Int J Pharm Pharm Sci, Vol 7, Issue 11, 21-26Original Article


SIMULTANEOUS SPECTROPHOTOMETRIC ESTIMATION OF MOXIFLOXACIN HYDROCHLORIDE AND DOXORUBICIN HYDROCHLORIDE

NAYAN PRADHAN*, HIYASHREE RAJKHOWA, HEMANT GIRI, BHUPENDRA SHRESTHA

Department of Pharmaceutical Analysis and Quality Assurance, Himalayan Pharmacy Institute, Majhitar 737132, East Sikkim, India
Email: nayanpradhan7@gmail.com     

 Received: 12 Dec 2014 Revised and Accepted: 10 Sep 2015

ABSTRACT

Objective: To develop a simple, accurate, and precise spectrophotometric method for the simultaneous estimation of moxifloxacin hydrochloride (MOX) and doxorubicin hydrochloride (DXR).

Methods: MOX and DXR solution were simultaneously determined in 0.1M HCl at their respective λmax. The absorbance λmax of MOX and DXR was 295 nm and 480 nm, respectively. The developed method was validated according to ICH guidelines for parameters like linearity, accuracy, precision, ruggedness and robustness.

Results: Molar absorptivities of MOX and DXR were found to be more in 0.1M HCl with compared to water, methanol and 0.1M NaOH. Linearity was obtained over the range 0.5–20.0 μg/ml and 1.5-50 μg/ml with a lower limit of quantitation of 0.25 μg/ml and 1.5 μg/ml for MOX and DXR, respectively. For each level of samples, inter-and intra-day precision ( % RSD) was<1.3 % and<1.4 % for MOX and<2.5 and 2.4 % for DXR, respectively. The mean recovery of MOX and DXR was in the range 96.21 %-98.77 % and 97.13 %-99.64 %, respectively.

Conclusion: The method developed was validated as per ICH guidelines for parameters like linearity, accuracy, method precision, robustness and ruggedness. The results obtained were well within the acceptable criteria. The method can be used for routine analysis of MOX and DXR.

Keywords: Moxifloxacin hydrochloride, Doxorubicin hydrochloride, Simultaneous estimation, Method development, Validation.

© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

INTRODUCTION

Moxifloxacin hydrochloride (MOX) is a synthetic fluoroquinolone antibiotic agent, chemically 1-Cyclopropyl-6-fluoro-1, 4-dihydro-8-methoxy-7-((4as, 7as)-octahydro-6H-pyrrolo (3, 4-b) pyridin-6-yl)-4-oxo-3-quinolinecarboxylic acid with the empirical formula C21H24FN3O4 [1]. MOX is a fourth-generation synthetic  fluoroquinolone  antibacterial agent.  Its antibacterial spectrum includes enteric Gram-(−) rods (Escherichia coli, Proteusspecies, Klebsiella species),  Haemophilus influenzae, atypical bacteria (Mycoplasma,  Chlamydia,  Legionella), and Streptococcus pneumoniae, and anaerobic bacteria. It differs from earlier antibacterials of the fluoroquinolone class such as levofloxacin and ciprofloxacin in having greater activity against  Gram-(+) bacteria and anaerobes. Because of its potent activity against the common respiratory pathogen Streptococcus pneumoniae, it is considered a respiratory quinolone. Moxifloxacin binds DNA and forms DNA gyrase (topoisomerase II) complex and blocks further DNA replication; it also blocks topoisomerase IV interferes with the separation of interlocked replicated DNA molecules [2].

Doxorubicin hydrochloride (DXR) is a cytotoxic anthracycline antibiotic isolated from cultures of the fungus Streptomyces peucetiusvar. caesius. The most common uses of DXR in cancer therapy is for various types of testicular cancer, leukemia, Ewing’s sarcoma, Hodgkin’s disease, and Kaposi’s sarcoma [3]. DXR is chemically 5, 12-Naphthacenedione, 10-[(3-amino-2, 3, 6-trideoxy-a-Llyxo-hexopyranosyl) oxy]-7, 8, 9, 10-tetrahydro-6, 8, 11-trihydroxy -8-(hydroxyacetyl)-1-methoxy, hydrochloride. Its empirical formula is C27H29NO11 [4].

DXR is a cytotoxic anthracycline antibiotic [5, 6], closely related to the natural product Daunomycin [7, 8] and like all anthracyclines, it works by inter calating DNA [9-10] with the most serious adverse effect being life-threatening heart damage. It is prescribed in the treatment of a wide variety of malignant neoplastic diseases, including leukemias, lymphomas, sarcomas, germ cell tumors, and carcinomas [12].

Literature showed anticancer and flouroquinolones are frequently prescribed in clinical practice and interaction may result without recognition. There are evidences of use of adjunct antibiotics like flouroquinolones with anticancer drugs enhancing the cytotoxic effects while, at the same time, decreasing chemotherapy-induced pro-inflammatory cytokine secretion from cells, which

Fig. 1: Structure of moxifloxacin hydrochloride


Fig. 2: Structure of doxorubicin hydrochloride

May be harmful during chemotherapeutic treatment. [13-15] Chemotherapy regimen used in clinical practice are empiric drug combinations [16-17], maximum doses prescribed for DXR is 75 mg/m2 [18] and for MXR is 400 mg [19]. Few spectrophotometric methods [20-29] and high-performance liquid chromatography methods [29-42] have been reported for the determination of moxifloxacin and DXR in single or combined pharmaceutical dosage forms or biological fluids.

But till date there is no report of simultaneous estimation of these two drugs in pharmaceutical dosage form or in biological fluid. The combined drug related toxicities or change in therapeutic efficacy should be monitored. There is a need of developing method for the simultaneous determination of these drugs body fluids. Thus, the authors have developed this novel method for simultaneous estimation of DXR and MOX, which could aid in determining those drugs in plasma or body fluids.

MATERIALS AND METHODS

Chemicals and reagents

MOX hydrochloride and DXR hydrochloride were obtained as a gift sample from Alkem Laboratories (Sikkim) and Yarrow Chem. Ltd (Mumbai, India). Methanol used was spectrophotometric grade and all other reagents employed were of analytical grade (S. D Fine Chem. Ltd., Mumbai, India). Distilled water was used throughout the experiment.

Instrumentation

Analytical balance model CP225D (Sartorius, Germany) was used. Simultaneous estimation was performed using a UV-Visible spectrophotometer (UV-1800, Shimadzu, Japan).

Selection of solvent and ratio of drugs to be analyzed

Individual sample of pure DXR and MOX was checked for their solubility in different solvents i.e. methanol, water, 0.1HCl and 0.1M NaOH. Then molar absorptivity of the respective drug in each of the four solvents was calculated taking the concentration of 5 µg/ml. The solvent having the highest molar absorptivity was selected as the choice of solvent for the rest of the experiment.

Preparation of analytical solution

A stock solution of DXR and MOX was prepared by dissolving 50 mg of the drug in 50 mL of 0.1M HCl.The final concentration of 3 µg/ml of DOX and 5 µg/ml of MOX was prepared by suitably diluting the stock solution with 0.1M HCl.

Method validation

Linearity

The linearity of the method was established by preparing different concentration of drug ranging from 0.5 to 30 µg/ml and 0.5 to 40 µg/ml of MOX and DXR, respectively. Absorbance against the corresponding analyte concentration was plotted and slope, intercept and correlation co-efficient were determined using linear regression analysis.

Precision

Intra-day precision was reported as % RSD for three replicate samples at three different concentrations (different ratio of drugs) levels against a qualified standard drug. Inter day precision was also carried out similarly but in two different days and the % RSD was calculated.

Accuracy

The accuracy was evaluated in triplicate by adding a pure drug of MOX and DXR in already analyzed sample solution consisting 3 µg/ml of DXR and 6 µg/ml of MOX. Known amount of DXR (0.6 µg/ml, 1.8 µg/ml and 2.4 µg/ml) and MOX (1.2 µg/ml, 3.6 µg/ml and 4.8 µg/ml) standard solutions was added to the already analyzed sample solution and the analysis was carried out. The total amount of drug present was determined by the proposed method and the % recovery of pure drug was calculated.

Limit of detection

LOD was determined by using a formula

LOD= 
Where, SD of observance is obtained from 6 replicates of absorbance obtained from the sample solution and the slope is obtained from the linearity curve.

Limit of quantification

LOQ was determined by using a formula

LOQ=

Where, SD of observance is obtained from 6 replicates of absorbance obtained from the sample solution and the slope is obtained from the linearity curve

Robustness

Robustness was carried out by changing the wavelength by±2 nm, the strength of the solvent, i.e. 0.1M HCl, and room temperature 25±2οc.

Ruggedness

Ruggedness was performed by carrying out an analytical procedure with the different analyst.

RESULTS

Method development

The molar absorptivity of the DXR was found to be maximum i. e 2×103 at 480 nm in acidic (0.1M HCl) solution than in water, methanol and 0.1 M NaOH. Similarly molar absorptivity of MOX was found to be maximum i. e 4×104 at 290 nm in basic (0.1M NaOH) solution than in water, methanol and 0.1M HCl. Comparison of molar absorptivity is shown in table 1 and 2.

Though MOX shows maximum absorptivity in a basic solution, its molar absorptivity in acidic solution is still 20 times higher than DXR and the λmax is also very far from one another making it possible to carry out simultaneous estimation. DXR hydrochloride solution showed absorption λmax at 480 nm and the MOX hydrochloride solution showed absorption λmax at 295 nm in 0.1M HCl as shown in fig. 3 and 4.

Fig. 3: Absorbance spectrum of MOX in 0.1M HCl


Fig. 4: Absorbance spectrum of DXR in 0.1M HCl


Fig. 5: Absorbance spectrum of MOX (λmax at 295 nm) and DXR (λmax at 480 nm) in 0.1M HCl

Method validation

Linearity

The calibration curve for DXR was linear over the concentration range of 1.5-50 µg/ml. The correlation coefficient value obtained was 0.998 with the regression equation y = 0.019x+0.024.

Fig. 6: Standard calibration curve for DXR


Fig. 7: Standard calibration curve of MOX

Similarly the calibration curve for MOX was linear over the concentration range of 0.2-20 µg/ml. The correlation coefficient value obtained was 0.998 with the regression equation y = 0.114x+0.031. The high value of correlation coefficient indicates the method is linear over the concentration range.

Precision

The precision of the method was determined by intra-day and inter-day precision studies by taking three different concentrations of the sample. Values of % RSD for intra-day were 2.4, 1.3, and 1.7 for 1.5, 3 and 4.5 µg/ml concentration of DXR and 0.95, 1.0 and 1.3 for 2.5, 5 and 7.5 µg/ml concentration of MOX; for inter-day 2.5, 0.7 and 2.1 for 1.5, 3 and 4.5 µg/ml concentration of DXR and 0.7, 1 and 1.3 for 2.5, 5 and 7.5 µg/ml concentrations of MOX respectively, as shown in table 3 and 4.

Accuracy

Recovery studies by the standard addition method were performed with a view to justify the accuracy of the proposed method. Previously analyzed sample was spiked with known amounts of standard DXR and MOX so as to get three different levels (20 %, 60 % and 80 %) and the mixture was analyzed by the proposed method. The experiment was performed in triplicate % recovery, mean % recovery and RSD ( %) was calculated for each concentration. The method has shown good and consistent recoveries ranging from 96.21 %-98.77 % and 97.13 %-99.64 % for DXR and MOX respectively confirming the accuracy of the method, as shown in table 5.

Table 1: Absortivity of DXR in different solvent

 

NaOH

HCl

Water

Methanol

λmax (nm)

489

480

496

495

Molar conc.

11.510-5

2.8710-5

2.8710-5

2.8710-5

Absorbance

0.045

0.498

0.390

0.405

Molar absorptivity

392

2.021103

1.463103

1.847103

Table 2: Absortivity of MOX in different solvent

 

NaOH

HCl

Water

Methanol

λmax (nm)

290

295

289

292

Molar conc.

1.2510-5

1.2510-5

1.2510-5

1.2510-5

Absorbance

0.512

0.498

0.390

0.405

Molar absorptivity

4.096104

3.984104

3.12104

3.24104

Table 3: Intra-day precision data of DXR and MOX by simultaneous equation method

Parameters

DXR

MOX

Drug concentration (µg/ml)

A

B

C

A

B

C

1.5

3

4.5

2.5

5

7.5

 %Assay

103.1

101.5

102.2

99.1

99.4

99.2

106.4

99.9

100.1

97.8

98.6

101.2

103.1

99.8

98.5

98.0

100.3

99.5

 % Mean

104.2

100.4

100.1

98.3

98.7

99.9

 % RSD

2.4

1.3

1.7

0.95

1.0

1.3

Table 4: Inter-day precision data of DXR and MOX by simultaneous equation method

Parameters

DXR

MOX

Drug concentration (µg/ml)

A

B

C

A

B

C

1.5

3

4.5

2.5

5

7.5

 %Assay

106.20

98.21

102.00

97.04

98.32

99.01

104.10

97.23

98.00

96.89

97.30

102.21

100.10

99.03

103.01

98.32

96.03

100.34

 % Mean

104.20

98.16

101.00

97.42

97.22

100.51

 % RSD

2.5

0.75

2.1

0.66

0.96

1.31

Robustness

Robustness was performed by small variations in spectro photometric conditions like solvent strength, wavelength and room temperature. It can be observed from the table 6 that the method is unaffected by the small variation in the spectro photometric conditions.

Limit of detection and limit of quantification

Typically the concentration level that generates a signal to noise (S/N) of 10 is regarded as the LOQ and the concentration level that generates S/N=3 is regarded as the LOD. The limits of detection and quantification were determined from the calibration curve.

The LOD and LOQ were 0.46 µg/ml and 1.4 µg/ml, respectively, for DXR and 0.082 µg/ml and 0.25 µg/ml, respectively for MOX.

Ruggedness

The results of ruggedness were well within acceptable limits of 98 % -102 % for both the drugs as shown in table 7.

Table 5: Accuracy data for the determination

Drug

Conc. of sample

(µg/ml)

Conc. of standard added (µg/ml)

Amt. added

 %

Total concentration found (µg/ml)

Recovery % (n=3)

Mean Recovery %(n=9)

 %RSD

MOX

5

1

20 %

5.76

96.02

97.13

1.05

5.88

98.04

5.84

97.32

5

3

60 %

7.86

98.30

98.45

0.56

7.84

97.98

7.92

99.06

5

4

80 %

8.92

99.09

99.64

1.27

8.91

99.02

8.89

98.80

DOX

1.5

0.3

20 %

1.71

95.02

96.21

1.09

1.74

96.6

1.75

97.00

1.5

0.6

60 %

2.06

98.30

97.70

0.72

2.06

97.88

2.04

96.93

1.5

1.2

80 %

2.65

98.30

98.77

1.07

2.64

98.02

2.70

99.98

Table 6: Robustness of simultaneous equation method for DXR and MOX

Parameters

 % RSD (n=3)

DOX

MOX

Temperature (23 and 30)

0.34

0.56

Solvent strength (0.1M)

0.03

0.05

Wavelength ()

2.63

3.84

Table 7: Ruggedness data of simultaneous equation method for DXR and MOX

Drug

Analyst I amount found %SD (n=3)

 % RSD

Analyst II amount found %SD (n=3)

 % RSD

DOX

98.031.20

1.22

98.321.89

1.92

MOX

101.100.76

0.75

99.051.55

1.56

DISCUSSION

DXR a prototype drug from the active class of anticancer agents used to treat solid cancers and hematological malignancies [43], while, MOX belongs to wide spectrum antibiotics fluoroquinolones group and is successfully used in clinical practice to treat a number of infections and as an antibacterial prophylactic in cancer patients [44]. Several clinical studies have adopted the different dose schedules of DXR alone or in combination with other drugs to improve the anticancer activity. [45] Some studies [13-15] show that, moxifloxacin significantly enhanced the anti-proliferative effect of chemotherapeutic agents, adding its significance to be used along with the chemotherapeutic agents. There is a need for the development of the method for clinical monitoring of these two drugs simultaneously, so that complication like cardio toxicity and relative effectiveness can be efficiently monitored when used in combination. Development of in vitro simultaneous method for the estimation of these two drugs aids for the development of method for clinical monitoring. For the first time, MOX and DXR have been simultaneously estimated by the spectroscopic method. Vierodt’s method was used for the estimation of the two drugs in solution which showed a relatively good degree of accuracy and precision. The developed method was validated in compliance with ICH guidelines for parameters like linearity, accuracy, method precision, robustness and ruggedness. The results obtained were well within the acceptable criteria. The method can be used for routine quality control analysis of MOX and DXR simultaneously.

ACKNOWLEDGEMENT

The authors are grateful to the management of the Himalayan Pharmacy Institute (HPI) for providing excellent laboratory facilities. We are grateful to Alkem Laboratories Ltd Kumrek, East Sikkim for a gift sample of MOX. We are thankful to Dr. Ankur Gupta, Ex. HOD of Department of Pharmaceutical Analysis and Quality Assurance, HPI for his encouragement and Mr. Jeewan Pradhan, Laboratory technician, HPI, for laboratory assistance.

CONFLICT OF INTERESTS

Declared None

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