TABLE OF CONTENTS
TABLE OF CONTENT VIII
LIST OF TABLE XI
LIST OF FIGURE XII
CHAPTER ONE: INTRODUCTION 1
1.0 Drug Interaction 1
1.1 Mechanisms of drug interaction 3
1.1.1 Pharmaceutical drug interactions 3
1.1.2 Pharmacokinetic drug interactions 4
1.1.3 Pharmacodynamic interaction 14
1.2 Basic concept of pharmacokinetic 17
1.2.1 Approach to pharmacokinetic 18
1.3 Pharmacokinetic parameters 24
1.4 Spectroscopy 27
1.4.1 Ultra-Violet/Visible absorption
1.4.2 Analytical method development 31
CHAPTER TWO: LITERATURE REVIEWS 36
2.1 Paracetamol 36
2.1.1 Chemistry 36
2.1.2 Structure activity relationship 38
2.1.3 Pharmacokinetics 39
2.1.4 Pharmacodynamics 49
2.1.5 Dosage 50
2.1.6 Saliva concentration of paracetamol
and assay methods 50
2.1.7 Assay method 52
2.1.8 Paracetamol interaction 54
2.2 Tramadol 57
2.2 Introduction and chemistry 57
2.2.1 Pharmacokinetics 58
2.2.2 Pharamcodynamics 61
2.2.3 Indications 62
2.2.4 Toxicity and adverse effects 62
2.2.5 Drug interaction 63
2.3 Cimetidine 64
2.3.1 Introduction and chemistry 64
2.3.2 History and development 66
2.3.3 Pharmacokinetics 67
2.3.4 Pharmacological properties and
2.3.5 Administration and dosage 73
2.3.6 Adverse effects 74
2.3.7 drug interaction 74
3.1 Materials and methods 81
3.1.1 Chemicals and standard sample 81
3.1.2 Glass wares 83
3.1.3 Equipment 83
3.2 Methods 85
3.2.1 Quality control 85
3.2.2 Analytical method 88
3.2.3 Precision of the analytic method 89
3.2.4 Extraction procedure and percentage
extraction recovery 89
3.2.5 Preparation and validation of
calibration curve 90
3.2.6 In-vivo pharmacokinetic studies 93
4.0 Results 97
4.1 Quality control assessments 97
4.2 Validation of analytic method 98
4.3 Percentage extraction recovery 99
4.4 Construction and validation of 100
4.4.1 Calibration curve 100
4.4.2 Validation of the calibration curve 101
4.5 In-vivo pharmacokinetic studies 102
4.6 Pharmacokinetics parameters 105
5.0 Discussions 109
5.1 Quality control assessments 109
5.2 Validation of analytic method 111
5.3 Percentage extraction recovery 111
5.4 Construction and validation of
calibration curve 112
5.4.1 Calibration curve 112
5.4.2 Validation of the calibration curve 114
5.5 In-vivo pharmacokinetic studies 1115
5.5.1 Single dose salivary pharmacokinetic s
of paracetamol 115
5.5.2 Influence of Tramadol on the
pharmacokinetics of paracetamol 117
5.5.3 Influence of Cimetidine on the salivary
pharmacokinetics of paracetamol 121
5.6 Conclusion 125
1.0 DRUG INTERACTIONS
Klaus and Jouni, (2001) stated that when two or more drugs are given together, the
response may be greater or smaller than the sum of the effects of the drugs given
separately. They further said that one drug may potentiate or antagonise the effects of
the other and in some cases there may also be qualitative difference in their response.
Doctors have always practiced poly pharmacy and a sound combination of drugs help
to increase the efficacy and safety of drug treatment.
The true prevalence of undesirable drug interactions is substantial but largely
unknown. It has been estimated that the number of death attributed to adverse drug
reactions may be as high as 200,000 deaths per year in the United States (Chyka,
2000). A pharmacoepidemiological study conducted by Kennedy et al, (2000)
demonstrated that half the population of 1225 adult general surgical patients were
taking medicines that were not related to surgery. On average these patients received
nine different drugs which may interact. The Boston collaborative drug surveillance
programme reported a study of 9,900 patients with 83,200 drug exposures and found
3,600 adverse drug reactions, 234 (6.5%) of which were attributable to drug
interactions. In a study where the medical charts of 1,800 surgical patients were
reviewed researchers found at least one potential drug interaction in 17% of patient
(Durrence et al, 1985).
Many adverse drug interactions are the result of concomitant therapy with potent
drugs. Patients treated with phenothiazines, corticosteroids, antineoplastics and many
other drugs must frequently be subjected to certain adverse effects in order to obtain
A possible correlation has been noted between the significant increases in adverse
effects and the use of multiple drug therapy (Evaluation of drug interaction, 1997).
The incidence of drug reaction increased with the number of drugs prescribed
simultaneously, and drug interaction make a small but significant contribution to the
overall morbidity and mortality due to drugs.
Many of the drug interaction reported in the literature are anecdotal and have not been
confirmed, nor does there exist any well pharmacological basis for believing they
could occur (Griffin and D’Arsy, 1979). Nevertheless, individual variability is such
that factors like pharmacokinetic differences and effects of disease states may have
contributed to a unique reaction. Environmental factors such as smoking and
atmospheric pollution or even the hardness of the water supply have also been
reported to influence drug metabolism and may also be involved in contributing to a
drug interaction. Other causes are dietary factors and particularly herbal remedies
which there is increase in their usage by the population due to the mistaken belief that
they are free of adverse effect, when in fact their usage in surrounded by ignorance of
their pharmacology and toxicology (Dukes, 1973). Outside the hospital less
information is available on the multiple usage of drugs. Patients frequently use over
the counter drugs (OTC), which they prescribed for themselves along with the
doctor’s prescription. These drugs frequently interact thereby complicating drug
In the light of these informations it seems reasonable to speculate that a very large
number of patients may be at risk of having potentially harmful drug interaction and
that an important problem in modern therapeutics might exist (Graham, 1977)
1.1 MECHANISMS OF DRUG INTERACTIONS
Drugs may interact in a number of ways such as pharmaceutical, pharmacodynamic or
pharmacokinetic basis. A number of drugs may also interact at several different sites.
Such cases represents drug – drug interactions. Other forms of interactions also do
occur. Therefore, the concept of drug interactions also include the modification of
drug effects by food or dietary items (drug – food interactions). There may also be
drug – laboratory test interaction, where a drug causes alteration of diagnostic
Drug – disease interaction represents a situation where some drugs are contraindicated
in certain disease states.
1.1.1 Pharmaceutical drug interactions
Pharmaceutical interactions normally occur before the drug in given to the patient.
They may be caused by several different mechanisms. When injecting for instance
thiopentone with vecuronium through the same giving set, a precipitate will form
instantaneously. Numerous incompatibilities have been demonstrated, and drugs
should never be mixed in this fashion unless the absence of reaction has been clearly
established (Trisel et al, 1994).
Pharmaceutical interactions can also be described as physicochemical
incompatibilities, which are unintentional interactions that occur in vitro between
drug and other component of medicinal products during their preparation, storage or
administration. Drug-drug, drug-excipient, excipient-excipient, drug-packaging, and
excipient-packaging are all interactions that may cause adverse effects on
bioavailability, efficacy or toxicity.
Another important area of drug interaction of clinical important occurs when drugs
are added to intravenous infusion. Over the last ten years the practice of administering
drug by continuous intravenous infusion has become more common, particularly in
surgical unit. The problem is mainly produced by incompatibility between the drug
added and component of the infusion fluid (Kramer, Inglott and Cluxton, 1974).
1.1.2 Pharmacokinetic drug interactions.
Pharmacokinetic drug interactions can be divided into interactions that occur during
absorption, distribution or elimination. In some cases, drugs may interact
simultaneously during several different phases during passage of the drug through the
Drugs can influence the absorption of other drugs at least by changing the
gastrointestinal pH and motility, by intraluminal binding or the chelation of drug, by
changing the regional blood flow, by inhibition or stimulation of first pass
metabolism, or through toxic effects on the gastrointestinal canal. The subcutaneous
and intramuscular absorption of drugs can be delayed or decreased after the
administration of drugs affecting regional blood flow (vasoactive agents). However,
these mechanisms have no major importance in pharmacokinetics. (Klaus and Jouni,
Table 22.214.171.124 possible mechanisms of drug absorption interactions
– pH effect on dissolution and ionization
– Changes in gastric emptying and GI motility
– Formation of complexes, ion pairs and chelates
– Interference with active transport
– Disruption of liquid micelles
– Changes in portal blood flow
– Toxic effect on G.I mucosa
– Change in volume, composition and viscosity of secretion
– Effects on mucosal and bacterial drug metabolism
– Change in membrane permeability
Because the rate of absorption of orally administered drugs is directly proportional to
the rate at which drugs pass from the stomach to the intestine, some drugs or agents
by increasing gastrointestinal motility, may increase the rate at which another drug
passes through the gastrointestinal tract which could lead to an increase in absorption.
Conversely, muscarinic receptor blockers e.g. atropine will reduce gastrointestinal
motility, decreasing dissolution of drugs in the GIT and consequently absorption.
This may lead to increase in plasma levels of drugs due to prolong contact with the
absorptive surfaces (Nimmo et al 1973) show that propantheline and
metochlopramide delay and accelerate gastric emptying respectively.
Table 126.96.36.199 Drugs that might influence gastric emptying.
– Atropine and anticholinergics
– Tricyclic antidepressants
– Antiparkinson drugs
– Narcotic analgesics
Drugs are probably absorbed more rapidly from the upper small intestine than from
stomach because of the much greater surface area of the intestine (Levine, 1970). The
rate of gastric emptying may therefore limit the rate of drug absorption and is very
important in the context of drug interactions since it can be influenced by many drugs
(Morselli et al, 1974). Because of this, any factor influencing gastric emptying and
therefore drug movement into the small intestine will alter the rate and possibly the
extent of absorption.
Changes in GIT pH also affect the absorption of most drugs. Since many drugs occur
as acids or bases or contain more than one ionisable group it is expected that PH of
the stomach could influence the extent of their absorption. Drugs cross membranes
more readily in their un-ionised form due to higher lipophilicity of this species.
According to the pH – partition theory, weak organic acids are largely absorbed from
the stomach where as weak bases are absorbed best from the more alkaline contents of
the upper small intestine (Brodie, 1964). The absorption of weak acids is reduced if
they are given with alkali since fewer drugs would be present in the un-ionised lipid
soluble diffusible state (Binns et al, 1971).
However, an opposite effect may be observed. Aspirin, for example is absorbed more
rapidly from buffered alkaline solution than from unbuffered solution at pH 2.8
(Cooke and Hunt, 1970). This is because of the greater dissolution rate and aqueous
solubility of aspirin in alkaline solution and rapid gastric emptying caused by an
increase in the pH of the gastric contents. Contrary to the pH-Partition theory, aspirin
is absorbed much more slowly from the stomach than from the small intestine (Siurala
et al, 1969). The stimulatory effect of alkali on gastric emptying may explain the
apparent increase in propantheline absorption caused by sodium bicarbonate (Chaput
de Saintonge and Herxheimer, 1973). Alkalis and antacids on the other hand may
decrease the rate of absorption of other basic drugs through effect on solubility, and
the inhibitory effect of sodium bicarbonate on the absorption of tetracycline may be
cited as an example (Barr et al, 1970). Generally antacids alter absorption of drugs by
affecting their solubility and dissolution. Tetracycline is poorly absorbed in the
presence of aluminum hydroxide, due to chelation ( Walsbren and Hueckel, 1950 ).
Sodium bicarbonate, which raises pH without releasing polyvalent cations, also delays
tetracycline absorption. For highly soluble and rapidly absorbed drugs such as
phenobarbitals (Pka 7.6), sodium sulphadiazine (Pka 6.5), quinine (Pka 8.4) and
isoniazid, their passage from the stomach into the intestine is the rate-limiting step in
their absorption. Therefore, antacids such as aluminum hydroxides that delay gastric
emptying will delay their absorption.
Generally, administering oral medication along with food or at a mealtime is a
convenient manner of drug dosing. However, drug interactions can occur that modify
the activity of the drug (decrease or increase drug effects) or impair the nutritional
benefit of certain food (May, 1997). Examples of drugs whose absorption is
decreased when taken with food include Penicillin, Tetracycline, Erythromycin,
Levodopa, Phenytoin and Digoxin (May, 1997). Drugs whose absorption increase
when taken with food include spironolactone (Melander et al, 1997), griseoflulvin
(Crounse, 1961) and itraconazole (Kastrup, 1999). Calcium, Magnesium and
aluminium found in food supplements or antacid compounds bind (chelate) with
Tetracycline to form an insoluble complex resulting in significantly decreased
absorption of Tetracycline (Mc Evoy, 1999). Many drugs, before reaching the
systemic circulation are subjected to first pass metabolism by the liver.
Theoretically, drug distribution can be affected by numerous factors. Practically all
drugs are bound to plasma components, red blood cells and plasma proteins. Protein
binding interactions have been studied extensively in – vitro. Concomitantly
administered drugs compete for binding sites on blood and tissue proteins to produce
displacement interactions. A protein that is very easy to obtain in a relatively pure
form and which has been well studied with respect to drug binding is serum albumin.
Albumin interacts with a variety of drugs than do other plasma proteins or even many
of the intracellular proteins and this provides some clues to the interaction features of
proteins. For instance, the albumin molecule which is a single polypeptide chain is
spread out. Therefore, the surface area of the molecule is relatively large, which is the
reason for the high viscosity of albumin solution. A macromolecule in a single chain
exposes a large proportion of its reactive groups to the reactive groups of drug
molecules. Secondly, albumin differs from many other types of protein in that it
contains more hydroxy amino acids relative to its carboxy amino acid content, thus
leaving numerous unbounded cationic N groups availalable which can then bind
anionic drug molecules (Gourley, 1971).
Because only the unbound fraction of the drug is pharmacologically active, an
increase in the free concentration of drug increases its pharmacological effects. Many
anaesthetics, including volatile anaesthetics, seem to be able to displace drug from
plasma protein in-vitro, but this does not appear to have any significant clinical
consequences. (Wood, 1991, Grandison et al, 2000). Warfarin, an anticoagulant, is
highly bound to plasma protein, when combined with phenylbutazone, warfarin will
be displaced from protein binding sites and the concentration of the free warfarin will
be increased. The result is enhanced anticoagulant activity with a possibility of
Binding of drugs to non-receptor site macro-molecules is important because it limits
the access of free fraction of drug to receptor sites and to the biotransformation
enzymes and excretion process. Binding may be beneficial because it may result in a
diminished incidence of side effect and a prolonged duration of action (Brodie, 1965).
A number of acidic drugs are attached to only one or two sites on the albumin
molecule; for these drugs, the protein has a limited carrying capacity. Many of these
acidic drugs appear to compete for the same limited number of non-specific protein
binding sites. Hence, competition between co-administered drugs for binding sites in
the body can result in displacement of one drug by another, with a resulting rise in the
free and active fraction of a drug. It is widely believed that this displacement or
redistribution phenomenon caused enhanced clinical effects and toxicity seen when
certain drugs interact in man (Wardell, 1974). Highly bound acidic agents such as
phenybutazone, oxyphenbutazone, ethylbiscoumaroacetate dicoumarol, sulfinpyrazole
and saliciylic acid are able to displace the long lasting albumin bound sulphonamides
from plasma proteins. Since these sulphonamides are not rapidly metabolized or
excreted, the displaced molecules diffuse from plasma into tissues with resultant
enhanced antibacterial activity. By the same mechanism, pheny butazone increases
the antibacterial activity of acidic antibiotics such as penicillin (Hartshorn, 1973).
Drug Metabolism Interaction.
The biotransformation of drugs during the first – pass and during elimination from
the systemic circulations in the liver, is usually divided into phase I and phase II
reactions. Many drugs are lipophilic and cannot be excreted through the kidneys until
they have been transformed into more favourable water soluble forms.
Phase I reactions include oxidation, reduction and hydrolysis. Phase I reactions add a
functional group to the drug, where as phase II reaction are conjugation reactions in
which the drug or its metabolite is attached to a water – soluble molecule, such as
glucuronic acid, glutathione, sulphatic group, acetyl group, methyl group or
glucosamine, making the whole complex more hydrophilic. Oxidation is the most
important phase I reaction catalysed by cytochrome P450 (CYP450) enzymes. Most
metabolic drug interactions involve either the induction or inhibition of cytochrome
P450 enzymes (Levy et al, 2000).
Oxidation reactions include aromatic and aliphatic hydroxylation, oxide formation,
desulfirization, deamination, dehalogenation, N-O and S dealkylation and
sulfoxidation. Reduction reactions include azoreduction, aldehyde reduction, nitro
reduction while hydrolytic reactions include de-esterification and deamination.
Oxidative and reducing enzymes are found primarily in the liver microsomes.
Hydrolytic enzymes are located in the plasma, liver microsomes and many other
CYTOCHROME P450 ENZYMES
Cytochrome P450 (Cyp450) enzymes are characterised by a maximum absorption
wavelenght of 450nm in the reduced state in the presence of carbon monoxide.
According to the homology of their amino acid sequence, the CYP enzymes are
divided into families, sub families and specific iso enzymes. CYPI, CYP2 and CYP3
are involved mainly in the metabolism of drugs and other xenobiotics, where as those
belonging to the families CYP4, CYP5 and CYP7 have endogenous functions (Levy,
So many drugs and environmental chemicals are implicated in cytochrome P450
enzymes induction and inhibition. Cytochrome P450 enzyme system inducers include
phenobarbital and many other drugs and environmental chemicals, including
chlorinated hydrocarbon, insecticides carcinogenic hydrocarbons, food additives and
cigarette smoke (Conney and Burns, 1972). Inhibitors of cytochrome P450 enzymes
such as phenlybutazone and imidazole compounds have since been recognized
(Powell and Donn, 1983). The activities of the cytochrome P450 dependent system
are extremely sensitive to difference in sex, age strain and species and to differences
in the hormonal and nutritional state of animal (Coney, 1967).
Co-administration of the inhibitor and the substrate of any CYP enzyme will result in
an increase of the substrate concentrations. The magnitude of the increase depends on
the inhibitor and its dose. An example is ketoconazole which increased the AUC for
oral triazolam approximately 30times compared with the administration of triazolam
with placebo ( Varhe et al, 1994). But the AUC of oral midazolam was increased
approximately 16times (Olkkola et al, 1994).
Many lipid-soluble compounds such as barbiturates phenytoin, carbamazepine and
also ethanol cause a stimulation of drug metabolism through the induction of hepatic
microsomal enzymes. The administration of the inducing drug causes stimulation not
only of its own metabolism, but also the metabolism of many unrelated drugs which
are substrates for microsomal enzymes.
Ritonavir is a protease inhibitor used in the treatment of HIV infection. A 2-day
ritonavir treatment greatly increases the concentration of intravenous fentanyl by
reducing fentanyl clearance. Because fentanyl clearance was reduced by 67%, it can
be calculated that ritonavir treatment results in approximately threefold increase in
fentanyl concentrations (Olkkola et al, 1999). However, the AUC of norpethidine
was increased suggesting the induction of hepatic pethidine metabolism by ritonavir.
(Piscitelli et al, 2000). It was shown that paracetamol, a substrate of the CYP2E1 and
CYP3A family, does not affect the pharmacokinetics of fentanyl at clinically relevant
concentrations (Feierman, 2000).
Ropivacaine is a local anaesthetic, which is metabolised mainly by CYP1A2 but also
by CYP3A4. It’s clearance is reduced by 77% by concomitant CYP1A2 inhibitor,
fluvoxamine. Erythromycine a CYP3A4 inhibitor alone only had a minor effect on
the pharmacokinetics of ropivacaine. However, compared with fluvoxamine alone, the
combination of fluvoxamine and erythromycine further increased the area under the
drug plasma concentration time curve by 50% (Jokinen, 2000).
Recent studies have shown that many dietary supplements and natural products can
modify the pharmacokinetics of drug. For instance, St. Johns wort (Hyperuricum
perforatum) a plant used as an antidepressant in the United States, is a potent inhibitor
of CYP3A4 and can have potentially hazardous drug interactions when used with the
substrates for CYP3A4. (Fugh – Berman, 2000).
Drug excretion interaction.
Any interaction here will only be important if a drug or its active metabolite is
eliminated principally through the kidney.
Excretion of drug by the kidneys is net effect of 3 processes. Passive glomerualr
filtration, active tubular secretion and passive tubular diffusion. Glomerular filtration
of a drug is not usually affected by other drugs except in disease states.
However, pH modifies the tubular reabsorption. When the tubular fluid (urine) is
alkaline, basic drugs like amphetamine will mostly be in unionised from and will be
greatly reabsorbed from the tubular fluid into blood. In this way the action of the
drug is prolonged. Acidic drugs (e.g. Nalidixic acid) under the same condition will be
mostly be in ionised forms and will not be reabsorbed but excreted. The opposite
obtains when the filtrate is acidic. Urine pH effect are best on the elimination of a
drug that is filtered at the glomerulus and reabsorbed by non – ionic diffusion back
into the blood as the glomerular filtrate passes down the nephron.
Changes in urine pH that increase the ionized fraction of the drug in the urine increase
the excretion rate of the drug. This has been extensively investigated with
Phenobarbital and alkalinization of the urine (Waddel and Buter, 1957) and put to use
treating patients with phenobarbital poisoning (Lassen, 1961).
Some acidic drugs like penicllin, probenicid, phenylbutazone, chlorpropamide are
transported from blood into the tubular fluid by an active process which may involve
enzyme systems. When two drugs normally excreted by the same active transport
mechanisms are given together, one of them will delay the elimination of the other
and in this way prolong its action. The prolongation of the action of penicillin by
probenicid is well known.
Change in renal function can modify a number of pharmacokinetic processes in the
body and thereby lead to unanticipated drug effect or interaction (Rendenberg, 1974).
Drug excretion is slowed in patients with impaired renal function
1.1.3 PHARMACODYNAMIC INTERATION
These are drug interactions at the site of action. Many drugs produce their
pharmacological effect by combining with specific receptors. Other drugs can occupy
these specific receptors without producing any response and in this way prevent or
reverse the effect. Example is reversal of the antihypertensive effect of guanethidine
by amphetamine. Guainethidine act by blocking nerve transmission on adrenergic
neurounes. Amphetamine displaces it from these neurones and thus abolished its
Frequently overlooked is the multiplicity of effect of many drugs. Thus,
phenothiazines are effective α-adrenergic antagonists; many antihistamines and
tricyclic antidepressants, are potent antagonists at muscarinic receptors.
These “minor” actions of drugs may be the cause of drug interactions (Goodman and
Gilman, 1996). From the foregoing we see four different kind of pharmacodynamic
(a) Enhanced effects produced by two drugs acting at same site. An example is
streptomycine (weak depolarising properties) in the presence of a depolarising
muscle relaxant (Toivakka and Hokkanen, 1965).
(b) The increased effects produced by two drugs acting at different receptor sites
(potentiation). This usually results in an effect, which is greater than the sum
of the component effects. E.g Antihypertensive drugs
(c) Enhanced effects of a drug by one which is devoid of action itself. Example of
increased anticoagulant effect of warfarin with clofibrate (Solomon and
(d) Antagonsim of the effect of one drug by another. An example is naloxone
action at opioid receptors.
FORCES INVOLVED IN DRUG–MACROMOLECULAR INTERACTION
Generally, the interaction between a therapeutically useful drug and macromolecule is
Macromolecule + drug Macromolecule-drug complex
(Unbound) (Bound drug)
This means that covalent bonds, which are very stable at body temperature, are not
involved in most interactions between drug and macromolecules in humans. Because
of the high bond strengths, covalent bonds are not likely to be broken down, unless
specific enzyme is present to break the bond and this may lead to serious drug
interactions. However, there are two types of drug – macromolecule interaction that
involve the formation of covalent bonds. The first type is the alkylation or arylation of
cell constituents by certain drugs that are respectively alkylating or arylating agents.
The second type is the covalent binding of drug or its metabolite to a protein to form a
hapten-protein conjugate, called an antigen, which include synthesis of antibodies for
the hapten (drug).
The main forces that are involved in the interactions of most drugs with
macromolecules in the body are much weaker than the covalent bonds and involve the
1. Electrostatic forces:- These forces are formed between ions of opposite
charge. This force increase in strength with increasing ionic charge and decreasing
distance of approach. Electrostatic bonds are stronger in a medium of low dielectric
constant, such as the lipid medium of a membrane, than in a medium of high
dielectric constant, such as water. Electrostatic forces play an important role in
interaction involving ionizable drugs, and macromolecules having an ionic group such
as certain mucopolysaccharides.
2. Hydrogen bonds:- This is a weak or loose bond between the electron deficient
hydrogen atoms of hydroxyl, amino and thiol groups and the non-bonding electrons of
electronegative atoms such as Oxygen, Nitrogen, Sulphur and Chlorine. Examples are
O-HO or N-HO. Usually the atoms are in linear configuration and one hydrogen atom
can form only one hydrogen bond and cannot interact simultaneously with a second
atom. Hydrogen bond formation may be inter or intra-molecular. Intramolecular
hydrogen bonding give rise to ring formation or chelation and this is usually when the
formation of a 5,6,0r 7 – membered ring is possible. Hydrogen bonds are weaker than
electrostatic forces but stronger than Van der waals forces.
3. Dispersion forces and van der waals forces: Any two electronic systems, for
example two atoms or molecules are weakly attracted to each other owing to slight
correlations between the motions of the electrons in the interacting molecules. The
attractions arise from natural polarization in electron clouds induced by atoms as they
approach one another. The electron clouds are thereby distorted by this polarization
and as a result the nuclei of each atom are attracted to the electrons of the other atom.
In addition to the attractive interaction of dispersion force, a repulsive force develops
between two atoms or molecules when their interatomic distance decreases to the
point at which the electron clouds interpenetrate. This combination of attractive and
repulsive forces is known as the van der waals force. Altough Van der waals forces
are weak and short acting, they are still important element of the binding processes.
Hydrophobic interaction describes the van der waals attraction between atoms in the
non-polar parts of two molecules immersed in water. Hydrophobic bonding is very
important for the binding of small molecules to biological macromolecule.
Hydrophobic interaction are not primarily involved macro-molecular drug interaction
but are important for the structure of proteins and nucleic acid and can be disturbed by
the approzimation of drug molecules, leading to alteration in the tertiary structure of
the macromolecules (Gourley, 1971).
1.2 BASIC CONCEPTS OF PHARMACOKINETICS
When drugs are applied both in-vitro and in-vivo the magnitude of the response is a
function of the concentration of the drug in the fluids bathing the sites of action.
Therefore to obtain the therapeutic success, an adequate concentration of the drug at
the site of action for the duration of therapy is necessary. Pharmacokinetics is the
knowledge of the mechanism of drug absorption, distribution and elimination together
with the kinetic of these processes. In contrast clinical pharmacokinetics is the
application of pharmacokinetic principles to the therapeutic management of patients.
Clinical pharmacokinetics thus attempts to provide both a more quantitative
relationship between dose and effect and the framework with which to interpret
measurements of concentrations of drugs in biological fluids.
1.2.1 Approach to Pharmacokinetics.
Once a drug is absorbed, it is distributed to the various tissues of the body. The rate
and extent of distribution are determined by how well each tissue is perfused with
blood, the binding of drug to plasma proteins and tissue components and the
permeability of tissue membrane to the drug. Distribution is the process of the
reversible transfer of a drug to and from the site of measurement, usually the blood or
plasma, where as elimination is the irreversible loss of drug from the site of
measurement. Practical clinical pharmacokinetic involves the quantitation of a drug
in readity accessible body fluids, followed by attempts to define mathematically the
time – course of events.
The attempts to define this time – course of events in the body have produced three
philosophical approaches to practical pharmacokinetics:-
(i) Compartment model approach
(ii) Physiological model approach
(ii) Non – compartment approach
The compartment model approach is the conventional method of characterising the
pharmacokinetic of a drug. With this approach the body is considered as consisting
on one or more compartment with no real anatomical or physiological reality.
Another characteristic of this approach in the assumption that rates of drug
absorption, transfer among compartment and drug elimination from compartments all
obey first order linear kinetics.
Single – compartment model.
This is the simplest model and depicts the body as a single homogenous unit where
the drug entering the body is distributed instantly between the blood and the body
fluid or tissues. The exchange of drug between plasma and tissue proceeds more
rapidly than the rate of elimination.
Single – compartment open model is actually an approximation or simplification used
to describe the two – compartment open model when kab>>kel (Notari; 1975).
Characteristics of a single compartment open model
D = Dose of drug administered
F = Bioavailability
Kab = Absorption rate constant
B = Body, (Blood, body fluids, tissue)
Vd = Volume of distribution
Cp = Drug concentration in plasma
Kel = Elimination rate constant
E = Routes of drug elimination
The fact that the body behaves as a one compartment does not mean that the
concentration of drug in all body tissues at any given time is the same. However any
changes that occur in plasma quantitatively reflect changes in tissue drug levels.
Assuming instantaneous distribution after an intravenous injection of a drug into this
model, the concentration (Co) in the plasma immediately after the injection is equal to
the dose (D) divided by the volume of the compartment (vd)
Co = D/vd.
The apparent volume of distribution (vd) is not a true volume, but that volume into
which all the drug in the body would appear to be distributed to achieve a
concentration the same as that in plasma.
Elimination follows a first order kinetics after intravenous distribution. This means
that a constant fraction of a drug in eliminated per unit time. A plot of plasma drug
concentration against time on the abscissa with each unit representing the time for one
half of the drug to be removed, result in the exponential curve shown in Fig 188.8.131.52
Re-plotting the curve with concentration on a logarithmic scale, results in a straight
line (Fig 184.108.40.206). This plot can be extrapolated back to zero time to give Co, the
theoretical initial concentration. The elimination half-life (t1/2) of the drug is
Plasma fig: 220.127.116.11 Plasma concentration-time plot on a linear (Arithmetic) scale
following a rapid IV injection of a drug into a single-compartment open model.
Plasma fig: 18.104.22.168 Plasma concentration-time plot on a logarithmic scale following a
rapid IV injection of a drug into a single-compartment open model.
T1/2 T1/2 T1/2
Taking first order kinetics between drug concentration and time, then decline in drug
concentration may be expressed as :
dc/dt = βCt
Where Ct = concentration
β = rate constants of elimination
Integration and conversion to logarithms to the base 10 gives
Log Ct = Log Co – βt/2.303
If the distribution of drug is very slow, a two-compartment model must be considered.
This model contains a central and a peripheral compartment.
The central compartment, composed of blood and the well perfuse tissues, while the
peripheral compartment composed of the tissues or the rest of the body. With this
model all drug removed from the body, regardless of the route of elimination is from
the central compartment. The central compartment is open since elimination occurs
from it. Drugs distribute within a few minutes through this compartment and
equilibrium between plasma and tissue is rapidly achieved. The combined effect of
two compartments gives rise to a biphasic curve on IV injection with two distinct
linear portions when drawn on a semi-log scale (fig 22.214.171.124)
0 T1 T2 T3
Even though drug distribution is slow, it is usually faster than elimination. Thus the
initial rapid fall in the concentration (distribution phase) mainly represents the
relatively rapid process of distribution from central to peripheral compartment. After
the distribution, the curve now enters the relatively slow elimination phase (B) during
which there is irreversible elimination from the central compartment.
LV DOSE K12
Elimination TWO COMPARTMENT MODEL
Three – compartment model:
In reality a maximum of three compartments is allowed in assay technique
(Paxton.1931). The three – compartment model is a modified model in which an
additional compartment in incorporated to represent the volume (vo) from which
absorption occurs at a first order rate. (fig 126.96.36.199). It is assumed the entire dose in
rapidly introduced into the size of absorption, from which it is absorbed into the
K12 & K21 are the
transfers rate constants
between the two
Ka = Absorption rate constant.
K12 and k21 = Transfer rate constant between the two compartments.
A concentration – time curve for a single oral dose is shown in fig188.8.131.52 This is also
obtainable for other administration routes like intramuscular or subcutaneous, which
have a preliminary absorption phase.
Absorption & Distribution phase
A conc- time plot after single oral dose.
1.3 PHARMACOKINETIC PARAMETERS
Following drug administration, absorption and disposition are characterised by some
important pharmacokinetic parameters. These are; Half-life (absorption/ elimination
t1/2), Area under the curve (AUC), systemic and total body clearance. Volume of
distribution (vd), Absorption and elimination rate constants (praxton, 1981). Other
are lagtime, Cmax, Tmax.
Clearance is the most important concept to be considered when a rational regimen for
long-term drug administration is to be designed. (Goodman and Gilman, 1996). It is
the volume of plasma from which a drug is totally and irreversibly removed per unit
time and is a direct index of drug elimination. The elimination of most drugs follow
first order kinetics. That is, a constant fraction of the drug is eliminated per unit time.
If mechanism for elimination becomes saturated, the kinetics becomes zero order.
That is a constant amount of drug is eliminated per unit time.
Clearance of a drug is the rate of elimination by all routes normalised to the
concentration of drug (c) in some biological fluids
CL = Rate of elimination
Clearance by means of various organs of elimination is additive.
CL renal + CL hepatic + CL other = CL systemic
Meaning Total systemic clearance is the sum of hepatic, renal and other clearance like
Area under the curve (AUC).
This is the area under the concentration-time curve from time zero to infinity (AUC)
and is a measure of the extent of drug absorbed into the systemic circulation
(Bioavailability). Some of the ways AUC may be obtained are;
1. Use of a planimetre
2. Cut and weigh method
3. Trapezoidal triangular method.
In the trapezoidal method, AUC is estimated by dividing the curve into sections that
approximate a series of trapezoids with a triangle at each end. The individual area of
the trapezoids and triangles are summed to obtain the AUC (Notari, 1975)
Apparent Volume of distribution (vd)
This is the volume in which the amount of drug in the body would need to be
uniformly distributed to produce the observed plasma concentration. This volume
does not necessarily refer to an identifiable physiological volume, but merely to the
fluid volume that would be required to contain all of the drug in the body at the same
concentration as in the blood or plasma.
V = Amount of drug in body
Vd can be determined from the relationship.
Vd = F x D
F = Bioavailability
D = Dose
AUC = Area under the curve
As might be expected, the volume of distribution for a given drug can change as a
function of the patient’s age, gender, disease and body composition (Goodman and
Elimination Half – Life ( t1/2β)
This is the time taken for half the Amount of the drug present in the body to be
eliminated either by extraction or metabolism or both.It is infact the time taken for the
plasma concentration to reduce by fifty percent of its original value (Wagner, 1986)
and can be determined for either one or two compartment model.
Spectroscopy is the study of interactions of electro-magnetic radiation with matter.
Such interactions of energy with organic molecules lead to changes in either
electronic, vibrational or rotational energy inherent in the molecules all of which
constitute what is known as the internal energy of a molecules. A plot of percentage
transmittance (or of absorbance) against wavelengths gives a picture described as a
spectrum. Various regions of the electromagnetic spectrum have applications in
pharmaceutical sciences in general. In particular, the techniques frequently employed
in pharmaceutical analysis include ultra-violet (colorimetry), infrared and atomic
absorption and emission spectroscopy. The spectral range for these measurements can
for convenience be divided into ultra violet (190 to 380nm), the visible (380 to
780nm) the near infra red (780-3000nm) and the infra red (2.5 to 40mcm or 4000 to
1.4.1 Ultra-viloet/visible absorption spectrophotometry
The commonest physico-chemical method and with the most application in analysis
of drugs in Biological sample is uv/visible spectrometry. This provide characteristic
spectrum that provide information on the identity or structure of the analyte, thus
useful for qualitative analysis and give quantitative information as well. The
quantitative information depend on the extent of absorption. This can be described in
terms of Beer-lambert law; Lamberts law relates the total absorption to the optical
Absorption(A)= log10 ( I/I ) = kl
I= incident light intensity, I= transmitted light
K=proportionality constant for material
Beer law relates absorption to the concentration of the absorption solute, c, in
Log10 ( I/I ) =kc (the path length l, being constant)
Combining the two equation you get
Log10 = ecl
E=constant for a particular solvent at a particular wavelength
With the advent of continous scanning spectrophotometer, with the control of the
spectrphotometric functions (wavelengths, filter, radiation source etc.) are
incorporated with computer. The characteristic single spectrum, which gives a sharp
peak, is achieved at a particular wavelength called maxima (max). But compounds
with the same functional group will have a similar absorption spectra, the technique is
therefore not suitable for identifying unknown components in a sample. The technique
is also not suitable for quantitative analysis if metabolites with same functional groups
are present. The use of appropriate solvent at a particular PH ensures only parent drug
is extracted and this further ensures specificity of absorption at a particular
wavelength. If a specific extraction step is applied hence absorption methods are only
specific for unmetabolised drug, as most drug metabolites are more polar than the
parent drugs (Chamberland 1995)
Ultraviolet-visible spectrophotometry is best known as a tool for quantitative analysis.
Speed, simplicity and sensitivity have made the UV-visible spectrophotometry a
popular tool among analysts for the quantification of drugs and metabolites in
biologic samples. One of the main reasons for its popularity is that the sensitivity of
the method is in the range of 1-10 μg/ml, which is comparable to the concentration
level of many drug substances in biologic samples (Smith and Stewards, 1981). The
following are some applications of UV spectrophotometry in pharmaceutical analysis.
Mustapha et al (1996) reported a UV method to study the effect of Tamarindus indica
on the bioavailability of aspirin in healthy human volunteers. In this method, plasma
samples (1ml) were placed in a 20ml extraction tube and 2ml of 0.05M Hcl was
added. 10ml of ethylacetate was then added and the mixture shaken for 15min at 3000
rpm. The supernatant was then transferred into a second extraction tube and
evaporated to dryness on a water bath maintained at 40 +_10°C under a gentle stream
of nitrogen passing through the tube. The residue was reconstituted in 5ml methanol
and absorbance taken at 231nm and 276nm respectively. Concentrations of aspirin
and its metabolite, salicylic acid in the plasma were estimated using the simultaneous
equation for multiple component mixture.
Garba et al (1999) reported a UV method to study the influence of Cimetidine on the
pharmacokinetics of paracetamol in healthy subjects. According to the method, saliva
samples (2ml) were placed in 10ml extraction (centrifuge) tube using auto-pipette.
Ethyl acetate (5ml) were added to the content of the tubes and well stoppered. The
mixture was vortex mixed for one minute and centrifuged at 2500rpm for 5minutes.
The ethyl acetate layer (upper) was removed with pasture pipette and its absorbance
measured at 262nm, using 1cm Silica cuvettes using a double beam SP8-100 UV
Drugs can be determined either as intact compound or as a derivative formed by
chemical reaction with suitable reagents (Smith and Steward, 1981) the advantage of
chemical derivatization as an aid to UV analysis is that the sensitivity of the UV
procedure is increased significantly. Example, it has been reported that the sensitivity
of the UV procedures for amitryptylline was increased significantly by oxidation of
the drug to anthraquinone. Anthraquinone shows stronger absorption around 250nm
than the drug itself (Hamilton et al, 1975)
In a study to determine paracetamol in the urine. The UV spectra of investigated
samples were recorded over the wavelengths range 220-400nm ( step 0.21nm,scan
speed 60nm/min) and second order derivative spectra were calculated. Second order
derivative spectra of different blank urine samples displayed the presence of a zerocrossing
point at 245-247nm defined as zc. The zero order absorption of paracetamol
in water displays maximum absorption at 243nm, while in second derivative spectra,
a minimum peak at 246nm was observed. Therefore, the application of zero –
crossing technique to the second derivative UV absorption spectrum should be useful
for the determination of paracetamol using Dzc. The obtained results were in good
accordance with published data on cumulative urinary excretion after per oral
administration of paracetamol obtained applying different spectrophotometric
methods of determination (Jelena et al, 2003).
Another type of chemical derivatization method uses conversion of a drug to a
stronger UV absorbing species by an acid catalyzed re-arrangement (Fellenberg and
Pollard, 1976). In the procedure carbamazepine is extracted from blood, with
dichloromethane, the solvent is then evaporated to dryness and the residue treated
with HCl at 1500C. After extraction of the acidic solution with n-heptane, its
absorbance is measured as 258nm.
In the UV assay of methadone, the compound is subjected to an alkaline extraction
into n-hexane and back extraction into a ceric acid-H²SO4 solution. After refluxing,
the generated benzophenone is measured in the n-heptane layer by UV
spectrophotometer. Amount of methadone as low as 5 μg can be detected in
Second-derivative UV spectrophotometric method for the determination of naproxen
in the presence of its metabolite, 6-0- desmethylnaproxen was reported (Panderi and
Parissi, 1994). According to the procedure, plasma (1ml) was acidified with 0.5ml of
1M HCl and 5ml of diethyl ether were added. The mixture was vortex mixed for one
minute and centrifuged at 4000 revolution per minute for 10minutes. After
refrigeration at -170C, the organic layer was placed in a glass vial and evaporated to
dryness at 370C, under a stream of nitrogen. The residue was dissolved in 2ml of
0.1M–NaOH and the second derivative UV spectrum of the solution was recorded
from 280 to 350nm and the amplitude at 328.2nm was measured. The calibration
graph was linear from 5-100mg/L of naproxen, with a determination limit of
2.42mg/L. The result obtained agreed with those obtained by HPLC (Panderi and
1.4.2 ANALYTICAL METHOD DEVELOPMENT
One of the first practical steps in the development of a method is to examine and
know the chemical characteristics of the drugs you want to analyze. Structural
information on the drug including NMR, and MASS-spectra are very vital.
Information on the UV-spectra are also very important both for qualitative and
quantitative applications, like in chromatography.
Another very important consideration, which may well dictate a method to be adopted
is the sensitivity required, that is the lowest concentration which could be measured
after optimization of analytical conditions. Concentration range for which various
technique are applicable has been developed by (De Silva, 1995). A diagram showing
sensitivity of the difference technology is presented in Fig 184.108.40.206
Gas Chrom FID
Gas Chrom EC-det
Gas Chrom N-det
Gas Chrom – Mass Spec
1 pg/ml 1 ng/ml 1 ug/ml 1 mg/ml
Diagramme of concentration range for which various techniques are applicable.
This diagram helps in the selection of the technique available for development of
analytical method within sensitivity range. There is a considerable improvement in the
sensitivities of the analytical methods over time. From mg/ml in the 1950s to
femtogrames per ml being claimed in the 1990s.
The most sensitive detector yet developed appears to be the laser induced
fluorescence detector, which in conjunction with capillary electrophoresis
chromatography can detect 10 Yoctomytes of analyte, or sire molecules ( Chambland,
VALIDATION OF ANALYTIC METHOD
Analytical method validation includes all the procedures followed to ensure that a
particular method for the quantitative determination of an analyte or series of analytes
in biological material is reliable for the intended application.
The objectives of analytical method validation include the following:
1. To build confidence in analytical data generated
2. To ensure that a selected analytical procedure will give reproducible
and reliable result. It is therefore, necessary to validate a method according to the well
established criteria of precision, accuracy, sensitivity, specificity and reproducibility.
The precision of an analytical method is the degree of agreement among individual
tests when the procedure is applied repeatedly. Precision is usually expressed as
standard deviation (Coefficient of variation) C.V. Where a small standard deviation
indicate higher precision.
Precision in usually determined by assaying sufficient number of aliquots of a
homogenous sample in order to be able to calculate statistically valid estimate of
mean (x), standard deviation (SD) or the coefficient of variation (CV). Both withinday
and between-day precisions are relevant in the development of analytical method.
PERCENTAGE EXTRACTION RECOVERY
The percentage extraction recoveries of analytical method give the efficiency of the
extraction procedure to be employed in the analyses. It also gives assurance on the
reproducibility of the extraction method employed.
The percentage extraction recovery is calculated with the following equation.
Amount in conc. recovered after sample extraction
Amount in conc. Recovered after extraction in O.1N Hcl
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