10 years of evolution in UHPLC-MS chromatography. Current trends.

Eduard Rogatsky Eduard Rogatsky Albert Einstein College of MedicineAlbert Einstein College of Medicine

Einstein-Montefiore Institute for Clinical & Einstein-Montefiore Institute for Clinical & Translational ResearchTranslational Research

Bronx, NYBronx, NY

National Automobile MuseumThe Harrah Collection, Reno, NV

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

1960s-1980sHPLC

1980s-2000s “Conventional”

HPLC

2004 - PresentAcquity UPLC (2004)

HPLC Instrument Development Milestones

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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

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

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.

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.

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

Column guard: contamination by rotor seal wear debris

LC column: Increase backpressure

Rotor seal dust

Agilent 1290 UHPLC. Adjustable fitting in the column compartment

Heat exchanger outlet tubing design

1/16 OD

1/32 capillary, that have limited resistance to mechanical stress during column installation

Delay volume-pressure impact:15% drop of mixing efficiency

min5 5.5 6 6.5 7

Norm.

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25

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75

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175

200

VWD1 A, Wavelength=254 nm (081908\LPUPND02.D) VWD1 A, Wavelength=254 nm (081908\HPUPND02.D)

Upchurch filter in the pump, no damper, valve, restrictor, Flow rate 0.7 ml/min Blue: Low Pressure V0 0.318 ml; V50 0.497mlRed: High Pressure 344Bar, V0 0.305 ml; V50 0.580 mlThe more shallow slope results in a larger V50, [poor mixing] as well as smaller V0

Low Pressure

High Pressure 344Bar

V50 0.497mlV50 0.580 ml

IDEXPEEKLinedSS

V50 increase with pressure

Pressure, bar

Summary

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

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.

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

UHPLC 2D/MS Agilent 1290 series

Matrix effect reduction - One column methodSPE AcN/Phospate sample

XIC of +Q1 SIM (3 ions): 1007.7 amu from Sample 10 (Luna only, Eduard plasma SPE/KPi) of 7222... Max. 2.1e4 cps.

9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6 10.8 11.0 11.2 11.4 11.6 11.8 12.0 12.2 12.4 12.6 12.8Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

Inte

ns

ity, c

ps

11.55

9.16 10.27 11.1310.60 10.75 12.5710.199.77 12.3312.2611.349.629.37 12.68

S/N p/p =16.1

Matrix effect reduction - Two column methodSPE AcN/Phospate sample

XIC of +Q1 SIM (3 ions): 1007.7 amu from Sample 14 (2D, seppack KPi) of 7222003SET1.wiff Max. 3.3e4 cps.

20.6 20.8 21.0 21.2 21.4 21.6 21.8 22.0 22.2 22.4 22.6 22.8 23.0 23.2 23.4 23.6 23.8 24.0Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

Inte

ns

ity, c

ps

22.19

12.6 foldimprovement

S/N p/p =201.4

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?

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.

LCxUHPLC

Online SPE design

LCxUHPLC design based on HPLC loading pump and UHPLC LC/MS pump

u u

Standard LC pump – loading step

fused core column fused core column

Human C-peptidePlasma SPE

2x10

0

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0.8

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+ESI MRM Frag=300.0V CID@21.0 (1007.6 -> 147.1) qc 1d 10 ul006.d Noise (RMS) = 1.30; SNR (2.229min) = 122.5

321159

52

1 1 22

Counts vs. Acquisition Time (min)0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9

Urinary cortisol and testosterone –single column

Testosterone

10 ul urine, 1:3 diluted

cortisol

Urinary cortisol and testosterone -trap

TestosteroneRT 3.5 min; was 5

10 ul urine, 1:3 diluted

cortisol

MMA from plasma 1D: trap +column

MMA

MMA from plasma 1D: trap +column

MMA

MMA from plasma 2D: trap loading +column switching

XIC of +MRM (4 pairs): 234.000/122.000 Da ID: qual from Sample 11 (2D Plasma 7 ul) of 08042014SET1.wiff (Turbo Spray) Max. 5.7e4 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

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Inte

ns

ity, c

ps

-0.36

3.59

-1.62

-2.77

! !- Noise -

S/N = 213.6Peak Int.(Subt.)=3.8e+4

3xStd.Dev.(Noise)=1.8e+2

! !- Noise -

S/N = 334.3

Peak Int.(Subt.)=4.0e+43xStd.Dev.(Noise)=1.2e+2

Column switching improves S/N from 214 to 334; aprox. 40% gain

MMA

33

Fused-Core Ascentis Express Particles

• Comparison of architecture of Fused-Core™ particles vs. porous 3 µm particles

2x50 Polaris C18 3µm 180 A versus 2x30 Ascentis Express Peptide 160A

XIC of +Q1 MI (4 ions): 1007.7 amu from Sample 40 (Fresh solvent 4.9 -5.7 35 ul) of 07062010SET1.wiff (Turbo Spray) Max. 9.7e4 cps.

6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

7.0e4

7.5e4

8.0e4

8.5e4

9.0e4

9.5e4

Inte

ns

ity, c

ps

11.50

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.

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

Hybrids typically have:More functionalityHigher cost, compare to its components, Only basic control and featuresLess ruggednessLimited support

LCxUHPLC is free from these disadvantages

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

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

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 TechnologiesIDEX corporation/Upchurch scientificOptimize Technologies

Acknowledgements