16, June, 2014
International congress on Neutron Capture Therapy in Helsinki
Parameter optimization for the
determination of BSH
in whole blood by 10B-NMR
Keijiro Saito
K. Yoshino, M. Muto, A. Ishikawa and H. Ohki
Department of chemistry, Faculty of Science
Shinshu University
Introduction
2
✓Clinically utilized boron compounds
2-
BPA
BSH
✓ Well-used for the boron part of new boron compounds
2-
2-
Boron clusters
Linker
Introduction
3
The linkage between two parts could be cloven in vivo after
administration.
ex.
B-C bond cleavage of boronic acid (such as BPA)
Sulfide formation
2-
2-
Introduction
4
The boron-10 (10B) concentration in vivo is important
factor for the effective treatment on BNCT.
10B
concentration have been determined by
F ICP-AES
F Prompt g ray spectroscopy
F
18F-BPA-PET
etc….
ICP-AES
18F-BPA-PET
Introduction
5
Traditional methods cannot determine concentrations
of each boron compound.
Table boron determination methods and their analytical
characteristic
method
determine 10B concentration as
ICP-AES
whole B atom concent
PG-SPECT
whole 10B atom content
BPA-PET
whole 18F atom content
B-NMR
concentrations of each boron compound
Introduction: 10B-NMR
6
Chemical species = peak position
Concentration = peak area
Boric acid
(d = 20 ppm)
BPA
(d = 30 ppm)
Na2B12H12
(d = -13 ppm)
BPA-Fructose
(d = 8 ~ 9 ppm)
Fig.1. B-NMR spectrum of BPA, boric acid, BPA-Fr and Na2B12H12containing water sample (Peaks of BPA, Boric acid and BPA-Fr are
mathematically separated)
Introduction
7
Example
BPA + BSH combination
whole boron conc.
= BPA + BSH + Metabolites
Boron concentration in tissue
80
70
60
whole B or
10B
atom
content
7% metabolites
13% BSH
50
40
30
80% BPA
20
10
0
1
Traditional
analysis
2
This method
Metabolite
BSH
BPA
Whole conc.
Introduction
8
✓ The measurement of BPA and BPA-Fr in water
and blood derivative.[1]
✗ Measurements of BSH in water and blood.
*Peak position, shape and intensity are completely different from
other boron compounds.
✗Resolution and determination performance of
NMR analysis were significantly reduced in the
case of whole blood samples.[2]
[1] K.Yohisno et al. Appli. Radiat. Isot. 2014
[2] K. Saito et al. poster presentation at YBNCT in Spain, 2013
Introduction
9
✓
The
and
water
BPA
+ Frmeasurement of BPA
BPA
+ FrBPA-Fr
in wholeinblood
blood derivative.[1]
in and
water
✗ Measurement of BSH in water and blood.
✗reduced intensity
*Peak position, shape and intensity are completely different
and sensitivity
other boron compounds.
BPA
from
BPA-Fr
✗Resolution and determination performance of
NMR analysis were significantly reduced in the
case of whole blood samples.[2]
[1] K.Yohisno et al. Appli. Radiat. Isot. 2014
[2] K. Saito et al. poster presentation at YBNCT in Spain, 2013
Introduction: The purpose and issues of this study
10
1) BSH measurement is one of our interest.
2) The issue of reduced analytical performance is
expected in the whole blood sample.
Measurement parameters of NMR was optimized to
improve analytical performance of NMR method.
Then, quantitative measurability of BSH in whole blood
was evaluated.
Experiment: Materials and methods
11
Chemicals
10B-BSH:
provided from Peof. M. Kirihata
Whole blood: donated from my boss
Water: deionized and degassed* water
(*refluxed with Ar bubbling to remove oxygen)
Apparatus
NMR spectrometer: JEOL ECA500 (500 MHz for 1H, 11.7 T)
NMR frequency for 10B: 53.7 MHz
Sample tube: f = 5 mm quartz NMR tube
Receiver gain: 60
Experiment: Sample preparation
12
Water samples
adequate mount
of mother sol.
Sampling to
NMR tube
6 samples
3-50 mg10B/mL
600 µL
100 mg 10B/mL
BSH mother sol.
diluted with
water
Blood samples
adequate mount
of mother sol.
Sampling to
NMR tube
600 µL
100 mg 10B/mL
BSH mother sol.
Diluted with
whole blood
NMR analysis
Results and discussion
SH
2-
Area ratio
: : :
dimer
13
50 µg 10B/mL water sample
peaks splitting
↓
B-H coupling
JB-H = 46.2 Hz
in whole blood sample…
40% decreased intensity
peak broadening
Experiment: Parameter optimization
14
Sample
F 15 µg 10B/mL BSH in whole blood
NMR sample tube
Table Screened parameters
Parameters
Values
FT-scan number
50000, 100000, 200000
X (data) points
1024, 2048, 4096
Temperature / oC
25, 35, 45, 55
sample spinning
RF irradiation
Relaxation delay / ms
NMR signal
30, 100
sample heating
Results and discussion: Temperature optimization
T2: spin-spin relaxation time
h: viscosity, T: temperature
25 °C
peak width D1/2 = 346 Hz
peak intensity I = 0.74
35 °C
D1/2 = 265 Hz
I = 0.85
55 °C
D1/2 = 228 Hz
I = 1.22
45 °C
D1/2 = 239 Hz
I = 0.88
15
Experiment: Optimal parameters for 10B-NMR
Table Optimized parameters
Parameters
Values
Scan number
100000
X (data) points
2048
Temperature / oC
55
Relaxation delay / ms
30
F Detection limit = Signal-Noise ratio (S/N) > 3
F Obtained calibration curves were compared
with those of other boron compounds
16
Results and discussion: water sample
17
S/N
ratio
Integrated Value
3
5:1
0.258 ± 0.02
6.5
5
8:1
0.421 ± 0.04
5.3
10
21:1
0.972
8 ± 0.03
2.3
15
34:1
1.50
7 ± 0.05
2.3
20
42:1
2.05
6 ± 0.05
1.7
25
65:1
2.73
5 ± 0.12
3.3
50
119:1
5.34
4 ± 0.11
1.7
detection limit*
c.a. 2.5 mg10B mL-1
*calculated
value
Peak area
Conc.
/ mg10B mL-1
RSD / %
quantitation limit
> 3 µg10B/mL
BSH in water
R² = 0.99791
3
2
1
0
0
10
10B
20
30
concentration/ µg
40
10B
mL-1
50
Results and discussion: blood sample
9
18
BSH in blood
8
R² = 0.9965
detection limit*
c.a. 3.0 mg10B mL-1
Peak area
7
6
5
4
Conc.
/ mg10B mL-1
S/N
ratio
3
Integrated
Value
3
4:1
0.192
1 ± 0.04
21
5
9:1
0.497
0 ± 0.06
12
10
15:1
0
1.10 ±
0.05
15
23:1
1.82 ± 0.07
4.1
20
30:1
2.54 ± 0.10
4.1
25
40:1
3.23 ± 0.07
2.1
50
88:1
7.43 ± 0.14
1.9
*calculated
value
RSD / %
2
10
20
30
40
4.9concentration/
10
µg B mL-1
10B
50
quantitation limit
> 5 µg10B/mL
Results and discussion
19
Comparison of calibration curve of BSH and BPA
9
8
BSH in blood
BSH in water
BPA in blood der.
BPA in water
Peak area
7
6
5
4
3
2
1
0
0
5
10
15
20 25 30 35 40 45
10B concentration/ µg 10B mL-1
50
55
Fig. Calibration curves of BPA and BSH (calibration curves of BPA were referred to [1])
Conclusion
20
We have succeeded in determination of 10B-BSH
concentration in whole blood for the first time!!
Peak broadening and reduced intensity were improved by
parameter (temperature) optimization.
Using this method, the concentration BPA, boric acid, BPAFr, BSH and BSH dimer can be measured simultaneously.
Introduction: 10B-NMR
21
Chemical species = peak position
Concentration = peak area
Boric acid
(d = 20 ppm)
BPA
(d = 30 ppm)
Na2B12H12
(d = -13 ppm)
BPA-Fructose
(d = 8 ~ 9 ppm)
Fig.1. B-NMR spectrum of BPA, boric acid, BPA-Fr and Na2B12H12containing water sample (Peaks of BPA, Boric acid and BPA-Fr are
mathematically separated)
Acknowledge
22
I would like to thank
My boss, K. Yoshino for his blood donation and support for
me.
Prof. M. Kirihata for donating 10B-BSH
Laboratory students for their helping to this study.
Daio wasabi farm
Kamikochi