# A Review: Sample Preparation and Chromatographic Technologies for Detection of Aflatoxins in Foods
## 3 Chromatography
### Thin Layer Chromatography
Attempts to couple TLC with mass spectrometry for more specific identification were reported by [PERSON] et al. (2018, 2018); [PERSON] et al. (2018); [PERSON] et al. (2018) and [PERSON] et al. (2018). However, aflat were unable to be directly analyzed by MS requiring extensive clean-up steps to remove aflat from the plate and purified prior to MS analysis. These procedures were time consuming and instrument setups were cumbersome and expensive. In terms of quantitation, TLC visual estimation cannot generate accurate quantitative results. A collaborative study also reported large variability among participating labs using TLC (K  
quench the fluorescence of aflatoxin B\({}_{1}\) and B\({}_{2}\). The method could detect aflat in foods as low as 10-20 ppb. Alternatively, [PERSON] [198] and [PERSON] and [PERSON] [199] used a flow cell of the fluorometric detector filled with silica-gel particles to extract aflat from the mobile phase after LC separation. In this work, FLD detection had the same physical characteristics as that on a TLC plate. Aflatoxins were absorbed and detected on a silica-gel particles solvent, thereby minimizing quenching of fluorescence. Although the addition of the silica gel particles into the flow cell slightly impacted chromatography (e.g., peak broadening), the approach was commercialized and widely integrated with LC-FLD as an indispensable feature for aflatoxin analysis in foods [69; 200; 201; 202]. In the 1980s, reversed phase LC rapidly replaced normal phase LC for aflatoxin analysis. LC separation of aflat on reversed phase columns was straightforward. Generally, mixtures of water, methanol and/or acetonitrile were used as the mobile phase, promoting aflatoxin separation using isocratic conditions. Fluorescence quenching in mobile phases remained a challenge for the detection of aflat at sub ppb levels in foods. The fluorescence emission of aflatoxin B\({}_{1}\) and G\({}_{1}\) could be significantly quenched by aqueous mobile phases utilized in reverse phase LC. To overcome this limitation, a common practice is to derivatize aflat, minimizing the impact of signal quenching. Either precolumn derivatization with trifluoroacetic acid (TFA) or post-column derivatization with a halogen (bromine or iodine), are commonly employed, adding one hydroxyl group or two atoms of the halogen at the 8,9-double bond of the dihydrofuran moiety of aflat. Alternatively, post-column photochemical derivatization can be used for aflatoxin analysis. The findings that aflatoxin B\({}_{1}\) can react with TFA, forming a fluorescing product, aflatoxin B\({}_{2}\)a in aqueous solvents [203; 204] paved the way for TFA-based derivatization, which was mainly used prior to LC separation. [PERSON] et al. [205] thoroughly investigated the hydration of aflatoxin B\({}_{1}\) and G\({}_{1}\) in acidic aqueous solutions in terms of pH, reaction times/rates and product stability. The study confirmed that the double bonds in the dihydrofuran moieties of the aflat B\({}_{1}\) and G\({}_{1}\) could be hydrated, generating reaction products with sufficient fluorescence for detection. However, the study also demonstrated these products were not stable and gradually degraded into non-fluorescing compounds. [PERSON] [188] developed and optimized a TFA-based derivatization for the analysis of aflat in wine. Aflatoxins were extracted from spiked wine using methylene chloride and the concentrated sample extracts were cleaned up on a silica gel-alumina column. After evaporation, the remaining extracts were reacted with TFA for a few seconds, followed by the addition of 10% acetonitrile and LC-FLD analysis. Derivatization products, aflatoxin B\({}_{2a}\), G\({}_{2a}\) and two native aflat, aflatoxin B\({}_{2}\) and G\({}_{2}\) were separated on a 25 cm \(\times\) 3.2 mm I.D., 10 \(\upmu\)m ODS-bonded silica gel column within 15 min. A mixture of methanol and water was used as the mobile phase. Recoveries of the four aflat spiked at 1 ppb ranged from 88%-106%.