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Experimental
N
H
H
N
CD2
CD
CD3
N
O
Formoterol-D6
Intensity, cps
Calibration Range
Accuracy & Precision
400
Accuracy
Precision
QC Conc. (pg/mL)
RE%
CV%
LLOQ
0.4
2.00
13.57
Low
1
5.00
8.94
Medium
40
9.00
3.81
High
90
8.78
3.49
Compared with Nominal Value (%)
> 71.95
Accuracy
320
Inter-Batch (n=18)
240
160
80
20
800
720
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
(B)
Method Recovery
640
560
480
400
240
160
0.5
1.0
1.5
2.0
2.5
3.0
Time, min
3.5
4.0
4.5
5.0
5.5
149.1
1.8e6
1.2e6
(A)
100 pg/mL of
Formoterol in Plasma
60
4.0e6
351.3
155.1
Intensity, cps
120
180
155.3
240
300
1.9e4
Formoterol-D6
In previous methods larger plasma sample volumes (12 mL) were needed in order to achieve a lower detection
limit. These sample volumes would make preparation
much longer requiring large individual SPE cartridges and
introduce more error in the preparation procedure. The
current method employed special SPE elution solution by
which the extraction method recovery was dramatically
increased. Therefore, only 0.5 mL of human plasma was
required to reach the requisite sensitivity .
6.0e5
4.17
Mass Spectrometry
MS System:
AB Sciex API-4000
Condition:
LC/(+)ESI-MS/MS (MRM)
MRM Transition:
Formoterol:
345.2
149.1
RE%
<9.89
< 11.1
< 9.75
< 5.30
327.5
345.3
2.3e4
Liquid Chromatography:
HPLC System
Shimadzu LC-10AD
Analytical Column: C8 column, 2.1 x 50 mm, 3 m
Mobile Phase A)
10 mM ammonium Acetate in water
(pH4)
Mobile Phase B)
Methanol
Gradient
Flow rate:
0.5 mL/min
Injection Volume:
25 L
Condition
3 Cycles, < -70 C
4 hrs, Room Temperature
4 Days, Room Temperature
117 Days, < -70 C
Freeze/Thaw
Bench-Top
Autosampler Extract Stability
Long-Term Storage Stability
320
360
333.4
351.6
420
2.5e6
1.5e4
1.1e4
1.0e6
7000.0
60
120
180
0.5
4.8e4
(B)
1.5
2.5
3.5
4.12
4.5
5.5
Formoterol-D6 in Plasma
240
m/z, amu
3000.0
300
360
420
1.14
4.0e4
0.95
3.2e4
2.4e4
1.6e4
8000.0
Formoterol
OH
480
HO
H
Control plasma
560
0.5
1.5
2.5
3.5
Time, min
4.5
5.5
Structures
640
Intensity, cps
OH
(A)
80
20
Introduction
HO
Sample Preparation
720
Intensity, cps
Intensity, cps
Overview
A highly sensitive and specific liquid chromatography- mass
spectrometry (LC-MS/MS) method capable of quantifying
formoterol in human plasma is described.
Conclusions
0.75
0.60
0.40
0.20
0.05
0
15
30
45
6
75
Analyte Conc.0(pg/mL).
90
100