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.     

Analysis of matrix-bound nitrofuran residues in worldwide-originated honeys by isotope dilution high-performance liquid chromatography-tandem mass spectrometry

A sensitive and selective isotope dilution liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMS/MS) method is presented for the simultaneous analysis of the metabolites of four nitrofuran veterinary drugs, that is, furazolidone, furaltadone, nitrofurantoin, and nitrofurazone, in honey samples. The method entails a combined hydrolysis of protein-bound drug metabolites and derivatization of the resulting metabolites with 2-nitrobenzaldehyde (NBA) during an overnight incubation, followed by a liquid-liquid extraction and a cleanup on a polymeric solid-phase extraction cartridge. Mass spectral acquisition is carried out in the positive ion mode by applying multiple reaction monitoring (MRM) of three diagnostic transition reactions for each analyte under survey. A reliable quantification is obtained by the use of one deuterated analogue per analyte (NBA-d(4) derivative). The method has been validated in honey according to the European Union criteria for the analysis of veterinary drug residues in food. Expressed in underivatized nitrofuran metabolite concentrations, the decision limits (CCalpha) ranged within 0.07-0.46 microg/kg, and the detection capabilities (CCbeta) were within 0.12-0.56 microg/kg. The method has been successfully applied in a survey of honeys of various geographical origins, showing that furazolidone is the main nitrofuran antibiotic administered to treat bacterial diseases of bees.     

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.     

Nitrogen isotope analysis of free glycine in soil using isotope dilution mass spectrometry

To enable the estimation of production and consumption rates of free glycine in soils through ¹⁵N isotope dilution experiments, an isotope dilution mass spectrometric method was developed. The method, which enabled high precision N isotope ratio determination of glycine in soil extracts at δ¹⁵N levels up to 4000‰ and concentrations from approximately 2 μM, is based on the following steps: (i) addition of glycine spike to the soil extract, (ii) removal of humic substances and pre-concentration of glycine using solid phase extraction, (iii) derivatization of amino acids, (iv) separation of the derivatives using gas chromatography (GC), (v) their combustion to yield sample N₂ gas, and (vi) finally the use of N isotope ratio mass spectrometry (IRMS). Judging by uncertainty budget calculations, the precision obtained (SD=0.01-0.06 at% ¹⁵N) is sufficient for detecting differences in N isotopic ratios obtained in ¹⁵N isotope dilution experiments.     

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.     

Development of a liquid chromatography-tandem mass spectrometry method using capillary liquid chromatography and nanoelectrospray ionization-quadrupole time-of-flight hybrid mass spectrometer for the detection of milk allergens

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the tryptic digest of a cleaned-up food matrix extract was used for the detection of milk allergens. The emphasis of this study was on casein, which is the most abundant milk protein and is also considered the most allergenic. A sample cleanup method was developed using an ion exchange column and centriprep device. Cookies spiked with milk powder from 0 to 1250 ppm were extracted, cleaned up, and either digested directly by trypsin or further cleaned up by gel electrophoresis before digestion. The peptide mixture was analyzed on a capillary LC-quadrupole time-of-flight system. Two marker peptides from alphaS1-casein were identified and used for prescreening. The MS/MS data from the mass spectrometry system were processed with Masslynx v4.0 and submitted for database search using either ProteinLynx Global Server or Mascot for protein identification. The LC-MS/MS method, using casein enzyme-linked immunosorbent assay as a reference, was tested on the cookie matrix and was extended to other sample matrices. There were good agreements between the two. This LC-MS/MS method provides a valuable confirmatory method for the presence of casein. It also allows the simultaneous detection of other milk allergens.     

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).     

Quantitative precursor studies on di- and trihydroxybenzene formation during coffee roasting using "in bean" model experiments and stable isotope dilution analysis

The objective of this study was to investigate the potential of various raw bean components as precursors of pyrogallol (1), hydroxyhydroquinone (2), catechol (3), 4-ethylcatechol (4), 4-methylcatechol (5), and 3-methylcatechol (6) under quasi "natural" roasting conditions by using the recently developed "in bean" model roast experiments. Freeze-dried, fully extracted bean shells were loaded with aqueous solutions of either single coffee compounds or fractions isolated from the raw bean solubles. After freeze-drying, these reconstituted beans were roasted, aqueous coffee brews were prepared, and the target phenols were quantified by means of a stable isotope dilution assay with LC-MS/MS detection. On the basis of the quantitative data, it can be concluded that upon coffee bean roasting, catechol (3) is primarily formed by degradation of caffeoylquinic acids from both the caffeic acid and the quinic acid moiety of the molecule, as well as from Maillard-type reactions from carbohydrates and amino acids. In contrast, pyrogallol (1) and hydroxyhydroquinone (2) are efficiently generated from carbohydrates and amino acids and, in addition, from free or chlorogenic acid bound quinic acid moieties. 4-Ethylcatechol (4) is exclusively generated upon thermal breakdown of caffeic acid moieties. 3-Methylcatechol (6) is formed primarily from the Maillard reactions and, to a minor extent, also from various phenolic precursors, whereas 4-methylcatechol (5) is produced in trace amounts only from all of the different precursors investigated. On the basis of this precursor study, reaction routes explaining the formation of the target phenols are proposed.     

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.     

Tylosin detection in animal feed by liquid chromatography-tandem mass spectrometry with enzymatic hydrolysis of the tylosin urea adduct

When the use of tylosin as a feed additive was forbidden by Council Regulation 2821/98, the necessity of a chemical confirmation method for the monitoring of the ban was created. Recently a method was developed for the detection of tylosin in animal feed by means of LC-MS/MS. During the validation high deviating values for the decision limit, detection capability, and repeatability for tylosin in cattle feed were observed, and the presence of urea and the formation of a tylosin urea adduct (TUA) were suggested as possible explanations. In this study two hydrolysis approaches for the TUA adduct were compared, namely, a chemical hydrolysis and an enzymatic hydrolysis with urease. The latter yielded a more complete hydrolysis of urea and was used for further validation. The recovery increased by approximately 15-25% depending on the amount of urea present in the feed (0.5-2%). The decision limit and detection capability were hardly influenced by the enzymatic hydrolysis.     

Optimization of a liquid chromatography-tandem mass spectrometry method for quantification of the plant lignans secoisolariciresinol, matairesinol, lariciresinol, and pinoresinol in foods

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of the four major enterolignan precursors [secoisolariciresinol, matairesinol, lariciresinol, and pinoresinol] in foods. The method consists of alkaline methanolic extraction, followed by enzymatic hydrolysis using Helix pomatia (H. pomatia) beta-glucuronidase/sulfatase. H. pomatia was selected from several enzymes based on its ability to hydrolyze isolated lignan glucosides. After ether extraction samples were analyzed and quantified against secoisolariciresinol-d8 and matairesinol-d6. The method was optimized using model products: broccoli, bread, flaxseed, and tea. The yield of methanolic extraction increased up to 81%, when it was combined with alkaline hydrolysis. Detection limits were 4-10 microg/(100 g dry weight) for solid foods and 0.2-0.4 microg/(100 mL) for beverages. Within- and between-run coefficients of variation were 6-21 and 6-33%, respectively. Recovery of lignans added to model products was satisfactory (73-123%), except for matairesinol added to bread (51-55%).     

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.     

液相色谱-大气压化学电离串联质谱法测定蔬菜中百菌清残留

建立了液相色谱-串联质谱法测定蔬菜中百菌清残留的方法.以乙腈提取样品中的百菌清,提取液无需净化,过滤膜后采用大气压化学电离源(APCI)负离子多反应监测(MRM)模式进行测定,基质匹配外标法定量.结果表明,蔬菜中百菌清的回收率与基质种类有关.在0.01、0.1、0.5 mg/kg3个添加水平下,番茄、西葫芦、大白菜和芹...     

Analysis of pesticides in dried hops by liquid chromatography-tandem mass spectrometry

An analytical method was developed for the determination of eleven agrochemicals [abamectin (as B1a), bifenazate, bifenthrin, carfentrazone-ethyl, cymoxanil, hexythiazox, imidacloprid, mefenoxam, pymetrozine, quinoxyfen, and trifloxystrobin] in dried hops. The method utilized polymeric and NH2 solid phase extraction (SPE) column cleanups and liquid chromatography with mass spectrometry (LC-MS/MS). Method validation and concurrent recoveries from untreated dried hops ranged from 71 to 126% for all compounds over three levels of fortification (0.10, 1.0, and 10.0 ppm). Commercially grown hop samples collected from several field sites had detectable residues of bifenazate, bifenthrin, hexythiazox, and quinoxyfen. The control sample used was free of contamination below the 0.050 ppm level for all agrochemicals of interest. The limit of quantitation and limit of detection for all compounds were 0.10 and 0.050 ppm, respectively.     

Determination of perfluorochemicals in cow's milk using liquid chromatography-tandem mass spectrometry

This study describes a new method developed for detection of 10 different perfluorochemicals (PFCs) in cow's milk, seven perfluorinated carboxylates and three perfluorinated sulfonate salts. After attempting multiple methods employing both acidic and basic extractions, a basic extraction using 10 mM sodium hydroxide in methanol digestion along with weak anion-exchange solid-phase extraction was employed. Vortex mixing and varying sonication times were compared as part of sample processing. Results show that sonication during sample processing yield decreased recovery of longer chain perfluorinated carboxylates. The final method developed was used to determine the concentration of PFCs in 12 raw and 49 retail milk samples from across the United States. With the exception of a single raw milk sample obtained from a dairy farm that had applied PFC containing biosolids to its fields, there were no milk samples containing PFCs.     

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.     

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.     

Quantification of lignans in food using isotope dilution gas chromatography/mass spectrometry

The optimization and validation of a protocol for the quantification of six dietary lignans, i.e., secoisolariciresinol, matairesinol, lariciresinol, pinoresinol, medioresinol, and syringaresinol, in food are presented. The method incorporates isotope dilution to ensure correct accuracy and precision, introducing the utilization of individual stable 13C3-labeled lignans. To demonstrate the potential of this new method, preliminary results of the levels of dietary lignans in selected foods are presented. It is concluded that the method fulfils the reliability criteria and can be applied to the analysis of the most common lignans in human food, being an essential asset to establish the intake of lignans in a determined population and their relation with disease prevention.     

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.     

Determination of cranberry phenolic metabolites in rats by liquid chromatography-tandem mass spectrometry

The glycosides of flavonoid, anthocyanins and A type proanthocyanidins in cranberry concentrate were characterized and quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cranberry concentrate (1 g/body weight) was orally gavaged to Fischer-344 rats (n = 6), and blood and urine samples were collected over 24 h periods. Quercetin, 3'-O-methylquercetin (isorhamnetin), myricetin, kaempferol, and proanthocyanidin dimer A2, together with thirteen conjugated metabolites of quercetin and methylquercetin and intact peonidin 3-O-galactoside and cyanidin 3-O-galactoside were identified in the rat urine after cranberry treatment. Very low levels of isorhamnetin (0.48 ± 0.09 ng/mL) and proanthocyanidin dimer A2 (0.541 ± 0.10 ng/mL) were found in plasma samples after 1 h of cranberry administration. Although no quercetin was detected in plasma, MRM analysis of the methanolic extract of urinary bladder showed that chronic administration of cranberry concentrate to rats resulted in accumulation of quercetin and isorhamnetin in the bladder. These results demonstrate that cranberry components undergo rapid metabolism and elimination into the urine of rats and are present in the urinary bladder tissue potentially allowing them to inhibit urinary bladder carcinogenesis.