LC-MS/MS法同时测定人血浆中氯吡格雷及其羧酸代谢物的浓度

目的建立快速测定人血浆中氯吡格雷及其羧酸代谢物SR26334含量的LC-MS/MS法。方法乙醚-正己烷(4:1,V:V)2次液-液提取法(中性和酸化条件下),采用Teknokroma C_(18)色谱柱,以那格列奈和吡格列酮为内标,同时测定血浆中氯吡格雷和SR26334的浓度。流动相:甲醇-0.1%甲酸(80:20,V:V);流速:0.2mL·min~(-1);以多反应离子监测方式检测:氯吡格雷[M+H]~+,m/z 322.1→212.1;那格列奈[M+H]~+,m/z 318.3→166.2;氯吡格雷羧酸代谢物SR26334[M+H]~+,m/z 308.1→q98.1;吡格列酮[M+H]~+,m/z 357.2→134.2。结果氯吡格雷和内标那格列奈的保留时间分别在4.4和3.7min,SR26334和内标吡格列酮的保留时间分别在1.3和1.7min,氯吡格雷的线性范围为5～5000ng·L~(-1);SR26334的线性范围为20～2500μg·L~(-1)。提取回收率大于75%,方法回收率大于90%,日内、日间RSD小于10%(n=5)。结论本方法简便快速,适用于氧吡格雷制剂的新药临床研究和临床长期治疗病人血药浓度的常规监测。     

Clopidogrel activities in patients with renal function impairment

OBJECTIVES: To assess the tolerability, pharmacodynamic effects and pharmacokinetic parameters after repeated doses of clopidogrel (Plavix((R))) in patients with moderate or severe renal failure. PATIENTS: Eight patients with severe renal failure (endogenous creatinine clearance 5 to 15 ml/min) and eight patients with moderate renal impairment (endogenous creatinine clearance 30 to 60 ml/min) were included. STUDY DESIGN: An open, uncontrolled, parallel-group study over 8 days' administration of 75mg once-daily clopidogrel. METHODS: Measurement of changes in ADP-induced platelet aggregation and skin bleeding time and of plasma concentrations and urinary excretion of clopidogrel and its main metabolite, SR 26334. Assessment of clinical tolerance and serial haematological and biochemical investigations. RESULTS: At the end of the dosage period, platelet aggregation was equally inhibited, by about 25%, and bleeding time equally extended, by a factor of about 2, in the two groups. There were no tolerability concerns. Maximum plasma concentration (C(max)) and time to reach C(max ) (t(max)) for clopidogrel were not significantly different between the two groups. SR 26334 excreted into the urine and renal clearance rate were significantly lower in the severely impaired group, while plasma elimination half-lives were not significantly different. C(max) and t(max) did not differ significantly between the two groups, but trough levels and area under the plasma concentration-time curve from zero to 24 hours (AUC(0-24h)) after the last dose were significantly higher in the moderately impaired group. CONCLUSIONS: Clopidogrel 75mg once daily was well tolerated in patients with either moderate or severe renal failure, and provided good inhibition of ADP-induced platelet aggregation without excessive extension of bleeding time. Dose adjustment in such patients does not appear to be required.     

Cirrhosis does not affect the pharmacokinetics and pharmacodynamics of clopidogrel

Clopidogrel, a new platelet ADP receptor antagonist used for the prevention of vascular ischemic events, is converted to an active metabolite via the cytochrome P450 system. Patients with cirrhosis may not metabolize drugs normally and may, in addition, have a number of defects in the coagulation system. To assess the effect of cirrhosis on the pharmacokinetics and pharmacodynamics of clopidogrel, the authors performed an open-label, parallel-group study of 12 patients with Child-Pugh Class A or B cirrhosis and 12 matched controls. All 24 subjects received clopidogrel 75 mg PO QD for 10 days. Pharmacokinetics of clopidogrel and the major metabolite SR 26334 were analyzed on Days 1 and 10; pharmacodynamics were assessed by the inhibition of ADP-induced platelet aggregation and by bleeding time prolongation factor. Pharmacokinetic analysis of clopidogrel was limited due to low plasma concentrations arising from rapid hydrolysis to SR 26334. The Cmax at SS for clopidogrel was higher in cirrhotics than in normals. However, exposures to the metabolite SR 26334, as measured by AUC(tau), were comparable. At Day 10, there was not a statistically significant difference in mean inhibition of platelet aggregation (49.2% +/- 38.6% in cirrhotics vs. 66.7% +/- 7.5% in normals) or in bleeding time prolongation factor (1.64 +/- 0.49 in cirrhotics vs. 1.54 +/- 0.87 in normals) between groups. No significant adverse events, including bleeding events, were reported. In conclusion, there were no significant differences in the pharmacokinetics and pharmacodynamics of clopidogrel in this group of subjects with cirrhosis and matched normals. Therefore, no dosage adjustment of clopidogrel is required in patients with Child-Pugh Class A or B cirrhosis.     

Discrimination of dimethylamphetamine and methamphetamine use: simultaneous determination of dimethylamphetamine-N-oxide and other metabolites in urine by high-performance liquid chromatography-electrospray ionization mass spectrometry

A simple and sensitive method by high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) has been investigated for the simultaneous determination of dimethylamphetamine (DMA), its specific yet labile main metabolite dimethylamphetamine-N-oxide (DMAO), and other metabolites, methamphetamine (MA) and amphetamine (AP), in urine. A combination of Bond Elut SCX columns for the solid-phase extraction of urine and a semi-micro SCX column for LC separations provided satisfactory results. The use of acetonitrile/5mM ammonium acetate buffer adjusted to pH 4 (65:35, v/v) as the mobile phase at a flow rate of 0.2 mL/min was found to be the most effective. The detection limits were 5 ng/mL for DMAO, 10 ng/mL for DMA and MA, and 50 ng/mL for AP in the SIM mode.     

HPLC determination and steady-state bioavailability study of levodropropizine sustained-release tablets in dogs

A simple HPLC method using UV detection was developed and validated for the determination of levodropropizine (LDP) in dog plasma. The sample was prepared for injection using a liquid-liquid extraction method with 1-phenypiperazine as the internal standard. The mobile phase was methanol-diethylamine solution (0.05 M) (20:80, v/v, pH adjusted to 3.0 with H3PO4) with a detection wavelength of 240 nm. The limit of quantitation (LOQ) of LDP in a biological matrix was determined to be 25.25 ng/mL. The calibration curve was linear across the concentration range of 25.25 to 2020 ng/mL. The intra-day and inter-day precision values (CV %) were within 7% and accuracy (R.E. %) was within 6% of the nominal values for medium (252.5 ng/mL) and high (2020 ng/mL) LDP concentrations. For the LDP concentration at the LOQ, the intra-day and inter-day precision and accuracy were within 20% and 10%, respectively. The average absolute recovery for LDP was 70.28%. This method was successfully used to analyze plasma samples in a steady-state bioavailability study of a newly developed sustained-release LDP tablets (SR) using immediate-release tablets (IR) as the reference. The relative bioavailability of the SR was determined to be 106.3 +/- 12.8% (n=6). The Cmax of the SR was significantly lower (P<0.05), and the tmax was significantly longer than that of the IR (P<0.05). The results of ANOVA and two one-sided tests indicated that the SR exhibited acceptable sustained release properties and was bioequivalent to the IR.     

Use of solid-phase microextraction (SPME) for the determination of methadone and its main metabolite, EDDP, in plasma by gas chromatography-mass spectrometry

A simple, rapid method for the determination of methadone and its metabolite 2-ethylene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in plasma using solid-phase microextraction (SPME) and gas chromatography-mass spectrometry is proposed. A 100-microm polydimethylsiloxane film fiber was exposed by immersion for 30 min in a diluted plasma solution (1:4 with buffer pH 9) containing both compounds and an internal standard (proadifen). Calibration curves were linear over the concentration range 50-2000 ng/mL. The analysis time was 45 min per sample. The determination of methadone and EDDP was subject to no interference. The performance of SPME was compared with that of liquid-liquid extraction, obtaining lower limits of detection for EDDP. The method using the two extraction procedures was applied to 10 plasma samples from methadone-treated patients.     

Quantification of flupirtine and its active metabolite D-13223 in human plasma by LC-MS/MS: application to a clinical trial of flupirtine maleate capsules in healthy male Chinese volunteers

In the present study, we developed a simple, sensitive, precise, and accurate liquid chromatography-tandem mass spectrometric (LC-MS/MS) method and validated such approach for simultaneous determination of flupirtine and its active metabolite D-13223 in human plasma. The flupirtine, D-13223, and stable isotope internal standard (IS) were extracted from plasma samples by liquid-liquid extraction and chromatographed on a C18 column with a mobile phase consisting of acetonitrile–water–ammonia (55:45:0.1, v/v/v) at a flow rate of 0.25 mL/min. Detection was performed on a triple quadrupole tandem mass spectrometer with an electrospray ionization source (ESI) by multiple reaction monitoring (MRM) in positive ion mode. The linear calibration curves were obtained within the concentration range of 10.00–2000.00 ng/mL for flupirtine and 2.00–400.00 ng/mL for D-13223. The intra- and inter-run RSD, calculated from quality control (QC) samples, was less than 9.26% for flupirtine and D-13223. The accuracy was –5.80%–3.31% for flupirtine and D-13223. The extraction recoveries of flupirtine, D-13223, and their IS were all between 88.3%–97.2%. The method was successfully applied to investigate the pharmacokinetic profiles of flupirtine and its active metabolite D-13223 in human plasma following peroral administration of 100 mg flupirtine maleate capsules in healthy male Chinese volunteers.     

A simple method for the determination of ethylene-thiourea (ETU) in biological samples

A direct, simple, and rapid high-performance liquid chromatographic method has been developed for the determination of ethylene-thiourea (ETU) in biological fluids. Samples were chromatographed on a Lichrosorb RP8 (5 pm) column after extraction with dichloromethane. The mobile phase was a mixture of hexane/isopropyl alcohol/ethyl alcohol (93:6:1 v/v) added with 0.6 mL/L butylamine. Detection was done with a UV detector set at 243 nm. This method was validated to standard criteria. Calibration curves for ETU in 100 microL of 0.9% saline, 500 microL plasma, and 10 mL urine were linear (r2 > 0.99) from 0.05 to 30 microg/mL, 0.025 to 30 microg/mL, and 1 to 100 ng/mL, respectively. The lower limit of detection was 20 ng/mL in plasma, 25 ng/mL in 0.9% saline, and 0.5 ng/mL in urine.     

Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of zofenopril and its active metabolite zofenoprilat in human plasma

A novel, sensitive and rapid liquid chromatographic-electrospray ionization mass spectrometric method was developed and validated for the determination of zofenopril and its active metabolite zofenoprilat in human plasma. The method was based on a single extraction step using methyl tert-butyl ether and did not require chemical derivatization. The chromatographic conditions were optimized; separation was performed on a phenyl-hexyl column (5μm, 250mm×4.6mm i.d.) with a mobile phase consisting of a solution of methanol and water (95:5, v/v) that also contained 0.1% of formic acid. A flow rate of 1.0mL/min was used. Zofenopril, zofenoprilat and the internal standard (IS) fosinopril sodium were measured using an electrospray ion source in a positive reaction monitoring mode. Linear calibration curves were generated for zofenopril concentrations between 0.1052 and 1052ng/mL and for zofenoprilat concentrations between 0.2508 and 2508ng/mL. In both cases, the coefficients of determination were greater than 0.995. The extraction recovery for zofenopril was 93.5% on average. It was 92.5% for zofenoprilat. The inter- and intra-batch precision and accuracy for both zofenopril and zofenoprilat were higher than 14%. The method was applied to measure the concentrations of zofenopril and zofenoprilat in plasma samples.     

Development of a rapid RP-HPLC method for the determination of clonazepam in human plasma

A rapid and sensitive high-performance liquid chromatography method with UV detection was developed for the determination of clonazepam in human plasma using 3-methylclonazepam, as internal standard. A one-step extraction of both compounds was performed with a mixture of hexane/ethyl acetate (90:10, v/v). The HPLC analysis was carried out on a Nova Pak((R)) C(18) reversed-phase column with a mobile phase of acetonitrile-0.01 M sodium acetate adjusted to pH 7 with dilute acetic acid (40:60, v/v). A linear response was observed over the concentration range 5-100 ng/mL. Intra- and inter-day assay precision and accuracy fulfilled the international requirements. The lower limit of quantification was 5 ng/mL without interference of endogenous components. For analytical purpose, the stability of clonazepam in bidistilled water and plasma has been studied. A rapid degradation was noticed when clonazepam was stored in bidistilled water at the daylight following a first-order kinetic rate with a 87 min half life whereas no significant degradation was observed in plasma. This method was applied to measure plasma concentrations of clonazepam either in patients receiving therapeutic doses or in poisoning cases.     

Effect of <Emphasis Type="Italic">CYP3A5*3</Emphasis> genotype on the pharmacokinetics and antiplatelet effect of clopidogrel in healthy subjects

Objective Clopidogrel is activated by cytochrome P450 3A (CYP3A) to generate an active metabolite that inhibits adenosine diphosphate (ADP)-induced platelet aggregation through irreversible binding to the platelet P2Y12 receptor. The objective of this study was to assess the effect of the CYP3A5 genotype on the pharmacokinetics and antiplatelet effect of clopidogrel in healthy subjects. Methods Twenty-two healthy subjects (CYP3A5*1/*1, n = 6; CYP3A5*1/*3, n = 8; CYP3A5*3/*3, n = 8) were recruited. After the administration of a loading dose of 300 mg of clopidogrel followed by 75 mg once daily for 6 days, plasma concentrations of clopidogrel and SR26334, an inactive metabolite, were measured for 24 h. The antiplatelet effect of clopidogrel was also measured, by determining the inhibition of ADP-induced platelet aggregation for 168 h, according to CYP3A5 genotype. Results Mean plasma concentration profiles of clopidogrel and SR26334 were comparable between CYP3A5 genotype groups. In addition, the CYP3A5 genotype did not affect the pharmacokinetics of either clopidogrel or SR26334. CYP3A5 genotype also did not modulate the inhibitory effect of clopidogrel on platelet aggregation. Conclusion The CYP3A5*3 genotype plays a minor role in causing interindividual variability of the disposition of clopidogrel and its antiplatelet effect in humans.     

瑞格非尼片在健康志愿者体内的药动学研究

目的利用固相萃取–高效液相色谱,建立测定人血浆中瑞格非尼及其代谢产物N-氧化瑞格非尼和去甲-N-氧化瑞格非尼的方法,研究其人体药动力学。方法采用固相萃取–高效液相色谱法进行检测,以索拉非尼为内标,Oasis HLB型固相萃取柱(200 mg×6 m L);流动相:0.5%磷酸二氢钾溶液–乙腈(30∶70);检测波长:260 nm;体积流量:0.5 m L/min;柱温:30℃;进样量:5μL。健康志愿者空腹条件下用50 m L温水送服40、160 mg/片瑞格非尼片。利用DAS 2.1.1软件对药–时数据进行统计学分析,并得到主要药动学数据。结果瑞格非尼及其代谢产物N-氧化瑞格非尼和去甲-N-氧化瑞格非尼在10~10 000 ng/m L线性关系良好;检测限(LOD)分别为0.67、0.72、0.57 ng/m L;定量限(LOQ)分别为1.98、2.32、1.61 ng/m L;得到瑞格非尼及其代谢产物N-氧化瑞格非尼和去甲-N-氧化瑞格非尼药–时曲线和主要药动学数据。40、160mg/片规格瑞格非尼片中瑞格非尼的Cmax、AUClast、AUCmf、t1/2分别为(2 215.7±216.9)、(5 721.2±672.1)ng/m L,(13 772.9±10 231.1)、(51 182.3±43 219.2)ng·h/m L,(12 858.3±12 510.2)、(66 531.8±51 093.1)ng·h/m L,(6.58±1.31)、(13.12±4.23)h。结论采用固相萃取–高效液相色谱法考察瑞格非尼片在健康志愿者体内的药动学研究具有操作简便、可靠性高等优点,对该药的应用具有重要意义。     

Simultaneous determination of albendazole metabolites, praziquantel and its metabolite in plasma by high-performance liquid chromatography-electrospray mass spectrometry

The analysis of albendazole sulfoxide, albendazole sulfone, praziquantel and trans-4-hydroxypraziquantel in plasma was carried out by high-performance liquid chromatography-mass spectrometry ((LC-MS-MS). The plasma samples were prepared by liquid-liquid extraction using dichloromethane as extracting solvent. The partial HPLC resolution of drug and metabolites was obtained using a cyanopropyl column and a mobile phase consisting of methanol:water (3:7, v/v) plus 0.5% of acetic acid, at a flow rate of 1.0 mL/min. Multi reaction monitoring detection was performed by electrospray ionization in the positive ion mode, conferring additional selectivity to the method. Method validation showed relative standard deviation (precision) and relative errors (accuracy) lower than 15% for all analytes evaluated. The quantification limit was 5 ng/mL and the linear range was 5-2500 ng/mL for all analytes. The method was used for the determination of drug and metabolites in swine plasma samples and proved to be suitable for pharmacokinetic studies.     

Determination of omeprazole in human plasma by liquid chromatography-electrospray quadrupole linear ion trap mass spectrometry

An analytical method for the determination of omeprazole in human plasma has been developed based on liquid chromatography mass spectrometry. The analyte and internal standard sildenafil are extracted from plasma by liquid-liquid extraction using diethyl ether:dichloromethane (60:40, v/v) and separated by reversed phase high-performance liquid chromatography (HPLC) using acetonitrile:methanol:10 mM ammonium acetate (37.5:37.5:25, v/v/v) as mobile phase. Detection is carried out by multiple reaction monitoring on a Q TRAP LC/MS/MS system (Q TRAP). The method has a chromatographic run time of 3.5 min and is linear within the range 0.50-800 ng/mL. Intra- and inter-day precision expressed as relative standard deviation ranged from 0.4 to 8.5% and from 1.2 to 6.8%, respectively. Assay expressed as relative error was <5.7%. The method has been applied in a bioequivalence study of two capsule formulations of omeprazole.     

High throughput assay for the determination of piperaquine in plasma

A high throughput assay for the determination of the antimalarial piperaquine in plasma has been developed and validated. The assay utilises 96-wellplate formats throughout the whole procedure, and easily enables a throughput of 192 samples a day using a single LC system. Buffer (pH 2.0; 0.05 M) containing internal standard was added to 0.25 mL plasma in a 96-wellplate (2 mL wells). The samples were extracted on a MPC solid phase extraction deep well 96-wellplate (3M Empore). Piperaquine and internal standard were analysed by liquid chromatography with UV detection on a Chromolith Performance (100 mm x 4.6 mm) column with a mobile phase containing acetonitrile-phosphate buffer (pH 2.5; 0.1 M) (8:92, v/v) at a flow rate of 3.0 mL/min. The within-day precisions for piperaquine were 3.3 and 2.3% at 40 and 1250 ng/mL, respectively. The between-day precisions for piperaquine were 5.8 and 1.3% at 40 and 1250 ng/mL, respectively. The total assay precisions using 29 replicates over 5 days were 6.7, 4.5 and 2.7% at 40, 200 and 1250 ng/mL, respectively. The lower limit of quantification (LLOQ) and the limit of detection (LOD) were 10 and 5 ng/mL, respectively using 0.25 mL plasma. Using 1 mL of plasma, it was possible to decrease LLOQ and LOD to 2.5 and 1.25 ng/mL, respectively.     

Simultaneous determination of risperidone and 9-hydroxyrisperidone enantiomers in human blood plasma by liquid chromatography with electrochemical detection

Risperidone is a commonly prescribed antipsychotic drug. An enantioselective HPLC method with electrochemical detection was developed and validated for simultaneous determination of plasma concentrations of risperidone and its active metabolites, 9-hydroxyrisperidone enantiomers. Following solid phase extraction of 1.0 mL blood plasma, a baseline separation of the analytes was achieved on an AGP (α₁ acid glycoprotein) column using isocratic mobile phase consisting of methanol–phosphate buffer (pH 6.2; 0.1 M) (15:85, v/v). Total analysis run time was 11 min. For the detection of the analytes analytical cell potentials were set at 500, 650, 950, and 950 mV. The method linearity was attained in the range from 1.0 to 100 ng/mL for risperidone and both 9-hydroxyrisperidone enantiomers. The limit of detection was 0.5 ng/mL for all three analytes. The method was precise and accurate and was successfully applied in a clinical study investigating the stereoselectivity of risperidone 9-hydroxylation.     

氯吡格雷单次和多次给药在大鼠体内的药动学特征

目的建立并确证氯吡格雷羧酸代谢物SR26334血浆药物浓度的HPLC检测法,研究氯吡格雷单次和多次大剂量给药后在大鼠体内的药动学特征。方法12只SD大鼠随机分成2组（单次给药组和多次给药组）,单次和多次灌胃给予氯吡格雷30mg·kg-1,多次给药为每天1次,共7d。采用HPLC法测定给药后不同时间点血浆中SR26334浓度,用DAS3．0处理经时血药浓度数据,计算主要药动学参数。结果单次给药和多次给药后SR26334血药浓度均于给药后约1h达峰,pmax分别为（35．57±10．25）mg·L。和（54．28±10．39）mg·L-1;两者g1／2z相近,分别为（6．88±1．54）h和（6．54±1．04）h;AUC0—24h分别为（304．63±63．07）mg·h·L-1和（543．81±43,27）mg·h·L-1;AUC0-∞分别为（334．00±66．24）mg·h·L-1和（594．91±46．84）mg·h·L-1;Vz／F分别为（0．92±0．23）L·kg。和（0．48±0．07）L·kg-1;CLz／F分别为（0．09±0．02）L·h-1·kg-1和（0．05±0．00）L·h-1·kg-1。结论氯吡格雷单次与多次给药后的主要药动学参数AUC0-24h、AUC0-∞、Vz／F、ck／F和pmax差异具有统计学意义（P〈0．01,P〈0．05）,说明多次大剂量给药时其代谢物SR26334在体内有蓄积。     

LC-MS／MS测定人血浆中甘草次酸的浓度及其在药代动力学中的应用(英文)

建立一种灵敏度高、重现性好的高效液相色谱-质谱法（LC-MS/MS）测定人血浆中甘草次酸的浓度,并用于甘草酸铵的人体药动学研究。采用液-液萃取法进行血浆样品预处理,色谱柱为C₁₈柱,流动相为乙腈-5mmol/L乙酸铵（70:30,v/v）,流速0.8mL/min。采用负离子模式,多离子监测（MRM）方式进行检测,甘草次酸与内标甲砜霉素离子对质核比（m/z）分别为469.3→355.3、354.1→185.0。方法学验证表明此方法特异性强、灵敏度高、准确度与精密度符合要求,线性范围为0.5-500ng/mL,血浆样本经两次冻融及冷冻稳定良好,可以用于甘草酸铵的人体药代动力学研究。健康志愿者口服75mg甘草酸铵后,活性代谢产物甘草次酸的药动学参数如下:AUC_0-t（3457.26±1999.01）ng·h/mL、AUC_0-∞（3708.85±2428.36）ng·h/mL、MRT_0-t（19.69±4.03）h、MRT_0-∞（22.83±8.45）h、t_1/2Z（11.71±7.77）h、T_max（13.40±4.84）h、CLz/F（29±17±19±82）L/h、Vz/F（487.38±518.07）L、C_max（215.85±99.88）ng/mL。     

High-performance liquid chromatographic analysis of the anticancer drug irinotecan (CPT-11) and its active metabolite SN-38 in human plasma

A rapid, sensitive, and specific high-performance liquid chromatography (HPLC) method for the simultaneous determination of irinotecan (CPT-11) and its active metabolite SN-38 in human plasma is described. The analytes are quantified as the totals of their carboxylate and lactone form. The sample pretreatment consisted of a simple protein precipitation with acetonitrile-methanol (1:1, v/v), after which CPT-11 and SN-38 were quantitatively converted to their carboxylate form by adding 0.01 mol/L sodium tetraborate (pH, 9). Chromatography was carried out on a Zorbax SB-C18 column with fluorescence detection. The method has been validated, and stability tests under various clinically relevant conditions have been performed. The lower limit of quantification (LLOQ) was 5.0 ng/mL for CPT-11 and 0.5 ng/mL for SN-38. Standard concentration ranges were linear between 5 and 1,500 ng/mL for CPT-11 and between 0.5 and 100 ng/mL for SN-38. This assay is simple, rapid, and very useful for therapeutic monitoring of CPT-11 and SN-38.     

Rapid determination of tramadol in human plasma by headspace solid-phase microextraction and capillary gas chromatography-mass spectrometry

A simple, rapid and sensitive method for determination of tramadol in plasma samples was developed using headspace solid-phase microextraction (HS-SPME) and gas chromatography with mass spectrometry (GC-MS). The optimum conditions for the SPME procedure were: headspace extraction on a 65-microm polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber; 0.5 mL of plasma modified with 0.5 mL of sodium hydroxide (0.1 M); extraction temperature of 100 degrees C, with stirring at 2000 rpm for 30 min. The calibration curve showed linearity in the range of 1-400 ng mL(-1) with regression coefficient corresponding to 0.9986 and coefficient of the variation of the points of the calibration curve lower than 10%. The detection limit for tramadol in plasma was 0.2 ng mL(-1). The proposed method was successfully applied to determination of tramadol in human plasma samples from 10 healthy volunteers after a single oral administration.