Quantification of Sugars and Organic Acids in Biological Matrices Using GC-QqQ-MS.

Gas chromatography coupled with triple quadrupole mass spectrometry (GC-QqQ-MS) can be used to accurately quantify endogenous small molecules extracted from biological samples such as plants and human fluids including sera and urine. In order to quantify primary metabolites typically from central carbon metabolism such as sugars from glycolysis and the pentose phosphate pathway; and organic acids involved in the tricarboxylic acid (TCA) cycle; polar endogenous metabolites must be extracted from the samples of interest, chemically derivatized and quantified against a linear calibration curve to a corresponding authentic standard. This chapter describes how to quantify a combination of 48 primary metabolites belonging to classes of sugars, sugar alcohols, sugar acids, sugar phosphates, and organic acids using a robust, optimized, multiple reaction monitoring (MRM)-based GC-QqQ-MS method.

Comparative metabolic and ionomic profiling of two cultivars of Stevia rebaudiana Bert. (Bertoni) grown under salinity stress.

This study provides a comprehensive investigation on the impact of increasing NaCl concentrations on hydroponically grown Stevia rebaudiana cultivars (Shoutian-2 and Fengtian). Growth parameters including plant height, biomass and physiological responses including osmotic potential were measured. In addition, the levels of steviol glycosides, elements and primary metabolites were measured and statistically evaluated. The cultivar Fengtian grew faster, accumulated less Na+ and compatible organic solutes, and more K+ in the leaves, as compared to the cv. Shoutian-2. Metabolite analysis identified 81 differentially accumulated metabolites, indicating an alteration in the metabolite phenotype of both cultivars upon exposure to salinity A general increase in many amino acids, amines, sugars and sugar phosphates with a concurrent decrease in most organic acids; including tricarboxylic acid (TCA) cycle intermediates, was observed. In the more salt tolerant cv. Fengtian, the levels of hexose phosphates and metabolites involved in cellular protection increased in response to salinity. These metabolites remained unchanged in the sensitive cv. Shoutian-2. Interestingly, salt treatment notably increased the rebaudioside A concentration by 53% while at the same time stevioside decreased by 38% in Fengtian which has important implications for controlling the relative amounts of reboudioside A and stevioside. The findings of this study leads to the conclusion that mild salinity stress can increase the yield of sweetener compounds, which is dependent on the cultivar and the level of salinity stress.

Quantitative profiling of polar primary metabolites of two chickpea cultivars with contrasting responses to salinity.

This study reports a GC-QqQ-MS method for the quantification of forty-eight primary metabolites from four major classes (sugars, sugar acids, sugar phosphates, and organic acids) which can be applied to a number of biological systems. The method was validated in terms of linearity, reproducibility and recovery, using both calibration standards and real samples. Additionally, twenty-eight biogenic amines and amino acids were quantified using an established LC-QqQ-MS method. Both GC-QqQ-MS and LC-QqQ-MS quantitative methods were applied to plant extracts from flower and pod tissue of two chickpea (Cicer arietinum L.) cultivars differing in their ability to tolerate salinity, which were grown under control and salt-treated conditions. Statistical analysis was applied to the data sets using the absolute concentrations of metabolites to investigate the differences in metabolite profiles between the different cultivars, plant tissues, and treatments. The method is a significant improvement of present methodology for quantitative GC-MS metabolite profiling of organic acids and sugars, and provides new insights of chickpea metabolic responses to salinity stress. It is applicable to the analysis of dynamic changes in endogenous concentrations of polar primary metabolites to study metabolic responses to environmental stresses in complex biological tissues.

A robust GC-MS method for the quantitation of fatty acids in biological systems.

Fatty acids (FAs) are involved in a wide range of functions in biological systems. It is important to measure the exact amount of fatty acids in biological matrices in order to determine the level of fatty acids and understand the role they play. The ability to quantify fatty acids in various systems, especially plant species and microbes has recently paved the way to the mass production of pharmaceuticals and energy substitutes including biodiesel. This chapter describes an efficient method to quantify the total fatty acids (TFAs) in biological systems using gas chromatography-mass spectrometry (GC-MS) and a commercially available standard mix of fatty acid methyl esters (FAMEs) using a step-by-step methodology to setup a quantitation method using the Agilent Chemstation software.