Research article Received: 19 January 2014,
Revised: 12 March 2014,
Accepted: 23 March 2014
Published online in Wiley Online Library: 7 May 2014
(wileyonlinelibrary.com) DOI 10.1002/bio.2685
Flow injection determination of diclofenac sodium based on its sensitizing effect on the chemiluminescent reaction of acidic potassium permanganate–formaldehyde Jingjing Song,a,b Pulv Sun,a,b Zhongling Jic and Jianguo Lia,b* ABSTRACT: A sensitive and simple chemiluminescent (CL) method for the determination of diclofenac sodium has been developed by combining the flow injection technique and its sensitizing effect on the weak CL reaction between formaldehyde and acidic potassium permanganate. A calibration curve is constructed for diclofenac sodium under optimized experimental parameters over the range 0.040–5.0 μg/mL and the limit of detection is 0.020 μg/mL (3σ). The inter-assay relative standard deviation for 0.040 μg/mL diclofenac sodium (n = 11) is 2.0%. This method is rapid, sensitive, simple, and shows good selectivity and reproducibility. The proposed method has been successfully applied to the determination of the studied diclofenac sodium in pharmaceutical preparations with satisfactory results. Furthermore, the possible mechanism for the CL reaction has been discussed in detail on the basis of UV and CL spectra. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: flow-injection; chemiluminescence; diclofenac sodium; potassium permanganate; formaldehyde
Introduction
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Diclofenac sodium (DS), [2–2,6-dichlorophenyl amino]-sodium phenylacetate (Fig. 1) is a new type of non-steroidal antiinflammatory drug (NSAID) advocated for use in painful and inflammatory rheumatic and certain non-rheumatic conditions. DS is the preferred treatment for acute and chronic pain and inflammatory conditions. It is also widely used in veterinary medicine in the treatment of food-producing animals, especially dairy animals (1). However, NSAIDs can cause adverse health effects in humans such as aplastic anemia, gastrointestinal disorders and agranulocytosis, and changes in renal function (1,2). Different analytical procedures have been proposed for the determination of DS, for example: liquid chromatography (LC) (3–8), thin-layer chromatography (TLC) (3,9,10), electrochemistry (11), capillary electrophoresis (12,13), Fourier transform (FT)-Raman spectrometry (14,15), gas chromatography-mass spectrometry (GC-MS) (3), flow injection analysis (16,17) and continuous injection-potential measurement and continuous injectionfluorescence measurement (18). However, each of the above methods had its own disadvantages, for example, chromatographic techniques are slow and expensive, and some spectroscopic techniques are time consuming and laborious. Therefore, it is very important to develop a rapid, simple, sensitive and accurate method for the determination of DS in order to obtain optimal therapeutic concentrations for quality assurance in pharmaceutical preparations. Chemiluminescence (CL) is the production of light from a chemical reaction when the vibronically excited product of an exoergic reaction relaxes to its ground state with the emission of photons. It is a chemical reaction that emits light without the need for an external energy supply. CL is an attractive means
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of detection for trace analysis in various fields (19–26), especially combined with flow injection. It has many advantages for pharmaceutical determinations such as high sensitivity, high selectivity, wonderful reproducibility, low levels of chemical consumption, cost effectiveness, simple sample preparation and instrumentation. These features make it superior to other detection principles. To our knowledge, no work on the CL behavior, reaction mechanism and sensitive determination of DS by flow injection chemiluminescence (FI–CL) has been previously reported, except for a study in which DS acts as a sensitizer for the determination of DS (27). The study deals with the development of a simple, rapid and sensitive method for the determination of DS in its pure form and in its pharmaceutical preparations. We found that a weak CL signal was produced on the oxidation of formaldehyde by potassium permanganate in acidic solution, and that the weak CL signal was remarkably increased in the presence of DS. Based on these observations, a new FI–CL method has been developed for the determination of trace amount of DS. Combined with flow injection, this effect provides a sensitive and convenient * Correspondence to: J.-G. Li, College of Chemistry, Chemical Engineering and materials science, Soochow University, Suzhou 215123, People’s Republic of China. E-mail: [email protected] a
College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, People’s Republic of China
b
The Key Lab of Health Chemistry and Molecular Diagnosis of Suzhou, Suzhou 215123, People’s Republic of China
c
School of Chemistry Biology and Material Engineering, Suzhou University of Science and Technology, Suzhou 215009, People’s Republic of China
Copyright © 2014 John Wiley & Sons, Ltd.
Flow injection CL determination of diclofenac sodium
Figure 1. Chemical structure of DS.
method for the determination of DS in pharmaceutical preparations. It has been used to determine DS in tablets and capsules, and the possible CL reaction mechanism has also been discussed on the basis of UV and CL spectra.
Experimental
injection was made via a six-way injection valve fitted with a sample loop of 60 μL. The mixed liquid flow cell was placed close to the photomultiplier tube (PMT; CR-105, Hamamatsu, Beijing, China). The CL signal was detected by the PMT with no wavelength discrimination and recorded by a computer using IFFM-E FI–CL analysis system software. The CL spectra were recorded using a computer-controlled Acton SP2300 CL spectrometer with a PIXIS100 CCD detector (Princeton Instrument Corporation, Princeton, NJ, USA). The fluorescence and absorption spectra were monitored using a F-4500 fluorescence spectrometer (Hitachi, Tokyo, Japan) and a Shimadzu UV—2450 UV/vis recording spectrophotometer (Shimadzu, Kyoto, Japan), respectively. Procedure
Reagents and chemicals Pure powder of DS was obtained from Nanjing Institute for Drug Control (Nanjing, China). Potassium permanganate, perchloric acid and formaldehyde were purchased from Shanghai Chemical Reagent Limited Company (Shanghai, China). All other reagents and chemicals were commercially available and of analytical reagent (AR) grade. All solutions were prepared with sub-boiling distilled deionized water. A standard solution of DS (1.000 mg/mL) was prepared by dissolving 0.5000 g of DS in water, diluting the solution to 50 mL in a volumetric flask with water, and protecting the sample from light. A stock solution (0.01 mol/L) of potassium permanganate was prepared in water (by dissolving in boiled water and filtering through glass wool) and protected from light. Perchloric acid solution (3.0 mol/L) and formaldehyde (3.0%) (v/v) were also prepared. These standard solutions were stored in a refrigerator at 4°C). All reagents were analytical grade, and all solutions were prepared using sub-boiling distilled deionized water.
Apparatus All CL measurements were carried out using an IFFM-E flow injection chemiluminescence analyzer (Remex Electronic Institute Limited Co., Xi’an, China) equipped with an IFFS-A multifunction chemiluminescence detector (Remex Electronic Institute Limited Co., Xi’an, China). A schematic diagram of the flow system used in this work is shown in Fig. 2. Two peristaltic pumps were used to deliver the flow streams, one to deliver the sample (DS) and formaldehyde at a flow rate of 1.5 mL/min (P1), and the other to deliver the acidic potassium permanganate (KMnO4 and HClO4) at a flow rate of 4.0 mL/min (P2). PTFE tubing (0.8 mm i.d.) was used to connect all the components in the flow system. Sample
The FI manifold was designed and fabricated as shown in Fig. 2. The solutions of DS, formaldehyde, potassium permanganate and perchloric acid were pumped continuously into the mixing element using two peristaltic pumps. DS and formaldehyde were pumped into the six-way valve after mixing with acidic potassium permanganate. The final stream was introduced into the flow CL cell. The full CL intensity vs time curve was then recorded. The concentration of DS was quantified by the CL intensity (peak height).
Results and discussion Kinetic characteristics of the CL reaction The kinetic characteristics were studied using a stop-flow injection method after the baseline had been steadily recorded. Formaldehyde (3.0% v/v) was mixed with 3.0 mol/L perchloric acid solution containing 6.0 × 105 mol/L potassium permanganate and the CL kinetic curve (curve b in Fig. S1) was simultaneously recorded using the IFFM-E luminometer. Curve a in Fig. S1 was the CL kinetic curve obtained when a mixture of 5.0 μg/mL of DS and 3.0% (v/v) formaldehyde was injected into 1.6 mol/L sulfuric acid solution containing 6.0 × 105 mol/L KMnO4. Changes in the CL intensity were seen after different solutions had flowed through the column. The CL intensity in the presence of DS (curve a) was approximately five times that in the absence of DS (curve b). Furthermore, it was found that the rate of the reaction was so fast that the CL intensity reached a peak only 2 s after reagent mixing, and it took ~ 4 s for the signal to return to the base line. The results indicate that the weak CL signal of KMnO4–HCHO increased remarkably in the presence of DS. Optimization of the experimental conditions
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Figure 2. Schematic diagram of CL flow system: a, diclofenac sodium; b, formaldehyde; c, acidic potassium permanganate; P1/P2, peristaltic pump; V, sixway injection valve; F, chemiluminescence flow cell; HV, negative high voltage supply; AMP, amplitude; R, recorder; W, waste solution.
The influence of chemical variables affecting the CL reaction was studied by controlling various parameters. The variables were reagent concentration, the conditions of the reaction medium and reagent flow rate. The first variable to be examined was the potassium permanganate concentration (Fig. S2), the effect of which was studied over a concentration range from 0.20 to 2.0 mol/L. The CL signal increased with the increase in the potassium permanganate concentration up to 6.0 × 105 mol/L, above which it showed an abrupt decrease. The intensity of the chemical reaction is heightened on increasing the concentration of potassium permanganate, which leads to signal enhancement. When the
J. Song et al. concentration of potassium permanganate reaches a certain level, there is no extra reducing substance in the system and the intensity of the CL signal will not change. Therefore, an oxidant concentration of 6.0 × 105 mol/L was selected as optimal for subsequent experiments. The KMnO4–DS system could only produce a weak CL emission. Various compounds, such as Rhodamine B, HCHO, HCOOH, H2O2, Na2SO3, Na2S2O3 and Na2S2O4, were tested as sensitizers for the CL system of KMnO4–DS. It was found that only HCHO and HCOOH enhanced the CL signal in the KMnO4–DS system, which is in agreement with the results reported by Townshend and co-workers (28,29). The relationship between the concentration of formaldehyde and the CL intensity was then tested. The results are shown in Fig. S3. The relative CL intensity increased as the concentration of formaldehyde increased, and trended to a plateau at 3.0% (v/v). Because HCOOH is a sensitizer for the system, the signals increase with the concentration of HCOOH. Thus, 3.0% (v/v) formaldehyde was optimal in further experiments. The variety and concentration of acids in the reaction system influence the CL intensity. Therefore, the CL emission intensities of 5.0 μg/mL of DS, 3.0% (v/v) formaldehyde, and 6.0 × 105 mol/L of potassium permanganate were tested in the presence of four different, namely HNO3, HClO4, H3PO4 and H2SO4. The experimental results showed that the strongest signal was obtained with HClO4. On increasing the concentration of perchloric acid, the CL intensity increased and reached a maximum value at 3.0 mol/L (Fig. 3). Potassium permanganate has strong oxidizing ability in an acidic medium. Therefore, as the concentration of HClO4 increased, the CL signal was enhanced. Nevertheless, perchloric acid at high concentrations also has an oxidizing ability, which would consume the reducing substances in the system. Thus, 3.0 mol/L of perchloric acid was selected as the acidic medium for the potassium permanganate solution. Flow rate is an important factor in CL detection as the time taken to transfer the excited product into the flow cell for maximum collection of the emitted light. In order to achieve a maximum CL intensity for the 5.0 μg/mL DS, 3.0% (v/v) formaldehyde, 3.0 mol/L perchloric acid solution and 6.0 × 105 mol/L
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Figure 3. Relationship between HClO4 concentration and relative CL intensity of 5 6.0 × 10 mol/L KMnO4–3.0% (V/V) HCHO–5.0 μg/mL diclofenac sodium.
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KMnO4, flow rates were studied in the range of 1.0–6.0 mL/min for each channel of both pumps. When the flow rate for P2 was 4.0 mL/min, the relative CL intensity, reproducibility of the signal, peak shape and signal-to-noise ratio were optimal. Therefore, a P2 flow rate of 4.0 mL/min was employed throughout the experiment. Since the sampling time is sufficient, the CL signal is unchanged when the flow rates of P1 exceeded 1.5 mL/min. The considering the reagent consumption, 1.5 mL/min was chosen as the flow rate of the sample and formaldehyde solution. At the flow rate of 1.5 mL/min, the determination of DS, including sampling and washing, could be performed in 30s, giving a sample measurement frequency of about 120 sample injections per hour. Accordingly, the solutions consumption per analysis was about 1.5 mL. In FI analysis, it is necessary to optimize the injection volume to achieve the desired sensitivity. The influence of sample injection volume on CL intensity was tested using 40, 60, 80, 100 and 120 μL of 2.0 μg/mL DS. The highest relative CL intensity and the best signal-to-noise ratio were obtained when the sample injection volume was 60 μL, this quantity was injected into the carrier stream.
Analytical characteristics A calibration curve (Fig. 4) was constructed for the DS assay under optimized experimental parameters over the range 0.040–5.0 μg/mL. Fig. 4 shows the FI–CL signals for DS. It was found that relative CL intensity increased linearly with increasing DS concentration. The linear regression equation was ΔI (relative units) = 1.828 c (108g/ml) 6.989 (r = 0.9995, n = 7). The limit of detection (LOD) for DS was 0.020 μg/mL (3σ). The intra-assay relative standard deviation (RSD) for 11 repetitive determinations at 0.40 μg/mL DS was 1.43%, showing good reproducibility. The inter-assay RSD for 0.040 μg/mL DS (n = 11) was 2.0%.
Interference Common excipients used in pharmaceutical preparations of DS and metal ions in the human body were tested as potential interferents. A series of solutions containing potential interfering
Figure 4. Calibration graphs of diclofenac sodium.
Copyright © 2014 John Wiley & Sons, Ltd.
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Flow injection CL determination of diclofenac sodium compounds and 1.0 μg/mL DS were prepared. The CL signals of the prepared solutions were recorded and compared with that obtained for standard DS solution (1.0 μg/mL). The tolerance limit was taken as the amount which caused a relative error of ± 5% in the peak height. The results are listed in Table 1. Some ions and the studied excipients in tablets did not interfere with the determination of DS in this system. The results indicated that this method can be used for the determination of DS in pharmaceutical preparations.
Application The proposed method was successfully applied to the determination of DS in a commercial pharmaceutical formulation. The DS tablets and capsules were bought from a local market in Suzhou. They were prepared as described in the Chinese Pharmacopoeia (30). A solution of DS pharmaceutical preparations was prepared as follows: three tablets (or the contents of four DS capsules) were finely ground, homogenized and a portion of the powder equivalent to 300 mg of DS was weighed accurately and diluted with 70 mL of ethanol. The mixture was vibrated and filtered. Two milliliters of filtrate was diluted further with ethanol to 100 mL. The sample solution was diluted appropriately with water so that the final concentration of DS was within the detection range. To evaluate the reliability and the accuracy of the method, certain quantities of standard DS solution were added to different batches of sample solutions and analyzed using the proposed method. The recoveries (shown in Table 2) of DS in different batches of tablets and capsules were calculated by comparing the results obtained before and after the addition of standard DS solution. The recoveries were all between 98 and 103%, indicating that the results were accurate. The t-test (listed in Table 3) indicated that there were no significant differences between the results obtained using the proposed method
Table 1. Tolerance of different substances in the determination of 1.0 μg/mL diclofenac sodium Interferent
Maxium tolerable ratio (relative error ± 5% )
Al3+, SO42, Cu2+, NH4+ Zn2+, Mg2+, Ca2+, Cl, CH3COO, PO43 Starch, glucose, sucrose, Pb2+ Citric acid, β-cyclodextrin, tryptophan Tyrosine, ascorbic aid, lactose
500 250 100 50 10
and those obtained using the Chinese Pharmacopoeia method (30) at a confidence level of 95%. Possible CL mechanism To obtain more information about the enhanced CL mechanism, we designed two types of detection method. First of all, the CL spectra (shown in Fig. 5) were determined about KMnO4–HCHO (curve a) and KMnO4–HCHO–DS (curve b). As shown in Fig. 5, the two CL systems possessed parallel CL spectra and had a maximum spectral peak at 710 ± 5 nm, revealing that the adding of DS could increase the CL signal without changing the CL process. The fluorescence spectra of the KMnO4–HCHO system and reaction products were scanned within the range 250–700 nm using a fluorescence spectrometer. No fluorescence could be found, highlighting that DS can not produce fluorescence. In order to investigate the possible reaction mechanism of CL enhancement, UV/vis absorption spectra of DS solution (curve a), KMnO4–HClO4 (curve b), KMnO4–HClO4–DS (curve c) and KMnO4–HClO4–DS (curve d) were recorded, as shown in Fig. 6. The absorption spectrum showed that DS has stronger absorption in 273 nm (curve a), whereas manganese(VII) has three characteristic absorption peaks at 309, 523 and 544 nm (as shown by curve b), respectively. The absorbance peaks of manganese(VII) decrease significantly when DS is added, although an absorption peak appears at 246 nm (curve c). This means that the concentration of MnO4 was diminished and a new intermediate was formed in the solution. The absorption peak at 246 nm increased remarkably in the presence of DS and formaldehyde (curve d). It can be concluded that formaldehyde promoted the CL reaction. These results indicated that an oxidation–reduction reaction occurred between DS and the potassium permanganate–perchloric acid–formaldehyde system, and energy was released, the reactant disappeared and samples corresponding oxidation products were generated. Electronically excited ‘singlet oxygen’ (1O2) species are thought to be generated in many CL systems (31). It has been suggested previously (32–34) that KMnO4 could react with some reductants in the presence of formaldehyde or formic acid to produce 1O12O2 (1Δ1gΔg). The principle emission bands of 1O2 occur in the near-infrared (λmax = 1270 nm) from a monomeric species (1Δg → 3Σg;) and in the visible region (λmax = 703 nm) from its dimeric aggregate [(1Δg)ν=0(1Δg)ν=0 → (3Σg)ν=1(3Σg)ν=0 and (1Δg)ν=0(1Δg)ν=0 → (3Σg)ν=0(3Σg)ν=0]. CL spectra were seen at 710 ± 5nm, which are similar to singlet oxygen chemiluminescence (35–39). Thus, it is possible that the singlet excited molecular oxygen species is an emitter in the present system, formed by the transfer of energy from oxidized DS to
Table 2. Results of recovery from different batches of diclofenac sodium Sample no.
Sample (μg/mL)
Found (μg/mL)
H20090492
0.20
0.21
H20067776
0.40
0.39
Diclofenac sodium added (μg/mL) 0.0 0.20 0.40 0.0 0.20 0.40
Diclofenac sodium found (μg/mL) 0.2 0.39 0.62 0.41 0.59 0.79
Recovery ± RSD (%) 100 ± 6.2 98 ± 6.0 103 ± 1.6 102 ± 0.7 98 ± 1.7 98 ± 2.1
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J. Song et al. Table 3. Comparative results for the determination of diclofenac sodium in different methods Samples H20090492 H20067776
Nominal content Proposed method RSD (%, n = 5) Titrimetry method RSD (%, n = 5) (mg/capsule or mg/tablet) (mg/capsule or mg/tablet) (mg/capsule or mg/tablet) 75.0 100.0
74.3 98.2
1.76 1.02
75.6 97.7
0.93 1.45
Conclusion Diclofenac sodium can significantly enhance the CL intensity of an acidic potassium permanganate–formaldehyde system, and based on this, a sensitive and simple flow-injection CL method was established. The experimental conditions affecting the CL reaction were optimized and the analytical characteristics for the determination of DS are presented here. The possible CL mechanism was also studied in detail based on the CL and UV spectra. The proposed method has been applied to the determination of DS in tablets and capsules with satisfactory results. This method is practical and valuable in clinical and biochemical laboratories for the determination of DS. Acknowledgements 5
Figure 5. CL spectrum: a, 6.0 × 10 mol/L KMnO4–3.0 mol/L HClO4–3.0% HCHO; 5 b, 6.0 × 10 mol/L KMnO4–3.0 mol/L HClO4–15.0 μg/mL diclofenac sodium–3.0% HCHO.
This work was supported by the Science Fund from the National Natural Science Foundation of China (No. 21075087), the Project of Scientific and Technologic Infrastructure of Suzhou (SZS201207), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
References
5
Figure 6. UV spectra of: a, DS (5.0 μg/mL); b, KMnO4 (6.0 × 10 mol/L)–HClO4 5 (3.0 mol/L); c, KMnO4 (6.0 × 10 mol/L)–HClO4 (3.0 mol/L)–DS (5.0 μg/mL); d, 5 KMnO4 (6.0 × 10 mol/L)–HClO4 (3.0 mol/L)–DS (5.0 μg/mL)–HCHO (3.0%).
dissolved oxygen. Based on the above discussion and previous reports (32–34), a possible CL mechanism could be expressed as follows:
36
MnO4 þ Hþ þ HCHO þ DS→H2 O þ Mn2þ þoxidized DS intermediate; 3 O2 ð3 ΣgÞ þ oxidized DS intermediate→ 1 O2 ð1 ΔgÞ 1 þoxidized DS product; 21 O2 ð1 ΔgÞ→ 1 O2 1 O2 1 Δg1 Δg ; O2 1 O2 1 Δg1 Δg →23 O2 ð3 ΣgÞ þ hνðλmax ¼ 710±5 nmÞ
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Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web site.
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Revised: 12 March 2014,
Accepted: 23 March 2014
Published online in Wiley Online Library: 7 May 2014
(wileyonlinelibrary.com) DOI 10.1002/bio.2685
Flow injection determination of diclofenac sodium based on its sensitizing effect on the chemiluminescent reaction of acidic potassium permanganate–formaldehyde Jingjing Song,a,b Pulv Sun,a,b Zhongling Jic and Jianguo Lia,b* ABSTRACT: A sensitive and simple chemiluminescent (CL) method for the determination of diclofenac sodium has been developed by combining the flow injection technique and its sensitizing effect on the weak CL reaction between formaldehyde and acidic potassium permanganate. A calibration curve is constructed for diclofenac sodium under optimized experimental parameters over the range 0.040–5.0 μg/mL and the limit of detection is 0.020 μg/mL (3σ). The inter-assay relative standard deviation for 0.040 μg/mL diclofenac sodium (n = 11) is 2.0%. This method is rapid, sensitive, simple, and shows good selectivity and reproducibility. The proposed method has been successfully applied to the determination of the studied diclofenac sodium in pharmaceutical preparations with satisfactory results. Furthermore, the possible mechanism for the CL reaction has been discussed in detail on the basis of UV and CL spectra. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: flow-injection; chemiluminescence; diclofenac sodium; potassium permanganate; formaldehyde
Introduction
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Diclofenac sodium (DS), [2–2,6-dichlorophenyl amino]-sodium phenylacetate (Fig. 1) is a new type of non-steroidal antiinflammatory drug (NSAID) advocated for use in painful and inflammatory rheumatic and certain non-rheumatic conditions. DS is the preferred treatment for acute and chronic pain and inflammatory conditions. It is also widely used in veterinary medicine in the treatment of food-producing animals, especially dairy animals (1). However, NSAIDs can cause adverse health effects in humans such as aplastic anemia, gastrointestinal disorders and agranulocytosis, and changes in renal function (1,2). Different analytical procedures have been proposed for the determination of DS, for example: liquid chromatography (LC) (3–8), thin-layer chromatography (TLC) (3,9,10), electrochemistry (11), capillary electrophoresis (12,13), Fourier transform (FT)-Raman spectrometry (14,15), gas chromatography-mass spectrometry (GC-MS) (3), flow injection analysis (16,17) and continuous injection-potential measurement and continuous injectionfluorescence measurement (18). However, each of the above methods had its own disadvantages, for example, chromatographic techniques are slow and expensive, and some spectroscopic techniques are time consuming and laborious. Therefore, it is very important to develop a rapid, simple, sensitive and accurate method for the determination of DS in order to obtain optimal therapeutic concentrations for quality assurance in pharmaceutical preparations. Chemiluminescence (CL) is the production of light from a chemical reaction when the vibronically excited product of an exoergic reaction relaxes to its ground state with the emission of photons. It is a chemical reaction that emits light without the need for an external energy supply. CL is an attractive means
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of detection for trace analysis in various fields (19–26), especially combined with flow injection. It has many advantages for pharmaceutical determinations such as high sensitivity, high selectivity, wonderful reproducibility, low levels of chemical consumption, cost effectiveness, simple sample preparation and instrumentation. These features make it superior to other detection principles. To our knowledge, no work on the CL behavior, reaction mechanism and sensitive determination of DS by flow injection chemiluminescence (FI–CL) has been previously reported, except for a study in which DS acts as a sensitizer for the determination of DS (27). The study deals with the development of a simple, rapid and sensitive method for the determination of DS in its pure form and in its pharmaceutical preparations. We found that a weak CL signal was produced on the oxidation of formaldehyde by potassium permanganate in acidic solution, and that the weak CL signal was remarkably increased in the presence of DS. Based on these observations, a new FI–CL method has been developed for the determination of trace amount of DS. Combined with flow injection, this effect provides a sensitive and convenient * Correspondence to: J.-G. Li, College of Chemistry, Chemical Engineering and materials science, Soochow University, Suzhou 215123, People’s Republic of China. E-mail: [email protected] a
College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, People’s Republic of China
b
The Key Lab of Health Chemistry and Molecular Diagnosis of Suzhou, Suzhou 215123, People’s Republic of China
c
School of Chemistry Biology and Material Engineering, Suzhou University of Science and Technology, Suzhou 215009, People’s Republic of China
Copyright © 2014 John Wiley & Sons, Ltd.
Flow injection CL determination of diclofenac sodium
Figure 1. Chemical structure of DS.
method for the determination of DS in pharmaceutical preparations. It has been used to determine DS in tablets and capsules, and the possible CL reaction mechanism has also been discussed on the basis of UV and CL spectra.
Experimental
injection was made via a six-way injection valve fitted with a sample loop of 60 μL. The mixed liquid flow cell was placed close to the photomultiplier tube (PMT; CR-105, Hamamatsu, Beijing, China). The CL signal was detected by the PMT with no wavelength discrimination and recorded by a computer using IFFM-E FI–CL analysis system software. The CL spectra were recorded using a computer-controlled Acton SP2300 CL spectrometer with a PIXIS100 CCD detector (Princeton Instrument Corporation, Princeton, NJ, USA). The fluorescence and absorption spectra were monitored using a F-4500 fluorescence spectrometer (Hitachi, Tokyo, Japan) and a Shimadzu UV—2450 UV/vis recording spectrophotometer (Shimadzu, Kyoto, Japan), respectively. Procedure
Reagents and chemicals Pure powder of DS was obtained from Nanjing Institute for Drug Control (Nanjing, China). Potassium permanganate, perchloric acid and formaldehyde were purchased from Shanghai Chemical Reagent Limited Company (Shanghai, China). All other reagents and chemicals were commercially available and of analytical reagent (AR) grade. All solutions were prepared with sub-boiling distilled deionized water. A standard solution of DS (1.000 mg/mL) was prepared by dissolving 0.5000 g of DS in water, diluting the solution to 50 mL in a volumetric flask with water, and protecting the sample from light. A stock solution (0.01 mol/L) of potassium permanganate was prepared in water (by dissolving in boiled water and filtering through glass wool) and protected from light. Perchloric acid solution (3.0 mol/L) and formaldehyde (3.0%) (v/v) were also prepared. These standard solutions were stored in a refrigerator at 4°C). All reagents were analytical grade, and all solutions were prepared using sub-boiling distilled deionized water.
Apparatus All CL measurements were carried out using an IFFM-E flow injection chemiluminescence analyzer (Remex Electronic Institute Limited Co., Xi’an, China) equipped with an IFFS-A multifunction chemiluminescence detector (Remex Electronic Institute Limited Co., Xi’an, China). A schematic diagram of the flow system used in this work is shown in Fig. 2. Two peristaltic pumps were used to deliver the flow streams, one to deliver the sample (DS) and formaldehyde at a flow rate of 1.5 mL/min (P1), and the other to deliver the acidic potassium permanganate (KMnO4 and HClO4) at a flow rate of 4.0 mL/min (P2). PTFE tubing (0.8 mm i.d.) was used to connect all the components in the flow system. Sample
The FI manifold was designed and fabricated as shown in Fig. 2. The solutions of DS, formaldehyde, potassium permanganate and perchloric acid were pumped continuously into the mixing element using two peristaltic pumps. DS and formaldehyde were pumped into the six-way valve after mixing with acidic potassium permanganate. The final stream was introduced into the flow CL cell. The full CL intensity vs time curve was then recorded. The concentration of DS was quantified by the CL intensity (peak height).
Results and discussion Kinetic characteristics of the CL reaction The kinetic characteristics were studied using a stop-flow injection method after the baseline had been steadily recorded. Formaldehyde (3.0% v/v) was mixed with 3.0 mol/L perchloric acid solution containing 6.0 × 105 mol/L potassium permanganate and the CL kinetic curve (curve b in Fig. S1) was simultaneously recorded using the IFFM-E luminometer. Curve a in Fig. S1 was the CL kinetic curve obtained when a mixture of 5.0 μg/mL of DS and 3.0% (v/v) formaldehyde was injected into 1.6 mol/L sulfuric acid solution containing 6.0 × 105 mol/L KMnO4. Changes in the CL intensity were seen after different solutions had flowed through the column. The CL intensity in the presence of DS (curve a) was approximately five times that in the absence of DS (curve b). Furthermore, it was found that the rate of the reaction was so fast that the CL intensity reached a peak only 2 s after reagent mixing, and it took ~ 4 s for the signal to return to the base line. The results indicate that the weak CL signal of KMnO4–HCHO increased remarkably in the presence of DS. Optimization of the experimental conditions
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Figure 2. Schematic diagram of CL flow system: a, diclofenac sodium; b, formaldehyde; c, acidic potassium permanganate; P1/P2, peristaltic pump; V, sixway injection valve; F, chemiluminescence flow cell; HV, negative high voltage supply; AMP, amplitude; R, recorder; W, waste solution.
The influence of chemical variables affecting the CL reaction was studied by controlling various parameters. The variables were reagent concentration, the conditions of the reaction medium and reagent flow rate. The first variable to be examined was the potassium permanganate concentration (Fig. S2), the effect of which was studied over a concentration range from 0.20 to 2.0 mol/L. The CL signal increased with the increase in the potassium permanganate concentration up to 6.0 × 105 mol/L, above which it showed an abrupt decrease. The intensity of the chemical reaction is heightened on increasing the concentration of potassium permanganate, which leads to signal enhancement. When the
J. Song et al. concentration of potassium permanganate reaches a certain level, there is no extra reducing substance in the system and the intensity of the CL signal will not change. Therefore, an oxidant concentration of 6.0 × 105 mol/L was selected as optimal for subsequent experiments. The KMnO4–DS system could only produce a weak CL emission. Various compounds, such as Rhodamine B, HCHO, HCOOH, H2O2, Na2SO3, Na2S2O3 and Na2S2O4, were tested as sensitizers for the CL system of KMnO4–DS. It was found that only HCHO and HCOOH enhanced the CL signal in the KMnO4–DS system, which is in agreement with the results reported by Townshend and co-workers (28,29). The relationship between the concentration of formaldehyde and the CL intensity was then tested. The results are shown in Fig. S3. The relative CL intensity increased as the concentration of formaldehyde increased, and trended to a plateau at 3.0% (v/v). Because HCOOH is a sensitizer for the system, the signals increase with the concentration of HCOOH. Thus, 3.0% (v/v) formaldehyde was optimal in further experiments. The variety and concentration of acids in the reaction system influence the CL intensity. Therefore, the CL emission intensities of 5.0 μg/mL of DS, 3.0% (v/v) formaldehyde, and 6.0 × 105 mol/L of potassium permanganate were tested in the presence of four different, namely HNO3, HClO4, H3PO4 and H2SO4. The experimental results showed that the strongest signal was obtained with HClO4. On increasing the concentration of perchloric acid, the CL intensity increased and reached a maximum value at 3.0 mol/L (Fig. 3). Potassium permanganate has strong oxidizing ability in an acidic medium. Therefore, as the concentration of HClO4 increased, the CL signal was enhanced. Nevertheless, perchloric acid at high concentrations also has an oxidizing ability, which would consume the reducing substances in the system. Thus, 3.0 mol/L of perchloric acid was selected as the acidic medium for the potassium permanganate solution. Flow rate is an important factor in CL detection as the time taken to transfer the excited product into the flow cell for maximum collection of the emitted light. In order to achieve a maximum CL intensity for the 5.0 μg/mL DS, 3.0% (v/v) formaldehyde, 3.0 mol/L perchloric acid solution and 6.0 × 105 mol/L
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Figure 3. Relationship between HClO4 concentration and relative CL intensity of 5 6.0 × 10 mol/L KMnO4–3.0% (V/V) HCHO–5.0 μg/mL diclofenac sodium.
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KMnO4, flow rates were studied in the range of 1.0–6.0 mL/min for each channel of both pumps. When the flow rate for P2 was 4.0 mL/min, the relative CL intensity, reproducibility of the signal, peak shape and signal-to-noise ratio were optimal. Therefore, a P2 flow rate of 4.0 mL/min was employed throughout the experiment. Since the sampling time is sufficient, the CL signal is unchanged when the flow rates of P1 exceeded 1.5 mL/min. The considering the reagent consumption, 1.5 mL/min was chosen as the flow rate of the sample and formaldehyde solution. At the flow rate of 1.5 mL/min, the determination of DS, including sampling and washing, could be performed in 30s, giving a sample measurement frequency of about 120 sample injections per hour. Accordingly, the solutions consumption per analysis was about 1.5 mL. In FI analysis, it is necessary to optimize the injection volume to achieve the desired sensitivity. The influence of sample injection volume on CL intensity was tested using 40, 60, 80, 100 and 120 μL of 2.0 μg/mL DS. The highest relative CL intensity and the best signal-to-noise ratio were obtained when the sample injection volume was 60 μL, this quantity was injected into the carrier stream.
Analytical characteristics A calibration curve (Fig. 4) was constructed for the DS assay under optimized experimental parameters over the range 0.040–5.0 μg/mL. Fig. 4 shows the FI–CL signals for DS. It was found that relative CL intensity increased linearly with increasing DS concentration. The linear regression equation was ΔI (relative units) = 1.828 c (108g/ml) 6.989 (r = 0.9995, n = 7). The limit of detection (LOD) for DS was 0.020 μg/mL (3σ). The intra-assay relative standard deviation (RSD) for 11 repetitive determinations at 0.40 μg/mL DS was 1.43%, showing good reproducibility. The inter-assay RSD for 0.040 μg/mL DS (n = 11) was 2.0%.
Interference Common excipients used in pharmaceutical preparations of DS and metal ions in the human body were tested as potential interferents. A series of solutions containing potential interfering
Figure 4. Calibration graphs of diclofenac sodium.
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Flow injection CL determination of diclofenac sodium compounds and 1.0 μg/mL DS were prepared. The CL signals of the prepared solutions were recorded and compared with that obtained for standard DS solution (1.0 μg/mL). The tolerance limit was taken as the amount which caused a relative error of ± 5% in the peak height. The results are listed in Table 1. Some ions and the studied excipients in tablets did not interfere with the determination of DS in this system. The results indicated that this method can be used for the determination of DS in pharmaceutical preparations.
Application The proposed method was successfully applied to the determination of DS in a commercial pharmaceutical formulation. The DS tablets and capsules were bought from a local market in Suzhou. They were prepared as described in the Chinese Pharmacopoeia (30). A solution of DS pharmaceutical preparations was prepared as follows: three tablets (or the contents of four DS capsules) were finely ground, homogenized and a portion of the powder equivalent to 300 mg of DS was weighed accurately and diluted with 70 mL of ethanol. The mixture was vibrated and filtered. Two milliliters of filtrate was diluted further with ethanol to 100 mL. The sample solution was diluted appropriately with water so that the final concentration of DS was within the detection range. To evaluate the reliability and the accuracy of the method, certain quantities of standard DS solution were added to different batches of sample solutions and analyzed using the proposed method. The recoveries (shown in Table 2) of DS in different batches of tablets and capsules were calculated by comparing the results obtained before and after the addition of standard DS solution. The recoveries were all between 98 and 103%, indicating that the results were accurate. The t-test (listed in Table 3) indicated that there were no significant differences between the results obtained using the proposed method
Table 1. Tolerance of different substances in the determination of 1.0 μg/mL diclofenac sodium Interferent
Maxium tolerable ratio (relative error ± 5% )
Al3+, SO42, Cu2+, NH4+ Zn2+, Mg2+, Ca2+, Cl, CH3COO, PO43 Starch, glucose, sucrose, Pb2+ Citric acid, β-cyclodextrin, tryptophan Tyrosine, ascorbic aid, lactose
500 250 100 50 10
and those obtained using the Chinese Pharmacopoeia method (30) at a confidence level of 95%. Possible CL mechanism To obtain more information about the enhanced CL mechanism, we designed two types of detection method. First of all, the CL spectra (shown in Fig. 5) were determined about KMnO4–HCHO (curve a) and KMnO4–HCHO–DS (curve b). As shown in Fig. 5, the two CL systems possessed parallel CL spectra and had a maximum spectral peak at 710 ± 5 nm, revealing that the adding of DS could increase the CL signal without changing the CL process. The fluorescence spectra of the KMnO4–HCHO system and reaction products were scanned within the range 250–700 nm using a fluorescence spectrometer. No fluorescence could be found, highlighting that DS can not produce fluorescence. In order to investigate the possible reaction mechanism of CL enhancement, UV/vis absorption spectra of DS solution (curve a), KMnO4–HClO4 (curve b), KMnO4–HClO4–DS (curve c) and KMnO4–HClO4–DS (curve d) were recorded, as shown in Fig. 6. The absorption spectrum showed that DS has stronger absorption in 273 nm (curve a), whereas manganese(VII) has three characteristic absorption peaks at 309, 523 and 544 nm (as shown by curve b), respectively. The absorbance peaks of manganese(VII) decrease significantly when DS is added, although an absorption peak appears at 246 nm (curve c). This means that the concentration of MnO4 was diminished and a new intermediate was formed in the solution. The absorption peak at 246 nm increased remarkably in the presence of DS and formaldehyde (curve d). It can be concluded that formaldehyde promoted the CL reaction. These results indicated that an oxidation–reduction reaction occurred between DS and the potassium permanganate–perchloric acid–formaldehyde system, and energy was released, the reactant disappeared and samples corresponding oxidation products were generated. Electronically excited ‘singlet oxygen’ (1O2) species are thought to be generated in many CL systems (31). It has been suggested previously (32–34) that KMnO4 could react with some reductants in the presence of formaldehyde or formic acid to produce 1O12O2 (1Δ1gΔg). The principle emission bands of 1O2 occur in the near-infrared (λmax = 1270 nm) from a monomeric species (1Δg → 3Σg;) and in the visible region (λmax = 703 nm) from its dimeric aggregate [(1Δg)ν=0(1Δg)ν=0 → (3Σg)ν=1(3Σg)ν=0 and (1Δg)ν=0(1Δg)ν=0 → (3Σg)ν=0(3Σg)ν=0]. CL spectra were seen at 710 ± 5nm, which are similar to singlet oxygen chemiluminescence (35–39). Thus, it is possible that the singlet excited molecular oxygen species is an emitter in the present system, formed by the transfer of energy from oxidized DS to
Table 2. Results of recovery from different batches of diclofenac sodium Sample no.
Sample (μg/mL)
Found (μg/mL)
H20090492
0.20
0.21
H20067776
0.40
0.39
Diclofenac sodium added (μg/mL) 0.0 0.20 0.40 0.0 0.20 0.40
Diclofenac sodium found (μg/mL) 0.2 0.39 0.62 0.41 0.59 0.79
Recovery ± RSD (%) 100 ± 6.2 98 ± 6.0 103 ± 1.6 102 ± 0.7 98 ± 1.7 98 ± 2.1
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J. Song et al. Table 3. Comparative results for the determination of diclofenac sodium in different methods Samples H20090492 H20067776
Nominal content Proposed method RSD (%, n = 5) Titrimetry method RSD (%, n = 5) (mg/capsule or mg/tablet) (mg/capsule or mg/tablet) (mg/capsule or mg/tablet) 75.0 100.0
74.3 98.2
1.76 1.02
75.6 97.7
0.93 1.45
Conclusion Diclofenac sodium can significantly enhance the CL intensity of an acidic potassium permanganate–formaldehyde system, and based on this, a sensitive and simple flow-injection CL method was established. The experimental conditions affecting the CL reaction were optimized and the analytical characteristics for the determination of DS are presented here. The possible CL mechanism was also studied in detail based on the CL and UV spectra. The proposed method has been applied to the determination of DS in tablets and capsules with satisfactory results. This method is practical and valuable in clinical and biochemical laboratories for the determination of DS. Acknowledgements 5
Figure 5. CL spectrum: a, 6.0 × 10 mol/L KMnO4–3.0 mol/L HClO4–3.0% HCHO; 5 b, 6.0 × 10 mol/L KMnO4–3.0 mol/L HClO4–15.0 μg/mL diclofenac sodium–3.0% HCHO.
This work was supported by the Science Fund from the National Natural Science Foundation of China (No. 21075087), the Project of Scientific and Technologic Infrastructure of Suzhou (SZS201207), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
References
5
Figure 6. UV spectra of: a, DS (5.0 μg/mL); b, KMnO4 (6.0 × 10 mol/L)–HClO4 5 (3.0 mol/L); c, KMnO4 (6.0 × 10 mol/L)–HClO4 (3.0 mol/L)–DS (5.0 μg/mL); d, 5 KMnO4 (6.0 × 10 mol/L)–HClO4 (3.0 mol/L)–DS (5.0 μg/mL)–HCHO (3.0%).
dissolved oxygen. Based on the above discussion and previous reports (32–34), a possible CL mechanism could be expressed as follows:
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MnO4 þ Hþ þ HCHO þ DS→H2 O þ Mn2þ þoxidized DS intermediate; 3 O2 ð3 ΣgÞ þ oxidized DS intermediate→ 1 O2 ð1 ΔgÞ 1 þoxidized DS product; 21 O2 ð1 ΔgÞ→ 1 O2 1 O2 1 Δg1 Δg ; O2 1 O2 1 Δg1 Δg →23 O2 ð3 ΣgÞ þ hνðλmax ¼ 710±5 nmÞ
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Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web site.
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