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) m 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 50C and at
20 min yielding red- brown p-benzoquinone which absorbed at a Amax of 420nm. Similarly, optimum conditions for the oxidation of paracetamol by V(V) were pH of 1.0, temperature of 70C at 8 min, and V(V) reduced to bluish-violet vanadium(II) ions which absorbed at a Amax 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.
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 A and 400 m. 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 m), mid infrared (2.5 – 50 m), and far infra red (50 – 1000 m) 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
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=3
Where, A= absorbance or optical density
= absorptivity or extinction coefficient
b = path length of radiation through sample (cm)
c = concentration of solute in solution (mol/dm3).
Both b and are constants, so is directly proportional to the concentration, C. When C is in gm/ 100ml, then the constant is called A (1 %,
lcm). i. .e., A= % 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 ( ( ) .
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(l %, 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.
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 concentration.
C = (A x C ) /Astd
Where Ces and Cst are the concentrations in the sample and standard solutions respectively; and Atest and Asta are the absorbances of the sample and standard solutions respectively4•
For assay of substances m 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 genenc 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 CsHoNO and a molecular weight of 151. 1 7. Hence, its molar mass is 151. 1 7 g/ mol.
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. 37°C). 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 hours\. Although, it is used to treat inflammatory pain, it is not generally classified as a non• steroidal anti-inflammatory drug (NSAID) because it exhibits only weak anti• inflammatory activity. Paracetamol is part of the class of drugs known as “aniline analgesics”, and it is the only such drug still in use today!I. 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.
Scheme 1.3: 4-hydroxyacetanilide (paracetamol)
The mam 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 H, an unstable molecule that is, in turn, converted to numerous other pro-inflammatory compounds. Classical anti• inflammatories 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 chemicals!7.18, This leads to a reduced amount
of prostaglandin E 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 thromboxanes?20. 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.
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-p• benzo-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.
This material content is developed to serve as a GUIDE for students to conduct academic research
SPECTROPHOTOMETRIC DETERMINATION OF PARACETAMOL USING ZIRCONIUM (IV) OXIDE AND AMMONIUM TRIOXOVANADATE (V)
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