**2. Comparison of six sample preparation methods for analysis of four preservatives and two artificial sweeteners in milk powders**

 It is not very easy to determine the trace residues or contaminants in infant milk powder for its complex matrix [7]. So, the sample pretreatment is the key step in the whole analytical procedures. According to the literatures, there are two kinds of sample preparation methods for analysis of contaminants in dairy products. One is to extract the targeted analytes, and the other is to remove the interferents. Usually, solid-phase extraction had been widely used as a milk sample preparation method. Sometimes, liquid-liquid extraction followed by a SPE cleanup step was served to remove the macromolecular protein prior to determination of the target analytes [8]. Removal of the protein in milk could be done by precipitating them with heavy metallic salt [9] or sodium tungstate [10].

 Our group developed six sample preparation methods based on the literatures, that is, liquid-liquid extraction, organic precipitation, heavy precipitation, and three different solid-phase extraction methods (C18, HLB, MAX). In order to obtain the higher recovery and reduce the time cost and organic solvent dosage, the six different sample preparation methods were compared.

### **2.1 Sample preparation and extraction**

## *2.1.1 Method A (liquid-liquid extraction)*

This method is based on the study of preservatives in cheeses [11]. Around 2.0 g milk powder was mixed with 4.0 mL of deionized water (60°C). After 10 min of ultrasonication, 5.0 mL of ethyl acetate and 1.0 mL 10 mmol L<sup>−</sup><sup>1</sup> of formic acid were added. The samples were extracted for 40 min on a rotary mixer at 400 rpm. After that, they were centrifuged for 5 min at 3200 rpm. The supernatant was transferred to another tube, and the sediment was extracted with 5.0 mL of ethyl acetate once again. The second supernatant obtained was combined with the one from the first extraction. Then they were filtered and evaporated to dryness at ambient temperature. The residues were dissolved in 500 μL mixture solution (0.1 M acetate buffer: methanol = 2:1, v/v) and vortexed for 20 s.

### *2.1.2 Method B (precipitation based on sodium tungstate)*

This method is based on the study of five macrolide antibiotics in milk [10]. The sample dissolving steps were the same as method A (liquid-liquid extraction). After 10 min of ultrasonication, this solution was centrifuged at 6000 rpm for 15 min. The defatted milk was transferred to a new centrifuge tube, and then 1.0 mL 10% sulfuric acid and 5.0 mL 10% sodium tungstate solutions were added. The resulting

 solution was vigorously shaken for 2 min and diluted to 10 mL with water and centrifuged at 4000 rpm for 10 min. The supernatant was filtered and evaporated to dryness at ambient temperature. The residues were dissolved in 500 μL mixture solution (0.1 M acetate buffer:methanol = 2:1, v/v) and vortexed for 20 s.
