Métodos de conservação e determinação de triptofano e serotonina em sangue por HPLC-FLD

Abstract

Tryptophan follows three metabolic pathways, including the serotonin pathway (5-HT), which acts in intestinal, immunological, endocrine, and metabolic processes, especially in ruminants. The detection of 5-HT in biological fluids is performed by chromatographic techniques, with HPLC-FLD being promising but still poorly established for blood fractions. As 5-HT is unstable and easily degraded, it requires antioxidants and controlled storage for reliable analysis. This study was conducted with the aim of developing a rapid and accurate methodology for storing and quantifying 5-HT together with tryptophan (TRP) using highperformance liquid chromatography with fluorescence detection (HPLC-FLD) and evaluating temporal variations and variations in blood fractions. The analytical method was isocratic with fluorescence detection. The mobile phase was evaluated with 50 mM potassium phosphate buffer at different pH values (3.5, 4.0, 4.5) and methanol:buffer ratios (25:75, 20:80, 15:85). The stability of analytes in blood serum samples with different preservatives was evaluated for tryptophan and serotonin stability in blood serum samples. To analyze the temporal dynamics of serotonin in blood fractions, an experiment was conducted with six male Holstein cattle (436.8 ± 36.4 kg). Blood samples were collected at time points 0 (before feeding), 6, 12, and 18 hours after the first feeding. The mobile phase composed of 80% KH₂PO₄ buffer (pH 3.5) and 20% methanol showed the best results with excellent correlation for TRP (R² = 1.0000) and 5-HT (R² = 0.9997). The limits of quantification (LOQ) and detection (LOD) found were 14.54 pmol/mL and 4.80 pmol/mL for TRP and 12.27 pmol/mL and 4.058 pmol/mL for 5-HT. The recoveries obtained were 105% for TRP and 103% for 5-HT. Treatment of serum samples with HClO₄ and methanol (40%) resulted in better preservation of TRP and 5-HT stored at -20°C and -80°C for 56 days. Serum 5-HT levels remained stable throughout the day, while tryptophan levels fluctuated. The results demonstrate that the method developed is sensitive and reliable for the quantification of serotonin and tryptophan in biological matrices. The joint addition of HClO4 and methanol allows TRP and 5-HT stored at -80°C for at least 56 days.