Original Article Novel validated stability-indicating UPLC method for the determination of Metoclopramide and its degradation impurities in API and pharmaceutical dosage form Prathyusha Sowjanya a, *, Palani Shanmugasundaram a , Petla Naidu b , Sanjeev Kumar Singamsetty b a Department of Pharmaceutical Analysis, School of Pharmaceutical Sciences, Vels University, Chennai 600117, India b Analytical Research & Development, Hospira Health Care India Pvt Ltd., Irungattukottai, Chennai 602105, India article info Article history: Received 9 May 2013 Accepted 5 July 2013 Available online 29 July 2013 Keywords: LCMS Metoclopramide Stress degradation products Ultra performance liquid chroma- tography (UPLC) Validation abstract Aim: To develop a stability-indicating reversed phase ultra performance liquid chromato- graphic (RP-UPLC) method for the determination of related substances in Metoclopramide bulk drugs and pharmaceutical dosage form. Method: The chromatographic separation was achieved using a Waters X-terra RP18 (150 4.6 mm), 3.5 mm particle size column using the gradient program with mobile phase consisting of solvent A: 30 mM monobasic sodium phosphate and 2.3 mM of pentane-1- sulphonic acid sodium salt (pH 3.0 buffer) and solvent-B (Acetonitrile). A flow rate of 1.2 mL/ min and UV detector at 273 nm was used. The runtime was 18 min within which Metoclopra- mide and its four impurities, ACETYLMETO, ACMA, CLEE and ACME were well separated. Results and discussion: The drug was subjected to stress conditions such as oxidative, acid & base hydrolysis, thermal and photolytic degradation. Metoclopramide was found to degrade signif- icantly in photolytic, oxidative & thermal stress conditions and stable in acid, base, hydrolytic & humidity stress conditions. The major degradation impurities in oxidation and photolytic degradation were identified by LCMS. The degradation products were well resolved from the main peak and its impurities, thus proved the stability-indicating power of the method. Conclusion: The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. The calibration curves obtained for the four impurities were linear over the range 0.062e3.040 mg/mL. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. * Corresponding author. Tel.: þ91 4427141358; fax: þ91 4427156816. E-mail address: [email protected](P. Sowjanya). Available online at www.sciencedirect.com journal homepage: www.elsevier.com/locate/jopr journal of pharmacy research 6 (2013) 765 e773 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.07.004
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j o u r n a l o f p h a rm a c y r e s e a r c h 6 ( 2 0 1 3 ) 7 6 5e7 7 3
Available online at w
journal homepage: www.elsevier .com/locate/ jopr
Original Article
Novel validated stability-indicating UPLC methodfor the determination of Metoclopramide and itsdegradation impurities in API and pharmaceuticaldosage form
aDepartment of Pharmaceutical Analysis, School of Pharmaceutical Sciences, Vels University, Chennai 600117, IndiabAnalytical Research & Development, Hospira Health Care India Pvt Ltd., Irungattukottai, Chennai 602105, India
Table 2 e Intra day e Inter day precision studies ofMetoclopramide related substances.
Name of impurity Intra day precision Inter day precision
a% Ofimpurity
a%RSD
a% Ofimpurity
a%RSD
ACETYLMETO 0.217 0.3 0.219 0.9
ACMA 0.211 0.0 0.211 0.2
CLEE 0.210 0.3 0.210 0.6
ACME 0.223 0.0 0.222 0.9
a Mean of six replicates.
j o u r n a l o f p h a rm a c y r e s e a r c h 6 ( 2 0 1 3 ) 7 6 5e7 7 3 771
3.1.6. Flow rate optimizationDifferent mobile phase flow rates (1.0, 1.2 and 1.4 mL/min)
were investigated. The optimum flow rate for which the col-
umn plate number was maximum, with the best resolution
between all compounds and a short runtime (18min) observed
was 1.2 mL/min.
3.1.7. Column temperature optimizationColumn thermostat temperatures were used at 30 �C, 35 �Cand 40 �C for better peak shapes, baseline and resolution. At
the column oven temperature of 35 �C the finest baseline
resolution was observed between all the components.
After an extensive study, the method has been finalized on
Waters X-terra RP18, 150 mm � 4.6 mm, 3.5 m using variable
composition of solvent A: NaH2PO4 (3.4 g/L), pentane-1-
sulfonic acid sodium salt (0.4 g/L), pH adjusted to 3.0 with
orthophosphoric acid and solvent B: acetonitrile. The flow rate
of the mobile phase was 1.2 mL/min. The UPLC gradient pro-
gram (T/%B) was set as 90/0, 90/1, 85/2, 83/5, 80/7, 75/8, 70/9,
75/13, 90/15 and 90/18. The column compartment temperature
was kept at 35 �C and the injection volume was 10 mL. The
detector response for all the components found maximum at
Table 4 e Linearity study of Metoclopramide Related substanc
% Spikelevel
ACETYLMETO ACMA
aAdded aRecovered aAdded aRecovere
LOQ 0.107 0.107 0.062 0.069
50 1.073 1.100 1.041 1.077
75 1.502 1.543 1.458 1.473
100 2.146 2.195 2.083 2.105
150 3.005 3.035 3.040 3.040
r 0.999924 0.999962
a mg/mL; r ¼ correlation coefficient.
Table 3 e Limit of quantification & Limit of detection.
Name of impurity Limit of quantification
Conc. mg/mL % Of impurity
ACETYLMETO 0.104 0.010
ACMA 0.067 0.007
CLEE 0.112 0.011
ACME 0.112 0.011
273 nm; hence the typical chromatogramwas recorded at this
wavelength. The typical UPLC chromatograms (Fig. 3) repre-
sent the satisfactory separation of all components among
each other.
3.2. Results of forced degradation studies/specificity
Forced degradation studies were performed on Metoclopra-
mide Injection USP to demonstrate selectivity and stability-
indicating capability of the proposed RP-UPLC method.
Accordingly the degradation stress studies were conducted by
stressingwith acid, base, peroxide, water, photolytic, heat and
humidity as mentioned in the Section 2.3.
Degradation was not observed in a Metoclopramide sam-
ple during acid, base, hydrolytic and humidity stress. About
1.36%, 5.6% and 8.10% of degradation were observed in
thermal, oxidative and photolytic stress respectively (Fig. 4).
The major impurity observed in peroxide degradation was
found to be N-oxide of Metoclopramide with molecular mass
of 315. LCMS data of the oxidation impurity is shown in Fig. 5.
The impurity was reported as a new metabolite earlier.7
Metoclopramide was highly photo labile in solution. Major
impurity of molecular mass 562 was observed in photolytic
degradation. LCMS data of photo degradation impurity is
shown in Fig. 6. The structures of the photo degradation
impurities were reported earlier based on LC-MS character-
ization.8 Dissociation of chlorine is the major photo degra-
dation pathway of Metoclopramide and is generally followed
by coupling of the products to generate high molecular
weight products.
Peakpurity test results fromthePDAdetectorconfirmedthat
the Metoclopramide peak obtained from all of the stress sam-
ples analyzed, was homogenous and pure. Peak purity results
from the PDA detector for the peaks produced by the degrada-
tion of Metoclopramide, confirmed that all these peaks were
j o u r n a l o f p h a rm a c y r e s e a r c h 6 ( 2 0 1 3 ) 7 6 5e7 7 3 773
phase and column temperature), the resolution between im-
purities and analyte was found to be more than 2.0 (Table 6).
3.3.7. Solution stability and mobile phase stabilityThe %RSD values of the four impurities during solution sta-
bility and mobile phase stability experiments were within
1.0%. No significant change was observed in the content of
impurities during solution stability and mobile phase stability
experiments confirm that sample solutions and mobile phase
used during the study were stable up to 48 h.
4. Conclusion
The simple UPLC method developed for the quantitative
determination of related compounds of Metoclopramide and
its possible degradation products is precise, accurate and
specific for the analysis of bulk material and formulation
samples. The method was fully validated, showing satisfac-
tory results for all the parameters tested. The developed
method is stability indicating and can be used for the routine
analysis of production samples.
Conflicts of interest
All authors have none to declare.
Acknowledgment
The authors thank Hospira Health Care India Pvt Ltd Man-
agement for encouragement and support. Cooperation
extended by all colleagues of Analytical Research Division is
gratefully acknowledged.
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