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.