Metabolite Apps

8/4/2019 Metabolite Apps

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Applications of LC/MS inQualitative Drug Metabolite

Studies


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Case Study: Identifying in vivo metabolites in human urine -

sample cleanup and LC/MS/MS strategies

Application

detection of drug (Vanlev) metabolites in human urine

limited sample need to identify major metabolites in high matrix background

Approach

on-line sample cleanup using small molecule trap column

(maximize S/N ) Use instrument control language (ICL) on TSQ to construct

unique data-dependent acquisition modes using combinedprecursor, neutral loss, and product ion scan modes (maximizeinformation per run)


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Dont forget about samplecleanup!


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Column Switching System for Metabolite CleanupSample Load

Electrically Actuated Divert ValveControlled by the Data System

1

2

3

4

5

6

Finnigan TSQ 700

Waters HPLCWaste

Analytical ColumnYMC-AQ C18 4.6 x 150 mm

UV

HPLCPump

AS

Wash solvent0.1% TFA/H2O

Flow: 0.1 mL/min

YMC-AQC18

2.1x33mm

Gradient system

1 mL/min


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100

80

60

40

20

0

9.991

e+0

1.345e+07

100

80

60

40

20

0

8:20 16:40 25:00 33:20

LC/UV and Full-Scan LC/MS Chromatograms of BMS-186716 and its

Metabolites in Human Urine Without Prior Sample Clean-up

Time (minutes)

UV

220 nm

RIC

Full-Scan MS200-800 amu

NormalizedIntensity


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LC/UV and Full-Scan LC/MS Chromatograms of BMS-186716 and its

Metabolites in Human Urine Using an On-Line Clean-up Column

UV

RIC

100

80

60

40

20

0100

80

60

40

20

0

8:20 16:40 25:00 33:20

Time (minutes)

2.801e+07

9.991e+0

220 nm

Full-Scan MS200-800 amu

Normalize

dIntensity


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Time (minutes )8:20 16:40 25:00 33:20

m/z= 423

m/z= 528

m/z= 585

m/z= 599

m/z= 615

m/z= 704

6.693e+04

2.637e+05

1.272e+04

5.981e+04

3.856e+04

5.451e+04

Extracted Ions of Metabolites of BMS-186716 in Human Urine from Full-

Scan LC/MS Chromatograms: Before Use of Clean-up Column


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8:20 16:40 25:00 33:20

Time (minutes )

m/z= 423

m/z= 528

m/z= 585

m/z= 599

m/z= 615

m/z= 704

2.282e+05

6.401

e+05

3.669e+05

1.958e+05

1.073e+05

1.867e+05

Extracted Ions of Metabolites of BMS-186716 in Human Urine from

Full-Scan LC/MS Chromatograms: After Use of Clean-up Column


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Effect of Clean-up Column on Observed Molecular Ion

Species (Rt ~ 20min)

M+Na +

599.1

621.1

637.0

M+H+

M+K+

637.0

599.1

500 550 600 650 700

621.1

m/z

M+Na+

M+K+

M+H+

SNH

O

N

S

OO2CO6H8C6

H3C

Glucuronide

Direct Injection onAnalytical Column

Injection onClean-up Column


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Metabolite LC/MS profiling in complex mixtures

Traditional Approach Run LC/MS and catalog potential metabolites (+16, +32, etc).

Set up additional runs to collect product ion MS/MS data on all ofthese signals

Or, possibly collect both MS and product ion MS/MS in a data-

dependent mode in a single run Watch out: data-dependent product scans can be problematic invivo metabolite samples due to endogenous material. A peak atevery mass can lead to collection many useless product ion spectrawhich are not drug-related.

Can perform precursor and neutral loss scans to search for relevant

metabolites, but collection of data and sorting through signals isvery labor intensive and time-consuming. If these are in vivometabolites, you may run out of sample first!


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Metabolite LC/MS profiling in complexmixtures

General precursor and neutral loss scanning approach for metabolite

screening Carefully study fragmentation of parent drug

Look for characteristic neutral losses or characteristic fragment ions in theproduct ion spectrum

Use characteristic fragment ions to set up precursor scans

Use characteristic neutral losses to set up neutral loss scans

Add other neutral loss scans (e.g., NL of 176 for glucuronidation)

Run these LC/MS/MS experiments

Perform product ion scans on any new potential metabolites with newLC/MS/MS experiments

Can iterate through this approach as new metabolites are discovered

Even more efficient!

Use the precursor and neutral loss scans to trigger product ion scans

Collect only potentially drug-related product ion scans

Get more done with less sample in less time


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Neutral Loss of 176 gives glucuronide conjugates

Parents of 128, other metabolites

HS

N

H

O

N

S

O

HO2C

128

BMS-186716, DMPI

Parent Ion/Neutral Loss Selection


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Data-Dependent MS/MS Using Pre-Cursor Ion and

Neutral Loss Scanning to Detect Drug Metabolites

LC/MS/MS

Chromatogram

Pre-setThreshold

Time

1

32 4

5Scans 1-2: Detect peak in neutral

loss or pre-cursor ion scan mode

Scans 3-5: Collect product-ion scans

The TSQ is scanned in either the neutral loss or pre-cursor ion scan modes

The ICL procedure requires that the ion intensity of a detected mass

surpass a pre-set threshold for two consecutive scans.

When this condition is met, three product ion spectra for that mass are

acquired.


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BMS-186716 and Selected Metabolites

HS

N

H

O

N

S

O

O2C

O6H

8C6

Glucuronide

HS

N

H

O

N

S

O

HO2C

S

N

H

O

N

S

O

HO2C

H3C

S

N

H

O

N

S

O

HO2C

S

NH 2

HO2

C

H

S

N

H

O

N

S

O

O2CO6H8C6

H3C

Glucuronide

S

N

H

O

N

S

O

O2CO6H8C6

CH 3

O

Glucuronide

S

N

H

O

N

S

O

O2C

S

NH 2

HO2C

H

O6H8C6

Glucuronide

(M+H)+ = 585

(M+H)+ = 615

(M+H)+ = 528

BMS-186716(M+H)+ = 409

(M+H)+ = 423

(M+H)+ = 599

(M+H)+ = 704


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Metabolite ID software can beused to assist with metabolite

characterization


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Metabolite ID

input expected drug modifications


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Metabolite IDMetabolite Data Browser - Extracted Modifications

parent

+16

+32

MS MS/MS


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Metabolite IDMetabolite Data Browser - MS/MS Correlation

**


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High-Throughput LC/MS

Metabolic Stability


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High Throughput Metabolic Stability

Analyze many (thousands) of drug candidates to evaluate and

rank compounds on the basis of metabolic stability. Sacrificedetail to obtain throughput.

Perform in vitro incubations w/ and wo/activator

Use a fast LC/MS system and method (1.5 min/sample)

Export integrated peaks areas to spreadsheet and/or database

Ratio the signals from activated vs. non-activated in vitro

metabolite incubations Visualize data using color coded sample status


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Autosampler(Alcott AS)

Ion Trap MS(Finnigan LCQ Duo)

Integrated HPLC(MAGIC 2002)

MAGIC 2002 Integrated HPLC, Finnigan LCQ

Duo, Alcott AS, integrated via Finnigan /custom software

Flow: 1.5 mls/min Column: YMC ODS-AQ, 2X20mm, 5u MS: Data directed, FS and MS/MS

UV: 200 and 220 nm, VWD

1.1 min method, 1.4 min full cycle, 750samples/18 hr

0.05 min wide peaks 18-23 fully resolved peaks possible!

Low flow rate, low delay volume, low psi Only slight decrease in theoretical peaks Similar cost to single quadrupole system Custom programming interface

High Throughput Metabolic Stability System


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Metabolic StabilityBuspirone - Human Liver Microsomes - Extracted Ions

Non-activated - NActivated - A

Parent

InternalStandard

InternalStandard

Parent


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Plate Level Screening ViewADME/TOX - Screen Summary - Metabolic Stability Calculation

PA - Parent-ActivatedPN - Parent-Non-ActivatedSA - Standard-ActivatedSN - Standard-Non-Activated

(PA/SA)

(PN/SN)1 -

Green = OK

YellowYellow == ??Red = Bad


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Conclusions

In vivo metabolite samples still pose significant challengesfor LC/MS/MS elucidation

Simple sample cleanup steps can result in dramaticimprovement

Intelligent data-dependent scanning can help toefficiently locate metabolites in the presence of matrixbackground

High-throughput LC/MS can be used to rank metabolicstability of compounds

New software tools (e.g., Metabolite ID) are useful inspeeding the identification and characterization of drugmetabolites


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Acknowledgments

Mark Sanders, Mike Nedved, RichGedamke (BMS)

Jeff Whitney (Novatia)

Documents

Metabolite Apps