The correct identification of pesticide residues in food by mass spectrometry is a fundamental requirement for laboratories to produce accurate and valid results that protect consumers and support international food trade. One might expect that analysts using exactly the same model of mass spectrometer to analyse exactly the same pesticide compounds would use the same analyte identification criteria. Surprisingly, this is not the case because the guidelines in different regions are not harmonised. Each time the guidelines are updated is an opportunity to align the identification criteria. So, did the recent updating of the EU SANTE guidelines represent an opportunity missed or a step towards harmonisation?
The latest updated version of the EU SANTE guidelines (SANTE/11813/2017) for the Analytical Quality Control and Method Validation Procedures for Pesticides Residues Analysis in Food and Feed became effective on 1st January 2018. The document, which is updated every 2-3 years to reflect new analytical developments and advancements in technology, has become an essential reference document for laboratories in Europe and beyond. The latest version adopted changes to the criteria for identification of analytes using data acquired by employing High Resolution Accurate Mass (HRAM) Mass Spectrometry (MS), but not triple quadrupole MS, which remained unaltered. The updated EU SANTE guidelines and the U.S. FDA guidelines Acceptance Criteria for Confirmation of Identity of Chemical Residues using Exact Mass Data for the FDA FVM Program still contain some subtle differences for identification criteria.
In the case of GC-Orbitrap MS with electron ionisation, compliance with both sets of identification criteria is relatively straightforward. For example, the Thermo Scientific™ Q Exactive™ GC Orbitrap™ GC-MS/MS system and the Thermo Scientific™ Exactive™ GC Orbitrap™ GC-MS system each produce compliant data as outlined in an application note titled Routine Quantitative Method of Analysis for Pesticides using GC Orbitrap Mass Spectrometry in accordance withSANTE/11945/2015 Guidelines. However, in the case of LC-HRAM, compliance with the SANTE identification criteria has historically proved more challenging than compliance with the FDA guidelines.
The Differences for LC-HRAM MS
For LC-HRAM MS data, the FDA guidelines require the presence of two ions, preferably the precursor ion measured with a mass accuracy of ≤ 5ppm and a fragment ion (irrespective of fragmentation mode) with a mass accuracy of ≤ 10ppm. There is no requirement for an ion ratio unless the mass accuracy requirement cannot be achieved. The Thermo Scientific™ Q Exactive™ Focus hybrid quadrupole-Orbitrap MS with typically 3 ppm mass accuracy for both precursor and fragment ions can easily meet these requirements.
Prior to the latest update, the SANTE guidelines required an ion ratio for HRAM MS data, preferably a precursor ion and fragment ion. However, when HRAM MS data is acquired with alternating full scan and fragmentation scan events the precursor ion the fragment are derived from two separate scan events. Under these circumstances, the ± 30% tolerance applied for triple quadrupole data is not applicable.
The latest update to SANTE Guidelines state that in the case of HRAM MS with acquisition in full scan, limited m/z, SIM, fragmentation with or without precursor ion selection, or combinations thereof the requirement for identification is: the presence of two ions (preferably including a precursor ion and one fragment ion) both with mass accuracy ≤ 5ppm. The mandatory requirement for an ion ratio is removed, since with the added value of accurate mass measurement, the need for matching ion ratios is less critical. Nonetheless, it is recommended that ion ratios should be used as indicative with deviations exceeding 30% to be further investigated and judged with care. Thereby returning the onus back to the analyst to monitor the ratios and, where necessary, provide evidence for confident identification such as additional accurate mass fragments, isotope pattern, adduct ions, etc. Since the liquid chromatography (LC)-Orbitrap technology can produce the additional mass spectrometric data; full compliance for identification is easily achieved.
Why is this important?
In the context of food safety, there is increasing pressure from the regulatory authorities to increase the scope of the methods to allow the simultaneous quantitation of residues expected to be present, and proactive screening for compounds not expected to be present. HRAM MS such as Orbitrap MS is the ideal technology to improve the chance of early detection of the contamination of the food supply, as recently occurred with fipronil in eggs produced in Europe. To date, the mandatory requirement of the need for ratios has proved to be a hindrance to wider adoption. But now that this requirement is removed, regulators and analysts will perhaps more encouraged adopting HRAM MS technology to provide greater protection to the consumers.
And yes, I would consider the latest updating of the SANTE guidelines a small step to harmonisation of guidelines, although ongoing further work is still required.