WO2006090428A2 - Method for the separation and simultaneous direct determination of compounds belonging to at least one of the groups chosen among purines and pyrimidines, n- acetylated amino acids, mono and dicarboxylic acids, sulphurylated compounds, nitrosylated compounds, bifunctional aldehydes, vitamins of group b, and derivatives ther - Google Patents

Abstract

The present invention concerns a method for the separation and simultaneous determination of purines and pyrimidines, N-acetylated amino acids, mono and dicarboxyliσ acids, sulphurylated compounds, nitrosylated compounds, bifunctional aldehydes, vitamins of group B, and derivatives thereof, in a biological sample by high performance liquid chromatography (HPLC) and related kits. In particular, the invention refers to a method for the simultaneous determination in biological samples of compounds of clinical biochemistry interest, for example, to diagnose hereditary dismetabolic pathologies, acute and chronic alterations of metabolism, tumoral pathologies.

Description

METHOD FOR THE SEPARATION AND SIMULTANEOUS DIRECT DETERMINATION OF COMPOUNDS BELONGING TO AT LEAST ONE OF THE GROUPS CHOSEN AMONG PURINES AND PYRIMIDINES, N- ACETYLATED AMINO ACIDS, MONO AND DICARBOXYLIC ACIDS, SULPHURYLATED COMPOUNDS, NITROSYLATED COMPOUNDS, BIFUNCTIONAL ALDEHYDES, VITAMINS OF GROUP B, AND DERIVATIVES THEREOF, IN A BIOLOGICAL SAMPLE BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) AND RELATED KITS

The present invention concerns a method the separation and simultaneous determination of purines and pyrimidines, N-acetylated amino acids, mono and dicarboxylic acids, sulphurylated compounds, nitrosylated compounds, bifunctional aldehydes, vitamins of group B, and derivatives thereof, in a biological sample by high performance liquid chromatography (HPLC) and related kits. In particular, the invention refers to a method for the simultaneous determination in biological samples of compounds of clinical biochemistry interest, for example, to diagnose hereditary dismetabolic pathologies, acute and chronic alterations of metabolism, states of vitamin decrease, pathological conditions of oxidative and nitrosative stress, tumoral pathologies, among which, N- acetylaspartate, N-acetylglutamate, N-acetylaspartylglutamate, cytosine, cytidine, uracil, uridine, β-pseudouridine, adenine, 3-methyladenine, hypoxanthine, xanthine, xanthosine, inosine, guanosine, adenosine, S- adenosylmethionine, S-adenosylhomocysteine, adenylosuccinate, 5- aminoimidazole-4-carboxamide ribonucleotide, ascorbic acid (vitamin C), thymine, thymidine, uric acid, 1-methyluric acid, orotic acid, malonic acid, methylmalonic acid, propionic acid, succinic acid, NO₂ ^', NO₃ ^", malondialdehyde,, vitamin B1 , (thiamine and thiamine pyrophosphate), vitamin B2 (riboflavin, flavin mono nucleotide, flavin adenine dinucleotide), vitamin B6 (pyridoxine, pyridoxamine, pyridoxale, pyridoxic acid, pyridoxine phosphate, pyridoxamine phosphate, pyridoxale phosphate), vitamin B12 (cobalamine), folic acid, creatinine, reduced glutathione and oxidized glutathione. The determination of purine and pyrimidine derivatives, of N- acetylated amino acids and of mono and dicarboxylic acids in biological fluids is very relevant for the chemical diagnosis of numerous inborn errors of metabolism (IEM). A short list of some of these genetic disorders, very often characterized by fatal prognosis, and of the compounds showing the most significant variations with respect to normal values, is reported below: Lesch-Nyhan syndrome (hypoxanthine, xanthine, uric acid, guanosine, inosine, adenine); Phosphoribosylpyrophosphate synthetase (hypoxanthine, xanthine, uric acid); Purine nucleoside phosphorylase deficiency (inosine, guanosine, uric acid); Xanthine dehydrogenase deficiency (hypoxanthine, xanthine, uric acid);

Adeninephosphoribosyltransferase deficiency (adenine); Uridine monophosphate synthetase deficiency (orotic acid); Dihydropyrimidine dehydrogenase deficiency (uracil, thymine); Mitochondrial encephalopathy neuro-gastro-intestinal due to thymidine phosphorylase deficiency (thymine, thymidine, uracil); Hepatocerebral type of the myopathy from mitochondrial DNA depletion due to thymidine kinase deficiency (β- pseudouridine); Diskeratosis congenita (β-pseudouridine); Canavan disease (N-acetylaspartate, N-acetylglutamate, N- acetylaspartylglutamate); Neurometabolic disorder of still unknown origin with clinical aspects similar to those of the Pelizaeus-Merzbacher syndrome (N-acetylaspartylglutamate); Adenylosuccinate lyase deficiency (adenylosuccinate); S-adenosylhomocysteine hydrolase deficiency (S- adenosylhomocysteine); 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and IMP cyclohydrolase deficiency (5-aminoimidazole-4- carboxamide ribonucleoside, 5-aminoimidazole-4-carboxamide ribonucleotide); Methylmalonic acidemia or aciduria from malonyl CoA decarboxylase deficiency (methylmalonic acid, malonic acid, succinic acid); Propionic aciduria or acidemia from propionyl CoA carboxylase deficiency (propionic acid); Succinic aciduria or acidemia (succinic acid).

It can be therefore affirmed that the chemical analysis of biological fluids for the screening of IEM is certainly crucial to obtain a prenatal or neonatal diagnosis, to monitor the development of the disease, to evaluate the effectiveness of eventual therapeutic interventions, as well as to evaluate pathological states characterized by the increase of one or more of the aforementioned compounds (1-5). For these reasons, analytical methods for the determination of the compounds of interest must be characterized by sensitivity, reproducibility, specificity and, in order to have a wide application in the clinical biochemistry laboratories, they should also be characterized by an ease of execution and by relatively moderate costs. Last but not least, in order to ensure reliable analytical results, any method considered must reduce to the minimum sample manipulation.

The aforementioned compounds, the concentration of which is significantly modified in various genetic metabolic disorders, are of very different chemical nature. In fact, N-acetylaspartate (NAA), N- acetylglutamate (NAG), N-acetylaspartylglutamate (NAAG) and dicarboxylic acids are ionizable substances, with two (or three, as in the case of NAAG) negative charges at neutral pH, purine and pyrimidine nucleosides are polar non-ionizable compounds, free purines and pymidines (and most derivatives thereof) are scarcely polar or non-polar compounds. Both in nucleosides and in free purines and pyrimidines, polarity (nucleosides) and apolarity can significantly vary depending on the type of base (purines are more electronegative than pyrimidines) and the chemical nature of the eventual substituting groups linked to the heterocyclic ring(s) (with electron-attractor or electron-repulsor effect). Also for these reasons, analytical methods capable of simultaneously separating compounds of classes with so different chemical properties are not available at present. Several methods to determine in biological fluids for screening purposes purines and pyrimidines (6-16), NAA and NAAG (17-20), dicarboxylic acids (21-25) have been described. No one of these methods, however, allow to separate and quantify synchronously the compounds of these different classes.

The analytical instruments mainly used in the majority of the methods reported in literature are HPLC (for purines and pyrimidines) and gas-chromatography/mass spectrometry (GC/MS, mostly utilized for purines and pyrimidines derivatives, NAA, mono and dicarboxylic acids). The most effective methods are those based on the use of GC/MS, thanks to the high separating efficiency of the gas-chromatographic column and to the high selectivity of the mass spectrometer detector. If GC/MS may be somehow preferable with respect to HPLC because of its efficiency and selectivity, it is not certainly the method of preferential choice for separating polar compounds. Consequently, to allow its application in the separation of ionizable and/or polar compounds (N-acetylated amino acids, mono and dicarboxylic acids, nucleosides) a rather complex sample treatment is required (trimethylsylanization for samples from any origin, preceded by urease pretreatment in the case of urine). In addition, it can also be necessary a stable isotopic dilution for detection of compounds having very low concentration. It is also worth underlining that GC/MS it is not absolutely utilizable for the determination of group B vitamins and for all thermo-labile substances (sulphurylated compounds such as GSH).

The analytical method preferentially chosen for the separation of ionizable and polar compounds it is not gas-chromatography but electrophoresis (capillary electrophoresis) or ion exchange chromatography or ion-pairing chromatography (HPLC), i.e. those techniques based on the principle of the different electric charge for separating ionizable and polar molecules. Reversed phase HPLC, widely used to analyze purines and pyrimidines (6-17), does not allow satisfying results in the separation of ionizable compounds, unless using chromatographic conditions not very convenient to separate purines and pyrimidines (addition of chaotropic agents in the eluents). The difficulty to apply HPLC (with UV-visible spectrophotometric detector) for the separation of N-acetylated amino acids, of mono and dicarboxylic acids, of sulphurylated compounds (GSH and GSSG), of nitrosylated compounds (NO₂ ^" and NO₃ ^"), synchronously with purines and pyrimidines separation, is due to the lack of any characteristic maximum of absorption except that in the far-UV region (below 215 nm wavelength), where unspecific absorbance of unknown compounds might often be revealed. It is however worth recalling that the aforementioned methods have the severe disadvantage of not allowing the simultaneous separation of all these compounds of the different chemical classes (N-acetylated amino acids, purines and pyrimidines, mono and dicarboxylic acids, sulphurylated compounds, nitrosylated compounds, bifunctional aldehydes, group B vitamins).

Among the metabolites that can be analyzed, N-acetylaspartate is of particular importance also because of its correlation with Canavan disease. In the group of N-acetylated amino acids, N-acetylaspartate (NAA) is certainly the most representative one. In mammals, it is mainly localized in cerebral tissue, specifically in neurons (26), where it reaches, under physiological conditions, relevant concentrations (at up to 10 μmol/g wet weight) comparable to those of the neurotransmitter glutamate (27, 28). Notwithstanding it is known that NAA is synthesized in mitochondria by the enzyme N-acetylasparto synthase and degraded by the enzyme N- acetylasparto acylase, its precise biological role is still under intense evaluation. Recent results seem to confirm the hypothesis originally proposed by Baslow (29-31) which suggests that NAA mainly act as a "molecular water pump", being thereby involved in the mechanisms of water extrusion from neurons towards the blood stream.

NAA can be determined in animals by using different analytical methods (HPLC, capillary electrophoresis, GC/MS) and in human beings by using non-invasive techniques such as proton nuclear magnetic resonance spectroscopy (¹H-NIvIRS). The clinical interest for the determination of this compound is related to its direct correlation with neuronal vitality, so that it is considered a valid biological marker for the evaluation of a correct cerebral metabolism. To this purpose, pre-clinical and clinical studies have demonstrated that NAA decreases in various states of acute (32-34) and chronic (35-37) cerebral sufferance. These studies have demonstrated that NAA variations are in close correlation with the patient clinical status, therefore having a relevance even as a prognostic index. In particular, NAA determination in the cerebrospinal fluid (CSF) or in microdialysis samples of patients suffering from different acute and/or chronic cerebral pathologies, such as head trauma, ischemia, post-traumatic coma, concussions, sub-arachnoid hemorrhage, etc., would be of great clinical importance because it may supply indications on the patient clinical evolution and/or it may give information on the effectiveness of eventual pharmacological treatments.

At present, the only exception wherein a dramatic increase of NAA cerebral concentration occurs is represented by the autosomic recessive genetic disease with fatal prognosis known as Canavan disease (38, 39). In this pathological condition, high level of NAA are accumulated in the brain already during fetus gestation and moreover after the birth. This phenomenon occurs because of the synthesis of an inactive (or partially active) form of the N-acetylasparto acylase responsible for NAA degradation (40, 41). Patients suffering from Canavan disease are affected by frequent seizure crisis and show very relevant increase of plasma and urinary NAA concentration. For this reason, the clinical diagnosis is also based on NAA determination in biological fluids (17-19, 42, 43). Similarly, pre-natal diagnosis of Canavan disease is also performed on the basis of the results of NAA quantification in amniotic fluid (44, 45).

High performance liquid chromatography (HPLC) for separating low molecular weight compounds in biological samples has been utilized in the last decades in a great number of pre-clinical and clinical studies, thanks to the remarkable versatility of this technique to be adapted for separating substances of different chemical nature.

In general, chromatographic conditions most frequently adopted to determine low molecular weight compounds require the use of HPLC column wherein the stationary phase is represented by silica gel bonded with carbon chains of 18 C atoms (octadecilsylane, ODS C-18), with particle size of 3 or 5 μm, length of 150 or 250 mm, internal diameter of 4,6 or 2,1 mm, pore size of 80 or 120 A. It is very important to note that the majority of the HPLC methods adopt chromatographic condition characteristic of the reversed phase, i.e. those conditions wherein the stationary phase is less polar than the mobile phase. Therefore, reversed phase chromatography allows to separate analytes depending on their different hydrofobicity, with the most hydrophobic ones having the highest affinity for the stationary phase. Hence, it easy to imagine that polar and ionizable compounds are not retained by the column and are eluted with the void volume of the system. To try to reduce this phenomenon and to try to increase their retention times, polar and ionizable compounds are preferentially separated by reversed phase HPLC using pH values of the eluents in such a way to eliminate charges of the molecules, thus slightly increasing their interaction with the stationary phase. To this purpose, several examples of separation and quantification of polar and highly polar compounds in biological fluid by the use of reversed phase HPLC can be found in literature (48-51). It should however be recalled that the capacity to separate this type of compounds but having similar values of k' (k' = retention factor = V-V₀ZVo, where V the elution volume and Vo is the void volume) is certainly decreased under these chromatographic conditions (reversed phase).

Numerous reversed phase HPLC methods have been developed to separate and quantify in biological fluids, with reasonable separating efficiency, compounds related to IEM such as purine and pyrimidine derivatives (6-16). Obviously, polar and ionisable compounds were not considered in these studies (because of the inability of reversed phase HPLC to separate simultaneously apolar, polar and ionisable substances), thereby limiting the number of hereditary metabolic pathologies, and of dismetabolisms in general, that can be evaluated by these analyses using reversed phase HPLC. Having to measure compounds of variable polarity (from non- polar to ionisable), ion-pairing HPLC certainly offers higher separating capacity. In this type of chromatography a normal reversed phase ODS C- 18 (having any of the characteristics reported above) is used. The difference is that variable concentration of a so called pairing reagent is added to the eluents. The pairing reagent is, by definition, a hydrophobic molecule with a chemical group practically dissociated at any pH value. In the case of the pairing reagent cationic exchanger, a short saturated hydrocarbon chain of 5-8 C atoms (these chains interacting with the C-18 hydrocarbon chains of the stationary phase) is functionalized with a sulphonic group. In the case of an anionic exchanger, a quaternary ammonium is tetra-substituted with 4 short saturated hydrocarbon chains of 4-8 C atoms (these chains interacting with the C-18 hydrocarbon chains of the stationary phase). In both cases, the addition of the pairing reagent to the eluents allows to cover the stationary phase surface of negative or positive charges. The difference with the normal ion exchanger HPLC columns is that under the chromatographic conditions used for ion-pairing HPLC the net charge on the stationary phase (either positive or negative) is partially masked by the presence of the hydrophobic component, with the final result of having a "moderate to weak" ionic exchanger. Furthermore, the advantage with respect to the phases for the classical ion-exchange HPLC is that the anionic pairing reagents (such as tetrabutylammonium) allow, because of the presence of 4 hydrophobic substituting groups, the interaction with non polar and/or low polar analytes. Such a peculiarity tremendously increases the possibility to effect synchronous separations of compounds having among them very different polarities (46-49).

By applying the ion pairing HPLC technique, chromatographic methods for the simultaneous separation and quantification of malondialdehyde and adenylic derivatives (50), oxypurines, nucleotides, ribonucleotides and deoxyribonucleotides (51), NAA and NAG (52), representative compounds of the redox and energy states (53), in cell or tissue samples, have been set up in the past. In the last paper cited, it has also been set up a deproteinization procedure with minimal sample manipulations, capable of preserving either the oxido-reductive state of compounds with different oxidation states or the concentrations of compounds with high turn over velocity, thanks to a rapid and efficient protein precipitation. Several of these methods have been successfully applied in numerous pre-clinical and clinical studies (54-60). It is however worth underlining that the aforementioned methods have evident limitations that significantly reduce the possibility of their application: in fact, the method described in (50) is only referred to the separation of compounds of a same chemical class (adenylic derivatives) but malondialdehyde and does not allow the separation of N-acetylated amino acids, of mono and dicarboxylic acids, of sulphurylated compounds, of nitrosylated compounds, of group B vitamins; the method described in (51) is devoted to the separation of ribonucleotides from the corresponding deoxyribonucleotides, plus some additional compounds (nucleosides and oxypurines) and it does not allow the separation of N-acetylated amino acids, of mono and dicarboxylic acids, of sulphurylated compounds, of nitrosylated compounds, of group B vitamins; the method described in (52) allows the separation of two N-acetylated amino acids only and hinders (for the chromatographic conditions adopted) the separation of mono and dicarboxylic acids, of sulphurylated compounds, of nitrosylated compounds, of group B vitamins and of purines and pyrimidines; the method described in (53) is exclusively finalized to the description of a deproteinization procedure of the biological sample capable of preserving compounds particularly exposed to change in their concentration caused by drastic deproteinizing conditions.

On the other hand, in the clinical biochemistry field the determination of nitrosylated compounds such as NO₂ ^"and NO₃ ^" has acquiring increasing importance in several pathological states characterized by the so called nitrosative stress (61-63). In fact, NO₂ ^"and NO₃ ^' are among the main products deriving from the transformation of NO (nitric oxide). NO is generated in relevant concentrations by the inducible form of nitric oxide synthase. This enzyme is activated in various pathological disorders including tissue ischemia and reperfusion, diabetes, head trauma, acute and chronic inflammations, degenerative neuropathies (Parkinson's disease, Alzheimer' disease, multiple sclerosis, etc.). It is therefore clear that NO₂ ^"and NO₃ ^" are considered as dosimeters of NO production and have a diagnostic and prognostic significance as far as the aforementioned diseases is concerned. To testify the importance of NO₂ ^" and NO₃ ^" determination it should be mentioned that it has been recently marketed an HPLC device exclusively devoted to the dosage of these compounds in biological samples. It is however worth underlining that both this instrumentation and the analytical kits of the spectrophotometric type suffer of some limitations. First of all, all the commercially available methods, as well as those described in literature, allow the quantification of Nθ2^"and NO₃ ^" only excluding from the analysis compounds of the other chemical classes comprised in the present Patent of invention, i.e. purines, pyrimidines, mono and dicarboxylic acids, suplhorylated compounds, bifunctional aldehydes, group B vitamins. In addition, both spectrophotometric methods and the most recent HPLC technique require sample derivatization with a specific reagent (Griess reagent) which gives rise to an optically active adduct that, unfortunately, increases remarkably sample manipulation thus rendering doubtful the analytical datum obtained in complex biological samples.

As in the case of nitrosative stress, the use of biological matkers of oxidant stress caused by reactive oxygen species (ROS) is becoming a necessity for diagnostic purposes. In fact, the only technique presently capable of directly measuring ROS is electron spin (paramagnetic) resonance (ESR or EPR), but unfortunately concentrations of these species in vivo are not sufficient to obtain a detectable ESR signal. To overcome this problem are generally utilized as markers of already occurred oxidative stress diverse molecules deriving from the reaction of ROS with target compounds such as DNA, proteins and polyunsaturated lipids. MDA, the bifunctional aldehyde deriving from ROS oxidized polyunsaturated fatty acids, seems to be a stable and reliable index for an indication of oxidant stress, when MDA is determined with appropriate analytical methods. In fact, its analysis in clinical biochemistry correlated to the importance that ROS have in the pathogenesis of several pathologies as well as in physiological processes (aging), is of primary importance even without a visible clinical amelioration. The determination of this marker can be particularly important in ischemia and reperfusion phenomena (for example in myocardial and cerebral ischemia) (64), in cerebral pathologies characterized by a remarkable ROS production (stroke, head injury, etc.) in degenerative neuropathies (Parkinson's disease, Alzheimer's disease, multiple and lateral amyotrophic sclerosis) (65), in diabetes, in acute and chronic inflammatory states, etc. Even in the aging process and in mental retardation present in the Down syndrome, characterized by a ROS overproduction oxidative stress monitoring, and hence of MDA, has a fundamental importance. But the analysis of MDA can also be adopted as an easy diagnostic technique that can be used by competent physicians to check the health status in working places. In fact, the constant control of the oxidant state is very important in some worker categories, such as of those workers involved in benzene-containing fuel industry, or of those workers of specific chemical industries. The presently available methods, based on the determination of the adducts formad between MDA and thiobrabituric acid, have all the same common defect of the lack of specificity in the reaction MDA- thiobarbituric acid, giving therefore rise to analytical errors, even of a great order of magnitude, concerning the real levels of this compound. Up to now, some HPLC methods based on the direct MDA determination in biological samples allowing a reliable quantification of this compound have been described (66, 67). However, these methods do not permit to synchronously measure the compounds of interest separated and quantified by the present Patent of invention.

The importance of the determination of group B vitamins and of folic acid in biological fluids lies in the role that these compounds (and derivatives thereof) have for the correct functioning of the cell metabolism, due to their fundamental activity of coenzymes and cofactors of key enzymes of metabolic cycles and pathways. For example, vitamin B1 thiamine and its phosphorylated derivative thiamine pyrophosphate (TPP) is crucial for the correct functioning of pyruvate dehydrogenase (PDH), a key enzyme for the oxidative decarboxylation of pyruvate deriving from the glucose catabolism via glycolysis, and the concomitant acetyl CoA formation which will then be condensed with oxaloacetate to form citric acid in the tricarboxylic acids cycle (Krebs cycle). Differently, vitamin B2 or riboflavin is the precursor necessary for the formation of the flavinic coenzymes flavin mono nucletotide (FMN) and flavin adenin dinucleotide (FAD). FMN and FAD have the function to transfer electrons in the oxido- reductive reactions and are cofactors of the enzymes of the oxidoreductase class, known as flavoenzymes or flavoproteins. Among them, we mention succinate dehydrogenase which plays the double role of fundamental enzyme of the tricarboxylic acid cycle and of electron transporter in the electron transport chain that, coupled with the mitochondrial oxidative phosphorylation and hence with adenosintriphosphate (ATP) production, is determinant for the activity of enzymes involved in biosynthetic reactions of biological macromolecules, having the function of carbon group transporter. In general, it should be reminded that the aforementioned vitamins and congeners profile has a wide application in nutritional medicine (both in pedriatic and adult age), in ginecology (for monitoring the pregnant state), in cardiology (for the possible correlation that the vitamin profile might have with ischemic cardiopathies). Furthermore, folic acid, thiamine, pyridoxine, cobalamine, are used in the pharmacological treatment of several IEM, wherein the monitoring of their plasma concentrations is often necessary to calibrate their correct dosage. Given the clinical relevance of the analysis of group B vitamins and of folic acid, several are the analytical methods available in literature, particularly those based on the use of HPLC (68-73). It must again be recalled and underlined that all these methods allow the determination of one or more subclasses of group B vitamins but no one of them is capable of performing the whole profile of B1 (thiamine and TPP), B2 (riboflavin, FMN, FAD), B6 (pyridoxine, pyridoxale, pyridoxamine, pyridoxic acid and their phosphorylated derivatives, first of all PLP), B12 (cobalamine) and folic acid. Moreover, all these methos do not allow the separation and quantification of the other compounds reported in the present Patent of invention (purines a,d pyrimidines, N-acetylated amino acids, mono and dicarboxylic acids, sulphurylated compounds, nitrosylated compounds, bifunctional aldehydes).

According to what reported above, it is therefore manifest the necessity to have new methods for the determination of analytes belonging to the different chemical classes that are present in biological samples.

The Authors of the present invention have now set up an original method for the synchronous separation and determination of purines, pyrimidines, N-acetylated amino acids, mono and dicarboxylic acids, sulphurylated compounds, nitrosylated compounds, bifunctional aldehydes, group B vitamins, and derivatives thereof, in biological samples by using high performance liquid chromatography (HPLC). The method according to the present invention allow the concomitant separation and determination in a single chromatographic run only of compounds present in biological samples such as NAA, NAG, NAAG, cytosine, cytidine, uracil, uridine, β-pseudouridine, adenine, 3-methyladenine, hypoxanthine, xanthine, xanthosine, inosine, guanosine, adenosine, SAM, S- adenosylhomocysteine, adenylosuccinate, 5-aminoimidazole-4- carboxamide ribonucleotide (AICAR), ascorbic acid (vitamin C), thymine, thymidine, uric acid, 1-methyluric acid, orotic acid, malonic acid, methylmalonic acid, propionic acid, succinic acid, NO₂ ^", NO₃ ^', MDA, vitamin B1 , (thiamine and TPP), vitamin B2 (riboflavin, FMN, FAD), vitamin B6 (pyridoxine, pyridoxamine, pyridoxale, pyridoxic acid, pyridoxine phosphate, pyridoxamine phosphate, PLP), vitamin B12 (cobalamine), folic acid, creatinine, reduced glutathione (GSH) and oxidized glutathione (GSSG). The method according to this invention, besides of introducing the significant innovation of permitting the concomitant separation of the aforementioned compounds, it ameliorates the sensitivity relatively to the determination of NAA, cytosine, cytidine, uracil, uridine, adenine, hypoxanthine, xanthine, inosine, guanosine, ascorbic acid, thymine, thymidine, uric acid, orotic acid, GSH, GSSG and allows the determinationof compounds like NAG and NAAG for which no advantageous analytical chromatographic HPLC method was available at present.

The method according to the present invention is characterized by very high sensitivity, reproducibility, ease of execution, sample preparation free of any manipulation altering the low molecular metabolite content and, last but not least, low cost. Furthermore, the new method permits the determination of the aforementioned compounds, each of them related to one or more IEM or acute and chronic dismetabolic states, in either biological fluids or tissues. The method can therefore be applied for the pre-natal and neonatal clinical-biochemicaldiagnosis of patients suffering from various IEM₁ the screening of at risk population, as well as the monitoring of patients suffering from acute and chronic pathological states able to alter the physiological concentrations in corporeal fluids and tissues of one or more compounds separated with this method, with respect to values detected in control subjects, the monitoring of the nutritional state and of the concentrations of the congeners belonging to the group B vitamins and of folic acid as a consequence of the therapeutic administration of pharmaceutical preparations containing the aforementioned vitamins.

Among the main pathologies or pathological states for which to apply the method according to the invention we can mention Canavan disease (NAA); methylmalonic aciduria (mathylmalonic acid, malonic acid); vitamin B12 deficiency (methylmalonic acid); Hypoxanthine phopshporibosyl transferase (HPRT) deficiency or Lesch-Nyhan syndrome (hypoxanthine, xanthine, uric acid, guanosine, inosine, adenine); Phosphoribosylpyrophosphate synthetase deficiency (hypoxanthine, xanthine, uric acid); Purine nucleoside phosphorylase deficiency (hypoxanthine, xanthine, uric acid); Xanthine dehydrogenase deficiency (hypoxanthine, xanthine, uric acid); Adenine phosphoribosyltransferase deficiency (adenine); Uridine monophosphate synthase deficiency δorotic acid); Dihydropyrimidine dehydrogenase deficiency (uracil, thymine); Pelizaeus-Merzbacher's disease (NAAG); Diskeratosis congenital (β- pseudouridine); Mitochondrial myopathy and sideroblastic anemia by defective β-pseudouridine synthase (β-pseudouridine); Neuro-gastro- intestinal mitochondrial encephalopathy by thymidine phosphorylase deficiency (thymine, thymidine, uracil); the Hepato-cerebral form of the myopathy caused by mitochondrial DNA depletion caused by thymidine kinase deficiency (thymidine); Adenylosuccinate lyase deficiency (adenylosuccinate); S-adenosylhomocysteine hydrolase deficiency (S- adenosylhomocysteine); 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and IMP cyclohydrolase deficiency (AICAR); Methylmalonic aciduria by methylmalonyl CoA decarboxylase deficiency (methylmalonic acid, malonic acid, succinic acid); Propionic acidemia or aciduria by propionyl CoA carboxylase deficiency (propionic acid); Succinic acidemia or aciduria (succinic acid); Ciliac disease (1-methyluric acid); the Diagnosis of numerous tumoral form in association with specific hematological antibodies (β-pseudouridine, 3-methyladenine); Head injury (NAA, hypoxanthine, xanthine, uric acid, inosine, adenosine, ascorbic acid, GSH, GSSG, NO₂ ^', NO₃ ^" and MDA); Stroke (NAA, hypoxanthine, xanthine, uric acid, inosine, adenosine, ascorbic acid, GSH, GSSG, NO₂ ^", NO₃ ^" and MDA); Sub-arachnoid hemorrhage (NAA, hypoxanthine, xanthine, uric acid, inosine, adenosine, ascorbic acid, GSH, GSSG, NO₂ ^", NO₃ ^" and MDA); Acute myocardial infarction (hypoxanthine, xanthine, uric acid, inosine, adenosine, ascorbic acid, GSH, GSSG, NO₂ ^", NO₃ ^" and MDA); all the pathological states characterized by the depletion of folic acid or one or more of the compounds belonging to the group B vitamins; alia the pathological states, acute and chronic, capable of altering the concentrations in the biological fluids of one or more of the compounds that can be separated and quantified by means of the present chromatographic method.

It is evident from what reported that also the monitoring of patients suffering from Canavan disease, as well as the screening for the pre-natal and neonatal diagnosis of subjects considered at risk, can be effected by using this original HPLC method, in place of the time- consuming methodologies used until now for this purpose.

Moreover, as far as the use of this analytical method for the chemical diagnosis of IEM is concerned, it is also possible its application, in a diagnostic step immediately successive to the determination of the metabolite levels in biological fluids, for the evaluation ex vivo of the activity of the enzymes involved in those particular IEM. For example, the hypoxanthine phsphoribosyltransferase (HPRT) deficiency, responsible for the Lesch-Nyhan syndrome, can be evaluated following the reaction hypoxanthine + phosphoribosylpyrophosphate (PRPP) -> AMP + ribose-5- phosphate, by assaying the concentration of AMP (purine derivative that can be separated and quantified by the present analytical method) wherein the AMP generated in a given amount of time is directly proportional to the HPRT activity; the deficiency of purine nucleoside phosphorylase (PNP) can be evaluated by following the reaction inosine + inorganic phosphate τ> hypoxanthine + ribose-1 -phosphate, by means of the determination of hypoxanthine (purine derivative that can be separated and quantified by the present analytical method) wherein the hypoxanthine generated in a given amount of time is directly proportional to the PNP activity. By following any time is necessary this principle, it is therefore possible to diagnose xanthine dehydrogenase deficiency, adenine phosphoribosyltransferase deficiency, uridine monophosphate synthetase deficiency, dihydropyrimidine dehydrogenase deficiency, adenylosuccinate lyase deficiency, S-adenosylhomocysteine hydrolase deficiency, 5- aminoimidazole-4-carboxamide formyltransferase and IMP cyclohydrolase deficiency, malonyl CoA decarboxylase deficiency, propionyl CoA carboxylase deficiency.

It is therefore specific object of the present invention a method for the simultaneous direct separation and quantification of compounds belonging to at least one group, preferably to at least two groups, more preferably to several groups, chosen among purines, pyrimidines, N- acetylated amino acids, mono and dicarboxylic acids, sulphurylated compounds, nitrosylated compounds, bifunctional aldehydes, group B vitamins, and derivatives thereof, in a biological sample by ion-pairing high performance liquid chromatography (HPLC) which uses a reversed phase column, said method requiring the formation of a step gradient obtained by subsequent elution phases with the use of two buffer solutions, A and B, having, respectively, essentially neutral pH and essentially acidic pH both buffers comprising an anionic exchanger pairing reagent, the first elution phase being an isocratic phase with 100% of buffer A having essentially neutral pH. In particular, purines and derivatives thereof can be chosen from the group consisting in adenine, 3-methyladenine, hypoxanthine, xanthine, xanthosine, inosine, guanosine, adenosine, SAM, S- adenosylhomocysteine, adenylosuccinate, uric acid, 1-methyluric acid. Pyrimidines and derivatives thereof can be chosen from the group consisting in cytosine, cytidine, uracil, uridine, β-pseudouridine, thymine, thymidine, orotic acid, AICAR. N-acetylated amino acids and derivatives thereof can be chosen from the group consisting in NAA, NAG, NAAG, creatinine. Mono and dicarboxylic acids and derivatives thereof can be chosen from the group consisting in ascorbic acid, malonic acid, methylmalonic acid, propionic acid, succinic acid, folic acid. Nitrosylated compounds and derivatives thereof can be chosen from the group consisting in NO₂ ^" e NO₃ ^". Bifunctional aldehydes and derivatives thereof can be chosen from the group consisting in MDA. Group B vitamins and derivatives thereof can be chosen from the group consisting in vitamin B1 (thiamine and TPP), vitamin B2 (riboflavin, FMN, FAD), vitamin B6 (pyridoxine, pyridoxamine, pyridoxale, pyridoxic acid, pyridoxine phosphate, pirydoxamine phosphate, PLP), vitamin B12 (cobalamine). Sulphurylated compounds and derivatives thereof can be chosen from the group consisting in GSH and GSSG. The biological sample that can be subjected to the method according to the present invention can be chosen from the group consisting in urine, plasma, amniotic fluid, cerebrospinal fluid or tissues, Preferably, biological sample is previously treated to protein removal prior to the chromatographic run. Sample deproteinization can be carried out with any known method, including:

1. Dialysis filtration on membrane with proper molecular weight cut-off, for example a membrane with a molecular weight cut-off of 3 kDa, capable of retaining proteins and allowing the passage of a filtrate containing all the molecular weight compounds, comprised those of interest.

2. Precipitation by addition of organic solvent (acetonitrile, primary, secondary and tertiary alcohols, phenols, etc,), followed by centrifugation to remove precipitated proteins, organic solvent removal by evaporation or extraction with different organic solvent not mixable with water, in such a way to leave an aqueous phase containing low molecular weight compounds, comprised those of interest. 3. Precipitation by addition of concentrated acids or bases

(perchloric acid, hydrochloric acid, trichloroacetic acid, trifluoroacetic acid, sulphuric acid, phosphotungstic acid, phosphoric acid, nitric acid, formic acid, etc.; sodium hydroxide, potassium hydroxide, calcium hydroxide etc.), followed by centrifugation to pellet precipitated proteins, neutralization of the resulting aqueous phase and containing the low molecular weight compounds, comprised those of interest.

4. Precipitation by the addition of concentrated salts (sodium chloride, potassium chloride, magnesium chloride, ammonium sulphate, uranyl acetate, etc.) followed by centrifugation to pellet precipitated proteins and subsequent excess salt removal (filtration through membranes, gel-filtration, etc.) from the aqueous solution obtained and containing the low molecular weight compounds, comprised those of interest.

The aqueous phase obtained with any proper method for protein removal from the starting biological fluid is directly injected in the

HPLC chromatographic device for the analysis of the compounds of interest. It is worth noting that protein removal should be effected on recently withdrawn samples, once performed sample deproteinization it can be saved at low temperature (at least -20 ⁰C) without any alteration in the metabolite contente for at least three weeks.

Preferably, the reversed phase column used according the method of the present invention is an ODS C-18.

Buffer solutions that can be used according to the present invention comprise a quaternary ammonium ion, a polar organic solvent, a salt of a weak acid.

According to a preferred embodiment, the method can require the use of a reversed phase column ODS C-18; a buffer A comprising a quaternary ammonium ion ranging from 8 to 15 mM, preferably from 10 to 13 mM, more preferably equal to 12 mM, a polar organic solvent ranging from 0.01 to 1%, preferably from 0.1 to 0.5%, more preferably equal to 0.125%, a salt of weak acid ranging from 5 to 15 mM, preferably from 8 to 13, more preferably equal to 10 mM, pH ranging form 6.5 to 7.5, preferably 7.00; a buffer B comprising a quaternary ammonium ion ranging from 0.01 to 3.5 mM, preferably from 2 to 3 mM, more preferably 2.8 mM, a polar organic solvent ranging from 20 to 80%, preferably from 25 to 35%, more preferably 30%, a salt of a weak acid from ranging 1 to 130 mM, preferably, from 80 to 110 mM, more preferably 100 mM, pH ranging from 4.5 to 6.5, preferably 5.50; a step gradient obtained by an initial isocratic phase with 100% of buffer A having a minimal duration of 5 minutes and maximal of 35 minutes, preferably 25 minutes; 5 to 20 minutes at up to have a percentage of buffer A ranging from 95 and 70%, preferably 8 minutes at up to 80% of buffer A; 5 to 30 minutes at up to have a percentage of buffer A ranging from 85 and 50%, preferably 10 minutes at up to 70% of buffer A; 8 to 30 minutes at up to have a percentage of buffer A ranging from 80 and 40%, preferably 12 minutes at up to 55% of buffer A; 4 to 25 minutes at up to have a percentage of buffer A ranging from 70 and 30%, preferably 11 minutes at up to 40% of buffer A; 5 to 25 minutes at up to have a percentage of buffer A ranging from 50 and 10%, preferably 9 minutes at up to 15% of buffer A; 5 to 20 minutes at up to have a percentage of buffer A ranging from 30 and 0%, preferably 10 minutes at up to 0% of buffer A; 10 to 90 minutes with a percentage of buffer A of 0%, preferably 60 minutes at 0% of buffer A; column washing, at the end of the chromatographic run, for an additional time interval ranging from 5 to 40 minutes, preferably 10 minutes, with 100% of buffer B and re-equilibration of the column with 100% of buffer A for at least 20 minutes, preferably 20 minutes. As far as the buffer solutions is concerned, the quaternary ammonium ion can be chosen in the group consisting in tetrabutylammonium, tetramiristylammonium, tetraoctylammonium; the polar organic solvent can be chosen in the group consisting in acetonitrile, phenols, primary, secondary and tertiary alcohols; the weak acid salt can be chosen in the group consisting in potassium, sodium or ammonium acetate, potassium, sodium or ammonium phosphate, potassium, sodium or ammonium formiate. The reversed phase ODS C-18 column can be chosen in the group consisting in columns having particle size, length and diameter, respectively, of 5 μm, 15 cm, 2.1 mm; 5 μm, 25 cm, 2.1 mm; 3 μm, 25 cm, 2.1 mm; 5 μm ,15 cm, 3.0 mm; 5 μm, 15 cm, 4.0 mm; 5 μm, 25 cm, 3.0 mm; 5 μm, 25 cm, 4.0 mm; 3 μm, 15 cm, 4.6 mm; 3 μm, 15 cm, 3.0 mm; 3 μm, 15 cm, 4.0 mm; 5 μm, 30 cm, 4.0 mm; 5 μm, 10 cm, 4.6 mm; 5 μm, 25 cm, 4.6 mm; 5 μm, 10 cm, 3.0 mm; 5 μm, 10 cm, 4.0 mm; 5 μm, 30 cm, 1.0 mm.

The flow of eluent that must be used varies with the column type, in particular, for column having particle size, length and diameter, respectively, of 5 μm, 15 cm, 2.1 mm; 5 μm, 25 cm, 2.1 mm or 5 μm ,15 cm, 3.0 mm the flow ranges from 0.1 to 0.8 ml/min; for column having particle size, length and diameter, respectively, of 5 μm, 15 cm, 4.0 mm or 5 μm, 25 cm, 3.0 mm the flow ranges from 0.2 to 1 ml/min; for column having particle size, length and diameter, respectively, of 3 μm, 25 cm, 2.1 mm; 5 μm, 25 cm, 4.6 mm; 5 μm, 25 cm, 4.0 mm; 3 μm, 15 cm, 4.6 mm; 3 μm, 15 cm, 3.0 mm; 3 μm, 15 cm, 4.0 mm; 5 μm, 30 cm, 4.0 mm; 5 μm, 10 cm, 4.6 mm; 5 μm, 10 cm, 3.0 mm; 5 μm, 10 cm, 4.0 mm; 5 μm, 30 cm, 1.0 mm, the flow ranges, respectively, from 0.05 to 0.6 ml/min, from 0.5 to 1.5 ml/min, from 0.2 a 1.2 ml/min, from 0.5 a 1.2 ml/min, from 0.4 to 1 ml/min, from 0.4 to 1.1 ml/min, from 0.3 to 1.2 ml/min, from 0.5 to 2 ml/min, from 0,5 to 1 ,5 ml/min, from 0.5 to 1.8 ml/min, from 0.01 to 0.5 ml/min.

The columns can be maintained at a temperature ranging from 5 to 25 ⁰C, preferably from 8 to 18 ⁰C, more preferably temperature is equal to 10 ⁰C.

A particular embodiment of the method according to the present invention is realized by the use of a ODS- C-18 reversed phase column with 5 μm particle size, 250 mm length and 4.6 mm diameter; a buffer A containing 12 mM tetrabutylammonium hydroxide, 0.125% methanol, 10 mM monobasic potassium phosphate, pH 7.00; a buffer B containing 2.8 mM tetrabutylammonium hydroxide, 30% methanol, 100 mM monobasic potassium phosphate, pH 5.50; a step gradient obtained by 25 minutes of 100% buffer A; 8 minutes at up to 80% buffer A; 10 minutes at up to 70% buffer A; 12 minutes at up to 55% buffer A; 11 minutes at up to 40% buffer A; 9 minutes at up to 15% buffer A; 10 minutes at up to 0% buffer A; 60 with 0% buffer A; column washing, at the end of the chromatographic run, for 10 additional minutes with 100% buffer B and column re-equilibration with 100% buffer A for 20 minutes; an eluent flow maintained at 1.2 ml/min and a temperature of the chromatographic column kept at 10 °C.

The detection of the compounds according to the method of the present invention is carried out by means of a spectrophotometric detector for HPLC at a wavelength ranging from 200 to 225 nm, preferably 206, for NAA, NAG, NAAG, malonic acid, methylmalonic acid, propionic acid, succinic acid, NO₂ ^", NO₃ ^", GSH, GSSG; at a wavelength ranging from 240 to 300 nm, preferably 260 nm, for cytosine, cytidine, uracil, uridine, β- pseudouridine, adenine, 3-methyladenine, hypoxanthine, xanthine, xanthosine, inosine, guanosine, adenosine, SAM, S- adenosylhomocysteine, adenylosuccinate, MDA, ascorbic acid, thymine, thymidine, uric acid, 1-methyluric acid, orotic acid, AICAR; at the wavelength ranging from 220 to 240 nm, preferably 234 nm, for creatinine; at the wavelength ranging from 300 to 500 nm, preferably 340 nm, for vitamin B1 (thiamine, TPP), vitamin B2 (riboflavin, FMN, FAD), vitamin B6 (pyridoxine, pyridoxamine, pyridoxale, pyridoxic acid, pyridoxine phosphate, pyridoxamine phosphate, PLP), vitamin B12 (cobalamin), folic acid.

Detection can be performed by means of a detector selected from the group consisting of high sensitivity diode array spectrophotometric detector, with a 5 cm light path flow cell, diode array spectrophotometric detector, variable wavelength UV-visible spectrophotometric detector, spectrofluorimetric detector, mass spectrometric detector. The diode array spectrophotometric detector can be connected in parallel (and/or in series) with a spectrofluorimetric or a mass spectrometric detector.

A preferred embodiment applies a method specifically devoted to the separation and quantification of purines, pyrimidines and derivatives thereof wherein the reversed phase column is an ODS-C18 with 5 μm particle size, 250 mm length and 4.6 mm diameter; buffer A comprises 12 mM tetrabutylammonium hydroxide, 0.125% methanol, 10 mM monobasic potassium phosphate, pH 7.00; buffer B comprises 1 mM tetrabutylammonium hydroxide, 70% methanol, 10 mM monobasic potassium phosphate, pH 5.50; a step gradient obtained by 25 minutes of 100% buffer A; 15 minutes at up to 80% buffer A; end of the chromatographic run after 45 minutes from sample injection, washing with 100% buffer B for 15 minutes and subsequent column equilibration with 100% buffer A for 20 minutes before the injection of the next sample.

A further preferred embodiment consists in a method specifically devoted to the separation and quantification of mono and dicarboxylic acids and derivatives thereof wherein the reversed phase column is an ODS-C18 with 5 μm particle size, 250 mm length and 4.6 mm diameter; buffer A comprises 12 mM tetrabutylammonium hydroxide, 0.125% methanol, 10 mM monobasic potassium phosphate, pH 7.00; buffer B comprises 1 mM tetrabutylammonium hydroxide, 80% methanol, 1 mM monobasic potassium phosphate, pH 5.50; a step gradient obtained by 23 minutes of 100% buffer A; 5 minutes at up to 80% buffer A; end of the chromatographic run after 30 minutes from sample injection, washing with 100% buffer B for 15 minutes and subsequent column equilibration with 100% buffer A for 20 minutes before the injection of the next sample. Furthermore, it is a matter of the present invention a method specifically devoted to the separation and quantification of N-acetylated amino acids and derivatives thereof wherein the reversed phase column is an ODS-C18 with 5 μm particle size, 250 mm length and 4.6 mm diameter; buffer A comprises 12 mM tetrabutylammonium hydroxide, 0.125% methanol, 10 mM monobasic potassium phosphate, pH 7.00; buffer B comprises 0.2 mM tetrabutylammonium hydroxide, 80% methanol, 1 mM monobasic potassium phosphate, pH 5.50; a step gradient obtained by 28 minutes of 100% buffer A; 5 minutes at up to 90% buffer A; end of the chromatographic run after 35 minutes from sample injection, washing with 100% buffer B for 15 minutes and subsequent column equilibration with 100% buffer A for 20 minutes before the injection of the next sample.

The present invention concerns, in addition, a method specifically devoted to the separation and quantification of nitrosylated compounds and derivatives thereof wherein the reversed phase column is an ODS-C18 with 5 μm particle size, 250 mm length and 4.6 mm diameter; buffer A comprises 12 mM tetrabutylammonium hydroxide, 0.125% methanol, 10 mM monobasic potassium phosphate, pH 7.00; buffer B comprises 0.3 mM tetrabutylammonium hydroxide, 80% methanol, 1 mM monobasic potassium phosphate, pH 5.50; a step gradient obtained by 30 minutes of 100% buffer A; 5 minutes at up to 80% buffer A; end of the chromatographic run after 35 minutes from sample injection, washing with 100% buffer B for 15 minutes and subsequent column equilibration with 100% buffer A for 20 minutes before the injection of the next sample.

According to a further preferred embodiment, the invention refers to a method specifically devoted to the separation and quantification of purines, sulphurylated compounds, nitrosylated compounds, mono and dicarboxylic acids, bifunctional aldehydes and derivatives thereof wherein the reversed phase column is an ODS-C18 with 5 μm particle size, 250 mm length and 4.6 mm diameter; buffer A comprises 12 mM tetrabutylammonium hydroxide, 0.125% methanol, 10 mM monobasic potassium phosphate, pH 7.00; buffer B comprises 0.1 mM tetrabutylammonium hydroxide, 80% methanol, 1 mM monobasic potassium phosphate, pH 5.50; a step gradient obtained by 35 minutes of 100% buffer A; 5 minutes at up to 85% buffer A; end of the chromatographic run after 40 minutes from sample injection, washing with 100% buffer B for 15 minutes and subsequent column equilibration with 100% buffer A for 20 minutes before the injection of the next sample.

It is an additional object of the present invention a method specifically devoted to the separation and quantification of group B vitamins, mono carboxylic acids and derivatives thereof wherein the reversed phase column is an ODS-C18 with 5 μm particle size, 250 mm length and 4.6 mm diameter; buffer A comprises 12 mM tetrabutylammonium hydroxide, 0.125% methanol, 10 mM monobasic potassium phosphate, pH 7.00; buffer B comprises 0.1 mM tetrabutylammonium hydroxide, 80% methanol, 1 mM monobasic potassium phosphate, pH 5.50; a step gradient obtained by 10 minutes of 100% buffer A; 5 minutes at up to 85% buffer A; 13 minutes at up to 70% buffer A; 7 minutes at up to 53% buffer A; 11 minutes at up to 38% buffer A; 9 minutes at up to 15% buffer A; 5 minutes at up to 0% buffer A; end of the chromatographic run after 70 minutes from sample injection, washing with 100% buffer B for 10 minutes and subsequent column equilibration with 100% buffer A for 20 minutes before the injection of the next sample.

It represents a further object of the present invention a device for the simultaneous direct separation and quantification of compounds belonging to at least one of the groups, preferably to at lest two groups, more preferably to various groups, selected among purines and pyrimidines, N-acetylated amino acids, mono and dicarboxylic acids, bifunctional aldehydes, nitrosylated compounds, sulphurylated compounds, group B vitamins, and derivatives thereof, according to the aforementioned described method, comprising: an HPLC system with low pressure eluent mixing formed by a high performance pump or an HPLC system with a high pressure eluent mixing formed by two high performance pumps, an eluent mixer, an eluent degasser, a variable volume injection valve ranging from 10 to 500 μl or a fixed volume injection valve with injection loops ranging from 10 to 500 μl, a refrigerated autosampler, a thermostatic system capable to keep the column temperature constant even below the room temperature, a detector, a software for the acquisition and analysis of chromatographic data, a hardware with an operative system capable of managing the software for the acquisition and analysis of chromatographic data and equipped with a printer. The detector can be selected in the group consisting of a high sensitive spectrophotometric diode array detector, with a 5 cm light path flow cell, a spectrophotometric diode array detector, a variable wavelength UV-visible spectrophotometric detector, a spectrofluorimetric detector, a mass spectrometric detector. In particular, the spectrophotometric diode array detector can be connected in parallel (and or in series) with a spectrofluorimetric detector or with a mass spectrometric detector. The mass spectrometric detector can be of the "ion trap" type.

The device according to the present invention is composed by an HPLC comprising an ODS-C-18 chromatographic column. In particular, the reversed phase ODS C-18 column can be chosen in the group consisting in columns having particle size, length and diameter, respectively, of 5 μm, 15 cm, 2.1 mm; 5 μm, 25 cm, 2.1 mm; 3 μm, 25 cm, 2.1 mm; 5 μm ,15 cm, 3.0 mm; 5 μm, 15 cm, 4.0 mm; 5 μm, 25 cm, 3.0 mm; 5 μm, 25 cm, 4.0 mm; 3 μm, 15 cm, 4.6 mm; 3 μm, 15 cm, 3.0 mm; 3 μm, 15 cm, 4.0 mm; 5 μm, 30 cm, 4.0 mm; 5 μm, 10 cm, 4.6 mm; 5 μm, 25 cm, 4.6 mm; 5 μm, 10 cm, 3.0 mm; 5 μm, 10 cm, 4.0 mm; 5 μm, 30 cm, 1.0 mm.

It is a further object of the present invention a kit for the direct simultaneous separation and quantification of compounds belonging to at least on of the groups, preferably to at least two groups, more preferably to various groups, chosen among purines, pyrimidines, N-acetylated amino acids, mono and dicarboxylic acids, sulphurylated compounds, nitrosylated compounds, bifunctional aldehydes, group B vitamins, and derivatives thereof, in a biological sample according to the aforementioned method, comprising to buffer solutions A and B having, respectively, essentially neutral pH and essentially acidic pH, both containing an anionic exchanger ion-pairing reagent. Buffer solutions can comprise a quaternary ammonium ion, a polar organic solvent, a salt if weak acid. In particular, the concentration of the quaternary ammonium ion in buffer A can range from 8 to 15 mM, preferably from 10 to 13 mM, more preferably equal to 12 mM.

The concentration of the polar organic solvent in buffer A can range from 0.01 to 1%, preferably from 0.1 to 0.5%, more preferably equal to 0.125%.

The concentration of the salt of a weak acid in buffer A can range from 5 to 15 mM, preferably from 8 to 13, more preferably equal to

1O mM. The concentration of the quaternary ammonium ion in buffer B can range from 0.01 to 3.5 mM, preferably from 2 to 3 mM, more preferably 2.8 mM.

The concentration of the polar organic solvent in buffer B can range from 20 to 80%, preferably from 25 to 35%, more preferably 30%. The concentration of the salt of a weak acid in buffer B can range 1 to 130 mM, preferably, from 80 to 110 mM, more preferably 100 mM.

Buffer solutions according to the present invention can contain a quaternary ammonium ion such as tetrabutylammonium, tetramiristylammonium, tetraoctylammonium; a polar organic solvent such as acetonitrile, phenols, primary, secondary and tertiary alcohols; a salt of a weak acid such as potassium, sodium or ammonium acetate, potassium, sodium or ammonium phosphate, potassium, sodium or ammonium formiate. Buffer A can have a pH value ranging from 6.5 to 7.5, preferably equal to 7.00, while pH of buffer B can range from 4.5 to 6.5, preferably equal to 5.50.

The kit according to the present invention can further comprise a reversed phase column such as an ODS C-18 column. The ODS C-18 column can be selected in the group consisting of column having particle size, length and diameter, respectively, of 5 μm,

15 cm, 2.1 mm; 5 μm, 25 cm, 2.1 mm; 3 μm, 25 cm, 2.1 mm; 5 μm ,15 cm, 3.0 mm; 5 μm, 15 cm, 4.0 mm; 5 μm, 25 cm, 3.0 mm; 5 μm, 25 cm, 4.0 mm; 3 μm, 15 cm, 4.6 mm; 3 μm, 15 cm, 3.0 mm; 3 μm, 15 cm, 4.0 mm; 5 μm, 30 cm, 4.0 mm; 5 μm, 10 cm, 4.6 mm; 5 μm, 25 cm, 4.6 mm; 5 μm, 10 cm, 3.0 mm; 5 μm, 10 cm, 4.0 mm; 5 μm, 30 cm, 1.0 mm. According to a particular type of realization of the present invention, the kit can comprise a buffer A containing 12 mM tertabutylammonium hydroxide, 0,125% methanol, 10 mM monobasic potassium phosphate, pH 7.00 and a buffer B containing 2.8 mM tertabutylammonium hydroxide, 30% methanol, 100 mM monobasic potassium phosphate, pH 5.50. In addition, this kit might further comprise an ODS C-18 reversed phase column having 5 μm particle size, 250 mm length and 4.6 mm diameter.

The kit according to the present invention can further comprise tubes equipped with a filtering membrane hving a 3 kDa molecular weight cut-off for deproteinize biological fluid samples.

According to a preferred embodiment, the invention refers to a kit specifically devoted to the separation and quantification of purines, pyrimidines, and derivatives thereof, comprising an ODS-C18 reversed phase column having 5 μm particle size, 250 mm length and 4.6 mm diameter; the buffer A comprises 12 mM tertabutylammonium hydroxide, 0,125% methanol, 10 mM monobasic potassium phosphate, pH 7.00, the buffer B comprises 1 mM tertabutylammonium hydroxide, 70% methanol, 10 mM monobasic potassium phosphate, pH 5.50.

The invention further refers to a kit for the separation and quantification of mono and dicarboxylic acids and derivatives thereof, comprising an ODS-C18 reversed phase column having 5 μm particle size, 250 mm length and 4.6 mm diameter; the buffer A that comprises 12 mM tertabutylammonium hydroxide, 0,125% methanol, 10 mM monobasic potassium phosphate, pH 7.00, the buffer B comprising 1 mM tertabutylammonium hydroxide, 80% methanol, 1 mM monobasic potassium phosphate, pH 5.50.

According to a further preferred embodiment, the invention concerns to a kit specifically dedicated to the separation and quantification of N-acetylated amino acids and derivatives thereof, comprising an ODS- C18 reversed phase column having 5 μm particle size, 250 mm length and 4.6 mm diameter; the buffer A comprising 12 mM tertabutylammonium hydroxide, 0,125% methanol, 10 mM monobasic potassium phosphate, pH 7.00, the buffer B comprising 0.2 mM tertabutylammonium hydroxide, 80% methanol, 1 mM monobasic potassium phosphate, pH 5.50.

Moreover, the invention concerns to a kit specifically dedicated to the separation and quantification of nitrosylated compounds and derivatives thereof, comprising an ODS-C18 reversed phase column having 5 μm particle size, 250 mm length and 4.6 mm diameter; the buffer A comprising 12 mM tertabutylammonium hydroxide, 0,125% methanol, 10 mM monobasic potassium phosphate, pH 7.00, the buffer B comprising 0.3 mM tertabutylammonium hydroxide, 80% methanol, 1 mM monobasic potassium phosphate, pH 5.50.

It is a further object of the present invention a kit specifically dedicated to the separation and quantification of purines, sulphurylated compounds, nitrosylated compounds, monocarboxylic acids, bifunctional aldehydes, and derivatives thereof, comprising an ODS-C18 reversed phase column having 5 μm particle size, 250 mm length and 4.6 mm diameter; the buffer A comprising 12 mM tertabutylammonium hydroxide, 0,125% methanol, 10 mM monobasic potassium phosphate, pH 7.00, the buffer B comprising 0.1 mM tertabutylammonium hydroxide, 80% methanol, 1 mM monobasic potassium phosphate, pH 5.50. According to a preferred embodiment, the invention concerns a kit specifically dedicated to the separation and quantification of group B vitamins, monocarboxylic acids, and derivatives thereof, comprising an ODS C-18 reversed phase column having 5 μm particle size, 250 mm length and 4.6 mm diameter; buffer A that comprises 12 mM tetrabutylammonium hydroxide, 0.125% methanol, 10 mM monobasic potassium phosphate, pH 7.00; buffer B that comprises 0.1 mM tetrabutylammonium hydroxide, 80% methanol, 1 mM monobasic potassium phosphate, pH 5.50.

It represents a further object of the present invention the use of buffer solutions A, comprising 12 mM tertabutylammonium hydroxide, 0,125% methanol, 10 mM monobasic potassium phosphate, pH 7.00, and B comprising 2.8 mM tertabutylammonium hydroxide, 30% methanol, 100 mM monobasic potassium phosphate, pH 5.50, in sequential mode, for the realization of a step gradient in separations methods. The present invention will now be described as an illustrative, but not limitative, scope, according to its preferred embodiment, with specific reference to figures and draws attached, wherein: Figure 1 reports an example of a chromatogram reporting the separation of a ultrapure standard mixture containing NAA, NAG₁ NAAG, cytosine, cytidine, uracil, uridine, β-pseudouridine, adenine, 3- methyladenine, hypoxanthine, xanthine, xanthosine, inosine, guanosine, adenosine, SAM, S-adenosylhomocysteine, adenylosuccinate, MDA₁ ascorbic acid, thymine, thymidine, uric acid, 1-methyluric acid, orotic acid, AICAR, malonic acid, methylmalonic acid, propionic acid, succinic acid, NO₂ ^", NO₃ ^', thiamine, TPP, riboflavin, FMN, FAD, pyridoxine, pyridoxamine, pyridoxale, pyridoxic acid, pyridoxine phosphate, pyridoxamine phosphate, PLP, cobalamine, folic acid, creatinine, GSH and GSSG having known concentrations. The figure shows three traces: one at 260 nm wavelength (panel A) and used for the quantification (or detection) of cytosine, cytidine, uracil, uridine, β-pseudouridine, adenine, 3-methyladenine, hypoxanthine, xanthine, xanthosine, inosine, guanosine, adenosine, SAM, S-adenosylhomocysteine, adenylosuccinate, MDA, ascorbic acid, thymine, thymidine, uric acid, 1-methyluric acid, orotic acid, AICAR, creatinine; one at 206 nm wavelength (panel B) and used for the quantification of NAA, NAG, NAAG, malonic acid, methylmalonic acid, propionic acid, succinic acid, NO₂ ^", NO₃ ^', GSH an GSSG; one at 340 nm wavelength (panel C) and used for the quantification of thiamine, TPP, riboflavin, FMN, FAD, pyridoxine, pyridoxamine, pyridoxale, pyridoxic acid, pyridoxine phosphate, pyridoxamine phosphate, PLP, cobalamine and folic acid.

Figure 2 shows examples of concentration-instrumental response relationship for some of the compounds of interest that evidence not only a very high degree of linearity (correlation coefficients very close to 1) but also a high sensitivity as far as the lower limit of detection is concerned.

Figure 3 reports an example of a normal human plasma sample (panel A) wherein are evident some of the compounds of interest, in comparison with the same plasma sample (panel B) supplemented with known concentrations of some of the compounds of interest (NAA, uracil, β-pseudouridine, hypoxanthine, uridine, inosine, thymidine, orotic acid, malonic acid, methylmalonic acid, GSSG). In both panels, the 260 nm (for the detection of purine and pyrimidine derivatives) and 206 nm (for the detection of NAA, malonic acid, methylmalonic acid, GSSG) chromatographic traces are reported. Figure 4 reports an example of a normal human urinary sample (panel A) wherein are evident some of the compounds of interest, in comparison with the same plasma sample (panel B) supplemented with known concentrations of some of the compounds of interest (NAA, uracil, β-pseudouridine, hypoxanthine, uridine, inosine, thymidine, orotic acid, malonic acid, methylmalonic acid, GSSG). In both panels, the 260 nm (for the detection of purine and pyrimidine derivatives) and 206 nm (for the detection of NAA, malonic acid, methylmalonic acid, GSSG) chromatographic traces are reported. Figure 5 reports an example of a normal human amniotic fluid sample (panel A) wherein are evident some of the compounds of interest, in comparison with the same plasma sample (panel B) supplemented with known concentrations of some of the compounds of interest (NAA, uracil, β-pseudouridine, hypoxanthine, uridine, inosine, thymidine, orotic acid, malonic acid, methylmalonic acid, GSSG). In both panels, the 260 nm (for the detection of purine and pyrimidine derivatives) and 206 nm (for the detection of NAA, malonic acid, methylmalonic acid, GSSG) chromatographic traces are reported.

Figure 6 reports an example of plasma sample of a patients suffering from Canavan disease evidencing high NAA levels.

Figure 7 shows an example of a urinary sample of patient suffering from Canavan disease evidencing very high NAA levels.

Example 1 : Analysis of biological fluid samples of normal or pathological subjects by using the method according to the invention and validation of the method.

Materials and Methods Reagents

Ultrapure HPLC methanol was obtained from Carlo Erba (Rodano, Milano, Italia); the ion-pairing tetrabutylammonium hydroxide was purchased as a 55% ultrapure aqueous solution from Nova Chimica (Cinisello Balsamo, Milano, Italia); ulrapure HPLC standards were obtained from ICN Biomedicals (Irvine, California, USA) e dalla Sigma (St. Louis, Missouri, USA).

Ultrapure standard containing solutions with known concentrations were prepared in doubly distilled water subsequently filtered on 0.22 μm filters. These standard mixtures were freshly prepared daily and immediately injected into the HPLC chromatographic system. Results obtained from the separation of these mixtures were utilized to determine reproducibility, linearity, limit of detection and limit of quantification of the HPLC method, as well as to calculate the concentrations of the compounds of interest in biological fluid samples with unknown concentration.

Withdrawn and treatment of samples

Biological fluid samples from healthy donors (plasma and urines) were obtained from volunteers among the people working in the laboratories, after informed consensus. Urinary and plasma samples of patients suffering from

Canavan disease were obtained by the Robert Wood Johnson Medical School for Gene Therapy for Canavan Disease, University of Medicine and Dentistry of New Jersey, U.S.A. Immediately after withdrawal, samples of both controls and patients were properly diluted, transferred in a 0.5 ml tube equipped with a filtering membrane by 3 kDa molecular weight cut-off (Nanosep® Centrifugal Devices, Pall Gelman Laboratory, Ann Harbor, Ml, USA) and subjected to centrifugation at 15,000 x g at 4 ⁰C for 20 minutes. This protein-free filtrate was directly injected (200 μl) into the HPLC chromatographic system. Stability of deproteinized samples was checked by dividing the same sample in different aliquots that were then saved at -80 ⁰C and assayed for 5 times once every two days. High performance liquid chromatography (HPLC) The HPLC system was composed by a single pump connected to gradient former device and to a membrane degasser for the eluents degassing. The valve for sample injection had a fixed volume loop (200 μl). To the outlet of the injection valve an ODS C-18 reversed phase column, 250 mm length, 4.6 mm internal diameter, 5 μm particle size, equipped with a guard column filled with the same stationary phase than the separative column, was connected. The HPLC system was equipped with a highly sensitive spectrophotometric diode array detector with a 5 cm light path flow cell, set up to detect light absorption between 200 and 500 nm wavelength. Both pump and detector were connected to a PC controlling either gradient formation or acquisition of chromatographic runs. The PC had a dedicated software allowing the subsequent qualitative and quantitative analyses of chromatographic traces, permitting to evaluate chromatographic peak purity according to the critical examination of the absorption spectra. Assignment of the different peaks in deproteinized biological fluid samples was effected by comparing absorption spectra and retention times of ultrapure standards. The quantification of the different compounds in chromatographic traces of biological samples was effected by comparing different peak areas with corresponding peak areas of standard chromatographic runs with known concentration. This calculation was carried out at the wavelength of: 206 nm for NAA, NAG, NAAG, malonic acid, methylmalonic acid, propionic acid, succinic acid, NO₂ ^", NO₃ ^", GSH and GSSG; 260 nm for cytosine, cytidine, uracil, uridine, β-pseudouridine, adenine, 3-methyladenine, hypoxanthine, xanthine, xanthosine, inosine, guanosine, adenosine, SAM, S-adenosylhomocysteine, adenylosuccinate, ascorbic acid, MDA, thymine, thymidine, uric acid, 1-methyluric acid, orotic acid, AICAR; 340 nm for thiamine, TPP, riboflavin, FMN, FAD, pyridoxine, pyridoxamine, pyridoxale, pyridoxic acid, pyridoxine phosphate, pyridoxamine phosphate, PLP, cobalamine and folic acid; 234 nm for creatinine.

Description of analysis

The separation of the various compounds within the column containing the stationary phase occurred according to the ionic pairing principle and thanks to the formation of a step gradient. To this purpose we used two different buffers having the following composition:

- Buffer A, composed by 12 mM tetrabutylammonium hydroxide (as the pairing reagent), 0.125% methanol, 10 mM monobasic potassium phosphate, pH 7.00. - Buffer B, composed by 2.8 mM tetrabutylammonium hydroxide (as the pairing reagent), 30% methanol, 100 mM monobasic potassium phosphate, pH 5.50.

Buffers were properly filtered through 0.22 μm membranes and degassed. The chromatographic column was equilibrated for about 20 minutes with buffer A at a flow of 1.2 ml/minute. Aliquotes of 200 μl of ultrapure standard mixtures or of deproteinized and properly diluted biological fluid samples, as above described, were loaded onto the column through the injection valve. The selective separation of the various compounds occurred by means of the following step gradient: 25 minutes of 100% buffer A; 8 minutes at up to 80% buffer A; 10 minutes at up to

70% buffer A; 12 minutes at up to 55% buffer A; 11 minutes at up to 40% buffer A; 9 minutes at up to 15% buffer A; 10 minutes at up to 0% buffer A; 60 with 0% buffer A. At the end of the chromatographic run (145 minutes from the sample injection), column was washed for 10 additional minutes with 100% buffer B and subsequently re-equilibrated with 100% buffer A for 20 more minutes. During the afore described phases, particularly those referring to the chromatographic run, the eluent flow was maintained at 1.2 ml/min and the temperature of the chromatographic column kept constant at 10 ⁰C, using a water jacket connected to a thermostatic re-circulating water bath. The above described gradient was interrupted when needed (and consequently was interrupted the chromatographic run), i.e. immediately after the elution from the column of the last compound of interest (for example, in the case of analysis dedicated to the determination of purines and pyrimidines, N-acetylated amino acids, mono and dicarboxylic acids, bifunctional aldehydes, nitrosylated compounds and sulphurylated compounds) chromatographic runs of samples of plasma, urines and amniotic fluid were interrupted 40 minutes after the sample injection since no traces of the compounds belonging to the chemical classes reported in parentheses having a higher retention times were found.

Sensitivity, reproducibility, recovery The validation of the chromatographic separation, object of the present patent of invention, was effected by determining the limit of detection (LOD), the limit of quantification (LOQ), the linearity and the reproducibility. We also performed the evaluation of accuracy, precision and recovery. The aforementioned parameters were estimated by injecting 200 μl of standard mixtures and was carried out, for any of the parameters considered and relative to the validation, on no less than 5 chromatographic runs.

Results Results are reported in Figures 1-7. In particular, Figure 1 is a representative chromatogram of a separation of a ultrapure standard mixture containing NAA, NAG, NAAG, cytosine, cytidine, uracil, uridine, β- pseudouridine, adenine, 3-methyladenine, hypoxanthine, xanthine, xanthosine, inosine, guanosine, adenosine, SAM, S- adenosylhomocysteine, adenylosuccinate, MDA, ascorbic acid (vitamin C), thymine, thymidine, uric acid, 1-methyluric acid, orotic acid, AICAR, malonic acid, methylmalonic acid, propionic acid, succinic acid, NO₂ ^", NO₃ ^" , vitamin B1 (thiamine and TPP), vitamin B2 (riboflavin, FMN and FAD) vitamin B6 (pyridoxine, pyridoxamine, pyridoxale, pyridoxic acid, pyridoxine phosphate, pyridoxamine phosphate and PLP), vitamin B12 (cobalamine), folic acid, creatinine, reduced glutathione (GSH) and oxidized glutathione (GSSG), with known concentration. The figure reports three traces: one at 260 nm wavelength and used for the quantification (or detection) of cytosine, cytidine, uracil, uridine, β-pseudouridine, adenine, 3-methyladenine, hypoxanthine, xanthine, xanthosine, inosine, guanosine, adenosine, SAM, S-adenosylhomocysteine, adenylosuccinate, MDA, ascorbic acid, thymine, thymidine, uric acid, 1-methyluric acid, orotic acid, AICAR, creatinine; one at 206 nm wavelength and used for the quantification of NAA, NAG, NAAG, malonic acid, methylmalonic acid, propionic acid, succinic acid, NO₂ ^", NO₃ ^", GSH and GSSG; one at 340 nm wavelength and used for the quantification of vitamin B1 (thiamine and TPP), vitamin B2 (riboflavin, FMN and FAD) vitamin B6 (pyridoxine, pyridoxamine, pyridoxale, pyridoxic acid, pyridoxine phosphate, pyridoxamine phosphate and PLP), vitamin B12 (cobalamine), folic acid. Figure 2 illustrates examples of concentration-instrumental response relationship (calibration curves) for some of the compounds of interest that evidence not only a very high degree of linearity (correlation coefficients very close to 1) but also a high sensitivity as far as the lower limit of detection is concerned.

Figure 3 reports an example of a normal human plasma sample (panel A) wherein are evident some of the compounds of interest, in comparison with the same plasma sample (panel B) supplemented with known concentrations of some of the compounds of interest (NAA, uracil, β-pseudouridine, hypoxanthine, uridine, inosine, thymidine, orotic acid, malonic acid, methylmalonic acid, GSSG). In both panels, the 260 nm (for the detection of purine and pyrimidine derivatives) and 206 nm (for the detection of NAA, malonic acid, methylmalonic acid, GSSG) chromatographic traces are reported.

Figure 4 reports an example of a normal human urinary sample (panel A) wherein are evident some of the compounds of interest, in comparison with the same plasma sample (panel B) supplemented with known concentrations of some of the compounds of interest (NAA, uracil, β-pseudouridine, hypoxanthine, uridine, inosine, thymidine, orotic acid, malonic acid, methylmalonic acid, GSSG). In both panels, the 260 nm (for the detection of purine and pyrimidine derivatives) and 206 nm (for the detection of NAA, malonic acid, methylmalonic acid, GSSG) chromatographic traces are reported.

Figure 5 reports an example of a normal human amniotic fluid sample (panel A) wherein are evident some of the compounds of interest, in comparison with the same plasma sample (panel B) supplemented with known concentrations of some of the compounds of interest (NAA, uracil, β-pseudouridine, hypoxanthine, uridine, inosine, thymidine, orotic acid, malonic acid, methylmalonic acid, GSSG). In both panels, the 260 nm (for the detection of purine and pyrimidine derivatives) and 206 nm (for the detection of NAA, malonic acid, methylmalonic acid, GSSG) chromatographic traces are reported.

Figure 6 reports an example of plasma sample of a patients suffering from Canavan disease evidencing high NAA levels whilst, Figure 7 refers to a urinary sample of patient suffering from Canavan disease evidencing very high NAA levels.

Sensitivity and linearity

In Table 1 minimal and maximal concentration values between which a linear analytical response was observed, as well as the value of the correlation coefficient of straight lines of the respective perameters of interest, are reported. Examples of linearity of the analysis are illustrated in Figure 2.

Table 1

Reproducibility

The high reproducibility of the present HPLC method was demonstrated by the low variability of the retention times of the compounds of interest (maximal coefficient of variation detected for orotic acid = 0.51 %) and of the peak areas (maximal coefficient of variations recorded for cytidine = 1.65%) determined on 5 chromatographic runs referring to 5 standard mixtures prepared and analyzed on 5 consecutive days. Data are summarized in Table 2.

Table 2

Recovery

The reliability of the analytical method in HPLC chromatography, object of the present patent of invention, was ultimately assessed through the evaluation of the efficiency in the recovery of the compounds of interest. To this purpose, non-pathological plasma and urinary samples were spiked with an appropriate amount of a mixture containing ultrapure standards with known concentrations. These resulting samples were then injected into the HPLC system. Data reported in Table 3 clearly demonstrate the very high efficiency in the recovery of the various metabolites assayed, with percent of recovery ranging from a minimum of 96.7% (uridine) to a maximum of 107.6% (NAG).

Table 3

Analysis of control biological fluids Values of the compounds of interest separated by the HPLC method object of the present patent of invention, determined in 15 plasma samples assayed in duplicate, obtained from healthy adults with age range 25-35 years, are illustrated in Table 4. Some of the compounds were absent in all the samples analyzed, or anyway below the LOD of the method. The comparison or these results with those reported in the specialized medical literature, and obtained with analytical methods different form that here described, shows a substantial similitude.

Table 4

Values of the compounds of interest separated with HPLC method object of the present patent of invention, determined in 6 amniotic fluid samples assayed in duplicate, withdrawn from pregnants between the 12^th and the 16^th week of pregnancy, are reported in Table 5. Some of the compounds were absent in all the samples analyzed, or anyway below the LOD of the method. The comparison or these results with those reported in the specialized medical literature, and obtained with analytical methods different form that here described, shows a substantial similitude.

Table 5

An amniotic fluid sample resulted with no detectable ascorbic acid.

** 1-methyluric acid was detected in 3/10 samples only. *** In an amniotic fluid sample a thymidin concentration of

7.25 μmol/l amniotic fluid was recorded. By eliminating this suspected anomalous sample, the resulting mean value would be of 1.52 μmol/l amniotic fluid (standard deviation = 1.21).

Values of the compounds of interest separated with the HPLC method object of the present patent of invention, determined in 50 urine samples assayed in duplicate, obtained from 50 control subjects and subdivided in different classes of age (10 subjects 1-3 years old, 10 subjects 4-6 years old, 10 subjects 8-10 years old, 10 subjects 1-3 years old, 10 subjects 12-18 years old, 10 subjects 25-35 years old) are reported in Table 6. Some of the compounds were absent in all the samples analyzed, or anyway below the LOD of the method, whilst other compounds (among which NAA, β-pseudouridin, hypoxanthine, xanthine, uric acid) are present in decreasing concentrations with the increasing of the age. The comparison or these results with those reported in the specialized medical literature, and obtained with analytical methods different form that here described, shows a substantial similitude. Tabella 6

Analysis of biological fluid samples of patients suffering from Canavan disease Values of the compounds of interest separated with HPLC method object of the present patent of invention, determined in 30 plasma samples assayed in duplicate, obtained from Canavan disease patients with age ranging from 0.5 to 6 years, are reported in Table 7. In these samples all the metabolites, but NAA, are present in concentrations not significantly different from those of controls Differently, NAA, undetectable in plasma of controls, has a concentration of 16.96 μmol/mmol di creatinine (D. S. = 19.57). It is worth underlining that NAA has never been detected previously in plasma of Caravan disease patients (or in control subjects) because of the scarcely sensitive methodologies available up to now, thereby rendering more important the clinical-biochemicalsignificance of the present HPLC method.

Table 7

The significantly higher level of ascorbic acid detected in Canavan disease patients was due to the administration of 1 g/die vitamin C. In a Canavan disease patient a plasma concentration of NAA of

1246.71 μmol/l plasma was recorded. This exceptionally high value (whose correctness was controlled by assaying this sample in quadruplicate) was omitted from the mean and standard deviation calculations. Values of the compounds of interest separated with HPLC method object of the present patent of invention, determined in 30 urinary samples assayed in duplicate, obtained from Canavan disease patients with age ranging from 0.5 to 6 years, are reported in Table 8. In these samples all the metabolites, but NAA and β-pseudouridine, are present in concentrations not significantly different from those of controls NAA has a mean concentration of 1872.03 μmol/mmol di creatinine (D. S. = 631.86), i.e. 843 times higher than that recorded in urine of controls, (2,22 μmoli/mmole di creatinina; D. S. = 1 ,85), whilst β-pseudouridine has a mean concentration of 319.27 μmol/mmol creatinine (D. S. = 198.36), i.e. 5.8 times higher than that recorded in urine of controls, (55.37 μmol/mmol di creatinine; D.S. = 21.44).

Table 8

Discussion of the results

Data reported un the present patent of invention demonstrate the validity of the mew ion-pairing HPLC method that allows the simultaneous separation and quantification of NAA, NAG, NAAG, cytosine, cytidine, uracil, uridine, β-pseudouridine, adenine, 3-methyladenine, hypoxanthine, xanthine, xanthosine, inosine, guanosine, adenosine, SAM, S-adenosylhomocysteine, adenylosuccinate, MDA, ascorbic acid (vitamin C), thymine, thymidine, uric acid, 1-methyluric acid, orotic acid, AICAR, malonic acid, methylmalonic acid, propionic acid, succinic acid, NO₂ ^", NO₃ ^" , vitamin B1 (thiamine and TPP), vitamin B2 (riboflavin, FMN and FAD) vitamin B6 (pyridoxine, pyridoxamine, pyridoxale, pyridoxic acid, pyridoxine phosphate, pyridoxamine phosphate and PLP), vitamin B12 (cobalamine), folic acid, creatinine, reduced glutathione (GSH) and oxidized glutathione (GSSG), in biological fluid samples, as well as in cell and/or tissue extracts. The method is particularly suitable to detect these compounds in patients suffering from IEM, such as patients suffering from Canavan disease (N-acetylasparto acylase enzyme deficiency), or to carry out the neonatal screening through the chemical diagnosis of these pathologies as well as to evaluate the efficacy of eventual therapeutic approaches, or for the pre-natal chemical diagnosis of IEM related to these compounds, as well as in all acute and chronic pathological states wherein one or more of these compounds are subjected to significant alterations of their respective physiological concentrations. In comparison with the analytical methods utilized at present, most of which are based on the use of GC/MS, the present method offers noteworthy advantages: i) in the case of the deproteinization by filtration through a membrane with appropriate molecular weight cut-off sample preparation does not require any type of manipulation, and any other deproteinization method can be alternatively applied without altering the analytical result; ii) the sensitivity of the proposed HPLC method is such to permit the detection of the concentration of the metabolites of interest using minimal amounts of biological fluid samples (20 μl for urine and 100 μl for plasma and amniotic fluid); iii) the proposed HPLC method allows the synchronous separation and quantification of 50 different metabolites in biological samples, the concentration of which increases o decreases in numerous pathological states thus resulting of applicative interest in the clinical-biochemical field; iiii) the method is applicable with the same analytical characteristics of resolution, reproducibility and sensitivity, to any type of biological sample, independently from the origin of the sample itself (deproteinized biological fluid, cell or tissue extracts).

The clear advantage of the HPLC method, object of the present patent of invention, is that to allow, by using an instrument common in all the analytical laboratories, the concomitant separation and quantification of low polar compounds (purines, pyrimidines, creatinine), polar non-dissociable (purine and pyrimidine nucleosides, bifunctional aldehydes) and polar dissociable (N-acetylated amino acids, mono and dicarboxylic acids, sulphurylated compounds, nucleotides, nitrosylated compounds, group B vitamins), all of diagnostic relevance. The analytical methods used at present are based on separations effected by GC/MS that, for the principle of gas-chromatography, can not be adapted to simultaneous separation of non-polar, non dissociable polar and dissociable polar compounds. In fact, to apply GC/MS to the separation of these classes of substances delicate, complex and time-consuming sample manipulation prior to the analysis, based on the chemical derivatization of the compounds so to render them non-polar, are necessary. Since the yield of these derivatization processes is never equal to 100% the final result is a remarkable loss of the sensitivity of the method and the impossibility of its application anytime very high analytical sensitivity is required. An example is NAA determination effected by GC/MS which can detect this compound in urine only. As far as the other biological fluids, such as plasma and amniotic fluid, is concerned GC/MS is applicable only after a further complicated sample manipulation represented by stable isotopic dilution (17-19). This procedure is not of routine practice in clinical biochemical laboratories and it is also so expensive in terms of both time and cost to hinder its application for the large scale screening of pathologies related to dismetabolism of this compound such as Canavan disease. In general, it should also be reminded that GC/MS can not be applied in the case of thermo-labile compounds such as GSH, ascorbic acid and group B vitamins.

The HPLC method applied to the concomitant separation of NAA, NAG, NAAG, cytosine, cytidine, uracil, uridine, β-pseudouridine, adenine, 3-methyladenine, hypoxanthine, xanthine, xanthosine, inosine, guanosine, adenosine, SAM, S-adenosylhomocysteine, adenylosuccinate, MDA, ascorbic acid (vitamin C), thymine, thymidine, uric acid, 1-methyluric acid, orotic acid, AICAR, malonic acid, methylmalonic acid, propionic acid, succinic acid, NO₂ ^", NO₃ ^", vitamin B1 (thiamine and TPP), vitamin B2 (riboflavin, FMN and FAD) vitamin B6 (pyridoxine, pyridoxamine, pyridoxale, pyridoxic acid, pyridoxine phosphate, pyridoxamine phosphate and PLP), vitamin B12 (cobalamine), folic acid, creatinine, reduced glutathione (GSH) and oxidized glutathione (GSSG), demonstrates to possess all the characteristics of sensitivity, reproducibility, resolution capacity, ease of sample preparation, type of instrumentation, time of execution, costs, to propose it as a valid analytical system to be used for the large scale screening of biological fluid samples for the pre-natal and neonatal chemical diagnosis of IEM related to malfunctioning of metabolic pathways involved in the biosynthesis and degradation of the compounds assayed with the present method. Furthermore, it is also possible to perform the monitoring of therapeutic approach aimed to modify the clinical status of patients suffering by IEM disturbances related to the metabolism of the aforementioned compounds, or affected by acute and chronic pathological states capable of modifying, even temporarily, the concentrations of one or more of these compounds in biological fluids, Data reported as an example of practical application in plasma and urinary samples of Canavan disease patients clearly demonstrate the validity of the HPLC mehod we set up. Particularly, the use of a highly sensitive spectrophotometric diode array detector allows to determine synchronously light absorption by compounds having maxima of absorption in differentiated regions of the UV-visible spectra. This is the case for cytosine, cytidine, uracil, uridine, β-pseudouridine, adenine, 3- methyladenine, hypoxanthine, xanthyne, xanthosine, inosine, guanosine, adenosine, SAM, S-adenosylhomocysteine, adenylosuccinate, AICAR, MDA, ascorbic acid, thymine, thymidine, uric acid, 1-methyluric acid, orotic acid, creatinine that are easily detectable between 230 and 300 nm wavelength, whilst NAA, NAG, NAAG, malonic acid, methylmalonic acid, propionic acid, succinic acid, NO₂ ^", NO₃ ^", GSH e GSSG are detectable around 206 nm wavelength only, and vitamin B1 (thiamine e TPP), vitamina B2 (riboflavin, FMN, FAD), vitamin B6 (pirydoxine, pirydoxamine, pirydoxale, pirydoxic acid, pirydoxine phosphate, pirydoxamine phosphate, PLP), vitamina B12 (cobalamine), folic acid are simultaneously detectable around 340 nm wavelength, where the other compounds do not show any type of interference. In addition, the use of a proper software allow the evaluation of the absorbance spectra purity and to indicate eventual impurities due to substances tha might co-elute within the same chromatographic peak, thereby guaranteeing the correctness of the analytical data.

Obviously, many variations and changes can be effected to the above described method, as well as other compounds can be recognized, separated and quantified under the chromatographic conditions described, without however modify its meanings and aims, as defined in the claims reported in appendix.

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Claims

1. Method for the direct simultaneous separation and quantification of compounds belonging to at least one of the groups chosen among purines and pyrimidines, N-acetylated amino acids, mono and dicarboxylic acids, sulphurylated compounds, nitrosylated compounds, bifunctional aldehydes, group B vitamins, and derivatives thereof, in a biological sample by ion-pairing high performance liquid chromatography (HPLC) which utilizes a reversed phase column, said method utilizing the formation of a discontinuos gradient obtained by subsequent elution steps with the use of two buffer solutions A and B having, respectively essentially neutral pH and essentially acidic pH both buffers comprising an anionic exchanger pairing reagent with different concentration in the two buffers, the first elution phase being an isocratic phase with 100% buffer A having essentially neutral pH.

2. Method according to claim 1 , wherein purines and derivatives thereof are chosen in the group consisting of adenine, 3-methyladenine, hypoxanthine, xanthine, xanthosine, inosine, guanosine, adenosine, SAM, S-adenosylhomocysteine, adenylosuccinate, uric acid, 1-methyluric acid.

3. Method according to claim 1 , wherein pyrimidines and derivatives thereof are chosen in the group consisting of cytosine, cytidine, uracil, uridine, β-pseudouridine, thymine, thymidine, orotic acid, AICAR.

4. Method according to claim 1 , wherein N-acetylated amino acids and derivatives thereof are chosen in the group consisting of NAA,

NAG, NAAG, creatinine.

5. Method according to claim 1 , wherein mono and dicarboxylic acids and derivatives thereof are chosen in the group consisting of ascorbic acid, malonic acid, methylmalonic acid, propionic acid, succinic acid, folic acid.

6. Method according to claim 1 , wherein sulphurylated compounds and derivatives thereof are chosen in the group consisting of GSH and GSSG.

7. Method according to claim 1 , wherein nitrosylated compounds and derivatives thereof are chosen in the group consisting of

NO₂ ^" and NO₃ ^". 8. Method according to claim 1 , wherein bifunctional aldehydes and derivatives thereof are MDA.

9. Method according to claim 1 , wherein group B vitamins and derivatives thereof are chosen in the group consisting of vitamin B1 , vitamin B2, vitamin B6, vitamin B12.

10. Method according to claim 1 , wherein the biological sample is chosen in the group consisting of urine, plasma, amniotic fluid, cerebrospinal fluid or tissues.

11. Method according to anyone of the preceding claims wherein the biological sample is preliminarily subjected to deproteinization.

12. Method according to claim 11 , wherein deproteinization is performed by dialysis filtration on molecular membrane with proper cut-off.

13. Method according to claim 12, wherein the molecular cut-off is of 3 kDa. 14. Method according to claim 11 , wherein deproteinization is carried out by the addition of organic solvent followed by centrifugation and removal of the organic solvent.

15. Method according to claim 14, wherein organic solvent are chosen in the group consisting of acetonitrile, phenols, primary, secondary and tertiary alcohols.

16. Method according to claim 11, wherein deproteinization is carried out by the addition of concentrated acids or bases, followed by centrifugation and neutralization of the deproteinized aqueous solution.

17. Method according to claim 16, wherein acids are chosen in the group consisting of perchloric acid, hydrochloric acid, nitric acid, formic acid, trichloroacetic acid, trifluoroacetic acid, sulphuric acid, phosphotungstic acid, phosphoric acid.

18. Method according to claim 16, wherein bases are chosen in the group consisting of sodium hydroxide, potassium hydroxide, calcium hydroxide.

19. Method according to claim 11 , wherein deproteinization in carried out by the addition of concentrated salts, followed by centrifugation and salt removal by the aqueous solution.

20. Method according to claim 19, wherein salts are chosen in the group consisting of sodium, potassium, magnesium, calcium hydrochloride, ammonium sulphate, uranyl acetate. 21. Method according to claim 1 , wherein the column is an ODS C-18 column.

22. Method according to claim 1 , wherein buffer solutions comprise a quaternary ammonium ion, a polar organic solvent, a salt of a weak acid.

23. Method according to anyone of the preceding claims, wherein the reversed phase column is an ODS C-18; buffer A comprises a quaternary ammonium ion ranging from 8 to 15 mM, a polar organic solvent ranging from 0.01 to 1%, a salt of a weak acid ranging from 5 to 15 mM, pH ranging from 6.5 to 7.5; buffer B comprises a quaternary ammonium ion ranging from 0.01 to 3.5 mM, a polar organic solvent ranging from 20 to 80%, a salt of a weak acid ranging from 1 to 130 mM, pH ranging from 4.5 to 6.5; the step gradient is obtained by an initial isocratic phase with 100% buffer A with minimal duration of 5 minutes and maximal of 35 minutes; from 5 to 20 minutes at up to a percent buffer A ranging form 95 to 70%; from 5 to 30 minutes at up to a percent buffer A ranging form 85 to 50%; from 8 to 30 minutes at up to a percent buffer A ranging form 80 to 40%; from 4 to 25 minutes at up to a percent buffer A ranging form 70 to 30%; from 5 to 25 minutes at up to a percent buffer A ranging form 50 to 10%; from 5 to 20 minutes at up to a percent buffer A ranging form 30 to 0%; from 10 to 90 minutes at a 0% buffer A; column washing, at the end of the chromatographic run, for an additional time interval ranging from 5 to 40 minutes with 100% buffer B and column re- equilibration with 100% buffer A for at least 20 minutes. 24. Method according to claim 23, wherein the concentration of the quaternary ammonium ion in buffer A ranges from 10 to 13 mM.

25. Method according to claim 24, wherein the concentration of the quaternary ammonium ion in buffer A is 12 mM.

26. Method according to claim 23, wherein the concentration of the polar organic solvent in buffer A ranges from 0.1 to 0.5%.

27. Method according to claim 26, wherein the concentration of the polar organic solvent in buffer A is 0.125%.

28. Method according to claim 23, wherein the concentration of the salt of a weak acid in buffer A ranges from 8 to 13 mM. 29. Method according to claim 28, wherein the concentration of the salt of a weak acid in buffer A is 10 mM. 30. Method according to claim 23, wherein the concentration of quaternary ammonium ion in buffer B ranges from 0.01 to 3 mM.

31. Method according to claim 30, wherein the concentration of quaternary ammonium ion in buffer B is 2.8 mM. 32. Method according to claim 23, wherein the concentration of the polar organic solvent in buffer B ranges from 25 to 80%.

33. Method according to claim 32, wherein the concentration of the polar organic solvent in buffer B is 30%.

34. Method according to claim 23, wherein the concentration of the salt of a weak acid in buffer B ranges from 1 to 110 mM.

35. Method according to claim 34, wherein the concentration of the salt of a weak acid in buffer B is 100 mM.

36. Method according to anyone of the preceding claims, wherein the quaternary ammonium ion is chosen in the group consisting of tetrabutylammonium, tetramiristylammonium, tetraoctylammonium.

37. Method according to anyone of the preceding claims, wherein the polar organic solvent is chosen in the group consisting of acetonitrile, phenols, primary, secondary and tertiary alcohols.

38. Method according to anyone of the preceding claims, wherein the salt of a weak acid is chosen in the group consisting of potassium, sodium or ammonium acetate, potassium, sodium or ammonium phosphate, potassium, sodium or ammonium formiate.

39. Method according to claim 23, wherein the step gradient is obtained by an initial isocratic phase with 100% buffer A of 25 minutes duration; 8 minutes at up to 80% buffer A; 10 minutes at up to 70% buffer A; 12 minutes at up to 55% buffer A; 11 minutes at up to 40% buffer A; 9 minutes at up to 15% buffer A; 10 minutes at up to 0% buffer A; 60 minutes with 0% buffer A; column washing, at the end of the chromatographic run, for 10 additional minutes with 100% buffer B and re- equilibrated with 100% buffer A for 20 minutes.

40. Method according to claim 23, wherein the ODS C-18 reversed phase column is chosen in the group consisting of columns having particle size, length and diameter, respectively, of 5 μm, 15 cm, 2.1 mm; 5 μm, 25 cm, 2.1 mm; 3 μm, 25 cm, 2.1 mm; 5 μm ,15 cm, 3.0 mm; 5 μm, 15 cm, 4.0 mm; 5 μm, 25 cm, 3.0 mm; 5 μm, 25 cm, 4.0 mm; 3 μm, 15 cm, 4.6 mm; 3 μm, 15 cm, 3.0 mm; 3 μm, 15 cm, 4.0 mm; 5 μm, 30 cm, 4.0 mm; 5 μm, 10 cm, 4.6 mm; 5 μm, 25 cm, 4.6 mm; 5 μm, 10 cm, 3.0 mm; 5 μm, 10 cm, 4.0 mm; 5 μm, 30 cm, 1.0 mm.

41. Method according to anyone of the preceding claims, wherein for the columns having particle size, length and diameter, respectively, of 5 μm, 15 cm, 2.1 mm; 5 μm, 25 cm, 2.1 mm o 5 μm, 15 cm, 3.0 mm ranges from 0.1 to 0.8 ml/min; for the columns having particle size, length and diameter, respectively, of 5 μm, 15 cm, 4.0 mm o 5 μm, 25 cm, 3.0 mm ranges from 0.2 to 1 ml/min; for the columns having particle size, length and diameter, respectively, of 3 μm, 25 cm, 2.1 mm; 5 μm, 25 cm, 4.6 mm; 5 μm, 25 cm, 4.0 mm; 3 μm, 15 cm, 4.6 mm; 3 μm, 15 cm, 3.0 mm; 3 μm, 15 cm, 4.0 mm; 5 μm, 30 cm, 4.0 mm; 5 μm, 10 cm, 4.6 mm; 5 μm, 10 cm, 3.0 mm; 5 μm, 10 cm, 4.0 mm; 5 μm, 30 cm, 1.0 mm, the flow of the eluent ranges, respectively, from 0.05 to 0.6 ml/min, from 0.5 to 1.5 ml/min, from 0.2 to 1.2 ml/min, from 0.5 a 1.2 ml/min, from 0.4 to 1 ml/min, from 0.4 to 1.1 ml/min, from 0.3 to 1.2 ml/min, from 0.5 to 2 ml/min, from 0.5 to 1.5 ml/min, from 0.5 to 1.8 ml/min, from 0.01 to 0.5 ml/min.

42. Method according to anyone of the preceding claims, wherein the column temperature is maintained between 5 and 25 ⁰C. 43. Method according to claim 42, wherein the column temperature is maintained between 8 and 18 ⁰C.

44. Method according to claim 42, wherein the column temperature is maintained constant at 10 ⁰C during the chromatographic analysis. 45. Method according to anyone of the preceding claims, wherein the reversed phase column is an ODS C_18 with 5 μm particle size, 250 mm length and 4.6 mm diameter; buffer A comprises 12 mM tetrabutylammonium hydroxide, 0.125% methanol, 10 mM monobasic potassium phosphate, pH 7.00; buffer B comprises 2.8 mM tetrabutylammonium hydroxide, 30% methanol, 100 mM monobasic potassium phosphate, pH 5.50; the step gradient is obtained by 25 minutes with 100% buffer A; 8 minutes at up to 80% buffer A; 10 minutes at up to 70% buffer A; 12 minutes at up to 55% buffer A; 11 minutes at up to 40% buffer A; 9 minutes at up to 15% buffer A; 10 minutes at up to 0% buffer A; 60 with 0% buffer A; column washing, at the end of the chromatographic run, for 10 additional minutes with 100% buffer B and re- equilibrated with 100% buffer A for 20 minutes; the flow of the eluent is maintained at 1.2 ml/min and the temperature of the chromatographic column is maintained at 10 ⁰C.

46. Method according to anyone of the preceding claims, wherein the detection is carried out at a wavelength ranging from 200 to 225 nm for NAA₁ NAG, NAAG, malonic acid, methylmalonic acid, propionic acid, succinic acid, NO₂ ^", NO3^", GSH and GSSG; at a wavelength ranging from 240 to 300 nm for cytosine, cytidine, uracil, uridine, β-pseudouridine, adenine, 3-methyladenine, hypoxanthine, xanthine, xanthosine, inosine, guanosine, adenosine, SAM, S-adenosylhomocysteine, adenylosuccinate, MDA, ascorbic acid, thymine, thymidine, uric acid, 1-methyluric acid, orotic acid, AICAR; at a wavelength ranging from 220 to 240 nm for creatinine; at a wavelength ranging from 300 to 500 nm for vitamin B1 , vitamin B2 vitamin B6, vitamin B12.

47. Method according to anyone of the preceding claims, wherein the detection is carried out at a wavelength of 206 nm for NAA,

NAG, NAAG, malonic acid, methylmalonic acid, propionic acid, succinic acid, NO₂ ^", NO₃ ^", GSH and GSSG; at a wavelength of 260 nm for cytosine, cytidine, uracil, uridine, β-pseudouridine, adenine, 3-methyladenine, hypoxanthine, xanthine, xanthosine, inosine, guanosine, adenosine, SAM, S-adenosylhomocysteine, adenylosuccinate, MDA, ascorbic acid, thymine, thymidine, uric acid, 1-methyluric acid, orotic acid, AICAR; at a wavelength of 234 nm for creatinine; at a wavelength of 340 nm for vitamin B1 , vitamin B2, vitamin B6, vitamin B12.

48. Method according to anyone of the preceding claims, wherein the detection is carried out by the use of a detector selected in the group consisting of high sensitive spectrophotometric diode array detector, with 5 cm light path flow cell, spectrophotometric diode array detector, variable wavelength UV-visible spectrophotometric detector, fluorimetric detector, mass spectrometric detector. 49. Method according to claim 48, wherein the spectrophotometric diode array detector is connected in parallel (ad/or in series) to a florimetric detector and/or to a mass spectrometric detector.

50. Method specifically devoted to the separation and quantification of purines, pyrimidines and derivatives thereof wherein the reversed phase column is an ODS-C18 with 5 μm particle size, 250 mm length and 4.6 mm diameter; buffer A comprises 12 mM tetrabutylammonium hydroxide, 0.125% methanol, 10 mM monobasic potassium phosphate, pH 7.00; buffer B comprises 1 mM tetrabutylammonium hydroxide, 70% methanol, 10 mM monobasic potassium phosphate, pH 5.50; a step gradient obtained by 25 minutes of 100% buffer A; 15 minutes at up to 80% buffer A; end of the chromatographic run after 45 minutes from sample injection, washing with 100% buffer B for 15 minutes and subsequent column equilibration with 100% buffer A for 20 minutes before the injection of the next sample.

52. Method specifically devoted to the separation and quantification of mono and dicarboxylic acids and derivatives thereof wherein the reversed phase column is an ODS-C18 with 5 μm particle size, 250 mm length and 4.6 mm diameter; buffer A comprises 12 mM tetrabutylammonium hydroxide, 0.125% methanol, 10 mM monobasic potassium phosphate, pH 7.00; buffer B comprises 1 mM tetrabutylammonium hydroxide, 80% methanol, 1 mM monobasic potassium phosphate, pH 5.50; a step gradient obtained by 23 minutes of 100% buffer A; 5 minutes at up to 80% buffer A; end of the chromatographic run after 30 minutes from sample injection, washing with 100% buffer B for 15 minutes and subsequent column equilibration with 100% buffer A for 20 minutes before the injection of the next sample. 53. Method specifically devoted to the separation and quantification of N-acetylated amino acids and derivatives thereof wherein the reversed phase column is an ODS-C18 with 5 μm particle size, 250 mm length and 4.6 mm diameter; buffer A comprises 12 mM tetrabutylammonium hydroxide, 0.125% methanol, 10 mM monobasic potassium phosphate, pH 7.00; buffer B comprises 0.2 mM tetrabutylammonium hydroxide, 80% methanol, 1 mM monobasic potassium phosphate, pH 5.50; a step gradient obtained by 28 minutes of 100% buffer A; 5 minutes at up to 90% buffer A; end of the chromatographic run after 35 minutes from sample injection, washing with 100% buffer B for 15 minutes and subsequent column equilibration with 100% buffer A for 20 minutes before the injection of the next sample.

54. Method specifically devoted to the separation and quantification of nitrosylated compounds and derivatives thereof wherein the reversed phase column is an ODS-C18 with 5 μm particle size, 250 mm length and 4.6 mm diameter; buffer A comprises 12 mM tetrabutylammonium hydroxide, 0.125% methanol, 10 mM monobasic potassium phosphate, pH 7.00; buffer B comprises 0.3 mM tθtrabutylammonium hydroxide, 80% methanol, 1 mM monobasic potassium phosphate, pH 5.50; a step gradient obtained by 30 minutes of 100% buffer A; 5 minutes at up to 80% buffer A; end of the chromatographic run after 35 minutes from sample injection, washing with 100% buffer B for 15 minutes and subsequent column equilibration with 100% buffer A for 20 minutes before the injection of the next sample.

55. Method specifically devoted to the separation and quantification of purines, sulphurylated compounds, nitrosylated compounds, mono and dicarboxylic acids, bifunctional aldehydes and derivatives thereof wherein the reversed phase column is an ODS-C 18 with 5 μm particle size, 250 mm length and 4.6 mm diameter; buffer A comprises 12 mM tetrabutylammonium hydroxide, 0.125% methanol, 10 mM monobasic potassium phosphate, pH 7.00; buffer B comprises 0.1 mM tetrabutylammonium hydroxide, 80% methanol, 1 mM monobasic potassium phosphate, pH 5.50; a step gradient obtained by 35 minutes of 100% buffer A; 5 minutes at up to 85% buffer A; end of the chromatographic run after 40 minutes from sample injection, washing with 100% buffer B for 15 minutes and subsequent column equilibration with 100% buffer A for 20 minutes before the injection of the next sample. 56. Method specifically devoted to the separation and quantification of group B vitamins, mono carboxylic acids and derivatives thereof wherein the reversed phase column is an ODS-C18 with 5 μm particle size, 250 mm length and 4.6 mm diameter; buffer A comprises 12 mM tetrabutylammonium hydroxide, 0.125% methanol, 10 mM monobasic potassium phosphate, pH 7.00; buffer B comprises 0.1 mM tetrabutylammonium hydroxide, 80% methanol, 1 mM monobasic potassium phosphate, pH 5.50; a step gradient obtained by 10 minutes of 100% buffer A; 5 minutes at up to 85% buffer A; 13 minutes at up to 70% buffer A; 7 minutes at up to 53% buffer A; 11 minutes at up to 38% buffer A; 9 minutes at up to 15% buffer A; 5 minutes at up to 0% buffer A; end of the chromatographic run after 70 minutes from sample injection, washing with 100% buffer B for 10 minutes and subsequent column equilibration with 100% buffer A for 20 minutes before the injection of the next sample.

57. Device for the direct simultaneous separation and quantification of compounds belonging to at least one of the groups chosen among purines and pyrimidines, N-acetylated amino acids, mono and dicarboxylic acids, sulphurylated compounds, nitrosylated compounds, bifunctional aldehydes, group B vitamins, and derivatives thereof, by means of the method described in claims from 1 to 56, consisting of: an HPLC system with low pressure eluent mixing formed by a high performance pump or an HPLC system with a high pressure eluent mixing formed by two high performance pumps, an eluent mixer, an eluent degasser, a variable volume injection valve ranging from 10 to 500 μl or a fixed volume injection valve with injection loops ranging from 10 to 500 μl, a refrigerated autosampler, a thermostatic system capable to keep the column temperature constant even below the room temperature, a detector, a software for the acquisition and analysis of chromatographic data, a hardware with an operative system capable of managing the software for the acquisition and analysis of chromatographic data and equipped with a printer. 58. Device according to claim 57, wherein the detector is selected in the group consisting of high sensitive spectrophotometric diode array detector, with 5 cm light path flow cell, spectrophotometric diode array detector, variable wavelength UV-visible spectrophotometric detector, fluorimetric detector, mass spectrometry detector. 59. Device according to claim 58, wherein the spectrophotometric diode array detector is connected in parallel (ad/or in series) to a florimetric detector and/or to a mass spectrometric detector.

60. Device according to claim 57, wherein the mass spectrometric detector is of the "ion trap" type. 61. Device according to each of the claims from 57 to 60, wherein the HPLC system is equipped with an ODS C-18 reversed phase HPLC column.

62. Device according to claim 61 , wherein the column is chosen in the group consisting of columns having particle size, length and diameter, respectively, of 5 μm, 15 cm, 2.1 mm; 5 μm, 25 cm, 2.1 mm; 3 μm, 25 cm, 2.1 mm; 5 μm ,15 cm, 3.0 mm; 5 μm, 15 cm, 4.0 mm; 5 μm, 25 cm, 3.0 mm; 5 μm, 25 cm, 4.0 mm; 3 μm, 15 cm, 4.6 mm; 3 μm, 15 cm, 3.0 mm; 3 μm, 15 cm, 4.0 mm; 5 μm, 30 cm, 4.0 mm; 5 μm, 10 cm, 4.6 mm; 5 μm, 25 cm, 4.6 mm; 5 μm, 10 cm, 3.0 mm; 5 μm, 10 cm, 4.0 mm; 5 μm, 30 cm, 1.0 mm.

63. Kit for the direct simultaneous separation and quantification of compounds belonging to at least one of the groups chosen among purines and pyrimidines, N-acetylated amino acids, mono and dicarboxylic acids, sulphurylated compounds, nitrosylated compounds, bifunctional aldehydes, group B vitamins, and derivatives thereof, in a biological sample by means of the method described in the claims from 1 to 56, comprising two buffer solutions A and B having, respectively, essentially neutral pH and essentially acidic pH both containing an anionic exchanger pairing reagent.

64. Kit according to claim 63, wherein buffer solutions contain a quaternary ammonium ion, a polar organic solvent, a salt of a weak acid. 65. Kit according to claim 64, wherein wherein the concentration of the quaternary ammonium ion in buffer A ranges from 8 to 15 mM.

66. Kit according to claim 65, wherein wherein the concentration of the quaternary ammonium ion in buffer A ranges from 10 to 13 mM.

67. Kit according to claim 66, wherein the concentration of the quaternary ammonium ion in buffer A is 12 mM.

68. Kit according to claim 63, wherein the concentration of the polar organic solvent in buffer A ranges from 0.01 to 1 %.

69. Kit according to claim 68, wherein the concentration of the polar organic solvent in buffer A ranges from 0.1 to 0.5%. 70. Kit according to claim 69, wherein the concentration of the polar organic solvent in buffer A is 0.125%.

71. Kit according to claim 63, wherein the concentration of the salt of a weak acid in buffer A ranges from 5 to 15 mM.

72. Kit according to claim 71 , wherein the concentration of the salt of a weak acid in buffer A ranges from 8 to 13 mM.

73. Kit according to claim 72, wherein the concentration of the salt of a weak acid in buffer A is 10 mM.

74. Kit according to claim 63, wherein the concentration of quaternary ammonium ion in buffer B ranges from 0.01 to 3.5 mM. 75. Kit according to claim 74, wherein the concentration of quaternary ammonium ion in buffer B ranges from 2 to 3 mM.

76. Kit according to claim 75, wherein the concentration of quaternary ammonium ion in buffer B is 2.8 mM.

77. Kit according to claim 63, wherein the concentration of the polar organic solvent in buffer B ranges from 20 to 80%.

78. Kit according to claim 77, wherein the concentration of the polar organic solvent in buffer B ranges from 25 to 35%. 79. Kit according to claim 78, wherein the concentration of the polar organic solvent in buffer B is 30%.

80. Kit according to claim 63, wherein the concentration of the salt of a weak acid in buffer B ranges from 1 to 130 mM. 81. Kit according to claim 80, wherein the concentration of the salt of a weak acid in buffer B ranges from 80 to 110 mM.

82. Method according to claim 81, wherein the concentration of the salt of a weak acid in buffer B is 100 mM.

83. Kit according to each of the claims from 63 to 82, wherein the quaternary ammonium ion is chosen in the group consisting of tetrabutylammonium, tetramiristylammonium, tetraoctylammonium.

84. Kit according to each of the claims from 63 to 82, wherein the polar organic solvent is chosen in the group consisting of acetonitrile, phenols, primary, secondary and tertiary alcohols. 85. Kit according to each of the claims from 63 to 82, wherein the salt of a weak acid is chosen in the group consisting of potassium, sodium or ammonium acetate, potassium, sodium or ammonium phosphate, potassium, sodium or ammonium formiate.

86. Kit according to each of the claims from 63 to 85, wherein pH of buffer A ranges from 6.5 to 7.5.

87. Kit according to claim 86, wherein pH of buffer A is 7.00.

88. Kit according to each of the claims from 63 to 87, wherein pH of buffer B ranges from 4.5 to 6.5.

89. Kit according to claim 88, wherein pH of buffer B is 5.50. 90. Kit according to each of the claims from 63 to 89, further comprising a reversed phase column.

91. Kit according to claim 90, wherein the reversed phase column is an ODS C-18 column.

92. Kit according to claim 91 , wherein the column is chosen in the group consisting of columns having particle size, length and diameter, respectively, of 5 μm, 15 cm, 2.1 mm; 5 μm, 25 cm, 2.1 mm; 3 μm, 25 cm, 2.1 mm; 5 μm ,15 cm, 3.0 mm; 5 μm, 15 cm, 4.0 mm; 5 μm, 25 cm, 3.0 mm; 5 μm, 25 cm, 4.0 mm; 3 μm, 15 cm, 4.6 mm; 3 μm, 15 cm, 3.0 mm; 3 μm, 15 cm, 4.0 mm; 5 μm, 30 cm, 4.0 mm; 5 μm, 10 cm, 4.6 mm; 5 μm, 25 cm, 4.6 mm; 5 μm, 10 cm, 3.0 mm; 5 μm, 10 cm, 4.0 mm; 5 μm, 30 cm, 1.0 mm. 93. Kit according to each of the claims from 63 to 92, wherein buffer A comprises 12 mM tetrabutylammonium hydroxide, 0.125% methanol, 10 mM monobasic potassium phosphate, pH 7.00; buffer B comprises 2.8 mM tetrabutylammonium hydroxide, 30% methanol, 100 mM monobasic potassium phosphate, pH 5.50.

94. Kit according to claim 93, further comprising an ODS C-18 reversed phase column with 5 μm particle size, 250 mm length and 4.6 mm diameter.

95. Kit according to each of the claims from 63 to 94, that further comprise tubes for deproteinization of biological fluid samples equipped with a filtering membrane with a molecular cut-off of 3 kDa.

96. Kit according to each of the claims from 63 to 95, specifically devoted to the separation and quantification of purines, pyrimidines, and derivatives thereof, comprising an ODS-C18 reversed phase column having 5 μm particle size, 250 mm length and 4.6 mm diameter; the buffer A comprising 12 mM tertabutylammonium hydroxide, 0,125% methanol, 10 mM monobasic potassium phosphate, pH 7.00, the buffer B comprising 1 mM tertabutylammonium hydroxide, 70% methanol, 10 mM monobasic potassium phosphate, pH 5.50. 97. Kit according to each of the claims from 63 to 95 specifically devoted to the separation and quantification of mono and dicarboxylic acids and derivatives thereof, comprising an ODS-C18 reversed phase column having 5 μm particle size, 250 mm length and 4.6 mm diameter; the buffer A comprising 12 mM tertabutylammonium hydroxide, 0,125% methanol, 10 mM monobasic potassium phosphate, pH 7.00, the buffer B comprising 1 mM tertabutylammonium hydroxide, 80% methanol, 1 mM monobasic potassium phosphate, pH 5.50.

98. Kit according to each of the claims from 63 to 95 specifically dedicated to the separation and quantification of N-acetylated amino acids and derivatives thereof, comprising an ODS-C18 reversed phase column having 5 μm particle size, 250 mm length and 4.6 mm diameter; the buffer A that comprises 12 mM tertabutylammonium hydroxide, 0,125% methanol, 10 mM monobasic potassium phosphate, pH 7.00, the buffer B that comprises 0.2 mM tertabutylammonium hydroxide, 80% methanol, 1 mM monobasic potassium phosphate, pH 5.50.

99. Kit according to each of the claims from 63 to 95 specifically dedicated to the separation and quantification of nitrosylated compounds and derivatives thereof, comprising an ODS-C18 reversed phase column having 5 μm particle size, 250 mm length and 4.6 mm diameter; the buffer A that comprises 12 mM tertabutylammonium hydroxide, 0,125% methanol, 10 mM monobasic potassium phosphate, pH 7.00, the buffer B that comprises 0.3 mM tertabutylammonium hydroxide, 80% methanol, 1 mM monobasic potassium phosphate, pH 5.50.

100. Kit according to each of the claims from 63 to 95 specifically dedicated to the separation and quantification of purines, sulphurylated compounds, nitrosylated compounds, monocarboxylic acids, bifunctional aldehydes, and derivatives thereof, comprising an ODS-C18 reversed phase column having 5 μm particle size, 250 mm length and 4.6 mm diameter; the buffer A that comprises 12 mM tertabutylammonium hydroxide, 0,125% methanol, 10 mM monobasic potassium phosphate, pH 7.00, the buffer B that comprises 0.1 mM tertabutylammonium hydroxide, 80% methanol, 1 mM monobasic potassium phosphate, pH 5.50.

101. Kit according to each of the claims from 63 to 95 specifically dedicated to the separation and quantification of group B vitamins, monocarboxylic acids, and derivatives thereof, comprising an ODS C-18 reversed phase column having 5 μm particle size, 250 mm length and 4.6 mm diameter; buffer A that comprises 12 mM tetrabutylammonium hydroxide, 0.125% methanol, 10 mM monobasic potassium phosphate, pH 7.00; buffer B that comprises 0.1 mM tetrabutylammonium hydroxide, 80% methanol, 1 mM monobasic potassium phosphate, pH 5.50. 102. Use of buffers A and B, as specified in claim 93, in a sequential mode for the preparation of a step gradient in separation methods.