Introduction & Literature Review Chapter 1

CHAPTER 1

INTRODUCTION &

LITERATURE REVIEW

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Introduction & Literature Review Chapter 1

Table of Contents

1.1 Formulation Excipients ....................................................................................... 3

1.1.1 Solution Vehicles ........................................................................................... 4
1.1.2 Suspension Vehicles ...................................................................................... 5
1.1.3 Formulation excipients selected for the research ........................................... 5
1.1.3.1 Cremophor EL ....................................................................................... 5
1.1.3.2 Polyethylene glycol 400 (PEG 400) ...................................................... 7
1.1.3.3 Solutol HS15 .......................................................................................... 8

1.2 Spiky plasma concentration profiles ................................................................ 10

1.3 Matrix Effect ...................................................................................................... 12

1.4 Literature Review .............................................................................................. 15

1.5 References ........................................................................................................... 21

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1.1 Formulation Excipients

Biological properties of molecules will depend on the composition of formulation

vehicle. The main objective of formulation preparation is to maximise exposure in
preclinical pharmacology studies, such that the PK/PD and toxicology readouts can be
correlated to targets biological response. Effective formulation preparation and drug
delivery strategies are important to achieve consistency in exposure. Formulation
development was much more challenging due to differences in the physiology
between various animal species, specific pharmacology models and different routes of
administration. While optimising a formulation vehicle combination, the
physicochemical properties of compounds should be taken in to consideration
(Neervannan S et al, 2006).
The goal of researchers is to obtain optimal oral bioavailability due to economical and
clinical reasons. Intersubject variability is very high with respect to plasma
concentrations if drugs have less oral bioavailability, which in turn results in
suboptimal regulation of drugs activities. In case of drugs with low bioavailability
more amount of material will be wasted which is certainly a economic disadvantage
for some of the costly drugs. Reasons for low bioavailability could be due to poor
permeability, poor solubility or first pass metabolism. But, major reason for low oral
bioavailability could be attributed to poor aqueous solubility of compounds that can
be dealt in with different kinds of formulation strategies. Oral bioavailability was
optimised by taking in to consideration the selection process, compound design and
also the effect of formulation. The limitations in achieving optimal oral bioavailability
are effectively overcome by drug discovery researchers with the aid of different
formulation strategies (QPS Application Note, 2008). In drug discovery programs,
active compounds with good pharmacokinetic properties were shortlisted by
performing pharmacokinetic (PK) studies in rats or mice. PK studies can be a bottle
neck in compound selection process due to requirement of both analytical and animal
resources, and complexity of understanding the PK data. If a new chemical entity
(NCE) in a series shows poor PK properties it will not be advanced to the next level
of screening, but to avoid discarding compounds that has good pharmacologic
properties, a cautious approach is recommended to understand the physicochemical

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properties of the compounds and design the best suitable formulation that completely
solubilises the compounds. For increasing oral absorption of low soluble compounds a
better formulation design will enable to solubilise the compounds and helps in
screening and short listing active compounds in the series studied.
One of the major challenges for drug discovery researchers is to select vehicles for
invivo studies. Researchers must keenly understand the objectives of the invivo
studies as each vehicle has different type of role and objective in enabling a good
formulation. If the objective of study is to understand the oral exposure of
compounds, then the excipients selected should not interfere with the end point of
measurement, and this can be achieved by preparing solution formulation than
suspension formulation. If the main objective of study is to understand whether the
compounds has developable properties, then a more complex suspension formulations
can be designed (Xue QC et al, 2006).

1.1.1 Solution Vehicles

To identify various vehicles that are optimal for solubilising the compounds, initially
the compounds solubility should be tested in both aqueous and non aqueous systems.
Along with solubility, also the chemical stability should be looked at. Various
strategies are available on hand to increase the solubility of poorly soluble
compounds. pH adjustment can be used to increase solubility, if the compounds are
ionisable in nature. pH adjustment along with cosolvents approach also can be tried if
pH adjustment alone does not produce desired results. The commonly used cosolvents
for solubilisation is polyethyleneglycol 400 (PEG 400) due to its inert nature and
good solubilisation of low soluble compounds that are administered in both oral and
intravenous routes (Raymond CR et al, 2009). Other cosolvents that could be tested
were Labrafil, N-methyl-2-pyrrolidone and vitamin E TPGS (d-alpha tocopheryl
polyethylene glycol 1000 succinate). Apart from use of cosolvents, complexing agents
(cyclodextrins) can be tested to increase the solubility. For drug discovery
programmes with diversity in molecular structures, extra care should be emphasized
to ensure the solubility of all compounds in cyclodextrin solutions.
Intravenous administration of compounds require the compound in solution form
before administration and there are various approaches to solubilise the compounds
beyond its aqueous solubility (Sweetana S et al, 1996). When cosolvents were used
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for solubilisation, extra care needs to be ascertained not to use them beyond toxic
levels. Use of high concentration of cosolvents can cause hemolysis and precipitation
of the compound immediately after administration. An invitro precipitation technique
was developed by yalkowsky and his group to predict the compounds liability to
precipitate after intravenous administration (Johnson JLH et al, 2003).

1.1.2 Suspension Vehicles

Majority of drugs are developed to be administered in oral route as solid dosage form.
Due to limited compound availability and amount of time required to prepare solid
dosage form, it is not desired to dose solid dosage forms as it delays the process of
compound screening in drug discovery and is quite costly affair. So instead of solid
dosage forms it is desirable to dose compound in suspension formulation. Suspension
formulations can be prepared by using various excipients such as emulsifying agents,
suspending agents and surfactants.
List of commercially available oral and injectable solution/suspension formulations
(Robert S, 2000) that are used as solubilizing excipients in preclinical formulations
include water soluble organic solvents, non-ionic surfactants, water insoluble lipids,
organic liquids/semisolids, various cyclodextrins, and phospholipids. Apart from the
solubilising excipients, various other chemical techniques to solubilise insoluble drugs
include cosolvents, pH adjustment, micro emulsions, complexation, micelles, self
emulsifying drug delivery systems, emulsions and liposomes.

1.1.3 Formulation excipients selected for the research

1.1.3.1 Cremophor EL

Reaction between varying amounts of ethylene oxide and castor oil/hydrogenated

castor oil, produces polyoxyethylene castor oil derivatives. Different types of castor
oil derivatives are commercially available, the best-known being the cremophor series
(Raymond CR et al, 2009). Polyoxyl 35 castor oil (CrEL) is produced by reacting 1
mole of castor oil with 35 moles of ethylene oxide. In the case of cremophor ELP, this
is followed by a purification process. These derivatives are complex mixtures
consisting of both lipophilic and hydrophilic components. Each derivative have
different amount of ethoxylation moles per PEG units indicated with the numerical
suffix (n). The only difference in chemical structures of polyethoxylated castor oil and
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polyethoxylated hydrogenated castor oil derivatives is double bond in fatty acid side
chain. Ethoxylated glycerol ricinoleate with 30 to 50 molecules of ethylene oxide is
the main component of cremophor EL (CrEL). In CrEL, the relatively lipophilic
component (glycerol polyethylene glycol ricinoleate) constituents comprise about 80-
83% of the total mixture (Figure 1.1). Unchanged castor oil, fatty acid ester of PEGs
are the minor hydrophobic constituents. Free polyethylene glycols and glycerol
ethoxylates are the main components of hydrophilic part that accounts for 17% of
total CrEL. Cremophor ELP, a purified grade of Cremophor EL is also a polyoxyl 35
castor oil; it has a lower content of water (<0.5%), potassium (<15 ppm), and free
fatty acids (C12C18 <1%), particularly ricinoleic (<0.2%), oleic (<0.1%) and
palmitic (<0.1%) acids, and hence is claimed to contribute to improved stability of
some specific active ingredients. Functionally polyoxyethylene castor oil derivatives
act as emulsifying agent, solubilizing agent and wetting agent. Polyoxyl 35 castor oil
occurs as a pale yellow, oily liquid that is clear at temperatures above 268 oC. It has a
faint but characteristic odour and can be completely liquefied by heating to 268oC.

Figure 1.1: Molecular structure of Cremophor EL ( x,y,z = 30-33)

Polyoxyethylene castor oil derivatives are non-ionic solubilizers and emulsifying

agents used in parenteral, topical and oral pharmaceutical formulations. CrEL is used
as a solubilising and emulsifying agent in the production of liquid preparations
containing fat soluble vitamins, volatile oils and other hydrophobic substances. CrEL
emulsifies or solubilises the fat-soluble vitamins A, D, E and K in aqueous solutions
for oral and topical administration. In 1mL of a 25% v/v aqueous solution it is
possible to incorporate approximately 10 mg of vitamin A palmitate, approximately
10 mg of vitamin D, approximately 120mg of vitamin E acetate, or approximately 120
mg of vitamin K1. In aqueous alcoholic solutions, it also very readily solubilises

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essential oils. Aqueous solutions of hydrophobic drugs (e.g. miconazole, hexetidine,

clotrimazole, benzocaine) can be prepared with CrEL. CrEL has also been used as a
solubilizing agent for drugs like cyclosporin A (Yingging R et al, 2001), paclitaxel
(Gelderblom H et al, 2001) and cisplatin (Gelderblom H et al, 2002). Polyoxyl 35
castor oil forms stable and clear aqueous solutions. It is miscible with other castor oil
derivatives when heated with fatty alcohols, fatty acids and certain vegetable, animal
oils. The solubility of CrEL will be reduced and the solution becomes turbid on
heating. If heated for prolonged periods and cooled, aqueous solutions of
hydrogenated castor oil derivatives will separate into solid and liquid phases. The
only way to restore the product to its original state is by homogenisation. Whereas the
aqueous solutions of castor oil derivatives are quiet stable in the presence of less
concentration of electrolytes such as salts or acids, but they are quite unstable in
presence of mercuric chloride.

1.1.3.2 Polyethylene glycol 400 (PEG 400)

General formulae used to represent polyethylene glycol (PEG) is

HOCH2(CH2OCH2)nCH2OH (Figure 1.2) where n represents the average number of
ethylene oxide groups. The number that follows after PEG represents the average
molecular weight of the polymer. Polyethylene glycols are widely used in a variety of
pharmaceutical formulations, namely, topical, parenteral, oral, ophthalmic and rectal
preparations. PEG has been used experimentally in biodegradable polymeric matrices
and controlled-release systems (Mohl S et al, 2004). PEGs are hydrophilic and stable
substances that are non-irritant in nature to the skin. As PEGs do not penetrate the
skin readily and can be easily washed away, they are the excipient of choice for the
preparation of ointment bases.

Figure 1.2: Molecular structure of polyethylene glycol 400

There are few disadvantages with using polyethylene glycols as formulation

excipients and they are as follows: chemically more reactive than fats; irritates the
mucous membranes than fats; rate of release of medication decreases with the

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increase in molecular weight of PEG. PEGs can be used to adjust the viscosity of
suspending vehicles or themselves can be used as suspending agents. Also when used
along with emulsifiers it can stabilize emulsions. It can also be used as water miscible
solvents in the preparation of soft gelatin capsules, but the only disadvantage is they
can cause hardening of capsule due to moisture absorption from gelatin in the shell.
Poorly soluble compounds aqueous solubility can be increased by making solid
dispersion with relevant PEG. PEGs were used in the oral delivery of insulin, to assist
in aerosilsation, cyclosporine oral bioavailability enhancement, as a drug carrier, as a
bio adhesive in controlled delivery formulations. Polyethylene glycols 200-600 are
liquids; grades 1000 and above are solids at ambient temperatures. PEGs are
chemically stable in solution and in air, whereas grades with molecular weight less
than 2000 are hygroscopic in nature. Polyethylene glycols do not become rancid and
do not support microbial growth. Polyethylene glycol polymers are formed by the
reaction of ethylene oxide and water under pressure in the presence of a catalyst.
PEGs are widely used as an excipient in a variety of preclinical/pharmaceutical
formulations. PEGs can be termed as non irritant and nontoxic materials. (Henry FS et
al, 2006).
Administration of higher doses of PEG in oral administration might result in laxative
effect. Liquid polyethylene glycols will be absorbed when administered orally, but
with increase in molecular weight there is a decrease in absorption from the
gastrointestinal tract. PEGs with low molecular weight are partially metabolised but
the PEGs with high molecular weight are almost excreted unchanged in the urine.

1.1.3.3 Solutol HS15

It is also called as macrogol 15 hydroxystearate. It is a mixture of mainly monoesters

and diesters of 12-hydroxystearic acid (Figure 1.3). Macrogols are obtained by
hydroxylation of 12-hydroxy stearic acid. The number of moles of ethylene oxide
reacted per mole of 12-hydroxystearic acid is 15. Macrogol 15 hydroxystearate is
frequently used in preclinical testing of drugs, mainly for IV and other parenteral
applications.(Von CC et al, 1998; Buszello K et al, 2000; Bittner B et al, 2002) The
solubilising capacity for some tested drugs (clotrimazole, carbamazepine, 17b-
estradiol, sulfathiazole and piroxicam) increases almost linearly with increasing
concentration of solubilising agent. This is due to the formation of spherical micelles
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even at high concentrations of macrogol 15 hydroxystearate. Similarly, tests have

revealed that viscosity increases with increasing amount of solubiliser, but the amount
of solubilised drugs does not have any additional influence on the kinematic viscosity.
Lipid nanocapsules comprising macrogol 15 hydroxystearate and soybean
phosphatidylcholine containing 3% docetaxel have been successfully prepared by a
solvent-free inversion process. Macrogol 15 hydroxystearate has been used in the
manufacture of aqueous parenteral preparations with vitamin A, D, E, K and a number
of other lipophilic pharmaceutical active agents, such as propanidid, miconazole,
alfadolone and alfaxalone. It is very efficient at solubilizing substances like fat-
soluble vitamins and active ingredients of hydrophobic nature. It is also an excellent
solubiliser for parenteral use at a concentration of 20% and the water solubility of
different drugs may be enhanced by a factor of 10-100, depending on the structure of
the drug molecule.

Figure 1.3: Molecular structure of Solutol HS15

Macrogol 15 hydroxystearate is used in parenteral pharmaceutical preparations in

concentrations up to 50% to solubilise diclofenac, propanidid and vitamin K1. It has
also been used in preclinical formulations in preparing supersaturated injectable
formulations of water-insoluble molecules. It is generally regarded as a relatively non
irritant and nontoxic excipient. Macrogol 15 hydroxystearate is not restricted solely to
parenteral use, but is also suitable for oral applications. Macrogol 15 hydroxystearate
has been investigated as a co-emulsifier in the preparation of parenteral o/w emulsions
and micro emulsions. It has also been investigated as a weak inhibitor of cytochrome
P450 3A activity on the metabolism of colchicine and midazolam (Bravo GRC et al,
2004). Oral bioavailability of the highly lipophilic and poorly water-soluble
immunosuppressive agent, cyclosporin A, showed twofold higher bioavailability with
a macrogol 15 hydroxystearate-based formulation compared to a micro suspension

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(Bravo GRC et al, 2002). It has also been studied along with microcrystalline
cellulose to prepare self emulsifying pellets using an extrusion/spheronization
technique to increase the bioavailability of lipophilic drugs. Macrogol 15
hydroxystearate has been incorporated as a solubility-increasing additive in rectal
suppository dosage form to study the increase in bioavailability of poorly water-
soluble drugs. Macrogol 15 hydroxystearate has been investigated as a therapeutic
agent in the preparation of lipid nanoparticles of an anticancer drug, and has also been
shown to be effective for reversing multidrug resistance, with low toxicity in vivo.

1.2 Spiky plasma concentration profiles

In early stages of drug discovery, rat is the most commonly studied animal in
pharmacokinetic and drug metabolism and disposition studies as it is relatively
inexpensive and can be easily acquired and handled (Tse FLS et al, 1991). In a typical
pharmacokinetic (PK) study new chemical entities (NCEs) are administered to rats via
intravenous and oral routes. Serial blood samples are collected and analysed by liquid
chromatography/tandem mass spectrometry (LC-MS/MS). Some NCEs have spiky
plasma concentration profiles and spiky profiles in elimination phase will lead to
inaccurate quantification of PK parameters.

Spiky plasma concentration profiles of compounds could be due to reabsorbption

through intestine by biliary excretion. The entire process of compound traveling from
systemic circulation to intestine through bile and getting reabsorbed from intestine in
to systemic circulation is termed as enterohepatic circulation (EHC). EHC of test
compounds produces multiple peaks in the plasma concentration-time profile, which
causes increased exposure. The bile acids and salts cause emulsification of non polar
compounds which causes reabsorbption back in the colon. The process of recycling of
bile and non polar components of bile is termed as EHC. It is with this process, the
bile acids reabsorb to the circulation and to the liver, to be used several times a day.
Various endogenous substances such as hormones (Groh H et al, 1993), steroid
hormone such as estrogen (Barone D et al, 2003), thyroid hormones such as thyroxine
(T4), triiodothyronine (T3) (Azezli AD et al, 2007), insulin growth factors (Philipps
AF et al, 2000), flavonoids such as luteolin (Sarawek S et al, 2008), pharmaceutical

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compounds like ezetimibe, verapamil, diclofenac, morphine, ranitidine, ketoprofen,

roquinimex, irinotecan (Karen R, 2010) has the tendency to show EHC.

Various methodologies were proposed to characterise the EHC behavior of test

compounds (Alvarez BL et al, 1998; Ezzet F et al, 2001; Fukuyama T et al, 1994;
Moriwaki T et al, 2003; Ploeger BA et al, 2000; Schaiquevich et al, 2002). Extensive
studies needed to be carried out to characterize enterohepatic circulation behavior of
test compounds.

The other reason for spiky plasma concentration-time profile could be due to
discrepancies in sample handling, sample collection and sample processing. In order
to address the spiky plasma concentration profiles of both these natures, there should
be a quality control check for the in vivo pharmacokinetic assays. For invitro assays
there exists quality control check process to assess if the performance of the assay
system was as per the specifications designed. In this process various known
molecules are selected and validated for the assay systems performance. Based on the
validation data, acceptance criteria can be fixed for the assay systems performance.
This enables the generation of acceptable and quality results from the invitro assay
systems. But, in case of invivo studies its difficult to have a quality control check as
the assay system was not pooled (invitro), instead it was animal specific. Multiple
compounds (both test compound and quality control compound) cannot be dosed to
one animal as it will rise for the potential for drug-drug interaction. Dosing multiple
compounds to one animal will not provide reliable pharmacokinetic results.

Formulation excipients are used at high concentrations in order to solublise the

compounds with varying lipophilicities. So here is an option where we can measure
the excipient as quality control compound. The novel approach that we tried to
address in our research is the analysis of formulation excipient along with test
compound that can act as quality control check for the invivo pharmacokinetic
studies. As formulation excipients have fixed plasma concentration profiles
irrespective of the NCE dosed, monitoring the plasma concentration levels of
excipient along with NCE will help to take a decision on the spiky plasma
concentration profiles of NCEs. A thoroughly developed and validated bioanalytical
method is required to fix the plasma concentration profile and understand the

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pharmacokinetic disposition of formulation excipient studied. Integrity of results from

pharmacokinetic studies can be cross verified if formulation excipients that have fixed
plasma concentration profile/PK parameters are monitored along with the test
compound studied.

1.3 Matrix Effect

High throughput pharmacokinetic screening plays an important role in pharmaceutical
research to rapidly identify pharmacokinetic profiles of potent and selective
compounds (Heath TG et al, 1997; Olah TV et al, 1997; Watt AP et al, 2000). Liquid
chromatography/tandem mass spectrometry (LC-MS/MS) with either electrospray
ionisation (ESI) or atmospheric pressure chemical ionisation (APCI) provides a
sensitive and selective detection method for the quantitation of drug candidates in
biological matrices (Covey TR et al, 1986). Analysis of samples with minimal sample
clean up and without rapid LC separation makes it as a widely used technique for the
analysis of NCEs. Although LC-MS/MS is extremely sensitive and robust in terms of
performance there is potential for ion suppression which leads to incorrect data
interpretation. Irrespective of the advantages that the mass spectrometer brings in as a
detection technique, when compared to HPLC, it suffers from matrix effect produced
by the matrix components. The matrix refers to all components in the sample other
than analyte(s) of interest (Terence GH et al). Matrix effects are defined as
interference from matrix components that are unrelated to the analyte. Matrix
effects produce significant variations in accuracy and precision of analytical results,
which in turn have significant impact on the assessment of pharmacokinetic
parameters of NCEs. When matrix effects cause variable ionisation enhancement or
suppression in between study samples and calibration samples, the precision and
accuracy of the analytical results will be significantly affected (Walter A.
Korfmacher). Matrix effect alters the reproducibility, sensitivity and reliability of the
analytical techniques.
Electrospray Ionisation (ESI) is more prone to matrix effects than the other ionisation
sources. In the ESI source, analytes first acquire charge in solution state and then
transform to gas phase. Charge acquisition in solution phase and transformation to the
gaseous phase makes electrospray ionisation source more vulnerable to matrix effects

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than compared to other ionisation sources namely, APPI or APCI (Jessome LL et al,
2006; King R et al., 2000; Trufelli H et al., 2011). ESI is more prone to ion
suppression effects than APCI (Pommier F et al, 2003; Dams R et al, 2003). Ion
suppression could originate from endogenous compounds such as phospholipids
(Little JL et al, 2006), metabolites, coadministered drugs, internal standards (Sojo LE
et al, 2003), dosing vehicles (Tong XS et al, 2002; Shou WZ et al, 2003;
Schuhmacher J et al, 2003), mobile phase additives (Mallet CR et al, 2004) and
plastic tubes (Mei H et al, 2003).
Apart from the much spoken endogenous components causing matrix effects,
exogenous components (formulation excipients) used in the preparation of
formulations were also concern as they could potentially cause suppression or
enhancement of the analyte and internal standard which in turn will have impact on
the accuracy of measured concentrations. A lot variety of formulation excipients
ranging from cosolvents, complexing agents, lipid based vehicles and surfactants will
be used in the preparation of formulations at preclinical level. Formulation excipients
used at high concentrations to solubilise NCEs in early preclinical pharmacokinetic
screening studies can interfere with analysis. The plasma concentration of excipients
will be high in the initial sampling points. Some formulation vehicles cause 80-90%
ion suppression when administered in both intravenous and oral administration routes
(XU X et al., 2005; Larger PJ et al., 2005; Shou WZ et al., 2003; Tong XS et al.,
2002). The presence of higher concentration of formulation excipient in early time
point samples after intravenous or oral administration can cause significant ion
suppression on the analytes (Tong XS et al, 2002; Shou WZ et al, 2003; Weaver R et
al, 2006). This effect is more pronounced with the use of ultrafast gradients that
causes co-elution of many analytes. Ion suppression effects are complicated to handle
in a drug discovery environment where hundreds of molecules with differing
physicochemical properties (logD7.4, logP, pKa) are handled. These molecules may be
differentially ion suppressed depending on their elution on a typical liquid
chromatography (LC) gradient, compared with ion suppressing agent and their ability
to compete with charge from the suppressing agent. The U.S food and drug
administration (FDA) guidance for industry on bioanalytical method validation insists

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upon the assessment of matrix effects during method validation for quantitative
LC-MS/MS methods (FDA, 2001).
The exact mechanism by which matrix components causes matrix effects is not
known. Matrix effects occur at the interface between the MS system and LC system
(King R et al., 2000).
Various mechanisms by which the matrix components cause ion
suppression/enhancement are as follows:
Charge competition between analyte and matrix components (Bennett P et al.,
2004; Chambers E et al., 2007).
Change in droplet surface tension leading to formation of large droplets and
insufficient desolvation (Bonfiglio R et al., 1999; King R et al., 2000).
Preferential ion evaporation due to matrix components gathering at droplet
surface.
Change in mass of analyte ion due to ion pairing and adduct formation
Co-precipitation with non-volatile matrix components (Van Hout MW et al.,
2003).
Gas phase deprotonation.
In the present thesis various strategies to reduce matrix effects caused by formulation
excipients were studied, which includes a) different sample preparation methods b)
different analytical conditions c) different ionisation sources. The mechanism of ion
suppression by which the formulation excipients cause matrix effects was proposed.

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1.4 Literature Review

Annesley T.M. (2007) had tested the effect of methanol from 9 different
sources for ionisation suppression on immunosuppressants. The decrease in
signal for the immunosuppressant drugs was shown to result from differential
ionization associated with the selected methanols. Post column sirolimus
infusion evaluation revealed that a 1000-fold analyte concentration difference
did not affect ionization. Organic solvents used in mobile phases and extract
preparation of biological samples may be associated with ion suppression,
affecting adduct formation and assay sensitivity.
Aurand C. et al., (2009) had evaluated the performance of recently developed
sample prep hybrid SPE precipitation for the purpose of matrix removal and
analyte recovery from spiked samples. Chromatographic run times can be
brought down to a few minutes, instead of longer run times which in turn
increase the throughput of analysis. Good recoveries were obtained by using
this precipitation technique.
Bennett P. et al., (2004) had worked on selective extractions in quantitative
LC-MS/MS for removing phospholipids and reduce the matrix effects. A
major source of matrix effects in positive ESI mode was due to the
phospholipids. Acidic pH organic LLE resulted in higher removal of
phospholipids than at other pHs. SPE types tested (optimized for analyte
recovery) resulted in significantly more phospholipids than LLE. LLE method
with MTBE, hexane or a mixture of these two at pH 2.0 has removed 200
times more phospholipids than commonly used SPE.
Dams R. et al., (2002) had investigated the synergistic effect of sample
preparation type, ionization type and biological fluid in causing the matrix
effect in quantitative LC-MS/MS analysis of drugs. It was demonstrated that
ESI was more susceptible to matrix effects than APCI.
De Nardi C. et al., (2006) described the steps taken to move from a fast to a
ballistic gradient in routine liquid chromatography/tandem mass spectrometry
(LC-MS/MS) analysis of plasma samples from pharmacokinetic (PK) profiling

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of new chemical entities. The ballistic method was successfully cross-

validated with the conventional fast gradient chromatographic assay.
Eva saar et al., (2009) had compared extraction efficiency and matrix effects
using common liquidliquid and solid-phase extraction procedures in both
ante-mortem and post-mortem specimen using LCMS/MS for analysis of
antipsychotics. The extraction comparison of ante-mortem and post-mortem
blood showed considerable differences, in particular the extraction efficiency
was quite different between ante-mortem and post-mortem blood.
Gelderblom H. et al, (2001) recommended alternative formulation
approaches without cremophor EL, as it is not an inert vehicle and produces
toxicological effects, most of which have important clinical relevance. Various
side effects reported for CrEL were hyperlipidemia, abnormal lipoprotein
patterns, severe anaphylactoid hypersensitivity reactions, peripheral
neuropathy and aggregation of erythrocytes. CrEL can change the
pharmacokinetic profile of various drugs by changing the free fraction through
micellar encapsulation.
Guo X. et al., (2006) investigated the structures and origins of typical
chemical background noise ions in positive atmospheric pressure ionization
liquid chromatography/mass spectrometry (API-LC/MS). One of the other
interesting conclusions is that there is a clear difference in structures between
the chemical background ions and the protonated analytes generated under
atmospheric pressure ionization.
Hsieh Y. et al., (2008) had developed a higher-throughput bioanalytical
method based on fast-gradient (1 min run time) high-performance liquid
chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS)
for screen-type analyses of plasma samples from early drug discovery studies
in support of exploratory pharmacodynamic studies. The use of the described
methods provided advantages such as a shorter chromatographic region of ion
suppression, less solvent consumption and shorter run times in comparison
with standard analytical column HPLC-MS/MS methods.
Jessome L.L. et al., (2006) presented two commonly used techniques to
detect the presence of the matrix effect. Modifying instrumental components

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and parameters, chromatographic separation, and sample preparation are all

considered as means of reducing or possibly eliminating ion suppression.
Johnson J.L.H. et al, (2003) had validated dynamic injection apparatus for
predicting mechanical phlebitis invitro instead of invivo experiments which is
a tedious process. Twenty one currently marketed IV products were injected in
to isotonic sorenson's phosphate buffer flowing at 5 mL/min. With the results
generated it was proven that the use of the dynamic model in place of animals
for preliminary phlebitis testing of new IV injectables.
King R. et al., (2000) had discussed in detail the mechanism of ion
suppression in relation to both gas phase and solution phase. It was concluded
that gas phase reactions resulting in loss of net charge on the compound is not
an important process in ionization suppression. The main factor behind ion
suppression is due to changes in solution properties due to presence of non
volatile solutes in electrospray ionization.
Larger P.J. et al., (2005) observed strong signal suppression effect with a
polysorbate co-solvent, that produced erroneous pharmacokinetic results
which, if undetected, could have false eliminated a compound that was a
promising drug candidate. Method optimisation with various chromatographic
conditions indicated that the differential elution can remove the ion
suppression effects. Matrix dilution was also proposed as a strategy to
eliminate the signal suppression effects.
Liang HR. et al., (2003) had studied the mechanism of ionization suppression
in electrospray ionization and enhancement in atmospheric pressure chemical
ionization in single ion monitoring and multiple reaction monitoring modes for
nine drugs with stable-isotope-labelled compounds as internal standards. The
nine drugs investigated had shown suppression in electrospray ionisation and
enhancement in APCI mode of ionisation.
Matuszweski B.K. et al., (2003) stated that determination of the matrix effect
allows the assessment of the reliability and selectivity of an existing HPLC-
MS/MS method. It was demonstrated that, for the investigational drug under
study, the matrix effect was clearly observed when ISP interface was utilized
but it was absent when the HPN interface was employed.

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Mei H. et al., (2005) performed a series of studies to investigate some of the

causes of matrix effect in bioanalytical assays. They had proposed various
strategies such as use of same brand of plastic tubes for sample
collection/sample processing; avoid using lithium heparin as anticoagulant,
switching the ionisation mode, switching the mass spectrometer.
Mohl S. et al, (2004) stated that the use of biodegradable polymeric matrices
as controlled release systems is known to be associated with various
drawbacks. So they had studied to develop an alternative delivery system
based on triglycerides, thereby aiming for sustained continuous protein
release.
Muller C. et al., (2002) had studied the relation between sample preparation
procedure and signal suppression effects in electrospray ionisation mass
spectrometry. It was concluded that signal suppression will not be present at
any RT in RP HPLC when longer gradient programmes were used, but could
be a problem in HT bioanalysis where the analyte will not be separated from
the supression zone.
Neervannan S. (2006) stated that biological properties of molecules will
depend on the composition of formulation vehicle in drug discovery. The main
objective of formulation preparation is to maximise exposure in preclinical
pharmacology studies, such that the PK/PD and toxicology readouts can be
correlated to targets biological response. He also stressed the importance of
knowing the physicochemical properties of compounds in drug discovery
while screening for formulation vehicles. The review discusses about the
problems faced at different stages of drug discovery in the formulation of
compounds.
QPS Application note (2008) have proposed four tired approach for
effectively preparing the formulation vehicles in preclinical drug discovery.
Various approaches proposed include the use of cosolvents, surfactants, pH
adjustment and lipid based vehicles. However combination of these
approaches will result in a good formulation without solubility issues.
Raymond C.R. et al, (2009) had summarized and discussed about the
physicochemical properties, stability and storage conditions, handling

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precautions, safety precautions, incompatibilities and applications of the

formulation vehicles used in the industry so far.
Robert S (2000) had discussed the role of biopharmaceutics in candidate drug
selection; preformulation development with small amounts of compound; the
role of preformulation as an aid to product design in early drug development.
Also various formulation dosage forms ranging from parenteral, inhalation,
oral, ophthalmic, nasal, topical and transdermal delivery systems were
discussed in detail.
Sandrine S. et al., (2004) had investigated matrix effect on mass spectrometry
response with commercially available electrospray ionisation (ESI) and
atmospheric pressure chemical ionisation (APCI) sources coupled with a
single quadruple mass spectrometer. With any of the sample preparation
procedures, APCI source appeared to be less liable to matrix effect than ESI
source. Among the different off-line sample preparations, LLE was the most
efficient extraction procedure.
Shou WZ. et al., (2003) presented various means by which the 'dosing vehicle
effect' can be minimised include better sample cleanup, better
chromatographic separations and alternative ionization methods.
Smeraglia J. et al., (2002) had proposed a number of approaches to improve
reproducibility and robustness of LC-MS/MS methods that are subjected to
matrix effect. The modifications described are related to instrumentation and
methodological issues and include modified ionisation, ionisation switching,
extraction modification and gradient high pressure liquid chromatography
(HPLC) techniques and have demonstrated significantly improved robustness
of complex bioanalytical methods to avoid matrix-related issues.
Sweetana S. et al, (1996) stated that solubilization of insoluble drugs to be
dosed in intravenous route is a very tricky task to the pharmaceuticist. A
detailed decision tree and different approaches to solubilise the intravenous
administered drugs was discussed in the review.
Tong XS. et al., (2002) had studied the plasma concentration time profile of
polyethyleneglycol 400 in rats. The plasma concentration of excipient can
produce an increase in clearance values on analytes by a factor of 2-5-fold.

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The compromised PK parameters can result in false rejection of lead

candidates in drug discovery.
Trufelli H. et al., (2011) stated that matrix effects were a major bottleneck to
the quantitative analysis of drugs using LC-MS/MS. The main strategies to
overcome the matrix effects were discussed. Special emphasis is devoted to
the sample preparation procedures, chromatographic and mass spectrometric
conditions. Various means of compensating the matrix effects was discussed
in detail.
Van Hout M.W. et al., (2003) observed ion suppression effects during the
determination of clenbuterol in APCI mode. Use of polymeric stationary phase
in SPE extraction helped to remove the ion suppression effects observed
Xue Q.C. et al, (2006) stated that, lead candidates are identified based on
pharmacological and toxicological data in drug discovery. They also discussed
that poor biopharmaceutical properties are a result of bad physicochemical
properties. The suboptimal biopharmaceutical properties often lead to higher
cost of developing the drug candidates. So physicochemical parameters such
as lipophilicity, stability and solubility should be understood at an early phase.
Along with the physicochemical properties, preformulation approaches for
developing the compounds formulation should be optimized.
Yingging R. et al, (2001) investigated the solubilization of cyclosporin A with
various solubilization techniques such as cosolvency, micellization and
complexation. Various excipients such as surfactants, cosolvents and
cyclodextrins were tested as solubilizing agents. Overall, compared to other
excipients, surfactants had a considerable effect on cyclosporin solubility.

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