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: shahidjamil07@gmail.com

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

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)

558 Chromatographia 2008, 68, October (No. 7/8) Original

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

Original Chromatographia 2008, 68, October (No. 7/8) 559

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

560 Chromatographia 2008, 68, October (No. 7/8) Original

(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

Original Chromatographia 2008, 68, October (No. 7/8) 561

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

562 Chromatographia 2008, 68, October (No. 7/8) Original

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)

Original Chromatographia 2008, 68, October (No. 7/8) 563

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

564 Chromatographia 2008, 68, October (No. 7/8) Original

Pharmaceutical Ltd, New Delhi, India,

for providing facilities in their organi-

zation.

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