MOOC: Validation of liquid chromatography mass spectrometry (LC-MS) methods (analytical chemistry) course

10.1 Robustness and ruggedness relation to LC-MS method development

Evaluation of and is very important in the case of LC-MS, as there are large number of system parameters, some of which are difficult to control. As a result, some of the key performance characteristics are also difficult to control or are sensitive to small changes in system parameters (or sample properties), resulting in poor instrument between the runs (between samples with formally identical matrix).

Furthermore, LC-MS is very often used for the determination of very low levels of analytes in highly complex matrices. Analysis of complex matrices often requires complex multi-step sample preparation procedures, which contribute to the overall complexity of the method.

Robustness and ruggedness relation to LC-MS method development

http://www.uttv.ee/naita?id=23687

https://www.youtube.com/watch?v=qM0R4OGYI5E&feature=youtu.be

As to which parameters should be varied during robustness studies, the guidelines vary in their recommendations []. In this course we recommend the following list of method parameters and changes in these parameters, that should be investigated during robustness studies:

Table 1. Recommended method parameters to be investigated during robustness studies.

Parameter (1)	Likelihood of uncontrollable change (2)	Possible extent of variation (3)	Comments
Liquid chromatography
Mobile phase pH	Medium	± 0.5 units	pH will have a strong effect on the retention time (and possibly resolution) if the analyte’s pK_a value is within ± 1.5 units of the mobile phase pH
Concentration of additives in the eluent	Medium	± 10% (relative)	Salts, ion-pair reagents, modifiers can suppress/enhance analyte’s ionization in the ion source and change its retention time and possibly resolution from other compounds
Organic solvent content in the eluent	Low to Medium	± 2% (relative)	Organic solvent content influences the retention time (and possibly resolution) and analyte signal in LC-MS
Column temperature	Low	± 5 °C	Column temperature influences the retention time (and possibly resolution)
Eluent flow rate	Low	± 20%	Eluent flow rate influences the retention time (and possibly resolution)
Column batch and age	Medium	–	Changes in the column can influence the retention time (and possibly resolution) and peak shape. There can be also influence on the ionization: because of different retention time there can be different co-eluting compounds and, consequently, different ionization suppression/enhancement.
Samples and sample preparation
Analyte extraction time; solvent amount and composition (in liquid/liquid and solid phase extraction, etc)	High	± 20%	Influences and /
Injection solvent composition	Low/High	± 10% (relative)	This is the solvent in which the analyte is taken up during the last stage of the sample preparation. This composition can influence the retention time and recovery and therefore also the matrix effect (in broad sense), LoQ/LoD and . The effect can be very serious in the case of UHPLC (4) and is usually not that critical in the conventional HPLC.
Matrix effect in broad sense (sample matrix source)	High	6 different sources	Can be assessed under studies. Influences (recovery and ionization suppression), LoQ/LoD
Mass spectrometry
Drying gas temp	Low	± 10 °C	Drying gas temperature can influence the analyte ionization efficiency in the ion source
Nebulizer gas pressure/flow rate	Low	± 5 psi / ± 1 L/min	Nebulizer gas pressure/flow rate can influence the analyte ionization efficiency in the ion source
Ion source configuration (Nebulizer position)	High (if configurations can vary) Not applicable (if fixed source)	According to the ion source design. Should be varied if the source is used in different configurations.	Ion source configuration can influence the spray and ionization efficiency in the ion source
Ion source condition (Nebulizer aging, ion source contamination)	High	After analysis of samples versus cleaned system	Contamination can spontaneously accumulate when analyzing a series of samples.

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(1) This list is not exhaustive, there can be different other parameters that, depending on the situation, could be changed.

(2) This grading is generic, corresponding to most common situations. In specific cases the grading can be different.

(3) These extents of parameter variation can be considered as possible defaults. In practice different extents can be used, but they should be realistic.

(4) In the case of UHPLC the smaller changes in the injection solvent have stronger influence, especially if larger injection volume is used.

10.1_robustness_and_ruggedness_relation_to_lc-ms_method_development.pdf