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Dioxin analysis in human serum of small sized sample by using concentration at
the inlet of the GC capillary column for 100 μL large volume injection method
Kimiyoshi Kitamura 1, Yoshiyuki Takei 2, Jae-Won Choi1, Shunji Hashimoto1, Hiroyasu Ito1 and Masatoshi Morita 1
1 National Institute for Environmental Studies, Onogawa 16-2, Tsukuba, Ibaraki 305-8506, Japan
2 GL Sciences, 237-2 Sayamagahara, Iruma, Saitama, 358-0032, Japan
Objective ： We aimed to apply concentration at the inlet of the GC capillary column, named “At-column concentration”, for 100 μL large volume injection for
dioxins analysis in small sized human serum sample.
Sample
： Reference material for interlaboratory calibration
studies made by the National Institute for
Environmental Studies (NIES, Japan), ( 5g VS 25g )
Extraction ： Liquid-liquid extraction （saturated ammonium sulfate
solution and 25%(v/v)-ethanol-hexane）
Cleanup ： Multi-layer silica- gel column
Active carbon-dispersed silica gel column
Apparatus ： 5g : HRGC-HRMS equipped with a Large volume
injection system (At-column kit)
25g : HRGC-HRMS ( splitless mode )
Table 1 The condition of injection of 100 ml by using large volume injection system
equipped with an At-column kit
Large volume injection system (OPTIC2, ATAS GL, Japan)
Equilibration time : 30sec
Initial Temperature : 105℃
Ramp Rate
: 16℃/sec
Final Temperature : 280℃
Vent time
: Auto (level 10)
Purge Pressure
: 25kPa
Transfer Pressure : 100kPa
Split Open Time : 2.5min
Transfer Time
: 3min
Initial Pressure : 100kPa
Final Pressure : 310kPa
End Time
: 49min
Split Flow
: 20ml/min
Vent Flow
: 100ml/min
Purge Flow
: 3ml/min
Table 2 The condition of HRGC-HRMS
HRGC (6890 series GC system, Agilent, USA)
GC Pre-column * Deactivated Capillary Tubing (GL Sciences), 30cm×0.53mm ID
GC Capillary column
CP-Sil 8 CB low bleed/MS (VARIAN), 60m×0.25mm ID, 0.25μm film thickness
Ramp of Oven Temp.
Large Volume Inj.
140℃(5℃min)-(15℃/min)-200℃(0min)-(3℃/min)-300℃(0min)-(5℃/min)310℃(5min)
Table 3 Reproducibility of peak area using At-column concentration (100μL) or
splitless (2μL)
Congener
PCDDs
2,3,7,8-TCDD
1,2,3,7,8-PeCDD
1,2,3,4,7,8-HxCDD
1,2,3,6,7,8-HxCDD
1,2,3,7,8,9-HxCDD
1,2,3,4,6,7,8-HpCDD
OCDD
PCDFs
2,3,7,8-TCDF
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCDF
1,2,3,4,7,8-HxCDF
1,2,3,6,7,8-HxCDF
1,2,3,7,8,9-HxCDF
2,3,4,6,7,8-HxCDF
1,2,3,4,6,7,8-HpCDF
1,2,3,4,7,8,9-HpCDF
OCDF
Splitless (2μL)
Mean
RSD(%)
At-column (100μL)
Mean
RSD(%)
283
148
103
125
102
86
136
10
6.4
15
8.3
10
11
10
284
158
103
124
102
87
139
9.1
6.7
15
11
9.3
12
12
396
290
271
182
223
148
180
154
104
170
10
10
11
12
9.4
11
8.3
8.8
4.9
8.9
414
285
270
182
217
150
181
156
101
172
11
8.8
11
9.0
10
10
7.6
8.7
6.5
9.3
7254
5943
2837
2272
15
12
11
8.0
Non-ortho-PCBs
3,3',4,4'-TCB (#77)
6912
9.1
3,4,4',5-TCB (#81)
5940
12
3,3',4,4',5-PeCB (#126)
2902
9.0
3,3',4,4',5,5'-HxCB (#169)
2124
6.4
These tests were repeated five times.
At-column: T-HpCDDs/Fs, 25 fg/100μL in toluene
OCDD/F, 50fg/100μL in toluene
Non-ortho-PCBs, 250fg/100μL in toluene
Splitless: T-HpCDDs/Fs, 25 fg/2μL in n-nonane
OCDD/F, 50fg/2μL in n-nonane
Non-ortho-PCBs, 250fg/2μL in n-nonane
Splitless Inj.
Injection Port Temp. (260℃)
120℃(1.5℃min)-(15℃/min)-200℃(0min)-(3℃/min)-300℃(0min)-(5℃/min)310℃(5min)
Table 4 Comparison between dioxin levels in 5 g serum using At-column
concentration and 25 g serum using splitless mode
HRMS (JMS 700, JEOL, Japan)
pg/g lipid
Ionizing current : 700μA
Ionizing energy : 42 eV
Measurement Mass : Selected Ion Monitor (SIM) using
12C - and 13C - TCB, TCDD/F, PeCDD
12
12
12C - and 13C - Pe～HxCB, PeCDF, Hx～OCDDF
12
12
Accelerating voltage : 10kV
Resolution : R>10 000
PFK
M+, (M+2)+
(M+2)+, (M+4)+
* For At-column concentration
Carrier gas
Hole for removing
solvent
Split purge
Injector
Valve
Target compounds
Column Oven
Concentrating point
Pre-column
Compounds
5g (At-column)
25g (Splitless)
Mean RSD(%) LOD
Mean RSD(%) LOD
PCDDs
2.2
5
0.16
2.3
2
0.30
2,3,7,8-TCDD
7.6
5
0.19
7.9
1
0.36
1,2,3,7,8-PeCDD
6.3
1
0.22
6.3
1
0.40
1,2,3,4,7,8-HxCDD
61
1
0.22
62
3
0.40
1,2,3,6,7,8-HxCDD
7.5
3
0.22
7.8
1
0.40
1,2,3,7,8,9-HxCDD
1,2,3,4,6,7,8-HpCDD
56
1
0.22
56
1
0.51
OCDD
470
4
0.65
470
4
1.1
PCDFs
2,3,7,8-TCDF
0.75 15
0.13
<0.25
0.25
1,2,3,7,8-PeCDF
(0.23) 21
0.16
<0.29
0.29
2,3,4,7,8-PeCDF
7.4
3
0.19
7.4
3
0.29
1,2,3,4,7,8-HxCDF
8.2
2
0.19
8.4
2
0.34
1,2,3,6,7,8-HxCDF
6.2
5
0.19
6.3
3
0.34
1,2,3,7,8,9-HxCDF
<0.19
0.19
<0.34
0.34
1.3
7
0.20
1.4
6
0.34
2,3,4,6,7,8-HxCDF
21
2
0.20
22
1
0.43
1,2,3,4,6,7,8-HpCDF
1,2,3,4,7,8,9-HpCDF
(0.37) 23
0.23
<0.43
0.43
<0.55
0.55
<1.1
1.1
OCDF
Non- ortho-PCBs
3,4,4',5-TCB (#81)
1.4
4
0.16
1.5
8
0.25
3,3',4,4'-TCB (#77)
2.9
2
0.16
2.8
3
0.25
3,3',4,4',5-PeCB (#126)
24
6
0.25
23
1
0.49
3,3',4,4',5,5'-HxCB (#169)
36
2
35
2
0.54
0.27
These tests were repeated three times. Parentheses are values beyond LOD
under LOQ and values under LOQ are shown by <LOD
Fig. 1. Scheme of solvent evaporation and concentrating target
compounds
Results and discussion
・ No significant difference was found in all congener levels measured in both the 5 g and 25 g samples.
・ Dioxin congeners could be quantitated with At-column concentration within the highest RSD of 15%.
・ The LOD was lower for the 5 g sample using At-column concentration than the 25 g using splitless.
→A volume of 100 μL of the final sample solution of 110 μL was introduced to HRGC-HRMS using At-column concentration. This is considerable in 4.5 g
when it is calculated as an amount of serum. On the other hand, 2 μL of the final sample solution of 20 μL was introduced to HRGC-HRMS using splitless,
this is considerable in 2 g when it is calculated as an amount of serum.
→As a result, the injection of a greater amount of sample solution onto the capillary column using At-column concentration achieved an LOD that was lower
than 5 g of serum. Moreover, the noise level with an injection of 100 μL using At-column concentration was almost the same as that with a 2 μL
injection using splitless.
We examined this application looking at quantitation levels of less than ppb for each congener in a serum sample of 5 g using At-column-HRGC-HRMS, this
succeeded as the most practical application.