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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). Improper balance of GH and IGF-I can lead to conditions such as acromegaly, dwarfism, and increased risk of cancer. Thus, there is significant interest in measuring IGF-I as a biomarker.2

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 and 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 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 of HRMS 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 using an

analytical LC and a tandem quadrupole instrument. We

further compare its performance characteristics to a

targeted HRMS approach for quantification.

LC-MS/MS QUANTIFICATION OF INTACT INSULIN-LIKE GROWTH FACTOR I (IGF-I) FROM SERUM

Authors: Nikunj Tanna, Mary Lame, Erin Chambers, Anthony Marcello, Ian Edwards and Mark Wrona Affiliations: Waters Corporation

Figure 1. Structure of Insulin-like Growth Factor I

METHODS

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

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. Hemamalini Ketha, Ravinder J. Singh; Clinical assays for quantitation of insulin-like-growth-factor-1

(IGF1); Methods 81 (2015) 93–98.

3. 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).

CONCLUSION

The 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 sensitivity, specificity, and assay robustness.

Low LLOQ’s were achieved without the use of nano-flow LC, increasing robustness and reproducibility of the assay 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 sensitivity and robustness of the HRMS platform (Xevo G2-XS) which meet the FDA guidelines implemented in most regulated bioanalysis laboratories.

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

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

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

Figure 2. Sample preparation Workflow

Table 1a. Instrument Conditions

Table 1b. LC Gradient

Table 1c. MRM parameters

Table 3. QC Statistics for IGF-I

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

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) 10.8 107.5 794.1 26.8 134.3 434.4 776.2 24.2 89.5 682.7

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

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

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

Blank Serum (Endogenous) 27 ng/mL

LQC - 127 ng/mL

MQC - 427 ng/mL

HQC - 827 ng/mL

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 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

Table 2. Calibration curve statistics for IGF-I

Figure 4. Representative chromatograms from Xevo TQ-XS for IGF-I spiked in A) Mouse Plasma and B) Human Serum

Figure 3. Sample preparation method development data showing >90% recovery for SDS+PPT+SPE

Figure 5. Representative chromatograms from Xevo G2-XS for IGF-I spiked in Mouse Plasma

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MOUSE PLASMA HUMAN SERUM