Lipid-metabolome-related biomarkers for adhd

Abstract

The amount of certain metabolomic markers from the lipid metabolism can be used for the highly specific and sensitive in-vitro diagnosis of ADHD.

Description

[0001] Lipid-metabolome-related biomarkers for ADHD

[0002] The present invention relates to an in-vitro method for assessing whether a person has ADHD or is at risk of developing ADHD, a method of selecting a patient fora therapy of ADHD, an in-vitro method of monitoring a patient suffering from ADHD or being treated for ADHD, a computer-implemented method assessing whether a person has ADHD or is at risk of developing ADHD, an in-vitro method of determining an ADHD biomarker in a blood sample from a person and the use of mass spectroscopy for the in-vitro determination of an ADHD biomarker in a dried blood sample of a person as well as the use of a biomarker for diagnosing ADHD in a blood sample.

[0003] Background of the Invention

[0004] Attention Deficit Hyperactivity Disorder (ADHD) is a very serious disease. ADHD causes significantly poorer school performance and leads to impairments in social, academic, and work environments in adulthood. In Germany, around 4 million persons are affected, including around 600,000 children aged 3 to 17 years. The prevalence (number of cases) of attention deficit hyperactivity disorder (ADHD) in childhood is 5 - 7%. Persistence with functional impairment into adulthood is common and the prevalence in adults is 2.8 - 4% worldwide. However, it can be assumed that the number of adults affected is higher, since ADHD is often not diagnosed in adulthood (Rivas-Vazquez, Diaz et al. 2023). If left untreated or treated ineffectively, ADHD increases the risk of drug abuse, antisocial behavior, divorce, accidents and increases the risk of death. Despite these often-serious consequences, the disease remains undetected in up to 80% of affected adults.

[0005] Today, the diagnosis of ADHD is carried out according to ICD-10 (WHO, 1992) and Diagnostic and Statistical Manual of Mental Disorders-5 (DSM-5) (American Psychiatric 2022) These include routine diagnostics based on self-history, family history, third-party history, physical and neurological diagnosis, and motoscopic / motometric development status.

[0006] The diagnosis for ADHD is therefore complex, is based on subjective criteria, is often questionnaire-based and does not reliably differentiate between normal variation and a disease that “requires treatment”. However, the most precise diagnostics possible are a prerequisite to avoid overdiagnosis and misdiagnosis, as otherwise limited resources are misallocated. As a result of these personnel-intensive diagnostics, there is a lack of capacity, which leads to long search and waiting times. To make matters worse, public attention to ADHD leads to a more frequent suspected diagnosis and thus to an increased demand for ADHD diagnostics.

[0007] Since the capacity for the initial diagnosis in Germany is too low, diagnostics accompanying therapy to check the response to the various therapy options and, if necessary, stratify them is another bottleneck.

[0008] Transcriptome-associated studies (TWAS) found a highly relevant association of dopaminergic and noradrenaline pathways with the ADHD phenotype. Among other findings, the genes for dopaminergic (dopamine transporter genes, dopamine p-hydroxylase, dopamine D4 receptor) and catecholamine pathways (catechol-O-methyltransferase (COMT), monoamine oxidases MAOA and MAOB, tryptophan hydroxylases) are altered (Bonvicini C, Faraone SV, Scassellati C. 2016). Attention-deficit hyperactivity disorder in adults: a systematic review and meta-analysis of genetic, pharmacogenetic and biochemical studies (Mol Psychiatry 21 : 1643). Other metabolic pathways are affected by mutations (for an overview Faraone and Larsson, Molec. Psychiatry. 2019, 24, 562-75; Brikell et al., Psychol. Med. 2021 51 , 2274- 2286).

[0009] Various meta-analyses and review articles, e.g. Scassellati et al. (J. Am. Acad. Child Adolesc. Psychiatry. 2012; 51 :1003-1019) and Takahashi et al. (J. Psychiatr. Res. 2021 , 137, 465-470), critically summarized which metabolites / biomarkers could be helpful in the diagnosis of ADHD. These were noradrenaline, 3- methoxy-4-hydroxyphenylethylene glycol [MHPG], monoamine oxidase [MAO], zinc and cortisol, as well as increased arginine concentration in the blood. Furthermore, sphingomyelins were significantly reduced in the ADHD cohort (Henriquez-Henriquez MP, Solari S, Quiroga T, Kim Bl, Deckelbaum RJ, Worgall TS: Front. Neurosci. 9: 300,2015: Low serum sphingolipids in children with attention deficit-hyperactivity disorder).

[0010] Due to the polygenetic structure with 76 ADHD-associated genes, human genetic tests are currently of little help in the diagnosis of ADHD (Nat Genet 55: 198-208 and Tomasik J, Harrison SJ, Rustogi N, Olmert T, Barton-Owen G, et al. 2024. Metabolomic Biomarker Signatures for Bipolar and Unipolar Depression. JAMA Psychiatry 81 : 101-06). Polygenic risk scores (PRS) have been used to predict symptom severity and comorbidities (accompanying diseases) of ADHD as well as to predict the persistence of symptoms into adulthood. As an example, WO2018 / 195184 proposes the nucleic acid-based determination of a copy number variation (CNV) in a subset of mGluR network genes. However, a substantial contribution of genetics to the diagnosis of ADHD in a large number of individuals is not realistic in the foreseeable future. Apart from the lack of sequencing capacity, interpreting the data is complex and not very precise for the affected individual. In order for an existing set of ADHD risk gene variants to also cause ADHD symptoms, usually "negative" environmental influences are required to be considered.

[0011] For the determination of Bipolar Disorder (BD) Tomasik et al. (JAMA Psychiatry 2024, 81 (1), 101-106) describe a method based on metabolomic profiling of a dried blood sample (DBS) using mass spectrometry for bipolar disorder. However, even using a panel of several biomarkers in combination, this method shows a low specificity of results.

[0012] Boretti et al. describe the effect of different diets fed to dogs based on the alteration of certain phospholipids, sphingolipids and diacylglycerols (Metabolomics (2020) 16:1). Giilec et al. speculate on the relationship between sphingomyelin and ceramide levels as determined by ELISA on soft neurological signs in ADHD (J Neur. Transmission (2025) 132:157).

[0013] The invention described herein is useful for the assessment of the health status of a patient regarding Attention Deficit Hyperactivity Disorder (ADHD) in diagnostics. Particularly, the markers found to be indicative of ADHD show high specificity and sensitivity for ADHD in humans.

[0014] Short description of the figures

[0015] FIG. 1 A) shows an ROC Curve of marker SPB d17: 1

[0016] FIG. 1 B) shows an average of predicted class probabilities of each sample across the 100 cross- validations. FIG. 2. shows an ROC Curve of a combination of markers SPB d17:1 and SPB d17:0.

[0017] FIG. 3 shows an ROC Curve of a combination of markers SPB d17:1 , SPB d17:0 and C3-DC (C4-OH).

[0018] FIG. 4 shows an ROC Curve of a combination of markers SPB d17:1 , SPB d17:0 and C16:2.

[0019] FIG. 5 shows an ROC Curve of a combination of markers SPB d17:1 , SPB d17:0 and C16:2 OH.

[0020] FIG. 6 shows an ROC Curve of a combination of markers SPB d17:1 , SPB d17:0 and PC O 44:4.

[0021] FIG. 7 shows an ROC Curve of SPB d17:1 , PC 0-44:4, C16:2, SPHK activity (3), LPP3 activity (3), C3- DC (C4-OH), PE P-18:0 / 17:1 , LDH Activity and PC 0-36:4.

[0022] FIG. 8 shows an ROC Curve of markers SPB d17:1 , PC 0-44:4, SPB d17:0, LPP3 activity, PE P- 18:0 / 17:1 , C3-DC (C4-OH), PC 0-38:0 and PC 0-36:4.

[0023] FIG. 9 shows an ROC Curve of markers SPB d17:1 , PC 0-44:4, SPB d17:0 and PE P-18:0 / 17:1 .

[0024] FIG. 10 shows an ROC Curve of markers PA 18:0_18:1 , PC 0-36:4, PC 0-38:0 and TG 14:0_36:4.

[0025] FIG. 11 shows an ROC Curve of markers SPB d17:1 , C3-DC (C4-OH), PA 18:0_18:1 and C16:2.

[0026] Object of the invention

[0027] Hence, there is a need for a highly specific and sensitive in-vitro method for assessing whether a person has ADHD. In other aspects, it should avoid any of the above difficulties, particularly can be easily applied to small amounts of sample collected in the field either in patient self-care or in routine diagnostics. There is also a need for downstream uses and applications of such in-vitro method desperately waited for by persons suffering from ADHD.

[0028] This object is achieved by the present invention in its various aspects. In particular, single markers and combinations are provided that enable more specific and sensitive assessment of ADHD.

[0029] Summary of the invention

[0030] The invention has found with high confidence that certain low molecular weight compounds and enzymatic activities in samples of blood or samples derived from blood are indicative as markers of ADHD. This leads to various beneficial methods and applications using this discovery.

[0031] The first aspect of the invention is directed to an in-vitro method of assessing whether a person has ADHD or is at risk of developing ADHD, comprising

[0032] - determining in a sample from the person the amount, concentration or enzymatic activity of an analyte selected from a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d 17: 1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3- DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or b) a combination of any of the markers under a) and

[0033] - comparing the determined amount, concentration or enzymatic activity to a reference. In a second aspect the invention is directed to a method of confirming that a patient has ADHD, comprising the assessment method above.

[0034] In a third aspect, the invention is directed to a method of selecting a patient for a therapy of ADHD, comprising

[0035] 1) determining in a sample from the person the amount, concentration or enzymatic activity of an analyte selected from a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d 17: 1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3- DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or b) a combination of any of the markers under a),

[0036] 2) comparing the determined amount, concentration or enzymatic activity to a reference, and

[0037] 3) marking the sample for therapy, if the sample differs in amount, concentration or enzymatic activity from the reference by more than 50 %.

[0038] In a fourth aspect, the invention is directed to an in-vitro method of monitoring a patient suffering from ADHD or being treated for ADHD, comprising determining in a sample from the person the amount, concentration or enzymatic activity of an analyte selected from a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d 17: 1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3- DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or b) a combination of any of the markers under a), and comparing the determined amount, concentration or enzymatic activity to a reference.

[0039] In a fifth aspect, the invention is directed to a computer-implemented method assessing whether a person has ADHD or is at risk of developing ADHD, said method comprising

[0040] 1) obtaining a measurement value for the level of an analyte selected from a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d 17: 1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3- DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, b) a combination of any of the markers under a),

[0041] 2) storing said measurement value in a data base in one or more computer or data base,

[0042] 3) comparing said value to a minimum or a maximum threshold value for the level of said marker or said combination stored in said data base, and

[0043] 4) outputting a positive signal in case said measurement value exceeds said minimum threshold value or undercuts that maximum threshold value, wherein said positive signal is indicative of the person having ADHD.

[0044] In a sixth aspect, the invention is directed to an in-vitro method of determining in a sample from the person the amount, concentration or enzymatic activity of an analyte selected from a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d 17: 1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3- DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or b) a combination of any of the markers under a), wherein the sample is provided from a dried blood spot into a mass spectrometer, and a measurement value for the signal strength of the mass of said marker or the result of said enzymatic activity of said analyte is obtained by mass spectrometry.

[0045] In a seventh aspect, the invention is directed to the use of mass spectroscopy for the in-vitro determination of the amount or concentration or enzymatic activity of any metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d17:1 , SPB d18:0, DG 18:0_20:0, PE P- 20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3-DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity or a combination of any of these markers in a sample from dried blood of a person for the assessment of ADHD.

[0046] In an eighth aspect, the invention is directed to the use of any metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d17:1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3-DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or a combination of any of these markers for the determination of ADHD in a human person.

[0047] In a nineth aspect, the invention is directed to a method for determining from which metabolomic subgroup of ADHD a patient is suffering comprising determining in a sample from the person the amount, concentration or enzymatic activity of an analyte selected from a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d 17: 1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3- DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or b) a combination of any of the markers under a), wherein the amount, concentration or enzymatic activity of an analyte selected from a1) a metabolomic marker selected from the group consisting of SPB d17:1 , SPB d17:0, C16:2, PC O- 44:4, C3-DC (C4-OH), LDH Activity and SPHK activity, b1) a combination of any of the markers under a1) is indicative of a first metabolomic ADHD group and wherein the amount, concentration or enzymatic activity of an analyte selected from a2) a metabolomic marker selected from the group consisting of PA 18:0_18:1 , PC 0-36:4, PC 0-38:0 and G 14:0_36:4, b2) a combination of any of the markers under a2) is indicative of a second metabolomic ADHD group.

[0048] In a tenth aspect, the invention is directed to a method for determining the treatment scheme of ADHD for a patient comprising

[0049] - determining in a sample from the person the amount, concentration or enzymatic activity of an analyte selected from a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d 17: 1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3- DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or b) a combination of any of the markers under a),

[0050] - comparing the determined amount, concentration or enzymatic activity to a reference, and

[0051] - selecting for said patient a treatment scheme from the group of available treatment schemes based upon said comparison.

[0052] In an eleventh aspect, the invention is directed to a method for determining the amount, concentration or enzymatic activity of any metabolomic marker selected from the group consisting of SPBP d17:0, PE P- 18:0 / 17:1 , SPB d17:1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3-DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P- 16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity or a combination of any of the markers in a sample from a human person, wherein said amount, concentration or enzymatic activity is determined by a method selected from the group consisting of LC-MS, Immunoassay and Enzyme Activity Assay for the diagnosis of ADHD.

[0053] In a twelfth aspect, the invention is directed to a method for determining whether a person has ADHD or is at risk of developing ADHD comprising assessing ADHD or the risk of developing ADHD by a psychiatric method and assessing ADHD or the risk of developing ADHD by an in-vitro method using a sample from the person, wherein the determination is positive, if the assessments in both steps are positive regarding ADHD.

[0054] In a thirteenth aspect, the invention is directed to an in-vitro method for screening a multiplicity of samples for persons having ADHD or the risk of developing ADHD, wherein each sample of said multiplicity of samples is subjected to determining the amount, concentration or enzymatic activity of an analyte selected from a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d 17: 1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3- DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity or b) a combination of any of the markers under a), and comparing the determined amounts, concentrations or enzymatic activities to a reference, wherein any differences from the results obtained to a reference value are indicative of ADHD or the risk of developing ADHD for the respective persons the samples showing differences were collected from.

[0055] In a fourteenth aspect, the invention is directed to the use of mass spectroscopy for the assessment of whether a person has ADHD or is at risk of developing ADHD based on a dried blood sample from said person.

[0056] In a fifteenth aspect, the invention is directed to the use of mass spectroscopy for the in-vitro determination of a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d17: 1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3- DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or b) a combination of any of the markers under a) in the assessment of ADHD in a sample from dried blood of a human person suspected to have ADHD.

[0057] In a sixteenth aspect, the invention is directed to the use of a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d17: 1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3- DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or b) a combination of any of the markers under a) for the determination of ADHD.

[0058] Detailed description of the invention

[0059] Terms and definitions

[0060] The below terms and definitions apply to all aspects of the present invention, if not mentioned otherwise.

[0061] An in-vitro method is a method performed on a sample outside of the living body, as compared to an in- vivo method, performed in or on a living body. Any in-vitro method therefore must be performed after taking a sample from the living body. In order to prepare the sample ready for the following determination, the sample may be subjected to a variety of sample preparation steps, dependent upon the kind of sample and the nature of the method of determination. In the present invention, an in-vitro method is not limited to, but includes research methods as well as diagnostic methods underlying regulated in-vitro methods.

[0062] In the present invention, a variety of samples can be used. However, the invention is particularly indicative of ADHD, if human blood or samples derived from human blood are used. The blood is taken from any person forwhich the determination of ADHD is intended. This can be a person which has not obtained an earlier diagnosis of ADHD (this is also useful in methods for screening a multiplicity of samples for hitherto unknown occurrence of ADHD in persons), or a patient which has been diagnosed before, e.g., by a psychiatric method (this is useful for confirming ADHD) or by an in-vitro method, such as a method according to the invention (this is useful for monitoring progression or reduction of ADHD, e.g. during therapeutic treatment).

[0063] The present invention can be applied to any human blood sample, but the invention is particularly useful for whole blood samples, particularly dried whole blood samples (DBS), or EDTA-blood samples, or dried EDTA-blood samples. This is very helpful, as this allows the samples to be transported in reasonable time to a laboratory for routine and large-scale testing with less risk of decomposition. Within such laboratories, these samples can be subjected to well-known methodologies, such as sample preparation and diagnostic methods, like mass spectrometry, immunoassays or assays for measuring enzymatic activities. The use of dried blood samples is particularly useful, as most of the markers used in the invention are not stable in original whole blood for longer periods of time and the method of the invention is relying on determining the amount, concentration or enzymatic activity of the markers, as opposed to the mere presence of any compounds as in other methods. The present invention includes methods wherein the original sample (e.g. from dried blood, EDTA-blood or dried EDTA-blood) is subjected to preparation steps, such as separation of the analytes from other material, such as cells or compounds not intended to be determined, prior to the actual determination of the analytes, to obtain the sample to be analyzed.

[0064] Diagnostic methods for the determination of compounds to be determined (in the following also called analytes) are well known. In immunoassays, the sample as prepared is contacted with reagents, at least one of which has an affinity to the analyte, e.g. an antibody directed to the analyte, and can thus bind to the analyte. This or another reagent is labelled, so any binding to the analyte can be measured and taken as an indication for the presence or amount of analyte present. In enzymatic assays, the prepared sample is contacted with a substrate for the enzymatic activity. In case the enzymatic activity is present in the sample, the substrate undergoes a chemical reaction, e.g. a cleavage reaction, which results in a color development or change. This reaction is measured using the color change and taken as an indication of the presence of the enzymatic activity or the analyte to be determined. In Mass Spectrometry (MS), the most beneficial in-vitro diagnostic method according to the invention, the prepared sample is subjected to an energy field to create a flying beam of molecules, including the analyte(s) or any fragments thereof. The analyte(s) or / and any fragments are separated according to their respective masses and the occurrences of masses characteristic for the analyte(s) to be determined, such as mass peaks, are measured and taken as an indication of the presence of the analyte in the sample. Enzymatic activity can for example be measured by mass spectrometry via the occurrence of a mass resulting from the specific enzymatic reaction catalyzed by the enzyme, e.g. a result of a cleavage of a substrate by the enzymatic activity.

[0065] In useful aspects of the present invention, the combination of chromatographic separation followed by mass spectroscopy has proven very helpful, as then the determination is more specific than other methods. In this method, the prepared sample is subjected to Liquid Chromatography (LC) in order to separate and / or concentrate any analyte to be determined from the other constituents of the sample that may interfere with the determination or may overcrowd the sample. A fraction resulting from the chromatographic separation, which should contain the analyte, if the analyte is present in the sample, is subjected to removal of any solvents used in the chromatographic step and the sample is then introduced into a mass spectrometer to determine the analyte. In the spectrometer, the fraction is subjected to an electric field and molecules and fragments are separated according to their mass and electric charge. The intensity of the mass peaks is a relative, but quantitative indication of the amount or concentration of the analyte. The combined method using liquid chromatography and mass spectrometry is called LC-MS. Instruments for LC-MS are commercially available for research and diagnostics.

[0066] An in-vitro method of assessing whether a person has ADHD or is at risk of developing ADHD can be used to assist a physician in concluding the determination of the health status of a person regarding ADHD and decide on a potential therapeutic treatment of the person. Thus, the physician may send a sample from a particular person to a laboratory for performing the in-vitro method according to the invention. The physician may use the result from the assessment to compare it to the psychiatric diagnosis and confirm the diagnosis or may compare the result to the result of an earlier assessment to conclude on the progression or reduction of ADHD, e.g. as result of the therapeutic treatment. The method according to the invention allows for the first-time large-scale testing of persons, as used in screening methods for samples obtained from a multiplicity of persons with known or unknown ADHD status. This is very useful, as a large number of individuals having by now unrecognized ADHD can now be motivated to obtain treatment.

[0067] A person is any human being irrespective of health and personal status. A patient in the context of the present invention is a person which is under surveillance by a physician for diagnosis or treatment of ADHD or shows unclear symptoms or received a diagnosis in which ADHD must differentially be included or excluded. The present invention has been proven to be reliable for adult humans. In samples from adolescents and children other thresholds or combinations of said markers might be useful.

[0068] While the methods of the invention are perfectly suitable to assess the ADHD status of person already showing psychiatric symptoms of ADHD, it may also be applicable for assessing the status of persons already showing a metabolomic status of ADHD, but not yet showing psychiatric symptoms (but bearing the risk to develop ADHD.

[0069] An assessment regarding ADHD or the risk to develop ADHD is based on a certain sensitivity and specificity. On the first hand, an assessment is dependent upon the quality of the sample used. Depending on the sensitivity of the method used for analyzing large volume samples, e.g. 10 ml, may seem to bear a smaller risk of wrong negative results than small volume samples. However, given the sensitivity of LC-MS, a single drop of blood, as can be used in the present invention, is highly sensitive. On the other hand, the heterogen icity of the disease ADHD and the unknown specificity and sensitivity of the gold standard diagnostic method (American Psychiatric, Diagnostic and statistical manual of mental disorders: DSM-5-TR: 2022 text revision. Washington, DC: American Psychiatric Association Publishing. Ixix, 1050 pages pp.) limits the reliability of the results. Despite these challenges the present invention allows assessing small volume samples with high specificity and sensitivity.

[0070] In-vitro methods according to the invention are performed on diagnostic instruments which provide the result as a measurement value, based on a signal measured. The signal may be measured as a light absorption, a light extinction, an electric impulse or a light flash. The measurement value derived from the intensity of the signal usually is an indication for the presence or amount of the compound to be determined by the method. This value therefore can be used for qualitative (presence or absence) or quantitative (amount) determination. In mass spectrometry the value measured is the signal strength obtained for the mass peak of a particular compound or a fragment thereof.

[0071] In order to assess the significance of the measurement value, the value is compared to a reference value. The reference value can be any value that is indicative of the result of the assessment, i.e. whether a person has ADHD or the risk of developing ADHD or not and may be taken from earlier assessments of the same or different kind. For example, using the method according to the invention, the actual measurement value of a person subject to the assessment is compared to corresponding measurement values for persons having been positively or negatively diagnosed by other methods (such a clear psychiatric indicia or a method according to the invention) as reference value. If the results are more similar to the values for positive reference diagnosis than for negative diagnosis, the assessment can conclude that the result of the actual assessment is positive, and vice versa. The reference value can also be a threshold value. If the threshold value is indicative of a minimum value for positive assessment of ADHD, any measurement value exceeding the threshold is indicative of a positive result on ADHD. In case of reduction or lack of measurement values in positively diagnosed ADHD, the threshold value may be a value lower than the reference in persons unaffected by ADHD, i.e. a maximum value for positive assessment.

[0072] The principles described above for an amount of an analyte in the sample are applicable to determining concentrations, based on the volume of the sample taken from the person and taking into consideration any dilution or concentration during sample preparation. Quantitative determination can also be achieved using artificial samples having a defined content (amount or concentration, i.e. a standard) of the analyte and performing a relative determination versus such standard. In this case, simple calculations can be applied.

[0073] An enzyme is characterized in that it has an enzymatic activity which catalyses chemical reactions yielding compounds that can be determined as a measure of the presence or quantity of the enzymatic activity and therefore the enzyme. Enzymatic activities having proved useful as an indication of ADHD via the present invention are L-Lactatdehydrogenase, Sphingosine Kinase and Lipid Phosphate Phosphatase 3.

[0074] The markers as used in the invention are part of the so-called metabolome. While there are different definitions of the term metabolome in the present literature, the present invention uses the term to cover any compound that is a product of metabolic processes in the human body. As the metabolism of each individual is individual, dependent upon the status of the individual, including age, sex, health status and genomic disposition, the metabolomic profile of each human individual is different from other individuals. Therefore, the metabolome of any human individual can be individually characterized by its metabolomic profile, i.e. the amount and concentration of constituents of its metabolome, relative to the other constituents of the metabolome or other constituents derived from the human body. This is why the present invention uses a comparison to a reference, i.e. a value for an analyte not affected by the ADHDpositive status.

[0075] A marker is generally defined as a measurable indicator of some biological state or condition. The present invention has found that persons having ADHD show a particular metabolomic profile characterized by the amount or concentration of certain markers selected from a subset of (general) markers, i.e. a group consisting of particular compounds involved in the lipid metabolism, called metabolomic markers. The particular metabolomic markers as determined in the present invention can be used in the methods of the invention.

[0076] The following list 1 is a list of the group of particular metabolomic markers as presently determined by the invention:

[0077] List 1

[0078] *AUC is obtained in a multivariate explorative ROC-analysis when a subsample of ADHD2 is compared to control patients

[0079] **The mass peaks of these compounds are so close to each other that they can hardly be distinguished.

[0080] Column 1 of List 1 shows the group (“ADHD1” or “ADHD2”, respectively) the biomarker has a preference for and thus is indicative for or whether the biomarker is not selective for a group (“ADHD” in general).

[0081] Column 2 lists the markers as used. The abbreviations of the markers follow the nomenclature according to the LIPID MAPS classification system as published in Liebisch et al. in J. Lipid Res. (2020) 61 (12) 1539-1555 and Journal of Lipid Research Volume 54, 2013 1523 as well as on the LIPID MAPS homepage https: / / www.lipidmaps.org / resources / education / classification, in the Human Metabolome Database (https: / / hmdb.ca / ) and / or the Metabolite Database in the Metabolomics Workbench (http: / / metabolomicsworkbench.org / databases / RefMet). It can be taken from these publications that the peaks in mass spectrometry in some cases can be the result of fragmentation of different markers leading to the same mass. In this case, the peak is attributed to any of the markers as mentioned. The peak therefore taken alone cannot be used to distinguish between the two markers. However, as both markers are indicative of ADHD, the appearance of a peak (signal) common to both markers is indicative of ADHD. For markers with two or more lipid chains, where only one of the chains is defined above (e.g.

[0082] C16:2), the nature (length, amount and position of double bonds) of the other chain(s) does not matter for the present invention. The markers are described in the brochure for the Biocrates assay used: https: / / biocrates.eom / wp-content / uploads / 2022 / 12 / biocrates-Quant500-XL-list-of-meta bolites-v3-2022.pdf

[0083] In Column 3 systematic names or alternative names of the markers of Column 2 are shown, if available.

[0084] Column 4 shows the RefMet ID the marker has been designated to by the Metabolomics Workbench, if available in the database.

[0085] Column 5 shows the AUC value of the respective marker for ADHD, ADHD1 or ADHD2, respectively, if taken alone (though the AUC for discriminating ADHD2 from neurotypical, TG 14:0_36:4 discriminates ADHD2 well from ADHD1 and, for this purpose, the AUC when used as single marker is 0.83). These AUCs are surprisingly high. The higher the AUC, the better is the significance of the marker, and thus, the preference of the marker for the assessment of ADHD in the method of the invention. An AUC of more than 0.8 is preferred because the has defined in a consensus of the World Federation of Societies of Biological Psychiatry on biomarkers for ADHD a threshold of 80 % (Thome J, Ehlis AC, Fallgatter AJ, Krauel K, Lange KW, et al. 2012. Biomarkers for attention-deficit / hyperactivity disorder (ADHD). A consensus report of the WFSBP task force on biological markers and the World Federation of ADHD. World J Biol Psychiatry 13: 379-400). The AUCs of the single markers SPBP d17:0, PE P-18:0 / 17:1 , SPB d17:1 , DG 18:0_20:0, PE P-20:0 / 20:5 and C16:2-OH exceed the value of 0.820 and thus are particularly indicative for ADHD1 . It is further remarkable, that the markers with the highest AUC for ADHD / ADHD1 , SPBP d17:0, PE P-18:0 / 17:1 and SPB d17:1 , contain one fatty acid chain of exactly 17 C-Atoms.

[0086] The present invention also found that the significance of the assessment according to the invention can be further improved by utilizing the determination of two or more markers as listed above. In this assessment, the result is positive, if both or more results for the two or more markers are positive. While also combinations of markers with relatively low AUCs exhibit AUCs higher than the single markers, it is further evident, that a combination of markers, particularly of the markers showing the highest AUC, fulfils the object of the invention at best. The invention provides the opportunity to increase specificity to the degree as desired by selecting and increasing the number of markers to be included in the assessment. Combinations showing an AUC of more than 0.82, and even more preferred of 0.9, are preferred. Particularly selective combinations are described below.

[0087] The selectivity can be further increased when performing a determination of two or more of these markers on the same sample in combination. The markers are chosen fortheir collective ability to distinguish between a diseased state and a healthy state with higher specificity than individual markers alone. Raw marker concentration values of the selected markers are normalized to reduce for technical variability, including but not limited to batch effects and sample dilution). In certain embodiments, the normalized data are subjected to mathematical transformations, such as logarithmic transformation or scaling in order to approximate statistical assumptions required for subsequent analysis including normality and homoscedasticity). The normalized and optionally transformed marker data are combined into composite variables by application of multivariate algorithms, such as principal component analysis (PCA) or partial least squares (PLS). This is performed in tools, such as in ANOVA / MetaboAnalyst 6.0, to create composite variables. The creation of composite variables allows for capturing synergistic or antagonistic interactions between markers, thereby amplifying the diagnostic signal. Statistical analysis of the composite variables is conducted using an analysis of variance (ANOVA) framework. The ANOVA test is applied to compare the composite marker values between ADHD positive samples and neurotypical controls and which combinations of markers exhibit statistically significant differences between diseased subjects (ADHD) and neurotypical controls as well as groups / sub-types. This approach enables the identification of marker combinations that exhibit discriminatory power, thereby increasing diagnostic specificity relative to the use of single marker measurements. In Column 6 it is indicated, whether the increase or decrease of the concentration or enzymatic activity relative to the concentration or enzymatic activity of the marker in a sample from a neurotypical person is indicative of ADHD or a group thereof.

[0088] Column 7 shows the Confidence Interval (Cl) for the determination of the markers. The Cl for the markers according to the invention was high compared to other markers.

[0089] These markers thereof can be found in any blood sample of human origin. Therefore, the presence of such markers itself is not sufficient to diagnose ADHD. The present invention has found, that the level of these markers or fragments thereof is altered in samples from persons affected by ADHD compared to persons not affected. Therefore, the present invention is directed to embodiments, where the level, either by amount or concentration or enzymatic activity, of these markers or fragments in the sample is determined and compared to a reference. A deviation of the result from the usual amount, concentration or enzymatic activity from the marker or fragment thereof to the reference by a certain threshold is taken as an indication for ADHD.

[0090] In order to be indicative, the deviation from the reference must be substantial; it is substantial if the deviation is more than factor 1 .5.

[0091] Particularly, the invention has found, that the amount, concentration or enzymatic activity of the markers PE P-18:0 / 17:1 , SPB d17:1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, C3DC (C4OH) (aka C3-DC (C4-OH)), C16:2, PA 18:0_18:1 , SPHK activity or LPP3 activity in an ADHD person compared to a neurotypical (non-ADHD) person is increased or / and the amount, concentration or enzymatic activity of the markers SPBP d17:0, PC 0-44:4, LPE 14:0, PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4 or LDH activity is decreased.

[0092] Depending upon the nature and design of the assessment method, the reference for the assessment of a particular marker above can be selected from the group of the same marker, but from a different sample from a non-ADHD or an ADHD affected person, a standard sample containing a known and defined amount or concentration of the same marker representing a non-ADHD or an ADHD level, another compound in the same sample which is present in that sample in defined and known amount or concentration, irrespective of ADHD, another compound in a different sample from a non-ADHD or an ADHD affected person, a standard sample containing a known and defined amount or concentration of a compound, irrespective of ADHD, the same marker, but from a sample taken at a different time from the same person, and a defined and normalized instrument standard for the measurement of the same marker.

[0093] Assays for each of the above amounts, concentrations or enzymatic activities are commercially available, for example in the “Biocrates MxP® quant 500 XL” kit and corresponding instrument. Assays for amounts, concentrations or enzymatic activities of the markers are also available commercially as stand-alone reagents or kits. The analytical method should be able to differentiate the analyte(s) of interest and internal standard from endogenous components in the matrix or other components in the sample. The definitions of the characteristics of an assay can be taken from Parikh R, Mathai A, Parikh S, Chandra Sekhar G, Thomas R. 2008. Understanding and using sensitivity, specificity and predictive values. Indian J Ophthalmol 56: 45-50. and EMEA. 2022. ICH guideline M10 on bioanalytical method validation. https: / / www.ema.europa.eu / en / ich-m10-bioanalytical-method-validation-scientific-guideline.

[0094] According to that the following applies:

[0095] The ability of a test to correctly classify an individual as disease-free is called the test's specificity.

[0096] Specificity (Probability of being test negative when disease absent) = d (true negative) I b (false positive) +d (true negative); X-axis in the ROC diagram.

[0097] Sensitivity is the ability of a test to correctly classify an individual as 'diseased'.

[0098] Sensitivity (Probability of being test positive when disease present) = a (true positive) I a (true positive) +c (false negative); Y-axis in the ROC diagram.

[0099] These terms are applied to all kinds of tests, i.e. tests for both ADHD in general and sub-types (groups) of ADHD. In the present invention, the terms sensitivity, specificity and selectivity are used for different tests and for different biomarkers in different context, i.e. for ADHD in general and for the ADHD groups in particular. A biomarker with high specificity for ADHD in general distinguishes ADHD from other diseases with high likelihood, while a biomarker with high specificity for an ADHD group distinguishes the ADHD group from one or more other ADHD groups with high likelihood. A biomarker with high specificity for ADHD in general and for a group of ADHD therefore distinguishes the ADHD group from the other group(s) and from other diseases. A biomarker with high specificity for ADHD in general and low or no specificity distinguishes ADHD from other diseases, but not the ADHD group from other ADHD group(s). A test with high specificity for ADHD in general distinguishes ADHD from other diseases with high likelihood. This can be a test using one or more biomarkers. In case of a single biomarker in the test, the biomarker is preferably a biomarker with high specificity for ADHD in general and low or no specificity for a group of ADHD. In case of more than one biomarker in the test, the biomarkers preferably are a combination of biomarkers, each having a specificity for at least one ADHD group. A test with high specificity for an ADHD group must contain at least one biomarker with high specificity for the ADHD group to be determined. In the present invention, tests are preferred comprising determining the enzymatic activity, amount or concentration of more than one biomarker individually and using the result of the individual determinations to conclude both the ADHD in general and the ADHD group. This is done currently done by different „manually“ applied algorithms. In brief, single samples are analyzed with ADHD1 and ADHD2 specific markers. If the samples are found to have group denominations of ADHD these samples are analyzed by ADHD1 and ADHD2 specific markers and depending on meeting certain thresholds grouped to either ADHD1 or ADHD2. The threshold is set to be 150 % of the reference value for the respective marker. As soon as the measurement value exceeds the threshold, the condition for grouping into the respective group is considered to be met. On the other hand, combination of a marker with high specificity for ADHD1 and a marker with high specificity for ADHD2 have a high likelihood for covering all ADHD positive samples, i.e. the combination is very sensitive. The specificity of the markers in the assessment of ADHD can be and was determined by determination of the marker in persons positively diagnosed for ADHD compared to persons negatively diagnosed for ADHD. The positive and negative diagnosis in this scenario is made vis-a-vis a scientifically acknowledged ADHD standard. Presently, the best available standard is the Diagnostic and Statistical Manual of Mental Disorders (DSM-5, American Psychiatric A. 2022. Diagnostic and statistical manual of mental disorders: DSM-5-TR: text revision. Washington, DC: American Psychiatric Association Publishing. Ixix, 1050 pages pp.) supplemented by the Research Domain Criteria (RDoC) approach. The selectivity (AUC) is usually visualized in a ROC diagram showing the dependency of the true positive rates versus the false positive rates. The AUC is obtained as the area under the curve relative to the full square.

[0100] For the assessment of ADHD1 AUCs markers with an AUC above 0,74 have been found effective when a marker is used as single marker, better results are provided by markers with an AUCs above 0,820, most preferable by the markers of the group having an AUC of 0,824 and above, as the first 7 markers mentioned in list 1 . For the assessment of ADHD2 markers with an AUC above 0,50 have been found effective when a marker is used as single marker, better results are provided by AUCs above 0,60, Using combinations of markers, the AUCs can be improved to above 0.83. Combinations of at least 3 markers drive the AUC to above 0.84, and even above 0.95. Thus, combinations of 3 or more markers are particularly useful in the invention. See above for the selection of markers for the combination.

[0101] Based on psychiatric diagnosis 3 main types of ADHD (sometimes also called sub-types or presentations) have been established: ADHS-HI (predominant hyperactivity), ADHS-I (predominant inattentiveness) and ADHS-C (both inattentiveness and hyperactivity). The invention may provide further insight into the relevance of this grouping as detailed below.

[0102] As can further be seen from the above table the markers have different specificity for different groups (sub-types) of ADHD, SPBP d17:0, PE P-18:0 / 17:1 , SPB d17:1 , SPB d18:0, DG 18:0_20:0, PE P- 20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3-DC (C4-OH)), C16:2 and LPE 14:0 are indicative of ADHD group 1 whereas PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0 and TG 14:0_36:4 are indicative of group 2 ADHD. As to the moment it is still open whether or how group 1 ADHD and group 2 ADHD determined by the present invention correspond to the clinically defined subtypes ADHD-C, I or HI. Multivariate analyses with subgroups from the DSM-V classification gave no overlapping of inattentive or hyperactivity scores with the metabolome classified groups 1 , 2, or 3. The new grouping defined by the invention may open a new classification of ADHD based on altered biochemical pathways mirrored in metabolites.

[0103] A combination of markers with selectivity for different subtypes may also be helpful in diagnosis and / or therapy, e.g. for the stratification of first line therapy with methylphenidate, Atomoxetin or behavioral therapy. If the psychiatric diagnosis does not need to differentiate between groups, a combination of SPB d17:1 , PC 0-44:4, SPB d17:0, LPP3 activity, PE P-18:0 / 17:1 , C3-DC (C4-OH), PC 0-38:0, PC 0-36:4 is especially selective.

[0104] A combination of the markers above with other markers, even markers for ADHD which are hitherto unknown, may be used to further provide an even better assessment. The method described above will be useful to select markers to create composite values indicative of ADHD or the sub-types. Aspects of the invention

[0105] Each of the aspects as described below take advantage of the features as described above in the terms and definitions section. In particular, the method according to the first aspect, as further exemplified or detailed by the embodiments as described in the terms and definitions provides the basis for the other aspects.

[0106] In the first aspect of the invention, an in-vitro method of assessing whether a person has ADHD or is at risk of developing ADHD, the amount, concentration or enzymatic activity of an analyte selected from a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d17: 1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3- DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or b) a combination of any of the markers under a), is determined in a sample from the person and the determined amount, concentration or enzymatic activity is compared to a reference.

[0107] Any significant deviation of the result from a non-ADHD (i.e. neurotypical) reference or reference sample is taken as an indication of ADHD or a nonsignificant deviation from or substantial similarity to a non- ADHD reference or reference sample is taken as an indication of absence of ADHD in the person the sample originated from. Therefore, the assessment of positive assessment of ADHD is derived from the fact that the result of the comparison of the determined amount, concentration or enzymatic activity to the reference is indicative of ADHD. A result that the determined amount, concentration or enzymatic activity is similar to the positive ADHD reference is indicative of ADHD positive. A result that the determined amount, concentration or enzymatic activity exceeds or falls short of the negative ADHD reference is indicative of ADHD positive, depending upon the marker. A result that the determined amount, concentration or enzymatic activity is similar to the negative ADHD reference is indicative of ADHD negative.

[0108] As can be seen from List 1 each of the markers has a very high AUC for ADHD when used alone. Therefore, they are already very indicative of ADHD alone. Fig. 1 shows the area under the curve (AUC) for SPB d 17:1 . The AUC is a convenient way to plot sensitivity for the biomarker analyzed. Despite high specificity and sensitivity 13 controls and 11 ADHD patients are grouped differing from their presumed class properties.

[0109] The selectivity can be further increased when performing a determination of two or more of these markers on the same sample in combination.

[0110] In embodiments comprising determination of a combination of markers or / and fragments each marker or / and fragment is determined individually as described above. In case two or more of the markers or / and fragments exhibit a significant deviation from the reference, the likelihood of the person having ADHD or being at risk of developing ADHD is increased, thus providing an improved assessment of the ADHD status of the person. In one embodiment of all aspects of the invention using a combination of markers, the determination is performed by using a first group of markers, including two or more of the markers of the invention, and comparing the determined amount, concentration or enzymatic activity to a reference for each marker of the first group, wherein the deviation from a reference of one marker, a second group of more than one, but less than all markers of the first group, or all markers of said first group is taken as an indication of ADHD or a sub-type thereof for the person assessed. This embodiment is particularly helpful to provide accurate results even for the assessment of persons having different metabolomic ADHD profiles, i.e. in case the concentration, amount or enzymatic activity of a certain marker of the invention is not indicative of ADHD in that person. This embodiment reflects the degree of variability of the significance of markers of the invention as evidenced by the different AUCs. This embodiment is particularly useful in the thirteenth aspect of the invention, as in different persons to be screened may exhibit different metabolomic profiles.

[0111] Based on the results of the present invention, combinations of one or more sphingosine derivatives with fatty acids of 17 carbon atoms (such as SPB d17: 1 or SPB d17:0) with other markers or / and fragments, particularly carnitine derivatives (such as C3-DC or C4-OH) have proven to allow for very high specificity and selectivity.

[0112] According to an embodiment of the present invention with high significance, the sample is subjected to a multiplicity of determinations, e.g. 10, 50 or even more than 100 determinations of different markers or / and fragments, and the single results of the determinations are used to derive a combined result. Due to the natural variability of the metabolome of individuals, the result of single markers alone may be sufficient for a positive assessment, but in other cases, the results of markers in combination may be more helpful. For example, in case the result for a single marker does not deviate from the reference clearly enough to allow for a reliable result, the fact, that other markers deviate as well, may assist in coming to an assessment with high significance.

[0113] As combination of two markers within a group, combinations of SPB d17:1 and SPB d17:0 show increased specificity and sensitivity (Fig. 2). For a combination of three compounds, a combination of SPB d17:1 , SPB d17:0 and C3-DC (C4-OH) increases specificity and sensitivity (Fig. 3). This was increased to an AUC of 0.859 with a combination of SPB d17:1 and SPB d17:0 with C16:2 (Fig. 4) and with a combination of SPB d17:1 , PE P-18:0 / 17:1 and PC 0-44:4 to an AUC of 0.861 (Fig. 5). The biomarker PC 0-44:4 was especially helpful. In a combination with SPB d17:1 , SPB d17:0, PC 0-44:4 increased the AUC to 0.887 (Fig. 6).

[0114] Using further biomarkers for ADHD1 gave no further increase of the AUC compared to the latter combination. A combination of SPB d17:1 , PC 0-44:4, C16:2, SPHK activity (3), LPP3 activity (3), C3-DC (C4-OH), PE P-18:0 / 17:1 , LDH Activity, PC 0-36:4 gave a comparable AUC of 0.906 (Fig. 7).

[0115] The highest AUC and highest specificity and sensitivity was reached when combining markers from ADHD1 and ADHD2. Without separation of presumed ADHD1 and ADHD2 highest AUC of 0.93 was obtained with a combination of SPB d17:1 , PC 0-44:4, SPB d17:0, LPP3 activity, PE P-18:0 / 17:1 , C3-DC (C4-OH), PC 0-38:0 and PC 0-36:4 (Figure 8). ADHD1 markers SPB d 17:1 , PC 0-44:4, SPB d17:0, PE P-18:0 / 17:1 differentiate ADHD1 patients accurately with an AUC of 0.961 from controls (Figure 9), while ADHD2 markers PA 18:0_18:1 , PC O- 36:4, PC 0-38:0 and TG 14:0_36:4 differentiate ADHD2 patients accurately with an AUC of 0.934 from controls (Figure 10).

[0116] ADHD1 patients can be differentiated from ADHD2 patients with markers SPB d 17: 1 , C3-DC (C4-OH), PA 18:0_18:1 , C16:2 with an AUC of 0.985 (Figure 11).

[0117] The determination of the markers as listed is most preferred and was tested successfully in the best mode of the invention.

[0118] This aspect of the invention is particularly useful for any determination of ADHD, either ADHD1 or / and ADHD2, including the de-novo determination of ADHD, i.e. in case the person has not been tested for ADHD before.

[0119] The present invention preferably uses a dried blood sample or EDTA blood. Dependent upon the kind of analyte and determination method, the sample may need further sample preparation steps as described above, like purification, separation and concentration steps. A purification achieves purifying the analyte to be detected to enhance amount or concentration of the analyte compared to other constituents of the sample. This can be done by established chromatographic methods. Separation achieves separating the analyte to be detected to remove the other constituents of the sample, particularly constituents disturbing the determination of the analyte. Again, established chromatographic methods are useful. Separation is a purification to a higher purity of the analyte. Concentration achieves enhancing the concentration of the analyte to be determined to higher concentration than in the original sample, e.g. large sample volumes are reduced in volume by removing constituents of the original sample, e.g. by evaporation. As an example, whole blood samples may be concentrated by drying the sample to remove water. The preparation steps can be combined as required by the sample and kind of determination steps.

[0120] In a second aspect the invention is directed to a method of confirming that a person has ADHD, comprising the assessment method above.

[0121] This aspect will be useful if an (earlier) psychiatric diagnosis was not conclusive or if psychiatric and method of the invention are run in parallel.

[0122] In a third aspect, the invention is directed to a method of selecting a patient for a therapy of ADHD, comprising

[0123] - determining the amount, concentration or enzymatic activity of an analyte selected from a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d 17: 1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3- DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity b) a combination of any of the markers under a) in a sample from the person,

[0124] - comparing the determined amount, concentration or enzymatic activity to a reference, and - marking the sample for therapy, if the sample differs in amount, concentration or enzymatic activity from the reference by more than 50 %.

[0125] This method includes the method of the first aspect of the invention wherein the result can be used to propose a therapy to the patient. In order to realize such proposal, the laboratory having determined the amount, concentration or enzymatic activity marks the sample by any means, such as putting a label indicating the result of the determination onto a container containing the sample, writing the result of the determination into an entry in a database specifying the sample or directly informing the client having provided the sample of the result. The marking can be a qualitative marking, i.e. that the difference to the reference is larger than 50%, such as “ADHD probable, further consultations by specialists recommended”, “therapy recommended” or “ADHD positive”, or quantitative, i.e. specifying percentage of the difference to the reference, such as “exceeds the reference by X %” or “X % compared to the reference” or “is within a range of X % an Y % compared to the reference”. This result can be used by a physician or psychiatrist to apply an appropriate medical or psychiatric treatment to the patient. This is particularly useful when applying the determination of the sub-type (group) of ADHD according to nineth aspect of the invention, as the different sub-types (groups) may require different therapeutic treatment.

[0126] In a fourth aspect, the invention is directed to an in-vitro method of monitoring a patient suffering from ADHD or being treated for ADHD, comprising

[0127] - determining in a sample from the person the amount, concentration or enzymatic activity of an analyte selected from a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d 17: 1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3- DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or b) a combination of any of the markers under a), and

[0128] - comparing the determined amount, concentration or enzymatic activity to a reference.

[0129] The reference in this case may be the amount, concentration or enzymatic activity determined in an earlier determination from the same patient.

[0130] In the method of monitoring according to this aspect the method of the first aspect is repeated as required to determine, whether the disease has proceeded or is retained or is decreased, e.g. following therapeutic treatment. Therefore, this aspect of the invention preferably comprises comparing the result of the actual determination to the result of an earlier determination of the same patient. This can be in the form of a medical history showing the actual and earlier result(s). Additionally, the method can include indicating a trend in the results towards improving or worsening of the patient health status deduced from altered concentration of ADHD specific metabolites on ADHD. The repetition can be in regular or in irregular intervals.

[0131] In the fifth aspect, the invention is directed to a computer-implemented method assessing whether a person has ADHD or is at risk of developing ADHD, said method comprising 1) obtaining a value for the level of an analyte selected from a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d 17: 1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3- DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or b) a combination of any of the markers under a),

[0132] 2) storing said value in a data base in one or more computer or data base,

[0133] 3) comparing said value to a minimum or a maximum threshold value for the level of said marker or said combination stored in said data base, and

[0134] 4) outputting a positive signal in case said value exceeds said minimum threshold value or undercuts that maximum threshold value, wherein said positive signal is indicative of the person having ADHD.

[0135] This method is particularly useful when a combination of markers is used for the determination. Then the different levels in amount, concentration or enzymatic activity of each analyte can be compared to a respective reference value for said analyte and the various deviations can be compared and used for the assessment. The results can further be related to other medical information, such as psychiatric diagnosis and other clinical parameters, such a concentration of other constituents or physiological parameters, e.g. EEG etc., in the same or other patient’s bodily materials. In this method, comparison to the reference can be made by comparing the value obtained to a threshold. For markers being indicative of ADHD, if the amount, concentration or enzymatic activity of the marker in ADHD-positive persons is decreased compared to a neurotypical person (see list 1), a value is indicative of ADHD if the value is lowerthan a set threshold value, which in this case is set to be lowerthan the value for a neurotypical person. On the other hand, for a marker being indicative of ADHD, if the amount, concentration or enzymatic activity of the marker in ADHD-positive persons is increased compared to a neurotypical person (see list 1), a value is indicative of ADHD if the value is higher than a set threshold value, which in this case is set to be higher than the value for a neurotypical person. Furthermore, this aspect of the invention can be used in combination with the nineth aspect of the invention to determine which sub-type of ADHD the person should be allocated to.

[0136] A particular advantage can be achieved, if the determination of the analytes in the combination is done in parallel using a single test kit for testing all possible combinations. While the Biocrates MxP quant 500 XL test only contains some of the potential assays for markers according to the invention, future kits could contain components for selectively testing markers for ADHD. Particularly, a kit useful for an assessment according to the present invention contains any marker or marker combination as outlined above and one or more reference analyte which is not related to ADHD.

[0137] In the sixth aspect, the invention is directed to an in-vitro method of determining in a sample from the person the amount, concentration or enzymatic activity of an analyte selected from a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d 17: 1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3- DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or b) a combination of any of the markers under a), characterized by - providing a sample from a dried blood spot into a mass spectrometer, and

[0138] - determining a measurement value for the signal strength of the mass of said marker or the result of said enzymatic activity of said analyte by mass spectrometry.

[0139] In mass spectrometry, the presence of an analyte is determined by determining the occurrence of one or more peaks related to the presence of the analyte such as the mass peak of the analyte or a mass derived from a fragment of the analyte created within the mass spectrometer by applying an electric field and electromagnetic energy and separating the molecules and fragments according to their (remaining) mass. The relative strength of the signal of a peak is related to the concentration of the analyte or a group of analytes yielding in fragments with the same peak. The quantification is done by comparing the measurement value for the signal strength of the sample from the patient with a reference value for the signal strength containing a known and defined amount or concentration of the same analyte in a standard sample having undergone the same sample preparation procedure as the patient sample. By that, the amount, concentration, or enzymatic activity can be calculated from the difference of the measurement values.

[0140] Currently, for quantification, the standards supplied with the Biocrates Kit 500XL are used. In a future protocol which shall measure only ADHD specific metabolites these standards shall be in house made and quantified using good laboratory procedures, quality management and wherever possible external quality tests.

[0141] This aspect can also be part of the computer implemented method as described below.

[0142] In the seventh aspect, the invention is directed to the use of mass spectroscopy for the in-vitro determination of the amount or concentration or enzymatic activity of any metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d17:1 , SPB d18:0, DG 18:0_20:0, PE P- 20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3-DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity or a combination of any of these markers in a sample from dried blood of a person for the assessment of ADHD.

[0143] Mass spectrometry is a known method for determining analytes in samples. The above metabolomic markers have been proposed together with several hundred other markers in a panel for analyzing the health status of patients on bipolar disorders (Tomasik J, Harrison SJ, Rustogi N, Olmert T, Barton-Owen G, et al. 2024. Metabolomic Biomarker Signatures for Bipolar and Unipolar Depression. JAMA Psychiatry 81 : 101-06) or schizophrenia (Shi M, Du X, Jia Y, Zhang Y, Jia Q, et al. 2024. The identification of novel schizophrenia-related metabolites using untargeted lipidomics by mass spectrometry: Cerebral Cortex 34). However, the markers allegedly indicative for bipolar disorders or schizophrenia are others than those of the invention.

[0144] These works and others not cited show that metabolomic analyses give metabolomic signatures that are more or less specific to a certain psychiatric diagnosis, i.e. the metabolites analyzed are specific for bipolar disorders or schizophrenia.

[0145] In the eighth aspect, the invention is directed to the use of any metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d17:1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3-DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity or a combination of any of these markers for the in-vitro determination of ADHD in a sample form a human person.

[0146] The markers as used in the present invention have not been brought into connection with ADHD before. That those markers could be indicative of ADHD and even the classification of ADHD into groups could not have been expected.

[0147] Other studies have analyzed ADHD samples with targeted metabolic analysis (Henriquez-Henriquez MP, Solari S, Quiroga T, Kim Bl, Deckelbaum RJ, Worgall TS. 2015: Low serum sphingolipids in children with attention deficit-hyperactivity disorder, Front Neurosci 9: 300, and Brunkhorst-Kanaan N, Trautmann S, Schreiber Y, Thomas D, Kittel-Schneider S, et al. 2021 : Sphingolipid and Endocannabinoid Profiles in Adult Attention Deficit Hyperactivity Disorder. Biomedicines 9). However, the metabolite panel used for analysis was not as comprehensive as the panel used in the present invention and did not contain the metabolites from List 1 . SPB d17: 1 , as well as other lipids with odd carbon chain number were not analyzed by these two studies. Sphingosine bases with odd chain lengths such as SPB d17:1 have been detected in human tissues in keratin cells (Berdyshev E, Goleva E, Bronova I, Bronoff AS, Streib JE, et al.

[0148] 2022. Signaling sphingolipids are biomarkers for atopic dermatitis prone to disseminated viral infections (J Allergy Clin Immunol 150: 640-48) and in brain tissue (Zeng K, Zhou X, Liu W, Nie C, Zhang Y. 2023: Determination of endogenous sphingolipid content in stroke rats and HT22 cells subjected to oxygenglucose deprivation by LC-MS / MS. Lipids Health Dis 22: 13). Despite an extensive literature search, we could not find any association of SPB d17:1 with other diseases. Only in eczema herpeticum C17 sphingosines were described in tissue ((Berdyshev et al. see above). No changes for SPB d17:1 have been described, for example, in recent metabolomically comprehensive studies on autism (Liu J, Tan Y, Zhang F, Wang Y, Chen S, et al. 2024. Metabolomic analysis of plasma biomarkers in children with autism spectrum disorders: MedComm (2020) 5: e488) and depression (Tomasik et al., see above). Sphingolipids with 17 carbon atoms have been described as increased in the caudate nucleus in Huntington's disease (Phillips GR, Saville JT, Hancock SE, Brown SHJ, Jenner AM, et al. 2022. The long and the short of Huntington's disease: how the sphingolipid profile is shifted in the caudate of advanced clinical cases. Brain Commun 4: fcab303). Similarly, a decrease of ceramide 17:2 in grey matter has been described in Alzheimer's disease (Obis E, Sol J, Andres-Benito P, Martin-Gari M, Mota-Martorell N, et al.

[0149] 2023. Lipidomic Alterations in the Cerebral Cortex and White Matter in Sporadic Alzheimer's Disease. Aging Dis 14: 1887-916). Thus, the finding of increased concentrations of lipids with odd chain numbers in blood / EDTA-plasma from ADHD patients was totally unexpected. Furthermore, genetic analyses have described 76 ADHD risk-genes (Demontis D, Walters GB, Athanasiadis G, Walters R, Therrien K, et al. 2023. Genome-wide analyses of ADHD identify 27 risk loci, refine the genetic architecture and implicate several cognitive domains. Nat Genet 55: 198-208). The involvement of the 76 genes in biological pathways had been analyzed, but no significant pathways showed significant enrichment. Thus, even deduction from genetic data would not have allowed a prediction of our set of metabolic markers (List 1). Taken together, the described (List 1) of ADHD specific markers is new, unexpected and could not have been predicted. Similarly, the group of markers with a high AUC of more than 0.82 bear the possibility to provide highly specific and sensitive methods. In the nineth aspect, the invention is directed to a method for determining from which group of ADHD a patient is suffering comprising determining in a sample from the person the amount, concentration or enzymatic activity of an analyte selected from a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d17:1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3- DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or b) a combination of any of the markers under a), wherein the amount, concentration or enzymatic activity of an analyte selected from a1) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d17:1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3-DC (C4-OH)), C16:2 and LPE 14:0, or b1) a combination of any of the markers under a1) is indicative of a first ADHD group and wherein the amount, concentration or enzymatic activity of an analyte selected from a2) a metabolomic marker selected from the group consisting of PA 18:0_18: 1 , PC 0-36:4, PC 0-38:0, LPE P-16:0 and TG 14:0_36:4, or b2) a combination of any of the markers under a2) is indicative of a second ADHD group.

[0150] If analytes from each group (including at least one marker with selectivity for the first ADHD group and at least one marker with selectivity for the second ADHD group) are tested for, the method will assess ADHD irrespective of the group.

[0151] This aspect of the invention is very special, as it can differentiate two metabolically different ADHD subtypes, i.e. sub-types exhibiting different and distinguishable metabolomes using this aspect of the invention. As pointed out above, psychiatric diagnosis presently acknowledges at least 3 sub-types (ADHS-HI, ADHS-I and ADHS-C), according to a behavioral classification. The present invention has only found two subtypes, but can more readily distinguish between these two subtypes. Those two metabolically distinguishable subtypes (metabolomic subgroups) have not yet been mapped to the presently acknowledged sub-types ADHS-HI (predominant hyperactivity) and ADHS-C (predominant inattentiveness) and are herein called ADHD1 (predominant hyperactivity) and ADHD2 (predominant inattentiveness), respectively. In addition, it appears possible that ADHD1 and ADHD2 may differ in response to medication, e.g. methylphenidate. Further studies with larger subgroups will allow to map metabolomical ADHD1 and ADHD2 to specific clinical ADHD phenotypes.

[0152] In a first embodiment of this this aspect of the invention, persons belonging to metabolomic group ADHD1 are preferably assessed using SPBP d17:0, PE P-18:0 / 17:1 , SPB d17:1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3-DC (C4-OH)), C16:2 or LPE 14:0 or a combination of these markers.

[0153] In a second embodiment of this this aspect of the invention, persons belonging to metabolomic group ADHD2 are preferably assessed using PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4 or a combination of these markers.

[0154] In the tenth aspect, the invention is directed to a method for determining the treatment scheme of ADHD for a patient comprising - determining in a sample from the person the amount, concentration or enzymatic activity of an analyte selected from a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d 17: 1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3- DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or b) a combination of any of the markers under a),

[0155] - comparing the determined amount, concentration or enzymatic activity to a reference, and

[0156] - selecting a treatment scheme from the group of available treatment schemes based upon said comparison.

[0157] This method includes the method of the first aspect of the invention wherein the result can be used to propose a therapy to the patient. This is particularly useful when applying the determination of the group of ADHD according to the nineth aspect of the invention, as the different groups may require different therapeutic treatment, e.g. a first group ADHD patient will need pharmaceutics to slow down activity, such as methylphenidate, and a second group ADHD patient may need help to focus the attention. A subgroup of ADHD patients does not respond to treatment by methylphenidate. Concentrations of ratios of biomarkers from SPBP d17:0, PE P-18:0 / 17:1 , SPB d17:1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3-DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity will be used to predict best treatment from the available ADHD treatment options. Which markers will turn out to be most helpful to predict treatment response or to adapt treatment doses is unknown at the moment as only treatment-naive participants were included in our study. However, such markers could be identified in a study using 200 newly diagnosed ADHD patients who will undergo treatment according to current guidelines. About 20 % of patients treated with methylphenidate will not respond.

[0158] The invention concludes that markers like SPB d 17:1 , PC O 44:4, PC 0-36:4, and PC 0-38:0 will be most specific for predicting treatment responses.

[0159] In the eleventh aspect, the invention is directed to a method for determining the amount, concentration or enzymatic activity of any metabolomic marker selected from the group consisting of SPBP d17:0, PE P- 18:0 / 17:1 , SPB d17:1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3-DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P- 16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity or a combination of any of these markers in a sample from said person, wherein said amount, concentration or enzymatic activity is determined by a method selected from the group consisting of LC-MS, Immunoassay and Enzyme Activity Assay for the diagnosis of ADHD.

[0160] Based on the findings of the present invention, that particular metabolites are indicative of ADHD, it will be possible to design specifically directed assays for the occurrence of these metabolites by any other known means using characteristics of the metabolites, such as immunological exposure of epitopes, e.g. in proteins or haptens, or as substrates for enzymes.

[0161] In the twelfth aspect, the invention is directed to a method for determining whether a person has ADHD or is at risk of developing ADHD comprising a) assessing ADHD or the risk of developing ADHD by a psychiatric method, and b) assessing ADHD or the risk of developing ADHD by an in-vitro method using a sample from the person, wherein the determination is positive, if the assessments in steps a) and b) are both positive regarding ADHD. The in-vitro method conveniently is a method according to first, sixth or seventh aspect of the invention.

[0162] This aspect of the invention can be used in databases for collecting personal health care data to conclude and provide an indication to persons evaluating the health care data that the person may suffer from ADHD. In this way, the psychiatric diagnosis, and the diagnosis according to the present invention can be independently provided to the database and evaluated by another independent person, such as the individual tested or a physician having access to the database. Using artificial intelligence results from in vitro and clinical data can be interpreted with an acknowledged algorithm to make ADHD diagnosis investigator-independent and more comparable.

[0163] Computer programs for the purpose of relating in-vitro results and results of clinical studies are well known, e.g. in the fields of linking genetic data to phenotypic data or in the field of cancer classification by sequencing data of the cancer cells. Well known examples for genetic databases are ClinVar of the NIH (ClinVar (nih.gov)), HGMD from Qiagen. Both databases combine genetic variations to human phenotypes and diagnosis. For supporting cancer treatment and linking the genetic signature of a patient to a cancer type and establish a personalized treatment plan the databases from Foundation medicine, Flatiron or Molecular Health are well known examples.

[0164] In the thirteenth aspect, the invention is directed to an in-vitro method for screening a multiplicity of samples for persons having ADHD or the risk of developing ADHD, wherein each sample of said multiplicity of samples is subjected to determining the amount, concentration or enzymatic activity of an analyte selected from a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d 17: 1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3- DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or b) a combination of any of the markers under a), and

[0165] - comparing the determined amounts, concentrations or enzymatic activities to a reference, wherein any differences from the results obtained to a reference value are indicative of ADHD or the risk of developing ADHD for the respective persons the samples showing differences were collected from.

[0166] As the present invention provides for the first time an in-vitro diagnostic test based on markers in a sample, the invention opens the possibility to screen populations for persons having unrecognized ADHD or having a risk of developing serious forms of ADHD. In addition, such a test used for screening can detect individuals which often are not suspected of having treatable ADHD. With an easily scalable test at hand, it appears possible to screen whole age groups at school; or all inmates in a correctional institution. 1

[0167] Obviously, persons, wherein the amounts, concentrations or enzymatic activities of the markers or combinations do not differ significantly from the reference values, can be attributed a neurotypical status.

[0168] In addition, the test can be used to check whether prescriptions for methylphenidate are valued. There is a known misuse of methylphenidate among students or even managers who want to obtain better grades / working results with the assumed doping by Ritalin.

[0169] In the fourteenth aspect, the invention is directed to the use of mass spectroscopy for the assessment of whether a person has ADHD or is at risk of developing ADHD based on a dried blood sample from said person.

[0170] Due to the ready availability of mass spectrometric tests for analytes in and from the lipid metabolome this aspect provides a very convenient way to utilize mass spectrometry for ADHD assessment and confirm or rule out the necessity for a drug based medical intervention. Given the partly subjective factors used for ADHD assessment an unknown number of ADHD diagnoses and treatments are not necessary. For major depressive disorders (MDD) a complex and heterogenous disorder, which is difficult to diagnose accurately among individuals due to different subtypes of depression the use of biomarkers before start of treatment has been demanded (Suseelan S, Pinna G. 2023. Heterogeneity in major depressive disorder: The need for biomarker-based personalized treatments. Adv Clin Chem 112: 1-67) the use of the marker-based test of this invention seems warranted to guide prescription of ADHD drugs.

[0171] In a fifteenth aspect, the invention is directed to the use of mass spectroscopy for the in-vitro determination of a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d17: 1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3- DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or b) a combination of any of the markers under a) in the assessment of ADHD in a sample from dried blood of a person suspected to have ADHD.

[0172] Dried blood samples are prepared by applying blood, e.g. capillary blood or EDTA plasma, or serum onto a filter paper capable of absorbing a liquid blood sample by capillarity and drying the applied blood sample. Devices for collecting blood on an absorbent filter paper are well known. In general, a few drops of blood are sufficient for conducting this aspect of the method of the invention. The dried blood sample is then transported to the laboratory for performing the methods of the invention. The collection of the sample as a dried blood sample can be done offsite, near a patient to be assessed, so the patient does not need to travel to provide the sample. Furthermore, the dried blood sample is more stable than a liquid sample. This avoids undefined destruction of the contents of the sample during transport of the sample to the laboratory where the testing is done. The dried blood sample is then subject to a sample preparation including dissolving the dried sample from the filter paper and conducting the preparation steps as explained above in the terms and definitions section and finally performing the methods according to the aspects of the present invention from the sample obtained.

[0173] The use of this aspect of the invention can be made in a laboratory, receiving the dried blood sample through an organization providing a sample collection device containing an absorbent fleece to the person, where the patient is further instructed on where to send the dried blood sample to. The result of the assessment according to the invention is then provided directly or via an organization to the person interested in the result. This can be done by letter or by online portals with secured access. Digital systems for data exchange between the lab and the physician are in the market for decades. But also, digital platforms, which provide medical information to both the patient and the treating physician, are increasingly being used. During the Covid-19 pandemic, advanced laboratories offered solutions to deliver results to patients and relay important information to health authorities.

[0174] In the sixteenth aspect, the invention is directed to the use of a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d17:1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-01-1, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3-DC (C4-OH)), C16:2, LPE 14:0, PA

[0175] 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or a combination of any of these markers for the determination of ADHD.

[0176] Due to the availability of commercial tests for the metabolomic markers mentioned this aspect provides a very convenient and objective way to assess whether a patient has ADHD or is at risk for developing ADHD in samples collected from any person. The use can be materialized in any of the methods of the aspects of the invention above.

[0177] The improved significance (specificity and sensitivity) of the alternative markers and groups of markers of the present invention can be used in all methods described above.

[0178] Description of the Figures

[0179] The Receiver Operating Characteristics (ROC) Curves as shown in the figures show the True Positive Rate (TPR, Y-axis) versus the False Positive Rate (FPR, X-axis) of the markers or combinations according to the invention. The Area Under the Curve (AUC) is indicative of the achievable differentiation. In case of a value of 0.5 there would be no differentiation, in case of 1 .0 there is complete differentiation. Statistical analysis was done using MetaboAnalyst (Pang et al Nucleic Acids Research, Volume 52, Issue W1 , 5 July 2024, Pages W398-W406) for Multivariate Exploratory ROC Analysis with support vector machines (SVM) parameter for multilevel analysis.

[0180] For the multivariate exploratory ROC analysis, ROC curves were generated by Monte Carlo cross- validation (MCCV) using balanced sub-sampling. For each MCCV, two-thirds (2 / 3) of the samples were used to assess the importance of characteristics. The top 2, 3, 5, 10 ...100 (max) features were then used to build classification models, which were validated on one-third of the samples that were not included. ROC curve analyses were performed based on three multivariate algorithms - Support Vector Machines (SVM), Partial Least Squares Discriminant Analysis (PLS-DA) and Random Forests.

[0181] FIG. 1 A) shows an ROC Curve of marker SPB d17:1 with an AUC of 0,831. FIG. 1 B) shows an average of predicted class probabilities of each sample across the 100 cross-validations. The algorithm uses a balanced sub-sampling approach, the classification boundary is located at the center (x = 0.5, the dotted line). The confusion matrix (cross validation: open circles: ’’ADHD”, full circles: “control”) is provided on the right side of the image:

[0182] FIG. 2. shows an ROC Curve of a combination of markers SPB d17:1 and SPB d17:0 with an AUC of 0,840.

[0183] FIG. 3 shows an ROC Curve of a combination of markers SPB d17:1 , SPB d17:0 and C3-DC (C4-OH) with an AUC of 0,847.

[0184] FIG. 4 shows an ROC Curve of a combination of markers SPB d 17:1 , SPB d17:0 and C16:2 with an AUC of 0.859

[0185] FIG. 5 shows an ROC Curve of a combination of markers SPB d 17:1 , PE P-18:0 / 17:1 and PC 0-44:4 with an AUC of 0.861.

[0186] FIG. 6 shows an ROC Curve of a combination of markers SPB d17:1 , SPB d17:0 and PC O 44:4 with an AUC of 0.887.

[0187] FIG. 7 shows an ROC Curve of SPB d17:1 , PC 0-44:4, C16:2, SPHK activity, LPP3 activity, C3-DC (C4- OH), PE P-18:0 / 17:1 , LDH Activity and PC 0-36:4 with an AUC of 0.906.

[0188] FIG. 8 shows an ROC Curve of markers SPB d17:1 , PC 0-44:4, SPB d17:0, LPP3 activity, PE P- 18:0 / 17:1 , C3-DC (C4-OH), PC 0-38:0 and PC 0-36:4 with an AUC of 0.931 . 0212-2024-WC

[0189] 30

[0190] FIG. 9 shows an ROC Curve of markers SPB d17:1 , PC 0-44:4, SPB d17:0 and PE P-18:0 / 17:1 with an AUC of 0.961 for the differentiation of ADHD1 from control.

[0191] FIG. 10 shows an ROC Curve of markers PA 18:0_18:1 , PC 0-36:4, PC 0-38:0 and TG 14:0_36:4 with an AUC of 0.934 for the differentiation of ADHD2 from control. FIG. 11 shows an ROC Curve of markers SPB d17: 1 , C3-DC (C4-OH), PA 18:0_18:1 and C16:2 with an AUC of 0.985 for the differentiation of ADHD2 from control.

[0192] It can be deduced from the results as shown in the figures that any combination of markers SPB d17: 1 and SPB d17:0 is particularly useful in the present invention. By combining these markers with others from the list, the selectivity can be further improved. The marker combinations as used in figures 1 to 11 are therefore particularly preferred in the invention.

[0193] Examples

[0194] Example 1 - Sample collection and selection

[0195] For the clinical study clearance of the study protocol from the ethics committee of the university of Greifswald was received. 87 persons were selected by Universitatsmedizin Greifswald, Fleischmannstr. 8, 17489 Greifswald, from patients seeking medical advice and volunteers from supposed non-ADHD public to participate in the study. These persons were informed of the purpose of the study and consent was obtained according to the required standards for clinical studies. Each person was subjected to psychiatric diagnosis according to the “gold standard” DSM-V by experienced psychiatrists of the Universitatsmedizin based on questionnaires. The results were pseudonymized. 43 persons with positive ADHD diagnosis and 44 persons with negative ADHD diagnosis (control) were selected and samples of blood were collected strictly according to a written protocol. The samples were pseudonymized using the same key as used in the psychiatric diagnosis in order to map the result of the psychiatric diagnosis to the respective sample for each patient. The study nurse had been instructed and supervised by one of the inventors in all steps of the protocol. Samples collected were frozen at- 20 °C directly after sampling. Transport from Greifswald to the Dietrich Bonhoeffer Klinikum Neubrandenburg was done on dry ice within 2 h to 7 d after sampling. After arrival in Neubrandenburg samples were aliquoted and stored at - 80 ° C prior to analysis.

[0196] Example 2 - Metabolomic testing

[0197] After closing of the enrolment, the samples were sent on dry ice to Centogene GmbH, Rostock for metabolomic analysis.

[0198] For metabolomic analysis the Kit MxP® Quant 500 XL from Biocrates life sciences ag, Eduard-Bodem- Gasse 8, A-6020 Innsbruck, Austria was used https: / / biocrates.com / wp- content / uploads / 2022 / 12 / biocrates-Quant500-XL-list-of-meta bolites-v3-2022.pdf downloaded August 28, 2024). The MxP® Quant 500 XL Kit offers quantitative, standardized, quality-controlled, and reproducible metabolomics technology. It analyses more than 1000 metabolites, more than 400 thereof in quantitative manner. Centogene itself is a laboratory certified by Biocrates.

[0199] Metabolomic analysis on the pseudonymized samples in Rostock was done according to SOP in Centogene’s accredited laboratory (College of American pathologists). The employed techniques have been published (Rus et al., Metabolites 11 (6):382 (2021)).

[0200] The test results were provided to the inventors.

[0201] Example 3 - Evaluation of test results

[0202] Evaluation was done by the inventors combining the psychiatric diagnosis data and the result of the metabolomic profile of each pseudonymized sample individually. Data analysis was mainly done using MetaboAnalyst (Pang et al Nucleic Acids Research, Volume 52, Issue W1 , 5 July 2024, Pages W398- W406). MetaboAnalyst offers several modules for statistical analysis. We analyzed our data mainly with the module called “biomarker analysis”. For Multivariate Exploratory ROC Analysis ROC curves were generated by Monte-Carlo cross validation (MCCV) using balanced sub-sampling. In each MCCV, two thirds (2 / 3) of the samples were used to evaluate the feature importance. The top 2, 3, 5, 10 ...100 (max) important features were then used to build classification models which were validated on the 1 / 3 the samples that were left out. ROC curve analyses were performed based on three multivariate algorithms - support vector machines (SVM), partial least squares discriminant analysis (PLS-DA), and random forests.

[0203] Multivariate analysis consistently revealed outliers in our two defined groups ADHD and control. Two independent analyzers repeatedly found an identical set of outliers using different statistical parameters. By splitting ADHD into two groups of 33 and 10 named ADHD1 and ADHD2 respectively, the number of outliers could be reduced significantly. ADHD1 and 2 could be separated from the control by divergent sets of metabolites indicated in list 1 .

[0204] Example 4 - Metabolomic assessment according to the invention

[0205] Patient’s blood is received on the DBS device CentoCard® from Centogene GmbH, Am Strande 7, 18055 Rostock, Germany, according to the manufacturer’s instructions and sent by mail to a laboratory experienced in performing the Biocrates Kit MxP® Quant 500 XL assay such as Centogene.

[0206] The results obtained from the testing are evaluated for an increase or decrease of the amount or concentration or enzymatic activity of the above biomarkers relative to the reference values. In case of a significant difference from the reference it is concluded that the person having provided the blood sample has ADHD.

[0207] Example 4a: One-Biomarker-Analysis

[0208] This analysis is based on a cut-off value for a biomarker. The measured values of the instrument are analyzed and processed with the software tool Biocrates WeblDQ-Workflow Manager. If the calculated value for this specific biomarker is above the threshold, we conclude that the patient has ADHD, otherwise this is not the case. Thresholds and cutoffs can only be set after ongoing analyses. Currently we must assume that due to an unknown specificity of the psychiatric gold standard diagnostic assay an unknown number of ADHD patients will be contained within the control group of our study. Taking specificity values for psychiatric diagnoses from other diseases like Autisms or depression we expect about 20 % of false negatives in our control cohort. For SPB d17: 1 the median concentration in the control group was found to be 12 pM. We expect this reference value to be corrected to ~ 10 pm once better diagnosis standards for ADHD have been found. With the current imprecise reference values a working cutoff for the distinction of ADHD from controls is 24 pM. For PC 0-44:4 the median for the control group was determined to be 160 pM. An excellent cutoff for distinguishing ADHD from controls was set at 75 pM. However, best distinction between ADHD and controls was found with multiple biomarker analysis. The strength of biomarker analysis is that multiple metabolites are compared to a dataset of group of controls without ADHD. With this experimental setting the medians and cut offs used by e.g. PLS-DA may vary slightly with growing number of controls in our data bank.

[0209] Example 4b: Multiple biomarker Analysis A combination of biomarkers is used in this analysis. The instrument readings are also analyzed and processed using the Biocrates WeblDQ-Workflow Manager software tool. Using statistical methods (in our case MetaboAnalyst), we calculated a multidimensional function that separates the ADHD patients from a control group based on the biomarkers identified. To calculate this function, the software uses various non- linear classifiers (e.g. Support Vector Machines, Partial Least Squares Discriminant Analysis (PLS-DA) and Random Forests). By entering these biomarker values (calculated by Biocrates WeblDQ-Workflow Manger) for a specific patient, the program determines whether the patient has ADHD or not and to which ADHD sub-cohort the patient belongs (if ADHD-positive).

Claims

Claims1 . An in-vitro method of assessing whether a human person has ADHD, comprising- determining in a sample from dried whole blood or EDTA-plasma from the person the amount, concentration or enzymatic activity of an analyte selected from a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d17:1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3-DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or b) a combination of any of the markers under a, and- comparing the determined amount, concentration or enzymatic activity to a reference by mass spectrometry.

2. The method according to claim 1 , characterized in that said markers are selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d17:1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5 and C16:2-OH.

3. The method according to claim 1 , characterized in that an increased presence of PE P-18:0 / 17:1 , SPB d17:1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, C3DC (C4OH) (aka C3-DC (C4-OH)), C16:2, PA 18:0_18:1 , SPHK activity or LPP3 activity is indicative of ADHD.

4. The method according to claim 1 , characterized in that a decreased presence of SPBP d17:0, PC O- 44:4, LPE 14:0, PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4 or LDH activity is indicative of ADHD.

5. A method of confirming that a human patient has ADHD, comprising the method of any of claims 1 to 4.

6. A method of selecting a human patient for a therapy of ADHD, comprising- performing the method according to any of claims 1 to 5 and- marking the sample for therapy of the patient, if the sample differs in amount, concentration or enzymatic activity from the reference by more than 50 %.

7. An in-vitro method of monitoring a human patient suffering from ADHD or being treated for ADHD, comprising repeatedly performing the method of any of claims 1 to 5.

8. A computer-implemented method assessing whether a human person has ADHD, said method comprising1) obtaining by mass spectrometry a measurement value for the level of an analyte in a sample from dried blood or EDTA-plasma from said person selected from a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d17:1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3-DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or b) a combination of any of the markers under a),2) storing said measurement value in a data base in one or more computer or data base,3) comparing said measurement value to a minimum or maximum threshold value for the level of said marker, compound, metabolite or said combination stored in said data base, and4) outputting a positive signal in case said measurement value exceeds said minimum threshold value or undercuts that maximum threshold value, wherein a positive signal is indicative of the person having ADHD.

9. An in-vitro method of determining by mass spectrometry in a sample from blood or EDTA-plasma from a human person the amount, concentration or enzymatic activity of an analyte selected from a) a metabolomic marker selected from the group consisting of SPB d17: 1 , SPB d17:0, PE P-18:0 / 17:1 , C16:2, PC 0-44:4, C3-DC (C4-OH), LDH Activity, SPHK activity, LPP3 activity, PA 18:0_18:1 , PC O- 36:4, PC 0-38:0 and TG 14:0_36:4, or b) a combination of any of the markers under a), characterized by- providing said sample into a mass spectrometer, and- determining a measurement value for the signal strength of the mass of said marker or the result of said enzymatic activity of said analyte by mass spectrometry.

10. A method for determining a treatment scheme of ADHD for a human patient comprising- determining by mass spectrometry in a sample from dried blood or EDTA-plasma from said patient the amount, concentration or enzymatic activity of an analyte selected from a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d17:1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3-DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or b) a combination of any of the markers under a),- comparing the determined amount, concentration or enzymatic activity to a reference, and- selecting a treatment scheme from the group of available treatment schemes based upon said comparison.11 .A method for determining the amount, concentration or enzymatic activity of any metabolomic marker selected from the group consisting of SPB d17: 1 , SPB d17:0, PE P-18:0 / 17:1 , C16:2, PC 0-44:4, C3- DC (C4-OH), LDH Activity, SPHK activity, LPP3 activity, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0 and TG 14:0_36:4 or a combination of any of these markers in a sample from dried blood or EDTA-plasma from a human person, wherein said amount, concentration or enzymatic activity is determined by LC-MS for the diagnosis of ADHD.

12. A method for determining whether a human person has ADHD comprising a) assessing ADHD by a psychiatric method, and b) assessing ADHD by an in-vitro method using a sample from dried blood or EDTA-plasma from said person, wherein the determination is positive, if the assessments in steps a) and b) are both positive regarding ADHD.

13. A method for screening a multiplicity of samples from dried blood or EDTA-plasma from human persons, wherein each sample of said multiplicity of samples is subjected to determining the amount, concentration or enzymatic activity of an analyte selected from a) a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d17:1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3-DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity, or b) d) a combination of any of the markers under a), and- comparing the determined amounts, concentrations or enzymatic activities to a reference, wherein the differences from the results obtained to the reference value is indicative of ADHD for the respective persons the samples were collected from.

14. The use of mass spectroscopy for the in-vitro determination of a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d17:1 , SPB d18:0, DG 18:0_20:0, PE P- 20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3-DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity or a combination thereof from a sample from dried blood or EDTA-plasma of a human person in the assessment of ADHD in samples of patients suspected to have ADHD.

15. The use of a metabolomic marker selected from the group consisting of SPBP d17:0, PE P-18:0 / 17:1 , SPB d17:1 , SPB d18:0, DG 18:0_20:0, PE P-20:0 / 20:5, C16:2-OH, SPB d17:0, PC 0-44:4, C3DC (C4OH) (aka C3-DC (C4-OH)), C16:2, LPE 14:0, PA 18:0_18:1 , PC 0-36:4, PC 0-38:0, LPE P-16:0, TG 14:0_36:4, LDH activity, SPHK activity and LPP3 activity or a combination thereof for the determination of ADHD in a human person.

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