A Method for Measuring a Concentration of a Biomarker-Analyte In Blood from Mammals

Abstract

The present invention relates to a method for measuring a concentration of one or more biomarker in blood from a mammal comprising: —providing a kit of parts for sampling blood comprising a lancet and a capillary, a vial comprising an extraction fluid and one or more internal standard, which is a radioisotope of one or more biomarker adapted to assess a quality and a concentration of the one or more biomarker in the blood sample, —distributing the kit of parts to the mammal, —receiving the vial comprising a blood sample inserted into the vial, —analysing the sample to determine the quality of the sample and the concentration of the one or more biomarker in the blood of the mammal by centrifuging the vial and performing a direct analysis of the supernatant.

Claims

1. A method for measuring a concentration of one or more biomarker/analyte in blood from a mammal comprising: providing a kit of parts for sampling blood, the kit of parts comprising a lancet and a capillary adapted to sample a defined volume of blood from the mammal and a vial comprising an extraction fluid together with one or more internal standard, wherein the one or more internal standard is a radioisotope of one or more biomarker selected from a group comprising one or more of 2H 3H, 11C, 13C, 14C, 13N, 15N, 15O, 17O and 18O radioisotope of said biomarker adapted to assess a quality and a concentration of the one or more biomarker in the blood sample, distributing the kit of parts to a location of the mammal, receiving the vial comprising a blood sample from the mammal inserted into the vial, analysing the blood sample to determine the quality of the sample and the concentration of the one or more biomarker in the blood of the mammal by centrifuging the vial and performing a direct analysis of the supernatant, and normalizing the analyzed results between different samples and a standard curve.

2. The method according to claim 1, wherein the one or more internal standard is a 2H, 3H, 11C, 13C, 14C, radioisotope of one or more biomarker.

3. The method according to claim 1, wherein the one or more internal standard is a 13C and/or 2H radioisotope of one or more biomarker.

4. The method according to claim 1, wherein the one or more internal standard is a 2H radioisotope of one or more biomarker.

5. The method according to claim 1, wherein the one or more internal standard is a 13C radioisotope of one or more biomarker.

6. The method according to claim 1, wherein the one or more internal standard is adapted for measuring a quality and a concentration of one or more pharmaceutical active compound (API) that can be stabilized in an extraction fluid, whereby the API is selected from the group comprising antifungal API, antiviral API, immunodepression API, anticonvulsant API, antidepressant API, antibiotic API, anticancer API, vitamins, cardiovascular API, painkilling API and opiates.

7. The method according to claim 1, wherein the one or more internal standard is adapted for measuring or monitoring a quality and a concentration of one or more pharmaceutical active compound (API) in the blood of a mammal that can be stabilized in an extraction fluid, whereby the one or more API is selected from the group comprising tamoxifen, 4-hydroxytamoxifen, CPA-cyclophosphamide and its active metabolite PAM-hb, DOC-docetaxel, DOX-doxorubicine, PAC-paclitaxel, EPI-epirubicin, statins, steroids, such as testosterone, or vitamins, such as vitamin B or D, or compounds related to addictions, such as opioids, cocaine, heroin, fentanyl, cannabinoids, marijuana, benzodiazepines, hallucinogens and methamphetamine, codeine, ibuprofen, dihydrocodeine, morphine, dextropropxyphene napsylate, aminorex, lidocaine, iso-LSD, norcocaine, amitriptyline, pemoline, prazepam, imipramine, propafenone, pheniramine, chlorpromazine, amphetamine sulfate, lofexidine hydrochloride, clozapine, ecgonine methyl ester, diphenylhydramine, estazolam, 3,4-methylenedioxy-amphetamine, melatonin, doxepin, ketamine, mescaline, aprobarbital, buprenorphine, benzoylecgnine, trifluoperazine, methadone, ecgonine ethyl ester, midazolam, fentanyl, norketamine, chlordiazepoxide, caffeine, hydrocodone, fenfluramine, tramadol, lorazepam, phenylpropanolamine, flunitrazepam, 2C—B, amobarbital, flurazepam, phencyclidine (PCP), barbital, carbamazepine, vancomycin and phenobarbital.

8. The method according to claim 1, wherein the one or more internal standard is a radioisotope of tamoxifen, z-endoxifen and/or 4-hydroxytamoxifen adapted for measuring the amount of tamoxifen, z-endoxifen and/or 4-hydroxytamoxifen in the blood of a mammal.

9. The method according to claim 8, wherein the internal standard is a 13C-radioisotope of tamoxifen, z-endoxifen and 4-hydroxytamoxifen.

10. The method according to claim 1, wherein the one or more internal standard is phosphatidylethanol 16:0/18:1 (PEth-16:0/18:1, PEth 16:0/18:2, PEth 18:1/16:0, and/or PEth 18:2/16:0) adapted for measuring long-term alcohol consumption.

11. The method according to claim 1, wherein the one or more internal standard is a radioisotope of methylmalonic acid adapted for measuring B-vitamin deficiency in a mammal.

12. The method according to claim 1, wherein the one or more internal standard is a radioisotope of dihydrotachysterol adapted for measuring D-vitamin deficiency in a mammal.

13. The method according to claim 1, wherein the one or more internal standard is adapted for measuring or monitoring a quality and a concentration of one or more pharmaceutical active compound (API) in the blood of a mammal that can be stabilized in an extraction fluid, whereby the one or more API is selected from the group comprising compounds related to addictions, such as opioids, cocaine, heroin, fentanyl, cannabinoids, marijuana, benzodiazepines, hallucinogens and methamphetamine, amphetamine, codeine, dihydrocodeine, morphine, iso-LSD, phencyclidine (PCP), norcocaine, fentanyl, methadone, hydrocodone and tramadol.

14. The method according to claim 1, wherein the one or more internal standard is adapted for measuring or monitoring a quality and a concentration of one or more statins selected from the group comprising atorvastatin, fluvastatin, cerivastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin, or mixtures thereof.

15. The method according to claim 1, wherein the one or more internal standard is adapted for measuring or monitoring a quality and a concentration of one or more steroid hormones selected from the group comprising alclometasone, prednisone, dexamethasone, triamcinolone, cortisone, fludrocortisone, oxandrolone, oxabolone, testosterone, nandrolone, diethylstilbestrol (DES) and estradiol, norethisterone, medroxyprogesterone acetate, hydroxyprogesterone caproate, cyproterone acetate, mifepristone and gestrinone, or mixtures thereof.

16. The method according to claim 1, wherein the one or more internal standard is adapted for measuring or monitoring a quality and a concentration of one or more antidepressant API selected from the group comprising bupropiontrazodone, nefazodone, vilazodone, vortioxetine, mitriptyline, bupropion, bupropion, bupropion, bupropion, bupropion, citalopram, desvenlafaxine, duloxetine, escitalopram, fluoxetine, mirtazapine, nortriptyline, paroxetine, sertraline, trazodone, venlafaxine, or mixtures thereof.

17. The method according to claim 1, wherein the blood sample is analysed using mass spectrometry (MS), liquid chromatography-mass spectrometry (LC-MS, LC-MS/MS, gas chromatography-mass spectrometry (GC-MS) or GC-MS/MS.

18. The method according to claim 1, wherein the extraction fluid is selected from the group comprising 2-propanol, methanol and acetonitrile, formic acid or mixtures thereof, which fluid is adapted to substantially stop all enzyme activity in the blood sample, stabilize the sample and extract the biomarker from the blood sample.

19. The method according to claim 1 for use in measuring or monitoring a quality and a concentration of one or more active pharmaceutical compound in the blood of a mammal, whereby the pharmaceutical active compound has a narrow therapeutic window selected from the group comprising or consisting of tamoxefin, digoxin, digitoxin, fosphenytoin, phenytoin, ethosuximide, alfentanil, tacrolimus, meperidine, temsirolimus, sirolimus, thiopental, fentanyl, alfentanil, theophylline, cyclosporine, clonidine, amitriptyline, protriptyline, imipramine, nortriptyline, quinidine, levothyroxine, carbamazepine, phenobarbital, ergotamine, dihydroergotamine and heparin.

20. The method according to claim 1, for use in Tailor Dose Monitoring (TDM).

21-22. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The invention will now be explained more closely by the description of different embodiments of the invention and with reference to the appended figures.

[0062] FIG. 1 shows an example of a kit of parts.

[0063] FIG. 2 shows a schematic overview of the method.

[0064] FIG. 3 shows a correlation between intravenous versus capillary blood sampling.

[0065] Tables 4 to 8, listing pharmaceutical active compounds and suitable internal standards.

DETAILED DESCRIPTION

[0066] A “narrow therapeutic window” of a pharmaceutical active compound (API)/drug is defined as a small difference in dose or blood concentration between a therapeutic effective dose/concentration and a concentration that may lead to serious therapeutic failures or adverse drug reactions. Serious events are those, which are persistent, irreversible, slowly reversible, or life-threatening, possibly resulting in hospitalization, disability, or even death, or compounds having little difference between toxic and therapeutic doses of said compound. These types of compounds often need frequent monitoring of the concentration of the API in the blood of a mammal.

[0067] An “extraction fluid” means a solution comprising an analytical reference fluid with a known concentration of the internal standard. Preferably, said fluid can be directly used after centrifugation in an analytical instrument to measure the quality and concentration of the one or more biomarker.

[0068] An “internal standard” means a radioisotope of a biomarker in a known concentration of the biomarker in the extraction fluid.

[0069] An “analyte” means a molecule/biomarker/active pharmaceutical compound (API) present in the blood sample that will be analysed and quantified in the method.

[0070] A “radioisotope” means radioactive isotopes of an element, which are atoms that contain an unstable combination of neutrons and protons, or an excess energy in their nucleus. The radioactive isotope may be 13C or 2H (also written as “D” for deuterium, which is the same as the term “1H” used in the priority document). Further examples of suitable radioisotopes that may be incorporated include 3H (also written as “T” for tritium), 11C, 14C, 13N, 15N, 15O, 17O, 18O, 18F, 35S, 36Cl, 82Br, 75Br, 76Br, 77Br, 123I, 124I, 125I and 131I. The radioisotope that is used will depend on the specific application of that radio-labelled derivative. In some aspects, the radioisotope is 2H. In some embodiments, the radioisotope is 3H. In some aspects, the radioisotope is 13C. In some aspects, the radionuclide is 14C. In some aspects, the radioisotope is 11C. And in some aspects, the radioisotope is 180. The internal standard may be one or more radioactive biomarkers, whereby each biomarker may have one or more radioactive isotopes.

[0071] A “mammal” means a human or an animal, such as a horse, dog, cat, cow or pig. The human may be a patient.

[0072] The term “addiction or equivalents thereof” as used herein includes addiction as a disease.

[0073] The term “disease” as used herein is meant to include disorders, illnesses and other sicknesses.

[0074] As shown in FIG. 1, the capillary kit 1 consists of a lancet 2, a capillary or capillary tube 3 that can take an exact volume of whole blood from 10 to 100 μl, or 50 μl and a vial 4 with a cap 6 comprising or consisting of an extraction fluid 5 with the one or more internal standard, a transport vial 7 with a cap 6, a plaster 8 and an address label 9.

[0075] The extraction fluid is optimized for the one or more specific biomarker, API or API metabolite to be measured or analyzed. Optimization is done so that the one or more biomarker is preferably stable for 7 or 14 days at room temperature, in the extraction fluid. In addition to a solvent, the extraction fluid contains an exact amount of one or more internal standard, that is, the one or more biomarker labeled with one or more radioisotope, such that only the mass of the internal standard differs from the biomarker/analyte. The one or more internal standard is adapted to assess a quality and concentration of the one or more biomarker in the blood sample.

[0076] The one or more internal standard is a radioisotope selected from a group comprising or consisting of one or more of 2H 3H, 11C, 13C, 14C, 13N, 15N, 15O, 17O and 18O radioisotope. The radioisotope may be a 2H 3H, 11C, 13C, 14C radioisotope. The radioisotope may be a 13C radioisotope. The radioisotope may be a 2H radioisotope. When more than one internal standard is used a combination of different radioisotopes may be used. For example, one of the internal standards may be a 2H radioisotope for one biomarker and another internal standard may be a 13C radioisotope of a different biomarker.

[0077] Tamoxifen, z-endoxifen and 4-OH tamoxifen may all be 13C labeled. If the radiolabeled molecular mass does not differ significantly from the none-radiolabeled molecular mass, the same molecule is labeled with both 13C- and 2H-radioisotopes. For methylmalonic acid (MMA) for example, the 13C MMA-radioisotopes and the 2H-MMA-radioisotopes differ only by one atom. Therefore, the same molecule may be labeled with both 13C— and 2H-MMA-radioisotopes to improve the resolution in the analyze.

[0078] To avoid interference with endogenous MMA, a standard curve is made by a labelled 13C-MMA and the internal standard is labelled both with 13C— and 2H-MMA

[0079] As shown in FIG. 2, the blood sample is taken by a stick of the lancet 2 into a finger 11 giving a wound from which 10 to 100 μL of blood 10 is collected. The blood sample is immediately transferred into the vial 4 comprising or consisting of the extraction fluid 5 and one or more internal standard and shaken vigorously. The extraction fluid stops all enzyme activities in the blood 10 and an extraction of the one or more biomarker from the blood sample to the extraction fluid occurs.

[0080] The blood sample 10 will then be transported to a laboratory for analysis. The known concentration and behavior of the internal standard(s) in the analyze will immediately indicate if the sample has not been properly handled, such as low blood volume or degradation due to heat.

[0081] The quality assurance that the internal standard(s) provides for the sample is unprecedented to any other method available on the market. Which is particularly important if the sample is taken in a home environment by a layman.

[0082] When the vial 4 with the extraction fluid containing the sample arrives at the laboratory, the vial is vortexed vigorously and centrifuged. The supernatant is transferred to a tube, which can be directly used in an e.g. LC-MS/MS instrument for analysis and concentration determination of the biomarker. The blood sample may be analysed using mass spectrometry (MS), liquid chromatography-mass spectrometry (LC-MS, LC-MS/MS, gas chromatography-mass spectrometry (GC-MS) or GC-MS/MS. The analysis can be atomized in a robotic setting.

[0083] The one or more internal standard in the extraction fluid is the same as that used in the standard curve in the analysis method. Preferably, the one or more internal standard is from the same production batch, although this is not necessary since it is possible to normalize different batches in relation to each other.

[0084] The one or more internal standard may be a biomarker for measuring a quality and concentration of any pharmaceutical active compound that can be stabilized in an extraction fluid. Examples of APIs are shown in tables 4 to 8. Suitable APIs may be selected from the group comprising tamoxifen, z-endoxifen, 4-hydroxytamoxifen, statins, steroids, such as testosterone, or vitamins, such as vitamin B or D, or compounds related to addictions, such as opioids, cocaine, heroin, fentanyl, cannabinoids, marijuana, benzodiazepines, hallucinogens and methamphetamine.

[0085] The one or more internal standard may be a biomarker for measuring a quality and concentration of a statin selected from the group comprising or consisting of atorvastatin, fluvastatin, cerivastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin or mixtures thereof.

[0086] The one or more internal standard may be a biomarker for measuring a quality and concentration of a steroid hormone, such as glucocorticoids, mineralocorticoids, androgens, oestrogens and progestogens. In one aspect, the steroid hormones is selected from the group comprising or consisting of alclometasone, prednisone, dexamethasone, triamcinolone, cortisone, fludrocortisone, oxandrolone, oxabolone, testosterone, nandrolone, diethylstilbestrol (DES) and estradiol, norethisterone, medroxyprogesterone acetate, hydroxyprogesterone caproate, cyproterone acetate, mifepristone and gestrinone or mixtures thereof.

[0087] The one or more internal standard may be a biomarker for measuring a quality and concentration of an anti-depressive selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, serotonin modulators and stimulators, serotonin antagonists and reuptake inhibitors, norepinephrine reuptake inhibitors, norepinephrine-dopamine reuptake inhibitors, tricyclic antidepressants, tetracyclic antidepressants, monoamine oxidase inhibitors and NMDA receptor antagonists or mixtures thereof.

[0088] The anti-depressant API may be selected from the group comprising or consisting of bupropiontrazodone, nefazodone, vilazodone, vortioxetine, mitriptyline, bupropion, bupropion, bupropion, bupropion, bupropion, citalopram, desvenlafaxine, duloxetine, escitalopram, fluoxetine, mirtazapine, nortriptyline, paroxetine, sertraline, trazodone and venlafaxine, or mixtures thereof.

[0089] The one or more internal standard may be a radioisotope of tamoxifen, z-endoxifen and/or 4-hydroxytamoxifen for measuring the amount of tamoxifen in blood of a patient. The API may be tamoxifen and the internal standards may be a radioisotope of tamoxifen, z-endoxifen and/or 4-hydroxytamoxifen. The API may be tamoxifen and the internal standards may be a radioisotope of tamoxifen, z-endoxifen and 4-hydroxytamoxifen. The radioisotope(s) may be a 13C radioisotope. The radioisotope(s) may be a 2H radioisotope and 13C radioisotope.

[0090] The one or more internal standard may be a radioisotope of phosphatidylethanol 16:0/18:1, which is a biomarker for measuring long-term alcohol consumption. The radioisotope may be a 2H radioisotope.

[0091] The one or more internal standard may be a radioisotope of methylmalonic acid for measuring vitamin B insufficiency. The radioisotope may be a 2H radioisotope. The radioisotope may be a 13C radioisotope. The radioisotope may be a 2H and 13C radioisotope.

[0092] The one or more internal standard may be a radioisotope of dihydrotachysterol (Vitamin D) adapted for measuring D-vitamin deficiency. The radioisotope may be a 2H radioisotope. The radioisotope may be a 13C radioisotope. The radioisotope may be a 2H and 13C radioisotope.

[0093] The one or more internal standard may be a biomarker for measuring a quality and concentration of one or more pharmaceutical active compound having a small therapeutic window. The API may be selected from the group comprising or consisting of tamoxifen, digoxin, digitoxin, fosphenytoin, phenytoin, ethosuximide, alfentanil, tacrolimus, meperidine, temsirolimus, sirolimus, thiopental, fentanyl, alfentanil, theophylline, cyclosporine, clonidine, amitriptyline, protriptyline, imipramine, nortriptyline, quinidine, levothyroxine, carbamazepine, phenobarbital, ergotamine, dihydroergotamine and heparin. The pharmaceutical active compound that has a narrow therapeutic window may be selected from the group comprising or consisting of tamoxifen, digoxin, digitoxin, phosphenytoin, levothyroxine and carbamazepine. The pharmaceutical active compound may be tamoxifen.

[0094] In an example of the method the following step are taken; [0095] providing a kit of parts for sampling of blood, the kit of parts comprising a lancet and a capillary adapted to sample a defined volume of blood from the human and a vial comprising an extraction fluid, which is 2-propanol or tetrahydrofuran, together with an internal standard, which is a 2H radioisotope of phosphatidylethanol 16:0/18:1, [0096] distributing the kit of parts to a location of the mammal, [0097] taking a blood sample from the mammal by creating a wound with the lancet, [0098] collecting a 50 μl volume of the blood in the capillary, [0099] entering the blood sample or the capillary into the vial, [0100] receiving the vial comprising the blood sample from the mammal inserted into the vial, [0101] optionally, extracting the fluid from the vial, [0102] centrifuging the vial, and [0103] analysing the supernatant to determine the quality of the sample and the concentration of phosphatidylethanol in the blood of the mammal.

[0104] In another example of the method the following step are taken; [0105] providing a kit of parts for sampling of blood, the kit of parts comprising a lancet and a capillary adapted to sample a defined volume of blood from the human and a vial comprising an extraction fluid, which is acetonitrile, together with an internal standard, which is a 2H radioisotope of methylmalonic acid, [0106] distributing the kit of parts to a location of the mammal, [0107] taking a blood sample from the mammal by creating a wound with the lancet, [0108] collecting a 50 μl volume of the blood in the capillary, [0109] entering the blood sample or the capillary into the vial, [0110] receiving the vial comprising the blood sample from the mammal inserted into the vial, [0111] optionally, extracting the fluid from the vial, [0112] centrifuging the vial, and [0113] analysing the supernatant to determine the quality of the sample and the concentration of methylmalonic acid in the blood of the mammal.

[0114] In a further example of the method the following step are taken; [0115] providing a kit of parts for sampling of blood, the kit of parts comprising a lancet and a capillary adapted to sample a defined volume of blood from the human and a vial comprising an extraction fluid, which is methanol, acetonitrile or formic acid, together with an internal standard, which is a 13C radioisotope of tamoxifen, z-endoxifen and 4-hydroxytamoxifen, [0116] distributing the kit of parts to a location of the mammal, [0117] taking a blood sample from the mammal by creating a wound with the lancet, [0118] collecting a 50 μl volume of the blood in the capillary, [0119] entering the blood sample or the capillary into the vial, [0120] receiving the vial comprising the blood sample from the mammal inserted into the vial, [0121] optionally, extracting the fluid from the vial, [0122] centrifuging the vial, and [0123] analysing the supernatant to determine the quality of the sample and the concentration of tamoxifen, z-endoxifen and 4-hydroxytamoxifen in the blood of the mammal.

[0124] In a further example of the method the following step are taken; [0125] providing a kit of parts for sampling of blood, the kit of parts comprising a lancet and a capillary adapted to sample a defined volume of blood from the human and a vial comprising an extraction fluid, which is methanol, acetonitrile or formic acid, together with an internal standard, which is a 13C radioisotope of an antidepressant API (e.g. seratralin, citalpram, excitaopram, venlafaxin and flouxetin, or a statin (e.g. pravastin, rosuvastatin, simvastatin and atorvastatin) or a steroid hormone (e.g. aldosteron, androsteron, crotisol, progetsteron and testosteron), [0126] distributing the kit of parts to a location of the mammal, [0127] taking a blood sample from the mammal by creating a wound with the lancet, [0128] collecting a 50 μl volume of the blood in the capillary, [0129] entering the blood sample or the capillary into the vial, [0130] receiving the vial comprising the blood sample from the mammal inserted into the vial, [0131] optionally, extracting the fluid from the vial, [0132] centrifuging the vial, and [0133] analysing the supernatant to determine the quality of the sample and the concentration of said anti-depressant API, statin and steroid hormone (e.g. sertralin, atorvastatin or testosterone), in the blood of the mammal.

[0134] Centrifuging may be done at 16400 rpm. Analysis may be performed in an LC-MS/MS instrument. Other examples of biomarkers of interest are shown in table 1.

TABLE-US-00001 TABLE 1 Drugs Codeine Ibuprofen Dihydrocodeine Morphine Dextropropxyphene Aminorex napsylate Lidocaine Iso-LSD Norcocaine Amitriptyline Pemoline Prazepam Imipramine Propafenone Pheniramine Chlorpromazine Amphetamine sulfate Lofexidine hydrochloride Clozapine Ecgonine methyl ester Diphenylhydramine Estazolam 3,4-Methylenedioxy- Melatonin amphetamine Doxepin Ketamine Mescaline Aprobarbital Buprenorphine Benzoylecgnine Trifluoperazine Methadone Ecgonine ethyl ester Midazolam Fentanyl Norketamine Chlordiazepoxide Caffeine Hydrocodone Fenfluramine Tramadol Lorazepam Phenylpropanolamine Flunitrazepam Amobarbital Flurazepam PCP Barbital Carbamazepine Phenobarbital Vancomycin

[0135] Further pharmaceutical active compounds may be CPA-cyclophosphamide and its active metabolite PAM-hb, or DOC-docetaxel, DOX-doxorubicine, PAC-paclitaxel, EPI-epirubicin.

[0136] The solvent used in the extraction fluid comprises an organic solvent or a combination of two or more organic solvents. The solvents may be selected from the group comprising or consisting of 2-propanol, methanol and acetonitrile, formic acid, or mixtures thereof. For the analyte PEth-16:0/18:1, the solvent may be 2-propanol or tetrahydroferan. For the analyte metylmalonic acid, the solvent may be acetonitrile. For the analyte tamoxifen, z-enoxifen, 4-hydroxytamoxifen, the solvent may be methanol, acetonitrile, formic acid, or mixtures thereof. Table 2 lists other examples of suitable solvents.

[0137] Other examples of pharmaceutical active compounds and internal standards are shown in tables 4 to 8 in the attached drawings. (Adaway J E, Therapeutic drug monitoring and LC-MS/MS, J. Chromatogr. B. 2012, 883-884, 33-49.)

TABLE-US-00002 TABLE 2 Solvents Methanol Chloro-butane Ethanol Ethyl-ether Isopropanol Methyl tert-butyl ether Butanol Ethanol Acetonitrile Ethyl acetate Tetrahydrofuran Acetic acid Acetone Ammonium hydroxide Dichloromethane Ammonium acetate Formic acid

[0138] The method and the kit of parts may be used for diagnosing a disease. The disease may be any disease related to the analyzed biomarker/analyte. Example of diseases may be any form of addiction or abuse, such as alcoholism, opiate abuse, or cancer, such as breast cancer, prostate cancer, or cardiovascular diseases, such as high blood pressure, or steroid deficiency diseases.

[0139] The method and the kit of parts may be used for Tailor Dose Monitoring (TDM).

[0140] The method and the kit of parts may be used for diagnosing and/or monitoring drug abuse, or alcoholism.

Example 1, Method of Measuring Long Term Alcohol Use and Validation of the Method

[0141] The kit as defined above was used. The kit comprised a vial that contained 200 μl of extraction solution 80% isopropanol with 20% tetrahydrofuran and 0.1 μM D5-PEth 16:0/18:1 (i.e. 2H radioisotope of phosphatidylethanol 16:0/18:1).

[0142] 50 μl blood was collected from human volunteers using the lancet and added to the extraction solution in the vial. Or 50 μl blood was collected from human volunteers using venous blood. The blood from seven different human volunteers have been collected.

[0143] The vials were transported to the laboratory for analysis.

[0144] The vials were vortexed and centrifuged. 20 μl of the supernatant was directly injected into the LC-MS/MS system. The LC-MS/MS system contains: Mobile phase A; 20% acetonitrile, 0.5 mM ammonia and 1 mM acetic acid in water, mobile phase B; 20% acetonitrile, 20% tetrahydrofuran and 60% 2-propanol were used.

[0145] The test showed that the same results were obtained independent how the blood samples were collected from the human volunteers.

[0146] The analysis allows for the quantification of low levels of 0.005 mmol/ml.

[0147] The results in table 3 and FIG. 3 show that there are no differences between a venous blood sample and a sample taken by lancet in the finger.

TABLE-US-00003 TABLE 3 Individual Conc. % CV 1 0.16 7 2 0.78 4 3 0.55 3 1 0.18 4 3 0.52 4 5 0.19 5 6 0.11 7 7 0.08 3 8 0.18 2

[0148] Table 1 Intravenous vs. capillary blood sampling: The concentration of triplicates from an intravenous sample and capillary blood sampled in triplicate are compared. Samples are collected from 7 healthy individuals, in a total of nine sample extractions, at two different days.

Example 2, Monitoring Amount of Tamoxifen in Blood of a Patient

[0149] The kit as defined above was used. The kit comprised a vial that contained 150 μl extraction solution containing acetonitrile with 0.2% formic acid and 0.1 ng/mL each of 13C-labeled tamoxifen, 13C-labeled tamoxifen, 13C-labeled z-endoxifen and 13C-labeled 4-OH tamoxifen, 0.1 μM of each. 50 μl of blood from the patient was collected using the lancet. The blood was added to 150 μl extraction solution.

[0150] The vial was transported to the laboratory for analysis.

[0151] The vial was vortexed and centrifuged. 150 μl of the supernatant was collected and transferred to glass vials. The protein precipitation solution was evaporated to dryness and reconstituted in 60 μl 20% acetonitrile in water. 20 μl of the solution was injected into the LC-MS/MS system. The LC-MS/MS system: the mobile phases A; 0.1% formic acid in water and mobile phase B; 0.1% formic acid in acetonitrile.

[0152] Three validation batches were prepared and analyzed at three different occasions. Each validation batch comprised two sets of all calibration standards, two sets of all quality control samples and a number of blank samples.

[0153] The results show that there are no differences between a venous blood sample and a sample taken by a lancet in the finger.

Example 3, Monitoring Amount of Methylmalonic Acid in Blood of a Patient

[0154] The kit comprised a vial that contained 100 μl extraction solution containing 0.5 μmol/L labelled MMA in water. 50 μl of blood from the patient was collected using the lancet. The blood was added to 100 μl extraction solution.

[0155] The vial was transported to the laboratory for analysis.

[0156] The vial was vortexed and transferred to a SPE column. The column is eluted by 150 μl 3% formic acid in acetonitrile. The eluate is evaporated to dryness. 100 μl water is added 20 μl is transferred to the LC-MS/MS system.

[0157] The mobile phases A: 0.05% acetic acid in 90% acetonitrile in water and mobile phase B: 0.5% formic acid in 20% acetonitrile in water.

[0158] The results show that there are no differences between a venous blood sample and a sample taken by a lancet in the finger