STABILITY INDICATING RP-HPLC METHOD FOR COMBINATION OF AMBROXOL HYDROCHLORIDE AND LEVOFLOXACIN HEMIHYDRATE IN PHARMACEUTICAL FORMULATION

STABILITY INDICATING RP-HPLC METHOD FOR COMBINATION OF AMBROXOL HYDROCHLORIDE AND LEVOFLOXACIN HEMIHYDRATE IN PHARMACEUTICAL FORMULATION

J. A. Goswami ^{* 1} and N. J. Shah ²

Department of Quality Assurance ¹, School of Pharmacy, RK University, Rajkot - 360020, Gujarat, India.

Indubhai Patel College of Pharmacy and Research Centre ², Dharmaj - 388430, Gujarat, India.

ABSTRACT: Ambroxol hydrochloride (AMB) and Levofloxacin hemihydrate (LVF) in combination were separated using Reverse-Phase - High-Performance Liquid Chromatographic (RP-HPLC) method. Mobile phase acetonitrile and 0.05 M potassium di-hydrogen orthophosphate buffer (pH 7.0 adjusted with sodium hydroxide solution) (50: 50, v/v) was selected for this chromatographic method. The separation was achieved in Zorbax Eclipse XDB -C18 column with (250 × 4.5 mm i.d), 5mm particle size with a flow rate of 1.0 ml/min. At 248 nm wavelength, 10 µl of 60 µg/ml Ambroxol hydrochloride and 400 µg/ml Levofloxacin hemihydrate (LVF) was injected for 15 min runtime, and an individual peak was obtained for LVF at retention time 2.61 min and for AMB at retention time 7.69 min. Linearity was achieved for Ambroxol hydrochloride in the range of 48 mcg/ml to 72 mcg/ml and Levofloxacin hemihydrate in the range of 320 mcg/ml to 480 mcg/ml. For stress degradation, AMB and LVF were subjected to acid hydrolysis, base hydrolysis, thermal degradation, UV light degradation, oxidation and analyzed with this chromatographic method. The results obtained with this method are useful for assay of this pharmaceutical formulation; hence this method can be used in the pharmaceutical industry.

Ambroxol hydrochloride (AMB), Levofloxacin hemihydrate (LVF), Stability indicating method, Assay, Reverse-Phase - High-Performance Liquid Chromatography (RP - HPLC)

INTRODUCTION: Ambroxol hydrochloride and Levofloxacin hemihydrate in combination used for the treatment and relief of symptoms of both upper and lower respiratory tract infections. Ambroxol hydrochloride is a mucolytic agent. Its IUPAC name is Trans- 4- [(2-amino-3, 5-dibromo benzyl) amino] cyclohexanol HCl Fig. 1 ^{1, 2}. Levofloxacin hemihydrate is a fluoroquinolone antibacterial agent. Its IUPAC name is t(-)-(S)-9 fluoro-2, 3- dihydro- 3- Methyl- 10- (4-methyl piperazine-1yl) -7-oxo -7H-pyrido [1, 2, 3 de]-1, 4-benzoxazine-6-carboxylic acid hemihydrates Fig. 2 ^{3, 4}.

This marketed formulation is available in 75 mg of Ambroxol hydrochloride and 500 mg of Levofloxacin hemihydrate dose.

To develop such an accurate, sensitive, rapid, precise and economical method for routine analysis is inevitable of Ambroxol hydrochloride and Levofloxacin hemihydrate combination in their pharmaceutical dosage form successfully.

MATERIALS AND METHODS:

Instrumentation: Agilent technologies 1260 infinity module HPLC system with photodiode array detector was used. Analytical balance Mettler Toledo model MS105DU was used to weigh chemicals. pH meter Systronics- model µpH system 361 was used to measure pH.

Reagents and Chemicals: Acetonitrile and methanol of HPLC grade, potassium dihydrogen phosphate of analytical reagent grade were procured from Merck Pvt. Ltd., India. Rankem Pharmaceuticals India Ltd. was supplied analytical grade sodium hydroxide, hydroxide peroxide, hydrochloride acid.

Preparation of Buffer: Accurately measured potassium di-hydrogen orthophosphate buffer 6.8 gm was dissolved in 1000 ml of HPLC grade water pH was adjusted to 7 with the help of sodium hydroxide solution.

Mobile Phase Preparation: Mobile phase was prepared by taking equal amount (50:50) of prepared buffer solution and acetonitrile. It was sonicated for 20 min to degas in the mixture mobile phase. The obtained solution was further used in the analysis.

Standard Preparation: Weighed accurately 75 mg of Ambroxol hydrochloride and 500 mg of Levofloxacin hemihydrate and transferred into 50 ml volumetric flask. Added 25 ml of mobile phase. Sonicated to dissolve. Made volume up to the mark with mobile phase and mixed. (1500 µg/ml AMB and 10000 µg/ml LVF). Pipetted 10 ml of above solution into 100 ml volumetric flask, made the volume up to the mark with mobile phase and mixed well. (150 µg/ml AMB and 1000 µg/ml LVF). Further pipetted 10 ml of the above solution into 25 ml volumetric flask, made volume to the mark with mobile phase and mixed well. (60 µg/ml AMB and 400 µg/ml LVF).

Sample Preparation: Weighed twenty tablets and crushed them into a fine powder with the help of mortar and pestle. Weighed and transferred sample powder equivalent to 75 mg AMB and 500 mg LVF into 50 ml volumetric flask. Added 10 ml mobile phase and sonicated for 10 min with continuous shaking. Made volume up to the mark with mobile phase and mixed well and filtered through Whatmann filter paper (0.45 µ). (1500 µg/ml AMB and 10000 µg/ml LVF). Further pipetted 10 ml of above filtrate into 100 ml volumetric flask, made volume up to the mark with mobile phase and mixed well. (150 µg/ml AMB and 1000 µg/ml LVF). Further pipetted 10 ml of the above solution into 25 ml volumetric flask, made volume up to the mark with mobile phase and mixed well. (60 µg/ml AMB and 400 µg/ml LVF).

Wavelength Selection: Solution of 60 µg/ml AMB and 400 µg/ml LVF were scanned in the UV region of 200-400 nm at room temperature, and the obtain spectra was by software Fig. 3.

FIG. 3: OVERLAY ZERO ORDER UV SPECTRUM OF AMB AND LVF IN DILUENTS

Chromatography Method Development: Various trials were made with reference of methods used for the assay of anti-histamine drugs. In each trial peak shape, the resolution between two components and tailing factor were observed. Buffer and acetonitrile in different proportions were tried and finally 0.05 M potassium dihydrogen orthophosphate with pH 7 adjusted with sodium hydroxide solution and acetonitrile in the ratio of 50: 50% v/v selected with better peak shape and resolution. In the mobile phase both the drugs were found to be soluble and stable, so the mobile phase is selected as a diluent. Chromatographic method selected with a Eclipse XDB column - C18, (250 × 4.6 mm i.d) and particle size 5 mm. AMB and LEV peak is obtained at 7.69 min and 2.61 min respectively with a flow rate of 1.0 ml/min and injection volume 10 µl for 15 min run time. The method was further validated under the chromatographic conditions.

Method Validation: The established chromato-graphic method was validated in compliance with ICH guidelines. The parameters like system suitability along with precision, linearity, specificity, and accuracy, LOQ and LOD were performed for validation.

Forced Degradation Studies (FDS):

Degradation with 3% H₂O₂: Accurately weighed 1118 mg of sample powder and transferred into 50 ml volumetric flask. Added 10 ml mobile phase and sonicated for 10 min with continuous shaking. Added 5.0 ml 3% v/v hydrogen peroxide to the flask. Kept the volumetric flask at 60 °C for 12 h. After the specified time remove the flask from the water bath and allow to cool the flask at room temperature. Made volume up to the mark with mobile phase and mixed well. Filtered the solution with 0.45 µm PVDF filter. Further pipetted 10 ml of above filtrate into 100 ml volumetric flask, made volume up to the mark with mobile phase and mixed well. Further dilution was made by taking 10 ml previous solution to 25 ml with mobile phase. (400 µg/ml LVF and 60 µg/ml AMB).

FIG. 4: CHROMATOGRAPH OF FDS ON SAMPLE SOLUTION CONTAINING AMBROXOL HYDRO-CHLORIDE AND LEVOFLOXACIN HEMIHYDRATE USING 3% HYDROGEN PEROXIDE SOLUTION

Degradation with 0.1M HCl: Accurately weighed 1118 mg of sample powder and transferred into 50 ml volumetric flask added 10 ml mobile phase and sonicated for 10 min with continuous shaking. Added 5.0 ml 0.1 M hydrochloric acid to the volumetric flask. Store flask at 60 °C for 12 h. After the specified time remove the flask from the water bath and allow to cool the flask at room temperature. Added 5 ml 0.1 M sodium hydroxide. Made volume up to the mark with mobile phase and mixed well. Filtered the solution with 0.45 µm PVDF filter. Further pipetted 10 ml of above filtrate into 100 ml volumetric flask, made volume to the mark with mobile phase and mixed well. Further dilution was made by taking 10 ml previous solution to 25 ml with mobile phase. (400 µg/ml LVF and 60 µg/ml AMB).

FIG. 5: CHROMATOGRAPH OF FDS ON SAMPLE SOLUTION CONTAINING AMBROXOL HYDRO-CHLORIDE AND LEVOFLOXACIN HEMIHYDRATE USING 0.1 M HCl SOLUTION

Degradation with 0.1M NaOH: Accurately weighed 1118 mg of sample powder and transferred into 50 ml volumetric flask added 10 ml mobile phase and sonicated for 10 min with continuous shaking. Added 5 ml 0.1 M sodium hydroxide to the volumetric flask. Store flask at 60°C for 12 h. After the specified time remove the flask from the water bath and allow to cool the flask at room temperature. Added 5 ml 0.1 M hydrochloric acid. Made volume up to the mark with mobile phase and mixed well. Filtered the solution with 0.45 µm PVDF filter. Further pipetted 10 ml of above filtrate into 100 ml volumetric flask, made volume up to the mark with mobile phase and mixed well. Further dilution was made by taking 10 ml previous solution to 25 ml with mobile phase.  (400 µg/ml LVF and 60 µg/ml AMB).

FIG. 6: CHROMATOGRAPH OF FDS ON SAMPLE SOLUTION CONTAINING AMBROXOL HYDRO-CHLORIDE AND LEVOFLOXACIN HEMIHYDRATE USING 0.1 M NaOH SOLUTION

Exposed to Heat: Accurately weighed 1118 mg of sample powder and transferred into 50 ml volumetric flask Added 10 ml Mobile phase and sonicated for 10 min with continuous shaking. Volumetric flask exposed under heat at 80 ºC for 12 h. After the specified time remove the flask from heat and allow to cool the flask at room temperature. Made volume up to the mark with mobile phase and mixed well. Filtered the solution with 0.45 µm PVDF filter. Further pipetted 10 ml of above filtrate into 100 ml volumetric flask, made volume up to the mark with mobile phase and mixed well. Further dilution was made by taking 10 ml previous solution to 25 ml with mobile phase.  (400 µg/ml LVF and 60 µg/ml AMB).

FIG. 7: CHROMATOGRAPH OF FDS ON SAMPLE SOLUTION CONTAINING AMBROXOL HYDRO-CHLORIDE AND LEVOFLOXACIN HEMIHYDRATE USING HEAT

Exposed to UV Light: Accurately weighed 1118 mg of sample powder and transferred into 50 ml volumetric flask added 10 ml mobile phase and sonicated for 10 min with continuous shaking. Volumetric flask exposed for UV radiation for 12 h. After the specified time made volume up to the mark with mobile phase and mixed well. Filtered the solution with 0.45 µm PVDF filter. Further pipetted 10 ml of above filtrate into 100 ml volumetric flask, made volume up to the mark with mobile phase and mixed well. Further dilution was made by taking 10 ml previous solution to 25 ml with mobile phase. (400 µg/ml LVF and 60 µg/ml AMB).

FIG. 8: CHROMATOGRAPH OF FDS ON SAMPLE SOLUTION CONTAINING AMBROXOL HYDRO-CHLORIDE AND LEVOFLOXACIN HEMIHYDRATE USING UV LIGHT

RESULTS AND DISCUSSION:

System Suitability: The system suitability was performed on a standard solution (60 µg/ml AMB and 400 µg/ml LVF).

TABLE 1: SYSTEM SUITABILITY TEST PARAMETER

All system suitability parameters were obtained by six repeated injections, and all the parameters were found to be within specified limits.

Specificity: Specificity was performed by determining peak purity. There were no peaks observed to interfere with our analytes.

Linearity: Linearity was developed by generating graph between analyte sample concentration versus average peak area of the analytes. Data as per Table 2 and graphically represented in Graph Fig. 9 and 10 indicate that the linearity for AMB was 48 to 72 (µg/ml) and for LVF was 320 to 480 (µg/ml).

TABLE 2: LINEARITY

FIG. 9: CALIBRATION CURVE OF LVF AT 248 nm

FIG. 10: CALIBRATION CURVE OF AMB AT 248 nm

Accuracy: Accuracy (recovery) study was performed on a known amount of placebo by spiking in API. The Samples were prepared by adding 80% to 120% of the sample concentration. As per the data, it was indicated that the method has an acceptable level of accuracy.

Precision:

System Precision: The standard solution of AMB (60 ppm) and LVF (400 ppm) were injected into the HPLC system with the same condition for six times. The % Relative standard deviation for six samples was found to be in the limits.

Method Precision: By analyzing assay for six individual samples prepared from the same batch precision test was evaluated for the proposed method. The average % assay and the % RSD (Relative Standard Deviation) for the six sample preparation were found to be in the specified limits.

Intermediate Precision (Ruggedness): Intermediate precision of the method was performed on various HPLC, columns, and analyst on different days. Six samples of the standard solution of AMB (60 ppm) and LVF (400 ppm of the same batch were prepared and analyzed. The mean, SD, and % relative standard deviation for the two sets of data are shown in Table 5.

TABLE 3: ACCURACY

TABLE 4: METHOD PRECISION

TABLE 5: INTERMEDIATE PRECISION

Robustness: Robustness of the method was investigated Table 6 by varying the analytical estimation operating conditions such as flow rate of mobile phase (± 0.2 ml/min), column oven temperature (± 2% ºC), and pH of buffer (0.2). The standard solution of AMB (60 ppm) and LVF (400 ppm) was prepared as per the standard method described above and estimated as per the developed procedure.

Stability of Sample Solution: The Prepared sample solution was stable up to 24 h. The Data for Forced degradation are tabulated in Table 7.

From blank and placebo, the interference was negligible in the analyte peaks, Peak purity of all FD samples was obtained and found in the specified limit. The obtained data reveal that the developed method was highly specific and stability indicating for the simultaneous estimation of Ambroxol hydrochloride and Levofloxacin hemihydrate, in their marketed tablet formulation.

Forced Degradation Study: For this chromatographic method following data were obtained for forced degradation study.

TABLE 6: ROBUSTNESS STUDY FOR AMB AND LVF

TABLE 7: SOLVENT STABILITY

TABLE 8: SUMMARY OF VALIDATION PARAMETERS OF RP-HPLC

TABLE 9: ANALYSIS OF MARKETED TABLET FORMULATION

TABLE 10: FD STUDY

CONCLUSION: It is concluded that the developed stability indicating analytical RP-High Performance Liquid Chromatography method is highly fast, very sensitive, enough economic and reliable and is complying with all validation parameter ad per ICH guidelines. So, for routine estimation of tablets containing AMB and LVF, this method can be employed. Due to the reduction of the cost of analysis and time and more effective than reported analytical methods may be replaced by our developed stability indicating Reverse-phase high-performance liquid chromatography method.

This validated method can be used for faster samples testing routinely in QC lab.

ACKNOWLEDGEMENT: The authors highly appreciate the support from the management of AARTI, Ahmedabad, Gujarat, India for providing Analytical instrumentation facility for the analytical work.

CONFLICT OF INTEREST: Nil

REFERENCES:

1. Indian pharmacopeia, - Ambroxol hydrochloride, Govt. of India, “Ministry of Health and Family Welfare.” Controller and publication, Delhi 2007; 2: 83.
2. British pharmacopeia, - Ambroxol hydrochloride, British Pharmacopoeia Volume I and II Monographs, Medicinal and Pharmaceutical Substances, Ph Eur Monograph 2009, 1489.
3. http://www.chemicalland21.com/lifescience/phar/AMBROXOL%20HYDROCHLORIDE.htm Chemicalland21 – Ambroxol Hydrochloride;[cited 2018 Jan 28].
4. Indian Pharmacopoeia, - Levofloxacin hemihydrate, Govt. of India, “Ministry of Health and Family Welfare.” Controller and Publication, Delhi, Vol. 2, 2007: 678-80.
5. United State Pharmacopoeia, - Levofloxacin hemihydrate, United States Pharmacopeial Convention, 2018: 2395-2397.
6. http://www.nexuspharma.net/Levo%20MSDS.pdf. Pack Pharmaceuticals LLC, Material Safety Data Sheet, Levofloxacin;[cited 2012 Oct 2].
7. https://pubchem.ncbi.nlm.nih.gov/compound/levofloxacin#sectionChemical-and-Physical-Properties PubChem Open chemistry Database, Levofloxacin;[cited 2018 Jan 28].
   

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   How to cite this article:

   Goswami JA and Shah NJ: Stability indicating RP-HPLC method for combination of Ambroxol hydrochloride and Levofloxacin hemihydrate in pharmaceutical formulation. Int J Pharm Sci & Res 2019; 10(1): 356-62. doi: 10.13040/IJPSR.0975-8232.10(1).356-62.

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