Development of rapid and precise boron isotope analysis

in whole blood by HR-ICP-MS

Yurie YamaguchiResearch and Development Office,

Japan Chemical Analysis Center

Required conditions for10B/11B isotope ratios

①Convenience, rapidity②High precision③High sensitivity

Objectives

Measurement methods

Satisfy the above

conditions

HR-ICP-MS

8.99060 11.01294

Q-ICP-MSSlight overlapping

of Ar4 ＋

on 10B

9.98797 10.01557

Ar4 ＋ 10B

HR-ICP-MSAr4 ＋ &10B

are well separated

・ To develop precise & accurate measurement method of concentration and isotope ratios of B by HR-ICP-MS.・ To apply this method to actual blood samples.・ To improve accuracy of analysis while shortening an analysis time.

JCAC

Optimization of Analytical Conditions

>2 ppb boron elution measured by HR-ICP-MS was obtained using borosilicate glass labware （ Fig. a ） .

0 5 10 15 20 25 300

2

4

6

88

4

2

0

6

0 　　　 10 20 30Time ［ hour ］

B (

ppb

)

(a)

Ultrapure water in a glass

volumetric flask

Int.

Inte

nci

ty［

cps

］

Time ［ min ］

0.1MHNO3

2.5minUltrapurewater

2.5min

0 1 2 3 4 5

5432

01

76

8

(b)After introduction

25ppb boron solution

*106

Prevention of pollution from labware

Elimination of boron memory effect

Plastic labwares were used to avoid potential contamination from borosilicate glass.

For an accurate determination of B isotope ratios,it is necessary to clean the sample introduction units in order to eliminate the memory effect.

4

Only Ultrapurewater 　Only 0.1M HNO3

： 14min： 11min

0.1M HNO3 ⇒Ultrapurewater(continuous rinsing) （ Fig. b ）

： 5min

＊ Rinsing method ＊Rinsing solution Rinsing time

speedup↑

10B/Y

11B/Y

11B/Sc

10B/Sc

Sc

11B/Be

10B/Be

Be

4

Selection of internal standard

Concentration(ppb)0 5 10 15 20 25 30 35 40

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

R² = 0.999655243229722Inte

nsity

ratio

Y

Inte

nsity

ratio

0 5 10 15 20 25 30 35 400.0

0.1

0.2

0.3

0.4

R² = 0.999903196376138

R² = 0.999338186753168

Concentration(ppb)

0 10 20 30 40 500.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

R² = 0.999486041399768

R² = 0.999607519063121Inte

nsity

ratio

Concentration(ppb)

High correlation coefficient and accuracy were obtained using Y standard solution as internal standard.

Y was chosen as the internal standard for the B determination.

5

R 2= 0.9990

Optimization of Measurement ConditionsResolution Sensitivity Accuracy Spectral Interference

【 Low 】 ◎ ◎ △

【 Medium 】

○ ○ ○

★Sensitivity★Accuracy

At Low-resolution measurement, providing flat-topped peaks and maximum instrument accuracy.

【 L 】 【 M 】

★Spectral Interference【 L 】 【 M 】

10B 10B40Ar4+ 40Ar4+

Signal intensity decreases as the resolution increases due to narrow slit.

●The precision was improved when the mass scanning window was reduced from 20%.

●In this study, isotope measurements for boron determination were carried out at low resolution(R=300)for accurate measurement.

【 Comparison between HR-ICP-MS measuring at Low and Medium resolution 】

10B is hindered by 40Ar4+ interference at m/z = 10 at low resolution.

6

●In measuring isotope ratios by ICP-MS, measured values will deviate from true values due to mass bias effect.●Therefore, mass bias correction is important for highly accurate isotope ratios determination. ●A correction factor is calculated as the mean of the measured isotope ratios of the standard of known isotopic composition, compared to the measured isotope ratios.

◆Mass bias correction

Katahira (2005), 　Nagao (2011)

Measurement of 10B/11B

Mass bias factor ＝

◆Validation of mass bias factor1. Calculation of mass bias factor using the standard of known isotopic

composition.2. Measurement of B isotope ratios of the standard of known concentration. 　　　　　　　　　　　→ Correction using mass bias factor calculated in 1 　　　　　　　　　　　　　

　　　　　　　　　　　　 → Comparison between correction value and natural abundance of B isotope ratios

②Measurement of B isotope ratios

10B/11B 　　　　before correction

10B/11B 　　　　 after

correction

RSD(%)

Relative error(%)

0.229 0.247 0.867 － 0.75×1.077Mass bias factor

Boron Isotope Ratio Analysis on the ELEMENT2 （ measured by Thermo Fisher Scientific ）　　　　 　　★ Relative error ： 1.31%, ★RSD value ： 0.062%（ n=5)

n= Number of analyses performed on each sample(average values shown).

NIST-SRM 1643e, standard multi-element solution was used to validate of the isotopic ratio measurements

B isotope ratios of NIST-SRM 1643e (10B/11B=0.2484*) was measured using mass bias correction(n=3).

*IUPAC Inorganic Chemistry Division, CIAAW(2011)

【 Reference values 】

●Determined B isotope ratios were in good agreement with the certified value. (The difference between certified values and measurement values were better than ±1%.)●The RSD values obtained for B isotope ratios was better than 1%.

Measurement of B concentration and 10B/11B isotope ratios of BSH and BPA

Concentration10

B /

tota

l B(%

)D

iffer

ence

from

ev

alua

ted

valu

e(%

) 0 5 10 15 20 25 30

-30

-25

-20

-15

-10

-5

0 B (ppb)

BSH

BPA BSH

BPA

B (ppb)

B (ppb) B (ppb)

>0.2ppb

>98.8% >98.9%

● ：✔● ： ×

>2%

>2.4ppb >2.9ppb

0 5 10 15 20 25 30-5

0

5

10

15

20

25

30

Isotope ratios

0 5 10 15 20 25 3075

80

85

90

95

100

0 5 10 15 20 25 3075

80

85

90

95

100

Determination of B in simulated plasmaBefore using whole blood, simulated plasma was used to confirm the effect of matrix elements concentration on the determination of B.

B : 5ppb

Matrix elements

Simulated plasma

Matrix elements were added in stages while keeping constant the concentration of boron as 5ppb.

〈 Changes of concentration of B and matrix elements in plasma 〉 〈 Methods 〉

Na-conc/ B-conc 0 25 50 100 200 300

B-conc (ppb) 5 5 5 5 5 5

Na-conc (ppb) 0 125 250 500 1000 1500

Cl-conc (ppb) 0 193 385 771 1542 2313

～ e.g. Na solution ～

～～

～～

～～

～～Na

KCa

MgP

×109 ×272

Cl

Time after onset of infusion(min)LAAKSO, 2001

3600

3200

150100

40353025201510

50

Con

cent

ratio

n(pp

m)

0 35050 100 150 200 250 300

Infusion BNCT BNCT

■ Plasma ▲ Whole blood

● Erythrocytes

B concentration (n=3)

0 50 100150200250300350400450500

-10.00

-5.00

0.00

5.00

10.00 ◆ ：Na ◆ ：Cl

10

5

0

-5

-10

0 100 200 300 400 500

-10

-5

0

5

1010

5

0

-5

-10

◆ ：K ◆ ：Cl

0 100 200 300 400 500

-10

-5

0

5

1010

5

0

-5

-10

Na/BCl/B

0 100 200 300 400 500

-10

-5

0

5

1010

5

0

-5

-10

Diff

eren

ce fr

om n

atur

al a

bund

ance

(%)

Diff

eren

ce fr

om e

valu

ated

val

ue(%

)

◆ ：Na ◆ ：Cl

◆ ：K ◆ ：Cl

Na/BCl/B

K /BCl/B

K /BCl/B

(n=3)B isotope ratios

Accurate and precise values of B concentration and B isotope ratios could be obtained despite the presence ofhigh concentration of matrix elements.

Mg (Mg/B=25)

Difference from evaluated value(%) 0.04

RSD(%) 0.16

P (P/B=25)

Difference from evaluated value(%) 0.49

RSD(%) 1.10

Mg (Mg/B=25)

Difference from natural abundance(%) 0.20

RSD(%) 1.32

P (P/B=25)

Difference from natural abundance(%) -0.12

RSD(%) 0.67

B concentration

Mg Mg

P P

0 100 200 300 400 500

-10

-5

0

5

10

0 100 200 300 400 500

-10

-5

0

5

10

Diff

eren

ce fr

om n

atur

al a

bund

ance

(%)

Diff

eren

ce fr

om e

valu

ated

val

ue(%

)

◆ ：Ca

◆ ：Ca

(n=3) (n=3)B isotope ratios

Determination of B in whole bloodTo find optimum digestion method of whole blood sample,B recovery test using hotplate digestion and microwave digestion were performed by the following method.

Hotplate digestion

Teflon Quartz

HR-ICP-MSmeasurement

Whole Bloodconc. HNO3

Microwave digestion(Ethos TC, Milestone)

Microwave digestion

TeflonQuartz

B/BPA/BSH

Hotplate digestion

Teflon

10B

/ to

tal B

(%)

Recovery ■ ■>98% with low RSD expect for BSH solutions. 10B / totalB isotope ratios ■ ■As for B, good agreement with the natural abundance. As for BPA/BSH, favorable B isotope ratios were obtained. Comparison between Teflon and Quartz inserts ■Thermal conductivity ： Teflon << Quartz Handling ： Teflon > QuartzEase of rinsing : Teflon << Quartz

Microw

ave digestion

B BPA BSH B BPA BSHTeflon Quartz

0

20

40

60

80

100

RecoveryRe

cove

ry(%

)

B BPA BSH B BPA BSHTeflon Quartz

0

20

40

60

80

100

Isotope ratio

■ ： Hotplate 　　 (Digestion procedure×3, Measurement×3)■ ： Microwave (Digestion procedure×3, Measurement×3)Results ～ closed system ～

There is a possibility of boron analyte loss in the case of open system digestion due to the chemical property of boron volatility.Thus, it is necessary to explore relationship between 【 Recovery － Sample amount 】 and 【 Recovery ―Digestion time 】

B BPA BSH B BPA BSHTeflon Quartz

0

20

40

60

80

100

B BPA BSH B BPA BSHTeflon Quartz

0

20

40

60

80

100

Recovery

Isotope ratio10

B /

tota

l B(%

)

Reco

very

(%)

■ ■ ： 0.05g■ ： 0.1g

■ ■ ： 0.05g■ ： 0.1g

Sample amount 0.05g 0.05g 0.1g

Heating time

Teflon 28min. 28min.+20min.(After completion of digestion) 45min.

Quartz 22min. 22min.+20min.(After completion of digestion) 30min.

Results ～ open system ～

B BPA BSH B BPA BSHTeflon Quartz

0

20

40

60

80

100

B BPA BSH B BPA BSHTeflon Quartz

0

20

40

60

80

100

Recovery

Isotope ratio10

B /

tota

l B(%

)

Reco

very

(%)

■ ■ ： 0.05g■ ： 0.1g

■ ■ ： 0.05g■ ： 0.1g

Comparison between Teflon and Quartz insertsThermal conductivity : Teflon << Quartz Handling : Teflon < QuartzEase of rinsing : Teflon << Quartz

Depends on sample amount and

heating time.

Sample amount/ Heating time

― 　 independentRequires further investigation

to be verified.

Results ～ open system ～

0 10 20 30 40 50 60 700

25

50

75

100

0 10 20 30 40 50 60 700

25

50

75

100

◆ 　： 0.05g◇ 　 ： 0.05g◆ ： 0.1g◆ ： 0.5g

◆ 　： 0.05g◇ 　 ： 0.05g◆ ： 0.1g◆ ： 0.5g

B(Quartz)

BPA(Quartz)

【 Relationship between recovery and heating time 】… Focus on the case of using quartz as vessel.

Reco

very

(%)

Reco

very

(%)

Heating time(min.) Heating time(min.)

After completion of digestion

0 10 20 30 40 50 60 700

25

50

75

100【 The degree of boron Volatilization rate 】

B > BPA >>> BSH◆ 　： 0.05g◇ 　 ： 0.05g◆ ： 0.1g◆ ： 0.5g

BSH(Quartz)

Reco

very

(%)

Heating time(min.)

BSH has a greater binding energy than that of B4H16N2O11 (Rational formula of B standard solution) and BPA.Thus, B-B binding is hard to be dissociated,and therefore, volatilization of boron is hardly occurred.

0 10 20 30 40 50 60 700

25

50

75

100

0 10 20 30 40 50 60 700

25

50

75

100

Sample amount Boron loss

◆ 0.05g -2.8%◆ 0.1g -9.1%

◆ 0.5g -34.2%

Sample amount Heating time Boron loss

◇ 0.05g 40min. -6.8%

◆ 0.1g 30min. -9.1%

◆ 　： 0.05g◇ 　 ： 0.05g◆ ： 0.1g◆ ： 0.5g

◆ 　： 0.05g◇ 　 ： 0.05g◆ ： 0.1g◆ ： 0.5g

B(Quartz)

BPA(Quartz)

Boron analyte loss occurs with the process of blood digestion.

Volatilization rate of boron is higher during digestion than after digestion.

It is therefore necessary to pay attention to the volatilization of boron using hotplate digestion in open system

【 Relationship between recovery and heating time 】… Focus on the case of using quartz as vessel.

Reco

very

(%)

Reco

very

(%)

Heating time(min.) Heating time(min.)

After completion of digestion

Digestion methods Hotplate digestion Microwave digestionSystem open closed

Preparation time(Teflon/Quartz)

~1min./~1min.~3min./ － ~6min./~6min.

Digestion time(0.05g, Teflon)

~28min. ~15min. ~25min.

Cooling time(Teflon/ Quartz)

7min./3min.10min./ － 20min./20min.

Portability ◎ △

Handling △ ◎

Safety △ ○ ○

Contamination × ◎

Vaporization △ ◎

Homogeneity of digestion × ○

Reliability ofdigestion △ ○

【 Methods comparison 】

Digestion methods Hotplate digestion Microwave digestionSystem open closed

Preparation time(Teflon/Quartz)

~1min./~1min.~3min./ － ~6min./~6min.

Digestion time(0.05g, Teflon)

~28min. ~15min. ~25min.

Cooling time(Teflon/ Quartz)

7min./3min.10min./ － 20min./20min.

Portability ◎ △

Handling △ ◎

Safety △ ○ ○

Contamination × ◎

Vaporization △ ◎

Homogeneity of digestion × ○

Reliability ofdigestion △ ○

【 Methods comparison 】

【 Hotplate-open 】✔Complicated operation 　 is not necessary.×Boron analyte loss may occur when it takes a long time 　　 to complete the digestion.

Digestion methods Hotplate digestion Microwave digestionSystem open closed

Preparation time(Teflon/Quartz)

~1min./~1min.~3min./ － ~6min./~6min.

Digestion time(0.05g, Teflon)

~28min. ~15min. ~25min.

Cooling time(Teflon/ Quartz)

7min./3min.10min./ － 20min./20min.

Portability ◎ △

Handling △ ◎

Safety △ ○ ○

Contamination × ◎

Vaporization △ ◎

Homogeneity of digestion × ○

Reliability ofdigestion △ ○

【 Methods comparison 】【 Hotplate-closed 】✔Enable to carry out digestion within the shortest time.×There is a possibility that the decomposition efficiency depends on a place for installation.

Digestion methods Hotplate digestion Microwave digestionSystem open closed

Preparation time(Teflon/Quartz)

~1min./~1min.~3min./ － ~6min./~6min.

Digestion time(0.05g, Teflon)

~28min. ~15min. ~25min.

Cooling time(Teflon/ Quartz)

7min./3min.10min./ － 20min./20min.

Portability ◎ △

Handling △ ◎

Safety △ ○ ○

Contamination × ◎

Vaporization △ ◎

Homogeneity of digestion × ○

Reliability ofdigestion △ ○

【 Methods comparison 】

【 Microwave-closed 】✔With microwave digestion, the samples are enclosed, so cross contamination and loss of volatiles are eliminated.×Rapidity is required for the clinical BNCT, thus it is necessary to further shorten the cooling time.

Conclusions●This work has clearly shown that precise and 　 accurate B ratios can be measured by HR-ICP-MS.

●Analytical method has been successfully applied to ①the determination of B in BPA expect for BSH. ②the determination of B in blood sample.

● Digestion method in closed system was applied to the determination of B in whole blood samples.

● It is more necessary to develop more rapid and efficient method suitable for clinical applications by the following methods. 【 Hotplate-close 】 To improve the efficiency of digestion. 【 Hotplate-open 】 To expand a range of sample amount. 【 Microwave 】 To further shorten the cooling time.

Acknowledgement

This work was supported by JSPS KAKENHI Grant Number 26870866.

Thank you for

your kind attention.