Development, Optimization and Validation of LC-MS/MS Method for Multi-Mycotoxin Detection in Cereals

Mycotoxins are fungal natural metabolites that have a wide range of toxic effects. Among hundreds of mycotoxins, aflatoxins (AFs) (AFB1, AFB2, AFG1, and AFG2), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB1 and FB2,), T2 and HT2-toxins are the major health concerns for humans and domestic animals. First, an HPLC method has been developed to investigate the separation of mycotoxins in liquid chromatography. Two derivatization systems, photochemical and chemical methods were applied for derivatization of AFB1 and AFG1, as well as FB1 and FB2, respectively. Then, a LC-MS/MS method has been developed by evaluating the effect of LC column (50 and 150 mm), organic modifier (methanol and acetonitrile) ionization process (ESI, APCI) and ionization mode (positive and negative) on separation and determination of mycotoxins. Then the developed method was optimized for simultaneous determination of the 11 mycotoxins. Response surface methodology (RSM) was used to optimize the LC conditions. The effect of organic solvent percentage at the beginning (0-20%) and end (75-95%) of gradient mobile phase, acid concentration in aqueous phase (0-1%), and flow rate (100-300 μl/min) have been investigated for optimization of LC responses peak area and signal to noise ratio (S/N). The optimized responses obtained using following conditions: organic solvent of 5% at start and 95% at the end of gradient mobile phase, 0.1% acid concentration, and 250 μl/min flow rate. In addition, best sample preparation procedure have been selected by evaluating the effects of two different common types of solvent extraction methods (one step and two step extraction) and four types of clean-up methods including Oasis HLB, MycoSep, immunoaffinity column (IAC) and no clean-up on mycotoxins recoveries. The results of the study showed that the best recoveries (79-109%) for all mycotoxins would be obtained by using one step extraction with no clean up. Finally, the optimized LC-MS/MS method was validated by measuring the selectivity, sensitivity, linearity, accuracy and precision. Limit of Detection (LOD) for AFB1, AFB2, AFG1, AFG2, DON, T2-Toxin, HT2-Toxin, FB1, FB2, OTA and ZEA was 0.05, 0.25, 0.05, 0.5, 5, 2, 2, 10, 10, 0.01, and 0.1, whereas the Limit of Quantification (LOQ) was 0.1, 0.5, 0.1, 1, 10, 4, 4, 20, 20, 0.02, and 0.2 ppb, respectively. Finally, the optimized and validated LC-MS/MS method was applied on real cereal samples (rice, barley, oat, wheat and maize) collected from Malaysian markets. The results showed applicability of the aforementioned method for being used as fast routine method with high accuracy and precision for simultaneous determination of mycotoxins in cereal

Validation of the procedure for the simultaneous determination of aflatoxins ochratoxin A and zearalenone in cereals using HPLC-FLD

Method validation for quantitative analysis of aflatoxins (AFs), ochratoxin A (OTA) and zearalenone (ZEA) in cereals using HPLC with fluorescence detector (FLD) is described. Mycotoxins were extracted with methanol : water (80 : 20) and purified with a multifunctional AOZ immunoaffinity column before HPLC analysis. The validation of the analytical method was performed to establish the following parameters: specificity, selectivity, linearity, limits of detection (LOD) and quantification (LOQ), accuracy, precision (within- and between-day variability), stability, robustness, measurement of performance, and measurement of uncertainty. Calibration curves were linear (r > 0.999) over the concentration range, from the LOQ to 26, 40 and 400 ng/g for AFs, OTA and ZEA, respectively. LOD and LOQ were 0.0125 and 0.05 ng/g for aflatoxin B1 (AFB1) and G1 (AFG1), 0.0037 and 0.015 ng/g for aflatoxin B2 (AFB2) and G2 (AFG2), as well as 0.05 and 0.2 ng/g for OTA and 0.5 and 2 ng/g for ZEA, respectively. The mean recovery values were 77-104% for different concentrations of AFs, OTA and ZEA in spiked cereal samples. Both intra- and inter-day accuracy and precision were within acceptable limits. This method was successfully applied for the simultaneous determination of mycotoxins for 60 cereal samples collected from Malaysian markets. Fifty per cent of the cereal samples were contaminated with at least one of these mycotoxins, at a level greater than the LOD. Only one wheat sample and two rice samples were contaminated with levels greater than the European Union regulatory limits for AFs and OTA (4 and 5 ng/g). The means and ranges of mycotoxins obtained for the cereal samples were 0.4 ng/g and 0.01-5.9 ng/g for total AFs; 0.18 ng/g and 0.03-5.3 ng/g for OTA; and 2.8 ng/g and 2.4-73.1 ng/g for ZEA, respectively. The results indicate that the method is suitable for the simultaneous determination of AFs, OTA and ZEA in cereals and is suitable for routine analysis

Optimization and validation of a HPLC method for simultaneous determination of aflatoxin B1, B2, G1, G2, ochratoxin A, and zearalenone using experimental design.

A reversed-phase HPLC optimization strategy is presented for investigating the separation and retention behavior of aflatoxin B 1, B 2, G 1, G 2, ochratoxin A and zearalenone, simultaneously. A fractional factorial design (FFD) was used to screen the significance effect of seven independent variables on chromatographic responses. The independent variables used were: (X1) column oven temperature (20-40°C), (X2) flow rate (0.8-1.2 ml/min), (X3) acid concentration in aqueous phase (0-2%), (X4) organic solvent percentage at the beginning (40-50%), and (X5) at the end (50-60%) of the gradient mobile phase, as well as (X6) ratio of methanol/acetonitrile at the beginning (1-4) and (X7) at the end (0-1) of gradient mobile phase. Responses of chromatographic analysis were resolution of mycotoxin peaks and HPLC run time. A central composite design (CCD) using response surface methodology (RSM) was then carried out for optimization of the most significant factors by multiple regression models for response variables. The proposed optimal method using 40°C oven temperature, 1 ml/min flow rate, 0.1% acetic acid concentration in aqueous phase, 41% organic phase (beginning), 60% organic phase (end), 1.92 ratio of methanol to acetonitrile (beginning) and 0.2 ratio (end) for X1-X7, respectively, showed good prediction ability between the experimental data and predictive values throughout the studied parameter space. Finally, the optimized method was validated by measuring the linearity, sensitivity, accuracy and precision parameters, and has been applied successfully to the analysis of spiked cereal samples

An econometrics method to estimate demand of sugar

Sugar is one of the strategic goods in the basket of households in each country and it plays an important role in supplying the required energy. On the other hand, it is one of the goods, which Iranian government is about to change its subsidy strategies. To design useful sugar subsidy strategies, it is necessary to know sugar position in the basket of households and be familiar with households' sugar demand or consumption behavior. This research estimates sugar demand for Iranian households by using time series of 1984-2008, which is taken from central bank of Iran. In this paper, first independent and dependent variables of household sugar demand model are chosen based on the literature review and theory of demand. Then, sugar demand is estimated by OLS technique and linear regression. The preliminary statistical observations such as Durbin-Watson, F statistic and R2 indicate that the regression is admissible. The results seem plausible and consistent with theory and show that sugar demand in Iranian households is associated with household expenditure, relative sugar price, family size and indicate that demand of sugar is affected during the war time. The results also show the income elasticity is 0.8 and price elasticity is -0.2 which means sugar is essential good for Iranian households and is inelastic to price

Multidetachment analogue models of fold reactivation in transpression : the NW Persian Gulf

Two deformation events have been documented in the NW Persian Gulf during the Late Cretaceous and the Late Cenozoic. The most distinctive feature in this part of the Persian Gulf is the reactivation of the Late Cretaceous NNE-SSW Arabian trending folds by NE-SW shortening during the Late Cenozoic Zagros orogeny. In general, if a set of folds with horizontal axes is shortened roughly parallel to its fold axis, a dome-basin fold interference pattern is produced. In the NW Persian Gulf, reactivation of some old folds occurred instead of a fold interference pattern. Reactivation may be influenced by the following factors: i) the presence of incompetent layers (i.e. evaporites), ii) a variable overburden, iii) basement faults, and iv) obliquity between the younger deformation shortening axis and fold axis. It is this last factor that we investigated by means of analogue modelling. The experimental apparatus is similar to that commonly used for experiments with brittle-ductile systems at the Laboratory of Experimental Tectonics of Géosciences Rennes. The model consisted of an alternation of ductile and brittle horizontal layers with a stratigraphy similar to the one found in the NW Persian Gulf. The model was deformed by two deformation events with an angle a between the two directions of shortening. After deformation, the resulting structure resembled a fold facing the static wall with internal thrust faults and detachment faults arranged into a geometry similar to a fish tail. In the second shortening event, the fold was reactivated without formation of an interference pattern. Moreover, the displacement on both the reactivated and newly formed faults varied between almost pure thrust faults for low a and oblique thrust faults with a strike-slip component for high a. The models suggest that the presence of incompetent layers plays an important role in fold reactivation and confirm that basement faults are not necessary