Cocaine is chief alkaloid found in the leaves of a South American shrub, Coca. In olden days, coca leaves were used for their stimulant properties (chewed as it produced a numbing effect on mouth and tongue) by natives of south America, particularly those living at high altitude ,who used it to reduce fatigue during work at these altitudes .Considerable mystical significance was attached to the powers of cocaine to boost the flagging human spirit.
Chemist Freud tested cocaine extensively on his patients and his family. Observing the result of Freud’s experiments with cocaine, his ophthalmologist colleague, Koller, obtained supplies of the drug and discovered its local anaesthetic action, later its psycho stimulant effects was proved. These discoveries led to cocaine becoming at one time, the most frequently abused substances in Western countries. Though its use has decreased somewhat since 1990. In the International context, UNODC (United Nations Office on Drugs and Crime) report some 13.4 million people (0.3%) of the world’s population aged between 15 and 64 years have used cocaine in the year 2009.
The first clinical use of cocaine as the local anaesthetic agent occurred in 1884, where cocaine was employed as a topical agent for eye surgery as well as being used to produce a nerve block. The idea was rapidly taken up within a few years and cocaine as anaesthesia was introduced in dentistry and general surgery. In Britain, The Rolleston Act of 1926 gives doctor the right to prescribe narcotics on their own judgement, it become primary basis of narcotic legislation until 1967. A synthetic substitute, procaine a potent anaesthetic was discovered in 1905. Many other useful compounds were later developed and effective Narcotic Laws were passed.
Generally, local anaesthetic molecule like cocaine consists of an aromatic parent linked by an ester bond to the basic side chain. It is a weak base, with pKa values mainly in the range 8-9. Consequently, it is mainly but not completely, ionised at physiological pH. This is an important in relation to its ability to penetrate the nerve sheath and axon membrane. The presence of the ester bond in cocaine is important because of its susceptibility to metabolic hydrolysis. The ester-containing compounds are usually inactivated in the plasma and tissue (mainly liver) by non –specific esterases. The metabolism of Cocaine leads to productions of mainly Benzoylecgonine along with Ecgonine and Ecgonine methyl ester.
Pharmacodynamics:
The primary action of the cocaine is blocking of Na+ channel conductance.In the nerve that is actively conducting impulses, it inhibits the inward migration of Na+ Ions. Its blocks the initiation and propagation of action potentials by preventing the voltage dependent increase in Na+ conductance. Its main action is to block sodium channels, which they do by physically plugging the transmembrane pore, interacting with the residues of the S6 trans membrane helical domain. In general, conduction in small- diameter nerve fibres is blocked more readily than in large fibres. Since noninceptive impulses are carried by A delta-and C-fibres (small- diameter nerve fibres), pain sensation is blocked more readily than other sensory modalities (touch, proprioception, etc.). Motor axons, being large in diameter, are relatively resistant. Thus in the nerve that is actively conducting impulses, it inhibits the inward migration of Na+ Ions. This results in elevation of the threshold for electrical excitation, reduction in the rate of rise of the action potential, slowing of the propagation of the impulse. If the drug concentration is sufficiently high, there is complete block of conduction. Thus cocaine interferes with the process fundamental to the generation of the action potential, namely, the large transient, voltage dependent rise in the permeability of the membrane to Na+ ions.
Neuronal reuptake of released noradrenaline is blocked by Cocaine .This enhance the effects of both sympathetic nerve activity and circulating noradrenaline. Cocaine enhances sympathetic transmission, causing increased arterial pressure, excitement etc. It strongly potentiates the action of noradrenaline in experimental animals or in isolated tissues provided the sympathetic nerve terminals are intact. Thus cocaine acts as a local anaesthetic.
Negative Body effects:
Cocaine enhances the peripheral effects of sympathetic nerve activity producing euphoria, garrulousness, increased motor activity and a magnification of pleasure. With excessive dosage, tremors and convulsions followed by respiratory and vasomotor depression may occur. They produce a mixture of stimulant and depressant effects on the CNS, often resulting in restlessness and tremor, with subjective effects ranging from confusion to extreme agitation The peripheral sympathomemitic actions leads to tachycardia, vasoconstriction and increase in the blood pressure . The main acute dangers are cardiac dysrhythmias and coronary or cerebral thrombosis. Slowly developing damage to the myocardium can also occur, leading to heart failure, even in absence of acute cardiac effects. Cocaine produces no clear cut physical dependence syndrome but tends to depression and dysphoria coupled with craving for the drug following the initial stimulant effect. Withdrawal of cocaine after administration for a few days causes a marked deterioration of motor performance and learned behaviour, which is restored by resuming dosage of the drug. There is therefore a considerable degree of psychological dependence. The pattern of dependence evolving from the occasional use through escalating dosage to compulsive binges. The duration of action of cocaine about 30 minutes when given intravenously. Other unwanted effects that are specific to particular drug include mucosal irritation in case of cocaine.
Cocaine can severely impair brain development in utero. The brain size is significantly reduced in babies exposed to cocaine in pregnancy, and the incidence of neurological and limb malformations also increased. The incidence of ischemic and haemorrhagic brain lesions, and of sudden infant death, is also higher in cocaine exposed babies.Cocaine has rapid onset of action (1min) and duration up to 2 hrs and Plasma half life of 1 hr, depending on the dose and route of administration used.
Pharmacokinetics
- Administration:
Cocaine is mainly available in two forms, cocaine powder (hydrochloride salt) and crack (freebase). It is readily absorbed by many routes. Cocaine powder (Cocaine-HCl) is cut into ‘lines’ (ca 25mg) and is usually administered by snorting through the nose (IN) via straw, spoon ,rolled up banknote etc. Cocaine may also be made into a solution and injected either on its own or in combination with heroin (known as a ‘Speedball’). Cocaine use increased dramatically when the free base form “crack” is produced by ‘washing’ the salt with ammonia or mixing it with sodium bicarbonate and is smoked in a pipe. As per the report, there were three main methods of consuming cocaine in 2003 and these were: snorting or sniffing (70%), injecting (17%), and smoking (11%).
- Absorption and Distribution:
The rate of absorption of a drug into the bloodstream is affected by the dose administered, the vascularity at the site of injection, and the specific physiochemical properties of the drug itself.Bioavailability or Peak plasma concentration of cocaine for different route of administration is different.
For e.g. Route of Administration--- Bioavailability--- Peak Plasma Concentration
Snorting (IN)--------------- 25-95%---------- 0.20-0.40mg/L(100mg dose)
Smoking (SM)-------------- 57-70%---------- 0.20-0.40mg/L (100mg dose)
Orally (PO) ---------------- 20%------------- 0.10-0.50mg/L (100mg dose)
Table 1: Bioavailability or Peak plasma concentration of cocaine for different route of administration. Plasma concentration of 5.0mg/L observed in fatalities.
All tissues will be exposed to cocaine after absorption, but the concentration achieved will vary among the different organs. Although the highest concentrations appear to occur in the more highly perfused organs (i.e. brain, kidney and lung), factors such as degree of protein binding and lipid solubility also affect drug distribution. The lung can absorb as much as 90 percent of cocaine during the first pass. Consequently, it acts as a buffer to prevent higher and, therefore, more toxic concentrations that would occur otherwise. Ester drug, cocaine is almost completely metabolized by liver before excretion. However, the total dose administered and the degree of drug accumulation resulting from the initial and subsequent doses are still of concern.
Placental transfer of local anaesthetics is known to occur rapidly, the foetal blood concentrations generally reflects those found in the mother. However, the quantity of drug crossing to the foetus is also related to the time of exposure, that is, from the time of injection to delivery.
- Decomposition and Clearance:
It is mainly by means of pseudocholinesterase of the blood and partly by liver enzymes resulting into a number of metabolites like benzoylecgonine, ecgonidine, norecgonidine methyl ester and m-hydroxyl-benzoylecgonine. Among these benzoylecgonine is the major metabolite, which is formed both non enzymatically as well as through the action of esterase found in a number of tissues including hepatocytes, the enzymatic mechanism being the dominant one, after which the metabolite is excreted via the kidney. Hence urine is majorly used as sample for detection.
A cocaine metabolite is also deposited in hair, and analysis of its content along the hair shaft allows the pattern of cocaine consumption to be monitored, a technique which has revealed a much higher incidence of cocaine use than was voluntarily reported. Cocaine metabolites are also found in sweat of the abuse.
Drug Analysis:
- Need of Analysis:
If there is a ‘new’ patient we need to: Determine the subjects current drug use. If an established patient we need to: First, check compliancy with prescription or abstinence. Second, determine any ‘extra-curricular’ drug use and then check for any ‘Sample adulteration’ aimed at minimising positive analytical results. If a patient presenting in a hospital A&E, the drug analysis is done to: First treat the patient’s symptoms.Then to obtain biological specimens to allow determination if any Drugs of Abuse are present. For Forensic purposes such as Screen analysis of drug is done to identify if any abused drugs are present and to determine concentrations in blood/tissues to potentially identify a cause of death.
a) Sample used for testing:
The studies carried out by Pascal Kintz in Strasbourg, France have shown that cocaine metabolites were present in greater concentrations than the parent drug for urine, and that in sweat and hair the parent drug was usually present in greater amounts. The duration of analyte is much higher in sweat and hair compared to urine. The appropriate and available sample should be used for the analysis of the drug.
b) Spot Test:
There are a number of presumptive tests for cocaine available from the literature. One of the Spot Test for Cocaine is done by using 5% (0.125M) NaOH in methanol. It reacts with cocaine to release methyl benzoate-detectable by its odour (oil of wintergreen). It is available in test kit form. Most widely used test is Cobalt Isothiocyanate test. It is simple test where there is the addition of the material under investigation to a 2% (wt/vol) solution of cobalt isothiocyanate in water. The presence of cocaine and related alkaloids results in blue colour being formed. The scott test is the modification of the cobalt isothiocyanate test and involves a 2% (wt/vol) solution of cobalt isothiocyanate in water, dilutes in equal volume of glycerine, concentrated HCl and chlororform. In order to the test the sample for the presence of cocaine, a small amount of material is placed in a test- tube and 5 drops of the first reagent are added. A blue colour develops if cocaine is present. One drop of concentrated HCl is then added and the blue colour, if it results from the presence of cocaine, should disappear, leaving a pink solution. Several drops of chloroform should then be added. An intense blue colour forms in the chloroform (lower) layer if cocaine is present. Negative and positive controls should be performed concurrently with analysis of the test sample. The Scott test gives greater discrimination and specificity towards cocaine.
- Method of analysis: Methods of Analysis
a) Immunoassay:
Immunoassays are tests that use antibodies to identify and measure analytes. In forensic science typically a biological sample is screened for the presence of an antigen such as a drug. Immunoassays are commonly uses for screening purposes because they are readily amenable to large batch analysis, are relatively sensitive, and require little or no sample preparation. Because immunoassays are targeted to detect benzoylecgonine, they are particularly well suited for screening urine specimens. There are several types of immunoassays on the market, depending on the product selected, for e.g. Fluorescence polarization immunoassay (FPIA), Enzyme immunoassay (EIA), Micropartical immunoassay (KIMS), Radioimmunoassay (RIA), Cloned Enzyme Donor Immunoassay (CEDIA), Enzyme Linked Immunosorbant Assay (ELISA). In addition to their use for urine analysis, some of these assays have been successfully adapted to blood tissue analysis. Although, all immunoassay techniques are targeted on benzoylecgonine, cross reactivities varies considerably by manufacturers and analytical principle, thus Immunoassay is chosen according to need. For e.g. Immunoassays that possess substantial cross reactivity to cocaine and ethylcocaine, such as the Daignostic Products corporation (DPC, Los Angeles) RIA assay, are particularly useful for screening blood. The detection limit for cocaine and ethyl cocaine using the DPC RIA is as low as 0.010mg/L depending on lot. In addition the assay can readily detect benzoylecgonine at 0.100 mg /L. The FPIA assay show considerable cross reactivity to m –hydroxybenzoylecgonine, making it well suited to screening meconium. Although the cut off concentration for benzoylecgonine in federal workplace drug test is 0.300 mg/L, most immunoassays can detect lower concentration reliably. Cocaines most common cross reactive substance are ecgonine and ecgonine methyl ester.
b) Chromatographical Methods:
1. Sample preparation:
For the cocaine and its metabolites to be analyzed using chromatographic techniques , the drugs must be separated form the biological matrix. Hence, the sample preparation is required. This may be accomplished either by liquid-liquid or solid-phase extraction procedures. The latter techniques can be readily adapted to laboratory automation devices.
There are several important issues that must be considered before selecting an extraction procedure. Cocaine is ester that is susceptible to hydrolysis in alkaline conditions and at elevated temperatures. In addition, plasma and liver esterases also contribute to hydrolysis. During sample preparation, it is critical that the amount of time the biological specimen remains in conditions that are unfavourable to cocaine stability be minimized; otherwise, esters may hydrolize in vitro and complicate the interpretation of analytical results. Consideration must also be given to the targeted analytes because there is considerable range in polarity for cocaine and its metabolites.
Other than stability concerns, the extraction of cocaine and ethylcocaine is straightforward. These compounds can be readily extracted into n- butyl chloride at the pH of 8-9 by Liquid/ liquid phase extraction. Here chloroform isopropanol mixture (9:1) is used. These conditions will also extract bezoylecgonine and ecgonine methyl ester, although not at optimal recoveries. Solid phase extraction (SPE) procedures, also used for these analytes commonly use a protein precipitation step before a buffered supernatant is applied to the extraction column. Elution is typically accomplished with mixture of methylene chloride, isopropanol and ammonium hydroxide (78:20:2). Ecgonine requires SPE containing anion exchange media. Their elution solvents have also been used, depending on the analytes targeted and the column used.
2. Methods:
- Thin Layer Chromatography (TLC):
Thin layer chromatography (TLC) is a simple technique that can be used to analyze for both cocaine and benzoylecgonine. It is generally limited to analysis of urine specimens due to lack of sensitivity. In fact, TLC has higher detection limits than immunoassay. After extracting 5 ml of urine for basic drugs and spotting the concentrated extract on a silica TLC plate, one can expect a sensitivity of about 1-2 mg/ml (3.4-6.9 micromol/L) for benzoylecgonine using the standard Davidow solvent with visualisation by either Dragendorff’s reagent and/or iodoplatinate. Sensitivity for the cocaine by TLC is generally better than for benzoylecgonine using most basic drug extraction schemes. High performance TLC results in considerably improved detection limits. As a qualitative tool, TLC may be used to confirm immunoassay results when positive forensic identification is not required.TLC is particularly useful for benzoylecgonine because it does not migrate up the plate very far, allowing it to be readily distinguished from many other basic drugs.In TLC of cocaine , the Liquid/Liquid extraction of urine/bile is done .The Spot concentration extract on silica TLC plate used is 1-2mg/L.It is developed with 1.5%NH3/methanol and visualized with UV254 or acidified potassium iodoplatinate.
- Gas Chromatography:
Gas chromatography is a separation technique most frequently used for the analysis of cocaine and its metabolites .Cocaine can be readily assayed without derivatisation using nitrogen- phosphorus detection and detection by Mass spectrometry (MS) in both the electron impact and positive chemical ionization modes when coupled with Gas Chromatography. Flame ionization detection may also be used, but is not nearly as sensitive. Chromatography may be improved by derivatisation of cocaine to the p-nitrococaine or p-fluorococaine derivative using p- nitrobenzoylchloride or p-flourorbenzoylchloride reply, in modification of the Schotten- Baumann reaction.
Electron capture detectors are very sensitive and have been used for analysis of benzoylecgonine after acylation.The major disadvantage to this detector is that cocaine must be reduced before acylation. Detection by MS provides the highest specificity of all GC detectors and is nearly requirement for the forensic confirmation of cocaine and metabolites today.
- Liquid Chromatography:
Liquid chromatography is very useful because cocaine, ethylcocaine and benzoylecgonine can be analysed simultaneously without derivatisation when using UV detection. UV has a sensitivity of 0.05 mg/L(0.17 micromol/L) for cocaine. But there is low selectivity and it does not allow the use of deuterated internal standards.
On the other hand ,LC coupled with MS may be the ideal separation and detection techniques for cocaine and its metabolites without need for derivatisation.LC/MS is still not commonly used due to cost and maintenance issues.
- Mass spectrometry:
Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles. Thus it is used for determining masses of particles, for determining the elemental composition or chemical structures of a sample or molecule and for detection and Quantification of drug in the sample.
Generally, MS instruments consist of three modules: An ion source, which can convert gas phase sample molecules into ions by fragmentation. A mass analyzer, which sorts the ions by their masses by applying electromagnetic fields. A detector, which measures the value of an indicator quantity, provides data for calculating the abundances of each ion present. Thus the technique has both qualitative and quantitative uses.
- Cocaine Fragmentation:
MS is now in very common use in analytical laboratories that study physical, chemical, or biological properties of a great variety of compounds. To determine the presence of cocaine and/or these metabolites, urine samples are screened using enzyme immunoassay, and positive results are confirmed using gas chromatography/mass spectrometry. Mass spectrometry is widely used for confirmation because MS data provide positive identification based on mass spectral information, and can be used as confirming evidence in courts of law. A particular advantage with MS detection is that deuterated analogs of cocaine e.g Norcocaine and its major metabolite are now readily available and can be used as internal standards by virtue of having chemical characteristics nearly identical to their non deutereated analogs. They allow for brilliant reproducibility and accurate analysis. Thus MS is coupled with GC or LC give excellent results.
In GC/MS ,cocaine and ethylcocaine assayed without derivatization while Benzoylecgonine/ecgonine require derivatization (Trimethylsilyl).The detection is carried out using Chemical ionization or Electron impact detection. The assay used helium as carrier gas, a 30-m bonded phase fused silica OV-1 capillary column, and solid injection at 290°C evaporator temperature. Sensitivity obtained was <0.01mg/ml when sample was human hair. Although GC/MS methods are well established, and provide excellent confirmation data, analysis time is long and sample preparation, which includes derivatization, is labour-intensive and time-consuming. However LC-MS benzoylecgonine and ecgonine can be directly assayed, thus save time and efforts. The limits of detection for LC with Tandem MS ranged from 3 to 23 ng /ml and the limits of quantitation ranged from 7 to 69 ng/ml.
A lot research is going on in improving the detection resulting into more sensitive and accurate method. One of such successful endeavour is Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI.). This method was used to image the distribution of cocaine and its metabolites in intact single hair samples from chronic users down to a concentration of 5 ng/mg. A less commonly used, but equally valuable, chromatographic method is high performance liquid chromatography coupled with mass spectrometry (HPLC/MS). The analytes do not have to be derivatized for this approach, making HPLC/MS more attractive to the analyst. A well-chosen stationary phase and a mobile phase with a high organic content allow efficient desolvation and ionization in the MS, and permit detection of cocaine and its metabolites at low picogram levels on-column. Under optimal screening conditions, limits of detection of 1ng/ml for cocaine and 5ng/ml for benzoylecgonine have been reported to be 10pg and 50pg on-column, respectively (10µL injection) for the C18 stationary phase. HPLC/MS, like GC/MS, provides reproducible, reliable data that can be used in court. The HPLC/MS method developed in this investigation provides symmetric peaks and excellent sensitivity in a reduced analysis time.
In Drug analysis these are devices that include a biological component and a transducer. The transducer converts the detection made by the biological component into an electrical signal that is a function of the concentration of the analyte. Often this achieved by immobilising the biological component on to the transducer. Because these biological components are specific to an analyte this gives specificity to the biosensor and means that they can used on samples that have not been pre-treated (to remove interfering compounds).Enzymes were the first catalysts used in biosensors. In Bioreceptors, the binding is non-catalytic and irreversible. The development of these biosensors took off with the increasing availability of monoclonal antibodies. One of the papers discussing this new technique was regarding Analyte 2000, a four-channel fibre optic biosensor (FOB) for analysis of cocaine and its metabolites (COC) in human urine using a competitive fluorescence immunoassay where fluorescence is produced by evanescent excitation of bound cyanine dye-tagged antimouse antibody (CY5-Ab). The effective concentration range of benzoylecgonine (BE) for inhibiting the fluorescent signals was 0.75-50 ng/ml, with IC50 of 9.0 ng/ml.
Use of Drug Analysis:
Both bulk samples and trace materials may be encountered by the forensic scientist. For the bulk samples, the analytical sequence includes Physical description, presumptive tests, TLC and confirmation using GC/MS. Interestingly, although the presumptive testing stage may include colour tests, another method can also be employed where the odour is noted following the addition of sodium hydroxide in methanol.
Under Sports law, cocaine is classified under stimulants leading to performance enhancement. Most drugs are banned outright in Olympic competitions. The World Anti-Doping Agency oversees world-wide doping and has a list of prohibited substances. Sample testing for first time carried out in1972 Munich Olympic Games was carried out with GC with nitrogen-selective detection. Then in following years GC/MS was used to confirm the presence of anabolic steroids. In ordered to be accredited a laboratory must be able to carry out: GC, GC-MS, HPLC, TLC, Immunoassays.
Cocaine has negative effects on body especially CNS, heart etc leading to driving fatalities due lack of concentration, coordination etc. The field tests are designed accordingly. Field Impairment Tests currently in use checks for physical signs: Pupil Size should be between 3 and 6.5 mm. Romberg Test- Involves standing with feet together, Test checking balance and co-ordination. Further confirmation is done by Testing Urine for the Presence of abused drugs. Primary screening which is usually by immunoassay. Cocaine is confirmed by TLC.
An individual who ingests wrapped packets of illicit drugs to transport them by internal concealment is called Body Pack. Generally, the analyses is carried out by CEDIA immunoassay screening of Urine and colon faeces for Cocaine and confirmed by GC/MS.Blood was screened for Basic drugs, stimulant drugs, acidic drugs, benzodiazepines and morphine.
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