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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.

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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 1.89

3.0 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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