Project Abstract

<p> </p><p>A simple and sensitive spectrophotometric method for the determination<br>of paracetamol was explored, using zirconium(IV) and vanadium(V) oxides. The<br>method was based on the oxidation of paracetamol by zirconium(IV) and<br>vanadium(V) in alkaline and acidic media respectively. The stoichiometric<br>studies indicated a mole-ratio of 11 for the reactions of paracetamol with both<br>zirconium(IV) and vanadium(V). Effects of other variables like pH, temperature<br>and time were determined and showed that the optimum conditions for the<br>oxidation of paracetamol by zr(IV) were pH of 9.0, temperature of 50ËšC and at<br>20 min yielding red- brown p-benzoquinone which absorbed at a λmax of 420<br>nm. Similarly, optimum conditions for the oxidation of paracetamol by V(V)<br>were pH of 1.0, temperature of 70ËšC at 8 min, and V(V) reduced to bluish-violet<br>vanadium(II) ions which absorbed at a λmax of 600 nm. The Beer-Lambert’s law<br>was obeyed at a concentration range of 5.0-40.0 μg/cm3 for paracetamol with<br>both Zr(IV) and V(V) respectively; and the correlation coefficients for both<br>oxidants were 0.997 and 0.999 respectively. The mean % recovery of<br>paracetamol in dosage form with Zr(IV) was 99.06 %, while V(V) gave 100.17 %.<br>Hence, the recovery studies had proved the method to be accurate, simple and<br>precise.</p><p>&nbsp;</p><p><strong>&nbsp;</strong></p> <br><p></p>

Project Overview

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