UV-VISIBLE SPECTROPHOTOMETRIC METHOD DEVELOPMENT AND VALIDATION OF ASSAY

OF PARACETAMOL TABLET FORMULATION

INTRODUCTION :

Spectroscopy Methods: It is the branch of science dealing with the study of interaction between
Electromagnetic radiation and matter. It is a most powerful tool available for the study of
atomic and molecular structure/s and is used in the analyses of wide range of samples. Optical
spectroscopy includes the region on electromagnetic spectrum between 100 Å and 400 μm.
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.5 The onset of analgesia is approximately 11 minutes after oral administration of
paracetamol,6 and its half-life is 1–4 hours. Though acetaminophen is used to treat
inflammatory pain, it is not generally classified as an NSAID 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), 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.

Materials and Reagents

Materials: Paracetamol standard of was provided by Torque Pharmaceuticals (P) Ltd. (India).
Paracetamol tablets containing 500mg Paracetamol and the inactive ingredient used in drug
matrix were obtained from market. Analytical grade methanol and water were obtained from
Spectrochem Pvt. Ltd., Mumbai (India).

Diluent Preparation: Methanol and Water (15:85, v/v) used as a diluents.

Standard Preparation: 10mg drug was dissolved in 15ml methanol and was shaked well. Then
85ml water was added to it to adjust the volume upto 100ml (100ppm). From that 5ml was
taken and volume was adjusted upto 50ml with diluents.

Test Preparation: 20 tablets were weighed and powdered. Powdered tablet equivalent to 100mg
of

paracetamol was weighed and taken into 100ml volumetric flask then 15ml of methanol was
added and shaked well to dissolve it after that 85ml of water was added to adjust the volume
upto 100ml . From that 1ml of solution was withdrawn and taken in 100ml volumetric flask. The
volume was adjusted with diluent up to 100ml.

Instrumentation: UV-Visible double beam spectrophotometer with matched quartz cells (1cm),
Model: Evolution 201, Make: Thermo Scientific, 81 Wyman Street Waltham, Massachusetts, U.S

RESULTS AND DISCUSSION:

Development and Optimization of the Spectrophotometric Method: Proper wave length

selection of the methods depends upon the nature of the sample and its solubility. To develop a
rugged and suitable spectrophotometric method for the quantitative determination of
paracetamol, the analytical condition were selected after testing the different parameters such
as diluents, buffer, buffer concentration, and other chromatographic conditions.

Our preliminary trials using different composition of diluents consisting of water with buffer and
methanol. By using diluent consisted of methanol – water (15:85, v/v) best result was obtained
and degassed in an ultrasonic bath (Enertech Electronics Private Limited). Below figures
represent the spectrums of blank, standard and test preparation respectively.

Selection of Wavelength: Scan standard solution in UV spectrophotometer between 200 nm to

400 nm on spectrum mode, using diluents as a blank.

Method Development:

Preparation of Standard Solutions:

• Accurately weigh approximately 100 mg of paracetamol standard and transfer it into a 100 mL
volumetric flask.

• Dissolve the standard in the chosen solvent and make up to the mark with the solvent. This is
the stock standard solution (1 mg/mL).

• Prepare working standard solutions by diluting the stock standard solution with the solvent to
obtain appropriate concentrations for calibration.

Spectrophotometric Scan:

• Prepare a blank solution using the chosen solvent. • Scan the UV-Vis spectrum of the working
standard solution from 200 nm to 400 nm. • Identify the maximum absorbance wavelength
(λmax) of paracetamol.
Linearity Study:
• Prepare a series of working standard solutions covering the expected concentration range of
the tablet formulation.

• Measure the absorbance of these solutions at λmax using the UV-Vis spectrophotometer.

• Plot a graph of absorbance against concentration.

• Determine the linearity range by ensuring that the graph shows a linear relationship.

Optimization of Method Parameters:

• Optimize the chosen solvent, concentration range, and wavelength for maximum sensitivity
and accuracy.

Method Validation:

• Validation is concerned with assuring that a measurement process produces valid

measurements;

• Results from method validation can be used to judge the quality, reliability and consistency of
analytical results. It is an integral part of any good analytical practice.

• A measurement process producing valid measurements for an intended application is fit for
purpose.

Method validation is the process used to confirm that the analytical procedure employed for a
specific test is suitable for its intended use. Results from method validation can be used to
judge the quality, reliability and consistency of analytical results; it is an integral part of any
good analytical practice.

Analytical methods need to be validated or revalidated

Before their introduction into routine use;

Whenever the conditions change for which the method has been validated (e.g., an instrument
with different characteristics or samples with a different matrix); and

Whenever the method is changed and the change is outside the original scope of the method.

ICH Guidelines (ICH Q2R1) for Analytical Procedure and ValidatioN 21: The analytical procedure
refers to the way of performing the analysis. It should describe in detail the steps necessary to
perform each analytical test. This may include but is not limited to: the sample, the reference
standard and the reagents preparations, use of the apparatus, generation of the calibration
curve, use of the formulae for the calculation, etc.

Types of Analytical Procedures to be validated: The discussion of the validation of analytical

procedures is directed to the four most common types of analytical procedures:

- Identification tests;

- Quantitative tests for impurities' content;

- Limit tests for the control of impurities;

- Quantitative tests of the active moiety in samples of drug substance or drug product or other
selected component(s) in the drug product.

The objective of the analytical procedure should be clearly understood since this will govern the
validation characteristics which need to be evaluated.Typical validation characteristics which
should be considered are listed below:

Accuracy Precision Repeatability Intermediate Precision Specificity Detection Limit

Quantitation Limit Linearity Range
Furthermore revalidation may be necessary in the following circumstances:
Changes in the synthesis of the drug substance; Changes in the composition of the finished
product; Changes in the analytical procedure.

Specificity:

• Verify that the method is specific for paracetamol by ensuring that no other components in the
tablet formulation interfere with the absorbance at λmax.

• Use a blank sample containing all the excipients in the tablet formulation but not paracetamol
to confirm this.

Linearity:

six points calibration curve were obtained in a concentration range from 0-150 ppm for
Paracetamol The response of the drug was found to be linear in the investigation concentration
range and the linear regression equation was y =0.004x+0.007 with correlation coefficient
0.998.

TABLE 1: CONC. VS ABS. TABLE FOR LINEARITY STUDY Conc. (ppm) Absorbance 0 0 50 0.246
75 0.338 100 0.456 125 0.582 150 0.67

Accuracy:

• Determine the accuracy of the method by analyzing known concentrations of paracetamol and
comparing the results to the actual values.

• Prepare three replicate samples at different concentrations within the linearity range.

• Calculate the percentage recovery for each concentration.

• The recovery should be within 98% to 102% of the expected value.

Accuracy of the method is ascertained by standard addition method at 3 levels. Standard
quantity equivalent to 50%, 100% and 125% is to be added in sample.

Precision:

Precision: Precision of the analytical method is ascertained by carrying out the analysis as per
the procedure and as per normal weight taken for analysis. Repeat the analysis six times.
Calculate the % assay, mean assay, % Deviation and % relative standard deviation and %RSD.
The developed method was found to be precise as the %RSD values for the repeatability and
intermediate precision studies were < 0.98 % and < 0.79 %, respectively.

Limit of Detection (LOD):

• Determine the LOD by analyzing a series of very dilute solutions of paracetamol until the
absorbance signal is just detectable above the noise level.

• Calculate the LOD as 3.3 times the standard deviation of the blank absorbance divided by the
slope of the calibration curve.

Limit of Quantitation (LOQ):

• Determine the LOQ by analyzing a series of solutions until the signal is reliably quantifiable.
• Calculate the LOQ as 10 times the standard deviation of the blank absorbance divided by the
slope of the calibration curve.

Sample Preparation:

Preparation of Sample Solutions:

• Weigh a certain amount of Paracetamol tablet and dissolve it in a suitable solvent (e.g.,
distilled water or a suitable buffer solution) to prepare a solution with a known concentration

Assay Procedure:

• Blank: Run a blank with the chosen solvent to zero the spectrophotometer.

• Standard: Run a standard solution of known concentration at λmax to obtain the absorbance
reading.

• Sample: Measure the absorbance of the sample solution at λmax.

Calculation:

• Calculate the concentration of paracetamol in the tablet formulation using the standard curve
or the formula:

> Concentration of Paracetamol (mg/mL) = (Absorbance of Sample / Absorbance of Standard)

* Concentration of Standard

• Calculate the amount of paracetamol in the tablet:

> Amount of Paracetamol (mg/tablet) = Concentration of Paracetamol (mg/mL) Volume of

Solution (mL) Weight Factor
Reporting:

• Report the results in a clear and concise manner, including:

* Name of the method * Assay method * Validation parameters

* Results of the analysis * Standard deviation and RSD * LOD and LOQ * Conclusion

Conclusion:

This analytical procedure outlines a validated UV-Vis spectrophotometric method for

quantifying paracetamol in tablet formulations. The method demonstrates good linearity,
accuracy, precision, specificity, LOD, and LOQ. It can be used for routine quality control analysis
of paracetamol tablets.

By following these steps, you can successfully perform the UV-visible spectrophotometric
determination and validation of Paracetamol tablet formulation.

REFERENCES

Chatwal. R,. Anand S. K, “Instrumental Methods of Chemical Analysis”, 5th Edn., Himalaya
Publishing House, New Delhi, 2002; 566-587, 624-639.