Quantitative analysis of piroxicam using temperature-controlled ionic liquid dispersive liquid phase microextraction followed by stopped-flow injection spectrofluorimetry
- Mohsen Zeeb1Email author,
- Parisa Tayebi Jamil†1,
- Ali Berenjian†1,
- Mohammad Reza Ganjali†2 and
- Mohamad Reza Talei Bavil Olyai†1
© Zeeb et al.; licensee BioMed Central Ltd. 2013
Received: 13 April 2013
Accepted: 23 July 2013
Published: 29 July 2013
Piroxicam (PXM) belongs to the wide class of non-steroidal anti-inflammatory drugs (NSAIDs). PXM has been widely applied in the treatment of rheumatoid arthritis, gonarthrosis, osteoarthritis, backaches, neuralgia, mialgia. In the presented work, a green and benign sample pretreatment method called temperature-controlled ionic liquid dispersive liquid phase microextraction (TCIL-DLPME) was followed with stopped-flow injection spectrofluorimetry (SFIS) for quantitation of PXM in pharmaceutical formulations and biological samples.
Temperature-controlled ionic liquid dispersive liquid phase microextraction (TCIL-DLPME) was applied as an environmentally friendly sample enrichment method to extract and isolate PXM prior to quantitation. Dispersion of 1-hexyl-3-methylimidazolium hexafluorophosphate ([Hmim][PF6]) ionic liquid (IL) through the sample aqueous solution was performed by applying a relatively high temperature. PXM was extracted into the extractor, and after phase separation, PXM in the final solution was determined by stopped-flow injection spectrofluorimetry (SFIS).
Results and Major Conclusion
Different factors affecting the designed method such as IL amount, diluting agent, pH and temperature were investigated in details and optimized. The method provided a linear dynamic range of 0.2-150 μg l-1, a limit of detection (LOD) of 0.046 μg l-1 and a relative standard deviation (RSD) of 3.1%. Furthermore, in order to demonstrate the analytical applicability of the recommended method, it was applied for quantitation of PXM in real samples.
Keywords1-Hexyl-3-methylimidazolium hexafluorophosphate Stopped-flow injection spectrofluorimetry Pharmaceutical formulations Biological samples
Piroxicam (PXM, 4-hydroxy-2-methyl-N-(pyridine-2-yl)-2H-1, 2-benzo-thiazine-3-carboxamide-1,2-dioxide) belongs to the wide class of non-steroidal anti-inflammatory and analgesic drugs (NSAIDs). This drug has been widely used to treat podagrous and rheumatoid arthritis, gonarthrosis, osteoarthritis, backaches, neuralgia, mialgia, and other diseases accompanied by the pain syndrome or an inflammatory process[2, 3]. To our knowledge, until now, some analytical techniques such as high performance liquid chromatography (HPLC), capillary electrophoresis, liquid chromatography-mass spectrometry (LC/MS), spectrometry, electrochemistry have been reported for the quantitative analysis of PXM. The data obtained in these works reveal that the sensitivity and selectivity is not acceptable which is due to low amount of analyte in real sample and matrix impact. Thus, development and application of practical and benign sample pretreatment procedures prior to quantitation are important tasks of chemists. Combination of sample enrichment procedures with inexpensive, selective and sensitive determination tools such as spectrofluorimetry makes it possible to determine trace levels of analytes, provide better selectivity and extremely reduce the cost of analysis.
Previous studies reveal that ionic liquids (ILs) are suitable materials in sample enrichment procedures due to their special properties. One of the practical advantages of ILs application in microextraction methods is the removal of toxic extraction materials. The most popular extraction techniques based on ILs are ionic liquid-based dispersive liquid-liquid microextraction (IL-DLLME)[10–13], cold-induced aggregation microextraction (CIAME) and temperature-controlled ionic liquid dispersive liquid phase microextraction (TCIL-DLPME).
In TCIL-DLPME procedure, dispersion of IL through the sample aqueous solution is occurred by applying a relatively high temperature. This phenomenon increases the chance of analyte extraction into extractor phase. By cooling the solution and centrifugation, it is possible to collect the IL-phase and transfer it to analytical tool for subsequent analysis. Based on the data obtained in our previous works, the out put of ionic liquid-sample enrichment methods meaningfully depends on variations in the values of ionic strength[16–19]. It is obvious that this factor can affect on the solubility of extractor. In order to obtained stable results, a common ion of extractor solvent (PF6-) was dissolved in the studied solution. Using this way, the volume of the extractor was not affected by changes occurred in the value of ionic strength.
In this study, spectrofluorimetric method was utilized for quantitation owning to some advantages including good selectivity and sensitivity, low cost of analysis and high response speed. To our knowledge, for the first time, TCIL-DLPME was combined with stopped-flow injection spectrofluorimetry (SFIS) for trace determination of PXM. The factors influencing the proposed method were studied in details and optimized. Finally, In order to demonstrate the analytical advantage of TCIL-DLPME-SFIS, It was utilized for quantitation of PXM in pharmaceutical and biological samples.
Material and methods
Fluorescence signals were recorded using FP-6200 spectrofluorimeter (JASCO Corporation, Tokyo, Japan). Xenon discharge lamp, peristaltic sipper unit (model SHP-292), and micro-cell (path length of 3 mm and volume of 15 μL) were used as the accessories. The PC-based Windows® Spectra Manager™ software was applied for recording and processing of the analytical signals. A centrifuge was purchased from Hettich (Tuttlingen, Germany) and used for accelerating the phase separation. The pH-meter model 692 (Herisau, Switzerland) supplied with a glass-combined electrode was used for pH measuring.
Reagents and materials
Analytical-reagent grade of chemicals was used in all experiments. All aqueous solutions were prepared using ultra pure water. Piroxicam hydrochloride was obtained from Alhavi Pharmaceutical Company (Tehran, Iran). 1-Hexyl-3-methylimidazolium hexafluorophosphate [Hmim][PF6], acetone, acetonitrile, methanol, ethanol, NH3, HCl, NaOH and sodium hexafluorophosphate (NaPF6) were purchased from Merck (Darmstadt, Germany). A stock solution of 200 mg ml-1 of NaPF6 was obtained by dissolving required amount of NaPF6 in water. Piroxicam has a low solubility in water (0.00004 M). Hence, a stock solution of piroxicam (1000 mg l-1) was obtained by dissolving 10 mg of pure drug in 10 ml of 5 M NH3. The obtained stock solution was stored at 5°C in the dark until use. Working solutions of lower concentrations were prepared daily from the above stock solution as required. Piroxicam capsules and tablets were obtained from a local pharmacy.
In the presented microextraction procedure, aliquots of 10.0 ml sample solution (pH = 3) containing PXM in the concentration range of 0.2-150 μg l-1 were placed into a screw-glass test tube with conic bottom. Then, 55 mg of [Hmim][PF6] IL and 0.9 ml of NaPF6 (200 mg ml-1) was added. After shaking, the conical tube was heated in a water bath with the temperature controlled at 40 °C for 5 min. Under this condition, the extractor was dissolved completely and the PXM was effectively extracted into the IL phase. After this step, the resulting solution was placed in ice-water bath and cooled for 7 min. After this process, a turbid condition was formed due to the decrease of the solubility of the extractor. The obtained solution was centrifuged for 5 min at 4000 rpm. The aqueous phase was removed using a proper syringe. For conditioning the extractor prior to quantitation by SFIS, the residue in the vessel was diluted to 250 μL by adding required amount of ethanol. Finally, the diluted IL-phase in the vessel was transferred to the spectrofluorimeter by the peristaltic sipper unit.
Stopped-flow injection spectrofluorimetry
Preparation of pharmaceutical formulations and biological samples
In order to obtain analyzable pharmaceutical solution, six piroxicam capsules or powdered tablets were entirely mixed, and afterwards a proper amount of powder containing 10 mg of PXM was dissolved in 5 M NH3. To prepare a clear solution, the resulting sample was filtered into a 100 ml volumetric flask by Whatman No. 42 filter paper, and made up to the mark using ultra pure water. Prior to quantitation, a proper dilution was performed, in order to ensure the concentration of the pharmaceutical solution was in the dynamic range.
In order to obtain analyzable human plasma samples, 1.0 ml of this real sample were spiked with PXM and deproteinized by addition of 5 ml acetonitrile. After this process, the resulting biological real sample was centrifuged at 4000 rpm for 15 min, and 2.0 ml of the clear upper pahse was diluted to 100 ml. Aliquot of 10 ml of this sample was utilized for each test. 10 ml of human urine samples were transferred into centrifuge tubes. Urine samples were centrifuged for 4 min at 4000 rpm. Afterwards, aliquots of 2 ml from clear upper phase were transferred into new centrifuge tubes, spiked with different concentrations of PXM and diluted to 20 ml. In order to determine the trace levels of PXM, aliquot of 10 ml of this solution was subjected to TCIL-DLPME-SFIS.
Results and discussion
In this equation, Csed and C0 show the concentration of PXM in the enriched phase and initial concentration of PXM in the aqueous phase, respectively. Csed, for extractor solvents and diluting agents, was measured using the calibration graph obtained from direct injection of PXM in enriched phase.
Spectrofluorimetric calibration curve and spectral characteristics
Selection of IL
Following consideration can ease the selection of IL: (a) the density of extractor must be higher than aqueous phase, (b) extractor must be liquid through the experiments, (c) IL must show proper hydrophobic manner and (d) IL must be certainly inexpensive. ILs containing (CF3SO2)2N− are relatively expensive and those containing PF6- are relatively inexpensive. According to the mentioned points, [Hmim][PF6] was selected as a microextraction solvent in all experiments.
Type of diluting agent
Kind of diluting agent is one the important factors in TCIL-DLPME. Since the density of ionic liquid is relatively high, this extractor must be diluted prior to transfer and quantitation. In the present test, some organic diluting agent involving methanol, ethanol, acetone and acetonitrile were investigated. Because of the better performance of ethanol and its better safety, this diluting material was utilized in all the tests.
Influence of IL amount
Influence of common ion amount and ionic strength
Based on the results obtained in our previous studies, in traditional sample enrichment methods based on ILs, the volume of remaining enriched phase depends on the value of the ionic strength. As it was explained above, in order to overcome this phenomenon, a common ion of extractor phase was dissolved in the sample under study. NaNO3 was utilized as an electrolyte to test the impact of this factor. This factor was carefully evaluated over the range of 0–35% (w/v) and no measurable impact was observed in this range.
Influence of pH
Influence of temperature
Complete dispersion of IL-phase in the sample solution is occurred by applying a relatively high temperature. The obtained data revealed that the solubility of extractor at temperature above 30°C significantly increased. A series experiments were performed in order to optimize the temperature in the range of 30–70°C. A better stability and sensitivity was obtained at 40°C, therefore this temperature was used an optimum value. In the next step, sample solutions were cooled in the temperature range of 0-25°C. By applying a low temperature, the analytical sensitivities improve which is due to the solubility decrease of extractor phase at low temperatures. Hence, a temperature of 0°C was utilized through the rest of the work.
Influence of centrifuge conditions
Evaluated and optimized experimental conditions of TCIL-DLPME-SFIS
Amount of [Hmim][PF6] ionic liquid (mg)
Amount of common ion (NaPF6) (mg)
Centrifugation rate (rpm)
Centrifugation time (min)
Suction time (s)
Transferring time (s)
Delay time (s)
Excitation wavelength (nm)
320 ± 5
Emission wavelength (nm)
455 ± 5
Excitation and emission slit widths (nm)
Selectivity of the method
In the present study, in order to demonstrate the selectivity of the method, the impact of some possible interfering substances such as Na+, Ca2+, Zn2+, Mg2+, Cl-, PO43-, SO42-, citric acid, starch, glucose, lactose, sucrose, ascorbic acid, uric acid, oxalic acid and lactic acid on the determination of PXM at 75 μg L-1 was tested. 100-fold the mentioned substances have no observable impact on the fluorescence responses (fluorescence response change below 5%).
Analytical performance of the proposed methodology
Dynamic range (μg L-1)
Correlation coefficient (R2)
LODa (μg L-1)
RSDb (%) (n = 4 ) (CPXM = 75 μg L-1)
PFc (CPXM = 75 μg L-1)
Sample volume (mL)
In the mentioned equation, the value of K is 3, sbl shows the standard deviation of the blank signals and m shows the calibration slope. By this way, the LOD found was 0.046 μg l-1. In order to define the repeatability of the designed system, four 75 μg l-1 standard solutions of PXM were analyzed and by this way the relative standard deviation (RSD) was 3.1%.
Analysis of PXM in real samples
Determination of PXM in real samples using TCIL-DLPME-SFIS a
Added (μg l-1)
Found (μg l-1)
In this study, a benign and simple sample enrichment method called temperature- controlled ionic liquid dispersive liquid phase microextraction (TCIL-DLPME) was followed by stopped-flow injection spectrofluorimetry (SFIS) for quantitation of piroxicam in pharmaceutical and biological samples. Ionic liquid was used an extractor instead of toxic solvents, in order to protect the environment against harmful material and provide a better safety for chemists during the experiments. Traditional sample enrichment methods based on the application of ionic liquid suffer from some limitations such as the dependence of extraction efficiency on ionic strength values. To remove the latter problem, the microextraction procedure was assisted by a common ion of extractor. The application of stopped-flow injection mode makes it possible to increase the speed of quantitative measurements, decrease the required enriched phase and provide a better reproducibility and automation. Fluorimetry was applied as a determination technique because of some advantages including good selectivity and sensitivity, low cost of analysis and high response speed. For quality control of PXM, the present methodology is an efficient, benign, simple and inexpensive analytical tool.
Support of this investigation by the Islamic Azad University Tehran south branch through grant is gratefully acknowledged.
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