# 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
On the LC-MS, recoveries for the aflat spiked at 2 ppb in figs, peanut butter, peanuts, chili, pepper and curry ranged between 40 and 270%. Variability of individual aflat was also unusually high. This study is a reminder of the challenges associated with LC-MS, especially matrix effects, demonstrating how clean-up procedures that work well for LC-FLD might not work for LC-MS. [PERSON] et al. [262] used aflatoxin M\({}_{1}\) as IS for the determination of aflat in corn. Samples were extracted using acetonitrile-water (80:20, _v/v_), followed by dilution and SPE cleanup. Aflatoxins were detected by LC-MS/MS. Quantitation was achieved by matrix-matched standards. Recoveries ranged from 81 to 101% with RSDs \(<12\%\) and the method LODs were from 0.1 to 0.6 ppb. Using internalstandards is a good practice, but whether the selected internal standard would behave similarly to target analytes in different matrices should be carefully evaluated. Without an appropriate internal standard or matrix-matched calibration standard, one would not be able to effectively minimize the impact of matrix effects on quantitation. [PERSON] et al. [263] synthesized and characterized deuterated aflat B\({}_{2}\) and G\({}_{2}\) and used them to compensate the loss of analytes in the course of extraction and matrix effects for LC-MS-based aflatoxin analysis. The two deuterated IS were spiked into samples (almonds and wheat flour) prior to extraction so excellent recoveries and precisions were achieved. Recoveries were between 90% and 105% for all the four aflat with coefficients of variation (CV) of 3.6%, 4.8%, 12% and 14% for aflatoxin B\({}_{1}\), B\({}_{2}\), G\({}_{1}\) and G\({}_{2}\), respectively. As expected, labeled B\({}_{2}\) and G\({}_{2}\) were more suited for the quantitation of aflatoxin B\({}_{2}\) and G\({}_{2}\) than B\({}_{1}\) and G\({}_{1}\) due to similar physicochemical properties between the labeled IS and the native aflat (B\({}_{2}\) and G\({}_{2}\)) and retention times on LC. [PERSON] et al. [264] developed an isotope dilution LC-MS method for the determination of aflat in feeds. Samples were extracted using methanol-water (70:30, _v/v_) or acetonitrile-water (84:16, _v/v_) depending on feed matrices, followed by filtration and dilution. Prior to LC-MS analysis, the extracts were spiked with \({}^{13}\)C-aflatoxin B\({}_{1}\) and aflat were separated on a C18 (2.1 mm \(\times\) 50 mm, 1.7 \(\upmu\)m) column. Detection was performed using a triple quadruple MS. Identification of aflat was achieved by comparing the retention times and relative ion ratios of the two MRM transitions in standards and samples [265]. Quantitation was based on linear least squares calibration of the relative response ratio of an aflatoxin and \({}^{13}\)C-aflatoxin B\({}_{1}\) plotted versus concentration. Recoveries of aflat range from 78 to 122% with intra-day and inter-day precisions of 2-15% and 3-17%, respectively. In this study due to the concern of cost, only one \({}^{13}\)C-aflatoxin was used and spiked into the sample extracts right before LC-MS analysis. Had \({}^{13}\)C-IS been spiked into the samples prior to extraction, probably only one extraction solvent would have been used. As to the cost of \({}^{13}\)C-IS, people often overlook the savings in labor cost and increase in sample throughput [255]. To take advantage of the sensitivity of LC-MS, [PERSON] and [PERSON] [266] used a \"dilute-and-shoot\" procedure to prepare samples for aflatoxin analysis in Brazil nut. Homogenized samples (25 g) were first extracted using 100 mL of acetonitrile-water (80:20, _v/v_) and further diluted 4\(\times\) using water prior to LC-MS analysis. Matrix-matched calibration standards were used for quantitation. Recoveries of spiked aflat at 1-10 ng/g ranged between 92 and 100% with RSDs \(<\) 10%. The method combined a simple sample preparation procedure and an effective solution to matrix effects together in a practical manner. Similar approaches have been reported in other studies [267; 268].