Multistage fragmentation (MS) 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) overcomes the limitation of triple quadrupole systems restricted to MS, 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 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 are less affected by matrix effect than those of MS. The established multistage fragmentation quantitative method showed good linearity (R ≥ 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.