Multistage fragmentation (MS3) detection enhances quantitative sensitivity and accuracy for LC–MS analysis of serum steroid hormones

A determination method using a home-built quadrupole-linear ion trap tandem mass spectrometry (Q-LIT) multistage fragmentation function to accurate measure ultra-trace steroid hormones in human serum was established. The capability of ion trap for multistage fragmentation (MS 3 ) overcomes the limitation of triple quadrupole systems restricted to MS 2 , enabling deeper structural characterization. Multistage fragmentation (MS3) enables the accurate detection of steroid hormones in serum samples without requiring extensive derivatization or time-consuming sample preparation and improves sensitivity by effectively reducing noise. Hydrocortisone, 4-androstenedione, 11-deoxycortisol, 21-deoxycortisol, and 17α-hydroxyprogesterone were quantified by MS 3 transitions m/z 363.2 → 327.2→309.2, m/z 287.2 → 97.0→79.0, m/z 347.1 → 311.2→293.2, m/z 347.0 → 311.1→293.1, and m/z 331.2 → 313.3→295.2, respectively. Multistage fragmentation mass spectrometry effectively prevents matrix interference, and the quantitative results of MS 3 are less affected by matrix effect than those of MS 2 . The established multistage fragmentation quantitative method showed good linearity (R 2  ≥ 0.99), and the limits of detection and quantification of 5 steroid hormones were lower than 0.06 and 0.20 ng/mL, respectively. The recovery was 86.56 %–123.14 %, with an RSD of ≤13.57 %. A total of 22 real serum samples were accurately and sensitively measured using the Q-LIT multistage fragmentation method. Therefore, Q-LIT based multistage fragmentation has great potential for applications, such as detecting steroid hormones with severe matrix effects and distinguishing among related isomers in complex biological samples.