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Identification of Degradation Products in Cyclophosphamide API by LC-QTOF

Mass Spectrometry

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DOI: 10.1080/10826076.2014.896817

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Identification of Degradation Products in

Cyclophosphamide API by LC-QTOF Mass Spectrometry
a b
Gudlawar Shivakumar & Jaya Dwivedi
a
Application Department , Waters India , Hyderabad , India
b
Department of Chemistry , Bansthali Vidyapeeth , Jaipur , India
Accepted author version posted online: 18 Mar 2014.Published online: 10 Oct 2014.

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To cite this article: Gudlawar Shivakumar & Jaya Dwivedi (2015) Identification of Degradation Products in Cyclophosphamide
API by LC-QTOF Mass Spectrometry, Journal of Liquid Chromatography & Related Technologies, 38:2, 190-195, DOI:
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 Journal of Liquid Chromatography & Related Technologies, 38: 190–195, 2015
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ISSN: 1082-6076 print/1520-572X online
DOI: 10.1080/10826076.2014.896817

Identification of Degradation Products in Cyclophosphamide

API by LC-QTOF Mass Spectrometry
GUDLAWAR SHIVAKUMAR1 and JAYA DWIVEDI2
1
Application Department, Waters India, Hyderabad, India
2
Department of Chemistry, Bansthali Vidyapeeth, Jaipur, India
Downloaded by [Mr Gudlawar Shivakumar] at 23:33 16 January 2015

A rapid, specific, and novel gradient LC–MS method has been developed for the identification of degradation products (DPs) of
cyclophosphamide (CY) under different stress conditions using liquid chromatography combined with quadrupole time-of-flight
electrospray ionization tandem mass spectrometry (LC-QTOF–ESI-MS=MS). CY was subjected to hydrolytic (acidic, alkaline,
and neutral) as per ICH guidelines Q1A (R2). The drug showed extensive degradation in hydrolytic stress conditions. In total, eight
DPs were formed, and the chromatographic separation of drug and its DPs was achieved on a C-8 column (2.1
100 mm, 1.7 mm)
using gradient elution method. The elemental composition of the degradation products was elucidated by using LC–ESI–MS=MS
combined with accurate mass measurements.
Keywords: cyclophosphamide, degradation studies, ESI, ICH guidelines, QTOF, UPLC

Introduction degradation products. Figure 1 presents the structures of

Cyclophosphamide (CY) is an important alkylating agent
that has been widely used in treating various malignant
tumors. DNA interstrand cross-links appear to be the
primary cytotoxic adduct and the base of antitumor activity
of phosphoramide compounds. Many studies have suggested
that DNA interstrand cross-links induced by alkylating
agents prevent the separation of the strands of the DNA
helix and thus inhibit DNA replication. In addition to its
toxicity to lymphocytes, it is an important immunosuppres-
sant agent in hematopoietic stem cell transplant therapies,
and more recently in the treatment of inflammatory con-
ditions such as systemic lupus erythematosis and rheumatoid
arthritis.[1–3] Further clarification of the molecular interac-
tions between DNA and cross-linking agents is necessary
understand the cellular responses to DNA interstrand
cross-links and the manner by which such lesions are recog-
nized and repaired in mammalian cells. An alkylating agent
adds an alkyl group to the guanine base of DNA at number
seven nitrogen of the imidazole ring.[4] High-performance
liquid chromatography method was reported previously,
but this method does not show separation of all known
impurities.[5] In the present work, degradation studies of
CY drug product including room temperature, acid, and
base were carried out using LC-QTOF-MS to identify the
Address correspondence to: Gudlawar Shivakumar,
Application Department, Waters India Pvt. Ltd., Plot No. 11,
Rukmini Towers, S.P Road, Begumpet, Secunderabad 500003,
India. E-mail: shivagudlawar@rediffmail.com
Color versions of one or more of the figures in the article
can be found online at www.tandfonline.com/ljlc.
CY and known impurities.
Experimental
Chemicals and Reagents
CY was purchased from (Sigma Aldrich, Bengaluru, India),
and impurities were obtained as gift samples from Cure Lab.
(Hyderabad, India), methanol (LCMS grade) was purchased
from Fluka Chemicals (Hyderabad, India), and ammonium
formate was purchased from Sigma Aldrich (Bengaluru,
India). Sodium hydroxide, hydrochloric acid, and ammonia
were obtained from Merck Laboratories Ltd. (Mumbai, India).
The 0.22 m PTFE filter was purchased from Advanced Micro
devices Pvt. Ltd. (Ambala Cantt, Haryana, India). Milli-Q
water was used throughout the experiment. Other chemicals
used were LCMS grade.
Instrumentation
Ultra Performance Liquid Chromatography[6,7]
The analyses were performed on a Waters Aquity UPLC-H
class instrument (Waters, Milford, MA, USA) equipped with
a quaternary pump, a de-gasser, an autosampler, a column
compartment, and Milli Q system (Millipore Corp.,
Bedford, MA, USA). The samples were separated on a
Waters BEH C8 (2.1
100 mm, 1.7 mm). The mobile phase
consists of 10 mM ammonium formate in water pH adjusted
to 9.25 with ammonia (solvent A), methanol (solvent B).
A gradient elution program is described in the context of
optimization of LC–MS conditions. A gradient flow rate was
used. The column temperature was maintained at 35 C.
 Identification of DPs in CY
Downloaded by [Mr Gudlawar Shivakumar] at 23:33 16 January 2015
Fig. 1. Structures of cyclophosphamide and its known impurities.
Injection volume was 1 mL, and autosampler temperature was
maintained at 8 C.
Mass Spectrometry
The MS analysis was performed on waters Xevo G2 QTOF
instrument (Waters, MA, USA) equipped with electrospray
ionization source (ESI). The data acquisition was under
the control of Masslynx software (Waters, Mildord, MA,
USA). Analyses were performed in positive and negative
ion modes. But ionization was good in positive ion mode.
The typical MS parameters in positive ion mode were cone
voltage which was set at 30 V; capillary voltage at 3.0 kv;
source temperature at 150 C; desolvation gas flow at
1000 L=hr, and desolvation temperature at 450 C. All spec-
tra were recorded under identical conditions.
Stressed Degradation Studies[8,9]
Stress degradation studies were carried out on the active
pharmaceutical ingredient (CY) to provide the ability of
the stability-indicating property and specificity of the pro-
posed method according to ICH guidelines Q1A (R2). About
10.0 mg of drug was subjected to stress degradation under
acidic, basic, and room temperature conditions by refluxing
with 10.0 mL of 0.1 N HCl, 0.1 N NaOH, and H2O at 80 C
for 24 hr, respectively. After the completion of stress
degradation, all the samples were kept in refrigerator at 5 C.
Sample Preparation
A 10 mg of CY standard and impurities were accurately
weighed, transferred in to 10 mL volumetric flask each,
and dissolved in water (1000 mg=mL). Further, above
1.0 mL solutions were transferred into 100 mL volumetric
flask, and dissolved in water (10 mg=mL).
191
Ten milligram of CY API powder was transferred into
100 mL volumetric flask and added 70 mL of water sonicated
and made up to the volume by water.
All the stressed samples were diluted with water
10 times.
Results and Discussion
Optimization of LC-MS Conditions[10–12]
The main aim of this work was to separate CY from its
known impurities and degradation products and identify
using QTOF. A waters BEH C8 column (2.1
100 mm,
1.7 mm) was found to be suitable for analysis after tried dif-
ferent columns like BEHC18, CSH Fluro-phenyl, and BEH
amide. Acetonitrile and methanol were tried, and methanol
was chosen, as it was giving good separation with sensi-
tivity. Different pHs were tried acidic, neutral, and basic,
basic pH gave good separation and sensitivity. So basic
pH was chosen, as all peaks were well resolved. pH of the
mobile phase was important as little change in pH resulted
in the loss of resolution between Impurity-C and
Impurity-D. So ammonium formate was chosen as buffer,
and pH was adjusted to 8.5 by ammonia. A gradient pro-
gram was adopted with mobile phase consisting of (A)
10 mM ammonium formate in water pH adjusted to 8.5
with ammonia and (B) methanol. Program was set as fol-
lows: flow rate mL=min=%A=time min: 0.2=98=0; 0.2=98=
1.5; 0.4=98=2; 0.4=15=6; 0.4=5=6.5; 0.2=98=7 and different
column temperatures 30 C, 40 C, and 50 C were tried,
but at 50 C Impurity-C and Impurity-D were merging,
and there was not much difference in selectivity at 40 C
and 35 C, so column temperature was kept constant
at 35 C.
 192 G. Shivakumar and J. Dwivedi

Fig. 2. Representative chromatogram showing separation of cyclophosphamide from its known impurities.
Downloaded by [Mr Gudlawar Shivakumar] at 23:33 16 January 2015

Degradation Behavior Impurity-B, and other degradation products were DP2,

The optimized LC–MS method is applicable to identify the
DPs, and the LC–ESI-MS total ion chromatograms (TIC)
obtained for CY under various stress conditions are given
in Figure 2. A total of eight DPs were identified, and their
elemental compositions are given in Table 1.
Acid and Base Degradations
The acid and basic degradation studies were carried out by
adding 10 mL of 0.1 N HCl and 0.1 N NaOH, respectively,
for 24 hr. Under these degradation conditions CY degraded
up to 14% and 13%, respectively. Major degradation product
in acidic conditions was Impurity-D, and other degradation
products were DP1, DP2, DP4, Impurity-C, Impurity-B,
and Impurity-A. Major degradation products in basic con-
ditions were Impurity-D and DP-4, and other degradation
products were DP1, DP2, DP3, Impurity-C, Impurity-B,
and Impurity-A. No further degradation was observed even
though study was extended for 48 hr.
Room Temperature
The room temperature degradation was carried out by
adding 10 mL of water and kept on the bench for 24 hr.
Major degradation products were DP4, Impurity-D, and
DP3, Impurity-A, and Impurity-C.
Identification of Known and Unknown Impurities by
QTOF-MS[13,14]
To confirm known impurities retention times and m=z values,
each impurity was spiked at 1.0% level to the CY drug, and
analysis was carried out. Figure 3 presents the chromatogram
showing separation of all impurities from CY. The impurities
were identified by mass accuracy and elemental formulae.
From the degradation studies four major unknown degra-
dants were observed at RTs 1.3, 3.8, 4.2, and 4.9 min. The
peak at RT 1.3 min showed m=z at 225.0999 with elemental
formula C7H18N2O4P, ms=ms spectrum shows abundant
ion at m=z 127.1233 (loss of –H3PO4, 98 Da, phosphoric acid)
with elemental formula C7H15N2, the fragmentation pattern
was similar to that of Impurity-D, suggests that the structure
of Dp1 may be related to Impurity-D. Dp1 was identified as
(3-f[2-(aziridin-1-yl) ethyl] aminogpropyl) phosphonic acid.
The peak at RT 4.2 min showed m=z at 207.0889 with
elemental formula C7H16N2O3P, ms=ms spectrum shows
abundant ions at m=z 164.0475 (loss of NHCH2CH2,
43 Da, aziridine) with elemental formula C5H11NO3P and

Table 1. Elemental composition of cyclophosphamide and its degradation products

RT Proposed Observed Calculated Error Proposed Error

Compound (min) formulae mass mass (ppm) MS=MS formulae (ppm)

Cyclophosphamide 5.92 C7H16Cl2N2O2P 261.0326 261.0327 0.4 140.0036 C4H8NCl2 2.9

IMP-A 5.49 C4H10Cl2N 142.0191 142.0190 0.7 63.0002 C2H4Cl 0.0
IMP-B 1.66 C7H17ClN2O3P 243.0669 243.0665 1.6 164.0472 C5H11NO3P 3.0
136.0168 C3H7NO3P 2.9
IMP-C 1.05 C3H11NO4P 156.0428 156.0426 1.3 58.06570 C3H8N 0.0
IMP-D 1.41 C7H18ClN2O4P 261.0774 261.0771 1.1 127.1236 C7H15N2 1.6
182.0584 C5H13NO4P 1.1
Unknown IMP-1 1.22 C7H18N2O4P 225.0999 225.1004 2.2 127.1233 C7H15N2 0.8
Unknown IMP-2 3.87 C4H11NOCl 124.0530 124.0529 0.8 106.0421 C4H9NCl 2.8
Unknown IMP-3 4.26 C7H16N2O3P 207.8890 207.8890 0.0 164.0475 C5H11NO3P 1.2
136.0168 C3H7NO3P 2.9
Unknown IMP-4 4.99 C7H18N2O3Cl2P 279.0432 279.0432 0.0 142.0193 C4H10NCl2 2.1
138.0317 C3H9NO3P 2.2
 Identification of DPs in CY 193
Downloaded by [Mr Gudlawar Shivakumar] at 23:33 16 January 2015

Fig. 3. Chromatogram of cyclophosphamide and degradation products under different stress conditions.
136.0168 (loss of CH3CH2NCH2CH2, 71 Da, suggests that the structure of Dp3 may be related to
1-ethylaziridine) with elemental formula C3H7NO3P, the Impurity-B. Dp3 was identified as (aziridin-1-yl)
fragmentation pattern was similar to that of Impurity-B, [3-(aziridin-1-yl)propyl] phosphinic acid.

Fig. 4. MS spectra of degradation products Dp-2 (top) and Dp-1(bottom).

 194 G. Shivakumar and J. Dwivedi
Downloaded by [Mr Gudlawar Shivakumar] at 23:33 16 January 2015

Fig. 5. MS spectra of degradation products Dp-4 (top) and Dp-3 (bottom).

The peak at RT 3.8 min showed m=z 124.0530 with 108 that was 30% height of m=z 106, suggests that fragment
elemental formula C4H11NOCl, ms=ms spectrum shows contains chlorine, and parent spectrum also shows the
abundant ion at m=z 106.0421 (loss of H2O, 18 Da) with presence of peaks at m=z 124 and m=z 126 at 3:1 ratio, sug-
elemental formula C4H9NCl, it also shows peak at m=z gests the presence of one chlorine, and loss of water indicates

Fig. 6. Proposed structures of degradation products formed under various stress conditions.
 Identification of DPs in CY
the presence of alcoholic group, which was confirmed by
elemental formula. Dp2 was identified as 2-[(2-chloroethyl)
amino] ethanol.
The peak at 4.9 min showed m=z at 279.0432 with
elemental formula C7H18N2O3PCl2, ms=ms spectrum
shows abundant ions at 142.0193 (loss of 137 Da, 3-
[(aminophosphanyl)oxy]propan-1-ol), with A þ 2 and A þ 4
peaks at m=z 144 (66%) and 146 (10%), respectively, suggests
presence of two chlorine atoms, further confirmed by
elemental formula (C4H10NCl2), and it also shows the pres-
ence of m=z 221 and 223 corresponding to phosphoramide
mustard formed from phosphate ester hydrolysis and loss
of n-propanol radical. Dp4 was identified as 3-hydroxy
propyl-N, N-bis (2-chloroethyl)phosphorodiamidate. All
fragment ions were confirmed by accurate mass measure-
ments. The MS, MS=MS of CY, all known impurities and
Downloaded by [Mr Gudlawar Shivakumar] at 23:33 16 January 2015
degradants were presented in Table 1; MS spectra of all
the degradation products were present in Figures 4 and 5;
and proposed structures of all unknown impurities in
Figure 6.
Conclusion
In this study, it was possible to develop a fast and selective
UPLC-MS method for CY on BEH C8 column. This could
separate drug and degradation products formed under var-
iety of stress conditions. The MS and MS=MS studies will
help to identify known impurities and degradants.
Total of four degradation products was formed under
various stress conditions. This method can be used for rou-
tine analysis due to its shorter runtime, and will be helpful
for the multiple generic manufacturers of the drug across
the globe by saving them from unnecessary repetition of
same studies. According to our knowledge, it is the first
method showing separation of CY from its known impuri-
ties and degradants formed under different stress conditions.
Acknowledgments
The authors wish to thank Waters India for providing the
analytical equipment and encouragement.
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