Spectrofluorimetric study of the charge-transfer complexation of certain fluoroquinolones with 7,7,8,8-tetracyanoquinodimethane

Spectrochimica Acta Part A 61 (2005) 281–286

Spectrofluorimetric study of the charge-transfer complexation of certain fluoroquinolones with 7,7,8,8-tetracyanoquinodimethane Li Ming Du∗ , Hai Yan Yao, Mi Fu Analytical and Testing Center, Shanxi Normal University, Shanxi linfen 041004, PR China Received 3 February 2004; accepted 7 April 2004

Abstract Simple, rapid and sensitive spectrofluorimetric methods are described, for the first time, for the determination of ciprofloxacin (CIP), norfloxacin (NOR), pefloxacin (PEF) and fleroxacin (FLE). The methods are based on the charge-transfer (CT) reaction of these drugs as n-electron donors with 7,7,8,8-tetracyanoquinodimethane (TCNQ) as ␲-electron acceptor. TCNQ was found to react with these drugs to produce intensely transfer reaction complexes and the fluorescence intensity of the complexes was enhanced in 21–35 fold higher than that of the studied fluoroquinolones itself. The formation of such complexes was also confirmed by both infrared and ultraviolet-visible measurements. The different experimental parameters that affect the fluorescence intensity were carefully studied. At the optimum reaction conditions, the drug-TCNQ complexes showed excitation maxima ranging from 277 to 284 nm and emission maxima ranging from 451 to 458 nm. Rectilinear calibration graphs were obtained in the concentration range of 0.03–0.9, 0.04–1.2, 0.04–1.3 and 0.08–2.4 ␮g ml−1 for CIP, NOR, PEF and FLE, respectively. The developed methods were applied successfully for the determination of the studied drugs in their pharmaceutical dosage forms with a good precision and accuracy compared to official and reported methods as revealed by t- and F-tests. © 2004 Elsevier B.V. All rights reserved. Keywords: Fluoroquinolones; 7,7,8,8-Tetracyanoquinodimethane; Charge-transfer complexes; Spectrofluorimetry

1. Introduction Fluoroquinolones (FQs) are a class of important synthetic antibiotics, which are active against both Gram (+) and Gram (−) bacteria through inhibition of their DNA gyrase [1], also they have some activity against mycobacteria, mycoplasmas and rickettsias. Several chromatographic methods have been reported for determination of these compounds, ciprofloxacin [2–4], norfloxacin [5–8], pefloxacin [9] and fleroxacin [10–12] were determined by high-performance liquid chromatography (HPLC). Various spectrophotometric methods were described for determination of CIP [13,14], PER [15] and NOR [16–18] by charge-transfer complex formation with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ), chloranilic acid (CL) and TCNQ. In addition, several methods have been reported for their determination such as fluorimetry [19–21], polarography [22] and voltammetric [23] and capillary electrophoresis [24]. HPLC methods generally require complex and expensive equipment, ∗

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1386-1425/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2004.04.016

provision for use and disposal of solvents, labor-intensive sample preparation procedure and personnel skilled in chromatographic techniques. Charge-transfer spectrofluorimetry (CTF) has been found to be useful for the determination of quinolone in real samples [25–27], showing several advantages such as low interference level, low detection limit, high sensitivity, good analytical selectivity, easy and less time consuming comparing with the above methods. A new spectrofluorimetric method for determination of CIP, NOR, PER and FLE was reported through CT complexation with TCNQ in this paper, having been satisfactorily applied to the determination of studied drugs in commercial formulations samples.

2. Experimental 2.1. Reagents All solvents used were of analytical reagent grade. Methanol (redistillation) and chloroform (Shanghai Chemical Reagent Co., China), TCNQ (Sigma Chemical Co.,

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USA) was prepared as 1 × 10−3 mol l−1 in methanol, solution was found be stable for at least 1 week at 4 ◦ C. CIP, NOR, PER and FLE of drug standard samples were kindly provided by Chinese National Institute for the Control of Pharmaceutical and Biological Products. Stock standard solution of 100 ␮g ml−1 was prepared by dissolving four drugs standard samples in methanol as needed. Working standard solutions were prepared by dilution of stock standard solution with methanol. Stock standard solutions were stable for several weeks at room temperature. 2.2. Pharmaceutical formulation The following available commercial preparations were analyzed: (1) Ciprofloxacin hydrochloride tablets® (Jingdu Pharmaceutical Co. Ltd., Zhejiang Xinchang China), labeled to contain 250 mg ciprofloxacin per tablet. (2) Jiafuning capsules® (Jinli Pharmaceutical Co., Fujian, China), labeled to contain 200 mg pefloxacin per grain. (3) Norfloxacin capsules® (Sanjin Pharmaceutical Industries Co., Taiyuan, China), labeled to contain 100 mg norfloxacin per grain. (4) Tianfang Luoxin tablet® (Tianfang Pharmaceutical Co., Ltd., Henan, China), labeled to contain 100 mg fleroxacin per tablet. 2.3. Apparatus Fluorescence signals were measured on an LS-50B luminescence spectrometer (Perkin-Elmer, USA) equipped with a xenon-lamp and computer working with FL WinLab software. All the measurements took place in a standard 10 mm path-length quartz cell, thermostated at 25.0 ± 0.5 ◦ C, with 2.5 nm bandwidths for the emission and excitation monochromators. A UV-2201 ultraviolet-visible spectrophotometer (Shimadzu Tokyo Japan) was used for the absorbance measurements. An infrared spectrometer IMPACT-410 (Nicolet, USA) was used for recording the IR spectrum. 2.4. Procedure 2.4.1. General procedure A suitable amount of drug solution was pipetted into a 10 ml volumetric flask, 1.0 ml of TCNQ solution was added, and the solution was diluted to volume with methanol and mixed thoroughly. The solution were thermostated at 25.0 ± 0.5 ◦ C and the fluorescence intensities of CT complexes of CIP, NOR, PER and FLE were measured at 458, 453, 451 and 456 nm using an excitation wavelength of 279, 277, 278 and 284 nm against a blank solution, respectively. The calibration graph was constructed in the same way with stud-

ied drugs solutions of known concentrations. The amount of drugs was computed from their calibration graphs. 2.4.2. Analysis of tablets and capsules The contents of 10 tablets or capsules of each drug were pulverized carefully or evacuated. Weigh accurately an amount of the powdered tablet equivalent to contain average weight of the grain and transferred into a 100 ml calibrated flask, dissolved in 2 ml methanol, swirled and sonicated for 3 min, the solution was diluted to volume with methanol. The first 10 ml of the filtrate was discarded, the 10 ml of continuation of sample solution was diluted to 100 times volume with methanol. Mix well and make serial dilution with methanol so that the solution contains 10 ␮g ml−1 CIP, 12 ␮g ml−1 NOR, 13 ␮g ml−1 PER and 24 ␮g ml−1 FLE and was proceed as under Section 2.4.1. 2.4.3. Preparation of the complexes for infrared To 5 ml of 0.05 mol l−1 TCNQ of in methanol and 5 ml of 0.05 mol l−1 each investigated drug in methanol was added in around bottom flask containing 50 ml of methanol and stirred for 30 min. The solvent was evaporated under reduced pressure and the resulting oily residues were dried over calcium chloride.

3. Results and discussion 3.1. Excitation spectra and emission spectra Solution of the studied drugs have weak native fluorescence, however in presence of TCNQ, the fluorescence intensity increases substantially, the sensitivity is enhanced by 21–32-fold (Fig. 1). Indicated CT complexes formation between the investigated drugs and TCNQ, these drugs were probably through the lone pair of electron donated by the N atom in piperazinyl of FQs (n-donor) to TCNQ (␲-acceptor). 3.2. Effect of reaction temperature The effect of temperature on the formed CT complexes was studied in the range of 10–60 ◦ C. All the formed complexes were stable up to 40 ◦ C, at temperatures higher than 40 ◦ C, the relative fluorescence intensity decreases due to dissociation of the complexes at higher temperatures. Similarly, The fluorescence intensity was found to depend on temperature of CT complexes with studied drugs is small in the range of 10–40 ◦ C, thus the determination of studied drugs were carried out at 25 ± 0.5 ◦ C. It was further found that takes 30 min to form the complexes completely which were stable for at least 24 h. 3.3. Effect of TCNQ concentration The influence of CT reagent concentration was studied in the range 1 × 10−5 –1 × 10−3 mol l−1 , The relative flu-

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Fig. 1. Fluorescence spectra of: (1, 1 ) CIP (0.842 ␮g ml−1 )–TCNQ. (2, 2 ) NOR (0.990 ␮g ml−1 )–TCNQ. (3, 3 ) PEF (0.995 ␮g ml−1 )–TCNQ. (4, 4 ) FLE (0.995 ␮g ml−1 )–TCNQ. (5, 5 ) NOR (0.990 ␮g ml−1 ). (6, 6 ) CIP (0.842 ␮g ml−1 ). (7, 7 ) PEF (0.995 ␮g ml−1 ). (8, 8 ) FLE (0.995 ␮g ml−1 ). (9, 9 ) TCNQ (1 × 10−4 mol l−1 ). (1–9) Excitation spectra; (1 –9 ) emission spectra.

orescence intensity increased with increasing TCNQ concentration up to 1 × 10−4 mol l−1 but leveled off at higher concentrations. Experiment indicated that 1.0 ml TCNQ solution is enough for each drug, thus the final TCNQ concentration of 1 × 10−4 mol l−1 was used for all of the studied drugs.

3.6. Investigations on the structure of the charge-transfer complexes The fluorescence intensity increases substantially indicated the possible CT complexes formation of the type n–␲ complexes. The formation of such complexes was also con-

3.4. Effect of solvent Fluorescence spectral characteristics of CIP, NOR, PER and FLE in different solvents are compared. The studied solvents involved water, methanol, ethanol, isopropanol, acetone, acetonitrile and chloroform. Experimental results indicated that methanol gave the maximum and stable fluorescence emission for studied drugs. 3.5. Effect of CT reagent The influence of the CT reagent on the relative fluorescence intensity of all the formed CT complexes with FQs (NOR was chosen as a representative example) was studied at their respective maxima using TCNQ, TCBQ, CL and DDQ as model electron acceptors. The results show that TCNQ is most sensitive CT reagent for the studied drugs. In general, the order of decreasing sensitivity is TCNQ > TCBQ > CL > DDQ (see Fig. 2).

Fig. 2. Fluorescence emission spectra of: (1) NOR (1.0 ␮g ml−1 ) with TCNQ (1 × 10−4 mol l−1 ). (2) NOR (1.0 ␮g ml−1 ) with TCBQ (1×10−4 mol l−1 ). (3) NOR (1.0 ␮g ml−1 ) with CL (1×10−4 mol l−1 ). (4) NOR (1.0 ␮g ml−1 ) with DDQ (1×10−4 mol l−1 ). (5) NOR (1.0 ␮g ml−1 ).

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Table 1 Characteristic bands (cm−1 ) of IR spectra of TCNQ and FQ–TCNQ complexes

Table 2 Wavelength (nm) of absorption maximum for FQs and FQ–TCNQ complexes

Compounds

ν C ≡N

νC=C

δ=C–H

Drugs

FQs

FQ–TCNQ

TCNQ CIP–TCNQ NOR–TCNQ PER–TCNQ FLE–TCNQ

2220 2190 2180 2190 2170

1540 1500 1510 1520 1510

860 810 810 830 820

CIP NOR PER FLE

310 276 312 292

762 772 768 743

firmed by both IR and UV measurements. The majority of infrared measurements on such CT complexes have been concerned with the shifts in the vibrational frequencies of donors or acceptors. Decreases in the vibration frequency of a particular band have been used as evidence for a particular site of a CT interaction [29]. The infrared spectra of the complexes show some differences compared with the sum of the spectra of the two components. This was used to distinguish between weak CT complexes and the products of electron-transfer [29]. The IR spectra of TCNQ shows strong bands at 2220, 1540 and 860 cm−1 corresponding to νC≡N , aromatic νC=C and 1,4-disubstituted benzene stretching, respectively (Fig. 2). These bands were shifted in the spectra of the complexes with the investigated compounds to 2190, 2180, 2170, 1500, 1510, 1520 and 810, 830 and 820 cm−1 (Table 1). Fig. 3 and Table 2 show the maximum absorbance of four FQs in methanol ranging from 276 to 312 nm. When TCNQ solution was added to studied drug solution, the studied drugs solution with TCNQ cause an immediate change in the absorption spectrum with new characteristic bands at

850 847 844 856

761–772 and 844–850 nm. The appearance of a new band in the visible region of the spectrum was evidence for the formation of a CT complex between the studied components and TCNQ. 3.7. Mechanism of reaction TCNQ is an ␲-acceptor, CIP, NOR, PER and FLE are nitrogenous compounds. So CT complexes can be formed with these drugs. Molar ration of the reactants in the CT complex was determined by molar ratio method and curved intersection method and it was found to be 1:1 for studied drugs with TCNQ. This ratio may be due to the presence of the fluorine atom acting as an electron drawing group in the molecule of FQS . The benzene ring has lower electron density, but nitrogen atom in 4 of piperazinyl has more electron density and less sterically hindered. So n–␲ CT complexes were formed (Table 3). 3.8. Analytical parameters Under the experimental conditions described, standard calibration curves of CT complexes for CIP, NOR, PER and FLE with TCNQ were constructed by plotting fluorescence intensity versus concentration, the linear regression equation for each method are listed in Table 4. The correlation coefficients ranged from 0.9992 to 0.9999, indicating good linearity. The small value of variance confirmed the small degree of scattering of the experimental data points around the regression line. Precision of the proposed methods was determined in each concentration range, by 11 measurements carried out on difTable 3 Structures of the investigated drugs CT complexes with TCNQ

10−4

mol l−1 )

Fig. 3. Absorption spectra: (a) fleroxacin (2.71 × against methanol blank; (b) fleroxacin (2.71 × 10−4 mol l−1 )–TCNQ (4.90 × 10−3 mol l−1 ) against reagent blank.

Drugs

R1

R2

R3

R4

Ciprofloxacin Norfloxacin Pefloxacin Fleroxacin

C 2 H5 C 2 H5 C 2 H4 F

H H F F

H H CH3 CH3

H H H H

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Table 4 Statistic and analytical parameters Parameters

CIP–TCNQ

NOR–TCNQ

PER–TCNQ

FLE–TCNQ

λex /λem (nm) Linear range (␮g ml−1 ) Limit of detection (␮g ml−1 ) Limit of quantitation (␮g ml−1 ) Slope (b) S.D. of slope (Sb ) Intercept on the ordinate (a) S.D. of the intercept on the ordinate (Sa ) Variance (S02 ) Number of points (n) Correlation coefficients (r)

279/458 0.03–0.90 0.008 0.027 1084.17 1.14 3.471 0.12 0.019 21 0.9992

277/453 0.04–1.20 0.011 0.037 825.07 1.01 2.218 0.05 0.004 19 0.9996

278/451 0.04–1.30 0.012 0.040 732.83 1.15 7.589 0.04 0.012 27 0.9994

284/456 0.08–2.40 0.024 0.080 351.76 1.46 2.156 0.02 0.006 28 0.9999

Table 5 Precision results of fluoroquinolone (n = 11) Concentration (␮g ml−1 )

0.01 0.10 1.00 a

Within-day R.S.D.a (%)

Between-day R.S.D.a (%)

CIP

NOR

PER

FLE

CIP

NOR

PER

FLE

1.1 1.5 0.8

1.7 0.6 1.1

2.1 0.8 0.9

1.5 1.3 1.0

1.6 0.9 1.2

1.8 1.2 0.8

1.1 0.7 1.2

1.3 1.0 1.8

Average value ±S.D. of 11 determinations.

ferent days within 1 week of different solution of CIP, NOR, PER and FLE. Target concentrations corresponded to middle values in each range. Table 5 gives a R.S.D. (within-day and between-day) of solutions of 0.01, 0.10, and 1.00 ␮g ml−1 were determined by using the proposed procedure. 3.9. Analysis of pharmaceutical formulations The proposed methods were applied to the determination of CIP, FLE in commercial tablets and NOR, PER in capsules. Five replicate determinations were made. Satisfactory results were obtained for studied drugs (Table 6). Moreover, to check the validity of the proposed methods, the standard addition method was applied by adding CIP, NOR, PER and FLE to the previously analyzed tablets or capsules. The recovery of each drug was calculated by comparing the concentration obtained from the (spiked) mixtures with those of the pure drugs. Table 6 shows the results of analysis of the commercial tablets, capsule and the recovery study (standard addition method) of studied drugs. Comparison of the results

obtained by the proposed method with those obtained by official method [28] and literature method [9,12]. The accuracy is satisfactory. The obtained high-intensity fluorescence bands and the very low reagent background make these procedures suitable for the routine quality control analysis of the investigated compounds with minimum interference. The proposed and reference methods were applied to the determination of the studied drugs in tablets and capsules containing different FQs (Table 6). The obtained mean values (±S.D.) of the labeled amounts ranged from 94.43 ± 1.23 to 99.12 ± 0.64, the recoveries ranged from 99.04 ± 0.72 to 99.29 ± 1.41. In the t- and F-tests, no significant differences were found between the calculated and theoretical values (95% confidence) of both the proposed and reference methods. This indicates similar precision and accuracy. 3.10. Effect of interfering substances The assay results were unaffected by the presence of excipients as shown by the excellent recoveries obtained when

Table 6 Determination of drugs in pharmaceutical formulation using TCNQ (n = 5) Drugs

CIP tablets NOR capsules PER capsules FLE tablets

Present method

Reference method

Found (mg per grain)

Equivalent nominal content (%, ±S.D.)a

247.1 99.1 196.3 97.4

98.84 99.12 98.15 97.43

± ± ± ±

1.09 0.64 0.78 1.23

(t, (t, (t, (t,

1.68; 0.97; 1.26; 2.13;

Recovery (%) F, F, F, F,

2.69) 3.21) 3.27) 2.46)

99.12 99.17 99.04 99.29

± ± ± ±

The tabulated values of t and F at the 95% confidence limit are t = 2.78 and F = 6.39. a Average value ±S.D. of five determinations.

1.32 0.81 0.72 1.41

Found (mg per grain)

Equivalent nominal content (%, ±S.D.)a

246.9 99.1 196.1 97.7

98.76 99.11 98.10 97.72

± ± ± ±

1.51 0.26 0.76 0.57

[28] [28] [9] [12]

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Table 7 Effect of commonly used excipients on the determination of FLE (0.1 ␮g ml−1 ) Drug (0.1 ␮g ml−1 )

Excipient (50 ␮g ml−1 )

Recovery (%, ±S.D.)a

Fleroxacin Fleroxacin Fleroxacin Fleroxacin Fleroxacin Fleroxacin

Starch Lactose Glucose Fructose Sucrose Magnesium stearare

99.2 100.4 99.3 98.8 99.1 99.3

a

± ± ± ± ± ±

0.8 1.4 1.1 0.9 0.6 1.2

Average value ±S.D. of five determinations.

analyzing the studied drugs in presence of commonly encountered excipients. Samples containing a fixed amount of the FQS (0.1 ␮g ml−1 ) and excipients (50 ␮g ml−1 ) were measured. No interference was observed from commonly used excipients such as starch, lactose, glucose, fructose, sucrose and magnesium stearare (Table 7). This fact indicates good selectivity of the method for determination of the studied drugs in raw material and in their dosage forms. 4. Conclusion The results obtained from the present study indicate that complex formation between the studied FQS and TCNQ be employed in the spectrofluorimetric assay of CIP, NOR, PER and FLE in its dosage forms. The proposed methods are suitable for the routine quality control of the drug alone and in tablets or capsules without fear of interference caused by the excipients expected to be present in tablets or capsules. Acknowledgements This research was supported by the Natural Science Foundation of Shanxi (No. 20041030). References [1] Martindale, The Extra Pharmacopoeia, 33rd ed., Royal Pharmaceutical Society, London, 2002.

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