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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:
10.1080/10826076.2014.896817
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Journal of Liquid Chromatography & Related Technologies, 38: 190–195, 2015
Copyright # Taylor & Francis Group, LLC
ISSN: 1082-6076 print/1520-572X online
DOI: 10.1080/10826076.2014.896817
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
Injection volume was 1 mL, and autosampler temperature was Ten milligram of CY API powder was transferred into
maintained at 8 C. 100 mL volumetric flask and added 70 mL of water sonicated
and made up to the volume by water.
Mass Spectrometry
All the stressed samples were diluted with water
The MS analysis was performed on waters Xevo G2 QTOF 10 times.
instrument (Waters, MA, USA) equipped with electrospray
ionization source (ESI). The data acquisition was under
the control of Masslynx software (Waters, Mildord, MA, Results and Discussion
USA). Analyses were performed in positive and negative
ion modes. But ionization was good in positive ion mode. Optimization of LC-MS Conditions[10–12]
The typical MS parameters in positive ion mode were cone
The main aim of this work was to separate CY from its
voltage which was set at 30 V; capillary voltage at 3.0 kv;
known impurities and degradation products and identify
source temperature at 150 C; desolvation gas flow at
using QTOF. A waters BEH C8 column (2.1 100 mm,
1000 L=hr, and desolvation temperature at 450 C. All spec-
1.7 mm) was found to be suitable for analysis after tried dif-
tra were recorded under identical conditions.
ferent columns like BEHC18, CSH Fluro-phenyl, and BEH
amide. Acetonitrile and methanol were tried, and methanol
Stressed Degradation Studies[8,9] was chosen, as it was giving good separation with sensi-
Stress degradation studies were carried out on the active tivity. Different pHs were tried acidic, neutral, and basic,
pharmaceutical ingredient (CY) to provide the ability of basic pH gave good separation and sensitivity. So basic
the stability-indicating property and specificity of the pro- pH was chosen, as all peaks were well resolved. pH of the
posed method according to ICH guidelines Q1A (R2). About mobile phase was important as little change in pH resulted
10.0 mg of drug was subjected to stress degradation under in the loss of resolution between Impurity-C and
acidic, basic, and room temperature conditions by refluxing Impurity-D. So ammonium formate was chosen as buffer,
with 10.0 mL of 0.1 N HCl, 0.1 N NaOH, and H2O at 80 C and pH was adjusted to 8.5 by ammonia. A gradient pro-
for 24 hr, respectively. After the completion of stress gram was adopted with mobile phase consisting of (A)
degradation, all the samples were kept in refrigerator at 5 C. 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=
Sample Preparation
1.5; 0.4=98=2; 0.4=15=6; 0.4=5=6.5; 0.2=98=7 and different
A 10 mg of CY standard and impurities were accurately column temperatures 30 C, 40 C, and 50 C were tried,
weighed, transferred in to 10 mL volumetric flask each, but at 50 C Impurity-C and Impurity-D were merging,
and dissolved in water (1000 mg=mL). Further, above and there was not much difference in selectivity at 40 C
1.0 mL solutions were transferred into 100 mL volumetric and 35 C, so column temperature was kept constant
flask, and dissolved in water (10 mg=mL). 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
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.
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 195
degradants were presented in Table 1; MS spectra of all performance liquid chromatographic method for the estimation of
the degradation products were present in Figures 4 and 5; cyclophosphamide in bulk drug. Int. J. Pharm. Pharm. Sci. 2013,
and proposed structures of all unknown impurities in 5 (Suppl 2), 184–187.
6. Wren, S. A. C.; Tchelitcheff, P. Use of Ultra performance liquid
Figure 6. chromatography in pharmaceutical development. J. Chromatogr.
A 2006, 1119,
140–146.
Conclusion 7. Swartz, M. E. UPLC: An introduction and review. J. Liq.
Chromatogr. Related Technol. 2005, 28, 1253–1263.
In this study, it was possible to develop a fast and selective 8. ICH Q1A (R2), Stability Testing of New Drug Substances and
UPLC-MS method for CY on BEH C8 column. This could Products, 2003.
separate drug and degradation products formed under var- 9. ICH Guideline Q3A (R2), Impurities in New Drug Substances,
iety of stress conditions. The MS and MS=MS studies will October 25, 2006.
help to identify known impurities and degradants. 10. Dejaeqher, B.; Hayden, V. V.; Dumarey, M. Method develop-
Total of four degradation products was formed under ment for drug impurity profiling: Part 1. LC-GC 2010, 23 (4),
various stress conditions. This method can be used for rou- 218–225.
11. Dejaeqher, B.; Hayden, V. V.; Dumarey, M. Method development
tine analysis due to its shorter runtime, and will be helpful for drug impurity profiling: Part 2. LC-GC 2010, 23 (10), 536–542.
for the multiple generic manufacturers of the drug across 12. Pramanik, B. N.; Lee, M. S.; Chen, G. Characterization of
the globe by saving them from unnecessary repetition of Impurities and Degradants using Mass Spectrometry; Wiley, 2011,
same studies. According to our knowledge, it is the first pp 183–208.
method showing separation of CY from its known impuri- 13. Reddy, G. V. R.; Kumar, A. P.; Reddy, B. V.; Kumar, P.;
ties and degradants formed under different stress conditions. Gautam, H. D. Identification of degradation products in Aripipra-
zole tablets by LC-QToF mass spectrometry. Eur. J. Chem. 2010,
1(1), 20–27.
Acknowledgments 14. Bharadwaj, S. P.; Singh, S. Study of forced degradation behavior of
enalapril maleate by LC and LC–MS and development of a validated
The authors wish to thank Waters India for providing the stability-indicating assay method. J. Pharm. Biomed. Anal. 2008, 46,
analytical equipment and encouragement. 113–120.