A Essential Appraisal In the Analytical Ways Available For Qualitative And Quantitative Analysis Of Cocaine.
Introduction:
Cocaine is an alkaloid extracted inside leaves with the cocoa plants Erythroxylum coca and Erythroxylum novogranatense. It is a powerful stimulant that targets the Central Nervous Method (CNS) and also the Cardio-Vascular procedure (CVS). It is a vasoconstrictor and activates the CNS, hence increasing the heart rate and blood pressure and causing a sensation of euphoria, energy and mental alertness. Cocaine is very addictive, due to the fact its effect wears off right after 20 to 40 minutes and the person always feels depressed, and normally opts for an additional dose to try to regain the condition of well-being. (Gold, Mark S. (2006)).
Cocaine is offered in 2 forms: the hydrochloride salt and the “freebase”. The hydrochloride salt, or powdered form of cocaine, can be taken intravenously or intra-nasally. Freebase is really a compound that has not been neutralized by an acid to produce the hydrochloride salt. The freebase type of cocaine is smokable.
Physical properties:
Cocaine hydrochloride crystals are colourless or white, hygroscopic, odourless and bitter-tasting. They are soluble in water and alcohol with solubility 200gm/100ml and 25gm/100ml respectively. These crystals are insoluble in ether. Their melting factor is 197°C including a 1% product is of neutral pH.
The freebase type (or crack-cocaine) has white crystals that are slightly volatile, anhydrous and bitter-tasting. They are soluble in water, alcohol and ether with solubility 0.17gm/100ml, 15.4gm/100ml and 28.6gm/100ml respectively. They melt at a lower temperature than the cocaine hydrochloride crystals - 98°C and boil at about 187-188°C.
Street cocaine employed by addicts can be mixed having a quantity of diluents ("cut"), and these include amphetamines, anti-histamines, benzocaine, inositol, lactose, lidocaine, mannitol, opioids, phencyclidine, procaine, sugars, tetracaine, and sometimes arsenic, caffeine, quinidine, and even flour or talc (Claustre, A. (1993)).
Chemical properties and metabolism:
The chemical name of cocaine is 3-tropanylbenzoate-2-carboxylic acid methyl ester. Its molecular pounds is 303.4 and molecular formula C17 H21 NO4. (Clarke, (1986))
Molecular structure of cocaine.
Cocaine is metabolized mainly to benzoylecgonine and ecgonine methylester. Cocaethylene is an indication of concurrent cocaine and alcohol consumption. Hydroxybenzoylecgonine is often a metabolite mainly present in meconium. Serum half-life of cocaine is 1-5 hours. Benzoylecgonine seems in circulation 15-30 minutes right after cocaine administration. Elimination half-life of benzoylecgonine is 5-6 hours. Benzoylecgonine will be detectable in urine following 2-3 hours and urine remains certain for 2-3 days (longer for chronic abusers) after cocaine use.
Cocaine in bodily fluids:
1 to 9% of cocaine is eliminated unchanged inside urine, having a greater proportion in acid urine. The metabolites ecgonine methyl ester, benzoylecgonine, and ecgonine are recovered in variable proportions which depend on a route of administration (Clarke, 1986). At the end of 4 hours, most from the drug is eliminated from plasma, but metabolites could be identified as much as 144 hours after administration (Ellenhorn & Barceloux, 1988).
Unchanged cocaine is excreted in the stool and in saliva (Clarke, 1986; Cone & Weddington, 1989). Cocaine and benzoylecgonine is also detected in maternal milk up to 36 hours right after administration, and in the urine of neonates for as much as Five days. (Chasnoff et al., 1987, 1989).
Freebase cocaine crosses the placenta, and norcocaine persists for 4 to Five days in amniotic fluid, even as soon as it's no longer detectable in maternal blood (Stinus, 1992).
Detection limits:
The detection of Cocaine in serum/plasma is limited for approx. 4-6 h (HT: 42-90 min.). In stored blood samples it decomposes with out stabilizing addition of NaF and cooling within A couple of days completely. As metabolites benzoylecgonine (HT: 5-7 h) and methylecgonine (HT: 4-5 h), that are each pharmacologically inactive, are detected. Inside urine is feasible through immunological quick tests (as Benzoylecgonine) for 3 to 5 days following the last consumption.
By means of hair analysis with GC/MS-detection, Cocaine and its metabolites are to become found as well as Coca ethylene as much as 6 months.
Analysis of cocaine:
Qualitative tests:
a. Solubility tests:
The sample is dissolved in water and ethanol separately. Dissolving in ethanol confirms presence of any ethanol insoluble carbohydrates in the sample. Solubility test is very best once large quantity of sample is available.
b. Thin-layer chromatography:
TLC relies on the reproducible migration pattern by drug of the thin layer adsorbent (eg: silica gel coated glass plates). Characterization of a specific drug is achieved by color reaction created by spraying the plate with coloring reagents. (Margoob, MA. et al. (2004)).
Quantitative tests:
1. Immunoassay tests
These testing are widely used from the medico-legal laboratories and are in accordance with the competitive binding of drug (that can be in a specimen) including a labeled drug to an antibody. A known quantity of an antibody is added towards the urine specimen. In addition, a known amount of labeled drug or drug metabolite (antigen) is added on the specimen. Any drug or drug metabolite offer inside the specimen will compete of the labeled drug or metabolite to bind from the antibodies forming antigen-antibody complexes. The amount of labeled antigen that's in a position to bind with an antibody is really a function in the amount of drug or drug metabolite during the urine. Spectrophotometric endpoints of these reactions are utilized to semi-quantitatively identify drugs and/or drug metabolites in each urine specimen.
Introduction:
Cocaine is an alkaloid extracted inside leaves with the cocoa plants Erythroxylum coca and Erythroxylum novogranatense. It is a powerful stimulant that targets the Central Nervous Method (CNS) and also the Cardio-Vascular procedure (CVS). It is a vasoconstrictor and activates the CNS, hence increasing the heart rate and blood pressure and causing a sensation of euphoria, energy and mental alertness. Cocaine is very addictive, due to the fact its effect wears off right after 20 to 40 minutes and the person always feels depressed, and normally opts for an additional dose to try to regain the condition of well-being. (Gold, Mark S. (2006)).
Cocaine is offered in 2 forms: the hydrochloride salt and the “freebase”. The hydrochloride salt, or powdered form of cocaine, can be taken intravenously or intra-nasally. Freebase is really a compound that has not been neutralized by an acid to produce the hydrochloride salt. The freebase type of cocaine is smokable.
Physical properties:
Cocaine hydrochloride crystals are colourless or white, hygroscopic, odourless and bitter-tasting. They are soluble in water and alcohol with solubility 200gm/100ml and 25gm/100ml respectively. These crystals are insoluble in ether. Their melting factor is 197°C including a 1% product is of neutral pH.
The freebase type (or crack-cocaine) has white crystals that are slightly volatile, anhydrous and bitter-tasting. They are soluble in water, alcohol and ether with solubility 0.17gm/100ml, 15.4gm/100ml and 28.6gm/100ml respectively. They melt at a lower temperature than the cocaine hydrochloride crystals - 98°C and boil at about 187-188°C.
Street cocaine employed by addicts can be mixed having a quantity of diluents ("cut"), and these include amphetamines, anti-histamines, benzocaine, inositol, lactose, lidocaine, mannitol, opioids, phencyclidine, procaine, sugars, tetracaine, and sometimes arsenic, caffeine, quinidine, and even flour or talc (Claustre, A. (1993)).
Chemical properties and metabolism:
The chemical name of cocaine is 3-tropanylbenzoate-2-carboxylic acid methyl ester. Its molecular pounds is 303.4 and molecular formula C17 H21 NO4. (Clarke, (1986))
Molecular structure of cocaine.
Cocaine is metabolized mainly to benzoylecgonine and ecgonine methylester. Cocaethylene is an indication of concurrent cocaine and alcohol consumption. Hydroxybenzoylecgonine is often a metabolite mainly present in meconium. Serum half-life of cocaine is 1-5 hours. Benzoylecgonine seems in circulation 15-30 minutes right after cocaine administration. Elimination half-life of benzoylecgonine is 5-6 hours. Benzoylecgonine will be detectable in urine following 2-3 hours and urine remains certain for 2-3 days (longer for chronic abusers) after cocaine use.
Cocaine in bodily fluids:
1 to 9% of cocaine is eliminated unchanged inside urine, having a greater proportion in acid urine. The metabolites ecgonine methyl ester, benzoylecgonine, and ecgonine are recovered in variable proportions which depend on a route of administration (Clarke, 1986). At the end of 4 hours, most from the drug is eliminated from plasma, but metabolites could be identified as much as 144 hours after administration (Ellenhorn & Barceloux, 1988).
Unchanged cocaine is excreted in the stool and in saliva (Clarke, 1986; Cone & Weddington, 1989). Cocaine and benzoylecgonine is also detected in maternal milk up to 36 hours right after administration, and in the urine of neonates for as much as Five days. (Chasnoff et al., 1987, 1989).
Freebase cocaine crosses the placenta, and norcocaine persists for 4 to Five days in amniotic fluid, even as soon as it's no longer detectable in maternal blood (Stinus, 1992).
Detection limits:
The detection of Cocaine in serum/plasma is limited for approx. 4-6 h (HT: 42-90 min.). In stored blood samples it decomposes with out stabilizing addition of NaF and cooling within A couple of days completely. As metabolites benzoylecgonine (HT: 5-7 h) and methylecgonine (HT: 4-5 h), that are each pharmacologically inactive, are detected. Inside urine is feasible through immunological quick tests (as Benzoylecgonine) for 3 to 5 days following the last consumption.
By means of hair analysis with GC/MS-detection, Cocaine and its metabolites are to become found as well as Coca ethylene as much as 6 months.
Analysis of cocaine:
Qualitative tests:
a. Solubility tests:
The sample is dissolved in water and ethanol separately. Dissolving in ethanol confirms presence of any ethanol insoluble carbohydrates in the sample. Solubility test is very best once large quantity of sample is available.
b. Thin-layer chromatography:
TLC relies on the reproducible migration pattern by drug of the thin layer adsorbent (eg: silica gel coated glass plates). Characterization of a specific drug is achieved by color reaction created by spraying the plate with coloring reagents. (Margoob, MA. et al. (2004)).
Quantitative tests:
1. Immunoassay tests
These testing are widely used from the medico-legal laboratories and are in accordance with the competitive binding of drug (that can be in a specimen) including a labeled drug to an antibody. A known quantity of an antibody is added towards the urine specimen. In addition, a known amount of labeled drug or drug metabolite (antigen) is added on the specimen. Any drug or drug metabolite offer inside the specimen will compete of the labeled drug or metabolite to bind from the antibodies forming antigen-antibody complexes. The amount of labeled antigen that's in a position to bind with an antibody is really a function in the amount of drug or drug metabolite during the urine. Spectrophotometric endpoints of these reactions are utilized to semi-quantitatively identify drugs and/or drug metabolites in each urine specimen.
A schematic diagram with the technique is as follows:
(Source: )
2. High performance liquid chromatography:
High-performance liquid chromatography (HPLC) is often a type of liquid chromatography to separate compounds which are dissolved in solution. HPLC instruments consist of the reservoir of mobile phase, a pump, an injector, a separation column, along with a detector. Compounds are separated by injecting a plug of the sample mixture onto the column. The different components in the mixture pass through the column at numerous rates due to differences in their partitioning behavior in between the cellular liquid phase as well as the stationary phase.
(Diagram taken from: )
HPLC can detect cocaine enantiomers. Recent development in HPLC coupled with electrospray (ESI) time-of-flight mass spectrometry provides a sensitive chromatographic confirmation program for cocaine and its metabolites. Use of the particular PFP Propyl column in combination using a high-organic cellular phase has resulted in short analysis times and allows detection limits at low picogram levels. (Sellers, K. (2005a)).
3. An FT-IR (Fourier Transform- Infrared spectroscopy):
This technique presumptively identifies unknown liquids and solids. It also distinguishes the cocaine salt within the free-base form because the presence of chloride ion during the salt type alters the absorption pattern in the cocaine molecule in FTIR.
4. Gas Chromatography-Mass Spectrometry (GC-MS)
GC/MS is really a combination of two numerous analytical techniques. Gas chromatography relies on physically separating the drug or drug metabolites inside the extract from one an additional as they pass via a long, small diameter column. The drug or drug metabolites migrate at various rates along the column and, therefore, exit the column at several times ahead of passing through a mass spectrometer which can positively identify a certain drug or drug metabolite. A mass spectrometer converts a drug or drug metabolite into charged particles and also the mass-to-charge (m/z) ratios on the particles generated create a pattern that provides a certain identification with the drug or drug metabolite at the measured retention time compared to a standard. (Analytical Testing Methods. (2005))
Gas Chromatography:
Gas Chromatography stands out as the separation of the mixture of compounds (solutes) into separate components, which then can me analyzed by a Mass Spectrometer to give us detailed empirical molecular info concerning the chemistry of the samples. In gas chromatography (GC), the sample is vaporized and injected onto chromatographic columns and then separate into many components. The elution is brought about by the flow of an inert gaseous cellular phase. Carrier gases, which compose the cellular phase of GC, include helium, argon, and nitrogen. As well as the stationary phase of GC is often a solid or liquid having a big surface where the absorption from the solutes takes place.
The schematic diagram of a Gas Chromatograph is as follows:
Below is really a typical flow chart of Gas Chromatography
Mass Spectrometery:
In mass spectroscopy, the sample (liquid, solid, solution, or vapor) enters the vacuum chamber via an inlet and depending on the sample, it may be ionized if it already isn’t. The ions are sorted during the mass analyzer in accordance with their mass-to-charge ratios after which collected by a detector wherever the ion flux is converted to a proportional electrical contemporary that's utilized to make a mass spectrum.
A regular flow chart of Mass Spectrometry is as follows:
(All diagrams are taken from: )
Sample preparation for GC-MS:
The drug or drug metabolite is selectively extracted from a urine specimen and it is then concentrated to a smaller volume (e.g., 0.5 mL or less) of an appropriate solution. The extraction technique is required to eliminate the drug or drug metabolite of interest from other “interfering” substances provide in urine. Once the drug or drug metabolite has been isolated and concentrated into an appropriate extract with a solid phase or solvent-solvent extraction procedure, the extract is injected into a GC/MS. (Analytical Tests Methods. (2005))
Advantages and disadvantages with the methods of analysis of cocaine:
The issue of false sure benefits creates the colour and odour tests unreliable. The presence of harmless white powders that could possibly be controlled drugs like methaqualone or synthetic local anaesthetics which are usually substituted for cocaine in the illicit site visitors usually hinder with the detection of cocaine within the colour test.
Thin layer chromatography (TLC) has been applied as a broad spectrum screen for detection of cocaine. Although this method is rapid and cheap, the outcomes of TLC can not be quantified. The major drawback of TLC is its low sensitivity and low specificity, thus unfavorable effects of TLC aren't always damaging by other methods. (Margoob, Mushtaq A. et al. (2004)).
Immunoassay tests, HPLC, GC-MS, LC/MS-MS all require sophisticated instruments and technical expertise and are hence expensive. Immunoassay diagnostic tests are readily automated and simple to perform, but they have many disadvantages. These diagnostic tests are slow and may well have some cross-reactivity of the drug or drug-metabolite apart during the 1 it's primarily screening for.
HPLC ways are also appropriate for detection of samples of “street cocaine” containing sugars since sugars aren't volatilized effortlessly and are difficult to analyse by GC. However, the ways are not yet well-developed and hence are not used widely. (Sellars, K. (2005b)).
GC-MS is time consuming as a result of numerous sample preparation steps, and poses the risk of exposure to hazardous solvents. However, the program is well established and it is still the process of choice. Confirmation of any certain cocaine result by this system is crucial in any capacity legal situation on each serum and urine samples. (Stimmel, B. (ed.), pp. 138).
Conclusion:
Of all the techniques available, GC-MS may be the most possible and reputable to date. Forensic screening laboratories about the globe use GC-MS for ones confirmation of cocaine in a sample. The procedures of analysis are well-established and have been shown to detect cocaine at picogram levels with accuracy. It provides undisputable identification of cocaine, its metabolites and adulterants and is hence generally preferred upon all of the other methods available.
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(Source: )
2. High performance liquid chromatography:
High-performance liquid chromatography (HPLC) is often a type of liquid chromatography to separate compounds which are dissolved in solution. HPLC instruments consist of the reservoir of mobile phase, a pump, an injector, a separation column, along with a detector. Compounds are separated by injecting a plug of the sample mixture onto the column. The different components in the mixture pass through the column at numerous rates due to differences in their partitioning behavior in between the cellular liquid phase as well as the stationary phase.
(Diagram taken from: )
HPLC can detect cocaine enantiomers. Recent development in HPLC coupled with electrospray (ESI) time-of-flight mass spectrometry provides a sensitive chromatographic confirmation program for cocaine and its metabolites. Use of the particular PFP Propyl column in combination using a high-organic cellular phase has resulted in short analysis times and allows detection limits at low picogram levels. (Sellers, K. (2005a)).
3. An FT-IR (Fourier Transform- Infrared spectroscopy):
This technique presumptively identifies unknown liquids and solids. It also distinguishes the cocaine salt within the free-base form because the presence of chloride ion during the salt type alters the absorption pattern in the cocaine molecule in FTIR.
4. Gas Chromatography-Mass Spectrometry (GC-MS)
GC/MS is really a combination of two numerous analytical techniques. Gas chromatography relies on physically separating the drug or drug metabolites inside the extract from one an additional as they pass via a long, small diameter column. The drug or drug metabolites migrate at various rates along the column and, therefore, exit the column at several times ahead of passing through a mass spectrometer which can positively identify a certain drug or drug metabolite. A mass spectrometer converts a drug or drug metabolite into charged particles and also the mass-to-charge (m/z) ratios on the particles generated create a pattern that provides a certain identification with the drug or drug metabolite at the measured retention time compared to a standard. (Analytical Testing Methods. (2005))
Gas Chromatography:
Gas Chromatography stands out as the separation of the mixture of compounds (solutes) into separate components, which then can me analyzed by a Mass Spectrometer to give us detailed empirical molecular info concerning the chemistry of the samples. In gas chromatography (GC), the sample is vaporized and injected onto chromatographic columns and then separate into many components. The elution is brought about by the flow of an inert gaseous cellular phase. Carrier gases, which compose the cellular phase of GC, include helium, argon, and nitrogen. As well as the stationary phase of GC is often a solid or liquid having a big surface where the absorption from the solutes takes place.
The schematic diagram of a Gas Chromatograph is as follows:
Below is really a typical flow chart of Gas Chromatography
Mass Spectrometery:
In mass spectroscopy, the sample (liquid, solid, solution, or vapor) enters the vacuum chamber via an inlet and depending on the sample, it may be ionized if it already isn’t. The ions are sorted during the mass analyzer in accordance with their mass-to-charge ratios after which collected by a detector wherever the ion flux is converted to a proportional electrical contemporary that's utilized to make a mass spectrum.
A regular flow chart of Mass Spectrometry is as follows:
(All diagrams are taken from: )
Sample preparation for GC-MS:
The drug or drug metabolite is selectively extracted from a urine specimen and it is then concentrated to a smaller volume (e.g., 0.5 mL or less) of an appropriate solution. The extraction technique is required to eliminate the drug or drug metabolite of interest from other “interfering” substances provide in urine. Once the drug or drug metabolite has been isolated and concentrated into an appropriate extract with a solid phase or solvent-solvent extraction procedure, the extract is injected into a GC/MS. (Analytical Tests Methods. (2005))
Advantages and disadvantages with the methods of analysis of cocaine:
The issue of false sure benefits creates the colour and odour tests unreliable. The presence of harmless white powders that could possibly be controlled drugs like methaqualone or synthetic local anaesthetics which are usually substituted for cocaine in the illicit site visitors usually hinder with the detection of cocaine within the colour test.
Thin layer chromatography (TLC) has been applied as a broad spectrum screen for detection of cocaine. Although this method is rapid and cheap, the outcomes of TLC can not be quantified. The major drawback of TLC is its low sensitivity and low specificity, thus unfavorable effects of TLC aren't always damaging by other methods. (Margoob, Mushtaq A. et al. (2004)).
Immunoassay tests, HPLC, GC-MS, LC/MS-MS all require sophisticated instruments and technical expertise and are hence expensive. Immunoassay diagnostic tests are readily automated and simple to perform, but they have many disadvantages. These diagnostic tests are slow and may well have some cross-reactivity of the drug or drug-metabolite apart during the 1 it's primarily screening for.
HPLC ways are also appropriate for detection of samples of “street cocaine” containing sugars since sugars aren't volatilized effortlessly and are difficult to analyse by GC. However, the ways are not yet well-developed and hence are not used widely. (Sellars, K. (2005b)).
GC-MS is time consuming as a result of numerous sample preparation steps, and poses the risk of exposure to hazardous solvents. However, the program is well established and it is still the process of choice. Confirmation of any certain cocaine result by this system is crucial in any capacity legal situation on each serum and urine samples. (Stimmel, B. (ed.), pp. 138).
Conclusion:
Of all the techniques available, GC-MS may be the most possible and reputable to date. Forensic screening laboratories about the globe use GC-MS for ones confirmation of cocaine in a sample. The procedures of analysis are well-established and have been shown to detect cocaine at picogram levels with accuracy. It provides undisputable identification of cocaine, its metabolites and adulterants and is hence generally preferred upon all of the other methods available.
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