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International Journal of ChemTech Research

 

 

 

CODEN( USA): IJCRGG

ISSN : 0974-4290

 

 

 

 

 

 

Vol.2, No.1, pp 79-87,

Jan-Mar 2010

 

 

 

SIMULTANEOUS ESTIMATION OF CEFIXIME TRIHYDRATE AND ERDOSTEINE IN PHARMACEUTICAL DOSAGE FORM BY USING REVERSE PHASE - HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Madhura V. Dhoka*, Vandana T. Gawande, Pranav P. Joshi

Department of Quality Assurance,

A.I.S.S.M.S. College of Pharmacy, Pune University, Pune-01,India.

*Corres.author: madhura1777@yahoo.com

ABSTRACT: A simple, precise, accurate and sensitive Reverse phase high performance liquid chromatographic (RP- HPLC) method for simultaneous estimation of Cefixime trihydrate and Erdosteine in combined capsule dosage form have been developed and validated. Drugs were resolved on a HiQ Sil C8 column (25cm X 4.6mm, 5μm), utilizing mobile phase of TetraButyl Ammonium Hydroxide (0.1 N aqueous) pH adjusted to 6.5 with Orthophosporic acid (10% aqueous) : ACN in a ratio of 2:1. Mobile phase was delivered at the flow rate of 1.0 ml/minute. Ultra violet Detection was carried out at 254nm. Separation was completed within 11 minutes. Calibration curves were linear with correlation coefficient 0.998 and 0.997 over a concentration range of 2-22 μg/ml for Cefixime trihydrate and 3- 33 μg/ml for Erdosteine respectively. Recovery was between 99.92-101.12 percent and 100.01-100.34 percent for Cefixime trihydrate and Erdosteine respectively. Method was found to be reproducible with relative standard deviation (R.S.D) for intra and interday precision to be <1.5% over the said concentration range.

Key words: Cefixime Trihydrate, Erdosteine, High Performance Liquid Chromatography, Capsules.

INTRODUCTION

Cefixime trihydrate, is the third generation cephalosporin antibiotic. Cefixime is given orally in the treatment of susceptible infections including respiratory tract infections like acute exacerbations of chronic bronchitis, gonorrhoea, otitis media, pharyngitis, lower respiratory-tract infections such as bronchitis, and urinary-tract infections(1). It official in USP. Chemially it is 5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-corboxylic acid,7-[[2-amino-4- thiazolyl) [(carboxymethoxy)imino]acetyl] amino]-3- ethenyl-8-oxo-,trihydrate,[6R-[6µ,7b(Z)] ]-(6R,7R)-7- [2-(-amino-4-thiazolyl) glyoxylamido]-8-oxo-3-vinyl- 5-thia-1-azabicyclo[4,2,0]oct-2-ene-2-carboxylic acid, 72-(Z)-[O-carboxymethyl)oxime](2) Erdosteine [2-Oxo-2-[(tetrahydro-2-oxo-3-thienyl)amino] ethyl] thio]acetic acid is a mucolytic and is official

in Martindale(3). It Modulates mucus production, viscosity and increases mucociliary transport, thereby improving expectoration and thus it shows mucolytic and antitussive activity(4-5).

Investigations are done to study effect of mucolytic on antibiotic penetration in sputum and it reveals that mucolytics improve the same(6-8). Hence combination of an antibiotic with a mucolytic is a treatment of choice for acute exacerbations of chronic bronchitis. Also comparative evaluation of cefixime plus erdosteine and amoxicillin plus bromhexine shows that former gives faster and better symptomatic relief and was also better tolerated than later(9). As Combination is not available in the market it was also developed.

There are several investigations concerning the determination of cefixime alone and in combination with other drugs in pharmaceutical preparations and

Madhura V. Dhoka et al /Int.J. ChemTech Res.2010,2(1)

plasma by UV , HPLC,LC-MS, HPTLC methods(10- 14)

One stability indicating HPTLC method is reported for erdosteine(15). Erdosteine and its optical active metabolite have been analyzed by high-performance liquid chromatography using a fluorescent chiral tagging reagent(16). Sensitive determination of erdosteine in human plasma has been achieved by automated 96-well solid-phase extraction and LC– MS–MS(17).

No references have been found for simultaneous quantitative determination of Cefixime trihydrate and Erdosteine in pharmaceutical preparations. Hence attempts were made to develop Simultaneous HPLC method.

In this paper we report simple, accurate, precise and sensitive Reverse phase high performance liquid chromatography method for simultaneous determination of Cefixime trihydrate and Erdosteine in combined capsule dosage form.. The proposed method is optimized and validated according to ICH guidelines(18).

EXPERIMENTAL Reagents

All chemicals and reagents used were of a HPLC grade or an analytical grade. Cefixime trihydrate was kindly supplied as gift sample by Maxim Pharmaceuticals, Pune and Erdosteine was also obtained as a gift sample. Rest of all chemicals and reagents were obtained as follows.

TetraButyl Ammonium Hydroxide solution (0.1 N aq solution) and Acetonitrile were obtained from Sisco Reasearch Laboratories and Merck Laboratories Pvt Ltd, Mumbai respectively.

Orthophosporic acid (10% aqueous), Dibasic Sodium phosphate and Monobasic Potassium Phosphate solution were obtained from S.d.Fine Chem Ltd. Mumbai.

Equipment

1. HPLC was performed using a Jasco HPLC system

2000 consisting of

a pump PU2080 Plus ,

Rheodyne sample injection port with 20 microlitre loop,

UV detector 2075 plus. Borwin Software version 1.

Column used was C-8 (4.6 ´ 250mm, 5m).

2.Shimadzu Model AY-120 balance

3.Delux 101 pH meter

4.Calibrated glassware were used for the study

Chemicals and materials

Preparation of TetraButyl Ammonium Hydroxide solution

80

15ml of 0.1 N TetraButyl Ammonium Hydroxide solutions was diluted to 150 ml with HPLC grade water, pH adjusted to 6.5 with Orthophosporic acid (10% aqueous).

Preparation of monobasic potassium phosphate solution

Solution was prepared by weighing 6.8 gm of monobasic potassium phosphate and dissolving it in to 500 ml of water.

Preparation of phosphate Buffer pH 7.0

Buffer Solution was prepared by weighing 7.1 gm of Dibasic Sodium phosphate and dissolving it in to 500mL of water and adjusting its pH to 7 with monobasic potassium phosphate solution

Praparation of Standard Stock Solution

Standard Stock Solution of each drug having concentration of 1mg/ml was prepared by dissolving pure drugs, Cefixime trihydrate and Erdosteine separately in phosphate buffer pH 7.0.

Preparation of solutions for calibration curve Standard Stock solutions of both drugs were diluted as 1ml to 10ml with phosphate buffer pH 7.0. These solutions were further diluted to get solutions of concentrations 2,6,10,14,18,22 μg/ml and 3,9,15,21,27,33 μg/ml of Cefixime trihydrate and Erdosteine respectively.

Procedure for Sample Preparation /capsule analysis

Sample details:

Composition: Each hard gelatin capsule contains Cefixime trihydrate equivalent to Cefixime USP 200mg

Erdosteine 300mg.

Gross weight of one capsule: 734 mg Fill weight of one capsule: 617mg

Contents of 20 capsules were emptied and powdered and capsule powder equivalent to 10 mg of Cefixime trihydrate and 15 mg of Erdosteine was taken in 10mL volumetric flask, and dissolved in sufficient phsophate buffer solution with aid of sonication and volume was made up to 10mL with phosphate buffer solution. Resultant solution was first filtered through whatman filter paper No. 41 and then through 0.45m filter paper in order to remove the excipients. 1ml of the filtrate was diluted to 10ml with phsophate buffer pH 7.0. Again 1ml of this solution is diluted to 10 ml with phosphate buffer solution to get final concentration of 10 μg/ml and 15 μg/ml for Cefixime trihydrate and Erdosteine respectively.

Dilutions for Precision studies

Precision of the method was checked by 3 replicate readings at 3 concentration levels. Concentration levels

Madhura V. Dhoka et al /Int.J. ChemTech Res.2010,2(1)

used for Cefixime trihydrate were 10, 14, 18 μg/ml and that for Erdosteine were 15, 21, 27 μg/ml.

Dilutions for Recovery studies

To study accuracy of the method, recovery studies were carried out by addition of standard drug solution to sample at 3 different levels, 80%, 100% and 120% of the test concentration ( test concentration is 10 μg/ml for Cefixime trihydrate and 15 μg/ml for Erdosteine)

Robustness studies

Robustness of the method was determined by small, deliberate changes in flow rate, mobile phase ratio, Wavelength of detection and pH of mobile phase. Flow rate was changed to 1 + 0.5 ml/min. The mobile phase ratio was changed to + 3% for both components. Wavelength of detection was changed to 254+ 2nm; pH was changed to 6.5 + 0.1

LOD and LOQ Determination

Limit of detection can be calculated by using following formula:

3.3 s

LOD =

S

Limit of quantitation can be calculated based on standard deviation of the response and the slope.

10 s

LOQ =

S

Where s = Standard deviation of the response S = Slope of the calibration curve

System Suitability Testing

System Suitability Testing is used to verify that the resolution and reproducibility of the system are adequate for the analysis to be performed. Parameters such as therotical plates, tailing factor, resolution and reproducibility (%RSD for retention time and for area of six replicates) are determined and compared against the specifications. The results of system suitability and system precision were presented in table no-2.

RESULTS AND DISCUSSION Method Development and Optimisation

Various mobile phases were tried containing methanol, phosphate buffer, acetic acid, but as chemical nature of both drugs is same (acidic) both drugs were eluting out nearly at same retention time. Litreature survey revealed the importance of ion pairing reagents to resolve chemically similar drugs. Ion-exchange chromatography systems have previously been utilized in HPLC analysis of ionic samples. Recently, reversed phase partition chromatography using ion-pair reagents has been developed and utilized. The ionic samples form an ion-pair with ion-pair reagents in the mobile phase to become electrically neutral. The increase in

81

hydrophobic character of the ion-pair results in a greater affinity for the reverse stationary phase and leads to sample resolution. For acidic samples, analysis is performed with pH adjusted to 7.5 with the addition of quaternary ammonium salts to the mobile phase. Acidic samples form an electrically neutral

ion-pair

 

with

the

quaternary

ammonium

salt

eg.

 

Tetrabutylammonium

Phosphate,

TetrabutylammoniumHydroxide(19).

 

Use of TetraButyl Ammonium Hydroxide was preferred as it is used as mobile phase component of pharmacopoeial method for Cefixime. Its use with ACN provided good resolution and peak shape. Thus TetraButyl Ammonium Hydroxide pH 6.5: ACN was finalized as mobile phase. By trying various proportions of TetraButyl Ammonium Hydroxide solution and ACN, final proportion selected was 2:1 ( TetraButyl Ammonium Hydroxide solution : ACN )

UV absorption spectra of both drugs in the range of 200-400nm showed considerable absorbance for both drugs at 254nm and at the same wavelength, diluents showed no interference. Therefore it was selected as detection wavelength as shown in figure 1.

Flow rate of 1ml/min provided runtime of about 10 minutes and was used as flow rate.

C8 is common stationary phase suitable for many pharmaceuticals and was found to be suitable in this case also.

With above selected method parameters, system suitability testing provided good resolution and reproducibility and was adequate for analysis to be performed. The results of system suitability are shown in table no.2

The method was validated for various parameters as per ICH Guidelines. The results of method validation are shown in table no. 3

Method validation

The linear relationship was observed between the AUC and concentration over the range of 2-22 μg/ml for cefixime trihydrate and 3- 33 μg/ml for Erdosteine. The linearity was expressed as correlation coefficient, which was 0.998 for Cefixime trihydrate and 0.997 for Erdosteine. Correlation coefficient, y- intercept, slope of regression line are shown in table no.3 and Figure -3 and 4

As per ICH guidelines, for assay procedure of active substance or finished product, range should be 80 – 120% of the test concentration. Therefore range of 2- 22 μg/ml was selected for Cefixime trihydrate and 3- 33 μg/ml was selected for that of Erdosteine.

Precision was carried out as repeatability as per ICH guidelines. It was determined at 3 concentration levels with 3 replicates at each level. For all three

Madhura V. Dhoka et al /Int.J. ChemTech Res.2010,2(1)

82

concentration levels % RSD obtained was less than 1.5

The proposed method was evaluated in the assay of

% for both Cefixime trihydrate and Erdosteine. The

capsule formulation containing Cefixime trihydrate

results of precision are given in table no.6

and Erdosteine. Five replicate determinations were

Considering composition of the dosage form, 10 μg/ml

carried out on capsules. Mean % content was 99.38 %

of Cefixime trihydrate and 15 μg/ml of Erdosteine was

for Cefixime trihydrate and that for Erdosteine was

selected as test concentration. The recovery studies

100.33 %. Results of capsule analysis was shown in

were carried out at 80, 100, and 120% of test

table no. 7

concentration. The results ranged from 98.88 -100.99

 

% for Cefixime trihydrate and 99.09 – 101.59 % for

CONCLUSION

Erdosteine. Results of recovery studies are shown in

The method described enables the quantification of

table no.4

Cefixime trihydrate and Erdosteine in combined

Robustness studies were carried out after deliberate

capsule dosage form. The validation data demonstrate

alterations of flow rate, mobile phase compositions,

good precision and accuracy, which prove the

wavelength of detection and mobile phase pH. It was

reliability of the proposed method. Hence, this HPLC

observed that the small changes in these operational

method can be used routinely for quantitative

parameters, did not lead to changes of retention times

estimation of both components in capsule dosage form.

of peak of interest. Results of robustness studies are

 

shown in table no.5

Table 1. Conditions Used for Chromatographic Analysis

Sr

Parameter

Conditions used for Analysis

No

 

 

1

Mobile phase

TetraButyl Ammonium Hydroxide ( 0.1 N

 

 

aqueous) pH 6.5 with Orthophosporic acid

 

 

(10% aqueous): ACN (2:1)

 

 

 

2.

Flow rate

1 ml/min

 

 

 

3.

Detection Wavelength

254 nm

 

 

 

4.

Sample injector

50 µl loop

 

 

 

5.

Column

HiQ Sil C8 Kya tech (4.6 ´ 250mm, 5m)

 

 

 

Table 2. System Suitability Testing

Drug

Retention

Area

No Of

Resolution

Asymmetry

time

Plates

 

(min)

 

 

 

 

 

 

 

 

Cefixime

10.00

451091.4

5836.73

0.0

1.1

trihydrate

 

 

 

 

 

 

Erdosteine

5.4

133777.1

4316.31

2.9

1.4

 

 

 

 

 

 

 

Madhura V. Dhoka et al /Int.J. ChemTech Res.2010,2(1)

83

Table 3. Results of validation Parameters

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sr No

 

Parameters

 

 

 

Results

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cefixime trihydrate

 

 

Erdosteine

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1.

 

Linearity (R2)

0.9982

 

 

 

 

0.9979

 

 

 

 

 

 

Y – intercept

24792

 

 

 

 

1032

 

 

 

 

 

 

Slope of regression line

44216

 

 

 

 

9216

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2.

%RSD (Indicates

< 2 %

 

 

 

 

< 2 %

 

 

 

 

 

 

 

precision)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3.

Mean % Recovery

100.52

 

 

 

 

100.12

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4.

Limit of Detection

 

0.31 µg/ml

 

 

 

0.28µg/ml

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5.

Limit of Quantitation

 

0.94µg/ml

 

 

 

0.84µg/ml

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6.

 

 

Range

 

 

2-22 mg/ml

 

 

 

3-33mg/ml

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 4. Results for Recovery study

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

% Added (%

 

Amount of drug

 

 

 

 

 

 

 

 

 

 

 

 

of test conc.)

 

after std.

 

Mean Area*

Amount Found

 

 

 

 

 

 

 

 

addition

 

 

 

 

 

 

 

 

 

(µg/ml)

 

 

 

 

 

(µg/ml)

 

% Recovery

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

CEF

 

ERDO

 

CEF

 

ERDO

CEF

ERDO

 

CEF

 

ERDO

 

80

 

 

18

 

27

 

829645.4

 

250731.7

18.20

27.09

 

101.12

 

100.34

 

 

 

 

 

 

 

 

 

 

100

 

 

20

 

30

 

908421.2

 

277569.1

19.98

30.003

 

99.92

 

100.01

 

 

 

 

 

 

 

 

 

 

120

 

 

22

 

33

 

1002734

 

305277.2

22.11

33.007

 

100.53

 

100.02

 

 

 

 

 

 

 

 

 

 

 

 

*mean area of

three replicates

 

 

 

 

 

 

 

 

 

 

 

Madhura V. Dhoka et al /Int.J. ChemTech Res.2010,2(1)

 

 

 

 

84

Table 5. Results for Robustness Study

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

% RSD *Found For Robustness Study

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Flow Rate

 

 

pH (6.5)

Mobile phase ratio

Wavelength of

 

(1mLmin-1)

 

 

 

 

(2:1)

 

detection 254nm

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Drug used

0.9

1.1

 

6.4

 

6.6

+3%

 

-3%

252

256

 

Cefixime

1.50

1.36

 

1.21

 

0.96

1.07

 

0.81

1.61

1.22

 

Trihydrate

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Erdosteine

1.80

1.62

 

1.28

 

1.17

1.22

 

1.59

1.00

1.33

 

 

 

 

 

 

 

 

 

 

 

 

 

 

*%RSD for three replicates

Table 6. Results for Method Precision

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sr No

Conc. in

Mean Area

 

Std Dev

 

%RSD

 

 

 

 

µgmL-1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cefixime

10

 

465939

4195.32

 

0.90

 

 

 

 

 

 

 

 

 

 

 

Trihydrate

14

 

654518.7

7180.94

 

1.097

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

18

 

829784.6

3122.34

 

0.37

 

 

 

 

 

 

 

 

 

 

 

 

 

Erdosteine

15

 

 

 

 

 

 

 

 

 

 

 

 

 

139187.3

782.18

 

0.56

 

 

 

 

 

 

21

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

198401.1

3095.89

 

1.56

 

 

 

 

 

 

27

 

248091.3

3565.04

 

1.43

 

 

 

 

 

 

 

 

 

 

 

 

Table 7. Results for Capsule Assay Study

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sr.

 

 

 

 

 

 

 

 

 

 

 

 

 

No.

 

Label Claim

 

Amount Found

 

% of Label Claim

 

 

 

(μg mL-1)

 

( μg mL-1)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cefixime

 

Erdosteine

 

Cefixime

Erdosteine

 

Cefixime

Erdosteine

 

 

Trihydrate

 

 

 

Trihydrate

 

 

 

Trihydrate

 

 

1.

 

10

 

15

 

9.87

15.16

 

98.76

101.10

 

 

 

 

 

 

 

 

 

 

 

 

 

2.

 

10

 

15

 

10.16

14.83

 

101.63

98.87

 

 

 

 

 

 

 

 

 

 

 

 

 

3.

 

10

 

15

 

10.01

15.13

 

100.11

100.90

 

 

 

 

 

 

 

 

 

 

 

 

 

4.

 

10

 

15

 

9.81

15.02

 

98.13

100.13

 

 

 

 

 

 

 

 

 

 

 

 

 

5.

 

10

 

15

 

9.82

15.09

 

98.24

100.63

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Madhura V. Dhoka et al /Int.J. ChemTech Res.2010,2(1)

85

Figure 1. Overlain spectra of Cefixime Trihydrate (CEF) and Erdosteine(ERDO)

CEF

ERDO

Figure 2. Resolution Study for Cefixime trihydrate and Erdosteine

Figure 3. Calibration Curve for Cefixime trihydrate

1200000

 

 

 

y = 44216x + 24792

 

 

 

 

 

 

 

1000000

 

 

 

R2

= 0.9982

 

 

 

 

 

800000

600000

400000

200000

0

0

10

20

30

Madhura V. Dhoka et al /Int.J. ChemTech Res.2010,2(1)

86

Figure 4. Calibration Curve for Erdosteine

350000

 

 

 

y = 9216.7x - 1032.3

 

 

 

 

300000

 

 

 

R2 = 0.9979

250000

 

 

 

 

200000

 

 

 

 

150000

 

 

 

 

100000

 

 

 

 

50000

 

 

 

 

0

 

 

 

 

0

10

20

30

40

ACKNOWLEDGEMENTS

The authors are grateful to Dr. A.R.Madgulkar, Principal, AISSMS College of Pharmacy, Pune.and Dr. K.G. Bothara for continuous support and guidance.

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