A simple and rapid assay for analyzing residues of carbamate insecticides in vegetables and fruits: hot water extraction followed by liquid chromatography-mass spectrometry

A simple, specific, and rapid analytical method for determining seven largely used carbamate insecticides in tomato, spinach, lettuce, zucchini, pear, and apple is here presented. This method is based on the matrix solid-phase dispersion technique, with heated water as extractant followed by liquid chromatography (LC)-mass spectrometry (MS) equipped with a single quadrupole and an electrospray ion source. Target compounds were extracted from the vegetal matrixes by water heated at 50 degrees C. After acidification and filtration, 0.25 mL of any aqueous extract was injected in the LC column. MS data acquisition was performed in the selected ion monitoring mode, selecting three ions for each target compound. Heated water appeared to be an excellent extractant because recovery data ranged between 76 (carbaryl in spinach) and 99% (pirimicarb in spinach), with RSDs not larger than 10%. Using trimethacarb (an obsolete carbamate insecticide) as a surrogate internal standard, the accuracy of the analysis varied between 84 and 110%, with RSDs not larger than 9%. On the basis of a signal-to-noise ratio of 10, limits of quantification were estimated to range between 2 (pirimicarb) and 10 ppb (oxamyl) and were not influenced by the type of matrix. When trying to fractionate analytes by using a short chromatographic run time, marked weakening of the ion signals for oxamyl, methomyl, and aldicarb were observed. This effect was traced to polar endogenous co-extractives eluted in the first part of the chromatographic run that interfered with gas-phase ion formation for carbamates. Adopting more selective chromatographic conditions eliminated this effect.     

Simple confirmatory method for the determination of erythromycin residues in trout: a fast liquid-liquid extraction followed by liquid chromatography-tandem mass spectrometry

In recent years, erythromycin has received considerable attention for its therapeutic efficacy against some bacterial kidney diseases in aquaculture and, therefore, suitable and sensitive analytical methods to monitor erythromycin residues in fish are required. A fast sample treatment followed by an LC-ESI-MS/MS method is described for the purification, identification, and quantification of erythromycin A residues in fish. After two extractions with acetonitrile, samples were defatted with n-hexane, filtered, and analyzed by tandem mass spectrometry. Three characteristic transition reactions (m/z 734 --> 716, 734 --> 576, and 734 --> 558) in multiple reaction monitoring were tested for the determination and confirmation of erythromycin A. The method was in-house validated through the determination of precision, accuracy, specificity, stability, calibration curve, decision limit (CCalpha), and detection capability (CCbeta), in accordance with European Commission Decision 657/2002. The coefficients of variation ranged from 1.8 to 9.4% and from 7.5 to 10.9% for intra- and interday repeatability, respectively. Recovery data were also satisfactory, with values varying from 85 to 97%. The method was specific, stable, and robust enough for the required purposes. The calibration curve showed a good linearity in the whole range of the tested concentrations (0-1000 microg kg(-1)) with a correlation coefficient (r2) equal to 0.9956. CCalpha and CCbeta were found to be 220 and 238 microg kg(-1), respectively.     

Rapid assay for analyzing biogenic amines in cheese: matrix solid-phase dispersion followed by liquid chromatography-electrospray-tandem mass spectrometry

A new rapid and sensitive method based on matrix solid-phase dispersion (MSPD) followed by liquid chromatography-electrospray-tandem mass spectrometry was devised for the determination of biogenic amines at trace levels in cheese samples. The method required 0.25 g of sample, CN-bonded silica as a dispersant sorbent, and a formic acid aqueous solution/methanol mixture as an eluting solvent. Extraction recoveries from soft cheese products were calculated in the 98 +/- 4-110 +/- 6% range. A procedure based on solid-phase extraction was also evaluated for the extraction of these compounds in cheese. Chromatographic separation was performed using a C18 column with an aqueous ammonium acetate/methanol mixture as the mobile phase under gradient conditions. The method was validated in terms of detection limits (LOD), quantitation limits (LOQ), linearity, recovery, precision, and trueness. Results in the 0.05-0.25 mg kg(-1) range were obtained for the LOD of histamine, tyramine, and beta-phenylethylamine in soft cheese samples. Linearity was established over 2 orders of magnitude. Excellent precision in terms of intra-day repeatability was calculated (RSD% < 5). The applicability of the method to the determination of biogenic amines in cheese products was demonstrated.     

Determination of dapsone in muscle tissue and milk using high-performance liquid chromatography-tandem mass spectrometry

A precise and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of dapsone in muscle tissue and milk has been developed. The sample preparation was based on extraction with organic solvent and automated solid-phase extraction (SPE) cleanup. At least three product ions were monitored for the analyte. The method was validated according to the European Decision 2002/657/EC. Estimated analytical limits were 0.0018 ng/g for CCα and 0.0031 ng/g for CCβ in meat and milk. An excellent linear concentration range was observed for both matrices with a correlation coefficient better than 0.997. Recoveries were 105-117% in meat and 101-108% in milk, with satisfactory precision and coefficients of variance (CV) less than 8%. Additionally, a simplified quantification approach was successfully evaluated depending only on the response factor (F) without the use of calibration curve. The developed method provides reliable and sensitive identification and quantification of dapsone in meat and milk.     

A rapid confirmatory method for analyzing tetracycline antibiotics in bovine, swine, and poultry muscle tissues: matrix solid-phase dispersion with heated water as extractant followed by liquid chromatography-tandem mass spectrometry

A rapid, specific, and sensitive procedure for determining four widely used tetracycline antibiotics and three related epimers in bovine, swine, and poultry muscle tissues is presented. The method is based on the matrix solid-phase dispersion technique with heated water as the extractant followed by liquid chromatography (LC)-tandem mass spectrometry (MS) equipped with an electrospray ion source. Target compounds were extracted from tissues with 5 mL of water heated at 70 degrees C. After acidification and filtration, 100 microL of the aqueous extract was injected in the LC column. MS data acquisition was performed in the multireaction monitoring mode, selecting two precursor ion to product ion transitions for each target compound. Heated water appeared to be an excellent extractant, since the absolute recovery data ranged between 70 and 78%. The accuracy of the method was determined at three spike levels, using minocycline as a surrogate analyte, in any different kind of muscle tissues considered and varied between 88 and 109% with relative standard deviations ranging between 3 and 11%. Limits of quantification were estimated to range between 1 (chlortetracycline) and 9 ng/g (4-epioxytetracycline), based on a signal-to-noise ratio of 10, and are well below the tolerance levels set by the European Union. The effects of the extraction temperature, volume of the extractant, and washing of the material supporting the biological matrix with ethylenediamine tetraacetic disodium salt on the analyte recovery were studied.     

Simple and rapid liquid chromatography-tandem mass spectrometry confirmatory assay for determining amoxicillin and ampicillin in bovine tissues and milk

A simple specific and rapid confirmatory method for determining the two amphoteric penicillins, that is, amoxicillin and ampicillin, in bovine muscle, liver, kidney, and milk is presented. This method is based on the matrix solid-phase dispersion technique with hot water as extractant followed by liquid chromatography (LC)-tandem mass spectrometry. With this instrumentation, the selected reaction monitoring acquisition mode with two fragmentation reactions for each analyte was adopted. After acidification and filtration of the aqueous extracts, 25 microL of the tissue final extracts and 50 microL of the milk final extract were injected into the LC apparatus. Absolute recovery of the two analytes in any biological matrix at the 50 ppb level in tissues and the 4 ppb level in milk was 74-95% with relative standard deviations (RSDs) of no larger than 9%. When penicillin V was used as surrogate internal standard, relative recovery of the targeted compounds present in bovine tissues and milk at, respectively, 25 and 2 ppb levels ranged between 100 and 106% with RSDs of no larger than 11%. When fractionation of analytes by using a short chromatographic run was attempted, remarkable signal weakening for the two analytes was experienced. This effect was traced to polar endogenous coextractives eluted in the first part of the chromatographic run that interfered with the gas-phase ion formation of the two penicillins. Slowing the chromatographic run eliminated this unwelcome effect. Limits of quantification of the two analytes in bovine milk were estimated to be <1 ppb, whereas amoxicillin and ampicillin could be quantified in bovine tissues down to 3.1 and 0.8 ppb levels, respectively.     

Determination of benzoxazinone derivatives in plants by combining pressurized liquid extraction-solid-phase extraction followed by liquid chromatography-electrospray mass spectrometry

A new analytical method based on the use of pressurized liquid extraction (PLE) followed by solid-phase extraction with LiChrolut RP C18 cartridges was evaluated for the sample preparation, extraction, and cleanup of eight naturally occurring benzoxazinone derivatives, 2-beta-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one, 2-beta-D-glucopyranosyloxy-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one, 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA), 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one, 2-hydroxy-1,4-benzoxazin-3-one, 2-hydroxy-7-methoxy-1,4-benzoxazin-3-one, benzoxazolin-2-one, and 6-methoxybenzoxazolin-2-one in plant samples. Afterward, liquid chromatography-electrospray mass spectrometry, using the selected ion monitoring mode and internal standard (2-MeO-DIBOA, indoxyl-beta-D-glucoside, and quercetin-3-O-rutinoside) quantification method was performed. This paper demonstrates the effectiveness of the PLE method, in conjunction with sensitive and specific mass spectrometric detection, for the quantitative recovery of compounds of the benzoxazinone class from plants. The recoveries of the analytes ranged from 66 to 110% with coefficients of variation ranging from 1 to 14%. This method gave detection limits between 1 and 27 microg/g. The method was applied to foliage and roots of three different wheat cultivars, and the analytes were detected in the range of 11-3261 microg/g of dry weight.     

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.     

Identification of puerarin and its metabolites in rats by liquid chromatography-tandem mass spectrometry

Puerarin (daidzein-8-C-glucoside) is the major bioactive isoflavone of kudzu root (the root of Pueraria lobata). Its metabolic fate, however, is not well-known. In this study, a sensitive and specific LC-ESI-MS/MS method for the determination of puerarin and its metabolites daidzein, dihydrodaidzein, and equol was developed for their analysis in biological samples. Two new metabolites of puerarin, mono- and dihydroxylated derivatives, were detected in the urine and feces of rats after oral administration. The persistence of puerarin in blood and urine as the principal metabolic form for the period of 4-72 h after oral administration suggested that puerarin is rapidly absorbed from the intestine without metabolism. Its presence in organs such as the brain suggests that this glucoside may enter tissues by specific transport pathways. Study of these metabolites may provide further understanding of the health beneficial effects of puerarin in kudzu dietary supplements.     

Quantitation of dityrosine in wheat flour and dough by liquid chromatography-tandem mass spectrometry

A method for the quantitation of dityrosine in wheat flour and dough by high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) using an isotope dilution assay with the internal standard 3,3'-(13)C(2)-dityrosine in the single-reaction monitoring mode was developed. The method consisted of the release of protein-bound dityrosine by hydrolysis in 4 mol/L hydrochloric acid/8.9 mol/L propionic acid for 24 h at 110 degrees C after addition of the internal standard, cleanup by C(18) solid-phase extraction, and HPLC-MS/MS. The limit of detection of dityrosine was 80 ng/g of sample (0.22 nmol/g), and the limit of quantitation was 270 ng/g of sample (0.75 nmol/g). The method was sensitive enough to analyze wheat flour and dough and to study the effect of flour improvers on the dityrosine content. Furthermore, the effect of the mixing time was studied. The dityrosine concentration in the flour was 0.66 nmol/g. After we mixed a dough to peak consistency, the dityrosine concentration doubled and remained constant on further mixing. Overdoses of hydrogen peroxide and hexose oxidase (HOX, E.C. 1.1.3.5) resulted in a strongly increased dityrosine content, whereas no increase of the dityrosine concentration was found after the addition of ascorbic acid and potassium bromate. Calculation of the percentage of dimeric tyrosine showed that less than 0.1% of the tyrosine residues of wheat protein were cross-linked. Therefore, dityrosine residues seem to play only a very minor role in the structure of wheat gluten.     

Solid phase extraction and liquid chromatography-tandem mass spectrometry for the evaluation of 4-hydroxy-2-nonenal in pork products

This research was aimed at setting up an analytical method for the determination in pork products of 4-hydroxy-2-nonenal (4-HNE), an aldehyde produced from the oxidation of omega-6-polyunsaturated fatty acids. Such a compound mediates various biological effects, but it is considered to be very toxic to mammalian cells at levels higher than physiological ones. The methods used for the determination of 4-HNE in biological fluids, such as blood, were found to be unsuitable for meat samples because both the repeatability and the recovery in spiked samples were unsatisfactory. A new method, based on solid phase extraction and HPLC-MS/MS, was therefore developed and validated. The limit of detection of 4-HNE in spiked samples was 0.043 mg/kg, and the recovery was approximately 60% depending on the concentration. Good linearity was observed in the range of 0.1-10 mg/kg, and repeatability and interday and intraday precision expressed as relative standard deviation were <10%. The method has been successfully applied to the determination of the aldehyde in samples of various pork products. 4-HNE was present in some products, especially the smoked and/or cooked ones, at levels that might not be a real risk for human health.     

Multianalysis of 35 mycotoxins in traditional chinese medicines by ultra-high-performance liquid chromatography-tandem mass spectrometry coupled with accelerated solvent extraction

A generic procedure, which involved accelerated solvent extraction and homemade cleanup cartridges, has been developed for the extraction and purification of 35 mycotoxins in various traditional Chinese medicine (TCM) matrixes, i.e., rhizomes and roots, seeds, flowers, and grasses and leaves, for subsequent analysis by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). All target analytes could be simultaneously quantitated in less than 17 min per run, showing narrow symmetrical peaks. The developed method was also successfully applied in routine monitoring programs, which implied a significant reduction of both effort and time, to investigate the contamination of TCMs. Among 60 commercial TCMs analyzed, 50 were positive. The achieved data underpin the practical application of the UHPLC-MS/MS method as a valuable tool for the trace analysis of multiple mycotoxins in TCMs.     

Analysis of flonicamid and its metabolites in dried hops by liquid chromatography-tandem mass spectrometry

An analytical method was developed for the determination of the neo-nicotinoid insecticide flonicamid ( N-cyanomethyl-4-trifluoromethylnicotinamide) and its metabolites N-(4-trifluoronicotinoyl) glycine (TFNG), 4-trifluoronicotinic acid (TFNA), and 4-trifluoromethylnicotinamide (TFNA-AM) in dried hops. The method utilized C18 and polymeric solid phase extraction (SPE) column cleanups, liquid-liquid partitioning, and liquid chromatography (LC) with mass spectrometry (MS/MS). Method validation and concurrent recoveries from untreated dried hops ranged from 66 to 119% for all compounds over five levels of fortification (0.005, 0.02, 0.2, 2.0, and 4.0 ppm). Flonicamid-treated hop samples collected from three field sites had the following residues: flonicamid levels of 0.561-2.85 ppm, TFNA levels of 0.302-0.470 ppm, TFNA-AM levels of 0.038-0.177 ppm, and TFNG levels of 0.098-0.204 ppm. Untreated hop samples from all fields had residues <0.005 ppm for flonicamid, TFNA, TFNA-AM, and TFNG. The limit of quantitation and limit of detection for all compounds were 0.005 and 0.0025 ppm, respectively.     

Simultaneous analysis of free and conjugated estrogens, sulfonamides, and tetracyclines in runoff water and soils using solid-phase extraction and liquid chromatography-tandem mass spectrometry

The ability to monitor multiple analytes from various classes of compounds in a single analysis can increase throughput and reduce cost when compared to traditional methods of analyses. This method for analyzing free (parent estrogen) and conjugated estrogens (metabolites) along with sulfonamides and tetracyclines utilizes a high pH (10.4) mobile phase with an ammonium hydroxide buffer for both positive- and negative-mode electrospray ionization. A single-step sample preparation by solid-phase extraction (SPE) was used to isolate and concentrate all analytes simultaneously. The analytical method was developed and validated for recoveries at 3 concentration levels for water and soil and produced recoveries of 42-123% and 21-105% respectively. Method detection limits ranged from 0.3 to 1.0 ng/L for water samples and 0.01 to 0.1 ng/g for soils. The method quantification limit ranged from 0.9 to 3.3 ng/L for water samples and 0.06 to 0.7 ng/g for soils. The single-point standard addition calibration procedure was validated across a linear range of MQL to 100 ng/L with ≥82% accuracy against a matrix matched standard curve. Furthermore, sorption of tetracyclines onto glassware was investigated and minimized by 10% using nitric acid-rinsed glassware, while separation parameters were further optimized based on retention time and signal responses. This method has been used for the quantification of estrogens, tetracyclines, and sulfonamides in soil and runoff waters with multiple compounds detected simultaneously in a single analysis.     

Analysis of fenhexamid in caneberry, blueberry, and pomegranate by liquid chromatography-tandem mass spectrometry

An analytical method for the determination of fenhexamid [N-(2,3-dichloro-4-hydroxyphenyl)-1-methylcyclohexanecarboxamide] in caneberry, blueberry, and pomegranate was developed utilizing acetone extraction, column cleanup, liquid-liquid partitioning, and liquid chromatography-tandem mass spectroscopy (LC-MS/MS) for detection. Method validation recoveries ranged from 91 to 96% for caneberry, from 80 to 91% for blueberry, and from 74 to 95% for pomegranate. Control samples collected from IR-4 trials for all matrixes had residue levels of <0.020 ppm. Fenhexamid-treated field samples had residue levels that ranged from 0.46 to 16.11 ppm (caneberry), from 0.87 to 2.91 ppm (blueberry), and from 1.59 to 1.85 ppm (pomegranate). The method was validated to a limit of quantitation of 0.020 ppm, and the limit of detection was 0.009 ppm.     

Determination of aflatoxin M1 in milk and milk powder using high-flow solid-phase extraction and liquid chromatography-tandem mass spectrometry

Animal feeds occasionally have some degree of contamination by Aspergillus spp. Even pasteurized milk at times contains the toxic liver carcinogen aflatoxin M1 (AFM1). Confirmation of its presence is now done with solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC)-fluorescence, using a small enough sample that SPE time is reasonable. In this study 200 mL of milk was extracted using a C18 disk at a flow rate of approximately 100 mL/min and AFM1 quantified by HPLC-tandem mass spectrometry with negative electrospray ionization. The effectiveness and cleanup efficacy of immunoaffinity columns (IAC) was compared with that of Mycosep multifunctional cleanup columns (MFC). Average recovery and detection limits of whole milk and low-fat milk cleaned up by IAC were significantly superior to those obtained with the MFC (78-87% and 0.59-0.66 ng/L, respectively). The new procedure improves extraction speed, sensitivity, and specificity.     

Solid-phase extraction followed by liquid chromatography-mass spectrometry for trace determination of beta-lactam antibiotics in bovine milk.

A confirmatory assay able to unambiguously identify and quantify 10 approved-for-use beta-lactam antibiotics in milk below stipulated U.S. and EU tolerance levels is presented. beta-Lactams are extracted from 10 mL of intact milk by a Carbograph 4 cartridge. After solvent removal, residue reconstitution, and filtration, a completely transparent and uncolored extract is injected into a liquid chromatography -mass spectrometry (LC-MS) instrument equipped with an electrospray (ES) ion source and a single quadrupole. During the chromatographic run, the ES/MS system is operated first in the positive-ion mode (PI) and then in the negative-ion (NI) mode. This is done to circumvent matrix interferences resulting in remarkable signal weakening of the last-eluted analytes, when detecting them as [M+H]+ adduct ions. MS data acquisition is performed by a time-scheduled three-ion selected ion monitoring program. At the 5 ng/mL level, recoveries of the beta-lactams are between 70 (nafcillin) and 108% (cephalin), with relative standard deviations ranging between 5 (oxacillin) and 11% (amoxicillin and ceftiofur). The response of the ES/MS detector is linearly related to injected amounts up to 500 ng, irrespective of the chemical characteristics of the beta-lactams and the acquisition mode selected (PI or NI modes). Limits of quantification, based on a minimal value of the signal-to-noise ratio of 10, were estimated to be within 0.4 (cephalin) and 3 ng/mL (dicloxacillin). Analyses of milk samples taken after intramammary application of amoxicillin showed that 1.2 ng/mL of this penicillin was still present 6 days after treatment. At this concentration level, the identification power of the method is not weakened, as signals of the three product ions of amoxicillin are still well distinguishable from the background noise.     

Simultaneous determination of five plant growth regulators in fruits by modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction and liquid chromatography-tandem mass spectrometry

An effective method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and optimized to obtain a complete separation of five representative plant growth regulators (PGRs) [gibberellic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), thidiazuron, forchlorfenuron, and paclobutrazol] in fruits. Extraction was performed with acetonitrile containing 0.1% (v/v) acetic acid, applying modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) methodology. LC-MS/MS conditions including composition of mobile phases and mass spectrometry (MS) conditions were evaluated to achieve the highest sensitivity in MS detection. All of the data acquisition was employed in the segmented multiple-reaction monitoring mode for the selected negative and positive transition ions. The octadecylsilyl (C18) dispersive solid-phase extraction (SPE) sorbent was found to provide the more satisfied recoveries than primary secondary amine (PSA) and graphitized carbon black (GCB) for five target PGRs. The optimized method allowed for recoveries of 76-112% for the five PGRs from fruit samples with relative standard deviation (RSD) values less than 10%. Limits of quantification (0.5-16.5 μg/kg) were lower than the maximum limit of residues established for PGRs. The results demonstrated that the developed LC-MS/MS and QuEChERS extraction method is highly effective for analyzing trace amounts of target PGRs in fruit samples. Finally, the method was successfully used to detect residual PGRs in Beijing, China, in 2010. The concentrations of 2,4-D (5.1-1503 μg/kg) and paclobutrazol (1-1381 μg/kg) found in orange and peach, respectively, suggesting that the use of these PGRs in these fruits should be regulated in China in the future.     

Determination of 17alpha-methyltestosterone in muscle tissues of tilapia, rainbow trout, and salmon using liquid chromatography-tandem mass spectrometry

An analytical method was developed to quantitate and confirm the presence of 17alpha-methyltestosterone in the muscles of tilapia, rainbow trout, and salmon. The method employed two liquid-liquid partitioning steps and two solid-phase extraction columns for sample cleanup. The final extracts were analyzed on an isocratic reverse-phase liquid chromatography-tandem mass spectrometry system with atmospheric-pressure chemical ionization in the positive ion mode. The method was validated at levels from 0.40 to 1.6 ng/g, with MT-d3 used as an internal standard. The accuracy was between 100% and 110%, and coefficients of variation of <10% were obtained for all three fish species. Muscle tissues from dosed fish were also assayed to demonstrate the effectiveness of the method for recovering the parent drug.     

Masked mycotoxins: determination of a deoxynivalenol glucoside in artificially and naturally contaminated wheat by liquid chromatography-tandem mass spectrometry

Conjugated mycotoxins, in which the toxin is usually bound to a more polar substance like glucose, are referred to as masked mycotoxins, as these substances escape routine detection methods but can release their toxic precursors after hydrolysis. This is the first report on the natural occurrence of a glucoside of deoxynivalenol (DON) in Fusarium-infected wheat and maize. To obtain appropriate standards, we chemically synthesized deoxynivalenol-3-beta-D-glucopyranoside (DON-3-glucoside) and deoxynivalenol-15-beta-D-glucopyranoside (DON-15-glucoside). The synthesis products were characterized by liquid chromatography-tandem mass spectrometry. The DON-glucosides showed different collision-induced dissociation (CID) fragmentation behaviors and could therefore be distinguished. Wheat plants were either treated with DON (n = 52) or with Fusarium spp. (n = 4) at anthesis, and after harvest, wheat ears were analyzed for DON and DON-glucosides. All 56 treated wheat samples contained DON and a DON-glucoside with the same retention time, molecular mass, and CID fragmentation behavior as the synthetic DON-3-glucoside. Moreover, the DON-glucoside was also found in two out of three analyzed naturally DON-contaminated maize and in five out of five naturally contaminated wheat samples, in a range from 4 to 12% of the DON concentration. To further confirm the identity of the DON-glucoside, the compound was isolated from wheat extracts and characterized as DON-3-glucoside with NMR. The results of this study indicate the importance to consider both DON and DON-3-glucoside with regard to food and feed safety.