ABSTRACT
A simple and sensitive spectrophotometric method for the determination
of paracetamol was explored, using zirconium(IV) and vanadium(V) oxides. The
method was based on the oxidation of paracetamol by zirconium(IV) and
vanadium(V) in alkaline and acidic media respectively. The stoichiometric
studies indicated a mole-ratio of 1:1 for the reactions of paracetamol with both
zirconium(IV) and vanadium(V). Effects of other variables like pH, temperature
and time were determined and showed that the optimum conditions for the
oxidation of paracetamol by zr(IV) were pH of 9.0, temperature of 50˚C and at
20 min yielding red- brown p-benzoquinone which absorbed at a λmax of 420
nm. Similarly, optimum conditions for the oxidation of paracetamol by V(V)
were pH of 1.0, temperature of 70˚C at 8 min, and V(V) reduced to bluish-violet
vanadium(II) ions which absorbed at a λmax of 600 nm. The Beer-Lambert’s law
was obeyed at a concentration range of 5.0-40.0 μg/cm3 for paracetamol with
both Zr(IV) and V(V) respectively; and the correlation coefficients for both
oxidants were 0.997 and 0.999 respectively. The mean % recovery of
paracetamol in dosage form with Zr(IV) was 99.06 %, while V(V) gave 100.17 %.
Hence, the recovery studies had proved the method to be accurate, simple and
precise.
TABLE OF CONTENTS
Title page ——————————————————————— i
Certification——————————————————————- ii
Dedication ——————————————————————– iii
Acknowledgment ————————————————————- iv
Table of Contents ———————————————————– v
List of Tables —————————————————————– vi
List of Figures —————————————————————- vii
Abstract ———————————————————————- viii
CHAPTER ONE
1.0 Introduction ————————————————————- 1
1.1 Ultraviolet – visible spectrophotometry (UV – visible
spectrophotometry). —————————————————- 1
1.2 Paracetamol ————————————————————- 4
1.3 The structure of paracetamol —————————————– 5
1.4 Mechanism of action of paracetamol ——————————— 6
1.5 Metabolism ————————————————————– 10
1.6 Medical uses of paracetamol —————————————– 10
1.7 Adverse effects/toxicity ———————————————— 11
1.8 Statement of the problem ——————————————— 12
1.9 Objectives of the study ————————————————- 13
CHAPTER TWO:
2.0 Literature review ——————————————————– 14
2.1 A brief historical background of paracetamol ———————- 14
2.2 Methods of determining paracetamol. ——————————- 17
2.2.1 Chromatographic methods of determination ——————- 17
2.2.2 UV-Visible spectrophotometric methods ———————— 21
2.2.3 Fluorescence spectrometric methods —————————- 27
2.3 Spectrophotometric determination of the stoichiometry of
metal to ligand in a complex —————————————– 30
vi
CHAPTER THREE
3.0 Materials and methods ———————————————— 33
3.1 Materials —————————————————————– 33
3.1.1 Aparatus/Equipment ———————————————– 33
3.2.0 Preparation of Reagents ——————————————- 33
3.2.1 Preparation of 0.1 M paracetamol ——————————– 33
3.2.2 Preparation of 0.1 M Zirconium(IV) oxide, (Zirconia) ———- 33
3.2.3 Preparation of 0.1 M ammonium trioxovanadate(V) ———– 34
3.3.0 Absorption spectra ————————————————– 35
3.3.1 Absorption spectrum of paracetamol —————————- 35
3.3.2 Absorption spectrum of zirconium(IV) in sodium hydroxide
Medium —————————————————————– 35
3.3.3. Absorption spectrum of mixture of paracetamol and Zr(IV)
in sodium hydroxide medium ————————————- 36
3.3.4 Absorption spectrum of vanadium(V) in
tetraoxosulphate(VI) acid medium ——————————— 36
3.3.5 Absorption spectrum of mixture of paracetamol and
vanadium(V) in tetraoxosulphate(VI) acid medium ————- 36
3.4.0 Determination of the stoichimetry of the reactions
between paracetamol and the oxidants ————————– 37
3.4.1 Stoichiometry of reaction between paracetamol and
zirconium(IV) ——————————————————— 37
3.4.2 Stoichiometry of reaction between paracetamol and
vanadium(V) ———————————————————- 37
3.5.0 Determination of optimal conditions —————————– 38
3.5.1 Effect of pH on Zr(IV)-paracetamol reaction ———————- 38
3.5.2 Effect of pH on V(V)-paracetamol reaction ———————- 38
3.5.3 Effect of time on the reaction of paracetamol with
zirconium(IV) ——————————————————— 38
3.5.4 Effect of time on the reaction of paracetamol with V(V) ——- 39
3.5.4 Effect of temperature on the reaction paracetamol with
vii
Zirconium(IV) ——————————————————— 39
3.5.6 Effect of temperature on the reaction of paracetamol
with vanadium(V) —————————————————- 39
3.6.0 Beer’s calibration plots ——————————————– 39
3.6.1 Calibration curve for paracetamol-Zr(IV) reaction ————- 39
3.6.2 Calibration curve for paracetamol-V(V) reaction ————— 40
3.7.0 Quantitative assay of the drugs———————————– 40
3.7.1 Assay of paracetamol with Zirconium(IV) ———————– 40
3.7.2 Assay of paracetamol with vanadium(V) ————————- 41
CHAPTER FOUR
4.0 Results and discussion ———————————————– 42
4.1 Absorption spectrum of paracetamol. —————————— 42
4.2 Absorption spectrum of zirconium(IV) in NaOH medium.——- 42
4.3 Absorption spectrum of a mixture of paracetamol and
zirconium(IV) in NaOH medium ———————————— 42
4.4 Absorption spectrum of vanadium(V)
in tetraoxosulphate(VI) acid medium ——————————- 47
4.5 Absorption spectrum of the product of paracetamol-V(V)
reaction in H2SO4 medium ——————————————- 47
4.6.1 Stoichiometry of reaction between paracetamol and Zr(IV) — 49
4.6.2 Stoichiometry of reaction between paracetamol and
vanadium(V) ———————————————————- 50
4.7.0 Effect of pH on the reaction of paracetamol and Zr(IV) ——- 51
4.7.1 Effect of pH on paracetamol-V(V) reaction ———————- 52
4.7.2 Effect of time on the reaction of paracetamol with Zr(IV) —– 53
4.7.3 Effect of time in the reaction of paracetamol with
vanadium(V) ———————————————————- 54
4.7.4 Effect of temperature on paracetamol-Zr(IV) reaction ——— 55
4.7.5 Effect of temperature on paracetamol-vanadium(V) reaction 56
4.8 Beer’s calibration plot for the reaction of paracetamol
with zirconium(IV)—————————————————— 57
viii
4.8.2 Beer’s calibration plot for the reaction of paracetamol
with vanadium(V) —————————————————– 58
4.9.0 Validation of paracetamol in dosage form with zirconium(IV)-59
4.9.1 Validation of paracetamol in dosage with vanadium(V) ——- 60
CHAPTER FIVE
Conclusion ——————————————————————– 61
References ——————————————————————– 62
ix
CHAPTER ONE
1.0 INTRODUCTION
Spectroscopy involves the study of the absorption and emission of light
and other radiations as related to wavelength of the radiation. Hence,
spectroscopy is the branch of science dealing with the study of interaction
between electromagnetic radiation and matter. It is the most powerful tool
available for the study of atomic and molecular structures, and is used in the
analysis of wide range of samples. Optical spectroscopy includes the region on
electromagnetic spectrum between 100 Ǻ and 400 ım. Hence, the regions of
electromagnetic spectrum are thus – far (or vacuum) ultraviolet (10-200 nm),
near ultraviolet (200-400 nm), visible (400 – 750 nm), near infrared (0.75 –
2.2 ım), mid infrared (2.5 – 50 ım), and far infra red (50 – 1000 ım) region.2, 3
1.1 Ultraviolet – visible spectrophotometry (UV-visible spectrophotometry).
UV – visible spectrophotometry is one of the most frequently employed
techniques in pharmaceutical analysis. It involves measuring the amount of
ultraviolet or visible radiations absorbed by a substance in solution.4
Instruments which measure the ratio, or function of ratio, of the intensity of
two beams of light in the UV-visible region are called ultraviolet-visible
spectrophotometers.4
A spectrophotometer consists of two instruments, a spectrometer and a
photometer, both housed in one cabinet. The spectrometer is used to split or
resolve light in bands of wavelength before it is fed to the photometer. To
achieve the designed resolution, a spectrometer is specially equipped with a
2
high resolution wavelength selector known as monochromator. This
monochromator can isolate an extremely narrow bandwidth almost comparable
to a single wavelength.5
In qualitative analysis, organic compounds can be identified by the use of
spectrophotometer; if any recorded data is available; and quantitative
spectrophotometric analysis is used to ascertain the quantity of molecular
species absorbing the radiation.4
Spectrophotometric technique is simple, rapid, moderately specific and
applicable to small quantities of compounds. The fundamental law that
governs the quantitative spectophotometric analysis is the Beer-Lambert’s law.
Beer’s Law: it states that the intensity of a beam of parallel
monochromatic radiation decreases exponentially with the number of
absorbing molecules. In other words, absorbance is proportional to the
concentration.
Lambert’s law: It states that the intensity of a beam of parallel
monochromatic radiation decreases exponentially as it passes through a
medium of homogeneous thickness. A combination of these two laws yields the
Beer – Lambert law.4
Beer – Lambert’s Law: When a beam of light is passed through a
transparent cell containing a solution of an absorbing substance, reduction of
the intensity of light may occur. Mathematically, Beer – Lambert’s law is
expressed as –
A = ııı
3
Where, A = absorbance or optical density
ı = absorptivity or extinction coefficient
b = path length of radiation through sample (cm)
c = concentration of solute in solution (mol/dm3).
Both b and ı are constants, so ı is directly proportional to the
concentration, C. When C is in gm/100ml, then the constant is called A (1 %,
1cm). i.e., A = ı%
ııı ıı 4.
Quantification of medicinal substance using spectrophotometer may be
carried out by preparing solution in transparent solvent and measuring its
absorbance at suitable wavelength. The wavelength normally selected is
wavelength of maximum absorption ((ıı ıı ).
The assay of single component sample, which contains other absorbing
substances is then calculated from the measured absorbance by using one of
the three principal procedures. However, these three principal procedures are –
use of standard absorptivity value, calibration graph; and single or double
point standardization. In standard absorptive value method, the use of
standard A(1 %, 1 cm) is used in order to determine its absorptivity. It is
advantageous in situations where it is difficult or expensive to obtain a sample
of the reference substance.
In calibration graph method, the absorbances of a number of standard
solutions of the reference substance at concentrations encompassing the
sample concentrations are measured and a calibration graph is constructed.
4
The concentration of the analyte in the sample solution is read from the graph
as the concentration corresponding to the absorbance of the solution.
The single point standardization procedure involves the measurement of
the absorbance of a sample solution and of a standard solution of the reference
substance. The concentration of the substances in the sample is calculated
from the proportional relationship that exists between absorbance and
concentration4.
Cıııı = (Aıııı x Cııı)/Astd
Where Ctest and Cstd are the concentrations in the sample and
standard solutions respectively; and Atest and Astd are the absorbances of the
sample and standard solutions respectively4.
For assay of substances in multi component samples by
spectrophotometer; the following methods are being used routinely, which
include – simultaneous equation method, derivative spectrophotometric
method, absorbance ratio method (Q – Absorbance method), difference
spectrophotometry and solvent extraction method6.
1.2 Paracetamol
Paracetamol has the following generic names – acetaminophen7,
paracetamol or acetophenum8. However, chemical names by which it is
identified are: 4-hydroxyacetanilide, p-hydroxy acetanilide, p-acetaminophenol,
p-acetylaminophenol or N-acetyl-p- aminophenol7. It is a white, odorless,
crystalline powder with a bitter taste. It has a molecular formula of C8H9NO2
and a molecular weight of 151.17. Hence, its molar mass is 151.17 g/mol.
5
Paracetamol or acetaminophen is a widely used analgesic and
antipyretic. An antipyretic analgesic is a remedial agent or drug that lowers the
temperature of the body in pyrexia, i.e., in situation when the body
temperature has been raised above normal, (i.e. 370C). Hence, paracetamol has
been found to be significantly effective in reducing fever to normal levels in
human 9.
However, the onset of analgesia is approximately 11 to 29.5 minutes after
oral administration of paracetamol and its half-life is 1 – 4 hours10. Although, it
is used to treat inflammatory pain, it is not generally classified as a nonsteroidal
anti-inflammatory drug (NSAID) because it exhibits only weak antiinflammatory
activity. Paracetamol is part of the class of drugs known as
“aniline analgesics”, and it is the only such drug still in use today11. This is
because the other aniline derivatives – acetanilide and phenacetin
(acetophenatidin), commonly used as antipyretic agents have been withdrawn
completely from being used due to their numerous toxic and undesirable
effects, such as skin manifestations, jaundice, cardiac irregularities, hemolytic
anemia, kidney and liver cancer9.
1.3 The structure of paracetamol.
O
N CH3
HO
H
Scheme 1.3: 4-hydroxyacetanilide (paracetamol)
6
The main mechanism proposed is the inhibition of cyclooxygenase
(COX), and recent findings suggest that it is highly selective for cyclooxygenase-
2 (COX-2)13. Because of its selectivity for COX – 2, it does not significantly
inhibit the production of the pro-clotting thromboxanes13. While it has
analgesic and antipyretic properties comparable to those of aspirin or other
non-steroidal anti-inflammatory drugs, its peripheral anti-inflammatory activity
is usually limited by several factors, one of which is the high level of peroxides
present in inflammatory lesion. However, in some circumstances, even
peripheral anti-inflammatory activity comparable to NSAIDS can be observed.
However, Anderson et al14 had reported the analgesic mechanism of
acetaminophen (paracetamol), being that the metabolites of acetaminophen,
e.g., N-acetyl-p-benzo-quinone imine (NAPQI) act on (transient receptor
potential sub family A, member I) TRPAI – receptors in the spinal cord to
suppress the signal transduction from the superficial layers of the dorsal horn,
to alleviate pain.
The COX family of enzymes is responsible for the metabolism of
arachidonic acid to prostaglandin H2, an unstable molecule that is, in turn,
converted to numerous other pro-inflammatory compounds. Classical antiinflammatories
such as the NSAIDs block this step. Only when appropriately
oxidized is the cyclooxygenase, (COX) enzyme highly active15, 16. Paracetamol
reduces the oxidized form of the cyclooxygenase (COX) enzyme preventing it
from forming pro-inflammatory chemicals17,18. This leads to a reduced amount
7
of prostaglandin E2 in the central nervous system (CNS), thus lowering the
hypothalamic set point in the thermoregulatory center.
Also, there is another possibility that paracetamol blocks cyclooxygenase
(as in aspirin), but in an inflammatory environment where the concentration of
peroxides is high, the high oxidation state of paracetamol prevents its actions.
Therefore, paracetamol has no direct effect at the site of inflammation, rather it
acts in the central nervous system (CNS) where the environment is not
oxidative; to reduce temperature19.
However, it should be noted that cyclooxygenase (COX), officially known
as prostaglandin-endoperoxide synthase (PTGS) is an enzyme that is
responsible for the formation of important biological mediators called
prostanoids, including prostaglandin, prostacyclin and thromboxanes20.
Pharmacological inhibition of cyclooxygenase (COX) can provide relief from the
symptoms of inflammation and pain20. At present, the three COX iso enzymes
are COX-1, COX-2, and COX-3; and in humans, it has been discovered that
acetaminophen works by inhibiting COX-221. There is much less gastric
irritation associated with COX-2 inhibiters, with a decreased risk of peptic
ulceration. However, the selectivity of COX-2 does not seem to negate other
side-effects of NSAIDS, notably an increased risk of renal failure21.
8
1.5 Metabolism
Paracetamol is metabolized primarily in the liver, into non-toxic
products. There are three metabolic pathways involved and they include
glucuronidation which is believed to account for 40 % to two-thirds of the
metabolism of paracetamol22; sulfation (sulfate conjugation) which may account
for 20-40 % 22; and thirdly, N-hydroxylation and rearrangement, then
glutathione sulfhydryl (GSH) conjugation which accounts for less than 15 %.
The hepatic cytochrome P450 enzyme system metabolizes paracetamol, forming
a minor yet significant alkylating metabolite known as NAPQI (N-acetyl-pbenzo-
quinone imine) 23. N-acetyl-p-benzo-quinone imine is then irreversibly
conjugated with the sulfhydryl groups of glutathione23. All the three pathways
yield final products that are inactive, non-toxic and eventually excreted by the
kidneys. In the third pathway, however, the intermediate product –NAPQI, is
toxic. N-acetyl-p-benzo-quinone imine (NAPQI) is primarily responsible for the
toxic effects of paracetamol.
9
The metabolic pathways
O
N
HO
H
O
N
O
G/CA
H
Paracetamol
Glucuronidation
O
N
O O
N
HO
GSH
H
S
O O
HO O
O
N
H
N-hydroxylation
and
Rearrangement
Acetaminophen
glucuronide
Sulfation
Acetaminophen
Sulphate
GSH Conjugation
Scheme 1.5: Toxic Reactions with proteins
and Nucleic acids.
NAPQI
NAPQI N-acetyl-P-benzo-quinone imine
1.6 Medical uses of paracetamol
Paracetamol is approved for reducing fever in people of all ages24. The
World Health Organization (WHO) recommends that paracetamol should only
be used to treat fever in children if their temperature is greater than 38.50C.
It is also used for the relief of pains associated with many parts of the
body. For example, backache, headache, migraine, muscle strains, menstrual
10
pain, toothache and aches and pains due to cold and flu. It has analgesic
properties comparable to those of aspirin, while its anti-inflammatory effects
are weaker. Paracetamol is used in the treatment of headaches and can relieve
pain in mild arthritis, but has no effect on underlying inflammation, redness
and swelling of the joints.
In combination with opioid analgesics, paracetamol is used to reduce
post-surgical pains and to provide palliative care in advanced cancer
patients25. Psychologically, paracetamol acts on, and suppresses pain through
the central nervous system rather than the peripheral nervous system, thereby
reducing the neural response that causes the pain of social rejection as well as
neural responses related to physical pain26.
However, it should be noted that the recommended dose of paracetamol
for adults is one or two 500mg tablets in every 4-6 h; up to a maximum of 8
tablets in 24 h.
1.7 Adverse effects/Toxicity
In recommended doses and for a limited course of treatment, the side
effects of paracetamol are mild to non existent27. However, paracetamol being
metabolized by the liver is hepatotoxic, and side effects are multiplied when
combined with alcoholic drinks, especially in chronic alcoholics or patients
with liver damage27, 28. A high dose-usage (greater than 2000mg per day),
increases the risk of upper gastrointestinal complications such as stomach
bleeding,29, and may cause kidney or liver damage30. Chronic users of
paracetamol many have a high risk of developing blood cancer31.
11
Paracetamol is generally believed to be safe in pregnancy32, as it does not
affect the closure of the fetal ductus arteriosus as NSAIDS do32. However, its
use has been linked to infertility in the subsequent adult life of the male
fetus25. It was found that pregnant women especially in the second trimester
(14 to 27 weeks of pregnancy)33, who used more than one pain killer
simultaneously, such as paracetamol and ibuprofen, had increased risk of
giving birth to sons with some form of undescended testes, or cryptochidism,
compared to women who took nothing33.
The first symptoms of overdose of paracetamol usually begin several
hours after ingestion, with nausea, vomiting, sweating and pain as acute liver
failure starts34. Paracetamol hepatotoxicity is the most common cause of acute
liver failure35, 36. The toxicity arises often due to its quinine metabolite37.
Untreated overdose can lead to liver failure and probably death, but treatment
is aimed at removing the paracetamol from the body and replacing
glutathione37. Activated charcoal can be used to decrease the absorption of
paracetamol if the patient presents for treatment soon after the overdose.
However, an antidote, acetylcysteine (N-acetylcysteine or NAC) acts as a
precursor for glutathione, helping the body regenerate enough to prevent
damage to the liver. Also, N – acetylcysteine helps in neutralizing the
imidoquinone metabolite of paracetamol37. For a severe liver damage, a livertransplant
is often requried37.
12
1.8 Statement of the problem
Owing to the widespread use of paracetamol in different kinds of
pharmaceutical preparations, rapid and sensitive methods for the
determination of paracetamol are being investigated. Many spectrophotometric
methods of determination of paracetamol as reported were based on the
hydrolysis of the compound leading to the formation of a Schiff base with a
substituted benzaldehyde, or reaction with o-cresol35. Others available in
literature include the use of iron(III) salts, cerium(IV)tetraoxosulphate(VI),
potassium tetraoxomanganate(VII), to bring about the oxidation of paracetamol
usually in highly concentrated acidic medium to p-benzoquinone, which is then
determined spectrophotometrically.
However, most of these methods require lengthy treatments and lack the
simplicity and sensitivity needed for the routine analysis. Given this scenario, it
becomes pertinent to look into some other spectrophotometric methods that
would present a more sensitive, simple, less expensive and involving not too
highly concentrated acidic media for their reactions.
In the present work therefore, two different compounds which are
oxidants, would be used to determine paracetamol spectrophotometricaly both
in the alkaline and acidic media respectively. The compounds are zirconium
(IV) oxide, and ammonium trioxovanadate(V).
13
1.9 Objectives of the study.
The objectives of this work are:
· To develop and validate a rapid, simple and sensitive spectrophotometric
method of analysis for the determination of paracetamol.
· To use the proposed method to quantitatively assay paracetamol in its
pharmaceutical preparation
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