LC2D-MS semi-preparative platform : A tool for Metabolite isolation

26 April 2016

Claessens Jehan

ן In vivo studies : global process

ן Is it possible to isolate metabolites of interest ?

ן Purification platform : Challenges

ן LC2D/MS platform description

ן Case study : drug isolation from rat urine

ן Limitations and perspectives

ן Conclusion

Agenda

2

ן Process overview

In vivo studies : global process

3

Metabolite profiling (Oxidation,

Hydroxylation, reduction,

glucuronidation, …)

0

2000

4000

6000

8000

10000

0 20 40

Ab

un

dan

ce

Time

Compound1

Parent (5.969)

Hydroxylation (4.372)

Hydroxylation (4.619)

HRMS analysis Compound injection Biofluids recovery

Metabolite Selection

Time2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%0

20

40

60

80

100

9.90e3Combined Metabolite Peaks (Found Expected Peaks only) [Analyte]

387

5.37

387

5.21

371

7.04

335

6.66385

6.20

Time2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%0

20

40

60

80

100

ן Identify finer structural details

Understand compound reactivity and identify potential soft points

Need additional NMR analysis for complete characterization

Low sensitivity

Need high purity compound

Non destructive tool

Quantification

ן Recover metabolites produced during in vivo studies

Avoid heavy synthesis

First functional test

Impact quickly therapeutic project

Is it possible to isolate metabolites of interest ?

4

ן Desalting and concentration module At-column dilution process

Purification platform : Challenges

5

Dilution pump

10 ml/min

100 % H2O

Loading pump

2 ml/min

50/50 % ACN/H2O

Mixing

Tee

Preparative

LOOP (10 ml)

Waste

Prestacking

column

10x50 mm

Metabolite concentration ( ~ 100 µg compound ~10 ml urine)

Avoid chromatographic distortion

ן Multiple metabolite isolation during the purification step

Metabolites triggering by mass spectrometer based on molecular weight

SQD2 mass spectrometer detector

Goal :

Limited collection volume

High sensitivity detection in vitro studies

ן Couple analytical and purification platform

Cycle time optimization

Pre- and post-purification analytical control

Internal GO/NO GO purification step

Purity evaluation

Purification platform : Challenges

6

ן High purity and selectivity purification

Obtain enough pure compound for NMR analysis

purity post purification > 95 %

Two purification cycles ( 2D purification)

Two elution modes ( ACIDIC – BASIC)

Two high resolution semi-preparative columns

(5 µm – 10 mm ID – 150 mm length)

Purification platform : Challenges

7

First

purification

Analytical

control

Initial

Analysis

Final post

purification

control

Second

purification

CYCLE 2 CYCLE 1

ן All purification cycles fully managed by software

Minimize human intervention

Masslynx Software FractionLynx Autopurify Method

8

Purification platform : Challenges

Possibility to select global process

Managed specific purification

methods based on analytical steps

LC2D/MS platform description

ן Masslynx sample Queue

One line with all the useful information

Case study : drug isolation from rat urine

11

150 µg Efletirizine in 8 ml rat urine

UCB compound

Preparative

injection

volume

Collection

method

Analytical

injection

volume

Autopurify

method

Case study : drug isolation from rat urine

12

Time %A %B Flow

(ml/min)

0 98 2 1.8

1 98 2 1.8

7 10 90 1.8

7.1 10 90 2.3

9 10 90 2.3

First

purification

Analytical

control

Initial

Analysis

Final post

purification

control

Second

purification

CYCLE 2 CYCLE 1

1st Step : Initial Analysis:

Acidic Elution

Column : Waters Sunfire MS C18 5 µm 150 x 4.6 mm

Injection volume : 20 µl

A : H2O/ACN/Formic acid (95/5/0.05 %)

B : ACN / Formic acid 0.075 % UV

MH+ 391 FOUND

Case study : drug isolation from rat urine

13

Time %A %B Flow

(ml/min)

0 98 2 7

5 98 2 7

7 98 2 7

16 10 90 7

20 10 90 7

Column : Waters Sunfire MS C18 5 µm 150 x 10 mm

Injection volume : 9000 µl

A : H2O/ACN/Formic acid (95/5/0.05 %)

B : ACN / Formic acid 0.075 %

Fraction

Collection

volume : ~ 3 ml

At-

co

lum

n

dil

uti

on

MH+ 391

UV

2nd Step : First purification

dimension : Acidic Elution

First

purification

Analytical

control

Initial

Analysis

Final post

purification

control

Second

purification

CYCLE 2 CYCLE 1

Case study : drug isolation from rat urine

14

Time %A %B Flow

(ml/min)

0 98 2 1.8

1 98 2 1.8

7 5 95 1.8

7.1 5 95 2.3

9 5 95 2.3

Column : Waters Xbridge MS C18 5 µm 150 x 4.6 mm

Injection volume : 20 µl

A : H2O/ACN/Ammonium formate/ Ammonia (95/5/10mM/0.1%)

B : ACN

First

purification

Analytical

control

Initial

Analysis

Final post

purification

control

Second

purification

CYCLE 2 CYCLE 1

3rd Step : First analytical control :

Basic Elution

compound

Purity 15 %

Impurities

MH+ 391

UV

Case study : drug isolation from rat urine

15

Time %A %B Flow

(ml/min)

0 98 2 7

5 98 2 7

7 98 2 7

16 10 90 7

20 10 90 7

Column : Waters Xbridge MS C18 5 µm 150x 10 mm

Injection volume : 9000 µl (fixed volume)

A : H2O/ACN/Ammonium formate/ Ammonia (95/5/10mM/0.1%)

B : ACN

At-

co

lum

n

dil

uti

on

MH+ 391

UV

Fraction

Collection

volume : ~ 3 ml

First

purification

Analytical

control

Initial

Analysis

Final post

purification

control

Second

purification

CYCLE 2 CYCLE 1

4th Step : Second purification

dimension : Basic Elution

Case study : drug isolation from rat urine

16

Time %A %B Flow

(ml/min)

0 98 2 1.8

1 98 2 1.8

7 10 90 1.8

7.1 10 90 2.3

9 10 90 2.3

Column : Waters Sunfire MS C18 5 µm 150 x 4.6 mm

Injection volume : 20 µl

A : H2O/ACN/Formic acid (95/5/0.05%)

B : ACN / Formic acid 0.075%

Purity 100%

Post purification recovery : 142 µg (95%)

Global cycle time : 77 minutes

MH+ 391

UV

First

purification

Analytical

control

Initial

Analysis

Final post

purification

control

Second

purification

CYCLE 2 CYCLE 1

5th Step : Final analytical

control : Acidic Elution

ן Limitations

Platform not fully automatized - Human intervention between purification cycles

ן Perspectives

Purification platform for in vitro studies

Limitations – Perspectives

17

Improve chromatographic separation

« Narrow methods » based on

analytical retention time

Fraction collection post at-column dilution

Avoid potential loss during the stacking step

Dilution pump

10 ml/min

100% H2O

Loading pump

2 ml/min

50/50% ACN/H2O

Mixing

Tee

Preparative LOOP (10ml)

Prestacking

column

10x50 mm

UV detctor

Fraction collector

CONCLUSION

18

We develop an chromatographic purification system able to :

Isolate low amount of compound out of complex mixture with

high recovery and sufficient purity for subsequent NMR

characterization and biological testing

Execute two consecutive cycles of purification avoiding

concentration steps in an automated environment limiting

human intervention

Thanks! UCB team

DELL’ AIERA Sylvie

DERWA Christelle

DELATOUR Claude

Waters Team

PETIT Davy

LIMAUGE Frederic

Questions?

20