Research article Received: 11 October 2013,
Revised: 14 February 2014,
Accepted: 2 April 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2696
Application of silver nanoparticles to the chemiluminescence determination of cefditoren pivoxil using the luminol–ferricyanide system Nawal A. Alarfaj, Fatma A. Aly and Maha F. El-Tohamy* ABSTRACT: A new simple, accurate and sensitive sequential injection analysis chemiluminescence (CL) detection method for the determination of cefditoren pivoxil (CTP) has been developed. The developed method was based on the enhancement effect of silver nanoparticles on the CL signal arising from a luminol–potassium ferricyanide reaction in the presence of CTP. The optimum conditions relevant to the effect of luminol, potassium ferricyanide and silver nanoparticle concentrations were investigated. The proposed method showed linear relationships between relative CL intensity and the investigated drug concentration at the range 0.001–5000 ng/mL, (r = 0.9998, n = 12) with a detection limit of 0.5 pg/mL and quantification limit of 0.001 ng/mL. The relative standard deviation was 1.6%. The proposed method was employed for the determination of CTP in bulk drug, in its pharmaceutical dosage forms and biological fluids such as human serum and urine. The interference of some common additive compounds such as glucose, lactose, starch, talc and magnesium stearate was investigated. In addition, the interference of some related cephalosporins was tested. No interference was recorded. The obtained sequential injection analysis-CL results were statistically compared with those from a reported method and did not show any significant differences. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: silver nanoparticles; cefditoren pivoxil; luminol-ferricyanide system; sequential injection analysis; chemiluminescence
Introduction Cefditoren pivoxil (CTP; Fig. 1) is a third-generation cephalosporin with antibacterial activity against gram-positive and gram-negative pathogens. It is chemically known as (6R)-7-[[(2Z)2-(2-amino-1,3-thiazol-4-yl)-2-methoxyiminoacetyl]amino]-3-[(Z)-2(4-methyl-1,3-thiazol-5-yl) ethenyl]-8-oxo-5-thia-1-aza bicyclo [4.2.0] oct-2-ene-2-carboxylic acid (1). It is a prodrug, which can be hydrolyzed by esterase during absorption to the active drug, cefditoren, and the drug is distributed in the circulating blood as an active cefditoren. CTP is used in the treatment of mild to moderate pharyngitis, tonsillitis, uncomplicated skin, skin structure infections and acute exacerbations of chronic bronchitis (2). A literature survey revealed that few analytical methods have been reported including high-performance liquid chromatography (3–5), gas chromatography (6–8), high-performance thinlayer chromatography (9), spectrophotometry (10,11) and potentiometry (2). In recent years, much attention has been given to using nanotechnology in analytical methods, in particular the use silver nanoparticles (AgNPs)/or gold NPs. (12,13) to enhance various chemical reactions. The objective of using nanotechnology in analytical chemistry is the evaluation of pharmaceutical drugs on a very small scale, increase the efficiency of analytical methods and reducing the cost of analysis. The literature survey revealed no chemiluminescence (CL) methods. The aim of the present study is the development of a simple sequential injection analysis (SIA)-CL method for the determination of CTP in bulk, dosage forms and in biological fluids. The developed method was based on the enhancement
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effect of AgNPs on the CL signal emission produced from the luminol–ferricyanide reaction in alkaline medium.
Experimental Materials and reagents All reagents were of analytical grade and were used without further purification. Distilled water was used throughout the experiments. Pure grade of CTP and its tablets (MeiactW 200 mg/tablet) were supplied from Tabuk Pharmaceuticals (MFG. Co., Tabuk, Saudi Arabia). Luminol stock solution 5.0 × 10–4 mol/L (Sigma Chemical Co., Sigma-Aldrich, Germany) was prepared in 100 mL of 1.0 × 10–2 mol/L sodium hydroxide (WINLAB). Potassium ferricyanide (WINLAB suppliers-New Jersey, USA) was used to prepare 1.0 × 10–3 mol/L solution by dissolving 0.033 g in 100 mL distilled water. Silver nitrate (BDH Laboratory Supplies, London, UK) was used to prepare 3.0 × 10–3 mol/L solution by dissolving 0.051 g in 100 mL distilled water. Sodium citrate dihydrate (WINLAB) was used to prepare 3.0 × 10–3 mol/L solution by dissolving 0.0882 g in 100 mL distilled water. Sodium borohydride (BDH Laboratory Supplies) was used to prepare 3.0 × 10–3 mol/L by dissolving 0.011 g in 100 mL distilled water. Sodium hydroxide and zinc sulfate (WINLAB) were used for * Correspondence to: Maha F. El-Tohamy, Department of Chemistry, College of Science, King Saud University, PO Box 22452, Riyadh 11495, Saudi Arabia. E-mail: [email protected] Department of Chemistry, College of Science, King Saud University, PO Box 22452, Riyadh 11495, Saudi Arabia
Copyright © 2014 John Wiley & Sons, Ltd.
N. A. Alarfaj et al. concentration of CTP in the range 0.001–5000 ng/mL. No further pretreatment was required for urine samples. SIA-CL detection was employed, peak heights of CL signals recorded and the percentage recovery was calculated by comparing the obtained results in serum and urine with the same concentration levels of the drug in water.
Figure 1. Chemical structure of cefditoren pivoxil.
preparation of 0.1 mol/L and 5.0% in distilled water, respectively. Urine samples were obtained from healthy volunteers and serum samples (Multi-Serum Normal, Randox Laboratories, Northern Ireland-UK) were obtained from commercial sources. Apparatus The SIA system (FIAlab-3500 instrument, FIAlab Instruments Inc., Bellevue, USA) comprised of a CAVRO XL 3000 syringe pump volume 2.5 mL (Cavro Scientific Instrument Int., USA) and Vici Valco Cheminer RTW 125-0718 eight-port manifolds. A fluorimetric/CL detector (ultraviolet [UV] lamp switched off) equipped with a lab-made CL module with spiral geometry was used and the photomultiplier tube voltage was 320 V. Autosampler model ALM 3200 is a separate unit and connected with the main instrument (FIAlab Instruments Inc. Bellevue, USA). The SIA system involved a holding coil (length 70 cm, i.d. 0.8 mm, PTFE tubing volume 1.2 mL). The same tubing was spirally coiled on a 52 mm × 52 mm Perspex plate, which substituted the secondary filter in the fluorimeter; this CL module had a central inlet, peripheral outlet and the diameter of the spiral was 24 mm. The SIA unit was PC controlled and data acquisition was performed with (FIAlab for windows version 5.9.321) software. The solution stability monitoring and UV spectrophotometry was performed on a UV-visible spectrophotometer Ultrospec (model 2100 pro, electronic Co. UK). Sample preparation Standard drug solution. A stock standard CTP solution (100 μg/mL) was prepared by dissolving 10 mg of pure drug in 100 mL distilled water. Serial solutions were prepared daily by appropriate dilution. The employed working solutions were in the range 0.001–10,000 ng/mL. Tablets. Ten tablets (MeiactW 200 mg/tablet) were finely powdered and weighed. An amount of powder equivalent to 10 mg CTP was dissolved in methanol and then sonicated for 10 min. The sonicated solution was filtered using membrane filter (pore size 5.0 μm). The working solutions were prepared by serial dilution in the range 0.001–5000 ng/mL. The proposed SIA-CL method was employed to determine the investigated drug in each concentration. The mean percentage recoveries were calculated using a calibration graph. Urine and serum. Enhanced AgNPs SIA-CL technique was proposed for the determination of CTP in human serum and urine. The spiking technique was used to prepare human serum and urine samples. One mL of serum was spiked with CTP standard drug solution to achieve 1.8 μg/mL and deprotonated by adding 1.0 mL acetonitrile. Then 0.1 mL of NaOH (0.1 mol/L), 1.0 mL of ZnSO4.7H2O (5.0% w/v) were added, where most of the interfering species (mainly proteins) were removed by precipitation (14). The prepared solution was centrifuged at 2500 rpm for 5 min. The treated sample was diluted with distilled water to obtain a
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Synthesis of silver nanoparticles. The synthesis of AgNPs was carried out by the reduction of silver nitrate using sodium borohydride as a reducing agent in aqueous solution according to the procedure described in the literature (15). Twenty-five mL of 3.0 × 10–3 mol/L sodium borohydride was added to 70 mL of 3.0 × 10–3 mol/L silver nitrate dropwise with continuous stirring for 10 min. The mixture turned yellow, which indicated silver nitrate reduction and the formation of AgNPs. Five mL sodium citrate 3.0 × 10–3 mol/L was added to the resultant solution to stabilize the AgNPs. The prepared AgNPs were stirred for 20 min. The average number of silver atoms per NPs was calculating according to the following formula N = π ρ D3NA/6 M, where N is the number of atoms per NP, ρ density of face countered cubic silver = 10.5 g/cm3, D3 the average diameter of NPs, NA number of atoms per mole and M the atomic mass of silver. Then the concentration of the prepared AgNPs was calculated using the following equation C = NT/NVNA where C the molar concentration of AgNPs, NT the total number of NP in solution, N is the number of atoms per NP, V the volume of the reaction solution in liters and NA number of NPs in moles (16). Figure 2 showed the characterization of AgNPs using transmission electron microscopy.
Procedure The SIA-CL setup as shown in Fig. 3 was used for automated aspiration of appropriate defined volumes of standard and test solutions of the investigated drug and reagents. All experiments were computer controlled to ensure precise, timing of pump and valve movements. For each experiment, all lines were first filled with carrier solution and air bubbles were removed. The Prim-port program was used first to fill in the lines connected with the test solution and reagents. The sequence of the aspirated sample and reagents was automatically controlled. Mixture of 40 μL of 5.0 × 10–4 moL/L luminol, 40 μL of 3.0 × 10–3 mol/L AgNPs, 48 μL sample solution and 30 μL of 1.0 × 10–3 mol/L potassium ferricyanide was aspirated into the holding coil through the eight-way injection valve at a flow rate of 100 μL/s and then the mixed solution was flushed continuously into the
Figure 2. Silver nanoparticles using transmission electron microscopy.
Copyright © 2014 John Wiley & Sons, Ltd.
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Chemiluminescence determination of cefditoren pivoxil
Figure 3. Schematic diagram of sequential injection analysis injection system for chemiluminescence determination of cefditoren pivoxil; carrier stream (water); re–4 –3 agent 1 (luminol 5.0 × 10 mol/L); reagent 2 (potassium ferricyanide 1.0 × 10 mol/ –3 L); reagent 3 (silver nanoparticles 3.0 × 10 mol/L, sample [cefditoren pivoxil]).
flow-through cell located in front of the detection cell of the photomultiplier tube. The resulting transient CL signal was recorded in the form of peaks, the peak heights were calculated automatically by our original (FIAlabW supported software, Version 5.9.321) and the data were stored by the PC for subsequent processing. All measurements were carried out at ambient temperature 25 ± 1 °C.
Calibration Under optimum conditions, the calibration curve for the determination of CTP was obtained. The graph related the CL intensity vs. the concentration of tested drug solutions was plotted at 12 experimental points. The mean peak heights were obtained after triplicate sample aspiration. Conventional linear regression was utilized for fitting the curve.
Results and discussion Optimization studies Selection of potassium ferricyanide as an oxidizing agent. To select the most suitable oxidizing agent, various oxidants including potassium ferricyanide, potassium permanganate, potassium periodate and hydrogen peroxide were carefully examined. No CL signal was recorded using potassium permanganate and potassium periodate, while hydrogen peroxide or potassium ferricyanide produced CL signals. Potassium ferricyanide gave a higher CL intensity signal than that of hydrogen peroxide. Therefore, the luminol–potassium ferricyanide–CL system was selected and the effect of luminol and potassium ferricyanide concentrations was further investigated and optimized. Effect of luminol and potassium ferricyanide concentrations. The effect of luminol and potassium ferricyanide concentrations on the CL signal was carefully investigated. This was carried out by using various concentrations in the range of 1.0 × 10–5–1.0 × 10–1 mol/L for both reagents. Figure 4 shows that the CL intensity showed a significant increase at 5.0 × 10–4 and 1.0 × 10–3 mol/L for luminol and potassium ferricyanide, respectively. Therefore, these concentrations were chosen for the subsequent experimental analysis.
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Figure 4. Effect of luminol and potassium ferricyanide concentration on CL –3 intensity, for luminol concentration (silver nanoparticles 3.0 × 10 mol/L and –3 potassium ferricyanide 1.0 × 10 mol/L) and for potassium ferricyanide concentra–3 –4 tion (silver nanoparticles 3.0 × 10 mol/L, luminol 5.0 × 10 mol/L) and 1.0 ng/mL cefditoren pivoxil. CL, chemiluminescence.
Effect of silver nanoparticles concentration. The effect of the AgNP concentration can greatly affect the CL intensity of luminol– ferricyanide system. The AgNP concentration was investigated over a range of 1.0 × 10–4–1.0 × 10–1 mol/L. Figure 5 showed that the CL intensity was sharply increased on using 3.0 × 10–3 mol/L. Hence, 3.0 × 10–3 mol/L was selected in further studies. Optimization of alkaline medium. To study the effect of alkaline medium on the luminol–ferricyanide CL reaction, three kinds of alkaline media including ammonium hydroxide, sodium carbonate and sodium hydroxide in the range 1.0 × 10–4–1.0 × 10–1 mol/L were investigated. As shown in Fig. 6, it was found that the use of 1.0 × 10–2 mol/L sodium hydroxide gave a sharp CL signal while in the cases of ammonium hydroxide and sodium carbonate a significant decrease in CL signal was observed. Therefore, 1.0 × 10–2 mol/L sodium hydroxide was used in the proposed method. The pH of the selected sodium hydroxide solution was tested in the range of 6–8. It was found that the suitable pH was 6.5. The selected pH was used for further experiments. Optimization of aspirated volumes of sample and reagents. The main critical parameter that should be carefully optimized in SIA-CL detection was the aspirated volume of sample and reagents. The optimum aspirated volume for luminol, AgNPs and
Figure 5. Effect of AgNPs concentration on the CL signal of luminol–potassium –4 –3 ferricyanide system (luminol 5.0 × 10 mol/L, potassium ferricyanide 1.0 × 10 mol/L) and 1.0 ng/mL cefditoren pivoxil. AgNPs, silver nanoparticles; CL, chemiluminescence.
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N. A. Alarfaj et al. Effect of foreign substances
Figure 6. Effect of sodium hydroxide, ammonium hydroxide and sodium carbonate concentration on CL intensity of luminol–potassium ferricyanide system (silver –3 –3 nanoparticles 3.0 × 10 mol/L, potassium ferricyanide 1.0 × 10 mol/L, luminol –4 5.0 × 10 mol/L) and 1.0 ng/mL cefditoren pivoxil. CL, chemiluminescence.
potassium ferricyanide and CTP was 40, 40, 30 and 48 μL, respectively and 48 μL for the CTP sample. The time was extended to about 45 s for complete flushing through the holding cell with carrier in between analysis cycles. In addition, the effect of flow rate on CL intensity was investigated in the range of 10–150 μL/s. It was noticed that the CL intensity was increased with the increase of flow rate. As shown in Fig. 7, the optimum flow rate was found to be 100 μL/s, which was used for further studies.
Sequential injection analysis control program To utilize an SIA control program for performing all calibration measurements and experimental analysis of CTP, the proposed program was employed for the determination of tested drug in its dosage forms and biological fluids. Table 1 shows the typical steps of the program. The single cycle takes about 45 s; therefore, the sample throughput of 80/h can be recorded.
The effect of some foreign substances such as common cations, some additive excipients, some common species such as amino acids, sugars and some related pharmacological action drugs was examined. To evaluate the interferences a test solution of 0.01 ng/mL of CTP was treated with an appropriate foreign substance to contain ≈ 1.0 μg/mL. The mean peak heights were compared with those obtained with pure 0.01 ng/mL analyte solution. The tolerable level was defined as the amount of foreign species that produced an error not exceeding 5% in determining the tested drug. The results of concentration levels for studied interferences are presented in Table 2. It can be seen that there is no influence on the determination of CTP in pharmaceutical dosage forms. While, for serum samples the possible interference may arise from ascorbic acid, uric acid, urea and heavy metal ions such as Fe3+, Fe2+, Cd2+, Co2+, Cu2+, Al3+ and Ni2+. The latter ions can be eliminated by the addition of EDTA. After the 1000-fold dilution, the interference from such substances could be greatly minimized to negligible levels. In addition, the recorded results showed interference due to the presence of some related drugs such as cephalexin and cefotaxime sodium in the determination of CTP. Method validation Method validation was carried out with respect to linearity, lower limit of detection, quantification limit, accuracy, precision and robustness according to ICH guidelines (17). Linearity. The proposed SIA-CL method for the determination of CTP using the AgNP–luminol–ferricyanide system was successfully applied for evaluation of the linear concentration range. The recorded signals were plotted as a function of the tested drug concentrations. Twelve standard solutions were subjected to SIA-CL detection. The regression line was calculated using the least squares statistical method. The results obtained showed that the proposed SIA-CL method exhibits a linear concentration range at 0.001–5000 ng/mL (Table 3). Lower limit of detection. The signal-to-noise ratio was performed to evaluate the lower limit of detection of the proposed SIA-CL method for the determination of CTP. The lower limit of detection was calculated by S/N = 3, as the concentration of CTP has a CL signal equal to three times that of the blank signal. The recorded signals showed a lower limit of detection of 0.5 pg/mL. Quantification limit. To determine the quantification limit of CTP by the proposed AgNP–luminol–ferricyanide SIA-CL method, a signal-to-noise ratio equal to 10 was assessed and the quantification limit was 0.001 ng/mL. Accuracy. The standard addition method was used for investigating the accuracy of the proposed AgNP–luminol–ferricyanide SIA-CL method. It was carried out using the tested drug and the results were calculated in terms of mean percentage recoveries. The calculated percentage recovery was 99.38 ± 1.2%.
Figure 7. The influence of flow rate on the relative CL intensity. Conditions: 40 μL –4 –3 of 5.0 × 10 mol/L luminol; 40 μL of 3.0 × 10 silver nanoparticles mol/L, 30 μL of –3 1.0 × 10 mol/L ferricyanide and 48.0 μL of 1.0 ng/mL cefditoren pivoxil. CL, chemiluminescence.
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Precision. To evaluate the precision of the proposed AgNP– luminol–ferricyanide SIA-CL method for the determination of CTP, intraday and interday precision was assessed. The calculated percentage relative SD (RSD) values were 1.3%, for the determination of CTP (nine replicates). The above percentage RSD value is less than 2% indicating good precision.
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Chemiluminescence determination of cefditoren pivoxil Table 1. The control program for the determination of cefditoren pivoxil using the silver nanoparticle–luminol- potassium ferricyanide sequential injection analysis-chemiluminescence system Device Loop Start (♯) 1 Next sample Peristaltic pump Detector Syringe pump Syringe pump Syringe pump Syringe pump Multiposition valve Syringe pump Syringe pump Multiposition valve Syringe pump Syringe pump Multiposition valve Syringe pump Syringe pump Syringe pump Multiposition valve Syringe pump Syringe pump Syringe pump Multiposition valve Syringe pump PMT Syringe pump Syringe pump PMT Refresh plat Loop end
Command
Parameter
Counter clockwise Delay (s) Peristaltic pump off ON Valve position IN Set flow rate (μL/s) Aspirate (μL) Delay until done Set valve position Set flow rate (μL/s) Aspirate (μL) Set valve position Set flow rate (μL/s) Aspirate (μL) Set valve position Set flow rate (μL/s) Aspirate (μL) Delay until done Set valve position Set flow rate (μL/s) Aspirate (μL) Delay until done Detector Set flow rate (μL/s) Start scan Empty Delay until done Stop scans
50% 25
120 1500 3 100 40 2 100 40 5 100 48 4 100 30
Tolerable level (μg/mL)
Na+, K+, Mg2+, Cl , NO3–, NH4+ , EDTA and SO42– Glucose, sucrose, lactose, talc, starch, magnesium stearate, citric acid Uric acid, ascorbic acid and urea Adrenaline, dopamine, cystine, histamine, tyrosine, glucosamine Al3+, Cd2+, Co2+, Fe2+, Fe3+, Mn2+, Ni2+ and Cu2+ Cephalexin and cefotaxime sodium
1000 800 50 100
(Luminol 5.0 × 10–4 mol/L) (Silver nanoparticles 3.0 × 10–3 mol/L)
Sample (cefditoren pivoxil)
(Potassium ferricyanide 1.0 × 10–3 mol/L)
Table 3. Performance data obtained from the determination of cefditoren pivoxil using the silver nanoparticle–luminol– potassium ferricyanide system Analytical characteristics Linear range, ng/mL Detection limit, pg/mL Quantification limit, ng/mL Intercept on the ordinate Slope %RSD (n = 12) Correlation coefficient, r
Value 0.001–5000 0.5 0.001 2780.03 30.02 1.6% 0.9998
45 25
Robustness. The robustness of the SIA-CL method for determination of CTP was investigated by introducing small changes in method parameters. The robustness of the method was carried out by changing the flow rate, aspiration rate or reagents and volume of sample using 100 ± 10 μL/s, 40 ± 5 μL and 30 ± 5 μL, respectively. The calculated percentage recovery for
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Pump filled with carrier
7 100
Table 2. Tolerable concentration level of interferents to 0.01 ng/mL cefditoren pivoxil Interferents
Action
the proposed method was 99.18 ± 0.7%. The obtained results were closely in agreement with those obtained from standard drug solutions. Analytical applications From the above previously mentioned results, it was evident that the proposed method gave satisfactory results for the
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N. A. Alarfaj et al. determination of CTP in pure form. Thus, its pharmaceutical dosage form (MeiactW 200 mg/tablet) was subjected to the analysis of its CTP content by the proposed enhanced AgNP SIA-CL method. The obtained results were presented in Table 4 and statistically compared by Student’s t-test and F-test (18) with those obtained from the reported spectrophotometric method (10). The results did not reveal any significant difference
between them at 95% confidence level proving similar accuracy and precision. The content uniformity assay for CTP tablets was investigated and the results were presented as the mean percentage recovery ± SD (99.25 ± 0.8%). The extremely high sensitivity of the proposed method prompted us to check its applicability to the determination of CTP in biological fluids such as human serum and urine. The
Table 4. Determination of cefditoren pivoxil using silver nanoparticle–luminol–potassium ferricyanide sequential injection analysis-chemiluminescence detection in pure form, dosage forms and biological fluids Sample
Taken ng/mL
Found ng/mL
Recovery (%)
0.001 0.01 10 100 1000 5000
0.00101 0.01 9.999 98.65 978.8 4998 99.58 ± 1.1 6 1.21 0.44 1.11 0.001 0.009873 10.181 99.28 993.8 4883.5 99.48 ± 1.3 6 1.69 0.53 1.31 0.45 (2.228)a 2.7(5.05)a 0.0009986 0.009916 9.914 97.18 995.6 5000
101.00 100.00 99.99 98.65 97.88 99.96
Pure solution
Mean% ± SD n Variance %SEM %RSD MeiactW 200 mg/tablet
Mean% ± SD n Variance %SEM %RSD t F Urine sample
Mean% ± SD n Variance SEM% %RSD Serum sample
0.001 0.01 10 100 1000 5000
0.001 0.01 10 100 1000 5000
100.00 98.73 101.81 99.28 99.38 97.67
Reported method (10)
99.74 ± 0.8 6 0.64 0.33 0.80
99.86 99.16 99.14 97.18 99.56 100.00 99.15 ± 1.1 6 1.21 0.44 1.11
0.001 0.01 10 100 1000 5000
0.00101 0.009914 9.917 97.24 986 4994.5
Mean% ± SD n Variance %SEM %RSD
101.00 99.14 99.17 97.24 98.60 99.89 99.17 ± 1.2 6 1.44 0.48 1.21
a
Figures in parentheses are the tabulated values of t- and F-test at 95% confidence limits (18).
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Chemiluminescence determination of cefditoren pivoxil obtained results (Table 4) were satisfactory in terms of accuracy and precision as the recovery values were 99.17 ± 1.2% and 99.15 ± 1.1% with low percentage RSD for human serum and urine, respectively.
Conclusion A new sequential injection SIA-CL method for the determination of CTP was developed. The method was based on the enhancement effect of AgNPs on the luminol–ferricyanide system. The method was found to be sensitive, reproducible and accurate for the determination of the drug in its bulk powder, dosage forms and biological fluids. The enhancement effect of CTP on the employed system was proportional to its concentration, which showed good results relevant to the linear concentration range 0.001–5000 ng/mL. The proposed method has been proved to be fast, inexpensive, highly sensitive and precise. Acknowledgments This project was supported by King Saud University, Deanship of Scientific Research, College of Science Research Center.
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5. Rieck W, Platt D. Determination of cefditoren (ME 1206) in the plasma of elderly patientswith multiple diseases using highperformance liquid chromatography. Clin Lab 2000;46:477–82. 6. Ohta M, Buckton G. The use of inverse gas chromatography to assess the acid-base contributions to surface energies of cefditoren pivoxil and methacrylate copolymers and possible links to instability. Int J Pharm 2004;272:121–8. 7. Ohta M, Oguchi T, Yamamoto K. Evaluation of solubility parameter to predict apparent solubility of amorphous and crystalline cefditoren pivoxil. Pharm Acta Helv 1999;74:59–64. 8. Ohta M, Buckton GA. Study of the difference between two amorphous spray-dried samples of cefditoren pivoxil which exhibited different physical stabilities. Int J Pharm 2005;289:31–8. 9. Vishal DM, Anurath SP, Shwini MA, Umesh N. Determination of cefditoren pivoxil in human plasma using high performance thin layer chromatography. Int J Res Ayur Pharm 2011;2:1582–4. 10. Niraimathi V, Aruna A, Suresh AJ, Prema V. Spectrophotometric estimation of cefditoren pivoxil in pharmaceutical oral solid dosage form. Int J Chem Sci 2010;8:724–8. 11. Narala SR, Saraswathi K. Validated spectrophotometric methods for determination of cefditoren pivoxil in drug formulations. Int J Chem Tech Res 2011;3:1025–7. 12. Behzad H, Somayyeh B. Flow injection chemiluminescence determination of isoniazide using luminol and silver nanoparticles. Microchem Acta 2010;95:192–7. 13. Li S, Tao S, Wang F, Hong J, Wei X. Chemiluminescence reactions of luminol system catalyzed by nanoparticles of a gold/silver alloy. Microchem Acta 2010;169:73–8. 14. Al-Ghamdi AF, Hefnawy MM, Almaged AA, Belal FF. Development of square-wave adsorptive stripping Voltammetric method for determination of acebutolol in pharmaceutical formulations and biological fluids. Chem Cent J 2010;6:15. 15. Solomon SD, Bahadory M, Jeyarajasingam AV, Rutkowsky SA, Boriz C. Synthesis and study of silver nanoparticles. J Chem Educ 2007;8:322–4. 16. Liu X, Atwater M, Wang J, Huo Q. Extinction coefficient of gold nanoparticles with different sizes and different capping ligands. Colloids Surf B Biointerfaces 2007;58:3–7. 17. ICH technical requirements for registration of pharmaceuticals for human use, complementary guidelines on methodology. Washington, DC 1996;13. 18. Miller JC, Miller JN. Statistics for analytical chemistry, 3rd edn. Chichester: Ellis Horwood-Prentice Hall, 1993.
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Revised: 14 February 2014,
Accepted: 2 April 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2696
Application of silver nanoparticles to the chemiluminescence determination of cefditoren pivoxil using the luminol–ferricyanide system Nawal A. Alarfaj, Fatma A. Aly and Maha F. El-Tohamy* ABSTRACT: A new simple, accurate and sensitive sequential injection analysis chemiluminescence (CL) detection method for the determination of cefditoren pivoxil (CTP) has been developed. The developed method was based on the enhancement effect of silver nanoparticles on the CL signal arising from a luminol–potassium ferricyanide reaction in the presence of CTP. The optimum conditions relevant to the effect of luminol, potassium ferricyanide and silver nanoparticle concentrations were investigated. The proposed method showed linear relationships between relative CL intensity and the investigated drug concentration at the range 0.001–5000 ng/mL, (r = 0.9998, n = 12) with a detection limit of 0.5 pg/mL and quantification limit of 0.001 ng/mL. The relative standard deviation was 1.6%. The proposed method was employed for the determination of CTP in bulk drug, in its pharmaceutical dosage forms and biological fluids such as human serum and urine. The interference of some common additive compounds such as glucose, lactose, starch, talc and magnesium stearate was investigated. In addition, the interference of some related cephalosporins was tested. No interference was recorded. The obtained sequential injection analysis-CL results were statistically compared with those from a reported method and did not show any significant differences. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: silver nanoparticles; cefditoren pivoxil; luminol-ferricyanide system; sequential injection analysis; chemiluminescence
Introduction Cefditoren pivoxil (CTP; Fig. 1) is a third-generation cephalosporin with antibacterial activity against gram-positive and gram-negative pathogens. It is chemically known as (6R)-7-[[(2Z)2-(2-amino-1,3-thiazol-4-yl)-2-methoxyiminoacetyl]amino]-3-[(Z)-2(4-methyl-1,3-thiazol-5-yl) ethenyl]-8-oxo-5-thia-1-aza bicyclo [4.2.0] oct-2-ene-2-carboxylic acid (1). It is a prodrug, which can be hydrolyzed by esterase during absorption to the active drug, cefditoren, and the drug is distributed in the circulating blood as an active cefditoren. CTP is used in the treatment of mild to moderate pharyngitis, tonsillitis, uncomplicated skin, skin structure infections and acute exacerbations of chronic bronchitis (2). A literature survey revealed that few analytical methods have been reported including high-performance liquid chromatography (3–5), gas chromatography (6–8), high-performance thinlayer chromatography (9), spectrophotometry (10,11) and potentiometry (2). In recent years, much attention has been given to using nanotechnology in analytical methods, in particular the use silver nanoparticles (AgNPs)/or gold NPs. (12,13) to enhance various chemical reactions. The objective of using nanotechnology in analytical chemistry is the evaluation of pharmaceutical drugs on a very small scale, increase the efficiency of analytical methods and reducing the cost of analysis. The literature survey revealed no chemiluminescence (CL) methods. The aim of the present study is the development of a simple sequential injection analysis (SIA)-CL method for the determination of CTP in bulk, dosage forms and in biological fluids. The developed method was based on the enhancement
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effect of AgNPs on the CL signal emission produced from the luminol–ferricyanide reaction in alkaline medium.
Experimental Materials and reagents All reagents were of analytical grade and were used without further purification. Distilled water was used throughout the experiments. Pure grade of CTP and its tablets (MeiactW 200 mg/tablet) were supplied from Tabuk Pharmaceuticals (MFG. Co., Tabuk, Saudi Arabia). Luminol stock solution 5.0 × 10–4 mol/L (Sigma Chemical Co., Sigma-Aldrich, Germany) was prepared in 100 mL of 1.0 × 10–2 mol/L sodium hydroxide (WINLAB). Potassium ferricyanide (WINLAB suppliers-New Jersey, USA) was used to prepare 1.0 × 10–3 mol/L solution by dissolving 0.033 g in 100 mL distilled water. Silver nitrate (BDH Laboratory Supplies, London, UK) was used to prepare 3.0 × 10–3 mol/L solution by dissolving 0.051 g in 100 mL distilled water. Sodium citrate dihydrate (WINLAB) was used to prepare 3.0 × 10–3 mol/L solution by dissolving 0.0882 g in 100 mL distilled water. Sodium borohydride (BDH Laboratory Supplies) was used to prepare 3.0 × 10–3 mol/L by dissolving 0.011 g in 100 mL distilled water. Sodium hydroxide and zinc sulfate (WINLAB) were used for * Correspondence to: Maha F. El-Tohamy, Department of Chemistry, College of Science, King Saud University, PO Box 22452, Riyadh 11495, Saudi Arabia. E-mail: [email protected] Department of Chemistry, College of Science, King Saud University, PO Box 22452, Riyadh 11495, Saudi Arabia
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N. A. Alarfaj et al. concentration of CTP in the range 0.001–5000 ng/mL. No further pretreatment was required for urine samples. SIA-CL detection was employed, peak heights of CL signals recorded and the percentage recovery was calculated by comparing the obtained results in serum and urine with the same concentration levels of the drug in water.
Figure 1. Chemical structure of cefditoren pivoxil.
preparation of 0.1 mol/L and 5.0% in distilled water, respectively. Urine samples were obtained from healthy volunteers and serum samples (Multi-Serum Normal, Randox Laboratories, Northern Ireland-UK) were obtained from commercial sources. Apparatus The SIA system (FIAlab-3500 instrument, FIAlab Instruments Inc., Bellevue, USA) comprised of a CAVRO XL 3000 syringe pump volume 2.5 mL (Cavro Scientific Instrument Int., USA) and Vici Valco Cheminer RTW 125-0718 eight-port manifolds. A fluorimetric/CL detector (ultraviolet [UV] lamp switched off) equipped with a lab-made CL module with spiral geometry was used and the photomultiplier tube voltage was 320 V. Autosampler model ALM 3200 is a separate unit and connected with the main instrument (FIAlab Instruments Inc. Bellevue, USA). The SIA system involved a holding coil (length 70 cm, i.d. 0.8 mm, PTFE tubing volume 1.2 mL). The same tubing was spirally coiled on a 52 mm × 52 mm Perspex plate, which substituted the secondary filter in the fluorimeter; this CL module had a central inlet, peripheral outlet and the diameter of the spiral was 24 mm. The SIA unit was PC controlled and data acquisition was performed with (FIAlab for windows version 5.9.321) software. The solution stability monitoring and UV spectrophotometry was performed on a UV-visible spectrophotometer Ultrospec (model 2100 pro, electronic Co. UK). Sample preparation Standard drug solution. A stock standard CTP solution (100 μg/mL) was prepared by dissolving 10 mg of pure drug in 100 mL distilled water. Serial solutions were prepared daily by appropriate dilution. The employed working solutions were in the range 0.001–10,000 ng/mL. Tablets. Ten tablets (MeiactW 200 mg/tablet) were finely powdered and weighed. An amount of powder equivalent to 10 mg CTP was dissolved in methanol and then sonicated for 10 min. The sonicated solution was filtered using membrane filter (pore size 5.0 μm). The working solutions were prepared by serial dilution in the range 0.001–5000 ng/mL. The proposed SIA-CL method was employed to determine the investigated drug in each concentration. The mean percentage recoveries were calculated using a calibration graph. Urine and serum. Enhanced AgNPs SIA-CL technique was proposed for the determination of CTP in human serum and urine. The spiking technique was used to prepare human serum and urine samples. One mL of serum was spiked with CTP standard drug solution to achieve 1.8 μg/mL and deprotonated by adding 1.0 mL acetonitrile. Then 0.1 mL of NaOH (0.1 mol/L), 1.0 mL of ZnSO4.7H2O (5.0% w/v) were added, where most of the interfering species (mainly proteins) were removed by precipitation (14). The prepared solution was centrifuged at 2500 rpm for 5 min. The treated sample was diluted with distilled water to obtain a
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Synthesis of silver nanoparticles. The synthesis of AgNPs was carried out by the reduction of silver nitrate using sodium borohydride as a reducing agent in aqueous solution according to the procedure described in the literature (15). Twenty-five mL of 3.0 × 10–3 mol/L sodium borohydride was added to 70 mL of 3.0 × 10–3 mol/L silver nitrate dropwise with continuous stirring for 10 min. The mixture turned yellow, which indicated silver nitrate reduction and the formation of AgNPs. Five mL sodium citrate 3.0 × 10–3 mol/L was added to the resultant solution to stabilize the AgNPs. The prepared AgNPs were stirred for 20 min. The average number of silver atoms per NPs was calculating according to the following formula N = π ρ D3NA/6 M, where N is the number of atoms per NP, ρ density of face countered cubic silver = 10.5 g/cm3, D3 the average diameter of NPs, NA number of atoms per mole and M the atomic mass of silver. Then the concentration of the prepared AgNPs was calculated using the following equation C = NT/NVNA where C the molar concentration of AgNPs, NT the total number of NP in solution, N is the number of atoms per NP, V the volume of the reaction solution in liters and NA number of NPs in moles (16). Figure 2 showed the characterization of AgNPs using transmission electron microscopy.
Procedure The SIA-CL setup as shown in Fig. 3 was used for automated aspiration of appropriate defined volumes of standard and test solutions of the investigated drug and reagents. All experiments were computer controlled to ensure precise, timing of pump and valve movements. For each experiment, all lines were first filled with carrier solution and air bubbles were removed. The Prim-port program was used first to fill in the lines connected with the test solution and reagents. The sequence of the aspirated sample and reagents was automatically controlled. Mixture of 40 μL of 5.0 × 10–4 moL/L luminol, 40 μL of 3.0 × 10–3 mol/L AgNPs, 48 μL sample solution and 30 μL of 1.0 × 10–3 mol/L potassium ferricyanide was aspirated into the holding coil through the eight-way injection valve at a flow rate of 100 μL/s and then the mixed solution was flushed continuously into the
Figure 2. Silver nanoparticles using transmission electron microscopy.
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Chemiluminescence determination of cefditoren pivoxil
Figure 3. Schematic diagram of sequential injection analysis injection system for chemiluminescence determination of cefditoren pivoxil; carrier stream (water); re–4 –3 agent 1 (luminol 5.0 × 10 mol/L); reagent 2 (potassium ferricyanide 1.0 × 10 mol/ –3 L); reagent 3 (silver nanoparticles 3.0 × 10 mol/L, sample [cefditoren pivoxil]).
flow-through cell located in front of the detection cell of the photomultiplier tube. The resulting transient CL signal was recorded in the form of peaks, the peak heights were calculated automatically by our original (FIAlabW supported software, Version 5.9.321) and the data were stored by the PC for subsequent processing. All measurements were carried out at ambient temperature 25 ± 1 °C.
Calibration Under optimum conditions, the calibration curve for the determination of CTP was obtained. The graph related the CL intensity vs. the concentration of tested drug solutions was plotted at 12 experimental points. The mean peak heights were obtained after triplicate sample aspiration. Conventional linear regression was utilized for fitting the curve.
Results and discussion Optimization studies Selection of potassium ferricyanide as an oxidizing agent. To select the most suitable oxidizing agent, various oxidants including potassium ferricyanide, potassium permanganate, potassium periodate and hydrogen peroxide were carefully examined. No CL signal was recorded using potassium permanganate and potassium periodate, while hydrogen peroxide or potassium ferricyanide produced CL signals. Potassium ferricyanide gave a higher CL intensity signal than that of hydrogen peroxide. Therefore, the luminol–potassium ferricyanide–CL system was selected and the effect of luminol and potassium ferricyanide concentrations was further investigated and optimized. Effect of luminol and potassium ferricyanide concentrations. The effect of luminol and potassium ferricyanide concentrations on the CL signal was carefully investigated. This was carried out by using various concentrations in the range of 1.0 × 10–5–1.0 × 10–1 mol/L for both reagents. Figure 4 shows that the CL intensity showed a significant increase at 5.0 × 10–4 and 1.0 × 10–3 mol/L for luminol and potassium ferricyanide, respectively. Therefore, these concentrations were chosen for the subsequent experimental analysis.
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Figure 4. Effect of luminol and potassium ferricyanide concentration on CL –3 intensity, for luminol concentration (silver nanoparticles 3.0 × 10 mol/L and –3 potassium ferricyanide 1.0 × 10 mol/L) and for potassium ferricyanide concentra–3 –4 tion (silver nanoparticles 3.0 × 10 mol/L, luminol 5.0 × 10 mol/L) and 1.0 ng/mL cefditoren pivoxil. CL, chemiluminescence.
Effect of silver nanoparticles concentration. The effect of the AgNP concentration can greatly affect the CL intensity of luminol– ferricyanide system. The AgNP concentration was investigated over a range of 1.0 × 10–4–1.0 × 10–1 mol/L. Figure 5 showed that the CL intensity was sharply increased on using 3.0 × 10–3 mol/L. Hence, 3.0 × 10–3 mol/L was selected in further studies. Optimization of alkaline medium. To study the effect of alkaline medium on the luminol–ferricyanide CL reaction, three kinds of alkaline media including ammonium hydroxide, sodium carbonate and sodium hydroxide in the range 1.0 × 10–4–1.0 × 10–1 mol/L were investigated. As shown in Fig. 6, it was found that the use of 1.0 × 10–2 mol/L sodium hydroxide gave a sharp CL signal while in the cases of ammonium hydroxide and sodium carbonate a significant decrease in CL signal was observed. Therefore, 1.0 × 10–2 mol/L sodium hydroxide was used in the proposed method. The pH of the selected sodium hydroxide solution was tested in the range of 6–8. It was found that the suitable pH was 6.5. The selected pH was used for further experiments. Optimization of aspirated volumes of sample and reagents. The main critical parameter that should be carefully optimized in SIA-CL detection was the aspirated volume of sample and reagents. The optimum aspirated volume for luminol, AgNPs and
Figure 5. Effect of AgNPs concentration on the CL signal of luminol–potassium –4 –3 ferricyanide system (luminol 5.0 × 10 mol/L, potassium ferricyanide 1.0 × 10 mol/L) and 1.0 ng/mL cefditoren pivoxil. AgNPs, silver nanoparticles; CL, chemiluminescence.
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N. A. Alarfaj et al. Effect of foreign substances
Figure 6. Effect of sodium hydroxide, ammonium hydroxide and sodium carbonate concentration on CL intensity of luminol–potassium ferricyanide system (silver –3 –3 nanoparticles 3.0 × 10 mol/L, potassium ferricyanide 1.0 × 10 mol/L, luminol –4 5.0 × 10 mol/L) and 1.0 ng/mL cefditoren pivoxil. CL, chemiluminescence.
potassium ferricyanide and CTP was 40, 40, 30 and 48 μL, respectively and 48 μL for the CTP sample. The time was extended to about 45 s for complete flushing through the holding cell with carrier in between analysis cycles. In addition, the effect of flow rate on CL intensity was investigated in the range of 10–150 μL/s. It was noticed that the CL intensity was increased with the increase of flow rate. As shown in Fig. 7, the optimum flow rate was found to be 100 μL/s, which was used for further studies.
Sequential injection analysis control program To utilize an SIA control program for performing all calibration measurements and experimental analysis of CTP, the proposed program was employed for the determination of tested drug in its dosage forms and biological fluids. Table 1 shows the typical steps of the program. The single cycle takes about 45 s; therefore, the sample throughput of 80/h can be recorded.
The effect of some foreign substances such as common cations, some additive excipients, some common species such as amino acids, sugars and some related pharmacological action drugs was examined. To evaluate the interferences a test solution of 0.01 ng/mL of CTP was treated with an appropriate foreign substance to contain ≈ 1.0 μg/mL. The mean peak heights were compared with those obtained with pure 0.01 ng/mL analyte solution. The tolerable level was defined as the amount of foreign species that produced an error not exceeding 5% in determining the tested drug. The results of concentration levels for studied interferences are presented in Table 2. It can be seen that there is no influence on the determination of CTP in pharmaceutical dosage forms. While, for serum samples the possible interference may arise from ascorbic acid, uric acid, urea and heavy metal ions such as Fe3+, Fe2+, Cd2+, Co2+, Cu2+, Al3+ and Ni2+. The latter ions can be eliminated by the addition of EDTA. After the 1000-fold dilution, the interference from such substances could be greatly minimized to negligible levels. In addition, the recorded results showed interference due to the presence of some related drugs such as cephalexin and cefotaxime sodium in the determination of CTP. Method validation Method validation was carried out with respect to linearity, lower limit of detection, quantification limit, accuracy, precision and robustness according to ICH guidelines (17). Linearity. The proposed SIA-CL method for the determination of CTP using the AgNP–luminol–ferricyanide system was successfully applied for evaluation of the linear concentration range. The recorded signals were plotted as a function of the tested drug concentrations. Twelve standard solutions were subjected to SIA-CL detection. The regression line was calculated using the least squares statistical method. The results obtained showed that the proposed SIA-CL method exhibits a linear concentration range at 0.001–5000 ng/mL (Table 3). Lower limit of detection. The signal-to-noise ratio was performed to evaluate the lower limit of detection of the proposed SIA-CL method for the determination of CTP. The lower limit of detection was calculated by S/N = 3, as the concentration of CTP has a CL signal equal to three times that of the blank signal. The recorded signals showed a lower limit of detection of 0.5 pg/mL. Quantification limit. To determine the quantification limit of CTP by the proposed AgNP–luminol–ferricyanide SIA-CL method, a signal-to-noise ratio equal to 10 was assessed and the quantification limit was 0.001 ng/mL. Accuracy. The standard addition method was used for investigating the accuracy of the proposed AgNP–luminol–ferricyanide SIA-CL method. It was carried out using the tested drug and the results were calculated in terms of mean percentage recoveries. The calculated percentage recovery was 99.38 ± 1.2%.
Figure 7. The influence of flow rate on the relative CL intensity. Conditions: 40 μL –4 –3 of 5.0 × 10 mol/L luminol; 40 μL of 3.0 × 10 silver nanoparticles mol/L, 30 μL of –3 1.0 × 10 mol/L ferricyanide and 48.0 μL of 1.0 ng/mL cefditoren pivoxil. CL, chemiluminescence.
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Precision. To evaluate the precision of the proposed AgNP– luminol–ferricyanide SIA-CL method for the determination of CTP, intraday and interday precision was assessed. The calculated percentage relative SD (RSD) values were 1.3%, for the determination of CTP (nine replicates). The above percentage RSD value is less than 2% indicating good precision.
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Chemiluminescence determination of cefditoren pivoxil Table 1. The control program for the determination of cefditoren pivoxil using the silver nanoparticle–luminol- potassium ferricyanide sequential injection analysis-chemiluminescence system Device Loop Start (♯) 1 Next sample Peristaltic pump Detector Syringe pump Syringe pump Syringe pump Syringe pump Multiposition valve Syringe pump Syringe pump Multiposition valve Syringe pump Syringe pump Multiposition valve Syringe pump Syringe pump Syringe pump Multiposition valve Syringe pump Syringe pump Syringe pump Multiposition valve Syringe pump PMT Syringe pump Syringe pump PMT Refresh plat Loop end
Command
Parameter
Counter clockwise Delay (s) Peristaltic pump off ON Valve position IN Set flow rate (μL/s) Aspirate (μL) Delay until done Set valve position Set flow rate (μL/s) Aspirate (μL) Set valve position Set flow rate (μL/s) Aspirate (μL) Set valve position Set flow rate (μL/s) Aspirate (μL) Delay until done Set valve position Set flow rate (μL/s) Aspirate (μL) Delay until done Detector Set flow rate (μL/s) Start scan Empty Delay until done Stop scans
50% 25
120 1500 3 100 40 2 100 40 5 100 48 4 100 30
Tolerable level (μg/mL)
Na+, K+, Mg2+, Cl , NO3–, NH4+ , EDTA and SO42– Glucose, sucrose, lactose, talc, starch, magnesium stearate, citric acid Uric acid, ascorbic acid and urea Adrenaline, dopamine, cystine, histamine, tyrosine, glucosamine Al3+, Cd2+, Co2+, Fe2+, Fe3+, Mn2+, Ni2+ and Cu2+ Cephalexin and cefotaxime sodium
1000 800 50 100
(Luminol 5.0 × 10–4 mol/L) (Silver nanoparticles 3.0 × 10–3 mol/L)
Sample (cefditoren pivoxil)
(Potassium ferricyanide 1.0 × 10–3 mol/L)
Table 3. Performance data obtained from the determination of cefditoren pivoxil using the silver nanoparticle–luminol– potassium ferricyanide system Analytical characteristics Linear range, ng/mL Detection limit, pg/mL Quantification limit, ng/mL Intercept on the ordinate Slope %RSD (n = 12) Correlation coefficient, r
Value 0.001–5000 0.5 0.001 2780.03 30.02 1.6% 0.9998
45 25
Robustness. The robustness of the SIA-CL method for determination of CTP was investigated by introducing small changes in method parameters. The robustness of the method was carried out by changing the flow rate, aspiration rate or reagents and volume of sample using 100 ± 10 μL/s, 40 ± 5 μL and 30 ± 5 μL, respectively. The calculated percentage recovery for
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Pump filled with carrier
7 100
Table 2. Tolerable concentration level of interferents to 0.01 ng/mL cefditoren pivoxil Interferents
Action
the proposed method was 99.18 ± 0.7%. The obtained results were closely in agreement with those obtained from standard drug solutions. Analytical applications From the above previously mentioned results, it was evident that the proposed method gave satisfactory results for the
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N. A. Alarfaj et al. determination of CTP in pure form. Thus, its pharmaceutical dosage form (MeiactW 200 mg/tablet) was subjected to the analysis of its CTP content by the proposed enhanced AgNP SIA-CL method. The obtained results were presented in Table 4 and statistically compared by Student’s t-test and F-test (18) with those obtained from the reported spectrophotometric method (10). The results did not reveal any significant difference
between them at 95% confidence level proving similar accuracy and precision. The content uniformity assay for CTP tablets was investigated and the results were presented as the mean percentage recovery ± SD (99.25 ± 0.8%). The extremely high sensitivity of the proposed method prompted us to check its applicability to the determination of CTP in biological fluids such as human serum and urine. The
Table 4. Determination of cefditoren pivoxil using silver nanoparticle–luminol–potassium ferricyanide sequential injection analysis-chemiluminescence detection in pure form, dosage forms and biological fluids Sample
Taken ng/mL
Found ng/mL
Recovery (%)
0.001 0.01 10 100 1000 5000
0.00101 0.01 9.999 98.65 978.8 4998 99.58 ± 1.1 6 1.21 0.44 1.11 0.001 0.009873 10.181 99.28 993.8 4883.5 99.48 ± 1.3 6 1.69 0.53 1.31 0.45 (2.228)a 2.7(5.05)a 0.0009986 0.009916 9.914 97.18 995.6 5000
101.00 100.00 99.99 98.65 97.88 99.96
Pure solution
Mean% ± SD n Variance %SEM %RSD MeiactW 200 mg/tablet
Mean% ± SD n Variance %SEM %RSD t F Urine sample
Mean% ± SD n Variance SEM% %RSD Serum sample
0.001 0.01 10 100 1000 5000
0.001 0.01 10 100 1000 5000
100.00 98.73 101.81 99.28 99.38 97.67
Reported method (10)
99.74 ± 0.8 6 0.64 0.33 0.80
99.86 99.16 99.14 97.18 99.56 100.00 99.15 ± 1.1 6 1.21 0.44 1.11
0.001 0.01 10 100 1000 5000
0.00101 0.009914 9.917 97.24 986 4994.5
Mean% ± SD n Variance %SEM %RSD
101.00 99.14 99.17 97.24 98.60 99.89 99.17 ± 1.2 6 1.44 0.48 1.21
a
Figures in parentheses are the tabulated values of t- and F-test at 95% confidence limits (18).
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Chemiluminescence determination of cefditoren pivoxil obtained results (Table 4) were satisfactory in terms of accuracy and precision as the recovery values were 99.17 ± 1.2% and 99.15 ± 1.1% with low percentage RSD for human serum and urine, respectively.
Conclusion A new sequential injection SIA-CL method for the determination of CTP was developed. The method was based on the enhancement effect of AgNPs on the luminol–ferricyanide system. The method was found to be sensitive, reproducible and accurate for the determination of the drug in its bulk powder, dosage forms and biological fluids. The enhancement effect of CTP on the employed system was proportional to its concentration, which showed good results relevant to the linear concentration range 0.001–5000 ng/mL. The proposed method has been proved to be fast, inexpensive, highly sensitive and precise. Acknowledgments This project was supported by King Saud University, Deanship of Scientific Research, College of Science Research Center.
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