Bioanalytical method development and validation of anticancer drugs using

LCMS and pharmacokinetic studies in animal model

Analytical chemistry is used to determining the qualitative and quantitative composition of
material under study and is divided into two branches quantitative and qualitative. A
qualitative analysis gives us the information about the nature of sample by knowing about the
presence or absence of certain components. A quantitative analysis provides numerical
information as to the relative amount of one or more of this component. Analytical method
development is essential for routine analysis of the pharmaceutical formulations, to determine
the percentage purity of drug in its bulk form, for therapeutic drug monitoring in clinical
settings and so on1&2. In non-instrumental methods, the conventional and physicochemical
property are used to analyse the sample. The instrumental methods of analysis are based upon
the measurements of some physical property of substance using instrument to determine its
chemical composition. The instrumental methods are simple, precise and reproducible as
compared to classical methods. Therefore, analytical methods developed using sophisticated
instruments such as spectrophotometer, HPLC, GC and HPTLC have wide applications in
assuring the quality and quantity of raw materials and finished products.

Chromatography: Chromatography is a laboratory technique for the separation of a mixture.

The mixture is dissolved in a fluid called the mobile phase, which carries it through a
structure holding another material called the stationary phase. The various constituents of the
mixture travel at different speeds, causing them to separate. The separation is based on
differential partitioning between the mobile and stationary phases. Subtle differences in a
compound's partition coefficient result in differential retention on the stationary phase and
thus affect the separation3

Chromatography may be preparative or analytical. The purpose of preparative

chromatography is to separate the components of a mixture for later use, and is thus a form of
purification. Analytical chromatography is done normally with smaller amounts of material
and is for establishing the presence or measuring the relative proportions of analytes in a
mixture. The two are not mutually exclusive4

High Pressure Liquid Chromatography (HPLC): High Performance Liquid Chromatography

(HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a
solvent (known as the mobile phase) at high pressure through a column with chromatographic
packing material (stationary phase). HPLC has the ability to separate, and identify
compounds that are present in any sample that can be dissolved in a liquid in trace
concentrations as low as parts per trillion. Because of this versatility, HPLC is used in a
variety of industrial and scientific applications, such as pharmaceutical, environmental,
forensics, and chemicals.

Types of HPLC: There are following variants of HPLC, depending upon the phase system
(stationary) in the process:
1. Normal Phase HPLC: This method separates analytes on the basis of polarity. NP-HPLC
uses polar stationary phase and non-polar mobile phase. Therefore, the stationary phase is
usually silica and typical mobile phases are hexane, methylene chloride, chloroform, diethyl
ether, and mixtures of these. Polar samples are thus retained on the polar surface of the
column packing longer than less polar materials.
2. Reverse Phase HPLC: The stationary phase is nonpolar (hydrophobic) in nature, while the
mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile. It
works on the principle of hydrophobic interactions hence the more nonpolar the material is,
the longer it will be retained.
3. Size-exclusion HPLC: The column is filled with material having precisely controlled pore
sizes, and the particles are separated according to their molecular size. Larger molecules are
rapidly washed through the column; smaller molecules penetrate inside the porous of the
packing particles and elute later.
4. Ion-Exchange HPLC: The stationary phase has an ionically charged surface of opposite charge to
the sample ions. This technique is used almost exclusively with ionic or ionizable samples. The
stronger the charge on the sample, the stronger it will be attracted to the ionic surface and thus, the
longer it will take to elute. The mobile phase is an aqueous buffer, where both pH and ionic strength
are used to control elution time.

Literature review:

1.Konatham TK Reddy et al.,2022 The present study was conducted to establish a convenient,
economical, robust, and accurate technique for assessing Ipilimumab in LC-MS/MS utilizing
cetuximab as a reference standard. The overall chromatographic operating time was 7 minutes, with a
retention time of 3.143 minutes for ipilimumab. The approach is verified across a dynamically linear
range between 12.50 and 100 ng/mL for ipilimumab having a coefficient of correlation (r2) of 0.999.
The intra-batch, as well as inter-batch accuracy (%CV), was below 15% for all five levels (LLOQ,
HQC, MQC, ULOQ, and LQC). This may be confirmed based on USFDA regulations
2.Pravallika KE.et al.,2020 A bioanalytical LC-MS/MS method for the entrectinib was developed and
validated with entrectinib D4 as IS. The method has excellent accuracy, precision, and recovery
compared with existed methods for the analysis of drug in rat plasma. The methods developed in our
laboratory are very simple, utilizing liquid-liquid extraction procedure, which makes the method high
throughput for analysis. Entrectinib was eluted within 6 min using RP-high-performance liquid
chromatography Luna, 250×4.6 mm, 5 µm column and the mobile phase containing 0.1%

Need of study
Bioanalysis describes quantitative estimation of chemicals or drug substances as well as their
metabolites in large variety of bio-samples. It is an integrated technique that has been
employed in preclinical stages of drug-discovery process to further support the clinical phases
of drug discovery. However, a bioanalytical method must be optimized, characterized, and
validated following documented procedures according to United States Pharmacopeia
(USP)/International Council for Harmonisation of Technical Requirements for
Pharmaceuticals for Human Use (ICH) guidelines to comply with the regulatory guidelines
and acceptance criteria. Bioanalytical studies should therefore provide accurate and
reproducible estimation of drugs or metabolites in biological samples at great level of
sensitivity, selectivity, and specificity1&2.
Prevalence of cancer is increasing worldwide and as per American cancer society (ACS) the
number of cancer cases will increase as high as 27 million by 2040. But the cancer can be
cured and managed by early detection and targeted drug therapies. Therefore sensitive,
effective bioanalytical methods are important to assess the pharmacokinetics of drugs and to
ensure whether the patients are receiving correct dose of medication in order to achieve
therapeutic effect and minimal or no toxic effect.
Liquid chromatography with mass spectrometer detector (LCMS) is a suitable approach for

the detection of compounds at nanogram and picogram concentrations and hence reliable for

therapeutic drug monitoring in clinical settings. LCMS methods are supercilious compared to

other bioanalytical methods due to their accuracy, specificity and selectivity. Hence the

proposed study is aimed at developing economical, accurate, sensitive, specific and simple

bioanalytical methods using LCMS for the determination of anticancer drugs in biological

samples.
Pharmacokinetics are the study of the movement of drug substances in living
organisms

using delineative models and mathematical rate equations to describe the

absorption,

distribution, metabolism or degradation, and elimination (clearance) of drugs

from the body

Pharmacokinetic assessments have become an essential component in the

preclinical

development and translation of nanomaterial-based drug delivery systems and

are routinely

studied in preclinical and clinical nanomaterials literature. The motivations for

leveraging

nanoscale materials for drug delivery are rooted in the distinct kinetic
behaviours of

nanomaterial-based drugs in vivo, such as improved delivery of encapsulated

drug into

target tissues (e.g., tumours) and reduced toxicities associated with off-target
drug

accumulation (e.g., cardiotoxicity, nephrotoxicity cutaneous phototoxicity, etc.) .

Important

achievements have been made towards our understanding of the complex and
multifactorial

relationships between physicochemical properties, species-dependent

physiological factors,

and the resulting disposition and pharmacokinetic profiles of many

nanomaterials .

Meaningful progress continues to be made in both the preclinical and clinical

realms with

comprehensive evaluations of the biological and immunological interactions

affecting

nanomaterial ADME in vivo . Translating these research discoveries into the

rational design

of innovative nanomaterials has yielded a spectrum of structurally and

functionally diverse

products wherein evaluation of pharmacokinetic profiles in animal models plays

an

important role for deciding whether to continue with preclinical development.

Historically, the leading cause behind the termination of almost 40% of new
small molecule

drug candidates in 1988 was inappropriate pharmacokinetic profiles in clinical

patients . By

2003 this rate had dropped to under 10% with the increased

standardisation of strategies and methods for collecting preclinical

pharmacokinetic data,

and the proliferation of in vitro screens for ADME and mathematical modelling
tools .

Although similar figures do not exist for complex nanomaterial-based drugs, a

comprehensive survey of the clinical failures in cancer nanomedicine revealed

that poor

efficacy—an indicator of inappropriate pharmacokinetic profiles in patients—

was a

significant and recurring factor in the decision to terminate further clinical

investigation of

many products . In fact, the pharmacokinetic variability of anti-cancer

nanomedicines

between patients (i.e., interindividual variability) has been greatly

underestimated from

preclinical studies and is increasingly recognised as the principle contributor to

treatment

failure of nanomedicines in oncology , challenging the role of nanomaterials as

superlative

drug delivery systems. Thus, interest has been renewed for understanding the
obstacles

limiting the overall predictiveness of pharmacokinetic data in preclinical animal

models and

how the choice of design framework and key experimental factors might
improve the

predictive power of these studies for humans.

Unfortunately, by one account an estimated 80% of preclinical studies with

therapeutic

nanomaterials published over the past decade have failed to report basic
characterisations of
their nanomaterials, including pharmacokinetic parameters . The prevalence of
poor

pharmacokinetic characterisations in nanomaterials reporting creates

uncertainty for

investigators over the accurate interpretation of reported pharmacokinetic data,

and

ultimately diminishes experimental reproducibility in nanomaterials research.

Therefore, the

motivation for this review is to provide researchers with the theoretical

understanding, and

the practical tools and techniques needed for performing appropriate

pharmacokinetic

assessments of nanomaterials in laboratory animals. This is accomplished by

examining the

state-of-the-art in industry research practices, the principle strategies and

important

considerations for collecting pharmacokinetic data of nanomaterials in

preclinical animal

models. These lessons are refined into simple guidelines that provides
researchers with

practical knowledge and pragmatic recommendations for selecting the most

appropriate

experimental design framework with which to study the pharmacokinetic

profiles of their

nanomaterials in animals. The objectives for these guidelines are to improve the
consistency

and quality of pharmacokinetic data in nanomaterials reporting with proper

study designs

and to advance the predictive power of preclinical pharmacokinetic profiles for

accurately

anticipating nanomaterial behaviours in humans. Succeeding these objectives

will accelerate

the discovery and development of innovative nanomaterials and maximise the

chances of

translational success of these products into patients.

Pharmacokinetic studies will provide information about the absorption, distribution,
metabolism, excretion and protein drug binding. Safe pharmacological and toxicological
studies are carried out in animals to select starting dose and dosing regimens for phase 1
human studies, dose-escalation schemes for clinical studies and to assess the toxic effects
with respect to target organs. The US Food and Drug Administration (FDA) and the
European Medicines Agency (EMA) recommend that new anti-cancer agents be evaluated in
both rodent and non-rodent species before undergoing human phase I evaluation3.
Aim and Objectives

The aim of the current research proposal is to develop and validate simple, economic,
reliable, specific and sensitive bioanalytical methods for anticancer drugs in biological
matrices
- The primary objective is to develop a new, simple, sensitive and specific analytical
methods for the selected drugs
- Optimization of instrumental conditions and mobile phase
- Extraction of drugs from biological matrices and sample preparation
- To validate proposed methods according to ICH guidelines
- The secondary objective is to carry out the pharmacokinetic studies in animal model
applying the developed and validated method.
Plan of work
 Extensive literature survey for selection of drugs, appropriate solvents to dissolve
respective selected drugs and preparation of stock solutions.
 Procurement of samples, standards and other chemicals
 Selection of chromatographic conditions & mobile phase
 Method trials on LC-MS by using different solvents and columns.
 Optimization of the developed method by varying mobile phase conditions,
temperature.
 Validation of the developed method as per ICH guidelines
 Selection of animal model and carrying out Pharmacokinetic studies after getting
IAEC approval.
References
1. Shivani S, Swapnil G, Kalindi C. A review on analytical method development and
validation. Interanational journal of applied pharmaceutics 2018:10.
2. Sonali GT, Rupesh VC and Madhukar RT. Development and validation of HPLC and
HPTLC methods for therapeutic drug monitoring of capecitabine in colorectal cancer
patients. Journal of chromatographic science 2020:57;892-900.
3. https://oncologypro.esmo.org/education-library/esmo-books/esmo-handbooks/
clinical-pharmacology-of-anti-cancer-agents/studies-in-animal-models dated
28/02/2024