1. 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 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  l by using large volume injection system equipped with an At-column kit 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 Large volume injection system (OPTIC2, ATAS GL, Japan) 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) Splitless Inj. Injection Port Temp. (260 ℃ ) 120 ℃ (1.5 ℃ min)-(15 ℃ /min)-200 ℃ (0min)-(3 ℃ /min)- 300 ℃ (0min)-(5 ℃ /min)-310 ℃ (5min) * For At-column concentration HRMS (JMS 700, JEOL, Japan) Ionizing current : 700μA Ionizing energy : 42 eV Accelerating voltage : 10kV Resolution : R>10 000 Measurement Mass : Selected Ion Monitor (SIM) using PFK 12 C 12 - and 13 C 12 - TCB, TCDD/F, PeCDDM+, (M+2)+ 12 C 12 - and 13 C 12 - Pe ～ HxCB, PeCDF, Hx ～ OCDDF (M+2)+, (M+4)+ 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 Table 3 Reproducibility of peak area using At-column concentration (100μL) or splitless (2μL) Congener MeanRSD(%)MeanRSD(%) PCDDs 2,3,7,8-TCDD283102849.1 1,2,3,7,8-PeCDD1486.41586.7 1,2,3,4,7,8-HxCDD1031510315 1,2,3,6,7,8-HxCDD1258.312411 1,2,3,7,8,9-HxCDD102101029.3 1,2,3,4,6,7,8-HpCDD86118712 OCDD1361013912 PCDFs 2,3,7,8-TCDF3961041411 1,2,3,7,8-PeCDF290102858.8 2,3,4,7,8-PeCDF2711127011 1,2,3,4,7,8-HxCDF182121829.0 1,2,3,6,7,8-HxCDF2239.421710 1,2,3,7,8,9-HxCDF1481115010 2,3,4,6,7,8-HxCDF1808.31817.6 1,2,3,4,6,7,8-HpCDF1548.81568.7 1,2,3,4,7,8,9-HpCDF1044.91016.5 OCDF1708.91729.3 Non-ortho-PCBs 3,3',4,4'-TCB (#77)69129.1725415 3,4,4',5-TCB (#81)594012594312 3,3',4,4',5-PeCB (#126)29029.0283711 3,3',4,4',5,5'-HxCB (#169)21246.422728.0 At-column (100μL) Splitless (2μL) These tests were repeated five times. Compounds MeanRSD(%)MeanRSD(%) PCDDs 2,3,7,8-TCDD 2.252.32 1,2,3,7,8-PeCDD 7.657.91 1,2,3,4,7,8-HxCDD 6.31 1 1,2,3,6,7,8-HxCDD 611623 1,2,3,7,8,9-HxCDD 7.537.81 1,2,3,4,6,7,8-HpCDD561 1 OCDD4704 4 PCDFs 2,3,7,8-TCDF 0.7515 1,2,3,7,8-PeCDF (0.23)21 2,3,4,7,8-PeCDF 7.43 3 1,2,3,4,7,8-HxCDF 8.228.42 1,2,3,6,7,8-HxCDF 6.256.33 1,2,3,7,8,9-HxCDF 2,3,4,6,7,8-HxCDF 1.371.46 1,2,3,4,6,7,8-HpCDF 212221 1,2,3,4,7,8,9-HpCDF (0.37)23 OCDF Non- ortho-PCBs 3,3',4,4'-TCB (#77) 1.441.583,4,4',5-TCB (#81) 2.92 2.83 3,3',4,4',5-PeCB (#126) 246231 3,3',4,4',5,5'-HxCB (#169)362352 <0.19<0.34 <0.55 <0.43 <1.1 <0.25 <0.29 pg/g lipid 5g (At-column)25g (Splitless) Table 4 Comparison between dioxin levels in 5 g serum using At- column concentration and 25 g serum using splitless mode LOD 0.16 0.19 0.22 0.65 0.13 0.16 0.19 0.20 0.23 0.55 0.16 0.25 0.27 0.30 0.36 0.40 0.51 1.1 0.25 0.29 0.34 0.43 1.1 0.25 0.49 0.54 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. 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 Fig. 1. Scheme of solvent evaporation and concentrating target compounds Valve Column Oven Target compounds Split purge Carrier gas Injector Concentrating point Hole for removing solvent Pre-column These tests were repeated three times. Parentheses are values beyond LOD under LOQ and values under LOQ are shown by <LOD