**4. Conclusions**

The extraction and clean-up methods play a very important role in the analysis of kanamycin. A series of information on methodologies for extraction and clean-up of kanamycin have been published. The extraction and clean-up methods for kanamycin have been applied to a variety of matrices, including animal feeds, liver and kidney tissues, and serum, among others. When the sample contains protein, as milk and serum, protein precipitation is an initial and key step. After protein precipitation, liquid-liquid extraction can be performed to remove fats by using hexane. SPE can be used to remove salts that might affect the ionization of the MS detector.

Much progress has been achieved in kanamycin detection. However, numerous problems still exist and need to be addressed. The UV and fluorescence derivatization methods are time consuming, and the reaction by-products often cause difficulties in quantitation. Therefore, simpler and direct detection methods are preferred, such as PED [10] and ELSD [50–52]. Nevertheless, ELSD is less sensitive than PED, needs to use volatile additives, and does not display a direct linear relation with the amount injected [10]. Some are semi-quantitative determination methods. LC-MS/MS methods can ensure good sensitivity and separation ability to detect kanamycin in animal-origin food [21,30]. However, the required instruments are not commonly available in many laboratories owing to their high cost. The IPLC is also suitable for MS-MS detector, while the IPR must be volatile and compatible with MS detector with low ionization suppression. Besides IPLC, HILIC is fully compatible with MS systems and free from IPR in the mobile phase. Meanwhile, the HILIC method can achieve lower detection limits [47]. Therefore, the HILIC-MS-MS offers further direction. Moreover, the MRLs of kanamycin residues defined by the EU Commission Decision is still not quite comprehensive, such as the absence of honey; thus, more sample materials needed to be included. We hope that this paper provides some help for kanamycin detection.

**Author Contributions:** X.Z. wrote the paper and organized data; J.W. revised the paper; Q.W. and L.L. collected relevant literature; Y.W. provided language modification; H.Y. provided the ideas about the paper and financial support.

**Funding:** The research was funded by the National Natural Science Foundation of China (31601536).

**Acknowledgments:** The research was supported by the Science and Technology Research Project from Hubei Provincial Education Department (Q20181321), the Yangtze Fund for Youth Teams of Science and Technology Innovation (2016cqt02).

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