Glucagon Bioanalysis by LC-MS: “Unprecedented Level of Sensitivity (10pg/mL) for a Novel Formulation”
Jean-Nicholas Mess1, Louis-Philippe Morin1, Mauro Aiello2, Xavier Misonne2, Gary Impey2, Johnny Cardenas2, Josée Michon1 and Fabio Garofolo1*
1Algorithme Pharma Inc., Laval (Montréal), QC, Canada
2AB SCIEX, Concord, ON, Canada
METHODS
• Purpose
– The challenging development of an LC-MS/MS
method for the quantification of glucagon at the low
pg/mL level to support bioanalysis of a new
formulation.
SAMPLE EXTRACTION
• Range of 10 – 10 000 pg/mL in human plasma
• 225 µL of sample extracted by solid phase extraction using
Oasis µElution MAX 96 well plates.
• Method
– Samples were extracted by SPE and analysed by
LC-MS on an AB SCIEX QTRAP®5500,
QTRAP®6500 and TripleTOFTM5600 operated in
ESI+
– The triple quadrupoles were used in MRM (unit
resolution) while the Q-ToF was used in TOF MS
(30K Resolution) or MRMHS (15K resolution)
High QC
7500.0 pg/mL
98.3
104.1
101.1
123.7
102.0
76.6
101.0
15.0
14.9
101.0
332.800
359.100
269.200
286.800
298.700
266.900
302.3
36.77
12.2
100.8
2115.00
1935.30
1943.40
2128.90
1988.90
2261.10
2062.1
128.36
6.2
103.1
6827.10
7254.40
7596.80
6799.80
7262.10
7205.90
7157.7
300.92
4.2
95.4
2000
3.50
S/N = 29
1500
3.11
1000
Figure 3: Representative Chromatograms of a Glucagon
on QTRAP®6500
3.37
2.90
3.57
0.45
0.34
0.52
0.88
1.52
2.22
1.63
3.73
2.84
Sampl
e Ind
ex:
33
Sampl
e Typ
e:
Unkn
own
Conce
ntrat
ion:
N
/A
Calcu
lated Conc:
0
.00
ng/m
L
Acq. Date:
2012
/05/10
Acq. Time:
21:5
2:40
2.0
Time, min
3.0
Max. 8029.6 cps.
2.35
700
600
S/N = 62
0.87
1.78
0.51
0
0.2
1.51
0.61
1.84
2.52
1.68
3.13
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Time, m
in
2.0
2.2
2.4
2.6
2.8
3.0
3.50
2.83
B) Extracted LLOQ (10.0 pg/mL)
3.43
3.11
3000
Sampl e Name: "QCLOQ
04-10"
S
ample ID: "1"
File: "Gluc_10MAY2012B_CE22.wi ff"
Peak Name: "Glucagon"
Mass(es): " 697.502/693.900 Da"
Comment: "1"
Annotation: ""
2.74
3.72
0.46
3.76
2.22
0.51
2.66
Sampl
e Ind
ex:
15
Sampl
e Typ
e:
Unkn
own
Conce
ntrat
ion:
N
/A
Calcu
lated Conc:
1
0.5
ng/m
L
Acq. Date:
2012
/05/10
Acq. Time:
20:0
9:51
Int. Type:
Val
ley
Reten
tion Time:
1
.70
Area:
30
80.51
628
Heigh
t:
1.12e
+003
Start Time
:
1
.63
End T
ime:
1
.74
1.70
1300
Modif
ied:
N
o
Proc. Algo
rithm: Spec
ify Pa
ramet
ers - MQ
Noise Perc
entage
:
50
Base. Sub. Windo
w:
1.00
min
PeakSplit
. Fact
or:
2
Repor
t Lar
gest P
eak:
Yes
Min. Peak Height
:
0.00
cp
s
Min. Peak Width:
0.00
se
c
Smoot
hing Width:
3
p
oints
RT Wi
ndow:
30
.0
sec
Expec
ted R
T:
1
.69
min
Use R
elati
ve RT:
N
o
2.89
II
I
1200
0.21
1100
min
count
s
cps
min
min
1000
900
2.0
Time, min
3.0
4.0
800
C) MRMHS: Sum of the 3 Most Intense Isotopomers of
Product Ion [M+5H-NH3]5+: MEW 20 mDa
XIC of +TOFProduct (697.4): 693.728 to 693.748 D
a fromSample 4 (Glucagon QC3 MR
MHR enhance) of 1.w
iff (Turbo Spray), Summed <XI...
Intensity, cps
2.27
500
400
3.08
1.12
0.40
1.75
300
200
Max. 717.4 cps.
0
0.2
700
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Time, m
in
2.0
2.2
2.4
2.6
2.8
3.0
C) Extracted ULOQ (10000.0 pg/mL)
600
S/N = 254
2.0
1.8
1.6
1.4
1.2
Regression type: Linear 1/x2
Coefficient Correlation: r = 0.9990
0.8
0.6
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
Analyte Conc. / ISConc.
7000.0
8000.0
9000.0
1.0e4
This research demonstrated that:
• The TripleTOFTM5600, when specifically used for
targeted quantification (MRMHS) can match, but not
exceed the sensitivity of the QTRAP®5500 triple
quadrupole.
• The QTRAP®6500 significantly improved the sensitivity
of our assay in order to reach an LLOQ of 10.0 pg/mL
The outcome of this research showed that it is possible to
develop an LC-MS/MS method more sensitive than the
standard LBA for the challenging quantification of Glucagon at
low pg/mL level.
Sampl e Name: "CR04-P13A"
Sample ID: "1"
File: "Gluc_10MAY2012B_CE22.wi ff"
Peak Name: "Glucagon"
Mass(es): " 697.502/693.900 Da"
Comment: "1"
Annotation: ""
Sampl
e Ind
ex:
1
Sampl
e Typ
e:
Stan
dard
Conce
ntrat
ion:
1000
0.0000
0
ng/mL
Calcu
lated Conc:
102
00.
ng/m
L
Acq. Date:
2012
/05/10
Acq. Time:
18:4
9:44
400
II
I
Int. Type:
Val
ley
Reten
tion Time:
1
.70
min
Area:
13
11066
.95587
co
unts
Heigh
t:
5.57e
+005
cps
Start Time
:
1
.64
min
End T
ime:
1
.78
min
300
ACKNOWLEDGEMENTS
1.70
5.5e5
Modif
ied:
N
o
Proc. Algo
rithm: Spec
ify Pa
ramet
ers - MQ
Noise Perc
entage
:
50
Base. Sub. Windo
w:
1.00
min
PeakSplit
. Fact
or:
2
Repor
t Lar
gest P
eak:
Yes
Min. Peak Height
:
0.00
cp
s
Min. Peak Width:
0.00
se
c
Smoot
hing Width:
3
p
oints
RT Wi
ndow:
30
.0
sec
Expec
ted R
T:
1
.69
min
Use R
elati
ve RT:
N
o
5.0e5
4.5e5
The authors would like to acknowledge Suma Ramagiri from
AB Sciex for her support and valuable advice during the
completion of the TripleTOFTM 5600 experiments.
4.0e5
200
3.5e5
100
3.0e5
2.5e5
2.0e5
1.0
2.2
2.51
100
0
2.4
-1.9
-1.3
7.9
6.7
3.5
2.4
-0.3
-2.7
-4.1
-5.1
-3.6
-3.9
1.3
0.88
600
1.81
500
2.6
Deviation
(% )
2.38
0.48
700
2.81
Intensity, cps
The TripleTOFTM5600 was tested in different acquisition
modes to determine optimal conditions. First, the full scan
TOFMS mode (30K resolution) was evaluated. This mode is
generic and is the easiest to use. The 3 most abundant
isotopomers of the +5 charged state of glucagon were
summed using a mass extraction window (MEW) of 20mDa
(Figure 2A). Moreover, this acquisition mode also allows to
sum different charged states in order to increase sensitivity
and assay ruggedness. The 3 most abundant isotopomers
of each of the +3 to +5 charged states were summed using
a MEW of 20 mDa (Figure 2B). This led to a 2 fold increase
in sensitivity.
2.8
BackCalculated
Conc.
9.8
24.7
53.9
106.7
206.9
512.1
997.2
1460.4
2398.4
3796.6
6819.5
8169.4
10125.2
1.14
100
1.0
Nominal
Calibrant ID
Conc.
(pg/mL)
P1
10.0
P2
25.0
P3
50.0
P4
100.0
P5
200.0
P6
500.0
P7
1000.0
P8
1500.0
P9
2500.0
P10
4000.0
P11
7000.0
P12
8500.0
P13
10000.0
3.0
0.22
400
4000
0
3.1
CONCLUSION
800
200
0.34
Glucagon.rdb (Glucagon): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000301 x + 0.00173 (r = 0.9990)
900
7000
RESULTS
7170.5
7527.0
7204.9
6612.3
7320.2
7520.0
7225.8
336.4
4.7
96.3
1000
300
1000
2049.9
1820.4
1952.3
1997.4
1917.8
1890.5
1938.1
80.9
4.2
96.9
Figure 4: Calibration Curve for Glucagon
0.0
1100
1.86
2000
46.6
43.7
48.0
43.9
47.9
43.2
45.5
2.2
4.9
91.1
0.0
1200
500
5000
7.0
9.7
9.5
8.4
10.9
7.7
8.8
1.4
16.3
88.4
1
2
3
4
5
6
Mean
S.D.
% C.V.
% Nominal
2.90
1300
4.0
XIC of +TOFMS: 697.33 to 697.35 D
a fromSample 1 (Glucagon QC3 TOFMS) of 1.w
iff (Turbo Spray), Summed <X
IC of +TOF M
S: 697.53 ...
6000
High QC
7500.0 pg/mL
0.2
Y
es
B) TOF MS: Sum of the 3 Most Intense Isotopomers of
Each of the +3 to +5 Charged States : MEW 20 mDa
8000
Mid QC
2000.0 pg/mL
Sampl e Name: "BL-3S"
Sam
ple ID: " 1"
F
i le: "G
l uc_10MAY2012B_CE22.wi ff"
Peak Name: "Glucagon"
Mass(es): " 697.502/693.900 Da"
Comment: "1"
Annotation: ""
Modif
ied:
1.0
Low QC
50.0 pg/mL
0.4
A) Extracted Blank
2.87
500
LLOQ QC
10.0 pg/mL
1.0
Intensity, cps
The present work describes the challenges and solutions
encountered using novel mass spectrometers to reach
unprecedented level of sensitivity for the analysis of low
plasma concentration of glucagon.
Figure 1: Structure of Glucagon
Max. 2530.9 cps.
1.86
0
Intensity, cps
As previously published by our group, a bioanalytical
method for glucagon in human plasma (lower limit of
quantification (LLOQ): 100 pg/mL) was validated under
FDA guidelines on an QTRAP®5500 mass spectrometer.
However, this LLOQ is not adequate enough to cover the
pharmacokinetic profile of this novel glucagon formulation.
Therefore, the method was re-developed on two different
instruments (QTRAP®6500 and TripleTOFTM5600) to
reach higher level of selectivity and sensitivity.
A) TOF MS: Sum of the 3 Most Intense Isotopomers of
the +5 Charged States : MEW 20 mDa
2500
Mid QC
2000.0 pg/mL
The validated assay was simply transferred from the
QTRAP®5500 to QTRAP®6500 to evaluate its performance.
The QTRAP®6500 was able to reach an LLOQ of 10 pg/ml
which is 10 times lower than our validated LLOQ on
QTRAP®5500 (Figure 3). The calibration curve was linear
(weighted 1/x2) over three orders of magnitude with a
coefficient of correlation of 0.9990 (Figure 4). Precision and
Accuracy for Glucagon obtained on the QTRAP®6500 for a
range of 10.0 to 10 000.0 pg/mL is shown in Table 2.
Figure 2: TripleTOFTM 5600 Acquisition Mode Evaluation
on a Mid-QC of Glucagon (2000 pg/mL)
XIC of +TOFMS: 697.33 to 697.35 D
a fromSample 1 (Glucagon QC3 TOFMS) of 1.w
iff (Turbo Spray), Summed <X
IC of +TOF M
S: 697.53 ...
Low QC
300.0 pg/mL
1
2
3
4
5
6
Mean
S.D.
% C.V.
% Nominal
Overall, the data showed that it is possible to have the
same sensitivity and performance on a TripleTOFTM5600 as
on an QTRAP®5500 but not to reach the targeted LLOQ of
10 pg/mL. Precision and Accuracy for Glucagon obtained on
the TripleTOFTM 5600 is shown in Table 1.
0.73
Intensity, cps
Ligand Binding Assay (LBA) is currently the most common
approach for Large Molecule quantification. However, LBA
may suffer from cross reactivity, lack of specificity and
selectivity for very low quantitation analysis. In the past
several years, LC-MS based assays showed to be as
effective and also complementary to LBA for Large
Molecule quantification. Currently, we are working on a
study for a novel formulation of glucagon, which should be
easier to administer than the currently available
formulations, resulting in a higher standard of Type 1
diabetes patient care.
Intensity, cps
INTRODUCTION
DETECTION
• Triple Quadrupoles:
• AB SCIEX QTRAP®5500 and QTRAP®6500
• MRM mode ESI(+) (unit resolution)
• The [M+5H]5+ (m/z 697.5 → 694.1) was monitored
• Q-ToF
• AB SCIEX TripleTOFTM5600
• TOF MS mode (ESI+)
• 100 – 1000 m/z, 100 ms accumulation
• Mass extraction window: 20 mDa
• MRMHS mode (ESI+)
• Q1 set at 697.5 ([M+5H]5+)
• Sum of 3 most abundant isotopes of [M+5H-NH3]5+
product ion
• Mass extraction window: 20 mDa
LLOQ QC
100.0 pg/mL
Table 2: Glucagon Precision and Accuracy by MRM on
QTRAP®6500
A n a lyte Are a / IS Are a
• Results
– On the Q-ToF, the use of a targeted approach
(MRMHS) led to optimal sensitivity over generic
TOFMS mode, but did not allow to decrease the
validated LOQ (100 pg/mL) achieved on the
API5000TM.
– On the QTRAP®6500, the LLOQ of 10 pg/ml was
achieved with good precision and accuracy.
CHROMATOGRAPHY
• Agilent Technology Series 1100 pumps and autosampler
• Zorbax 300 SB-C18, 50x2.1mm, 3.5µm
• Gradient elution of 0.1% HCOOH and ACN
• 4.0 minutes run time
Table 1: Glucagon Precision and Accuracy by MRMHS on
TripleTOFTM 5600
Peak Area Ratio
OVERVIEW
Finally, MRMHS mode (15K resolution), which can be
considered as a targeted approach and resemble standard
MRM experiment performed on a triple quadrupole, was
also evaluated and the 3 most abundant isotopomers of the
+5 charge state product ion (neutral loss of NH3) were
summed (Figure 2C). Over 4-fold gain in sensitivity was
obtained in MRMHS compared to TOF MS mode which
allowed to reach an LLOQ of 100.0 pg/mL.
2.0
Time, min
3.0
4.0
1.5e5
1.0e5
5.0e4
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Time, m
in
2.0
2.2
2.4
2.6
2.8
3.0
* CORRESPONDING AUTHOR