2.5. Identity confirmation: retention time

Retention time and peak shape of an analyte in a standard solution and in a sample must match (within some tolerance limit, see below). If they do, then the analyte may be present in the sample, but additional confirmation is still required. On the other hand, if the retention time or the peak shape differ, then the peak under question cannot be due to the analyte (assuming properly functioning LC system).

For a proper chromatographic separation from fast-eluting sample components (which are often abundant), the analyte’s retention time should be at least twice the dead time of the chromatographic system. This means that the retention factor k (aka capacity factor, k’) should be ≥ 1. This recommendation is general in chromatography and is specifically important in case of LC-MS, because the matrix effects (ionization suppression/enhancement) are most probable close to a dead time of the system (see sections 5.3 and 5.4).

According to the most recent SANTE/SANCO validation guideline, retention times of an analyte in a sample and in a standard solution must not differ more than by 0.1 min (1). This requirement is valid in the case of classical HPLC as well as UHPLC. The European Commission Decision 2002/657/EC specifies a tolerance criterion for an analyte retention time relative to the standard substance retention time. The relative retention time of an analyte in a standard solution must correspond to that in a sample within tolerance of 2.5%.

The shape of the chromatographic peak may also be used to evaluate identity of an analyte. If peaks in a standard solution and in a sample differ in shape, it may indicate that the compound detected in the sample is not the analyte or that some compound coelutes with an analyte. It is possible that the analyte peak is distorted in a sample because of the sample matrix. In order to avoid this, matrix matched standard solutions or isotopically labelled internal standard (ILIS) should be used. ILIS, although expensive, are rather common in LC-MS analysis. If ILIS are used, then the peak shape of an analyte and a respective ILIS shall match.

 

Identity confirmation: retention time

http://www.uttv.ee/naita?id=23631

https://www.youtube.com/watch?v=LWAeC2Xr2f0

 

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(1) The previous SANCO guidelines established the retention time tolerance as ± 0.2 min [ref 4].