*2.1. Protein Precipitation*

Deproteinization was commonly used in the extraction of kanamycin from biological matrices because removal of interferences is necessary to retain good recoveries. Acetonitrile, acidified methanol, and trichloroacetic acid were commonly used precipitation reagents.

In human plasma sample, the simple organic solvent of acetonitrile was used for deproteinization with kanamycin recovery range from 92.3% to 100.8% [12,13]. The acidified methanol with a final concentration of 0.13 mol/L hydrochloric acid (HCl) can also be used for deproteinization of human plasma, and kanamycin recovery ranges from 91.2% to 93.4% [14].

In rat plasma samples, trichloroacetic acid (TCA) with a final concentration of 25–30% was a good precipitation reagen<sup>t</sup> and offers best recovery [15].

In human serum sample, the acidified methonal with a final concentration of 0.14 mol/L HCl can be used to extract kanamycin [16]. Meanwhile, TCA with a final concentration of 40% can be applied in human serum deproteinization, and recovery of kanamycin ranges from 93.9% to 98.4% [17].

Dried blood spots (DBSs) were more convenient than traditional venous blood sampling. In one anti-TB drug analysis, 0.1 mol/L HCl in mixed methonal solution was used for deproteinization of DBS samples [18].

Pig feeds samples were extracted with 0.1 mol/L HCl and kanamycin recovery ranged from 89.4% to 92.8% [19].

In bovine milk, swine and poultry muscle, samples were first precipitated by 15% TCA and then purified with bulk C18 resin. The recoveries of kanamycin were 92% in milk and 36.8–67% in muscle [20].

The chicken meat samples were extracted and precipitated with a mixture of acetonitrile (ACN)-2% TCA (45:55, *v*/*v*), followed by on-line clean-up using turbulent flow chromatography [21]. This automated on-line technique enabled a larger number of samples to be analyzed per day than the traditional clean-up technique. Kanamycin recovery ranged from 109% to 120% in chicken meat [21].
