# A Review: Sample Preparation and Chromatographic Technologies for Detection of Aflatoxins in Foods
## 2 Sample Preparation
### Sample Homogenization--Dry, Wet and Cryogenic Grinding
#### 2.1.2 Wet Grinding  
Wet grinding is performed by mixing and blending samples and water or extraction solvents together. Careful efforts should be invested to optimize solvent/sample ratios, which is critical to homogeneity and extraction efficiency. [PERSON] and [PERSON] [36] evaluated wet grinding procedures for aflat analysis in peanut, whole seeds, peanut and cottonseed meals and copra. Using a matrix-dependent sample to water ratios (e.g., 1:1.5 for peanut butter, 1:2.25 for whole seeds, 1:4 for commercial corn and cottonseed meals and 1:5 for copra), the authors demonstrated that making slurries of aflatoxin samples led to more uniform distribution of particles and thus better reproducibility among test portions than conventional dry millings. In order to improve extraction efficiency of aflatoxin from raw peanuts, [PERSON] et al. [49; 50] conducted a series of tedious studies by testing 49 different combinations of methanol concentrations and solvent/peanut ratios. It was concluded that a combination of 60% methanol and a 10.8 mL solvent/g peanuts ratio might be used to achieve optimal extraction efficiency for aflat. The distribution of aflat in heterogeneous test matrices was evaluated by [PERSON] and [PERSON] (1996) using pistachio samples in the shell [51]. In this work, pistachios samples (up to 3 kg per sample) were homogenized with water and sodium chloride (150 mL tap water and 4 g NaCl/100 g sample) and blended for 30-45 min. The formation of a wet slurry provided a homogenous matrix, which is difficult to achieve for pistachios using dry milling. Another benefit of this procedure was to avoid manually shelling pistachios, a painstaking effort to prepare tree nuts samples with shells for dry milling. [PERSON] et al., 2006 [48] reported the milling and homogenization processes for cocoa, green coffee, almonds and pistachio, where the authors demonstrated the effect of dry milling and wet milling. The homogenization process was evaluated in terms of the analytical results, coefficients of variation for different mills, sample and subsample sizes and particle size distributions. The study concluded that the coefficients of variation were higher for dry milling than those for slurry mixing. As an extension of the study in 2006, [PERSON] et al. [43] applied the developed wet milling method to matrices with sample sizes greater than 1.5 kg to ensure homogeneity of the final samples for LC-MS/MS analysis. In a similar study, [PERSON] et al., 2003 [52] compared the slurry mixing method to dry grinding procedures in pistachios. The results demonstrated that after mixing the pistachio sample with water (sample/water mass ratio, 1/2), the resulting coefficient of variation is less than half that of the dry grinding. Interestingly, the study also suggested that wet grinding would enhance the efficiency of extraction of aflat from smaller particles. Therefore, aflatoxin concentrationsdetermined in some matrices using wet grinding could be higher than those done by dry milling. Similar observations were reported in other studies [36; 53], underlying the effects of sample sizes, matrix compositions, as well as grinding devices/conditions should be carefully evaluated. In a recent study, [PERSON] et al. [54] demonstrated the analytical variances associated with testing a 25.0 g slurry (1:1, sample:water), 50.0 g dry grind and 12.5 g dry grind sample portion in the ratio of 1:5:15, suggesting that the wet grinding could provide improved homogeneity for cereals. In recent years, LC-MS-based mycotoxin analysis has been increasingly used. Due to its superior sensitivity, test portions (0.5-5 g) for LC-MS analysis are much smaller than those of LC-Ultraviolet/Fluorescence detection (LC-UV/FLD) (25-50 g). To ensure test portions would give representative information of the sample, [PERSON] et al., 2017 [55] developed a secondary fine-milling procedure for LC-MS/MS-based multimycotoxin analysis.