Determination of Multiple Free Nucleotides in Meat Products by High Performance Liquid Chromatography

This study developed and validated a method to detect adenosine monophosphate (AMP), inosine monphosphate (IMP), guanosine monophosphate (GMP), hypoxanthine riboside (HxR), and hypoxanthine (Hx) in meat products by high performance liquid chromatography (HPLC). The optimized procedure was as follows: samples were extracted twice with 5% perchloric acid with shaking for 30 min; the pooled extract was adjusted to pH 6.5 and made up to 50 mL with deionized water; an appropriate amount of the extract was taken and purified by liquid-liquid extraction with n-hexane followed by centrifugation; the bottom layer was harvested for analysis by HPLC. Chromatographic separation was accomplished on a C₁₈ column using isocratic elution with a mobile phase composed of 0.01 mol/L aqueous potassium dihydrogen phosphate and methanol (98:2, V/V) at a flow rate 1.0 mL/min, and injection volume was set at 20 μL. Detection wavelength was set at 254 nm. Quantification was carried out using the external standard method. The results showed that the five nucleosides had a good linear relationship within the concentration range of 0.5–80 μg/mL, with correlation coefficients greater than 0.999. The limit of detection (LOD) was 5.0 mg/kg, the limit of quantitation (LOQ) was 15.0 mg/kg. Recoveries from spiked samples was 80.1%–109.5% with relative standard deviation (RSD) ≤ 9.6% (n = 3). In summary, this method is simple to operate, accurate, repeatable, and suitable for the detection of the five free nucleotides in the complex meat matrix.