PowerPoint Presentation€¦ · PPT file · Web view2017-10-02 · Brad Ackermann from Eli Lilly....

B IOANALY

T ICAL C

RO

THE LC /MS EXPERTST M

Alturas Analytics, Inc.1324 Alturas DriveMoscow, ID 83843

(208) 883-3400

Microspray and Microflow Liquid Chromatography: The Perfect Fit for

Modern LC-MS Bioanalysis

Shane Needham, Ph.D.Co-Founder and Laboratory DirectorAlturas Analytics, Inc.

AlturasAnalytics.com The LC/MS ExpertsTM

100 mL/min, 1.00 mm ID

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9

Time, min

0.0e0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

4.0e5

4.2e5

4.4e5

4.6e5

4.8e5

5.0e5

Inte

nsity

10.0 mL/min, 0.300 mm ID

30.0 mL/min, 0.500 mm ID

400 mL/min, 2.00 mm ID

HPLC-MS/MS and MFLC-MS/MS chromatograms from the analysis of a 1.5 µL injection of a 5.00 ng/mL Propranolol solution on an AB SCIEX QTRAP® 5500

Notice band broadening on this first “prototype” MFLC ESI source


MFLC-MS/MS IN REGULATED BIOANALYSIS

• Can MFLC-MS/MS methods be validated?

• Are the MFLC-MS/MS methods rugged?

• Do the MFLC-MS/MS method results compare well to HPLC-MS/MS methods?


METHOTREXATE (MTX)

• MW = 454

• Used in treatment of cancer, autoimmune diseases,ectopic pregnancy

•Alturas previously validated a LC-MS/MS method in human plasma from 1-500 ng/mL

•Acetonitrile precipitation


LC-MS/MS Overlayed Chromatograms (HPLC vs. MFLC) from Bioanalysis of Methotrexate (400 ng/mL) from Human Plasma Using

Eksigent ekspert microLC 200 and a QTRAP 5500


COMPARISON OF THE LC-MS/MS BIOANALYTICAL VALIDATION OF METHOTREXATE (MTX) FROM HUMAN PLASMA

LC-MS Inter A/P Intra A/P MF

Conv. 105±10.2 *112±13.7 1.02

MFLC 93.3±12.9 *87.1±13.2 1.07

Dynamic range of 1-1000 ng/mL *Includes LLOQ values


MFLC-MS/MS Chromatograms Overlayed fromBeginning and End of Run of >350 injections of MTX

Extracted from Human Plasma


QTRAP® 5500 interface plate clean, before any injections


QTRAP® 5500 interface plate after approximately 400 extracted human plasma injections from the MFLC system – no switching valve

Slight halo effects


QTRAP® 5500 interface plate after approximately 150 extracted human plasma injections from the conventional HPLC system – no switching valve

Notice deposits and source contamination


FUTURE OF LC-MS/MS: COUPLING THE MS SOURCE AND MFLC

Why? Because it works! – simplifies LC-MS

Natural progression of coupling analytical techniques

Less connections-less band broadening

Source is column heater No switching valve needed to

divert waste SAMPLING ADVANTAGE


“The source is the column, the column is the source”

PicoFuze™ to be inserted into ESI-MS probe

MFLC “column” in the PicoFuze™

QTRAP® 6500 with the Microflow Eksigent ekspert microLC 200 System

PicoFuze™ MS/MS on QTRAP 6500

ESI-MS Probe


SIGNAL COMPARISON: MTX

0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4Time, min

0.00

5.00e4

1.00e5

1.50e5

2.00e5

2.50e5

3.00e5

3.50e5

4.00e5

4.50e5

5.00e5

5.50e5

6.00e5

6.50e5

7.00e5

7.50e5

8.00e5

8.50e5

9.00e5

9.50e5

1.00e6

Intensity, cps

700 µL/min

PicoFuze™, 12 µL/min

PicoFuze™, 7 µL/min

Notes:•Maximum signal was achieved at 7 µl/min, but the RT and signal was inconsistent at this flow rate.•Optimal signal, run time and robustness were achieved at 12 µL/min


2.05 2.10 2.15 2.20 2.25 2.30 2.35 2.40 2.45 2.50 2.55 2.60 2.65 2.70 2.75 2.80 2.85 2.90 2.95 3.00Time, 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

Intensity, cps

1st Injection

After ~500 injectionsMTX 400 ng/mL extracted from

human plasma

“Plug and Play”

“First try at full bioanalytical validation on PicoFuze™”

PicoFuze™ in Bioanalysis


MTX INTRA/INTERBATCH ACCURACY AND PRECISION


ELECTROSPRAY “SWITCHING VALVE”

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1100

1150

Intensity, cps

TIC: from Sample 2 (Methotrexate 1 ng/mL) of ES_Switching.wiff (Turbo Spray IonDrive) Max. 1.3e5 cps.

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0Time, min

0.00

5000.00

1.00e4

1.50e4

2.00e4

2.50e4

3.00e4

3.50e4

4.00e4

4.50e4

5.00e4

5.50e4

6.00e4

6.50e4

7.00e4

7.50e4

8.00e4

8.50e4

9.00e4

9.50e4

1.00e5

1.05e5

1.10e5

1.15e5

1.20e5

1.25e5

Inte

ns

ity, cp

s

2.83

Blank solvent injection showing ESI voltage “turned on” at 2.3 minutes and “turned off” at 3.4 minutes


≈1990-API III

≈1996-API 3000

≈2001-API 4000

≈2012-API 6500

PicoFuze™ - Future of the LC-MS Source

4.6 mm ID, 1.0 ml/min, split, 100 µL injections

2.1 mm ID, 0.4 ml/min, 40 µL injections

<1.0 mm ID, <100 µl/min, 1-10 µL injections

<0.3 mm ID, <10 µl/min, 1-2 µL injections

But wait, I can improve LLOQ by injecting more on my 2.1 mm ID and I can only inject 1-10 µL on my MFLC!!


Consumer Driven “Convenience”!

Ease of use is “HIGH PERFORMANCE!”

Inject less volume with better signal for MFLC gives “peace of mind” – SAMPLING

ADVANTAGE


OTHER BENEFITS OF MFLC-MS/MS

• Reduced source contamination due to less flow and lower injection volumes

• More up time-increased PRODUCTIVITY• No need for a switching valve… -increased EFFICIENCY

• Reduced matrix effects?• Develop methods faster?• Less potential failures?

• Saves solvent – “Goes Green”• 5 mL of organic mobile phase per day at 24/7 operation

(normally 500 mL!) -increased EFFICIENCY

More research needed….


CONCLUSION: MFLC-MS/MS AND PICOFUZE™ BUZZ WORDS

It works! – “Ease of use is high performance”

“Peace of mind” – More injections per sample due to increased sensitivity – SAMPLING ADVANTAGE

“Plug and play” – minimal optimization for transferring methods from conventional LC or MFLC

Efficient, Productive and Convenient – “Source is the column, column is the source”

Less connections-less band broadening, easier to use Source is column heater No switching valve needed to divert waste


CONCLUSION

MFLC-MS/MS and PicoFuze™ -MS/MS coupled chromatography and MS will continue to develop

More research needed Expand user base, DATA, DATA, DATA APCI??? Vendors develop HPLC pumps that work for micro and

conventional flows in the same pump Cost of “source column” should compete with cost of

HPLC column


ACKNOWLEDGEMENTS

Those on Microflow Team at AlturasChad Christianson and Jennifer Zimmer

SCIEX and EksigentGary Valaskovic and entire team at New

Objective Inc.Mike Lee from Milestone DevelopmentNathan Yates from University of PittsburghBrad Ackermann from Eli Lilly


Contact Us:AlturasAnalytics.com

Alturas Analytics, Inc.1324 Alturas DriveMoscow, ID 83843

(208) 883-3400


PowerPoint Presentation€¦ · PPT file · Web view2017-10-02 · Brad Ackermann from Eli Lilly....

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