2.5.1. Selectivity

The good resolution between the chromatographic peaks of analytes and the absence of interferences in the spiked milk samples indicate that a good selectivity was achieved.

#### 2.5.2. Linearity and Sensitivity

Standard solutions showed linearity for all of the target analytes within the range of 0.5 to 10 ng μL−<sup>1</sup> and showed and good correlation coefficients (0.981–0.999). Moreover, calibration curves were constructed using fortified milk samples after sample preparation, and good coefficients of determination between 0.9969 and 0.999 were achieved over the examined range. (Table 3). Limit of quantification for all analytes in milk was 50 μg kg−1.


**Table 3.** Linearity data in standard solutions and spiked milk samples. (SN = sulfanilamide, SDZ = sulfadiazine, STZ = sulfathiazole, SMT = sulfamethizole).

#### 2.5.3. Precision and Accuracy

The precision of the method was based on within-day repeatability and between-day precision. The former was assessed by replicate (*n* = 4) measurements from a spiked milk sample at the MRL level for all examined sulfonamides. The recoveries of spiked samples were calculated by comparison of the peak area ratios for extracted compounds toward the values derived from spiked calibration curves. In Between-day reproducibility a triplicate determination was performed for a period of three days (Table 4). Precision and accuracy was determined at three concentration levels according to the 657/2002/EC decision [40].



#### 2.5.4. Decision Limit and Capability of Detection

Decision limit (CCα) is defined as "the limit at and above which it can be concluded with an error probability" and it was calculated after the analysis of 20 spiked milk samples at the MRLs of each compound. The decision limits CCa were 100.2 μg kg−<sup>1</sup> for SN, 100.3 μg kg−<sup>1</sup> for SDZ, 100.4 μg kg−<sup>1</sup>

for STZ, and μg kg−<sup>1</sup> for 100.3 SMT. Capability of detection (CCb) defined as "the smallest content of the substance that may be detected, identified, and/or quantified in a sample with an error probability of b" and it was calculated after the spiking of 20 blank milk samples at the CCa level of each compound. The capability of detection (CCb) were 110.7 μg kg−<sup>1</sup> for SN, 109.3 μg kg−<sup>1</sup> for SDZ, 115.4 μg kg−<sup>1</sup> for STZ, and 114.3 μg kg−<sup>1</sup> for SMT.

#### *2.6. Application to Real Samples*

The method was applied for the determination of the examined analytes in cow milk samples from local food stores. Five random samples of three different types of milk were collected and analyzed, including full-fat (3.5%), semi-skimmed (1.5%), and skimmed (0%) milk. All analyzed samples were negative in the presence of examined analytes.

#### *2.7. Comparison with Other Methods*

The method described in this study was compared with previous analytical approaches for the determination of SAs in milk. The analysis' results are comparable with those attained by other methods, with fairly good recoveries and quite satisfactory sensitivity. Although it provides higher LODs and LOQs than previously reported methods, it is a less costly (no commercial SPE products are needed) and less time-consuming method with easy handling of sponge and does not require highly sophisticated equipment since no MS is used (Table 5).


**Table 5.** Performance of the presented method in comparison with previously reported analytical methods.

#### **3. Materials and Methods**

## *3.1. Chemicals and Reagents*

Sulfathiazole (STZ), sulfamethizole (SMT), sulfadiazine (SDZ), and sulfanilamide (SN) were purchased from Sigma-Aldrich (Steinheim, Germany). HPLC grade acetonitrile and methanol obtained from Chem-Lab (Zedelgem, Belgium). Formic and acetic acid were of analytical grade and purchased from Chem-Lab (Zedelgem, Belgium) and Merck (Darmstadt, Germany) respectively. Ethanol, reagen<sup>t</sup> grade (Chem-Lab, Zedelgem, Belgium) and ammonia, 25% solution (PANREAC QUIMICA SA, Barcelona, Spain) were used for the sponge optimization. Polyvinyl alcohol high molecular weight solid, (PVA 98–99 hydrolized) was purchased from A Johnson Company (New Brunswick, NJ, USA).

Graphite was purchased from Sigma Aldrich (St. Louis, MO, USA). Double-deionized water was filtered with 0.45 μm filter membrane before use.

Milk samples were collected from local market (Thessaloniki, Greece). Di fferent fresh milk types were analyzed including skimmed (0% fat), semi-skimmed (1.5% fat), and full-fat milk (3.5% fat). All milk samples were kept refrigerated (at 4 ◦C) until use.
