Determination of synephrine in bitter orange raw materials, extracts, and dietary supplements by liquid chromatography with ultraviolet detection: single-laboratory validation

Abstract

A method has been developed to quantify synephrine in bitter orange raw material, extracts, and dietary supplements. Single-laboratory validation has been performed on the method to determine the repeatability, accuracy, selectivity, limit of detection/limit of quantification (LOQ), ruggedness, and linearity for p-synephrine and 5 other biogenic amines: octopamine, phenylephrine (m-synephrine), tyramine, N-methyltyramine, and hordenine, which may be present in bitter orange. p-Synephrine was found to be the primary biogenic amine present in all materials tested, accounting for >80% of the total biogenic amine content in all samples except a finished product. Repeatability precision for synephrine was between 1.48 and 3.55% RSD. Synephrine recovery was between 97.5 and 104%. The minor alkaloids were typically near the LOQ of the method (300-900 microg/g) in the test materials, and between-day precision for the minor compounds was poor because interferences could sometimes be mistakenly identified as one of the minor analytes. Recoveries of the minor components ranged from 99.1 to 103% at approximately 6000 microg/g spike level, to 90.7 to 120% at 300 microg/g spike level.

Figure 1

Structures of biogenic amines.

Figure 2

Stock standard solution chromatogram peak assignments and approximate retention times: (1) Octopamine (11.7 min), (2) p-synephrine (14.6 min), (3) phenylephrine (18.0 min), (4) tyramine (18.7 min), (5) N-methyltyramine (20.1 min), and (6) hordenine (22.8 min).

Figure 3

Bitter orange powdered fruit chromatograms: (A) NIST bitter orange fruit material and (B) C. aurantium powdered immature fruit material from Nutratech.

Figure 4

Bitter orange extract chromatograms: (A) NIST bitter orange extract and (B) C. aurantium 30% synephrine extract from Nutratech.

Figure 5

Dietary supplement finished products containing bitter orange chromatograms: (A) Product A dietary supplement capsules and (B) Product B dietary supplement tablets.

Figure 6

Product C negative control chromatogram.

Figure 7

Synephrine linearity residual plot.

Figure 8

Expanded chromatograms showing octopamine interferences.