WO2020094846A1

WO2020094846A1 - Skin print sweat analysis for drug detection

Info

Publication number: WO2020094846A1
Application number: PCT/EP2019/080693
Authority: WO
Inventors: Melanie Jane BAILEY; Mahado ISMAIL; Jeremy Nigel Burgess WALKER
Original assignee: University Of Surrey
Priority date: 2018-11-09
Filing date: 2019-11-08
Publication date: 2020-05-14
Also published as: GB201818285D0; GB2578871A

Abstract

A method of determining whether a subject has taken a drug, the method comprising: analysing a first sample of sweat obtained from the subject in the form of a skin- print to determine the presence therein of the drug and/or a metabolite thereof; and analysing a second sample of sweat obtained from the subject in the form of a skin- print to determine the presence of at least one amino acid or derivative thereof; wherein the first sample of sweat and the second sample of sweat are the same sample of sweat; or wherein the first sample of sweat and the second sample of sweat are different samples obtained from the same place on the subject.

Description

SKIN PRINT SWEAT ANALYSIS FOR DRUG DETECTION

TECHNICAL FIELD

The present invention relates to a method of determining whether a subject has taken a drug through analysis of sweat obtained from the subject, in the form of a skin-print. The invention may improve quantitation of analytes, improve precision and accuracy of analysis from sample to sample taken from the same donor or between donors, or simply provide greater confidence in the integrity of any sample tested.

BACKGROUND TO THE INVENTION

An impression left by the friction ridges of human skin, such as the skin of a human finger, contains information regarding the identity of the human. It is widely known that the appearance of the impression of the human finger, known as a fingerprint, is unique to each human and may be used to confirm the identity of the human. The appearance of the impression of the skin of other human body parts may also be unique to each human and so may also be used to confirm the identity of the human. Impressions of human skin, including but not limited to the skin of the human finger, may be called skin-prints.

In addition to the appearance of the impression left by human skin, the impression may contain chemical species which themselves may be detected in order to obtain further information.

For example, when a human intakes a substance (e.g. by ingestion, inhalation or injection) the substance may be metabolised by the human body giving rise to secondary substances known as metabolites. The presence of a particular metabolite can be indicative of a specific intake substance. The intake substance and/or metabolites may be present in sweat and, as such, may be left behind in a skin-print, e.g. a fingerprint. Detection of such metabolites in a skin-print can be used as a non-invasive method of testing for recent lifestyle activity such as (but not limited to) drug use, or compliance with a pharmaceutical or therapeutic treatment regime.

Importantly, the taking of a skin-print is much simpler than obtaining other body fluids such as blood, saliva and urine, and is more feasible in a wider range of situations. Not only this but since the appearance of the skin-print itself provides confirmation of the identity of the person providing the skin-print, there can be greater certainty that the substance or substances in the skin-print are associated with the individual. This is because substitution of a skin-print, particularly a fingerprint, is immediately identifiable from appearance whereas substitution of, for example, urine, is not immediately identifiable from

appearance. As such, testing for one or more substances in a skin-print provides a direct link between the one or more substances and the identity of the human providing the skin- print.

Techniques for chemical analysis of skin-prints, including the use of mass spectrometry, for example paper spray mass spectrometry, are known. Lateral flow skin-print analysis is also known, for example as described in WO 2016/012812, published 28 January 2016.

Techniques that facilitate chemical analysis of skin-prints have a wide variety of

applications. One example application may be to check for complicity with a particular dosage regime. For example, it may be expected that a quantity of a particular analyte present in a skin-print may be expected to be within predetermined bounds at a specific interval following the patient taking the drug. In the event that the quantity of analyte measured by the chemical analysis is outside the predetermined bounds at the requisite testing time, this may indicate that the patient has deviated from the dosage regime (e.g. the quantity of drug and/or the timing of the dose). For example, drug resistant

tuberculosis is a continuing threat to eradication of the disease. Drug resistance can emerge when a patient is either infected with M. tuberculosis resistant to a particular antibiotic or M. tuberculosis acquires resistance during treatment. Acquired resistance can develop when patients stop taking their medication (non-adherence to treatment) or due to suboptimal in vivo drug concentration in treatment regimens. Treatment for multi-drug resistance M. tuberculosis infection takes longer (sometimes over 20 months) and requires more expensive and toxic drugs, which can cause severe side effects and make adherence to treatment more difficult for patients. Success rates can be as low as 33%.

Various strategies have been employed to increase adherence to treatment and improve treatment success rates (30). Current methods to monitor adherence include regular engagement with medical and nursing teams, including provision of direct observation therapy (DOTS), tablet count and assessment of clinical improvement. A urine dipstick test (based on a colorimetric test) can also be used on the spot if there are concerns or doubts regarding treatment efficacy. However, each of these approaches have their limitations: tablet count is ineffective because it is easily altered by the patient, engagement with the patient through direct observation therapy (DOTS) is labor-intensive and expensive to administer and a urine dipstick test requires clinic access or supervised sample collection.

In addition to adherence monitoring, it is of interest to clinicians to be able to carry out therapeutic drug monitoring, to ensure that the drugs used to treat tuberculosis have been delivered at an appropriate therapeutic dose. The risk of multi drug resistant tuberculosis increases with sub-optimal drug concentrations, which may arise due to non-adherence as well as other factors (malabsorption for example). This can be done by testing of blood samples using liquid chromatography mass spectrometry (LC-MS), and dried blood spots have also been proposed for this. For this approach, it is necessary to extract blood samples, which requires trained staff, the discomfort of a skin-prick and there is complexity around the health and safety of transporting blood samples. The approach is also prohibitively expensive for the majority of countries with the highest burden of infection.

Another application may be to confirm whether or not there are analytes present in a skin- print that would indicate that the user has taken a dose of a specific drug, such as an illicit and/or addictive recreational drug that might, for example, impair their ability to perform a particular function (e.g. a narcotic, such as heroin).

While it is known that drugs and/or metabolites thereof can be detected in sweat samples, current methods known in the art may return too many false positives or false negatives for the results to be reliably trusted. Furthermore, there is currently no method to measure or account for the volume or quality of sweat collected in the form of a skin-print. This means that currently, skin-prints can only be used qualitatively as a“yes/no” test, which restricts their use to adherence monitoring, rather than the broader area of therapeutic drug monitoring.

It is therefore one object of the present invention to provide a more reliable/trustworthy method of analysing sweat samples for the presence of drugs and/or their metabolites.

It is a further object of the invention to provide a more quantitative method of analysing sweat samples for the presence of drugs and/or their metabolites. It is a further object of the present invention to provide a method of authenticating whether or not a sample is a sweat sample.

It is a further object of the present invention to overcome at least some of the

disadvantages of the prior art or to provide a commercially useful alternative thereto.

SUMMARY OF THE INVENTION

In a first aspect the invention provides a method of determining whether a subject has taken a drug, the method comprising:

analysing a first sample of sweat obtained from the subject in the form of a skin- print to determine the presence therein of the drug and/or a metabolite thereof; and analysing a second sample of sweat obtained from the subject in the form of a skin- print to determine the presence of at least one amino acid or derivative thereof; wherein the first sample of sweat and the second sample of sweat are the same sample of sweat; or

wherein the first sample of sweat and the second sample of sweat are different samples obtained from the same place on the subject.

The present inventors have surprisingly found that amino acids or derivatives thereof may be detected within sweat samples and used as a reliability indicator for drug detection carried out on the same sweat sample or on a sweat sample taken from the same place on the subject.

Without wishing to be bound by theory, detecting the presence of amino acids or derivatives thereof in a sweat sample, or another sweat sample obtained from the same place on the subject, may indicate that the sweat sample is of sufficient volume or quality to be reliably analysed for the presence of a drug. Conversely, if no amino acids or derivatives thereof are detected in a sweat sample, or another sweat sample obtained from the same place on the subject, this may mean that the sweat sample is not of sufficient volume or quality to be reliably analysed for the presence of a drug. Using the method of the present invention therefore reduces the number of false positives and/or false negatives. In a further aspect of the invention there is provided a method of authenticating whether or not a sample is a sweat sample, the method comprising analysing using mass

spectrometry the sample to determine the presence or absence therein of at least one amino acid or derivative thereof.

Without wishing to be bound by theory, the presence of at least one amino acid or derivative thereof may be used to indicate the authenticity of the sample as a true sweat sample and not a substitute, e.g. an artificial substitute.

Preferred embodiments of the methods according to the invention appear throughout the specification and in particular in the examples.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition.

As used in the specification and the appended claims, unless specified to the contrary, the following terms have the meaning indicated:

As used herein, the term“skin-print" refers to sweat deposited as an impression of a skin’s ridge pattern. Skin-prints include, for example, fingerprints and toe-prints. The term ‘fingerprint" refers to sweat deposited as an impression of a finger’s ridge pattern or a thumb’s ridge pattern. The term“toe-print” refers to sweat deposited as an impression of a toe’s ridge pattern.

The term“taken”, in the context of“a subject has taken a drug” includes a subject having swallowed, absorbed, injected and/or inhaled a drug recreationally or in accordance with the requirements of the subject’s course of medication.

Isoniazid refers to the compound having the formula:

Acetylisoniazid refers to the compound having the formula:

Pyrazinamide refers to the compound having the formula:

Rifampicin refers to the compound having the formula:

Ethambutol refers to the compound having the formula:

The invention provides a method of determining whether a subject has taken a drug, the method comprising:

It is understood that the sample of first sample of sweat and/or the second sample of sweat is or‘has been’ obtained in the form of a skin-print, that is, the method is directed to the analysis of a first sample of sweat and/or a second sample of sweat that have previously been provided. Put explicitly, the method is an ex-vivo method. For example, the first sample of sweat and/or the second sample of sweat may have been obtained by the subject contacting a substrate with an area of skin. The substrate/sample may then be provided or transported for analysis and subjected to the method as described herein.

Preferably the subject is a human.

Preferably the first sample of sweat and/or the second sample of sweat obtained from the subject are in the form of a fingerprint or a toe-print. More preferably the first sample of sweat and/or the second sample of sweat obtained from the subject are in the form of a fingerprint. A fingerprint, unlike other biological matrices, offers a unique opportunity for subjects to provide samples non-invasively, saving clinical resources and improving patient experience, as they are easily collected and transported. Furthermore, the identity of the donor is encapsulated in the sample, making it difficult to falsify and assuring chain of custody.

Preferably the sample of sweat in the form of a skin-print is obtained without requiring the skin to be washed and cleaned beforehand.

In an alternative embodiment, the first sample of sweat and/or the second sample of sweat in the form of a skin-print is obtained after the skin has been washed and cleaned, for example by using soap and water, and before the washed and cleaned skin contacts any other surface. Preferably the first sample of sweat and/or the second sample of sweat is obtained at least 15 minutes after the skin has been washed and cleaned and left uncovered, more preferably at least 30 minutes after the skin has been washed and cleaned and left uncovered.

Preferably the first sample of sweat and/or the second sample of sweat in the form of a skin-print are obtained on a substrate. Suitable substrates are known in the art. Preferably the substrate comprises or is chromatography paper or plastic, for example chemically inert plastic. More preferably the substrate comprises or is chromatography paper.

Alternatively, preferably, the substrate comprises or is chemically inert plastic.

Preferably the first sample of sweat and/or the second sample of sweat are obtained from 1 to 48 hours after the subject has (or is meant to have) taken the drug. More preferably, the first sample of sweat and/or the second sample of sweat are obtained from 1 to 36, or 1 to 24, or 1 to 18, or 1 to 12, or 1 to 8, or 1 to 6, or 1 to 5, or 1 to 4 hours after the subject has (or is meant to have) taken the drug. Most preferably the first sample of sweat and/or the second sample of sweat are obtained from 1 to 3, or 1.5 to 2.5, or around 2 hours after the subject has (or is meant to have) taken the drug.

Preferably the at least one amino acid or derivative thereof is taurine, lysine, ornithine and/or pyroglutamic acid. More preferably the at least one amino acid or derivative thereof is taurine, lysine and/or ornithine. More preferably the at least one amino acid or derivative thereof is taurine and/or lysine. Most preferably the at least one amino acid or derivative thereof is taurine.

Preferably the first sample of sweat and the second sample of sweat are the same sample of sweat, i.e. the method preferably comprises analysing a sample of sweat obtained from the subject in the form of a skin-print to determine the presence therein of the drug and/or a metabolite thereof; and analysing the sample of sweat to determine the presence of at least one amino acid or derivative thereof. Analysis of the same sample of sweat for the presence of a drug and/or metabolites thereof and an amino acid and/or derivatives thereof is advantageous because the presence of an amino acid and/or derivatives thereof directly indicates that drug analysis on the same sample is more likely to be reliable and

trustworthy. For example, if a fingerprint sweat sample is found to contain an amino acid and/or derivative thereof, this may indicate that drug analysis on the (same) fingerprint sweat sample is reliable. When the first sample of sweat and the second sample of sweat are the same sample of sweat, the sample may be analysed to determine the presence therein of the drug and/or a metabolite thereof at the same time as analysing the sample to determine the presence of at least one amino acid or derivative thereof. Alternatively, when the first sample of sweat and the second sample of sweat are the same sample of sweat, the sample may be analysed to determine the presence therein of the drug and/or a metabolite thereof before or after the sample is analysed to determine the presence of at least one amino acid or derivative thereof.

In an alternative embodiment, the first sample of sweat and the second sample of sweat are different samples obtained from the same place on the subject. As used herein, the term“same place on the subject" denotes substantially the same area of the subject’s skin, and includes, but is not limited to, the same finger of the subject. Preferably the first sample of sweat and the second sample of sweat are obtained from the same finger. In particular, analysing a second (different) sweat sample obtained from the same place on the subject (as the first sample) may give an indication of the typical constituents / volume / quality of the subject’s skin-print sweat in the specified place. This information may be compared to information obtained during analysis of the first sample. For example, if a second fingerprint sweat sample is found to contain an amino acid and/or derivative thereof, this may indicate that drug analysis on a first (different) fingerprint sweat sample obtained from the same finger is reliable.

The first sample of sweat and the second sample of sweat may be analysed concurrently. Alternatively, the first sample of sweat may be analysed before the second sample of sweat. Alternatively, the second sample of sweat may be analysed before the first sample of sweat.

Preferably, determining the presence of the drug and/or a metabolite thereof in the first sample comprises determining the amount of the drug and/or a metabolite thereof. If present, the amount may be provided as, for example, a volume, a mass, a relative signal intensity or a peak area (for example in a mass spectrum).

Preferably, determining the presence of one or more amino acid or derivative thereof in the second sample comprises determining the amount of the at least one amino acid or derivative thereof. If present, the amount may be provided as, for example, a volume, a mass, a relative signal intensity or a peak area (for example in a mass spectrum).

Preferably the method further comprises normalising the amount of the drug and/or a metabolite thereof to the amount of the at least one amino acid or derivative thereof. The concept of normalisation is known in the art. For example, the amount of the drug and/or a metabolite thereof may be normalised to the amount of the at least one amino acid by simply dividing one value by the other. Advantageously, normalising the amount of the drug and/or a metabolite thereof to the amount of the at least one amino acid or derivative thereof provides more reliable and/or more quantitative results in the method of determining whether or not a subject has taken a drug.

Preferably the method further comprises confirming the identity of the subject by checking the skin-print obtained from the subject. Confirming the identity of the subject may involve photographic imaging of the skin-print in order to facilitate a photographic or optical comparison with a database.

Drugs that may be detected using the method of the present invention include, but are not limited to:

A. ANABOLIC AGENTS. These include, but are not limited to:

1. Anabolic Androgenic Steroids (AAS)

a. Exogenous^* AAS, including:

1-androstendiol (5oandrost-1-ene-33,173-diol ); 1-androstendione (5oandrost-1- ene-3,17-dione); bolandiol (19-norandrostenediol); bolasterone; boldenone;

boldione (androsta-1 ,4-diene-3,17-dione); calusterone; clostebol; danazol (17a- ethynyl-173-hydroxyandrost-4-eno[2,3-d]isoxazole);

dehydrochlormethyltestosterone (4-chloro-173-hydroxy- 17a-methylandrosta-1 ,4- dien-3-one); desoxymethyltestosterone (17a-methyl-5a-androst-2-en-17b-oI); drostanolone; ethylestrenol (19-nor-17a-pregn-4-en-17-ol); fluoxymesterone;

formebolone; furazabol (173-hydroxy- 17a-methyl-5a-androstano[2,3-c]-furazan); gestrinone; 4-hydroxytestosterone (4,173-dihydroxyandrost-4-en-3-one);

mestanolone; mesterolone; metenolone; methandienone (173-hydroxy- 17 a- methylandrosta-1 ,4-dien-3-one); methandriol; methasterone (2a, 17a-dimethyl-5a- androstane-3-one-17b-oI); methyldienolone (173-hydroxy-17a-methylestra-4,9-dien- 3-one); methyl-1 -testosterone (173-hydroxy-17omethyl-5oandrost-1-en-3-one); ethylnortestosterone (173-hydroxy-17a-methylestr-4-en-3-one); ethyltrienolone (173-hydroxy-17a-methylestra-4,9,1 1-trien-3-one); methyltestosterone; mibolerone; nandrolone; 19-norandrostenedione (estr-4-ene-3,17-dione); norboletone;

norclostebol; norethandrolone; oxabolone; oxandrolone; oxymesterone;

oxymetholone; prostanozol ([3,2-c]pyrazole-5a-etioallocholane-17b- tetrahydropyranol); quinbolone; stanozolol; stenbolone; 1 -testosterone (17b- hydroxy-5oandrost-1-en-3-one); tetrahydrogestrinone (18a-homo-pregna-4,9,1 1- ίpbh-17b-oI-3-ohb); trenbolone, and other substances with a similar chemical structure or similar biological effect(s). b. Endogenous^** AAS:

androstenediol (androst-5-ene-3b,17b-diol); androstenedione (androst-4-ene-3,17- dione); dihydrotestosterone (17b-hydroxy-5a-androstan-3-one) ; prasterone

(dehydroepiandrosterone, DHEA); testosterone and the following metabolites and isomers:

5oandrostane-3a,17odiol; 5a-androstane-3a,17b-diol; 5a-androstane^,17a-diol; 5a-androstane-3b,17b-diol; androst-4-ene-3a,17a-diol; androst-4-ene-3a,17b-diol; androst-4-ene-3b, 17odiol; androst-5-ene-3a, 17a-diol; androst-5-ene-3a, 17b^ίoI; androst-5-ene^,17a-diol; 4-androstenediol (androst-4-ene-3b,17b-diol); 5- androstenedione (androst-5-ene-3,17-dione); epi-dihydrotestosterone; 3ohydroxy- 5a-androstan-17-one; 3b-hydroxy-5a-androstan-17-one; 19-norandrosterone; 19- noretiocholanolone. “exogenous” refers to a substance which is not ordinarily capable of being produced by the body naturally.

“endogenous” refers to a substance which is capable of being produced by the body naturally.

2. Other Anabolic Agents. These include, but are not limited to:

Clenbuterol, tibolone, zeranol, zilpaterol.

B. Hormones. These include, but are not limited to: 1. Erythropoietin (EPO);

2. Growth Hormone (hGH), Insulin-like Growth Factors (e.g. IGF-1 ),

Mechano Growth Factors (MGFs);

3. Gonadotrophins (LH, hCG), prohibited in males only;

4. Insulin;

5. Corticotrophins.

C. BETA-2 AGONISTS, including their D- and L-isomers.

D. AGENTS WITH ANTI-ESTROGENIC ACTIVITY. These include, but are not limited to:

1. Aromatase inhibitors including, but not limited to, anastrozole, letrozole, aminoglutethimide, exemestane, formestane, testolactone.

2. Selective Estrogen Receptor Modulators (SERMs) including, but not limited to, raloxifene, tamoxifen, toremifene.

3. Other anti-estrogenic substances including, but not limited to, clomiphene, cyclofenil, fulvestrant.

E. DIURETICS AND OTHER MASKING AGENTS. These include, but are not limited to:

Diuretics^*, epitestosterone, probenecid, alpha-reductase inhibitors (e.g. finasteride, dutasteride), plasma expanders (e.g. albumin, dextran, hydroxyethyl starch) and other substances with similar biological effect(s).

Diuretics include:

acetazolamide, amiloride, bumetanide, canrenone, chlorthalidone, etacrynic acid, furosemide, indapamide, metolazone, spironolactone, thiazides (e.g.

bendroflumethiazide, chlorothiazide, hydrochlorothiazide), triamterene, and other substances with a similar chemical structure or similar biological effect(s).

F. AGENTS FOR THE ENHANCEMENT OF OXYGEN TRANSFER. These include, but are not limited to:

1. Autologous, homologous or heterologous blood or red blood cell products of any origin.

2. perfluorochemicals, efaproxiral (RSR13) and modified haemoglobin products (e.g. haemoglobin-based blood substitutes, microencapsulated haemoglobin products). G. STIMULANTS (including both their (D- & L-) optical isomers where relevant). These include, but are not limited to:

Adrafinil, adrenaline^**, amfepramone, amiphenazole, amphetamine, amphetaminil, benzphetamine, benzylpiperazine, bromantan, cathine^***, clobenzorex, cocaine, cropropamide, crotetamide, cyclazodone, dimethylamphetamine, ephedrine^****, etamivan, etilamphetamine, etilefrine, famprofazone, fenbutrazate, fencamfamin, fencamine, fenetylline, fenfluramine, fenproporex, furfenorex,

heptaminol sometheptene, levmethamfetamine, meclofenoxate, mefenorex, mephentermine, mesocarb, methamphetamine (D-),methylenedioxyamphetamine, methylenedioxymethamphetamine, pmethylamphetamine, methylephedrine^****, methylphenidate, modafinil, nikethamide, norfenefrine, norfenfluramine, octopamine, ortetamine, oxilofrine, parahydroxyamphetamine, pemoline, pentetrazol, phendimetrazine, phenmetrazine, phenpromethamine, phentermine, 4- phenylpiracetam (carphedon), prolintane, propylhexedrine, selegiline, sibutramine, strychnine, tuaminoheptane and other substances with a similar chemical structure or similar biological effect(s).

H. NARCOTICS. These include, but are not limited to:

Buprenorphine, dextromoramide, diamorphine (heroin), fentanyl and its derivatives, hydromorphone, methadone, morphine, codeine, oxycodone, oxymorphone, pentazocine, pethidine.

I. CANNABINOIDS. These include, but are not limited to:

Hashish, marijuana.

J. GLUCOCORTI COSTEROIDS

K. ALCOHOL (ethanol).

L. BETA-BLOCKERS. These include, but are not limited to:

acebutolol, alprenolol, atenolol, betaxolol, bisoprolol, bunolol, carteolol, carvedilol, celiprolol, esmolol, labetalol, levobunolol, metipranolol, metoprolol, nadolol, oxprenolol, pindolol, propranolol, sotalol, timolol. M. AMPHETAMINES. These include, but are not limited to:

methamphetamine and MDMA (3,4-methylenedioxy-N-methylamphetamine); LSD (lysergic acid diethylamide); PCP (Phencyclidine), ketamine and derivatives;

N. ALKALOIDS AND THEIR DERIVATIVES. These include, but are not limited to: nicotine, cocaine, ephedrine, mescaline; opium alkaloids (opiods), including morphine and codeine, and semi-synthetic opoids such as diamorphine (heroin); tryptamine alkaloids such as dimethyltriptamine and alpha-methyltryptamine;

O. BENZODIAZEPINES. These include, but are not limited to:

Alprazolam, Diazepam, Lorazepam, Clonazepam, Temazepam, Oxazepam, Flunitrazepam, Triazolam, Chlordiazepoxide, Flurazepam, and Nitrazepam, and nonbenzodiazepines, including Imidazopyridines, Pyrazolopyrimidines,

Cyclopyrrones.

P. GHB (gamma-Hydroxybutyric acid) and derivatives

Q. TUBERCULOSIS (TB) MEDICATION, including, but not limited to, isoniazid, pyrazinamide, rifampicin and/or ethambutol.

R. ANTIPSYCHOTICS, including, but not limited to, olanzapine, respiridone, quetiapine or clozapine

Preferably the drug comprises isoniazid, pyrazinamide, rifampicin, ethambutol, morphine, heroin, olanzapine, respiridone, quetiapine and/or clozapine. More preferably the drug comprises isoniazid, pyrazinamide, rifampicin, ethambutol, morphine and/or heroin. More preferably the drug comprises isoniazid, morphine and/or heroin. More preferably still the drug comprises isoniazid or heroin and the metabolite comprises acetylisoniazid, morphine or 6-acetylmorphine

In one embodiment, preferably the drug comprises Tuberculosis medication. Preferably the Tuberculosis medication comprises isoniazid, pyrazinamide, rifampicin and/or ethambutol. More preferably the Tuberculosis medication comprises isoniazid and/or pyrazinamide. Most preferably the Tuberculosis medication comprises isoniazid. Preferably the Tuberculosis medication comprises isoniazid and the metabolite comprises acetylisoniazid.

In a further embodiment, preferably, the drug comprises morphine and/or heroin. More preferably the drug comprises heroin and the metabolite thereof comprises 6- acetylmorphine and/or morphine.

In a further embodiment, preferably, the drug comprises an antipsychotic drug, preferably wherein the antipsychotic drug comprises olanzapine, respiridone, quetiapine or clozapine.

Preferably the analysis of the first sample of sweat and/or the second sample of sweat is carried out using mass spectrometry. Preferably the mass spectrometry is liquid chromatography mass spectrometry (LC-MS) or paper spray mass spectrometry.

More preferably the mass spectrometry is liquid chromatography mass spectrometry. More preferably still the mass spectrometry is liquid chromatography high resolution mass spectrometry. Analysis using LC-MS is known in the art. LC-MS is known to be quantitative, highly sensitive and selective. LC-MS involves extracting and preparing the first sample of sweat and/or the second sample of sweat prior to analysis by mass spectrometry.

Alternatively, preferably, the mass spectrometry is paper spray mass spectrometry. Paper spray mass spectrometry is described in“Rapid, Secure Drug Testing Using Fingerprint Development and Paper Spray Mass Spectrometry” Catia Costa, Roger Webb, Vladimir Palitsin, Mahado Ismail, Marcel de Puit, Samuel Atkinson, Melanie J. Bailey, DOI:

10.1373/clinchem.2017.275578, published September 2017. Advantageously, paper spray mass spectrometry does not require extraction and sample preparation.

Preferably the method further comprises determining the volume and/or the mass of the first sample of sweat and/or the second sample of sweat. Determining the volume and/or the mass of the first sample of sweat and/or the second sample of sweat is advantageous because it provides a further confirmation that the sample is of sufficient volume/mass to be reliably analysed for the presence of a drug and/or metabolite thereof. More preferably, the method further comprises determining the volume of the first sample of sweat and/or the second sample of sweat.

Preferably the volume of the first sample of sweat and/or the second sample of sweat is determined using an electromagnetic detector to obtain an optical image.

Preferably the first sample of sweat and/or the second sample of sweat has a volume of about 1 to about 100 nl, more preferably about 5 to about 75 nl, most preferably about 5 to about 50 nl.

In a specific arrangement, the optical image may be analysed to determine an image density of the skin-print. Skin-print quantity may be calculated as a function of image density of the optical image.

The method may further comprise transmitting electromagnetic radiation towards the skin- print, wherein the step of using an electromagnetic detector to obtain the image comprises detecting a reflection of the transmitted electromagnetic radiation.

Optical images may be processed to determine a region of interest equivalent to the region of the skin-print on a substrate. Examples of such processing include use of one of more of: a rolling ball background subtraction algorithm; a second order polynomial least squares fit; frequency based modelling; gradient threshold algorithms; standardised substrate subtraction; and pixel intensity threshold.

The invention further provides a method of authenticating whether or not a sample is a sweat sample, the method comprising analysing using mass spectrometry the sample to determine the presence or absence therein of at least one amino acid or derivative thereof.

Preferably the sample is obtained from a subject in the form of a skin-print, preferably in the form of a fingerprint.

Preferably the method comprises analysing using mass spectrometry the sample to determine the presence or absence therein of at least two amino acids or derivatives of one or more thereof. More preferably the method comprises analysing using mass

spectrometry the sample to determine the presence or absence therein of at least three amino acids or derivatives of one or more thereof. More preferably still the method comprises analysing using mass spectrometry the sample to determine the presence or absence therein of at least four amino acids or derivatives of one or more thereof.

Preferably the method comprises analysing using mass spectrometry the sample to determine the presence or absence therein of taurine, and, optionally, at least one further amino acid or derivative thereof. More preferably the method comprises analysing using mass spectrometry the sample to determine the presence or absence therein of taurine, and, optionally, lysine, ornithine and/or pyroglutamic acid. More preferably still the method comprises analysing using mass spectrometry the sample to determine the presence or absence therein of taurine, and at least one of lysine, ornithine and pyroglutamic acid.

Preferably the method further comprises determining the volume of the sample.

Preferably the sample has a volume of about 1 to about 100 nl, more preferably about 5 to about 75 nl, most preferably about 5 to about 50 nl.

Preferably the method is a method of authenticating whether or not a sample is a human sweat sample.

When introducing elements of the present disclosure or the preferred embodiments(s) thereof, the articles“a”,“an”,“the” and“said” are intended to mean that there are one or more of the elements. The terms“comprising”,“including” and“having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

The foregoing detailed description has been provided by way of explanation and illustration, and is not intended to limit the scope of the appended claims. Many variations in the presently preferred embodiments illustrated herein will be apparent to one of ordinary skill in the art, and remain within the scope of the appended claims and their equivalents.

These and other aspects of the invention will now be described with reference to the accompanying Figures, in which:

Figure 1 : is a diagram showing relative amounts of Acetylisoniazid in fingerprint sweat samples obtained from sixteen subjects who had taken Isoniazid. Figure 2: is a diagram showing the average level of Taurine in fingerprint samples obtained from six subjects.

Figure 3: is a diagram showing the average level of Lysine in fingerprint samples obtained from six subjects.

Figure 4: is a diagram showing the average level of Pyroglutamic acid in fingerprint samples obtained from six subjects.

Figure 5: is a diagram showing the average level of Ornithine in fingerprint samples obtained from six subjects.

Figure 6: is a diagram showing the relative standard deviations for fingerprint samples obtained from 14 subjects.

Figure 7: is a diagram showing a comparison per donor (subject) of the coefficient of variability (CV) of acetylisoniazid measurements from fingerprints using only the internal standard for normalisation (diamonds) and using normalisation to taurine (triangles).

Figure 8: is a box plot diagram showing the distribution (range, upper and lower quartile and median) of CVs obtained for the different fingerprint sets using different normalisation strategies.

The following non-limiting examples further illustrate the present invention.

EXAMPLES

The present invention will now be described in relation to several examples.

Experimental Methods

Fingerprint collection method

All subjects from whom fingerprint samples were obtained were human. Fingerprints were obtained from the five fingers (i.e. the four fingers and the thumb) of each subject’s right hand. Fingerprint samples were collected on 2 x 2 cm squares of Whatman 1-Chr grade chromatography paper. Samples were collected using kitchen scales (Sainsbury’s Colour) to measure the pressure applied during sample collection (800 - 1200 g for 10 seconds).

Liquid Chromatography Mass Spectrometry Analysis of Samples

The chromatography paper with fingerprint samples thereon were placed in a 2 ml

Eppendorf microcentrifuge tube, following which an extraction solution (1.5 ml of 10% dichloromethane in methanol) was added. The tube was then centrifuged for 2 min (at 9.5 centrifugal force). The solvent extract was evaporated to dryness under a stream of nitrogen at room temperature (20 °C) and reconstituted in 100 pi mobile phase solution (50:50 (v/v) water/acetonitrile + 0.1% formic acid + 50 ng/ml isoniazid-d₄ as the internal standard) before being vortexed and transferred to a 300 mI glass micro-insert vial, with 5 mI being injected onto an LC-MS/MS system.

Chromatographic separation was performed on a Thermo Scientific™ Ultimate3000 UHPLC system equipped with a binary solvent manager, column manager and

autosampler. The injection volume was 5 pL. Separation was performed on a Kinetex XB- Ci₈ column (100 x 2.1 mm, 5 pm) operated at 30 °C at a flow rate of 0.25 ml/min. Mobile phase comprises 95% FhO (0.1 % formic acid) and 5% acetonitrile (ACN) (0.1% formic acid).

Analysis of 0.5-10 ng isoniazid and acetylisoniazid standards extracted from paper produced R² values >0.999 and within-day precision <3% (n = 5). The lower limit of detection was 10 pg for both analytes. To evaluate matrix effects, 10 pL of analyte standard at 100, 500 and 1000 ng/mL in methanol was pipetted onto fingerprints (n=5 donors) from non-isoniazid-taking subjects collected“as presented” and after handwashing, as well as blank paper. For all standards, the presence of a fingerprint suppressed the isoniazid signal by -20%. This matrix effect varied by <9% for the five different donors.

For acetylisoniazid, no overall signal suppression was observed, and the corresponding variability between donors was <14%.

The prepared samples were introduced to a Thermo Orbitrap Q-Exactive Plus mass spectrometer using the standard ESI interface with a capillary temperature of 320 °C and ESI spray voltage 4 kV. Positive mass spectra were acquired in full scan mode within a range of m/z 120-1000 at a mass resolution of 70 000 at m/z 200.

Example 1 : Tuberculosis drugs

Fingerprint sweat samples were obtained from sixteen subjects (patients) who had taken 200 to 300 mg Isoniazid within the previous 24 hours. The samples were analysed using liquid chromatography high resolution mass spectrometry to determine the presence and amounts therein of Acetylisoniazid, which is a metabolite of Isoniazid. The results are displayed in Figure 1.

The limit of detection (LOD) was determined from five blank extractions from paper and represents the lowest amount of analyte (for example Acetylisoniazid) detectable (10 pg).

The samples were also analysed to determine the presence and relative amounts therein of Taurine, Lysine, Pyroglutamic acid and Ornithine.

Results for five of the samples, as well as samples taken from a negative control subject (who had not taken Isoniazid), are shown in Figures 2 to 5.

As shown in Figures 1 to 5, each of the samples obtained from Patient 37 tested negative for the presence of acetylisoniazid. All of these samples also tested negative for the presence of Taurine, Lysine, Pyroglutamic acid and Ornithine. The absence of Taurine, Lysine, Pyroglutamic acid and Ornithine indicates that the samples obtained from patient 37 were not of sufficient volume and/or quality for the acetylisoniazid results to be reliable.

Patient 30 tested positive for the presence of acetylisoniazid in the sample obtained from the right middle finger. Taurine, Lysine, Pyroglutamic acid and Ornithine was also detected in the right middle finger sample. Patient 30 also returned a low amount (close to the LOD) of acetylisoniazid in the right index fingerprint. No taurine or pyroglutamic acid was detected in this sample; however, lysine and ornithine were detected. With regard to the remaining three samples obtained from Patient 30, all of these tested negative for the presence of acetylisoniazid (lower than the LOD). Taurine was also not detected in any of these samples. However, lysine and ornithine were detected, whilst pyroglutamic acid was detected in one (right ring finger) of the three. Each of the samples obtained from Patient 33 tested positive for the presence of acetylisoniazid. All of these samples also tested positive for the presence of T aurine, Lysine and Pyroglutamic acid. Four out of the five samples also tested positive for the presence of Ornithine. However, the sample obtained from the right thumb of Patient 33 tested negative for the presence of Ornithine.

Each of the samples obtained from Patient 38 tested positive for the presence of acetylisoniazid. All of these samples also tested positive for the presence of T aurine, Lysine, Pyroglutamic acid and Ornithine.

Four out of the five samples obtained from Patient 39 tested positive for the presence of acetylisoniazid. The same four samples obtained also tested positive for the presence of Taurine, Lysine, Pyroglutamic acid and Ornithine. The sample taken from the right thumb tested negative for the presence of acetylisoniazid. The sample taken from the right thumb also tested negative for the presence of T aurine, Lysine, Pyroglutamic acid and Ornithine. The absence of T aurine, Lysine, Pyroglutamic acid and Ornithine from the right thumb sample indicates that this sample was not of sufficient volume and/or quality for acetylisoniazid to be reliably detected.

With regard to the negative control samples, all samples tested positive for the presence of Taurine, Lysine, Pyroglutamic acid and Ornithine.

The results show that amino acids and/or derivatives thereof such as Taurine, Lysine, Pyroglutamic acid and Ornithine are useful in showing qualitatively that a fingerprint of sufficient volume and/or quality for the reliable detection of acetylisoniazid has been deposited and extracted. Furthermore, the results show that amino acids and/or derivatives thereof such as Taurine, Lysine, Pyroglutamic acid and Ornithine are useful in determining the authenticity of a sample provided (i.e. whether or not the sample is a genuine sweat sample).

Example 2: Normalization to Taurine

Fingerprint sweat samples (five per isoniazid-taking subject) were analysed using liquid chromatography high resolution mass spectrometry to determine the presence and amounts therein of Acetylisoniazid and Taurine. The relative standard deviations for acetylisoniazid across the five samples for each subject were calculated. The results are shown in Figure 6 (diamond data points). The amount of Acetylisoniazid in each sample was also normalized to the amount of Taurine in the same sample. The relative standard deviations of the normalized amounts across the five samples for each subject were calculated. The results are shown in Figure 6 (‘x’ data points).

The samples were also normalised to an internal standard. Figure 7 shows how by first rejecting samples for which taurine is not detected and then normalizing the acetylisoniazid signal to taurine reduces the coefficient of variation (CV) (i.e. the amount of variation) of fingerprint samples deposited by the same donor (subject), in every case.

In Figure 8, box plots, depicting the median, upper and lower quartiles as well as the ranges of CVs of fingerprint sample sets, with different normalization strategies are presented. The box plot on the left depicts the CVs of the fingerprint sample sets produced by normalizing only to the internal standard and without any consideration for the quality/volume of the fingerprint. The middle box plot shows the same data, but now rejecting (excluding) samples for which taurine is not detected. The far right box plot shows the effect of rejecting samples for which taurine is not detected and additionally normalizing to taurine.

The median CV reduces from 65%, to 55% and then 26% using these normalization strategies. The results thus show that normalizing to taurine reduces the intra sample variability.

Example 3: Recreational drugs

Five fingerprint sweat samples were obtained from each of three subjects (A, B and C) who had taken heroin. The samples were analysed using mass spectrometry to detect the relative amounts of heroin and its metabolites, 6-acetylmorphine and morphine.

All samples tested positive for the presence of heroin and its metabolites. The relative standard deviation for the respective subjects’ five samples was determined for each drug/metabolite. These‘not normalised’ results can be seen in Table 1 below. The samples were also analysed to detect the presence of T aurine. All of the samples tested positive for the presence of Taurine.

For each sample, the relative amount of each drug or metabolite thereof was normalised to the relative amount of Taurine. The relative standard deviation for the respective subjects’ five samples was determined for the normalised drug/metabolite values. These normalised results can be seen in Table 1 below.

Table 1

As can be seen in Table 1 , normalising to Taurine reduces the relative standard deviation compared to no normalisation across 5 fingerprints for each of the subjects A, B and C.

The data presented in Examples 1 to 3 above shows that amino acids and/or derivatives thereof, such as lysine, ornithine, pyroglutamic acid and taurine, can be used as internal markers to demonstrate that sufficient fingerprint material has been deposited by a subject such that drug analysis undertaken on the same or a similar sample can be trusted to a higher level of reliability. Furthermore, normalization to amino acids and/or derivatives thereof, such as taurine, provides more reliable and quantitative results from skin-print analysis. Additionally, amino acids and/or derivatives thereof can be used to authenticate whether or not a sample is a genuine sweat sample.

Claims

CLAIMS:

1. A method of determining whether a subject has taken a drug, the method comprising:

2. The method of claim 1 , wherein the first sample of sweat and/or the second sample of sweat obtained from the subject is in the form of a fingerprint.

3. The method of claim 1 or claim 2, wherein the at least one amino acid or derivative thereof is taurine, lysine, ornithine and/or pyroglutamic acid.

4. The method of any of the preceding claims, wherein the at least one amino acid or derivative thereof is taurine.

5. The method of any of the preceding claims, wherein the first sample of sweat and the second sample of sweat are the same sample of sweat.

6. The method of any of claims 1 to 4, wherein the first sample of sweat and the second sample of sweat are different samples obtained from the same place on the subject.

7. The method of claim 6, wherein the first sample of sweat and the second sample of sweat are obtained from the same finger.

8. The method of any of the preceding claims, wherein determining the presence of the drug and/or a metabolite thereof in the first sample comprises determining the amount of the drug and/or a metabolite thereof.

9. The method of any of the preceding claims, wherein determining the presence of one or more amino acid or derivative thereof in the second sample comprises determining the amount of the at least one amino acid or derivative thereof.

10. The method of any of the preceding claims, further comprising normalising the amount of the drug and/or a metabolite thereof to the amount of the at least one amino acid or derivative thereof.

1 1. The method of any of the preceding claims, further comprising confirming the identity of the subject by checking the skin-print obtained from the subject.

12. The method of any of the preceding claims, wherein the drug comprises

Tuberculosis medication, preferably wherein the Tuberculosis medication comprises isoniazid, pyrazinamide, rifampicin and/or ethambutol.

13. The method of claim 12, wherein the Tuberculosis medication comprises isoniazid and/or pyrazinamide.

14. The method of claim 12 or claim 13, wherein the Tuberculosis medication comprises isoniazid and/or wherein the metabolite comprises acetylisoniazid.

15. The method of any of claims 1 to 11 , wherein the drug comprises morphine and/or heroin.

16. The method of claim 15, wherein the drug comprises heroin and the metabolite thereof is 6-acetylmorphine and/or morphine.

17. The method of any of the preceding claims, wherein the analysis of the first sample of sweat and/or the second sample of sweat is carried out using mass spectrometry.

18. The method of claim 17, wherein the mass spectrometry is liquid chromatography mass spectrometry or paper spray mass spectrometry.

19. The method of any of the preceding claims, further comprising determining the volume of the first sample of sweat and/or the second sample of sweat.

20. The method of claim 19, wherein the volume of the first sample of sweat and/or the second sample of sweat is determined using an electromagnetic detector to obtain an optical image of the first sweat sample in the form of the skin-print and/or the second sweat sample in the form of the skin-print.

21. The method of claim 20, further comprising analysing the optical image to determine an image density relating to the skin-print and calculating skin-print quantity as a function of image density of the optical image.

22. The method of claim 20 or claim 21 , further comprising transmitting electromagnetic radiation towards the skin-print, wherein the step of using an electromagnetic detector to obtain the image comprises detecting a reflection of the transmitted electromagnetic radiation.

23. A method of authenticating whether or not a sample is a sweat sample, the method comprising analysing using mass spectrometry the sample to determine the presence or absence therein of at least one amino acid or derivative thereof.

24. The method claim 23, wherein the sample is obtained from a subject in the form of a skin-print, preferably in the form of a fingerprint.

25. The method of claim 23 or 24, wherein the method comprises analysing using mass spectrometry the sample to determine the presence or absence therein of taurine, and, optionally, at least one further amino acid or derivative thereof.