The invention discloses a method for detecting multiple lipid-soluble vitamins in a blood sample simultaneously. The method comprises the steps of preprocessing a biological sample by a simple liquid-liquid extraction method, then carrying out
chromatographic separation and
mass spectrometric detection, respectively selecting a pair of qualitative ions and a pair of quantitative ions according to each
vitamin, wherein the relative
retention time and qualitative
ion pairs of the various vitamins are taken as qualitative basis, and a
standard curve for quantitation is formed by standard substances. Meanwhile, according to the method, three levels of
quality control substances are used for inspecting the accuracy and effectiveness of the method, and the
distortion of the detection result is avoided. According to the method, the purpose that six lipid-soluble vitamins in one serum sample can be simultaneously detected can be realized for the first time by applying an LC-MS technology, the influence caused by interferents can be alleviated, the operation is simple, convenient and rapid, the analysis time is only 4.8min, the
throughput is high, the cost is low, the levels of lipid-soluble vitamins in a
human body are effectively monitored, and the method has guiding significance on the reasonable and safe supplementation of vitamins, and is liable to clinical promotion and popularization.