Internal Standard Application to Dried Blood Spots ... - lc-ms.de

DBS Workshop 06.09.2011
FH Nordwestschweiz, Basel
Harlan Laboratories
Internal Standard Application to Dried Blood Spots -
Evaluation of a Novel Automated Application System
from CAMAG
Dieter Zimmer
1

Introduction
• IS are added in the extraction solvent for DBS analysis so far
– IS not in liquid blood, nor penetrating into dried blood
– IS does not compensate for losses during sample preparation
– IS compensates for matrix effects (SIL) and injection problems
• Alternative methods for IS addition are desirable, to
– Mimick all effects acting on analyte as close as possible
– Result in IS recovery similar to analyte
• “IS Addition” subteam formed within the EBF “DBS topic team”
• Addition of IS by spraying was evaluated

CAMAG DBS-MS 16
• Occasionally lent by CAMAG to HARLAN for the investigations performed there
• First results presented at EBF Conference 2010 and ASMS 2011 (posters)

Homogeneity of Spraying on HPTLC Plates
For equipment validation in HPTLC the following is done
• 0.5 mg/mL Oracet Red G [1-(methylamino)anthracene-9,10-dione] in methanol
used as a test dye for spraying
• Spraying with the automated high precision sprayer ATS4 (CAMAG)
• Merck 60 F254 HPTLC plates to measure the intensity variation of the
spraying process
• Analysis with an optical scanner (CAMAG TLC Scanner 4), detection
collimator 4 x 0.3mm (length x width) in remission mode at 546 nm
A similar process was used for DBS, but with 14C coffeine mimicking an IS, and
determination by Imaging like QWBAR
ATS4 HPTLC Sprayer DBS-MS 500 Sprayer

Homogeneity of Spraying on HPTLC Plates
A finer graded HPTLC substrate was used
• A centred circular area of 4.0 mm is used for extraction with the DBS-MS
• Profile plots of the spray pattern (bars indicate 4 mm region).
• Table with CVs of the integrated response of centred 4 mm area

Blood Spotting
• Fresh rat whole blood (EDTA) stored up to 10 days in freezer (5 o C)
• 15 µL blood per spot (approx. 7 mm diameter)
• Ahlstrom 226 cards, green (ID Biological Systems)
• Drying for about 2 hrs at ambient temperature
• Storage in plastic bags at ambient temperature until analysis

IS Distribution by Spraying – The Concept
• Scan von Rat slice und DBS Karte
•

Homogeneity of IS Distribution on DBS cards
• Cards were spotted with blank whole blood of rats
• [1-Methyl 14 C] Caffeine (mimicking an IS) sprayed onto dried blood
spots on cards with the high precision sprayer ATS4 (CAMAG)
• Radioactivity and its distribution were determined by Imaging
• Homogeneity of IS distribution across the blood spot
• Reproducibility between spots
• Effect of the sprayed solution volume (5 – 40 µL) on reproducibility

Spraying with ATS4 (Automatic TLC Sampler, CAMAG)
• [1-Methyl- 14 C] Caffeine, MW 194.2 g/mol, mimicking an IS
H 3
C*
O
N
O
N
CH 3
N
N
CH 3
• Specific Activity: 55 mCi/mMol = 11241 kBq/mg
• Concentration in ethanol: 0.1 mCi/mL = 3.7 MBq/mL = 222 x 106 dpm/mL
• Spray Solution: Target 14C concentration of 40000 dpm/20 µL
• 60 µl [1-Methyl- 14 C] Caffeine Standard in 6 mL Methanol
• Concentration measured: 3 Aliquots a 20 µl = 39448 dpm ± 0.9 %
corresponding to 98.6% of Target
• 10 x 10 mm area microsprayed (5 – 40 µL) covering the whole blood
spot (see figures)

Determination of Radioactivity
• BAS 5000 (Fuji Photo Film Co. Ltd.)
• Imaging Plate Type BAS III (20x25 cm) (Fuji)
• Exposure: 18 hours, Resolution: 25 µm
• Carbon-14 decays into nitrogen-14 through beta decay (156 keV)
• Betas are detected up to a depth of approximately 100 µm

Profiles Across Spots
Intensity [PSL]
2
1
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Position [mm]

Homogeneity of IS Distribution on DBS cards
12.0 mm
Intensity [PSL]
20
10
12.0 mm
4.0 mm
4.0 mm
0 1 2 3 4 5 6 7 8 9 10 11 12
Position [mm ]
Spray pattern analysis for 14C Caffeine on Alstrom 226 cards
as function of sprayed volume: 2-dim plot with profiles for 20 µL/cm 2
4.0 mm
20
Intensity [PSL]
10
0 1 2 3 4 5 6 7 8 9 10 11 12
P osition [m m ]
• 15 µL blood spotted ~7 mm
• 10 x 10 mm sprayed with IS
• 4 mm circle online extracted
• Center located by laser beam

Radioactivity (PSL/mm²)
Homogeneity of IS Distribution on DBS cards
800
700
600
500
400
300
200
100
0
Homogenity of Internal Standard applied as [14C] Spray
Solution to DBS
5 10 20 40
Spay Volume [µL/cm²]
Homogenity of Radiolabeled Standard applied as Spray Solution to DBS
Spray Volume
Radioactivtity
Mean (n=8) S.D. CV
[µL/cm²] [PSL/mm²] [PSL/mm²] [%]
5 184.83 3.45 1.87
10 274.83 7.40 2.69
20 403.55 11.23 2.78
40 664.79 39.23 5.90
Spray pattern analysis for 14 C
Caffeine on Alstrom 226 cards as
function of sprayed volume:
2 cards (8 spots) per volume
Card B7 (5 µL/100 mm², 10000 dpm)
Card B8 (5 µL/100 mm², 10000 dpm)
Card B9 (10 µL/100 mm², 20000 dpm)
Card B10 (10 µL/100 mm², 20000 dpm)
Card B11 (20 µL/100 mm², 40000 dpm)
Card B12 (20 µL/100 mm², 40000 dpm)
Card B13 (40 µL/100 mm², 80000 dpm)
Card B14 (40 µL/100 mm², 80000 dpm)

Spray Drift Investigation by Extraction & LC-MS/MS
Spraying of IS-mix solution (Nortryptiline-D 3 , Loratadine-D 3 , Formoterol-D 3 )
spraying onto neat untreated cards:
• IS sprayed onto alternating spots on 4 cards
• Card 1 – 1st spot, card 2 – 2nd spot etc.
• IS Drift into adjacent blankspots in [%] of sprayed IS peak area, means (N= 3 - 4)
[%] of peak area of sprayed IS spot
Blank spot left from IS
Blank spot right from IS
2nd 2 blank spot right/left from IS
nd blank spot right/left from IS
NOR-D3
[%]
0.07
0.21
0.00
LOR-D5
[%]
0.09
0.54
0.00
FOR-D6
[%]
0.00
0.00
0.00
Card
1
2
3
4
��
��
��
��
��
��
��
��
��
��
��
�� IS spray
�� blank spot left/right
�� 2nd blank spot left/right
��
��
��

Variability of IS Peak Area After Extraction & LC-MS/MS
• Tested with a prototype sprayer, not optimized for DBS (EBF Poster 2010)
• IS-mix solution either
- sprayed onto neat card before blood spotting
OR
- sprayed onto dried blood spot
• Extraction with DBS-MS 16 prototype, Analysis by LC-MS/MS
Means N = 4
IS sprayed before blood
spotting
IS sprayed onto dried
blood spot
Card 1
Card 2
Card 1
Card 2
NOR-D3
Mean Area
x103 x103 814
815*
3229
2763
CV [%]
12.8*
*N=3: data of 4th spot considered as outlier, since IS solution was run out.
• CVs of peak areas of extracted IS
- 0.5 – 7.3% if IS sprayed onto dried blood
- 5.6 – 6.9% if IS sprayed onto neat card
- Precision of 15 % (20% @ LLOQ) accepted in Bioanalysis
• More detailed investigations are ongoing
6.8
5.8
0.5
Mean Area
x103 x103 367
370*
1011
789
LOR-D5
CV [%]
5.6
16.7*
7.3
4.6
Mean Area
x103 x103 76
72*
250
202
FOR-D6
CV [%]
6.9
10.0*
4.7
3.0

Conclusion
• The maximum capacity of the filter paper was < 40 µL of spray solution /cm 2
• Overfilling leads to uncontrolled spreading and higher variability, explaining
the higher CV at 40 µL spray volume
• Recommended spray application volumes are 5 - 20 µL/cm2 • The homogeneity within the 4 mm extraction area of the blood spot was high
• Precision and accuracy of the spray process were high
• The imaging analysis with 14C Caffeine confirmed that the blood spot absorbs
the sprayed solution homogeneously
• Boundary effects appeared outside the 4 mm region which is extracted
• Spray-drift of IS into adjacent blank spots was well below 1 %
• CVs of areas of extracted IS were 0.5 – 7.3% (N=3-4) with prototype sprayer
• Spraying of IS is a very good alternative to addition of IS in extraction solvent

Acknowledgements
• Matthias Loppacher, Christoph Fankhauser, Urs Schranz, CAMAG
• Stephan Hassler, ADME Dept., HARLAN (Imaging)
• From the Bioanalytical & LC/MS Dept., HARLAN:
– Bea Betschart
– Joelle Brodman
– Petra Hill
– Esther Müller
– Stephan Sack
– Stephanie Weber

Presentations
For download from www.lc-ms.de

Life Demo of DBS-MS 500
• API QTRAP 2000 available here at FH Nordwestschweiz
– Sensitivity (MRM) not fully optimized, similar to API 3000
• DBS-MS 500 interfaced to QTRAP yesterday
• Test Compounds: Formoterol, Loratadine, Nortriptyline
• Calibration range 10 – 5000 ng/mL blood
– 15 µL blood spotted, less than 5 days stored at 5 o C
• IS-Mix was spiked parallel to analyte mixture into blood
OR
• IS-Mix solution of 1000 ng/mL each was sprayed onto blood spot

Life Demo - Test Compounds
Formoterol
t R = 0.8 min
Nortriptyline
t R = 1.1 min
IS: Formoterol-D 3, Nortriptyline-D 3, Loratadine-D 5
Total LC/MS run time 2.4 min – rate limiting!
Loratadine
t R = 1.3 min

Back Up

Principle of Online Extraction with DBS-MS
Inlet Outlet
4 mm ∅
seal
DBS card