1. Three years of experience with backflush and retention time locking on Agilent 7000B GC-MS/MS Hans Ragnar Norli Bioforsk, Plant Health and Plant Protection Pesticide Team

2. The History of GC-MS Technology at Bioforsk -Agilent 5973 MS, full scan, Targeted -Agilent 5890 GC with 30 m HP5 MSI kolonne -Pre column 2.5 m. -Gerstel PTV injector air cooled, 20 uL inj. volume -Liner: sintred -RT-lock. -Dataanalysis: ChemStation with -DRS system and AMDIS database; 196 compounds - Injection cycle: 55 min (injection to injection) -Agilent 5973 MS, full scan, Targeted -Agilent 5890 GC with 30 m HP5 MSI kolonne -Pre column 2.5 m. -Gerstel PTV injector air cooled, 20 uL inj. volume -Liner: sintred -RT-lock. -Dataanalysis: ChemStation with -DRS system and AMDIS database; 196 compounds - Injection cycle: 55 min (injection to injection) -Agilent 7000 B GC/MS Triple Quad -Agilent 7890 GC with 2X 15m HP5 MSUI -Pre column 2.5 m. -MMI inlet, air cooled, 5 uL inj. volume -Liner: 2mm DIMPLED -RT-lock -Back-flush in post run (column midpoint) -Data analysis: Masshunter; 122 compounds -Injection cycle: 30 min -Agilent 7000 B GC/MS Triple Quad -Agilent 7890 GC with 2X 15m HP5 MSUI -Pre column 2.5 m. -MMI inlet, air cooled, 5 uL inj. volume -Liner: 2mm DIMPLED -RT-lock -Back-flush in post run (column midpoint) -Data analysis: Masshunter; 122 compounds -Injection cycle: 30 min 2012 2005 2010: Introduction of backflush on GC-MS

3. FLOW New Column Column after several injections Column after backflush FLOW The Idea behind Backflush Technology

4. Quechers of Leek 10.0015.0020.0025.0030.0035.0040.00 1.25e+07 Time--> Abundance TIC: 00501007.D\data.ms 10.0015.0020.0025.0030.0035.0040.00 280000 TIC: 00401008.D\data.ms 10.0015.0020.0025.0030.0035.00 8500000 TIC: 00501001.D\data.ms 10.0015.0020.0025.0030.0035.00 280000 TIC: 00401002.D\data.ms Leek, no back flush ACN, injected after Leek, with back flush ACN, injected after

5. Control chart examples of Ortho-phenylphenol and Boscalid at spiking level 0.05 mg/kg LettuceAppleOrange Back Flush introduced

6. Decreasing the Measurement Uncertainty Measurement Uncertainty (2*CV%) Pesticide (0.05 mg/kg) Three months before BF installation, N=49 Three months after BF installation, N=49 Change (Before-After) Orto-phenylphenol7238-34 Diphenylamine5628-28 Pirimiphos-methyl6431-33 Chlorpyriphos5722-35 Cyprodinil4816-32 Prothiophos4520-25 Boscalid5823-35 Cypermethrin7965-14 MatricesBean(2) Apple (8) Lettuce (27) Wheat (8) Rice (4) Bean(3) Apple (13) Lettuce (13) Wheat (5) Rice (3) Orange (13)

7. - Number of analytes: 119 -Number of time segments: 19 -Largest segment consist of 30 transitions (15 analytes) 3 cycles/s -RT-Lock with ditalimfos at 14.4 min -PCM:10 psi (50 psi in post run) -Column head pressure 16 psi -Backflush time 2.7 min (column hold up time x 5) -Injection cycle: 30 min - Number of analytes: 119 -Number of time segments: 19 -Largest segment consist of 30 transitions (15 analytes) 3 cycles/s -RT-Lock with ditalimfos at 14.4 min -PCM:10 psi (50 psi in post run) -Column head pressure 16 psi -Backflush time 2.7 min (column hold up time x 5) -Injection cycle: 30 min Agilent 7000 B GC-MS/MS: BACK- FLUSH: -After last eluting compound postrun is enabled: The pressure in the injector is lowered ->0.5 psi PCM pressure and temp in oven and injector are increased-> 50 psi, 280 °C, 280 °C

8. Establishing a Constant Pressure Method with Backflush in combination with RT-Lock For improving peak shape of early eluting peaks; include a pre column Establish method with constant pressure P: Make test runs of a high standard with all pesticides and establish your chromatography with as few time segments as possible! Enable backflush in post run and set a pressure at your pressure controller (PCM) Make a run and ensure that during the run the flow in Column 2 (Column 1+ 0.04 ml/min) is always higher than Column 1 Eventually adjust column head pressure or PCM pressure For improving peak shape of early eluting peaks; include a pre column Establish method with constant pressure P: Make test runs of a high standard with all pesticides and establish your chromatography with as few time segments as possible! Enable backflush in post run and set a pressure at your pressure controller (PCM) Make a run and ensure that during the run the flow in Column 2 (Column 1+ 0.04 ml/min) is always higher than Column 1 Eventually adjust column head pressure or PCM pressure

9. Implementing RT-Lock Put the transitions of your Rt-lock compound into the neighboring time segments RT-Lock program: select the pressures at P+2 and P-2 Make runs at P+2 and P-2 and ensure that flow in Column 2 > Column 1 Eventually adjust the P+2 or P-2 and re-inject Run the RT-Lock setup program with your RT-Lock compound Increase the number of time segments to improve the sensitivity Put the transitions of your Rt-lock compound into the neighboring time segments RT-Lock program: select the pressures at P+2 and P-2 Make runs at P+2 and P-2 and ensure that flow in Column 2 > Column 1 Eventually adjust the P+2 or P-2 and re-inject Run the RT-Lock setup program with your RT-Lock compound Increase the number of time segments to improve the sensitivity P+1 =P+10% P Rt P P-1 P-2 P+2 P+1 Due to increased injector pressure, the flow in column 1 could be higher than in column 2 The PCM module may also not maintain its pressure Due to reduced injector pressure, the flow in column 1 could be too low; constant backflush

10. Maintenance Routines Re-Lock with Ditalimfos Changing liner Changing pre- column Changing column 1 Changing column 2 Cleaning ion source Tuning Before starting samples 50-60 samples After 3-6 months Once a yearAfter 6-8 months Every month There is no need for venting the MS when changing column 1

11. Changing Columns UltiMetal Ferrules SilTite http://www.chem.agilent.com/Library/usermanuals/Public/G3188-90001.pdf The UltiMetal ferrules are not leaking, simpler to install and cause no problems with snapping off the column when tightening!

12. Conclusion Backflush: Protects the ion source Decrease the measurement uncertainty for several compounds No need for venting the MS when changing the first column #1 Column #2 can last twice as long as column #1 Easy change of columns with UltiMetal Ferrules Robust system: First installed and tuned it works without technical problems; «hassle-free»

13. Conclusion Don't solve problems with traditional difficult pesticides like captan, folpet, pyrethroids… We still have problems with certain matrices: – Tea -> increased retention times – Extraction at pH 1 for captan, folpet and chlorothalonil -> reduced sensitivity You have to invest time and effort to establish and understand how the system work

14. Thanks to: Kari Stuveseth Anita Glosli Application notes from Agilent: 5990-4643EN, 5990-3578EN and 5990-9317EN