*2.3. Chromatography*

The target analytes were separated by gradient elution. Optimum gradient program was chosen as providing good analytes' resolution, at the shortest analysis. A typical chromatogram is shown in Figure 5. The retention times were observed at 6.345, 7.566, 8.748, and 12.899 min for SN, SDZ, STZ, and SMT respectively.

**Figure 5.** A typical HPLC chromatogram of standard solution of examined analytes at the concentration of 5 ng μL−1. Peaks are as follows: SN: 6.345 min, SDZ: 7.566 min, STZ: 8.748 min, and SMT: 12.899 min.

## *2.4. Sample Preparation Optimization*

All initial optimization experiments were performed using standard solutions of sulfonamides. The optimum conditions established were further checked for their appropriateness to the milk matrix.

In the loading and elution step different methods were tested. Although stirring showed the best results in the tests with the standard solutions, as shown in Table 1 the extraction declined sharply when the milk samples were tested and the recovery rates ranged from 7 to 14%. Thus, centrifugation in low rates was selected. Centrifugation at low rates had two purposes: (1) sufficient sample interaction with the material, and (2) preventing the adsorbent from escaping from the structure of the sponge. High centrifugation rates hindered the extraction process. With regards to sonication, GO particles were released from the sponge and sample handling was difficult.

**Table 1.** Effect of the loading/elution time and the extraction procedure on the efficiency of the method. (SN = sulfanilamide, SDZ = sulfadiazine, STZ = sulfathiazole, SMT = sulfamethizole)


Additionally, the volume of the sample, the elution solvents, the size of the sponge, loading and elution time, and the pH were optimized. The extraction was conducted with two different volume samples (1.5 and 3 g) that were spiked with the same amount of the target analytes. The results revealed a decrease in the extraction efficiency by increasing the volume of the sample.

With regards to the elution, methanol (MeOH) and acetonitrile (ACN) were tested both separately and in mixture. It is obvious from the results that the mixed solution increases the efficiency of the elution. In order to succeed better results, 1% acetic acid was added. The addition of acetic acid was successful and the optimum volume ratio for the CH3COOH/ACN/MeOH solution was 50:40:10.

As for the loading and elution time 10, 15, 20 min were tested. From the results it is observed that 10 min are not enough for the loading and the extraction of the target analytes. However, 15 and 20 min yielded similar results, and the shortest time was selected to reduce the process time.

The effect of the pH in the extraction efficiency was tested, adding 0.5 mL of buffer solution into the sample. Table 2 presents the results obtained from the addition of pH 3, 5, 7, and 9 buffer solution in milk sample. It is obvious from the results that the optimum pH is 5, whereas lower or higher pH values results in decrease in the adsorption for all SAs.

**Table 2.** Effect of the pH on the adsorption efficiency of the four sulfonamides. (SN = sulfanilamide, SDZ = sulfadiazine, STZ = sulfathiazole, SMT = sulfamethizole). Optimum pH value is given in bold.


The proposed sample preparation protocol is very simple and rapid, with low consumption of organic solvents and very clean background signal. Figure 6 illustrates the simple pretreatment procedure. Typical chromatograms of a blank and a spiked milk sample are shown in Figure 7a,b. It is clear that the peaks of the substrate do not interfere with the analysis as they elute at different times.

**Figure 6.** Steps of sample preparation procedure.

**Figure 7.** Chromatogram of (**a**) blank milk sample and (**b**) spiked milk sample at a concentration of 300 μg kg−1.
