1. C.Bi 1, Y.Yamaguchi 1, S.Bamba 1, H.Kumada 2, K.Nakai 3 and T.Morimoto 1 1 Research and Development Office, Japan Chemical Analysis Center 2 Proton Medical Research Center, University of Tsukuba, Japan 3 Faculty of Medicine, Department of Neurosurgery, University of Tsukuba, Japan A method for individual quantitation of the combined BPA and BSH by LC/MS the combined BPA and BSH by LC/MS

2. 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. Introduction 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 10 B 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 10 B 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 10 B origin. ● Thus, a suitable method by LC/MS for the individual quantitation of BSH and BPA is studied in this work.

3. Why LC/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).+ LC/MS = HPLCMS

4. 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. Keywords: Separation and Quantitation to confirm applicability for blood by LC/MS.

5. Standard solution in water of 10 B-enriched BSH and BPA were used to investigation of the analytical condition for LC/MS. Experiment (1) ～ Preparation of standard solution ～ 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. ※ Saturated solubility for BPA is 1.6mg/ml in water （ 25 ℃） 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. Preliminary experiment for solubility condition Stock solution preparation ※ Methanol, acetonitrile are popularly used as LC/MS solvents.

6. (1) The mobile phase for LC is selected methanol loaded with 5mM DHAA, as an ion-pair regent. (2) The chromatography was performed by a Shim-pack FC-ODS column, packing with small particle size of 3μm for high resolution. Experiment (2) ～ Selection of mobile phase and column ～ Dihexylammonium Acetate (0.5mol/L in Water) Molecular formula: C 14 H 31 NO 2 Molecular weight : 245.41 ・ CH 3 COOH DHAA (For LC/MS) R 2 － NH 2 X - + To improve ESI response for increasing sensitivity. To enhance the hydrophobic of BSH. To provide sufficient retention and separation selectivity.

7. Apparatus (LC/MS) Liquid Chromatograph Mass Spectrometery (LC/ MS 2020, Shimadzu Corporation, Japan)

8. 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. 10 B-BPA m/z 207 10 B-BSH m/z 723 1 2 Time (min.) ～ Effectiveness of DHAA for LC separation ～ Sample: Mixed standard solution Flow rate: 0.2ml/min. Injection: 3μl 35min. for gradient analysis LC analytical condition: Mobile phase: DHAA-Methanol Column: FC-ODS(150mmL×2mmI.D.,3μm ） Result (1) Inten.

9. Mass spectrum of BSH by ESI-SCAN(+) for the mass range 50-1000 m/z Mass spectrum of 10 B-BPA by ESI-SCAN(-) for the mass range 50-1000 m/z The positive ion [ 10 B 12 H 11 SH ・ 3C 12 H 28 N] + 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). Inten.(×100000) Inten.(×1000000) ～ Mass spectrum of BSH and BPA ～ Result (2) Inten.*10 6 Inten.*10 5 m/z BSH BPA

10. BSH target concentration (ppm) BPA target concentration (ppm) 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. Calibration carve for BSH Result (3) ～ Calibration curve for Standard solution ～ Calibration carve for BPA Peak area*10 6 Peak area*10 5 [n=6] BSH BPA [n=6]

11. Applicability for Human plasma Red blood cells(45%) Plasma(55% contains proteins and lipids) Buffy coat(1%) Human whole blood (COSMO BIO CO.LTD) Centrifuge (3000rpm×10min. ） Human Plasma was collected Spiked with mixed standard [BSH+BPA] Deproteinization and delipidation 【 Methanol/(Plasma+Standard) 】 =3:1 （ clear samples obtained ） Deproteinization and delipidation operation from human blood 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. LC/MS ●Human plasma samples preparation●

12. Target concentration for BSH (ppm) 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. Plasma calibration curve for BSH Target concentration for BPA (ppm) Plasma calibration curve for BPA Result (4) ～ Calibration standard for human plasma ～ Peak area*10 6 Peak area*10 5 BSH BPA

13. Result (5) ～ BSH & BPA determination in plasma ～ 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%. 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.

14. Conclusions 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. 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. 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.

15. This research is supported in part by Grant-in-Aid for Scientific Research ( 80647321 ) from the Japan Society for the Promotion Science(JSPS). Acknowledgement Thank you for your kind attention !