Quantitative Determination of Fumonisins B1 and B2 by High-Performance Liquid Chromatography with Fluorescence Detection

Abstract

A high-performance liquid chromatographic method has been developed for the quantitative determination of the recently described mycotoxins, fumonisins B₁ (FB₁) and B₂ (FB₂) utilizing pre-column derivatization with o-phthaldialdehyde, isocratic elution, and fluorescence detection. Prior to analysis, sample extracts were purified on strong anion exchange cartridges. The method has been applied to the analysis of naturally contaminated corn and mixed horse feed samples as well as fungal culture material, for the presence of the mycotoxins. Detection limits are approximately 50 ng g^?1 for FB₁ and 100 ng g^?1 for FB₂. The method proved to be highly reproducible and recoveries of the toxins from the purification steps were found to be 99.5% and 85.9% for FB₁ and FB₂, respectively.     

Determination of hexavalent chromium by using speciated isotope-dilution mass spectrometry after microwave speciated extraction of environmental and other solid materials

Precise and accurate determination of hexavalent chromium in different types of solid environmental sample is regarded as a technical challenge with significant potential error if historically accepted methods are used. Microwave-assisted alkaline extraction (0.5 mol L^–1 NaOH+0.28 mol L^–1 Na₂CO₃) followed by anion-exchange chromatographic separation and inductively coupled plasma mass spectrophotometric detection has been shown to provide accurate and precise results. To obtain a better understanding of potential species conversion during and/or after extraction steps, speciated isotope-dilution mass spectrometry (SIDMS) (EPA Method 6800) metrology has been successfully applied as a diagnostic tool with the modified accompanying extraction version of EPA Method 3060A. In our study, aggregate materials distributed over a large area of a major western US state were found to contain a high concentration of total chromium (195±13 to 709±19 g g^–1) and significant amounts of Cr⁶⁺ (141±6 to 341±29 g g^–1) which are at least three orders of magnitude higher than the US EPA threshold limit (0.5 g g^–1). Sediment samples from a major western US state, studied independently, were found to contain less (1.77±0.34 g g^–1) or no Cr⁶⁺ in the presence of significant total chromium.     

Detection and characterization of twenty‐eight isomers of fumonisin B1 (FB1) mycotoxin in a solid rice culture infected with Fusarium verticillioides by reversed‐phase high‐performance liquid chromatography/electrospray ionization time‐of‐flight and ion trap mass spectrometry

Fumonisin mycotoxins which are hazardous to humans and animals were produced in a Fusarium verticillioides‐infected solid rice culture. To decrease the possibility of the formation of artifacts, the fumonisins were analysed by reversed‐phase high‐performance liquid chromatography/electrospray ionization time‐of‐flight (RP‐HPLC/ESI‐TOFMS) and ion trap mass spectrometry (RP‐HPLC/ESI‐ITMS) immediately after the extraction of the culture material, without any further sample clean‐up. The fumonisin isomers were separated by using a flat gradient on a special, high‐coverage C₁₈, narrow‐bore HPLC column (YMC‐Pack J'sphere ODS H80) suggested for the separation of structural isomers by the manufacturer. Exact mass measurements (TOFMS) of the protonated molecules and extraction of the ion chromatogram corresponding to the empirical formula (C₃₄H₅₉NO₁₅) of FB₁ toxins led to the identification of 29 peaks and shoulders, including those of FB₁. The FB₁ toxin and 28 of its isomers were also detected by ITMS after separation with RP‐HPLC. The characteristic m/z values of the product ions, including the backbones obtained by ITMS², undoubtedly indicated the structures of the FB₁ isomers for 28 peaks and shoulders. In the MS² spectra of the protonated molecules of the FB₁ isomers, with some exceptions, 15 characteristic product ions including the hydrocarbon backbone at m/z 299 were observed. The abundance ratio of the cation at m/z 299 ranged up to 5.8%. The relative quantities of the isomers found in the sample extract were expressed as percentages of the FB₁ content (0.001–0.579%). The total amount of the 28 FB₁ isomers was 2.803% of the quantity of FB₁ that is important from the aspect of food and feed safety. Copyright © 2009 John Wiley & Sons, Ltd.     

A Highly Sensitive Electrochemical Immunosensor for Fumonisin B1 Detection in Corn Using Single‐Walled Carbon Nanotubes/Chitosan

A sensitive electrochemical immunosensor was developed for detecting fumonisin B₁ (FB₁) in corn using the single‐walled carbon nanotubes/chitosan. The detection mechanism of immunosensor was based on an indirect competitive binding to a fixed amount of anti‐FB₁ between free FB₁ and FB₁‐bovine serum albumin, which was conjugated on covalently functionalized nanotubes/chitosan laid on the glass carbon electrode. The anti‐rabbit immunoglobulin G secondary antibody labeled with alkaline phosphatase was then bound to the electrode surface through reactisubstrate α‐naphthyl phosphate, which produced electrochemical signal. Under optimized conditions, this method could detect FB₁ from 0.01 to 1000 ng mL^?1 with a detection limit of 2 pg mL^?1. This is well below the detection limit required from European Union legislation, 2–4 mg L^?1. Moreover, good recoveries were obtained for the detection of spiked corn samples and actual corn samples. As the method has good sensitivity and recovery for detecting FB₁, it is a practical detection method.     

Preparation and properties of [PtII(SS)(NN)]-type complexes and their iodine-doped analogues

Summary Complexes of the general formula [Pt(SS) (NN)], where SS is dddt (5,6-dihydro-1,4-dithiin-2,3-dithiolate) or pddt (6,7-dihydro-5H-1,4-dithiepin-2,3-dithiolate) and NN is bipy (2,2-bipyridine) or phen (1,10-phenanthroline), were prepared by the reaction of [PtCl₂(NN)] with dithiolate ligands. The¹H-n.m.r. spectra shows upfield shifts in the bipy or phen signals upon substitution of the chlorides in [PtCl₂(bipy)] or [PtCl₂(phen)] by dddt or pddt. The u.v.-vis. spectra exhibits intense intramolecular ligand-to-ligand charge transfer bands ca. 600 nm. Cyclic voltammograms show a reversible oxidation step, assigned to [Pt(SS) (NN)]⁰/[Pt(SS)(NN)]⁺. When the complexes were partially oxidized by I₂, two broad e.s.r. signals atg = 1.91,g = 2.02 appeared. Raman spectra show the presence of I ₃ ^– and I^5/– in the iodine-doped complexes. The electrical conductivities of the neutral mixed ligand complexes (10^–9-10^–10S cm^–1) are raised to 10^–7–10^–8S cm^–1 by I₂ doping.     

Synthesis and properties of disulfonated (2-benzimidazolyl)(phenyl)methanone 5-nitro-2-pyridylhydrazone (S2BINPH) and spectrophotometric determination of trace amounts of nickel with S2BINPH

Disulfonated (2-benzimidazolyl)(phenyl)methanone 5-nitro-2-pyridylhydrazone (S₂BINPH) has been synthesized and its reactivity with metal ions investigated. A sensitive and selective spectrophotometric method for the determination of nickel with this reagent has been developed. S₂BINPH reacts with nickel(II) to form a stable 12 (metal ligand) complex with an absorption maximum at 501 nm. The complex formation is quantitative in the pH range 7.2–8.5. Beer's law is obeyed over the range 60–700 ng ml^–1 of nickel and the apparent molar absorptivity of the complex is 8.86 × 10⁴ mol^–1 1 cm^–1 at 501 nm. The proposed method was applied to the determination of nickel in a standard iron- and -steel sample with satisfactory results. Furthermore, proton dissociation constants of S₂BINPH and the overall formation constant of its nickel complex were also determined spectrophotometrically.     

Electrical properties of poly(bis(β-phenoxyethoxy)phosphazene) and its complexes

Electrical and dielectrical properties of poly(bis(-phenoxyethoxy)phosphazene) (I) and its complexes with various content ratios of AgSO₃CF₃ to monomeric unit (0.25/1 (II) and 0.5/1 (III) in molar ratio) were investigated.Dc conductivity of respective samples at 18 °C were 6.1×10^–12, 4.4×10^–9, and 7.1×10^–8 S/m.Dc conduction was considered to be due to ion hopping. Charge mobility ranged from 3×10^–12 to 6× 10^–11 m²/Vs depending on the applied field in sample II. In sample I, a tan peak was found which can be ascribed to molecular relaxation of main chains. The peak vanished upon introducing AgSO₃ CF₃. Temperature dependence of total conductivity ( _T ) measured byac method in the temperature range between –150 °C and 50 °C showed several peaks at the temperatures corresponding to the peak temperatures of tan. Total conductivities of respective samples at 100 kHz were 4.9×10^–7 (69 °C), 1.7×10^–4 (45 °C), and 1.5×10^–4(40°C)S/m.     

Bacterial biosorption and retention of thorium and uranyl cations by Mycobacterium smegmatis

Biosorption of Th⁴⁺ and UO ₂ ²⁺ ions, both separately and in mixed equimolar ratio, was carried out using nitrate-buffered solutions of the cations at pH 1 in the presence of 5%w/w non-proliferative cell suspensions of Mycobacterium smegmatis. At equilibrium following a 3 h treatment, specific adsorption for 2 mM Th and U was, respectively, 102 and 115 mol g^–1 dry biomass for individual solutions and 102 and 42 mol g^–1 for the mixed 2/2 mM solution. Desorption studies of the cation-loaded biomass preparations in aqueous media and in soilbacterial suspensions within the pH range <1 to 11 showed that leaching of throium was generally less than 1% at pH 1–11 after 7 d, whereas uranium was leached to the extent of 2% at pH 1 and up to 10% under the same conditions in Th–U mixtures.     

Assessment of the essential element and heavy metal content of edible fish muscle

The aim of this work was to determine the concentrations of some essential and toxic elements in the muscle of ten species of commercial fish consumed in Portugal. We combined two different techniques for determination of the elements—energy-dispersive X-ray fluorescence (EDXRF) was used to quantify K, Ca, Fe, Zn, Se, Rb, and Sr and flame atomic-absorption spectrometry for analysis of Cr, Ni, Cu, Cd, Hg, and Pb. The latter technique was used because of its higher sensitivity, because these elements were not detected by EDXRF. The results obtained show a similar pattern for the trace elements. K and Ca are present at the highest concentrations in all the samples studied, from 0.6–1.3% and from 0.04–0.08%, respectively, followed by Zn, Fe, Sr, Se, and Rb. Sr is present at higher concentrations than Rb in all the species studied except meagre. Concentrations of the elements in octopus do not follow this pattern—Fe is present at a higher concentration than Zn. Low concentrations of Cr (0.66–1.5 g g^–1), Ni (0.11–0.24 g g^–1), Cd (0.01–0.08 g g^–1), Hg (0.49–2.74 g g^–1), and Pb (0.02–0.06 g g^–1) were observed in all the samples analysed. The concentration of Hg was highest in Helicolenus dactylopterus—5.4 g g^–1 in one sample.     

Matrix Solid-Phase Dispersion Micro-Extraction of Pesticides from Single Insects with Subsequent GC–MS Analysis

A simple multiresidue method based on matrix solid-phase dispersion (MSPD) micro-extraction was studied to determine various organophosphorus (OPPs) and pyrethroid pesticides in single insects. Optimisation of the relevant parameters showed that, in the case of OPPs and permethrin, extraction from 40 mg of isopod (Porcellio scaber) and the use of C₈-bonded silica and 100 L of ethyl acetate were the best conditions, while silica and 3.5 mL of n-hexane were optimal for the less polar pyrethroids. Subsequently, 1 L (ethyl acetate) or 20 L (n-hexane) of the extracts were analysed by GC–MS without any further clean-up. The limits of detection (LODs), in the SIM mode, were 5–80 ng g^–1 for most OPPs and trans-permethrin (cis-permethrin 0.3 ng g^–1) when using the C₈-bonded silica/ethyl acetate procedure, and 5–35 ng g^–1 for the pyrethroids when using the silica/n-hexane procedure. The recoveries of the OPPs and pyrethroids from the isopods were 52–94%. The practicality of the proposed method was studied for pesticides in isopods which had been fed polluted leaves.     

Electronic and ESR spectroscopic study of thiocyanate-containing coordination compounds of titanium(III) in alcohol solutions

In an investigation of electronic and ESR spectra, it has been established that titanium(III) with the thiocyanate ion (in ethanol) forms distorted tetrahedral and octahedral complexes. The tetrahedral compounds, which are hydroxo complexes of titanium(III) that contain one hydroxyl group and three thiocyanate ions in the coordination sphere, give signals in the ESR spectra with g_o=1.960, A_o=15.0·10^–4 cm^–1 g=1.958, A ^Ti =26.4·10^–4cm^–1, g= 1.691, A^Ti=9.0·10^–4 cm^–1. In the ESR spectra of liquid solutions, additional splitting on the nitrogen atoms is manifested(A _o ^N =2.2·10^–4 cm^–1), which indicates bonding of thiocyanate ions through the nitrogen atom. The pseudo-octahedral complexes of titanium(III), containing from two to six thiocyanate ions in the coordination sphere, are characterized by d-d absorption bands in the 520–590 nm region; and at 77°K, they give anisotropic ESR signals. On the basis of the temperature dependences of the equilibrium constants for the reaction of conversion of the tetrahedral complexes to the octahedral complexes, and also the lifetimes of the tetrahedral complexes, values have been estimated for the heat of reaction (H=26.8 kJ/mole) and the entropy change (S=–1.66 kJ/mole·°K) of the equilibrium process, and also the activation energy for the reaction of titanium(III) thiocyanate formation (E=37 kJ/mole).Translated from Teoreticheskaya i Éksperimental'naya Khimiya, Vol. 21, No. 5, pp. 560–567, September–October, 1985.     

Determination of lutetium by fluorimetry, using BPMPHD and CTMAB

Lutetium(III) forms an association compound with a new synthetic reagent, 1,6-bi(1-phenyl-3-methyl-5-pyrazolone-4)hexandione (BPMPHD), and cetyltrimethylammonium bromide (CTMAB). The compound enhances the natural fluorescence of BPMPHD remarkably, upon which a new fluorescence method was developed for determining lutetium in rare earth (RE) samples. The determination range was 1.80 × 10^–7–8.8 × 10^–6 g/ml. The determination limit was 29 ng/ml. The composition of the ion associate was [Lu(BPMPHD)₂]–CTMAB⁺.     

Quantum-chemical calculations and interpretations of electronic spectra of phthalocyanines of transition metals and of their ionic forms

The INDO/S method with Zerner's parametrization has been used to calculate the energies and intensities of electronic transitions for a number of metallophthalocyanines of transition metals and their negatively charged ions: CoPC, FePC·L₂, MnPC·L₂, CoPC^–, FePC^–, MnPC^–·L₂, FePC^2–, FePC^–·L₂, and COPC^2– (PC is the macrocyclic phthalocyanines ligand, and L the molecules of axial ligands, in the present instance ammonia molecules). It is shown that within an isoelectronic series of compounds, having the same total number of electrons and the same disposition of pairs of d orbitals, the laws governing the changes in the electronic spectra, related to changes in the d- interactions, can be described theoretically by allowing for configuration interactions of doubly excited configurations in the basis. It is established that in the visible and near UV and IR regions of the spectrum the allowed electronic transitions in compounds of an isoelectronic series, corresponding to an uncharged state of the phthalocyanine ligand, consist of a superposition of the ^* configuration (2a_1u 6e_g) and two types of intramolecular CT configurations: (d 3b_1u) and the double excitation (2a_1u, d 6e_g, 6e_g). Data are obtained for the energy levels of the even electronic states which are responsible for the rapid radiationless deactivation of excited molecules of the phthalocyanines of transition metals.Translated from Teoreticheskaya i Éksperimental'naya Khimiya, Vol. 26, No. 6, pp. 651–658, November–December, 1990.     

High-Throughput Analysis of Sofalcone in Human Plasma by Use of Automated 96-Well Protein Precipitation and LC-MS-MS

A sensitive and selective method for the determination of sofalcone in human plasma was established by use of protein precipitation and liquid chromatography-tandem mass spectrometry. Plasma samples were transferred into 96-well plate using an automated sample handling system and spiked with 10 L of 2 g mL^–1 internal standard solution (d₃-sofalcone). 0.5 mL of acetonitrile was added to the 96-well plate and the plasma samples were then vortexed for 30 sec. After centrifugation, the supernatant was transferred into another 96-well plate and completely evaporated at 40 °C under a stream of nitrogen. The dry residue was reconstituted with mobile phase. All sample transfer and protein precipitation was automated through the application of both the PerkinElmer MultiPROBE II HT and TOMTEC Quadra 96 workstation. The limit of quantitation of sofalcone was 2 ng mL^–1 using a sample volume of 0.2 mL for the analysis. The reproducibility of the method was evaluated by analyzing five samples at nine quality control (QC) levels over the nominal concentration range from 2 ng mL^–1 to 1000 ng mL^–1. Validation of the method showed that the assay has good precision and accuracy. Sofalcone and internal standard produced a protonated precursor ion ([M+H]⁺) at m/z 451 and 454, and both gave a corresponding product ion at m/z 315. The high sample throughput of the method has been successfully applied to a pharmacokinetic study of sofalcone in human plasma.     

Determination of Glimepiride in Human Plasma by LC-MS-MS and Comparison of Sample Preparation Methods for Glimepiride

A sensitive and selective method for quantitation of glimepiride in human plasma was established using liquid chromatography-electrospray ionization tandem mass spectrometry. Three different methods for the sample preparation of glimepiride and an internal standard were investigated (liquid-liquid extraction, solid-phase extraction and protein precipitation). Glipizide was used as an internal standard. Compounds were separated on a C₁₈ column with 80% acetonitrile and 20% deionized water (adjusted to pH 3.5 with acetic acid), as mobile phase at a flow rate of 200 L min^–1. By use of multiple reaction monitoring mode in MS-MS with liquid-liquid extraction and solid-phase extraction, glimepiride and glipizide were detected without severe interference from the human plasma matrix. Glimepiride produced a protonated precursor ion ([M+H]⁺) at m/z 491 and a corresponding product ion at m/z 352, and the internal standard produced a protonated precursor ion ([M+H]⁺) at m/z 446 and a corresponding product ion at m/z 321. The limit of quantitation was 0.1 ng mL^–1, 0.5 ng mL^–1 and 1.0 ng mL^–1 when using liquid-liquid extraction, solid-phase extraction and protein precipitation, respectively. The validation, reproducibility, stability, and recovery of the different sample preparation methods were comparable and all the methods gave reliable results. The method has been successfully applied to pharmacokinetic study of glimepiride in human plasma.     

Electronic absorption spectra,ESR spectra,and structure of coordination polyhedrons of titanium(III) in molten and “instantly cooled” systems of sodium phosphates and halides

The structure of Ti(III) coordination polyhedrons in both individual NaPO₃ and 70 mole % NaPO₃-30 mole % Na_nX mixtures (X=PO₃ ^3–, P₂O₇ ^4–, F^–, Cl^–) in the molten and solid states has been investigated on the basis of data from electronic absorption and ESR spectra. The formation of tetragonally distorted Ti(III) coordination polyhedrons with D_4h symmetry coordinated only by (PO₃ ^–)_n chains with the following values of the spectroscopic parameters for the molten (and solid) states has been established in all the phosphate systems investigated: 10Dq=17,300 (18,000) cm^–1 (Ds=50 cm^–1, Dt=5600, g=1.959, g₁=1.930, =93. cm^–1). It has been shown that the greater is the extent of depolymerization of the phosphate chains in the melt, the stronger is the Ti(III) coordination polyhedron formed; the following relative series of the depolymerizing strength of the anions has accordingly been established: PO₄ ^3–2O₇ ^4–––.Translated from Teoreticheskaya i Éksperimental'naya Khimiya, Vol. 25, No. 5, pp. 610–614, September–October, 1989.     

Selective Solid Phase Extraction and Pre-Concentration of Heavy Metals from Seawater by Physically and Chemically Immobilized 4-Amino-3-Hydroxy-2-(2-Chlorobenzene)-Azo-1-Naphthalene Sulfonic Acid Silica Gel

4-Amino-3-hydroxy-2-(2-chlorobenzene)-azo-1-naphthalene sulfonic acid (AHCANSA) was used as a chelating modifier to improve the reactivity of the silica gel surface in terms of selective binding and extraction of heavy metal ions. The surface coverage values were found to be 0.488 and 0.473mmolg^–1 for the newly modified physically adsorbed silica gel phase (I) and chemically immobilized-AHCANSA phase (II), respectively. The modified silica gel phases (I, II) were tested for stability in different acidic buffer solutions (pH 1–6) and found to be highly resistant to hydrolysis and leaching by buffer solutions above pH 2. The application of these two phases as solid extractors for a series of mono-, di-, and tri-valent metal ions from aqueous solutions was also performed with different controlling factors such as the pH value of metal ion solutions and equilibrium shaking time. The mmolg^–1 metal capacity values determined by silica gel phases (I, II) were found to confirm high affinity and selectivity characters for binding with heavy metal ions such as Cr³⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺ and Pb²⁺ in a range of 0.250–0.483. The tested alkali and alkaline earth metals, Na⁺, K⁺, Mg²⁺ and Ca²⁺, were found to exhibit little interaction and binding ability with the modified silica gel phases. The selectivity characters incorporated into the modified silica gel phases were further utilized and applied in solid phase extraction and pre-concentration of trace concentration levels (1.0µgmL^–1 and 2.00–2.50ngmL^–1) from real seawater samples. The percentage recovery values determined for Cr³⁺, Cu²⁺, Zn²⁺, Cd²⁺ and Pb²⁺ were found to be in the range of 95.2–98.1±2.0–5.0%, and the pre-concentration recovery values for the same tested heavy metal ions were found to be in the range of 92.5–97.1±3.0–6.0% for the two newly modified silica gel phases with a pre-concentration factor of 500.Received December 20, 2002; accepted May 14, 2003 published online September 1, 2003     

New books

Summary Micromolar analyses of the nitrogen species NH₃, NO ₂ ^– , and NO ₃ ^– in soil and other samples are usually accomplished by extracting several samples and testing each for a different species. This procedure is not viable when the quantity of the initial sample is limited. An improved method of separating and analysing for ammonia NH₃(aq), nitrite NO ₂ ^– (aq), and nitrate NO ₃ ^– (aq) from a single small sample with concentrations of 0–50 mol/l is reported. No interferences or carryovers among the three nitrogen containing species were found. Uncertainties were ±2–5 mol/l and accuracies with respect to standards were ±3 mol/l.     

Application of isotope-dilution laser ablation ICP–MS for direct determination of Pu concentrations in soils at pg g<Superscript>−1</Superscript> levels

The methods available for determination of environmental contamination by plutonium at ultra-trace levels require labor-consuming sample preparation including matrix removal and plutonium extraction in both nuclear spectroscopy and mass spectrometry. In this work, laser-ablation inductively coupled plasma mass spectrometry (LA–ICP–MS) was applied for direct analysis of Pu in soil and sediment samples. Application of a LINA-Spark-Atomizer system (a modified laser ablation system providing high ablation rates) coupled with a sector-field ICP–MS resulted in detection limits as low as 3×10^–13 g g^–1 for Pu isotopes in soil samples containing uranium at a concentration of a few g g^–1. The isotope dilution (ID) technique was used for quantification, which compensated for matrix effects in LA–ICP–MS. Interferences by UH⁺ and PbO₂⁺ ions and by the peak tail of ²³⁸U⁺ ions were reduced or separated by use of dry plasma conditions and a mass resolution of 4000, respectively. No other effects affecting measurement accuracy, except sample inhomogeneity, were revealed. Comparison of results obtained for three contaminated soil samples by use of -spectrometry, ICP–MS with sample decomposition, and LA–ICP–IDMS showed, in general, satisfactory agreement of the different methods. The specific activity of ^239+240Pu (9.8±3.0 mBq g^–1) calculated from LA–ICP–IDMS analysis of SRM NIST 4357 coincided well with the certified value of 10.4±0.2 mBq g^–1. However, the precision of LA–ICP–MS for determination of plutonium in inhomogeneous samples, i.e. if "hot" particles are present, is limited. As far as we are aware this paper reports the lowest detection limits and element concentrations yet measured in direct LA–ICP–MS analysis of environmental samples.Sergei F. Boulyga is on leave from The Radiation Physics and Chemistry Problems Institute, 220109 Sosny, Minsk, Belarus.     

Fast Determination of Fumonisin B1 and B2 in Corn Using a Modified QuEChERS Method and LC–MS–MS

A fast analytical method for the simultaneous determination of fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) in corn using a novel QuEChERS method and LC–MS–MS was developed and validated. Samples were extracted with methanol–water (3:1 v/v) by means of ultrasonic extraction. The extract was purified with a novel modified QuEChERS method. Firstly, FB₁ and FB₂ in the extract were retained with primary secondary amine (PSA). Then, FB₁ and FB₂ were released from PSA with 1.0 % formic acid in methanol. The final eluate was diluted with water, and analyzed by LC–MS–MS on a Waters Acquity BEH C₁₈ column with 0.1 % formic acid in water/methanol as mobile phase with gradient elution. Mean recoveries of 83.5–102.4 % with CVs of 3.6–10.5 % were obtained at fortification levels of 2, 50 and 1,000 μg kg⁻¹. The limit of quantification was 2.0 μg kg⁻¹.