Rui Guo, Miao Zhu, Fei Huang, Feng Cheng, Zhonghua Lu, Song Xu, Li He, Guang Xu, Heng Wang, Jie Zhang
Serum neurotransmitter profiling is analytically challenging because the target panel includes highly polar compounds with markedly different ionization behaviors, while several catecholamine-related analytes are also susceptible to oxidation during thawing and handling. Here, we developed and validated a derivatization-free hydrophilic interaction liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of 15 serum neurotransmitters and related metabolites within a 12-min run. To improve the quantitative reliability of oxidation-prone analytes, serum samples were stabilized immediately after thawing with ascorbic acid before protein precipitation. The final workflow combined hydrophilic interaction liquid chromatography separation, stable isotope dilution, and single-step protein precipitation. The method showed good linearity across the validated concentration ranges for all analytes, with coefficients of determination above 0.99, limits of detection of 0.01-0.30 ng/mL, recoveries of 85.19% to 107.41%, and acceptable intra- and inter-day precision. Stability experiments further demonstrated marked signal loss for labile catecholamine-related analytes in the absence of antioxidant protection, whereas immediate post-thaw ascorbic acid stabilization substantially improved preservation of dopamine, norepinephrine, epinephrine, serotonin, and L-dopa during sample handling. Application of the method to serum samples from 188 pregnant women identified higher gamma-aminobutyric acid together with lower acetylcholine, choline, norepinephrine, L-dopa, serotonin, and 3-methoxytyramine in the antenatal depression group than in non-depressed controls. These findings support the use of this stability-aware hydrophilic interaction liquid chromatography-tandem mass spectrometry platform for cohort-scale serum neurotransmitter profiling and biomarker-oriented studies in pregnancy.