International Journal of Chromatography and Separation Techniques (ISSN: 2577-218X)

Article / research article

"Validated Stability Indicating RP-HPLC Method for Simultaneous Determination of Cefixime and Acetylcysteine in Pharmaceutical Dosage Form"

A. Lakshmana Rao*, T. Prasanthi, V. Aswini

Department of Pharmaceutical Analysis, VV Institute of Pharmaceutical Sciences, Gudlavalleru, Andhra Pradesh, India

*Corresponding author: A. Lakshmana Rao, Department of Pharmaceutical Analysis, VV Institute of Pharmaceutical Sciences, Gudlavalleru, Andhra Pradesh 521356, India. Tel: +919848779133; +918674274649; Email: dralrao@gmail.com

Received Date: 01 July, 2018; Accepted Date: 30 July, 2018; Published Date:  03 August, 2018

1.                   Abstract

A simple stability indicating RP-HPLC method has been developed for the simultaneous determination of Cefixime in combination with Acetylcysteine using ODS C18 column (250 × 4.6 mm, 5 μm) with UV detection at 274 nm. The mobile phase consisting of 0.1% Ortho Phosphoric Acid (OPA) and acetonitrile in a ratio of 58:42, v/v and at a flow rate of 1.0 mL/min. The method was linear over the concentration range for Cefixime 50-375 μg/mL and for Acetylcysteine 75-400 μg/mL. The retention times for Cefixime and Acetylcysteine were found to be 2.018 and 5.141 min respectively. The average percentage recoveries of Active Pharmaceutical Ingredient (API) Cefixime and Acetylcysteine were found to be in the range of 99.23% and 100.13% respectively. %RSD of the Cefixime and Acetylcysteine were found to be 0.9 and 0.8 respectively. %Assay obtained as 99.23% and 100.13% for Cefixime and Acetylcysteine respectively. The method was validated and was successfully employed for the routine quantitative analysis of pharmaceutical formulations containing Cefixime and Acetylcysteine in combined tablet dosage form.

2.                   Keywords: Acetylcysteine; Cefixime; HPLC; Validation

1.                   Introduction

Cefixime (Figure 1), an antibiotic, is a third-generation oral bactericidal cephalosporin. Cefixime is chemically known as (6R,7R)-7-[(2Z)-2-(2-amino-1,3-thiazol-4-yl)-2-[(carboxymethoxy) imino] acetamido]-3-ethenyl-8-oxo-5-thia-1-azabicyclooct-2-ene-2-carboxylic acid [1]. The antibacterial effect of Cefixime results from inhibition of mucopeptide synthesis in the bacterial cell wall. Cefixime is extremely stable in presence of β-lactamase enzymes. Cefixime is used in the treatment of uncomplicated urinary tract infections caused by Escherichia coli and Proteus mirabilis, otitis media caused by Haemophilus influenzae, pharyngitis and tonsillitis caused by S. pyogenes and uncomplicated gonorrhea (cervical/urethral) caused by Neisseria gonorrhoeae etc.

Acetylcysteine (Figure 2), is primarily used as a mucolytic agent and in the management of acetaminophen poisoning. It is chemically known as (2R)-2-acetamido-3-sulfanylpropanoic acid [2]. It is a derivative of cysteine with an acetyl group attached to the amino group of cysteine. NAC is essentially a prodrug that is converted to cysteine (in the intestine by the enzyme aminoacylase 1) and absorbed in the intestine into the blood stream. Acetylcysteine can also be used as a general antioxidant which can help mitigate symptoms for a variety of diseases exacerbated by Reactive Oxygen Species (ROS). N-acetylcysteine is now widely used in the treatment of HIV. Acetylcysteine is also being successfully used to treat a variety of neuropsychiatric and neurodegenerative disorders including cocaine, cannabis, and smoking addictions, Alzheimer's and Parkinson's diseases, autism, compulsive and grooming disorders, schizophrenia, depression, and bipolar disorder.

Cefixime is an antibiotic which kills bacteria by preventing them from forming their own protective covering which is required for their survival in the human body. Acetylcysteine is a mucolytic which thins and loosens mucus (phlegm) making it easier to cough out. The combination of these two drugs can be used effectively to treat respiratory tract infection. In literature there are various methods were reported for individual estimation of Cefixime and Acetylcysteine in pharmaceutical dosage forms using UV Spectrophotometry [3-5], HPTLC [6,7] and HPLC [8,9], but very few HPLC methods [10,11] were reported for simultaneous estimation of these drugs in combined dosage form. The main objective of the present work is to develop, and valid a simple, sensitive, rapid, accurate and precise method developed for determination of Cefixime and Acetylcysteine in combined dosage form.

2.                   Materials and Methods

2.1                Materials

Cefixime and Acetylcysteine Pure Drugs (API) are procured from Spectram Labs, Hyderabad, Combination of Cefixime and Acetylcysteine tablets (Mucomelt Forte bearing the label claim Cefixime 200 mg, Acetylcysteine 300 mg) were procured from local market. Distilled water, Acetonitrile, Methanol, Potassium dihydrogen ortho phosphate, OPA is purchased from Rankem Chemicals Ltd., Mumbai, India.

2.2                Instrumentation

The analysis of drugs was carried out on a Waters HPLC system on an ODS C18 column (250 x 4.6 mm, 5 µm). The instrument is equipped with a 2695 pump with inbuilt degasser, 2998 photodiode array detector and a Rheodyne injector with 20 µL sample loop. A 20 µL Hamilton syringe was used for injecting the samples. Data was analysed by using Waters Empower 2 software. A double-beam Shimadzu UV-Visible 2450 spectrophotometer was used for spectral studies. Degassing of the mobile phase was done by using an ultrasonic bath sonicator. A Shimadzu balance was used for weighing the materials.

2.3                Mobile Phase

A mobile phase consisting of mixture of 0.1% OPA and acetonitrile in the ratio of 58:42, v/v was prepared.

2.4                Diluent

Based up on the solubility of the drugs, diluent was selected, Acetonitrile and Water taken in the ratio of 50:50, v/v.

2.4.1           Preparation of Standard Stock Solutions

Accurately weighed 20 mg of Cefixime, 30 mg of Acetylcysteine and transferred to 10 mL volumetric flask. Add 3/4thof diluent and sonicated for 10 minutes. Flasks were made up with diluent and labeled as standard stock solution (2000 µg/mL of Cefixime and 3000 µg/mL of Acetylcysteine).

2.4.2           Preparation of Standard Working Solution (100% solution)

1 mL from each stock solution was pipetted out and taken into a 10mL volumetric flask and made up with diluent (200 µg/mL of Cefixime and 300 µg/mL of Acetylcysteine).

2.4.3           Preparation of Sample Stock Solutions

5 tablets were weighed, and the average weight of each tablet was calculated, then the weight equivalent to 1 tablet was transferred into a 100 mL volumetric flask, 50 mL of diluent was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC filters (2000 µg/mL of Cefixime and 3000 µg/mL of Acetylcysteine).

2.4.4           Preparation of Sample Working Solutions (100% solution): 1mL of filtered sample stock solution was transferred to 10 mL volumetric flask and made up with diluent (200 µg/mL of Cefixime and 300 µg/mL of Acetylcysteine).

3.                   Results and Discussion

3.1                Determination of λmax and Optimized wavelength

λmax of Cefixime and Acetylcysteine was 279.1 nm and 246.0 nm respectively. Overlay spectra (Figure 3) gave the 274 nm as the optimized wavelength for these two drugs.

3.2                Method development

Various trails were performed by using different mobile phases and based on peak parameters the following chromatographic conditions (Table 1) were optimized and optimal chromatogram was shown in (Figure 4).

3.3                Validation

3.3.1           System Suitability Parameters

The system suitability parameters were determined by preparing standard solutions of Cefixime (200 µg/mL) and Acetylcysteine (300 µg/mL) and the solutions were injected six times and the parameters like peak tailing, resolution and USP plate count were determined. All the system suitability parameters (Table 2) were within the range and satisfactory as per ICH guidelines [12].

3.4                Specificity

Specificity is the parameter used to check the interference in the optimized method. We should not find interfering peaks in blank (Figure 5), placebo (Figure 6), standard (Figure 7) and sample (Figure 8) at retention times of these drugs in this method. So, this method was said to be specific.

3.5                Linearity

Accurately weighed 20 mg of Cefixime, 30 mg of Acetylcysteine and transferred to 10 mL volumetric flask. Add 3/4th of diluent to these flasks and sonicated for 10 minutes. Flasks were made up with diluent and labeled as standard stock solution (2000 µg/mL of Cefixime and 3000 µg/mL of Acetylcysteine). From the above solutions transfer 0.25,0.5,0.75,1,1.25 and 1.5 mL to 10 mL volumetric flask and diluted to mark with diluent to prepare 50,100,150,200,250,300 µg/mL solutions of Cefixime and 75, 150, 225, 300, 375, 400 µg/mL solutions of Acetylcysteine. The results were furnished in (Table 3) and calibration curves were shown in (Figure 9&10).

3.6                Precision

Precision of method was studied by performing intra-day and inter-day precision. Intra-day precision (Table 4) and inter-day precision (Table 5) was studied by injecting the 6 replicates of standard solution in a single day and six days. Calculate the %RSD and it should not be more than 2.0.

3.7                Accuracy

The accuracy of the method was established by calculating percentage recovery of Cefixime and Acetylcysteine by the method of addition. Known amount of Cefixime and Acetylcysteine at 50%, 100%, and 150% was added to a pre-quantified sample solution. The recovery studies (Tables 6,7) were carried out in the tablet in triplicate each in the presence of placebo. The mean percentage recovery at each level was not less than 99% and not more than 101.

3.8                Sensitivity

Limit of Detection was calculated by intercept method and LOD for Cefixime and Acetylcysteine were found to be 0.50 and 1.53 µg/ml respectively.

Limit of Quantification was calculated by intercept method and LOQ for Cefixime and Acetylcysteine were found to be 0.49 and 1.47 µg/ml respectively.

3.9                Robustness

Small deliberate changes in method like flow rate, mobile phase ratio and temperature are made but there was no recognized change in the result and are within range as per ICH Guide lines. Robustness conditions like flow minus (0.9 mL/min), flow plus (1.1 mL/min), mobile phase minus, mobile phase plus, temperature minus (25°C) and temperature plus (35°C) was maintained and samples were injected in duplicate manner. System suitability parameters were not much effected, and all the parameters were passed. %RSD was within the limit. The results were shown in (Table 8).

3.10             Assay

Mucomelt Forte, bearing the label claim Cefixime 200 mg, Acetylcysteine 300 mg. Assay was performed with the above formulation. Average % Assay for Cefixime (Table 9) and Acetylcysteine (Table 10) obtained was 99.23 and 100.13%respectively.

3.10.1        Degradation Studies

3.10.2        Acid Degradation Studies

To 1 mL of stock solution Cefixime and Acetylcysteine 1mL of 2N Hydrochloric acid was added and refluxed for 30 mins at 60ºC. The resultant solution was diluted to obtain 200 µg/mL & 300 µg/mL solution and 10µL solutions into the system and the chromatograms were recorded to assess the stability of sample.

3.10.3        Alkali Degradation Studies

To 1 mL of stock solution Cefixime and Acetylcysteine, 1 mL of 2N sodium hydroxide was added and refluxed for 30 min at 60ºC. The resultant solution was diluted to obtain 200 µg/mL & 300 µg/mL solution and 10 µL were injected into the system and the chromatograms were recorded to assess the stability of sample.

3.11             Oxidation

To 1 mL of stock solution of Cefixime and Acetylcysteine, 1 mL of 20% hydrogen peroxide (H2O2) was added separately. The solutions were kept for 30 min at 60ºC. For HPLC study, there resultant solution was diluted to obtain 200 µg/mL & 300 µg/mL solution and 10µL were injected into the system and the chromatograms were recorded to assess the stability of sample.

3.11.1        Dry Heat Degradation Studies

The standard drug solution was placed in oven at 105°C for 1h to study dry heat degradation. For HPLC study, the resultant solution was diluted to 200 µg/mL & 300 µg/mL solution and 10 µL were injected into the system and the chromatograms were recorded to assess the stability of the sample.

3.11.2        Photo Stability Studies

The photochemical stability of the drug was also studied by exposing the 2000 µg/mL & 3000 µg/mL solution to UV Light by keeping the beaker in UV Chamber for 1day or 200-Watt hours/m2 in photo stability chamber. For HPLC study, the resultant solution was diluted to obtain 200 µg/mL & 300 µg/mL solutions and 10 µL were injected into the system and the chromatograms were recorded to assess the stability of sample.

3.11.3        Neutral Degradation Studies

Stress testing under neutral conditions was studied by refluxing the drug in water for 1 hr at a temperature of 60ºC. For HPLC study, the resultant solution was diluted to 200 µg/mL & 300 µg/mL solution and 10 µL were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Degradation studies results were computed in (Table 11,12).

4.                   Discussion

A stability indicating RP-HPLC method was developed for the simultaneous estimation of Cefixime and Acetylcysteine by using mobile phase consisting of 0.1% OPA and Acetonitrile in the ratio of 58:42, v/v. Retention times for Cefixime and Acetylcysteine were found to be 2.018 and 5.141 min respectively. The method was validated as per ICH guidelines. Linearity range was found to be 50-300 µg/mL for Cefixime and 75-400 µg/mL for Acetylcysteine. Mean recoveries were found to be satisfactory. This method also passed the test for repeatability, intermediate precision and robustness for both drugs.

Degradation studies are also carried out in acid, base, peroxide, heat, UV light and neutral stressed conditions. The results revealed that both the drugs are stable in described conditions. Thus, it is evident that the described method can be adopted for routine estimation of Cefixime and Acetylcysteine in combined dosage form.

5.                   Conclusion

The present method was proposed for the simultaneous estimation of the Cefixime and Acetylcysteine by using RP-HPLC in tablet dosage form is found to be simple, accurate, rapid and precise. Retention times were decreased, and the run time was decreased. The method developed was simple and economical therefore can be applied in regular quality control tests in industries.


Figure 1: Structure of Cefixime.





Figure 2: Structure of Acetylcysteine.




Figure 3: Overlay spectra of Cefixime and Acetylcysteine.




Figure 4: Optimized Chromatogram of Cefixime and Acetylcysteine.




Figure 5: Chromatogram of blank.




Figure 6: Chromatogram of placebo.




Figure 7: Chromatogram of working standard solution.




Figure 8: Chromatogram of working sample solution.





Figure 9: Calibration curve of Cefixime.




Figure 10: Calibration curve of Acetylcysteine.


Mobile phase

OPA (0.1%): Acetonitrile 58:42(v/v)

Flow rate

 1 mL/min

Column

 ODS C18 (250 x 4.6 mm, 5 µm)

Detector wave length

  274 nm

Column temperature

30°C

Injection volume

 10 µL

Run time

 8 min

Diluent  

Water: acetonitrile in the ratio 50:50(v/v)

Retention time

Cefixime-----2.018 min Acetylcysteine----5.141 min

Table 1: Optimized Chromatographic conditions.


S.No.

 

Cefixime

Acetylcysteine

 

Resolution

RT (min)

USP Plate Count

Tailing

RT (min)

USP Plate Count

Tailing

1

2.017

3478

1.04

5.096

9415

0.98

17.6

2

2.017

3486

1.04

5.106

9656

0.97

17.6

3

2.018

3555

1.04

5.123

9725

0.98

17.7

4

2.02

3641

1.05

5.126

9677

0.97

17.6

5

2.021

3590

1.06

5.135

9512

0.97

18

6

2.028

3735

1.04

5.141

9650

0.97

17.7

Table 2: System suitability parameters for Cefixime and Acetylcysteine.


Cefixime

Acetylcysteine

Conc (μg/mL)

Peak area

Conc (μg/mL)

Peak area

0

0

0

0

50

90458

75

243098

100

157126

150

427624

150

236651

225

645398

200

312067

300

872071

250

386173

375

1065936

300

462348

450

1284061

Slope

1523

Slope

2829.7

Intercept

6394

Intercept

11637

R2

0.999

R2

0.9994

Table 3: Linearity results for Cefixime and Acetylcysteine.


S. No.

Area of Cefixime

Area of Acetylcysteine

1

308617

871018

2

307253

866153

3

309971

878547

4

304898

868728

5

309887

872930

6

309719

877729

Mean

308391

872518

S. D

2002.3

4915.3

%RSD

0.6

0.6

Table 4: Intra-day Precision results of Cefixime and Acetylcysteine.


S. No.

Area of Cefixime

Area of Acetylcysteine

1

325919

924692

2

326311

929501

3

328279

923486

4

326918

934770

5

323091

915533

6

331546

934992

Mean

327011

927162

S.D

2801.1

7476.8

%RSD

0.9

0.8

Table 5: Inter-day Precision results of Cefixime and Acetylcysteine.


% Level

Amount Spiked (μg/mL)

Amount recovered (μg/mL)

% Recovery

Mean % Recovery

50%

100

100.38

100.38

99.67%

100

100.17

100.17

100

99.58

99.58

100%

200

198.86

99.43

200

199.07

99.53

200

200.1

100.05

150%

300

300.11

100.04

300

296.2

98.73

300

297.27

99.09

Table 6: Accuracy results of Cefixime.


% Level

Amount Spiked (μg/mL)

Amount recovered (μg/mL)

% Recovery

Mean % Recovery

50%

150

150.08

100.05

99.66%

150

151.14

100.76

150

148.01

98.67

100%

300

297.8904

99.3

300

297.7296

99.24

300

297.275

99.09

150%

450

448.6034

99.69

450

447.1729

99.37

450

453.4871

100.77

Table 7: Accuracy results of Acetylcysteine.


S. No.

Condition

%RSD of Cefixime

%RSD of Acetylcysteine

1

Flow rate (-) 1.1mL/min

1

0.7

2

Flow rate (+) 1.3mL/min

0.6

0.3

3

Mobile phase (-) 35B:65A

1

0.6

4

Mobile phase (+) 45B:55A

0.9

0.6

5

Temperature (-) 25°C

0.8

0.5

6

Temperature (+) 35°C

0.5

0.5

Table 8: Robustness data for Cefixime and Acetylcysteine.


S. No.

Standard area

Sample area

% Assay

1

309272

308617

99.3

2

309026

307253

98.86

3

309782

309971

99.74

4

310161

304898

98.11

5

311398

309887

99.71

6

311342

309719

99.66

Avg

310164

308391

99.23

St.dev

1014.6

2002.3

0.644

%RSD

0.3

0.6

0.65

Table 9: Assay Data of Cefixime.


S. No.

Standard area

Sample area

% Assay

1

867368

871018

99.95

2

869486

866153

99.4

3

871302

878547

100.82

4

874116

868728

99.69

5

870462

872930

100.17

6

870588

877729

100.72

Avg

870554

872518

100.13

St.dev

2215.8

4915.3

0.564

%RSD

0.3

0.6

0.56

Table 10: Assay Data of Acetylcysteine.


S. No.

Degradation Condition

% Drug Degraded

Purity Angle

Purity Threshold

1

Acid

3.34

0.832

1.398

2

Alkali

2.97

1.041

1.492

3

Oxidation

2.31

0.877

1.404

4

Thermal

1.68

0.041

0.492

5

UV

0.83

0.819

1.384

6

Neutral

0.05

0.169

0.094

Table 11: Degradation Data of Cefixime.


S. No.

Degradation Condition

%Drug Degraded

Purity Angle

Purity Threshold

1

Acid

4.72

0.115

0.286

2

Alkali

3.45

0.079

0.295

3

Oxidation

1.67

0.112

0.294

4

Thermal

1.16

0.079

0.295

5

UV

0.95

0.1

0.275

6

Neutral

0.59

0.094

0.279

Table 12: Degradation Data of Acetylcysteine.

1.       Indian Pharmacopoeia (2014) Indian Pharmacopoeial Commission, Controller of Publication, Government of India, Ministry of health and Family Welfare, Ghaziabad, India. 1012.

2.       The United States Pharmacopoeia (2014) National Formulary. Asian Edition. Rockville, MD: United States Pharmacopoeia Convention, Inc. 1608.

3.       Suddhasattya D, Prasanna KP, Upadhayay UM, Shah S, Goswami K (2012) UV Spectrophotometric Determination of Cefixime in Bulk and its Dosage Form. J Pharm Res 5: 5419-5422.

4.       Nayon AU, Nesa JU, Uddin N, Shah A, Bushra U (2013) Development and validation of UV Spectrometric Method for the Determination of Cefixime trihydrate in Bulk and Pharmaceutical Formulation. Asian J Biomed Pharm Sci 3: 1-5.

5.       Toussaint B, Pitti C, Streel B, Ceccato A, Hubert P, et al. (2000) Quantitative analysis of N-acetylcysteine and its pharmacopeial impurities in a pharmaceutical formulation by liquid chromatography-UV detection-mass spectrometry. J Chromat. Analysis 896: 191-199.

6.       Madhura VD, Vandana TG, Pranav PJ (2013) Validated HPTLC Method for Determination of Cefixime Trihydrate and Erdosteine in Bulk and combined Pharmaceutical Dosage Form. Eurasian J Ana Chem 8: 99-106.

7.       Nanda RK, Bhagwat VV, Potawale SE, Hamane SC (2010) Development and Validation of A HPTLC Method for Simultaneous Densitometric Analysis of Cefixime and Potassium Clavulanate as the Bulk Drugs and in the Tablet Dosage Form. Asian J Res Chem 3: 998-1001.

8.       Shaikh S, Rajani A, Pravin P, Nadkar S, Naik S (2012) Development and Validation of RP-HPLC Method for the Estimation of N-Acetylcysteine in Wet Cough Syrup. Int J Drug Dev Res 4: 284-293.

9.       Talebpour Z, Rafati H, Abdollahpour A, Pourabdollahi H, Bashour Y (2013) Determination of Cefixime by a validated stability indicating HPLC method and identification of its related substances by LC-MS/MS Studies. Scientia Pharmaceutica 81: 493-496.

10.    Lalit BR, Vasanti S, Shinde NK (2015) Development and validation of a stability indicating RP-HPLC method for simultaneous determination of Acetylcysteine and Cefixime in pharmaceutical formulation. Int J Pharm Sci Res 6: 4886-4894.

11.    Janhavi D, Elizabeth MM, Sudheer M (2016) Analytical Method Development and Validation of RP-HPLC Method for Simultaneous Estimation of N-acetylcysteine and Cefixime from its fixed dose combination. Res J Pharm Tech 9: 542-548.

12.    ICH, Validation of analytical procedures: Text and Methodology (1996) International Conference on Harmonization, IFPMA, Geneva.


Citation: Rao AL, Prasanthi T, Aswini V (2018) Validated Stability Indicating RP-HPLC Method for Simultaneous Determination of Cefixime and Acetylcysteine in Pharmaceutical Dosage Form. Int J Chromatogr Sep Tech: IJCST-117. DOI: 10.29011/2577-218X.000017

free instagram followers instagram takipçi hilesi