Journal of Chromatographic Science Advance Access published January 22, 2015 Journal of Chromatographic Science 2015;1– 7 doi:10.1093/chromsci/bmu214

Article

Simultaneous Determination of Tramadol and Its Metabolite in Human Urine by the Gas Chromatography– Mass Spectrometry Method Bilal Yilmaz1* and Ali Fuat Erdem2 1

Department of Analytical Chemistry, Faculty of Pharmacy, Ataturk University, Erzurum 25240, Turkey, and 2Department of Anesthesiology and Reanimation, Faculty of Medicine, Sakarya University, Sakarya 54187, Turkey

*Author to whom correspondence should be addressed. Email: [email protected] Received 16 April 2014

A sensitive and efficient method was developed for determination of tramadol and its metabolite (O-desmethyltramadol) in human urine by gas chromatography –mass spectrometry. Tramadol, O-desmethyltramadol and medazepam (internal standard) were extracted from human urine with a mixture of ethylacetate and diethylether mixture (1 : 1, v/v) at basic pH with liquid– liquid extraction. The calibration curves were linear (r 5 0.99) over tramadol and O-desmethyltramadol concentrations ranging from 10 to 200 ng/mL and 7.5 to 300 ng/mL, respectively. The method had an accuracy of >95% and intra- and interday precision (relative standard deviation %) of 4.93 and 4.62% for tramadol and O-desmethyltramadol, respectively. The extraction recoveries were found to be 94.1 + 2.91 and 96.3 + 3.46% for tramadol and O-desmethyltramadol, respectively. The limit of quantification using 0.5 mL human urine was 10 ng/mL for tramadol and 7.5 ng/mL for O-desmethyltramadol. After oral administration of 100 mg of tramadol hydrochloride to a patient, the urinary excretion was monitored during 24 h. About 15% of the dose was excreted as unchanged tramadol. Introduction Tramadol (Figure 1), (+)-trans-2-[(dimethylamino) methyl]1-(3-methoxyphenyl) cyclohexanol, is an analgesic used in treatment of mild pain (1). The plasma concentration in therapeutic terms has a domain between 100 and 300 ng/mL, being almost completely and quickly absorbed through a metabolism giving different metabolites due to the action of cytochrome P450 isozyme CYP2D6 (2 –6). The bio-availability ranges from 65 to 70% because of the early stage of metabolism, knowing also that 6% of population has a slow CPY2D6 activity giving a slightly reduced analgesic effect. Tramadol is transformed to O- and N-desmethyl compounds (7 –9). Several analytical methods for determination of tramadol and its metabolites were used, starting from gas chromatography (GC) with nitrogen selective or mass spectrometry (MS) detection (10 –12), but recently the new separation methods were introduced, capillary electrophoresis (13) and high-performance liquid chromatography (HPLC) methods with electrochemical (14), MS (15 –18) or fluorescence detection (19 –24). El-Sayed et al. (10) have developed a GC– MS method for the determination of tramadol and O-desmethyltramadol in human urine with positive electron impact ionization following b-glucuronidase hydrolysis and liquid – liquid extraction. The proposed method has a total run time of 12.6 min. A linear calibration curve was obtained over the range 10 – 1,000 ng/mL. Intra-assay precision was within 1.29–6.48% and inter-assay precision

was within 1.28–6.84% for tramadol and O-desmethyltramadol. Intra-assay accuracy was within 91.79 – 106.89% for tramadol and O-desmethyltramadol. This method is also the most comprehensive method which can extract tramadol in a single extraction procedure. Our recovery is better than those of the studies reported by El-Sayed et al. Our method has a total run time 8.0 min which is faster than previously published GC – MS methods for tramadol and its metabolites (10). Ghasemi (11) has developed a GC– MS method for the determination of tramadol in different biological samples using solvent bar microextraction. The detection limit was 0.02 mg/L with 4.5% relative standard deviation (RSD). Tramadol was extracted from plasma with a solid-phase extraction (SPE) procedure. This method is also the most comprehensive method, which can extract tramadol in a single extraction procedure. The method has a total run time of 15 min. In our study, the retention time of tramadol was much shorter than that studied by Ghasemi (11). The UV detection from biological samples is not suitable because of low sensitivity and selectivity, although there is a benzene ring present in tramadol and its metabolite molecules (25 – 30). For the moment, only two types of detectors have reached low quantification levels: MS and fluorescence. By using any of them, the lowest level for tramadol quantification was 1.0 ng/mL (12, 19, 21), meaning that the lowest limit of detection was 0.2 –0.5 ng/mL (12, 19). Ebrahimzadeh et al. (31) have developed multivariate optimization of surfactant-assisted directly suspended droplet microextraction combined with GC for the preconcentration and determination of tramadol in biological samples. In this technique, a free microdroplet of solvent is transferred to the surface of an immiscible aqueous sample containing Triton X-100 and tramadol while being agitated by a stirring bar placed on the bottom of the sample vial. After the predetermined time, the microdroplet of solvent is withdrawn by a syringe and analyzed. Pinho et al. (32) have developed simultaneous quantification of tramadol and O-desmethyltramadol in hair samples by GC – MS. In this study, a methodology aimed at the simultaneous quantification of tramadol and metabolite in human hair was developed and validated. After decontamination of hair samples (60 mg), tramadol and metabolite were extracted with methanol in an ultrasonic bath (5 h). Purification was performed by SPE using mixed-mode extraction cartridges. Subsequently to derivatization, analysis was performed by GC –MS. Several methods have been reported for determination of tramadol, including the GC–MS method, human plasma, saliva, hair and biological materials. But, on extensive survey of the literature, no

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Figure 1. Structural formula of tramadol (A), O-desmethyltramadol (B) and medazepam (IS) (C).

method is reported till date for determination of tramadol and metabolite by the GC –MS method without derivatization in patient human urine. Therefore, the aim of our study was to develop a specific, sensitive, precise and accurate GC– MS method for analysis of tramadol and its metabolite in patient human urine. The proposed method was fully validated in respect to limits of detection (LOD) and limits of quantification (LOQ), precision, accuracy, linearity, specificity, stability and extraction recovery parameters according to International Conference on Harmonization (ICH) guidelines (33). The advantages of the present method include a simple and single-step extraction procedure using inexpensive chemicals and short run time. Besides, this method was used to assay the tramadol and O-desmethyltramadol in urine samples obtained from a patient. At the same time, this method was used to a pharmacokinetic study after therapeutic doses of tramadol and O-desmethyltramadol.

Experimental Chemicals and reagents Tramadol hydrochloride and O-desmethyltramadol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Medazepam was obtained from Criminal Police Laboratory (Erzurum, Turkey). Sodium carbonate, ethylacetate, hexane, diethylether, dichloromethane, acetonitrile, butanol and chloroform were purchased from Sigma-Aldrich. Contramal tablet drug (100 mg tramadol hydrochloride) was obtained from the pharmacy (Erzurum, Turkey).

Apparatus and analytical conditions Chromatographic analysis was carried out on an Agilent 6890N GC system equipped with 5973 series mass selective detector, 7673 series autosampler and chemstation (Agilent Technologies, Palo Alto, CA, USA). A HP-5 MS column with 0.25 mm film thickness (30 m  0.25 mm ID) was used for separation. Splitless injection was used and the carrier gas was helium at a flow rate of 1 mL/ min. The injector and detector temperatures were 2508C. The temperature programs of the GC oven were as follows: initial temperature 708C, held for 1 min, increased to 2508C at 358C/min held for 1 min and finally to 2908C at 208C/min with a final hold of 1 min. The MS detector parameters were transfer line temperature 2908C, solvent delay 3 min and electron energy 70 eV. The MS was run in scan mode (m/z 40–500) for qualitative analysis 2 Yilmaz and Erdem

and selected ion monitoring mode for quantitative analysis [(m/ z 58 for tramadol and O-desmethyltramadol, and m/z 207 for internal standard (IS)] (Figure 2).

Preparation of stock and standard solutions Stock solutions of tramadol and O-desmethyltramadol were prepared by dissolving the accurately weighed reference compounds in methanol to give a final concentration of 10 mg/mL of both. The solutions were then serially diluted with methanol to achieve standard working solutions at concentrations of 10, 25, 50, 100, 150, 175 and 200 ng/mL and 7.5, 15, 25, 50, 100, 200 and 300 ng/mL for tramadol and O-desmethyltramadol, respectively. Stock solution of IS was prepared in methanol at the concentration of 5 mg/mL. All the solutions were stored at 48C and were brought to room temperature before use. The quality control (QC) solutions were prepared by adding aliquots of standard working solution of tramadol and O-desmethyltramadol to final concentrations of 12.5, 75 and 175 ng/mL and 7.5, 75 and 250 ng/mL containing 0.1 mL IS (500 ng/mL)

Extraction procedure The liquid – liquid extraction method was used in this study. Several solvents (ethylacetate, diethylether, dichloromethane, acetonitrile, butanol and chloroform) were tested for the extraction. Finally, the ethylacetate and diethylether mixture (1 : 1, v/v) proved to be the most efficient in extracting tramadol and O-desmethyltramadol from human urine. A 0.5 mL of the urine samples was spiked with 1 mL of tramadol or O-desmethyltramadol, 0.1 mL of IS and 1 mL of 0.1 M sodium carbonate were added. After vortex mixing for 5 s, 4 mL of ethylacetate and diethylether mixture (1 : 1, v/v) were added. The mixture was vortexed for 30 s and then centrifuged at 3,000  g for 3 min. The organic layer was transferred into another tube and evaporated to dryness at room temperature under nitrogen gas. The dry residue was dissolved in 100 mL of methanol. The mixture was vigorously shaken and then 1 mL sample was injected into the GC – MS system.

Results Method development and optimization The method development for the assay of tramadol and O-desmethyltramadol was based on their chemical properties.

Figure 2. Structural formula and MS spectra of tramadol (A), O-desmethyltramadol (B) and medazepam (IS) (C).

In this study, the capillary column coated with 5% phenyl and 95% dimethylpolysiloxane is a good choice for separation of these analytes because they elute as symmetrical peaks at a wide range of concentrations. Different temperature programs were investigated for GC oven. At the end of this investigation, the best temperature program was selected for a good separation. The splitless injection mode was chosen. Additionally, preliminary precision and linearity studies performed during the development of the method showed that the 1 mL injection volume was reproducible and the peak response was significant at the analytical concentration chosen. Validation of the method To evaluate the validation of the present method, parameters as selectivity, linearity, precision, accuracy, LOD and LOQ, recovery and stability were investigated according to the ICH validation guidelines (33). Selectivity The selectivity of the assay was checked by comparing the chromatograms of batches of blank urine with the corresponding spiked urine. Each blank sample was tested for the observation of interference, and no endogenous interferences were encountered (Figure 3a). The retention time of tramadol and O-desmethyltramadol in human urine was 6.2 and 6.5 min with good peak shape (Figure 3b).

Linearity Calibration curves for the urine assay developed with a peak-area ratio (y) of tramadol and O-desmethyltramadol to IS versus drug concentration were found to be linear over the concentration range 10–200 and 7.5–300 ng/mL using a weighted least squares method. The linear regression equations of the calibration curves of tramadol and O-desmethyltramadol are summarized in Table I. The correlation coefficients were found to be .0.99. Precision and accuracy The precision of the analytical method was determined by repeatability (intraday) and intermediate precision (interday). Repeatability was evaluated by analyzing spiked blank urine six times per day, at three different concentrations which were urine QC samples. The intermediate precision was evaluated by analyzing the same urine samples once daily for 3 days. The RSD of the predicted concentrations from the regression equation was taken as precision. The accuracy of this analytical method was assessed as the percentage relative error. For all the concentrations studied, intra- and interday RSD values were 4.93% and for all concentrations of tramadol and O-desmethyltramadol the relative errors were 5.89%. These results are given in Table II. LOD and LOQ The sensitivity was evaluated by the LOQ, the lowest concentration of the urine spiked with tramadol or O-desmethyltramadol Determination of Tramadol and Its Metabolite in Human Urine by GC– MS 3

in the calibration curve. The LOD was determined as the lowest concentration, which gives a signal-to-noise ratio of 3 for tramadol or O-desmethyltramadol. Under the experimental conditions, the LOQ values were 10 and 7.5 ng/mL for tramadol and O-desmethyltramadol, respectively. Furthermore, the LOD

Figure 3. Representative chromatograms of (a) drug-free human urine, (b) the human urine spiked with tramadol (200 ng/mL), O-desmethyltramadol (150 ng/mL) and IS (500 ng/mL), (c) after 12 h intravenous administration of 100 mg tramadol hydrochloride, (1) tramadol and (2) O-desmethyltramadol.

Table I Linearity of Tramadol and O-Desmethyltramadol in Human Urine Parameter

Tramadol

O-Desmethyltramadol

Linearity (ng/mL) Regression equationa Standard deviation of slope Standard deviation of intercept Correlation coefficient Standard deviation of correlation coefficient Limit of detection (ng/mL) Limit of quantification (ng/mL)

10 –200 y ¼ 0.025x þ 0.0711 5.2  1023 1.5  1023 0.999 5.5  1024 3.0 10

7.5 –300 y ¼ 0.034x þ 0.134 2.8  1024 2.1  1022 0.998 4.6  1023 2.5 7.5

a

Based on six calibration curves, y is the peak-area ratio and x is the concentration (ng/mL).

values were 3.0 ng/mL for tramadol and 2.5 ng/mL for O-desmethyltramadol (Table I). Recovery For extraction of tramadol and O-desmethyltramadol from human urine, the liquid – liquid extraction technique was tried using ethylacetate, diethylether, dichloromethane, acetonitrile, butanol and chloroform. It was observed that extraction yields were very low. The urine sample was done alkaline by 1 mL of 0.1 M sodium carbonate solution and then tramadol and O-desmethyltramadol were extracted from urine using 4 mL of ethylacetate and diethylether mixture (1 : 1, v/v). The analytical recovery of tramadol and O-desmethyltramadol from human urine was assessed by direct comparison of concentrations of tramadol and O-desmethyltramadol obtained after the whole extraction procedure by using six replicates at different concentration levels in the calibration graph versus standard tramadol and O-desmethyltramadol solutions. The extraction recoveries of tramadol and O-desmethyltramadol from human urine were between 88 and 104% as summarized in Table III. Matrix effect The blank urines used in this study were from three different batches of healthy human urine. If the ratio ,85 or .115%, a matrix effect was implied. The relative matrix effect of tramadol and O-desmethyltramadol at three different concentrations was less than +1.2% (Table IV). The results showed that there was no matrix effect of the analytes observed from the matrix of urine in this study. Stability The stability of tramadol and O-desmethyltramadol in human urine was studied under a variety of storage and handling conditions at low (50 ng/mL) and high (300 ng/mL) concentration levels. The short-term temperature stability was assessed by analyzing three aliquots of each of the low and high concentration samples that were thawed at room temperature and kept at this temperature for 8 h. Freeze– thaw stability (2208C in urine) was checked through three cycles. Three aliquots at each of the low and high concentrations were stored at 2208C for 24 h and thawed unassisted at room temperature. When completely

Table II Precision and Accuracy of Tramadol and O-Desmethyltramadol in Human Urine Added (ng/mL)

Tramadol 12.5 75 175 O-Desmethyltramadol 7.5 75 250

Intraday

Interday

Found + SD

Precision % RSDa

Accuracyb

Found + SD

Precision % RSDa

Accuracyb

11.92 + 0.223 71.47 + 1.251 168.54 + 1.635

1.87 1.75 0.97

24.64 24.71 23.69

12.09 + 0.596 78.02 + 2.956 171.09 + 4.414

4.93 3.79 2.58

23.28 4.03 22.23

7.41 + 0.342 70.58 + 1.532 242.62 + 3.164

4.62 2.17 1.30

21.20 25.89 22.95

7.34 + 0.286 71.48 + 1.823 228.16 + 5.924

3.89 2.55 2.59

22.13 24.69 24.74

SD, standard deviation of six replicate determinations; RSD, relative standard deviation. Average of six replicate determinations. Accuracy: (% relative error) (found 2 added)/added  100.

a

b

4 Yilmaz and Erdem

thawed, the samples were refrozen for 24 h under the same conditions. The freeze–thaw cycles were repeated three times and then analyzed on the third cycle. The long-term stability was determined by analyzing three aliquots of each of the low and high concentrations stored at 2208C for 1 week. The results of the stability studies are given in Table V and no significant degradation of tramadol and O-desmethyltramadol was observed under the tested conditions. Application of the method Prior to the study, the clinical protocol was approved by the Ethics Committee of Faculty of Medicine, Ataturk University. Table III Recovery of Tramadol and O-Desmethyltramadol in Human Urine Added (ng/mL)

Found (mean + SD)

Tramadol 10 25 50 100 150 175 200 O-Desmethyltramadol 7.5 15 25 50 100 200 300

% RSDa

% Recovery

8.8 + 0.44 24 + 0.98 47 + 2.41 97 + 3.84 141 + 5.23 162 + 6.17 193 + 6.42

88 96 94 97 94 93 97

5.0 4.1 5.1 3.9 3.7 3.8 3.3

7.4 + 0.32 14 + 0.46 26 + 0.74 48 + 1.24 97 + 1.73 196 + 2.67 292 + 3.86

99 93 104 96 97 98 97

4.3 3.3 2.8 3.6 1.8 1.4 1.3

SD, standard deviation of six replicate determinations; RSD, relative standard deviation. a Average of six replicate determinations.

Table IV Matrix Effect Evaluation of Tramadol and O-Desmethyltramadol and IS in Human Urine (n ¼ 3) Samples

Concentration level (ng/mL)

A (mean + SD)

B (mean + SD)

% Matrix effect

Tramadol

50 100 200 50 150 250 500

47 + 2.86 97 + 6.98 202 + 8.67 46 + 3.74 136 + 8.28 232 + 9.14 504 + 8.37

52 + 3.29 103 + 8.45 198 + 9.73 54 + 2.87 145 + 6.62 253 + 8.64 513 + 7.76

1.11 1.06 20.98 1.17 1.07 1.09 1.02

O-Desmethyltramadol

IS

The patient was given written informed consent to participate in the study according to the principles of the Declaration of Helsinki. The patient who submitted the agreements to attend this project was medically examined and a volunteer was selected (42.2 years; 71.4 kg; 172 cm) for tramadol and O-desmethyltramadol. Then, he was allowed to drink water. The total amount of water drunk during the day was 1,500 mL. The patient was sitting during lunch. He had normal activity (standing or sitting) during the study, but was never in a supine position during the 24 h after administration. Urine samples were collected at the following times: 0, 0 –2, 2 –4, 4 –6, 6 –12 and 12 – 24 h. Representative chromatograms obtained after 12 h administration of the drug are shown in Figure 3c. Amounts of excreted tramadol and O-desmethyltramadol compounds were obtained by multiplication of the calculated concentration by the measured excreted urine volume during the time interval (Figure 4).

A, the amount of tramadol, O-desmethyltramadol and IS in blank urine sample’s reconstituted solution (the final solution of blank urine after extraction and reconstitution); B, the amount of tramadol and O-desmethyltramadol and IS.

Dilution integrity The dilution integrity experiment was performed with an aim to validate the dilution test to be carried out on higher analyte concentration above the upper limit of quantification, which may be encountered during real subject sample analysis. The precision and accuracy values for one-fifth and one-tenth dilution ranged from 3.42 to 7.28% and 98.2 to 102.7 for tramadol and O-desmethyltramadol.

Discussion Today, GC – MS is a powerful technique for highly specific and quantitative measurements of low levels of analytes in biological samples. As compared with HPLC, high-resolution capillary GC has been less frequently used (34, 35). However, it has inherently high resolving power and high sensitivity with excellent precision and accuracy allowed simultaneous detection of tramadol and O-desmethyltramadol. In addition, the detection limits were lowered to ng levels by GC combined with MS. The specificity of the method was verified by investigating the peak interference from the endogenous urine substances. Representative chromatograms of blank urine and urine samples spiked with tramadol, O-desmethyltramadol and IS are shown in Figure 3b. The samples were extracted by an SPE cartridge (C18, 3 mL, 500 mg; Bond-elut, Agilent). The cartridge was conditioned with 3 mL methanol and 3 mL water. The frozen urine samples

Table V Stability of Tramadol and O-Desmethyltramadol in Human Urine Under Various Storage Conditions (n ¼ 3)

Tramadol

Storage conditions

Concentration (ng/mL)

Calculated concentration (ng/mL)

% RSD

% Relative error

Room temperature for 8 h

50 150 50 150 50 150 150 300 150 300 150 300

48 143 49 141 51 144 145 313 143 316 141 319

2.8 4.4 3.1 5.9 3.4 5.8 5.8 4.4 7.4 6.5 5.6 6.7

24.0 24.7 22.0 26.0 2.0 24.0 23.3 4.3 24.7 5.3 26.0 6.3

Three freeze – thaw cycles 1 week at 2208C

O-Desmethyltramadol

Room temperature for 8 h Three freeze – thaw cycles

1 week at 2208C

Determination of Tramadol and Its Metabolite in Human Urine by GC– MS 5

The proposed method has a total run time of 8.0 min, which is faster than previously published GC – MS methods for tramadol and its metabolites (37, 38). Moreover, adequate sensitivity is obtained for tramadol and O-desmethyltramadol without derivatization compared with the reported previous methods (39, 40). When this method is applied to urine samples, its sensitivity was found to be adequate for pharmacokinetic studies. The present method has the following advantages over the reported method. The method is as good or superior to that reported in the other papers (23, 36–40). One of the objectives of this study was targeted to determine for how long tramadol and its main metabolite O-desmethyltramadol remain detectable in human urine. The method was sensitive enough to detect tramadol and O-desmethyltramadol in human urine samples up to 24 h. Figure 3 shows the cumulative urinary excretion curve for tramadol and O-desmethyltramadol obtained from a patient with a single dose of Contramal tablet (100 mg tramadol hydrochloride). Although our results were obtained from only one subject, our results are in agreement with the literature (13). Figure 4. Cumulative excretion curves of tramadol and O-desmethyltramadol of a patient during 24 h after oral administration of 100 mg of tramadol hydrochloride.

(0.5 mL) were thawed at a temperature of 258C (room temperature) and 0.75 mL water and IS (0.5 mL at 1.0 mg/mL concentration) were added to the urine. The mixture was vortexed and transferred to the SPE cartridge. Then, the cartridge was washed with a mixture of acetonitrile–water (2 mL, 15/85) and 3 mL hexane. The eluate was collected from with 3 mL acetonitrile. The eluate was evaporated at 508C under nitrogen. The residue was dissolved in methanol and 1 mL volume was injected into GC–MS system. The extraction recoveries of tramadol and O-desmethyltramadol from human urine were between 53.2 and 61.8%. Therefore, the liquid– liquid extraction method was used in this study. The presented extraction procedure offers a rapid way to isolate tramadol and its main metabolite O-desmethyltramadol from the human plasma. Rouini and Ardakani (23, 36) published a method for simultaneous extraction of tramadol and O-desmethyltramadol in human plasma, using ethyl acetate and 1.0 M sodium hydroxide as alkaline medium. In this work, a very poor recovery for O-desmethyltramadol (5%) was resulted by the use of 1.0 M sodium hydroxide. The poor recovery could be interpreted due to the character of the phenoloic group in O-desmethyltramadol that reacts with sodium hydroxide to form the sodium phenoxide salt, which is very soluble in aqueous layer. There are reported LC methods with tandem mass detection for the analysis of tramadol and O-desmethyltramadol in human plasma (15 –18). Ceccato et al. (15) have reported an LC method with tandem mass detection for the analysis of tramadol and O-desmethyltramadol in human plasma. The calibration curve of the LC – MS-MS method was linear for of tramadol in the range 20 to 10,000 ng/mL. Intra- and interday precision, expressed as the RSD, were ,6.4%, and accuracy (relative error) was better than 6.1%. Detection using LC–MS-MS would be a more sensitive approach but is costly and not yet available for every laboratory. Tramadol was extracted from plasma with an SPE procedure in the literature (27, 32, 40). This methods are also the most comprehensive method which can extract tramadol in a single extraction procedure. The mean recovery is better than those of the reported previous studies (27, 32, 40) 6 Yilmaz and Erdem

Conclusion A novel, simple, specific and sensitive GC – MS method was described for analysis of tramadol and O-desmethyltramadol in human urine. Furthermore, the method has good linearity, precision, accuracy and sensitivity according to the results obtained from validation data. Additional advantages of this method include small sample volume (0.5 mL), good extraction recovery from urine and a readily available IS. In addition, the extraction procedure in this study was simple. No significant interferences and matrix effect caused by endogenous compounds were observed. Therefore, the method can be very useful and an alternate to performing pharmacokinetic studies in determination of tramadol and O-desmethyltramadol for clinical use. Acknowledgments The authors are thankful to Vedat Akba (Criminal Police Laboratory, 25060, Erzurum, Turkey) for their help in GC – MS analyses. The authors are also thankful to the patient who participated in this study.

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Determination of Tramadol and Its Metabolite in Human Urine by GC– MS 7

Simultaneous Determination of Tramadol and Its Metabolite in Human Urine by the Gas Chromatography-Mass Spectrometry Method.

A sensitive and efficient method was developed for determination of tramadol and its metabolite (O-desmethyltramadol) in human urine by gas chromatogr...
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