11_PO-CON1751E - 第4页
4 Quantitation of plasma metanephrine and normetanephrine by derivatization using an integrated LC-MS/MS analyzer equipped with fully-automated sample preparation device Metanephrine (MN) MN-d3 Normetanephrine (NMN) NMN-…
3
Quantitation of plasma metanephrine and normetanephrine
by derivatization using an integrated LC-MS/MS analyzer equipped
with fully-automated sample preparation device
Fig. 2 Appearance of CLAM-LCMS system, and the pretreatement steps carried out for derivatization.
Fig. 2 illustrates the general appearance of the instrument setting, with CLAM-2000 connected to LCMS-8060 to
comprise an integrated “turnkey” type analyzer system. The pretreatment steps carried out are shown in the ow chart
that include 5 steps of reagent addition and 4 steps of vortexing. The procedure completes within 6 minutes, and it can
be ran parallelly with LC-MS/MS measurement so resulting in no wait-time between sequential analyses.
Automated sample preparation
Filtration
Vortex
120 sec
Vortex
60 sec
Plasma
sample
in collection
tube
CLAM-2000 LCMS-8060
Automatically
transferred to
autosampler for
seamless injection
s
e
c
Pretreatment steps (6 min)
Reagent
Dispensing
60 µL
Reagent1
Vortex
10 sec
IS
Dispensing
30 µL
IS
Sample
Dispensing
30 µL
plasma
Reagent
Dispensing
30 µL
Reagent2
Reagent
Dispensing
90 µL
Reagent3
Vortex
60 sec
Commercially available pooled plasma was used as sample matrix. IS solution (1 ppb of standard compounds and
deuterated internal standard in water), Reagent 1 (butanal/acetic acid = 25:75), Reagent 2 (7% 2-picolineborane in
EtOH, w/v) and Reagent 3 (5% aq. ammonia) were prepared in 6 mL glass container and placed in CLAM-2000 as
reagent reservoir.
Sample and reagents
Methods
4
Quantitation of plasma metanephrine and normetanephrine
by derivatization using an integrated LC-MS/MS analyzer equipped
with fully-automated sample preparation device
Metanephrine (MN)
MN-d3
Normetanephrine (NMN)
NMN-d3
254.15
257.15
296.20
299.20
Precursor m/z
(derivatized)
197.23
200.23
183.20
186.20
MW
-14
-21
-17
-21
CE (V)
236.15
154.10
278.20
154.10
Product m/zCompound
Fig. 3 MRM chromatograms of MN and MN-d3 spiked in control plasma at 1 ng/mL concentration.
Shown below are the list of target compounds, their MRM transitions and the HPLC condition for LC/MS/MS analysis.
Analytical conditions
Results
MN
(run 1)
MN
(run 2)
MN-d3
(run 1)
MN-d3
(run 2)
Column : Shimpack GISS C18 (100 mm x 2.0 mm, 3 μm)
Mobile phase A : 0.1% formic acid in water
Mobile phase B : Methanol
Flow rate : 0.4 mL/min
Column temp. : 40 °C
Injection volume : 1 μL
8.09e4ISTD 257.15>154.10
A=171010
H=80171
RT =2.446
2.0 2.5 3.0
0.00
%
100.00
8.34e4ISTD 257.15>154.10
A=171613
H=81989
RT =2.443
2.0 2.5 3.0
0.00
%
100.00
7.27e4Q 254.15>236.15
A=147336
H=68771
RT =2.426
2.0 2.5 3.0
0.00
%
100.00
8.02e4Q 254.15>236.15
A=158391
H=76320
RT =2.430
2.0 2.5 3.0
0.00
%
100.00
5
Quantitation of plasma metanephrine and normetanephrine
by derivatization using an integrated LC-MS/MS analyzer equipped
with fully-automated sample preparation device
Fig. 4 MRM chromatograms of NMN and NMN-d3 spiked in control plasma at 1 ng/mL concentration.
NMN
(run 1)
NMN
(run 2)
NMN-d3
(run 1)
NMN-d3
(run 2)
1.54e6ISTD 299.20>154.10
A=5958581
H=1534491
RT =3.313
2.5 3.0 3.5
0.00
%
100.00
1.57e6ISTD 299.20>154.10
A=6077891
H=1574332
RT =3.310
2.5 3.0 3.5
0.00
%
100.00
8.42e6Q 296.20>278.20
A=31185517
H=8346933
RT =3.322
2.5 3.0 3.5
0.00
%
100.00
8.56e6Q 296.20>278.20
A=31474446
H=8478530
RT =3.317
2.5 3.0 3.5
0.00
%
100.00
Two consecutive measurement of MN and NMN were made, each starting fresh sample pretreatment. High intensity
signal was detected demonstrating that direct one-pot derivatization in plasma was successful, and that there was
minimal difference between the two runs. Under the given condition, NMN showed higher signal; derivatization
condition was likely more favorable for NMN than for MN.