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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