http://www.chemistrymag.org/cji/2000/023016pe.htm |
Mar.28,
2000 Vol.2 No.3 P. 16 Copyright |
Du Liming#, Jin Weijun, Dong
Chuan, Liu Changsong, Xu Qingqin #
(Department of Chemistry, Shanxi University, Taiyuan 030006; #Center of
Analysis and Test, Shanxi Normal University, Linfen 041004, China)
Received Dec. 3, 1999; Supported by the National Natural Science Foundation of China
(No. 29875016) and Shanxi NSF (No.981006)
Abstract A new method for fluorescence enhancement of Sparfloxacin (SPFX) was first
established. In acid media Sparfloxacin is oxidized by nitrous acid then react with
potassium iodide further to form a new fluorescence substance, which fluoresces strongly
with excitation maximal and emission wavelength at 286nm and 464nm, respectively. The
fluorescence intensity is 134 fold more than that of SPFX itself. By this, a new sensitive
method for the determination of SPFX in human urine by derivative-synchronous fluorescence
is presented with a good result. The linear range is 2*10-8
mol /L-1*10-6mol /L with a detection
limit of 1.5*1010mol/L. Finally , the
fluorescence behavior and the reaction mechanism are explored in detail.
Keywords sparfloxacin, nitrous acid, potassium iodide, derivative-synchronous
fluorescence.
1. INTRODUCTION
Sparfloxacin is the fourth generation of the antibacterial
quinolones. Due to its intensive and efficient antibacterial activity compared to the
common quinolones [1] , it has been widely used in clinical practice.
Nevertheless, because of the structural differences between SPFX and other quinolones, the
fluorescence of SPFX is so weak that it can not be determined by means of flu orescence
spectra. Hence, it will be of great interest to make a detailed study on its fluorescence
enhancement whether in clinical practice or in pharmacokinetics. As to the analytical
methods in determining quinolones by spectrophotometry[2-4], there have been
reported a lot, such as HPLC [5-6] , and thin-layer chromatography [7] etc.,
but using fluorescence spectra as a means to determine trace amount of SPFX in human body
has not been reported.
Herein, a detailed investigation on SPFX―
NaNO2― KI system is reported. It has been found
that SPFX can be oxidized promptly by nitrous acid, then reacts with KI further to yield a
new fluorescence substance, which can emit strong fluorescence. Based on this, a new
sensitive method for the determination of SPFX by derivative- synchronous fluorescence was
established. Meanwhile, the structure of this new fluorescence substance was characterized
and the mechanism of reaction was discussed. Experiments showed that the method presented
in this work was not only high-sensitive but also easy to operate and good in selection,
is very convenient to carry out.
2. EXPERIMENTAL
2.1 Apparatus and reagents
The fluorescence spectra were obtained on a Hitachi F-4500
spectrofluorometer equipped with a 450w Xenon source and a R928 Photomultiplier tube.
Excitation and emission of bandpass 5nm were employed and a 1cm cuvette was employed for
measurements. The acidity of the system was measured on a pHs-2 meter from Shanghai
second Analytical Instrument Factory.
Sparfloxacin from the National Institute for the Control of
Pharmaceutical and Biological Products was prepared as 5*10-5 mol/L stock solution. NaNO2 was prepared as 5*10-3 mol/L stock solution which
was stored in a brown reagent bottle and kept in refrigerator. 0.2mol/L of KI and HCl were
also prepared as stock solutions. All the reagents used in this work were of analytical
grade. Water was doubly distilled in a sub-boiling distiller.
2.2 Procedures
An aliquot of SPFX and 0.5ml HCl stock solutions were in turn piped into a comparison tube
of 25mL,into which 0. 25mL NaNO2 solution was added dropwise in an ice-bath at
the temperature range of 0-5°C then 0.3mL KI was added and shaken to mix well the solution. The
mixture solution was kept for 30min within the above temperature range, then heated for 2h
over a hot water bath at 100°C and
diluted to final volume with pH 3.3 NaAc-HAc buffer solution.
After setting for 10min, fluorescence of the solution was measured. The excitation and
emission wavelengths were fixed at 286nm and 464nm, respectively.
 |
Fig. 1 Excitation(A) and emission(B) of
SPFX and SPFX-NaNO2-KI a,b:lex/lem=284nm/538nm; [SPFX]=5´ 10-5moL/L;a',b':lex/lem=286nm/464nm; [SPFX]=5´ 10-5moL/L;[HCl]=4´ 13moL/L; [NaNO2]=5´ 10-5moL/L; [KI] =2.4´ 10-3moL/L; a ,b ,a', b' in NaAc-HAc buffer pH=3.3; |
3. RESULTS AND DISCUSSION
3.1 Excitation and emission spectra
As shown in Fig.1, SPFX can emit only very weak fluorescence at 538nm.While after it was
oxidized by nitrous acid and reacted with KI, the fluorescence intensity of solution was
enhanced in 134 fold higher than that of SPFX itself. Meanwhile, the wavelength of
fluorescence emission is blue-shifted to 464nm, which indicates that a new fluorescence
substance is formed. With quinine sulfate (0.05mol/L of H2SO4,
Ff =0.55) as standard
solution, the fluorescence quantum yield of SPFX and that of the new substance were
determined as 0.0072 and 0.1529, respectively.
3.2 Effect of dosage of reagent
Experiments show that when 0.15-0.3mL of 5*10-3 mol/L NaNO2, 0.25-0.8mL of 0.2mol/L HCl, 0.2-0.5mL of 0.2
mol/L KI are used, the fluorescence intensity of the system is highest and stable. So in
this experiment, the choices of 0.5mL HCl, 0.25mLNaNO2 and 0.3mL KI was
adopted.
3.3 Effect of time for Ice-bathing and Heating
The time of SPFX―NaNO2―KI
kept in the ice-bath as well as the heating time would directly affect the fluorescence
intensity of the reaction system.It was shown by experiments that the fluorescence
intensity reached its maximum and the apparent fluorescence quantum yield was the highest
when the ice-bathing time and heating time were 30min and 2 hours, respectively.
3.4 Effect of pH
Four buffer solutions ( KCl-HCl, NaAc-HAc,KH2PO4-Na2B4O7,and
NaOH-Na2B4O7)were used to control the pH value of the
system with SPFX concentration at 5*10-6mol/L. The
fluorescence intensity was varied with pH value increased, and reached the maximum at pH
value of 3.3-3.4. This may be indicated that the protonation of the atom N and O in -C=O,
-NH2 and -COOH groups of molecules can directly affect the electron properties
and the distribution of electron cloud in the quinolones conjugated system, thus affect
the electron- transition probability.
3.5 Calibration graph of SPFX
Under the optimum experimental condition, the linear equation of SPFX can be obtained as Fem=26.05*107Cspfx+ 15. 7, of which the correlation
coefficient is 0.9997.When the concentration of SPFX within the range 2*10-8-1*10-6mol/L,there exists a good
linear relationship between the concentration and the fluorescence intensity of SPFX.
3.6 Determination of SPFX tablets
 |
Fig.2 1st order derivative
synchronous fluorescence spectra a. Internal Hormones in human urine ; b. SPFX-NaNO2-KI; [HCl] =4´ 10- 3 moL/L; [NaNO2]=5´ 10-5moL/L; [KI] =2.4´ 10-3moL/L; pH=3.3; CSPFX =2.5mg/mL |
10 tablets of SPFX was exactly
weighted and ground, then a moderate amount of powder (about 100mg SPFX) was charged into
a beaker of 250mL,dissolved with NaOH solution of 0.01mol/L and filtered. The above
filtered solution and SPFX standard of 50mg carefully charged into a flask of 500mL,and
diluted to final volume. Then transferred the above mixture solution 10mL into a flask of
100mL to obtain a mixture as working solution. At last, 2.5mL of above mixed solution was
operated according to the procedures described previously, from which the average of SPFX
in tablet was determined as 96.94% by five times determining. This is a good agreement
with the result obtain by non-water titration. The recovery is 97.2% and the RSD 0.8%.
3.7 Calibration graph of determination of SPFX by derivation synchronous
fluorimetry
According to the above experimental procedure, the mixture solution of standard SPFX and
blank urine were scanned by fixing the excitation wavelength within the range of
200-450nm, Fig.2 gives the first-order derivative synchronous spectra of mixture of SPFX
with blank urine fixed as Dl=80nm,then measured using peak-zero method with 361nm(-)as
determining wavelengths. The results show that the plot of dF/dl versus [SPFX]
possesses a good linearity in the concentration range of 0. 2mg/mL-4. 0mg/mL, the linear equation was dF/dl = -8.987 Cspfx-0.750, with a
correlation coefficient of 0.9995 and detection limit 0.2mg/mL
3.8 Measurement of urine sample
2.5mL of urine sample of healthy person was exactly piped and SPFX was determined five
times in parallel. As show in Table 1, the RSD are 0.7-1.2% and
the recovery, 93.8-101.2%.
Table 1 Analytical results of sample (n=5)
| Sample | SPFX Found (mg/mL) |
SPFX Added (mg/mL) |
Total SPFX Found |
Recovery (%) |
RSD (%) |
| Urine 1 | 0.198 | 0.3 | 0.467 | 93.8 | 1.2 |
| Urine 2 | 0.424 | 0.6 | 0.977 | 95.4 | 0.7 |
| Urine 3 | 0.701 | 0.8 | 1.519 | 101.2 | 1.1 |
| Urine 4 | 1.099 | 1.0 | 2.040 | 97.2 | 0.8 |
No. 1,
2,3,4 were urinary excretions of SPFX four oral dose of SPFX tablet(100,200,300,400mg) to four healthy volunteers after two hours, respectively.3.9 Reaction mechanism
A reaction flask (150mL) was charged with SPFX (1.0g) and HCl solution (0.2mol/L,4mL).To the mixture was added NaNO2 (0.2mol/L,15mL) drop wise with agitation over an ice bath for 10min ,into which KI solution (0.2mol/L,15mL) being added slowly. After setting for 30min under cooling condition, the mixture was refluxed for 2 hours. They were washed with HCl(0.2mol/L)and then dried over a bath to afford product.
According to the results measured by IMPACT-410 (NICOLET) IR spectrum (America), It can be seen that (KI)peaks at n
N-H3435 cm-1, 3350 cm-1, nC- N 1285cm-1, dN-H 1565cm-1 all disappear. This indicates that -NH2 group of the molecular has been substituted by atom I. It can be demonstrated from the strong absorption peak at nC-I 1056 cm-1. So the fluorescence enhancement mechanism of SPFX derivatizing reaction can be propesed as follows:
The structures of SPFX and other quinolones were
compared. It is found that the 5-NH2is the ultimate reason for SPFX
fluorescence disappearing. In SPFX molecules, 5-NH2 is easily protonized in
acid media to form NH3+, herefor,it can be translated from a strong
electron donating groups to a strong electron-attacting groups. On the other hand, in SPFX
molecules, 5-NH2 and 4-C=O (or-OH) easily form intramolecular hydrogen bond,
which reduces the degree of freedom of plane p-bond.
Finally it leads to the great reduction of fluorescence yield. From our investigation, as
the 5-NH2 is substituted by I, the electron cloud of benzoate is not further
reduced and the intramolecular hydrogen bond is also disappeared. Therefore, it can cause
the freedom of plane p-bond
enhance and cause the fluorescence of SPFX molecules enhance.
REFERENCES
[1] Xu T N. Biochemical Pharmacy, Preparation Branch, 1992, 13
(5): 285-289.
[2] Meyyanathan S N, Sebastion M, Uresh B. Indian Drugs, 1998, 35 (6): 344.
[3] Chetna T. Indian Drugs, 1998, 35 (4): 229.
[4] Ministry of sanitary of the People's Republic of China.
Standards issued by Ministry of Sanitary of P.R.C. 1997, WS-508 (X-440).
[5] Klaus B, Ellen B. J. Chromatogr. 1992, 579: 285.
[6] Bhavani K, Srivastave C M R. East Pharm, 1997, 40 (476): 161.
[7] Mody V D. J. Pharm. Biomed. Anal., 1998, 16 (8): 1289.