The use of Dried Plasma Spots (DPS) and Dried Urine Spots (DUS) for

LC/MS/MS assays

Matt Barfield

PTS DMPK, GlaxoSmithKline, Ware, UK

(Matthew.Barfield@gsk.com)

Status of DBS Sampling at GSK*

231 DBS bioanalytical methods validated for 105

compounds

Studies Completed

Studies Analytes Samples

Pre-Clinical

nonGLP168 85 20,357

Pre-Clinical GLP 86 35 18,829

Clinical 9 15 4,027

* as of 20 May 2010 (does not include incurred sample reanalysis)

Have the benefits of DBS been realised at GSK

Safety

Assessment

Refinement

No warming of

rat or miceReduction

Non GLP TK Satellites

removed

70uL bleeds UK

45uL bleeds US

Data quality

Serial not composite

Test substance

Ease & Speed

Have the benefits been realised - cont.

Clinical

Simplified bleeding &

processing

Cost of

shipping/storage

Paediatric

studies

Have the benefits been realised - cont.

Bioanalysis

No real advantage

yet!!

Direct Elution Direct extraction

What can we do to help Clinical?

• Cost savings for clinical will come after phase 1

-Shipping and storage

GSK estimate if all TK and Clinical samples utilised

card technologies we could save between 5 and 8

million pounds a year

• We can’t change easily from plasma to blood

• Why not use Dried Plasma Spots?

• Why not use Dried Urine Spots?

DPS advantages

DPS– Cost savings

– Ease

– Utilize new technologies

Direct elusion/ionisation

DUS– Cost savings

– Scientific

– Solubility

– Homogeneity

– Ease

– Utilize new technologies

Direct elusion/ionisation

– Its much nicer

Full DPS validation for Paroxetine in human plasma

O

N

F

O

O

Chiral

H

• 20uL spots

• 6mm punch

• 100ul I.S. extract (70:30 MeOH/H20)

• Shake and decant supernatant

• Inject on reverse phase

chromatography

• LC/MS/MS API-5000

•Assay range

•0.2-200ng/mL

Validation contents

Linearity

– Duplicate calibration lines (front & back)

Precision, accuracy, sensitivity & reproducibility

– 5 concentrations, 6 replicates

– 3 occasions for 1st pre-clin species & human

– Single occasion for subsequent species

On card stability

– 2 concentrations (VC2 & 4), 6 replicates desiccated at room temp for 35days

Processed sample stability

– Re-inject validation QCs with fresh calibrants after storage at room temp for 24 hrs

Selectivity

– Total blanks & blanks from 6 different sources

Assay robustness to pipetting error (10 - 20μL)

– 2 concentrations (VC2 & 4), 6 replicates

– Apply precision / accuracy acceptance criteria

Dilution with control matrix extract

Recovery & suppression

Assessment of indicating papers

The use of indicating papers

Accuracy, Precision & Sensitivity

Nominal

concentration

(ng/mL)

0.2 0.8 10 160 200

Mean

concentration

(ng/mL)

0.22 0.81 9.53 167.26 212.59

SD. 0.03 0.08 0.67 11.21 5.88

Overall precision

(%CV)

13.2 10.1 7.0 6.7 2.8

Average

accuracy (%

bias)

9.0 0.9 -4.7 4.5 6.3

Average intra-run

precision (%)

12.9 9.0 7.4 6.9 2.5

Inter-run

precision (%)

3.8 5.4 Negligible Negligible 1.4

Nominal

concentration

(ng/mL)

0.2 0.8 10 160 200

mean

concentration

(ng/mL) 0.18 0.88 9.60 161.82 199.51

SD. 0.01 0.05 0.63 9.14 11.13

precision (%CV) 6.4 5.9 6.5 5.6 5.6

accuracy (%

bias) -10.7 10.2 -4.0 1.1 -0.2

DPS 226 paper

3 runs

A developmental grade of

untreated FTA™ DMPK

1 run

Plasma spot size using 226

Nominal

concentration

0.8 ng/mL 160 ng/mL

Volume of human

blood spotted onto

FTA paper

15 μL 20 μL 25 μL 15 μL 20 μL 25 μL

Mean concentration

(ng/mL)

0.76 0.83 0.76 167.0 172.8 167.7

SD. 0.061 0.076 0.042 6.4 7.8 13.1

Precision (%CV) 8.13 9.13 5.59 3.80 4.50 7.80

Accuracy (% bias) -5.5 4.0 -5.0 4.4 8.0 4.8

Difference from

20μL Spot (%)

-9.2 -8.7 -3.4 -3.0

Post column infusion

0.5 1.0

Time, min

0.0

0.5xe4

1.0e4

1.5e4

2.0e4

2.5e4

2.8e4

226 Paper

Indicating FTA™

Plasma

Paroxetine extract

Real pooled sample comparison – DPS v Plasma

0,00

0,50

1,00

1,50

2,00

2,50

3,00

3,50

4,00

4,50

5,00

0 2 4 6 8 10 12 15 18 24 32 48 72 120 168

Co

ncen

trati

on

(n

g/m

L)

Time (hours)

Type of Sample

Fresh Plasma Dried Plasma Spot

AUC (ng.hr/mL) 83.52 76.56

Cmax (ng/mL) 3.51 3.75

Tmax (hr) 12 12

DUS Data – precision & accuracy 1 run

Concentration (ng/mL) Mean Standard Deviation %CV Accuracy

0.1 0.09763 0.007428 7.6 97.6

0.4 0.377821 0.022761 6.0 94.5

5 4.720766 0.379967 8.0 94.4

40 36.509379 1.787507 4.9 91.3

50 48.024223 1.244762 2.6 96.0

Real pooled sample comparison – DUS v Urine

Time (hour)

Concentration

(ng/mL)

0

2

4

6

8

10

12

0 1 2 3 4 5 6 7

DUS

Urine

Sitamaquine : Direct Elution and Manual extraction

XIC of +MRM (2 pairs): 344.4/271.1 amu from Sample 5 (BLOOD STD 100) of BLOOD PLASMA URINE SPOT COMP SITAMAQUINE 226 07MA... Max. 4.7e5 cps.

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

4.0e5

4.2e5

4.4e5

4.6e5

Inte

ns

ity

, c

ps

0.74

XIC of +MRM (2 pairs): 344.4/271.1 amu from Sample 13 (PLASMA STD 100) of BLOOD PLASMA URINE SPOT COMP SITAMAQUINE 226 07M... Max. 1.3e6 cps.

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7Time, min

0.00

5.00e4

1.00e5

1.50e5

2.00e5

2.50e5

3.00e5

3.50e5

4.00e5

4.50e5

5.00e5

5.50e5

6.00e5

6.50e5

7.00e5

7.50e5

8.00e5

8.50e5

9.00e5

9.50e5

1.00e6

1.05e6

1.10e6

1.15e6

1.20e6

1.25e6

Inte

ns

ity

, c

ps

0.73

XIC of +MRM (2 pairs): 344.4/271.1 amu from Sample 22 (URINE STD 100) of BLOOD PLASMA URINE SPOT COMP SITAMAQUINE 226 07MA... Max. 1.4e6 cps.

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

1.3e6

1.4e6

Inte

ns

ity

, c

ps

0.73

Paracetamol : Direct Elution and Manual extraction

XIC of +MRM (2 pairs): 151.9/110.0 amu from Sample 8 (BLOOD STD 100) of BLOOD PLASMA URINE SPOT COMP PARACETAMOL CAMAG ... Max. 1.9e6 cps.

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

1.3e6

1.4e6

1.5e6

1.6e6

1.7e6

1.8e6

1.9e6

Inte

ns

ity

, c

ps

1.54

XIC of +MRM (2 pairs): 151.9/110.0 amu from Sample 15 (PLASMA STD 100) of BLOOD PLASMA URINE SPOT COMP PARACETAMOL CAMA... Max. 1.9e6 cps.

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

1.3e6

1.4e6

1.5e6

1.6e6

1.7e6

1.8e6

1.9e6

Inte

ns

ity

, c

ps

1.54

XIC of +MRM (2 pairs): 151.9/110.0 amu from Sample 24 (URINE STD 100) of BLOOD PLASMA URINE SPOT COMP PARACETAMOL CAMAG ... Max. 1.6e6 cps.

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

1.3e6

1.4e6

1.5e6

1.6e6

Inte

ns

ity

, c

ps

1.55

Summary

The use of DPS and DUS could accelerate the cost benefit offered by

card technologies

Many of the benefits of DBS apply to DPS and DUS

DBS is still the preferred option but for late stage compounds and

compounds that are not applicable for DBS then DPS has its uses

DUS in my opinion is better scientifically and practically than urine

Both DUS and DPS can utilise new technologies which offer

advantages to the bioanalyst

Acknowledgements

GSK

Neil Spooner

Paul Abu-Rabie

Rob Wheller

GE

Mark Green