Quantification of three neurotransmitters in cerebrospinal fluid, serum and random urine using a robust and...

Imbalance in the monoamine neurotransmitters has been implicated in a variety of neurological disorders, making it necessary to quantify neurotransmitters accurately. Thus, the study aims to develop a robust and validated method for simultaneously quantifying serotonin, 5-Hydroxyindole Acetic Acid (5-HIAA), and homovanillic acid (HVA) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). One-step precipitation with methanol containing 0.2 % formic acid was used for pretreating serum and cerebrospinal fluid samples, while dilution with deionized water was used for pretreating urine samples. Neurotransmitters were detected using an Exion AD liquid chromatography-tandem Qtrap 6500 Plus mass spectrometer, with a total run time of 6.5 mins. The linearity range was 0.5-500.0, 0.2-100.0, 2.0-1000.0 ng/mL for serotonin, 5-HIAA, and HVA in serum and cerebrospinal fluid, and 2.0-500.0, 40.0-10,000.0, 100.0-10,000.0 ng/mL in urine (R²≥0.997). Recovery rate was 81.5-114.4 %, 80.3-114.6 %, and 85.0-115.6 % for serotonin, 5-HIAA, and HVA in three matrices. The matrix effect was compensated by using internal standards. Acceptable intra-assay and inter-assay precision were achieved for all analytes and the total coefficients of variation were 4.9-14.4 %, 6.1-11.2 %, and 4.5-10.5 % for serotonin, 5-HIAA, and HVA. Additionally, we also explored the distribution of neurotransmitters. Patients with motor impairment had higher HVA levels than those without symptoms (P < 0.05), while serotonin and 5-HIAA concentrations were insignificant. Accordingly, a robust LC-MS/MS method combined with easy sample preprocessing was established and systematically validated for quantifying three important neurotransmitters in multiple matrices in a single run, allowing for accurate identification of motor impairment.