WO2024052243A1 - Metabolisable control marker for the endogenous marking of urine - Google Patents

Metabolisable control marker for the endogenous marking of urine Download PDF

Info

Publication number
WO2024052243A1
WO2024052243A1 PCT/EP2023/074099 EP2023074099W WO2024052243A1 WO 2024052243 A1 WO2024052243 A1 WO 2024052243A1 EP 2023074099 W EP2023074099 W EP 2023074099W WO 2024052243 A1 WO2024052243 A1 WO 2024052243A1
Authority
WO
WIPO (PCT)
Prior art keywords
urine
polyethylene glycols
curcumin
marker
drinking solution
Prior art date
Application number
PCT/EP2023/074099
Other languages
German (de)
French (fr)
Original Assignee
Ruma Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ruma Gmbh filed Critical Ruma Gmbh
Publication of WO2024052243A1 publication Critical patent/WO2024052243A1/en

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
    • G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/493—Physical analysis of biological material of liquid biological material urine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors

Definitions

  • a urine sample is most commonly used to detect the ingestion of illegal substances. These tests are used in many areas, e.g. addiction therapy, substitution therapy, driving ability tests, sports, employee screening, etc.
  • Diagnostic procedures, procedures for monitoring the progress of a therapeutic measure, routine prophylactic examinations and forensic medical examinations on humans generally include the laboratory analysis of samples, such as: B. hair, blood or serum samples taken from the test subject, as well as the examination of the test subject's excretions, such as. B. urine.
  • urine is the most suitable because the illegal substances can be detected in urine for longer than in the others, for example in blood.
  • urine is susceptible to tampering. By adding liquid or chemicals, such as dishwashing liquid, an attempt is made to disguise the illegal ingestion of unauthorized substances. It is also often attempted to exchange the "contaminated" sample for an uncontaminated sample. To rule out such manipulations, the urine sample is still taken under direct visual inspection with a view of the subject's genitals.
  • non-metabolizable substances is polyethylene glycols.
  • the subject's urine is marked, meaning that the administered polyethylene glycols can be detected in the urine.
  • EP 1 410 014 describes a diagnostic method and polyethylene glycols used for this as marker substances, through which deception can be revealed in the context of endogenous marking, and which does not require visual inspection of the test person's genitals.
  • Polyethylene glycols were particularly preferred as marker substances in 1,410,014. In order to ensure security against deception, either a polyethylene glycol with one molecular weight or a combination of polyethylene glycols of different molecular weights are used as marker substances.
  • Polyethylene glycols can be poly- or monodisperse.
  • An obvious deception is that the subject gives the drinking solution with the marker substances that was given to him to another person who takes the marker substances, so that the subject sees the urine produced by this third person as his/her own. can pass their own urine. This deception can be prevented by taking the solution under supervision.
  • Another possible deception is that a sponge hidden in the mouth absorbs the marker substances while taking the drinking solution and is then expressed into another person's clean urine.
  • control marker substance a small amount of a metabolizable substance, hereinafter referred to as "control marker substance”, is added to the drinking solution containing the marker substances and given to the test subject to drink, so that in the urine only the non-metabolizable marker substances are present, but not the control marker. So if the drinking solution, which contains both marker substances and the control marker substances, is mixed with someone else's urine, the detection of the marker substances together with the control marker substance in the urine can be recognized as a sure attempt at deception.
  • Another option is to add a metabolizable sugar, such as sucrose, to the drinking solution with polyethylene glycols.
  • a metabolizable sugar such as sucrose
  • the added sugar is ideally completely metabolized and is not found in the urine.
  • sucrose has the disadvantage that, although it is highly likely to indicate manipulation by spitting on the marker solution, it ultimately does not necessarily provide evidence of this.
  • this is the injection of a solution containing sucrose, as is the case with the abusive use of methadone, but slightly elevated sucrose levels can also be measured in the urine in some stomach/intestinal diseases. These can be, for example, stomach ulcers in which the stomach wall is permeable to sucrose.
  • Sucrose is only broken down in the stomach/intestinal tract. If sucrose enters the blood through any route, it is excreted in the urine. The limit value for a suspicious sucrose finding (manipulation by spitting the marker) must therefore be chosen slightly higher than would actually be necessary in order to detect manipulations in which marker was added in the lower concentration range.
  • sucrose When using sucrose, it is also necessary that the person sending the urine sample strictly follows the instructions. The amount of sucrose added must be exactly right and no fruit juices or lemonades may be used for administration, otherwise it will make it more difficult to evaluate the findings. Furthermore, the sucrose can be broken down by yeast, for example. If yeasts are added to manipulated urine that contains sucrose, the sucrose is broken down. Yeast or bacteria can occur in urine even without manipulation, through bladder disease or through non-sterile urine output, so direct detection of yeast or bacteria in urine is not helpful. (The "natural yeasts or bacteria would hardly be sufficient to break down the sucrose under normal conditions.) The indirect detection of yeasts by measuring the breakdown of an added amount of sucrose before analysis in the laboratory has not proven to be reliable.
  • 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.
  • curcumin is ideal as a control marker substance to replace sucrose or can be used in addition to sucrose.
  • the subject of the present invention is a drinking solution for the endogenous marking of urine, the solution also containing 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 particular analyte by using the aforementioned drinking solution.
  • Analytes can be intoxicants, drugs, doping substances, or metabolites of the aforementioned substances, the detection of which in the sample allows information about the behavior or treatment of the test subjects.
  • the analyte can be heroin, methadone, cocaine, THC, barbiturate, nicotine, alcohol or a doping substance on the list of doping substances.
  • Curcumin is non-toxic and is the main component of the seasoning, medicinal and coloring agent turmeric, which is used together with other spices in curry powder (https://de.wikipedia.org/wiki/Curcumin).
  • curcumin can be detected in very small amounts, but is completely metabolized in the body and is therefore well suited as a control marker substance.
  • curcumin By adding curcumin to the drinking solution with a polyethylene glycol or with a combination of polyethylene glycols as marker substances, deception can be easily detected by adding the drinking solution to someone else's urine, as even very small amounts of curcumin can be easily detected.
  • curcumin with the liquid/liquid extraction used is approx. 10 ng/ml.
  • the detection limit for alternatively tested shoot and dilute methods was approximately 50-100 ng/ml.
  • the sample is hydrolyzed before extraction to convert any analyte glucuronides present into the free analyte.
  • the internal standard IS is added before hydrolysis. During the liquid extraction, 10 ⁇ l of IS are added.
  • glucuronides 10 ⁇ l of acetate buffer and 10 ⁇ l of a ß-glucuronidase solution and 40 ⁇ l of methanol are added to each sample. The solution is incubated overnight or for 1 hour at 60°C and only then extracted.
  • Hydrolysis is not necessary for the detection of curcumin and the PEGs, but is carried out because under certain circumstances the drugs can also be determined at the same time.
  • the sample is placed in a screw-top glass tube with 1.6 ml of saturated NaCl solution and 25 ⁇ l of 3 M NH3 and 2 ml of ethyl acetate/dichloromethane (1:1) are added.
  • the sample is mixed for 10 minutes on an overhead shaker. Then it is centrifuged and the supernatant is placed in a glass tube.
  • the sample is then evaporated in a stream of nitrogen or with air until it dries.
  • the residue is first mixed with 25 pl Methanol and then dissolved with 75 pl H2O.
  • the dissolved samples are filled into vials with microinserts and sealed. 5 ⁇ l of sample are injected into the LCMS/MS.
  • curcumin Since no commercial controls with curcumin are available, a separate control is produced which, in addition to the drugs, narcotic substances and PEGs, also contains curcumin in a concentration of 20 - 50 ng/ml. The detection of curcumin is qualitative, therefore no calibration is carried out. However, every laboratory is free to quantify curcumin.
  • Samples in which no marker was found are repeated from the original urine tube.
  • a confirmed negative marker result is considered negative with the indication that a negative result can result even if the waiting time between taking the marker substances and urine output is too short.
  • Positive curcumin urines are also measured again from the original vessel.
  • a confirmed positive curcumin result is assessed as manipulation by spitting the marker solution into another urine.

Abstract

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

Description

Metabolisierbare Kontroll-Marker für die endogene Markierung von Urin Metabolizable control markers for endogenous urine labeling
Beschreibung Description
Zum Nachweis der Einnahme illegaler Substanzen wird am häufigsten eine Urinprobe verwendet. Diese Tests finden Anwendung in vielen Bereichen, z.B. Suchttherapie, Substitutionstherapie, Fahreignungsprüfung, Sport, Arbeitnehmer-Screening usw. A urine sample is most commonly used to detect the ingestion of illegal substances. These tests are used in many areas, e.g. addiction therapy, substitution therapy, driving ability tests, sports, employee screening, etc.
Diagnostische Verfahren, Verfahren zur Überwachung des Verlaufs einer therapeutischen Maßnahme, prophylaktische Routineuntersuchungen sowie gerichtsmedizinische Untersuchungen am Menschen beinhalten in der Regel die laboranalytische Untersuchung von Proben, wie z. B. Haar, Blut- oder Serumproben, die dem Probanden entnommenen wurden, als auch die Untersuchung von Ausscheidungen des Probanden, wie z. B. Urin. Diagnostic procedures, procedures for monitoring the progress of a therapeutic measure, routine prophylactic examinations and forensic medical examinations on humans generally include the laboratory analysis of samples, such as: B. hair, blood or serum samples taken from the test subject, as well as the examination of the test subject's excretions, such as. B. urine.
Von allen Analyseverfahren eignet sich Urin am besten, da die illegalen Substanzen im Urin länger nachweisbar sind als in den anderen z.B. in Blut. Of all the analysis methods, urine is the most suitable because the illegal substances can be detected in urine for longer than in the others, for example in blood.
Allerdings ist Urin anfällig für Manipulationen. Durch Zugabe von Flüssigkeit oder Chemikalien, wie z.B. Spülmittel, wird versucht, die illegale Einnahme nicht erlaubter Substanzen zu verschleiern. Es wird ebenfalls oft versucht, die „belastete" Probe gegen eine unbelastete Probe auszutauschen. Zum Ausschluss solcher Manipulationen wird die Urinprobe immer noch unter direkter Sichtkontrolle mit Blick auf das Genital des Probanden/der Probandin gewonnen. However, urine is susceptible to tampering. By adding liquid or chemicals, such as dishwashing liquid, an attempt is made to disguise the illegal ingestion of unauthorized substances. It is also often attempted to exchange the "contaminated" sample for an uncontaminated sample. To rule out such manipulations, the urine sample is still taken under direct visual inspection with a view of the subject's genitals.
Es ist daher wünschenswert, ein Verfahren zur Verfügung zu stellen, das einerseits auf die Aufsicht bei der Probenentnahme verzichtet, aber andererseits sicherstellt, dass keine Täuschung möglich ist. Ein solches Verfahren ist die so genannten „endogene Markierung", bei der dem Probanden/der Probandin vor der Probeentnahme mindestens eine nicht metabolisierbare Substanz verabreicht wird, die später in der Urinprobe identifiziert werden kann. It is therefore desirable to provide a method that, on the one hand, dispenses with supervision when taking samples, but on the other hand ensures that no deception is possible. One such method is the so-called "endogenous labeling", in which the test subject is administered at least one non-metabolizable substance before the sample is taken, which can later be identified in the urine sample.
Ein Beispiel für die nicht metabolisierbaren Substanzen sind z.B. Polyethylenglycole. Durch das Trinken einer mit Polyethylenglycolen versetzten Lösung wird der Urin des Probanden/der Probandin markiert, d.h. die verabreichten Polyethylenglycole sind im Urin nachweisbar. An example of non-metabolizable substances is polyethylene glycols. By drinking a solution containing polyethylene glycols, the subject's urine is marked, meaning that the administered polyethylene glycols can be detected in the urine.
In EP 1 410 014 werden ein Diagnoseverfahren und hierfür eingesetzte Polyethylenglycole als Marker-Substanzen beschrieben, durch welche sich Täuschung im Rahmen der endogenen Markierung aufdecken lassen sollen, und welches ohne die Sichtkontrolle auf das Genital des Probanden/der Probandin auskommt. Als Marker-Substanzen wurden in 1 410 014 Polyethylenglycole besonders bevorzugt. Um die Täuschungssicherheit zu gewährleisten, werden als Marker-Substanzen entweder ein Polyethylenglycol mit einem Molekulargewicht, oder eine Kombination von Polyethylenglycolen verschiedener Molekulargewichte verwendet. Polyethylenglycole können poly- oder monodispers sein. EP 1 410 014 describes a diagnostic method and polyethylene glycols used for this as marker substances, through which deception can be revealed in the context of endogenous marking, and which does not require visual inspection of the test person's genitals. Polyethylene glycols were particularly preferred as marker substances in 1,410,014. In order to ensure security against deception, either a polyethylene glycol with one molecular weight or a combination of polyethylene glycols of different molecular weights are used as marker substances. Polyethylene glycols can be poly- or monodisperse.
Bei der Verwendung von Marker-Substanzen muss aber sichergestellt werden, dass die Marker-Substanzen tatsächlich von der Person eingenommen wurden, die getestet werden soll. When using marker substances, however, it must be ensured that the marker substances were actually taken by the person being tested.
Eine offensichtliche Täuschung besteht darin, dass der Proband die Trinklösung mit den Marker-Substanzen, die ihm ausgehändigt wurde, einer anderen Person gibt, die die Marker- Substanzen einnimmt, sodass der Proband/die Probandin den von dieser dritten Person produzierten Urin als seinen/ihren eigenen Urin ausgeben kann. Diese Täuschung kann dadurch verhindert werden, dass die Einnahme der Lösung unter Aufsicht erfolgt. An obvious deception is that the subject gives the drinking solution with the marker substances that was given to him to another person who takes the marker substances, so that the subject sees the urine produced by this third person as his/her own. can pass their own urine. This deception can be prevented by taking the solution under supervision.
Eine weitere mögliche Täuschung besteht darin, dass ein im Mundraum verstecktes Schwämmchen während der Einnahme der Trinklösung die Marker-Substanzen aufsaugt und anschließend in den sauberen Urin einer anderen Person ausgedrückt wird. Another possible deception is that a sponge hidden in the mouth absorbs the marker substances while taking the drinking solution and is then expressed into another person's clean urine.
Um die o.g. Täuschung zu erfassen, wird der mit den Marker-Substanzen versetzten Trinklösung eine geringfügige Menge einer metabolisierbaren Substanz zugesetzt, im Nachfolgenden „Kontroll-Marker-Substanz" genannt, und dem Probanden/der Probandin zu Trinken gegeben, so dass im Urin nur die nicht metabolisierbaren Marker-Substanzen vorhanden sind, aber nicht der Kontroll-Marker. Wenn also die Trinklösung, die sowohl Marker-Substanzen als auch die Kontroll-Marker- Substanzen enthält, einem fremden Urin zugemischt wird, lässt sich der Nachweis der Marker-Substanzen zusammen mit der Kontroll-Marker-Substanz in dem Urin als sicherer Täuschungsversuch erkennen. In order to detect the above-mentioned deception, a small amount of a metabolizable substance, hereinafter referred to as "control marker substance", is added to the drinking solution containing the marker substances and given to the test subject to drink, so that in the urine only the non-metabolizable marker substances are present, but not the control marker. So if the drinking solution, which contains both marker substances and the control marker substances, is mixed with someone else's urine, the detection of the marker substances together with the control marker substance in the urine can be recognized as a sure attempt at deception.
In EP 1 563 311 wurde als Kontroll-Marker-Substanz Methyl-4-hydroxybenzoat vorgeschlagen. Diese Substanz ist wegen der geringen Toxizität in bestimmten Mengen lediglich als Lebensmittel- und Arzneimittelzusatzstoff zugelassen. In EP 1 563 311, methyl 4-hydroxybenzoate was proposed as a control marker substance. Because of its low toxicity, this substance is only approved in certain quantities as a food and drug additive.
Eine weitere Möglichkeit besteht darin der Trinklösung mit Polyethylenglycolen einen metabolisierbaren Zucker, z.B. Saccharose, zuzugeben. Der zugegebene Zucker wird idealerweise vollständig metabolisiert und wird im Urin nicht gefunden. Another option is to add a metabolizable sugar, such as sucrose, to the drinking solution with polyethylene glycols. The added sugar is ideally completely metabolized and is not found in the urine.
Die Saccharose hat den aber Nachteil, dass sie zwar mit hoher Wahrscheinlichkeit auf eine Manipulation durch Zuspucken der Markerlösung hinweist, aber letzten Endes nicht zwingend beweisend dafür ist. Es gibt noch andere Quellen, die eine erhöhte Saccharosekonzentration im Urin verursachen können. Das ist zum einen die Injektion von Saccharose haltiger Lösung, wie bei der missbräuchlichen Verwendung von Methadon, aber auch bei einigen Magen/Darmerkrankungen können leicht erhöhte Saccharosewerte im Urin gemessen werden. Das können zum Beispiel Magengeschwüre sein, bei denen die Magenwand durchlässig für Saccharose ist. Saccharose wird nur im Magen/Darmtrakt gespalten. Gelangt Saccharose auf irgendeinen Weg ins Blut, wird sie über den Urin ausgeschieden. Der Grenzwert für einen verdächtigen Saccarosebefund (Manipulation durch Zuspucken des Markers), muss daher etwas höher gewählt werden, als eigentlich nötig wäre, um Manipulationen zu erkennen, bei denen Marker im unteren Konzentrationsbereich zugegeben wurde. However, sucrose has the disadvantage that, although it is highly likely to indicate manipulation by spitting on the marker solution, it ultimately does not necessarily provide evidence of this. There are other sources that can cause increased sucrose concentration in urine. On the one hand, this is the injection of a solution containing sucrose, as is the case with the abusive use of methadone, but slightly elevated sucrose levels can also be measured in the urine in some stomach/intestinal diseases. These can be, for example, stomach ulcers in which the stomach wall is permeable to sucrose. Sucrose is only broken down in the stomach/intestinal tract. If sucrose enters the blood through any route, it is excreted in the urine. The limit value for a suspicious sucrose finding (manipulation by spitting the marker) must therefore be chosen slightly higher than would actually be necessary in order to detect manipulations in which marker was added in the lower concentration range.
Bei der Verwendung von Saccharose ist es ebenfalls nötig, dass sich der Einsender der urinprobe streng an die Anleitung hält. Die zugegebene Saccharosemenge muss exakt stimmen und es dürfen keine Fruchtsäfte oder Limonaden für die Verabreichung verwendet werden, da sonst die Bewertung der Befunde erschwert wird. Weiterhin ist die Saccharose z.B. durch Hefen abbaubar. Werden Hefen zu einem manipulierten Urin, der Saccharose enthält gegeben, so wird die Saccharose abgebaut. Hefen oder Bakterien können auch ohne Manipulation im Urin vorkommen, durch Blasenerkrankungen oder durch die nicht sterile Urinabgabe, daher ist der direkte Nachweis von Hefen oder Bakterien im Urin nicht hilfreich. (Die "natürlichen Hefen oder Bakterien würden unter normalen Bedingungen kaum ausreichen, um die Saccharose ab zu bauen.) Der indirekte Nachweis von Hefen durch die Messung des Abbaus einer zugesetzten Saccharosemenge vor der Analyse im Labor, hat sich nicht als zuverlässig erwiesen. When using sucrose, it is also necessary that the person sending the urine sample strictly follows the instructions. The amount of sucrose added must be exactly right and no fruit juices or lemonades may be used for administration, otherwise it will make it more difficult to evaluate the findings. Furthermore, the sucrose can be broken down by yeast, for example. If yeasts are added to manipulated urine that contains sucrose, the sucrose is broken down. Yeast or bacteria can occur in urine even without manipulation, through bladder disease or through non-sterile urine output, so direct detection of yeast or bacteria in urine is not helpful. (The "natural yeasts or bacteria would hardly be sufficient to break down the sucrose under normal conditions.) The indirect detection of yeasts by measuring the breakdown of an added amount of sucrose before analysis in the laboratory has not proven to be reliable.
Die Aufgabe der vorliegenden Erfindung besteht darin, eine Substanz als Kontroll-Marker vorzuschlagen, die nicht toxisch, in sehr kleinen Mengen nachweisbar, durch Hefe nicht abbaubar und einfach zu handhaben ist. 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.
Es wurde überraschenderweise festgestellt, dass Curcumin als Kontroll-Marker-Substanz hervorragend geeignet ist, Saccharose zu ersetzen oder zusätzlich zur Saccharose verwendet werden kann. It was surprisingly found that curcumin is ideal as a control marker substance to replace sucrose or can be used in addition to sucrose.
Der Gegenstand der vorliegenden Erfindung eine Trinklösung zur endogenen Markierung von Urin, wobei die Lösung neben einem oder mehreren Polyethylenglycolen auch Curcumin enthält. Darüber hinaus stellt die vorliegende Erfindung ein Verfahren zur Analyse einer Urinprobe auf die Anwesenheit von mindestens einem bestimmten Analyten durch Verwendung der vorgenannten Trinklösung. Analyten können Rauschmittel, Arzneimittel, Dopingsubstanzen, oder Metabolite der vorgenannten Substanzen sein, deren Nachweis in der Probe über das Verhalten oder eine Behandlung der Probanden zulässt. Beispielsweise kann der Analyt Heroin, Methadon, Kokain, THC, Barbituratem Nikotin, Alkohol oder ein auf der Liste der Dopingmittel stehendes Dopingmittel sein. The subject of the present invention is a drinking solution for the endogenous marking of urine, the solution also containing 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 particular analyte by using the aforementioned drinking solution. Analytes can be intoxicants, drugs, doping substances, or metabolites of the aforementioned substances, the detection of which in the sample allows information about the behavior or treatment of the test subjects. For example, the analyte can be heroin, methadone, cocaine, THC, barbiturate, nicotine, alcohol or a doping substance on the list of doping substances.
Curcumin ist nicht toxisch und ist der Hauptbestandteil des Würz-, Heil- und Färbemittels Kurkuma, das zusammen mit anderen Gewürzen im Currypulver verwendet wird (https://de.wikipedia.org/wiki/Curcumin). Curcumin is non-toxic and is the main component of the seasoning, medicinal and coloring agent turmeric, which is used together with other spices in curry powder (https://de.wikipedia.org/wiki/Curcumin).
Es wurde festgestellt, dass Curcumin in sehr geringen Mengen nachweisbar ist, aber im Körper vollkommen verstoffwechselt wird und dadurch als Kontroll-Marker-Substanz gut geeignet ist. Durch die Zugabe von Curcumin in die Trinklösung mit einem Polyethylenglycol oder mit einer Kombination von Polyethylenglycolen als Marker-Substanzen kann eine Täuschung durch Zugabe von der Trinklösung in den fremden Urin sehr leicht ermittelt werden, da schon sehr kleine Mengen an Curcumin leicht nachweisbar sind. It was found that curcumin can be detected in very small amounts, but is completely metabolized in the body and is therefore well suited as a control marker substance. By adding curcumin to the drinking solution with a polyethylene glycol or with a combination of polyethylene glycols as marker substances, deception can be easily detected by adding the drinking solution to someone else's urine, as even very small amounts of curcumin can be easily detected.
Durch die Messung mehrerer tausend nativer Urine der Drogenabhängigen und Messung - auch nach hoher eingenommener Menge Curcumin - konnte man Curcumin im Urin nicht finden. Die Nachweisgrenze für Curcumin liegt bei der eingesetzten flüssig/flüssig Extraktion bei ca. 10 ng/ml. Die Nachweisgrenze bei alternativ getesteten Shoot- und Dilute-Verfahren lag bei ca. 50-100 ng/ml. By measuring several thousand native urines of drug addicts and measuring - even after high amounts of curcumin had been ingested - curcumin could not be found in the urine. The detection limit for curcumin with the liquid/liquid extraction used is approx. 10 ng/ml. The detection limit for alternatively tested shoot and dilute methods was approximately 50-100 ng/ml.
Beispiel Example
Die Probe wird vor der Extraktion hydrolysiert, um vorhandene Analyt-Glucuronide in den freien Analyten zu überführen. Vor der Hydrolyse wird der interne Standard IS zugegeben. Bei der Flüssig-Extraktion werden 10 pl IS zugegeben. The sample is hydrolyzed before extraction to convert any analyte glucuronides present into the free analyte. The internal standard IS is added before hydrolysis. During the liquid extraction, 10 μl of IS are added.
Pro Probe werden zur Hydrolyse der Glucuronide 10 pl Acetatpuffer und 10 pl einer ß- Glucuronidase-Lösung und 40 pl Methanol zugesetzt. Die Lösung wird über Nacht oder 1 Stunde bei 60°C inkubiert und erst dann extrahiert. To hydrolyze the glucuronides, 10 μl of acetate buffer and 10 μl of a ß-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 only then extracted.
Die Hydrolyse ist für den Nachweis von Curcumin und den PEGs nicht erforderlich, wird aber durchgeführt, da unter Umständen simultan auch die Drogen bestimmt werden können. Hydrolysis is not necessary for the detection of curcumin and the PEGs, but is carried out because under certain circumstances the drugs can also be determined at the same time.
Flüssig-EXtraktion Liquid EXtraction
100 pl Urinprobe werden wie oben beschrieben hydrolysiert. 100 μl urine sample is hydrolyzed as described above.
Nach der Hydrolyse wird die Probe mit 1,6 ml gesättigter NaCI-Lösung und 25 pl 3 M NH3 in ein verschraubbares Glasröhrchen gegeben und mit 2 ml Ethylacetat/Dichlormethan (1:1) versetzt. Die Probe wird 10 min. am Überkopfschüttler vermischt. Dann wird zentrifugiert und der Überstand in ein Glasröhrchen gegeben. Anschließend wird die Probe bis zur Trocknung im Stickstoffstrom oder mit Luft abgedampft. Der Rückstand wird zuerst mit 25 pl Methanol und dann mit 75 pl H2O gelöst. Die gelösten Proben werden in Vials mit Mikroinsert gefüllt und verschlossen. 5 pl Probe werden in die LCMS/MS injiziert. After hydrolysis, the sample is placed in a screw-top 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 minutes on an overhead shaker. Then it is centrifuged and the supernatant is placed in a glass tube. The sample is then evaporated in a stream of nitrogen or with air until it dries. The residue is first mixed with 25 pl Methanol and then dissolved with 75 pl H2O. The dissolved samples are filled into vials with microinserts and sealed. 5 μl of sample are injected into the LCMS/MS.
Kontrollmaterial Control material
Da keine kommerziellen Kontrollen mit Curcumin erhältlich sind, wird eine eigene Kontrolle hergestellt, die neben den Drogen, Suchtstoffen und PEGs auch Curcumin in einer Konzentration von 20 - 50 ng/ml enthält. Der Nachweis des Curcumins ist qualitativ, daher wird keine Kalibration durchgeführt. Es steht aber jedem Labor frei, Curcumin zu quantifizieren. Since no commercial controls with curcumin are available, a separate control is produced which, in addition to the drugs, narcotic substances and PEGs, also contains curcumin in a concentration of 20 - 50 ng/ml. The detection of curcumin is qualitative, therefore no calibration is carried out. However, every laboratory is free to quantify curcumin.
Ergebnisse Results
Proben, in denen kein Marker gefunden wurde, werden aus dem original Urinröhrchen wiederholt. Ein bestätigtes negatives Markerergebnis wird als negativ befundet mit dem Hinweis, dass auch bei zu kurzer Wartezeit zwischen Einnahme der Markersubstanzen und Urinabgabe ein negativer Befund die Folge sein kann. Ebenso werden positive Curcumin- Urine erneut aus dem Originalgefäß gemessen. Ein bestätigtes positives Curcuminergebnis wird als Manipulation durch Zuspucken der Markerlösung zu einem anderen Urin bewertet. Samples in which no marker was found are repeated from the original urine tube. A confirmed negative marker result is considered negative with the indication that a negative result can result even if the waiting time between taking the marker substances and urine output is too short. Positive curcumin urines are also measured again from the original vessel. A confirmed positive curcumin result is assessed as manipulation by spitting the marker solution into another urine.

Claims

Ansprüche Expectations
1. Trinklösung enthaltend eine Kombination von einem oder mehreren Polyethylenglycolen und Curcumin, wobei die Polyethylenglykole unterschiedliche Molekulargewichte aufweisen, und wobei die Molekulargewichte der Polyethylenglykole zwischen 200 und 5000 Da betragen. 1. Drinking solution containing a combination of one or more polyethylene glycols and curcumin, the polyethylene glycols having different molecular weights, and the molecular weights of the polyethylene glycols being between 200 and 5000 Da.
2. Trinklösung nach Anspruch 1, wobei die Polyethylenglycole monodispers sind. 2. Drinking solution according to claim 1, wherein the polyethylene glycols are monodisperse.
3. Trinklösung nach Anspruch 1, wobei die Polyethylenglycole polydispers sind. 3. Drinking solution according to claim 1, wherein the polyethylene glycols are polydisperse.
4. Trinklösung nach einem der vorhergehenden Ansprüche, wobei einer der Polyethylenglycole (PEGs) ein Molekulargewicht von 200, 300, 400, 500, 600, 1000 oder 1500 aufweist. 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. Trinklösung nach einem der vorhergehenden Ansprüche, wobei die Lösung drei Polyethylenglycole mit verschiedenen Molekulargewichten enthält. 5. Drinking solution according to one of the preceding claims, wherein the solution contains three polyethylene glycols with different molecular weights.
6. Trinklösung nach einem der vorhergehenden Ansprüche, wobei die Lösung drei Polyethylenglycole mit verschiedenen Molekulargewichten enthält. 6. Drinking solution according to one of the preceding claims, wherein the solution contains three polyethylene glycols with different molecular weights.
7. Verwendung von Curcumin als metabolisierbarer Kontroll-Marker bei der endogenen Markierung. 7. Use of curcumin as a metabolizable control marker in endogenous labeling.
8. Verwendung von Curcumin als metabolisierbarer Kontroll-Marker bei der endogenen Markierung zusammen mit einer Kombination von Polyethylenglycolen in einer Trinklösung. 8. Use of curcumin as a metabolizable control marker in endogenous labeling along with a combination of polyethylene glycols in a drinking solution.
9. Verfahren zur Analyse einer Urinprobe auf die Anwesenheit von mindestens einem bestimmten Analyten, wobei das Verfahren das Verabreichen der Trinklösung gemäß einem der Ansprühe 1-8 umfasst. 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 one of the sprays 1-8.
10. Kit zur Verwendung in dem Verfahren gemäß Anspruch 9, enthaltend eine Trinklösung nach einem der Ansprüche 1-8. 10. Kit for use in the method according to claim 9, containing a drinking solution according to any one of claims 1-8.
PCT/EP2023/074099 2022-09-07 2023-09-02 Metabolisable control marker for the endogenous marking of urine WO2024052243A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102022122731.6A DE102022122731A1 (en) 2022-09-07 2022-09-07 Metabolizable control markers for endogenous urine labeling
DE102022122731.6 2022-09-07

Publications (1)

Publication Number Publication Date
WO2024052243A1 true WO2024052243A1 (en) 2024-03-14

Family

ID=87972010

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2023/074099 WO2024052243A1 (en) 2022-09-07 2023-09-02 Metabolisable control marker for the endogenous marking of urine

Country Status (2)

Country Link
DE (1) DE102022122731A1 (en)
WO (1) WO2024052243A1 (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1410014A1 (en) 2001-03-15 2004-04-21 KELLER, Ruprecht Method for sample identification in a mammal as well as a kit for performing this method
EP1563311A2 (en) 2002-11-18 2005-08-17 Gisela Gauchel Marker substances and the use of the same in diagnostic methods
US20170143646A1 (en) * 2014-03-28 2017-05-25 Omniactive Health Technologies Limited Effect of lipophilic nutrients on diabetic eye diseases
DE202018103853U1 (en) * 2018-07-05 2018-08-07 Viktoria Apotheke Dr. Ludwig Trennheuser Inhaber Dr. Fritz Trennheuser Composition for application of curcumin
US20190302098A1 (en) * 2014-06-18 2019-10-03 Ruprecht Keller Method for identifying of a biological sample of a mammal, composition for use in this method and kit for performance of this method

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1410014A1 (en) 2001-03-15 2004-04-21 KELLER, Ruprecht Method for sample identification in a mammal as well as a kit for performing this method
EP1563311A2 (en) 2002-11-18 2005-08-17 Gisela Gauchel Marker substances and the use of the same in diagnostic methods
EP1563311B1 (en) * 2002-11-18 2007-01-03 Gisela Gauchel Marker substances and the use of the same in diagnostic methods
US20170143646A1 (en) * 2014-03-28 2017-05-25 Omniactive Health Technologies Limited Effect of lipophilic nutrients on diabetic eye diseases
US20190302098A1 (en) * 2014-06-18 2019-10-03 Ruprecht Keller Method for identifying of a biological sample of a mammal, composition for use in this method and kit for performance of this method
DE202018103853U1 (en) * 2018-07-05 2018-08-07 Viktoria Apotheke Dr. Ludwig Trennheuser Inhaber Dr. Fritz Trennheuser Composition for application of curcumin

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HUPPERTZ BERND ET AL: "Urine labeling with orally applied marker substances in drug substitution therapy", CLINICAL CHEMISTRY AND LABORATORY MEDICINE, vol. 42, no. 6, 7 January 2004 (2004-01-07), pages 621 - 626, XP055282632, ISSN: 1434-6621, DOI: 10.1515/CCLM.2004.107 *

Also Published As

Publication number Publication date
DE102022122731A1 (en) 2024-03-07

Similar Documents

Publication Publication Date Title
Balíková Hair analysis for drug abuse. Plausibility of interpretation
DE60217351T3 (en) METHOD FOR IDENTIFYING SAMPLES OF ANIMAL MUG AND A KIT FOR CARRYING OUT THE METHOD
DE202005020939U1 (en) In vitro measurement of neutrophil cell activation status in a mammalian sample comprises (immuno)capturing myeloperoxidase (MPO) present in the sample; measuring and comparing the MPO values with normal MPO levels
DE2801216A1 (en) DIAGNOSTIC METHODS AND MEANS FOR DETECTION OF MALABSORPTION
DE2819574A1 (en) METHOD AND DEVICE FOR PROCESSING A BODY FLUID SAMPLE
WO2024052243A1 (en) Metabolisable control marker for the endogenous marking of urine
Wormley Micro-chemistry of Poisons: Including Their Physiological, Pathological, and Legal Relations; with an Appendix on the Detection and Microscopic Discrimination of Blood: Adapted to the Use of the Medical Jurist, Physician, and General Chemist
DE202022002742U1 (en) Metabolizable control markers for endogenous labeling of urine
Cittadini et al. Prevalence of alcohol and other drugs in injured drivers and their association with clinical outcomes.
US7674625B2 (en) Method for determining improper manipulation of urine samples using marker compounds
Stehr-Green et al. An evaluation of serum pesticide residue levels and liver function in persons exposed to dairy products contaminated with heptachlor
DE69635091T2 (en) Prevention of sudden infant death syndrome
DE69730606T2 (en) TEST STRIPS FOR IDENTIFYING STOMACH PROBLEMS BASED ON THE PRESENCE OF UREASE
MD et al. Workplace drug testing
EP1102993A1 (en) Method for detecting drugs, and a solvent and a decomposition solution therefor
WO2000047990A2 (en) Gas analyser and the use thereof in medical diagnostics
DE10331379A1 (en) Sensory method for evaluating breath
Botero Clinical trial methodology in intestinal parasitic diseases
Omran Systematic Review of Postmortem Examination in Toxicological Fatalities
Khadadah et al. Studies of the relationship between allergen-specific IgE antibodies and skin test reactivity in patients with asthma in Kuwait
DE102008061174A1 (en) Assigning biological sample to donor of sample, comprises obtaining DNA sample, determining DNA sequences of non-coding regions, obtaining biological sample, isolating DNA-containing cells, determining and comparing DNA sequences
Lucas Evaluation of Effectiveness of Door Locks pre 1951 and post 1955 Automobiles
Blenden et al. Applications of laboratory technology in the evaluation of the risk of rabies transmissions by biting dogs and cats
Oliver et al. Amitriptyline and metabolites in biological fluids
Whitaker et al. CHEMISTRY (including Chemical Toxicology)