3.1.4 Reproducibility of metabolite quantification
To evaluate the reproducibility of the methods detected, we used average
relative standard deviation (RSD) as an indicator to evaluate the
reproducibility of the extraction solvent. The RSD results of 43
metabolites obtained using 12 combinations of quenching and extraction
methods were shown in Table 2 . The introduction of13C internal standards allowed us to quantitatively
evaluate extraction methods with regard to biological reproducibility.
In 2D Hela cells, the difference between the average RSD values of
different methods was not significant, mostly between 20%-30%. S-A
method had the lowest average RSD (18.9%) while the
N2-M/C method had the largest average RSD (39.9%). The
average RSD of the N2-A method which behaved excellent
regarding metabolite coverage and abundance was 27.8%. The RSD values
of 11 methods (except N2-M/C) were below 30%,
indicating good reproducibility independent of quenching and extraction
procedures. In addition, in all methods (except N2-M/C),
the percentage of individual metabolites with RSD <30% was
higher than 58% (Table 3 ). Guenther et al. found that in
HCT116 cells, the percentages of metabolites with RSD < 30%
cultured in 10% and 20% fetal calf serum (FCS) conditions using
different quenching solvents, extraction ratios were ranging from 0% to
79% which had a large error [20]. In our study, the percentage of
individual metabolites with RSD <30% for the optimal
N2-A method could reach 61.0%, and the largest
percentage of individual metabolites with RSD <30% of all
methods was 69.9%, which proved that our methods were reproducible for
most metabolites and N2-A method was relatively stable
for the extraction of metabolites detected. By analyzing the RSDs of 12
methods, we found that the sample preparation methods using liquid
N2 as the quenching agent had higher RSDs than the other
two quenching agents, and the proportion of metabolites with RSDs less
than 30% was also lower. We speculated that this might be caused by the
difficulty in accurately determining the added volume and the
volatilization time of liquid nitrogen, which made it impossible to
ensure complete consistency of the quenching time (the contact time
between samples and quenching agents) between parallel samples. However,
for the metabolites with RSDs>50%, there were only three metabolites
in N2-A method, which was the fewest among all 12
methods.