Challenges in the determination of total vitamin B12 by cyanidation conversion: insights from stable isotope dilution assays.

Previous methods for vitamin B12 (B12) analysis have extensively used cyanidation conversion with the intention of converting all cobalamins to cyanocobalamin (CNCbl) for total B12 determination. This approach has been favored for its advantages in reducing the number of analytes, increasing analyte concentration, and improving analyte stability. However, the present study revealed underlying limitations associated with this approach. First, a stable isotope dilution assay (SIDA) determining total B12 as CNCbl after cyanidation conversion (conversion SIDA method) was developed. Method validation demonstrated good sensitivity, recovery, accuracy, and reproducibility for the target analyte CNCbl. However, subsequent application of the conversion method to real meat samples showed incomplete conversions of cobalamins. These inconsistencies revealed day-to-day variability and reliability challenges associated with the cyanidation process. It was not possible to identify this issue during method validation as CNCbl was spiked as the sole analyte and it requires no further cyanidation conversion. The application of LC-MS/MS enabled the detection of trace amounts of unconverted cobalamins. Nevertheless, this approach remains restricted by instrument sensitivity and stability as well as the performance of immunoaffinity purification for different vitamers. Further development of a reliable monitoring method is a prerequisite for further optimization of the cyanidation process. However, significant improvements of analytical instrumentation in terms of sensitivity and stability are required to overcome the current limitations.

Development of analytical methods to study the effect of malting on levels of free and modified forms of Alternaria mycotoxins in barley.

A liquid chromatography tandem mass spectrometry (LC-MS/MS) multi-mycotoxin method was developed for the analysis of the Alternaria toxins alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), altertoxin I (ATX I), altertoxin II (ATX II), alterperylenol (ALTP), and altenuene (ALT), as well as the modified toxins AOH-3-glucoside (AOH-3-G), AOH-9-glucoside (AOH-9-G), AME-3-glucoside (AME-3-G), AOH-3-sulfate (AOH-3-S), and AME-3-sulfate (AME-3-S) in barley and malt. The toxin tenuazonic acid (TeA) was analyzed separately as it could not be included into the multi-mycotoxin method. Quantitation was conducted by using a combination of stable isotope dilution analysis (SIDA) for AOH, AME, and TeA, and matrix-matched calibration for all other toxins. Limits of detection were between 0.05 µg/kg (AME) and 2.45 µg/kg (ALT), whereas limits of quantitation ranged from 0.16 µg/kg (AME) to 8.75 µg/kg (ALT). Recoveries between 96 and 107% were obtained for the analytes when SIDA was applied, while recoveries between 84 and 112% were found for analytes quantified by matrix-matched calibration. The method was applied for the analysis of 50 barley samples and their respective malts from the harvest years 2016-2020 for their mycotoxin content, showing the overall potential of toxin formation during the malting process. The toxins ALTP and ATX I were mainly found in the malt samples, but not in barley.

Production of Four 15N-Labelled Cobalamins via Biosynthesis Using Propionibacterium freudenreichii.

Cobalamins (vitamin B12) are required by humans for their essential roles as enzyme cofactors in diverse metabolic processes. The four most common cobalamin vitamers are hydroxocobalamin (OHCbl), adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), and cyanocobalamin (CNCbl). Humans are not able to synthesise cobalamins de novo and thus must acquire them from external sources. Therefore, a reliable and robust analytical method to determine the cobalamins in dietary sources is highly required. For such a purpose, stable isotope dilution assays (SIDAs) with LC-MS/MS are most suited due to their superior sensitivity, specificity, and ability to compensate for matrix effects and analyte loss during sample work-up. However, a critical bottleneck for developing a SIDA method for cobalamins is the availability of stable isotope-labelled internal standards. In the present study, we harnessed the potential of Propionibacterium (P.) freudenreichii for the biosynthesis of 15N-labelled cobalamins. First, we developed a chemically defined medium (CDM) containing ammonium sulphate as a single nitrogen source except three essential vitamins that supported long-term stable growth of P. freudenreichii throughout continuous transfers. The CDM was further optimised for cobalamin production under different incubation schemes. With the optimised CDM and incubation scheme, fully 15N-labelled cobalamins were obtained in P. freudenreichii with a final yield of 312 ± 29 µg/L and 635 ± 102 µg/L, respectively, for [15N]-OHCbl and [15N]-AdoCbl. Additionally, an optimised incubation process under anaerobic conditions was successfully employed to produce specifically labelled [15N, 14N2]-cobalamins, with a yield of 96 ± 18 µg/L and 990 ± 210 µg/L, respectively, for [15N, 14N2]-OHCbl and [15N, 14N2]-AdoCbl. The labelled substances were isolated and purified by solid phase extraction and semi-preparative HPLC. Chemical modifications were carried out to produce [15N]-CNCbl and [15N]-MeCbl. Eventually, 15N-labelled compounds were obtained for the four cobalamin vitamers in high chromatographic and isotopic purity with desired 15N-enrichment and labelling patterns, which are perfectly suited for future use in SIDAs or other applications that require isotopologues.

Development of Stable Isotope Dilution Assays for the Analysis of Natural Forms of Vitamin B12 in Meat.

The first multiple stable isotope dilution assay method was developed for the simultaneous determination of four cobalamins, namely, hydroxocobalamin (OHCbl), adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), and cyanocobalamin (CNCbl), in their native forms. The sample preparation was optimized with enzyme treatment and immunoaffinity purification. The analysis was performed by LC-MS/MS using respective 15N-labeled cobalamins as internal standards. Method validation resulted in limits of detection ranging from 0.19 to 0.58 ng/g and limits of quantification ranging from 0.68 to 1.73 ng/g. Recoveries at three levels were between 82 and 121%. Intra-day and inter-day precisions were below 6% and 11% RSD, respectively. The analysis of a reference material resulted in a variance of <1% from the certified value. The newly developed method demonstrated excellent sensitivity, recovery, accuracy, and reproducibility and was further applied to quantitate the four cobalamins in various meats.

Analysis of 13 Alternaria mycotoxins including modified forms in beer.

A multi-mycotoxin LC-MS/MS method was developed to quantify 13 free and modified Alternaria toxins in different beer types by applying a combination of stable-isotope dilution assays (SIDAs) and matrix-matched calibration. With limits of detection (LODs) between 0.03 µg/L (alternariol monomethyl ether, AME) and 5.48 µg/L (altenuene, ALT), limits of quantitation (LOQs) between 0.09 µg/L (AME) and 16.24 µg/L (ALT), and recoveries between 72 and 113%, we obtained a sensitive and reliable method, which also covers the emerging toxins alternariol-3-glucoside (AOH-3-G), alternariol-9-glucoside (AOH-9-G), alternariol monomethyl ether-3-glucoside (AME-3-G) and alternariol-3-sulfate (AOH-3-S) and alternariol monomethylether-3-sulfate (AME-3-S). Furthermore, 50 different beer samples were analyzed, showing no contamination with Alternaria toxins apart from tenuazonic acid (TeA) in concentrations between 0.69 µg/L and 16.5 µg/L. According to this study, the exposure towards TeA through beer consumption can be considered as relatively low, as the threshold of toxicological concern (TTC) value of 1500 ng/kg body weight per day might not be reached when consuming reasonable amounts of beer.

Enzymatic Synthesis of Modified Alternaria Mycotoxins Using a Whole-Cell Biotransformation System.

Reference standards for Alternaria mycotoxins are rarely available, especially the modified mycotoxins alternariol-3-glucoside (AOH-3-G), alternariol-9-glucoside (AOH-9-G), and alternariol monomethylether-3-glucoside (AME-3-G). To obtain these three glucosides as analytical standards for method development and method validation, alternariol and alternariol monomethylether were enzymatically glycosylated in a whole-cell biotransformation system using a glycosyltransferase from strawberry (Fragaria x ananassa), namely UGT71A44, expressed in Escherichia coli (E. coli). The formed glucosides were isolated, purified, and structurally characterized. The exact amount of the isolated compounds was determined using high-performance liquid chromatography with UV-detection (HPLC-UV) and quantitative nuclear resonance spectroscopy (qNMR). This method has proved to be highly effective with biotransformation rates of 58% for AOH-3-G, 5% for AOH-9-G, and 24% for AME-3-G.

Comprehensive Analysis of the Alternaria Mycobolome Using Mass Spectrometry Based Metabolomics.

SCOPE: Alternaria fungi are widely distributed plant pathogens infecting grains and vegetables and causing major harvest losses in the field and during postharvest storage. Besides, consumers are endangered by the formation of toxic secondary metabolites. Some of these secondary metabolites are chemically characterized as mycotoxins, but the majority of the Alternaria mycobolome still remains unknown. METHODS AND RESULTS: Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) and LC-MS/MS are combined for the non-targeted and targeted analysis of the metabolome of three A. alternata isolates and one A. solani isolate. Due to the ultra-high resolution of FTICR-MS, unique molecular formulae are assigned to measured m/z signals. The molecular formulae are matched to entries of the databases Antibase and Kyoto Encyclopedia of Genes and Genomes. The non-targeted analysis of the fungal extracts reveals variations in the secondary metabolite profile of A. alternata and A. solani. Differences in the biosynthesis of dibenzo-α-pyrones, perylene quinones, tentoxin, and tenuazonic acid of the A. alternata and A. solani isolates are determined applying targeted LC-MS/MS. CONCLUSION: FTICR-MS analyses reveal clear differences in the metabolic profile of the A. solani and the A. alternata isolates.

Quantitation of Six Alternaria Toxins in Infant Foods Applying Stable Isotope Labeled Standards.

Alternaria fungi are widely distributed saprophytes and plant pathogens. As pathogens, Alternaria fungi infect crops and vegetables and cause losses in the fields and during postharvest storage. While farmers suffer from declining yields, consumers are endangered by the formation of secondary metabolites, because some of these exhibit a pronounced toxicological potential. The evaluation of the toxicological capabilities is still ongoing and will contribute to a valid risk assessment. Additionally, data on the incidence and the quantity of Alternaria mycotoxins found in food products is necessary for dietary exposure evaluations. A sensitive LC-MS/MS method for the determination of the Alternaria mycotoxins alternariol (AOH), alternariol monomethylether (AME), tentoxin (TEN), altertoxin I (ATX I), alterperylenol (ALTP), and tenuazonic acid (TA) was developed. AOH, AME, and TA were quantified using stable-isotopically labeled standards. TEN, ATX I, and ALTP were determined using matrix matched calibration. The developed method was validated by using starch and fresh tomato matrix and resulted in limits of detection ranging from 0.05 to 1.25 µg/kg for starch (as a model for cereals) and from 0.01 to 1.36 µg/kg for fresh tomatoes. Limits of quantification were determined between 0.16 and 4.13 µg/kg for starch and between 0.02 and 5.56 µg/kg for tomatoes. Recoveries varied between 83 and 108% for starch and between 95 and 111% for tomatoes. Intra-day precisions were below 4% and inter-day precisions varied from 3 to 8% in both matrices. Various cereal based infant foods, jars containing vegetables and fruits as well as tomato products for infants were analyzed for Alternaria mycotoxin contamination (n = 25). TA was the most frequently determined mycotoxin and was detected in much higher contents than the other toxins. AME and TEN were quantified in many samples, but in low concentrations, whereas AOH, ATX I, and ALTP were determined rarely, among which AOH had higher concentration. Some infant food products were highly contaminated with Alternaria mycotoxins and the consumption of these individual products might pose a risk to the health of infants. However, when the mean or median is considered, no toxicological risk was obvious.

Evaluation of an enzyme immunoassay for the detection of the mycotoxin tenuazonic acid in sorghum grains and sorghum-based infant food.

An enzyme-linked immunosorbent assay (ELISA) for the Alternaria mycotoxin tenuazonic acid (TeA) was evaluated by comparative analysis of naturally contaminated sorghum grains and sorghum-based infant food, using a stable isotope dilution LC-MS assay (SIDA; limit of detection (LOD) 1.0 µg/kg) as the reference method. LODs of the ELISA were 30 µg/kg in sorghum grains and 220 µg/kg in sorghum-based infant cereals. With SIDA, 100% of the samples (n = 28) had been positive for TeA in a concentration range of 6-584 µg/kg (mean 113 µg/kg). The ELISA consistently detected TeA in all naturally contaminated samples at cut-off levels of 30-60 µg/kg (sorghum) and 200-300 µg/kg (infant cereals), as based on corresponding to SIDA values. Although the ELISA was much less sensitive than the SIDA method, it may be useful as a screening method for sorghum and sorghum-based infant foods and can be employed to identify samples containing elevated concentrations of TeA in food, well below the proposed level of concern (500 µg/kg).

Validated UPLC-MS/MS Methods To Quantitate Free and Conjugated Alternaria Toxins in Commercially Available Tomato Products and Fruit and Vegetable Juices in Belgium.

Ultraperformance liquid chromatography tandem mass spectrometry and Quick, Easy, Cheap, Effective, Rugged, and Safe based analytical methodologies to quantitate both free (alternariol (1), alternariol monomethyl ether (2), tenuazonic acid (3), tentoxin (4), altenuene (5), altertoxin-I (6)) and conjugated (sulfates and glucosides of 1 and 2) Alternaria toxins in fruit and vegetable juices and tomato products were developed and validated. Acceptable limits of quantitation (0.7-5.7 µg/kg), repeatability (RSDr < 15.7%), reproducibility (RSDR < 17.9%), and apparent recovery (87.0-110.6%) were obtained for all analytes in all matrices investigated. 129 commercial foodstuffs were analyzed, and 3 was detected in 100% of tomato product samples (

Recent developments in stable isotope dilution assays in mycotoxin analysis with special regard to Alternaria toxins.

Stable isotope dilution assays (SIDAs) are becoming ever commoner in mycotoxin analysis, and the number of synthesized or commercially available isotopically labelled compounds has greatly increased in the 7 years since our last review dealing with this topic. Thus, this review is conceived as an update for new applications or improvements of SIDAs for compounds discussed earlier, but the main focus is on newly introduced labelled substances and the development of SIDAs for, for example, fusarin C, moniliformin or the enniatins. Mycotoxin research has concentrated on the emerging group of Alternaria toxins in recent years, and a series of SIDAs have been developed, including ones for tenuazonic acid, alternariol, altertoxins and tentoxin that are discussed in detail in this review. Information about synthetic routes, isotopic purity and mass-spectrometric characterization of labelled compounds is given, as well as about the development and validation of SIDAs and their application to foods, feeds or biological samples. As the number of commercially available labelled standards is increasing continuously, a general tendency for the use of analytical methods based on liquid chromatography coupled with mass spectrometry capable of identifying a series of mycotoxins simultaneously ("multimethods") and using one or more labelled internal standards can be observed. An overview of these applications is given, thus demonstrating that SIDAs are increasingly being used in routine analysis.

Development and validation of an ultra-high-performance liquid chromatography tandem mass spectrometric method for the simultaneous determination of free and conjugated Alternaria toxins in cereal-based foodstuffs.

A UPLC-ESI+/--MS/MS method for the simultaneous determination of free (alternariol, alternariol monomethyl ether, altenuene, tenuazonic acid, tentoxin, altertoxin-I) and conjugated (sulfates and glucosides of alternariol and alternariol monomethyl ether) Alternaria toxins in cereals and cereal products (rice, oat flakes and barley) was developed. Optimization of the sample preparation and extraction methodology was achieved through experimental design, using full factorial design for extraction solvent composition optimization and fractional factorial design to identify the critical factors in the sample preparation protocol, which were in turn subjected to optimization. Final extracts were analysed using an Waters Acquity UPLC system coupled to a Quattro Premier XE mass spectrometer equipped with an electrospray interface operated in both positive and negative ionization mode. Chromatographic separation was achieved using an Acquity UPLC HSS T3 column, and the applied gradient elution programme allowed for the simultaneous determination of 10 Alternaria toxins in a one-step chromatographic run with a total run time of only 7min. Subsequently, the method, applying isotopically labelled internal standards ([2H4]-alternariol monomethyl ether and [13C6,15N]-tenuazonic acid), was validated for several parameters such as linearity, apparent recovery, limit of detection, limit of quantification, precision, measurement uncertainty and specificity (in agreement with the criteria mentioned in Commission Regulation No. 401/2006/EC and Commission Decision No. 2002/657/EC). During validation, quality of the bioanalytical data was improved by counteracting the observed heteroscedasticity through the application of weighted least squares linear regression (WLSLR). Finally, 24 commercially available cereal-based foodstuffs were subjected to analysis, revealing the presence of tenuazonic acid in both rice and oat flake samples (

Development of analytical methods for the determination of tenuazonic acid analogues in food commodities.

Analogues of the Alternaria mycotoxin tenuazonic acid (TA, biosynthesized by the fungus from the amino acid isoleucine) derived from valine (ValTA), leucine (LeuTA), alanine (AlaTA) and phenylalanine (PheTA) were synthesized and characterized by mass spectrometry (MS) and (1)H nuclear magnetic resonance (NMR) spectroscopy. Concentrations of stock solutions were determined by quantitative (1)H NMR (qHNMR). Two analytical methods based on high performance liquid chromatography (HPLC) and MS detection were developed, one with derivatization with 2,4-dinitrophenylhydrazine (DNPH) and one without derivatization. Limits of detection (LODs) were between 1-3 µg/kg (with derivatization) and 50-80 µg/kg (without derivatization). Respective limits of quantitation (LOQs) were about three times higher. Beside TA, the analogues LeuTA (about 4% of TA content) and ValTA (about 10% of TA content) were found in highly contaminated sorghum infant cereals and sorghum grains. Other analogues were not detected. Quantification of LeuTA and ValTA was performed using [(13)C6,(15)N]-TA as internal standard and matrix matched calibration. Recovery was between 95±11% and 102±10% for both compounds. Precision (relative standard deviation of triplicate sorghum cereal analyses three times during 3 weeks) was 7% for TA (912±60 µg/kg), 17% for LeuTA (43±8 µg/kg) and 19% for ValTA (118±22 µg/kg). These results indicate that several TA-like compounds, which are not yet characterized in aspects of their toxic properties, were detected in sorghum based infant food highly contaminated with TA, already.

Determination of tenuazonic acid in human urine by means of a stable isotope dilution assay.

The content of tenuazonic acid in human urine was determined by a stable isotope dilution assay (SIDA) that was recently developed for the analysis of food commodities and extensively re-validated for urine matrix in this study. Linearity of the response curve was proven between molar ratios n(labeled standard)/n(analyte) of 0.02-100. The limits of detection and determination were 0.2 and 0.6 µg/L, respectively. The mean recovery of the stable isotope dilution assay was 102 ± 3 % in the range between 1.0 and 100 µg/L. Interassay precision was 6.7 % (relative standard deviation of three triplicate analyses of a human urine sample during 3 weeks). The method was applied to two studies dealing with urinary excretion of tenuazonic acid: In the first study, tenuazonic acid was quantified in the 24-h urine of six volunteers from Germany (three female, three male) in a concentration range of 1.3-17.3 µg/L or 2.3-10.3 ng/mg(-1) creatinine, respectively. In the second study, two volunteers (one female, one male) ingested 30 µg tenuazonic acid by consumption of naturally contaminated whole meal sorghum infant cereals and tomato juice, respectively. The urinary excretion of the ingested tenuazonic acid was 54-81 % after 6 h, depending on matrix and volunteer. After 24 h, 87-93 % of the ingested amount of tenuazonic acid was excreted, but the fate of the remaining about 10 % is open. Thus, it is not possible to exclude potential health hazards for the consumer, completely.

Content of the Alternaria mycotoxin tenuazonic acid in food commodities determined by a stable isotope dilution assay.

The Alternaria mycotoxin tenuazonic acid (TA) was quantified in fruit juices (n = 50), cereals (n = 12) and spices (n = 38) using a recently developed stable isotope dilution assay (SIDA). [(13) C6,(15) N]-TA was used as the internal standard. Method validation revealed low limits of detection (LODs) of 0.15 µg/kg (fruit juices), 1.0 µg/kg (cereals) and 17 µg/kg (spices). The respective limits of quantitation were about three times higher. Recovery was about 100% for all matrices. The precision (relative standard deviation of replicate analyses of naturally contaminated samples) was 4.2% (grape juice; 1.7 µg/kg), 3.5% (whole wheat flour; 36 µg/kg) and 0.9% (curry powder; 215 µg/kg). The median content of TA in the analyzed samples was 1.8 µg/kg (fruit juices), 16 µg/kg (cereals) and 500 µg/kg (spices). Positive samples amounted to 86% (fruit juices), 92% (cereals) and 87% (spices).

Development of a stable isotope dilution assay for tenuazonic acid.

A stable isotope dilution assay (SIDA) for the Alternaria mycotoxin tenuazonic acid was developed. Therefore, [(13)C(6),(15)N]-tenuazonic acid was synthesized from [(13)C(6),(15)N]-isoleucine by Dieckmann intramolecular cyclization after acetoacetylation with diketene. The synthesized [(13)C(6),(15)N]-tenuazonic acid was used as the internal standard for determination of tenuazonic acid in tomato products by liquid chromatography tandem mass spectrometry after derivatization with 2,4-dinitrophenylhydrazine. Method validation revealed a limit of detection of 0.1 µg/kg and a limit of quantitation of 0.3 µg/kg. Recovery was close to 100% in the range of 3-300 µg/kg. Determination of tenuazonic acid in two samples of different tomato ketchups (naturally contaminated) was achieved with a coefficient of variation of 2.3% and 4.7%. Different tomato products (n = 16) were analyzed for their content of tenuazonic acid using the developed SIDA. Values were between 15 and 195 µg/kg (tomato ketchup, n = 9), 363 and 909 µg/kg (tomato paste, n = 2), and 8 and 247 µg/kg (pureed tomatoes and comparable products, n = 5).

Precise determination of the Alternaria mycotoxins alternariol and alternariol monomethyl ether in cereal, fruit and vegetable products using stable isotope dilution assays.

Cereal, fruit and vegetable products were analyzed for contamination with the Alternaria mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) using stable isotope dilution assays (SIDAs). Both toxins were practically not detected in cereals and cereal products: AOH-one out of 13 samples at a content of 4.1 µg/kg; AME-two out of 13 samples at contents ranging between 0.2 and 0.6 µg/kg. However, if cereals for animal nutrition were analyzed, much higher values were found: AOH-five out of six samples (13-250 µg/kg); AME-six out of six samples (3-100 µg/kg). This finding may pose a potential problem concerning animal health. AOH and AME were frequently detected in vegetable products: AOH-5 out of 10 samples (2.6-25 µg/kg); AME-6 out of 10 samples (0.1-5 µg/kg). Tomato products were affected, especially. The highest content of AOH (25 µg/kg) and AME (5 µg/kg) were found in triple concentrated tomato paste. Special wines like "Trockenbeerenauslese" or "Spätlese" (affected by noble rot in the vineyard) contained AOH (4/6 samples; 1.2-4.9 µg/kg) and AME (4/6 samples; 0.1-0.3 µg/kg), but the values did not exceed the values of both toxins that were found generally in wines.

Stable isotope dilution assays of alternariol and alternariol monomethyl ether in beverages.

Stable isotope dilution assays (SIDAs) for the determination of the most important mycotoxins of the black mold Alternaria, namely, alternariol and alternariol monomethyl ether, have been developed. For this purpose, deuterated alternariol and alternariol methyl ether were synthesized by palladium catalyzed protium-deuterium exchange from the unlabeled toxins. Reaction conditions were chosen in such a manner that the formation of the [(2)H(4)]-isotopologues was favored. The synthesized products were characterized by LC-MS, NMR, and UV-spectroscopy. On the basis of the use of [(2)H(4)]-alternariol and [(2)H(4)]-alternariol methyl ether as internal standards, SIDAs were developed and applied to the determination of alternariol and alternariol methyl ether in beverages using LC-MS/MS. Method validation revealed a high sensitivity, i.e., low limits of detection (alternariol, 0.03 microg/kg; alternariol methyl ether, 0.01 microg/kg) and limits of quantitation (alternariol, 0.09 microg/kg; alternariol methyl ether, 0.03 microg/kg), respectively. Recovery from spiked apple juice was 100.5 +/- 3.4% for alternariol (range 0.1-1 microg/kg) and 107.3 +/- 1.6% for alternariol methyl ether (range 0.05-0.5 microg/kg). Interassay precision (expressed as coefficient of variation, CEV) for alternariol was 4.0% (7.82 +/- 0.31 microg/kg; vegetable juice, naturally contaminated) and 4.6% (1.04 +/- 0.05 microg/kg; grape juice, naturally contaminated). For alternariol methyl ether, a CEV of 2.3% (0.79 +/- 0.02 microg/kg; vegetable juice, naturally contaminated) was obtained. Analysis of fruit juices showed low contamination with alternariol and alternariol methyl ether in general, but higher values of both toxins were found in wine and vegetable juices. The values for alternariol were higher than those for alternariol methyl ether in nearly any case. However, the developed SIDA has proven to be optimally suited for further studies on alternariol and alternariol methyl ether content in food samples to obtain further insight into possible health hazards for the consumer.

Concentrations of total glutathione and cysteine in wheat flour as affected by sulfur deficiency and correlation to quality parameters.

A method for the simultaneous quantitation of total glutathione and total cysteine in wheat flour by a stable isotope dilution assay using high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) was developed. As internal standards, L-[(13)C3, (15)N]cysteine and L-gamma-glutamyl-L-[(13)C3, (15)N]cysteinyl-glycine were used. The method consisted of the extraction and reduction of flour with tris(2-carboxyethyl) phosphine after the addition of internal standards, protection of free thiol groups with iodoacetic acid, derivatization of free amino groups with dansyl chloride, and HPLC-MS/MS. The limits of detection and quantitation for glutathione were 0.75 nmol/g and 2.23 nmol/g flour, respectively. For cysteine, the limits of detection and quantitation were 0.72 nmol/g and 2.12 nmol/g flour, respectively. The developed method was found to be sensitive enough for quantitation of total glutathione and cysteine levels in wheat flour. This method was then utilized to investigate the effect of sulfur (S) deficiency on the amount of total glutathione and cysteine in flour. In S-deficient wheat, the concentrations of total glutathione and cysteine were proportional to the amount of S supplied during growth. The calculation of correlations revealed that GSH and Cys concentrations influenced the rheological dough properties and the baking performance at least as much as protein parameters. Thus, the low concentration of GSH and Cys in flour from S-deficient wheat had a similar effect on the technological properties as the altered composition of gluten proteins.

Use of isotope-labeled aflatoxins for LC-MS/MS stable isotope dilution analysis of foods.

Aflatoxins are a group of very carcinogenic mycotoxins that can be found on a wide range of food commodities including nuts, cereals, and spices. In this study, the first LC-MS/MS stable isotope dilution assay (SIDA) for the determination of aflatoxins in foods was developed. The development of this method was enabled by easily accessible isotope-labeled (deuterated) aflatoxins B2 and G2, which were synthesized by catalytic deuteration of aflatoxin B1 and G1, purified, and well-characterized by NMR and MS. All four aflatoxins of interest (B1, B2, G1, and G2) were quantified in food samples by using these two labeled internal standards. The response factors (RF) of the linear calibrations were revealed to be matrix independent for labeled aflatoxin B2/aflatoxin B2 and labeled aflatoxin G2/aflatoxin G2. For labeled aflatoxin B 2/aflatoxin B 1 and labeled aflatoxin B2/aflatoxin G1 matrix-matched calibration was performed for the model matrices almonds and wheat flour, showing significant differences of the RFs. Limits of detection (LOD) were determined by applying a statistical approach in the presence of the two model matrices, yielding 0.31 microg/kg (aflatoxin B1), 0.09 microg/kg (aflatoxin B2), 0.38 microg/kg (aflatoxin G1), and 0.32 microg/kg (aflatoxin G2) for almonds (similar LODs were obtained for wheat flour). Recovery rates were between 90 and 105% for all analytes. Coefficients of variation (CV) of 12% (aflatoxin B1), 3.6% (aflatoxin B2), 14% (aflatoxin G1), and 4.8% (aflatoxin G2) were obtained from interassay studies. For further validation, a NIST standard reference food sample was analyzed for aflatoxins B1 and B2. The method was successfully applied to determine trace levels of aflatoxins in diverse food matrices such as peanuts, nuts, grains, and spices. Aflatoxin contents in these samples ranged from about 0.5 to 6 microg/kg.