The beneficial effects of soybean isoflavones for the human body have been confirmed, and such as antioxidant activity and suppresses cancer occurrence. Therefore, soybean is widely used as food in the world. In the past few decades, several soybean varieties have been developed. A high-performance liquid chromatography electro-spray ionization with tandem mass spectrometry (HPLC-ESI-MS/MS) approach was developed and validated to determine isoflavone in 11 soybean varieties of Taiwan. The separation of 12 isoflavones was realized with a Phenomenex F5 column (2.1 × 50 mm, 1.7 µm) and using acetonitrile as mobile phase over a total analytical run time of 15 min. An Agilent 6470 triple quadrupole mass spectrometer (QqQ) with multiple reaction monitoring (MRM) was employed in ESI positive mode was achieved for isoflavone analysis. In this method validation includes the linear relationship in the range of 3.0 to 800 ng/mL (R2 > 0.99); extraction recovery (71.2% to 93.6%); matrix effect (71.3% to 84.9%); intra- and inter-day accuracy were within 81 to 118% and intra- and inter-day precision below 15%. The LC-MS approach was successfully achieved to assess isoflavone of 11 soybean varieties, and the average total isoflavone content was 7150 ng/mL. In addition, this approach can contribute to robust applications as a workflow for the stable and sensitive detection of soybean seeds.
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