A new high resolution approach to the detection of insulin
degradation products
Stephen Lock
SCIEX, Warrington, United Kingdom
Introduction
In drug manufacturing, storage studies are needed for safety andefficacy reasons to identify toxic breakdown products and determine theshelf life of drugs such as insulin. In this study we have looked tocompare and contrast capillary electrospray ionization mass
spectrometry (CESI-MS) and high flow liquid chromatography-mass
spectrometry (LC-MS) in the analysis of degraded insulin.
Standard reverse phase liquid chromatography separates constituents
based on their differential interactions with the column stationary phaseand an organic mobile phase. CESI-MS is a different separation
technique and is the integration of CE and ESI into one dynamic process
and provides highly efficient peptide and protein separations based on
their size and charge (1). The ionization is at the ultra-low nanoflow
regime (~25 nL/min) and it simply uses an open capillary (Figure 1)
which eliminates some of the challenges of stationary phase based
separations such as protein recovery or conformational changes using
reverse phase gradient elution solvents.
The purpose of this work was to compare the breakdown products
detected in the presence of high levels of the parent drug using both
CESI-MS and LC-MS techniques. In this comparison the MS method
remained the same with the exception of the source conditions which
had to be optimized for the different flow rates used by CESI and LC.
The peak height of the parent insulin peak was adjusted by modifying theinjection conditions so levels of the impurities detected were not biased.
In the case of the LC method, this meant reducing the injection volume to2 µL which also helped reduce peak tailing of the parent insulin.

LC-MS method: For the LC-MS analysis 2 µL of the insulin sample wasinjected onto an Aeris peptide HPLC column (XB-C18, 2.7µm, 2.1 x100mm) which has been developed for the analysis of peptides. For thisanalysis the SCIEX TripleTOF® 5600+ mass spectrometer was fitted withthe DuoSpray™ source. Gas 1 was 50 psi, Gas 2 was 60 psi, curtaingas was 30 psi, Temperature 450 ⁰C, and the ion-spray voltage was setat 5500 V. The HPLC separation used the gradient elution shown in
Table 1 where mobile phase A was water containing 0.1% formic acidand mobile phase B was acetonitrile containing 0.1% formic acid with thecolumn held at 25 ºC. Data was acquired using a TOF-MS method overthe mass range 600 – 2000 amu with DP set to 100 V and CE set to 10V.
Step0123456Figure 1: OptiMS - Sheathless ESI Interface.

Materials and Methods
Chemicals: All chemicals were Reagent Grade and were purchasedfrom Sigma Aldrich.
Sample Preparation: Insulin (Sigma I2643: 5.44 mg dissolved) wasdissolved in 2 mM phosphate buffer (1 mL). Hydrochloric acid (2 M, 15µL) was added and mixed until the insulin had dissolved. Sodiumhydroxide (2 M, 15 µL) was added followed by phosphate buffer (4.4mL). The sample was then degraded for 3½ days (50°C). Beforeanalysis the sample was diluted with an equal volume of ammonium
acetate (200 mM).

Flow Rate(µL/min)
400
400
400
400
400
400400% MobilePhase A9595
5010109595% MobilePhase B
55509090
55Table 1:- HPLC Conditions used for Reverse phase separation ofdegradation products of insulin
CESI-MS method: Sample was injected by pressure (3 psi, 5 s) onto a30 μm ID x 95 cm bare-fused-silica capillary (including porous spray tip)housed in an OptiMS CESI cartridge. The capillary was covalently
coated with Polyethylenimine (PEI) (2) and thermostatted using
recirculating liquid coolant regulated at 25oC. For this analysis, theSCIEX TripleTOF® 5600+ mass spectrometer was fitted with theNanoSpray® III source. Gas 1, gas 2 and temperature were not applied(set to 0, 0 and 50 respectively) because ionization at these very low flow
rates occurs by simply applying the ionspray voltage set at 2000 V. The
curtain was set very low at 5psi (set automatically in the CESI software).
The CE separation used the condition shown in Table 2 with abackground electrolyte of 10% acetic acid. Data was acquired by TOFMS over the mass range 600 – 2000 amu, conditions identical to thoseused for the LC-MS analysis.
Action®

Time(mins)
01
10
12
13
13.2
15

Rinse
Rinse
Rinse
Rinse
Rinse
Rinse
Injection
Injection
Separation
VoltageTime(mins)
31323210s
30s
153

Pressure(psi)757575757575
30.5
0.50.5

Direction
Forward
Forward
Forward
Forward
Forward
ReverseForward
Forward
Forward
ForwardVoltage(kV)
00000000251

Solution
MethanolWater
1 M NaCl
Water
10% Acetic acid
10% Acetic acid
Sample Vial
10% Acetic acid
10% Acetic acid
10% Acetic acid

Table 2:- CESI separation method used for the separation ofdegradation products of insulinp1

Results
Analytes separate in CE based on their mobility (m/z)and elute in HPLCCESI-MS

based on hydrophobic interactions with the stationary phase in thecolumn. Both techniques showed complementary specificity with peaks
moving in opposite directions. In the LC-MS experiment, insulin elutes at
6.8 minutes accompanied by an early eluting sharp peak corresponding
to the solvent front and a late eluting broad peak corresponding to theLC-MS

high organic section of the gradient elution (Fig 2). In both of theseareas only singly charged peaks could be found.

Neither of thesefeatures was present in the CESI-MS electropherogram where the broadpeak at 7.5 minutes contained the majority of the singly charged species

Figure 3:- Expanded region around insulin parent peak to compare

and insulin migrated at 9.4 minutes.

resolution of major degradation product.

In these experiments, the injection pressure and time in the CESI-MS

Figure 4 shows the region where products from the breakage of theanalysis together with the injection volume of the LC-MS analysis wasinsulin backbone elute and migrate. In the CESI analysis these cleavage

adjusted so that the response for the insulin peak (the most prominent

products migrate in a sharp peak easily detected at 7.2 minutes . In thepeak) was the same in both injections.

LC-MS analysis these constituents elute in a less distinguishable peak at
6.3 minutes and in addition is missing several of the diagnostic ions(notable peaks at 2434 and 2460 amu) which were present in the CESIMS analysis.

Components move by charge
and size

CESI-MS

Components move by interaction
with stationary phase

LC-MS

Figure 2:- Direct comparison on a CESI-MS electropherogram and a LCMS chromatogram from the injection of the same insulin sample.

Figure 4:- Expanded region containing cleavage products from breakageof the insulin backbone.

Both CESI-MS and LC-MS data were studied in depth to compare
resolution and the capability of both techniques to identify low level

In Figure 5 the peak at 8.9 minutes was the result of loss of terminal

impurities. In Figure 3 the region around the parent peak for insulin has

amino acids which generated a component of mass 5430 amu. In thebeen expanded to show the resolution for the major impurity peak which

HPLC analysis this component co-eluted under the parent mass and its

was formed as a result of a deamination modification. In CESI-MS thisretention time was identified using extracted ion chromatograms.

peak migrates before the parent peak and for LC-MS this peak elutes
after the parent. In both cases the deamination resulted in a small masschange of 1 amu but also resulted in a charge alteration. The CESI-MS

CESI-MSgave better resolution than HPLC-MS with a baseline separation of thisproduct from the parent insulin. Separation of this major product isimportant as this small mass change would otherwise be masked by thespectra of the parent ions.

LC-MSFigure 5:- Detection of the loss of terminal amino acidsp2

Finally in Figure 6 two components, resulting from the degradation
process, were identified in the CESI-MS which were not detected by LCMS. One was a dimer of two insulin molecules which migrated as aminor peak at 10.35 minutes.

This peak as expected migrated after

insulin (9.4 minutes) as it was twice as big (11615 amu). In addition

Conclusions
LC-MS and CESI-MS have been shown to becomplementary techniques because of their differing
mechanism of separation.

there was a shoulder eluting before the parent ion at 9.1 minutes. Thiswas identified as an additional deamination product which under theseparation conditions used had a slightly different charge which caused it
to migrate after the major deamination product. This 2nd deamination
product could not be distinguished in the LC-MS analysis.

Both techniques were capable of detecting low level
degradation products which were less than 0.5% of the areaof the insulin peak.
In this study, CESI-MS exhibited better peak resolution fordegradation products of insulin and was capable of detecting
additional features not detected by the LC-MS method.

Both LC-MS and CESI-MS analysis were capable of detecting
degradation products which were less than 1% the size of the parentpeak.

References
1.

2.

2nd deamination

New dimer

product

product

Busnel, J-M., et. al., “High Capacity Capillary
Electrophoresis-Electrospray Ionization Mass
Spectrometry: Coupling a Porous Sheath lessInterface with Transient-Isotachophoresis”, Anal.
Chem. 82 9476-9483 2010.
Santos, M. R., et al.” A covalent, cationic polymer
coating method for the CESI-MS analysis of intact
proteins and polypeptides”, SCIEX Separations
application note, 2015.

Figure 6:- Components identified in the CESI-MS analysis which were
not found in the LC-MS analysis.

Document number: RUO-MKT-02-2586-A© 2015 AB Sciex. For Research Use Only. Not for use in diagnostic procedures. AB Sciex doing business as SCIEX.
The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license

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