4. Calibration and Validation

T/F? The sensitivity of an analytical method is best represented by the limit of detection. (1.3b)

F; best represented by slope of calibration

T/F? For standard addition calibration, it is best to add the deuterated form of an analyte in increasing amounts for each successive calibration point. (1.3f+3.3f)

F; (deuterated form = replace H with D) this is used for internal standard calibration

It should be adding more of the analyte itself, not its deuterated form

In a comprehensive method validation, explain how one should determine and report PRECISION for an analytical method. (1.4a)

Measure high, medium, and low concentration fortified (spiked) matric-matched samples in triplicate to quintuplicate. Report % RSD at each concentration level.

Formula:

Standard deviation / Average = % RSD

In a comprehensive method validation, explain how one should determine and report ACCURACY for an analytical method. (3.4a)

Measure high, medium, and low concentration fortified (spiked) matric-matched samples in triplicate to quintuplicate. Report the average results and compare with prepared amount.

Formula:

((Value measured - Value prepared) / Value prepared) * 100% = % error in accuracy

What are some common requirements of a good internal standard? (1.4c+6.3)

• Available in high purity

• Inert (unreactive) 

• Behaves similarly to analyte (physiochemically)

• Not present in sample

• Easily detected and differentiated from analyte

What is the difference between external standard and internal standard calibration? Why is an internal standard calibration often preferred? (2.2)

For external standard calibration, a series of standard samples of the analyte are prepared at different concentrations and analyzed. We relate analyte response to analyte concentration to build a calibration curve. 

For internal standard calibration, we do the same as above, but a different compound which is similar to the analyte is incorporated in a fixed concentration in all calibration standards, quality control sampler, and unknown samples. All measured instrumental responses for analyte are normalized by the response of the internal standard. These normalized responses are plotted against analyte concentration to create the calibration curve. 

I.S. calibration is preferred bc it provides better precision. Many systematic error can be accounted with the IS procedure.

What is an internal standard, what are three requirements for a good internal standard, and how is an internal standard calibration and analysis performed? (4.2)

An internal standard is a chemical compound which is different than the target analyte but is added to solutions analyzed for quantitative analysis. The IS is added to all calibrations, controls, and unknowns at a consistent, fixed concentration.

The IS must be:

• Available in high purity

• Inert (unreactive) 

• Behaves similarly to analyte (physiochemically)

• Not present in sample

• Easily detected and differentiated from analyte

A calibration is performed using solutions containing different amounts of analyte and a fixed concentration of IS. The response of analyte/response of IS is plotted on the y-axis vs analyte concentration on the x-axis. The analyte and IS response in the unknown sample is measured and the calibration curve is used to determine analyte concentration in the unknown.

When validating an analytical method, how are accuracy and precision typically determined? (4.3)

Sample matrix matched standards are prepared at low, medium, and high concentrations for target analyte. The samples are measured 3-5 times. 

The % RSD (Standard deviation / Average = % RSD) is determined to report precision at each concentration level. 

The % error in accuracy (= ((Value measured - Value prepared) / Value prepared) * 100%) is determined to report accuracy at each concentration level.

What is one advantage and one disadvantage of performing a standard addition calibration (5.2)

Advantage:

Calibrate in the actual matrix solution of the sample; best possible match of background matrix for calibration, in case of matrix effects

Disadvantages:

- No presence of internal standard to improve precision.

- Need a larger amount of sample if separate sample/calibration solutions are prepared for each standard addition