Sunday, July 21, 2019

Cocaine Abuse Detection with Double Confirmation Technique

Cocaine Abuse Detection with Double Confirmation Technique Introduction Throughout history, natural and synthetic drugs have been used for many uses. The first drugs were based on natural sources [1] and it is common practice to isolate a drug from its natural sources before synthesis. Usage of these drugs is regulated by governmental organizations, in the U.S.A. the Food and Drug Organization achieves this goal, and any use that is not approved is commonly frowned upon. The deviation of the use of drugs from their approved medical practice is termed drug abuse. The practice of drug abuse is as old as drugs itself. Drugs that alter mood have always existed and the approved usage is partially constructed by society. For example, alcohol use is normal, and sometimes excessively celebrated, in western culture but is prohibited in some religious cultures. While there are several other drugs that are abused daily, this review will be mainly focused on cocaine and its abuse [2]. Originally derived from the coca plant, the history of cocaine use can be detailed as far back as 600 AD. It was common practice for Peruvian Indians to digest the leaves for its euphoric effects. In the 1880s, it was first isolated to use in anesthetic agents due to its abilities as a vasoconstrictor. By 1885, coca could be found in various products such as cigarettes and Coca Cola. Other soft drinks and alcoholic beverages also contained cocaine at the time. However, once news surfaced of cocaine addiction, the perspectives regarding cocaine began to shift. Laws were enacted against cocaine use and by the 1950s, the earlier cocaine abuse was forgotten. However, the more potent version of cocaine, crack, became popular in the 80s and a rise of cocaine abuse followed. It was popular to administer the alkaloid via inhalation or intravenous injections. It was also placed under the tongue (oral sublingual), rubbed on the skin, or inserted into the vagina/anus [2]. Due to its rampant usa ge, it became evident that a method to test for cocaine presence in the human body was necessary to curb its abuse. The purpose of general drug testing is to identify and detect the drug in question, and in turn, deter its usage. An unlimited amount of drugs can be detected in drugs and the sample can be re-tested several times. The analytical techniques utilized in drug testing are separated into two classes: assays based on molecule recognition and separation techniques such as gas chromatography with mass spectrometry (GC-MS), liquid chromatography (LC-MS), and capillary electrophoresis (CE). The art of drug testing combines different analytical techniques into a two-tier system where the first step is a quick screening followed by a confirmation step to confirm the results. While the quick on-site screening reduces costs and errors from delays between sampling and analysis, the possibility of false results is lowered with a confirmation step [3]. The biological specimen normally used in these tests is blood and urine; each with its benefits and downfalls. The use of urine as a biological speci men is the cheapest option and as a result, it is most commonly used. However, it is difficult to handle and it is easy to cheat the testing system since people are not watched when the sample is collected. The use of blood offered the advantage of monitoring the patient while the sample is collected. However, blood is also difficult to handle and store and isolating the sample from blood complicates drug testing [4]. As a result, other biological specimens such as hair, sweat, and nasal mucus have been researched as alternatives. In the case of cocaine, this article shows that mucus can be used as an alternative specimen to test for cocaine presence. A two-tier system was developed that utilizes ion mobility spectrometry (IMS) and infrared spectroscopy(IR) to test for cocaine in mucus was developed and refined. Mucus was the specimen of interest because it is a non-invasive collection process and unlike blood and urine, the parent compound is present. IMS is a technique with high sensitivity so it will be able to identify positive samples and IR possesses high selectivity so it will be able to confirm these positive results. As a result, IMS is used to detect if the sample is present and IR is used to confirm the original result. Methods and Results Twelve seized cocaine samples with concentrations varying from 25-80% w/w were utilized in conjunction with mucus specimen collected from cocaine users. These users were male from 25 years old to 40 years old. Cocaine-free mucus was obtained from males and females from 25 40 years old. Note that the seized cocaine samples were utilized to develop the method and the collected samples were used to test the method. A liquid chromatography procedure was utilized as the reference procedure. A LC Dionex P680 system was utilized and an acetonitrile-phosphate buffer was used as the mobile phase. For the IMS procedure, an IONSCAN-LS system was used to analyze the samples. Nicotinamide acted as an internal calibrant in order to collect a plasmagram for the sample being tested. A Teflon membrane was used to insert the sample into the machine and to prevent any systematic errors, a blank Teflon membrane was inserted in the machine beforehand. For the IR procedure, a Tensor 27 FT-IR spectrometer was used to obtain the spectra and this was analyzed using the OPUS program. The transmission cell utilized to insert the sample had an open upper side in order to strengthen sensitivity. As a result, a normal cell with 2 mm windows now had Teflon spacers that were 0.5mm thick [3]. The IMS results comparing cocaine containing samples to non-cocaine samples are shown in figure 1a. The highest peak is from the internal calibrant with a K0 of 1.860 cm2 V-1sec-1 . Despite needing a mass spectrometer to assign the peaks, it can be hypothesized that the main peak present in the cocaine seized samples that is absent in non-cocaine users can be attributed to cocaine. Cocaine presents a peak at 15.07 ms drift time with a reduced mobility of 1.16 cm2 V-1sec-1 which matches the literature values. As a result, the characteristics of this peak were used to formulate a criterion that determines the presence of cocaine. Within each plasmagram in question, one must look for the K0 value, a variability value of 50  µg of the peak drift time, a peak amplitude of 1.5, a threshold value of 20, and a full width value at the 1/2maximul amplitude of the peak of 200  µs. Once the IMS results were formulated, two seized samples that tested positive underwent an IR procedu re. The resulting spectra presented a plethora of absorption bands but three are of interest: the stretching of the carbonyl group causes a band at 1726 cm-1; the mono-substituted benzene stretches and causes a band at 1017 cm-1; and out-of-plane bending vibrations cause a band at 965 cm-1. Therefore, a criterion must also be formulated to determine the presence of cocaine. Within an IR spectrum, one should look for peaks within three regions: 1786 to 1701 cmà ¢Ã‹â€ Ã¢â‚¬â„¢1, from 1363 to 1257 cmà ¢Ã‹â€ Ã¢â‚¬â„¢1, and 1166 to 950 cmà ¢Ã‹â€ Ã¢â‚¬â„¢1 [3]. In order to determine the most effective means in collecting mucus, an artificial sniffer to replicate the nose was developed and two different types of swabs were tested. In these studies, a benzocaine, lidocaine, and lactose mixture was utilized. 50 mg of the mixture was sniffed with the artificial sniffer and different swabs were used to collect the samples. The procedure was repeated thrice in order to determine recovery efficiency. The two swabs tested were a double cotton tipped polystyrene stick and single cotton tipped wood stick. Looking at figure 1b and 1c, it is evident that the double cotton tipped polystyrene stick is better for powdered compounds mixed in with mucus so this stick is recommended for mucus collection [3]. If one is to develop a technique to detect and identify an analyte, thresholds must be determined for these assays. By using successive dilutions of a cocaine standard of 1000 mg/L, the limit of detection (LOD) of the IMS technique was determined to be 15 pg. This means that if cocaine is present at any lower amounts, the IMS technique cannot be used to generate a true positive value. The limit of concentration (LOC) was used to determine the threshold for the IR technique. This value was determined to be 100  µg and if any sample is tested to be below this concentration, it cannot be confirmed to be cocaine[3]. Another factor that must be considered is the time delay between administration of drug and drug testing. As a result, the IMS+IR method was performed on these samples with varying times from 15 minutes up until 120 mins, and then after 24 hours. In figure 2c, cocaine was still identifiable up until 120 mins and even after 24 hours, traces were still detectable via the IMS technique [3]. In order for this new method to be viable, the possibility of interference from other compounds must be ruled out. In street cocaine, the product is cut in order to maximize profit. In other words, other anesthetics are mixed in to reduce the amount of cocaine needed to make a profit. In order to show that these common cutting agents do not interfere with the method, these compounds were analyzed by IMS and IR. Evident in figure 3, almost all cutting agents do not overlap with the distinct cocaine peak except for tetracine that has a peak at 15.25 ms with a K0 of 1.151 cm2 V-1sec-1, oxymetazoline at 14.90 ms with a K0 of 1.165 cm2 V-1sec-1, and xylometazoline at 14.77 ms with a K0 of 1.168 cm2 V-1sec-1. These three were analyzed by IR and their spectra (Figure 3) can be distinguished from cocaines spectra. The correlation coefficents were calculated to be less than 90% so the probability of false positives is close to zero. The worry that competitive ionization would also create false positives was also quenched by evaluating different mixtures of cocaine and cutting agents and in most cases, cocaine was detected. The use of other compounds such as a xylomethazoline-containing nasal spray and a topical ointment were tested using the double confirmation method. The only exception is the 1:5 and 1:10 ratio of tetracaine and cocaine. It seems like when cocaine is diluted, the cutting agent causes a shift in cocaines peak in the IMS plasmagram. However, the IR spectra can confirm the presence of this sample. Throughout this process, only one false positive showed up, the 1:10 ratio of lidocaine and cocaine due it the correlatio n coefficient being less than 90% [3]. Now that the method has been fully developed, it was tested using the cocaine samples collected from individuals. 35 samples were collected, nine samples were from individuals who did not use cocaine and the rest used. It is a bit difficult to quantify the amount of cocaine in the mucus due to the matrix but the purpose of the method to detect and identify the analyte. The samples also underwent a liquid chromatography procedure as a reference for the data. As evident in figure 4, the number of false positives is zero using the double confirmation technique. Even when the nose was cleaned with saline before sample collection, the method was still able to detect and identify the analyte afterwards [3]. Discussion This double confirmation technique has been proven to be a successful method for cocaine abuse. The thresholds for each step has been determined and it is recommended that a double cotton tipped polystyrene stick should be used for extractions. The laboratory equipment used can be found in smaller sizes, in order to use outside of lab. As mentioned earlier, some advantages with this technique include easier specimen collection, high selectivity and sensitivity, low risk of interference, and its ability to generate a true positive response up to two hours of exposure [3]. Other techniques with similar advantages are currently being developed as well. Scientists in Europe have developed a surface mass spectrometry technique that detects cocaine from fingerprints [5]. Researchers have suggested sweat patch testing as an alternative to urinalysis for cocaine abuse [6]. However, the possibility of cheating this new method via nasal irrigation and neti pots raises some questions about its application. In other words, if someone has the ability to flush out the main analyte from their nose, in what cases should this technique be utilized? Is it viable for drug testing for employers since people can just flush out their nose in succession a few days before the test? Is this technique more viable in a forensic sense? These are questions that need further research. References (Note:re-format for all of them to have the same format and re-order alphabetically) [1] Goodman Gilmans: The Pharmacological Basis of Therapeutics, 12e Eds. Laurence L. Brunton, et al. New York, NY: McGraw-Hill, , http://accessmedicine.mhmedical.com.ezproxy.fiu.edu/content.aspx?bookid=1613sectionid=102124003. [2] Das, Gopal. Cocaine abuse in North America: a milestone in history. The Journal of Clinical Pharmacology 33.4 (1993): 296-310. [3] Noninvasive Double Confirmation of Cocaine Abuse Sergio Armenta, Miguel de la Guardia, Manel Alcalà  , and Marcelo Blanco. Analytical Chemistry. 2013. 85. (23), 11382-11390 [4] Klaassen, Curtis D., and Mary O. Amdur, eds. Casarett and Doulls toxicology: the basic science of poisons. Vol. 8. New York: McGraw-Hill, 2013. [5] Bailey, Melanie J., et al. Rapid detection of cocaine, benzoylecgonine and methylecgonine in fingerprints using surface mass spectrometry. Analyst 140.18 (2015): 6254-6259. [6] Liberty, Hilary James, Bruce D. Johnson, and Neil Fortner. Detecting Cocaine Use Through Sweat Testing: Multilevel Modeling of Sweat Patch Length-of-Wear Data. Journal of analytical toxicology 28.8 (2004): 667-673. Print.

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