A New Heart-Cutting Method for a Multiplex Quantitative Analysis of Steroid Hormones in Plasma Using 2D-LC/MS/MS Technique.

The aim of the current research was to develop a simple and rapid mass spectrometry-based assay for the determination of 15 steroid hormones in human plasma in a single run, which would be suitable for a routine practice setting. For this purpose, we designed a procedure based on the 2D-liquid chromatography-tandem mass spectrometry with a minimalistic sample pre-treatment. In our arrangement, the preparation of one sample takes only 10 min and can accommodate 40 samples per hour when tested in series. The following analytical run is 18 min long for all steroid hormones. In addition, we developed an independent analytical run for estradiol, significantly increasing the assay accuracy while taking an additional 10 min to perform an analytical run of a sample. The optimized method was applied to a set of human plasma samples, including chylous. Our results indicate the linearity of the method for all steroid hormones with squared regression coefficients R2 ≥ 0.995, within-run and between-run precision (RSD < 6.4%), and an accuracy of 92.9% to 106.2%. The absolute recovery for each analyzed steroid hormone ranged between 101.6% and 116.5%. The method detection limit for 15 steroid hormones ranged between 0.008 nmol/L (2.88 pg/mL) for aldosterone and 0.873 nmol/L (0.252 ng/mL) for DHEA. For all the analytes, the lowest calibration point relative standard deviation was less than 10.8%, indicating a good precision of the assay within the lowest concentration of interest. In conclusion, in this method article, we describe a simple, sensitive, and cost-effective 2D-LC/MS/MS method suitable for the routine analysis of a complex of steroid hormones allowing high analytical specificity and sensitivity despite minimal sample processing and short throughput times.

A novel accurate LC-MS/MS method for quantitative determination of Z-lumirubin.

Although phototherapy (PT) is a standard treatment for neonatal jaundice, no validated clinical methods for determination of bilirubin phototherapy products are available. Thus, the aim of our study was to establish a such method for clinical use. To achieve this aim, a LC-MS/MS assay for simultaneous determination of Z-lumirubin (LR) and unconjugated bilirubin (UCB) was conducted. LR was purified after irradiation of UCB at 460 nm. The assay was tested on human sera from PT-treated neonates. Samples were separated on a HPLC system with a triple quadrupole mass spectrometer detector. The instrument response was linear up to 5.8 and 23.4 mg/dL for LR and UCB, respectively, with submicromolar limits of detection and validity parameters relevant for use in clinical medicine. Exposure of newborns to PT raised serum LR concentrations three-fold (p < 0.01), but the absolute concentrations were low (0.37 ± 0.16 mg/dL), despite a dramatic decrease of serum UCB concentrations (13.6 ± 2.2 vs. 10.3 ± 3.3 mg/dL, p < 0.01). A LC-MS/MS method for the simultaneous determination of LR and UCB in human serum was established and validated for clinical use. This method should help to monitor neonates on PT, as well as to improve our understanding of both the kinetics and biology of bilirubin phototherapy products.

Saffron authentication based on liquid chromatography high resolution tandem mass spectrometry and multivariate data analysis.

Saffron is one of the oldest and most expensive spices, which is often target of fraudulent activities. In this research, a new strategy of saffron authentication based on metabolic fingerprinting was developed. In the first phase, a solid liquid extraction procedure was optimized, the main aim was to isolate as maximal representation of small molecules contained in saffron as possible. In the second step, a detection method based on liquid chromatography coupled with high-resolution mass spectrometry was developed. Initially, principal component analysis (PCA) revealed clear differences between saffron cultivated and packaged in Spain, protected designation of origin (PDO), and saffron packaged in Spain of unknown origin, labeled Spanish saffron. Afterwards, orthogonal partial least square discriminant analysis (OPLS-DA) was favorably used to discriminate between Spanish saffron. The tentative identification of markers showed glycerophospholipids and their oxidized lipids were significant markers according to their origin.

Allium Discoloration: Color Compounds Formed during Pinking of Onion and Leek.

Structures and formation pathways of compounds responsible for pink discoloration of onion and leek were studied. A procedure was developed for the isolation and purification of the color compounds from various model systems and their identification by HPLC-DAD-MS/MS. In total, structures of 15 major color compounds were tentatively determined. It was found that the pigment is a complex mixture of highly conjugated species composed of two N-substituted 3,4-dimethylpyrrole-derived rings linked by either a methine or a propenylidine bridge. These two-ring units are further modified by various C1- and C3-side chains. Experiments with isotope-labeled thiosulfinates revealed that the methine bridge and C1-side chains originate from the methyl group of methiin, whereas the C3 units are derived from the propenyl group of isoalliin.

Multi-analyte method for the analysis of various organohalogen compounds in house dust.

In the present study, a novel analytical approach for the simultaneous determination of 27 brominated flame retardants (BFRs), namely polybrominated diphenyl ethers (PBDEs), isomers of hexabromocyclododecane (HBCD), tetrabromobisphenol A (TBBPA) and several novel BFRs (NBFRs), together with 18 perfluoroalkyl substances (PFASs) in indoor dust was developed and validated. To achieve integrated isolation of analytes from the sample and their fractionation, a miniaturized method based on matrix solid phase dispersion (MSPD) was employed. Principally, after mixing the dust (<0.1 g) with the Florisil(®), the mixture was applied on the top of a sorbent (Florisil(®)) placed in glass column and then analytes were eluted using solvents with different polarities. For the identification/quantification of target compounds largely differing in polarity, complementary techniques represented by gas and liquid chromatography coupled to tandem mass spectrometry (GC-MS/MS and LC-MS/MS) were used. The results of validation experiments, which were performed on the SRM 2585 material (for PBDEs, HBCDs and TBBPA), were in accordance with the certified/reference values. For other analytes (NBFRs and PFASs), the analysis of an artificially contaminated blank dust sample was realized. The method recoveries for all target compounds ranged from 81 to 122% with relative standard deviations lower than 21%. The quantification limits were in the range of 1-25 ng g(-1) for BFRs and 0.25-1 ng g(-1) for PFASs. Finally, 18 samples (6 households × 3 sampling sites) were analyzed. The high variability between concentrations of PFASs and BFRs in the dust samples from various households as well as collecting sites in a respective house was observed. The total amounts of PFASs and BFRs were in the range of 1.58-236 ng g(-1) (median 10.6 ng g(-1)) and 39.2-2320 ng g(-1) (median 325 ng g(-1)), respectively. It was clearly shown that dust from the indoor environment might be a significant source of human exposure to various organohalogen pollutants.

Metabolic fingerprinting based on high-resolution tandem mass spectrometry: a reliable tool for wine authentication?

Ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (MS) and an alternative technology represented by direct analysis in real time coupled with quadrupole time-of-flight MS were investigated for metabolic fingerprinting of 343 red and white wine samples. Direct injection of pure wine and an extraction procedure optimized for isolation of polyphenols were used to compare different analytical and data handling strategies. After data processing and data pretreatment, principal component analysis was initially used to explore the data structure. Initially, the unsupervised models revealed a notable clustering according to the grape varieties, and therefore supervised orthogonal partial least squares discriminant analysis models were created and validated for separation of red and white wines according to the grape variety. The validated orthogonal partial least squares discriminant analysis models based on data (ions) recorded in positive ionization mode were able to classify correctly 95% of samples. In parallel, authentication parameters, such as origin and vintage, were evaluated, and they are discussed. A tentative identification of markers was performed using accurate mass measurement of MS and MS/MS spectra, different software packages and different online libraries. In this way, different flavonol glucosides and polyphenols were identified as wine markers according to the grape varieties.

Analytical strategies for controlling polysorbate-based nanomicelles in fruit juice.

This study focused on the detection and quantification of organic micelle-type nanoparticles (NPs) with polysorbate components (polysorbate 20 and polysorbate 80) in their micelle shells that could be used to load biologically active compounds into fruit juice. Several advanced analytical techniques were applied in the stepwise method development strategy used. In the first phase, a system consisting of ultrahigh-performance liquid chromatography employing a size exclusion column coupled with an evaporative light scattering detector (UHPLC-SEC-ELSD) was used for the fractionation of micelle assemblies from other, lower molecular weight sample components. The limit of detection (LoD) of these polysorbate micelles in spiked apple juice was 500 µg mL(-1). After this screening step, mass spectrometric (MS) detection was utilized to confirm the presence of polysorbates in the detected micelles. Two alternative MS techniques were tested: (i) ambient high-resolution mass spectrometry employing a direct analysis in real time ion source coupled with an Orbitrap MS analyzer (DART-Orbitrap MS) enabled fast and simple detection of the polysorbates present in the samples, with a lowest calibration level (LCL) of 1000 µg mL(-1); (ii) ultrahigh-performance reversed-phase liquid chromatography coupled with high-resolution time-of-flight mass spectrometry (UHPLC-HRTOF-MS) provided highly selective and sensitive detection and quantification of polysorbates with an LCL of 0.5 µg mL(-1).

A rugged high-throughput analytical approach for the determination and quantification of multiple mycotoxins in complex feed matrices.

We have developed and optimized high throughput method for reliable detection and quantification of 56 Fusarium, Alternaria, Penicillium, Aspergillus and Claviceps mycotoxins in a wide range of animal feed samples represented by cereals, complex compound feeds, extracted oilcakes, fermented silages, malt sprouts or dried distillers' grains with solubles (DDGS). From three tested extraction approaches (acetonitrile, acetonitrile/water, and QuEChERS), the QuEChERS-based method (Quick, Easy, Cheap, Effective, Rugged and Safe) was selected as the best in terms of analytes recoveries and low matrix effects. For separation and detection of target mycotoxins, method based on ultra-high performance liquid chromatography coupled with sensitive tandem mass spectrometry (U-HPLC-MS/MS) was employed. With regards to a high complexity of most of investigated feed samples, optimization of extraction/purification process was needed in the first phase to keep the method as rugged as possible. A special attention was paid to the pH of extraction solvents, especially with regard to the pH-sensitive silages. Additionally, purification of the acetonitrile extract by dispersive solid phase clean-up was assessed. Significant elimination of lipidic compounds was observed when using C18 silica sorbent. Matrix co-extracts were characterized by ultra-high performance liquid chromatography coupled with ultra-high resolution mass spectrometry (U-HPLC-HRMS). Large variability of matrix effects depending on the nature of examined feed was demonstrated in depth on a broad set of samples. Simple and unbiased strategies for their compensation were suggested.

The determination of perfluoroalkyl substances, brominated flame retardants and their metabolites in human breast milk and infant formula.

In the present study, a novel analytical approach for the simultaneous determination of 18 perfluoroalkyl substances (PFASs) and 11 brominated flame retardants (BFRs) including their hydroxylated metabolites and brominated phenols has been developed and validated for breast milk and infant formula. The sample preparation procedure based on extraction using acetonitrile and subsequent purification by dispersive solid-phase extraction (d-SPE) employing C18 sorbent is rapid, simple and high-throughput. Ultra-high performance liquid chromatography (UHPLC) interfaced with a tandem mass spectrometry (MS/MS) was employed for the identification/quantification of these compounds. The method recoveries of target compounds for both matrices ranged from 80% to 117% with relative standard deviations lower than 28% and quantification limits in the range of 3-200 pg/mL for milk and 5-450 pg/g for infant formula. Within the pilot study, the new method was used for the analysis of PFASs and BFRs in 50 human breast milks and six infant formulas. In the breast milk samples the total contents of PFASs and BFRs were in the range of 38-279 and 45-16,200 pg/mL, respectively. The most abundant PFASs detected in all tested breast milk samples were perfluorooctanoic acid (PFOA) and perfluorooctanesulfonate (PFOS), the latter contaminant was present not only as a linear form but also as a branched isomers. The incidence of BFRs was lower, the only representatives of this group, tetrabromobiphenol A (TBBPA) and α-hexabromocyclododecane (α-HBCD), were detected in less than 30% of breast milk samples. None of the infant formulas contained BFRs, traces of either PFOS, PFOA or PFNA were found in three samples.

Rapid and simple method for determination of hexabromocyclododecanes and other LC-MS-MS-amenable brominated flame retardants in fish.

In this study, a novel analytical approach for simultaneous determination of hexabromocyclododecane isomers (HBCDs), tetrabromobisphenol A (TBBPA), three brominated phenols, and four hydroxylated derivatives of polybrominated diphenyl ethers (OH-PBDEs) was developed and validated for muscle tissue of both lean and fatty fish. The rapid, simple, and high-throughput sample-preparation procedure was based on acetonitrile extraction then purification by dispersive solid-phase extraction (d-SPE) with a combination of C18 and primary-secondary amine (PSA) sorbents. Ultra-high performance liquid chromatography (UHPLC) coupled to tandem mass spectrometry (MS-MS) was used for identification and quantification of the analytes. Method recovery for both matrices ranged from 80 to 115% with relative standard deviations (RSDs) <13% for all analytes. Limits of quantification (LOQs) were in the range 0.1-1 µg kg(-1) wet weight. The validated method was used for analysis of brominated compounds in 32 fish and five bivalve samples collected from different European markets within the monitoring survey organized in the framework of the CONffIDENCE project. Of the 12 targeted analytes, only α-HBCD, 2,4-dibromophenol (2,4-DBP), and 2,4,6-tribromophenol (2,4,6-TBP) were quantified in the samples. α-HBCD was found in six fish samples (herring and mackerel) in the range of 0.8-2.5 µg kg(-1) wet weight. 2,4-DBP and 2,4,6-TBP were found in three blue mussel samples in the range of 19.6-43.5 and 2.3-7.5 µg kg(-1) wet weight, respectively.

Critical assessment of extraction methods for the simultaneous determination of pesticide residues and mycotoxins in fruits, cereals, spices and oil seeds employing ultra-high performance liquid chromatography-tandem mass spectrometry.

This study addresses a current trend in chemical food safety control represented by an effort to integrate analyses of various groups of food contaminants/toxicants into a single, high-throughput method. The choice of optimal sample preparation step is one of the key conditions to achieve good performance characteristics. In this context, we investigated the possibility to expand the scope of the three multi-analyte extraction procedures employed earlier in other studies for rapid isolation of either pesticides or mycotoxins from plant matrices. Following procedures were tested: A - aqueous acetonitrile extraction followed by partition (QuEChERS-like method), B - aqueous acetonitrile extraction, and C - pure acetonitrile extraction. On the list of target analytes, we had 288 pesticides (including 'troublesome' acidic, basic and base-sensitive compounds) together with 38 mycotoxins (including all EU regulated ones and many 'emerging' toxins on the European Food Safety Authority (EFSA) list). The matrices selected for the experiments, apple baby food, wheat flour, spices and sunflower seeds, represented various composition categories in terms of moisture, fat and extractable compounds (e.g. pigments and essential oils) content. In preliminary experiments, acceptable recoveries (70-120%) for most of analytes were obtained by the analysis of spiked matrices, regardless which extraction procedure was used. However, when analysing dry samples with incurred pesticide residues/mycotoxins, the method C did not enable efficient extraction of some common contaminants. Procedure A, thanks to a higher matrix equivalent compared to the method B and relatively less pronounced matrix effects, enabled lower quantification limits for all analyte/matrix combinations, with the exception of polar mycotoxins and/or pesticides. Higher recoveries for the latter group of analytes could be achieved by the method B; on the other hand, extraction efficiency of non-polar pesticides from fatty matrix was rather poor by this method.

Deoxynivalenol oligoglycosides: new "masked" fusarium toxins occurring in malt, beer, and breadstuff.

The co-occurrence of deoxynivalenol-3-glucoside with its parent toxin, deoxynivalenol, has been recently documented in many cereal-based foods, especially in those produced by enzyme-catalyzed processes. The presence of this masked mycotoxin in the human diet has become an issue of health concern, mainly because of its assumed bioavailability. A selective immunoaffinity-based preconcentration strategy, followed by ultrahigh-performance liquid chromatography coupled with high-resolution orbitrap mass spectrometry, revealed that, in addition to the most common deoxynivalenol-3-glucoside, also oligoglycosylated deoxynivalenols with up to four bound hexose units were present in cereal-based products. The structure, origination, and fate of these deoxynivalenol conjugates during malt/beer production and bread baking have been thoroughly investigated. Special attention has been paid to the changes of deoxynivalenol conjugates enabled by industrial glycosidase-based enzymatic preparations. To the authors' best knowledge, this is the first study documenting the complexity of masked deoxynivalenol issue.

Novel approaches to analysis of 3-chloropropane-1,2-diol esters in vegetable oils.

A sensitive and accurate method utilizing ultrahigh performance liquid chromatography (U-HPLC) coupled to high resolution mass spectrometry based on orbitrap technology (orbitrapMS) for the analysis of nine 3-chloropropane-1,2-diol (3-MCPD) diesters in vegetable oils was developed. To remove the interfering triacylglycerols that induce strong matrix effects, a clean-up step on silica gel column was used. The quantitative analysis was performed with the use of deuterium-labeled internal standards. The lowest calibration levels estimated for the respective analytes ranged from 2 to 5 µg kg(-1). Good recovery values (89-120%) and repeatability (RSD 5-9%) was obtained at spiking levels of 2 and 10 mg kg(-1). As an alternative, a novel ambient desorption ionization technique, direct analysis in real time (DART), hyphenated with orbitrapMS, was employed for no separation, high-throughput, semi-quantitative screening of 3-MCPD diesters in samples obtained by chromatographic fractionation. Additionally, the levels of 3-MCPD diesters measured in reallife vegetable oil samples (palm oil, sunflower oil, rapeseed oil) using both methods are reported. Relatively good agreement of the data generated by U-HPLC-orbitrapMS and DART-orbitrapMS were observed. With regard to a low ionization yield achieved for 3-MCPD monoesters, the methods presented in this paper were not yet applicable for the analysis of these contaminants at the naturally occurring levels.

Simple, high throughput ultra-high performance liquid chromatography/tandem mass spectrometry trace analysis of perfluorinated alkylated substances in food of animal origin: milk and fish.

The present study documents development and validation of a novel approach for determination of 23 perfluorinated alkylated substances (PFASs) in food of animal origin represented by milk and fish. The list of target analytes comprises four classes of PFASs, both ionic and non-ionic: 11 perfluorocarboxylic acids (PFCAs), 4 perfluorosulphonic acids (PFSAs), 5 perfluorosulphonamides (FOSAs) and 3 perfluorophosphonic acids (PFPAs). Fast sample preparation procedure is based on an extraction of target analytes with acetonitrile (MeCN) and their transfer (supported by inorganic salts and acidification) into the organic phase. Removing of matrix co-extracts by a simple dispersive solid phase extraction (SPE) employing ENVI-Carb and C18 sorbents is followed by an efficient sample pre-concentration performed by acetonitrile evaporation and subsequent dilution of residue in a small volume of methanol (matrix equivalent in the final extracts was 16 and 8 g mL(-1), for milk and fish respectively). Using modern instrumentation consisting of ultra-high performance liquid chromatography (UHPLC) hyphenated with a tandem mass spectrometer (MS/MS), limits of quantification (LOQs) as low as 0.001-0.006 µg kg(-1) for milk and 0.002-0.013 µg kg(-1) for fish can be achieved. Under these conditions, a wide spectrum of PFASs, including minor representatives, can be determined which enables collecting data required for human exposure studies. The pilot study employing the new method for examination of milk and canned fish samples was realized. Whereas in majority of canned fish products a wide spectrum of PFCAs, perfluorooctanesulphonic acid (PFOS) and perfluoro-1-octanesulphonamide (PFOSA) was detected, only in a few milk samples very low concentrations (LOQ levels) of PFOS and perfluorooctansulphonic acid (PFDS) were found.

The study of deoxynivalenol and its masked metabolites fate during the brewing process realised by UPLC-TOFMS method.

The co-occurrence of deoxynivalenol (DON) and deoxynivalenol-3-glucoside (DON-3-Glc) has been recently reported in malt and beer. In this study, the concentration changes were monitored within the brewing process of four beer brands: light, dark tap and two lagers, produced from ground malt mixtures differing in composition, and also mycotoxins content. A simple and rapid method employing DON-dedicated immunoaffinity columns (IAC) for the selective pre-concentration, followed by ultra-performance liquid chromatography coupled to a time-of-flight mass spectrometer (UPLC-TOFMS) system for the reliable quantification at (ultra)trace levels, was validated for all experimental matrices. The results document the key role of the malt contamination nature. While in the first monitoring period a significant increase of both DON and DON-3-Glc occurred (up to 250% and 450%, respectively), fairly different trends were observed when new malts were used for identical technological processing (in some beers a decrease of DON and only a small increase of DON-3-Glc occurred). Worth noticing, that the outcome of the brewing process was surprisingly reproducible for a particular malt mixture. In the final phase, a small monitoring study comparing Czech and Austrian alcohol-free and conventional beers was carried out.

The use of high performance liquid chromatography-quadrupole time-of-flight mass spectrometry coupled to advanced data mining and chemometric tools for discrimination and classification of red wines according to their variety.

In this study, the potential of high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (HPLC-QTOFMS) for metabolomic profiling of red wine samples was examined. Fifty one wines representing three varieties (Cabernet Sauvignon, Merlot, and Pinot Noir) of various geographical origins were sourced from the European and US retail market. To find compounds detected in analyzed samples, an automated compound (feature) extraction algorithm was employed for processing background subtracted single MS data. Stepwise reduction of the data dimensionality was followed by principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) which were employed to explore the structure of the data and construct classification models. The validated PLS-DA model based on data recorded in positive ionization mode enabled correct classification of 96% of samples. Determination of molecular formula and tentative identification of marker compound was carried out using accurate mass measurement of full single MS spectra. Additional information was obtained by correlating the fragments obtained by MS/MS accurate mass spectra using the QTOF with collision induced dissociation (CID) of precursor ions.

Identification/quantification of multiple pesticide residues in food plants by ultra-high-performance liquid chromatography-time-of-flight mass spectrometry.

In this study, the potential of ultra-high-performance liquid chromatography coupled with the time-of-flight mass spectrometry (UHPLC-TOF MS) to enable rapid and comprehensive analysis of 212 pesticide residues in QuEChERS extracts obtained from four plant matrices has been investigated. Method optimization is discussed in detail. In addition to molecular adducts, also fragment ions were provided for all target pesticides, thus obtaining at least three identification points required by European Decision 2002/657/EC was achieved. To get maximum information on analytes present in the extracts, each sample was examined within two injections, the first in a positive and the next one in a negative ionization mode. Under UHPLC conditions, both analyses were completed within 24min. For more than 96% of pesticides involved in this study, the limit of quantification was < or =10micro/kg. As a part of the work, strategy enabling screening of non-target pesticides and their metabolites is demonstrated on analysis of real-life samples.

Control of strobilurin fungicides in wheat using direct analysis in real time accurate time-of-flight and desorption electrospray ionization linear ion trap mass spectrometry.

Ambient mass spectrometry has been used for the analysis of strobilurin residues in wheat. The use of this novel, challenging technique, employing a direct analysis in a real time (DART) ion-source coupled with a time-of-flight mass spectrometer (TOF MS) and a desorption electrospray ionization (DESI) source coupled with a linear ion trap tandem MS (LIT MS(n)), permitted a direct screen of the occurrence of target fungicides in treated grains in less than 1 min. For quantification purpose by DART-TOF MS, an ethyl acetate extract had to be prepared. With the use of a prochloraz as an internal standard, the performance characteristics obtained by repeated analyses of extract, spiked at 50 microg kg(-1) with six strobilurins (azoxystrobin, picoxystrobin, dimoxystrobin, kresoxim-methyl, pyraclostrobin, and trifloxystrobin), were in the following range: recoveries 78-92%, repeatability (RSD) 8-15%, linearity (R(2)) 0.9900-0.9978. The analysis of wheat with incurred strobilurin residues demonstrated good trueness of data generated by the DART-TOF MS method; the results were in a good agreement with those obtained by the conventional approach, i.e., by the QuEChERS sample handling procedure followed by identification/quantification employing high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Tandem mass spectrometry using DESI-LIT MS(n) provided a sufficient number of product ions for confirmation of the identity of azoxystrobin and pyraclostrobin in incurred wheat samples.

Rapid analysis of multiple pesticide residues in fruit-based baby food using programmed temperature vaporiser injection-low-pressure gas chromatography-high-resolution time-of-flight mass spectrometry.

A rapid method using programmed temperature vaporiser injection-low-pressure gas chromatography-high-resolution time-of-flight mass spectrometry (PTV-LP-GC-HR-TOF-MS) for the analysis of multiple pesticide residues in fruit-based baby food was developed. The fast and inexpensive buffered QuEChERS (quick, easy, cheap, effective, rugged, and safe) extraction method and "conventional" approach that employs ethyl acetate extraction followed by gel permeation chromatography (GPC) cleanup were employed for sample preparation. A PTV injector in solvent venting mode was used to reduce volume of acetonitrile and acetic acid (from the buffered QuEChERS extracts) that caused higher column bleed without their elimination. Otherwise, the time-to-digital converter would become saturated in HR-TOF-MS. For fast GC separation allowing analysis of 100 analytes within a 7 min runtime, both a high temperature programming rate and vacuum conditions in a megabore GC column were employed. The use of HR-TOF-MS allowed the unbiased identification and reliable quantification of target analytes through the application of a narrow mass window (0.02 Da) for extracting analyte ions and the availability of full spectral information even at very low levels. With only a few exceptions, the lowest calibration levels for the pesticides tested were