Identification point

The MS detection system can be such that the full mass spectrum is observed (at least five peaks) or just selected ions monitored (SIM) with three or four identification points. For some analyses, it may be necessary to use MS-MS" techniques [8]. In LC-MS, it is important to make sure that ionization of the compounds of interest has been achieved. For all of these approaches, the criteria for matching of the analyte with the standard should be established during validation studies. 

Tandem mass spectrometry is considered as necessary to provide unequivocal identification and quantification of analytes, as pointed out by Maralikova et al. [70], It is also clear that monitoring of a single transition could lead to false compound identifications [71]. This had been previously predicted in the 2002 document issued by the European Union Commission that contains detailed information about mass spectrometric identification of drug residues in foodstuffs [57], where a minimum of three so-called Identification Points (IPs) is required for compound identification. 

Multidimensional and multihyphenated techniques may become increasingly useful, particularly for the analysis of drugs in biological fluids where LC-GC interfacing has a great deal of promise with respect to sample cleanup and preparation timeJ GC-MS applications continue to grow in number from the qualitative structural identification point of view, for quantitative analysis that uses and for other... 

F. Hernandez, M. Ibanez, J.V. Sancho, O.L. Pozo, Comparison of different MS techniques combined with EC for confirmation of pesticides in environmental water based on the use of identification points. Anal. Chem., 76 (2004) 4349. 

These eriteria veiy mueh meet the ones reeently established in the EU guidelines [2], These eriteria ate based on the principle of identification points . One point is earned for each ion in the mass spectrum and/or for the precursor ion in a production MS-MS spectrum, and one-and-a-half point for a product ion in the MS-MS spectrum. For the confirmation of Group A (illegal) compounds four identification points are required, while for the confirmation of Group B (legal, but with maximum residue level, MRL) compounds three identification points are required. In addition, ion ratios must be within 20-30% of the ratios in the reference spectrum. 

If a number of narrow bands in one group are relatively close, the most clearly defined band for this absorption range is the one that dictates identification. An example of this principle is tolbutamide, spectrum no. 508. Identification point I is the most clearly defined band in the long-wave range 263 nm (not 273 nm), and point II is the considerably more clearly defined short-wave band in methanol at 228 nm. 

For qualification the principle of identification points is used (European Commission 2002). Identification points can be obtained from MS-data, but also from other sources of information. The following classification can be obtained. The flow scheme is presented in Figure 8.3 ... 

Identification The target compound is present in the analysed extract. At least three identification points are obtained ... 

Absence (below detection limit) No identification points are obtained using MS. 

Firstly the MS results are evaluated by comparing the relative intensities of the selected ion peaks. For every ion peak an identification point is obtained, when its relative intensity (compared to the base peak in %) measured in the sample does not deviate more than ((0.1 x Istd) +10)% from the relative intensity of the diagnostic ion in the external standard solution. Three identification points give a positive identification. If less than three ion peaks are available (due to sensitivity (S/N < 3) or absence of fragments (PAH)), additional identification points should be gathered using additional analytical evidence. The possibilities are given in Table 8.3. 

Table 8.3 gives the analytical possibilities but it is also possible to use additional evidence. Interpretation of environmental data is always a combination of data analyses, knowledge about the origin of the sample, knowledge on the behaviour of contaminants and processes that occur or may occur. This is also true for the interpretation of GC-MS analysis. As stated a component is identified if three identification points are obtained. If only one or two diagnostic ions are present additional identification points are necessary. An extra identification point is obtained if one of the following criteria is fulfilled ... 

Table 8.3 Examples of number of identification points (n=integer). Derived from European Commission...

Decision 2002/657/EC has set relative abundance criteria that are dependent on the relative intensities of two transitions (Table 6.2), and has also established an identification points (IPs) system in order to confirm organic residues and contaminants in live animals and animal products (Table 6.3). For instance, where the relative intensity of the confirmation MRM is >50%, the maximum permitted tolerance is 20% relative. Therefore, at 60% relative abundance, the acceptable range would be 48-72%. With regard to the assignment of IPs, one precursor ion and one transition product are assigned I IP and 1.5 IPs, respectively, from a low-resolution (unit mass) mass spectrometer. Therefore, one precursor and two transitions from a QqQ mass spectrometer earn a total of 4 IPs. For the confirmation of banned substances listed in group A in Council Directive 96/23/EC, a minimum of 4 IPs are required. For substances listed in the group B in the directive, a minimum of 3 IPs are mandatory. 

TABLE 6.3 Relationships between MS Data, Mass Accuracy, and Identification Points (IPs)... 

Schurmann A, Dvorak V, Crtlzer C, Butcher P, Kaufmann A, False-positive liquid chromatography/tandem mass spectro-metric confirmation of sebuthylazine residues using the identification points system according to EU Directive 2002/657/EC due to a biogenic insecticide in tarragon, Rapid Commun. Mass Spectrom. 2009 23 1196-1200. 

The decision limit (CCa) is defined in point 1.11 of the Annex to Commission Decision 2002/657/EC. The decision limit (CCa) means the limit at and above which it can be concluded with an error probability of a that a sample is non-compliant. During a screening process, a substance detected above the CCP must be declared suspect (or screening non-compliant). In some laboratories, the CCa is used as a threshold to establish the suspicion, especially for forbidden substances. During a confirmatory process, a substance detected above the CCa must be declared non-compliant, on the condition that the appropriate identification criteria are fulfilled, for example, using the identification point system described in Commission Decision 2002/657/EC and CAC/GL 71-2009. ... 

However, although high chromatographic resolution is one of the features of utmost importance in food analysis and other complex matrices by LC-MS, the identification and confirmation of an analyte can also be carried out by complanen-tary identification points described in the EU directive 2002/657/EC [53], by nsing other analytical approaches. 

Table 9.1 Identification points (IPs) assigned (European Commission 2002) for different SIM/MRM monitoring techniques for chromatography, subject to satisfaction of relative abundance criteria (Table 9.2) ...

Mass Spectrometric Technique Identification Points per Ion Monitored... 

Another positive aspect was the explicit concern for confirmation of analyte identity that used the identification points (IPs) approach (European Contunission 2002, see discussion of Tables 9.1-9.3 in Section 9.3.3b). For low resolution MS/MS detection using an ion trap as was done here, one IP is awarded for the m/z selected precursor ion and 1.5 IPs for each MRM transition monitored, with three IPs required (European Commission 2002) for acceptable confirmation of analyte identity. To qualify for the IPs, at least one ion abundance ratio for two MRM product ions must be measured and fall within tolerance intervals of contemporary values measured for an authentic analytical standard, defined (European Commission 2002) as 20 % for a relative abundance of >50 %, to 25 % for 10-50 % and to 50 % for < 10 %, although the US FDA recommends (FDA 2003) that these tolerances should be within 10% regardless of the absolute values of the relative abundances. The present work (Yang 2006) satisfied both tolerance criteria over the calibration range, although it was considered unlikely that they could be satisfied at the MDL. 

A QqQtiap mass spectrometer (Section 6.4.6) was used in conventional triple quadrupole MRM mode for routine quantitation, but in some experiments this was combined using the information dependent acquisition (IDA) software facility function with the ion trap function for identification of unknowns and/or confirmation of target analytes. Only one MRM transition was monitored for each analyte, thus gaining only 2.5 identification points rather than the specified three (European Commission 2002, see Table 9.1) this was undoubtedly a consequence of the large number of analytes monitored in this multiresidue analysis. A retention time within 5 % of the standard value determined for pure analytes in distilled water was also a required criterion (Hua 2006) for confirmation of identity. 

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