Article pubs.acs.org/ac
Noninvasive Double Confirmation of Cocaine Abuse Sergio Armenta,*,† Miguel de la Guardia,† Manel Alcalà,‡ and Marcelo Blanco‡ †
Department of Analytical Chemistry, Research Building, University of Valencia, 50th Dr. Moliner St., E-46100 Burjassot, Valencia, Spain ‡ Department of Chemistry, Faculty of Sciences, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain S Supporting Information *
ABSTRACT: A double confirmation procedure, based on the combined application of Ion Mobility Spectrometry (IMS) and Infrared Spectroscopy (IR), has been developed for the noninvasive unambiguous identification of cocaine consume. The use of nasal mucus as a biological specimen for cocaine abuse confirmation has been proposed as an alternative to the use of blood and urine due to its noninvasive character and the presence of the parent compound instead of its metabolites. Sampling conditions, interferences caused by cutting agents and other substances, and limits of identification (LOI) and confirmation (LOC) have been deeply evaluated. The procedure combines the high sensitivity of the IMS to identify positive samples with the high selectivity of the IR procedure to confirm positive results. Thus, the proposed two tier method has been applied to the detection and identification of cocaine in the nasal mucus of different individuals, consumers, and nonconsumers, providing results comparable with those obtained by a reference procedure.
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spectrometry (MS),8 liquid chromatography (LC-MS),9 and capillary electrophoresis (CE).10 To date, the most common drug-testing practices are based on a two-tier approach of a rapid on-site screening method followed by a confirmatory testing of preliminary positive screen results performed in official laboratories, especially in situations where a rapid decision is needed.11,12 The rapid “yes/ no” response analytical tools that indicates whether the target analytes are present above or below a preset concentration threshold provides some important advantages; such as reduction of costs, rapidity, simplicity, and minimization of errors owing to delays between sampling and analysis. However, the unreliability problem associated with qualitative testing, the relative proportion of wrong yes/no responses (false positive and false negative respectively), is one of the most important aspects that needs to be systematized.13 Moreover, in cases where the legal implications of a false positive carry penalties, that range from economic sanctions to prison sentences, seems to be evident the imperative need to reduce to the minimum the possibility of a false positive in the screening tests. On the other hand, the consequences of a false negative in cases of on road traffic controls could be fatal, and consequently, it should be also reduced to the minimum. In this paper, we propose the use of two analytical technologies based on different chemical principles, sequentially or in combination, to accomplish fast and accurate detection, identification, and semiquantification of the drugs from nose swab samples. It is expected an improvement of on-
rug abuse remains a significant public health issue worldwide. According to data reported by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA),1 cocaine remains the second most commonly used illicit drug in Europe overall. It is estimated that about 15.5 million Europeans have used cocaine at least once in their life (4.6% of adults aged from 15 to 64, data reported correspond to 2010). Moreover, cocaine is the most trafficked drug in the world after cannabis, with global seizures largely stable at about 694 tonnes per year.2 It can be assessed that the main objectives of drug-of-abuse testing are the detection, identification, and/or deterrence of substance abuse or misuse. Drug testing has been conducted primarily on blood and urine. However, recently, there is a high interest in their replacement by alternative biological specimens which can be collected using noninvasive sampling techniques.3 Hair, sweat, and oral fluids have reached a sufficient level of scientific credibility to be considered for use in drug testing.4 However, for drugs consumed by nasal insufflations, commonly called sniffing or snorting, as cocaine is, the nasal mucus could be the most interesting body fluid to be analyzed. The main function of nasal mucus is to trap small particles such as dust, particulate pollutants, and allergens and avoid that they enter in the respiratory system. An additional advantage of nasal mucus over other biological fluids is that the parent snorted compound is present instead of its metabolites. On the other hand, the choice of analytical drug-testing technologies can be mainly grouped into two major categories: 1) assays that are based on molecular recognition and ligand binding, with immunoassays being the most popular techniques for drugs-of-abuse screening,5−7 and 2) separation methodologies, including gas chromatography (GC) with mass © 2013 American Chemical Society
Received: July 25, 2013 Accepted: October 31, 2013 Published: October 31, 2013 11382
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Article
Before analysis, Teflon membranes were introduced into the IMS instrument to remove any possible interference. Desorption, inlet, and drift tube temperatures were adjusted to 260, 275, and 232 °C, respectively. Using a 10 s postdispense delay, the sample tray containing the Teflon membrane was inserted in the heated zone, and the sample was held in this position for 30 s. Infrared Spectroscopy Procedure. IR spectra were recorded using a Tensor 27 FT-IR spectrometer from Bruker (Karlsruhe, Germany) equipped with a DLaTGS detector. Spectra were obtained by coadding 10 scans at a resolution of 4 cm−1 and a scanner velocity of 10 kHz HeNe frequency, from 4000 to 900 cm−1. For instrumental and measurement control, spectra treatment, and data manipulation, the OPUS program (version 6.5) from Bruker was employed. In this study, a transmission cell with an open upper side has been used to improve the sensitivity of the method without sacrificing the simplicity. Thus, a standard transmission flow cell with 2 mm thick CaF2 windows has been equipped with two Teflon spacers providing a path length of 0.5 mm and an internal volume of approximately 35 μL. Once the cell was assembled, standard and sample absorbance were measured by transmission mode using manual introduction of solutions inside the cell, using a Hamilton 50 μL syringe (Bonaduz, Switzerland) and chloroform as background. Cleaning of the cell was achieved by three sequential injections of chloroform blank solutions. Nose swab samples, recognized as positive from the IMS study, were evaporated to dryness under a nitrogen flow, reconstituted in 100 μL of chloroform and analyzed by IR. Liquid Chromatography Reference Procedure. The LC system Dionex P680 (Sunny Vale, CA, USA), equipped with Chromeleon software from Dionex and equipped with a quaternary pump, a thermostatted column compartment, and an UVD 170U variable wavelength UV−vis detector, was used for chromatographic analysis. All separations were carried out on a Kromasil 100 C18 column (250 mm × 2.0 mm, 5.0 μm), and the column temperature was maintained at 25 °C. The isocratic mobile phase, pumped at a flow rate of 1.0 mL min−1, consisted of acetonitrile-phosphate buffer pH 2.5 (50:50 v/v), which was freshly prepared, filtered through a 0.22 μm filter, and degassed by sonication for 15 min prior to use. The injection volume was 20 μL, and the detection wavelength was 225 nm. Standards and Samples. Seized samples (12), containing cocaine from 25 to 80% w/w, were kindly provided by the “Unidad de Inspección de Farmacia y Control de Drogas” from the Valencia Health Service Area. All the solvents used in this study were HPLC grade or higher. Methanol, acetone, and chloroform were purchased from Scharlau Chemie S.A. (Barcelona, Spain). A double-ended cotton tipped, regular size swab with a polystyrene handle was used for biological nose fluid collection. The swab was inserted into the nostril, approximately 2 or 3 cm, rotated twice (2 × 360° turns) collecting the biological fluid, and slowly removed, and finally it was inserted in a 2 mL amber glass vial containing 1.5 mL of methanol. The polystyrene handle was cut, and the vial was closed, named with an appropriate code and stored until analysis. The sampling was repeated by inserting the other swab end in the second nostril of the nose. Mucus specimens were obtained from cocaine chronic consumers who provided their consent after appropriate
site drug testing by the application of two analytical methods of categories A and B of the Scientific Working Group for the Analysis of Seized Drugs14 such as infrared spectroscopy (IR) and ion mobility spectrometry (IMS). It will reduce the number of false positive results implying a reduction of costs and maintaining the high reliability currently associated with actual drug testing methods. IMS has been becoming an indispensable tool in drug enforcement departments for detecting trace amounts of drugs in mail, imported articles, suspects clothing, luggage, and so on.15,16 IMS is an analytical technique based on the gas-phase separation of ionized analytes under a weak electric field at ambient pressure. The analytical potential of IMS is derived from its high sensitivity, its operational speed that substantially shortens the analysis time, and its simplicity.17 IR spectroscopy has a long history in illicit drug analysis.18 The computerized drug library produced at the Georgia State Crime Laboratory (GSCL) is a standard in forensic analysis. Recently, a drug library of 455 spectra measured by ATR was produced at the Illinois State Police Laboratory.19 The choice of sampling technique is largely dependent on the information required about the sample. For identification work, a singlebounce diamond ATR crystal is often used for quick analysis of solid powder samples. However, in the case of biological samples, sensitivity has been traditionally considered the Achilles Heel of the technique, and different methodologies have been previously proposed to solve this problem.20 The use of transmission measurements with increased optical path length cells combined with reduced internal volume has demonstrated to provide detection limits at the low parts per million level,21 being appropriate to analyze nose swabs in driving under the influence cases. In summary, the main objective of this study has been the use of two analytical technologies, based on different chemical principles, sequentially or in combination, to accomplish fast and accurate detection and identification of the drugs from nose swab samples. The order of the analytical methodologies in the aforementioned sequence was fixed taking into consideration the high sensitivity of the IMS to identify positive samples combined to the high selectivity of the IR procedure to confirm positive results.
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EXPERIMENTAL SECTION Ion Mobility Spectrometry Procedure. An IONSCANLS (Smiths Detection, Morristown, US) equipped with a 63Ni foil radioactive ionization source was used to separate and identify the different compounds involved in this study. IM station software (version 5.389) was used for data acquisition and processing. Plasmagrams were acquired in positive ion mode using nicotinamide, with a reduced mobility (K0) of 1.860 cm2 V−1 s−1, as internal calibrant. The number of segments per analysis was 56, containing every plasmagram 779 data points. The shutter grid width was 0.2 ms (the value optimized by the manufacturer), and plasmagrams were collected with a scan period of 40 ms. A counterflow of dry air, set to 300 mL min−1, was introduced as drift gas at the end of the drift region. The electric field strength in the drift region was 252 V cm−1 with a total drift voltage of 1763 V and a drift tube length of 7 cm. Thermal desorption from a Teflon membrane was used for sample introduction. In this strategy, one microliter of sample was placed onto the Teflon membrane and heated to vaporize the analyte which was transferred to the ionization region. 11383
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Figure 1. IMS and IR signals for cocaine seized samples and nasal mucus from cocaine users and nonconsumer subjects. Cocaine seized (i. and ii.), cocaine consumers (iii., iv., and v.), and nonconsumers (vi. and vii.).
around 11.65 and 12.02 ms, probably due to cutting agents, can be observed. On the other hand, in negative samples cocaine peak is absent, and only small peaks around that of the calibrant peak can be observed, probably due to components of the nose mucus, such as amino acids or protein fragments. An alarm was generated to alert the presence of cocaine in the nose swab samples, using the following peak descriptors: (i) the K0 value, (ii) a variability value of 50 μs of the peak drift time, (iii) a peak amplitude of 1.5, (iv) a signal threshold value of 20, and (v) a full width value at the half-maximum amplitude of the peak of 200 μs. FT-IR Spectra of Nasal Mucus Samples. Figure 1b shows the IR spectra of two seized samples dissolved in chloroform with a concentration of cocaine of 500 mg L−1 considering a purity of 60 and 45% w/w, respectively, and different positive and negative nose swab samples. In those samples in which cocaine is present, typical absorption bands at 1726 cm−1 (stretching vibration of the carbonyl group), 1271, 1180, and 1115 cm−1 (acetate C−O stretching), 1071, 1029, and 1017 cm−1 (monosubstituted benzene stretching and the last one an out-of-plane bending), and 965 cm−1 (attributable to bending vibrations out-of-plane) can be observed. Those absorption bands perfectly match with previously reported cocaine IR spectra.24,25 Using the Quick Compare function of the OPUS software, it is possible to compare nose swab sample spectra with a cocaine reference spectrum or a set of seized cocaine sample spectra, calculating the corresponding correlation coefficient. To perform an accurate comparison, the spectral regions from
information about the study. Cocaine consumers were males with ages ranging from 29 to 40. Samples were collected at different times after abuse of the drug using the aforementioned swab technique and stored in methanol at −4 °C until analysis. Cocaine-free mucus were collected from nonconsumer subjects, males and females with ages ranging from 25 to 40. It should be highlighted that under no circumstances have the authors trafficked or provided illegal substances, aimed, promoted, facilitated, stimulated, or forced in any way the consuming of illegal substances during this study.
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RESULTS AND DISCUSSION IMS Plasmagrams of Nasal Mucus Samples. The ion mobility plasmagrams of cocaine and positive and negative swab samples are depicted in Figure 1a. The most intense peak in all plasmagrams is due to the internal calibrant, nicotinamide (K0 = 1.860 cm2 V−1 s−1), used in the positive ionization mode to correct for variations in temperature, pressure, and drift field and to increase the selectivity of measurements. Although precise assignment of the peaks needs a MS coupled to the IMS, it could be speculated that the main peak of the plasmagram, excluding that of the reactant ion, is due to the analyte molecular mass peak. Cocaine plasmagram provides a peak at 15.07 ms drift time with a reduced mobility of 1.16 cm2 V−1 s−1, which is consistent with previously reported values.22,13,23 Figure 1a contains the plasmagrams of two cocaine seized samples and several positive and negative nose swab samples. In seized samples and positive swab plasmagrams, cocaine can be easily identified, and additional peaks 11384
dx.doi.org/10.1021/ac4023583 | Anal. Chem. 2013, 85, 11382−11390
Analytical Chemistry
Article
Figure 2. Limit of detection of employed methodologies a) LOD of the IMS procedure. b) LOI of the IR method considering a threshold of 90%. c) IMS plasmagrams of nasal mucus swabbed at different times after cocaine abuse. Inset: Capability of the IR method to discriminate positive and negative cocaine samples.
1786 to 1701 cm−1, from 1363 to 1257 cm−1, and 1166 to 950 cm−1 were selected. The correlation coefficient (r) of two spectra (y1 and y2) was calculated. It is recalculated as a percentage being the −1 ≤ r
