TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2018 Waters Corporation | LL-pdf FOR RESEARCH USE ONLY, NOT FOR USE IN DIAGNOSTIC PROCEDURES LC-MS/MS QUANTIFICATION OF INTACT INSULIN- LIKE GROWTH FACTOR I (IGF-I) FROM SERUM FOR CLINICAL RESEARCH Nikunj Tanna 1 , Khalid Khan 2 , Mary Lame 1 , Anthony Marcello 1 , Ian Edwards 1 , Mark Wrona 1 1 Waters Corporation, Milford, MA 2 Waters Corporation, Wilmslow, UK INTRODUCTION Insulin-like Growth Factor I (IGF-I) is a 70 amino acid (7.6 kDa) peptide hormone, with 3 internal di-sulphide bonds (Figure 1). It plays a significant role in mediating the effects of Growth Hormone (GH), circulates at ng/mL levels, and is strongly bound to Insulin-like Growth Factor Binding Protein (IGFBP). Historically, immunoassays have been used for quantification of IGF-I. In recent years, use of LC-MS for quantification of IGF-I has increased. Most labs using LC-MS use the surrogate peptide approach, with or without immuno-affinity extraction, followed by quantification of resulting signature peptides by analytical scale LC with a tandem quadrupole (TQ) instrument. Although widely accepted, digestion may not always be required for proteins under 10 kDa on a TQ. However, achieving relevant analytical sensitivity levels for such intact proteins does require meticulous attention to sample preparation. The immuno-affinity extraction and the surrogate peptide workflows described in literature are complex and laborious, adding cost and complexity to the analysis. High resolution mass spectrometry (HRMS) systems are usually the preferred platforms to perform intact protein analysis, but have rarely been used routinely for quantitative bioanalytical applications. With recent advances in MS instrumentation and software capabilities, use ofHRMS for bioanalytical quantification is on the rise. Some labs have reported quantifying IGF-I using immuno-affinity extraction and followed by nano-flow LC and a HRMS system 2 . Here, we highlight a simplified sample preparation workflow using sample pretreatment, protein precipitation, and solid phase extraction (SPE) for the quantification of intact IGF-I from human serum for clinical research using an analytical LC with a tandem quadrupole instrument. We further compare its performance characteristics to a targeted HRMS approach for quantification. Figure 1. Structure of Insulin-like Growth Factor I Parameter Conditions LC System ACQUITY UPLC ® I-Class MS System Waters Xevo TQ-XS Mass Spectrometer, ESI+ Column ACQUITY UPLC CORTECS C18+, 90Å, 1.6 μm, 2.1 mm x 50 mm Column Temperature 60 o C Sample Temperature 5 o C Injection Volume 10 µL Mobile Phases A: 0.1% Formic Acid in H 2 O B: 0.1% Formic Acid in ACN Table 1a. Instrument Conditions Time (mins) Flow Rate (mL/min) %A %B Curve 0.0 0.400 95 5 6 2.5 0.400 70 30 6 3.5 0.400 50 50 6 3.6 0.400 5 95 6 4.0 0.400 5 95 6 4.1 0.400 95 5 6 5.0 0.400 95 5 6 Table 1b. LC Gradient Precursor (m/z) Product (m/z) Collision Energy (eV) Cone Voltage (V) 1093.0 (+7) 1196.4 35 30 956.4 (+8) 1196.4 30 30 Table 1c. MRM parameters METHODS References 1. Filipe Lopes, David A. Cowan, Mario Thevis, Andreas Thomas and Mark C. Parkin; Quantification of intact human insulin-like growth factor-I in serum by nano-ultra high-performance liquid chromatography/ tandem mass spectrometry; Rapid Commun. Mass Spectrom. 2014, 28, 1426–1432. 2. Martin Bidlingmaier, Nele Friedrich, Rebecca T. Emeny, Joachim Spranger, Ole D. Wolthers, Josefine Roswall, Antje Körner, Barbara Obermayer-Pietsch, Christoph Hübener, Jovanna Dahlgren, Jan Frystyk, Andreas F. H. Pfeiffer, Angela Doering, Maximilian Bielohuby, Henri Wallaschofski, and Ayman M. Arafat; Reference Intervals for Insulin-like Growth Factor-1 (IGF-I) From Birth to Senescence: Results From a Multicenter Study Using a New Automated Chemiluminescence IGF-I Immunoassay Conforming to Recent International Recommendations; J Clin Endocrinol Metab, May 2014, 99(5):1712–1721 RESULTS & DISCUSSION Circulating IGF-I binds very strongly to its binding partner, Insulin-like Growth Factor Binding Protein (IGFBP). Effectively disrupting this binding and preventing reformation of this complex during sample preparation was crucial for successful IGF-I recovery in serum/ plasma. Prior to sample extraction, IGF-II, which also binds strongly to IGFBP, was added in excess to prevent IGF-I-IGFBP complex reformation. Various serum pretreatment options were evaluated. Treatment with acid, base, denaturing reagents and protein precipitation (PPT) alone, or in combination were tested. Total IGF-I recovery (SDS pretreatment, PPT, and SPE) using the optimized protocol was >90% (Figure 3). LLOQ of 5 ng/mL of IGF-I (human sequence) was achieved in mouse plasma using the Xevo TQ-XS. Calibration curves, in both mouse plasma and human serum, were linear with r 2 values > 0.99 (1/x 2 weighted regression) with mean accuracies of and 101.76, and 99.98, respectively (Table 2). Precision and accuracy for both the mouse and human QC samples were excellent with mean % RSDs <7% and QC accuracy ranges of 93.9-107.7 (Table 3 and Figure 4, panel A). Calculated endogenous IGF-I human serum level (26.81 ng/mL) is shown in Table 3 and is also illustrated in Figure 4, panel B. Additionally, IGF-I was accurately quantified from 10-1000 ng/mL using the targeted mode of the Xevo G2-XS. Calibration curves were linear (1/x 2 weighting) with r 2 values >0.99 and mean accuracies between 98-101% (Table 2). QC performance was comparable to the TQ-XS, with accuracy ranges between 89-97% and CV’s < 10% (Table 3, Figure 5). Xevo TQ-XS (Mouse Plasma) Xevo TQ-XS (Human Serum) Xevo G2-XS (Mouse Plasma) Range (ng/mL) 5-1000 100-1000 10-1000 Weighting 1/x 2 1/x 2 1/x 2 Linear Fit (r 2 ) 0.991 0.994 0.993 Mean Accuracy (%) 101.76 99.98 100.03 Table 2. Calibration curve statistics for IGF-I Table 3. QC Statistics for IGF-I Xevo TQ-XS (Mouse Plasma) Xevo TQ-XS (Human Serum) Xevo G2-XS (Mouse Plasma) Expected Conc (ng/mL) 10 100 750 27 127 427 827 25 100 750 Cal Conc (ng/mL) 11 108 794 27 134 434 776 24 90 683 Mean Accuracy (%) 107.7 107.5 105.9 98.6 105.7 101.8 93.9 96.9 92.0 89.8 Mean CV (%) 6.5 5.6 5.4 3.3 5.5 1.6 2.0 9.3 3.2 3.4 0 10 20 30 40 50 60 70 80 90 100 H3PO4+SPE NH4OH+SPE PPT+SPE SDS+SPE SDS+PPT+SPE % Recovery % IGF-1 Recovery Figure 3. Sample preparation method development data showing >90% recovery for SDS+PPT+SPE CONCLUSION The clinical research method described employs a simple pretreatment and SPE sample preparation strategy combined with analytical flow LC and tandem-quadrupole MS for the direct analysis and quantification of intact IGF-I from serum/plasma. Sample preparation with simple SPE was < 1.5 hours, which is 3X faster than the complex sample preparation with protein digestion or affinity chromatography. Protein dissociation followed by PPT and a mixed-mode SPE strategy with OASIS MAX, effectively removes denaturant reagents, and improves analytical sensitivity, selectivity, and method robustness. Low LLOQ’s were achieved without the use of nano-flow LC, increasing robustness and reproducibility of the method and decreasing the run times. This method achieves analytical sensitivity of 5 ng/mL, linear dynamic range of 5-1,000 ng/mL, and accurately quantifies endogenous IGF-I with excellent robustness and reproducibility. Additionally, this work also highlights the analytical sensitivity and robustness of the HRMS platform (Xevo G2-XS) which meet the FDA guidelines implemented in most regulated bioanalysis laboratories. 100 µL of mouse plasma and human serum was spiked at various concentrations (5-1,000 ng/mL) with IGF-I. Samples were pretreated with 0.6% sodium dodecyl sulphate (SDS), incubated, precipitated with 5% acetic acid in acetonitrile and centrifuged. The supernatant was then pretreated with NH 4 OH and passed through an Oasis MAX µElution 96 well plate & eluted with 2x25 µL aliquots of 60% methanol containing 10% acetic acid. The eluate was diluted with 50 µL water and injected (Figure 2). LC-MS/MS conditions used are described below in tables 1a, 1b and 1c Figure 4. Representative chromatograms from Xevo TQ-XS for IGF-I spiked in A) Mouse Plasma and B) Human Serum Human IGF-I - Xevo TQ-XS Time 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 % 0 100 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 % 0 100 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 % 0 100 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 % 0 100 MRM of 7 Channels ES+ 1093 > 1196.4 (1093_1) 1.73e5 Area 418 MRM of 7 Channels ES+ 1093 > 1196.4 (1093_1) 1.73e5 Area 890 MRM of 7 Channels ES+ 1093 > 1196.4 (1093_1) 1.73e5 Area 1315 MRM of 7 Channels ES+ 1093 > 1196.4 (1093_1) 1.73e5 Area 6159 LOD - 2.5 ng/mL LLOQ - 5 ng/mL LQC - 10 ng/mL 50 ng/mL MOUSE PLASMA Time 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 % 0 100 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 % 0 100 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 % 0 100 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 % 0 100 MRM of 7 Channels ES+ 1093 > 1196.4 (1093_1) 1.73e6 Area 4282 MRM of 7 Channels ES+ 1093 > 1196.4 (1093_1) 1.73e6 Area 12402 MRM of 7 Channels ES+ 1093 > 1196.4 (1093_1) 1.73e6 Area 37287 MRM of 7 Channels ES+ 1093 > 1196.4 (1093_1) 1.73e6 Area 60647 Blank Serum (Endogenous) 27 ng/mL LQC - 127 ng/mL MQC - 427 ng/mL HQC - 827 ng/mL HUMAN SERUM Figure 5. Representative chromatograms from Xevo G2-XS for IGF-I spiked in Mouse Plasma Time 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 % 0 100 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 % 0 100 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 % 0 100 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 % 0 100 3: TOF MSMS ES+ 1093.716 0.0500Da 4.00e4 Area 3: TOF MSMS ES+ 1093.716 0.0500Da 4.00e4 Area 3: TOF MSMS ES+ 1093.716 0.0500Da 4.00e4 Area 3: TOF MSMS ES+ 1093.716 0.0500Da 4.00e4 Area Human IGF-I - Xevo G2-XS Blank LLOQ - 10 ng/mL LQC - 25 ng/mL MQC - 100 ng/mL 114 254 1134 Figure 2. Sample preparation Workflow
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TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2018 Waters Corporation | LL-pdf
FOR RESEARCH USE ONLY, NOT FOR USE IN DIAGNOSTIC PROCEDURES
LC-MS/MS QUANTIFICATION OF INTACT INSULIN- LIKE GROWTH FACTOR I (IGF-I) FROM SERUM FOR CLINICAL RESEARCH Nikunj Tanna
1, Khalid Khan
2, Mary Lame
1, Anthony Marcello
1, Ian Edwards
1, Mark Wrona
1
1 Waters Corporation, Milford, MA
2 Waters Corporation, Wilmslow, UK
INTRODUCTION Insulin-like Growth Factor I (IGF-I) is a 70 amino acid (7.6 kDa) peptide hormone, with 3 internal di-sulphide bonds (Figure 1). It plays a significant role in mediating the effects of Growth Hormone (GH), circulates at ng/mL levels, and is strongly bound to Insulin-like
Growth Factor Binding Protein (IGFBP).
Historically, immunoassays have been used for quantification of IGF-I. In recent years, use of LC-MS for quantification of IGF-I has increased. Most labs using LC-MS use the surrogate peptide approach, with or without immuno-affinity extraction, followed by quantification of resulting signature peptides by analytical scale LC with a tandem quadrupole (TQ) instrument. Although widely accepted, digestion may not always be required for proteins under 10 kDa on a TQ. However, achieving relevant analytical sensitivity levels for such intact proteins does require meticulous attention to sample preparation. The immuno-affinity extraction and the surrogate peptide workflows described
in literature are complex and laborious, adding cost and complexity to the analysis.
High resolution mass spectrometry (HRMS) systems are usually the preferred platforms to perform intact protein analysis, but have rarely been used routinely for quantitative
bioanalytical applications. With recent advances in MS instrumentation and software capabilities, use ofHRMS for bioanalytical quantification is on the rise. Some labs have reported quantifying IGF-I using immuno-affinity extraction and
followed by nano-flow LC and a HRMS system2.
Here, we highlight a simplified sample preparation workflow using sample pretreatment, protein precipitation, and solid phase extraction (SPE) for the quantification of intact IGF-I from human serum for clinical research using an analytical LC with a tandem quadrupole instrument. We further compare its performance characteristics to a
targeted HRMS approach for quantification. Figure 1. Structure of Insulin-like Growth Factor I
Parameter Conditions
LC System ACQUITY UPLC® I-Class
MS System Waters Xevo TQ-XS Mass Spectrometer, ESI+
Column ACQUITY UPLC CORTECS C18+, 90Å, 1.6 μm, 2.1 mm x 50 mm
Column Temperature 60 oC
Sample Temperature 5 oC
Injection Volume 10 µL
Mobile Phases A: 0.1% Formic Acid in H2O B: 0.1% Formic Acid in ACN
Table 1a. Instrument Conditions
Time (mins) Flow Rate (mL/min)
%A %B Curve
0.0 0.400 95 5 6
2.5 0.400 70 30 6
3.5 0.400 50 50 6
3.6 0.400 5 95 6
4.0 0.400 5 95 6
4.1 0.400 95 5 6
5.0 0.400 95 5 6
Table 1b. LC Gradient
Precursor (m/z) Product (m/z) Collision Energy (eV) Cone Voltage (V)
1093.0 (+7) 1196.4 35 30
956.4 (+8) 1196.4 30 30
Table 1c. MRM parameters
METHODS
References
1. Filipe Lopes, David A. Cowan, Mario Thevis, Andreas Thomas and Mark C. Parkin; Quantification of intact human insulin-like growth factor-I in serum by nano-ultra high-performance liquid chromatography/tandem mass spectrometry; Rapid Commun. Mass Spectrom. 2014, 28, 1426–1432.
2. Martin Bidlingmaier, Nele Friedrich, Rebecca T. Emeny, Joachim Spranger, Ole D. Wolthers, Josefine Roswall, Antje Körner, Barbara Obermayer-Pietsch, Christoph Hübener, Jovanna Dahlgren, Jan Frystyk, Andreas F. H. Pfeiffer, Angela Doering, Maximilian Bielohuby, Henri Wallaschofski, and Ayman M. Arafat; Reference Intervals for Insulin-like Growth Factor-1 (IGF-I) From Birth to Senescence: Results From a Multicenter Study Using a New Automated Chemiluminescence IGF-I Immunoassay Conforming to Recent International Recommendations; J Clin Endocrinol Metab, May 2014, 99(5):1712–1721
RESULTS & DISCUSSION Circulating IGF-I binds very strongly to its binding partner, Insulin-like Growth Factor Binding Protein (IGFBP). Effectively disrupting this binding and preventing reformation of this complex during sample preparation was crucial for successful IGF-I recovery in serum/plasma. Prior to sample extraction, IGF-II, which also binds strongly to IGFBP, was added in excess to prevent IGF-I-IGFBP complex reformation. Various serum pretreatment options were evaluated. Treatment with acid, base, denaturing reagents and protein precipitation (PPT) alone, or in combination were tested. Total IGF-I recovery (SDS pretreatment, PPT, and SPE) using the optimized protocol was >90% (Figure 3).
LLOQ of 5 ng/mL of IGF-I (human sequence) was achieved in mouse plasma using the Xevo TQ-XS. Calibration curves, in both mouse plasma and human serum, were linear with r2 values > 0.99 (1/x2 weighted regression) with mean accuracies of and 101.76, and 99.98, respectively (Table 2). Precision and accuracy for both the mouse and human QC samples were excellent with mean % RSDs <7% and QC accuracy ranges of 93.9-107.7 (Table 3 and Figure 4, panel A). Calculated endogenous IGF-I human serum level (26.81 ng/mL) is shown in Table 3 and is also illustrated in Figure 4, panel B.
Additionally, IGF-I was accurately quantified from 10-1000 ng/mL using the targeted mode of the Xevo G2-XS. Calibration curves were linear (1/x2 weighting) with r2 values >0.99 and mean accuracies between 98-101% (Table 2). QC performance was comparable to the TQ-XS, with accuracy ranges between 89-97% and CV’s < 10% (Table 3, Figure 5).
Xevo TQ-XS
(Mouse Plasma) Xevo TQ-XS
(Human Serum) Xevo G2-XS
(Mouse Plasma)
Range (ng/mL) 5-1000 100-1000 10-1000
Weighting 1/x2 1/x2 1/x2
Linear Fit (r2) 0.991 0.994 0.993
Mean Accuracy (%) 101.76 99.98 100.03
Table 2. Calibration curve statistics for IGF-I
Table 3. QC Statistics for IGF-I
Xevo TQ-XS
(Mouse Plasma) Xevo TQ-XS (Human Serum)
Xevo G2-XS (Mouse Plasma)
Expected Conc (ng/mL)
10 100 750 27 127 427 827 25 100 750
Cal Conc (ng/mL)
11 108 794 27 134 434 776 24 90 683
Mean Accuracy (%)
107.7 107.5 105.9 98.6 105.7 101.8 93.9 96.9 92.0 89.8
Mean CV (%) 6.5 5.6 5.4 3.3 5.5 1.6 2.0 9.3 3.2 3.4
0
10
20
30
40
50
60
70
80
90
100
H3PO4+SPE NH4OH+SPE PPT+SPE SDS+SPE SDS+PPT+SPE
% R
eco
ve
ry
% IGF-1 Recovery
Figure 3. Sample preparation method development data showing >90% recovery for SDS+PPT+SPE
CONCLUSION The clinical research method described employs a simple pretreatment and SPE sample preparation strategy combined with
analytical flow LC and tandem-quadrupole MS for the direct analysis and quantification of intact IGF-I from serum/plasma.
Sample preparation with simple SPE was < 1.5 hours, which is 3X faster than the complex sample preparation with protein
digestion or affinity chromatography.
Protein dissociation followed by PPT and a mixed-mode SPE strategy with OASIS MAX, effectively removes denaturant
reagents, and improves analytical sensitivity, selectivity, and method robustness.
Low LLOQ’s were achieved without the use of nano-flow LC, increasing robustness and reproducibility of the method and
decreasing the run times.
This method achieves analytical sensitivity of 5 ng/mL, linear dynamic range of 5-1,000 ng/mL, and accurately quantifies
endogenous IGF-I with excellent robustness and reproducibility.
Additionally, this work also highlights the analytical sensitivity and robustness of the HRMS platform (Xevo G2-XS) which meet
the FDA guidelines implemented in most regulated bioanalysis laboratories.
100 µL of mouse plasma and human serum was spiked at various concentrations (5-1,000 ng/mL) with IGF-I. Samples were pretreated with 0.6% sodium dodecyl sulphate (SDS), incubated, precipitated with 5% acetic acid in acetonitrile and centrifuged. The supernatant was then pretreated with NH4OH and passed through an Oasis MAX µElution 96 well plate & eluted with 2x25 µL aliquots of 60% methanol containing 10% acetic acid. The eluate was diluted with 50 µL water and injected (Figure 2).
LC-MS/MS conditions used are described below in tables 1a, 1b and 1c
Figure 4. Representative chromatograms from Xevo TQ-XS for IGF-I spiked in A) Mouse Plasma and B) Human Serum
Human IGF-I - Xevo TQ-XS
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80
%
0
100
MRM of 7 Channels ES+ 1093 > 1196.4 (1093_1)
1.73e5Area
418
MRM of 7 Channels ES+ 1093 > 1196.4 (1093_1)
1.73e5Area
890
MRM of 7 Channels ES+ 1093 > 1196.4 (1093_1)
1.73e5Area
1315
MRM of 7 Channels ES+ 1093 > 1196.4 (1093_1)
1.73e5Area
6159
LOD - 2.5 ng/mL
LLOQ - 5 ng/mL
LQC - 10 ng/mL
50 ng/mL
MOUSE PLASMA
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80
%
0
100
MRM of 7 Channels ES+ 1093 > 1196.4 (1093_1)
1.73e6Area
4282
MRM of 7 Channels ES+ 1093 > 1196.4 (1093_1)
1.73e6Area
12402
MRM of 7 Channels ES+ 1093 > 1196.4 (1093_1)
1.73e6Area
37287
MRM of 7 Channels ES+ 1093 > 1196.4 (1093_1)
1.73e6Area
60647
Blank Serum (Endogenous) 27 ng/mL
LQC - 127 ng/mL
MQC - 427 ng/mL
HQC - 827 ng/mL
HUMAN SERUM
Figure 5. Representative chromatograms from Xevo G2-XS for IGF-I spiked in Mouse Plasma
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40
%
0
100
3: TOF MSMS ES+
1093.716 0.0500Da
4.00e4Area
3: TOF MSMS ES+
1093.716 0.0500Da4.00e4
Area
3: TOF MSMS ES+
1093.716 0.0500Da4.00e4
Area
3: TOF MSMS ES+ 1093.716 0.0500Da
4.00e4
Area
Human IGF-I - Xevo G2-XS
Blank
LLOQ - 10 ng/mL
LQC - 25 ng/mL
MQC - 100 ng/mL
114
254
1134 Figure 2. Sample preparation Workflow
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