INTRODUCTION:

Paracetamol is the active metabolite of Acetanilide and Phenacetin. Chemically, it is N-Acetyl-p-aminophenol having molecular formula C₈H₉NO₂. The word Acetaminophen comes from a chemical name for the compound, para-acetylaminophenol. In some contexts, it is simply abbreviated as APAP, for acetyl-para-aminophenol. Acetaminophen is a part of the class of drugs known as “aniline analgesics”; it is the only such drug still in use today^1,2. It is not considered as an NSAID because it does not exhibit significant anti-inflammatory activity (it is a weak COX inhibitor). This is despite the evidence that Acetaminophen and NSAIDs have some similar pharmacological activity^3,4.

Paracetamol also known as Acetaminophen is a widely used non-prescription over-the-counter analgesic (pain reliever) and antipyretic (fever reducer) agent. 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. It is a commonly used analgesic compound combined with centrally acting compounds such as caffeine, codeine and dextropropoxyphene as well as with oral decongestants in a variety of formulations for the relief of the symptoms of common cold, influenza, and sinusitis. In combination with opioid analgesics, paracetamol can also be used in the management of more severe pain such as post-surgical pain and in providing palliative care to cancer patients⁵. The onset of analgesia is approximately 11-30 minutes after oral administration of paracetamol, and its half-life is 1-4 hours. Though paracetamol is used to treat inflammatory pain, it is not generally classified as NSAID because it exhibits only weak anti-inflammatory activity^6,7.

While generally safe for use at recommended doses (500 mg per single dose and up to 4000 mg per day for adults), acute overdoses of paracetamol can cause potentially fatal liver damage and renal failure and, in rare individuals, a normal dose can do the same, the risk is heightened by alcohol consumption⁵. Paracetamol is quickly and completely absorbed from GIT and uniformly distributed throughout the body fluids. It almost shows 100% bio-availability^8,9. The mechanism of action involves the inhibition of cyclooxygenase (COX) and it is highly selective for COX-2. Metabolized and excreted through hepatic and renal routes respectively. Three metabolic pathways are notable that are glucuronidation, sulfation, and N-hydroxylation. Paracetamol can cross the placenta and excrete in breast milk^10,11.

Table 1: Paracetamol drug Profile^12-14

Common Name	Paracetamol/ Acetaminophen
Chemical Name	N-Acetyl-p-aminophenol/ p-Hydroxy-acetanilide
Structure
IUPAC Name	N-(4-hydroxyphenyl) acetamide
Molecular Formula	C₈H₉NO₂
Molecular Weight	151.2 g/mol
Description	White crystalline solid
Melting point	169 - 172°C
Boiling point	420°C
pH	5.5 – 6.5
Solubility	Soluble in water and alcohol.
Category	Analgesic, Antipyretic.
Dose	500 mg to 1 g every 4 to 6 hours, up to 4 g daily, in divided doses.
Storage	Store protected from light and moisture.

UV- Visible Spectroscopy is concerned with the study of the absorption of UV and visible radiations which ranges from 200-800 nm. Absorption of UV radiation causes the promotion of a valence electron from bonding to anti-bonding orbital’s¹⁵. The analyte concentration in the solution can be determined by measuring the absorbance at some wavelength and by applying Beer-Lambert’s Law. UV-Visible spectroscopy can be employed for both qualitative and quantitative analysis of chemical compounds¹⁶.

Beer’s Law:

According to this law, when a beam of monochromatic radiation is passed through a solution of absorbing species, the intensity of a beam of monochromatic light decreases exponentially witan h increase the in concentration of absorbing species.

-dI/dC α I

Lambert’s Law:

Lambert’s law states that the rate of decrease of intensity of monochromatic light with the thickness of the medium is directly proportional to the intensity of incident light.

-dI/dt α I

Beer-Lambert’s Law:

This law states that when a beam of monochromatic light is passed through a medium with an absorbing substance, the decreasing rate of the radiation intensity along with the thickness of the absorbing solution is proportional to the concentration of the solution and the incident radiation^17-19.

A = log (I₀/I) = εbC

Where, A = Absorbance

ε = Molar absorptivity (L/mol.cm)

b = Path length (cm)

C = Concentration (Mol/L).

An Assay is an investigative procedure for qualitatively assessing or quantitatively measuring the presence or amount or the functional activity of a target entity which can be a drug or biochemical substance or organic sample. Pharmaceutical manufacturers are required to follow strict regulatory guidelines and prove their products are “high quality, safe, effective, and free of contamination and defects.” Assays play an important role in this process by determining the concentration of a drug compared to its labelled amount^{20, 21}.

Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. The Assay of an absorbing substance may be quickly carried out by preparing a solution in a transparent solvent and measuring its absorbance at a suitable wavelength^22,23.

Single Component Analysis:

The analysis of a sample containing a single component can be carried out using one of the following methods^24-26

1. Using standard absorptivity value (IP method)

2. Calibration curve method

3. Single-point standardization

4. Double-point standardization.

Using Standard Absorptivity value:

This procedure is adopted by official compendia, e.g. Indian Pharmacopoeia, for stable substances that have reasonably broad absorption bands and which are practically unaffected by variation of instrumental parameters, e.g. slit width, scan speed. The use of standard A (1%, 1cm) or ε values avoids the need to prepare a standard solution of the reference substance to determine its absorptivity. The absorptivity value A (1%, 1cm) of a standard at a selected wavelength in n particular solvent is established and the concentration of the sample is determined by comparison with the standard value²⁷.

Using Standard Calibration Graph:

In this method, the absorbances of a number of standard solutions (typically 4-6) of the reference substance at concentrations encompassing the sample concentrations are measured and a calibration graph is constructed. The concentration of analyte in the sample solution is read from the graph as the concentration corresponding to the absorbance of the solution. If the absorbance values and concentrations bear a linear relationship, the regression line y = mx + c may be estimated by the method of least squares²⁸.

Where y = Absorbance of sample solution

m = Slope of a line

x = Concentration of sample solution

c = Intercept.

Assay limit as per IP: Paracetamol tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of Paracetamol, C₈H₉NO₂²⁹.

MATERIALS AND METHODS:

Materials:

1. Paracetamol API- Mfg. by Loba Chemie Laboratory Reagents and Fine Chemicals Pvt. Ltd.

2. Sodium Hydroxide Flakes- Mfg. by Loba Chemie Laboratory Reagents and Fine Chemicals Pvt. Ltd.

3. Paracetamol Tablets 500mg- Ten different brands of Paracetamol 500mg Tablets were purchased from the market and used as a sample.

4. Distilled Water.

Instruments:

1. UV-Visible double beam Spectrophotometer with matched quartz cells (1 cm)

2. Model- Shimadzu UV-1800

3. Analytical balance Model- ACZET CY 124C.

Selection of Methods:

The methods employed for this study are the UV-Visible Spectrophotometric methods.

1. Using standard absorptivity value (IP method)

2. Calibration curve method.

Determination of λmax of Paracetamol:

10 µg/ml of Paracetamol solution was scanned between 400nm to 200nm. The absorption maxima (λmax) were found to be at 257nm. 257nm λmax was used for the Calibration curve method (as shown in figure 1).

Figure 1: Absorption Maxima (λmax) of Paracetamol at 257nm.

Absorption maxima of Paracetamol in 0.1M sodium hydroxide solution was found to be at 257nm.

Preparation of 0.1M NaOH:

0.4g of Sodium Hydroxide was taken into a 100ml volumetric flask and dissolved in a sufficient amount of distilled water to produce 100ml. The followed concentration was 0.1M NaOH³⁰.

Assay procedure:

All brands of paracetamol tablets were assayed spectrophotometrically by using the following methods

1. Using standard absorptivity value (IP Method):

20 Tablets of Paracetamol from each brand were weighed and finely powdered by using a mortar and pestle. An accurately weighed quantity of powder equivalent to 75mg of Paracetamol was transferred to a 100ml volumetric flask, 25ml of 0.1M NaOH and 50ml of distilled water were added and mechanically shaken for 15 minutes, then diluted with a sufficient amount of distilled water to produce 100ml. The resulting solution was then filtered by passing through Whatman filter paper No. 41. 10ml of the filtrate was transferred to a 100ml volumetric flask and further diluted to 100ml with distilled water. Again to 10ml of the resulting solution, 10ml of 0.1M NaOH was added and diluted to 100ml with distilled water and mixed thoroughly. The UV Spectrophotometer was put at zero by running a baseline (between 400-200nm) using 0.1M NaOH solution as blank. The absorbance of each sample was determined at 257nm. The content of Paracetamol was calculated taking 715 as the specific absorbance at 257nm λmax of Paracetamol^31,32.

2. Calibration Curve Method:

Preparation of standard stock solution:

Standard stock solution of Paracetamol (100µg/ml) was prepared by dissolving 100mg of paracetamol pure powdered drug in 50ml of 0.1M NaOH solution and diluted to 100ml with distilled water. 10ml of the above solution was transferred to a 100ml volumetric flask and further diluted to 100ml with distilled water³³.

Preparation of standard dilutions:

6 standard dilutions were preparedfrom the above stock solution of 100 µg/ml by diluting 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, 1.0 ml, and 1.2 ml to 10 ml with 0.1M NaOH. The absorbance of 6 standard dilutions of concentrations 2 µg/ml, 4 µg/ml, 6 µg/ml, 8 µg/ml, 10 µg/ml, and 12 µg/mlwere determined against 0.1M NaOH solution as a blank. The calibration curve was plotted between absorbance vs concentration at 257nm³⁴.

Preparation of Sample solution:

20 tablets were weighed and finely powdered by using a mortar and pestle. An accurately weighed quantity of powder equivalent to 75mg of Paracetamol was placed in a 100ml volumetric flask, to which 25ml of 0.1M NaOH and 50ml of distilled water were added and shaken by mechanical means for 15 minutes, then diluted with sufficient amount of distilled water to produce 100 ml. The resulting solution was then filtered by passing it through Whatman filter paper No. 41. 10 ml of the filtrate was transferred to a 100ml volumetric flask and further diluted to 100ml with distilled water. Again to 10ml of the resulting solution, 10ml of 0.1M NaOH was added and diluted to 100ml with distilled water and mixed thoroughly. The UV Spectrophotometer was put at zero by running a baseline (between 400-200nm) using 0.1M NaOH solution as blank. The absorbance of each sample was determined at 257nm^35,36. The percentage content of paracetamol was calculated using a linear regression equation obtained from the standard calibration plot (as shown in figure 2).

Figure 2: Standard Calibration Curve of Paracetamol.

The standard calibration curve of series of standard dilutions of Paracetamol is plotted against absorbance and concentration in ppm.

RESULT AND DISCUSSION:

The assay of ten brands of paracetamol tablets manufactured by different manufacturers was performed in this study. The average weight and percentage content of paracetamol in different brands of paracetamol tablets were calculated and evaluated by using UV- Visible Spectroscopic methods.

The results obtained by the IP method are tabulated below in Table 2.

Table 2: Comparison of different brands of Paracetamol Tablets 500mg by IP method

Sr. No.	Brand Name	Label Claim (in mg)	Average Weight (in mg)	Percentage Content (in %)	IP Specifications (in %)	Inference
1.	B1	500	634.7	102	95.0-105.0	PASS
2.	B2	500	620.55	104.4		PASS
3.	B3	500	612.2	112		FAIL
4.	B4	500	636.6	104		PASS
5.	B5	500	606.05	104		PASS
6.	B6	500	845.75	94.13		FAIL
7.	B7	500	572.1	98		PASS
8.	B8	500	617.7	100		PASS
9.	B9	500	600.05	98.64		PASS
10.	B10	500	587.8	104.4		PASS

The percentage content of Paracetamol in various brands of Paracetamol Tablets is compared by using Standard Absorptivity Value (IP Method).

From the results obtained using the IP method, it was observed that eight out of ten brands of paracetamol tablets passed the assay since the percentage content of all of them are within the limit specified by the Indian Pharmacopoeia, while two brands failed where one of them contain below and one contains above the specified limit.

Figure 3: Comparison of various brands of Paracetamol tablets by IP method.

The chart shows the graphical representation of the comparison between results obtained by the standard absorptivity value method (IP Method) and calibration curve method.

Figure 3 shows the graphical representation of the comparison of the percentage content of paracetamol in various brands of paracetamol tablets by IP Method.

The percentage content of Paracetamol in different brands of Paracetamol tablets was successfully estimated by using standard absorptivity value (assay method given in IP) and by Calibration curve method. The results obtained by both methods were compared and tabulated in the following table (Table 3).

Table 3: Comparison of Assay Methods

Sr. No.	Brand Name	% Content by IP Method	% Content by Calibration Curve Method
1.	B1	102	100
2.	B2	104.4	97.33
3.	B3	112	105.3
4.	B4	104	97.33
5.	B5	104	97.33
6.	B6	94.13	88
7.	B7	98	92
8.	B8	100	93.33
9.	B9	98.64	92
10.	B10	104.4	97.33

The results obtained by Standard Absorptivity Value (IP Method) and Calibration Curve Method is compared.

According to the results obtained by both methods, there is a difference in the percentage content of paracetamol in paracetamol tablets obtained by two methods. IP method shows a slightly higher percentage content of paracetamol than the Calibration curve method.

It was observed that the difference between the assay of paracetamol tablets by two methods is in the trend as follows:

1. The IP method shows the results on a higher side than the calibration curve method.

2. The IP method shows higher percentage content of paracetamol than the calibration curve method by 3-5%.

Figure 4 Shows the comparison between results obtained by the standard absorptivity value method (IP Method) and calibration curve method.

Figure 4: Comparison of assay methods.

The chart shows the graphical representation of the comparison of the percentage content of Paracetamol in various brands of Paracetamol tablets by IP Method.

CONCLUSION:

From the above study, it was found that the branded formulations marketed by reputed pharmaceutical industries show that the percentage content of paracetamol in paracetamol tablets complies with the specifications given in Indian Pharmacopoeia. Some generic tablets also comply with the assay limit given in IP, however, some generic tablets did not comply with the assay limit as per IP. So this study suggests that there is a need for improvement related to regulations during the approval of pharmaceutical products.

This study also shows that there is a noticeable difference between the results obtained by the standard absorptivity value method i.e. IP method and the Calibration Curve Method. IP method shows a slightly higher percentage content of paracetamol in all brands of paracetamol tablets compared to the Calibration curve method.

DECLARATIONS:

Consent for publication:

All the authors approved the manuscript for publication.

Availability of data and material:

All required data is available.

Competing interests:

All authors declare no competing interests.

ACKNOWLEDGEMENT:

The authors are thankful to Divine College of Pharmacy, Satana, Nashik, Maharashtra, India for providing experimental facilities to carry out this work. I am sincerely thankful to my guide for providing such research scenario in the department of pharmaceutical chemistry, and for his valuable guidance, inspiration, encouragement and moral support throughout our entire work.

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Received on 06.02.2023 Modified on 22.04.2023

Asian J. Pharm. Ana. 2023; 13(3):155-161.

DOI: 10.52711/2231-5675.2023.00025

Vaishali N. Sonawane¹, Chaitali A. Yeola¹, Vijayraj N. Sonawane¹*,

Khemchand R. Surana¹, Dhananjay M. Patil²,Deepak D. Sonawane²

Vaishali N. Sonawane1, Chaitali A. Yeola1, Vijayraj N. Sonawane1*,

Khemchand R. Surana1, Dhananjay M. Patil2, Deepak D. Sonawane2

Vaishali N. Sonawane¹, Chaitali A. Yeola¹, Vijayraj N. Sonawane¹*,

Khemchand R. Surana¹, Dhananjay M. Patil²,Deepak D. Sonawane²