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Reverse Homogeneous Liquid–Liquid Extraction as a Miniaturized Method for Extraction of Aflatoxins from Pistachio and Wheat and LC Post-column Derivatization-Fluorescence Detection
Abstract
A rapid and simple quantitative method for the simultaneous determination of the four aflatoxins (AFs) B1, B2, G1 and G2 was
developed using reverse homogeneous liquid–liquid extraction (RHLEE) and HPLC post-column derivatization-fluorescence (HPLC-FL)
detection. The method based on the rapid extraction of AFs from a methanolic sample into chloroform after addition of water
containing KBr, with a method we called reverse homogeneous liquid–liquid extraction. Recoveries were in the range of 88–125%
and the limits of detection were between 0.001–0.042ngg−1 for different AFs. Wheat and pistachio were chosen for the analysis of real samples and the method was also successfully
applied to a FAPAS® TEST MATERIAL (T04151).
KeywordsHPLC-FL–RHLLE–Aflatoxins–Preconcentration
... The mixture was injected to an immune-affinity column with specific antibodies of AFs B1, B2, G1, and G2. The AFs were eluted with methanol from immune-affinity columns into acid-washed vials, and finally, 20 µl was applied into HPLC (Hassan & Habibi, 2011). ...
... Methanol, acetonitrile, and water (20:20:60 v/v) with 119 mg of potassium bromide, 100 µl of 65% Nitric acid were the mobile phase and filtered through a Millipore 0.22 µm membrane filter before use. At the same fluorescence emission wavelength in a single run, AFs elute in the order of G 2 , G 1 , B 2 , and B 1 (Hassan & Habibi, 2011). ...
This study aimed to investigate the potential ability of simultaneously used L. acidophilus(LA‐5), L.rhamnosus(LGG), and L.casei(LC‐01) in encapsulated (E) and nonencapsulated (NE) forms in mycelial growth of Aspergillus spp and aflatoxin production by A. flavus. In order to assess the zone of fungal growth inhibition by E and NE lactic acid bacteria, the agar well diffusion method was applied. Quantification of aflatoxin was performed using a high‐performance liquid chromatography technique. Lactic acid bacteria exhibited high antifungal activity and significantly reduced AFB1, AFB2, AFG1, and AFG2 production in both E and NE forms compared to the control group. The percentage of reduction in total AFs production in treated samples with E and NE lactic acid bacteria was 94.1% and 95.5%, respectively. These results suggested that simultaneously used lactic acid bacteria in E and NE forms can prevent growth and decrease aflatoxin production of toxigenic aspergilla.
... The main disadvantage of the DLLME technique is the use of chlorinated solvents as extractants that are heavier than water and more toxic than hydrocarbons. Recently, we reported our experiments for employing low-density extraction solvents in DLLME that we called, miniaturized dispersive liquid-liquid extraction (MDLLE) [10][11][12]. Traditionally, selective detectors in gas chromatography (GC) have been used to detect individual classes of GC-amenable pesticides, such of organochlorines, organophosphates, and organonitrogens. In recent years, GC/mass spectrometry (MS) has become the primary approach to analyze all classes of GC-amenable pesticides in the same chromatogram. ...
... In the experiment, 8.0 mL of deionized water spiked with 10.0 ng mL −1 each of the OPPs was used to study the extraction performance under different experimental conditions. The theory of RMDLLE is similar to that of LLE and can find elsewhere [10][11][12]. ...
In this work, the application of reverse miniaturized dispersive liquid–liquid
extraction techniques for determination of some semi-polar pesticides (diazinon, chlorpyrifos,
and butachlor) in water samples has been evaluated. Reverse miniaturized dispersive
liquid–liquid extraction method based on extraction with methanol containing
butyl acetate as a solvent was added sample and after phase separation, butyl acetate injected
to the gas chromatography/mass spectrometry (GC/MS) instrument. GC/MS in
selected ion storage mode (SIS) was employed for the identification and quantification of
diazinon, chlorpyrifos, and butachlor. Important parameters affecting both extraction
and reverse miniaturized dispersive liquid–liquid extraction procedures were investigated
and optimized. Analytical method provides enrichment factors in the range of 11–
18 for these pesticides. The calibration plots were linear in the ranges 0.06–15.0 μg L−1,
μg L−1, and 0.4–15.0 μg L−1, and limit of detection was 0.02, 0.03, and 0.1 μg L−1 for diazinon,
chlorpyrifos, and butachlor, respectively.
... [1]. Due to their severe toxicity as well as their high abundance, much attention has been devoted to AFs [1][2][3]. These toxic compounds are generated in various agricultural products such as dried fruits, cereals and nuts [4]. ...
Herein, a novel method entitled magnetic solid-phase extraction based on MIL-101(Cr)/Fe3O4@SiO2@propylthiouracil composite was developed using the magnetite MIL-101(Cr) for separation and determination of aflatoxins (B1, B2, G1, G2) in pistachio samples. Characterization of the nanoadsorbent was conducted by various techniques such as FTIR, SEM, TEM, XRD, VSM and elemental analysis. Determination of AFs was carried out with a HPLC–fluorescence instrument. Experimental design methodology was utilized to investigate and find the optimum values of affecting parameters. Afterward, figures of merit of the new method were evaluated and LODs were attained in the range of 0.02–0.09 ng g−1 with the linearity domain of 0.1–4.0 ng g−1. Precision of the method was evaluated as RSD %, which was 6.4%-12.8% (n = 5). Eventually, MSPE method was employed for separation and determination of AFs in pistachio samples.
... However, this technique frequently requires a clean-up and preconcentration procedure to improve the detection sensitivity due to the influence of matrix components and the trace level of target compounds (Yang and An 2011). A variety of sample preparation techniques including supercritical-fluid extraction (Holcmb et al. 1996), reverse homogeneous liquid-liquid extraction (Hassan and Habibi 2011), dispersive liquid-liquid microextraction (DLLME) (Campone et al. 2012;Rezaee et al. 2006), combination of hollow fiber supported liquid membrane and DLLME (Simão et al. 2016), solid phase extraction (SPE) (Augusto et al. 2013;Dos Reis et al. 2017), liquid-liquid extraction (Andrade et al. 2013), and magnetic solid phase extraction (MSPE) (Hashemi et al. 2014) has been applied for extraction of AFs from different matrices. ...
In this study, the new approach of dispersive micro-solid phase extraction using a magnetic sheet was developed. A circle magnetic sheet (diameter, 25 mm; thickness, 0.4 mm) was perforated and used as a support for fixing and re-collecting three-dimensional magnetic graphene nanoparticles as an adsorbent. The method was coupled with high-performance liquid chromatography-fluorescence detector (HPLC-FLD) and utilized for extraction of aflatoxins (B1, B2, G1, and G2). All steps of the extraction procedure including sample loading, sorbent dispersion, and re-collecting, and elution were performed using a syringe. The significant parameters such as adsorbent amount, desorption conditions, flow rates, ionic strength, and pH which influence extraction efficiency was investigated. The linear dynamic range was 0.2–1000 μg kg⁻¹, with a coefficient of determinations > 0.9914. The limits of detection and quantification were 0.06–0.1 and 0.20–0.33 μg kg⁻¹, respectively. The developed method was applied to the analysis of aflatoxins in real white and moldy bread samples, and the related extraction recoveries of the four aflatoxins were obtained in the range of 59–69% (RSD, 4.4–5.6%).
BACKGROUND
Magnetic three‐dimensional graphene‐based nanoadsorbents have unique characteristics such as large surface area, good thermal and chemical stability, and high adsorption capacity that make them efficient materials in sorbent‐based extraction techniques. In this study, four aflatoxins (AFs) were analyzed in bread samples using magnetic three‐dimensional graphene as the adsorbent phase in dispersive micro solid‐phase extraction.
RESULTS AND CONCLUSIONS
In‐syringe magnetic sheet solid‐phase extraction based on magnetic three‐dimensional graphene in tandem with dispersive liquid–liquid microextraction was used for the extraction and preconcentration of the target AFs. The effect of significant parameters of the method was investigated and the optimum conditions were determined as follows: adsorbent dosage, 20 mg; desorption/disperser solvent (methanol) volume, 700 μL; desorption solvent flow rate, 0.7 mL min⁻¹; pH, neutral; salt (NaCl) concentration, 10% (w/v); extraction solvent (chloroform) volume, 250 μL; and centrifugation rate (and time), 4000 rpm (5 min). The limits of detection and quantification were in the ranges 0.043–0.083 and 0.14–0.28 μg kg⁻¹, respectively. The extraction method was followed by the HPLC technique with fluorescence detection and applied to the determination of the AFs in four different Iranian fresh and moldy bread samples. The relative recoveries were in the range 84–107% with relative standard deviations of 3.9–8.6%. © 2019 Society of Chemical Industry
A simple method based on miniaturized dispersive liquid–liquid extraction is reported for extraction and preconcentration of formaldehyde in water samples. Eight milliliters of an aqueous sample containing 2,4-dinitrophenylhydrazine were added to a 10.0 mL volumetic flask containing 1.0 mL of methanol–butyl acetate (2: 1) mixture. After phase separation the analyte was determined by HPLC-UV. No clean-up or evaporation were required after extraction. Several factors influencing the extraction efficiency, such as the type and volume of organic solvent, type and volume of co-solvent, pH of the sample solution, ionic strength, and extraction time were evaluated and optimized. Under optimal experimental conditions, good linearity was obtained in the range of 1.5 × 10–4–10 µg/mL (r = 0.996), and the limit of detection was 5 × 10–5 µg/mL. The method produced 20-fold enrichment of the formaldehyde. The average recovery of spiked formaldehyde in real samples was in the range of 90–110% with RSD in the range of 3–8%.
Post Column derivatisation (PCD) coupled with high performance liquid chromatography or ultra-high performance liquid chromatography is a powerful tool in the modern analytical laboratory, or at least it should be. One drawback with PCD techniques is the extra post-column dead volume due to reaction coils used to enable adequate reaction time and the mixing of reagents which causes peak broadening, hence a loss of separation power. This loss of efficiency is counter-productive to modern HPLC technologies, -such as UHPLC. We reviewed 87 PCD methods published from 2009 to 2014. We restricted our review to methods published between 2009 and 2014, because we were interested in the uptake of PCD methods in UHPLC environments. Our review focused on a range of system parameters including: column dimensions, stationary phase and particle size, as well as the geometry of the reaction loop. The most commonly used column in the methods investigated was not in fact a modern UHPLC version with sub-2-micron, (or even sub-3-micron) particles, but rather, work-house columns, such as, 250 × 4.6 mm i.d. columns packed with 5 μm C18 particles. Reaction loops were varied, even within the same type of analysis, but the majority of methods employed loop systems with volumes greater than 500 μL. A second part of this review illustrated briefly the effect of dead volume on column performance. The experiment evaluated the change in resolution and separation efficiency of some weak to moderately retained solutes on a 250 × 4.6 mm i.d. column packed with 5 μm particles. The data showed that reaction loops beyond 100 μL resulted in a very serious loss of performance. Our study concluded that practitioners of PCD methods largely avoid the use of UHPLC-type column formats, so yes, very much, PCD is incompatible with the modern HPLC column.
The application of miniaturized homogeneous liquid-liquid extraction (MHLLE) technique as a simple, inexpensive, quick and efficiency clean up method has been evaluated for determination of diazinon, alachlor, chlorpyrifos and butachlor in cow milk samples. Methanol was used as extraction solvent for the extraction of analytes from cow milk samples and then, methanol phase was extracted and cleaned up by MHLLE method. In this method, butyl acetate was added to methanol phase and after addition of water, butyl acetate was separated from methanol phase and injected to the GC/TSD instrument. The concentration ranges were from 1.0-1000.0 ng/mL for diazinon and chlorpyrifos and from 5.0-1000.0 ng/mL for alachlor and butachlor. The limits of detection were 0.4, 1.6, 0.3 and 1.4 ng/mL for diazinon, alachlor, chlorpyrifos and butachlor, respectively. Finally, the extraction method was successfully applied to the analysis of raw cow milk samples.
This work describes the application of ultrasound-assisted matrix solid-phase dispersion as an extraction and sample preparation
approach for aflatoxins (B1, B2, G1 and G2) and subsequent determination of them by high-performance liquid chromatography–fluorescence detection. A Box–Behnken design
in combination with response surface methodology was used to determine the affecting parameters on the extraction procedure.
The influence of different variables including type of dispersing phase, sample-to-dispersing phase ratio, type and quantity
of clean-up phase, ultrasonication time, ultrasonication temperature, nature and volume of the elution solvent was investigated
in the optimization study. C18, primary–secondary amine (PSA) and acetonitrile were selected as dispersing phase, clean-up
phase and elution solvent, respectively. The obtained optimized values were sample-to-dispersing phase ratio of 1 : 1, 60
mg of PSA, 11 min ultrasonication time, 30°C ultrasonication temperature and 4 mL acetonitrile. Under the optimal conditions,
the limits of detection were ranged from 0.09 to 0.14 ng g−1 and the precisions [relative standard deviation (RSD%)] were <8.6%. The recoveries of the matrix solid-phase dispersion process
ranged from 78 to 83% with RSD <10% in all cases. Finally, this method was successfully applied to the extraction of trace
amounts of aflatoxins in rice samples.
Liquid–liquid extraction (LLE) is widely used as a pre-treatment technique for separation and preconcentration of both organic and inorganic analytes from aqueous samples. Nevertheless, it has several drawbacks, such as emulsion formation or the use of large volumes of solvents, which makes LLE expensive and labour intensive. Therefore, miniaturization of conventional liquid–liquid extraction is needed. The search for alternatives to the conventional LLE using negligible volumes of extractant and the minimum number of steps has driven the development of three new miniaturized methodologies, i.e. single-drop microextraction (SDME), hollow fibre liquid-phase microextraction (HF-LPME) and dispersive liquid–liquid microextraction (DLLME). The aim of this paper is to provide an overview of these novel preconcentration approaches and their potential use in analytical labs involved in inorganic (ultra)trace analysis and speciation. Relevant applications to the determination of metal ions, metalloids, organometals and non-metals are included.
The European Union (EU) has established demanding regulatory limits for controlling aflatoxins B1, B2, G1 and G2, in cereals, nuts, nut products and dried fruit, aflatoxin M1 in milk, and ochratoxin A in cereals. These limits are likely to be extended in the future to additional commodities and other mycotoxins. For enforcement purposes and in particular for resolving any disputes between parties, it is essential that validated methods are available, with performance characteristics that meet certain minimum criteria. As such methods were not available and had not previously been validated either for matrices of interest in Europe or at the low European limits compared to the USA, the EU funded a method-validation project to fulfil this requirement. Immunoaffinity column clean-up methods with HPLC determination were established for aflatoxins B1, B2, G1 and G2 in peanut butter, pistachios, fig paste and paprika, aflatoxin B1 in baby food, aflatoxin M1 in liquid milk, and ochratoxin A in roasted coffee and baby food. For patulin in apple juice and apple puree, solvent extraction and solid-phase clean-up HPLC methods were developed. To undertake collaborative studies, particular care was taken in preparation of naturally-contaminated test materials containing the toxins at levels close to regulatory limits and in demonstrating the homogeneity of batches of material. To ensure that participants in the validation exercise could follow the procedures to be tested, videos of the methods were prepared showing, in particular, any critical steps. Prior to undertaking the method validation, participants were invited to collaborative study workshops to ensure that they fully understood the methods and their role in the study. This care in planning and executing the collaborative studies led to impressive performance characteristics and adoption of six procedures by AOAC International as First Action Methods and seven methods by CEN as European standards. The valuable lessons learned in undertaking these validation exercises are now being put to further use in studies aimed at validating methods for mycotoxins in foodstuffs, which are appropriate for developing countries based on TLC as the end determination but use more modern sample clean-up techniques.
A novel technique called miniaturized homogeneous liquid-liquid extraction (MHLLE) followed by high performance liquid chromatographic-fluorescence detection (HPLC-FL) was developed for the extraction and determination of some polycyclic aromatic hydrocarbons (PAHs) as model for analytical problem in sediment samples. The method is based on the rapid extraction of PAHs from a methanolic sample solution into 0.5 mL n-hexane, as a solvent of lower density than water. After addition of water, the extracting solvent immediately forms a distinct water-immiscible phase at the top of the vial, which can be easily separated, evaporated and re-dissolved in 25 microL of methanol and injected to the HPLC instrument. The parameters affecting the extraction process such as type and volume of organic extraction solvent, extraction time, and salt addition were investigated and the partition coefficient between methanol/water-n-hexane phases was evaluated and used to predict the extraction efficiency. Under optimal conditions, the limits of detection were estimated for the individual PAHs as 3Sb (three times of the standard deviation of baseline) of the measured chromatogram, are in the range of 0.003-0.04 ng g(-1) for sediment samples. The relative recoveries of PAHs at spiking levels of 1.0 ng g(-1) for sediment samples were in the range of 81-92%. The method was also applied to a corresponding standard references materials (IAEA-408) successfully. The proposed method is very fast, simple, and sensitive without any need for stirring and centrifugation.
Dispersive liquid-liquid microextraction (DLLME) has become a very popular environmentally benign sample-preparation technique, because it is fast, inexpensive, easy to operate with a high enrichment factor and consumes low volume of organic solvent. DLLME is a modified solvent extraction method in which acceptor-to-donor phase ratio is greatly reduced compared with other methods. In this review, in order to encourage further development of DLLME, its combination with different analytical techniques such as gas chromatography (GC), high-performance liquid chromatography (HPLC), inductively coupled plasma-optical emission spectrometry (ICP-OES) and electrothermal atomic absorption spectrometry (ET AAS) will be discussed. Also, its applications in conjunction with different extraction techniques such as solid-phase extraction (SPE), solidification of floating organic drop (SFO) and supercritical fluid extraction (SFE) are summarized. This review focuses on the extra steps in sample preparation for application of DLLME in different matrixes such as food, biological fluids and solid samples. Further, the recent developments in DLLME are presented. DLLME does have some limitations, which will also be discussed in detail. Finally, an outlook on the future of the technique will be given.
Single-drop microextraction (SDME) has become a very popular liquid-phase microextraction technique because it is inexpensive, easy to operate and nearly solvent-free. Essentially, SDME combines extraction (and conceivably, cleanup) and concentration in a minimum number of steps, and thereafter, direct extract introduction into an analytical system. In this review, in order to encourage further development of SDME, we focus on its recent developments in its various guises. Its applications when used in combination with different analytical techniques, such as gas chromatography, high-performance liquid chromatography, inductively-coupled plasma mass spectrometry, capillary electrophoresis, mass spectrometry and electrothermal atomic absorption spectrometry, are summarized. SDME does have some limitations, and these are also discussed as well. Finally, an outlook on the future of the technique is given.
Homogeneous liquid-liquid extraction method was studied based on a phase separation phenomenon in a ternary solvent system. According to this procedure, mononitrotoloenes were extracted by single-phase extraction in a water/methanol/chloroform, homogeneous ternary solvent system. Methanol and chloroform were used as consolute and extraction solvents, respectively. The homogeneous solution was broken by the addition of salt and a cloudy solution was formed. After centrifugation, the fine droplets of the extraction solvent were sedimented in the bottom of the conical test tube. Analysis of the extracts was carried out by gas chromatography. The optimization procedure was performed using Box-Behnken design. The variables involved were: sample and extraction solvent volumes, consolute solvent volume and phase separator reagent concentration. Optimum results were obtained under the following conditions: sample volume of 5 mL, extraction solvent volume of 55 microL, consolute solvent volume of 1 mL and phase separator reagent concentration; 5% (w/v). Under these conditions, the enrichment factors of 354, 311 and 300, dynamic linear ranges of 0.5-500, 1-500 and 1-500 microg L(-1), and limit of detections (LODs) of 0.09, 0.09 and 0.1 microg L(-1) were obtained for o-nitrotoluene, m-nitrotoluene and p-nitrotoluene, respectively. Finally, the method was successfully applied to the extraction and determination of MNTs in the waste water samples in the range of micrograms per liter with R.S.Ds.<13.2%.
Mycotoxins likely have existed for as long as crops have been grown but recognition of the true chemical nature of such entities of fungal metabolism was not known until recent times. Conjecturally, there is historical evidence of their presence back as far as the time reported in the Dead Sea Scrolls. Evidence of their periodic, historical occurrence exists until the recognition of aflatoxins in the early 1960s. At that time mycotoxins were considered as a storage phenomenon whereby grains becoming moldy during storage allowed for the production of these secondary metabolites proven to be toxic when consumed by man and other animals. Subsequently, aflatoxins and mycotoxins of several kinds were found to be formed during development of crop plants in the field. The determination of which of the many known mycotoxins are significant can be based upon their frequency of occurrence and/or the severity of the disease that they produce, especially if they are known to be carcinogenic. Among the mycotoxins fitting into this major group would be the aflatoxins, deoxynivalenol, fumonisins, zearalenone, T-2 toxin, ochratoxin and certain ergot alkaloids. The diseases (mycotoxicoses) caused by these mycotoxins are quite varied and involve a wide range of susceptible animal species including humans. Most of these diseases occur after consumption of mycotoxin contaminated grain or products made from such grains but other routes of exposure exist. The diagnosis of mycotoxicoses may prove to be difficult because of the similarity of signs of disease to those caused by other agents. Therefore, diagnosis of a mycotoxicoses is dependent upon adequate testing for mycotoxins involving sampling, sample preparation and analysis.
In this study, a new method was developed for analyzing malathion, cypermethrin and lambda-cyhalothrin from soil samples by using homogeneous liquid-liquid extraction (HLLE) and gas chromatography with electron capture detector (GC-ECD). Acetone was used as extraction solvent for the extraction of target pesticides from soil samples. When the extraction process was finished, the target analytes in the extraction solvent were rapidly transferred from the acetone extract to carbon tetrachloride, using HLLE. Under the optimum conditions, linearity was obtained in the range of 0.05-40 microg kg(-1) for malathion, 0.04-10 microg kg(-1) for lambda-cyhalothrin and 0.05-50 microg kg(-1) for cypermethrin, respectively. Coefficients of correlation (r(2)) ranged from 0.9993 to 0.9998. The repeatability was carried out by spiking soil samples at concentration levels of 2.5 microg kg(-1) for lambda-cyhalothrin, and 10 microg kg(-1) for malathion and cypermethrin, respectively. The relative standard deviations (RSDs) varied between 2.3 and 9.6% (n=3). The limits of detection (LODs), based on signal-to-noise ratio (S/N) of 3, varied between 0.01 and 0.04 microg kg(-1). The relative recoveries of three pesticides from soil A1, A2 and A3 at spiking levels of 2.5, 5 and 10 microg kg(-1) were in the range of 82.20-91.60%, 88.90-110.5% and 77.10-98.50%, respectively. In conclusion, the proposed method can be successfully applied for the determination of target pesticide residues in real soil samples.
doi:10.1016/j.ijfoodmicro
- Richard
doi:10.1016/j.chroma
- M Shamsipur
- J Hassan