3.1.3 Absolute quantitation of intracellular metabolite concentrations
The absolute concentration of the intracellular metabolites was used to compare the efficiency of extraction solvents. We found that different extraction solvents differed in extraction efficiencies of specific intracellular metabolites. In terms of amino acid recovery, we analyzed 18 amino acids by GC-MS and observed a clear trend for amino acids. The largest amount of amino acids was extracted when the liquid nitrogen as the quenching agent and 50% cold acetonitrile as the extractant. However, the extraction of amino acids with cold methanol as the quenching agent was very poor, which was caused by the serious leakage of amino acids mentioned above. Also, methanol/chloroform (M/C) had a poor extraction effect on intracellular amino acids. This may be ascribed to the weak polarity of methanol/chloroform and low solubility of amino acids. M/C extraction, which was originally used to extract non-polar metabolites [59], such as fatty acids [18]. Hot ethanol extraction was proved to be as effective as acetonitrile for amino acid recovery, but there were differences in the selective recovery of specific amino acids such as serine (Ser), aspartate (Asp), asparagine (Asn) (Figure 7 ). Overall, amino acids were more soluble in water-based extractions (methanol, acetonitrile, and hot ethanol) which was consistent with previous study [26].
For organic acids and sugar phosphates, cold methanol as the quencher also behaved poor extraction effects and extracted the least amount. The recovery of fumarate (FUM), oxaloacetate (OAA) and phosphoenolpyruvic acid (PEP) was relatively low regardless of the type of extractants. For the rest organic acids such as pyruvic acid (PYR), malate (MAL) and α-ketoglutarate (α KG), using cold methanol and hot ethanol as the extractant can achieve better extraction results. Among sugar phosphates, the extraction effects of glyceraldehyde 3-phosphate (G3P), 3-phosphoglycerate (3PG), erythrose-4-phosphate (E4P) and fructose-1,6-bisphosphate (FBP) were generally poor. When liquid nitrogen and acetonitrile was used as the quencher and the extractant respectively, the extraction amount of 6-phosphogluconate (6PG) and sedoheptulose-7-phosphate (S7P) was the highest, and the extracted S7P even reached about 5.89 nmol/million cells, which was 8.96 times to 295.69 times higher than other extraction methods. This method was also suitable for the extraction of glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) which was only slightly less than normal saline quenching - 80% methanol extraction (S-M) and liquid nitrogen quenching - 80% methanol extraction (N2-M) (Figure 8 ).
Finally, high temperature in some extraction methods might affect the recovery of thermally unstable metabolites. Nevertheless, in our study, we did not observe that hot ethanol obviously caused the adverse effect regarding the number and amount of detected metabolites (Figure 8 ). One previous study has reported that several metabolites (PYR, nucleotides and sugar phosphates) were unstable in hot ethanol [22]. However, Sellick et al. found that hot ethanol had a good performance on recovering fatty acids such as stearic acid and palmitic acid because of the increased solubility of fatty acids [26]. To demonstrate the specific effects of heat extraction, we focused on the levels of the labile metabolite nicotinamide adenine dinucleotide (NAD) (Figure 8 ). The recovery of intracellular NAD clearly showed that liquid nitrogen quenching - 50% acetonitrile extraction (N2-A) and normal saline quenching - 50% acetonitrile extraction (S-A) recovered relatively larger amounts of NAD, hot ethanol extraction was slightly inferior, but better than methanol and M/C as extractants. In our study, M/C was poor in the extraction of each metabolite although previous study showed certain advantages in the extraction of cholesterol and fatty acids [57]. Therefore, these data emphasized the importance of using the appropriate quenching-extraction process for the extraction of specific metabolites. An ideal extraction method should recover the metabolites as much as possible; however, one extraction method in general was not capable of allowing a complete metabolite extraction. For this purpose, a combined extraction process might be an alternative to increase the number and amount of metabolites. However, the reduction of accuracy and reproducibility of the combination method in extraction was also an undisputable fact, which largely offsets its advantage in enhancing the metabolite spectrum [57].