1. 10 years of evolution in UHPLC-MS chromatography. Current trends. Eduard Rogatsky Albert Einstein College of Medicine Einstein-Montefiore Institute for Clinical & Translational Research Bronx, NY

2. National Automobile Museum The Harrah Collection, Reno, NV

3. 1912 BAKER - electric 1913 STANLEY -steam 1912 SELDEN - gasoline In July 1886 the newspapers reported on the first public outing of the three-wheeled Benz Patent Motor Car, model no. 1. Reproduced with permission

4. 1960s-1980s HPLC 1980s-2000s “Conventional” HPLC 2004 - Present Acquity UPLC (2004) HPLC Instrument Development Milestones ?

5. HPLC Instrumentation Development UHPLC was introduced 10+ years ago. This new technology, while having great promise for improved efficiency and productivity, has not to date displaced HPLC. This transition will be slow process. Currently, both HPLC and UHPLC can be working in the same lab. 1992, MS-DOS 1998, Windows 95 2001, Windows XP

6. UHPLC it is not only a system that’s capable of operating at higher pressures. UHPLC is optimized to work with sub 2 micron ultra – high resolution columns to produce very narrow sharp chromatographic peaks. UHPLC –in contrast to conventional HPLC, is a low dwell volume system designed for fast runs at high pressure. UHPLC vs HPLC

7. Current trends in chromatography Transition from HPLC to UHPLC Evolution of conventional HPLC for full support of Fused-Core chromatographic columns (delay volume reduction) Evolution of UHPLC Chromatography with Mass Spectrometry analysis.

8. UHPLC performance UHPLC performance is mostly based upon correct choice of flow rate, operating pressure, dwell/post-column volume and chromatographic column. Choice of these parameters is not module-based. Correct choice is system-based.

9. UHPLC : pressure and flow rate impact Autosampler Pump Column thermostat Detector Injector valve, wash port, sample loop size Purge valve, mixer Heat exchanger Switching valve Flow-cell design, pressure and volume LC devices Critical components and parameters

10. Column guard: contamination by rotor seal wear debris LC column: Increase backpressure

11. Rotor seal dust

12. Agilent 1290 UHPLC. Adjustable fitting in the column compartment

13. Heat exchanger outlet tubing design 1/16 OD 1/32 capillary, that have limited resistance to mechanical stress during column installation

14. Delay volume-pressure impact: 15% drop of mixing efficiency Upchurch filter in the pump, no damper, valve, restrictor, Flow rate 0.7 ml/min Blue: Low Pressure V 0 0.318 ml; V 50 0.497ml Red: High Pressure 344Bar, V 0 0.305 ml; V 50 0.580 ml The more shallow slope results in a larger V 50, [poor mixing] as well as smaller V 0 Low Pressure High Pressure 344Bar V 50 0.497ml V 50 0.580 ml

15. IDEX PEEK Lined SS

16. V50 increase with pressure Pressure, bar

17. Summary New experience: moving parts and mixers are less rugged and efficient in high pressure applications.

18. UHPLC and MS UHPLC/UV requires baseline separation In contrast, MS detection utilizes ions resolution by m/z and chromatographic peaks overlay is typical in LC/MS analysis.

19. 2D chromatography – efficient way to increase peak capacity. It is important for UV applications, but in general, not for LC/MS applications 2D and LCMS Benefits of 2D in MS: Minimum of signal suppression Better background Better peak shape More clean instrument

20. UHPLC 2D/MS Agilent 1290 series

21. Matrix effect reduction - One column method SPE AcN/Phospate sample S/N p/p =16.1

22. Matrix effect reduction - Two column method SPE AcN/Phospate sample 12.6 fold improvement S/N p/p =201.4

23. Chromatography myth Single column chromatography is always more robust, compared to column switching applications. That’s true – if we are comparing standard pressure ranges. High pressure operations are less robust and more expensive - compare to standard LC Is it possible to combine more robust standard pressure/larger volume sample injection with UHPLC?

24. New paradigm: sample injection via standard LC followed by UHPLC chromatography. The sample was injected by a standard (not UHPLC) autosampler into a pre-analytical column, where it was desalted and partially purified. A standard pressure range binary HPLC pump was used. The fraction containing analytes of interest was transferred through a UHPLC valve to a fused core column. A fast gradient was performed using a UHPLC 1290 pump.

25. LCxUHPLC

26. Online SPE design LCxUHPLC design based on HPLC loading pump and UHPLC LC/MS pump uu Standard LC pump – loading step fused core column

27. Human C-peptide Plasma SPE

28. Urinary cortisol and testosterone –single column Testosterone 10 ul urine, 1:3 diluted cortisol

29. Urinary cortisol and testosterone -trap Testosterone RT 3.5 min; was 5 10 ul urine, 1:3 diluted cortisol

30. MMA from plasma 1D: trap +column MMA

31. MMA from plasma 1D: trap +column MMA

32. MMA from plasma 2D: trap loading +column switching Column switching improves S/N from 214 to 334; aprox. 40% gain MMA

33. 33 Fused-Core Ascentis Express Particles Comparison of architecture of Fused- Core™ particles vs. porous 3 µm particles

34. 2x50 Polaris C18 3µm 180 A versus 2x30 Ascentis Express Peptide 160A S/N 170 S/N 40 Low loading capacity –the main limitation factor of the Fused-Core material – was overcome by using 2D chromatography. Only small, C-Peptide containing fraction was loaded [transferred] onto the column.

35. Column comparison in the second dimension under the same conditions Peak 1: Aeris Peptide C18, 3.6 um 2.1 x50 mm (fused-core); Peak 2: XBridge C18, 3.5 um 2.1x30 mm; Peak 3: Ascentis Express Peptide C18, 2.7 um 2.1 x 30mm (fused-core)

36. Summary 1 LCxUHPLC is more rugged column switching system, compare to 2D UHPLC UHPLC sample loading is limited Sample loading to trap, using standard LC pump and autosampler is more convenient and cost efficient Loading and wash step using trap greatly improve ruggedness of mass spectrometer operations

37. Hybrids typically have: More functionality Higher cost, compare to its components, Only basic control and features Less ruggedness Limited support LCxUHPLC is free from these disadvantages

38. We successfully used LCxUHPLC platform for LC/MS analysis. Instead of retiring an entire functioning Agilent 1100 LC system, we just added one UHPLC pump to achieve much greater overall performance, functionality and lower cost, compared to a single pump UHPLC system purchase. Take Home Message

39. Summary 2 The addition of a UHPLC valve and pump to a standard 1100/1200 series LC system (autosampler, pump and column compartment) greatly extends operational flexibility including column selection, while standard LC – which is already available in the lab, performs the initial steps of sample loading and clean-up

41. Funding: This research was supported in part by a Clinical and Translational Science Award (CTSA) from the National Institutes of Health (UL11RR025740) awarded to the Albert Einstein College of Medicine of Yeshiva University. NIH: R01 DK61644-01 Technical Support: Agilent Technologies IDEX corporation/Upchurch scientific Optimize Technologies Acknowledgements