Application NotePharma & Biopharma

How to Catch a Potential
Mutagenic Impurity
Using an Agilent LC/MSD XT and Agilent InfinityLabPoroshell 120 HILIC-Z column for sensitive andreliable detection of dalfampridine impurities
AuthorKyle Covert
Agilent Technologies, Inc.

Abstract
This Application Note demonstrates the enhanced analytical sensitivity of using
a mass selective detector (MSD) over UV detection alone, and the excellent
separation and reliability of the Agilent InfinityLab Poroshell 120 HILIC-Z column,
with compliance features provided by OpenLab CDS software. The United States
Food and Drug Administration (FDA) has set the guidance threshold of toxicologicalconcern (TTC) for potential mutagenic impurities to 1.5 µg/day. An Agilent 1260
Infinity II Prime LC coupled to an Agilent LC/MSD XT mass selective detector wasused to detect two potential mutagenic impurities (PMIs) of a daily dose (20 mg)of dalfampridine with an instrument detection limit (IDL) of less than 16 pg for bothcompounds. This is nearly 100 times more sensitive than UV detection alone, andensures that impurities can be detected for even small doses of pharmaceuticaldrugs. An InfinityLab Poroshell 120 HILIC-Z column was able to easily separate thecompounds in less than five minutes. Injections over multiple days showed a %RSDof less than 7% for peak area, and less than 1% for retention times.

Introduction
Dalfampridine (4-aminopyridine (4AP))
is a novel drug used to treat patientswith multiple sclerosis or other diseases
affecting motor function. It has helped
improve walking ability in adults byinhibiting augmented potassiumchannels. These channels release toomuch potassium, causing a decrease inaction potential duration and amplitude.
Dalfampridine may contain impurities
from the manufacturing process of theactive pharmaceutical ingredient (API),
which are potential mutagenic impurities.
Specifically, two PMIs were targeted inthis work: isonicotinamide and an isomerof the API itself, 3-aminopyridine, whosestructures are shown in Figure 1. With a
TTC for PMIs at 1.5 µg/day, a standard
daily dose of 20 mg dalfampridine
corresponds to no more than 75 ppm ofPMIs in relation to the API1,2. Therefore,
detection of these PMIs requires asensitive, precise, and high-throughput
methodology and instrumentation tomeet the requirements.
NH2NH2ONH2N
4-Aminopyridine

NIsonicotinamide

N3-Aminopyridine

Figure 1. API and two potential mutagenic
impurities.

The API and PMIs are hydrophilic,and do not retain well on a typical
reversed‑phase liquid chromatography
column. To obtain sufficientchromatographic separation,
reversed‑phase ion pairing can be used.

2However, this method requires reagentsthat are hard to remove from the system,
and can cause ion suppression in themass spectrometer, and this requires adedicated LC. Therefore, a hydrophilicinteraction liquid chromatography (HILIC)
method was employed.
The InfinityLab Poroshell 120 HILIC-Z(zwitterionic) column offers the perfectsolution for hydrophilic compoundseparation and detection. It is designed
to retain polar compounds with superior
robustness. The typical mobile phases
are acetonitrile (ACN) and water withappropriate additives.
The Agilent single quadrupole LC/MSD
XT can easily be incorporated into an
HPLC stack, and is the perfect choicefor impurity detection with massconfirmation alongside UV detection. It
is specifically designed to be integrated
seamlessly into the Agilent 1260
Infinity II LC or 1290 Infinity II LC system.

Experimental
Standards and chemicals
All reagents and solvents were HPLCor LC/MS grade. ACN was purchasedfrom Honeywell (Morristown, NJ, USA).
4-Aminopyridine, isonicotinamide,3-aminopyridine, ammonium formate,and formic acid were all purchased from
Millipore-Sigma (Merck, Darmstadt,Germany). Ultrapure water was produced
using a Milli-Q Integral system equippedwith a LC-Pak Polisher and a 0.22 µmpoint-of-use membrane filter cartridge(EMD Millipore, Billerica, MA, USA).

Instrumentation
The LC/MSD XT system consists of thefollowing modules:•Agilent 1260 Infinity II Flexible Pump
(G7104C)•Agilent 1260 Infinity II Vialsampler(G7129A)•

Agilent 1260 Infinity II Multicolumn
Thermostat (G7116A)•

Agilent 1260 Infinity II Diode ArrayDetector HS (DAD) (G7117C)•

Agilent LC/MSD XT system(G6135CA)

The high-sensitivity diode array detector
was used with a 60 mm Max-Light
Cartridge Cell to use the maximum
sensitivity of the UV detector incombination with the LC/MSD XT.

Sample preparation
API samples were prepared by dissolving
20 mg of 4AP into 1 mL of ACN in a 2 mLscrew top sample vial (20,000 µg/mL).
Stock solutions of the impurity standards
were prepared by dissolving 10 mg ofeach into 100 mL of ACN in a volumetricflask (100 µg/mL). A series of serial
dilutions was performed to make up
10, 1.0, 0.1, and 0.01 µg/mL solutionsof the impurity standards. Separate
samples of 20,000 μg/mL API standard
were spiked with 150 µL of the previous
impurity standards, respectively, tomake ~1,150, 115, 11.5, and 1.15 ng/mLof impurity spiked API samples. The
relation of impurities to the API in thesamples was 75 to 0.075 ppm. This
concentration range was chosen to cover
the respective detection limits of the UV
detector and LC/MSD XT in scan andselected ion monitoring (SIM) modes.

OpenLab CDS
Agilent OpenLab CDS software wasused for data acquisition, processing,and reporting. OpenLab CDS provides
compliance features that support data
integrity with US FDA 21 CFR Part
11, EU Annex 11, and other similar
regulations. The 1260 Infinity II LC andLC/MSD XT are designed to ensure
reliable and robust LC/MS for routine
applications.

Results and discussion
The integrated divert valve ensuressystem robustness
MS spectral information facilitates
the detection and confirmation ofimpurities. However, repeated sample
injection of high API concentrations
may slowly cause contamination of
the MS ionization source over time.
This challenge can be minimized using
the built-in diverter valve includedin Agilent mass spectrometers. Thevalve may be set to divert the column
eluent flow to waste during the elutionof the highly concentrated main peak
(20,000 µg/mL). During LC method
development, we ensured that the main
API peaks were well separated from therest of the impurities. The diverter valve
switching is smooth, and maintains
retention time reproducibility.

Table 1. 1260 Infinity II LC method parameters.
Parameter

HPLC Set Value

ColumnInfinityLab Poroshell 120 HILIC-Z, 2.1 × 150 mm, 2.7 µm, PEEK lined at 35 ºC

Mobile Phase A

10 mM ammonium formate + 0.02 % formic acid (pH 5.0)

Mobile Phase B

0.1% formic acid in acetonitrile (ACN)

GradientTime0245

Post Run

5 minutes

Flow Rate

0.5 mL/min

Injection Volume1 µL

Detection UV(265, 10/ref. 360, 80) nm%B
95906060

Table 2. Agilent LC/MSD XT parameters.
Parameter

Single Quadrupole Set ValueIon Source

ESI+

Peak Filter

0.02 minutes

Scan/Dwell Time

Scan 250 ms, SIM 50 ms

Drying Gas Temperature

350 °C

Gas Flow

10 L/min

Nebulizer Pressure

40 psiCapillary Voltage

3 kV

Fragmentor Voltage

135 V

Scan Range

m/z 80 to 300

SIM Ions

m/z 95.2, 123.1

Divert to Waste

3.5 to 5 minutes

Table 3. List of compounds of interest with their m/z, retention time,and log P values.
Compoundm/z

Retention Timelog P

API (4-Aminopyridine)

95.2

3.98 (onset)

0.26

Impurity I (Isonicotinamide)

123.1

1.14

-0.28

Impurity II (3-Aminopyridine)

95.2

1.96

-0.02

Nicotinamide

123.1

1.52

-0.11

3

There are two peaks detected in the pure
API at 1.523 and 3.146 minutes. The
first peak has an m/z of 123.1, and issuspected to be nicotinamide, an isomerof the precursor (isonicotinamide),commonly used in the reaction tosynthesize dalfampridine. This is further
confirmed by the log P values, a measureof a compound’s hydrophobicity, whichplaces nicotinamide between impurity I
and impurity II. The second peakdetected in the API standard has an m/zof 95.2, which gives it the same massas the API and impurity II. However, theretention time for this peak does notmatch with the API or impurity II, so it
is possibly another isomer of the API,
2-aminopyridine.

mAU
3600
3400
3200
3000
2800
2600
2400
2200
2000
1800
1600
1400
1200
1000
800
600
400
200
0API20 mg/mL

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0
Time (min)Figure 2. UV chromatogram of the API (20 mg/mL).
m/z 123.1

m/z 95.2

×105AbundanceA 1 µL injection of 20,000 µg/mL
API standard without impurities wasperformed with only UV detection.
Figure 2 shows the UV profile of theAPI to determine the time window todivert the LC flow to waste, and protect
the MS detector. This same sample isinjected again, shown in blue in Figure 3,
diverting the API to waste from 3.5 to5.0 minutes. This was performed to see
if there was any detectable abundance ofthe target impurities in the API standardalone.

3.6
3.4
3.2
3.0
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
09510511512595105115

125

Pure API (20 mg/mL)
API spiked with 11.5 ng/mL
impurity standards

Impurity II
1.959

1.523

3.146
API diverted to waste

Impurity I
1.136

ONH2Nm/z 123.1
1.528

m/z 95.2
3.097

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0
Mass-to-charge (m/z)

Figure 3. Mass chromatogram (SIM TIC), from mass detector, of the pure API (20 mg/mL) shown in blue,
and the same API spiked with 11.5 ng/mL of impurities, relative to the API, shown in black for comparison.
MS spectra of the impurity peaks are inlayed. The peak at 1.528 has been identified as nicotinamide,present in the API standard.

4

A 1 µL injection containing 11.5 ng/mLof impurities shows no discernablepeaks for the impurities in theUV chromatogram shown in Figure 4A.
Impurities were only detected inthe UV at concentrations above
20 ng/mL. An inset in Figure 4A shows
the UV chromatogram of a 1 µL injection
at a concentration of 115 ng/mL ofimpurities. Impurity II and the API
are well separated by the InfinityLab
Poroshell 120 HILIC-Z column.
Figure 4B features the SIM TIC, andthe impurities are easily distinguishedfrom the noise as peaks at 1.136
and 1.950 minutes. SIM mode only
allows single m/z ions to pass in thequadrupole. Therefore, the quadrupole
spends significantly more time analyzing
select ions, enabling greatly enhanced
analytical sensitivity versus scanning
across an m/z range.

AmAU

UV Chromatogram at 11.5 ng/mL

1.0
0.8
0.6

Impurity I

Impurity II

0.4
0.2
0-0.2

Impurity I-0.4
UV Chromatogram at 115 ng/mL-0.6
Impurity II-0.8-1.0

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0
Time (min)B×104 Mass chromatogram (SIM TIC) at 11.5 ng/mL
5.5
1.950

3.295

5.0
4.5

1.136

4.0
3.5
CountsEnhanced analytical sensitivity ofLC/MSD XT

3.0

Impurity Im/z 123.1

Impurity IIm/z 95.2
API diverted to waste

2.5
2.0
1.5
1.0
0.5
0
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0
Time (min)Figure 4. Two chromatograms of the API spiked with 11.5 ng/mL of impurities: A) Zoomed-in region ofthe UV chromatogram with the expected retention time of the impurities highlighted with dashed lines.
The inset is a UV chromatogram from a higher concentration of impurities, confirming retention times.
B) Mass chromatogram (SIM TIC), m/z 123.1 and 95.2, impurity I and II, respectively, at 11.5 ng/mL.

5

The API was analyzed with a series of1 µL injections where the impurities
were spiked into the API at 115, 11.5,
and 1.15 ng/mL concentrations, and areshown in Figure 6. At the 115 ng/mLlevel, the impurities can be determined
by UV at approximately the limit ofquantitation, and are easily distinguishedby the LC/MSD XT in scan mode. At the11.5 ng/mL level, the impurities cannot
be seen in the UV chromatogram, andare not distinguishable in the scan modeof the mass spectrometer. However, theLC/MSD XT is easily able to detect theimpurities in SIM mode.
The lowest concentration analyzed was
1.15 ng/mL for both impurities. The
signal-to-noise ratio (S/N) was 12 and 28
for impurities I and II, respectively, using
SIM mode. This demonstrates that theLC/MSD XT in SIM mode has a detection
limit 100x greater than UV detection forthese compounds.

61.970
Impurity I only
Impurity II only
Spiked API

3.6
3.2
2.8
2.4
CountsBecause the masses of impurity II
and the API are the same, standards
for these compounds were analyzed
individually to obtain their specificretention times. The retention timesof the impurities were determined bya 1 µL injection of 150 ng/mL of thestandard impurity, as shown in Figure 5.
An injection containing 11.5 ng/mL ofimpurities is also shown to demonstratethat the spiked samples have the same
retention times.

1.141×105

2.0
1.6
1.2
0.8
1.136

0.4

1.950

00

0.2

0.4

0.6

0.8

1.0

1.2

1.4
1.6
1.8
Time (min)

2.0

2.2

2.4

2.6

2.8

3.0

Figure 5. Stacked mass chromatogram (SIM TIC) of the individual impurities and a 11.5 ng/mL spiked
sample. The pure impurity RTs were compared to the spiked sample to ensure that the RT remains
consistent in the presence of a large amount of API.

×105
3.6

Impurity II
1.959

Impurity I
1.140

Impurity II
1.959

3.2
2.8
2.4
CountsLC/MSD XT with improved analyticalsensitivity and higher precision for
low-level impurities

2.0

115 ng/mL
11.5 ng/mL
1.15 ng/mL

1.136
Impurity I

1.6
1.2
0.8

1.136

1.950

0.4
00 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6Time (min)Figure 6. Stacked mass chromatogram (SIM TIC) of the impurities spiked into the API at 115, 11.5, and1.15 ng/mL concentrations. The inlay is a zoomed portion of the SIM chromatogram at 1.15 ng/mL(1.15 pg on‑column) to show that the impurities are still detectable at this concentration.

The reproducibility of the 1260 Infinity II
Prime LC coupled to the LC/MSD XT with
the InfinityLab Poroshell 120 HILIC-Z
column shows excellent performance,
as shown in Figure 8, by performing
three injections of the same sample
over three days, approximately 24 hours
apart. The %RSD of the peak areas were
6.7% for impurity I and 4.1% impurity II.
Both impurities have a %RSD of retention
times of less than 1%.

Amount measured = 100 pg
n = 10 injections

CountsA good measure of the overall analyticalsensitivity of a method can beestablished by calculating the instrument
detection limit (IDL)3,4. The IDL not only
takes the detector of choice into account,but also the entire HPLC system frompump to column by comparing peak
RSDs over multiple injections3,4. The
IDL was determined for the LC/MSD XToperating in SIM mode for the impurity
peaks. The IDL was determined tobe 15.40 pg for impurity I and 5.65
pg for impurity II from 10 replicate 5µL injections of a 20 ng/mL sample
containing only the impurities, as shownin Figure 7.

×105
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0Impurity II
Peak area %RSD = 2.004
IDL = 5.65 pg

Impurity I
Peak area %RSD = 5.465
IDL = 15.40 pg

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6Time (min)Figure 7. SIM chromatograms of 100 pg injections containing only the impurities. Ten injections were
performed to calculate the IDL for the individual impurities. The IDL was determined to be 15.40 pg for
impurity I and 5.65 pg for impurity II.

×104

CountsInfinityLab Poroshell 120 HILIC-Zand LC/MSD XT detection sensitivity(IDL <16 pg)

9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.04.54.0
3.5
3.0
2.5
2.0

Three injections over three days
Impurity I
Peak area %RSD = 6.7
1.135

Impurity II
Peak area %RSD = 4.1
1.979

1.128
1.980

1.950
1.136

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6Time (min)Figure 8. Stacked mass chromatogram (SIM TIC) of a 11.5 ng/mL spiked API sample injected over thecourse of three days. %RSDs of the peak areas are shown to display the reproducibility of the LC/MSD XT
system with the Agilent InfinityLab Poroshell 120 HILIC-Z column.

7Conclusion

References

Identifying potential mutagenic
impurities requires a sensitive andreliable instrument with the capabilityof detecting high concentrations of API.
The InfinityLab series of consumables,LC modules, and the LC/MSD XT
offers a reliable, sensitive, and robust
system that can easily be incorporated
into an impurity analysis workflow,
in a compliant environment using
OpenLab CDS. UV detection alone is
not sensitive enough to detect the lowlevels of PMIs, underlining the need for
an MSD, as demonstrated in this study.
Together with the 1260 Infinity II PrimeLC System, the LC/MSD XT obtained
instrument detection limits of 15 pg
for impurity I, and 6 pg for impurity II.
The InfinityLab Poroshell 120 HILIC-Z
column is perfectly suited for separationof polar compounds, and it has beenshown to produce consistent and reliable
separations.

1.

US Department of Health andHuman Services; FDA; CDER; CBER.
M7(R1) Assessment and Control ofDNA Reactive (Mutagenic) Impurities
in Pharmaceuticals to Limit Potential
Carcinogenic Risk. ICH, March 2018.

2.

Jain, M.; et al. Determination ofFive Potential Genotoxic Impurities
in Dalfampridine Using Liquid
Chromatography. J. Pharm. Biomed.
Anal. 2016, 133, 27–31.

3.

Parra, N. P.; Taylor, L. Why
Instrument Detection Limit(IDL) Is a Better Metric for
Determining The Sensitivity ofTriple Quadrupole LC/MS Systems.
Agilent Technologies Technical
Overview, 2014.

4.

Sheehan, T. L.; Yost, R. A. What’s theMost Meaningful Standard for Mass
Spectrometry: Instrument Detection
Limit or Signal-to-Noise Ratio?
Spectroscopy 2015, 13(4), 16–22.

www.agilent.com/chem
For Research Use Only. Not for use in diagnostic procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc. 2019
Printed in the USA, September 20, 20195994-0864EN