1. No category

A Sensitive LC/MS/MS Method for the Quantification of Telmisartan in Human Plasma
ASMS 2011
MP 10- 181
Srividya Kailasam, Life Sciences Center, Agilent Technologies, India
I t d ti
Introduction
E
Experimental
i
t l
Results
R
lt
Di
i
l andd Discussion
Results
R
lt andd Discussion
Di
i
In this study we describe a sensitive LC/MS/MS method
System
Tuning of the mass spectrometer was optimized using
g
No peak was seen for the telmisartan transition in the
Each calibration and QC sample was injected three times. The mean response ratios of telmisartan to telmisartan-d3 were
for quantification of telmisartan in human plasma.
G1312B 1260 Infinity binary pump
MassHunter Optimizer software, which identifies the mostt
first plasma blank injected after injecting the ULOQ
pplotted against
g
the concentrations of telemisartan to obtain the calibration curve. A linear curve fitting
g was used and
abundant product ions together with the optimum fragmentorr
sample (5000 pg/mL) thrice as can be seen in figure 3.
3
weighted (1/x).
(1/x) A linear dynamic range of 50 - 5000 pg/mL was achieved for telmisartan in human plasma with an R2 value
voltages and collision energies. A simple protein precipitation
n
The figure 4 shows the overlay of the telmisartan peaks in
greater than
h 0.995.
0 995 Figure
Fi
6 shows
h
a representative
i calibration
lib i curve for
f telmisartan
l i
i plasma
in
l
using
i telmisartan-d3
l i
d3 as the
h
sample
p ppreparation
p
method was used to extract telmisartan
n
the LLOQ and blank samples.
p
It can be seen that the area
internal standard. From Table 1 it can be seen that the calculated concentrations for all calibration standards and the QC
and the internal standard,
standard telmisartan-d3 from 200 μL of spikedd
of the MRM signal in the blank is less than 20% of the
samples show good precision and accuracy.
plasma
l
samples.
l
area off analyte
l
peakk in
i the
h LLOQ sample
l confirming
fi i that
h
acts by blocking angiotensin II receptor. The deuterated
analog,
g telmisartan-d3 was used as the internal standard
in this study.
study The structures of the analyte and the internal
standard
d d are shown
h
i figure
in
fi
1 The
1.
Th high
hi h sensitivity
i i i off the
h
6460 mass spectrometer with the Agilent Jet Stream ESI
source enabled the quantification of 50 pg/mL of
telmisartan in human plasma.
plasma This makes the method
suitable for the analysis of sample from low dose studies.
G1379B 1260 Infinityy micro degasser
g
G1367D 1260 Infinity autosampler
G1330B 1260 Infinity
I fi it Thermostat
Th
t t
G1316A 1260 Infinity Thermostatted Column Compartment
G6460
Triple Quadrupole Mass Spectrometer with the
Agilent Jet Stream Technology
Software MassHunter B.03.01 (Acquisition and Qualitative
Analysis)
MassHunter B.04.00 ((Quantitative analysis
y
1
6
criteria.
5.5
5
45
4.5
Column: ZORBAX Eclipse Plus - C8 3.0
3 0 X 50 mm,
mm 1.8
1 8 m
C l
Column
temperature: 45 C
05
0.5
3
04
0.4
Telmisartan-d3
300 pg/mL
15
1.5
03
0.3
02
0.2
1
01
0.1
0.5
Mobile phase B: Acetonitrile
0
0
0.5
Flow rate: 0.5
0 5 mL/min
1
1.5
2
2.5
3
3.5
Counts vs. Acquisition Time (min)
4
4.5
Figure
g
2: MRM chromatogram
g
of the Mid-QC sample
p
Time (min)
% Acetonitrile
All plasma samples were treated with 500 μL of cold
0
20
acetonitrile
aceto
t e aandd tthen
e vortexed
o te ed for
o 1 min. 66755 μμL supe
supernatant
ata t
05
0.5
95
was transferred to a new microcentrifuge tube and dried by
25
2.5
95
vacuum concentration. The residues were resuspended in 200
2.6
20
μL of 9:1 (v/v) water:methanol, and centrifuged prior to
Stop time
5 min
-200
Figure 4: Telmisartan peak in the LLOQ (50 pg/mL)
sample
analysis
analysis.
Source: ESI with Jet stream (+)
Figure 1: Instrumentation and analytes
Product Fragmentor
ion
voltage (V)
( /)
(m/z)
Collision
energy
( )
(eV)
515.2 ((Telmisartan))
275.6
180
52
518.44 (Telmisartan-d3)
518
(Telmisartan d3)
(ISTD)
279 2
279.2
180
50
Figure
g
3: First blank injected
j
after the ULOQ sample
p
injections
0
200
400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200 5400
Concentration
oncentration(pg/m
(pg/ml)
Figure 6:Standard curve in plasma with 3 replicate injections at each calibration level
Telmisartan-d3
Telmisartan
d3
Precursor ion (m/z)
07
0.7
3.5
2.5
Mobile pphase A: 10 mM Ammonium acetate
08
0.8
06
0.6
Telmisartan,750 pg/mL
4
2
Telmisartan
the carryover meets the FDA bioanalytical recommended
x10 2 + MRM (515.2000 -> 275.6016) Pl_750Telmisartan300Tel-d3-r003.d
6.5 1
Telmisartan - 8 Levels, 8 Levels Used, 23 Points, 23 Points Used, 0 QCs
y=0001913*x
= 0.001913 x +0003180
+ 0.003180
x10 1 y
R^2 = 0.99679385
09
0.9
Rela
ative Resp
ponse
es
Telmisartan is used for the treatment of hypertension and
Figure
g
5: Overlay of the telmisartan peaks in the LLOQ
and blank samples
Concentration
(pg/mL)
Mean
Calculated
C
Concentration
i
(n=3)
Precision
(% R.S.D.)
Accuracyy ((%))
50
46 93
46.93
11 11
11.11
93 86
93.86
A sensitive and selective LC/MS/MS method for the
75
77.08
10.40
102.77
250
251.12
2.18
100.45
qquantification of telmisartan in human pplasma using
g an
500
510.31
6.41
102.06
1000
994.50
7.49
99.45
1500
1519 06
1519.06
6 54
6.54
101 27
101.27
2500
2531 09
2531.09
8 34
8.34
101 24
101.24
5000
4617.22
11.35
92.34
internal standard from plasma.
plasma The response ratio of the
150 (LQC)
130.68
5.80
87.12
analyte and the internal standard was found to be linear
750 ((MQC))
773.39
4.17
103.12
over the range
g 50 - 5000 pg
pg/mL.
4000 (HQC)
3746 79
3746.79
7 95
7.95
93 67
93.67
C
Conclusions
Table 1: Response ratios, precision and accuracy at the various
concentrations
Agilent 1260 Infinity LC system and 6460 Triple Quadrupole
M
Mass
S
Spectrometer
with
i h the
h Agilent
A il
J Stream
Jet
S
ESI
source has been described. A simple protein precipitation
method was used for extracting the analyte and the