I
Objective:
To provide a procedure for the Validation of the method used in the analysis for the trace level
determination of Efavirenz in Rinse samples by UV Spectroscopy.
II.
Scope:
This protocol applies to the In -house method (common method by UV Spectroscopy) used
For the trace level determination of Efavirenz
content in Rinse samples.
III.
Responsibility:
It is the responsibility of Quality Control department to carry out the Validation test for the
method as per the procedure mentioned in the protocol.
IV.
Method details:
Standard preparation: Weigh accurately about 10 mg of Efavirenz working standard in a
100 mL of volumetric flask. Dissolve and dilute to volume with methanol. Further dilute
10.0 mL of this solution to 100 mL with methanol.
Sample solution: Filter the sample through filter paper and analyze.
Instrument conditions:
Mode
: Spectrometric
Range
: 200 nm to 400 nm
Wavelength of detection : 247 nm
Measuring mode
: Absorbance
Procedure: Scan separately the standard and sample solutions between 200 and 400 nm using water
as blank. Measure the UV absorbance of standard and sample solutions at 247 nm.
Calculate the content in % taken by the formula:
Calculation:
Content of Efavirenz = Abs of sample x C/Abs of standard
C is the concentration of standard solution in %
Page 1 of 6
Weight of standard (grams) x 10 x 100
C (%) = -------------------------------------------------------100x 100
VI. Experimental design:
The following parameters should be considered for the Validation:
1.
Specificity
2.
Precision
3.
Limit of Detection
4.
Limit of Quantitation
5.
Linearity & Range
6.
Accuracy
7.
Ruggedness
1. Specificity
Standard preparation: Weigh accurately about 10 mg of Efavirenz working standard in a 100 mL
of volumetric flask. Dissolve and dilute to volume with methanol. Further dilute 10.0 mL of this
solution to 100 mL with methanol.
Blank: Methanol
Procedure: Scan separately standard preparation, and blank in the range of 200 to 400 nm.
Measure the absorption maxima of standard solution and measure the absorbance of blank solution
and blank swab at about 247 nm and record the spectrum.
Acceptance criteria
I.
The standard solution should have the absorption maxima at about 247nm.
II.
The absorbance at about 247nm of methanol (blank) should be less than 0.005.
2) Limit of Detection (LOD):
Determine the Limit of Detection of Efavirenz based on Signal to Noise ratio.
Derive the LOD concentration, which will give Signal to Noise ratio is between 3 to 5
Preparation of reference solution for LOD/LOQ: Prepare 10 ppm Efavirenz standard solution
as detailed in method details and use as reference solution for LOD/LOQ.
Page 2 of 6
Procedure:
1) Scan the reference solution and draw the Signal to Noise ratio of Efavirenz and consider
Signal to Noise ratio value is between 3 to 5 as Limit of Detection and find out the LOD
concentration in ppm considering the noise (h) =0.005
2) After finding the LOD concentration prepare the LOD solution and evaluate
the actual Signal to Noise ratio of LOD solution
3) Report the concentration and Signal to Noise ratio of Efavirenz obtained
from LOD solution.
Acceptance criteria:
The Signal to Noise ratio of Efavirenz should be between 3 to 5
3) Limit of Quantitation:
Determine the Limit of Detection of Efavirenz based on Signal to Noise ratio. Derive the
Limit of Quantitation level of Efavirenz based on the Concentration arrived for LOD which
should give Signal to noise ratio is between 10 and 15.
Preparation of LOQ solution: Prepare 3 to 3.5 times concentration to that of the
concentration of LOD solution using the stock solution. (Prepared in LOD study for reference
solution).
Procedure:
1) Calculate the Signal to Noise ratio of ratio from the LOQ solution.
2) Scan the LOQ solution in 6 replicates and calculate the % RSD of Efavirenz.
3) Report the concentration and Signal to Noise ratio and concentration of Efavirenz
obtained from LOQ solution.
Acceptance criteria: The Signal to Noise ratio of Efavirenz should be between 10 and 15 and
% RSD for the absorbance obtained from 247 nm from 6 replicates of LOQ solution should be
less than 5.0.
4) Linearity &Range
Standard stock solution preparation: Weigh accurately about 50 mg of Efavirenz WS in 50 mL
volumetric flask. Dissolve and dilute to volume with methanol.
Dilute the standard stock solution as follows to get various linearity level solutions.
Page 3 of 6
Preparation of Linearity level-1 solution: Prepare the solution in a concentration so as so to
obtain the concentration of LOQ concentration of Efavirenz (Note: Values can be taken from
LOQ study when the linearity study is performed followed by the LOQ study).
Preparation of Linearity level-2 solution: Pipette out 0.5 mL of standard stock solution in a
100 mL volumetric flask, dissolve and make up to the volume with methanol. (This solution
contains 5ppm of Efavirenz standard).
Preparation of Linearity level-3 solution: Pipette out 0.8 mL of standard stock solution in a
100 mL volumetric flask, dissolve and make up to the volume with methanol. (This solution
contains 8ppm of Efavirenz standard).
Preparation of Linearity level-4 solution: Pipette out 1.0 mL of standard stock solution in a
100 mL volumetric flask, dissolve and make up to the volume with methanol. (This solution
contains 10ppm of Efavirenz standard).
Preparation of Linearity level-5 solution: Pipette out 1.2 mL of standard stock solution in a
100 mL volumetric flask, dissolve and make up to the volume with methanol. (This solution
contains 12ppm of Efavirenz standard).
Procedure: Scan each level 1and Level 5 for 6 replicates and Level 2 to Level 4 for 3 replicates
in the range of 200nm to 400nm and measure the absorbance at 247 nm and record the spectrum.
Plot the linearity curve of Efavirenz working standard concentration (ppm) against the absorbance
at 247 nm and determine the correlation coefficient linearity curve.
Acceptance criteria: The correlation coefficient should be more than 0.995
Range:
Lower level: Scan the linearity solution of 1st level in replicates (6 times) in the range of 200nm to
400nm and measure the absorbance at 247 nm from 6 replicates
Upper level: Scan the linearity solution of 6th level in replicates (6 times) in the range of 200 nm to
400nm and measure the absorbance at 247 nm from 6 replicates.
Page 4 of 6
Acceptance criteria
The RSD of absorbance at 247nm from 6 replicates of standard solution should be less than
5.0% at each level.
5) Accuracy:
Preparation of Accuracy level-1 solution: Transfer a known quantity of standard stock
solution (prepare for linearity) which should gives to equal concentration of LOQ level on SS
plate having curved edges to hold the solution, wash the plate with methanol. Collect the
combined washings in a dish. And reconstitute to the known volume and carry out
determination.
Preparation of Accuracy level-2 solution: Transfer 0.5 mL of standard stock solution (prepare
for linearity) on SS plate having curved edges to hold the solution. Wash the plate with
methanol. Collect the combined washings in a dish. And reconstitute to 100 mL with methanol
and carry out determination.
Preparation of Accuracy level-3 solution: Transfer 1.0 mL of standard stock solution (prepare
for linearity) on SS plate having curved edges to hold the solution. Wash the plate with
methanol. Collect the combined washings in a dish. And reconstitute to 100 mL with methanol
and carry out determination.
Preparation of Accuracy level-4 solution: Transfer 1.2 mL of standard stock solution (prepare
for linearity) on SS plate having curved edges to hold the solution. Wash the plate with
methanol. Collect the combined washings in a dish. And reconstitute to 100 mL with methanol
and carry out determination.
Procedure: Prepare and scan each level, Level 2 and Level 4 for 3 replicates and Level 4 for 6
replicates(as part of precision) in the range of 200nm to 400nm and measure the
absorbance at Record the Spectrum.
Calculate the % recovery for each level by using the following formula.
Recovered absorbance of individual accuracy level
% Recovery = ----------------------------------------------------------------------------------- x 100
Mean absorbance obtained from the corresponding linearity level
Note: Report the lowest recovery value of accuracy study as the recovery factor of the method.
Page 5 of 6
Acceptance criteria: The % recovery should be between 75 to 115 % at each level
6) PRECISION:
Procedure: Calculate the % content of the Efavirenz using the absorbance values from each
preparation of Accuracy level-3 as per method details and calculate the %RSD for the results
obtained from 6 preparations of accuracy level-3 (100% concentration of standard=10 ppm).
Acceptance criteria
1. % RSD of the content of Efavirenz should not be more than 10.0 %
Ruggedness(Intermediate precision):
Carry out the precision study on a different day, with different chemist, using different lot of reagents
with freshly prepared standard and sample preparations and determine the reproducibility from 6
replicate determinations as detailed in precision study.
Acceptance criteria
The % RSD of the content of Efavirenz obtained from ruggedness study should not be more than 10.0%
The cumulative % RSD of content of Efavirenz obtained from ruggedness study and the precision study
should not be more than 15.0%.
Page 6 of 6