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    Home > Biochemistry News > Biotechnology News > GX-274 ASPEC Solid Phase Extraction System Application: Extraction of Deoxysonic Fusarium Enol from Wheat

    GX-274 ASPEC Solid Phase Extraction System Application: Extraction of Deoxysonic Fusarium Enol from Wheat

    • Last Update: 2022-08-30
    • Source: Internet
    • Author: User
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    Apply | Extraction of Deoxyscarbate Fusarenol (DON) from Wheat by Gilson GX-274 ASPEC® Automatic Solid Phase Extraction System



    Fig.


    On February 8, 2017, Brazil established RDC No.


    The determination of Fusarium deoxystenol in food includes the following steps: extraction, purification, elution, and chromatographic quantification



    sample

    Control sample B-MYC0856 naturally contaminated wheat flour with a DON standard value of 877 μg/kg±23 μg/kg was purchased from LGCStandards (Wesel, Germany


    Main instruments and reagents

    • Gilson GX-274 ASPEC® Fully Automated Solid Phase Extraction System (Gilson, Inc.


    • DonStar™ R Free Affinity Column (PN COIAC5004).


    Chromatographic analysis conditions:

    Alliance 2695 HPLC System

    • Mobile Phase: Water: Methanol: Acetonitrile (80:15:5; v/v/v)


    method

    Add 100 mL of deionized water (resistivity 18 MΩ.


    Figure 2 Sample pretreatment steps in trilution®LH software: sample purification and DON elution


    Step 1: Sample purification

    • Add 2 mL filtrate to the immunoaffence column at a flow rate of 0.


    • Wash the immune affinity column with 10 mL of ultrapure water at a flow rate of 2 mL/min and wash twice


    • Due to the low pressure of the nitrogen source in the laboratory, the immunoaffency column


    Step 2: Elution

    • Put the immunoaffe column back into the Gilson GX-274 ASPEC® fully automated solid phase extraction system


    Remove the tubes from the Gilson GX-274 ASPEC® fully automated solid phase extraction system and place in a 45 °C water bath
    .
    Under the nitrogen gas stream, evaporate until dry
    .
    Dissolve the distilled extract in 1.
    0 mL of water, vortex, and subsequently filter
    with a 0.
    45 μm PTFE membrane.
    Collect filtrates for further liquid chromatography analysis
    .
    DON is quantified
    by the external standard method.


    Results and discussion

    In this application, we report sample preparation methods for automated separation of DON from wheat samples
    .
    Sample pretreatment and elution steps were automated using the GX-274 ASPEC® Fully Automated Solid Phase Extraction System, and DON
    was detected using hplc method.
    HPLC assay results show that purification using an immunoaffence column can effectively remove the matrix background (Figure 3
    ).
    Based on the recommendations of the Brazilian National Institute of Metrology, Standardization and Industrial Quality, we have verified
    parameters such as accuracy, precision, detection limits and quantitative limits.
    ³
     

    Fig.
    3 High performance liquid chromatography-UV detection of DO content in wheat samples (λ = 220 nm, DON retention time: 10.
    1 min).


    The detection limit and the quantitative limit are approximately 3 and 10 times the signal-to-noise ratio (S/N), respectively (Table 1
    ).
    The quantitative limits match the first point of the calibration curve (Figure 4
    ).

     

    Table 1 LC/MS analysis conditions
     

    Fig.
    4 Calibration curve (concentration range 50-1000 ng/mL, corresponding to final sample concentration 200-4000 μg/kg)
     

    The accuracy
    of the method was evaluated by the recovery rate test.
    Six replicates of the control sample were performed at a single concentration level to calculate the mean concentration, standard deviation, and coefficient of variation of
    the DON.
    The measured DON recovery rate ranged from 99.
    5% to 104.
    9%, and the average recovery rate of 102.
    0% was calculated, indicating that the method had good accuracy (Table 2
    ).

     

    Table 2 Recovery rates of Fusarenol (DON) for deoxyspermum
     

    Accuracy refers to the degree of dispersion of the results obtained by independent and repeated measurements of the same sample under certain conditions
    .
    It is usually expressed
    as a standard deviation or coefficient of variation value.
    The measurement of accuracy requires the same instrument under the same measuring procedure, the same measuring personnel and the same parameters
    .
    Control samples were tested
    six times on the same day.
    The mean, standard deviation and coefficient of variation for the DON determination are shown in Table 3
    .
    These data indicate that the method has good reproducibility
    .
    Intermediate precision is assessed
    by two inspectors performing six tests on the same sample in two different days.
    The coefficient of variation for the same and different days is below 3% (Table 3
    ).
    The results were analyzed for variance, and the results of different testers in the same laboratory did not differ significantly
    from day to day.

     

    Table 3 Determination of the concentration of Fusarium deoxystenol (DON) in control samples for detection accuracy


    Summarize and apply the advantages

    Automating the step of extracting Fusarium fusarenol (DON) from wheat enables precise control of loading volume and flow rate
    .
    The coefficient of variation is less than 3%, ensuring consistent
    results across batches of samples.
    The average recovery rate was 102%, demonstrating the accuracy
    of the method.

    The methodology is in line with current Brazilian legislation
    .
    A manual step can be easily integrated into the automation process to meet laboratory requirements
    .
    In the same application, TRILUTION®LH software can balance sequential and batch requirements to improve repeatability
    .
    The GX-274 ASPEC® fully automated solid phase extraction system is capable of processing up to 4 samples simultaneously, increasing efficiency and throughput
    .


    Ordering information

    *For other accessories, please consult an authorized Gilson salesperson
    .

     

    References:

    1.
    Almeida, A.
    P.
    , Lamardo, L.
    C.
    , Shundo, L.
    , Silva, S.
    A.
    ,Navas, S.
    A.
    , Alaburda, J.
    , Ruvieri, V.
    , Sabino, M.
    Occurrence of deoxynivalenol in wheat flour, instant noodle and biscuits commercialized in Brazil.
    Food Addit.
    Contam.
    Part B Surveill.
    , 9, 251-255 (2016).

    2.
    Resolução RDC No.
    138 de 8 de fevereiro de 2017.
    Altera a Resolução da Diretoria Colegiada – RDC No.
    7, de 18 de fevereiro de 2011, que dispõe sobre limites máximos tolerados (LMT) para micotoxinas em alimentos, para alterar os LMT da micotoxina deoxinivalenol (DON) em trigo e produtos de trigo prontos para oferta ao consumidor e os prazos para suaaplicação.

    http://bvsms.
    saude.
    gov.
    br/bvs/saudelegis/anvisa/2017/rdc0138_08_02_2017.
    pdf

    3.
    Orientação sobre Validação de Métodos de Ensaios Químicos (Instituto Nacional de Metrologia, Normalização e Qualidade Industrial, 2011).

    4.
    Vocabulário Internacional de Metrologia, Conceitos fundamentais e gerais de termos associados (Instituto Português da Qualidade, 2012).

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