Methods for storage and determination of tryptophan and serotonin in bovine blood by high-performance liquid chromatography with fluorescence detection

Tryptophan (TRP) and serotonin (5-HT) play important biological roles, including neurotransmission. However, the preservation and quantification of TRP and 5-HT in blood samples are challenging due to their instability. In addition, methods for their determination in biological samples using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) remain limited. This study aimed to develop methods for the preservation and quantification of 5-HT and TRP in blood using HPLC-FLD. Mobile phases with different pH values (3.5, 4.0, and 4.5) and methanol-to-buffer ratios (25:75, 20:80, and 15:85) were evaluated. A stability study using different preservatives (perchloric acid, oxalic acid, oxalic acid + glycine, EDTA, EDTA + perchloric acid, Na 2 S 2 O 5 , Na 2 S 2 O 5 + EDTA, 15% methanol, and 30% methanol) and storage temperatures was conducted. TRP and 5-HT concentrations were determined in whole blood, serum, heparinized plasma, and EDTA plasma. The optimized mobile phase consisted of methanol and KH 2 PO 4 buffer (pH 3.5) at a 20:80 ratio. Intraday and interday precision were 2.20% and 4.43% for TRP, and 2.36% and 4.96% for 5-HT, respectively. Mean accuracy was 105.2% for TRP and 99.4% for 5-HT. Serum samples preserved with 30% methanol showed no significant changes (P > 0.05) in TRP and 5-HT concentrations when stored at −20°C or −80°C. TRP exhibited minor variation among blood fractions, whereas 5-HT was higher in serum. The proposed analytical method is sensitive and reliable for quantifying TRP and 5-HT in blood. Methanol (30%) effectively preserved TRP and 5-HT during frozen storage for at least 56 days.