Binary Solvents Dispersive Liquid—Liquid Microextraction (BS-DLLME) Method for Determination of Tramadol in Urine Using High-Performance Liquid Chromatography
© Kiarostami et al.; licensee BioMed Central Ltd. 2014
Received: 16 July 2013
Accepted: 25 January 2014
Published: 3 February 2014
Tramadol is an opioid, synthetic analog of codeine and has been used for the treatment of acute or chronic pain may be abused. In this work, a developed Dispersive liquid liquid microextraction (DLLME) as binary solvents-based dispersive liquid-liquid microextraction (BS-DLLME) combined with high performance liquid chromatography (HPLC) with fluorescence detection (FD) was employed for determination of tramadol in the urine samples. This procedure involves the use of an appropriate mixture of binary extraction solvents (70 μL CHCl3 and 30 μL ethyl acetate) and disperser solvent (600 μL acetone) for the formation of cloudy solution in 5 ml urine sample comprising tramadol and NaCl (7.5%, w/v). After centrifuging, the small droplets of extraction solvents were precipitated. In the final step, the HPLC with fluorescence detection was used for determination of tramadol in the precipitated phase.
Various factors on the efficiency of the proposed procedure were investigated and optimized. The detection limit (S/N = 3) and quantification limit (S/N = 10) were found 0.2 and 0.9 μg/L, respectively. The relative standard deviations (RSD) for the extraction of 30 μg L of tramadol was found 4.1% (n = 6). The relative recoveries of tramadol from urine samples at spiking levels of 10, 30 and 60 μg/L were in the range of 95.6 – 99.6%.
Compared with other methods, this method provides good figures of merit such as good repeatability, high extraction efficiency, short analysis time, simple procedure and can be used as microextraction technique for routine analysis in clinical laboratories.
KeywordsDispersive liquid-liquid microextraction Tramadol HPLC Urine
Tramadol ((±) cis-2-[(dimethylamino) methyl]-1-(3methoxyphenyl) cyclohexanol hydrochloride) is an opioid, synthetic analog of codeine and is not currently classified as a controlled substance[1–3]. Tramadol has been used since 1977 like other narcotics applied for the treatment of acute or chronic pain may be abused. In Iran, this drug is easily available for patient without prescription and according to annual reports of ministry of health; 350 million tramadol tablets (100 mg) were sold in 2006–2007 and recently, it has become one of the most widely dispensed analgesics in Iran’s essential drugs list[5, 6]. The extraction of tramadol from biological samples has usually been carried out by using liquid-liquid extraction (LLE) and solid phase extraction (SPE)[4, 7–9]. However, LLE is time consuming and requires large amounts of organic solvent and SPE uses much less than LLE, but can be relatively expensive. Recently, other extraction methods as free solvent and miniaturized extractions, such as liquid phase microextraction (LPME)[10, 11], solid phase microextraction (SPME), solvent bar microextraction (SBME), liquid phase microextraction with back extraction (LPME-BS), three - phase hollow fiber liquid phase microextraction (HF-LPME), have successfully been developed for determination of tramadol from different matrices. Dispersive liquid-liquid microextraction (DLLME) is a miniaturized liquid extraction that was introduced in 2006 by Rezaee and coworkers. However, in this method, the selection of extraction solvents is limited to one type of heavier or lighter extraction solvent than water.
In our previous work, a new method based on DLLME methodology as binary solvents–based dispersive liquid-liquid microextraction (BS-DLLME) was developed for determination of patulin from apple juice samples. In this method, two kinds of extraction solvents (mixture of low and high density solvents) can be used simultaneously. In the present study, a rapid, sensitive and simple BS-DLLME and high performance liquid chromatography coupled with florescence detection has been carried out for the extraction and pre-concentration of tramadol in urine samples.
HPLC-grade methanol, acetonitrile, acetone, chloroform, analytical grade ethyl acetate and deionized water were obtained from Merck chemical co (Darmstadt, Germany). Carbon tetrachloride (CCl4) with grade of trace analysis was obtained from Merck. The pure substances of tramadol were kindly gifted by Grünenthal chemical co (Stolberg, Germany). Phosphoric acid, sodium hydroxide and sodium chloride were all of analytical grade from Merck and were used without further purification. All the glassware used in experiments first washed with HPLC grade water and acetone and then dried in an oven. For calibration studies, blank urine samples were kindly obtained from one female healthy volunteer in our lab which not exposed to the mentioned drug. The use of tramadol in healthy subjects has been approved in Tehran University of Medical Sciences ethics committee (Ethics board code 4233).
For recovery studies, fresh urine samples from three male volunteers with abuse of tramadol were kindly provided by the Loghman hospital (Tehran, Iran). The study of tramadol pharmacokinetics in drug abused subjects has been approved in Tehran University of Medical Sciences ethics committee (Ethics board code 20324).
Standard solution of tramadol in concentration of 1 mg/mL was prepared by dissolving 10 mg of this compound in 10 mL HPLC-grade water. Working standard solutions of tramadol were prepared by dilution of the stock solution using HPLC-grade water. Stock and standard solutions of this compound were stored at 4°C.
For separating and analyzing the drug a WellChrom HPLC instrument from Knauer Company (Berlin, Germany) was applied. The chromatographic apparatus equipped with a fluorescence RF-10AXL detector (excitation wavelength of 200 nm and emission wavelengths of 301 nm). Gradient HPLC K-1001 pump and online K-5020 degasser. A Rheodyne model 7725i injector with a 20 μL loop was applied to inject the samples. Chromatographic data were acquired and analyzed using ChromGate Chromatography Software from Knauer Company. Separation was carried out on a ChromolithTM Performance RP-18e, 100 mm × 4.6 mm column (Merck, Darmstadt, Germany) protected by a ChromolithTM Guard Cartridge RP-18e 5 mm × 4.6 mm. A mixture of water and methanol (81:19 v/v) adjusted to pH 2.5 by phosphoric acid at a flow rate of 2 mL/min in isocratic elution mode was used as a mobile phase. Eppendorf centrifuge 4515c (Netheler-Hinz GMBH Germany) was used for sedimentation of the dispersive phase.
Standard solutions containing 100 μg/mL of tramadol was prepared in HPLC grade water at (4°C) and brought to room temperature just prior to use. The pH of urine sample containing 100 μg/mL tramadol (spiked urine sample) at pH = 10 by addition of 5 M NaOH. Then 5 mL of this sample was placed in centrifuge tube, after centrifugation at 1133 × g for 10 min, upper solution was separated from sediment and was transferred to a 10 mL glass test tube.
A mixture of a disperser solvent and binary extraction solvents (ethyl acetate and chloroform) were injected rapidly into the mentioned pretreated urine solution (5 ml) by 1.00 mL syringe and immediately a cloudy solution was formed. After centrifuging the cloudy solution for 10 min at 1133 × g, the dispersed fine droplets of ethyl acetate and chloroform were settled in the bottom of conical test tube. The deposited phase was transferred to another glass tube and evaporated to dryness under a gentle air stream. The residue was dissolved in 70 μL mobile phase and 20 μL was injected into the HPLC with fluorescence detection system for analysis.
Results and Discussion
In order to optimize the BS-DLLME for determination of tramadol in urine samples, the effective parameters on extraction efficiency such as the type and volume of high density extraction solvent, the volume of ethyl acetate as low density extraction solvent, the type and volume of disperser solvent, salt addition and extraction time were studied. Statistical calculations (single factor analysis of variance and unpaired t-test) carried out with Microsoft® excel 2007 for comparing of data. Significant difference was pretended if the probability level (p) was less than of 0.05.
Selection of high density extraction solvent
Effect of extraction solvent volume
Effect of ethyl acetate and its volume
Selection of disperser solvent
Effect of disperser solvent volume
Effect of salt addition
Effect of extraction time
Overall, according to optimum condition, the values of studied factors were as follows: 0.6 mL acetone as disperser solvent, 100 μL binary extraction solvents (70 μL of chloroform and 30 μL of ethyl acetate) and 7.5% (w/v) NaCl.
Figures of merit in the BS-DLLME
RSD, %(N = 6)
Y = 78382x + 24663
Application in real sample
Relative recoveries of tramadol in urine samples
Concentration determined mean ± SDb/μg/ L
9.96 ± 0.2
28.67 ± 1.5
59.03 ± 3.48
Comparison of BS-DLLME with other methods
Comparison of the proposed method with other methods for the extraction of tramadol
Liquid extraction HPLC
Human oral fluid
Human plasma, urine
LLE-ion pair formation
The present study has proposed a new method for determination of tramadol in urine samples using the BS-DLLME coupled with HPLC-fluorescence detection. BS-DLLME method provides good repeatability and higher recoveries within a short time. The comparison of this method with others demonstrated that BS-DLLME was very fast, simple and inexpensive with good figures of merit. In comparison to conventional DLLME, the selection of extraction solvents in BS-DLLME is not limited to high density solvents. In summary, the developed methodology shows good performance of analytical protocol, exhibits excellent recoveries for tramadol in urine samples.
Binary solvents dispersive liquid liquid microextraction
Solid phase extraction
Solid phase microextraction
Molecular imprinting solid phase extraction
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