柱后衍生-高效液相色谱法测定玉米中伏马菌素B1和B2

建立了邻苯二甲醛(OPA)柱后衍生-高效液相色谱测定玉米中伏马菌素B1和B2(FB1和FB2)的方法。采用ZORBAX SB-C18色谱柱,以0.1 mol/L磷酸二氢钠溶液(pH 3.3)-甲醇为流动相,梯度洗脱。流动相流速为0.8 mL/min,柱温40 ℃;衍生剂的流速为0.4 mL/min,衍生温度为室温。实验对衍生剂缓冲液的pH、衍生剂的浓度和流速、激发和发射波长等重要条件进行了优化。结果表明,衍生剂的pH在10.5、OPA的质量浓度为2 g/L、流速为0.4 mL/min、激发波长335 nm、发射波长440 nm时测定效果良好,FB1、FB2在0.2～20 mg/L范围内线性关系良好,相关系数大于0.999; FB1和FB2的检出限均为0.02 mg/kg;在0.1～ 4.0 mg/kg范围内,3个添加水平的平均回收率为82.5%～89.8%。该方法精确、简单、快速,适合玉米中FB1和FB2的测定。     

Determination of fumonisins B1 and B2 in corn-based foods for infants and young children by LC with immunoaffinity column cleanup: interlaboratory validation study

A liquid chromatographic method for the determination of fumonisins B1 (FB1) and B2 (FB2) in corn-based foods for infants and young children was subjected to an interlaboratory validation study involving 11 laboratories. Five blind duplicate sample pairs of each matrix were analyzed to establish the accuracy, repeatability, and reproducibility of the method. Mass fractions in the baby food samples ranged from 89.1 to 384.4 microg/kg FB1 and from 22.5 to 73.6 microg/kg FB2. The method involved a warm extraction with citrate phosphate buffer-methanol-acetonitrile (50 + 25 + 25, v/v/v), a cleanup through an immunoaffinity column, and an end-determination of fumonisins by LC after automated precolumn derivatization with o-phthaldialdehyde reagent. RSDs for within-laboratory repeatability (RSDr) ranged from 6.8 to 23.5% for FB1 and 7.6 to 22.9% for FB2. RSDs for between-laboratory reproducibility (RSDR) ranged from 15.4 to 26.2% for FB1 and 21.6 to 36.3% for FB2. Mean FB1 recoveries from baby foods spiked at 100.0 and 250.0 microg/kg were 89 and 96%, respectively; for FB2 spiked foods at 25.0 and 62.5 microg/kg recoveries were 90 and 85%, respectively. HorRat values ranged from 0.8 to 1.2 for FB1, whereas for FB2 they ranged from 0.9 to 1.4 when calculated according to Horwitz, and from 1.0 to 1.7 when calculated according to Thompson, indicating an acceptable among-laboratory precision for all matrixes (HorRat values <2).     

Analysis of fumonisins B1 and B2 in spices and aromatic and medicinal herbs by HPLC-FLD with on-line post-column derivatization and positive confirmation by LC-MS/MS

Fumonisins are produced by the fungus Fusarium verticillioides, which are known to cause fatal diseases in some animals and humans. Here, we describe a sensitive, reproducible and reliable analytical method for the quantitative determination of fumonisins B(1) (FB(1)) and B(2) (FB(2)) in 112 spices and aromatic and medicinal herbs marketed in China. This method is based on high performance liquid chromatography and fluorescence detection (HPLC-FLD) coupled to a new on-line post-column derivatization using ortho-phthaldialdehyde with 2-mercaptoethanol and immunoaffinity column clean-up. Under the optimized experimental conditions, a complete separation of FB(1) and FB(2) was obtained using a Synergi C(18) column and a gradient elution at 0.8 mL min(-1) with methanol and 0.1 M phosphate buffer at pH 3.15. The limits of detection for FB(1) and FB(2) were both 40 μg kg(-1). Good recoveries were found for spiked samples with FB(1) and FB(2), ranging from 82.34% to 98.16% for FB(1) and from 72.58% to 97.10% for FB(2), with relative standard deviation (RSD) < 7.0%. 5 spices, 11 aromatic herbs and 96 medicinal herbs including 93 normal samples and 19 visibly moldy samples, which were spoiled artificially, were analyzed. The results showed that 8 (42.1%) visibly moldy samples and 8 (8.6%) normal samples were contaminated with FB(1) at mean contents of 129.0 and 165.9 μg kg(-1), and with FB(2) at 1745.0 and 256.8 μg kg(-1), respectively. Positive confirmation of detected samples was performed by liquid chromatography tandem electrospray ionization mass spectrometry (LC-ESI-MS/MS), using a triple quadrupole analyzer and operated in the multiple reaction monitoring mode.     

Determination of fumonisins B1 and B2 in corn and corn flakes by liquid chromatography with immunoaffinity column cleanup: collaborative study.

A liquid chromatographic (LC) method for the determination of fumonisins B1 (FB1) and B2 (FB2) in corn and corn flakes was collaboratively studied by 23 laboratories, which analyzed 5 blind duplicate pairs of each matrix to establish the accuracy, repeatability, and reproducibility characteristics of the method. Fumonisin levels in the corn ranged from <0.05 (blank) to 1.41 microg/g for FB1 and from <0.05 to 0.56 microg/g for FB2, whereas in the corn flakes they ranged from <0.05 to 1.05 microg/g for FB1 and from <0.05 to 0.46 microg/g for FB2. The method involved double extraction with acetonitrile-methanol-water (25 + 25 + 50), cleanup through an immunoaffinity column, and LC determination of the fumonisins after derivatization with o-phthaldialdehyde. Relative standard deviations for the within-laboratory repeatability (RSDr) of the corn analyses ranged from 19 to 24% for FB1 and from 19 to 27% for FB2; for the corn flakes analyses, RSDr ranged from 9 to 21 % for FB1 and from 8 to 22% for FB2. Relative standard deviations for the between-laboratories reproducibility (RSDR) of the corn analyses ranged from 22 to 28% for FB1 and from 22 to 30% for the FB2; for corn flakes analyses, RSDR ranged from 27 to 32% for FB1 and from 26 to 35% for FB2. Mean recoveries of FB1 and FB2 from corn spiked with FB1 at 0.80 microg/g and with FB2 at 0.40 microg/g were 76 and 72%, respectively; for corn flakes spiked at the same levels recoveries were 110 and 97% for FB1 and FB2, respectively. HORRAT ratios for the analyses of corn ranged from 1.44 to 1.53 for FB1 and from 0.96 to 1.48 for FB2, whereas for corn flakes they ranged from 1.60 to 1.82 for FB1 and from 1.39 to 1.68 for FB2.     

免疫亲和柱净化-柱后光化学衍生-高效液相色谱法同时检测粮谷中的黄曲霉毒素、玉米赤霉烯酮和赭曲霉毒素A

建立了同时检测粮谷中黄曲霉毒素(B1、B2、G1和G2)、玉米赤霉烯酮和赭曲霉毒素A的免疫亲和柱净化-柱后光化学衍生-高效液相色谱方法。样品经过甲醇-水(体积比为80∶20)提取,通过免疫亲和柱富集和净化,采用Waters Nova-Pak色谱柱(3.9 mm i.d.×150 mm,4 μm),以甲醇、乙腈和1%的磷酸溶液为流动相,梯度洗脱,柱后光化学衍生、改变波长荧光检测。黄曲霉毒素(B1、B2、G1和G2)、玉米赤霉烯酮和赭曲霉毒素A检出限分别为0.24,4.0和0.5 μg/kg,标准曲线的线性范围分别为0.24~6.0,4.0~100.0和0.5~40.0 μg/L;在小麦、玉米、黑麦样品中,平均加标回收率为70.8% ~94.0%,相对标准偏差为2.79% ~9.38%。     

Determination of fumonisins B1 and B2 in Portuguese maize and maize-based samples by HPLC with fluorescence detection

Fumonisins B₁ (FB₁) and fumonisin B₂ (FB₂) are the main members of a family of mycotoxins produced by Fusarium verticillioides, Fusarium proliferatum, and other fungi species of the section Liseola. The present work shows the results of comparative studies using two different procedures for the analysis of fumonisins in maize and maize-based samples. The studied analytical methods involve extraction with methanol/water, dilution with PBS, and clean-up through immunoaffinity columns. Two reagents (o-phthaldialdehyde and naphthalene-2,3-dicarboxaldehyde) were studied for formation of fluorescent derivatives. The separation and identification were carried out by high-performance liquid chromatography with fluorescence detection. The optimized method for analysis of fumonisins in maize involved extraction with methanol/water (80:20), clean-up with an immunoaffinity column, and derivatization with naphthalene-2,3-dicarboxaldehyde (NDA). The limit of detection was 20 μg kg⁻¹ for FB₁ and 15 μg kg⁻¹ for FB₂. Recoveries of FB₁ and FB₂ ranged from 79% to 99.6% for maize fortified at 150 μg kg⁻¹ and 200 μg kg⁻¹, respectively, with within-day RSDs of 3.0 and 2.7%. The proposed method was applied to 31 samples, and the presence of fumonisins was found in 14 samples at concentrations ranging from 113 to 2,026 μg kg⁻¹. The estimated daily intake of fumonisins was 0.14 μg kg⁻¹ body weight per day.     

Development of a liquid chromatography/tandem mass spectrometry method for the quantification of fumonisin B1, B2 and B3 in cornflakes

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of fumonisin B1 (FB1), B2 (FB2) and B3 (FB3) in cornflakes is described. During method development, special attention was paid to the selection of a suitable internal standard (IS) in order to offer a good alternative for deuterated FB1. In this respect, the C12-sphinganine analogue (2S,3R)-2-aminododecane-1,3-diol was chosen because of its structural similarity to the fumonisin backbone and its chromatographic elution between the target analytes. For the extraction of the fumonisins from the cornflakes matrix, MeOH/H2O (adjusted to pH 4 with 0.1 M HCl; 70:30, v/v), ACN/MeOH/H(2)O (25:25:50, v/v/v) and acidified ACN/MeOH/H2O (25:25:50, v/v/v; pH 4) were evaluated. Preference was given to acidified MeOH/H2O (70:30, v/v) with mean recoveries (n=12) for FB1, FB2 and FB3 of, respectively, 84+/-10, 78+/-7 and 85+/-9%. Cleanup was performed using immunoaffinity columns (FumoniTest, VICAM). The chromatography was performed under isocratic conditions at a flow of 0.3 mL min-1 with a mobile phase consisting of ACN/H2O (60:40, v/v) containing 0.3% formic acid. The mass spectrometer was operated in the positive electrospray ionization (ESI+) mode using multiple reaction monitoring (MRM). An intralaboratory validation was conducted with fortified samples determining limits of detection (LOD), limits of quantification (LOQ), precision, trueness, specificity and measurement uncertainty. The LOD concentrations for FB1, FB2 and FB3 were 20, 7.5 and 12.5 microg/kg. The LOQs were 40 microg/kg for FB1, 15 microg/kg for FB2 and 25 microg/kg for FB3. The coefficients of variation (CVs) under repeatability conditions varied from 11 to 13% for FB1, from 9 to 14% for FB2 and from 7 to 10% for FB3. Under within-laboratory reproducibility conditions, the CVs ranged from 12 to 17% for FB1, from 9 to 16% for FB2 and from 7 to 13% for FB3. The percent bias for FB1 varied from -12 to -10%, while for FB2 and FB3 bias ranged, respectively, from -4 to -2% and from -12 to -5%. The expanded measurement uncertainties for FB1, FB2 and FB3 were, respectively, 19, 18 and 22%.     

Analysis of fumonisins B(1), B(2) and B(3) in corn-based baby food by pressurized liquid extraction and liquid chromatography/tandem mass spectrometry

A sensitive and reliable method using pressurized liquid extraction (PLE) and liquid chromatography (LC)/electrospray ionization (ESI) tandem mass spectrometry with a triple quadrupole (QqQ) analyzer has been developed for the analysis of fumonisin B(1) (FB(1)), fumonisin B(2) (FB(2)) and fumonisin B(3) (FB(3)) in corn-based baby foods. Influence of several extraction parameters that affect PLE efficiency such as temperature, pressure, solvent extraction, number of cycles and dispersant/clean-up agents were studied. The selected PLE operating method was: 3g of sample was packed into 11 ml stainless-steel cell and fumonisins were extracted with methanol at 40 degrees C, 34 atm in one cycle of 5 min at 60% flush. The analytes were ionized in ESI operating with positive ion mode and identified by selecting two monitoring transitions, permitting quantification and confirmation in a single injection. Recoveries ranged from 68% to 83% at fortification levels of 200 microg kg(-1) with relative standard deviation (RSD) from 4% to 12%. The limits of quantification were from 2 microg kg(-1) for FB(1) and FB(2), and 5 microg kg(-1) for FB(3), which are below the maximum residue level established by the European Union legislation in infant formulas. The proposed method was successfully applied to the analysis of twenty seven samples of baby food products collected from different markets, and one positive sample with a content of 15.9 microg kg(-1) for FB(1), 9.2 microg kg(-1)for FB(2) and 5.8 microg kg(-1) for FB(3) was obtained. Given the simplicity and potential of the proposed procedure, its application for safety control is recommended.     

液相色谱-串联质谱法测定牛奶中伏马菌素FB1和FB2及其水解代谢物

建立了MAX混合阴离子固相萃取柱净化-高教液相色谱-串联质谱法测定牛奶中伏马菌素FB1和FB2及其水解代谢产物HFB1和HFB2的方法.牛奶样品经水稀释后,经MAX柱直接净化,甲醇洗脱得到FB1和FB2,经液相色谱-串联质谱负离子扫描测定,1％乙酸甲醇洗脱得到HFB1和HFB2,经液相色谱-串联质谱正离子扫描测定.结果表明,添加浓度为0.1～5.0 μg/L,牛奶中FB1和FB2及其水解代谢产物的回收率为76.4％～92.3％;相对标准偏差(RSD,n=5)为5.9％～12.5％;方法检出限(LOD)均为0.03 μg/L;定量限(LOQ)均为0.1 μg/L.本方法操作简单,灵敏度、回收率和重复性均良好.     

Simultaneous determination of fumonisins B1, B2 and B3 contaminants in maize by ultra high-performance liquid chromatography tandem mass spectrometry

The present work developed an analytical method for simultaneous determination of fumonisins B(1), B(2) and B(3) residues in maize by ultra high-performance liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) under the multiple reaction monitoring (MRM) mode, and especially focused on the optimization of extraction, clean-up, UHPLC separation and MS/MS parameters. The method involves addition of fumonisins isotope internal standards, extraction with a mixture of acetonitrile and water and clean-up with solid-phase extraction (SPE) cartridges before UHPLC-MS/MS analysis. A single-laboratory method validation was conducted by testing three different spiking levels for repeatability and recovery according to International Union of Pure and Applied Chemistry (IUPAC) guidelines. The LOQ of FB(1), FB(2) and FB(3) were 1.50, 1.65 and 0.4 μg kg(-1), respectively, which were lower than the criteria of EU, USA and other countries regarding minimum residue limits of fumonisins in foods including baby foods and feedstuffs. Recoveries of three fumonisins ranged from 80.9% to 97.0% with RSD values of 2.4-11.1%.The advantages of this method include simple pretreatment, rapid determination and high sensitivity, and it fulfills the requirements for food analysis with respect to minimum residue limits of fumonisins in various countries.     

Robotic automated clean-up for detection of fumonisins B1 and B2 in corn and corn-based feed by high-performance liquid chromatography

A high-performance liquid chromatography (HPLC) system with fluorescence detection and an automated on-line solid-phase extraction procedure for fumonisins B1 and B2 in corn and corn-based products is described. Different amounts of strong anion-exchange, C18 and end-capped C18 (C(18 ec)) silicas were tested for sample clean-up. Various HPLC parameters were analyzed. The best methodology was found to be extraction with acetonitrile-water and clean up on C(18 ec) disposable extraction cartridges. The system has the advantage of running in an unattended mode of operation and allows processing of 40 samples without system refuel, performing clean-up, o-phthaldialdehyde derivatization, injection and fumonisin detection by fluorescence detection linked to a computer integrator for automated data processing. Recoveries were performed with corn and corn-based feed samples (n=3) spiked with 0.1, 0.5, 1.0, 5.0 and 10 microg/g. Average recoveries for corn and corn-based feed were, respectively, 92.6 and 88.3% with relative standard deviations (RSDs) of 5.04 and 6.22%, for fumonisin B1 and 91.2 and 89.0% with RSDs of 5.84 and 7.88% for fumonisin B2. Detection limits (S/N=3) for corn and corn-based feed were approximately 0.03 microg/g for fumonisin B1 and 0.05 microg/g for fumonisin B2     

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.     

Determination of fumonisins B1 and B2 in corn by liquid chromatography/mass spectrometry with immunoaffinity column cleanup: single-laboratory method validation

A single-laboratory method validation was conducted to establish the effectiveness of an immunoaffinity column cleanup procedure followed by liquid chromatography/mass spectrometry (LCIMS) for the determination of fumonisins B1 and B2 (FBI + FB2) in corn. The test portion is extracted with acetonitrile-methanol-water (25 + 25 + 50). The extract is filtered, diluted with phosphate-buffered saline solution, and applied to an immunoaffinity column. FB1 + FB2 are removed with methanol and directly determined by reversed-phase LC with MS detection using selected-ion monitoring of 2 characteristic ions in each case. Test portions of blank corn samples were spiked with a mixture of FB1 + FB2 to give total levels of 200 and 500 ng/g, respectively. Recoveries of both FB1 and FB2 from spiked samples averaged 90.4-101%. Based on results for spiked raw corn (triplicates at 2 levels), the relative standard deviation for repeatability ranged from 2.8 to 7.1%. The accuracy of the method was demonstrated by analysis of Food Analysis Performance Assessment Scheme (FAPAS) test material. The method was also applied to a small survey of processed corn products such as corn chips, cornflakes, and popcorn.     

A reversed phase high performance liquid chromatography method for the determination of fumonisins B1 and B2 in food and feed using monolithic column and positive confirmation by liquid chromatography/tandem mass spectrometry

The development of a reversed phase high performance liquid chromatography fluorescence method for the determination of the mycotoxins fumonisin B(1) and fumonisin B(2) by using silica-based monolithic column is described. The samples were first extracted using acetonitrile:water (50:50, v/v) and purified by using a C(18) solid phase extraction-based clean-up column. Then, pre-column derivatization for the analyte using ortho-phthaldialdehyde in the presence of 2-mercaptoethanol was carried out. The developed method involved optimization of mobile phase composition using methanol and phosphate buffer, injection volume, temperature and flow rate. The liquid chromatographic separation was performed using a reversed phase Chromolith(?) RP-18e column (100 mm × 4.6 mm) at 30 °C and eluted with a mobile phase of a mixture of methanol and phosphate buffer pH 3.35 (78:22, v/v) at a flow rate of 1.0 mL min(-1). The fumonisins separation was achieved in about 4 min, compared to approximately 20 min by using a C(18) particle-packed column. The fluorescence excitation and emission were at 335 nm and 440 nm, respectively. The limits of detections were 0.01-0.04 μg g(-1) fumonisin B(1) and fumonisin B(2), respectively. Good recoveries were found for spiked samples (0.1, 0.5, 1.5 μg g(-1) fumonisins B(1) and B(2)), ranging from 84.0 to 106.0% for fumonisin B(1) and from 81.0 to 103.0% for fumonisin B(2). Fifty-three samples were analyzed including 39 food and feeds and 14 inoculated corn and rice. Results show that 12.8% of the food and feed samples were contaminated with fumonisin B(1) (range, 0.01-0.51 μg g(-1)) and fumonisin B(2) (0.05 μg g(-1)). The total fumonisins in these samples however, do not exceed the legal limits established by the European Union of 0.8 μg g(-1). Of the 14 inoculated samples, 57.1% contained fumonisin B(1) (0.16-41.0 μg g(-1)) and fumonisin B(2) (range, 0.22-50.0 μg g(-1)). Positive confirmation of selected samples was carried out using liquid chromatography-tandem mass spectrometry, using triple quadrupole analyzer and operated in the multiple reaction monitoring mode.     

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。     

Analysis of phenols in water by high-performance liquid chromatography using coumarin-6-sulfonyl chloride as a fluorogenic precolumn label

A simple, sensitive and rapid reversed-phase high-performance liquid chromatography (RP-HPLC) method is proposed for the analysis of some environmentally important phenols in water. The use of coumarin-6-sulphonyl chloride (C6SCl) as a fluorescence-labeling reagent has been investigated. The compound reacts with phenols within 20 min under mild conditions (ambient temperature, pH 9.0) to give sulphonates that can be separated by RP-HPLC employing fluorescence detection at lambda(ex) = 360 and lambda(em) = 460 nm. The optimum conditions for fluorescence, derivatization and chromatographic separation have been established and detection limits in the range 0.1-0.9 microg l(-1) were obtained for the studied compounds. The calibration curves were linear for the range 6-200 microg l(-1) for phenol, 3-200 microg l(-1) for 2-chlorophenol, 4-chlorophenol and 2,3,5-trichlorophenol and for the range of 3-100 microg l(-1) for 2,3-dichlorophenol and 3,5-dichlorophenol. The practical applicability of the method to environmental samples was demonstrated by analyzing drinking and industrial water samples spiked with the phenolic compounds.     

Determination of type B fumonisin mycotoxins in maize and maize-based products by liquid chromatography/tandem mass spectrometry using a QqQlinear ion trap mass spectrometer

A sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for determining the type B fumonisin mycotoxins in corn-based foodstuffs is described. Fumonisins FB1 and FB2 were extracted from a 1 g sample by homogenization with acetonitrile/water (75:25, v/v, 50 mmol/L formic acid, 25 mL final volume) and the extract was defatted on C18 phase. Volumes of 5 mL of crude extracts were cleaned up on Carbograph-4 cartridges. The final solution was analyzed by HPLC with electrospray ionization mass spectrometry in positive ion mode using multiple reaction monitoring with a QqQ linear ion trap mass spectrometer. Recoveries for spiked corn-based foodstuffs ranged from 91-105% (RSD% < or =8%), and method detection limits were < or =2 ng/g for FB1 and < or =1 ng/g for FB2. Two different spiking levels were tested (5000 and 100 ng/g for FB1, 1000 and 20 ng/g for FB2). Quantitation was achieved by an external calibration procedure using matrix-matched standards, with diclofenac added post-cleanup as internal standard for the LC/MS/MS analyses. Calibration curves showed linearity in the concentration range 0.005-5 ng/microL of final extract (0.992 < or = R2< or =0.995). Two other fumonisins, FB3 and FB4, were identified in naturally contaminated samples of corn meal using an information-dependent acquisition protocol that looped three experiments, including neutral loss scan, enhanced resolution scan, and enhanced product ion scan. FB3 and FB4 quantitation was estimated as peak area ratios relative to the FB2 response in view of the lack of both standards. This work also includes an application of the present LC/MS/MS method to some maize and maize-based product samples (corn meal, cornflakes and popcorn) collected from Italian stores. FB1 and FB2 contamination levels exceeding the European Union recommendation were found in 8 out of 15 corn meal samples.     

Determination of fumonisin B1 in botanical roots by liquid chromatography with fluorescence detection: single-laboratory validation

The accuracy, repeatability, and reproducibility characteristics of a published method for measuring levels of fumonisin B1 (FB1) in botanical root products were determined by an AOAC single-laboratory validation procedure. Replicates of 10 test portions of each powdered root product (black cohosh, echinacea, ginger, ginseng, valerian, dong quai, and turmeric) at each spiking level (FB1 at 0, 50, 100, and 200 ng/g) were analyzed on 3 separate days. Test samples were extracted with methanol-acetonitrile-water (25 + 25 + 100, v/v/v). The extracts were centrifuged, the supernatants diluted with phosphate-buffered saline (PBS) containing 1% Tween 20 and filtered, and the filtrates applied to an immunoaffinity column containing antibodies specific for fumonisins. After the column was washed sequentially with PBS and water, the toxin was eluted from the column with 80% methanol, and the eluate dried by lyophilization. The residue was reconstituted with 50% acetonitrile. FB1 was derivatized with a mixture of o-phthaldialdehyde and mercaptoethanol by using an LC autoinjector. Separations were performed with an RP-LC column, and the FB1 derivative was quantified by fluorescence detection. All root products were found to contain FB1 at <10 ng/g. Average within- and between-day recoveries of FB1 from the botanical roots ranged from 67 to 95% and from 68 to 100%, respectively. Total RSD values for within- and between-day repeatability ranged from 5.5 to 26.4%. HorRat values were <1.3 for all of the matrixes examined. The method meets the AOAC method performance criteria at levels of >50 ng/g for the seven botanical roots tested.     

Determination of fumonisins B1 and B2 in various maize products by a combined SAX + C18 column and immunoaffinity column

Fumonisins B1 and B2 were determined in 42 samples of different maize products from the Swedish market by 2 different methods based on cleanup steps using an immunoaffinity column and a combination of SAX + C18 columns, respectively. A simple "precipitation step" was included before the samples were added to the main column(s), giving less column clogging, fewer interfering peaks, and better recoveries for the different sample matrixes. Recovery, repeatability, and results from the survey showed comparable results with the methods. The limit of detection for both methods was 5 micrograms/kg for fumonisin B1 and 10 micrograms/kg for fumonisin B2. All 7 maize chips analyzed and 6 of 8 popcorn samples contained fumonisins (B1 + B2) with averages of 180 and 115 micrograms/kg, respectively. All other samples except a maize flour sample contained little or no fumonisins.     

Analysis of T-2 and HT-2 toxins in cereal grains by immunoaffinity clean-up and liquid chromatography with fluorescence detection

A sensitive, precise and accurate method has been developed for the simultaneous determination of T-2 and HT-2 toxins in cereal grains at ppb levels using high-performance liquid chromatography (HPLC) with fluorescence detection and 1-antroylnitrile (1-AN) as labeling reagent after immunoaffinity clean-up. Cereal samples were extracted with methanol/water (90:10, v/v), and the extracts were cleaned-up through commercially available immunoaffinity columns containing monoclonal anti-T-2 antibodies (T-2 test HPLC, Vicam). T-2 and HT-2 toxins were quantified by reversed-phase HPLC with fluorometric detection (excitation wavelength 381 nm, emission wavelength 470 nm) after derivatization with 1-AN. The monoclonal antibody showed 100% cross-reactivity with both T-2 and HT-2 toxin, and the immunoaffinity column clean-up was effective up to 1.4 microg of both toxins. The method was successfully applied to the analysis of T-2 and HT-2 toxins in wheat, maize and barley. Recoveries from spiked samples with toxin levels from 25 to 500 microg/kg ranged from 70% to 100%, with relative standard deviation generally lower than 8%. The limit of detection of the method was 5 microg/kg for T-2 toxin and 3 microg/kg for HT-2 toxin, based on a signal-to-noise ratio 3:1. HT-2 toxin was detected in ten naturally contaminated wheat samples out of 14 samples analyzed, with toxin levels ranging from 10 to 71 microg/kg; three of them contained also T-2 toxin up to 12 microg/kg.