Determination of zearalenone in cereal grains, animal feed, and feed ingredients using immunoaffinity column chromatography and liquid chromatography: interlaboratory study

A method using immunoaffinity column chromatography (IAC) and liquid chromatography (LC) for determination of zearalenone in cereal grains, animal feed, and feed ingredients was collaboratively studied. The test portion is extracted by shaking with acetonitrile-water (90 + 10, v/v) and sodium chloride. The extract is diluted and applied to an immunoaffinity column, the column is washed with water or phosphate-buffered saline or methanol-water (30 + 70, v/v), and zearalenone is eluted with methanol. The eluate is evaporated, the residue is dissolved in mobile phase and analyzed by reversed-phase LC with fluorescence detection. The presence of zearalenone can be confirmed using an alternate excitation wavelength or diode array detection. Twenty samples were sent to 13 collaborators (8 in Europe, 2 in the United States, one in Japan, one in Uruguay, and one in Canada). Eighteen samples of naturally contaminated corn, barley, wheat, dried distillers grains, swine feed, and dairy feed were analyzed as blind duplicates, along with blank corn and wheat samples. The analyses were done in 2 sample sets with inclusion of a spiked wheat control sample (0.1 mg/kg) in each set. Spiked samples recoveries were 89-116%, and for the 18 naturally contaminated samples, RSDr values (within-laboratory repeatability) ranged from 6.67 to 12.1%, RSDR values (among-laboratory reproducibility) ranged from 12.5 to 19.7%, and HorRat values ranged from 0.61 to 0.90.     

Rapid immunoaffinity-based method for determination of zearalenone in corn by fluorometry and liquid chromatography

An immunoaffinity-based method was developed to determine zearalenone in corn. Corn samples were extracted in acetonitrile-water (90 + 10, v/v), applied to an immunoaffinity column, and eluted with methanol. The isolated toxin was quantitated either by reaction with aluminum chloride hexahydrate (AlCl3.6H2O) prior to measurement with a fluorometer or injection into a liquid chromatographic (LC) system with a fluorescence detector. Performance was evaluated in terms of antibody specificity, limit of detection, percentage recovery, precision, column capacity, assay linearity, and comparison with AOAC Official Method 985.18. With the immunoaffinity column cleanup procedure, only zearalenone and its metabolites were recognized by the antibody (> or = 75% recovery). Limits of detection were 0.10 microgram/g for the fluorometer and 0.10 or 0.0025 microgram/g (sensitive method) for the LC method. Percentage recovery averaged 105% (fluorometer) and 93% (LC method), with average relative standard deviations (RSDs) of 15.7 and 9.3%. Naturally contaminated samples gave comparable RSDs of 8.3 and 9.9% for the fluorometer and LC methods, respectively. Column capacity was 4.0 micrograms with 89% recovery. Assay linearity was comparable for both methods (r2 = 0.998). Optimum assay ranges were 0.10-5.0 micrograms/g for the fluorometer and 0.10-50 or 0.0025-5.0 micrograms/g (sensitive method) for the LC method. Comparative analysis of 17 naturally contaminated corn samples using Zearala Test LC and the official AOAC LC method for detection of zearalenone showed that Zearala Test is statistically comparable to the AOAC Official Method 985.18 (r2 = 0.747).     

Sample clean-up by sol-gel immunoaffinity chromatography for determination of chloramphenicol in shrimp

The paper describes the characterisation and application of sol-gel columns prepared by entrapping anti-chloramphenicol (CAP) antibodies. Retention of CAP in the column was caused by specific interactions with the anti-CAP antibodies and not by non-specific adsorption to the sol-gel glass. After optimising important operation conditions, e.g. feeding medium, feeding flow-rate, elution medium, elution flow-rate and elution volume, the sol-gel columns were included in a clean-up procedure developed to determine CAP in shrimp. The selectivity of the columns was high enough to efficiently remove interfering matrix compounds. Due to the chromatographic conditions applied retention of cross-reacting substances in the immunoaffinity column did not pose a problem. CAP recovery of the analytical method was 68% with a relative standard deviation of 4% (n=4). In spite of applying highly complex shrimp extracts the columns could be used for clean-up of at least 12 samples. However, when detection of CAP is carried out with an UV detector the analytical method has a relatively poor sensitivity (LOD=1.8 ng/g, S/N=3). The most obvious way is to replace the UV detector by a detector based on an inherently more sensitive and selective detection principle, like a mass spectrometer.     

Simultaneous determination of aflatoxins, ochratoxin A, and zearalenone in grains by new immunoaffinity column/liquid chromatography

The simultaneous determination of mycotoxins was performed in 3 steps: extraction, cleanup, and detection. For extraction, a mixture of acetonitrile-water (60 + 40, v/v) was proved appropriate. For cleanup, a new Afla-Ochra-Zea immunoaffinity column was used. After derivatization with trifluoroacetic acid, the mycotoxins aflatoxins, ochratoxin A (OTA), and zearalenone (ZEA) were determined simultaneously by liquid chromatography with fluorescence detection. The detection limits in different matrixes after cleanup with the new immunoaffinity column were very low: aflatoxins, 0.002-0.7 microg/kg; OTA, 0.07-0.25 microg/kg; ZEA, 1-3 microg/kg. The limits of determination were: aflatoxins, 0.25 microg/kg; OTA, 0.5 microg/kg; ZEA, 5 microg/kg. The recovery rates for aflatoxins, OTA, and ZEA for rye and rice were between 86 and 93% when a 0.5 g sample matter per immunoaffinity column was used.     

Immunoaffinity column cleanup with liquid chromatography using postcolumn bromination for the determination of aflatoxins in black and white sesame seed: single-laboratory validation

A single-laboratory validation was conducted to establish the effectiveness of an immunoaffinity column cleanup procedure followed by LC with fluorescence detection for the determination of aflatoxins B1, B2, G1, and G2 in sesame seeds. The sample is homogenized with 50% water (w/w) to form a slurry, then the test portion is extracted with methanol-water (60 + 40, v/v) using a high-speed blender. The sample extract is filtered, diluted with 15% Tween 20 in phosphate-buffered saline solution, and applied to an immunoaffinity column. Aflatoxins are removed with neat methanol, then directly determined by RP-LC with fluorescence detection using postcolumn bromination (Kobra cell). Test portions of blank white sesame seed slurry were spiked with a mixture of aflatoxins to give total levels of 4 and 10 microg/kg. Recoveries for individual and total aflatoxins ranged from 92.7 to 110.3% for spiked samples. Based on results for spiked sesame paste (triplicates at two levels), the RSD for repeatability (RSD(r)) averaged 1.1% for total aflatoxins and 1.4% for aflatoxin B1. The method was demonstrated to be applicable to naturally contaminated samples of black and white sesame seeds obtained from local markets in China.     

Multiple mycotoxins analysis in animal feed with LC‐MS/MS: Comparison of extract dilution and immunoaffinity clean‐up

The aim of this study was a performance comparison of two clean‐up procedures (dilutions versus immunoaffinity columns) in the simultaneous determination of eight mycotoxins (aflatoxin B1, deoxynivalenol, fumonisin B1 & B2, ochratoxin A, toxin T‐2 & HT‐2 and zearalenone) in the animal feed. After extraction the analytes were separated on a Kinetex Biphenyl column with a gradient elution using methanol/0.01 M ammonium acetate as a mobile phase and analyzed with the LC‐MS/MS technique. Both of the procedures were validated by analysis of a series of spiked feed samples (n = 6) at three different concentration levels. Better signal to noise ratios were observed for immunoaffinity clean‐up. The recoveries of analyses were in the range 88–110% for the dilution procedure and 78–120% for the immunoaffinity clean‐up. The dilution procedure was more precise (coefficient of variation of the within‐laboratory reproducibility for it was 7.8–22.4% in comparison to 12–35.5% for the immunoaffinity clean‐up. The results show that both procedures fulfilled the requirements for mycotoxin analysis and can be used successfully in multi‐analyte determination. Although the dilution procedure shows better precision and trueness, the immunoaffinity clean‐up procedure can have advantages in more complex feed samples thanks to lower matrix effect and limits of detections.     

Molecularly imprinted polymers applied to the clean-up of zearalenone and α-zearalenol from cereal and swine feed sample extracts

A molecularly imprinted polymer prepared using 1-allylpiperazine (1-ALPP) as the functional monomer, trimethyltrimethacrylate (TRIM) as the crosslinker and the zearalenone (ZON)-mimicking template cyclododecanyl-2,4-dihydroxybenzoate (CDHB) has been applied to the clean-up and preconcentration of this mycotoxin (zearalenone) and a related metabolite, alpha-zearalenol (alpha-ZOL), from cereal and swine feed sample extracts. The extraction of ZON and alpha-ZOL from the food samples was accomplished using pressurized liquid extraction (PLE) with MeOH/ACN (50:50, v/v) as the extraction solvent, at 50 degrees C and 1500 psi. The extracted samples were cleaned up and preconcentrated through the MIP cartridge and analyzed using HPLC with fluorescence detection (lambda (exc)=271/ lambda (em)=452 nm). The stationary phase was a polar endcapped C18 column, and ACN/MeOH/water 10/55/35 (v/v/v, 15 mM ammonium acetate) at a flow rate of 1.0 mL min(-1) was used as the mobile phase. The method was applied to the analysis of ZON and alpha-ZOL in wheat, corn, barley, rye, rice and swine feed samples fortified with 50, 100 and 400 ng g(-1) of both mycotoxins, and it gave recoveries of between 85 and 97% (RSD 2.1-6.7%, n=3) and 87-97% (RSD 2.3-5.6%, n=3) for alpha-ZOL and ZON, respectively. The method was validated using a corn reference material for ZON.     

Co-isolation of deoxynivalenol and zearalenone with sol–gel immunoaffinity columns for their determination in wheat and wheat products

The paper describes a sample clean-up method for the co-isolation of deoxynivalenol (DON) and zearalenone (ZON), two mycotoxins naturally co-occurring in wheat. The method is based on immunoaffinity columns prepared by co-immobilising anti-DON and anti-ZON antibodies in a porous sol–gel glass. The main task in developing the method consisted in finding a loading medium allowing retention of both analytes as well as a common elution medium for the dissociation of both antigen–antibody complexes formed. This can be achieved by co-extracting DON and ZON with ACN–water (60:40, v/v), reducing the acetonitril concentration to 2.5% before loading an aliquot of the diluted sample extract onto the DON/ZON column. The columns are washed with 5 ml of MeOH–water (10:90, v/v) before DON and ZON are co-eluted with 4 ml of ACN–water (50:50, v/v). Concentrations of DON and ZON are determined with HPLC-UV and HPLC-fluorescence detection, respectively. The sample clean-up method was shown to be applicable to wheat and wheat products, e.g., cornflakes, milk wheat mash and rusk. Spiking experiments (spike level 500 μg DON/kg and 50 μg ZON/kg) resulted in recovery rates from 82% to 111%.     

Selective trace enrichment by immunoaffinity capillary electrochromatography on-line with capillary zone electrophoresis - laser-induced fluorescence

Limited by the lack of a sensitive, universal detector, many capillary-based liquid-phase separation techniques might benefit from techniques that overcome modest concentration sensitivity by preconcentrating large injection volumes. The work presented employs selective solid-phase extraction by immunoaffinity capillary electrochromatography (IACEC) to enhance detection limits. A model analyte, fluorescein isothiocyanate (FITC) biotin, is electrokinetically applied to a capillary column packed with an immobilized anti-biotin-IgG support. After selective extraction by the immunoaffinity capillary, the bound analyte is eluted, migrates by capillary zone electrophoresis (CZE), and is detected by laser-induced fluorescence. The column is regenerated and reused many times. We evaluate the performance of IACEC for selective trace enrichment of analytes prior to CZE. The calibration curve for FITC-biotin bound versus application time is linear from 10 to 300 seconds. Recovery of FITC-biotin spiked into a diluted urinary metabolites solution was 89.4% versus spiked buffer, with a precision of 1.8% relative standard deviation (RSD).     

Reversed-phase liquid chromatographic method for the determination of ochratoxin A in wine

In this paper, we propose a new, rapid, highly sensitive and reproducible RP-HPLC-FLD method for the detection of ochratoxin A (OTA) in wine, by directly injecting the liquid in the chromatographic system without any extraction or clean-up. An alkaline mobile phase (NH4Cl:CH-CN 85:15 (v/v), 20 mM, pH 9.8) was used to obtain a distinct fluorescence enhancement. This improvement allows to reach, without an immunoaffinity clean-up or concentration, a detection limit of 0.05 ng/ml, which is similar to those commonly obtained after immunoaffinity purification and acidic elution. The method was statistically validated and directly applied to a series of wine samples.     

Determination of quinolones in chicken tissues by liquid chromatography with ultraviolet absorbance detection

This paper presents an analytical method for the determination of quinolones in chicken tissues. The procedure involves pre-treatment by solid-phase extraction (SPE) and subsequent liquid chromatography (LC) with UV absorbance detection. Different SPE disposable cartridges and extractants of the tissue samples were tested, and various columns were systematically tested. The mobile phase was composed of acetonitrile and citric buffer at pH 4.5, with an initial composition of acetonitrile-water (12:88, v/v) and using linear gradient elution. Recoveries were 66-91% in the concentration range 30-300 microg kg(-1). The detector response was linear in this range. The limits of detection were 16-30 microg kg(-1). These values were lower than the maximum residue limits established by the European Union.     

Analysis of abamectin residues in avocados by high-performance liquid chromatography with fluorescence detection

In this work an analytical method for the determination of abamectin residues in avocados is developed using high-performance liquid chromatography (HPLC) with fluorescence (FL) detection. A pre-column derivatization with trifluoroacetic anhydride (TFAA) and N-methylimidazole (NMIM) was carried out. The mobile phase consisted of water, methanol and acetonitrile (5:47.5:47.5 v/v/v) and was pumped at a rate of 1 mL/min (isocratic elution). The fluorescence detector was set at an excitation wavelength of 365 nm and an emission wavelength of 470 nm. Homogenized avocado samples were extracted twice with acetonitrile:water 8:2 (v/v) and cleaned using C(18) solid-phase extraction (SPE) cartridges. Recovery values were in the range 87-98% with RSD values lower than 13%. The limits of detection (LODs) and quantification (LOQs) of the whole method were 0.001 and 0.003 mg/kg, respectively. These values are lower than the maximum residue limit (MRL) established by the European Union (EU) and the Spanish legislation in avocado samples.     

Stability-indicating high-performance liquid chromatographic assay of atorvastatin with fluorescence detection

The purpose of this work was to develop a sensitive, selective, and validated stability-indicating high-performance liquid chromatographic (LC) assay of atorvastatin (ATV) in bulk drug and tablet form. ATV was subjected to different stress conditions, including UV light, oxidation, acid-base hydrolysis, and temperature. ATV and its degradation products were analyzed on an Agilent Zorbax XDB C18 column using isocratic elution with acetonitrile-0.02 M sodium acetate, pH 4.2 (45 + 55, v/v) for 25 min. The samples were monitored with fluorescence (FL) detection at 282 nm (excitation)/400 nm (emission). The response ratio of FL to UV detection (at 247 nm) for ATV was 1.66. The method showed good resolution of ATV from its decomposition products. The photodegradation products were separated by silica gel thin-layer chromatography using double development with ethyl acetate-n-hexane-glacial acetic acid-methanol (40 + 55 + 0.5 + 4.5, v/v/v/v) followed by (39 + 55 + 0.5 + 5.5, v/v/v/v), and confirmed by LC-FL analysis. The FL response was linear over the investigated range for ATV. The linear range was 10-1200 ng/injection, and the limit of quantitation was 2.0 ng/injection.     

免疫亲和柱净化/柱前衍生化-高效液相色谱荧光检测法测定粮谷中的T-2毒素

建立了免疫亲和柱净化/柱前衍生化-高效液相色谱荧光检测器测定粮谷中T-2毒素含量的方法。样品经甲醇-水(体积比为80∶20)混合溶剂提取,通过免疫亲和柱(IAC)净化,以氰酸蒽(1-AN)为衍生化试剂、4-二甲基氨基吡啶(DMAP)为催化剂进行衍生,以ZORBAX Eclipse XDB-C18 柱为分离柱,乙腈-水(体积比为80∶20)为流动相进行高效液相色谱分离及荧光检测，荧光检测的激发波长为381 nm，发射波长为470 nm。T-2毒素的质量浓度为0.01~1.5 mg/L时与峰高呈良好的线性,相关系数为0.9985。在0.01~1.5 μg/g添加水平下，回收率为79.7%~94.5%,相对标准偏差小于7%；检出限（S/N＝3）为0.01 μg/g。该方法净化效果好,灵敏度高,操作简便快速。     

免疫亲和柱净化-高效液相色谱法同时检测鸡蛋中6种玉米赤霉醇类化合物残留量

建立了鸡蛋中6种玉米赤霉醇类化合物(α-玉米赤霉醇、β-玉米赤霉醇、α-玉米赤霉烯醇、β-玉米赤霉烯醇、玉米赤霉酮和玉米赤霉烯酮)残留量的免疫亲和柱净化-高效液相色谱检测方法。样品酶解后用叔丁基甲醚提取、氢氧化钠反萃取,经免疫亲和柱富集和净化后,采用高效液相色谱-紫外检测器进行测定。色谱柱: Agilent Eclipse XDB-C18(150 mm×4.6 mm, 3.5 μm);流动相: 甲醇-乙腈-水(50:15:35, v/v/v);流速: 1.0 mL/min;检测波长: 270 nm。结果表明,6种目标物在0.01～0.2 mg/L范围内线性关系良好,相关系数(r)≥0.9998,检出限(LOD,S/N≥3)为1.0 μg/kg,平均回收率为73.2%～95.7%,相对标准偏差小于8%。该方法灵敏度高、重现性好,适用于鸡蛋样品中痕量玉米赤霉醇类药物残留的测定。     

Determination of atropine (hyoscyamine) sulfate in commercial products by liquid chromatography with UV absorbance and fluorescence detection: multilaboratory study

A liquid chromatographic (LC) method with 2 detection systems for determining atropine (hyoscyamine) sulfate in commercial products was tested in a multilaboratory study. Depending on the type of product, sample solutions are prepared in methanol or methanol-water (1 + 1). The standard solution contains about 1.0 mg atropine sulfate/100 mL and is prepared in the same solvent used in sample preparation. LC separations are performed on a 7.5 cm Novapak silica column. The mobile phase is prepared by mixing 970 mL methanol with 30 mL of a 1% aqueous solution of 1-pentanesulfonic acid, sodium salt. Detection is by 2 systems, UV absorbance detection at 220 nm and fluorescence detection with excitation at 255 nm and emission at 285 nm. The injection volume is 100 or 200 microL. The following materials were used for the study: 2 separate samples of tablets labeled to contain 0.4 mg atropine sulfate, 2 separate samples of extended-release tablets labeled to contain 0.375 mg hyoscyamine sulfate, one sample of atropine sulfate injection labeled to contain 2 mg/mL, and one sample of 1% (v/v) atropine sulfate ophthalmic. Eight participants analyzed 2 separate portions of the 6 samples by both detection systems. A ninth participant analyzed the samples in duplicate but only by UV absorbance detection because of the unavailability of a fluorescence detector. The relative standard deviation (RSD) between laboratories ranged from 1.4 to 3.3% for samples of tablets and injections but higher for ophthalmic solutions (5.1-5.2%). A linearity study was conducted in the originating laboratory before the multilaboratory study with 5 solutions ranging in concentration from 0.80 to 1.20 mg atropine sulfate in 100 mL. Average recoveries were 100.0% by UV absorbance detection and 99.9% by fluorescence detection; the RSDs were 1.1 and 1.2%, respectively.     

Determination of ochratoxin A in beer marketed in Spain by liquid chromatography with fluorescence detection using lead hydroxyacetate as a clean-up agent

A new sample treatment for liquid chromatographic analysis of ochratoxin A (OTA) in beer is proposed. Degassed beer is mixed with lead hydroxyacetate, which precipitates some bulk components but does not remove OTA. The precipitate is separated and the acidified liquid is extracted with chloroform. The solvent is evaporated and the residue is dissolved in mobile phase (acetonitrile-water, 40:60, v/v; acidified at pH 3.0 with phosphoric acid) and separated by liquid chromatography using fluorescence detection. The limit of detection was 0.005 ng/ml. The average recovery rate and the average RSD of recovery in the spiking level range 0.01-0.5 ng/ml were 95.5% and about 5%, respectively. The method is cheaper that other alternative ones using immunoaffinity columns or other solid-phase extraction cleanup:The separation was optimised with regard to composition and flow of the mobile phase and no interference from the matrix was found. The method was applied to 88 samples of beer (domestic and imported) marketed in Spain. OTA was detected in 82.9% of them. The range for positive samples was 0.007-0.204 ng of OTA/ml.     

鸡肉中11种喹诺酮类药物多残留的高效液相色谱检测

建立了用荧光检测器同时测定11种喹诺酮类药物(包括诺氟沙星、培氟沙星、环丙沙星、恩诺沙星、氧氟沙星、达氟沙星、洛美沙星、二氟沙星、沙拉沙星、恶喹酸和氟甲喹)在鸡肉中的多残留的高效液相色谱检测方法。鸡肉样品用10%三氯乙酸-乙腈(体积比为7∶3)提取两次并稀释,随后用反相固相萃取柱净化。采用Hypersil BDS-C18色谱柱分离,以乙腈和水为流动相梯度洗脱,荧光检测器用程序编程检测波长检测。11种喹诺酮类药物标准曲线的线性范围为5～1200 μg/L,相关系数大于0.998。在高、中、低三个添加水平下的回收率为56%～119%,批内相对标准偏差为0.4%～16.1%,批间相对标准偏差为1.4%～23.0%。检出限和定量限分别为1～23 μg/kg和4～40 μg/kg。该方法快速、灵敏,达到了兽药残留检测的要求。     

Quantitative determination of coenyzme Q10 by liquid chromatography and liquid chromatography/mass spectrometry in dairy products

The dietary sources of CoQ10 and the evaluation of CoQ10 in dairy products were characterized. For quantitation of CoQ10 in food samples, 2 liquid chromatography (LC) methods with UV and mass spectrometry (MS) detections were developed. LC with UV detection was performed at 25 degrees C on a Hyperclone ODS 5 microm 150 x 4.6 mm column with mobile phase consisting of methanol-ethanol-2-propanol (70 + 15 + 15, v/v/v). Flow rate was 1.0 mL/min. Retention time of CoQ10 was 10.9 +/- 0.1 min. The method was sensitive [limit of detection (LOD) = 0.2 mg/kg], reproducible [relative standard deviation (RSD) = 3:0%), and linear up to 25 mg/kg (R > 0.999). LC/MS analysis was performed on a LUNA C18 3 microm, 150 x 4.6 mm column, using mobile phase consisting of ethanol-dioxane-acetic acid (9 + 1 + 0.01, v/v/v), flow rate was 0.6 mL/min, and the retention time of CoQ10 was 4.1 +/- 0.1 min. Identification and quantitation were performed with a Finnigan-LCQ mass detector in positive atmospheric pressure chemical ionization mode. Mass spectra were obtained in selected-ion monitoring mode; molecular mass (M+H)+ m/z 863.4 +/- 1 was used for quantitative determination. MS detection is more sensitive than UV detection (LOD = 0.1 mg/kg), less reproducible (RSD = 4.0%), and linear in selected range. Analytical recoveries are 75-90% and depend on the ratio between the amount of fat in the matrix and the concentration of CoQ10 in the sample. Some soybean milk products were analyzed together with different cow, goat, and sheep milk products. Concentrations obtained with LC and LC/MS were compared with a few accessible results available from the literature. Concentrations varied from 0 ppm in soybean milk to nearly 2 ppm in fresh milk from local farms.     

Liquid chromatographic-tandem mass spectrometric determination of nonylphenol polyethoxylates and nonylphenol carboxylic acids in surface water

Various liquid chromatographic methods used in the analysis of mycotoxins (zearalenone, trichothecenes and fumonisins) produced by Fusarium species were compared in this work. The results demonstrate the suitability of modern clean-up procedures employing multifunctional MycoSep and immunoaffinity columns although these methods are more expensive than conventional methodologies for clean-up. HPLC with both fluorescence and photodiode array detection is a suitable technique for the analysis of toxic secondary metabolites produced by Fusarium species; different derivatisation strategies have been studied to improve the sensitivity of the technique because of the low concentration of these metabolites in contaminated food. The utility of the proposed methodology was assessed in cereal cultures of various Fusarium strains.