1. Parameter optimization for the determination of BSH in whole blood by 10 B-NMR Keijiro Saito K. 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

2. Introduction ✓ Clinically utilized boron compounds ✓ Well-used for the boron part of new boron compounds 2 BPABSH Boron clusters 2- Linker

3. Introduction 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 3 2-

4. Introduction The boron-10 ( 10 B) concentration in vivo is important factor for the effective treatment on BNCT. 10 B concentration have been determined by  ICP-AES  Prompt  ray spectroscopy  18 F-BPA-PET etc…. 4 ICP-AES 18 F-BPA-PET

5. Introduction Traditional methods cannot determine concentrations of each boron compound. Table boron determination methods and their analytical characteristic 5 methoddetermine 10 B concentration as ICP-AESwhole B atom concent PG-SPECTwhole 10 B atom content BPA-PETwhole 18 F atom content B-NMR concentrations of each boron compound

6. Introduction: 10 B-NMR 6 Fig.1. B-NMR spectrum of BPA, boric acid, BPA-Fr and Na 2 B 12 H 12 - containing water sample (Peaks of BPA, Boric acid and BPA-Fr are mathematically separated) BPA (  = 30 ppm) Boric acid (  = 20 ppm) BPA-Fructose (  = 8 ~ 9 ppm) Na 2 B 12 H 12 (  = -13 ppm) Chemical species = peak position Concentration = peak area

7. Introduction 7 Example BPA + BSH combination whole boron conc. = BPA + BSH + Metabolites Traditional analysis This method 7% metabolites 13% BSH 80% BPA whole B or 10 B atom content

8. Introduction ✓ 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 8

9. Introduction ✓ 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 9 BPA + Fr in water BPA + Fr in whole blood ✗ reduced intensity and sensitivity BPA-FrBPA

10. Introduction: 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. 10 Measurement parameters of NMR was optimized to improve analytical performance of NMR method. Then, quantitative measurability of BSH in whole blood was evaluated. Measurement parameters of NMR was optimized to improve analytical performance of NMR method. Then, quantitative measurability of BSH in whole blood was evaluated.

11. Experiment: Materials and methods Chemicals 10 B-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 1 H, 11.7 T) NMR frequency for 10 B: 53.7 MHz Sample tube:  = 5 mm quartz NMR tube Receiver gain: 60 11

12. Experiment: Sample preparation 12 Water samples Blood samples 100  g 10 B/mL BSH mother sol. diluted with water Sampling to NMR tube Sampling to NMR tube 100  g 10 B/mL BSH mother sol. Diluted with whole blood NMR analysis 6 samples 3-50  g 10 B/mL 600 µL adequate mount of mother sol. adequate mount of mother sol.

13. Results and discussion 13 dimer peaks splitting ↓ B-H coupling J B-H = 46.2 Hz 50 µg 10 B/mL water sample peak broadening 40% decreased intensity in whole blood sample…

14. Experiment: Parameter optimization Sample  15 µg 10 B/mL BSH in whole blood Table Screened parameters 14 ParametersValues FT-scan number50000, 100000, 200000 X (data) points1024, 2048, 4096 Temperature / o C25, 35, 45, 55 Relaxation delay / ms30, 100 RF irradiationNMR signal sample heating NMR sample tube sample spinning

15. Results and discussion: Temperature optimization 15 T 2 : spin-spin relaxation time  : viscosity, T: temperature 25 °C35 °C 45 °C55 °C peak width  1/2 = 346 Hz peak intensity  = 0.74  1/2 = 265 Hz  = 0.85  1/2 = 239 Hz  = 0.88  1/2 = 228 Hz  = 1.22

16. Experiment: Optimal parameters for 10 B-NMR Table Optimized parameters  Detection limit = Signal-Noise ratio (S/N) > 3 16 ParametersValues Scan number100000 X (data) points2048 Temperature / o C55 Relaxation delay / ms30  Obtained calibration curves were compared with those of other boron compounds

17. Results and discussion: water sample 17 Conc. /  g 10 B mL -1 S/N ratio Integrated ValueRSD / % 35:10.258 ± 0.026.5 58:10.421 ± 0.045.3 1021:10.972 ± 0.032.3 1534:11.50 ± 0.052.3 2042:12.05 ± 0.051.7 2565:12.73 ± 0.123.3 50119:15.34 ± 0.111.7 detection limit* c.a. 2.5  g 10 B mL -1 quantitation limit > 3 µg 10 B/mL * calculated value

18. Results and discussion: blood sample 18 Conc. /  g 10 B mL -1 S/N ratio Integrated ValueRSD / % 34:10.192 ± 0.0421 59:10.497 ± 0.0612 1015:11.10 ± 0.054.9 1523:11.82 ± 0.074.1 2030:12.54 ± 0.104.1 2540:13.23 ± 0.072.1 5088:17.43 ± 0.141.9 detection limit* c.a. 3.0  g 10 B mL -1 quantitation limit > 5 µg 10 B/mL * calculated value

19. Results and discussion Comparison of calibration curve of BSH and BPA 19 Fig. Calibration curves of BPA and BSH (calibration curves of BPA were referred to [1])

20. Conclusion 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. 20 We have succeeded in determination of 10 B-BSH concentration in whole blood for the first time!!

21. Introduction: 10 B-NMR 21 Fig.1. B-NMR spectrum of BPA, boric acid, BPA-Fr and Na 2 B 12 H 12 - containing water sample (Peaks of BPA, Boric acid and BPA-Fr are mathematically separated) BPA (  = 30 ppm) Boric acid (  = 20 ppm) BPA-Fructose (  = 8 ~ 9 ppm) Na 2 B 12 H 12 (  = -13 ppm) Chemical species = peak position Concentration = peak area

22. Acknowledge I would like to thank My boss, K. Yoshino for his blood donation and support for me. Prof. M. Kirihata for donating 10 B-BSH Laboratory students for their helping to this study. Daio wasabi farm Kamikochi 22