Using UHPLC-Triple Quadrupole MS/MS to Detect the Presence of Bark Extract and Yohimbine Adulteration in Dietary Supplements and Botanicals

Significance of the Topic

This study addresses the critical need for reliable analytical methods to detect and quantify yohimbine and related alkaloids in dietary supplements. Yohimbine, a primary indole alkaloid from Pausinystalia johimbe bark, is widely marketed for athletic and sexual performance enhancement, yet its safety and regulatory status vary globally. Effective differentiation between synthetic yohimbine HCl and natural bark extracts is essential to ensure consumer safety and comply with regional regulations.

Goals and Study Overview

The primary objective was to develop and validate a rapid, sensitive, and selective UHPLC–triple quadrupole MS/MS method, combined with QuEChERS sample preparation, for sub-ppb detection of yohimbine and ajmalicine. A secondary goal was to chromatographically resolve diastereomers, enabling distinction between synthetic yohimbine and yohimbe bark extract. Comprehensive qualitative profiling with Q-TOF MS/MS was included to confirm the identity of targeted alkaloids and to screen for any unknown analogs.

Methodology and Instrumentation

Sample preparation employed a modified QuEChERS protocol on tablets, capsules, gels, and powders, involving aqueous extraction, acetonitrile partitioning, salt addition, and dispersive C18 cleanup. Final extracts were diluted and filtered prior to analysis.

Instrumentation:

• UHPLC system with binary pump, autosampler, and thermal column compartment
• Agilent Poroshell 120 EC-C18 column (2.1×100 mm, 2.7 µm) for quantitative analysis
• Agilent 6490 Triple Quadrupole MS/MS with Jet Stream ESI in positive mode
• Agilent 6550 Q-TOF MS for qualitative confirmation and analog screening

Chromatographic gradient: formic acid in water/methanol, 0.4 mL/min, 12 min run. Dynamic MRM transitions were optimized for yohimbine (m/z 355.2→211.9,144.0) and ajmalicine (m/z 353.2→117.1,144.0).

Main Results and Discussion

Calibration covered 0.1–100 ppb with R²>0.9999. Limits of quantitation at 0.1 ppb yielded S/N ratios >190. Spike recoveries ranged 99.7–103.3% with RSD 2.0–3.6%. In contrast, simpler dilute-and-shoot prep showed greater variability (RSD up to 6.4%). Analysis of ten commercial supplements revealed three products free of target alkaloids, one with three times the declared yohimbine level, and others containing varying levels of both compounds.

MRM chromatograms of yohimbe bark extract displayed multiple diastereomer peaks absent in synthetic standards. Comparison of product profiles indicated that some supplements contained synthetic yohimbine (lacking diastereomers), while others mirrored the natural bark pattern. Q-TOF All Ions and Auto MS/MS scans detected no previously unknown indole alkaloid analogs.

Benefits and Practical Applications

• High sensitivity and specificity for yohimbine and ajmalicine at trace levels
• Rapid analysis (<15 min) suitable for high-throughput QA/QC
• Ability to differentiate synthetic adulterants from natural bark extracts using diastereomer separation
• Comprehensive screening for unknown analogs enhances consumer safety and regulatory compliance

Future Trends and Opportunities

Emerging opportunities include integration of high-resolution mass spectrometry for non-targeted screening of novel alkaloids, development of automated data analysis workflows for rapid pattern recognition, and expansion of the methodology to other botanical adulterants. Further work on standardized reference materials and interlaboratory validation will strengthen method adoption in regulatory and industrial laboratories.

Conclusion

A validated UHPLC–triple quadrupole MS/MS method with QuEChERS sample preparation enables sub-ppb quantitation of yohimbine and ajmalicine, robust diastereomer separation, and clear differentiation between synthetic and natural sources. Complementary Q-TOF MS/MS profiling confirmed the absence of undescribed analogs. This analytical approach supports regulatory enforcement, quality control, and consumer safety in the dietary supplement industry.

References

1. Ernst E., Pittler M. H. “Yohimbine for Erectile Dysfunction: A Systematic Review and Meta-Analysis of Randomized Clinical Trials.” Journal of Urology, 159(2):433–436 (1998).
2. Lucas D., Neal-Kababick J., Zweigenbaum J. “Characterization and Quantitation of Yohimbine and Its Analogs in Botanicals and Dietary Supplements Using LC/QTOF-MS and LC/QQQ-MS for Determination of the Presence of Bark Extract and Yohimbine Adulteration.” Journal of AOAC International, 98(2):330–335 (2015).