## 1 Introduction  
Mastitis is a major endemic disease of dairy animals, characterized by the inflammation of all structures forming the mammary tissue and the surrounding connective tissues. Mastitis significantly influences the quality and quantity of mammary parenchyma, leading to pathological changes in the glandular tissue of the udder and inducing physical, chemical and bacteriological alteration in the milk [1; 2]. Mastitis occurs worldwide with a high incidence and has various underlying causes and severity degrees which reduce milk production and adversely impact on farm animal welfare. Several bacterial species have been identified as mastitis-causing pathogens; among others, _Escherichia coli_, _Streptococcus uberis_, _Staphylococcus aureus_, _Streptococcus agalaciae_, are worth being mentioned [3; 4; 5; 6]. Bacterial contamination of milk from the affected cows renders it unsuited to human consumption as it can provide a spread of diseases like tuberculosis, Q-fever and brucellosis, just to cite some [3]. In this scenario, the early diagnosis of mastitis, especially at its subclinical stages, becomes a pivotal factor for a prompt treatment of the animal, thus minimizing mammary lesions and the related consequences. While clinical mastitis can be readily diagnosed from either abnormality in milk flavour, taste and colour or through the occurrence of secondary clinical signs (such as swollen quarters), the diagnosis of subclinical states is more problematic. Indeed, in the latter case, clinical signs are not evident (milk appears normal) and the use of specific tests is required for effective detection [3; 7; 8]. Although several methods have been so far developed for rapid diagnosis of subclinical mastitis [9], the somatic cells count (SCC) still remains the elective test carried out on milk samples to recognize early stage of intramammary infections. Generally, elevated SCC values indicate low milk quality and might be prognostic for further aggravation. The results of many studies [3; 10; 11] suggest that cows with milk SCC levels lower than 200,000 have not been infected with major mastitis pathogens (namely, _Staphylococcus aureus_ and _Streptococcus agalaciae_), while SCC values above 300,000 cells/mL are sign of probable infection. However, about 15% of infected cows have SCC milk levels \(<\) 200,000 cells/mL, thus behaving as false negative samples. At the same time, a quite relevant percentage (about 15%) of uninfected milk samples is misclassified according to SCC, indicating the potential occurrence of false positives [3]. EU Regulation 98 (Regulation (EC) No 853/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific hygiene rules for food of animal origin) stipulates that food business operators must initiate procedures to ensure that raw milk meets the following criteria: plate count at 30 \({}^{\circ}\)C \(\leq\) 100,000 colony forming unit (cfu)/mL and SCC \(\leq\) 400,000 cells/mL. The concept that metabolite concentrations change during mastitis (even at a subclinical stage) is well-known [7; 12]. Accordingly, in previous studies [13; 14; 15; 16] it was found that total amino acid content significantly increases in mastitis milk compared to normal, with a particular enhancement in the content of some specific species, including branched-chain and aromatic amino acids (referred to as BCAAs and AAAs, respectively). In these studies, indirect reversed-phase (RP) chromatography methods based on the use of derivatizing reagents were applied. It is well established that indirect method can incur into misquantification of the actual amino acid content, as a consequence of the required multi-step derivatization processes. Indeed, for different amino acids, derivatization reaction can go to completion in a different time; the high purity of the derivatizing reagent is critical since the presence of impurities (along with the excess of the derivatizing reagent itself) can generate side products that could interfere with separation; derivatization can be time consuming.