Automated 2-Dimensional Fingerprint Analysis for Routine Botanical Authentication Using the ACQUITY QDa Mass Detector

Importance of the Topic

The authentication of botanical ingredients is critical to ensure product safety, protect consumer health, and maintain regulatory compliance. Black cohosh (Actaea racemosa) is widely used in dietary supplements and herbal medicines, but its complex phytochemical profile and similarity to related species can lead to misidentification or adulteration.

Objectives and Study Overview

This study demonstrates an automated two-dimensional fingerprint analysis for routine authentication of North American black cohosh using a cost-effective mass detector and streamlined data processing. The approach integrates chromatographic and mass spectral patterns of a key marker compound to distinguish authentic samples from closely related species and commercial products.

Methodology and Instrumentation

Sample Preparation:

• Authentic extracts: three North American (NA1–NA3) and three Asian (A1–A3) black cohosh, plus four commercial products (U1–U4) and two mixed samples (M5, M10).
• Diluted in 70% methanol (~5 mg/mL) and filtered (0.2 µm PTFE).
• Four reference standards (cimifugin, cimiracemoside C, 27-deoxyactein, actein) at ~5 µg/mL.

Chromatography and Detection:

• ACQUITY UPLC H-Class System with BEH C18 column (2.1 × 100 mm, 1.7 µm).
• Mobile phases: 0.1% formic acid in water (A) and in ACN/MeOH 7:3 (B); gradient from 20% to 100% B over 6 min.
• ACQUITY QDa Mass Detector, ESI+ scan (200–1000 Da), capillary voltage 1.5 kV, cone voltage 10 V.
• Automated data processing in Empower 3 CDS Software.

Main Results and Discussion

Chromatographic Fingerprint:

• Cimiracemoside C selected as marker (m/z 621 Da, RT ~5.77 min).
• NA samples exhibit two major peaks of similar height; marker peak RT and relative area used as quantitative parameters.
• Thresholds set at RT ±1% and % area ±3×SD based on triplicate analyses.

Mass Spectral Fingerprint:

• Top five ions at RT 5.77 min: m/z 621 (base peak), 603 (fragment), 643 (Na adduct), and isotopic peaks at 622 and 644.
• Relative intensities compared to base peak with lower limits at 3×SD below the average.

Two-Dimensional Authentication:

• Empower method extracts ion chromatogram at m/z 621, integrates peaks, verifies RT and % area, performs library match, and checks ion intensity criteria.
• Automated flags identify authentic versus adulterated samples.
• Validation with commercial and home-made mixtures correctly classified all samples.

Benefits and Practical Applications

This approach leverages a simple marker-based fingerprint and low-cost MS detector to enable rapid, routine botanical authentication. Automated Empower processing reduces operator expertise requirements, accelerates throughput, and ensures consistent decision criteria for quality control laboratories, dietary supplement manufacturers, and regulatory testing.

Future Trends and Potential Applications

Extending this method to other botanicals will broaden its utility. Potential developments include:

• Integration with chemometric tools for multi-marker analyses.
• Expansion of MS libraries with high-resolution reference spectra.
• Real-time in-line monitoring using compact detectors.
• Cloud-based data sharing and AI-assisted anomaly detection.

Conclusion

An automated two-dimensional fingerprint strategy using UPLC-QDa and Empower software provides an efficient, objective, and affordable solution for routine authentication of North American black cohosh. The combination of chromatographic and mass spectral patterns of a single marker compound ensures high selectivity and robustness in distinguishing authentic materials from adulterants.

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