Pyrrolizidine alkaloids in food: downstream contamination in the food chain caused by honey and pollen

In recent years, there has been a steadily growing number of published data on pyrrolizidine alkaloids (PAs) in honey and pollen. This raises the question whether honey and/or pollen used as ingredients in food processing might provoke a downstream contamination in the food chain. Here we addressed two different facets in connection with PAs in honey and pollen. First, we analysed the PA content of several food types such as mead (n?=?19), candy (n?=?10), fennel honey (n?=?9), soft drinks (n?=?5), power bars and cereals (n?=?7), jelly babies (n?=?3), baby food (n?=?3), supplements (n?=?3) and fruit sauce (n?=?1) that contained honey as an ingredient in the range of 5% to approximately 37%. Eight out of 60 retail samples were tested as being PA-positive, corresponding to 13%. Positive samples were found in mead, candy and fennel honey, and the average PA content was calculated to be 0.10?µg?g^–1 retronecine equivalents (ranging from 0.010 to 0.484?µg?g^–1). Furthermore, we investigated the question whether and how PAs from PA pollen are transferred from pollen into honey. We conducted model experiments with floral pollen of Senecio vernalis and PA free honey and tested the influence of the quantity of PA pollen, contact time and a simulated honey filtration on the final PA content of honey. It could clearly be demonstrated that the PA content of honey was directly proportional to the amount of PA pollen in honey and that the transfer of PAs from pollen to honey was a rather quick process. Consequently, PA pollen represents a major source for the observed PA content in honey. On the other hand, a good portion remains in the pollen. This fraction is not detected by the common analytical methods, but will be ingested, and it represents an unknown amount of ‘hidden’ PAs. In addition, the results showed that a technically and legally possible honey filtration (including the removal of all pollen) would not be an option to reduce the PA level of the final product significantly.     

Pyrrolizidine alkaloids in food and feed on the Belgian market

Pyrrolizidine alkaloids (PAs) are widely distributed plant toxins with species dependent hepatotoxic, carcinogenic, genotoxic and pneumotoxic risks. In a recent European Food Safety Authority (EFSA) opinion, only two data sets from one European country were received for honey, while one feed data set was included. No data are available for food or feed samples from the Belgian market. We developed an LC-MS/MS method, which allowed the detection and quantification of 16 PAs in a broad range of matrices in the sub ng g^?1 range. The method was validated in milk, honey and hay and applied to honey, tea (Camellia sinensis), scented tea, herbal tea, milk and feed samples bought on the Belgian market. The results confirmed that tea, scented tea, herbal tea and honey are important food sources of pyrrolizidine alkaloid contamination in Belgium. Furthermore, we detected PAs in 4 of 63 commercial milk samples. A high incidence rate of PAs in lucerne (alfalfa)-based horse feed and in rabbit feed was detected, while bird feed samples were less contaminated. We report for the first time the presence of monocrotaline, intermedine, lycopsamine, heliotrine and echimidine in cat food.     

Rapid multi-class multi-residue method for the confirmation of chloramphenicol and eleven nitroimidazoles in milk and honey by liquid chromatography-tandem mass spectrometry (LC-MS)

A confirmatory method was developed to allow for the analysis of eleven nitroimidazoles and also chloramphenicol in milk and honey samples. These compounds are classified as A6 compounds in Annex IV of Council Regulation 2377/90 (European Commission 1990) and therefore prohibited for use in animal husbandry. Milk samples were extracted by acetonitrile with the addition of NaCl; honey samples were first dissolved in water before a similar extraction. Honey extracts underwent a hexane wash to remove impurities. Both milk and honey extracts were evaporated to dryness and reconstituted in initial mobile phase. These were then injected onto a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system and analysed in less than 9 min. The MS/MS was operated in multiple reaction monitoring (MRM) mode with positive and negative electrospray ionization. The method was validated in accordance with Commission Decision 2002/657/EC and is capable of analysing metronidazole, dimetridazole, ronidazole, ipronidazole and there hydroxy metabolites hydroxymetronidazole, 2-hydroxymethyl-1-methyl-5-nitroimidazole, and hydroxyipronidazole. The method can also analyse for carnidazole, ornidazole, ternidazole, tinidazole, and chloramphenicol. A recommended level of 3 µg l^?1/µg kg^?1 for methods for metronidazole, dimetridazole, and ronidazole has been recommended by the Community Reference Laboratory (CRL) responsible for this substance group, and this method can easily detect all nitroimidazoles at this level. A minimum required performance level of 0.3 µg l^?1/µg kg^?1 is in place for chloramphenicol which the method can also easily detect. For nitroimidazoles, the decision limits (CCα) and detection capabilities (CCβ) ranged from 0.41 to 1.55 µg l^?1 and from 0.70 to 2.64 µg l^?1, respectively, in milk; and from 0.38 to 1.16 µg kg^?1 and from 0.66 to 1.98 µg kg^?1, respectively, in honey. For chloramphenicol, the values are 0.07 and 0.11 µg l^?1 in milk and 0.08 and 0.13 µg kg^?1 in honey. Validation criteria of accuracy, precision, repeatability, and reproducibility along with measurement uncertainty were calculated for all analytes in both matrices.     

Pyrrolizidine alkaloids in honey: comparison of analytical methods

Pyrrolizidine alkaloids (PAs) are a structurally diverse group of toxicologically relevant secondary plant metabolites. Currently, two analytical methods are used to determine PA content in honey. To achieve reasonably high sensitivity and selectivity, mass spectrometry detection is demanded. One method is an HPLC-ESI-MS-MS approach, the other a sum parameter method utilising HRGC-EI-MS operated in the selected ion monitoring mode (SIM). To date, no fully validated or standardised method exists to measure the PA content in honey. To establish an LC-MS method, several hundred standard pollen analysis results of raw honey were analysed. Possible PA plants were identified and typical commercially available marker PA-N-oxides (PANOs). Three distinct honey sets were analysed with both methods. Set A consisted of pure Echium honey (61-80% Echium pollen). Echium is an attractive bee plant. It is quite common in all temperate zones worldwide and is one of the major reasons for PA contamination in honey. Although only echimidine/echimidine-N-oxide were available as reference for the LC-MS target approach, the results for both analytical techniques matched very well (n = 8; PA content ranging from 311 to 520 μg kg(-1)). The second batch (B) consisted of a set of randomly picked raw honeys, mostly originating from Eupatorium spp. (0-15%), another common PA plant, usually characterised by the occurrence of lycopsamine-type PA. Again, the results showed good consistency in terms of PA-positive samples and quantification results (n = 8; ranging from 0 to 625 μg kg(-1) retronecine equivalents). The last set (C) was obtained by consciously placing beehives in areas with a high abundance of Jacobaea vulgaris (ragwort) from the Veluwe region (the Netherlands). J. vulgaris increasingly invades countrysides in Central Europe, especially areas with reduced farming or sites with natural restorations. Honey from two seasons (2007 and 2008) was sampled. While only trace amounts of ragwort pollen were detected (0-6.3%), in some cases extremely high PA values were detected (n = 31; ranging from 0 to 13019 μg kg(-1), average = 1261 or 76 μg kg(-1) for GC-MS and LC-MS, respectively). Here the results showed significantly different quantification results. The GC-MS sum parameter showed in average higher values (on average differing by a factor 17). The main reason for the discrepancy is most likely the incomplete coverage of the J. vulgaris PA pattern. Major J. vulgaris PAs like jacobine-type PAs or erucifoline/acetylerucifoline were not available as reference compounds for the LC-MS target approach. Based on the direct comparison, both methods are considered from various perspectives and the respective individual strengths and weaknesses for each method are presented in detail.     

Quantification of pyrrolizidine alkaloids in North American plants and honey by LC-MS: single laboratory validation

Pyrrolizidine alkaloids (PAs) are a class of naturally occurring compounds produced by many flowering plants around the World. Their presence as contaminants in food systems has become a significant concern in recent years. For example, PAs are often found as contaminants in honey through pollen transfer. A validated method was developed for the quantification of four pyrrolizidine alkaloids and one pyrrolizidine alkaloid N-oxide in plants and honey grown and produced in British Columbia. The method was optimised for extraction efficiency from the plant materials and then subjected to a single-laboratory validation to assess repeatability, accuracy, selectivity, LOD, LOQ and method linearity. The PA content in plants ranged from1.0 to 307.8 µg/g with repeatability precision between 3.8 and 20.8% RSD. HorRat values were within acceptable limits and ranged from 0.62 to 1.63 for plant material and 0.56–1.82 for honey samples. Method accuracy was determined through spike studies with recoveries ranging from 84.6 to 108.2% from the raw material negative control and from 82.1–106.0 % for the pyrrolizidine alkaloids in corn syrup. Based on the findings in this single-laboratory validation, this method is suitable for the quantitation of lycopsamine, senecionine, senecionine N-oxide, heliosupine and echimidine in common comfrey (Symphytum officinale), tansy ragwort (Senecio jacobaea), blueweed (Echium vulgare) and hound’s tongue (Cynoglossum officinale) and for PA quantitation in honey and found that PA contaminants were present at low levels in BC honey.     

Carry-over of pyrrolizidine alkaloids from feed to milk in dairy cows

Pyrrolizidine alkaloids are toxins present in many plants belonging to the families of Asteraceae, Boraginaceae and Fabaceae. Particularly notorious are pyrrolizidine alkaloids present in ragwort species (Senecio), which are held responsible for hepatic disease in horses and cows and may lead to the death of the affected animals. In addition, these compounds may be transferred to edible products of animal origin and as such be a threat for the health of consumers. To investigate the possible transfer of pyrrolizidine alkaloids from contaminated feed to milk, cows were put on a ration for 3 weeks with increasing amounts (50–200?g day^?1) of dried ragwort. Milk was collected and sampled twice a day; faeces and urine twice a week. For milk, a dose-related appearance of pyrrolizidine alkaloids was found. Jacoline was the major component in milk despite being a minor component in the ragwort material. Practically no N-oxides were observed in milk, notwithstanding the fact that they constituted over 80% of the pyrrolizidine alkaloids in ragwort. The overall carry-over of the pyrrolizidine alkaloids was estimated to be only around 0.1%, but for jacoline 4%. Notwithstanding the low overall carry-over, this may be relevant for consumer health considering the genotoxic and carcinogenic properties demonstrated for some of these compounds. Analysis of the faeces and urine samples indicated that substantial metabolism of pyrrolizidine alkaloids is taking place. The toxicity and potential transfer of metabolites to milk is unknown and remains to be investigated.     

Pyrrolizidine alkaloids in honey: risk analysis by gas chromatography-mass spectrometry

Recently, contamination of honey with pyrrolizidine alkaloids (PA) has been reported as potential health risk. Therefore, it was of interest to develop a reliable tool for selective and quantitative determination of PA in honey. Sample preparation of the novel method comprises strong cation exchange SPE (SCX-SPE), followed by two reduction steps using zinc and LiAlH(4), as well as subsequent silylation. During this procedure the separated PA are converted into the necin backbone, the common structural feature of PA toxicity, which is analyzed by GC-MS in the SIM mode. The procedure was validated using PA from extracts of Senecio vernalis as well as authentic PA standards including their corresponding N-oxides. The PA content of honey samples was quantified with heliotrine as internal standard. The method was applied to generate a dataset in order to evaluate the potential risk of PA contamination especially for retail honeys available on the German/European market. No selection criteria in terms of floral or geographical origin were applied on the samples before analysis. In total, 216 commercially available floral honey samples were analyzed. Among them 19 samples contained PA, in the range of 0.019-0.120 microg/g, calculated as retronecine equivalents. The reported method facilitates the selective determination of PA without the need to identify each individual PA independently. The PA contamination of honey is expressed in terms of a single sum parameter and no background information such as foraged plants and pollen analysis is necessary. The LOQ is 0.01 ppm with a S/N of 7:1.     

Pyrrolizidine alkaloids in floral honeys of tropical Ghana: a health risk assessment

ABSTRACT

There is a vast amount of information about the nutritional and medicinal properties of honey as a result of its numerous benefits. However, honeys have been found to be contaminated with hepatotoxic and carcinogenic pyrrolizidine alkaloids (PAs) on account of bees foraging on PA-containing plants. This study deals with the analysis of PAs in tropical honeys emanating from different agro-ecological zones of Ghana in order to assess its potential health risk. PAs of 48 honey samples were analysed using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results show that a total of 85% of the honeys from various agro-ecological zones were PA positive including all honeys from supermarkets. The highest concentration of PAs was 2639 μg kg^?1, while the average PA concentration of the samples was 283 μg kg^?1. The study also found Chromolaena odorata pollens in majority of the honeys, thus indicating the plant as major source of PA contamination of honeys in the tropical regions.     

Pyrrolizidine alkaloids in food: a spectrum of potential health consequences

Contamination of grain with 1,2-dehydropyrrolizidine ester alkaloids (dehydroPAs) and their N-oxides is responsible for large incidents of acute and subacute food poisoning, with high morbidity and mortality, in Africa and in central and south Asia. Herbal medicines and teas containing dehydroPAs have also caused fatalities in both developed and developing countries. There is now increasing recognition that some staple and widely consumed foods are sometimes contaminated by dehydroPAs and their N-oxides at levels that, while insufficient to cause acute poisoning, greatly exceed maximum tolerable daily intakes and/or maximum levels determined by a number of independent risk assessment authorities. This suggests that there may have been cases of disease in the past not recognised as resulting from dietary exposure to dehydroPAs. A review of the literature shows that there are a number of reports of liver disease where either exposure to dehydroPAs was suspected but no source was identified or a dehydroPA-aetiology was not considered but the symptoms and pathology suggests their involvement. DehydroPAs also cause progressive, chronic diseases such as cancer and pulmonary arterial hypertension but proof of their involvement in human cases of these chronic diseases, including sources of exposure to dehydroPAs, has generally been lacking. Growing recognition of hazardous levels of dehydroPAs in a range of common foods suggests that physicians and clinicians need to be alert to the possibility that these contaminants may, in some cases, be a possible cause of chronic diseases such as cirrhosis, pulmonary hypertension and cancer in humans.     

Identification,mycotoxin risk and pathogenicity of Fusarium species associated with fig endosepsis in Apulia,Italy

In a survey carried out on 87 rotted fig fruits samples collected in the Apulia region of Italy, the authors isolated 126 Fusarium strains identified as F. ramigenum (69 strains), F. solani (49), F. proliferatum (five) and three not identified. Investigation on the fertility of the strains belonging to F. proliferatum and F. ramigenum revealed that only strains of F. proliferatum were fertile. The identity of F. ramigenum strains was confirmed by sequencing a portion of the translation elongation factor-1α gene. When Fusarium species were analysed for their toxigenicity, 37/69 strains of F. ramigenum produced fusaric acid (FA) up to 525 mg kg^?1; 30 strains produced beauvericin (BEA) up to 190 mg kg^?1; 60 strains produced fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) up to 1575 mg kg^?1 of total FBs; and two strains produced fusaproliferin (FUP) up to 345 mg kg^?1; all five strains of F. proliferatum produced FA at low levels; two strains produced BEA up to 205 mg kg^?1; one strain produced FB₁ and FB₂, 1100 and 470 mg kg^?1, respectively; and one strain produced FUP, 820 mg kg^?1; F. solani (30 strains) produced FA, 13 strains up to 215 mg kg^?1. Few fungal extracts showed high toxicity toward brine shrimp larvae and in some cases in relation to BEA and FA content. A pathogenic assay on fig fruits showed that all three species were pathogenic, with higher virulence of F. ramigenum. These data report for the first time the production of BEA and FB₁/FB₂ by F. ramigenum and show that it is a main agent of fig endosepsis in Apulia and can contribute to fumonisin contamination of fresh and dried figs.     

Determination of pyrrolizidine alkaloids in tea,herbal drugs and honey

Honey was previously considered to be one of the main food sources of human pyrrolizidine alkaloid (PA) exposure in Europe. However, comprehensive analyses of honey and tea sampled in the Berlin retail market revealed unexpected high PA amounts in teas. This study comprised the analysis of 87 honey as well as 274 tea samples including black, green, rooibos, melissa, peppermint, chamomile, fennel, nettle, and mixed herbal tea or fruit tea. Total PA concentrations in tea ranged from < LOD to 5647 µg kg^?1, while a mean value of about 10 µg kg^?1 was found in honey samples. Additionally, herbal drugs were investigated to identify the source of PA in teas. Results suggest that PA in tea samples are most likely a contamination caused by co-harvesting of PA-producing plants. In some cases such as fennel, anise or caraway, it cannot be excluded that these plants are able to produce PA themselves.     

Quantitative determination of mycotoxins in urine by LC-MS/MS

Naturally occurring mycotoxins are responsible for a wide array of adverse health effects. The measurement of urinary mycotoxin levels is a useful means of assessing an individual's exposure, but the development of sensitive and accurate analytical methods for detecting mycotoxins and their metabolites in urine samples is challenging. Urinary mycotoxins are present in low pg ml^–1 concentrations, and the chromatographic identification of their metabolites can be obscured by other endogenous metabolites. We developed an analytical method focused on the selection of two appropriate multiple-reaction monitoring transition for unambiguous identification and quantification of carcinogenic aflatoxin M₁ (AFM₁), ochratoxin A (OTA) and fumonisin B₁, B₂ (FB₁, FB₂) in urine samples from a small volunteer group in a pilot study. AFM₁, OTA, FB₁ and FB₂ were concentrated selectively, interfering substances were removed using an immunoaffinity column (IAC), and mycotoxins were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in combination with a stable-isotope standard-dilution assay (SIDA). The method was sensitive enough to measure mycotoxins and their metabolites at pg ml^–1 levels in urine. The combination of LC-MS/MS and SIDA was critical to distinguishing pseudo-OTα interference from genuine OTα. Twelve urine samples contained OTA ranging from 0.013 to 0.093 ng ml^?1 (mean = 0.031 ng ml^?1). AFM₁ were detected in one sample at a 0.002 ng ml^?1 level, while FB₁ and FB₂ were undetectable in all 12 samples. None of the samples in this pilot study contained a detectable level of OTα, despite the presence of OTA, and this may suggest the need for further epidemiological investigation of OTA exposure in the Korean population.     

Methods for the analysis of melamine and related compounds in foods: a review

In recent years, two adulteration incidents concerning the addition of melamine (MEL) and related compounds to dairy products and vegetable proteins have occurred in China. These episodes prompted numerous governmental and private laboratories to develop or implement methods for the analysis of a wide variety of food products for MEL and related compounds, including cyanuric acid, ammeline, and ammelide. Methods have been developed for both screening and quantitation purposes; procedures used in the methods range from sensitive hyphenated chromatographic-mass spectrometric techniques to immunoselective assays. Various issues are encountered during the analysis of MEL and related compounds in food products. These issues include contamination, matrix effects, and analyte instability, and their severity varies according to the method used, and matrices and analytes examined.     

UNE-EN ISO/IEC 17025:2005-accredited method for the determination of pesticide residues in fruit and vegetable samples by LC-MS/MS

A rapid, simple and sensitive multi-residue method was developed and validated for the simultaneous quantification and confirmation of 69 pesticides in fruit and vegetables using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The samples were extracted following the quick, easy, cheap, effective, rugged and safe method known as QuEChERS. Mass spectrometric conditions were individually optimised for each analyte in order to achieve maximum sensitivity in multiple reaction monitoring (MRM) mode. Using the developed chromatographic conditions, 69 pesticides can be separated in less than 17 min. Two selected reaction monitoring (SRM) assays were used for each pesticide to obtain simultaneous quantification and identification in one run. With this method in SRM mode, more than 150 pesticides can be analysed and quantified, but their confirmation is not possible in all cases according to the European regulations on pesticide residues. Nine common representative matrices (zucchini, melon, cucumber, watermelon, tomato, garlic, eggplant, lettuce and pepper) were selected to investigate the effect of different matrices on recovery and precision. Mean recoveries ranged from 70% to 120%, with relative standard deviations (RSDs) lower than 20% for all the pesticides. The proposed method was applied to the analysis of more than 2000 vegetable samples from the extensive greenhouse cultivation in the province of Almería, Spain, during one year. The methodology combines the advantages of both QuEChERS and LC-MS/MS producing a very rapid, sensitive, accurate and reliable procedure that can be applied in routine analytical laboratories. The method was validated and accredited according to UNE-EN-ISO/IEC 17025:2005 international standard (accreditation number 278/LE1027).     

Analysis of isothiazolinone biocides in paper for food packaging by ultra-high-performance liquid chromatography–tandem mass spectrometry

A novel and simple method to detect isothiazolinone-type biocides (2-methyl-3-isothiazolinone (MI), 5-chloro-2-methyl-3-isothiazolinone (CMI), 1,2-benzisothiazolinone (BIT) and 2-octyl-3-isothiazolinone (OIT)) in paper used for food packaging by ultrasonic extraction coupled with UPLC-MS/MS was developed. Parameters affecting process efficiency such as extraction solvents, UPLC mobile phase, gradient elution procedure and MS/MS conditions were studied to optimise the operating conditions. Using the optimised gradient elution procedure, the retention time was less than 6?min. The limits of detection (LODs) were found to be between 0.001 and 0.010?mg?kg^?1, which was validated using actual concentrations. After diluting the standard solution with a blank matrix, the linear calibration curve ranges were 0.002–1.000?mg?kg^?1 for BIT and OIT, 0.005–1.000?mg?kg^?1 for MI, and 0.020–1.000?mg?kg^?1 for CMI, with correlation coefficients higher than 0.9985 (n?=?6). A good level of precision with a mean recovery greater than 81.3% and a relative standard deviation (RSD) less than 6.2% were also obtained. A methodology has been proposed for the analysis of isothiazolinones in paper.     

气相色谱-质谱法测定酱油中氯丙醇类化合物

建立了酱油中两种氯丙醇类化合物检测的气相色谱-质谱分析方法.样品中加入两种稳定性氘代同位素内标,经基质固相分散萃取(MSPD)分步提取、净化,七氟丁酰基咪唑衍生化后进行GC/MS测定.两种氯丙醇化合物的线性范围均为10～1600 ng,检出限为5μg/kg;回收率为99.5%～110%,RSD%为3.8%～6.6%.该方法测定灵敏度高,定性准确,选择性好,稳定性高,适合于样品中多种痕量氯丙醇类化合物的同时测定.     

Transfer of aflatoxin B1 and fumonisin B1 from naturally contaminated raw materials to beer during an industrial brewing process

The aim of this research was to determine the fate of aflatoxins (AFs) and fumonisins (FBs) naturally occurring in raw materials (maize grit and malted barley) during four industrial brewing processes. The aflatoxin B₁ (AFB₁) level in raw materials varied from 0.31 to 14.85 µg kg^?1, while the fumonisin B₁ (FB₁) level (only in maize grit) varied from 1146 to 3194 µg kg^?1. The concentration in finished beer ranged from 0.0015 to 0.022 µg l^?1 for AFB₁ and from 37 to 89 µg l^?1 for FB₁; the other aflatoxins and fumonisin B₂ were not found in beer samples. The average percentage of toxins recovered in finished beer, referring to the amounts contained in raw materials, were 1.5% ± 0.8% for AFB₁ and 50.7% ± 4.7% for FB₁. These results were mainly due to the different solubility of the two mycotoxins during the mashing process. If raw materials comply with the limits fixed by European Commission Regulations, the contribution of a moderate daily consumption of beer to AFB₁ and FB₁ intake does not contribute significantly to the exposure of the consumer.     

Antioxidant capacity of potato chips and snapshot trends in acrylamide content in potato chips and cereals on the Canadian market

The concentration of acrylamide was measured in selected varieties of five brands of potato chips and breakfast cereals over a 5-year period. Most of the products were purchased in one locality in Canada. Samples were analysed by an isotope dilution (¹³C₃) acrylamide method. They were extracted with water, partitioned with dichloromethane, filtered through a 5 kDa centrifuge filter, cleaned-up on HLB Oasis polymeric and Accucat mixed mode anion and cation exchange SPE columns, and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The acrylamide concentration in potato chips varied from 106 to 4630 ng g^?1, while values in cereals varied from 50 to 347 ng g^?1. Wide variations were observed between brands, within brands over time, and between lots of the same brand. A subset of potato chip samples was analysed for in vitro antioxidant activity. No relationship was found between antioxidative capacity of potato chips and their acrylamide content.     

Validation by collaborative trial of an isotope dilution liquid chromatographic tandem mass spectrometric method to determine the content of acrylamide in roasted coffee

A method for the determination of acrylamide in roasted coffee was subjected to method validation by collaborative trial. The aim was to extend the scope of a method already standardized for the determination of acrylamide in bakery and potato products to include roasted coffee. Modifications of the already standardized method were therefore kept to a minimum. The method was based on aqueous extraction of the roasted coffee matrix and solid-phase extraction (SPE) clean-up followed by isotope dilution high-performance liquid chromatography with tandem mass spectrometric detection (LC-MS/MS). The test portion of the sample was spiked with stable isotope-labelled acrylamide and extracted on a mechanical shaker with n-hexane and water for 1 h. The sample extract was centrifuged, the organic phase discarded, and a portion of the aqueous extract further cleaned-up by SPE on an Isolute Multimode cartridge followed by a second clean-up step on an Isolute ENV + cartridge. The volume of the acrylamide-containing fraction eluted from the second SPE column was reduced by evaporation and analysed by LC-MS/MS. Three coffee samples and one aqueous acrylamide standard solution were sent to eleven laboratories from eight European Union Member States. All samples were sent as blind duplicates. Based on the reported results, the relative standard deviations for reproducibility (RSD_R) were 11.5% at a level of 160 µg kg^?1, 10.1% at a level of 263 µg kg^?1, and 9.6% at a level of 585 µg kg^?1. The values for repeatability (RSD_r) in those materials ranged from 1.0% to 3.5%. The method performance parameters satisfied internationally accepted criteria. Hence, the method would be suitable for the enforcement of regulatory limits.     

Occurrence of deoxynivalenol and its 3-β-D-glucoside in wheat and maize

Deoxynivalenol-3-β-D-glucoside (D3G), a phase II plant metabolite of the mycotoxin deoxynivalenol (DON), occurs in naturally Fusarium-contaminated cereals. In order to investigate the frequency of occurrence as well as the relative and absolute concentrations of D3G in naturally infected cereals, 23 wheat samples originating from fields in Austria, Germany and Slovakia as well as 54 maize samples from Austrian fields were analysed for DON and D3G by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Both analytes were detected in all the 77 field samples. DON was found at levels from 42 to 4130 ng g^?1 (977 ± 1000 ng g^?1 on average). The D3G concentrations in all cereal samples were in the range 10–1070 ng g^?1 (216 ± 253 ng g^?1 on average), corresponding to about 5–46 mol% of their DON concentrations (15 ± 8 mol% on average).