Analysis of tryptophan in spinach after hydrolysis

The proteins must be hydrolyzed for the determination of the total amounts of amino acids in the biological samples. Hydrolysis usually is carried out either in 6M HCl or various alkalies (NaOH, KOH, LiOH) solutions at 100-140⁰C often with the addition of oxidation inhibitors (phenol, thioglycolic acid, or other). Not all amino acids survive harsh hydrolysis conditions. Glutamine and asparagine are totally converted into the corresponding amino acids. Recovery of some other amino acids (for example, methionine, serine, and threonine) may be lower due to partial decomposition.

The most challenging analysis is that of tryptophan which is modified chemically during the hydrolysis to other compounds and cannot be detected as a free tryptophan. Even trace amounts of oxygen lead to the destruction of tryptophan. In the classical 6M HCl hydrolysis the overwhelming part of tryptophan gets destroyed. Preserving tryptophan from destruction by sealing the samples in the inert nitrogen atmosphere and adding inhibitors is difficult to control and validate.

There was received a request for determination of tryptophan in spinach.

The Solution

Instrumental: Orbitrap Discovery hybrid FT mass spectrometer coupled with Accela UPLC (Thermo Scientific). Chromatographic separations were carried out using Gemini C18 column (2×150 mm, 3µ particle size, Phenomenex) using linear gradients of methanol/water (with 0.1% AcOH) or ACN/water (with 0.1% AcOH).

First, there were determined the products of tryptophan degradation during hydrolysis. For that purpose, free tryptophan was heated in 6M HCl solution with the addition of phenol at 100⁰C for 12 hours.

On the other side degradation products of tryptophan were not detected when hydrolysis was carried out in 5M NaOH though absolute recovery was much lower than expected. Therefore, for the quantitative determination of tryptophan in spinach, the hydrolysis was carried out with the addition of isotopically labeled tryptophan-D5 as an internal standard.

Summary and conclusions

Quantitative analysis of tryptophan in spinach was carried out after NaOH hydrolysis. To ensure the highest possible recovery of tryptophan the biological sample was hydrolyzed in the presence of an internal standard namely, tryptophan-D5.