term stability studies per ICH, for example, in treatment for common cold formulations containing acetaminophen, phenylephrine hydrochlo-ride, and chlorpheniramine maleate. Degradation prod-ucts for these formulations were isolated and found to be an addition compound of phenylephrine and maleic acid (Wong et al. 2006). The definition of degradation product in accordance with the ICH guideline is “any chemical change occur[ing] due to overreaction or over heating or changing in condition of solution, i.e., change in pH, exposure to light, etc. or reaction of final product with container or closure or excipients used in making product” [Gorog 2003, ICH Q-3A (R2) 2006].




Reagents, ligands, and catalysts

Reagents, ligands, and catalysts are seldom present in the final products (Ahuja 1998, Roy 2002). For the synthesis of the drug substance or any excipient catalysts, chemical reagents and ligands are used that can be conveyed to the concluding products as impurities in minute levels. For example, carbonic acid chloromethyl tetrahydro-pyran-4-yl ester (CCMTHP) (Gorog 2003), is an alkylating agent that was observed as an impurity in the synthesis of a β lactam drug substance.

Products of overreaction

Products of overreaction form when reactions for the syn-thesis are not selective as much as necessary, so nonse-lective interaction at an undesired site will produce an incorrect compound. For example, the last step for the syn-thesis of nanodralone decanoate is the decanoylation of the 17-OH group. Enol compound 3, 17 β-dihydroxyestra-3, 5-diene disdecanoate was formed because of overreaction at the 4ene-3 oxo group site (Gorog 2000, 2003).

Contamination by organic impurities

Contamination with organic impurities is not related to a drug but might unknowingly be present in the drug. For example, for drug substances derived from plants, her-bicides used to protect plants may be present, such as diquat and glyphosate, or pesticides such as carbofuran and endrin sprayed into the environment (Bauer et al. 2001).

Inorganic impurities

Inorganic impurities include filter aids, color removing agents such as charcoal, reaction rate modifiers (cata-lysts), ligands, and heavy metals. One example would be a catalyst used in a substitution reaction during the synthesis of the API or raw materials. Inorganic impuri-ties might have toxic effects, so they should be removed or controlled to a minimum level. Batch-to-batch varia-tion in impurity levels suggests that the manufacturing or synthesis process of the drug product is not controlled (Roy 2002, Basak et al. 2007, Hulse et al. 2008, ICH Q-3D 2009).

Inorganic impurities normally known and identified are as follows.

Contamination by inorganic impurities

These are unforeseen impurities found in final product. Contaminant impurities detected in drugs have been con-trolled in many ways. For example, previously used glass vessels for reaction are now replaced with acid/alkali resisted glass (Bauer et al. 2001). So, impurities that might be present due to leaching from glass vessel is minimized to safer levels.

Reagents, ligands, and catalysts

Reagents, Ligands and Catalysts are well defined under organic impurity of this paper. However, catalysts used in decomposition of intermediates (iodide catalysts), and monodentate ligand such as chloride ions might remain in the final product as inorganic impurities.

Residual solvents

Residual solvents in pharmaceuticals are the volatile chemicals that are produced as a result of side reactions or used in the manufacturing of API or excipients, or in the formulation [ICH Q-3C (R4) 2009]. Theoretically they can be removed from the final product but practically they can not. Therefore, it may be a vital parameter in the process for making a drug product.

Polymorphic forms

Solid material that subsists in two or more forms or in a crystalline structure is said to be polymorphic. Some organic and inorganic compounds form different crystal-line structures called polymorphs or polymorphic forms. The resulting change of intermolecular interactions gives rise to different pharmacokinetic properties of medical drugs, as well as to different properties of organic and inorganic materials. Therefore, the unambiguous identifi-cation and characterization of polymorphs is very impor-tant, especially from the economic point of view. In 2006 a new crystal form of maleic acid had arisen when solution of caffeine and maleic acid (2:1) in chloroform is set aside to evaporate slowly (Day et al. 2006).

Enantiomeric impurities

To determine purity of the chiral compound term enantio-meric excess (EE) is used. These impurities present in the




Table 1 Threshold for organic impurities [ICH Q-3A (R2)].

Maximum daily dosea (g/day)

Reporting thresholdb,c

(%)

Identification thresholdc

Qualification thresholdc

≤ 2 0.05 0.10% or 1.0 mg/day intake (whichever is lower)

0.15% or 1.0 mg/day intake (whichever is lower)

> 2 0.03 0.05% 0.05%

aThis is the amount of drug substance administered per day.bHigher reporting thresholds should be scientifically justified.cLower thresholds can be appropriate if the impurity is unusually toxic.

Table 2 Minimum degradation threshold for daily intake of drug product.

Max daily dose Qualification threshold

Identification threshold

Reporting threshold

< 1 mg 1.0% or 50 μg/TDI 1.0% or 5 μg/TDI 0.10%1 mg–10 mg 1.0% or 50 μg/TDIa 0.5% or 20 μg/TDI 0.10% > 10 mg–100 mg 0.5% or 200 μg/TDI 0.2% or 2 mg/TDI 0.10% > 100 mg-1 g 0.2% or 3 mg/TDI 0.2% or 2 mg/TDI 0.10% > 1 g–2 g 0.2% or 3 mg/TDI 0.2% or 2 mg/TDI 0.05% > 2 g 0.15% 0.10% 0.05%

aQualification threshold for 10 mg/day is 0.5%/200 μg TDI.

drug are due to change in the critical parameter of mol-ecules during synthesis. The following equation is used to determine enantiomeric excess (EE):

EE (( R-S) /( R R)) 100= + ×

where R and S stand for the individual optical isomer in the mixture (and R+S = 1).

These determinations are important particularly when we are talking about efficacy of the drug, because in the case of optical isomers of a drug only one isomer has therapeutic efficacy while the rest of them have either a toxic effect or have no effect at all (Armstrong et al. 1998, Roy 2002, Gorog 2003, Qiu and Narwood 2007).

Control of impuritiesAccording to theory, all impurities should be removed from the final product, but in practice, impurities cannot be entirely abolished from the final product. So, for a quality product, impurities should be kept within the limits. According to a study carried out for impurity, very low amount of impurities in the product should be allowed. However, in special cases, rather high quantities of impurities are permitted, for example, biotechnologi-cally derived products that have biological activity.

Most of the bulk pharmaceutical chemicals (BPCs) are obtained from various sources. Therefore, it is crucial that impurities in BPCs be monitored and controlled very carefully.

Various controlling authorities for impurity (USP 1995, ICH Q-6A 1999, ICH Q-6B 1999) are mentioned in monographs and specifications about maximum tolerable limits.

Control of organic impurity

Most often, reduction in quantity of by-products in the reaction can be carried out by tightly controlled reaction conditions at crucial steps of the reaction to preclude a new impurity or diverging level of impurity. Another approach to reduce the quantity of impurity in the final product is to use superior quality starting materials. Like-wise, the use of high-grade solvents also imparts its effort to obviate the production of by-products or any unknown entity. The thresholds for allowable organic impurities are shown in Table 1.

Control of degradation impurity

This particular impurity covers degradation products of active substance, including reaction products with excipi-ent or container system [ICH Q-3A (R2) 2006, ICH Q-3B (R2) 2006]. Degradation products observed in stability studies performed at recommended storage conditions should be identified, qualified, and reported when the following thresholds exceeded (Table 2).

Control of inorganic impurities

Oral/parenteral concentration limits (ppm) have been proposed for 14 metals in active substances or excipients: Pt, Pd, Ir, Rh, Ru, Os, Mo, V, Ni, Cr, Cu, Mn, Zn, and Fe. Metals are divided into three classes as follows, and limits have been summarized in Table 3.

Class 1: Metals of significant safety concern

Some metals are known or suspected human carcinogens, genotoxic, and sometimes nongenotoxic carcinogens or




Table 3 Limits of inorganic impurities in oral and injectable (CPMP/SWP/QWP/4446/00).

Classes of metals Oral Injectable

PDE (μg/day) Concentration (ppm) PDE (μg/day) Concentration (ppm)

Class 1A: Pt, Pd 100 10 10a 1a

Class 1B: Ir, Rh, Ru, Os 100b 10b 10b 1b

Class 1C: Mo, Ni, Cr, V 300 30 30a 3a

Class 2: Cu, Mn 2500 250 250 25Class 3: Fe, Zn 13,000 1300 1300 130

aSeparate limits for inhalation exposure to Pt, Cr (VI) and Ni.bSubclass limit.PDE, permitted daily exposure.

Table 4 Limits of initially controlled residual solvents in pharmacopoeias.

Organic volatile impurities

Limit (ppm)

USP 22 3rd edition

BP (1993) EP 3rd edition

ChP 1995 edition

Chloroform 50 50 50 50Benzene 100 100 100 1001,4-Dioxane 100 100 100 100Dichloromethane 100 100 100 100Trichloroethene 100 100 100 100Acetonitrile – 50 50 –Pyridine – 100 100 100Toluene – – – 100

potential contributory agents which produce irreversible toxicity, for example, neurotoxicity or teratogenicity. A few of them produce significant but reversible toxicity, such as Ir, Pd, Pt, Ru, Rh, Os, Mo, V, Cr, and Ni.

Class 2: Metals with low safety concern

Trace metals required for nutritional purposes can be present in foodstuffs or as readily available supplements, for example, Cu and Mn.

Class 3: Metals with minimal safety concern

Metals, omnipresent in the environment or plant and animal kingdoms, as such have high tolerable toxic values for humans. The nutritional intakes of ≥ 10 mg/day is rec-ommended. Examples are Fe and Zn.

Control of residual solvents

Various regulatory authorities have been concerned about toxicity of the residual solvent in the pharmaceutical world. At most, various pharmaceutical provide guide-lines (USP 1990, BP 1996, EP 1997, EMEA 2009) for the control of residual solvents and with different categories in pharmaceuticals gives acceptance limits (Table 4). In addition, for solvents that are used in pharmaceuticals, there are only a few residual solvents that are controlled (Hu and Liu 2011). So globally there is a need for a stand-ard guideline to be established for the control of residual solvents. Therefore, the harmonized guidelines for control of residual solvents by ICH has been released.

For pharmaceutical production, organic solvents invariably remain present in the processes. The pharma-ceutical industry is one of the largest users of organic sol-vents per amount of the final product (Slater et al. 2006,

Smith and Webb 2007, Katarzyna and Andrzej 2010). The synthesis of an active or inactive pharmaceutical ingredi-ent usually requires large amounts of solvent and some-times during the drug product formulation process, as well as during the formulation process methylene chloride, is used as solvent in large amounts for coating process. Residual solvents are placed in following classes based on their toxic effects to human health.

Class 1 solvents: solvents to be avoided

Solvents in class 1, due to their known carcinogenicity and hazardousness to environment should not be utilized in the manufacturing of active and inactive materials, or drug products. Even so, in any circumstances, if we can avoid use of this class of solvents, they should be limited in the final product as shown in Table 5.

Class 2 solvents: solvents to be limited

Solvents listed in Table 6 might be less toxic than class 1 sol-vents, but because of their inherent toxicity they should be limited as PDEs. This class is very much higher than class 1.




Table 5 Solvents in pharmaceutical products that should be avoided (ICH Q3-C).

Solvent Concentration limit (ppm)

Concern

Benzene 2 CarcinogenCarbon tetrachloride 4 Toxic and environmental

hazard1,2-Dichloroethane 5 Toxic1,1-Dichloroethene 8 Toxic1,1,1-Trichloroethane 1500 Environmental hazard

Class 3 solvents: solvents with low toxic potential

Solvents in this class have low toxic potential to humans, as these solvents have PDEs of 50 mg or more per day.

Class 3 solvents which should be limited by GMP are as under (per ICH Q3C):

1-Butanol Methyl acetate 1-Pentanol EthanolHeptanes 1-Propanol Propyl acetateAcetone Isobutyl acetate Tert-Butylmethyl

ether Ethyl acetate

2-Butanol 3-Methyl-1-butanol

Methyl isobutyl ketone

Ethyl ether

Anisole Isopropyl acetate 2-Methyl-1-propanol 2-PropanolAcetic acid Ethyl format CumeneMethyl ethyl ketone Pentane Formic acidButyl acetate Dimethyl sulfoxide

Other class: solvents for which no adequate toxicological data was found

This class lists additional solvents for which no adequate toxicological data available to generate a PDE. Some examples are (ICH Q-3C 2009)

Isooctane 1, 1-Dimethoxymethane Petroleum ether

Methyl isopropyl ketone

1, 1-Diethoxypropane Trifluoroacetic acid

2, 2-Dimethoxypropane Isopropyl ether Methyltetrahydrofuran Trichloroacetic acid

Control of genotoxic impurities

Existing ICH Q-3 guidelines do not provide acceptable toxicological limits of genotoxic impurities in active

Table 6 Solvents in pharmaceutical products that should be limited (ICH Q-3C).

Solvent PDE (mg/day) Concentration limit (ppm)

Acetonitrile 4.1 410Chlorobenzene 3.6 360Chloroform 0.6 60Dichloromethane 6.0 600N,N-imethylformamide 8.8 8801,4-Dioxane 3.8 380Ethyleneglycol 6.2 620Formamide 2.2 220Hexane 2.9 290Methanol 30.0 30002-Methoxyethanol 0.5 50Methylbutyl ketone 0.5 50Methylcyclohexane 11.8 1180N-Methylpyrrolidone 5.3 530Nitromethane 0.5 50Sulfolane 1.6 160Tetrahydrofuran 7.2 720Tetralin 1.0 100Toluene 8.9 8901,1,2-Trichloroethene 0.8 80Xylenea 21.7 2170

aUsually 60% m-xylene, 14% p-xylene, 9% o-xylene with 17% ethyl benzene.

pharmaceuticals. Determination of genotoxic effects of impurities without any data is very difficult for assess-ing impurity. Most of the pharmaceutical and other concerned industries accept the approach of thresh-old of toxicological concern (TTC). This approach gives an acceptable risk value (a TTC value of 1.5 μg/day intake) for intake of genotoxic impurity for most pharmaceuticals.

Various classes of genotoxic impurities are as follows (McGovern and Jacobson-Kram 2006):

– Class 1: Impurities known to be genotoxic (mutagenic) and carcinogenic

– Class 2: Impurities known to be genotoxic (mutagenic) but with unknown carcinogenic potential

– Class 3: Alerting structure, unrelated to the parent structure and of unknown genotoxic (mutagenic) potential

– Class 4: Alerting structure, related to the parent API – Class 5: No alerting structure or indication of genotoxic

potential.

During clinical trials some data that signifies the allow-able daily intake is summarized in Table 7 (EMEA 2006, McGovern and Jacobson-Kram 2006).




Isolation and characterization of impuritiesA number of methods can be used for isolating and char-acterizing impurities. The application of any given method depends on the nature of the impurity, in other words, its structure, physical and chemical properties, and avail-ability (the amount present in the original material from which it must be isolated). The following methods may be useful in this context:

– Extraction – Chromatography – Preparative separations.

Extraction

Extraction is one of the most useful methods for isola-tion of an impurity. For this the following methods can be helpful:

– Liquid/solid extraction – Supercritical fluid extraction – Liquid/liquid extraction or solvent extraction.

Liquid/solid extraction or solid-phase extraction (SPE)

Solid-liquid extraction allows soluble components to be removed from solids using a solvent. The same principle is applied here to choose a solvent for dissolving the impu-rity of interest present in the solid matrix. For example, if we want to determine salt in sand, we would simply use water to dissolve it and filter the solution, which on evaporation will produce salt in a reasonably pure form.

Table 7 Permissible limit for daily intake of genotoxic impurities on clinical development.

Duration of exposure

≤ 1 month

> 1–3 months

> 3–6 months

> 6–12 months

> 12 months

Allowable daily intake (μg/day) for all phases of development

120 40 20 10 1.5

or or or or Alternative maximum based on percentage of impurity in API

0.5% 0.5% 0.5% 0.5%

If, conversely, other water-soluble impurities were present in the sand, then it would be necessary to select a different solvent or it would be necessary to manipulate the solu-tion further.

It is noticed that when we are talking about the impu-rities that are already present in pharmaceuticals, it will be harder to isolate the impurity in its pure form. We have to use an organic solvent or mixtures of organic solvents to deal with the impurity. Moreover, organic solvents are volatile in nature, so we can evaporate them under low temperature to get a concentrated product.

Solid-phase extraction (SPE) (Thurman and Mills 1998, Fritz 1999, Dobo et al. 2006) is normally done with the use of cartridges and disks, available with a variety of stationary phases.

Normal phase SPEThe theory involved in normal phase SPE generally require mid- to nonpolar solvent mixtures (e.g., n-hexane, methylene dichloride, acetic acid, diethyl ether, etc.), a polar substrate (e.g., drug molecule, excipients, etc.) and a polar stationary phase. For the normal phase, various stationary phase mate-rials are used. One of them is silica, which can be modified further with polar heads (e.g., Si-C4-CN, Si-C4-NH2, etc.). Other adsorbents used are florisil, alumina, etc.; the mecha-nism involved in retention of substrate in normal phase SPE is principally the interaction with a polar analyte functional group and polar heads in the stationary phase.

Reversed phase SPEThe mechanism involved in reversed phase SPE requires a polar mobile phase (e.g., methanol, ethanol, water, etc.) or a semi-polar solvent mixture and a nonpolar stationary phase. In the reverse phase SPE modified silica is used as the stationary phase, in other words alkyl- or aryl-bonded silicas (Si-C-18, Si-C-8, Si-C-4, and Si-C-Ph).

Ion exchange SPEThe main rationale of the ion exchange SPE is to separate oppositely charged ions in a solution. Different types of exchangers have been used to separate the charged moi-eties. Commercially available ion exchangers contain resinous parts having amine or quaternary ammonium groups or other ionic groups for the separation of anionic or cationic compounds. The retention mechanism for the analyte is at the exchanger surface for the diffusion of ions. This depends on the concentration of the solution and the degree of cross linking of ion exchangers.




Anion exchange SPEMaterial used in anion exchange SPE for the stationary phase is having a positively charged group (e.g., an ali-phatic quaternary amine group or amino group). Posi-tively charged groups such as quaternary amines are strong bases that will draw anionic molecules into the solution and strongly attach to the exchanged group. As it strongly binds to the anionic group, it is termed a strong anion exchanger (SAX). Because of its strong binding capacity, it is generally used when recovery of anion is no longer required. However anions that can be displaced by another anion shall be eluted by changing the pH of the solution.

The stationary phase containing amino group, used in the normal phase SPE, can be used as a weak anion exchanger (WAX). The advantage of WAX utilization for separation of species is that we can isolate and recover strong as well as weak anions.

Cation exchangeThe materials used for cation exchange are high molecu-lar weight cross-linked polymers having carboxylic, phe-nolic, or aliphatic sulfonic acid groups. Among these groups sulfonic acid pulls in cationic species strongly present in solution and so is termed a strong cation exchanger (SCX). Moreover, materials containing a car-boxylic or phenolic group that is a weak anion can be used as weak cation exchanger (WCX). By the use of WCX, strong and weak cations can be isolated and recovered easily.

Supercritical fluid extraction

In the field of supercritical fluid extraction (SFE) (Hedrick et al. 1992, Wai and Laintz 1994, Simpson 2000, McHugh and Krukonis 2008) various researchers proposed the use of supercritical carbon dioxide (CO2) as an extractant for separating various components.

The procedure involved in SFE is very convenient for novices. A sample thimble is used to handle a sample through which supercritical fluid is pumped. The extrac-tion of the soluble compounds is allowed to take place as the supercritical fluid passes into a collection trap through a restricting nozzle. After passing through the nozzle, it is recompressed by venting in the collection trap for future use. The material left behind in the collection trap is the product of the extraction. Characteristics of gases nor-mally using SFE are given in Table 8.

Table 8 Solvents for SFE.

Solvent Pressure (atm)

Temperature (°C)

Density (g/ml)

n-Pentane 33.6 196.6 0.232CO2 72.9 – 0.448NH3 111.3 132.3 0.24

Liquid/liquid extraction or solvent extraction

In liquid-liquid extraction components are separated based on their solubility in two slightly miscible or com-pletely immiscible solvents, where mass transfer occurs at the interface and components separate by their affin-ity to the solvents (Qiu and Narwood 2007, Aguilar and Cortina 2008).

Partition coefficient plays an important role in this extraction process, by which the amount of solute that is distributed between two immiscible solvents a and b can be easily found:

d a bK C /C=

where, Kd is the distribution coefficient or partition coefficient.

Cais the concentration of component in solvent a.Cb is the concentration of component in solvent b.By the use of this technique the solution containing

impurity can be concentrated and thus the impurity can be easily detected.

Chromatography

Most recently, organic drug substance impurities are measured using chromatographic procedures, as they give more accurate results. These procedures should involve a separation mode that allows for the resolu-tion of impurities from the drug substance and a detec-tion mode that allows for the accurate measurement of impurities.

Owing to the polar and nonvolatile nature of most compounds used as medicinal drugs, reversed-phase HPLC is the most common technique for monitoring the drug substance and its impurities. GC is also used, par-ticularly for residual solvents, and capillary electrophore-sis (CE) has been introduced in more recent times. Some older methods use thin-layer chromatography (TLC), but use of this methodology for the quantitative measurement of impurities is not common.




HPLC-MS or HPLC-NMR

The most common technique for monitoring impurities is HPLC with UV detection. Quantification of impurities is achieved by reference standards, when available, or by area percent or height percent relative to the parent com-pound (Lee and Kerns 1999, Kostiainen et al. 2003). Impor-tant application for impurity identification with HPLC is by the use of MS as detector.

Recently, HPLC-MS has become the popular tech-nique for structural elucidation and confirmation of impurities. During the synthesis it is necessary to iden-tify the various types of impurities for maintaining the quality of the product. Because of its selectivity, sensitiv-ity, and compatibility with LC, LC-MS and LC-NMR have become absolutely necessary analytical techniques for the analysis of impurities present in various drugs and drug products and have become the first choice method. As it provides some structural information about frag-ments, empirical formula, and molecular weight, it has become a popular and advantageous method for the impurity analysis.

Coupling of LC and NMR (Treiber 1987, Albert 2002) has recently attracted research because of reduction in tedious preparative steps and substantially acquires higher efficiency and precision when handling complex mixtures.

Thin-layer chromatography (TLC)

For isolation and purification of compounds, TLC has gained importance because of its simplicity and utility. No major equipment is required, and the method of devel-opment is relatively easy (Sherma and Fried 1991, Ahuja 2003, Smith and Webb 2007). The primary limitation is the small number of theoretical plates that are obtained with this method as compared to GC or HPLC.

Detection frequently is performed visually or by UV (e.g., 366 nm). The fluorescence-quenching substances absorbing UV light in the short-wavelength region also can be detected if the layer is impregnated with a fluo-rescent substance. Iodine vapors can help detect most organic substances. A number of techniques can be used to recover the sample from the plate. The most simple and convenient method for obtaining the desired material is scraping the sorbent from the adsorbent site and shifting it to an extraction vessel, where different solvents are used for extraction of a compound.

Capillary electrophoresis (CE)

CE is not used much for impurity identification, but it offers the advantage that CE procedures can be employed when HPLC procedures have failed to measure the impu-rities adequately. CE is particularly important for the separation of chiral compounds that have closely related structures.

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dium of Chemical Terminology; 2nd Edition. McNaught, A. D.; Wilkinson, A., Comp. Blackwell Scientific Publications: Oxford, UK, 1997.

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Kovaleski, J.; Kraut, B.; Mattiuz, A.; Giangiulio, M.; Brobst, G.; Cagno, W.; Kulkarni, P.; Rauch, T. Impurities in generic pharmaceutical development. Adv. Drug Deliv. Rev. 2007, 59, 56–63.

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Chan, T. M. Major degradation product identified in several pharmaceutical formulations against the common cold. Anal. Chem. 2006, 78, 7891–7895.

Yadvendra K. Agrawal is the Director of the Institute of Research & Development, Gujarat Forensic Sciences University. He was the first to receive a grant for excellence in nanotechnology from Gujcost in 2004. He is also the recipient of the Russian Science Academy Award, 1985, Hari Om Ashram Award, 1989 and 1991, Royal Society of Chemistry Research Award in 1997 and 1998 (on supramolecules in nanotechnology), and the H. K. Sen Memorial Award in Pharma-ceutical Science, 1998. His main area of research is in pharmaceuti-cal sciences, supramolecules and nanotechnology, fullerenes and analytical chemistry.

Prajesh Prajapati works as an Assistant Professor at Institute of Research & Development, Gujarat Forensic Sciences University, Gandhinagar. He is pursuing his PhD and has completed his Mas-ter’s of Pharmacy in Quality Assurance in 2009. His main areas of research work are pharmaceutical method development, impurity identification, and degradation studies.



J. of Supercritical Fluids 95 (2014) 597–602

Contents lists available at ScienceDirect

The Journal of Supercritical Fluids

j our na l ho me page: www.elsev ier .com/ locate /supf lu

SFC–MS/MS for identification and simultaneous estimation of theisoniazid and pyrazinamide in its dosage form

Prajesh Prajapati ∗, Y.K. AgrawalInstitute of Research and Development, Gujarat Forensic Sciences University, Sector-18 A, Nr. Police Bhavan, Gandhinagar 382007, Gujarat, India

a r t i c l e i n f o

Article history:Received 22 May 2014Received in revised form 9 September 2014Accepted 11 September 2014Available online 22 October 2014

Keywords:Supercritical fluid chromatographyMass spectrometryGreen chemistryIsoniazidPyrazinamide

a b s t r a c t

An ecofriendly and sensitive SFC–MS/MS method (using TurboIonSpray probes) has been developedto measure Isonaizid (INH) and pyrazinamide (PYZ) in fix dosage combination (FDC) by dissolving indichloromethane:methanol:formic acid (50:50:0.1 v/v/v). Supercritical carbon dioxide (SC-CO2) is usedas mobile phase at a flow rate of 2 ml/min and modifier used is dichloromethane:methanol:formicacid (50:50:0.1 v/v/v) at a flow rate of 0.3 ml/min. High penetration power, low viscosity, negligibletoxicity makes the obvious choice for environment friendly mobile phase. The separation of INH andPYZ was achieved in less than 5 min using a C18 reverse-phase fused-core column (Inertsil ODS-5 �mC18, 150 mm × 4.6 mm). The method was validated as per international standards in terms of selec-tivity, linearity, precision and recovery. The method was found to be linear and % recovery was foundto be 98.89–100.33% and 99.27–100.06% for INH and PYZ, respectively. Lower limits of detection andquantification could be achieved for INH was 11.87 ng/ml and 35.97 ng/ml, respectively, and for PYZwas 35.42 ng/ml and 107.36 ng/ml, respectively. The proposed method was applied to the marketedformulation of different manufacturers.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

The concept of “Green Separation” was taken for the environ-mentally consented chemical process [1]. In this process, we arelimiting the use of hazardous chemicals as given in the list of ICHguidelines [2].

Isoniazid (INH) and pyrazinamide (PYZ) (Fig. 1) has been clas-sified as the recommended first-line antitubercular drugs forprevention and treatment of tuberculosis in adults [3]. In tuber-culosis, first line agents have to be taken for longer periods at aprescribed time interval and hence the quality of the product shouldbe maintained for longer period. A safe, reliable and economicalmethod has to be incorporated to find out the concentration ofthese drugs simultaneously as they are available in combined aswell as single dosage.

Extended review reveals that various analytical methods basedon amperometric [4], voltametric [5,6], spectrophotometric [7–13],spectrofluorometric [14], HPLC [15–23], HPTLC [24–26], RP-HPTLC[27] determinations are reported for identification and estima-tion of these drugs in fixed dosage form. The International

∗ Corresponding author. Tel.: +91 9825318996; fax: +91 07923247465.E-mail address: [email protected] (P. Prajapati).

Pharmacopoeia [28] has reported the simultaneous determinationof INH and PYZ using RP-HPLC. So with this in view, this paperdescribes the method on supercritical fluid chromatography (SFC)and mass spectrometry for simultaneous determination of INH andPYZ and also to check if any impurity is present in the API as wellas the interference from the excipient present in the fixed dosecombination (FDC).

2. Experimental

2.1. Instruments

2.1.1. Supercritical fluid chromatographA JASCO-2000 series (Japan Spectroscopic Co. Ltd., Hachioji,

Japan) of supercritical fluid chromatograph was used for the sepa-rations in this study. It was equipped with two pumps (PU-2080and PU-2080 CO2), which were capable to adjust the flow rate(0.001 to 10 ml/min.) for both Supercritical CO2 and modifier. Thesystem pressure was maintained electronically by back-pressureregulator (BP-2070), which allowed the flow rate and pressure tobe controlled independently. An external loop with a capacity of20 �l was equipped with rheodyne injector, capable to inject liq-uid sample accurately into the analytical column. The temperatureof the column was thermostatically controlled in a column oven

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598 P. Prajapati, Y.K. Agrawal / J. of Supercritical Fluids 95 (2014) 597–602

Fig. 1. Molecular structures of (a) isoniazid (INH) and (b) pyrazinamide (PYZ).

(Jasco-Co-965), while inbuilt with a cooling circulator. Detection ofanalyte was done by using a UV detector (Jasco-UV-2070). The efflu-ent coming from the SFC was injected in the MS/MS for detectionof any impurity if present.

2.1.2. Mass spectrometerAn AB Sciex (Canada) QTRAP-4500 series mass spectrome-

ter was used in the present investigation. It was equipped withexclusive TurboVTM source contained TurboIonSpray probes whichprovide advanced linear ion trap technology for the highest level ofsensitivity. The TurboV ion chamber has embedded ceramic heatertechnology with improved gas dynamics. Data acquisition and inte-gration were done by Windows-based analyst software.

2.1.3. SFC/MS/MS conditionsAnalysis was performed on fused silica column (inertsil

ODS-5 �m C18, 150 mm × 4.6 mm) protected by precolumn fil-ter cartridges. After optimization, mobile phase consisting ofdichloromethane:methanol:formic acid (50:50:0.1 v/v/v) was usedat flow rate of 0.3 ml/min and Supercritical CO2 was flowed at2 ml/min.

The optimized value for MS/MS analyses were as follows: ESIpositive ion mode; capillary voltage, 3.5 kV; cone voltage, 40 V; Gas1 (nebulizing gas) and Gas 2 (cone gas) were set to 50 units eachand the source temperature was set at 550◦. High-purity nitrogenwas used as nebulizer and cone gas.

The injection volume and column temperature were set at20 �l and 40 ◦C, respectively. Full-scan SFC–MS/MS spectra wereobtained by scanning from m/z 50 to 500.

2.2. Materials

Isoniazid and pyrazinamide standard was obtained as endow-ment samples from Sunij Pharma Pvt. Ltd. (Vatva GIDC,Ahmedabad) and tablet containing both the drug were procuredfrom local market. Dichloromethane (HPLC Grade) and Methanol(HPLC Grade)—Lichrosolv®- was purchased from E. Merck (India)Ltd., Mumbai. Whatmann filter paper no. 42 (0.45 �m) was used tofilter the solutions.

2.3. Method

2.3.1. Selection of analytical wavelengthThe overlay spectra of INH and PYZ were taken and found that

at �max 262 nm, both the drug exhibit linear correlation and can bedetected at the nano-gram level. So 262 nm was chosen as detectionwavelength in SFC.

2.3.2. Preparation of mobile phaseA blend of 50 ml Methanol, 50 ml of Dichloromethane and 0.1 ml

Formic acid was filtered through 0.45 �m filter paper. After filtra-tion, the blend was sonicated for 10 min to degas the mixture andused as mobile phase.

2.3.3. Preparation of INH and PYZ stock standard and workingsolutions

The INH and PYZ stock standard was prepared by dissolving10 mg of INH and 25 mg of PYZ in 10 ml mobile phase contain-ing dichloromethane:methanol:formic acid (50:50:0.1 v/v/v). Thissolution was kept in refrigerator at 2–8 ◦C. The working solutionswere obtained by suitably diluting the INH and PYZ stock solution.

2.3.4. Preparation of calibration standards and quality control(QC) samples

The appropriate volume of aliquots from standard INH and PYZwas transferred to volumetric flasks of 10 ml capacity to prepare sixcalibration standards. The volume was adjusted to the mark withmobile phase give a solution containing 1–6 �g/ml for INH and 2.5,5, 7.5, 10, 12.5 and 15 �g/ml for PYZ.

2.3.5. Determination of INH and PYZ from dosage formFor the analysis of dosage forms, not less than 20 tablets were

weighed and make uniform powder. To prepare assay sample solu-tion, weigh powder equivalent to 1 and 5 mg of INH and PYZ,respectively, was transferred to a clean and dry 10 ml of volumet-ric flask containing 5 ml of mobile phase as diluting solution andshaken thoroughly to extract the drug from the excipients and thensonicated for 10 min for complete dissolution of the drug (standardaddition can be done to make sure the complete integrity of finalconcentration). The solution was allowed to cool at room tempera-ture and then the volume was made up to the mark with the samediluting solution. The solution was filtered through Whatman filterpaper (no. 42) and sonicated for 10 min. The appropriate volume ofthis prepared solution was taken and transferred to the volumet-ric flask of 10 ml capacity and volume was made up to the markwith the mobile phase to give a solution containing 3 �g/ml and7.5 �g/ml INH and PYZ, respectively. This solution was used for theestimation of INH and PYZ (Table 2).

2.4. Method validation

Method validation was performed by using international guide-lines [29,30] for determining selectivity, limits of quantification(LOQ) and detection (LOD), linearity and recovery. To assess theselectivity of the proposed method, spiked and non-spiked sam-ples were injected into the SFC/MS system. The detection (LOD)and quantification limits (LOQ) were experimentally determinedby injecting a number of non-spiked samples (n = 6) and measuringthe magnitude of the background analytical response. The LOD andLOQ were estimated as three and ten times the signal-to-noise (S/N)ratio, respectively. The recovery was determined in 6 replicates at3 concentrations (low, medium and high QC levels).

3. Results and discussion

The goal of this work was to provide an ecofriendly selectivealternate method for determination of the INH and PYZ in FDC bySFC–MS/MS. In earlier work, we found that the addition of a lowlevel of a volatile acid, such as formic acid, to the mobile-phasemodifier made a dramatic improvement in the elution of polarand ionic molecules by SFC–MS/MS [31,32], We therefore added1 mM formic acid to the methanol:dichloromethane (50:50 v/v) asa modifier in the SFC/MS work.

3.1. Selection of mobile phase

For the selection of mobile phase, we have varied the concentra-tion of modifier Methanol and Dichloromethane with the additionof 0.1% of formic acid ranging from 30% to 100% at a flow rates

P. Prajapati, Y.K. Agrawal / J. of Supercritical Fluids 95 (2014) 597–602 599

Table 1Regression analysis data of calibration curves prepared by the proposed method.

Concentration of INHand PYZ (�g/ml)

Area INH Area PYZ

Mean ± S.D. (n = 6) (%)RSD Mean ± S.D. (n = 6) (%)RSD

(1) 2.5 298,614 ± 768 0.26 390,347 ± 449 0.11(2) 5 542,435 ± 1217 0.22 692,194 ± 1165 0.17(3) 7.5 734,623 ± 714 0.10 947,274 ± 1092 0.12(4) 10 936,962 ± 1673 0.18 1172,508 ± 1521 0.13(5) 12.5 1126,940 ± 649 0.06 1417,901 ± 1252 0.09(6) 15 1296,514 ± 2031 0.16 1689,852 ± 3149 0.19

ranging from 0.1 to 0.3 ml/min and supercritical carbon dioxide (SC-CO2) with flow rates from 1.5 to 2.0 ml/min and chromatogramswere recorded.

Amongst the all result obtained, the optimized system con-taining dichloromethane:methanol:formic acid (50:50:0.1 v/v/v) at0.3 ml/min and CO2 at 2 ml/min, was found to be satisfactory andgave well separate peak for INH and PYZ mixture (Fig. 2). Calibra-tion data for INH and PYZ is shown in Table 1. The calibration curvefor INH and PYZ were prepared by plotting area and concentration(Fig. 3).

The following equations for straight line were obtained for INHand PYZ:

Linear equation for INH : y = 198, 439x + 128, 146

with regression coefficient R2 = 0.9971.

Linear equation for PYZ : y = 101, 713x + 161, 692

with regression coefficient R2 = 0.9983.

3.2. SFC–MS/MS optimization

The positive ionization mode in ESI was selected for ion produc-tion due to the presence of an amine group in the structures of INHand PYZ. Under UV trace no any other significant peak is observedin SFC, and as no any change in mass spectra of standard drugsobserved, so we can ensure about the purity of the standard drug forfurther use in identification of these drugs in FDC. After collision-induced dissociation, the most abundant and stable product ionswere at m/z 138.1 for INH and at m/z 124.1 for the PYZ (Fig. 4).Thus, the MRM transitions of m/z 160 → 100 and m/z 130 → 60 wereselected for INH and PYZ, respectively. The mono-isotopic massesof INH and PYZ are 137.1 and 123.1, respectively. As a result, the

Fig. 2. Chromatogram of standard INH and PYZ in proposed mobile phase.

masses of their protonated molecular ions were found to be 138.1and 124.1 for INH and PYZ, respectively. After the analysis of thesedrugs in FDC, when compared with mass spectrum of standard drugit was observed that there is no any interference observed from theexcipient. So, we can routinely use this method for determinationof INH and PYZ in FDC.

3.3. Application of method

The validated method was successfully applied to determine thecontent of INH and PYZ in three different FDC. All the samples wereanalyzed in triplicate and percentage of content was determinedand found to be lies within the limit (Table 2). Method validationhas been discussed in following section.

Fig. 3. Calibration curve of (a) INH and (b) PYZ in proposed mobile phase.


Fig. 4. MS/MS spectrum of (a) INH and (b) PYZ.

Table 2Analysis of marketed formulation.

Formulation INH (mg) PYZ (mg)

Mean ± S.D. (n = 3) (%) RSD Mean ± S.D. (n = 3) (%) RSD

FORECOX, Macleods Pharma (INH 100 mg, PYZ 500 mg) 98.53 ± 0.51 0.52 496.75 ± 3.75 0.75RF-3, Sunij Pharma (INH 100 mg, PYZ 500 mg) 99.64 ± 0.96 0.96 497.29 ± 2.69 0.54R-CINEX-EZ, Lupin Lab. (INH 150 mg, PYZ 750 mg) 148.32 ± 1.25 0.84 745.51 ± 5.98 0.80

Table 3Results of recovery study of INH and PYZ.

Amount of drug Amount of drug added Total amount recovered (%) Recovery

INH(�g/ml)

PYZ(�g/ml)

INH(�g/ml)

PYZ(�g/ml)

INHMean ± S.D.*

(�g/ml)

PYZMean ± S.D.*

(�g/ml)

INHMean ± S.D.*

(�g/ml)

(%) RSD PYZMean ± S.D.*

(�g/ml)

(%) RSD

3 7.5 2.4 6 5.34 ± 0.04 13.43 ± 0.09 98.89 ± 0.73 0.74 99.51 ± 0.63 0.633 7.5 3 7.5 6.02 ± 0.07 14.89 ± 0.10 100.33 ± 1.17 1.17 99.27 ± 0.67 0.683 7.5 3.6 9 6.53 ± 0.06 16.51 ± 0.08 98.94 ± 0.91 0.92 100.06 ± 0.49 0.49

* n = 3.

3.4. Method validation

The developed SFC–MS/MS method was validated as per inter-national guidelines [29,30].

3.4.1. AccuracyTo investigate the accuracy in sample preparation (i.e., extrac-

tion efficiency), we prepare a spiked solution by adding knownamounts of related substances into a sample matrix. Thereafterresponses of the spike solutions and the neat standard solutionswere taken to assess the recovery from the sample preparation. Atthis stage, 20 tablets were taken and analysis of the sample wascarried out. Recovery studies were carried out by the addition ofstandard drug to the sample at 3 different concentration levels(80%, 100%, 120%) taking into consideration percentage purity ofadded bulk drug samples. The method was found to be accuratewith % recovery 98.89%–100.33% for INH and 99.27%–100.06% forPYZ (Table 3).

3.4.2. PrecisionPrecision was calculated as repeatability (Table 4) and intra-

day and interday variation for both INH and PYZ. The method wasfound to be precise with %RSD 0.10–0.20 for intraday (n = 3) and%RSD 0.11–0.21 for interday (n = 3) for INH and %RSD 0.13–0.16

for intraday (n = 3) and %RSD 0.13–0.16 for interday (n = 3) for PYZ(Table 5).

3.4.3. RobustnessRobustness of the method is determined by two operators (2 and

3) other than operator writing this paper, using standard methodas described in this paper under different chromatographic condi-tions than those used in the present method. The chromatographicconditions and the results obtained are listed in Table 6.

3.4.4. Other validation parameterThe method was found to be specific as no interference observed

when the drug was estimated in the presence of the excipients. Themethod was also rugged as there was no change in area up to 48 h ofpreparation of solution in Mobile phase. The LOD and LOQ for INHwere 11.87 ng/ml and 35.97 ng/ml, respectively, and for PYZ was35.42 ng/ml and 107.36 ng/ml, respectively. Summary of validation

Table 4Repeatability data for INH and PYZ.

Name of drug Conc. (�g/ml) Area Mean ± S.D. (n = 6) (%) RSD

INH 3 734,586 ± 589 0.08PYZ 7.5 947,048 ± 1129 0.12

P. Prajapati, Y.K. Agrawal / J. of Supercritical Fluids 95 (2014) 597–602 601

Table 5Precision data for INH and PYZ.

Name ofdrug

Conc.(�g/ml)

Intraday precision Interday precision

Area Mean ± S.D. (n = 3) (%)RSD Area Mean ± S.D. (n = 3) (%) RSD

INH 2 542,721 ± 1069 0.20 542,567 ± 1124 0.213 734,126 ± 725 0.10 734,458 ± 816 0.114 936,782 ± 1476 0.16 936,364 ± 1256 0.13

PYZ 5 692,148 ± 978 0.14 692,751 ± 1123 0.167.5 947,512 ± 1509 0.16 947,764 ± 1347 0.1410 1172,364 ± 1491 0.13 1171,953 ± 1574 0.13

Table 6Robustness for INH and PYZ.

Operator Pharmaceutical INH (mg) PYZ (mg)

Mean ± S.D. (n = 3) (%)RSD Mean ± S.D. (n = 3) (%)RSD

1 Tablet 99.15 ± 0.59 0.60 498.56 ± 5.69 1.142* Tablet 98.63 ± 0.54 0.55 501.15 ± 3.78 0.753# Tablet 100.07 ± 0.89 0.89 497.52 ± 2.31 0.46

* Conditions: [mobile phase—methanol:dichloromethane (40:60 v/v), flow-rate 0.2 ml min−1, SC-CO2 2 ml min−1 column temperature 38 ◦C and UV detection, at 257 nm)].# Conditions: [mobile phase—methanol:dichloromethane (60:40 v/v), flow-rate 0.4 ml min−1, SC-CO2 2.5 ml min−1 column temperature 35 ◦C and UV detection, at 267 nm)].

Table 7Summary of validation parameter for INH and PYZ.

PARAMETERS INH PYZ

Linearity (�g/ml) 1–6 2.5–15Slope 198,439 101,713Intercept 128,146 161,692R2 0.9971 0.9983

% Recovery 98.89–100.33 99.27–100.06Precision (% RSD)

Repeatability (n = 6)Intraday (n = 3)Inter day (n = 3)

0.080.10–0.200.11–0.21

0.120.13–0.160.13–0.16

Robustness (%RSD) 0.55–0.89 0.46–1.14LOD (ng/ml) 11.87 35.42LOQ (ng/ml) 35.97 107.36

parameters is tabulated in Table 6. Final optimized conditions aretabulated in Table 7.

4. Conclusion

To the best of our knowledge this is the first method thatuses the principle of green chemistry with the support of tandemmass spectroscopy to determine isoniazid and pyrazinamide in fixdose combination. The method has been successfully applied onthree pharmaceutical dosage forms of different manufacturer. Thereported method offers the advantage of the usage of ecofriendlymethod which separates both the component within the 5 minwithout any interference from the excipient.

Acknowledgements

The authors are grateful to Sunij Pharma Pvt. Ltd. (Vatva GIDC,Ahmedabad, Gujarat, India), for rendering isoniazid and pyrazi-namide as gift sample and to the Gujarat Council of Science andTechnology (GUJCOST) Gujarat, India, for devoting financial assis-tance.

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