Patent Description:
Illegal drug use is a globally recognized phenomenon affecting thousands of victims every year. New illegal drugs are continuously coming to the market motivating society to search for tools to combat the production, trafficking, distribution and use of these illegal drugs. The use of drugs is found giving rise to many illegal activities that consume a lot of resources of the countries. Therefore, European Union, Norway, Australia, USA, Canada and other countries have already stringent laws for drug abuse and have mandated the drug testing.

Detection of illegal drugs can be broadly categorized into either non-confirmatory or confirmatory analysis. Non-confirmatory analysis deals with the analysis and identification of illegal drugs at the point where such chemicals have been presumably consumed. The confirmatory analysis is performed in the laboratory, which involves the identification of the use of drug by suspect with some kind of device and is employed in sample preparation, storage and transport to corresponding central lab using cargo entry points and secure facilities for transport.

While both categories of detection are important, the preventative nature of confirmatory test on a site makes it extremely useful.

Generally, drug testing is conducted using urine, blood, sweat, hair or oral fluid (saliva). Urine provides retrospective information about past drug use, but provide little information about the current effect of the drug on a person and/or their ability to drive. Blood and oral fluid (OF) are likely to give the most accurate measurement of an illegal drug's active form concentration, which is what affects driving behavior. The analysis of oral fluid for illegal drug abuse determination offers different advantages compared to blood and urine. Nonmedical personnel can collect it in a simple, inexpensive, and non-invasive manner. Oral fluid sampling can be closely supervised without an invasion of privacy and to prevent substitution, adulteration, or dilution of the sample, which could happen with urine analysis. Oral fluid sampling also avoids the risk of infection, which is possible during a blood draw.

Several approaches for detecting drugs in OF have been developed. They can be categorized as non-confirmatory and confirmatory methods. The majority of non-confirmatory methods are used in situ and they are based on immunological procedures. The present immunoassay tests are simple and easy to use, but these kind of tests have high error rates due to the ambiguity of detection (in the form of faint stripes), degradation of antibodies used, and cross reactivity with other analytes. Some examples of the cross-reactivity are listed here: Remnants of poppy seed roll give false positive for heroin due to the morphine and codeine naturally found in poppy seeds, for example morphine and codeine concentrations are from <NUM> to <NUM> ng/mL and from <NUM> to <NUM> ng/mL, respectively, [<NPL>]. Ecstasy (MDMA) and its analogue MDEA cannot be differentiated from methamphetamine. Tyramine found naturally in OF metabolized by a monoamine oxidase (MAO) and/or coming from food (meat, fish, cheese, alcoholic beverages, and protein rich food) can give false positive for amphetamine testing. MDEA's and MDMA's metabolite MDA gives false positive for amphetamine, for example, cross-reactivity <NUM> ng/ml for Dräger Drug Test <NUM>, (Dräger DrugTest <NUM> STK IVD User Manual Table <NUM> Specificity), Reference: <NPL>. Noscapine and lidocaine give false positive for opiate test and helional for ecstasy test, Biosens <NUM>, <NPL>). The immunoassay tests would fall to identify poly-drug mixtures of amphetamine, MDMA, MDEA, MDEA and/or methamphetamine. According to the DRUID study performed during <NUM>-<NUM> in Spain, <NUM>% of the samples tested contained two or more drugs.

This fact results to the situation that there is a great probability of obtaining a false negative or false positive result. Some studies performed with various commercially available assays revealed a <NUM>% false positive and sometimes <NUM>% false negative detection accuracy. Other independent case studies showed that the error for being punished while not using illegal drugs was <NUM>-<NUM>% and error rates for not being punished while drugged was <NUM>-<NUM>%, respectively. Moreover, these immunoassay tests are only qualitative and, therefore, cannot give an estimation of the impairment level neither the indication of the recent drug use. The immunoassay tests have certain non-adjustable cut off limits, varying from manufacturer to manufacturer. Therefore, when the 'per se' threshold approach is implemented, the threshold limits can be adjusted to the cut-off of immunoassay strips, not vice versa.

Other pitfalls of immunoassay tests are well known:.

Thus, immunoassays are used as preliminary screening approaches, in situ, which are then followed by a chromatographic technique to confirm the results. Well-known chromato-mass-spectroscopic methods like gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry have been described for determination of banned chemicals. In comparison to the immunoassays, chromatographic techniques are not suitable for field analysis, in general, require sophisticated sample pre-treatment, qualified personal for performing measurements which makes the overall process of analysis time-consuming.

All the reasons above encourage and promote more and more attention to the development of alternative method approaches. Technological advancements and product portfolio expansion is the key trend witnessed in the market.

Implementation of robust, reproducible, user friendly technology is critical to meet the testing suspects of using illegal drugs in situ (roadside, public events) placed on today's law enforcement institutions. Upgrades in technology are necessary to facilitate increased output, while continuing to generate quality analytical data and attempting to minimize the number of invalid test results and instrument - related investigations. It is desirable to achieve adequate resolution between analytes, and separations within reasonable timeframes, and with reliable reproducibility. The instrument and method must be robust and completely automatic so it could be operated by a layperson (e.g. a law enforcement officer). Thus, it is an object of the invention to provide improved illegal drug tester for using on the site but free of immunoassay pitfalls and thus, having confirmatory power.

There is considerable interest in the development of such fast and reliable analytical instrumentation for the identification of illegal drugs and other banned chemicals since the results provided by these analyses constitute an indispensable tool for law enforcement agencies during the investigations and prevention of use of illegal drugs and other banned chemicals. While electrophoresis has historically been used in quality control for product purity and fragmentation analysis, the methodology has transformed from gel - based, to capillary - based, and more recently, to the portable instruments. Capillary electrophoresis (CE) is alternative technology to immunoassay. It is, undoubtedly, one of the easiest methods to be miniaturized and automatized. Portable capillary electrophoresis allows for dramatically reduced sample analysis times, while maintaining the performance and reproducibility standards required for forensic analysis, (<NPL>).

Until now, CE has received less attention as a tool for determination of illegal drugs. CE requires extremely low volumes of sample and is quick and cost-effective.

The small sample size and the small detection path length (<NUM>-<NUM>) makes the detection limits of the CE several orders higher than in the case of other chromatographic and spectroscopic techniques. This, however, can be overcome by using advanced detection technologies such as fluorescence and impedance. One attractive feature of CE is the compactness and robustness of the equipment, which would open the opportunity for the construction of portable instruments. These could be used as a confirmation tool by law enforcement agencies in situ, at the point of interest (in street, roadside, public events). If the detection limits of the CE could be reduced to the required cut off level then CE instruments could become an attractive alternative to the immunoassays.

CE with native fluorescence detection capability offers an attractive combination having potential for the confirmatory identification of illegal drug consumption on the site. A portable, CE instrument with miniature flash Xe-lamp with excitation broadband from <NUM> to <NUM> have greater flexibility for detection of illegal drugs in suspected saliva which has been demonstrated at the several electronic music festival (in Estonia, between <NUM> to <NUM> years and roadside testing, (<NPL>). Known is <CIT>, comprising an apparatus for the separation and determination of banned compounds in biological sample using electrophoresis, comprising a separation capillary and a fluorescence detector. This can be considered as the closest prior art.

The present invention relates to a roadside analyzer for the determination of illegal drugs abuse, including, but not limiting to detection of explosives, toxic industrial chemicals and other banned or regulated compounds, biomarkers and phytochemicals in a sample in situ in at least one human body fluid sample, specifically in oral fluid (saliva), but not limiting to other clinical samples of interest (urine, blood, exhaled breath, exhaled breath condensate, etc.) It consists of automatic processor for preparing samples suitable for analysis. Analysis part of the instrument implements three technologies, namely solid phase extraction prior to analysis, capillary electrophoresis for separation of analytes from the sample matrix and impedance (contactless conductivity) or fluorescence or both impedance (contactless conductivity) and fluorescence for detection of analytes of interest. Contrary to the sensors based on the molecular recognition, the analyzer identifies not the class of illegal drugs (e. g "Amphetamines") but the illegal drug itself (e.g. amphetamine, methamphetamine, ecstasy (MDMA) and its analogues (MDA, MDEA, PMA, PMMA). It determines the use of other drugs like cocaine, marijuana cannabinoids (THC, CBD), LSD, morphine and others, including, but not limited to other drugs and banned or regulated compounds, and estimates their concentration in a sample of interest, in particular in oral fluid (saliva sample) at the confidence needed for confirmatory power and can be used at the point of interest (street, roadside, public events). Performance of the analyzer is superior over commercially available testers (based on immunoassay) because it is more selective than those testers and gives more information regarding the real drugged level of alleged person and the recent use. The analyzer is simple enough to be used in the field and handled by various professionals (police, custom workers, prison guards and various transport situations).

The present invention provides a highly sensitive and selective illegal drug testing device as claimed in claim <NUM>.

A method is provided as claimed in claim <NUM>.

The invention is described in detail with references to the drawings where in.

The general concept of an apparatus according to the invention is illustrated in <FIG>, where items <NUM> to <NUM> correspond to the analyser sampler comprising a first stepper motor <NUM> controlling height of the lift of the vial, a vial lift <NUM>, a second stepper motor <NUM>, controlling the position of the sampler carousel, a stand <NUM> for the inlet electrode <NUM> and capillary <NUM> through the inlet electrode <NUM>, an inlet vial <NUM>, a sampler carousel <NUM>, the separation capillary <NUM>, the inlet electrode <NUM>. The outlet part of the analyser comprises outlet elctrode <NUM>, stand <NUM> for the outlet electrode <NUM> and capillary <NUM> passing through the electrode <NUM>. The stand <NUM> is connected to BGE replenishment and rinsing system <NUM> and outlet vial <NUM>. The first channel <NUM> connects the outlet vial <NUM> with the vacuum pump (not shown in drawings). The capillary <NUM> passes through fluorescence or impedance detector <NUM>.

Sample extractor part comprises an extract vial <NUM>, a syringe <NUM> for tampon/swab with the collected sample of interest, a solenoid valve <NUM> for extra saliva removal, a solenoid valve <NUM> for directing extracted sample to a sample vial <NUM>, a second channel <NUM> to a vacuum pump (not shown in drawings), mentioned above second channel <NUM> connects a vial for extra saliva collection <NUM> to a vacuum pump (not shown in drawings), a solid phase extractor <NUM>, the vial for extra saliva collection <NUM>, a sample vial <NUM>, third channel <NUM> connecting to the sample vial <NUM> to vacuum pump (not shown in drawings), a micro peristaltic pump <NUM> connected via inlet conduit <NUM> to sample vial <NUM> and via outlet conduit <NUM> to inlet vial <NUM> for directing sample from sample vial <NUM> to inlet vial <NUM>.

The assembly of the apparatus according to the present invention (<FIG>) comprises fluorescence detector <NUM> attached to the support frame <NUM> of the analyser, a sampler carousel <NUM> with vial adapter <NUM> with vial <NUM>, base of carousel <NUM>, lift mechanism <NUM>, cooling system using Peltier elements <NUM>.

The sample carousel (<FIG>) according to an embodiment of invention comprises the first stepper motor <NUM> for lifting vial <NUM>. The stepper motor <NUM> is connected via shaft <NUM>, connecting sleeve <NUM> and connecting plate <NUM> to the vial lift <NUM>. The vial lift <NUM> comprises head of the lifting mechanism <NUM>, which connects to the vial <NUM> located in vial adapter <NUM> to rise it to the level enabling electrode <NUM> with separation capillary <NUM> to be drawn into conditioning liquid <NUM> in vial <NUM>. In addition the lifting mechanism comprises supporting rods <NUM> for the lift stepper motor, linear guides <NUM> providing smooth vertical movement of the vial lift <NUM>. Vial remover of lifting mechanism <NUM> removes the vial <NUM> from inlet electrode <NUM>, keeping the vial against head of lifting mechanism <NUM>. Inlet electrode <NUM> is mounted on the stand for the inlet electrode <NUM> and stand base <NUM>. The separation capillary <NUM> (see drawing <FIG>) is guided through capillary chamber <NUM> by capillary guides <NUM>, <NUM>, <NUM>, <NUM>, where first and last capillary guide <NUM> and <NUM> are attached to the housing of the capillary chamber <NUM> by connection elements <NUM>. The capillary chamber <NUM> is closed with housing of the capillary chamber <NUM>. The capillary chamber and fluorescence detector <NUM> are attached to the support frame <NUM> of the analyser (shown in <FIG>).

In <FIG> is illustrated schematically a fluorescence detector part of the apparatus according to the invention, where a fluorescence detector <NUM> comprises a xenon lamp <NUM>, aspherical collimator lens <NUM>, excitation filters <NUM>, excitation focusing lens <NUM>, separation capillary <NUM>, aspherical emission collecting lens <NUM>, emission filters <NUM>, a photomultiplier tube <NUM>, emission focusing lens <NUM>, a first neutral filter <NUM>, a beam splitter <NUM>, reference beam focusing lens <NUM>, a second neutral filter <NUM>, a reference photodiode <NUM>.

The apparatus according to invention is controlled by a computer (personal computer) via conventional connecting means (bluetooth, wi-fi, cable etc.) where in <FIG> is shown the functional schematic of the device control according to the invention where a computer program controls carousel auto-sampler and controller, carousel stepper with lift stepper, vacuum pump, and high voltage power supply to electrodes, sample extractor controller and extra saliva solenoid and vacuum pump.

In <FIG> is an example electropherogram of illegal drug standards shown. It is suitable for determination of amphetamine type stimulants and other common narcotics. Peak numbers in the figure correspond to the following Identified compounds: <NUM> and <NUM> - internal standards, <NUM> - AMP, <NUM> - tyramine, <NUM> - METH, <NUM> - MDA, <NUM> - MDMA, <NUM> - MDEA, <NUM> - cocaine, <NUM> - cocaethylene (cocaine metabolite), <NUM> - metoprolol (simulant of LSD) and <NUM> - fentanyl.

Conditions: uncoated, fused-silica capillaries, i. <NUM> were used for the analyses. The fluorescence detector was positioned <NUM> to capillary end with total length of <NUM>. Prior to injection, the capillary was rinsed sequentially with <NUM> NaOH, deionized water and the BGE for <NUM> each. Separations were performed at +<NUM> kV. Before the measurements, new capillaries were conditioned by rinsing them sequentially with <NUM> sodium hydroxide and deionized water. Between analyses, the capillaries were rinsed with the BGE solution for <NUM>.

In <FIG> is another electropherogram shown. Capillary conditioning procedures are the same as described for <FIG>. BGE2 was applied for analysis of cannabinoids. Peaks, <NUM>- electroosmotic flow, <NUM> - THC, <NUM> - CBD, <NUM> - internal standard.

In <FIG> is illustrated a typical electropherogram of suspects' oral fluid samples. The oral fluid samples were provided to us by police officers from the Police and Border Guard Board (PBG) of Estonia. The suspected users' OF sample contained <NUM> and <NUM> - internal standards. <NUM> - AMP and <NUM> - tyramine (the compound is associated with smoking and some foods).

The instrument consisted of a sample preparation unit (<FIG>, <NUM>-<NUM>) or without it (if manual sample preparation is performed), autosampler carousel (<FIG>, <FIG>), separation capillary (<FIG>, <FIG>), and detector (<FIG>, <NUM>). During the analysis the electrodes (<FIG>, <FIG> and <NUM>) are powered by high voltage power supply (not shown in drawings) and the detector signal is recorded by a built in computer. The built in computer sends control signals to the control board, which in turn controls stepper motors (<FIG> and <FIG>), solenoid valves (<FIG>, <NUM>, <NUM>) and switches on/off the vacuum pump or pumps (not shown in drawings).

The present device and methods can operate with an automatic sample extraction unit (<FIG>, <NUM>-<NUM>) or without it. The work of the sample extraction unit is controlled by a built in computer. The built in computer sends control signals to the control board, which in turn delivers commands to the solenoid valves and vacuum pump or pumps. A tampon/swab with suspect's oral fluid is placed into syringe <NUM> which is then sealed.

Solenoid valve <NUM>, which is initially in OFF position, allows excess saliva to be delivered into excess saliva vial <NUM> when the experiment starts after switching on the vacuum pump. The vacuum pump creates low pressure in the excess saliva vial <NUM>. This facilitates removal of the superfluous saliva removed from the tampon/swab and retaining constant amount of sample into the tampon/swab. After a preset interval of time, the solenoid valve <NUM> is set to the ON position and because solenoid valve <NUM> is initially in OFF position the extractant in the vial <NUM> flows to the syringe <NUM> which retains low pressure. The sample is extracted from the tampon/swab, which is in the syringe <NUM>, and during extraction atmospheric pressure establishes in the syringe <NUM>.

After a preset time the solenoid valve is set to ON position, which facilitates flow of the extracted sample into sample vial <NUM> through the filter as solid phase extractor <NUM> due to the low pressure which has established there through third channel <NUM>. Filter in solid phase extractor <NUM> removes peptides and proteins from the sample. When the transport of the sample to the sample vial <NUM> has been completed, the solenoids <NUM> and <NUM> are set to OFF position and vacuum pump is switched off. By initiating the work of the peristaltic pump <NUM> the sample is transported via inlet conduit <NUM> and outlet conduit <NUM> to the input vial <NUM> in the sampler carousel <NUM>.

The work of the carousel autosampler unit (<FIG>) is controlled by a built in computer. The built in computer sends control signals to a control board, which in turn controls the lift stepper motor <NUM> and brushless DC motor <NUM> of carousel <NUM> and the vacuum pump (not shown in the drawings). Some of the input vials <NUM> in the carousel autosampler <NUM> are prefilled with capillary conditioning (wash) liquid and BGE, not limiting to other liquids, and the rest of the vials are filled with oral fluid extracts from the sample extraction unit or manually extracted samples.

Fluorescence detector <NUM> is shown in <FIG>. The xenon flash lamp <NUM> in Xenon lamp housing <NUM> delivers <NUM> light pulses to the detection window of the separation capillary <NUM> at a repetition frequency of <NUM>-<NUM>. An aspherical lens <NUM> is used to collect the excitation light and a spherical lens <NUM> to focus the light to the capillary <NUM> with high efficiency. The xenon flash lamp has strong emission bands in the region of <NUM>-<NUM>, but it emits also in the broad-spectrum range until near infrared. Therefore, a set of three bandpass filters or excitation filters <NUM> is used to block emission outside that region. Radiation emitted by the solution inside the capillary <NUM> is collected by an aspherical emission collecting lens <NUM> and focused by an emission focusing lens <NUM> on the cathode of the PMT <NUM>, which is located perpendicularity to the excitation beam but at an angle approximately of <NUM> degree to the capillary <NUM>. This angle is introduced to minimize the intensity of the refracted and reflected in the capillary parasitic radiation from the Xenon lamp <NUM>. Doubled emission filters <NUM> or first neutral filters <NUM> are mounted within <NUM> - <NUM> wavelength range for detection of analytes of interest, <NUM> - <NUM> wavelength range is useful for illegal drugs native fluorescence detection. An optical reference channel is introduced to eliminate the xenon lamp <NUM> aging effect on measurement accuracy. A beam splitter <NUM> reflects a part of the excitation beam and directs it through a reference beam focusing lens <NUM> to the reference photodetector or reference photodiode <NUM>. A second neutral filter <NUM> is used to attenuate the reference flux. The reference signal is measured each time after turning on the detector, and its value was recorded in the memory and used for correction of measurement results.

The fluorescence detector <NUM> can be replaced with other detectors of need, for example, the contactless conductivity detector. The cell of the contactless conductivity detector can have different designs. For instance, the cell can be built into a rectangular piece of alumina. Two tubular electrodes and an operational amplifier are placed inside the cage. Two tubular electrodes can have a length of <NUM> and a gap of <NUM>, not limiting to other sizes and materials. Electrodes are shielded from each other by the grounded conductive layer. One of the electrodes is excited with a voltage (<NUM> V or different) peak-to-peak sine wave oscillating in a frequency range of <NUM>-<NUM> (or different). The signal is picked up by the second electrode and further amplified. The software allows to control the hardware by changing the excitation frequency and amplification amount.

A first method according to the invention uses BGE1 which consisted of <NUM>% (<NUM> tris(hydroxymethyl) methylamine, <NUM> phosphoric acid, <NUM>% triethylamine, pH <NUM>) and <NUM>% methanol as an organic modifier. Method <NUM> was used for separation of common narcotics (except THC and CBD). An example of separation is presented in the <FIG>.

A second method according to the invention implements nonaqueous capillary electrophoresis (NACE). It was used for the separation of THC and CBD cannabinoids. BGE2 consisted of <NUM> NaOH dissolved in MeOH/ACN (<NUM>:<NUM>) at pH=<NUM>. An example of separation is presented in the <FIG>. The background electrolyte composition is not limited to the compounds mentioned in method <NUM> and method <NUM>.

To test the feasibility of the invention a prototype of the instrument was build. Details of the prototype are presented in the drawings of <FIG>. The present invention will be first described by the following examples.

The specificity of the CE-FD analyzer was assured by the properly utilized excitation/emission filters in FD and which properties were suited to the native fluorescence characteristics of illegal drugs in the specific region under excitation within the wavelength range of <NUM>-<NUM>, not limiting to lower wavelength range up to <NUM>. Moreover, the specificity was achieved by utilized CE mode with the specific electrophoretic separation conditions and a special sampling/extraction/preconcentration procedure. Therefore, the probability of co-migrating of the fluorescing interference from another substance and their registering at the certain region of emission wavelength controlled by filters and CE conditions was minimized.

The instrumental detection (IDL) and quantification (IQL) limits of the illegal drugs were evaluated in acetonitrile using developed and optimized CE methodologies, excluding the matrix effect of OF and sampling/extraction/pre-concentration procedure recoveries. The instrumental detection and quantitation limits were found using the signal-to-noise (S/N) approach. The S/N ratio for IDL level equaled <NUM>:<NUM>, proving the presence of the analyte in the test sample with a probability larger than <NUM>%. The S/N ratio for the IQL level was set to <NUM>:<NUM>, respectively. The analysis of samples containing the analytes at the level of IDL was performed and the results showed that the designed CE-FD instrument was able to detect amphetamine, methamphetamine, MDMA, MDA, MDEA, cocaine, cocaethylene, fentanyl, morphine, LSD, THC and other illegal drugs and banned or regulated compounds at the recommended by DRUID project cut-off limits for illegal drug abuse determination in oral fluid.

Claim 1:
An apparatus for the separation and determination of banned compounds in a biological sample using electrophoresis and consisting of a separation capillary (<NUM>) constituting a separation channel, a fluorescence detector (<NUM>) for characterizing electrophoretic zones of compounds passing through a detection zone of the separation channel,
an injection system for introducing fluids, including sample solutions and background electrolyte into an inlet end of the separation channel, to conduct the sample processing sequence, prior sample analysis sequence,
high voltage power supply,
a controlling solution for commanding the injection system, flow of fluids through the separation channel, and operation of the detector, characterized in that said apparatus comprising a sample preparation and extraction device for processing oral fluid consisting of a compartment for swab/pad/tampon with oral fluid, an excess saliva vial for collecting excess oral fluid and a sample vial for collecting a sample solution, solenoid valves for controlling oral fluid transport through the device vessel containing extractant first into said excess oral fluid vial and then into said sample vial, a controlling solution for commanding the solenoid valves that facilitate flow of fluids through the separation channel, to conduct the sample processing sequence, prior to the sample analysis sequence so as to allow the sample analysis sequence to be conducted, after conducting the sample processing sequence which comprises introduction of a sample and a background electrolyte and applying a voltage potential across the separation capillary to effect a separation of banned compounds.