Development of two stable isotope dilution assays for the quantitation of acrolein in heat-processed fats

Two stable isotope dilution assays were developed for the quantitation of acrolein in fats and oils using [(13)C(3)]-acrolein as the internal standard. First, a direct GC-MS headspace method, followed by an indirect GC-MS method using derivatization with pentafluorophenyl hydrazine, was established. Analysis of six different types of oils varying in their pattern of fatty acids showed significant differences in the amounts of acrolein formed after heating at various temperatures and for various times. For example, after 24 h at 140 °C, coconut oil contained 6.7 mg/kg, whereas linseed oil was highest with 242.3 mg/kg. A comparison of the results showed that the extent of acrolein formation seemed to be correlated with the amount of linolenic acid in the oils. Although the acrolein concentrations were lowered in all six oils after frying of potato crisps, linseed and rapeseed oil still contained the highest amounts of acrolein after frying. By applying both methods on different thermally treated fats and oils, nearly identical quantitative data were obtained.     

Synthesis of deuterated volatile lipid degradation products to be used as internal standards in isotope dilution assays. 1. Aldehydes.

The isotopically labeled compounds [5,6-(2)H(2)]hexanal (d-I), [2, 3-(2)H(2)]-(E)-2-nonenal (d-II), [3,4-(2)H(2)]-(E,E)-2,4-nonadienal (d-III), and [3,4-(2)H(2)]-(E,E)-2,4-decadienal (d-IV) were prepared in good yields using new or improved synthesis procedures. Labeling position, chemical purity, and isotopic distribution of the compounds were characterized by various MS and NMR techniques. These molecules are used as internal standards in quantification experiments based on isotope dilution assay. Synthesis of d-I, d-III, and d-IV has not yet been reported in the literature.     

Synthesis of deuterated volatile lipid degradation products to be used as internal standards in isotope dilution assays. 2. Vinyl ketones.

The isotopically labeled compounds [5,6-(2)H(2)]-(Z)-1, 5-octadien-3-one (d-I) and [1-(2)H(1;2),2-(2)H(1;1)]-1-octen-3-one (d-II), as well as the unlabeled reference compound (Z)-1, 5-octadien-3-one (I) were prepared by improved synthesis procedures. Labeling position, chemical purity, and isotopic distribution of the compounds were characterized by various MS and NMR techniques. These molecules are used as internal standards in quantification experiments based on isotope dilution assay. The newly prepared compound d-II was synthesized in a simple two-step procedure, and formation of the main isotopomers was studied in model systems.     

Quantitation of odor-active compounds in rye flour and rye sourdough using stable isotope dilution assays

Application of the aroma extract dilution analysis on a flavor distillate prepared from freshly ground rye flour (type 1150) revealed 1-octen-3-one (mushroom-like), methional (cooked potato), and (E)-2-nonenal (fatty, green) with the highest flavor dilution (FD) factors among the 26 odor-active volatiles identified. Quantitative measurements performed by stable isotope dilution assays and a comparison to the odor thresholds of selected odorants in starch suggested methional, (E)-2-nonenal, and hexanal as contributors to the flour aroma, because their concentrations exceeded their odor thresholds by factors >100. Application of the same approach on a rye sourdough prepared from the same batch of flour revealed 3-methylbutanal, vanillin, 3-methylbutanoic acid, methional, (E,E)-2,4-decadienal, 2,3-butanedione, and acetic acid as important odorants; their concentrations exceeded their odor thresholds in water and starch by factors >100. A comparison of the concentrations of 20 odorants in rye flour and the sourdough made therefrom indicated that flour, besides the fermentation process, is an important source of aroma compounds in dough. However, 3-methylbutanol, acetic acid, and 2,3-butanedione were much increased during fermentation, whereas (E,E)-2,4-decadienal and 2-methylbutanal were decreased. Similar results were obtained for five different flours and sourdoughs, respectively, although the amounts of some odorants in the flour and the sourdough differed significantly within batches.     

Synthesis of four carbon-13-labeled type a trichothecene mycotoxins and their application as internal standards in stable isotope dilution assays

The first stable isotope dilution assay (SIDA) for the simultaneous quantitation of the most abundant type A trichothecenes in foods and feeds was developed. Synthesis of carbon-13-labeled T2-toxin, HT2-toxin, diacetoxyscirpenol, and monoacetoxyscirpenol was accomplished by [13C2]-acetylation of T2-triol and scirpentriol, respectively. Scirpentriol was prepared from diacetoxyscirpenol by complete alkaline hydrolysis and subsequently was converted to [13C6]-triacetoxyscirpentriol by peracetylation with [13C4]-acetic anhydride. The latter compound was selectively hydrolyzed using ammonium hydroxide to give [13C4]-diacetoxyscirpenol and [13C2]-monoacetoxyscirpenol in reasonable yields. Analogously, [13C6]-T2-triacetate was prepared from T2-triol and subjected to controlled hydrolysis to yield [13C4]-T2-toxin and [13C2]-HT2-toxin. All synthesized products were characterized by NMR and MS experiments. Using the prepared isotopically labeled standards, SIDAs were developed for the quantitation of type A trichothecenes in food and feeds. The mycotoxins were quantified by LC-single and tandem MS after cleanup on multifunctional columns. The method revealed good sensitivity with low detection and quantification limits along with excellent recovery and good precision in interassay studies. Food samples were analyzed using the developed SIDA and showed substantial contamination of oat products with T2-toxin and HT2-toxin. Diacetoxyscirpenol was detected on potatoes, whereas monoacetoxyscirpenol was not present in the analyzed samples.     

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).     

Syntheses of labeled vitamers of folic acid to be used as internal standards in stable isotope dilution assays

[2H4]Folic acid was synthesized by deuterating p-aminobenzoic acid, which was then coupled to glutamic acid and 6-formylpterin. Using [2H4]folic acid as starting component enabled the preparation of labeled vitamers tetrahydrofolate, 5-formyltetrahydrofolate, 5-methyltetrahydrofolate, and 10-formylfolate which were characterized by electrospray mass spectrometry and collision-induced dissociation. The mass spectrometric studies confirmed that the compounds could be used as internal standards in stable isotope dilution assays.     

Biosynthesis of 15N3-labeled enniatins and beauvericin and their application to stable isotope dilution assays

The first stable isotope dilution assay for the determination of enniatins A, A1, B, and B1 and beauvericin was developed. The (15)N(3)-labeled enniatins and beauvericin were biosynthesized by feeding two Fusarium strains Na(15)NO(3) and subsequently isolated from the fungal culture. The chemical structures of the biosynthesized products were characterized by LC-MS/MS and (1)H NMR. Standard solutions of (15)N(3)-labeled beauvericin, enniatin A, and enniatin A1 were accurately quantitated by quantitative NMR. On the basis of the use of the labeled products as internal standards, stable isotope dilution assays were developed and applied to various food samples using LC-MS/MS. The sample extracts were directly injected without any tedious cleanup procedures. The limits of detection were 3.9, 2.6, 3.7, 1.9, and 4.4 μg/kg for enniatins A, A1, B, and B1 and beauvericin, respectively. Limits of quantitation were 11.5 (enniatin A), 7.6 (enniatin A1), 10.9 (enniatin B), 5.8 (enniatin B1), and 13.1 μg/kg (beauvericin). Recoveries were within the range between 90 and 120%, and good intraday and interday precisions with coefficients of variation between 1.35 and 8.61% were obtained. Thus, the stable isotope dilution assay presented here is similarly sensitive and precise but more accurate than assays reported before. Analyses of cereals and cereal products revealed frequent contaminations of barley, wheat, rye, and oats with enniatins B and B1, whereas beauvericin was not quantifiable.     

Development of stable isotope dilution assays for the simultaneous quantitation of biogenic amines and polyamines in foods by LC-MS/MS

Microbial amino acid metabolism may lead to substantial amounts of biogenic amines in either spontaneously fermented or spoiled foods. For products manufactured with starter cultures, it has been suggested that certain strains may produce higher amounts of such amines than others; however, to support efforts of food manufacturers in mitigating amine formation, reliable methods for amine quantitation are needed. Using 10 isotopically labeled biogenic amines as the internal standards, stable isotope dilution assays were developed for the quantitation of 12 biogenic amines and of the 2 polyamines, spermine and spermidine, in one LC-MS/MS run. Application of the method to several foods revealed high concentrations of, for example, tyramine and putrescine in salami and fermented cabbage, whereas histamine was highest in Parmesan cheese and fermented cabbage. On the other hand, ethanolamine was highest in red wine and Parmesan cheese. The results suggest that different amino acid decarboxylases are active in the respective foods depending on the microorganisms present. The polyamine spermine was highest in salami and tuna.     

Labile lead in polluted soils measured by stable isotope dilution

It is well known that lead (Pb) is strongly immobilized in soil by adsorption or precipitation. However, the reversibility of these reactions is poorly documented. In this study, the isotopically exchangeable Pb concentration in soils (E‐value) was measured using a stable isotope (²⁰⁸Pb). Soils were collected at three industrialized sites where historical Pb emissions have resulted in elevated Pb concentrations in the surrounding soil. Lead concentrations ranged from background values, in the control soils collected far from the emission source, to highly elevated concentrations (5460–14440 mg Pb kg^?1). The control soil of each site was amended in the laboratory with Pb(NO₃)₂ to the same total Pb concentrations as the field‐contaminated soils. The %E values (E‐value relative to total Pb content) were greater than 84% in the laboratory‐amended soils, and ranged from 45% to 78% (mean 58%) in the field‐contaminated soils. The relatively large labile fractions of Pb in the field‐contaminated soils show that the majority of Pb is reversibly bound despite the fact that the binding strength is large. The Pb concentrations in soil solution were up to 3500‐fold larger for the laboratory‐amended soils than for field‐contaminated soils at corresponding total Pb concentrations. These differences cannot be explained by differences in labile fractions of Pb but are attributed to the decrease in soil solution pH upon addition of Pb²⁺‐salt.     

Determination of key aroma compounds in the crumb of a three-stage sourdough rye bread by stable isotope dilution assays and sensory studies

An investigation of the volatile fraction of a freshly prepared sourdough rye bread crumb by means of the aroma extract dilution analysis (AEDA), followed by identification experiments, revealed 22 flavor compounds in the flavor dilution (FD) factor range of 128 to 2048. Quantitations performed by stable isotope dilution assays (SIDA) and a calculation of odor activity values (OAV; ratio of concentration to odor threshold) revealed the following as contributors to the overall crumb flavor: 3-methylbutanal (malty), (E)-2-nonenal (green, fatty), (E,E)-2,4-decadienal (fatty, waxy), hexanal (green), acetic acid (sour, pungent), phenylacetaldehyde (honey-like), methional (boiled potato-like), vanillin (vanilla-like), 2,3-butandione (buttery), 3-hydroxy-4,5-dimethyl-2(5H)-furanone (spicy), and 2- and 3-methylbutanoic acid (sweaty). Using either citrate buffer, starch, or deodorized crumb as model matrixes, the typical malty and sour rye bread crumb flavor was reproduced by adding a mixture of 20 reference odorants in the "natural" concentrations as quantitatively determined in the fresh crumb.     

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.     

Quantification of free coumarin and its liberation from glucosylated precursors by stable isotope dilution assays based on liquid chromatography-tandem mass spectrometric detection

A stable isotope dilution assay for the quantification of free coumarin and glucosylated coumarin precursors has been developed using [13C2]-coumarin as the internal standard. The doubly labeled coumarin was synthesized by reacting [13C2]-acetic anhydride with salicylic aldehyde and characterized by means of mass spectrometry and nuclear magnetic resonance (NMR) experiments. The specifity of liquid chromatography-tandem mass spectrometry enabled unequivocal determination and sensitive quantitation of the odorant. Because of the very simple extraction procedure, free coumarin could be analyzed within 1h. For quantification of total coumarin, the odorant was liberated from its precursors by an incubation with hydrochloric acid or beta-glucosidase. In analyses of breakfast cereals, the intra-assay coefficient of variation was 9.9% ( n = 5) for total coumarin. When coumarin was added to butter cookies at a level of 10 microg/kg, a recovery of 94.1% was found. Further addition studies revealed a detection limit of 2.9 microg/kg and a quantification limit of 8.6 microg/kg. Application of the stable isotope dilution assay to several plants, foods, and essential oils revealed high contents in cassia products and those foods in which cassia has been used as an ingredient. In contrast to this, Ceylon cinnamon contained much less coumarin. The odorant was also quantified in woodruff, clover seeds, and the essential oils of lavender, citron, and chamomile. Only trace amounts were detected in carrots and the essential oils of peppermint and dill, whereas in bilberries, black raspberries, and Angelica roots, coumarin was below detectable levels. In Ceylon cinnamon and cassia, the odorant occurred mainly in its free form, whereas in fenugreek seeds and woodruff, 68 and 88% of the total coumarin content was liberated from glucosylated precursors, respectively.     

Quantification of the mycotoxin patulin by a stable isotope dilution assay.

Two stable isotope dilution assays for the quantification of patulin [4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one] in foods were developed using (13)C-labeled patulin as the internal standard. One method was performed by means of LC/MS in negative electrospray ionization mode without derivatization; the other used HRGC/HRMS after trimethylsilylation of the patulin isotopomers. In comparison with previously reported methods based on high-performance liquid chromatography with UV detection, HRGC/HRMS of the derivatized samples showed better repeatability, higher recovery rates (96% at a spike level of 200 ng/L), and a 100 times lower detection limit (12 ng/L). In contrast, LC/MS showed a much lower performance as compared to HPLC/UV or HRGC/HRMS. Using HRGC/HRMS, the mycotoxin was quantified in many different fruit products and in molded wheat bread.     

Improved approach for analyzing bromophenols in seafood using stable isotope dilution analysis in combination with SPME

An analytical method for the measurement of five naturally occurring bromophenols of sensory relevance in seafood (barramundi and prawns) is presented. The method combines simultaneous distillation-extraction followed by alkaline back extraction of a hexane extract and subsequent acetylation of the bromophenols. Analysis of the bromophenol acetates was accomplished by headspace solid phase microextraction and gas chromatography-mass spectrometry using selected ion monitoring. The addition of (13)C 6 bromophenol stable isotope internal standards for each of the five congeners studied permitted the accurate quantitation of 2-bromophenol, 4-bromophenol, 2,6-dibromophenol, 2,4-dibromophenol, and 2,4,6-tribromophenol down to a limit of quantification of 0.05 ng/g of fish flesh. The method indicated acceptable precision and repeatability and excellent linearity over the typical concentration range of these compounds in seafood (0.5-50 ng/g). The analytical method was applied to determine the concentration of bromophenols in a range of farmed and wild barramundi and prawns and was also used to monitor bromophenol uptake in a pilot feeding trial.     

Evaluation of the key aroma compounds in beef and pork vegetable gravies a la chef by stable isotope dilution assays and aroma recombination experiments

Although the aroma compounds of meat processed as such have been studied previously, data on complete homemade dishes containing beef and pork meat were scarcely studied. Recently, 38 odor-active compounds were characterized in beef and pork vegetable gravies using GC-olfactometry. In the present investigation, the most odor-active compounds were quantitated in a freshly prepared stewed beef vegetable gravy (BVG) as well as a stewed pork vegetable gravy (PVG) by means of stable isotope dilution assays. Calculation of odor activity values (OAVs; ratio of concentration to odor threshold) revealed 3-mercapto-2-methylpentan-1-ol, (E,E)-2,4-decadienal, (E,Z)-2,6-nonadienal, (E)-2-decenal, (E)-2-undecanal, and 3-hydroxy-4,5-dimethyl-2(5H)-furanone as the most potent odorants in both gravies. However, significantly different OAVs were found for 12-methyltridecanal, which was much higher in the BVG, whereas (E,Z)-2,4-decadienal showed a clearly higher OAV in the PVG. Aroma recombination experiments performed on the basis of the actual concentrations of the odorants in both gravies revealed a good similarity of the aromas of both model mixtures containing all odorants with OAVs > 1 with those of the original gravies.     

Improved folate extraction and tracing deconjugation efficiency by dual label isotope dilution assays in foods

A dual label stable isotope dilution assay was developed to trace the deconjugation efficiency of polyglutamic folate vitamers converted to their monoglutamic analogues. For this purpose, [(13)C(5)]-pteroylheptaglutamate was synthesized and added during extraction of foods as a tracer isotopologue along with [(2)H(4)]-5-methyltetrahydrofolate, [(2)H(4)]-5-formyltetrahydrofolate, [(2)H(4)]-tetrahydrofolate, [(2)H(4)]-10-formylfolate, and [(2)H(4)]-folic acid. The [(2)H(4)]-labeled folates were used as internal standards for the monoglutamates. Deconjugation converted the addition tracer [(13)C(5)]-pteroylheptaglutamate to the detection tracer [(13)C(5)]-folic acid, which was quantified along with unlabeled folic acid using [(2)H(4)]-folic acid as the internal standard. LC-MS/MS enabled the unequivocal differentiation of the three isotopologues. This tracing was used to optimize deconjugation efficiency, which was achieved by using 4-morpholineethanesulfonic acid buffer for extraction at pH 5.0 . The optimized assay revealed limits of detection for the folate vitamers ranging between 2.0 and 5.6 pmol per assay (equivalent to 2.2-6.6 μg/100 g dry mass), recoveries ranging between 98 and 105% and relative standard deviations in inter-assay precision ranging between 2 and 6%. The assay was applied to quantitate folates in spinach, beans, cheeses, bread, wheat germs, and yeast .     

Quantification of free and bound pantothenic acid in foods and blood plasma by a stable isotope dilution assay

A stable isotope dilution assay for quantification of pantothenic acid in food and blood plasma uses a 4-fold labeled isotopomer of the vitamin as an internal standard. Pantothenic acid and its labeled analogue were detected as trimethylsilyl derivatives by gas chromatography-mass spectrometry, showing a minimized spectral overlap. In starch a detection limit of 44 microg/kg, an intrasample relative standard deviation of 6.7%, and recovery values ranging between 97.5 and 99.4% were determined. Total pantothenic acid content was determined in rice, milk powder, apple juice, and blood plasma after enzymatic hydrolysis of the vitamin's conjugates; free pantothenic acid was quantified prior to enzyme treatment. Almost all results were found to be in good agreement with literature data.     

Synthesis and stable isotope dilution assay of ethanethiol and diethyl disulfide in wine using solid phase microextraction. Effect of aging on their levels in wine

Ethanethiol and diethyl disulfide (DEDS) most often occurred at levels above their olfactive threshold in wines with nauseous sulfur-linked smells. As ethanethiol is very oxidizable and chemically reactive, a stable isotopic dilution analysis of both ethanethiol and its disulfide in wines using solid phase microextraction and GC-MS was developed. The latter involved the determination of the proportion of DEDS formed by oxidation of the thiol during the analysis conditions, which was obtained by the use of two differently labeled disulfide standards. An original synthesis of labeled ethanethiol standards in conditions minimizing oxidation was developed, and the corresponding labeled diethyl disulfides were obtained from these thiols. This analytical method was used to follow the levels of these sulfur compounds during aging in a young red wine spiked with ethanethiol and added with enological tannins, with or without oxygen addition. The total levels of these two sulfur compounds were shown to decrease steadily after 60 days of aging, up to 83%. The effect of oxygen sped this decrease, but the effect of enological tannins was very slight. Residual ethanethiol was detected in its disulfide form from approximately 36% in the nonoxygenated wines to 69% in the oxygenated samples.     

Development of a stable isotope dilution assay for the quantitation of glycidamide and its application to foods and model systems

On the basis of a stable isotope dilution assay and derivatization with 2-mercaptobenzoic acid, the presence of the carcinogenic glycidamide ( 2) in processed foods was verified for the first time. Using (13)C-labeled 2 as the internal standard and the formation of the thioether derivatives, a new stable isotope dilution assay for the quantitation of 2 was developed. Application of the method on several potato samples revealed amounts between 0.3 and 1.5 mug/kg depending on the processing conditions. In a model experiment, the formation of 2 by an epoxidation of the double bond in acrylamide, that is, by a reaction with linoleic acid hydroperoxides, was established. This result was in good agreement with data showing that French fries processed in sunflower oil, which is high in linoleic acid, contained more 2 as compared to fries prepared in coconut oil. The derivatization procedure allows the simultaneous quantitation of acrylamide and glycidamide in foods.