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High performance liquid chromatographic

assay of indomethacin and its related
substances in tablet dosage forms

Article in International Journal of Pharmacy and Pharmaceutical Sciences · June 2012

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 Academic Sciences International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 4, Suppl 3, 2012

Research Article
HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC ASSAY OF INDOMETHACIN AND ITS
RELATED SUBSTANCES IN TABLET DOSAGE FORMS

BOYKA TSVETKOVA*, IVANKA PENCHEVA, ALEXANDER ZLATKOV, PLAMEN PEIKOV

Department of Pharmaceutical chemistry, Medical University, Sofia, Faculty of Pharmacy, 2 Dunav st., 1000 Sofia, Bulgaria.
Email: [email protected]
Received: 3 Feb 2012, Revised and Accepted: 19 March 2012
ABSTRACT
A reversed-phase high performance liquid chromatographic (RP-HPLC) method with UV detection was proposed for separation of indomethacin
and its impurities from tablet dosage forms. The best separation was achieved on a LiChrosorb C18, 250 mm x 4.6 mm, 5 μm column at a detector
wavelength of 240 nm. The utilization of mixture of 40 volumes 0.5 % v/v orthophosphoric acid, 20 volumes of methanol and 40 volumes of
acetonitrile as mobile phase with a flow rate of 2ml/min enabled acceptable resolution of indomethacin, in large excess, from possible impurities, in
a short elution time (9 min). Analytical parameters linearity, accuracy, precision and specificity were determined by validation procedure and found
to be satisfactory. Overall, the proposed method was found to be simple, rapid, precise and accurate for quality control of indomethacin and its
impurities in dosage forms and in raw materials.
In this work the kinetic investigation of the alkaline hydrolysis of indomethacin was also carried out. The degradation reaction was monitored by
means of HPLC method developed and was found to follow first-order kinetics. The rate constant and half-life of the hydrolytic decomposition were
estimated.
Keywords: Liquid chromatography, Validation, Indomethacin, Stability, Impurities.

INTRODUCTION MATERIALS AND METHODS

Indomethacin 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3- Chemicals and Reagents
acetic acid, is a non-steroidal anti-inflammatory, analgetic and
antipyretic drug 1. Despite its high toxicity, indomethacin is a primary Indomethacin RS, 4-chlorobenzoic acid RS and 5-methoxy-2-
medicine used for the treatment of rheumatoid arthritis, gout, and indoleacetic acid RS were used as standards. The tablets containing
collagen disease 2. It is a potent inhibitor of cyclooxygenases, reducing 25 mg of indomethacin were obtained commercially. LC-grade
prostaglandin synthesis, relieving pain and reducing fever in febrile methanol and acetonitrile were supplied from Merck (Germany). All
patients. The drug is usually administered orally. It can also be other chemical reagents were of analytical grade.
administered as a suppository and topical gel. By decomposition Instrumentation and chromatographic conditions
indomethacin forms two degradation products: 4-chlorobenzoic acid,
mentioned in European Pharmacopoeia as impurity A and 5-methoxy- Chromatographic separation was performed on modular HPLC
2-methylindoleacetic acid. They have to be monitored together with system LC-10A Shimadzu (Japan) arranged with a LC-10A pump,
an active substance both during manufacturing process and storage of solvent degasser DGU-3A, Rheodyne injector, column oven CTO-10A,
pharmaceuticals with aim to control the quality and quantity of the SPD-M10A fixed wavelength detector and communication bus
pharmaceutical product. module CBM-10A. A LiChrosorb C18, 250 mm x 4.6 mm, 5 μm
column was used as a stationary phase. The components were
The European Pharmacopoeia (Ph. Eur. 5) 3 uses a titration method separated isocratically with a mobile phase consisting of 40 volumes
for the determination of indomethacin. The substance is titrated 0.5 % v/v orthophosphoric acid, 20 volumes of methanol and 40
with 0.1 M sodium hydroxide and blank titration has to be carried volumes of acetonitrile at a flow rate of 2.0 ml/min. The analysis was
out simultaneously. That procedure is time consuming and carried out at an ambient temperature and injection volume was 20
impractical for routine analyses of pharmaceutical samples, μl. The UV detector was set at 240 nm.
especially during stability studies and quality control in the
manufacturing process, where there could be many samples to be Calibration solutions
controlled, often in replicates. The amount of indomethacin active
Reference stock solutions of 4-chlorobenzoic acid (0.1 mg/ml), 5-
substance in capsules and suppositories is determined by means of
methoxy-2-indoleacetic acid (0.2 mg/ml) and indomethacin (0.5
absorption spectrophotometry. The European Pharmacopoeia
mg/ml) were prepared in the mobile phase and filtered through
recommends thin-layer chromatography for determination of
related substances in active substance and capsules as well as HPLC 0.45-μm membrane filter. Calibration solutions for indomethacin
for purity assessment of suppositories. Some analytical methods were prepared by diluting the reference stock solution to furnish
have been reported for assaying indomethacin in pure as well as in concentrations in the range 25.00-200.0 μg/ml. Calibration solutions
pharmaceutical dosage forms. The methods include: densitometry 4, for 4-chlorobenzoic acid were prepared by diluting the reference
LC-MS 5, HPLC with UV detection 6, phosphorimetic method 7, stock solution to obtain concentrations in the range 5.00-40.00
polarography 8, potentiometry 9, fluorimetry 10, spectrophotometry μg/ml. Calibration solutions for 5-methoxy-2-indoleacetic acid were
11-17. It is important to emphasize that there is a few analytical prepared by diluting the stock reference stock solution to achieve
procedures for determinination of indomethacin and its two concentrations in the range 2.50-20.00 μg/ml. Working standard
degradation products in one analysis simultaneously 18-20. To achieve solutions for analysis of related substances contained 4.00 mg/ml
this aim there is a need to develop a new, simple and fast analytical indomethacin, 20.00 μg/ml 4-chlorobenzoic acid and 10.00 μg/ml 5-
method for the simultaneous determination of these substances in methoxy-2-indoleacetic acid. Working standard solution for the test
pharmaceutical formulations. In our study, HPLC with UV detection is assay contained 100.0 μg/ml indomethacin.
chosen for separation, identification and quantitation of indomethacin Sample preparation
active substance and its two degradation products 4-chlorobenzoic
acid and 5-methoxy-2-indoleacetic acid in the tablet dosage form. The For the investigation indomethacin gastro-resisitant tablets were
analytical method described is validated 21-23 and also applied to used. One tablet contained 25 mg active substance. Sample solutions
monitor hydrolysis of indomethacin in alkaline media 24-28. were prepared by first preparing stock solutions. Twenty tablets
 Tsvetkova et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 3, 549-552

were weighed and finely powdered. An amount of the powder at 25±0.2 °C. Samples were taken at suitable time intervals during 6
equivalent to 25.0 mg indomethacin for assay and 200.0 mg for hr of incubation. The progress of hydrolysis was monitored by
analysis of related substances were weighed into 50.0 ml volumetric means of HPLC method developed. 20 µl of sample examined was
flasks and approximately 30 ml mobile phase were added to each. analysed for remaining indomethacin. First order rate constant for
The samples were sonicated for 10 min and the solutions were then the hydrolysis was determined from the slope of linear plot of the
diluted to volume with mobile phase, mixed well, and filtered. For logarithm of residual indomethacin against time.
assay, 5.00 ml stock solution were diluted to 25.00 ml with mobile
phase to give a solution containing 100 μg/ml indomethacin. The RESULTS AND DISCUSSION
solution used for analysis of related substances contained 4.0 mg/ml Under the proposed chromatographic conditions the obtained
indomethacin. retention times were 2.41 min for 5-methoxy-2-indoleacetic acid,
Stability of indomethacin 3.31 min for 4-chlorobenzoic acid and 7.42 min for indomethacin.
From the chromatogram shown in Fig. 1, it is evident, that both of
In the kinetic run, the reaction was initiated by adding 10.0 ml related substances were completely separated from each other (Rs =
reference stock solutions of indomethacin to 50.00 ml of preheated 0.95), which indicated that the method is selective and could be used
sodium hydroxide buffer solution (pH 9.0; 1mM), the final for their simultaneously identification, quantification and in purity
concentrations being 100 μg/ml. The reaction flask was maintained tests.
m V(x10)
2.414/1245646
8.0 Detector A:240nm

7.416/2133626
7.0

6.0

5.0
3.331/634103

4.0

3.0

2.0

1.0

0.0

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 m in

Fig. 1: Chromatogram of working standards: 4-chlororbenzoic acid RS, 5-methoxy-2-indoleacetic acid RS and indomethacin RS

Method validation Calibration and linearity

The proposed method was validated with respect to specificity, The linearity of the method was determined at six concentration
linearity, precision, accuracy, limit of quantification (LOQ) and limit levels ranging from 25.00 to 200.0 μg/ml for indomethacin, 5.00 to
of detection (LOD). 40.00 μg/ml for 4-chlorobenzoic acid and 2.50 to 20.00 μg/ml for 5-
methoxy-2-indoleacetic acid.
Specificity
The calibration curves were constructed by plotting peak areas
The specificity of the HPLC method was confirmed by injecting versus concentrations of compounds, and the regression equations
placebo as well as reference solutions. No other peaks were were calculated. Each response was the average of three
observed at the retention times of indomethacin and its degradation determinations. Linear regression data for calibration curves were
products, indicating that interfering substances were not present. shown in Table 1.

Table 1: Linear regression data for calibration curves

Drugs Indomethacin 4-Chlorobenzoic acid 5-Methoxy-2-indoleacetic acid
Concentration range (μg/ml ) 25.00-200.0 5.00-40.00 2.50-20.00
Slope 28233.16 74766.1 56476.1
Intercept 8325.3 658.8 513.2
Correlation coefficient (r) 0.9999 0.9992 0.9986

Precision and Accuracy Limit of quantification and limit of detection

The precision of the analytical system was investigated by The limits of quantitation and limits of detection were calculated
performing six consecutive replicate injections of the same standard from the standard deviation of responses and slopes using signal-to-
solution. The standard deviation (S d ) and relative standard noise ratio. The quantitation limits for indomethacin, 4-
deviation (RSD) obtained are listed in Table 2. The low RSD values chlorobenzoic acid and 5-methoxy-2-indoleacetic acid were 0.2
indicated that the method is precise. µg/ml, 1.0 µg/ml and 0.8 µg/ml, respectively, while detection limits
were 0.05 µg/ml, 0.2 µg/ml and 0.25 µg/ml, respectively.
The accuracy of the method was investigated by determination of
Alkaline hydrolysis of indomethacin
both impurities in the presence of indomethacin. A solution
containing indomethacin (C = 4.0 mg/ml) with no detectable At constant pH value and temperature the formation of the
impurities was spiked with the reference substances at appropriate degradation products 4-chlorobenzoic acid and 5-methoxy-2-
concentrations. The recovery and relative standard deviations (RSD) indoleacetic acid was found to be linear function of the initial
obtained (Table 3) confirmed the satisfactory accuracy of the indomethacin concentration indicating first-order degradation
method. kinetics. First-order rate constant for the hydrolysis was calculated

550
 Tsvetkova et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 3, 549-552

from the slope of semilogarithmic plot of percent indomethacin first order plot for degradation of indomethacin was presented. The
remaining versus time and was found to be 8.4.10-3. On Fig. 2 the corresponding half-life was 82.51 min.

Table 2: Precision of the method

Compound Precision
Mean (μg/ml) Sd RSD (%)
Indomethacin 99.77 0.37 0.37
4-Chlorobenzoic acid 19.61 0.34 1.73
5-Methoxy-2-indoleacetic acid 9.78 0.17 1.74

Table 3: Results of recovery studies

Compound Recovery (%) Sd RSD (%)
Indomethacin 99.66 0.18 0.18
4-Chlorobenzoic acid 99.14 0.45 0.45
5-Methoxy-2-indoleacetic acid 99.53 0.36 0.36

5,0

4,5

4,0

3,5
ln %remaining

3,0

2,5

2,0

1,5

0 50 100 150 200 250 300 350 400

Time (min)

Fig. 2: First-order plot for the degradation of indomethacin

CONCLUSION 4. Krzek J, Starek M. Simultameous densitometric determination

of indomethacin and its degradation products, 4-chlorobenzoic
HPLC procedure for quality control of indomethacin was performed. acid and 5-methoxy-2-methyl-3-indoleacetic acid in
The method was validated in respect of purposes of pharmaceutical pharmaceutical preparation. J. AOAC Int. 2001; 84: 1703-1707.
practices. The developed RP-HPLC procedure was suitable for 5. Abdel-Hamid M, Novotny L, Hamza H. Determination of
simultaneous qualitative and quantitative determination of diclofenac sodium, flufenamic acid, indomethacin and
indomethacin and its related substances in tablet dosage forms and ketoprofen by LC-APCI-MS. J. Pharm. Biomed. Anal. 2001; 24:
in raw materials. 587-594.
AKNOWLEDGMENT 6. Plakogiannis F, Ali A, Kazmi S. High performance liquid
chromatographic determination of indomethacin in capsules.
The present study was kindly supported by Project № I-5/2010 Drug Dev. Ind. Pharm. 1981; 7: 215-221.
from Medical Science Council. 7. Arrunda A, Capiglia A. Phosphorimetric determination of
indomethacin in pharmaceutical formulations. Analyst. 1997;
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