## 4 Discussion
Furthermore, bulk samples typically contain flowers and other organs at various developmental stages, limiting reproducibility due to organ- and stage-specific metabolite profiles. To address these limitations, we tested two extraction procedures that share a common workflow: collecting flowers at the same developmental stage, rapid freezing in liquid nitrogen, cryogenic ball milling at low temperature, and allowing for long-term storage before processing. The two differ in sampling strategy and offer complementary advantages. The \"bulk-flower\" method involves collecting 50-60 stage 4 flowers per plant, immediately freezing them, milling them to a fine powder, and extracting metabolites using the developed protocol. In contrast, the \"single-flower\" method entails collecting individual stage 4 flowers (in several replicates) per plant, placing each in an Eppendorf tube, and following the same freezing, milling, and extraction steps. Our findings showed that while the \"single-flower\" method (Table 3) had slightly but significantly lower reproducibility (\(p<0.001\)) compared to the \"bulk-flower\" approach (Tables S1 and S2), it still exhibited low intra-plant variability. The relative standard deviation (RSD) for 12 out of 16 identified compounds was below 15%, indicating limited variation among single flowers from the same genotype. However, elevated RSD values were observed for protopseudohypericin and protohypericin (compounds **13** and **15**), exceeding 20% on average and reaching 34.92% and 36.51%, respectively, in genotype 139_28. Table 3 also presents combined data for the protoforms and their stable counterparts, pseudohypericin and hypericin (compounds **13+14** and **15+18**). When protoform peak areas were summed with their corresponding stable forms, the RSD values decreased significantly and fell below 15% (Table 3). The protoforms protopseudohypericin and protohypericin are known to be highly sensitive to light, rapidly converting to pseudohypericin and hypericin upon sunlight exposure [69]. The increased variation observed for these compounds may be attributed to differential light exposure among individual flowers in the field. Similar variability in the levels of protohypericins was previously reported in fresh buds, flowers, and leaves exposed to sunlight [69]. A common practice in _H. perforatum_ extract analysis involves light exposure of extracts to promote the conversion of protoforms to stable forms, thereby improving consistency in naphthodianthrone quantification [70]. However, this also induces compositional changes, including hyperforin degradation, which necessitates the analysis of both light-exposed and unexposed samples for accurate profiling [68]. Alternatively, [PERSON] et al. proposed a single assay based on mathematical models that estimates hypericin composition from protoform levels, avoiding light exposure and preserving hyperforin from oxidative degradation [68]. Our study demonstrated that combining the peak areas of protoforms with their stable counterparts significantly reduces variability, and we recommend this approach, particularly when using the \"single-flower\" method in comparative metabolomic analyses. While the \"bulk-flower\" method offers lower variability, it also requires larger flower quantities and slower sample processing. The choice between methods should depend on the specific research objectives. Both approaches are suitable for comparative analysis of metabolite composition in individual plants. Given that secondary metabolite accumulation reflects the plant's biosynthetic network activity, microscale extraction and comparative analysis of relative compound levels can be valuable for various research applications, including biodiversity assessment [30], QTL mapping, and the identification of loci linked to specific compound accumulation [71,72].