Research Article Open Access
A Validated Stability Indicating RP-HPLC Method Development and Validation for Simultaneous Estimation of Cefixime Trihydrate and Levofloxacin Hemihydrate in Pharmaceutical Dosage Form
Patel Dhara*1, Meshram Dhananjay1, Parmar Vandana1, Pathak Devanshi1 and Patel Hiral1
1Department of Quality Assurance, Pioneer Pharmacy Degree College, Vadodara, Gujarat, India
*Corresponding author: Dhara Patel, Ph D, Associate Professor, Department of Quality Assurance, Pioneer Pharmacy Degree College, Vadodara,Gujarat, India; E-mail: @
Received:08 May, 2017; Accepted: 08 June, 2017; Published: 19 June, 2017
Citation: Patel Dhara, Meshram Dhananjay, Parmar Vandana, et.al. (2017) A Validated Stability Indicating RP-HPLC Method Development and Validation for Simultaneous Estimation of Cefixime Trihydrate and Levofloxacin Hemihydrate in Pharmaceutical Dosage Form. Int J Analytical Techn 3(1):1-12.
Abstract
The present study describes the stability indicating RP-HPLC method for simultaneous estimation of Cefixime trihydrate and Levofloxacin hemihydrate in pharmaceutical dosage forms. The proposed RP-HPLC method was developed by using Shimadzu (LC- 20 AD) system equipped with PDA detector and chromatographic separation was carried out on Phenomenex Luna C18 (250 x 4.6 mm x 5μ) column at a flow rate of 1 mL/min. The mobile phase consisted of 0.5 % Glacial acetic acid in water pH adjusted to 4.5 with ammonia solution: Methanol (45:55 % v/v) and eluents were scanned using PDA detector at 290 nm. The retention time of Cefixime trihydrate and Levofloxacin hemihydrate was found to be 3.07 and 5.40 min, respectively. The method has been validated for linearity, accuracy and precision, LOD, LOQ and system suitability according to ICH Q2R1 Guideline. The validated lowest limit of detection was 1.0990 and 1.0008 μg/mL and lowest limit of quantification was 3.331 and 3.032 μg/ml for Cefixime trihydrate and Levofloxacin hemihydrate respectively. Mean assay was found to be 98.5 % and 100.4 % for Cefixime trihydrate and Levofloxacin hemihydrate. The stability indicating method was developed by subjecting the drugs to stress conditions such as acid and base hydrolysis, oxidation and photoand thermal degradation and the degraded products formed were resolved successfully from the samples.

Keywords: Cefixime Trihydrate; Levofloxacin hemihydrates; RPHPLC Method; Forced Degradation
Introduction
Cefixime trihydrate (CEF) is an oral third generation cephalosporin class of antibiotic. Chemically, it is (6R, 7R)-7- {[2-(2-amino-1,3- thiazol-4-yl)-2(carboxymethoxyimino)acetyl] amino}-3-ethenyl-8-oxo-5-thia-1 azabicyclo-[4.2.0]oct-2-ene-2 carboxylic acid, clinically used in the treatment of susceptible infections including gonorrhea, otitis media, pharyngitis, lower respiratory-tract infections such as bronchitis, and urinary-tract infections [1,2](Figure 1). It is official in Indian Pharmacopoeia (IP), British Pharmacopoeia (BP), United States Pharmacopoeia (USP), European Pharmacopoeia (EP), Japanese Pharmacopoeia (JP) [3-7]. Literature survey reveals spectrophotometric, TLC, HPTLC, HPLC and HPCE method for estimation of CEF individually and in combination with other drugs in bulk drugs and human plasma [8-22].
Figure 1: Chemical Structure of CEF
Levofloxacin hemihydrates (LEVO) chemically (-)-(S)-9- fluoro2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7- oxo-7H-pyrido[1,2,3-de]-1,4benzoxazine-6-carboxylic acid hemihydrate, is a fluoroquinolone antimicrobials, is the active S-isomer isolated from the racemic ofloxacin [23] (Figure 2). It possesses wide spectrum of antibacterial activity against both Gram positive and Gram-negative bacteria, as well as atypical pathogens such as Mycoplasma, Chlamydia and Legionella [24]. Levofloxacin hemihydrate is official in IP [25]. Numerous HPLC, UV and HILIC⁄MS⁄MS has been used to determine drugs in dosage form and biological fluids [26-34].

The International Conference on Harmonization (ICH) guideline entitled “Stability testing of new drug substances and
Figure 2: Chemical structure of LEVO
products” requires that stress testing be carried out to elucidate the inherent stability characteristics of the active substance. An ideal stability-indicating method is one that resolves the drug and its degradation products efficiently. Consequently, the implementation of an analytical methodology to determine CEF and LEVO simultaneously, in presence of its degradation products is rather a challenge for pharmaceutical analyst. Therefore, it was thought necessary to study the stability of CEF and LEVO under acidic, alkaline, oxidative, UV and photolytic conditions. This paper reports validated stability-indicating HPLC method for simultaneous determination of CEF and LEVO in presence of their degradation products. The proposed method is simple, accurate, reproducible, stability-indicating and suitable for routine determination of CEF and LEVO in combined dosage form. The method was validated in compliance with ICH guidelines. The purpose of this study was to develop a stabilityindicating method for the simultaneous determination of CEF and LEVO in bulk drugs and to apply the developed method for the quantitative determination of these drugs from tablets. The RPHPLC technique was chosen because of its previously mentioned advantages. The proposed method was able to separate the compounds of interest and their degradation products within 10 min. Thereafter, this method was validated as per International Conference on Harmonization (ICH) guidelines [35-37]. A literature survey has shown that a stability-indicating HPLC method for the simultaneous determination of CEF and LEVO has not been developed. The previously developed methods have been able to separate both the drugs during a minimum run time, but they were not stability-indicating i.e., the separation of various degradation products, employing ICH prescribed stress conditions, was not achieved [38-40].
Materials and Method
Chemicals and Reagents
CEF and LEVO of pharmaceutical grade were kindly supplied as gift samples by Sunrise Remedies, Ahmedabad and Cadila healthcare, Ahmadabad, respectively. Acetonitrile (ACN), methanol, and water used were of HPLC grade and were purchased from Merck specialist Pvt. Ltd, India. Hydrochloric acid, Sodium hydroxide, Hydrogen peroxide was purchased from Suvidhinath Laboratories, India.
Instruments and Equipments
The liquid chromatographic system was of Shimadzu (LC-20 AD) system and was manufactured by Shimadzu, Kyoto, Japan, equipped with Injector (Rheodyne, 20 μL), UV and photodiode array (PDA) detector. The chromatographic analysis was performed using LC Solution software on a Phenomanex Luna C18 (150X4.6) mm, 5μ column. In addition, Digital weighing balance (Shimadzu ATX 224, Japan) pH meter (Janki impex Pvt. Ltd), fast clean ultrasonic cleaner (Toshco instrument), hot air oven (Thermolab, Mumbai), humidity cum photostability chamber (Thermolab, Mumbai) were used in this study.
Chromatographic Conditions
Mobile phase: 0.5% Glacial acetic acid in Water pH adjusted to 4.5 with
ammonia solution: methanol (45:55 % v/v)
Column: Phenomanax Luna C18 (150X4.6) mm, 5μ Column
temperature: 30oC
Injection volume: 20μL Flow rate: 1.0 mL/min
Wavelength: 290 nm
Diluent: Diluent-1: Water: Methanol (50:50 % v/v) Stock
Solution Diluent-2: Mobile phase for 2 dilution
Preparation of Mobile Phase
450 mL of pH 4.0 Glacial acetic acid and 550 mL of methanol were mixed. This mixture was sonicated for 10 min and filtered through 0.22 μm membrane filter and used as mobile phase.
Preparation of 0.5 % Glacial acetic acid (pH 4.5)
5 mL of Glacial acetic acid was added in 1000 mL of water. Adjusted ph 4.5 with ammonia solution.
Standard Stock Solution Preparation of Cefixime & Levofloxacin: (100 μg/mL & 125 μg/mL respectively)
Accurately weighed 25 Mg of Cefixime and 31.25 Mg Levofloxacin were transferred in 50 mL volumetric flask, then 35ml of Diluent-1 added and sonicated for 10 minutes to dissolve it completely. The volume made up with Diluent-1. Pipette out 5 mL of solution in 25 mL of volumetric flask and make up the volume with Diluent-2.
Preparation of Sample Solution
Twenty tablets for combined dosage form of CEF and LEVO were weighed and grind to a fine powder, take label claim quantities of powder equivalent to 25 Mg CEF and 31.25 Mg LEVO were weighed, mixed, and transferred to a 50 ml volumetric flask. The solution was sonicated to dissolve the powder in 30 ml diluents 1 and diluted up to mark with diluents 1. The solution was filtered through a Whatmann filter paper no. 41. Take 5 mL of the above solution and make up to 50 ml with diluents 2 to get 100 μg/mL CEF and 125 μg /mL LEVO. A total of 20 μL volume of the above sample solution was injected into HPLC and peak areas were measured under optimized chromatographic conditions.
Method Validation
The method of analysis was validated as per the recommendations of ICH and USP for the parameters like specificity, accuracy, linearity, precision, detection limit, quantification limit, and robustness. Specificity was determined by evaluating the ability of the proposed method to separate CEF and LEVO from its potential degradation products. Forced degradation studies were performed for bulk drug and formulation to provide an indication of the stability-indicating property and specificity of the proposed method. The accuracy of the method was determined by calculating the percentage recovery of CEF and LEVO. For both the drugs, recovery studies were carried out by applying the method to drug sample to which known amount of CEF and LEVO corresponding to 80,100 and 120 % of label claim had been added (standard addition method). Intraday and interday precision study of CEF and LEVO was carried out as per guideline. The Limit of Detection (LOD) and Limit of Quantification (LOQ) were calculated using the following formula:

LOD=3.3(SD)/S and LOQ=10(SD)/S

Where,
SD = standard deviation of response (peak area)
S = average of the slope of the calibration curve.

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 drug solution to check the reproducibility of the system. For robustness evaluation of HPLC method a few parameters like flow rate, percentage of methanol in the mobile phase and pH of mobile phase were deliberately changed. One factor was changed at one time to estimate the effect.
Forced Degradation Studies
Forced degradation studies of both the drugs were carried out under conditions of hydrolysis, dry heat, oxidation, and photolysis.
Sample Stock Preparation for Forced Degradation Study
Transferred 5 intact tablets in to 200 mL of volumetric flask, added about 150 mL of Diluent-1 in to it, sonicated for 30 minutes with intermittent shaking, cooled to attain room temperature and made up to volume with Diluent-1 and filtered the solution with 0.45μ nylon filter.
Sample Preparation for Acidic Degradation
1mL of above sample stock solution filtrate was transferred to 100 mL of Volumetric Flask; added 5 mL of 1N HCl to it and it was kept for 3 hours at room temperature. Then added 5 mL of 1N NaOH to neutralize it and volume was made up to mark with Diluent-2, mixed well and injected.
Sample Preparation for Basic Degradation
1 mL of above sample stock solution filtrate was transferred to 100 mL of Volumetric Flask; added 5 mL of 1N NaOH to it and it was kept for 2 hours at room temperature. Then added 5 mL of 1N HCl to neutralize it and volume was made up to mark with Diluent-2, mixed well and injected.
Sample Preparation for Peroxide Degradation
1 mL of above sample stock solution filtrate was transferred to 100 mL of Volumetric Flask; added 5 mL of 3% H2O2 to it and it was kept for 2 hours at room temperature. Then volume was made up to mark with Diluent-2 and mixed well and injected.
Sample Preparation for Thermal Degradation
1 mL of above sample stock solution filtrate was transferred to 100 mL of Volumetric Flask; it was kept for 3 hours at 80°C temperature. Then volume was made up to mark with Diluent-2 and mixed well and injected.
Sample Preparation for Sunlight Degradation
1 mL of above sample stock solution filtrate was transferred to 100 mL of Volumetric Flask; it was kept for 12 hours in sunlight. Then volume was made up to mark with Diluent-2 and mixed well and injected.
Results and Discussions
Method Development
A series of trials was conducted with different columns like Phenomanax Luna C18 and and C-8 columns with different mobile phases to develop a suitable RP-HPLC method for estimation of CEF and LEVO in tablet dosage form, and finally a typical chromatogram was obtained with isocratic elution of mobile phase consisting of 0.5% Glacial acetic acid in Water pH adjusted to 4.5 with ammonia solution : Methanol (45:55 % v/v), and at flow rate of 1.0 mL/min The chromatographic separation was performed on Phenomanax Luna C18 (150X4.6) mm, 5μ by injecting 20 μL and analytes were detected with PDA detector at 290 nm. The retention time of CEF and LEVO was found to be 3.012 min and 5.40 min, respectively (Figure 3). Forced degradation studies were also carried using the developed method and the degraded compounds were effectively resolved from the CEF and LEVO in tablet dosage form. The optimized conditions were given in (Table 1).
Method Validation
System Suitability
System suitability was performed to verify the acceptability of the resolution and repeatability of the system. System suitability was performed by injecting six replicate injections of the standard solution (100 %) and parameters such
Figure 3: Optimised chromatogram of CEF and LEVO
Table 1: Optimized Chromatographic Conditions

Sr. no

Parameters

Optimized Chromatographic Condition

1

Column

Phenomanax Luna C18 (150X4.6) mm, 5µ

2

Mobile Phase

0.5% Glacial acetic acid in Water pH adjusted to 4.5 with ammonia solution : Methanol (45:55 %v/v)

3

Flow rate

1.0 mL/min

4

Detector

PDA detector at 290 nm

5

Injection Volume

20 μL

6

Temperature

30oC

7

Retention time

CEF 3.012 min
LEVO 5.40 min

as peak area, USP tailing, theoretical plates, retention time, and peak asymmetry were evaluated. The % RSD was determined and reported within the limits. The results were shown in (Table 2).
Linearity
The peak area was dynamic-linear in the concentration ranges of 80.1-120.1 mg mL−1 for CEF and 100.1-150.1 mg mL−1 for LEVO, respectively. Highly significant correlation coefficient demonstrated the linearity of the method (Table 3 and Figure 4).
Table 2: Data of system suitability

Parameters

CEF

LEVO

Retention time (minute)

3.072

5.401

Resolution

7.9

Theoretical Plates

11248

10254

Tailing Factor

1.1

1.1

Table 3: Parameters of regression analysis
Parameters CEF LEVO
Linearity ( mg mL−1 ) 80.1-120.1 100.1-150.1
Correlation coefficient (R2) 0.9998 0.9990
Slope 65592.486 80164.266
Intercept 86857.404 111778.210
Figure 4: Overlain Chromatogram of CEF and LEVO (Linearity)
Specificity
The chromatograms of blank, placebo, test sample, and standard were used to justify the specificity of target analyte. The method was specific since excipients in the formulation did not interfere in the estimation of CEF and LEVO (Figure 5).
Accuracy
The accuracy of the proposed method was evaluated by calculating the recovery studies of the test drug at three different concentration levels (80 %, 100 %, and 120 %) by standard addition method. A known amount of CEF and LEVO was added to prequantified sample solution and three replicates of each concentration were injected in developed chromatographic conditions. The % recovery results were shown in (Table 4).
Precision
The values of %RSD for intraday and interday variation were found very well and within 2 % limit, indicating that the current method is repeatable (Table 5).
Figure 5: Chromatogram of CEF and LEVO (a) Blank (b) Placebo (c) Standard and (d) formulation
Table 4: % Recovery results of CEF and LEVO

Spiked Level

%Recovery

%RSD

CEF

LEVO

CEF

LEVO

80%

98.5

101.3

 

0.7

 

0.3

99.3

101.4

99.8

100.8

100 %

99.3

101.5

 

0.5

 

0.7

98.8

100.9

98.3

100.1

120 %

99.0

100.7

 

0.4

 

0.5

98.4

100.2

99.0

101.2

Table 5: Result of precision of CEF and LEVO

Drug

Sample Concentration (mg mL−1 )

Peak Area

(Day 1)

Overall ± %RSD

Peak Area

(Day 2)

Overall ± %RSD

CEF

90.1

 

 

5820537

5820089± 0.011

5812345

5814440± 0.074

 

5820414

5819413

5819315

5811563

100.1

6471128

6434480±0.49

6469852

6427121±0.58

 

6421088

6411256

6411225

6400256

110.1

7115843

7136223±0.79

7101236

7106106±0.24

 

7092813

7091452

7200013

7125631

LEVO

112.6

8907099

8943751±0.66

 

8906512

8907659±0.02

 

 

8912135

8910235

 

9012018

8906231

125.1

9913240

9902636±0.17

 

9902563

 

9865203

±0.46

 

9882151

9878456

 

9912516

9814589

137.6

10100862

10135213
±0.40

 

10800730

 

10864134

±0.50

 

10181215

10891458

 

10123561

10900214

Sensitivity
The Data for the LOD and LOQ for CEF and LEVO Shown in (Table 6).
Robustness
The effects of robustness study under different altered conditions of this proposed method are satisfactory (Table 7). The mean recovery and % RSD of analyzed sample indicate that the current method is robust.
Assay of Marketed Formulation
The % assay of the marketed formulation was found to be 98.5 % for CEF and 100.4 % for LEVO (Table 8).
Table 6: LOD and LOQ data for CEF and LEVO

Limit of Detection (LOD)

CEF

LEVO

1.10 μg/Ml

1.00 μg/mL

Limit of Quantitation

3.33 μg/mL

3.03 μg/Ml

Table 7: Robustness data for CEF and LEVO

At Normal Range (CEF)

Peak Area ± %RSD Are

Flow rate 1ml/min

6422932 ±0.21

Mobile phase (45:55)

pH 4.5

Sr. No.

Flow rate
+0.1

Flow rate
-0.1

M.P + 2

M.P - 2

pH + 0.2

pH - 0.2

1

5847283

7068145

8897643

6438437

9843851

6420128

2

5835432

7021756

8881317

6418546

9836270

6392656

3

5792291

7007583

8897218

6448751

9792430

6430960

%RSD

0.5

0.5

0.5

0.5

0.3

0.3

At Normal Range (LEVO)

Peak Area ± %RSD

Flow rate 1ml/min

9849881± 0.14

Mobile phase (45:55)

pH 4.5

1

8833112

10864522

9765496

9876432

9838124

9757021

2

8877220

10874268

9833987

9890048

9859416

9836270

3

8881317

10834869

9784174

9865372

9865372

9792430

%RSD

0.5

0.2

0.5

0.2

0.2

0.4

Table 8: % Assay of marketed formulation

Drug

Label claim

Amt. of drug estimated

% Label claim

CEF

400mg

394mg

98.5%

LEVO

500mg

502mg

100.4 %

Forced Degradation Studies
In the present study forced degradation studies were carried out to ensure the effective separation of CEF and LEVO from degradation products. Degradation was observed by decreasing the peak areas of the drug substances with same drug molecules of degraded peak areas. The percentage assay of degradation was calculated from the peak area obtained in degradation conditions and it was compared with assay of nondegraded conditions. Acidic and alkali degradation was carried out by treating the sample solution with 1N HCl and 1N NaOH solutions. Oxidative degradation studies were performed by treating 3 % H2O2 solution and keeping it at room temperature for 3 h min. For thermal stress studies the drug solutions were placed in oven at 80°C for 3 h and then injected into HPLC system and sunlight testing was carried out by keeping the drug solutions in sunlight for 12 hrs from the chromatograms, it was found that both the molecules are susceptible to acidic, alkali, oxidative, thermal and sunlight degradation and percentage assay degradation in all the conditions was found to be within the limits (Figure 6-10). The forced degradation studies were performed without intending to identify the degradation products but merely to show that they are not interfering with active molecules if any present. The results of stress studies were shown in (Table 9 and 10).
Figure 6: Acidic stress degradation chromatogram of (a) Blank (b) Formulation
Table 9: Retention time of degradant product of CEF and LEVO (Stress Degradation Study)

Conditions

Retention time (Rt) (minute)

CEF

LEVO

Degradant1

Degradant2

Degradant3

Acid Degradation

3.03

5.43

2.15

6.00

-----------

Base Degradation

3.05

5.48

2.15

5.03

--------------

Peroxide Degradation

3.05

5.46

3.56

5.01

-------------

Thermal Degradation

3.04

5.46

2.30

3.59

4.99

Sunlight Degradation

3.04

5.4

3.60

5.11

-------------

Figure 7: Alkaline stress degradation chromatogram of (a) Blank (b) Formulation
Figure 8: Oxidative stress degradation chromatogram of (a) Blank (b) Formulation
Figure 9: Thermal stress degradation chromatogram of Formulation
Figure 10: Sunlight stress degradation chromatogram of Formulation
Table 10: Results of Forced degradation studies

Stress type

Stress conditions

CEF

LEVO

% Assay

%Degradation

% Assay

% Degradation

Control Sample

Sample itself

98.5

NA

100.4

NA

Acid Degradation

1 N HCL,
5mL for 3 hours

87.1

11.4

85.9

14.5

Base Degradation

1N NaOH,
5ml for 5 hours

80.9

17.6

87.2

13.2

Peroxide Degradation

5mL 3% H2 O2 at RT
for 3 hours

88.3

10.2

88.1

13.2

Thermal Degradation

At 80ºC for 3 Hours

87.2

11.3

86.9

13.5

Sunlight Degradation

At sunlight for 12 hours

86.8

11.7

87.1

13.3

Conclusion
The reported RP-HPLC method was proved to be simple, rapid, and reproducible. The validation data indicate good precision, accuracy, and reliability of the method. The developed method offers several advantages in terms of simplicity in mobile phase, isocratic mode of elution, easy sample preparation steps, and comparative short run time which makes the method specific and reliable for its intended use in simultaneous determination of CEF and LEVO in tablet dosage form. Quick stability indicating RP-HPLC method was developed for the simultaneous estimation of CEF and LEVO in the presence of its degradation products, generated from forced degradation studies. The developed method separates CEF and LEVO in impurities/degradation products. There were no reported stability indicating methods for this combination of drugs in liquid dosage form; hence, this method has an advantage of being unique and novel.
Acknowledgement
The authors greatly thankful to Sunrise Remedies, Ahmedabad and Cadila healthcare, Ahmedabad, for providing the gift sample of Cefixime trihydrate and Lecofloxacin hemihydrates. Authors also extend their thanks to the management, Pioneer Pharmacy Degree College, Vadodara for providing the facilities to carry out the present work.
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