Quantitative liquid chromatographic method using fluorescence detection for determining zearalenone and its metabolites in blood plasma and urine

The liquid chromatographic (LC) method described, suitable for use with both blood plasma and urine, is applicable for determination of zearalenone and alpha-zearalenol at levels as low as 0.5 ng/mL plasma and 5 ng/mL urine. The sample is incubated overnight with beta-glucuronidase to analyze for both conjugated and unconjugated forms of zearalenone. The next day, the sample is acidified with H3PO4, extracted with chloroform, and evaporated to dryness. The residue is dissolved in toluene and loaded onto a silica gel cartridge which is washed with toluene and eluted with toluene-acetone (88 + 12). The eluate is evaporated, and the residue is dissolved in chloroform, extracted with 0.18M NaOH, neutralized with H3PO4, and re-extracted with chloroform. The chloroform extract is evaporated, dissolved in mobile phase for LC, and injected onto a normal phase column under the following chromatographic conditions: mobile phase of water-saturated dichloromethane containing 2% 1-propanol, and fluorescence detector, excitation wave-length 236 nm, and 418 nm cut-off emission filter. Recoveries of zearalenone and its metabolites from blood plasma and urine are 80-89% in the range 2.0-10 ng standard/mL plasma, and 81-90% in the range 10-30 ng standard/mL urine. This method was used to analyze blood and urine samples from a pig fed zearalenone-contaminated feed (5 mg/kg), corresponding to 80 micrograms/kg body weight. Zearalenone was rapidly metabolized to alpha-zearalenol, which appeared in the blood only 30 min after feeding. Almost all zearalenone and alpha-zearalenol was found conjugated with glucuronic acid in both blood plasma and urine.     

Concentration levels of zearalenone and its metabolites in urine, muscle tissue, and liver samples of pigs fed with mycotoxin-contaminated oats

The content of zearalenone and its metabolites in urine and tissue samples from pigs fed zearalenone-contaminated oats was established by analytical methods combining solid-phase extraction cleanup of the samples with highly selective liquid chromatography-mass spectrometry (LC-MS)/MS detection. Investigation of the urine samples revealed that approximately 60% of zearalenone was transformed in vivo to alpha-zearalenol and its epimer beta-zearalenol in a mean ratio of 3:1. Zeranol and taleranol as further metabolites could only be detected in trace amounts. Zearalanone was identified at considerable concentrations, though only in a couple of samples. In contrast, liver samples contained predominantly alpha-zearalenol, and to a minor extent beta-zearalenol and zearalenone, with a mean ratio of alpha-/beta-zearalenol of 2.5:1, while zeranol, taleranol, or zearalanone could not be identified in any of the investigated samples. The degree of glucoronidation was established for zearalenone as 27% in urine and 62% in liver; for alpha-zearalenol as 88% in urine and 77% in liver; and for beta-zearalenol as 94% in urine and 29% in liver. Analyses of muscle tissue revealed relatively high amounts of nonglucuronidated zeranol and alpha-zearalenol together with traces of taleranol and zearalenone, indicating that the metabolism of zearalenone and its metabolites is not restricted to hepatic and gastrointestinal metabolic pathways.     

Liquid chromatographic determination of alpha-zearalenol and zearalenone in corn: collaborative study

The liquid chromatographic determination of alpha-zearalenol and zearalenone in corn was collaboratively studied. Each of 13 collaborators received 7 corn samples; 2 were blanks and 5 were spiked to contain 50, 100, and 200 ng alpha-zearalenol/g and 50, 100, 500, 1000, and 4000 ng zearalenone/g. Four sets (including blanks) of blind duplicates were included in the study. Five naturally contaminated corn samples (one in duplicate) were also provided. All collaborators detected both mycotoxins at 50 ng/g. Average recoveries reported by all collaborators ranged from 81.9% at 200 ng/g to 100.3% at 50 ng/g for alpha-zearalenol and from 77.8% at 1000 ng/g to 123% at 50 ng/g for zearalenone. Three collaborators reported false positives for both alpha-zearalenol and zearalenone. The within-laboratory CV values based on blind duplicates were 22.6% for alpha-zearalenol and 31.4% for zearalenone. The CV values based on laboratory-sample interaction were 25.6 and 33.8% for alpha-zearalenol and zearalenone, respectively. The CV values for naturally contaminated samples (including duplicates) were 47.0% for alpha-zearalenol and 37.7% for zearalenone. The method has been adopted official first action.     

Rapid, sensitive liquid chromatographic method for determination of zearalenone and alpha- and beta-zearalenol in wheat

A rapid, sensitive liquid chromatographic (LC) method is described for quantitative determination of zearalenone and alpha- and beta-zearalenol in wheat. The procedure incorporates an internal standard, zearalenone oxime, to facilitate quantitation and automated analysis. A sample, buffered with pH 7.8 phosphate, is extracted with water-ethanol-chloroform (2 + 50 + 75) and cleaned up. The final residue is dissolved in LC mobile phase and injected onto a reverse phase RP-18 column under the following conditions: water-methanol-acetonitrile (5 + 3 + 2) mobile phase; fluorescence (excitation wavelength 236 nm, 418 nm cut-off emission filter) and UV (254 nm, range 0.0025 AU) detectors. The limit of detectability (twice background) is 0.5 ng for zearalenone and alpha-zearalenol standards on the fluorescence detector and 4 ng for beta-zearalenol on the UV detector, which is equivalent to 20 micrograms zearalenone and 20 micrograms alpha-zearalenol/kg, and 160 micrograms beta-zearalenol/kg feed. Standard curves are linear over the range 0-35 ng zearalenone and alpha-zearalenol on the fluorescence detector and 0-50 ng beta-zearalenol on the UV detector. Recoveries of all compounds are 87.5-101% in the range 0.1-3.0 mg/kg (ppm).     

Use of deuterated internal standards for quantitation of T-2 and HT-2 toxins in human blood by tandem mass spectrometry

Deuterated acetyl derivatives (3-trideutero-acetyl-T-2 and 15-trideutero-HT-2) were prepared for use as internal standards for the quantitation of T-2 and HT-2 in blood by tandem mass spectrometry. The method used was multiple reaction monitoring (MRM), which essentially involves the selection of a parent ion for analysis followed by monitoring of the daughter ions generated by collision activated decomposition. The parent ions chosen for the trifluoroacetate derivative of T-2 and HT-2 were m/z+ 478 and 532, respectively. Both parents yield the same daughter ions, i.e., 180, 138, and 121. HT-2 and T-2 were added to blood extracts in amounts ranging from 1 to 20 ppb. The limit of detection is about 0.5 ppb with an effective detection limit of 1.0 ppb in a range of 1-20 ppb. The recovery is about 90%. This method can be used by veterinarians for purposes of diagnostics. It can be used for urine as well as blood.     

Liquid chromatographic determination of zearalenone and alpha- and beta-zearalenols in milk

Previous research has demonstrated transmission of zearalenone and alpha- and beta-zearalenols into the milk of cows and other animals. Since human intake of zearalenone and its metabolites via milk is an unknown factor in risk assessment of zearalenone and because appropriate methodology for their determination in milk is not available, a rapid and sensitive analytical method has been developed. Essentially, the method includes extraction with basic acetonitrile, acidification, partition into methylene chloride on a hydrophilic matrix, cleanup on an aminopropyl solid phase extraction column, and reverse-phase liquid chromatography with fluorescence detection. Recoveries from milk averaged 84% for zearalenone, 93% for alpha-zearalenol, and 90% for beta-zearalenol at spiking levels of 0.5 to 20 ng/mL. As little as 0.2 ng/mL of zearalenone and alpha-zearalenol and 2 ng/mL of beta-zearalenol can be detected in milk. These 3 compounds are stable in refrigerated milk for at least 2 weeks and in milk brought to boiling. Enzymes (beta-glucuronidase and aryl sulfatase) may be added to milk prior to extraction to hydrolyze any conjugates.     

Liquid chromatographic determination of aflatoxins, ochratoxin A, and zearalenone in grains, oilseeds, and animal feeds by post-column derivatization and on-line sample cleanup

A liquid chromatographic method using on-line sample cleanup, reverse flow analytical column loading, gradient elution, and postcolumn derivatization with iodine permits direct, rapid determination of aflatoxins B1, B2, G1, and G2, as well as ochratoxin A and zearalenone. Limits of quantitation are 5 ppb for the aflatoxins and ochratoxin A and 30 ppb for zearalenone. This procedure performs well as a multimycotoxin screen for cereal grains and oilseeds, with more limited success in complete animal feeds.     

Rapid immunochemical screening method for aflatoxin B1 in human and animal urine

A method has been developed to determine the presence of aflatoxin B1 in the urine of animals (including humans) by utilizing commercial immunochemical kits that can be used in the field. Urine is treated with diatomaceous earth and filtered to clarify the sample; 2-3 ppb aflatoxin B1, corresponding to about 300 ppb in the ingested feed/food, can be detected in the filtered urine without further purification. To improve sensitivity, the urine filtrate is passed through a C18 solid phase column to extract the aflatoxin. The column is washed with acetonitrile-water (15 + 85) and water, aflatoxin B1 is eluted with methanol-water (7 + 3), and water is added to the eluate, which is then tested for aflatoxin with the test kit. The limit of detection is 0.2 ppb, reflecting consumption of 40 ppb or more aflatoxin in the feed/food. When the initial sample volume is adequate, purification through the C18 column step is usually sufficient. For limited sample volumes, the eluate from the C18 column is mixed with water, added to an immunosorbent affinity column, and washed with water to remove excess sample matrix and impurities. Aflatoxin B1 is eluted with acetonitrile. The extract is evaporated under nitrogen and the residue is redissolved in methanol-water (25 + 75). At this purification stage, the limit of detection is reduced to 0.05 ppb.     

High performance liquid chromatographic method using fluorescence detention for quantitative analysis of zearalenone and alpha-zearalenol in blood plasma

A sensitive, high performance liquid chromatographic method is described for quantitative determination of zearalenone and alpha-zearalenol in blood plasma. Blood plasma is extracted with 2-propanol in ether, the extract is evaporated to dryness, and the residue is dissolved in 0.18N NaOH. The aqueous phase is washed with chloroform, dichloromethane, and benzene, neutralized with 0.10M H3PO4, and extracted with benzene. The extract is evaporated, dissolved in methanol, and injected onto a reverse phase column containing LiChrosorb RP-8 under the following conditions: methanol-acetonitrile-water mobile phase, fluorescence detector, excitation wavelength 236 nm, and 418 nm cut-off emission filter. The limit of detectability (twice background) is 0.5 ng standard which is equivalent to 0.6 ng standard/mL blood plasma. Linear standard curves are observed over the range of 0-35 ng of injected zearalenone and alpha-zearalenol. The recoveries from blood plasma are 76-101% in the range of 1.5-6.0 ng standard/mL blood.     

Quantitation and confirmation of sulfamethazine residues in swine muscle and liver by LC and GC/MS

The present paper describes a liquid chromatographic (LC) method for purification of crude swine tissue extracts before gas chromatographic/mass spectrometric (GC/MS) quantitation and confirmation of sulfamethazine at low ppb levels. Fractions corresponding to sulfamethazine were collected, evaporated to dryness, N-methylated with diazomethane, concentrated, and analyzed by GC/MS. A mass spectrometer was set to selected ion monitoring (SIM) mode. Ions 233, 227, 228, and 92 m/z were detected. Ratio 227/233 m/z (sulfamethazine/internal standard, [phenyl 13C6] sulfamethazine) was used for quantitation, while ratios 228/227 and 92/227 m/z, respectively, were used for confirmation. Quantitation in spiked blank muscle tissue was tested from 100 to 1 ppb and found acceptable at all concentrations studied; coefficients of variations ranged from 4.9 to 14.4%. Similar results were obtained for liver tissue from 5 to 20 ppb; coefficients of variation ranged from 1.2 to 9.1%.     

Determination of grayanotoxins in biological samples by LC-MS/MS

A rapid LC-MS/MS method was developed for the quantitative determination of grayanotoxins I, II, and III in rumen contents, feces, and urine. The grayanotoxins were extracted from solid samples with methanol. The methanol extract was diluted with water and cleaned up using a reversed phase solid phase extraction column. HPLC separation was performed by reversed phase HPLC using a gradient of water and methanol containing 1% acetic acid. Determination was by positive ion electrospray ionization and ion trap tandem mass spectrometry. Grayanotoxin I quantitation was based on fragmentation of the sodium adduct ion at m/z 435 to a product ion at m/z 375. Grayanotoxins II and III were quantitated on the basis of fragmentation of the ion at m/z 335 to the product ion at m/z 299. The method detection limits were 0.2 microg/g in rumen contents and feces and 0.05 microg/g in urine. Fortifications at the detection limits and 10 times the detection limits of bovine rumen contents, caprine feces, and ovine urine were recovered in the range 80-114%. The diagnostic utility of the method was tested by analyzing samples submitted to the veterinary toxicology laboratory.     

Method for determination of pentobarbital in dry dog food by gas chromatography/mass spectrometry

A procedure has been developed and validated for measuring the concentration of pentobarbital residues in dry, extruded animal feed in the range of 3-200 ng/g (ppb) with an estimated limit of quantitation of 2 ppb. The method was developed for surveillance purposes: to measure the concentration of euthanizing agent which might be present in feeds incorporating rendered products which themselves might include some fraction of euthanized animals. A previously published qualitative procedure was modified by adding isotopically labelled pentobarbital as an internal standard. Dry feed was ground and extracted with methanol. The extract was loaded on a mixed-mode (C-18, anion exchange) solid-phase extraction cartridge designed for barbiturate residues. Pentobarbital was eluted and derivatized for gas chromatography/mass spectrometry in positive ion chemical ionization mode. Quantitation was based on the ratio of dimethyl-pentobarbital MH+ (m/z 255) vs dimethyl-pentobarbital-d(5) (m/z 260) in standards and extracts. Accuracy ranged from 112% at 3 ppb to 96% at 200 ppb, with relative standard deviations ranging from 4% at 3 ppb to 2% at 200 ppb.     

Detection of twelve mycotoxins in mixed animal feedstuffs, using a novel membrane cleanup procedure.

A multimycotoxin thin layer chromatographic screening method is described which is applicable to most animal feedstuffs. Interference from nonspecific lipid, pigment, and other components of simple and mixed feeds is reduced to a minimum by using a membrane cleanup step. Aflatoxins B1, B2, G1, and G2, citrinin, diacetoxyscirpenol, ochratoxin A, patulin, penitrem A, sterigmatocystin, T-2 toxin, and zearalenone may be reliably detected. The sensitivity of the method is generally low for mixed feeds but even so aflatoxin B1 can be detected at a level of 3 ppb and ochratoxin A at 80 ppb. While the basic method is less sensitive for sterigmatocystin (330 ppb), patulin (600 ppb), zearalenone (1000 ppb), and the trichothecenes (1000-4000 ppb), it may be adapted so as to reduce the above detection limits when the presence of these toxins is suspected. Lower levels may be detected in extracts of simple feeds.     

Creatinine measurements in 24 h urine by liquid chromatography--tandem Mass Spectrometry

A simple, sensitive, and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determining urinary creatinine was developed and used to evaluate 24 h urine samples collected during an exposure study. Urine (1 microL) was diluted with methanol and then directly applied to LC-MS/MS. Under electrospray ionization (ESI) conditions, the transition molecules of creatinine and creatinine- d3 were observed at m/ z 114 > 44 and m/ z 117 > 47, respectively. The retention time of creatinine was 0.59 min. The linear range was 1-2000 ng/mL, with a detection limit in urine of 1 ng/mL. LC-MS/MS and colorimetric end-point methods were significantly associated ( R2 = 0.8785, p < 0.0001). The LC-MS/MS method to determine creatinine in 24 h urine samples had shorter retention times, was more sensitive, reliable, reproducible, simple, selective, and used a smaller sample size than other LC-MS/MS or commercial methods.     

Mycoflora and multimycotoxin detection in corn silage: experimental study

Agricultural activities involve the use of crop preservation such as "trench-type" silo, which can sometimes be contaminated by fungi. To investigate the exposure of livestock and farm workers to fungal spores and mycotoxins, a multimycotoxin analysis method has been developed. Six mycotoxins (aflatoxin B1, citrinin, deoxynivalenol, gliotoxin, ochratoxin A, and zearalenone) were quantified by high-performance liquid chromatography coupled to mass spectrometry after solid-phase extraction. An experimental study of fungal species and mycotoxins was conducted in corn silage (Normandy, France) during 9 months of monitoring. The results indicated the recurrence of around 20 different species, with some of them being potentially toxigenic fungi such as Aspergillus fumigatus, Aspergillus parasiticus, Fusarium verticillioides, and Monascus ruber, and the detection of aflatoxin B1 (4-34 ppb), citrinin (4-25 ppb), zearalenone (23-41 ppb), and deoxynivalenol (100-213 ppb). This suggested a possible chronic exposure to low levels of mycotoxins.     

Development of liquid chromatography-electrospray mass spectrometry for the determination of patulin in apple juice: investigation of its contamination levels in Japan

Patulin is a mycotoxin produced by mainly Penicillium species, for example, P. expansum, and Aspergillus species. There are several reports of patulin contamination in apple juice. Last year, the Ministry of Health, Labour and Welfare of Japan set the maximum allowable level of patulin in apple juice at 50 ppb and decided that the measurement of patulin levels in apple juice products should be conducted. To this end, a simple, accurate, and selective analytical method for the detection of patulin at levels lower than 5 ppb, the detection limit, is desired. This paper reports the development of an analytical method that employs solid-phase extraction-liquid chromatography-mass spectrometry (SPE-LC-MS). When MS measurements were conducted with the selected ion monitoring (SIM) mode, the pseudomolecular ions at m/z 153 and 156 were used to monitor patulin and (13)C(3)-labeled patulin, respectively. The detection limit (S/N = 3) and the quantification limit (S/N = 10) of patulin at injection levels into LC-MS were 12.5 and 25 pg, respectively. However, when the actual sample was applied for the analysis based on the developed method including the sample preparation, the detection limit (S/N = 3) and quantification limit (S/N = 10) were 2.5 and 5 pg in sample, respectively. The calibration curve obtained for concentrations ranging from 5 to 500 ppb showed good linearity with a coefficient of determination (r (2)) of 0.999. In addition, the recovery was >95% when an internal standard was used. The method was applied to the analysis of 76 apple juice samples from Japan, and as a result, patulin levels ranging from <1.0 to 45 ppb (detection frequency = 15/76) were detected. In this study, it was found that patulin was a greater contaminant in concentration/reduction than in "not from concentrate" apple juice.     

南美白对虾空气油炸过程中脂质组学快检技术研究

为实时监测南美白对虾在空气油炸过程中脂质组学轮廓变化,本研究采用iKnife-REIMS联用技术、主成分分析(PCA)和正交偏最小二乘判别分析(OPLS-DA)探究了不同空气油炸温度(140、170、200℃,10 min)对南美白对虾肌肉组织脂质组成的影响。结果表明,经结构鉴定和相对含量测定,南美白对虾样品中共检出10种脂肪酸与31种磷脂分子,其中,亚油酸(m/z 279,21.88%)、EPA(m/z 301,16.59%)与DHA(m/z 327,15.14%)为主要脂肪酸离子;[PE 36:1-H]^-(m/z 744,20.16%)与[PE 38:5-H]^-/[PC O-36:5-H]^-(m/z 764,15.92%)为主要磷脂离子。随着油炸温度的升高,油炸南美白对虾中饱和脂肪酸与甘油磷脂酸(PA)分子的相对含量呈上升趋势,而不饱和脂肪酸、磷脂酰乙醇胺(PE)、磷脂酰胆碱(PC)与磷脂酰肌醇(PI)分子的相对含量不断减少。通过共享与特有化合物结构分析图(SUS-plot)确定了6个潜在标记物(m/z 277、m/z 770、m/z 810、m/z 818、m/z 844及m/z 836),可用于空气油炸样品的实时鉴别。经方法学验证,该iKnife-REIMS联用实时检测方法的灵敏度和精密度均可满足空气油炸过程中南美白对虾脂肪酸和磷脂的脂质组学轮廓分析测试要求。本研究结果为食品加工过程中脂质组学变化研究提供了新的检测技术手段。     

Gas-liquid chromatographic-mass fragmentographic determination of low levels of diethylcarbonate in beverages.

Sodium chloride and ethanol (omitted for samples with greater than 10% alcohol) are added to the beverage sample and the sample is allowed to equilibrate in a 30 degrees C water bath. An aliquot of the headspace is injected into a gas chromatography containing a column packed with 0.2% Carbowax 1500 on 80--100 mesh Carbopack C. During the elution of diethylcarbonate (DEC), an impurity that is present in diethylpyrocarbonate, the column effluent is vented to a mass spectrometer with a multiple ion detection system and operated in the electron impact mode. The ions at m/e 63 and 91 are monitored. Lemonade, fruit drinks, wine, and beer samples (138 total) were analyzed for DEC. Sixteen samples had greater than 30 ppb DEC. Eight analyses of a lemonade sample gave a mean of 88 ppb with a coefficient of variation of 11%.     

Capillary gas chromatographic-mass spectrometric determination of fluoroacetate residues in animal tissues

A method for the quantitative determination of fluoroacetate (FAC) residues in animal tissues is described. The procedure involves tungstic acid extraction, partitioning into ethyl acetate, evaporation of ethyl acetate, derivatization with pentafluorobenzyl bromide (PFB), and analysis of the resulting derivative (PFB-FAC) by capillary gas chromatography-mass spectrometry (CGC-MS) with specific ion monitoring (SIM). The tungstic acid system extracted 96.8 +/- 4.2% of the endogenous 14C-1080 residues in rat tissues. Recovery of FAC during the extraction, purification, and derivatization procedures is established by use of a 14C-FAC spike. 1,2-Dibromobenzene is used as an internal standard for the CGC-MS analysis. PFB-FAC is identified on the basis of comparative retention times and the relative intensities of m/z 257.9 and 181.0. PFB-FAC is quantitated by comparing the response at m/z 257.9 to a PFB-FAC standard curve. Routine sensitivity of the method allows determination of 10 ppb fluoroacetate in tissue.     

Preparative enzymatic synthesis of glucuronides of zearalenone and five of its metabolites

The resorcylic acid lactones zearalenone ( 1), alpha-zearalenol ( 2), beta-zearalenol ( 3), alpha-zearalanol (zeranol) ( 4), beta-zearalanol (taleranol) ( 5), and zearalanone ( 6) were converted to their glucuronides on a preparative scale in good yields. Reactions were conducted with bovine uridine 5'-diphosphoglucuronyl transferase (UDPGT) as catalyst and uridine 5'-diphosphoglucuronic acid (UDPGA) as cofactor. The glucuronides were isolated by column chromatography and characterized by NMR spectroscopy and mass spectrometry. Although the principal products were 4- O-glucuronides (i.e., linkage through a phenolic hydroxyl), significant quantities of the 6'- O-glucuronides (i.e., linkage through the aliphatic hydroxyl) of alcohols 2, 4, and 5 were also isolated. In the case of 3, the 2- O-glucuronide was isolated as the minor product. Overall isolated yields of glucuronides, performed on a 20-50 mg scale, were typically ca. 80% based on the resorcylic acid lactone starting material. LC-UV-MS (2) analysis of purified specimens revealed MS (2) fragmentations useful for defining the point of attachment of the glucuronide moiety to the zearalenone nucleus.