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Chapter 1 Introduction

Department of Pharmaceutics Jamia Hamdard

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1. Introduction

Combination therapy with two or more agents having complementary mechanisms of action

represents a type of incremental innovation that has extended the range of therapeutic options in

the treatment of almost every human disease. Fixed-dose combination products, with two or

more drugs combined or co-formulated in a single dosage form, are becoming popular because

of simplified treatment regimens, improved clinical effectiveness, enhanced patient adherence

and reduced costs. The development of fixed dose combination drug products is becoming

increasingly high either to improve patients compliance or to benefit from the added effects of

the two or more active drugs given together. They are being used in the treatment of a wide

range of conditions and are particularly useful in the management of chronic conditions.

1.1 Fixed dose combination (FDC) products A fixed dose combination (FDC) is a formulation of two or more active ingredients combined

in a single dosage form available in certain fixed doses.

WHO definition

New fixed-ratio combination products are regarded as new drugs in their own right. They are

acceptable only when (a) the dosage of each ingredient meets the requirements of a defined

population group, and (b) the combination has a proven advantage over single compounds

administered separately in terms of therapeutic effect, safety or compliance. They should not be

treated as generic versions of single-component products (WHO Guidelines for registration of

fixed-dose combination medicinal products, 2005).

Combination therapy is commonly used in treatment of almost every area of diseases, especially

hypertension, HIV/AIDS, tuberculosis, malaria, diabetes and pain management, etc. Ideally,

combination products can provide a synergistic effect of individual drugs with reduced side

effects. From a compliance point of view, combination products provide a single pill, reducing

the number of pills taken on a daily basis and therefore enhancing patient compliance. It is

cheaper to purchase an FDC product than to purchase the products separately. The logistics of

procurement and distribution are simpler, which can be especially important in remote areas.

List of some marketed FDCs approved by DCGI are listed in Table 1.



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Table 1: List of some marketed FDCs approved by DCGI

Category Fixed dose combination (FDC) products

Antihypertensive Losartan + Hydrochlorothiazide tablets

Valsartan + Hydrochlorothiazide tablets

Telmisartan + Hydrochlorothiazide tablets

Atenolol + Hydrochlorothiazide tablets

Hypercholesterolemia

(Lipid lowering)

Atorvastatin + Ezetmibe tablets

Simbastatin + Ezetmibe tablets

Antidiabetics Glipizide + Metformin tablets

Glimepiride + Metformin tablets

Pioglitazone + Metformin tablets

Pioglitazone +Glimepiride + Metformin tablets

Antitubercular Isoniazide + Rifampicin tablets

Isoniazide + Ethambutol tablets

Isoniazide + Rifampicin + Ethambutol tablets

Antimalarials Sulfadoxine + Pyrimethamine tablets

Artemether + Lumefantrine tablets

NSAIDS, Analgesic

and antipyretic

Diclofenac + Paracetamol tablets

Ibuprofen + Paracetamol tablets

Aceclofenac + Paracetamol tablets



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Advantages of FDC products

1. Reducing the number of pills diminishes the complexity of the regimen, so that improved

patient adherence is expected with combination products.

2. Combination products make particular sense in the treatment of infectious disease, where

partial adherence can lead to the development of drug-resistant strains and a threat to public

health.

3. In hypertension treatment, combination rationale for the most commonly employed FDC

products is to counterbalance drug regulatory mechanisms and increase drug

pharmacological effectiveness, i.e., to combine drugs belonging to different classes and

having mechanisms of action that are complementary to each other. Example: Telmisartan

and hydrochlorothiazide.

4. Using FDC products in tuberculosis and malaria control simplifies the doctor's prescription

and patient's drug intake, helps patients adhere to treatment, precludes inadvertent mono-

therapy by the patient and therefore prevents the emergence of drug resistance due to missed

dose of a constituent drug. Example: isoniazide and rifampicin.

5. Several fixed-dosed FDC products are commercially available with complementary

mechanisms of action to improve glycemic control in type 2 diabetes patients. Example:

Metformin and glimepiride, Pioglitazone and glimepiride etc.

6. FDC products of two or more analgesics with different modes of action activate multiple

pain-inhibitory pathways and thus provide more effective pain relief for a broader spectrum

of pain. Example: Aceclofenac and paracetamol, diclofenac and paracetamol etc.

Disadvantages of FDC products

1. It can be argued that the FDC discourages adjustment of doses to the patient’s needs.

Dosage alteration of one drug is not possible without alteration of the other drug.

2. Some FDCs when combined lead to increased toxicity. Example, the anti-TB drugs, they

have the side effects (oto and nephro-toxicity).

3. If the biological half-life of different compounds in FDC are different, it may considerably

affect the pattern of drug availability in the plasma, and hence the over all efficacy of the

preparation (rifampicin fixed dose antitubercular formulations).



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Regulation of FDC products

The Drug Controller General of India (DCGI) had given marketing approvals for 40 FDCs in

January 2002. It is an accepted fact that FDC is treated as a new drug, because by combining two

or more drugs, the safety, efficacy, and bioavailability of the individual active pharmaceutical

ingredient (API) may change. As per the Drugs and Cosmetic Act, 1940, any new drug and the

permission to market a drug is to be given by the DCGI. Before the approval of any drug, the

Central Drugs Standard Control Organization (CDSCO) undergoes a process with respect to their

quality, safety and efficacy. The DCGI monitors the drug formulations, including the

combinations of drugs, from the angle of safety, effectiveness and rationality. Internationally,

there is an increasing trend to license fixed-dose combination products for the market. Currently

there are no specific international guidelines for FDCs. Some national authorities have developed

their own guidelines, some for specific classes of medicines. Guidelines for registration of FDCs

products are summarized in Table 2.

Table 2: Guidelines for registration of FDCs products

Title, publisher and date Notes

Scientific and technical principles for FDC

roducts. Botswana, 22 April 2004

Described the registration, quality, efficay, and

safety requirements for FDCs.

Fixed-combination medicinal products. CPMP

April 1996, CPMP/EWP/240/95, III/5773/94

formerly known as Testing combination; and

licensing criteria for FDC products.

Require circumstances in which FDCs are

acceptable, describe the considerations of

pharmacokinetic and pharmcodynamic

interactions, evidence for safety and efficacy.

WHO guidelines for registration of fixed-dose

combination medicinal products. (Annex 5,

39th report). TRS No. 929, Geneva, 2005

Described the advantages, disadvantages,

quality, safety and marketing authorization of

FDCs.

Guidance for industry on fixed dose

combinations (FDCs), CDSCO, New Delhi,

April 2010

Guidelines to manufacture, import, and

marketing approval of FDCs as per Drugs and

Cosmetics Act and Rules.



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Approval of FDCs The FDCs in which active ingredients already approved/marketed individually are combined for

the first time (for marketing in India), for a particular claim and where the ingredients are likely

to have significant interaction of a pharmacokinetic or pharmacodynamic nature. For approval of

such FDCs, following documents have to be submitted.

1. Complete chemical and pharmaceutical data.

2. Rationale for combining them in the proposed ratio and therapeutic justification.

3. Summary of drug-drug interactions (known and/or expected) among the active ingredients

present in the FDC.

4. Clinical trials data showing safety and efficacy of the FDC in the same strength that has been

carried out in other countries.

Information on active ingredients 1. Drug information (Generic name, chemical name).

2. Physicochemical data.

3. Physical properties- Description, solubility, partition coefficient and dissociation constant.

4. Analytical data- Elemental analysis, UV spectra, IR spectra, mass spectra and NMR spectra.

5. Complete monograph specification- Identification, identity/quantification of impurities, assay

method and impurity estimation method.

6. Stability studies- Final release specification, reference standard characterization and material

safety data.

Data on formulation 1. Dosage form, composition and details of formulation.

2. Excipient compatibility and content uniformity of active ingredients.

3. Master manufacturing formula.

4. Finished product specification.

5. In process quality control check.

6. Validation of analytical method,

7. Assay, impurities and forced degradation study

8. Stability evaluation in market intended pack at proposed storage conditions.



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Complexities in the development of FDC Products In relation to quality and stability, very similar principles apply to fixed dose combination

finished pharmaceutical products as apply to single entity products. However, there are

additional complexities arising due to the presence of two or more drug substances. These

complexities are related to compatibility of drug substances, assay, stability, physicochemical

properties (for example dissolution rate) and bioavailability/bioequivalence, analytical methods

and acceptance criteria for impurities, stress testing and determination of shelf lives of FDC

products (WHO Guidelines for registration of fixed-dose combination medicinal products, 2005).

1. Chemical and physicochemical compatibility of the APIs in an FDC with one another as well

as with possible excipients.

2. The degradability of each API under stress conditions in the presence of the others.

3. Uniformity of content of each active prior to compression (tablets) or filling (for instance

capsules, sachets and suspension dosage forms). This study determines whether mixing

during manufacture is adequate.

4. Analytical methods should be validated for each active ingredient in the presence of related

process impurities and degradation products. The interference by degradation products can be

controlled by peak purity testing by HPLC-UV, HPLC-PDA, HPLC-MS, UPLC-PDA and

UPLC/QTOF techniques.

5. The acceptance criteria for impurities in fixed dose combination products are expressed with

reference to the parent active ingredient and not with reference to the total content of active

ingredients. During stress testing, the active ingredients are combined in the same ratio as in

the final product. The expiry date is determined on the basis of stability of the least stable

active ingredient.

6. The dissolution rate of each active in pilot formulations. Multipoint limits should normally be

established for routine quality control of each active. For some FDC products, different

dissolution media may be acceptable for the different actives.

Analytical method development for FDCs Developing and validating a stability-indicating assay method becomes more challenging when

multiple drugs are present in a drug product. Since developing and marketing new chemical

entities (NCEs) for multiple indications is difficult task, pharmaceutical companies are looking



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into creating products by combining two or more known, compatible APIs to treat multiple

diseases and achieve better patient compliance. Method development for two or more

compounds and their related impurities becomes very complex if the solubility and the pKa

values vary greatly and the UV profiles are not similar. If the solubility and the pKa values of the

APIs involved are similar, where all the active components are totally soluble in water, the

method development is much easier. In the case of two APIs, new impurities seen in stability

samples, the samples must be evaluated carefully and the impurities reported to their origins. For

routine analysis in a stability program, a stability-indicating method is required for analzing both

the API and impurities. From a scientific or medical perspective, FDCs are more likely to be

useful when several of the following factors apply:

1. There is a medical rationale for combining the actives. The combination has a greater

efficacy than any of the component actives given alone at the same dose.

2. The incidence of adverse reactions in response to treatment with the combination is lower

than in that response to any of the component actives given alone, for example as a result of a

lower dose of one component or a protective effect of one component, and particularly when

the adverse reactions are serious.

3. For antimicrobials, the combination results in a reduced incidence of resistance.

4. One drug acts as a booster for another (for example in the case of some antiviral drugs).

5. The component actives have compatible pharmacokinetics and/or pharmacodynamics.

6. The active pharmaceutical ingredients are chemically and physicochemically compatible, or

special formulation techniques have been used that adequately address any incompatibility.

1.2 Stability testing of pharmaceutical products

Stability testing is an essential part of pharmaceutical development program and is required by

regulatory agencies for establishing and sustaining the high quality products. The pharmaceutical

products will not be approved without adequate stability information. Stability is an important

factor which is directly related with the quality, safety and efficacy of a drug product. A product

which is not having sufficient stability can result various changes in physical as well as chemical

properties that are ultimately harmful to the patients. In general the physical changes might affect

the appearance, clarity and color of solution, water content, crystal modification, and particle

size whereas the chemical changes can be observed in an increase in the degradation products or



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decrease of assay. Stability, as defined by USFDA, “is the capacity of a drug substance or drug

product to remain within established specifications to maintain its identity, strength, quality, and

purity throughtout the retest or expiration dating period.” (Yoshioka and Stella, 2002)

WHO definition: “Ability of a pharmaceutical product to retain its properties within its

specified limits throughout its shelf life. The chemical, physical, microbiological and

biopharmaceutical aspects of stability are all to be considered.” The stability testing is performed

by employing certain tests which are known as stability tests. Stability tests are a series of tests

designed to obtain an assurance of stability of a drug product in order to define its utilization

period and expiration dating period (shelf life).

Overall development stages and stability program

The stability studies are incorporated at all stages of drug product life cycle from early stages of

product development to final stage follow-up stabilities. The role of stability studies at different

stages of pharmaceutical development is shown in Fig. 1. The overall development stages of

pharmaceutical product and stability related with each stage are divided in to six steps which are

as follows (Carstensen and Rhodes, 2002),

1. Early stage stress and accelerated testing with drug substances for investigation of effect of

temperature, humidity, oxidation, light and then identification of degradation products.

2. Stability on preformulation batches for compatibility tests of excipients with drug and

optimization of final dosage form.

3. Stress and accelerated testing with final formulation and registration batches for investigation

of stability-indicating power of analytical procedure, selection of packaging, establishment of

shelf life and storage instructions.

4. Accelerated and long term testing with products of registration batches for confirmation of

results of stress and acceclerated testing and derivation of shelf lives.

5. Ongoing stability testing for confirmation and extension of shelf life.

6. Follow up stability testing for monitoring of continuous production and confirmation of

derived stability information.



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Fig. 1: Stability studies at various stages of drug product development

Drug Product • Drug Product Stability • Excipients Compatibility • Formulation Interactions

Formulation Development

Drug Substance

• Drug Substance Stability • Process Impurity

Packaging Selection

Final Packaged Product • Packaging Interactions • Storage conditions

Excipients



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Stress testing/forced degradation study Stress testing of the drug substance can help in the identification of degradation products which

can help in establishing the degradation pathways and to investigate the stability-indicating

power of the analytical procedures applied for the drug substance and drug product. The ICH

stability guidelines Q1A (R2) defines stress testing as for drug substances “these tests are studies

undertaken to elucidate the intrinsic stability of the drug substance. Such testing is part of the

development strategy and is normally carried out under more severe conditions than those used

for acceclerated testing.” For drug product “stress tests are studies undertaken to assess the effect

of severe cnditions on the drug products.” A stability-indicating method is an analytical

procedure that is capable of discriminating between the active pharmaceutical ingredients (API)

from any degradation product formed under defined storage conditions during the stability

evaluation period. In addition, it must also be sufficiently sensitive to detect and quantify one or

more degradation products.

Details regarding the design and strategy of stress testing studies are not covered by any

regulatory guidance document. Stress testing is carried out on a single batch of the drug

substance. It should include the effect of temperatures in 10°C increments (e.g., 50°C, 60°C etc.)

above that of accelerated testing, humidity (e.g., 75% RH or greater), oxidation, photolysis and

acid base hydrolysis. Stress testing should induce not more than 5-15 % degradation of the main

compound. The standard conditions for photostability testing are described in ICH Q1B. The

most common analytical technique for monitoring forced degradation experiments is HPLC with

UV and/or MS detection for peak purity, mass balance, and identification of degradation

products. The UPLC, with QTOF or integrated multi-detection by PDA/MS, allows for faster

and higher peak capacity separations. Combining the chromatographic speed, resolution, and

sensitivity of UPLC separations with the high-speed scan rates of UPLC-specific photodiode

array and MS detection will give you confidence that you are thoroughly identifying degradation

products and thus shortening the time required to develop stability-indicating methods.

Information provided by stress testing at various stages of pharmaceutical product development

is summarized in Table 3. The most common analytical technique for monitoring forced

degradation experiments is HPLC with UV and/or MS detection for peak purity, mass balance,

and identification of degradation products. HPLC-based methodologies are time-consuming and



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provide only medium resolution to ensure that all of the degradation products are accurately

detected. The UPLC, with QTOF or integrated multi-detection by PDA/MS, allows for faster and

higher peak capacity separations, no matter how complex your degradation product profiles can

be. Combining the chromatographic speed, resolution, and sensitivity of UPLC separations with

the high-speed scan rates of UPLC-specific photodiode array and MS detection will give you

confidence that you are thoroughly identifying degradation products and thus shortening the time

required to develop stability-indicating methods.

Table 3: Information provided by stress testing at various stages

Development Stages Purpose of stress testing

Preformulation

1. Selection of compounds and excipients.

2. Formulation optimization.

3. Selection of proper packaging.

4. Registration application dossiers.

Formulation, registration

batches and manufacturing

1. Stability-indicating analytical method.

2. Understanding impurity profile.

3. Structure elucidation of degradation products.

4. Establishment of degradation pathways.

5. Establishment of shelf life.

6. Selection of packaging and storage instructions.

1.3 Impurities in pharmaceutical products

Impurities in pharmaceuticals are the unwanted chemicals that remain with the active

pharmaceutical ingredients (APIs), or develop during formulation, or upon storage of both API

and formulated APIs. According to ICH Q3A (R) “Impurities in the New Drug Substance” and

ICH Q3B (R) “Impurities in the New Drug Product”, a drug substance impurity is “any

component of the new drug substance that is not the chemical entity defined as the new drug

substance,” and a drug product impurity is “any component of the new drug product that is not

the drug substance or an excipient in the drug product”. ICH guidelines classify impurities into

three categories: organic impurities, inorganic impurities, and residual solvents. These impurities

can be from a variety of sources, as given in Table 4. According to the ICH guidelines impurities



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in new drug product, degradation products observed in stability studies conducted at

recoomended storage conditions should be identified when present at a level greater than the

identification thresolds (0.1% for a maximum daily dose of >2g). Identification of impurities

below the 0.1% level is generally not considered to be necessary unless the potential impurities

are expected to be unusually potent or toxic. However, if the level is at or above the 0.1% limit,

then effort should be put forth to identify it (Roy and Ahuja, 2006; Qiu and Norwood, 2007).

Classification of impurities is summarized in Table 4.

Table 4: Impurity classification based on ICH guidelines

Organic Impurities • Starting materials

• Intermediates

• Related products

• Degradation products

Inorganic Impurities • Heavy metals

• Inorganic salts

Residual solvents • Organic or inorganic liquids

Identification of impurities and/or degradation products For the drug development and formulation process detecting and quantifying drug substances

and their impurities in raw materials and final product testing is an essential part. Impurities may

influence the safety and efficacy of the pharmaceutical products. An easy way of doing this is to

compare the retention times of known process-related compounds to that in question. If this

analysis confirms that the compound is an unknown, the next step would be to obtain an LC-MS

on the compound. Mass spectrometry provides structural information which aids in determining

structure. In some cases, mass spectrometry will be enough to identify the compound. In other

cases, more complicated methods like liquid chromatography coupled to nuclear magnetic

resonance (LC-NMR) are needed or the impurity will need to be isolated in order to obtain

additional information (Qiu and Norwood, 2007).



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Identification of impurities by HPLC Once a decision has been made to identify an unknown compound, the next step is to evaluate all

known process-related impurities, precursors, intermediates, and degradation products. By

observing the relative retention times (HPLC) of all known process-related impurities,

precursors, and intermediates, one can quickly determine whether or not the impurity of interest

is truly unknown. If the relative retention time of the unknown impurity matches that of a

standard, then it can be identified using HPLC with UV or photodiode array detection and LC-

MS, and GC-MS for volatile impurities. The identity is confirmed by correlating the retention

time, ultraviolet spectra, and mass spectra of the unknown impurity with that standard. The

process outlined in Fig. 2. illustrates the overall strategy used for identification of unknown

impurities. If the relative retention time doesnot match that of a standard. The next step is to

obtain molecular mass and fragmentation data via HPLC-MS. It is essential to dertermine the

molecular mass of the unknown impurity. To run LC-MS, a mass spectrometry-compatible

HPLC method must be developed. If the mass spectrometry data evaluation yields sufficient

structural information, this eliminates the need to isolate the impurity. If standards are not

available, which is usually the case, the proposed structures can be discussed with the project

team. The project team can then decide if the information is suitable for their needs, or if

isolation is required. A number of methods can be used for isolating impurities and/or

degradation products. Three of the most utilized techniques are TLC, flash chromatography

(column chromatography), and preparative HPLC.

Mass spectrometry (MS) Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of

charged particles. In the technique of mass spectrometry, the compound under investigation is

bombarded with a beam of electrons which produce an ionic molecule or ionic fragments of the

original species. The resulting assortment of charged particles is then separated according to their

masses. The spectrum produced, known as mass spectrum is a record of information regarding

various masses produced and their relative abundances. When a sample substance is bombarded

with electrons of energies of 9 to 15 eV, the molecular ion is produced by loss of a single

electron. This will give rise to a very simple mass spectrum with essentially all of the ions

appearing in one peak called molecular ion peak.



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Applications of mass spectrometry

1. Molecular mass determination Mass spectrometry is one of the best methods to determine the molecular mass accurately. When

a substance is bombarded with moving electrons and the mass spectrum is recorded, the mass of

the peak at the highest m/e reveals the molecular mass accurately. This method is only accurate

when no ions heavier than the parent ion are formed.

2. Impurity detection Mass spectrometry is one of the best methods to detect impurities. The detection of impurities in

trace amount is only possible if their structures differ considerably from those of the major

components. Impurities present can be detected by the additional peaks, highest value of mass

peaks than compound itself and from the fragmentation pattern.

3. Identification of an unknown compound From fragmentation pattern, one may find out very easily a preliminary indication about

functional groups and also partial structural information about the compound. The final

identification of unknown compound may be done by comparing its mass spectrum with that of

an authentic sample.

Mass spectrometry in identification of impurities Mass spectromery, by itself and in various combinations with other analytical instrumentation, is

the first logical technique to use to probe unknown structures. It is structurally sensitive

technique, giving the molecular mass and structure-indicating pieces of information in one

observation. Mass spectrometry requires small amounts of sample to obtain significant amounts

of structural information on the target compound. One of the powerful tools of impurity profile is

liquid chromatography (LC) coupled with mass spectroscopy (MS), and it is employed for the

identification of impurities, natural products, drug metabolites, and proteins. LC-MS is steadily

applied to scrutinize impurity during pharmaceutical product development and manufacturing

process to support the safety evaluation of batches used in clinical studies. After a step-by-step

investigation using various LC-MS techniques, it is often possible to propose a possible

structure(s) for an unknown impurity (Qiu and Norwood, 2007; Lee et al., 2008).



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Mass Analyzers for LC-MS

Single quadrupole analyzer: The quadrupole mass analyzer is very popular for LC-MS, due

to its relative simplicity and relatively low cost. The primary information available from a LC-

MS experiment using a single quadrupole analyzer is the molecular weight of the unknown

impurity. Molecular weight is the most important structural information required for unknown

structure elucidation.

Triple quadrupole (QqQ): A triple quadrupole mass spectrometer is comprised of two

separate quadrupole mass analyzers (Q1 and Q3) and an RF (radio frequency) only quadrupole

(q2, note that newer instrument may use a hexapole or octapole or other device as the collision

cell, yet the name of the triple quadrupole still remains.) which is used as a collision cell. MS/MS

scan modes on a triple quadrupole instrument include product ion scan, precursor ion scan and

neutral loss scan. Product ion scan generates fragments (product ions) which are crucial in

structural elucidation. The molecular ion (e.g., [M+H]+) of the impurity of interest can be mass

selected by the first quadrupole and then fragmented in the collision cell. The induced fragments

are then mass analyzed using the second quadrupole analyzer. Since the structure of the drug

substance is known, by comparing the resulting spectrum of the drug substance with that of the

impurity, one can find common ions and different ions from the drug substance and the impurity.

The common ions indicate the common substructure of the impurity and the drug substance,

whereas the different ions indicate the portion of the structure that is modified in the impurity.

Time-of-flight (TOF): Time-of-flight (TOF) is one of the most widely used mass analyzers

for accurate mass measurement using LC-MS. In a TOF analyzer, the mass of an ion is

determined based on the time it takes to reach a detector through an evacuated flight tube;

therefore, physically, there is no limit on the molecular size. Because of this feature, TOF is an

ideal mass analyzer for large biomolecules that are ionized by MALDI (Matrix assisted laser

desorption ionization). The newer generation LC-TOF can routinely generate data with a mass

accuracy of 3 ppm or better. Quadrupole-TOF is a hybrid instrument that combines a quadrupole

mass analyzer, a collision cell, and a high resolution TOF analyzer.



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PRI= Product related impurity; LC= Liquid chromatography

RRT = Relative retention time; UV= Ultraviolet spectrometry

Deg. STD= Degradation standard; MW = Molecular weight

MS = Mass spectrometry; NMR= Nuclear magnetic resonance;

Fig. 2: Impurity/degradant isolation and identification process flow chart

UnknownDegradant/ Impurity Identified

No

Impurity Level>0.1%

PRI/Deg. STD RRT

MS Data MW

Possible Structures

Isolate degradant/impurity

for NMR Studies

Confirm Structure by RRT/UV/MS

HPLC/UV LC/MS

HPLC/UV LC/MS

Yes

YesYesYes

Develop LC-MS Method and

Run LC-MS

No

No

No

Yes



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1.4 LC/MS method development

Requirements for developing effective LC/MS analytical methods.

1. Mobile phase requirements

Mobile phase degassing is an important step in the LC/MS experiment and can be accomplished

by sonication, helium sparging or as a part of mobile phase filtration step.

(b) Organic components

Acetonitrile and methanol are almost exclusely chosen in LC/MS methods as organic mobile

phase components. Methanol has a greater gas phase acidity, polarity, and volatility than

acetonitrile and may be preferred for some types of separations. In positive ion mode, methanol

has been shown to deliver 10 to 50% better sensitivity than acetonitrile, while in negative ion

mode there is little difference in sensitivities for most analytes. A typical mobile phase to start

the experiments can be 90/10 mixtures of MeOH/H2O or ACN/ H2O can be used until the desired

capacity factor is achieved. Higher organic composition is desired in LC/MS due to improved

effluent evaporation at a given temperature, thereby decreasing the background.

(c) Aqueous components

Nonvolatile aqueous components, whether salts, acids, bases, or buffers, will greatly decrease

and even prevent the detection of analyte ions. These nonvolatile buffers can also foul ion

sources and vacuum regions of mass spectrometers. Nonvolatile phosphate or citrate buffers are

not recommended for both ionization and practical reasons.

(d) Buffers

Ammonium acetate or formate buffers can be used with concentration ranging from 2 to 50 mM,

although a maximum concentration of 10-20 mM is recommended to avoid ion suppression. A

useful rule is to use as low a concentration of buffers as possible to give reasonable

chromatographic performance.

(e) Acids and bases

Formic or acetic acid concentrations of 0.1-1% (v/v) are recommended when preparing low pH

mobile phase to enhance ionization in electrospray. Ammonium hydroxide is recommended for



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high pH mobile phases. For basic compounds, 0.1% acid should be mixed with the organic

compoent, whereas water or neutral buffer should be used for neutral or acidic species.

2. Mobile phase components that are not recommended

Certain types of traditional LC mobile phase additives should be avoided due to nonvolatility and

ion suppression effects. These additives includes nonvolatile salts or buffers such as phosphates,

citrates, and carbonates; inorganic acids such as hydrochloric, sulphuric, phosphoric, and

sulfonic acids; and strong bases. Complete suppression of ionization as well as interferences in

both positive and negative ion mode will occur when these agents are utilized.

1.5 Method validation

Method validation is a regulatory requirement. Validation parameters required for validation of

analytical procedures as per international conference on harmonization (ICH) guidelines are

described as follows [Validation of analytical procedures: text and methodology, Q2 (R1), 2005].

1. Linearity and Range

The linearity of an analytical procedure is its ability to obtain test results which are directly

proportional to the concentration of analyte in the sample within a given range. Range of an

analytical method is the interval between the upper and lower concentration of analyte for which

the method has been shown to be precise, accurate, and linear. For linearity studies a minimum

of five concentrations is recommended. The least squares method is recommended for evaluation

of the regression line. A correlation coefficient, intercept, slope of regression line should be

reported.

2. Detection limit (DL)

The detection limit (DL) is the lowest concentration of the analyte that can be detected, but not necessarily quantitated as an exact value. The minimum concentration at which the analyte can be reliably detected is the limit of detection. According to ICH, several approaches are used depending on whether the procedure is instrumental or noninstrumental. These approaches are as follows.



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1. Based on visual evaluation Visual evaluation may be used both for instrumental and noninstrumental methods. It requires

analysis of samples with concentrations of analyte and establishing the minimum level at which

analyte can be reliably detected.

2. Based on the standard deviation of response and the slope The detection limit may be calculated based on the standard deviation (SD) of the response and

slope (S) of the calibration curve. Detection limit (DL) = 3.3 × SD

The SD of the response can be determined from the SD of the y-intercept of the regression line.

3. Quantification limit (QL) The quantitation limit is the lowest concentration of analyte in a sample that can be

quantitatively determined with acceptable precision and accuracy. This is a parameter of the

quantitative assays for low levels of compounds in sample matrices such as determination of

impurities and/or degradation products. For better precision and accuracy, a higher concentration

must be reported for the QL. The ICH lists the same two options that can be used to determine

the QL. They are visual evaluation for both noninstrumental and instrumental; the later method

can be based on the standard deviation of the response and the slope. The formula is changed to

SD = 10 ×SD/S. 4. Precision Precision is the measure of how close the data values are to each other for a number of

measurements under the same experimental conditions. Precision is defined as “the degree of

agreement among individual test results obtained by repeatedly applying the analytical method to

multiple samplings of a sample.” Thus the precision refers to the distribution of individual test

results around their average. The precision is usually expressed as RSD (%) for a statstically

significant number of samples. Precision may be considered at three levels: repeatability,

intermediate precision and reproducibility. 4.1 Repeatability Repeatability is also termed intra-assay precision. For analysis repeatability, determinations are made on multiple measurements of a sample by the same analyst under the same analytical conditions. The ICH recommends that repeatability should be determined from a minimum of



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nine determinations covering the specified range for the procedure (e.g., three levels, three replicates each), or from a minimum of six determinations at 100% of the test or target concentration. The target concentration is defined as the concentration of the compound of interest given in the analytical method.

4.2 Intermediate precision Intermediate precision expresses within-laboratory variations. This parameter evaluates the reliability of the method in an environment different from that used during the method development phase. The method can be evaluated on different days, with different analysts and equipment etc. 4.3 Reproducibility Reproducibility expresses the precision between laboratories (collaborative studies, usually applied to standardization of methodology). This is assessed by means of an inter-laboratory trial.

5. Accuracy Accuracy is the measure of how close the experimental value is to the true value. It is measured as the percent of analyte recovered by assay or by spiking samples. For the drug product, this is performed by analyzing synthetic mixtures spiked with known quantities of drug. Accuracy should be established across the specified range of the analytical procedure. For quantification of impurity, accuracy is determined by spiking drug substance or drug product with known amounts of available impurities.

6. Robustness The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but delibarate variations in the method parameters and provides an indication of its reliability during normal usage. The robustness of the method is investigated by varying some or all conditions, e.g., organic composition of the mobile phase, pH of mobile phase, different columns (different lots and/or suppliers), temperature and flow rate. 7. Specificity/Selectivity The terms specificity and selectivity are often used interchangeably. The specificity of the

method is the ability to measure accurately and specifically the analyte of interest in the presence



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of other components in the sample matrix. These components may include other active

ingredients, excipients, impurities, and degradation products. According to ICH, the validation

procedure should be able to demonstrate the ability of the method to assess the analyte in the

presence of impurities, matrix components, and degradation products. The impurity test is shown

by spiking drug substance or drug product with appropriate levels of impurities and

demonstrating the separation of these impurities individually and/or from other components in

the sample matrix. For stability-indicating assays where potency and impurities are determined

simultaneously mass balance must be taken in to consideration. Any decrease in potency should

be explained by mass balance. The following equation can be used to account for any loss of

potency: 100 % = Drug % + Related substance % + Degradation products %

Specificity of the method is also determined by forced degradation study. For these studies, acid

and base hydrolysis, temperature, photolysis, and oxidation are recommended.

8. System Suitability Test to verify the proper functioning of the operating system, i.e., the electronics, the equipment, and the analytical operations. According to ICH, system suitability testing is an integral part of chromatographic procedures. These tests are used to dertermine that the resolution and reproducibility of the system are adequate for the analysis to be performed. As stated earlier, system suitability involves checking a system to ensure it is performing adequately before or during the analysis. To establish, the reproducibility (%RSD) of five or six replicates is calculated and compared to predetermined specification limits. System suitability tests are performed prior to analysis of actual samples. These parameters are studied by analysis of a system suitability sample that is a mixture of main active drug and expected degradation product or a known impurity. 1.6 UPLC/Q-TOF/MS

Ultra-performance liquid chromatography (UPLC) has been investigated as an alternative to

HPLC for the analysis of pharmaceutical development compounds. UPLC produced significant

improvements in method sensitivity, speed, and resolution. Sensitivity increases with UPLC,

which were found to be analyte-dependent, were as large as 10-fold and improvements in

method speed were as large as 5-fold under conditions of comparable peak separations. Acquity

UPLC is specially designed to resist higher back-pressures, with the advantages of fast injection



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cycles, low injection volumes, negligible carryover and temperature control (4–40 ºC), which

collectively contributes to speedy and sensitive analysis. Furthermore, acquity UPLC columns

contain hybrid X-Terra sorbent, which utilizes bridged ethylsiloxane/ silica hybrid (BEH)

structure, ensures the column stability under the high pressure and wide pH range (1–12). UPLC

is a novel chromatographic technique utilizing high linear velocities, which is based on concept

using columns with smaller packing (1.7-1.8 µm porous particles) and operated under high

pressure (up to 15000 psi). This is an extremely powerful approach which dramatically improves

peak resolution, sensitivity and speed of analysis. In addition to UPLC, the use of orthogonal

quadrupole time-of-flight mass spectrometry (Q-TOF-MS) with low and high collision energy

full scans acquisition simultaneously performed, allows the generation of mass information with

higher accuracy and precision, which is ultimately helpful in structure elucidation and

identification of fragmentation pattern of the compounds. It also confidently detects impurities in

compounds even at trace levels. The rapid switching of the collision cell energy produces both

precursor and product ions of all of the analytes in the sample while maintaining a sufficient

number of data points across the peak for reliable quantification. The sensitivity and flexibility of

exact mass time-of-flight mass spectrometry with alternating collision cell energies, combined

with the high resolving power of the UPLC system, allows for the rapid profiling and

identification of impurities and/or degradation products. Its most popular applications are in drug

discovery, samples characterization, structural elucidation, etc., determination of degradation

products, impurities, by-products, break-down products, stability testing, etc., where accurate

mass determinations are required. Also, or it is used more widely in vitro and in vivo

bioanalytical samples for metabolite research and identification with accurate mass. A summary

of the applications of the UPLC/Q-TOF system is listed below (Plumb et al., 2004; Novakova et

al., 2006; Swartz, 2008).

• High resolution mass spectrometry • Accurate mass determinations • Drug discovery with accurate mass • Bioanalytical applications with accurate mass • Metabolite ID with accurate mass • High throughput screening (HTS) with accurate mass • Peptide and protein research with accurate mass

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