Bioanalysis of Exenatide: Intact Versus Signature Peptide Approach
to Reach Optimal Sensitivity in Large Molecule Quantification by LC-MS
Jean-Nicholas Mess, Daniel Villeneuve and Fabio Garofolo*
Algorithme Pharma Inc., Laval (Montréal), QC, Canada
3.5e4
1.5e4
XIC of +MRM (2 pairs): 474.800/688.400 Da from Sample 10 (CR04-ZRA) of MSMS27.wiff (Turbo Spray), Smoothed
2.672.68
1.0e4
1.0
1.5
2.0
Time, min
2.5
3.0
Figure 3: Tryptic Digestion Efficiency of Exenatide to its
Signature Peptide in Human Plasma SPE Extracts
from Sam
ple 40 (REF 25ug ACN) of Digestion.wiff (Turbo Spray)
2.04
Intensity, cps
Intensity (cps)
160
140
M
ax. 160.0 cps.
0.22
120
3.00
0.37
1.70 1.78
2.46
2.75
3.03
60
0.85
40
0.99 1.36
1.65
3.19
20
0.5
XIC of +M
RM (10 pairs): 838.300/396.200 Da
1.0
1.5
2.0
Time, min
2.5
QMEEEAVR
LFIEWLK
NGGPSSGAPPPS-NH2
Monoisotopic
(m/z)
1278.5961
639.8017
426.8702
991.4513
496.2293
331.1553
948.5553
474.7813
316.8566
1023.4854
512.2463
341.8333
Not Detected
2.33
5.37e6
2.33
5.74e4
Not Detected
0.34
7.30e5
0.34
1.56.e4
2.3
2.4
2.5
2.6
Time, min
2.7
2.8
2.9
Digestion Process Yield
75.3% at Low QC
85.5% at High QC
Total Process Yield
48.7% at Low QC
48.8% at High QC
Matrix Effect
Acceptable for 9/10 donors of human
plasma including lipemic samples.
Hemolysed samples under investigation.
Selectivity
No significant interference at analyte and
IS retention times for 10 donors including
lipemic and hemolysed samples
Dilution Integrity
up to 4 000 pg/mL, diluted 5-fold
Processed Stability
45.8 hrs at 4°C
Bench-Top Stability (K2EDTA + Inhibitor)
20.0 hrs on ice/water bath
2.2 hrs at room temperature
Long-Term Storage Stability (K2EDTA + Inhibitor)
On going
Freeze-Thaw Stability (K2EDTA + Inhibitor)
3 Freeze Thaw Cycles
Stability in Whole Blood (K2EDTA)
2.0 hr on ice/water
Stock Solution Stability 100 µg/mL (Days)
48 days at 4°C in aq.solution
3.0
B) Extracted LLOQ (10.0 pg/mL)
Max. 6100.0 cps.
2.68
350
300
250
200
150
100
50
0
2.0
2.1
2.2
2.3
2.4
2.5
2.6
Time, min
2.7
2.8
2.9
3.0
C) Extracted ULOQ (2000.0 pg/mL)
XIC of +MRM (2 pairs): 474.800/688.400 Da from Sample 6 (CR04-P10A) of MSMS27.wiff (Turbo Spray)
Max. 4.8e4 cps.
2.45
4.0e4
3.5e4
3.0e4
2.5e4
2.0e4
1.5e4
2.0
2.1
2.2
2.3
2.4
2.5
2.6
Time, min
2.7
2.8
2.9
3.0
Exenatide
7000
The calibration curve was linear (weighted 1/x2) from
10.0 to 2000.0 pg/mL with a coefficient of correlation of
0.9951 (Figure 5). Summary of the preliminary data
obtained during method development is shown in Table 2.
6000
5000
4000
3000
0
0.0
0.5
1.0
1.5
2.0
Time, min
2.5
3.0
Figure 5: Calibration Curve for Exenatide
B) Following Digestion (3 hrs at 37°C)
6.00
Calibrant
XIC of +M
RM (10 pairs): 475.100/688.400 Da
from Sam
ple 63 (REF 25ug M
eOH) of Digestion.wiff (Turbo Spray)
2.5e4
M
ax. 3.2e4 cps.
1.73
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
5.00
Signature Peptide
2.0e4
4.00
1.5e4
1.0e4
5000.0
0.0
0.0
0.5
XIC of +M
RM (10 pairs): 838.300/396.200 Da
400
1.0
1.5
2.0
Time, min
2.5
3.0
from Sam
ple 63 (REF 25ug M
eOH) of Digestion.wiff (Turbo Spray)
M
ax. 400.0 cps.
0.39
Nominal
(pg/mL)
10.0
20.0
40.0
100.0
300.0
500.0
800.0
1400.0
1700.0
2000.0
Back-Calculated
(pg/mL)
10.7
17.7
37.4
105.2
301.8
537.9
776.1
1477.9
1655.4
1956.8
Deviation
(%)
6.6
-11.3
-6.5
5.2
0.6
7.6
-3.0
5.6
-2.6
-2.2
Intensity (cps)
350
300
2.00
0.46
200
1.87
1.20 1.271.61
Regression type: Linear 1/x2
Coefficient Correlation: r = 0.9951
1.00
1.76 2.00
• This study demonstrates that the tryptic digestion approach is
suitable for the quantification of exenatide in human plasma by
LC-MS/MS.
• The digestion step was efficient (within 3 hours) and
reproducible.
• The tryptic peptide generated allowed to decrease the
LLOQ of the exenatide assay.
3.00
Exenatide
250
CONCLUSION
• The LLOQ achieved by this LC-MS/MS assay is comparable,
if not lower, to the commercial LBA assay kits.
1.63
When comparing the synthetic reference standard material
for exenatide and its tryptic peptide prepared at an
equimolar concentration (Figure 2), the detection of
exenatide tryptic peptide was 5 times more sensitive than
the intact exenatide.
64.7% at Low QC
57.1% at High QC
2.01
3.2e4
3.0e4
Not Detected
2.2
M
ax. 8160.0 cps.
2000
tR
Intensity
(min)
(cps)
Outside calibration
0.63
8.84e5
7.73e5
0.63
2.1
XIC of +MRM (2 pairs): 474.800/688.400 Da from Sample 11 (CR04-P1A) of MSMS27.wiff (Turbo Spray), Smoothed
0.0
from Sam
ple 40 (REF 25ug ACN) of Digestion.wiff (Turbo Spray)
Extraction Process (SPE) Yield
8000
Intensity (cps)
HGEGTFTSDLSK
Charge
State
+1
+2
+3
+1
+2
+3
+1
+2
+3
+1
+2
+3
2.0
5000.0
3.0
1000
Peptide
100
1.0e4
0
0.0
Inter Batch Precision (%CV) and Accuracy (%Bias) 8.2% and 11.7% at LLOQ QC
7.1% and 2.2% at Low QC
3.7% and 1.7% at Mid QC
4.6% and 3.0% at High QC
150
4.8e4
4.5e4
100
80
200
0
2.77
Signature Peptide
Results
50
A) No Trypsin Added
XIC of +M
RM (10 pairs): 475.100/688.400 Da
Evaluation
250
Intensity, cps
(cps)
Intensity
0.5
Table 2: Summary of the Evaluations Performed
Max. 3296.5 cps.
300
Peak Area Ratio
Table 1: Exenatide Tryptic Digestion
Intact exenatide
S/N = 84.2
The extraction efficiency of exenatide (SPE process) was
on average 60.9% and the digestion efficiency was 80.4%.
No intact exenatide was found in the extracted samples
following tryptic digestion (Figure 3). Overall, the entire
extraction efficiency process (SPE + digestion) was found to
be 49.0%.
Intensity, cps
Exenatide amino acids sequence analysis showed 3
theoretical trypsin cleavage sites which would result in the
generation of 4 tryptic peptides. However, experimental
digestion data performed in solution showed the presence
of only 3 tryptic peptides. One of the tryptic peptide
(LFIEWLK) had good characteristic for quantification
(retention on C18 column and MS sensitivity) (Table 1).
This peptide mainly ionized to the +2 charge state
(m/z 474.8) and clearly fragmented to one singly charged
product ion (m/z 688.4).
2.0e4
0.0
0.0
Intensity (cps)
LC-MS is an emerging technique for the quantification of
large molecules such as therapeutic peptides, proteins
and biomarkers. However, due to the limited mass range
of triple quadrupole mass spectrometers, these analytes
are typically monitored as multicharged species. This
dispersion of MS signal combined with poor fragmentation
(CID) efficiency may affect the achievable LOQs. Another
approach is to generate smaller signature peptides
through tryptic digestion. These tryptic peptides will
generally show less charge state distribution and clearer
CID pattern than the intact analyte. However, the
efficiency of the digestion process could be poor and
compromise its sensitivity. In this study, both approaches
will be compared in order to reach optimal sensitivity for
the quantification of exenatide in human plasma.
Figure 1: Structure of Exenatide and Signature Peptide
2.5e4
5000.0
DETECTION
• AB SCIEX QTRAP®5500
• MRM mode ESI(+)
• The [M+5H]5+ (m/z 838.3 → 396.2) was monitored for
exenatide and the [M+2H]2+ (m/z 475.1 → 688.4) was
monitored for the signature peptide.
RESULTS
Figure 4: Representative Chromatograms of Extracted
and Digested Exenatide in Human Plasma
A) Extracted Blank
Intensity, cps
CHROMATOGRAPHY
• Agilent Technologies Series 1100 pumps and autosampler
• Ascentis Express C18, 50x2.1mm, 2.7µm
• Gradient elution of 0.1% HCOOH and ACN
• 3.5 minutes run time
INTRODUCTION
Signature peptide
S/N = 431.5
3.0e4
Intensity, cps
• Results
– The detection of the signature peptide was 5 times
more sensitive than the intact exenatide.
– The digestion efficiency was evaluated to be >80%
– Using the digestion approach, an LLOQ of 10 pg/mL
was achieved with an assay showing good precision
and accuracy.
Max. 3.8e4 cps.
1.73
3.8e4
Intensity, cps
• Method
– Sensitivity comparison was performed using
reference standard material.
– Samples were extracted by SPE, digested with
trypsin and analysed by LC-MS/MS on an AB SCIEX
QTRAP®5500 operated in ESI+
XIC of +MRM (10 pairs): 475.100/688.400 Da from Sample 11 (NE) of Digestion.wiff (Turbo Spray)
Intensity (cps)
SAMPLE EXTRACTION
• Range of 10 – 2000 pg/mL in human plasma
• 500 µL of sample extracted by solid phase extraction using
Oasis ion exchange SPE.
• Following elution and drying step, the samples were
reconstituted with trypsin (Sigma T1426) and incubated for
3 hours at 37°C
The lower limit of quantification (LLOQ) achievable using the
tryptic digestion approach was 10 pg/mL (Figure 4), which is
significantly lower than the reachable LLOQ for the intact
exenatide.
Intensity, cps
• Purpose
– Direct comparison of two different quantification
approaches: intact versus signature peptide to reach
optimal sensitivity for the quantification of exenatide
in human plasma.
Figure 2: Sensitivity comparison of Intact Exenatide and
Exenatide Signature Peptide Prepared at Equimolar
Concentration (1 nM)
Intensity (cps)
METHODS
Intensity
Intensity,(cps)
cps
OVERVIEW
• On-going work is currently being performed on the novel triple
quadrupole QTRAP®6500 to decrease the LLOQ to the low
pg/mL level.
2.44
2.32
2.59
150
1.55
100
0.27
0.50
0.86
50
0
0.0
1.24
2.16
2.84
1.57
0.00
2.88
1.16
3.04
0.0
200.0
400.0
600.0
800.0
1000.0
1200.0
1400.0
1600.0
1800.0
2000.0
Concentration (pg/mL)
0.5
1.0
1.5
2.0
Time, min
2.5
3.0
* CORRESPONDING AUTHOR