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WO1991007661A1 - Procede et kit de detection de medicaments dans la peau de mammifere vivants et morts - Google Patents

Procede et kit de detection de medicaments dans la peau de mammifere vivants et morts Download PDF

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Publication number
WO1991007661A1
WO1991007661A1 PCT/US1990/006657 US9006657W WO9107661A1 WO 1991007661 A1 WO1991007661 A1 WO 1991007661A1 US 9006657 W US9006657 W US 9006657W WO 9107661 A1 WO9107661 A1 WO 9107661A1
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WO
WIPO (PCT)
Prior art keywords
drugs
stratum corneum
skin
living
layer
Prior art date
Application number
PCT/US1990/006657
Other languages
English (en)
Inventor
Vincent E. Hill
Original Assignee
Hill Vincent E
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 Hill Vincent E filed Critical Hill Vincent E
Publication of WO1991007661A1 publication Critical patent/WO1991007661A1/fr

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Classifications

    • 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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G01N33/567—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds utilising isolate of tissue or organ as binding agent

Definitions

  • the present invention relates to a method of detecting drugs in living and post-mortem skin and a kit therefor.
  • the particular mode of drug testing which is used may depend upon whether the user is tested alive or post-mortem. For example, if the drug user is tested while living for the presence of one or more drugs, it is most common to test the blood. In fact, blood is the
  • HIV immunodeficiency virus
  • tissue sample used depends on a variety of factors, including mode of administration. It is known, for example, that drugs administered by injection accumulate in the kidneys.
  • invention to provide one or more kits for practicing the above method.
  • a method for detecting one or more drugs in the stratum corneum layer of living or post-mortem mammalian skin which entails: a) removing, a portion of the stratum corneum skin layer of living or post-mortem mammalian skin, and b) detecting the presence of said one or more drugs in or from said stratum corneum skin layer.
  • Figure 1 illustrates the detection in an autopsy specimen of cocaine metabolite and phencyclidine (PCP) using a stratum corneum sample. The internal standard is also shown.
  • Figure 2 illustrates the detection in an autopsy specimen of cocaine, cocaine metabolite and phencyclidine (PCP) using a stratum corneum sample.
  • PCP phencyclidine
  • the skin is composed of two distinct structures the dermis, a connective tissue layer covered by the epidermis, and the epidermis which is an epithelial layer. Each of the layers confers special properties on the skin.
  • The. dermis provides mechanical strength to the skin by virtue of the presence of collagen and elastic fibers and provides a reservoir of defense and regenerative capabilities.
  • the primary role of the epidermis is to function as the primary barrier to mechanical damage, microbial invasion and desiccation.
  • the epidermis is non-vascular and consists of
  • the epidermis forms a defensive covering on the surface of the true skin, and limits the evaporation of water vapor from its free
  • stratum corneum The more superficial layer of epidermal cells, forming a horny layer, is called the stratum corneum.
  • the epidermis consists of several distinct layers of epithelial cells agglutinated together and having a laminated arrangement. These several layers may be described as composed of four different strata from within outward: (1) the Stratum Malpighi, (2) the Stratum granulosum, (3) the Stratum lucidum and (4) the Stratum corneum. Due to the development of fresh layers
  • the cells contain no discernable nucleus. See Gray's Anatomy, (15th Edition, 1977).
  • the stratum corneum layer of the skin can serve as a useful sample source for the detection of drugs in either living or post-mortem mammalian subjects. That is, the stratum corneum, by itself, suffices as a drug reservoir and it is, therefore, no longer necessary to utilize full thickness skin samples in drug testing.
  • the method of the present invention may be practiced with either living or post-mortem mammalian subjects.
  • the present method may be easily applied in the toxicological analyses of human bodies which are in an advanced stage of decomposition.
  • the present invention may be used in the routine drug screening of living subjects for a variety of purposes such as workplace drug testing or the monitoring of
  • stratum corneum skin specimens may be obtained from anywhere on the hairless regions of the body.
  • a portion of stratum corneum may be scrapped off the skin surface, after the removal of hair if necessary, with a rough-edged object or one or more pieces of adhesive material of any variety may be applied to the selected area of the stratum corneum in order to strip a sample from the stratum corneum.
  • the scrapping or stripping is conducted anywhere from 1 to 10 times with from 1 to 10 different pieces of adhesive material for stripping. More commonly, the scrapping or stripping of the stratum corneum occurs about four times with four different sheets.
  • the adhesive material containing the stratum corneum is cut into strips and then a suitable organic solvent, and a suitable buffer and an internal standard is added thereto. Alternatively, the scrapped sample may be added to this mixture. Then, the mixture is agitated for anywhere from 30 seconds to 5 minutes. Thereafter, the drug to be analyzed is extracted from the mixture, wherein after an alkaline substance is added to the extracted drug in order to form the free base-of the drug.
  • any organic solvent can be used which is capable of dissolving the drug or drugs to be detected.
  • solvents such as diethyl ether, methanol, acetone or chloroform are used.
  • any solvent which is capable dissolving the drug of interest may be used.
  • any alkaline substance may be used in an aqueous solution provided that it is capable of releasing the free drug base.
  • examples of such an alkaline substance is sodium and potassium carbonates and bicarbonates.
  • the drug is extracted from the alkalized-mixture using a suitable organic solvent.
  • any buffer having a pH of about 7.5 to 9.0 may be used.
  • the drug is analyzed by a number of different analytical techniques, one of-which is gas chromatography/mass spectrometry.
  • analytical techniques one of-which is gas chromatography/mass spectrometry.
  • Enzyme-based assays are of particular interest in the present invention.
  • antibodies such as those disclosed in U.S. Patents 4,151,268 (barbituric acid and derivatives thereof), and 4,197,237 and 4,123,431 (cocaine and
  • the stratum corneum sample may be worked up as described previously and the extracted drug containing solvent mixture may be applied to the antibody-containing surface and then detected using the techniques described in any of the incorporated U.S. Patents 4,151,268, 4,123,431 and 4,197,237.
  • opiate receptors are used, the same can be obtained from synaptic membranes of mammalian brain tissue in accordance with known techniques.
  • the opiate receptors may be adhered to a solid support, such as a glass plate or glass beads or the wall of a container which is compatible with a scintillation counter.
  • the drug or drugs present may then be detected using the technique described in U.S. Patent 4,257,773.
  • the presence of one or more drugs may be detected using any of the above-described detection means without extracting the same from the stratum corneum samples.
  • the detecting antibodies either monoclonal or polyclonal, may be adhered to the surface of the adhesive material used to extract stratum corneum samples in any manner such that stratum corneum samples may be adhered to the surface antibodies.
  • opiate receptors or enzyme-containing materials may be adhered to the surface of the adhesive material.
  • any substance may be used which effectively binds the same to the adhesive.
  • fluorimetric and colorimetric techniques may be used. Any of the above procedures are extremely expeditious inasmuch as many samples can be routinely analyzed in only several hours.
  • drugs which can be detected using the present method are as diverse as
  • the present method is effective for drugs having a wide disparity of
  • Anterior torso skin specimens were obtained from the hairless regions (by showing the area if necessary), along a "Y" shaped incision.
  • the skin specimens were trimmed of grossly visible adipose tissue, with surgical scissors. After removal of subcutaneous fat the specimens were kept at -10 to -20°C. Storage time prior to extraction varied from 4 hours to 4 months. Following freezing the specimens were cut into 0.5 cm cubes.
  • the dissected samples were blended in 20 ml of distilled water for 2 minutes. The blended mixture was placed in a 1000 ml flask. 150 ml of ether, 1.0 ml of AMP (alkaline mono phosphate) such as sodium or potassium mono phosphate buffer and 1.0 ml of internal standard were added.
  • AMP alkaline mono phosphate
  • the sample and reagents were agitated for 3 minutes. After shaking, the ether layer was poured into a 150 ml erlenmeyer flask. Add 5.0 ml of buffer (pH 8.5) and shake for 3 minutes, decant and
  • AMP buffer then vortexed and the pH was tested.
  • the pH should be about 9.0.
  • the aqueous extraction was centrifuged at 8,000 to 10,000 GS for 1-minute. 100 ul aliquot of the lower chloroform layer was removed for GC/MS analysis of cocaine and phencyclidine.
  • GC/MS parent drug identification was based on the detection of all ions, i.e., cocaine: 303, 82 and 182; and phencyclidine: 243,
  • spectrometry cocaine analysis only detected the parent drug.
  • the skin and blood results were statistically analyzed using Baye's rule. See Tables 2 and 3.
  • Four of the 44 cases were not included in the predictive value analysis for lack of adequate history.
  • Cases 463, 566, 592 and 832 are John Does.
  • the 16 morphine cases were all from decedents with a positive history of drug use. See Table 4.
  • Blood from case 463 was not available due to decomposition. All morphine concentrations were obtained by radioimmunoassay.
  • Statistical analyses for morphine was also carried out using the predictive value tables. See Tables 5 and 6.
  • Diphenhydramine was found in a diphenhydramine overdose showing moderate decomposition, i.e., epidermal-dermal separation.
  • Lidocaine was found in decedents who received bolus lidocane injections during resuscitation and quinine in heroin abusers.
  • Table 1 evidences the result (in mg/ml) of cocaine detected in blood and skin samples of the forty-four subjects tested as described above.
  • Tables 2 and 3 illustrate the high sensitivity and specificity of the present method for the detection of cocaine. Notably, the use of the stratum corneum, in accordance with the present invention, affords a much greater sensitivity than the use of blood.
  • Table 4 evidences the result (in ng/ml) of morphine detected in blood and skin samples of the forty-four subjects tested as described above.
  • Tables 5 and 6 illustrate the high sensitivity and specificity of the present method for the detection of morphine.
  • the use of the stratum corneum, in accordance with the present invention affords a much greater sensitivity than the use of blood.
  • Table 7 lists the molecular weight and octanol/water partition coefficients of a few of the drugs which may be detected by the present method.
  • the present method is operable in detecting drugs having a variety of molecular weights and octanol/water partition coefficients.
  • the stratum corneum can be used for both living and post-mortem analysis.
  • stratum corneum samples may be used to detect the presence of bacteria and viruses, in particular the HIV virus.
  • the present invention may be used to detect any endogenous substance such as glucose, sodium and potassium ions, chloride ions, proteins.
  • proteins encompass many diverse substances such as enzymes and hormones.
  • the present invention may also be used to detect steroids.
  • kits for practicing the present invention are provided.
  • any standard kit for the detection of one or more drugs may be used in accordance with the present invention provided that either a means for
  • scrapping stratum corneum samples or adhesively removing stratum corneum samples are provided in the kit.
  • the kit of the present invention may include adhesive material for removing stratum corneum samples to which is adhered antibodies (monoclonal or polyclonal), opiate receptors or enzymes or enzyme-- containing materials.
  • one of these detecting means may be immobilized on the surface of the adhesive material.
  • a protective, substantially non-adhesive coating or cover may be placed over the immobilized detecting means until it is ready for use.
  • kit of the present invention also contains various fluorimetric or
  • the present invention may be used advantageously with humans, it may also be used in numerous veterinary applications. For example, it may be used in conjunction with dogs and horses, such as in racing.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

Selon un procédé de détection d'un ou plusieurs médicaments dans la peau de mammifères vivants ou morts, (a) on enlève une partie de la couche du stratum corneum d'un mammifère vivant ou mort, et (b) on détecte la présence d'un ou plusieurs médicaments dans ladite couche du stratum.
PCT/US1990/006657 1989-11-20 1990-11-20 Procede et kit de detection de medicaments dans la peau de mammifere vivants et morts WO1991007661A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US438,084 1982-11-01
US43808489A 1989-11-20 1989-11-20

Publications (1)

Publication Number Publication Date
WO1991007661A1 true WO1991007661A1 (fr) 1991-05-30

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0836407A4 (fr) * 1995-06-07 1999-09-22 Sudor Partners Timbre transdermique sans un materiau absorbant separe
US8585971B2 (en) 2005-04-05 2013-11-19 The General Hospital Corporation Devices and method for enrichment and alteration of cells and other particles
US8895298B2 (en) 2002-09-27 2014-11-25 The General Hospital Corporation Microfluidic device for cell separation and uses thereof
US8921102B2 (en) 2005-07-29 2014-12-30 Gpb Scientific, Llc Devices and methods for enrichment and alteration of circulating tumor cells and other particles

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3965888A (en) * 1975-02-12 1976-06-29 Brenner And Bender, Inc. Specimen collector and holder
US4495281A (en) * 1982-10-21 1985-01-22 Miles Laboratories, Inc. Tricyclic antidepressant drug immunogens, antibodies, labeled conjugates, and related derivatives
US4771005A (en) * 1983-06-27 1988-09-13 Erez Forensic Technology Ltd. Reagents, test kits and methods for the detection of cannabinoids

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3965888A (en) * 1975-02-12 1976-06-29 Brenner And Bender, Inc. Specimen collector and holder
US4495281A (en) * 1982-10-21 1985-01-22 Miles Laboratories, Inc. Tricyclic antidepressant drug immunogens, antibodies, labeled conjugates, and related derivatives
US4771005A (en) * 1983-06-27 1988-09-13 Erez Forensic Technology Ltd. Reagents, test kits and methods for the detection of cannabinoids

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOLOGICAL ABSTRACTS, Volume 88, No. 6, 69055, issued 15 September 1989, OFFIDANI et al., "Drugs in Hair: A New Extraction Procedure"; & SCI. INT., 41(1/2), 35-40, see page 1243. *
CHEMICAL ABSTRACTS, Volume 108, No. 21, 181598t, issued 23 May 1988, NANJI et al., "Detection of Drugs in Patients with Overdose: Comparison Between Skin Surface Air Sampling and thin Layer Chromatography"; & INT. J. CLI. PHARMACD., Ther. Toxical. 261(1), 1-3. see page 218. *
JOURNAL OF CLINICAL CHEMISTRY AND CLINICAL BIOCHEMISTRY, Volume 27, No. 11, issued October 1989. H. SACHS, "Results of Comparative Determination of Morphine in Human Hair using RIA and GC/MS", pages 873-877. *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0836407A4 (fr) * 1995-06-07 1999-09-22 Sudor Partners Timbre transdermique sans un materiau absorbant separe
US8895298B2 (en) 2002-09-27 2014-11-25 The General Hospital Corporation Microfluidic device for cell separation and uses thereof
US8986966B2 (en) 2002-09-27 2015-03-24 The General Hospital Corporation Microfluidic device for cell separation and uses thereof
US10081014B2 (en) 2002-09-27 2018-09-25 The General Hospital Corporation Microfluidic device for cell separation and uses thereof
US11052392B2 (en) 2002-09-27 2021-07-06 The General Hospital Corporation Microfluidic device for cell separation and uses thereof
US8585971B2 (en) 2005-04-05 2013-11-19 The General Hospital Corporation Devices and method for enrichment and alteration of cells and other particles
US9174222B2 (en) 2005-04-05 2015-11-03 The General Hospital Corporation Devices and method for enrichment and alteration of cells and other particles
US9956562B2 (en) 2005-04-05 2018-05-01 The General Hospital Corporation Devices and method for enrichment and alteration of cells and other particles
US10786817B2 (en) 2005-04-05 2020-09-29 The General Hospital Corporation Devices and method for enrichment and alteration of cells and other particles
US12409457B2 (en) 2005-04-05 2025-09-09 The General Hospital Corporation Devices and method for enrichment and alteration of cells and other particles
US8921102B2 (en) 2005-07-29 2014-12-30 Gpb Scientific, Llc Devices and methods for enrichment and alteration of circulating tumor cells and other particles

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