Robust NMR Data Analysis Pipeline

NMR spectra were phased, and baseline corrected using an ACDlab Processor Academic Edition (Advanced Chemistry Development, 12.01, 2010) and chemical shifts referenced internally to TSP at d = 0.0 ppm. Next, the spectral range containing the residual water signal (4.7–4.9 ppm) was excluded. The final analyzed spectral regions were defined between 0.7–4.7 ppm and 4.9–9.5 ppm. Spectral integration was carried out using the ACD Labs intelligent bucketing method [58 (link)]. A bucket width of 0.04 ppm with a 50% looseness factor was used. This flexibility allows the bucket width to deviate slightly from the set value. The intelligent bucketing technique identifies local minima within the spectra and adjusts the bucket positions accordingly. This approach ensures that peaks are integrated into their respective buckets, even when minor chemical shift differences are present due to factors like pH variations. The areas of the bucketed regions were normalized using Median Fold Change Normalization (MFC) [59 (link)], a method particularly favored when working with urine samples compared to total sum normalization.