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. 2021 Mar 4;26(5):1384.
doi: 10.3390/molecules26051384.

Cyanogenic Glycoside Analysis in American Elderberry

Michael K Appenteng  1 , Ritter Krueger  1 , Mitch C Johnson  1 , Harrison Ingold  1 , Richard Bell  2 , Andrew L Thomas  3 , C Michael Greenlief  1
Affiliations

Cyanogenic Glycoside Analysis in American Elderberry

Michael K Appenteng et al. Molecules. .

Abstract

Cyanogenic glycosides (CNGs) are naturally occurring plant molecules (nitrogenous plant secondary metabolites) which consist of an aglycone and a sugar moiety. Hydrogen cyanide (HCN) is released from these compounds following enzymatic hydrolysis causing potential toxicity issues. The presence of CNGs in American elderberry (AE) fruit, Sambucus nigra (subsp. canadensis), is uncertain. A sensitive, reproducible and robust LC-MS/MS method was developed and optimized for accurate identification and quantification of the intact glycoside. A complimentary picrate paper test method was modified to determine the total cyanogenic potential (TCP). TCP analysis was performed using a camera-phone and UV-Vis spectrophotometry. A method validation was conducted and the developed methods were successfully applied to the assessment of TCP and quantification of intact CNGs in different tissues of AE samples. Results showed no quantifiable trace of CNGs in commercial AE juice. Levels of CNGs found in various fruit tissues of AE cultivars studied ranged from between 0.12 and 6.38 µg/g. In pressed juice samples, the concentration range measured was 0.29-2.36 µg/mL and in seeds the levels were 0.12-2.38 µg/g. TCP was highest in the stems and green berries. Concentration levels in all tissues were generally low and at a level that poses no threat to consumers of fresh and processed AE products.

Keywords: American elderberry; UHPLC-MS/MS; cyanogenic glycosides; picrate method; solid phase extraction; total cyanogenic potential.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Generic structure for a cyanogenic glycoside, where R1 is often methyl or a proton and R2 is a variable organic group.
Figure 2
Figure 2
Calibration curve for the amount of CN− eq. produced by reaction of picric acid with HCN using amygdalin as a CNG standard measured by UV-Vis spectrophotometry at λmax = 510 nm.
Figure 3
Figure 3
Total cyanogenic potential for different types of tissue of Ozone and Ozark AE genotypes. The amounts of CNGs in these genotypes were determined using UV-Vis spectrophotometry. The error bars represent the standard deviation of at least three replicate samples.
Figure 4
Figure 4
Total cyanogenic potential for different types of AE tissue of pooled samples made up of five different genotypes. The amounts of CNGs in pooled samples were determined using UV-Vis spectrophotometry. The error bars represent the standard deviation of at least three replicate samples.
Figure 5
Figure 5
ESI positive mode product ion (296 m/z, product ion) spectrum for amygdalin (465 m/z, precursor ion).
Figure 6
Figure 6
Ion chromatograms for (A) amygdalin (MRM), (B) amygdalin, (C) dhurrin, (D) prunasin, and (E) linamarin (SIR). Retention times in min. are: 4.61, 4.61, 2.54, 5.37, and 1.18, respectively.
Figure 7
Figure 7
Elution profile for amygdalin as a function of methanol content in the extraction solvent. (AUC is the area under the curve).
Figure 8
Figure 8
Amounts of CNGs (µg/g) in tissues (seeds, juice, skin and stem) of Ozone elderberry samples as measured by UHPLC-MS/MS.
Figure 9
Figure 9
Amounts of CNGs (µg/g) in tissues (seeds, juice, skin and stem) of Ozark elderberry samples as measured by UHPLC-MS/MS.
Figure 10
Figure 10
Cyanogenic glycosides standards used in this study: amygdalin, dhurrin, prunasin, and linamarin.
Figure 11
Figure 11
A picrate-paper cyanide color chart for qualitative analysis of CNGs for the range of 0–100 µg CN− eq.
See this image and copyright information in PMC

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