Assessment and validation of the p-QuEChERS sample preparation methodology for the analysis of >200 veterinary drugs in various animal-based food matrices.

The need remains for veterinary multi-residue methods that reliably quantify and identify veterinary drugs in the various animal-based food matrices. Such a method should not only show good method performance parameters (e.g. recoveries of analytes) but must also be fast and cheap. The proposed method focused on the following points: acceptable analyte trueness (recovery) and precision for a large number (200) of diverse veterinary drugs in the relevant animal-based food matrices (egg, muscle, fatty fish, liver, kidney, and honey). The sample preparation method termed p-QuEChERS uses a salt mixture consisting of potassium phosphates to induce phase separation. The avoidance of conventional QuEChERS salts (e.g. magnesium sulphate) significantly improves recoveries of several critical analytes. Analyte recoveries were further improved by adding a centrifugation and a defatting step before initiating the salt-induced phase separation. This combined clean-up removes a large fraction of the potentially interfering matrix compounds. As a result, matrix effects in the electrospray interface were minimized. These factors were the basis for the obtained good validation data. Two types of high-resolution mass spectrometers coupled to liquid chromatography were compared for analysis. In comparison with conventional QuEChERS, the proposed p-QuEChERS concept improved the recovery of polar analytes such as penicillins, tetracyclines and quinolones. The simplicity of the procedure and the low consumable expenses make the method ideal for the routine control of veterinary drugs in all evaluated animal-based food matrices.

Simplifying Nontargeted Analysis of PFAS in Complex Food Matrixes.

BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) are a class of toxic environmental contaminants that are characterized by their high chemical stability and enormous structural diversity. OBJECTIVE: The limited availability of PFAS reference standards is the main motivation for developing nontargeted analytical methods. Current concepts are complex and rely on multiple filtering steps (e.g., assumption of homologous series, detection of mass defects, generic fragments, and spectra obtained from web-based sources). METHOD: High-resolution mass spectrometry (HRMS)-based chromatograms of fish liver extracts were deconvoluted. Based on the ion abundance between the monoisotopic and the first isotopic peak, the number of carbons (C) was estimated for each extracted feature. A mass over carbon (m/C) and mass defect over carbon (md/C) ratio was calculated. RESULTS: PFAS-related peaks are strongly discriminated from matrix peaks when plotting m/C versus md/C. This enables nontarget detection of PFAS present at low µg/kg concentration in complex food matrixes. CONCLUSIONS: The proposed concept is highly selective by revealing a relatively small number of high-probability PFAS candidates (features). The small number of surviving candidates permits the MS/MS-based confirmation of each feature. This strategy led to the finding of one PFAS not present in the reference standard solution, as well as the detection of an unexpected set of PFAS adducts. HIGHLIGHTS: The proposed concept of mass over carbon versus mass defect over carbon is suited for the nontarget detection of low amounts of PFAS in complex matrixes. It should be capable of detecting any PFAS (F/H ratio should be >1:1) regardless of the ionization mode.

Improving the QuEChERS Liquid/Liquid Extraction of Analytes With Widely Varying Physicochemical Properties: Example of 201 Veterinary Drugs in Milk.

BACKGROUND: QuEChERS is an extraction and sample processing technique widely used for multiresidue methods (e.g., pesticides or veterinary drugs). OBJECTIVE: QuEChERS recovers a wide analyte polarity range, and yet low or even very low recoveries are observed for some polar analytes. This is especially relevant when analyzing veterinary drug residues in milk. METHOD: Based on the Hoffmeister row, potassium hydrogenphosphate/dihydrogenphosphate were selected as salting-out agents. The methodology was termed p-QuEChERS. RESULTS: The avoidance of weak complexing agents (magnesium and probably sodium), as present in conventional QuEChERS salt mixtures, improved the partition of critical analytes into the organic phase. Significantly higher recoveries were obtained for analytes belonging to the families of tetracyclines, penicillins, and cephalosporins. CONCLUSIONS: The proposed p-QuEChERS concept, compared to conventional QuEChERS, improves the recovery of various polar analytes. Analytes benefiting from this methodology are drugs, which are particularly relevant in the specific field of multiresidue analysis in milk. On the other hand, none of the total 201 investigated analytes showed a reduced recovery. In addition, neither the sample handling nor the co-partition of involatile salts into the extracts was negatively affected. HIGHLIGHTS: The p-QuEChERS based method has been fully validated according to European Community guidelines, using liquid chromatography coupled to high-resolution mass spectrometry instrumentation (time of flight (TOF) as well as Orbitrap). The developed multiresidue method quantifies 201 different veterinary drugs at relevant residue concentration in milk and is highly suitable for high throughput routine control.

High resolution mass spectrometry-based detection and quantification of ß-agonists at relevant trace levels in a variety of animal-based food matrices.

ß-agonists have been illegally used for growth promoting purposes in animal husbandry, leading to residue concentrations capable of inducing acute toxic reactions among consumers of animal-based food. There is not only a need for detecting ß-agonist residues at low concentrations, but also to increase the number of compounds to be monitored. It was therefore the aim of this paper to develop a unified method capable of detecting a wide range of different ß-agonists (20 analytes including some metabolites) in a variety of matrices (muscle, liver, plasma, milk and urine). The developed procedure permits the quick processing of samples with limited labour input and consumption of consumables. The method has been validated according to the Commission Decision 98/536/EC. Detection is based on ultrahigh-performance chromatography coupled to high-resolution mass spectrometry. Validation was performed on two different instruments (Orbitrap and time of flight). The obtained limit of quantification (0.05 to 0.5 µg/kg and the average recovery of 78% in the most complex matrix (liver) satisfies current regulatory requirements.

Partially overlapping sequential window acquisition of all theoretical mass spectra: A methodology to improve the spectra quality of veterinary drugs present at low concentrations in highly complex biological matrices.

RATIONALE: Residues of veterinary drugs in food matrices have to be detected, identified and confirmed at low concentrations. Data-independent acquisition (DIA) methods such as the sequential window acquisition of all theoretical spectra (SWATH) permit the extraction of relatively clean spectra out of complex matrices. Such spectra can be significantly improved by using a modified SWATH algorithm which provides several times narrower mass isolation windows without affecting the cycle time. METHODS: A quadrupole-time-of-flight mass spectrometer was operated in a partially overlapping SWATH mode. Unlike in conventional SWATH, acquisition sequences are not identically repeated, but each sequence is initiated with a mass intercept (mass shift). Pearson correlation is used to assign HRMS-resolved product ions to the precursor mass of interest. Trace analytes (veterinary drugs) in a complex matrix extract (bovine liver) were investigated. RESULTS: Utilizing identical cycle times, the partially overlapping SWATH mode produced for the investigated small molecules a significantly higher selectivity than the conventional SWATH acquisition mode. The acquisition strategy enables long ion accumulation times and therefore the required high sensitivity of detection. The study investigates the quality of the obtained product ion spectra and compares it with that in conventional acquisition modes. CONCLUSIONS: The modified SWATH mode permits high duty cycles in combination with narrow virtual mass windows. The technique is a further step toward harvesting a HRMS product ion spectrum out of a data-independent acquisition which moves closer towards the quality of a dedicated precursor unit isolation product ion spectrum.

Analysis of a variety of inorganic and organic additives in food products by ion-pairing liquid chromatography coupled to high-resolution mass spectrometry.

A reversed-phase ion-pairing chromatographic method was developed for the detection and quantification of inorganic and organic anionic food additives. A single-stage high-resolution mass spectrometer (orbitrap ion trap, Orbitrap) was used to detect the accurate masses of the unfragmented analyte ions. The developed ion-pairing chromatography method was based on a dibutylamine/hexafluoro-2-propanol buffer. Dibutylamine can be charged to serve as a chromatographic ion-pairing agent. This ensures sufficient retention of inorganic and organic anions. Yet, unlike quaternary amines, it can be de-charged in the electrospray to prevent the formation of neutral analyte ion-pairing agent adducts. This process is significantly facilitated by the added hexafluoro-2-propanol. This approach permits the sensitive detection and quantification of additives like nitrate and mono-, di-, and triphosphate as well as citric acid, a number of artificial sweeteners like cyclamate and aspartame, flavor enhancers like glutamate, and preservatives like sorbic acid. This is a major advantage, since the currently used analytical methods as utilized in food safety laboratories are only capable in monitoring a few compounds or a particular category of food additives. Graphical abstract Deptotonation of ion pair agent in the electrospray interface.

Coalescence and self-bunching observed in commercial high-resolution mass spectrometry instrumentation.

RATIONALE: Self-bunching and coalescence are well-known effects in Fourier transform ion cyclotron resonance (FTICR) and multi-reflection time-of-flight (TOF) mass spectrometry. These detrimental effects can also be observed in currently more frequently used high-resolution mass spectrometry (HRMS) instruments, such as the Orbitrap and single-reflection TOF. METHODS: A modern single-reflection TOF and a Q-Orbitrap were used to produce conditions in which self-bunching and coalescence were observed. This was done by infusion experiments of several isobaric compounds. The peak widths of some low-mass isobaric ions as well as the mass resolution of such mixtures were investigated. Attention was paid to possible self-bunching and coalescence effects. RESULTS: For the utilized TOF mass spectrometer, the measured peak widths of the ions become significantly narrower (self-bunching) when increasing the ion abundance. On the other hand, isobaric ion pairs (delta <30 milli-m/z units) became unresolvable above a certain ion abundance (coalescence). The tested Orbitrap shows similar behavior, although coalescence appeared only at delta <15 milli-m/z units. Coalescence was shown to affect the quantitative data, while self-bunching can lead to biased relative isotopic ratios. CONCLUSIONS: The conventional measurement of a peak width does not truly reflect the mass resolving power of modern HRMS instrumentation. The mass resolving power is better demonstrated by resolving a mixture of isobaric compounds. Measurements obtained at low and high ion abundances should be investigated. Coalescence and self-bunching can reduce the truly available mass resolving power and therefore negatively affect quantitative and qualitative measurements.

Comparison of linear intrascan and interscan dynamic ranges of Orbitrap and ion-mobility time-of-flight mass spectrometers.

RATIONALE: The linear intrascan and interscan dynamic ranges of mass spectrometers are important in metabolome and residue analysis. A large linear dynamic range is mandatory if both low- and high-abundance ions have to be detected and quantitated in heavy matrix samples. These performance criteria, as provided by modern high-resolution mass spectrometry (HRMS), were systematically investigated. METHODS: The comparison included two generations of Orbitraps, and an ion mobility quadrupole time-of-flight (QTOF) system In addition, different scan modes, as provided by the utilized instruments, were investigated. Calibration curves of different compounds covering a concentration range of five orders of magnitude were measured to evaluate the linear interscan dynamic range. The linear intrascan dynamic range and the resulting mass accuracy were evaluated by repeating these measurements in the presence of a very intense background. RESULTS: Modern HRMS instruments can show linear dynamic ranges of five orders of magnitude. Often, however, the linear dynamic range is limited by the detection capability (sensitivity and selectivity) and by the electrospray ionization. Orbitraps, as opposed to TOF instruments, show a reduced intrascan dynamic range. This is due to the limited C-trap and Orbitrap capacity. The tested TOF instrument shows poorer mass accuracies than the Orbitraps. In contrast, hyphenation with an ion-mobility device seems not to affect the linear dynamic range. CONCLUSIONS: The linear dynamic range of modern HRMS instrumentation has been significantly improved. This also refers to the virtual absence of systematic mass shifts at high ion abundances. The intrascan dynamic range of the current Orbitrap technology may still be a limitation when analyzing complex matrix extracts. On the other hand, the linear dynamic range is not only limited by the detector technology, but can also be shortened by peripheral devices, where the ionization and transfer of ions take place.

Using In Silico Fragmentation to Improve Routine Residue Screening in Complex Matrices.

Targeted residue screening requires the use of reference substances in order to identify potential residues. This becomes a difficult issue when using multi-residue methods capable of analyzing several hundreds of analytes. Therefore, the capability of in silico fragmentation based on a structure database ("suspect screening") instead of physical reference substances for routine targeted residue screening was investigated. The detection of fragment ions that can be predicted or explained by in silico software was utilized to reduce the number of false positives. These "proof of principle" experiments were done with a tool that is integrated into a commercial MS vendor instrument operating software (UNIFI) as well as with a platform-independent MS tool (Mass Frontier). A total of 97 analytes belonging to different chemical families were separated by reversed phase liquid chromatography and detected in a data-independent acquisition (DIA) mode using ion mobility hyphenated with quadrupole time of flight mass spectrometry. The instrument was operated in the MSE mode with alternating low and high energy traces. The fragments observed from product ion spectra were investigated using a "chopping" bond disconnection algorithm and a rule-based algorithm. The bond disconnection algorithm clearly explained more analyte product ions and a greater percentage of the spectral abundance than the rule-based software (92 out of the 97 compounds produced ≥1 explainable fragment ions). On the other hand, tests with a complex blank matrix (bovine liver extract) indicated that the chopping algorithm reports significantly more false positive fragments than the rule based software. Graphical Abstract.

Practical application of in silico fragmentation based residue screening with ion mobility high-resolution mass spectrometry.

RATIONALE: A screening concept for residues in complex matrices based on liquid chromatography coupled to ion mobility high-resolution mass spectrometry LC/IMS-HRMS is presented. The comprehensive four-dimensional data (chromatographic retention time, drift time, mass-to-charge and ion abundance) obtained in data-independent acquisition (DIA) mode was used for data mining. An in silico fragmenter utilizing a molecular structure database was used for suspect screening, instead of targeted screening with reference substances. METHODS: The utilized data-independent acquisition mode relies on the MSE concept; where two constantly alternating HRMS scans (low and high fragmentation energy) are acquired. Peak deconvolution and drift time alignment of ions from the low (precursor ion) and high (product ion) energy scan result in relatively clean product ion spectra. A bond dissociation in silico fragmenter (MassFragment) supplied with mol files of compounds of interest was used to explain the observed product ions of each extracted candidate component (chromatographic peak). RESULTS: Two complex matrices (fish and bovine liver extract) were fortified with 98 veterinary drugs. Out of 98 screened compounds 94 could be detected with the in silico based screening approach. The high correlation among drift time and m/z value of equally charged ions was utilized for an orthogonal filtration (ranking). Such an orthogonal ion mobility based filter removes multiply charged ions (e.g. peptides and proteins from the matrix) as well as noise and artefacts. Most significantly, this filtration dramatically reduces false positive findings but hardly increases false negative findings. CONCLUSIONS: The proposed screening approach may offer new possibilities for applications where reference compounds are hardly or not at all commercially available. Such areas may be the analysis of metabolites of drugs, pyrrolizidine alkaloids, marine toxins, derivatives of sildenafil or novel designer drugs (new psychoactive substances). Copyright © 2017 John Wiley & Sons, Ltd.

Nested data independent MS/MS acquisition.

Data independent acquisition (DIA) attempts to provide comprehensive MS/MS data while providing a cycle time that is capable of following the elution profile of chromatographic peaks. Currently available MS technology is not yet fully capable of fulfilling these expectations. This paper suggests a new multiplex-based approach to more closely achieve this objective. Customized scans have been programmed for a Q Orbitrap instrument. Multiple nonadjacent mass range segments are sequentially collected (cut out) by the quadrupole. These combined mass ranges undergo fragmentation, and the resulting product ions are analyzed as a whole by the Orbitrap analyzer. The systematical variation of the mass range segments (nested design) permits the mathematical assignment of the observed product ions within a narrow precursor mass range. The proposed approach allows the use of mass windows that are narrower than those in conventional DIA (SWATH). A unique aspect of the proposed approach is the fact that halving the mass window width requires the addition of only a single multiplexed scan. This is different from conventional DIA, which requires the number of mass windows to be doubled in order to achieve the same objective. This paper shows that for a given cycle time, the proposed nested DIA technique produces significantly less chimeric product ion spectra than conventional DIA. However, further improvements from the programming, and most likely the hardware side, are still required in order to achieve the aim of comprehensive MS/MS. Graphical Abstract Schematic of nested design.

Improved performance of multiplexed targeted tandem mass spectrometry scans using customized Q Orbitrap data acquisition.

RATIONALE: The Q Orbitrap permits multiplexed targeted selected ion monitoring (SIM) or tandem mass spectrometry (MS/MS) scans. Such scans provide a significantly higher sensitivity than conventional full scan acquisition. However, due to the multiplexing, a monitored product ion extracted from a MS/MS scan can no longer be linked to the precursor ion from which it was derived. Furthermore, due to the automatic gain control, quantification based on targeted product ions acquired in the multiplexed MS/MS mode can become questionable. METHODS: The application programming interface (API) of the Q Exactive has been used to program a multiplexed targeted MS/MS mode that permits the establishment of a link (based on digital coding) between the product ion and the responsible precursor ion. Furthermore, switching off the automatic gain control feature and the definition of appropriate C-trap settings were tested to improve the quantification performance. RESULTS: The use of dedicated decoding scans permitted the clear assignment of all monitored product ions to the responsible precursor ion. Furthermore, the customized multiplexed targeted scan used for quantification showed good sensitivity and linearity for a maximum number of eight co-eluting analytes. CONCLUSIONS: Multiplexed targeted MS/MS scans can be optimized to provide better selectivity (correct linking of an observed product ion to the responsible precursor ion) as well as improved quantitative performance (enforcement of an identical ion injection time for all targeted precursor ions). These two improvements are relevant for quantitative residue analysis.

Determination of corticosteroids, anabolic steroids, and basic nonsteroidal anti-inflammatory drugs in milk and animal tissues.

A quantitative LC/MS/MS method was developed for the determination of 14 steroidal compounds and three basic nonsteroidal anti-inflammatory drugs (detected as metabolites) in bovine milk and animal muscle tissue. The proposed method is sufficiently sensitive and highly selective for residue applications. The described approach offers the possibility to detect, quantify, and confirm anti-inflammatory drugs belonging to two widely diverging chemical categories. The employed single-stage SPE step (mixed mode cation exchange) retains both steroids and basic metabolites of nonsteroidal anti-inflammatory drugs. The method is capable of handling widely diverging relevant concentration ranges (0.1 microg/kg for dexamethasone and 100 microg/kg for metamizol metabolites) for the individual compounds with a single extraction, cleanup, and LC/MS/MS procedure. It provides good analyte precision and accuracy data.

Study of high-resolution mass spectrometry technology as a replacement for tandem mass spectrometry in the field of quantitative pesticide residue analysis.

A validated LC/MS/MS-based multiresidue pesticide method was converted to an LC high-resolution MS single-stage Orbitrap platform. No changes regarding the cleanup and LC were made. Optimization of high-resolution MS-specific parameters and interface settings was kept at a minimum. The aim was to explore the capability of current Orbitrap technology to substitute for LC/MS/MS technology. The test included the quantitative performance (sensitivity, selectivity, linearity, accuracy, and precision) of some 240 analytes in three different matrixes. The LC/MS/MS instrumentation was operated at the edge of its technical limitations. A further extension of the number of analytes for LC/MS/MS would require the use of even narrower dwell times, significantly reducing sensitivity and reproducibility of measurement. No such limitations exist for the high-resolution technology. Similar performance was observed for both technologies. A current drawback of the high-resolution technology is the speed of data processing, which took significantly longer than for LC/MS/MS data due to the limited capabilities of the software.

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.