latest developments in microsampling for regulated...
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Latest developments in microsampling for regulated quantitative bioanalysis
Neil Spooner EBF 6th Open Meeting November 2013
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Dried Blood Spots
2 November 2013
• Collect blood in small
‘pot’, or directly into
capillary
• Spot 5 µL onto DBS card
• Dry for >2 hours
• Store at ambient
• Punch fixed diameter
disc of entire spot – Sub-punch approach
also possible
• Place disc in tube
• Extract with 200 µL
solvent (60 min) Can automate the analysis
Barfield et al (2008) J. Chrom. B 870, 32-37 Latest Developments in
Microsampling
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The Ups and Downs of DBS...
• Understanding of the technique is continually evolving
– Learning where the technique is and is not suitable, as well as potential
hurdles
– More difficult for BA group; advantages are to preclinical and clinical groups
• ‘Validation’ is more extensive than for a plasma based assay
• Regulatory agencies are taking conservative approach to its use
for clinical studies
– For the time being, we are expected to employ sparse sampling approach of
wet blood to compliment DBS samples and show continued concordance
• One of the biggest concerns with the technology is the
hematocrit effect
3 November 2013 3 Latest Developments in
Microsampling
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Impact of Haematocrit Effect Continuing along the Microsampling Road
Courtesy Sue Sparrow, GSK 4 November 2013 4 Latest Developments in
Microsampling
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Blood / Water - Capillary
5
Courtesy Amanda Wilson, AZ
November 2013 Latest Developments in
Microsampling
• Collect exact
volume (≥5 µL)
using EDTA
capillary • Place in tube • Dilute with
known volume
of water
• Mix
• Freeze
• Analyse aliquot
Can automate the analysis
Jonsson et al (2012) Bioanalysis 4, 661-674
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Blood / Water - Pipette
6 November 2013
• Collect exact volume
(≥5 µL) using pipette
Can automate the analysis
• Dilute with known
volume of water
• Mix
• Freeze
• Extract aliquot
Latest Developments in
Microsampling
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DSM – Experimental DBS Substrate
Novel substrate material
• Preliminary data for material which is
still under investigation
• Minimises effect of hematocrit on spot
size
• Minimises effect of hematocrit on
recovery
However;
• For paracetamol a bias in the data
was observed with HCT when the sub-
punch approach was used
• This was not observed for codeine
7
Courtesy Ynze Mengerink, DSM & Philip Denniff
November 2013 Latest Developments in
Microsampling
20% HCT 45% HCT 70% HCT
-20%
-10%
0%
10%
20%
30%
15 25 35 45 55 65 75
Bia
s r
ela
tiv
e 4
5%
HC
T
HCT (%)
Assay bias (sub punch)
Assay bias (Whole spot)
Area Bias
Recovery bias
15 µL blood containing paracetamol
spotted onto DSM card
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Solid Phase Microextraction
8
Courtesy Supelco SPME Team & Sheelan Ahmad
November 2013 Latest Developments in
Microsampling
0
500
1000
1500
2000
2500
3000
3500
0 5 10 15 20
Blo
od
Co
ncen
trati
on
of
Dis
op
yra
mid
e (
ng
/mL
)
Time (Hours)
Protein Precipitation
SPME
Disopyramide concentration in ex vivo samples
from male beagle dog after 20 mg/kg oral dose
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What happens if you want plasma concentrations?
9 November 2013 Latest Developments in
Microsampling
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Plasma – Hematocrit Tube Approach
10
Courtesy Amanda Wilson, AZ
November 2013
• Collect 32 µL blood in
EDTA HCT tube
• Plug with wax
• Place in labelled tube
• Centrifuge 1500 xg for
10 min
• Cut using capillary cutter
• Collect exact volume
(8 µL) plasma with
capillary
• Freeze
• Analyse whole aliquot
Can automate the analysis
Jonsson et al (2012) Bioanalysis
4, 1989-1998
Wax
plug
Centrifuge
tube Plasma
fraction
Filled micopipette
placed in sample tube
Latest Developments in
Microsampling
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Drummond Plasma Microsampling Capillary
11
Capillary
• Glass capillary
• Mylar film coat (increased strength and
safety)
• Internal EDTA coating
• Porous plug
• Thixotropic gel
Operation Overview
• Fill capillary with blood (~70 μL) by
capillary action
• Porous plug swells & seals capillary on
contact with blood
• Centrifuge capillary
• Gel migrates to blood plasma interface
• Push porous plug with Wiretrol to
expel plasma
• Collect plasma (20-30 μL) & freeze
• Analyse 7 μL aliquot
November 2013 Latest Developments in
Microsampling
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Sample Collection & Processing
12
Collect blood sample into a capillary
using a labelled 1.1ml tube as a holder
Centrifuge
samples at
3000–4000
xg for 10
mins at
ambient
temp
C
E
Use Wiretrol to expel the
plasma from the capillary into a
labelled tube and freeze
Defrost and analyse 7 µL
aliquot
November 2013 Latest Developments in
Microsampling
Bowen et al (2013) Bioanalysis
5(9), 1131-1135
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Noviplex Plasma Separator Card
• Add 20 - 75 µL whole blood to test area
– Control spot indicates whether enough blood
has been added
– Lateral spreading layer rapidly spreads blood so
it enters the filtration layer as a front
– Filtration layer captures blood cells by a
combination of filtration and adsorption
– Collection layer loads with a specific aliquot of
plasma
– The isolation screen precludes lateral wicking
along the card surface
• After 3 mins, peel back the top layer
• The collection disc contains 2.5 µL of
plasma
• Air dry for 15 minutes and store/ship in
original packaging with desiccant
• For analysis, remove the collection disc
from the card prior to extraction
13
Courtesy Neil Loftus, Shimadzu
November 2013 Latest Developments in
Microsampling
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Yorktest Plasma Separator Card
14
Courtesy Robert Sturm & Jack Henion, Quintiles Bioanalytical Labs and Richard
Abbott & Phil Wang, Shire Pharmaceuticals
November 2013 Latest Developments in
Microsampling
• 25 µL blood containing SIL-IS applied to upper membrane
• Control spot indicates when upper layer can be removed (5 min after
spotting)
• Lower membrane air dried for ≥1 hr
See Y Li et al (2012) Rapid Commun. Mass Spectrom., 26, 1208-1212
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Digital Microfluidics
15
Courtesy Aaron Wheelers Group at University of Toronto
November 2013 Latest Developments in
Microsampling
Method Parameter Sitamaquine Proguanil Benzethonium
Chloride
Ibuprofen
- [Actual Drug]
ng/mL
900 450 1000 200,000
conventional [Drug] ng/mL 978 468 1120 197,000
conventional % Accuracy 109 104 112 98.8
conventional %CV of Blind 3.2 2.4 3.1 5.7
conventional No. replicates 6 6 6 6
DMF [Drug] ng/mL 814 457 669 240000
DMF % Accuracy 90 102 67 120
DMF %CV of Blind 2.4 16 16 29
DMF No. replicates 3 4 8 8
Lefreniere et al (2014) Bioanalysis In Press (Feb)
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• Site of sampling
• Composite vs serial • Acceptance
• Method validation
• Technology available
• Complexity of Method development
• Applicable to all drugs?
• Small volumes
• Accurate volumes
• Wet / dry
• Handling issues & automation
• HCT Issues
• Ability to perform repeats
• Samples for MIST Sample type &
handling Analytical sensitivity
Toxicokinetics Regulatory
Microsample
Microsampling – Issues to Consider Courtesy Amanda Wilson, AZ
November 2013 Latest Developments in
Microsampling
16
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The Problem......
Will any of these approaches be
acceptable to regulators????
Will any of these approaches be
acceptable to the practitioners???
• Conservative nature of the industry
– Are WE our own worst enemies for the
adoption of innovative approaches???
17 November 2013 Latest Developments in
Microsampling
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The Solution......
• Cross company high
quality science as the best
way forwards
– NC3Rs
– EBF Topic Teams
– IQ Consortium
– Scientific publications
– White papers
– etc........
18 November 2013 Latest Developments in
Microsampling
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The Future??!!
19 November 2013 Latest Developments in
Microsampling
The mould can be broken
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Acknowledgements
• Sheelan Ahmad
• Paul Abu-Rabie
• Chet Bowen
• Philip Denniff
• Wesley Dopson
• Nella Rebisz
• Susan Sparrow
• Amanda Wilson
• Joe Siple
• Ynze Mengerink
• Jack Henion
• Robert Sturm
• Neil Loftus
• Richard Abbott
• Phil Wang
• SPME Team
• Aaron Wheeler
• Nelson Lafreniere
20 November 2013 Latest Developments in
Microsampling
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Thank you
All animal studies were ethically reviewed and carried out in accordance with
European Directive 2010/63/EU