From HPLC to UHPLC and how to continue?

Transcription

1 From HPLC to UHPLC and how to continue? Van HPLC naar UHPLC en hoe nu verder? Arjen Gerssen

2 Outline RIKILT? Marine biotoxins HPLC UHPLC NanoUHPLC microuhplc

3 RIKILT Institute of Food Safety Wageningen UR RIKILT contributes to European food and feed safety and health with high-quality, independent research and consultancy Results of RIKILT are more often found in the media

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5 Marine biotoxins Natural toxinsproduced by algae whichcan end-up in filter feeders

6 Marine biotoxins In Europe three different intoxication syndromes ASP (Amnesic Shellfish Poisoning) PSP (Paralytic Shellfish Poisoning) DSP (Diarrhoea Shellfish Poisoning)

7 Goal of this research

8 Conventional LC-MS/MS Traditional: LC-MS/MS C 18 andacidicchromatographic conditions Problems; Overlap ESI -/+, Peak shape YTX OA PTX2 AZA1 ESI - ESI + GYM SPX1 YTX Retention time (min)

9 Like always with multi methods Okadaic acid Azaspiracids Yessotoxins Pectenotoxins Cyclic imines

10 Conventional LC-MS/MS More polymeric based columns and alkaline conditions Separation in ESI + and ESI - Somewhat poor peak shape AZAs Excellent sensitivity for acid containing compounds (OA, YTX) negatief positief AZA1 OA PTX2 YTX GYM SPX Retention time (min) Gerssen et al. 2009, J Chrom. A

11 Finished!... Or not? Problem with reference methods in legislation Improvements are not formally allowed and changing legislation is a tedious procedure

12 UHPLC instead of UPLC Increase the throughput of the method (LC ~ 20 min) Upgrade the conventional method towards latest generation equipment Improve in speed of analysis Include more toxins in same procedure Pin-E Pin-F Pin-G SPX1 GYM

13 UHPLC method developed Analytical column: BEH C mm x 2.1mm, 1.7µm Flow of 0.6 ml/min Analytical column temp 40C Detection with Xevo TQ-S ESI + and ESI - 27 toxins (50 transitions) well time 3msec Time A (%) B (%)

14 New faster method 5 min per analysis Extreme valuable additional information i.e. EFSA But more data analysis

15 Application of improved method Rapid method allowed us to do additional research on these samples Analysis of 100 samples HPLC = 33h UPLC = 8h

16 Conclusion for HPLC and UHPLC Both conventional LC and UHPLC can be used for routine monitoring With UHPLC method possible to incorporate more toxins UHPLC is incorporated in the Dutch routine program Needs Cheaper solutions saving valuable standards and solutions REDUCTION OF COSTS

17 Moving to µlc and nlc devices Cons Sample-size is usually not limited Nano equipment does not comply with the requirements we want to have in small molecule world Pros Increase costs of high-grade solvents Less contamination of source etc. Sample size limited i.e. forensic applications or bioactivity-driven hazard identification

18 Our definition (UPLC - µuplc nuplc) UPLC UPLC µ n 2.1mm 1mm 0.15mm 0.085mm High flow >600µl/min Conventional flow µl/min Low flow µl/min 1-10µl/min <1µl/min Gerssen et al. LC-GC North America, Special Issue 2014, 32-5, 18-23

19 Start with nanouhplc (2009) Application to > 100 veterinary drugs in porcine, succesfull (column 75µm i.d. flow 600nl/min) Improved sensitivity (180 fold increase peak area) Drift sensitivity low Green chemistry But Analysis time doubled (16 to 33 min) And..

20 Drawback of the NanoUPLC Manual connections introduces void volume Difficult to rapidly change columns

21 Plug-and- play µuplc (Ikey) Ceramic device 50mm x 150µm, 1.7µm particles (BEH C 18 ) Max pressure psi Integrated ESI emitter No manual connections when changing a column!!

22 UHPLC compared to µuhplc (steroids) UPLC (urine spiked at 2ppb) µl injection Relative intensity (%) Intensity β-t t r 7.16 min Area: α-t t r 7.95 min Area: t r (min) Trizaic (urine spiked at 2ppb) t r (min) Intensity β-t t r 8.44 min Area: µl injection α-t t r 9.18 min Area: t r (min)

23 UHPLC compared to µuhplc (steroids) Asymmetry factor UPLC sym = 1.37±0.34 µuplc sym = 1.39±0.11 Effective plate number Relative intensity (%) W 1/2 W 10% UPLC t r 7.16 min b a t r (min) Trizaic µuplc UPLC N eff = µuplc N eff = Relative intensity (%) W 1/2 W 10% b a t r 8.44 min t r (min)

24 Differences for marine biotoxins methods Mobile phase (µl) Injection volume (µl) HPLC UHPLC µuhplc Analysis time(min) AZA3 AZA1 AZA2

25 Conclusions Currently majority of the methods changed from HPLC to UHPLC New possibilities of µuhplc more and more applicable for routine use Chemisch Weekblad(C2W) 26 September 2014 UHPLC µuhplc

26 UHPLC µuhplc