Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 292299

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular and

Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa

Ultraviolet spectrophotometry (dual wavelength and chemometric)

and high performance liquid chromatography for simultaneous
estimation of meropenem and sulbactam sodium in pharmaceutical
dosage form
Kuldip R. Marwada a,, Jigar B. Patel a, Nisarg S. Patel b, Bhargav D. Patel a, Dharnant V. Borkhatariya a,
Archita J. Patel a
a
b

Department of Quality Assurance, K.B. Institute of Pharmaceutical Education and Research, Kadi Sarva Vishwavidhyalaya, Sector-23, Gh-6, Gandhinagar, Gujarat, India
Ramanbhai Patel College of Pharmacy, CHARUSAT, Changa, Gujarat, India

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Development of chemometric

methods for simultaneous estimation

of MERM and SB.
 Development of dual wavelength
method for simultaneous estimation
of MERM and SB.
 Development of RP-HPLC method for
simultaneous estimation of MERM
and SB.
 Comparison of developed methods
using ANOVA test.

a r t i c l e

i n f o

Article history:
Received 15 October 2013
Received in revised form 30 December 2013
Accepted 3 January 2014
Available online 17 January 2014
Keywords:
Meropenem
Sulbactam sodium
Dual wavelength ultraviolet
spectrophotometric method
Chemometrics
High performance liquid chromatography

a b s t r a c t
UV spectrophotometric and high performance liquid chromatography (HPLC) methods were developed
for simultaneous determination of meropenem (MERM) and sulbactam sodium (SB) in injection. UV spectrophotometric methods were developed using 0.1 N sodium hydroxide as solvent. The Beers plot for
dual wavelength method was linear in the range of 424 lg mL1 and 212 lg mL1 for MERM and
SB, respectively. The percent recoveries were found to be 98.52 1.23% for MERM and 101.45 1.1%
for SB. Chemometrics assisted UV spectrophotometry was performed using Partial Least Square (PLS)
analysis model and Principal Component Regression (PCR) analysis model. The % recoveries of the MERM
were found to be 100.61 0.06% and 101.31 0.12% using PLS and PCR, respectively. The % recoveries of
the SB were found to be 98.29 0.09% and 97.61 0.13% using PLS and PCR, respectively. Chromatography was performed on Hypersil BDS C18 column using methanol:acetonitrile:water (10:20:70 v/v/v) as
mobile phase. The retention times of MERM and SB were found to be 2.9 min and 2.25 min, respectively.
Developed HPLC method was found to be linear in the range of 50250 lg mL1 and 25125 lg mL1 for
MERM and SB, respectively. The % recoveries were found to be 98.85 0.25% and 98.63 0.34% for MERM
and SB, respectively. The developed analytical methods did not show any interference of the excipients
when applied to pharmaceutical dosage form.
2014 Elsevier B.V. All rights reserved.

Corresponding author. Address: K.B. Institute of Pharmaceutical Education and Research, Sector 23, Near Gh-6 Circle, Gandhinagar 382023, Gujarat, India. Tel.: +91
9427458555.
E-mail addresses: kuldip_marwada@yahoo.com (K.R. Marwada), jigas2181983@gmail.com (J.B. Patel), n.nisarg@yahoo.com (N.S. Patel), bhargav.patel@live.in (B.D. Patel),
bdharanant@yahoo.com (D.V. Borkhatariya), architajpatel@gmail.com (A.J. Patel).
1386-1425/$ - see front matter 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2014.01.008

K.R. Marwada et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 292299

293

Fig. 1. Structure of (A) MERM and (B) SB.

Introduction
Meropenem (MERM) is chemically (4R, 5S, 6S)-3-{[(3S, 5S)-5(dimethylcarbamoyl)
pyrrolidin-3-yl]
sulfanyl}-6-[(1R)-1hydroxyethyl]-4-methyl-7-oxo-1-azabicyclo [3.2.0] hept-2-ene-2carboxylic acid (Fig. 1(A)) is a broad-spectrum carbapenem
antibiotic [1]. It is active against Gram-positive and Gram-negative
bacteria. MERM exerts its action by penetrating bacterial cells
readily and interfering with the synthesis of vital cell wall
components (inhibition of peptidoglycan synthesis), which leads
to cell death [2]. MERM is ofcial in Indian pharmacopoeia [3]
and United State Pharmacopoeia 35/National Formulary 30 [4].
Sulbactam sodium (SB) is chemically sodium (2S-cis)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylate
4,4-dioxide (SB) (Fig. 1(B)) which is a competitive, irreversible
beta-lactamase inhibitor. Its binding to penicillin-binding proteins
imparts weak intrinsic antibacterial activity. SB is ofcial in British
Pharmacopoeia [5] and United States pharmacopoeia 35/National
Formulary 30 [4].
The pharmaceutical formulation (injection) of these drugs contains 1000 mg of MERM, 500 mg of SB and sodium carbonate as
excipients. This dosage form is used in severe meningitis and infection caused by Pseudomonas aeruginosa, Escherichia coli, Klebsiella
pneumoniae [6].
Based on literature review it was found that number of methods
have been reported for estimation of MERM and SB individually as
well as in combination with other drugs. The spectrophotometric
[7,8], HPLC [916], LCMS/MS [17], LCMS [18], capillary zone
electrophoresis [19] methods have been reported for estimation
of MERM and spectro-photometric [2022], RP-HPLC [2331],
HPTLC [32], LCMS [33], capillary isotachophoresis [34] methods
have been reported for estimation of SB. So far one UV spectrophotometric method [35] has been reported for simultaneous estimation of MERM and SB in the synthetic mixture, but the developed
UV method did not show any application to pharmaceutical dosage
form and it was not fully validated. Hence, an attempt was made to
develop and validate a dual wavelength and chemometrics based
UV spectrophotometric methods and RP-HPLC method for simultaneous estimation of MERM and SB in injection. All the developed
methods were compared statistically using ANOVA.
Dual wavelength spectrophotometric method is used to calculate the unknown concentration of a component of interest present
in a mixture containing another component of interest and an unwanted interfering component, by utilizing the absorbance difference at two wavelengths in spectra of the mixture. This directly
gives the concentration of the component of interest without any
interference from other components present in mixture or formulation. The basis for dual wavelength method is the selection of
two such wavelengths where the interfering component shows
same absorbance the difference in its absorbance is zero, whereas
the component of interest shows signicant concentration dependent difference in absorbances at the selected wavelengths [36].

Chemometrics-spectrophotometry is science of extracting

information from chemical system by data driven means [37]. A
combination of chemometrics with analytical chemistry can enhance the signal-to-noise (S/N) ratio, improve selectivity of determination, optimize experimental conditions, raise analytical
operation efciency. The approach is useful in simultaneous determination of two or more components in pharmaceutical dosage
form with overlapping spectra [38].
The Partial Least Square (PLS) approach has been used in
chemometrics for extracting chemical information from complex
spectra which contain interference effects from other factors
(noise) than those of primary interest [3945]. The main advantages of PLS regression are higher speed of processing data concerning the values of concentrations and absorbance of compounds
with strongly overlapping spectra, errors of the calibration model
are minimized by measuring the absorbance values at many points
in the wavelength range of spectra. Principal components regression (PCR) is a method for combating multi-colinearity and results
in estimation and prediction better than ordinary least squares
when used successfully. This transformation ranks the new orthogonal variables in order of their importance and the procedure then
involves eliminating some of the principal components to effect a
reduction in variance. After elimination of the least important principal components, a multiple regression analysis of the response
variable against the reduced set of principal components is performed [46]. Various statistical parameters like predicted residual
sum of squares (PRESS), standard error of prediction (SEP), root
mean square error of calibration (RMSEC), root mean square error
of cross validation (RMSECV) and gure of merits (FOM) like sensitivity, analytical sensitivity, limit of detection (LOD), and limit of
quantitation (LOQ) values are needed to be determined to check
chemometric model (PLS and PCR in our case) suitability [47].
RP-HPLC is a well established technique for separation of component of interest present in pharmaceutical dosage form, plasma,
bulk drug etc. It is more specic method for determination of components in pharmaceutical dosage form [48,49]. The developed RPHPLC method was validated as per ICH guideline Q2R1 [5052].

Experimental
Materials and methods
Standards for drugs and reagents
MERM was obtained as gift sample from Montage Laboratories,
Himatnagar (Gujarat, India). SB was obtained as gift sample from
Montage Laboratories, Himatnagar (Gujarat, India) Astral pharmaceuticals, Vadodara (Gujarat, India) and Aurobindo pharma Ltd.,
(Bangalore, India). Sodium hydroxide pellets were purchased from
Finar chemicals, Vadodara (Gujarat, India). 0.1 N sodium hydroxide
solution (0.1 N) was prepared freshly in laboratory during study
using distilled water prepared in-house. HPLC grade water

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(Milli-Q water) was used for HPLC study. The solvents methanol
(Merck specialities Pvt. Ltd. Mumbai, India), acetonitrile (Fisher
scientic India Pvt. Ltd., Mumbai, India) were of HPLC grade.
Instruments
The dual wavelength UV spectrophotometric and chemometric
assisted UV spectrophotometric methods were developed using UV
Spectrophotometer (SHIMADZU 1800, Japan), having a pair of
10 mm matched quartz cuvettes. An analytical balance (CP 124S,
Sartorius, Germany) was used to weigh accurately the standard
and test samples. An ultrasonic bath (Fast clean, Ajmer, India)
was used for sonication. The solutions were ltered using Whatman lter paper No. 41.
The RP-HPLC method was developed using an analytical HPLC
(Analytical, PU-2080, India), Quarternary Gradient with detector
UV 2230. Milli-Q water system (Q-Guard, France) was used to prepare HPLC grade water.
Softwares
UV probe software was used in UV spectrophotometric methods
for recording spectra and absorbance. PLS_Toolbox solo demo software (Eigenvector) version 7.0.2. was used for the analysis of the
absorbance spectra by chemometric method (PLS and PCR based
regression analysis). Analchrom software was used for recording
the peaks by using RP-HPLC method.
Dual wavelength UV spectrophotometric method
Standard solutions
Accurately weighed quantity of MERM (10 mg) and SB (10 mg)
were transferred in separate 10 mL volumetric ask, dissolved in
5 mL of 0.1 N sodium hydroxide, and volume was made up to the
mark with 0.1 N sodium hydroxide (1000 lg mL1). Suitable dilution of standard stock solution was done to have concentration
of 100 lg mL1 and 50 lg mL1 of MERM and SB, respectively.
Then different aliquots were taken and suitably diluted to prepare
a mixture containing solutions in the working range of 4
24 lg mL1 of MERM and 212 lg mL1 of SB using 0.1 N sodium
hydroxide into 10 mL volumetric ask.
Sample solution
Sample solution was prepared by taking accurately weighed
quantity of powder equivalent to 10 mg of MERM and 5 mg of SB
from injection vial containing powder for injection into 10 ml volumetric ask and dissolved in 5 mL of 0.1 N sodium hydroxide.
Then volume was made up to mark using 0.1 N sodium hydroxide.
The resulting solution was appropriately diluted to get
100 lg mL1 of MERM and 50 lg mL1 of SB. From the above solution suitable dilutions were made to get nal concentration of
10 lg mL1 of MERM and 5 lg mL1 of SB using 0.1 N sodium
hydroxide.
Study of overlain spectrum and selection of wavelength
Suitable dilutions from the working standard solutions of
100 lg mL1 of MERM and 100 lg mL1 of SB were done to prepare
the solutions of MERM (8 lg mL1) and SB (8 lg mL1). The prepared solutions were scanned over the range of 200400 nm. The
wavelengths were selected on the basis of absorbance at two different wavelengths for both the drugs.
Assay of pharmaceutical formulation by dual wavelength
spectrophotometry
An accurately weighed powder from injection vial equivalent to
about 10 mg of MERM and 5 mg of SB was transferred to 10 mL
volumetric ask and dissolved in 5 mL of 0.1 N sodium hydroxide.
The volume was made up to the mark using 0.1 N sodium

hydroxide as solvent. The resulting solution was ltered through

Whatman lter paper No. 41 and 1 mL of this ltrate was appropriately diluted up to mark in 10 mL volumetric ask. The above
solution was further diluted 10 times with 0.1 N sodium hydroxide. The absorbance was measured at the selected wavelengths
and concentration of each component was determined.
Method validation
Linearity of the method was determined by plotting calibration
curve in the range of 424 lg mL1 for MERM and 212 lg mL1
for SB. The study was performed ve times and average absorbance
was calculated for respective wavelengths. Precision of the method
was determined by performing method repeatability studies, intraday variation, and interday variation. In repeatability study, one
concentration (MERM 16 lg mL1 and SB 8 lg mL1) of both the
drugs were analyzed six times. Accuracy of the proposed method
was studied using standard addition method at three different levels 80%, 100% and 120% of test concentration. The LOD and LOQ of
developed method were determined by standard deviation
method.
Chemometric method
Preparation of solutions and selection of chemometric data set
Accurately weighed quantity of MERM (10 mg) and (SB 10 mg)
were transferred in 10 mL volumetric ask, separately, dissolved in
5 mL of 0.1 sodium hydroxide and volume was made up to the
mark with 0.1 N sodium hydroxide (1000 lg mL1). Appropriate
aliquots of standard stock solutions were diluted with 0.1 N
sodium hydroxide to get concentrations of 100 l g mL 1 and
50 lg mL1 of MERM and SB, respectively. Then suitable dilutions
were made to prepare a mixture containing a working range
of 424 lg mL1 of MERM and 2.512 lg mL1 of SB using 0.1 N
Sodium hydroxide into 10 mL volumetric ask.
To obtain a suitable calibration set for PLS and PCR; a random
design was made utilizing 25 experiments where the lowest
and highest amount for MERM was 5 lg mL1 and 24 lg mL1.
The lowest and highest amount for SB was 2.5 l g mL 1 and
12 lg mL1. The data set was containing total 25 mixtures. From
calibration datasets 9 mixtures were removed and used as validation dataset for prediction of true values and to nd out the model
suitability as well as sensitivity. The calibration data set contained
16 and the validation data set contained 9 standard mixtures. The
selected data set sequence and its information is given in Table 1.

Table 1
Calibration and validation (concentration) data set for chemometrics.
Sr. No.

Concentration (lg mL1)

Calibration data set
MERM

SB

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

5
5.5
7.5
9
12
13.5
15
16.5
21
22.5
6
9
12
15
16.5
18

2.5
4
5.5
7
5
7
10
8.5
12
12
4
8
12
5
7
9

Sr. No.

Concentration (lg mL1)

Validation data set
MERM

SB

1
2
3
4
5
6
7
8
9

10
18
19.5
24
5.5
7.5
12.5
13.5
19.5

5.5
11.5
10
2.5
12
6
9.5
3.5
11

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Data processing by PLS and PCR

Various parameters like correlation coefcient, PRESS, RMSEC,
RMSECV and standard error of prediction, were determined by
developing a chemometric model. Correlation coefcient determines the degree of correlation between concentration and observed signal. Standard error of prediction refers to uncertainty
in the concentration or reference values and it should be negligible.
Various gure of merits like sensitivity, analytical sensitivity, LOD
and LOQ were also determined. Sensitivity for a given analyte is
dened as the slope of the analytical calibration curve. Analytical
sensitivity refers to the ratio between sensitivity and instrumental
noise. The instrumental noise and slope of the calibration curve
were used to calculated limit of detection and limit of quantitation.

P
jj

P
jj

LOD 3:3  jjbjj  jj

LOQ 10  jjbjj  jj

Where; jjbjj slope of calibration curve

P
jj jj Noise
RP-HPLC method
Preparation of solution
Standard solution. An accurately weighed quantity of MERM
(10 mg) and SB (5 mg) was transferred in 10 mL volumetric ask,
dissolved in 5 mL of HPLC grade water and volume was made up
to the mark with Milli Q water (1000 lg mL1 MERM and
500 lg mL1 SB). An aliquot of 1 mL of standard stock solution
was taken and further diluted to 10 mL with HPLC grade water
to get the concentration of 100 lg mL1 and 50 lg mL1 for MERM
and SB respectively. 20 ll of the nal solution was injected by
autosampler into column and the chromatogram was recorded.
Sample solution. Accurately weighed quantity of powder for injection equivalent to 10 mg of MERM and 5 mg of SB, respectively,
was transferred into 10 mL volumetric ask and dissolved in HPLC
grade water with vigorous shaking. The volume was made up to
mark with the same solvent. The solution was ltered through
Whatman lter paper No. 41. An aliquot of 1 mL was taken and further diluted to 10 mL with the HPLC grade water. The prepared
solution (20 lL) was injected into column using autosampler and
chromatograms were recorded.

295

Results and discussion

Initially, the solubility of both the drugs was checked in water,
methanol, sodium hydroxide and hydrochloric acid. Both drugs
were freely solubility in water and methanol. The spectra were recorded for drugs in methanol and water but they did not show better absorbance for SB in comparison with MERM. When the spectra
were recorded using sodium hydroxide, spectrum of SB showed
about 56 times higher absorbance than that in water or methanol.
This may be due to non-enzymatic conversion of SB to 5 carboxy6-methyl-6-sulno-4-aza-2-heptanoic acid II (Fig. 2) at high pH,
resulting in opening of b lactam ring. This structure showed high
absorbance in UV region above 200 nm [53,54]. Behavior of MERM
was unaffected in alkaline medium. Hence, 0.1 N sodium hydroxide was selected as solvent system.
Validation of dual wavelength method
The overlain spectra of the standard solutions of MERM and SB
(Fig. 3) indicated that a dual wavelength spectrophotometric
method was a suitable method for simultaneous determination
of MERM and SB. In dual wavelength method, wavelengths
242.0 nm and 274.0 nm were selected for determination of MERM,
as SB showed same absorbance at these wavelengths. For determination of SB wavelengths 285.0 nm and 306.0 nm were selected, as
MERM showed same absorbance at these wavelengths.
The calibration curves of MERM and SB were linear in the range
of 424 lg mL1 and 212 lg mL1, respectively. The % relative
standard deviation (% RSD) values for repeatability studies, intraday and interday precision were found to be less than 2. The LOD
and LOQ for MERM were found to be 1.16 lg mL1 and

Fig. 2. Structure formed from SB at high pH.

Preparation of calibration curves for MERM and SB. The calibration

curves were plotted over a concentration range of 50250 lg mL1
for MERM and 25125 lg mL1 for SB. Accurately measured aliqouts of standard stock solutions of MERM and SB were transferred
to a series of 10 mL volumetric asks and the volume in each ask
was adjusted to 10 mL with HPLC grade water. The resulting solutions were injected into HPLC system and various conditions of
mobile phase composition, ow rate, peak area were determined.
Calibration curves were constructed for MERM and SB by plotting
peak area vs concentration at 220 nm.
Method validation
The method was validated using ICH guideline Q2 R1 [52]. Various parameters like linearity, precision, accuracy, LOD, LOQ,
robustness and specicity were determined. Linearity was performed in triplicate and % R.S.D. was determined at each level.
Repeatability, intraday and interday variation were studied to
determine the precision of the method. Accuracy was measured
using standard addition method at 80%, 100% and 120% level. Percent recoveries were determined at each level of addition. LOD and
LOQ values were determined based on standard deviation method.

Fig. 3. Overlain spectra of standard solution of MERM and SB in 0.1 N sodium

hydroxide (8 lg mL1).

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3.52 lg mL1, respectively and 0.9 lg mL1 and 2.73 lg mL1,

respectively for SB. The percent recoveries were found to be
98.52 1.23% for MERM and 101.45 1.1% for SB.
Chemometric method
Selection of spectral region and calibration parameters for PLS and PCR
From the spectra (Fig. 4) of mixture of MERM and SB, absorbance data from spectral region of 200 nm to 215 nm was removed
as it contained noise and region of 340400 nm was removed as no
absorbance due to drugs was seen in it. Then further smoothing
was done and absorbance data of spectral region from 235 nm to
320 nm was selected as chemometric region.
The absorbance data from the selected region was installed to
the model and developed for their suitability in the PLS_Toolbox
demo software. From various models, PLS and PCR were found to
t the best. The nal calibration and validation dataset were applied to both models and the values of correlation coefcient,
RMSEC, RMSECV, PRESS, and SEP were determined. The details of
the obtained data are given in Table 3.
Fig. 5A and B shows the variation of RMSECV as a function of
Number of components and RMSECV as a function of latent variables for PCR and PLS, respectively. Two factors were found to be
optimum for both components in the mixture by PCR and number
of latent variables were three for both components in the mixture
by PLS.
From the calibration data set some data set having high Q residual value were extracted out and used for validation data set. Their
concentrations were predicted from absorbance data of the same.
The results of the methods displayed % recovery >97% for MERM
and SB from the mixture. This showed that there is a very good
compliance between measured values and predicted values. Results of analysis for various calibration statistical parameters and
% recoveries are shown in Table 2. Analytical gures of merit
(FOM) like sensitivity, analytical sensitivity, LOD, and LOQ were
determined (Table 3).
RP-HPLC method
The experimental conditions used for UV spectrophotometric
methods could not be extended to HPLC system as 0.1 N sodium

Fig. 4. UV spectra of calibration mixture for selection of chemometric region.

Fig. 5. (A) Plot of RMSEC value vs principal component number for MERM and SB
for PCR. (B) Plot of RMSEC value vs latent variable number for MERM and SB for PLS.

hydroxide (pH 11) would raise the pH above the working pH

range (pH28) for the C-18 column. Hence even though small
absorbance values were obtained for SB using HPLC grade water,
it was used along with methanol and acetonitrile for method
development. The solutions (25 lg mL1) of MERM and SB individually scanned in the UV region. The UV overlay spectra of MERM
and SB in water is as shown in Fig. 6. From the spectra, 220 nm
was selected as detection wavelength for both drugs. Various
parameters of HPLC system were optimized (Table 4) and the
standard mixture containing MERM (100 l g mL 1 ) and SB
(50 lg mL1) were injected into the column and the chromatograms were recorded. The recorded chromatograms for standard
and test solutions are shown in Figs. 7 and 8. The chromatogram
of test solution was similar with out of the standard solution and
no interfering peaks were observed suggesting the specicity of
the method. The method was further validated as per ICH guideline
Q2(R1) and system suitability parameters were also studied as an
integral part of validation study as per USP NF 31.
Method validation
The method was found to be linear in the range of 50
250 lg mL1 for MERM and 25125 lg mL1 for SB. Peak area
was directly proportional to the concentrations of drugs in this region. Accuracy was performed by standard addition method at
three different levels 80%, 100%, and 120%. The % recoveries were

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Table 2
Results of calibration statistics obtained by chemometric analysis.
Parameters

PLS regression analysis

MERM

Media
r2c
r2cv
PRESS
RMSEC
RMSECV
SEP
r2 (Correlation coefcient)
% Recovery

0.1 N Sodium hydroxide

0.996
0.997
1.21
0.436
0.815
0.027
0.996
100.61 0.06%

PCR regression analysis

SB

MERM

SB

0.996
0.996
0.40
0.186
0.280
0.020
0.997
98.29 0.09%

0.1 N Sodium hydroxide

0.993
0.997
2.35
0.442
0.810
0.029
0.997
101.31 0.12%

0.996
0.998
0.55
0.186
0.282
0.050
0.996
97.61 0.13%

Table 3
Figure of merits.
Parameters

Sensitivity
Analytical sensitivity
LOD
LOQ
Bias

PLS regression analysis

PCR regression analysis

MERM

SB

MERM

SB

1.01
2.62
1.26
3.81
0

1.00
6.24
0.53
1.60
0

1.005
2.57
1.28
3.89
0

1.1
6.23
0.5
1.90
0

Fig. 7. Chromatogram of standard solution of MERM (100 lg mL1) and SB

(50 lg mL1).

Fig. 6. UV spectra of MERM and SB in HPLC grade water.

Table 4
Optimized chromatographic conditions for HPLC.
Sr.
No.

Parameter

Chromatographic conditions

1
2

Stationary phase
Mobile phase

3
4
5
6
7

Mode of elution
Temperature
Flow rate
Injection volume
Wavelength of
detection

Hypersil BDS C18 (250  4.60 mm i.d., 5l)

Methanol:acetonitrile:water (10:20:70 v/
v/v)
Isocratic
Room temperature
1 mL min1
20 lL
220

Fig. 8. Chromatogram of test solution of MERM (100 lg mL1) and SB (50 lg mL1).

found to be 98.85 0.25% and 98.63 0.34% for MERM and SB,
respectively. The precision of developed method was expressed
as a % of relative standard deviation (%RSD) for repeatability (%
R.S.D. values were 0.83 and 0.79 for MERM and SB, respectively),
intraday precision (% R.S.D. values were 0.90 and 0.98 for MERM
and SB, respectively), and interday precision (% R.S.D. values were
1.07 and 0.74 for MERM and SB, respectively). The LOD and LOQ
values were found to be 5.40 lg mL1 and 16.30 lg mL1 for
MERM and 3.26 lg mL1 and 9.89 lg mL1 for SB, respectively.
The robustness of the method was studied by changing the composition of mobile phase (methanol:acetonitrile:water; 9:19:72,
11:21:68, v/v/v), detection wavelength from 220 5 nm and ow
rate from 1 0.1 mL/min % R.S.D. was found to be less than 2% in
each case.

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Table 5
Analysis of pharmaceutical formulation containing MERM and SB by developed analytical methods.
Drug

Drug amount

Dual wavelength UV spectrophotometric method

Chemometric assisted method

RP-HPLC

PLS

PCR

MERM

Amount taken (mg)

Amount found (mg)
% Label claim

10
9.95
99.50 0.13%

20
20.05
100.28 0.32%

20
20.31
101.57 0.45%

100
98.12
98.12 0.25%

SB

Amount taken (mg)

Amount found (mg)
% Label claim

5
4.86
97.36 0.24%

10
9.82
98.2 0.39%

10
9.872
98.72 0.28%

50
47.28
97.5 0.51%

Table 6
Summary of comparison of the developed analytical methods by single factor ANOVA.
Anova: Single factor
Methods

Count

Sum

Average

Variance

Summary
Dual wavelength method
PLS analysis
PCR analysis
RP-HPLC method

2
2
2
2

196.86
198.48
200.29
195.62

98.43
99.24
100.15
97.81

2.29
2.16
4.06
0.19

Source of variation

SS

df

MS

P-value

F crit

ANOVA
Between methods
Within methods
Total

6.15
8.71
14.86

3
4
7

2.05
2.18

0.94

0.50

6.59

Applications of developed methods

References

To determine the applicability of the developed methods to the

analysis of samples, they were applied to pharmaceutical formulation. Three replicate measurements were made. The results are
shown in Table 5. The results obtained were in good accordance
with the label claim for the drugs.

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Comparison of developed methods

All developed methods were compared by using ANOVA and the
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Conclusion
Three different methods were developed and validated for
simultaneous estimation of MERM and SB in injection dosage form.
All the methods were comparable to each other, suggesting that
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Acknowledgements
The authors are thankful to K.B. Institute of Pharmaceutical
Education and Research, Gandhinagar for providing the facilities
for work. The authors are also thankful to Montage Laboratories,
Astral Pharmaceuticals, Aurobindo Pharma Ltd. for providing gift
sample of MERM and SB.

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