Advanced Analytical Chemistry

Quantitative analysis of active constituent of Paracetamol

OBJECTIVE: To develop a HPLC method for the determination of active constituent of Paracetamol. METHOD: HPLC analysis was carried out by using a C18 (150mm, 4.6mm and 3m) column with gradient program. Mobile phase composedof KH2PO4-methamol-acetonitrile-isopropyl alcohol (20: 20: 30: 30) (v/v) . Flow for mobile phase elution is 1.0ml per min; column oven temperature is maintained at 40C and measured the absorbance at 210nm. HPLC water is used as diluent. RESULTS: Paracetamol was eluted at the outlet. The method was validated with specificity, precision, linearity, accuracy, ruggedness and robustness. The response was linear over the concentration range of 10 to 60 g per mL for ingredient. Recovery results were between 98 percent to 102 percent. CONCLUSION: The developed RP-HPLC method is single and reproducible, with high resolution and has been successfully applied for the analysis of Paracetamol pharmaceutical drug products.

Hina Aslam
03-May-12

Quantitative analysis of active constituents of Paracetamol

Hina Aslam

Table of contents:
Quantitative analysis of active ingredients of Paracetamol 04 Introduction . 04 Molecular structure . 04 Physical data . 05 Ingredients of Paracetamol tablet ... 05 Paracetamol 500mg Tablet Formulation per tablet .. 06 Methodology for quantitative determination of Paracetamol .. 06 Review of HPLC technique .. 06 Purpose of analysis 07 Material and method . 08 Reagents and solutions .. 08 Apparatus ... 08 Chromatographic conditions .... 08 Procedure ... 09 Literature review ... 09 Preparation of Standard Solutions ... 09 Preparation of Samples . 10 Internal standard ... 10 Mobile Phase Optimization ... 11 Calibration .. 11 Chemical analysis ... 11 Interpretation of result .. 12 Quantification . 12 Conclusion ... 12

References 13

Quantitative analysis of active ingredients of Paracetamol

Introduction:
Paracetamol or acetaminophen is a widely used over-the-counter analgesic (pain reliever) and antipyretic (fever reducer). It is commonly used for the relief of headaches and other minor aches and pains and is a major ingredient in numerous cold and flu remedies. In combination with opioid analgesics, paracetamol can also be used in the management of more severe pain such as post-surgical pain and providing palliative care in advanced cancer patients.[1] The onset of analgesia is approximately 11 minutes after oral administration of paracetamol,[2] and its half-life is 14 hours. Though acetaminophen is used to treat inflammatory pain, it is not generally classified as an NSAID (non-steroidal anti-inflammatory drugs) because it exhibits only weak anti-inflammatory activity. While generally safe for use at recommended doses (1,000 mg per single dose and up to 4,000 mg per day for adults),[3] acute overdoses of paracetamol can cause potentially fatal liver damage and, in rare individuals, a normal dose can do the same; the risk is heightened by alcohol consumption. Paracetamol toxicity is the foremost cause of acute liver failure in the Western world, and accounts for most drug overdoses in the United States, the United Kingdom, Australia and New Zealand.[4][5][6][7] It is the active metabolite of phenacetin, once popular as an analgesic and antipyretic in its own right, but unlike phenacetin and its combinations, paracetamol is not considered carcinogenic at therapeutic doses.[8] The words acetaminophen (used in the United States, Canada, Japan, South Korea, Hong Kong, and Iran[9] ) and paracetamol (used elsewhere) both come from a chemical name for the compound: para-acetylaminophenol and paraacetylaminophenol. In some contexts, it is simply abbreviated as APAP, for acetyl-paraaminophenol.

Molecular structure:
Paracetamol consists of a benzene ring core, substituted by one hydroxyl group and the nitrogen atom of an amide group in the para (1,4) pattern.[10] The amide group is acetamide (ethanamide). It is an extensively conjugated system, as the lone pair on the hydroxyl oxygen, the benzene pi cloud, the nitrogen lone pair, the p orbital on the carbonyl carbon, and the lone pair on the carbonyl oxygen are all conjugated. The presence of two activating groups also
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makes the benzene ring highly reactive toward electrophilic aromatic substitution. As the substituents are ortho, para-directing and para with respect to each other, all positions on the ring are more or less equally activated. The conjugation also greatly reduces the basicity of the oxygens and the nitrogen, while making the hydroxyl acidic through delocalisation of charge developed on the phenoxideanion.

N-(4-hydroxyphenyl)ethanamide N-(4-hydroxyphenyl)acetamide

Physical data:
Formula: Mol. Mass: Density: Melt. Point: Solubility in water: C8H9NO2 151.17 g/mol 1.263 g/cm 169 C (336 F) [11][12] 12.78[13] mg/mL (20 C)

Ingredients of Paracetamol tablet:

Paracetamol/Acetaminophen ( Active ingredients) Maize Starch (Binder) Croscarmellose (Desintegrant) Nipagin/Methyl Paraben (Preservative) Nipasol/Propyl Paraben (Preservative) Purified Water Talc (antisticking) Silicon Dioxide Colloidal (anti adherent) Magnesium Stearate (Lubricants)
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Paracetamol 500mg Tablet Formulation per tablet:

Paracetamol/Acetaminophen ( Active ingredients) Maize Starch (Binder) 5% w/w Croscarmellose (Desintegrant) 3% w/w Nipagin/Methyl Paraben (Preservative) 0.03% w/w Nipasol/Propyl Paraben (Preservative) 0.07% w/w Microcrystalline Cellulose (Filler) 10% w/w Purified Water Microcrystalline Cellulose 102 (Filler)qs ad Talc (antisticking) 0.5% w/w Silicon Dioxide Colloidal (antiadherent) 1% w/w Magnesium Stearate (Lubricants) 0.5% w/w

500 mg 42.5 mg 25.5 mg 0.255 mg 0.595 170 mg 94.15 mg 4.25 mg 8.5 mg 4.25 mg

Methodology for quantitative determination of Paracetamol:

Numerous methods have been reported for the analysis of paracetamol and its combination in pharmaceutical or in biological fluids. Paracetamol has been determined in combination with other drug using titrimetry [14,15] , voltammetry [16], fluorimetry [17], colorimetry [17], UV-spectrophotometry [18-20], quantitative thin layer chromatography (TLC) [21], high pressure liquid chromatography (HPLC) [22-27] and gas chromatography (GC) [28] in pharmaceutical preparation.

Review of HPLC technique:

High Performance Liquid Chromatography (HPLC) is the most important analytical technique employed during the drug manufacturing and analysis. The key to a proper HPLC system operation is knowledge of the principles of the chromatographic process, as well as understanding the reasons behind the choice of the components of the chromatographic systems such as column, mobile phase and detectors. That HPLC became undoubtedly the most important analytical method for identification and quantification of drugs, either in their active pharmaceutical ingredient or in their formulations during the process of their discovery, development and manufacturing.

Schematic diagram of HPLC

Purpose of analysis:
The main purpose of this experiment is to quantify the active ingredients in commercial analgesic tablets Paracetamol by a simple, rapid and accurate HPLC technique and to compare it with the quantity of active ingredient as per label claims. The objective of this study is to develop and validate a special, rapid, accurate, precise and reproducible quality control method for paracetamol in their combinations.

Material and method:

Reagents and solutions:
High pure (not less than 98.5%) standards Paracetamol is used for analysis. HPLC grade acetonitrile and water, AR grade Potassium di-hydrogen ortho-phosphate are used for this study.[29]

Apparatus:
Mobile Phase: Mix and filter 0.01 M KH2PO4-methamol-acetonitrile-isopropyl alcohol (20: 20: 30: 30) (v/v) with 0.45 filter.[30] Diluent : Paracetamol: HPLC water. Label Claim per tablet: acetaminophen 500 mg

Internal Standards: Nicotinic acid; 0.5mL has a concentration of 5mg/mL. Standards: Acetaminophen

All reagents and chemicals are of analytical grade.

Chromatographic conditions:
Column: Detector: Detection: Injection Volume: Flow rate: Column temp: Run time: C18, 15 cm x 4.6 mm, 3 m. UV/VIS detector UV 210 nm 10 l 1 ml/min. 40C 25min [29]

Procedure: Literature review:

Start experiment with the internet search of analytical methods in quantitative determination of paracetamol using some technique like HPLC and find articles regarding this experiment. Following procedure is helpful in quantitative analysis of paracetamol using HPLC.

Preparation of Standard Solutions:

Stock of Internal Standard solution: 1. Transfer about 500.0 mg of nicotinic acid, accurately weighed, into a 100 ml volumetric flask. 2. Half fill it with mobile phase. 3. Sonicate until dissolve then dilute to volume with mobile phase.

Stock Solution of Acetaminophen: 1. To prepare stock solution of acetaminophen (0.5mg/mL) transfer about 50 mg of Acetaminophen, accuately weighed, into a 100 ml volumetric flask. 2. Add 0.5 ml Internal STD Stock Solution. 3. Half fill with mobile phase then sonicate until dissolve. 4. Dilute it to volume with mobile phase.

Working standard solution: Working standard solution is prepared by suitable dilution of stock solution. These solutions are stable for 1 week if kept in the refrigerator. 1. Transfer 10 mL of the stock solution into 100 ml volumetric flask. 2. Dilute to the mark with mobile phase to give final concentration of 100 g/ml. 3. Aliquots of 0.5, 1, 3, 5, and 7 mL of acetaminophen from its working solution (100 g/mL) are transferred into a series of 10 mL volumetric flasks. 4. Dilute to volume with mobile phase and mix well.

Preparation of Samples:
The most important step in any chemical analysis is obtaining a representative sample to measure. This process is called sampling. 1. Weigh 10 tablets of paracetamol for analysis and the average weight per tablet is 570 mg. 2. The 10 tablets, which form the composite is pulverized in a mortar by a pestle. It is used the composite because one tablet can have a wrong amount of active product. 3. After that it is weighted out one tablet equivalent. 4. The solvent use to dissolve the sample is a solution of acetonitrile and the dissolution is accomplished by USP.

Preparation of the Stock Solution of the Sample: 1. Add 50 mg of Acetaminophen in a volumetric flask of 100 ml. 2. Sonicate the volumetric flask for 3 minutes in half fill of mobile phase. 3. Add 0.5 ml of internal standard, which has a concentration of 5mg/ml. 4. After that the volumetric flask is completed to the mark with mobile phase. 5. Consequently, the final concentration Acetaminophen becomes 0.50mg/ml.

Internal standard:
An internal standard is a known amount of a compound, different from analyte, that is added to the unknown. Signal from analyte is compared with signal from the internal standard to find out how much analyte is present.

Reason for use of internal standard: Internal standards are especially useful for analysis in which the quantity of sample analyzed or the instrument response varies slightly from run to run for reasons that are difficult to control. Internal standards are widely used in HPLC because the small quantity of sample solution injected into the HPLC is not very reproducible in some experiments. Internal standards are also desirable when samples loss can occur during sample preparation steps prior to analysis. If a known quantity of standard is added to the unknown prior to any manipulation, the ratio of standard to analyte remains constant because the same fraction of each is lost in any operation.

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Required features of internal standard: Sample is chemically inert Similar chemical structure from the analyte The internal standard should elute out faster, in about 3 minutes It needs to add the same amount of same compound to all calibration and samples. The internal standard must have resolution and detectability and mimics analytes in pretreatment steps.

Mobile Phase Optimization:

The mobile phase optimizations is done by plotting in a graph the capacity factor (k) versus different concentrations of mobile phase and observing the separation of internal standard (IS) and the active ingredient (acetaminophen).

Calibration:
The method of calibration is concerned with the calibration curve, which shows the response of a chemical analysis to known quantities (standard solutions) of analyte. There is a linear response; so the corrected analytical signal is proportional to the quantity of analyte. Throughout the study, the suitability of chromatographic system was monitored by calculating capacity factor (k0), the resolution (R), the selectivity nad the peak symmetry (T).

Chemical analysis:
HPLC is performed using isocratic gradient elution[30] . The final step is the injection of solution in chromatographic column which measure the quantity of analyte. The column is packed with tiny particles of silica to which a long hydrocarbons are attached. 10 l of sample solution is injected into column. Analyte are detected at the outlet by their ability to absorb UV light from lamp. The graph of detectors response verses time is called chromatogram. Acetaminophen is the major peak in the chromatogram. Small peaks can also arise from other substances. For quantitative analysis we assume that the area (or the height) of our peak in the chromatogram is proportional to the amount of substance that produced the peak. In the simplest method we measure areas or heights, which are then normalized (this means that each area or height is expressed as a percentage of the total). The normalized hieghts or areas give the composition of our mixture.
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There are two problems with this approach, which are: a. We has to be sure that we has counted all the components, i.e. that each component appears as a separate peak on the chromatogram. Components may co-elute, or be retained on the column, or may elute without being detected. b. We are assuming that we get the same detector response for equal amounts of each component.

Interpretation of result:
By knowing how much acetaminophen is in the sample, we can calculate how much acetaminophen is in the paracetamol tablet.

Quantification:
% of content = Area of test solution x Std. Concentration x average weight x Potency of standard Area of standard solution x sample concentration x Label claim

Conclusion:
This method represents a fast analytical procedure for the simultaneous quantitation of acetaminophen. The sample preparation is simple, the analysis time is short and the elution is isocratic. The method is amenable to the analysis of large numbers of samples with excellent precision and accuracy. The complete results proved that the developed method to be convenient and effective for the determination of all active ingredients during the analysis of the bulk as well as pharmaceutical dosage forms. It has advantages of simplicity, reliability, repeatability, sensitivity, short analysis time and cheap reagents. The use of nicotinic acid as internal standard lowers the possible analytical errors which are brought by the sample dilution and injection steps. As a result the proposed method can be used to carry out quantitative analysis acetaminophen.

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