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