Addressing a major interference in the quantification of psilocin in mouse plasma: Development of a validated liquid chromatography tandem mass spectrometry method.

Psilocybin is a psychedelic compound found in some hallucinogenic "magic mushrooms". Psilocin is the active metabolite of Psilocybin, and it is the subject of several studies for the treatment of psychological disorders, such as anxiety, depression, and post-traumatic stress disorder. As such, the pharmacokinetic properties of psilocin should be evaluated to ensure its safety and efficacy as part of the drug development process. Based on the previously published studies, reversed-phase liquid chromatography (LC) was tested for psilocin quantification. The analysis, however, showed a major interference in mouse plasma that was not, to the best of our knowledge, reported previously. We, therefore, aimed to identify and separate the interference, using various chromatographic columns, mobile phase conditions, and mass spectrometers (MS) instruments. Chromatographic separation was achieved on an ultra high performance liquid chromatography (UHPLC) system, and a quadrupole-linear ion trap equipped with an electrospray ionization (ESI) source was used in positive ion mode with multiple reaction monitoring (MRM). Several chromatographic conditions and column chemistries, including C-18 and Phenyl-hexyl were initially tested, and failed to separate the interference. Exact mass measurement and MS/MS analysis were used to determine the structure of the interfering compound, which was confirmed to be tryptophan. Using the identified structure of the interfering compound, a fast and reliable hydrophilic interaction liquid chromatography (HILIC)-MS/MS method was developed and validated, that was capable of separating psilocin from the interference while achieving a 0.5 ng/ml lower limit of quantification (LLOQ). The validated method was successfully applied to a pharmacokinetic study where psilocin was orally administered to C57BL/6 mouse subjects. Psilocin concentration in all the analyzed mouse plasma samples was successfully determined.

Simplified Liquid Chromatography-Mass Spectrometry Methods for Gestagen Analysis in Animal Fat and Liver.

Gestagens, a class of veterinary drugs also called progestogens, are synthetic hormones used to increase feed efficiency and rate of gain in heifers. The Canadian Food Inspection Agency analyzes progestogens melengestrol acetate (MGA), megestrol acetate, and chlormadinone acetate using liquid chromatography-mass spectrometry (LC-MS). Our conventional gestagen method for kidney fat has many time-consuming steps, including solid-phase extraction. A sample preparation procedure having fewer clean-up steps was developed for routine diagnostic analysis of kidney fat and provided similar results faster, and at lower cost. A confirmatory liver method for gestagens, developed using salt-assisted extraction, employed minimal clean-up steps that resulted in high chemical background at the desired lower limit of quantification (LLOQ). Differential ion mobility spectrometry, specifically high-field asymmetric waveform ion mobility spectrometry (FAIMS), was used to filter chemical background in the gas phase. The effect of the ionization probe position on FAIMS parameters, including sensitivity, is described. With LC-FAIMS-MS, chemical background for each gestagen was virtually eliminated, resulting in a quantitative liver method having the desired 0.6 ng/g LLOQ and estimated limits of detection (LODs) up to 140 times lower than LC-MS. Incurred MGA samples, analyzed using kidney fat and liver methods from the same animal, show levels within the quantitative ranges of both methods.

Biochemical changes after cold acclimation in Nordic red clover (Trifolium pratense L.) accessions with contrasting levels of freezing tolerance.

The ability to tolerate low freezing temperatures is an important component of winter survival and persistence of red clover. Cold acclimation (CA) allows plants to acquire higher levels of freezing tolerance. However, the biochemical responses to cold and the importance of such changes for the plant to acquire adequate freezing tolerance have not been investigated in red clover of Nordic origin, which has a distinct genetic background. To shed light on this, we selected five freezing tolerant (FT) and five freezing susceptible (FS) accessions and studied the effect of CA on the contents of carbohydrates, amino acids, and phenolic compounds in the crowns. Among those compounds which increased during CA, FT accessions had higher contents of raffinose, pinitol, arginine, serine, alanine, valine, phenylalanine, and one phenolic compound (a pinocembrin hexoside derivative) than FS accessions, suggesting a role for these compounds in the freezing tolerance in the selected accessions. These findings, together with a description of the phenolic profile of red clover crowns, significantly add to the current knowledge of the biochemical changes during CA and their role in freezing tolerance in Nordic red clover.

A comparative metabolomics investigation of flavonoid variation in faba bean flowers.

INTRODUCTION: Faba bean (Vicia faba L.) flowers are edible and used as garnishes because of their aroma, sweet flavor and attractive colors. Anthocyanins are the common plant pigments that give flowers their vivid colors, whereas non-anthocyanin flavonoids can serve as co-pigments that can modify the color intensity of flowers. OBJECTIVES: To explore the polyphenol diversity and differences in standard and wing petals of faba bean flowers; and identify glycosylated flavonoids that contribute to flower color. METHODS: Flower standard and wing petals from 30 faba bean genotypes (eight color groups with a total of 60 samples) were used for polyphenol extraction. Samples were analyzed using a targeted method and a semi-untargeted analysis using liquid chromatography-high resolution mass spectrometry (LC-HRMS) combined with photodiode array (PDA) detection. Compound Discoverer software was used for polyphenol identification and multivariate analysis. RESULTS: The semi-untargeted analysis guided by the PDA detected 90 flavonoid metabolites present in faba bean flower petals. Ten anthocyanins largely influenced the flower colors, but other flavonoids (63 flavonols and 12 flavones) found with variable levels in different flower color groups appeared to also influence color, especially in mixed colors. CONCLUSION: Analysis of the different colored faba bean flowers confirmed that the color variation between the flowers was mainly controlled by anthocyanins in brown, red and purple-red flowers. Of the other flavonoids, multiglycosylated kaempferols were abundant in white and brown flowers, monoglycosylated kaempferols were common in red and purple-red flowers, and quercetin and apigenin glycosides were abundant co-pigments in purple-red flowers.

Untargeted profiling of secondary metabolites and phytotoxins associated with stemphylium blight of lentil.

MAIN CONCLUSION: Stemphylium botryosum alters lentil secondary metabolism and differentially affects resistant and susceptible genotypes. Untargeted metabolomics identifies metabolites and their potential biosynthetic pathways that play a crucial role in resistance to S. botryosum. The molecular and metabolic processes that mediate resistance to stemphylium blight caused by Stemphylium botryosum Wallr. in lentil are largely unknown. Identifying metabolites and pathways associated with Stemphylium infection may provide valuable insights and novel targets to breed for enhanced resistance. The metabolic changes following infection of four lentil genotypes by S. botryosum were investigated by comprehensive untargeted metabolic profiling employing reversed-phase or hydrophilic interaction liquid chromatography (HILIC) coupled to a Q-Exactive mass spectrometer. At the pre-flowering stage, plants were inoculated with S. botryosum isolate SB19 spore suspension and leaf samples were collected at 24, 96 and 144 h post-inoculation (hpi). Mock-inoculated plants were used as negative controls. After analyte separation, high-resolution mass spectrometry data was acquired in positive and negative ionization modes. Multivariate modeling revealed significant treatment, genotype and hpi effects on metabolic profile changes that reflect lentil response to Stemphylium infection. In addition, univariate analyses highlighted numerous differentially accumulated metabolites. By contrasting the metabolic profiles of SB19-inoculated and mock-inoculated plants and among lentil genotypes, 840 pathogenesis-related metabolites were detected including seven S. botryosum phytotoxins. These metabolites included amino acids, sugars, fatty acids and flavonoids in primary and secondary metabolism. Metabolic pathway analysis revealed 11 significant pathways including flavonoid and phenylpropanoid biosynthesis, which were affected upon S. botryosum infection. This research contributes to ongoing efforts toward a comprehensive understanding of the regulation and reprogramming of lentil metabolism under biotic stress, which will provide targets for potential applications in breeding for enhanced disease resistance.

Mass Spectrometry-Based Untargeted Metabolomics Reveals the Importance of Glycosylated Flavones in Patterned Lentil Seed Coats.

Lentil seed coats are rich in antioxidant polyphenols that are important for plant defense and have potential as valorized byproducts. Although biochemical differences among lentil seed coat colors have been previously studied, differences among seed coat patterns remain largely unexplored. This study used mass spectrometry-based untargeted metabolomics to investigate polyphenol differences among lentil seed coat patterns to search for biochemical pathways potentially responsible for seed coat pattern differences. Comparing patterned with non-patterned green lentil seed coats, 28 significantly upregulated metabolites were found in patterned seed coats; 19 of them were identified as flavones. Flavones were virtually absent in non-patterned seed coats, thereby strongly suggesting a blockage in their flavone biosynthetic pathway. Although the black pattern is not readily discernible on black seed coats, many of the same flavones found in green marbled seed coats were also found in black seed coats, indicating that black seed coats likely have a marbled pattern.

Water-soluble phenolic compounds and their putative antioxidant activities in the seed coats from different lentil (Lens culinaris) genotypes.

The seed coat is a major byproduct of lentil processing with potential as a sustainable source of antioxidant polyphenols. Profiles of water-soluble phenolic compounds and antioxidant activities of seven genotypes of lentil which includes both normal-tannin and low-tannin seed coats were investigated. Antioxidant activities were assessed using four antioxidant assays, and phenolic compounds were quantified using liquid chromatography mass spectrometry (LC-MS). Total phenolic content (TPC) varied significantly among genotypes and ranged between 1519 ± 140 and 6502 ± 154 µg/g. Thirty phenolic compounds were identified with kaempferol tetraglycoside, catechin-3-glucoside and procyanidins being the dominant compounds in normal-tannin seed coats. Kaempferol tetraglycoside predominated (80-90%) in low-tannin seed coats. Antioxidant activities strongly correlated with TPC (r > 0.93) with a 6-9 times higher activity in normal-tannin than that of low-tannin lentils. Without flavan-3-ols and procyanidins, low-tannin seed coat may not exert strong antioxidant potential, whereas normal-tannin seed coat contains water-extractable natural phenolic compounds with promising antioxidant potential.

Performance of indirect enzyme-linked immunosorbent assay using Trichinella spiralis-derived Serpin as antigen for the detection of exposure to Trichinella spp. in swine.

Indirect enzyme-linked immunosorbent assay (ELISA) utilizing excretory-secretory (E-S) antigens of Trichinella spiralis is currently the method of choice for testing pigs and wild boars for exposure to Trichinella spp. The E-S proteins are released by first-stage larvae (L1) of this parasitic nematode maintained in vitro. However, the production of these antigens is cumbersome and time-consuming. The process requires animals to be experimentally infected with the parasite as the source of L1. Antigen production using recombinant technology would be more time- and cost-effective. In this study, we produced a Serpin of T. spiralis as a recombinant protein secreted by the yeast Pichia pastoris. The diagnostic performance of indirect ELISA with purified Serpin antigen was compared to that of E-S ELISA. Both Serpin ELISA and E-S ELISA demonstrated 98 % diagnostic specificity in testing 1056 pigs from the Canadian Trichinella-free commercial herd. Twenty of 21 pigs with non-negative test results in E-S ELISA tested negative by the confirmatory Western blot (WB) assay. Therefore, the diagnostic specificity of combined E-S ELISA and WB was 99.9 %. Forty-five sera collected at or after six weeks from 34 pigs experimentally infected with various numbers of T. spiralis L1 produced positive results in both E-S and Serpin ELISA, resulting in 100 % diagnostic sensitivity. However, testing of sera serially collected from four pigs experimentally infected with various low doses of T. spiralis L1 demonstrated a delayed Serpin-specific antibody response compared to seroconversion detected by E-S ELISA in three animals. Moreover, Serpin ELISA demonstrated significantly lower sensitivity for detecting antibodies induced by experimental infections of pigs with T. britovi, T. nativa, Trichinella T6 and T. pseudospiralis, suggesting that it will not provide consistent detection of exposure to sylvatic Trichinella spp. The validation data support the application of Serpin ELISA in seroepidemiological surveys for detecting exposure to T. spiralis in swine.

Improved Thyreostatic Drug Detection in Animal Tissues Using Liquid Chromatography-High-Field Asymmetric Waveform Ion Mobility Spectrometry-Mass Spectrometry.

Thyreostatic drugs (thyreostats) interfere with thyroid function and have been used illegally in animals slaughtered for food. Thyreostat use leads to poorer quality meat, and the drug residues can cause adverse effects in humans. These drugs, with the exception of thiouracil, do not occur naturally and require sensitive methodologies for their detection in animal tissues. Because thyreostats are low-molecular-weight polar analytes, liquid chromatography-mass spectrometry (LC-MS) is typically used for detection and, in particular, triple quadrupole mass spectrometry with selective reaction monitoring (i.e., LC-SRM). However, LC-SRM thyreostat methods suffer from chemical background noise and endogenous interferences arising from the complex tissue matrix. An improved high-field asymmetric waveform ion mobility spectrometry interface (FAIMS Pro), which separates ions based on differential ion mobility, was combined with LC-SRM to minimize these interferences. Using the same samples and conditions, LC-FAIMS-SRM showed improvements in the signal-to-noise ratio (S/N) of up to 50 times compared with our validated LC-SRM method. In addition, wider linear ranges, including substantial improvements in the lower limit of quantification (approximately an order of magnitude for tapazole and methylthiouracil), were observed with LC-FAIMS-SRM.

Exploring the structure of atom-precise silver-palladium bimetallic clusters prepared via improved single-pot co-reduction synthesis protocol.

Designing atom-precise bimetallic clusters with a relatively cost-effective and more abundant metal than Au (i.e., Ag) is desirable for the development of heterogeneous bimetallic cluster catalysts for industrial applications. Atom-precise Ag-based bimetallic clusters, which are analogs of the well-studied Au based clusters, are yet to be fully explored as catalysts. Establishing the Pd loading limit and the position of the Pd dopant in AgPd bimetallic clusters will further give an insight into the structure-activity relationships for these atom-precise AgPd heterogeneous catalysts. In this study, an improved single-pot co-reduction strategy was employed to prepare the bimetallic clusters, which were then characterized by mass spectrometry, x-ray photoelectron spectroscopy (XPS), and x-ray absorption spectroscopy (XAS) to identify the loading and position of the dopant metal. Our results show that only a single dopant Pd atom can be incorporated, and in comparison with monometallic Ag25 clusters, the absorption peaks of Ag24Pd1(SPhMe2)18 2- bimetallic clusters are blue shifted due to the incorporation of Pd. The XPS and XAS results show that the Ag24Pd1(SPhMe2)18 2- bimetallic clusters have multivalent Ag(0) and Ag(I) atoms and surprisingly show Pd(II) species with significant Pd-S bonding, despite the prevailing wisdom that the Pd dopant should be in the center of the cluster. The XAS results show that the singly doped Pd atom predominantly occupies the staple position, albeit we cannot unambiguously rule out the Pd atom in an icosahedral surface position in some clusters. We discuss the ramifications of these results in terms of possible kinetically vs thermodynamically controlled cluster formation.

An Untargeted Metabolomics Approach for Correlating Pulse Crop Seed Coat Polyphenol Profiles with Antioxidant Capacity and Iron Chelation Ability.

Pulse crop seed coats are a sustainable source of antioxidant polyphenols, but are typically treated as low-value products, partly because some polyphenols reduce iron bioavailability in humans. This study correlates antioxidant/iron chelation capabilities of diverse seed coat types from five major pulse crops (common bean, lentil, pea, chickpea and faba bean) with polyphenol composition using mass spectrometry. Untargeted metabolomics was used to identify key differences and a hierarchical analysis revealed that common beans had the most diverse polyphenol profiles among these pulse crops. The highest antioxidant capacities were found in seed coats of black bean and all tannin lentils, followed by maple pea, however, tannin lentils showed much lower iron chelation among these seed coats. Thus, tannin lentils are more desirable sources as natural antioxidants in food applications, whereas black bean and maple pea are more suitable sources for industrial applications. Regardless of pulse crop, proanthocyanidins were primary contributors to antioxidant capacity, and to a lesser extent, anthocyanins and flavan-3-ols, whereas glycosylated flavonols contributed minimally. Higher iron chelation was primarily attributed to proanthocyanidin composition, and also myricetin 3-O-glucoside in black bean. Seed coats having proanthocyanidins that are primarily prodelphinidins show higher iron chelation compared with those containing procyanidins and/or propelargonidins.

Do Faba Bean Genotypes Carrying Different Zero-Tannin Genes (zt1 and zt2) Differ in Phenolic Profiles?

Faba bean is a cool season grain legume that produces seeds with a high protein content. Seed coat tannins limit its use in food and feed. A low-tannin phenotype is controlled by either of two unlinked recessive genes zt1 and zt2. Liquid chromatography-mass spectrometry was used to characterize phenolic profiles of seed coat and flower tissue of three faba bean genotypes: CDC Snowdrop (zt1 gene), Disco/2 (zt2 gene), and ILB 938/2 (tannin-containing). For both tissues, clear differences in phenolic profiles of ILB 938/2 were observed in comparison to both low-tannin lines. Although seed coat phenolic profiles of zt1 and zt2 genotypes were similar, distinct differences were evident in flower tissue, suggesting that the gene action results in some different end products of the phenolic biosynthetic pathway. These distinctive compounds could be used as biochemical markers to distinguish between low-tannin phenotypes.

Fast Quantification Without Conventional Chromatography, The Growing Power of Mass Spectrometry.

Mass spectrometry (MS) in hyphenated techniques is widely accepted as the gold standard quantitative tool in life sciences. However, MS possesses intrinsic analytical capabilities that allow it to be a stand-alone quantitative technique, particularly with current technological advancements. MS has a great potential for simplifying quantitative analysis without the need for tedious chromatographic separation. Its selectivity relies on multistage MS analysis (MSn), including tandem mass spectrometry (MS/MS), as well as the ever-growing advancements of high-resolution MS instruments. This perspective describes various analytical platforms that utilize MS as a stand-alone quantitative technique, namely, flow injection analysis (FIA), matrix assisted laser desorption ionization (MALDI), including MALDI-MS imaging and ion mobility, particularly high-field asymmetric waveform ion mobility spectrometry (FAIMS). When MS alone is not capable of providing reliable quantitative data, instead of conventional liquid chromatography (LC)-MS, the use of a guard column (i.e., fast chromatography) may be sufficient for quantification. Although the omission of chromatographic separation simplifies the analytical process, extra procedures may be needed during sample preparation and clean-up to address the issue of matrix effects. The discussion of this manuscript focuses on key parameters underlying the uniqueness of each technique for its application in quantitative analysis without the need for a chromatographic separation. In addition, the potential for each analytical strategy and its challenges are discussed as well as improvements needed to render them as mainstream quantitative analytical tools. Overcoming the hurdles for fully validating a quantitative method will allow MS alone to eventually become an indispensable quantitative tool for clinical and toxicological studies.

Polyphenol profile comparisons of seed coats of five pulse crops using a semi-quantitative liquid chromatography-mass spectrometric method.

INTRODUCTION: Pulse crops are nutritious and therefore widely grown. Pulse seed coats are typically discarded, despite their high content of polyphenols that are known for their antioxidant properties and health benefits. A better understanding of polyphenol diversity and biochemical pathways will ultimately provide insight into how polyphenols are linked to health benefits, which will help to better utilise these seed coats. OBJECTIVES: To explore polyphenol profiles among seed coats of diverse genotypes of five pulse crops using a targeted liquid chromatography mass spectrometry (LC-MS) method. METHODS: Four genotypes of each of common bean, chickpea, pea, lentil and faba bean seed coats were selected for analysis. Following extraction, polyphenols were quantified using LC-MS. RESULTS: An LC-MS method was developed to quantify 98 polyphenols from 13 different classes in 30 min. The low-tannin seed coats had the lowest concentrations of all polyphenols. Chickpea and pea seed coats had the most similar polyphenolic profiles. The black common bean showed the most diverse seed coat polyphenol profile, including several anthocyanins not detected in any of the other seed coats. CONCLUSION: The LC-MS method reported herein was used to show polyphenol diversity within several polyphenol classes among the pulse crop seed coats. Detected in all seed coats, flavonols and hydroxybenzoic acids appear well-conserved in the edible Fabaceae. The presence of anthocyanins, flavan-3-ols and proanthocyanins in the coloured seed coats suggests that unique divergent branches were introduced in the flavonoid biosynthetic pathway, possibly in response to environmental stressors.

Improved folate monoglutamate extraction and application to folate quantification from wild lentil seeds by ultra-performance liquid chromatography-selective reaction monitoring mass spectrometry.

Folates are important micronutrients in lentils (Lens culinaris Medik.). In this work, the folate extraction workflow in ascorbate-containing buffer was optimized and validated, and the concentrations of eight folate monoglutamates in cultivated and six wild lentil species, grown under field or greenhouse conditions, were quantified by ultra-performance liquid chromatography and mass spectrometry (UPLC-MS). In general wild lentil species had higher folate concentrations than cultivated genotypes. Lens tomentosus had the highest folate concentration with median values of 439.7 and 360.9 µg/100 g in the field and greenhouse, followed by Lens orientalis with 416.6 and 327.6 µg/100 g, respectively. A significant effect (P < 0.05) of growing conditions was observed in four out of six wild lentil species, with seeds from the field having higher folate concentration (6% to 45%) compared with the greenhouse. MeFox, an oxidation product of 5-methyltetrahydrofolate, was present in all lentil species at concentrations 2.2 to 5.6 times higher than the total folates.

Quantitative determination of potential urine biomarkers of respiratory illnesses using new targeted metabolomic approach.

The diagnosis of asthma and chronic obstructive pulmonary disease (COPD) can be challenging due to the overlap in their clinical presentations in some patients. There is a need for a more objective clinical test that can be routinely used in primary care settings. Through an untargeted 1H NMR urine metabolomic approach, we identified a set of endogenous metabolites as potential biomarkers for the differentiation of asthma and COPD. A subset of these potential biomarkers contains 7 highly polar metabolites of diverse physicochemical properties. To the best of our knowledge, there is no liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that evaluated more than two of the target metabolites in a single analytical run. The target metabolites belong to the families of monosaccharides, organic acids, amino acids, quaternary ammonium compounds and nucleic acids, rendering hydrophilic interaction liquid chromatography (HILIC) an ideal technology for their quantification. Since a clinical decision is to be made from patients data, a fully validated analytical method is required for biomarker validation. Method validation for endogenous metabolites is a daunting task since current guidelines were designed for exogenous compounds. As such, innovative approaches were adopted to meet the validation requirements. Herein, we describe a sensitive HILIC-MS/MS method for the quantification of the 7 endogenous urinary metabolites. Detection was achieved in the multiple reaction monitoring (MRM) mode with polarity switching, using quadrupole-linear ion trap instrument (QTRAP 6500) as well as single ion monitoring in the negative-ion mode. The method was fully validated according to the regulatory guidelines. Linearity was established between 6 and 21000 ng/mL and quality control samples demonstrated acceptable intra- and inter-day accuracy (85.7%-112%), intra- and inter-day precision (CV% <11.5%) as well as stability under various storage and sample processing conditions. To illustrate the method's applicability, the validated method was applied to the analysis of a small set of urine samples collected from asthma and COPD patients. Preliminary modelling of separation was generated using partial least square discriminant analysis (R2 0.752 and Q2 0.57). The adequate separation between patient samples confirms the diagnostic potential of these target metabolites as a proof-of-concept for the differentiation between asthma and COPD. However, more patient urine samples are needed in order to increase the statistical power of the analytical model.

Polyphenolic Composition of Lentil Roots in Response to Infection by Aphanomyces euteiches.

Polyphenols comprise the largest group of plant secondary metabolites and have critical roles in plant physiology and response to the biotic and abiotic environment. Changes in the content of polyphenols in the root extracts and root tissues of wild (Lens ervoides) and cultivated (Lens culinaris) lentil genotypes were examined in response to infection by Aphanomyces euteiches using liquid chromatography mass spectrometry (LC-MS). Genotype, infection and their interaction determined the composition of polyphenols in lentil roots. The levels of several polyphenols were lower in the root extract of the low-tannin genotype L. culinaris ZT-4 compared to L. ervoides L01-827A. Kaempferol derivatives including kaempferol dirutinoside and kaempferol 3-robinoside 7-rhamnoside were more concentrated in the healthy root tissues of L. ervoides L01-827A than in L. culinaris genotypes. Infection increased the concentration of kaempferol, apigenin, and naringenin in the root tissues of all genotypes, but had no effect on some polyphenols in the low-tannin genotype L. culinaris ZT-4. The concentrations of apigenin, naringenin, apigenin 4-glucoside, naringenin7-rutinoside, diosmetin, and hesperetin 7-rutinoside were higher in the infected root tissues of L. ervoides L01-827A compared with the L. culinaris genotypes. Organic acids including coumaric acid, vanillic acid, 4-aminosalicylic acid, 4-hydroxybenzoic acid, and 3,4-dihydroxybenzoic acid effectively suppressed the in-vitro hyphal growth of A. euteiches. Some of these bioactive polyphenols were more concentrated in roots of L. ervoides L01-827A but were low to undetectable in ZT-4. This study shows that genotypic differences exist in the composition of root polyphenols in lentil, and is related to the response to infection caused by A. euteiches. Polyphenols, particularly the organic acid content could be useful for selection and breeding of lentil genotypes that are resistant to Aphanomyces root rot (ARR) disease.

Toward a high-throughput method for determining vicine and convicine levels in faba bean seeds using flow injection analysis combined with tandem mass spectrometry.

Although faba bean provides environmental and health benefits, vicine and convicine (v-c) limit its use as a source of vegetable protein. Crop improvement efforts to minimize v-c concentration require low-cost, rapid screening methods to distinguish between high and low v-c genotypes to accelerate development of new cultivars and to detect out-crossing events. To assist crop breeders, we developed a unique and rapid screening method that uses a 60 s instrumental analysis step to accurately distinguish between high and low v-c genotypes. The method involves flow injection analysis (FIA) coupled with tandem mass spectrometry (i.e., selective reaction monitoring, SRM). Using seeds with known v-c levels as calibrants, measured v-c levels were comparable with liquid chromatography (LC)-SRM results and the method was used to screen 370 faba bean genotypes. Widespread use of FIA-SRM will accelerate breeding of low v-c faba bean, thereby alleviating concerns about anti-nutritional effects of v-c in this crop.

Quantification of vicine and convicine in faba bean seeds using hydrophilic interaction liquid chromatography.

Faba bean (Vicia faba L.) provides environmental and health benefits; however, the presence of the pyrimidine glycosides vicine and convicine (v-c) in its seeds limits consumption. Low v-c genotypes have been introduced, but the convicine levels in these genotypes have not been quantified. To improve detection, the polar nature of v-c was exploited by implementing hydrophilic interaction liquid chromatography (HILIC). A sample preparation method using a two-step extraction was developed for use with UV and/or tandem mass spectrometry (SRM) detection. The HILIC-UV method was suitable for over three orders of magnitude, covering the range of v-c concentrations in faba bean seeds across all genotypes tested. The linear range of HILIC-SRM was slightly less (∼3 orders of magnitude), but improved sensitivity and selectivity make it more suitable for quantifying low v-c samples. The analysis of 13 genotypes suggests that v-c concentrations in faba bean seeds may be independent quantitative traits.

Profiling the Phenolic Compounds of the Four Major Seed Coat Types and Their Relation to Color Genes in Lentil.

Phenolic compounds can provide antioxidant health benefits for humans, and foods such as lentils can be valuable dietary sources of different subclasses of these secondary metabolites. This study used LC-MS analyses to compare the phenolic profiles of lentil genotypes with four seed coat background colors (green, gray, tan, and brown) and two cotyledon colors (red and yellow) grown at two locations. The mean area ratio per mg sample (MARS) values of various phenolic compounds in lentil seeds varied with the different seed coat colors conferred by specific genotypes. Seed coats of lentil genotypes with the homozygous recessive tgc allele (green and gray seed coats) had higher MARS values of flavan-3-ols, proanthocyanidins, and some flavonols. This suggests lentils featuring green and gray seed coats might be more promising as health-promoting foods.