THIN-LAYER CHROMATOGRAPHY SYSTEM AND METHOD FOR ASSESSING ANALYTE CONCENTRATIONS IN SAMPLES

Abstract

The invention relates to cartridges for thin layer chromatography (TLC) which hermetically seal the TLC mobile phase during loading, performance of the TLC process, and analysis of the chromatogram. It further relates to associated methods of usefor example for detecting or quantifying an analyte in a sample. It further relates to processes for assembling the cartridge, to associated equipment adapted for use with them, and kits of parts.

Claims

1. A method for detecting or quantifying an analyte in a sample, the method comprising: (i) providing said sample; (ii) preparing an analysis sample from said sample; (iii) providing a thin layer chromatography (TLC) cartridge comprising the TLC plate and a sealed reservoir containing the TLC mobile phase; (iv) loading the analysis sample and one or more reference standards onto the TLC plate of the cartridge; (v) exposing the TLC plate to the TLC mobile phase to perform TLC on said analysis sample in the TLC cartridge to produce a chromatogram; (vi) optically analysing analyte and reference standards bands on the chromatogram, with an imaging device to generate optical signals corresponding to the analyte and reference standards; (vii) detecting or quantifying the analyte in the sample according to said optical signals, characterised in that the TLC cartridge hermetically seals the TLC mobile phase during loading, performance of the TLC process, and analysis of the chromatogram.

2. A method as claimed in claim 1 wherein step (v) comprises in any appropriate order: (v-1) breaking a barrier between the mobile phase reservoir and the TLC plate to allow fluid access between them; (v-2) orientating the cartridge to a first orientation, which first orientation permits the base of the plate to be exposed to the released mobile phase solvent and start the TLC process; (v-3) during or after production of the chromatogram, orientating the cartridge to a second orientation, which second orientation ceases exposure of the base of the plate to the released mobile phase, to stop or slow the TLC process.

3. A method as claimed in claim 1 or claim 2 wherein: (a) first orientation is substantially vertical, and (b) the second orientation is substantially horizontal.

4. A method as claimed in any one of claims 1 to 3 wherein step (v) further comprises: (v-4) orientating the cartridge to a third orientation, which third orientation permits the mobile phase solvent to run into an absorbent material present in the cartridge.

5. A method as claimed in any one of claims 1 to 4 wherein step (iv) comprises loading the analysis sample onto a lane of the TLC plate by penetration through a septum of self-sealing material.

6. A method as claimed in any one of claims 1 to 5 wherein step (iv) comprises loading one or more reference standards onto respective lanes of the TLC plate by penetration through a septum of self-sealing material.

7. A method as claimed in any one of claims 1 to 6 wherein said one or more reference standards are incorporated into said TLC cartridge in sealed reservoirs

8. A method as claimed in claim 7 wherein the reference standards are accessed by penetration through a septum of self-sealing material.

9. A method as claimed in any one of claims 1 to 8 wherein an internal standard is introduced into the analysis sample, whereby the difference between the recovered or quantified amount and the known amount of internal standard can be used to scale the analyte if quantified in step (vii).

10. A method as claimed in claim 9 wherein the internal standard is incorporated into said TLC cartridge in a sealed reservoir, which is optionally accessed by penetration through a septum of self-sealing material.

11. A method as claimed in any one of claims 5 to 10 wherein the septum of self-sealing material, if present, is an elastomeric material.

12. A method as claimed in any one of claims 1 to 10 wherein following stages (v) and (vi), the pH of the plate is adjusted, and the optical analysis of stage (vi) repeated at this new pH in order to identify an analyte in a sample wherein the optical properties of the analyte are pH dependent.

13. A method as claimed in claim 12 wherein the cartridge comprises a sealed reservoir for a pH modifying agent whereby in use a barrier between the pH modifying agent reservoir and TLC plate is broken to allow fluid access between them, such that the pH of the plate is adjusted.

14. A method as claimed in any one of claims 1 to 13 wherein the cartridge comprises one or more asymmetric wells for sample centrifugation, wherein the or each sample preparation well includes a side region adapted to trap or retain particulate or higher density material directed into the side region when the cartridge is centrifuged.

15. A method as claimed in any one of claims 1 to 14 wherein the cartridge comprises one or more of the following additional functionalities: (a) one or more wells for receiving the sample in step (i); (b) one or more wells for syringe cleaning or rinsing; (c) one or more wells for syringe disinfecting.

16. A method as claimed in any one of claims 1 to 15 wherein the sample in (i) is selected from the list consisting of: an environmental sample; a chemical synthesis sample; a body fluid.

17. A method as claimed in claim 1 wherein the sample preparation of step (ii) comprises removal of particulate or higher density material from the original source sample, optionally by one or more precipitation, filtration or centrifugation steps.

18. A method as claimed in claim 16 or claim 17 wherein the analyte is a drug and\or drug metabolite, and the sample is a body fluid from a subject to whom said drug has been administered.

19. A method as claimed in claim 18 wherein the body fluid is blood, and the analysis sample is prepared by removing the red blood cells by centrifugation, and optionally protein is precipitated and removed by centrifugation.

20. A method as claimed in claim 18 or claim 19 wherein the drug and\or drug metabolite is quantified in step (vii) for one or more of the following purposes (i) therapeutic drug monitoring; (ii) measuring or monitoring the pharmacokinetics or pharmacodynamics of the drug; (iii) assisting a physician to devise or adjust a dosage regimen of the drug, optionally in conjunction with observational data of side effects or therapeutic benefit of the drug in the subject; (iv) designing or performing a regulatory trial of said drug.

21. A method as claimed in claim 18 or claim 19 wherein the drug and\or drug metabolite is detected or quantified in step (vii) for assessing compliance by the subject with a dosage regimen of the drug.

22. A method as claimed in any one of claims 18 to 21 wherein the drug is a chemotherapeutic.

23. A method as claimed in any one of claims 18 to 21 wherein the drug is a selected from the list consisting of: an antiepileptic; an antiarrhythmic; an antibiotic; an antifungal; an immunosuppressant; a dermal medicine; a bronchodilator; a psychoactive drug.

24. A method as claimed in any one of claims 18 to 23 wherein the drug is a selected from the drugs set out in Table D.

25. A method as claimed in any one of claims 1 to 24 wherein the analyte is either: (a) natively fluorescent; (b) non-natively fluorescent, but labelled with a fluorescent tag; (c) non-natively fluorescent, which is assessed by absorbance or fluorescence quenching of a fluorescent TLC plate, and the fluorescence of the plate is optically analysed in step (vi).

26. A method as claimed in any one of claims 1 to 25 wherein the optical analysis in step (vi) generates optical signals proportionate to the concentrations of analyte and reference standards on the developed chromatogram.

27. A method as claimed in any one of claims 1 to 26 wherein the method is for quantifying the analyte in the sample and step (vi) comprises optically analysing analyte and reference standards bands on the chromatogram to generate optical signals proportionate to the concentrations of analyte and reference standards and step (vii) comprises converting the optical signals into an analyte concentrations in the sample.

28. A method as claimed in claim 27 wherein in step (vii) optical signal of the analyte is compared to a standard curve derived from the optical signals of the reference standards to quantify the analyte concentration, which is optionally scaled according to an internal standard introduced into the analysis sample.

29. A method as claimed in claim 27 or claim 28 wherein steps (vi) and (vii) comprise: (a) optically analysing analyte and reference standards bands on the chromatogram to generate providing an image of the TLC plate including said band; (b) determining an intensity of the portion of the image representing the band; (c) determining the concentration of the compound represented the band using the determined intensity.

30. A method as claimed in claim 29 wherein step (b) and (c) are performed using boundary definition of the band and then summation of pixel intensities within a defined area of the band.

31. A method as claimed in claim 30 wherein step (b) includes: (b-1) receiving an input identifying an approximate region of the image, the approximate region including the band; (b-2) identifying the boundaries of the band; (b-3) determining the intensity of the band based on the intensities of a plurality of pixels located within the boundaries of the band.

32. A method as claimed in claim 31 wherein the boundaries of the band are determined by defining an imaging area of the band representing the analyte or standard in terms of pixels of varying intensity differing from a background pixel intensity of the plate where the analyte or standard is not present.

33. A method as claimed in any one of claims 29 to 32 wherein step (a) comprises receiving the image from a suitable imaging device, which is optionally selected from a dedicated TLC plate reader; a digital camera; a smartphone; a flatbed scanner; and a tablet computer.

34. A method as claimed in any one of claims 29 to 33 including the step of converting the image into black and white, or grayscale, and optionally inverting the colours in the image.

35. A thin layer chromatography (TLC) cartridge comprising the TLC plate and a sealed reservoir for the TLC mobile phase characterised in that the TLC cartridge is adapted to hermetically seal the TLC mobile phase during loading, performance of the TLC process, and analysis of the resulting TLC chromatogram.

36. A cartridge as claimed in claim 35 comprising a breakable barrier between the mobile phase reservoir and the TLC plate to allow fluid access between them.

37. A cartridge as claimed in claim 35 or claim 36 comprising a septum of self-sealing material for loading an analysis sample onto a lane of the TLC plate.

38. A cartridge as claimed in any one of claims 35 to 37 comprising a plurality a septa of self-sealing material for loading a plurality of reference standards onto respective lanes of the TLC plate.

39. A cartridge as claimed in any one of claims 35 to 38 wherein a plurality of reference standards are incorporated into said TLC cartridge in sealed reservoirs, which are optionally accessed by penetration through a septum of self-sealing material, and optionally selected from the drugs defined in any one of claims 22 to 24.

40. A cartridge as claimed in any one of claims 35 to 39, wherein an internal standard is incorporated into said TLC cartridge in a sealed reservoir, which is optionally accessed by penetration through a septum of self-sealing material.

41. A cartridge as claimed in any one of claims 37 to 40 wherein the septum of self-sealing material is an elastomeric material.

42. A cartridge as claimed in any one of claims 35 to 41 wherein the cartridge comprises a sealed reservoir for a pH modifying agent whereby in use a barrier between the pH modifying agent reservoir and TLC plate is broken to allow fluid access between them, such that the pH of the plate is adjusted.

43. A cartridge as claimed in any one of claims 35 to 42 which comprises one or more asymmetric wells for sample centrifugation, wherein the or each sample preparation well includes a side region adapted to trap or retain particulate or higher density material directed into the side region when the cartridge is centrifuged.

44. A cartridge as claimed in any one of claims 35 to 43 wherein the cartridge comprises one or more of the following additional functionalities: (a) one or more wells for receiving a sample in step; (b) one or more wells for syringe cleaning or rinsing; (c) one or more wells for syringe disinfecting.

45. A cartridge as claimed in any one of claims 35 to 44 comprising: (i) a face unit incorporating an aperture to view a TLC plate; (ii) a window covering the aperture in the face unit; (iii) a rear unit defining a mobile phase reservoir; (iv) a TLC plate located between the face unit and the rear unit; (v) a breakable barrier between the mobile phase reservoir and the TLC plate to allow fluid access between them. (vi) a plurality of gaskets for collectively sealing at least (a) the mobile phase reservoir; and (b) a volume defined by the face unit and/or rear unit in which the TLC plate can be exposed to the mobile phase once the mobile phase is released from the reservoir, such as to prevent mobile phase entering the external environment; wherein: the face unit further incorporates a plurality a septa of self-sealing material for loading reference standards and/or sample onto respective lanes of the TLC plate; and the face unit further incorporates a septum of self-sealing material for accessing and breaking the barrier to release the mobile phase from reservoir into the volume in which the TLC plate can be exposed to the mobile phase.

46. A cartridge as claimed in claim 45 comprising a window gasket which is compressed between the window and the face unit by an outer window sealing plate mountable on the face unit, optionally be means of a plurality of screws.

47. A cartridge as claimed in claim 45 or claim 46 wherein the septa of self-sealing material to allow loading reference standards and/or sample onto respective lanes of the TLC plate are held between the face unit and an injection plate, wherein the face unit and injection plate incorporate corresponding apertures to permit said loading of the TLC plate.

48. A cartridge as claimed in any one of claims 45 to 47 comprising a rotatable paddle for breaking the barrier to release the mobile phase from reservoir into the volume in which the TLC plate can be exposed to the mobile phase, wherein in use the paddle is actuated by penetrating the septum of self-sealing material which permits access to the barrier with a needle, and rotating the paddle through the barrier.

49. A cartridge as claimed in any one of claims 35 to 48 further comprising mobile phase in the reservoir.

50. A cartridge as claimed in any one of claims 35 to 49 wherein the cartridge is adapted for use in a method of any one of claims 1 to 36.

51. A process for preparing a cartridge as claimed in any one of claims 45 to 48, or claim 49 or 50 as dependent on any one of claims 45 to 48, the process comprising providing: (i) a face unit incorporating an aperture to view a TLC plate; (ii) a window for covering the aperture in the face unit; (iii) a rear unit defining a mobile phase reservoir; (iv) a TLC plate; (v) a breakable barrier between the mobile phase reservoir and the TLC plate to allow fluid access between them. (vi) a plurality of gaskets for collectively sealing at least (a) the mobile phase reservoir; and (b) a volume defined by the face unit and/or rear unit in which the TLC plate can be exposed to the mobile phase once the mobile phase is released from the reservoir, such as to prevent mobile phase entering the external environment; (vii) a plurality of a septa of self-sealing material for incorporating into the face unit for loading reference standards and/or sample onto respective lanes of the TLC plate; and (viii) a further septum of self-sealing material for incorporating into the face unit for accessing and breaking the barrier to release the mobile phase from reservoir into the the volume into which the TLC plate can be exposed to the mobile phase; (ix) a mobile phase assembling (i) to (viii) into a cartridge which hermetically seals the TLC mobile phase (ix) during loading, performance of the TLC process, and analysis of the resulting TLC chromatogram.

52. A process as claimed in claim 51, wherein the cartridge further comprises the window gasket and outer window sealing plate as defined in claim 46, the injection plate of claim 47, and/or the rotatable paddle of claim 48.

53. A kit of parts comprising parts (i) to (viii) as defined in claim 51, and optionally the window gasket and outer window sealing plate as defined in claim 46, the injection plate of claim 47, and/or the rotatable paddle of claim 48.

54. A method of using a cartridge as claimed in any one of claims 35 to 50, the method comprising: (i) loading an analysis sample and references standards onto the TLC plate; (ii) allowing fluid access between the mobile phase reservoir and the TLC plate; (iii) orientating the cartridge into a vertical orientation to let the mobile phase flow into contact with the base of the TLC plate allowing the plate to develop normally by capillary forces; (iv) orientating the cartridge back down to a flat orientation to stop the TLC plate development.

55. A companion fluid handling device for use with, and optionally comprising, the cartridge of any one of claims 35 to 50, which device comprises: (i) a centrifuge for centrifugation of the cartridge and sample for separation of particulate or higher density material from the original source sample; (ii) a cartridge holder adapted to receive said cartridge and keep it firmly held during centrifugation; (iii) a tiltable stage for altering the orientation of the cartridge to perform the centrifugation, the TLC process, and optionally reading the TLC plate of the cartridge; wherein elements (i) to (iii) are optionally comprised within a single housing.

56. A companion TLC plate reader and data processor for use in the method of any one of claims 1 to 34, or the cartridge of any one of claims 35 to 50; or the device of claim 55, which reader and processor comprises: (i) a light or fluorescence excitation source; (ii) an imaging device; (iii) a data processor for processing the image and converting the optical signals into an analyte concentration in the sample, optionally according to the method of any one of claims 27 to 33.

57. A system or kit which comprises: (i) the TLC cartridge of any one of claims 35 to 50 or kit of parts of claim 53, and optionally one or more of: (ii) a TLC plate reader adapted to read the bands on the plate in said cartridge; (iii) a data processor for converting the optical signals from the reader into an analyte concentrations; (iv) a centrifuge for sample preparation, optionally adapted to seat the cartridge, or alternatively to be used directly on a receptacle for said sample; (v) a hypodermic needle for transferring solutions; (vi) instructions for use according to the method of any one of claims 1 to 34.

Description

FIGURES

[0260] FIG. 1Separation of EPI, DOX, and DOL on the TLC plate.

[0261] FIG. 2Separation of EPI, DOX, and DOL from spiked samples of healthy human plasma.

[0262] FIG. 3Separation of EPI, DOX and DOL from clinical samples of two different cancer patients undergoing DOX treatment (Patient 1, left; Patient 2, right).

[0263] FIG. 4Linearity of the optical quantification of DOX and DOL. B Graph showing the measured fluorescence intensity versus concentration for DOX and DOL concentrations ranging from 0.01 to 10 M. B. Zoomed in view of panel A showing data points at the lower concentration range.

[0264] FIG. 5Quantification of DOX and comparisons to HPLC derived values. A Average values with errors bars showing standard error of the mean (n=3). The 15% error range allowed the by FDA for a bioanalytical method is shown by the blue lines. B All the data points are shown

[0265] FIG. 6Quantification of DOL and comparisons to HPLC derived values. A Average values with errors bars showing standard error of the mean (n=3). The 15% error range allowed the by FDA for a bioanalytical method is shown by the blue lines. B All the data points are shown

[0266] FIG. 7Quantification of Irinotecan and comparisons to HPLC derived values. A Average values with errors bars showing standard error of the mean (n=2). The 15% error range allowed the by FDA for a bioanalytical method is shown by the blue lines. B All the data points are shown.

[0267] FIG. 8TLC run from plasma extract of different plasma volumes showing the EPI, DOX and DOL can all be successfully separated from one another with starting plasma volumes as low as 5 l.

[0268] FIG. 9aLayered prototype design of the TLC cartridge in practice. It is hermetically sealed so there is no leaking of the mobile phase even when tipped up. The sample has been loaded onto the TLC plate and the cartridge tipped up to let the mobile phase touch the bottom of the TLC plate. The mobile phase naturally wicks up the plate and as can be seen after 13 minutes it has made it over of the way up the plate. This demonstrates that there is nothing inherent about the hermetically sealed environment that prevents normal TLC development from occurring.

[0269] FIG. 9bA fluorescent image of the developed TLC plate inside the cartridge. The plate is still wet with the mobile phase even though it has been laid down flat and the bulk mobile phase has drained away from the plate. This demonstrates normal development of the TLC plate with well separated lanes and bands. There is a full calibration curve and a patient plasma extract sample. The DOX, Dol, and EPI are clearly visible in all instances. Again, this demonstrates that the hermetically sealed environment does not interfere with the normal development of the TLC plate, nor does it interfere with the fluorescence imaging of the plate.

[0270] FIG. 9cAnother instance of a successfully developed TLC plate in the hermetically sealed cartridge. Here the DOX in the patient plasma extract was quantified using the analysis software and it was found to have 12.4% error with the gold standard HPLC derived concentration value.

[0271] FIG. 9dA calibration curve from the fluorescence intensities measured on a TLC plate developed inside the hermetically sealed cartridge. The R.sup.2 value is 0.987 which shows that the curve is linear across the desired concentration range.

[0272] FIG. 10aSchematic representation of the self-contained TLC cartridge. The cartridge is based on a two chamber concept where the mobile phase and the TLC plate are kept in spate chambers for transport and storage. A glass coverslip barrier between the two chambers is breached and the cartridge tipped up to stand straight up to allow the mobile phase to come into contact with the lower edge of the TLC plate allowing it to develop. The development process is stopped by tipping the cartridge back to lie flat allowing the mobile phase to drain back into the second chamber. The TLC plate can then be imaged from above by a CCD camera system.

[0273] FIG. 10bFurther embodiment of the invention, including asymmetric spin chambers for removal of red blood cells.

[0274] FIG. 10cFurther embodiment of the invention, permitting treatment with basic chloroform to manipulate pH of the plate. A pan structure holds the basic chloroform when released allowing the whole TLC plate to be soaked with it. It prevents mixing with the acidic mobile phase until after treatment and imaging of the TLC plate. Preferably the mobile phase is not a strong acid allowing them to mix later when discarded without harmful reactions. The pan structure still allows the mobile phase to come into contact with the plate when tipped up to do the initial developing of the plate.

[0275] FIG. 10dIllustration of layers used to prepare one embodiment cartridge of the invention.

[0276] FIG. 11Image of assembled embodiment of the invention illustration wells and access points.

[0277] FIG. 12Design of the device that automates sample preparation, loading and chromatography.

[0278] FIG. 13a-dMatLab Quantification Code Demonstration for image preparation and quantitative analysis. A optical image taken of Sudan IV dye that has been run on a silica gel TLC plate. B The picture has been inverted by the program to make the bands appear as light bands on a dark background.

[0279] TLC lane selection: the user defines the lane where the analyses will take place as the red square. It is important to define the entire length of the lane beyond just the bands of interest so the background subtraction algorithm has the most data to work with to make the background as flat as possible. C. The selected region now appears as a new figure that is stretched out in the X direction to allow better viewing of the band.

[0280] TLC band selection: the upper and lower bounds of the band are defined by the user by drawing the red rectangle. D. After the band has been quantified a visual conformation of the area that has been integrated for fluorescence intensity is shown by the red overlay. Additional bands can be sequentially defined and quantified.

[0281] FIG. 14Bathochromic and hypsochromic shift. FIG. 14A shows a general idealised case. FIG. 14B shows Absorbance spectra (left) and fluorescence spectra (right) of Irinotecan (top), and SN-38 (bottom) at the pH values specified.

[0282] FIG. 15IR TLC analysis. The Figure shows the different fluorescent properties of the same TLC chromatogram when imaged under acid and basic conditions. CPT=carbonyloxycamptothecin (Irinotecan). SN-38=an active metabolite of CPT.

[0283] FIG. 16Quantification of Irinotecan and SN-38 from spiked plasma (n=3). The graph shows the average value and the standard deviation of the measurements.

[0284] FIG. 17Cartridge embodiment having a primarily bipartite design comprising upper (face) and lower (base) body units.

[0285] FIG. 18Illustration of the components of the further cartridge embodiment of FIG. 17(01) Window screw; (02) Window sealing plate; (03) Window; (04) Window o-ring; (05) Septum screw; (06) Injection plate; (07) Septum; (08) Body screw; (09) Upper (face) body; (10) TLC plate; (11) Bridge; (12) Pivot pin; (13) Paddle; (14) Bridge pin; (15) Segregator foil; (16) Body o-ring; (17) Reservoir o-ring; (18) Lower (base) body.

[0286] FIG. 19Illustration showing transmission of force through the bridge.

[0287] FIG. 20Illustration showing removal of elements to allow filling or refilling of mobile phase reservoir.

[0288] FIG. 21Illustration showing location of solid fill modification regions which can be used to modify the mass of the cartridge.

[0289] FIGS. 22 and 23Illustration showing penetration of the segregator by a needle to release the mobile phase.

[0290] FIG. 24Illustration showing how rotation of cartridge exposes the TLC place to mobile phase after the reservoir is pierced.

[0291] FIG. 25Illustration showing detail of support posts in rear unit which support the TLC plate, and separation of front and rear of TLC plate to prevent wicking.

[0292] FIGS. 26 and 27Illustration showing detail of stand-off posts in front unit which help to prevent wicking across the TLC plate.

[0293] FIG. 28Illustration showing detail of support posts in rear unit which support the TLC plate, and separation of front and rear of TLC plate to prevent wicking.

[0294] FIG. 29Illustration showing cross section of the bridge and injection, illustrating how the edge extrusions assist in preventing wicking.

[0295] FIG. 30Illustration showing profile of mobile phase reservoir when vertical: the reservoir follows a tapering v contour below the TLC plate (in both the face and rear units) but enters a rectilinear profile before the lowest point of the TLC plate.

EXAMPLES

Example 1Materials and Methods

[0296] Materials

[0297] Doxorubicin hydrochloride, epirubicin hydrochloride, irinotecan, CPT, and acetic acid were all purchased from Sigma Aldrich (Dorset, United Kingdom). Doxorubicinol hydrochloride was purchased from Toronto Research Chemicals (Toronto, Canada). The Analytical Chromatography TLC Silica gel 60 F.sub.245 used for the DOX experiments and the Analytical Chromatography TLC Silica gel 60 for the irinotecan experiments were both purchased from Merck/Millipore (Hertfordshire, United Kingdom). The methanol and the acetonitrile were purchased from Fisher Scientific (Loughborough, United Kingdom). The chloroform was purchased from VWR (Leicestershire, United Kingdom). Water was purified using the MilliQ filtration system from Merck/Millipore (Hertfordshire, United Kingdom). Healthy plasma samples and the irinotecan clinical patient samples were generously donated from at the Centro di Riferimento Oncologico di Aviano, Italy. The doxorubicin clinical patient samples were generously donated from the Universitat Munster Klinik fur Kinder and Jugendmedizin, Germany. Imaging of the TLC plates was performed using a SynGene PXI imaging system (SynGene, Cambridge, United Kingdom). MatLab R2015A was used to write the custom optical quantification program (Mathworks, Cambridge, United Kingdom). The cartridge materials consisted on chemically resistant Teflon plastic and flexible Teflon tape.

[0298] Mobile Phase PREPARATION

[0299] The mobile phase consisted of chloroform:methanol:aceticacid:water (80:20:14:6) (v:v:v:v)[12, 23]. The mixture was shaken to ensure proper dissolution of any water droplets that formed. If the water would not dissolve giving the solution a cloudy white appearance then the solution was placed in a 37 C. water bath until the solution became clear. The running of the TLC plate was performed at room temperature.

[0300] Preparation of Spiked Plasma Samples

[0301] A 100 l sample of plasma from healthy donors was spiked with 10 l of a prepared solution of EPI, DOX and DOL in methanol in various concentrations. The spiked sample was vortexed at max speed for 10 seconds to ensure proper mixing. The plasma was prepared beforehand using centrifugation to pellet out the red blood cells.

[0302] Calibration Curve Samples

[0303] A solution of DOX, DOI, and EPI (each at 1 M) in methanol, was prepared and then diluted samples from this were made at 0.5, 0.1 0.05 M. These four concentrations were placed onto each TLC plate to prepare the standard curve for that plate.

[0304] Spiking Internal Standard into Clinical Samples

[0305] A 10 l sample of 0.5 M Epirubicin (EPI) in methanol was spiked into 100 l clinical plasma samples to provide an internal standard. EPI was chosen because it is an isomeric form of DOX [24] that has very similar chemical and fluorescence properties to both DOX and DOL. The difference between the recovered amount of EPI and the known amount of EPI spiked into the plasma sample helped to scale the DOX and DOL concentration values to account for variability in the extraction efficiency as well as effects on overall fluorescence levels that could potentially be influenced by the buffering properties of the plasma.

[0306] Drug Extraction from the Plasma with Protein Precipitation

[0307] The proteins needed to be precipitated from the plasma solution because they would interfere with the running of the TLC plate due to their hydrophilicity and high concentration. A mixture of acetonitrile:methanol (2:1) (v:v) was prepared[19]. A 100 l sample of plasma was placed in a 15 ml tube followed by a sample of 250 l of the above solution. The sample was aspirated 3 times, vortexed at maximum speed for 10 min and finally spun down at 5000 rpm for 5 min. The resulting supernatant containing the extracted drugs was carefully removed leaving the precipitant pellet at the bottom of the tube. This supernatant was then placed in a glass vial for analysis.

[0308] TLC Plate Loading and Development

[0309] A 10 l sample of each of the calibration curve solutions was loaded into the first 4 lanes of the TLC plate. A single 10 l sample of the plasma extract was loaded onto the 1 sample lane. The 3 sample lane had three sequential 10 l samples of the plasma extract loaded on top of each other allowing the solution to dry between applications. Loading three samples like this increased the signal which was important for low concentration samples. Loading more than 3 spots caused observed distortions to the bands that could cause trouble with quantification.

[0310] The TLC plate was allowed to air dry for 5 min while being covered with aluminum foil to prevent photobleaching of the sample.

[0311] For the DOX samples the plate was then placed into a beaker containing the mobile phase where the liquid level covered the bottom of the TLC plate with the upper level of the mobile phase below the level of the sample spots on the TLC plate. The container was covered to increase the vapor pressure of the solvent in the container and speed the development of the plate. The plate was then run for 30 min until the advancing phase was just below the top of the plate. This covered a distance of approximately 8.5 cm.

[0312] For the irinotecan samples the mobile phase was diluted 50:50 (v:v) with chloroform and then treated the same as for the DOX samples.

[0313] Optical Analysis of the TLC Plate

[0314] After the plate was developed and before the solvent had a chance to evaporate, the TLC plate was placed into a SynGene PXI Imaging System and the plate was imaged at different exposure times using the blue mLED illumination unit coupled with a UV06 filter. An exposure time of 30 seconds was used to image the DOX plates and 30 seconds was used to image the irinotecan plates.

[0315] A custom MatLab program was then used to quantify the fluorescence level of each band in both the calibration curve samples and in the 1 and 3 sample lanes. The intensity values determined for the DOX and DOL bands in the 3 sample lane were compared to the calibration curve generated using the 0.1 and 0.05 M concentrations of DOX and DOL. These values were then scaled by the difference in the measured and expected results of the EPI internal standard concentration.

[0316] Analysis Program

[0317] The custom MatLab program analysed each band by first allowing the user to define the boundaries of the lane in which the bands were located. The program then allowed the user to define an upper and a lower bound in which a particular band of interest was located. The program started by analyzing a single line of pixels running the length of the lane and did so sequentially for each line of pixels starting from left to right covering the width of the lane. For each line of intensity data, the MatLab function msbackadj was used to flatten the uneven background created by the lighting source. This created a flat background level at zero fluoresce intensity from which the band peaks could be detected along the length of the defined line. The program would then define the physical dimensions of band in the y direction by defining the full-width-half-max of the peak. The area under this defined portion of the curve was calculated using the trapezoidal approximation using the MatLab function trapz. This process was repeated for the next line of pixels until the entire width of the user defined lane was analysed. The X dimensions of the band were determined by setting a lower threshold limit for peaks to be defined by using a Min Peak Prominence of 1500 for DOX and for irinotecan. Lower concentrations of irinotecan required the thresholds be adjusted downward compared to the highest concentrations to make sure they were properly detected. The area under the curve defined by each line of pixels was added together to produce a total intensity for the fluorescence band.

[0318] It was important that the program objectively defined the actual boundaries of each band to prevent user error from influencing the obtained intensity values. The user's only influence on the program was to define the rough boundaries of a band in which the program could then search for and define the actual band.

[0319] In practice the program was used as follows:

[0320] 1 The program asks the user how many lanes and how many bands in each lane are to be analysed.

[0321] 2 The MinPeakProminence then needs to be determined, which defines the prominence of the peak maximum with respect to the noise floor. The default setting is set to 1500 which works well for most application.

[0322] 3 The program then asks for the divisor value. This is the value that the program uses to divide the peak max value to define the lower limit of the peak. The default is set to 2, so the full width half max of the peak will be analysed.

[0323] 4 The program will ask the user to select a file of the picture to analyse. The user then draws a rectangle to define the entirety of a single lane as shown in FIG. 13B.

[0324] 5 The program then compiles a new picture using just the band (FIG. 13C). The user draws a small rectangle to define the upper and lower bounds of the first band. The left and right dimension of the rectangle are not used for the analysis.

[0325] 6 The program will analyse the band and return the integrated fluorescence intensity. It will also cover in red the region that was analysed (FIG. 13D). This can take up to 20 seconds to complete. 2 parameters can be adjusted in case the identified region does not overlap well with the band of interest. If that red region extends significantly beyond the left and right of the band itself, then the MinPeakProminence value needs to be increased. If the program does not register a band and there is no red region then this value needs to be lowered.

[0326] This is repeated for other lanes and bands.

[0327] 7 Once all the band intensities have been quantified they can be used to determine the concentration of the analyte. The fluorescence intensity versus known concentration of the standard curve samples is plotted and fitted with a linear curve fit. The equation for that line can then be used to calculate the concentration of the unknown sample based on its measured fluorescence intensity. This is done by utilising the fluorescence intensity of the band of interest in the equation for the fluorescence intensity variable and then solving the equation for the concentration variable.

[0328] HPLC Quantification of DOX and DOL from the Same Clinical Plasma Samples

[0329] Each of the clinical samples analysed using the TLC method described above was also assessed using traditional gold standard HPLC method. A 100 l sample of patient plasma was spiked with 50 l of ethanol containing 120 g/L EPI as the internal standard. Then 100 l of a solution of phosphate buffer (pH 8.5) and 1000 l chloroform was added followed by mixing on a rotation shaker for 5 min at max intensity. The solution was then centrifuged for 5 min at 20,800 g. The organic phase was then transferred to a new tube and evaporated with nitrogen at 35 C. The resulting residue was redissolved in H.sub.2O/acetonitrile 3:1 (v:v) with 0.1% formic acid for injection into the HPLC.

[0330] The HPLC-method used a C18 column with a dual gradient elution. Solution A was 0.1% formic acid in H.sub.2O and solution B was 0.1% formic acid in acetonitrile. The elution protocol started with 75% A and 25% B changing to 70% A and 30% B over 7 minutes. Then the solution changed to 58% A and 42% B over 3 minutes. Detection was fluorescence based using 488 nm excitation and 555 nm emission wavelengths.

[0331] HPLC Quantification of Irinotecan from the Same Clinical Plasma Samples

[0332] The HPLC protocol used to determine the gold standard values of irinotecan in the clinical patient samples was previously established and is described in Marangon et al.[25].

[0333] Clinical Plasma Sample Collection from DOX Patients

[0334] The plasma samples used in the study were obtained during a larger clinical study as described by Krischke et al. [26].

Example 2Results

[0335] Separation in Spiked and Clinical Plasma Samples

[0336] The mobile phase was successful in being able to separate the EPI, DOX, and DOL from one another using the Silica TLC plate as shown in FIG. 1. The separation between EPI and DOX was sufficient to resolve the two bands from one another for successful quantification.

##STR00001##

[0337] The TLC method was also able to successfully separate the EPI, DOX and DOL from spiked plasma samples as shown in FIG. 2. Hemoglobin present in the plasma sample is also separated into a band below DOL. Hemoglobin has an absorption and emission spectra that is very similar to DOX and DOL which makes its physical separation from the drugs essential to prevent interference.

[0338] The process also successfully separated the EPI, DOX, and DOL from clinical plasma samples collected from patients undergoing chemotherapy treatment with DOX as shown in FIG. 3. TLC plates from two different patients is shown. As can be seen there is a large variation in the amount of contaminating plasma residue from patient to patient.

[0339] Optical Analysis of TLC Plates

[0340] The fluorescence imaging of the TLC plates of DOX and DOI was performed using the SynGene PXI gel reader system. DOX, DOL, and EPI are all naturally fluorescent compounds allowing the fluorescence mode of the PXI system to easily distinguish the drugs from the background. FIG. 4 shows the linearity of the system in quantifying the fluorescence intensity over a range of DOX and DOL concentrations from 0.01 to 10 M.

[0341] A custom MatLab program (described in more detail below) was used to obtain intensity values for the different bands. The results of the quantification and their comparison to the gold standard HPLC values obtained for the same DOX clinical samples is shown in FIG. 5. The samples were run in triplicate and were compared to the concertation obtained using HPLC for the same sample. The error between the HPLC and the TLC derived values is shown in FIG. 5A as the mean with the error bars showing the standard error of the mean. Out of a total of 12 samples studied 10 fit within this 15% error resulting in an 83% success rate. FIG. 5B shows all the data points individually. Here 28 out of a total of 36 samples fell within the 15% error resulting in a 78% success rate.

TABLE-US-00001 TABLE 1 Summary of the data shown in FIG. 5. Individual Average Data Points Within Range 9 27 Outside Range 3 9 Total # Points 12 36 Percent within 0.75 0.75 15%

[0342] The results for Dol are shown in FIG. 6. In the depicted instance, background fluorescent levels created by light leakage through the optical filter used in the experiment resulted in a lower correlation but nevertheless the experiment confirms the utility of the hermetically sealed cartridge.

TABLE-US-00002 TABLE 2 Summary of the data shown in FIG. 6. Individual Average Data Points Within Range 5 12 Outside Range 7 24 Total # Points 12 36 Percent within 0.42 0.33 15%

[0343] This TLC technical was also applied to a second chemotherapy drug, irinotecan, which is also inherently fluorescent. FIG. 7 shows the agreement between the TLC method and the gold standard HPLC analysis for 7 different clinical patient samples.

TABLE-US-00003 TABLE 3 Summary of the data shown in FIG. 7. Individual Average Data Points Within Range 6 10 Outside Range 1 4 Total # Points 7 14 Percent within 0.86 0.71 15%

[0344] To better understand the minimum amount of plasma necessary to conduct the protein precipitation and TLC separation a study was conducted where spiked samples of plasma were separated into different volumes followed by the protein precipitation and spotting onto the TLC plate. As shown in FIG. 7 a strong signal can be collected even from a 5 l sample of plasma. A typical finger stick blood drop is on the order of 50 L [27, 28] which should produce about 25 L of plasma [29, 30]. This shows that it is possible to successfully conduct a TLC separation using the plasma volume that could recovered by a finger stick.

Example 3Self-Contained Cartridge Design

[0345] A self-contained cartridge was designed and built to fully contain the TLC plate and the mobile phase to prevent release of fumes or silica dust. The cartridge comprised the following components.

TABLE-US-00004 Component TLC plate Casing Insulation Breakable glass barrier Chloroform MeOH Acetic acid

[0346] Plastics components are readily prepared by injection molding or 3-D printing, according to methods well known in the art. Plates, glass elements and solutions are available commercially, for example from Sigma Aldrich.

[0347] Access to the inside of the hermetically sealed cartridge is achieved by using hypodermic needles to pass through rubber septa placed at different locations in the cartridge as shown in FIGS. 9, 10 and 11. The design incorporates two separate chambers, one for the TLC plate and the second for the mobile phase. These two chambers keep the TLC plate separate from the mobile phase before use during transport and storage. A weak point in the separation between the chambers has been incorporated in the form of a glass coverslip which can be easily broken by inserting a needle through a designated rubber septa.

[0348] The use of the TLC cartridge use the following simple protocol: [0349] 1. Load the samples onto the TLC plate using a hypodermic needle to penetrate through the rubber septa. This step will be automated in the future. [0350] 2. Use the hypodermic needle to break the glass coverslip separating the two chambers [0351] 3. Tip up the cartridge to let the mobile phase flow into the TLC chamber and come into contact with the bottom of the TLC plate allowing the plate to develop normally by capillary forces. [0352] 4. Tip the cartridge back down to a flat orientation to stop the TLC plate development by allowing the mobile phase to drain back into the second chamber. [0353] 5. In this flat orientation the TLC plate can be imaged using a CCD camera system.

[0354] The cartridge is designed with readily available solvent resistant materials that allow it to be disposable. This prevents any cross contamination between patient samples and also reduces the maintenance burden for an automated system.

Example 4Discussion of Examples 1 to 3

[0355] TLC has been used for drug quantification for many years [11-14], and the process has been partially automated for high throughput applications [15].

[0356] However, prior to the present invention, the TLC process has never been adapted for use in a bedside clinical setting. This is advantageous because many drugs can have significant degradation within 15 min of collection if not immediately spun down to remove red blood cells and cooled on ice [16]. Requiring the sample to be sent to the hospital lab for analysis usually incurs delay which can adversely affect the accuracy of the final blood concentration estimate.

[0357] The TLC based separation and quantification method described here has been successful in quantifying actual clinical patient samples with 83% the DOX content and 86% of the irinotecan content falling within 15% of the HPLC derived values. This method meets the FDA recommendations for the accuracy of a bioanalytical method [31]. It also shows that the process can be adapted for use with different drug compounds. The only changes that need to be made to the system to be applied to a different drug is the choice of the proper internal standard (as would be required for HPLC analysis) and the right adjustments to the single mobile phase.

[0358] The process described herein may also be used with drugs that are not inherently fluorescent. The use of TLC plates coated with an appropriate inorganic fluorescent compound (such as commercially available F254 plates) allows photon absorption of the drugs to be optically monitored using the same TLC process, except looking for blocked fluorescence from the TLC plate. The analysis of the greyscale image of the TLC plate is then inverted so the bands would show up as varying degrees of grey against a black background to allow the analysis software to quantify the band fluorescence intensity.

[0359] The custom written MatLab image analysis and quantification software was successfully applied to both the DOX and the irinotecan samples. The only changes necessary in the program parameters were to adjust the cutoff intensity values used to determine the boundaries of the actual bands because the fluorescence intensity behavior of the two compounds was different.

[0360] We also demonstrate that this simple extraction method and single mobile phase development make this TLC based technique applicable for a self-contained cartridge design. The prototype cartridge described in the preferred embodiment shown here consists of different PVC plastic layers that under compression create a sealed cartridge using fluorocarbon rubber seals. It will be appreciated that injection molding techniques could be used to create cartridges that have fewer layers and use less material.

[0361] In Summary, the TLC method described here successfully quantified over 80% of both the DOX and irinotecan clinical samples by being within 15% of established HLC values which meets the FDA criteria for accuracy of a bioanalytical method [31].

REFERENCES FOR DESCRIPTION AND EXAMPLES 1-4

[0362] 1. Wilkinson, G. R., Drug metabolism and variability among patients in drug response. New England Journal of Medicine, 2005. 352(21): p. 2211-2221. [0363] 2. Klotz, U., Pharmacokinetics and drug metabolism in the elderly. Drug metabolism reviews, 2009. 41(2): p. 67-76. [0364] 3. Sawyer, M. and M. J. Ratain, Body surface area as a determinant of pharmacokinetics and drug dosing. Investigational new drugs, 2001. 19(2): p. 171-177. [0365] 4. Gurney, H., Dose calculation of anticancer drugs: a review of the current practice and introduction of an alternative. Journal of Clinical Oncology, 1996. 14(9): p. 2590-2611. [0366] 5. de Jonge, M. E., et al., Individualised cancer chemotherapy: strategies and performance of prospective studies on therapeutic drug monitoring with dose adaptation. Clinical pharmacokinetics, 2005. 44(2): p. 147-173. [0367] 6. Galpin, A. J. and W. E. Evans, Therapeutic drug monitoring in cancer management. Clinical chemistry, 1993. 39(11): p. 2419-2430. [0368] 7. Petros, W. P., et al., Associations between drug metabolism genotype, chemotherapy pharmacokinetics, and overall survival in patients with breast cancer. Journal of clinical oncology, 2005. 23(25): p. 6117-6125. [0369] 8. Robert, J., et al., Pharmacokinetics of adriamycin in patients with breast cancer: correlation between pharmacokinetic parameters and clinical short-term response. European Journal of Cancer and Clinical Oncology, 1982. 18(8): p. 739-745. [0370] 9. Touw, D., et al., Cost-effectiveness of therapeutic drug monitoring: a systematic review. Therapeutic drug monitoring, 2005. 27(1): p. 10-17. [0371] 10. Bardin, C., et al., Therapeutic drug monitoring in cancerAre we missing a trick? European Journal of Cancer, 2014. 50(12): p. 2005-2009. [0372] 11. Yesair, D., et al., Comparative pharmacokinetics of daunomycin and adriamycin in several animal species. Cancer research, 1972. 32(6): p. 1177-1183. [0373] 12. Brenner, D. E., et al., Improved high-performance liquid chromatography assay of doxorubicins detection of circulating aglycones in human plasma and comparison with thin-layer chromatography. Cancer chemotherapy and pharmacology, 1985. 14(2): p. 139-145. [0374] 13. Chan, K. K. and C. D. Wong, Quantitative thin-layer chromatography: thin-film fluorescence scanning analysis of adriamycin and metabolites in tissue. Journal of Chromatography A, 1979. 172(1): p. 343-349. [0375] 14. Watson, E. and K. Chan, Rapid analytic method for adriamycin and metabolites in human plasma by a thin-film fluorescence scanner. Cancer treatment reports, 1976. 60(11): p. 1611-1618. [0376] 15. Fredriksson, S., K. Elwinger, and J. Pickova, Fatty acid and carotenoid composition of egg yolk as an effect of microalgae addition to feed formula for laying hens. Food Chemistry, 2006. 99(3): p. 530-537. [0377] 16. Kontny, N. E., et al., Minimization of the preanalytical error in plasma samples for pharmacokinetic analyses and therapeutic drug monitoring-using doxorubicin as an example. Therapeutic drug monitoring, 2011. 33(6): p. 766-771. [0378] 17. Therasse, P., et al., New guidelines to evaluate the response to treatment in solid tumors. Journal of the National Cancer Institute, 2000. 92(3): p. 205-216. [0379] 18. Lennard, L., Therapeutic drug monitoring of cytotoxic drugs. British journal of clinical pharmacology, 2001. 52(S1): p. 75-87. [0380] 19. Ibsen, S., et al., Extraction protocol and mass spectrometry method for quantification of doxorubicin released locally from prodrugs in tumor tissue. Journal of Mass Spectrometry, 2013. 48(7): p. 768-773. [0381] 20. Paci, A., et al., Review of therapeutic drug monitoring of anticancer drugs part 1cytotoxics. European Journal of Cancer, 2014. 50(12): p. 2010-2019. [0382] 21. Gurney, H., How to calculate the dose of chemotherapy. British journal of cancer, 2002. 86(8): p. 1297-1302. [0383] 22. Besse, B., et al., 2nd ESMO Consensus Conference on Lung Cancer: non-small-cell lung cancer first-line/second and further lines of treatment in advanced disease. Annals of oncology, 2014. 25(8): p. 1475-1484. [0384] 23. Licata, S., et al., Doxorubicin metabolism and toxicity in human myocardium: role of cytoplasmic deglycosidation and carbonyl reduction. Chemical research in toxicology, 2000. 13(5): p. 414-420. [0385] 24. Hortobagyi, G., Anthracyclines in the treatment of cancer. Drugs, 1997. 54(4): p. 1-7. [0386] 25. Marangon, E., et al., Development and Validation of a High-Performance Liquid ChromatographyTandem Mass Spectrometry Method for the Simultaneous Determination of Irinotecan and Its Main Metabolites in Human Plasma and Its Application in a Clinical Pharmacokinetic Study. PloS one, 2015. 10(2): p. e0118194. [0387] 26. Krischke, M., et al., Pharmacokinetic and pharmacodynamic study of doxorubicin in children with cancer: results of a European Pediatric Oncology Off-patents Medicines Consortium trial. Cancer chemotherapy and pharmacology, 2016. 78(6): p. 1175-1184. [0388] 27. McDade, T. W., S. Williams, and J. J. Snodgrass, What a drop can do: dried blood spots as a minimally invasive method for integrating biomarkers into population-based research. Demography, 2007. 44(4): p. 899-925. [0389] 28. Robison, E. H., et al., Whole genome transcript profiling from fingerstick blood samples: a comparison and feasibility study. BMC genomics, 2009. 10(1): p. 617. [0390] 29. Haeberle, S., et al., Centrifugal extraction of plasma from whole blood on a rotating disk. Lab on a Chip, 2006. 6(6): p. 776-781. [0391] 30. Brun, J., et al., The paradox of hematocrit in exercise physiology: which is the normal range from an hemorheologist's viewpoint? Clinical hemorheology and microcirculation, 2000. 22(4): p. 287-303. [0392] 31. Health, U.D.o. and H. Services, Guidance for industry, bioanalytical method validation. http://www.fda.gov/cder/guidance/index.htm, 2001.

Example 5pH-Mediated Molecular Differentiation for Fluorimetric Quantification of Chemotherapeutic Drugs in Human Plasma

[0393] Irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyl-oxycamptothecin, CPT-11) is commonly used as an antitumor drug. The drug is considered a prodrug since it undergoes cleavage of the bispiperidino-side chain by carboxyesterase to form SN-38 (7-ethyl-10-hydroxycampto-thecin), an active metabolite that has shown to be up to 1,000 more potent at inhibiting topoisomerase I than the parent Irinotecan.

[0394] Some of the main challenges for the simultaneous quantification of Irinotecan and SN-38 reside in the fact that their absorption and fluorescence properties are almost identical under physiological conditions, and the fact that the concentration of SN-38 can be over 30 times lower than that of Irinotecan.

[0395] The pH of the media in which molecules are dissolved can play a major role not only in properties such as their solubility, but also in their optical performance.

[0396] The premise of the pH-mediated molecular differentiation is illustrated as follows and in FIG. 14A:

[0397] Generic Case:

##STR00002##

Example

[0398] ##STR00003##

[0399] For Irinotecan and SN-38, this was investigated by characterising the absorbance as well as the emission behaviour at excitation wavelengths of 370 nm and 430 nm under acidic (pH=1.4) and basic conditions (pH=12.1). As shown in FIG. 14B, Irinotecan exhibited near identical optical properties for both absorbance and fluorescence under acidic and basic conditions, while SN-38 displayed a pronounced shift for absorbance as well as emission when basifying.

[0400] A rationalisation of the molecular structure under acidic and basic conditions is depicted in the following scheme shows the effect of pH in the chemical structure of Irinotecan (top), and SN-38 (bottom):

##STR00004##

[0401] A solid phase extraction protocol was used to assess the effectiveness of a pH-mediated molecular differentiation using real patient sample compositions.

[0402] While the fluorescence of Irinotecan was measured in acidic conditions (pH=1.4) using an excitation wavelength of 370 nm, the fluorescence of SN-38 was determined in basic conditions (pH=12.1) with an excitation wavelength of 430 nm. The results are summarised in FIG. 16. In total seven samples were run in triplicate, six spiked and one reference sample. For Irinotecan, 17 out of 18 obtained data points were within 15% error (94%). In the case of SN-38, 16 out of 18 data points were within this tolerance (89%). We note that for samples with concentrations below 100 nM for Irinotecan and 15 nM for SN-38, the measured background signal of the reference sample became significant, resulting in higher errors below this level, which is at the bottom end of the relevant clinical range.

[0403] In order to show the applicability of this approach to other commonly used chemotherapeutic drugs with similar molecular structure, we compared the pH-dependent optical properties of SN-38 to Epirubicin and Methotrexate. With the former containing an alcohol and the latter an amine group attached to the aromatic core, a basic pH is likely to cause deprotonation resulting in a change of the optoelectronic properties of the molecules. The absorbance and fluorescence spectra of the three compounds were recorded in acidic (pH=1.4) and basic (pH=12.1) conditions (data not shown). In all cases, a change from acidic to basic conditions resulted in a modification in the optical properties of the drugs, with the absorption spectrum shifting to longer wavelengths, from 30 nm for Methotrexate to almost 85 nm for Epirubicin, which compared to 40 nm for SN-38. The bathochromic shift in the absorbance spectra of the molecules in basic conditions was determined to be 15 nm in the case of Methotrexate and 97 nm for Epirubicin in comparison to 137 nm for SN-38.

[0404] In conclusion, we have demonstrated that pH-mediated molecular differentiation can be utilised to quantify the amount of a chemotherapeutic prodrug and its active metabolite spiked in human plasma at clinically relevant concentrations.

Example 6Utility of Real-Time Point-of-Care (POC) Device of the Invention in Cancer Therapeutic Drug Monitoring C-TDM

[0405] Global Cancer: Scale of the Problem

[0406] In 2012, there were 8.2 million deaths worldwide attributable to cancer and 14.1 million new cancer cases. 1.8 million new cases were in North America, 1 million in Europe and 4.1 million in East Asia. Despite accounting for only 20% of the global population, 43% of new cancer cases occur in developed countries. The most common cancers in the developed world are lung, colorectal, prostate and breast with lung cancer claiming the highest fatality rate for both men and women [1].

[0407] The World Health Organisation predicts that cancer cases will increase globally by 70% over the next two decades [2] and that the number of deaths will reach 13.1 million [3]. Despite increases in cancer research funding, some cancers have seen little to no progress in the last decade: brain, lung, cancer and oesophageal cancers continue to prove difficult to treat [3].

[0408] After the first administration of a chemotherapeutic agent, practitioners typically monitor drug efficacy based on the cancer or tumour's response to the drug. Methods to do this involve feeling external lumps, tumours and nodes to see if the size has decreased, doing the same but with various imaging techniques for internal cancers, blood tests such as ones that measure organ function, and tests for biomarkers that are specific to certain types of cancer. It is typical for these measurements to be taken every two to three cycles of chemotherapy, with each cycle lasting between two to six weeks [25]. This means that many patients will go for months without knowing if they are responding to treatment. There is likely a large demand for methods that will either provide more timely response data or allow drug optimisation sooner in the treatment process.

[0409] Precision Medicine and Therapeutic Drug Monitoring

[0410] Precision medicine (not limited to cancer) currently has a market size of $43 billion and is expected to grow rapidly to $71 billion by 2021 [9].

[0411] One form of precision medicine involves use of therapeutic drug monitoring (TDM). For a select variety of drugs, the serum or blood concentration correlates with efficacy [11]. The need for TDM arises when there is high inter-individual variability in the metabolism, distribution, absorption and excretion of the drug, which is a common feature of chemotherapeutic agents [11]. Plasma drug concentrations typically vary by as much as 14-fold for most chemotherapy drugs and as much as 100-fold for 5-fluorouracil. Despite this, chemotherapy dosage levels are based on estimations of the patients surface area and TDM is not commonplace; it has only seen clinical use in the monitoring of methotrexate and busulfan [11].

[0412] For a drug to benefit significantly from TDM, it must have significant variability in inter- or intra-individual pharmacokinetics, a defined relationship between blood concentration and pharmacological efficacy, a narrow therapeutic index and a precise and accurate assay to measure it. The introduction of widespread TDM for chemotherapy has been slowed primarily by the fact that there are no available assays for the drugs. However, there have also been problems in defining the relationship between blood/serum concentrations and efficacy, and the advent of combination therapies has made this even more challenging [11].

[0413] Almost all serum/blood drug concentration monitoring is carried out using HPLC or LC-MS. Methotrexate is monitored every 24 h for a period of 3 days and this is typically performed by immunoassay, though chromatography methods do exist. Plasma busulfan concentrations are routinely monitored in high dose, paediatric patients. A calibration curve consisting of seven to twelve distinct time-points is taken at the first administration of the drug with the concentration being determined by chromatography, though new ELISA methods have been developed [11].

[0414] Bach et al. [11] highlighted a number of chemotherapy drugs which they think are prospective candidates for TDM: mTOR inhibitors, 5-flourouracil, imatinib, EGF receptor inhibitors, platinum based agents, etopisode, doxorubicin and suramin. The authors highlight that many of these drugs are not subject to TDM due to poor understanding of the relationship between dose and efficacy and that strong evidence and understanding of this relationship is paramount to justify monitoring.

[0415] Point of Care Testing Market

[0416] Point of care (POC) medicine is a growing field of medical testing devices that can be used be used at, or near, the point of care, i.e. the bedside or GP surgery instead of in a medical laboratory. The benefits of point of care testing are that it brings the test immediately to the patient, meaning that the caregiver receives the results of the test faster, thereby enabling timely diagnosis and more dynamic monitoring and treatment of patients. POC testing is exceptionally important in the field of personalised, targeted medicine as it allows cheap, easy and effective ways to diagnose and monitor patients.

[0417] However POC monitoring of cancer treatment has not yet been widely accepted. This is part of a wider trend in TDM POC devices which lag behind other fields in which POC devices have been successfully deployed. Sanavio and Krol [17] suggest a variety of reasons for this in their 2015 review on the matter. One major problem is the lack of evidence to support significantly superior clinical performance, in terms of cost-effectiveness, granted by TDM for most drugs. As healthcare providers are trying to provide services at reduced cost wherever possible, TDM must be supported by strong data that it will significantly improve care to justify the cost (or it must be cheap enough that this justification is unnecessary). Another cited issue is that most drug development pipelines and clinical trials only require dose tolerance and dose response data with pharmacokinetics and pharmacodynamics data being a secondary objective at best. There is therefore often very little pharmacokinetic and pharmacodynamic data on the drug and so rarely any commentary on the efficacy of TDM. Thus when pharmaceutical companies design clinical trials, they do not presently consider TDM as an option, either when choosing which drugs to trial or when designing the format of the trial, meaning that most of the drugs on the market have little need for TDM.

[0418] The availability of a new cheaper method of performing TDM as described herein therefore provides an important contribution to the art.

Example 7Utility of Real-Time POC Medical Device of the Invention in Other Therapeutic Areas

[0419] As explained above, the present invention opens up new utilities for POC TDM analysis of drugs.

[0420] Typical examples of drugs that may typically be monitored are antiepileptics, antiarrhythmics, immunosuppressants, and antibiotics.

[0421] Some bronchodilators, psychoactive drugs and chemotherapeutics are also monitored.

[0422] Three examples of drug classes in which the invention can show particular utility are: chemotherapeutics, antiarrhythmics and fungicidal agents as all have a large portion of drugs that are recommended for monitoring or already are monitored. Furthermore these drugs have a large portion of natively fluorescent compounds which facilitates detection within the TLC cartridge platform

[0423] The following table consists of drugs that are fluorescent. The drugs that are not currently monitored were identified from a range of reviews highlighting drugs that should be considered for TDM [19] [20] [21] [22] [23] [24]:

Example 8Further Cartridge Embodiment

[0424] A further embodiment of a cartridge of the present invention is shown in FIGS. 18 to 30.

[0425] The first embodiment shown in FIGS. 10 and 11 was based on a planar (16 layer) design formed from acrylic, Viton and PTFE sheeting with an integrated glass liquid-shield that segregated the mobile phase from the TLC plate prior to elution.

[0426] The further embodiment shown in FIGS. 18 and 19 has a primarily bipartite design comprising upper (face, or front) and lower (base, or back) body units, with further elements as shown in FIGS. 19 to 29.

[0427] In this further embodiment as illustrated, the two body units are aluminium and the window is float glass.

[0428] The liquid mobile phase is segregated from the face chamber prior to elution. In this example the segregator foil (15) is aluminium foil.

[0429] O-rings (or gaskets) or septa are used to hermetically seal different fluid or vapour holding volumes of the cartridge as shown: specifically the cartridge illustrated includes a body o-ring (16), reservoir o-ring (17), window o-ring (04) and septa for injection of analytes and standards, and releasing the mobile phase (07). In this example these elements are fluoroelastomers (FKM).

[0430] This cartridge is designed so that the total mass and centre of mass of the cartridge can be adjusted by modifying the solid fill in the upper and lower units, see FIG. 21. The TLC plate is mounted and secured using the support posts shown in FIG. 25. The dimensions of the TLC plate are shown in FIG. 26.

[0431] Additional support along the backside and the incorporation of stand-off posts on the internal side of the face unit prevent both front-side wicking and rear-to-front side wicking of the liquid mobile phase during elution, see FIG. 27. These are also shown in FIG. 28.

[0432] A cross section of the bridge and injection ports shows the edge extrusions that prevent wicking, see FIG. 29.

[0433] To prepare the cartridge, the elements shown in FIG. 18 were provided.

[0434] Tension was applied by body screws (08) between the upper and lower units to form two seals. A unit seal around the outer edge of the cartridge preventing leakage of mobile phase into the working environment i.e. compression of body o-ring (16). In addition, the same tension applied by the body screws simultaneously forms a seal around the liquid mobile phase reservoir in the lower unit through compression of the reservoir o-ring (17). This is achieved through translation of force through the bridge, see FIG. 19.

[0435] The cartridge can be charged or is recharged upon removal and replenishment of the TLC plate (10), segregator (15) and liquid mobile phasesee FIG. 20.

[0436] In use samples and standards can be loaded via the injection ports shown in FIGS. 17, 18, 22, 26 and 28.

[0437] The TLC plate is fully supported under each injection port such that a hypodermic needle entering the device will not deform or damage the plate whilst spotting.

[0438] The segregator foil is pierced by inserting a needle through the septum (FIG. 22, 19). The hypodermic needle is captured/engages with a well or pocket in the paddle (20) and follows a curved arc downwards (21).

[0439] This motion results in a large aperture being formed in the foil segregator, see FIG. 23. The length/height of this aperture is important as air must be allowed to back-fill the reservoir upon rotation, see FIG. 24.

[0440] The mobile phase reservoir follows a tapering v contour below the TLC plate (in both the face and rear compartments) but enters a rectilinear profile before the lowest point of the TLC plate, see FIG. 30. This ensures the mobile phase wicks up the plate evenly.

TABLE-US-00005 TABLE D Leading candidate drugs for TDM. Drug Class Drug Therapeutic window (g/mL) Current TDM method Antiepileptics Stiripentol 10-15 AED monitoring Antiarrhythmics NAPA 4-8 Homogenous enzyme immunoassay Amiodarone 0.5-2.sup. HPLC, LC-MS, ELISA Flecainide 0.2-1.sup. HPLC, Immunoassay Mexiletine 0.5-2.5 HPLC, GC Quinidine 2-5 Homogenous enzyme immunoassay Procainamide 4-8 Homogenous enzyme immunoassay Propafenone 0.06-1 Not currently monitored Sotalol 0.5-3.sup. Not currently monitored Verapamil 0.025-0.25 Not currently monitored Antibiotics Linezolid 1.8-7.5 Not currently monitored Ciprofloxacin 0.1-8.3 Not currently monitored Fluconazole 1-5 Not currently monitored Antifungals Itraconazole 0.5-17 Not currently monitored Voriconazole 1-5 Not currently monitored Posaconazole Maintain at 0.7 Not currently monitored Immunosuppressants Mycophenolic Acid 1-4 Homogenous enzyme immunoassay Dermal medicines Salicylate 15-30 Spectrophotometric Chemotherapeutics 5-fluoruoracil 2000-3000 Not currently monitored Imatinib 1-3 Not currently monitored Etopisode 1-10 Not currently monitored Doxorubicin 10-58 Not currently monitored Suramin Maintain at 500 Not currently monitored Sunitinib Not yet determined Not currently monitored In-depth list of candidate drugs Analyte for existing Natively Class Drug Window Test method test Fluorescent? Antiepileptic Carbamazepine 5-10 ug/mL Homogenous enzyme immunoassay Serum, plasma Possibly, might require acid activation Phenobarbital 10-35 ug/mL Homogenous enzyme immunoassay Serum, plasma No, requires labelling Phenytoin 10-20 ug/mL Homogenous enzyme immunoassay Serum, plasma No, requires analog Clobazam Varies, 0.2-5 HPLC Plasma No, requires ug/mL additional processing Tiagibine 20-200 ng/mL HPLC Serum Unknown Ethosuximide 40-100 ug/mL Enzyme immunoassay Serum, plasma Unknown Gabapentin 2-20 ug/mL Homogenous enzyme immunoassay Serum, plasma requires processing Lacosamide 5-10 ug/mL Homogenous enzyme immunoassay Serum, plasma Unknown Oxcarbazepine 3-35 mg/mL Homogenous enzyme immunoassay Serum, Unknown Plasma Primidone 5-12 ug/mL Fluoroimmunoassay serum, plasma No Vigabratin 0.8-36 ug/mL HPLC Plasma, urine Requires processing Zonisamide 10-40 ug/ml Homogenous enzyme immunoassay Serum, plasma Unknown, Leveltiracetam 20-40 ug/mL Homogenous enzyme immunoassay Serum, plasma No, requires derivitsation Valproic acid 50-125 ug/mL Homogenous enzyme immunoassay Serum, plasma Probably not, requires a probe Clonazepam 20-80 ug/mL No existing test N/A Requires processing Nitrazepam 0.03-0.1 ug/mL No existing test N/A Requires additions and processing Felbamate 50-110 ug/mL No existing test N/A No required probe Levetiracetam 10-40 ug/mL No existing test N/A No, requires derivitisation Topiramate 2-10 ug/mL No existing test N/A No, requires labelling Rufinamide ~15 ug/mL No existing test N/A Can't find anything Stiripentol 10-15 ug/mL No existing test N/A Yes Lamotrigine 2.5-15 ug/mL Homogenous enzyme immunoassay Serum, plasma Requires processing Antiarrhythmics Digoxin 0.0005-0.002 ug/mL Homogenous enzyme immunoassay Serum, plasma Requires processing Lidocaine 1.5-5 ug/ml Homogenous particle enhanced Serum, plasma Probably not, turbidimetric immunoassay requires a probe NAPA 4-8 ug/mL Homogenous enzyme immunoassay Serum, plasma Yes Amiodarone 0.5-2 ug/mL HPLC, LC-MS, ELISA Serum, plasma Yes Flecainide 0.2-1 ug/mL HPLC, immunoassay Serum Yes Mexiletine 0.5-2.5 ug/mL HPLC, GC Serum, plasma Yes Quinidine 2-5 ug/mL Homogenous enzyme immunoassay Serum, plasma Yes Procainamide 4-8 ug/ml Homogenous enzyme immunoassay Serum, plasma Yes Disopyramide 2-4 ug/ml No existing test N/A No requires immunoassay Propafenone 0.064-1.044 ug/mL No existing test N/A Yes Sotalol 0.5-3 ug/mL No existing test N/A Yes Verapamil 0.025-0.25 ug/mL No existing test N/A Yes Antibiotics Gentamicin 0.5-2/5-10 ug/mL Homogenous enzyme Serum, plasma No, required tagging immunoassay Tobramicin 2-10 ug/mL Homogenous enzyme Serum, plasma No, requires tagging immunoassay Vancomycin 10-20 ug/mL Homogenous enzyme Serum, plasma No, requires tagging immunoassay Amikacin 5-15 ug/mL Homogenous particle enhanced Serum, plasma No, requires tagging turbidimetric immunoassay Teicoplanin 10-60 ug/mL No existing test N/A No, requires immunoassay Linezolid 1.75-7.53 ug/ml No existing test N/A Yes Ciprofloxacin 0.1-8.3 ug/mL No existing test N/A Yes Fluconazole 1-5 ug/mL No existing test N/A Yes Antifungals Itraconazole 0.5-17 ug/mL No existing test N/A Yes Voriconazole 1-5 ug/mL No existing test N/A Yes Posaconazole ~0.7 ug/mL No existing test N/A Yes Flucytosine 70-100 ug/mL No existing test N/A No Antimanics Lithium 0.6-1.2 mEq/L Spectrohptoemetrically with Serum No enzyme assay system Bronchodilators Theophylline 5-15 ug/mL Homogenous enzyme Serum, plasma No, requires tagging immunoassay Immunosupressants Cyclosporin 0.1-0.4 ug/mL Chemiluminescent Blood No, requires analog microparticle immunoassay Mycophenolic 1-4 ug/mL Homogenous enzyme Plasma Yes Acid immunoassay Tacrolimus 5-20 ug/mL Affinity chrome- mediated Blood No, requires analog immunoassay Sirolimus 5-10 ng/mL Chemiluminescent Blood Unknown microparticle immunoassay Azathioprine can't find No existing test N/A Steroids Varies No existing test N/A anti-lymphocyte can't find No existing test N/A globulin OKT3 can't find No existing test N/A Daclizumab can't find No existing test N/A Basiliximab can't find No existing test N/A Antianginal Perhexeline 0.3-4 ug/mL HPLC, LC-MS Plasma Requires processing Anticonvulsant Lamotrigine 2.5-15 ug/mL Homogenous enzyme Serum, plasma Requires processing immunoassay Dermal treatment Salicylate 15-30 ug/mL Spectrophotometric Urine, Yes serum, plasma Cancer Methotrexate Varies with use Homogenous Serum, plasma No enzyme immunoassay Busulfan 0.9-1 ug/ml HPLC, LC-MS Serum, plasma Requires processing mTOR inhibitors 0.005-0.015 ug/mL No existing test Unknown 5-flourouracil 2000-3000 ug/ml No existing test Yes imatinib 1-3 ug/ml No existing test Yes EGF Various No existing test Unknown receptor inhibitors platinum Various No existing test No requires analog based agents etopisode 1-10 ug/mL but No existing test Yes highly varied from patient to patient doxorubicin 10.4-57.7 mg/m{circumflex over ()}2 No existing test Yes suramin max 500 ug/ml No existing test Yes nilotinib ~0.5 ug/ml No existing test No requires hybridisation dasatinib ~0.05 ug/ml No existing test No requires hybridisation erlotinib 100-150 mg/day No existing test No - causes quenching of BSA sunitinib Still being No existing test Yes determined sorafenib 800 mg/day No existing test No - required fluorescent agents retuximab Maintain at 375 No existing test No-required label mg/m.sup.2 cetuximab Maintain dose No existing test No-required label at 250 mg/m{circumflex over ()}2 Antibodies Adalimumab 3.5-7 ug/mL No existing test No - required label for autoimmune diseases Certolizumab 3-84 ug/mL No existing test No - required label pegol Infliximab ~3.8 ug/mL No existing test No - required label

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