Simultaneous hydrophilic interaction liquid chromatography-tandem mass spectrometry quantification of serum...

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.