Determination of levulinic acid in soy sauce by liquid chromatography with mass spectrometric detection

A novel method using liquid chromatography coupled with mass spectrometry (LC–MS) was developed for the determination of levulinic acid (LV) in soy sauces to identify the addition of acid hydrolyzed vegetable protein (acid-HVP). One hundred percentage naturally brewed soy sauce (NBS) and enzymatic hydrolyzed vegetable protein (enzymatic HVP) did not contain LV (<0.01 mg/mL). There was apparent detection of LV in acid-HVP and blended soy sauce with acid-HVP (2.98–21.66 mg/mL). Gas chromatography (GC) and high performance liquid chromatography (HPLC) at 430 nm methods were also investigated. The results by GC gave similar data to those by LC–MS. In enzymatic HVP, LV was detected by HPLC at 430 nm, but it was confirmed that the detected component was not in fact LV by mass spectrometric identification of the isolated peak compound. We found that LV is under the detection limit in enzymatic HVP. This study showed that LV is below the detection limit in NBS and enzymatic HVP and is apparently detected in acid-HVP and blended soy sauces with acid-HVP. These results indicate that LV is a practical index of blend with acid-HVP and also corresponds with the JAS (Japanese Agricultural Standard) or CNS (Chinese National Standard) criteria in which the presence of LV in soy sauces indicates adulteration with acid-HVP. The LC–MS method is analytically optimal for the precise determination of LV, because peak misidentification may be practically eliminated.