A method using solid phase
extraction (SPE) for sample preparation and followed with high performance
liquid chromatography coupled with diode array detector and fluorescence
detector (HPLC-DAD/FLD) analysis was developed to determine 9 polymethoxyflavones
(PMFs) compounds in orange juice. Orange juice was filtered and went through a
C18 SPE cartridge; the cartridge was washed in series with ultra-pure water and
30% of acetonitrile; the adsorbed components were then eluted with ethyl
acetate, dried under nitrogen current and re-dissolved in
methanol [l1] [ql2] to produce the PMFs sample. Using 0.05% of phosphoric
acid solution, methanol and 50% of tetrahydrofuran
as the
mobile phase[l3] [ql4] , the sample was separated under gradient elution
condition by reverse phase HPLC. Meanwhile, sample PMFs were qualitatively and
quantitatively determined using the ultraviolet (UV) and fluorescence (FL)
spectra and the peak area obtained by DAD and FLD. All the nine PMFs separated
in 10 min under the solvent gradient composed. Using 330 nm for UV
quantification, 340 nm and 450/500 nm for FL excitation and emission, the
external standard calibration showed good linearity (R2>0.999).
The converted limit of quantitation for juice
was [l5] 4.00-61.2 μg/L and 0.024-0.61 mg/L respectively for UV and FL signal; recovery study showed good
accuracy for both UV (96.4%-104.2%) and FL (94.2%-102.1%) examination; furthermore, UV and FL
determination showed well accordance with each other.
This
method possessed the advantages including simple preparation, short
time-consuming and low cost, which enabled it applicable for the rapid
determination of PMFs contained in orange juice. As an instance application,
fresh squeezed juices from eleven
juice-processing sweet orange cultivars were analyzed; the results showed that
Yuzaocheng contained the highest amount of PMFs, exhibiting prospective potency
for further exploitation.