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Parameter optimization for the determination of BSH
in whole blood by 10B-NMR
Keijiro SaitoK. Yoshino, M. Muto, A. Ishikawa and H. Ohki
Department of chemistry, Faculty of Science
Shinshu University
16, June, 2014
International congress on Neutron Capture Therapy in Helsinki
2Introduction
✓Clinically utilized boron compounds
✓ Well-used for the boron part of new boron compounds
BPA BSH
Boron clusters
2- 2-
2-
Linker
3Introduction
The linkage between two parts could be cloven in vivo after administration.
ex.
B-C bond cleavage of boronic acid (such as BPA)
Sulfide formation2- 2-
4Introduction
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
18F-BPA-PET
etc….
ICP-AES 18F-BPA-PET
5Introduction
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
6Introduction: 10B-NMR
Fig.1. B-NMR spectrum of BPA, boric acid, BPA-Fr and Na2B12H12-containing water sample (Peaks of BPA, Boric acid and BPA-Fr are mathematically separated)
BPA( d = 30
ppm)
Boric acid( d = 20
ppm)
BPA-Fructose( d = 8 ~ 9
ppm)
Na2B12H12
( d = -13 ppm)
Chemical species = peak positionConcentration = peak area
7
1 20
10
20
30
40
50
60
70
80
MetaboliteBSHBPAWhole conc.
Boro
n co
ncen
trati
on in
tiss
ue
Introduction
ExampleBPA + BSH combination
whole boron conc. = BPA + BSH + Metabolites
Traditional analysis This method
7% metabolites13% BSH
80% BPA
whole B or 10B atom content
8Introduction
✓ 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
9Introduction
✓ The measurement of BPA and BPA-Fr in water and blood derivative.[1]
✗ Measurement 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
BPA + Fr in water
BPA + Fr in whole blood
✗reduced intensity and sensitivity
BPA-FrBPA
10Introduction: The purpose and issues of this study
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.
11Experiment: Materials and methods
Chemicals10B-BSH: provided from Peof. M. Kirihata
Whole blood: donated from my boss
Water: deionized and degassed* water
(*refluxed with Ar bubbling to remove oxygen)
ApparatusNMR 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
12Experiment: Sample preparation
Water samples
Blood samples
100 mg 10B/mLBSH mother sol.
diluted with water
Sampling toNMR tube
Sampling toNMR tube
100 mg 10B/mLBSH mother sol.
Diluted withwhole blood
NMR analysis
6 samples3-50 mg10B/mL
600 µL
600 µL
adequate mount of mother sol.
adequate mount of mother sol.
13Results and discussion
SH 2- Area ratio1:5:5:1
dimer
peaks splitting↓
B-H couplingJB-H = 46.2 Hz
50 µg 10B/mL water sample
peak broadening
40% decreased intensity
in whole blood sample…
14Experiment: Parameter optimization
Sample
F 15 µg 10B/mL BSH in whole blood
Table Screened parameters
Parameters Values
FT-scan number50000, 100000,
200000
X (data) points 1024, 2048, 4096
Temperature / oC 25, 35, 45, 55
Relaxation delay / ms 30, 100
RF irradiation NMR signal
sample heating
NMR sample tube
sample spinning
15Results and discussion: Temperature optimizationT2: spin-spin relaxation timeh: viscosity, T: temperature
25 °C 35 °C
45 °C55 °C
peak width D1/2 = 346 Hz peak intensity I = 0.74
D1/2 = 265 Hz I = 0.85
D1/2 = 239 Hz I = 0.88
D1/2 = 228 Hz I = 1.22
Experiment: Optimal parameters for 10B-NMR
Table Optimized parameters
F Detection limit = Signal-Noise ratio (S/N) > 3
16
Parameters Values
Scan number 100000
X (data) points 2048
Temperature / oC 55
Relaxation delay / ms 30
F Obtained calibration curves were compared with those of other boron compounds
17Results and discussion: water sample
Conc. / mg10B mL-
1
S/N ratio Integrated Value RSD / %
3 5:1 0.258 ± 0.02 6.5
5 8:1 0.421 ± 0.04 5.3
10 21:1 0.972 ± 0.03 2.3
15 34:1 1.50 ± 0.05 2.3
20 42:1 2.05 ± 0.05 1.7
25 65:1 2.73 ± 0.12 3.3
50 119:1 5.34 ± 0.11 1.7
detection limit*c.a. 2.5 mg10B mL-1
quantitation limit> 3 µg10B/mL
0 10 20 30 40 500
1
2
3
4
5
6
7f(x) = 0.133681506849315 x + 0.0424429223744278R² = 0.997907819117863
BSH in water
10B concentration/ µg 10B mL-1
Peak
are
a
*calculated value
18Results and discussion: blood sample
Conc. / mg10B mL-
1
S/N ratio Integrated Value RSD / %
3 4:1 0.192 ± 0.04 21
5 9:1 0.497 ± 0.06 12
10 15:1 1.10 ± 0.05 4.9
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
detection limit*c.a. 3.0 mg10B mL-1
quantitation limit> 5 µg10B/mL
0 10 20 30 40 500
1
2
3
4
5
6
7
8f(x) = 0.153537187399729 x − 0.409026379118846R² = 0.996499288959538
BSH in blood
10B concentration/ µg 10B mL-1
Peak
are
a
*calculated value
19Results and discussion
Comparison of calibration curve of BSH and BPA
Fig. Calibration curves of BPA and BSH (calibration curves of BPA were referred to [1])
0 5 10 15 20 25 30 35 40 45 50 550
1
2
3
4
5
6
7
8
BSH in bloodLinear (BSH in blood)BSH in water
10B concentration/ µg 10B mL-1
Peak
are
a
20Conclusion
Peak broadening and reduced intensity were improved by parameter (temperature) optimization.
Using this method, the concentration BPA, boric acid, BPA-Fr, BSH and BSH dimer can be measured simultaneously.
We have succeeded in determination of 10B-BSH concentration in whole blood for the first time!!
21Introduction: 10B-NMR
Fig.1. B-NMR spectrum of BPA, boric acid, BPA-Fr and Na2B12H12-containing water sample (Peaks of BPA, Boric acid and BPA-Fr are mathematically separated)
BPA( d = 30
ppm)
Boric acid( d = 20
ppm)
BPA-Fructose( d = 8 ~ 9
ppm)
Na2B12H12
( d = -13 ppm)
Chemical species = peak positionConcentration = peak area
22Acknowledge
I would like to thankMy 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