Development and Validation of RP-HPLC
for Simultaneous Estimation of Cefpodoxime Proxetil and Dicloxacillin Sodium
Tablets
G.Kumaraswamy1*, M.A. Zeeshan
Hamza2, R.Suthakaran2
1Research
scholar, Pharmaceutical Analysis, Jawaharlal Nehru Technological University
Kakinada,
Kakinada -533003.A.P.
2Department
of Pharmaceutical analysis & QA, Teegala Ramreddy College of Pharmacy,
Meerpet-Hyderabad-500097.Telangana.
*Corresponding Author E-mail: kumaraswamy.gandla@gmail.com
ABSTRACT:
The present work deals with the development
of a precise, accurate, simple, specific, reliable and less time consuming
RP-HPLC method for the estimation of Cefpodoxime Proxetil and Dicloxacillin Sodium
tablets .The chromatographic separation was achieved on a Inertsil
C18 ODS(4.6 x 250mm, 5µm) with a mobile
phase combination of methanol and water (50:50) v/v at a flow rate of 1.0 ml/min, and the detection was carried
out by using PDA detector at 290 nm.
Ambient column temperature has maintained. The total run time was 10mins.The
retention time of Dicloxacillin
Sodium and Cefpodoxime Proxetil
were found to be 2.9 min. and 3.5 min. respectively. The performance of the
method was validated according to the present ICH guidelines.
KEYWORDS: RP-HPLC, Cefpodoxime
Proxetil, Dicloxacillin
Sodium; Tablet dosage forms, RP-HPLC Method.
1. INTRODUCTION:
Cefpodoxime proxetil
(CEF) is chemically 1-(isopropoxy carbonyloxy)
ethyl (6R,7R)-7-[2-(2-amino-4-thiazolyl)-(z)-2-(methoxyimino)
acetamido]-3-methoxymethyl-3-cephem-4-carboxylate [1] (is shown in fig 1), is a third generation
cephalosporin anti-biotic. It is used for infections of the respiratory tract,
urinary tract and skin and soft tissues. It has greater activity against Staphylococcus
aureus[2]. CEF is
official in IP[3] and USP[4],
which describes liquid chromatography method for its estimation. Literature
survey reveals the high-performance thin layer chromatography (HPTLC)[5] method for the determination of CEF individually.
Literature survey also reveals the reversed-phase high-performance liquid
chromatographic (RP-HPLC)[6] and spectrophotometric[7] methods for determination of CEF with other drugs. Dicloxacillin (DCX) is chemically 9 (2S,5R,6R)-6-[3-(2,6-dichlorophenyl)-5-methyl-1,2-oxazole-4-amido]-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]
heptane-2-carboxylic acid (is shown in figure 2).[8],
and is a penicillinase resistant penicillin, used in
the treatment of bacterial infections such as pneumonia and bone, ear, skin and
urinary tract infection [9].
It is official in IP [10]
and USP [11] , and describe RP-HPLC method for its
estimation. Literature survey reveals HPLC [12]
method for determination of DCX in pharmaceutical dosage forms as well as in
biological fluids. Literature survey also reveals spectrofluorimetric
[13] and RP-HPLC [14],[15],[16]
methods for determination of DCX with other drugs.
The combined dosage form of CEF and DCX are
available in the market for the treatment of infections caused by susceptible
microorganisms like urinary tract infections and gonococcal
urethritis. The combination of these two drugs is not
official in any pharmacopoeia, hence, no official method is available for the
simultaneous estimation of CEF and DCX in their combined dosage forms.
Literature survey does not reveal any simple spectrophotometric or other method
for simultaneous estimation of CEF and DCX in combined dosage form. The present
communication describes simple, sensitive, rapid, and accurate RP-HPLC method
for simultaneous estimation of both drugs in their combined tablet dosage forms.
A
literature survey reveals that there are few analytical methods reported for
the estimation of Cefpodoxime Proxetil
alone and in combination with Haloperidol [11, 12] or in combination
with other antihypertensive drugs. However the reported methods have several
limitations.
Fig.No.1.Chemical structure of Cefpodoxime
proxetil
Fig.No.2.chemical structure of Dicloxacillin
In
one of the reported method retention time for HPD was not found to be
significant which limits its use and in another; flow rate for separation of
both the drugs found to be >1ml/min which means excess of solvent is
required throughout the analysis compared to usual flow rates (1±0.2ml/min)
which is ideal for good column performance. Therefore in order to overcome the
drawbacks of the reported methods; need arise to develop a new method which
should be suitable for routine analysis of these drugs in combination. The
present study is able to overcome the drawbacks in the sense of being
economical and with significant retention time for both the drugs which proves
that present method is perfect compared to reported methods.
2.
EXPERIMENTAL
2.1 MATERIALS AND METHODS
Reagents and chemicals
Methanol HPLC grade was procured from E. Merck Ltd.,
Mumbai. Methanol, orthophosphoric acid, Triethyl amine buffer AR grade were procured from S.D. fine
chemicals, Hyderabad. Water HPLC grade was prepared using Millipore
purification system. Cefpodoxime Proxetil
and Dicloxacillin Sodium reference standards procured
from Dr. Reddy’s laboratories, Hyderabad.
Instrumentation
The HPLC system consists of water Empower 2695 having
photodiode array detector system, which was connected with the help of
Empower-2 software for data integration and processing. Xbridge
ODS‐3V (250 X 4.6 mm) 5μ column was used
for the analysis.
HPLC conditions
he contents of the mobile phase
were consisting of methanol: water in the ratio of 50:50. These were filtered
through 0.45μ membrane filter and degassed by sonication before use. The
flow rate of mobile phase was optimized to 1.0 ml / min. The run time was set
at 10 min and column temperature was maintained at ambient. The volume of
injection was 10μl, and the eluent was detected
at 290nm. Each of standard and test preparations was injected into the column
and the responses recorded.
Preparation of stock solution:
The solution was prepared by
dissolving 20.0 mg of accurately weighed Cefpodoxime Proxetil RS and 25.0 mg Dicloxacillin
Sodium RS in Mobile phase, in two 100.0 mL volumetric
flasks separately and sonicate for 20min. From the
above solutions take 10.0 mL from each solution into
a 50.0 mL volumetric flask and then makeup with
mobile phase and sonicate for 10min.
Preparation of working standard solution:
The stock solutions equivalent
to 20ppm to 80ppm with respect to both drugs were prepared in combination of Cefpodoxime Proxetil and Dicloxacillin Sodium as above, sonicated
and filtered through 0.45µ membrane.
Preparation of sample drug solution for pharmaceutical formulations:
Twenty tablets were weighed
accurately and a quantity of tablet powder equivalent to 20 mg Dicloxacillin Sodium l and 25 mg Cefpodoxime
Proxetil was weighed and dissolved in the 70 mL mobile phase with the aid of ultrasonication
for 20 min. The content was diluted to 100 mL with
mobile phase to furnish a stock test solution. The stock solution was filtered
through a 0.45 µm Nylon syringe filter and 10.0 mL of
the filtrate was diluted into a 50.0 mL volumetric
flask to give a test solution containing 40 µg/mL Cefpodoxime Proxetil and 50 µg/mL Dicloxacillin Sodium
Procedure for calibration curve:
The
contents of the mobile phase were filtered before use through 0.45micron
membrane and pumped from the respective solvent reservoirs to the column at a
specified flow rate. Prior to injection
of the drug solutions, the column was equilibrated for at least 30min with the
mobile phase flowing through the system. The chromatographic separation was
achieved using a mobile phase consisting of Methanol: water at 50:50V/V the eluent was monitored using PDA detector at a wavelength of
290nm.
Table.1. Optimized
chromatographic conditions.
Parameters
|
Chromatographic Conditions
|
|
Mobile phase |
Methanol : Water ( 50 : 50 )%V/V |
|
Stationary phase (column) |
Inertsil -ODS C18(250 x 4.6
mm, 5 µ) |
|
Detection wavelength (nm) |
290nm |
|
Flow rate (ml/min) |
1.0 ml/min |
|
Volume of injection loop (µl) |
20µl |
|
Column temperature (°C) |
Ambient |
|
Run Time (min ) |
7min |
|
Drug RT (min) |
2.9min for Dicloxacillin
Sodium and 3.5 for Cefpodoxime Proxetil |
Fig 03.Typical chromatogram
mixture of Cefpodoxime Proxetil
and Dicloxacillin Sodium.
The column was maintained at ambient temperature (270c)
and an injection volume of 20µl of each of standard and sample solutions were
injected into the HPLC system to get the chromatograms. The retention time,
peak areas of drug was recorded graph was plotted by taking concentration of
the drug on x-axis and peak area on y-axis. A typical chromatogram of Dicloxacillin Sodium and Cefpodoxime
Proxetil combination.
Calculation:
The amount of drugs present in each pharmaceutical formulation was
calculated by using the standard calibration curves (concentration in ppm was taken on x-axis and peak area on y-axis). A typical chromatogram of Dicloxacillin
Sodium and Cefpodoxime Proxetil(100ppm) (formulation)
Optimized Chromatographic
Conditions:
A Xbrdge C18 [4.6 x 150 mm] column was
used for the separation of drugs. The mobile phase comprised of water: Methanol
(50:50) .Injection volume was 20μl and run time was 15min
and flow rate 1.0 ml/min. The column was maintained at ambient temperature and
the eluent was detected at 290nm. The separation
of CEF and DCX under optimized
condition is shown in Figure 3.
Method:
Preparation of stock solution:
Reference solution: The solution
was prepared by dissolving 20.0 mg of accurately weighed Cefpodoxime
Proxetil RS and 25.0 mg Dicloxacillin
Sodium RS in Mobile phase, in two 100.0 mL volumetric
flasks separately and sonicate for 20min. From the
above solutions take 10.0 mL from each solution into
a 50.0 mL volumetric flask and then makeup with
mobile phase and sonicate for 10min.
Preparation of working standard solution:
The stock solutions equivalent
to 20ppm to 80ppm with respect to both drugs were prepared in combination of Cefpodoxime Proxetil and Dicloxacillin Sodium as above, sonicated
and filtered through 0.45µ membrane.
Preparation of sample drug solution for pharmaceutical
formulations:
Twenty tablets were weighed
accurately and a quantity of tablet powder equivalent to 20 mg Dicloxacillin Sodium l and 25 mg Cefpodoxime
Proxetil was weighed and dissolved in the 70 mL mobile phase with the aid of ultrasonication
for 20 min. The content was diluted to 100 mL with
mobile phase to furnish a stock test solution. The stock solution was filtered
through a 0.45 µm Nylon syringe filter and 10.0 mL of
the filtrate was diluted into a 50.0 mL volumetric
flask to give a test solution containing 40 µg/mL Cefpodoxime Proxetil and 50 µg/mL Dicloxacillin Sodium
Fig.No.04. calibration curve of Cefpodoxime Proxetil
Procedure for calibration curve:
The
contents of the mobile phase were filtered before use through 0.45micron
membrane and pumped from the respective solvent reservoirs to the column at a
specified flow rate. Prior to injection
of the drug solutions, the column was equilibrated for at least 30min with the
mobile phase flowing through the system. The chromatographic separation was
achieved using a mobile phase consisting of Methanol : water at 50:50V/V the eluent was monitored using PDA detector at a wavelength of
290nm.The column was maintained at ambient temperature (270c) and an
injection volume of 20µl of each of standard and sample solutions were injected
into the HPLC system to get the chromatograms. The retention time, peak areas
of drug was recorded graph was plotted by taking concentration of the drug on
x-axis and peak area on y-axis. A typical chromatogram for the linear responses
of Dicloxacillin Sodium and Cefpodoxime
Proxetil combination was shown in Fig 4 and5.
Fig.No.05. calibration curve of Dicloxacillin Sodium
2.6 System suitability
System
suitability is a pharmacopoeial requirement and is
used to verify, whether the resolution and reproducibility of the
chromatographic system are adequate for analysis to be done. The tests were
performed by collecting data from 5 replicate injections of standard solutions.
The values obtained
demonstrated the suitability of the system for the analysis of this drug
combination and the system suitability parameters fall within ±2% standard
deviation range during performance of the method. Here tailing factor for peaks
of CEF and DCX was less than 2% and
resolution was satisfactory.
3. RESULTS
AND DISCUSSION:
The
chromatographic conditions were optimized to develop RP-HPLC method for
simultaneous determination of CEF and
DCX with adequate resolution and rapid analysis time.
3.1 Method Validation
The developed chromatographic
method for simultaneous estimation of CEF and DCX was validated according ICH
guidelines for linearity, accuracy, precision, specificity, robustness and
ruggedness.
3.1.1 Linearity
According to USP; tablet powder equivalent to
20, 30, 40, 50, 60, 70 and 80 µg/ml of label claim was taken and dissolved in
Methanol diluted appropriately with Methanol to obtain a concentration in the
range of 20-80 µg/ml of the test concentration. Each of this concentration was
injected to get reproducible response. The calibration curve was plotted as
concentration of the respective drug versus the response at each level. The
proposed method was evaluated by its correlation coefficient and intercept
value calculated in the statistical study.
3.1.2
Recovery
The
accuracy of the method was determined by recovery experiments. The recovery
studies were carried out using standard addition method at 50, 100 and 150 %
level; known amount of standards was added to reanalyzed sample and subjected
them to the proposed HPLC method. Percentage recovery was calculated from the
amount found and actual amount added. The mean recovery is within acceptable
limits which indicate that the method is accurate .The results of recovery
studies are shown in Table 2
3.1.3 Precision
The
precision of an analytical method is expressed in terms of SD or RSD of series
of measurements. It was ascertained by replicate estimation of CEF and DCX by
proposed method. Percentage relative standard deviation (%RSD) was found to be
less than 2% which proves that method is precise. The results of precision
study are shown in Table 3.
Preparation of standard stock solution
Standard stock
solutions Cefpodoxime Proxetil
and Dicloxacillin Sodium of strength 1mg/ml were
prepared using dichloromethane. Appropriate amounts of these stock solutions
were then further diluted to get the required concentrations of standard stock
solutions.
System suitability studies
The resolution, number of theoretical plates, retention
time and peak asymmetry were calculated for the working standard solutions and
is as shown in Table 2. The values obtained demonstrated the suitability of the
system for the analysis of these drugs in combination.
Table 2: Recovery Studies.
|
Drug |
Sample No. |
Amount present (mg/ml) |
Amount added (mg/ml) |
Amount estimated* (mg/ml) |
% Recovery* |
S.D |
% R.S.D |
|
CEF |
1 |
2.06 |
3 |
5.0486 |
97.94 |
0.6433 |
0.6533 |
|
2 |
2.06 |
6 |
8.99 |
98.71 |
|||
|
3 |
2.06 |
9 |
11.97 |
99.22 |
|||
|
DCX |
1 |
40.09 |
2.5 |
42.621 |
100.1 |
0.475 |
0.4766 |
|
2 |
40.09 |
5 |
42.521 |
99.75 |
|||
|
3 |
40.09 |
7.5 |
42.521 |
99.16 |
Table 3: System suitability Studies.
|
Parameters |
Cefpodoxime Proxetil |
Dicloxacillin Sodium |
|
Theoretical plates |
10978.86 |
9550.677 |
|
Asymmetry Factor |
1.05 |
1.15 |
|
HETP (cm) |
0.00075 |
0.00162 |
|
Resolution* |
|
4.63 |
3.1.4
ASSAY
Preparation of sample solutions
Twenty tablets were weighed and powdered. Powder
equivalent to 10 mg of Cefpodoxime Proxetil Sodium was weighed and transferred to 10 ml
volumetric flask. Cefpodoxime Proxetil about 8 ml was added and sonicated
for 10 min, volume was made up with the same solvent. This solution was then
filtered through membrane filter paper. Further dilutions were made in
dichloromethane to get concentrations in Beers law range. The retention times
of Cefpodoxime Proxetil and
Dicloxacillin Sodium were found to be 2.62 ± 0.02 and
3.96 ± 0.03 respectively. The assay was calculated from the equation of
regression line for each drug. The percentage assay of individual drug was
calculated.
4. CONCLUSION
The results of the analysis of pharmaceutical dosage
forms by the proposed methods are highly reproducible, reliable, and are in
good agreement with the label claims of the drug. The additives usually present
in the pharmaceutical formulations of the assayed samples did not interfere
with Cefpodoxime Proxetil
and Dicloxacillin Sodium. It may be said that the
proposed methods are precise, sensitive, and accurate, so that these can be
used as standard pharmacopoeial methods for the
simultaneous determination of Cefpodoxime Proxetil and Dicloxacillin Sodium
in tablets using the HPLC systems. The advantages of the proposed method
involve a simple procedure for sample preparation and relatively short time of
analysis. Apart from this, it can be used for assays of Cefpodoxime
Proxetil and Dicloxacillin
Sodium in biological fluids or in pharmacokinetic investigations.
5. ACKNOWLEDGEMENTS:
The authors would like to thank Ms Active Pharma Labs Pvt., Hyderabad, for providing the gift samples
of Cefpodoxime Proxetil and
Dicloxacillin Sodium for the project work. The
authors are thankful to Principal and Chairman of Teegala
Ram Reddy College of Pharmacy-Hyderabad for their kind help and providing all
necessary facilities.
6.
REFERENCES:
1.
O'Neill MJ. Editor. The Merck Index. An Encyclopedia of Chemicals,
Drugs and Biologicals. 14 th
ed. Whitehouse Station, New Jersey: Merck Research Laboratories, Division of
Merck and Co., Inc.; 2006. p. 319.
2.
Sweetman SC, editor. The Martindale: The Complete
Drug Reference. 35 th ed. London:
Pharmaceutical Press; 2007. p. 207.
3.
Indian Pharmacopeia. Vol. 3. New Delhi: The Controller
Publication, Govt. of India; 2010. p. 1018.
4.
The United State Pharmacopeia. USP28-NF23. Rockville, MD: United
State Pharmacopeial Convention, Inc.; 2005. p. 397.
5.
Darji BH, Shah NJ, Patel AT, Patel NM.
Development and validation of a HPTLC method for the estimation of cefpodoxime proxetil. Indian J Pharm Sci 2007; 69:331-3.
6.
Singh S, Dubey N, Jain D, Tyagi L, Singh M. Spectrophotometric and RP-HPLC methods
for simultaneous determination of cefpodoxime proxetil and clavulanate
potassium in combined tablet dosage form. Am Eurasian J Sci
Res 2010; 5:88-93.
7.
Gandhi SV, Patil UP, Patil
NG. Simultaneous spectrophotometric determination of cefpodoxime
proxetil and potassium clavulanate.
Hindustan Antibiot Bull 2009; 51:24-8.
8.
O'Neill MJ. Editor. The Merck Index. An Encyclopedia of Chemicals,
Drugs and Biologicals. 14th ed. Whitehouse
Station, New Jersey: Merck Research Laboratories, Division of Merck and Co.,
Inc.; 2006. p. 523.
9.
Sweetman SC, editor. The Martindale: The Complete
Drug Reference. 35 th ed. London:
Pharmaceutical Press; 2007. p. 237.
10.
Indian Pharmacopeia. Vol. 3. New Delhi: The Controller
Publication, Govt. of India; 2010. p. 1201-2.
11.
The United State Pharmacopeia. USP28-NF23. Rockville, MD: United
States Pharmacopeial Convention, Inc.; 2005. p.
451-2.
12.
Alderete O, González-Esquivel
DF, Del Rivero LM, Castro Torres N. Liquid
chromatographic assay for dicloxacillin in plasma. J Chromatogr B Analyt Technol Biomed Life Sci 2004;
805:353-6.
13.
Morelli B. Second-derivative spectrophotometric
assay of mixtures of dicloxacillin sodium and ampicillin sodium in pharmaceuticals. J Pharm
Sci 1988; 77:1042-6.
14.
Barot T, Patidar K, Kshartri N, Vyas N. Development
and validation of LC method for the determination of ampicillin
and dicloxacillin in pharmaceutical formulation using
an experimental design. Eur J Chem
2009; 6:955-64.
15.
Kathiresan K, Murugan R, Hameed SM, Inimai GK, Kanyadhara T. Analytical method development and validation
of cefixime and dicloxacillin
tablets by RP-HPLC. Rasayana J Chem
2009; 2:588-92.
16.
Zhang MJ, Zhang HJ, Guan X, Xiao ZH. HPLC determination of dicloxacillin and amoxicillin in human plasma. Yaowu Fenxi Zazhi
2006; 26:228-31.
17.
The International Conference on Harmonization, Q2 (R1) Validation
of Analytical Procedure: Text and Methodology, 2005. Available from: http://www.ich.org/fileadmin/
Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__Guideline.pdf
[Last Accessed on 2012 Dec 28].
Received on 05.11.2014 Accepted on 25.11.2014
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Asian J. Pharm. Ana. 4(4): Oct. - Dec. 2014; Page 151-155