# A Review: Sample Preparation and Chromatographic Technologies for Detection of Aflatoxins in Foods
## 3 Chromatography
### Liquid Chromatography and Mass Spectrometry
#### 3.3.2 LC-MS for Quantitative Analysis  
Since the later 1990s, modern LC-MS instruments, especially LC-triple quadrupole (LC-QQQ), has been developed with increasing sensitivity for quantitative analysis of various organic chemicals in foods. Despite high capital costs of LC-MS instruments, many efforts have been exerted to quantitate aflat using this technique [253]. One of the challenges of LC-MS based mycotoxin analysis is matrix effects. In the course of the LC-MS analysis, signals of aflat could be significantly suppressed by coeluting matrix components, an observation often referred to as \"matrix-effects\" [254]. If solvent-only calibration standards are used for quantitation, the differences in signal suppression or enhancement in solvent, which is free of co-eluted matrix components, compared to a sample matrix could lead to inaccurate quantitative results. Suppression is mainly caused by insufficient ionization of aflat due to the competition of charges from co-eluted matrix components in the ion source. Matrix effects can be experimentally circumvented by diluting the sample matrix, in-depth cleanup, standard addition, matrix-matched calibration and/or the use of internal standards, especially \({}^{13}\)C-labeled aflat [255], but the solution is a balance of data quality, sample throughput and cost [256]. Dilution seems to be a convenient approach to mitigate matrix effects, but it requires sensitive LC-MS [257] and dilution factors are matrix-, instrument- and analyte-dependent. Additionally, regulatory levels of aflat should not be overlooked. For example, the current FDA action level of aflatoxin M\({}_{1}\) is 0.5 ppb in milk. If a 10\(\times\) dilution is necessary to eliminate matrix effects, the LC-MS instrument must be able to quantitate the aflatoxin below 0.05 ppb [89]. When factoring in EU's regulatory levels of aflatoxin M\({}_{1}\) (0.025-0.05 ppb), more sensitivity is required if samples are diluted prior to LC-MS analysis. While such sensitivity is not possible for the majority of commercial LC-MS instruments, concentration and clean-up steps are necessary to ensure the detection of aflatoxin M\({}_{1}\) at and below the regulatory levels [258,259]. In-depth sample clean-up could be used to eliminate matrix-components/interferences but very often the process is lengthy and laborious. Sometime, even with immunoaffinity columns, LC-MS analysis was not free from matrix effects [142]. Matrix-matched calibration standards are commonly used to achieve satisfactory quantitative results. In this approach, known amounts of aflat are spiked into \"clean\" matrix and used as calibration standards. This is expected to bring same/similar impact of matrix effects on the response of aflat in both calibration standards and samples. The preparation of matrix-matched calibration standards is tedious, especially when one needs to deal with a wide variety of food matrices [57,260]. Standard addition would be straightforward only if one analyte needs to be quantitated. When using the approach to quantitate the four aflat at various concentrations in a sample, it becomes a challenging operation for analysts. The ideal solution for matrix effects would be stable isotope dilution assay (SIDA). Using commercially available \({}^{13}\)C-aflatoxins or deuterated aflat as internal standards (IS), one could quantitate native aflat with solvent only calibration standards [256]. Very often extraction solvents and cleanup procedures have to be optimized depending on the matrix to ensure extraction efficiency. The incorporation of labeled IS into the sample prior to extraction and clean-up provides greater flexibility in sample extraction conditions across multiple sample matrices. [PERSON] and [PERSON] [261] developed an LC-MS method for the determination of aflat B\({}_{1}\), B\({}_{2}\), G\({}_{1}\) and G\({}_{2}\) in food and interestingly, they used aflatoxin M\({}_{1}\) as the internal standard. Samples were extracted with methanol and further cleaned by column chromatography, previously developed for LC-LFD.