Metabolisable control marker for the endogenous marking of urine

Abstract

The present invention describes the use of curcumin in endogenous marking together with one or more polyethylene glycols.

Description

[0001] Metabolizable control markers for endogenous labeling of urine

[0002] Description

[0003] A urine sample is most commonly used to detect illegal substance use. These tests are used in many areas, including addiction treatment, substitution therapy, driving aptitude testing, sports, employee screening, etc.

[0004] Diagnostic procedures, procedures for monitoring the course of a therapeutic measure, prophylactic routine examinations and forensic examinations on humans usually involve the laboratory analysis of samples, such as hair, blood or serum samples taken from the subject, as well as the examination of the subject's excretions, such as urine.

[0005] Of all the analytical methods, urine is the most suitable because the illegal substances can be detected longer in urine than in other methods, such as blood.

[0006] However, urine is susceptible to tampering. Attempts are made to conceal the illegal use of prohibited substances by adding liquid or chemicals, such as dish soap. There are also often attempts to swap the "contaminated" sample for an uncontaminated one. To rule out such tampering, the urine sample is still collected under direct visual inspection, with the subject's genitals in view.

[0007] It is therefore desirable to provide a procedure that, on the one hand, eliminates the need for supervision during sample collection, but, on the other hand, ensures that no deception is possible. One such procedure is so-called "endogenous labeling," in which the subject is administered at least one non-metabolizable substance prior to sample collection, which can later be identified in the urine sample.

[0008] An example of non-metabolizable substances is polyethylene glycols. Drinking a solution containing polyethylene glycols will stain the subject's urine, meaning the administered polyethylene glycols are detectable in the urine.

[0009] EP 1 410 014 describes a diagnostic method and the polyethylene glycols used therefor as marker substances, which are intended to detect deception during endogenous marking and which does not require visual inspection of the subject's genitals. Polyethylene glycols were particularly preferred as marker substances in EP 1 410 014. To ensure resistance to deception, either a polyethylene glycol with a single molecular weight or a combination of polyethylene glycols with different molecular weights is used as marker substances. Polyethylene glycols can be polydisperse or monodisperse.

[0010] When using marker substances, however, it must be ensured that the marker substances were actually taken by the person to be tested.

[0011] An obvious deception is when the subject gives the drinking solution containing the marker substances to another person who ingests the marker substances, allowing the subject to pass off the urine produced by this third person as their own urine. This deception can be prevented by ensuring that the ingestion of the solution takes place under supervision.

[0012] Another possible deception is that a sponge hidden in the mouth absorbs the marker substances while the drinking solution is taken and then squeezed out into the clean urine of another person.

[0013] To detect the above-mentioned deception, a small amount of a metabolizable substance, hereinafter referred to as the "control marker substance," is added to the drinking solution containing the marker substances and given to the test subject to drink, so that only the non-metabolizable marker substances are present in the urine, but not the control marker. Thus, if the drinking solution, which contains both the marker substances and the control marker substances, is mixed with a foreign urine sample, the detection of the marker substances together with the control marker substance in the urine can be recognized as a sure attempt at deception.

[0014] In EP 1 563 311, methyl 4-hydroxybenzoate was proposed as a control marker substance. Due to its low toxicity, this substance is only approved as a food and pharmaceutical additive in certain amounts.

[0015] Another option is to add a metabolizable sugar, such as sucrose, to the drinking solution containing polyethylene glycols. Ideally, the added sugar is completely metabolized and not detected in the urine.

[0016] Sucrose has the disadvantage that, while it highly likely indicates tampering with the marker solution by spitting it in, it ultimately isn't conclusive proof. There are other sources that can cause elevated sucrose concentrations in urine. One is the injection of sucrose-containing solutions, as in the case of methadone abuse. Slightly elevated sucrose levels can also be measured in urine in some gastrointestinal diseases. These can include, for example, stomach ulcers, where the stomach lining is permeable to sucrose. Sucrose is only broken down in the gastrointestinal tract. If sucrose enters the bloodstream by any means, it is excreted in the urine.The threshold value for a suspicious sucrose finding (manipulation by spitting in the marker) must therefore be set somewhat higher than would actually be necessary to detect manipulations in which marker was added in the lower concentration range.

[0017] When using sucrose, it is also necessary for the person submitting the urine sample to strictly adhere to the instructions. The amount of sucrose added must be exactly correct, and no fruit juices or sodas may be used for administration, as otherwise the evaluation of the results would be more difficult. Furthermore, sucrose can be degraded, for example, by yeast. If yeast is added to manipulated urine containing sucrose, the sucrose will be degraded. Yeast or bacteria can also be present in urine without manipulation, due to bladder diseases or non-sterile urine collection, therefore the direct detection of yeast or bacteria in urine is not helpful. (Under normal conditions, natural yeast or bacteria would hardly be sufficient to degrade sucrose.) Indirect detection of yeast by measuring the degradation of an added amount of sucrose prior to analysis in the laboratory has not proven reliable.

[0018] The object of the present invention is to propose a substance as a control marker that is non-toxic, detectable in very small amounts, not degradable by yeast and easy to handle.

[0019] Surprisingly, it was found that curcumin is an excellent control marker substance that can replace sucrose or be used in addition to sucrose.

[0020] The present invention relates to a drinking solution for the endogenous labeling of urine, wherein the solution contains curcumin in addition to one or more polyethylene glycols. Furthermore, the present invention provides a method for analyzing a urine sample for the presence of at least one specific analyte using the aforementioned drinking solution. Analytes can be narcotics, pharmaceuticals, doping substances, or metabolites of the aforementioned substances, the detection of which in the sample allows for information about the behavior or treatment of the test subjects. For example, the analyte can be heroin, methadone, cocaine, THC, barbiturates, nicotine, alcohol, or a doping agent listed as doping agents.

[0021] Curcumin is non-toxic and is the main component of the spice, medicinal and coloring agent turmeric, which is used together with other spices in curry powder (https: / / de.wikipedia.org / wiki / Curcumin).

[0022] It has been found that curcumin is detectable in very small amounts, but is completely metabolized in the body, making it well-suited as a control marker substance. By adding curcumin to the drinking solution with a polyethylene glycol or a combination of polyethylene glycols as marker substances, deception caused by adding the drinking solution to the urine sample can be easily detected, as even very small amounts of curcumin are easily detectable.

[0023] By measuring several thousand native urine samples from drug addicts and measuring them—even after ingesting large amounts of curcumin—curcumin could not be detected in the urine. The detection limit for curcumin using the liquid / liquid extraction method used is approximately 10 ng / ml. The detection limit for alternatively tested shoot and dilute methods was approximately 50–100 ng / ml.

[0024] Example

[0025] The sample is hydrolyzed prior to extraction to convert any existing analyte glucuronides into the free analyte. The internal standard (IS) is added prior to hydrolysis. During liquid extraction, 10 μl of IS is added.

[0026] For hydrolysis of the glucuronides, 10 μl of acetate buffer, 10 μl of a β-glucuronidase solution, and 40 μl of methanol are added to each sample. The solution is incubated overnight or for 1 hour at 60°C and then extracted.

[0027] Hydrolysis is not required for the detection of curcumin and PEGs, but is performed because the drugs can also be determined simultaneously under certain circumstances.

[0028] Liquid extraction

[0029] 100 μl of urine sample is hydrolyzed as described above.

[0030] After hydrolysis, the sample is transferred into a screw-cap glass tube with 1.6 ml of saturated NaCl solution and 25 μl of 3 M NH3, and 2 ml of ethyl acetate / dichloromethane (1:1) are added. The sample is mixed for 10 min on an overhead shaker. Centrifuge the sample, and the supernatant is transferred to a glass tube. The sample is then evaporated to dryness in a stream of nitrogen or with air. The residue is dissolved first with 25 μl of methanol and then with 75 μl of H2O. The dissolved samples are transferred to vials with microinserts and sealed. 5 μl of sample is injected into the LCMS / MS.

[0031] Control material

[0032] Since no commercial curcumin controls are available, a proprietary control is produced that contains curcumin at a concentration of 20–50 ng / ml in addition to the drugs, narcotic substances, and PEGs. Curcumin detection is qualitative, so no calibration is performed. However, each laboratory is free to quantify curcumin.

[0033] Results

[0034] Samples in which no marker was detected are repeated from the original urine tube. A confirmed negative marker result is recorded as negative, with the caveat that a negative result may also result if the delay between ingestion of the marker substances and urine collection is too short. Likewise, positive curcumin urine samples are retested from the original tube. A confirmed positive curcumin result is considered tampering with the marker solution by spitting it into another urine sample.

Claims

Claims 1. Drinking solution containing a combination of one or more polyethylene glycols and curcumin, wherein the polyethylene glycols have different molecular weights, and wherein the molecular weights of the polyethylene glycols are between 200 and 5000 Da.

2. Drinking solution according to claim 1, wherein the polyethylene glycols are monodisperse.

3. Drinking solution according to claim 1, wherein the polyethylene glycols are polydisperse.

4. Drinking solution according to one of the preceding claims, wherein one of the polyethylene glycols (PEGs) has a molecular weight of 200, 300, 400, 500, 600, 1000 or 1500.

5. Drinking solution according to one of the preceding claims, wherein the solution contains three polyethylene glycols with different molecular weights.

6. Drinking solution according to one of the preceding claims, wherein the solution contains three polyethylene glycols with different molecular weights.

7. Use of curcumin as a metabolizable control marker in endogenous labeling.

8. Use of curcumin as a metabolizable control marker in endogenous labeling together with a combination of polyethylene glycols in a drinking solution.

9. A method for analyzing a urine sample for the presence of at least one specific analyte, the method comprising administering the drinking solution according to any one of claims 1-8.

10. Kit for use in the method according to claim 9, comprising a drinking solution according to any one of claims 1-8.