Quantitation of estragole by stable isotope dilution assays

Various calibration strategies for the quantitation of the phenylpropane estragole by gas chromatography-mass spectrometry were developed and compared. For application in stable isotope dilution assays, two deuterium labelled estragole isotopologues were synthesized. Of these, [3′,3′-²H₂]estragole was prepared by Wittig reaction of 4-methoxy-phenylacetaldehyde with [²H₃]methyl-triphenyl-phosphonium bromide, whereas [1″,1″,1″-²H₃]estragole was obtained by demethylation of estragole and deuteromethylation of the resulting 4-allylphenole.Besides estragole isotopologues, 1,2,4-trimethoxybenzene and 4-propylanisole were also tested as internal standards (I.S.) for the determination of estragole in fennel tea.[1″,1″,1″-²H₃]Estragole, 1,2,4-trimethoxybenzene, and 4-propylanisole revealed linear calibration functions and, therefore, were suitable for estragole quantitation. In contrast to this, [3′,3′-²H₂]estragole could only be applied as I.S. if it was added to the extracts in stoichiometric deficiency compared to unlabelled estragole. Moreover, due to its different chemical and physical properties, 1,2,4-trimethoxybenzene showed a recovery as low as 77%, whereas the other I.S. revealed recovery rates close to 100%. Considering the “real” values of estragole in fennel tea, the choice of the I.S. obviously is less important than the way of preparing the tea. In contrast to the common method for tea preparation, squeezing of the teabags increased the estragole content significantly by 50%.     

Stable isotope dilution analysis of the Fusarium mycotoxins deoxynivalenol and 3-acetyldeoxynivalenol

Trichothecenes are secondary metabolites produced by several fungi of the Fusarium genus during their growth period. They inhibit protein biosynthesis in eukaryotic cells resulting in numerous toxic effects such as diarrhea, vomiting, and gastro-intestinal inflammation. Considering its occurrence in food and feedstuff, deoxynivalenol (DON) is one of the most important trichothecenes. We report the synthesis of stable isotope labeled 15-d(1)-deoxynivalenol (15-d(1)-DON) from its natural precursor 3-acetyldeoxynivalenol (3-AcDON) as starting material. Furthermore, a method for the analysis of DON and 3-AcDON using HPLC-MS/MS with stable isotope labeled 15-d(1)-DON and 3-d(3)-AcDON as internal standards has been developed. In total, 18 cereal product samples were analyzed with contamination levels ranging from 10-301 microg/kg for DON and 5-14 microg/kg for 3-AcDON. This is the first report of an isotope dilution MS method for the analysis of type B-trichothecenes.     

Determination of acrylamide in potato chips by a reversed-phase LC–MS method based on a stable isotope dilution assay

Potato-based products represent an important part of the daily intake of food-derived acrylamide, mainly on adolescent population from western countries. A reversed-phase liquid chromatography-mass spectrometry based on a stable isotope dilution assay was used for acrylamide analysis. Aqueous sample extraction, cleaning with Carrez solution and solid phase extraction with methanol was applied. The ratio potato/NaCl solution is critical during extraction where the optimum ratio is 0.125 g/ml NaCl 2 M solution. The use of virgin olive oil, as retaining matrix, during methanol desiccation was critical to achieve high recoveries. The method performance was validated for limit of detection (23.2 μg/kg) and quantitation (91.8 μg/kg), linearity (r > 0.999, 25–1000 μg/kg), recovery (98.8%). The method was applied on commercial potato chips; the intra-day repeatability was set at 3.9% and values were corrected with a labeled internal standard (¹³C₃-acrylamide). No significant differences on the acrylamide content were observed between industrial-scale and local-scale processed potato chips.     

Quantification of 1,8‐cineole and of its metabolites in humans using stable isotope dilution assays

The metabolism of 1,8‐cineole after ingestion of sage tea was studied. After application of the tea, the metabolites 2‐hydroxy‐1,8‐cineole, 3‐hydroxy‐1,8‐cineole, 9‐hydroxy‐1,8‐cineole and, for the first time in humans, 7‐hydroxy‐1,8‐cineole were identified in plasma and urine of one volunteer. For quantitation of these metabolites and the parent compound, stable isotope dilution assays were developed after synthesis of [²H₃]‐1,8‐cineole, [9/10‐²H₃]‐2‐hydroxy‐1,8‐cineole and [¹³C,²H₂]‐9‐hydroxy‐1,8‐cineole as internal standards. Using these standards, we quantified 1,8‐cineole by solid phase microextraction GC‐MS and the hydroxyl‐1,8‐cineoles by LC‐MS/MS after deconjugation in blood and urine of the volunteer. After consumption of 1.02 mg 1,8‐cineole (19 μg/kg bw), the hydroxycineoles along with their parent compound were detectable in the blood plasma of the volunteer under study after liberation from their glucuronides with 2‐hydroxycineole being the predominant metabolite at a maximum plasma concentration of 86 nmol/L followed by the 9‐hydroxy isomer at a maximum plasma concentration of 33 nmol/L. The parent compound 1,8‐cineole showed a low maximum plasma concentration of 19 nmol/L. In urine, 2‐hydroxycineole also showed highest contents followed by its 9‐isomer. Summing up the urinary excretion over 10 h, 2‐hydroxycineole, the 9‐isomer, the 3‐isomer and the 7‐isomer accounted for 20.9, 17.2, 10.6 and 3.8% of the cineole dose, respectively.     

A reference isotope dilution headspace GC/MS method for the determination of nitrite and nitrate in meat samples

A novel method for the determination of nitrite and nitrate in meat products is presented. The samples were ground and extracted in hot water with the presence of and internal standards. The solution was buffered with sodium bicarbonate and reacted with triethyloxonium tetrafluoroborate to convert nitrite and nitrate into EtNO₂ and EtONO₂. Such derivatives could be detected by headspace GC/MS in positive chemical ionisation mode with 0.05 µg g⁻¹ and 1.0 µg g⁻¹ LOD. The method was used for and quantitation in the 0.5-300 and 2.5-300 µg g⁻¹ ranges. The method was applied for the analysis of fifteen meat products. Despite minimal sample preparation, the headspace sampling ensured a clean chromatography for over 135 analyses (throughput ten samples per hour). The proposed method offers selective GC/MS detection combined with high-precision isotope dilution calibration, it is suitable for metrological applications and can support regulations on meat safety (European Commission, 2011).     

Glucosinolate and folate content in sprouted broccoli seeds

HPLC analysis of broccoli seeds and laboratory-grown broccoli sprouts revealed that three aliphatic glucosinolates (glucoraphanin, glucoiberin and glucoerucin) and a group of indol-glucosinolates including 4-hydroxy-glucobrassicin are preformed in the seeds. In the early stage of sprouting a reduction (approx. 20%) of the aliphatic glucosinolates was measured. During further growing, until day 8, and subsequent cold storage up to the 12th day the amounts of the aliphatic glucosinolates levelled off. While 4-hydroxy-glucobrassicin declined continuously, three minor indole-derivatives increased steadily, but remained at a comparatively low level. Besides glucosinolates, folates were quantified in broccoli sprouts by stable isotope dilution assays (SIDAs). During germination, the contents of total folates increased to 72 μg/100 g fresh mass and 546 μg/100 g dry mass on the 4th day, which was equivalent, respectively, to a 3-fold and a 24-fold increase in the seed’s content. Thereafter, total folates decreased again to 13 μg/100 g fresh mass until the 8th day of germination and remained at this low level. The folate pattern measured by SIDA revealed 5-methyltetrahydrofolate as the predominant vitamer at each stage.     

Determination of melamine and its analogues in egg by gas chromatography-tandem mass spectrometry using an isotope dilution technique

A method was developed for the simultaneous determination of melamine, ammeline, ammelide, and cyanuric acid in egg using gas chromatography-tandem mass spectrometry (GC-MS/MS). The samples were first extracted by the solution of diethylamine–water–acetonitrile (10:40:50, v/v/v). Clean-up employed an ‘On Guard II’ RP cartridge, and the dried elute was derivatised using bis-(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS). Derivatised samples were analysed by GC-MS/MS using multiple-reaction monitoring (MRM) with ¹³C₃-¹⁵N₃-labelled melamine and cyanuric acid as internal standards. Blank samples of egg were spiked with the four analytes at concentration level of 0.1, 0.5, 1.0 mg kg^?1, and the intra-day and inter-day recoveries were in the range 75.7–122.5% with the relative standard deviation (RSD) from 2.6% to 22.8%. Decision limits (CCα, α = 0.01) for melamine, ammeline, ammelide, and cyanuric acid in egg samples and milk powder were 3.5–5.9 and 2.5 to 3.8 µg kg^?1, and the detection capabilities (CCβ, β = 0.05) were 4.9–8.4 and 3.6–9.5 µg kg^?1, respectively. The method was successfully applied to egg samples and milk products as well. Satisfactory results were obtained as part of the 2009 European Union melamine proficiency test.     

同位素稀释-超高效液相色谱-串联质谱法检测蜂蜜中泛酸含量

建立了同位素稀释-超高效液相色谱-串联质谱法（UPLC-MS/MS）检测蜂蜜中泛酸（维生素B5）的快速定量和确证方法。样品经0.1%氨水（V/V）提取,MAX固相萃取柱净化后,以0.1%甲酸乙腈-0.1%甲酸水为流动相梯度洗脱,使用HSS T3色谱柱进行分离,电喷雾正离子多反应监测（MRM）模式检测,同位素内标法定量。在优化条件下,泛酸在0.500～500 μg/L范围内线性良好,相关系数（R2）为0.999 6;方法检出限为1.0 μg/kg（S/N=3）,定量限为3.0 μg/kg（S/N=10）;回收率在98.3%～98.7%之间,相对标准偏差（RSD）为2.4%～3.1%。通过市售蜂蜜样品测试表明,该方法操作简单、检测结果准确,可用于蜂蜜中泛酸的快速检测。     

Analysis of coumarin in various foods using liquid chromatography with tandem mass spectrometric detection

Recently authorities and the media have published reports of increased coumarin levels in Christmas biscuits, gingerbread, cinnamon cookies and other foods. The maximum tolerance limits for coumarin in food are set out in EC Directive 88/388/ECC. Since the standard use of analytical techniques, such as HPLC–UV, are only limitedly suitable for verifying compliance with the regulatory limit of 2 mg/kg for coumarin in food, a selective, sensitive isotope dilution LC–MS/MS method for determining coumarin content in foods was developed, tested and validated, and the results then compared to the HPLC–UV method. This LC–MS/MS method increased selectivity and raised sensitivity by a factor of 100 compared to the HPLC–UV technique. The respective limits of determination and quantification for the method were 0.03 and 0.05 mg/kg, respectively, with an RSD of 1–8% and a linearity range of 0.1–500 ng/mL (corresponding to 0.05–250 mg/kg in food). In addition, a selection of around 500 food samples was also tested for coumarin content using the LC−MS/MS technique.     

Absence of 2'-deoxyguanosine-carbon 8-bound ochratoxin A adduct in rat kidney DNA monitored by isotope dilution LC-MS/MS

The contribution of DNA adduct formation in the carcinogenic action of the mycotoxin ochratoxin A (OTA) has been subject to much debate. Recently, a carbon-bonded ochratoxin A-2'-deoxyguanosine adduct (dGuoOTA) formed by photochemical reaction in vitro has been shown by 32P-postlabeling/TLC to comigrate with a spot detected in DNA isolated from rat and pig kidney following exposure to OTA. Considering the large body of evidence arguing against covalent DNA binding of OTA and the poor resolution and specificity of postlabeling analysis, we developed a stable isotope dilution LC-MS/MS method to analyze dGuoOTA in kidney DNA isolated from rats treated with OTA. dGuoOTA and nitrogen-15-labeled dGuoOTA (15N(5)-dGuoOTA) were prepared by photoirradiation of OTA in the presence of dGuo or nitrogen-15-labeled dGuo. Conditions for DNA hydrolysis were optimized using a synthetic oligonucleotide containing dGuoOTA to ensure complete release of dGuoOTA. The LOD of the method (S/N > 3) was 10 fmol dGuoOTA on-column. However, dGuoOTA was not detected in DNA samples isolated from male F344 rats treated with OTA for up to 90 days at doses known to cause renal tumor formation. Detection limits, calculated for each individual sample based on the absolute LOD and the amount of DNA injected, were as low as 3.5 dGuoOTA/10(9) nucleotides. These data are consistent with previous results showing lack of DNA adduct formation by OTA and demonstrate that dGuoOTA is not formed in biologically relevant amounts under physiological conditions in vivo.     

Determination of acrylamide in roasted chestnuts and chestnut-based foods by isotope dilution HPLC-MS/MS

A collaboratively trial tested isotope dilution liquid chromatographic method with positive electrospray ionisation tandem mass spectrometry for the analysis of acrylamide in bakery ware and potato products has been extended to the determination of acrylamide in roasted chestnuts and chestnut-based foods. As chestnuts have a similar composition to potatoes, considerable amounts of acrylamide can be expected, especially in roasted chestnut products. This paper presents the concentrations of acrylamide in 31 different chestnut samples (fresh, roasted, flour, cooked, glazed) that were collected in nine European countries during 2005/2006. The influence of the roasting time on the acrylamide content was also experimentally investigated. A test portion was extracted after homogenisation with water and isotopically labelled acrylamide was added. The extract was centrifuged and the supernatant was cleaned-up in two consecutive solid phase extraction steps. The final extract was analysed by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). An HPLC column based on graphitised carbon was applied for chromatographic separation. Acrylamide concentrations in purchased roasted chestnuts were in the range of <8–1278 μg/kg whereas only low amounts (<4–159 μg/kg) were found in chestnut products. However, the median acrylamide content of the commercial roasted chestnut samples was 90 μg/kg. The influence of the roasting time on the acrylamide content in roasted chestnuts was evaluated too. As with roasted and fried potato products, the roasting time has a significant influence on the acrylamide formation. Therefore, the consumers might be exposed to significant amounts of acrylamide by eating roasted chestnuts, especially when a batch remains in the roasting vessel for too long time.     

Fate of enniatins and beauvericin during the malting and brewing process determined by stable isotope dilution assays

The fate of enniatins A, A1, B, B1 and beauvericin during the malting and brewing process was investigated. Three batches of barley grains were used as starting material, one was naturally contaminated, two were artificially inoculated with Fusarium fungi. Samples were taken from each key step of the malting and brewing procedure, the levels of the toxins were determined with stable isotope dilution assays using liquid chromatography–tandem mass spectrometry detection. Significant increases of the toxins were found during germination of two batches of barley grains, resulting in green malts contamination up to a factor of 3.5 compared to grains before germination. Quantitative PCR analyses of fungal DNA revealed in all batches growth of Fusarium avenaceum during germination. After kilning, only 41–72% of the total amounts of the toxins in green malts remained in kilned malts. In subsequent mashing stage, the toxins in kilned malts predominantly were removed with spent grains. In the final beer, only one batch still contained 74 and 14 μg/kg of enniatin B and B1, respectively. Therefore, the carryover of these enniatins from the initial barley to final beer was less than 0.2% with the main amounts remaining in the spent grains and the malt rootlets.     

Quantification of persistent organic pollutants in dietary supplements using stir bar sorptive extraction coupled with GC-MS/MS and isotope dilution mass spectrometry

ABSTRACT

In this work, we describe a method developed to quantify persistent organic pollutants (POPs) including polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides (OCPs) in dietary supplement samples using stir-bar sorptive extraction (SBSE)-GC-MS/MS-isotope dilution mass spectrometry (IDMS). This method enables accurate, precise, and sensitive quantification of POPs in plant-extract based dietary supplement products commercially available in the United States. When compared with calibration curves, IDMS provided more accurate and precise measurements. The mean error of measurements using this method was 7.24% with a mean RSD of 8.26%. The application of GC-MS/MS enabled approximately two-order-of-magnitude lower limit of quantifications compared with GC-MS. 12 commercially available plant-extract based dietary supplement samples were analysed using this method. PAHs including naphthalene, acenaphthene, fluorene, phenanthrene, fluoranthene, pyrene, chrysene, and benzo[a]pyrene were detected in most of the products and had average concentrations over 1 ng/g. OCPs were detected less frequently than PAHs in these products, and none of the OCPs had mean concentrations over 1 ng/g. The mean toxin concentration of each product was calculated, and the highest value was 3.20 ng/g. These results were compared with existing guidelines and none of the analytes in the samples were found to be above the daily allowable limits.     

Characterisation of the key aroma compounds in a freshly reconstituted orange juice from concentrate

Application of an aroma extract dilution analysis on the entire volatile fraction isolated from an orange juice freshly reconstituted from concentrate revealed 40 odour-active constituents in the flavour dilution (FD) factor range of 4–2,048. Among them, ethyl butanoate and linalool showed the highest FD factor of 2,048, followed by octanal with an FD factor of 512. Thirty-six of the 40 odour-active compounds detected could be identified, all of which have previously been reported as volatile constituents of various orange juices. Quantification of 17 key odorants by stable isotope dilution assays followed by a calculation of odour activity values (OAVs) on the basis of odour thresholds in water or citrate buffer (pH 3.8), respectively, revealed the following most important odorants in the overall aroma of the freshly reconstituted juice: (R/S)-linalool, (R)-limonene and (S)-ethyl 2-methylbutanoate with the highest OAVs (>1,000) followed by octanal, (R)-α-pinene, ethyl butanoate, myrcene, acetaldehyde, decanal and (E)-β-damascenone with OAVs > 100. A model mixture containing all 14 aroma compounds with OAVs > 1 in their actual concentrations in the juice showed a good similarity with the aroma of the original orange juice under investigation, thus corroborating that the key odorants of a freshly reconstituted orange juice were characterised for the first time.     

Influence of provenance and roast degree on the composition of potent odorants in Arabica coffees

 Twenty-eight potent odorants were quantified by stable isotope dilution assays of medium-roasted Arabica coffee blends from Colombia (Col), Brazil (Bra), El Salvador (El) and Kenya (Ken) as well as the varieties Tipica (Tip) from Colombia and Caturra from the regions Pinas (Cat-P) and Quevedo (Cat-Q) in Ecuador. The blends from Col and Bra were similar in their composition of odorants. Ken contained higher amounts of 3-mercapto-3-methylbutylformate than Col and Bra, while 2-furfurylthiol, guaiacol and 4-ethylguaiacol were present in lower concentrations. El contained less 4-vinylguaiacol and 2-furfurylthiol, but more 3-methyl-2-buten-1-thiol. Tip, Cat-P and Cat-Q differed from Col, Bra and Ken in having lower amounts of 2,3-butanedione, 2,3-pentanedione and 4-hydroxy-2,5-dimethyl-3(2H)-furanone and higher contents of guaiacol, 3-mercapto-3-methylbutylformate and 3-methyl-2-buten-1-thiol. The largest amount of 2-furfurylthiol was found in Tip and Cat-P. The blends from Col and Ken and the variety Tip showed a significant increase of guaiacol, 4-ethylguaiacol and 3-methyl-2-buten-1-thiol when the roast degree was raised. 2-Furfurylthiol and 2(5)-ethyl-4-hydroxy-5(2)-methyl-3(2H)furanone increased only in Col and Ken, while in Tip the amounts of carbonyl compounds, 4-hydroxy-2,5-dimethyl-3(2H)furanone, methional and (E)-β-damascenone decreased during roasting from medium to dark. Received: 29 December 1998     

Deoxynivalenol and its acetyl derivatives in bread and biscuits in Shandong province of China

In the present study, 100 commercial breads and biscuits were investigated for the occurrence of deoxynivalenol (DON) and its acetylated derivatives 3-acetyldeoxynivalenol (3-Ac-DON) and 15-acetyldeoxynivalenol (15-Ac-DON). The target mycotoxins were determined by isotope dilution ultrahigh performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). DON was determined in 95% of the test samples with a mean value of 153.3 µg/kg. Compared with DON, 3-Ac-DON and 15-Ac-DON were far less frequently detected, with mean values of 1.14 and 0.37 µg/kg, respectively. The estimated daily intakes of the sum of DON and its derivatives in breads and biscuits were 0.0059 and 0.0313 µg/kg bw/day, respectively, which was within the group provisional tolerable daily intake of 1.0 µg/kg bw/day set by the Joint FAO/WHO Expert Committee on Food Additives. In the future, systematic monitoring programmes of DON and its derivatives in relevant foodstuffs are still necessary for food safety and human health.     

A new solid phase extraction clean-up method for the determination of 12 type A and B trichothecenes in cereals and cereal-based food by LC-MS/MS

A new reliable and cost-efficient solid phase extraction-based clean-up method for the determination of 12 type A and B trichothecenes [deoxynivalenol (DON), nivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, fusarenon-X, T-2 toxin, HT-2 toxin, neosolaniol, monoacetoxy-scirpenol, diacetoxyscirpenol, T-2 triol and T-2 tetraol] in cereals and cereal-based food is presented. Furthermore, the suitability for the simultaneous determination of zearalenone is examined. Toxins were extracted from cereal samples using ACN/water (80/20, v/v), purified by means of a new Bond Elut Mycotoxin column and analyzed via liquid chromatography-electrospray ionization tandem mass spectrometry. Limits of detection were calculated for the matrix wheat and ranged from 0.3 to 5 ng/g, depending on the toxin. Average recovery rates for the tested compounds in seven cereal-based matrices have been determined ranging from 65 to 104%. The relative standard deviations of the complete method ranged from 2.67 (DON, wheat) to 20.0% (T-2 toxin, oats).     

奶粉中微量铅的螯合柱预分离富集同位素稀释电感耦合等离子体质谱测定方法

建立了离线微柱预分离富集的同位素稀释电感耦合等离子体质谱法(ID-ICP-MS)测定奶粉中微量铅含量。样品经灰化处理,采用螯合阳离子树脂(D401)柱去除奶粉中大量复杂基体,同时对微量铅进行富集,用铊作为内标的ID-ICP-MS准确测定奶粉中的微量铅。其平均加标回收率为99%,以空白值的3倍标准偏差考察方法的检出限为0.228μg/kg(n=8);测量精密度(RSD)为1.7%(n=4)。利用该法测定了市售的几种奶粉中的铅含量,结果表明,这几种知名品牌的奶粉中的铅含量远低于国家有关标准。     

Development and application of a method for the analysis of two trichothecenes: deoxynivalenol and T-2 toxin in meat in China by HPLC-MS/MS

A reliable and sensitive method was developed and successfully applied for the determination of deoxynivalenol and T-2 toxin simultaneously in pig dorsal muscle, pig back fat and chicken muscle by high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) analysis. Limit of detection of deoxynivalenol and T-2 was 0.02 μg/kg and 0.007 μg/kg, and limit of quantification of deoxynivalenol and T-2 was 0.07 μg/kg and 0.02 μg/kg, respectively. Sixty-six meat samples were analyzed and deoxynivalenol was detected in the samples of pig back fat, with concentrations lower than 0.5 μg/kg, and T-2 toxin was detected in the samples of pig dorsal muscle, pig back fat and chicken muscle, with concentrations lower than 0.5 μg/kg. The results of sample analysis show that only trace residues of deoxynivalenol and T-2 toxin were detected in the samples analyzed.     

Interference-free determination of acrylamide in potato and cereal-based foods by a laboratory validated liquid chromatography–mass spectrometry method

A simple and rapid method was developed and validated for the determination of acrylamide in potato and cereal-based foods by using a single quadrupole liquid chromatography–mass spectrometry (LC–MS) interfaced with positive atmospheric pressure chemical ionization (APCI+). Acrylamide was simply extracted with 0.01 mM acetic acid in a vortex mixer prior to LC–MS analysis. The applicability of validated method was shown for a wide range of processed foods including chips, fries, crisps, breads, biscuits and cookies. The mean recovery was found to be 99.7 with a repeatability of 1.8% in the range 100–1000 ng/g. During LC–MS analyses, the major interfering co-extractive was identified as valine which yields characteristic [M + H]⁺ and compound specific product ions having m/z of 118 and 72, respectively. Valine increased the baseline signal preventing accurate and precise quantitation, and resulted in poorer sensitivity in selected ion monitoring mode. The adverse effect of valine could be limited by instrumentally adjusted delay time or by solid-phase extraction with strong cation-exchanger sorbent.