QUANTITATIVE HPTLC CANNABINOID FIELD TESTING DEVICE AND METHOD

Abstract

A field testing device is provided for quantitating components of marijuana such as THC, THC-A, CBD, CBDA, and/or CBN. Quantitation may be made, e.g. from biological fluids such as saliva, or from plant extracts. A device according to the invention may include an HPTLC plate for spatially separating interferents and analytes, and may also include fluorometric components for quantitating analytes. The device may include a microprocessor adapted to relate fluorescent intensity to analyte concentration through one or more calibration curves. Devices may optionally include microfluidics for carrying out HPTLC on biological samples including sample reservoirs, reagent reservoirs, micro-pumps, mixers, and the like.

Claims

1. A cannabinoid quantitation device, comprising: a cartridge including: a sample receptacle adapted to receive a volume of liquid sample; a micropump having an intake in fluid communication with the sample receptacle; a development chamber adapted to contain a thin layer chromatography mobile phase; a stationary phase disposed within the development chamber and suitable for conducting thin layer chromatography; a sample deposit area of the stationary phase in fluid communication with an output of the micropump; a mobile phase reservoir adapted to contain a thin layer chromatography mobile phase; and a casing combining, into the form of a cartridge, the sample receptacle, the micropump, the development chamber, the stationary phase, the sample deposit area, and the mobile phase reservoir; an excitation source emitting light suitable for measurably exciting electrons in a cannabinoid UV absorption band, the excitation source being in optical communication with a stationary phase of the cartridge, wherein the excitation source comprises an ultraviolet light emitting diode having operably sufficient spectral output between 310 nm and 390 nm to quantitate the cannabinoid; an emission detection component operatively sensitive to cannabinoid emission resulting from relaxation of the excited electrons, the emission detection component being in optical communication with the stationary phase of the cartridge; and a microprocessor adapted to receive spectral data collected by the emission detection component and calculate a cannabinoid concentration from predefined calibration curves.

2. The cannabinoid quantitation device of claim 1, wherein the excitation source comprises an ultraviolet light emitting diode having operably sufficient spectral output between 365 nm and 385 nm to quantitate the cannabinoid.

3. The cannabinoid quantitation device of claim 2, wherein the excitation source simultaneously illuminates all analyte spots on the stationary phase.

4. The cannabinoid quantitation device of claim 1, wherein the emission detection component is an image-forming device operably sensitive to light between 400 nm and 450 nm to quantitate the cannabinoid.

5. The cannabinoid quantitation device of claim 4, wherein light impinging the emission detection component is filtered to exclude light from the excitation source and pass light emitted by analytes.

6. The cannabinoid quantitation device of claim 1, wherein the excitation source serially illuminates analyte spots.

7. The cannabinoid quantitation device of claim 6, wherein the excitation source comprises an ultraviolet LED laser having operably sufficient spectral output between 310 nm and 390 nm to quantitate the cannabinoid.

8. The cannabinoid quantitation device of claim 6, wherein the excitation source comprises a non-laser collimated ultraviolet LED having operably sufficient spectral output between 310 nm and 390 nm to quantitate the cannabinoid.

9. The cannabinoid quantitation device of claim 6, further comprising a moveable mask adapted to expose analyte spots serially to light from a non-laser un-collimated ultraviolet LED having operably sufficient spectral output between 310 nm and 390 nm to quantitate the cannabinoid.

10. The cannabinoid quantitation device of claim 6, wherein the emission detection component is a non-image-forming device operably sensitive to light between 400 nm and 450 nm to quantitate the cannabinoid.

11. The cannabinoid quantitation device of claim 1 further comprising an imaging chamber adapted to exclude ambient light and adapted to support the cartridge, the excitation source, and the emission detection component in an optically aligned relation to each other.

12. The cannabinoid quantitation device of claim 1, wherein the stationary phase comprises a reverse phase medium.

13. The cannabinoid quantitation device of claim 12, wherein the mobile phase comprises methanol at nine parts by volume, water at one part by volume, and acetic acid 0.3 parts by volume.

14. The cannabinoid quantitation device of claim 1, wherein the stationary phase comprises a normal phase medium.

15. The cannabinoid quantitation device of claim 14, wherein the mobile phase comprises one part chloroform to one part hexane by volume.

16. A method for quantitating cannabinoids comprising the steps of: providing a quantitation device according to claim 1; applying a sample mixture to a sample deposit area of the quantitation device; actuating a micropump of the quantitation device causing an analytically effective amount of the sample mixture to contact a stationary phase of the quantitation device; causing a mobile phase to flow from a mobile phase reservoir of the quantitation device to the stationary phase and contact the stationary phase; waiting for a solvent front of the mobile phase to move a predetermined distance on the stationary phase; exciting a cannabinoid analyte of the sample mixture with an excitation source of the quantitation device; detecting emission of the cannabinoid analyte with an emission detection component of the quantitation device; and calculating a cannabinoid concentration from predefined calibration curves of the quantitation device.

17. The method for quantitating cannabinoids of claim 16, wherein the sample mixture comprises oral fluid.

18. The method for quantitating cannabinoids of claim 16, wherein the sample mixture comprises plant extract.

Description

III. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The invention may take physical form in certain parts and arrangement of parts, embodiments of which will be described in detail in this specification and illustrated in the accompanying drawings which form a part hereof, wherein like reference numerals indicate like structure, and wherein:

[0034] FIG. 1A is a pair of graphs illustrating the linearity of THC and CBD fluorescence with respect to concentration when excited at 240 nm;

[0035] FIG. 1B is a graph showing the linearity of HPTLC/Fluorescence measurements according to an embodiment of the invention;

[0036] FIG. 1C is a photograph of an HPTLC plate illustrating an embodiment's capacity to separate an important interferent from THC;

[0037] FIG. 2A is a plan view of a lab-on-chip microfluidic device according to one embodiment of the invention;

[0038] FIG. 2B is a side cross sectional view of the device shown in FIG. 2A taken along line 2B-2B;

[0039] FIG. 3 illustrates an embodiment separating fluorescent analytes by high performance thin layer chromatography (HPTLC) while showing the relative positions of an excitation light source and detector-side optics including a long-pass filter;

[0040] FIG. 4 illustrates an embodiment showing a fluorescent background embodiment where the analytes are either non-emitting or their emission can be neglected without the need for filter optics;

[0041] FIG. 5 illustrates a transmission mode embodiment; and

[0042] FIG. 6 illustrates an embodiment TLC plate with marks showing imaging landmarks.

IV. DETAILED DESCRIPTION OF THE INVENTION

[0043] All drawings and figures contained herein are for purposes of illustrating embodiments of the invention only and not for purposes of limiting the same.

[0044] Headings are used herein for convenience purposes only. Paragraphs under differing headings may include similar or even overlapping teachings. Accordingly, headings are not meant to limit the invention and should not be used to construe the meaning of teachings thereunder.

[0045] As used herein the terms embodiment, embodiments, some embodiments, other embodiments and so on are not exclusive of one another. Except where there is an explicit statement to the contrary, all descriptions of the features and elements of the various embodiments disclosed herein may be combined in all operable combinations thereof.

[0046] Language used herein to describe process steps may include words such as then which suggest an order of operations; however, one skilled in the art will appreciate that the use of such terms is often a matter of convenience and does not necessarily limit the process being described to a particular order of steps.

[0047] Conjunctions and combinations of conjunctions (e.g. and/or) are used herein when reciting elements and characteristics of embodiments; however, unless specifically stated to the contrary or required by context, and, or and and/or are interchangeable and do not necessarily require every element of a list or only one element of a list to the exclusion of others.

[0048] Except where otherwise required by context, the word may and the phrase may be are used herein to indicate that a particular act or structure is within the scope of the present invention, is consistent with one or more elements of the invention, and/or supports patentability of the claimed invention. With particular regard to acts, statements indicating that an act may be performed are used herein to indicate that carrying out the act is expected to produce results supporting patentability of the invention claimed herein based on experimental data and/or the inventor's experience and expertise in the art. The word may and the phrase may be do not mean that the indicated act was actually carried out, nor should it be construed to suggest that the act was not carried out, or that the applicant is unsure whether the act or structure is or is not within the scope of the invention, or that the applicant is unsure whether the recited act or structure is permissible, possible, or practical.

[0049] Where the word fluorescent is used herein, its use is not meant to exclude phosphorescence or bioluminescence where phosphorescent or bioluminescent alternatives are available to the person having ordinary skill in the art. Such variations are contemplated to be within the scope of the present invention.

[0050] All solvents and chemicals are of analytical grade. Drug standards of .sup.9-tetrahydrocannabinol (THC), CBD, CBN, and THC-COOH may be purchased from Cerilliant (Round Rock, Tex., USA). Cannabinoid solutions may be prepared in methanol and diluted to required concentrations. All drug compounds may be stored at 20 C. Saliva, urine, and blood from volunteer donors may be used to show viability of quantifying THC in bodily fluids. Comparison of results with certified reference standards may be used to assure the identity of the THC analyte. The potential interferents commonly found in oral fluid such as food particles, tobacco, caffeine and other drugs of abuse may be considered and included in test samples at appropriate levels during the validation procedure to assure that results are unaffected.

[0051] Calibration solutions may be prepared using certified reference solutions of THC, CBD, CBN and 11-nor-9-carboxy-9-THC; each of 1 mg/mL in methanol, may be purchased from Cerilliant Corporation (USA). Individual calibration solutions of THC may be prepared and tested as well as solutions with mixtures of the other cannabinoids commonly found in oral fluid using methanol as the diluent. Fresh negative oral fluid samples spiked with purchased standards may be tested. Self-identified, declassified, drug-positive oral fluid samples from volunteers may be tested. Method validation may be performed with pure cannabinoid standards. Method performance was evaluated using spiked oral fluid as well as native drug-positive samples.

[0052] Sample preparation and pre-conditioning, is desirable or even necessary in some pre-existing analytical tests; however, embodiments of the present invention may use raw or neat oral fluid with little or no pre-purification or preparation. Proceeding without preparative steps may increase the speed of carrying out sample analyses, and may decrease the apparent complexity from the point of view of the user. However, embodiments may include preparative steps after the user introduces a sample. For instance, automated microfluidics may be preconfigured to carryout preparative steps that are unseen by the user.

[0053] Linearity of Cannabinoid Concentration Versus Florescent Intensity

[0054] .sup.9-Tetrahydrocannabinol (THC) and cannabidiol (CBD) are demonstrated to have a linear relationship between concentration and fluorescent intensity over an analytically useful range for quantifying intoxicating drug levels. Standard solutions of 1 mg/mL in methanol each may be purchased from Cerilliant Corporation (USA) and diluted to make calibration standards. The stock solutions may be stored at 20 C. Calibration solutions of 0, 5, 20, 50, 100 and 200 ng/mL may be prepared using methanol as the diluent. This range encompasses the current legal standards for intoxication (5 ng/mL-20 ng/mL). Fluorescence emission spectral measurements at an excitation range from 230 nm to 400 nm may be taken three times with three replicates at each concentration using a Perkins Elmer LS50B Luminescence Spectrometer. Solutions may be mixed for 30 seconds before each measurement is taken to assure homogeneity. The results displayed in FIG. 1A illustrate liquid phase measurements of the fluorescence emission intensity versus sample concentration using an excitation of 240 nm. Standards may be spotted on an embodiment, dried, and then measured according to fluorescent methods described herein to establish a calibration curve for quantitating THC or other compounds of interest.

[0055] The data shown in FIG. 1A illustrates that fluorescence may be used to measure the concentration of THC and CBD in solution with acceptable linearity and sensitivity in concentration ranges suitable for assessing intoxication, i.e. 5-100 ng/mL. Furthermore, the data illustrate that an off-peak excitation of 240 nm, as opposed to 230 nm (on-peak), can be used as the excitation frequency, which permits the use of a commercially available and relatively low-cost UV-LED source.

[0056] Measurements similar to those shown in FIG. 1A may be made using oral fluid rather than methanol. Oral fluid was shown to have a matrix effect caused by light scattering. Thus, liquid phase fluorescence measurements of such analytical samples require a correction factor that is expected to vary from one oral fluid to another. However, no such correction factor is necessary for measurements made with embodiments of the invention comprising TLC separation devices and methods.

[0057] FIG. 1B illustrates the linearity of detection and measurement methods of the invention. Particularly, THC concentration in oral fluid is determined by HPTLC in combination with fluorescence intensity measurements. Excitation wavelength was 366 nm. The HPTLC method is linear between 0.2 ng/spot and 30 ng/spot. This range includes THC concentrations expected in oral fluid after smoking marijuana.

[0058] FIG. 1C is a photograph of an HPTLC plate illustrating adequate separation of THC and CBD according to methods of the invention taught herein. The plate is visualized with an aqueous solution of 0.1% 4-Benzoylamino-2,5-dimethoxyaniline, Azoic Diazo No. 24. (also referred to herein as Fast Blue) THC and CBD standards are shown having been run against mixtures of CBD and THC.

[0059] Chromatography

[0060] It is demonstrated that HPTLC is useful for quantitating THC in mixtures of THC and CBD. Samples and standards may be spotted on at least three types of commercially available plates: Si60 HPTLC glass plates, Si60 HPTLC glass plates coated with fluorescent indicator F254, Si60 UTLC glass plates, and C-18 HPTLC plates coated with fluorescent indicator F254. Application of 5 L samples to the plates may be carried out using a capillary liquid dispensing system such as, and without limitation, CAMAG Nanomat 4. Advantageously, samples may be applied 1 cm from the bottom of the plate and 2 cm from the sides. Sample volumes of 100 L to 400 L may be used to increase the concentration of cannabinoids available for spot detection leading to increased sensitivity.

[0061] Plates may be developed in a twin trough chamber and/or a horizontal development chamber such as, and without limitation, CAMAG Horizontal Developing Chamber 2, without vapor saturation or humidity control. One developing solvent system within the scope of the invention used for normal phase plates is heptane/diethyl ether/formic acid (or acetic acid); 75/25/3 drops, v/v/v. Another developing solvent system within the scope of the invention but used for reverse phase plates is methanol/DI water; 80/20, v/v. The developing distance for the normal phase plates is 5 cm from the spot application position and which occurs in approximately eight minutes. The developing distance for the reverse phase plates is 3.5 cm and occurs in approximately four minutes. Results such as these are consistent with environmental conditions in the lab of around 16-17 C. and relative humidity <50%. The person having ordinary skill in the art will readily understand that a variety of solvent systems humidity and temperature conditions may be suitable and it would be within the skill of the art to make such a selection.

[0062] The developed plates are dried with a stream of warm air for approximately three minutes to remove residual solvents. See Table 1 for various derivatizing reagents and conditions used to enhance fluorescence. Results are observed under UV 254, UV 366 and visual light.

TABLE-US-00001 TABLE 1 TLC Experiment Conditions for Derivatization Reagents for Enhanced Visualization. Sample Tested Derivatization Regent Preparation, use 1 mg/mL THC in Methanol Diphenylboric Acid 2-Amino Dipping & dry for 5 min. with Ethyl Ester hair dryer 1 mg/mL THC in Methanol Polyethylene Glycol 400 Dipping & dry for 5 min. with hair dryer 1 mg/mL ea. THC, CBD, CBN, Fast Blue Salt Dipping & dry for 5 min. with THC-COOH in Methanol- hair dryer concentration of 100 g/mL

[0063] Enhancing Sensitivity

[0064] Derivatizing THC and THC-COOH with dansyl chloride (5 Dimethylamino-naphthanlene-1-sulphonyl Chloride) enhances their fluorescent quantum yield and thus leads to improved sensitivity and limits of detection. Dansylation of samples may be achieved by adding 100 L NaOH to 1 mL of test fluid to adjust the sample pH to 10+ followed by adding 1 mg/mL of dansyl chloride solution (1 mg/mL in acetone). The samples may be heated for approximately three minutes at 60 C. and cooled at room temperature. Derivatized samples may be applied according to procedures disclosed herein for non-derivatized samples.

[0065] Device

[0066] A device for the roadside quantitation of THC in oral fluid or other bodily fluids may include HPTLC components to separate and isolate cannabinoids, and may include fluorescence detection components to measure the native fluorescence or enhanced fluorescence (e.g. dansylated, derivatized, or conjugated) of THC and/or THC derivatives.

[0067] Embodiments of the invention may comprise a device adapted to quantitatively identify psychoactive cannabinoids and/or metabolites thereof in biological samples including, without limitation, human oral fluid and plant tissue preparations. Devices according to embodiments of the invention may include a cartridge adapted to support a stationary phase suitable for thin layer chromatography (TLC), and may include microfluidics adapted to apply analytical samples to the stationary phase and contact the stationary phase with a suitable mobile phase. Cartridges according to some embodiments may include adaptations capable of receiving biological fluid samples, and/or contacting the biological fluid samples with analytical sample deposit areas of the stationary phase. Moreover, cartridges according embodiments of the invention may be adapted to cooperate with optical components thereof to fluorometrically quantitate analyte concentrations, e.g. in oral fluid.

[0068] Biological samples such as oral fluid may be introduced or applied to an embodiment without dilution or preparation, i.e. neat. However, diluting samples and/or adding reagents to samples may also be compatible with embodiments of the invention. Diluted samples may include buffers such as, without limitation, phosphate buffered saline (PBS) or physiological saline, or may include solvents such as, without limitation, methanol or water. Reagents may include one or more of derivatizing agents, luminescent tags or markers including luminescent and phosphorescent chemical species, antigens, bioluminescent chemical species, catalysts, enzymes, substrates, and the like. Moreover, reagents may include solid phase reagents as well as liquid phase reagents. Embodiments may include one or more reservoirs and microfluidic components for introducing diluents to a sample and/or mixing diluents into a sample.

[0069] Cannabinoids are known to have solution phase absorption peaks around 210-250 nm, and some particularly useful cannabinoids, including .sup.9-tetrahydrocannabinal (THC) and cannabidiolic acid (CBDA), have absorptions peaks between 220-240 nm with useful absorption bands up to 400 nm. More specifically, a particularly advantageous solution phase absorption peak is 230 nm with a corresponding emission peak wavelength of 305 nm. The skilled artisan will appreciate that the specific wavelength may shift by a few nm according to experimental conditions including solvent effects, pH, and temperature. Absorption and emission shifts are also seen in connection with stationary phase media. Accordingly, absorption bands may shift from 210-250 nm to about 340-390 nm, and emission may shift from about 305 nm to about 430 nm.

[0070] In view of the known absorption characteristics of cannabinoids, embodiments may include an appropriate ultraviolet source to excite cannabinoids, and a suitable optical arrangement may be applied to detect native fluorescent emission of cannabinoids. Embodiments may employ one of several optical arrangements including detecting emission at 90 degrees relative to the emission source using filters to remove excitation radiation while passing emission radiation, and using various photosensitive detectors to quantify emission signals. As used herein, detection at 90 degrees may include front-face detection modes including without limitation where the emitter and detector are 90 degrees from each other, but each is 45 degrees from the front face of the sample. Such a front face detection mode may be particularly advantageous for taking spectral measurements of TLC spots. Embodiments may also include structures for detecting emission in transmission mode, e.g. at 180 degrees relative to the emission source.

[0071] With regard to determining the degree of current intoxication by quantitating cannabinoids, oral fluid has been found to be particularly useful. Notably, both THC and CBD are found in oral fluid, but only THC is psychoactive.

[0072] Since THC and CBD have overlapping absorption and emission spectra, these compounds must be spatially separated in order to accurately quantitate THC. Spatial separation is achieved by embodiments of the invention through thin layer chromatographic features and adaptations.

[0073] FIG. 2A is a plan view diagram of a cartridge 100 according to one embodiment. The cartridge 100 includes a casing or enclosure 102 adapted to contain and/or comprise various fluidic and analytical adaptations. One such adaptation is a sample receptacle 104 adapted to receive a sample fluid such as oral fluid or plant extract. The sample receptacle 104 of this embodiment optionally includes a mixer 106. The receptacle 104 is in fluid communication with a peristaltic micropump 108 which is adapted to draw fluid from the sample receptacle 104 and distribute it to a plurality of analytical sample deposit areas 116 of a TLC stationary phase 114. The stationary phase 114 is disposed within a development chamber 112 which is in fluid communication with a mobile phase reservoir 110.

[0074] According to the embodiment shown in FIG. 2A, a sample fluid sample may be deposited in the receptacle 104 and a predetermined volume of the sample may be delivered to each of the plurality of analytical sample deposit areas 116. The mobile phase may then be released from the reservoir 110 and development may be allowed to proceed for a predetermined amount of time. The TLC plate may then be dried thus stopping separation, and fixing the analytes in place on the plate. The analytes may then be quantitated using spectroscopic methods of the invention.

[0075] FIG. 2B is a side view of the cartridge 100 taken along line 2B-2B, which is intended to illustrate that the components of this embodiment are contained within the casing 102, or are recessed. For instance, the TLC stationary phase 114 must optically communicate with components external to the cartridge 100 and thus cannot be enclosed within opaque walls; however, it may be advantageous to protect the stationary phase 114 from abrasion. Therefore, one suitable arrangement may include a recessed cavity containing the stationary phase 114. The recessed cavity may optionally include a cover 101 that may be retractable, removable, or optically clear to UV-Vis spectrum light. The cover may serve to retain solvent vapors during development, but permit optical communication during quantitation. It may be desirable to have a retractable or removable cover so that solvent and/or solvent vapors can be vented quickly to stop the separation at a well-defined predetermined time.

[0076] FIG. 3 illustrates an embodiment 200 that includes optical elements external to the cartridge 100 where a light source 210, long-pass filter 212 and detector 214 lie on an optical path forming a front-face detection arrangement. Arc .sub.1 illustrated in FIG. 3 corresponds to the angle between the surface of the stationary phase 114 and an incident ray 211i from the light source 210. Arc .sub.2 illustrated in FIG. 3 corresponds to the angle between the surface of stationary phase 114 and an emitted ray 211e traveling along a line of sight of the detector 214. Arc corresponds to the angle between the light source 210 and the detector 214 as defined by the incident ray 211i and the emitted ray 211e. The particular values of .sub.1, .sub.2, and are non-critical however embodiments may include setting to 90 degrees and setting both .sub.1 and .sub.2 to 45 degrees.

[0077] The cartridge 100 of the embodiment 200 includes three deposit areas 116 forming three lanes 216 so that samples may be run in triplicate. One skilled in the art will appreciate that the embodiment 200 would be enclosed so as to exclude ambient light, and that the detector 214 would be isolated from the light source 210 so that it only receives filtered light. Enclosures and optical isolation structures are omitted from FIG. 3 to more clearly show the relative position of the cartridge 100 and external optical components 210, 212, and 214; however, the skilled artisan will readily appreciate how such structures can be fashioned and incorporated without undue experimentation.

[0078] The light source 210 of FIG. 3 may emit at a wavelength coinciding with the absorption (i.e. excitation) bands of the various analytes. Light sources may include, for instance and without limitation, an ultraviolet light emitting diode (UV LED) having an emission peak between 350-390 nm, or otherwise having analytically useful emission intensity in the 350 nm to 390 nm wavelength range. Depending on the nature of the detector 214, the source 210 may illuminate all of the analyte spots at once, or it may illuminate them one at a time. More specifically, if the detector 214 is a charge coupled device (CCD), or other image-forming device such as a mobile phone camera, then the light source 210 can illuminate the entire stationary phase 114 at once. The CCD detector or other camera can be relied upon to relate light signals to particular analyte spots.

[0079] However, if the detector 214 is a photodiode or other non-image-forming light detection device then the light source 210 may illuminate one spot at a time so that the detected light may be attributed to a particular spot. Illumination of one analyte spot at a time may be accomplished with focusing and/or collimating optics, by using a UV diode laser as source 210, or by interposing a movable mask between the source 210 and the stationary phase 114. Furthermore, the photodiode detector may optionally include a long-pass filter 212 as an optical layer deposited on the photodiode.

[0080] With continuing regard to FIG. 3, the sample spots 220, 222, and 224 may emit at characteristic wavelengths, or the same wavelength, and a portion of the emission may pass through long-pass filter 212 which may remove excitation radiation from the optical path. The brightness of the spots 220, 222, and 224 is directly proportional to the amount of analyte present, and is thus relatable to the concentration of analyte in the original sample. A suitable filter may be used to remove light originating from the light source 210 while passing the cannabinoid emission peak. For instance and without limitation, in the case of cannabinoid quantitation, the light source may have an emission peak around 370 nm, and the analyte may have an emission peak centered around 430 nm. The filter 212 may be free-standing, or the filter 212 may be deposited on the detector 214 as an optical layer according to known methods. Depositing the filter as an optical layer may eliminate the need for otherwise isolating the detector 214 from the light source 210.

[0081] FIG. 4 illustrates an embodiment 300 where the native fluorescence of the analyte spots 220, 222, and 224 is neglected in favor of a fluorescent background. In this embodiment, the emission characteristics of the stationary phase itself, or of a compound bound to or otherwise impregnated into the stationary phase, are used to generate a fluorescent background. Advantageously, the background may absorb and/or emit at a wavelength different from that of the analyte spots. Where the analyte and background absorb at non-overlapping wavelengths the analyte spots present as dark spots against a fluorescent background. Moreover, the darkness of the spot is directly related to the amount of analyte present and therefore is relatable to analyte concentration in the original sample. Light sources suitable for such embodiments may emit advantageously around 254 nm, and since all of the emission is attributable to the background emission field no filter optics are necessary. The detector can comprise any image-forming detector such as a dedicated CCD, or an off-the-shelf cell phone or digital camera. The skilled artisan will appreciate that these detectors are merely exemplary and not limiting. Optical detection hardware within the scope of the invention includes cell phone cameras such as the Samsung-SM-G930V, CCD cameras such as TRAX UV, and CMOS (complementary metal-oxide semiconductor) sensors any or all of which may be integrated with an imaging chamber that may exclude ambient light and may also include structures for supporting optical components in an optically aligned relation relative to each other and/or the sample.

[0082] FIG. 5 illustrates a transmission mode embodiment. An embodiment cartridge 100 is shown interposed between a light source 210 and a detector 214. Excitation ray 510 is illustrated with a solid arrow being emitted from the source 210 and transmitting through an optically clear sample cover 101. Excitation ray 510 is incident upon a stationary phase 114 and possibly a sample. An emission ray is illustrated by dashed line 512 being emitted either from the stationary phase 114 as in embodiments operating according background emission field methods, or from a sample as in embodiments operating according to native or enhanced fluorescence methods. A long pass optical filter 514 is shown integrated into the back panel of the cartridge 100 to filter out excitation ray 510, while passing emission ray 512.

[0083] Imaging

[0084] Processing and quantitation of sample images may be carried out according to similar algorithms for both color (e.g. RGB) and monochrome images. In either case an image stacking procedure may be used whereby multiple images are averaged together to improve signal-to-noise ratio.

[0085] Each image includes one standard spot 621 of 1 mg/mL THC (or other cannabinoid) and one unknown sample spot 623 to be analyzed in a grid as shown in FIG. 6. The plate 600 is analyzed by drawing a horizontal line 614i marking the initial position of the sample 623 and standard spots 621. The ordinarily skilled artisan will readily appreciate that such lines and other markings described herein need not be literally drawn, but rather may be logically inferred especially in instrumental analysis. A mobile phase carries the standard along path 613a and the sample components along 613b. The centroid of the THC standard 620 is located, and a horizontal line 614f is drawn through the standard centroid 620. Any THC contained in the sample spot 623 will have a centroid 622 coinciding with that of the THC standard 620. The skilled artisan will readily appreciate that other components of the sample will have centroids at other locations along path 613b. An analysis area 612a for the THC standard spot is generated by using a predetermined threshold value for pixel intensity, a similar analysis area 612b is generated for the unknown sample. Three separate background readings are taken just outside of the analysis regions 612a, 612b around the samples 620, 622. The fluorescence due to analyte in the unknown sample is calculated according to the basic background substation formula: I.sub.analyte=Iintegrated density-[(Analysis Area)(I.sub.background)]. A similar value I.sub.standard for the standard may also be calculated according to the same method.

[0086] This intensity value is then compared to calibration curves for cannabinoids to obtain the concentration of the unknown sample. Thresholding and subtraction of background fluorescence may correct for non-uniform illumination and variations between individual analytical devices and plates. The use of cannabinoid standards along with the retardation factor (R.sub.f) value provide positive identification of the cannabinoid. This allows analysis of spot intensity on the sub-visual level, meaning that intensity that is too low for visual inspection can be detected by the image processing algorithm. As used herein R.sub.f is defined as distance traveled by sample 614f divided by distance traveled by solvent 614s.

[0087] Certain analytical metrics may be used to validate embodiments of the invention including an embodiment's ability to chromatographically separate THC from interferents, its ability to identify and quantify THC in unknown samples of body fluids such as oral fluid, as well as the accuracy, precision, linearity, specificity for THC, limits of detection, limits of quantitation, and range of measurement.

[0088] Calibration curves may be generated according to known methods to relate fluorescent intensity to THC analyte concentration. Calibration solutions may be prepared at 0-500 g/mL to establish the linear range. Quality control samples may be prepared from a different lot of certified reference standards and verified by GC-MS. All testing is done in triplicate at each concentration level. Calibration data may be subjected to a linear regression analysis to obtain a calibration curve and a linear correlation coefficient (R.sup.2).

[0089] The range of measurements that an embodiment may reliably and accurately obtain may be defined as the range of measurements that produce a linear response within a predefined acceptable limit of accuracy and/or precision. Devices suitable for detecting intoxication in human subjects may have a range encompassing legally defined intoxication levels. For instance, and without limitation, a range of approximately 2 ng/mL to 500 g/mL would be suitable.

[0090] Specificity of an embodiment may be established by comparing test results of adulterated samples having common impurities as well as known levels of THC, and THC concentration standards. Impurities may include, for instance, tobacco, alcohol, food products, and non-psychoactive cannabinoids such as CBD, CBN, and/or 11-nor-9-carboxy-9-THC. Embodiments reliably identify THC by its HPTLC R.sub.f value. Visualization and quantitation of sample and standard spots may be accomplished according to several different methods including by native fluorescence, enhanced fluorescence as previously described herein, as well as densitometric methods. Embodiments relying on densitometry for quantitation may derivatize the analyte so as cause it to absorb in the visible spectral range. For instance and without limitation, aqueous solutions of 0.1% 4-Benzoylamino-2,5-dimethoxyaniline, Azoic Diazo No. 24 (CAS Number 6268-05-9) is one such derivatizing agent.

[0091] The Limit of Detection (LOD) is the smallest amount of analyte that can be detected. This quantity may be established for an embodiment using the standard deviation of the fluorescence intensity response and the slope of the calibration curve according to equation 1, where a is the standard deviation of the fluorescence intensity response and m is the slope of the calibration curve.

[00001]$\begin{array}{cc}\mathrm{LOD}=\frac{3.3.Math..Math.}{m}& \mathrm{Equation}.Math..Math.\left(1\right)\end{array}$

[0092] Limit of Quantitation (LOQ) is the smallest amount of analyte that can be quantitatively measured. This quantity may be established for an embodiment using the standard deviation of the fluorescence intensity response and the slope of the calibration curve according to equation 2, where a is the standard deviation of the fluorescence intensity response and m is the slope of the calibration curve.

[00002]$\begin{array}{cc}\mathrm{LOQ}=\frac{10.Math..Math.}{m}& \mathrm{Equation}.Math..Math.\left(2\right)\end{array}$

[0093] The reliability of embodiments may be referred to as robustness. This depends on factors that may vary significantly during real world use of an embodiment such as pH, temperature, and viscosity of the test sample; ambient temperature, ambient light, and the storage temperature of the cartridge of an embodiment and/or the reader of an embodiment.

[0094] Validation methods described herein may be consistent with the ICH Harmonized Tripartite Guideline (1996/2005). The guideline was formulated for use in the validation of analytical procedures for the identification and quantitation of the active moiety in samples containing a drug substance (among other procedures). This document was prepared to establish common characteristics to be used during validation of analytical procedures to bridge the differences found in regulations of different countries. This has been proposed by the European Union, Japan and the USA. It closely follows the requirements established by the Food and Drug Administration (FDA) for bioanalytical method validation.

[0095] Crop Grading

[0096] Embodiments of the invention may also find application in grading the potency of marijuana by quantifying the amount of psychoactive components in plant tissues. According to such applications, plant tissue may be sampled from a marijuana plant and THC and/or other psychoactive compounds may be extracted from the tissue according to known methods. For example, and without limitation, plant tissue samples may be extracted with solvents such as ethanol, methanol, tetrahydrofuran (THF), naphtha, petroleum ether; or edible oils such as olive oil, coconut oil, or canola oil. Other known extraction methodologies use carbon dioxide, or volatile hydrocarbons such as butane or propane. Plant tissues may be extracted as-sampled or may be prepared for extraction through known mastication or homogenization methods. The plant extract derived according to these methods may be spotted on a stationary phase of an embodiment with or without dilution depending on concentration. Highly concentrated samples may require dilution in order to prevent streaking on the TLC plate and/or to obtain linear measurements of concentration.

[0097] Crop Grading: Example Extraction

[0098] 100 mg of crushed or ground plant material is mixed with 1 mL of solvent in a flat-bottomed collection vial. The solvent may be ethanol, methanol, naphtha, or isopropyl alcohol. Invert the vial 10 times and allow to rest for five minutes. A plunger with an attached filter layer is pressed into the collection vial to separate the plant material from the matrix and extract a maximum amount of cannabinoids. An aliquot of the extract may be communicated to a sample cartridge of an embodiment for separation and analysis.

[0099] Crop Grading: Chromatographic Analysis

[0100] Chromatographic separation of the following cannabinoids: cannabidiolic acid (CBDA), cannabinol (CBN), 9 tetrahydrocannabinolic acid A (THC-A), and .sup.9tetrahydrocannabinal (THC); is achieved by high performance thin-layer chromatography (HPTLC) using C18-W silica gel 60 plates and a mobile phase of methanol/water/acetic acid (9:1:0.3, v/v/v), according to ICH guidelines. A custom photometric procedure is used for in situ identity verification and quantitative analysis of the isolated cannabinoids using an image capture system followed by image analysis of the plate by fluorescence detection. Results indicate that an inexpensive, rapid detection system using an HPTLC test cartridge combined with a custom image analysis system and method provides an accurate test for establishing cannabinoid levels in marijuana or hemp extracts, or cannabinoid-based products.

[0101] Analytical reagent-grade methanol and acetic acid are purchased from Sigma-Aldrich (Milwaukee, Wis., USA). Certified cannabinoid reference standards for 9-THC, CBN, CBDA, are dissolved in reagent-grade methanol, and the THC-A is dissolved in acetonitrile. All reference standards are purchased from Cerilliant Corporation (Round Rock, Tex.). The pure reference standards are used for method development and validation. Testing is performed on commercially available, pre-coated normal phase (NP) and reverse phase (RP-18W) silica gel HPTLC plates without fluorescent indicators (Merck, Steinheim, Germany) to determine the best stationary phase to meet the research objectives. Tests are repeated using reagent-grade ethanol as the diluent with similar results. Iso-propyl alcohol may also be used.

[0102] Crop Grading: Standard Solutions

[0103] External standards are prepared using cannabinoid reference standards of THC, CBDA, CBN and THC-A, using methanol as the diluent. The concentration of the external standards used is 100 g/mL. Individual calibration solutions of each cannabinoid are prepared and used for identity verification of sample mixtures. Reference standards are stored at 20 C. prior to use.

[0104] Crop Grading: Sample Preparation

[0105] Sample mixtures are prepared to evaluate the separation efficiency of the methodologies. Cannabinoid mixtures are prepared with cannabinoid reference standards of THC, CBDA, CBN and THC-A with methanol as the diluent. The concentration of each cannabinoid in the mixture used is 100 g/mL.

[0106] The NP plates used are 1010 cm, glass-backed HPTLC, silica gel 60 (Merck, Steinheim, Germany). The RP-18W plates used are 1010 cm, glass-backed HPTLC, silica gel 60 RP-18W (Merck, Steinheim, Germany). Reverse phase plates greatly reduce the impact of humidity on the separations which is advantageous for a field test in uncontrolled environments. Additionally, reverse phase plates are compatible with less toxic mobile phase solvents leading to greener chemistry. Still further, reverse phase plates can provide enhanced fluorescence response of some analytes which is advantageous to improve sensitivity. An appropriate stationary phase is incorporated into the test cartridge for use in a field-testing device according to the invention.

[0107] Mobile phases of the invention meet criteria including adequate separation of cannabinoids, rapid elution, and low toxicity. The chemical properties of polarity and lipophilicity of the cannabinoids are considered when selecting possible solvents. According to one embodiment a suitable mobile phase for use with NP plates is chloroform/hexane (1:1, v/v) and a suitable mobile phase for use with RP plates is methanol/water/acetic acid (9:1:0.3, v/v/v). The mobile phase volume used is 10 mL for each system.

[0108] Development to a solvent front of 70 mm is performed in CAMAG Twin Trough development chambers without chamber saturation as a pre-conditioning phase. This is followed by drying with an air dryer.

[0109] Three cannabinoid mixture samples are applied to each plate as spots with a constant volume of 5 L, using multiple applications per spot to assure compactness of spots. Cannabinoid external standards are applied to each plate for identity verification based on Rf value. Each plate type (NP & RP) is run in triplicate for each system. The plates containing the mixtures are derivatized by spraying with a 0.5% Fast Blue B solution to allow for visualization.

[0110] Fluorescence detection offers enhanced selectivity as compared to absorbance methods due to the involvement of the two different wavelengths (excitation and emission). The use of excitation/emission profiles of molecules that display the property of intrinsic or native fluorescence can be used for label-free identification and quantification with limits of detection down to the picogram per millimeter (parts per trillion) range. Some of the cannabinoids found in marijuana, including THC, CBDA, CBN and THC-A exhibit this unique quality. Fluorescent signals from certain interfering compounds can be blocked so as not to overlap with fluorescent signals from THC, CBDA, CBN, and THC-A. Interfering fluorescent signals can be largely blocked by placing a bandpass filter in the optical path of the excitation beam thereby isolating the absorption bands of the analytes, e.g. THC, CBDA, CBN, and/or THC-A. Accordingly, most compounds that would otherwise emit interfering fluorescence are not excited and therefore cannot emit interfering fluorescence.

[0111] Quantitative evaluation is performed using a basic image processing ImageJ macro of the present invention to provide automated data analysis of the image. Images captured using the test apparatus are then used to relate image pixel intensity values to concentration levels and assess linearity. Using this technique, a linear regression calibration curve is created which relates the intensity of the fluorescence emission response (y-value) to the concentration of the analyte in solution (x-value). By using the calibration curve along with the fluorescence response of an unknown sample while precisely measuring the amount of sample applied, a highly accurate and sensitive quantification of cannabinoid concentration in methanol can be achieved. The image captured also provides documentation of the test results and can be re-evaluated at any time. Preliminary results show the potential of this methodology to provide excellent separation, good analyte recovery and good linearity (r.sup.2=0.9996) of the image capture system and optical quantitation algorithm over a wide range of concentrations.

TABLE-US-00002 TABLE 2 Cannabinoid R.sub.f values Normal Phase (NP) Plates R.sub.f Value Cannabinoid Cannabinoid Mixture THC 0.65 CBD 0.64 CBN 0.62 THCA 0.39

TABLE-US-00003 TABLE 3 Cannabinoid R.sub.f values Reverse Phase (RP) Plates R.sub.f Value Cannabinoid Cannabinoid Mixture THC 0.43 CBDA 0.57 CBN 0.48 THCA 0.41

[0112] Both plate systems show good analyte separation. There is less variation between the Rf values of the two mixtures in the RP system, although some of the spots are slightly different colors between the cannabinoid mixture and the interference mixture, suggesting there may be some interaction with one or more of the interferents. The specificity of fluorescence detection ensures that any interferents are not detected during quantitation.

[0113] The analytical results presented indicate a methodology suitable for the rapid and robust separation of major cannabinoids in methanol or ethanol solutions. This method is selective and sensitive enough to provide objective data about active drug levels when combined with photometric detection procedures of the invention. The RP plates are preferred but not required. Test cartridges in accord with the invention may have a suitable width to accommodate a desired number of chromatographic separation lanes and external standards. For example, a device detecting THC, CBDA, CBN, and THC-A may have four external standard lanes and three sample lanes for averaging test results in triplicate.

[0114] The solution phase fluorescence spectra for most major cannabinoids dissolved in solvents such as ethanol or methanol are well known and available in the literature. Known peak excitation wavelengths for solution phase THC-A, THC and CBDA are around 210-250 nm and corresponding emission peaks are reported at 305 nm. When certain fluorescent species interact with solid phase silica, e.g. during HPTLC, a fluorescent shift towards longer wavelengths is observed. Thus, excitation/emission profiles are different than for the same analytes in solution.

[0115] Due to solid state related fluorescence shifts, embodiments may advantageously use excitation peak wavelengths between about 340 nm and 390 nm and may detect emission peaks centered at about 430 nm+/5%. Accordingly, embodiments may incorporate less costly glass optics rather than fused silica or quartz which would otherwise be required for transmitting excitation beams below 305 nm. Moreover, commercially available LEDs are available for use as excitation sources emitting at, e.g. 310 nm, 320 nm, 340 nm, 350 nm, 365 nm, 370 nm, 385 nm to optimize for signal to noise ratio. LEDs emitting between 365 nm and 385 nm are particularly advantageous for use as an excitation source of the invention.

[0116] It will be apparent to those skilled in the art that the above methods and apparatuses may be changed or modified without departing from the general scope of the invention. The invention is intended to include all such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

[0117] Having thus described the invention, it is now claimed: