Method Validation - Limit of Detection, Quantitation limits and Robustness

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