# A Review: Sample Preparation and Chromatographic Technologies for Detection of Aflatoxins in Foods
## 2 Sample Preparation
### Sample Homogenization--Dry, Wet and Cryogenic Grinding
#### 2.1.3 Cryogenic Grinding  
In aflatoxin determination, cryogenic milling (cryo-milling) has potential applications as it permits heat sensitive materials to be effectively ground into very small particle sizes [56]. The utility of cryogenic milling has been demonstrated for predominantly fatty food and feed (e.g., groundnuts and derived products, almonds, pistachios, Brazil nuts, maize, rice, figs, cotton seed and spices) matrices [57]. Furthermore, cryogenic milling also retains the nature of the matrix more than dry or wet milling. This is very important for the preparation of proficiency test samples and reference materials [58; 59]. The excessive heat generated by dry milling or the addition of water in wet milling modifies the physical composition of starting materials, compromising of the quality of the final reference material. For example, to prepare starting materials for a National Institute of Standards and Technology (NIST) standard reference material (SRM 1565), [PERSON] et al., 2019 [59] used cryogenic milling to homogenize and blend 70 kg of blank corn and 14 kg of incurred corn. The analysis of particle size distribution shows that in the final blank and incurred corn, the average particle sizes are 588 \(\upmu\)m with a standard deviation of 17 \(\upmu\)m (\(n=10\)) and 676 \(\upmu\)m with a standard deviation of 92 \(\upmu\)m (\(n=10\)), respectively. Due to the homogeneity of the material, test portions ranging from 1 to 5 g would be enough to ensure analytical results are in a good agreement with certified values. Currently, to ensure homogeneity of test samples and accommodate a wide range of analytical procedures used by participating labs, many proficiency test organizers use cryogenic milling to prepare mycotoxin materials [58]. Cryogenic milling requires special milling devices and dry ice or liquid nitrogen as the cooling agent. To achieve better grinding, samples are preferred to be stored in freezers overnight before grinding. After grinding, samples cannot be directly extracted to allow sufficient time for CO\({}_{2}\) to sublime. Prior to extraction or weighing, samples also need to equilibrate to room temperature otherwise moisture in the air could condense on the samples. Though dry milling has been preferred by many labs due to it being easy for use, it is noted that wet or cryogenic grinding could be successfully applied to accommodate food matrices that are not suited for dry milling. The heat generated during dry grinding resulted in the formation of clogging, which may hinder the formation of homogenous particle size across the matrix. Whereas, in the case of wet or cryogenic grinding, the addition of water and dry ice to the matrix allows the formation of particles with smaller and more uniform sizes and therefore homogenous distribution of aflat. Wet grinding appears superior in providing a uniform particle size distribution and a homogeneous matrix, but it has certain disadvantages, which includes difficulties in cleaning the grinding apparatus. Regardless of grinding techniques used,one of the continuing challenges is to characterize the correlation between homogeneity of the final sample and grinding conditions (devices, milling time, sample type, sample size, subsample size and test portion size). Use of sample sizes too small can lead to biased analysis of aflat with high variability. On the other hand, using unnecessary large sample sizes could increase operation cost and impact throughput when a large number of samples need to be processed. Therefore, in the course of routine sample analysis or evaluation of new homogenization devices/protocols, a homogeneity test and quality control should be performed to ensure that the homogenization process could meet the predefined degree of fineness in the final sample. In some situations, conventional homogeneity protocols may be cumbersome [60], so it is important to explore and adopt existing technologies such as light scattering to monitor the homogeneity of processed samples in an efficient manner [59; 61].