HPLC-MS Methods for Quantification of Yohimbine in Yohimbe Bark and to Distinguish Synthetic Yohimbine HCl from Yohimbine-Containing Bark Extracts

Reviewed: 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. J AOAC Int. 2015;98(2):330-335.

Extracts of the bark of the yohimbe tree (Pausinystalia johimbe, Rubiaceae) are used in dietary supplements to enhance physical and sexual performance. The yohimbe bark contains 1-6% indole alkaloids, e.g., yohimbine, ajmalicine, corynantheidine, and epimers of yohimbine (Figure 1).¹ The major alkaloid of the bark, yohimbine, is also produced via total synthesis and sold as the HCl salt. However, pure yohimbine HCl is generally considered a drug (it is used as a remedy for male erectile dysfunction) and use of this compound in an herbal product violates the dietary supplement current Good Manufacturing Practices (cGMPs) in the USA. The paper describes HPLC-MS methods for the analysis of two samples of authentic yohimbe bark extracts provided by Flora Research Laboratories (Grants Pass, OR; one of the laboratories that is collaborating in the Botanical Adulterants Program) and 10 dietary supplement products purchased in retail stores in Grants Pass, OR. The initial method, using a triple-quadrupole (QQQ) mass spectrometer, enables the accurate quantification of yohimbine and ajmalicine at concentrations above 0.1 ng/mL, while the use of a quadrupole time-of-flight (QTOF) mass spectrometer allows for the acquisition of the accurate mass for further confirmation of the correct structure of the analytes (although the high-resolution mass spectrometer does not allow distinction of diastereomeric/epimeric compounds). Products that contain yohimbine without its associated epimers and without other indole alkaloids likely have synthetic yohimbine as an ingredient.

The yohimbe bark samples were found to contain 10.8 mg/g and 13.6 mg/g yohimbine, and 59.9 μg/g and 12.0 μg/g ajmalicine, respectively. The 10 dietary supplements analyzed contained between 0 and 29.5 mg/g yohimbine and between 0 and 580.3 μg/g ajmalicine. Three supplements did not contain any alkaloids at all. There was no evidence of admixture of synthetic yohimbine HCl to any of the products.

Comment: Dietary supplements containing yohimbe bark remain popular and have ranked as number seven of the top-selling products in 2014 in the US retail mainstream multi-outlet channel.² It is not clear why some of the commercial products did not contain any yohimbe alkaloids, but substitution with a different ingredient is one possible explanation. Most of the published methods for analysis have used HPLC-MS or HPLC-UV/MS, but there is one paper that also included a microscopic description of yohimbe bark.^3-5 The HPLC-QQQ method by Lucas et al. has been thoroughly validated and as such is a reliable way to quantify yohimbine and ajmalicine in the material. The use of a mass spectrometer rather than a UV detector helps in the identification of the compounds, and also allows detection of the many co-eluting indole alkaloids in the materials by extracting ion chromatograms of different molecular weights. For those companies that cannot afford the high-end QQQ and QTOF instruments, entry-level HPLC-MS instruments should be sufficient to authenticate the material and quantify the main indole alkaloids, even if the limits of quantification may not be as low as those of the QQQ mass spectrometer.

References

1.     Betz JM, White KD, der Marderosian AH. Gas chromatographic determination of yohimbine in commercial yohimbe products. J AOAC Int. 1995;78(5):1189-1194.

2.     Smith T, Lynch ME, Johnson J, Kawa K, Bauman H, Blumenthal M. Herbal dietary supplement sales in US increase 6.8% in 2014. HerbalGram. 2015;(107):52-59. Available at: http://cms.herbalgram.org/herbalgram/issue107/hg107-mktrpt-2014hmr.html. Accessed October 7, 2015.

3.     Raman V, Avula B, Galal AM, Wang Y-H, Khan IA. Microscopic and UPLC-UV-MS analyses of authentic and commercial yohimbe (Pausinystalia johimbe) bark samples. J Nat Med. 2013;67(1):42-50.

4.     Sun J, Chen P. Chromatographic fingerprint analysis of yohimbe bark and related dietary supplements using UHPLC/UV/MS. J Pharm Biomed Anal. 2012;61:142-149.

5.     Sun J, Baker A, Chen P. Profiling the indole alkaloids in yohimbe bark with ultra-performance liquid chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry. Rapid Commun Mass Spectrom. 2011;25(18):2591-2602.