Cocaine is an illicit drug that is made from the leaves of the coca plant. Cocaine can be manufactured in several. A common method involves copping the coca leaves and mixing this with ammonia and lime. Following this, the mixture is placed into barrels with diesel gasoline. The active component of cocaine dissolves into the gasoline and the gasoline is filtered out from the rest of the mixture that is discarded. Sulfuric acid is then mixed with the diesel mixture. This allows for the formation of cocaine sulfate. The cocaine sulfate is then mixed with more lime to neutralize the acid. What is left is cocaine paste, which is further processed by being once again dissolved in sulfuric acid. An oxidizing agent is then added and the product filtered and treated with ammonia to neutralize the acid. Once it is dried, the resultant product is cocaine hydrochloride, which is the street drug (Guzman, 2012)
Figure 1 left: cocaine
right: benzoylecgonine
Cocaine is a commonly abused drug throughout the world. It is a sympathomimetic that can be snorted, smoked, or injected. In vivo, it forms benzoylecgonine and ecognine methyl ester metabolites (Pilgrim, Woodford, Drummer, 2013). When ingested with alcohol a third metabolite is formed, cocaethylene, which is more toxic than both products used alone. The metabolites all have much longer half-lives than cocaine itself. Cocaine causes an increase in adgrenergic activity, and sodium channel inhibition, and alterations in the calcium flux across a cell membrane. Users may suffer acute fatal consequences in the form of various heart aberrations (Pilgrim, et al., 2013).
The drug’s use in Australia is an increasing problem and approximately 7% of the population admits to having used it at some point in their lives (Pilgrim, Woodford, Drummer, 2013). Certain local anesthetics for minor ear, nose and throat surgeries may contain cocaine or derivatives that may be prescribed only by a doctor, however, all other forms of the drug are illegal (Fucci, 2011). The Single Convention on Narcotic Drugs is an international convention that bans the production and supply of cocaine except under very specific licensed purposes. Thus, cocaine is basically illegal the world-over except for a very narrow set of medicinal uses.
Testing for cocaine takes many forms. When testing an unknown substance, CYP450 biosensors put onto screen-printed carbon electrodes provide an electrochemical test for the determination of cocaine powder (Asturias-Arribas, et al., 2011).
In vivo, Cocaine and its metabolite benzoylecgonine can be detected from blood, hair, urine, or saliva samples (Vindenes, et al., 2012). Oral fluid sampling is very popular among police forces worldwide. Oral samples are non-invasive, fast, and relatively cheap to take. Furthermore, concern has grown about the potential for adulteration of urine samples and oral sampling provides a fast method of testing that does not offer an opportunity for adulteration (Bosker & Huestis, 2009). Cocaine appears quickly in the oral cavity after injection, smoking, or intranasal ingestion and oral samples give a good estimation of actual concentrations of the drug and the ratio of oral fluid to blood concentration is approximately 3 (Kolbrich, et al., 2003). Several systems currently exist to conduct oral testing, including: Drugwipe by Securetec, Oralscreen by Avitar, inc., Cozart RapiScan Oral Fluid Drug Testing System by Cozart Bioscience, Ltd and the Concateno Alere DDS 2 (Strano-Rossi, et al., 2012; Drummer 2006).
Oral fluid testing relies on enzyme immunoassays to screen for the drugs. Alkaline phosphatase, horseradish peroxidase, -galactosidase, and glucose oxidase have all been used as labels. Glucose oxidase produces hydrogen peroxide in a catalytic process of -D-glucose to D-glucono--lactone in the presence of oxygen. Chromogens are used to determine the amount of hydrogen peroxide produced, the best being 3,3′,5,5′-tetramethylbenzidine because it is non-carcinogenic. The amount of cocaine or benzoylecgonine is assessed by competitive inhibition of an anti-benzoylecgonine antibody by benzoylecgonine conjugated glucose oxidase. With the minimum levels of cocaine and its metabolites present, it takes twelve minutes for a color change in the indicator to occur (Yamaguchi, et al., 2001).
The Concateno Alere DDS 2 system is a portable device that uses a five-cartridge drug panel and can test for opiates, benzodiazepines, methadone, cocaine, and amphetamines simultaneously using either enzyme linked immunosorbent assay or cloned enzyme donor immunoassay techniques. The method is very good because it also takes a reserve sample of oral fluid which can be tested again later if need be. It is a qualitative detection method and does not give a specific quantity present, but only indicates the presence of the drug. Saliva samples are collected by a swab and are place in the test cartridge with a run fluid. In a few minutes, if the sample is negative for a drug, a red line appears in the cartridge slot for that drug. The test is only valid if a red line appears in the control zone (De Giovanni, et al., 2002). The cartridge is then placed into a device that performs an electronic reading and a digital readout of is given for each drug that was tested. Visual reading is less sensitive than the electronic reading due to a variety of reasons. Furthermore, gas chromatography-mass spectrometry can confirm the results depending on the size of the sample obtained. Concateno Alere DDS 2 is sensitive, specific, and reproducible allowing for the detection of low levels of drugs. In studies performed by De Giovanni, et al, no false positives were obtained using a similar earlier system. With the lowest concentrations of drug samples a few false negatives were obtained which were corrected by using the electronic reader. False negatives are mostly problems linked to visual detection of the indicator line, rather than a problem with the assay. However, during testing of the kits, gas chromagraphy-mass spectrometry did reveal two cases of cocaine use that were not picked up by the immunoassay. This was probably linked to a low cocaine and metabolite concentration (2002). Thus it appears that a threshold level of cocaine must be present in order for it to be detected by the Rapiscan system that is higher than what is required for gas chromagraphy-mass spectrometry.
Ultimately, quality control principles in use for any enzyme immunoassay systems would be the appropriate controls – for instance, there must be serial dilution of high positive stock samples, suitable dilutions selected, large batches produced, stability testing, variation testing, and proper labeling, storage and dispensing of the products (Green, et al., 1997). Furthermore, control samples must be performed on the assays, replicate analysis can be conducted, prevision testing can be performed, storage conditions should be tested, and finally, laboratory technique may be a source for variation (Green, et al., 1997). Concateno has a comprehensive quality control regime outlined in a document of technical specifications. They will take samples from the positive oral fluid test and do a comprehensive analysis of the sample in their laboratory. In order for them to verify the presence of cocaine in the oral sample, a complete chain of custody for the sample must be present and there must be no damage to the kit. Missing tapper seals, lack of signatures, incorrect collector in the tube, delay of over a month from collection to delivery to the laboratory, even the sample pad placed incorrectly in the container, will result in a refusal to analyze the sample (Concateno, 2011). However, direct quality control and quality assurance data is a guarded secret that is only presented in the case of a challenged analytical result. In the case of a challenge, an unopened B sample is made available for an independent analysis.
There is an ongoing myth that amoxicillin a beta lactam antibiotic has the ability to produce false positives on screening tests for cocaine and its metabolites. It is important to note that there is no other pathway for the creation of benzoylecgonine other than cocaine ingestion, whether it is intentional or otherwise. Amoxicillin and its metabolites bare very little resemblance to the chemical structures of cocaine an its metabolites. Reisfield, et al., put the theory of amoxicillin producing false positives in urine screenings for cocaine metabolites to rest in a study where they conclusively showed that amoxicillin did not produce false positives in immunoassaying methods of cocaine detection (2008).
A properly performed immunoassay using the Concateno Alere DDS 2system will be very hard to challenge in court. Enzyme immunoassay is scientifically valid and accepted as a method of detection for cocaine use. Niedbala, et al., performed a study attempting to adulterate oral fluid samples with various foodstuffs and they were never able to produce a false positive. Furthermore, cocaine metabolites are stable in stored samples and therefore if they need to be tested in the future, it is easily possible. Finally, pH was shown to also have no effect on the detectability of drugs and their metabolites in the oral fluid (2001).
Oral fluid is a mix of saliva, cellular material, and food residue. The concentration of cocaine in oral fluid is very well correlated to the plasma concentration (Crouch 2006; Kadehjian, 2005). One possible issue with oral fluid sampling is the size of the sample obtained. Various drugs may increase or decrease salivation and acutely, cocaine, due its sympathomimetic properties will decrease salivation and this may hinder an adequate test and reserve sample being made (Crouch 2006; Kadehjian, 2005). Furthermore, anxiety and personality factors also may cause decreased amounts of oral fluid. This particular problem can be overcome by using acidic stimulation, which places a small amount of an acidic substance such as citrate into the mouth to induce salivation (Kadehjian, 2005). Any substance used during collection, be it an acid to stimulate salivation or the plate or tubes used in collecting the sample mustn’t interfere with the immunoassay (Kadehjian, 2005). Another issue is the correlation between the levels of drug in the oral fluid versus the concentration in the plasma. If drugs were recently snorted, smoked, or eaten, the concentration will be drastically higher in the oral fluid and out of approximation with the plasma levels; however, within a few hours this problem is solved by the natural physiology of the mouth.
The real legal challenge to an enzyme immunoassay oral fluid drug detection system will be in the handling of the specimen by the authorities. The chain of custody of the sample is important evidence to present in court and it must not have any inconsistencies, abnormalities, or alterations (Kadehjian, 2005). Another possible challenge to the validity of the collected sample is with regards to the collector and his reliability. Improper initial handling may result in the invalidity of the test. As noted previously, Concateno may refuse to validate a sample in their laboratory if the absorbent pad used in the fluid collection is not properly stored within the case.
Interpretation of the test results may also be a substantial question for the lab personnel, toxicologists, and physicians when called upon at trial of a suspect. The question will arise whether or not the sample indicates a current, recent, or incidental exposure to the drug tested for and what that means regarding the levels of impairment of the tested subject (Kadehjian, 2005). This is of particular concern when the testing is being done roadside in a suspected drugged-driving case.
Oral fluid drug testing would have been invaluable in the case of Monica Knight on March 30th, 2012 in Massachusetts, United States. Ms. Knight was driving and crashed head on into a school bus leaving Ms. Knight’s passengers severely injured including one in a coma. The police found many drugs in her vehicle, including: cocaine, marijuana, amphetamines and “spice.” Ultimately she was charged with reckless operation of a motor vehicle, failure to wear a seatbelt, and possession of a controlled substance. Notably missing from her charge sheet was the fact that she was probably driving while high on drugs (Springer, 2013). Had the police had an oral fluid collection and sampling kit present they would have been able to test her for recent drug use which would have at least confirmed what seemed woefully obvious to the community at large. Instead, justice was not served, nor society adequately protected because the offender was not even charged with the most serious of offenses she probably committed because no one had available a cheap, fast, accurate, non-invasive sampling method. It is important to note that the oral tests for drug intoxication is now standard practice in much of Europe under the DRUID project which seeks to limit the loss of life due to driving under the influence of drugs, alcohol, and medicines.
Oral fluid sampling provides a fast, reliable, cheap, non-invasive method of cocaine intoxication detection in either the intoxicated driving context or in any other context where it becomes necessary to perform an instantaneous drug test to discover the possibility of impairment. In order for this method of sample determination to be useful in a court of law confirmation of the sample must be attained in order to determine the quantitative evaluation that will be open to interpretation by expert witnesses. The science behind the detection methodology is relatively unassailable and challenges to the results validity will revolve around technical legal issues such as the chain of custody, and potential tampering. Particularly in the case of cocaine, oral fluid samples provide an accurate and reliable ratio to the plasma concentrations of the drug ingested. Any inaccuracy in the levels detected by the oral sampling method is probably a result of recent ingestion of drugs depositing a residue in the oral mucosa. In sum, this is an excellent method of drug intoxication.
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