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.     

Synthesis of deuterated gamma-lactones for use in stable isotope dilution assays

Two syntheses of deuterated gamma-lactones for use as internal standards in stable isotope dilution assays (SIDA) were developed. [2,2,3,3-2H4]-gamma-Octa-, -gamma-deca-, and -gamma-dodecalactones with >89% deuterium incorporation were prepared in 27, 17, and 19% overall yields, respectively, by the reduction of a doubly protected hydroxypropiolic acid with deuterium gas. [3,3,4-2H3]-gamma-Octa- and -gamma-dodecalactones were prepared in 6 and 23% yields with >92% deuterium incorporation by the free radical addition of 2-iodoacetamide to [1,1,2-2H3]-1-hexene and [1,1,2-2H3]-1-decene, respectively. Reaction yields were highly dependent upon the purity of the 1-alkene starting material. The deuterated gamma-lactones were evaluated as internal standards for SIDA.     

Application of a tri-enzyme extraction for total folate determination in foods

A tri-enzyme digestion procedure using chicken pancreas conjugase, alpha-amylase, and Pronase was evaluated to determine its usefulness in the microbiological quantitation of total folate in foods. Folate values obtained by traditional conjugase digestion were compared to those obtained by the tri-enzyme method for 12 food products that represent diverse matrixes. The tri-enzyme treatment increased measurable folate from most foods when compared to levels found after conjugase digestion. Largest increases were noted for tuna fish (51%) and yogurt (33%) after tri-enzyme digestion. For the 12 foods, a mean increase of 19% in measurable folate was obtained with tri-enzyme treatment. The study shows that traditional conjugase treatment does not completely free folate from complex food matrixes before microbiological analysis. Further, as other investigations have suggested, current accepted methods for folate analysis may be underestimating folate levels in foods.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Sulphur immobilization and availability in soils assessed using isotope dilution

Increasing recognition of S deficiency in soils has raised the need for understanding processes governing S cycling and availability in soils. However, the quantification of the two main processes of S cycling, i.e. mineralization and immobilization, remains difficult as these processes occur simultaneously. A modified isotope ³⁵SO₄ dilution technique was developed and used to measure the effect of sulphate (SO₄) fertilization on S mineralization and immobilization in planted (pot experiment with ryegrass (Lolium multiflorum L.)) and unplanted soils (incubation). The immobilization and mineralization of S was calculated from the dynamics of stable and labelled S in soil KH₂PO₄ extracts containing an anion exchange membrane that concentrates SO₄ and mainly excludes other S species. The mathematical analysis of the isotope dilution data differs from methods proposed earlier. The radiolabile S in unplanted soil (E value) and in ryegrass (L value) were used as a measure of total available S in soils. Sulphate immobilization rate significantly declined during incubation. Sulphate application reduced gross mineralization but surprisingly reduced SO₄ immobilization. The E value significantly increased during the incubation in all soils as a result of gross mineralization, e.g. from 3.8 mg S kg⁻¹ at day 0 to 11.5 mg S kg⁻¹ at day 43 in the sandy soil with no sulphate addition. A full recovery in the E value of S added in (+S) treatments was achieved. Similarly, radiolabile S in the above-ground ryegrass biomass (L value) increased with S addition, with a full recovery of added S. The E and L values nearly fit a 1:1 line suggesting identical S dynamics in a planted and unplanted soil. The method proposed has operational advantages compared to methods used earlier.     

Certification of lead concentration in standard reference materials by isotope dilution mass spectrometry

In response to needs for analytical standards by researchers studying the exposure of humans to lead, a wide variety of environmental and "food" Standard Reference Materials have been prepared and certified for lead as well as for many other elements. Among the food types are SRM 1571, Orchard Leaves, 45 ppm; SRM 1575, Pine Needles, 10.8 ppm; SRM 1573, Tomato Leaves, 6.3 ppm; SRM 1566, Oyster Tissue, 0.48 ppm; SRM 1577, Bovine Liver, 0.34 ppm; SRM 1568, Rice Flour, 0.045 ppm; and SRM 1567, Wheat Flour, 0.020 ppm. These materials, intended for use in calibrating instruments and methods, have been certified by a definitive method, isotope dilution mass spectrometry. The advantages and disadvantages of this technique are discussed and some suggestions for the use of its isotopic selectivity in the study of lead in the human environment are presented.     

Detection of poultry and pork in cooked and canned meat foods by enzyme-linked immunosorbent assays

Enzyme-linked immunosorbent assays (ELISA) are described for the detection of poultry and pork in cooked and canned meat foods. These assays are based on species-specific, polyclonal antibodies raised against heat-resistant antigens. The heat-resistant antigens were isolated from raw skeletal muscle tissue of pork and chicken and were found to be immunoreactive even after heating to 120 degrees C for 15 min. The poultry ELISA could detect chicken or turkey at the 126 ppm level, and the pork ELISA could detect pork at the 250 ppm level. Samples of frankfurters, bolognas, pressed meats, canned baby foods, and canned spreads were prepared by simple aqueous extractions.     

A method for the analysis of natural and synthetic folate in foods

The essentiality of dietary folates for human beings has been known for many years. Over the shorter term, biological activities associated with several human maladies and the attenuation of biomarkers for several chronic diseases also have been assigned to folates. In the United States, these observations have led to the addition of folic acid to several foods and food ingredients (food fortification) and to dietary recommendations that assign biological activity to each of the forms of folate in the food supply. There currently is unavailable a robust, instrumental procedure that will distinguish between naturally occurring food folates and synthetic folic acid as part of the routine analysis of foods. The procedure proposed in this publication is unique in that it uses "off-the-shelf" supplies and instrumentation, to the extent possible, and was developed with "normal" corporate work schedules in mind. This method takes advantage of the tri-enzyme food digestion and folate deconjugation steps but was optimized with a commercially available rat plasma as the source of conjugase. A high-capacity styrene-divinylbenzene-based solid-phase extraction column was identified, and conditions were developed for quantitative recovery of 5-methyltetrahydrofolate and folic acid (FA) with it. The various forms of food folates are separated on a C-18 high-performance liquid chromatography (HPLC) column which is resistant to degradation at low pH. As a result, the mobile phase was simplified to a gradient of low-pH phosphate buffer (pH 2.2) and acetonitrile. Although FA does not exhibit fluorescence, a UV-induced photolysis system was added, which is controlled by the HPLC system, so that an appropriate segment of the HPLC column effluent is subjected to photolytic conditions and, thereby, FA can be measured as a fluorescent product. The application of the system was verified by analyzing several certified reference materials and foods and comparing results with certified values and/or total folate values as determined by microbiological assay.     

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.