AU2016101553A4

AU2016101553A4 - Novel methods and kits for detecting of urea cycle disorders using mass spectrometry

Info

Publication number: AU2016101553A4
Application number: AU2016101553A
Authority: AU
Inventors: Géraldine Carrard; Ralph Fingerhut
Original assignee: Labsystems Diagnostics Oy
Current assignee: Labsystems Diagnostics Oy
Priority date: 2015-09-02
Filing date: 2016-09-02
Publication date: 2016-10-06
Anticipated expiration: 2024-09-02
Also published as: DE202016104839U1

Abstract

The present invention relates to newborn screening kits, methods and internal standard solution for high throughput screening and analysis of metabolic disorders using liquid chromatography mass spectrometry (LC-MS) are provided. The metabolic disorders can be amino acid, organic acid or fatty acid oxidation disorders, and particularly urea cycle disorders or deficiencies, hyperammonemia, Hyperornithinemia hyperammonemia-homocitrullinuria (HHH), and/or argininosuccinic aciduria.

Description

NOVEL METHODS AND KITS FOR DETECTING OF UREA CYCLE DISORDERS USING MASS SPECTROMETRY

FIELD OF THE INVENTION

Methods, reagents, internal standard solutions, and kits for high throughput screening and analysis of metabolic disorders using liquid chromatography mass spectrometry (LC-MS) are provided. The methods, reagents, internal standard solutions, and kits are particularly useful for conducting a plurality of in vitro screening tests in newborns and detecting a panel of metabolic disorders at high speeds, for confirmation and/or follow up of the same diseases.

BACKGROUND OF THE INVENTION

Particular newborn metabolic deficiencies to be detected in newborns are urea cycle deficiencies. The urea cycle is a metabolic pathway for disposal of the toxic metabolite ammonia and surplus nitrogen as urea in mammals. The urea cycle plays an important role in prevention of the accumulation of toxic nitrogenous compounds and further contains several of the biochemical reactions required for the de novo biosynthesis and degradation of arginine. As shown in Figure 1, the urea cycle is catalyzed by five enzymes: carbamoyl phosphate synthetase 1 (CPS1), ornithine transcarbamoylase (OTC), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and arginase (ARG1). The first two enzymes of the urea cycle are located within the mitochondrial matrix and the remaining three enzymes are cytosolic. CPS1 promotes the formation of carbamoyl phosphate from ammonium ions and carbon dioxide in the first step. The next reaction is catalyzed by OTC which transfers a carbamoyl group from carbamoyl phosphate to ornithine to form citrulline. ASS is involved in the formation of argininosuccinate from citrulline and aspartate in the third step and ASL facilitates the formation of arginine and fumarate from argininosuccinate in the fourth step. ARG1 catalyzes the formation of ornithine and urea from arginine in the fifth step. In addition, N-acetyl glutamate synthetase (NAGS) catalyses the formation of N-acetyl glutamate which activates the CPS1. Therefore, interruptions in the metabolic pathway for urea synthesis may be caused by the deficiency or inactivity of any one of several enzymes involved in specific steps in the cascade.

There is however an existing clinical situation that challenges the introduction of universal neonatal screening for UCDs. To this regard, a recent UCD guideline concluded that NBS for proximal disorders cannot currently be recommended, but it may be considered for the distal UCDs. In effect, attempts to develop UCDs screening tests which are based on the direct measurement of enzymes or accumulated substrates in blood or urine are limited to the last steps in the cycle, since the enzymes are restricted to the mitochondria, for the first steps, e.g.. NAGS, CPS1 and OTC, and there is thus no substrate accumulation (Figure 1).

One of the most common and severe defect of the urea cycle, OTC deficiency, was considered for inclusion in the panel, but it did not meet the assigned evaluation criteria, due to the lack of a screening test that had been validated in the general newborn population. The laboratory indications for OTC deficiency are elevated concentrations of glutamine and ammonia, low citrulline, and elevated excretion of orotic acid in the urine. NBS laboratories have thus attempted to use low citrulline and several ratios to identify infants at risk for OTC deficiency, but have had so far limited success. Also, highlighting the difficulty to use low citrulline as a marker for the detection of proximal UCDs, a study in Tuscany applying LC-MS/MS was performed between 2001 and 2008. The authors concluded that hypocitrullinemia was not a reliable marker for OTCD newborn screening, especially for late-onset forms that may exhibit normal citrulline levels. Low citrulline concentrations may also be found in other metabolic disorders further challenging its use as screening marker.

Several other caveats regarding newborn screening for urea cycle defects are that CPS1 deficiency, OTC deficiency, and NAGS deficiency currently cannot be reliably detected. Furthermore, although hyperargininemia or ARG1 deficiency has been detected by these methods, newborn screening cannot be expected to reliably detect all cases.

The common factor to all five enzyme deficiencies in this pathway and that is the extreme elevation of the plasma ammonia level (hyperammonemia). Hyperammonemia is typically associated with an increase in ammonia buildup are acute episodes of vomiting, abnormal liver enzyme levels, lethargy, convulsions and coma. Even treated, protracted severe hyperammonemia leads to mental and physical retardation. Detection of hyperammonemia would thus be most important for early diagnosis and effective treatment. However, technically, the direct measurement of ammonia in a dried blood spot is nearly impossible.

Since the direct measurement of ammonia is not feasible in NBS, glutamine would be another metabolite that is generally elevated in UCDs. However, numbers of difficulties have also been reported for using glutamine as a marker for UCDs. Indeed, glutamine is highly unstable in plasma and serum, and thus spontaneously convert into glutamate and pyroglutamate, which formations lead to false low glutamine levels, rendering glutamine currently not a suitable screening parameter for NBS using LC-MS.

The use of mass spectrometry (MS) in clinical laboratories has very much increased with time. This development is obviously due to great advances in mass spectrometry applications in the last fifteen years. Mass spectrometry permits a very rapid measurement of different metabolites in different biological specimens using filter paper spots or directly in different biological fluids. Because of its high sensitivity, this technique can be used for qualitative and quantitative analysis of many analytes or metabolites such as amino acids and acylcamitines, homocysteine, orotic acid, succinylacetone, etc... with appropriate internal standards. Particularly, MS is extensively used for analysis of metabolites from dried blood spots taken at birth (Guthrie-cards) but among the detected metabolites those due to UCDs are not effectively detected because the defects discussed in the earlier paragraphs.

While the inclusion of UCD screening into newborn screening (NBS) is highly desirable, it is hampered by the fact that there is not a specific marker for every single UCD, by the fact that so far the common feature of UCDs, e. i.'.. hyperammonemia, is not directly detectable in dried blood spots (DBS), and by the fact that the detection of secondary elevations of glutamine seemed not feasible, because of the proposed instability of glutamine in DBS.

Hence, the aim of the present invention is to provide an analytical method, internal standard solutions, and reagent kits that could allow the determination of metabolites correctly indicative of UCDs, particularly of glutamine, along with the determination of other metabolites that are commonly determined for metabolites screening, especially those screening performed on dried blood spots taken at birth. The present invention thus relates to a newborn screening kit allowing for a fast and reliable determination and detection of a plurality of UCDs as well other metabolic deficiencies using tandem mass spectrometry (tandem-MS NBS).

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

SUMMARY OF THE INVENTION

According to a first aspect, the present invention provides a newborn screening method of detecting the presence and/or measuring the levels of one or more metabolites in a blood sample, optionally a dried spot blood, of a newborn, by tandem mass spectrometry, said metabolites comprising at least glutamine and said method comprising the following steps: (i) extracting said metabolites from the sample with an extraction solution; (ii) providing, before or after the step (i), an internal standard solution comprising a known amount of one or more stable-isotope labeled internal standards corresponding to said metabolites to be detected in the sample, (iii) ionizing said metabolites and said one or more stable-isotope labeled internal standards to generate ions, (iv) acquiring the mass to charge (m/z) ratio of said one or more ions in the mass spectrum in the Multiple Reaction Monitoring (MRM) mode, and (v) determining the sum of glutamine and lysine in MRM mode, (vi) detecting and quantifying the amount of glutamine with stable-isotope labeled internal standard corresponding to lysine.

According to a second aspect, the present invention provides a newborn screening kit when used in detecting the presence and/or measuring the levels of glutamine in a blood sample of a newborn, by tandem mass spectrometry, allowing diagnosis of one or more metabolic deficiencies selected among urea cycle disorders, and/or hyperammonemia, Hyperomithinemia-hyperammonemia-homocitrullinuria (HHH), and/or argininosuccinic aciduria, said kit comprising an internal standard solution comprising at least a stable isotopically-labeled internal standard corresponding to lysine.

According to a third aspect, the present invention provides an internal standard solution comprising at least a lysine stable isotopically-labeled internal standard, and further comprising one or more stable isotopically-labeled internal standards corresponding to alanine, citrulline, homocitrulline glycine, leucine, methionine, ornithine, glutamine, phenylalanine, valine, glutamic acid, aspartic acid, glutamic acid, tyrosine, serine, oxoproline, proline, argininosuccinate, succinylacetone, orotic acid, free carnitine, acetylcamitine, propionylcamitine, butyrylcamitine, isovalerylcaminitine, glutarylcamitine, hexanoylcamitine, octanoylcamitine, decanoylcamitine, lauroylcamitine, myristoylcamitine, palmitoylcamitine, and/or stearoylcamitine, wherein said standards are optionally dried.

The present invention thus relates to newborn screening kits useful to assess the levels of glutamine metabolite in the blood sample from a newborn, comprising a testing tray which itself comprises a plurality of cells, and individually stored internal standards, comprising at least lysine stable isotopically-labeled internal standard, which are placed in the plurality of cells of the testing tray, and optionally a solvent dispenser such as micropipette or any other mean to dispense the solvent. Such individually stored standards may be preferably dried.

The present invention also relates to newborn screening kits, internal standard solutions, and methods for measuring the levels of glutamine in a sample, such as a dried blood spot obtained from the newborns. The kits, internal standard solutions, and methods are particularly useful for newborn screening (NBS) and particularly for detecting urea cycle disorders or deficiencies (UCDs), OTC deficiency, and/or hyperammonemia, HHH, and/or argininosuccinic aciduria. Such kits, internal standard solutions, and methods are also useful for detecting and/or further quantifying metabolites including amino acids, organic acids, a plurality of carnitines, and/or succinyl acetone in said sample.

The present invention also relates to newborn screening kits, internal standard solutions, and methods for screening or diagnosing UCDs, hyperammonemia, HHH, and/or argininosuccinic aciduria in newborns comprising detecting and/or measuring the levels of glutamine in a sample, such as a dried blood spot obtained from newborns.

The present invention further relates to a novel set of internal standards or internal standard solution which may be used in the method or with the kits of the present invention, comprising at least a lysine stable isotopically-labeled internal standard, and further comprising one or more additional stable isotopically-labeled internal standard corresponding to additional metabolites to be tested in said newborn screening kit. The novel set of internal standards or the internal standard solution may be present in dried form.

FIGURES

Figure 1: shows a schematic of the urea cycle.

Figure 2: is a table showing pathological values for the main enzymatic deficiencies within the urea cycle. Figure 3: is a table providing some control levels of endogenous amino acids.

Figure 4: is a graph showing the linearity of the measurement of glutamine, lysine, arginino succinic acid, and orotic acid and standard solutions of the respective analyte using tandem mass spectrometry.

Figure 5: is a graph showing the linearity of the measurement of glutamine, lysine, arginino succinic acid, and orotic acid and standard solutions of the respective analyte using tandem mass spectrometry.

Figure 6: is a graph showing the linearity of the measurement of glutamine, lysine, arginino succinic acid, and orotic acid and standard solutions of the respective analyte using tandem mass spectrometry.

Figure 7: is a graph showing the linearity of the measurement the sum of lysine and glutamine in dried blood spots (DBS) using ion exchange chromatography.

Figure 8: shows the proposed results of the measurement of the sum of glycine and lysine, as well as measured values from 180 DBS of healthy newborns, measured values from 2 samples of a patient with proven OTC deficiency, and expected range of patients with urea cycle defects (UCDs). In this Figure, the legends are as follows: Lysin = Reference Range from Literature, Glutamine = Reference Range from

Literature, Sum(Gln + Lys) = Combined Reference Range, Normal = 180 normal NBS samples OTC = 2 DBS from a patient with proven OTC deficiency, UCD = expected range for newborns with UCD.

Figure 9: is a table showing the physiological levels of amino acids in plasma (pmol/L) in men, women, adolescents and children.

Figure 10: is a flowchart showing the decision tree starting from the detection of elevated sum of glutamine and lysine as early markers to subsequent diagnosis steps for detecting more precisely the type of UCD in the subject.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is thus directed a newborn screening kit, method and novel sets of internal standards or internal standard solutions for detecting the presence and/or measuring the levels of metabolites in a sample obtained from a subject, by tandem mass spectrometry, said metabolites comprising at least glutamine, and/or sum of lysine and glutamine, wherein said kit and method comprise the following steps: (i) extracting said metabolites from the sample with an extraction solution; (ii) providing, before or after the step (i), a solution comprising a known amount of one or more stable isotopically-labeled internal standards corresponding to said metabolites to be detected in the sample, (iii) ionizing said metabolites and said one or more stable isotopically-labeled internal standards to generate ions, (iv) acquiring the mass to charge (m/z) ratio of said one or more ions in the mass spectrum in the Multiple Reaction Monitoring (MRM) mode, and (v) determining the sum of glutamine and lysine in MRM mode, (vi) detecting and quantifying the amount of glutamine and/or glutamine and lysine with stable isotopically-labeled internal standard corresponding to lysine, and optionally glutamine.

In vitro method newborn screening kit, and novel sets of internal standards or internal standard solutions of the present invention allow quantifying target metabolites having same isobaric properties, and the quantitative concentration of one or more isobars in a sample can be determined by using the sum or ratios. The methods thus provides for high throughput analysis of metabolites in complex mixtures for unresolved chromatographic peaks and or unstable metabolites. Such methods allow for deconvoluting contributions of a plurality of metabolites in a sample from a mass spectrometer signal, preferably a tandem mass spectrometry signal. Therefore, quantitative concentrations of metabolites, such as, for example, isobars or structural isomers may be obtained. Because of the isobaric natures of glutamine and lysine, sum and or ratios of such isobars can be assessed from the chromatographic signal, subsequently the levels of glutamine may then be deduced by comparing with the stable isotopically-labeled internal standard corresponding to lysine or to alternatives to lysine as mentioned above.

Therefore, according to the present invention, lysine is used to quantify the amount of glutamine. However, as indicated above, it is understood that the present methods, kits and internal standards are not limited to lysine. Any metabolite having a similar mass structure as that of lysine and being isobaric to glutamine may also be used as an alternative to lysine in the methods and reagent kits of the present invention. The quantitative concentration of one or more isobars in a sample can be determined by using the sum or ratios, and to quantify the amount of glutamine in the sample of subjects. By way of examples of alternatives for lysine, we can cite glutamate, methionine, methyl-, dimethyl-, trimethy-, hydroxy-, acetyl-, sumoyl-, glocosilated- lysine, as well as precursors of lysine synthesis, such as aspartic acid, etc... The present invention also relates to a newborn screening kit, in vitro method and novel sets of internal standards or internal standard solutions for the diagnosis and/or the detection of metabolic disorders such as UCDs, hyperammonemia, argininosuccinic aciduria, and/or Hyperomithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, comprising detecting the presence and/or measuring the levels of metabolites in a sample obtained from a newborn, by tandem mass spectrometry, said metabolites comprising at least glutamine, and said kit and method comprising the following steps: (i) extracting the metabolites from the sample with an extraction solution; (ii) providing, before or after the step (i), a solution comprising a known amount of one or more stable isotopically-labeled internal standards corresponding to the metabolites to be detected in the sample, (iii) ionizing said metabolites and said one or more stable isotopically-labeled internal standards to generate ions, (iv) acquiring the mass to charge (m/z) ratio of said one or more ions in the mass spectrum in the Multiple Reaction Monitoring (MRM) mode, (v) determining the sum of glutamine and lysine in MRM mode, and (vi) detecting and quantifying the amount of glutamine with stable isotopically-labeled internal standard corresponding to lysine, and optionally (vii) determining whether the level of glutamine as obtained in step (vi) is elevated compared to physiological levels of glutamine.

Expected plasmatic physiological levels of glutamine for patients having such deficiencies are well known in the art (See for example Physician’s Guide to the Laboratory Diagnosis of Metabolic Diseases, Editors: Nenad Blau, Marinus Duran, Milan E. Blaskovics, K. Michael Gibson, Springer-Verlag Berlin Heidelberg, 2nd Edition 2003, which is incorporated herein by reference). Such levels of glutamine in plasma of healthy subjects are provided in Figures 2 and 3. For example, physiological levels of glutamine in children may range from around 200 to 900pmol/L, or from 250 to 850pmol/L, or from around 330 to 809 pmol/L. Determining the presence and quantifying the amount of glutamine correlates with the presence or absence of urea cycle disorders and/or hyperammonemia, and/or argininosuccinic aciduria, and/or Hyperomithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome.

Hyperammonemia refers to a clinical condition associated with elevated ammonia levels manifested by a variety of symptoms and signs, including significant central nervous system (CNS) abnormalities, ft is crucial to include diagnostic method for early assessment of neonatal predisposition to UCDs, hyperammonemia, and/or argininosuccinic aciduria in a human neonatal patient, before the physiologic and behavior symptoms become apparent.

Hyperomithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is characterized by high plasma concentrations of glutamine and ammonia. A characteristic clinical biochemical abnormality in this case is significant elevations in plasma glutamine concentrations. However, as plasma ammonia concentrations return to normal, plasma glutamine concentrations may remain mildly elevated (1.5- to twofold the upper limits of control values). Generally ammonia formed is trapped by glutamate to form glutamine by the enzyme, Glutamine Synthetase, hence any elevation of glutamine would lead to a higher trapping of ammonia. Hence glutamine would serve as a useful marker for detection and for planning further treatment of HHH by administration of pharmacological agents that can act as glutamine trap in particular and amino acid trap in general, hence diverting nitrogen from urea synthesis to alternatives routes of excretion.

As shown in Figure 1, Urea Cycle Disorders (UCDs) may include a variety of genetic defects due to one or more enzymatic deficiencies within the urea cycle: (a) carbamyl phosphate synthetase deficiency (CPSD or CPS 1), (b) N-acetyl glutamate synthetase deficiency (NAGS), (c) ornithine transcarbamylase deficiency (OTCD), (d) argininosuccinic acid synthetase deficiency (ASD), (e) argininosuccinate lyase deficiency or Citrullinemia type 1 (ALD or ASS 1), and (f) arginase deficiency (ARG1). Each of these diseases represents a defect in the biosynthesis of one of the normally expressed enzymes of the urea cycle and is characterized by signs and symptoms induced by the accumulation of precursors of urea, principally ammonium and glutamine.

The present invention is thus particularly useful for detection of proximal markers like glutamine, which serve as diagnostic marker for proximal urea cycle disorders defects. The current invention helps to detect additional markers like glutamine which helps to discriminate between the proximal and distal urea cycle defects, as such markers could be products of the proximal enzymes and a substrate for the distal enzymes. Prompt replacement of glutamine and other amino acids is then possible once it is determined whether the defect is in a proximal or distal urea cycle disorder.

According to a second embodiment of the invention, newborn screening kits and methods as well as the novel set of standards or internal standard solutions are useful for further detecting the presence and/or measuring the levels of additional metabolites alongside to glutamine in a blood sample obtained from a newborn, by tandem mass spectrometry, and is thus particularly useful to detect all UCDs, including proximal abnormalities along with classical distal deficiencies. According to this embodiment, said further metabolites may comprise arginine, citrulline, argininosuccinate, ornithine, lysine, and/or orotic acid. Indeed as showed in the diagnosis decision tree in Figure 10, once an elevated level of glutamine has been detected according to the present invention, a precise determination of the type of UCDs may be further diagnosed, such as in particular, new proximal urea cycle deficiencies including N-acetylglutamate synthase (NAGS) deficiency, carbamyl phosphate synthetase (CPS) deficiency, ornithine transcarbamylase (OTC) deficiency, and/or ornithine translocase deficiency (HHH).

According to this embodiment, the present invention thus relates to newborn screening kits and in vitro methods as well as novel set of standards or internal standard solutions are useful for further detecting the presence and/or measuring the levels of additional metabolites alongside to glutamine, selected among arginine, citrulline, argininosuccinate, ornithine, lysine, and/or orotic acid, in a blood sample obtained from a newborn, by tandem mass spectrometry, and is thus particularly useful to conduct a plurality of in vitro tests to assay further metabolites in said blood sample, said kit and method comprising: (i) extracting said metabolites from the sample with an extraction solution; (ii) providing, before or after the step (i), a solution comprising a known amount of one or more stable isotopically-labeled internal standards corresponding to the metabolites to be detected in the sample; (iii) ionizing the extracted metabolites and said one or more stable isotopically-labeled internal standards to generate ions; (iv) acquiring the mass to charge (m/z) ratio of said one or more ions in the mass spectrum in the Multiple Reaction Monitoring (MRM) mode; (v) determining the sum of glutamine and lysine in MRM mode, (vi) detecting and quantifying the amount of glutamine with stable isotopically-labeled internal standard corresponding to lysine; (v) determining the presence and/or the levels of said one or more metabolites by analyzing the mass MRM spectrum as obtained in step (iv).

According to this embodiment, the present invention thus provides reliable and sensitive newborn screening kits and methods to evaluate the predisposition, presence and severity of a broader number of UCDs including OTC deficiency, argininosuccinate synthetase deficiency (citrullinemia), argininosuccinate lyase deficiency (argininosuccinicaciduria), arginase deficiency and hyperammonemia-hyperomithinemia-homocitrullinemia syndrome (HHH), at an improved detection and/or precision level than the methods typically practiced at the present time, by the determination and quantification of a combination of various indicator metabolites in a biological sample.

According to a first aspect of this embodiment, the present invention relates to newborn screening kits, newborn screening in vitro methods, and novel set of internal standards or internal standard solution for detecting or diagnosing in newborns a suspicion of arginase deficiency (ARG1), comprising detecting the presence and/or measuring the levels of metabolites in a sample obtained from a newborn, by tandem mass spectrometry, said metabolites comprising at least glutamine and arginine, and said kit and method comprising the following steps: (i) extracting the metabolites from the sample with an extraction solution; (ii) providing, before or after the step (i), a solution comprising a known amount of one or more stable isotopically-labeled internal standards corresponding to the metabolites to be detected in the sample, (iii) ionizing said metabolites and said one or more stable isotopically-labeled internal standards to generate ions, (iv) acquiring the mass to charge (m/z) ratio of said one or more ions in the mass spectrum in the Multiple Reaction Monitoring (MRM) mode, (v) determining the sum of glutamine and lysine in MRM mode, and (vi) detecting and quantifying the amount of glutamine with stable isotopically-labeled internal standard corresponding to lysine, (vii) determining whether the amount of glutamine as obtained in step (vi) is elevated compared to physiological levels of glutamine, and (viii) if the level of glutamine as obtained in step (vii) is elevated, further determining the level of arginine and whether the level of arginine is elevated compared to physiological levels of arginine, thereby allowing to diagnose a suspicion of arginase deficiency.

According to a second aspect of this embodiment, the present invention relates to newborn screening kits, newborn screening in vitro methods, and novel set of internal standards or internal standard solution for detecting or diagnosing in newborns a suspicion of N-acetylglutamate synthetase (NAGS) deficiency, comprising detecting the presence and/or measuring the levels of metabolites in a sample obtained from a newborn, by tandem mass spectrometry, said metabolites comprising at least glutamine, arginine, citrulline, said kit and method comprising the following steps: (i) extracting the metabolites from the sample with an extraction solution; (ii) providing, before or after the step (i), a solution comprising a known amount of one or more stable isotopically-labeled internal standards corresponding to the metabolites to be detected in the sample, (iii) ionizing said metabolites and said one or more stable isotopically-labeled internal standards to generate ions, (iv) acquiring the mass to charge (m/z) ratio of said one or more ions in the mass spectrum in the Multiple Reaction Monitoring (MRM) mode, (v) determining the sum of glutamine and lysine in MRM mode, and (vi) detecting and quantifying the elevated level of glutamine with stable isotopically-labeled internal standard corresponding to lysine, (vii) detecting the level of arginine and citrulline with stable isotopically-labeled internal standards corresponding to arginine and citrulline, respectively, and (viii) if the level of glutamine as obtained in step (vi) is elevated, and the level of arginine is decreased compared to physiological levels of arginine, further determining whether the level of citrulline is decreased compared to physiological levels of citrulline, thereby suspecting a deficiency of the N-acetyl glutamate synthetase (NAGS).

According to third aspect of this embodiment, the present invention relates to newborn screening kits and newborn screening in vitro methods, and novel set of internal standards or internal standard solution for detecting or diagnosing in newborns of argininosuccinate synthase (ASS) deficiency, argininosuccinate aciduria (ASA) and/or citrullinemia, comprising detecting the presence and/or measuring the levels of metabolites in a sample obtained from a newborn, by tandem mass spectrometry, said metabolites comprising at least glutamine, arginine, citrulline, argininosuccinate, and said kit and method comprising the following steps: (i) extracting the metabolites from the sample with an extraction solution; (ii) providing, before or after the step (i), a solution comprising a known amount of one or more stable isotopically-labeled internal standards corresponding to the metabolites to be detected in the sample, (iii) ionizing said metabolites and said one or more stable isotopically-labeled internal standards to generate ions, (iv) acquiring the mass to charge (m/z) ratio of said one or more ions in the mass spectrum in the Multiple Reaction Monitoring (MRM) mode, (v) determining the sum of glutamine and lysine in MRM mode, and (vi) detecting and quantifying the elevated level of glutamine with stable isotopically-labeled internal standard corresponding to lysine, (vii) detecting the level of arginine, citrulline, and argininosuccinate with an internal standard solution corresponding to stable isotopically-labeled internal standards corresponding to arginine, citrulline, and argininosuccinate, and (viii) if the level of glutamine as obtained in step (vi) is elevated and the level of arginine is decreased compared to physiological levels of arginine, and further determining whether the level of citrulline is increased compared to physiological levels of citrulline, thereby diagnosing a suspicion of citrullinemia, and further determining whether the level of argininosuccinate is elevated compared to physiological levels of argininosuccinate, thereby suspecting a deficiency of the argininosuccinate aciduria (ASA).

According to fourth aspect of this embodiment, the present invention relates to newborn screening kits, newborn screening in vitro methods, and novel set of internal standards or internal standard solution for detecting or diagnosing in newborns a deficiency of ornithine transcarbamylase deficiency (OTCD) and/or of carbamyl phosphate synthetase (CPS) deficiency, comprising detecting the presence and/or measuring the levels of metabolites in a sample obtained from a newborn, by tandem mass spectrometry, said metabolites comprising at least glutamine, arginine, citrulline, and orotic acid, and said kit and method comprising the following steps: (i) extracting the metabolites from the sample with an extraction solution; (ii) providing, before or after the step (i), a solution comprising a known amount of one or more stable isotopically-labeled internal standards corresponding to the metabolites to be detected in the sample, (iii) ionizing said metabolites and said one or more stable isotopically-labeled internal standards to generate ions, (iv) acquiring the mass to charge (m/z) ratio of said one or more ions in the mass spectrum in the Multiple Reaction Monitoring (MRM) mode, (v) determining the sum of glutamine and lysine in MRM mode, and (vi) detecting and quantifying the elevated level of glutamine with stable isotopically-labeled internal standard corresponding to lysine, (vii) detecting the level of arginine, citrulline, and orotic acid with an internal standard solution comprising stable isotopically-labeled internal standards corresponding to arginine, citrulline, and orotic acid, and (viii) if the level of glutamine as obtained in step (vi) is elevated and the levels of arginine and citrulline are decreased compared to physiological levels of arginine and citrulline, further determining whether the level of orotic acid is increased compared to physiological levels of orotic acid, thereby diagnosing a suspicion of deficiency of the ornithine transcarbamylase (OTCD), or whether the level of orotic acid is normal, thereby diagnosing a suspicion of carbamoyl phosphate synthetase (CPS) deficiency.

According to fifth aspect of this embodiment, the present invention relates to newborn screening kits, newborn screening in vitro methods, and novel set of internal standards or internal standard solution for screening or diagnosis in newborns a suspicion of Lysinuric protein intolerance (LPI) or Hyperomithinemia-hyperammonemia-homocitrullinuria (HHH), comprising detecting the presence and/or measuring the levels of metabolites in a sample obtained from a newborn, by tandem mass spectrometry, said metabolites comprising at least glutamine, lysine, and ornithine, and said kit and method comprising the following steps: (i) extracting the metabolites from the sample with an extraction solution; (ii) providing, before or after the step (i), a solution comprising a known amount of one or more stable isotopically-labeled internal standards corresponding to the metabolites to be detected in the sample, (iii) ionizing said metabolites and said one or more stable isotopically-labeled internal standards to generate ions, (iv) acquiring the mass to charge (m/z) ratio of said one or more ions in the mass spectrum in the Multiple Reaction Monitoring (MRM) mode, (v) determining the sum of glutamine and lysine in MRM mode, and (vi) detecting and quantifying the amount of glutamine and lysine with stable isotopically-labeled internal standard corresponding to lysine, (vii) determining whether the amount of glutamine as obtained in step (vi) is elevated and whether the level of lysine is decreased as compared to physiological levels of glutamine and lysine; (viii) if the level of glutamine as obtained in step (vii) is elevated and level of lysine is decreased compared to physiological levels, further determining whether the level of ornithine is elevated compared to physiological levels of ornithine, thereby allowing to diagnose a suspicion of Hyperomithinemia-hyperammonemia-homocitrullinuria (HHH), or whether the level of ornithine is normal, thereby allowing to diagnose a suspicion of a Lysinuric protein intolerance (LPI).

Physiological levels of arginine, citrulline, argininosuccinate, orotic acid, and ornithine are well known in the art. Those are inter alia described in Physician’s Guide to the Laboratory Diagnosis of Metabolic Diseases, Editors: Nenad Blau, Marinus Duran, Milan E. Blaskovics, K. Michael Gibson, Springer-Verlag Berlin Heidelberg, 2nd Edition 2003, ISBN 978-3-642-62709-5). Levels of amino acids in plasma of healthy subjects are provided in Figure 9.

Additional metabolites which may be detected using newborn screening kits, methods and internal standard solutions of the present invention comprise for example N-acetyl glutamate, proline, serine, aspartate, and/or homocitrulline, thereby allowing a more precise diagnosis of the UCD. Indeed, it has been reported so far that a low level of N-acetyl glutamic acid correlated with a deficiency of the NAGS enzyme, or that low level of proline correlated with a deficiency of the pyrroline-5-carboxylase synthase (P5CS), or again that homocitrulline detection in blood correlate with the hyperomithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome.

Therefore, according to this second embodiment, newborn screening kits and methods, as well as novel set of internal standards or internal standard solution, advantageously provide a more complete metabolic profile allowing for a more reliable and efficient in vitro diagnosis or prediction of UCDs, hyperammonemia, HHH, and/or argininosuccinic aciduria in a subject, since the accumulation of glutamine, which is an early marker of the urea cycle is now detected in a biological sample of a newborn subject alongside to further metabolites indicative of UCDs, hyperammonemia, and/or argininosuccinic aciduria. As shown in the table of Figure 2, glutamine is one of the most important markers as it is indicative of a wide range of UCDs. Therefore, detecting and measuring glutamine in combination with other marker metabolites is crucial for the screening and detection of UCDs.

According to a third embodiment, newborn screening kits and in vitro methods as well as novel set of internal standards or internal standard solution according to the present invention may further be used to determine the presence and/or levels of further additional metabolites which have been commonly used as markers of metabolic diseases, especially in newborns, thereby ensuring a much broader detection of disease conditions in newborns at once.

Said further additional metabolites may include without any limitations, amino acids, organic acids, carnitines or a plurality of carnitines, as well as succinylacetone (SUAC). Elevated amino acids, free carnitine and acylcamitine levels are examples of metabolites that can be indicative of one or more of several metabolic disorders. Free carnitine and acylcamitines are markers for disorders that are classified as fatty acid oxidation (FAO) disorders and organic acidurias (OA). Similarly, amino acids are used as markers for several metabolic disorders collectively known as amino acidopathies.

Therefore according to this third embodiment, the newborn screening kits, in vitro methods, and novel set of internal standards or internal standard solution may be used to identify, detect and/or quantitate clinically relevant amino acids which are commonly used as markers of metabolic diseases in newborns. The identity and amount of amino acids in a patient's body fluid is important in a patient's health for a number of reasons. Aberrant amino acid levels can be used to diagnose disease or illness. The presence, absence, identity, amount or modification of an endogenous amino acid as well as its presence and amount in comparison to other amino acids (i.e., the overall profile of free amino acids) are important parameters in assessing a subject's metabolic state. Figure 3 provides some control levels of endogenous amino acids. Aberrant amino acid levels or increased/decreased levels of certain amino acids in comparison to other amino acids can indicate a metabolic disturbance. The qualitative and quantitative analysis of free amino acids in blood is central to the diagnosis and management of a wide variety of metabolic disturbances including primary amino acid enzymopathies (e.s.. phenylketonuria, maple syrup urine disease) and disorders of amino acid transport (e.s.. cystinuria).

By way of examples, these metabolites may be selected within proteinogenic amino acids plus non-proteinogenic amino acids. More precisely, they may be chosen among alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, γ-aminobutyric acid (GABA), L-Dopa, hydroxyproline, selenomethionine, phosphoserine, α-aminoadipic acid, phosphoethanolamine, sarcosine, β-alanine, taurine, β-aminoisobutyric acid, camosine, methyl histidine, alpha-aminobutyric acid, anserine, ethanolamine, cystathionine, hydroxylysine, ornithine, argininosuccinate, s-sulfocysteine, homocitrulline, hawkinsin. Amino acid metabolites are preferably chosen among valine, alanine, leucine, lysine, ornithine, phenylalanine, tyrosine, glycine, aspartate, glutamate, citrulline, arginine, proline, methionine, serine, homocitrulline, asparagine, and/or 5-oxoproline.

Other further additional metabolites may also comprise carnitines such as free carnitine, acetyl carnitine, propionylcamitine, butyrylcamitine, isovalerylcaminitine, glutarylcamitine, hexanoylcamitine, octanoylcamitine, decanoylcamitine, lauroylcamitine, myristoylcamitine, palmitoylcamitine, and/or stearylcamitine.

Acylcamitine profile (ACP) analysis is performed for the biochemical screening of disorders of fatty acid oxidation (FAO) and organic acid metabolism. Inherited FAO disorders are inborn errors of metabolism (IEM) of relatively recent discovery. They may present at any age, from birth to adulthood, frequently leading to life-threatening episodes of metabolic decompensation after a period of inadequate caloric intake and intercurrent illness. Typical manifestations are hypoketotic hypoglycemia, liver disease, skeletal and cardiomyopathy, and sudden unexpected death. Organic acidemias are a more heterogeneous group of IEM. They typically present with recurrent episodes of acute life-threatening illness, hypo- or hypertonia, failure to thrive, and developmental delay. Common acute manifestations include vomiting, lethargy, coma, and seizures.

In Organic Acidurias (OA), the metabolic pathways of organic acids are disrupted and thus accumulation of the acids in blood and urine alters the acid-base balance of the body. Resulting modifications or adaptations to intermediary metabolic pathways may cause numerous clinical symptoms, including metabolic acidosis, ketosis, hyperammonemia, failure to thrive, sepsis or coma. In particular, determination of succinylacetone (SUAC) from a sample is also particularly useful. Newborn screening for tyrosinemia type I (Tyr-I) is mandatory to identify infants at risk before life-threatening symptoms occur. The analysis of tyrosine alone is limited, and might lead to false-negative results. Consequently, the analysis of SUAC is needed. According to the present invention, there is no need for a separate derivatization step for SUAC.

Therefore, further additional metabolites are preferably be detected and quantified according to the third embodiment of present invention are chosen among comprise argininosuccinate, succinylacetone, valine, alanine, leucine, lysine, ornithine, phenylalanine, tyrosine, glycine, aspartate, glutamate, citrulline, arginine, proline, methionine, serine, free carnitine, acetyl carnitine, propionylcamitine, butyrylcamitine, isovalerylcaminitine, glutarylcamitine, hexanoylcamitine, octanoylcamitine, decanoylcamitine, lauroylcamitine, myristoylcamitine, palmitoylcamitine, and/or stearoylcamitine.

Newborn screening kits, in vitro methods, and novel set of internal standards or internal standard solution of the present invention are thus useful for the diagnosis of all UCDs, as well as routine metabolic newborn screening for metabolic disorders which are generally performed in laboratories early on in life in newborns, thereby providing an expanded but single neonatal screening. Metabolic disorders are generally inborn errors of metabolism in a newborn.

In particular, newborn screening kits, in vitro methods, and novel set of internal standards or internal standard solution of the present invention allow for early assessment of neonatal predispositions to UCDs, hyperammonemia, HHH, and/or argininosuccinic aciduria, as well as additional metabolic deficiencies which are generally tested in human newborns. Said further metabolic disorders include, but are not limited to, amino acid disorders, fatty acid oxidation (FAO) disorders, and organic acidurias (OA).

Amino acid disorders can include, for example but are not limited to, phenylketonuria (PKU) and other hyperphenylalaninemias, maple syrup urine disease (MSUD), homocysteinemia, citrullinemia (types I and II), argininemia, tyrosinemia (types I and II), methionine adenosyltransferase (MAT) deficiency, biopterin deficiencies, hyperprolinemia, hypermethioninemia, and gyrate atrophy of choroid and retina. Screening for amino acid disorders by MS/MS usually involves making quantitative or semi-quantitative measurements of amino acids. FAO disorders can include, for example but are not limited to, medium chain acyl-CoA dehydrogenase (MCAD) deficiency, very long chain acyl-CoA dehydrogenase (VLCAD) deficiency, short chain acyl-CoA dehydrogenase (SCAD) deficiency, multiple acyl-CoA dehydrogenase (MAD) deficiency or glutaric academia type II (GA-II), long chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency, medium/short chain L-3-hydroxyacyl-CoA dehydrogenase (M/SCHAD) deficiency, trifunctional protein deficiency (TFP), carnitine palmitoyltransferase deficiencies of types I and II (CPT-I, CPT-II), camitine-acylcamitine translocase (CACT) deficiency, carnitine transporter deficiency/camitine uptake defect, short chain 3-ketoacyl-CoA thiolase (SKAT) deficiency, medium chain 3-ketoacyl-CoA thiolase (MCKAT) deficiency, and 2,4-dienoyl-CoA reductase deficiency. Screening for fatty acid disorders by MS/MS usually involves making quantitative or semi-quantitative measurements on acylcamitines. Free carnitine is not an acylcamitine, but as those skilled in the art will understand, use of the term “acylcamitines”, in the context of making measurements for the purpose of NBS, often includes free carnitine along with true acylcamitines.

Furthermore, organic acid disorders can include, for example but are not limited to, 3-methylcrotonyl-CoA carboxylase (3-MCC) deficiency, glutaric acidemia type I (GA-I), methylmalonic acidemias (MMA), propionic acidemia (PA), isovaleric acidemia (IVA), malonic aciduria (MA), multiple carboxylase deficiency (MCD), 2-methyl-3-hydroxybutyrl-CoA Dehydrogenase (MHBD) deficiency, 3-hydroxy-3-methylglutaryl-CoA lyase (HMG) deficiency, 2-methylbutyryl-CoA dehydrogenase (2 MBCD) deficiency, 3-methylglutaconic acidurias (MGA), isobutyryl-CoA dehydrogenase (IBD) deficiency, beta-ketothiolase deficiency (BKT), and ethylmalonic encephalopathy (EE).

Preferably, such diagnosis is performed in neonates is between 1 to 5 days of life. Blood samples of newborns, most preferably in the form of a dried blood spot (DBS). The dried blood samples may be obtained from the newborns for example, by pricking the patient's skin a heel prick) and depositing whole blood on filter paper, or filter card (or Guthrie cards) as one or more spots. The design of sampling cards varies, but typically the sampling cards contain from b 2 to 10 spots per card. The spot or spots may then be punched (e.s.. with a diameter in the range of about 3/16 inches to 2/16 inches) and placed into a container. For example, different spots may be placed within different wells of a microtiter plate. Alternatively, the blood sample may be provided in any other form appropriate to the desired test(s) and/or application, such as in the form of hemosylate, stored liquid blood or blood products or freeze dried samples, etc...

Newborn screening kits according to the previous embodiments may comprise at least one plate or microplate, dried blood spot controls, stored stable isotopically-labeled internal standards of known concentrations, extraction solution, eluent solution, and covers. Preferably, newborn screening kits comprise one or more of the following elements: extraction plates, such as for example U-bottomed microplates, or any appropriate microplates for extraction; analysis plates, such as for example V-bottomed microplates, or any appropriate microplates for analysis; dried blood spot Controls; stored stable isotopically-labeled internal standards of known concentrations, comprising labeled amino acids, and/or labeled acylcamitines, and/or labeled succinyl acetone, and/or labeled argininosuccinic acid; extraction solution; eluent solution; adhesive plastic covers; optionally aluminium foil covers.

Most preferably, the newborn screening kits comprise the following elements: (a) extraction plates (U-bottomed microplates), (b) analysis plates (V-bottomed microplates), (c) dried blood spot Controls, (d) individually stored stable isotopically-labeled internal standards of known concentrations (labeled amino acids); (e) individually stored stable isotopically-labeled internal standards of known concentrations (labeled acylcamitines); (f) individually stored stable isotopically-labeled internal standards of known concentrations (labeled succinyl acetone); (g) individually stored stable isotopically-labeled internal standards of known concentrations (labeled argininosuccinic acid); (h) extraction solution; (i) eluent solution; (j) adhesive plastic covers; and (k) aluminium foil covers.

Said internal standard solutions comprises internal stable isotopically-labeled standards may be stored individually in separate vials or in a common vial. The stable isotopically-labeled internal standards are as described above in the various embodiments and aspects of the present invention. They are preferably stored in dried form.

The extraction microplates and analysis microplates may be made of any appropriate material, such as plastic or metal or combinations, and may contain cells of varying size and number. Also, test kit cover pieces may be aseptic. Extraction and eluent solutions contained in the kit can be stored in for example silanized glass vials, or any appropriate containers, vials or flasks.

The newborn screening kit may optionally comprises a solvent or extraction solution and a dispenser such as micropipette, multi-channel micropipettes and robotic dispensers or any other mean to dispense the extraction and eluent solutions. One or more components of the kit can be stored in a container that prevents or minimizes loss of material or evaporation of a solvent.

Said diagnostic newborn screening kits and in vitro methods may comprise a technical information sheet and/or instruction protocols describing that the diagnosis steps which may include i) obtaining a blood sample of said neonatal patient immediately, in particular 1 minute to 6 hours after birth, (ii) measuring the levels of endogenous metabolites in a dried blood sample obtained from the newborn, via tandem mass spectrometry, said metabolites comprising at least glutamine and optionally any other endogenous metabolites of interest for detecting a plurality of additional metabolic deficiencies in the metabolic pathways of amino acids, acylcamitine, and or organic acids of the newborn as described above, in order to produce a metabolic profile of the endogenous metabolites in the newborn, (iii) comparing the measured levels of the endogenous metabolites with the corresponding levels of the endogenous metabolites in a biological sample obtained from a control, in order to assess the predisposition and/or the presence or absence of UCD, hyperammonemia, HHH, and/or argininosuccinic aciduria, and optionally of the presence the plurality of additional metabolic deficiencies which are generally tested in newborns.

Said technical information sheet and/or instruction protocols may preferably describe in details the steps for using the screening kits, wherein said steps comprise a step of preparation of a working solution comprising said one or more reconstituted labeled internal standards with the extraction solution, and a step of sample extraction, wherein said steps may be performed in any order.

Preferably, the newborn screening kit according to the present invention comprises instructions for the following steps: - a step of preparation of internal standards (labeled amino acids, labeled acylcamitines, etc..), - a step of preparation of a working solution comprising reconstituted labeled internal standards with the extraction solution, and/or - a step of sample extraction.

The newborn screening kit may further comprise instructions for instrument settings according to the instrument manufacturer, and/or for the calculation of the results according to the instrument manufacturer. Most preferably, the newborn screening kit comprises instructions for the following steps:

Step 1: preparation of internal standard solution comprising the stable isotopically-labeled standards, such as stable isotopically-labeled amino acids, stable isotopically-labeled acylcamitines, etc. ) are dissolved in 1 ml extraction solution.

Step 2: preparation of a “working solution” A working solution may be prepared in any appropriate container, for example a 10 ml volumetric flask. The working solution is prepared by a. adding 100 μΐ reconstituted labeled amino acids, 100 μΐ reconstituted labeled acyl carnitines, etc.. b. filling up to 10 ml with extraction solution, and c. mixing thoroughly.

Step 3: Sample extraction. A blood sample of a newborn is obtained and deposited on a Dried Blood Spot (for example a Guthrie card or similar). Blood spot punches, each of them bearing aliquots of newborn dried blood sample.

Sample extraction may be conducted by: i. punching 3 mm disks of dried blood controls into the U-bottomed microplate (a), ii. punching 3 mm disks from patient samples into the U-bottomed microplate (a), iii. adding 100 μΐ of “working solution” as prepared in step 2, iv. ensuring that disks are properly soaked, v. covering the plate with adhesive plastic cover (j), vi. shaking at room temperature (RT) for 20 min (+/- 4 min) at 650 rpm vii. removing plastic cover (j), viii. transferring 70 μΐ of the content of each well to a V-bottomed microplate (b), ix. covering the plate with aluminium foil cover (k), x. placing the covered microplate to the autosampler of the tandem mass spectrometer and injecting the sample.

Screening kit may further contain instructions regarding: the instrument settings according to the instrument manufacturer, and optionally the calculation of the results according to the instrument manufacturer.

The blood spot extracts, containing the isotopically-labeled standards may be subject to chemical modification or analyzed directly. All conditions to which the sample is exposed also apply to the internal standards. Kits and methods may or may not require derivatization of the metabolites. The derivatization step is performed by covalently modifying, Le_., by methylation or ester formation, one or more metabolites of the sample, in order to detect and measure said metabolites. Typically, a derivatizing agent such as butanol-n-HCl is used for this purpose, or other appropriate derivatizing agent. The samples must then be dried again to remove excess derivatizing agent. The methods and kits according to the present invention preferably do not require any derivatization.

Dried blood samples may be treated with an extraction and eluent solutions, before or after the addition of the stable isotopically-stable internal standards. Newborn screening kits according to the present invention may thus comprise a solvent for addition to the cells already containing dried stable isotopically-labeled internal standards. The sample of the newborn’s blood may thus be extracted from the punched blood spot with a solvent or an extraction solution which also contains stable isotopically-labeled standards of known concentrations. As described above, such solvent comprises a lysine stable isotopically-labeled internal standard and any appropriate additional stable isotopically-labeled internal standards which may be further added in the kits to perform the desired and/or required further additional plurality of tests of the blood sample from the newborns.

Alternatively, the test tray having cells may already provide with one or more dried stable isotopically-labeled internal standards. In both cases, said stable isotopically-labeled internal standards comprise at least a lysine stable isotopically-labeled internal standard, and may further comprise one or more stable isotopically-labeled internal standards as appropriate for determining the amount of any further metabolites of interest which are also tested with the newborn screening kits according to the present invention. The amount of material measured in the blood spot extract by tandem mass spectrometry is inferred by observation of the ratio of instrument response of the known standard to the unknown substance undergoing analysis.

Preferred newborn screening kits and methods of the present invention utilizes dried stable isotopically-labeled standards for newborn screening which are dissolved and used on a sample-by-sample basis. The standards may be dried according to known methods including heat drying and freeze drying (lyophilization).

According to a fourth embodiment, the present invention comprises an internal standard solution comprising a novel set of stable isotopically-labeled internal standards for detection of UCDs, hyperammonemia, HHH, and/or argininosuccinic aciduria, comprising at least a stable isotopically-labeled internal standard for lysine, and may further comprise one or more stable isotopically-labeled internal standards corresponding to arginine, citrulline, argininosuccinate, orotic acid, and/or ornithine. Said stable isotopically-labeled internal standards are preferably present in dried form.

Preferably, the set of stable isotopically-labeled internal standards allows the detection of all UCDs as well as additional routine neonatal screening as described above, and thus comprises a lysine dried stable isotopically-labeled internal standard, and additional dried stable isotopically-labeled internal standards corresponding to one or more metabolites of interest to be further detected in the newborn screening kit. Such internal standard solution may thus comprise at least a lysine stable isotopically-labeled internal standard, optionally a glutamine stable isotopically-labeled internal standard, and one or more stable isotopically-labeled internal standards corresponding to one or more metabolites selected among argininosuccinate, succinylacetone, orotic acid, ornithine, valine, alanine, leucine, phenylalanine, tyrosine, glycine, aspartate, glutamate, citrulline, arginine, proline, methionine, serine, free carnitine, acetyl carnitine, propionylcamitine, butyrylcamitine, isovalerylcaminitine, glutarylcamitine, hexanoylcamitine, octanoylcamitine, decanoylcamitine, lauroylcamitine, myristoylcamitine, palmitoylcamitine, and/or stearoylcamitine. Said internal standards are preferably present in dried form.

Preferably, internal standard solution may comprise a lysine dried stable isotopically-labeled internal standard in combination with a stable isotopically-labeled internal standard corresponding to citrulline, argininosuccinate, orotic acid and ornithine, thereby allowing for the detection of all UCDs as well as NAGS deficiency, CPS deficiency, OTC deficiency, HHH, citrullinemia as described above.

The lysine dried stable isotopically-labeled internal standard may be used in combination with a stable isotopically-labeled internal standard corresponding to leucine and/or valine for detecting all UCDs and at the same time of MSUD disorder, and/or with phenylalanine for the detection of PKU and hyperphenylalaninemias as well.

Other combinations according to this fourth embodiment of the present invention are lysine stable isotopically-labeled internal standard a stable isotopically-labeled internal standard corresponding to tyrosine for the detections of all UCDs and tyrosinemia, alternatively or in addition with a stable isotopically-labeled internal standard corresponding to argininosuccinic acid for further detecting argininesuccinyl-CoA-lyase (ASAL) deficiency.

Said internal standard solution may still comprise a lysine dried stable isotopically-labeled internal standard in combination with a stable isotopically-labeled internal standard corresponding to citrulline, argininosuccinate, orotic acid, ornithine, leucine, phenylalanine, valine and/or alanine.

To any of the above combinations may be added a a stable isotopically-labeled internal standard corresponding to free carnitine, acetyl carnitine, propionylcamitine, butyrylcamitine, isovalerylcaminitine, glutarylcamitine, hexanoylcamitine, octanoylcamitine, decanoylcamitine, lauroylcamitine, myristoylcamitine, palmitoylcamitine, and/or stearoylcamitine, for further detecting fatty acid oxidation disorders. A stable isotopically-labeled internal standard is separately detectable from the molecule based on unique physical characteristics, such as a unique mass or mass-to-charge ratio. Stable isotopically labeled form of the metabolite of interest represents commonly used internal standards. For example, if the analyte was MPP, the internal standard can be an isotopically labeled MPP. Stable isotope labeled analogs can be used to quantitate the corresponding metabolite of interest using the technique known as isotope dilution mass spectrometry wherein the metabolite and internal standards are processed in the same sample.

As used herein, isotopic labeled, isotopically-labeled, and other similar terms are used as is understood in the art. Specifically, an isotopically-labelled compound is a compound in which at least one atom of known position is enriched with an isotope other than the most abundant naturally-occurring isotope for that element. For example, methane may be 13C-isotopically-labelled, and have the structure 13CH4, or deuterium-labelled. Deuterium-labelled methane may refer to a compound in which one or more of the four hydrogen atom positions associated with methane are enriched with 2H (D). Common deuterium-labelled methane structures include CDH3 and CD4. Isotopically-labelling refers to isotopic enrichment above natural abundance. Preferably, the isotopic purity at the enriched position is greater than 50%, or greater than 80%, greater than 90%, greater than 95%, greater than 97%, greater 98%, or greater than 99%.

Internal standards can be designed such that (1) the labeling causes a shift in mass of at least 1 mass unit and (2) that none of the stable isotope labels are located in labile sites to prevent exchange. The actual location of the labels on the molecule can vary provided the prerequisite (2) above is satisfied. Moreover, the position of the labels and the potential change in the mass of the fragment ions can also be used to confirm separation of the internal standard and metabolites.

According to the current invention for the internal standards, one or more isotopic labels can be used, and when more than one is used, multiple of the same label (e.s. deuterium) or different labels (e.s.. deuterium and 13C) can be present. Exemplary isotopically-labeled internal standards are those derivatives that can be clearly differentiated from the isotope peaks of the metabolite of interest.

Any appropriate isotopic labels can be used including, for example, H, C, N, and 0 or combination thereof. While not being bound by any theory, the physicochemical behavior of such stable isotopically-labeled derivative with respect to sample preparation and signal generation would be expected to be identical to that of the unlabeled analyte, but clearly differentiable on the mass spectrometer.

Amino acid standards which are labeled with one or more of stable isotopes preferably have three or more of mass difference relative to unlabeled amino acid. By labeling to an unlabeled amino acid with stable isotope so as to bring about 3 or more of mass differences, the influence due to natural existence ratio of the isotope in the unlabeled object may be reduced, and highly precise analysis can be performed. For example, distribution of the natural isotopes in alanine (molecular formula; C3H7N02, molecular mass 88) are molecular weight 88 (95.8%), 89 (3.74%), 90 (0.34%), and 91 (0.02%), and by making the mass difference into 3 or more, the influence of natural isotopes is avoided.

Therefore, according to this fourth embodiment, internal standard solution useful in the newborn screening kits and methods may comprise at least lysine-13C6-15N2, and one or more of the stable isotopically- labeled amino acid standards chosen among H4-alanine, H4- C-arginine, H2-citrulline, N- C-glycine, H3-leucine, H3-methionine, H6-omithine, Ce-phenylalanine, Hg-valine, "H3-aspartic acid, H3-glutamic acid, 13C3-serine,13C6-tyrosine, 2H5-proline, 13C5-succinylacetone, and/or 15N4-13C6-argininosuccinate, and/or one or more labeled carnitine standards chosen among 2H9-camitine, 2H3-acetylcamitine, 2H3-propionylcamitine, 2H3-butyrylcamitine, 2H9-isovalerylcamitine, 2H3-glutarylcamitine, 2H3- hexanoylcamitine, 2H3-octanoylcamitine, 2H3-decanoylcarnitine, 2H3-lauroylcamitine, 2H9- myristoylcamitine, 2H3-palmitoylcamitine, 2H3-stearoylcamitine. Preferred concentrations of amino acids range from 0.2 to 50 μιηοΙ/L. Preferred concentrations of (acyl) carnitines in the kit reagents of the present invention range from 0.001 to 1 μιηοΙ/L Said internal standards are preferably present in dried form. According to the methods and kits of the present invention, ionizing step (iii) is performed by delivering said metabolites extracted from DBS samples and dried stable isotope internal standards to an ion source of the mass spectrometer. The extracted metabolites and dried stable isotopically-labelled internal standards used as controls, are sprayed through a small tube into a strong electric field in the presence of a flow of warm nitrogen gas to assist desolvation and formation of ions. The ionization process typically involves transfer of a charge to the solvent droplets, evaporation of the solvent and, finally, production of positively and negatively charged ions. Such ion formation process is a starting point for mass spectrum analysis.

Several ionization methods are available and the choice of ionization method depends on the sample to be analyzed. For example, for the analysis of polypeptides, a relatively gentle ionization procedure such as electrospray ionization (ESI) may be used. For ESI, a solution containing the sample is passed through a fine needle at high potential which creates a strong electrical field resulting in a fine spray of highly charged droplets that is directed into the mass spectrometer. Other ionization procedures include, for example, fast-atom bombardment (FAB) which uses a high-energy beam of neutral atoms to strike a solid sample causing desorption and ionization. Matrix-assisted laser desorption ionization (MALDI) is a method in which a laser pulse is used to strike a sample that has been crystallized in an UV-absorbing compound matrix. Other ionization procedures known in the art include, for example, plasma and glow discharge, plasma desorption ionization, resonance ionization, and secondary ionization.

In the preferred embodiment of the invention, the solvated test sample/standards are directly introduced into a tandem mass spectrometer that is adapted to process the test sample/standards using an electrospray. The tandem mass spectrometer may be any appropriate spectrometer commercially available.

Electrospray ionization (ESI) has several properties that are useful for the invention described herein. For example, ESI can be used for biological molecules such as polypeptides that are difficult to ionize or vaporize. In addition, the efficiency of ESI can be very high which provides the basis for highly sensitive measurements. Furthermore, ESI produces charged molecules from solution, which is convenient for analyzing reporters that are in solution. In contrast, ionization procedures, such as MALDI, require crystallization of the sample prior to ionization. Since ESI can produce charged molecules directly from solution, it is compatible with samples from liquid chromatography systems. For example, a mass spectrometer can have an inlet for a liquid chromatography system, such as an HPLC, so that fractions flow from the chromatography column into the mass spectrometer. This in-line arrangement of a liquid chromatography system and mass spectrometer is sometimes referred to as LC-MS. A LC-MS system can be used, for example, to separate un-cleaved or partially cleaved tag reporters from cleaved tag reporters before mass spectrometry analysis. In addition, chromatography can be used to remove salts or other buffer components from the tag reporter sample before mass spectrometry analysis. For example, desalting of a sample using a reversed-phase HPLC column, in-line or off-line, can be used to increase the efficiency of the ionization process and thus improve sensitivity of detection by mass spectrometry. Preferably, the ionizing step (iii) is performed by delivering said extracted metabolites and standards to an ion source of the mass spectrometer by liquid chromatography system.

This vaporized and ionized mixture enters the first MS which functions as a separating device and allows only the ion(s) of interest to pass through. Ions passing through the first MS are called precursors or parent ions which enter the collision cell where the fragmentation takes place. Fragmentation is achieved by putting an inert collision gas such as nitrogen or argon into the collision cell. The fragments generated in the collision cell are called products or daughter ions. The mass of these fragments are measured in the second MS. Fragments in the second MS are correlated with the intact molecules produced in the first MS. This process enables unique MS such as precursor ion scans and neutral loss scans.

According to the present invention, the acquisition step (iv) of the mass to charge (m/z) ratio is performed in Multiple Reaction Monitoring (MRM) mode and refers to a tandem spectrometry (MS-MS) experiment where one or more specific products of a selected precursor ion, Le., a parent ion, a molecular ion or a daughter ion, is monitored. A variety of configurations of mass spectrometers can be used in a method of the invention. In general, a mass spectrometer has the following major components: a sample inlet, an ion source, a mass analyzer, a detector, a vacuum system, and instrument-control system, and a data system. Differences in the sample inlet, ion source, and mass analyzer generally define the type of instrument and its capabilities. For example, an inlet can be a capillary-column liquid chromatography source or can be a direct probe or stage such as used in matrix-assisted laser desorption. Common ion sources are, for example, electrospray, including nanospray and microspray or matrix-assisted laser desorption. Common mass analyzers include a quadrupole mass filter, ion trap mass analyzer and time-of-flight mass analyzer. Preferably, such data acquisition step (iv) may be performed with a triple-quadrupole mass spectrometer or high-resolution mass spectrometer.

The test sample is scanned using a mass spectrometer to produce one or more mass spectra. Any mass spectrometer that can detect a signal from the extracted metabolites and the standards can be used in the inventive methods. A tandem mass spectrometer is preferably used according to the present invention as it identifies different molecular entities that produce a common fragment when subjected to collision-induced dissociation (CID). In tandem mass spectrometry, two mass analyzers are linked in series via a collision cell. The first mass analyzer (first quadrupole) is used to select an ion of interest (e.s.. an ion of a particular mass-to-charge ratio (m/z)). The selected ions are then transferred to a collision cell where they are fragmented by collision with an inert gas (e.s., nitrogen or helium or argon). This process is called collisionally activated dissociation (CAD) and is performed in the collision cell of the mass spectrometer. Once the precursor ions have been fragmented, the second mass analyzer (third quadrupole) is used to either scan or detect all of the produced product ions, or to select and detect particular fragment ions. Tandem mass spectrometry is used to ionize lysine and/or glutamine as well as any additional metabolites to be detected in the newborn screening kits and methods of the present invention Such tandem mass spectrometry allows ion fragmentation and detection specific peaks that are indicative of the presence of these molecules in the sample.

The tandem mass spectrometry detection can be accomplished in a number of ways. In one type of tandem mass spectrometry (commonly performed on triple quadrupole tandem mass spectrometers), ions that fragment to produce common product (fragment) ions can be detected as a class by performing a "precursor ion scan", where by selecting the appropriate mass for the common fragmentation in the collision cell, all ions that produce the common fragment ions are detected. In a different form of tandem mass spectrometry, ions that fragment to produce a common neutral loss can be detected as a class by performing a so called neutral loss scan where by setting an appropriate mass offset equal to the common neutral loss between first and third quadrupoles all ions that fragment to produce the specified neutral loss are detected. In yet another type of tandem mass spectrometry known as multiple reaction monitoring (MRM), a precursor ion of interest is selected in the first quadrupole, fragmented in the collision cell and a specific fragment ion resulting from the collisional activation is selected in the third quadrupole and finally detected.

First and third quadrupoles are fixed to respectively select the corresponding precursor and fragment ion pairs of interest for a predetermined amount of time (a few milliseconds). If additional analytes or metabolites need to be detected, additional detection transitions can be introduced in the experiment. The data from all selected mass transitions can be acquired sequentially to obtain the desired information. A quadrupole mass analyzer, as well as the other mass analyzers described herein, can be programmed to analyze a defined m/z or mass range. Since the mass range of cleaved tag reporters will be known prior to an assay, a mass spectrometer can be programmed to transmit ions of the projected correct mass range while excluding ions of a higher or lower mass range. The ability to select a mass range can decrease the background noise in the assay and thus increase the signal-to-noise ratio. In addition, a defined mass range can be used to exclude analysis of molecules. Therefore, the mass spectrometer can accomplish an inherent separation step as well as detection and identification of metabolites.

Ion trap mass spectrometry utilizes an ion trap mass analyzer. In these mass analyzers, fields are applied so that ions of all m/z are initially trapped and oscillate in the mass analyzer. Ions enter the ion trap from the ion source through a focusing device such as an octapole lens system. Ion trapping takes place in the trapping region before excitation and ejection through an electrode to the detector. Mass analysis is accomplished by sequentially applying voltages that increase the amplitude of the oscillations in a way that ejects ions of increasing m/z out of the trap and into the detector. In contrast to quadrupole mass spectrometry, all ions are retained in the fields of the mass analyzer except those with the selected m/z. One advantage to ion traps is that they have very high sensitivity, as long as one is careful to limit the number of ions being trapped at one time. Control of the number of ions can be accomplished by varying the time over which ions are injected into the trap. The mass resolution of ion traps is similar to that of quadrupole mass filters, although ion traps do have low m/z limitations.

Time-of-flight mass spectrometry utilizes a time-of-flight mass analyzer. For this method of m/z analysis, an ion is first given a fixed amount of kinetic energy by acceleration in an electric field (generated by high voltage). Following acceleration, the ion enters a field-free or “drift” region where it travels at a velocity that is inversely proportional to its m/z. Therefore, ions with low m/z travel more rapidly than ions with high m/z. The time required for ions to travel the length of the field-free region is measured and used to calculate the m/z of the ion.

One consideration in this type of mass analysis is that the set of ions being studied be introduced into the analyzer at the same time. For example, this type of mass analysis is well suited to ionization techniques like MALDI which produces ions in short well-defined pulses. Another consideration is to control velocity spread produced by ions that have variations in their amounts of kinetic energy. The use of longer flight tubes, ion reflectors, or higher accelerating voltages can help minimize the effects of velocity spread. Time-of-flight mass analyzers have a high level of sensitivity and a wider m/z range than quadrupole or ion trap mass analyzers. Also data can be acquired quickly with this type of mass analyzer because no scanning of the mass analyzer is necessary.

Tandem mass spectrometry can utilize combinations of the mass analyzers described above. Tandem mass spectrometers can use a first mass analyzer to separate ions according to their m/z in order to isolate an ion of interest for further analysis. The isolated ion of interest is then broken into fragment ions (called collisionally activated dissociation or collision induced dissociation) and the fragment ions are analyzed by the second mass analyzer. These types of tandem mass spectrometer systems are called tandem in space systems because the two mass analyzers are separated in space, usually by a collision cell. Tandem mass spectrometer systems also include tandem in time systems where one mass analyzer is used, however the mass analyzer is used sequentially to isolate an ion, induce fragmentation, and then perform mass analysis. Mass spectrometers in the tandem in space category have more than one mass analyzer. For example, a tandem quadrupole mass spectrometer system can have a first quadrupole mass filter, followed by a collision cell, followed by a second quadrupole mass filter and then the detector. Another arrangement is to use a quadrupole mass filter for the first mass analyzer and a time-of-flight mass analyzer for the second mass analyzer with a collision cell separating the two mass analyzers. Other tandem systems are known in the art including reflectron-time-of-flight, tandem sector and sector-quadrupole mass spectrometry.

Mass spectrometers in the tandem in time category have one mass analyzer that performs different functions at different times. For example, an ion trap mass spectrometer can be used to trap ions of all m/z. A series of rf scan functions are applied which ejects ions of all m/z from the trap except the m/z of ions of interest. After the m/z of interest has been isolated, an rf pulse is applied to produce collisions with gas molecules in the trap to induce fragmentation of the ions. Then the m/z values of the fragmented ions are measured by the mass analyzer. Ion cyclotron resonance instruments, also known as Fourier transform mass spectrometers, are an example of tandem-in-time systems.

Several types of tandem mass spectrometry experiments can be performed by controlling the ions that are selected in each stage of the experiment. The different types of experiments utilize different modes of operation, sometimes called “scans,” of the mass analyzers. In a first example, called a mass spectrum scan, the first mass analyzer and the collision cell transmit all ions for mass analysis into the second mass analyzer. In a second example, called a product ion scan, the ions of interest are mass-selected in the first mass analyzer and then fragmented in the collision cell. The ions formed are then mass analyzed by scanning the second mass analyzer. In a third example, called a precursor ion scan, the first mass analyzer is scanned to sequentially transmit the mass analyzed ions into the collision cell for fragmentation. The second mass analyzer mass-selects the product ion of interest for transmission to the detector. Therefore, the detector signal is the result of all precursor ions that can be fragmented into a common product ion. Other experimental formats include neutral loss scans where a constant mass difference is accounted for in the mass scans. The use of these different tandem mass spectrometry scan procedures can be advantageous when large sets of metabolites are measured in a single experiment as with multiplex experiments.

These techniques are well-known to skilled person in the art who can readily recognize that different mass spectrometry methods, for example, quadrupole mass spectrometry, ion trap mass spectrometry, time-of-flight mass spectrometry and tandem mass spectrometry, can use various combinations of ion sources and mass analyzers which allows for flexibility in designing customized detection protocols. In addition, mass spectrometers can be programmed to transmit all ions from the ion source into the mass spectrometer either sequentially or at the same time. Furthermore, a mass spectrometer can be programmed to select ions of a particular mass for transmission into the mass spectrometer while blocking other ions. The ability to precisely control the movement of ions in a mass spectrometer allows for greater options in detection protocols which can be advantageous when a large number of metabolites, for example, from a multiplex experiment, are being analyzed.

Different mass spectrometers have different levels of resolution, that is, the ability to resolve peaks between ions closely related in mass. The resolution is defined as R=m/delta m, where m is the ion mass and delta m is the difference in mass between two peaks in a mass spectrum. For example, a mass spectrometer with a resolution of 1000 can resolve an ion with m/z of 100.0 from an ion with m/z of 100.1. Those skilled in the art will therefore select a mass spectrometer having a resolution appropriate for the analyte(s) to be detected.

Mass spectrometers can resolve ions with small mass differences and measure the mass of ions with a high degree of accuracy. Therefore, metabolites of similar masses can be used together in the same experiment since the mass spectrometer can differentiate the mass of even closely related molecules. The high degree of resolution and mass accuracy achieved using mass spectrometry methods allow the use of large sets of metabolites because they can be distinguished from each other.

The level of metabolites in the test sample is then determined by comparing a peak in the one or more mass spectra that corresponds to the metabolites with a peak in the one or more mass spectra that corresponds to the isotopically enriched standard. In one embodiment, relative peak heights are used. In another embodiment, the areas under the peaks are integrated and compared.

Once the metabolite level in a test sample or dried blood sample has been determined it can be compared with a range of normal levels. If the level is outside this normal range then the dried blood sample or the patient from whom it was obtained may be referred for further analysis. For example, the test could be repeated with one or more additional blood spots to obtain an average level. Additionally or alternatively the metabolite level could be measured using an alternative method known in the art, g.g.. an immunochemical method or a mass spectral method using a serum sample.

In a preferred embodiment the system includes the MRM mode of operation of a triple quadrupole (tandem) mass analyzer. In MRM mode of operation, the first mass analyzing quadrupole is set to select a specific “precursor” ion from the ions passing through the first mass analyzing quadrupole, a second nonmass analyzing quadrupole is used to cause controlled dissociation of the precursor ion, and the third quadrupole (the second mass analyzing quadrupole) is set to select only a specific fragment, or “product”, ion of the precursor ion. The precursor and product combination is referred to as the MRM ion pair. According to some embodiments of the present invention, MRM data can be acquired in several different manners, with the differences being in how the laser is allowed to interact with the sample on the target plate, for example, depending on the ablation mode that is used. A “raster” ablation mode involves the laser beam cutting a straight line swath across one or more samples. To accomplish this “rastering” the laser beam is fired at a non-sample location prior to a sample spot of interest, and the target plate can then be moved continuously to present new samples to the laser impingement point. This means that the laser desorption point will encounter an alternating series of sample/no sample sections of the target plate. The resulting MRM data contains a series of ion signal “peaks”, indicated by ion counts per second as a function of mass spectrometer analysis time, with non-zero signal where sample was encountered, interspersed with regions of zero-signal baseline in between sample spots. Depending on laser power, speed of target plate movement under the laser beam, number of metabolites being monitored and sample composition, it can be typical to acquire the data for a single sample (monitoring one, or several, MRM ion pairs) in approximately 0.25 to 5 seconds for this mode of ablation.

In a “discrete” mode of operation, with the laser in a non-firing state the target plate can be positioned under the laser impingement point and the laser can then be turned on for some specific period of time. While the laser is not firing, the mass spectrometer records a signal of zero (since no ions are generated). When the laser commences firing the laser beam desorbs material from this specific location, creating an MRM signal “peak”. The MRM signal intensity rises from zero and quickly maximizes, and it then decreases as the sample is depleted from that specific location on the target plate. The MRM signal level returns to the zero baseline level either by turning off the laser, or by permitting the laser to fire until the sample is completely consumed from the particular location and there is no more sample from which to generate ions. After the laser firing is stopped, the sample plate can then be moved so as to present a new sample location for the next ablation. Depending on laser power, sample composition, and the length of time the laser is fired, it can be typical to acquire the data for a single sample in well under one second for this mode of ablation.

Other ablation modes involve the movement of a sample spot according to some pattern, such that the continually firing laser generates a pattern across the sample spot. This “pattern raster” mode generates a steady stream of ions from a few seconds to several minutes, depending on the form and speed of the pattern within the sample spot. One or more sample spots can be included in such a mode, with proper accommodation when moving from one sample spot to the next. The mass spectrometer method can be constructed to perform a number of different measurements, even including the mixing of mass spectrometer scan types. Pattern rasters are useful for measurements which require ablation times longer than those typically used for MRM analyses with this technique (e.s.. precursor or neutral loss scans).

In the preferred reagent kits and methods of the present invention, the extraction step (ii) is performed by using an extraction buffer comprising one or more organic solvents, ionizing step (iii) is performed by delivering said extracted metabolites and internal standard solution to an ion source of the mass spectrometer by liquid chromatography system, data acquisition step (iv) is performed with a triple-quadrupole mass spectrometer or high-resolution mass spectrometer, the step (v) is performed by selecting at least one accurate mass-to-charge (m/z) ratio ions corresponding to an at least one calculated mass-to-charge (m/z) ratio of said metabolites present in said sample. Also, a direct tandem mass spectrometric analysis of metabolites in dried blood spots is performed without chemical derivatization for neonatal screening. Most preferably, multiple reaction monitoring (MRM) technique IS applied for tandem mass spectrometric measurements, such that each metabolite has its individual precursor and product ion settings.

Optionally, prior to mass spectrometric analysis, the sample is rapidly preprocessed, for example by chromatography, ultrafiltration, electrophoresis or dialysis. Examples of chromatography include ion exchange chromatography, affinity chromatography, hydrophobic chromatography, hydrophilic chromatography and reverse phase chromatography.

In the most preferred reagent kits and methods of the present invention, comprise a step of separating the one or more metabolites by liquid chromatography (LC) prior to proceeding to the mass spectrometry (MS) analysis as described above. Therefore most preferred kits and methods are liquid chromatography-mass spectrometry methods and kits.

High pressure liquid chromatography (HPLC) is a form of column-based chromatography that is routinely used in analytical chemistry to separate, identify or detect and quantify molecules. HPLC utilizes a column that holds chromatographic packing material (stationary phase), a pump that moves the mobile phase(s) through the column, and a detector that detects the abundance of the molecules and shows their retention on the chromatographic column in relation to the elapsed time (retention time). Retention times vary depending on the interactions between the stationary phase, the molecules being analyzed, and the solvent(s) used. A sample containing the metabolites is injected into the mobile phase manually or by an automated autosampler. The polarity of the metabolites, the stationary phase of the column(s) used and the mobile phase(s) determine the retention time of the metabolite as well as its separation from interferences and extent of quantifiability. Amino acid separation using HPLC may be performed with any commercially available LC apparatus using automated or manual sample injection and adjustable, consistent and reproducible solvent flow rates.

Columns suitable for liquid chromatography contain packing materials that include very small and usually spherical particles, e.g., silica particles, having a diameter of 3-50 microns and a pore size of 100-1000 angstroms. Commonly, HPLC is performed with a stationary phase attached to the outside surface of such small particles; such stationary phase may provide that surface hydrophobic properties or enable ion change or ion pairing. A chromatographic column typically includes two ports, one inlet port for receiving a sample and one outlet port for discharging an effluent that may or may not include the sample.

In some embodiments of the present invention, the one or more amino acids in a sample enter a column from the inlet port, are then eluted with a solvent or solvent mixture, and eventually discharged at the outlet port. In preferred embodiments, one or more amino acids in a sample enter a column from the inlet port where after the flow across said column is reversed and one or more amino acids are then eluted with a solvent or solvent mixture, and eventually discharged back at the inlet port (flow-reverse). Using a chromatographic column flow-reverse, and specifically using the first chromatographic column of two successive chromatographic columns flow-reverse, proved beneficial in delaying the elution of hydrophilic amino acids and in improving their ionization in the mass detector, leading to increased analytical sensitivity.

Different solvents or solvent mixtures may be selected for eluting the amino acids. For example, liquid chromatography may be performed using a gradient mode with differing amounts of solvents in the mixture, an isocratic mode with continuously fixed amounts of solvents in the mixture or a partially isocratic, partially gradient mixed mode. Suitable solvents and solvent mixtures include sodium or lithium buffers (for cation exchange HPLC) or acetonitrile (for reverse phase HPLC).

The internal diameter of an HPLC column is an important parameter that influences the detection sensitivity and separation selectivity. Column dimensions in preferred embodiments of the present invention include a column internal diameter of 2.1-3.0 mm and a column length of 3-10 cm.

Liquid chromatography is based on the principle that a metabolite is adsorbed to a stationary phase and eventually desorbed and eluted with the mobile phase into a detection unit for proper detection and/or quantitation. The choice of both stationary and mobile phase greatly influences the success of chromatographic separation.

Reversed phase HPLC (RP-HPLC or RPC) has a non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary phase is treated silica. With these stationary phases, retention time is longer for molecules which are more non-polar, while polar molecules elute more rapidly. The mobile phase is generally a binary mixture of water and a miscible polar organic solvent like methanol, acetonitrile or tetrahydrofuran (THF). Reversed phase chromatography is based on partition and is typically used for separations by non-polar differences.

In contrast to reversed phase HPLC, normal phase HPLC (NP-HPLC) uses a polar stationary phase and a non-aqueous, non-polar mobile phase, and works effectively for separating metabolites readily soluble in non-polar solvents. The metabolites associate with and are retained by the polar stationary phase until final elution. Typical stationary phases for normal phase chromatography are silica or organic moieties with cyano- and/or amino-functional groups. In NP-HPLC, the most nonpolar molecules elute first and the most polar molecules elute last. The mobile phase consists of a very nonpolar solvent like hexane or heptane mixed with a slightly more polar solvent like isopropanol, ethyl acetate or chloroform. Retention increases, as the amount of nonpolar solvent in the mobile phase increases. NP-HPLC is based on adsorption and is typically employed for the analysis of solutes readily soluble in organic solvents, based on their polar differences such as amines, acids, metal complexes, etc...

Use of ion exchange chromatography may also be within the scope of the invention, since amino acids, by definition, contain at least one amino-group and one carboxyl acid group, they are ionizable and consequently carry a positive or negative charge, when the pH of the mobile phase differs from the amino acid's pKa. Below the neutral pH of 7.0, amino acids with primarily basic groups (e.g., amino groups) are positively charged, whereas above the neutral pH of 7.0, amino acids with primarily acidic groups (e.g., carboxylic acid groups) are negatively charged. The 20 proteinogenic amino acids that represent the building blocks of proteins differ in their side-chain groups, which influence the amino acids' chemical reactivity, ionic charge, relative hydrophilicity or hydrophobicity and polarity. Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on the charge properties of the metabolites. Charged amino acids may be either acidic or basic. The stationary phase surface displays ionic functional groups (R—X) that interact with analyte ions of opposite charge. This type of chromatography is further subdivided into cation exchange chromatography and anion exchange chromatography. The target metabolites (anions or cations) are retained on the stationary phase but can be eluted by increasing concentrations of similarly charged species that will displace the analyte ions from the stationary phase. For example, in cation exchange chromatography, the positively charged analyte could be displaced by the addition of positively charged sodium ions.

Ion-pairing chromatography may also be contemplated for use within the scope of this invention. Similar to ion-exchange chromatography, ion pairing chromatography utilizes the ionizability and charge properties of the metabolites for the chromatographic separation. However, instead of exchanging ions, ion-pairing systems are established using perfluorinated carboxylic acids such as tridecafluoroheptanoic acid (TDFHA) or trifluoroacetic acid (TFA) as mobile phase constituents and a stationary phase that can accept or donate electrons or both. Specifically, ion pairing chromatography can be used to separate ionic metabolites on a reversed-phase column in order to suppress the ionic characteristic of charged organic compounds. Ion pair reagents have a charge opposite of the metabolites and a hydrophobic region to interact with the stationary phase. The charge of the absorbed ion pair reagent interacts electrostatically with the charge of the metabolites. As an example, amines can produce a serious tailing chromatographic peak on a reversed phase column, while addition of an ion pair agent such as trifluoroacetic acid curtails tailing. With advances in column phases and a better selection of ion pair reagents, ion pair chromatography not only sharpens chromatographic peaks but also modulates the retention of ionic metabolites on reverse-phase columns. Typical ion pair reagents include tetra-alkylammonium ions and perfluorinated organic acids. The type of ion pair reagent, the concentration of ion pair reagent, the type of organic modifier in the mobile phase, the concentration (gradient) of the organic modifier, and the proper selection of the columns are critical to a successful ion pair chromatography experiment. In preferred embodiments, ion pair chromatography was used.

According to a fifth embodiment, newborn screening kits and methods according to the present invention may also be extended to include detection of variant proteins and polypeptides implicated in serious diseases. For example, many variant or mutant forms of the polypeptide sub-units of hemoglobin are known to result in various forms of anemia, and many such mutations are of only one amino acid.

Preferred kits and methods would be targeting specific selected ionised species. Kist and methods may be applied with advantage to detect not only variants containing amino acids which differ from the norm, as in the many possible known hemoglobinopathies described hereinafter, but also to detect variants in glycosylation patterns of polypeptides, as well as deletions and/or additions of one or more amino acid residues from the expected polypeptide sequence.

According to this fifth embodiment, kits and methods are particularly useful for testing a sample by mass spectrometry, where the ionisation technique produces a multiply-charged spectrum, to further detect (a) the presence or absence of a known polypeptide or derivative of a polypeptide, or (b) the presence or absence of a variant of a known polypeptide or derivative of a polypeptide, in which scanning of the sample is targeted to selected ionised species of known mass/charge ratio, the absence of the expected value of mass/charge ratio being indicative of the absence of the known polypeptide or derivative thereof, or the presence of the variant polypeptide or derivative thereof being indicated by a shift in mass/charge ratio from the expected value. Such method thus allows concentrating data acquisition on a restricted mass/charge range (mass window) to include the normal polypeptide and the variant polypeptide or polypeptides of interest. Targeting in this manner is more reproducible and reveals peaks corresponding to the normal mass/charge ratio and any shifts from the norm due to variants present in samples taken from patients who are either homozygous or heterozygous in this respect. Thus the method may frequently require only one restricted mass window to be targeted. The use of separate windows for the normal and for the variant polypeptide is also possible. Additional mass windows may be used to target other variants or other polypeptides.

The kits and methods according to this embodiment of the present invention may thus be used to detect any protein variant such as a protein mutation or an abnormal concentration of a wild-type protein. Any inherited disorder leading to variant protein production may therefore be detected using kits and method according to this embodiment. Preferably hemoglobin variants, albumin, myoblobin, cytochromes, and variant proteins associated with various congenital disorders like for example disorders of glycosylation are detected.

Preferred kits and methods of the present invention are used to detect clinically important hemoglobin variants such as Hb S, Hb C, Hb Dp""|ab. Hb 0Arab, Hb Lepore, Hb E, delta beta thalassemia, hereditary persistence of fetal hemoglobin trait (HPFH) and alpha zero thalassemia trait.

Hemoglobin variants analysis may be conducted simultaneously or sequentially with the analysis of metabolites described above, Le_., glutamine, and further amino acids, carnitines, acylcamitines, SUAC, ASA either from the same sample like dried blood spot or from a different sample like different lot of dried blood spot.

EXAMPLES

Example 1: Determination of the levels of glutamine, lysine, arginino succinic acid, and orotic acid

To determine the linearity of the measurement of glutamine, lysine, arginino succinic acid, and orotic acid, standard solutions of the respective analyte up to 20 mM were prepared and 10 pL injected into the tandem mass spectrometer and the respective ions/transitions were measured. (Fig. 6-8)

Example 2: Determination of the levels of glutamine and lysine in dried blood samples To determine the linearity of the measurement of the sum of lysine and glutamine in dried blood spots (DBS), blood of a healthy donor was spiked with lysine. Endogenous concentrations of lysine and glutamine were determined from an aliquot by standard amino acid determination using ion exchange chromatography. (Fig. 9)

Example 3: Determination of the sum of glutamine and lysine in dried blood samples

Figure 10 shows the proposed results of the measurement of the sum of glycine and lysine. For lysine and glutamine the reference range of lysine and glycine is plotted, together with the calculated sum of those 2 reference ranges (sum of lower - sum of upper reference range), measured values from 180 DBS of healthy newborns, measured values from 2 samples of a patient with proven OTC deficiency, expected range of patients with urea cycle defects (UCD).

Claims

1. Newborn screening method of detecting the presence and/or measuring the levels of one or more metabolites in a blood sample, optionally a dried spot blood, of a newborn, by tandem mass spectrometry, said metabolites comprising at least glutamine and said method comprising the following steps: (i) extracting said metabolites from the sample with an extraction solution; (ii) providing, before or after the step (i), an internal standard solution comprising a known amount of one or more stable-isotope labeled internal standards corresponding to said metabolites to be detected in the sample, (iii) ionizing said metabolites and said one or more stable-isotope labeled internal standards to generate ions, (iv) acquiring the mass to charge (m/z) ratio of said one or more ions in the mass spectrum in the Multiple Reaction Monitoring (MRM) mode, and (v) determining the sum of glutamine and lysine in MRM mode, (vi) detecting and quantifying the amount of glutamine with stable-isotope labeled internal standard corresponding to lysine.

2. Newborn screening kit when used in detecting the presence and/or measuring the levels of glutamine in a blood sample of a newborn, by tandem mass spectrometry, allowing diagnosis of one or more metabolic deficiencies selected among urea cycle disorders, and/or hyperammonemia, Hyperomithinemia-hyperammonemia-homocitrullinuria (HHH), and/or argininosuccinic aciduria, said kit comprising an internal standard solution comprising at least a stable isotopically-labeled internal standard corresponding to lysine.

3. Newborn screening method or kit of claim 1 or claim 2, wherein said internal standard solution further comprises one or more stable isotopically-labeled internal standards corresponding to one or more metabolites selected among amino acids, organic acids, acylcamitines, a plurality of carnitines, and/or succinylacetone, wherein said standards are optionally dried, wherein said internal standards are stored in separate vials or in a common vial, allowing for the diagnosis in said newborns of one or more metabolic deficiencies selected among urea cycle disorders, and/or hyperammonemia, Hyperomithinemia-hyperammonemia-homocitrullinuria (HHH), and/or argininosuccinic aciduria, as well as further metabolic newborn disorders, such as amino acid disorders, fatty acid oxidation (FAO) disorders, and organic acidurias (OA).

4. Newborn screening method or kit according to any one of claims 1 to 3, wherein said internal standard solution comprises one or more stable isotopically-labeled internal standards corresponding to alanine, arginine, citrulline, homocitrulline, glycine, leucine, methionine, ornithine, phenylalanine, valine, aspartic acid, glutamic acid, tyrosine, serine, oxoproline, proline, glutamic acid, argininosuccinate, succinylacetone, orotic acid, free carnitine, acetyl carnitine, propionylcamitine, butyrylcamitine, isovalerylcaminitine, glutarylcamitine, hexanoylcamitine, octanoylcamitine, decanoylcamitine, lauroylcamitine, myristoylcamitine, palmitoylcamitine, and/or stearoylcamitine, or wherein said stored stable isotopically-labeled internal standards of known concentrations comprise labeled amino acids, and/or labeled acylcamitines, and/or labeled succinyl acetone; and/or labeled argininosuccinic acid, and wherein said standards are optionally dried.

5. An internal standard solution comprising at least a lysine stable isotopically-labeled internal standard, and further comprising one or more stable isotopically-labeled internal standards corresponding to alanine, citrulline, homocitrulline glycine, leucine, methionine, ornithine, glutamine, phenylalanine, valine, glutamic acid, aspartic acid, glutamic acid, tyrosine, serine, oxoproline, proline, argininosuccinate, succinylacetone, orotic acid, free carnitine, acetylcamitine, propionylcamitine, butyrylcamitine, isovalerylcaminitine, glutarylcamitine, hexanoylcamitine, octanoylcamitine, decanoylcamitine, lauroylcamitine, myristoylcamitine, palmitoylcamitine, and/or stearoylcamitine, wherein said standards are optionally dried.