Biomedical Engineering Reference
In-Depth Information
methanol and acetonitrile were from Sigma-Aldrich (St. Louis, MO, USA). A.C.S.
grade ammonium formate and formic acid were also from Sigma-Aldrich. Water
was purified through a Milli-Q water purifier from Millipore (Billerica, MA, USA).
Human blood was purchased from Biological Specialty Corporation (Colmar, PA,
USA).
2.2
Sampling Paper/Cards
Nine kinds of sampling paper/cards were used. FTA Elute (Micro), FTA DMPK-B,
FTA DMPK-A, Indicating FTA (pink), FTA Plant, Blood Stain, Protein Saver 903
(previously Guthrie Card or Schleicher & Scheull Card in different times), and 31
ET were from Whatman (now GE Healthcare, Piscataway, NJ, USA) and VWR 237
filter paper from VWR International (West Chester, PA, USA).
2.3
Instruments
Harris Uni-Core 1.00, 3.00, 6.00 mm cutters and cutting mats were from
Whatman. A Sil-10 HTc autosampler and two LC-10AD pumps were from
Shimadzu (Kyoto, Japan). An API-4000 mass spectrometer with turbo ionspray
ionization probe was from AB Sciex (Foster City, CA, USA). The software for
data acquisition and processing was Analyst ver. 1.5 from Applied Biosystems
as well. An automated liquid handler was MicroLab AT 2 Plus from Hamilton
(Reno, NV, USA). A MixMate vortexer was from Eppendorf (Hauppauge, NY,
USA). A CR412 centrifuge was from Jouan (now Thermo Fischer). Column
selection valve was from Valco Instruments (Houston, TX, USA). An Atlantis
dc18 column (100 × 2.1 mm, 5 mm) was purchased from Waters (Milford, MA,
USA).
2.4
Sample Preparation
Dried blood spot on different paper/cards were punched and dispensed into the
wells of a 96-well plate, 20 mL of internal standard in approximately 50 % ace-
tonitrile was added. Three hundred microliters of 70 % acetonitrile (v/v) was added
and the plate was vortexed for 5 min at 800 rpm. Fifty microliters of 6 M ammonium
formate was added and the plate was vortexed at 850 rpm for 10 min. The plate was
then centrifuged at 3,500 rpm and 10ºC for 5 min. The upper layer was transferred
to a new plate and the samples were evaporated by a nitrogen stream at room tem-
perature. The dried samples were reconstituted with 30 % acetonitrile and 5 m L of
reconstituted sample were injected.
