Division 2.3.3.2 Mass spectrometric detection
United Kingdom
Mass-spectrometric detection shall be carried out by employing MS-techniques such as recording of full mass spectra (full scans) or selected ion monitoring (SIM), as well as MS-MSn techniques such as Selected Reaction Monitoring (SRM), or other suitable MS or MS-MSn techniques in combination with appropriate ionisation modes. In high-resolution mass spectrometry (HRMS), the resolution shall typically be greater than 10000 for the entire mass range at 10 % valley.
Full scan: When mass spectrometric determination is performed by the recording of full scan spectra, the presence of all measured diagnostic ions (the molecular ion, characteristic adducts of the molecular ion, characteristic fragment ions and isotope ions) with a relative intensity of more than 10 % in the reference spectrum of the calibration standard is obligatory.
SIM: When mass spectrometric determination is performed by fragmentography, the molecular ion shall preferably be one of the selected diagnostic ions (the molecular ion, characteristic adducts of the molecular ion, characteristic fragment ions and all their isotope ions). The selected diagnostic ions should not exclusively originate from the same part of the molecule. The signal-to-noise ratio for each diagnostic ion shall be ≥ 3:1.
Full scan and SIM: The relative intensities of the detected ions, expressed as a percentage of the intensity of the most intense ion or transition, shall correspond to those of the calibration standard, either from calibration standard solutions or from spiked samples, at comparable concentrations, measured under the same conditions, within the following tolerances:
Table 4
Maximum permitted tolerances for relative ion intensities using a range of mass spectrometric techniques
| Relative intensity (% of base peak) | EI-GC-MS (relative) | CI-GC-MS, GC-MSn LC-MS, LC-MSn (relative) |
|---|---|---|
| > 50 % | ± 10 % | ± 20 % |
| > 20 % to 50 % | ± 15 % | ± 25 % |
| >10 % to 20 % | ± 20 % | ± 30 % |
| ≤ 10 % | ± 50 % | ± 50 % |
Interpretation of mass spectral data: The relative intensities of the diagnostic ions and/or precursor/product ion pairs have to be identified by comparing spectra or by integrating the signals of the single mass traces. Whenever background correction is applied, this shall be applied uniformly throughout the batch (see 2.3.1, paragraph 4) and shall be clearly indicated.
Full scan: When full scan spectra are recorded in single mass spectrometry, a minimum of four ions shall be present with a relative intensity of ≥ 10 % of the base peak. The molecular ion shall be included if it is present in the reference spectrum with a relative intensity of ≥ 10 %. At least four ions shall lie within the maximum permitted tolerances for relative ion intensities (Table 5). Computer-aided library searching may be used. In this case, the comparison of mass spectral data in the test samples to that of the calibration solution has to exceed a critical match factor. This factor shall be determined during the validation process for every analyte on the basis of spectra for which the criteria described below are fulfilled. Variability in the spectra caused by the sample matrix and the detector performance shall be checked.
SIM: When mass fragments are measured using other than full-scan techniques, a system of identification points shall be used to interpret the data. For the confirmation of substances listed in Group A of Annex I of Directive 96/23/EC, a minimum of 4 identification points shall be required. For the confirmation of substances listed in Group B of Annex I of Directive 96/23/EC, a minimum of 3 identification points are required. The table below shows the number of identification points that each of the basic mass spectrometric techniques can earn. However, in order to qualify for the identification points required for confirmation and the sum of identification points to be calculated:
a minimum of at least one ion ratio shall be measured, and
all relevant measured ion ratios shall meet the criteria described above, and
a maximum of three separate techniques can be combined to achieve the minimum number of identification points.
Table 5
The relationship between a range of classes of mass fragment and identification points earned
| MS technique | Identification points earned per ion |
|---|---|
| Footnotes: | |
(1) Each ion may only be counted once. | |
(2) GC-MS using electron impact ionisation is regarded as being a different technique to GC-MS using chemical ionisation. | |
(3) Different analytes can be used to increase the number of identification points only if the derivatives employ different reaction chemistries. | |
(4) For substances in Group A of Annex 1 to Directive 96/23/EC, if one of the following techniques are used in the analytical procedure: HPLC coupled with full-scan diode array spectrophotometry (DAD); HPLC coupled with fluorescence detection; HPLC coupled to an immunogram; two-dimensional TLC coupled to spectrometric detection; a maximum of one identification point may be contributed, providing that the relevant criteria for these techniques are fulfilled. | |
(5) Transition products include both daughter and granddaughter products. | |
| Low resolution mass spectrometry (LR) | 1,0 |
| LR-MSn precursor ion | 1,0 |
| LR-MSn transition products | 1,5 |
| HRMS | 2,0 |
| HR- MSn precursor ion | 2,0 |
| HR-MSn transition products | 2,5 |
Table 6
Examples of the number of identification points earned for a range of techniques and combinations thereof (n = an integer)
| Technique(s) | Number of ions | Identification points |
|---|---|---|
| GC-MS (EI or CI) | N | n |
| GC-MS (EI and CI) | 2 (EI) + 2 (CI) | 4 |
| GC-MS (EI or CI) 2 derivatives | 2 (Derivative A) + 2 (Derivative B) | 4 |
| LC-MS | N | n |
| GC-MS-MS | 1 precursor and 2 daughters | 4 |
| LC-MS-MS | 1 precursor and 2 daughters | 4 |
| GC-MS-MS | 2 precursor ions, each with 1 daughter | 5 |
| LC-MS-MS | 2 precursor ions, each with 1 daughter | 5 |
| LC-MS-MS-MS | 1 precursor, 1 daughter and 2 granddaughters | 5,5 |
| HRMS | N | 2 n |
| GC-MS and LC-MS | 2 + 2 | 4 |
| GC-MS and HRMS | 2 + 1 | 4 |