Prepared By: Shruti Vij (Senior Analyst) , Geeta Mathur(Senior Scientist) ,Khushbu ( Analyst) This slide show contains detailed explanation of three characteristics of method validation- Limit of detection, Quantitation limits and Robustness. Limit of detection is the minimum amount of substance that can be detected but not measured, quantitation limit is the minimum amount of substance which can be detected and measured. Common approach to these procedures- signal to noise ratio has also been covered. Robustness is a characteristic which determines a method’s reliability when deliberate variations are induced in parameters.

1. METHOD VALIDATION
Prepared By:
Sigma Test & Research Centre
Shruti Vij (Senior Analyst)
Geeta Mathur (Senior Scientist)
Khushbu ( Analyst)

2. Ratio of the height of the analyte to the
height of the noise measured on a blank
OR
S/N ratio is a measure that compares the
level of a desired signal to the level of a
background noise

4. Limit of Detection
(LOD)

5. It can be defined as the
lowest amount of the
analyte in a sample that
can be detected but not
necessarily quantified.

6. How is it determined ?

7. 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

8. 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.

9. LOD can be determined as a signal to
noise ratio ≥ 3
Where S=Height of Signal
N=Height of Noise

10. QUANTITATION
LIMIT
(QL OR LOQ)

11. Minimum amount of analyte in a
sample that can be quantified
with acceptable precision and
accuracy under the stated
operational conditions of the
method.

12. How is it determined ?

13. 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

14. 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.

15. LOQ can be determined as a signal to
noise ratio ≥ 10
Where S=Height of Signal
N=Height of Noise

16. LOQ can also be calculated
by this formula:-
LOQ = 3.3 x LOD

17. THANK YOU

18. ROBUSTNESS
/RUGGEDNESS

19. CAPACITY OF A METHOD TO REMAIN
UNAFFECTED BY SMALL DELIBERATE
VARIATIONS IN METHOD PARAMETER

20. THE EVALUATION OF ROBUSTNESS
SHOULD BE CONSIDERED DURING THE
DEVELOPMENT PHASE AND DEPENDS
ON THE TYPE OF PROCEDURE UNDER
STUDY

21. 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

22. Why should we do Robustness Parameter
?

23. It is a Series Of System
Parameter Which is Established
To Ensure That, The Validity Of
Analytical Procedure is
Maintained Whenever it is Used.

24. THANK YOU