Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 216–222
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Development and validation of sensitive kinetic spectrophotometric method for the determination of moxifloxacin antibiotic in pure and commercial tablets Safwan Ashour a,⇑, Roula Bayram b a b
Department of Chemistry, Faculty of Sciences, University of Aleppo, Aleppo, Syria Department of Pharmaceutical Chemistry, Pharmacy Program, Batterjee Medical College, Jeddah, Saudi Arabia
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A novel kinetic method for analysis of
moxifloxacin based on the reaction with MBTH was developed. We found that acidic medium is necessary to success the spectrophotometric method. The method validation demonstrated good recoveries and low detection limit. The method was successfully applied for analysis of real pharmaceutical samples.
a r t i c l e
i n f o
Article history: Received 3 May 2014 Received in revised form 22 December 2014 Accepted 23 December 2014 Available online 3 January 2015 Keywords: Moxifloxacin Kinetic spectrophotometry 3-Methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (MBTH) Tablets
a b s t r a c t New, accurate, sensitive and reliable kinetic spectrophotometric method for the assay of moxifloxacin hydrochloride (MOXF) in pure form and pharmaceutical formulations has been developed. The method involves the oxidative coupling reaction of MOXF with 3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (MBTH) in the presence of Ce(IV) in an acidic medium to form colored product with lambda max at 623 and 660 nm. The reaction is followed spectrophotometrically by measuring the increase in absorbance at 623 nm as a function of time. The initial rate and fixed time methods were adopted for constructing the calibration curves. The linearity range was found to be 1.89–40.0 lg mL1 for initial rate and fixed time methods. The limit of detection for initial rate and fixed time methods is 0.644 and 0.043 lg mL1, respectively. Molar absorptivity for the method was found to be 0.89 104 L mol1 cm1. Statistical treatment of the experimental results indicates that the methods are precise and accurate. The proposed method has been applied successfully for the estimation of moxifloxacin hydrochloride in tablet dosage form with no interference from the excipients. The results are compared with the official method. Ó 2015 Elsevier B.V. All rights reserved.
Introduction
⇑ Corresponding author at: Analytical Biochemistry Laboratory, Department of Chemistry, Faculty of Sciences, University of Aleppo, Aleppo, Syria. Tel.: +963 933 604016. E-mail addresses: [email protected], [email protected] (S. Ashour). http://dx.doi.org/10.1016/j.saa.2014.12.111 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
Moxifloxacin, 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy7-[(4aS,7aS)-octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl]-4-oxo-3quinoline carboxylic acid, is a new fourth generation 8-methoxy fluoroquinolone antibacterial agent with a broad spectrum and improved activity against Gram-positive bacteria (including staphylococci, streptococci, enterococci), anaerobes and atypical
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bacteria [1,2]. Several methods have been described for the quantitative determination of moxifloxacin hydrochloride in pharmaceutical dosage forms by flow injection with chemiluminescence detection [3], atomic absorption spectrometry [4], atomic absorption spectroscopy, conductometry and colorimetry [5], spectrophotometry [4,6–10], kinetics spectrophotometry [4,11], voltammetry [12,13], differential pulse polarography [14], spectrofluorimetry [15–17] and capillary electrophoresis [18,19]. Chromatographic methods such as reversed-phase HPLC/fluorescence [20–22], HPLC with ultra violet detection [23–25] and high-performance thin-layer chromatography [26] have been reported for the estimation of moxifloxacin hydrochloride in pharmaceutical products. Voltammetry [12,13], spectrofluorimetry [15], high-performance liquid chromatography with fluorescence [27–29] or UV detection [30–34], liquid chromatography tandem mass spectrometry (LC/MS/MS) [35,36] and capillary electrophoresis [37,38] are also reported for determination of moxifloxacin hydrochloride from human body fluids. Literature survey reveals that moxifloxacin hydrochloride is official in British Pharmacopeia (BP) [39]. The official procedure in pharmaceutical preparations utilize liquid chromatographic method. 3-Methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) is one of the widely used chromogenic reagents for spectrophotometric analysis of phenols [40]. It undergoes an interesting reaction with phenolic, amino, ketonic and aldehydic compounds in the presence of oxidizing agent such as H2O2, cerium(IV), iron(III), chromium(VI) yielding a highly colored reaction products [40]. MBTH had been used for spectrophotometric determination of caffeine and theophylline [41], acetaminophen and phenobarbital [42], ritodrine hydrochloride [43], metronidazole and tinidazole [44] and ketoprofen [45]. Kinetic methods have certain advantages in pharmaceutical analysis regarding selectivity and elimination of additive interferences, which affect direct spectrophotometric methods. The literature is still poor in analytical assay methods based on kinetics for the determination of moxifloxacin in dosage forms. Some specific advantages that the kinetic methods possess are as follows: simple and fast methods because some experimental steps such as filtration, extraction, etc., are avoided prior to absorbance measurements, high selectivity since they involve the measurement of the absorbance as a function of reaction time instead of measuring the concrete absorbance value, other active compounds present in the commercial dosage forms may not interfere if they are resisting the chemical reaction conditions established for the proposed kinetic method and colored and/or turbid sample background may possibly not interfere with the determination process [46,47]. In this work, the reaction between MBTH and moxifloxacin was kinetically studied in an attempt to develop a reliable and specific spectrophotometric method for the determination of moxifloxacin in pure form and pharmaceutical preparations. The method is based on oxidation of MBTH with Ce(IV) then coupling with moxifloxacin in presence of H2SO4, the colored condensation product is measured at 623 nm kinetically using the Initial rate and fixed time methods.
Experimental
217
800 nm and slit width 0.1 nm. Electronic balance (Kern, Germany) was used for weighing the samples. Materials Working reference standard of moxifloxacin hydrochloride (MOXF) was obtained from Matrix Laboratories Limited (India). Its purity was found to be 99.8%. 3-Methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (MBTH) 97% was from Aldrich Chemical Co., St. Louis (USA). All other chemicals and reagents used were of analytical grade and were of Merck (Germany). All solutions were prepared with double distilled water. The commercial formulations containing moxifloxacin hydrochloride 400 mg per tablet were subjected to the analytical procedures. Solutions Standard solution of MOXF was prepared by direct weighing of standard substance with subsequent dissolution in double distilled water. The concentration of the stock standard solution was 0.5 mg mL1. A series working standard solutions of MOXF (1.89– 40.0 lg mL1) were prepared by diluting the stock standard solution with the double distilled water. Standard solutions were found to be stable for one month at least when stored in the dark at 2–8 °C. 1 102 M MBTH solution was prepared with double distilled water and 1% Ce(SO4)2 solution was prepared with sulfuric acid (0.360 M) medium. Freshly prepared solutions were always used. General procedures Initial rate method Aliquots of standard MOXF solution (0.038–0.8 mL, 0.5 mg mL1) were transferred into a series of 10 mL calibrated volumetric flasks. Then 0.75 mL of MBTH solution was added. After that, 1.0 mL of Ce(SO4)2 solution was added. The volume was made up to the mark with distilled water. After mixing, the contents of each flask were immediately transferred to the spectrophotometric cell and the increase in absorbance was recorded at 623 nm as a function of time between 0 and 45 min against reagent blank treated similarly. The initial rate of the reaction (m) at different concentrations was obtained from the slope of the tangent to the absorbance–time curve. The calibration curve was constructed by plotting the logarithm of the initial rate (log m) versus the logarithm of the molar concentration of the MOXF (log C). The amount of the drug was obtained either from the calibration graphs or the regression equation. Fixed time method Aliquots of standard MOXF solution (0.038–0.8 mL, 0.5 mg mL1) were transferred into a series of 10 mL calibrated volumetric flasks. Then 0.75 mL of MBTH solution was added. After that, 1.0 mL of Ce(SO4)2 solution was added. The volume was made up to the mark with distilled water. After mixing, the absorbance was measured after 40 min at 623 nm against reagent blank treated similarly. The calibration curve was constructed by plotting the absorbance against the final concentration of the drug. The amount of the drug in each sample was computed either from calibration curve or regression equation.
Instrumentation Double beam UV/Visible spectrophotometer (Shimadzu, model 1800, Japan) with matched 10 mm quartz cells was used to measure absorbance of all the solutions. Spectra were automatically obtained by UV-Probe system software ver.2.43 under the following operating conditions: scan speed medium, scan range 500–
Procedure for formulations Twenty tablets containing MOXF were weighed and finely powdered. An amount of the powder equivalent to 25 mg of the cited drug was dissolved in a 25 mL of methanol and mixed for about 5 min and then filtered through Whatman filter paper number 40. The methanol was evaporated to about the dryness. The
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remaining portion of solution was diluted in a 50 mL volumetric flask to the volume with double distilled water to achieve a concentration of 0.5 mg mL1. The general procedure was then followed in the concentration range mentioned above. Results and discussion Absorption spectra MOXF reacts with MBTH in the presence of Ce(SO4)2 in an acidic medium to form green colored oxidative coupling product that can be measured at 623 and 660 nm. Under the experimental conditions the pure drug showed a negligible absorbance at the corresponding maximum (Fig. 1). Optimization of reaction conditions The development of the color product depends on the reaction conditions and was optimized as follows. The effect of various parameters such as volume of MBTH and Ce(SO4)2, addition of buffer solutions, waiting time, order of addition of reagents and the stability of colored oxidative coupling product were studied at room temperature. The applicability of MBTH in combination with various oxidizing agents such as FeCl3, KIO4, NaIO4, Ce(SO4)2, K2Cr2O7 and KMnO4 were examined. Ce(SO4)2 was found to be optimal to form colored oxidative coupling product (MOXF–MBTH) and enhanced the final color. Effect of MBTH concentration The maximum conversion of the analyte into absorbing specie depends on the amount of the reagent available in the solution for reaction and the equilibrium involved. So, the reagent concentration in solution was studied by varying the MBTH volume of 0.01 M in the presence of 1.0 mL of 1% Ce(SO4)2, while the MOXF concentration was maintained constant at 20 lg mL1. The result is shown in Fig. 2. The study revealed that the reaction was dependent on MBTH reagent. The absorbance of the reaction solution increased as the MBTH concentration increased, and the highest absorption intensity was attained when the volume of MBTH was
Fig. 2. Effect of volume of MBTH 0.01 M in the presence of 1.0 mL of 1% Ce(SO4)2 and volume of 1% Ce(SO4)2 in the presence of 0.75 mL of 102 M MBTH on the formation of colored product MOXF (20 lg mL1)–MBTH at 623 nm, temperature: 25 ± 0.5 °C; reaction time: 40 min. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
0.75 mL of 0.01 M MBTH. Increasing the volume of MBTH leads to decrease in the absorbance; this may be due to the high background absorbance of the reagent. Effect of Ce(IV) concentration Fig. 2 shows that a volume of 1.0 mL of 1% Ce(SO4)2 was found to be optimal for maximum color development in the presence of 0.75 mL of 0.01 M MBTH, since the absorbance was found to be maxima at the mentioned volume. Effect of buffer Addition of KCl–HCl or Britton buffer solutions effected negativity on the formation of the colored oxidative coupling product. Addition of drug, MBTH and Ce(SO4)2 in that order gave maximum absorbance. Effect of temperature and time
Fig. 1. Absorption spectra of (A) MOXF against distilled water, (B) reagent blank against distilled water and (C) MOXF–MBTH–Ce(SO4)2 system against reagent blank. CMOXF = 20 lg mL1 + 0.75 mL of 102 M MBTH + 1 mL of 1% Ce(SO4)2.
The effect of temperature and time on the reaction of MOXF with MBTH in acidic aqueous medium was studied at different values (20–45 °C, 0–60 min) by continuous monitoring of the absorbance at 623 nm. It was found that the reaction with MBTH at laboratory ambient temperature (25 ± 5 °C) went to completion within 40 min. The results revealed that increasing the temperature had negative effect on the absorption values of the reaction solution. This was probably attributed to the instability of the MOXF–MBTH derivative. Under the aforementioned optimum conditions, increase absorbance values were observed from the beginning of the experiment up to 40 min. After this time and up to 60 min, absorbance suffered a slight increase, reaching values up to 0.6% higher than those observed after 40 min of the reaction. In view of these results, all measurements were carried out after 40 min of mixing of the reagent in order to make the method faster. The effect of time on the stability of the chromogen was studied by following the absorption intensity of the reaction solution (after dilution) at different time intervals. It was found that the absorbance of the chromogen remains stable for at least 2 h. This allowed the processing of large batches of samples and their comfortable measurements with convenience. This increased the convenience of the methods as well as made it applicable for large number of samples.
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Quantitation methods
Rate constant method
Because the intensity of the color increased with time (Fig. 3), this was used as the basis for a useful kinetic method for the determination of MOXF. The initial rate, rate constant, variable time (fixed concentration or fixed absorbance) and fixed time methods [48,49] were tested and the most suitable analytical methods were chosen regarding the applicability, sensitivity, the values of the intercept and correlation coefficient (R).
Graphs of log absorbance versus time for MOXF concentration in the range of 15–30 lg mL1 (3.42 105–6.85 105 M) were plotted and all appeared to be rectilinear. Pseudo order rate constant (k0 ) corresponding to different MOXF concentrations were calculated from the slopes multiplied by 2.303 and are presented in Fig. 5 and Table 1. Regression of C versus k0 gave the following equation: 0
k ¼ 2:5878C 0:00052ðR ¼ 0:9877Þ Initial rate method The initial rate of reaction would follow a pseudo order rate constant and obeyed the following rate equation:
m ¼ DA=Dt ¼ k0 C n where m is the reaction rate, A is the absorbance, t is the measuring time, k0 is the pseudo order rate constant, C is the concentration of the drug mol L1 and n is the order of the reaction. A calibration curve was constructed by plotting the logarithm of the initial rate of reaction (log m) versus logarithm of drug concentration (log C) which showed a linear relationship over the concentration range of 1.89–40 lg mL1 (Fig. 4). The logarithmic form of the above equation is written as follows: 0
log m ¼ log DA=Dt ¼ log k þ n log C log m ¼ log DA=Dt ¼ 0:0289 þ 0:9063log½MOXFðR ¼ 0:9972Þ
Fixed absorbance method Reaction rate data were recorded for different MOXF concentrations in the range 20–40 lg mL1 (4.56 105–9.13 105 M). A preselected value of the absorbance 0.35 was fixed and the time was measured in the seconds (Table 2). The reciprocal of time (1/ t) versus the initial concentration of MOXF was plotted (Fig. 6) and the following equation of calibration graph was obtained:
1=t ¼ 38:0819C 0:0013ðR ¼ 0:9963Þ The range of MOXF concentration giving the most satisfactory results was limited 20–40 lg mL1. Fixed time method At preselected fixed time, the absorbance of green colored solution containing varying amounts of MOXF was measured at 25 °C
Thus, k0 = 1.0688 S1, and the reaction is the first order (n = 0.9063 1) with respect to MOXF concentration.
Fig. 5. Calibration plot of MOXF for rate constant method.
Fig. 3. Absorbance–time curve for the reaction of MOXF with MBTH in aqueous acidic medium at 623 nm; CMOXF = 1.89–40 lg mL1.
Table 1 Values of rate constant k0 for different rates of variable concentration of MOXF. CMOXF (M)
k0 (S1)
3.42 105 4.56 105 5.70 105 6.85 105
44.00 105 41.08 105 37.37 105 35.39 105
Table 2 Values of reciprocal time taken at fixed absorbance (0.35) for the different rates of variable concentration of MOXF at MBTH constant concentration.
Fig. 4. Calibration plot of logarithm rate of the reaction against logarithm molar concentration of MOXF for initial rate method.
CMOXF (M)
1/t (S1)
4.56 105 5.70 105 6.85 105 7.99 105 9.13 105
46.8 105 87.4 105 123.8 105 173.3 105 221.3 105
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a
Parameters
MOXF
kmax (nm) Beer’s law range (lg mL1) Molar absorptivity (L mol1 cm1) Optimum photometric range (lg mL1) Detection limit (lg mL1) Quantification limit (lg mL1) Sandell’s sensitivity (lg cm2 per 0.001 absorbance unit) Regression equationa Correlation coefficient, R
623 1.89–40.0 0.89 104 8.3–40.0 0.043 1.89 0.049 A = 0.0180C + 0.0014 0.9974
A = mC + b, where A is the absorbance and C is the concentration in lg mL1.
Fig. 6. Calibration plot of MOXF for fixed absorbance method.
Table 3 Regression equations for MOXF at fixed time (40 min) and 25 °C. Time (min)
Regression equation
Correlation coefficient
Linear range (lg mL1)
5 10 15 20 25 30 35 40 45
A = 0.0066C 0.0175 A = 0.0108C 0.0114 A = 0.0132C 0.0048 A = 0.0147C 0.0016 A = 0.0159C + 0.0006 A = 0.0167C + 0.0017 A = 0.0173C + 0.0032 A = 0.0180C + 0.0014 A = 0.0179C + 0.0063
0.9860 0.9938 0.9937 0.9938 0.9940 0.9942 0.9944 0.9974 0.9947
1.89–40 1.89–40 1.89–40 1.89–40 1.89–40 1.89–40 1.89–40 1.89–40 1.89–40
A, absorbance; C, concentration.
and 623 nm. Calibration graphs were constructed by plotting the absorbance against the initial concentration of MOXF at fixed time 5, 10, 15, 20, 25, 30, 35, 40 and 45 min. The regression equations, correlation coefficients and linear ranges are given in Table 3. Correlation coefficient, intercept and slope values for the calibration data calculated using the least squares method [50]. It is clear that, both the slopes and intercepts increase with time. The most acceptable values of the correlation coefficient and more reaction products (indicated by higher absorbance readings) as shown in Fig. 3 were obtained for a fixed time of 40 min, which was, therefore chosen as the most suitable time interval for measurements. After optimizing the reaction conditions, the most acceptable values of the correlation coefficients were obtained for the initial rate and fixed time (40 min) methods. Thus, they were applied for the determination of MOXF in pure form and pharmaceutical formulations over the concentration range of 1.89– 40.0 lg mL1. The limit of detection (LOD) and limit of quantification (LOQ) for fixed time (40 min) method were determined and for more accurate analysis, Ringbom optimum concentration range was calculated as shown in Table 4.
Stoichiometric relationship The stoichiometry of colored oxidative coupling product was studied adopting the limiting logarithmic method [51]. The ratio of the reaction between MOXF and MBTH in acidic aqueous medium was calculated by dividing the slope of MBTH curve over the slope of the MOXF curve (Fig. 7a and b). It was found that the ratio was 1:1 (MBTH to MOXF). Under the reaction conditions, on oxidation, MBTH loses two electrons and one proton forming an electrophilic intermediate,
Fig. 7. Stoichiometry of the reaction between MOXF and MBTH adopting limiting logarithmic method. (a) CMOXF = 5 104 M, CMBTH = 1.25 104–2.5 103 M and (b) CMBTH = 7.5 104 M, CMOXF = 5.71 106–9.13 105 M.
which is the active coupling species. The coupling of the oxidized form of the drug with electrophilic intermediate of MBTH results in the formation of intensely colored product [52]. The reaction mechanism for the method is shown in Scheme 1. Analytical methods validation The accuracy and precision of the proposed method were carried out by six determinations at four different concentrations. Percentage relative standard deviation as precision and percentage relative error (Er%) as accuracy of the suggested methods were calculated. Table 5 shows the values of relative standard deviations for different concentrations of MOXF determined from the calibration curves. These results of accuracy and precision show that the proposed method has good repeatability and reproducibility. The proposed method was found to be selective for the estimation of MOXF in the presence of various tablet excipients. For this purpose, a powder blend using typical tablet excipients was prepared along
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Scheme 1. Proposed oxidative coupling reaction of MOXF with MBTH.
Table 5 Accuracy and precision for the determination of MOXF in bulk powder by the proposed initial rate and fixed time methods. MOXF (lg mL1)
Method
a
Er%
RSD%
%Recovery ± SD
a
Taken
Found
Initial rate
5.00 10.00 20.00 40.00
5.01 10.04 20.04 40.40
0.20 0.40 0.20 1.00
2.50 1.45 0.99 0.75
100.20 ± 2.48 100.40 ± 1.46 100.20 ± 1.00 101.00 ± 0.76
Fixed time
5.00 10.00 20.00 40.00
5.14 10.10 20.04 40.06
2.89 0.99 0.22 0.15
2.76 2.29 1.71 0.91
102.89 ± 2.84 100.99 ± 2.32 100.22 ± 1.71 100.15 ± 0.92
Average of six determinations.
Table 6 Application of the proposed method to the determination of MOXF in tablet dosage form using MBTH. Sample
Manufacturer
Content (mg/tablet)
%Recoverya ± SD Proposed methods
a
Official method [6]
Initial rate
Fixed time
Avalox
Bayer, Germany
400
102.52 ± 1.97 t = 1.32 F = 1.34
101.80 ± 1.59 2.53 1.14
101.18 ± 1.70 1.55
Moxaquin
Oubari Pharma, Syria
400
101.12 ± 2.05 t = 1.22 F = 1.13
101.20 ± 2.42 1.11 1.57
100.18 ± 1.93 0.20
Moxicin
Ibn Al Haytham, Syria
400
102.04 ± 2.09 t = 2.18 F = 1.11
101.12 ± 1.85 1.35 1.41
100.62 ± 2.20 0.63
Maxim
Jamjoom Pharma, Saudi Arabia
400
101.64 ± 2.21 t = 1.66 F = 1.05
101.00 ± 2.24 1.00 1.08
100.19 ± 2.15 0.20
Average of five determinations. At 95% confidence limit the theoretical t- and F-values at five degree of freedom are t = 2.776 and F = 6.26.
with the drug and then analyzed. The recoveries were not affected by the excipients and the excipients blend did not show any absorption in the range of analysis.
ing no significant difference between the methods compared. The proposed method has the advantages of being virtually free from interferences by excipients such as glucose, lactose, and starch or from common degradation products.
Analysis of moxifloxacin in tablet dosage forms Conclusion The proposed procedure was applied to determine MOXF in its pharmaceutical formulations. The results obtained were compared statistically by the Student’s t-test (for accuracy) and the variance ratio F-test (for precision) with those obtained by the reference method [6] on samples of the same batch (Table 6). Mean values were obtained with a Student’s t- and F-tests at 95% confidence limit for five degrees of freedom [50]. The results showed comparable accuracy (t-test) and precision (F-test), since the calculated values of t- and F-tests were less than the theoretical data indicat-
The developed kinetic spectrophotometric method for the determination of MOXF was sensitive, accurate and precise and hence can be used for the routine analysis of MOXF in bulk and pharmaceutical formulations with a limit of detection of 0.043 lg mL1. MBTH was used as a reagent in acidic medium. The fixed time method for the proposed kinetic spectrophotometric method can be easily applied to the determination of MOXF in its pure form and tablets. The proposed method is compared with
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the previously reported methods in terms of accuracy and precision. The proposed method is more selective and higher sensitivity than the sophisticated spectrophotometric techniques and similar reported methods and has a wider range of linearity. The sample recoveries from all formulations were in good agreement with their respective label claims, which suggested non-interference of formulations excipients in the estimation. Moreover, all the analytical reagents are inexpensive, have good shelf life and are available in any analytical laboratory along with the lower reagents consumption leads to an environmentally friendly spectrophotometric procedure, which makes it especially suitable for routine quality control analysis work, as alternatives for the existing methods.
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Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Development and validation of sensitive kinetic spectrophotometric method for the determination of moxifloxacin antibiotic in pure and commercial tablets Safwan Ashour a,⇑, Roula Bayram b a b
Department of Chemistry, Faculty of Sciences, University of Aleppo, Aleppo, Syria Department of Pharmaceutical Chemistry, Pharmacy Program, Batterjee Medical College, Jeddah, Saudi Arabia
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A novel kinetic method for analysis of
moxifloxacin based on the reaction with MBTH was developed. We found that acidic medium is necessary to success the spectrophotometric method. The method validation demonstrated good recoveries and low detection limit. The method was successfully applied for analysis of real pharmaceutical samples.
a r t i c l e
i n f o
Article history: Received 3 May 2014 Received in revised form 22 December 2014 Accepted 23 December 2014 Available online 3 January 2015 Keywords: Moxifloxacin Kinetic spectrophotometry 3-Methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (MBTH) Tablets
a b s t r a c t New, accurate, sensitive and reliable kinetic spectrophotometric method for the assay of moxifloxacin hydrochloride (MOXF) in pure form and pharmaceutical formulations has been developed. The method involves the oxidative coupling reaction of MOXF with 3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (MBTH) in the presence of Ce(IV) in an acidic medium to form colored product with lambda max at 623 and 660 nm. The reaction is followed spectrophotometrically by measuring the increase in absorbance at 623 nm as a function of time. The initial rate and fixed time methods were adopted for constructing the calibration curves. The linearity range was found to be 1.89–40.0 lg mL1 for initial rate and fixed time methods. The limit of detection for initial rate and fixed time methods is 0.644 and 0.043 lg mL1, respectively. Molar absorptivity for the method was found to be 0.89 104 L mol1 cm1. Statistical treatment of the experimental results indicates that the methods are precise and accurate. The proposed method has been applied successfully for the estimation of moxifloxacin hydrochloride in tablet dosage form with no interference from the excipients. The results are compared with the official method. Ó 2015 Elsevier B.V. All rights reserved.
Introduction
⇑ Corresponding author at: Analytical Biochemistry Laboratory, Department of Chemistry, Faculty of Sciences, University of Aleppo, Aleppo, Syria. Tel.: +963 933 604016. E-mail addresses: [email protected], [email protected] (S. Ashour). http://dx.doi.org/10.1016/j.saa.2014.12.111 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
Moxifloxacin, 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy7-[(4aS,7aS)-octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl]-4-oxo-3quinoline carboxylic acid, is a new fourth generation 8-methoxy fluoroquinolone antibacterial agent with a broad spectrum and improved activity against Gram-positive bacteria (including staphylococci, streptococci, enterococci), anaerobes and atypical
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bacteria [1,2]. Several methods have been described for the quantitative determination of moxifloxacin hydrochloride in pharmaceutical dosage forms by flow injection with chemiluminescence detection [3], atomic absorption spectrometry [4], atomic absorption spectroscopy, conductometry and colorimetry [5], spectrophotometry [4,6–10], kinetics spectrophotometry [4,11], voltammetry [12,13], differential pulse polarography [14], spectrofluorimetry [15–17] and capillary electrophoresis [18,19]. Chromatographic methods such as reversed-phase HPLC/fluorescence [20–22], HPLC with ultra violet detection [23–25] and high-performance thin-layer chromatography [26] have been reported for the estimation of moxifloxacin hydrochloride in pharmaceutical products. Voltammetry [12,13], spectrofluorimetry [15], high-performance liquid chromatography with fluorescence [27–29] or UV detection [30–34], liquid chromatography tandem mass spectrometry (LC/MS/MS) [35,36] and capillary electrophoresis [37,38] are also reported for determination of moxifloxacin hydrochloride from human body fluids. Literature survey reveals that moxifloxacin hydrochloride is official in British Pharmacopeia (BP) [39]. The official procedure in pharmaceutical preparations utilize liquid chromatographic method. 3-Methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) is one of the widely used chromogenic reagents for spectrophotometric analysis of phenols [40]. It undergoes an interesting reaction with phenolic, amino, ketonic and aldehydic compounds in the presence of oxidizing agent such as H2O2, cerium(IV), iron(III), chromium(VI) yielding a highly colored reaction products [40]. MBTH had been used for spectrophotometric determination of caffeine and theophylline [41], acetaminophen and phenobarbital [42], ritodrine hydrochloride [43], metronidazole and tinidazole [44] and ketoprofen [45]. Kinetic methods have certain advantages in pharmaceutical analysis regarding selectivity and elimination of additive interferences, which affect direct spectrophotometric methods. The literature is still poor in analytical assay methods based on kinetics for the determination of moxifloxacin in dosage forms. Some specific advantages that the kinetic methods possess are as follows: simple and fast methods because some experimental steps such as filtration, extraction, etc., are avoided prior to absorbance measurements, high selectivity since they involve the measurement of the absorbance as a function of reaction time instead of measuring the concrete absorbance value, other active compounds present in the commercial dosage forms may not interfere if they are resisting the chemical reaction conditions established for the proposed kinetic method and colored and/or turbid sample background may possibly not interfere with the determination process [46,47]. In this work, the reaction between MBTH and moxifloxacin was kinetically studied in an attempt to develop a reliable and specific spectrophotometric method for the determination of moxifloxacin in pure form and pharmaceutical preparations. The method is based on oxidation of MBTH with Ce(IV) then coupling with moxifloxacin in presence of H2SO4, the colored condensation product is measured at 623 nm kinetically using the Initial rate and fixed time methods.
Experimental
217
800 nm and slit width 0.1 nm. Electronic balance (Kern, Germany) was used for weighing the samples. Materials Working reference standard of moxifloxacin hydrochloride (MOXF) was obtained from Matrix Laboratories Limited (India). Its purity was found to be 99.8%. 3-Methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (MBTH) 97% was from Aldrich Chemical Co., St. Louis (USA). All other chemicals and reagents used were of analytical grade and were of Merck (Germany). All solutions were prepared with double distilled water. The commercial formulations containing moxifloxacin hydrochloride 400 mg per tablet were subjected to the analytical procedures. Solutions Standard solution of MOXF was prepared by direct weighing of standard substance with subsequent dissolution in double distilled water. The concentration of the stock standard solution was 0.5 mg mL1. A series working standard solutions of MOXF (1.89– 40.0 lg mL1) were prepared by diluting the stock standard solution with the double distilled water. Standard solutions were found to be stable for one month at least when stored in the dark at 2–8 °C. 1 102 M MBTH solution was prepared with double distilled water and 1% Ce(SO4)2 solution was prepared with sulfuric acid (0.360 M) medium. Freshly prepared solutions were always used. General procedures Initial rate method Aliquots of standard MOXF solution (0.038–0.8 mL, 0.5 mg mL1) were transferred into a series of 10 mL calibrated volumetric flasks. Then 0.75 mL of MBTH solution was added. After that, 1.0 mL of Ce(SO4)2 solution was added. The volume was made up to the mark with distilled water. After mixing, the contents of each flask were immediately transferred to the spectrophotometric cell and the increase in absorbance was recorded at 623 nm as a function of time between 0 and 45 min against reagent blank treated similarly. The initial rate of the reaction (m) at different concentrations was obtained from the slope of the tangent to the absorbance–time curve. The calibration curve was constructed by plotting the logarithm of the initial rate (log m) versus the logarithm of the molar concentration of the MOXF (log C). The amount of the drug was obtained either from the calibration graphs or the regression equation. Fixed time method Aliquots of standard MOXF solution (0.038–0.8 mL, 0.5 mg mL1) were transferred into a series of 10 mL calibrated volumetric flasks. Then 0.75 mL of MBTH solution was added. After that, 1.0 mL of Ce(SO4)2 solution was added. The volume was made up to the mark with distilled water. After mixing, the absorbance was measured after 40 min at 623 nm against reagent blank treated similarly. The calibration curve was constructed by plotting the absorbance against the final concentration of the drug. The amount of the drug in each sample was computed either from calibration curve or regression equation.
Instrumentation Double beam UV/Visible spectrophotometer (Shimadzu, model 1800, Japan) with matched 10 mm quartz cells was used to measure absorbance of all the solutions. Spectra were automatically obtained by UV-Probe system software ver.2.43 under the following operating conditions: scan speed medium, scan range 500–
Procedure for formulations Twenty tablets containing MOXF were weighed and finely powdered. An amount of the powder equivalent to 25 mg of the cited drug was dissolved in a 25 mL of methanol and mixed for about 5 min and then filtered through Whatman filter paper number 40. The methanol was evaporated to about the dryness. The
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remaining portion of solution was diluted in a 50 mL volumetric flask to the volume with double distilled water to achieve a concentration of 0.5 mg mL1. The general procedure was then followed in the concentration range mentioned above. Results and discussion Absorption spectra MOXF reacts with MBTH in the presence of Ce(SO4)2 in an acidic medium to form green colored oxidative coupling product that can be measured at 623 and 660 nm. Under the experimental conditions the pure drug showed a negligible absorbance at the corresponding maximum (Fig. 1). Optimization of reaction conditions The development of the color product depends on the reaction conditions and was optimized as follows. The effect of various parameters such as volume of MBTH and Ce(SO4)2, addition of buffer solutions, waiting time, order of addition of reagents and the stability of colored oxidative coupling product were studied at room temperature. The applicability of MBTH in combination with various oxidizing agents such as FeCl3, KIO4, NaIO4, Ce(SO4)2, K2Cr2O7 and KMnO4 were examined. Ce(SO4)2 was found to be optimal to form colored oxidative coupling product (MOXF–MBTH) and enhanced the final color. Effect of MBTH concentration The maximum conversion of the analyte into absorbing specie depends on the amount of the reagent available in the solution for reaction and the equilibrium involved. So, the reagent concentration in solution was studied by varying the MBTH volume of 0.01 M in the presence of 1.0 mL of 1% Ce(SO4)2, while the MOXF concentration was maintained constant at 20 lg mL1. The result is shown in Fig. 2. The study revealed that the reaction was dependent on MBTH reagent. The absorbance of the reaction solution increased as the MBTH concentration increased, and the highest absorption intensity was attained when the volume of MBTH was
Fig. 2. Effect of volume of MBTH 0.01 M in the presence of 1.0 mL of 1% Ce(SO4)2 and volume of 1% Ce(SO4)2 in the presence of 0.75 mL of 102 M MBTH on the formation of colored product MOXF (20 lg mL1)–MBTH at 623 nm, temperature: 25 ± 0.5 °C; reaction time: 40 min. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
0.75 mL of 0.01 M MBTH. Increasing the volume of MBTH leads to decrease in the absorbance; this may be due to the high background absorbance of the reagent. Effect of Ce(IV) concentration Fig. 2 shows that a volume of 1.0 mL of 1% Ce(SO4)2 was found to be optimal for maximum color development in the presence of 0.75 mL of 0.01 M MBTH, since the absorbance was found to be maxima at the mentioned volume. Effect of buffer Addition of KCl–HCl or Britton buffer solutions effected negativity on the formation of the colored oxidative coupling product. Addition of drug, MBTH and Ce(SO4)2 in that order gave maximum absorbance. Effect of temperature and time
Fig. 1. Absorption spectra of (A) MOXF against distilled water, (B) reagent blank against distilled water and (C) MOXF–MBTH–Ce(SO4)2 system against reagent blank. CMOXF = 20 lg mL1 + 0.75 mL of 102 M MBTH + 1 mL of 1% Ce(SO4)2.
The effect of temperature and time on the reaction of MOXF with MBTH in acidic aqueous medium was studied at different values (20–45 °C, 0–60 min) by continuous monitoring of the absorbance at 623 nm. It was found that the reaction with MBTH at laboratory ambient temperature (25 ± 5 °C) went to completion within 40 min. The results revealed that increasing the temperature had negative effect on the absorption values of the reaction solution. This was probably attributed to the instability of the MOXF–MBTH derivative. Under the aforementioned optimum conditions, increase absorbance values were observed from the beginning of the experiment up to 40 min. After this time and up to 60 min, absorbance suffered a slight increase, reaching values up to 0.6% higher than those observed after 40 min of the reaction. In view of these results, all measurements were carried out after 40 min of mixing of the reagent in order to make the method faster. The effect of time on the stability of the chromogen was studied by following the absorption intensity of the reaction solution (after dilution) at different time intervals. It was found that the absorbance of the chromogen remains stable for at least 2 h. This allowed the processing of large batches of samples and their comfortable measurements with convenience. This increased the convenience of the methods as well as made it applicable for large number of samples.
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Quantitation methods
Rate constant method
Because the intensity of the color increased with time (Fig. 3), this was used as the basis for a useful kinetic method for the determination of MOXF. The initial rate, rate constant, variable time (fixed concentration or fixed absorbance) and fixed time methods [48,49] were tested and the most suitable analytical methods were chosen regarding the applicability, sensitivity, the values of the intercept and correlation coefficient (R).
Graphs of log absorbance versus time for MOXF concentration in the range of 15–30 lg mL1 (3.42 105–6.85 105 M) were plotted and all appeared to be rectilinear. Pseudo order rate constant (k0 ) corresponding to different MOXF concentrations were calculated from the slopes multiplied by 2.303 and are presented in Fig. 5 and Table 1. Regression of C versus k0 gave the following equation: 0
k ¼ 2:5878C 0:00052ðR ¼ 0:9877Þ Initial rate method The initial rate of reaction would follow a pseudo order rate constant and obeyed the following rate equation:
m ¼ DA=Dt ¼ k0 C n where m is the reaction rate, A is the absorbance, t is the measuring time, k0 is the pseudo order rate constant, C is the concentration of the drug mol L1 and n is the order of the reaction. A calibration curve was constructed by plotting the logarithm of the initial rate of reaction (log m) versus logarithm of drug concentration (log C) which showed a linear relationship over the concentration range of 1.89–40 lg mL1 (Fig. 4). The logarithmic form of the above equation is written as follows: 0
log m ¼ log DA=Dt ¼ log k þ n log C log m ¼ log DA=Dt ¼ 0:0289 þ 0:9063log½MOXFðR ¼ 0:9972Þ
Fixed absorbance method Reaction rate data were recorded for different MOXF concentrations in the range 20–40 lg mL1 (4.56 105–9.13 105 M). A preselected value of the absorbance 0.35 was fixed and the time was measured in the seconds (Table 2). The reciprocal of time (1/ t) versus the initial concentration of MOXF was plotted (Fig. 6) and the following equation of calibration graph was obtained:
1=t ¼ 38:0819C 0:0013ðR ¼ 0:9963Þ The range of MOXF concentration giving the most satisfactory results was limited 20–40 lg mL1. Fixed time method At preselected fixed time, the absorbance of green colored solution containing varying amounts of MOXF was measured at 25 °C
Thus, k0 = 1.0688 S1, and the reaction is the first order (n = 0.9063 1) with respect to MOXF concentration.
Fig. 5. Calibration plot of MOXF for rate constant method.
Fig. 3. Absorbance–time curve for the reaction of MOXF with MBTH in aqueous acidic medium at 623 nm; CMOXF = 1.89–40 lg mL1.
Table 1 Values of rate constant k0 for different rates of variable concentration of MOXF. CMOXF (M)
k0 (S1)
3.42 105 4.56 105 5.70 105 6.85 105
44.00 105 41.08 105 37.37 105 35.39 105
Table 2 Values of reciprocal time taken at fixed absorbance (0.35) for the different rates of variable concentration of MOXF at MBTH constant concentration.
Fig. 4. Calibration plot of logarithm rate of the reaction against logarithm molar concentration of MOXF for initial rate method.
CMOXF (M)
1/t (S1)
4.56 105 5.70 105 6.85 105 7.99 105 9.13 105
46.8 105 87.4 105 123.8 105 173.3 105 221.3 105
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a
Parameters
MOXF
kmax (nm) Beer’s law range (lg mL1) Molar absorptivity (L mol1 cm1) Optimum photometric range (lg mL1) Detection limit (lg mL1) Quantification limit (lg mL1) Sandell’s sensitivity (lg cm2 per 0.001 absorbance unit) Regression equationa Correlation coefficient, R
623 1.89–40.0 0.89 104 8.3–40.0 0.043 1.89 0.049 A = 0.0180C + 0.0014 0.9974
A = mC + b, where A is the absorbance and C is the concentration in lg mL1.
Fig. 6. Calibration plot of MOXF for fixed absorbance method.
Table 3 Regression equations for MOXF at fixed time (40 min) and 25 °C. Time (min)
Regression equation
Correlation coefficient
Linear range (lg mL1)
5 10 15 20 25 30 35 40 45
A = 0.0066C 0.0175 A = 0.0108C 0.0114 A = 0.0132C 0.0048 A = 0.0147C 0.0016 A = 0.0159C + 0.0006 A = 0.0167C + 0.0017 A = 0.0173C + 0.0032 A = 0.0180C + 0.0014 A = 0.0179C + 0.0063
0.9860 0.9938 0.9937 0.9938 0.9940 0.9942 0.9944 0.9974 0.9947
1.89–40 1.89–40 1.89–40 1.89–40 1.89–40 1.89–40 1.89–40 1.89–40 1.89–40
A, absorbance; C, concentration.
and 623 nm. Calibration graphs were constructed by plotting the absorbance against the initial concentration of MOXF at fixed time 5, 10, 15, 20, 25, 30, 35, 40 and 45 min. The regression equations, correlation coefficients and linear ranges are given in Table 3. Correlation coefficient, intercept and slope values for the calibration data calculated using the least squares method [50]. It is clear that, both the slopes and intercepts increase with time. The most acceptable values of the correlation coefficient and more reaction products (indicated by higher absorbance readings) as shown in Fig. 3 were obtained for a fixed time of 40 min, which was, therefore chosen as the most suitable time interval for measurements. After optimizing the reaction conditions, the most acceptable values of the correlation coefficients were obtained for the initial rate and fixed time (40 min) methods. Thus, they were applied for the determination of MOXF in pure form and pharmaceutical formulations over the concentration range of 1.89– 40.0 lg mL1. The limit of detection (LOD) and limit of quantification (LOQ) for fixed time (40 min) method were determined and for more accurate analysis, Ringbom optimum concentration range was calculated as shown in Table 4.
Stoichiometric relationship The stoichiometry of colored oxidative coupling product was studied adopting the limiting logarithmic method [51]. The ratio of the reaction between MOXF and MBTH in acidic aqueous medium was calculated by dividing the slope of MBTH curve over the slope of the MOXF curve (Fig. 7a and b). It was found that the ratio was 1:1 (MBTH to MOXF). Under the reaction conditions, on oxidation, MBTH loses two electrons and one proton forming an electrophilic intermediate,
Fig. 7. Stoichiometry of the reaction between MOXF and MBTH adopting limiting logarithmic method. (a) CMOXF = 5 104 M, CMBTH = 1.25 104–2.5 103 M and (b) CMBTH = 7.5 104 M, CMOXF = 5.71 106–9.13 105 M.
which is the active coupling species. The coupling of the oxidized form of the drug with electrophilic intermediate of MBTH results in the formation of intensely colored product [52]. The reaction mechanism for the method is shown in Scheme 1. Analytical methods validation The accuracy and precision of the proposed method were carried out by six determinations at four different concentrations. Percentage relative standard deviation as precision and percentage relative error (Er%) as accuracy of the suggested methods were calculated. Table 5 shows the values of relative standard deviations for different concentrations of MOXF determined from the calibration curves. These results of accuracy and precision show that the proposed method has good repeatability and reproducibility. The proposed method was found to be selective for the estimation of MOXF in the presence of various tablet excipients. For this purpose, a powder blend using typical tablet excipients was prepared along
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Scheme 1. Proposed oxidative coupling reaction of MOXF with MBTH.
Table 5 Accuracy and precision for the determination of MOXF in bulk powder by the proposed initial rate and fixed time methods. MOXF (lg mL1)
Method
a
Er%
RSD%
%Recovery ± SD
a
Taken
Found
Initial rate
5.00 10.00 20.00 40.00
5.01 10.04 20.04 40.40
0.20 0.40 0.20 1.00
2.50 1.45 0.99 0.75
100.20 ± 2.48 100.40 ± 1.46 100.20 ± 1.00 101.00 ± 0.76
Fixed time
5.00 10.00 20.00 40.00
5.14 10.10 20.04 40.06
2.89 0.99 0.22 0.15
2.76 2.29 1.71 0.91
102.89 ± 2.84 100.99 ± 2.32 100.22 ± 1.71 100.15 ± 0.92
Average of six determinations.
Table 6 Application of the proposed method to the determination of MOXF in tablet dosage form using MBTH. Sample
Manufacturer
Content (mg/tablet)
%Recoverya ± SD Proposed methods
a
Official method [6]
Initial rate
Fixed time
Avalox
Bayer, Germany
400
102.52 ± 1.97 t = 1.32 F = 1.34
101.80 ± 1.59 2.53 1.14
101.18 ± 1.70 1.55
Moxaquin
Oubari Pharma, Syria
400
101.12 ± 2.05 t = 1.22 F = 1.13
101.20 ± 2.42 1.11 1.57
100.18 ± 1.93 0.20
Moxicin
Ibn Al Haytham, Syria
400
102.04 ± 2.09 t = 2.18 F = 1.11
101.12 ± 1.85 1.35 1.41
100.62 ± 2.20 0.63
Maxim
Jamjoom Pharma, Saudi Arabia
400
101.64 ± 2.21 t = 1.66 F = 1.05
101.00 ± 2.24 1.00 1.08
100.19 ± 2.15 0.20
Average of five determinations. At 95% confidence limit the theoretical t- and F-values at five degree of freedom are t = 2.776 and F = 6.26.
with the drug and then analyzed. The recoveries were not affected by the excipients and the excipients blend did not show any absorption in the range of analysis.
ing no significant difference between the methods compared. The proposed method has the advantages of being virtually free from interferences by excipients such as glucose, lactose, and starch or from common degradation products.
Analysis of moxifloxacin in tablet dosage forms Conclusion The proposed procedure was applied to determine MOXF in its pharmaceutical formulations. The results obtained were compared statistically by the Student’s t-test (for accuracy) and the variance ratio F-test (for precision) with those obtained by the reference method [6] on samples of the same batch (Table 6). Mean values were obtained with a Student’s t- and F-tests at 95% confidence limit for five degrees of freedom [50]. The results showed comparable accuracy (t-test) and precision (F-test), since the calculated values of t- and F-tests were less than the theoretical data indicat-
The developed kinetic spectrophotometric method for the determination of MOXF was sensitive, accurate and precise and hence can be used for the routine analysis of MOXF in bulk and pharmaceutical formulations with a limit of detection of 0.043 lg mL1. MBTH was used as a reagent in acidic medium. The fixed time method for the proposed kinetic spectrophotometric method can be easily applied to the determination of MOXF in its pure form and tablets. The proposed method is compared with
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the previously reported methods in terms of accuracy and precision. The proposed method is more selective and higher sensitivity than the sophisticated spectrophotometric techniques and similar reported methods and has a wider range of linearity. The sample recoveries from all formulations were in good agreement with their respective label claims, which suggested non-interference of formulations excipients in the estimation. Moreover, all the analytical reagents are inexpensive, have good shelf life and are available in any analytical laboratory along with the lower reagents consumption leads to an environmentally friendly spectrophotometric procedure, which makes it especially suitable for routine quality control analysis work, as alternatives for the existing methods.
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