A New High-resolution Approach to the Detectionof Insulin Degradation Products
Stephen Lock
SCIEX, Warrington, UKIntroduction
In drug manufacturing, storage studies are needed for safetyand efficacy reasons to identify toxic breakdown products anddetermine the shelf life of drugs such as insulin. In this study,
we have looked to compare and contrast capillary electrosprayionization mass spectrometry (CESI-MS) and high flow liquid
chromatography-mass spectrometry (LC-MS) in the analysis ofdegraded insulin.

Figure 1. The PA800 P

Standard reverse phase liquid chromatography separates

Figure 1. The OptiMS cartridge provides an ultra-low flow ESI interface.

constituents based on their differential interactions with thecolumn stationary phase and an organic mobile phase. CESI-Materials and methods

MS is a different separation technique and is the integration ofChemicals: All chemicals were Reagent Grade and wereCE and ESI into a single dynamic process and provides highly
efficient peptide and protein separations based on their sizeand 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 phasebased separations such as protein recovery or conformationalpurchased from Sigma Aldrich.
Sample Preparation: Insulin (Sigma I2643, 5.44 mg dissolved)
was dissolved in 2 mM phosphate buffer (1 mL). Hydrochloric
acid (2 M, 15 μL) was added and mixed until the insulin haddissolved. Sodium hydroxide (2 M, 15 μL) was added followed bychanges using reverse phase gradient elution solvents.

phosphate buffer (4.4 mL). The sample was then degraded for

The purpose of this work was to compare the breakdown

an equal volume of ammonium acetate (200 mM).

products detected in the presence of high levels of the parent
drug using both CESI-MS and LC-MS techniques. In this3 1⁄2 days (50 °C). Before analysis, the sample was diluted with

StepTime (min)Flow rate(µL/min)% mobile
phase A% mobile
phase B

exception of the source conditions, which had to be optimized for

00400955

the different flow rates used by CESI and LC. The peak height of11400955

the parent insulin peak was adjusted by modifying the injection

2104005050conditions, so levels of the impurities detected were not biased.

3124001090

In the case of the LC method, this meant reducing the injection

4134001090

513.240095

5615400955comparison, the MS method remained the same with thevolume to 2 μL, which also helped reduce peak tailing of theparent insulin.

Table 1. HPLC conditions used for reverse phase separation ofdegradation products of insulin.

p1

LC-MS method: For the LC-MS analysis, 2 μL of the insulin

charged peaks could be found. Neither of these features wassample was injected onto an Aeris peptide HPLC column

present in the CESI-MS electropherogram where the broad peak

(XB- C18, 2.7 μm, 2.1 x 100 mm) that has been developed forat 7.5 min contained the majority of the singly charged species

the analysis of peptides. For this analysis, the SCIEX TripleTOF®

and insulin migrated at 9.4 min.

5600+ LC-MS/MS System was fitted with the DuoSpray™ Turbo
V Ion Source. Gas 1 was 50 psi, gas 2 was 60 psi, curtain gas
was 30 psi, temperature was 450 °C and the ion-spray voltage
was set at 5500 V. The HPLC separation used the gradientelution shown in Table 1, where mobile phase A was watercontaining 0.1% formic acid, and mobile phase B was acetonitrile
containing 0.1% formic acid with the column held at 25 °C. Data
was acquired using a TOF-MS method over the mass range
600–2000 Da with DP set to 100 V and CE set to 10 V.
CESI-MS method: Sample was injected by pressure (3 psi,5 s) onto a 30 μm ID x 95 cm bare fused-silica capillary(including porous spray tip) housed in an OptiMS cartridge.
The capillary was covalently coated with polyethylenimine (PEI)2and thermostatted using recirculating liquid coolant regulated
at 25 °C. For this analysis, the SCIEX TripleTOF® 5600+ System
was fitted with the NanoSpray® III Ion Source. Gas 1, gas 2 andtemperature were not applied (set to 0, 0 and 50, respectively).
In order to generate ionization at these very low flow rates,
simply apply ion-spray voltage below 2000 V (typically
900–1800 V is sufficient). The curtain gas was set very low
at 5 psi (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 acquiredby TOF MS over the mass range 600–2000 Da, with conditions
identical to those used for the LC-MS analysis.

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

In these experiments, the injection pressure and time in theCESI-MS analysis together with the injection volume of theLC-MS analysis was adjusted so that the response for the insulin
peak (the most prominent peak) was the same in both injections.
Both CESI-MS and LC-MS data were studied in depth tocompare resolution and the capability of both techniques toidentify low-level impurities. In Figure 3, the region around theparent peak for insulin has been expanded to show the resolution
for the major impurity peak, which was formed as a result of adeamination modification. In CESI-MS, this 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 mass changeof 1 Da, but also resulted in a charge alteration. The CESI-MS
gave better resolution than HPLC-MS with a baseline separation• A separation current above 5 μA might cause permanent
damage to the separation capillary.

of this product from the parent insulin. Separation of this major• Generally, please do not apply >2000V to generate
electrospray as it may result in capillary damage.

be masked by the spectra of the parent ions.

product is important as this small mass change would otherwise

Results
Analytes separate in CE based on their mobility (m/z) and elutein HPLC based on hydrophobic interactions with the stationary
phase in the column. Both techniques showed complementary
specificity with peaks moving in opposite directions. In the LC-MS
experiment, insulin elutes at 6.8 min accompanied by an early
eluting sharp peak corresponding to the solvent front and a lateeluting broad peak corresponding to the high organic section of
the gradient elution (Figure 2). In both of these areas, only singly

Figure 3. Expanded region around insulin parent peak to compare
resolution of major degradation product.
p2Finally, in Figure 6, 2 components resulting from the degradation
process were identified in the CESI-MS that were not detectedby LC-MS. The first was a dimer of 2 insulin molecules that
migrated as a minor peak at 10.35 min. This peak, as expected,
migrated after insulin (9.4 min) as it was twice as big (11615 Da).
In addition, there was a shoulder eluting before the parent ion
at 9.1 min. This was identified as an additional deamination
product that, under the separation conditions used, had a slightly
different charge, which caused it to migrate after the major
Figure 4. Expanded region containing cleavage products from breakageof the insulin backbone.

Figure 4 shows the region where products from the breakageof the insulin backbone elute and migrate. In the CESI analysis,
these cleavage products migrate in a sharp peak easily detecteddeamination product. This second deamination product could
not be distinguished in the LC-MS analysis.
Both LC-MS and CESI-MS analysis were capable of detecting
degradation products that were less than 1% of the size of theparent peak.

at 7.2 min. In the LC-MS analysis, these constituents elute in aless distinguishable peak at 6.3 min and in addition is missing
several of the diagnostic ions (notable peaks at 2434 and2460 Da) that were present in the CESI-MS analysis.

Figure 6. Components identified in the CESI-MS analysis that were notfound in the LC-MS analysis.

Conclusions
Figure 5. Detection of the loss of terminal amino acids.

In Figure 5, the peak at 8.9 min was the result of loss of terminal
amino acids, which generated a component of mass 5430 Da.
In the HPLC analysis, this component co-eluted under the parent
mass and its retention time was identified using extracted
ion chromatograms.

LC-MS and CESI-MS have been shown to be complementary
techniques because of their differing mechanism of separation.
Both techniques were capable of detecting low-level degradation
products that were less than 0.5% of the area of the insulin peak.
In this study, CESI-MS exhibited better peak resolution for
degradation products of insulin and was capable of detecting
additional features not detected by the LC-MS method.

p3

References
1. Busnel, J.-M. et al. High Capacity Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry: Coupling aPorous Sheath less Interface with Transient-Isotachophoresis
Anal. Chem. 2010, 82, 9476–9483.
2. Santos, M.R. et al. A covalent, cationic polymer coating
method for the CESI-MS analysis of intact proteins andpolypeptides. SCIEX separations application note, 2015.

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