Spectrofluorimetric Estimation of Some Fluoroquinolones.

 

Rakesh Patel*¹, Anil Bhandari²

¹Indore Institute of Pharmacy, Indore M P

²Goenka College of Pharmacy, Lachhmangarh, Sikar (Raj) India

 

ABSTRACT:

A highly sensitive spectrofluorimetric procedure is developed for the analysis of certain fluoroquinolones: norfloxacin (NFX), sparfloxacin (SFX) and ofloxacin (OFX) in their pharmaceutical dosage forms and in biological fluids. This procedure is based upon the intrinsic fluorescence of selected drugs in 0.1N H₂SO₄. The excitation (l_ext ) and emission wavelengths (l_emis.) are 270 nm and 464 nm, 276 nm and 442 nm, 280 nm and 510 nm for NFX, SFX and OFX respectively. The calibration curve was rectilinear from the 0.8-2 mg mL^-1, 0.4-2.8 mg mL^-1 and 0.01-0.4 mg mL^-1 for NFX, SFX and OFX respectively. The accuracy and precision of the proposed method was confirmed by estimating six replicates within Beer-Lambert limits for each of the investigated drug with correlation coefficient not less than 0.999. The developed method was applied successfully for the determination of the studied drugs in their pharmaceutical dosage forms and in the biological fluids with a good precision and accuracy compared to official and reported methods as revealed by statistical analysis.

 

 

 

INTRODUCTION:

The antibacterial activity of 4-quinolones is greatly increased by the addition of 6-fluoro and 7-piperazinyl groups hence named fluoroquinolones. Selected drugs are second-generation member of quinolones and greatly effective against both gram positive and gram-negative bacteria. The antibacterial action is due to the ability to inhibit DNA gyrase and topoisomerase IV that is required for DNA replication and transcription. They are rapidly absorbed from gastrointestinal tract and metabolized in liver before being excreted as inactive glucuronides.¹

Norfloxacin is official in IP 1996², USP XXIV³ and BP 2003⁴. These pharmacopoeias recommend non-aqueous titration method for the determination of bulk material and HPLC for the determination in dosage forms. Ofloxacin is official in USP XXIV³ and BP 2003⁴.

 

Both pharmacopoeias recommend non-aqueous titration method for the determination of bulk material and USP XXIV recommend HPLC for the determination in dosage forms. Other analytical methods are also reported in the literatures, including HPLC, RPHPLC, LC-MS, HPTLC, Spectrophotometry, Derivative Spectrophotometry, Spectrofluorimetry, Voltammetric, Non-aqueous titrimetry, etc. ^5-20

 

These methods are time consuming and require sophisticated instruments and special grade chemicals, which are too costly to afford especially by small-scale industries and for routine experimentation in academic institutes. Thus there is a need for a simple and sensitive method for the determination of selected drugs. Fluorimetry, by virtue of its high sensitivity, meets these requirements.

 

MATERIAL AND METHODS:

Jasco Spectrofluorimeter (FP 6200) was used for all measurements with excitation and emission slits at 5nm and 1cm quartz cells. All the chemicals used were of analytical grade and the solvents were of spectroscopic grade.

 

Preparation of stock solutions:

100 mg of each drug was weighed accurately, transferred into three separate 100 mL volumetric flasks and dissolved in 0.1N H₂SO₄ to give a stock solutions having strength of 1mg mL^-1. Serial dilutions were made with 0.1N H₂SO₄ to cover the working concentration range.

 

Determination of excitation and emission wavelengths:

Each drug solution having the conc. of 1.0 mg mL^-1 was used to obtain the excitation and emission wavelengths.

 

Calibration curves of the studied pure compound:

Standard drug solutions of selected drugs (containing the concentration range cited in table 1) were prepared by diluting the stock solution with 0.1N H₂SO₄ and fluorescent intensities were measure at their respective l_emis. and l_ext. Standard calibration curves were prepared by plotting the concentration verses relative fluorescent intensity (RI).

 

Procedure for tablet dosage form:

An accurately weighed amount, equivalent to 10 mg of each drug from composite of 20 powdered tablets, were transferred into 100 mL volumetric flasks and diluted to the mark with the 0.1N H₂SO₄. These solutions were sonicated for 15 min and filtered off to obtain solutions of 100 mg mL^-1. Further dilutions were made with the same solvent to obtain sample solution and measure the fluorescent intensity at their respective l_emis. and l_ext.

 

Procedure for synthetic mixtures:

Synthetic mixtures were prepared by mixing various common excipients; sucrose (10 mg), glucose (10 mg), starch (5 mg), talk (5 mg) and magnesium stearate (10 mg) with per 50mg of each drug. An accurately weighed amount, equivalent to 10 mg of drug from these mixtures, were transferred into 100 mL volumetric flasks and diluted to the mark with the 0.1N H₂SO₄. These solutions were sonicated for 15 min and filtered off to obtain solutions of 100 mg mL^-1. Further dilutions were made to obtain sample solutions and the fluorescent intensities were measured at their respective l_emis. and l_ext.

 

Procedure for biological fluids

For Urine:

Urine samples were centrifuged at 4000 rpm for 5 min and then 1 mL of clear supernatant was spiked with 1 mL of the drug stock solution. Appropriate dilutions were made with 0.1N H₂SO₄ to obtain the working sample solutions and measure the fluorescent intensity at their respective l_emis. and l_ext.

 

For plasma:

10 mL acetonitrile was added in 5 mL of plasma and centrifuged at 4000 rpm for 5 min for the deproteination. One mL of clear supernatant was spiked with 1 mL (having concentration of 1mg mL^-1 for NFX and 0.1mg mL^-1 SFX and 1.0 mg mL^-1 for OFX) of standard drug solution. The mixture was then extracted with 2 portions; each of 5 mL chloroform. The chloroform extract was collected, evaporated and then residue was dissolved in 0.1N H₂SO₄ to obtain the working sample solutions. Fluorescent intensity was measured at their respective l_emis. and l_ext.

 

RESULTS AND DISCUSSION:

The native fluorescent of fluoroquinolones is due to the high degree of conjugation in the structure. Different solvents were tried for measuring the intrinsic fluorescence of drugs. These include water, 0.1N sodium hydroxide, 0.1N hydrochloric acid, 0.1N Glacial acetic acid and 0.1N sulphuric acid. Maximum fluorescence intensity was observed in 0.1N sulphuric acid.

 

Linear regression analysis of the results show that a rectilinear relationship exists between the fluorescent intensity and the concentration; in the range from 0.8-2.0 mg mL^-1, 0.4-2.8 mg mL^-1and 0.01-0.4 mg mL^-1for NFX, SFX and OFX respectively. The correlation coefficient was 0.9997, 0.9995 and 0.9992 for NFX, SFX and OFX respectively. The concentration of different samples of selected drugs in bulk, pharmaceutical dosage form and biological fluids were calculated from the following regression equation:

For norfloxacin:

RI = 83.63C + 30.705

For sparfloxacin:

RI = 64.192C - 6.9713

For ofloxacin:

RI = 1460.4C - 6.4338

 

Where C = Concentration in mg mL^-1and RI = Relative fluorescent intensity.

 

Method Validation:

The developed spectrofluorimetric method was validated according to ICH guidelines. The value of correlation coefficient indicates the good linearity in range studied. Linearity was also checked by calculating the variance of the slope and intercept. The accuracy of method were determined by investigating recovery of each studied drug at 3 concentration levels covering the specified range including 100% test concentration (3 replicates of each concentration). The results show excellent recoveries. The limit of detection (LOD) and limit of quantitation (LOQ) indicates the high sensitivity of the proposed method. The assay results were unaffected by the presence of excipients as shown by the excellent recoveries obtained when analyzing the studied drugs in the presence of common excipients. This indicates proper selectivity of the method for the estimation of selected drugs in bulk and pharmaceutical dosage forms.

 

Table 1: Optical and validation parameters.

Parameters	NFX	SFX	OFX
Wavelength of maximum excitation (nm) Wavelength of maximum emission (nm) Linearity Range (mg mL-1) Correlation coefficient Regression equation (y*)                    Slope (a)   Intercept (b) Precision (% CV)                Repeatability (n= 6)        Intraday (n =3)        Interday (n =3) % Recovery (mean ± SD) LOD (mg mL-1) LOQ (mg mL-1)	270   464   0.80-2.00 0.9997   83.63 30.71   0.0157 0.5409 0.2991 99.64 ± 0.39 0.0559 0.1694	276   442   0.40-2.80 0.9995   64.19 -6.97   0.0474 0.6921 0.6221 99.89 ± 0.56 0.0728 0.2207	280   510   0.01-0.40 0.9972   1460.40 -6.43   0.0290 0.3313 0.3848 100.01 ± 1.04 0.0031 0.0097

 

APPLICATIONS:

The proposed method was applied successfully for the estimation of selected drugs in pharmaceutical dosage forms. Five replicate measurements were made in each case. Statistical analysis of the results obtained from the proposed method and reference method revealed no significant difference (Table 2). Common excipients such as sucrose, glucose, starch, talk and magnesium stearate did not interfere with the assay of selected drugs     (Table 3). The proposed method was further applied for the determination of studied drugs in biological fluids as urine and plasma (Table 4). Only plasma samples required deproteination and extraction steps while untreated urine samples are processed directly.

 

Table 2: Results of Assay of tablets.

Drugs	By developed method		By reference method		t-value
	% Found*	Mean ± SD	% Found*	Mean ± SD
NFX	99.80 99.80 99.73	99.78 ± 0.04	99.60 99.70 97.90	99.07± 1.01a	1.27
SFX	99.40 100.03 98.90	99.44 ± 0.57	99.20 99.60 99.30	99.43± 0.32b	0.05
OFX	98.80 99.90 100.01	99.57 ± 0.67	98.60 99.50 99.90	99.33± 0.67a	2.76

*Average of 5 measurements of three different brands. The tabulated value of t at 95% confidence limit is 2.92.

^a Ref. [5],  ^b Ref. [19].

 

Table 3:  Assay of drugs in presence of some common excipients.

Excipients/ amount	% Recovery* ± S.D.
	NFX	SFX	OFX
Sucrose 10 mg Glucose 10 mg Starch 5 mg Talk 5 mg Magnesium stearate 10 mg	98.8 ± 0.9 99.3 ± 0.4 99.4 ± 0.8 99.3 ± 0.2 9           99.6 ± 0.8	98.9 ± 0.6 99.8 ± 1.1 99.5 ± 0.4 99.2 ± 0.9 10         100.2 ± 0.8	99.6 ± 0.7 98.9 ± 0.6 99.8 ± 0.7 98.7 ± 0.9 9        99.4 ± 0.8

* Recovered from 50 mg of drugs.

 

Table 4: Determination of drugs in spiked urine and plasma samples.

Drugs	Spiked (mg mL-1)	Spiked urine sample		Spiked plasma sample
		Found *(mg mL-1)	% Recovery	Found *(mg mL-1)	% Recovery
NFX SFX  OFX	1.00 0.10 1.00	1.0122 0.09915 0.9897	101.22 99.15 98.97	0.9908 0.09894 0.9967	99.08 98.94 99.67

* Average of 5 measurements.

Table 5: Results of recovery (spiking).

Drug	Spiking of pure drug (mg mL-1)	By developed method		By reference method		t-value
		% Recovery	Mean ± SD	% Recovery	Mean ± SD
NFX	1.00 1.50 2.00	99.275 100.05 99.60	99.64 ± 0.39	99.24 100.01 98.86	99.37 ± 0.59a	1.16
SFX	1.00 1.50 2.00	100.53 99.55 99.58	99.89 ± 0.56	99.60 99.56 99.48	99.55 ± 0.06b	1.15
OFX	0.10 0.20 0.30	98.85 100.33 100.85	100.01 ± 1.04	99.34 100.26 99.75	99.78 ± 0.46a	0.49

The tabulated value of t at 95% confidence limit is 2.92. ^a Ref. [5],  ^b Ref. [19].

 

Table 6: Statistical analysis the results obtained using reference and developed methods for analysis of authentic samples (% estimated).

Drugs	Reference method	Mean ± SD	Developed method	Mean ± SD	t-value
NFX	99.66 99.89 99.85	99.98 ± 0.12a	99.96 99.94 99.93	99.94 ± 0.02	1.82
SFX	100.10 99.82 99.97	99.96 ± 0.14b	100.08 100.01 99.99	100.03 ± 0.05	0.98
OFX	99.18 98.91 99.88	99.32 ± 0.50a	99.75 99.93 99.98	99.89 ± 0.12	2.12

The tabulated value of t at 95% confidence limit is 2.92.

^a Ref. [5],  ^b Ref. [19].

 

CONCLUSION:

The proposed method is advantageous than many of the reported methods because of its simplicity, accuracy, precision, selectivity, less expensiveness, and time saving nature. Thus, this method can be adopted for the routine analysis in pharmaceutical dosage form and in biological fluids and also in pharmacokinetic studies of these drugs.

 

ACKNOLOGEMENT:

Authors are heartly thankful to the Dr. Reddy Laboratory, Hyderabad, Karnataka, India for providing the pure (reference) drug samples.

 

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Received on 13.08.2017                Accepted on 18.09.2017               

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