Research article Received: 29 April 2014,
Revised: 2 December 2014,
Accepted: 28 December 2014
Published online in Wiley Online Library: 18 February 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.2864
Simple chemiluminescence determination of ketotifen using tris(1,10 phenanthroline) ruthenium(II)- Ce(IV) system Ali Mokhtari,a* Mehrgan Ghazaeian,a Mahdieh Maghsoudi,a Mohsen Keyvanfardb and Iraj Emamic ABSTRACT: A new method using chemiluminescence (CL) detection has been developed for the simple determination of ketotifen fumarate (KF). The method is based on the catalytic effect of KF in the CL reaction of tris(1,10 phenanthroline)ruthenium(II), Ru(phen)32+, with Ce(IV) in sulfuric acid medium. The CL response was detected using a lab-made chemiluminometer. Effects of chemical variables were investigated and under optimum conditions, the CL intensity was proportional to the concentration of the drug over the range 0.34-34.00 μg mL1 KF. The limit of detection (S/N=3) was 0.09 μg mL1. Effects of common ingredients were investigated and the method was applied successfully for determining KF in pharmaceutical formulations and human plasma. The percent of relative standard deviation (n=11) at level of 3.4 μg mL1 of KF was 4.6% and the minimum sampling rate was 70 samples per hour. The possible CL mechanism is proposed based on the kinetic characteristic of the CL reaction, CL spectrum, UV-Vis and phosphorescence spectra. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: chemiluminescence; ketotifen; human plasma; pharmaceuticals
Introduction
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Ketotifen is a second-generation H1-antihistamine and mast cell stabilizer (1). The structure of ketotifen is shown in Fig. 1. It is most commonly available as a salt of fumaric acid (ketotifen fumarate, KF) and is accessible in two forms. In its ophthalmic form, it is used to treat allergic conjunctivitis, or the itchy red eyes caused by allergies (2). In its oral form, it is used to prevent asthma attacks. Side effects include drowsiness, weight gain, dry mouth, irritability, and increased nosebleeds (3). The therapeutic importance of the drug has prompted to develop various techniques for its assay in pharmaceuticals and body fluids. It is quantified by high performance liquid chromatography (4–11), high performance thin layer chromatography (12), liquid chromatography with mass spectrometry (13–15), spectrofluorimetry (16), direct differential pulse polarographic and adsorptive-stripping voltammetry (17), UV spectrophotometry (18–20), potentiometric titration (21,22) and coulumetric titration (23). Chemiluminescence (CL) is a powerful analytical tool because of its high sensitivity, wide dynamic range and simple instrumentation (24). The CL involving Ru(II) complexes is one of the most interesting series of CL reactions. It involves the oxidation of Ru (II) in the complex to Ru(III), which is following by reduction with an analyte species to produce CL emission (25,26). Tertiary amines can reduce Ru(III) complex and produce intense emission. Three CL methods (24,27,28) also proposed for the determination of KF up to now. The first method was on the basis of a europium (III)-doped Prussian blue analog film modifying platinum electrode as the working electrode and a Ru(bpy)32+-based electrochemiluminescence (ECL) assay coupled with capillary electrophoresis (24). The method was used for the determination of KF in urine and pharmaceutical samples. In the second CL
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method, potassium hexacyanoferrate(III) reacted with the mixture of calcein and KF (27). The CL intensity enhanced by trace amounts of Mg2+ and it was strongly dependent on KF concentration. The method was used to determine KF in tablets. The third method (28) was based on the catalytic effect of KF on the reaction of luminol with ferricyanide in sodium hydroxide medium. The significance of this work is developing a new simple chemiluminescence method for the determination of KF in pharmaceuticals (tablets, syrups and drops) and spiked human plasma. It is based on the catalytic effect of KF in the CL reaction of tris(1,10 phenanthroline)ruthenium(II), Ru(phen)32+, with Ce(IV) in sulfuric acid medium. The method has been applied to determine KF in drug formulations and human plasma with satisfactory results.
Experimental Reagents All the solutions were prepared using reagent grade chemicals and doubly distilled water. Acetonitrile was HPLC-grade (Caledon, Canada). KF standard solution (425.0 μgmL1) was daily * Correspondence to: A. Mokhtari, Laboratory of Analytical Chemistry, Department of Science, Golestan University, Gorgan, I.R. Iran. E-mail: [email protected] a
Department of Science, Golestan University, Gorgan, I.R. Iran
b
Department of Chemistry, Majlesi Branch, Islamic Azad University, Isfahan, I.R. Iran
c
Department of Physics, Isfahan University of Technology, Isfahan, I.R. Iran
Copyright © 2015 John Wiley & Sons, Ltd.
Simple chemiluminescence determination of Ketotifen Preparation of tablets
Figure 1. Chemical structure of ketotifen.
prepared by dissolving 0.0425 g of KF (Behsa, Iran) in 100.0 mL water. Working solutions were prepared by appropriately diluting the stock solution when used. Ru(phen)32+ solution (1.0×102 mol L1) was prepared by dissolving 0.3640 g of dichlorotris(1,10phenanthroline) ruthenium(II)hydrate (Sigma-Aldrich, Steinheim, Germany) in 50.0 mL water. Ce(IV) solutions (2.0×104-7.0×103 mol L1) were prepared by dissolving proper amount of ceric ammonium nitrate (Riedel-de Haën, Germany) in proper volumes of 1.0 mol L1 H2SO4 and diluting to the mark with distilled water in 100.0 mL volumetric flasks. In this way, H2SO4 concentrations of 0.03, 0.05, 0.08, 0.12 and 0.16 mol L1 were prepared. Plasma samples were taken from the health center of Gorgan (Iran). KF pharmaceuticals were purchased from local drugstores.
Ten tablets of KF (1 mg per tablet) were weighed and powdered. An accurately weighed portion of the powder, including active ingredients equivalent to 1.00 mg was transferred into a 100.0 mL calibrated flask containing 50 mL of water and the mixture was sonicated for 10 minutes. Then the volume was adjusted to 100.0 mL with water and the suspension was filtered. Each time 5.0 mL of this solution and a proper volume of the standard solution of KF were transferred into a 25.0 mL volumetric flask. Then it was diluted to the mark in order to obtain the appropriate concentration for the analysis.
Preparation of drops Two types of ketotifen drops were investigated, including oral and aphthalmic drugs. For each drop, contents of two drops were transferred into a beaker. An aliquot (1.0 mL) of the oral drug and an aliquot (4.0 mL) of the aphthalmic drug were transferred into 100 mL flasks and they were diluted to the mark with water. For each drug, 5.0 mL of this solution and a proper volume of the standard solution of KF were transferred into a 25.0 mL volumetric flask. Then it was diluted to the mark to obtain the appropriate concentration for the analysis.
Apparatus CL analysis was applied using a 0.50 cm light path length quartz cell. The CL signal was measured with a CL analyzer with PMT (Hammamatso, model R212, Japan) using a low pass filter which its output was connected to a data processing system with a Pentium IV PC. A schematic block diagram of the used instruments is shown in Fig. 2.
Preparation of the syrup samples 5.0 mL of the syrup drug was directly transferred into a 100.0 mL volumetric flask and diluted to the mark. Each time 5.0 mL of this solution and a proper volume of the standard solution of KF were transferred into a 25.0 mL volumetric flask and diluted to the mark.
General procedure An aliquot (200 μL) of standard solution consisting of KF with 400 μL of 2.0×103 mol L1 of Ru(phen)32+ were transferred into the 0.50 cm path light length quartz cell. Then, the cell was placed at its location in front of PMT and the program was started. After a few seconds, 200 μL of acidic Ce(IV) was injected into the cell by a microsyringe and the peak-like CL emission was recorded by a computer (with interval times of 100 ms). Those data information were collected into Excel software. Maximum CL response of KF appeared about 7 seconds after injection of Ce(IV) solution. For obtaining the analytical signal, response from the blank at second 7 after injection of Ce(IV) solution, was subtracted from maximum peak height of each sample.
Procedure for spiked plasma Only a deproteination process was carried out by using acetonitrile as a sample pretreatment and extraction procedure was not necessary (29). The standard addition method was used for the determination of KF in the plasma samples. Therefore each time, 1.0 mL of plasma sample was transferred into a centrifuge tube including 2 mL of acetonitrile and the mixture centrifuged at 4000 r/min for 15 min. The protein-free supernatant was transferred into a small conical flask and evaporated to dryness under a stream of nitrogen at room temperature. The dry residue was transferred into a 25.0 mL flask using double distilled water, then the standard solution was added into the flask and the mixture was diluted to the mark.
Results and discussion Kinetic curve of the CL reaction
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Figure 2. Schematic block diagram of the CL instrument.
Typical CL profiles for KF (3.4 μg mL1) and blank samples are shown in Fig. 3. Maximum CL response of KF appeared about 7 seconds after injection of Ce(IV) solution and the response declined to base slowly. CL profile of KF demonstrated that the CL reaction was slow. It took about 7 seconds to achieve the maximum peak, compared with 150-250 seconds for the signal to decline to base.
A. Mokhtari et al. To explore the possible CL mechanism, some experiments performed and following results were obtained.
Figure 3. Typical CL responses of a) blank and b) 3.4 μg mL
1
of KF.
Detailed CL mechanism Method is based on rapid reduction of Ru(phen)33+ produced in the reaction between Ru(phen)32+ and acidic Ce(IV) by KF that it produces strong CL. Solution of Ru(phen)32+ is orange and its color changes to green immediate after mixing with oxidizing agent, Ce(IV) solution, and production of Ru(phen)33+ (30,31). During about 3 minutes after mixing Ru(phen)32+ with Ce(IV), the color of the mixture changes slowly from green to orange. In Fig. 4, UV-Vis spectrum of the mixture of Ru(phen)32+-Ce(IV) is shown immediately after mixing (spectrum a) and 3 minutes after mixing (spectrum b). As can be seen in Fig. 4, Maximum absorbance at 450 nm which is related to Ru(phen)32+, disappeared immediately after mixing the solutions (spectrum a) and it appeared again after 3 minutes (spectrum b). The reason is that, the resulting Ru (phen)33+ produced in the reaction of Ru(phen)32+ with acidic Ce(IV), is a powerful oxidant and oxidizes water into O2 and protons (32). Therefore, it returns slowly to its reduced state. If there was a reducing agent in the reaction media, it can reduce Ru(phen)33+ very fast. The electrons from reducing agent transfer to the π*-orbital of phenanthroline ligand and the Ru (phen)32+ π* metal-to-ligand charge transfer (MLCT) excited state can be produced (33). The excited electron then undergoes intersystem crossing to the lowest triplet state of Ru (phen)32+, from where emission occurs (34). In order to confirm the mechanism proposed above, some CL pathways might be investigated for the Ru(phen)32+-Ce(IV)-KF CL system, involving the formation of Ce(III)*, oxidation products in excited state and [Ru(phen)32+]*.
(1) A weak CL intensity was observed when Ru(phen)32+ solution was mixed with acidic Ce(IV) solution. Enhancement in CL intensity was detected when KF solution was present in the mixture of Ru(phen)32+ and acidic Ce(IV). (2) Phosphorescence spectrum of KF (λex = 310 nm) was scanned using spectrofluorometer (Jasco, model FP-750) using batch mode. Phosphorescence of KF (λmax = 485 nm) was disappeared when Ce(IV) solution was added into the cuvette and new peak was appeared at 368 nm. These are due to oxidation of KF and formation of Ce(III) that is a well known fluorescent ion (35). (3) Phosphorescence emission spectrum of Ru(phen) 3 2+ (λex = 450 nm), had a maximum at 575 nm. (4) CL spectra of mixtures including Ce(IV)-KF (Fig. 5a), Ce(IV)-Ru(phen)32+ (Fig. 5b) and Ce(IV)-Ru(phen)32+-KF (Fig. 5c) were obtained using spectrofluorometer (Jasco, model FP750). No detectable CL intensity obtained for the first mixture. This suggests that oxidation products and Ce(III)* are not main emitters. Moreover Both spectra of second and third mixtures had same maximum emission wavelength at 575 nm which is same as maximum phosphorescence emission of Ru(phen)32+. This indicates that the CL spectra are independent of KF and the emitter is [Ru(phen)32+]*. KF is a tertiary amine and from previous studies, the oxidation of tertiary amines is understood to produce a short-lived radical cation. The α-carbon is then deprotonated, yielding a strongly reducing intermediate. This reduces the Ru(phen)33+ (produced by oxidant) to the excited state that subsequently emits light (29,36–38). According to the above discussion, the following mechanism is proposing for the CL reaction of KF. RuðphenÞ3 2þ þ CeðIVÞ → CeðIIIÞ þ RuðphenÞ3 3þ ketotifen þ CeðIVÞ → CeðIIIÞ þ ketotifen•þ ketotifen•þ → ketotifen• þ Hþ
RuðphenÞ3 3þ þ ketotifen• þ H2 O →
h i RuðphenÞ3 2þ *
þketotifen fragments h i h i 2þ RuðphenÞ3 * → RuðphenÞ3 2þ þ hυ
2+
2+
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Figure 4. UV-Vis spectrum of the mixture of Ru(phen)3 -Ce(IV), a) immediately after mixing b) 3 minutes after mixing.
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2+
Figure 5. CL spectrum of a) Ce(IV)-KF, b) Ce(IV)-Ru(phen)3 , c) Ce(IV)-Ru(phen)3 -KF. 1 3 1 1 Conditions: a) 2 mL KF (3.4 μg mL ) and 400 μL Ce(IV) (3.0×10 mol L in 0.1 mol L 3 1 3 2+ of H2SO4), b) 2 mL Ru(phen)3 (2.0×10 mol L ) and 400 μL Ce(IV) (3.0×10 1 1 3 1 2+ mol L in 0.1 mol L of H2SO4), c) 2 mL Ru(phen)3 (2.0×10 mol L ), 200 μL KF 1 3 1 1 (3.4 μg mL ) and 400 μL Ce(IV) (3.0×10 mol L in 0.1 mol L of H2SO4).
Copyright © 2015 John Wiley & Sons, Ltd.
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Simple chemiluminescence determination of Ketotifen Influence of chemical variables To study the influence of chemical variables, influence of Ru (phen)32+, Ce(IV) and H2SO4 concentrations on the CL intensity were investigated. The influence of concentration of Ru(phen)32+ on the sensitivity was studied in the range 1.0×103-7.0×103 mol L1 by injecting concentration of 1.0×103 mol L1 of Ce(IV) prepared in 0.10 mol L1 of H2SO4. The CL signal increased with increasing Ru(phen)32+ concentration until 2.0×103 mol L1 and then decreased. Therefore, concentration of 2.0×103 mol L1 was selected as the optimum concentration for the complex of Ru(phen)32+. The influence of concentration of Ce(IV) on the CL sensitivity was investigated in the range 1.0×103 to 1.0×102 mol L1 of Ce(IV) in 0.10 mol L1 of H2SO4. For this variable sensitivity increased to concentration of 3.0×103 mol L1 and then decreased at higher concentrations. Therefore, 3.0×103 mol L1 of Ce(IV) was selected as the optimum concentration. The influence of concentration of H2SO4 on the CL intensity was also studied in the range 0.04 to 0.14 mol L1 of H2SO4. The CL response increased with increasing the concentration of H2SO4 to 0.1 mol L1 and then decreased. Therefore, concentration 0.1 mol L1 H2SO4 was selected for further studies. Analytical features Under optimum conditions, a long series of standard solutions of KF were subjected to the optimized CL method for the purpose of calibration. CL response was found to be linear in the concentration ranges of 0.34-34.00 μg mL1 (Fig. 6). The correlation equation between CL intensity and concentration of KF in linear range was: ICL = 4.50 + 12.74 CKF (R2 = 0.9979) where CKF is concentration of KF (μg mL1). The limit of detection (LOD) was calculated as 3σ/m where σ is the standard deviation existing in 10 times determination of the blank response and m is slope of the correlation equation between CL intensity and concentration of KF (12.74 in calibration equation mentioned above). The LOD obtained was 0.09 μg mL1, indicating good detectability. The reproducibility was investigated using 3.4 μg mL1 of KF (n=11) and the percent of relative standard deviation (%RSD) was 4.6%. The minimum sampling rate could be about 70 samples per hour.
pharmaceutical preparations and some amino acids were studied by recovering 3.4 μg mL1 (8.0×106 mol L1) of KF in presence of each substance. The tolerance of each substance was taken as the largest amount yielding an error of less than 3σ in the analytical signal of 3.4 μg mL1 KF (σ is the standard deviation in the response obtained from 11 times determination of 3.4 μg mL1 of KF). Because of interference effects from some substances in the real samples, such as citric acid, ascorbic acid, oxalic acid, cysteine, glutathione etc., CL intensity at 50 seconds after injection of Ce(IV) solution was chosen as the analytical signal for interference and application studies (instead of CL intensity at 7 seconds after injection of Ce(IV) solution when maximum response of KF appears). For example ascorbic acid and oxalic acid (5 μg mL1) had a sharp peak which their maximum appeared at 0.7 and 1.7 seconds after the injection of Ce(IV) solution respectively, and their CL intensity decreased to baseline after about 10 seconds. Citric acid had a relatively broad peak but its peak decreased to baseline after about 40 seconds. In the CL system proposed in this study, KF had a broad time profile with detectable response at 50 seconds after injection of Ce(IV) solution. Therefore for decreasing the effect of some interfering substances in real samples, interference effect from all substances were investigated at 50 seconds after injection of Ce(IV) solution. The results have been shown in Table 1. It is known that compounds having a tertiary amine or carboxylic acid group, such as some drugs, amino acids and organic acids and also some ions, emit light when they react with Ru (phen)33+ in an acidic solution (29,39–42). In spite of CL light emission produced in presence of some of the investigated compounds, these compounds have little or zero interference effect for the determination of KF, because most of them have a zero or very weak CL intensity at 50 seconds after injection of Ce(IV) solution. Nonetheless some of investigated substances such as tyrosine, morphine and I had a quenching effect on the CL intensity and their interference effect remained until 50 seconds after starting the CL reaction.
Table 1. Effect of foreign substances on the determination of 3.4 μg mL1 KF
Influence of interfering substances In order to validate of the possible analytical application of the method, interferences from some common ions, excipients in
Substance/KFa
Substance Threonine, Serine, Lactose, Sucrose, Glucose, Fructose, Saccharin, Starch, Urea, Valine, Leucine, K+, Cl, Na+, Zn2+, SO42, Br, NO3 Glycine, Cystine Oxidized glutathione, Proline, Alanine, HCO3, CO32, PO43, CH3COO, Phenylanaline, Cu2+ Histidine, Aspartic acid, Tryptophane, Ascorbic acid, Fe2+, Mg2+, Ca2+ Tyrosine Glutathione, Cysteine, Citric acid, Mn2+, I Oxalic acid, Noscapine Codeine, Morphine
1000
500 100
50 10 5 1 0.1
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Molar ratio of substance to KF.
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a
Figure 6. Calibration curve of KF.
A. Mokhtari et al. Application
have been summarized in Table 4. In the CE-ECL method, a separating technique (CE) has been used and therefore, it is more favorable than other mentioned methods for analyzing of biological samples. However in ECL detection system, oxidations of KF and Ru(II) complex have been performed by electrochemical instruments and on a platinum microelectrode modified with Europium(III)-doped Prussian analog film (Eu-PB). Moreover, electrochemical instrumentation has been used for electrodepositing of Eu-PB on the platinum electrode. Therefore the ECL detection system is more expensive and more complex than the chemical oxidation performed in the proposed method. As can be seen in Fig. 3, KF produced a broad CL time profile in the Ru(phen)32+-Ce(IV) CL system. It had a detectable response even after 50 seconds after injection of Ce(IV) solution. Moreover many of the investigated compounds mentioned in the interference study section had a zero or very weak CL intensity at that time. Therefore, for many of the substances that they can enhance the CL response, the extent of interference effect at 50 seconds after injection of Ce(IV) solution is less than their
In order to evaluate the applicability of the proposed method, KF syrup, drops and tablets were analyzed to determine their KF contents. For all samples, CL intensity at 50 seconds after injection of Ce(IV) solution was chosen as the analytical signal. Also recovery of KF from human plasma was investigated. The results are shown in Table 2. The obtained results from analyzing of syrup sample were also certified by the British Pharmacopoeia (BP) method. BP provided a potentiometric titration method using perchloric acid as titrant (43). Statistical analysis of the results using student t-test and the variance ratio F-test showed no significant difference between the performance of two methods as regards to accuracy and precision. The results are presented in Table 3.
Response characteristics The analytical parameters of the previously reported CL methods and proposed method for the determination of KF
Table 2. Determination of KF in real samples Real Sample
Sample No.
Added (μg mL1)
Total Found (μg mL1)
Recovery (%)
1 2 3 4 1 2 3 1 2 3 1 2 3 1 2 3
0.00 4.00 8.00 12.00 0.00 4.00 8.00 0.00 4.00 8.00 0.00 4.00 8.00 0.00 1.00 2.00
2.69±0.22 6.68±0.34 10.53±0.52 14.25±0.46 2.83±0.17 6.87±0.42 11.04±0.61 2.38±0.39 6.60±0.42 10.65±0.81 2.94±0.22 7.00±0.37 10.87±0.58 0.11±0.17 1.07±0.24 2.14±0.19
99.8 98.0 96.3 101.0 102.6 105.5 103.4 101.5 99.1 96.0 101.5 100.4±2.94
a
Tablet (1mg per tab )
Syrup (1mg per 5mLb)
Drop (1mg per mLc)
Drop (0.25%d)
Plasma
Mean±S.D. a
Ketotifen Amin 1mg tab (Amin Co., Iran). Each tablet contains 1.38 mg KF equivalent to 1mg ketotifen base. Amiten 60 mL syrup (Amin Co., Iran). Each 5 mL solution contains 1.38 KF equivalent to 1mg ketotifen base. c Ketotifen-Behsa 1 mg mL1 oral drop (Behsa, Iran). Each 1 mL solution contains 1.38 mg KF equivalent to 1mg ketotifen base. d Ketotifen 0.25% ophthalmic drop (Daru Pakhsh, Iran). Each 1 mL contains 0.345 mg ketotifen hydrogen fumarate corresponding to 0.25 mg ketotifen. b
Table 3. Analysis of a formulation containing KF using the proposed method and the official method Sample
KF syrup
Nominal value
1.38 mg per 5 mL
Analytical Resultsa Proposed method
BP methodb
1.42±0.10
1.49±0.07d
t-testc
F-testd
1.15
2.04
a
Mean values of four replications. British Pharmacopoeia, a potentiometric titration method. c Student t-test calculated, theoretical value=3.182 (P=0.05). d F-test calculated, theoretical value=9.28 (P=0.05). b
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Simple chemiluminescence determination of Ketotifen Table 4. Analytical features of CL methods proposed for the determination of KF CL reagent
Oxidation method
Ru(bpy)32+
Electrochemistry
CalceineMg2+
Luminol
Ru(phen)32+
Methodology of CL reaction
D.L. (g mL1)
Oxidation of Ru(bpy)32+ 2.1×108 on the platinum microelectrode modified with europium (III)-doped Prussian blue analog film (Eu-PB) and reduction of produced Ru(bpy)33+ by KF 3.0×109 hexacyanoferrate(III) Reaction of potassium hexacyanoferrate(III) with the mixture of calcein and KF. A stronger CL signal was observed when a trace amount of Mg2+ was added into KF solution Hexacyanoferrate Catalytic effect of KF on 5.7×109 (III) the oxidation rate of luminol with ferricyanide in sodium hydroxide medium Ce(IV) Oxidation of Ru(phen)32+ 9.0×108 using Ce(IV) and reduction of produced Ru(phen)33+ by KF
LDR Speed Samples (g mL1) (samples per h) 3.0×108– 2.0×106
15
6.0×109– 2.0×107
Ref.
Tablet, Drop, Urine
(24)
About 70
Tablet
(27)
1.0×108– 1.0×106
N/A
N/A
(28)
3.4×107– 3.4×105
70
Tablet, Proposed Drops, Method Syrup, Plasma
a
Capillary Electrophoresis- Electrochemiluminescence.
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Conclusions A new method based on the CL of Ru(phen)32+ and acidic Ce(IV), was proposed for the quantification of KF. The method is simple, rapid and adequately sensitive for the determination of KF in pharmaceuticals. Some common sugars, amino acids and ions hadn’t significant interference effect in the quantification of KF indicating high accuracy and suitability for determining of KF in human fluids and quality assurance in drug formulations. The proposed CL system is a simple and rapid analytical tool for obtaining of preliminary chemical information about KF prior the use of more complex instrumental techniques. One future trend might be the combination of the proposed CL system, (Ru(phen)32+ and acidic Ce(IV)) (used previously for the determination of some drugs), with liquid chromatography and developing a technique for the determination of KF in various matrixes and pharmaceuticals. Acknowledgements The authors are grateful to the Campus of Golestan University for supporting this work.
References 1. Kidd M, McKenzie SH, Steven I, Cooper C, Lanz R. Efficacy and safety of ketotifen eye drops in the treatment of seasonal allergic conjunctivitis. Br J Ophthalmol 2003;87:1206–11.
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interference extent at 7 seconds after injection of Ce(IV) solution, when maximum response of KF appears. Therefore, in this study we used CL intensity at 50 seconds after injection of Ce(IV) solution as the analytical signal for interference and application investigations. As can been in Table 1, in the proposed method, sugars, some amino acids and metal ions such as Ca2+, Mg2+, Zn2+ and Fe2+ had not interference effect for the determination of KF. In the method in which Calceine-Mg2+ had been used as CL reagent, good sensitivity was obtained, but the effect of amino acids had not been checked and they found that some metal ions including Ca2+, Zn2+, Cd2+, Cu2+ and Fe2+ could enhance the CL signal of potassium hexacyanoferrate(III)-calceinMg-KF reaction and they can interfere for the determination of KF at concentrations higher than 1×107 g mL1. In the ECL method, no interference study had been performed and only they mentioned that excipients and additives in pharmaceuticals, uric acid and other matrices in urine samples had little interference in the detection with the use of the E-PB electrode. The proposed CL system is an easy and fast analytical tool for obtaining preliminary chemical information about KF, prior the use of more complex instrumental techniques. In the proposed method oxidation has been performed using chemical oxidation which is simpler and cheaper than electrochemical oxidation method. Moreover, interference effect from some ions such as Ca2+, Zn2+, Cu2+ and Fe2+ is less than the method in which Calceine-Mg2+ had been used as CL reagent.
A. Mokhtari et al. 2. Jack V, Greiner OD, Clifford Michaelson DO, Cecilia L, Mc Whirter MS, Naveed BK, et al. Single dose of ketotifenfumarate.025%vs 2 weeks of cromolyn sodium 4% for allergic conjunctivitis. Adv Ther 2002;19:185–93. 3. Bustos GJ, Bustos D, Bustos GJ, Romero O. Prevention of asthma with ketotifen in pre asthmatic children: a three-year follow-up study. Clin Exp Allergy 1995;25:568–73. 4. Chen GL, Qu H. HPLC determination of ketotifen fumarate in compound metamizol sodium tablets. Yaowu Fenxi Zazhi 2003;23:123–5. 5. Nnane IP, Damani LA, Hutt AJ. Development and validation of stability indicating high-performance liquid chromatographic assays for ketotifen in aqueous and silicon oil formulations. Chromatographia 1998;48:797–802. 6. Zarapkar SS, Bhounsule NJ, Halkar UP. High performance liquid chromatographic determination of ketotifen hydrogen fumarate in pharmaceuticals. Indian Drugs 1992;29:365–6. 7. Hoogewijs G, Massart DL. Development of a standardized analysis strategy for basic drugs, using ion-pair extraction and highperformance liquid chromatography-III. Analysis of pharmaceutical dosage forms. J Pharm Biomed Anal 1984;2:449–63. 8. Muralidharan S, Han LB, Ming Y, Lau J, Sailishni K, Arumugam S. Simple and accurate estimation of ketotifen fumarate by RP-HPLC. Int J Pharm Chem Biol Sci 2012;2:392–6. 9. Semreen MH. Optimization and validation of HPLC method for the analysis of ketotifen fumarate in a pharmaceutical formulation. Bull Pharm Sci 2005;28:291–6. 10. Elsayed MMA. Development and Validation of a Rapid HPLC Method for the Determination of Ketotifen in Pharmaceuticals. Drug Dev Ind Pharm 2006;32:457–61. 11. Ansari M, Kazemipour M, Mohammadi A. Development and validation of a simple HPLC determination of ketotifen fumarate for quality control purposes. J Pharm Pharmacol 2006;58:106–11. 12. Sanghavi NM, Puranik KA, Samarth MM. Analysis of ketotifen fumarate by high performance thin layer chromatography. Indian Drugs 1995;32:53–4. 13. Kyung-Don N, Sung-Kwon T, Ji-Sun P. Bioequivalence assessment of Fumatifen tablet to Zaditen tablet (ketotifen fumarate 1mg) by liquid chromatography tandem mass spectrometry. J Pharm Invest 2012;42:221–8. 14. Alali FQ, Najib NM, Tashtoush BM. Determination of ketotifen in human plasma by LC–MS. J Pharm Biomed Anal 2004;34:87–94. 15. Chen X, Zhong D, Liu D, Wang Y, Han Y, Gu J. Determination of ketotifen and its conjugated metabolite in human plasma by liquid chromatography/tandem mass spectrometry: application to a pharmacokinetic study. Rapid Commun Mass Spectrom 2003;17:2459–63. 16. Sastry CSP, Naidu PY. Spectrofluorimetric estimation of ketotifen and terfenadine in pharmaceutical formulations. Indian Drugs 1998;35:147–9. 17. Bersier PM, Szczepaniak W, Ren M. Direct differential pulse polarographic and adsorptive stripping voltammetric assay of ketotifen in tablets. Archiv Der Pharmazie 1992;325:253–9. 18. Mohamed ME, Aboul-Enein HY. Spectrophotometric and differential pulse polarographic methods of analysis for ketotifen hydrogen fumarate. Drug Dev Ind Pharm 1986;12:733–46. 19. Szczepaniak W, Cychowska T, Przadka T. Spectrophotometric determination of ketotifen in pharmaceutical preparations after isolation on ion-exchanger. Acta Pol Pharm 1992;49:3–5. 20. Amanlou M, Hoseinzadehnazlou M, Azizian H, Souri E, Farsam H. Determination of Ketotifen Fumarate in Raw Material and Pharmaceutical Products Using Ion-pair Formation. Anal Lett 2007;40:3267–79. 21. Hopkała H, Drozd J. Ion-selective electrodes for the determination of antihistaminic drug ketotifen. Chem Anal 1999;44:603–9. 22. Frag EYZ, Mohamed GG, Khalil MM, Hwehy MA. Potentiometric determination of ketotifen fumarate in pharmaceutical preparations
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and urine using carbon paste and PVC membrane selective electrodes. Int J Anal Chem 2012;2012:154907. Ciesilski W, Zakrzewski R, Złobińska U. Coulometric titration of ketotifen in tablets. Pharmazie 2005;60:237–8. Zhou M, Li YJ, Ma YJ, Wang WF, Mi J, Chen H. Determination of ketotifen fumarate by capillary electrophoresis with tris(2,2′bipyridyl) ruthenium(II) electrochemiluminescence detection. Luminescence 2011;26:319–23. Gorman BA, Francis PS, Barnett NW. Tris(2,2′-bipyridyl)ruthenium(II) chemiluminescence. Analyst 2006;131:616–39. Han HY, He ZK, Zeng YE. A direct chemiluminescence method for +2 the determination of nucleic acids using [Ru(phen)3] - Ce(IV) system. Fres J Anal Chem 1999;364:782–4. Fei N, Jiuru L. Determination of ketotifen by using calcein as chemiluminescence reagent. Anal Chim Acta 2007;592:168–72. He SH, Tian KJ, Zhang SQ, Yu WY. Determination of ketotifen fumarate based on the chemiluminescence reaction of luminol with ferricyanide. J Instrum Anal 2005;24:98–9. Aly FA, Al-Tamimi SA, Alwarthan A. Chemiluminescence determination of some fluoroquinolone derivatives in pharmaceutical formula2+ tions and biological fluids using [Ru(bipy)3 ]-Ce(IV) system. Talanta 2001;53:885–93. Yoshida R, Ueki T. Evolution of self-oscillating polymer gels as autonomous polymer systems. NPG Asia Mater 2014;6:107. Hong D, Jung J, Park J, Yamada Y, Suenobu T, Lee YM, et al. Watersoluble mononuclear cobalt complexes with organic ligands acting as precatalysts for efficient photocatalytic water oxidation. Energy Environ Sci 2012;5:7606–16. Hara M, Waraksa CC, Lean JT, Lewis BA, Mallouk TE. Photocatalytic Water Oxidation in a Buffered Tris(2,2-bipyridyl)ruthenium Complex-Colloidal IrO2 System. J Phys Chem A 2000;104:5275–80. Wallace WL, Bard AJ. Electrogenerated chemiluminescence. 35. Temperature dependence of the ECL efficiency of tris (2, 2’bipyridine) rubidium (2+) in acetonitrile and evidence for very high excited state yields from electron transfer reactions. J Phys Chem 1979;83:1350–7. Bolton E, Richter MM. Chemiluminescence of Tris (2, 2’-bipyridyl) ruthenium (II): A glowing experience. J Chem Educ 2001;78:47. Campiglio A. Determination of naproxen with chemiluminescence detection. Analyst 1998;123:1571–4. Thongpoon C, Liawruangrath B, Liawruangrath S, Wheatley RA, Townshend A. Flow injection chemiluminescence determination of cephalosporins in pharmaceutical preparations using tris (2, 2′bipyridyl) ruthenium (II)-potassium permanganate system. Anal Chim Acta 2005;553:123–33. Greenway G, Dolman SL. Analysis of tricyclic antidepressants using electrogenerated chemiluminescence. Analyst 1999;124:759–62. Chen X, Yi C, Li M, Lu X, Li Z, Li P, et al. Determination of sophoridine and related lupin alkaloids using tris (2, 2′-bipyridine) ruthenium electrogenerated chemiluminescence. Anal Chim Acta 2002;466:79–86. Suliman FEO, Al-Hinai MM, Al-Kindy SM, Salama SB. Chemiluminescence determination of chlorpheniramine using tris (1, 10phenanthroline)–ruthenium (II) peroxydisulphate system and sequential injection analysis. Luminescence 2009;24:2–9. Zorzi M, Pastore P, Magno FA. Single calibration graph for the direct determination of ascorbic and dehydroascorbic acids by 2+ electrogenerated luminescence based on Ru(bpy)3 in aqueous solution. Anal Chem 2000;72:4934–9. He Z, Gao H. Simultaneous determination of oxalic and tartaric acid with chemiluminescence detection. Analyst 1997;122:1343–6. Wu F, He Z, Luo Q, Zeng Y. HPLC determination of oxalic acid using tris (1, 10-phenanthroline) ruthenium (II) chemiluminescenceapplication to the analysis of spinach. Food Chem 1999;65:543–6. British Pharmacopoeia Volume I & II, The British Pharmacopoeia Commission, London. 2012, Ketotifen Hydrogen Fumarate, monograph 1592.
1100 wileyonlinelibrary.com/journal/luminescence
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2015; 30: 1094–1100
Revised: 2 December 2014,
Accepted: 28 December 2014
Published online in Wiley Online Library: 18 February 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.2864
Simple chemiluminescence determination of ketotifen using tris(1,10 phenanthroline) ruthenium(II)- Ce(IV) system Ali Mokhtari,a* Mehrgan Ghazaeian,a Mahdieh Maghsoudi,a Mohsen Keyvanfardb and Iraj Emamic ABSTRACT: A new method using chemiluminescence (CL) detection has been developed for the simple determination of ketotifen fumarate (KF). The method is based on the catalytic effect of KF in the CL reaction of tris(1,10 phenanthroline)ruthenium(II), Ru(phen)32+, with Ce(IV) in sulfuric acid medium. The CL response was detected using a lab-made chemiluminometer. Effects of chemical variables were investigated and under optimum conditions, the CL intensity was proportional to the concentration of the drug over the range 0.34-34.00 μg mL1 KF. The limit of detection (S/N=3) was 0.09 μg mL1. Effects of common ingredients were investigated and the method was applied successfully for determining KF in pharmaceutical formulations and human plasma. The percent of relative standard deviation (n=11) at level of 3.4 μg mL1 of KF was 4.6% and the minimum sampling rate was 70 samples per hour. The possible CL mechanism is proposed based on the kinetic characteristic of the CL reaction, CL spectrum, UV-Vis and phosphorescence spectra. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: chemiluminescence; ketotifen; human plasma; pharmaceuticals
Introduction
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Ketotifen is a second-generation H1-antihistamine and mast cell stabilizer (1). The structure of ketotifen is shown in Fig. 1. It is most commonly available as a salt of fumaric acid (ketotifen fumarate, KF) and is accessible in two forms. In its ophthalmic form, it is used to treat allergic conjunctivitis, or the itchy red eyes caused by allergies (2). In its oral form, it is used to prevent asthma attacks. Side effects include drowsiness, weight gain, dry mouth, irritability, and increased nosebleeds (3). The therapeutic importance of the drug has prompted to develop various techniques for its assay in pharmaceuticals and body fluids. It is quantified by high performance liquid chromatography (4–11), high performance thin layer chromatography (12), liquid chromatography with mass spectrometry (13–15), spectrofluorimetry (16), direct differential pulse polarographic and adsorptive-stripping voltammetry (17), UV spectrophotometry (18–20), potentiometric titration (21,22) and coulumetric titration (23). Chemiluminescence (CL) is a powerful analytical tool because of its high sensitivity, wide dynamic range and simple instrumentation (24). The CL involving Ru(II) complexes is one of the most interesting series of CL reactions. It involves the oxidation of Ru (II) in the complex to Ru(III), which is following by reduction with an analyte species to produce CL emission (25,26). Tertiary amines can reduce Ru(III) complex and produce intense emission. Three CL methods (24,27,28) also proposed for the determination of KF up to now. The first method was on the basis of a europium (III)-doped Prussian blue analog film modifying platinum electrode as the working electrode and a Ru(bpy)32+-based electrochemiluminescence (ECL) assay coupled with capillary electrophoresis (24). The method was used for the determination of KF in urine and pharmaceutical samples. In the second CL
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method, potassium hexacyanoferrate(III) reacted with the mixture of calcein and KF (27). The CL intensity enhanced by trace amounts of Mg2+ and it was strongly dependent on KF concentration. The method was used to determine KF in tablets. The third method (28) was based on the catalytic effect of KF on the reaction of luminol with ferricyanide in sodium hydroxide medium. The significance of this work is developing a new simple chemiluminescence method for the determination of KF in pharmaceuticals (tablets, syrups and drops) and spiked human plasma. It is based on the catalytic effect of KF in the CL reaction of tris(1,10 phenanthroline)ruthenium(II), Ru(phen)32+, with Ce(IV) in sulfuric acid medium. The method has been applied to determine KF in drug formulations and human plasma with satisfactory results.
Experimental Reagents All the solutions were prepared using reagent grade chemicals and doubly distilled water. Acetonitrile was HPLC-grade (Caledon, Canada). KF standard solution (425.0 μgmL1) was daily * Correspondence to: A. Mokhtari, Laboratory of Analytical Chemistry, Department of Science, Golestan University, Gorgan, I.R. Iran. E-mail: [email protected] a
Department of Science, Golestan University, Gorgan, I.R. Iran
b
Department of Chemistry, Majlesi Branch, Islamic Azad University, Isfahan, I.R. Iran
c
Department of Physics, Isfahan University of Technology, Isfahan, I.R. Iran
Copyright © 2015 John Wiley & Sons, Ltd.
Simple chemiluminescence determination of Ketotifen Preparation of tablets
Figure 1. Chemical structure of ketotifen.
prepared by dissolving 0.0425 g of KF (Behsa, Iran) in 100.0 mL water. Working solutions were prepared by appropriately diluting the stock solution when used. Ru(phen)32+ solution (1.0×102 mol L1) was prepared by dissolving 0.3640 g of dichlorotris(1,10phenanthroline) ruthenium(II)hydrate (Sigma-Aldrich, Steinheim, Germany) in 50.0 mL water. Ce(IV) solutions (2.0×104-7.0×103 mol L1) were prepared by dissolving proper amount of ceric ammonium nitrate (Riedel-de Haën, Germany) in proper volumes of 1.0 mol L1 H2SO4 and diluting to the mark with distilled water in 100.0 mL volumetric flasks. In this way, H2SO4 concentrations of 0.03, 0.05, 0.08, 0.12 and 0.16 mol L1 were prepared. Plasma samples were taken from the health center of Gorgan (Iran). KF pharmaceuticals were purchased from local drugstores.
Ten tablets of KF (1 mg per tablet) were weighed and powdered. An accurately weighed portion of the powder, including active ingredients equivalent to 1.00 mg was transferred into a 100.0 mL calibrated flask containing 50 mL of water and the mixture was sonicated for 10 minutes. Then the volume was adjusted to 100.0 mL with water and the suspension was filtered. Each time 5.0 mL of this solution and a proper volume of the standard solution of KF were transferred into a 25.0 mL volumetric flask. Then it was diluted to the mark in order to obtain the appropriate concentration for the analysis.
Preparation of drops Two types of ketotifen drops were investigated, including oral and aphthalmic drugs. For each drop, contents of two drops were transferred into a beaker. An aliquot (1.0 mL) of the oral drug and an aliquot (4.0 mL) of the aphthalmic drug were transferred into 100 mL flasks and they were diluted to the mark with water. For each drug, 5.0 mL of this solution and a proper volume of the standard solution of KF were transferred into a 25.0 mL volumetric flask. Then it was diluted to the mark to obtain the appropriate concentration for the analysis.
Apparatus CL analysis was applied using a 0.50 cm light path length quartz cell. The CL signal was measured with a CL analyzer with PMT (Hammamatso, model R212, Japan) using a low pass filter which its output was connected to a data processing system with a Pentium IV PC. A schematic block diagram of the used instruments is shown in Fig. 2.
Preparation of the syrup samples 5.0 mL of the syrup drug was directly transferred into a 100.0 mL volumetric flask and diluted to the mark. Each time 5.0 mL of this solution and a proper volume of the standard solution of KF were transferred into a 25.0 mL volumetric flask and diluted to the mark.
General procedure An aliquot (200 μL) of standard solution consisting of KF with 400 μL of 2.0×103 mol L1 of Ru(phen)32+ were transferred into the 0.50 cm path light length quartz cell. Then, the cell was placed at its location in front of PMT and the program was started. After a few seconds, 200 μL of acidic Ce(IV) was injected into the cell by a microsyringe and the peak-like CL emission was recorded by a computer (with interval times of 100 ms). Those data information were collected into Excel software. Maximum CL response of KF appeared about 7 seconds after injection of Ce(IV) solution. For obtaining the analytical signal, response from the blank at second 7 after injection of Ce(IV) solution, was subtracted from maximum peak height of each sample.
Procedure for spiked plasma Only a deproteination process was carried out by using acetonitrile as a sample pretreatment and extraction procedure was not necessary (29). The standard addition method was used for the determination of KF in the plasma samples. Therefore each time, 1.0 mL of plasma sample was transferred into a centrifuge tube including 2 mL of acetonitrile and the mixture centrifuged at 4000 r/min for 15 min. The protein-free supernatant was transferred into a small conical flask and evaporated to dryness under a stream of nitrogen at room temperature. The dry residue was transferred into a 25.0 mL flask using double distilled water, then the standard solution was added into the flask and the mixture was diluted to the mark.
Results and discussion Kinetic curve of the CL reaction
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Figure 2. Schematic block diagram of the CL instrument.
Typical CL profiles for KF (3.4 μg mL1) and blank samples are shown in Fig. 3. Maximum CL response of KF appeared about 7 seconds after injection of Ce(IV) solution and the response declined to base slowly. CL profile of KF demonstrated that the CL reaction was slow. It took about 7 seconds to achieve the maximum peak, compared with 150-250 seconds for the signal to decline to base.
A. Mokhtari et al. To explore the possible CL mechanism, some experiments performed and following results were obtained.
Figure 3. Typical CL responses of a) blank and b) 3.4 μg mL
1
of KF.
Detailed CL mechanism Method is based on rapid reduction of Ru(phen)33+ produced in the reaction between Ru(phen)32+ and acidic Ce(IV) by KF that it produces strong CL. Solution of Ru(phen)32+ is orange and its color changes to green immediate after mixing with oxidizing agent, Ce(IV) solution, and production of Ru(phen)33+ (30,31). During about 3 minutes after mixing Ru(phen)32+ with Ce(IV), the color of the mixture changes slowly from green to orange. In Fig. 4, UV-Vis spectrum of the mixture of Ru(phen)32+-Ce(IV) is shown immediately after mixing (spectrum a) and 3 minutes after mixing (spectrum b). As can be seen in Fig. 4, Maximum absorbance at 450 nm which is related to Ru(phen)32+, disappeared immediately after mixing the solutions (spectrum a) and it appeared again after 3 minutes (spectrum b). The reason is that, the resulting Ru (phen)33+ produced in the reaction of Ru(phen)32+ with acidic Ce(IV), is a powerful oxidant and oxidizes water into O2 and protons (32). Therefore, it returns slowly to its reduced state. If there was a reducing agent in the reaction media, it can reduce Ru(phen)33+ very fast. The electrons from reducing agent transfer to the π*-orbital of phenanthroline ligand and the Ru (phen)32+ π* metal-to-ligand charge transfer (MLCT) excited state can be produced (33). The excited electron then undergoes intersystem crossing to the lowest triplet state of Ru (phen)32+, from where emission occurs (34). In order to confirm the mechanism proposed above, some CL pathways might be investigated for the Ru(phen)32+-Ce(IV)-KF CL system, involving the formation of Ce(III)*, oxidation products in excited state and [Ru(phen)32+]*.
(1) A weak CL intensity was observed when Ru(phen)32+ solution was mixed with acidic Ce(IV) solution. Enhancement in CL intensity was detected when KF solution was present in the mixture of Ru(phen)32+ and acidic Ce(IV). (2) Phosphorescence spectrum of KF (λex = 310 nm) was scanned using spectrofluorometer (Jasco, model FP-750) using batch mode. Phosphorescence of KF (λmax = 485 nm) was disappeared when Ce(IV) solution was added into the cuvette and new peak was appeared at 368 nm. These are due to oxidation of KF and formation of Ce(III) that is a well known fluorescent ion (35). (3) Phosphorescence emission spectrum of Ru(phen) 3 2+ (λex = 450 nm), had a maximum at 575 nm. (4) CL spectra of mixtures including Ce(IV)-KF (Fig. 5a), Ce(IV)-Ru(phen)32+ (Fig. 5b) and Ce(IV)-Ru(phen)32+-KF (Fig. 5c) were obtained using spectrofluorometer (Jasco, model FP750). No detectable CL intensity obtained for the first mixture. This suggests that oxidation products and Ce(III)* are not main emitters. Moreover Both spectra of second and third mixtures had same maximum emission wavelength at 575 nm which is same as maximum phosphorescence emission of Ru(phen)32+. This indicates that the CL spectra are independent of KF and the emitter is [Ru(phen)32+]*. KF is a tertiary amine and from previous studies, the oxidation of tertiary amines is understood to produce a short-lived radical cation. The α-carbon is then deprotonated, yielding a strongly reducing intermediate. This reduces the Ru(phen)33+ (produced by oxidant) to the excited state that subsequently emits light (29,36–38). According to the above discussion, the following mechanism is proposing for the CL reaction of KF. RuðphenÞ3 2þ þ CeðIVÞ → CeðIIIÞ þ RuðphenÞ3 3þ ketotifen þ CeðIVÞ → CeðIIIÞ þ ketotifen•þ ketotifen•þ → ketotifen• þ Hþ
RuðphenÞ3 3þ þ ketotifen• þ H2 O →
h i RuðphenÞ3 2þ *
þketotifen fragments h i h i 2þ RuðphenÞ3 * → RuðphenÞ3 2þ þ hυ
2+
2+
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Figure 4. UV-Vis spectrum of the mixture of Ru(phen)3 -Ce(IV), a) immediately after mixing b) 3 minutes after mixing.
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2+
Figure 5. CL spectrum of a) Ce(IV)-KF, b) Ce(IV)-Ru(phen)3 , c) Ce(IV)-Ru(phen)3 -KF. 1 3 1 1 Conditions: a) 2 mL KF (3.4 μg mL ) and 400 μL Ce(IV) (3.0×10 mol L in 0.1 mol L 3 1 3 2+ of H2SO4), b) 2 mL Ru(phen)3 (2.0×10 mol L ) and 400 μL Ce(IV) (3.0×10 1 1 3 1 2+ mol L in 0.1 mol L of H2SO4), c) 2 mL Ru(phen)3 (2.0×10 mol L ), 200 μL KF 1 3 1 1 (3.4 μg mL ) and 400 μL Ce(IV) (3.0×10 mol L in 0.1 mol L of H2SO4).
Copyright © 2015 John Wiley & Sons, Ltd.
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Simple chemiluminescence determination of Ketotifen Influence of chemical variables To study the influence of chemical variables, influence of Ru (phen)32+, Ce(IV) and H2SO4 concentrations on the CL intensity were investigated. The influence of concentration of Ru(phen)32+ on the sensitivity was studied in the range 1.0×103-7.0×103 mol L1 by injecting concentration of 1.0×103 mol L1 of Ce(IV) prepared in 0.10 mol L1 of H2SO4. The CL signal increased with increasing Ru(phen)32+ concentration until 2.0×103 mol L1 and then decreased. Therefore, concentration of 2.0×103 mol L1 was selected as the optimum concentration for the complex of Ru(phen)32+. The influence of concentration of Ce(IV) on the CL sensitivity was investigated in the range 1.0×103 to 1.0×102 mol L1 of Ce(IV) in 0.10 mol L1 of H2SO4. For this variable sensitivity increased to concentration of 3.0×103 mol L1 and then decreased at higher concentrations. Therefore, 3.0×103 mol L1 of Ce(IV) was selected as the optimum concentration. The influence of concentration of H2SO4 on the CL intensity was also studied in the range 0.04 to 0.14 mol L1 of H2SO4. The CL response increased with increasing the concentration of H2SO4 to 0.1 mol L1 and then decreased. Therefore, concentration 0.1 mol L1 H2SO4 was selected for further studies. Analytical features Under optimum conditions, a long series of standard solutions of KF were subjected to the optimized CL method for the purpose of calibration. CL response was found to be linear in the concentration ranges of 0.34-34.00 μg mL1 (Fig. 6). The correlation equation between CL intensity and concentration of KF in linear range was: ICL = 4.50 + 12.74 CKF (R2 = 0.9979) where CKF is concentration of KF (μg mL1). The limit of detection (LOD) was calculated as 3σ/m where σ is the standard deviation existing in 10 times determination of the blank response and m is slope of the correlation equation between CL intensity and concentration of KF (12.74 in calibration equation mentioned above). The LOD obtained was 0.09 μg mL1, indicating good detectability. The reproducibility was investigated using 3.4 μg mL1 of KF (n=11) and the percent of relative standard deviation (%RSD) was 4.6%. The minimum sampling rate could be about 70 samples per hour.
pharmaceutical preparations and some amino acids were studied by recovering 3.4 μg mL1 (8.0×106 mol L1) of KF in presence of each substance. The tolerance of each substance was taken as the largest amount yielding an error of less than 3σ in the analytical signal of 3.4 μg mL1 KF (σ is the standard deviation in the response obtained from 11 times determination of 3.4 μg mL1 of KF). Because of interference effects from some substances in the real samples, such as citric acid, ascorbic acid, oxalic acid, cysteine, glutathione etc., CL intensity at 50 seconds after injection of Ce(IV) solution was chosen as the analytical signal for interference and application studies (instead of CL intensity at 7 seconds after injection of Ce(IV) solution when maximum response of KF appears). For example ascorbic acid and oxalic acid (5 μg mL1) had a sharp peak which their maximum appeared at 0.7 and 1.7 seconds after the injection of Ce(IV) solution respectively, and their CL intensity decreased to baseline after about 10 seconds. Citric acid had a relatively broad peak but its peak decreased to baseline after about 40 seconds. In the CL system proposed in this study, KF had a broad time profile with detectable response at 50 seconds after injection of Ce(IV) solution. Therefore for decreasing the effect of some interfering substances in real samples, interference effect from all substances were investigated at 50 seconds after injection of Ce(IV) solution. The results have been shown in Table 1. It is known that compounds having a tertiary amine or carboxylic acid group, such as some drugs, amino acids and organic acids and also some ions, emit light when they react with Ru (phen)33+ in an acidic solution (29,39–42). In spite of CL light emission produced in presence of some of the investigated compounds, these compounds have little or zero interference effect for the determination of KF, because most of them have a zero or very weak CL intensity at 50 seconds after injection of Ce(IV) solution. Nonetheless some of investigated substances such as tyrosine, morphine and I had a quenching effect on the CL intensity and their interference effect remained until 50 seconds after starting the CL reaction.
Table 1. Effect of foreign substances on the determination of 3.4 μg mL1 KF
Influence of interfering substances In order to validate of the possible analytical application of the method, interferences from some common ions, excipients in
Substance/KFa
Substance Threonine, Serine, Lactose, Sucrose, Glucose, Fructose, Saccharin, Starch, Urea, Valine, Leucine, K+, Cl, Na+, Zn2+, SO42, Br, NO3 Glycine, Cystine Oxidized glutathione, Proline, Alanine, HCO3, CO32, PO43, CH3COO, Phenylanaline, Cu2+ Histidine, Aspartic acid, Tryptophane, Ascorbic acid, Fe2+, Mg2+, Ca2+ Tyrosine Glutathione, Cysteine, Citric acid, Mn2+, I Oxalic acid, Noscapine Codeine, Morphine
1000
500 100
50 10 5 1 0.1
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Molar ratio of substance to KF.
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a
Figure 6. Calibration curve of KF.
A. Mokhtari et al. Application
have been summarized in Table 4. In the CE-ECL method, a separating technique (CE) has been used and therefore, it is more favorable than other mentioned methods for analyzing of biological samples. However in ECL detection system, oxidations of KF and Ru(II) complex have been performed by electrochemical instruments and on a platinum microelectrode modified with Europium(III)-doped Prussian analog film (Eu-PB). Moreover, electrochemical instrumentation has been used for electrodepositing of Eu-PB on the platinum electrode. Therefore the ECL detection system is more expensive and more complex than the chemical oxidation performed in the proposed method. As can be seen in Fig. 3, KF produced a broad CL time profile in the Ru(phen)32+-Ce(IV) CL system. It had a detectable response even after 50 seconds after injection of Ce(IV) solution. Moreover many of the investigated compounds mentioned in the interference study section had a zero or very weak CL intensity at that time. Therefore, for many of the substances that they can enhance the CL response, the extent of interference effect at 50 seconds after injection of Ce(IV) solution is less than their
In order to evaluate the applicability of the proposed method, KF syrup, drops and tablets were analyzed to determine their KF contents. For all samples, CL intensity at 50 seconds after injection of Ce(IV) solution was chosen as the analytical signal. Also recovery of KF from human plasma was investigated. The results are shown in Table 2. The obtained results from analyzing of syrup sample were also certified by the British Pharmacopoeia (BP) method. BP provided a potentiometric titration method using perchloric acid as titrant (43). Statistical analysis of the results using student t-test and the variance ratio F-test showed no significant difference between the performance of two methods as regards to accuracy and precision. The results are presented in Table 3.
Response characteristics The analytical parameters of the previously reported CL methods and proposed method for the determination of KF
Table 2. Determination of KF in real samples Real Sample
Sample No.
Added (μg mL1)
Total Found (μg mL1)
Recovery (%)
1 2 3 4 1 2 3 1 2 3 1 2 3 1 2 3
0.00 4.00 8.00 12.00 0.00 4.00 8.00 0.00 4.00 8.00 0.00 4.00 8.00 0.00 1.00 2.00
2.69±0.22 6.68±0.34 10.53±0.52 14.25±0.46 2.83±0.17 6.87±0.42 11.04±0.61 2.38±0.39 6.60±0.42 10.65±0.81 2.94±0.22 7.00±0.37 10.87±0.58 0.11±0.17 1.07±0.24 2.14±0.19
99.8 98.0 96.3 101.0 102.6 105.5 103.4 101.5 99.1 96.0 101.5 100.4±2.94
a
Tablet (1mg per tab )
Syrup (1mg per 5mLb)
Drop (1mg per mLc)
Drop (0.25%d)
Plasma
Mean±S.D. a
Ketotifen Amin 1mg tab (Amin Co., Iran). Each tablet contains 1.38 mg KF equivalent to 1mg ketotifen base. Amiten 60 mL syrup (Amin Co., Iran). Each 5 mL solution contains 1.38 KF equivalent to 1mg ketotifen base. c Ketotifen-Behsa 1 mg mL1 oral drop (Behsa, Iran). Each 1 mL solution contains 1.38 mg KF equivalent to 1mg ketotifen base. d Ketotifen 0.25% ophthalmic drop (Daru Pakhsh, Iran). Each 1 mL contains 0.345 mg ketotifen hydrogen fumarate corresponding to 0.25 mg ketotifen. b
Table 3. Analysis of a formulation containing KF using the proposed method and the official method Sample
KF syrup
Nominal value
1.38 mg per 5 mL
Analytical Resultsa Proposed method
BP methodb
1.42±0.10
1.49±0.07d
t-testc
F-testd
1.15
2.04
a
Mean values of four replications. British Pharmacopoeia, a potentiometric titration method. c Student t-test calculated, theoretical value=3.182 (P=0.05). d F-test calculated, theoretical value=9.28 (P=0.05). b
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Luminescence 2015; 30: 1094–1100
Simple chemiluminescence determination of Ketotifen Table 4. Analytical features of CL methods proposed for the determination of KF CL reagent
Oxidation method
Ru(bpy)32+
Electrochemistry
CalceineMg2+
Luminol
Ru(phen)32+
Methodology of CL reaction
D.L. (g mL1)
Oxidation of Ru(bpy)32+ 2.1×108 on the platinum microelectrode modified with europium (III)-doped Prussian blue analog film (Eu-PB) and reduction of produced Ru(bpy)33+ by KF 3.0×109 hexacyanoferrate(III) Reaction of potassium hexacyanoferrate(III) with the mixture of calcein and KF. A stronger CL signal was observed when a trace amount of Mg2+ was added into KF solution Hexacyanoferrate Catalytic effect of KF on 5.7×109 (III) the oxidation rate of luminol with ferricyanide in sodium hydroxide medium Ce(IV) Oxidation of Ru(phen)32+ 9.0×108 using Ce(IV) and reduction of produced Ru(phen)33+ by KF
LDR Speed Samples (g mL1) (samples per h) 3.0×108– 2.0×106
15
6.0×109– 2.0×107
Ref.
Tablet, Drop, Urine
(24)
About 70
Tablet
(27)
1.0×108– 1.0×106
N/A
N/A
(28)
3.4×107– 3.4×105
70
Tablet, Proposed Drops, Method Syrup, Plasma
a
Capillary Electrophoresis- Electrochemiluminescence.
Luminescence 2015; 30: 1094–1100
Conclusions A new method based on the CL of Ru(phen)32+ and acidic Ce(IV), was proposed for the quantification of KF. The method is simple, rapid and adequately sensitive for the determination of KF in pharmaceuticals. Some common sugars, amino acids and ions hadn’t significant interference effect in the quantification of KF indicating high accuracy and suitability for determining of KF in human fluids and quality assurance in drug formulations. The proposed CL system is a simple and rapid analytical tool for obtaining of preliminary chemical information about KF prior the use of more complex instrumental techniques. One future trend might be the combination of the proposed CL system, (Ru(phen)32+ and acidic Ce(IV)) (used previously for the determination of some drugs), with liquid chromatography and developing a technique for the determination of KF in various matrixes and pharmaceuticals. Acknowledgements The authors are grateful to the Campus of Golestan University for supporting this work.
References 1. Kidd M, McKenzie SH, Steven I, Cooper C, Lanz R. Efficacy and safety of ketotifen eye drops in the treatment of seasonal allergic conjunctivitis. Br J Ophthalmol 2003;87:1206–11.
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interference extent at 7 seconds after injection of Ce(IV) solution, when maximum response of KF appears. Therefore, in this study we used CL intensity at 50 seconds after injection of Ce(IV) solution as the analytical signal for interference and application investigations. As can been in Table 1, in the proposed method, sugars, some amino acids and metal ions such as Ca2+, Mg2+, Zn2+ and Fe2+ had not interference effect for the determination of KF. In the method in which Calceine-Mg2+ had been used as CL reagent, good sensitivity was obtained, but the effect of amino acids had not been checked and they found that some metal ions including Ca2+, Zn2+, Cd2+, Cu2+ and Fe2+ could enhance the CL signal of potassium hexacyanoferrate(III)-calceinMg-KF reaction and they can interfere for the determination of KF at concentrations higher than 1×107 g mL1. In the ECL method, no interference study had been performed and only they mentioned that excipients and additives in pharmaceuticals, uric acid and other matrices in urine samples had little interference in the detection with the use of the E-PB electrode. The proposed CL system is an easy and fast analytical tool for obtaining preliminary chemical information about KF, prior the use of more complex instrumental techniques. In the proposed method oxidation has been performed using chemical oxidation which is simpler and cheaper than electrochemical oxidation method. Moreover, interference effect from some ions such as Ca2+, Zn2+, Cu2+ and Fe2+ is less than the method in which Calceine-Mg2+ had been used as CL reagent.
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