Characterizing “nature’s Ozempic ”: Non-targeted screening of berberine supplements using ion mobility high-resolution mass spectrometry

Importance of the topic

Berberine is a plant-derived isoquinoline alkaloid investigated for metabolic, cardiovascular, neurological and gastrointestinal benefits and has gained attention as a natural alternative to GLP-1 therapies (colloquially "nature's Ozempic"). Variable composition, low oral bioavailability, and the presence of co-occurring alkaloids in commercial supplements can influence efficacy and safety. Reliable, high-resolution chemical profiling is therefore essential for consumer information, quality control, and risk assessment.

Objectives and overview of the study

The study applied a non-targeted analytical workflow combining high-resolution mass spectrometry (HRMS) with ion mobility spectrometry (IMS) to qualitatively characterize the alkaloid composition of seven commercially available powder-form berberine/berberis supplements. The goals were to (1) identify and tentatively assign isoquinoline alkaloids beyond berberine, (2) evaluate sample complexity and inter-product variability, and (3) demonstrate the benefit of IMS for resolving co-eluting or isobaric species.

Methods

• Samples: Seven over-the-counter powder supplements (root, root-bark, whole-herb, berberine-labeled and more "purified" formulations) purchased online; a pooled sample was also prepared.
• Sample preparation: ~100 mg sample sonicated in 50:50 water:methanol, centrifuged, filtered and diluted 100× prior to analysis. Authentic standards of berberine and palmatine were analyzed for confirmation.
• Data processing: MassLynx for acquisition and UNIFI within the waters_connect platform for data processing, library searching and neutral loss-based tentative identifications.

Used instrumentation

• UPLC: ACQUITY Premier system with ACQUITY Premier CSH Phenyl Hexyl column (1.7 µm, 2.1 × 100 mm), 0.3 mL/min, 30 °C, 1 µL injection; mobile phases water + 2 mM ammonium acetate + 0.1% acetic acid (A) and acetonitrile (B).
• Ion mobility / MS: SELECT SERIES Cyclic IMS (Waters) coupled to a TOF HRMS operated in positive electrospray HDMSE (data-independent acquisition) mode. Parameters: capillary voltage ~2 kV, mass range 50–1200 Da, transfer cell collision energy 40–60 V, MS analyzer V-mode (~60,000 FWHM resolution).

Main results and discussion

• Alkaloid speciation: Berberine was the dominant alkaloid across all samples but its contribution to the total measured alkaloid peak area ranged from ~57% to ~98% among products. The remaining fraction consisted of structurally related isoquinoline alkaloids such as palmatine, jatrorrhizine, berberrubine, berberastine and hydroxy-palmatine, many identified tentatively by common fragmentation patterns and neutral losses (CH4, C2H4O, C3H8O).
• Inter-product variability: Considerable variability in the number and relative abundance of co-occurring alkaloids was observed across products. Product label claims (e.g., whole-herb vs purified berberine) did not reliably predict measured chemical complexity.
• Analytical advantages of IMS+HRMS: Ion mobility added an orthogonal separation dimension that resolved co-eluting and isobaric species. A key example was separation of a minor component at m/z 352.1183 (C20H18NO5+) from palmatine at m/z 352.1549 (C21H22NO4+), demonstrating IMS capacity to distinguish species with nearly identical m/z in complex matrices. HDMSE fragmentation and library/neutral-loss searches supported both positive identifications (berberine, palmatine) and tentative assignments for related alkaloids.
• Figures and tables (summarized): Table 1 lists tested products and basic attributes. Figure 2 shows the SELECT SERIES Cyclic IMS schematic. Figure 4 presents fragmentation spectra for selected alkaloids. Figure 5 plots retention time vs drift time to illustrate sample complexity and IMS separation; Figure 6 displays pie charts of relative peak-area contributions for identified/tentatively identified compounds per sample.

Benefits and practical applications of the method

• Comprehensive profiling: Non-targeted IMS-HRMS enables detection and tentative identification of multiple isoquinoline alkaloids in a single analysis, improving the chemical understanding of botanical supplements.
• Quality control and authentication: The approach can detect undeclared components, variable speciation and sample complexity that are not evident from labels, informing manufacturers, regulators and consumers.
• Risk assessment: Identification of potentially toxic alkaloids (e.g., palmatine has reported toxic effects) or combinations that could alter bioactivity supports safety screening and post-market surveillance.
• Analytical performance: Ion mobility improves resolution of isobars and co-eluting compounds, reducing false identifications and enabling more reliable structural assignments in complex natural product matrices.

Future trends and possibilities for application

• Standardization and CCS libraries: Expanding collision cross section (CCS) libraries for isoquinoline alkaloids will improve confidence in IMS-based identifications and facilitate cross-laboratory comparability.
• Routine non-targeted surveillance: Integration of IMS-HRMS into routine supplement screening pipelines for manufacturers and regulatory agencies to detect adulteration and composition variability.
• Quantitative follow-up: Development of targeted quantitative methods (e.g., validated LC-MS/MS assays) for berberine and co-occurring alkaloids to translate non-targeted findings into dose and exposure metrics.
• Pharmacological linkage: Coupling chemical speciation with in vitro bioassays or metabolomics to relate product composition to bioactivity, bioavailability, and safety outcomes.
• Improved informatics: Use of robust spectral libraries and automated neutral-loss workflows to accelerate tentatively assigning structurally related natural products in complex extracts.

Conclusion

Non-targeted IMS-HRMS profiling of seven commercial berberine/berberis supplements revealed substantial variability in isoquinoline alkaloid composition. Berberine was generally predominant but samples ranged from nearly pure berberine to complex mixtures containing palmatine, jatrorrhizine and other related alkaloids. Ion mobility provided critical separation of isobaric/co-eluting species, improving the reliability of structural assignments in complex botanical matrices. These results underline the need for advanced analytical approaches in supplement quality control and for caution when inferring product composition from labeling alone. A combined strategy of non-targeted screening, targeted quantification and expanded spectral/CCS libraries will strengthen product characterization, safety assessment, and regulatory oversight.

References

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Note: Authors disclosed employment at the instrument vendor. The study demonstrates analytical strengths of IMS-HRMS for natural product speciation but readers should consider conflict of interest when interpreting vendor-presented data.