Validation of liquid chromatography mass spectrometry (LC-MS) methods
2.4. Introduction to identity confirmation
Confirmation of the analyte’s identity is closely related to the assessment of the method. Validation guidelines usually discuss these topics together and it might be confusing. In case of a selectivity assessment, we know where the analyte peak is, or if it is missing. In case of an , the problem is reversed – there is a peak on the chromatogram, and the analyst must provide evidence that this peak belongs to the analyte. The information required to collect such evidence is obtained during a validation.
The following evidence is used in chromatographic methods for confirming the identy of an analyte:
- Chromatographic retention time. The basic requirement in chromatography is that the retention time of the compound must be the same in the standard solution and in the sample. But similar retention time alone does not confirm the identity of the analyte as there are potentially many compounds (sample constituents) that may elute at the same (or very similar) retention time.
- Spectrometric methods used for detection can provide evidence that the detected compound is the same as the analyte. UV-Vis and fluorescence detectors are classical spectrometric LC detectors, which provide some information about the structural features of compounds, but UV-Vis and fluorescence spectra are usually not sufficiently characteristic to the analytes for their positive identification. Mass spectrometric techniques, especially high resolution MS and tandem MS (MS/MS), are the most powerful methods for identity confirmation.
Most of the validation guidelines consider LC and some recognize the power of MS for identity confirmation. In contrast, ICH is more general and summarizes general requirements instead of considering specific techniques (LC, spectrophotometry, etc). In order to demonstrate that the developed method is able to discriminate between compounds of closely related structures, positive results must be obtained for samples containing the analyte and negative results for samples which do not. Also, negative results must be demonstrated for compounds, which are structurally similar or closely related to an analyte. Proper selection of potentially interfering compounds requires scientific judgement, experience and knowledge.
The following two sections look in more detail at the identity confirmation via retention time and MS, respectively.
