Dried Blood Spot Analysis From The Clinic To The Laboratory · Dried Blood Spot Analysis –From...

©2011 Waters Corporation 1

Dried Blood Spot Analysis – From The Clinic To The Laboratory

Joanne Mather,

Pharmaceutical Life Sciences

Waters Corporation


From The Clinic To The Lab

Blood Spot Sampling First Used In Neonatal Screening


Dried Blood Spots – Timeline of Implementation

>10 yrs ago

DBS were exclusively used for neonatal, infectious disease screening, therapeutic drug monitoring

5 Years Ago

2 manuscripts – DBS to discovery-stage PK and metabolite ID

(Merck Frost)

Present Day

Potential application to routine drug development was realized within GlaxoSmithKline in 2006

Quickly saw the broadly applicable advantages offered by DBS

Other pharmaceutical companies (sanofi-aventiset. al.) becoming heavily involved with DBS (as well as CROs and vendors)

Dried Blood Spots have been around for over 40 years


DBS Uptake

On Board

To date GSK has developed >150 validated DBS methods for nearly 75 compounds >200 studies supported using DBS >10 compounds have reached phase 1 pre-clin GLP studies


Issues and Advantages of Dried Blood Spot Analysis

[Card based sample collection]

— Reduced animal usage/ cost/ 3 R‘s

— Lower shipping costs

— Reduced compound needed for tox studies

— Improved data- PK data from toxicology animals

[Small sample volumes 10-20µL]

— Assay sensitivity

[Background card interference]

— More complicated methods development

[Resolution from metabolites and matrix needed]

— High LC separation efficiency


Analytical Issues

Non – liquid format

Current work flow will not work with this format

Current approach requires punching, either manual or

automated

Card background may cause issues

Matrix effects from blood components

— Resolution from metabolites and matrix needed for quality


Analytical Issues



Current approach require punching, either manual or

automated




Blood Spotting

Aliquot 15μL blood per spot

—3 spots per sample plus

‗spare‘

—Using a pipette or

capillary

o Do NOT allow tip to

touch card surface!

Dry for 2 hours at room

temperature

Ship & store in sealable

bags containing desiccant


Sample Prep & Analysis

Analytical sample obtained by

punching small circular disc

(typically 3 mm) from centre

of DBS

—Manually

—Automation – BSD1000

Extract disc in organic solvent

(typically methanol)

containing internal standard

Quantification by validated LC-

MS/MS assay


Chromatography Requirements

Long separation

Short Separations


Analysis with LC/MS/MS

Quantification by validated LC-MS/MS assay

Use of UPLC:

• Increased sensitivity

• Increased resolution

• Increased speed of analysis

Time0.00 0.50 1.00 1.50 2.00

%

0

0.00 0.50 1.00 1.50 2.00

%

0

0.00 0.50 1.00 1.50 2.00

%

0

0.00 0.50 1.00 1.50 2.00

%

0

170210_JoM_DBS_138 2: MS2 ES+ TIC

1.50e10

1.91

1.791.67

0.810.610.15 1.301.12

2.13 2.26

170210_JoM_DBS_138 1: MRM of 3 Channels ES+ 325 > 297 (Alprazolam Metabolite)

2.55e7

0.74

170210_JoM_DBS_138 1: MRM of 3 Channels ES+ 314.2 > 286.1 (AlprazolamD5)

4.15e7

0.80

170210_JoM_DBS_138 1: MRM of 3 Channels ES+ 309.2 > 281 (Alprazolam)

5.20e7

0.81


Time0.00 0.50 1.00 1.50 2.00

%

0

0.00 0.50 1.00 1.50 2.00

%

0

0.00 0.50 1.00 1.50 2.00

%

0

0.00 0.50 1.00 1.50 2.00

%

0

170210_JoM_DBS_138 2: MS2 ES+ TIC

1.50e10

1.91

1.791.67

0.810.610.15 1.301.12

2.13 2.26

170210_JoM_DBS_138 1: MRM of 3 Channels ES+ 325 > 297 (Alprazolam Metabolite)

2.55e7

0.74

170210_JoM_DBS_138 1: MRM of 3 Channels ES+ 314.2 > 286.1 (AlprazolamD5)

4.15e7

0.80


5.20e7

0.81

m/z80 100 120 140 160 180 200 220 240 260 280 300 320

%

0

100

170210_JoM_DBS_040 3 (0.790)

3.79e7296.81

278.81175.81 251.76226.76

324.86

PIC Scan Acquisition

Rapid Mode Switching Precursor Ion Confirmation Scan

Confirm peak identity in the presence of a complex matrix in one analytical run.


Analytical Issues




automated




Dried blood spot microvolume sampling for DMPK

Three types are available (Whatman, GE),

which have different chemistries.

— FTA DMPK-A (treated)

— FTA DMPK-B (treated)

— FTA DMPK-C (untreated)

FTA DMPK A & B cards contain proprietary

chemical mixtures that lyse cells, inactivate

pathogens and denature degradative

enzymes and other proteins.

FTA DMPK-C cards contain only cellulose.

http://www.whatman.com/dmpk.aspx


Rapid MS to MRM Switching


Using RADAR to Evaluate Card Type To Be Used

DMPK ADMPK A

DMPK BDMPK B

DMPK CDMPK C

Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

010210_JoM_DBS_cards_013 2: MS2 ES+ TIC

5.98e91.18

1.030.22

0.32

0.94

1.55

1.421.29

2.411.911.76 2.08 2.24


5.98e9

1.54

0.98

0.22 0.63

1.28

1.40

1.63

2.341.78

1.88 2.052.24

2.41


5.98e91.54

0.23 1.45

1.281.030.90

0.58

2.34

1.73

1.872.07

DMPK ADMPK A

DMPK BDMPK B

DMPK CDMPK C

Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100


5.98e91.18

1.030.22

0.32

0.94

1.55

1.421.29

2.411.911.76 2.08 2.24


5.98e9

1.54

0.98

0.22 0.63

1.28

1.40

1.63

2.341.78

1.88 2.052.24

2.41


5.98e91.54

0.23 1.45

1.281.030.90

0.58

2.34

1.73

1.872.07

Full scan blank card -background signal


Using RADAR to Evaluate Card Type To Be Used

DMPK ADMPK A

DMPK BDMPK B

DMPK CDMPK C

Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

020210_JoM_DBS_cards_010 1: MRM of 3 Channels ES+ 309.2 > 281 (Alprazolam)

9.61e5

1.10


9.61e5

1.09


9.61e5

1.09

DMPK ADMPK A

DMPK BDMPK B

DMPK CDMPK C

Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100


9.61e5

1.10


9.61e5

1.09


9.61e5

1.09

AnalyteResponse


Analytical Issues




automated





Gain adequate resolution from matrix interferences and

other analytes/ metabolites

Maximize sensitivity

Maintain or improve productivity

Removal of matrix components

Matrix – The Challenge


RADAR - Monitor matrix complexity

Time0.50 1.00 1.50 2.00

%

0

0.50 1.00 1.50 2.00

%

0

170210_JoM_DBS_135 2: MS2 ES+ TIC

1.54e10

2.01

1.90

1.561.42

1.28

2.24


6.36e7

1.42

Time0.50 1.00 1.50 2.00

%

0

0.50 1.00 1.50 2.00

%

0

170210_JoM_DBS_138 2: MS2 ES+ TIC

1.50e10

1.91

1.79

1.67

1.61

0.810.611.30

2.132.26


5.20e7

0.81

Gradient A Gradient B

Full Scan

MRMResolved more effectively from the endogenous blood and card matrix peaks


Alternate Approach for Calculating Matrix Factors

AN and ISinjected through the column

2) Inject Solvent Blank

3) Inject Extracted Matrix Blank

LC

Column

1) Determine Analyte Profile

Solvent Blank injected with post column infusion of AN and IS

Extracted Blank injected with post column infusion of AN and IS

MRM Signal

AN and IS infused

post-column

Analyte = ANInternal Standard = IS


Automating Matrix Factor Calculations: Integrated Intellistart Fluidics


MassLynx TargetLynx Simplifies Calculating The Matrix Effects

Results Presentedin Final Report

TargetLynx™ automaticallycalculates matrix effects

from 6 injections


Removal of Phospholipids


Sensitivity and Robustness

Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00

%

0

100

040210_JoM_DBS_QC_216 MRM of 3 Channels ES+ 309.2 > 281 (Alprazolam)

2.57e4

1.47

1.351.21

1.82

1.641.90

2.25

040210_JoM_DBS_QC_301 MRM of 3 Channels ES+ TIC

2.57e4

2.370.37 2.62

Concentration

(ng/mL) Average Std Dev

QC 1 0.25 0.23 2.09

QC 2 0.4 0.38 4.84

QC 3 3 3.25 2.56

QC 4 30 31.9 4.97

QC 5 300 300.3 3.81

Compound name: Alprazolam

Correlation coefficient: r = 0.999680, r^2 = 0.999360

Calibration curve: 0.00110984 * x + 0.000605461

Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )

Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

ng/mL0 50 100 150 200 250 300 350 400 450 500

Re

sp

on

se

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

0.400

0.450

0.500

0.550

Alprazolam (benzodiazepine)

100pg/mL - 500ng/mL

Alprazolam100pg/mL

Blank

Published- Journal Bioanalysis Feb 2011, Vol 3:4 p411-420Rapid Analysis of DBS samples with sub 2µm LC/MS/MS Mather J et al.


Sitamaquine From Dried Blood Spot Compound name: Sitamaquine

Correlation coefficient: r = 0.999910, r^2 = 0.999820

Calibration curve: 15.233 * x + 38.3894

Response type: External Std, Area

Curve type: Linear, Origin: Exclude, Weighting: Null, Axis trans: None

ng/mL0 50 100 150 200 250 300 350 400 450 500

Re

sp

on

se

0

2000

4000

6000

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

22FEB2010_PR_118 MRM of 2 Channels ES+ 344.3 > 271.2 (Sitamaquine)

5.44e3

1.41

1.000.78 1.12 1.31

1.841.53

Sitamaquine (WR-6026) is an orally active 8-

aminoquinoline analog for the potential treatment of

visceral leishmaniasis (also known as kala-azar, black fever, and Dumdum fever).

LLOQ of 50pg/mL with a linear dynamic range of 4 orders of magnitude.

LLOQ of 50pg/mL


Heart Cut Configuration – 2D UPLC/MS/MS

Trapping – Decrease Matrix Effects and Increase Sensitivity

At-Column Dilution –Inject Large Volume Samples in Strong Solvent

No evaporation & reconstitution


2 Dimensional Dried Blood Spot Analysis of Rosuvastatin

No ―At Column Dilution‖ With ―At Column Dilution‖

• Direct injection of methanol extract from DBS sample• 80uL injection of methanol• At column dilution


2 Dimensional UPLC/MS/MSBioanalysis

Blank100pg/mL


Conclusions

Dried Blood Spots offer a significant scientific and expense advantage.

The lower sample volume derived from the dried blood spots and the background card matrix poses a significant analytical challenge.

Information rich LC/MS/MS (RADAR) can simplify the process of DBS analysis/ method development.

Analysis times as low as 3 minutes possible.

2D LC to remove Matrix components (trap) and allow injection of large volume samples in high organic solvent.


Acknowledgements

Paul Rainville

Jennifer Simeone

Robert Plumb

David Little

Jessalynn Wheaton

Erin Chambers

Christopher Evans – GSK

Neil Spooner - GSK

Ian Wilson - Astra Zeneca

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