Improved liquid-liquid extraction followed by HPLC-UV for accurate and eco-friendly determination of tetramethylpyrazine in vinegar products

Tetramethylpyrazine (TMP) is an important bioactive compound in vinegars, contributing to their health-enhancing attributes. It serves as a crucial benchmark for the assessment of vinegar quality. Unfortunately, inaccuracies have arisen due to incomplete extraction techniques and the use of an inappropriate standard substance. These challenges have significantly curtailed comprehensive exploration into the underlying TMP formation mechanisms, impeding advancements within prevailing benchmarks and methodologies governing vinegar products. To address these challenges, several critical parameters, encompassing pH, solvent type, centrifugal force, extraction times and reference materials were investigated and optimized. The TMP content was determined by adjusting the pH to 9 using a sodium hydroxide solution, followed by extraction with ethyl acetate and subsequent re-extraction of the ethyl acetate layer with 0.2 mol/L HCl. A high-performance liquid chromatography method with an ultraviolet detector (UV) was developed and validated. This method demonstrated superior sensitivity compared to existing methods, with a limit of detection (LOD) of 0.0237 μg/g, limit of quantification (LOQ) of 0.0829 μg/g, method limit of detection (MLOD) of 0.10 μg/g and method limit of quantitation (MLOQ) of 0.25 μg/g. The modified method exhibited excellent linearity for TMP in the range of 0.1-118.4 μg/mL, with a good correlation coefficient (R2 > 0.999). The recovery rate of TMP in vinegar products ranged from 82.4 to 96.2%. Consequently, the proposed method exhibits substantial promise for systematic inquiry into TMP formation mechanisms and for ensuring consistent quality control during the production of premium-grade vinegars.