WO2008122805A1

WO2008122805A1 - Analysis method

Info

Publication number: WO2008122805A1
Application number: PCT/GB2008/001250
Authority: WO
Inventors: James Gordon Campbell; Suesse Silke; Boris Lasarow Avrom
Original assignee: Surescreen Diagnostics Limited; Trimega Laboratories Limited
Priority date: 2007-04-10
Filing date: 2008-04-10
Publication date: 2008-10-16
Also published as: EP2150820A1; GB0910214D0; GB0706831D0

Abstract

An analysis method consisting of the accurate determination of human fatty acid ethyl esters in hair, skin secretions and/or skin patches, toenails and fingernails by extraction and determination by gas chromatography/mass spectrometry or liquid chromatography/mass spectrometry to determine fatty acid ethyl esters (FAEE's) amongst others the concentrations of ethyl myristate, ethyl palmitate, ethyl oleate compared with deuterised internal standards which allow accurate quantification of these species, in conjunction with the determination of ethyl glucuronide (EtG) to show that the alcohol has been metabolized and is not from contamination with alcohol and comparison of individual levels of FAEE's with real-life data from alcohol consumers, and comparison of the cumulative sum of FAEE's with real-life data from alcohol consumers to give a reliable comparison to the amount of alcohol that has been consumed, whether overall or month by month or such other historical period as may be required from the sample.

Description

ANALYSIS METHOD

FIELD OF INVENTION

The present invention relates to a method of analyzing specially selected human lipids and other markers that have been produced in the body in conjunction with alcohol such that their analysis provides a reliable marker for the level of alcohol consumption by that person. This method is unique in that it has been developed to allow long term analysis of alcohol consumption for that individual and it may be applied by collecting hair, fingernail, toenail or skin secretions, the latter either by a swab applied directly to the skin or else by a patch which is worn on the surface of the skin.

The analysis method assays a range of fatty acid ethyl esters (FAEE's) and ethyl glucuronide (EtG) and the quantities detected are compared to control levels determined by testing a series of individuals with reported alcohol consumption. Analysis uses EtG controls and deuterised fatty acid ethyl esters (d FAEE) as internal standards so that precise identification of the markers is made and the quantity detected may be reliably determined.

Moreover, by sectioning hair into different lengths and comparing these lengths with the rate of hair growth, the analysis of these lipid acid derivatives may be compared with a period in history with the capability of historical analysis dictated only by the extent of the length of hair.

There is a distinct benefit in analyzing both FAEE and EtG from the same sample source because each species is a direct alcohol marker that is metabolized by the body of an alcohol consumer in proportion to the amount of alcohol that is consumed. Since both FAEE's and EtG are excreted on skin or deposited in the hair shaft or fingernail or toenail during its growth in different ways, by determining both markers at the same time the_. accuracy of the technique is greatly enhanced. Analytical accuracy is enhanced by adding together the FAEE's because this normalizes any deviations in one or other FAEE in the sample. This analytical process also guarantees the absence of any contamination effects from alcohol-derived substances such as shampoos, hair dye or medication such as head lice treatments because only alcohol that has been ingested and metabolised by the body can result in FAEE's and EtG. False positives cannot therefore occur with this method of analysis.

Aspects of the present invention are defined in the claims attached to this application.

BACKGROUND TO THE PROBLEMS OF ALCOHOL TESTING ADDRESSED BY THIS PATENT

Alcohol (chemical name: ethanol or ethyl alcohol) is the most abused drug in Western society. In Britain alone, there are 5 million cases of hospital accident and emergency visits that are alcohol related every year. The population of many countries has developed a binge drinking culture, and because alcohol is widely available in most countries each one has a significant proportion of the population that is addicted to alcohol. More recently alcohol has been classified as the chemical which is the fifth most serious in terms of harm after only heroin, cocaine, barbiturates and street methadone.

It has long been acknowledged by the medical profession that a reliable test for overindulgent alcohol consumption is required. Currently, there exist a number of simple alcohol tests but each one has severe limitations.

Long-term alcohol abuse affects the liver and so there are various liver function tests based on blood or urine to determine whether alcohol abusers have an affected liver. However, quite apart from there being a number of medical conditions that can produce false results, a correctly diagnosed positive result means the liver is already affected and this test is therefore conducted too late in the patient's history. By the time such tests register positive results, it may be too late to begin preventive treatment. These tests do not relate directly to the quantity of alcohol consumed so their effectiveness is limited as a diagnostic tool. In the early stages of an alcoholic's career these tests will probably give negative results. Breath alcohol tests exist in many forms but alcohol is lost from breath relatively quickly and so this is not a reliable test when conducted more that a few hours after the last drinking session. These tests are only suitable for detecting the level of current intoxication.

There is a biological marker in blood, called carbohydrate deficient transferrin (CDT) which increases in quantity due to compromises in the breakdown of glucose in the presence of ethanol. This marker is present in people who have an alcohol dependence, and when they abstain it deteriorates slowly over several weeks. However, CDT is of limited use medically, because people abusing alcohol often have irregular alcohol consumption patterns, or may be having intermittent alcohol drinking relapses; something which often happens with alcoholics.

BRIEF PRINCIPLE

Seeing that alcohol is a big social problem, and realizing that alcohol may change the chemistry of some of the fundamental lipids in the body, the inventor has researched and isolated a group of lipids (fatty acids) that change fundamentally in proportion to the quantity of ethanol that is present in the body over a period of time. These fatty acids are always present in the body and are relatively consistent in their presence throughout the population. When alcohol is consumed, fatty acid markers that have metabolized in the presence of alcohol in the body have been combined with another alcohol derivative, ethyl glucuronide, EtG, which shows categorically that the alcohol was ingested, to produce reliable results when these substances are extracted from a sample of hair and treated according to the invention.

Alcohol has a simple molecular structure and it leaves the body quite quickly. Alcohol is broken down in complex ways depending on the genetic and physiological make up of the person, and their ethnic background. Our research into the metabolism has shown us that there are five basic biotypes.

Biotype 1 is the THIQ Biotype. He has a defective alcohol dehydrogenase Il enzyme (ADH2*2 variant) which converts alcohol 40% faster to acetaldehyde resulting in leakage of acetaldehyde into bloodstream and brain and its condensation with neurotransmitter dopamine forms addictive morphine analogues - tetrahydropapoverilone and salsolinol which are tetrahydroisoquinoiines (THIQ). Its condensation with 5HT also produces beta-Carboline another addictive THIQ which is a psychotogen. All of these end products are dipostropic or stimulants of alcohol consumption because they displace and destroy the natural mood transmitters in the brain making him dependent on alcohol to feel normal. His fast ADHlI enzyme activity ensures he receives a constant supply of "biologically rewarding" mood euphoriant dipsotropics and whilst he needs several drinks to get the feeling he seeks, there is little accompanying intoxication and no hangover. This category is made up of Type-A personalities, compulsive with a strong sex drive, requiring little sleep to function properly. Alcohol gives him energy and improves his performance. They are the big drinkers who can consume alcohol all day and who stay on a high with little signs of inebriation or negative effects. They have a high tolerance for alcohol and after many years of drinking are more prone to develop liver problems than psychiatric symptoms. ^*This biotype also include the minority group affected by the mu opioid receptor mutants N40D+118A, -172G/T, -111C/T, -38C/A, A6V, 4OD, and S147C.

Biotype 2 is the Allergic-Addicted biotype: He is allergic to grapes, grains, amyl alcohols, phenols or "congeners" in alcohol and addicted to the endogenous opioids they liberate in the brain resulting in short term high followed by bad physical withdrawal. Once opioids are exhausted the pleasant "biological reward" effects are interrupted and he will experience a rebound depression, aggression, or crying spells and allergic, "toxic" withdrawals the morning after. This biotype may develop cravings for alcohol after exposure to chemicals like gasoline, formaldehyde, printers ink and hydrocarbons which activate the same processes in the brain as alcohol congeners. Often arising from Northern European or Amerindian ancestors, this category is made up of the typical "bad-starters" who learn how to drink. They are often moody, changeable and unpredictable alcoholics who experience very bad hangovers and who may become socially disruptive, engaging in fights and arguments, dangerous driving, irrational or bizarre behaviour and even engage in criminal acts after drinking.

Biotype 3 is the PGE 1 Deficient biotype. He suffers with a lifelong depression resulting from a genetically low biosynthesis of the neurotransmitter PGE1. Alcohol releases PGE1 until depleted and then depression returns. Drinking also inhibits the already compromised manufacture of PGE1 from the starting materials Linoleic acid and GLA thus worsening the rebound depression when drinking ceases. Often arising from Scottish, Welsh, Irish, and Scandinavian ancestors this category is made up of lifelong somber, introverted and depressed individuals who cheer up after drinking. They drink to banish their depression and may become suicidal once a tolerance builds up to the effects of the drug.

Biotype 4 is the Hypoglycaemic biotype. He is addicted to the sugars in alcohol because his body ordinarily produces excess insulin (hyperinsulinism) which starves his brain of the glucose it needs. He feels a rapid lift after drinking followed shortly therafter by an exaggeration in his original hypoglycaemic symptoms like light-headedness, spaciness, confusion, weakness, sleepiness and lack of co-ordination and gets very hungry after drinking. He has a low tolerance for alcohol and can easily be misidentified as an out of control drinker. He usually experiences hangovers because alcohol triggers insulin shock which causes fatigue, lethargy, confusion, brain fog, depression and irritiability. This category is made up of individuals who cannot handle too much alcohol and who despite feeling a temporary increase in wellbeing after a drink or two, quickly show signs of neuroglycopenia (glucose starvation) resembling intoxication.

Biotype 5 is the Dopamine deficient biotype. He inherits a genetic defect in the Dopamine D2 inhibitory autoreceptor and possibly other receptors and transporters (D1 receptor A48G, D3 receptor S9G, D4 receptor VNTR, DA Transporter SLC6A3-9) in the Dopamine neurotransmitter system which leads to low Dopamine tone and "Reward Deficiency Syndrome." The presence of the DRD2A1 allele is associated with reduced expression of D2 receptors, indicates a genetic susceptibility to low dopamine levels and consequently an increased likelihood of addictive behaviours that temporarily raise Dopamine levels like drinking. (There are four Dopamine receptor gene Taq 1A alleles known, the A1 , A2, A3 and A4 alleles. The A3 and A4 alleles are rare, whereas the A2 allele is found in nearly 75 percent of the general population and the A1 allele in about 25 percent of the population. Of 35 severe liver-damaged alcoholics 69 percent will have the A1 allele and 31 percent will have the A2 allele whereas in 35 nonalcoholics 20 percent have the A1 allele and 80 percent have the A2 allele) The science suggests that A1 carriers may not be sufficiently rewarded by stimuli that A2 carriers, for instance, find satisfying or calming. This category then is made up of the risk takers, gamblers, sexually promiscuous, compulsive overaters, drug takers, and alcoholics who (unlike the temporary alcohol-induced behavioural effects of the allergic addicted alcoholic biotype) may possess a general genetic propensity for antisocial behaviour, conduct disorders, violent behaviour and criminal tendencies. Many prisoners and re-offenders will probably fall into this category. *This product will also be suitable for the minority biotype affected by the Tyrosine Hydroxylase enzyme mutant VNTR intron 1

Recessive Biotypes: The Monoamine Oxidase A (MAOA) dinucleotide repeat polymorphism and Catechol-O-Methyl Transferase (COMT) L(Met) mutants are additional minority biotypes which will also be catered for by the patent.

These biotytpes have been described to demonstrate that alcohol affects different people in different ways. Nevertheless all of these biotypes are catered for in the present invention because irrespective of their metabolism, our research has shown that they will all produce sebaceous excretions on skin and will lay down hair in the same manner in the form of FAEE and they all produce typical amounts of EtG which demonstrates the alcohol was metabolised. FAEE's and EtG are better markers than any others hitherto because although there are five biotypes the formation of FAEE and EtG are believed to be constant and unaffected by the genetic alcohol biotype.

APPLICATION OF FAEE'S AND EtG TO HAIR, FINGERNAILS AND TOENAILS AS A SAMPLE SOURCE

Concerning hair in particular, hair consists of a protein core or cortex of keratin and a cuticle sheath of protein, protected by scales. The hair is manufactured in the root and when there are substances in the body such as drugs or lipids, these are captured within the hair cortex as it grows. Since the drugs or alcohol have been metabolized by the body they generally exist as glucuronides or esters

The hair grows in most people at a predictable rate and it takes about one week to ten days for the hair forming the root to be visible on the scalp. Once the hair has formed, chemicals such as drugs or lipid glucuronides or esters are trapped in the hair in minute quantities and cannot be removed. They remain in the hair until the hair is cut away. Concerning toenails and fingernails in particular, nail consists of keratin protein manufactured in the nail bed and when there are substances in the body such as drugs or lipids, these are captured within the nail keratin as it grows. Since the drugs or alcohol have been metabolized by the body they generally exist as glucuronides or esters. The sample may be cut from the end of the nail or else nail scrapings may be taken with a blade for example held at right angles to the surface of the nail and scraped across the surface of the nail.

These substances can be removed from the hair or nails for analysis by several processes. They can be digested in accordance with U.S. patents 5,324,642 and 6,582,924 proposed by Psychemedics Inc. Los Angeles USA. They can be extracted by supercritical steam extraction, or they can be extracted by soaking in several consecutive solvent solutions such as methanol, or n-heptane, or dimethyl sulphoxide (DMSO) and n-heptane mixture. To speed up the extraction process the hair or nail can be milled, and heated and subjected to ultrasonic sonification.

Once the lipids have been extracted from the hair they are concentrated by partial evaporation and mixed in a known proportion with deuterised controls of those substances under investigation. This mixture is then subjected to headspace solid phase microextraction and analysed by gas chromatography/mass Spectrometry or liquid chromatography/mass spectrometry, such as those made by Hewlett Packard. These instruments separate all the volatile organic compounds and produces an identifiable decomposition spectrum that unequivocally identifies the compounds that have been detected. The use of deuterised standards of those FAEE's ensures the identification is precise and the concentration is assayed accurately.

APPLICATION TO SKIN SWABS OR SKIN PATCHES AS A SAMPLE SOURCE

Concerning sebaceous skin samples in particular, substantially blunt scrapers such as those made from plastic or metal may be used to collect skin secretions, or swabs such as those consisting of cotton or polymeric material, or collecting sticks such as those made of cotton or polymeric material, or sponges made from cotton or polymeric material may be readily used to collect sebaceous deposits from the skin. Suitable locations for samples are forehead, temples, and neck amongst others. These deposits may be removed from the swabs etc. using solvent such as n-heptane.

Alternatively a skin patch may be applied to the skin such as the upper arm, thigh or chest using an impervious or microporous patch having a padded liner in contact with the skin. The pad is preferably manufactured from cotton or some other absorbent material. The patch preferably has some form of tamper evident system to prevent the patch from being peeled off and reapplied at a later date to attempt to avoid an accurate quantity of fatty acid from being collected by the donor. One such tamper evident system consists of pressure sensitive tape manufactured by The Le Mrk Group, 32 Stephenson Road, St. Ives, Cambridgeshire, PE27 3WJ, UK. Typically the patch remains in place for one week or longer.

CHEMISTRY

In addition to oxidative metabolism that breaks down ethanol into water and carbon dioxide, it can be metabolized by esterification with fatty acids to form fatty acid ethyl esters. Our laboratory has focused its efforts on various aspects of fatty acid ethyl esters and their production and our research group has studied the effect of alcohol consumption of varying proportions on the production of fatty acid ethyl esters in hair and on the skin of a number of individuals.

Fatty acid ethyl esters are esterification products of fatty acids in the presence of bodily alcohol. These compounds are believed to be generated through the action of an enzyme known as fatty acid ethyl ester synthase. Substantial evidence exists that fatty acid ethyl esters are the toxic mediators of ethanol metabolism responsible for the organ damage generated by ethanol abuse. Variations in the presence and performance of ester synthase is thought to be one genetic factor responsible for the generation of various biotypes as previously described and these variable traits are believed to be genetically inherited.

Notwithstanding these genetic variants, FAEE's represent a reliable set of markers for identifying alcohol abuse, and their deposition in keratinaceous material (hair, nails) and skin excretions provide a historical assay capability, because FAEE production has been found to be reasonably consistent in all individuals when the sum total of FAEE's is used as an alcohol marker.

THE INVENTION

Hair extraction, nail sampling, and skin secretion analysis as described, forms one of the many laboratory procedures available to the scientist practiced in the art of detecting substance abuse. What is not currently known is the method used to identity of the particular markers that reveal the history of alcohol abuse for that person.

Using their skill as research scientists the inventors have determined through regressional analysis that certain FAEE substances are reliable markers for alcohol abuse when found in hair and they can be analysed using special procedures when combined with ethyl glucuronide (EtG).

Fatty Acids are aliphatic carboxylic acids with varying hydrocarbon lengths at one end of the chain joined to terminal carboxyl (-COOH) group at the other end. The general formula is R-(CH₂)_n-COOH. Fatty acids are predominantly unbranched and those with even numbers of carbon atoms between 12 and 22 carbons long react with glycerols to form lipids (fat-soluble components of living cells). Fatty acids ail have common names such as lauric (C12), Myristic (C14), palmitic (C16), oleic (C18, unsaturated), stearic (C 18) and linoleic (C 18, polyunsaturated) acids. The saturated fatty acids have no double bonds, while oleic acid is an unsaturated fatty acid with one double bond (also described as olefinic) and polyunsaturated fatty acids like linolenic acid contain two or more double bonds. Each of these fatty acids form ethyl derivatives in the presence of blood alcohol.

The compounds which may be involved in this metabolism process are primarily esters of fatty acids, for example ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate plus one direct metabolite ethyl glucuronide. However the important markers identified by analysis include the following fatty acids and derivatives such as ethyl esters of the following substances:

• 10-Nitrooleate 11 ,14-Eicosadienoic Acid

113-Docosenamide

1-Arachidonoyl Glycerol-d5

1-LinoleoyI Glycerol

1-Palmitoyl-2-linoleoyl PE

20-HETE

20-HETE Ethanolamide

2-Arachidonoyl Glycerol

2-Arachidonoyl Glycerol-d5

2-Arachidonyl Glycerol ether

2-FIuoropalmitic Acid

2-Hydroxymyristic Acid

2-Linoleoyl Glycerol

2-thioacetyl MAGE

3-Thiatetradecanoic Acid

4,5-dehydro Docosahexaenoic Acid

5,6-dehydro Arachidonic Acid

7,7-dimethyI-5,8-Eicosadienoic Acid

8,11 ,14-Eicosatriynoic Acid

8,11-Eicosadiynoic Acid

9, 12-Octadecadiynoic Acid

9-Nitrooleate

9-Octadecenamide

9-Thiastearic Acid

9Z, 11 E, 13E-Octadecatrienoic Acid

9Z,11 E,13E-Octadecatrienoic Acid ethyl ester

9Z,11 E,13E-Octadecatrienoic Acid methyl ester

Adrenic Acid α-Linolenic Acid α-Linolenic Acid ethyl ester α-Linolenic Acid methyl ester α-Linolenoyl Ethanolamide

AM404

Arachidonamide

Arachidonic Acid

Arachidonic Acid-biotinimide

Arachidonic Acid-d8

Arachidonic Acid ethyl ester

Arachidonic Acid Leelamide

Arachidonic Acid methyl ester

Arachidonic Acid N-methyl amide

Arachidonic Acid N,N-dimethyl amide

Arachidonic Acid (peroxide free)

Arachidonic Acid Quant-PAK

Arachidonic Acid (sodium salt)

Arachidonoyl Ethanolamide

Arachidonoyl Ethanolamide-d8

Arachidonoyl Glycine

Arachidonoyl Glycine-d8

Arachidonoyl m-Nitroaniline

Arachidonoyl p-Nitroaniline Arachidonyl Trifluoromethyl Ketone

Arachidoyl Ethanolamide

Cerulenin cis-4, 10,13,16-Docosatetraenoic Acid cis-4,10-13,16-Docosatetraenoic Acid methyl ester cis-7-Hexadecenoic Acid cis-7-Hexadecenoic Acid methyl ester cis-Δ2-11-methyl-Dodecenoic Acid cis-Parinaric Acid

Conjugated Linoleic Acid (10E.12Z)

Conjugated Linoleic Acid (9E.11E)

Conjugated Linoleic Acid (9Z,11E)

CP 24,879 (hydrochloride)

Decanoyl m-Nitroaniline

Decanoyi p-Nitroaniline

Δ2-cis Eicosenoic Acid

Δ2-trans Eicosenoic Acid

Dihomo-γ-Unolenic Acid

Dihomo-γ-Linolenic Acid ethyl ester

Dihomo-γ-Lino!enic Acid methyl ester

Dihomo-γ-Linolenoyl Ethanolamide

Docosahexaenoic Acid

Docosahexaenoic Acid-d5

Docosahexaenoic Acid methyl ester

Docosahexaenoic Acid Quant-PAK

Docosahexaenoyl Ethanolamide

Docosanoyl Ethanolamide

Docosapentaenoic Acid

Docosatetraenoyl Ethanolamide

Docosatrienoic Acid

Eicosapentaenoic Acid

Eicosapentaenoic Acid-d5

Eicosapentaenoic Acid (peroxide free)

Eicosatetraynoic Acid

Eicosatrienoic Acid (11Z,14Z,17Z)

Eicosatrienoic Acid (5Z,8Z,11Z)

Eicosatriynoic Acid

Elaidic Acid

Ethyl Tricosanoate

Fatty Acid ethyi ester Standard Mixture γ-Linolenic Acid

Laurie Acid

Laurie Acid ethyl ester Laurie Acid Leelamide ϋnoelaidic Acid Linoleic Acid Linoleic Acid-d4 Linoleic Acid ethyl ester Linoleic Acid (peroxide free)

Linoleic Acid Quant-PAK

Linolenic Acid ethyl ester

Linoleoyl Ethanolamide

Mead Acid Ethanolamide

Methyl α-Linolenyl Fluorophosphonate

Methyl Arachidonyl Fluorophosphonate

Methyl γ-l_inolenate

Methyl γ-Linolenyl Fluorophosphonate

MS-PPOH

Myristic Acid ethyl ester

N-(3-hydroxyphenyl)-Arachidonoylamide

N-Oleoylglycine

N-Oleoyl Taurine

N-Palmitoyl Taurine

N-Stearoyl Taurine

Oleic Acid

Oleic Acid-2,6-diisopropylanilide

Oleic Acid ethyl ester

Oleoyl Ethanolamide

Oleoyl Ethanolamide-d2

Oleyl Anilide

Oleyl Trifluoromethyl Ketone ω-3 Arachidonic Acid ω-3 Arachidonic Acid-d8 ω-3 Arachidonic Acid ethyl ester ω-3 Arachidonic Acid methyl ester ω-3 Arachidonic Acid Quant-PAK

Palmitic Acid

Palmitic Acid ethyl ester

Palmitic Acid methyl ester

Palmitoleic Acid ethyl ester

Palmitoyl Ethanolamide

Palmitoyl Ethanolamide-d4

Palmityl Trifluoromethyl Ketone

Phytanic Acid

Pinolenic Acid

Pinolenic Acid ethyl ester

Pinolenic Acid methyl ester

Resolvin E1

Ricinoleic Acid

Sesamin

Stearic Acid ethyl ester

Stearidonic Acid

Stearidonic Acid ethyl ester

Stearidonic Acid methyl ester

Stearoyl Ethanolamide trans-Δ2-11 -methyl-Dodecenoic Acid Ethyl glucuronide (ethyl β-D-6-glucosiduronic acid) is a product of ethanol metabolism which is produced in the liver directly from alcohol. Ethyl glucuronide is a minor metabolite of ethanol, and those practiced in the art will appreciate that its presence in urine can be used as a laboratory test to detect recent alcohol intake, even for some time after the ethanol is no longer measurable in the breath or blood.

The present invention therefore looks for these special markers called fatty acid ethyl esters (FAEE) as well as ethyl glucuronide (EtG) in the hair, nails or skin secretions.

Both marker groups are only produced when there is alcohol in the bloodstream, and the more alcohol there is, the greater the proportion of FAEE and EtG. The advantage of looking at both FAEE and EtG is that they are direct alcohol markers that possess the unchanged ethyl group of alcohol in contrast to the indirect biochemical markers currently in use. As both are deposited in the hair shaft or nails or skin secretions in different ways, by looking at both we can be certain of obtaining accurate results.

Moreover, having determined that these lipid derivatives are reliable indicators for alcohol abuse, the inventors have set about producing a protocol for analysis of hair whereby internal control markers have been specially manufactured. The purpose of these markers is to introduce a particular concentration of these markers into the simple extract being studied. When analysed in a gas chromatography / mass spectrometer any lipid derivative will be revealed by the instrument, together with an accurately known amount of the internal control marker, thereby allowing the concentration of the lipid derivative by comparison with the concentration of the internal control marker.

Internal markers may be manufactured for example by taking a supply of the fatty acid that is of interest and allowing deuterised (heavy hydrogen) ethanol to form deuterised fatty acid ethyl esters in the presence of a suitable fatty acid ethyl ester synthase.

This deuterised (dFAEE) standard has the same chemical structure as the derivative and therefore reacts in the same way during transport in the gas chromatography column, but it has a slightly heavier molecular structure and so it lags behind in the analysis and produces two peaks, one for the derivative under investigation, and one for the deuterised standard. Knowing the concentration of the dFAEE standard that was added to the sample containing FAEE's, the concentration of the FAEE may now be calculated either by peak heights, or more accurately, by integrating the curve or by any other statistical means available with the analysis instrument, comparing dFAEE with FAEE.

The aforementioned also applies to samples obtained from skin secretions or from nail clippings or scrapings as appropriate.

It will also be appreciated by those proficient in the art that the aforementioned methods of analysis also are equally applicable to other alcohol species. In particular the method may prove valuable in cases of long-term methanol (methylated spirits) use, or in forensic instances where methanol has been supplied over a period to an individual as a poison. In other applications where alcohols have been used in industrial processes the adaptation of the method to include these fatty acid alcohol esters (for example fatty acid propanol ester) may be used to determine whether a person's exposure to the industrial process has been harmful (such as from overexposure to propyl alcohol). The patent therefore applies to these situations in the spirit of the application of the method.

RESULTS OF ANALYSIS

Once FAEE results have been obtained they may be compared with trial data to establish the likely pattern of alcohol abuse, or the absence of alcohol consumptions, as may be the case.

One suitable method of interpretation of results is to take the value of EtG and apply this to a drinking pattern. A more accurate method is to analyse for the dominant ester, ethyl oleate and relate drinking pattern to this value. However our experience is that it is eminently more accurate to add the results from the four main FAEE derivatives, namely ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate, and compare the sum of these derivatives with mean data derived from real life drinking incidents. For example, cumulative values in the range 0 - 2ng/10mg for alcohol abstainers, 2 - 9ng/10mg for moderate to heavy social drinking and above 9ng/10mg for alcohol abuse were obtained. In each case EtG was detected and corroborated the above results. EXAMPLE OF USE OF THE INVENTION

In one example a medical consultant had to establish whether a patient was suitable for a particularly expensive treatment which would lose its effectiveness if alcohol was subsequently consumed in any significant quantity. This individual had a past history of alcohol abuse. Analysis using liver function and carbohydrate deficient transferring indicated no recent alcohol dependence but in view of the possibility of complications with the medical procedure the consultant decided to carry out a hair test covering a period of six months history. The results confirmed the individual had abstained from alcohol over that six months giving the consultant confidence to commence the treatment.

In another example two individuals had a past history of alcohol dependence and the courts had to decide which parent should have custody of children. Hair analysis covering a protracted history enabled the courts to consider the results of alcohol use month by month as part of their decision making process.

In a third example an executive being considered for promotion chose to demonstrate his reported alcohol problems with a hair test analysis that showed him to be a social drinker but with levels of alcohol markers that indicated his steady but moderate drinking pattern. As part of his new appointment he agreed to future monitoring and to moderate his drinking habits.

In a fourth example an employee in a critical job in the oil industry was screened for pre- employment prior to working on site and was found to have a severe alcohol problem. The results allowed his employers to take appropriate action to minimize the risk to their installation from a person who may not otherwise have been safe on site.

In a fifth example individuals at alcohol treatment centres agreed to subject themselves to regular screening using skin secretion samples to show improvements in their alcohol habit. Results were mostly favourable and the screening process was thought to have assisted in the determination needed by these individuals to abstain from alcohol. ADVANTAGES

The present invention has a number of advantages over the prior art. These advantages will be apparent in the following description.

By way of example, the present invention is advantageous because alcohol abuse can be quantified over an extended period while ever the hair has not been cut from the donor. For example it would be possible to record the donor's history of alcohol abuse over a period of one year or more if the hair is long enough. This provides valuable evidence in cases where it is important to establish whether the donor is alcohol dependent or not. Such instances may, for example, include child custody cases, and the choosing suitable subjects for liver transplants.

By way of further example the history is locked into the hair sample as it grows and no other system can provide the long term evidence of alcohol abuse in this way because a history of this sort is not recorded. This history allows the hair to be sectioned, for example, month by month to give a reliable history of quantity consumed. This approach may be advantageous for alcohol rehabilitation centres.

By way of further example, provided the hair is intact it is not possible to contaminate, add or remove these markers from the hair because they are contained within the hair shaft and are unable to be attacked or modified by the application of shampoos, medication, deliberate drug-reducing treatment applications or the like, nor are they able to be substantially altered by the application of heat, or cosmetic treatments. This provides confidence in the results and the method in general.

By way of further example, the present invention is advantageous because alcohol abuse can be quantified over an extended period even when the donor is long-term dead, where in particular, alcohol determination by other means presents a problem because of fermentation of bodily fluids through bacterial infection (such fermentation causing the generation of alcohol in these fluids) or alternatively through the thorough drying out of the body. Hair is not affected in this way. Indeed the technique may be demonstrated on those long-term dead such as mummified remains provided the hair has remained relatively cold. Such a procedure is therefore a valuable forensic tool. By way of example, the present invention is advantageous because alcohol abuse can be quantified over an extended period month-by-month or period-by-period so that the investigator can determine whether the level of alcohol use is increasing or decreasing in that subject.

By way of example, the present invention is advantageous because alcohol abuse can be quantified over an extended period in hair that is collected and kept for an extended period. Such occasions might occur in a forensic case, such as a fatal car accident in which it later transpires that there is an accusation that the driver was drunk or was an alcoholic. Such an application might, for example apply to Henri Paul, Princess Diana's chauffeur who has posthumously been shown to have been regularly under the influence of alcohol which has been a matter of dispute.

By way of example, the present invention is advantageous because alcohol abuse determined in hair in this way is not affected by the use of alcoholic solutions such as may exist in hair spray or hair lacquer, because the method only detects the fatty acid ethyl ester derivatised from the hair core together with ethyl glucuronide, which has been metabolized by the body. Alcohol from other sources will not appear as the glucuronide. The proposed method is therefore failsafe.

By way of further example the system is applicable to hair, fingernails, toenails and skin secretions.

By way of further example the skin secretion method is applicable to collections by scraping or swabbing the skin, and also by collection with a skin patch (incorporating evidential tamper-evident seal) for medium to long term collection of a sample.

By way of further example the system is adaptable for methanol and other alcohols that might be encountered in poisoning, or alcohol misuse or industrial accidents or poisonings.

Claims

1. An analysis method consisting of the accurate determination of human fatty acid ethyl esters in hair, skin secretions and/or skin patches, toenails and fingernails by extraction and determination by gas chromatography/mass spectrometry or liquid chromatography/mass spectrometry to determine fatty acid ethyl esters (FAEE's) amongst others the concentrations of ethyl myristate, ethyl palmitate, ethyl oleate compared with deuterised internal standards which allow accurate quantification of these species, in conjunction with the determination of ethyl glucuronide (EtG) to show that the alcohol has been metabolized and is not from contamination with alcohol and comparison of individual levels of FAEE's with real-life data from alcohol consumers, and comparison of the cumulative sum of FAEE's with real-life data from alcohol consumers to give a reliable comparison to the amount of alcohol that has been consumed, whether overall or month by month or such other historical period as may be required from the sample.

2. A method according to claim 1 , wherein a patient's ingestion of alcohol is based on the level of ethyl glucuronide (EtG) from one or other samples of hair, toenail, fingernail or skin secretion when compared top real life analyses of EtG from individuals with known drinking habits.

3. A method according to claim 1, wherein the individual level of ethyl oleate is determined from one or other samples of hair, toenail, fingernail or skin secretion when compared to real life analyses of ethyl oleate from individuals with known drinking habits.

4. A method according to claim 1 , wherein the individual level of the four main FAEE's (ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate) are determined from one or other samples of hair, toenail, fingernail or skin secretion when compared to real life analyses of ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate from individuals with known drinking habits.

5. A method according to claim 1 , wherein the individual level of the four main FAEE's (ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate) are determined from one or other samples of hair, toenail, fingernail or skin secretion when compared to real life analyses of ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate from individuals with known drinking habits, such as cumulative values for hair analysis in the range 0 - 2ng/10mg represent alcohol abstainers, 2 - 9ng/10mg represent moderate to heavy social drinking and above 9ng/10mg represent alcohol abuse.

6. A method according to claim 1 , wherein the individual level of the four main FAEE's (ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate) are added together to provide an FAEE factor from one or other samples of hair, toenail, fingernail or skin secretion when compared to real life analyses of the sum of ethyl myristate, ethyl palmitate, ethyl oleate, ethyl stearate and ethyl glucuronide from individuals with known drinking habits, with the presence of these fatty acid ethyl esters and ethyl glucuronide proving that the presence of alcohol could not have arisen from environmental contamination of alcohol (for example from shampoos, hair dyes, head lice treatments, nail varnish and the like).

7. A method according to claim 1, wherein the individual level of the four main FAEE's (ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate) and the metabolite ethyl glucuronide are determined from one or other samples of hair, toenail, fingernail or skin secretion when compared to real life analyses of ethyl myristate, ethyl palmitate, ethyl oleate, ethyl stearate and ethyl glucuronide from individuals with known drinking habits.

8. A method according to claim 1 , wherein the individual level of the four main FAEE's (ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate) are added together to provide an FAEE factor from one or other samples of hair, toenail, fingernail or skin secretion when compared to real life analyses of the sum of ethyl myristate, ethyl palmitate, ethyl oleate, ethyl stearate from individuals with known drinking habits.

. A method according to claim 1 , wherein the individual level of the four main FAEE's (ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate) are added together to provide an FAEE factor from one or other samples of hair, toenail, fingernail or skin secretion when compared to real life analyses of the sum of ethyl myristate, ethyl palmitate, ethyl oleate, ethyl stearate and ethyl glucuronide from individuals with known drinking habits, and compared with the level of ethyl glucuronide (EtG) as evidence of alcohol metabolism and proof that the levels could not have occurred from environmental contamination by alcohol.

10. A method according to claim 1 , wherein the levels of several or many FAEE's are measured, individually or added together to provide an FAEE factor, as may be appropriate, from one or other samples of hair, toenail, fingernail or skin secretion when compared to real life analyses of the equivalent values from individuals with known drinking habits.

11. A method according to claim 1 incorporating any of the aforementioned claims wherein a hair sample is sectioned to give discrete period of history (for example month by month) for the purposes of determining increasing or decreasing levels of alcohol ingestion or even complete abstinence over particular periods.

12. A method according to claim 1 , wherein the individual levels of FAEE's and/or EtG are extracted and analysed using deuterised control standards forming an internal calibrator for accuracy in assay determination.

13. A method according to claim 1 , wherein the individual levels of FAEE's and/or EtG are extracted and analysed using deuterised control standards forming an internal calibrator for accuracy in assay determination from a sample collected with a skin patch.

14. A method according to claim 1, wherein the individual levels of specific FAEE's and/or alcohol glucuronides are extracted and analysed using deuterised control standards forming an internal calibrator for accuracy in assay determination in cases where poisoning or industrial exposure is concerned or where other exposure to alcohols is suspected.

15. A method according to claim 1, wherein the individual levels of FAEE's and/or EtG are extracted and analysed some time after death of the individual.

16. An analysis method for determining an alcohol consumption history for individual, the method comprising

a) determining a set of fatty acid ethyl ester (FAEE) markers; b) comparing a level of each respective FAEE marker relative to ethyl glucuronide (EtG) and/or each other in a growth media of a reference host having a known alcohol consumption history at desired intervals over a growth period for the growth media to define an analysis template; and, c) taking a growth media from an individual and determining a level of each respective FAEE marker relative to EtG and/or each other as a result profile and comparing the result profile with the analysis template to provide at least part of a probable alcohol consumption history for the individual.

17. A method as claimed in claim 16 wherein the desired intervals relate to expected growth markers in the growth media for comparison.

18. A method as claimed in claim 17 wherein the growth markers are indicative of time.

19. A method as claimed in any of claims 16 to 18 wherein the method includes monitoring the individual and/or the host for specific rate of growth of the growth media to calibrate the analysis template and/or the result profile relative to each other.

20. A method as claimed in any of claims 16 to 19 wherein the FAEE markers are fatty acid fatty acid ethyl esters as listed above.

21. A method as claimed in any of claims 16 to 20 wherein the growth media is human hair, human skin, human skin secretions or human nail.

22. A method as claimed in any of claims 16 to 21 wherein the level of each FAEE marker is combined in an accumulative value to provide a combined FAEE marker level within the analysis template.

23. A method as claimed in any of claims 16 to 22 wherein the FAEE marker level is defined by a threshold for comparison in the analysis template.

24. A method as claimed in any of claims 16 to 23 wherein the growth media is sectioned to define time periods for comparison between the analysis template and the result profile.

25. A method as claimed in any of claims 16 to 24 wherein the analysis template is an example of an alcohol consumption history for comparison with the result profile.

26. A method for determining an alcohol consumption history substantially as herein before described with reference to the accompanying drawings.

27. Any novel subject matter or combination including novel subject matter disclosed herein, whether or not within the scope of or relating to the same invention as any of the preceding claims.