Characterization of Humulus lupulus glycosides with porous graphitic carbon and sequential high performance liquid chromatography quadrupole time-of-flight mass spectrometry and high performance liquid chromatography fractionation.

Monoterpenes contribute to the characteristic aroma of several hop varieties and may occur as nonvolatile glycosides. Upon hydrolysis, the volatile terpenes are released from the glycoside precursors. Little is known, however, about the glycoside composition of hops. Seven pentose-hexose monoterpene alcohol glycosides from dried Humulus lupulus L. cv. Citra cones were isolated using high performance liquid chromatography separation and fractionation on a reverse phase phenyl-hexyl column. Further evaluation of each isolated fraction through HPLC qTOF MS with porous graphitic carbon (PGC) showed that the seven isolated monoterpenyl glycoside fractions could be further resolved into 20 isomers. Isolation on phenyl-hexyl followed by separation on PGC was needed to distinguish each isomer present. Additionally, the hop cones were screened for potential aroma glycosides. Using the PGC column combined with a database of over 900 potential glycosides, the identification of 21 additional monoterpene-polyol, norisoprenoid, volatile phenol, and aliphatic alcohol glycosides is reported.

Characterization of Free and Bound Monoterpene Alcohols during Riesling Fermentation.

The isomeric nature of monoterpenyl glycosides makes unambiguous identification of intact glycosides difficult. As a result, it is challenging to relate the changes in free monoterpenol concentrations to the corresponding glycosides during wine fermentation and storage. In this study, we isolated and identified linalool, nerol, and geraniol monoterpenyl glycosides fromVitis viniferacv. Riesling grapes through fractionation followed by acid or enzyme hydrolysis. Changes in the composition of identified monoterpenyl glycosides and their respective free volatiles were then monitored during alcoholic fermentations of Riesling juice with four different yeast strains across two successive years. The relative concentrations of the volatiles were monitored by solid-phase microextraction gas chromatography mass spectrometry, while ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry was used for intact glycosides. Glycoside hydrolysis during fermentation could be related to relative concentrations of the corresponding free aglycones. However, other sources of free monoterpenols were also observed. Differences in glycoside hydrolysis among yeast strains and across years were observed and may be related to grape maturity and/or nutrient levels.

Wildfires: Identification of a new suite of aromatic polycarboxylic acids in ash and surface water.

Ash and surface water samples collected after wildfires in four different geographical locations (California, Colorado, Kansas and Alberta) were analyzed. The ash samples were leached with deionized water, and leachates were concentrated by solid phase extraction and analyzed by liquid chromatography/time-of-flight mass spectrometry. In addition, three surface water samples and a lysimeter water sample were collected from watersheds recently affected by fire in California and Colorado, and analyzed in similar fashion. A suite of benzene polycarboxylic acids (BPCAs), with two and three carboxyl groups and their corresponding isomers were identified for the first time in both ash leachates and water samples. Also found was a pyridine carboxylic acid (PCA), 3,5-pyridine dicarboxylic acid. Furthermore, putative identifications were made for other carboxylated aromatic acids: quinolinic, naphthalenic, and benzofuranoic acid carboxylates. The wildfire ashes, a controlled wood ash, and post-fire surface water samples suggest that burned woody material, along with surface plant-material and heated o-horizon soil organic matter, contribute to both BPCAs and PCAs in runoff. This study is the first of its kind to identify this suite of aromatic acids in wildfire ash and surface water samples. These data make an important contribution to the nature of dissolved organic matter from wildfire and are useful to better understand the impact of wildfire on water quality and drinking water sources.

Nontargeted Screening of Water Samples Using Data-Dependent Acquisition with Similar Partition Searching.

A rapid screening method for the detection of nontargeted compounds in surface water samples was developed using MS-MS high-resolution mass spectrometry and data-dependent acquisition. The key parameters for the acquisition method were optimized using five model compounds with diverse chemical characteristics. The parameter selection required optimization between the total number of precursor ions that could be selected in an LC-MS run, the quality of each MS (full range) spectrum, and the quality of each MS-MS fragmentation spectrum. After the acquisition method was optimized, 18 surface water samples from rivers, reservoirs, and effluents from wastewater treatment plants were analyzed, generating 41625 MS-MS spectra in about 14 h. The raw data were then converted into two generic formats using the open-access program MSConvert. A combinatorial approach, similar partition searching (SPS), was then used to putatively identify analytes from the accurate mass of each analyte (adjusted for the adduct mass) and the corresponding MS-MS spectra were obtained. In this approach, the structures of about 250000 common compounds, stored in a large database as mathematical partitions of their exact mass, were compared directly to each MS-MS spectrum. Compounds with a similar mass and retention time were grouped together and labeled as "Analytes", using an Excel Add-In. The isotope ratio data from the MS spectrum, the corresponding MS-MS spectra, and the putative identifications were then imported into an Access relational database to facilitate sorting, searching, filtering, and querying the results. This allowed final inspection to assess confidence of the identifications made through the nontargeted screening.

Direct Analysis of Glycosidic Aroma Precursors Containing Multiple Aglycone Classes in Vitis vinifera Berries.

Ultra-high-performance liquid chromatography (UHPLC) accurate mass tandem mass spectrometry is a powerful tool for identifying and profiling plant metabolites. Here, we describe an approach to characterize glycosidically bound precursors of monoterpenoids, norisoprenoids, volatile phenols, aliphatic alcohols, and sesquiterpenoids in grapes. Chromatographic separation of glycosylated compounds was evaluated using phenyl-hexyl (reverse phase), glycan/hydrophilic interaction, and porous graphitic carbon (PGC) stationary phases. PGC provided the best UHPLC separation for 102 tentatively identified aroma precursors in Vitis vinifera L. cv. Riesling and Muscat of Alexandria berries. Monoterpene-triol, monoterpene-tetraol, and sesquiterpenol glycosides were tentatively identified for the first time in grapes, and a C6-alcohol trisaccharide was tentatively identified for the first time in any plant. Comparison of glycosylated aroma molecules in Riesling and Muscat of Alexandria grapes showed that the two varieties were distinguishable based on relative abundances of shared glycosides and the presence of glycosides unique to a single variety.

Direct injection high performance liquid chromatography coupled to data independent acquisition mass spectrometry for the screening of antibiotics in honey.

The targeted analysis of veterinary drug residues in honey traditionally involves a series of extraction and purification steps prior to quantification with high performance liquid chromatography coupled to high resolution or tandem mass spectrometry. These steps, designed to separate the target analytes from interferences, are generally time-consuming and costly. In addition, traditional cleanup steps are likely to eliminate other compounds whose analysis could prove decisive in current or future assessment of the honey sample. Alternatively, direct injection without complex sample preparation steps has been introduced for the fast analysis of trace compounds in environmental and food matrices. The aim of this study was to develop a rapid method for the targeted analysis of 7 key veterinary drug residues in honey based on direct injection high performance liquid chromatography coupled to quadrupole time-of-flight, while simultaneously recording data-independent MS/MS (e.g. All Ions MS/MS data) for future re-examination of the data for other purposes. The new method allowed for the detection of the target residues at levels approximately 20-100 times lower than current regulatory limits, for a total analysis time of about 45 min. The recoveries (103-119%), the linearity (R ≥ 0.996) and the repeatability (RSD ≤ 7%) were satisfactory. The method was then applied to 35 honey samples from the Canadian market. Residues of tylosin A, tylosin B, sulfamethazine and sulfadimethoxine were detected in 6, 9, 6 and 23% of the samples respectively, at levels below the regulatory limits in Canada. The possibility of adding a hydrolysis step to study sulfonamides in honey was tested, which provided good results for this family of compounds but lead to degradation of some of the other analytes. Finally, the non-targeted identification of several compounds was demonstrated as a proof of concept of future re-examination of All Ions MS/MS data. This paper illustrates the capacity of this novel method to combine targeted and non-targeted screening of chemical residues in honey.

Changes in glycosylation patterns of monoterpenes during grape berry maturation in six cultivars of Vitis vinifera.

Plants conjugate monoterpenoids to sugars, rendering them non-volatile. Hydrolysis of these glycosidic precursors frees the volatile aroma compounds. Here, we profile intact monoterpenyl glycosides in six Vitis vinifera grape berry cultivars. Relative concentrations of twenty-six monoterpenyl glycosides, including nine new putatively identified compounds, were analyzed by UHPLC-QTOF MS/MS at three times during grape maturation (pre-véraison, véraison, and post-véraison). Total glycoside content reached a maximum in Muscat cultivars post-véraison but remained relatively constant in all other cultivars. Three types of monoterpenyl glycosides predominated in all samples: malonylated monoterpenol glucosides, monoterpenol hexose-pentoses, and monoterpendiol hexose-pentoses. The two Muscat cultivars were not differentiated at the earlier developmental stages but could be differentiated post-véraison. In contrast, similarities between Chardonnay and Pinot noir glycoside profiles developed post-véraison. Overall monoterpene glycoconjugation patterns may align with underlying genetic relationships among cultivars. By understanding monoterpene glycoconjugation in wine grapes, scientists and winemakers can better understand grape and wine aromas.

Chemical Characteristics of Sangiovese Wines from California and Italy of 2016 Vintage.

Sangiovese is the most widespread Italian red cultivar and constitutes the basis of internationally known wines such as Chianti and Brunello di Montalcino. Outside of Europe, Argentina is the largest producer, followed by the United States. This study sought to define and compare 2016 vintage Sangiovese wine composition from various production regions in California and Italy. Forty-six commercial Sangiovese wines from California and Italy were analyzed for volatile profile, color, phenolic, and elemental content. This study demonstrates that it is possible to determine regional differences among wines based on these chemical profiles. However, some Californian and Italian wine had similar chemical compositions. In order to compare Californian and Italian wines, Californian wine reference models were developed using the chemical parameters from Sangiovese wines, performing a Soft Independent Modeling of Class Analogy (SIMCA). To our knowledge, this is the first time that an extensive regionality study has been attempted for Sangiovese wines.

Characterization of Red Wine Proanthocyanidins Using a Putative Proanthocyanidin Database, Amide Hydrophilic Interaction Liquid Chromatography (HILIC), and Time-of-Flight Mass Spectrometry.

Proanthocyanidins are complex polymers of flavan-3-ol monomers and play a key sensory and health role in foods and beverages. We describe here a novel method for characterizing wine proanthocyanidins using a theoretical database comprised of the chemical formula and exact mass of 996 compounds. The database was constructed using the four primary grape and wine proanthocyanidin monomers: (epi)catechin, (epi)catechin-3-O-gallate, (epi)gallocatechin, and (epi)gallocatechin-3-O-gallate, each combined in all possible combinations up to a polymerization of 10. The database was queried against spectra collected using ultrahigh performance liquid chromatography (UHLPC) with a hydrophilic interaction liquid chromatography (HILIC) column and coupled to a high-resolution accurate mass quadrupole time-of-flight mass spectrometer (Q-TOF MS). Two wine samples produced with different post fermentation maceration were analyzed using the presented method to demonstrate application for analysis of diverse proanthocyanidins. The first sample was pressed immediately at the end of fermentation when all sugar had been utilized and the second received eight weeks of post fermentation maceration. The HILIC column combined with high resolution tandem mass spectrometry and database matching provided tentative identification of 89 compounds with excellent resolution and without the need for two-dimensional separations. The identified compounds were visualized with Kendrick mass analysis, a simple technique allowing for rapid visualization of which compounds are present in a given sample.

Quantitation of Oleuropein and Related Phenolics in Cured Spanish-Style Green, California-Style Black Ripe, and Greek-Style Natural Fermentation Olives.

Oleuropein, ligstroside, and related hydrolysis products are key contributors to olive bitterness, and several of these phenolics are implicated in the prevention of lifestyle age-related diseases. While table olive processing methods are designed to reduce oleuropein, the impact of processing on ligstroside and related hydrolysis products (e.g., oleacein, oleocanthal, hydroxytyrosol glucoside, ligstroside aglycone, and oleuropein aglycone) is relatively unknown. Herein, levels of these compounds were measured in Spanish-style green (SP), Californian-style black ripe (CA), and Greek-style natural fermentation (GK) olives using rapid ultrahigh-performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS). GK olives had the highest concentration of all compounds measured, with the exception of oleocanthal, which was highest in SP olives (0.081 mg kg-1 wet weight (w.wt)). CA olives had the lowest levels of most compounds measured, including ligstroside (0.115 mg kg-1 w.wt) and oleuropein (0.974 mg kg-1 w.wt). Hydroxytyrosol was the predominate compound in all three styles of commercial olives, with similar concentrations observed for GK and SP olives (134.329 and 133.685 mg kg-1 w.wt, respectively) and significantly lower concentrations observed for CA olives (19.981 mg kg-1 w.wt).

The Influence of pH and Sodium Hydroxide Exposure Time on Glucosamine and Acrylamide Levels in California-Style Black Ripe Olives.

Acrylic acid, N-acetyl-glucosamine and glucosamine were investigated for their role in the formation of acrylamide in California-style black ripe olives [CBROs]. Levels of acrylic acid and glucosamine are reported for the first time in fresh (333.50 ± 21.88 and 243.59 ± 10.06 nmol/g, respectively) and in brine-stored olives (184.50 ± 6.02 and 165.88 ± 11.51 nmol/g, respectively). Acrylamide levels significantly increased when acrylic acid (35.2%), N-acetyl-glucosamine (29.9%), and glucosamine (124.0%) were added to olives prior to sterilization. However, isotope studies indicate these compounds do not contribute carbon and/or nitrogen atoms to acrylamide. The base-catalyzed degradation of glucosamine is demonstrated in olive pulp and a strong correlation (r2 = 0.9513) between glucosamine in olives before sterilization and acrylamide formed in processed CBROs is observed. Treatment with sodium hydroxide (pH > 12) significantly reduces acrylamide levels over 1 to 5 d without impacting olive fruit texture.

Occurrence of Ochratoxin A in Infant Foods in the United States.

Ochratoxin A (OTA) is a possible human carcinogen and occurs frequently in cereal grain, soy, and other agricultural commodities. Infants and young children may be more susceptible to contaminants than adults because of their lower body weight, higher metabolic rate, reduced ability to detoxify food toxicants, and more restricted diet. The purpose of this study was to investigate the occurrence and levels of OTA in infant formula and infant cereal products available in the U.S. market. In the present study, 98 powdered infant formula (milk- and soy-based) samples and 155 infant cereal (barley-, rice-, oat-, wheat-, and mixed grain-based) products were collected from different retail locations in the United States over a 2-year period. OTA levels were determined by liquid chromatography-tandem mass spectrometry. Although OTA was not detected in any of the infant formula samples, 47 (30%) of 155 infant cereals were contaminated with OTA in the range of 0.6 to 22.1 ng/g. At present, there is no regulatory limit for OTA in the United States. However, all of the positive samples were above the maximum level set by the European Commission (0.5 ng/g) for OTA in baby foods. OTA was detected in all types of infant cereals, but the highest incidence and concentrations were found in oat-based infant cereals (59%), followed by mixed grain cereals (34%). Increased surveillance and monitoring of OTA levels in grains used in infant foods may be needed to reduce exposure of infants and young children to OTA from cereal products.

Comparison of veterinary drug residue results in animal tissues by ultrahigh-performance liquid chromatography coupled to triple quadrupole or quadrupole-time-of-flight tandem mass spectrometry after different sample preparation methods, including use of a commercial lipid removal product.

Veterinary drug residues in animal-derived foods must be monitored to ensure food safety, verify proper veterinary practices, enforce legal limits in domestic and imported foods, and for other purposes. A common goal in drug residue analysis in foods is to achieve acceptable monitoring results for as many analytes as possible, with higher priority given to the drugs of most concern, in an efficient and robust manner. The U.S. Department of Agriculture has implemented a multiclass, multi-residue method based on sample preparation using dispersive solid phase extraction (d-SPE) for cleanup and ultrahigh-performance liquid chromatography-tandem quadrupole mass spectrometry (UHPLC-QQQ) for analysis of >120 drugs at regulatory levels of concern in animal tissues. Recently, a new cleanup product called "enhanced matrix removal for lipids" (EMR-L) was commercially introduced that used a unique chemical mechanism to remove lipids from extracts. Furthermore, high-resolution quadrupole-time-of-flight (Q/TOF) for (U)HPLC detection often yields higher selectivity than targeted QQQ analyzers while allowing retroactive processing of samples for other contaminants. In this study, the use of both d-SPE and EMR-L sample preparation and UHPLC-QQQ and UHPLC-Q/TOF analysis methods for shared spiked samples of bovine muscle, kidney, and liver was compared. The results showed that the EMR-L method provided cleaner extracts overall and improved results for several anthelmintics and tranquilizers compared to the d-SPE method, but the EMR-L method gave lower recoveries for certain ß-lactam antibiotics. QQQ vs. Q/TOF detection showed similar mixed performance advantages depending on analytes and matrix interferences, with an advantage to Q/TOF for greater possible analytical scope and non-targeted data collection. Either combination of approaches may be used to meet monitoring purposes, with an edge in efficiency to d-SPE, but greater instrument robustness and less matrix effects when analyzing EMR-L extracts. Graphical abstract Comparison of cleanup methods in the analysis of veterinary drug residues in bovine tissues.

U.S. domestic cats as sentinels for perfluoroalkyl substances: Possible linkages with housing, obesity, and disease.

Perfluoroalkyl substances (PFAS), such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), are persistent, globally distributed, anthropogenic compounds. The primary source(s) for human exposure are not well understood although within home exposure is likely important since many consumer products have been treated with different PFAS, and people spend much of their lives indoors. Herein, domestic cats were used as sentinels to investigate potential exposure and health linkages. PFAS in serum samples of 72 pet and feral cats, including 11 healthy and 61 with one or more primary disease diagnoses, were quantitated using high-resolution time-of-flight mass spectroscopy. All but one sample had detectable PFAS, with PFOS and perfluorohexane sulfonate (PFHxS) ranging from

Nontargeted Screening of Food Matrices: Development of a Chemometric Software Strategy To Identify Unknowns in Liquid Chromatography-Mass Spectrometry Data.

The ability to identify contaminants or adulterants in diverse, complex sample matrixes is necessary in food safety. Thus, nontargeted screening approaches must be implemented to detect and identify unexpected, unknown hazardous compounds that may be present. Molecular formulas can be generated for detected compounds from high-resolution mass spectrometry data, but analysis can be lengthy when thousands of compounds are detected in a single sample. Efficient data mining methods to analyze these complex data sets are necessary given the inherent chemical diversity and variability of food matrixes. The aim of this work is to determine necessary requirements to successfully apply data analysis strategies to distinguish suspect and control samples. Infant formula and orange juice samples were analyzed with one lot of each matrix containing varying concentrations of a four compound mixture to represent a suspect sample set. Small molecular differences were parsed from the data, where analytes as low as 10 ppb were revealed. This was accomplished, in part, by analyzing a quality control standard, matrix spiked with an analytical standard mixture, technical replicates, a representative number of sample lots, and blanks within the sample sequence; this enabled the development of a data analysis workflow and ensured that the employed method is sufficient for mining relevant molecular features from the data.

Discovery of highly conserved unique peanut and tree nut peptides by LC-MS/MS for multi-allergen detection.

Proteins unique to peanuts and various tree nuts have been extracted, subjected to trypsin digestion and analysis by liquid chromatography/quadrupole time-of-flight mass spectrometry, in order to find highly conserved peptides that can be used as markers to detect peanuts and tree nuts in food. The marker peptide sequences chosen were those found to be present in both native (unroasted) and thermally processed (roasted) forms of peanuts and tree nuts. Each peptide was selected by assuring its presence in food that was processed or unprocessed, its abundance for sensitivity, sequence size, and uniqueness for peanut and each specific variety of tree nut. At least two peptides were selected to represent peanut, almond, pecan, cashew, walnut, hazelnut, pine nut, Brazil nut, macadamia nut, pistachio nut, chestnut and coconut; to determine the presence of trace levels of peanut and tree nuts in food by a novel multiplexed LC-MS method.

Profiling monoterpenol glycoconjugation in Vitis vinifera L. cv. Muscat of Alexandria using a novel putative compound database approach, high resolution mass spectrometry and collision induced dissociation fragmentation analysis.

In this work we present a novel approach for the identification of plant metabolites using ultrahigh performance liquid chromatography coupled to accurate mass time-of-flight mass spectrometry. The workflow involves developing an in-house compound database consisting of exact masses of previously identified as well as putative compounds. The database is used to screen accurate mass spectrometry (MS) data to identify possible compound matches. Subsequent tandem MS data is acquired for possible matches and used for structural elucidation. The methodology is applied to profile monoterpene glycosides in Vitis vinifera cv. Muscat of Alexandria grape berries over three developmental stages. Monoterpenes are a subclass of terpenes, the largest class of plant secondary metabolites, and are found in two major forms in the plant, "bound" to one or more sugar moieties or "free" of said sugar moieties. In the free form, monoterpenes are noted for their fragrance and play important roles in plant defense and as attractants for pollinators. However, glycoconjugation renders these compounds odorless, and it is this form that the plant uses for monoterpene storage. In order to gain insight into monoterpene biochemistry and their fate in the plant an analysis of intact glycosides is essential. Eighteen monoterpene glycosides were identified including a monoterpene trisaccharide glycoside, which is tentatively identified here for this first time in any plant. Additionally, while previous studies have identified monoterpene malonylated glucosides in other grapevine tissue, we tentatively identify them for the first time in grape berries. This analytical approach can be readily applied to other plants and the workflow approach can also be used for other classes of compounds. This approach, in general, provides researchers with data to support the identification of putative compounds, which is especially useful when no standard is available.