ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2011, том 66, № 8, с. 842-847
ОРИГИНАЛЬНЫЕ СТАТЬИ
УДК 543
FLUORESCENT MICROSCOPIC DETERMINATION OF GATIFLOXACIN IN MILK, INJECTION, HUMAN URINE AND RABBIT SERUM SAMPLES BY SELF-ORDERED RING TECHNIQUE ON GLASS SLIDES SUPPORT
© 2011 Yan Min Yu, Ying Liu
College of Life and Environmental Sciences, Minzu University of China Beijing 100081, the People's Republic of China Received 15.04.2010; in final form 10.12.2010
A simple and sensitive self-ordered ring (SOR) technique, which was based on the capillary effect of solvent on a hydrophobic glass slide, was successfully applied to the determination of gatifloxacin in milk, injection, human urine and rabbit serum samples. In a medium of pH 3.20 (HAc-NaAc) with the aid of poly(vinyl alcohol)-124 (PVA-124), when 0.50 pL aluminum-sensitized gatifloxacin was dropped on glass slide with dimethyl dichlorosilane (DMCS) pretreated, a typical fluorescent SOR with diameter (2R) of the ring less than ca. 1.77 mm and the belt width (28) less than 29.3 pm can be obtained. The solute on the ring belt had strong fluorescence. Data of the imaged SOR showed that the gatifloxacin molecule across the SOR belt section follows a Gaussian distribution. The assay showed that when the droplet volume is 0.1 p.L, the SOR method coued be used to determine gatifloxacin in the range of 5.61 x 10-14 ~ 1.50 x 10-12 mol/ring (5.61 x 10-8~1.50 x 10-5 M) and the limit of determination (LOD) reached 5.61 x 10-15 mol/ring (5.61 x 10 8 M) with three-fold signal-to-noise ratio (S/N = 3).
Keywords: self-ordered ring technique, gatifloxacin, fluorescence microscopic analysis.
With the social development, people made widely use of (fluoro)quinolones as clinical durg for humans and as veterinary drugs for animals to treat and prevent various infectious diseases [1, 2]. Gatifloxacin (1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-[3-methyl-1-piperazi-nyl]-4-oxo-3-quinolinecarboxylicacid, GFLX), a synthetic broad-spectrum and advanced generation antimicrobial fluoroquinolone belonging to a group of recently developed quinolone antimicrobials that is active against both gram-negative and gram-positive bacteria, is used in the treatment of a wide range of infections [3]. In recent years, more and more clinical studies have shown that gatifloxacin can cause glucose abnormality for patients [4— 6]. Because of the wide use of gatifloxacin in agriculture and aquaculture [7], the presence ofresidues in esculent products will influence human health. So, more and more countries are establishing MRLs (maximum residue levels) and withdrawal periods. The European Union (EU) established legislation (Council Regulation 2377/90/EEC [8]). Therefore, methods with high sensitivity, high selectivity and small sample consumption for the determination of gatioxacin are urgently needed.
The methods ofchoice for the analysis ofgatioxacin in formulations are liquid chromatography [9—12], capillary electrophoretic [13], fluorescence detection [14], pressurized capillary electrochromatography [8] and microbiological assay [15]. The methods are time consuming, expensive and complex as many steps are involved in sample preparation and analysis. The SOR fluorescent microscopy technique in present assay is a new trace
analysis method with high sensitivity and low interference of coexisting substances due to the fluorescent analytes enrichment in the ring belt of the SOR [16]. It was successfully applied to the determination of gatifloxacin in milk, injection, human urine in healthy volunteers and rabbit serum samples with the recoveries of97.0 ~ 108.0%.
O
Gatifloxacin
EXPERIMENTAL
Apparatus and software. An Olympus IX81 inverted microscope (Olympus, Japan) equipped with a 100-W mercury arc lamp and a U mirror cube unit with excitation filter of 330—385 nm, was used to observe the SORs formed on a solid support, 4 x objective and 10 x objective lens were used. The SOR image was captured by using a Cohu 4910 series cooled CCD (Cohu, USA) coupled with Image-Pro Plus 6.0 software package (Media Cybernetics, USA) to measure the fluorescence of the SORs. A GL-88B vortex mixer (Haimen Instrumental Ltd., Jiangsu, China) was used to blend the solutions. A Galanz microwave oven (G8023CSL-2C, 800 W Foshan
120 100 80 60 40 20
0
100 Pixels
200
Fig. 1. A typical blue SOR planform of aluminum—gatioxacin chelate formed on a DMCS pretreated glass slide (A), the blue three-dimensional SOR image of fluorescein (B), and the digital distributed presentation of fluorescence molecules (C). Spotted solution: GFLX, 6.0 x 10-6 M; Al3+, 6 x 10-6 M; PVA-124, 0.2% (w/v); pH, 3.20. Droplet volume, 0.50 ^L. A 4 x objective was used.
Shunde Galanz Microwave Oven Electrical Appliance Co., t, Guangdong), a PB-10 digital pH meter (Sartorius Corporation, Germany), BS224S electronic balance (Sartorius Corporation, Germany) were also used. Origin 7.5 software package and Drug and Statistics for Windows 2.0 (DAS2.0) were used for linear fitting, Gaussian fitting and the pharmacokinetic parameters.
Reagents. A 1 x 10-3 M stock solution of gatifloxacin (China National Institute for the Control of Pharmaceuf-ical , Beijing, China) was prepared by dissolving 0.0380 g and diluted to 100 mL with doubly distilled water and kept in the refrigerator at 4°C. The working solution was diluted to 1.01 x 10-4 M. 1 x 10-3 M aluminum ion stock solution was prepared by dissolving aluminum chloride anhydrous (Sinopharm Chemical Reagent Co., Ltd, Beijing China) in doubly distilled water. The HAc-NaAc (pH3.20) buffer solution and 1%(w/v) solution of poly(vinyl alcohol)-124 (PVA-124, Beijing's chemical reagents) were used. All reagents were analytical-reagent grade. Doubly distilled water was used throughout.
Pretreatment of samples. The samples of milk were 200-fold directly diluted with doubly distilled water before general analysis procedures. Gatifloxacin injection was purchased from Brilliant Pharmaceutical Co., Ltd., Chengdu. About 1mL of the sodium chloride injection was directly dissolved in distilled water to prepare a 250-mL analysis sample.
The human urine samples were collected at 0, 1.0, 3.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 12.0, 24.0 and 40.0 h after a single 400 mg orally dose was taken by two healthy volunteers of the Minzu University of China. After centrifuged for 10 min at the speed of 3000 r/min, the supernatant
fluid of the samples was collected and 200-fold diluted with doubly distilled water, then directly subjected to general procedures for detection.
The fresh serum samples were collected from healthy rabbit at 0, 15, 30, 60, 90, 120 and 180 min, respectively, by using single-use vacuum storage vessel after injection of a therapeutic dosage of 3.9 mg/kg. After standing for 10 min, the blood was centrifuged for 10 min at 3000 r/min, thus the supernatant was taken out. The proteins of supernatant was cleaned with an equal volume of methanol and re-centrifugation for 10 min at 3000 r/min to remove proteins, the supernatant was taken as the serum analysis sample.
Procedures. Into a 0.5 mL micro-tube appropriate working solutions of gatifloxacin or sample solutions, 30.0 ^L of 1.0 x 10-4 M aluminum ion solution and 50.0 ^L HAc—NaAc buffer solution were added in turn, then vortex-mixed. After standing for 60 min, 100 ^L of 0.60% PVA-124 solution was added and the mixture was diluted with doubly distilled water to 0.5 mL, and mixed thoroughly. Then (0.1, 1.0 ^L) of the mixture was spotted on the surfaces of the pretreated glass slides. The glass slides were immediately dried for about 1.5 min in the microwave oven, then the formed SOR image were observe and measured under Olympus IX 81 inverted microscope system.
RESULTS AND DISCUSSION
Features of SORs. Fluorescence intensity of gatifloxacin is enhanced when gatioxacin reacts with Al3+. Fig. 1A shows a planform of the blue aluminum-gatioxacin
Table 1. Influence of coexistent foreign substances*
Substance Concentration, M Change of M, % Substance Concentration, M Change of AI, %
K+, NO- 1.0 x 10-4 +0.3 Fe3+, Cl- 1.1 x 10-5 +9.0
Na+, NO- 1.0 x 10-3 +2.5 Sucrose 1.0 x 10-4 +0.8
Mg2+, Cl- 1.0 x 10-4 +3.6 Amylum* 1.52 x 10-2 +5.5
Cu2+, Cl- 1.0 x 10-5 + 1.3 Lysine 1.0 x 10-5 +1.0
Ca2+, NO- 1.0 x 10-4 +5.4 Glycin 1.0 x 10-5 +3.5
Zn2+, Cl- 1.0 x 10-4 + 10.3 CTMAB 1.0 x 10-4 +0.9
NH+, Cl- 1.0 x 10-3 -2.5 SDBS 1.0 x 10-4 -2.0
Co2+, NO- 1.0 x 10-5 -9.7 VitB5 1.0 x 10-4 +1.9
Ba2+, Cl- 1.0 x 10-4 -10 HSA* 0.1 -2.1
Cr3+, Cl- 1.0 x 10-5 +2.7 Urea 1.1 x 10-3 -6.2
* Concentration ofAmylum and HSA are evinced as mg/mL; CTMAB: cetyltrimethylammonium bromide; SDBS: sodiumdodecyl benzene sulfonate; Spotted solution: GFLX, 6.0 x 10-6 M; Al3+, 6 x 10-6 M; PVA-124, 0.2%; pH, 3.20; droplet volume, 0.50 ^L; A4 x objective was used.
chelate SOR. The diameter (2R) ofthe ring is ca. 1.77 mm and the SOR belt width (25) is 29.3 ^m. Figure 1B provids a three-dimensional image. Figure 1C displays the digital distributed presentation of the molecules through the ring center. In Fig. 1C, it can be seen that the SOR is symmetrical. By fitting the data, we can found that the distribution of aluminum-gatioxacin chelate molecules across the SOR belt section follows a Gaussian function and the fluorescence intensity inside and outside SOR is almost zero. It is obvious that the contribution of the background is very small, which is a big challenge for the sensitivity improvement for spot analysis on solid support such as thin film substrate including octadecylsilanized silica and poly(vinyl chloride) plate [16]. In Fig. 2, a fluorescent material radial distribution and fluorescence intensity (/max) of the radial distance (pixels) for a Gaussian fitting are shown.
t n ir 84 3.67 -2(x - 28.09)2/6.932 . 2
la = 12.16 + -7=- e " ' (Xa = 3.H) Vn/2
Ib = 10.70 + 84752 e-2(x - 315 74)2/ 6 752 (x2b = 0.83). Jn/2
Optimization of the general procedure. The effect ofpH. It is proved that uorescence inte
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