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Photochemical fluorescence enhancement of the terbium–lomefloxacin complex and its application
Talanta 49 (1999) 77 – 82
Photochemical fluorescence enhancement of the terbium–lomefloxacin complex and its application Zhang Tieli, Zhao Huichun *, Jin Linpei Department of Chemistry, Beijing Normal Uni6ersity, P.O. Box 55, Beijing, 100875, PR China Received 30 July 1998; received in revised form 27 October 1998; accepted 9 November 1998
Abstract The sensitized fluorescence intensity of the terbium ion (Tb3 + ) can be notably enhanced after the Tb3 + – lomefloxacin(LFLX) complex system was irradiated by 365nm ultraviolet light. A photochemical reaction occurs to the irradiated Tb3 + –LFLX complex. A new Tb3 + system with intense fluorescence is obtained. On this basis a new sensitive and selective photochemical fluorimetry for the determination of LFLX was established. Under the optimal experimental conditions, the linear range of the determination is 2.0 – 500 ×10 − 8 mol l − 1 for LFLX, and the detection limit is 6.0 × 10 − 9 mol l − 1.Without any pre-treatment the recoveries of LFLX in human urine and serum were determined. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Terbium complex; Lomefloxacin; Photochemical fluorimetry; Body fluids
1. Introduction Lomefloxacin (LFLX) is one of the synthetic antibacterial fluoroquinolone agents of the third generation (its chemical structure shown in Fig. 1.), which exhibits high activity against a broad spectrum of gram-negative and gram-positive bacteria. In consequence, it is of great importance to determine its contents in various biological samples, such as blood, urine and tissues. At the moment, the detection of LFLX in biological fluids is usually performed by liquid chromatography method [1 – 3] and microbiologi-
* Corresponding author. Tel. +86-10-6220-0567.
cal assay method [4], which either needs an expensive equipment or is too time-consuming. Because the fluoroquinolone nucleus has the a-keto carboxylic skeleton, we attempted to explore the chelate formation between LFLX and lanthanides to give the narrow and intense fluorescence of the lanthanide ions [5,6] with the aim of improving the sensitivity and specificity by fluorimetric method. Of the lanthanide complexes with LFLX giving the native fluorescence of Ln3 + , the Tb3 + –LFLX complex can enhance the characteristic fluorescence of the Tb3 + . On investigating the sensitized fluorescence of the Tb3 + –LFLX complex, we surprisingly found that the sensitized fluorescence intensity of the Tb3 + was enhanced with the excitation time going on. This is because a photochemical reaction in the Tb3 + –LFLX
0039-9140/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 9 8 ) 0 0 3 6 4 - 6
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Z. Tieli et al. / Talanta 49 (1999) 77–82
complex system takes place and the new fluorescent terbium complex formed facilitates energy transfer. On this basis, a new method —photochemical fluorimetry of lanthanide complexes was proposed for the determination of LFLX. The method has the advantages of both sensitized fluorescence of lanthanide ions and photochemical fluorimetry, with high selectivity and sensitivity. In this paper, detailed study of the appropriate conditions has been conducted for the determination of LFLX by this method, and satisfaction with the recoveries of LFLX in urine and serum samples was obtained. 2. Experimental
2.1. Apparatus Fluorescence spectra were recorded with a Hitachi-850 spectrofluorimeter (Japan) equipped with a 150W xenon lamp. Absorption spectra were recorded on a Shimadzu-UV250 spectrophotometer, Japan. The pH measurements were made with a pHS-2 meter, Shanghai. A ZF-1 Ultraviolet analytical meter (Shanghai) was used as the light source for irradiation.
2.2. Reagents All of the reagents used were of analytical grade and distilled, de-ionized water was used throughout. Stock standard solution (1.00×10 − 3 mol l − 1) of LFLX (Institute of Medical Biotechnology, Beijing) was prepared by dissolving 38.8 mg LFLX in an appropriate amount of water containing three drops of 0.1 mol l − 1 NaOH solution and diluting to 100 ml with water, kept at 4°C and protected from light. The working standard solutions were obtained by appropriate dilution of the stock standard solution with water. Stock standard solution of the Tb3 + (1.00× 10 − 2 mol l − 1) was prepared by dissolving 186.9 mg Tb4O7 (purity, 99.99%) in hot (75°C) 1:1 HCl and evaporating the solution to be almost dry before diluting to 100 ml with water, stored in a plastic bottle, kept at 4°C. It was diluted to the desired concentration when used.
Fig. 1. Chemical structure of lomefloxacin.
0.2 mol l − 1 acetic acid–sodium acetate buffer solution was prepared for pH 3.5–6.5, and pH 6.5–8.5 was obtained by buffering with 0.2 mol l − 1 ammonium acetate–ammonia.
2.3. Procedure Add a proper amount of standard LFLX solution, 0.40 ml of 1.00 × 10 − 2 mol l − 1 standard Tb3 + solution and 5.0 ml of NH4Ac–NH3 (pH= 7.0) buffer solution to a 10 ml calibrated tube with a stopper, then dilute up to the mark with water before shaking. After the sample was irradiated for 30 min under a ultraviolet lamp at 365 nm with the irradiation intensity of 30 mw cm − 2, the difference of fluorescence intensities DIF between sample and blank was determined using an excitation wavelength of 320 nm and monitored at the emission wavelength of 545 nm, with excitation and emission slits of 6 and 8 nm in width, respectively.
Fig. 2. Absorption spectra of LFLX and Tb3 + – LFLX systems in the ultraviolet region.
Z. Tieli et al. / Talanta 49 (1999) 77–82
79
Fig. 3. Fluorescence excitation (a) and emission (b) spectra of LFLX and the Tb3 + – LFLX systems.
3. Results and discussion
3.1. Spectroscopic characteristics The absorption spectra of LFLX and Tb3 + – LFLX systems with and without irradiation are shown in Fig. 2. From the curves 1 and 2, it can be seen that there are two absorption bands for each of them, but the absorption peak position has a slight red shift, from 280 nm of LFLX to 283 nm of the Tb3 + – LFLX, and the intensity of the latter increases. This suggests that the complexation of the Tb3 + and LFLX has occurred. From the curve 3, we can see that two absorption bands of the irradiated Tb3 + – LFLX system at 273 and 324 nm are observed, which are different from that of the non-irradiated Tb3 + – LFLX system. It implies that the absorption property of the Tb3 + –LFLX complex has been changed after it is irradiated by 365 nm ultraviolet light, i.e. the complex may have undergone photochemical reactions and some new absorption substances may have been produced in the process. Fig. 3. shows the fluorescence excitation and emission spectra of LFLX and the Tb3 + –LFLX systems with and without irradiation. It can be
seen from the excitation spectra that the maximum excitation wavelengths are in the range of 270–280 nm for the three systems. But in order to avoid the direct excitation of the Tb3 + and the disturbance of the scattered excitation light at these wavelengths, the excitation was performed at the shoulder of the main excitation band, i.e. at 320 nm [7]. The emission spectra show that the native fluorescence emission wavelength of LFLX is 420 nm. After the Tb3 + –LFLX complex is formed, the band is red-shifted to 430 nm and this broad emission band decreases in intensity greatly while the narrow emission bands of the Tb3 + appear at 490, 545, 585 and 620 nm, corresponding to the transitions of the Tb3 + 5D4 7F6, 5D4 7 F5, 5D4 7F4 and 5D4 7F3, respectively. Therefore, it can be concluded that the intramolecular energy transfer has occurred between LFLX and the Tb3 + . From the curve 3%, it can be seen that in the irradiated Tb3 + –LFLX system the broad emission band at 420 nm arising from the ligand is further decreased and the sensitized fluorescence emission of the terbium ion is enhanced notably. This suggests that the new Tb3 + complex, the photoproduct, has a higher energy transfer effi-
Z. Tieli et al. / Talanta 49 (1999) 77–82
80
Fig. 4. Influence of irradiation time on the fluorescence intensity.
ciency than the Tb3 + – LFLX complex, i.e. the photoproduct is more favorable to intramolecular energy transfer. The further research is in progress. The detailed discussion will be reported in another paper.
3.2. Experimental conditions Through our experiments it was found that the sensitized fluorescence intensity of the Tb3 + was increasing with lengthening the excitation time. In order to get the highest IF value, the following factors were tested and the best experimental conditions were obtained.
3.2.1. The irradiation wa6elength Under the same conditions, the two wavelengths of the ultraviolet lamp, 254 and 365 nm, were tested as the irradiation wavelengths. It was found that the IF value of the latter was about three times as high as the former. So 365 nm was selected as the irradiation wavelength in this work. 3.2.2. Influence of irradiation time Fig. 4. Shows the influence of irradiation time
on IF value. Irradiation intensity was set to 30 mw cm − 2. As shown in Fig. 4, the IF value is increased notably with the irradiation time at the beginning, then slowly and becomes almost constant after 20–30 min. Two different concentrations of LFLX were tested. It was found that a little longer time was required when the concentration was higher. 30 min was selected as the irradiation time in this work.
3.2.3. Influence of pH and medium Fig. 5. gives the relationship between the fluorescence intensity of the irradiated Tb3 + – LFLX complex and pH value. As shown, the maximum emission intensity was observed in the pH range of 7.0–7.5. At pH \ 8.0, the sensitized fluorescence of the Tb3 + decreases, probably owing to the precipitation of terbium hydroxide[8]. And we also tested the effect of the following buffer solution media on the fluorescence intensity, NH4Ac–NH3, Tris–HCl and (CH2)6N4 –HCl. It was found that the sensitivity in the NH4Ac–NH3 medium is the highest. Therefore, 5.0 ml of 0.2 mol l − 1 NH4Ac–NH3 of pH 7.0 buffer solution was chosen.
Z. Tieli et al. / Talanta 49 (1999) 77–82
81
Fig. 5. Influence of pH on the fluorescence intensity.
3.2.4. Influence of the terbium concentration Fig. 6. Shows the influence of the mole ratio of the terbium ion to LFLX on the fluorescence intensity. As we can see, the sensitized fluorescence intensity of the terbium ion is related to the mole ratio. The intensity increases with the increasing of the mole ratio and tends to be constant at a certain ratio ( ]200). When the
concentration of LFLX is different, the ratio to get the maximum intensity is different, too. The lower the concentration of LFLX is, the higher the ratio required. For a fixed 2.00 × 10 − 6 mol l − 1 LFLX concentration, 4.00× 10 − 4mol l − 1 solution of the terbium ion gives an optimal emission. It was used in the calibration curve drawing.
Fig. 6. Influence of Tb3 + concentration on the fluorescence intensity.
Z. Tieli et al. / Talanta 49 (1999) 77–82
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Table 1 Recovery of lomefloxacin added to urine and serum samples Samples
LFLX added (mol l − 1, ×10-6)
LFLX found (mol l − 1, ×10-6)
Mean recovery (%)
R.S.D. (%)
Urine 1 Urine 2 Serum
0.40 0.80 0.020
0.383a 0.777a 0.0203b
95.8 97.2 101.3
3.4 3.2 5.3
a b
Average of nine experiments. Average of five experiments.
3.3. Calibration cur6e and detection limit
4. Conclusion
According to the experimental method described above, values of DIF were determined and the calibration curve was drawn, within the range of 2.00 × 10 − 8 – 5.00 × 10 − 6 mol l − 1 a linear relation was found between DIF value and the LFLX concentration. The linear equation obtained by the least-square analysis was found to be DIF = 0.0545 + 1.773 ×106CLFLX (mol l − 1), with correlation coefficient r = 0.9996. The detection limit for LFLX was calculated from the standard deviation of the blank (the reagent blank without LFLX, n= 19) (3s) as 6.0 × 10 − 9 mol l − 1LFLX.
A new photochemical fluorimetric method for determination of LFLX has been established. Because of its high sensitivity, selectivity, accuracy and good repeatability, it has been successfully used in the determination of LFLX in urine and serum samples without any pre-handling but only by appropriate dilution of the samples. It is a simple and rapid method for determination of LFLX in body fluids.
3.4. Sample determination After an oral administration of 400 mg of LFLX within 24 h the average concentrations of LFLX in the urine and serum samples were in the ranges of 332 – 41 and 3.98 – 0.20 mg l − 1 [9], i.e. 8.6–1.1× 10 − 4 mol l − 1 and 100.0 – 5.1 ×10 − 7 mol l − 1, respectively. In order to make the sample concentrations of the drug within the linear range of determination, urine and serum samples were diluted 500-fold and 50-fold, respectively, and the recoveries of artificially synthetic urine and serum samples containing LFLX were determined by calibration curve method and standard addition method, respectively, and the spectral calibration was made to eliminate the background emission of the serum. The results obtained are shown in Table 1.
.
Acknowledgements The authors thank the National Natural Science Foundation of China for the financial support of this research. References [1] A.M. Shibl, A.F. Tawfik, S. El-Houfy, F.J. Al-Shammary, J. Clin. Pharm. Ther. 16 (1991) 353. [2] G. Carlucci, A. Cilli, M. Liberato, P. Mazzeo, J. Pharm. Biomed. Anal. 11 (1993) 1105. [3] Y. Huang, Zh.W. Li, Zh. Hong, Chin. Pharm. J. 31 (1996) 417. [4] J.S. Lou, J.L. Zhang, C.L. Zhang, Chin. J. Antibiotics 19 (1994) 253. [5] T. Moeller, D.F. Martin, L.C. Thompson, R. Ferrus, G.I. Feistel, W.J. Rendall, Chem. Rev. 62 (1965) 1. [6] S. Schreurs, J.P.C. Vissers, C. Gooijer, N.H. Velthorst, Anal. Chim. Acta 262 (1992) 201. [7] A. Rieutord, L. Vazquez, M. Soursac, P. Prognon, J. Blais, Ph. Bourget, G. Mahuzier, Anal. Chim. Acta 290 (1994) 215. [8] J.X. Duggan, J. Liq. Chromatogr. 14 (1991) 2499. [9] Y.G. Shi, Y.J. Cao, L. Wang, Q.J. Zhang, Y.Y. Zhang, Chin. J. Antibiotics 19 (1994) 248.
Photochemical fluorescence enhancement of the terbium–lomefloxacin complex and its application Zhang Tieli, Zhao Huichun *, Jin Linpei Department of Chemistry, Beijing Normal Uni6ersity, P.O. Box 55, Beijing, 100875, PR China Received 30 July 1998; received in revised form 27 October 1998; accepted 9 November 1998
Abstract The sensitized fluorescence intensity of the terbium ion (Tb3 + ) can be notably enhanced after the Tb3 + – lomefloxacin(LFLX) complex system was irradiated by 365nm ultraviolet light. A photochemical reaction occurs to the irradiated Tb3 + –LFLX complex. A new Tb3 + system with intense fluorescence is obtained. On this basis a new sensitive and selective photochemical fluorimetry for the determination of LFLX was established. Under the optimal experimental conditions, the linear range of the determination is 2.0 – 500 ×10 − 8 mol l − 1 for LFLX, and the detection limit is 6.0 × 10 − 9 mol l − 1.Without any pre-treatment the recoveries of LFLX in human urine and serum were determined. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Terbium complex; Lomefloxacin; Photochemical fluorimetry; Body fluids
1. Introduction Lomefloxacin (LFLX) is one of the synthetic antibacterial fluoroquinolone agents of the third generation (its chemical structure shown in Fig. 1.), which exhibits high activity against a broad spectrum of gram-negative and gram-positive bacteria. In consequence, it is of great importance to determine its contents in various biological samples, such as blood, urine and tissues. At the moment, the detection of LFLX in biological fluids is usually performed by liquid chromatography method [1 – 3] and microbiologi-
* Corresponding author. Tel. +86-10-6220-0567.
cal assay method [4], which either needs an expensive equipment or is too time-consuming. Because the fluoroquinolone nucleus has the a-keto carboxylic skeleton, we attempted to explore the chelate formation between LFLX and lanthanides to give the narrow and intense fluorescence of the lanthanide ions [5,6] with the aim of improving the sensitivity and specificity by fluorimetric method. Of the lanthanide complexes with LFLX giving the native fluorescence of Ln3 + , the Tb3 + –LFLX complex can enhance the characteristic fluorescence of the Tb3 + . On investigating the sensitized fluorescence of the Tb3 + –LFLX complex, we surprisingly found that the sensitized fluorescence intensity of the Tb3 + was enhanced with the excitation time going on. This is because a photochemical reaction in the Tb3 + –LFLX
0039-9140/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 9 8 ) 0 0 3 6 4 - 6
78
Z. Tieli et al. / Talanta 49 (1999) 77–82
complex system takes place and the new fluorescent terbium complex formed facilitates energy transfer. On this basis, a new method —photochemical fluorimetry of lanthanide complexes was proposed for the determination of LFLX. The method has the advantages of both sensitized fluorescence of lanthanide ions and photochemical fluorimetry, with high selectivity and sensitivity. In this paper, detailed study of the appropriate conditions has been conducted for the determination of LFLX by this method, and satisfaction with the recoveries of LFLX in urine and serum samples was obtained. 2. Experimental
2.1. Apparatus Fluorescence spectra were recorded with a Hitachi-850 spectrofluorimeter (Japan) equipped with a 150W xenon lamp. Absorption spectra were recorded on a Shimadzu-UV250 spectrophotometer, Japan. The pH measurements were made with a pHS-2 meter, Shanghai. A ZF-1 Ultraviolet analytical meter (Shanghai) was used as the light source for irradiation.
2.2. Reagents All of the reagents used were of analytical grade and distilled, de-ionized water was used throughout. Stock standard solution (1.00×10 − 3 mol l − 1) of LFLX (Institute of Medical Biotechnology, Beijing) was prepared by dissolving 38.8 mg LFLX in an appropriate amount of water containing three drops of 0.1 mol l − 1 NaOH solution and diluting to 100 ml with water, kept at 4°C and protected from light. The working standard solutions were obtained by appropriate dilution of the stock standard solution with water. Stock standard solution of the Tb3 + (1.00× 10 − 2 mol l − 1) was prepared by dissolving 186.9 mg Tb4O7 (purity, 99.99%) in hot (75°C) 1:1 HCl and evaporating the solution to be almost dry before diluting to 100 ml with water, stored in a plastic bottle, kept at 4°C. It was diluted to the desired concentration when used.
Fig. 1. Chemical structure of lomefloxacin.
0.2 mol l − 1 acetic acid–sodium acetate buffer solution was prepared for pH 3.5–6.5, and pH 6.5–8.5 was obtained by buffering with 0.2 mol l − 1 ammonium acetate–ammonia.
2.3. Procedure Add a proper amount of standard LFLX solution, 0.40 ml of 1.00 × 10 − 2 mol l − 1 standard Tb3 + solution and 5.0 ml of NH4Ac–NH3 (pH= 7.0) buffer solution to a 10 ml calibrated tube with a stopper, then dilute up to the mark with water before shaking. After the sample was irradiated for 30 min under a ultraviolet lamp at 365 nm with the irradiation intensity of 30 mw cm − 2, the difference of fluorescence intensities DIF between sample and blank was determined using an excitation wavelength of 320 nm and monitored at the emission wavelength of 545 nm, with excitation and emission slits of 6 and 8 nm in width, respectively.
Fig. 2. Absorption spectra of LFLX and Tb3 + – LFLX systems in the ultraviolet region.
Z. Tieli et al. / Talanta 49 (1999) 77–82
79
Fig. 3. Fluorescence excitation (a) and emission (b) spectra of LFLX and the Tb3 + – LFLX systems.
3. Results and discussion
3.1. Spectroscopic characteristics The absorption spectra of LFLX and Tb3 + – LFLX systems with and without irradiation are shown in Fig. 2. From the curves 1 and 2, it can be seen that there are two absorption bands for each of them, but the absorption peak position has a slight red shift, from 280 nm of LFLX to 283 nm of the Tb3 + – LFLX, and the intensity of the latter increases. This suggests that the complexation of the Tb3 + and LFLX has occurred. From the curve 3, we can see that two absorption bands of the irradiated Tb3 + – LFLX system at 273 and 324 nm are observed, which are different from that of the non-irradiated Tb3 + – LFLX system. It implies that the absorption property of the Tb3 + –LFLX complex has been changed after it is irradiated by 365 nm ultraviolet light, i.e. the complex may have undergone photochemical reactions and some new absorption substances may have been produced in the process. Fig. 3. shows the fluorescence excitation and emission spectra of LFLX and the Tb3 + –LFLX systems with and without irradiation. It can be
seen from the excitation spectra that the maximum excitation wavelengths are in the range of 270–280 nm for the three systems. But in order to avoid the direct excitation of the Tb3 + and the disturbance of the scattered excitation light at these wavelengths, the excitation was performed at the shoulder of the main excitation band, i.e. at 320 nm [7]. The emission spectra show that the native fluorescence emission wavelength of LFLX is 420 nm. After the Tb3 + –LFLX complex is formed, the band is red-shifted to 430 nm and this broad emission band decreases in intensity greatly while the narrow emission bands of the Tb3 + appear at 490, 545, 585 and 620 nm, corresponding to the transitions of the Tb3 + 5D4 7F6, 5D4 7 F5, 5D4 7F4 and 5D4 7F3, respectively. Therefore, it can be concluded that the intramolecular energy transfer has occurred between LFLX and the Tb3 + . From the curve 3%, it can be seen that in the irradiated Tb3 + –LFLX system the broad emission band at 420 nm arising from the ligand is further decreased and the sensitized fluorescence emission of the terbium ion is enhanced notably. This suggests that the new Tb3 + complex, the photoproduct, has a higher energy transfer effi-
Z. Tieli et al. / Talanta 49 (1999) 77–82
80
Fig. 4. Influence of irradiation time on the fluorescence intensity.
ciency than the Tb3 + – LFLX complex, i.e. the photoproduct is more favorable to intramolecular energy transfer. The further research is in progress. The detailed discussion will be reported in another paper.
3.2. Experimental conditions Through our experiments it was found that the sensitized fluorescence intensity of the Tb3 + was increasing with lengthening the excitation time. In order to get the highest IF value, the following factors were tested and the best experimental conditions were obtained.
3.2.1. The irradiation wa6elength Under the same conditions, the two wavelengths of the ultraviolet lamp, 254 and 365 nm, were tested as the irradiation wavelengths. It was found that the IF value of the latter was about three times as high as the former. So 365 nm was selected as the irradiation wavelength in this work. 3.2.2. Influence of irradiation time Fig. 4. Shows the influence of irradiation time
on IF value. Irradiation intensity was set to 30 mw cm − 2. As shown in Fig. 4, the IF value is increased notably with the irradiation time at the beginning, then slowly and becomes almost constant after 20–30 min. Two different concentrations of LFLX were tested. It was found that a little longer time was required when the concentration was higher. 30 min was selected as the irradiation time in this work.
3.2.3. Influence of pH and medium Fig. 5. gives the relationship between the fluorescence intensity of the irradiated Tb3 + – LFLX complex and pH value. As shown, the maximum emission intensity was observed in the pH range of 7.0–7.5. At pH \ 8.0, the sensitized fluorescence of the Tb3 + decreases, probably owing to the precipitation of terbium hydroxide[8]. And we also tested the effect of the following buffer solution media on the fluorescence intensity, NH4Ac–NH3, Tris–HCl and (CH2)6N4 –HCl. It was found that the sensitivity in the NH4Ac–NH3 medium is the highest. Therefore, 5.0 ml of 0.2 mol l − 1 NH4Ac–NH3 of pH 7.0 buffer solution was chosen.
Z. Tieli et al. / Talanta 49 (1999) 77–82
81
Fig. 5. Influence of pH on the fluorescence intensity.
3.2.4. Influence of the terbium concentration Fig. 6. Shows the influence of the mole ratio of the terbium ion to LFLX on the fluorescence intensity. As we can see, the sensitized fluorescence intensity of the terbium ion is related to the mole ratio. The intensity increases with the increasing of the mole ratio and tends to be constant at a certain ratio ( ]200). When the
concentration of LFLX is different, the ratio to get the maximum intensity is different, too. The lower the concentration of LFLX is, the higher the ratio required. For a fixed 2.00 × 10 − 6 mol l − 1 LFLX concentration, 4.00× 10 − 4mol l − 1 solution of the terbium ion gives an optimal emission. It was used in the calibration curve drawing.
Fig. 6. Influence of Tb3 + concentration on the fluorescence intensity.
Z. Tieli et al. / Talanta 49 (1999) 77–82
82
Table 1 Recovery of lomefloxacin added to urine and serum samples Samples
LFLX added (mol l − 1, ×10-6)
LFLX found (mol l − 1, ×10-6)
Mean recovery (%)
R.S.D. (%)
Urine 1 Urine 2 Serum
0.40 0.80 0.020
0.383a 0.777a 0.0203b
95.8 97.2 101.3
3.4 3.2 5.3
a b
Average of nine experiments. Average of five experiments.
3.3. Calibration cur6e and detection limit
4. Conclusion
According to the experimental method described above, values of DIF were determined and the calibration curve was drawn, within the range of 2.00 × 10 − 8 – 5.00 × 10 − 6 mol l − 1 a linear relation was found between DIF value and the LFLX concentration. The linear equation obtained by the least-square analysis was found to be DIF = 0.0545 + 1.773 ×106CLFLX (mol l − 1), with correlation coefficient r = 0.9996. The detection limit for LFLX was calculated from the standard deviation of the blank (the reagent blank without LFLX, n= 19) (3s) as 6.0 × 10 − 9 mol l − 1LFLX.
A new photochemical fluorimetric method for determination of LFLX has been established. Because of its high sensitivity, selectivity, accuracy and good repeatability, it has been successfully used in the determination of LFLX in urine and serum samples without any pre-handling but only by appropriate dilution of the samples. It is a simple and rapid method for determination of LFLX in body fluids.
3.4. Sample determination After an oral administration of 400 mg of LFLX within 24 h the average concentrations of LFLX in the urine and serum samples were in the ranges of 332 – 41 and 3.98 – 0.20 mg l − 1 [9], i.e. 8.6–1.1× 10 − 4 mol l − 1 and 100.0 – 5.1 ×10 − 7 mol l − 1, respectively. In order to make the sample concentrations of the drug within the linear range of determination, urine and serum samples were diluted 500-fold and 50-fold, respectively, and the recoveries of artificially synthetic urine and serum samples containing LFLX were determined by calibration curve method and standard addition method, respectively, and the spectral calibration was made to eliminate the background emission of the serum. The results obtained are shown in Table 1.
.
Acknowledgements The authors thank the National Natural Science Foundation of China for the financial support of this research. References [1] A.M. Shibl, A.F. Tawfik, S. El-Houfy, F.J. Al-Shammary, J. Clin. Pharm. Ther. 16 (1991) 353. [2] G. Carlucci, A. Cilli, M. Liberato, P. Mazzeo, J. Pharm. Biomed. Anal. 11 (1993) 1105. [3] Y. Huang, Zh.W. Li, Zh. Hong, Chin. Pharm. J. 31 (1996) 417. [4] J.S. Lou, J.L. Zhang, C.L. Zhang, Chin. J. Antibiotics 19 (1994) 253. [5] T. Moeller, D.F. Martin, L.C. Thompson, R. Ferrus, G.I. Feistel, W.J. Rendall, Chem. Rev. 62 (1965) 1. [6] S. Schreurs, J.P.C. Vissers, C. Gooijer, N.H. Velthorst, Anal. Chim. Acta 262 (1992) 201. [7] A. Rieutord, L. Vazquez, M. Soursac, P. Prognon, J. Blais, Ph. Bourget, G. Mahuzier, Anal. Chim. Acta 290 (1994) 215. [8] J.X. Duggan, J. Liq. Chromatogr. 14 (1991) 2499. [9] Y.G. Shi, Y.J. Cao, L. Wang, Q.J. Zhang, Y.Y. Zhang, Chin. J. Antibiotics 19 (1994) 248.