lc-ms/ms quantification of intact insulin-like …andreas f. h. pfeiffer, angela doering, maximilian...

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

Nikunj Tanna, Caitlin Dunning, Mary Lame, Mark Wrona Waters Corporation, Milford, MA

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

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 2. Sample preparation Workflow

METHODS 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

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.

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.

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


1.73e5Area

890


1.73e5Area

1315


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


1.73e6Area

4282


1.73e6Area

12402


1.73e6Area

37287


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

Human IGF-I - Xevo G2-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 0.0500Da

4.00e4Area

3: TOF MSMS ES+ 1093.716 0.0500Da

4.00e4Area

3: TOF MSMS ES+ 1093.716 0.0500Da

4.00e4Area

Blank

LLOQ - 10 ng/mL

LQC - 25 ng/mL

MQC - 100 ng/mL

114

254

1134

FOR RESEARCH USE ONLY, NOT FOR USE IN DIAGNOSTIC PROCEDURES

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