Michael H Court 2005
Internal standard: Alprazolam (0.25
 g/ml solution in methanol)
Triazolam: 0.01 and 0.1
 g/ml in methanol
Extraction solvent: toluene: isoamyl alcohol (IAA) (98.5:1.5)
Mobile Phase: Ammonium Acetate (10mM) : Acetonitrile : Methanol (40:40:20)
Triazolam, IAA is purchased from Sigma (St. Louis, MO)
Toluene is from Fisher Scientific (Fair Lawn, NJ)
Preparation:
1.
Take the stock solution of TRZ and Alprazolam out of the freezer (-20

C), wait 30-
60min until the solutions reach room temperature.
2.
Wash tubes using methanol/acetone and let it dry in the hood.
Procedure:
Calibration curve volumes:
Test tube
#
Amount of
TRZ
(ng/tube)
Solution
Conc.
(
 g/ml)
1
2
3
4
0
0.1
0.25
0.5
0.01
``
``
``
Volume of
TRZ (
0
10
25
50
 l)
Amount of
Alprazolam
(ng/tube)
25
25
25
25
Solution conc.
(
 g/ml)
0.25
``
``
``
Volume of
Alprazolam
(
 l)
100
``
``
``
1.
5
6
7
8
9
1.0
2.5
5.0
7.5
10
0.1
``
``
``
``
100
25
50
75
100
25
25
25
25
25
``
``
``
``
``
``
``
``
``
``
Pipette I.S. and TRZ to 1--9 tubes as the volumes shown above, put the solution back to freezer after use.
2.
3.
Remove calf serum and patient samples from the freezer.
Dry down the solutions in vacuum oven. (oven: ~50

C)
4.
Add 1ml drug-free bovine calf serum or patient plasma to each tube.
5.
Add 2ml Toluene: isoamyl alcohol (IAA) mix (98.5:1.5).
6.
Cap the tubes. Vortex the mix for 1 min.

Observe the tubes, make sure the mix is vortexed.

Michael H Court 2005

If emulsion happens, put the sample to the freezer (-20

C). After frozen, take it out and let it thaw, and then re-centrifuge.
7.
Centrifuge for 10 min at 1 speed (1200g).
8.
TRZ and Alprazolam are extracted to the organic phase of the solvent, which is on the top. Under the hood, use the disposable plastic pipette to transfer the top layer into
2ml auto-sampling vials (GC vials).

Be sure not to take any inter-phase.
9.
Dry extracts in vacuum oven.
10.
Dissolve residue in 100
 l of mobile phase, vortex the vials, and transfer to the insert.
Finally, run the samples with LC/MS.