The experimental design included eight individual quality controls, designated as QC1 through QC8, which were composed of pooled samples (a combination of all 76 NEs). The objective was to assess the uniformity and constancy of the UHPLC-HRMS 2 metabolite profiling runs. The QCs were injected following batches of ten consecutive samples, resulting in a total of eight QCs being injected once within the set (QC1 to QC8). The entire set, comprising samples, blanks, and QCs, underwent three rounds of measurements denoted as injections A, B, and C. This entailed a cyclical analysis of the complete set, with alternation between PI and NI modes. After data processing in MZmine3, the response areas and retention times of all peaks in the aligned features table of only QC samples (all technical replicates (QC1-8) in the three analytical replicates (injections A, B, and C) were compared. This showed that the detected features exhibited consistent retention times, with a maximum deviation of Δ 0.05 minutes across all injections for both ionization modes (see Figure ). The variation of the total sum area (addition of the areas of all features detected in a sample) between the eight QCs technical replicates (1-8) within each analytical replicate (injection A, B, and C) ranged from 3% to 8% for PI and from 4% to 7% for NI (see Figure ). Comparison of the total sum area of the same QCs technical replicates between analytical replicates (e.g. QC-A1, QC-B1, QC-C1) showed a variation range from 2% to 7% for the PI and 6% to 9% for NI (see Figure ). Additionally, a visual assessment of the peak areas from all the QCs across the three analytical replicates of the dataset was conducted using an interactive heat map plot (PI, NI). A Principal Component Analysis (PCA) constructed with the complete dataset in both ionization modes, validated these findings. The PCA plots exhibited defined clustering of all QCs samples and distinct separation of the diverse NEs (see Figure ), along with a fairly good grouping of the three analytical replicates of the NEs. These visual results corroborate the reproducibility of the analyses. and demonstrate that there is no apparent batch effect in either the chromatographic or MS dimensions. Further comparative analyses were conducted on specific samples within the dataset. Figure shows the comparison of UHPLC-HRMS 2 metabolite profiles across the analytical replicates (injections A-C) for ethyl acetate extracts of P. indica roots (Figure ) and T. wilfordii roots (Figure ). Like the behavior observed in Figure -b for the QCs, these NEs, in each ionization mode, exhibited slight shifts in retention times, with an ∆RT of 0.05 minutes between injections. The three replicates of both samples were processed and aligned using MZmine3, using identical parameters for the entire dataset (see Methods). The outcomes regarding the total detected features in each replicate for both ionization modes are provided in Table . The observed number of features between replicates displays a notable similarity. This comprehensive analysis underscores the robustness of the methodology.