Determination of Aflatoxins G₁ and G₂ Using Ion Mobility Spectrometry

Introduction

Aflatoxins (AF) belong to the group of mycotoxins, toxic fungal metabolites found as contaminants in various agricultural commodities under favourable temperature and humidity. AFs are difuranocumarine derivatives produced by Aspergillus Flavus and Aspergillus Parasiticus through a polyketid path way^1–3. These compounds are carcinogenic, mutagenic and teratogenic to the most animals and humans. Among 20 identified AFs, the four major AFs are B₁, B₂, G₁ and G₂ (Fig. 1) named based on their fluorescence under UV light (blue or green) and relative chromatographic mobility during TLC⁵. Those AFs occur naturally in wide variety of agricultural products including cereals, nuts and dried fruits. Pistachio nuts are especially sensitive samples to AFs contamination. Some reports indicate natural contamination with AFB and AFG mainly in countries with warm and humid climate^6-8.

In order to control AFs levels in agricultural products, several analytical methods have been developed TLC [9], HPLC with different detectors^10-13 and enzyme linked immunosorbent assay¹⁴ are the most commonly used. These methods are usually time consuming and require harmful solvents and well equipped laboratories. Therefore, it is still necessary to develop a sensitive, rapid and low cost method for the determination of AFs.

Here we report a method for determination of AFs ion mobility spectrometry (IMS). IMS is a gas phase ion separation technique that allows analytes to be identified on the basis of ion mobility. The principles of IMS are well described in book and literature^15,16. In brief IMS characterizes chemical compounds using gas phase motilities of ions in weak eclectic fields at ambient pressure. The mobility of an ion is determined by the ion velocity that is measured in the drift tube of the spectrometer where an electric filed is applied. During their drift, ions are separated based on their size, shape, and charge. Therefore, different ions reach the detector at different drift times, which are their characteristic. The number of ions reach to the detector is considered as a measure of the analytes concentration. IMS has applied for the rapid and sensitive detection of trace amounts of a broad range of compounds.^17-20

We previously, reported the capability of IMS equipped with positive corona discharge ionization source in determination of AFB₁ and AFB₂ in pistachio nut as a real sample¹⁸. Here, the method was extended for the determination of AFG₁ and AFG₂ and also the simultaneous determination of AFB and AFG in pistachio sample investigated.

Figure 1 Click here to View figure

 

Experimental

Materials and Chemicals

Pistachio samples were supplied by Pistachio Co. (Rafsanjan, Iran). The materials and solvents used for this research were purchased from Merck. AFs (B and G) were obtained from sigma Co. Stock standard solutions were prepared by dissolving analytes in methanol. The concentration of the solutions was determined using a UV-vis instrument as explained in Ref.²¹ and stored below 5 ^ºC until use. Working standard solutions were prepared by diluting stock standard in methanol. Immunoaffinity columns (Afla Test) were supplied by Vicam of USA.

Instrumentation

The ion mobility spectrometer used in this work was constructed in Isfahan University of Technology. The spectrometer, equipped with corona discharge ionization source was operating in the positive mode. The instrument was described in details in Ref.¹⁷. The newly designed injection port¹⁸ for direct introducing liquid samples into the IMS was used. The injection port was filled with steel wool to enhance evaporation rate of the solvent and analyte. A UV-vis spectrophotometer (Shimadzu, 160-A, Japan) with 1 cm matched quartz cells was also used to verify the concentration of AF standard solutions.

Figure 2 Click here to View figure

Sample Preparation

The extraction and purification of AFs were performed based on the AOAC method²². 25 g grounded pistachio sample was used for each experiment. 5 g of NaCl and 125 mL of methanol solution (70%) were added to the grounded pistachio sample. The mixture was then blended at high speed for 2 min and filtered. 15 mL of the filtered solution was then mixed with 30 mL of water and filtration repeated. 15 mL of filtered solution was passed through Afla test affinity column at a flow rate of 1–2 drops per second. The column was twice washed with 10 mL water and dried with air. Finally, it was eluated by passing 1 mL methanol. This solution was then used for determination of AFs using IMS method.

Figure 3 Click here to View figure

 

Results and Discussion

Optimization of the IMS Operation Parameters

Preliminary studies indicated the possibility of using IMS to determine AFG. AFG₁ and AFG₂ were separately injected into the IMS cell. Fig. 2 shows the ion mobility spectrum of AFG1 which contains only one peak. The spectrum of AFG₂ was similar to AFG₁, i.e, a single peak appeared at a drift time identical to that of AFG₁. Based on their structures (Fig. 1) and molecular weight (328 and 330), this behavior was predictable. IMS is not able to resolve two ions with similar structure that differ by only 2 amu. This was the case in our previous work, where it was shown that AFB₁ and AFB₂ have identical spectra¹⁸.

Figure 4 Click here to View figure

 

To obtain the best sensitivity for determination of AFG the parameters such as the corona and drift voltage, the injection and cell temperature, the flow rates of drift and carrier gas, and the shutter grid pulse width were investigated and optimized. The optimized experimental conditions for determining of AFG are given in Table 1 which are the same as those previously obtained for determination of AFB¹⁸. Among all parameters, temperature is the most important one. The spectrum was recorded at different temperature for injection port as well as for the IMS cell. Figs 3 and 4 show the effect of temperature (injector and cell, respectively) on the sensitivity. As shown, increasing the injection port temperature enhances the peak height. Similarly the cell temperature improves the sensitivity.

Figure 5 Click here to View figure

 

Table 1: The optimized experimental conditions of IMS for determination of AFG

Parameter	Setting
Length of drift tube	11 (cm)
Drift field	600 (V cm-1)
Corona voltage	2200 (V)
Drift gas flow (N2)	600 (mL min-1)
Carrier gas flow (N2)	300 (mL min-1)
Injection port temperature	220 (oC)
IMS cell temperature	190 (oC)
Typical shutter grid pulse width	100 (µs)

Analytical Parameters

The calibration curves for AFG were prepared with spiking different volumes of the analyte standard solutions into uncontaminated pistachio sample as blank. The blank was analyzed by IMS in which no AFG peak was observed. The analytical parameters for the determination of AFG were given in Table 2. According to this Table and Ref.¹⁸, the results show the wide linear range in comparison with the determination of AFB. Furthermore, the precision is also improved for determination of AFG.

Table 2: The analytical parameters for the determination of AFG₁ and AFG₂

 

Determination of AFG₁ and AFG₂

To evaluate the analytical applicability of the proposed method, it was applied to the determination of AFG₁ and AFG₂ in spiked pistachio nuts. The results are given in Table 3. The recoveries are close to 100 % and indicate that the developed method can be used to the determination of AFG in pistachio samples.

Table 3: Determination of AFG₁ or AFG₂ for spiked pistachio samples

 

The possibility of determination of the total AFG₁ and AFG₂ was investigated. As mentioned before, the product ions of AFG₁ and AFG₂ completely overlap and appear at same drift time (13.7 ms). This means that IMS cannot separate and distinguish between AFG₁ and AFG₂. This problem was also observed in determination of AFB₁ and AFB₂¹⁸. To achieve to the determination of total AFG₁ and AFG₂, the response factors of them were evaluated and compared based on the slopes on their calibration curves. Statistical calculations show that the response factors slightly differ. Therefore, the determination of total AFG can be performed by IMS method.

Analysis of Mixtures of AFB and AFG

The other objective of our work was the development of IMS method for the simultaneous determination of mixtures of AFB and AFG without pre-separation. Binary mixtures of AFB₁ and AFG₁ were injected into the instrument at conditions given at Table 1. The ion mobility spectrum of this mixture is shown in Fig. 5. The product ions were appeared in the range of 15 to 16.8 ms so that the corresponding peaks did not overlap. The peak appeared at lower drift time is originated from AFG₁ whereas the second peak originates from AFB₁. The obtained results were unacceptable due to influence AFB₁ and AFG₁ signal on another. Therefore, a separation technique such as HPLC is required in the simultaneous determination purposes.

Conclusions

IMS using corona discharge ionization source permits rapid, sensitive and reproducible determination of AFG₁ and AFG₂. The reasonable results were achieved that showed the developed method can be applied as a simple and low cost method comparison with to other methods for the determination of AFs.

Acknowledgments

The authors are thankful to the Islamic Azad University, Yazd branch for the financial support of this work.

References

1. Sherif, S.O, Salama, E.E., and Abdel-Wahhab, M.A., Int. J. Hyg. Environ. Health 212: 347 (2009).
2. Richard, J.L., Int. J. Food Microbiol. 119: 3 (2007).
3. Zhang, D.H., Li, P.W., Zhang, Q., Zhang, W., Huang, Y.L., Ding, X.X., and Jiang, J., Anal. Chim. Acta 636: 63 (2009).
4. Catharino, R.R., Marques, L.D.A., Santos, L.S., Baptista, A.S., Gloria, E.M., Calori-Domingues, M.A., Facco, E.M.P., and Eberlin, M.N., Anal. Chem. 77: 8155 (2005).
5. Bennett, J.W., and Klich, M., Clin. Microbiol. Rev. 16: 497 (2003).
6. Luttfullah, G., and Hussain, A., Food Control 22: 426 (2011).
7. Sulyok, M., Krska, R., and Schuhmacher, R., Food Chem. 119: 408 (2010).
8. Leong, Y.H., Ismail, N., Latif, A.A., and Ahmad, R., Food Control 21: 334 (2010).
9. Nawaz, S., Coker, R.D., and Haswell, S.J., Analyst 117: 67 (1992).
10. Afsah-Hejri, L., Jinap, S., Arzandeh, S., and Mirhosseini, H., Food Control 22: 381 (2011).
11. Becker, M., Degelmann, P., Herderich, M., Schreier, P., and Humpf, H., J. Chromatogr. A 818: 260 (1998).
12. Josephs, R.D., Krska, R., Grasserbauer, M., and Broekaert, J.A.C., J. Chromatogr. A 795: 297 (1998).
13. Wang, Y., Chai, T., Lu, G., Quan, C., Duan, H., Yao, M., Zucker, B.A., and Schlenker, G., Env. Res. 107: 139 (2008).
14. Gonzalez, E., Nogueira, J.H.C., Fonseca, H., Felicio, J.D., Pino, F.A., and Corrêa, B., Int. J. Food Microbiol. 123: 184 (2008).
15. Eiceman, G.A., and Karpas, Z., Ion Mobility Spectrometry, CRC Press, Boca Raton, FL, 2005.
16. Creaser, C.S., Griffiths, J.R., Bramwell, C.J., Noreen, S., Hill, C.A., and Thomas, C.L.P., Analyst 129: 984 (2004).
17.  Tabrizchi, M., and ILbeigi, V., J. Hazard. Mater. 176: 692 (2010).
18. Sheibani, A., Tabrizchi, M., and Ghaziaskar, H.S., Talanta 75: 233 (2008).
19. Schmidt, H., Baumbach, J.I., and Klockow, D., Anal. Chim. Acta 484: 63 (2003).
20. Keller, T., Keller, A., Tutsch-Bauer, E., and Monticelli, F., Forensic Sci. Int. 161: 130 (2006).
21. AOAC Official Method, 1995, 970.44.
22. AOAC Official Method, 2000, 991.31.

---

This work is licensed under a Creative Commons Attribution 4.0 International License.