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Original Article

RP-HPLC determination of pazufloxacin in injectable dosageform

Kalaichelvi Ramalingam a,b,*, Jayachandran Ekambaram c

aDepartment of Pharmaceutical Analysis, K.C. Reddy Institute of Pharmaceutical Sciences, Jangamguntla Palem, Medikonduru Mandal,

Guntur 522 348, IndiabDepartment of Science and Technology, Jawaharlal Nehru Technological University Hyderabad, Kukatpally, Hyderabad 500 085,

Andhra Pradesh, IndiacDepartment of Pharmaceutical Chemistry, S.C.S. College of Pharmacy, Harapanahalli 583131, Devanagari Dist, Karnataka, India

a r t i c l e i n f o

Article history:

Received 31 January 2013

Accepted 16 February 2013

Available online 17 March 2013

Keywords:

Injection formulation

Pazufloxacin

RP-HPLC estimation

Validation

* Corresponding author. Tel.: þ91 (0) 8632327E-mail addresses: rkselvi1983@gmail.com

0974-6943/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.jopr.2013.02.016

a b s t r a c t

Aim: The present work aim to develop a simple, accurate and sensitive RP-HPLC method

and validated for the estimation of pazufloxacin in pure drug and it’s injectable dosage.

Methods: A reverse phase high performance liquid chromatographic method was per-

formed by using Kromasil C18, 150 � 4.6 mm, 5 mm column with UV detection at 246 nm.

The mobile phase consists of 0.05 M phosphate buffer (pH 3) and acetonitrile in the ratio of

80:20% v/v.

Results: The retention time for pazufloxacin was 3.7 min. The method was linear in the

concentration range of 12.5e150 mg/mL of pazufloxacin with the correlation coefficient of

0.999. In the intra-day and intermediate precision, coefficient of variation was less than 1%

showing high precision of the method. The method was highly accurate with a recovery in

the range 99.01e100.36%.

Conclusion: The developed reverse phase high performance liquid chromatographic method

was simple, sensitive, precise and accurate and the method was found suitable for esti-

mating pazufloxacin in injection formulation.

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction estimated in human plasma andurine by HPLC3 and a validated

Pazufloxacin is chemically, (3R)-10-(1-aminocyclopropyl)-9-

fluoro-2,3-dihydro-3-methyl-7-oxo7H-pyrido[1,2,3-de]1,4-benz

oxazine-6-carboxylic acid.1 Pazufloxacin is broad spectrum flu-

oroquinolone antibiotic and it exhibits antibacterial activity by

inhibiting DNA gyrase thus preventing DNA replication and

synthesis.2 The literature survey reveals that the drug can be

766., rkselvi123@rediffmail.c2013, JPR Solutions; Publi

stability-indicating RP-HPLC method for the estimation of

pazufloxacin in presence of its degradation products.4

Based on the literature survey authors found that there

was no any RP-HPLC method to quantify the drug in pure and

formulation. The aim of the study was to develop a simple,

precise and accurate RP-HPLC method for the estimation of

pazufloxacin in pure drug and in injectable dosage form.

om (K. Ramalingam).shed by Reed Elsevier India Pvt. Ltd. All rights reserved.

Fig. 1 e A model chromatogram of pazufloxacin standard solution.

j o u r n a l o f p h a rm a c y r e s e a r c h 7 ( 2 0 1 3 ) 1 7 2e1 7 4 173

2. Experimental methods

2.1. Instrumentation

Waters 2695 HPLC system equipped with Kromasil C18,

150 � 4.6 mm, 5 mm column, Rheodyne injector with 20 mL

loop, 2996 PDA detector and Empower-2 software was used.

2.2. Chemicals and reagents

The mobile phase consisted of 0.05 M phosphate buffer (pH 3)

and acetonitrile in the ratio of 80:20% v/v that was set at a flow

rate of 1 mL/min. All the regents and solvents used are

analytical and HPLC grade. The mobile phase buffer was pre-

pared by dissolving 6.8 g potassium dihydrogen orthophos-

phate in 1000 ml of water and pH adjusted to 3 with

orthophosphoric acid. The pure drug of pazufloxacin was

obtained from commercial supplier India. Injectable formu-

lation of the drug was obtained from local pharmacy.

2.3. Drug stock solution

Stock solution of pazufloxacin was prepared by dissolving

accurately weighed 100 mg of the drug in 100 mL of HPLC

grade water (final concentration, 1 mg/mL). The prepared

stock solution was stored at 4 �C protected from light.

Fig. 2 e A chromatogr

2.4. Construction of calibration plot

Calibration plot was constructed by analysis of appropriate

working solutions (concentration 12.5, 25, 50, 75, 100, 125 and

150 mg/mL) of pazufloxacin in the mobile phase and plotting

concentration against peak area response for each injection.

Unknown samples were quantified by reference to this cali-

bration plot.

2.5. Sample preparation

The pazufloxacin injectable dosage form was diluted with

mobile phase to get 50 mg/mL of drug and filtered through a

0.2 mm membrane filter. From this solution 20 mL was injected

for HPLC analysis.

3. Results and discussion

The developed method was optimised to obtain the best

chromatographic conditions, the wavelength for detection of

drug without any interference of additives used for the prep-

aration of formulation, the column and the mobile phase

composition must be effectively selected. Column chemistry,

solvent type, solvent strength, detection wavelength and flow

ratewere varied to determine the chromatographic conditions

giving the best separation of pazufloxacin. The mobile phase

am of excipients.

Table 1 e System suitability study.

Drug USP tailing USP plate count Retention time (min), (n ¼ 6) Peak area, (n ¼ 6)

Mean � S.D. %CV Mean � S.D. %CV

Pazufloxacin 50 mg/mL 1.56 3126 3.70 � 0.0615 1.662 1838099 � 3011 0.1637

j o u rn a l o f p h a rma c y r e s e a r c h 7 ( 2 0 1 3 ) 1 7 2e1 7 4174

conditions were optimised so there was no interference with

the drug peak from solvent or excipients peaks. An absor-

bancemaximum for drugwas 246 nm. Solutions of the drug in

the mobile phase were injected directly for HPLC analysis and

the responses (peak area) were recorded at 246 nm. The

retention time of the drug was 3.7 min (Fig. 1). A chromato-

gram of the excipients is shown in Fig. 2.

3.1. Validation of the method

3.1.1. System suitabilityThe system suitability was assessed by six replicate analyses

of the drug at a concentration of 50 mg/mL. The acceptance

criterion was �2% for the percent coefficient of variation (%

CV) for the peak area and retention time. The %CV of peak

area and retention time for drug is within 2% indicating the

suitability of the system (Table 1).

3.1.2. LinearityThe plot of peak areas of each sample against respective

concentration of pazufloxacin was found to be linear in the

range of 12.5e150 mg/mL with correlation coefficient of 0.999.

The regression of acipimox concentration over its peak area

was found to be Y ¼ 36114.33X þ 429.33, where Y is the mean

peak area and X is the concentration of pazufloxacin.

3.1.3. PrecisionThe precision of the method was demonstrated by repeat-

ability and intermediate precision studies. In the repeatability

Table 2 e Precision studies.

Repeatability(%RSD) (n ¼ 6)

Intermediate precision (%RSD) (n ¼ 6)

Day 1 Day 2

Analyst 1 Analyst 2 Analyst 1 Analyst 2

0.1743 0.0949 0.2375 0.2770 0.3768

Table 3 e Assay and recovery results.

Formulation Labelledamount

(mg/100 mL)

(%) Labelclaima � S.D.

% Recovery

Pazufloxacin

infusion

500 99.90 � 0.1742 99.01e100.36%

a Average of six determinations.

studies, solutions of sample were repeated six times in a day

and percentage relative standard deviation (%RSD) for

response factor was calculated. In the intermediate precision

studies, injections of sample solutions were made on 2

consecutive days with two different analyst and %RSD were

calculated. The results of precision studies are expressed in

Table 2. From the data obtained, the developed RP-HPLC

method was found to be precise.

3.1.4. Assay and recovery studies resultsThe HPLC method was applied to quantify the drug from

pharmaceutical formulation (injectable). The amount esti-

mated is tabulated in Table 3. Analytical recovery studieswere

carried out from a series of spiked concentrations added to the

preanalysed dosage form (Table 3).

3.1.5. Limit of detection (LOD) and limit of quantitation (LOQ)Limit of detection and limit of quantification were calculated

using standard deviation of the blank response and slope of

calibration curve. The LOD for pazufloxacin was found to be

0.0147 mg/mL. The LOQ was 0.0446 mg/mL.

4. Conclusion

The developed RP-HPLCmethodwas simple, sensitive, precise

and accurate and hence can be used in routine for the deter-

mination of pazufloxacin in pure as well as pharmaceutical

preparations.

Conflicts of interest

All authors have none to declare.

r e f e r e n c e s

1. The Merck Index. An Encyclopedia of Chemical, Drugs andBiologicals. Whitehouse Station, NJ: Merck Research Laboratory,Division of Merck Co. Inc; 2006. 1212.

2. Martindale. The Complete Drug Reference. vol. 35. London:Pharmaceutical Press; 2004. 813.

3. Li Q, Rui W. Determination of the pazufloxacin mesilate inhuman plasma and urine by HPLC. Asian J Drug Metab Pharm.2004;4:289e293.

4. Rewatkar NS, Umarkar AR, Chaple DR, Ghante MH. A validatedstability-indicating RP-HPLC method for the estimation ofpazufloxacin in presence of its degradation products. J PharmRes. 2011;4:3060e3062.