Determination of Key Compositions of Biogenic Hydrolysate Materials by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

Protein hydrolysate is a compositionally complex biogenic material, rich in a variety of amino acids, guanine, adenine, peptides and other bioactive substances, and therefore has been most commonly used in mammalian cell culturing for industrial production of recombinant protein therapeutics. A novel ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed in this work for accurate analysis of guanine, adenine and free amino acids of the hydrolysate sample. Samples with no need of any chemical derivatization were first subjected to a separation by a RESTEK Raptor Polar X column with an optimal elution procedure, and then detected in the multiple reaction monitoring (MRM) mode. Sixteen key compositions were thus quantitatively determined. These compositions showed good linearity in the individual mass content ranges, with all the determination coefficients (R2) above 0.99. The recoveries at different addition levels were satisfactory, ranging from 91.7% to 106.7%. The method was simple, highly sensitive and accurate, and therefore could be used for the purposes of quantitative determination of key compositions and quality evaluation of complex biogenic mixtures. Moreover, the change of compositions of samples was inspected when stored in dark at five temperatures during a period of 30 days. Results of analysis of variance (ANOVA) and principal component analysis (PCA) revealed that there was no significant change at all between the compositions of interest when samples were stored at low temperature of -80 degrees C,-40 degrees C and -20 degrees C over a period of 30 days. However, the content of most of these compounds significantly decreased under the storage condition of 4 degrees C on day 8 and 25 degrees C on day 2, respectively (p<0.05). It indicated that the compositions of hydrolysate materials were definitely degrading at relatively high temperature over a long storage time, which would influence their bioactivity. This study turned a spotlight on tackling the issues of unclear chemical composition, difficult quality control and unstable bioactivity of raw hydrolysate materials.