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Volume 62 2015 Number 21

JOURNAL OF THE BULGARIAN PHARMACEUTICAL SCIENTIFIC SOCIETY

Editorial Board:

Alexander Zlatkov (Faculty of Pharmacy, MU, Sofia, Bulgaria)

Bojka Tzvetkova (Faculty of Pharmacy, MU, Sofia, Bulgaria)
Christo Tsvetanov (Institute of Polymers, BAS, Sofia, Bulgaria)
Darvin Ivanov (Faculty of Pharmacy, MU, Sofia, Bulgaria)
Danka Obreshkova (Faculty of Pharmacy, MU, Sofia, Bulgaria)
Georgi Momekov (Faculty of Pharmacy, MU, Sofia, Bulgaria)
Guenka Petrova (Faculty of Pharmacy, MU, Sofia, Bulgaria)
Ilijana Jonkova (Faculty of Pharmacy, MU, Sofia, Bulgaria)
Nikolai Lambov (Faculty of Pharmacy, MU, Sofia, Bulgaria)
Nikolai Danchev (Faculty of Pharmacy, MU, Sofia, Bulgaria)
Stefan Nikolov (Faculty of Pharmacy, MU, Sofia, Bulgaria)
Bistra Angelovska (Goce Delcev University, Skopje, Macedonia)
Ebba Holme Hansen (University of Kbenhavns, Kbenhavn, Denmark)
Fabrice Clerfeuille (University of Nantes, Nantes, France)
Georg Heun (University of Applied Sciences, Koetten, Germany)
Luisa Pistelli (University of Pisa, Pisa, Italy)
Marion Schaefer (Institute of Clinical Pharmacology and Toxcology, Berlin, Germany)
Mecedes Unzeta (Autonomic University of Barselona, Barcelona, Spain)
Ruediger Groening (University of Muenster, Muenster, Germany)
Svjetlana Luterotti (University of Zagreb, Zagreb, Croatia)
Danijel Kikelj (University of Ljubljana, Ljubljana, Slovenia)

Editor in Chief: P. Peikov

Secretary: M. Georgieva

Indexed in: MEDLINE, CAplusSM/Chemical Abstracts, TOXCENTER, EMBASE/Excerpta Medica, PASCAL,

BIOTECHNOBASE, ExtraMEDTM, SCOPUS

Elsevier impact metrics SJR for Pharmacia for 2011: 0.122

Editorial/publishing policy: Manuscripts submitted to PHARMACIA are only accepted on the understanding, that they
are subject to editorial review and review of at least two independent referees, that they have not been and will not be
bublished whole or in part in any other journal and that recommendations to comply with with ethycal standards when
performing clinical and other biological experiments have been adhered to.

Publishing frequency is four times a year (volume). Only abstracts published in the Journal may be reproduced without
prior permission; reproduction of other materials requires publishers consent.

Address of Editorial Board

Faculty of Pharmacy Editor in Chief: (+359 2) 9236 505

2, Dunav str., Sofia 1000 E-mail: pharmacia_editor@pharmfac.net
Fax (02) 987 987 4 Secretary: (02) 9236 515
E-mail: pharmacia_secretary@pharmfac.net
 PHARMAcIA
Volume 62 2015 Number 2
Volume 60 2013 Number 1

contents
coNteNts

original articles
Original articles
I. Todorov, M. Christov, K. Stanoeva, K. Yakimova. Leptin and GABA interactions on body temperature of rats.............................................................................00
St. Pankova , D. Tsvetkova, K. Ivanov, St. Papanov, St. Ivanova. Validation of TLC densitometric method for
S. Harkov, D.identification and determination
Havrylyuk, V. Atamanyuk, of estradiol........................................................................................................................
B. Zimenkovsky, R. Lesyk. Synthesis and biological activity of isatines bearing thiazolidinone and pyrazoline 3
moieties..................00
A. Kougioumtzoglou, L. Peikova, M. Georgieva, Al. Zlatkov. Evaluation of the stability of indomethacin substance
L. Peikova, B. Tsvetkova. RP-HPLC method for simultaneous determination of Amlodipine besylate and Valsartan in pharmaceutical dosage form. 00
under a model of physiological conditions, using modified and validated RP-HPLC method.......................................... 10
Review articles
Kr. Makukh, T. Ryvak, O. Lopatynska. Patients compliance to phytotherapy prescribed and self-medicated
Georgi Momekov, Niko Benbassat. Pharmacological properties of Hawthorn leaf and flower as a cardiovascular agent................................................................00
with herbal medicines in Ukraine....................................................................................................................................... 18
L. Peikova, B. Tsvetkova. Amide-based prodrugs of nonsteroidal anti-inflammatory drugs.................................................................................................00

L. Andonova, M. Georgieva, Al. Zlatkov. Arylpiperazine derivatives new agents affecting mood disorders................00
Review articles
Georgi Momekov, Iliana Ionkova, Paraskev Nedialkov, Zlatina Kokanova-Nedialkova, Dimitrina Zheleva-Dimitrova, Ilina Krasteva, Yohana Ilieva,
Ilina Dineva,L.Gerassim
Andonova, M.Stefan
Kitanov, Georgieva,
Nikolov, Al. Zlatkov.
Spiro Free Overview
Konstantinov. radicals,ofoxidative stress, and diseases
the oncopharmacological studies of associated
plant-derived with
naturalthem. .................. 26
products
conducted at the Faculty of Pharmacy (MU-Sofia) 00
St. Ivanova, K. Ivanov, St. Pankova, Br. Zlatkov, K. Stoychev. Sport supplementation: beneficial effects of
D. Obreshkova. Reactive oxygen species induced neurodegeneration in Alzheimers disease............00
vitamin E and creatine on exercise performance................................................................................................................ 40
Instructions to authors00
E. Drakalska, D. Momekova, S. Rangelov, N. Lambov. Nanoparticles as platforms for delivery of curcumin.................. 50

From the Editorial board

Instructions to authors......................................................................................................................................................... 58
10 PHARMACIA, vol. 62, No. 2/2015 A. Kougioumtzoglou, L. Peikova, M. Georgieva, Al. Zlatkov

EVALUATION OF THE STABILITY OF INDOMETHACIN SUBSTANCE

UNDER A MODEL OF PHYSIOLOGICAL CONDITIONS, USING
MODIFIED AND VALIDATED RP-HPLC METHOD
A. Kougioumtzoglou, L. Peikova, M. Georgieva, Al. Zlatkov
Department of Pharmaceutical Chemistry, Medical University Sofia, Faculty of Pharmacy,
2 Dunav Street, 1000 Sofia, Bulgaria

Abstract. A fast, simple and fully automated RP-HPLC analytical method with UV detection for
determination of indomethacin substance and its degradation product 4-chlorobenzoic acid was
modified and validated. The analysis was performed at isocratic conditions, applying a mobile
phase of acetonitrile and 0.5% ortophosphoric acid (50:50, v/v) at flow rate 1.5 ml/min. The sys-
tems suitability parameters and validation parameters were set up.
The modified and validated method was successfully applied for determination of stability of
indomethacin substance under a model of physiological conditions. The analyzed indomethacin
substance was established to be stable in acid and neutral media, while a hydrolysis occurs in
alkali media. A first order rate constant for the degradation of indomethacin in alkali media was
determined.

Key words: stability, indomethacin, RP-HPLC, validation

Introduction an analgesic. Indomethacin can also affect warfarin

Indomethacin: a widely used non-steroid anti-in-
flammatory agent
Indomethacin is a non-steroidal anti-inflammato-
ry drug (NSAID) commonly used as a prescription
medication to reduce fever, pain, stiffness, and swell-
ing. It works by inhibiting the production of prosta-
glandins, molecules known to cause these symptoms
[1]. Indomethacin has also been used clinically to
delay premature labor [2], reduce amniotic fluid in
polyhydramnios [3, 4]. Indomethacin is also used in
closure of the ductus arteriosus in newborn humans
[5]. Indomethacin is a potent drug with many serious
side effects and should not be considered an analgesic
for minor aches and pains or fever. The medication is
better described as an anti-inflammatory, rather than
and subsequently raise INR.
Some conflicting data in its studies have led to a
reexamination of the substances stability. It has been
considered, that the lack of indomethacin activity in
some biological studies may have resulted from in-
jection of inactive solutions. [6].
As described in literature indomethacin forms
by decomposition two degradation products: 5-me-
thoxy- 2-methylindol-3-yl acetate (A) and 4-chlo-
robenzoic acid (B) (Fig. 1) [7, 8]. The latter has to
be monitored together with an active substance both
during manufacturing process and storage of phar-
maceuticals, due to some evidences of its hepato-
toxicity [9].

O
H
N OH

O
5-methoxy-2-indoleacetic acid

(A) (B)
Fig. 1. Chemical structure of the degradation products of Indomethacin.
Evaluation of the stability of indomethacin substance...
High performance liquid chromatography is the
method-of-choice enabling determination of active
substance and its degradation products during one-step
procedure simultaneously and automatically [10].
The aim of our study is to modify and validate an
automated RP-HPLC method for simultaneous iden-
tification and quantitation of indomethacin substance
and its degradation product 4-chlorobenzoic acid.
Materials and methods
Reagents
LC-grade methanol and acetonitrile were sup-
plied from Merck (Germany). All other chemicals
and reagents were of analytical grade. HPLC grade
water was purified using a Milli-Ro-15 Water Pu-
rification System (Millipore, Bedford,MA,USA),
which consists of a Super-C carbon cartridge, two
Ion-X ion-exchange cartridges and an Organex-Q
cartridge. The water was filtered through a 0.22m
Millipak stack filter prior to use.
Indomethacin RS and 4-chlorobenzoic acid RS
were used as standards.
HPLC system
A modular HPLC system LC-10A Shimadzu (Ja-
pan) arranged with a LC-10A pump, solvent degasser
DGU-3A, Rheodyne injector, column oven CTO-
10A, SPD-M10A fixed wavelength detector and
communication bus module CBM-10A was used. A
Luna C18 (2), 250 mm x 4.6 mm, 5 m column was
applied as stationary phase. The analysis was carried
out at an ambient temperature with injection volume
of 20 l. The UV detector was set at 240 nm.
Selection of a mobile phase.
As a result from the performed analysis a mobile
phase consisting of 50 volumes 0.5 % v/v orthophos-
phoric acid and 50 volumes of acetonitrile was used
in the current assay with a flow rate of 1.5 ml/min.
Preparation of buffers.
All buffer solutions were prepared according to
the requirements of European Pharmacopoeia [11].
Preparation of the standard solutions.
Reference stock solutions of 4-chlorobenzoic acid
(0.1 mg/ml) and indomethacin (0.5 mg/ml) were pre-
pared in acetonitrile and filtered through 0.45-m
membrane filter. Calibration solutions for indometh-
acin were prepared by diluting the reference stock
solution to furnish concentrations in the range 25.00-
200.0 g/ml. Calibration solutions for 4-chloroben-
PHARMACIA, vol. 62, No. 2/2015 11
zoic acid were prepared by diluting the reference
stock solution to obtain concentrations in the range
5.00-40.00 g/ml. The mobile phase was applied as
solvent for dilutions. 20 l of these solutions were
injected in triplicate into the HPLC system and the
peak areas were recorded.
Preparation of test solutions.
In the kinetic run, the reaction was initiated by
dissolving 0.001 g of the analyzed indomethacin sub-
stance to 10.00 ml of preheated corresponding buffer
solution pH 2.0; 1mM (Solution 1), pH 7.4; 1mM
(Solution 2) and pH 9.0; 1mM (Solution 3) obtain-
ing a final concentrations of 100 g/ml.
Stability evaluation
The obtained solutions were maintained at
370.2C for a total time of 120 min. Aliquot sam-
ples of 20 l from the analyzed solutions were taken
at definite time intervals (0, 30, 60, 90 and 120 min.)
and the corresponding chromatograms were record-
ed. The progress of hydrolysis was monitored by
means of the modified HPLC method.
The objective of the proposed study is to modify
and validate a stability indicating HPLC method for
simultaneous estimation of indomethacin substance
and its hydrolytic product 4-chlorobenzoic acid.
Results and discussion
Validation and chromatographic procedure
Validation of an analytical procedure is performed
in order to demonstrate that the procedure is suitable
for its intended use. [12-14]. This process is essen-
tial as it serves to ensure that the quality of analytical
data generated is both reliable and accurate, and is
also capable to identify potential problems with the
method [14-16].
In this assay the following eight parameters, out-
lined by the USP and ICH [12, 17] for evaluation
of an analytical procedure, like accuracy, precision,
specificity, limit of detection (LOD) and limit of
quantitation (LOQ), linearity and range were deter-
mined. These parameters are specific to the valida-
tion of a developed method, and forms a part of an
overall validation process, which includes validation
of hardware and software used and verification of
system suitability and performance [17].
In the presented work the selection of the mobile
phase was based on methods published in the liter-
ature [18] and on the literary methods for the indi-
vidual analysis of indomethacin and 4-chlorobenzoic
acid [8, 19, 20]. The applied modification was initi-
12 PHARMACIA, vol. 62, No. 2/2015 A. Kougioumtzoglou, L. Peikova, M. Georgieva, Al. Zlatkov

ated from the necessity to develop simple and fast
analytical method for simultaneous determination
of indomethacin and its possible hydrolytic product
4-chlorobenzoic acid.
For improvement of the chromatographic behav-
ior the influence of buffer molarity and pH were as-
sessed and considered. For the assessment of the ef-
fect of these variables, the analyzed compounds were
detected at the defined wavelength and 0.1 AUFS at
1.5 ml/minute [21].
Results from validation procedure.
The proposed method was validated with respect
to specificity, selectivity, linearity, precision, accu-
racy, limit of quantitation (LOQ) and limit of detec-
tion (LOD).
Specificity
Specificity is defined as the ability to measure the
analyte in the presence of other components that may
be expected in the sample matrix, accurately and spe-
cifically [12, 13, 17, 22, 23]. Specificity was assessed
by comparing chromatograms obtained from analy-
sis of a placebo solution and reference solutions. No
other peaks were observed at the retention times of
were completely separated from each other (Rs =
7.783), which indicated that the method is selective
and could be used for their simultaneous identifica-
tion, quantification and in purity tests.
Linearity
The linearity of the range of detectability is de-
pendent on Beers Law, such that the absorbance of
a solute is directly proportional to its concentration
in the solution [24]. Linearity in this range is depen-
dent on both the compound analyzed and the detec-
tor used [22].
In our study the linearity of the method was de-
termined at eight concentration levels ranging from
25.00 to 200.0 g/ml for indomethacin and 5.00 to
40.00 g/ml for 4-chlorobenzoic acid. The calibra-
tion curves were constructed by plotting peak areas
versus concentrations of the analyzed compounds,
and the regression equations were calculated. Each
response was the average of three determinations.
The obtained calibration curves for each of the ana-
lyzed compounds are presented on Fig. 3.
800

indomethacin and its degradation product 4-chloro- 700

600
benzoic acid, indicating that interfering substances
were not present. 500
Peak area

400

Selectivity 300

200
Under the proposed chromatographic conditions y = 3,8949x - 4,5002
100
the obtained retention times for standard solution 2
R = 0,9988
0
containing a mixture of the tested 4-chlorobenzoic 0 50 100 150 200
acid RS and indomethacin RS were 2.074 and 9.857 Concentration

min., respectively. From the chromatogram shown

in Fig. 2, it is evident, that the analyzed compounds a)

800

700

600

500
Peak area

400

300

200

100 y = 19,413x - 23,129

R2 = 0,9915
0
0 10 20 30 40 50
Concentration

b)

Fig. 2. Cromatogram of standard solution, containing Fig. 3. Calibration curves for linearity determination for
4-chlorobenzoic acid RS and indomethacin RS. Indomethacin (a) and 4-chlorobenzoic acid (b).
Evaluation of the stability of indomethacin substance... PHARMACIA, vol. 62, No. 2/2015 13

Table 1. Precision of the method

Precision
Compound
Mean (g/ml) SD %RSD
Indomethacin 100.05 0.33 0.33
4-chlorobenzoic acid 19.89 0.28 1.41

An r2 value of >0.990 was considered to be suf- Solution 2 with phosphate buffer giving a pH val-
ficient to demonstrate linearity of the method. The ue of 7.4 equal to the physiological pH in the blood
calibration curve was linear over the concentration plasma and
range studied, with r2 = 0.9988 for indomethacin and Solution 3 with solvent citrate buffer giving a pH
r2 = 0.9915 for 4-chlorobenzoic acid. The equations value of 9.0 close to the physiological pH in the
of the regression lines were y = 3.8949x - 4.5002 and small intestines.
y = 19.413x - 23.129 for indomethacin and 4-chloro- The solutions were kept at 37oC and aliquot sam-
benzoic acid, respectively. ples of 20 l were taken at definite time intervals (0,
30, 60, 90 and 120 min.). The drawn aliquots were
Precision analyzed with the modified HPLC method and the
According to ICH precision is usually expressed obtained chromatograms were recorded. The corre-
as the percentage relative standard deviation (%RSD) sponding peak areas are presented in Table 2, Table
[12, 13, 17]. 3 and Table 4 for Solution 1, Solution 2 and Solu-
The precision of the proposed analytical system tion 3 respectively.
was investigated by performing six consecutive rep-
licate injections of the same standard solution. The
standard deviation (SD) and relative standard devia- Table 2. Determined peak area for indomethacin and
tion (%RSD) obtained are listed in Table 1. 4-chlorobenzoic acid for Solution 1 for the indicated time
The low %RSD values indicated that the method intervals.
is precise. Rt (min) Peak area of
Number Time
indomethacin indomethacin
Limit of quantitation and limit of detection 1. 0 8.952 1090624
The limits of quantitation and limits of detection 2. 30 8.854 1142932
were calculated from the standard deviation of re-
3. 60 8.789 1129844
sponses and slopes using signal-to-noise ratio. The
quantitation limits for Indomethacin and 4-chloro- 4. 90 8.775 1025257
benzoic acid were 0.3 g/ml and 0.9 g/ml, respec- 5. 120 7.898 1151147
tively. The calculated detection limits were 0.06 g/
ml and 0.3 g/ml, respectively for the analyzed indo-
methacin and 4-chlorobenzoic acid. Table 3. Determined peak area for indomethacin and
The assessed system suitability parameters and val- 4-chlorobenzoic acid for Solution 2 for the indicated time
idation parameters determined that the method may be intervals.
successfully applied for practical determination of in- Rt (min) Peak area of
Number Time
domethacin and its degradation product 4-chloroben- indomethacin indomethacin
zoic acid for following and control of degradation of Standard 5.101 567181
the active substance during stability studies. 1. 0 5.208 578048
2. 30 5.267 584223
Results from stability evaluation of indomethacin RS.
Three solutions were prepared according to the 3. 60 5.273 569807
above mentioned procedure: 4. 90 5.297 572215
Solution 1 with hydrochloric acid buffer giving 5. 120 5.303 579153
a pH value of 1.2 close to the physiological pH in
the stomach;
14 PHARMACIA, vol. 62, No. 2/2015 A. Kougioumtzoglou, L. Peikova, M. Georgieva, Al. Zlatkov

Table 4. Determined peak area for indomethacin and lowed, using the modified RP-HPLC method. A high
4-chlorobenzoic acid for Solution 3 for the indicated time decrease in the peak area of the peak corresponding
intervals. by retention time to the analyzed Indomethacin sub-
Rt (min) Peak area of stance is observed in the 30th min of incubation at
Number Time 37oC (Fig. 6). In addition an increase in the peak area
indomethacin indomethacin
corresponding by retention time to the 4-chloroben-
1. 0 9.786 4286630 zoic acid is also observed (Fig. 6). Almost complete
2. 30 9.841 198798 exhaustion of the initial indomethacin was obtained
at the 60th min of the incubation.
3. 60 9.869 33791
The obtained chromatograms confirmed the ex-
4. 90 9.878 19646 pected results for rapid decomposition in alkaline
5. 120 9.883 14607 solutions. From the performed experiments is visi-
ble, that indomethacin and its degradation product
4-chlorobezoic acid are completely separated from
each other, which indicates that the modified and val-
The results show, that the incubation of indo-
idated proposed RP-HPLC method is selective and
methacin RS in buffer solution with pH 2.0 (close
could be used for their simultaneous identification,
to stomach pH) and temperature of 37oC showed no
quantitation and in purity testing.
changes in the peak area of the peak corresponding
The decrease in the measured peak areas observed
by retention time to that of the analyzed Indometha-
in the chromatogram of Solution 3 shows, that hy-
cin RS substance. Thus it may be concluded, that the
drolysis occurs in the first 30 min of the incubation
analyzed substance could be considered stable under
at the analyzed pH (pH = 9.0). On the other hand, no
these conditions (Fig. 4).
visible decrease in the peak areas was observed in
A similar kinetic profile was obtained, when incu-
Solution 1 and Solution 2 (1.2 for Solution 1 and
bated in buffer with pH = 7.4 and 37oC temperature
pH =7.4 for Solution 2), which indicates lack of hy-
for the same time interval. The corresponding chro-
drolysis at these conditions. This observation may be
matographic profile and retention times for the ana-
visualized with the following graph (Fig. 7).
lyzed substances are presented on Fig. 5.
From the followed formation of the degradation
The presented results that indomethacin may be
product 4-chlorobenzoic acid, monitored at constant
considered stable in solutions with pH 7.4 and below,
pH of 9.0 and temperature of 37oC was found, that
are confirmed by literary data [6].
the process may be considered linear, indicating
The kinetic profile of the stability of indometh-
first-order degradation kinetics (Fig. 8).
acin in buffer solution with pH = 9.0 was also fol-

(a) (b)
Fig. 4. Chromatographic profile of indomethacin RS of stability evaluation in acid media (pH = 2.0) at 0th min (a) and
120th min (b) of incubation under 37oC.
Evaluation of the stability of indomethacin substance... PHARMACIA, vol. 62, No. 2/2015 15

(a) (b)
Fig. 5. Chromatographical profile of indomethacin RS of stability evaluation in acid media (pH = 7.4) at 0th min (a) and
120th min (b) of incubation under 37oC.

(a) (b)
Fig. 6. Chromatograms obtained during the analysis of indomethacin RS in Solution 3 (buffer with pH 9.0) at 0th min (a)
and at 30th min (b) of incubation under 37oC.

Fig. 7. Time dependence of the calculated concentrations Fig. 8. First order plot of degradation of indomethacin in
for Solution 1, Solution 2 and Solution 3. alkali media.
16 PHARMACIA, vol. 62, No. 2/2015
This result was further confirmed in the literature,
where the apparent first-order rate constants have been
calculated [7, 18]. It has been established, that the rate
constant-hydroxide-ion concentration profile is linear
with a negative slope and these experimental data con-
firmed the proposed reaction of degradation [7].
Conclusion
A fast, simple and fully automated analytical meth-
od for determination of indomethacin substance and
its impurity 4-chlorobenzoic acid using RP-HPLC
with UV detection was modified and validated. The
chromatography was performed using isocratic elu-
tion with mobile phase composed of acetonitrile and
0.5% phosphoric acid (50:50, v/v) at flow rate 1.5
ml/min. System suitability parameters and valida-
tion parameters including method precision, accura-
cy, linearity, selectivity and robustness were set up.
Afterwards, the method was successfully applied for
the practical determination of stability study of In-
domethacin RS in conditions close to physiological.
A first order rate constant for the hydrolysis of
indomethacin in alkali media was determined from
the slope of linear plot of the logarithm of residual
Indomethacin against time.
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Corresponding author
Maya Georgieva
Faculty of pharmacy, Medical University, Sofia
2 Dunav str., 1000 Sofia, Bulgaria
Phone: +359 2 9236 515
e-mail: maya.bg77@gmail.com

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