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Can Nonmatching Mycotoxin SIDA Internal Standards Be Used for Stable Isotope Dilution Assay Calibration?

Mycotoxins are secondary metabolic products of fungi that can pose a health risk when present in the food supply. Among the many analytical techniques that have been developed to detect the presence of mycotoxins in a wide variety of matrices, LC-MS/MS has emerged as a very popular option for routine food safety analysis. However, any MS technique that utilizes electrospray ionization is challenged by the severe effects coeluting matrix components can have on the ionization efficiency of mycotoxins. Signal enhancement or suppression can occur, depending on the given mycotoxin, the matrix, and the LC separation of the two. Sample preparation techniques can be used to try to eliminate the presence of the matrix, but the need for time-consuming, matrix-specific sample preparation methods is an impediment to the streamlined workflows most food safety labs desire. One strategy to overcome the poor accuracy and precision that unchecked matrix effects cause is to account for those matrix effects through calibration techniques like stable isotope dilution assay (SIDA).

Detailed descriptions of SIDA calibrations are reported elsewhere [1], but the use of isotopically labeled internal standards (ISs) that are subjected to the same extraction, chromatographic separation, ionization, and detection conditions as the target mycotoxins offers an excellent way to account for any losses during sample preparation or ionization effects due to coeluting matrix components. Excellent precision and accuracy are typically observed when using mycotoxin SIDA calibration, but the technique is not without its drawbacks: isotopically labeled ISs are very expensive and ISs may not be commercially available for every target mycotoxin.

Because of those potential obstacles to using mycotoxin SIDA calibration, there may be an interest in using one isotopically labeled IS for the quantitation of multiple target mycotoxins, not just that one IS’s analogous target mycotoxin. While this is not an unusual practice, our analysis of two externally procured reference standards of maize flour, each containing four mycotoxins, illustrates the risk of trying to use a single labeled IS to quantify multiple target mycotoxins.

Table I compares the observed concentrations of the four different mycotoxins present in the reference standards with those reported by the reference standard supplier. For the three mycotoxins that were quantified using their corresponding isotopically labeled ISs, the agreement between the two values is excellent. However, in the case of the mycotoxin zearalenone, where we did not have its analogous labeled IS, we tried to quantify using the closely eluting 13C17-aflatoxin G1 IS, which we did have, even though aflatoxin-G1 was not one of the four mycotoxins in the reference material. For zearalenone quantified with another mycotoxin’s labeled IS, the agreement between the observed results and those reported by the reference material supplier is very poor, which illustrates that similar chromatographic retention alone is not enough to predict the effects of sample preparation and/or matrix-related changes in ionization efficiency. A more detailed description of Restek’s research on the LC-MS/MS analysis of mycotoxins in various foods comparing SIDA to matrix-matched calibration is published in a peer reviewed study [2].

In light of these results, it is strongly recommended that isotopically labeled ISs only be used to quantify matching target mycotoxins. If matching ISs are not available, other calibration approaches, like matrix matched calibration, should be used.

Table I: Mycotoxin SIDA calibration should only be used for matching analyte and IS pairs.

Reference
Material

Analyte

IS

Measured
Concentration
(ng/g), n=3

Assigned
Concentration
(ng/g)

Percent
Accuracy
(RSD, %)

TET017RM

Deoxynivalenol

13C15-Deoxynivalenol

1867.9 ± 37.36

1971 ± 195

94.8 (2)

TET017RM

Aflatoxin B1

13C17-Aflatoxin B1

8.68 ± 0.434

9.49 ± 0.85

91.4 (5)

TET017RM

Ochratoxin A

13C20-Ochratoxin A

4.48 ± 0.134

4.81 ± 0.75

93.2 (3)

TET017RM

Zearalenone

13C17-Aflatoxin G1

31.26 ± 2.19a

231 ± 25

13.5 (7)a

T04301Q

Deoxynivalenol

13C15-Deoxynivalenol

639.7 ± 19.19

649 ± 222

98.6 (3)

T04301Q

Aflatoxin B1

13C17-Aflatoxin B1

8.69 ± 0.348

9.21 ± 4.05

94.4 (4)

T04301Q

Ochratoxin A

13C20-Ochratoxin A

2.81 ± 0.197

3.03 ± 1.33

92.6 (7)

T04301Q

Zearalenone

13C17-Aflatoxin G1

16.2 ± 0.810a

138.5 ± 59.6

11.7 (5)a

a Results quantified using a nonmatched labeled IS.


References

[1] K. Zhang, K., M.R. Schaab, G. Southwood, E.R. Tor, L.S. Aston, W. Song, B. Eitzer, S. Majumdar, T. Lapainis, H. Mai, K. Tran, A. El-Demerdash, V. Vega, Y. Cai, J.W. Wong, A.J. Krynitsky, T.H. Begley, A collaborative study: determination of mycotoxins in corn, peanut butter, and wheat flour using stable isotope dilution assay (SIDA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), J. of Agric. Food Chem. 65 (33) (2017) 7138–7152. DOI: 10.1021/acs.jafc.6b04872 https://pubs.acs.org/doi/10.1021/acs.jafc.6b04872
[2] D. Li, J.A. Steimling, J.D. Konschnik, S. Grossman, T. Kahler, Quantitation of mycotoxins in four food matrices comparing stable isotope dilution assay (SIDA) with matrix-matched calibration methods by LC–MS/MS, J. AOAC Int. (2019) DOI: 10.5740/jaoacint.19-0028 https://doi.org/10.5740/jaoacint.19-0028

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