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Simultaneous Determination of Moxifloxacin Hydrochloride and Flavoxate Hydrochloride in A New Tablet Formulation by High Performance Liquid Chromatography


JASHRAJ SACHDEVA*1, YUVRAJ SINGH SARANGDEVOT2, PRIYADARSHANI KAMBLE3
*1 Bhupal Nobles College of Pharmacy, Udaipur, 313001, India
2Dept. of Pharmacognosy, Bhupal Nobles College of Pharmacy, Udaipur, 313001, India
3Dept. of Quality Assurance, Bhupal Nobles College of Pharmacy, Udaipur, 313001, India

 ABSTRACT

 A rapid, simple, precise, robust and accurate high performance liquid chromatographic method is described for the simultaneous determination of Moxifloxacin hydrochloride and Flavoxate hydrochloride in a new tablet formulation. Chromatographic separation of the two drugs was achieved on Agilent Technologies Thermo scientific, BDS hypersil C18 Column, (150 x 4.6 mm) 5µm particle size at 27oC temperature. The mobile phase consisting of a mixture of 0.1% formic acid in water: Acetonitrile (25:75 v/v) adjusted at pH 3 by 1M HCl was delivered at a flow rate of 1.0 ml/min. Detection was performed at 218 nm. Specificity, Accuracy, Precision, Linearity and Robustness were found to be acceptable over the concentration range. Separation was complete in less than 10 min. The proposed method can be used for the quality control of formulation products.

Key words: Moxifloxacin hydrochloride, Flavoxate hydrochloride, High Performance Liquid Chromatography

INTRODUCTION

Moxifloxacin hydrochloride is a fourth-generation synthetic fluoroquinolone antibacterial agent developed by Bayer AG (initially called BAY 12-8039). Its antibacterial spectrum includes – Enteric Gram-(−) rods (Escherichia coli , Proteus species, Klebsiella species)- responsible for Urinary Tract Infections. Haemophilus influenzae, atypical bacteria (Mycoplasma, Chlamydia, Legionella), and  Streptococcus pneumoniae, and anaerobic bacteria. It differs from earlier antibacterials of the fluoroquinolone class in having greater activity against such bacteria as compared to other Classes.

Flavoxate hydrochloride is a spasmolytic flavone derivative that acts by relaxing the smooth muscle in the urinary tract. Flavoxate is a competitive muscarinic receptor antagonist indicated for the treatment of overactive bladder with symptoms of urge urinary incontinence, urgency, and urinary frequency. Muscarinic receptors play an important role in several major cholinergically mediated functions, including contractions of urinary bladder smooth muscle and stimulation of salivary secretion.

An increased interest by pharmaceutical companies has been shown to develop a formulation containing both the drugs. In the process of the development, a fast and reliable analytical method is required for the simultaneous determination of both drugs in this compound formulation.

Some methods can be found for the individual determination of Moxifloxacin hydrochloride and Flavoxate hydrochloride. Reverse phase High performance liquid chromatography (RP-HPLC) with spectrometric detections has been used for the determination of the individual drugs. However, no references have been found for the simultaneous determination of Moxifloxacin hydrochloride and Flavoxate hydrochloride in pharmaceutical preparations.

The present paper describes the method development and Validation as per ICH guidelines for the simultaneous determination of Moxifloxacin hydrochloride and Flavoxate hydrochloride in a new tablet formulation by High performance liquid chromatography (HPLC). The performance of the developed method was validated in terms of specificity, accuracy, precision, linearity and robustness.

MATERIALS AND METHOD

Chemicals and Reagents

The Reference standards of Moxifloxacin hydrochloride (purity : 99.23%) and Flavoxate hydrochloride (purity : 99.70%) were obtained from Cris Pharmaceutical (India) Ltd. (Dehradun, India). Moxifloxacin hydrochloride and Flavoxate hydrochloride tablets were also from Cris Pharmaceutical (India) Ltd. (Dehradun, India). Each tablet contains 400 mg Moxifloxacin hydrochloride and 200 mg Flavoxate hydrochloride. HPLC grade methanol and acetonitrile were from E. Merck Ltd. (Mumbai, India). Formic acid of Analytical grade was from Rankem Company (Gurgaon, India). High purity water was prepared in-house using a Milli-Q water purification system obtained from Millipore (India) Pvt. Ltd. (Bangaluru, India).

Apparatus and Chromatographic conditions

Liquid chromatographic was performed on an Agilent 1260 Infinity Series equipped with a G1311C Quat pump VL and a variable UV- Vis detector.

A Thermo scientific, BDS hypersil C18 column  (150 x 4.6 mm, 5µm particle size) was used for separation at 27oC temperature and using mobile phase 0.1% formic acid in water : Acetonitrile  (25:75 v/v) adjusted at pH 3 by 1M HCl at a flow rate of 1.0 ml/min. and wavelength for detection was 218 nm. The injection volume was 20µL.

Preparation of Standard solutions

A stock solution with Moxifloxacin hydrochloride at about 4 mg/ml and Flavoxate hydrochloride at about 2 mg/ml was prepared with methanol followed by diluting 5ml from both the solutions with mobile phase separately. Standard solutions were prepared by dilution of the stock solution with the mobile phase to give solutions containing Moxifloxacin hydrochloride and Flavoxate hydrochloride in the concentration ranges of 40.0 – 400.0 µg/ml and 20.0 – 200.0 µg/ml respectively.

Preparation of sample solutions

Twenty tablets were accurately weighed and finely powdered. An accurate weighed portion of the powder equivalent to 400 mg of Moxifloxacin and 200 mg of Flavoxate was transferred in a 100 ml volumetric flask containing methanol, followed by its transfer to a 50 ml volumetric flask where about 10 ml of mobile phase was added, the mixture was shaken and brought to the volume with the mobile phase, which was used as the sample solution.

RESULTS AND DISCUSSION

Method Development

The primary target in developing this HPLC method is to achieve simultaneous determination of Moxifloxacin hydrochloride and Flavoxate hydrochloride in the compound formulation under common conditions that are applicable for the routine quality control of this product in ordinary laboratories. After several tests with the pH, the optimal pH value was found to be 3. In Ultraviolet region both the drugs exhibits significant absorption, their Isoabsorptive point was found to be at 218 nm wavelength.

Mobile phase was selected in terms of its components and proportions. This work began with a binary mixture of methanol and water in the ratio of 50:50(v/v at pH 3). After several trials, it was found that the incorporation of 0.1% formic acid in water with acetonitrile in the ratio 25:75 (v/v at pH 3) in the mobile phase favored the separation and elution of the two drugs. As it produced good resolution, reasonable retention and acceptable peak shapes for both drugs. No significant peaks were observed in chromatograms, indicating no interference from the formulation matrix. A typical chromatogram for tablet sample solution is shown in fig.1. The retention time is 5.85 min. for Moxifloxacin hydrochloride and 8.35 min. for Flavoxate hydrochloride. The run time is less than 10 min.

Fig.1: A typical Chromatogram for the tablet sample containing 400µg/ml of Moxifloxacin hydrochloride and 200µg/ml of Flavoxate hydrochloride

Method Validation

(A) System Suitability

A 20µl solution of Moxifloxacin hydrochloride (400µg/ml) and Flavoxate hydrochloride (200µg/ml) solution was injected and parameter like theoretical plate per column, tailing factor and resolution were calculated.

Table 1: Data for system suitability parameters for Moxifloxacin Hydrochloride & Flavoxate hydrochloride

Sr. No. Parameters Moxifloxacin Hydrochloride Flavoxate Hydrochloride
1 Resolution (USP) 0.00 2.68
2 Asymmetry

(NMT 10% – USP)

2.51% 3.02%
3 Theoretical Plates 4916.18 4937.98

 Report

The system suitability parameters determined for both Moxifloxacin Hydrochloride & Flavoxate hydrochloride were found to be within the acceptance criteria.

(B) Accuracy

Accuracy was determined by applying the developed method to mixtures of excipients to which known amounts of each drug corresponding to 80, 100 and 120 % of label claim had been added. The accuracy was then calculated as the % of analyst recovered from the formulation matrix.

 Table 2: Accuracy (Recovery) data for Moxifloxacin

Conc. In sample (µg) Conc. Added (µg/ml) Total Conc. (µg/ml) Peak Area Conc. Recovered (µg/ml) % Recovery Mean

% Recovery

400 0.8 320 43568319 322.521 100.787 100.537
400 0.8 320 43821470 324.394 101.373
400 0.8 320 42990898 318.246 99.452
400 1 400 53174669 393.633 98.408 100.489
400 1 400 54980104 406.998 101.749
400 1 400 54743391 405.246 101.311
400 1.2 480 64093510 474.461 98.846 99.224
400 1.2 480 63991478 473.706 98.688
400 1.2 480 64930710 480.659 100.137

 Table 3: Accuracy (Recovery) data for Flavoxate

Conc. In sample (µg) Conc. Added (µg/ml) Total Conc. (µg/ml) Peak Area Conc. Recovered (µg/ml) % Recovery Mean

% Recovery

200 0.8 160 97632141 161.046 100.654 99.697
200 0.8 160 96960104 159.938 99.961
200 0.8 160 95520997 157.564 98.477
200 1 200 122863741 202.666 101.333 99.873
200 1 200 120401193 198.604 99.302
200 1 200 120014901 197.967 98.983
200 1.2 240 145987963 240.810 100.337 100.407
200 1.2 240 146502014 241.658 100.691
200 1.2 240 145778901 240.465 100.194

 Table 4: Accuracy (Recovery) report for Moxifloxacin & Flavoxate

Total Drug Concentration % Recovery of Moxifloxacin % Recovery of Flavoxate Acceptance Criteria
80% 100.537 99.697 98% – 102%
100% 100.489 99.873 98% – 102%
120% 99.224 100.407 98% – 102%

 Table 5: System Precision Data for Moxifloxacin & Flavoxate

Sr. no. Moxifloxacin Flavoxate
RT Peak Area RT Peak Area
1 5.852 53174669 8.360 122863741
2 5.898 54980104 8.395 120401193
3 5.960 54743391 8.365 120014901
4 5.863 53982041 8.347 121366869
5 5.963 54690073 8.410 122032031
6 5.985 54906357 8.388 119985213
Average 5.920 54412773 8.3775 121110658
S.D. 0.056623 702869.08 0.0239395 1179705.28
% R.S.D. 0.956% 1.291 0.285% 0.974

 Report

The mean % Recovery for Moxifloxacin & Flavoxate was shown in above table, which were well within the acceptance limit of 98% – 102% and hence the method was found to be accurate.

(C) Precision

The system precision is a measure of the method variability that can be expected for a given analyst performing the analysis and was determined by performing six replicate analyses of the same working solution.

The Method Precision of the developed HPLC method was determined by injecting and three replicates each of three concentrations. The %RSD (Relative standard deviation) of the result, expressed as a percentage of the label claim which was used to evaluate the method precision.

Table 6: Method Precision (Repeatability) data for Moxifloxacin hydrochloride.

 

Concentration

( µg/mL)

 

Area Mean Area S.D % R.S.D
80 40568339 41246500 710672.35 1.722
80 41985741
80 41185420
100 54246398 54493122 618466.5 1.134
100 54036100
100 55196869
120 62924369 63207392 428025.2 0.677
120 63699801
120 62998006

Table 7: Repeatability report for Moxifloxacin hydrochloride

 

Concentration

( µg/mL)

%R.S.D – Area of Moxifloxacin Acceptance Criteria
80 1.722 Not more than 2%
100 1.134 Not more than 2%
120 0.677 Not more than 2%

 Table 8: Method Precision (Repeatability) data for Flavoxate hydrochloride

Concentration

( µg/mL)

Area Mean Area S.D % R.S.D
80 92633323 93331299 605221.33 0.648
80 93710541
80 93650032
100 116953489 118979858 1761880.7 1.480
100 120149874
100 119836210
120 145942598 147240394 2079176.6 1.412
120 149638512
120 147240394

 Table 9: Repeatability report for Flavoxate hydrochloride

 

Concentration

( µg/mL)

 

%R.S.D – Area of Moxifloxacin Acceptance Criteria
80 0.648 Not more than 2%
100 1.480 Not more than 2%
120 1.412 Not more than 2%

 Intermediate Precision (Ruggedness) of the developed HPLC method was determined by injecting six replicates of the same concentration but by different analyst in different days followed by calculating the % Recovery of the drugs.

Table 10: Intermediate Precision (Ruggedness) Data by Analyst 1

Sr. No. Analyst 1
Area of Moxifloxacin Area of Flavoxate % Recovery of Moxifloxacin % Recovery of Flavoxate
1 54298001 121245693 100.487 99.998
2 53140998 119396001 98.346 98.473
3 53174669 119774130 98.408 98.785
4 54980104 122863741 101.749 101.333
5 54743391 120401193 101.311 99.302
6 54863887 120014901 101.534 98.983
Average 54200175 120615943 100.306 99.479
S.D. 839877.95 1269130.2 1.554331 1.046731
% R.S.D 1.549 1.052

 Table 11: Intermediate Precision (Ruggedness) Data by Analyst

Sr. No. Analyst 2
Area of Moxifloxacin Area of Flavoxate % Recovery of Moxifloxacin % Recovery of Flavoxate
1 54982014 119638740 101.753 98.673
2 53620147 121400369 99.232 100.126
3 53966595 122739228 99.873 101.230
4 54001231 119446096 99.938 98.514
5 54802998 120479464 101.421 99.366
6 54787124 120339447 101.392 99.251
Average 54360018 120673891 100.602 99.527
S.D. 565057.29 1226544.74 1.04573029 1.011608
% R.S.D 1.039 1.016

 Table 12:  Intermediate Precision (Ruggedness) report for Moxifloxacin & Flavoxate

Parameters Moxifloxacin Flavoxate Acceptance Criteria
Analyst 1 Analyst 2 Analyst 1 Analyst 2
% Recovery 100.306% 100.602% 99.479% 99.527% 98% – 102%
% R.S.D 1.549 1.039 1.052 1.016 NMT 2%

 Report

The % R.S.D values of assay for Moxifloxacin & Flavoxate by Analyst 1 & Analyst 2 were shown in the above table which is well within the acceptance criteria limit.

(D) Linearity

The Linearity of the responses of the two drugs was verified at the 7 concentrations levels ranging from 80 to 320 µg/ml for Moxifloxacin hydrochloride & 40 to 160 µg/ml for Flavoxate hydrochloride, respectively. The calibration curve was constructed by plotting mean area response Y against concentration C of each drug.

The regression equations obtained for the two drugs were

Y = 52401x – 79777 (r2 = 0.999) for Moxifloxacin hydrochloride

Y = 168806x – 857706 (r2 = 0.999) for Flavoxate hydrochloride

(E) Robustness

This parameter was carried out to check the ability of the system to give unaffected results for small deliberate changes in the system parameters and method parameters.

Change in Wavelength

The set wavelength was changed from 218 nm to 216nm & 220 nm to check the ability of the system to give good results.

Table  13: Robustness Study Report of Moxifloxacin & Flavoxate for change in Wavelength

Change in Wavelength Decreased Increased Acceptance Criteria
% RSD for area Moxifloxacin 0.843 1.311 NMT 2.0
Flavoxate 1.372 1.093
Asymmetry factor for peak (USP) Moxifloxacin 2.51 2.33 NMT 10.0
Flavoxate 3.02 2.51
Theoretical plate Moxifloxacin 4123 4158 NLT 4000
Flavoxate 4332 4360

 Report

From the above Observation, it can be concluded that the method is Robust with respect to change in the wavelength.

Change in Flow Rate

Table 14: Robustness Study Report of Moxifloxacin & Flavoxate for change in Flow-rate

Change in Flow rate Decreased Increased Acceptance Criteria
% RSD for area Moxifloxacin 1.125 0.615 NMT 2.0
Flavoxate 1.335 1.403
Asymmetry factor for peak (USP) Moxifloxacin 2.53 2.20 NMT 10.0
Flavoxate 3.01 2.99
Theoretical plate Moxifloxacin 4120 4206 NLT 4000
Flavoxate 4302 4369

Report

From the above Observation, it can be concluded that the method is Robust with respect to change in the Flow-rate.

Assay of the Tablets

The developed and validated HPLC method was used to determine Moxifloxacin & Flavoxate in combined dosage form (Tablets).

Formulation Used : Tablet

Dosage : 400 mg of Moxifloxacin hydrochloride + 200 mg of Flavoxate hydrochloride

20 µL of the sample solutions were injected into HPLC system & the results obtained are presented below :-

Table 15: Determination of Assay of Moxifloxacin hydrochloride

Conc. Moxifloxacin hydrochloride
Peak Area Conc.(µg/ml) % Assay
400 µg/ml 53983680 399.622 99.90%
400 µg/ml 53290132 394.488 98.62%
400 µg/ml 53960363 399.449 99.86%

 Table 16: Determination of Assay of Flavoxate hydrochloride

Conc. Flavoxate hydrochloride
Peak Area Conc.(µg/ml) % Assay
200 µg/ml 119639685 197.348 98.67%
200 µg/ml 120401193 198.604 99.30%
200 µg/ml 120014901 197.967 98.98%

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