Determination of Plasma Protein Binding in Support of Early ADME Drug Discovery Efforts by LC/MS/MS

John P. Walsh, Reginald O. Angeles, Kelly M. Jenkins, Robyn A. Rourick

Deltagen Research Laboratories, Suite 400, 4570 Executive Drive, La Jolla, CA 92121
Equilibrium Dialysis (Multi-Equilibrium Dialyzer): Figure 3. Correlation between Refractive Index and Buffer Fraction Equilibrium Dialysis (Equilibrium Dialyzer-96):
OVERVIEW METHODS The low-throughput method was used as the standard Vertical lines represent the average volume shift observed for all four compounds with the
respective dextran concentration.
• Corresponds to literature and standard method
for other methods to be compared. for compounds with moderate to high binding
Nadolol, propranolol, verapamil and warfarin were 1.3520

• Introduction prepared in 10mM DMSO stocks and stored at 4°C. • Use of large volumes (1000µL) for each sample -17.5% • Precision of the method was acceptable for all
~ Background For use in the assays, the DMSO stock was diluted to 1.3500 compounds except nadolol
• Labor-intensive and time-consuming
• Methods 100µM and 1µM with methanol and 100mM potassium
• Membrane dislodged on approximately 15% of

Refractive Index
-3%

~ SIM vs. MRM

phosphate buffer or pooled human plasma, • Not amenable to automation
the samples

5% Dextran
1.3480

respectively. Calibration standards were prepared from

~ Volume Shift Equilibrium Dialysis (Equilibrium Dialyzer-96): 10.5%
the 1µM buffer and plasma stocks by serial dilution. All Ultrafiltration (Microcon-96):

3.5% Dextran
~ Multi-equilibrium Dialyzer
plasma samples were incubated for 5 hours, The high-throughput method was evaluated for
1.3460

~ Equilibrium Dialyzer-96 • Inconsistent accuracy when compared to the

precipitated with methanol, the supernatant

2.5% Dextran
~ Microcon-96 precision and accuracy.
concentrated and resuspended in dialysis buffer (see 1.3440
30%
literature values and the standard equilibrium
• Results Figure 1). • Use of smaller volumes (150µL) for each sample dialysis assay

0% Dextran
~ SIM vs. MRM
SIM vs. MRM (API 4000 triple quadrupole): • 96-well format increases throughput and assay 1.3420
• Unacceptable precision for nadolol and
~ Volume Shift
set-up verapamil
~ Binding Selected ion monitoring (SIM) was compared to multiple
• Conclusion reaction monitoring (MRM) for sensitivity and selectivity. • Can be semi-automated 1.3400 • Location of the sample on the plate contributed
-20.0 -10.0 0.0 10.0 20.0 30.0 40.0 50.0
to the widely variable precision
• Buffer and plasma samples were analyzed by Ultrafiltration (Microcon-96): Buffer Fraction (%)
both modes • The plates are not compatible with standard 8-
The high-throughput method was evaluated for channel pipettes or standard footprints
• MRM transitions were optimized prior to sample Figure 4. Percent Bound for Test Compounds Determined by the
precision and accuracy.
INTRODUCTION analysis
• Use of smaller volumes (250µL) for each sample
Listed Analysis Techniques
Error bars are shown for all techniques with n≥12. Literature error bars represent the
Volume Shift (Mark II Refractometer):
• 96-well format is not compatible with standard 8-
range of data reported previously.
100.0
CONCLUSION
The assessment of plasma protein binding for potential drug For equilibrium dialysis methods, the osmotic pressure channel pipettes
candidates in discovery programs has become increasingly differences between the phosphate buffer and the
• Can be fully automated SIM vs. MRM
important. It is generally accepted that only unbound drug or free plasma caused a portion of the buffer to cross the 80.0

fraction is responsible for pharmacological effects, thus the extent dialysis membrane to the plasma portion. High • MRM will be used when analyzing samples for
of binding to plasma proteins can have a profound impact on molecular weight dextran was used to reduce this the specificity and sensitivity it provides
therapeutic potential. Equilibrium dialysis is currently the most

% Bound
volume shift. 60.0
Volume Shift
accepted method for precise measurements. However, this
• Dextran concentrations of 0%, 2.5%, 3.5% and RESULTS
approach is labor intensive and limited to a throughput of 10 • Dextran is necessary to improve throughput by
5% w/v were evaluated
compounds per week for a single dialyzer unit. Additional plate 40.0
removing the refractive index measurement
based approaches include ultracentrifugation and ultrafiltration. • Volume shift was measured by refractive index
• A working buffer solution of 3-3.5% w/v dextran
While these methods provide less precise measurements, they
• Plasma samples were measured from both Figure 2. Chromatograms and Calibration Curves of Propranolol 20.0 should make any volume shifts negligible
have found utility as front-line screens due to their enhanced
equilibrium dialysis methods Analyzed by SIM and MRM
throughput. This poster presents information for an ultrafiltration Standard Equilibrium Dialysis
Representative chromatograms are shown for the LLOQ of the corresponding method
method in addition to a standard and plate based equilibrium Figure 1. Preparation Scheme for Equilibrium Dialysis Sample Name: "cs125_plasma" Sample ID: ""
Peak Name: "Peak 1" Mass(es): "TIC"
File: "Data041602_Verapamil_SIM_Plasma.wiff" Sample Name: "cs7_plasma" Sample ID: "" File: "Data041602_Verapamil_MRM_Plasma.wiff"
Peak Name: "Verapamil" Mass(es): "455.0/165.3 amu"
0.0
• The standard method offers good precision and
dialysis assay.
Comment: "" Annotation: "" Comment: "" Annotation: ""
Sample Index: 6 Sample Index: 2
Sample Type: Standard Sample Type: Standard

Nadolol Propranolol Verapamil Warfarin

1.5e7 2.01 1.30
Concentration: 125. nM Concentration: 7.80 nM 1500

Samples
Calculated Conc: 88.4 nM 1.5e7 Calculated Conc: 7.61 nM
Acq. Date: 04/17/02 Acq. Date: 04/17/02 1480

accuracy at the cost of throughput

Acq. Time: 06:29:21 AM 1.5e7 Acq. Time: 08:24:59 AM
1460
Modified: Yes 1.5e7 Modified: Yes

SIM
Bunching Factor: 3 Bunching Factor: 3 1440
Noise Threshold: 4.33e4 cps 1.4e7 Noise Threshold: 38.25 cps

MRM
Area Threshold: 2.16e5 cps Area Threshold: 191.24 cps 1420
Num. Smooths: 7 1.4e7 Num. Smooths: 7

10mM DMSO stock

RT Window: 30.0 sec RT Window: 30.0 sec 1400
Expected RT: 2.01 min 1.4e7 Expected RT: 1.32 min 1.34
Sep. Width: 0.20 Sep. Width: 0.20 1380
Sep. Height: 0.01 1.4e7 Sep. Height: 0.01
Exp. Peak Ratio: 5.00 Exp. Peak Ratio: 5.00 1360
Exp. Adj. Ratio: 4.00 1.4e7 Exp. Adj. Ratio: 4.00

Presently, we are applying the more precise equilibrium dialysis Microcon-96

Exp. Val. Ratio: 3.00 Exp. Val. Ratio: 3.00

Multi-equilibrium dialyzer
1340
Use Relative RT: No 1.3e7 Use Relative RT: No
1320
Int. Type: Manual 1.3e7 Int. Type: Manual
Retention Time: 1.33 min Retention Time: 1.30 min 1300
Area: 4.23e+007 counts 1.3e7 Area: 8.70e+003 counts

• Should be used as a follow-up assay when more

Height: 5.62e+006 cps Height: 1.48e+003 cps 1280
Start Time: 1.19 min 1.3e7 Start Time: 1.24 min
End Time: 1.46 min End Time: 1.53 min 1260
1.3e7
1240
1.2e7
1220

assay to assess protein binding of lead candidates. Plasma

1.2e7

1.2e7

1.2e7
1200

1180

1160 Equilibrium dialyzer-96 Literature

exact measurements are necessary
1.2e7
1140
1.1e7

100µM MeOH stock 1.1e7

1.1e7
1120

1100

protein binding determinations are made with a Multi-Equilibrium

1.1e7 1080

1.1e7 1060

1040
1.0e7
1020
1.0e7

SIM vs. MRM (API 4000 triple quadrupole):

1.0e7 1000

9.8e6 980

Dialyzer (Harvard Bioscience). Four highly characterized High Throughput Equilibrium Dialysis
9.6e6 960

9.4e6 940

9.2e6 920

9.0e6 900

880

1µM buffer 1µM plasma

8.8e6

8.6e6 860

8.4e6 840

compounds, which include nadolol, propranolol, verapamil and 8.2e6 820

• Selected ion monitoring (SIM) showed significant

8.0e6 800

working solution working solution 7.8e6 780

• Has reasonable precision and accuracy with

Intensity,cps

Intensity,cps
7.6e6 760
1.32
7.4e6 740

7.2e6 720

warfarin were chosen for comparison since they cover a broad

7.0e6 700

interference
6.8e6 680

6.6e6 660

moderate throughput
6.4e6 640

6.2e6 620

6.0e6 600

580

range of plasma protein binding.

5.8e6

5.6e6 560

5.4e6 540

5.2e6 520

5.0e6 500

Equilibrium Check Binding Sample • Multiple reaction monitoring (MRM) had reduced
4.8e6 480

4.6e6 460

• Can be used as a screen to “bin” compounds

4.4e6 440

4.2e6 420

4.0e6 2.78 400

3.8e6 380

interference and a lower limit of quantitation

3.6e6 360

3.4e6 340
1uM plasma solution

according to their approximate binding

2.58
3.2e6 320

Prepare calibration Prepare calibration 3.0e6 300

1uM buffer solution

2.8e6 280

260

Table 1. LC/MS Assay Parameters for all Methods

2.6e6

2.4e6 240

standards standards
phosphate buffer

phosphate buffer

2.2e6 220

2.0e6 200

1.8e6 180

• Both modes provided a linear response to

1.99 2 . 0 1
1.6e6 160

1.4e6 140

Ultrafiltration
1.2e6 120 1.48

1.0e6 100 1.52

2.79
8.0e5 80 2.13 2.16 2.81
2.67 2.71
6.0e5 60 1.64
1.73 2.27 2.52
2.44

concentration once a S/N threshold was passed

4.0e5 40 1.77
0.97
2.0e5 0.39 20
0.18 0.36 0.59 0.72 0 . 7 9 0.90 0 . 9 3 1 . 0 5 1.11 2.87 2.93
8.33e-5 0.07 0.13 0.22 0.54 1.22

HPLC
0.0 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 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
Time, min Time, min
Chamber 2:

Chamber 1:
Chamber 1:

Chamber 2:

Apparatus Shimadzu LC-10 ADVP pumps; HTS PAL autosampler; Valco Divert Valve Verapamil_SIM_Plasma.rdb (Peak 1): "Linear" Regression ("1 / x" weighting): y = 3.99e+005 x + 7.02e+006 (r = 0.9761) Verapamil_MRM_Plasma.rdb (Verapamil): "Linear" Regression ("1 / x" weighting): y = 1.14e+003 x + -0.686 (r = 0.9975)
• Unacceptable precision and accuracy for all four
A: 0.05% Formic acid in Water 3.8e8
1.19e6

Mobile Phase B: 0.04% Formic acid in Acetonitrile

3.7e8

SIM
1.15e6

Volume Shift (Mark II Refractometer): compounds

MRM
3.6e8
1.10e6
3.5e8

99% A: 1% B for 0.3min 3.4e8

3.3e8
1.05e6

Gradient
Ramp to 5% A: 95% B at 2.5min
Hold for 0.1min
3.2e8

3.1e8

3.0e8
1.00e6

9.50e5 • Samples without any dextran added to the buffer • Lack of standard 96-well footprint challenges
automation strategies
2.9e8

component showed a significant volume shift

9.00e5

Re-equilibrate 2.8e8

2.7e8
8.50e5

Flow rate 0.500mL/min Column Waters Xterra MS C18 2.1x30mm 2.6e8

2.5e8
8.00e5

Injection Volume 20uL Divert time 0.3min Precipitate: • Will not be used for the screening of compounds
2.4e8 7.50e5

Mass Spectrometry 100µL plasma

2.3e8

2.2e8

2.1e8
7.00e5 • A dextran concentration of 3.5% w/v had a
6.50e5

Apparatus PE Sciex API 4000 triple quadrupole mass spectrometer

+ -
300µL MeOH
2.0e8

1.9e8

1.8e8
6.00e5
negligible effect on the volume shift Overall Strategy
Ionization Type APCI (Warfarin: APCI ) Source Temp 500°C 1.7e8

1.6e8
5.50e5

5.00e5

Curtain Gas 20 Nebulizer Current 3

Nadolol Propranolol Verapamil Warfarin
Plate for injection Determine RI Shake 5min
1.5e8

1.4e8

1.3e8
4.50e5

4.00e5
• High molecular weight dextran can be used to • Use of the Equilibrium Dialyzer-96 for screening
counteract volume shifts of equilibrium dialysis
1.2e8

MRM Transition 310.4 - 254.1 260.1 - 155.1 455.0 - 165.3 307.0 - 160.8 1.1e8 3.50e5

Collision Energy 30 35 40 40
1.0e8

9.0e7
3.00e5
with a follow-up assessment by the standard
Centrifuge 10min 8.0e7

7.0e7

6.0e7
2.50e5

2.00e5
methods assay for leads
5.0e7
1.50e5
4.0e7

3.0e7 1.00e5

Remove 200µL supernatant

Equilibrium Dialysis (Multi-Equilibrium Dialyzer):
2.0e7
5.00e4
1.0e7

and evaporate to dryness 0.0

0 100 200 300 400 500

Concentration, nM
600 700 800 900 1000
0.00
0 100 200 300 400 500
Concentration, nM
600 700 800 900 1000
References
Reconstitute 150µL • Corresponds accurately to the literature data 1. Piotrovskii, VK, et al., J. Chromatography, 1984, 309(2): 421-425.
phosphate buffer
• Data obtained has a good degree of precision 2. Edeki, T., et al., American J. Therapeutics, 1996, 3(9): 611-615.
Plate for injection Shake 5min
3. Chan, E., et al., Br. J. Clin. Pharmacology, 1994, 37(6): 563-569.