Sample preparation: Standard aflatoxin M1 was purchased from Sigma Aldrich. All other chemicals used were commercially available and of LC-MS grade. Milk samples were purchased from local vendors and were stored in the refrigerator at 2 to 8 °C until sample analysis.
An optimized extraction procedure was developed in which 5 mL of milk was mixed with 10 mL of acetonitrile and vortexed for 10 min. Then, 2 g of NaCl was added, vortexed for 5 min and centrifuged at 3200 rpm for 5 min at 4 °C. The organic layer was collected and evaporated to dryness under nitrogen. The residue was reconstituted with 1 mL with 90:10 water/methanol, filtered and transferred into autosampler vials for LC-MS/MS analysis.
Chromatography: Separation was performed using an ExionLC system with a Kinetex C18 column (1.7 µm, 100 Å, 150 x 2.1 mm). An injection volume of 20 µL was used. The gradient program is shown in Table 1.
Mass spectrometry: The SCIEX QTRAP 4500 system was operated in multiple reaction monitoring (MRM) mode. The Turbo V ion source was used with an electrospray ionization (ESI) probe, in positive polarity at an ion spray voltage of 2800. The MRM transitions used are shown in Table 2. Analyst software 1.7 was used for method development and data acquisition.
Data processing: LC-MS/MS data was processed using the MultiQuant software 3.0.3.
First, the chromatography was optimized to provide a fast 7.5 min run time (Figure 1). Calibration curves were generated across a concentration range of 0.15 to 1.50 ppb of aflatoxin M1 in extracted milk matrix to determine the performance of the method. A LOD of 0.15 ppb was achieved in extracted milk and the %CV (n=10) was still <5%. Good linearity across the range was observed (Figure 2). Reproducibility was evaluated between and within days with the 6 replicates injections and the %CV was found to be <5%, which proves the method is robust and reproducible below its MRL level (0.50 ppb) (Figure 3, bottom). No significant matrix interferences were observed (Figure 3, top). Ion ratios were used to ensure that the correct peaks were integrated in each sample, for added confidence in detection (Figure 4).
The method developed here enables the fast and accurate quantification and confirmation of aflatoxin M1 in extracted milk samples by LC-MS/MS. The SCIEX QTRAP 4500 system provides good sensitivity and selectivity for this analysis, with minimal sample preparation, providing maximized throughput for the analysis of large sample batches in a short time period. The method was developed in accordance with the Commission Decision (2002/657/EC) directive recommendations and showed acceptable accuracies (70%-130%) across the calibration curves in matrix, had good linearity for both the MRM transitions and had high repeatability with %CV < 5%.
This rapid and sensitive LC-MS/MS method for the quantification of aflatoxin M1 in milk also easily meets the MRL of 0.5 ppb level for this toxin in milk.