The effects of cephalexin on fracture healing in a rat femur fracture model.

OBJECTIVES: The aim of this study was to examine the effects of cephalexin on the fracture union histomorphometrically, radiologically, biomechanically, immunohistochemically, and histopathologically in a rat femur fracture model and to evaluate the effects of the antibiotics to be used in the prophylaxis of fracture infection on the union of the fracture. MATERIALS AND METHODS: A total of 48 male Wistar rats were divided into four groups as two-week control (C2) and cephalexin (CEP2) and four-week control (C4) and cephalexin (CEP4). After establishment of standard fracture model on right femurs, 60 mg/kg/day of cephalexin was applied to CEP2 and CEP4 by oral gavage. Radiological, biomechanical, histopathological, immunohistochemical, and histomorphometric examinations were performed on amputated femurs. RESULTS: Callus volume of CEP4 group significantly increased compared to CEP2 group (p=0.005), while no significant difference was found in the bone mineral density and callus/bone volume among the groups (p>0.05). There was no significant difference in flexural strength between the C4 and CEP4 groups (p=0.093). Histological healing scores increased from Week 2 to Week 4 (p=0.002) and inflammation scores decreased in both control and cephalexin groups (p=0.010 and p=0.008); however, no significant difference was found in healing and inflammation scores (p>0.05). The CD34+ immunoreactivity in the CEP2 group was significantly higher than the C2 group (p=0.029). Collagen type III level was significantly lower in the CEP2 and CEP4 groups compared to the corresponding control groups (p=0.008 and p=0.016, respectively). CONCLUSION: Cephalexin did not exert any radiological, histopathological, histomorphometric, biomechanical, and immunohistochemical adverse effects on the femoral fracture healing model in rats; however, it showed positive effects on CD34 and Collagen type III levels. Based on these findings, antibiotherapy with cephalexin may be considered as a safe treatment for fracture union.

Preparation, Characterization, and Evaluation of Cytotoxicity of Fast Dissolving Hydrogel Based Oral Thin Films Containing Pregabalin and Methylcobalamin.

The oral availability of many drugs is problematic due to the pH of the stomach, enzymes, and first-pass effects through the liver. However, especially geriatric, pediatric, bedridden, or mentally handicapped patients and those with dysphagia have difficulty swallowing or chewing solid dosage forms. Oral Thin Films (OTFs) are one of the new drug delivery systems that can solve these problems. Pregabalin (PG) and Methylcobalamin (MC), which are frequently preferred for pain originating in the central nervous system, were brought together for the first time using OTF technology in this study. In this study, a quantification method for PG and MC was developed and validated simultaneously. Optimum formulations were selected with organoleptic and morphological controls, moisture absorption capacity, swelling capacity, percent elongation, foldability, pH, weight variability, thickness, disintegration time, and transparency tests on OTFs prepared by the solvent pouring method. Content uniformity, dissolution rate, determination of release kinetics, SEM, XRD, FT-IR, DSC, long-term stability, and cytotoxicity studies on the tongue epithelial cell line (SCC-9) were performed on selected OTFs. As a result, OTFs containing PG-MC, which are non-toxic, highly flexible, transparent, compatible with intraoral pH, with fast disintegration time (<30 s), and acceptable in taste and appearance, have been developed successfully.

Effects of Linagliptin and Pioglitazone on Fracture Healing in an Experimental Type 2 Diabetes Rat Model.

AIM: Our study aimed to examine the effects of Linagliptin, Pioglitazone, and their combination on fracture healing in a diabetes rat femur fracture model. MATERIAL AND METHODS: Type 2 diabetes mellitus (T2DM) induced rats were randomly divided into four groups: non-treated diabetes group (TD), Pioglitazone group (P), Linagliptin group (L), and Pioglitazone and Linagliptin group (PL). Daily oral dosage of pioglitazone (10 mg/kg/day), linagliptin (10 mg/kg/day), and their combination were administered. Femur fractures were stabilized intramedullary. At weeks 2 and 6, rats were sacrificed for evaluation radiologically, biomechanically, histopathologically, histomorphometrically, and immunohistochemically. RESULTS: Flexural strength of the L and PL groups were significantly higher compared to the P group. The highest healing score was in the L group and lowest in the P group, while the highest inflammation score was in the P group and lowest in the L group. A cluster of differentiation (CD) CD 34 reactivity was highest in the L group and lowest in the PL group. CONCLUSION: Linagliptin treatment significantly increased histological healing scores, callus volume, biomechanical strength, and vascularity, however, minimized the inflammatory process, which was increased by pioglitazone. The combination of linagliptin and pioglitazone restored BMD and increased biomechanical strength. Linagliptin monotherapy is rarely indicated; hence, T2DM patients with a high risk of bone fractures can be considered for combined therapy of pioglitazone and linagliptin.

Determination of neopterin in urine of industrial workers by HPLC.

This study aimed to determine neopterin levels in the urine of industrial workers by the high-performance liquid chromatography method. Intra- and inter-day precision values for neopterin in urine were less than 3.14, and accuracy (relative error) was better than 3.00%. The limits of detection and quantification of neopterin were 0.3 and 1.0 ng/mL, respectively. Also, the developed method was applied to real samples to determine the neopterin levels in the urines of industrial workers, who have been exposed to various chemicals such as formaldehyde, heavy metals and thinners. Urine neopterin levels of industrial workers including auto painters, bodywork and furniture workers were statistically compared with healthy volunteers. The highest and lowest values of urinary neopterin for industrial workers were obtained 908.96 and 119.86 µmol/mol, respectively. Our investigation demonstrates that there is a meaningful difference in urinary neopterin levels between the workers and the control groups (P<0.05). Workers in the auto paint, body and furniture business may have been exposed to a toxic environmental exposure in their occupation. As a result, an increase in the concentration of neopterin in the urine may be important in the diagnosis and treatment of various diseases.

Composition and bioactivity of essential oil from Stachys macrostachya (Wend.) Briq.

Stachys species belonging to Lamiaceae family have been used for medicinal purposes since ancient times. The aim of the present study was to investigate the chemical compositions and antibacterial, anti-tyrosinase activities of the essential oil of Stachys macrostachya. The essential oil was prepared by hydrodistillation method using a Clevenger-type apparatus and chemical composition was determined by gas chromatography (GC). The antibacterial activity of essential oil was performed by the disc diffusion and microdilution broth method against five Gram-positive and two Gram-negative bacteria. The tyrosinase inhibitory activity was evaluated by minor modifications of Masuda's method. According to the results of GC analyses, twenty-three compounds were identified representing 91.9% of the total volatile composition. The main compounds were germacrene D (12.2%), globulol (10.9%), α-pinene (9.7%), and valencene (7.6%). The present study showed that the tested essential oil of S. macrostachya exhibited antibacterial activity against Acinetobacter baumannii (MIC 62.50 µg/mL) and tyrosinase inhibition activity (IC50 22.86 ± 0.82 µg/mL). These results suggest that the essential oil could be exploited as a potential source of natural antimicrobial agents of this bacterium as well as tyrosinase inhibitors.

Disulfiram-loaded functionalized magnetic nanoparticles combined with copper and sodium nitroprusside in breast cancer cells.

Disulfiram (DSF), one of the members of the dithiocarbamate family, is a reactive species (RS) generator and is capable of inducing cancer cell death in breast cancer. However, it is hydrophobic and highly degradable in blood. Therefore, drug delivery systems would be of great benefit in supporting the selective accumulation of DSF in tumor cells. In this study, it was aimed to prepare a drug carrier system based on magnetic mesoporous silica nanoparticles (Fe3O4@mSiO2 MNPs) which are non-toxic, biocompatible, and have a mesoporous structure. The Fe3O4@mSiO2 MNPs were modified with folic acid linked polyethyleneimine (PEI-FA) to increase both their solubility in water and specificity for cancer cells. Thus, the cancer-selective DSF-carrier system (mMDPF) was synthesized with a high surface area but with dimensions of less than 160 nm, and were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM) and Brunauer-Emmett-Teller (BET) analysis. The drug-loading capacity of mMDPF was measured as 4.35% by high-performance liquid chromatography (HPLC) and the best drug release kinetics of mMDPF was observed at 37 °C and pH 6.0 which is the pH in the endosome. The cytotoxicity of the mMDPF on breast cancer (MCF-7) cells was improved by applying mMDPF with copper and/or sodium nitroprusside. It was observed that mMDPF was taken up more by MCF-7 cells and its toxicity on MCF-7 cells was much higher than non-tumorigenic (MCF-10A) cells.

Herbal medicine against genotoxicity of dimethoate, an insecticide, in mammalian somatic cells.

In this study, the genotoxic effects of dimethoate (DIM) were investigated with the in vitro micronucleus test in human peripheral lymphocytes. The ethanol extracts of Rosa canina and Salvia lavandulifolia were used to remove possible genotoxic effects of these substances. For this purpose, different concentrations (0.5-1-2 µg/mL) of dimethoate, DIM + RCeta and DIM + SLeta (1:1 v/v) application groups were prepared and applied to the blood culture. The obtained data were compared with the negative control group that was prepared with dimethyl sulfoxide (DMSO) as solvent and a well-known genotoxic effects of ethyl methanesulfonate (EMS) as positive control group. It was observed in lymphocyte cells that the frequency of MN considerably increased depending on the increasing dose of DIM whereas the nuclear division index (NBI)decreased according to the control group, especially in the last concentration (2 µg/mL). But, as the MN frequency decreased, NBI values approached to control group with 2µg/mL DIM + RCeta and 2µg/mL DIM + SLeta according to DIM application group (P < 0.05). Additionally, RCeta and SLeta were analyzed by gas chromotography-mass spectrometry (GC-MS).

Enhanced removal of basic violet 10 by heterogeneous sono-Fenton process using magnetite nanoparticles.

The removal of basic violet 10 (BV10), which is known as a cationic dye, from aqueous solution was studied by employing a heterogeneous sono-Fenton process over the nano-sized magnetite (Fe3O4) which had been prepared by the milling of magnetite mineral using a high-energy planetary ball milling process. The magnetite samples were characterized using the X-ray diffraction (XRD), high resolution scanning electron microscopy (HR-SEM), energy-dispersive X-ray spectroscopy (EDX), Brunauer-Emmett-Teller (BET), Fourier transform infrared spectroscopy (FTIR), and inductively couple plasma mass spectrometer (ICP-MS). It was found that the catalytic activity of the ball-milled magnetite sample was enhanced along with the improvement in its physicochemical properties; also, the ball-milled magnetite of 6 h displayed the highest catalytic activity in BV10 removal by the heterogeneous sono-Fenton process as compared with that for 4 h (66.12% after 120 min) and 2 h (48% after 120 min).The effect of operational parameters, namely, pH solution, catalyst dosage, the initial H2O2 concentration, ultrasonic power and the initial BV10 concentration, on the removal efficiency (RE%) of BV10 was investigated. The optimum conditions for the BV10 RE% were: the pH value of 3, the catalyst dosage of 1.5 g L-1, the initial H2O2 concentration of 36 mM, the ultrasonic power of 450 W L-1, and the initial BV10 concentration of 30 mg L-1. The RE% of BV10 was 75.94% at these conditions after the reaction time of 120 min. The trapping experiments revealed that OH radicals were the dominant oxidative species, but O2-/HO2 radicals also had a partial role in the removal of BV10.The reusability of the magnetite nanoparticles revealed about 28% decrease in the removal efficiency within five consecutive runs. The results obtained through GC-MS analysis also confirmed the efficient removal of BV10 molecules in the aqueous solution during the process.

Preparation of magnetite nanoparticles by high-energy planetary ball mill and its application for ciprofloxacin degradation through heterogeneous Fenton process.

In this study, the heterogeneous Fenton oxidation of ciprofloxacin (CIP) in an aqueous solution was examined over the nano-sized magnetite (Fe3O4) as a catalyst supplied through high-energy planetary ball milling process. To characterize the magnetite samples after and before ball milling operation, the X-ray diffraction (XRD), High-resolution scanning electron microscopy (HR-SEM), energy-dispersive X-ray spectroscopy (EDX), Brunauer-Emmett-Teller (BET) and Fourier transform infrared spectroscopy (FTIR) analysis were applied. The catalytic properties of the magnetite were considerably improved because of the enhancement in its physical properties, resulted from milling process. The findings also indicated that 6 h ball-milled magnetite demonstrated better properties for elimination of CIP of about 89% following 120 min reaction at optimal conditions of H2O2 12 mM, Fe3O4 1.75 g L-1, CIP 10 mg L-1 and pH 3.0. The effects of various operational parameters, including the initial pH of the solution, H2O2 initial concentration, catalyst dosage, milling time and CIP initial concentration was investigated. Application of organic and inorganic scavengers considerably decreased the CIP removal efficiency. Correspondingly, with respect to the leached iron values at pH 3, it was concluded that CIP elimination was mainly occurred through heterogeneous Fenton procedure. This process included the adsorption and oxidation phases in which the hydroxyl radicals (OH) played a significant role. GC-MS analysis was used for recording of the generated intermediates of the CIP removal in the course of heterogeneous Fenton process.

Simultaneous Determination of Arbutin and Hydroquinone in Different Herbal Slimming Products Using Gas Chromatography-mass Spectrometry.

OBJECTIVES: The aim of our study was to develop a simple, precise, sensitive and specific method for the simultaneous determination of arbutin and hydroquinone in different herbal slimming products using GC-MS. MATERIALS AND METHODS: The methanol and aqueous extracts of nine herbal slimming products in Turkey were evaluated for analysis of arbutin and hydroquinone using GC-MS method. RESULTS: The retention times of arbutin and hydroquinone were found as 11.32 and 5.44 min, respectively. The linear ranges in this method were 5-500 ng/mL for arbutin and hydroquinone, respectively. The intra- and inter-day precisions, expressed as the relative standard deviation, were less than 1.94 and 2.73%, determined from quality control samples for arbutin and hydroquinone, and accuracy was within 1.13 and 2.56% in terms of relative error, respectively. The limit of detection and quantification were 0.555 and 1.665 ng/mL for arbutin, and 0.031 and 0.093 ng/mL for hydroquinone, respectively. CONCLUSION: The developed method can be used for routine quality control analysis of arbutin and hydroquinone in different herbal slimming products.

Stability Indicating HPLC Method for the Determination of Fulvestrant in Pharmaceutical Formulation in Comparison with Linear Sweep Voltammetric Method.

This paper describes two rapid, sensitive and specific methods for the determination of fulvestrant in pharmaceutical preparations by high performance liquid chromatography (HPLC) and linear sweep voltammetry (LSV). HPLC method was used to study the degradation behaviour. Fulvestrant was subjected to degradation under the conditions of hydrolysis (acid and alkali), oxidation (30% H2O2). The linearity was established over the concentration range of 5-50 m g mL-1 for LSV and 0.5-20 m g mL-1 for HPLC method. The intra- and inter-day relative standard deviation (RSD) was less than 3.96 and 3.07% for LSV and HPLC, respectively. Limits of quantification were determined as 5.0 and 0.50 m g mL-1 for LSV and HPLC, respectively. No interference was found from tablet excipients at the selected assay conditions. The methods were applied for the quality control of commercial fulvestrant dosage form to quantify the drug and to check the formulation content uniformity.

Simple high-performance liquid chromatography method for formaldehyde determination in human tissue through derivatization with 2,4-dinitrophenylhydrazine.

A simple high-performance liquid chromatography method has been developed for the determination of formaldehyde in human tissue. FA Formaldehyde was derivatized with 2,4-dinitrophenylhydrazine. It was extracted from human tissue with ethyl acetate by liquid-liquid extraction and analyzed by high-performance liquid chromatography. The calibration curve was linear in the concentration range of 5.0-200 µg/mL. Intra- and interday precision values for formaldehyde in tissue were <6.9%, and accuracy (relative error) was better than 6.5%. The extraction recoveries of formaldehyde from human tissue were between 88 and 98%. The limits of detection and quantification of formaldehyde were 1.5 and 5.0 µg/mL, respectively. Also, this assay was applied to liver samples taken from a biopsy material.

HPLC/Fluorometric Detection of Carvedilol in Real Human Plasma Samples Using Liquid-Liquid Extraction.

A simple, rapid and sensitive high-performance liquid chromatography (HPLC) method has been developed to quantify carvedilol in human plasma using an isocratic system with fluorescence detection. The method included a single-step liquid-liquid extraction with diethylether and ethylacetate mixture (3 : 1, v/v). HPLC separation was carried out by reversed-phase chromatography with a mobile phase composed of 20 mM phosphate buffer (pH 7)-acetonitrile (65 : 35, v/v), pumped at a flow rate of 1.0 mL/min. Fluorescence detection was performed at 240 nm (excitation) and 330 nm (emission). The calibration curve for carvedilol was linear from 10 to 250 ng/mL. Intra- and interday precision values for carvedilol in human plasma were <4.93%, and accuracy (relative error) was better than 4.71%. The analytical recovery of carvedilol from human plasma averaged out to 91.8%. The limits of detection and quantification of carvedilol were 3.0 and 10 ng/mL, respectively. Also, the method was successfully applied to three patients with hypertension who had been given an oral tablet of 25 mg carvedilol.

MCP-1 and soluble TWEAK levels are independently associated with coronary artery disease severity in patients with chronic kidney disease.

PURPOSE: Patients diagnosed with chronic kidney disease (CKD) have a greater rate of cardiovascular mortality when compared with the general population. The soluble form of TNF-like weak inducer of apoptosis (TWEAK) and monocyte chemoattractan protein 1 (MCP-1) play important roles in cellular proliferation, migration and apoptosis. The current study aimed to analyze whether soluble TWEAK (sTWEAK) and MCP-1 levels are associated with the severity of coronary arterial disease (CAD) in CKD patients. METHODS: Ninety-seven patients diagnosed with CKD stages 2-3 according to their estimated glomerular filtration rate and the presence of kidney injury were included in the study. Plasma sTWEAK and MCP-1 concentrations were determined using commercially available ELISA kits. Coronary angiographies were performed through femoral artery access using the Judkins technique. RESULTS: Correlation analysis of sTWEAK and Gensini scores showed significant association (p < 0.01, r(2) = 0.287). Also significant correlation has been found in MCP-1 levels and Gensini scores (p < 0.01, r(2) = 0.414). When patients were divided into two groups with a limit of 17 according to their Gensini score, sTWEAK levels indicated a statistically significant difference (p < 0.01). CONCLUSIONS: Our findings support a relationship between sTWEAK and MCP-1 levels and CAD in CKD stages 2-3 patients.

Square Wave Voltammetric Determination of Diclofenac in Pharmaceutical Preparations and Human Serum.

In this study, a simple and reliable square wave voltammetric (SWV) method was developed and validated for determination of diclofenac in pharmaceutical preparations and human serum. The proposed method was based on electrooxidation of diclofenac at platinum electrode in 0.1 M TBAClO4/acetonitrile solution. The well-defined two oxidation peaks were observed at 0.87 and 1.27 V, respectively. Calibration curves that were obtained by using current values measured for second peak were linear over the concentration range of 1.5-17.5 µg mL(-1) and 2-20 µg mL(-1) in supporting electrolyte and serum, respectively. Precision and accuracy were also checked in all media. Intra- and inter-day precision values for diclofenac were less than 3.64, and accuracy (relative error) was better than 2.49%. Developed method in this study is accurate, precise and can be easily applied to Diclomec, Dicloflam and Voltaren tablets as pharmaceutical preparation. Also, the proposed technique was successfully applied to spiked human serum samples. No electroactive interferences from the endogenous substances were found in human serum.

Differential pulse voltammetric determination of diclofenac in pharmaceutical preparations and human serum / Pulso diferencial determinação voltamétrica de diclofenaco em preparações farmacêuticas e em soro humano

This article describes a differential pulse voltammetric (DPV) method for the determination of diclofenac in pharmaceutical preparations and human serum. The proposed method was based on electro-oxidation of diclofenac at platinum electrode in 0.1 M TBAClO₄/acetonitrile solution. The well-defined two oxidation peaks were observed at 0.87 and 1.27 V, respectively. Calibration curves that obtained by using current values measured for second peak were linear over the concentration range of 1.5-17.5 μg mL^-1and 2-20 μg mL^-1 in supporting electrolyte and serum, respectively. Precision and accuracy were also checked in all media. Intra- and inter-day precision values for diclofenac were less than 3.87, and accuracy (relative error) was better than 4.12%. The method developed in this study is accurate, precise and can be easily applied to Diclomec, Dicloflam and Voltaren tablets as pharmaceutical preparation. In addition, the proposed technique was successfully applied to spiked human serum samples. No electro-active interferences from the endogenous substances were found in human serum...

Este artigo descreve um método de voltametria de pulso diferencial (VPD) para a determinação de diclofenaco em preparações farmacêuticas e em soro humano. O método proposto foi baseado em eletroxidação de diclofenaco no eléctrodo de platina em solução 0,1 M TBAClO₄/acetonitrila. Dois picos de oxidação bem definidos foram observados em 0,87 e 1,27 V, respectivamente. As curvas de calibração obtidas utilizando-se valores de corrente medidos por segundo pico foram lineares no intervalo de concentração de 1,5-17,5 μg mL^-1e 2-20 μg mL^-1em eletrólito suporte e soro, respectivamente. Precisão e exatidão também foram verificadas em todos os meios. Valores de precisão intra- e inter-dia para o diclofenaco foram inferiores a 3.87 e a precisão (erro relativo) foi melhor do que 4,12%. O método desenvolvido neste estudo é exato, preciso e pode ser facilmente aplicado a Diclomec, Dicloflam e comprimidos Voltaren, como preparação farmacêutica. Além disso, a técnica proposta foi aplicada com sucesso em amostras de soro humano. Não se observaram interferências das substâncias endógenas no soro humano...

Determination of bosentan in pharmaceutical preparations by linear sweep, square wave and differential pulse voltammetry methods.

In this study, simple, fast and reliable cyclic voltammetry (CV), linear sweep voltammetry (LSV), square wave voltammetry (SWV) and differential pulse voltammetry (DPV) methods were developed and validated for determination of bosentan in pharmaceutical preparations. The proposed methods were based on electrochemical oxidation of bosentan at platinum electrode in acetonitrile solution containing 0.1 M TBACIO4. The well-defined oxidation peak was observed at 1.21 V. The calibration curves were linear for bosentan at the concentration range of 5-40 µg/mL for LSV and 5-35 µg/mL for SWV and DPV methods, respectively. Intra- and inter-day precision values for bosentan were less than 4.92, and accuracy (relative error) was better than 6.29%. The mean recovery of bosentan was 100.7% for pharmaceutical preparations. No interference was found from two tablet excipients at the selected assay conditions. Developed methods in this study are accurate, precise and can be easily applied to Tracleer and Diamond tablets as pharmaceutical preparation.

Simultaneous Determination of Tramadol and its Metabolite in Human Plasma by GC/MS.

A simple and sensitive GC/MS method for the determination of tramadol and its metabolite (O-desmethyltramadol) in human plasma was developed and validated. Medazepam was used as an internal standard. The calibration curves were linear (r=0.999) 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 (RSD%) of ≤4.83% and ≤4.68% for tramadol and O-desmethyltramadol, respectively. The extraction recoveries were 97.6±1.21% and 96.3±1.66% for tramadol and O-desmethyltramadol, respectively. The LOQ using 0.5 mL human plasma was 10 ng/mL for tramadol and 7.5 ng/mL for O-desmethyltramadol. Stability studies showed that tramadol and O-desmethyltramadol were stable in human plasma after 8 h incubation at room temperature or after 1 week storage at -20°C with three freeze-thaw cycles. Also, this method was successfully applied to six patients who had been given an intravenous formulation of 100 mg tramadol with Cmax results of 2018.1±687.8 and 96.1±22.7 ng/mL for tramadol and O-desmethyltramadol, respectively.

Determination of Flurbiprofen in Human Plasma by High-Performance Liquid Chromatography.

A simple high-performance liquid chromatography method has been developed for determination of flurbiprofen in human plasma. The method was validated on an Ace C18 column using UV detection. The mobile phase was acetonitrile-0.05 M potassium dihydrogen phosphate solution (60:40, v/v) adjusted to pH 3.5 with phosphoric acid. The calibration curve was linear between the concentration range of 0.10-5.0 µg/mL. Intra- and inter-day precision values for flurbiprofen in plasma were <4.47, and accuracy (relative error) was better than 3.67%. The extraction recoveries of flurbiprofen from human plasma were between 93.0 and 98.9%. The limits of detection and quantification of flurbiprofen were 0.03 and 0.10 µg/mL, respectively. In addition, this assay was applied to determine the pharmacokinetic parameters of flurbiprofen in six healthy Turkish volunteers who had been given 100 mg flurbiprofen.

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 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 = 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.