USRE44859E1 - Method for sample identification in a mammal as well as a kit for performing this method - Google Patents

Method for sample identification in a mammal as well as a kit for performing this method Download PDF

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USRE44859E1
USRE44859E1 US13/592,236 US200213592236A USRE44859E US RE44859 E1 USRE44859 E1 US RE44859E1 US 200213592236 A US200213592236 A US 200213592236A US RE44859 E USRE44859 E US RE44859E
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polyethylene glycols
sample
molecular weights
urine sample
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Ruprecht Keller
Gisela Gauchel
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    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/104165—Lipid, cholesterol, or triglyceride standard or control
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/105831—Protein or peptide standard or control [e.g., hemoglobin, etc.]

Definitions

  • the present invention relates to a method by which a sample which was taken from an excretion, a body fluid of a mammal or as a tissue sample, can be identified with relation to the origin of the sample and, in this way, can be unequivocally assigned to the donor of the sample, whereby the sample can be investigated for an analyte. Additionally, the object of the invention is a kit for performing this method.
  • Diagnostic methods, methods for monitoring the course of a therapeutic measure, prophylactic routine investigations as well as forensic medical investigations on man normally include the analytical investigation of samples in the laboratory, such as for example blood or serum samples which were taken from the subject, as well as the investigation of excretions of the subject, such as for example urine.
  • samples in the laboratory such as for example blood or serum samples which were taken from the subject
  • excretions of the subject such as for example urine.
  • a very wide variety of analytical methods with animal samples is today every bit as much common practice as well.
  • problems having arisen in connection with intensive livestock farming, such as BSE sicknesses due to the feeding of animal meal or the admixing of illegal food additives in the form of hormones and/or antibiotic preparations into the mast of livestock necessitate an extension of regular control investigations in animal herds in agriculture.
  • test results can be used as incriminating evidence against the sample donor or, in the case of a sample originating from a livestock animal, against the owner of the animal.
  • test results can be used as incriminating evidence against the sample donor or, in the case of a sample originating from a livestock animal, against the owner of the animal.
  • the urine must be checked at least once a week or, under certain circumstances, even more frequently.
  • submission of the urine sample under observation is not possible in normal doctors' offices since commonly only a small restroom is present and normally male medical personnel are not at adequate disposal to accompany the male methadone patients.
  • the construction of restrooms suitable for sample submission under observation requires a high financial outlay. Just the costs for such investment in the health office of Duesseldorf came to 50 TDM.
  • the goal of the present invention is therefore to ensure an unequivocal assignment of the samples to the donor and, in this way, to overcome the problems or disadvantages common to the prior art.
  • this goal is met by providing a method for the investigation of biological samples from a mammal for at least one component, wherein the method includes the following steps:
  • FIG. 1 shows the results of a chromatographic elution pattern of “Marker A” (PEG 400) of a urine sample as described in Example 2.
  • the results show that Marker A can be used as a marker substance for identification of a urine sample from a specified donor.
  • FIG. 2 shows the results of a chromatographic elution pattern of “Marker B” (PEG 600) of a urine sample as described in Example 2.
  • the results show that Marker B can be used as a marker substance for identification of a urine sample from a specified donor.
  • FIG. 3 shows the results of a chromatographic elution pattern of “Marker C” (mixture of PEG 400 and PEG 600) of a urine sample as described in Example 2.
  • the results show that PEG 400 and 600 can be used in combination as marker substances for identification of a urine sample from a specified donor.
  • the idea of the present invention was therefore to find a possibility with which the sample to be investigated can be marked while preventing this marker from being removed from the sample by methods accessible to a layperson.
  • the method is therefore suitable for example for monitoring methadone therapy as well as for doping checks.
  • Advantageous marking substances are in general characterized by a series of specific characteristics. These marker substances exert no pharmacological side effects on the organism of the mammal at the concentrations which are necessary for detection of these marker substances in the blood, in the urine or other body fluids or in body excretions according to the invention.
  • a derivative which is specifically formed from the at least one marker substance can also just as well be used in place of the latter.
  • derivatives are to be understood all subsequent products which arise as a result of a chemical transformation in the organism of the subject or in the removed sample, wherein however all subsequent products are excluded which are not exclusively attributable to the transformation of a specific marker in the subject organism or in the removed sample.
  • the marker substances are soluble in a liquid, that the normal taste of the liquid such as for example juice is not changed by the addition or that, following dissolving in water, no unpleasant taste of the resulting solution is caused by the marker substances and, therefore, the subject can willingly drink the liquid containing the markers.
  • marker substances are characterized in that they are absorbed quickly through the intestinal mucous membranes and are excreted from the subject in the urine. It is further advantageous if these marker substances in urine samples can be detected in as simple a manner as possible by detection methods already established in chemical investigation laboratories such as for example common methods of clinical analytical chemistry. According to the invention, it is preferable to use marker substances which are not metabolized following uptake by the subject.
  • Preferred marker substances are sugars or sugar derivatives such as for example arabinose, erythrulose, myo-inositol, cis-inositol, mannitol, sorbose, rhamnose, sorbitol, xylose and xylulose, which are soluble in water and which can be easily detected by enzymatic tests.
  • isoprenoids lipids, saccharides, polyols, polyethylene glycols, derivatives or mixtures of these substances as the marker substance.
  • the marker substance or a combination of multiple marker substances is dissolved in a liquid, and the liquid is orally administered in that the subject drinks the liquid approximately 30 to 60 minutes before the urine submission.
  • Polyethylene glycols or mixtures thereof are most preferably used as marker substances for the investigation of urine samples.
  • the administration of the marker substance can be accomplished in different ways.
  • administration is to be understood the introduction of one or a multitude of marker substances into the organism of the sample donor.
  • the marker substance or the multitude of marker substances can be administered to the sample donor preferably parenterally or orally. It is especially preferred that the marker substance or the multitude of marker substances be taken up via the digestive tract and that, during uptake, no metabolization of the marker substances takes places.
  • This length of time represents the time which the at least one marker substance requires to reach the location of sample removal.
  • the time is to be understood as being that time which is required until the at least one marker substance is present in the separable component and this component is separated from the sample donor.
  • the amount of time one must wait can be empirically determined, wherein however in most cases the corresponding values or methods for their determination are known in the prior art (van Rossum, J.
  • Sample removal occurs in different ways depending on the type of sample to be investigated.
  • part of the sample is taken up into a sample vessel and, after this time, is ready for further investigation.
  • the samples can usually be furnished by the subjects themselves in that the subject is simply given a sample vessel.
  • a direct operation on the subject is normally necessary.
  • obtention of blood from the subject can be accomplished using a suction pipette following pricking or cutting of the skin with a disposable lancet or—in larger quantities—using an injection syringe or blood collection tube (German: Venule) after puncture of the vein.
  • the latter is obtained by lumbar, suboccipital or ventricle puncture.
  • biological sample is meant the components of a mammal designated for the analytical investigation. Relevant here are body excretions, body fluids or tissue samples. The components making up the sample can include components of a mammalian organism which still exist in the mammal at the time of sample removal as well as previous components of the mammal.
  • body excretions or “excretion” are to be understood urine, stool, secretions from salivary, milk, tear and sweat glands.
  • body fluid are to be understood extracellular liquids of a mammalian organism like blood, serum and liquor.
  • the samples removed from or excreted by a mammal are body excretions, body fluids or tissue samples.
  • tissue sample is to be understood an organization of identically differentiated cells obtained by a direct operation into the living mammalian organism, as well as these cells' intercellular substance. Hair samples and samples of sloughed-off parts of skin are also to be understood as falling within the meaning of this term.
  • the respective sample has to be prepared prior to the analysis method.
  • the preparation steps can include centrifugation for the separation of solid, non-solubilized materials in a liquid sample such as for example urine, solubilization or suspension of solid samples such as for example stool, concentration by ion-exchange chromatography using Centricons, by precipitation with suitable reagents such as ammonium sulfate, adjustment of the pH value required for the analysis method, homogenization of the sample such as by ultrasonication or by using vibration cell mills in order to, for example, be able to investigate components from originally intact tissues, separation of materials used in lysing the sample such as for example detergents and other preparation steps known to one of ordinary skill in the art.
  • a number of enzymatic, immunological, mass-spectroscopic and electrophoretic detection methods as well as combinations of these methods are available for the determination of the presence or absence of at least one marker substance in a sample.
  • detection is accomplished by a coupled Gas Chromatography/Mass Spectrometry (GC/MS) or High Performance Liquid Chromatography/Mass Spectrometry (HPLC/MS) method or by High Performance Liquid Chromatography (HPLC) or Gas Chromatography (GC).
  • GC/MS Gas Chromatography/Mass Spectrometry
  • HPLC/MS High Performance Liquid Chromatography/Mass Spectrometry
  • HPLC High Performance Liquid Chromatography
  • GC Gas Chromatography
  • these detection methods allow a high degree of automatization so that a multitude of samples can be analyzed in a short time and, in as far the chromatograms and, as the case may be, mass spectroscopic fractionation patterns of reference substances already exist in the computer evaluation unit, the actual detection of the at least one marker substance is also greatly simplified.
  • analyte is to be understood at least one chemical substance, wherein the knowledge as to the presence or, as the case may be, also of its concentration in the sample, allows a conclusion as to a past, expected or present condition of the sample donor.
  • a conclusion as to an incorrectly functioning—because incomplete—resorption of glucose from the urine by the kidney tubules (glucosuria) in a subject is made possible on the basis of knowledge of the concentration of an analyte such as for example the glucose concentration in the urine of a urine sample, which was normally enzymatically determined by means of glucose oxidase (GOD) or hexokinase.
  • Analytes can further be intoxicants, medicines, metabolites of the previously named substances, the detection of which in the sample yields information as to the behavior or a treatment of the subject.
  • the method can advantageously be used in the monitoring of adherence to regulations for the use of feed additives in agricultural livestock mast farming.
  • the use of the method according to the invention can avoid the problem of a tampering with the samples to be investigated by the owner of the herd of mast pigs.
  • those marker substances are advantageous which remain in the animal over a long length of time—in the ideal case over the entire duration of masting—yet which are still continuously present in a detectable amount, for example in a body excretion.
  • those marker substances are advantageous which can be administered to the animal as a time-release agent, by virtue of which for example a time-delayed yet continuous resorption through the intestinal mucous membranes takes place and therefore the at least one marker substance is detectable over a longer length of time, for example in a body excretion like animal feces.
  • Especially suitable samples are samples with which both the investigation for the at least one marker substance as well as the detection or the concentration determination of at least one analyte takes place.
  • kits for performing the described method for sample identification in a mammal wherein the kit according to the invention includes a marker substance in a container such as a tablet vessel as well as, as the case may be, means for administering the at least one marker substance to the mammal.
  • this kit also contains at least one reference substance for the detection of the marker substance or the multitude of marker substances.
  • a kit according to the invention preferably contains, for the oral administration of the marker substances, these marker substances in the form of individual water-soluble effervescent tablets.
  • these effervescent tablets can also already contain the marker substances as mixtures of multiple marker substances.
  • the respective substance code can then be taken from the label of the tablet vessel.
  • the kit can comprise effervescent tablets with varying concentrations of marker substances corresponding to the circle of people to whom the marker is to be administered, so that these marker substances can be applied for example to children as well as adults without reaching a concentration of marker substances in the subject at which pharmacological side effects can arise.
  • Kits intended for the marking of urine samples of methadone patients preferably contain tablets, capsules, or similar application forms in which both the amount of methadone to be administered as well as the mixture of marker substances are available together.
  • kits according to the invention include multiple reference substances by means of which the marker substances can be easily identified in the chromatographic analysis of the sample, such as for example in the investigation of the urine sample.
  • an ampoule tube can also be present in the kit according to the invention, which ampoule tube contains a mixture of marker substances solubilized in a suitable carrier means according to the chosen chromatographic method, wherein this mixture corresponds exactly to the mixture present in the corresponding methadone tablets.
  • the embodiment relates to the marking of a urine sample to be investigated and its subsequent investigation.
  • the subject receives 100-300 ml of liquid to drink, in which 1 g polyethylene glycol 600 is solubilized as a marker substance.
  • Fruit juices, water, and other liquids palatable to humans can be used as liquids to drink.
  • monodisperse fractions or mixtures of monodisperse fractions can also be used.
  • the laboratory establishes a substance code. Such a code is given in the following as five monodisperse polyethylene glycol fractions. Here, “0” stands for not present and “1” stands for present.
  • the substances A, B, C, D and E correspond to polyethylene glycol fractions with molecular weights:
  • the subject After ingestion the subject was requested to wait at least 30 minutes and at the most 4 hours before urinating. The subject was allowed to consume further liquids or solid food during this waiting phase. The subject did not have to be supervised during the waiting time. The submission of urine by the subject took place without supervision.
  • the sample vessel was identified with a barcode label coding a job number also contained on the computer-readable accompanying tag.
  • the accompanying tag were noted the name of the subject, the desired investigation as well as the combination of marker substances or the substance code.
  • the sender is saved by the job number in the master data of the lab computer.
  • the samples were transported to the laboratory with the accompanying tag.
  • the accompanying tag was entered into the computer with a card reader. The job was recorded in this way.
  • the substance combination or the substance code was also entered into the computer.
  • the urine was centrifuged, 100 ⁇ l of the supernatant was given on Nucleosil C 100-(C18), 3 ⁇ m (4.6 ⁇ 125 mm) at a flow rate of 0.5 ml/min (methanol/water 5/95) and was investigated for polyethylene glycol by detection with an Refractive Index (RI) detector.
  • RI Refractive Index
  • each investigated urine sample could be unequivocally assigned to the respective subject via the substance code of the different polyethylene glycol fractions used.
  • the subject was subsequently investigated for the analyte, i.e. an intoxicant to be detected like heroin or its derivatives.
  • Sugars for marking body fluids can be used in the same manner as described for polyethylene glycol fractions. These are determined from urine or other body fluids via enzymatic detection reactions. The analytical detection methods required for this are known in the prior art (Methods of Enzymic Analysis, ed. Bergmeyer, H. U. VCH Verlagsgesellschaft mbH, Weinheim 1986).
  • the sample were prepared as follows: 10 ml urine was centrifuged at 10500 ⁇ g for 10 min.
  • the chromatograph was operated isocratically at ambient temperatures in the column-switching mode. Because RI detection limits to isocratic mobile phases, eluent of cleanup and analytical pump were identical, consisting of 44% methanol and 56% water. 100 ⁇ l supernatant of the centrifuged urine were injected automatically onto a (60 ⁇ 4.6 mm) precolumn filled with Nucleosil 100 C18, 5 ⁇ m. With the eluent delivered by the clean-up pump at a flow of 0.4 ml/min and a pressure of 36 bar, matrix impurities were discharged to the waste, while the polyethylene glycol (PEG) fractions were retarded on the stationary phase.
  • PEG polyethylene glycol
  • HPLC-Equipment sample injector S 5200 fitted with a 100 ⁇ l injection loop, precolumn clean-up pump S 2100, degaser integrated, six-port motor switching valve ProLAB, column oven SFD 125-600, refraction index detector of deflection type, inline filter element PATTM, for PEEK 3 ⁇ m inline filters was obtained from Schambeck SFD GmbH, Bad Honnef, Germany.
  • Analytical pump M480, degassing module degasys DG1310 and data acquisition system Chromeleon 6.11 under Windows NT 4.0 were purchased from Gynkotec.
US13/592,236 2001-03-15 2002-03-14 Method for sample identification in a mammal as well as a kit for performing this method Active 2025-04-12 USRE44859E1 (en)

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Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE10112470A DE10112470B4 (de) 2001-03-15 2001-03-15 Verfahren zur Proben-Identifizierung bei einem Säugetier sowie Kit zur Durchführung dieses Verfahrens
DE10112470 2001-03-15
US47181502A 2002-03-14 2002-03-14
PCT/EP2002/002868 WO2002075307A1 (en) 2001-03-15 2002-03-14 Method for sample identification in a mammal as well as a kit for performing this method
US13/592,236 USRE44859E1 (en) 2001-03-15 2002-03-14 Method for sample identification in a mammal as well as a kit for performing this method

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US (2) USRE44859E1 (de)
EP (1) EP1410014B2 (de)
JP (1) JP2004535554A (de)
CN (1) CN1630817B (de)
AT (1) ATE350659T1 (de)
AU (1) AU2002304857B2 (de)
BR (1) BR0208069A (de)
CA (1) CA2440045C (de)
CY (1) CY1107490T1 (de)
DE (2) DE10112470B4 (de)
DK (1) DK1410014T4 (de)
ES (1) ES2280540T5 (de)
HK (1) HK1064738A1 (de)
IL (1) IL157917A0 (de)
NZ (1) NZ527880A (de)
PT (1) PT1410014E (de)
WO (1) WO2002075307A1 (de)

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NO333424B1 (no) * 2008-07-10 2013-06-03 Resman As Et sporstoffsystem og en fremgangsmate for a spore en sporstofforbindelse i et petroleumsproduksjons-fluidsystem
DE102008061174A1 (de) 2008-12-09 2010-06-10 Ladr Gmbh Medizinisches Versorgungszentrum Dr. Kramer Und Kollegen Verfahren zur Identifikation von biologischen Proben sowie Kit mit einem Identifikationssystem für biologische Proben
WO2011032584A2 (en) * 2009-09-16 2011-03-24 Ruprecht Keller Method for sample identification in a mammal as well as a kit for performing this method
US20140342380A1 (en) * 2011-11-21 2014-11-20 Daniel Saal Verifying the source of biological samples; method, composition and kit therefor
ES2739351T3 (es) * 2012-10-10 2020-01-30 Ruprecht Keller Marcadores para productos farmacéuticos
WO2014210434A1 (en) * 2013-06-27 2014-12-31 KUHLMANN, Anthony Methods and compositions for marking urine samples to identify source
US20150369794A1 (en) * 2014-06-18 2015-12-24 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
CN104483426A (zh) * 2014-11-27 2015-04-01 黑龙江省乳品工业技术开发中心 一种奶牛泌乳中乙草胺含量消退的预测方法及应用
DE102015001872A1 (de) 2015-02-12 2016-08-18 Eberhard Wieland Verfahren zur Untersuchung einer biologischen Probe bei Doping- und/oder Drogentests
ES2952542T3 (es) 2019-07-04 2023-11-02 Ruma Gmbh Control de toma independiente de la ubicación
DE202022002742U1 (de) 2022-09-07 2023-03-24 Ruma Gmbh Metabolisierbare Kontroll-Marker für die endogene Markierung von Urin
DE102022122731A1 (de) 2022-09-07 2024-03-07 Ruma Gmbh Metabolisierbare Kontroll-Marker für die endogene Markierung von Urin

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US20040166532A1 (en) 2004-08-26
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CA2440045C (en) 2011-05-17
ES2280540T5 (es) 2012-10-22
PT1410014E (pt) 2007-03-30
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EP1410014B1 (de) 2007-01-03
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CA2440045A1 (en) 2002-09-26
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AU2002304857B2 (en) 2006-11-02
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