Self test 10.1 – Validation of liquid chromatography mass spectrometry (LC-MS) methods

1. Look at the list of compounds. Please indicate the compounds with increasing retention time in reversed phase stationary phase (C18) when the mobile phase pH is changed one unit from pH 10 to 11.

Compound	pKa
Bases
Pyrrolidine	11.27
Piperidine	11.12
2-methylpyridine	5.97
Acids
2-nitrophenol	7.17
2,5-dinitrophenol	5.15
3-nitroaniline	17.9

Pyrrolidine

Piperidine

2-methylpyridine

2-nitrophenol

2,5-dinitrophenol

3-nitroaniline

2. Indicate correct statements concerning influence of the changes in liquid chromatography.

Column batch and age can influence the retention time, resolution and possibly therefore also ionization.

Changes in eluent flow rate should be excluded in robustness tests since my LC-MS method is saved with fixed flow rate and therefore I do not need to evaluate the influence of this parameter.

Changes in pH have a high impact on method performance in if analyte’s pK_a value is very close to the eluent pH.

Changes in the salts and ion pair reagents concentrations do not have an effect to the method performance.

3. Indicate correct statements concerning influence of the changes in sample preparation.

Extraction time, amount of solvent used during the extraction and also the composition of the solvents in the sample preparation procedure influences recovery and LoQ/LoD.

Different matrices do not influence my method’s performance if I have evaluated matrix effects for my method.;

Injection solvent composition (the solvent used in the last step of sample preparation) do not influence my method’s performance, since I use the same solvent composition each and every time.

4. Indicate correct statements concerning influence of the changes in mass spectrometry.

Changes in the ion source cleanliness can affect method performance during the analytical run.

Ion source configuration can influence spray and ionization efficiency in the ion source.

Evaluation of the changes in the ion source configuration is not needed if the source is fixed.

Drying gas temperature does not influence my method performance.

Nebulizer gas pressure/flow rate can influence analyte ionization efficiency in the ion source.

5. Indicate the correct influence (Low, Medium, High) of the parameter to the method performance:

1. Liquid chromatography

Parameter	Change in the parameter	Influence
pH	- 1 unit
Organic solvent	+ 10%
Column temperature	+15 °C
Eluent flow rate	+ 20%

2. Mass spectrometry

Parameter	Change in the parameter	Influence
Drying gas temp	+ 10 °C
Nebulizer gas pressure/flow rate	+ 5 psi / + 1 L/min
Ion source configuration (Nebulizer position)	Variations in the configuration during the last usage