Boodida Sathyanarayana (Orcid ID: 0000-0003-3034-8609)

A Rapid RP-HPLC Stability Indicating Method Development and

Validation of Moxifloxacin Hydrochloride Related Substances in Finished

Dosage Forms

Krishna Rao Vankalapati *, Pallavi Algete and Sathyanarayana Boodida

Department of Chemistry, JNTUH College of Engineering Jagitial, Nachupally (Kondagattu),

Telangana State, 505501, India

Correspondence Author:

Dr. Sathyanarayana Boodida, Department of Chemistry, JNTUH College of Engineering,

Jagitial, Telangana State-505501, India; Mobile: 09010069233; mail id: bs14@jntuh.ac.in

Abstract:

A Reversed Phase High Performance Liquid Chromatography (RP-HPLC) method was

developed and validated for the identification and quantification of the Moxifloxacin

hydrochloride related substances in finished dosage forms. Efficient chromatographic separation

has been achieved on a Agilent C18 (150 x 4.6) mm, 5m column with mobile phase (0.01M

Potassium dihydrogen ortho phosphate as buffer and methanol in the ratio of 70:30) eluted in

isocratic mode. The HPLC flow rate was 1.0 mL/minute and peaks were monitored at 230 nm

with Photo Diode Array (PDA) detector. The column over temp was kept constant at 30° C and

the injection volume was 10µl. The run time of method was 16 min. The method was validated

as per the International Conference on Harmonization (ICH) guidelines. Linearity was recorded

at various concentrations ranging from 0.25 to 1.5 µg/mL for all the Moxifloxacin impurities.

Linearity, regression value, recovery, %Relative Standard Deviation (RSD) of method precision
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lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/bmc.5192

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values was found within the acceptance limits. The method for Related Substances (RS) in

Moxifloxacin was found to be specific, linear, accurate, precise, rugged and robust. The

validated method was suitable for quantification of the related substances in Moxifloxacin drug

product. The method was applied in Quality control lab for the analysis Moxifloxacin impurities

in stability analysis.

Keywords: Moxifloxacin, Related Substances, Method development, Validation, HPLC

Introduction:

Moxifloxacin is an antibiotic used to treat a number of bacterial infections. This includes

pneumonia, conjunctivitis, endocarditis, tuberculosis, and sinusitis. Moxifloxacin is in

the fluoroquinolone family of medications. It usually results in bacterial death through blocking

their ability to duplicate DNA. Moxifloxacin is an antibiotic examined as green corrosion

inhibitor for carbon steel in 1M HCl solution with chemical and electrochemical methods at 250

C. Moxifloxacin is a broad-spectrum antibiotic that is active against both Gram-

positive and Gram-negative bacteria. It functions by inhibiting DNA gyrase, a type

II topoisomerase, and topoisomerase IV, enzymes necessary to separate bacterial DNA, thereby

inhibiting cell replication (Fouda et al., 2016; Prof. Richard Wise 1999). The chemical

structures of Moxifloxacin and its related impurities were depicted in Figure 1.

Number of stability indicating HPLC methods for the self-determining analysis of Moxifloxacin

(Predrag Djurdjevic et al., 2009; M. Lalitha Devi & K. B. Chandrasekhar, 2009; Wu, Cai Sheng

et al., 2012; Razzaq, Syed Naeem et al., 2014; Syed Naeem Razzaq et al., 2012; Xander Van

Doorslaer et al., 2012; Attimarad et al., 2019; Momin, Mohammad A. M et al., 2018) reported in

the literature were followed good chromatographic integration practices as per USP <621>

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general chapter. The different combination formulations reported in the literature. To the best of

our knowledge, no HPLC related substances method has been described for separation of five

known impurities within 10 minutes from Moxifloxacin formulation.

The main objective of this study was to develop a rapid, sensitive and stability indicating HPLC

method to estimate the impurities and stability of Moxifloxacin in Moxifloxacin formulation.

According to analytical method validation guidelines (Yuwono & Indrayanto, 2005; ICH

Guidelines, 2003; 2005); stability indicating HPLC analytical method is critical in estimating

the stability of the drugs in final formulations (Bakshi & Singh, 2002). In the current study

Performed forced degradation as per ICH condition and proved the stability indicating nature.

The forced degradation study under different stressed conditions (acid, base, peroxide, thermal,

UV and water) applied to separation of drugs from degradation products (Jain et al., 2013;

Palakurthi et al., 2013; 2017; thirupathi et al., 2018; 2020).

Materials and Methods

Chemicals and Reagents:

HPLC grade of methanol was purchased from Merck Pvt. Ltd, Mumbai, India. Water purified

on Millipore was used for throughout the analysis. The AR grade ortho phosphoric acid (OPA,

purity, 85 %), Triethyl amine, and potassium dihydrogen ortho phosphate (KH2PO4, purity 99.9

%) were purchased from SD fine chemicals, Mumbai, India. HPLC grade water used throughout

the experiments. Any other reagents or chemicals used were of analytical or HPLC grade.

Instruments:

The analysis was carried out using a HPLC 2695 SYSTEM with PDA detector integrated with

Empower 2 Software. The HPLC instrument consisted of a binary pump, an online degasser, an

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auto-sampler, a thermostatically controlled column compartment and a photo diode array (PDA)

detector used ranging from 200-400 nm. The data were collected and processed using Empower-

2 software from waters. The HPLC column is (150 x 4.6) mm, 5m, manufactured by Agilent

Technologies. Sartorius analytical balance range from for the 0.1g to 220 g used for preparation

of standard and samples. Bransonic ultrasonic baths used for extraction impurities from sample

matrix.

Chromatographic conditions:

The chromatographic separation was achieved with isocratic mobile phase. Prepared 10mM

Potassium dihydrogen ortho phosphate buffer solution in water, added 1 mL of Triethylamine

and then finally adjusted pH to 4.8 with diluted orthophosphoric acid solution. Buffer and

methanol are mixed in the ratio of 70:30 v/v respectively. Mixture of buffer and methanol in the

ratio of 50:50 v/v was prepared for diluent purpose. The HPLC flow rate was 1.0 mL/min and

used Agilent (150 x 4.6) mm, 5m column, maintained temperature at 30° C. The injection

volume was 10 µL and UV detection was carried out at 230 nm.

Preparation of Moxifloxacin and its impurity standard mixture solution

Each 0.5mg of Moxifloxacin standard and its impurities dimethoxy, hydroxy acid, methyl

stearate, dimethoxy acid and gati acid were accurately weighed and transferred to a 10 mL

volumetric flask and dissolved in small amount of diluent. The solution was sonicated for 5min

and the solution was making up with diluent to 10mL which gives 50ppm solution for each

component. 0.2 mL of the above solution was transferred to another 10 mL volumetric flask and

was making up with the diluent to give 1ppm solution.

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Preparation of Sample solution (1000ppm)

Weigh and transferred 149 mg of sample powder (Equivalent to 100 mg of Moxifloxacin) into a

100 mL volumetric flask. Added about 70 mL of diluent and shaken for 20 minutes by

mechanical or manually and further sonicated for 30 minutes. The solution was diluted up to the

mark with the diluent and filtered the solution through 0.45 µm PVDF filter.

Results and Discussion:

The objective was to develop a new RS method to identify and separate close eluting

Moxifloxacin related substances like A, B, C, D and E with Moxifloxacin with smaller run time.

To achieve the objective, different methods were adopted by considering variables like column,

mobile phase composition and elution mode (Table 1). All the analytes are detected at 230 nm,

well resolved and system suitability chromatogram shows method specificity, hence the

developed method is applicable for intended purpose.

Method Validation:

The proposed method was validated as per the international conference on harmonization (ICH

2005, Ashok et al., 2019) guidelines for the parameters-system suitability, specificity, method

precision, linearity, range, accuracy, robustness and solution stability.

System Suitability:

The standard mixture containing Moxifloxacin standard and its impurities (1µg/mL) injected

and analyzed as six replicates. The tailing factor, theoretical plate counts, RT and Resolution for

Moxifloxacin and its impurities were determined at first injection and observed satisfactory

results which are presented in Table-2. The % RSD of peak areas observed for Moxifloxacin:

0.5; Imp-A: 0.6; Imp-B: 0.7; Imp-C: 0.4; Imp-D: 0.7 and Imp-E: 1.0. The system suitability

chromatogram represented in Figure 2.

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Linearity and Range:

Independent calibration curves for Moxifloxacin and its impurities were developed using the

optimized chromatographic conditions. Standard test solutions were prepared at concentration

levels ranging from 0.25 µg mL-1 to 1.5 µg mL-1 and generated by six concentration points. The

peak area versus concentration data was analyzed with least squares linear regression (Katakam

et al., 2020; Lakshmi et al., 2020). Correlation coefficient (r) and % y-intercept ratio to the

response of target concentration were found above 0.99 and below 5.0%, respectively,

indicating the good linearity of the method. Moxifloxacin and its impurity linearity results were

represented in Table-3.

Limit of Detection (LOD) and Limit of Quantification (LOQ) Establishment:

Detection of a lower level of an analyte in a sample that can be detected as per the developed

chromatographic conditions and no need to be quantified an exact value (Dongala et al., 2019a;

2019b). Quantification at the lower most of analyte in a sample shall be quantified with

acceptable accuracy and precision in the developed chromatographic conditions. Solutions at

LOQ level were injected six times, RSD value of areas was found below the acceptance limit of

5.0%. The results were tabulated in Table-3.

Accuracy / Recovery:

The accuracy of the optimized method was determined at three levels (LOQ, 50, 100 & 150%).

Prepared the samples at each level (n=3), then spiked with the impurity solution mixture at 0.5

µg mL-1, 1.0 µg mL-1 and 1.5 µg mL-1 for all impurities of Moxifloxacin. Each individual

impurity recovery was calculated by dividing the amount found in µg mL-1 with amount added

in µg mL-1. The % of recovery for each impurity was shown more than 95%. All the method

recovery results were shown in Table-3. Recovery RSD value of each related substance was

<2.0 % (n=9)

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Method Precision:

The method precision was evaluated by carrying out six independent preparations of a

homogenous test sample of Moxifloxacin drug product to specify the method reliability results

for a single batch with nominal concentrations spiking of impurities at 1ppm level. Six test

solutions of the single batch were analysed as per the proposed methodology and which was

considered as intra-day precision (Dongala et al., 2021; Subramnian et al., 2020). Inter-day

precision was evaluated from a total of six replicates with spiking of impurities at 1ppm level on

sample and analyzed on another day using different column and instrument. The precision was

expressed as the % RSD of measured responses. The results for Intra- and Inter-day precision

data for Moxifloxacin and its impurities in Moxifloxacin drug product were presented in Table-

3. The % RSD for all impurities in both intra- and inter- day precision data was < 10 and

difference between their mean of results was in the range of ±10, indicating that this method is

more precise. For both intra and inter precision study, peak tailing are observed 0.9 to 1.3 and

plate counts for Moxifloxacin, Imp-A, Imp-B, Imp-C, Imp-D and Imp-E were > 2000, >7000,

8000, > 8800, > 9500 and >9800 respectively. The developed method precision chromatograms

are shown in Figure 3.

Specificity:

Specificity is the ability of the method to measure the analyte response in the presence of its

potential impurities. Stress testing of the drug substance can help to identify the likely

degradation products, which can in turn help to establish the degradation path ways and the

intrinsic stability of the molecule and validate the stability indicating power of the analytical

procedure used (Dongala et al., 2019; Palakurthi et al., 2020). In specificity test, no overlapping

peak was observed from diluent and placebo chromatograms at the retention time of

Moxifloxacin and its impurities. All impurity peaks are well separated from each other and

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found spectrally pure (purity angle < purity threshold). Thus, the method was found specific for

determination of related impurities in Moxifloxacin drug product. Specificity chromatograms

with purity plot and purity threshold for Moxifloxacin and its impurities, indicating no

interference of blank, placebo and other peaks with the retention time of Moxifloxacin and its

impurities.

Forced Degradation Study:

The current regulatory frame work requires conducting forced degradation experiments under a

verity of conditions and separation of drugs from degradation products (Jain & Basniwal, 2013;

Subramanian et al., 2020; Thirupathi et al., 2018). Forced degradation study was evaluated for

acid, base, peroxide, thermal, UV and water. The purity angel and purity threshold data shows

that there are no co-eluting peaks for Moxifloxacin and its impurities in all degradation

conditions (Katakam et al., 2021a, 2021b). Mass balances show that the developed method is

specific. The forced degradation results were shown in Table 4.

Robustness:

In order to demonstrate the robustness of the method, system suitability parameters were

verified and test solution spiked with impurities at 1µg/mL limit, were tested by changing flow

rate by ±0.2 mL/min, column temperature by ±5 ◦C, mobile phase by ±10 units and mobile

phase pH variation with ±0.2 units. No significant difference was found between the normal and

altered conditions indicating the robustness of the method. The results showed that our

stability-indicating RS method was specific, precise, accurate and robust, and it could be used

for RS analyses of Moxifloxacin drug product during release analyses, stress-testing and

stability studies.

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Stability of standard and sample solutions:

Stability of the standard and sample solutions was assessed from 0 to 24 hours at room

temperature and refrigerator conditions. The stability study results showed that no additional

peaks were observed, and the samples were stable for 24 hours at room temperature and

refrigerator. These results were consistent with the acceptance criteria of the Moxifloxacin drug

product.

Conclusion:

The developed and validated RS method for related substances in Moxifloxacin was simple,

specific, stable, linear, accurate, precise, rugged and robust in the range of 25-150% of

specification level. The method was found to meet the entire acceptance criteria as per ICH

guidelines. Based on the validation study results, it has been concluded that the developed

HPLC method for related substances in Moxifloxacin was suitable for routine analysis to

identify and quantify the related substances in the pharmaceutical industry.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgement:

I express my sincere thanks to Department of Chemistry, JNTUH College of Engineering,

Jagitial, Telangana state for providing opportunity to carry out this research work.

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 Name Molecular Molecular Structure
formula weight
Moxifloxacin C21H24FN3O4 401.431
g/mol

N-Methyl tetrachloro C9H3Cl4NO2 298.94 g/mol

phthalimide (Imp-A):

Moxifloxacin Difluoro C13H9F2NO4 281.21 g/mol

Hydroxy Acid Impurity

(Imp-B)

Methyl Stearate C22H26FN3O4 415.46 g/mol

Impurity (Imp-C)

Di methoxy Acid C22H27N3O5 413.5 g/mol

Impurity (Imp-D)

Gotic Acid Impurity C14H11F2NO4 295.24 g/mol

(Imp-E)

Figure 1: Chemical Structures of Moxifloxacin and related impurites

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Figure 2: A Typical system suitability Chromatogram for Moxifloxacin and impurites

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Figure 3: A Typical method precision Chromatogram for Moxifloxacin and impurites

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 Table-1: Method optimization experiments and inference

Experiment Mobile phase Column Inference

no.
1 Methanol and KH2PO4 Discovery (250 x 4.6) mm, The resolution between the
(50:50 v/v) 5µm. 30oC impurities not adequate
2 Acetonitrile and 0.1% Waters (Symmetry) (250 x Imp-E was merging with main
OPA (40:60 v/v) 4.6) mm, 5µ. 30oC peak and observed poor
resolution
3 Acetonitrile and 0.1% Waters (Symmetry) (250 x Imp-E totally merged with
OPA (20:80 v/v) 4.6) mm, 5µ. 30oC main peak and other four
impurities resolution was not
satisfied
4 Methanol and KH2PO4 Agilent (250 x 4.6) mm, Better than previous method
(40:60 v/v) 5µm, 30oC but need to separate Imp-E with
better resolution
5 Acetonitrile and Agilent (250 x 4.6) mm, All separated with good
KH2PO4 (20:80 v/v) 5µm, 30oC resolution but Imp-E peak
shape was not satisfied
6 Acetonitrile and Agilent (250 x 4.6) mm, All peaks separated with main
KH2PO4 (30:70 v/v) 5µm, 30oC peak

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 Table-2: System Suitability Results Moxifloxacin and related impurities

Name of the component RT Plate count Tailing Resolution

Moxifloxacin 2.345 2362 1.22 -
Imp-A 5.997 7932 1.10 15.2
Imp-B 6.890 8178 1.08 3.2
Imp-C 7.614 9124 1.08 2.2
Imp-D 8.474 9736 1.10 2.6
Imp-E 9.811 8431 1.05 3.4

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 Table 3: Method validation Results and summary

Parameters Moxifloxacin Imp-A Imp-B Imp-C Imp-D Imp-E

RT 2.345 5.997 6.890 7.614 8.474 9.811
Plate count 2362 7332 8178 9124 9736 8431
Tailing 1.22 1.10 1.08 1.08 1.10 1.05
Resolution - 15.2 3.2 2.2 2.6 3.4
RRf 1.00 0.73 0.93 0.77 0.73 0.19
Range (µg /mL) 0.25-1.50 0.25-1.50 0.25-1.50 0.25-1.50 0.25-1.50 0.25-1.50
Slope 670213 490573 626308 516443 490796 126329
Intercept 3464.31 140.98 1266.31 5271.16 1118.04 302.42
% Y-intercept at bias 0.52 0.03 0.20 1.01 0.23 0.24
Correlation coefficient 0.9997 0.9998 0.9998 0.9999 0.9998 0.9998
LOD (µg /mL) 0.04 0.03 0.03 0.01 0.03 0.03
LOQ (µg /mL) 0.12 0.08 0.10 0.04 0.11 0.08
a
Accuracy (percentage of recovery)
LOQ Mean, % RSD 98.0, 0.4 99.8, 0.2 101.7, 0.2 98.0, 0.4 100.5, 1.0
50% Mean, % RSD 97.9, 0.3 98.8, 1.2 99.0, 0.4 98.2, 0.3 99.2, 0.5
100% Mean, % RSD 99.0, 0.2 98.2, 0.6 99.5, 0.6 98.5, 0.2 98.1, 0.1
150% Mean, % RSD 98.1, 0.3 96.7, 0.4 99.3, 0.6 98.4, 0.2 98.8, 0.5
b
Precision (RSD, %)
Repeatability 1.5 0.6 0.4 0.3 0.6 0.4
Intermediate precision 0.4 0.6 1.5 1.5 1.6 0.5
LOQ precision 0.9 1.6 2.5 0.9 1.2 1.5
a
Average of three determinations of each concentration levels.
b
RSD of six determinations of each component.

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 Table 4: Forced degradation condition and results

Forced Degradation % of total Mass Purity Purity

impurities Balance angle thresholds

Acid 4.83 95.1 0.344 0.346

Base 4.72 95.2 0.323 0.337
Peroxide 4.01 95.9 0.350 0.401
Thermal 3.3 96.7 0.387 0.429
UV 2.27 97.7 0.415 0.454
Water 0.84 99.2 0.458 0.532

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