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マイクロフローデバイス LC/MS を⽤いたヒト⾎漿中のオキシトシンの⾼感度定量
ULTRASENSITIVE QUANTIFICATION ASSAY FOR OXYTOCIN IN HUMAN PLASMA
USING A LC/MS MICROFLUIDICS PLATFORM
佐藤太 1、Catalin E. Doneanu2 and Paul Rainville2
1
⽇本ウォーターズ株式会社； 2 ウォーターズコーポレーション
OVERVIEW
LC-SRM LC/MS を⽤いたヒト⾎漿中のオキシトシンの⾼感度定量法を開発し、
5 pg/mL の検出下限が得られた。
INTRODUCTION
医薬品の探索と開発の様々な段階において、低分⼦医薬品のバイオアナリシスをサポートする
定量的な LC-SRM 法は、様々な⽣体試料において、通常、50-100 pg/mL の定量下限を
達成している。ペプチド医薬品については、測定はより困難で、さらに低い定量下限が求められ
ることが多い。
オキシトシン（OT）は、9 残基の環状ペプチドで、脳内で神経伝達物質として作⽤し、ヒト⾎
漿中の濃度は、通常、数 pg/mL である [1]。
これまで、内在性の OT 濃度測定の⽅法としては、ELISA [2-3] 及び質量分析法が報告さ
れている。しかしながら、商業的に利⽤可能な ELISA の定量下限は、内因性の濃度よりも⾼
い。近年、いくつかの質量分析計に基づく⽅法が開発されており、それらにおいては、アフィニティー
による保持 [3] や、2 次元 LC/タンデム MS が採⽤されており、必要な感度を達成するために、
⼤量注⼊(ヒト⾎漿 1.4 mL) [4] による抽出が前提になっている。
本研究では、ヒト⾎漿中の内在性 OT の検出を可能とするための、マイクロフローデバイス
（1-5 µL/min の流速で使⽤する）を⽤いた⼀次元の LCSRM 法について報告する。本法
は、300 µL のヒト⾎漿試料により、内在性 OT の検出が可能な感度を要しており、迅速で堅
METHODS
A
Sample Preparation
Oxytocin (OT, Sigma Aldrich, St. Louis, MO) was spiked in 300 µL of
K2-EDTA human plasma (Bioreclamation, East Meadow, NY) at the
following concentrations: 1, 2, 5, 10, 50, 100, 500, 1000, 5000,
13 15
C N-isotopically
labeled
OT
10000
and
100000
pg/mL.
(CYIQNCPLG-NH2, Sigma Aldrich) was added as an internal standard
at 10000 pg/mL in all samples and the plasma samples were diluted
1:1 with 4% H3PO4. Samples were clean-up using Oasis MCX µElution
Plates (Waters P/N 186001830BA, 30 µm particles). After sample
loading, the wells were washed with 200 µL of 2% formic acid (FA) in
DI water and with 5% CH3OH in DI water. Three elution solvents were
investigated in order to optimize the extraction protocol. First, wells
were eluted with 2x50 µL of 25% ACN, 2% NH4OH; followed by
elution with 2x50 µL of 50% ACN, 2% NH4OH; and finally the third
elution step was performed with 2x50 µL of 75% ACN, 2% NH4OH.
SPE eluates were diluted 1:1 with 0.1% FA in DI water and analyzed
by LC/MS.
LC/MS Conditions
Samples were analyzed on a 150 µm ID x 5 cm prototype microfluidic
device packed with 1.7 µm BEH C18 particles coupled to a XevoTM TQS mass spectrometer. Separations were performed at a flow rate of 3
µL/min at 60 oC, using a mobile phase containing 0.1% FA in DI water
(Solvent A) and 0.1% FA in acetonitrile (Solvent B). The analyte was
eluted using a “step” gradient from 0 to 20% Solvent B (in 0.2 min)
following by a 4 min isocratic step at 20% B. The washing step with
90% B was started after analyte elution.
SRM Assay
SAMPLE PREPARATION
OT and
13
C15N OT are spiked into plasma
1:1 dilution with H3PO4
2 washing steps
0
-1
+3
0
SRM analyses were performed on a XevoTM TQ-S mass spectrometer
equipped with a TRIZAIC ESI source. Operating parameters were: ESI
potential 3.2 kV, source temperature 100 oC, desolvation gas 0.2 bar,
MS1/MS2 isolation window 0.75 Da (FWHM). Collision energy and
cone voltage we optimized on the LC-timescale using a dwell time of
10 ms.
SPE Optimization
-2
A
B
C
2500000
5% CH3OH
R2=0.998
2000000
100 pg/mL
1,000 pg/mL
25% ACN, 2% NH4OH
1000000
0
+1
+2
500000
10,000 pg/mL
+5
C
50% ACN, 2% NH4OH
0
0
100,000 pg/mL
D
Figure 1. Optimization of tile positioning: left and right, along X-axis is
shown in panel A; up and down, along Y-axis, is displayed in panel B and Zaxis optimization (in and out) is presented in panel C. The numbers shown
on each slide correspond to the unit settings of the XYZ stage.
A
b6
B
[M+H]+1
20000
40000
60000
80000
100000
Conc (pg/mL)
75% ACN, 2% NH4OH
Figure 4. Optimization of the SPE protocol for oxytocin in human
plasma: (A) washing step with 5% methanol; (B) elution with 25%
ACN, 2% NH4OH; (C) elution with 50% ACN, 2% NH4OH; (D) elution
with 75% ACN, 2% NH4OH. The volume of sample injected was 1 µL
for each run.
Figure 7. Example of the linearity of the SRM assay: MRM chromatogram for five oxytocin concentrations spiked in plasma, covering a
dynamic range of four orders of magnitude (from 10 to 100,000 pg/
mL). The volume of each injection was 1 µL.
C-Y-I-Q-N-C-P-L-G-NH2
Figure 9. Calibration curve for oxytocin spiked in human plasma
in the range of 5 - 100,000 pg/mL.
CONCLUSIONS
Assay Reproducibility
b6
50% ACN, 2% NH4OH elution
Concentration
(pg/mL)
Run01
Run02
Run03
5
10
50
100
500
1000
5000
10000
100000
306
617
2614
5374
26588
56596
284592
622345
6351754
293
639
2529
5520
23519
59901
301425
597699
6573004
267
570
2578
5678
24815
55377
292805
610475
6748837
Peak Area
Run04
Run05
Mean
RSD %
248
575
2373
5466
24506
57642
299544
617184
6475522
278
593
2527
5466
25286
57973
296964
606467
6486703
8.1
5.5
3.7
2.7
5.8
3.7
2.9
2.5
2.8
・ ヒト⾎漿中のオキシトシン定量のための、迅速で、堅牢であり、かつ⾮常に感度
の⾼い LC-SRM 法を開発した
+2
[M+2H]
Figure 2. ESI-MS spectrum of oxytocin (A) and the MS/MS spectrum (B)
produced by the fragmentation of the singly charged precursor using a collision energy of 28 eV. Both spectra were recorded on the LC timescale.
Peak Area
60000
Figure 5. RADAR scans indicating the presence of sample matrix in
the SPE elution solvent. Full MS scans (m/z=400-1400, 200 ms scantime) were acquired along with SRMs throughout the entire LC run.
40000
CE (eV)
CYIQNCPLG-NH2
1007.4->723.3
100
28
20000
CYIQNCPLG-NH2
1014.4 -> 730.3
100
28
0
25% ACN,
2% NH4OH elution
28 eV
A
80000
CV (V)
CE (eV)
20
40
0.1% FA blank
60
plasma blank
100 V
B
150000
2 pg/mL OT in plasma
LC-SRM
たシグナルは、ヒト⾎漿の内在性オキシトシンに由来する可能性が考えられた
・ 測定法の直線性は、4 桁（5 - 100,000 pg/mL ヒト⾎漿にスパイク）で
あった
・ 測定法の再現性は、10%以下であった
Table I. Reproducibility of the oxytocin assay in human plasma across
the entire concentration range (5 - 100,000 pg/mL). Five replicate injections (5 µL injection volume) were performed for each concentration. Peak area RSD % was better than 10% for all concentrations.
References
1.Burd JM, Weightman DR, Baylis PH J. Immunoassay, 1985, 6,
2.Fisher LA, Fernstrom JD Life Sc., 1981, 28, 1471.
A
3.Cool DR, DeBroose D, J Chromatogr. B, 2003, 792, 375.
B
Peak Area RSD: 5.5%
4. Zhang G, Zhang Y, Fast DM, Lin Z, Steenwyk R Anal. Biochem.,
2011, 416, 45.
5. Sobhi HR, Vatansever B, Wortmann A, Grouzmann E, Rochat B
C
J Chromatogr. A, 2011, 1218, 8536.
6. Mabrouk OS, Kennedy RT J Neurosc. Meth., 2012, 209, 127.
100000
D
5 pg/mL OT in plasma-LLOQ
50000
CV(V)
0
0
20
40
60
80
100
120
140
Figure 3. Collision energy screening (CE, panel A) and cone voltage optimization (CV, panel B) performed on the LC time scale. Fifteen collision energy values in the range of 4-60 eV were optimized in a single LC-SRM run using a
fixed cone voltage of 40 V. Nineteen CV values (5-140 V range) were then optimized using a fixed CE of 28 eV in a subsequent injection.
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・ オキシトシン定量下限は 5 pg/mL であった。ヒト⾎漿ブランク試料で検出され
227.
Peak Area
200000
276
565
2539
5294
27004
60349
306453
584633
6284397
Assay LLOQ
0
Xevo TQ-S tandem quadrupole mass spectrometer equipped
with a TRIZAIC source.
10 pg/mL
1500000
MRM Transition
dilution with 0.1% FA
3000000
+2
-1
Peptide sequence
Elution with 25% and with 50% ACN, 2% NH4OH
Assay Linearity
+1
LC-SRM
The injection volumes were in the range of 1-5 µL and the total
runtime for the SRM assay was 10 min.
牢な⽅法である。
RESULTS
B
+2
+1
Peak Area
10 pg/mL OT in plasma
E
Figure 6. Establishing the LLOQ of the assay: solvent A blank injection (0.1% FA, panel A); human plasma blank (B); 2 pg/mL oxytocin
spiked in human plasma (C); 5 pg/mL oxytocin spiked in human
plasma (LLOQ, panel D) and 10 pg/mL oxytocin spiked in human
plasma (E). The volume of each injection was 1 µL.
Figure 8. Reproducibility of the SRM assay: five replicate injections
(2 µL volume) of 10 pg/mL oxytocin spiked in serum. Peak area RSD
was 5.5%.
©2014 Waters Corporation MKT14080