A method for individual quantitation of
the combined BPA and BSH by LC/MS
C.Bi1, Y.Yamaguchi1, S.Bamba1, H.Kumada2 , K.Nakai3 and T.Morimoto1
1Research
and Development Office, Japan Chemical Analysis Center
2Proton Medical Research Center, University of Tsukuba, Japan
3Faculty of Medicine, Department of Neurosurgery, University of Tsukuba, Japan
Introduction
In order to improve the drug effectiveness, especially for the requirement of
malignant brain tumors in BNCT clinical trial, it is reasonable to use BSH in
conjunction with BPA.
BSH has higher boron content, penetrate into the brain tumors but not normal
brain. It depends on the destruction of BBB (Blood-Brain Barrier) and 10B
concentration in the blood.
BPA is active uptake into tumors through amino acid transporter. When the
accumulation of BPA in the tumors is low, the combination of BSH is effective.
Although total 10B concentration from both of BSH and BPA accumulated in
the tumors can be calculated such as ICP method , PGA-SPECT, it is disable to
ascertain 10B origin.
● Thus, a suitable method by LC/MS for the individual quantitation of
BSH and BPA is studied in this work.
Why LC/MS?
LC/MS
=
HPLC
+
MS
LC/MS system schematic diagram
-Chemical compounds are separated by retention time depending on the
interactions of the sample with the mobile phase and column by LC.
-The mass spectrometer works by ionizing molecules and then sorting and
identifying the ions according to their m/z (mass-to-charge ratios).
Objectives
To develop an optimum separation condition for BSH and
BPA by LC, including the discussion of solubility condition of
standard solution, selection of mobile phase and column.
To perform the compounds identification and quantitation of
BSH and BPA.
to confirm applicability for blood by LC/MS.
Keywords:
Separation and Quantitation
Experiment (1)
～ Preparation of standard solution～
Standard solution in water of 10B-enriched BSH and BPA were
used to investigation of the analytical condition for LC/MS.
Preliminary experiment for solubility condition
BSH was able to dissolve into water, methanol, acetonitrile directly.
BPA was optimized for dissolution completely by using ultra- sonication for
6h into water and methanol, but not able to dissolve into acetonitrile.
※Methanol, acetonitrile are popularly used as LC/MS solvents.
Stock solution preparation
Both of the concentration for BSH and BPA were adjusted to 1000ppm as
a stock solution dissolved into ultrapure water and corresponding content
were adjusted by following dilution.
※Saturated solubility for BPA is 1.6mg/ml in water （25℃）
Experiment (2)
～ Selection of mobile phase and column～
(1) The mobile phase for LC is selected methanol loaded with 5mM
DHAA, as an ion-pair regent.
DHAA (For LC/MS)
 To enhance the hydrophobic
of BSH.
Dihexylammonium Acetate
(0.5mol/L in Water)
 To provide sufficient retention
and separation selectivity.
Molecular formula: C14H31NO2
Molecular weight : 245.41
 To improve ESI response for
increasing sensitivity.
+
・CH3COOH
R2－NH2X-
(2) The chromatography was performed by a Shim-pack FC-ODS
column, packing with small particle size of 3μm for high resolution.
Apparatus (LC/MS)
Liquid Chromatograph Mass Spectrometery
(LC/ MS 2020, Shimadzu Corporation, Japan)
Result (1)
～ Effectiveness of DHAA for LC separation～
Inten.
10B-BSH
m/z 723
2
LC analytical condition:
Mobile phase: DHAA-Methanol
Column: FC-ODS(150mmL×2mmI.D.,3μm）
Sample: Mixed standard solution
10B-BPA
1
m/z 207
Flow rate: 0.2ml/min.
Injection: 3μl
35min. for gradient analysis
Time (min.)
Represented ion chromatogram of
mixed standard solution for BSH and BPA
BSH and BPA were separated completely according to the different
retention times after loaded with DHAA.
Furthermore, the peak for BPA appears at about 5.0min. and the BSH
compound is monitored at about 18.0min.
Result (2)
～ Mass spectrum of BSH and BPA～
6
Inten.*10
Inten.(×1000000)
Inten.*105
Inten.(×100000)
BSH
m/z
Mass spectrum of BSH by ESI-SCAN(+)
for the mass range 50-1000 m/z
BPA
m/z
Mass spectrum of 10B-BPA by ESI-SCAN(-)
for the mass range 50-1000 m/z
The positive ion [10B12H11SH・3C12H28N]+ at m/z 723 for BSH (left fig.)
and the negative ion [M-H]- at m/z 207 for BPA (right fig.) were identified
from the total ions of range 50-1000 m/z(mass-to-charge ratio).
Result (3)
～ Calibration curve for Standard solution～
Peak area*105
Peak area*106
BPA
BSH
[n=6]
BSH target concentration (ppm)
Calibration carve for BSH
[n=6]
BPA target concentration (ppm)
Calibration carve for BPA
The concentration ranges are 0.05-5ppm for BSH and 0.5-50ppm for BPA respectively.
Linear calibration curves were obtained with a correlation coefficient higher than 0.999.
The relative standard deviation(for Precision) and the relative error(for Accuracy) were
calculated and the results are acceptable.
Applicability for Human plasma
●Human plasma samples preparation●
Normal human plasma collected from the whole blood through centrifugation, was used as
target samples. After loaded with mixed standard of BSH and BPA, samples were under
deproteinization and delipidation as a pretreatment. The concentration of samples
supplied for LC/MS analysis, were adjusted to 0.05-5ppm for BSH and 0.5-50ppm for BPA
respectively.
Deproteinization and delipidation operation from human blood
Plasma(55% contains proteins and lipids)
Buffy coat(1%)
Red blood cells(45%)
Human whole blood
(COSMO BIO CO.LTD)
Centrifuge
(3000rpm×10min.）
Spiked with
mixed standard
[BSH+BPA]
Human Plasma
was collected
Deproteinization
and delipidation
【Methanol/(Plasma+Standard)】
=3:1 （clear samples obtained）
LC/MS
Result (4)
～ Calibration standard for human plasma～
Peak area*106
BSH
Target concentration for BSH (ppm)
Plasma calibration curve for BSH
Peak area*105
BPA
Target concentration for BPA (ppm)
Plasma calibration curve for BPA
Linearity was proven between 0.05-5ppm for BSH and 0.5-50ppm for BPA
in human plasma samples according to the former analytical condition.
Result (5)
～ BSH & BPA determination in plasma～
Precision and accuracy for measurement of human plasma was calculated.
The precision was evaluated using three determinations at three concentration
levels spread over entire linear range.
BSH target
concentration
(ppm)
Mean
RSD
measured
(n=3)
value
(ppm)
(%)
RE
BPA target
concentration
(%)
(ppm)
Mean
RSD
measured
(n=3)
value
(ppm)
(%)
RE
(%)
0.5
0.53
1.8
5.0
5
4.20
2.6
-15.7
1
1.06
0.1
5.9
10
10.9
0.3
9.3
5
4.97
1.0
-0.6
50
49.3
0.9
-1.4
RSD: Relative standard deviation
RE: Relative error
Acceptable results were obtained for both BSH and BPA with overall
precision(RSD value) of 0.1~2.6% and accuracy(RE value) of -0.6~-15.7%.
Conclusions
 The calibration curves for standard solution were linear over a
concentration range of 0.05-5ppm for BSH and 0.5-50ppm for BPA.
Linearity was also proven normal human plasma loaded with BSH and BPA.
 Precision, accuracy for measurement of normal human plasma show the
pretreatment of blood and this analytical method for separation and
quantitation of BSH and BPA work very well.
From these observations, it can be concluded that this analytical technology
for separation and quantitation of BSH and BPA is available and we expect to
implement for the whole blood analysis.
Acknowledgement
This research is supported in part by Grant-in-Aid for
Scientific Research ( 80647321 ) from the Japan Society for
the Promotion Science(JSPS).
Thank you for your kind attention !