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Procedure for Whatman DMPK Cards

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May 19, 2009<br />

800 Centennial Avenue<br />

Piscataway, NJ 08855<br />

USA<br />

T 1 800 526 3593<br />

T 1 800 WHATMAN<br />

F 1 732 885 6529<br />

<strong>Procedure</strong> <strong>for</strong> <strong>Whatman</strong> <strong>DMPK</strong> <strong>Cards</strong><br />

Recommended guidelines <strong>for</strong> how to use <strong>Whatman</strong> <strong>DMPK</strong> <strong>Cards</strong>.<br />

1. Applying blood samples<br />

• Label the card.<br />

• Hold pipette tip or capillary a few mm above the card, not touching the card.<br />

• Expel the 15 µL sample smoothly and quickly (not spraying) as a drop that<br />

touches the card. The drop will quickly spread and soak in.<br />

• Dry the card thoroughly. Ship and store it desiccated at ambient temperatures.<br />

2. Analyzing samples<br />

• Cut a 3 mm disc from the sample area (avoiding the edge of the spot).<br />

• Transfer disc to labeled tube or multiwell plate.<br />

• Add 100 µL methanol (containing internal standard if desired) to elute<br />

analytes).<br />

• Vortex-mix gently 60 min and centrifuge briefly to remove any cellulose fibers.<br />

(See adjustments and additional in<strong>for</strong>mation below.)<br />

• Analyze supernatant by HPLC-MS/MS as usual.<br />

3. Possible adjustments <strong>for</strong> optimization<br />

• Test both <strong>DMPK</strong> card types initially, then focus on type giving better results <strong>for</strong><br />

the analytes of interest.<br />

• Adjust elution time – shorter <strong>for</strong> good analyte/solvent combinations or longer<br />

<strong>for</strong> weak ones. (See additional in<strong>for</strong>mation below.)<br />

• Larger blood volume, such as 40 µL, allows more discs per sample spot.<br />

• Select best elution solvent – HPLC mobile phase may be good.<br />

• Reduce elution volume, elute from 2 punches, or evaporate and re-dissolve<br />

extract in smaller volume <strong>for</strong> increased sensitivity.<br />

Please contact me with any additional questions.<br />

Regards,<br />

Jim Robbins<br />

James C. Robbins, Ph.D.<br />

Field Applications Scientist In Europe, please contact<br />

<strong>Whatman</strong>, Inc. Claire Dupont<br />

GE Healthcare Bio-Sciences Corp. <strong>DMPK</strong> Product Manager<br />

800 Centennial Ave - Bldg 1-244 Tel : +33 6 83 93 90 70<br />

Piscataway, NJ 08855 USA Claire.Dupont@ge.com<br />

<strong>Whatman</strong> Inc.


T +1 732 885 6519<br />

M +1 973 979 1372<br />

E Jim.Robbins@GE.com<br />

Additional In<strong>for</strong>mation<br />

<strong>Whatman</strong> Inc.<br />

• We strongly encourage new users to start with these recommended procedures<br />

and then to modify them if desired based on experience and goals.<br />

• The process is not difficult but some attention to the technique will maximize<br />

reproducibility of the results. The goal is to have the sample spread evenly on the<br />

card surface as it quickly soaks in, giving a spot area closely linked to the sample<br />

volume. The cards are designed and carefully manufactured <strong>for</strong> reproducible<br />

spread so it is only necessary to take advantage of that capability. The same<br />

technique can be used <strong>for</strong> serum or plasma. If the samples are colorless it may<br />

be useful to mark the sample area with a pencil or pen be<strong>for</strong>e it dries.<br />

• Normally the blood samples contain an anticoagulant such as EDTA or the<br />

sample capillary is coated with it. The cards contain components that rapidly lyse<br />

cells, inactivate pathogens and denature proteins. They do not require<br />

anticoagulants, as long as the blood has not already begun to clot, but are<br />

compatible with them.<br />

• The pipette or capillary tip should be held a little above the card, not touching it.<br />

Sample is then expelled smoothly and quickly, without spraying, into a drop that<br />

touches the card. This way the sample spreads quickly and evenly on the card as<br />

it soaks in. If the pipette or capillary tip were held in contact with the card during<br />

dispensing, the liquid flow would tend to be more through the card, encouraging<br />

local depletion of the card’s chemical components. The flow would probably be<br />

more directional, resulting in uneven sample distribution. These results would be<br />

more likely to vary between samples and between operators.<br />

• Carryover of analytes from punch to punch has not been a practical problem as<br />

long as the cards were thoroughly dry, preventing carryover of fibers. You can<br />

also clean the punching tool by cutting a waste disc between samples from an<br />

unused part of the card.<br />

• Results to date have shown little variation with where in the spot the discs are<br />

taken. We do not recommend sampling from the very edge of the blood spot. In<br />

some cases, particularly with the FTA Elute <strong>for</strong>mulation, a pink halo surrounds the<br />

blood spot. We recommend sampling from the dark, inner main spot.<br />

• Elution of analytes with organic solvents will leave protein precipitated in the<br />

cards. Some of the cards’ chemical components will be eluted with the analytes.<br />

Thus far these have not seriously interfered in analyte quantitation by HPLC-<br />

MS/MS.<br />

• Elution with a good solvent should be much faster than 60 min but time<br />

minimization experiments have not been done yet.

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