Method Validation Workflow #

1. pre-validation testing
   It could also be called method development

2. Validation
   determine accuracy, imprecision, sensitivity, linearity, interferences (specificity), matrix effects, and carryover

Order of Experiments #

1.  linearity, within-run imprecision, blank matrix, LLOQ
2.  matrix effect, recovery, interferences
3.  accuracy: certified reference materials, human samples, proficiency testing samples
4.  stability studies
5.  every run should include between-run imprecision samples
6.  carryover, dilutions

Accuracy #

evaluation of agreement between LC-MS/MS test results and true value.

Guidelines: FDA guidelines, CLSI EP9, CLSI EP15, CLSI C62

Imprecision #

evaluation of agreement between repeated analysis of same sample.
within-run imprecision
between-run imprecision

Sensitivity #

1.  depicted by lower limit of quantitation (LLOQ) of a method
2.  lowest concentration that can be reproducibly measured
3.  determined by the imprecision: < 20% between-run imprecision at LLOQ (at minimum use 20 matrix-based replicates over 20 days)
4.  determined by signal-to-noise ratio: >20:1 at LLOQ is recommended; 10:1 minimum
5.  ion ratios still have to pass

Linearity #

ability of LC-MS/MS method to provide counts per second values that are directly proportional to concentration of analyte

should carry out linearity experiment for every matrix that will be acceptable for a given method

do not use serial dilutions to create material for linearity experiments