Download The microflow LC method

For Research Use Only. Not for use in diagnostic procedures.
A highly sensitive, accurate LC-MS/MS quantitation method for glucagon peptide hormone in human plasma
Witold Woroniecki1; Lei Xiong1; Rahul Baghla1; Suma Ramagiri2
1SCIEX, Redwood City, California; 2SCIEX, Concord, ON
SCIEX QTRAP® LC-MS/MS System
A.
ABSTRACT
C.
Glucagon
The purpose of this study was to develop a highly sensitive and robust LC-MS/MS method that measures
glucagon levels in human plasma for accurate assessments of glucose homeostasis. Herein, we describe an
MRM acquisition method utilizing the QTRAP® 6500 System for achieving the high level of sensitivity and the
lowest limit of quantitation for the detection of glucagon peptide. Two liquid chromatography approaches were
developed: 1) a conventional LC method for high-throughput analysis and 2) a microflow LC method for limitedvolume samples.
Table 1
INTRODUCTION
Glucagon, a peptide hormone produced by alpha cells in the pancreas, raises blood glucose levels by
converting stored glycogen to glucose (Figure 1 shows the chemical structure), an activity complementary to
that of the peptide hormone insulin, which lowers elevated blood glucose levels. Typically, glucagon levels are
low in healthy patients (< 200 pg/mL), but during certain pathophysiological states, glucagon can be elevated. A
highly sensitive and robust LC-MS method for monitoring glucagon peptide levels in human plasma is needed
for accurate assessment of glucose homeostasis.
B.
We developed a traditional MRM acquisition method that utilizes the QTRAP® 6500 LC-MS/MS System to
detect glucagon in human plasma at high sensitivity and low limits of quantitation. Importantly, we demonstrate
an accurate and reproducible methodology for the preparation and analysis of glucagon in human plasma, and
we show that glucagon can be detected at levels as low as 10 pg/mL using either conventional high-flow or
microflow liquid chromatography separation strategies.
Figure 1. A. SCIEX QTRAP® 6500 LC-MS/MS System. B. Schematic overview of the MRM acquisition
technique. C. Chemical structure of glucagon, a non-steroid peptide hormone (29 amino acids, MW 3485 Da).
MATERIALS AND METHODS
RESULTS
Sample Preparation:
Human plasma was spiked with glucagon over a range of concentrations (10–10,000 pg/mL); internal standard,
([¹³C₆]Leu¹⁴)-glucagon was added to each sample. Glucagon was extracted from plasma using a strong anionexchange SPE process, prior to elution using 25/65 H2O/ACN with 10% acetic acid (50 μL). Samples were
diluted (50 μl H2O) prior to LC-MS/MS injection. Replicates (3) were performed for each concentration.
Achieving a robust method
HPLC Conditions:
Conventional Flow:
A Shimadzu Prominence LC system was used with an Phenomenex Kinetex C18, 100 x 3 mm, 2.6 μm column
coupled with a Phenomenex KrudKatcher pre-filter at 700 μL/min. Mobile phase consisted of 0.1% formic acid
in H2O (A) and in ACN (B). Sample (30 μL) was loaded at 5% B for 0.9 min and eluted by increasing B from 5–
45% in 4.5 min, followed by re-equilibration at 90% B for 0.7 min (cycle total, 7.5 min).
Non-specific binding to glass or stainless steel components can be a concern during peptide analysis,
particularly for longer, hydrophobic peptides, like glucagon. To evaluate sample absorption during peptide
extraction and quantitation, glucagon levels were monitored over time in different vial types, dilution solvents,
and LC components. We discovered that glucagon binds strongly to glass vials, resulting in a 50% loss in
peptide signal after 24 hours, compared to low-binding polypropylene vials. Similarly, storage in glass vials for
96 hours resulted in ~3x peptide losses compared to polypropylene. Neither loop type (PEEK or stainless steel)
nor acetonitrile content had any effect on sample absorption during chromatography.
A)
Table 2. Microflow quantitation statistics. Glucagon spiked into plasma was measured over a range of
concentrations (10–10,000 pg/mL) using the MRM transition of 697.6/813.4 under microflow conditions
(25 μL/min).
Quantitation Sensitivity
The conventional LC
The microflow LC method
Glucagon was measured in human plasma using a microflow LC approach that employed the same MRM
transitions (697.6/813.4) as the conventional approach. Calibration curves generated using the microflow
method were linear over the entire range of concentrations evaluated with a regression coefficient of 0.99964
(Figure 3A). Similarly to the conventional LC method, the LOQ obtained under microflow conditions was the
same as the lowest tested concentration (10 pg/mL) but was obtained using a smaller injection volume (5 μL).
Representative chromatograms of the blank sample, the LOQ, and replicates across the linear range are shown
(Figure 3B). Table 2 shows the quantitation statistics for the microflow method; the LOQ (10 ng/mL) displays a
CV of 1.41% and an accuracy of 98.2%. For the remaining concentrations, CV’s were less than 5%, and
accuracy was within 10%. For both chromatography methods, concentrations higher than 10,000 pg/mL were
also expected to be linear, but they were not evaluated because they were not physiologically relevant.
An MRM acquisition method on the QTRAP 6500 System was used to detect glucagon with high sensitivity in
human plasma. For conventional-flow LC, the calibration curve (MultiQuant™ Software) was linear over a
wide range of concentrations (10–10,000 pg/mL) with a regression coefficient (r 2) of 0.99934 (Figure 2A). The
limit of quantification (LOQ) was 10 pg/mL under conventional flow conditions. Representative MRM
chromatograms for glucagon are shown for blank samples, LOQ, and other concentrations in the range
evaluated (Figure 2B). Table 1 displays the quantitation statistics for the assay; a coefficient of variance (CV)
less than 7% and accuracy of 103% is shown for the LOQ sample (10 pg/mL). For all other calibration
standards, the CV was less than 5%, and accuracy was within 10%.
A)
r2 = 0.99964
r2 = 0.99934
MicroFlow:
An Eksigent 425 LC system was used with an Eksigent Halo C18, 50 x 0.5 mm, 3 μm column coupled with a
Thermo Scientific trap column at 25 μL/min. Mobile phase was 0.1% formic acid in H2O (A) and in ACN (B).
Sample (5 μL) was loaded onto the trap column of an HPLC column-switching system using 5% B at
0.25 mL/min (1 min). After desalting/cleanup and trap alignment with the analytical column, and sample was
eluted with gradient, the total cycle time is 7 min.
MS/MS Conditions:
MRM acquisitions were performed on a SCIEX QTRAP® 6500 LC-MS/MS system with IonDrive™ Turbo V ion
source. Samples were ionized by ESI in positive ion mode with an ISV of 5500 V, CAD gas was set to high, and
a dwell time of 100 ms was used.
Table 1. Conventional-flow quantitation statistics. Glucagon spiked into plasma was measured over a
range of concentrations (10–10,000 pg/mL) using the MRM transition of 697.6/813.4 under conventional LC
flow conditions (700 μL/min).
CONCLUSIONS
Microflow LC
Conventional flow LC
•
•
B)
0 pg/ml
10 pg/ml
20 pg/ml
100 pg/ml
B)
0 pg/ml
10 pg/ml
20 pg/ml
100 pg/ml
•
Glucagon levels were robustly quantified using a high-throughput, conventional, high-flow LC methodology
for larger sample volumes; a microflow LC method was developed for smaller injection volumes to
accommodate limited-volume samples. In each case, an accurate and reproducible LOQ of 10 pg/mL was
achieved for each chromatography approach.
Glucagon’s peptide properties, stability, and non-specific adsorption were considered during the method
development process, resulting in a robust quantitative assay.
High-throughput glucagon peptide quantitation for large or small sample volumes can be achieved on the
SCIEX QTRAP 6500 system due to the sensitivity gains realized with IonDrive™ technology.
REFERENCES
Data processing:
Data was acquired with Analyst® 1.6.2 Software, and quantitation of glucagon were performed with
MultiQuantTM 3.0 Software. Multiple analytical runs with various plasma sources demonstrated that transitions
697.6/813, and 697.6/940.5 have lowest baseline noise and are most suitable for reliable quantification of
glucagon.
1. Woroniecki, W. and Jonakin, K. “Highly sensitive and accurate quantification of glucagon peptide hormone in
human plasma.” SCIEX technical note 8540113-01.
2.Bi
TRADEMARKS/LICENSING
Figure 2. MRM data obtained under conventional flow LC conditions. A. The calibration curve for
glucagon was linear for a wide concentration range (10–10,000 pg/mL). B. Representative chromatograms of
glucagon concentrations (0, 10, 20 and 100 pg/mL) are shown for MRM transition 697.6/813.4.
Figure 3. MRM data obtained under microflow LC conditions. A. Calibration curve obtained from MRM
transition 697.6/813.4 B. Representative chromatograms of a range of glucose concentrations (0, 10, 20 and
100 pg/mL) were monitored using MRM transitions 697.6/813.4.
For Research Use Only. Not for use in diagnostic procedures.
The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SciexTM
is being used under license.
© 2015 AB Sciex.