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Official websites use. Share sensitive information only on official, secure websites. As drug overdose deaths across the United States continue to rise, there is increasing interest in field testing of illicit substances. This work discusses a paper-based analytical device idPAD that can run a library of 12 colorimetric tests at the same time, each detecting different chemical functional groups and materials found in illicit drugs, distractor substances, and cutting agents. The library approach used in the idPAD may provide the accuracy and robustness necessary for a presumptive field drug test. Keywords: paper analytical device and PAD, presumptive test, field test, illicit drug, controlled substance, field drug analysis. Street drugs consist of unregulated and highly variable mixtures of illicit drugs and cutting agents, making them difficult to evaluate under field conditions. We designed a paper analytical device idPAD to run multiple chemical color tests on illicit drugs at the same time. Libraries of chemical tests can differentiate between closely related solvents 1 — 3 , spoiled and wholesome meats 4 , and genuine and falsified formulations of medications 5. By running tests in parallel, a library generates a multidimensional response that encodes information about the components of the test substance. While library tests have proven useful in detecting components in complex mixtures, with few exceptions 6 — 8 , presumptive tests for illicit substances are still conducted in the field as individual assays. There are multiple field drug detection technologies on the market. There are no published error rates, no central agency regulates the manufacture or sale of the tests, and no comprehensive records are kept about their use 9. These pouches are marketed to identify particular illicit drugs, such as cocaine or heroin. A test is conducted by adding a small amount of solid substance to a plastic pouch that contains glass ampules of concentrated reagents, followed by the user breaking the ampules in a specific order. The liquid reagents then mix with the unknown and yield a specific color based on the analyte. These single-color tests have come under media scrutiny as they have shown unacceptable false positive rates due to the subjectivity of color interpretation and interference or false positives caused by a variety of household and over-the-counter substances 10 — For example, 15 false positives for methamphetamine were recorded in the first seven months of in one Florida study. This information prompted a statewide analysis in which data from the Florida Department of Law Enforcement showed that 21 percent of evidence listed as methamphetamine was falsely identified, and over half of these samples were not illicit substances at all. It is believed that most errors and mis-classifications are caused by user errors breaking the tubes in an incorrect order, using an inconsistent amount of solid , sensitivity of the test results to the environmental situation temperature, humidity , and poor lighting that impacts user ability to read the colors accurately flashing police lights, time of day, sun glare One private company, Detectachem, has developed a modified pouch test where the user must collect multiple swabs, apply them to separate cartridges, squeeze the cartridge to break the reagent ampules, and take separate photos which are evaluated separately by the phone software On the idPAD, all the tests are run simultaneously, and the results are considered as a gestalt. Other technologies such as Raman, infrared spectroscopy, and portable mass spectrometers are alternatives to the traditional pouch tests due to their ability to selectively, and with high discrimination, identify a suite of drugs. However, their high costs make them impractical to bring into the field in many jurisdictions. Additionally, low quality drug mixtures and adulterant or cutting agent interference can limit their usability 15 — The idPAD was modeled after the device made by Weaver et al. The idPAD is comprised of 12 different color tests, each designed to detect specific functional groups commonly found in opioids, amphetamines, and other substances of abuse. When run together, the 12 tests generate a color barcode that can be used to piece together the composition of illicit substances and cutting agents in a sample. The color barcodes can then be compared and identified using an image library. The test results can be archived by taking a photograph of the card; features such as fiducial marks and a set of printed color standards facilitate computer image analysis of the results, which can be performed to back up visual comparisons done in the field, and the cards are serialized to help link each test to other forensic records. The idPAD has been designed to be an alternative or supplement to the current field drug detection technologies. The idPAD was used to differentiate four common street illicit drugs heroin, cocaine HCl, crack cocaine, and methamphetamine from 64 distractor substances and cutting agents. We evaluated the limit of detection for the four controlled substances and performed blinded studies to measure sensitivity and specificity for detecting the targeted controlled substances in a lab setting. The wax features mark out twelve independent lanes. Test cards were then allowed to air dry prior to packaging in heat sealed aluminum foil bags. Detailed reagent preparation information can be found in S1. Copper II sulfate pentahydrate, cobalt II nitrate hexahydrate, iodine, 4-nitroaniline, and 4- dimethylamino cinnamaldehyde were procured from Sigma Aldrich. Polyvinylpyrrolidone povidone , p-toluenesulfonic acid monohydrate tosic acid , ninhydrin, and dimethylglyoxime were purchased from Alfa Aesar. Tris base and sodium nitrite were obtained from J. Ferric chloride hydrate, sodium hydroxide, and nickelous chloride were purchased from Fischer Scientific Chemical. All purchased chemicals were used without further purification. Corn starch was purchased from a local grocery store. Dimethyl sulfone and lactose were procured from Fisher Scientific. Acetaminophen, diphenhydramine, levamisole, quinine, enalapril, ciprofloxacin, and procaine were purchased through Sigma Aldrich. Two different batches of heroin, three of methamphetamine, three of cocaine HCl, and two of crack cocaine were used. FTIR spectra of street samples can be found in S2. Adulterants are defined as substances that mimic the physiological effects of the drugs. Cutting or bulking agents are used to increase stock volume S3 shows reproducibility of the solid-spreading method. The user then places the bottom edge of the paper test card in water. The water moves up the card due to capillary action and rehydrates the dried reagents, bringing the chemicals into contact with the unknown substance. Different color reactions take place in each lane depending on the specific functional groups present in the sample, as shown in FIG 1. After approximately 3 minutes, the user takes a photo for analysis and compares the image to a library of standards to identify the substance. After approximately 3 minutes, the bar code for the unknown has developed. The user takes a photo for analysis and compares the image to a library of standards. Image pixel values were inverted so that higher values indicated more intense color, then the R, G, and B channels were separated. The inverted mean R, G, and B, pixel values of the colored response area of the lane were divided by the inverted mean pixel value for the printed standard color box respectively on the idPAD to ensure comparable color values when the photos were taken under different light conditions. The blue channel data was used to study the reaction product of Lanes A, B, and C. Cocaine HCl, heroin, methamphetamine, and crack cocaine were selected as the targets of this study because of their prevalence in southwestern Michigan. All samples of a given compound were made from the same, relatively high purity, lot of street drug. Samples were created and blinded at the Berrien County Crime Lab. Solid solution mixtures were made by grinding the drug and cutting agent together in a mortar for at least 3 minutes to ensure a homogeneous mixture. Samples were put into 2mL Eppendorf tubes with a code number written on the tube. Blinded samples consisted of pure and diluted illicit substances. An additional 40 samples of crack cocaine included 20 of street crack cocaine with no detected cutting agents, and 20 where FTIR detected the cutting agent nicotinamide. A total of blinded samples were prepared. Lactose was used to cut cocaine HCl and heroin, and dimethyl sulfone was used to cut methamphetamine. Lactose and dimethyl sulfone are idPAD inactive do not produce colored reactions. Each idPAD was weighed before and after applying the sample. By taking two photos of each idPAD, colors that form and wash out during the capillary flow period were captured. Each reader was told that the sample could contain one of the proposed drugs, a diluted drug, or no drug at all. The readers were asked to only use the images in the idPAD reader when categorizing the samples. Solid samples were prepared by grinding a known weight of illicit substance and cutting agent together in a mortar for 3 minutes to create a homogeneous mixture; portions of mixtures were serially diluted by adding additional cutting agent to a known mass of the mixture and grinding. The mass of the idPAD was taken before and after application of the drug mixture to obtain the mass of the solid on the card. The total mass on the card was divided by twelve to determine the amount of solid in each of the twelve lanes on the idPAD. Twelve PADs of each color test were stamped. One idPAD of each of the 12 chemical tests were packaged together and heat sealed in an aluminum pack. At each time point one week, one month, and three months , a pack of idPADs was removed from each of the temperature conditions and tested with a known positive analyte for each color test. The idPAD was assessed to determine the consistency of color barcodes produced by heroin, methamphetamine, cocaine HCl, and crack cocaine. Each bar code involves results from 3—5 lanes. Although the individual color tests are not specific for a certain drug, readers can deduce chemical structure information based on the colored response of multiple lanes. Color barcodes for targeted illicit substances- cocaine HCl, crack cocaine, methamphetamine, and heroin. Samples were obtained from the Berrien County Forensic Lab. Each color barcode consists of reactivity with 3—5 idPAD lanes highlighted in the red box. A box-and-whisker plot illustrates the difference in lanes A-C between the royal-blue color produced by cocaine HCl to the teal-blue color produced by heroin FIG 3. A lane by lane statistical breakdown can be found in S8. To target tertiary amines and bulky secondary amines found in all the target drugs, lanes A, B, and C form colored ion pairs using a cobalt thiocyanate reagent and buffers at pH 2, 8, and This test reliably distinguishes heroin teal from cocaine HCl royal blue. Crack cocaine is made by mixing cocaine HCl and baking soda to form the free base. Crack cocaine can be distinguished from cocaine HCl as there is little to no reactivity with crack cocaine in basic lanes B and C where acid is not available to protonate the amines needed for color production. A mechanism for color formation in lanes A, B, and C has been proposed 21 with crystallographic data in support of the colored product formed 22 — In the presence of protonated secondary and tertiary amines, ion pairs are formed excluding water from the coordination sphere and pushing the geometry around cobalt tetrahedral, which is blue. The color-forming reaction is similar to the first step in the Scott Test used in many field pouch tests 25 , This is due to the smaller number of hydrophobic carbon groups, which can lead to increased hydrogen bonding between the amine and the mobile phase water. A characteristic wash-out of the royal blue color from methamphetamine and MDMA is observed in lanes A-C as water flows up the lane, leaving blue color at the sides of the hydrophobic wax lane boundaries. Lanes D and E on the idPAD target phenols which can be found in cutting agents such as acetaminophen. The reaction chemistry involves diazotization of 4-nitroaniline, followed by deprotonation of the phenol with NaOH and nucleophilic attack forming azo dyes 27 , Heroin also reacts with these lanes although it does not contain a phenol. However, the base present in the phenol lane partially deacetylates heroin into 6-monoacetylmorphine, which does have a phenol. Phenols are also present in incompletely acetylated material in impure street samples. An analytical standard of heroin was tested on the idPAD and faint reactivity was seen in the phenol lanes S9. Illicit drugs can sometimes be found as colored powders. In this work, brown powder heroin and white powder heroin were tested. The teal-blue color observed was consistent regardless of the powder color. We have not yet had access to items such as brightly colored MDMA tablets but the impact on the background powder color must be considered. Some of these compounds are used as adulterants or cutting agents in street drugs. Procaine, acetaminophen and other over the counter pain killers are also commonly added to methamphetamine and heroin Table 1 shows the range of colors developed from illicit substances, cutting agents, and adulterants for each of the lanes. The possibility for cutting agents, adulterants, and other household products to lead to false positive results on the idPAD is described in Blinded Study. Typical colors resulting from illicit substances and distractors on the 12 lanes of the idPAD. Each lane is shown sideways, with the swipe line oriented toward the left and the top of the lane oriented toward the right. After establishing the color bar codes for the target illicit substances, a blinded study was performed at the Berrien Country Forensic Lab to determine the sensitivity and specificity of the idPAD in identifying the targeted controlled substances. There was no difference in the expert vs. All 9 of the cocaine HCl samples that gave false negatives were read as crack cocaine. All of the incorrect categorizations for both samples of crack cocaine were identified as cocaine HCl. However, in the field a much greater array of distractor substances would be encountered in street samples, which is likely to lower the specificity. We therefore tested the ability of readers to distinguish between real illicit drugs and distractor substances. Images were read by two additional expert readers Expert 1 and Expert 2 utilizing the same idPAD reader and training from the first blinded study. Readers were told that each sample may contain a drug, a diluted drug, or no drug at all. If they believed a drug was present, they were asked to identify it using the idPAD reader. S12 and S13 display a full table of all sample identifications by Experts 1 and 2 and the corresponding idPAD images. This revealed that cutting agents, over-the-counter drugs, and some prescription drugs containing tertiary or secondary amines could be mistaken for illicit substances, and further testing on such mixtures is needed. However, these findings present a significant improvement over traditional pouch tests, where identification based off a single color result can be challenging even for trained investigators 9. The idPAD can correctly identify methamphetamine, heroin, cocaine HCl, and crack cocaine in high microgram levels when the drugs are diluted in a solid lactose matrix. NIJ Standard These stability values are comparable to other presumptive field devices in development 7. Additional stability information can be found in S A paper based analytical device can be used to classify a suite of illicit drugs. The idPAD does not require electricity, is inexpensive, requires minimal training, and continues to be explored as a tool for field detection of illicit substances. They differ from the idPAD because both devices require the samples to be in solution, which limits field usability. The RDM was designed to detect only cocaine and methamphetamine and uses a cell phone to read hue and saturation values. Companies such as Detectachem are also using phone programs to make sample identification off color analysis. Further advancements of the idPAD include exploring more complex mixtures drug-drug and drug-cutting agent and their interference with the determined drug color barcodes. Additional lane tests for detection of other cutting agents are also being explored. A cell phone application capable of reading idPAD color barcodes is also being developed to eliminate or limit human interpretation. Full validation using street samples from different geographical regions to account for the variations in adulterants and cutting agents used in drug mixtures will need to be completed prior to field deployment; however, the idPAD shows promise as a presumptive field drug test. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author s and do not necessarily reflect the official views of the Army STTR Program Office, the U. This section collects any data citations, data availability statements, or supplementary materials included in this article. As a library, NLM provides access to scientific literature. J Forensic Sci. Published in final edited form as: J Forensic Sci. Find articles by Tracy-Lynn E Lockwood. Tammy Leong , B. Find articles by Tammy Leong. Sarah L Bliese , B. Find articles by Sarah L Bliese. Alec Helmke , B. Find articles by Alec Helmke. Find articles by Alex Richard. Getahun Merga , Ph. Find articles by Getahun Merga. John Rorabeck , M. Find articles by John Rorabeck. Marya Lieberman , Ph. Find articles by Marya Lieberman. Issue date Jul. PMC Copyright notice. The publisher's version of this article is available at J Forensic Sci. Open in a new tab. Similar articles. Add to Collections. Create a new collection. Add to an existing collection. Choose a collection Unable to load your collection due to an error Please try again. Add Cancel. Secondary and Tertiary Amines — Acidic pH 21 , 25 , 26 , Secondary and Tertiary Amines- neutral pH 21 , 25 , 26 , Secondary and Tertiary Amines- basic pH 21 , 25 , 26 , Phenols with P-Nitroaniline 27 , Phenols with Sulfanilamide 27 , Nucleophiles 31 , Amylose 33 , Potato Starch. Nitrogen and Oxygen rich compounds Primary Amines Aromatic Amines Timer Lane
L-1 for cocaine detection. •. Adsorption of cocaine over the electrode surface was investigated by scanning electron microscopy (SEM) and energy dispersive.
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