2.3. Test and Reference Formulations: β-Alanine Content Verification

Both products were tested to verify their β-alanine content by HLPC with the following procedure: A 4-level calibration curve prepared with pure β-alanine as reference ({"type":"entrez-protein","attrs":{"text":"P01090","term_id":"112740","term_text":"P01090"}}P01090, Cambridge Commodities Ltd., Ely, UK) with known purity (99.6%, batch number 201704043) at different concentrations (10, 20, 50 and 100 ppm) was performed. Correlation r² > 0.990 was required to validate the calibration curve after regression analysis by the method of least squares. Additionally, verification after quantification of a known quantity prepared in a solution with the reference standard, must be within 90–110% of this quantity. A sample containing one single dose of each product—10 slow-release tablets, and the amount containing 8 g β-alanine of the controlled-release powder—were weighed in a precision weight scale (Mettler Toledo AT261, Marshall Scientific LLC, Hampton, NH, USA) for content quantification. Samples were diluted in deionized water and derivatized after mixing with reagents for 15 min at 40 °C, further buffered with phosphate. Afterwards, 10 μL of sample was analyzed by HPLC in a Agilent Technology 1200 series system with a 5 µm, 4.6 × 150 mm C18 column (Zorbax Eclipse Plus, Agilent Technologies Inc., Marshfield, MA, USA) and a UV-VIS detector set at 360 nm, flow rate 1 mL/min at 20 °C with a mobile phase of phosphate buffer (pH = 6) and acetonitrile using a gradient elution (t, %B: (0; 10), (10; 25), (15; 50), (19; 50), (20; 10) and (26; 10)). The assay complied with the validation criteria of the Association of Official Analytical Chemists (AOAC), Standard Method Performance Requirements (SMPRs) for Identification and Quantitation of Free Alpha Amino Acids in Dietary Ingredients and Supplements [47].