UHPLC Trapping Application with Optimize Technolgies EXP Traps

Improved Sensitivity, Selectivity and Robustness through Trace Enrichment on UHPLC-MS/MS Daniela Fraier Bioanalytical Group - Pharmacokinetics, Dynamics & Metabolism

Content • Background – Bioanalysis at Pfizer

• Opti-Trap EXP™* evaluation – System setup – Applications • Dry Blood Spot (DBS) • Brain µ-Dialysis • Small peptide analysis

– Robustness

• Conclusions * U.S. and Foreign Patents Pending

Bioanalysis at Pfizer • Supplies the bioanalytical solutions essential to bring compounds from early discovery through candidate nomination (CAN) & clinical phase.

• Developed a rational approach to deal with the physicochemical diversity of a large number of compounds and a wide range of biological matrices (e.g. plasma, blood, urine, tissues, etc.).

• Harness the synergies between general bioanalysis expertise and biomarker specialist knowledge to solve quantitation issues associated with challenging compound chemistries.

• Evaluate and implements novel analytical technologies and methodologies.

Opti-Trap EXP™ Evaluation

(U.S. and Foreign Patents Pending)

System Setup

• The Opti-Trap EXP™ was installed in the integrated column selection valve of the Agilent 1200RR thermostated column compartment

System Setup: 10-Port Valve (Load) AUTOSAMPLER WASTE

HPLC PUMP (LOADING) UHPLC PUMP

COLUMN HEATER

ANALYTICAL COLUMN

TRAP *

MS ∗ Opti-Trap EXP™ (U.S. and Foreign Patents Pending) ∗ Halo™ C18, 4.6 x 5mm (100µL bed volume) ∗ Designed to work with UHPLC up to 15,000 psi

System Setup: 10-Port Valve (Elute)

AUTOSAMPLER WASTE

HPLC PUMP (LOADING) UHPLC PUMP

COLUMN HEATER

ANALYTICAL COLUMN

TRAP*

MS ∗ Opti-Trap EXP™ (U.S. and Foreign Patents Pending)

System Design • •

Trapping column loaded in aqueous mobile phase and back flushed onto an Eclipse XDB-C18 3 x 50 mm,1.8µ column.

“Generic” rapid gradient from 10% methanol (mobile phase A) to 90 % (mobile Phase B) run through the analytical column to elute the analytes of interest.

Trap in line with analytical column

Trap diverted to waste

Applications

(U.S. and Foreign Patents Pending)

DBS: The Test Mixture r+

S O

s

O

O O

UK-112166

O

pKa 8.6/4.9 O

N O S N

F

F

F

F

O

F

O

F

Dofetilide

N

Cl

Mw 441

O

O O

s

N

UK-258300

+

N

s

Chiral N

O

O N S O

F

N

N

F O

N N

pKa 4.5 / 5.3

Midazolam

N

N

Mw 306

O

F

N

N

cLogP -0.11 N

Cl

HO HN

pKa 5.59

cLogP 3.93, Mw 325

O

Mw 730

F F

Fluconazole

UK-141495

N O

Propanolol pKa 13.84/9.14 cLogP 3.1 Mw 259

cLogP 3.94

O

N

O

pKa 6.1

N

N O

Mw 314 N

O

r

N

Mw 515

Cl

O s

cLogP 1.28

S

cLogP 4.2

cLogP 1.56

N

N

pKa 8.18/2.28

O

pKa NA, Mw 682

+

N

pKa 7 / 9.2

F

cLogP 2.94

O

Mw 365

Ranitidine

Candoxatril pKa 4.5

N

O

cLogP -0.05

r

O

O

O

Chiral

O

N

Gabapentin

pKa 3.7 cLogP 1.19 Mw 171

O

N

N N

O N

N

S

O

UK-338003 pKa 10.32/9.29 cLogP 0.69 Mw 506

DBS: Example Chromatogram • 25µL solution injection • Loading Mobile Phase: 2mM Ammonium Acetate/MeOH (90/10) + 0.1% TFA

Candoxatril (Lipophilic acid)

Ranitidine

DBS: Opti-Trap EXP™ Loading Capacity 900000 800000

Mean Peak Responce

700000 600000 500000

Optimal 11 compound mean

400000 300000 200000 100000 0 0

50

100

150

200

250

Injection Volume loaded to trap (µL)

• Enables larger injection volumes than conventional setup (linear increase in sensitivity up to 200µL injected).

• Reconstitution solvent and loading mobile phase can be optimised for retention purposes.

DBS: Polar Compounds 110 Percentage optimal responce

105 100 95 90

Optimal responce

85

Mean 4 most polar compounds

80

Mean all 11 compounds

75 70 65 60 0

50

100

150

200

250

Injection volum e loaded to trap (µL)

• • •

Data was plotted based on the response as a ”% optimal response”.

The very polar compounds gave an exponential increase of response with increased loading volume rather than a linear increase.

Overall, polar compounds displayed better sensitivity with larger load volumes.

DBS: Sample Extract Good peak shape and retention Blood spiked at 100 ng/mL. Spotted to dry, 3mm (~2.8 ÂľL) punch taken and extracted into 300 ÂľL methanol. Final sample concentration ~ 1ng/mL.

DBS: Matrix Effects

•

Matrix effect was looked at for all the compounds in the DBS extraction mix.

•

Matrix effects from the blood on the DBS card, the card itself and PPT fresh blood (25µL blood with 275µL Methanol) were investigated.

•

Most of the compounds gave a matrix effect that was no greater than usually reported experimental errors (±20%).

•

Those compounds giving greater matrix effects were still not unacceptably large.

Brain µ-Dialysis: In vitro Recovery of “Sticky CPDs” Compound A

Compound B

Compound C Propanolol (IS)

Mw 353.4 cLogP 0.7 pKA 9.99/6.89

Mw 488.3 cLogP 4.84 pKA 13.97/9.08

Mw 413.5 cLogP 5.29 pKA 12.77/0.52

Compound D

Compound E

Compound F

Mw 476.5 cLogP 2.29 pKA 14.92/7.42

Mw 357.4 cLogP 2.18 pKA 11.46/3.66

Mw 352 cLogP 5.29 pKA 4.85

Mw 259.3 cLogP 2.9 pKA 12.84/9.5

Brain µ-Dialysis: Example Chromatogram • 5 µL µ-dialysis sample (CSF) diluted with 200 µL reconstitution solvent.

• 100 µL injected onto the system.

Good peak shape & separation

Compound A

Compound D

Compound B

Compound E

Propanolol (IS)

Compound C

Compound F

Brain µ-Dialysis: System Carry-over

• Blank matrix injection following top standard.

• Strong solvent wash flushed through the trap during previous sample data collection.

0.47%

0.21%

N/A

0.22%

0.75%

0.23%

N/A

Small Peptide Assays (4-10 amino acids) •

•

In blood the octapeptide RFRP-3 (RFamiderelated peptide-3) degrades to a variety of smaller peptides (the hexapeptide, the pentapeptide and the tetrapeptide).

Assay requirement to simultaneously quantify the 8,6,5 and 4 amino acid peptides.

H3C

NH2

H3C

O N

O

NH

NH

•

High sensitivity (sub ng/mL) required due to low expected levels.

O

• •

The 8, 7 and 6 amino acid peptides well retained, 5 partially retained (with a % coming off at solvent front) and 4 not retained.

Signal at 4.4e5 for RFRP-3 for 100 ng/mL solution.

The best analytical conditions for RFRP-3 assay shown in next slide.

N

O

O

O H2N

O NH

O

NH2

Standard UHPLC: •

CH3

H3C

O

O NH H2N

NH NH NH H2N

Small Peptide Assays: Standard UHPLC X IC

o f + M R M

(6

p a ir s ) : 4 8 5 . 5 / 5 4 6 . 5

a m u

fro m

S a m p le

6

(S a m p le 0 0 3 ) o f F u ll s e t R F R P -3

_

1 0 - 6 - 9 . w iff ( T u r b o

S p ra y )

M a x . 4

1 .3 1

4 .3 e 5 4 .2 e 5

4.4e5

4 .0 e 5 3 .8 e 5 3 .6 e 5 3 .4 e 5 3 .2 e 5 3 .0 e 5 2 .8 e 5

Mob Ph A: 0.1 % Formic Acid

2 .6 e 5 2 .4 e 5 2 .2 e 5 2 .0 e 5 1 .8 e 5

Mob Ph B: Acetonitrile

1 .6 e 5 1 .4 e 5 1 .2 e 5 1 .0 e 5 8 .0 e 4 6 .0 e 4 4 .0 e 4 2 .0 e 4 0 .0

0 .2

0 .4

0 .6

0 .8

XIC of +MRM(6 pairs): 546.3/226.3 amu from Sample 6 (Sample003) of Full set RFRP-3 _ 10-6-9.wiff (Turbo Spray)

Max. 84

8440

1 .0

1 .2 T i m e , m in

1 .4

6

4.0e5

4

7500 7000 6500

1 .8

2 .0

2 .2

XIC of +MRM (6 pairs): 485.5/546.5 amu from Sample 6 (Sample003) of Full set RFRP-3 _ 10-6-9.wiff (Turbo Spray) 4.3e5 4.2e5

8000

1 .6

Max. 4 1.31

7

3.8e5 3.6e5

8

3.4e5 3.2e5

6000

3.0e5

5500

5

2.8e5

5000

2.6e5 2.4e5

4500

2.2e5

5

4000 3500 3000

2.0e5 1.8e5 1.6e5 1.4e5

2500

1.2e5

2000

1.0e5 8.0e4

1500

6.0e4

1000

4.0e4

500 0

2.0e4

0.22

0.24

0.26

0.28

0.30

0.32

0.34

0.36 0.38 Time, min

0.40

0.42

0.44

0.46

0.48

0.50

0.0

1.12

1.14

1.16

1.18

1.20

1.22

1.24

1.26

1.28 Time, min

1.30

1.32

1.34

1.36

1.38

1.40

1.42

Small Peptide Assays: Ion Pairing • Mobile phase altered to include 0.1% TFA to aid retention of the smaller peptides.

• The 8, 7, 6, 5 and 4 amino acid peptides all retained and separated.

• However large drop in signal intensity due to inclusion of TFA in mobile phase.

• Signal at 1.1 e5.

• The best analytical conditions for RFRP-3 assay shown in next slide.

Small Peptide Assays: Ion Paired Standard UHPLC X IC

o f + M R M

(7

p a ir s ) : 4 8 5 . 5 / 5 4 6 . 5

a m u

fro m

S a m p le

3 3

(0 .1 %

T F A ) o f F u ll s e t R F R P - 3

_

1 0 - 6 - 9 .w iff ( T u r b o

S p ra y )

M a x . 1

1 .5 0

1 .0 5 e 5 1 .0 0 e 5

1.1e5

9 .5 0 e 4 9 .0 0 e 4 8 .5 0 e 4 8 .0 0 e 4 7 .5 0 e 4 7 .0 0 e 4 6 .5 0 e 4

Mob Ph A: 0.1% TFA Mob Ph B: Acetonitrile

6 .0 0 e 4 5 .5 0 e 4 5 .0 0 e 4 4 .5 0 e 4 4 .0 0 e 4 3 .5 0 e 4 3 .0 0 e 4 2 .5 0 e 4 2 .0 0 e 4 1 .5 0 e 4 1 .0 0 e 4 5 0 0 0 .0 0 0 .0 0

0 .2

0 .4

0 .6

0 . 8

1 .0

XIC of +MRM(7 pairs): 546.4/226.3 amu from Sample 33 (0.1% TFA) of Full set RFRP-3 _ 10-6-9.wiff (Turbo Spray)

1 .2

1 .4

T i m e , m in

1 .6

1 .8

2 .0

2 .2

2 . 4

2 .6

XIC of +MRM(7 pairs): 485.5/546.5 amu from Sample 33 (0.1% TFA) of Full set RFRP-3 _ 10-6-9.wiff (Turbo Spray)

Max. 18

1.00e5

150

9.50e4

140

Max. 1

7

1.05e5

160

2 .8

1.50

9.00e4

6

8.50e4

130

8.00e4

120

7.50e4

110

8

7.00e4 6.50e4

100

5

6.00e4

90

5.50e4

80

5.00e4 4.50e4

70

4

60 50 40

4.00e4 3.50e4 3.00e4 2.50e4 2.00e4

30

1.50e4

20

1.00e4

10 0 0.16

5000.00 0.00

0.18

0.20

0.22

0.24

0.26

0.28

0.30 0.32 Time, min

0.34

0.36

0.38

0.40

0.42

0.44

1.26

1.28

1.30

1.32

1.34

1.36

1.38

1.40

1.42

1.44 1.46 Time, min

1.48

1.50

1.52

1.54

1.56

1.58

1.60

1

Small Peptide Assays: Ion Paired Trapping UHPLC X I C

o f

+ M

R M

( 8

p a ir s ) :

6 0 5 .6 /2 4 9 . 2

a m

u

f ro m

S a m

p le

4 3

( in j 2 5 0

lo a d

0 . 2 5

in

T F A

P

e p t id e

m

ix

in

3 0 %

re c o n

S

lo w

g ra d

o n

M

P 2 )

o f

M

IX

1 . ..

M a x .

7 .7 e 5

7 . 6 e 5

c p s .

1 . 6 1

7 .5 e 5

7.7e5

7 .0 e 5 6 .5 e 5 6 .0 e 5

Trap system set up:

5 .5 e 5 5 .0 e 5 4 .5 e 5

• Loading in 0.1%TFA

4 .0 e 5 3 .5 e 5 3 .0 e 5 2 .5 e 5

• Eluting in 0.1% HCOOH

2 .0 e 5 1 .5 e 5 1 .0 e 5 5 .0 e 4 0 .0

0 . 1

0 .2

0 . 3

0 .4

0 . 5

0 .6

0 . 7

0 .8

0 . 9 T im

XIC of +MRM(8 pairs): 605.6/249.2 amu fromSample 43 (inj 250 load 0.25 in TFA Peptide mix in 30% recon Slow grad on MP2) of MIX1...

1 .0 e , m

1 . 1

1 .2

1 . 3

1 .4

1 . 5

1 .6

1 . 7

1 .8

Leuprolide

8

Max. 7.6e5 cps.

1 . 9

in

XIC of +MRM(8 pairs): 605.6/249.2 amu fromSample 43 (inj 250 load 0.25 in TFA Peptide mix in 30% recon Slow grad on MP2) of MIX1...

Max. 7.6e5 cps.

1.61

1.00

7.5e5

0.95

6

0.90 0.85 0.80 0.75

7.0e5 6.5e5 6.0e5

0.70 5.5e5

0.65 0.60

5.0e5

0.55

Kisspeptin-10

4.5e5

0.50 0.45

4.0e5

0.40

5

0.35 0.30 0.25 0.20

3.5e5 3.0e5 2.5e5

0.15 2.0e5

0.10 0.05

1.5e5

0.00

4

-0.05 -0.10 -0.15 0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30 0.32 Time, min

0.34

0.36

0.38

0.40

0.42

0.44

0.46

0.48

0.50

1.0e5 5.0e4 0.0

1.28 1.30

1.32 1.34

1.36 1.38 1.40

1.42 1.44

1.46

1.48 1.50 1.52 1.54 Time, min

1.56

1.58 1.60

1.62 1.64 1.66

1.68 1.70 1.72

1.74 1.76

Small Peptide Assays: Trapping System •

The 8, 6, 5 and 4 amino acid peptides were all retained.

•

Signal at 7.7e5.

•

With the TFA not pumping into MS, the signal intensity did not suffer and in fact due to: – a) the ability to wash away some of the protein injected into the system;

and – b) the greater linearity of loading volume on the trap;

signal intensity from the same extract concentration almost doubled.

•

Additionally the endogenous peptide kisspeptin-10 and the modified peptide leuprolide were also retained.

Robustness •

All the work to date has been performed using a single Halo™ C18, 4.6 x 5mm Opti-Trap EXP™.

•

We have not experienced issues of leaking from the trap and no notable drop in retention, sensitivity or peak performance from the trap.

•

Additionally the UHPLC column has shown no notable increase in backpressure or deterioration of peak shape after injection of ~ 5000 biological samples, the majority of which dirty methanol crashed blood or ACN crashed plasma samples.

•

Previous work performed using the Eclipse XDB-C18 3 x 50 mm,1.8µ column UHPLC columns on the same systems has shown then to be prone to blocking and over pressuring after injection of multiple PPT samples.

•

The use of the Opti-Trap EXP™ enables routine PPT sample preparation, also desirable for retaining the most polar analytes such as metabolites which could be lost when performing sample extraction.

Conclusions •

A variety of significant gains can be made by integrating the Opti-Trap EXP™ technology into a UHPLC system.

•

The UHPLC system can be set up as a generic system to capture all analytes of interest (including more polar metabolites) or can be optimised for a single component by varying the loading mobile phase and the chemistry of the trap cartridge packing material.

•

The loading mobile phases can carry additives that you wouldn’t use with MS due to causing ion suppression (e.g. TFA, derivatisers, etc.).

•

Dirty sample extracts (e.g. PPT blood and plasma) can be directly injected without fear of blocking the narrow bore sub 2-micron particle column.

•

Column lifetime is greatly increased (ca 5000 samples introduced into the system with no need to replace either the trap cartridge or the analytical column).

Acknowledgements • • • • • •

Julian Haynes (PDM, Pfizer) Paul Turnpenny (PDM, Pfizer) Pooja Sood (University of Leicester) Mike J.E. Stephens (Optimize Technologies Inc.) Douglas W. Ford (Optimize Technologies Inc.) Hashim Javaid (Crawford Scientific Limited)