method validation - limit of detection, quantitation limits and robustness
TRANSCRIPT
METHOD VALIDATION
Prepared By:Sigma Test & Research Centre
Shruti Vij (Senior Analyst)Geeta Mathur (Senior Scientist)
Khushbu ( Analyst)
Ratio of the height of the analyte to the height of the noise measured on a blank
ORS/N ratio is a measure that compares the level of a desired signal to the level of a
background noise
Signal To Noise Ratio
Signal to noise ratio
Limit of Detection (LOD)
It can be defined as the lowest amount of the analyte in a sample that can be detected but not necessarily quantified.
How is it determined ?
LOD
Detection limit is determined by the analysis of sample with
known concentration of analyte and by establishing the
minimum level at which the analyte can be reliably detected
Based on visual approach
S/N ratio is performed by comparing measured signals from samples with
known low concentration of analyte with those of blank samples and by establishing the minimum concentration at which the
analyte can be reliably detected. A S/N ratio between 3:1 or 2:1 is generally
accepted.
Based on signal to noise approach
LOD can be determined as a signal to noise ratio ≥ 3
Where S=Height of Signal N=Height of Noise
QUANTITATION LIMIT
(QL OR LOQ)
Minimum amount of analyte in a sample that can be quantified with acceptable precision and
accuracy under the stated operational conditions of the
method.
How is it determined ?
LOQ
Quantitation limit is determined by the analysis of sample with known
concentration of analyte and by establishing the minimum level at which the analyte can be reliably
quantitated
Based on visual approach
S/N ratio is performed by comparing measured signals from samples with
known low concentration of analyte with those of blank samples and by establishing the minimum concentration at which the
analyte can be reliably quantitated.
A S/N ratio ≥ 10 is accepted.
Based on signal to noise approach
LOQ can be determined as a signal to noise ratio ≥ 10
Where S=Height of Signal N=Height of Noise
LOQ can also be calculated by this formula:-
LOQ = 3.3 x LOD
THANK YOU
ROBUSTNESS /RUGGEDNESS
CAPACITY OF A METHOD TO REMAIN
UNAFFECTED BY SMALL DELIBERATE
VARIATIONS IN METHOD PARAMETER
DEFINITION
THE EVALUATION OF ROBUSTNESS
SHOULD BE CONSIDERED DURING THE
DEVELOPMENT PHASE AND DEPENDS
ON THE TYPE OF PROCEDURE UNDER
STUDY
Cont….
1) FLOW RATE: It can be adjusted by as much as 50%
2) TEMPERATURE: It can be adjusted by as much as 10%
3) PH OF MOBILE PHASE: PH of an aqueous buffer used in the preparation of the mobile phase can be adjusted to within 0.2 units of the volume of range specified
EXAMPLES
Why should we do Robustness Parameter
?
It is a Series Of System
Parameter Which is Established
To Ensure That, The Validity Of
Analytical Procedure is
Maintained Whenever it is Used.
THANK YOU