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Development and Validation of Simultaneous UV Spectrophotometric Method for the Determination of Levofloxacin and Ambroxol in Tablets
Development and Validation of Simultaneous UV Spectrophotometric Method for the Determination of Levofloxacin and Ambroxol in Tablets
Journal of the Korean Chemical Society. 2008. Dec, 52(6): 622-629
Copyright © 2008, The Korean Chemical Society
  • Received : February 12, 2008
  • Published : December 20, 2008
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Tabassum Patil
Yogesh Pore

Abstract
An accurate, specific and precise UV spectrophotometric method was developed for the simultaneous determination of levofloxacin (LFX) and ambroxol (ABX) in pharmaceutical dosage forms. The method involves formation of Q-absorbance equation at 219 (isoabsorptive point) and at 287 nm, using distilled water as a solvent. The linearity for both levofloxacin and ambroxol was in the range of 2-20 μg/ml and 5-50 μg/ml respectively. The % recovery was found to be 100-101% and 99-102% for levofloxacin and ambroxol respectively indicating proposed method is accurate and precise for simultaneous estimation of levofloxacin and ambroxol in tablets.
Keywords
INTRODUCTION
Levofloxacin hemihydrate (LFX) ( . 1 A) chemically, [(-)(s)-9-fluro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl-7-oxo-7H-pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid is an optically L-isomer of ofloxacin. 1 It is a broad spectrum fluoroquinolone class of antibacterial agent and effective against many gram positive and gram negative bacteria. 2 , 3 It is a potent inhibitor of bacterial DNA gyrase enzyme (topoisomerase II & IV), which is necessary for negative supercoiling of DNA prior to replication. 4
Ambroxol hydrochloride (ABX) ( . 1 B) chemically, 4-[(2-amino-3,5-dibromophenyl)-methyl]-amino] cyclohexanol hydrochloride is a mucolytic expectorant and used to reduce the viscosity of mucous secretions. 5
A fixed dose combination of levofloxacin hemihydrate (LFX) and ambroxol hydrochloride (ABX) is available for the treatment of upper and lower respiratory tract infections.
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(A) Chemical structure of levofloxacin (LFX), (B) Chemical structure of ambroxol (ABX).
Literature survey reveals that several methods have been developed for the quantitative determination of LFX in formulations as well as in plasma and urine. These include capillary electrophoresis and UV spectrophotometry, 6 HPLC, 7 - 10 simultaneous HPTLC method with ornidazole 11 and flow injection analysis. 12
It has been reported that ambroxol hydrochloride has been estimated by capillary electrophoresis, 13 - 15 spectrophotometry, 16 gas chromatography, 17 , 18 liquid chromatography with potentiometric estimation, 19 MS detection, 20 UV detection, 21 - 24 RP HPLC, 25 , 21 Raman spectroscopy, 26 liquid chromatography with roxithromycin 27 and derivative UV and HPLC. 28 Simultaneous reversed phase high performance liquid chromatographic method for determination of LFX and ABX in pharmaceutical formulations has been also reported. 29
However, most of the analytical methods developed for the quantization of LFX and ABX involve analysis of single component, except HPTLC for LFX and HPLC for ABX, which are simultaneous and quite expensive. To our knowledge, no simultaneous UV spectrophotometric method is available for quantitative determination of LFX and ABX in pharmaceutical dosage form.
This work was aimed to investigate the utility of UV spectrophotometric method in the simultaneous determination of LFX and ABX in pharmaceutical preparations. The method had sufficiently good accuracy, precision and permitted a simple and cost effective assay for these compounds in mixtures.
RESULTS AND DISCUSSION
- Method development
LFX and ABX, both are freely soluble in water, hence double distilled water was chosen as a solvent for their determination in solid dosage forms. The UV spectra of standard solutions of LFX and ABX (10 μg/mL each) were determined separately in distilled water ( . 2 A and 2 B). The λ max of LFX was found to be 287 nm whereas the λ max of ABX was recorded at 245 nm.
Initially, simultaneous equation method was tried for the determination of drugs in their dosage forms, as ABX showed negligible absorbance at the λ max of LFX. However, LFX showed considerable absorbance at the λ max of ABX. Therefore, absorbance ratio (Q analysis) method was applied for the analysis of both the drugs in tablets.
The developed method for the simultaneous analysis of LFX and ABX was validated with respect to stability, linearity, sensitivity, precision, accuracy, specificity, robustness and ruggedness. 30 - 33
The stability of both the drugs in distilled water was checked by recording their UV spectra at an appropriate time interval for up to 36 hours. They were compared with freshly prepared solutions and not any difference was found between them. This indicated that both these drugs were highly stable in solution phase. Further, a UV spectrum of standard solution containing LFX and ABX (mixture) was also recorded to check any chemical interaction between these drugs. The λ max of both the drugs in a mixture was found to be similar as compared to individual drugs indicating no chemical interference with each other ( . 2 C).
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(A) λmax of levofloxacin (LFX) in distilled water, (B) λmax of ambroxol (ABX) in distilled water, (C) λmax of mixture of levofloxacin + ambroxol in distilled water, (D) Overlain spectra of levofloxacin and ambroxol in distilled water.
- Q analysis method
The ratio of two absorbance determined on the two solutions at two different wavelengths is constant. This constant is termed as Q value. The Q value is independent of concentration and thickness of solution and therefore is used to access the purity of compounds. The absorbance ratio method is a modification of the simultaneous equation procedure. Graphical absorption ratio method uses the ratio of observed absorbance at two selected wavelengths, one of which is isoabsorptive point. It depends on property for that substance which obeys Beer’s law at all wavelengths. The ratio of absorbance at any wavelength is constant value independent of concentration or path length.
For Q analysis method, the overlain spectra of LFX and ABX were recorded in the range of 400 to 200 nm. It showed that ( . 2 D) the peaks were well resolved, satisfying the criteria for obtaining maximum precision, based on absorbance ratios. 34 The criteria being the ratios, (A 2 /A 1 )/(a x2 /a x1 ) and (a y2 /a y1 )/(A 2 /A 1 ), should lie outside the range 0.1-2.0 for the precise determination of X (LFX) and Y (ABX), respectively. Where A 1 , A 2 represents the absorbance of the mixture at λ 1 (wavelength at isoabsorptive point) and λ 2 max of LFX), a x1 and a x2 denote absorptivities of X at λ 1 and λ 2 , and a y1 and a y2 denote absorptivities of Y at λ 1 and λ 2 , respectively. In the present work, the above criteria was found to be satisfied for LFX (X) and ABX (Y), where λ 1 was 219 nm and λ 2 287 nm for Q-absorbance method.
In the quantitative assay of LFX and ABX in an admixture by absorbance ratio method, absorbances were measured at any two wavelengths, one being iso-absorptive point (λ 1 ) and the other being λ max of one of the component i.e. LFX (λ 2 ). Two equations were constructed as described below (Eq. 1 and Eq. 2), using the relationship a x1 = a y1 at λ 1 and b= 1 cm. Equations are
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and
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Dividing Eq. 2 by Eq. 1
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Dividing each term by C X +C Y and let F X = C X / (C X +C Y ) and F Y = C Y /(C X + C Y ) where, F X and F Y are the fractions of X and Y respectively in the mixture of LFX and ABX.
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But F Y = 1− F X
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Let
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Eq. (3) gives the fraction of X in the mixture of LFX and ABX. For the determination of absolute concentration of X and Y the equation 5 was rearranged.
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From Eq. 3
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Similarly,
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Where, C X and C Y are concentrations of LFX and ABX, respectively. 34
- Linearity and precision
In quantitative analysis the calibration curve was constructed for both LFX and ABX after analysis of consecutively increased concentrations. To check the precision and reproducibility of the method, six samples of the same concentration (n=6) of LFX and ABX were prepared and analysed. The low % RSD values obtained for LFX (0.83) and ABX (0.29) indicated that the method had high precision and reproducibility. The regression equation, slope, intercept, correlation coefficient, precision and linearity range are given in 1 .
Validation parameters for standard LFX and ABX.
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LFX: Levofloxacin; ABX: Ambroxol hydrochloride; a: at 219 nm; b: at 287 nm; *Indicates mean of six determinations (n=6).
- Analysis in tablet formulations
For the determination of LFX and ABX from pharmaceutical tablet formulations by Q analysis method, the absorbance of sample solutions and absorptivity values at the particular wavelengths were calculated and substituted in the following equation (equations 4 and 5) to obtain the concentrations of two components.
C LFX =(Q M -Q Y )×A 1 /(Q X -Q Y )×a x1 , C ABX =(Q M -Q X )× A 1 /(Q Y -Q X )×a y1 where, C LFX and C ABX are concentrations of LFX and ABX, respectively, A 1 is the absorbance of sample at 219 nm, a x1 is the absorptivity of LFX at 219 nm, a x2 is the absorptivity of LFX at 287 nm, a y1 is absorptivity of ABX at 219 nm, a y2 is absorptivity of ABX at 287 nm, Q X was obtained by using the equation, (absorptivity of LFX at 287 nm a x2 )/(absorptivity of LFX at 219 nm a x1 ). Similarly, Q Y was obtained from (absorptivity of ABX at 287 nm a y2 )/(absorptivity of ABX at 219 nm a y1 ) and Q M from, (absorbance of sample at 287 nm A 2 )/(absorbance of sample at 219 nm A 1 ). The respective absorptivity values for LFX and ABX at λ 1 and λ 2 are represented in 2 . The results obtained from analysis of dosage forms are given in 4 .
Absorptivity values at 219 nm (isoobsorptive wavelength) and 287 nm (λmax of LFX).
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LFX: Levofloxacin; ABX: Ambroxol hydrochloride; * Indicates mean of three experiments; S.D.: Standard deviation.
Comparison of linearity, LOD, and LOQ of LFX and ABX with reported HPLC method.29
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LFX: Levofloxacin; ABX: Ambroxol hydrochloride; a: at 219 nm; b: at 287 nm.
Analysis of dosage forms and recovery studies.
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LFX: Levofloxacin; ABX: Ambroxol hydrochloride; * Indicates mean of six determinations (n=6).
- Limit of detection (LOD) and limit of quantification (LOQ)
Limit of detection (LOD) and limit of quantification (LOQ) decide about the sensitivity of the method. LOD is the lowest detectable concentration of the analyte by the method while LOQ is the minimum quantifiable concentration. LOD and LOQ of LFX and ABX were calculated according to the equations 6 and 7 respectively at both the λ max where, δ is the standard deviation of blank and s is slope of calibration. 35
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The limit of detection (LOD) for LFX was 0.1 μg/ml and 0.2 μg/ml at λ max of 219 nm and λ max of 287 nm respectively while, the limit of quantification (LOQ) was 0.31 μg/ml and 0.38 μg/ml at the respective λ max . For ABX, The LOD was found to be 0.11 μg/ml and 0.02 μg/ml at λ max of 219 nm and λ max of 287 nm respectively while, the LOQ was 0.35μg/ml and 3.5 μg/ml at the respective λ max ( 1 ). The linearity, LOD and LOQ values of LFX and ABX obtained from UV method have been compared with the reported method. 29 The data displayed in 3 , indicates high sensitivity of the UV method over reported one.
- Reproducibility
The accuracy and specificity of the proposed method was tested by recovery experiments. Recovery studies were carried out at 100 % level by adding a known quantity of pure drug to the preanalyzed formulation and the proposed method was followed. From the amount of drug found, percentage recovery was calculated ( 4 ). The % recovery for LFX and ABX were found to be in the range of 100.53-101% (% RSD 0.51-1.51) and 99.69-101.84% (% RSD 0.49-0.76) respectively for both the formulations tested. The high recovery rate with low % RSD values indicated that the method had a good accuracy and specificity, as there was no interference from the excipients present in formulations.
Intra-day precision and accuracy were evaluated by analyzing three samples of two different concentrations, prepared on same day. Inter-day variability was assessed by analyzing two concentrations on three different days, over a period of one week. No significant difference was found in these experiments, indicating accuracy and reproducibility of the assays. The % RSD values reported in 5 shows that proposed method provides acceptable intra-day and inter-day variation of LFX and ABX.
Ruggedness of the proposed methods was determined by analyzing LFX and ABX by different analysts, using similar operational and environmental conditions; the % RSD values are reported in 5 and found to be less than 2 %.
% RSD values for repeatability, intra- day, inter-day variation and ruggedness (n=3).
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LFX: Levofloxacin; ABX: Ambroxol hydrochloride; n: No. of experiments.
Robustness of the proposed method was checked by minor changes on the selected wavelength. Since the absorbance was not significantly affected, the proposed method could be considered as robust.
EXPERIMENTAL
- Instrumentation
A Shimadzu 1700 UV (Shimadzu, Japan) spectrophotometer with 1 cm matched quartz cells was used for the estimation.
- Chemicals and reagents
LFX and ABX were kindly supplied by Cipla Ltd., Mumbai, India, as gift samples. Tablets containing LFX and ABX were procured from local pharmacy. All the reagents were of analytical grade. Double distilled water was used throughout the experiment.
- Standard Preparation
Accurately weighed quantities (10 mg each) of LFX and ABX were dissolved separately in sufficient quantity of distilled water in a 100 ml volumetric flask. The solutions were sonicated and the volume was adjusted up to the mark with distilled water to obtain a stock solution of 100 μg/ml; each of LFX and ABX. For the selection of analytical wavelength for the Q absorbance method, the stock solutions of LFX and ABX were separately diluted in distilled water, to get concentrations of 10 μg/ml each, and scanned in the wavelength range of 200-400 nm. From the overlain spectra of both drugs, wavelengths 219 nm (isoabsorptive point) and 287 nm (λmax of LFX) were selected for the formation of Q-absorbance equation. For calibration curves, stock solutions of LFX and ABX were appropriately diluted to obtain concentration range of 2-20 μg/ml and 5-50 μg/ml respectively. The absorbance of LFX was measured at 287 nm and 219 nm, and calibration curves were plotted. Similarly the absorbance of ABX was measured at 219 nm and 287 nm, and calibration curves were plotted. The absorptivities (A1%, 1 cm) of each drug at both the wavelengths were also determined.
- Sample preparation
For the estimation of drugs from the commercial formulations, twenty tablets of two brands L-cin A (Lupin Ltd., Mumbai, India) and Mucosyn (Alembic Ltd., Vadodara, India) containing 500 mg of LFX and 75 mg of ABX were weighed, and finely powdered. For the analysis of drugs, a standard addition method was used. An accurately weighed 175 mg of pure ABX was added to finely powdered samples to bring the concentration of ABX in linearity range. With this addition, the ratio of LFX to ABX in samples was brought to 2:1. Quantity of powder equivalent to 20 mg of LFX and 10 mg of ABX was transferred to 100 ml volumetric flask, dissolved in sufficient quantity of distilled water, sonicated and the volume was adjusted up to the mark with distilled water to obtain a stock solution of 200 μg/ml of LFX and 100 μg/ml of ABX. The solution was then filtered through Whatman filter paper No. 41 and the filtrate was appropriately diluted to obtain final concentrations 10 μg/ml of LFX and 5 μg/ml of ABX. Absorbance of this solution was measured at appropriate wavelengths, and values were substituted in the respective formulae to obtain concentrations.
CONCLUSION
The proposed method was successfully applied to the simultaneous determination of LFX and ABX from pharmaceutical tablet formulation. The presented method was found to be simple, accurate, precise, rugged and robust. It can be directly and easily applied to the analysis of the combined pharmaceutical tablet formulation of LFX and ABX. Moreover, the present method is quick and costeffective as compared to chromatographic techniques. Therefore, it can be concluded that the proposed method provides an alternative procedure for the quality control of LFX and ABX in pharmaceutical formulations.
Acknowledgements
We are grateful to Cipla Ltd., Mumbai, India, for providing gift samples of drug for research work. We are thankful to Principal, Govt. College of Pharmacy, Karad for providing laboratory facilities and constant encouragement.
References
Sweetman S. C. 2002 Martindale The Complete Drug Reference 33rd ed. Pharmaceutical Press London 219 -
Bertino J. , Fish D. 2000 Clin. Ther. 22 798 -    DOI : 10.1016/S0149-2918(00)80053-3
Lorian V. , Williams L. 1996 Antibiotics in Laboratory Medicine 4th ed. 591 -
Hardman J. , Goodman A. 1996 The Pharmacological Basis of Therapeutics 9th ed. McGraw-Hill NY 1065 -
Budavari S. 1996 The Merck Index 12th ed. Merck and Co. Inc. Whitehouse Station, NJ 404 -
Belal F. , Al-Majed A. , Al-Obaid A. M. 1999 Talanta. 50 765 -    DOI : 10.1016/S0039-9140(99)00139-3
Ines M. , Santoro R. M. , Singh A. K. , Erika R. M. , Hackmam K. 2006 J. Pharm. Biomed. Anal. 40 179 -    DOI : 10.1016/j.jpba.2005.06.018
Neckle U. , Joukhadar C. , Mayer B. X. 2002 Anal. Chim. Acta. 463 199 -    DOI : 10.1016/S0003-2670(02)00429-4
Wong F. A. , Juzwin S. J. , Flor S. C. 1997 J. Pharm. Biomed. Anal. 15 765 -    DOI : 10.1016/S0731-7085(96)01890-0
Siewert S. 2006 J. Pharm. Biomed. Anal. 41 1360 -    DOI : 10.1016/j.jpba.2006.02.010
Chepurwar S. B. , Shirkhedkar S. B. , Bari S. B. , Fursule R. A. , Surana S. J. 2007 J. Chromatogr. Sci. 45 531 -    DOI : 10.1093/chromsci/45.8.531
Altiokka G. , Atkosar Z. , Can N. O. 2002 J. Pharm. Biomed. Anal. 30 881 -    DOI : 10.1016/S0731-7085(02)00354-0
Pospíilová M. , Poláek M. , Jokl V. 2001 J. Pharm. Biomed. Anal. 24 421 -    DOI : 10.1016/S0731-7085(00)00448-9
Perez-Ruiz T. , Martínez-Lozano C. C. , Sanz C. , Bravo E. 2000 J. Chromatogr. B. 742 205 -    DOI : 10.1016/S0378-4347(00)00150-X
Pérez-Ruiz T. , Martínez-Lozano C. , Sanz A. , Bravo E. 1997 J. Chromatogr. B. 692 199 -    DOI : 10.1016/S0378-4347(96)00482-3
Reddy N. M. , Kanna Rao K. V. , Swapna M. , Sarkar D. G. 1998 Indian J. Pharm. Sci. 60 249 -
Colombo L. , Marcucci F. M. , Marini G. M. , Pierfederici P. , Mussini E. 1990 J. Chromatogr. B. 530 141 -    DOI : 10.1016/S0378-4347(00)82313-0
Schmid J. 1987 J. Chromatogr. 414 65 -    DOI : 10.1016/0378-4347(87)80025-7
Bazylak G. , Nagels L. J. 2003 J. Pharm. Biomed. Anal. 32 887 -    DOI : 10.1016/S0731-7085(03)00191-2
Kim H. , Yoo J. Y. , Han S. B. , Lee H. J. , Lee K. R. 2003 J. Pharm. Biomed. Anal. 32 209 -    DOI : 10.1016/S0731-7085(03)00059-1
Heinänen M. , Barbas C. 2001 J. Pharm. Biomed. Anal. 24 1005 -    DOI : 10.1016/S0731-7085(00)00533-1
Koundourellis J. E. , Malliou E. T. , Broussali T. A. 2000 J. Pharm. Biomed. Anal. 23 469 -    DOI : 10.1016/S0731-7085(00)00318-6
Nobilis M. , Pastera J. , Svoboda D. , Kvtina J. , Macek K. 1992 J. Chromatogr. 581 251 -    DOI : 10.1016/0378-4347(92)80278-X
Brizzi V. , Pasetti U. 1990 J. Pharm. Biomed. Anal. 8 107 -    DOI : 10.1016/0731-7085(90)80015-H
Indrayanto G. , Handayani R. 1993 J. Pharm. Biomed. Anal. 11 781 -    DOI : 10.1016/0731-7085(93)80189-8
Hwang M. S. , Cho S. , Chung H. , Woo Y. A. 2005 J. Pharm. Biomed. Anal. 8 210 -    DOI : 10.1016/j.jpba.2004.12.031
Qi M. , Wang P. , Cong R. , Yang J. 2004 J. Pharm. Biomed. Anal. 35 1287 -    DOI : 10.1016/j.jpba.2004.04.001
Dinçer Z. , Basan H. , Göger N. G. 2003 J. Pharm. Biomed. Anal. 5 867 -    DOI : 10.1016/S0731-7085(02)00664-7
Kothekar K. M. , Balasundaram J. , Khandhar A. P. , Mishra R. K. 2007 Eurasian J. Anal. Chem. 2 21 -
ICH 1996 International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. ICH Harmonised Tripartite Guideline.
Ermer J. , Ploss H. J. 2005 J. Pharm. Biomed. Anal. 37 859 -    DOI : 10.1016/j.jpba.2004.06.018
Fabre H. , Altria K. D. 2001 LC-GC. 14 302 -
Braggio S. , Barnaby R. J. , Grossi P. , Cugola M. A. 1996 J. Pharm. Biomed. Anal. 14 375 -    DOI : 10.1016/0731-7085(95)01644-9
Beckett A. H. , Stenlake J. B. 1997 Practical Pharmaceutical Chemistry 4th ed. CBS Publisher New Delhi Part-II 275 -
Busaranon K. , Suntornsuk W. , Suntornsuk L. 2006 J. Pharm. Biomed. Anal. 41 158 -    DOI : 10.1016/j.jpba.2005.11.008