LC‐MS Fingerprinting Method for the Trace Amines Ratio and Synephrine Enantiomers Found in Citrus Aurantium ‐Listing Pre‐Workout Supplements

Background/Aim The popularity of weight‐loss and pre‐workout supplements (PWS) has been associated with adverse cardiovascular effects. Since 2004, bitter orange ( Citrus aurantium) extracts were listed as a natural stimulant in these supplements. Bitter orange is known to contain the sympathomimetics, p ‐synephrine, p ‐octopamine and p ‐tyramine. However, the origin of these amines is questionable due to discrepancies between the labelled and actual content found in previous analytical methods. This study aimed to produce an LC‐MS fingerprint profile for the three amines and enantiomeric ratios of p ‐(±)‐synephrine in standardized reference materials (SRMs) to compare to PWS. Methods Three SRMs and fifteen bitter orange‐listing PWS were analyzed under two reversed‐phase LC‐QDa‐MS chromatographic separation protocols. Quantitative method used an XBridge BEH C18 (100mm x 4.6mm, 2.5μm) with a mobile phase of water (A) and acetonitrile (B), both with ammonium carbonate buffer (pH 10.0; 5mM), under gradient conditions (1–45 %B over 10 minutes) at a flow rate of 0.8mL/min and UV detection at 242 nm. Quantifications were based on the racemic amine standards. Qualitative enantiomeric separations were conducted using a Chiral‐CBH column (100mm x 4.0mm, 5μm) under isocratic conditions with a mobile phase of methanol (15%, w/w) in ammonium acetate buffer (pH 7.0, 5mM) and a flow rate of 0.6 mL/min. UV detection was at 225nm. A QDa mass detector provided structural confirmation in ESI+ mode. Results Trace amines and enantiomeric ratios of the SRMs support the current literature where (−)‐synephrine was the main amine of bitter orange, whereas octopamine, tyramine and (+)‐synephrine were present in low concentrations . Quantitative methods were linear over a 1.0 – 250 μg/mL range. The limit of quantification (LoQ) for amines was 0.45 μg/mL. Synephrine was detected in the majority of supplements (11/15; 0.025mg – 38.8 mg/serve) whereas octopamine was only in 5/15 (0.013 mg – 0.54 mg/serve) and tyramine in 8/15 supplements (0.017 mg – 0.43 mg/serve). Only 3/15 PWS had trace amine ratios (octopamine: synephrine: tyramine) similar to that found in the plant SRMs. The enantiomeric separation was linear, ranging from 0.5 – 200 μg/mL. LoQ was 1.75 μg/mL for (−)‐synephrine and 1.62 μg/mL for (+)‐synephrine. Only 5/15 supplements showed similar enantiomeric ratios to that of the SRMs, 4/15 supplements showed racemic synephrine profiles while 2/15 supplements showed higher amounts of (+)‐synephrine . Conclusion This pilot study incorporated bitter orange SRMs to quantify and then establish a relative ratio for the trace amines, synephrine, octopamine and tyramine, as well as determine enantiomeric ratios for p‐synephrine. Only 3/15 PWS contained a matching profile of trace amines and an enantiomer ratio suggestive of natural origin. Furthermore, the amounts of (+)‐synephrine suggest that synephrine had been added synthetically or undergone racemization during manufacture. Variability of trace amine composition in PWS presents increased risks to consumers, especially when supplements often contain a cocktail of stimulants, including caffeine and other plant extracts. Support or Funding Information Research was supported by an Australian Government Research Training Program Scholarship Representative summary of the two LC‐MS methods to quantify and qualify trace amines in standardized bitter orange extract and pre‐workout supplements that had natural, racemic and synthetic profiles. Top row: Representative peaks of synephrine (Syn; green), octopamine (Oct; red) and tyramine (Tyr; blue). Bottom row: representative peaks of (±)‐synephrine enantiomers. Figure 1