Carrageenan analysis. Part 1: Characterisation of the carrageenan test material and stability in swine-adapted infant formula

A method was developed and validated in support of a 28-day feeding study of swine-adapted infant formula stabilised with carrageenan administered to neonatal piglets. Carrageenan concentrations in the test formulations were 0, 300, 1000 and 2250 mg kg^–1 formula. Extraction of carrageenan from swine-adapted infant formula was achieved by breaking carrageenan–protein cross-linkages using saturated sodium chloride, followed by separation of the non-gelling carrageenan fraction via centrifugation. The extraction of carrageenan from formula was successful with respect to consistent recovery of the non-gelling carrageenan fraction from both test and control formula samples. Molecular weight analysis (Mw) of the recovered carrageenan fractions from the test and control formula samples confirmed that the carrageenan used to manufacture the formula was not degraded during the infant formula production process and subsequent storage for 4 months covering the 28-day piglet dietary feeding study. Carrageenan has excellent stability in infant formulations.     

Measurement of ampicillin residue levels in chicken eggs during and after medicated feed administration by LC-MS/MS

A simple and sensitive LC-MS/MS method was developed and validated for the determination of ampicillin (ABPC) in chicken eggs. Residues were extracted by reverse-phase solid-phase extraction. Chromatographic separation was performed using a reverse-phase column with an elution gradient. The limits of detection and quantification were 0.01 and 0.1 ng g^?1, respectively. For the 0.1–50 ng g^?1 concentration range, mean recovery and accuracy values were 93.9–98.5% and 100.2–118.0%, respectively. ABPC residue concentrations in eggs before, during and after 7 days of medicated feeding of maximum dosage (40 mg kg^?1 body weight day^?1) of ABPC were determined with the LC-MS/MS method. The maximum concentration of ABPC in eggs was 3.6 ± 1.7 ng g^?1 (mean ± SD) on the last day of the administration period. Residue concentrations of ABPC in eggs during and after ABPC administration were not over the Japanese maximum residue limit of 0.01 mg kg^?1.     

Tissue deposition and residue depletion of melamine in fattening pigs following oral administration

The adulteration of animal feed as well as milk products with melamine has led to concerns about the ability to establish appropriate withdrawal intervals to ensure food safety. Two experiments were conducted in this study. The first was to investigate the deposition and depletion of melamine in blood and tissues of pigs exposed to adulterated feed with high doses of melamine. A total of 500 or 1000 mg kg^–1 melamine was added to the diet for fattening pigs (initial BW = ±60.24 kg). Melamine residues were detected in tissues (brain, duodenum, liver, heart, muscle and kidney) by LC-MS/MS. Dose-dependent effects were found between melamine residual concentration and its dose in feed. Five days after the withdrawal of melamine from the diets, the residue concentration in tissues fell below 2.5 mg kg^–1. In the second experiment, blood samples were taken at different time points from fattening pigs (BW = 100 kg) fed with adulterated feed with 1000 mg kg^–1 of melamine for 42 days. Results from the pharmacokinetics analysis showed that it would take 83 h for the melamine level in plasma depleting to the safe level of 50 ng ml^–1 after an expose of 1000 mg kg^–1 melamine contaminated feed for 42 days.     

Determination of halosulfuron-methyl residues and dissipation in wheat by QuEChERS and LC-MS/MS

A sensitive and rapid analytical method based on QuEChERS (quick, easy, cheap, effective, rugged and safe) sample preparation and LC-MS/MS detection was developed for the analysis of halosulfuron-methyl residues in wheat. The recoveries of halosulfuron-methyl in both the wheat plant and grain ranged from 87% to 119% and from 75% to 97%, respectively, with relative standard deviations (RSDs) of 3–9%. The limit of quantification (LOQ) was 0.005 mg kg^?1 for wheat plant and 0.001 mg kg^?1 for wheat grain. The half-life of halosulfuron-methyl in the wheat plant was 0.9–9.5 days. The terminal residue levels of halosulfuron-methyl in wheat grain were below 0.01 mg kg^?1 at harvest.     

Comparison of multiple methods for the determination of sulphite in Allium and Brassica vegetables

Sulphites are a family of additives regulated for use worldwide in food products. They must be declared on the label if they are present in concentrations greater than 10 mg kg^–1, determined as sulphur dioxide (SO₂). The current US regulatory method for sulphites, the optimised Monier–Williams method (OMW), produces false-positive results with vegetables from the Allium (garlic) and Brassica (cabbage) genera due to extraction conditions that are thought to cause endogenous sulphur compounds to release SO₂. Recently, modifications to the OMW method (2x MW) were published that reportedly reduced this false-positive in garlic. However, no other vegetables from these genera have been investigated. In addition, an LC-MS/MS method was developed for sulphite analysis, but it has not yet been tested with these problematic matrices. Ten vegetable species were analysed using these sulphite methods (OMW titration, OMW gravimetric, 2x MW and LC-MS/MS) to determine the false-positive rate. Sulphite concentrations > 10 mg kg^–1 SO₂ were observed with the OMW analyses. The 2x MW method reduced the measured concentration in unsulphited samples to ≤ 10 mg kg^–1 SO₂ for all matrices analysed. The LC-MS/MS method showed concentrations < 10 mg kg^–1 for the Brassica samples, but only displayed a slight reduction in the Allium matrices. Spiked recovery studies were conducted to determine if these methods can detect added sulphite. The 2x MW had recoveries of 17% and 42% for water and fresh garlic, respectively, and the LC-MS/MS had recoveries of 108%, 125%, 116% and 107% for water, fresh garlic, roasted garlic, and hummus, respectively. The low recoveries of the 2x MW may indicate that sulphur compounds cannot be properly quantified with this method. The ability to eliminate false-positives will enable accurate determination of added sulphite to ensure compliance with sulphite labelling requirements.     

Validation and quantification of neonicotinoid insecticide residues in rice whole grain and rice straw using LC-MS/MS

An efficient analytical method was developed and validated using a modified QuEChERS method and LC-MS/MS for the detection and quantification of neonicotinoid insecticide residues in rice whole grain and rice straw. The samples were extracted using acetonitrile and cleaned up by dispersive solid-phase extraction. Validation based on five fortification levels showed good recoveries of neonicotinoids ranging from 75% to 116 % and 60% to 105 % for rice whole grain and straw, respectively. The precision ranged between 3% and 17 %, and 2% and 10 % for grain and straw, respectively. The limit of detection was from 0.007 to 0.0084 mg kg^?1 and 0.005 to 0.15 mg kg^?1 and the limit of quantification was in the range of 0.024–0.028 mg kg^?1 and 0.016–0.051 mg kg^?1 for rice whole grain and rice straw, respectively. Monitoring of farm gate samples indicated that, out of 24 samples, 1 rice whole grain sample was contaminated with thiamethoxam residues (0.07 mg kg^?1).     

Carrageenan analysis. Part 3: Quantification in swine plasma

Development and validation of this method was conducted to support a 28-day piglet feeding study of swine-adapted infant formulations stabilised with carrageenan. The validation was performed in accordance with USFDA Good Laboratory Practice (GLP) Regulations and associated current bioanalytical guidelines. Separation of carrageenan from plasma protein was unsuccessful using saturated sodium chloride due to the extremely strong cross-linking interactions between carrageenan and protein. Poligeenan is the deliberately acid-hydrolysed low molecular weight polygalactan non-food product produced from carrageenan. Poligeenan molecules are nearly identical to carrageenan molecules with respect to molecular structure, the primary difference being molecular weight. These poligeenan molecules have similar molecular weight when compared with the lowest molecular weight fraction of carrageenan called the low molecular-weight tail (LMT). Poligeenan was separated from plasma protein using the salting procedure, this being due to the significantly weaker interaction with protein caused by its shorter molecular chain length. Thus, poligeenan was applied as a chemical analyte surrogate for the LMT of carrageenan solely for the development and validation of the method. This method was used to try to detect the LMT of the carrageenan test material during the 28-day piglet feeding study, and if such was absorbed into the bloodstream. Successful development and validation of the method was achieved using LC-MS/MS coupled with ESI in negative-ion mode. A standard curve of instrument response versus poligeenan concentration was developed using swine plasma spiked with a range of poligeenan concentrations. The lower level of quantification (LLOQ) of poligeenan was 10.0 µg ml^–1, and the quantification range was 10.0–100.0 µg ml^–1. No animals were fed poligeenan.     

A Robust Transferable Method for the Determination of Glyphosate Residue in Liver After Derivatization by Ultra-high Pressure Liquid Chromatography–Tandem Mass Spectrometry

Glyphosate determination in liver is challenging due to this particular molecule/matrix combination. Glyphosate is a very polar molecule and liver composition is highly variable between individuals and species. Since 2014, the Multiannual Control Program (MACP) of the European Union (EU) demands to analyse glyphosate in food of animal origin on a voluntary basis. Moreover, this analysis will be mandatory in 2017. This paper describes a robust and easily transferable method for glyphosate quantification in liver of animal origin by means of liquid chromatography–tandem mass spectrometry (LC-MS/MS). An intensive clean-up was used to eliminate matrix interferences and was combined with a derivatization step which ensures good retention of glyphosate on a conventional reverse-phase LC column. This method allows to meet the MACP requirements without a time-consuming change in the set-up of the routinely used LC-MS/MS system. Furthermore, it allows the use of an LC column and mobile phases often used in multi-residue analysis. The analytical method was validated according to the SANCO/12571/2013 criteria. Isotopic dilution was used to quantify glyphosate, leading to mean apparent recoveries of 115 and 101 % for the low (0.025 mg kg^?1) and the high (0.250 mg kg^?1) fortification levels, respectively. At both levels, the relative standard deviation was below 10 %. The limit of quantification of 0.025 mg kg^?1 was found to be satisfactory as it was below the maximum residue level (MRL) value set at 0.050 mg kg^?1 for glyphosate in liver. It is also the lowest MRL for all commodity types.     

Deposition and depletion of decoquinate in eggs after administration of cross-contaminated feed

Decoquinate, a chemical coccidiostat used as a feed additive, can occur in eggs due to cross-contamination of feedstuffs for laying hens. An experiment was designed to assess the transfer of decoquinate to hen eggs and its distribution between egg yolk and egg white. Hens were given the feed containing decoquinate at a cross-contamination level (0.34 mg kg^–1) and collected eggs were analysed using an LC-MS/MS method. The plateau level was reached on the eighth day of the experiment and averaged 8.91 µg kg^–1, which is far below the maximum level established at 20 µg kg^–1 for whole eggs. Decoquinate was deposited mostly in egg yolks (26.2 µg kg^–1) and did not deplete completely during 14 days of administration of decoquinate-free feed. The results confirmed that administration of cross-contaminated feed is associated with very low risk of non-compliant residue levels of decoquinate in eggs.     

Gluten contamination of naturally gluten-free flours and starches used by Canadians with celiac disease

A large national investigation into the extent of gluten cross-contamination of naturally gluten-free ingredients (flours and starches) sold in Canada was performed. Samples (n = 640) were purchased from eight Canadian cities and via the internet during the period 2010–2012 and analysed for gluten contamination. The results showed that 61 of the 640 (9.5%) samples were contaminated above the Codex-recommended maximum level for gluten-free products (20 mg kg^–1) with a range of 5–7995 mg kg^–1. For the ingredients that were labelled gluten-free the contamination range (5–141 mg kg^–1) and number of samples were lower (3 of 268). This picture was consistent over time, with approximately the same percentage of samples above 20 mg kg^–1 in both the initial set and the subsequent lot. Looking at the total mean (composite) contamination for specific ingredients the largest and most consistent contaminations come from higher fibre ingredients such as soy (902 mg kg^–1), millet (272 mg kg^–1) and buckwheat (153 mg kg^–1). Of the naturally gluten-free flours and starches tested that do not contain a gluten-free label, the higher fibre ingredients would constitute the greatest probability of being contaminated with gluten above 20 mg kg^–1.     

Quantitative determination of sodium monofluoroacetate “1080” in infant formulas and dairy products by isotope dilution LC-MS/MS

A fast and easy-to-use confirmatory liquid-chromatography tandem mass-spectrometry (LC-MS/MS) based-method was developed for the analysis of the pesticide sodium monofluoroacetate (MFA, also called “1080”) in infant formulas and related dairy products. Extraction of the compound encompassed sample reconstitution and liquid–liquid extraction under acidic conditions. Time-consuming solid-phase extraction steps for clean-up and enrichment and tedious derivatisation were thus avoided. Resulting sample extracts were analysed by electrospray ionisation (ESI) in negative mode. Quantification was performed by the isotopic dilution approach using ¹³C-labelled MFA as internal standard. The procedure was validated according to the European document SANCO/12571/2013 and performance parameters such as linearity (r² > 0.99), precision (RSD(r) ≤ 9%, RSD(iR) ≤ 11%) and recovery (96–117%) fulfilled its requirements. Limit of quantifications (LOQ) was 1 µg kg^?1 for infant formulas and related dairy products except for whey proteins powders with a LOQ of 5 µg kg^?1. Method ruggedness was further assessed in another laboratory devoted to routine testing for quality control.     

Toxic and essential elements in five tree nuts from Hangzhou market,China

In this study, a total of 35 tree nut samples of walnut, pecan, pine seed, hickory nut and torreya were obtained from 5 farm product markets in Hangzhou, China, and investigated for essential (Cr, Mn, Fe, Mo, Cu, Zn, Se and Sr) and toxic (Al, As, Cd and Pb) elements by inductively coupled plasma–mass spectroscopy. Mean elemental concentrations of different tree nuts were in the following ranges: Cr 0.26–0.78 mg kg^–1, Mn 42.1–174 mg kg^–1, Fe 33.7–43.9 mg kg^–1, Mo 0.11–0.48 mg kg^–1, Cu 10.3–17.6 mg kg^–1, Zn 21.6–56.1 mg kg^–1, Se 0.015–0.051 mg kg^–1, Al 1.44–37.6 mg kg^–1, As 0.0062–0.047 mg kg^–1, Cd 0.016–0.18 mg kg^–1 and Pb 0.0069–0.029 mg kg^–1. The estimated provisional tolerable daily intake of Al, As, Cd and Pb was much lower than the provisional tolerable daily intake.     

Occurrence of 3-MCPD and glycidyl esters in edible oils in the United States

Fatty acid esters of 3-monochloropropanediol (3-MCPD) and glycidol are processing contaminants found in a wide range of edible oils. While both 3 MCPD and glycidol have toxicological properties that at present has concerns for food safety, the published occurrence data are limited. Occurrence information is presented for the concentrations of 3-MCPD and glycidyl esters in 116 retail and/or industrial edible oils and fats using LC-MS/MS analysis of intact esters. The concentrations for bound 3-MCPD ranged from below the limit of quantitation (<LOQ) to 0.09 mg kg^?1 (ppm) in 22 unrefined oils and from 0.005 to 7.2 mg kg^?1 (ppm) in 94 refined oils. The concentrations for bound glycidol ranged from <LOQ to 0.03 mg kg^?1 (ppm) in unrefined oil samples and from <LOQ to 10.5 mg kg^?1 (ppm) in processed oil samples. The highest concentrations for both 3-MCPD and glycidol were seen in refined palm oil and palm olein samples. Palm olein samples also contained a higher percentage of 3-MCPD in mono-ester form than any other type of oil.     

Heavy metals in vegetables sold in the local market in Jordan

Heavy metals (As, Cd, Co, Cr, Cu, Mn, Ni, Pb and Zn) in various vegetables (cabbage, green onion, lettuce, parsley, rocket, spinach, carrot, onion, potato and cauliflower) from the market in Jordan were measured using inductively coupled plasma-mass spectrometry. As, Cd, Co, Cr, Cu, Mn, Ni, Pb and Zn ranged from 0.009–0.275 mg kg^?1 wet weight, 0.004–0.060 mg kg^?1, 0.003–0.401 mg kg^?1, 0.105–3.51 mg kg^?1, 0.15–1.15 mg kg^?1, 0.93–14.39 mg kg^?1, 0.044–0.702 mg kg^?1, 0.072–0.289 mg kg^?1 and 2.23–6.65 mg kg^?1, respectively. Parsley, followed by spinach, contained the highest concentration of heavy metals. Onion contained high levels of toxic heavy metals. The content of Cu in parsley and spinach and Pb in onion exceeded the Codex limits. However, the daily intake of heavy metals from the tested vegetables was lower than the maximum limits for allowable intake.     

Determination of Underivatized Glyphosate Residues in Plant-Derived Food with Low Matrix Effect by Solid Phase Extraction-Liquid Chromatography-Tandem Mass Spectrometry

A method was developed for the determination of glyphosate residues in plant-derived food using a two-step solid phase extraction (SPE) combined with mixed-mode liquid chromatography-tandem mass spectrometry (LC-MS/MS). The samples were extracted with water. Then, the extracting solution was pretreated by a C18 cartridge to remove protein and weak-polar interferences and further directly extracted using a strong anion exchange (SAX) cartridge to remove neutral and basic substances. The obtained glyphosate residues from the SPE were separated on a hydrophilic interaction/weak anion-exchange (HILIC/WAX) column and detected by mass spectrometry with negative electrospray ionization (ESI-) in multiple reaction monitoring (MRM) mode. This approach was evaluated by five different kinds of plant-derived food (soybean, corn, carrot, apple, and spicy cabbage) matrices in terms of matrix effect and recovery. Results showed that two-step SPE and mixed-mode chromatography separation provided the method with a very low matrix effect, and the spiked recoveries of glyphosate were satisfied in the range of 83.1 to 100.8 % at three spiked levels. The limit of quantification (LOQ) and detection (LOD) of the method in different matrices were 0.016–0.026 and 0.005–0.008 mg kg^?1, respectively. The procedure was validated and showed good accuracy and precision over a large linear range of 0.02–10 mg kg^?1.     

Use of 4-chloro-3, 5-dinitrobenzotrifluoride (CNBF) Derivatization and Ultrahigh-performance Liquid Chromatography Tandem Mass Spectrometry for the Determination of 20 Free Amino Acids in Chinese Jujube Date

Ultrahigh-performance liquid chromatography tandem mass spectrometry with 4-chloro-3,5-dinitrobenzotrifluoride (CNBF) derivatization was developed for simultaneous determination of 20 free amino acids in Chinese jujube date. The MS/MS conditions, choice of mobile phase, the extraction process, and matrix effects were studied with a view to a method of optimization. The limits of detection for measurement of the amino acids ranged from 0.8 to 600.0 μg L^?1. The correlation coefficients (r²?≥?0.9947) indicated good correlation between the concentrations of amino acids and the peak areas for the CNBF-derivatives. Recoveries for samples spiked at 25.0, 50.0, and 100.0 mg kg^?1 ranged from 66.3 to 106.6 % (except tryptophan spiked at 25.0 and 50.0 mg kg^?1, which provided recoveries of 61.2 and 60.4 %, and tyrosine spiked at three different concentrations, which provided recoveries of 54.8–58.6 %), with relative standard deviation values of less than 9.1 %.     

Occurrence of chloramphenicol in cereal straw in north-western Europe

Two surveys are presented of straw analysed for naturally occurring chloramphenicol (CAP), a drug banned for use in food-producing animals. In the first study, CAP was analysed by LC-MS/MS and detected in 37 out of 105 straw samples originating from the Netherlands, France, the UK, Germany and Denmark. The highest level found was 6.3 µg kg^?1, the average 0.6 µg kg^?1 and the median 0.2 µg kg^?1. The second study included a method comparison between ELISA and LC-MS/MS and a survey of CAP in cereal straw sampled at farms in all areas of Sweden. A total of 215 samples were screened by ELISA and a subset of 26 samples was also analysed by LC-MS/MS. Fifty-four of the samples contained more than 1 µg kg^?1 CAP and the highest level found was 32 µg kg^?1 (confirmed by LC-MS/MS). The highest contents of CAP in this study were allocated to the Baltic sea coast in the south-eastern part of Sweden (the county of Skåne and the Baltic Sea isle of Gotland). These results indicate a high incidence of CAP in straw in north-west Europe and have a severe impact on the enforcement of European Union legislation.     

Developing a microbiological growth inhibition screening assay for the detection of 27 veterinary drugs from 13 different classes in animal feedingstuffs

Many regulations prohibit using veterinary drugs in feedingstuffs to protect consumers and animals alike. Within this investigation we developed a simple, cost-efficient primary screening method for detecting antibiotics and coccidiostats in animal feeds. Thirty-two veterinary drugs were originally considered. Following matrix-free testing to optimise detection, an assay based on matrix extraction with methanol/acetonitrile/phosphate buffer followed by inoculation and diffusion in agar plates was developed. Final validation was performed with 14 representative drugs (one per drug class) and four bacteria (Escherichia coli ATCC11303 and ATCC27166, Staphylococcus aureus ATCC6538P, Micrococcus luteus ATCC9341) in bovine, lamb and swine fodder, measuring growth inhibition zones. Of the original drugs tested, 27 remained detectable in feed matrices at or below 20 mg kg^?1. Of the 14 validated representatives, two had estimated minimum detectable concentrations of 10–11 mg kg^?1, others of 5 mg kg^?1 or lower, an earlier minimum European Union inclusion rate for many veterinary drugs. No significant matrix effect on inhibition zones was detected. Per cent wrong negative deviations ranged from 0% (nine of 14 compounds) to 20–27% (two of 14), while inter-day precision based on inhibition zones had relative standard deviations (RSDs) of 6–109% (mean of 40%). When setting a 1 mm inhibition zone, the maximum observed for negative controls, as a cut-off level, no false-positives were found. While not all targeted antibiotics were detectable in complex matrices, the majority of veterinary drugs were detected with reasonable sensitivity, indicating that this method could be suitable for screening feedingstuffs prior to further confirmatory investigation of positive findings such as by LC-MS/MS.     

Distribution of semduramicin in hen eggs and tissues after administration of cross-contaminated feed

Semduramicin is an ionophore coccidiostat used in the poultry industry as a feed additive. Cross-contamination of feeds for non-target animals with semduramicin is unavoidable. However, it is not known whether undesirable residues of semduramicin may occur in food after cross-contaminated feed is administered to animals. The aim of the work was to determine the levels of semduramicin in hen eggs (yolks and albumen) and tissues (liver, muscle, spleen, gizzard, ovarian yolks and ovaries) after administration of feed contaminated with 0.27 mg kg^?1 of this coccidiostat. The residues were determined using LC-MS/MS. The distribution pattern confirmed the high lipophilicity of semduramicin. Residues were found mainly in egg yolks (28.8 µg kg^?1), ovarian yolks (19.5 µg kg^?1) and liver (2.57 µg kg^?1), while hens’ muscle was free from semduramicin (LOD = 0.1 µg kg^?1). Among edible tissues, the maximum level (2 µg kg^?1) was exceeded only in the liver.     

Analysis of quinclorac and quinclorac methyl ester in canola from the 2015 harvest using QuEChERS with liquid chromatography polarity-switching tandem mass spectrometry

A method using QuEChERS sample preparation with liquid chromatography polarity-switching tandem mass spectrometry was developed and validated for the analysis of quinclorac and its degradation product quinclorac methyl ester in canola seed. The method was used to analyse canola treated with quinclorac, harvest sample composites and samples of canola shipments. Quinclorac residues were present in all samples of canola treated with a quinclorac-containing herbicide that were analysed. Quinclorac was found in 93% of samples, with an average of 0.018 mg kg^–1. All samples contained quinclorac methyl ester, with an average of 0.061 mg kg^–1. The average concentration of total residues (as quinclorac equivalents) on treated canola was 0.075 mg kg^–1, with a range of 0.016–0.124 mg kg^–1. The observed residues were all at least 10 times lower than the Canadian maximum residue limit of 1.5 mg kg^–1. Quinclorac and quinclorac methyl ester were not found in any harvest and export composite samples, which represented the majority of canola grown in western Canada in 2015 and canola exported in late 2015. Even though usage of quinclorac-containing herbicide on canola can result in the presence of low concentrations of residues, the absence of quinclorac residues in harvest and shipment samples suggests that use of quinclorac-containing herbicide was not widespread, and that any residues present were diluted as canola was combined along the grain-handling chain into shipment lots, or segregated and prevented from entering shipment lots.