novel validated stability-indicating uplc method for the
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Original Article
Novel validated stability-indicating UPLC methodfor the determination of Metoclopramide and itsdegradation impurities in API and pharmaceuticaldosage form
Prathyusha Sowjanya a,*, Palani Shanmugasundarama, Petla Naidu b,Sanjeev Kumar Singamsetty b
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
a r t i c l e i n f o
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
* Corresponding author. Tel.: þ91 4427141358E-mail address: prathyusha.pchgs@gmail
0974-6943/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.jopr.2013.07.004
a b s t r a c t
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 nmwas used. The runtime was 18min within which Metoclopra-
mide and its four impurities, ACETYLMETO, ACMA, CLEE and ACME were well separated.
Results and discussion: Thedrugwassubjectedtostressconditionssuchasoxidative,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 themethod.
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.
; fax: þ91 4427156816..com (P. Sowjanya).2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
j o u rn a l o f p h a rma c y r e s e a r c h 6 ( 2 0 1 3 ) 7 6 5e7 7 3766
1. Introduction and acetonitrile were purchased from Ranbaxy Chemicals,
Metoclopramide is chemically 4-amino-5-chloro-N-[2-(dieth-
ylamino)ethyl]-2-methoxybenzamide, an antiemetic and
gastroprokinetic agent. It is commonly used to treat nausea
and vomiting, to facilitate gastric emptying in people with
gastroparesis, and as a treatment for gastric stasis often
associated withmigraine headaches. The antiemetic action of
Metoclopramide is due to its antagonist activity at D2 re-
ceptors in the chemoreceptor trigger zone (CTZ) in the central
nervous system (CNS)dthis action prevents nausea and
vomiting triggered by most stimuli.1 At higher doses, 5-HT3
antagonist activity may also contribute to the antiemetic ef-
fect. The gastroprokinetic activity of Metoclopramide is
mediated by muscarinic activity, D2 receptor antagonist ac-
tivity and 5-HT4 receptor agonist activity.2 Metoclopramide is
freely soluble inwater and ethanol and practically insoluble in
ether. The molecular formula is C14H22ClN3O2, which corre-
sponds to a molecular weight of 299.80.
Very few analytical methods have been reported for the
quantitative determination of Metoclopramide in formula-
tions as well as biological fluids. These include gas chroma-
tography3,4 and high performance liquid chromatography.5,6
These previously published methods comprise of compli-
cated mobile systems and are not directly applicable for this
novel type of dosage form which is prepared and need more
investigation for method development and validation. How-
ever, no stability indicating UPLC methods were reported to
estimate Metoclopramide and its degradation products
(Fig. 1). The proposed method was stability indicating by
which all the degradation products of Metoclopramide can be
estimated quantitatively at very low levels.
2. Experimental
2.1. Chemicals and reagents
Metoclopramide (purity 99.0%) and standard materials of
degradation productswere obtained fromHospira Health Care
India Pvt Ltd, Chennai, India. Monobasic sodium phosphate,
pentane-1-sulfonic acid sodium salt, orthophosphoric acid
Cl
NH2 O
O
NHN
Cl
NH2 O
O
OHCl
NH
O
Metoclopramide
ACMA CLEE
Fig. 1 e Structures of Metoclop
NewDelhi, India and all are of HPLC grade.Water was purified
by milli-Q-water purification system (Millipore, Bedford, MA,
USA) and used for preparation of all the solutions.
2.2. UPLC instrumentation and condition
The analysis was performed using Waters Acquity system
equipped with a binary solvent delivery pump and PDA de-
tector. Data acquisition and processing were done by using
Empower2 software version FR5 (Waters Corporation, USA).
The chromatographic separation was performed using a Wa-
ters X-terra RP18 column (150� 4.6mm), 3.5 m particle column.
Themobile phasewas amixture ofmobile phase A andmobile
phase B. Mobile phase Awasmono sodiumphosphate (3.4 g/L)
and pentane-1-sulfonic acid sodium salt (0.4 g/L) adjusted to
pH 3.0 with orthophosphoric acid and acetonitrile as mobile
phase B. The gradient program T (min) ¼ % B: 0 ¼ 10, 2 ¼ 15,
5 ¼ 17, 7 ¼ 20, 8 ¼ 25, 9 ¼ 30, 13 ¼ 25, 15 ¼ 10, and 18 ¼ 10, with
flow rate of 1.2 mL/min was employed. The injection volume
was 10 mL while the detector was set at 273 nm. The column
temperature was maintained at 35 �C.
2.2.1. Preparation of buffer, diluent, standard and samplesolutionAbout 3.4 g of monobasic sodium phosphate dissolved in
800 mL of water, adjusted to pH 3.5 � 0.05 with dilute
orthophosphoric acid solution was used as buffer. The di-
luent used was a mixture of buffer, acetonitrile and water in
the ratio of 80:15:5 (v/v/v).
A stock solution of Metoclopramide Hydrochloride (240 mg/
mL) was prepared by dissolving an appropriate amount in the
diluent. Standard solution containing 6 mg/mL was prepared
from this stock solution. 5 mL of Metoclopramide injection USP
solutioncontaining5000mg/mLwasdissolved in25mLofdiluent
to give a solution containing 1000 mg/mL as sample solution.
2.3. Forced degradation sample solution for specificitystudy
The study was intended to ensure the separation of Metoclo-
pramide and its degradation impurities. Forced degradation
Cl
NH O
O
NHN
O
O
O
O Cl
NH2 O
O
O
ACETYLMETO
ACME
ramide and its impurities.
Fig. 2 e UV Spectrum of Metoclopramide and its impurities.
Fig. 3 e Representative chromatogram of M
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 3 767
study was performed to evaluate the stability indicating
properties and specificity of the method. Multiple stressed
samples were prepared as indicated below.
2.3.1. Hydrolytic conditions: acid, base, water induceddegradationSolution containing 1 mg/mL of Metoclopramide was treated
with 1 NHCl, 1 NNaOH andwater respectively. These samples
were refluxed at 80 �C for 5 h. After cooling the solutions were
neutralized and diluted with diluent.
2.3.2. Oxidative condition: hydrogen peroxide-induceddegradationSolution containing 1 mg/mL of Metoclopramide was treated
with 6% w/v H2O2 at 40 �C for 6 h was cooled and diluted with
diluent.
2.3.3. Thermal degradation studyThe drug solution (5mg/mL)was subjected to heat at 105 �C for
24h.After cooling5mLof theabove solutionwas transferred in
a 25 mL volumetric flask, diluted to the volume with diluent.
2.3.4. Photolytic degradation studyThe drug solution (5mg/mL)was exposed to the UV light in the
photolytic chamber providing an overall illumination of
1.2 million lux h and ultraviolet energy of 200 W h/square
meters for 184 h. 5mL of the above solutionwas transferred in
25 mL volumetric flask, diluted to the volume with diluent.
2.3.5. Humidity degradation studyMetoclopramide injection USP (5 mg/mL) was subjected to
25 �C/90% RH for 7 days. 5 mL of the above solution was
transferred in 25 mL volumetric flask, diluted to the volume
with diluent.
3. Results and discussion
3.1. Method development and optimization
The development of selective method for determination of
Metoclopramide and its related substances is described as an
important issue in method development. Metoclopramide
and its related substances show different affinities for
etoclopramide spiked with impurities.
j o u rn a l o f p h a rma c y r e s e a r c h 6 ( 2 0 1 3 ) 7 6 5e7 7 3768
chromatographic stationary and mobile phases due to differ-
ences in their molecular structures. To obtain a good resolu-
tion among the impurities and main drug substance different
stationary phases were tested considering;
a. The feature of stationary phase.
b. The particle size of the column.
Considering the Metoclopramide and their related com-
pounds, buffer of acidic naturewas preferred for optimization;
the followingmobile phaseswith gradient elutionwere tested,
1. NaH2PO4$H2O (3.4 g/L) and pentane-1-sulphonic acid so-
dium salt (0.4 g/L) as a buffer (pH 2.5, 3, 3.5, 4) in combina-
tion with acetonitrile.
2. NaH2PO4$H2O (3.4 g/L) and pentane-1-sulphonic acid so-
dium salt (0.4 g/L) as a buffer (pH 2.5, 3, 3.5, 4) in combina-
tion with methanol.
Fig. 4 e Representative chromatograms of Metoclopramide on ac
(d), photolytic (e), thermal (f) and humidity (g) degradations.
3. NaH2PO4$H2O (3.4 g/L) and octane-1-sulphonic acid sodium
salt (0.4 g/L) as a buffer (pH 2.5, 3, 3.5, 4) in combinationwith
acetonitrile.
4. (NH4)H2PO4 (2.5 g/L) and pentane-1-sulphonic acid sodium
salt (0.4 g/L) as a buffer (pH 2.5, 3, 3.5, 4) in combinationwith
acetonitrile.
3.1.1. Selection of stationary phaseIt is clear from the molecular structure (Fig. 1), that all com-
pounds do not possess a functional group which can readily
ionize indicating polar in nature. Hence we started the devel-
opment activity with C8 stationary phase of various manu-
facturers using different mobile phases. The poor resolution
between Metoclopramide and ACETYLMETO and broad peak
shape for Metoclopramide implies that C8 stationary phase is
not suitable for this application. Hence C18 stationary phase
was chosen to improve resolution among the peaks and peak
id stress (a), base stress (b), peroxide stress (c), water stress
Fig. 4 e (continued).
Fig. 5 e LCMS data of Metoclopramide peroxide degradation impurity.
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 3 769
Fig. 6 e LCMS data of Metoclopramide photolytic degradation impurity.
j o u rn a l o f p h a rma c y r e s e a r c h 6 ( 2 0 1 3 ) 7 6 5e7 7 3770
shape for Metoclopramide. The peak shape for Metoclopra-
mide and resolution among all components improved with
Waters X-terra RP18, 150 mm � 4.6 mm, 3.5 m columns.
3.1.2. Influence of mobile phase buffer salt and surfactantsThe resolution among related impurities and Metoclopramide
was found poor using mobile phase with octane-1-sulfonic
acid sodium salt. Mobile phase containing pentane-1-sulfonic
acid sodium salt with ammonium phosphate instead of
octane-1-sulfonic acid sodium salt gives the better resolution.
However, one unknown impurity is merging with
ACETYLMETO. Ammonium phosphate is replaced with
sodium phosphate buffer keeping pentane-1-sulfonic acid so-
dium salt as such, gives the better separation among the
impurities.
3.1.3. Influence of organic modifierInitially methanol was used as an organic modifier which
gives the poor baseline with baseline drift. The retention for
all impurities was increased leading to inadequate resolution
among the peaks. To improve the resolution among the peaks
Table 1 e Forced degradation studies of Metoclopramide.
Condition % Degradation Purity angle
Acid 0.05 1.645
Base 0.09 1.596
Oxidation 5.60 1.693
Water 0.02 1.376
Photolytic 8.10 1.794
Heat 1.36 1.601
Humidity 0.03 2.676
and response, acetonitrile was tried as an organic modifier.
The baseline was found to be good and response for all com-
ponents was improved. The peak shape for all components
was also improved and hence acetonitrile was selected as the
organic modifier.
3.1.4. Influence of pH of the mobile phase bufferThe mobile phase was buffered because of the existence of
ionizable groups in the chemical structure of the drug, which
could ionize at different pH values. The pH values tested were
2.5, 3.0 and 3.5. Finally, the best results were obtained at pH
3.0 � 0.1 by adjusting with orthophosphoric acid solution. The
choice of this mobile phase is justified by the excellent sym-
metry of the peaks and adequate retention times of Metoclo-
pramide and its degradents.
3.1.5. Selection of wavelengthBased on the spectra of Metoclopramide and its related sub-
stances 273 nm was selected as detection wavelength for the
method. The UV spectrum of Metoclopramide and its impu-
rities were shown in Fig. 2.
Purity threshold Purity flag Mass balance
6.927 No 100.85%
6.955 No 100.65%
5.212 No 97.22%
6.790 No 99.05%
3.856 No 94.34%
6.741 No 98.82%
4.570 No 100.30%
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
es.
CLEE ACME
d aAdded aRecovered aAdded aRecovered
0.114 0.119 0.109 0.106
1.036 1.102 1.094 1.142
1.450 1.432 1.532 1.532
2.071 2.131 2.188 2.232
3.024 3.049 3.063 3.147
0.999914 0.999942
Limit of detection
% RSD Conc. mg/mL % Of impurity % RSD
1.6 0.034 0.003 4.9
2.8 0.022 0.002 4.8
1.8 0.037 0.001 2.9
1.4 0.037 0.001 4.1
Table 6 e Robustness study of Metoclopramide Relatedsubstances.
Parameter RRT of impurity
ACETYLMETO ACMA CLEE ACME
Column 30 �C 0.87 1.32 1.65 1.90
Temperature 35 �C 0.88 1.32 1.67 1.89
40 �C 0.89 1.31 1.72 1.90
j o u rn a l o f p h a rma c y r e s e a r c h 6 ( 2 0 1 3 ) 7 6 5e7 7 3772
homogenous and pure for all the stressed samples analyzed.
Themass balance results were calculated for all of the stressed
samples and were found to be more than 94% (Table 1). The
purity and assay of Metoclopramide were unaffected by the
presence of its impurities and degradation products, which
confirms the stability-indicating power of the developed
method. ACETYLMETO & ACMA are found to be degradation
impurities and CLEE and ACME are process related impurities.
pH of buffer 2.8 0.88 1.32 1.67 1.893.0 0.88 1.34 1.70 1.91
3.2 0.88 1.31 1.66 1.88
Flow rate 1.0 mL min�1 0.88 1.32 1.67 1.89
1.2 mL min�1 0.88 1.29 1.68 1.89
1.4 mL min�1 0.88 1.33 1.72 1.90
3.3. Results of method validation study
3.3.1. Method validationThe described method has been validated for the assay and
related substances by UPLC determination. According to FDA9
and ICH,10 the key analytical parameters that are required for
validation are accuracy, precision, linearity, recovery, LOD,
LOQ and ruggedness.
3.3.2. PrecisionThe repeatability of the developed UPLC method was checked
by a six-fold analysis of the Metoclopramide sample spiked
with the four impurities. The RSD of peak area was calculated
for each impurity. Inter and Intra-day variation and analyst
variation were studied to determine the intermediate preci-
sion of the developed method. The RSD of the area of Meto-
clopramide related compound ACETYLMETO, ACMA, CLEE
and ACME was within 0.3%. The RSD of results obtained in
intermediate precision studies was within 0.9% (Table 2).
3.3.3. Limit of detection and limit of quantificationLimit of detection (LOD) and limit of quantification (LOQ)
values were determined using the signal to noise ratio
method. The LOD of Metoclopramide and its impurities were
found to be in the range of 0.001e0.004 mg/mL (of analyte
concentration 1 mg/mL). The LOQ of Metoclopramide and its
impurities were found to be in the range of 0.07e0.1 mg/mL.
The precision for Metoclopramide and its impurities at LOQ
level was below 3.0% RSD (Table 3).
Table 5 e Accuracy eRecovery study of Metoclopramide Relate
% Spikelevel
ACETYLMETO
aAdded aRecovered % Recove
LOQ 0.107 0.107 100.0
50 1.073 1.100 102.5
75 1.502 1.543 102.7
100 2.146 2.195 102.3
150 3.005 3.035 101.0
% Spikelevel
CLEE
aAdded aRecovered % Recove
LOQ 0.114 0.119 104.4
50 1.036 1.036 100.0
75 1.450 1.450 100.0
100 2.071 2.130 102.9
150 3.024 3.049 100.8
a mg/mL.
3.3.4. LinearityThe linearity of the test methodwas established from the LOQ
to 150% of the test concentration for Metoclopramide and its
related substances. The correlation coefficients obtained were
greater than 0.9999. The result showed that an excellent cor-
relation existed between the peak area and concentration of
the analyte (Table 4).
3.3.5. AccuracyThe accuracy of an analytical procedure expresses the close-
ness of agreement between the reference value and the value
found. The percentage recovery of ACETYLMETO, ACMA, CLEE
and ACME ranged from 99 to 105% (Table 5). Chromatograms
of spiked samples at 0.2% level of all four impurities in a
Metoclopramide sample are shown in Fig. 3.
3.3.6. RobustnessThe robustness of an analytical procedure is a measure of its
capacity to remain unaffected by small but deliberate varia-
tions in chromatographicmethodparameters andprovided an
indication of its reliability during normal usage. In all the var-
ied chromatographic conditions (flow rate, pH of the mobile
d substances.
ACMA
ry aAdded aRecovered % Recovery
0.062 0.063 100.5
1.041 1.077 103.5
1.458 1.473 101.0
2.083 2.105 101.1
3.040 3.071 101.1
ACME
ry aAdded aRecovered % Recovery
0.109 0.109 100.0
1.094 1.142 104.4
1.532 1.570 102.5
2.188 2.232 102.0
3.063 3.147 102.7
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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