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Revi e w A r t i c l e

Bioanalysis in drug discovery and development

A b s t ra c t
Recent years have witnessed the introduction of several high-quality review articles
into the literature covering various scientific and technical aspects of bioanalysis.
Now it is widely accepted that bioanalysis is an integral part of the pharmacokinetic/
pharmacodynamic characterization of a novel chemical entity (NCE) from the time of
its discovery and during various stages of drug development, leading to its market
authorization. In this compilation, the important bioanalytical parameters and its
application to drug discovery and development approaches are discussed, which will
help in the development of safe and more efficacious drugs with reduced development
time and cost. It is intended to give some general thoughts in this area which will form
basis of a general framework as to how one would approach bioanalysis from inception
(i.e., discovery of a lead molecule) and progressing through various stages of drug
development.

Key words: Bioanalytical, method validation, metabolism, pharmacokinetics,

toxicokinetic

INTRODUCTION
The discovery and development of a new drug costs around $1 billion and
it may take approximately 10 years for the drug to reach the marketplace. [1]
Drug discovery and development is the process of generating compounds and
evaluating all their properties to determine the feasibility of selecting one novel
chemical entity (NCE) to become a safe and efficacious drug. Strategies in the
drug discovery and drug development processes are undergoing radical change.
For example, the contribution of pharmacokinetics (PK) to both processes is
increasing.[2,3] Furthermore, toxicokinetics has now become established as
an essential part of toxicity testing.[4,5] With this emphasis in the use of PK/
toxicokinetics and the greater potencies of newer drugs, a sensitive and specific
bioanalytical technique is essential.

The emergence of the field of bioanalysis as a critical tool during the process of
drug discovery and development is well understood and globally accepted.[6-9]
Over the past few decades, a plethora of assays has been continuously developed
for NCEs to support various stages of discovery and development, including
assays for important metabolites.[10-14] Additionally, multiple analytical procedures
Saurabh Pandey, Preeti
are available for prescription medicines (Rx) and/or generic products.[15-23]
Pandey, Gaurav Tiwari,
Bioanalytical data generated in discovery and pre-clinical programs are a valuable
Ruchi Tiwari
guide to early clinical programs. Plasma concentration–response data from these
Pranveer Singh
programs can be compared with those obtained in man. Such comparisons are
Institute of Technology,
Bhauti, Kanpur, particularly valuable during the phase one-initial dose escalation study. To
Uttar Pradesh, India maximize this, it is our practice to generate PK data between each dose increase.[24]
Address for correspondence:
Dr. Saurabh Pandey, BIOANALYSIS
Pranveer Singh
Institute of Technology, Kanpur,
Uttar Pradesh, India.
Bioanalysis is a term generally used to describe the quantitative measurement
E-mail: [email protected] of a compound (drug) or their metabolite in biological fluids, primarily blood,
plasma, serum, urine or tissue extracts.[25] A bioanalytical method consists of two
DOI: **** main components

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 Pandey, et al.: Bioanalysis in drug discovery and development

Sample preparation: Sample preparation is a technique (LOD), recovery, reproducibility and ruggedness
used to clean up a sample before analysis and/or to (robustness).[31-33] Validation involves documenting,
concentrate a sample to improve its detection. When through the use of specific laboratory investigations,
samples are biological fluids such as plasma, serum that the performance characteristics of the method
or urine, this technique is described as bioanalytical are suitable and reliable for the intended analytical
sample preparation. The determination of drug applications. The acceptability of analytical data
concentrations in biological fluids yields the data used corresponds directly to the criteria used to validate
to understand the time course of drug action, or PK, the method.[34]
in animals and man and is an essential component
of the drug discovery and development process.[26] In early stages of drug development, it is usually not
Most bioanalytical assays have a sample preparation necessary to perform all of the various validation
step to remove the proteins from the sample. Protein studies. Many researchers focus on specificity, linearity
precipitation, liquid–liquid extraction and solid phase and precision studies for drugs in preclinical through
extraction (SPE) are routinely used.[27] Phase II (preliminary efficacy) stages. The remaining
studies penetrating validation are performed
Detection of the compound: The detector of choice when the drug reaches the Phase II (efficacy) stage
is a mass spectrometer.[26] Currently, the principle of development and has a higher probability of
technique used in quantitative bioanalysis is high becoming a marketed product. Presently, Guidelines
performance liquid chromatography coupled with for pharmaceutical methods in United States
tandem mass spectrometry (HPLC-MS/MS) using pharmacopoeia (USP), International Conference on
either electrospray ionization (ESI) or atmospheric Harmonization (ICH) and FDA provide a framework
pressure chemical ionization (APCI) techniques.[28] for regulatory submission must include study on such
The triple quadrupole (QqQ) mass spectrometer (MS), fundamental parameters.
when operated in the selected reaction monitoring
(SRM) mode, offers a unique combination of sensitivity, Validation parameters
specificity and dynamic range. Consequently, the There is a general agreement that at least the
QqQ MS has become the instrument of choice for following validation parameters should be evaluated
quantitation within the pharmaceutical industry. for quantitative procedures: selectivity, calibration
Since ESI and APCI can be operated at flow rates model, stability, accuracy (bias, precision) and limit
as high as 1 and 2 mL/min, respectively, most of of quantification. Additional parameters which
the convenience columns (e.g., C18, C8, C4, phenyl, might have to be evaluated include LOD, recovery,
cyanopropyl) are compatible. Recent technological reproducibility and ruggedness (robustness).
advances have made 1.7 μm particle size packing
material available. Coupling with high pressure pump Specificity/selectivity
and high-speed acquisition MS, ultra-high pressure A method is specific if it produces a response for only one
liquid chromatography (UPLC) offers unique high- single analyte. Since it is almost impossible to develop a
throughput and resolving power to obtain maximum chromatographic assay for a drug in a biological matrix
chromatographic performance and superior assay that will respond to only the compound of interest, the
Origi
sensitivity. [29] nal Aritcle term selectivity is more appropriate. The selectivity
of a method is its ability to produce a response for
Before a bioanalytical method can be implemented for the target analyte which is distinguishable from all
routine use, it is widely recognized that it must first other responses (e.g., endogenous compounds such as
be validated to demonstrate that it is suitable for its protein, amino acids, fatty acids, etc).[35]
intended purpose. A GLP (Good Laboratory Practices)
validated bioanalytical method is needed to support Accuracy
all development studies (e.g., toxicology studies and Accuracy of an analytical method describes the
human clinical trials). According to the Food and closeness of mean test results obtained by the method
Drug Administration (FDA) GLP guidance,[30] there to the true value (concentration) of the analyte. This
is a general agreement that at least the following is sometimes termed as trueness . The two most
validation parameters should be evaluated for commonly used ways to determine the accuracy or
quantitative procedures: selectivity, calibration method bias of an analytical method are (i) analyzing
model, stability, accuracy (bias, precision) and limit control samples spiked with analyte and (ii) by
of quantification. Additional parameters which comparison of the analytical method with a reference
might have to be evaluated include limit of detection method.[36]

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Precision will be negatively affected at the extremes of the range

It is the closeness of individual measures of an analyte by extensively expanding the range beyond necessity.
when the procedure is applied repeatedly to multiple Correlation coefficients were most widely used to test
aliquots of a single homogenous volume of biological linearity. Although the correlation coefficient is of
matrix. [30] There are various parts to precision, benefit for demonstrating a high degree of relationship
such as repeatability, intermediate precision, and between concentration and response data, it is of
reproducibility (ruggedness). Repeatability means little value in establishing linearity.[38] Therefore, by
how the method performs in one lab and on one assessing an acceptable high correlation coefficient
instrument, within a given day. Intermediate alone the linearity is not guaranteed and further tests
precision refers to how the method performs, both on linearity are necessary, for example, a lack-of-fit
qualitatively and quantitatively, within one lab, but test.
now from instrument-to-instrument and from day-
to-day. Finally, reproducibility refers to how that Range
method performs from lab-to-lab, from day-to-day, The range of an analytical procedure is the interval
from analyst-to-analyst, and from instrument-to- between the upper and lower concentration
instrument, again in both qualitative and quantitative (amounts) of analyte in the sample (including these
terms.[35,36] The duration of these time intervals is not concentrations) for which it has been demonstrated
defined. Within/intraday, - assay, -run and -batch that the analytical procedure has a suitable level of
are commonly used to express the repeatability. precision, accuracy and linearity.[30]
Expressions for reproducibility of the analytical
method are between interday, -assay, -run and Robustness
-batch. The expressions intra/within-day and inter/ It is the measure of its capacity to remain unaffected by
between-day precision are not preferred because a set small, but deliberate, variations in method parameters
of measurements could take longer than 24 hours or and provides an indication of its reliability during
multiple sets could be analyzed within the same day.[37] normal usage.

Detection limit Extraction recovery

The LOD is the lowest concentration of analyte in It can be calculated by comparison of the analyte
the sample that can be detected but not quantified response after sample workup with the response of
under the stated experimental conditions.[37] The LOD a solution containing the analyte at the theoretical
is also defined as the lowest concentration that can maximum concentration. Therefore, absolute
be distinguished from the background noise with recoveries can usually not be determined if the
a certain degree of confidence. There is an overall sample workup includes a derivatization step, as
agreement that the LOD should represent the smallest the derivatives are usually not available as reference
detectable amount or concentration of the analyte of substances.
interest.
Stability
Quantitation limit It is the chemical stability of an analyte in a given
The quantitation limit of individual analytical matrix under specific conditions for given time
procedures is the lowest amount of analyte in a intervals.[30] The aim of a stability test is to detect any
sample, which can be quantitatively determined with degradation of the analyte(s) of interest during the
suitable precision and accuracy. entire period of sample collection, processing, storing,
preparing, and analysis.[39] All but long-term stability
Linearity studies can be performed during the validation of
According to the ICH definition, “the linearity of an the analytical method. Long-term stability studies
analytical procedure is its ability (within a given range) might not be complete for several years after clinical
to obtain test results which are directly proportional to trials begin. The condition under which the stability is
the concentration (amount) of analyte in the sample”. determined is largely dependent on the nature of the
The concentration range of the calibration curve analyte, the biological matrix, and the anticipated time
should at least span those concentrations expected to period of storage (before analysis). The ICH guidelines
be measured in the study samples. If the total range are summarized in Table 1.
cannot be described by a single calibration curve, two
calibration ranges can be validated. It should be kept The drug research can be divided functionally into two
in mind that the accuracy and precision of the method stages: discovery/design and development [Figure 1].

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Table 1: US FDA guidelines for bioanalytical method validation

Selectivity Analyses of blank samples of the appropriate biological matrix (plasma, urine or other matrix) should be obtained from at
(specificity) least six sources. Each blank should be tested for interference and selectivity should be ensured at LLOQ
Accuracy Should be measured using a minimum of six determinations per concentration. A minimum of three concentrations in
range of expected concentrations is recommended for determination of accuracy. The mean should be ±15% of the
actual value except at LLOQ, where it should not deviate by ±20%. This deviation of mean from the true values serves
as the measure of accuracy
Precision Should be measured using a minimum of five determinations per concentrations. A minimum of three concentrations in
the range of expected concentrations is recommended. The precision determined at each concentration level should not
exceed 15% of the coefficient of variation (CV) except for the LLOQ, where it should not exceed 20% of the CV
Recovery Recovery experiments should be performed at three concentrations (low, medium and high) with unextracted standards
that represent 100% recovery
Calibration curve Should consist of a blank sample (matrix sample processed without internal standard), a zero sample (matrix sample
processed with internal standard) and six to eight non-zero samples covering the expected range, including LLOQ
LLOQ Analyte response should be five times the response compared to blank response. Analyte peak should be identifiable,
discrete and reproducible with a precision of 20% and an accuracy of 80–120%
Freeze–thaw Analyte stability should be determined after three freeze–thaw cycles. At least three aliquots at each of the low and high
stability concentrations should be stored at the intended storage temperature for 24 hours and thawed at room temperature.
When completely thawed, refreeze again for 12–24 hours under same conditions. This cycle should be repeated two
more times, then analyze on third cycle. Standard deviation of error should be <15%. If the analyte is unstable, freeze at
–70oC for three freeze–thaw cycles
Short-term stability Three aliquots of each of the low and high concentrations should be thawed at room temperature and kept at this
temperature for 4–24 hours and analyzed. % Deviation should be <15%
Long-term stability At least three aliquots of each of low and high concentrations at same conditions as study samples. Analyze on three
separate occasions. Storage time should exceed the time between the date of first sample collection and the date of last
sample analysis
Stock-solution Stability of stock solutions of drug and the internal standard should be evaluated at room temperature for at least 6
stability hours. % Deviation should be <15%
Quality control QC samples in duplicates at three concentration levels (one near the 3× LLOQ, one in mid-range, one close to high
(QC) samples end) should be incorporated at each assay run. At least four out of every six should be within 15% of respective nominal
value. Two of six may be outside 15% but not both at the same concentration. Minimum number QCs should be at least
5% of total number of unknown samples or six total QCs, whichever is greater
Lower limit of Quantification

could be merely to provide reasonable values of

either concentrations and/or exposure which would
be used to form a scientific basis for lead series
identification and/or discrimination amongst several
lead candidates. Therefore, the aim of the analyst at
this stage should be to develop a simple, rapid assay
with significant throughput to act as a great screening
tool for reporting some predefined parameters of
several lead contenders across all the various chemical
scaffolds.
Origi nal Aritcle
The initial method of analysis developed during
the discovery phase of the molecule, with some
modifications, may sometimes serve as a method
of choice to begin with as the NCE enters the
preclinical development stage. Since the complexity
of development generally tends to increase as the lead
candidate enters the toxicological and clinical phase
of testing, it naturally calls for improved methods of
analytical quantization, improvement in selectivity
and specificity, and employment of sound and rugged
Figure 1: Showing different stages of discovery/design and development
validation tools to enable estimation of PK parameters
that would also aid in the decision-making of the
DRUG DISCOVERY/DESIGN drug molecule’s advancement in the clinic in addition
to safety and tolerability data gathered at all phases
Initially, in the discovery stage, the aim of bioanalysis

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of development. Additionally, it becomes necessary documented.[47-50] These important tests are collectively
to quantify active metabolite(s) in both animals and referred to as ADME characteristics (Absorption,
humans.[40] Distribution, Metabolism and Elimination).[26] Figure
2 shows an illustration of a possible scenario of
Drug discovery/design consists of identification and bioanalysis in discovering drugs which are active
characterization of new targets (enzymes or receptors), in vitro and improving these by modification of the
synthesis of new lead molecules, screening of new chemical structure optimized for in vivo activity.[51]
lead molecules for in vitro and/or in vivo biological
activities, and physicochemical characterization of
leads.[41] For discovery, the priority is to examine a
large number of compounds and determine which
pharmacologically active compounds are most
suitable for drug development. In practice, when
a compound is obtained which has the required
biological activity, a number of analogues or
chemically similar compounds will be synthesized
and tested to optimize the preferred characteristics of
the compound (a process known as lead optimization).
Using automated techniques, ultrahigh throughput
can be obtained by the most advanced laboratories and
tens of thousands of compounds can be screened in one
day. In the secondary screening stage, physiochemical
properties such as solubility, lipophilicity and stability
are determined by using octanol–water partition
coefficient and pKa. These measurements are useful
in predicting protein binding, tissue distribution
and absorption in gastrointestinal tract.[42] In parallel
studies, information is learned on a drug molecule’s
absorption, distribution (including an estimate of
protein binding), metabolism and elimination by
sampling from dosed laboratory animals (called in Figure 2: Showing possible scenario of bioanalysis in discovering drugs
vivo testing) and from working cells and/or tissues
removed from a living organism (called in vitro testing Table 2: List of experiments to assess ADME
since the cells are outside a living animal). For in Parameter examined Typical experiments
vivo characterization of PK and bioavailability, it is Absorption Caco-2 cells, MDCK cells, PgP transport
In vivo PK profiling
necessary to administer the drug to selected animal
Distribution In vitro protein binding
species both intravenously and by the intended
In vivo tissue distribution studies
route of administration (usually oral). Whole blood Metabolism Metabolic stability
samples are collected over a predetermined time Microsomes, subcellular fractions,
course after dosing, and the drug is quantified in the hepatocytes
harvested plasma by a suitable bioanalytical method. P450 inhibition studies
The use of in vitro drug metabolism approaches for Microsomes
the prediction of various in vivo PK characteristics is P450 induction studies
widely practiced in the pharmaceutical industry.[43-46] Gene chips, multiple dosing
Elimination Quantitation of drugs and metabolites in
In particular, in vitro metabolic stability assessment biological fluids
using hepatic subcellular fractions to predict in
vivo hepatic clearance is employed as part of the
initial screening of candidates in a lead optimization Table 3: Reasons for failure in drug development
program. This is because the liver is the main organ [57]
involved in the metabolism of xenobiotics, the process Reasons %
by which most drugs are cleared from the body. The Poor biopharmaceutical properties 40
correlation between in vivo hepatic clearance values Lack of efficacy 30
and the intrinsic clearance values determined from Toxicity 21
Commercial reasons 8
liver microsomal incubation experiments is also well

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ADME/PK screening is usually taken to mean in vitro Preclinical stage

systems for studying absorption and metabolism. Once a chemical is identified as a new drug candidate,
However, in vivo studies still provide the definitive extensive preclinical analyses must be completed
assessment of overall drug disposition, and progress before the drug can be tested in humans.[57] The main
has been made in overcoming some of the constraints goals of preclinical studies (also named nonclinical
associated with this approach. Previously, drug studies) are to determine a product’s ultimate
metabolism studies were performed at a late stage of safety profile. Each class of product may undergo
drug development process and very often not until different types of preclinical research. For instance,
the phase of clinical studies. Therefore, inadequate drugs may undergo pharmacodynamics (PD), PK,
metabolism and PK parameters were the major reason ADME, and toxicity testing through animal testing.
of failure for NCEs.[52] Nowadays, introduction of in During preclinical investigation, validation should
vitro approaches into drug metabolism enables the be formalized and mandated as per the required
characterization of the metabolic properties of drug norm. The validation should address as many
candidates at an earlier stage in the drug development parameters as possible which are relevant, to obtain
process, at early preclinical studies performed during unambiguous analytical data [the list could include
the drug discovery phase. Recently, the major reasons accuracy, precision, specificity, selectivity, linearity
for high attrition rates have instead been identified to range, lower limit of quantification (LLQ), upper
be lack of efficacy and safety, together accounting for limit of quantification (ULQ), dilution effect, stability
approximately 60% of the failures. Cassette dosing is or extraction recovery]. Since the data gathered
now an established method within the pharmaceutical during this stage, especially PK and toxicokinetic
industry as it provides a relatively quick way of properties of the NCE, would become part of the
ranking compounds according to their PK properties initial investigational new drug and clinical trial
and requires the use of fewer animals.[53,54] (IND/CTA) filings in several regions, the adherence
to certain rigid validation parameters and protocols
A list of experiments that are commonly performed becomes of paramount importance. The developed
to assess the ADME characteristics of potential lead assay at this stage may differ from the original assay
compounds in drug discovery is given in Table 2.[26] of the discovery phase in that an internal standard
addition may be used (in the event that an internal
DRUG DEVELOPMENT standard was not used before) to ensure reliability
of the quantitation. It is a common practice in some
It focuses on evaluation of safety/toxicity and efficacy pharmaceutical companies to incorporate a stable
of new drug molecules. However, majority of the isotope labeled compound of the parent as an
drug molecules fail in subsequent drug development internal standard (IS) to provide extra comfort in the
program because the efficacy and safety are not bioanalysis of NCEs at this stage.[58] It is especially
governed by its PD characteristics alone. It also valuable in lead optimization for studying the PK of
depends to a large degree on the biopharmaceutical multiple compounds administered simultaneously.
(e.g., solubility, stability, permeability and first pass Plasma levels of the drug are normally monitored to
effect) and PK (clearance rate, biological half-life, permit the calculation of PK parameters such as Cmax
Origi nal Aritcle
extent of protein binding and volume of distribution) [maximum plasma concentration (e.g., ng/mL)] and
properties of the drug, since these properties control AUC [area under the plasma concentration–time curve
the rate and the extent to which the drug can reach its (e.g., ng h/mL)]. Distribution parameters describe the
site of action, i.e., biophase [55]. Some data on reasons extent of drug distribution and are related to body
for withdrawal of candidate drugs from development volumes (e.g., mL or L), and time course parameters
have been published by the Center for Medicines are related to time (t1/2, Tmax). These PK parameters are
Research[56] [Table 3]. calculated from mathematical formulae, and specific

Table 4: Objectives of the four phases in clinical drug development and typical numbers of volunteers or
patients involved
Phase I Phase II Phase III Phase IV
Establish safe dosing range and Demonstrate efficacy, Gain data on safety and Expand on approved claims or
assess PK; also called first time in identify side effects and effectiveness in a larger demonstrate new claims; examine
man (FTIM) assess PK population of patients; assess PK special drug–drug interactions;
20–80 males 200–800 patients 1000–5000 patients assess PK
Volunteers Several A few thousand to thousand patients

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Table 5: Bioanalytical framework in drug discovery and development: Key considerations

Stage Key objectives for Suggested validation work with comments
bioanalysis
Discovery Screen leads for microsomal •Selectivity and specificity (no endogenous interference; lack of interference from other
stability, in vivo PK coadministered agents, if cassette dosing and/or cassette analysis is pursued)
screen; preliminary in vitro •Preliminary range specification for quantitation
cytochrome P450 inhibitory •Number of standards (n = 3–5): Although duplicate standards are recommended, the analyst
liability screens, caco-2 cell should make the judgment call if single standards are adequate
permeability •Reasonable accuracy and precision
•Early read on chromatographic and detection conditions
Preclinical: Characterization of PK •Complete assessment of accuracy and precision (3–5 validation runs): To cover intra- and
Stage 1 disposition in rodents/dogs/ inter-day variability assessments in both %bias and precision
monkeys (single dose, •Confirmation of selectivity and specificity
multiple dose, absolute •Defined range of standard calibration curve
bioavailability); route- •Fixing of LLQ and ULQ of the assay
dependent PK disposition; •Assessment of dilution effect on accuracy and precision
formulation screening for •Finalization of extraction scheme, chromatographic and detection conditions
toxicology work; definitive in •Extraction recovery assessment using acceptable methods (should cover the entire range);
vitro drug–drug interaction also, IS recovery should be assessed
liability; in vivo drug–drug •Stability experiments to cover various scenarios such as freeze–thaw, bench top and
interaction potential, processed sample stability, frozen conditions
toxicokinetics (and/or •In order to facilitate PK and toxicokinetic work in animals, it is suggested to have a frozen
exposure levels) in toxicology stability window in plasma/serum (or urine) established for 6–8 weeks
species Cross validation: Protocols may be developed to measure drug levels in a second species and/
or a second matrix using a previous fully validated method
Partial validation: As deemed appropriate, specific analytical protocols may be developed to
support this. This would aid in extending the analytical range for a very specific application
Preclinical: Characterization of PK Approaches to validation plan as described under Clinical stage 1 and stage 2 can be followed
Stage 2 disposition of parent and and modified as deemed necessary
metabolite in pharmacological
and toxicological species;
characterization of in vivo
drug–drug interaction studies
Clinical: Characterization of PK •Similar to preclinical stage 1 scheme, all elements need to be covered
Stage 1 disposition in healthy human •The fixing of LLQ (i.e., determination of lowest sensitivity) for the assay may be based on
subjects – single doses, anticipated concentrations following the lowest dose in humans
multiple doses; relative •Throughput of the assay should be optimized to obtain rapid analytical data to support the
bioavailability assessment next dose escalation stage
(solution/suspension vs. solid •The window for sample stability under frozen conditions needs to be for at least 6 months
dosage form); food effect •Unlike preclinical, a contingency plan to develop a second analytical laboratory to validate and
potential; characterization run the assay may be considered
of PK disposition in targeted •Verification of robustness/ruggedness of the assay needs to be established (covering both
patient population; delineation laboratory-to-laboratory variability as well as the analyst’s transfer of the assay)
of PK in special populations
(pediatric, geriatric, renal
impairment, hepatic
dysfunction)
Clinical: Characterization of the PK •The assay developed in clinical stage 1 may be used to support these activities. Since one
Stage 2 disposition of the parent and or more metabolites are introduced, it is imperative that the assay be validated to support the
its metabolites in plasma, quantitation of such metabolites
urine and serum •Separate assay procedure may be developed for the metabolites or the parent and
metabolites may be assessed in a single run (to be followed, validation is required)
•Validation includes the following: linearity, LLQ, ULQ, precision, selectivity, specificity and
stability assessment of metabolites
Clinical: Evaluate the drug–drug •Sponsor has two options in developing and validating the assays for these activities
Stage 3 interaction potential with •Option 1: Methods may be developed wherein the NCE and potential metabolites are
agents commonly prescribed simultaneously quantified with the coadministered agent (without and/or with appropriate
for such patient population; metabolites)
drug–drug interaction potential •Option 2: Separate methods may be developed for the coadministered agents, while earlier
with agents having narrow developed assays for NCE and metabolites (stages 1 and 2) may be used for quantitation of
safety window (digoxin, parent and metabolites as the need arises
warfarin, etc.) •It is recommended that separate methods be developed for the coadministered agents by the
sponsor because of the applicability of the assay for future
•NCEs without having to necessarily validate it again (if option 1 is used)
•Finalization of chromatographic detection conditions
•Validation includes the following: linearity, LLQ, ULQ, accuracy, precision, selectivity,
specificity and stability assessment of metabolites

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obtained in drug development in preclinical species,

the definitive kinetics is obtained in drug development
by conducting single dose experiments in preclinical
species and in humans. These data are essential in
defining the dosage regimen in man and ensuring that
the therapeutic benefit is maximized.[65-70]

Clinical stage
Clinical trials are used to judge the safety and efficacy
of new drug therapies in humans. Drug development
comprises of four clinical phases: Phase I, II, III and
IV [Table 4]. Each phase constitutes an important
juncture, or decision point, in the drug’s development
cycle. A drug can be terminated at any phase for any
valid reason. As the molecule advances into clinical
development, the developed assay for human sample
analyses (plasma, serum or urine matrix) needs to
be more rugged, robust and be able to withstand the
Figure 3 : Showing various steps of bioanalysis in the development test of time during this the longest phase of clinical
stage of drugs development.[71-79]
computer programs are usually used to do this (e.g., The requirements and adherence to specificity,
WinNonlin).[59-63] The parameters may be estimated selectivity and stability will become very important.
by compartmental or non-compartmental approaches Since it is likely that patients will be concomitantly
(or model-dependent and model-independent, ingesting other medications, the assay has to be
respectively). Figure 3 shows some possible steps flexible enough to accommodate minor alterations
of bioanalysis in the development stage of drugs. in chromatographic conditions to circumvent the
However, the determination of metabolite profiles interfering peaks, if necessary.
is usually performed for a limited number of lead
molecules in vivo and in vitro, and in these experiments Table 5 provides a framework of the various stages
the key issues are high specificity and sensitivity of bioanalytical assay development (discovery,
rather than speed.[64] preclinical and clinical), key objectives that assay
would support and some suggested validation
I n t h e p h a r m a c e u t i c a l i n d u s t r y, t h e t e r m workup that may be required to achieve the end goals.
“toxicokinetics” is generally used to describe the PK Based on the presentation [Table 1], it is apparent that
performed at the dose levels used in the toxicological assays developed in the early discovery stage may
risk assessment of drugs. The aims of the toxicokinetic find utility during the course of an NCE’s progress
evaluation are in development.
Origi
• to define nal
the Aritcle between systemic
relationship
exposure to test compound and the administered
dose, CONCLUSION
• to provide information on potential dose- and
time-dependencies in the kinetics, The need for sound bioanalytical methods is well
• to determine the effect of age on the PK in animals, understood and appreciated in the discovery phase
provide clearer delineation when there are sex- and during the preclinical and clinical stages of drug
related differences, determine whether there are development. Therefore, it is generally accepted
any changes in kinetics in pregnancy (during that sample preparation and method validation are
reproductive toxicology studies) and also provide required to demonstrate the performance of the
greater detail on interspecies comparisons. method and the reliability of the analytical results.
The acceptance criteria should be clearly established
However, the overall aim in conducting toxicokinetics in a validation plan, prior to the initiation of the
during safety studies is to extrapolate the risk validation study. The developed assay should be
assessment from the toxicity test species to humans. sufficiently rugged that it provides opportunities for
[51]
Whilst preliminary PK and toxicokinetic data are minor modifications and/or ease of adoptability to

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suit other bioanalytical needs such as applicability to and validation for simultaneous determination of five model
compounds—antipyrine, metoprolol, ketoprofen, furesemide and
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Source of Support: Nil, Conflict of Interest: None declared.
197/ONO-8025) and its metabolites in human plasma by liquid

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