APPARATUS AND METHOD FOR DETERMINATION OF BANNED SUBSTANCES

Abstract

The present invention relates to roadside analyzer for 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.

Claims

1. An apparatus for separation and determination of regulated compounds in biological sample using electrophoresis and comprising at least one separation capillary; at least one fluorescence detector for characterizing electrophoretic zones of compounds passing through a detection zone of the separation capillary; an injection system for introducing fluids, including sample solutions and background electrolyte into an inlet end of the at least one separation capillary to conduct a sample processing sequence, prior to a sample analysis sequence; a high voltage power supply; a computerized controlling system for commanding the injection system, flow of fluids through the separation capillary, and operation of the at least one fluorescence detector; the apparatus being configured to conduct the sample analysis sequence after to conducting the sample processing sequence, wherein the sample analysis sequence comprises introduction of a sample and a background electrolyte and applying a voltage potential across the at least one separation capillary to effect separation of the compounds.

2. The apparatus according to claim 1, wherein the apparatus comprises at least one contactless conductivity detector.

3. The apparatus according to claim 1, wherein the apparatus comprises multiple fluorescence and contactless conductivity detectors.

4. The apparatus according to claim 1, wherein the apparatus comprises a temperature control system for temperature stabilization in a capillary chamber.

5. An apparatus for the separation and determination of regulated compounds in biological fluid sample using electrophoresis and comprising at least one separation capillary; at least one fluorescence detector for characterizing electrophoretic zones of compounds passing through a detection zone of the at least one separation capillary; an injection system for introducing fluids, including sample solutions and background electrolyte into an inlet end of the at least one separation capillary, to conduct a sample processing sequence prior to a sample analysis sequence; a high voltage power supply; a computerized system for commanding the injection system, flow of fluids through the at least one separation capillary, and operation of the at least one fluorescence detector; a sample preparation and extraction device for processing a biological fluid sample, the device comprising a compartment for swab/pad/tampon comprising the biological fluid sample, vials for collecting excess oral fluid and sample solution, solenoid valves for controlling transport of the biological fluid sample through the device vessel containing extractant, a computerized controlling system for commanding the solenoid valves that facilitate flow of fluids through the separation channel, to conduct the sample processing sequence; the apparatus being configured to conduct the sample analysis sequence after conducting the sample processing sequence, wherein the sample analysis sequence comprises introduction of a sample and a background electrolyte and applying a voltage potential across the at least one separation capillary to effect separation of the compounds.

6. The apparatus according to claim 5, wherein the apparatus comprises at least one contactless conductivity detector.

7. The apparatus according to claim 5, wherein the apparatus comprises multiple fluorescence detectors.

8. The apparatus according to claim 5, wherein the apparatus comprises multiple contactless conductivity detectors.

9. The apparatus according to claim 5, wherein the apparatus comprises multiple fluorescence and contactless conductivity detectors.

10. The apparatus according to claim 5, wherein the apparatus comprises a temperature control system for temperature stabilization in a capillary chamber.

11. The apparatus according to claim 5 for detection of natively fluorescing compounds in a wavelength range from 220 nm to 600 nm, the compounds being selected from a group consisting of amphetamine, methamphetamine, MDMA (ecstasy), MDEA, MDA, cocaine, cocaethylene, fentanyl, heroin, morphine, LSD, psilocybin, MDPV, CPP, cannabinoids, BZP, TFMPP phenolic compounds, BTEX, and naphthalene derivatives.

12. The apparatus according to claim 5, wherein the biological sample is oral fluid, exhaled breath condensate, tears, hair, sweat, urine or blood sample.

13. The apparatus according to claim 5, wherein the biological sample is oral fluid, exhaled breath condensate, tears, hair, sweat, urine or blood sample.

14. A method for preparation of a biological sample for separation and determination of regulated compounds with the apparatus of claim 5, the method comprising the steps of: rinsing of a subject's mouth with 2-5 mL of mouth rinsing solution, physiological saline solution or deionized water for 30-60 seconds; introducing a mixture of oral fluid and mouth rinsing solution into a collection compartment and introducing a swab/pad/tampon into the compartment to be in contact with the mixture; introducing the tampon/swab/pad comprising the mixture into a vacuum container; applying extraction solvent to the tampon/swab/pad to extract compounds of interest; directing the extract to a sample vial through a solid phase extraction filter comprising unbound silica for removal of interfering peptides and proteins; and introducing the sample to apparatus of claim 5 with help of a peristaltic micropump.

15. The apparatus according to claim 5 wherein in the injection system the background electrolyte is water-based or based on a mixed aqueous-alcoholic solution including mineral acids organic acids.

16. The apparatus of claim 15 wherein the apparatus is suitable for determination of compounds selected from the group consisting of AMP, METH, MDMA, MDA, MDEA, PMA, and PMMA, cocaine and its metabolite cocaethylene, fentanyl, LSD, metoprolol, and morphine.

17. The apparatus according to claim 5, wherein the apparatus is configured to separate cannaboids by use of non-aqueous capillary electrophoresis (NACE) and wherein suitable organic solvents used for capillary electrophoresis-have high relative permittivity, so that the number density of charge carriers is given directly by the nominal concentration of the electrolyte, and such solvents include acetonitrile and methanol in which the analytes are dissociated.

18. The apparatus according to claim 17, wherein the background electrolyte comprises strong bases selected from sodium or potassium hydroxide dissolved in mixture of organic solvents.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0082] The invention is described in detail with references to the drawings where in

[0083] FIG. 1 is illustrated generally a functional schematic of the instrument;

[0084] FIG. 2 is top view to the assembly of the apparatus according to present invention;

[0085] FIG. 3 is a cross section view along the line A-A in FIG. 2 to illustrate main details of the sample carousel according to embodiment of invention;

[0086] FIG. 4 is a cross section view along the line B-B in FIG. 2 to illustrate sample vial, separation capillary, capillary outlet and outlet electrode according to embodiment of invention;

[0087] FIG. 5 is a cross section view along the line C-C in FIG. 4 to illustrate a schematic of the fluorescence detector according to embodiment of the invention;

[0088] FIG. 6 illustrates the functional schematic of the device control according to invention;

[0089] FIG. 7 is an example electropherogram of illegal drug standards for first method according to invention;

[0090] FIG. 8 is an electropherograms obtained for second method according to invention;

[0091] FIG. 9 illustrates a typical electropherograms of suspects' oral fluid samples.

DESCRIPTION OF EMBODIMENTS

[0092] The general concept of an apparatus according to the invention is illustrated in FIG. 1, where items 1 to 8 correspond to the analyzer sampler comprising a first stepper motor 1 controlling height of the lift of the vial, a vial lift 2, a second stepper motor 3, controlling the position of the sampler carousel, a stand 4 for the inlet electrode 8 and capillary 7 through the inlet electrode 8, an inlet vial 5, a sampler carousel 6, the separation capillary 7, the inlet electrode 8. The outlet part of the analyzer comprises outlet electrode 11, stand 9 for the outlet electrode 11 and capillary 7 passing through the electrode 11. The stand 9 is connected to BGE replenishment and rinsing system 24 and outlet vial 13. The first channel 12 connects the outlet vial 13 with the vacuum pump (not shown in drawings). The capillary 7 passes through fluorescence or impedance detector 10.

[0093] Sample extractor part comprises an extract vial 14, a syringe 15 for tampon/swab with collected sample of interest, a solenoid valve 16 for extra saliva removal, a solenoid valve 17 for directing extracted sample to a sample vial 21, a second channel 18 to vacuum pump (not shown in drawings), mentioned above second channel 18 connects a vial for extra saliva collection 20 to vacuum pump (not shown in drawings), a solid phase extractor 19, the vial for extra saliva collection 20, a sample vial 21, third channel 22 connecting to the sample vial 21 to vacuum pump (not shown in drawings), a micro peristaltic pump 23 connected via inlet conduit 23.1 to sample vial 21 and via outlet conduit 23.2 to inlet vial 5 for directing sample from sample vial 21 to inlet vial 5.

[0094] The assembly of the apparatus according to present invention (FIG. 2) comprises fluorescence detector 10 attached to the support frame 121 of the analyzer, a sampler carousel 6 with vial adapter 5.1 with vial 5, base of carousel 6.1, lift mechanism 2, cooling system using Peltier elements 25.

[0095] The sample carousel (FIG. 3) according to embodiment of invention comprises the first stepper motor 1 for lifting vial 5. The stepper motor 1 is connected via shaft 1.1, connecting sleeve 1.2 and connecting plate 1.3 to the vial lift 2. The vial lift 2 comprises head of the lifting mechanism 2.2, which connects to the vial 5 located in vial adapter 5.1 to rise it to the level enabling electrode 8 with separation capillary 7 to be drawn into conditioning liquid 5.2 in vial 5. In addition, the lifting mechanism comprises supporting rods 2.1 for the lift stepper motor, linear guides 2.4 providing smooth vertical movement of the vial lift 2, Vial remover of lifting mechanism 2.3 removes the vial 5 from inlet electrode 8, keeping the vial against head of lifting mechanism 2.2. Inlet electrode 8 is mounted on the stand for the inlet electrode 4 and stand base 4.1. The separation capillary 7 (see drawing FIG. 4) is guided through capillary chamber 119 by capillary guides 7.1, 7.2, 7.3, 7.4, where first and last capillary guide 7.1 and 7.4 are attached to the housing of the capillary chamber 120 by connection elements 7.5. The capillary chamber 119 is closed with housing of the capillary chamber 120. The capillary chamber and fluorescence detector 10 are attached to the support frame 121 of the analyzer (shown in FIG. 5).

[0096] In FIG. 5 is illustrated schematically fluorescence detector part of the apparatus according to invention, where a fluorescence detector 10 comprises a xenon lamp 101, aspherical collimator lens 102, excitation filters 103, excitation focusing lens 104, separation capillary 7, aspherical emission collecting lens 106, emission filters 107, a photomultiplier tube 108, emission focusing lens 109, a first neutral filter 110, a beam splitter 111, reference beam focusing lens 112, a second neutral filter 113, a reference photodiode 114.

[0097] The apparatus according to invention is controlled by computer (personal computer) via conventional connecting means (bluetooth, wi-fi, cable etc.) where in FIG. 6 is showed the functional schematic of the device control according to invention where 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.

[0098] In FIG. 7 is an example electropherogram of illegal drug standards for first method according to invention. It is suitable for determination of amphetamine type stimulants and other common narcotics. Peak numbers in the figure correspond to the following Identified compounds: 1 and 10—internal standards, 2—AMP, 3—tyramine, 4—METH, 5—MDA, 6—MDMA, 7—MDEA, 8—cocaine, 9—cocaethylene (cocaine metabolite), 11—metoprolol (simulant of LSD) and 12—fentanyl.

[0099] Conditions: uncoated, fused-silica capillaries, i.d. 75 μm were used for the analyses. Fluorescence detector was positioned 35 cm to capillary end with total length of 51 cm. Prior to injection, the capillary was rinsed sequentially with 0.1 M NaOH, deionized water and the BGE for 2 min each. Separations were performed at +20 kV. Before the measurements, new capillaries were conditioned by rinsing them sequentially with 1 M sodium hydroxide and deionized water. Between analyses, the capillaries were rinsed with the BGE solution for 2 min.

[0100] In FIG. 8 is an electropherograms obtained for second method according to invention. Capillary conditioning procedures are the same as described for FIG. 4. BGE2 was applied for analysis of cannabinoids. Peaks, 1—electroosmotic flow, 2—THC, 5—CBD, 4—internal standard.

[0101] In FIG. 9 is illustrated a typical electropherograms 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 1 and 4—internal standards. 2—AMP and 3—tyramine (the compound is associated with smoking and some foods).

[0102] Portable CE Instrument.

[0103] The instrument consisted of a sample preparation unit (FIG. 1, elements 14-23) or without it (if manual sample preparation is performed), autosampler carousel (FIG. 1, element 6), separation capillary (FIG. 1, element 7), and detector (FIG. 1, element 10). During the analysis the electrodes (FIG. 1, element 8 and 11) are powered by high voltage power supply (not shown in drawings) and detector signal is recorded by built in computer. Built-in computer sends control signals to control board, which in turn controls stepper motors (FIG. 1, element 1 and 3), solenoid valves (FIG. 1, element 16, 17) and switches on/off the vacuum pump or pumps (not shown in drawings).

[0104] Sample Extraction Unit

[0105] The present device with methods can operate with automatic sample extraction unit (FIG. 1, elements 14-23) or without it. The work of the sample extraction unit is controlled by a built-in computer. Built-in computer sends control signals to control board, which in turn delivers commands to the solenoid valves and vacuum pump or pumps. Tampon/swab with suspect's oral fluid is placed into syringe 15 which is then sealed. Solenoid valve 16, which is initially in OFF position, allows excess saliva to be delivered into excess saliva vial 20 when the experiment starts after switching on the vacuum pump. Vacuum pump creates low pressure in the excess saliva vial 20. This facilitates removal of the superfluous saliva removed from the tampon/swab and retaining constant amount of sample into tampon/swab. After preset interval of time, the solenoid valve 16 is set to the ON position and because solenoid valve 17 is initially in OFF position the extractant in the vial 14 flows to the syringe 15 which retains low pressure. Sample is extracted from the tampon/swab, which is in the syringe 15, and during extraction atmospheric pressure establishes in the syringe 15. After preset time the solenoid valve is set ON position, which facilitates flow of extracted sample into sample vial 21 through the filter as solid phase extractor 19 due to the low pressure which has established there through third channel 22. Filter in solid phase extractor 19 removes peptides and proteins from the sample. When the transport of the sample to the sample vial 21 has been completed, the solenoids 16 and 17 are set to OFF position and vacuum pump is switched off. By initiating the work of the peristaltic pump 23 the sample is transported via inlet conduit 23.1 and outlet conduit 23.2 to the input vial 5 in the sampler carousel 6.

[0106] Carousel Autosampler

[0107] The work of the carousel autosampler unit (FIG. 1) is controlled by a built: in computer. Built-in computer sends control signals to control board, which in turn controls the lift stepper motor 1 and brushless DC motor 3 of carousel 6 and the vacuum pump (not shown in the drawings). Some of the input vials 5 in the carousel autosampler 6 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. [0108] 1. Experiment starts by moving the vial 5 containing capillary conditioning liquid to the position under the electrode inlet 8 located in the stand 4. With the help of the vial lift 2 the stepper motor 1 rises the inlet vial 5 to the level which enables electrode 8 with separation capillary 7 to be drawn into conditioning liquid. The vacuum pump is switched ON and it creates low pressure in the output vial 13 through first channel 12. Due to the low pressure at the end of the separation capillary 7 the condition liquid flows through the capillary washing out impurities at the capillary inner wall and establishing permanent coverage of its inner surface with hydroxyl groups. The low pressure is set for the period, which enables to flow through the capillary amount of liquid equal to several volumes of inner volume of the capillary. [0109] 2. The procedure described in the p1 is repeated for the vial with BGE. [0110] 3. The procedure described in p1 is repeated for the vial, containing sample. However, now the capillary is only partially filled with sample at the inlet end. [0111] 4. High voltage is delivered to the inlet electrode 8 and outlet electrode 11. [0112] 5. During the electrophoresis run analytes pass before the detector window and are recorded by fluorescent detector 10. [0113] 6. After preset time the high voltage is switched off and all controls are reset. [0114] 7. For the following samples all the procedures 1-6 are repeated.

[0115] Fluorescence Detector.

[0116] Fluorescence detector 10 is shown in FIG. 5. The xenon flash lamp 101 in Xenon lamp housing 116 delivers 0.5 μs light pulses to the detection window of the separation capillary 7 at a repetition frequency of 300-700 Hz. An aspherical lens 102 is used to collect the excitation light and a spherical lens 104 to focus the light to the capillary 7 with high efficiency. The xenon flash lamp has strong emission bands in the region of 230-260 nm, but it emits also in the broad-spectrum range until near infrared. Therefore, a set of three bandpass filters or excitation filters 103 is used to block emission outside that region. Radiation emitted by the solution inside the capillary 7 is collected by an aspherical emission collecting lens 106 and focused by an emission focusing lens 109 on the cathode of the PMT 108, which is located perpendicularity to the excitation beam but at angle approximately of 55 degree to the capillary 7. This angle is introduced to minimize the intensity of the refracted and reflected in the capillary parasitic radiation from the Xenon lamp 101. Doubled emission filters 107 or first neutral filters 110 are mounted within 280-600 nm wavelength range for detection of analytes of interest, 280-340 nm wavelength range is useful for illegal drugs native fluorescence detection. An optical reference channel is introduced to eliminate the xenon lamp 101 aging effect on measurement accuracy. A beam splitter 111 reflects a part of the excitation beam and directs it through a reference beam focusing lens 112 to the reference photodetector or reference photodiode 114. A second neutral filter 113 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.

[0117] Contactless Conductivity Detector

[0118] The fluorescence detector 10 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 8 mm and a gap of 0.8 mm, 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 (60 V or different) peak-to-peak sine wave oscillating in a frequency range of 300 kHz-2 MHz (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.

[0119] Background Electrolytes for Separation

[0120] A first method according to invention uses BGE1 which consisted of 95% (20 mM tris(hydroxymethyl) methylamine, 50 mM phosphoric acid, 0.4% triethylamine, pH 3.3) and 5% methanol as an organic modifier. Method 1 was used for separation of common narcotics (except THC and CBD). Example of separation is presented in the FIG. 7.

[0121] A second method according to invention implements nonaqueous capillary electrophoresis (NACE). It was used for the separation of THC and CBD cannabinoids. BGE2 consisted of 2.5 mM NaOH dissolved in MeOH/ACN (1:1) at pH=12. Example of separation is presented in the FIG. 8.

[0122] The background electrolyte composition is not limited to the compounds mentioned in method 1 and method 2.

EXAMPLES

[0123] To test the feasibility of the invention a prototype of the instrument was build. Details of the prototype are presented in FIG. 2-FIG. 5. The present invention will be first described by the following examples. These examples are provided to illustrate one mode for practicing the present invention and are not to be construed as limiting the scope of the invention as defined by the appended claims.

[0124] Performance Characteristics of Roadside Analyzer of Illegal Drugs

[0125] 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 200-265 nm, not limiting to lower wavelength range up to 600 nm. 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.

[0126] 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 3:1, proving the presence of the analyte in the test sample with a probability larger than 99%. The S/N ratio for IQL level was set to 10:1, 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.

[0127] Analysis of Oral Fluid Samples.

[0128] The assembly of the apparatus according to present invention is utilized for the determination of illegal drugs of abuse in OF during police roadside drug testing and at various public events (for example, music festivals). FIG. 9 presents the OF sample with drugs intoxication evidence.

[0129] List of Details [0130] 1—stepper motor controlling height of the lift of the vial [0131] 1.1—shaft of the stepper motor [0132] 1.2—connecting sleeve [0133] 1.3—connecting plate [0134] 2—vial lift [0135] 2.1—supporting rod for lifting mechanism [0136] 2.2—head of lifting mechanism [0137] 2.3—vial remover of lifting mechanism [0138] 2.4—linear guides of lifting mechanism [0139] 3—brushless DC motor motor controlling the position of sampler carousel [0140] 3.1—carousel position feedback magnet [0141] 4—stand for the inlet electrode [0142] 4.1—base of the stand [0143] 5—inlet vial [0144] 5.1—vial adapter [0145] 5.2—sample [0146] 6—sampler carousel [0147] 6.1—base of carousel [0148] 7—separation capillary or set of capillaries [0149] 7.1, 7.2, 7.3, 7.4—capillary guide [0150] 7.5—connection element for capillary guide [0151] 8—inlet electrode [0152] 9—stand for the outlet electrode and capillary through the electrode [0153] 10—fluorescence detector [0154] 101—Xe lamp [0155] 102—aspherical collimator lens [0156] 103—excitation filters [0157] 104—excitation focusing lens [0158] 106—aspherical emission collecting lens [0159] 107—emission filters [0160] 108—photomultiplier tube (PMT) [0161] 109—emission focusing lens [0162] 110—first neutral filter [0163] 111—beam splitter [0164] 112—reference beam focusing lens [0165] 113—second neutral filter [0166] 114—reference photo diode [0167] 115—housing of the detector [0168] 116—housing of Xenon lamp [0169] 117—cover of the detector housing [0170] 118—circuit board for Xenon lamp, PMT, photo detector and other electronics [0171] 119—capillary chamber [0172] 120—cover of the capillary chamber [0173] 121—support frame of analyzer [0174] 11—capillary outlet and outlet electrode [0175] 11.1—capillary guide in outlet electrode [0176] 11.2—outlet chip [0177] 11.3—tube fitting [0178] 12—first channel to vacuum pump [0179] 13—outlet vial [0180] 14—extract vial [0181] 15—syringe for tampon/swab with collected sample of interest [0182] 16—first solenoid valve for extra saliva removal [0183] 17—second solenoid valve for directing extracted sample [0184] 18—second channel to vacuum pump [0185] 19—solid phase extractor [0186] 20—vial for extra saliva collection [0187] 21—sample vial [0188] 22—third channel to vacuum pump [0189] 23—micro peristaltic pump [0190] 23.1—inlet conduit from sample vial to peristaltic pump 23 [0191] 23.2—outlet conduit from peristaltic pump to sample vial 5 [0192] 24—BGE replenishment and rinsing system [0193] 25—cooling system using Peltier elements