WO2009054784A1

WO2009054784A1 - Method for assessing previous ethanol intake

Info

Publication number: WO2009054784A1
Application number: PCT/SE2008/051187
Authority: WO
Inventors: Arthur Varga; Staffan Nilsson
Original assignee: Anaxcess Ab
Priority date: 2007-10-23
Filing date: 2008-10-22
Publication date: 2009-04-30
Also published as: SE0702354L; SE531588C2

Abstract

The invention comprises a method for assessing previous intake of ethanol, the main alcohol constituent in alcoholic beverages, from a biological sample. Level of ethanol intake is determined by measuring phosphatidylethanol (Peth), which is formed in cell membranes via the enzyme phospholipase D, only in the presence of ethanol. The present invention mainly relates to an analytical method based on capillary electrophoresis in which Peth is separated from other compounds, enabling detection and quantification. The invention further relates to an analytical method in which non aqueous capillary electrophoresis and stacking is combined, in order to enhance detection and quantification of Peth. The present invention also relates to a method for detecting and/or quantifying ethanol intake by determining a ratio of at least two ethanol metabolites, preferably Peth and Ethyl phosphate.

Description

METHOD FOR ASSESSING PREVIOUS ETHANOL INTAKE

Technical Field

The invention relates to a method by which assessement of alcohol intake from a biological sample is undertaken. More specifically the invention relates to a method for determining previous ethanol intake from a human and/or non-human body sample, e.g. blood sample.

Background of the Invention

Hazardous and harmful alcohol use is a world-wide social and health related problem. According to estimations by WHO, about 76.3 million people in the whole world are suffering from alcohol-related diseases. A causal relationship exists between alcohol intake and more than 60 different types of diseases (WHO).

A significant number of patients in health centres and hospitals are heavy users of alcohol, whose diseases may be alcohol-induced. Since patients do not usually reveal heavy use of alcohol, the immoderate intake has to be recognised in other ways, e.g. through laboratory tests (Hannuksela et al. 2007). The currently used biological state markers of alcohol intake are carbohydrate-deficient transferrin (CDT), γ-glutamyltransferase (GGT) and mean corpuscular volume (MCV). The aforementioned are indirect markers which all have limitations in validity (Hannuksela et al. 2007).

The limitations include i) the time spectrum of detection they reflect, and ii) influences of age, gender and a variety of substances and non-alcohol-associated diseases (Laposata, 1999 and Allen et al. 2003). Direct biological state markers contain ethanol incorporated into the marker itself (e.g. ethanol, ethyl sulfate), and are thus more specific in displaying ethanol intake.

Body mass index (BMI) and medication also influence the expression of said markers, thereby limiting their value when measured. Therefore, a need to provide better tests for this purpose still exists. Phosphatidylethanol (Peth) is an abnormal phospholipid, formed in the body only in the presence of ethanol (Ailing et al., 1983). The enzymatic reaction resulting in the formation of Peth is catalyzed by phospholipase D, which replaces the choline part of phosphatidylcholine with ethanol by transphosphatidylation. The reaction is shown in Figure 1.

Studies have supported use of Peth in blood as a marker of alcohol abuse (for review, see Varga et al., 2001 ). Gunnarsson et al. (1998) and Varga et al. (1998) have described a method wherein Peth is determined directly in blood. In the method, a lipid fraction is isolated from blood and Peth is separated from other compounds with liquid chromatography and detected with mass spectrometry or evaporative light- scattering detection . A daily intake of about 50 g of ethanol for 2-3 weeks (1000 g cumulatively) is needed for Peth to be positive (i.e. above detection limit) in blood, when measured with liquid chromatography and evaporative light-scattering detection.

In two recent studies, each employing a considerable battery of different markers, an extraordinary high specificity for Peth was demonstrated when sober subjects of known alcoholic patients were tested (Wurst et al., 2003 and Wurst et al., 2004)

Gas chromatography with mass spectrometry, requiring derivatisation of Peth, a laborious procedure, has also been employed for Peth analysis (Yon and Han, 2000).

Neither of the above-mentioned methods for assaying Peth is very sensitive in detecting alcohol intake. Furthermore, the large amount of labour and complexity render the methods unsuitable for clinical routine work. This includes withdrawal of several milliliters of venous blood into sampling tubes, a process which has to be carried out by a trained professional.

In US 2004/0203070 an immunoassay method is disclosed. An antibody has been produced against phosphatidylalcohol enabling a considerably simpler assay for detecting heavy use of alcohol. This antibody is however not specific against Peth, since it binds to other phospholipids as well. Sensitivity and specificity may thus be questioned for this immunoassay, as well as the practical use of the assay. Still, a method that fullfills all important demands has not been developed. In the light of aforementioned problems it is thus highly desirable to detect alcohol intake in a valid, sensitive and less laborious way. Thus, new methods for detecting alcohol intake are needed, wich can be used alone or in concert with other methods of detecting alcohol intake. In this respect, the present invention addresses these needs and interests.

Summary of the Invention

In view of the foregoing disadvantages known in the art of detecting previous alcohol intake the present invention provides methods for assessing alcohol intake from a human and/or non-human animal's body sample. The method of the invention is characterized in that Peth is determined from the sample by means of photometric detection, the presence and level of Peth in the sample correlating with the human and/or non-human animal's ethanol intake. Further, a new analytical chemical separation method for Peth is presented. For the first time a capillary electrophoresis (CE) separation method for Peth has been developed.

The main aspect of the invention relates to a method in which Peth is separated and concentrated on-line, named "stacking", by use of capillary electrophoresis with specified chemical solutions, named "separation media".

The present invention thus provides a method for detecting ethanol intake in a human and/or a non-human animal from a biological sample, wherein phosphatidylethanol (Peth) is determined from the sample by means of capillary electrophoresis, the presence or level of Peth in said sample correlating to the accumulated ethanol intake of said human and/or non-human animal.

In one embodiment, the method according to the invention is a method wherein non aqueous capillary electrophoresis together with stacking obtained through lowering ionic strength in the sample, is employed in order to determine Peth. Further embodiments are wherein the presence or level of Peth in said biological sample is determined via a kit or via a microchip.

Another aspect of the present invention provides use of the methods according to the invention to screen and/or monitor the inake of ethanol in a human. Screening and/or monitoring the intake of ethanol in a human using the methods of the present invention may be performed 1 to 7 days, weeks, or even months after ethanol intake.

Still a further aspect of the present invention provides a method for detecting and/or quantifying ethanol intake in a human and/or a non-human animal comprising: obtaining a body sample from said human and/or non-human animal, determining a ratio of at least two ethanol metabolites in said body sample, and correlating said ratio of said at least two ethanol metabolites in said body sample to the ethanol intake of said human and/or non-human animal.

Further embodiments are wherein said at least two ethanol metabolites are Peth and Ethyl phosphate.

Brief Description of the Drawings

Fig. 1 shows formation of phosphatidylethanol (Peth). The enzymatic formation of Peth from phosphatidylcholine by transphosphatidylation via phospholipase D (PLD) is illustrated.

Fig. 2 shows an electropherogram of a Peth analysis. CE analysis of whole blood extract spiked with 10 micromolar of standard Peth is shows. Voltage was 15-20 kV, length of capillary was 20-25 cm, detection window in capillary at 15-17 cm from inlet. Injection of sample was performed hydrodynamically. Detection was performed with ultra violet light apsorption spectrometry (UV detection) at 200 nm. Fig. 3 illustrates the stacking effect in an electropherogram. The peak shapes of Peth standard (16:0/18:1 fatty acid chains) at concentrations 1-10 micromolar are shown. Detection was performed with UV at 200 nm.

Fig. 4 shows an electropherogram of a Peth analysis of a clinical blood sample, here comprising Peth. CE analysis of human whole blood lipid extract is shown in the electropherogram. Voltage was 15-20 kV, length of capillary was 20-25 cm, detection window in capillary was at 15-17 cm from inlet. Injection of sample was performed hydrodynamically. Detection was performed with UV at 200 nm. The concentration of Peth was determined to 3.9 micromolar, equalling 1.6 micromole/liter blood. The Peth peak is further illustrated in the enlarged figure.

Fig. 5 shows the results after an analysis of a clinical blood sample without Peth (zero sample) in an electropherogram. CE analysis of human whole blood lipid extract is shown. Voltage was 15-20 kV, length of capillary was 20-25 cm, detection window in capillary at 15-17 cm from inlet. Injection of sample was performed hydrodynamically. Detection was performed with UV at 200 nm. The sample contains no Peth, also illustrated in the enlarged figure.

Fig. 6 shows an electropherogram of Peth analysis with short capillary. CE analysis of whole blood extract spiked with 34 micromolar of standard Peth. Voltage was 15- 20 kV, length of capillary was 20-31 cm, detection window in capillary at 6-9 cm from inlet. Injection of sample was performed hydrodynamically. Detection was performed with UV detection at 200 nm. Peth elutes after 82 seconds = 1.36 minutes.

Detailed Description of the Invention

Capillary electrophoresis (CE) methodology has now been developed for the separation and determination of Peth. A method based on CE for Peth is not a straightforward solution of a problem, and is unexpected. Peth is an abnormal phospholipid with unordinary chemical properties, and requires special organic solvents to be dissolved. Molecules to be analysed with CE generally require to be charged, which is true for Peth only at higher pH. Ultra-Violet (UV) detection is considered as a relatively poor detection method for phospholipids, since these molecules have limited light absorbing moietys in their structure. The method which has now been developed comprises non-aqueous media, stacking and conventional UV detection. Peth may be determined in e.g. extracts of human blood samples, tissue biopsies or cells.

Separation of Peth from other compounds in the sample may be performed in less than 5 minutes, such as 4, 3, 2, 1 , or even less than 1 minute, as illustrated in Figure 2. The stacking increases the detection sensitivity about 200-fold for Peth, as illustrated in Figure 3. The method facilitates smaller sample volumes, microliters instead of milliliters of blood, and performs about 10 times faster compared to earlier chromatographical methods.

By using shorter capillaries (5-15 cm in length) performance may be even faster.

The described CE method offers sampling of e.g. blood or tissue into capillary instead of sampling into standard vacuum blood sample tubes and sample biopsies of larger sizes.

Advantages of the method of the invention over prior art include highest possible specificity together with high sensitivity, very small sample volumes and improved quickness.

As used herein "Peth" means the phosphatidylethanol molecule in which the two fatty acid moieties may vary, but mainly consist of 16:0 (palmitic acid), 18:0 (stearic acid), 18:1 (oleic acid), 18:2 (linoleic acid) and 20:4 (arachidonic acid).

As used herein "stacking" means if the conductivity of the introduced sample is lower than that of the electrolyte solution, the higher electric field strength and the higher migration velocities of the sample components result in the sample zones being sharpened when entering the electrolyte solution.

As used herein "Microchip" means a piece most commonly made of glass or plastic, with the size of a few cm (length) x a few cm (width) and a few millimeters in height. Also called lab-on-a-chip or μ-TAS (miniaturised total chemical analysis system). The microchip may contain reaction- and mixing chambers, separation channels and detection windows. Such commercial chips are provided by e. g. Micronit Microfluidics BV and Microfluidic ChipShop GmbH.

A method for assessing ethanol intake in a human and/or a non-human animal from a biological sample

A first aspect of the present invention provides a method for detecting ethanol intake in a human and/or a non-human animal from a biological sample, wherein phosphatidylethanol (Peth) is determined from the sample by means of capillary electrophoresis, the presence or level of Peth in said sample correlating to the accumulated ethanol intake of said human and/or non-human animal. Examples of non-human animals are cattle, swine, horse, sheep, dog, cat, rat, mouse, bird and fish.

A correlation between alcohol intake and Peth levels is known from several studies (Hansson et al. 1997, Varga et al. 1998, Gunnarsson et al. 1998, Varga et al. 2000). However, as described above, the lack of sensitivity and laborious demanding methods makes the situation unsatisfactory for an efficient, sensitive and time efficient analysis of alcohol intake. Since the mere presence of Peth correlates to ethanol intake in a human or a non-human animal, the need for a highly sensitive and reliable assay is appearant. A previous study on healthy volunteers revealed that a single dose of ethanol (32-47 g) did not produce measurable amounts of Peth (Varga et al. 1998). However, out of twelve volunteers who had consumed between 624 and 2134 g of ethanol during three weeks, eight persons had detectable levels of Peth (1.0-2.1 micromole/litre). A break point of total ethanol intake yielding detectable Peth seemed to be around 1000g, with a mean daily intake of 5Og. The correlation between alcohol intake and Peth levels is mainly a physiological phenomenon and very much independent of analytical procedures. Peth determined using different methods, e.g. with liquid chromatography (HPLC) or CE, all provides a similar correlation between Peth and alcohol intake, however the sensitivity and labour effort of all methods differ. In a further aspect, the non-aqueous capillary electrophoresis together with stacking obtained through lowering ionic strength in the sample, is employed in order to determine Peth.

In a further aspect, the presence or level of Peth in said biological sample is determined via a kit.

In yet a further aspect, the presence or level of Peth in said biological sample is determined via a microchip.

In further aspects the presence or level of Peth can be determined in said biological sample which has a volume of less than 100 microliters and a weight of less than 100 milligrams, a weight of less than 100 microgram or a volume of less than 100 microliters.

In further aspects the Peth is determined in any biological sample with potential of containing Peth, including blood, tissue or cells.

Thus, according to the first aspect, the invention provides a method for assessing ethanol intake in a human and/or a non-human animal from a biological sample, wherein phosphatidylethanol is determined from the sample by means of capillary electrophoresis, the presence and level of phosphatidylethanol in said sample correlating to the ethanol intake of said human and/or non-human animal.

A capillary electrophoresis (CE) method for Peth has now successfully been developed. It has been unexpectedly found that a capillary electrophoresis method leads to a more sensitive, to about submicromolar, and much faster, to about a few minutes, way of determining Peth in biological samples. The assessement of previous alcohol intake in biological samples by detection and quantification of Peth with the present method, requires pretreatment of samples by extraction, most commonly carried out with organic solvents. Examples of organic solvents for this purpose are hexane, iso-propanol, chloroform, methanol and ethyl acetate, either alone or in a mixture thereof. The extraction is necessary since Peth in biological samples occurs either bound to cell membranes, lipoproteins, other particles or in the form of lipid vesicles and thus needs to be liberated to occur as dissolved free molecules. After extraction, a concentration step of the sample is carried out through evaporation. Capillary electrophoresis (CE) is a family of related techniques that employ narrow- bore (20-200 μm inner diameter, i.d.) capillaries to perform high efficiency separations of both large and small molecules. These separations are facilitated by the use of high voltages, which may generate electroosmotic and electrophoretic flow of buffer solutions and ionic species, respectively, within the capillary (Mikkers et al. 1979, Jorgenson and Lukacs 1981 ). The basic methods encompassed by CE include capillary zone electrophoresis (CZE), capillary electrochromatography (CEC), micellar electrokinetic chromatography (MEKC), isotachophoresis (ITP), capillary isoelectric focusing (CIEF) and capillary gel electrophoresis (CGE). In the invention CZE is applied.

Until recent years surfactants have been used in electrophoresis in order to separate hydrophobic compounds like phospholipids, the technique being called MEKC. A few publications have described the use of non-aqueous capillary electrophoresis (NACE) for separation of phospholipids (Raith et al. 1998, Guo et al. 2005, Gao et al. 2007).

Non aqueous capillary electrophoresis to determine Peth

The present invention provides a method wherein non aqueous capillary electrophoresis together with stacking obtained through lowering ionic strength in the sample, is employed in order to determine Peth.

The capillary is a cylindrical tube of which the length may be between 1 and 100 cm, such as 1 , 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 100 cm, and the inner diameter may be between 1 and 200 micrometer, such as 1 , 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 170, 180, 190, or 200 micrometer. Suitable capillaries are e.g. fused silica capillaries provided by Polymicro Technologies (Phoenix, Arizona, USA) or by other manufacturers/suppliers. The determination of Peth is carried out with UV-detection, but may also be carried out with fluorescence detection. The determination of Peth may also be carried out via an internal or external standard. Detection may be improved by increase of the inner diameter (light path) of the capillary by up to 4 times, resulting in a "bubble cell" at the point of detection.

Another way of extending the light path is by introduction of a bend in the capillary at the point of detection, resulting in a so called Z-cell.

Modern CE systems e. g. provided by Beckman Coulter, Agilent etc. have built-in UV- detectors.

The invention describes for the first time a method where NACE together with stacking is used for separation of a lipid compound. Lower conductivity of the introduced sample is obtained through lower ionic strength in the described invention. Lower conductivity may however also be obtained through a) injection of water-plug into capillary before injection of sample, b) reversed polarity or c) adjustment of pH.

The major advantage of the invention is that charged lipid compounds, like phospholipids and Peth, can be separated electrophoretically and detected at sufficiently low levels (0.05 - 5 μM) with conventional UV-detection. UV-detection is considered as a simple standard detection method with low sensitivity for lipids, (submillimolar concentrations). UV-detection is the most common built-in detector in commercial CE separation systems. Therefore the invention offers immediate use with most commercial units (e. g. Beckman Coulter P/ACE™ or Agilent CE system). In the method, the separation medium comprises an electrolyte which may be ammonium formate, ammonium acetate, ammonium propionate or a mixture of two or more of these alternatives. The separation medium contains no water. The separation medium may comprise other chemical compounds.

In the invention the presence or level of Peth in said biological sample is determined via a kit. The kit comprises a fused silica capillary and separation media. Sample introduction may be carried out hydro-dynamically (pressure), electrokinetically (electroosmotic flow) or by flow-injection.

Separation voltage may vary between 40 V/cm and 5000 V/cm, such as 40, 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, and 5000 V/cm. Stacking in the context of the invention means if the conductivity of the introduced sample, containing Peth, is lower than that of the electrolyte solution. In the invention this is achieved by a lower concentration of the electrolyte (lower ionic strength, typically 1.5 to 4 times lower) in the introduced Peth sample compared to the electrolyte solution. The effect of stacking in the invention improves the detection limit by 150 - 300 times, compared to a similar system without stacking.

The method for determining phosphatidylethanol according to the invention is intended particularly for detecting and monitoring binge drinking and heavy prolonged use of alcohol in a reliable manner, behaviours associated with hazardous and harmful alcohol use.

The method in the following example is optimised particularly in order to measure Peth in human blood samples, but works well for other biological samples as well, e. g. tissues, cells including similar biological samples from non-human animals. The method may be used to measure other compounds than Peth, e. g. non-polar lipids.

A second aspect of the present invention provides screening and/or monitoring the intake of ethanol in a human or a non-human animal via the method of the present invention. Thus, the use of said methods to screen and/or monitor intake of ethanol in a human or a non-human is provided.

In the presence of alcohol in the body, an ethanol molecule incorporates into phospholipids in a reaction catalyzed by phospholipase D wherein ordinarily water incorporates. Peth accumulates in the body as long as ethanol is present. The amount of Peth measured in blood correlates with the total amount of alcohol (ethanol) intake in the previous days-weeks (Varga et al. 1998). The higher the amount of ethanol intake is, the higher the level of Peth gets. The results of an analysis of a human clinical blood sample containing Peth is shown in Figure 4. Analysis of a human clinical blood sample with no Peth is shown in Figure 5. The analysis is performed according to example 1.

Further aspects provides that said method is performed 1 to 7 days, weeks, or months after ethanol intake. Examples are 1 , 2, 3, 4, 5, 6,7 days, 1 , 2, 3, 4, 5, 6, 7, 8 weeks, or 1 , 2 months.

Detectable phosphatidylethanol remains in the body for 1 to 20 days after end of intake, depending on level at start of sobriety. These time frames however depend on the sensitivity of the method, and may thus be changed whenever a more sensitive method is developed. The sensitivity of the present invention is in the range of 0.2- 0.6 micromole Peth per liter of blood.

Even further aspects provide that said method is combined with at least one second method for determining ethanol intake in a human and/or non-human animal. Situations may occur where Peth alone is not sufficient for assessing ethanol intake or at occasions where Peth needs to be confirmed by another method. Analysis of ethyl glucuronide in urine (Stephanson et al. 2002) is an alternative in situations where blood may not be available.

Still even further aspects provide wherein the presence or level of Peth is measured in more than one type of sample and in any type of biological sample.

A method wherein Peth is determined in any biological sample with potential of containing Peth e.g. blood, tissue or cells, or any body fluid that may comprise Peth is provided in a further aspect. This may then be used to display hazardous and harmful drinking. Intake exceeding 24 doses of alcohol in men and 16 doses of alcohol in women per week or, alternatively, more than 7 doses in men and more than 5 doses in women at a single occasion can be considered as heavy use of alcohol. One dose corresponds with about 12 grams of pure ethanol. The method can also be utilized in finding and studying alcohol related diseases. It is estimated that the Peth method may display intake down to 25-40 doses of alcohol. Examples of alcohol related diseases are diabetes, hypertension and cerebrovascular diseases. A sample to be examined may be any biological sample with potential of containing Peth; e.g. blood, tissue or cells. The sample may also be a biopsy, a medico-legal sample, a post mortem sample or a sample taken from tissues of an embryo or a foetus. Indirectly, they may also be used for detecting the activity and location of phospholipase D e.g. from cell or tissue cultures or histological samples. It seems from previous studies on human cadavers, that organs like kidney, lung and spleen contain the highest levels of Peth (Aradottir et al. 2004)

Still further aspects provides for measurements in small volumes.

A method wherein the presence or level of Peth can be determined in said biological sample which has a volume of less than 100 microliters and a weight of less than 100 milligrams. One great advantage of the method is the possibility of analysing very small amounts and volumes of biological samples. Amounts and volumes that are 10- 15 times smaller (15-30 microliters) can be analysed, compared to established methods. Harm and suffering may be reduced in humans and non-human animals at withdrawal of smaller samples. Other advantages are reducing sampling labour and that sampling may be carried out by non-professionals.

Further aspects of the methods according to the invention are where the methods further comprise: correlating the ethanol intake and/or the level of Peth determined in said body sample to a level of Peth detected in a food, food-like substance, beverage or medication injested by said human and/or non-human animal prior to or at the time of obtaining said body sample.

It is known that Peth can be used as drug-carrier for specific drugs. Liposomes with Peth may e. g. be used for oral delivery of insulin (Kisel et al. 2001 ).

A third aspect of the present invention provides a method for detecting and/or quantifying ethanol intake in a human and/or a non-human animal comprising: obtaining a body sample from said human and/or non-human animal, determining a ratio of at least two ethanol metabolites in said body sample, and correlating said ratio of said at least two ethanol metabolites in said body sample to the ethanol intake of said human and/or non-human animal. Ethyl phosphate is a potential metabolite when Peth is degraded in vivo and may thus be directly coupled to Peth. The present invention is directed at detecting and/or quantifying the intake of ethanol over a wide time span, including days and weeks after intake. It is understood that at any point in time during this time span such a detection and/or quantification can be performed. Ethyl phosphate can be analysed with a liquid chromatography-tandem mass spectrometry based method (Bicker et al. 2006). A determined ratio of Peth and ethyl phosphate may give additional information about the ethanol intake pattern history.

The marker Peth may soon come to have important implication in fields of public health and public safety as a more objective method for confirming alcohol-related diseases, preventing addiction development and preventing traffic accidents and violent behaviour. The use of this marker either alone or together with other biological state markers like serum ethanol, ethyl glucuronide and ethyl phosphate, is expected to lead to significant improvement in treatment outcome, therapy effectiveness, and health, social and socio-economic benefits that will be hard to overestimate.

Thus, further aspects of the present invention provides the use of Peth- measurements using the provided methods herein either alone or together with other biological state markers like serum ethanol, ethyl glucuronide and ethyl phosphate.

Different biological state markers of alcohol intake reflect different time spectrums and levels of intake. By combining analysis of two or more markers, more descriptive information of intake history is revealed. Ethanol itself remains in body up to 18 h after intake, ethyl glucuronide remains in urine up to 80 h after intake, Peth in blood up to several weeks and GGT up to several months. The sensitivities are in the range of 0-100% and the specificities are in the range of 11-100% for these markers.

Further aspects provides wherein said at least two ethanol metabolites are Peth and Ethyl phosphate. A fourt object of the present invention is to provide a kit for detecting ethanol intake ub a human and/or a non-human animal from a biological samle.

The kit comprises at least one capillary, optionally at least one separation medium, examples herein, and optionally at least one extental or internal standard as commonly used in the art. The capillary is for measuring Peth by means of capillary electrophoreses. Examples of suitable capillars are given herein.

Further embodiments are wherein the capillary is for non-aqueous capillary electrophoresis as disclosed herein.

The kit may further comprise instructions, e.g. written on a leaflet or on a computer redable medium such as e.g. a compact disc, CD, wherein the instructions refer to the methods of the present invention as disclosed herein.

The following non-limiting examples below will further describe the invention. The results are given in short here below.

Example 1

CHEMICALS

Peth used as standard was from MP Biomedicals (OH, USA). Hexane was from Sigma-Aldrich (Steinheim, Germany). Methanol was from BDH Laboratory Supplies (Poole, England) and iso-propanol was from Labscan (Dublin, Ireland). Acetonitrile, ammonium acetate and sodium hydroxide were from Merck (Darmstadt, Germany). Deionized, sterile-filtered water was from an ELGA® Maxima ultrapure water system.

BLOOD SAMPLING

Either capillary or venous human blood is collected into a blood sampling tube containing anticoagulant. PREPARATION OF BLOOD SAMPLES

For human blood samples one volume of blood was added under agitation to 33 volumes of hexane : 2-propanol (3:2 v/v) and was further agitated for 5 s. Centrifugation of the mixture was done at 1500 x g for 10 min, and the supernatant was collected for further analysis.

Peth standard solution and human blood extracts were evaporated under nitrogen until dryness and dissolved in separation medium prior to analysis.

INSTRUMENTAL EQUIPMENT

NACE separations were performed on a capillary electrophoresis system equipped with UV detector. A fused silica capillary of 75 μm inner diameter, 375 μm outer diameter with a total length of 24 cm (17.2 cm to detector) was used.

SEPARATION CONDITIONS

Fourty to sixty percent of acetonitrile and 26 - 40 % of 2-propanol with 60 - 90 mM ammonium acetate as electrolyte, and with 5 - 15 % of hexane and 2 - 10 % of methanol as additional modifiers, is used as separation medium. Note: no water is included, thus NACE. Samples are dissolved in the separation medium, with the exception that 30 - 50 mM of ammonium acetate is used instead. Note: this reduction of electrolyte, lowering the ionic strength, causes sample stacking. Samples were introduced hydro-dynamically at 34 mbar for 2 - 4 s, but may also be introduced electrokinetically. Injection volume is 3 - 8 nL. UV detection was carried out at 200 nm. Separation voltage is 10 - 20 kV. Separations were run towards negative pole. The capillary temperature was set to 25°C in runs. Prior to daily use the capillary was successively rinsed with > 100 volumes of 0.5 M sodium hydroxide, water, methanol and separation medium respectively. Between runs the capillary was rinsed with 8 volumes of separation medium. All solutions were degassed in an ultra-sound water bath.

RESULTS AND CONCLUSIONS

An earlier established LC-based method, HPLC with ELSD detection, expressed an

LOD of 2 μM Peth (Varga et a!. 1998). In the NACE method the LOD was slightly better, 1 μM of Peth, by applying stacking and conventional UV-detection (Figure 3). This LOD level further improves the ability to display heavy drinking compared to earlier methods. The very small amount (about 8 fmol) of Peth and the injected volume of only about 5 nL, however, offers miniaturization of this NACE method. Theoretically, only 10 nL of blood is required to display Peth with the analysis. However the practical handling of blood sampling with such small volumes is not in common use at clinical laboratories today. Nevertheless, blood sampling with capillary from fingertip, instead of venous needlemonitored sampling, required for the HPLC-ELSD analysis, is conceivable. The total analysis time was 5 min per sample run (Figure 2, 4, 5), which is clearly faster compared to 60 min per run required for HPLC (Varga et al. 1998). In addition the NACE analysis consumes several orders of magnitude less organic solvents compared to HPLC, thus reducing the pressure on environment from organic waste.

The NACE method of Peth analysis offers an important additional tool to assess ethanol intake. The saves in analysis time and sample volume compared to earlier LC methods (Hansson et al. 1997, Varga et al. 1998), makes it an interesting new alternative. The present study further manifests Peth as an accurate and clinically practicable marker of hazardous and harmful alcohol consumption.

Example 2

Peth is measured with the method described in example 1 . Ethyl phosphate is measured with the method described by Bicker et al. 2006. A ratio of Peth and ethyl phosphate is determined. The ratio is obtained either by dividing concentration of Peth with concentration of ethyl phosphate or by vice versa. The ratio may provide additional information of alcohol intake, e. g. time spectrum and intake pattern.

Example 3

A microchip method

A method wherein the presence or level of Peth in said biological sample is determined via a microchip. The described method can be down-scaled into microchip format. This means a down-scaling of processes, particularly the separation of Peth into a device in which the length of the separation channel is < 5 cm (1 , 2, 3, 4 cm) and the inner width of this channel is < 50 micrometer (5, 10, 15, 20, 25, 30, 40). The example illustrated in Figure 6 shows separation of Peth from other blood lipids after migration in 6-9 cm of capillary.

Results and conclusions

Analysis of a whole blood extract spiked with 34 micromolar of standard Peth was carried out with NACE in a fused silica capillary of 75 μm inner diameter. The detection window in the capillary was at 6-9 cm from inlet. Peth was baseline separated and eluted already after 82 seconds = 1.36 minutes (Figure 6). A separation and detection of Peth after migration in 6-9 cm of capillary resembles the small scales commonly used in microchips. It is evident that Peth analysis may be carried out in capillaries with less than 50 μm inner diameter and less than 5 cm from inlet to detection window, dimensions that indeed are in the scale of microchips.

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Claims

1. A method for detecting ethanol intake in a human and/or a non-human animal from a biological sample, wherein phosphatidylethanol (Peth) is determined from the sample by means of capillary electrophoresis, the presence or level of Peth in said sample correlating to the accumulated ethanol intake of said human and/or non- human animal.

2. The method as claimed in claim 1 , wherein non aqueous capillary electrophoresis together with stacking obtained through lowering ionic strength in the sample, is employed in order to determine Peth.

3. The method of claim 1 , wherein the presence or level of Peth in said biological sample is determined via a kit.

4. The method of claim 1 , wherein the presence or level of Peth in said biological sample is determined via a microchip.

5. The method of claim 1 , wherein the presence or level of Peth can be determined in said biological sample which has a volume of less than 100 microliters and a weight of less than 100 milligrams.

6. A method as claimed in claim 1 , wherein the Peth is determined in any biological sample with potential of containing Peth, including blood, tissue or cells.

7. Screening and/or monitoring the intake of ethanol in a human via the method of claim 1.

8. The method of claim 1 , wherein said method is performed 1 to 7 days, weeks, or months after ethanol intake.

9. The method of claim 1 , wherein said method is combined with at least one second method for determining ethanol intake in a human and/or non-human animal.

10. The method of claim 1 , wherein the presence or level of Peth is measured in more than one type of sample.

11. A method for detecting and/or quantifying ethanol intake in a human and/or a non-human animal comprising: obtaining a body sample from said human and/or non-human animal, determining a ratio of at least two ethanol metabolites in said body sample, and correlating said ratio of said at least two ethanol metabolites in said body sample to the ethanol intake of said human and/or non-human animal.

12. The method of claim 10, wherein said at least two ethanol metabolites are Peth and Ethyl phosphate.

13. The method of claim 1 further comprising: correlating the ethanol intake and/or the level of Peth determined in said body sample to a level of Peth detected in a food, food-like substance, beverage or medication injested by said human and/or non-human animal prior to or at the time of obtaining said body sample.

14. A kit for detecting ethanol intake in a human and/or non-human a non-human animal from a biological sample, wherein the kit comprises at least one capillary for measuring Peth by means of capillary electrophoreses.