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Mycotoxin Analysis in Commercial Baby Foods using HPLC-MS/MS Method -…

- - - - (i) sample was spiked with working mixture of mycotoxin (ii) spiked sample left for 3 hours (RTP)
      - why spiked sample was left for 3h?
        
        (a) to let the solvent to evaporate (b) to establish equilibrium between the spiked mycotoxins and the baby food samples
  - - - on a system consist of
        
        Agilent 1200 chromatograph coupled to a 3200 QTRAP mass spectrometer equipped with a turbo ion-spray electrospray ionization (ESI) interface
    - - eluent A (mobile phase A) : slightly acidified with 0.1% formic acid & 5mM ammonium formate eluent B (mobile phase B): Methanol with 5mM ammonium formate
    - - Start: 0% eluent B
        in 10min: increased 100%
        in 5min: decreased to 80%
        in 2min: decrease to 70%
    - - ion spray voltage: 5500v
        curtain gas: 20 (arbitrary units)
        GS1 & GS2: 55 & 65 psi
        probe temperature (TEM): 500 degree celsius
      - performed using Turbo V ionspray in positive ionization mode (ESI+)
      - nitrogen: as nebulizer & collision gas
    - - MS operated in SRM mode
        
        optimization of MS parameters was performed by flow injection analysis for each compound; entrance potential (EP) (10V) & collision cell exit potential (CXP) (4V)
      - MS/MS data acquisition also performed in SRM mode for increased sensitivity & selectivity
  - - - Matrix matched calibration - used for reliable quantitative determinations
        
        the linearity was calculated using matrix-matched curves prepared by spiking 3 different baby foods
        
        analyzed them in triplicate at 6 concentration level: from LOQ to 100xLOQ
        
        the experiment was repeated for 10x within a day for intra-day precision test. the test performed for 5 days for inter-day test.
        
        LODs & LOQs: determined by the analysis of decreasing concentration of the spiked baby food
    - - Matrix effect (ME): matrix matched calibration slope (B)/ slope of the standard calibration in solvent (A)
    - - if >100% : there is signal enhancement
        if <100% : there is signal suppression
- - - - When the ESI+ mode was used, good sensitivity was obtained for a few mycotoxins.
      - For all mycotoxins, the base peak was [M+H]+except for trichothecene type A
      - Table 2 presents the optimum parameter values for each analyte as well as the two most important SRM transitions.
        
        1st transition - quantification purposes
        2nd transition - to confirm the presence of targeted compounds in the sample
        
        mycotoxin patulin has not been included
        
        Because mycotoxin is mostly found in apples and apple products. This fruit was merely a minor element in some of the baby food samples that were chosen. As a result, it was considered that if patulin was present, it would only be in trace amounts.
        
        One of the major purposes of this method was to identify as many mycotoxins as possible in a single run.
      - To compare the performance of the QTRAP's two operating modes (triple quadrupole and triple quadrupole linear ion trap)
        
        IDA method was developed
        
        The first experiment used an SRM approach that included the target compounds' most common transitions.
        
        The inclusion of this IDA experiment allowed for a clear identification of the mycotoxins included in the matrix.
  - - - The validation of the technique should be done in line with the analytical method's performance requirements.
        
        compared both of this in order to evaluate matrix effects in baby foods
        
        External matrix-matched calibration and Internal standard calibration
        
        These matrix effects were dependent not only on the cereal composition but also on how it was presented, based on the considerable differences in matrix effect values within and between baby foods.
        
        As a result of these findings, it became important to examine matrix effects as part of the method validation process in order to assure the results' reliability.
        
        The use of isotopically labelled internal standards, on the other hand, is the only option to assure great precision in the findings.
        
        The matrix-matched calibrations were performed for effective quantification to avoid any over-oror under-estimation of residues due to the variability of matrix influence for different chemicals among the samples tested and given in this research.
  - - - Validation parameters were obtained using 10 replicates for each of the three different sample presentations, taking into consideration the dependency of mycotoxin behavior on sample presentation and cereal composition, as well as the similarity of this dependence to matrix effects.
  - - - Every batch of samples was evaluated to internal quality control to ensure that the analytical system was in proper working order.
        
        To ensure the proposed method's validity, internal quality control includes a matrix-matched calibration, a reagent blank, a matrix blank, and a spiked blank at the LOQ concentration level.
        
        As an additional confirmation technique, IDA experiments were performed on positive mycotoxin samples.
      - A total of 17 baby food samples had positive results (i.e., samples that contained mycotoxins). The most often discovered mycotoxins were BEA (15 positive samples out of 35 total tests) and DON (9 positive samples out of 35 total samples).
        
        BEA has been verified in baby food by several authors, requiring its addition in a multi-mycotoxin analysis of baby food.
- - - - liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using a hybrid triple quadrupole-linear ion trap mass spectrometer (QTRAPÒ).