2.6 Data processing
Data processing was performed using the R statistical programming
language. Waters raw data files generated from the metabolomics analysis
were converted to mzData files using the convert.waters.raw R package.
The quality control pooled injections were used to find the optimal peak
picking parameters, retention time corrections and grouping parameters
with the isotopologue parameter optimization (IPO) package. The
resulting parameters were inputted into the XCMS package for
peak-picking, to integrate the area under the curve and to replace zero
values. The CAMERA package was applied to XCMS processed features to
annotate possible isotopes and adducts. XCMS and CAMERA packages were
used to combine data from both positive and negative ionization modes.
The resulting data was subsequently normalized to internal standards,
and features were filtered by applying a threshold of 30% relative
standard deviation within quality control injections. Urinary
metabolites were normalized to both urinary creatinine and internal
standards, to account for differences in urine concentration. Features
were grouped by retention time and correlation into “pcgroups” by the
CAMERA package. Within each pcgroup, only the feature with the highest
mean raw intensity was kept for further data analysis.