Research article Received: 10 November 2013,
Revised: 1 February 2014,
Accepted: 9 February 2014
Published online in Wiley Online Library: 22 April 2014
(wileyonlinelibrary.com) DOI 10.1002/bio.2659
Potassium permanganate–acridine yellow chemiluminescence system for the determination of fluvoxamine, isoniazid and ceftriaxone Jafar Abolhasani* and Javad Hassanzadeh ABSTRACT: Based on the oxidation of acridine yellow by permanganate in basic medium, a new chemiluminescence system was developed for the sensitive determination of some important drugs. The remarkable inhibiting effect of fluvoxamine, ceftriaxone and isoniazid on this reaction was applied to their detection. A possible mechanism was proposed for this system based on chemiluminescence emission wavelengths and experimental observations. Under optimum conditions, calibration graphs were obtained for 1 × 109 to 1 × 106 mol/L of fluvoxamine; 2 × 108 to 8 × 106 mol/L of ceftriaxone and 5 × 108 to 4 × 105 mol/L of isoniazid. This proposed method was satisfactorily used in the determination of these drugs in pharmaceutical samples and human urine and serum. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: chemiluminescence; permanganate; acridine yellow; drug
Introduction
Luminescence 2014; 29: 1053–1058
* Correspondence to: Jafar Abolhasani, Department of Chemistry, East Azerbaijan Science and Research Branch, Islamic Azad University, Tabriz, Iran. E-mail: [email protected] Department of Chemistry, East Azerbaijan Science and Research Branch, Islamic Azad University, Tabriz, Iran
Copyright © 2014 John Wiley & Sons, Ltd.
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Pharmaceutical analysis is important for detecting drug dosages in pharmaceutical preparations and biological fluids. It is important to achieve the quality assurance in pharmaceutical manufacturing and the best therapeutic concentrations in body fluids, thus reducing the risk of toxicity. A simple, fast and sensitive determination method for fluvoxamine (FLU), ceftriaxone (CEF) and isoniazid (ISO), drugs that have many applications, could be highly important for analysis. FLU (Fig. 1a), (E)-5-methoxy-1-[4-(trifluoromethyl)-phenyl]-1pentanone-O-2-aminoethyl) oxime maleate, is a well-tolerated antidepressant that functions as a selective serotonin reuptake inhibitor and is used in the treatment of depression and obsessivecompulsive disorders (1). Various methods have been developed for the quantification of FLU, and these are usually based on chromatographic analysis (1–7). CEF (Fig. 1b), 8-[2-amino-2-(4-hydroxyphenyl)-acetyl] amino-4methyl-7-oxo-2-thia-6-azabicyclo [4.2.0] oct-4-ene-5-carboxylic acid, is a third-generation cephalosporin antibiotic. CEF is often used for the treatment of pneumonia, typhoid fever, gonorrhea and bacterial meningitis, or to exclude sepsis. A great variety of analytical methods have been reported for the determination of CEF involving spectrophotometry (8,9), fluorometry (10), chemiluminescence (CL) (11,12) and high-performance liquid chromatography (13,14). ISO (Fig. 1c), isonicotinohydrazide, is an important antibiotic, which is the medication most widely used in the prevention and treatment of tuberculosis. Several different analytical methods have been developed for the determination of ISO, such as, spectrophotometry (15–17), fluorometry (18), CL (19–21), electrochemical (22) and high-performance liquid chromatography (23–25). The CL method (light production from a chemical oxidation reaction) has been frequently used for the analysis of pharmaceutical compounds in recent years (26–29), because of its promising
advantages, e.g., low detection limit, wide linear dynamic range and relatively simple and inexpensive instrumentation. Potassium permanganate (KMnO4) is the most common oxidant used in CL reactions, which was first applied in the analytical field by Stauff and Jaeschke (30); after that, there has been a significant rise in the number of applications, particularly those involving organic analytes. Comprehensive reviews by Hindson and Barnett and Adcock et al. have indicated a wide range of analytical applications of acidic KMnO4 in CL reactions (31,32). Alkaline media was scarcely applied for permanganate CL reactions; however, it can reduce the many interferences that exist in acidic media. The acridine family (anthracene derivatives containing a nitrogen atom in the central ring) is very large and includes diaminoacridines such as acridine yellow (AY). The CL behavior of some members of the diaminoacridines has been demonstrated when oxidized by strong inorganic oxidants such as KMnO4 (33,34). The CL efficiency of acridine-based reagents is often better than that of other common CL reactants such as luminol (35). Particular advantages of these compounds are the very low background light level, extreme simplicity of the light yielding reaction conditions and no other catalyst being required (36,37). In this study, we found that a strong CL signal was produced as a result of AY oxidation by KMnO4 in alkaline solution. To date only one article has addressed acidic medium for AY determination (33). On the other hand, it was recognized that some drugs have an inhibiting effect on CL signal, which is probably related to loss of intermediate radicals. Based on these
J. Abolhasani and J. Hassanzadeh to 50 mL and stored in dark at 4°C. A suitable aliquot of this solution was taken for the determination of drugs according to the recommended general procedure. Human urine sample containing the drug concerned was obtained by adding a suitable amount of standard drug solution to drug-free urine. An amount of 1 mL of this sample was gently vortex mixed with 1.8 mL of 0.1 mol/L Ba(OH)2 and 2 mL of 0.1 mol/L ZnSO4 solution was put aside at room temperature for 5 min, and then centrifuged 2700 g for 10 min). In this condition, any protein and reducing substances were precipitated to prevent any interfering effect. The clear supernatant was diluted to the appropriate concentration (38). The human serum sample was spiked by adding suitable concentration of drug standard solution. Two mL of the spiked serum sample was mixed with 0.5 mL of acetonitrile and centrifuged at 2700 g for 10 min. The resulting supernatant was used for analysis under the optimum conditions. General procedure for chemiluminescence detection
Figure 1. Chemical structure of (a) fluvoxamine, (b) isoniazid and (c) ceftriaxone.
observations, a new simple and sensitive CL method has been developed for the determination of trace amounts of three drugs: FLU, CEF and ISO. This method is simpler and less expensive than other reported techniques and offers good accuracy and precision. The method has also been used to determine FLU, CEF and ISO in tablets and human urine and serum.
CL measurements were carried out in the batch condition. One hundred μL of AY (0.01 mol/L), and 100 μL of NaOH (1.0 mol/L) were added into a 3 mL quartz tube. Then an appropriate volume of sample or standard analyte (FLU, CEF or ISO) solution was added and the final volume was reached to 1.3 mL with the distilled water. After injection of 200 μL of KMnO4 (0.005 mol/L) by an automatic injector, monitoring the CL signal versus time was started automatically. The maximum CL intensity was used as the analytical signal.
Results and discussion Acridine yellow–KMnO4 chemiluminescence system
Materials and methods Materials AY, KMnO4 and NaOH were obtained from Merck (Darmstadt, Germany) and used without further purification. Doubly distilled deionized water (Ghazi Serum Co., Tabriz, Iran) was used throughout. A stock standard solution of 100 mg/L for each analyte (FLU, CEF and ISO, obtained from Sobhan Darou, Tehran, Iran) was prepared in water and stored at 4°C in a sealed volumetric flask.
Permanganate can oxidize AY in basic media and produce a relatively high CL signal (Fig. 2). The oxidation of acridinium esters, which has been extensively studied by McCapra (39), is compatible with aqueous solution at neutral pH. Oxidation by KMnO4 in acid media has only been reported once for AY determination (33). Acidic KMnO4 is a powerful oxidant that can oxidize almost all oxidizable compounds. On the other hand, the CL emission produced is due to excited Mn2+ species that is independent of the oxidized reagents. Therefore, each oxidation reaction in the
15
The CL detection was conducted on LUMAT LB 9507 chemiluminometer (Berthold; www.berthold.com). The CL spectra were measured on a Shimadzu RF-5301 PC spectrofluorometer using a flow mode with the excitation light source being turned off.
10
5
0
Sample preparation
0
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Pharmaceutical tablets were obtained from Tehran Daru (Iran). Five tablets of each drug were weighed and powdered. Three hundred mg of this powder was dissolved in deionized water and filtrated for each drug separately. The filtrate was diluted
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CL intensity
Apparatus
25
50
75
100
Time (s) Figure 2. Kinetic curve for KMnO4–acridine yellow CL system, (a) alone and (b) in 3 presence of 1 μmol/L fluvoxamine (KMnO4, 1 × 10 mol/L; acridine yellow, 3 1 × 10 mol/L and NaOH 0.1 mol/L). CL, chemiluminescence.
Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2014; 29: 1053–1058
Alkaline permanganete chemiluminescece
Figure 3. CL spectrum of KMnO4–acridine yellow system in absence (a) and presence of 10 μmol/L fluvoxamine (b). CL, chemiluminescence. (Condition is like Fig. 2.)
Table 1. Effect of some drugs on chemiluminescence emission intensity of acridine yellow–KMnO4 ΔI 1288 1773 2566 538 1258 1183 6475 5684 1283 3698 5480
Drug
ΔI
Drug
Fluoxetine Tramadol Amitriptyline Clomipramine Safrazine Quercetin Alprazolam Amoxicillin Cefazolin Cefixime Clonazepam
364,627 286,695 301,173 2176 2458 8695 5366 9358 8158 3358 3682
Fluvoxamine Isoniazid Ceftriaxone Citalopram Escitalopram Norfluxacine Ofluxacine Iproniazid Ascorbic acid Folic acid Norephinephrine
presence of KMnO4 may result in CL emission and lead to high interferences and low selectivity. This is the reason that KMnO4 CL systems are often applied in the determination of drugs in
simple pharmaceutical systems. However, in alkaline conditions, the CL emission is related to oxidized species or analytes. In addition, the KMnO4 oxidation ability is low and thus interferences are reduced. We found that this method is successfully applied to the determination of drugs in biological samples without considerable interference. We believe that this research will lead to a wide variety of analytical applications for this type of CL reaction. The mechanism of light emission of the acridine-based reactants is very well understood in acidic conditions, which involves the protonation of the acridine and its oxidation by transference of two electrons. The formation of an excited acridone intermediate is proposed, which can emit blue light at 440 nm or transfer energy to other fluoresce molecules (34). However, in alkaline media it has reported that KMnO4 as an oxidizing agent can degrade the acridine forming quinoline-2,3-dicarboxylic acid (40). To determine the luminophor in the systems, the CL emission spectra for AY–KMnO4 and AY–KMnO4–drug systems were recorded (Fig. 3). The spectrum of these systems displayed a broad emission band in the range of 450–650 nm, with a maximum at ∼510 nm, which is similar to the fluorescence spectrum of AY (λem = 510 nm and λex = 450 nm). This reveals that the emitting species in the CL reaction is probably AY. On the other hand, to determine product of AY oxidation by KMnO4, the fluorescence spectrum of AY was plotted after oxidation. An emission band was observed in the range of 400– 500 nm, which is very similar to the fluorescence spectrum of acridone (35). This wavelength range is completely overlapped with the excitation spectrum of AY. Based on these observations, it can be considered that excited product of AY due to oxidation by KMnO4 (M*) can transfer energy to excess AY molecules, which then can emit as light: AY þ MnO4 – þ OH– →M M þ AY→M þ AY AY →AY þ hv ð510 nmÞ The kinetic profiles for the CL reaction in the absence and presence of FLU are shown in Fig. 2. As can be seen, the maximum CL signal was achieved at about 1 s. In addition, it showed that, in the absence of drugs, the CL intensity was relatively high. However, introduction of these drugs into this reaction system resulted in the CL intensity decrease, proportional to the amount of drugs introduced. This effect can be related to the competitive oxidation of drugs and consumption of KMnO4.
Table 2. Analytical characteristics of the proposed method for the determination of FLU, ISO and CEF Parameter Linear range, nmol/L Slope Intercept Correlation coefficient (r) Number of points LOD, nmol/L LOQ, nmol/L RSD% (10 nmol/L, n = 3) RSD% (100 nmol/L, n = 3) RSD% (800 nmol/L, n = 3)
FLU
ISO
CEF
1–1000 10,557 4652 0.9979 11 0.24 0.81 1.4 2.3 1.9
50-40,000 9855 3547 0.9988 11 14 47 2.8 1.4
20-8000 8750 1529 0.9991 11 4.9 17 3.2 1.6
Luminescence 2014; 29: 1053–1058
Copyright © 2014 John Wiley & Sons, Ltd.
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CEF, ceftriaxone; FLU, fluvoxamine; ISO, isoniazid; LOD, limit of detection; LOQ, limit of quantitation; RSD, relative standard deviation.
J. Abolhasani and J. Hassanzadeh Table 3. Interference study of some coexisting agents in the determination of fluvoxamine Coexisting agent
Tolerable concentration ratios
Ca2+, Mg2+, Fe3+ Na+, K+, Cl, NO3, SO2 4 2+ 2+ PO3 4 , Zn , Cu , vitamins B1 and B2 Oxalate, glucose, ascorbic acid Cysteine
1800 1000 500 150 50
The effect of the AY concentration over the range of 1 × 104– 3 × 103 mol/L was investigated to determine the maximal emission intensity. The highest CL intensity was obtained at a concentration of AY of 0.001 mol/L, which did not improve upon increasing the AY concentrations. Hence, 0.001 mol/L of AY was selected as the optimum concentration. To investigate the effect of the KMnO4 concentration, solutions with different concentrations of KMnO4 were prepared over the range of 1 × 104–5 × 103 mol/L. The CL signal was increased up to 0.001 mol/L and then did not increase further at high concentrations. At lower KMnO4 concentrations, the number of excited intermediates was decreased and the response was diminished.
Optimization of experimental parameters The optimum conditions for the FLU, CEF and ISO determinations were carried out using the 1 × 107 mol/L concentration of drug. In the proposed system, the CL intensity was mainly dependent on the concentration of NaOH. Therefore, to examine the effect of this parameter on the CL signal, NaOH at different concentrations was tested. The maximum CL response was obtained at 0.1 mol/L NaOH for all three drugs and higher concentrations had no significant effect.
Analytical parameters The effect of some drugs on the CL emission of AY–KMnO4 was studied. The results, which are shown in Table 1, show that many tested drugs had a minor diminishing effect, but only few drugs such as FLU, CEF and ISO had a major effect on light emission (Fig. 2). To determine drug concentration, the difference between the CL intensity (ΔI = I0 – I) in the absence (I0) and presence of each drug (I) was plotted versus the concentration of drug in
Table 4. Results for the determination of fluvoxamine, isoniazid and ceftriaxone in pharmaceutical and biological samples using the proposed method Sample Pharmaceutical samples
Fluvoxamine maleate (25 mg)
Isoniazid (100 mg)
Ceftriaxone (250 mg per 1 mL injection)
Urine samples
Isoniazid
Ceftriaxone
Fluvoxamine
Serum samples
Isoniazid
Ceftriaxone
Fluvoxamine
Adda
Foundb
Recovery %
t-statisticc
0 5.0 10.0 15.0 0 25 50 100 0 50 100 150 1.0 2.5 5.0 1.0 2.5 5.0 1.0 2.5 5.0 0.20 0.50 1.0 0.20 0.50 1.0 0.20 0.50 1.0
24.7 ± 0.3 295 ± 0.2 34.3 ± 0.2 39.4 ± 0.2 98.0 ± 1.6 123 ± 1.0 147 ± 1.1 197 ± 1.3 241 ± 4.0 288 ± 2.2 342 ± 2.2 394 ± 3.2 0.98 ± 0.03 2.44 ± 0.03 4.88 ± 0.06 1.02 ± 0.04 2.45 ± 0.03 5.02 ± 0.08 0.99 ± 0.03 2.46 ± 0.03 4.98 ± 0.08 0.20 ± 0.01 0.49 ± 0.01 0.97 ± 0.02 0.20 ± 0.01 0.49 ± 0.01 1.0 ± 0.04 0.19 ± 0.01 0.50 ± 0.01 0.99 ± 0.04
– 97 ± 3.1 96 ± 2.0 98 ± 1.4 – 101 ± 4.1 97 ± 2.1 100 ± 1.3 – 103 ± 2.2 103 ± 2.2 103 ± 2.1 98.3 ± 2.5 97.7 ± 1.2 97.6 ± 1.2 102 ± 3.5 98.1 ± 1.2 100 ± 1.6 98.7 ± 3.1 99.6 ± 1.2 99.6 ± 1.6 98.2 ± 3.8 97.1 ± 1.7 96.7 ± 1.5 99.7 ± 2.8 98.8 ± 1.8 101.7 ± 3.5 95.5 ± 2.3 99.7 ± 1.7 98.7 ± 3.8
2.3 1.7 3.6 2.9 2.2 0.6 2.0 0.4 3.8 1.0 2.0 2.3 1.1 3.2 3.3 0.8 2.6 0.4 0.8 2.3 0.42 0.68 2.4 3.1 0.17 0.93 0.67 2.8 0.22 0.50
a
The unit for added and found amounts is mg for tablet and mg/L for urine and serum samples. Mean of three determinations ± SD. c t-critical = 4.3 for n = 2 and P = 0.05. b
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Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2014; 29: 1053–1058
Alkaline permanganete chemiluminescece μmol/L. The analytical parameters for each of these drugs are summarized in Table 2. The results indicated that this CL system had good linearity, relatively high sensitivity and good precision. It should also be noted that AY–KMnO4 is a new CL system, which can have potential applications in the determination of some other important compounds.
Interference studies To test for interference, some potentially interfering substances in increasing amounts were added into a standard solution of 0.1 μmol/L for each drug. The tolerable concentration ratios for interferences with the relative error of
Revised: 1 February 2014,
Accepted: 9 February 2014
Published online in Wiley Online Library: 22 April 2014
(wileyonlinelibrary.com) DOI 10.1002/bio.2659
Potassium permanganate–acridine yellow chemiluminescence system for the determination of fluvoxamine, isoniazid and ceftriaxone Jafar Abolhasani* and Javad Hassanzadeh ABSTRACT: Based on the oxidation of acridine yellow by permanganate in basic medium, a new chemiluminescence system was developed for the sensitive determination of some important drugs. The remarkable inhibiting effect of fluvoxamine, ceftriaxone and isoniazid on this reaction was applied to their detection. A possible mechanism was proposed for this system based on chemiluminescence emission wavelengths and experimental observations. Under optimum conditions, calibration graphs were obtained for 1 × 109 to 1 × 106 mol/L of fluvoxamine; 2 × 108 to 8 × 106 mol/L of ceftriaxone and 5 × 108 to 4 × 105 mol/L of isoniazid. This proposed method was satisfactorily used in the determination of these drugs in pharmaceutical samples and human urine and serum. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: chemiluminescence; permanganate; acridine yellow; drug
Introduction
Luminescence 2014; 29: 1053–1058
* Correspondence to: Jafar Abolhasani, Department of Chemistry, East Azerbaijan Science and Research Branch, Islamic Azad University, Tabriz, Iran. E-mail: [email protected] Department of Chemistry, East Azerbaijan Science and Research Branch, Islamic Azad University, Tabriz, Iran
Copyright © 2014 John Wiley & Sons, Ltd.
1053
Pharmaceutical analysis is important for detecting drug dosages in pharmaceutical preparations and biological fluids. It is important to achieve the quality assurance in pharmaceutical manufacturing and the best therapeutic concentrations in body fluids, thus reducing the risk of toxicity. A simple, fast and sensitive determination method for fluvoxamine (FLU), ceftriaxone (CEF) and isoniazid (ISO), drugs that have many applications, could be highly important for analysis. FLU (Fig. 1a), (E)-5-methoxy-1-[4-(trifluoromethyl)-phenyl]-1pentanone-O-2-aminoethyl) oxime maleate, is a well-tolerated antidepressant that functions as a selective serotonin reuptake inhibitor and is used in the treatment of depression and obsessivecompulsive disorders (1). Various methods have been developed for the quantification of FLU, and these are usually based on chromatographic analysis (1–7). CEF (Fig. 1b), 8-[2-amino-2-(4-hydroxyphenyl)-acetyl] amino-4methyl-7-oxo-2-thia-6-azabicyclo [4.2.0] oct-4-ene-5-carboxylic acid, is a third-generation cephalosporin antibiotic. CEF is often used for the treatment of pneumonia, typhoid fever, gonorrhea and bacterial meningitis, or to exclude sepsis. A great variety of analytical methods have been reported for the determination of CEF involving spectrophotometry (8,9), fluorometry (10), chemiluminescence (CL) (11,12) and high-performance liquid chromatography (13,14). ISO (Fig. 1c), isonicotinohydrazide, is an important antibiotic, which is the medication most widely used in the prevention and treatment of tuberculosis. Several different analytical methods have been developed for the determination of ISO, such as, spectrophotometry (15–17), fluorometry (18), CL (19–21), electrochemical (22) and high-performance liquid chromatography (23–25). The CL method (light production from a chemical oxidation reaction) has been frequently used for the analysis of pharmaceutical compounds in recent years (26–29), because of its promising
advantages, e.g., low detection limit, wide linear dynamic range and relatively simple and inexpensive instrumentation. Potassium permanganate (KMnO4) is the most common oxidant used in CL reactions, which was first applied in the analytical field by Stauff and Jaeschke (30); after that, there has been a significant rise in the number of applications, particularly those involving organic analytes. Comprehensive reviews by Hindson and Barnett and Adcock et al. have indicated a wide range of analytical applications of acidic KMnO4 in CL reactions (31,32). Alkaline media was scarcely applied for permanganate CL reactions; however, it can reduce the many interferences that exist in acidic media. The acridine family (anthracene derivatives containing a nitrogen atom in the central ring) is very large and includes diaminoacridines such as acridine yellow (AY). The CL behavior of some members of the diaminoacridines has been demonstrated when oxidized by strong inorganic oxidants such as KMnO4 (33,34). The CL efficiency of acridine-based reagents is often better than that of other common CL reactants such as luminol (35). Particular advantages of these compounds are the very low background light level, extreme simplicity of the light yielding reaction conditions and no other catalyst being required (36,37). In this study, we found that a strong CL signal was produced as a result of AY oxidation by KMnO4 in alkaline solution. To date only one article has addressed acidic medium for AY determination (33). On the other hand, it was recognized that some drugs have an inhibiting effect on CL signal, which is probably related to loss of intermediate radicals. Based on these
J. Abolhasani and J. Hassanzadeh to 50 mL and stored in dark at 4°C. A suitable aliquot of this solution was taken for the determination of drugs according to the recommended general procedure. Human urine sample containing the drug concerned was obtained by adding a suitable amount of standard drug solution to drug-free urine. An amount of 1 mL of this sample was gently vortex mixed with 1.8 mL of 0.1 mol/L Ba(OH)2 and 2 mL of 0.1 mol/L ZnSO4 solution was put aside at room temperature for 5 min, and then centrifuged 2700 g for 10 min). In this condition, any protein and reducing substances were precipitated to prevent any interfering effect. The clear supernatant was diluted to the appropriate concentration (38). The human serum sample was spiked by adding suitable concentration of drug standard solution. Two mL of the spiked serum sample was mixed with 0.5 mL of acetonitrile and centrifuged at 2700 g for 10 min. The resulting supernatant was used for analysis under the optimum conditions. General procedure for chemiluminescence detection
Figure 1. Chemical structure of (a) fluvoxamine, (b) isoniazid and (c) ceftriaxone.
observations, a new simple and sensitive CL method has been developed for the determination of trace amounts of three drugs: FLU, CEF and ISO. This method is simpler and less expensive than other reported techniques and offers good accuracy and precision. The method has also been used to determine FLU, CEF and ISO in tablets and human urine and serum.
CL measurements were carried out in the batch condition. One hundred μL of AY (0.01 mol/L), and 100 μL of NaOH (1.0 mol/L) were added into a 3 mL quartz tube. Then an appropriate volume of sample or standard analyte (FLU, CEF or ISO) solution was added and the final volume was reached to 1.3 mL with the distilled water. After injection of 200 μL of KMnO4 (0.005 mol/L) by an automatic injector, monitoring the CL signal versus time was started automatically. The maximum CL intensity was used as the analytical signal.
Results and discussion Acridine yellow–KMnO4 chemiluminescence system
Materials and methods Materials AY, KMnO4 and NaOH were obtained from Merck (Darmstadt, Germany) and used without further purification. Doubly distilled deionized water (Ghazi Serum Co., Tabriz, Iran) was used throughout. A stock standard solution of 100 mg/L for each analyte (FLU, CEF and ISO, obtained from Sobhan Darou, Tehran, Iran) was prepared in water and stored at 4°C in a sealed volumetric flask.
Permanganate can oxidize AY in basic media and produce a relatively high CL signal (Fig. 2). The oxidation of acridinium esters, which has been extensively studied by McCapra (39), is compatible with aqueous solution at neutral pH. Oxidation by KMnO4 in acid media has only been reported once for AY determination (33). Acidic KMnO4 is a powerful oxidant that can oxidize almost all oxidizable compounds. On the other hand, the CL emission produced is due to excited Mn2+ species that is independent of the oxidized reagents. Therefore, each oxidation reaction in the
15
The CL detection was conducted on LUMAT LB 9507 chemiluminometer (Berthold; www.berthold.com). The CL spectra were measured on a Shimadzu RF-5301 PC spectrofluorometer using a flow mode with the excitation light source being turned off.
10
5
0
Sample preparation
0
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Pharmaceutical tablets were obtained from Tehran Daru (Iran). Five tablets of each drug were weighed and powdered. Three hundred mg of this powder was dissolved in deionized water and filtrated for each drug separately. The filtrate was diluted
wileyonlinelibrary.com/journal/luminescence
CL intensity
Apparatus
25
50
75
100
Time (s) Figure 2. Kinetic curve for KMnO4–acridine yellow CL system, (a) alone and (b) in 3 presence of 1 μmol/L fluvoxamine (KMnO4, 1 × 10 mol/L; acridine yellow, 3 1 × 10 mol/L and NaOH 0.1 mol/L). CL, chemiluminescence.
Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2014; 29: 1053–1058
Alkaline permanganete chemiluminescece
Figure 3. CL spectrum of KMnO4–acridine yellow system in absence (a) and presence of 10 μmol/L fluvoxamine (b). CL, chemiluminescence. (Condition is like Fig. 2.)
Table 1. Effect of some drugs on chemiluminescence emission intensity of acridine yellow–KMnO4 ΔI 1288 1773 2566 538 1258 1183 6475 5684 1283 3698 5480
Drug
ΔI
Drug
Fluoxetine Tramadol Amitriptyline Clomipramine Safrazine Quercetin Alprazolam Amoxicillin Cefazolin Cefixime Clonazepam
364,627 286,695 301,173 2176 2458 8695 5366 9358 8158 3358 3682
Fluvoxamine Isoniazid Ceftriaxone Citalopram Escitalopram Norfluxacine Ofluxacine Iproniazid Ascorbic acid Folic acid Norephinephrine
presence of KMnO4 may result in CL emission and lead to high interferences and low selectivity. This is the reason that KMnO4 CL systems are often applied in the determination of drugs in
simple pharmaceutical systems. However, in alkaline conditions, the CL emission is related to oxidized species or analytes. In addition, the KMnO4 oxidation ability is low and thus interferences are reduced. We found that this method is successfully applied to the determination of drugs in biological samples without considerable interference. We believe that this research will lead to a wide variety of analytical applications for this type of CL reaction. The mechanism of light emission of the acridine-based reactants is very well understood in acidic conditions, which involves the protonation of the acridine and its oxidation by transference of two electrons. The formation of an excited acridone intermediate is proposed, which can emit blue light at 440 nm or transfer energy to other fluoresce molecules (34). However, in alkaline media it has reported that KMnO4 as an oxidizing agent can degrade the acridine forming quinoline-2,3-dicarboxylic acid (40). To determine the luminophor in the systems, the CL emission spectra for AY–KMnO4 and AY–KMnO4–drug systems were recorded (Fig. 3). The spectrum of these systems displayed a broad emission band in the range of 450–650 nm, with a maximum at ∼510 nm, which is similar to the fluorescence spectrum of AY (λem = 510 nm and λex = 450 nm). This reveals that the emitting species in the CL reaction is probably AY. On the other hand, to determine product of AY oxidation by KMnO4, the fluorescence spectrum of AY was plotted after oxidation. An emission band was observed in the range of 400– 500 nm, which is very similar to the fluorescence spectrum of acridone (35). This wavelength range is completely overlapped with the excitation spectrum of AY. Based on these observations, it can be considered that excited product of AY due to oxidation by KMnO4 (M*) can transfer energy to excess AY molecules, which then can emit as light: AY þ MnO4 – þ OH– →M M þ AY→M þ AY AY →AY þ hv ð510 nmÞ The kinetic profiles for the CL reaction in the absence and presence of FLU are shown in Fig. 2. As can be seen, the maximum CL signal was achieved at about 1 s. In addition, it showed that, in the absence of drugs, the CL intensity was relatively high. However, introduction of these drugs into this reaction system resulted in the CL intensity decrease, proportional to the amount of drugs introduced. This effect can be related to the competitive oxidation of drugs and consumption of KMnO4.
Table 2. Analytical characteristics of the proposed method for the determination of FLU, ISO and CEF Parameter Linear range, nmol/L Slope Intercept Correlation coefficient (r) Number of points LOD, nmol/L LOQ, nmol/L RSD% (10 nmol/L, n = 3) RSD% (100 nmol/L, n = 3) RSD% (800 nmol/L, n = 3)
FLU
ISO
CEF
1–1000 10,557 4652 0.9979 11 0.24 0.81 1.4 2.3 1.9
50-40,000 9855 3547 0.9988 11 14 47 2.8 1.4
20-8000 8750 1529 0.9991 11 4.9 17 3.2 1.6
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CEF, ceftriaxone; FLU, fluvoxamine; ISO, isoniazid; LOD, limit of detection; LOQ, limit of quantitation; RSD, relative standard deviation.
J. Abolhasani and J. Hassanzadeh Table 3. Interference study of some coexisting agents in the determination of fluvoxamine Coexisting agent
Tolerable concentration ratios
Ca2+, Mg2+, Fe3+ Na+, K+, Cl, NO3, SO2 4 2+ 2+ PO3 4 , Zn , Cu , vitamins B1 and B2 Oxalate, glucose, ascorbic acid Cysteine
1800 1000 500 150 50
The effect of the AY concentration over the range of 1 × 104– 3 × 103 mol/L was investigated to determine the maximal emission intensity. The highest CL intensity was obtained at a concentration of AY of 0.001 mol/L, which did not improve upon increasing the AY concentrations. Hence, 0.001 mol/L of AY was selected as the optimum concentration. To investigate the effect of the KMnO4 concentration, solutions with different concentrations of KMnO4 were prepared over the range of 1 × 104–5 × 103 mol/L. The CL signal was increased up to 0.001 mol/L and then did not increase further at high concentrations. At lower KMnO4 concentrations, the number of excited intermediates was decreased and the response was diminished.
Optimization of experimental parameters The optimum conditions for the FLU, CEF and ISO determinations were carried out using the 1 × 107 mol/L concentration of drug. In the proposed system, the CL intensity was mainly dependent on the concentration of NaOH. Therefore, to examine the effect of this parameter on the CL signal, NaOH at different concentrations was tested. The maximum CL response was obtained at 0.1 mol/L NaOH for all three drugs and higher concentrations had no significant effect.
Analytical parameters The effect of some drugs on the CL emission of AY–KMnO4 was studied. The results, which are shown in Table 1, show that many tested drugs had a minor diminishing effect, but only few drugs such as FLU, CEF and ISO had a major effect on light emission (Fig. 2). To determine drug concentration, the difference between the CL intensity (ΔI = I0 – I) in the absence (I0) and presence of each drug (I) was plotted versus the concentration of drug in
Table 4. Results for the determination of fluvoxamine, isoniazid and ceftriaxone in pharmaceutical and biological samples using the proposed method Sample Pharmaceutical samples
Fluvoxamine maleate (25 mg)
Isoniazid (100 mg)
Ceftriaxone (250 mg per 1 mL injection)
Urine samples
Isoniazid
Ceftriaxone
Fluvoxamine
Serum samples
Isoniazid
Ceftriaxone
Fluvoxamine
Adda
Foundb
Recovery %
t-statisticc
0 5.0 10.0 15.0 0 25 50 100 0 50 100 150 1.0 2.5 5.0 1.0 2.5 5.0 1.0 2.5 5.0 0.20 0.50 1.0 0.20 0.50 1.0 0.20 0.50 1.0
24.7 ± 0.3 295 ± 0.2 34.3 ± 0.2 39.4 ± 0.2 98.0 ± 1.6 123 ± 1.0 147 ± 1.1 197 ± 1.3 241 ± 4.0 288 ± 2.2 342 ± 2.2 394 ± 3.2 0.98 ± 0.03 2.44 ± 0.03 4.88 ± 0.06 1.02 ± 0.04 2.45 ± 0.03 5.02 ± 0.08 0.99 ± 0.03 2.46 ± 0.03 4.98 ± 0.08 0.20 ± 0.01 0.49 ± 0.01 0.97 ± 0.02 0.20 ± 0.01 0.49 ± 0.01 1.0 ± 0.04 0.19 ± 0.01 0.50 ± 0.01 0.99 ± 0.04
– 97 ± 3.1 96 ± 2.0 98 ± 1.4 – 101 ± 4.1 97 ± 2.1 100 ± 1.3 – 103 ± 2.2 103 ± 2.2 103 ± 2.1 98.3 ± 2.5 97.7 ± 1.2 97.6 ± 1.2 102 ± 3.5 98.1 ± 1.2 100 ± 1.6 98.7 ± 3.1 99.6 ± 1.2 99.6 ± 1.6 98.2 ± 3.8 97.1 ± 1.7 96.7 ± 1.5 99.7 ± 2.8 98.8 ± 1.8 101.7 ± 3.5 95.5 ± 2.3 99.7 ± 1.7 98.7 ± 3.8
2.3 1.7 3.6 2.9 2.2 0.6 2.0 0.4 3.8 1.0 2.0 2.3 1.1 3.2 3.3 0.8 2.6 0.4 0.8 2.3 0.42 0.68 2.4 3.1 0.17 0.93 0.67 2.8 0.22 0.50
a
The unit for added and found amounts is mg for tablet and mg/L for urine and serum samples. Mean of three determinations ± SD. c t-critical = 4.3 for n = 2 and P = 0.05. b
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Luminescence 2014; 29: 1053–1058
Alkaline permanganete chemiluminescece μmol/L. The analytical parameters for each of these drugs are summarized in Table 2. The results indicated that this CL system had good linearity, relatively high sensitivity and good precision. It should also be noted that AY–KMnO4 is a new CL system, which can have potential applications in the determination of some other important compounds.
Interference studies To test for interference, some potentially interfering substances in increasing amounts were added into a standard solution of 0.1 μmol/L for each drug. The tolerable concentration ratios for interferences with the relative error of
