Lc 20a

Sample aliquots (5 μL) were subjected to chromatography in a C18 column (150 mm×4.6 mm; Waters Corp, Milford, MA, USA) at 0.8 mL/min and 50°C. The mobile phase consisted of 0.1% heptafluorobutyric acid+0.1% formic acid in water (A) and 0.1% heptafluorobutyric acid+0.1% formic acid in acetonitrile (B). Elution was performed during 20 min: 90% A for 5 min, 90% to 70% A for 2 min, 70% to 65% A for 3 min, 65% to 5% A for 5 min, 5% to 0% A for 0.5 min, and 90% A for 4.5 min.
Mass spectrometry was performed on a API 4000 QQQ (Applied Biosystems, Carlsbad, CA, USA) coupled to an HPLC system (LC20A, Shimadzu Instruments, Kyoto, Japan) operating in positive-ion mode. A standard curve was generated with different concentrations (5 to 1,000 mM) of pure carnosine (C9625, Sigma-Aldrich, St. Louis, MO, USA). The resulting linear regression was y = 0.000132x–3.45e⁻⁵ (r = 0.9935), of which x means carnosine concentration, y means mass spectrum response. The retention times and peak areas were analyzed by API analyst 1.5 software (LC20A, Shimadzu Instruments, Kyoto, Japan). Carnosine concentration was calculated from the standard curve.

Phenolics and flavonoids were separated by reverse phase HPLC (Shimadzu LC 20A; Shimadzu Corp.) fitted with a C₁₈ column (250 × 4.6, 5 μm, Waters Corporation, Milford, MA, USA) and UV detector (see ESI†).^{33 (link)}