Approaches to highest sensitivity analysis of selected Dioxin / Furan congeners:

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Agatha McKenzie
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1 Approaches to highest sensitivity analysis of selected Dioxin / Furan congeners: a.) Cryogenic Peak Zone Focusing (cyro - PZF) b.) Fast GC Dirk Krumwiede, Jeff Focant, Heinz Mehlmann, Daniela Cavagnino, Don Patterson

2 Why do this? / Whatfore? - reach sub fg levels for: - Blood spot analysis (low level / very limited sample amounts) Combine highest sensitivity with highest sensitivity GC technique = HR with GCxGC (fast GC) 2

3 Overview: presentation main topics 1.) Maximum sensitivity in standard GC-HR analysis? 2.) Basic concept and setup of Cyro PZF 3.) Cryo PZF results 4.) Fast GC setup & results 5.) Conclusions & outlook 3

4 What is the maximum sensitivity in standard GC-HR? (instrumental sensitivity) How to evaluate this? Based on what quality criteria? (e.g. SigNoise or precision at low levels) 4

m/z 322): Standard: - Welington Standard DMD2378 (product code) = 1 fg/ul native 2,3,7,8 tcdd and 5 pg/ul 13C 2,3,7,8 TCDD, plus 4 5 more native tetra dioxin congeners

5 system performance evaluation standard first sketch stair standard GC chromatogramm, tetra dioxins mass trace (e.g. m/z 322): Standard: - Welington Standard DMD2378 (product code) = 1 fg/ul native 2,3,7,8 tcdd and 5 pg/ul 13C 2,3,7,8 TCDD, plus 4 5 more native tetra dioxin congeners at different lower concentrations - tetra-dios should be easily separatable, if possible within time range of 2-3 mins - concentrations should be stepwise rising from earlier to later eluting tcdd congeners (on DB5); 2,3,7,8tcdd should be the last with conc. of 1 fg/ul x,x,x,x tcdd - 2 fg/ul - x,x,x,x tcdd - 5 fg/ul - x,x,x,x tcdd - 1 fg/ul - x,x,x,x tcdd - 2 fg/ul - 2,3,7,8 tcdd - 1 fg/ul - 5

6 RT: Instrument Performance Standards: 1.) Sensitivity standard One standard with 6 different native tcdd isomers at different concentrations (2 1 fg/ul) Qualifier (ratio) mass trace 1379-TCDD 1368-TCDD 1378-TCDD 1478-TCDD 1234-TCDD 2378-TCDD 4.9 8E _tcdd E3 8 Quantification mass trace 1 fg 6 5 fg 812_tcdd fg 2 fg 5 fg 1 fg Solvent: nonane

7 Instrument Performance Standards: 2.) Reproducibility standard One standard with 6 different native tcdd isomers at same concentrations (5 fg/ul) TCDD 1379-TCDD 1378-TCDD 1478-TCDD 1234-TCDD 2378-TCDD Here: 5 times diluted Std. 2 (= 1 fg) RT: 1.79 RT: 11.9 RT: RT: Solvent: nonane 7

8 Instrument Performance Standards: from CAMPRO Scientific 8

9 POPs CoE webpage download poster 9

10 POPs analysis with DFS sensitivity RT: Challenge 2 fg RT: 1.73 good DFS at 1 ul (= well maintained) 5 fg RT: ul injec. even on DFS with bad perform.. 1 fg RT: fg RT: fg RT: RT: fg DFS measurement Time (m in) 5.69E3 m /z= M S ICIS 812_tcdd1 14 DFS (HR) range for quantitative analysis 1

11 Basic concept of peak focusing

12 GC-DFS: peak compression by Cryo focusing and Fast GC first experiments in Bremen 21 November 8th 19th Main goals: 1.) First experiments to develop a working method get it going! 2.) evaluate potential for increased sensitvity (lowest reachable level?) 3.) optimize method for best performance (acceptable repeatability at lowest level) First of all: work on standards (2 fg/ul, 2 fg/ul, 5 ag/ul) and focused on TCDD only Basic instrumental configuration / setup: Thermo instumentation: Trace GCxGC (Cryo modulator) with HR (DFS) columns: TR-Dioxin 5ms: 2m x.18 (.18 um film) 3m x.25 (.1 um film) Cryo PZF: Fast GC: TR-Dioxin 5ms: 3m x.25 (.1 um film) no 2d dimension column! TR-Dioxin 5ms: 5-6m x.18 (.18 um film), cut off from 2 m 12

13 13 GCxGC -HR (DFS): peak compression by Cryo focusing Modulator with Jet 2 spraying modulator on one single column

14 Cyro PZF GC-DFS: What is it basically about? RT: RT: AA: 1441 AH: E3 ICIS 111_tcdd Cryo ca. 4 x higher Here: 2 fg TCDD 2.5E ICIS 111_tcdd8_ RT: AA: 1212 AH: 655 Standard (3 m col.)

Cyro PZF GC-DFS: (one) analytical method approach time event control 2 fg/ul 1 ul injected (time event control: jet on min 9-1 (precool), Cryofocus min 11-11.8) RT: 11.91-12.9 1 8 6 4 RT: 12.

15 Cyro PZF GC-DFS: (one) analytical method approach time event control 2 fg/ul 1 ul injected (time event control: jet on min 9-1 (precool), Cryofocus min ) RT: RT: AA: E _tcdd ) Jet ON: conditioning of the modulator (precooling) (9min) 2.) Jet OFF: (1min) (release trapped chemical noise) 3.) Jet ON: traping of target analyte (11min) 4.) Jet OFF: release of target analyte (11.8min) Only the GCxGC peak focusing feature is used RT: AA: 155 RT: AA: RT: 12.4 AA: 1454 RT: AA: E2 111_tcdd E _tcdd1 9 15

16 Cyro PZF GC-DFS: What is it basically about? RT: RT: AA: 1441 AH: E3 ICIS 111_tcdd Cryo Peak width:.57 sec at 5 % height, (1 sec at 1 %) Here: 2 fg TCDD 2.5E ICIS 111_tcdd8_ Peak shape is defined by 2 dimension column length (here ca. 4 m) RT: AA: 1212 AH: 655 Standard (3 m col.) (6 m column TCDD peak at e.g. 25 min = ca. 5 - > 1 sec) Peak width: 2 sec (5 %), (3.5 sec at 1 %)

17 Cyro PZF GC-DFS: Higher peak = higher sensitivity? RT: RT: AA: 1441 AH: E3 ICIS 111_tcdd9 1 Hz Cryo Here: 2 fg TCDD 2.5E ICIS 111_tcdd8_ Hz RT: AA: 1212 AH: 655 Standard (3 m col.)

18 Optimization between GC and sensitivity sensitivity GC peak width dwell time? Slow down GC Speed up HR Find optimum settings Time [sec] 18

19 Trumpet Curve for Analytical Instrument Performance Validation RSD e.g. ion ratios (or RRFs, etc.) +15% Variation curve at different levels amount -15% LOQ How much variation is acceptable? - Which minimum RSD value is required at the level of LOQ (e.g. here +/- 15%)? 19

20 Cryo PZF and Fast GC limitations and compromises combination of fast GC method (narrowest peaks) with slow technique - GC separation efficiency reduced: - no separation of e.g. tetra congeners - for samples with 2378 ers only - only monitor selected target masses: - only selected congeners analysed - Nr. of Data points over GC peak limited - precision / ratio quality limited at lowest levels? - What is acceptable? Makes still sense? 2

21 Cryo PZF results

22 Cryo PZF GC-DFS TCDD at 2 and 2 fg 4 m 2d col length, 1 Res. (unsmoothed data here) RT: fg/ul 1 ul injected (5 pg/ul IS) RT: AA: RT: AA: 1196 RT: AA: 134 RT: AA: RT: AA: RT: AA: RT: AA: 4144 RT: AA: RT: E _tcdd E3 111_tcdd E _tcdd E _tcdd fg/ul 1 ul injected (ca..5 pg/ul IS) (here: modulation 2 sec) RT: AA: RT: AA: 133 RT: AA: 186 RT: AA: RT: 12.4 AA: RT: AA: E _tcdd E2 111_tcdd E _tcdd E _tcdd1 8 RT: fg/ul 1 ul injected (time event control: jet on min 9-1 (precool), Cryofocus min ) RT: AA: 135 RT: AA: 155 RT: AA: RT: 12.4 AA: 1454 RT: AA: E _tcdd E2 111_tcdd E _tcdd E _tcdd1 9 22

23 Cryo PZF GC-DFS TCDD < 2 fg 4 m 2d col length, 8 K all chromatograms below scaled to 2 fg peak height, small 5 g smoothing D:\Xcalibur\...\111_tcdd11 11/11/21 11:25:18 AM 1 ul 2fg tcdd, traping, Res 8, retuned TR5ms 3m x.25 mm (.1 um), K = 1.55, column length mod - DFS increased RT: SM: 5G fg (1 ul 2 fg/ul) RT: MA: E _tcdd 11 RT: SM: 5G zoom RT: MA: E _tcdd fg (2 ul 5 ag/ul) RT: AA: E _tcdd RT: AA: E _tcdd solvent blank RT: 8.94 AA: RT: 1.45 AA: RT: 1.84 AA: RT: 11.4 AA: RT: AA: 7.73 RT: AA: E2 111_tcdd RT: AA: E2 111_tcdd

24 Cryo PZF GC-DFS TCDD < 2 fg - ratios no smoothing here, fixed scaling to 2 fg peak height blank 1 ul Toluene 5 ag/ul 2 ul injected 2 fg/ul 1 ul injected RT: E _tcdd1 12 RT: RT: MA: E _tcdd1 13 RT: RT: MA: E _tcdd E2 111_tcdd E2 111_tcdd RT: AA: E2 111_tcdd RT: AA: RT: AA:

25 Cryo PZF GC-DFS: TCDD 2 ul 5 ag/ul repeated injections 4 m 2d col length, 8 K, smoothed 5 g Note: different trapping methods (timing) used leading to different RT times RT: SM: 5G RT: MA: RT: SM: 5G 1.41E _tcdd RT: MA: 193 RT: E _tcdd RT: AA: E _tcdd RT: MA: E2 111_tcdd RT: MA: E _tcdd RT: 12.5 AA: E2 111_tcdd

26 Cryo PZF GC-DFS: real sample 4 m 2d col length, 8 K 2 ul SAMPLE D (Jef) RT: Relative e Abundance ul D RT: 9.79 AA: 7.59 RT: AA: 491 RT: AA: 683 RT: AA: RT: AA: RT: E _tcdd E3 111_tcdd E _tcdd zoom RT: AA: 629 RT: AA: 491 RT: AA: 683 RT: AA: ul D RT: AA: 334 RT: AA: RT: AA: 167 RT: E _tcdd E3 111_tcdd E _tcdd RT: AA: 2.2 RT: AA: 26.1 RT: AA: 19 RT: AA: 34 RT: AA: ul 2 fg/ul tcdd (.5 pg/ul IS) RT: AA: RT: AA: So sample concentration should be roughly 2 fg/ul native tcdd (IS ca. 2.5 pg/ul) RT: AA: 27 RT: AA: 1384 RT: AA: RT: AA: 188 RT: AA: RT: AA: E _tcdd E2 111_tcdd E _tcdd1 1 26

27 Cryo PZF GC-DFS: real sample 4 m 2d col length, 1 K 2 ul SAMPLE D (Jef) RT: D RT: 8.32 AA: 7.38 RT: MA: RT: 11.4 RT: AA: 38 AA: 3148 RT: MA: RT: AA: RT: E _tcdd E2 111_tcdd E _tcdd D RT: MA: zoom RT: MA: RT: AA: 3148 RT: AA: 2265 RT: AA: 251 RT: AA: RT: AA: E _tcdd E2 111_tcdd E _tcdd

28 Cryo PZF: problems with too short second dimension column length 13 C TCDD mass trace RT: Note: RTs shifted to overlay peaks 4 m 2 d col length RT: AA: AH: RT: AA: 5191 AH: E ICIS 111_tcdd9 Cryo PW:.57 sec (5%) (1 sec at 1 %) RT: AA: 1429 AH: 35 PH increase = ca. x 4 RT: AA: AH: Std. 3 m col PW (5 %): 2 sec (3.5 at 1 %) All Same scaling RT: m 2 d col length RT: 12.3 AA: AH: RT: 12.3 AA: AH: results not reproducible Cryo PW:.29 sec (5%) (.571 RT: sec at 1 %) RT: AA: 2227 AH: AA: 2493 AH: E ICIS 111_tcdd56 8.E ICIS 111_tcdd8_ E ICIS 111_tcdd8_ PH increase = ca. x 6 Std. 3 m col PW (5 %): 2 sec (3.5 at 1 %) 28

29 Fast GC setup & results

30 Fast GC: peak focusing with short columns & high heating ramps nonane as solvent, (same MID settings as for Cryo focus), R 1 K RT: ve Abundance Relativ 1 ul splitless 2fg /ul TCDD (non.), 12(2) RT: 4.9 AA: RT: 4.17 AA: RT: 4.22 AA: 842 RT: 4.22 AA: 123 RT: 4.22 AA: RT: 4.26 AA: 14 IS RT: 4.34 AA: RT: 4.43 AA: 12 RT: E _5m18 7 _tcdd E3 111_5m18 _tcdd E _5m18 _tcdd E _5m18 _tcdd32 1 ul splitless 2fg /ul TCDD (non.) 12(2)-1-29 Observations: RT: 4.2 MA: IS 4.16 RT: 4.2 MA: 16 RT: 4.2 AA: RT: 4.23 AA: RT: 4.27 AA: E _5m18 _tcdd E2 111_5m18 _tcdd E _5m18 _tcdd323 Some problems with nonreproducible RT s Higher (column) noise was obsevered. Probably due to higher GC elution temps. (Getting worse over time) Better column quality might be mandatory for this. Additionally column should be even shorter (= narrower peak and lower elution temp.) 5-6m x.18 (.18 um film), cut off from 2 m

31 Fast GC versus Cryo PZF peak focusing compared on 2 fg TCDD, R 1 K RT: RT: MA: 1321 MH: RT: RT: Time 4.22 (min) MA: MH: E3 111_tcdd9 Cryo (4m 2 d col length) PW:.57 sec (5%) (1 sec at 1 %) E3 111_5m1 8_tcdd32 Fast GC PW:.63 sec (5%) (1.125 sec at 1 %) No experiments with Fast GC < 2 fg due to higher noise RT: SM: 5G RT: 4.31 MA: RT: 4.31 AA: 25 RT: 4.31 AA: RT: 4.35 AA: 68 2 fg TCDD RT: 4.85 AA: 164 RT: 5.26 AA: RT: 5.75 AA: E _5m18_tc dd E2 ICIS 111_5m18_tc dd E ICIS 111_5m18_tc dd44 31

32 Fast GC different cycle times 2 fg/ul injections different cycle times, 1 ul, nonane, R 1 K RT: SM: 5G RT: 4.31 MA: sec E _5m18 _tcdd44 RT: SM: 5G RT: 4.23 MA: sec E _5m18 _tcdd45 RT: SM: 5G RT: 4.17 AA: sec 2.3E _5m18 _tcdd RT: 4.31 AA: E2 111_5m18 _tcdd RT: 4.23 MA: E2 111_5m18 _tcdd RT: 4.17 AA: 168 RT: 4.24 AA: RT: 4.37 AA: E2 111_5m18 _tcdd RT: 4.31 AA: E _5m18 _tcdd RT: 4.23 AA: E _5m18 _tcdd RT: 4.17 AA: E _5m18 _tcdd RT: 4.35 AA: RT: 4.32 AA: RT: 4.23 AA:

33 Fast GC different cycle times / data points 2 fg/ul injections (also note: variation of RTs at heating ramp 1) RT: SM: 5G sec (ca. 7 Hz).12 sec (ca. 8 Hz).1 sec (1 Hz) RT: 4.31 AA: 25 RT: 4.31 AA: RT: 4.35 AA: E2 111_5m18 _tcdd E _5m18 _tcdd44 RT: SM: 5G RT: 4.23 AA: 152 RT: 4.23 AA: E2 111_5m18 _tcdd45 4.8E _5m18 _tcdd45 RT: SM: 5G 6 6 IS IS IS RT: 4.17 AA: 168 RT: 4.17 AA: RT: 4.21 AA: E2 111_5m18 _tcdd E _5m18 _tcdd46 33

34 Fast GC: separation of tetra congeners 2-1 fg performance standard in Fast GC RT: RT: 4.26 AA: 338 RT: 4.31 AA: E _5m18 _tcdd49 Relative Abund dance RT: 4.12 AA: 312 RT: 4.12 AA: 342 RT: 4.24 AA: 1437 RT: 4.23 AA: 1754 RT: 4.26 AA: 4134 RT: 4.31 AA: E4 111_5m18 _tcdd RT: 4.3 AA: E _5m18 _tcdd RT: 4.34 AA:

35 Fast GC complete Dioxin / Furan standard, tetra Hexa IS traces complete CSL 1/1 (diluted 1/1 CSL, 9 % tol., 1% non.) in Fast GC RT: RT: 4.18 AA: RT: 4.2 AA: 2835 RT: 4.22 AA: (2)-6-29 (octa at 5.81) RT: 4.33 AA: 543 RT: 4.5 AA: 1979 RT: 4.49 AA: RT: 4.39 AA: 1197 RT: 4.59 AA: 1518 RT: 4.61 AA: 78 RT: 4.68 AA: 1629 RT: 4.61 AA: RT: 4.89 AA: 2197 RT: 4.63 AA: 1519 RT: 4.88 AA: RT: 4.97 AA: 2121 RT: 5.6 AA: 221 RT: 4.97 AA: RT: 5.5 AA: 1682 RT: 5.7 AA: 1228 RT: 5.3 AA: E _5m18_pcddf E _5m18_pcddf E _5m18_pcddf E _5m18_pcddf E _5m18_pcddf E _5m18_pcddf4 52 RT: RT: 5.51 AA: RT: 5.53 AA: 71 RT: 5.58 AA: (2)-3-29 (octa at 7.51) RT: 5.6 AA: 754 RT: 5.95 AA: 1431 RT: 6.9 AA: 1923 RT: 6.13 AA: 592 RT: 5.95 AA: RT: 5.82 AA: 62 RT: 6.12 AA: 1594 RT: 6.12 AA: RT: 6.7 AA: 1121 RT: 6.15 AA: 472 RT: 6.47 AA: 4714 RT: 6.61 AA: 273 RT: 6.69 AA: 2174 RT: 6.61 AA: 758 RT: 6.48 AA: 8486 RT: 6.69 AA: 766 RT: 6.4 AA: 1213 RT: 6.6 AA: RT: 6.9 AA: 262 RT: 6.67 AA: E _5m18_pcddf E _5m18_pcddf E _5m18_pcddf E _5m18_pcddf E _5m18_pcddf E _5m18_pcddf4 6 35

36 Fast GC TIC of complete Dioxin / Furan standard complete CSL 1/1 (diluted 1/1 CSL, 9 % tol., 1% non.) in Fast GC 12 (2) (2)-3-29 RT: E5 TIC 111_5m1 8_pcddf47 RT: E5 TIC 111_5m1 8_pcddf471 36

37 Conlusions & outlook

38 Conclusions: - Peak compression by Cryo focusing or Fast GC allows to improve sensitivity - further method development needed - achievable lowest limits still need to be evaluated - Both approaches feature some specific advantages / disadvantages - no routine technique (at this point of time) - need for optimum performance on GC and side - high operator experience for proper operation & maintenance mandatory 38

39 Outlook: Cryo PZF: - determin optimum 2 d column length (4 m > x > 1 m) - determin minimum dwell time Fast GC: - Find.18 ID column with lowest possible noise (bleed) - shorter coloumn length & lower ramps for more reproducible RTs Both: - Include more target components - Run real (low level) samples 39

40 Thanks for your attention!!? 4

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