3.6. Determination of Carnosine and Anserine Content

Both carnosine and anserine in the samples were extracted by water. Briefly, a 1.0 g portion of the sample was mixed with 9.0 mL of ultrapure water by a vortex mixer (XW-80A, Shanghai, China). The mixture was heated at 50 °C for 10 min in an electro-thermostatic water bath (HH-1, Jiangsu, China), and then was centrifuged at 10000 g and 4 °C for 10 min. Each sample was extracted twice by the above procedures. The supernatants were collected and diluted with water to 25 mL. The obtained solution was filtered through a 0.22 μm filter (Millipore, Bedford, MA, USA) prior to analysis.

The contents of carnosine and anserine in samples were determined by the HPLC method. The HPLC system consisted of a Waters e2695 series (Medford, MA, USA) equipped with a degasser, quaternary pump, column oven, ultraviolet detector, and automatic sampler. A reversed-phase chromatographic column (Capcell PAK C18 AQ S5, 250 mm × 4.6 mm i.d., 5 μm) was purchased from Osaka Soda Co. (Osaka, Japan). The mobile phase, consisting of 99% phosphate buffer solution (20 mmol/L, pH 9.0) and 1% methanol, was filtrated through a 0.22 μm filter and degassed ultrasonically prior to use. The chromatographic column was equilibrated at 35 °C. The flow rate was set at 1.0 mL/min. The injection volume was 5 μL. Carnosine and anserine were detected by the ultraviolet detector at 230 nm. Calibration curves were constructed of peak areas versus standard carnosine and anserine concentrations (1.0–500 mg/L). The limit of detection (LOD), the limit of quantification (LOQ), and the recovery and the relative repeatability standard deviation (RSDr) were calculated according to Snyder et al. [38]. LOD and LOQ were estimated as follows: LOD = 3.3 σ/S’ and LOQ = 10 σ/S’, where σ and S‘ are the standard deviation and slope of the calibration curve, respectively.