Rapid determination of fumonisins B1 and B2 in corn by liquid chromatography-tandem mass spectrometry with ultrasonic extraction

A simple, sensitive, and reliable analytical method is developed for the rapid determination of fumonisin B(1) and fumonisin B(2) in corn by high-performance liquid chromatography-positive electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS). Fumonisin B(1) and fumonisin B(2) are extracted from corn with methanol-water (3:1, v/v) by means of ultrasonic extraction, and directly injected into an LC-MS-MS system after centrifugation. Fumonisin B(1) and fumonisin B(2) are separated on a Zorbax Eclipse XDB-C(18) column with a solution of methanol-water-formic acid as the mobile phase. The method is validated with respect to linearity, accuracy, precision, specificity, and stability. Moreover, the method was applied to real samples and demonstrated to be suitable for the determination of fumonisin B(1) and fumonisin B(2) in corn. The total time required for the analysis of one sample was ~30 min.     

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.     

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     

Determination of Fumonisin B1 in Corn by High Performance Liquid Chromatography With Fluorescence Detection

Abstract

A method is described for the determination of fumonisin B1 in corn. The method involves sample extraction with methanol:water (75:25) and partial purification using a solid phase extraction. Fumonisin B1 is reacted with naphthalene-2,3-dicarboxaldehyde (NDA) to produce a highly fluorescent derivative, 1-cyano-2-alkyl-benz[f]isoindole (CBI) and then separated from the sample matrix on a reverse phase C-18 column with a mobile phase of acetonitrile:water:acetic acid (55:45:1). The NDA-derivative is quantitated by fluorescence detection at 410 nm excitation and a 440 nm, long past emission filter. Recoveries of fumonisin B1 added to corn at levels of 0.25–20.0 μg/g averaged 88.1% with a coefficient of variation of 10.3%. Confirmation of fumonisin B1 in corn samples was accomplished by fast atom bombardment (FAB) spectroscopy.     

Rapid determination of fumonisins in corn-based products by liquid chromatography/tandem mass spectrometry

A simple, fast, and robust method was developed for the determination of fumonisin B1 (FB1), fumonisin B2 (FB2), and fumonisin B3 (FB3) in corn-based human food and animal feed (cornmeal). The method involves a single extraction step followed by centrifugation and filtration before analysis by ultra-performance liquid chromatographylelectrospray ionization (UPLC/ESI)-MS/MS. The LC/MS/MS method developed here represents the fastest and simplest procedure (<30 min) among both conventional HPLC methods and other LC/MS methods using SPE cleanup. The potential for high throughput analysis makes the method particularly beneficial for regulatory agencies and analytical laboratories with a high sample volume. A single-laboratory validation was conducted by testing three different spiking levels (200, 500, and 1000 ng/g for FB1 and FB2; 100, 250, and 500 ng/g for FB3) for accuracy and precision. Recoveries of FB1 ranged from 93 to 98% with RSD values of 3-8%. Recoveries of FB2 ranged from 104 to 108%, with RSD values of 2-6%. Recoveries of FB3 ranged from 94 to 108%, with RSD values of 2-5%.     

Detection of new fumonisin mycotoxins and fumonisin-like compounds by reversed-phase high-performance liquid chromatography/electrospray ionization ion trap mass spectrometry

Fumonisins were produced in a rice culture infected with Fusarium verticillioides. To decrease the possibility of the formation of artifacts, the fumonisins were analyzed by reversed-phase high-performance liquid chromatography with electrospray ionization ion trap tandem mass spectrometry (RP-HPLC/ESI-IT-MS2) immediately after the extraction of the culture material without any sample clean-up. In addition to already known fumonisins, numerous new fumonisin mycotoxins and fumonisin-like compounds were detected. On the basis of the IT-MS2 data, detailed fragmentation pathways including new mechanisms were proposed for the different series of fumonisins. The retention times, the masses of the protonated molecules and of the product ions including the backbones and the characteristic neutral mass losses from the protonated molecules of the new compounds suggested their structures (applying the well-known designation): iso-FA1a,b, iso-FB1a-d, iso-FB2,3a-e, PHFB2a-c, PHFB4a-d, FB5/iso-FB5a-d, FBK1 2TCA, FBK4 2TCA, FC2, iso-FC2,3, PHFC4, FD and FBX series. The relative quantities of fumonisins and fumonisin-like compounds found in the sample extract were expressed as percentages of FB1 (0.02-100%). The backbone of the compound denoted FD contained fewer carbon atoms than the well-known fumonisins with the C19 or C20 backbone and may well be a precursor of the longer compounds. For the compounds denoted FBX (12 compounds), one or two OH groups attached to the fumonisin backbone were esterified by carboxylic acids other than tricarballylic acid, such as cis-aconitic acid, oxalylsuccinic acid and oxalylfumaric acid.     

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.     

A simple aptamer molecular beacon assay for rapid detection of aflatoxin B1

A simple aptamer molecular beacon assay for rapid detection of aflatoxin B1 (AFB1) was achieved. AFB1-binding induced formation of a hairpin structure and closeness of fluorophore label and quencher probe, causing fluorescence decrease.     

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.     

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。     

原料奶、婴儿配方奶粉及酸奶中蛋白去除效果的研究

建立了毛细管电泳法分析α-乳白蛋白、β-乳球蛋白A及β-乳球蛋白B的方法,考察了不同浓度冰乙酸(HAc)及三氯乙酸(TCA)对原料奶、婴儿配方奶粉A和B及酸奶中蛋白的去除效果。结果表明,TCA去除蛋白效果优于HAc,不同样品所需TCA浓度不同:2g原料奶需3mL100g/LTCA;0.5g婴儿配方奶粉A和B分别需5mL10g/L及20g/LTCA;2g酸奶则需3mL20g/LTCA才能将蛋白完全沉淀。为原料奶、婴儿配方奶粉及酸奶等乳制品的样品前处理提供了有价值的参考。     

Evaluating the performance of sampling plans to detect fumonisin B1 in maize lots marketed in Nigeria

Fumonisins are toxic and carcinogenic compounds produced by fungi that can be readily found in maize. The establishment of maximum limits for fumonisins requires the development of scientifically based sampling plans to detect fumonisin in maize. As part of an International Atomic Energy Agency effort to assist developing countries to control mycotoxin contamination, a study was conducted to design sampling plans to detect fumonisin in maize produced and marketed in Nigeria. Eighty-six maize lots were sampled according to an experimental protocol in which an average of 17 test samples, 100 g each, were taken from each lot and analyzed for fumonisin B1 by using liquid chromatography. The total variability associated with the fumonisin test procedure was measured for each lot. Regression equations were developed to predict the total variance as a function of fumonisin concentration. The observed fumonisin distribution among the replicated-sample test results was compared with several theoretical distributions, and the negative binomial distribution was selected to model the fumonisin distribution among test results. A computer model was developed by using the variance and distribution information to predict the performance of sampling plan designs to detect fumonisin in maize shipments. The performance of several sampling plan designs was evaluated to demonstrate how to manipulate sample size and accept/reject limits to reduce misclassification of maize lots.     

European intercomparison study for the determination of fumonisins in maize

Fumonisins are mycotoxins occurring largely in maize and maize products, which cause animal diseases, such as equine leukoencephalomalacia and porcine pulmonary edema, and may also induce liver cancer on experimental rats. The European Commission Standards, Measurements and Testing (SMT, formerly BCR) Programme, has sponsored a project to improve analytical methodologies for the determination of the two major fumonisins (fumonisin B1 and fumonisin B2) in maize materials. The project involved the following steps: i) the preparation of a blank and a maize material contaminated with fumonisins Bl and B2; ii) a preliminary study of the -irradiation conditions for sterilization; iii) homogeneity and stability studies of the maize materials; iv) an intercomparison study for fumonisins analysis in the above materials with the involvement of 24 European laboratories, most of which have national or international responsibilities for foodstuff and/or feedstuff quality control. Results of the intercomparison study are presented together with the homogeneity and stability data relevant to the maize materials.     

膜基质免疫分析法同时检测玉米中伏马菌素B1和呕吐毒素

建立了基于聚偏氟乙烯膜(Polyvinylidene fluoride,PVDF)基质的直接竞争免疫分析法,同时检测玉米中的伏马菌素B1(Fumonisin B1,FB1)及呕吐毒素(Deoxynivalenol,DON)。PVDF膜用甲醇浸湿、激活,用移液器将FB1及DON全抗原点阵于相应的膜反应区,同时采用三聚氰氯法和高碘酸钠法分别制备抗FB1、抗DON的辣根过氧化酶(Horseradish peroxidase,HRP)标抗体(monoclonal antibody,McAb),以直接竞争免疫检测方法的模式实现同时检测玉米中的FB1及DON。该方法对于FB1和DON的检出限分别为2.5和50μg/L,样品前处理简单,检测时间15 min,可肉眼辨别结果,随机检测了30份市售玉米样品,并与市售ELISA试剂盒进行方法学比较,结果无明显差异。     

Minimization of carryover for high‐throughput liquid chromatography with tandem mass spectrometry analysis of 14 mycotoxins in corn grits

A method for the simultaneous analysis of 14 mycotoxins with the minimization of carryover was developed. Our verification experiments suggested that the carryover occurred due to the chelation of fumonisins with the metal. To wash the fumonisins from the metal, the inner surface of the injection needle was rinsed with 10 mM trisodium citrate and 1% formic acid in water/methanol/acetonitrile/isopropanol after each injection, and the analysis was performed on a metal‐free Mastro C18 column. This approach remarkably minimized the carryover of fumonisins. Fourteen mycotoxins in samples were extracted with 2% acetic acid in water/acetonitrile and a quick, easy, cheap, effective, rugged, and safe extraction kit, purified on a MultiSep 229 Ochra, and then quantified by liquid chromatography with tandem mass spectrometry. Determinations performed using this method produced a linearity greater than 0.99 and recoveries ranging from 72.6 to 117.4%, with good intraday precision from 4.0 to 12.4%, and interday precision from 6.5 to 17.0%. The limits of detection ranged from 0.01 to 0.71 μg/kg, demonstrating that a highly sensitive method for the simultaneous analysis of mycotoxins over a wide range of concentrations was achieved with minimal carryover. When 12 samples of commercially available corn grits were analyzed with this method, deoxynivalenol, fumonisin B1, fumonisin B2, fumonisin B3, and zearalenone were present most frequently.     

Evaluation of Fumonitest Immunoaffinity Columns

Abstract

A method for the determination of fumonisins B1 and B2 in corn was developed. The method involves sample extraction with methanol:water (80:20) and the use of a commercially available Fumonitest column for sample cleanup. The capacity, selectivity, column-to-column and lot-to-lot reproducibility of the Fumonitest columns were evaluated. The total capacity of the column was found to be 1.2 μg fumonisin. Both fumonisins B1 and B2 had an equal affinity toward the Fumonitest column, with the sample matrix demonstrating little effect on the column performance. The maximum sample size was 0.5 g for samples containing total fumonisins of less than 2 ppm. After elution from the immunoaffinity column, fumonisins B1 and B2 were reacted with naphthalene-2, 3-dicarboxaldehyde (NDA) to produce a highly fluorescent derivative, 1-cyano-2-alkyl-benz[f]isoindole (CBI). The derivatives were then separated from the sample matrix on a reverse phase C-18 column with a mobile phase consisting of acetonitrile:water:acetic acid (55:45:1) Average recoveries of fumonisins B1 and B2 from corn samples spiked at a level of 1000 ng (500ng B1 + 500ng B2)/g were 85.4 and 87.1%, respectively. The detection limit for B1 and B2 was estimated to be 10 and 4 ppb, respectively. The coefficient of variations for fumonisins B1 and B2 were determined to be 10.2% and 10.6%, respectively.     

花生及其制品中多种霉菌毒素残留同时测定方法

利用液相色谱-质谱联用（LC-MS／MS）建立了花生及其制品中多种霉菌毒素包括黄曲霉毒素（B1,B2,G1,G2）、赭曲霉毒素A、伏马毒素B1、脱氧雪腐镰刀菌烯醇、T-2毒素、HT-2毒素及玉米赤霉烯酮的同时测定方法。样品经PBS溶液和甲醇-水溶液提取,提取液经稀释、过滤后,用免疫亲和柱净化,通过淋洗去除免疫亲和柱上的杂质,随后用洗脱液过柱,将目标物分离下来,氮吹干后定容。以液相色谱-质谱／质谱测定,外标法定量。方法的检出限黄曲霉毒素B1为0．0005mg／kg,黄曲霉毒素B2,G1,G2为0．001mg／kg,赭曲霉毒素A为0．002mg／kg,伏马毒素B1为0．020mg／kg,脱氧雪腐镰刀菌烯醇为0．050mg／kg,T-2毒素为0．010mg／kg,HT-2毒素为0．010mg／kg,玉米赤霉烯酮为0．002mg／kg。在样品中添加检出限水平的毒素混标溶液,加标回收率为72．35％-97．82％,测定结果的相对标准偏差为8．95％～18．41％（n=10）.     

Evaluation of silica- and sepharose-based immunoaffinity sorbents for sample cleanup in determination of fumonisins B1 and B2 in corn products

Anti-fumonisin B1 polyclonal antibodies were isolated from the serum of rabbits, immobilized onto the surface of glutaraldehyde-activated silica or Sepharose CL-4B particles, and placed into empty small plastic solid-phase extraction cartridges. The immobilized antibodies were evaluated for their ability to retain fumonisin B1 and fumonisin B2. Cartridge capacity and elution conditions were determined, and the results were compared to those obtained with a commercially available cartridge. The cartridges, which were tested for their effectiveness to isolate the fumonisins from extracts of corn flour and nacho chips, detected fumonisins down to levels of about 20 ng/g. However, additional cleanup was required for detection at lower concentrations. With the use of a strong anion-exchange cartridge as a preliminary cleanup before immunoaffinity chromatography, the detection limit reached 2-5 ng/g in the products tested. The silica sorbent material exhibited strong interactions with the fumonisins, requiring acidified ethanol-water mixtures for elution and resulting in an additional degree of selectivity in isolating fumonisins from sample extracts. The silica-based immunoaffinity cartridges were successfully reused more than 10 times; the Sepharose-based cartridges were less robust. Liquid chromatography with fluorescence detection was used after prechromatographic derivatization with o-phthaldialdehyde-mercaptoethanol.     

Use of ion-exchange chromatography coupled with TLC-laser scanning densitometry for the quantitation of fumonisin B(1)

A simple TLC-Laser scanning densitometric (TLC-LSD) method was developed for the quantitation of fumonisin B(1) (FB(1)) isolated from solid media cultures (corn) and liquid media cultures of toxigenic Fusarium moniliforme strains (F. moniliforme MRC 826, F. moniliforme 4223 and F. moniliforme 2927)). FB(1) was isolated from the cultures by solvent extraction (methanol:water, 3:1) and purified in a single step by ion-exchange chromatography using Dowex-1. FB(1) in the purified extracts was detected by TLC analysis using p-anisaldehyde as a post-chromatographic derivatizing agent. The major toxin identified was FB(1) (R(f) 0.51) along with traces of FB(2) (R(f) 0.57) and FB(3) (R(f) 0.60) based on their comparison with the reference standard fumonisins. The sensitivity of the TLC-LSD method for the quantitation of FB(1) was found to be 500 ng g(-1). The linear regression analysis performed for the quantitation of FB(1) by the TLC-LSD method showed a correlation coefficient (r) value of 0.9. Spiking studies revealed the recovery of standard FB(1) (5 and 10 mug g(-1)) loaded on to Dowex-1 in the range of 87-96%. The purity of FB(1) purified from the cultures was determined by the two-dimensional TLC analysis. Two-dimensional TLC-analysis of the purified FB(1) revealed the purity to be greater than 85%. The method developed may find wide application in the environmental monitoring of the FB(1) contaminations in the various agricultural commodities and screening fumonisin producing toxigenic strains of F. moniliforme.     

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%.