WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Chakravarthi et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 6.041

Volume 5, Issue 8, 1451-1464 Research Article ISSN 2278 – 4357

A NEW RP-HPLC METHOD DEVELOPMENT AND VALIDATION

FOR SIMULTANEOUS ESTIMATION OF PITOFENONE,
DICLOFENAC POTASSIUM AND FENPIVERINIUM BROMIDE IN
PHARMACEUTICAL TABLET DOSAGE FORM

K. S. Chakravarthi1* and N. Devanna2

1
Department of Technical Education, Govt. Polytechnic, Hyderabad, Telangana, India.
2
Head of Chemistry Department, JNTUA, Ananthapuramu, 515001, A.P, India.

Article Received on
ABSTRACT
15 June 2016, A simple, rapid, accurate, precise, specific and sensitive reverse phase-
Revised on 05 July 2016,
Accepted on 25 July 2016 HPLC method has been developed and validated for the simultaneous
DOI: 10.20959/wjpps20168-7439 estimation of Pitofenone, Diclofenac Potassium and Fenpiverinium
Bromide in bulk drug and pharmaceutical dosage form. The

*Corresponding Author
chromatographic separation was performed on the Kromasil C18
K. S. Chakravarthi column (250mm×4.6mm, 5μm particle size), using a mobile phase of
Department of Technical Buffer: Acetonitrile taken in the ratio 35:65 v/v, at a flow rate of 1.0
Education, Govt. ml/min at an ambient temperature of 30˚C with the detection wave
Polytechnic, Hyderabad,
length at 215nm. The retention times of Pitofenone, Diclofenac
Telangana, India.
Potassium and Fenpiverinium Bromide were 2.48 min, 3.85 min and
5.23 min respectively. The linearity was performed in the concentration range of 125-750
ppm, 12.5-87.5 ppm, 0.25-1.5 ppm each of Pitofenone, Diclofenac Potassium and
Fenpiverinium Bromide with a correlation coefficient of 0.999, 0.999 and 0.999 for
Pitofenone, Diclofenac Potassium and Fenpiverinium Bromide respectively. The percentage
purity of Pitofenone, Diclofenac Potassium and Fenpiverinium Bromide was found to be
99.43%, 100.07% and 99.66% respectively. The proposed method has been validated for
specificity, linearity, range, accuracy, precision and robustness were within the acceptance
limit according to ICH Q2 (R1) guidelines and the developed method can be employed for
routine quality control analysis in the bulk and combined pharmaceutical dosage form of
Pitofenone, Diclofenac Potassium and Fenpiverinium Bromide.
KEYWORDS: Pitofenone, Diclofenac Potassium, Fenpiverinium Bromide, RP-HPLC,
Validation.

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INTRODUCTION
Pitofenone (PIT) (Fig.1) is an antispasmodic found in the marketed formulation
SPASMALGON, a Bulgarian drug[1] in combination with Fenpiverinium Bromide,
and metamisol sodium used to relieves pain and spasms of smooth muscles. The IUPAC
name of PIT is Methyl 2-[4-(2-piperidin-1-ylethoxy)benzoyl]benzoate, Molecular formula
C22H25NO4, Molecular weight 367.45 g/mol. Literature survey indicate that it had been
validated by HPLC [2], Ion pair liquid chromatography [3] and by UV spectrophotometry[4].

Fig.1. Chemical structure of Pitofenone

Fenpiverinium Bromide (FEN) (Fig.2) is chemically 1-(4-amino-4-oxo-3,3-diphenylbutyl)-1-

methylpiperidinium used as an anti-cholinergic and anti-spasmodic. Molecular formula
C22H29N2O, Molecular weight 337.48 g/mol. Fenpiverinium bromide is official in United
States of Pharmacopoeia.[5] A detailed literature survey that very few methods like UV
method[6] and extraction spectrometry[7] are developed for the quantification of
Fenpiverinium bromide.

Fig.2. Chemical structure of Fenpiverinium Bromide

Diclofenac (DIC) (Fig.3)is available as sodium and potassium salt form in various marketed
dosage forms. The main difference between the two is that diclofenac potassium is absorbed
into the body more quickly than diclofenac sodium. A quick action is useful where immediate
pain relief is required. The IUPAC name of diclofenac is [(2,6-dicholorophenyl)amino]-
phenyl acetate. It is a sodium or potassium salt of an arylacetic acid derivative. It inhibits
prostaglandins synthesis by interfering with the action of prostaglandin synthetase
(Cyclooxygenase)[8]. It possesses analgesic,anti-inflammatory and antipyretic activity. It is

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widely used in various diseases including rheumatoid arthritis and osteoarthritis, soft tissue
disorders, renal colic, acute gout, migraine and dysmenorrhea[9]. UV spectrophotometric
methods[10-12] and high performance liquid chromatography (HPLC)[13-14] are often employed
in the analysis of diclofenac. Many HPLC methods with UV detection were adopted in the
literature for the determination of DIC[15-21].

In the present work, a stability indicating, simple, rapid, precise and sensitive reverse phase
HPLC method was developed and validated for the simultaneous estimation of Pitofenone
(PIT), Diclofenac Potassium(DIC) and Fenpiverinium bromide(FEN) in pharmaceutical
tablet dosage form.

Fig.3. Chemical structure of Diclofenac Potassium

MATERIALS AND METHODS

Instrumental and analytical conditions
Reagents and chemicals
The pharmaceutical drug samples Pitofenone, Diclofenac Potassium and Fenpiverinium
Bromide were obtained from Spectrum Pharma Pvt.Ltd, Hyderabad. All the chemicals used
of HPLC grade. The pharmaceutical dosage form (NOVASPAS from Ultramark Healthcare
Pvt Ltd.) was purchased from local pharmacy. The solvents used in this work were of HPLC
grade and obtained from Merck Specialties Private Limited, Mumbai. Milli Q Water was
used in the buffer preparation.

Equipment
A Waters e2695 gradient system with Empower-2 software and 2996 module Photo Diode
Array detectors equipped with a quaternary solvent delivery pump, automatic sample injector
and column thermostat was used for the analysis.

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Chromatographic conditions
The column used was Kromasil C18, (250mm×4.6mm, 5μm particle size) for analytical
separation. The mobile phase consists of an aqueous solution of 0.1% ortho-phosphoric acid
and acetonitrile in the ratio of (35:65%v/v). The flow was adjusted to 1ml/min. The
instrument was operated at an ambient temperature. The injection volume was 10μL. The UV
detection was achieved at 215 nm which is the isobestic point shown in Fig.4.

Fig.4. UV spectra showing Isobestic point of PIT,DIC and FEN.

Preparation of analytical solutions

Preparation of 0.1%OPA buffer solution: 1ml of Ortho phosphoric acid was pippeted out
and dissolved in a 500ml of Milli-Q water taken in a 1000ml Volumetric flask and final
volume was made up to the mark with Milli-Q water.

Preparation of mobile phase

Mixture of buffer and acetonitrile taken in the ratio 35:65 v/v was degassed in ultrasonic
water bath for 10min. Filtered through 0.45µ filtered under vacuum filtration.

Diluent preparation: Mixture of Water and acetonitrile taken in the ratio 50:50 v/v was used
as diluent.

Preparation of the Pitofenone, Diclofenac Potassium and Fenpiverinium Bromide

standard preparation
Accurately weighed and transferred 50mg of Pitofenone and 5mg of Diclofenac and 1 mg of
Fenpiverinium working Standards into 3 different 10ml clean dry volumetric flasks, added
3/4th volume of diluent, sonicated for 5 minutes and made up to the final volume with diluent.
From the above stock solutions 1ml of Pitofenone, Diclofenac and 0.1ml of Fenpiverinium

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stock solutions were pippeted into a 10ml volumetric flask and made up to 10ml with diluent
to get a mixed standard solution containing concentration of 500ppm, 50ppm and 1ppm of
Pitofenone, Diclofenac and Fenpiverinium respectively, so that the drugs Pitofenone,
Diclofenac and Fenpiverinium were in the ratio equal to that of the marketed formulation.

Preparation of sample solution

Twenty tablets were weighed accurately and finely powdered. A quantity of tablet powder
equivalent to 50mg of Pitofenone, 5mg of Diclofenac and 1mg of Fenpiverinium was
accurately weighed and transferred into a 10 ml volumetric flask, containing 5 ml of mobile
phase and ultrasonicated for 25 min; the volume was made up to the mark and mixed well.
The solution was filtered through a 0.2 μm filter to ensure the absence of particulate matter.
From the filtered solution 1ml was pipeted out into a 10 ml volumetric flask and made upto
10ml with diluent.

Method Development and Validation of HPLC

The suggested analytical method was validated according to ICH Q2 (R1) [22] with respect to
certain parameters such as system suitability, specificity, linearity, accuracy, precision,
robustness and ruggedness.

Specificity
The specificity was carried out to determine whether there are any interference of any
impurities (presence of components may be unexpected to present) in retention time of
analytical peak. The specificity of the method was determined by checking the interference of
any of the possible degradation products generated during the forced degradation study of the
drugs. The forced degradation of the drug was carried out with 2 N HCl, 2 N NaOH, 20% v/v
hydrogen peroxide, heat (80°C) and photolysis (365 nm) for determining the stability nature
of the drugs. The degradation samples were prepared by taking suitable aliquots of the drug
and drug product solutions, and then undertaking the respective stress testing procedures for
each solution. After the fixed time period the treated test solutions were diluted up to the
mark with mobile phase. For every stress condition three solutions were prepared as 500ppm
of PIT, 50ppm of DIC 1ppm of FEN. The specific stress conditions are described as follows.

Acidic degradation condition: Acidic degradation was carried out by adding 5 ml of 2N

HCl, and after 45 minutes neutralizing the mixture by adding 2N NaOH.

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Alkali degradation condition: Alkali degradation was carried out by adding 5 ml of 2N

NaOH, and after 45 minutes neutralizing the mixture by adding 2N HCl.

Oxidative degradation condition: Oxidative degradation was performed by exposing the

drug to 5 ml of 20% (v/v) H2O2 for 45 minutes.

Thermal degradation condition: Thermal degradation was performed by heating the drug
content at 80ºC on a thermostatically controlled water bath for 45 minutes.

Photolytic degradation condition: Photolytic degradation was carried out by exposing the
drug content to UV light (365nm) inside a UV chamber for 180 minutes.

Linearity and concentration ranges: The linearity of the proposed HPLC procedure was
evaluated by analyzing a series of different concentrations for each of the three analytes. The
linear regression equations were generated by least squares treatment of the calibration data.
Under the optimized conditions described above, the measured peak areas were found to be
proportional to concentrations of the analytes. Table 1 presents the performance data and
statistical parameters including linear regression equations, concentration ranges, correlation
coefficients. A stock solution of 1000 ppm of three analytes were prepared with diluent. From
it, various working standard solutions were prepared in the range of 125 to 750 ppm, 12.5
ppm to 87.5 ppm and 0.25 ppm to 1.5 ppm for PIT, DIC and FEN respectively and injected
into HPLC. It was shown that the selected drugs had linearity in stated range. The calibration
plot (peak area versus concentration) was generated by replicate analysis (n=3) at all
concentration levels and the linear relationship was evaluated using the least square method
within Microsoft Excel program.

The retention time of standards was 2.48 min for PIT, 3.85 min for DIC and 5.23 min for
FEN. A typical HPLC chromatogram of the standard mixtures is shown in Fig. 5.

Fig.5: Standard Chromatogram of Pitofenone, Diclofenac Potassium and

Fenpiverinium Bromide

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Accuracy
Accuracy was determined in terms of percentage recovery the accuracy study was performed
for 50%, 100% and 150 % for PIT, DIC and FEN. Standard and sample solutions were
injected in to HPLC system in triplicate and percentage recoveries of PIT, DIC and FEN were
calculated. The area of each level was used for calculation of % recovery.

Precision
Express the closeness of agreement between the series of measurement obtained from
multiple sampling of same homogeneous sample under the prescribed conditions. Method
precision was determined both in terms of repeatability (injection and analysis) and
intermediate precision/Ruggedness (It shows the degree of reproducibility of test results
obtained by analyzing the sample under variety of normal test conditions such as analyst,
instruments). The precision of the method was ascertained from the peak area obtained by
actual determination of six replicates of 500ppm, 50ppm and 1ppm of Pitofenone, Diclofenac
and Fenpiverinium respectively. The precision of the assay was also determined in terms of
intra- and inter-day variation in the peak areas of a set of drug solutions on three different
days. The intra and inter-day variation in the peak area of the drug solution was calculated in
terms of relative standard deviation (RSD).

Robustness
Robustness of the developed method was studied by evaluating the influence of small
deliberate variations in procedure variables like flow rate (±5%) and change in wave length
(±5nm). The robustness was performed for the flow rate variations from 0.8ml/min to
1.2ml/min and the method is robust only in less flow condition and even by change in the
mobile phase ±5% . The results are given in Table 7. Ruggedness of the method was carried
out by different analysts.

System Suitability
System suitability tests are an integral part of chromatographic method which are used to
verify reproducibility of the chromatographic system. To ascertain its effectiveness, certain
system suitability test parameters were checked by repetitively injecting the freshly prepared
standard stock solutions at the concentration level 500ppm, 50ppm and 1ppm of Pitofenone,
Diclofenac and Fenpiverinium respectively to check the reproducibility of the system and the
results are shown in Table 2.

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Stability of solutions The stability of standard working solutions as well as sample solutions
in distilled water was examined and no chromatographic changes were observed within 24 h
at room temperature. Also, the stock solutions prepared in HPLC-grade methanol were stable
for at least 2 weeks when stored refrigerated at 4°C. Retention times and peak areas of the
drugs remained unchanged and no significant degradation was observed during these periods.

Detection and quantification limits According to the pharmacopoeial recommendations

(USP, 2011), the limit of detection is defined as the concentration that has a signal-to-noise
ratio of 3:1, while for limit of quantification, the ratio considered is 10:1. The limit of
detection values for PIT, DIC and FEN were 0.07 ppm, 0.79 ppm and 0.004 ppm,
respectively. The limit of quantification values for PIT, DIC and FEN were 0.20 ppm, 2.39
ppm and 0.012 ppm, respectively.

RESULTS AND DISCUSSIONS

The present investigation reported is a new RP-HPLC method development and validation of
simultaneous estimation of PIT, DIC and FEN. The method developed was proceeded with
wavelength selection. The optimized wavelength was 215nm. In order to get the optimized
RP-HPLC method various mobile phases and columns were used. From several trials final
method is optimized with the following conditions:

The mobile phase consists of 0.1% ortho-phosphoric acid buffer and acetonitrile in the ratio
of 35:65%v/v and the column used was Kromacil C18 column (250mm×4.6mm,5μm particle
size). The flow rate was adjusted to 1ml/min. The instrument was operated at an ambient
temperature. The UV detection was achieved at 215nm and purity analysis was performed
over a wavelength range of 200-400nm. The injection volume was 10μL. The specificity of
the method was to determine whether there are any interference of any impurities (the
presence of components may be unexpected to present) in retention time of analytical peak.
The linearity was determined as linearity regression of the claimed analyte concentration of
the range 125 to 750 ppm, 12.5 ppm to 87.5 ppm and 0.25 ppm to 1.5 ppm for PIT, DIC and
FEN respectively. The calibration curve obtained by plotting peak area versus concentration
and presented in Table 1 was linear and the correlation coefficient was found to be 0.999,
0.999 and 0.999 for PIT, DIC and FEN respectively.

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The precision of the method was ascertained from determinations of peak areas of six
replicates of sample solution. The %Relative Standard Deviation for method precision was
found to be 0.63 and 0.81 and 0.9 for PIT, DIC and FEN respectively.

The accuracy study was performed in 50%, 100% and 150%. The percentage recovery
determined for PIT, DIC and FEN was presented in Table 6. The robustness were carried out
with minor but deliberate changes in parameters i.e., mobile phase, column temperature, and
flow rate as presented in Table 7. Theoretical plates and tailing factor were observed and
were found to be 7433, 7054 and 6783 (theoretical plates) and 1.11, 1.05 and 1.41 (tailing
factor) for PIT, DIC and FEN respectively. The system suitability parameters like theoretical
plates (N), tailing factor (T) were calculated and were found to be more than 2000 and not
more than 2 and ascertained that proposed RP-HPLC method was accurate and precise.

Forced degradation studies were performed to establish the stability indicating property and
specificity of the proposed method. Degradation studies were carried out under conditions of
acid hydrolysis, base hydrolysis, thermal, oxidation, and photolysis and the drug substances
were observed high degradation in acid (2N HCl) comparative remaining in all conditions.
Thermal degradation conditions were performed by heating the drug sample at 80˚C. Acid
and base hydrolysis showed slight (2-3%) degradation, very slight degradation observed in
oxidation, thermal, photolytic hydrolysis. The results of forced degradation studies were
given in table 8.

Table No. 1: Linearity results for PIT, DIC and FEN

Regression equation parameters
Parameter
Pitofenone Diclofenac Potassium Fenpiverinium Bromide
Linearity range (ppm) 125-750 12.5-87.5 0.25-1.5
Correlation co-efficient 0.999 0.999 0.999
Slope 2487 8920 21003
Y-intercept 35544 3026 4635

Fig .6.Showing linearity for Pitofenone

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Fig.7.Showing linearity for Diclofenac Potassium

Fig .8.Showing linearity for Fenpiverinium Bromide

Table No.2: System suitability Parameters for PIT,DIC and FEN.

Results
Parameter Limits
Pitofenone Diclofenac Potassium Fenpiverinium Bromide
RSD of peak area 0.31 0.52 1.29 <2.0
RSD of retention time 0.00 0.00 0.00 <1.0
USP tailing factor (T) 1.11 1.05 1.41 T<2
USP plate count (N) 7433 7054 6783 >2000
USP resolution (R) 9.2 6.3 -- >2

Table No. 3: System Precision values for Pitofenone

S.No Peak Name Rt (min) Area USP Plate Count USP Tailing
1 PIT _Injection -1 2.468 1253854 7615 1.08
2 PIT _Injection -2 2.473 1257633 7433 1.11
3 PIT _Injection -3 2.479 1262044 7502 1.06
4 PIT _Injection -4 2.483 1264913 8087 1.09
5 PIT _Injection -5 2.486 1262041 8068 1.09
6 PIT _Injection -6 2.486 1261377 7759 1.09
Mean 1260310
Std. Dev. 3928.9
% RSD 0.3

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Table No. 4: System Precision values for Diclofenac

S.No Peak Name Rt (min) Area USP Plate count USP tailing
1 DIC _Injection -1 3.834 445074 7141 1.03
2 DIC _Injection -2 3.841 449102 7054 1.05
3 DIC _Injection -3 3.848 446441 7386 1.04
4 DIC _Injection -4 3.85 443896 7276 1.04
5 DIC _Injection -5 3.854 444124 7319 1.04
6 DIC _Injection -6 3.854 442561 7291 1.04
Mean 3.847 445200
Std. Dev. 0.01 2306.69
% RSD 0.00 0.52

Table No. 5: System Precision values for Fenpiverinium Bromide

S.No Peak Name Rt (min) Area USP Plate count USP tailing
1 FEN _Injection -1 5.215 217420 7057 1.35
2 FEN _Injection -2 5.218 215793 7209 1.35
3 FEN _Injection -3 5.232 212056 7484 1.45
4 FEN _Injection -4 5.242 219863 6783 1.41
5 FEN _Injection -5 5.242 216062 7253 1.36
6 FEN _Injection -6 5.243 219163 7323 1.39
Mean 5.232 216726
Std. Dev. 0.01 2806.50
% RSD 0.00 1.30

Accuracy of the proposed HPLC method: Accuracy of the developed method was
determined standard addition method (n=average of 3 analyses). In the first method known
amounts of Pitofenone, Diclofenac Potassium and Fenpiverinium Bromide were
supplemented to the previously analysed sample solution and then experimental and true
values were compared. Three levels were made corresponding to 50%, 100% and 150% of
the nominal analytical concentration.

Table 6: Determination of Accuracy

Standard addition measured concentration
Drugs Spiked concentration (ppm)
(ppm) ± SD; RSD (%)
250 99.98±1.0; 0.01
Pitofenone 500 100.64±1.8; 0.01
750 100.13±1.2; 0.02
25 100.24±0.5; 0.01
Diclofenac Potassium 50 100.22±0.3; 0.00
87.5 100.31±1.2; 0.01
0.5 100.56±1.0; 0.01
Fenpiverinium Bromide 1 100.71±0.5; 0.00
1.5 99.52±0.5; 0.01

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Table 7: Robustness study of PIT, DIC and FEN.

Average area Rt(min)
Chromatographic conditions
PIT DIC FEN PIT DIC FEN
Buffer: Acetonitrile 30:70(v/v) 1314930 443718 215272 2.35 3.41 5.53
Buffer: Acetonitrile 35:65(v/v) 1260310 445200 216726 2.48 3.85 5.23
Buffer: Acetonitrile 40: 60(v/v) 1249077 444009 212500 2.69 4.39 5.15
Flow rate (0.8 mL/min) 1407463 473373 236927 2.64 4.10 5.45
Flow rate (1.0 mL/min) 1260310 445200 216726 2.48 3.85 5.23
Flow rate (1.2 mL/min) 1215054 427726 205108 2.43 3.77 5.04
Temperature 28°C 1245357 439694 210923 2.54 3.95 5.26
Temperature 30°C 1260310 445200 216726 2.48 3.85 5.23
Temperature 32°C 1326854 443825 204264 2.54 3.93 5.24

Table 8: Forced degradation study of PIT,DIC and FEN.

% Assay of active ingredients
Stress conditions PIT % Degradation DIC % Degradation FEN
% Degradation
Acid, 2N HCl, 96.38 3.62 96.74 3.26 97.20 2.80
Base, 2N NaOH 97.65 2.35 97.16 2.84 97.92 2.08
H2O2 (30%,v/v) 98.13 1.87 97.70 2.30 98.61 1.39
Dry heat (80 ºC) 99.13 0.87 99.14 0.86 99.40 0.60
UV (365 nm) 99.38 0.62 99.73 0.27 99.07 0.93
Water 99.57 0.43 99.81 0.19 99.31 0.69

CONCLUSION
In this study, a simple, specific and reliable procedure was developed for the assay of PIT,
DIC and FEN in their tablet dosage form. The three analytes were subjected to forced
degradation using several stress (hydrolytic, oxidative, photolytic and thermal) conditions,
and the proposed method was successfully employed for the resolution of the analyte peaks
from those of the forced degradation products. The most important feature in the proposed
method is the specificity and stability-indicating merits. To our present knowledge, no such
specific and stability indicating procedure has been reported for the assay of this triple drug
combination. The developed method made use of DAD as a tool for peak identity and purity
confirmation; however, it can be adapted to conventional HPLC with UV detection which is
the most popular in quality control laboratories. Finally, the method was thoroughly
validated, therefore, it can be recommended for routine analysis and for checking quality
during stability studies of the cited drugs.

ACKNOWLEDGEMENTS
The authors are thankful to Department of Chemistry, Govt. Polytechnic College, Hyderabad
providing necessary facilities to carry out the research work.

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