**9. Conclusions**

We report for the first time, the simultaneous CTS adsorption of 11 mycotoxins in PKC as an ingredient of animal feed. Statistical optimization using RSM (CCD) was a valuable tool for maximizing the e ffects and interactions of pH, time and temperature for removal of mycotoxins. The optimum condition for the removal of mycotoxins was at pH 4 for 8 h and at temperature of 35 ◦C, with high overall desirability (D 0.77). The overall coe fficient of determination values for the regression models were high (0.89 < *R<sup>2</sup>* < 0.98), as revealed by ANOVA. Results from the current study revealed the removal of eight target mycotoxins using Chitosan. Mycotoxin removal e fficiency was 45.90% and 94.35% for AFB2 and AFB1 respectively. In increasing order, removal e fficiency generally followed this trend: AFB1 > FB1 > FB2 > OTA > AFG2 > AFG1 > ZEA and AFB2. The present method o ffers clear advantages in terms of simplicity, speed, cost-e ffectiveness and ensuring low concentration of adverse mycotoxins in the sample. This finding is important as the mycotoxins were diminished to a greater extent than was reported using other adsorbents. The results clearly showed that pH is an important primary factor to be considered in the removal of mycotoxins with CTS. Lastly, CTS is relatively inexpensive and is thus a good candidate for practical applications involving simultaneous removal of mycotoxins in animal feed.

#### **10. Materials and Methods**

Palm kernel cake (PKC) samples were collected from local mills across di fferent regions in Malaysia (Shah Alam and Kelantan in Selangor and Kelantan state respectively). Representative samples of the PKC were prepared as described previously [46]. Analytical pure standards of the aflatoxins (AFB1, AFB2, AFG1, and AFG2), ochratoxin A (OTA) zearalenone (ZEA), trichothecenes (deoxynivalenol (DON), HT-2 and T-2 toxin) and fumonisins (FB1-FB2), were purchased from VICAM (Watertown, MA, USA). Chitosan (CTS > 85% deacetylation) was sourced from Sigma-Aldrich (St. Louis, MO, USA). Deionized water was prepared with a water purifier (Elga Classic UV MK2; Elga, Marlow, UK). HPLC-grade solvents (acetonitrile, methanol and formic acid) were from Merck (Darmstadt, Germany). Filtration of all eluents was done using 0.22-μm Whatman membrane filters (Whatman, 110 Maidstone, UK).

#### *Mycotoxins Analysis by LC–MS*/*MS*

The mass spectrometer used for the analyses was an Agilent 1290 Infinity UHPLC module LC/MS-MS with a Triple Quad LC/MS (Agilent 6410, Agilent technologies, Palo Alto, CA, USA). This system consisted of an auto sampler, a degasser and column oven. Separation was performed using a Zorbax Eclipse plus C18 column (2 × 150 mm, 3 μm) at column temperature of 30 ◦C manufactured by Agilent Technologies (Palo Alto, CA, USA). The analysis was operated in positive and negative modes with electrospray interface (ESI±) with the following parameters: capillary voltage of 4 kV, nitrogen as spray gas and desolvation temperature 40 ◦C. Mycotoxins were analyzed in multi reaction monitoring (MRM) mode while matrix-matched standard calibration was used for quantification. As shown in Table 6, the mobile phase consisted of a gradient of 2 solvents: mobile phase A (methanol) slightly acidified with mobile phase B (0.1% formic acid in water), at a flow-rate of 0.2 mL/min. Validation of the LC-MS/MS method was carried out by investigating the basic performance characteristics included linearity, limit of detection, limit of quantification and recovery in accordance with the European Commission regulation for the performance of analytical methods (EC 657/2002).

**Table 6.** Gradient elution program of the LC/MS-MS.


**Author Contributions:** A.A.P. carried out the entire experiments and wrote the manuscripts with support from J.S., A.A.P., J.S., S.Z.I. and N.I.P.S. designed the experiments. J.S. supervised the project. A.A.P., J.S., N.I.P.S. and S.Z.I. discussed the results and contributed to the final manuscript. All authors approved the final manuscripts. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Ministry of Education Malaysia [grant number 6369114] and HICOE environment set at the Institute of Tropical Agriculture and Food Security.

**Acknowledgments:** The authors would like to acknowledge the contribution of the Ministry of Education Malaysia [grant number 6369114] and HICOE environment set at the Institute of Tropical Agriculture and Food Security.

**Conflicts of Interest:** The authors declare no conflict of interest.
