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METHODS
In Vivo
Female
CD-1
Mouse
PK
Studies
Plasma
PK
followin
g
Discrete
Dosing
Water was
provided
ad libitum
and
animals
were not
fasted.
The dose
formulations were
prepared in 0.5%
aqueous \
methylcellulose
with 0.2%
polysorbate 80
(MCT)
(v/w).
Animals were given a
Blood
PK
followin
g
Discrete
andCass
ete
Dosing
Mice
Mice were
randomized into
groups (n = 9 or 12
mice/group to obtain
three or fouar blood
samples/time point)
Preparat
ion of
Working
Solution
s
forCalibr
ation
Curves
were
Mice
randomized
into
groups
groups (n
(n =
= 3
3 or
or 4
4
mice/ group) and
compounds
were
compounds
were
administered
administered
individually
or as a cassette
(five-in-one)
(five-in-one)
containing all five
one
compounds
compounds
Calibrati
on Curve
Preparat
ion
Sample
Preparat
ion
Mice
were
randomized
into
groups
groups (n
(n =
= 3
3 or
or 4
4
mice/ group) and
compounds
were
compounds
were
administered
administered
individually
or as a cassette
(five-in-one)
c
(five-in-one)
c
ontaining all five
compounds
compounds
Before sample
collection
collection mice
mice were
were
placed
placed under
under a
a
heat
heat lamp
lamp (for
(for
approximately 3 min)
until
until the
the lateral
lateral ail
ail
veins were dilated A
27G needle was used
to
to nickhe
nickhe lateral
lateral tail
tail
vein.
vein. Exactly
Exactly 10
10 or
or
15 :L of blood were
collected
collected
Chromat
ographic
Conditio
ns
blood
sample
via
Two blood samples
terminal cardiac
were withdrawn
puncture(appro
from each animal
ximately
0.2
following
mL) at 0.25 and
isoflurane
2 h, 0.5 and 4 h,
anesthesia
or 1 and 6 h
postdose.
Two blood samples
were withdrawn
A positive displacement pipette
from each animal
was used
to
following
to enhance
enhance sample
sample collection
collection and
and
Animals
transfer
accuracy
transfer
accuracy
isofluranOne blood
were
compared with air-displacement
sample was
pipettes.
euthanized
pipettes.
withdrawn
via
using CO2
retroorbital bleed
Blood samples were collected into
inhalation
(approximately
microtainer tubes with EDTA as
0.1 mL)e
anticoagulant and spun to harvest
anesthesia
plasma.
Mass
Spectro
metc
Conditio
ns
Blood
Stability
and
Extractio
n
Recovery
PK Data
Analysis
transferred to
polypropylene
Costar cluster
tubes (96-well
format) and stored
at approximately
80C until thawed
Sample
preparation
25 micro liters of smple (diluted blood or
plasma)
Instrumen LC-MS
Instrumen
Calibration
Curve
Preparatio
n
Dacarbazin,
gemcitabin,
gefinitib,
imatinib &
topotecan
dibuat 1 mg/ml
Diekstraksi
dengan
penambahan
200l protein
presipitation
(asetonitril)
Divortex 5 menit
& disentrifugasi
15 menit at 25ooC.
Supernatan
dibagi
menjadi 3
tempat injeksi
yg berbeda
Sample
Preparatio
n
25l
sampel
dipipet ke dlm
tabung cluster
protein
presipitation
(divortex
&
dilakukan sesuai
cara diatas)
35l dig untuk
kuantifikasi
(dacarbazine
&
gemcitabine)
35l kuantifikasi
(gefinitib,
imatinib &
topotecan)
Chromatogra
phic
Conditions
(gefinitib,imatinib,
topotecan)
PhenomenexKinetex
XB-C18
laju alir 0,8 ml/mnit. V
injeksi: 10 L, waktu: 2,3
mnit
(dacarbazine ,
gemcitabine)
SIELC Obelisc N
laju alir 0,8 ml/mnit, V
injeksi: 10 L, waktu: 2,5
mnit
PK Data
Analysis
Dilakukan pd darah &
plasma darah
Menggunakan data
konsentrasi waktu
rata (plasma) atau
waktu indivudual
(darah)
Konsentrasi di bawah
batas
kuantisasi
dianggap sebagai nol
dalam
perhitungan
nilai rata-rata untuk
setiap titik waktu.
MERCI