Assisted Reductive Amination for Quantitation of Tryptophan, 5-Hydroxytryptophan, and Serotonin by Ultraperformance Liquid Chromatography Coupled with Tandem Mass Spectrometry

Herein, we used isotopic formaldehyde and sodium cyanoborohydride via reductive amination to label two methyl groups on primary amine to arrange the standards (h(2)-formaldehyde-modified) and internal standards (ISs, d(2)-formaldehyde-modified) of tryptophan and its metabolites, such as serotonin (5-hydroxytryptamine) and 5-hydroxytryptophan. These derivatized reactions with a high yield are very satisfactory for manufacturing standards and ISs. This strategy will generate one or two methyl groups on amine to create different mass unit shifts with 14 vs. 16 or 28 vs. 32 in individual compounds for biomolecules with amine groups. In other words, multiples of two mass units shift are created using this derivatized method with isotopic formaldehyde. Serotonin, 5-hydroxytryptophan, and tryptophan were used as examples to demonstrate isotopic formaldehyde-generating standards and ISs. h(2)-formaldehyde-modified serotonin, 5-hydroxytryptophan, and tryptophan are standards to construct calibration curves, and d(2)-formaldehyde-modified analogs such as ISs spike into samples to normalize the signal of each detection. We utilized multiple reaction monitoring modes and triple quadrupole mass spectrometry to demonstrate the derivatized method suitable for these three nervous biomolecules. The derivatized method demonstrated a linearity range of the coefficient of determinations between 0.9938 to 0.9969. The limits of detection and quantification ranged from 1.39 to 15.36 ng/mL.

Automated summary

In this study, we used isotopic formaldehyde and sodium cyanoborohydride through reductive amination to label two methyl groups on primary amine for the preparation of standards and internal standards of tryptophan and its metabolites. The derivatized reactions showed high yields and were suitable for manufacturing standards and internal standards. This method created one or two methyl groups on amine to generate different mass unit shifts, providing a useful tool for biomolecules with amine groups. Using serotonin, 5-hydroxytryptophan, and tryptophan as examples, we successfully demonstrated the application of isotopic formaldehyde for generating standards and internal standards. The derivatized method showed good linearity, with coefficients of determination ranging from 0.9938 to 0.9969, and limits of detection and quantification ranging from 1.39 to 15.36 ng/mL.