Paracetamol:

• Paracetamol is classified as a mild analgesic. 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.
• The onset of analgesia is approximately 11–29.5 minutes after oral administration of paracetamol, and its half-
life is 1-4 hours
• While generally safe for use at recommended doses (1,000 mg per single dose and up to 4,000 mg per day for
adults), 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
• Systematic (IUPAC) name:N-(4-hydroxyphenyl)acetamide

• 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
• Paracetamol is part of the class of drugs known as “aniline analgesics”; it is the only such drug still in use today
 METHODOLOGY:
• The Analysis means the examination of something in details in order to understand it better or draw conclusions from it
• Sample Collection: Seven samples of paracetamol 500mg tablets were obtained from various pharmacy shops within Hyderabad, the
samples were obtained together with their packs and receipt.
• Practical Method: The methods employed for the purpose of this study are
1.UV Visible spectrophotometric
2.HPLC

• Practical Procedure:
• By UV Spectrophotometry: The tablets were assayed spectrophotometrically using the following procedures
• Instrument: HI-TECH
• Software: UV-Win
• The average weight of tablet from each sample was determined by weighing ten tablets and dividing the result by ten.
• Two tablets were then crushed using a clean pestle and mortar (i.e. from each sample).
• For each sample, powder containing 0.05g (50mg) of paracetamol was accurately weighed and transferred into different 100ml
volumetric flasks. All the 7 samples were labelled using a pen and a masking tape.
• To Each volumetric flask, 50ml of 0.1 M NaOH and 100ml of distilled water were added, and sonicated for few minutes to
dissolve the drug molecule. After sonicating, the volume was made to 100ml with distilled water.
• The mixture in each flask was then mixed well and filtered through a filter paper into clean beakers.
• From the filtrate, 10ml was taken using a pipette and transferred into a 100ml volumetric flask; distilled water was then added to
make up the volume.
• From the resultant solution above (6), 10ml was taken with a pipette into a 100ml volumetric flask and 10ml of 0.1M NaOH was
added, distilled water was then added and make up the volume (5µg/ml).
• The UV Spectrophotometer was put at zero by running a baseline (between 200-400nm) using 0.1 M NaOH solution as blank.
The absorbance of each sample was determined at 257nm, by putting small amount of the sample into a cuvette, and the cuvette
was put into the machine.
• The same procedure was repeated for the standard using 100mg of the powdered standard, and absorbance determined, which
was used to calculate the percentage content (in mg) of paracetamol from each brand.
• The concentration of each sample was also determined using Beer Lambert’s law according to IP.
• RESULTS: The average weight of different brands of Paracetamol tablets were calculated and tabulated blow (Used for UV and
HPLC).
• Samples weights
• SAMPLE C (Paracip) 605.59
• SAMPLE D (Calpol ) 636.19
• The absorbance and % content of different brands of Paracetamol tablets brands are evaluated by using the UV spectroscopy and results obtained are tabulated
in blow
SAMPLE SOLUTION CONCENTRATION(mg/ml) ABSORBANCE %CONTENT
SAMPLE A (Paracip) 0.000605 0.62 107.52
SAMPLE B (Calpol ) 0.000636 0.45 99.55
• Obtained results are subjected to calculation for evaluating standard deviation and coefficient variation of different brands of paracetamol were calculated and tabulated
blow:
Samples Mg content (X) X-X
SAMPLE A (Paracip) 495 101.74
SAMPLE B (Calpol ) 471 98.74

standard deviation = 108.48

coefficient variation = 22.58
 By HPLC method:
• The tablets were assayed chromatographic method using the following procedure
• Instrument: Cyber Labs;
• Software: LC-100
• The mobile phase containing methanol and water in the ratio of 60:40 was prepared. This was done by
measuring 600ml of methanol and 400ml of distilled water into a 1000ml measuring cylinder, and put on
to a sonicator for ten (10) minutes. This was then removed and filtered using a membrane filter and a
vacuum pump.
• From the powdered drug samples, powder containing 50mg of paracetamol was weighed from each
sample, and then transferred into a 100ml volumetric flask each, and was labeled.
• 100ml of the mobile phase was measured and added to each of the volumetric flask, and was put on to a
sonicator for five (5) minutes, for the drug molecules to dissolve.
• After sonicating for five minutes, the solutions were then filtered through a filter paper into clean
beakers.
• 10ml of each filtrate was taken and put into different 100ml volumetric flask, and the mobile phase was
added to make up the volume.
• From the above solutions (5), small portion of each was then put into different chromatographic sample
vial, and the vials were put into the machine at different locations.
• Enough of the mobile phase was put into the chromatographic tank, the machine was put on, and settings
were made to select the vial to be run. The connected computer displays the result of the analysis on the
screen (i.e. the chromatogram), and these were printed with the aid of a connected printer.
• The same procedure was carried out using 50mg of the standard Paracetamol powder, and the result was
used to calculate the percentage content and content (in mg) of each sample.
 RESULTS:
• The Peak area and %content obtained for prepared concentration of different brands of paracetamol tablets were
calculated and tabulated blow:
Sample solution Concentration (mg/ml) Peak Area % Content
Sample A 0.0495 35699.3 103.27
Sample B 0.047 34650.1 100.23
• The obtained results are subjected to calculation of standard deviation and coefficient variation was calculated by using
corresponding formula and results are:
Samples Mg content (X) X-X
Sample A 495 101.74
Sample B 471 98.74
 Compersion of results:

• The results for standard deviation and coefficient variance obtained by spectroscopic and chromatographic were
compared for evaluation of two methods are
Method Mean (X) Variance (S)2=[Σ(x-x)2/N-1] SD=√S2 CV= SD/X*100%
UV 480.22 11768.72 108.48 22.58
HPLC 507.5 11673.59 108.04 21.2
• DISCUSSION: According to the Indian Pharmacopoeia (I.P), paracetamol tablet should contain not less than
90% (495mg) and not more than 110% (550mg) of paracetamol
• From the results obtained using the spectrophotometric method, it can be seen that samples, both samples are within the
limit specified by the I.P
• From the results obtained using HPLC method shows that both samples are within the limit
 Analysis of aspirin:
• Aspirin, also known as acetylsalicylic acid (ASA), is a medication used to treat pain, fever, or inflammation
• Aspirin given shortly after a heart attack decreases the risk of death. Aspirin is also used long-term to help prevent further heart attacks, ischaemic strokes, and blood
clots in people at high risk.
• For pain or fever, effects typically begin within 30 minutes. Aspirin is a nonsteroidal anti-inflammatory drug (NSAID) and works similarly to other NSAIDs but also
suppresses the normal functioning of platelets.

• 2-acetoxybenzoic acid
acetylsalicylate

•
• Multidrug pharmaceutical preparations for the therapy of pain of weaker genesis contain the different components, usually acetylsalicylic acid and
paracetamol with caffeine, codeine, derivates of pyrazolones, barbiturates, vitamins, phenacetine, pentazocine which can improve the
pharmacological value of these preparations.
• For the assay of acetylsalicylic acid, paracetamol, caffeine and phenobarbital in the mixtures, the different methods have been reported, including
 spectrophotometry
 second derivative spectrophotometry
 planar chromatography
 IR spectroscopy
 and capillary chromatography
• Considering the properties of the compounds investigated, such as mid polarity, as well as thermolability and low volatility, HPLC methods have
been the most explored.
• The determinations of these drugs have been mainly performed with the fluorescent detection after postcolumn derivatisation, due to the different
UV absorption characteristics of compounds, which lead to a better sensitivity of an assay.
 Experiment

1. Chemicals
• All chemicals and solvents were of analytical reagent grade
 Acetonitrile and H3PO3
 Deinoized and distilled water was used.
 Standards for acetylsalicylic acid [2-(acetyloxy)benzoic acid], paracetamol [N-(4-hydroxyphenyl)acetamide], caffeine [3,7-dihydro-1,3,7-trimethyl-1H-purine- 2,6-
dione] and phenobarbital [5-ethyl-5-phenyl- 2,4,6(1H,3H,5H)pyrimidinetrione]
 Malophenum tablets
2. Chromatographic equipment and conditions
• HPLC analysis was performed on a chromatographic system equipped with a Bio Rad 18 01 solvent pump, Rheodine 71 25 injector, and Bio Rad 18 01 UV/Vis
detector. A Value Chrom Chromatography Software data system was used to collect, integrate and analyse the chromatographic data. UV detection was carried out
with set to 207 nm, at a 0.01 AUFS range.
• Bio SiL HL C18, 5 mm, 250/4.6 mm column was used for the separation of these compounds.
3. Mobile phase
• For the analysis, the mobile phase consisted of MeCN/water (25:75 v/v) adjusted to pH 2.5 with H3PO3 was used, at a flow rate of 2.0 ml min.
4. Standard preparations
• The stock solution containing 62.5 mg ml of acetylsalicylic acid and paracetamol, 12.5 mg ml of caffeine and 5 mg ml
of phenobarbital in mobile phase was used. The calibration curves were prepared by diluting the stock solution in the
mobile phase to furnish solutions with final concentrations of 0.05, 0.1, 0.2, 0.25, 0.3, 0.35, and 0.4 mg ml for
acetylsalicylic acid and paracetamol, 0.01, 0.02, 0.04, 0.05, 0.06, 0.07 and 0.08 mg ml for caffeine and 0.032 mg ml for
phenobarbital.
5.Sample preparation
• The mean weight of finely powdered Malophenum tablet containing 250 mg of acetylsalicylic acid and
paracetamol, 50 mg of caffeine and 20 mg of phenobarbital was accurately transferred into 100 ml calibrated
flask and 80 ml of mobile phase was added; the mixture was extracted in the ultrasonic bath for 10 min at room
temperature and diluted with mobile phase to the mark. The supernatant liquid was filtered through Anotop 25,
0.02 mm filter. One millilitre of this solution was transferred to the 10 ml calibrated flask and diluted with
mobile phase to the mark.
• To obtain satisfactory resolution and to avoid peak tailing of compounds, an optimization of the proposed method was
carried out using the different mobile phases. The use of mobile phase acetonitrile-/0.05 M phosphate buffer (pH 2.5,
adjusted with HCl) gave asymmetrical peaks with tailing.
• Mobile phases of various compositions of acetonitrile and water were also tested. Using the system acetonitrile/water
40:60 v/ v (pH 2.5 adjusted with phosphoric acid) the peaks of phenobarbital and acetylsalicylic acid were not well
separated.
• With an increase of polarity of mobile phase, all the compounds were well separated reducing peak tailing. The best
results were obtained using the mobile phase acetonitrile/water 25:75 v/v (pH 2.5 adjusted with phosphoric acid).
• The most reproductive results were obtained with octadecil stationary phase (Bio SiL HL C18, 5 mm, 250/4.6 mm
column) on the chromatographic system consisting Bio Rad 2800 solvent pump and Bio Rad 1801 UV/Vis Detector.
• The detection was performed at 207 nm in sensitivity range 0.01 AUFS. To find optimal detection conditions, the different
analytical wavelength corresponding to UV
• Maximums of acetylsalicylic acid (228 nm and 276 nm), paracetamol (240 nm), caffeine (273 nm) and pheno- barbital (254
nm) were tested, no UV detection to be satisfactory for all compounds in mixture was provided.
• After optimization, HPLC method was carried out on Bio SiL HL C18, 5 mm, 250/4.6 mm column using acetonitrial/water
(25:75 v/v) adjusted to pH 2.5 with phosphoric acid as mobile phase at a flow rate of 2.0 ml min.
• Typical chromatograms obtained are illustrated in Fig. 1.
• The retention times were 7.78 min for acetylsalicylic acid, 4.86 min for caffeine, 2.97 min for paracetamole, 9.89 min for
phenobarbital and 11.66 min for salicylic acid.
• System suitability tests were performed and chroma- tographic parameters calculated from experimental data, such as capacity
factor (k), peak asymmetry factor (A), selectivity factor (a) and resolution factor (Rs) are given in Table 1. The capacity factor,
calculated as a relation of the time of injection of sample to column (1B/kB/10), selectivity factor (a/1) and resolution factor
(Rs/1) were found to be satisfactory. The values obtained for peak asmmetry were significantly lower than theoretical values
(1B/AB/1.2).
• The retention times were 7.78 min for acetylsalicylic acid, 4.86 min for caffeine, 2.97 min for paracetamole,
9.89 min for phenobarbital and 11.66 min for salicylic acid.
• System suitability tests were performed and chroma- tographic parameters calculated from experimental data,
such as capacity factor (k), peak asymmetry factor (A), selectivity factor (a) and resolution factor (Rs) are
given in Table 1. The capacity factor, calculated as a relation of the time of injection of sample to column
(1B/kB/10), selectivity factor (a/1) and resolution factor (Rs/1) were found to be satisfactory. The values
obtained for peak asmmetry were significantly lower than theoretical values (1B/AB/1.2).
 Result:
• HPLC method is simple, rapid and sensitive and therefore suitable for the routine analysis of acetylsa- licylic acid,
paracetamol, caffeine and phenobarbital in tablets.