LC-MS/MS analyses have been reported as challenging for the reliable separation and quantification of cyanogenic glycosides (CNGs), especially (
R)-prunasin and sambunigrin isomers found in American elderberry (
Sambucus nigra L. subsp.
canadensis (L.) Bolli). Hence, a novel multiple reaction monitoring (MRM)-based
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LC-MS/MS analyses have been reported as challenging for the reliable separation and quantification of cyanogenic glycosides (CNGs), especially (
R)-prunasin and sambunigrin isomers found in American elderberry (
Sambucus nigra L. subsp.
canadensis (L.) Bolli). Hence, a novel multiple reaction monitoring (MRM)-based ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated in the present study for simultaneous separation and quantification of five CNGs, including amygdalin, dhurrin, linamarin, (
R)-prunasin, and (
S)-prunasin (commonly referred to as sambunigrin). Initially, the role of ammonium formate was investigated as an aqueous mobile-phase additive in developing MRM-based UHPLC-MS/MS. Later, chromatographic conditions for the resolved separation of (
R)-prunasin and sambunigrin were identified. Validation studies confirmed that the developed method has good linearity and acceptable precision and accuracy. A noticeable matrix effect (mainly signal enhancement) was observed in leaf samples only. This method was used to detect and quantify CNGs, including (
R)-prunasin and sambunigrin, in leaf and fruit samples of American elderberry. Among the studied CNGs, only (
R)-prunasin was detected in the leaf samples. Interestingly, (
S)-prunasin (sambunigrin) was not detected in the samples analyzed, even though it has been previously reported in elderberry species.
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