to perform analytical method validation of paracetamol tablets by uv-spectrophotometric method

Drug Regulation & Regulatory Authorities

Dept. Of Quality Assurance & Regulatory Affairs L. J. Institute of Pharmacy, Ahmedabad.

EXPERIMENT NO.: DATE:

AIM: To perform Analytical method validation of Paracetamol Tablets by UV-

spectrophotometric method.

REFERENCES:

1) ICH Q2 (R1) guidelines for validation of analytical procedures.

2) Indian Pharmacopoeia, 2010 vol. III, Govt. of India Ministry of health and Family

Welfare Published by The Indian Pharmacopoeia Commission , Ghaziabad; 1861-

1862.

REQUIREMETNS:

Apparatus: 100ml & 10ml volumetric flasks, pipettes, beakers.

Reagents: Paracetamol API, Paracetamol Tablet, Methanol, Distilled Water.

Equipments:

1) Shimadzu UV-1800

Sr. No.: A114550/08677

2) Shimadzu UV-1800

Sr. No.: A114549/08780

THEORY:

General Description of Paracetamol:

Name: Paracetamol INN or Acetaminophen USAN.

Dose: 500 mg.

Chemical name: N-Acetyl-p-aminophenol.

Structure:

Class: mild analgesic.

Use: 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. Though Paracetamol is

used to treat inflammatory pain, it is not generally classified as an NSAID because it

exhibits only weak anti-inflammatory activity.

Half-life: 1–4 hours.

Adverse effects: causes gastrointestinal problems or allergic skin reactions. Blood

dyscrasia (e.g. thrombocytopenia), methaemoglobinemia, and hemolytic anemia are very

rare.

Validation Of Analytical Methods

Validation of an analytical method is documented evidence which provide a high

degree of assurance that the given method will consistently produce a product meeting its

predetermined specifications and quality attributes.

Validation parameters as per ICH

1) Accuracy

The accuracy of an analytical method may be defined as the closeness of the test

results obtained by the method to the true value. It is the measure of the exactness of the

analytical method developed. Accuracy may often be expressed as percent recovery by the

assay of a known amount of analyte added. Accuracy may be determined by applying the

method to samples or mixture of excipients to which known amount of analyte have been

added both above and below the normal levels expected in the samples. Accuracy is then

calculated from the test results as the percentage of the analyte recovered by the assay.

Dosage form assays commonly provide accuracy within 3-5% of the true value. The ICH

documents recommend that accuracy should be assessed using a minimum of nine

determinations over a minimum of three concentration levels, covering the specified range

(i.e. three concentrations and three replicates of each concentration).

2) Precision

The precision of an analytical method is the degree of agreement among individual

test results when the method is applied repeatedly to multiple sampling of homogenous

samples. This is usually expressed as the standard deviation or the relative standard

deviation (coefficient of variation). Precision is a measure of the degree of reproducibility

or of the repeatability of the analytical method under normal operating circumstances.

Repeatability:

Repeatability can be defined as the precision of the procedure when repeated by

same analyst under the same operating conditions (same reagents, equipments, settings



and laboratory) over a short interval of time. Repeatability is also termed as intra-assay

precision.

The ICH documents recommend that repeatability should be assessed using:

a) A minimum of 9 determinations covering the specified range for the procedure (e.g., 3

concentrations/3 replicates each); or

b) A minimum of 6 determinations at 100% of the test concentration.

It is normally expected that at least six replicates be carried out and a table showing each

individual result provided from which the mean, standard deviation and co-efficient of

variation should be calculated for set of n values.

The RSD values are important for showing degree of variation expected when the

analytical procedure is repeated several times in a standard situation. (RSD below 1% for

bulk drugs and RSD below 2% for assays in finished product).

Intermediate precision:

Intermediate precision expresses within-laboratories variations: different days,

different analysts, different equipment, etc.

The extent to which intermediate precision should be established depends on the

circumstances under which the procedure is intended to be used. The applicant should

establish the effects of random events on the precision of the analytical procedure. Typical

variations to be studied include days, analysts, equipment etc. It is not considered

necessary to study these effects individually. The use of an experimental design (matrix) is

encouraged.

Reproducibility:

Reproducibility means the precision of the procedure when it is carried out under

different conditions-usually in different laboratories-on separate, identical samples taken

from the same homogenous batch of material. Comparisons of results obtained by

different analysts, by the use of different equipments, or by carrying out the analysis at

different times can also provide valuable information.

3) Linearity

The linearity of an analytical procedure is its ability (within a given range) to

obtain test results which are directly proportional to the concentration (amount) of analyte

in the sample.

A linear relationship should be evaluated across the range of the analytical procedure. It

may be demonstrated directly on the drug substance (by dilution of a standard stock



solution) and/or separate weighing of synthetic mixtures of the drug product components

using the proposed procedure.

Linearity should be evaluated by visual inspection of a plot of signals as a function of

analyte concentration or content. If there is a linear relationship, test results should be

evaluated by appropriate statistical methods, for example, by calculation of a regression

line by the method of least squares. The correlation coefficient, y-intercept, slope of the

regression line and residual sum of squares should be submitted. A plot of the data should

be included. In addition, an analysis of the deviation of the actual data points from the

regression line may also be helpful for evaluating linearity.

The linear range of detectability that obeys Beer’s law is dependent on the compound

analyzed and the detector used. The working sample concentration and samples tested for

accuracy should be in the linear range. The claim that the method is linear is to be justified

with additional mention of zero intercept by processing data by linear least square

regression. Data is processed by linear least square regression declaring the regression co-

efficient and b of the linear equation y= ax + b together with the correlation coefficient of

determination r. For the method to be linear the r value should be close to1.

For the establishment of linearity, a minimum of 5 concentrations is recommended. Other

approaches should be justified.

4) Range

The range of an analytical procedure is the interval between the upper and lower

concentration (amounts) of analyte in the sample (including these concentrations) for

which it has been demonstrated that the analytical procedure has a suitable level of

precision, accuracy and linearity.

The specified range is normally derived from linearity studies and depends on the intended

application of the procedure. The following minimum specified ranges should be

considered:

For the assay of a drug substance or a finished (drug) product: normally from 80 to 120%

of the test concentration;

For content uniformity, covering a minimum of 70 to 130% of the test concentration,

unless a wider more appropriate range, based on the nature of the dosage form (e.g.,

metered dose inhalers), is justified;

For dissolution testing: +/-20 % over the specified range;

e.g., if the specifications for a controlled released product cover a region from20%, after 1

hour, up to 90%, after 24 hours, the validated range would be 0-110% of the label claim.



5) Limit of Detection and Limit of Quantitation

Limit of Detection (LOD): - The limit of detection is the parameter of limit tests. It is the

lowest level of analyte that can be detected, but not necessarily determined in a

quantitative fashion, using a specific method under the required experimental conditions.

The limit test thus merely substantiates that the analyte concentration is above or below a

certain level.

Limit of Quantitation (LOQ): - Limit of quantitation is a parameter of quantitative

assays for low levels of compounds in sample matrices such as impurities in bulk drugs

and degradation products in finished pharmaceuticals.

The limit of quantitation is the lowest concentration of analyte in a sample that may be

determined with acceptable accuracy and precision when the required procedure is

applied. In many cases, the limit of quantitation is approximately twice the limit of

detection.

Several approaches for determining the detection limit are possible, depending on whether

the procedure is a non-instrumental or instrumental.

I. Based on Visual Evaluation

Visual evaluation may be used for non-instrumental methods but may also be used

with instrumental methods. The detection limit is determined by the analysis of

samples with known concentrations of analyte and by establishing the minimum

level at which the analyte can be reliably detected.

II. Based on Signal-to-Noise

The determination of the limit of detection of instrumental procedures is carried

out by determining the signal-to-noise ratio by comparing test results from the

samples with known concentration of analyte with those of blank samples and

establishing the minimum level at which the analyte can be reliably detected. A

signal-to-noise ratio of 2:1 or 3:1 (For LOD) and 10:1(For LOQ) is generally

accepted. The signal-to-noise ratio is determined by dividing the base peak by the

standard deviation of all data points below a set threshold. Limit of detection is

calculated by taking the concentration of the peak of interest divided by three times

the signal-to-noise ratio.

III. Based on the Standard Deviation of the Response and the Slope

The detection limit may be expressed as:

LOD = 3 * σ / S

LOQ = 10 * σ / S



Where, σ = the standard deviation of the intercept

S = the slope of the calibration curve

6) Selectivity and Specificity

The selectivity of an analytical method is its ability to measure accurately and

specifically the analyte of interest in the presence of components that may be expected to

be present in the sample matrix.

If an analytical procedure is able to separate and resolve the various components of a

mixture and detect the analyte qualitatively the method is called selective. On the other

hand, if the method determines or measures quantitatively the component of interest in the

sample matrix without separation, it is said to be specific.

Hence one basic difference in the selectivity and specificity is that, while the former is

restricted to qualitative detection of the components of a sample, the latter means

quantitative measurement of one or more analyte.

Selectivity may be expressed in terms of the bias of the assay results obtained when the

procedure is applied to the analyte in the presence of expected levels of other components,

compared to the results obtained on the same analyte without added substances. When the

other components are all known and available, selectivity may be determined by

comparing the test results obtained on the analyte with and without the addition of the

potentially interfering materials. When such components are either unidentified or

unavailable, a measure of selectivity can often be obtained by determining the recovery of

standard addition of pure analyte to a material containing a constant level of the other

components.

7) Robustness and Ruggedness

Robustness: - The robustness of an analytical method is a measure of its capacity to

remain unaffected by small but deliberate variation in method parameters and provides an

indication of its reliability during normal usage.

The evaluation of robustness should be considered during the development phase and

depends on the type of procedure under study. It should show there liability of an analysis

with respect to deliberate variations in method parameters.

Ruggedness: - The ruggedness of an analytical method is the degree of reproducibility of

test results obtained by the analysis of the same samples under a variety of normal test

conditions such as different laboratories, different analysts, using operational and

environmental conditions that may differ but are still within the specified parameters of

the assay.



For the determination of ruggedness, the degree of reproducibility of test result is

determined as function of the assay variable. This reproducibility may be compared to the

precision of the assay under normal condition to obtain a measure of the ruggedness of the

analytical method.

PROCEDURE:

Linearity

Stock solution:

Stock solution (100g/ml) of PCM was prepared by dissolving 0.01 g PCM in 100 ml

volumetric flasks and completing the volume distilled water.

Selection of analytical wavelength for PCM:

The solution of PCM was prepared in Water at a concentration of 15 µg /ml from the stock

solution. It was scanned in the wavelength range of 200-400 nm. Maximum absorbance

was obtained at 245 nm. This analytical wavelength was selected for determination of

PCM.

For calibration curve:

An aliquots of stock solution of PCM (0.5, 1, 1.5,2, and 2.5 ml) were pipettes out in 10ml

volumetric flasks and further diluted to attain concentration of about 5, 10, 15, 20, 25

µg/ml.

Now plot the graph of Absorbance Vs Concentration. Find out the r2 value from the

Graph.

Accuracy

API: From the stock solution (100µg/ml) pipette out 1 ml of solution in 3 volumetric

flasks, further add 80,100 & 120% of solution from the stock solution to each test tube

respectively. Similarly 2 other sets are prepared.

Measure the absorbance of each set i.e. total 3 concentrations of 3 sets. Take the Mean of

each concentration of each set. Find out the % recovery, Standard Deviation & % Relative

Standard Deviation.

TABLETS: From the stock solution of PCM tablets (100µg/ml) pipette out 1 ml of

solution in 3 volumetric flasks, further add 80,100 & 120% of solution from the Std. stock

solution to each volumetric flasks respectively. Similarly 2 other sets are prepared.

Measure the absorbance of each set i.e. total 3 concentrations of 3 sets. Take the Mean of

each concentration of each set. Find out the % recovery, Standard Deviation & % Relative

Standard Deviation.



Precision

Repeatability: Select the middle concentration i.e. 15µg/ml and carry out the repeatability

by taking the absorbance of the solution 6 times.

Calculate the mean of the absorbance and find the Standard Deviation and % Relative

Standard Deviation.

Intraday precision: Prepare 3 concentrations from the stock solution (i.e. 5, 15 &

25µg/ml), make another 2 sets similarly. Measure the absorbance of each concentration of

each set at the time interval of 2 hours.

Calculate the Mean, Standard Deviation & % Relative Standard Deviation.

Interday precision: Prepare 3 concentrations from the stock solution (i.e. 5, 15 &

25µg/ml), make another 2 sets similarly. Measure the absorbance of each concentration of

each set for 3 days.

Calculate the Mean, Standard Deviation & % Relative Standard Deviation.

LOD & LOQ

LOD & LOQ are found by using the following equation.

LOD = 3.3 * σ / S

LOQ = 10 * σ / S

Where, σ = the standard deviation of the intercept

S = the slope of the calibration curve

Robustness and Ruggedness

Robustness: It is carryout by doing deliberate variation in method parameters is done (i.e.

change in wavelength). Absorbance of any one concentration (i.e. 15µg/ml) is measured at

2 different wavelengths i.e. 244.8&245.2 nm. And calculate the % Assay or % Drug

Recovery. Repeat the procedure three times.

Ruggedness: It is carried out by the analysis of the sample (i.e. 15µg/ml) under a variety

of normal test conditions such as different laboratories, different analysts, using

operational and environmental conditions that may differ but are still within the specified

parameters of the assay.

Instruments used:

1. Shimadzu UV-1800

Sr. No.: A114550/08677

2. Shimadzu UV-1800

Sr. No.: A114549/08780



Measure the Absorbance and calculate Mean, Standard Deviation, % Relative standard

deviation & % Assay.

CALCULATIONS:

Equations

Std. Deviation =

Where, ∑= Sum across the value

X= Number of values

X= mean or Average

n = Number of values

% RSD = Std. Deviation * 100 / Mean

Linearity:

Concentrations

(µg/ml) Set

1

Set

2

Set

3 Mean

Std.

Deviation

%

RSD

5 0.385 0.391 0.379 0.385 0.006 1.558

10 0.798 0.819 0.811 0.809 0.010 1.309

15 1.159 1.152 1.157 1.156 0.003 0.311

20 1.591 1.587 1.579 1.585 0.006 0.385

25 1.932 1.946 1.924 1.934 0.011 0.575

Mean % RSD 0.827

y = 0.0777x + 0.0069 R² = 0.9989

0

0.5

1

1.5

2

2.5

0 5 10 15 20 25 30

Ab

sorb

ance

Concentration(µg/ml)

Calibration curve of PCM (set 1)



Accuracy(Std. addition method):

Sr.

No. Concentration 1 2 3 Mean

Std.

Deviation % RSD

% Drug

recovery

1 80% 1.215 1.202 1.195 1.204 0.01015 0.84293 85.92 %

2 100% 1.295 1.327 1.326 1.316 0.01819 1.38248 84.62 %

3 120% 1.425 1.472 1.465 1.454 0.02536 1.74398 84.62 %

Mean of % RSD 1.32313

85.03 % Average of % Drug Recovery

y = 0.0776x + 0.0156 R² = 0.9986

0

0.5

1

1.5

2

2.5

0 5 10 15 20 25 30

Ab

sorb

ance



y = 0.0772x + 0.0126 R² = 0.9986

0

0.5

1

1.5

2

2.5

0 5 10 15 20 25 30

Ab

sorb

ance





Precision:

Repeatability (15µg/ml)

Sr. No. Absorbance

1 1.145

2 1.145

3 1.151

4 1.141

5 1.149

6 1.147

Mean 1.1463333

Std. Deviation 0.0035024

%RSD 0.3055289

Intraday Precision

Sr.

No.

Concentrations

(µg/ml) 1 2 3 Mean

Std.

Deviation % RSD

1 5 0.385 0.391 0.379 0.385 0.006 1.55

2 15 1.159 1.152 1.157 1.156 0.00360555 0.311

3 25 1.932 1.946 1.924 1.934 0.01113553 0.575

Mean % RSD 0.812

Interday Precision

Sr.

No.

Concentrations

(µg/ml) 1 2 3 Mean Std. Deviation % RSD

1 5 0.409 0.405 0.405 0.40633 0.002309401 0.56835

2 15 1.172 1.171 1.169 1.17067 0.001527525 0.13048

3 25 1.92 1.896 1.895 1.90367 0.014153916 0.74351

Mean % RSD 0.48078

LOD & LOQ

LOD = 3.3 * σ / S

= 3.3 * 0.0305 / 0.0775

= 1.298µg/ml

LOQ = 10 * σ / S

= 10 * 0.0305 / 0.0775

= 3.935µg/ml



Robustness & Ruggedness

Robustness (15µg/ml)

Wavelength(nm) Absorbance % Assay

244.8 1.154 98.4 %

245 1.156 98.63 %

245.2 1.153 98.29 %

Average % Assay 98.44 %

Ruggedness

1. Instrument 1: Shimadzu UV-1800, Sr. No.- A114550/08677

Sr. No. Absorbance % Assay

1 1.125 95.92 %

2 1.125 95.92 %

3 1.131 96.44 %

4 1.141 97.30 %

5 1.134 97.13 %

6 1.138 97.04 %


2. Instrument 2: Shimadzu UV-1800, Sr. No.- A114549/08780

Sr. No. Absorbance % Assay

1 1.123 95.76 %

2 1.123 95.76 %

3 1.119 95.41 %

4 1.149 97.98 %

5 1.149 97.98 %

6 1.132 96.53 %


RESULT:

Sr. No. Parameters Normal range Result

1 Linearity 0.999 0.998

2 Accuracy % RSD < 2% 1.32313

3

Precision

Repeatability

% RSD < 2%

0.3055289

Intraday 0.812

Interday 0.48078

4 LOD - 1.298 µg/ml

5 LOQ - 3.935 µg/ml

6 Robustness (% RSD < 2%) % Assay 98.44 %

7 Ruggedness (% RSD < 2%) % Assay 96.59 %

CONCLUSION:

The method was validated as per ICH Q2 R1 and from the result table, we conclude that

method is linear, precise, accurate, robust and rugged.

to perform analytical method validation of paracetamol tablets by uv-spectrophotometric method

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