Determination of Tramadol in Liver Tissues Using HPLC-DAD

Tramadol prescribed narcotic analgesic; tramadol overdose was reported old male 35 years. Free tramadol and its metabolite isolated by two methods of extraction, Stas Otto and ammonium sulfate extraction from liver tissues and comparison between efficiency of the two methods. Liver extractions have tramadol and main metabolite O-desmethyltramadol was quantified by HPLC-DAD. Tramadol was determined in liver concentration 27.98 μg/g and 27.93 O-desmethyltramadol in Stas Otto. Liver concentration of tramadol 23.92 μg/g and O-desmethyltramadol 9.62 μg/g in ammonium sulfate extraction. Objective: To determination free tramadol and its metabolite in liver tissues by using two methods of extraction and comparison concentrations by HPLC-DAD of liver old male 35 years over dose in Upper Egypt. Determination of Tramadol in Liver Tissues Using HPLC-DAD


Introduction
Tramadol consists of two enantiomers (+) -, and chemically (+)-trans-2-(Dimethylaminomethyl)-1-(m-methoxyphenyl) cyclohexanol, was first synthesized in 1962 by Grünenthal GmbH in Germany. Tramadol is rapidly and almost completely absorbed after oral or parenteral administration. The main tramadol metabolic reactions are N-and O-demethylation and conjugation with glucuronic acid and sulfate. The major metabolites formed are O-monodesmethyltramadol, N,O-didesmethyltramadol and their conjugates, and N-monodesmethyltramadol. O-Monodesmethyltramadol is an active metabolite and has a greater analgesic activity than the parent drug.
Tramadol analgesic effects are due to the inhibition of norepinephrine and serotonin reuptake as well as agonism of mu receptors which cause blockage of the pain impulses in the spinal cord while its metabolite O-desmethyltramadol acts on the μ-opioid receptor.
The relation between serum level and analgesic effect is not the same among different people. It is estimated that the normal therapeutic serum level of tramadol and its metabolite are 0.1 to 0.3 mg/L and 0.03 to 0.04 mg/L, respectively [7][8][9][10]. It is advised that the drug be used with caution in those with liver or kidney failure, due to metabolism in the liver (to desmetramadol) and elimination via the kidneys [11].
Symptoms of tramadol intoxication are similar to those of other opioid analgesics but may include serotonergic and noradrenergic components. Symptoms include central nervous system (CNS) depression and coma, tachycardia, cardiovascular collapse, seizures, and respiratory depression up to respiratory arrest.
O-desmethyltramadol impairs consciousness and causes electrocardiographic (ECG) changes and seizure Seizure is more common in young males (mean age of 22-39.5 yrs.), however, some studies reported no significant difference in the frequency of seizure between different genders [17]. The most common mechanisms of death after tramadol overdose are cardio-respiratory depression, resistant shock, asystole, and liver failure [18]. Apnea may increase the risk of tramadol intoxication-related deaths [19].
The extractions converted to alkaline by ammonia and chloroform solvent was used.

Discussion
Tramadol is available as tablets, solution for injection, and suppository. Tramadol is the first distribution drug abuse in Egypt.
A fatal overdose case involving tramadol, all body tissues and blood, the drug concentration was greater than the metabolites N-desmethyl and O-desmethyltramadol, and O-desmethyltramadol was always greater concentration than the N-desmethyl metabolite [20].
Tramadol is extensively metabolized in the liver by demethylation, oxidation and conjugation (sulphation and glucuronidation). Tramadol is considered to be a safe drug so, most of the studies were performed according to the GLP guidelines on the basis of the guidelines on the quality, safety and efficacy of tramadol for human use of the commission of the European Communities (EC Guidelines).
However, mortality has been reported old male 35 years in Upper Egypt, his family mentioned that' he was orally unknown materials and dead at reached to emergency of hospital. Autopsy didn't find any signs and lab investigations of pesticides and insecticides and another routine work of toxic compounds it's negative results and other work of drugs except tramadol drug only by high concentrations was determined (over dose). Tramadol and its metabolite O-desmethyltramadol were determined in over dose by two famous methods of extractions and comparison between its Stas-Otto and ammonium sulfate are used to remove fatty components, impurities and other interference materials and precipitate proteins.
The results reported over dose in Table 1 explain the same and Stas-Otto had higher extraction efficiency of tramadol and Odesmethyltramadol more than in ammonium sulfate extraction in tramadol and metabolite's, hence Stas-Otto is preferred (Figures 1-4).

Conclusion
Physicians and experts in branches of medicine must be aware peoples from tramadol addiction and over dose of tramadol, the governments must be but control in used this compound and fights distribution between youth.