UPLC-QTOF-MS-based metabolomics and chemometrics studies of geographically diverse C. torulosa needles†

Cupressus torulosa, an evergreen tree commonly known as the Himalayan or Bhutan cypress, is a significant coniferous species native to the Himalayan regions of Bhutan, northern India, Nepal, and Tibet. In this study, we employed ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) integrated with chemometrics to analyze the metabolite profiles of C. torulosa needles collected from 14 distinct geographical regions. Advanced statistical tools, including Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA), were utilized to identify significant variations in the chemical composition across these locations. Our analysis identified 24 marker compounds consistently present in all samples, screened using stringent filtering criteria including One-way ANOVA, moderated T-test, and multiple testing correction with the Benjamini–Hochberg FDR method. Additionally, for the first time, we established the flavonoid biosynthesis pathway in C. torulosa, enhancing the understanding of its metabolic fingerprint. These findings provide critical insights into the phytochemical diversity of C. torulosa and offer valuable tools for quality control, authentication and advancing the application of UPLC-QTOF-MS in natural product research.