Signal suppression can bias selected reaction monitoring ratios. Implications for the confirmation of positive findings in residue testing.

RATIONALE: Selected reaction monitoring (SRM) ratios based on two or more transitions are commonly used to confirm the identity of a suspected finding in residue testing. International norms like the EU directive commission decision 2002/657/EC (CD) require the use of such ratios to prove the unequivocal identification of a particular compound detected at trace level (confirmation of a suspected residue). METHODS: In this study, the relative abundances of different precursor ions (e.g. protonated molecule, solvent adducts, characteristic fragment ions or multiply charged ions) derived from the same analyte molecule were found to be differently (asymmetrically) affected by matrix-related signal suppression effects. This observation was made when analyzing veterinary drug residues (colistin and amoxicillin) in animal tissue extracts. RESULTS: The term 'asymmetric signal suppression' was coined since different ionic species produced from the same analyte molecule are differently (asymmetrically) suppressed by co-eluting matrix compounds. In the case of the colistin assay, the extent of asymmetric signal suppression is such that the measured SRM ratios lie beyond the defined (CD) tolerances. Hence, the compound present in a sample cannot be confirmed. CONCLUSIONS: The [M+H](+) ion may be the most commonly used precursor ion in liquid chromatography coupled to electrospray operated in the positive ionization mode. However, the absence of a sufficiently intensive confirmation transition frequently leads to the selection of another precursor ion to be utilized for the confirmation transition. The SRM ratio derived from such transitions should not be compared to the SRM ratio derived from pure standard solutions but spiked blank matrix extracts.

Semi-targeted residue screening in complex matrices with liquid chromatography coupled to high resolution mass spectrometry: current possibilities and limitations.

Some twenty cultured fish samples were analyzed for possible residues of veterinary drugs with high resolution mass spectrometry (single stage Orbitrap) coupled to ultra performance liquid chromatography. Quantitative analysis based on external standards covered 110 analytes. Some 116 additional compounds were monitored without having access to reference materials. Detection was based on calculated exact masses and narrow mass windows. Furthermore, a number of semi-targeted techniques were evaluated and compared to corresponding triple quadrupole precursor scan experiments. Single stage high resolution mass spectrometry was used to monitor compound specific product ions (without relying on a previous precursor selection). The capabilities of neutral loss searches based on exact masses were shown by detecting small concentrations of incurred oxytetracycline residues. High resolution mass spectrometry provided more sensitivity and selectivity than corresponding tandem quadrupole precursor and neutral loss scans. The currently limiting factor is the not adequate performance of the available software used for data mining. The high number of false positives that were produced, when searching for chlorine isotopic patterns, was clearly linked to the fact that the utilized software does not perform a peak deconvolution, but simply investigates one individual spectrum after another.

Quantitative and confirmative performance of liquid chromatography coupled to high-resolution mass spectrometry compared to tandem mass spectrometry.

The quantitative and confirmative performance of two different mass spectrometry (MS) techniques (high-resolution MS and tandem MS) was critically compared. Evaluated was a new extraction and clean-up protocol which was developed to cover more than 100 different veterinary drugs at trace levels in a number of animal tissues and honey matrices. Both detection techniques, high-resolution mass spectrometry (HRMS) (single-stage Orbitrap instrument operated at 50 000 full width at half maximum) and tandem mass spectrometry (MS/MS) (quadrupole technology) were used to validate the method according to the EU Commission Decision 2002/657/EEC. Equal or even a slightly better quantitative performance was observed for the HRMS-based approach. Sensitivity is higher for unit mass resolution MS/MS if only a subset of the 100 compounds has to be monitored. Confirmation of suspected positive findings can be done by evaluating the intensity ratio between different MS/MS transitions, or by accurate mass based product ion traces (no precursor selection applied). MS/MS relies on compound-specific optimized transitions; hence the second, confirmatory transition generally shows relatively high ion abundance (fragmentation efficacy). This is often not the case in single-stage HRMS, since a generic (not compound-optimized) collision energy is applied. Hence, confirmation of analytes present at low levels is superior when performed by MS/MS. Slightly better precision, but poorer accuracy (fortified matrix extracts versus pure standard solution) of ion ratios were observed when comparing data obtained by HRMS versus MS/MS.