Quantification and Comparison of Opium (Morphine) and Tramadol from Biological Samples "Liquid - Liquid Extraction"

Symptoms of opiod intoxication are similar tramadol analgesics, Symptoms include central nervous system (CNS) depression and coma, tachycardia, cardiovascular collapse, seizures, and respiratory depression up to respiratory arrest.

Opioid is a term for a number of natural substances (originally derived from the opium poppy) and their semi-synthetic and synthetic analogues that bind to specific opioid receptors. The main components of opium is morphine has widespread effects in the central nervous system and on smooth muscle.

Opium was used as a sedative and hypnotic, but it was determined to be addictive, the effects by activating specific G protein-coupled receptors in the brain, spinal cord, and peripheral nervous system. There are three major classes of opioid receptors being delta δ-opioid, kappa κ-opioid and mu μ-opioid. Opium will generate an agonist activity which will later open the potassium channels and prevent the opening of voltage-gated calcium channels. This activity causes a reduction in neuronal excitability and inhibits the release of pain neurotransmitters ¹.

Opium and its derivatives and tramadol are the most commonly used medications for the treatment of acute and chronic pain. Opium and its alkaloid-derivatives can also be used as tranquilizers, antitussives and in the treatment of diarrhea ², but the direct use of opium is not common nowadays and its derivatives such as morphine and codeine etc...are used.

The unspecific effect of opium to the different opioid receptors produce the generation of various effects such as sedation, euphoria, dysphoria, respiratory depression, constipation, pruritus, nausea, and vomiting. It is reported that the secondary effects tend to be diminished as long-term use tolerance is developed. Some reports have also shown an opioid-driven impairment of the hypothalamic function that can result in a loss of libido, impotence, and infertility.

The addictive character of opium is related to the binding to the μ-opioid receptors, which will activate dopaminergic neurons in the ventral tegmental area of the midbrain and thus, enhance the dopamine release in the nucleus accumbens. This mechanism involves the reward activity of the mesolimbic dopaminergic pathway ³.

Opium contains tenths different alkaloid opiates. The most common metabolism of opiates is to be ultimately converted to morphine which is further converted to morphine-3,6-diglucuronide. In the cardiovascular system, there are reports of peripheral vasodilatation, including cutaneous causing flushing of the face, neck, and thorax, impaired sympathetic reflexes and postural hypotension. In the gastrointestinal and urogenital system, the increase in smooth muscle tone has been shown to produce reduced peristalsis, delayed gastric emptying and urinary retention ¹.

The main opium overdose toxicity effect is decreased respiratory rate and depth, which can progress to apnea and therefore result in death. Other complications (pulmonary edema, which usually develops within minutes to a few hours after opioid overdose) and death result primarily from hypoxia. Pupils are miotic. Delirium, hypotension, bradycardia, decreased body temperature, and urinary retention may also occur ¹.

Tramadol is rapidly absorbed after oral administration. The major metabolites of tramadol are O-monodesmethyltramadol, N,O-didesmethyltramadol and their conjugates, and N-monodesmethyltramadol. O-Monodesmethyltramadol is an active metabolite and has a effective analgesic than tramadol drug. The effective of tramadol due to inhibition of nervous transports such as norepinephrine and serotonin reuptake as well as agonism of mu receptors which cause blocking pain impulses of the spinal cord while the O-desmethyltramadol binding in μ-opioid receptor. This binding wit mu receptors causing effectives as opioid; while noradrenergic pathways causing inhibition of norepinephrine reuptake in Central Nervous System (CNS); Affecting serotonergic pathways causing inhibition of serotonin reuptake in the CNS; GABAergic pathways causing increased GABA neurotransmitter in the brain ^{4, 5, 6, 7, 8, 9, 10}.

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 (10-13). 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 ¹².

Protein Precipitation is the process in which protein is separated from tissues or any extra contaminants that may be mixed with it. It is an important part of downstream processing and can be done with a variety of different techniques. While there are a number of different methods of precipitation, in this study two methods were used to comparison in extraction opium as morphine and tramadol as free drug.

The extraction of drugs from solid tissues requires the tissue matrix be broken down to release drugs into an environment from which they are accessible for solvent extraction. This can be achieved by cutting tissues by scissor and homogenization. Many methods direct solvent extraction are used classic for protein precipitation.

Liquid-liquid extractions are predominates in most laboratories, The choice of an appropriate solvent is often a matter of experience or tradition. The chosen solvent should ideally extract as much of the target analyte as possible.

Many methods of extraction drugs from biological samples were used, but the most popular ones is Salt Induced Precipitation (Salting Out). with ammonium sulfate the first method of “salting out” precipitation protein can occur with a number of different types of neutral salts but ammonium is preferred salt because it is high on the Hofmiester series and has a high solubility rate.

Protein interacts with the salt as opposed to the water, which leads to less interaction between the water and the protein’s solvent layer, which in turn leads to more hydrophobic patches being exposed and to encourage those patches to interact with one another. This leads the proteins to aggregate and precipitate. The second methods is Precipitation with miscible solvents, such as ethanol to a solution cause proteins in the solution to precipitate. The solvent layer around the protein will decrease as the organic solvent progressively displaces water from the protein surface and binds it in hydration layers around the organic solvent molecules. With smaller hydration layers, the proteins can aggregate by attractive electrostatic and dipole forces.

Validation Study

Physicians and experts in branches of medicine must be aware peoples from opium and generally opoides compounds has similar effected such as tramadol addiction and over dose of opium and tramadol, the governments must be but control in used this compound and fights distribution it's materials between peoples specialties youth.

Two methods were used to isolation and precipitation protein (Stass-Otto and Ammonium Sulfate), quantitative of morphine and tramadol respectively are 176 u/g and 267 u/g, and 22.41 u/g and 26.18 u/g in Stas Otto and ammonium sulfate, So that results explained morphine extraction by Amm. Sulfate excellent while tramadol in Stas-Otto is preferred than Amm. Sulfate, and good conditions of GC-MS was used to quantification morphine and tramado.

Morphine and tramadol quantification in biological samples (liver) by two methods precipitations of protein using GC-MS and discuses methods of prefer by applied in liver tissues of old male 40 years over dose in Upper Egypt.

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