## 3 Results and Discussion  
APA preliminarily measured the SCC value in all 65 milk samples under investigation. Samples were then divided into two groups according to EU Regulation 853/2004 indications: Group I composed of milk samples with SCCs < 400,000 cells/mL (Table 1) and Group II encompassing samples with SCCs > 400,000 cells/mL (Table 2). In order to directly appraise the relationships between the incidental alterations of the amino acid content with the SCC value, the free (unbound) amino acid pool was extracted from each of the 65 milk samples and then submitted to purification through an ion-exchange resin, as described in Section 2.5. A direct ion-pairing reversed-phase method based on the use of the evaporative light-scattering detector (IP-RP-HPLC/ELSD) was applied for the analysis of the milk extracts. The method was previously developed in our laboratory and profitably employed for the direct analysis of amino acid content with the SCC value. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Sample \#** & **SCC (\(\times\)10\({}^{3}\))** & **Ile (\#g/mL)** & **Leu (\#g/mL)** & **Val (ug/mL)** & **Tyr (\#g  
acids in complex food matrices [18; 21]. Detailed information on the method is reported in the caption to Figure 1. The use of HFBA as IP reagent increases the residence time of the investigated polar analytes into the RP column, thereby enhancing the quality (both thermodynamic and kinetic) of the chromatographic performance as a whole. Moreover, differently from other perfluoroxylyl carboxylic acids, HFBA containing eleuents give the advantage to avoid prolonged re-equilibration times between consecutive runs [18] as a result of its negligible adsorption onto the C18 chains. As an additional benefit arising from HFBA use, its volatility makes it compatible with mass spectrometry detectors for accurate molecular investigations. The chromatogram of an extract is exemplarily shown in Figure 1.  
As expected [18; 21], the selected IP-RP-HPLC-ELSD method, produced the co-elution of Ala/Glu and Ser/Asn (data not shown). Moreover, interfering matrix effects were found in the first 18 min of each sample analysis, thus impairing the detection of certain amino acids (data not shown). Therefore, the attention was exclusively addressed to all the remaining species eluting after 20 min of analysis: Val, Met, Tyr, Ile, Leu, Phe, Trp (Figure 1A). The elution order observed in Figure 1A is not readily explained as it is strictly correlated to analyte charge and polarity at once [18]. The chromatogram in Figure 1B reveals a pronounced selectivity and efficiency of the applied HPLC method towards the selected amino acid pool. Notwithstanding, the selected IP-RP-HPLC method allowed the base-line separation of the two isomers Ile and Leu. Quantitative analysis of Met, Phe and Trp was made difficult by their undetectable level in the majority of milk samples. Therefore, these three amino acids were overlooked in the study. For the remaining species (namely, the BCAAs and Tyr), the result of the quantitative analysis in the 65 extracts is shown in Tables 1 and 2. In order to find a relationship between the SCC value associated to each milk sample and the concentration of Ile, Leu, Val and Tyr, the statistical method based on the analysis of variance (ANOVA) was pursued. Indeed, bar plot analysis coupled with ANOVA studies and Tukey HSD (honest significant difference) tests, were useful to highlight differences in the amino acid content among the main two groups (I and II) of the analysed 65 milk samples (Tables 1 and 2). More in depth, this analysis evidenced some statistically significant differences in the content of Ile, Leu and Tyr between the two milk sample groups.