Simultaneous analysis of tramadol, O-desmethyltramadol, and N-desmethyltramadol enantiomers in rat plasma by high-performance liquid chromatography-tandem mass spectrometry: application to pharmacokinetics

Tramadol (T) is available as a racemic mixture of (+)‐trans‐T and (−)‐trans‐T. The main metabolic pathways are O‐demethylation and N‐demethylation, producing trans‐O‐desmethyltramadol ( M1 ) and trans‐N‐desmethyltramadol ( M2 ) enantiomers, respectively. The analgesic effect of T is related to the opioid activity of (+)‐trans‐T and (+)‐ M1 and to the monoaminergic action of (+/−)‐trans‐T. This is the first study using tandem mass spectrometry as a detection system for the simultaneous analysis of trans‐T, M1 , and M2 enantiomers. The analytes were resolved on a Chiralpak® AD column using hexane:ethanol (95.5:4.5, v/v) plus 0.1% diethylamine as the mobile phase. The quantitation limits were 0.5 ng/ml for trans‐T and M1 and 0.1 ng/ml for M2 . The method developed and validated here was applied to a pharmacokinetic study in rats. Male Wistar rats (n = 6 at each time point) received a single oral dose of 20 mg/kg racemic trans‐T. Blood samples were collected up to 12 h after drug administration. The kinetic disposition of trans‐T and M2 was enantioselective (AUC_(+)/(−) ratio = 4.16 and 6.36, respectively). The direction and extent of enantioselectivity in the pharmacokinetics of trans‐T and M2 in rats were comparable to data previously reported for healthy volunteers, suggesting that rats are a suitable model for enantioselective studies of trans‐T pharmacokinetics. Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

Effects of chronic tramadol exposure on the zebrafish brain: a proteomic study

Tramadol hydrochloride (TH), has become the most prescribed opioid worldwide. However, its neurotoxicity and abuse potential are not well documented. In the present study, TH administration induced abnormal behavior and body and brain mean weight loss. Two principal metabolites O- and N-desmethyltramadol were detected in the brain tissue, and N-desmethyltramadol was the main metabolite produced. A total of 30 differential protein spots were identified using semi-quantitative 2D-PAGE and proteomic analyses, and classified into 13 categories, in which subtypes of 14-3-3 proteins, creatine kinase, ATP synthase beta chain, and tubulin were identified at the separated location on the gels 3, 3, 4, and 11 times respectively. Many TH responsive proteins have functions related to oxidative stress, including 14-3-3 proteins, creatine kinase BB, ubiquitin carboxy-terminal hydrolase L-1, ATP synthase, synaptosome-associated protein, tubulin and actin. Irrespective of oxidative damage, other pathways affected include apoptosis, energy metabolism, signal disorders, and cytoskeletal structure. Ultrastructural observation of mitochondria showed a series of morphological changes in the case of TH exposure.     

Use of hydrophilic natural gums in formulation of sustained-release matrix tablets of tramadol hydrochloride

The objective of this work was to develop matrix sustained-release tablets of highly water-soluble tramadol HCl using natural gums (xanthan [X gum] and guar [G gum]) as cost-effective, nontoxic, easily available, and suitable hydrophilic matrix systems compared with the extensively investigated hydrophilic matrices (ie, hydroxypropyl methylcellulose [HPMC]/carboxymethyl cellulose [CMC] with respect to in vitro drug release rate) and hydration rate of the polymers. Matrix tablets of tramadol (dose 100 mg) were produced by direct compression method. Different ratios, of 100∶0, 80∶20, 60∶40, 20∶80, 0∶100 of G gum (or X):HPMC, X gum:G gum, and triple mixture of these polymers (G gum, X gum, HPMC) were applied. After evaluation of physical characteristics of tablets, the dissolution test was, performed in the phosphate buffer media (pH 7.4) up to 8 hours. Tablets with only X had the highest mean dissolution time (MDT), the least dissolution efficiency (DE₈%), and released the drug following a zero-order model via swelling, diffusion, and erosion mechanisms. Guar gum alone could not efficiently control the drug release, while X and all combinations of natural gums with HPMC could retard tramadol HCl release. However, according to the similarity factor (f ₂), pure HPMC and H₈G₂ were the most similar formulations to Topalgic-LP as the reference standard. Published: March 17, 2006     

Liver and kidney toxicity in chronic use of opioids: An experimental long term treatment model

In this study, histopathological and biochemical changes due to chronic usage of morphine or tramadol in liver and kidney were assessed in rats. Thirty male Wistar rats (180–220 g) were included and divided into three groups. Normal saline (1 ml) was given intraperitoneally as placebo in the control group (n = 10). Morphine group (n = 10) received morphine intraperitoneally at a dose of 4, 8, 10 mg/kg/day in the first, second and the third ten days of the study, respectively. Tramadol group (n = 10), received the drug intraperitoneally at doses of 20, 40 and 80 mg/kg/day in the first, second and the third ten days of the study, respectively. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), creatinin, blood urea nitrogen (BUN) and malondialdehyde (MDA) levels were measured in the serum. Liver and kidney specimens were evaluated by light microscopy. Serum ALT, AST, LDH, BUN and creatinin levels were significantly higher in morphine group compared to the control group. Serum LDH, BUN and creatinin levels were significantly increased in the morphine group compared to the tramadol group. The mean MDA level was significantly higher in morphine group compared to the tramadol and control groups (P<0.05). Light microscopy revealed severe centrolobular congestion and focal necrosis in the liver of morphine and tramadol groups, but perivenular necrosis was present only in the morphine group. The main histopathologic finding was vacuolization in tubular cells in morphine and tramadol groups. Our findings pointed out the risk of increased lipid peroxidation, hepatic and renal damage due to long term use of opioids, especially morphine. Although opioids are reported to be effective in pain management, their toxic effects should be kept in mind during chronic usage Presented at the 10th XX Annual ESRA Congress, 6–9 April 2002, Warsaw, Poland.     

Analgesia induced by combined action of Tramadol and microwave irradiation in somatic pain in mice

In experiments on albino male mice weighing 24 to 32 g, we studied the suppression of the pain reaction induced by injection of formalin into the dorsal surface of the foot, using a combination of injection of tramadol hydrochloride (Tramal) in a dose of 0.42 mg/kg and irradiation of the acupuncture point (AP) E-36 with low-intensity microwaves (frequency 30 to 300 GHz and power rate density 3·10⁻⁹ W/cm²). The combined use of tramadol injection and the action of microwaves on the AP led to a decrease in the duration of the pain reaction (licking of the pain nidus), on average, by 58.2%, as compared with that in the control animal group. The analgesic effect was more strong in the case of such a combination than after isolated injection of tramadol in doses of 0.83 and 0.42 mg/kg (by 14.4 and by 10.0%, respectively) and isolated microwave irradiation of the AP TA E-36 (by 48.7%). The data obtained demonstrate the possibility of effective analgesia using a combination of microwave irradiation of the AP and the pharmacological influence with a significant decrease in the dose of the analgesic. Neirofiziologiya/Neurophysiology, Vol. 38, No. 4, pp. 314–319, July–August, 2006.     

Optimized HPLC method for tramadol and O-desmethyl tramadol determination in human plasma

The optimized method for HPLC determination of tramadol and its metabolite O-desmethyl tramadol in human plasma using sotalol as internal standard has been developed and validated by a new approach. The determination by fluorescence detection was performed on re-eluted solution, obtained after liquid–liquid extraction with ethyl acetate of the three analytes from plasma. The chromatographic separation of tramadol under a gradient elution was achieved at a temperature of 15 °C with a RP-18 column, guarded by a C18 precolumn. The mobile phase was a mixed aqueous solution containing ortho-phosphoric acid, triethylamine, acetonitrile and methanol in a complex gradient mode. The quantitative determination of tramadol was performed at different successive pairs of excitation/emission wavelengths (200/300 nm, 200/295 nm, 212/305 nm) with lower limits of quantification: LLOQ = 4.078 ng/ml for tramadol, respectively LLOQ = 3.271 ng/ml for O-desmethyl tramadol. For the LLOQ limits, were calculated the values of the coefficient of variation and difference between mean and the nominal concentration. For tramadol analyte they were CV% = 5.147% and bias% = − 7.273% in the intra-days and CV% = 4.894% and bias% = 0.836% in the between-days assay, respectively for the metabolite O-desmethyl tramadol they were CV% = 11.517% and bias% = 0.337% in the intra-days and CV% = 6.41% and bias% = 3.259% in the between-days assay.

In addition, the stabilities of the analytes were verified in different conditions. Both, tramadol and its metabolite proved to be stable in plasma for four weeks, frozen at − 20 °C, but also for 48 h at 15 °C in the re-eluted solution after liquid–liquid extraction.     

曲马朵与二氢埃托啡协同镇痛并推迟大鼠急性耐受

本文旨在研究曲马朵（tramadol,TRA）和二氢埃托啡（dihydroetorphine,DHE）联合用药是否可产生协同镇痛并延缓耐受的发生。TRA（mg,腹腔注射）与DHE（ng,皮下注射）按固定比率给药（1：6．25,1：12．5,1：25,1：50,l：100,1：200）,用热辐射甩尾法评价镇痛效应,采用等高线法评估药物的协同作用。在急性耐受实验中,连续6次注射TRA（20mg／kg）、DHE（1000ng／kg）或两药的组合（TRA20mg／kg＋DHE250ng／kg）。结果显示：（1）除1mg：6．25ng和1mg：50ng两个比例外,其他所有比例用药均产生显著的协同镇痛效应;（2）TRA与DHE联合用药的疗效在连续给药中持续时间明显延长,提示二者联合使用可延缓耐受。以上结果提示：TRA与DHE在一定剂量比范围内可产生协同镇痛效应,并可推迟耐受的形成。     

Polymeric Matrix System for Prolonged Delivery of Tramadol Hydrochloride, Part I: Physicochemical Evaluation

Management of moderate or severe chronic pain conditions is the burden of clinicians dealing with patients trying to improve their quality of life and diminish their suffering. Although not a new opioid, tramadol has been recently rediscovered and widely used; this may be due to its favorable chronic safety and dependence profiles together with its high potency. Tramadol is a centrally acting analgesic with half-life of ~6 h; therefore, it requires frequent dosing. It is freely soluble in water; hence, judicious selection of retarding formulations is necessary. The current study is focused on the innovation of a novel, simple, monolayer, easy-to-use, cost-effective, and aesthetically acceptable bioadhesive transdermal delivery system overcoming the defects of the conventional “patch” as carrier system for tramadol, ensuring its adequate delivery, along with the physicochemical evaluation of the designed formulations. Monolithic tramadol matrix films of chitosan, different types of Eudragit®, and binary mixtures of both were prepared. As a single-polymer film, chitosan film showed best properties except for somewhat high moisture uptake capacity, insufficient strength and rapid release, and permeation. Polymer blends were monitored in order to optimize both properties and performance. Promising results were obtained, with chitosan–Eudragit® NE30D (1:1) film showing the most desirable combined, sufficiently rapid as well as prolonged release and permeation profiles along with satisfactory organoleptic and physicochemical properties.     

Applying quality by design (QbD) concept for fabrication of chitosan coated nanoliposomes

In the present investigation, a quality by design (QbD) strategy was successfully applied to the fabrication of chitosan-coated nanoliposomes (CH-NLPs) encapsulating a hydrophilic drug. The effects of the processing variables on the particle size, encapsulation efficiency (%EE) and coating efficiency (%CE) of CH-NLPs (prepared using a modified ethanol injection method) were investigated. The concentrations of lipid, cholesterol, drug and chitosan; stirring speed, sonication time; organic:aqueous phase ratio; and temperature were identified as the key factors after risk analysis for conducting a screening design study. A separate study was designed to investigate the robustness of the predicted design space. The particle size, %EE and %CE of the optimized CH-NLPs were 111.3?nm, 33.4% and 35.2%, respectively. The observed responses were in accordance with the predicted response, which confirms the suitability and robustness of the design space for CH-NLP formulation. In conclusion, optimization of the selected key variables will help minimize the problems related to size, %EE and %CE that are generally encountered when scaling up processes for NLP formulations. The robustness of the design space will help minimize both intra-batch and inter-batch variations, which are quite common in the pharmaceutical industry.     

Pharmacokinetics of the enantiomers of trans-tramadol and its active metabolite, trans-O-demethyltramadol, in healthy male and female chinese volunteers

By using a high-performance capillary electrophoresis method, the pharmacokinetics of the enantiomers of trans-tramadol (trans-T) and its active metabolite, trans-O-demethyltramadol (M1), was studied in healthy male and female Chinese volunteers after oral administration of 100 mg trans-T hydrochloride. The values of Cmax for the enantiomers of trans-T and M1, and AUC0- infinity for (-)-trans T, (+)-M1, and (-)-M1 were higher in females than in males. The values of V(d)/F for the enantiomers of trans-T and CLr for (+)-M1 were lower in females than in males. The value of t(1/2) for (-)-M1 was longer in females than in males. There were significant differences in pharmacokinetic parameters of the two enantiomers of trans-T or M1 both in males and in females. The (+)/(-)-enantiomeric ratios of t(max), V(d)/F for trans-T in males were significantly different from those in females and the (+)/(-)-enantiomeric ratios of pharmacokinetic parameters for M1 in males were similar to those in females. There are gender-related differences in the pharmacokinetics of the enantiomers of trans-T and M1 which may be due to the greater body weights for men and/or the higher CYP2D6 activity in women. The pharmacokinetics of trans-T and M1 is stereoselective in men and women. There is a gender-related difference in the stereoselectivity in pharmacokinetics of trans-T in human and the stereoselectivity in pharmacokinetics of M1 in men is similar to that in women. Chirality 16:112-118, 2004.