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Equilibration time : 30sec

Large volume injection system (OPTIC2, ATAS GL, Japan). Table 1 The condition of injection of 100 m l by using large volume injection system equipped with an At-column kit. Equilibration time : 30sec. Purge Pressure : 25kPa. Final Pressure : 310kPa. Initial Temperature : 105℃.

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Equilibration time : 30sec

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  1. Large volume injection system (OPTIC2, ATAS GL, Japan) Table 1 The condition of injection of 100 ml by using large volume injection system equipped with an At-column kit Equilibration time : 30sec Purge Pressure : 25kPa Final Pressure : 310kPa Initial Temperature : 105℃ Transfer Pressure : 100kPa End Time : 49min Ramp Rate : 16℃/sec Split Open Time : 2.5min Split Flow : 20ml/min Final Temperature : 280℃ Transfer Time : 3min Vent Flow : 100ml/min Vent time : Auto (level 10) Initial Pressure : 100kPa Purge Flow : 3ml/min Carrier gas Hole for removing solvent Split purge Injector Valve Target compounds Concentrating point Column Oven Pre-column Fig. 1. Scheme of solvent evaporation and concentrating target compounds 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 2GL 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 3 Reproducibility of peak area using At-column concentration (100μL) or splitless (2μL) Splitless (2μL) At-column (100μL) Congener Mean RSD(%) Mean RSD(%) PCDDs 2,3,7,8-TCDD 283 10 284 9.1 1,2,3,7,8-PeCDD 148 6.4 158 6.7 1,2,3,4,7,8-HxCDD 103 15 103 15 1,2,3,6,7,8-HxCDD 125 8.3 124 11 1,2,3,7,8,9-HxCDD 102 10 102 9.3 1,2,3,4,6,7,8-HpCDD 86 11 87 12 OCDD 136 10 139 12 PCDFs 2,3,7,8-TCDF 396 10 414 11 1,2,3,7,8-PeCDF 290 10 285 8.8 2,3,4,7,8-PeCDF 271 11 270 11 1,2,3,4,7,8-HxCDF 182 12 182 9.0 1,2,3,6,7,8-HxCDF 223 9.4 217 10 1,2,3,7,8,9-HxCDF 148 11 150 10 2,3,4,6,7,8-HxCDF 180 8.3 181 7.6 1,2,3,4,6,7,8-HpCDF 154 8.8 156 8.7 1,2,3,4,7,8,9-HpCDF 104 4.9 101 6.5 OCDF 170 8.9 172 9.3 Non-ortho-PCBs 3,3',4,4'-TCB (#77) 6912 9.1 7254 15 Table 2The condition of HRGC-HRMS 3,4,4',5-TCB (#81) 5940 12 5943 12 3,3',4,4',5-PeCB (#126) 2902 9.0 2837 11 HRGC (6890 series GC system, Agilent, USA) 3,3',4,4',5,5'-HxCB (#169) 2124 6.4 2272 8.0 GC Pre-column * Deactivated Capillary Tubing (GL Sciences), 30cm×0.53mm ID These tests were repeated five times. GC Capillary column 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 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) 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 Accelerating voltage : 10kV 5g (At-column) 25g (Splitless) Ionizing energy : 42 eV Resolution : R>10 000 Compounds Mean RSD(%) Mean RSD(%) LOD LOD Measurement Mass : Selected Ion Monitor (SIM) using PFK 12C12- and 13C12- TCB, TCDD/F, PeCDD M+, (M+2)+ PCDDs 2.2 5 0.16 2.3 2 0.30 12C12- and 13C12- Pe～HxCB, PeCDF, Hx～OCDDF (M+2)+, (M+4)+ 2,3,7,8-TCDD 7.6 5 0.19 7.9 1 0.36 1,2,3,7,8-PeCDD * For At-column concentration 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 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.

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