A liquid chromatography⿿tandem mass spectrometry assay for the detection and quantification of trehalose in biological samples

Abstract

Trehalose is an important disaccharide that is used as a cellular protectant by many different organisms, helping these organisms better survive extreme conditions, such as dehydration, oxidative stress, and freezing temperatures. Methods to detect and accurately measure trehalose from different organisms will help us gain a better understanding of the mechanisms behind trehalose's ability to act as a cellular protectant. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay using selected reaction monitoring mode for the detection and quantification of trehalose using maltose as an internal standard has been developed. This assay uses a commercially available LC column for trehalose separation and a standard triple quadrupole mass spectrometer, thus allowing many scientists to take advantage of this simple assay. The calibration curve from 3 to 100μM trehalose was fit best by a single polynomial. This LC-MS/MS assay directly detects and accurately quantifies trehalose, with an instrument limit of detection (LOD) that is 2-1000 times more sensitive than the most commonly-used assays for trehalose detection and quantification. Furthermore, this assay was used to detect and quantify endogenous trehalose produced by Escherichia coli (E. coli) cells, which were found to have an intracellular concentration of 8.5±0.9mM trehalose. This method thus shows promise for the reliable detection and quantification of trehalose from different biological sources.

Quantification of the Disaccharide Trehalose from Biological Samples: A Comparison of Analytical Methods

“…Moreover, there is precedent for single polynomial fits for LC–MS/MS data. 34 , 37 − 40 For example, a single polynomial fit has been used to model the response of an LC–MS/MS instrument to a platinum anticancer drug in human plasma samples. 38 For the LC–MS/MS-based assay used here, a single polynomial gives the best fit.…”

“… 34 , 41 The matrix effect is a change in the response of the LC–MS/MS to a specific compound when complicated samples are analyzed; it is hypothesized to result from molecules originating from the sample mixture that coelute with the compound of interest, causing either ionization suppression or enhancement in the mass spectrometer. 34 , 41 For all three assays, the percent recoveries from samples prepared in E. coli lysates are within 7% of the expected concentrations, ranging from 93 to 106% ( Table 3 ). For samples prepared in Jurkat cell lysates, the percent recoveries are also within 7% of the expected concentrations, ranging from 93 to 100% ( Table 4 ).…”

“…Several methods have been reported for the quantification of trehalose from biological sources. − One of the most commonly used methods for quantifying trehalose is an enzymatic assay that uses trehalase, an enzyme that cleaves trehalose into two glucose monomers (Scheme ). − The enzyme hexokinase then catalyzes the phosphorylation of glucose to glucose-6-phosphate (G-6-P), and G-6-P is subsequently oxidized to gluconate-6-phosphate.…”

“…Another reported method for the quantification of trehalose is high-performance liquid chromatography (HPLC) coupled with detection by a pulsed electrochemical detector or a refractive index (RI) detector (RID). − In this method, trehalose is separated from other biological compounds via liquid chromatography and quantified by its refractive index (RI) or electrochemical signal. Liquid chromatography–mass spectrometry (LC–MS) has also been used to quantify trehalose; − recently, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for the quantification of trehalose has been developed by our laboratory . In this assay, trehalose is separated via liquid chromatography and quantified via tandem mass spectrometry using maltose as an internal standard.…”

Crotalaria spectabilis as a source of pyrrolizidine alkaloids and phenolic compounds: HPLC‐MS/MS dereplication and monocrotaline quantification of seed and leaf extracts

“…Peaks 1 ( m/z 341 [M–H] – ) and 2 ( m/z 549 [M+HCOO] – ), which were detected in the seeds extract only, could be attributed to the presence of the saccharides di‐hexose and tri‐hexose, respectively, especially due to the main fragment at m/z 179 being attributed to the neutral loss of 162 Da and two neutral losses of 162 Da from the precursor ion 16 . These sugars are widely present in plants, and can be extracted by polar solvents such as hydroalcoholic solutions.…”