Method Validation

Calibration

determines the relationship between the analytical response (typically of instrumentation) and the analyte concentration

analytical response

the measurable physical, chemical, or electrical signal produced by an analytical instrument (such as peak area, peak height, absorbance, or fluorescence intensity) when it interacts with a target analyte

analyte concentration

the known amount or concentration of an analytes (the target substance being measured) in standard solutions used to establish a quantitative relationship between an instrument's signal response and actual chemical presence

External standard calibration, standard addition method, internal standard calibration

Types of several possible strategies of calibration

External Standard Calibration

a series of standard solutions is prepared separately from the sample, and is measured for their response

Calibration curve of external standard calibration

typically a linear relationship, regression analysis established using the least-squares method

matrix of the standards behaves identically to the sample matrix

Assume what in external standard calibration curve

Ideal blank

an identical solution to the sample without the analyte (External standard calibration)

solvent blank

contains the same solvent in which the sample is dissolved (External standard calibration)

Reagent blank

solvent + all the reagents used in sample preparation (External standard calibration)

Standard Addition Method

Used when the matrix affects the instrumental response

Matrix effect

a change in the analytical signal of a target substance (analyte) caused by all other components (the matrix) present in a sample

Spiking

Known amouunts of standard analyte are added directly to aliquots of the sample matrix

Internal Standard Calibration

A known amount of a reference compound (distinct from the analyte, but chemically similar) is added to all standards, blanks, and unknown samples

Sensitivity

refers to an instrument or method' s ability to demonstrate a measurable change in response for a given change in analyte concentration

calibration sensitivity

change in the measured quantity per unit change in analyte concentration (slope of calib. curve)

analytical sensitivity

the ratio of the calibration curve slope to the standard deviation of the analytical signal at a given analyte concentration

specificity

focuses on how exclusive the method/instrument is to the particular analyte to be determined

Linearity

measures how well a calibration curve follows a straight line, showing that the response is directly proportional to the quantity of analyte

Accuracy

measures how close/faithful is the response of the analyte to a known accepted true value

Certified Reference materials

used to evaluate accuracy

Precision

measures how well replicate measurements agree with one another

Repeatablity

spread in results when one person uses one procedure to analyze the same sample by the same method multiple times

reproducibility

spread in results when different people in different labs using different instruments each try to follow the same procedure

Robustness

capacity of a method to remain unaffected by small, deliberate changes in internal operational parameters

Ruggedness

degree of reproducibility under normal operational variations between environments and personnel

limit of detection

lowest concentration of analyte that can be reliably detected, but not necessarily quantified

limit of quantification

lowest concentration of analyte that can be quantitatively determined with acceptable precision and accuracy