二重誘導体化-スターバー抽出-加熱脱着-GC/MS法を用いた河川水中エストラジオールの分析(独）産業技術総合研究所
計測標準研究部門
有機分析科 バイオメディカル標準研究室
川口 研
2014年5月30日GC研究懇談会：参考資料
薬業健保会館

Introduction
A novel method for the trace analysis of 17βestradiol (E2) in water sample was developed,
which involved stir bar sorptive extraction(SBSE) with in situ acylation followed bythermal desorption (TD) with quartz wool
assisted (QWA) in tube silylation and gas
chromatography-mass spectrometry (GC-MS).

Strategy

E2 has both an aromatic and an aliphatic hydroxyl group. In
this study, the aromatic hydroxyl group was derivatized by
the SBSE with in situ acylation. It was expected that therecovery of E2 by SBSE was increased. On the other hand,remaining the aliphatic hydroxyl group was derivatized by
TD with in tube silylation. It was expected that the volatilityof E2 was improved, and the high sensitivity GC-MS analysis
was achieved.
OHCH3

H

SBSE with in situacylation

OH
CH3

H

CH3COO

HOE2

Acyl derivative of E2

TD with in tubesilylation

OSi(CH3)3CH3
H

CH3COO

Dual derivative of E2

Chemical structures
OHCH3HOOHHH

CH3COOE2Acyl derivative of E2OSi(CH3)3CH3H(CH3)3SiO

CH3OSi(CH3)3CH3HCH3COO

Silyl derivative of E2

Dual derivative of E2

Hoshi University

Stir bar sorptive extraction
PDMS

Glass stir bar●Glass stir bar coated withpolydimethylsiloxane (PDMS)●Liquid-liquid extraction●Extraction, Concentration andPurification●Easy sample preparation
TDSTransfer line
CISSample
MSGC

Thermal desorption (TD)-GC-MS

SBSE with in situ acylation
Phenolic hydroxyl group (High polarity and low volatility)
Low sensitivity

Derivatization in sample solution (in situ)

Acyl derivative by acetic acid anhydride(Low polarity and high volatility)●Merits of SBSE・Low polarity→High recovery

・High volatility→

Improvement of TD efficiency

High sensitivity●Merits of GC/MS・Low polarity
・High volatility

Improvement of peak form

Extraction time profile
10 ng ml-1 standard solution (10 ml)
1M K2CO3 (1 ml)

Peak area (e+06)

Acetic acid anhydride (100 μl)

45403530252015
1050

TD-GC-MS

SBSE with in situ acylation

Optimum extraction time

Acyl derivative of E2120 min

060120180240300

Extraction time (min)Fig. Extraction time profiles of E2 by SBSE without derivatization
and acyl derivative of E2 obtained by SBSE with in situ acylation

TD with QWA in tube silylation
Glass capillary tube

PDMS stir bar

Quartz wool

FlowAbundance
45000
Heated part

Back portion

40000
35000

Dual derivative of E230000(0.5 ng ml-1)

25000
20000
15000
10000

QWA

5000
18.50

None QWA
19.00

19.50

20.00

20.50

21.00

21.50minIn tube derivatization reagent
Peak area (e+07)

252015

Optimal volume of BSTFA

101.0 μｌ

5000.511.5

2

Volume of BSTFA (μl)
Fig. Optimum volume of BSTFA for TD with QWA in tube silylation
A PDMS-coated stir bar and acylation reagents were added to 10 ml of standardsolution (10 ng ml-1) and stirring was performed. Then, a glass capillary tube filled withBSTFA as silylation reagent was inserted into the back portion of the glass TD tube withquartz wool. The TD tube was placed inside the TD system where the stir bar was
thermally desorbed and subjected to GC-MS thereafter. The optimum volume ofBSTFA was examined in the range of 0 to 2.0 µl.

Peak area (e+07)

TD temperature condition121086420The dual derivative of E2 did notreach equilibrium even after thetemperature reachedapproximately 280 oC.
180200220240

260280TD temperature (oC)Fig. Optimum TD temperature for analysis of E2 by dual derivatization method
A PDMS-coated stir bar and acylation reagents were added to 10 ml of standard
solution (10 ng ml-1) and stirring was performed. Then, a glass capillary tube filledwith BSTFA as silylation reagent was inserted into the back portion of the glass TDtube with quartz wool. The TD tube was placed inside the TD system where the stirbar was thermally desorbed and subjected to GC-MS thereafter. The TD hold time
was set at 5 min, and the optimum TD temperature was examined in the range of 180
to 280 oC.

Peak area (+e07)

TD hold time condition
20Optimum TD temperature

15280 oC

105Optimum TD hold time

05 min051015

TD hold time (min)Fig. Optimum TD hold time for analysis of E2 by dual derivatization method
A PDMS-coated stir bar and acylation reagents were added to 10 ml of standard
solution (10 ng ml-1) and stirring was performed. Then, a glass capillary tube filledwith BSTFA as silylation reagent was inserted into the back portion of the glass TDtube with quartz wool. The TD tube was placed inside the TD system where the stirbar was thermally desorbed and subjected to GC-MS thereafter. The TD temperature
was set at 280 oC, and the optimum TD hold time was examined in the range of 0 to15 min.

Method
Sample (10 ml)
Surrogate standard
1 M K2CO3 (1 ml)
Acetic acid anhydride (100 μl)

BSTFA: 1.0 μl

PDMS stir barTDS

Surrogate standard
Estradiol-13C4

1000 rpm, 120 minMSトランスファーラインCISGC

TD and GC-MS conditions
TDS conditions

CIS conditions

Flow Mode :Splitless

Initial Temp :－150 ℃

Initial Temp :20 ℃

Ramp Rate :12 ℃/sTransfer Temp:

300 ℃

Final Temp :300 ℃

Ramp Rate :60 ℃/minFinal Time :10 minFinal Temp :280 ℃

Final Time :

5 min

TDS

GC-MS conditions
Flow gas :Transfer line
CIS

Helium (99.9999 %)Flow rate : 1.2 mL/min
Injection : splitless
Column : DB-5ms (0.25 mm X 30 m, 0.5 μm)
Oven Temp : 60 ℃ - 15 ℃/min - 300 ℃ (4 min)Ionization : EI (70 eV)

MSGCValidation
Table Figures of merit of SBSE methods
CompoundDerivatization

ab

LOD-1

LOQ(pg ml-1)
500

Correlation coefficient(r )E2none(pg ml )100

Acyl derivative of E2

in situ derivatization

210

0.999 (10-10000)

Silyl derivative of E2

in tube derivatization

520

0.999 (20-10000)

Dual derivative of E2dual derivatization

0.5

2

0.999 (2-10000)

0.999 (500-10000)ca

LOD : limit of detection (S/N = 3)b

LOQ : limit of quantification (S/N > 10)

cValues in parentheses are the linear ranges of the calibration curves (pg ml-1).

The in situ acylation method exhibited approximately 50-fold higher sensitivity thanthe method without derivatization. Moreover, the QWA in tube silylation method
exhibited approximately 25-fold higher sensitivity than the method without
derivatization. Furthermore, the dual derivatization method exhibited approximately
5-fold higher sensitivity than the in situ acylation method, 10-fold higher sensitivity
than the in tube silylation method, and 250-fold higher sensitivity than the method
without derivatization.

Recovery test
Table Recovery test (n = 6)

CompoundAmount spiked
0.05 ng ml-1

E2



1.0 ng ml-1

Recovery (%)

RSD (%) Recovery (%)

RSD (%)

93.1

1.4

0.8

98.4

Therefore, the method is applicable to theprecise determination of trace amounts of E2
in river water samples.

Application

Abundance

SIM: m/z = 344
850

Dual derivative of E2(5.5 pg ml-1)

800
750
700
650
600
550
500
450
400
350
18.50
Abundance

19.00

19.50

20.00

SIM: m/z = 348

20.50

21.00

21.50

min

Dual derivative of E2-13C4(surrogate standard)

250000We measured the concentrations of E2
in two water samples collected from Tama River,Japan. Typical chromatograms of the river watersamples (Point A) are shown. E2 from Points Aand B was detected at concentrations of 5.5 and4.7 pg ml-1, respectively.

200000150000100000In the present study, the dual
derivatization method enabled the successful
determination of trace amounts of E2 in a small
volume of water sample (10 ml) with only one
PDMS stir bar.

50000

18.50

19.00

19.50

20.00

20.50

21.00

21.50

min

Fig. SIM chromatograms of dual derivatives of E2 and E2-13C4 inriver water sample (A).

Conclusions


Determination of E2 in river water samples by
SBSE with in situ acylation and TD with QWA in
tube silylation followed by GC/MSAcyl derivatives by using acetic acid anhydride Silyl derivative by using BSTFA→ sub pg/ml (ppt) This simple, accurate and highly sensitive method
is expected to have potential applications invarious water samples.
