Original Article

A validated and Stability Indicating HPLC Method

for Analysis of Diminazene Aceturate and Antipyrine
Combination in a Ready Injectable Solution

Authors M. N. Abualhasan1, N. Batrawi2, A. N. Zaid1, D. G. Watson3

1
Affiliations Department of Pharmacy, An-Najah National University, Nablus, Palestine
2
Advanced Veterinary Company, Ramallah, Palestine
3
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK

Key words Abstract of acetonitrile, methanol, phosphate buffer and

●
▶ HPLC
▼ hexane sulfonate; the flow rate was 0.6 mL/min
●
▶ diminazene
Diminazene aceturate and Antipyrine combina- and ultraviolet detection was at 291 nm. This
●
▶ antipyrine
tion therapy is widely used in veterinary medi- method was validated in accordance with FDA
●
▶ stability-indicating
cine. A simple reverse HPLC method for the and ICH guidelines and showed good linearity,
analysis of samples of a ready injectable formu- accuracy, precision, selectivity and the system
lation containing a mixture of active ingredients suitability results were within the acceptance
and inactive excipients has been developed. The criteria. A stability-indicating study was also car-
HPLC analysis was carried out using a reversed ried out and indicated that this method could be
phase (RP)-C18 (250 mm × 4.0 mm, 5 μm) column. used for purity and degradation evaluation of
The isocratic mobile phase consisted of a mixture these formulations.

Introduction stability and quality of the prepared dosage form

received 21.12.2012
accepted 17.02.2013
Bibliography
DOI http://dx.doi.org/
10.1055/s-0033-1337939
Published online: 2013
Drug Res
© Georg Thieme Verlag KG
Stuttgart · New York
ISSN 2194-9379
Correspondence
M. N. Abualhasan
Pharmacy
An-najah University
Juniad Street
Nablus 400
Palestinian Territory
Occupied
Tel.: + 970595472322
Fax: + 970595472322
m_abualhasan@najah.edu
▼ is still missing in most important pharmacopeias
Diminazene diaceturate (DD) is a [4,4’-(diazo-
amino) dibenzaamidine]; DD is a diamidine
derivative and is mainly used in the veterinary
field as a trypanocide and babesiacide in affected
areas of the world [1, 2]. It is still in common use
for the treatment of babesiosis in cattle, cats,
camel, goats, dogs, sheet, and swine [3]. It is also
of useful in the treatment of ileriousis in combi-
nation with other antibiotics (● ▶ Fig. 1a). DD
practically insoluble in water (6.67 mg/l) at room
temperature, slightly soluble in alcohol and very
slightly soluble in ether or chloroform [4].
Antipyrine was the first pyrazolin-5-one deriva-
tive used as an analgesic, antipyretic and anti-
inflammatory drug (● ▶ Fig. 1b). It is a water soluble
powder with slightly bitter taste [4, 5]. Besides hav-
ing antipyretic and analgesic activity [6], antipyrine
is also mixed with DD at a suitable concentrations
as a stabilizer in most formulations, since DD is
unstable in water on its own. Aqueous solutions of
these preparations may remain stable for 10–15
days at room temperature [7].
A DD and Antipyrine combination is commer-
cially available in the market as powder for injec-
tion and ready for use injectable solution [8, 9].
Despite the importance and the widespread use
of this combination, a suitable pharmaceutical
quality control method for the evaluation of the
[10]. However, during the past decades multiple
analytical methods have been developed to
quantify DD in dosage forms, plasma and animal
tissues. These methods include spectrophoto-
metric determination [11] and high performance
liquid chromatographic (UV and/or MS detector)
methods [12, 13].
In this study we succeeded in developing a novel
isocratic reversed phase HPLC method using a UV
detector to quantify a DD and Antipyrine combi-
nation in an injectable solution. To our knowl-
edge there is no available method for analysis of
this combination in liquid injectable dosage
form. Very few analytical methods are available
in the literature for analysis of diminazine [13]
and antipyrine separately [14–18]. Only one
method is available in the literature for quantifi-
cation of this combination in the form of a pow-
der for injection [10]. The advantage of our
method is that it is a simple method which can be
used for routine and quality control analysis of a
ready injectable solution. Moreover, the method
is a stability indicating and can be used for purity
and degradation evaluation of the formulation.A
single isocratic run is used for the assay of both
active ingredients in a relatively short time. In
addition, the method has been successfully used
for analysis of drug-excipient compatibility in

Abualhasan MN et al. Analysis of Diminazene & Antipyrine by HPLC … Drug Res

This document was prepared for the exclusive use of Mutasim Abu-Hasan. Unauthorized distribution is strictly prohibited.
 Original Article

injectable formulations and subsequently an in house stability culations. The HPLC grade water was prepared by Aquatron
indicating study. Our main objective in this study was to estab- equipment model A4000D. The chromatographic conditions are
lish a validated and stability indicating HPLC assay method for outlined in ●
▶ Table 1.

the determination of DD and Antipyrine in an injectable solu-

tion, formulated in the research laboratory at Advanced Veteri-
nary Company-Ramallah, in accordance with the requirements
of FDA and ICH guidelines [19].
Experimental
▼ and then 5 mL was diluted to 25 mL using the mobile phase to
Chemicals and reagents
DD and Antipyrine active ingredients were purchased from A. B.
Enterprises LTD, India and Zenita Life Science LTD, India respec-
tively. USP reference standards were used to validate the method.
The injection dosage form DD aceturate 70 mg per mL and Anti-
pyrine (Phenazone) 375 mg per mL was formulated in house at
our research laboratory. The methanol and acetonitrile used
were of HPLC grade. The water for HPLC was obtained by double
distillation. Other reagents such as Phosphate buffer, Phosphoric
acid, Hexanesulfonic acid sodium salt, hydrochloric acid, sodium
hydroxide, and hydrogen peroxide were purchased from Merck,
Sigma Aldrech and J. T. Baker reliable commercial sources and
were used as such.
Instrumentation
A Dionex-Ultimate 3000 HPLC system equipped with LPG-
3400 SD pump, WPS-3000SL autosampler, TCC-3000 column
oven, and DAD-3000 UV–VIS with diode array detector. Chrome-
leon Data system Software (Version 6.80 DU10A Build 2826
(171948)) was used for data acquisition and mathematical cal-
a H
N N
N
HN
NH2
NH 2
HN
Preparation of working solutions
DD (70 mg) and Antipyrine (375 mg) were weighed accurately
into a 100 mL volumetric flask and dissolved in distilled water
then 5 mL of this solution was diluted to 25 mL using the mobile
phase (standard stock solution). The samples were prepared by
diluting (1:10) of the injection formulation with distilled water
give solutions with concentrations equivalent to the standard
stock solution.
Method validation
The method was validated for parameters like specificity, linearity,
range, accuracy, precision, LOD/LOQ and Ruggedness/Robustness.
To evaluate the linearity and range of the method 5 different test
concentrations were prepared (based upon the original formula-
tion): 60 %, 80 %, 100 %, 120 % and 140 %. 10 separate injections
were analyzed under the same conditions.
The accuracy and precision were established on 3 concentra-
tions around the test concentration (80 %, 100 % and 120 %), 3
replicates of each concentration. The percentage recovery and %
RSD were calculated for each of the replicate samples.
The detection limit or LOD may be expressed as a concentration
that gives a signal to noise ratio of approximately 3:1. While the
Quantification limit or LOQ in sample can be determined with
acceptable precision and accuracy with a signal to noise ratio of
approximately 10:1.
The ruggedness/robustness of the method was determined by
performing the same trial using different mobile pH, detection
wavelength, flow rate, elapsed assay time and analyst. The
applied ruggedness parameters are illustrated in ● ▶ Table 2.
Forced degradation study
Forced degradation studies were performed to evaluate the sta-
bility indicating properties and specificity of the method. Inten-
tional degradation was carried out by exposing the formulation
to 5 stress conditions, the condition mentioned in ● ▶ Table 3 are

b CH 3
Table 2 The applied ruggedness/robustness conditions.
H 3C
N Robustness parameter Condition checked
Detection wavelength WL of 289, 291 and 293 nm
N
Flow rate of the mobile phase Flow rate of 0.5, 0.6 and 0.7 mL/min
pH values of the mobile phase pH of the mobile 5.0, 5.2&5.4
O Elapsed assay times The same analyst analyzed the same
trial on two different days
Analysts 2 analysts analyzed the same trial on
Fig. 1 Chemical structures of a Diminazine and b Antipyrine. the same day

Chromatographic conditions Table 1 HPLC chromatographic

conditions.
Mobile phase Isocratic elution of 100 mg sodium hexane sulfonate in 280 mL of 0.05M phosphate
buffer (pH 5.2 ± 0.1), 120 mL Methanol and 100 mL Acetonitrile
Flow rate 0.6 mL/min
Detection (λ) 291 nm, PDA detector 190–800 nm for spectra analysis
Stationary phase C18 (LiChrospher® 100 Merck Germany) 5μm, 250 × 4 mm
Column temperature 25 °C
Injection volume 20 μL
Run time 9 min

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 Original Article

Stress type Conditions Time Table 3 Stress conditions.

Acid hydrolysis 1 mg/mL in 0.1 N (up to 1 N), HCl at 40 °C 1–7 days

Base hydrolysis 1 mg/mL in 0.1 N (up to 1 N), NaOH at 40 °C 1–7 days
Oxidative/solution 0.3 % (up to 3 %) H2O2; at RT; protected from light Few hours to 7 days
Thermal 70 °C, RH 40 % Up to 3 weeks
Photo-degradation UV light (254 nm) at RT Few hours to 3 days

Sample No. Sample Peak Area Standard Peak Area Assay ( %) Table 4 Accuracy and precision
results of DD.
inj # 1 inj # 2 average inj # 1 inj # 2 average
80 %
1 380.00 377.00 378.50 388.00 388.00 388.00 97.55
2 378.00 378.00 378.00 97.42
3 382.00 382.00 382.00 98.45
100 %
1 486.00 481.00 483.50 488.00 483.00 485.50 99.59
2 483.00 483.00 483.00 99.49
3 484.00 485.00 484.50 99.79
120 %
1 604.00 606.00 605.00 603.00 603.00 603.00 100.33
2 590.00 592.00 591.00 98.01
3 590.00 587.00 588.50 97.60
Mean 98.69
SD 1.12
RSD 1.13

Sample No. Sample Peak Area Standard Peak Area Assay ( %) Table 5 Accuracy and precision
results of Antipyrine.
inj # 1 inj # 2 average inj # 1 inj # 2 average
80 %
1 1 470.00 1 474.00 1 472.00 1 505.00 1 507.00 1 506.00 97.74
2 1 467.00 1 466.00 1 466.50 97.38
3 1 476.00 1 476.00 1 476.00 98.01
100 %
1 1 820.00 1 824.00 1 822.00 1 863.00 1 867.00 1 865.00 97.69
2 1 843.00 1 840.00 1 841.50 98.74
3 1 821.00 1 822.00 1 821.50 97.67
120 %
1 2 207.00 2 205.00 2 206.00 2 218.00 2 228.00 2 223.00 99.24
2 2 178.00 2 184.00 2 181.00 98.11
3 2 170.00 2 184.00 2 177.00 97.93
Mean 98.06
SD 0.58
RSD 0.59

the general conditions which are generally followed in the stress
study protocol. Stressed samples were analyzed periodically and
the presence of related peaks and peak purity for the active
ingredients was checked.
desired range. The RSD was calculated for each recovery solution
of both active ingredients DD and Antipyrine, all the results are
within limits. Acceptable precision of ± 1.13 % and ± 0.59 % were
obtained for DD and Antipyrine respectively, accuracy was > 97 %
(●
▶ Table 4, 5).

Result and Discussion Stability indicating study

▼ Stress testing of the drug injection DD and Antipyrine combina-
Linearity and range tion was done to identify the likely degradation products, the
The linearity of the method was observed in the expected concen- stability of the molecule and also validate specificity of the ana-
tration range (60–140 %) for both DD and Antipyrine demonstrat- lytical procedures. The stability indicating study was performed
ing its suitability for analysis as shown. The goodness-of-fit (R2) under various stress conditions mentioned in section 2.5. The
was found to be 0.9998 and 0.9999 indicating a linear relationship results of the stability studies are listed in ●
▶ Table 6. The drug

between the concentration of analyte and area under the peak. active ingredients exposed for 2 days under alkaline condition
(0.2N NaOH) formed degradant A (● ▶ Fig. 2a) at about 20 % of the

Accuracy and precision original peak of the DD. Acidic conditions did not produce any
The results of accuracy studies done on both sample and refer- degradation. The formulation showed degradant B (● ▶ Fig. 2b) at

ence standard showed that the method is accurate within the about 14 % of the original peak of the DD under thermal stress

Abualhasan MN et al. Analysis of Diminazene & Antipyrine by HPLC … Drug Res

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 Original Article

conditions (70 °C) when he original drug was exposed for 4 days. System suitability
There was no evidence of degradation of the formulation stress System suitability parameters are used to verify that the system
with hydrogen peroxide or UV light. is adequate for the analysis to be performed. The UV spectrum of
DD and Antipyrine are shown in ● ▶ Fig. 3. Our method shows

Peak specificity that all the values for the system suitability parameters are
There was no evidence of interference between the chromato- within acceptable limits, The column efficiency was about 4055
graphic peaks for DD and Antipyrine and the excipients, impuri- and 10512 theoretical plates for DD and Antipyrine respectively.
ties and degradants products under the various stress conditions The tailing factors are about 2.0 and 1.6 for DD and Antipyrine
(●
▶ Fig. 2a, b). The purity of all the peaks was tested using photo- respectively. The resolution values for DD and Antipyrine are 4.3
diode array (PDA) and the results showed that none of the tested and 4.7 respectively.
peaks had a purity of less than 99 %.
Limit of detection and limit of quantification:
Ruggedness & robustness The detection limit or LOD is the lowest amount of analyte in a
The ruggedness and robustness of the method were examined sample that can be detected. While the Quantification limit
using some minor modifications listed in ● ▶ Table 2. The result (LOQ) is the lowest amount of analyte in a sample that can be
indicated that minor modifications to the experimental param- determined with acceptable precision and accuracy. Our method
eters did not affect the assay and its ability to accurately and showed an LOD of 0.03 & 0.18 mg/L for DD and Antipyrine
precisely detect/quantify the active ingredients. respectively. The LOQ was 0.11 &0.6 mg/L for DD and Antipyrine
respectively.

Table 6 The results of stability indicating studies.

Stress type Detectable change Conclusion

Acid hydrolysis No change ▼
Base hydrolysis Degradant A. A simple and efficient HPLC assay method utilizing UV detection
Oxidative/solution No change has been developed for the analysis of a ready injectable solution
Thermal Degradant B. of DD and Antipyrine combination. The method was novel,
Photo-degradation No change. robust and economical and can be used for rapid assay of the 2
Oxidative/solution No change ingredients simultaneously. The developed method was vali-

a st1-NaOH UV_VIS_1 Fig. 2 a Chromatogram of a well separated peaks

1.400 of the active ingredient and degradative peak A
mAU WVL:291 nm
and the injections formulation b Chromatogram
1.200 of well separated peaks of the active ingredient,
degradative peak b and the injections formulation
7
8.
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Abualhasan MN et al. Analysis of Diminazene & Antipyrine by HPLC … Drug Res

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 Original Article

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