????HPLC-UV????????????

1
????HPLC-UV????????????
???,???,???,???,??? (??????????,????
030006)
?? ?????????????HPLC??????????????,?????????????
HPLC-UV???SSa?SSd ????????,???????240?250nm,????
?-?(4060, vv),?? 1.0 mL/min ,????Hypersil ODS
C18???LC-TOF/MS?????????????????????????????????
,???SSa?SSd??????????SSb1?SSb2?????????????HPLC-U
V???????SSa?SSd?????????????SSa?SSd????????????,?
?????,???????SSb1?SSb2,??????????240?250nm?
????SSa?SSd ?HPLC??
Waters ???????
????
?????????TOF/MS??
??????
(1)
(2)
(3)
??Agilent G1969A LC/MSD-TOF-MS,??????????
Alltima ODS-C18?(4.6250 mm)?????????-0.1??????
Xuemei Qin, Yuanyuan Li,et al.(2007) Validation
of a New Optimized Method for the Determination
of Saikosaponin in Bupleurum Chinense DC. by
Liquid Chromatography Time-of-Flight Mass
Spectrometry (LC-TOF/MS)(In Press)
2
Validation of a New Optimized Method for the
Determination of Saikosaponin in Bupleurum
Chinense DC. by Liquid Chromatography
Time-of-Flight Mass Spectrometry (LC-TOF/MS)
Xuemei Qin, Yuanyuan Li, Xuehong Wei, Xiaoqing
Guo, Lizeng Zhang School of Chemistry and
Chemical Engineering, Shanxi University, Taiyuan
030006, China
Abstract Objective To establish a new optimized
method for the determination of Saikosaponin in
Bupleurum chinense DC. and validate the mechanism
of the acidification reaction. Method An
acidification reaction mechanism was investigated
and validated by LC-TOF/MS technique. Some
possible effects were discussed in detail, such
as temperature, reaction time, acidity and
quantity of base. Saikosaponin a(SSa) and
Saikosaponin d(SSd) were determined by HPLC
coupled with UV detector at 240 and 250nm with a
mobile phase of acetonitrile - water (4060, vv)
at a flow rate of 1.0 mLmin-1on a Hypersil ODS
C18 column. Results. The extraction was treated
by 4HCl-CH3OH. Temperature and acidity had some
influence on the product of acidification
reaction. The reaction was stopped immediately
when 2 KOH-CH3OH was added. Two major
derivatives SSa and SSd were quantified.

• The HPLC Picture of Bupleurum Chinense DC.

• Acidification Reaction Equation

As shown in Eq. (1) and (2), SSa turns to
different products in different temperature. When
the reaction temperature was down to 40?, SSa
turned to SSb1 only when it was up to 40? and
down to 80?, SSa would turn to SSb1 and SSg. Eq.
(3) shows that SSd turns to SSb2 only.
The reaction condition was at room temperature
(25?), 4 HCl-methanol, 4 hours, and acid
alkali (11.5). The UV-HPLC system consisted of a
Hypersil ODS-C18 column (200 mm4.6 mm), mobile
phases were CH3CN and H2O (40 60), the effluent
was delivered at 1.0 mL/min, and the UV
absorbance was monitored at 240 nm and 250 nm.

• The TOF/MS Picture of Bupleurum Chinense DC.

TOF-MS data were recorded with an Agilent G1969A
LC/MSD-TOF-MS equipped with Spray electro-spray
ionization source. A generic positive
electro-spray ionization method was used.
Capillary 4000v, sample cone 100v, extraction
cone voltages 250V, desolvation 11 L/min,
nebulization flows 55 psig, gas temperatures
340?. An Alltima ODS-C18 column (4.6250 mm) was
used. The LC mobile phases were CH3CN for solvent
A and CH3COOH/H2O (11000, v/v) for solvent B.
Conclusion A rapid, accurate and sensitive HPLC
method was set up for determining the content of
saikosaponins. Validation result was consist with
the present report. SSa and SSd could be regarded
as the main components that determine the quality
of the phytomedicine.