Research article Received: 21 May 2014,
Revised: 10 July 2014,
Accepted: 28 December 2014
Published online in Wiley Online Library: 12 February 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.2861
Spectroscopic study of the interaction between adenosine disodium triphosphate and gatifloxacin–Al3+ complex and its analytical application Mohammad Kamruzzaman,a A. Nayeem Faruqui,a Mohammed Ifteker Hossainb and Sang Hak Leec* ABSTRACT: A new and sensitive spectrofluorimetric method has been proposed to determine trace amount of adenosine disodium triphosphate (ATP). The method is based on the fluorimetric interaction between gatifloxacin (GFLX)–aluminium (III) (Al3+) complex and ATP and studied using UV-visible and fluorescence spectroscopy. Weak luminescence spectra of Al3+ were enhanced after complexation with GFLX at 423 nm upon excitation at 272 nm due to energy transfer from the ligand to the Al3+ ion. It was observed that the FL emission spectrum of GFLX–Al3+ was enhanced significantly by the addition of ATP. Under the optimal conditions, the enhancement of FL intensity at 423 nm was responded linearly with the concentration of ATP in the range 1.3 × 10–10 – 1.0 × 10–8 mol L–1 with correlation coefficient (r) of 0.9981. The limit of detection (LOD) was found to be 1.1 × 10–11 mol L–1 for ATP with the standard deviation (RSD) of 1.21% for five repeated measurement of 2.3 × 10–8 mol L–1 ATP. The presented method is simple, sensitive, free from coexisting interferents and can be applied successfully to determine ATP in the real samples. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: adenosine disodium triphosphate; aluminium (III); gatifloxacin; fluorescence; milk; pharmaceutical formulation
Introduction
Luminescence 2015; 30: 1077–1082
* Correspondence to: Sang Hak Lee, Department of Chemistry, Kyungpook National University, Daegu, 702–701, South Korea. E-mail: [email protected] a
Department of Textile Engineering, Daffodil International University, Dhaka-1207, Bangladesh
b
Department of Natural Science, Daffodil International University, Dhaka1207, Bangladesh
c
Department of Chemistry, Kyungpook National University, Daegu 702-701, South Korea Abbreviations: LOD, limit of detection.
Copyright © 2015 John Wiley & Sons, Ltd.
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Adenosine disodium triphosphate (ATP) is an important coenzyme used as universal energy carrier in the cells of all living organisms. It is often called the “molecular unit of currency” of the intracellular energy transfer (1). ATP acts as the mediator of energy exchange in all living cells, both catabolic, degradative processes and anabolic, biosynthesis process. Because of its ubiquitous presence in living cells, ATP participates in the metabolism of protein, fat, nucleic acid, nucleotide and sugar in the body, in the transport of molecules and ions throughout the living cells, muscle contraction and other cellular movement. ATP transports energy to the living organisms by converting ATP to adenosine diphosphate (ADP) and phosphoric acid during metabolic process. ATP possesses significant importance for the biomass determinations in clinical microbiology, food quality control and environmental analysis (2). ATP is used to treat the diseases caused by tissue injury and activity reduction of ecto enzyme, including heart failure, cardiac insufficiency, cardiac muscle disease and for the treatment of progressive amyotrophia, cerebral hemorrhage sequelae, hepatitis etc. Thus, determination of ATP is of great importance in view of pharmaceutical and biological activities. The most common methods used for the determination of ATP including bioluminometric (3–7), resonance light scattering (8), capillary electrophoresis (9), enzyme immobilized biosensor (10), high-performance liquid chromatography (11–14), luminescence (15,16), electrochemical (17), spectrofluorimetric (18–22) methods, etc. Some of these methods have suffered from
various drawbacks including being time consuming, sample separation and treatment, expensive instruments and reagents, low sensitivity, inconvenient reagent pretreatment, unable to assay real sample etc. Spectrofluorimetric method has been widely used for determining ATP because of its high sensitivity, selectivity, simple and inexpensive instrumentation. Gatifloxacin, GFLX [(±)-1-cyclopropyl-6-fluoro-1,4-dihydro-8methoxy-7-(3-methyl-1-pipe-razinyl)-4-oxo-3-quinoline carboxylic acid] is the fourth generation of a new class of synthetic antibacterial fluoroquinolone agents. GFLX containing an α-carbonyl carboxylic acid group is able to form a complex with the metal ions. Therefore, GFLX can form a stable complex with an Al3+ ion. GFLX is a good ligand for Al3+ and can bind with Al3+, which can be proved by the fluorescence emission and UV-visible spectra.
M. Kamruzzaman et al. In the present study, we presented the interaction between the GFLX–Al3+ complex and ATP using fluorescence and UV-vis spectra. GFLX exhibits a fluorescence (FL) emission peak at about 443 nm when excited at 283 nm. The FL emission intensity of GFLX was enhanced significantly when Al3+ was mixed with GFLX while Al3+ alone showed almost no emission peak. The FL emission intensity of the GFLX–Al3+ system was markedly increased in the presence of ATP. It was found that the fluorescence intensity of the GFLX–Al3+–ATP system increased proportionally to the ATP concentration. Based on this, a simple spectrofluorimetric method with high sensitivity and selectivity has been proposed to determine ATP based on the complexation of GFLX and Al3+ and its interaction with ATP using fluorescence and UV-visible spectra.
Experimental Apparatus All the fluorescence spectra were recorded using a spectrofluorimeter (Model F-4500, Hitachi, Japan) equipped with a 450-W Xenon lamp (Model XBO 450 W/1, Osram, Germany) as the excitation light source and a photomultiplier tube (Model R928, Hamamatsu, Japan) powered at 950V as the detector. The excitation and emission slits were 10 nm to measure all fluorescence spectra. pH was adjusted using a pH meter (Model Orion, 520A, USA). All the UV-visible spectra were measured using a UV-visible spectrophotometer (UV-1800, Shimadzu, Japan). Reagents Aluminium potassium sulphate dodecahydrate [KAl(SO4)2.12H2O], gatifloxacin (GFLX) and adenosine disodium triphosphate (ATP) were obtained from Sigma–Aldrich (USA). Double deionised (DI) water (Millipore, MilliQ Water System, USA) was used throughout. A stock standard solution (1 mmol L–1) of GFLX was prepared by dissolving an appropriate amount of GFLX in DI water and stored at 4°C. A primary stock solution (0.5 mmol L–1) of ATP was prepared using DI water. A stock solution of Al3+ (10 mmol L–1) was prepared by dissolving 0.237 g KAl(SO4)2.12H2O in DI water. All other reagents used were of analytical reagent grade. Working solutions were prepared daily from the stock solutions by appropriate dilution with DI water immediately before use. Sample preparation
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Five commercially available ATP coated tablets (Jeil Pharm. Co. Ltd, South Korea) were weighed and grounded to a fine powder by pestle and mortar. Then an accurate weight of the powder containing ATP 20 mg was dissolved into a 50 mL volumetric flask with DI water. The resulting solution was then filtered through a Millipore membrane filter paper (0.22 μm pore size) in order to remove the insoluble excipients. The solution was then diluted appropriately within the working range of ATP measurements. For injection, five ATP injections (Jeil Pharm. Co. Ltd, South Korea) collected from a local medicine store were mixed thoroughly in a dry beaker. Then, a volume equivalent to 20 mg of ATP was transferred to a 50 mL volumetric flask and diluted with DI water up to the mark. Milk samples were collected from a local firm and ATP was extracted from milk according to the procedure described in the
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literature (15) with slight modification. Briefly, 5 mL milk sample was mixed with 1 mL of trichloroacetic acid (1 mol L–1) in a centrifuge tube. The mixture was then shaken vigorously and put on the ice bath. The mixture was centrifuged for 15 min and 3 mL of the extract was neutralized with 2 mol L–1 sodium hydroxide. The mixture was then taken into a 25 mL volumetric flask and diluted with DI water up to the mark. Experimental procedure All the fluorescence measurements were made through the following procedure. To a 10 mL volumetric flask, a certain volume of Al3+ solution, buffer (pH = 5.2) solution and GFLX solution were added and diluted with 5 mL double DI water. All the reagents were mixed thoroughly and allowed to stand for several minutes. Then, a specified volume of ATP solution was mixed with the above mixture and stood for about 20 min. The solution was then put into the 1 cm quartz cell to measure all FL spectra of the system with an emission wavelength at 423 nm when excited at 272 nm. Both the excitation and emission slits were set to 10 nm and the voltage for the photomultiplier tube was set to 900 V.
Results and discussion Fluorescence spectra The fluorescence emission and excitation spectra of Al3+ , ATP, Al3+–ATP, GFLX, GFLX–ATP, GFLX–Al3+, GFLX–Al3+–ATP are shown in Fig. 1. GFLX exhibited maximum fluorescence emission at a wavelength of about 443 nm (Fig. 1a, curve 4) when excited at 283 nm (Fig. 1b, curve 4). When Al3+ was added to the GFLX solution, GFLX molecule and Al3+ could undergo a charge transfer reaction. The FL intensity was enhanced and the emission wavelength was blue shifted from 443 nm to 423 nm (Fig. 1a, curve 6), which indicates that the FL intensity of GFLX was increased in the presence of Al3+. The FL intensity of GFLX–Al3+ system was enhanced markedly when ATP was added into the GFLX–Al3+ system (Fig. 1a, curve 7), which indicates that ATP can form a stable ternary complex with the GFLX–Al3+ system. However, the Al3+–ATP system did not show an enhanced emission peak which may be due to the formation of an unstable complex. Therefore, the GFLX–Al3+–ATP system exhibited enhanced FL intensity at 423 nm. From the excitation spectra (Fig. 1b), it could be observed that the maximum FL excitation wavelength of the GFLX–Al3+–ATP system was about 272 nm while the emission wavelength was about 423 nm. Thus, 272 nm and 423 nm were selected as the excitation and emission wavelengths for the determination of ATP. Possible interaction mechanism of the presented system In order to understand the interaction mechanism of GFLX, Al3+, and ATP, absorption spectra of Al3+ , ATP, Al3+–ATP, GFLX, GFLX– ATP, GFLX–Al3+, and GFLX–Al3+–ATP were recorded and shown in Fig. 2. GFLX exhibited maximum absorbance at about 286 nm (Fig. 2, curve 4). When Al3+ was added into the GFLX solution, the absorbance peak of GFLX was blue shifted from 286 nm to 275 nm and the absorbance was increased (Fig. 2, curve 6), which indicated that a charge transfer reaction might occur between GFLX and Al3+ ion. According to the Förster’s non-
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2015; 30: 1077–1082
Spectroscopic determination of ATP
a
2100
a
1800
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Absorbance
FL Intensity (a.u.)
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0.25
7
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6 600
5 4
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50
300
450
500
550
FL Intensity (a.u.)
2000
1200
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400 0
1,2,3 240
260
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3+
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Figure 1. FL emission (a) and excitation (b) spectra of (1) Al ; (2) ATP; (3) Al – 3+ 3+ ATP; (4) GFLX; (5) GFLX–ATP; (6) Al –GFLX; and (7) Al –GFLX–ATP. Conditions: 8 –1 3+ –1 –1 [ATP], 3.5 × 10 mol L ; [Al ], 0.06 mmol L ; [GFLX], 7.0 μmol L ; NaAc–HAc –1 (0.1 mol L ); pH, 5.2, λex/λem = 272/422 nm.
7
Absorbance
1.6
6
1.2
5 0.8
0 540
Optimization of the reaction conditions
4
5
Wavelength (nm)
2.0
480
the wavelength was slightly blue shifted from 275 nm to 271 nm (Fig. 2, curve 7). The above results indicated that ATP can form a strong ternary complex with GFLX–Al3+. Therefore, the FL intensity of the GFLX–Al3+–ATP system at 423 nm was increased several times (Fig. 1a). The FL intensity of the system was enhanced proportionally with the concentration of ATP.
1600
800
420
Figure 3. Spectral overlap between (a) emission spectrum of GFLX and (b) ab+1 sorption spectrum of Al .
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Wavelength (nm)
Wavelength (nm) 2400
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1,2,3
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Effect of pH and buffer solution. The pH of a solution plays an important role in obtaining maximum FL intensity. Thus, the effect of pH on the FL intensity of the GFLX–Al3+–ATP system was investigated and the maximum FL intensity was obtained at pH of 5.2. When pH is higher, the hydration of Al3+ results in the formation of soluble hydroxo complexes, polycations or hydroxo polymers which are not conducive to chelating between Al3+ and GFLX. This might hamper the sensitivity of the FL intensity. Therefore, an optimal pH value of 5.2 was selected for the whole experiment. The experimental results indicated that buffer solutions had also a large effect on the FL intensity of the presented system. Thus, the effect of the following buffers on the FL intensity was studied: phosphate buffer, KH2PO4– Na2HPO4 (0.1 mol L–1), borate (acid–base) buffer, borax–HCl (0.1 mol L–1), NH4Cl–NH3 (0.1 mol L–1), acetate (acidic) buffer, NaAc–HAc (0.1 mol L–1), hydrogen phosphate (acidic) buffer, KH2PO4–NaOH (0.1 mol L–1), Tris–HCl (0.1 mol L–1). Among the above buffers, NaAc–HAc exhibited maximum FL intensity (Fig. 4). Thus, acetate buffer, NaAc–HAc (0.1 mol L–1) at pH5.2 was used to obtain the highest FL intensity.
radiation energy transfer theory (23), the rate of energy transfer depends upon the extent of overlap of the emission spectra of the donor with the absorption spectra of the accepter as well as the distance between them. Energy transfer easily occurs between Al3+ and GFLX because of the strong spectral overlap between the emission spectra of donor (GFLX) and the absorption spectra of accepter (Al3+) as shown in Fig. 3. After the addition of ATP into the GFLX–Al3+ system, the absorbance is enhanced and
Effect of GFLX concentration. GFLX solution might also influence the sensitivity of the presented method. Thus, the effect of GFLX concentration on the FL intensity was investigated and shown in Fig. 6. The maximum FL intensity of the system was obtained using GFLX at a concentration of 7 μmol L–1. Above this concentration, the FL intensity starts to decline due to the maximum complex formation at this concentration. Therefore, an excess amount of GFLX would absorb or quench the FL intensity of the system. Thus, 7 μmol L–1 GFLX was chosen for further study.
300
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Wavelength (nm) 3+
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3+
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Figure 2. Absorbance spectra of (1) Al ; (2) ATP; (3) Al –ATP; (4) GFLX; (5) GFLX– 3+ 3+ 8 –1 ATP; (6) Al –GFLX; and (7) Al –GFLX–ATP. Conditions: [ATP], 1.5 × 10 mol L ; 3+ –1 –1 –1 [Al ], 0.08 mmol L ; [GFLX], 11.0 μmol L ; NaAc–HAc (0.1 mol L ).
Effect of Al3+ ion concentration. The effect of Al3+ ion concentration on the FL intensity was examined in the range of 0.01–0.1 mmol L–1. The FL intensity was increased with increasing the concentration of Al3+ up to 0.06 mmol L–1 (Fig. 5). Therefore, an Al3+ concentration of 0.06 mmol L–1 was selected as optimum concentration for this experiment.
250
M. Kamruzzaman et al. IV
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GFLX, ATP showed the maximum FL intensity. The chelation reaction of the GFLX–Al3+–ATP system could complete in about 20 minutes at room temperature. Therefore, the FL intensity of the system reached a maximum in 20 min after all the reagents had been added and remained stable for at least 2 h.
FL Intensity (a.u.)
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Interference of foreign substances
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III
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Buffer solutions –1
Figure 4. Effect of buffers. I, KH2PO4–Na2PO4 (0.1 mol L ; pH, 7.2); II, borax–HCl –1 –1 (0.1 mol L ; pH, 8.0); III, NH4Cl–NH3 (0.1 mol L ; pH, 9.2); IV, NaHc–HAc (0.1 mol –1 –1 –1 L ; pH, 5.2); V, KH2PO4–NaOH (0.1 mol L ; pH, 7.2); and VI, Tris–HCl (0.1 mol L ; 8 –1 3+ –1 pH, 7.5). Conditions: [ATP], 1.5 × 10 mol L ; [Al ], 0.08 mmol L ; [GFLX], 11.0 –1 μmol L .
In order to investigate the selectivity of the proposed method, the effect of interfering substances that coexist with the analyte and which may suppress or enhance the FL intensity was examined. Therefore, the effect of potential foreign substances was studied by preparing a set of solutions, each one with 3.5 × 10 8 mol L–1 of ATP and different concentrations of the chemical species to be tested. A foreign species is considered as a noninterfering component if it produces an error less than ± 5% in the determination of ATP. The results of the effect of interferencing substances are summarized in Table 1. It was observed that the coexisting foreign substances did not generate a significant effect in the determination of ATP.
2100
Linear range and detection limit of ATP FL Intensity (a.u.)
1800
Under the aforementioned optimal condition, a calibration curve was constructed which showed a good linear relationship between the FL intensity and the concentration of ATP in the range of 1.3 × 10–10 to 1.0 × 10–8 mol L–1 with correlation coefficient (r) of 0.9981. The regression equation was Y = 6.19 × 1010CATP +357 where CATP is the concentration of ATP and Y is the fluorescence intensity in arbitrary unit (a.u.). The limit of detection (LOD) as defined by IUPAC, CLOD = 3 * Sb/m (where Sb is the standard deviation of the blank signals
1500 1200 900 600 300 0 0.00
0.02
0.04
0.06
0.08
0.10
0.12
Table 1. Effect of interfering substances on the GFLX–Al3+– ATP system
[Al3+] mmol L-1 Figure 5. Effect of aluminium (III) ion concentration. Conditions: [ATP], 3.5 × 10 –1 –1 –1 mol L ; [GFLX], 7.0 μmol L ; NaAc–HAc (0.1 mol L ); pH, 5.2.
8
2100
FL Intensity (a.u.)
1800 1500 1200 900 600 300 0
0
2
4
6
8
10
12
[GFLX], µmol L-1 Figure 6. Effect of GFLX concentration. Conditions: [ATP], 3.5 × 10–8 mol L–1; [Al3+], 0.06 mmol L–1; NaAc–HAc (0.1 mol L–1); pH, 5.2.
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Effect of the addition order of reagents. The addition order of the reagents also influences the FL intensity of the system. Thus, the effect of the reagent addition order was investigated and observed that the following addition order: Al3+, NaAc–HAc buffer,
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Interfering substances Cu2+ Zn2+ Al3+ Fe2+ Mg2+ Fe3+ Ca2+ Cd2+ Cr3+ Tryptophan Thymine Adenine Ascorbic acid L-Glutamic acid BSA Glucose Sucrose L-Lysine L-Cystine L-Histidine L-Phenylalanine
Copyright © 2015 John Wiley & Sons, Ltd.
Tolerable concentration (mol L–1) 1.2 2.1 1.4 1.6 2.2 2.5 3.1 2.7 3.2 3.7 3.9 1.9 2.3 2.3 2.4 1.5 1.7 3.6 4.1 5.7 3.1
× 10–6 × 10–6 × 10–6 × 10–6 × 10–5 × 10–7 × 10–5 × 10–6 × 10–8 × 10–5 × 10–6 × 10–8 × 10–6 × 10–5 × 10–7 × 10–4 × 10–4 × 10–5 × 10–5 × 10–6 × 10–5
Change in fluorescence intensity (%) +1.15 +0.71 +2.31 –4.1 –0.85 +2.15 –3.12 –1.35 –3.19 +1.55 +2.11 +1.85 –2.3 +1.4 –2.55 –2.75 –4.25 –3.21 +2.05 +1.37 –3.51
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Spectroscopic determination of ATP Table 2. Assays of ATP in pharmaceutical formulations by the proposed method Sample
Amount (mg)
Standard addition method
Active ingredient label
Found by the proposed method ± RSDa
Tablet
20 mg of ATP
21.25 ± 1.71
Injection
20 mg of ATP
19.55 ± 1.35
Added (×10
–7
–1
Recovery (%) –7
mol L )
–1
Observed (×10 mol L ) ± RSDa
1.0 3.0 5.0 1.0 3.0 5.0
1.03 3.14 4.89 0.97 3.01 5.07
± ± ± ± ± ±
1.32 1.05 1.45 1.35 0.92 0.92
103.00 104.67 97.80 97.00 100.33 101.40
Relative standard deviation for five replicate measurements.
a
Table 3. Determination of ATP in milk samples and results for recovery test Sample
Found by the proposed method (×10–7 mol L–1) ± RSDa
Milk
1.76 ± 1.55
Standard addition method Added (×10–7 mol L–1)
Found (×10–7 mol L–1) ± RSDa
2.0 4.0 6.0 8.0 10.0
2.09 3.93 6.11 8.13 9.81
± 1.11 ± 1.21 ± 1.32 ± 1.05 ± 1.73
Recovery (%)
104.50 98.25 101.83 101.62 98.10
Relative standard deviation for five replicate measurements.
a
and m is the slope of the calibration graph) was found to be 1.1 × 10–11 mol L–1 for ATP. The standard deviation (RSD) was 1.21% for five repeated measurements of 2.3 × 10–8 mol L–1 ATP.
Analytical application Determination of ATP in tablet and injection. In order to demonstrate the applicability of the proposed method, it was applied to determine ATP in a commercially available pharmaceutical tablet and injection (Jeil Pharm. Co. Ltd, South Korea). The results are listed in Table 2. As shown in Table 2, the amount of ATP in tablet and injection obtained by the proposed method did not show significant difference to the labeled contents. Recoveries performed by standard addition method were in the range of 97.00– 104.67% for ATP.
Luminescence 2015; 30: 1077–1082
A sensitive, simple and cost-effective spectrofluorimetric method has been developed to determine ATP based on the interaction of ATP with the GFLX–Al3+ complex. The FL intensity of the GFLX–Al3+ complex was enhanced in the presence of ATP by increasing the concentration of ATP in the range of 1.3 × 10–10 to 1.0 × 10–8 mol L–1. The proposed method offers a low LOD (1.1 × 10–11 mol L–1) compared with previously reported methods. The presented method has been successfully applied to determine ATP in pharmaceutical preparations and milk sample with good reproducibility and satisfactory results.
Acknowledgements This research was supported by Kyungpook National University Research Fund, 2013.
References 1. Knowles JR. Enzyme-catalyzed phosphoryl transfer reactions. Annu Rev Biochem 1980; 49: 877–919. 2. Hou F, Miao Y Jiang C. Determination of adenosine disodium tri3+ phosphate (ATP) using oxytetracycline–Eu as a fluorescence probe by spectrofluorimetry. Spectrochim Acta A 2005; 61: 2891–5. 3. Trajkovska S, Dzhekova-Stojkova S Kostovska S. Determination of validity for blood for transfusion by firefly bioluminescent assay for ATP. Anal Chim Acta 1994; 290: 246–8. 4. Kamidate T, Yanashita K, Tani H, Ishida A Notani M. Firefly bioluminescent assay of ATP in the presence of ATP extractant by using liposomes. Anal Chem 2006; 78: 337–42.
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Determination of ATP in milk sample. The presented method was applied to determine ATP in milk samples collected from a local firm. The results of determination of ATP by the proposed method are summarized in Table 3. The recovery of the method was determined by adding a known amount of ATP to the milk sample and subtracting the results obtained for the sample prepared in the same manner but without ATP. The recoveries were found in the range of 98.10–104.50% for ATP. From the results, it was assumed that no interfering materials were encountered which showed the applicability of the method to the determination of ATP in milk samples.
Conclusion
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15. Pérez-Ruiz T, Martínez-Lozano C, Tomás V Martín J. Determination of ATP via the photochemical generation of hydrogen peroxide using flow injection luminol chemiluminescence detection. Anal Bioanal Chem 2003; 377: 189–94. 16. Ronner P, Friel E, Czerniawski K Frankle S. Luminometric assays of ATP, phosphocreatine, and creatine for estimation of free ADP and free AMP. Anal Biochem 1999; 275: 208–16. 17. Yang X, Johansson G, Pfeiffer D Scheller F. Enzyme electrodes for ADP/ATP with enhanced sensitivity due to chemical amplification and intermediate accumulation. Electro-analysis 1991; 3: 659–63. 18. Hou F, Miao Y Jiang C. Determination of adenosine disodium tri3+ phosphate (ATP) using oxytetracycline–Eu as a fluorescence probe by spectrofluorimetry. Spectrochim Acta A 2005; 61: 2891–5. 19. Miao Y, Liu J, Hou F Jiang C. Determination of adenosine disodium 3+ triphosphate (ATP) using norfloxacin–Tb as a fluorescence probe by spectrofluorimetry. J Lumin 2006; 116: 67–72. 20. Yu F, Li L Chen F. Determination of adenosine disodium triphosphate using prulifloxacin–terbium (III) as a fluorescence probe by spectrofluorimetry. Anal Chim Acta 2008; 610: 257–62. 21. Patil SR, Mote US, Patil SR, Lee SH Kolekar GB. Fluorimetric study of 3+ the interaction between ATP and ciprofloxacin–Y complex and its application. J Rare Earth 2010; 28: 329–32. 22. Hou F, Wang X Jiang C. Determination of ATP as a fluorescence probe with europium (III)–doxycycline. Anal Sci 2005; 21: 231–4. 23. Lakowicz JR. Principles of fluorescence spectroscopy. New York: Plenum Press, 1983; p. 303.
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Luminescence 2015; 30: 1077–1082
Revised: 10 July 2014,
Accepted: 28 December 2014
Published online in Wiley Online Library: 12 February 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.2861
Spectroscopic study of the interaction between adenosine disodium triphosphate and gatifloxacin–Al3+ complex and its analytical application Mohammad Kamruzzaman,a A. Nayeem Faruqui,a Mohammed Ifteker Hossainb and Sang Hak Leec* ABSTRACT: A new and sensitive spectrofluorimetric method has been proposed to determine trace amount of adenosine disodium triphosphate (ATP). The method is based on the fluorimetric interaction between gatifloxacin (GFLX)–aluminium (III) (Al3+) complex and ATP and studied using UV-visible and fluorescence spectroscopy. Weak luminescence spectra of Al3+ were enhanced after complexation with GFLX at 423 nm upon excitation at 272 nm due to energy transfer from the ligand to the Al3+ ion. It was observed that the FL emission spectrum of GFLX–Al3+ was enhanced significantly by the addition of ATP. Under the optimal conditions, the enhancement of FL intensity at 423 nm was responded linearly with the concentration of ATP in the range 1.3 × 10–10 – 1.0 × 10–8 mol L–1 with correlation coefficient (r) of 0.9981. The limit of detection (LOD) was found to be 1.1 × 10–11 mol L–1 for ATP with the standard deviation (RSD) of 1.21% for five repeated measurement of 2.3 × 10–8 mol L–1 ATP. The presented method is simple, sensitive, free from coexisting interferents and can be applied successfully to determine ATP in the real samples. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: adenosine disodium triphosphate; aluminium (III); gatifloxacin; fluorescence; milk; pharmaceutical formulation
Introduction
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* Correspondence to: Sang Hak Lee, Department of Chemistry, Kyungpook National University, Daegu, 702–701, South Korea. E-mail: [email protected] a
Department of Textile Engineering, Daffodil International University, Dhaka-1207, Bangladesh
b
Department of Natural Science, Daffodil International University, Dhaka1207, Bangladesh
c
Department of Chemistry, Kyungpook National University, Daegu 702-701, South Korea Abbreviations: LOD, limit of detection.
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Adenosine disodium triphosphate (ATP) is an important coenzyme used as universal energy carrier in the cells of all living organisms. It is often called the “molecular unit of currency” of the intracellular energy transfer (1). ATP acts as the mediator of energy exchange in all living cells, both catabolic, degradative processes and anabolic, biosynthesis process. Because of its ubiquitous presence in living cells, ATP participates in the metabolism of protein, fat, nucleic acid, nucleotide and sugar in the body, in the transport of molecules and ions throughout the living cells, muscle contraction and other cellular movement. ATP transports energy to the living organisms by converting ATP to adenosine diphosphate (ADP) and phosphoric acid during metabolic process. ATP possesses significant importance for the biomass determinations in clinical microbiology, food quality control and environmental analysis (2). ATP is used to treat the diseases caused by tissue injury and activity reduction of ecto enzyme, including heart failure, cardiac insufficiency, cardiac muscle disease and for the treatment of progressive amyotrophia, cerebral hemorrhage sequelae, hepatitis etc. Thus, determination of ATP is of great importance in view of pharmaceutical and biological activities. The most common methods used for the determination of ATP including bioluminometric (3–7), resonance light scattering (8), capillary electrophoresis (9), enzyme immobilized biosensor (10), high-performance liquid chromatography (11–14), luminescence (15,16), electrochemical (17), spectrofluorimetric (18–22) methods, etc. Some of these methods have suffered from
various drawbacks including being time consuming, sample separation and treatment, expensive instruments and reagents, low sensitivity, inconvenient reagent pretreatment, unable to assay real sample etc. Spectrofluorimetric method has been widely used for determining ATP because of its high sensitivity, selectivity, simple and inexpensive instrumentation. Gatifloxacin, GFLX [(±)-1-cyclopropyl-6-fluoro-1,4-dihydro-8methoxy-7-(3-methyl-1-pipe-razinyl)-4-oxo-3-quinoline carboxylic acid] is the fourth generation of a new class of synthetic antibacterial fluoroquinolone agents. GFLX containing an α-carbonyl carboxylic acid group is able to form a complex with the metal ions. Therefore, GFLX can form a stable complex with an Al3+ ion. GFLX is a good ligand for Al3+ and can bind with Al3+, which can be proved by the fluorescence emission and UV-visible spectra.
M. Kamruzzaman et al. In the present study, we presented the interaction between the GFLX–Al3+ complex and ATP using fluorescence and UV-vis spectra. GFLX exhibits a fluorescence (FL) emission peak at about 443 nm when excited at 283 nm. The FL emission intensity of GFLX was enhanced significantly when Al3+ was mixed with GFLX while Al3+ alone showed almost no emission peak. The FL emission intensity of the GFLX–Al3+ system was markedly increased in the presence of ATP. It was found that the fluorescence intensity of the GFLX–Al3+–ATP system increased proportionally to the ATP concentration. Based on this, a simple spectrofluorimetric method with high sensitivity and selectivity has been proposed to determine ATP based on the complexation of GFLX and Al3+ and its interaction with ATP using fluorescence and UV-visible spectra.
Experimental Apparatus All the fluorescence spectra were recorded using a spectrofluorimeter (Model F-4500, Hitachi, Japan) equipped with a 450-W Xenon lamp (Model XBO 450 W/1, Osram, Germany) as the excitation light source and a photomultiplier tube (Model R928, Hamamatsu, Japan) powered at 950V as the detector. The excitation and emission slits were 10 nm to measure all fluorescence spectra. pH was adjusted using a pH meter (Model Orion, 520A, USA). All the UV-visible spectra were measured using a UV-visible spectrophotometer (UV-1800, Shimadzu, Japan). Reagents Aluminium potassium sulphate dodecahydrate [KAl(SO4)2.12H2O], gatifloxacin (GFLX) and adenosine disodium triphosphate (ATP) were obtained from Sigma–Aldrich (USA). Double deionised (DI) water (Millipore, MilliQ Water System, USA) was used throughout. A stock standard solution (1 mmol L–1) of GFLX was prepared by dissolving an appropriate amount of GFLX in DI water and stored at 4°C. A primary stock solution (0.5 mmol L–1) of ATP was prepared using DI water. A stock solution of Al3+ (10 mmol L–1) was prepared by dissolving 0.237 g KAl(SO4)2.12H2O in DI water. All other reagents used were of analytical reagent grade. Working solutions were prepared daily from the stock solutions by appropriate dilution with DI water immediately before use. Sample preparation
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Five commercially available ATP coated tablets (Jeil Pharm. Co. Ltd, South Korea) were weighed and grounded to a fine powder by pestle and mortar. Then an accurate weight of the powder containing ATP 20 mg was dissolved into a 50 mL volumetric flask with DI water. The resulting solution was then filtered through a Millipore membrane filter paper (0.22 μm pore size) in order to remove the insoluble excipients. The solution was then diluted appropriately within the working range of ATP measurements. For injection, five ATP injections (Jeil Pharm. Co. Ltd, South Korea) collected from a local medicine store were mixed thoroughly in a dry beaker. Then, a volume equivalent to 20 mg of ATP was transferred to a 50 mL volumetric flask and diluted with DI water up to the mark. Milk samples were collected from a local firm and ATP was extracted from milk according to the procedure described in the
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literature (15) with slight modification. Briefly, 5 mL milk sample was mixed with 1 mL of trichloroacetic acid (1 mol L–1) in a centrifuge tube. The mixture was then shaken vigorously and put on the ice bath. The mixture was centrifuged for 15 min and 3 mL of the extract was neutralized with 2 mol L–1 sodium hydroxide. The mixture was then taken into a 25 mL volumetric flask and diluted with DI water up to the mark. Experimental procedure All the fluorescence measurements were made through the following procedure. To a 10 mL volumetric flask, a certain volume of Al3+ solution, buffer (pH = 5.2) solution and GFLX solution were added and diluted with 5 mL double DI water. All the reagents were mixed thoroughly and allowed to stand for several minutes. Then, a specified volume of ATP solution was mixed with the above mixture and stood for about 20 min. The solution was then put into the 1 cm quartz cell to measure all FL spectra of the system with an emission wavelength at 423 nm when excited at 272 nm. Both the excitation and emission slits were set to 10 nm and the voltage for the photomultiplier tube was set to 900 V.
Results and discussion Fluorescence spectra The fluorescence emission and excitation spectra of Al3+ , ATP, Al3+–ATP, GFLX, GFLX–ATP, GFLX–Al3+, GFLX–Al3+–ATP are shown in Fig. 1. GFLX exhibited maximum fluorescence emission at a wavelength of about 443 nm (Fig. 1a, curve 4) when excited at 283 nm (Fig. 1b, curve 4). When Al3+ was added to the GFLX solution, GFLX molecule and Al3+ could undergo a charge transfer reaction. The FL intensity was enhanced and the emission wavelength was blue shifted from 443 nm to 423 nm (Fig. 1a, curve 6), which indicates that the FL intensity of GFLX was increased in the presence of Al3+. The FL intensity of GFLX–Al3+ system was enhanced markedly when ATP was added into the GFLX–Al3+ system (Fig. 1a, curve 7), which indicates that ATP can form a stable ternary complex with the GFLX–Al3+ system. However, the Al3+–ATP system did not show an enhanced emission peak which may be due to the formation of an unstable complex. Therefore, the GFLX–Al3+–ATP system exhibited enhanced FL intensity at 423 nm. From the excitation spectra (Fig. 1b), it could be observed that the maximum FL excitation wavelength of the GFLX–Al3+–ATP system was about 272 nm while the emission wavelength was about 423 nm. Thus, 272 nm and 423 nm were selected as the excitation and emission wavelengths for the determination of ATP. Possible interaction mechanism of the presented system In order to understand the interaction mechanism of GFLX, Al3+, and ATP, absorption spectra of Al3+ , ATP, Al3+–ATP, GFLX, GFLX– ATP, GFLX–Al3+, and GFLX–Al3+–ATP were recorded and shown in Fig. 2. GFLX exhibited maximum absorbance at about 286 nm (Fig. 2, curve 4). When Al3+ was added into the GFLX solution, the absorbance peak of GFLX was blue shifted from 286 nm to 275 nm and the absorbance was increased (Fig. 2, curve 6), which indicated that a charge transfer reaction might occur between GFLX and Al3+ ion. According to the Förster’s non-
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Spectroscopic determination of ATP
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Figure 1. FL emission (a) and excitation (b) spectra of (1) Al ; (2) ATP; (3) Al – 3+ 3+ ATP; (4) GFLX; (5) GFLX–ATP; (6) Al –GFLX; and (7) Al –GFLX–ATP. Conditions: 8 –1 3+ –1 –1 [ATP], 3.5 × 10 mol L ; [Al ], 0.06 mmol L ; [GFLX], 7.0 μmol L ; NaAc–HAc –1 (0.1 mol L ); pH, 5.2, λex/λem = 272/422 nm.
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0 540
Optimization of the reaction conditions
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Wavelength (nm)
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the wavelength was slightly blue shifted from 275 nm to 271 nm (Fig. 2, curve 7). The above results indicated that ATP can form a strong ternary complex with GFLX–Al3+. Therefore, the FL intensity of the GFLX–Al3+–ATP system at 423 nm was increased several times (Fig. 1a). The FL intensity of the system was enhanced proportionally with the concentration of ATP.
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Figure 3. Spectral overlap between (a) emission spectrum of GFLX and (b) ab+1 sorption spectrum of Al .
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Effect of pH and buffer solution. The pH of a solution plays an important role in obtaining maximum FL intensity. Thus, the effect of pH on the FL intensity of the GFLX–Al3+–ATP system was investigated and the maximum FL intensity was obtained at pH of 5.2. When pH is higher, the hydration of Al3+ results in the formation of soluble hydroxo complexes, polycations or hydroxo polymers which are not conducive to chelating between Al3+ and GFLX. This might hamper the sensitivity of the FL intensity. Therefore, an optimal pH value of 5.2 was selected for the whole experiment. The experimental results indicated that buffer solutions had also a large effect on the FL intensity of the presented system. Thus, the effect of the following buffers on the FL intensity was studied: phosphate buffer, KH2PO4– Na2HPO4 (0.1 mol L–1), borate (acid–base) buffer, borax–HCl (0.1 mol L–1), NH4Cl–NH3 (0.1 mol L–1), acetate (acidic) buffer, NaAc–HAc (0.1 mol L–1), hydrogen phosphate (acidic) buffer, KH2PO4–NaOH (0.1 mol L–1), Tris–HCl (0.1 mol L–1). Among the above buffers, NaAc–HAc exhibited maximum FL intensity (Fig. 4). Thus, acetate buffer, NaAc–HAc (0.1 mol L–1) at pH5.2 was used to obtain the highest FL intensity.
radiation energy transfer theory (23), the rate of energy transfer depends upon the extent of overlap of the emission spectra of the donor with the absorption spectra of the accepter as well as the distance between them. Energy transfer easily occurs between Al3+ and GFLX because of the strong spectral overlap between the emission spectra of donor (GFLX) and the absorption spectra of accepter (Al3+) as shown in Fig. 3. After the addition of ATP into the GFLX–Al3+ system, the absorbance is enhanced and
Effect of GFLX concentration. GFLX solution might also influence the sensitivity of the presented method. Thus, the effect of GFLX concentration on the FL intensity was investigated and shown in Fig. 6. The maximum FL intensity of the system was obtained using GFLX at a concentration of 7 μmol L–1. Above this concentration, the FL intensity starts to decline due to the maximum complex formation at this concentration. Therefore, an excess amount of GFLX would absorb or quench the FL intensity of the system. Thus, 7 μmol L–1 GFLX was chosen for further study.
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3+
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Figure 2. Absorbance spectra of (1) Al ; (2) ATP; (3) Al –ATP; (4) GFLX; (5) GFLX– 3+ 3+ 8 –1 ATP; (6) Al –GFLX; and (7) Al –GFLX–ATP. Conditions: [ATP], 1.5 × 10 mol L ; 3+ –1 –1 –1 [Al ], 0.08 mmol L ; [GFLX], 11.0 μmol L ; NaAc–HAc (0.1 mol L ).
Effect of Al3+ ion concentration. The effect of Al3+ ion concentration on the FL intensity was examined in the range of 0.01–0.1 mmol L–1. The FL intensity was increased with increasing the concentration of Al3+ up to 0.06 mmol L–1 (Fig. 5). Therefore, an Al3+ concentration of 0.06 mmol L–1 was selected as optimum concentration for this experiment.
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M. Kamruzzaman et al. IV
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GFLX, ATP showed the maximum FL intensity. The chelation reaction of the GFLX–Al3+–ATP system could complete in about 20 minutes at room temperature. Therefore, the FL intensity of the system reached a maximum in 20 min after all the reagents had been added and remained stable for at least 2 h.
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Figure 4. Effect of buffers. I, KH2PO4–Na2PO4 (0.1 mol L ; pH, 7.2); II, borax–HCl –1 –1 (0.1 mol L ; pH, 8.0); III, NH4Cl–NH3 (0.1 mol L ; pH, 9.2); IV, NaHc–HAc (0.1 mol –1 –1 –1 L ; pH, 5.2); V, KH2PO4–NaOH (0.1 mol L ; pH, 7.2); and VI, Tris–HCl (0.1 mol L ; 8 –1 3+ –1 pH, 7.5). Conditions: [ATP], 1.5 × 10 mol L ; [Al ], 0.08 mmol L ; [GFLX], 11.0 –1 μmol L .
In order to investigate the selectivity of the proposed method, the effect of interfering substances that coexist with the analyte and which may suppress or enhance the FL intensity was examined. Therefore, the effect of potential foreign substances was studied by preparing a set of solutions, each one with 3.5 × 10 8 mol L–1 of ATP and different concentrations of the chemical species to be tested. A foreign species is considered as a noninterfering component if it produces an error less than ± 5% in the determination of ATP. The results of the effect of interferencing substances are summarized in Table 1. It was observed that the coexisting foreign substances did not generate a significant effect in the determination of ATP.
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Linear range and detection limit of ATP FL Intensity (a.u.)
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Under the aforementioned optimal condition, a calibration curve was constructed which showed a good linear relationship between the FL intensity and the concentration of ATP in the range of 1.3 × 10–10 to 1.0 × 10–8 mol L–1 with correlation coefficient (r) of 0.9981. The regression equation was Y = 6.19 × 1010CATP +357 where CATP is the concentration of ATP and Y is the fluorescence intensity in arbitrary unit (a.u.). The limit of detection (LOD) as defined by IUPAC, CLOD = 3 * Sb/m (where Sb is the standard deviation of the blank signals
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Table 1. Effect of interfering substances on the GFLX–Al3+– ATP system
[Al3+] mmol L-1 Figure 5. Effect of aluminium (III) ion concentration. Conditions: [ATP], 3.5 × 10 –1 –1 –1 mol L ; [GFLX], 7.0 μmol L ; NaAc–HAc (0.1 mol L ); pH, 5.2.
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[GFLX], µmol L-1 Figure 6. Effect of GFLX concentration. Conditions: [ATP], 3.5 × 10–8 mol L–1; [Al3+], 0.06 mmol L–1; NaAc–HAc (0.1 mol L–1); pH, 5.2.
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Effect of the addition order of reagents. The addition order of the reagents also influences the FL intensity of the system. Thus, the effect of the reagent addition order was investigated and observed that the following addition order: Al3+, NaAc–HAc buffer,
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Interfering substances Cu2+ Zn2+ Al3+ Fe2+ Mg2+ Fe3+ Ca2+ Cd2+ Cr3+ Tryptophan Thymine Adenine Ascorbic acid L-Glutamic acid BSA Glucose Sucrose L-Lysine L-Cystine L-Histidine L-Phenylalanine
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Tolerable concentration (mol L–1) 1.2 2.1 1.4 1.6 2.2 2.5 3.1 2.7 3.2 3.7 3.9 1.9 2.3 2.3 2.4 1.5 1.7 3.6 4.1 5.7 3.1
× 10–6 × 10–6 × 10–6 × 10–6 × 10–5 × 10–7 × 10–5 × 10–6 × 10–8 × 10–5 × 10–6 × 10–8 × 10–6 × 10–5 × 10–7 × 10–4 × 10–4 × 10–5 × 10–5 × 10–6 × 10–5
Change in fluorescence intensity (%) +1.15 +0.71 +2.31 –4.1 –0.85 +2.15 –3.12 –1.35 –3.19 +1.55 +2.11 +1.85 –2.3 +1.4 –2.55 –2.75 –4.25 –3.21 +2.05 +1.37 –3.51
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Spectroscopic determination of ATP Table 2. Assays of ATP in pharmaceutical formulations by the proposed method Sample
Amount (mg)
Standard addition method
Active ingredient label
Found by the proposed method ± RSDa
Tablet
20 mg of ATP
21.25 ± 1.71
Injection
20 mg of ATP
19.55 ± 1.35
Added (×10
–7
–1
Recovery (%) –7
mol L )
–1
Observed (×10 mol L ) ± RSDa
1.0 3.0 5.0 1.0 3.0 5.0
1.03 3.14 4.89 0.97 3.01 5.07
± ± ± ± ± ±
1.32 1.05 1.45 1.35 0.92 0.92
103.00 104.67 97.80 97.00 100.33 101.40
Relative standard deviation for five replicate measurements.
a
Table 3. Determination of ATP in milk samples and results for recovery test Sample
Found by the proposed method (×10–7 mol L–1) ± RSDa
Milk
1.76 ± 1.55
Standard addition method Added (×10–7 mol L–1)
Found (×10–7 mol L–1) ± RSDa
2.0 4.0 6.0 8.0 10.0
2.09 3.93 6.11 8.13 9.81
± 1.11 ± 1.21 ± 1.32 ± 1.05 ± 1.73
Recovery (%)
104.50 98.25 101.83 101.62 98.10
Relative standard deviation for five replicate measurements.
a
and m is the slope of the calibration graph) was found to be 1.1 × 10–11 mol L–1 for ATP. The standard deviation (RSD) was 1.21% for five repeated measurements of 2.3 × 10–8 mol L–1 ATP.
Analytical application Determination of ATP in tablet and injection. In order to demonstrate the applicability of the proposed method, it was applied to determine ATP in a commercially available pharmaceutical tablet and injection (Jeil Pharm. Co. Ltd, South Korea). The results are listed in Table 2. As shown in Table 2, the amount of ATP in tablet and injection obtained by the proposed method did not show significant difference to the labeled contents. Recoveries performed by standard addition method were in the range of 97.00– 104.67% for ATP.
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A sensitive, simple and cost-effective spectrofluorimetric method has been developed to determine ATP based on the interaction of ATP with the GFLX–Al3+ complex. The FL intensity of the GFLX–Al3+ complex was enhanced in the presence of ATP by increasing the concentration of ATP in the range of 1.3 × 10–10 to 1.0 × 10–8 mol L–1. The proposed method offers a low LOD (1.1 × 10–11 mol L–1) compared with previously reported methods. The presented method has been successfully applied to determine ATP in pharmaceutical preparations and milk sample with good reproducibility and satisfactory results.
Acknowledgements This research was supported by Kyungpook National University Research Fund, 2013.
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Determination of ATP in milk sample. The presented method was applied to determine ATP in milk samples collected from a local firm. The results of determination of ATP by the proposed method are summarized in Table 3. The recovery of the method was determined by adding a known amount of ATP to the milk sample and subtracting the results obtained for the sample prepared in the same manner but without ATP. The recoveries were found in the range of 98.10–104.50% for ATP. From the results, it was assumed that no interfering materials were encountered which showed the applicability of the method to the determination of ATP in milk samples.
Conclusion
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