INTRODUCTION:

Sparfloxacin¹ belongs to abroad spectrum antibacterial fluoroquinolone which acts on certain microorganisms comprising Gram-positive and Gram-negative bacteria and signifies moderate activity on anaerobes and mycobacteria. Chemically Sparfloxacin (Fig.1) is 5-amino-1-cyclopropyl-7-(cis-3,5-dimethyl-piperazin-1-yl)-6,8-difluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid. It is generally used for antibacterial and anti-tubercular drug treatment. Sparfloxacin inhibits the growth of bacteria by acting on the topoisomerase II and topoisomerase IV which are responsible for the bacterial growth². Few visible spectroscopic methods,^3-6 UV spectroscopy,⁷an immune assay method,⁸few electrochemical methods^9-12 and some recently developed HPLC methods^13-17were reported in the literature for estimation of Sparfloxacin in tablets.

Since there are no official spectrophotometric methods reported, the present study has been taken up to develop simple and sensitive UV spectrophotometric methods for quantification of Sparfloxacin in bulk and dosage form.

Fig. 1: Structure of Sparfloxacin

MATERIALS AND METHODS:

A Shimadzu UV/Visible double beam spectrophotometer (model 1700) with 1cm matched quartz cells was used for all spectral measurements. Scanning range of 190-380nm for UV range. All chemicals (A.R. grade) from S.D. Fine-Chem., Mumbai. Reference standard of Sparfloxacin was kindly gifted by Dr. Reddy Labs Pvt. Ltd, Hyderabad.

Preparation of stock solution (1000μg/mL):

Accurately weighed about 50mg of sparfloxacin into a 50mL volumetric flask, dissolved in few mL of methanol and then made up the volume with the same solvent.

Preparation of the working standard solution (100 μg/mL):

5ml of stock solution of sparfloxacin was taken in different 50mL volumetric flasks and diluted with phosphate buffer- pH 6.8 and borate buffer – pH 9.0 and further analyzed.

Preparation of sample solution (from tablets):

20 tablets (Sparquin) each containing 200mg of sparfloxacin were weighed, crushed to powder and average weight was calculated. Powder equivalent to 50.0mg of Sparfloxacin (92.25mg) was weighed, the contents were transferred to a 50mL volumetric flask, dissolved in a few mL of methanol and sonicated for 15 min. The solution was made up to the mark, filtered and from the filtrate 1mL was taken and diluted with phosphate buffer pH 6.8 and borate buffer 9.0 in different 10ml volumetric flasks.

Optimized Method:

Two UV spectroscopic methods (Method A, D^o and Method B, AUC) were developed in phosphate buffer pH 6.8 and borate buffer pH 7.4 after a series of trials using various solvent systems. 10μg/mL solutions of sparfloxacin were prepared and scanned in the range of 200–400nm against blank. Sparfloxacin showed maximum absorption at 290nm in both the buffers. The area under curve was computed in the region between 250 – 312nm.

VALIDATION:

The developed UV spectroscopic methods were validated in terms of linearity, precision, accuracy, robustness, LOD and LOQ as per ICH guidelines¹⁸.

Linearity:

Linearity was performed by studying the Beer-Lambert’s law. From the working standard solution of sparfloxacin, a series of dilutions were prepared in the range of 1-50 μg/mL in phosphate 6.8 and borate 9.0 buffers. These solutions were scanned, and the absorbance of each solution was noted at 290nm and the AUC was measured between 250 – 312nm. The linearity graphs were plotted for absorbance Vs concentration (Method A), AUC Vs concentration (Method B), the regression analysis was performed and the correlation coefficients were calculated.

Molar absorptivity, also known as the molar extinction coefficient, is a measure of how well a chemical species absorbs a given wavelength of light. It allows to make comparisons between compounds without considering differences in concentrations or solution length during measurements. This is calculated from the linearity data.

Precision:

To check the degree of repeatability of both the methods, a series of aliquots of formulation samples were taken in triplicate in 10mL volumetric flasks to obtain a final concentration of 10, 20, and 30μg/mL of sparfloxacin in phosphate buffer 6.8 borate buffer and 9.0. The absorbance and AUC for each solution was measured on the same day at different time intervals and on a different day for intra and inter day study. Assay values were calculated for each solution from the data obtained and subsequently % RSD was calculated.

Accuracy:

Accuracy was studied by standard addition method. To a fixed concentration of the formulation, varying concentrations of pure sparfloxacin solution was added at 50%, 100% and 150% in triplicate at each level and the absorbance and AUC values were measured.

Robustness:

Change in pH of buffer:

3 different concentrations (10, 20, 30μg/mL) were prepared in replicates and absorbance was measured at 290 nm using phosphate buffers (6.7, 6.8 and 6.9) and borate buffers (8.9, 9.0 and 9.1). AUC was also measured for each solution in the region of 250 – 312 nm in both the buffers and the % RSD was calculated for the assay.

Change in wavelength:

3 different concentrations of the drug (10, 20, 30μg/mL) were prepared in replicates and absorbance was measured at different wavelengths (288, 290, 292nm). Similarly, the AUC was also measured at altered wavelength points (248 – 314, 250 – 312, 252 – 310 nm). The assay was calculated for which the % RSD was determined.

LOD and LOQ:

The LOD and LOQ were calculated based on the linearity data using the following formula.

LOD=3.3 σ/S, LOQ=10 σ/S

Where σ is the standard deviation of y-intercept and S is the slope obtained from calibration curve.

Sandell’s sensitivity:

The Sandell’s sensitivity is the concentration of the analyte (μg/mL) which will give an absorbance of 0.001 in a cell of path length 1cm and is expressed as μg/cm². It was calculated for sparfloxacin from the linearity data.

Assay procedure:

The developed methods were applied for the assay of sparfloxacin in tablets. From the sample solution prepared from tablets (SPARQUIN), aliquots were pipetted into 10ml volumetric flasks, suitably diluted with phosphate buffer pH 6.8 and borate buffer 9.0 in replicates. The absorbance and AUC of each solution was measured under the optimized conditions from which the assay was calculated.

RESULTS AND DISCUSSION:

Two simple UV spectrophotometric methods (fundamental and AUC) have been developed for the determination of sparfloxacin. These methods were validated according to the ICH guidelines and applied for determination of sparfloxacin in formulations and the results are discussed below. Various trials were performed using different buffers like phosphate buffer pH 7.0, 6.8 and 4.9, acetate pH 4.0, borate pH 9.0. Among the different solvents used phosphate buffer pH 6.8 and borate buffer 9.0 were chosen as the optimized solvents because the peaks obtained were perfect, they also provided superior recovery and precision for the analyte of interest. Solvent was also selected based on the solubility and pKa of the drug. Sparfloxacin exhibited maximum absorbance at 290 nm in both the buffers and the AUC was measured in between 250–312 nm.

Linearity was estimated by linear regression analysis of the calibration curve. The calibration curves were linear over the concentration range of 1-50μg/mL for both the buffers (phosphate 6.8, borate 9.0) in both methods A and B. The corresponding spectra and linearity plots in the fundamental and AUC methods are given in Fig. 2a to 2d, Fig. 3a to 3d and the data is given in table 1.

Fig. 2a: Overlain spectra in phosphate buffer 6.8 (A)

Fig. 2b: Linearity plot in phosphate buffer 6.8 (A)

Fig. 2c: Overlain spectra in borate buffer 9.0 (A)

Fig. 2d: Linearity plot in borate buffer 9.0 (A)

Fig. 3a: AUC Spectrum in phosphate buffer 6.8 (10 µg/mL)

Fig. 3b: Linearity plot in phosphate buffer 6.8 (B)

Fig. 3c: AUC Spectrum in borate buffer 9.0 (10 µg/mL)

Fig. 3d: Linearity plot in borate buffer 9.0 (B)

Table 1: Linearity data

Conc. (µg/mL)	D˚ Absorbance		AUC
Conc. (µg/mL)	Phosphate buffer 6.8	Borate buffer 9.0	Phosphate buffer 6.8	Borate buffer 9.0
1	0.065	0.017	1.136	0.165
2	0.125	0.114	2.148	2.029
5	0.333	0.252	5.935	5.017
10	0.636	0.536	11.305	10.019
20	1.207	0.972	21.377	20.139
30	1.807	1.505	32.026	30.599
40	2.429	2.053	43.01	40.619
50	3.033	2.607	53.38	50.017

D˚- Fundamental method

The methods were found to be precise as observed from the % RSD values calculated for the assay of sample solution as given in the below tables 2a and 2b in phosphate 6.8 and borate 9.0 buffers.

Table 2a: Precision data in phosphate buffer 6.8

Conc.
(µg/mL)

D˚

AUC

*Assay (% w/w) ± SD, %RSD

Intraday

Interday

Intraday

Interday

101.9±

0.36, 0.35

101.8±

0.2, 0.19

99.49±

0.57, 0.58

99.83±

1, 1.0

100.18±

0.05, 0.04

100.28±

0.12, 0.11

99.74±

0.28, 0.28

99.4±

0.08, 0.08

100.1±

0.1, 0.09

100±

0.05, 0.05

100.2±

0.88, 0.87

99.6±

0.07, 0.07

* Mean of three replicates

Table 2b: Precision data in borate buffer 9.0

Conc. (µg/mL)	D˚		AUC
	*Assay (% w/w) ± SD, %RSD
	Intraday	Interday	Intraday	Interday
10	101.5± 0.41, 0.40	101.06± 0.54, 0.53	101.01± 1.15, 1.14	101.13± 1.0, 0.98
20	101.03± 0.15, 0.14	101.13± 0.15, 0.14	100.8 0.28, 0.28	100.4± 0.37, 0.37
30	100.2± 0.1, 0.09	100.42± 0.6, 0.55	100.5± 0.44, 0.43	101.3± 1.2, 1.18

* Mean of three replicates

Accuracy:

Recovery studies were carried out at 50, 100, 150% on sparfloxacin tablets. The mean recoveries obtained (98.2 – 101.6%) as given in table 3 indicate the accuracy of the methods.

Table 3: Accuracy data

Level
(%)

D˚

AUC

*Recovery (%) ± SD, %RSD

Phosphate 6.8

Borate 9.0

Phosphate 6.8

Borate 9.0

101.5±

0.10, 0.10

99.9±

0.25, 0.25

98.2±

0.28, 0.28

100.3±

1.06, 1.05

100

101.6±

0.13, 0.12

99.95±

0.77, 0.7

99.9±

0.79, 0.79

99.9±

0.79, 0.79

150

100.05±

0.02, 0.02

100.7±

0.60, 0.59

99.1±

0.05, 0.05

100.7±

0.56, 0.55

* Mean of three replicates

Robustness:

The methods were proved to be robust as they were not much affected by the deliberate changes in pH of the buffer system and wavelength of measurement. The assay and % RSD were found to be within the limits as given in tables 4a and 4b. The overlain spectra for changes in pH of the buffers are given in Fig. 4a and 4b (Method A).

Assay:

The developed methods were used for quantification of sparfloxacin in tablets and the assay obtained as stated against the label claim are given in table 5.

Fig. 4a: Overlain spectra in phosphate buffer (pH 6.7, 6.8, 6.9)

Fig. 4b: Overlain spectra in borate buffer (pH 8.9, 9.0, 9.1)

Table 4a: Robustness data (change in pH)

Conc.
(µg/mL)

D˚

AUC

*Assay (% w/w) ± SD, %RSD

Phosphate 6.8

Borate 9.0

Phosphate 6.8

Borate 9.0

6.7

101.46±

0.15, 0.14

101.2±

0.2, 0.19

100.9±

0.6, 0.59

100.03±

0.95, 0.94

6.8

102.2±

0.45, 0.44

101.6±

0.4, 0.39

100.4±

0.4, 0.45

99.93±

1.10, 1.10

6.9

101.3±

0.2, 0.19

101.4±

0.2, 0.19

100.2±

0.7, 0.70

100.07±

1.00, 0.99

6.7

100.2±

0.07, 0.06

100.3±

0.1, 0.09

99.9±

0.2, 0.28

100.07±

0.55, 0.54

6.8

100.3±

0.12, 0.11

101.1±

0.15, 0.14

99.8±

0.2, 0.23

99.87±

0.56, 0.56

6.9

100.3±

0.07, 0.06

101.25±

0.52, 0.5

100±

0.55, 0.55

100.6±

0.26, 0.25

6.7

100.3±

0.05, 0.04

100.3±

0.1, 0.09

100.4±

0.95, 0.94

100.34±

0.30, 0.29

6.8

100±

0.05, 0.2

100.2±

0.1, 0.09

99.8±

0.23, 0.23

100.03±

0.34, 0.33

6.9

100.03±

0.05, 0.04

100.2±

0.05, 0.04

100.2±

0.58, 0.57

100.16±

0.52, 0.51

* Mean of two replicates

Table 4b: Robustness data (change in wavelength)

Conc. (µg/mL)	λ max (nm)	D˚		AUC region (nm)	AUC
		Assay (% w/w) ±* SD, %RSD			Assay (% w/w) ±* SD, %RSD
		Phosphate 6.8	Borate 9.0		Phosphate 6.8	Borate 9.0
10	288	101.1± 0.72, 0.71	100.2± 1.22, 1.21	248 – 314	101.4± 0.73, 0.71	99.9± 0.67, 0.67
	290			250 – 312
	292			252 - 310
20	288	100.2± 0.24, 0.23	101.1± 0.33, 0.32	248 – 314	100.1± 0.40, 0.39	99.89± 0.66, 0.66
	290			250 – 312
	292			252 - 310
30	288	99.98± 0.09, 0.09	99.6± 0.59, 0.59	248 – 314	100± 0.30, 0.39	100.1± 0.41, 0.40
	290			250 – 312
	292			252 - 310

* Mean of two replicates

Table 5: Assay

Brand name	Parameter	D˚		AUC
Brand name	Parameter	Phosphate 6.8	Borate 9.0	Phosphate 6.8	Borate 9.0
Sparquin (200 mg)	Amount obtained (mg)	198.63	197.52	199.78	196.34
Sparquin (200 mg)	*Assay (% w/w) ± SD	99.32 ± 0.98	98.76 ± 0.85	99.96 ± 1.38	98.17 ± 1.47

* Mean of two replicates

SUMMARY AND CONCLUSION:

An attempt was made to develop simple and specific UV spectrophotometric methods for the determination of sparfloxacin using phosphate buffer pH 6.8 and borate buffer 9.0. The methods were validated as per ICH guidelines and the summary of the optical and validation parameters is given in table 6. The methods were found to be linear, precise, accurate and robust in the working range. The methods were sensitive as calculated from the LOD, LOQ and Sandell’s sensitivity values. These methods were also applied for the assay of sparfloxacin in tablets without any interference from the excipients. These methods were economical with the use of buffer solutions and yet has good optical characteristics thus making them as alternatives to the existing methods. Hence it can be concluded that these methods can be conveniently used in the regular quality control analysis of sparfloxacin in bulk and dosage forms.

Parameter		D˚		AUC
Parameter		Phosphate 6.8	Borate 9.0	Phosphate 6.8	Borate 9.0
λ max (nm)		290		-	-
AUC region (nm)		-	-	250-312
Linearity (μg/mL)		1-50
Slope (y = mx + c)		0.0603x + 0.0118	0.0517x - 0.0125	1.0648x + 0.2308	1.0067x + 0.0647
Correlation coefficient (R²)		0.9998	0.9991	0.9998	0.9998
Molar absorptivity (ε) (L mol^-1 cm^-1)		23981.61	20078.18	-	-
Precision	Intraday	0.04-0.35	0.28-0.87	0.09-0.40	0.28-1.14
Precision	Interday	0.05-0.19	0.07-1.0	0.14-0.55	0.37-1.18
Accuracy (% Recovery)		100.04-101.75	99.62-101.29	98.1-100.8	99.3-101.6
Robustness (% RSD)	Change in pH	0.04-0.44	0.04-0.5	0.23-0.94	0.25-1.10
Robustness (% RSD)	Change in wavelength	0.09-0.72	0.33-0.59	0.39-0.71	0.40-0.67
Assay (% w/w)		99.32	98.76	99.96	98.17
LOD (μg/mL)		0.027	0.174	-	-
LOQ (μg/mL)		0.0834	0.5297	-	-
Sandell's sensitivity (µg/ cm2 /0.001abs.)		0.018	0.015	-	-