Identification and determination of osajin and pomiferin
Liquid chromatography-electrospray ionization-tandem mass spectrometry
(LC-ESI-MS) was used to identify osajin and pomiferin. A Thermo
Scientific ACCELA series HPLC system (ACCELA 1250 UHPLC pump, ACCELA1
HTC cool stack autoinjector, and an ACCELA 80 Hz Photodiode Array (PDA)
detector, Thermo Corp., Milford, MA, USA) running under Thermo
Scientific Xcalibur 2.1.0.1140 LC-MS software equipped with a Thermo
Electron LTQ Orbitrap Discovery Mass Spectrometer (MS) connected to a
high precision electrostatic ion trap (Orbitrap) was used. The MS was
run with the electrospray ionization (ESI) probe in the negative mode.
The source inlet temperature was 300 ˚C, the sheath gas rate was set at
50 arbitrary units, the auxiliary gas rate was set at 5 arbitrary units,
and the sweep gas rate was set at 2 arbitrary units. The maximal mass
resolution was set at 30,000, the spray voltage was 3.0 kV, and the tube
lens was set at -100 V. Other parameters are determined and set by the
calibration and tuning process. The column used was an Inertsil ODS-3
reverse phase C-18 column (3 µm, 150 x 3 mm from GL Sciences, Torrance,
CA, USA). The initial solvent system was
30% acetonitrile verses water,
with 0.1% formic acid in both solvents, at a flow rate of 0.25 ml per
minute. After injection (15-25 µl) the column was developed in a linear
gradient to 100% acetonitrile in 50 minutes. The column effluent was
monitored at 280 nm in the PDA detector. The software package was set to
collect mass data between 100-2000 AMUs. Generally, the most significant
sample ions generated under these conditions were
[M-1]- and [M+HCOO]-. The
two major mass peaks eluted at 42.21 and 46.56 minutes were with a m/z
of 419.14984 [M-H]- and 403.15440
[M-H]-, respectively. These correlate to a
molecular formula of
C25H23O6 (calculated
monoisotopic mass of 419.149465) with a delta mmµ of 0.28 and
C25H23O5 (calculated
monoisotopic mass of (403.154550) with a delta mmµ of 1.015.
HPLC analysis to determine the percent concentrations of osajin and
pomiferin was conducted on a Shimadzu LC-20 HPLC system (LC-20AT
quaternary pump, DGU-20A5 degasser, SIL-20A HT autosampler, and a SPD
M20A photodiode array detector, running under Shimadzu LCSolutions
version 1.22 chromatography software, Columbia, MD, USA). The column
used was an Inertsil ODS-3 reverse phase C-18 column (5 µm, 250 x 4.6
mm, GL Sciences, Torrance, CA). The initial conditions were 30%
methanol with 0.25% trifluroacetic acid and 70% water with 0.25%
trifluroacetic acid, at a flow rate of 1 ml per minute. The effluent was
monitored at 250 nm on the VWD. After injection (typically 25 µL), the
column was developed to 100% methanol with 0.25% trifluroacetic acid
in a linear gradient over 55 minutes. Five-point standard curves for the
evaluation of the concentration of osajin and pomiferin purified in this
laboratory was used for the determination of extinction coefficients at
250 nm. Quantitation was based on conversion of the absorbance peak
areas to mg/g based the extinction coefficient determined from the
standard curves. OOE was determined to consist of 42.9% osajin and 30%
pomiferin.