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.

Easy and Fast Method for the Determination of Biogenic Amines in Fish and Fish Products with Liquid Chromatography Coupled to Orbitrap Tandem Mass Spectrometry.

A quantitative method for the determination of biogenic amines was developed. The method is characterized by the virtual absence of sample cleanup and does not require a derivatization reaction. Diluted extracts are centrifuged, filtrated, and directly injected into an ultra-HPLC column, which is coupled to a single-stage high-resolution mass spectrometer (Orbitrap). The chromatography is based on a reversed-phase column and an eluent containing an ion-pairing agent (heptafluorobutyric acid). The high sensitivity of the instrument permits the injection of very diluted extracts, which ensures stable retention times and the virtual absence of signal suppression effects. In addition, the quantification of histamine (a regulated compound) is further aided by the use of an isotopically labeled internal standard. The method was validated for three fish-based matrixes. Both the sample processing and the analytical measurement are very fast; hence, the methodology is ideal for high-throughput work. In addition, the method is significantly more selective than conventional methods (i.e., derivatization followed by LC with UV/fluorescence (FL) detection) for biogenic amines. A comparison showed that LC-UV/FL methods can produce false-positive findings due to coeluting matrix compounds.

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.

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.

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.

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.

Are liquid chromatography/electrospray tandem quadrupole fragmentation ratios unequivocal confirmation criteria?

Multiple reaction monitoring (MRM) ratios as provided by tandem mass spectrometers are used to confirm positive residue findings (e.g. veterinary drugs or pesticides). The Commission Decision 2002/657/EEC defines tolerance levels for MRM ratios, which are intended to prevent the reporting of false positives. This paper reports findings where blank sample extracts have been spiked by a drug (difloxacin) and the corresponding measured MRM ratios significantly deviated from MRM ratios observed in matrix-free solution. The observation was explained by the formation of two different [M+H](+) analyte ions within the electrospray ionization (ESI) interface. These two ions vary only by the site of analyte protonation. Since they are isobaric, they are equally transmitted through the first quadrupole, but are differently fragmented in the collision chamber. The existence of two isobaric ions was deduced by statistical data and the observation of a doubly charged analyte ion. It was hypothesized that the combined presence of [M+H](+) and [M+2H](2+) implies the existence of two different singly charged ion species differing only by the site of protonation. Low- and high-energy interface-induced fragmentation was performed on the samples. The surviving precursor ion population was mass selected and again fragmented in the collision chamber. Equal product ion spectra would be expected. However, very different product ion spectra were observed for the two interface regimes. This is consistent with the assumption that the two postulated isobaric precursor ions show different stability in the interface. Hence the abundance ratio among the two types of surviving precursor ions will shift and change the resulting product ion spectra. The existence of the postulated singly charged ions with multiple chargeable sites was finally confirmed by successful ion mobility separation.

Quantitative multiresidue method for about 100 veterinary drugs in different meat matrices by sub 2-microm particulate high-performance liquid chromatography coupled to time of flight mass spectrometry.

A quantitative multiresidue method covering more than 100 veterinary drugs, belonging to different drug families, has been developed. The proposed approach uses an liquid-liquid-solid extraction technique (bi-polarity extraction) which is capable in recovering polar, medium polar and apolar compounds. A thorough generic reversed phase solid-phase extraction (SPE) clean-up removes interfering proteins and provides clean and stable extracts. Dedicated rinsing steps are proposed to reduce analyte adsorption on glass walls and on precipitating proteins. The resulting extract is analyzed by ultra-performance liquid chromatography (UPLC) coupled to time of flight mass spectrometry (TOF). The method was validated according to the Commission Decision 2002/657/EEC. Validation coved the relevant meat matrices (muscle, kidney and liver).

Ultra-performance liquid chromatography coupled to time of flight mass spectrometry (UPLC-TOF): a novel tool for multiresidue screening of veterinary drugs in urine.

A multiresidue method for the screening of veterinary drugs in urine with ultra-performance liquid chromatography (UPLC) coupled to time of flight mass spectrometry (TOF) is proposed. The method covers currently more than 100 analytes belonging to different families of veterinary drugs. Urine samples are simply diluted and injected unfiltered into the UPLC-TOF. The short run time permit high-throughput of large series under a routine environment. The suggested approach includes the detection of some metabolites which are relevant to the urine matrix. The paper discusses the usefulness of metabolites as additional confirmation criteria for positive findings and suggests a revisiting of the identification point system in the light of additional resolution, as provided by the TOF and the UPLC technology. The proposed multiresidue approach was further broadened by monitoring drug group specific collision induced fragments (CID). Examples show the monitoring of generic CID fragments typical for sulfonamides and penicillins.

Determination of 11 aminoglycosides in meat and liver by liquid chromatography with tandem mass spectrometry.

A method using liquid chromatography with tandem mass spectrometry was developed for the determination of 11 commonly used aminoglycoside antibiotics in meat. The proposed method is sufficiently sensitive (detection limits of 15 to 40 ppb for the various antibiotics) and highly selective. It is suitable for the quantitation and confirmation of aminoglycosides in a variety of matrixes (pork muscle, fish, and veal liver). Any multiresidue method for aminoglycosides must take into account their high affinity toward sample proteins and the significantly different pK values of the various analytes. The developed method uses a low-pH extraction with trichloracetic acid to ensure complete extraction of the analytes from the matrix. An anion-exchange step is used to remove the acid from the centrifuged extract. Aminoglycosides in this solution of low ionic strength can be quantitatively retained and afterwards eluted from a weak cation-exchanger solid-phase extraction (SPE) cartridge. The highly selective SPE steps produce clean extracts, which minimize possible suppression of the mass spectrometer signal.