Multilaboratory Collaborative Study of a Nontarget Data Acquisition for Target Analysis (nDATA) Workflow Using Liquid Chromatography-High-Resolution Accurate Mass Spectrometry for Pesticide Screening in Fruits and Vegetables.

Nontarget data acquisition for target analysis (nDATA) workflows using liquid chromatography-high-resolution accurate mass (LC-HRAM) spectrometry, spectral screening software, and a compound database have generated interest because of their potential for screening of pesticides in foods. However, these procedures and particularly the instrument processing software need to be thoroughly evaluated before implementation in routine analysis. In this work, 25 laboratories participated in a collaborative study to evaluate an nDATA workflow on high moisture produce (apple, banana, broccoli, carrot, grape, lettuce, orange, potato, strawberry, and tomato). Samples were extracted in each laboratory by quick, easy, cheap, effective, rugged, and safe (QuEChERS), and data were acquired by ultrahigh-performance liquid chromatography (UHPLC) coupled to a high-resolution quadrupole Orbitrap (QOrbitrap) or quadrupole time-of-flight (QTOF) mass spectrometer operating in full-scan mass spectrometry (MS) data-independent tandem mass spectrometry (LC-FS MS/DIA MS/MS) acquisition mode. The nDATA workflow was evaluated using a restricted compound database with 51 pesticides and vendor processing software. Pesticide identifications were determined by retention time (tR, ±0.5 min relative to the reference retention times used in the compound database) and mass errors (δM) of the precursor (RTP, δM ≤ ±5 ppm) and product ions (RTPI, δM ≤ ±10 ppm). The elution profiles of all 51 pesticides were within ±0.5 min among 24 of the participating laboratories. Successful screening was determined by false positive and false negative rates of <5% in unfortified (pesticide-free) and fortified (10 and 100 µg/kg) produce matrices. Pesticide responses were dependent on the pesticide, matrix, and instrument. The false negative rates were 0.7 and 0.1% at 10 and 100 µg/kg, respectively, and the false positive rate was 1.1% from results of the participating LC-HRAM platforms. Further evaluation was achieved by providing produce samples spiked with pesticides at concentrations blinded to the laboratories. Twenty-two of the 25 laboratories were successful in identifying all fortified pesticides (0-7 pesticides ranging from 5 to 50 µg/kg) for each produce sample (99.7% detection rate). These studies provide convincing evidence that the nDATA comprehensive approach broadens the screening capabilities of pesticide analyses and provide a platform with the potential to be easily extended to a larger number of other chemical residues and contaminants in foods.

Quantitation of Cannabinoids in Cannabis Dried Plant Materials, Concentrates, and Oils Using Liquid Chromatography-Diode Array Detection Technique with Optional Mass Spectrometric Detection: Single-Laboratory Validation Study, First Action 2018.11.

This paper describes a single-laboratory validation of a liquid chromatography-diode array detection (LC-DAD) method for quantification of 12 major cannabinoids in Cannabis dried plant materials, concentrates, and oils. The method met Standard Method Performance Requirements for quantitative analysis of cannabinoids in Cannabis concentrates and Cannabis dried plant materials. The LOQs were in the range 0.003-0.10% (w/w), depending on the analyte and matrix. Spike recoveries were between 96.7 and 101.3% with relative SDs (RSDs) ≤2.3%. Precision expressed as repeatability and intermediate precision was within 0.3-4.8 and 1.1-5.1%, respectively. The chromatographic separation conditions used in this versatile method are compatible with both DAD-UV and MS detection. During method validation, high-resolution quadrupole time-of-flight MS was employed as a secondary detector (connected in series to the LC-DAD instrument) to provide high confidence identification of target analytes and as a tool for monitoring other cannabinoids for which reference standards were not available. The obtained results demonstrate applicability of the method to quantitative analysis of important cannabinoids in dried plants, concentrates, and oils. Limited data were generated for a food matrix (Cannabis-containing cookies) using this method with LC coupled to a compact single quadrupole mass spectrometer.

Method development and validation for low-level propineb and propylenethiourea analysis in baby food, infant formula and related matrices using liquid chromatography-tandem mass spectrometry.

Two simple, selective and rugged liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed and validated for determination of propineb and propylenethiourea (PTU) in infant formula, fruit-based and cereal-based baby food and raw materials used in production of infant formula, including carbohydrates, protein isolates, vegetable oils and emulsifiers. The sample preparation procedure for propineb analysis was based on streamlined derivatisation to form and stabilise the target analyte (propylenebisdithiocarbamate-dimethyl), followed by extraction using a modified QuEChERS procedure with a dispersive solid phase extraction (d-SPE). The PTU determination employed an aqueous extraction with optimised protein precipitation and single-step SPE clean-up. To achieve maximum sensitivity, electrospray ionisation and atmospheric-pressure chemical ionisation were employed for LC-MS/MS analysis of propineb and PTU, respectively. Validation of the developed methods was performed in accordance with Document SANTE/11813/2017. Mean recoveries were in the range of 86-120% for propineb and PTU, respectively, with interday and intraday repeatabilities below 13%. A limit of quantification (LOQ) of 0.003 mg kg-1 was validated for most of the evaluated analyte/sample matrix combinations with the exception of PTU in soy protein isolate and soybean oil, for which an LOQ of 0.01 mg kg-1 was obtained. This is the first report that provides validated methods for monitoring propineb and PTU in infant formula and baby foods at concentrations compliant with the maximum residue levels established in the EU legislation.

Method development and validation for total haloxyfop analysis in infant formulas and related ingredient matrices using liquid chromatography-tandem mass spectrometry.

According to the European Commission directive 2006/141/EC, haloxyfop residue levels should not exceed 0.003 mg/kg in ready-to-feed infant formula, and the residue definition includes sum of haloxyfop, its esters, salts, and conjugates expressed as haloxyfop. A simple method for total haloxyfop analysis in infant formula and related ingredient matrices was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The sample preparation consisted of an alkaline hydrolysis with methanolic sodium hydroxide to release haloxyfop (parent acid) from its bound forms prior to the extraction with acetonitrile. A mixture of magnesium sulfate (MgSO4) and sodium chloride (NaCl) (4:1, w/w) was added to the extract to induce phase separation and force the analyte into the upper acetonitrile-methanol layer and then a 1-mL aliquot was subsequently cleaned up by dispersive solid phase extraction with 150 mg of MgSO4 and 50 mg of octadecyl (C18) sorbent. The analytical procedure was developed and carefully optimized to enable low-level, total haloxyfop analysis in a variety of challenging matrices, including infant formulas and their important high-carbohydrate, high-protein, high-fat, and emulsifier ingredients. The final method was validated in two different laboratories by fortifying samples with haloxyfop and haloxyfop-methyl, which was used as a model compound simulating bound forms of the analyte. Mean recoveries of haloxyfop across all fortification levels and evaluated matrices ranged between 92.2 and 114% with repeatability, within-lab reproducibility, and reproducibility RSDs ≤ 14%. Based on the validation results, this method was capable to convert the haloxyfop ester into the parent acid in a wide range of sample types and to reliably identify and quantify total haloxyfop at the target 0.003 mg/kg level in infant formulas (both powdered and ready-to-feed liquid forms). Graphical abstract LC-MS/MS-based workflow for the determination of the total haloxyfop in infant formula and related ingredients.

Impact of vacuum frying on quality of potato crisps and frying oil.

This research was focused on a critical assessment of vacuum frying as a technology enabling minimization of acrylamide formation in potato crisps and reducing undesirable chemical changes that occur in frying oil at high temperatures. The potato slices were fried in rapeseed oil under vacuum at 125°C and atmospheric pressure at 165°C. The experiments were performed on two potato varieties, Saturna and Impala. Vacuum frying reduced the formation of acrylamide by 98% and also other Maillard reaction products, specifically alkylpyrazines. Concurrently a lower extent of oxidative changes was observed in the frying oil, while 3-MCPD esters decreased fairly quickly during conventional frying. Sensory characteristics of the vacuum and conventionally fried potato crisps were evaluated by a 23-member panel. The majority of panellists preferred the flavour of 'conventional crisps', while only a few of them appreciated potato-like fresh flavour of 'vacuum crisps' and classified this product as 'tasty'.

Single-Laboratory Validation Study of a Method for Screening and Identification of Phosphodiesterase Type 5 Inhibitors in Dietary Ingredients and Supplements Using Liquid Chromatography/Quadrupole-Orbital Ion Trap Mass Spectrometry: First Action 2015.12.

A single-laboratory validation study of a method for screening and identification of phosphodiesterase type 5 (PDE5) inhibitors in dietary ingredients and supplements is described. PDE5 inhibitors were extracted from the samples using a 50:50 (v/v) mixture of acetonitrile and water and centrifuged. Supernatant was diluted, filtered, and analyzed by LC-high-resolution MS. Data were collected in MS acquisition mode that combined full-scan MS experiment with all-ion fragmentation and data-dependent MS/MS product from the ion scan experiment. This approach enabled collection of MS and tandem MS (MS/MS) data for both targeted and nontargeted PDE5 inhibitors in a single chromatographic run. Software-facilitated identification of targeted analytes was performed based on the retention time, accurate mass, and isotopic pattern of pseudomolecular ions, and accurate masses of fragment ions using an in-house compound database. Detection and identification of other PDE5 inhibitors and novel analogs were performed by retrospective evaluation of MS and MS/MS experimental data. The method validation results obtained for evaluated matrixes fulfilled the probability of identification requirements and probability of detection requirements (for the pooled data) set at 90% (95% confidence interval) in the respective AOAC Standard Method Performance Requirements for identification and screening methods for PDE5 inhibitors. Limited data demonstrating the quantification capability of the method were also generated. Mean recovery and repeatability obtained for the evaluated PDE5 inhibitors were in the range 69-90% and 0.4-1.8%, respectively.

Target and non-target analysis of migrants from PVC-coated cans using UHPLC-Q-Orbitrap MS: evaluation of long-term migration testing.

A simple, rapid and sensitive method for analyzing multi-target and non-target additives in polyvinyl chloride (PVC) food can coatings using ultra-high-performance liquid chromatography coupled to quadrupole-orbital ion-trap mass spectrometry was developed. This procedure was used to study the behaviour of a cross-linking agent, benzoguanamine (BGA), two slip agents, oleamide and erucamide, and 18 other commonly used plasticisers including phthalates, adipates, sebacates, acetyl tributyl citrate and epoxidised soybean or linseed oils. This optimised method was used to detect these analytes in food simulants (water and 3% acetic acid) in a long-term migration test of PVC-coated food cans for a period ranging from 1 day to 1.5 years at 40°C. Although very low detection limits (5 ng ml(-1)) were obtained for the majority of compounds, none of the monitored plasticisers and slip agents was detected in simulants extracted from cans over the period of the test. However, the presence of BGA in both aqueous food simulants was confirmed based on high-resolution mass spectrometry, product ion spectra and analysis of a reference standard. The BGA concentration in both simulants continued to increase with storage time: after 1.5 years storage in aqueous food simulants at 40°C, BGA was detected at concentrations up to 84 µg dm(-2). We believe this is the first study describing the long-term migration capacity of BGA from any vinyl coating material intended for use in PVC-coated food cans. Our results may have implications for migration test protocols for food cans that will be stored for extended time periods.

Prediction of acrylamide formation in biscuits based on fingerprint data generated by ambient ionization mass spectrometry employing direct analysis in real time (DART) ion source.

The objective of this study is the evaluation of the potential of high-throughput direct analysis in real time-high resolution mass spectrometry (DART-HRMS) fingerprinting and multivariate regression analysis in prediction of the extent of acrylamide formation in biscuit samples prepared by various recipes and baking conditions. Information-rich mass spectral fingerprints were obtained by analysis of biscuit extracts for preparation of which aqueous methanol was used. The principal component analysis (PCA) of the acquired data revealed an apparent clustering of samples according to the extent of heat-treatment applied during the baking of the biscuits. The regression model for prediction of acrylamide in biscuits was obtained by partial least square regression (PLSR) analysis of the data matrix representing combined positive and negative ionization mode fingerprints. The model provided a least root mean square error of cross validation (RMSECV) equal to an acrylamide concentration of 5.4 µg kg(-1) and standard error of prediction (SEP) of 14.8 µg kg(-1). The results obtained indicate that this strategy can be used to accurately predict the amounts of acrylamide formed during baking of biscuits. Such rapid estimation of acrylamide concentration can become a useful tool in evaluation of the effectivity of processes aiming at mitigation of this food processing contaminant. However, the robustness this approach with respect to variability in the chemical composition of ingredients used for preparation of biscuits should be tested further.

Mass spectrometric analysis of pharmaceutical adulterants in products labeled as botanical dietary supplements or herbal remedies: a review.

The increased availability and use of botanical dietary supplements and herbal remedies among consumers has been accompanied by an increased frequency of adulteration of these products with synthetic pharmaceuticals. Unscrupulous producers may add drugs and analogues of various classes, such as phosphodiesterase type 5 (PDE-5) inhibitors, weight loss, hypoglycemic, antihypertensive and anti-inflammatory agents, or anabolic steroids, to develop or intensify biological effects of dietary supplements or herbal remedies. The presence of such adulterated products in the marketplace is a worldwide problem and their consumption poses health risks to consumers. Analytical methods that allow rapid and reliable testing of dietary supplements for the presence of synthetic drugs are needed to address such fraudulent practices. Mass spectrometry (MS) and hyphenated techniques such as liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) have become primary tools in this endeavor. The present review critically assesses the role and summarizes the applications of MS in the analysis of pharmaceutical adulterants in botanical dietary supplements and herbal remedies. The uses of MS techniques in detection, confirmation, and quantification of known pharmaceutical adulterants as well as in screening for and structure elucidation of unexpected adulterants and novel designer drugs are discussed.

Quantification of aristolochic acids I and II in herbal dietary supplements by ultra-high-performance liquid chromatography-multistage fragmentation mass spectrometry.

A rapid, selective and sensitive ultra-high-performance liquid chromatography-multistage fragmentation mass spectrometry (UHPLC-MS³) method was developed and evaluated for the determination of aristolochic acids I and II (AA I and II) in herbal dietary supplements. A hybrid triple quadrupole/linear ion-trap mass spectrometry was used to monitor MS³ ion transitions m/z 359.2 > 298.1 > 268.0 and m/z 329.2 > 268.2 > 238.0 to detect AA I and II, respectively. The extraction and clean-up of target analytes from dry powdered samples was performed using the quick, easy, cheap, effective, rugged and safe (QuEChERS) procedure. Herbal liquid extracts were analysed directly. Average recoveries ranged from 89% to 112%, with relative standard deviations (RSDs) ranging from 3% to 16%. Limits of quantification (LOQs) estimated for three selected matrices were as follows (AA I/II): 5/10 ng g⁻¹ (tablets); 25/50 ng g⁻¹ (capsules); and 2.5/5.0 ng ml⁻¹ (liquid herbal extract). The method was applied in a limited survey of 30 herbal products marketed in the United States via the Internet. AA I and II were detected in 20% and 7%, respectively, of tested samples.

Rapid LC-MS-based metabolomics method to study the Fusarium infection of barley.

Ultra high performance liquid chromatography with quadrupole/time-of-flight mass spectrometry was applied to evaluate the potential of nontarget metabolomic fingerprinting in order to distinguish Fusarium-infected and control barley samples. First, the sample extraction and instrumental conditions were optimized to obtain the broadest possible representation of polar/medium-polar compounds occurring in extracts obtained from barley grain samples. Next, metabolomic fingerprints of extracts obtained from nine barley varieties were acquired under ESI conditions in both positive and negative mode. Each variety of barley was tested in two variants: artificially infected by Fusarium culmorum at the beginning of heading and a control group (no infection). In addition, the dynamics of barley infection development was monitored using this approach. The experimental data were statistically evaluated by principal component analysis, hierarchical clustering analysis, and orthogonal partial least-squares discriminant analysis. The differentiation of barley in response to F. culmorum infection was feasible using this metabolomics-based method. Analysis in positive mode provided a higher number of molecular features as compared to that performed under negative mode setting. However, the analysis in negative mode permitted the detection of deoxynivalenol and deoxynivalenol-3-glucoside considered as resistance-indicator metabolites in barley.

Targeted analysis of multiple pharmaceuticals, plant toxins and other secondary metabolites in herbal dietary supplements by ultra-high performance liquid chromatography-quadrupole-orbital ion trap mass spectrometry.

In this study, an ultra-high performance liquid chromatography-quadrupole-orbital ion trap mass spectrometry (UHPLC-Q-orbitrap MS) method was developed and validated for simultaneous determination of 96 pharmaceuticals, plant toxins, and other plant secondary metabolites in herbal dietary supplements. Target analytes were extracted from samples using the QuEChERS (quick easy cheap effective rugged safe) procedure. The instrument was operated in full MS-data dependent tandem mass spectrometry (full MS-dd-MS/MS) acquisition mode which enabled collection of quantitative high resolution (HR) full mass spectral data and confirmatory HR MS/MS data in a single run. The method provided excellent selectivity in both full MS and dd-MS/MS mode. Under optimized collision energy settings, product ion spectra containing both precursor and two or more product ions were obtained for most of the analytes. Limits of detection (LODs) and limits of quantification (LOQs) for the method differed significantly for the examined matrices. LODs≤10µg kg(-1) and LOQs≤50µg kg(-1) were obtained for 48 to 81% of target compounds across five different matrices. With the exception of highly polar analytes, the optimized QuEChERS extraction procedure provided acceptable recoveries in the range 70%-120%. The precision of the method, characterized as the relative standard deviation (RSD, n=5), was ≤25% and ≤18% at spiking concentrations of 50µg kg(-1) and 500µg kg(-1), respectively. Because of variations in matrix effects in extracts of herbal dietary supplements that differed in composition, the method of standard additions and an approach based on dilution of matrix components followed by quantification using solvent standards were applied for quantification. The procedure was used to examine commercial dietary supplements for the 96 analytes of interest. To the best of our knowledge, this is the first report of an integrated analysis and quantification of this wide range of compounds.

Determination of multiple mycotoxins in dietary supplements containing green coffee bean extracts using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).

An ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the determination of 34 mycotoxins in dietary supplements containing green coffee bean (GCB) extracts was developed, evaluated, and used in the analysis of 50 commercial products. A QuEChERS-like procedure was used for isolation of target analytes from the examined matrices. Average recoveries of the analytes were in the range of 75-110%. The precision of the method expressed as relative standard deviation was below 12%. Limits of detection (LODs) and limits of quantitation (LOQs) ranged from 1.0 to 50.0 µg/kg and from 2.5 to 100 µg/kg, respectively. Due to matrix effects, the method of standard additions was used to ensure accurate quantitation. Ochratoxin A, ochratoxin B, fumonisin B1 and mycophenolic acid were found in 36%, 32%, 10%, and 16% of tested products, respectively. Mycotoxins occurred in the following concentration ranges: ochratoxin A, <1.0-136.9 µg/kg; ochratoxin B, <1.0-20.2 µg/kg; fumonisin B1, <50.0-415.0 µg/kg; mycophenolic acid, <5.0-395.0 µg/kg. High-resolution mass spectrometry operated in full MS and MS/MS mode was used to confirm the identities of the reported compounds.

Rapid monitoring of heat-accelerated reactions in vegetable oils using direct analysis in real time ionization coupled with high resolution mass spectrometry.

Transmission-mode direct analysis in real time ionization coupled with high resolution mass spectrometry (TM-DART-HRMS) was used to monitor chemical changes in various vegetable oils (olive, rapeseed, soybean and sunflower oil) during their thermally-induced oxidation. This novel instrumental approach enabled rapid fingerprinting of examined samples and detection of numerous sample components, such as triacylglycerols (TAGs), phytosterols, free fatty acids (FFA), and their respective oxidation products. Mass spectra obtained from DART were processed with the use of principal component analysis (PCA) in order to assess the compositional differences between heated and non-heated samples. Good correlation was observed between the normalized intensities of the pre-selected ion corresponding to mono-oxidized TAG and 'classic' criterion represented by the levels of TAG polymers determined by high performance-size exclusion chromatography with refractometric detection (HP-SEC-RID).

Mass spectrometry-based metabolomic fingerprinting for screening cold tolerance in Arabidopsis thaliana accessions.

The availability of rapid and reliable tools for monitoring of plants' cold tolerance is a prerequisite for research aimed at breeding of cold-tolerant crop plants. Therefore, we have tested the capacity of metabolomics-based methods employing ultra-high-performance liquid chromatography (UHPLC)-mass spectrometry and direct analysis in real time-mass spectrometry for high-throughput screening of cold tolerance in eight differentially cold-tolerant accessions of Arabidopsis thaliana. Metabolomic fingerprinting of leaf tissues was performed in methanolic extracts for (1) 6-week-old non-acclimated (NAC) plants grown at room temperature, (2) NAC plants cold-acclimated (ACC) at 4 °C for 2 weeks, and (3) cold-acclimated plants given sub-zero-temperature treatments by slow cooling at -4 °C for 8 h. The generated chromatograms and mass spectra were processed with the use of multivariate statistical analysis employing principal component analysis (PCA) and linear discriminant analysis. The PCA of metabolomic fingerprints classified the investigated A. thaliana accessions into three categories with low, intermediate, and high cold tolerance for both the cold-acclimated and the sub-zero-temperature-treated plants. This indicates the potential application of metabolomics-based fingerprinting for measuring cold tolerance in the cold-acclimated state, i.e., without treating plants at freezing temperatures that is required by currently available methods. Furthermore, we employed UHPLC coupled to the quadrupole-time-of-flight mass spectrometry to identify characteristic metabolites in ACC state and found the abundance of gluconapin and flavon-3-ol glycosides, respectively, in the cold-sensitive and the cold-tolerant accessions.

Direct analysis in real time high-resolution mass spectrometry for high-throughput analysis of antiparasitic veterinary drugs in feed and food.

RATIONALE: Direct analysis in real time (DART) is a novel ionization technique that has been demonstrated in numerous applications as a useful tool for fast and convenient mass spectrometry (MS)-based analysis of complex samples. In this study, the feasibility of DART ionization coupled to a high-resolution mass spectrometer utilizing an orbitrap mass analyzer (orbitrap MS) for high-throughput analysis of antiparasitic veterinary drugs was explored. METHODS: To obtain the best DART-orbitrap MS performance, stepwise optimization of instrumental parameter settings, such as ionization gas temperature and mass resolving power, was performed. The optimized method was applied to feed and bovine milk samples previously extracted following a QuEChERS-like strategy. RESULTS: Most antiparasitic drugs could be analyzed following the described method. Positive DART ionization provided the protonated molecules [M+H](+); in negative DART ion mode, deprotonated molecules [M-H](-) were observed. As an exception, polyether ionophores could be observed as the sodiated adducts [M+Na](+). Samples of milk and feed were extracted using a modified QuEChERS method for the determination of benzimidazoles and coccidiostats respectively and quantification was carried out by matrix-matched calibration curves. CONCLUSIONS: The combination of an analysis time of less than 1 min per sample and the possibility to acquire accurate masses under high mass resolving power (HR) makes the DART-HRMS technique an effective tool for rapid qualitative screening of antiparasitic veterinary drugs. Additionally, the results obtained in this study demonstrated the feasibility of this approach to quantify target analytes at levels down to 1 µg kg(-1) for benzimidazolic compounds in milk and 0.25 mg kg(-1) for coccidiostats in chicken feed.

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.

Rapid analysis of caffeine in various coffee samples employing direct analysis in real-time ionization-high-resolution mass spectrometry.

The development and use of a fast method employing a direct analysis in real time (DART) ion source coupled to high-resolution time-of-flight mass spectrometry (TOFMS) for the quantitative analysis of caffeine in various coffee samples has been demonstrated in this study. A simple sample extraction procedure employing hot water was followed by direct, high-throughput (<1 min per run) examination of the extracts spread on a glass rod under optimized conditions of ambient mass spectrometry, without any prior chromatographic separation. For quantification of caffeine using DART-TOFMS, an external calibration was used. Isotopically labeled caffeine was used to compensate for the variations of the ion intensities of caffeine signal. Recoveries of the DART-TOFMS method were 97% for instant coffee at the spiking levels of 20 and 60 mg/g, respectively, while for roasted ground coffee, the obtained values were 106% and 107% at the spiking levels of 10 and 30 mg/g, respectively. The repeatability of the whole analytical procedure (expressed as relative standard deviation, RSD, %) was <5% for all tested spiking levels and matrices. Since the linearity range of the method was relatively narrow (two orders of magnitude), an optimization of sample dilution prior the DART-TOFMS measurement to avoid saturation of the detector was needed.

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.

Precursors and formation of pyrithione and other pyridyl-containing sulfur compounds in drumstick onion, Allium stipitatum.

Two novel, structurally unusual cysteine derivatives were isolated from the bulbs of Allium stipitatum (Allium subg. Melanocrommyum) and shown to be S-(2-pyridyl)cysteine N-oxide and S-(2-pyridyl)glutathione N-oxide. The former compound is the first example of a naturally occurring alliinase substrate that contains an N-oxide functionality instead of the S-oxide group. In addition, S-methylcysteine S-oxide (methiin) and S-(methylthiomethyl)cysteine 4-oxide (marasmin) were found in the bulbs. Presented data suggest that the previously reported identification of S-(2-pyridyl)cysteine S-oxide was most likely erroneous. The alliinase-mediated formation of pyridyl-containing compounds following disruption of A. stipitatum bulbs was studied by a combination of HPLC-MS, HPLC-PDA, DART-MS, and NMR techniques. It was found that no pyridyl-containing thiosulfinates are present in homogenized bulbs in detectable quantities. Instead, various pyridine N-oxide derivatives are formed, including N-hydroxypyridine-2(1H)-thione (pyrithione), 2-(methyldithio)pyridine N-oxide, 2-[(methylthio)methyldithio]pyridine N-oxide, di(2-pyridyl) disulfide N-oxide, and di(2-pyridyl) disulfide N,N'-dioxide. This represents the first report of pyrithione formation as a natural product.