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![Page 1: Development and Validation of a Stability-Indicating LC Method for Simultaneous Analysis of Aceclofenac and Paracetamol in Conventional Tablets and in Microsphere Formulations](https://reader031.fdocuments.us/reader031/viewer/2022020600/57506b9b1a28ab0f07bedca1/html5/thumbnails/1.jpg)
Development and Validation of a Stability-Indicating LC Method for SimultaneousAnalysis of Aceclofenac and Paracetamolin Conventional Tablets and in MicrosphereFormulations
Shahid Jamil&, Sushma Talegaonkar, Roop K. Khar, Kanchan Kohli
Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, M.B. Road, Hamdard Nagar, New Delhi 110062, India;E-Mail: [email protected]
Received: 9 April 2008 / Revised: 5 June 2008 / Accepted: 19 June 2008Online publication: 19 September 2008
Abstract
A stability-indicating reversed-phase LC method for analysis of aceclofenac and paracet-amol in tablets and in microsphere formulations has been developed and validated. Themobile phase was 80:20 (v/v) methanol–phosphate buffer (10 mM at pH 2.5 ± 0.02). UVdetection was at 276 nm. The method was linear over the concentration ranges 16–24 and80–120 lg mL-1 for aceclofenac and paracetamol, respectively, with recovery in the range100.9–102.22%. The limits of detection and quantitation for ACF were 0.0369 and0.1120 lg mL-1, respectively; those for PCM were 0.0631 and 0.1911 lg mL-1,respectively.
Keywords
Column liquid chromatographyParacetamolAceclofenacSimultaneous determinationMicrospheres
Introduction
Aceclofenac (ACF; o-(2,6-dichloroanili-
no)phenylacetate glycolic acid ester),
which is structurally related to diclofenac,
has anti-inflammatory and analgesic
properties [1] and is used for acute and
chronic treatment of the signs and symp-
toms of rheumatoid arthritis, osteoar-
thritis, ankylosing spondylitis, and
scapulohumeral periarthritis [2, 3]. Para-
cetamol (PCM;N-(4-hydroxyphenyl)acet
amide), also known as acetaminophen, is
a popular analgesic and antipyretic drug
widely used for management of pain and
fever [1]. Several commercially available
fixed-dose combination dosage forms of
aceclofenac and paracetamol are used for
relief from severe pain and inflammation
in osteoarthritis, rheumatoid arthritis,
ankylosing spondylitis, lower-back pain,
dental pain, gynaecological pain, and
painful and inflammatory conditions of
the ear, nose, and throat.
Whereas several LC methods have
been reported for analysis of paraceta-
mol combined with other drugs in phar-
maceutical preparations [4–10], most LC
methods for analysis of ACF have been
for the single compound, or for the
compound in combination with other
drugs or in the presence of its metabo-
lites, in biological fluids [11–16]. Only
one LC method has been reported for
analysis of ACF in a pharmaceutical
preparation [17]. Simultaneous analysis
of aceclofenac and paracetamol in tab-
lets by LC [18, 19] with 65:35 (v/v) ace-
tonitrile–phosphate buffer, pH 3.0, as
mobile phase [18] resulted in retention
times (tR) of 1.58 and 4.01 min for par-
acetamol and aceclofenac, respectively,
and resolution of 4.83. The linear ranges
were very short—1.8 to 4.2 lg mL-1 for
ACF and 9 to 21 lg mL-1 for PCM—
and quantitative determination of the
analytes in the presence of their degra-
dation products was not reported. The
very short retention time of PCM
(1.58 min), near solvent peak, led to
merging of the peaks and inaccurate
quantification of the drug. In another
LC method [19] with 60:40 (v/v) aceto-
nitrile–phosphate buffer, pH 5.0, as
mobile phase, the retention times (tR) of
2008, 68, 557–565
DOI: 10.1365/s10337-008-0797-x0009-5893/08/10 � 2008 Vieweg+Teubner | GWV Fachverlage GmbH
Original Chromatographia 2008, 68, October (No. 7/8) 557
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paracetamol and aceclofenac were 4.77
and 6.44 min. Linear ranges were, again,
very short—0.5 to 3.5 lg mL-1 for ACF
and 20–80 lg mL-1 for PCM; resolu-
tion, 1.32, was less than the value (2)
regarded as acceptable in the litera-
ture [20]. Another disadvantage of this
method was that it was not stability-
indicating. The literature contains no
reports of stability-indicating methods
for simultaneous determination for ACF
and PCM in tablet dosage forms and
microsphere formulations with methanol
as organic mobile phase component. For
reasons of economy, safety, and volatil-
ity methanol is preferable to acetonitrile.
This paper reports an attempt to de-
velop and validate a novel stability-indi-
cating LC method with UV detection for
simultaneous analysis of aceclofenac and
paracetamol in commercial tablet dosage
forms and in microsphere formulations.
The method was developed and validated
with the objective it should be simple,
economical, selective, and sensitive with a
short run time and simple mobile phase
composition. The method was validated
for the important characteristics—line-
arity, accuracy, precision, sensitivity,
robustness, etc.—in accordance with
International Conference on Harmoni-
zation (ICH) guidelines [21] and another
reported method [20].
Experimental
Materials and Reagents
Paracetamol was purchased from Orient
Pharma and Chemicals (New Delhi,
India). Pure aceclofenac was a gift
from Ranbaxy Research Laboratory
(Gurgaon, India). The commercial prep-
aration Acemil-P (batch no. CSEACD
6002; labelled to contain 100 mg ace-
clofenac and 500 mg paracetamol per
tablet) was purchased locally (New
Delhi, India). Methanol (HPLC-grade)
was purchased from Qualigen (Mumbai,
Maharashtra, India), Potassium dihy-
drogen phosphate and orthophosphoric
acid were purchased from Merck
(Mumbai, Maharashtra, India). High-
purity water was prepared using a
Millipore purification system.
Chromatography
LC was performed with Shimadzu
equipment comprising LC-10A VP qua-
ternary pumps, an SPD-10AVP variable-
wavelength programmable UV–visible
detector, a column oven, and an SCL
10AVP system controller with Class-VP
5.032 software.
Compounds were injected with a
Rheodyne injector fitted with a 20-lLloop and separated on a 25 cm 9 4.6 mm
i.d. stainless-steel analytical column con-
taining 5-lm particle LiChrosphere-100
RP 18 packing. The isocratic mobile
phase was 80:20 (v/v) methanol–potas-
sium dihydrogen phosphate buffer
(10 mM, pH 2.5 ± 0.02, adjusted with
orthophosphoric acid) at a flow rate of
1.0 mL min-1. The variable-wavelength
UV–visible detector was set at 276 nm.
Table 1. Results from testing of system suitability for analysis of aceclofenac and paracetamol(n = 3)
Method characteristic Paracetamol Aceclofenac
Theoretical plates 2062.28 4385.02Tailing factor 1.39 1.38Resolution 13.77 13.77Capacity factor 2.2 2.8Repeatabilitya 0.192 0.212
a RSD of retention time (%, n = 3)
Fig. 1. LC chromatogram of aceclofenac (tR 6.5 min) and paracetamol (tR 2.39 min) in thepresence of products of degradation of each analyte (tR 7.19 and 8.83 min and tR 4.7 min,respectively) under acidic conditions (0.1 M HCl)
Fig. 2. LC chromatogram of aceclofenac (tR 6.5 min) and paracetamol (tR 2.4 min) in thepresence of products of degradation of each analyte (tR 7.19 and 7.44 min and tR 4.72 min,respectively) under alkaline conditions (0.1 M NaOH)
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All analysis was performed at ambient
temperature.
Standard Solutions andCalibration Solutions
Stock solution of aceclofenac was pre-
pared by dissolving 50 mg of the drug,
accurately weighed, in methanol in a 50-
mL volumetric flask. For preparation of
mixed paracetamol and aceclofenac
standard solution, 50 mg paracetamol
was accurately weighed, dissolved in
methanol in a 50-mL volumetric flask,
10 mL aceclofenac stock solution was
added, and the solution was diluted to
volume with methanol. For preparation
of calibration plots, solutions containing
aceclofenac and paracetamol at different
concentrations in the ranges 16–24 and
80–120 lg mL-1, respectively, were pre-
pared by diluting 2, 2.25, 2.5, 2.75, and
3 mL of the mixed standard solution with
mobile phase in 25-mL volumetric flasks.
Preparation of Test Samplefor Assay of Aceclofenac andParacetamol in Tablets
Ten tablets from the same batch were
accurately weighed and powdered in a
mortar. A quantity of the powder equiv-
alent to approximately 50 mg paraceta-
mol or 10 mg aceclofenac was weighed
accurately, transferred to a 50-mL volu-
metric flask, and suspended in methanol,
with ultra-sonication for up to 10 min.
The solution was then diluted to volume
with the same solvent. For further dilu-
tion 2.5 mL suspension was diluted with
mobile phase in a 25-mL volumetric flask
and, after shaking, the sample was filtered
through a 0.45-lm membrane filter into
vials, ready for LC analysis.
Preparation of Test Samplefor Assay of Aceclofenacand Paracetamol in aMicrosphere Formulation
A quantity of reconstituted microspheres
powder equivalent to approximately
50 mg paracetamol or 10 mg aceclofenac
was accurately weighed, transferred to a
50-mL volumetric flask, and suspended in
the absolute ethanol. The sample was
placed in an ultrasonic bath for three
20-min periods separated by 60 min.
After complete extraction of the active
ingredients the solution was diluted to
volumewith the same solvent then filtered
through a 0.45-lm filter. The final sample
for analysis was prepared by placing
2.5 mL filtrate in a 25-mL volumetric
flask, diluting to volume with mobile
phase, and, after shaking, again filtered
through a 0.45-lm membrane filter into
vials ready for LC analysis.
Method Validation
Specificity
The specificity of a method is the extent
to which it can be used for analysis of a
particular analyte in a mixture or matrix
without interference from other com-
ponents. In this assay specificity was
tested by analysis of solutions contain-
ing degradation products produced in
forced degradation studies. A mixture of
ACF and PCM and separate pure
samples of the drugs (prepared by dis-
solving 100 mg of each in 20 mL
methanol) were subjected to forced
degradation under acidic and basic
conditions, by heating under reflux with
0.1 M HCl or 0.1 M NaOH, respec-
tively, at 70 �C for 6 h. The stability of
the drugs to oxidation was studied by
stirring a solution containing 1% (w/v)
of the drugs and 3% H2O2 for 2 h. The
resulting solutions were appropriately
diluted with mobile phase and injected
for LC analysis.
Linearity
Linearity is studied to determine the
range over which analyte response was a
Fig. 3. LC chromatogram of aceclofenac (tR 6.49 min) and paracetamol (tR 2.39 min) in thepresence of the product of degradation of aceclofenac (tR 7.18 min) under oxidizing conditions(3% H2O2)
Fig. 4. LC chromatogram obtained from a mixed standard of aceclofenac (tR 6.49 min) andparacetamol (tR 2.39 min) under normal conditions
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linear function of concentration. This
study was performed by preparing stan-
dard solutions at five concentrations
from 80 to 120% of the target analyte
concentrations, i.e. concentrations rang-
ing from 16 to 24 and from 80 to
120 lg mL-1 for aceclofenac and para-
cetamol, respectively. These analyses
were performed in triplicate.
Accuracy
The accuracy of an analytical method is
the closeness of results obtained by that
method to the true value for the sample.
It was expressed as recovery (%), which
was determined by the standard addition
method. Pre-analyzed samples were
spiked with an extra 80, 100, and 120%
of the standard and then re-analyzed.
The experiment was performed in tripli-
cate. Recovery (%), RSD (%), and
standard error of the mean (SEM) were
calculated for each concentration.
Precision
The precision of an analytical method is
the closeness of replicate results obtained
from analysis of the same homogeneous
sample. Precision was considered at two
levels, i.e. repeatability and intermediate
precision, in accordance with ICH
recommendations. Repeatability, or
intra-day precision, was determined by
performing nine analyses at three con-
centrations, in one laboratory, on the
same day. Intermediate precision was
determined out by analysing the same
sample in the same way on a second day.
Results from determination of repeat-
ability and intermediate precision were
expressed as SD, RSD (%), and SEM.
Limits of Detection and Quantitation
Limits of detection (DL) and quantitation
(QL) were determined by the standard
deviation method. For determination of
DL and QL, a blank sample was injected
in triplicate and the standard deviation
Table 2. Results from forced degradation of a mixture of aceclofenac and paracetamol in study of specificity
Method characteristica Stress condition
0.1 M HCl (6 h, 70 �C) 0.1 M NaOH (6 h, 70 �C) 3% H2O2 (2 h)
Aceclofenac (100%)Mean peak area ± SDb 15233.37 ± 1352.8 10520.58 ± 1228.1 92474.78 ± 1375.9SEM 781.02 709.05 794.39No. of degradation products, retention time(s) (tR) 2, 7.19 and 8.83 min 2, 7.19 and 7.44 min 1, 7.18 minConcn remaining (lg mL-1) 0.533 0.383 2.991Recovery (%) 2.665 1.915 14.955Paracetamol (100%)Mean peak area ± SDb 326927.28 ± 3005.0 358246.21 ± 3078.0 1729656.65 ± 3148.6SEM 1734.9 1777.6 1817.9No. of degradation products, retention time(s) (tR) 1, 4.7 min 1, 4.72 min –Concn remaining (lg mL-1) 18.24 19.94 94.56Recovery (%) 18.24 19.94 94.56
a SEM, standard error of the mean; SD, standard deviation; tR, retention timeb Average from three determinations (n = 3)
Table 3. Linear regression data for the calibration plota
Method characteristic Aceclofenac Paracetamol
Linear range (lg mL-1) 16–24 80–120Mean regression equation y = 31418x - 1525.3 y = 18394x - 8710.1Mean correlation coefficient 0.9996 0.9989Mean slope ± SDb 31,418 ± 152.321 18,394 ± 132.321Mean intercept ± SD -1525.3 ± 90.231 -8710.1 ± 89.31Mean slope without intercept ± SD 31,343 ± 121.954 18,308 ± 123.984Standard error of mean slope 89.7 77.22Standard error of mean intercept 52.36 51.7695% Confidence interval of slope 31959 to 31186 18860 to 1819595% Confidence interval of intercept -1209.2 to -1659.9 -8397.8 to -8843.3
a Average from three calibration plots (n = 3)b Standard deviation
Table 4. Accuracy of the methoda
Drug Excess drugadded toanalyte (%)
Theoreticalcontent(lg mL-1)
Concn found± SDb
(lg mL-1)
Recovery(%)
RSDb
(%)SEMb
Aceclofenac 80 28.8 29.44 ± 0.125 102.22 1.03 0.072100 40 40.6 ± 0.080 101.5 0.49 0.046120 52.8 53.28 ± 0.194 100.9 0.97 0.112
Paracetamol 80 144 146.4 ± 0.105 101.6 1.5 0.075100 200 222 ± 0.060 100.9 0.96 0.56120 264 267 ± 0.075 101.1 0.99 0.121
a Average from three determinations (n = 3)b SD, standard deviation; RSD, relative standard deviation; SEM, standard error of themean
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(Sy/x) of the blank response was calcu-
lated. DL and QL were then determined
from the slope, S, of the calibration plot
and Sy/x by use of the formulae:
DL ¼ 3:3� Sy=x=S andQL ¼ 10� Sy=x=S
Robustness
Robustness was assessed to evaluate
the effect of small but deliberate vari-
ation of the chromatographic condi-
tions on results from analysis of the
drugs. The mobile phase flow rate was
changed to 1.0 ± 0.25 mL min-1 and
the concentration of methanol in the
mobile phase was changed to 80 ± 2%
(v/v).
Assay of Aceclofenac andParacetamol in Tablets andin Microsphere Formulations
Quantification of the analytes in tablets
and in microsphere formulations was
achieved by recording peak areas and
calculating amounts, in milligrams per
formulation, by use of the equation:
Amount mg per dosage formð Þ¼ AT=ASð Þ � C=Wð Þ � A� F
where AT and AS are the peak areas of
ACF or PCM obtained from the test
solution and standard solution, respec-
tively, C is the weight (mg) of respective
ACF or PCM reference standards used
to prepare the standard solution, W is
the weight (mg) of sample taken to pre-
pare the test solution, A is the average
weight (mg) of each dosage form, and F is
the factor obtained from the dilution
factors of the standard and test solutions.
The significance of method for assay of
the analytes in commercial tablets and in
microsphere formulations prepared in
this laboratory was evaluated by use of
Student’s t test.
Stability Testing of Blister-Packed Tablets andMicrosphere FormulationsContaining ACF and PCM
For stability studies, commercial tablets
and microsphere formulations were
stored at high temperature and at high
and low relative humidity (50 �C/75%
Table 5. Intra-day and inter-day precision of the method
Amount of drug taken(lg mL-1)
Mean concnfound (n = 3)
±SDa RSD (%)a SEMa
Aceclofenac (ACF)Intra-day (1st day)16 15.95 0.04583 0.287 0.0264620 19.93 0.03512 0.176 0.0202824 24.04 0.05000 0.2079 0.02887
Inter-day (2nd day)16 16.079 0.06851 0.426 0.0395520 20.072 0.07353 0.366 0.0424524 24.14 0.07000 0.2899 0.04042
Paracetamol (PCM)Intra-day (1st day)80 80.33 0.1002 0.1245 0.05783100 100.35 0.1510 0.150 0.08718120 120.37 0.1947 0.1617 0.1124
Inter-day (2nd day)80 80.61 0.1266 0.1566 0.07311100 100.74 0.2150 0.2134 0.1241120 120.82 0.2606 0.2157 0.1504
a SD, standard deviation; RSD, relative standard deviation; SEM, standard error of the mean from triplicate samples (n = 3)
Table 6. Robustness of the method to deliberate alteration of mobile phase composition
Concn level Mobile phasecomposition(methanol–phosphatebuffer, pH 2.5)
Statistical data
Aceclofenac (20 lg mL-1) Paracetamol (100 lg mL-1)
Original Used Level Mean peakarea ± SDab
SEMb Mean tR(min) ± SDb
RSD(%)
Mean peakarea ± SDab
SEMb MeantR (min)± SDb
RSD(%)
100% 80:20 78:18 -2 602,669 ± 794.2 458.42 6.14 ± 0.03 0.119 1,799,234 ± 3199.6 1847.3 2.13 ± 0.05 0.22380:20 0 603,185 ± 775.6 447.8 6.48 ± 0.01 0.212 1,802,448 ± 3372.4 1947.2 2.38 ± 0.02 0.23682:22 +2 603,805 ± 799.4 461.6 6.73 ± 0.04 0.220 1,805,965 ± 3305.5 1908.3 2.67 ± 0.04 0.216
a Average from three determinations (n = 3)b tR, retention time; SD, standard deviation; SEM, standard error of the mean
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RH, 50 �C/50% RH), conditions which
might possibly lead to accelerated
degradation, and the drug content was
analyzed after 1, 3, and 6 months.
Results
System-Suitability Tests
System-suitability tests were performed
to measure the number of theoretical
plates, tailing factor, resolution, capacity
factor (k), and repeatability of retention
time (RSD, %) of the analytes. The
results is listed in Table 1.
Method Validation
It was decided to use an isocratic mobile
phase to achieve optimum resolution of
the analytes. In this method there was no
interference at the elution time of the
analytes from peaks of degradation
products produced under acidic, alka-
line, or oxidizing conditions. The reten-
tion times, tR, of ACF and PCM were
6.48 ± 0.02 min and 2.38 ± 0.02 min,
respectively. After treatment with acid
(0.1 M HCl), degradation products of
ACF were observed at tR 7.19 and
8.83 min and a degradation product of
PCM was observed at tR 4.7 min. After
treatment with alkali (0.1 M NaOH),
degradation products of ACF were
observed at tR 7.19 and 7.44 min and a
degradation product of PCM was ob-
served at 4.72 min. After treatment with
oxidizing agent (3% H2O2) a degrada-
tion product of ACF only was observed
at tR 7.18 min. Typical chromatograms
are illustrated in Figs. 1, 2, 3 and 4 and
complete results are listed in Table 2.
Response to the compounds was a
linear function of concentration in the
ranges 16–24 and 80–120 lg mL-1 for
ACF and PCM, respectively (correlation
coefficients 0.9996 and 0.9989, respec-
tively). Linear regression data are given
in Table 3. Test solutions containing
low, medium, and high concentrations
(80, 100, and 120%) of ACF and PCM
were used to determine recovery (accu-
racy) and intraday and intermediate
precision. Recovery of ACF and PCM
was in the range 100.9–102.22%. Results
from measurement of accuracy and
intraday and intermediate precision are
given in Tables 4 and 5. The limits of
detection and quantitation for ACF were
0.0369 and 0.1120 lg mL-1, respec-
tively; those for PCM were 0.0631 and
0.1911 lg mL-1, respectively. The reli-
ability of the method during normal use
was checked by assessment of robust-
ness. RSD of retention time (tR, min)
ranged from 0.119 to 0.255% when
mobile phase composition and flow rate
were varied slightly. Results from
assessment of robustness are given in
Tables 6 and 7.
Assay of Aceclofenac andParacetamol in Tablets andin Microsphere Formulations
The method was evaluated by assay of
the compounds in different samples and
calculation of analyte recovery. Mean
recovery from assay of the tablet dosage
form was 102.0 and 98.8% (RSD 0.21
and 0.33%) for ACF and PCM, respec-
tively. For the microsphere formulations
mean recovery was 99.0 and 99.6%
(RSD 0.32 and 0.42%) for ACF and
PCM, respectively. The results from
assay using the method were compared
Table 7. Robustness of the method to deliberate alteration of mobile phase flow rate
Concn level Flow rate(mL min-1)
Statistical data
Aceclofenac (20 lg mL-1) Paracetamol (100 lg mL-1)
Original Used Level Mean peakarea ± SDab
SEMb Mean tR(min) ± SDb
RSD(%)
Mean peakarea ± SDab
SEMb Mean tR(min) ± SDb
RSD(%)
100% 1.0 0.75 -0.25 603,097 ± 678.2 391.5 6.31 ± 0.03 0.245 1,801,599 ± 2774.73 1602.11 2.15 ± 0.02 0.2131.0 0 603,155 ± 672.3 381.5 6.48 ± 0.01 0.232 1,802,388 ± 2857.7 1649.7 2.38 ± 0.01 0.2321.25 +0.25 603,513 ± 673.4 386.8 6.52 ± 0.04 0.255 1,803,315 ± 3035.56 1752.6 2.43 ± 0.04 0.245
a Average from three determinations (n = 3)b tR, retention time; SD, standard deviation; SEM, standard error of the mean
Table 8. Results from assay of aceclofenac and paracetamol in commercial tablets and inmicrosphere formulations
Drug Aceclofenac Paracetamol
TabletsConcn taken (mg) 10 50Concn found (mg, n = 6)a 10.2 49.3Mean recovery (%, ±SD)b 102.03 ± 0.2210c 98.81 ± 0.3255c
Confidence limitd 101.4–102.13 97.708–99.22RSD (%)c 0.21 0.33
Microsphere formulationConcn taken (mg) 10 50Concn found (mg, n = 6)a 9.9. 49.8Mean recovery (%, ±SD)b 99.02 ± 0.321c 99.61 ± 0.413c
Confidence limitd 97.93–99.95 98.2–100.19RSD (%)c 0.32 0.42
a Average from six determination (n = 6)b SD, standard deviation; RSD, relative standard deviationc P < 0.0001 compared with theoretical zero value, Student’s t testd 95% Confidence limit
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with those from use of a literature
method [19]. Mean recovery from assay
of the tablet dosage form by use of the
literature method was 95.2 and 98.5%
(RSD 0.45 and 0.53%) for ACF and
PCM, respectively. Mean recovery from
assay of a microsphere formulation by
use of the literature method was 94.6 and
98.3% (RSD 0.43 and 0.51%) for ACF
and PCM, respectively. These results,
given in Tables 8 and 9, indicate the new
method enables successful simultaneous
quantitative analysis of the drugs in the
formulations.
Study of the Stabilityof Commercial Tablets andMicrosphere Formulations
The ACF and PCM content of samples
analyzed to study sample stability were
in the ranges 102.03–99.8 and 98.81–
96.57%, respectively, for tablets and in
the ranges 99.02–96.54 and 99.61–96.87,
respectively, for microsphere formula-
tions. The result is given in Table 10.
Discussion
In this paper we report a stability-indi-
cating LC method for simultaneous
analysis of aceclofenac and paracetamol
in tablets and microsphere formulations.
Among different isocratic and gradient
mobile phases investigated, use of the
isocratic mobile phase 80:20 (v/v) metha-
nol–phosphate buffer (10 mM at pH
2.5 ± 0.02) was selected on the basis of
simplicity, better column regeneration,
and appropriate system suitability
(theoretical plate number, tailing factor,
resolution, capacity factor, k, and
repeatability) for the analyte peak which
complied with limits stipulated by
USFDA [20]. The concentration of
methanol was selected on the basis of
resolution, capacity factor, and elution
time. The effect of the amount of metha-
nol on capacity factor was studied; the
dependence of the logarithm of capacity
factor on the amount (%) of methanol in
the mobile phase is shown in Fig. 5.
Because of the weakly acidic nature of
ACF, its capacity factor was affected by
the amount of methanol in the mobile
phase more than that of PCM. The
capacity factor of ACF decreased with
increasing methanol concentration, and
increasing the concentration of methanol
reduced the resolution of aceclofenac and
paracetamol, because reducing the con-
centration of methanol increased the
elution time of aceclofenac but had no
effect on that of paracetamol. We there-
fore selected methanol and buffer con-
centrations in the ratio 80:20 (v/v), which
resulted in better resolution, capacity
factor, and peak symmetry than other
ratios, and appropriate elution times.
The detection wavelength 244 nm is
specified for assay of acetaminophen in
USP 2000 [22]. Light of this wavelength
is strongly absorbed by PCM but
absorption of ACF is very low, which
prevents use of this wavelength. Because
both PCM and ACF absorb strongly at
276 nm this wavelength was selected as
the optimum for simultaneous detection
of PCM and ACF.
The specificity of the analytical
method was apparent from the absence
Table 9. Results from comparison of proposed method with literature method for determination of test samplea
Drug Proposed LC method Literature method [19]
Tablets Microspheres Tablets Microspheres
Recovery(%, mean ± SD)a
RSD(%)a
Recovery(%, mean ± SD)a
RSD(%)a
Recovery(%, mean ± SD)a
RSD(%)a
Recovery(%, mean ± SD)a
RSD(%)a
Aceclofenac 102.0 ± 0.2210 0.21 99.02 ± 0.321 0.32 95.2 ± 0.452 0.45 94.6 ± 0.432 0.43Paracetamol 98.81 ± 0.3255 0.33 99.61 ± 0.413 0.41 98.5 ± 0.531 0.53 98.3 ± 0.511 0.51
a Average from six determination (n = 6)
Table 10. Results from long-term study of the stability of blister-packed tablets and microsphere formulation (n = 6)
Duration(months)
Tablets Microspheres
Accelerated storage(50 �C/75% RH)
Accelerated storage(50 �C/50% RH)
Accelerated storage(50 �C/75% RH)
Accelerated storage(50 �C/50% RH)
ACF PCM ACF PCM ACF PCM ACF PCMDrug content(%) ± SD
Drug content(%) ± SD
Drug content(%) ± SD
Drug content(%) ± SD
Drug content(%) ± SD
Drug content(%) ± SD
Drug content(%) ± SD
Drug content(%) ± SD
Initial(control)
102.03 ± 0.2210 98.81 ± 0.3255 102.03 ± 0.2210 98.81 ± 0.3255 99.02 ± 0.321 99.61 ± 0.213 99.02 ± 0.321 99.61 ± 0.313
1 101.02 ± 0.541 97.80 ± 0.632 101.13 ± 0.712 97.91 ± 0.685 98.01 ± 0.423 98.65 ± 0.361 98.07 ± 0.391 98.57 ± 0.5133 101.00 ± 0.713 97.78 ± 0.823 101.03 ± 0.856 97.83 ± 0.796 97.91 ± 0.521 98.11 ± 0.423 97.87 ± 0.489 97.89 ± 0.4316 99.8 ± 0.912 96.57 ± 0.934 100.01 ± 0.912 96.78 ± 0.935 96.54 ± 0.612 96.87 ± 0.623 96.75 ± 0.632 96.91 ± 0.512
All results P > 0.05 compared with initial (control), Student’s t test, average from six determinations (n = 6)
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of significant changes in the retention
times of aceclofenac and paracetamol in
the presence of the degradation products
of the drugs in chromatograms obtained
after separate treatment of a mixture of
the drugs with 0.1 M HCl, 0.1 M
NaOH, or 3% H2O2. The degradation
product of PCM at tR 4.7 min after
acidic and basic hydrolysis, eluting after
the PCM peak, was chloracetanilide, a
known degradation product [6]; this was
confirmed by separate degradation of a
pure sample of PCM with 0.1 M HCl
and 0.1 M NaOH. After acidic and
alkaline hydrolysis of aceclofenac two
degradation products eluted at tR 7.1
and 8.8 min, i.e. after the ACF peak;
these have been shown by LC to be
diclofenac and 1-[2,6-dichlorophenyl]-2-
indolinone [17]; the identities were con-
firmed by separate degradation of pure
ACF with 0.1 M HCl and 0.1 M NaOH.
Separation and quantification of the
degradation products and analytes
showed the method is stability-indicat-
ing.
Construction of the calibration plots
revealed the response of the method to
aceclofenac and paracetamol standards
was linear over the ranges 16–24 and
80–120 lg mL-1, respectively—a broad
range compared with a previous meth-
od [18].
Intra-day and inter-day precision and
accuracy of the method were shown to be
acceptable, reliable, and in accordancewith
published acceptance limits [20]. The
detection limits were 0.0369 lg mL-1
for aceclofenac and 0.0631 lg mL-1 for
paracetamol, very close to the value for
paracetamol in a previously published
method [8]; that for aceclofenac was
0.1120 lg mL-1, very near the LOQ
obtained by use of an LC method for
plasma [12].TheLOQforparacetamolwas
0.1911 lg mL-1, also close to the value for
paracetamol in a previously published
method [8]. These values indicate the sen-
sitivity of this method is adequate.
The reliability of the method during
normal usage was checked by determi-
nation of robustness. The retention times
of ACF and PCM did not change sig-
nificantly when mobile phase composi-
tion (80:20 ± 2%) and flow rate
(1 ± 0.25 mL min-1) were deliberately
altered.
In recent years LC methods have
been published for individual analysis of
aceclofenac [17] or for simultaneous
analysis of aceclofenac and paraceta-
mol [18, 19] in tablet dosage forms only.
In this LC method aceclofenac and par-
acetamol were analysed simultaneously
both in tablets and microsphere formu-
lations. This method is novel because it
is the only stability-indicating method
reported for quantification of each ana-
lyte in the presence of its degradation
products. Resolution and recovery of the
drugs from microsphere formulations
containing different polymers are better
than in previously published methods
and there is no interference from the
polymers. Another advantage over pre-
viously published methods is the absence
of interfering peaks from excipients, en-
abling better analysis of PCM in mix-
tures; chromatograms obtained from a
placebo and from a test sample of PCM
are depicted in Figs. 6 and 7.
Evidence of the suitability of this LC
method was obtained by simultaneous
analysis of aceclofenac and paracetamol
in a commercial pharmaceutical prepa-
ration and in a product under develop-
ment; recoveries of the analytes were
better than for literature methods.
Student’s t test was used to determine
whether results obtained for a commer-
cial tablet and for a prepared micro-
sphere formulation were significantly
different before and after exposure to
accelerated storage conditions (i.e.
50 �C/75% RH and 50 �C/50% RH).
The test showed there was no significant
difference (P > 0.05). It may therefore
be inferred that degradation of ACF and
PCM had not occurred in the commer-
cial tablet or in the prepared micro-
sphere formulation during accelerated
storage.
Conclusions
This LC method is stability-indicating
and, as revealed by the validation data,
enables specific, accurate, robust, and
precise simultaneous analysis of ACF
and PCM in tablet and microsphere
pharmaceutical preparations. The meth-
od is sensitive enough for quantitative
detection of the analytes in pharmaceu-
tical preparations and can thus be used
for routine analysis, quality control, and
for studies of the stability of pharma-
ceutical preparations containing these
drugs.
Acknowledgments
The authors are grateful to Ranbaxy
Research Laboratory, Gurgaon, India,
for providing the gift sample of ace-
clofenac. The authors are also grateful to
the analytical Department of Arbro
0
0.2
0.4
0.6
0.8
1
0 20 40 60 80 100
Methanol (%)
log
K
PCM
ACF
Fig. 5. Plot of log k against amount (%) ofmethanol in the mobile phase
Fig. 6. LC chromatogram obtained fromplacebo
Fig. 7. LC chromatogram obtained fromPCM in a real sample
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Pharmaceutical Ltd, New Delhi, India,
for providing facilities in their organi-
zation.
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