Analysis of amino acids in body fluids by liquid chromatography-mass spectrometry

Abstract

To provide methods for identifying individual amino acids in various bodily fluids obtained from a human patient, and to provide reference ranges for normal amino acid levels in the various bodily fluids (e.g., blood plasma, urine, cerebrospinal fluid, and saliva) and for various age groups (e.g., neonates, infants, children, and adults).SOLUTION: The methods include: derivatizing amino acids in the body fluid; separating the derivatized amino acids by liquid chromatography (LC); and identifying the derivatized amino acids using mass spectrometry (MS) analysis and quantifying the derivatized amino acid by comparison with structurally similar amino acids from a set of amino acids standards.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention generally relates to the detection and analysis of amino acids, particularly amino acids contained in biological fluids.

Background Art

[0002] The types and amounts of amino acids in a patient's body fluid (e.g., plasma) are important for the patient's health for various reasons. The diagnosis of diseases or illnesses can be made using abnormal amino acid levels. For example, a decrease in plasma amino acid levels can occur in patients with cancer, eating disorders, arthritis, folliculitis, alcohol dependence, glucagon-producing tumors, and / or pregnancy. Patients under stress or in a depressive state may also have a decrease in plasma amino acid levels. In particular, patients with depression may experience a deficiency of phenylalanine, tyrosine, methionine, glycine, tryptophan, and / or taurine. Psychiatric patients may have a decrease in the levels of amino acids (e.g., glycine, tryptophan, and / or histidine) and an increase in the levels of amino acids (e.g., phenylalanine, tyrosine, and / or serine).

[0003] Amino acid levels can also decrease in patients with infectious diseases and / or fever, although some amino acids (e.g., phenylalanine) in these patients may show increased levels. In patients with renal insufficiency, the levels of amino acids (e.g., tyrosine, threonine, leucine, isoleucine, valine, lysine, and / or histidine) can decrease. In patients with Crohn's disease, ulcerative colitis, and chronic fatigue syndrome, the levels of cystine and glutamine in plasma can abnormally decrease.

[0004] Furthermore, higher-than-normal amino acid levels can also indicate a disease state. For example, elevated plasma amino acid levels may be observed in patients with liver disease, pancreatitis, heavy metal poisoning, vitamin C deficiency, and / or vitamin D deficiency. Wilson's disease patients, in particular, may show elevated levels of tryptophan and histidine. Cushing's disease or gout patients may show elevated levels of alanine. Diabetic patients may show elevated levels of valine, leucine, and / or isoleucine. Hyperactive children may show elevated levels of tyrosine and phenylalanine. Patients with maple syrup urine disease may show elevated levels of leucine, isoleucine, and valine in their plasma. Thus, methods of analyzing amino acids in body fluids (e.g., plasma) are useful in the fields of medicine and science.

[0005] Traditionally, amino acid analysis has involved a derivatization step. During derivatization, amino acids are reacted with derivatizing reagents that facilitate the analysis of amino acids in a sample. These derivatizing reagents generally react with the free amino groups of amino acids in the sample. Common substances used for derivatizing amino acids include isothiocyanates (e.g., phenyl isothiocyanate (PITC)), o-phthalidaldehyde (OPA), 2,4-dinitrofluorobenzene (DNFB), and Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA). The reagent may contain substituents that facilitate the analysis of derivatized amino acids, making it useful. For example, the derivatization reagent may contain a chromophore for UV absorption detection or a fluorophore for fluorescence detection.

[0006] Derivatized amino acids can be separated and detected by performing chromatography, such as liquid chromatography (LC) or gas chromatography (GC), in combination with mass spectrometry (i.e., LC-MS or GC-MS). However, amino acids have diverse chemical structures (e.g., basic, acidic, aromatic, polar, nonpolar, etc.), and the chemical structures of various amino acids that may be present in body fluids differ significantly, making it difficult for analysts to distinguish between these chemical structures. Derivatization / separation of the compound using LC-MS or GC-MS is a challenging task.

[0007] Methods for detecting amino acids using LC and MS have been reported to date, including, for example, Casetta et al., “Development of a method for rapid quantification of amino acids by liquid chromatrography, tandem mass spectrometry” (LC-MSMS) in plasma ”Clin Chem lab Med (2000) 38: 391-401; Hess et al., “Acid hydrolysis of silk fibroins and determination of the enrichment of isotopically labeled amino acids using precolumn derivitization and high performance liquid chromatography-electrspray ionization mass spectrometry” Anal Biochem (2002) 311:19-26; Ji et al., “Determination of phenethyl isothiocyanate in human plasma and urine by ammonia derivatization and liquid chromatography-tandem mass spectrometry” Anal Biochem (2003) 323:39-47;Van Eijik et al., “Determination of amino acid isotope enrichment using liquid chromatography-mass spectrometry” (1999) Anal Biochem 271:8-17; and Liu et al., “Derivitization of amino acids with N,N-dimethyl-2,4-dinitro-5-fluorobenzylamine for liquid chromatography / electrospray ionization mass spectrometry” (2004) Rapid Commun Mass Spectrom 18:1059-65. Improved methods for detecting amino acids in body fluids are desired. [Overview of the project] [Means for solving the problem]

[0008] Summary of the Invention A method for detecting various individual amino acids that may be present in the body fluids of an individual is disclosed. The detection of individual amino acids in body fluids can be used to determine whether the body fluid has one or more amino acid abnormalities. In some respects, the method of the present invention involves: derivatization of body fluid amino acids, separation of the derivatized amino acids by liquid chromatography (LC), identification of the derivatized amino acids by mass spectrometry (MS), and quantification of the derivatized amino acids by comparison with structurally similar amino acids in a group of amino acid standards. Preferably, the MS is not tandem MS (e.g., single MS). Since structurally similar amino acids share important structural features (e.g., major functional groups), the identification of one amino acid by mass spectrometry can be used to identify other structurally similar amino acids in the same manner. It is preferable to add the group of individual amino acid standards to the body fluid before processing and use them as a series of internal standards.

[0009] Suitable body fluids include, for example, plasma, serum, saliva, urine, and cerebrospinal fluid (CSF). Methods for detecting, identifying, and / or quantifying individual amino acids in CSF allow their levels to be compared to the reference ranges shown in Table 1. Plasma amino acid levels outside the reference ranges in Table 1 may be identified as abnormal. Methods for detecting, identifying, and / or quantifying individual amino acids in urine allow their levels to be compared to the reference ranges shown in Table 2. Urinary amino acid levels outside the reference ranges in Table 2 may be identified as abnormal. Individual amino acids are detected in CSF, and the same... In determinative and / or quantitative methods, the level can be compared to the reference range shown in Table 3. CSF amino acid levels outside the reference range in Table 3 may be identified as abnormal. Methods for detecting, identifying, and / or quantifying individual amino acids in saliva allow their levels to be compared to the reference ranges shown in Table 4. Salivary amino acid levels outside the reference ranges in Table 4 may be identified as abnormal. In some embodiments, the reference range for each amino acid varies depending on the age of the subject (i.e., neonatal, infant, child, or adult).

[0010] The standard substances may be added to the starting body fluid or any post-treatment step without derivatization, and then derivatized. In a preferred embodiment, the standard substances include individual deuterated amino acids (i.e., single amino acids containing one or more deuterium ions). The standard substances may be added to the body fluid amino acids after the derivatization step. In this case, the added standard substances should be derivatized in the same manner as the body fluid amino acids. In one method, the amino acids are converted to isothiocyanates (e.g., For example, derivatization is performed with phenyl isothiocyanate (PITC). In a preferred embodiment, the derivatization reagent is PITC. Other preferred derivatization reagents include o-phthalidaldehyde (OPA), 2,4- These include dinitrofluorobenzene (DNFB) and Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA).

[0011] The amount of each amino acid identified from a fixed volume of body fluid can be measured by comparing the MS signal with the signals of structurally similar amino acids in known quantities. Then, the amount of amino acids in the body fluid can be expressed relative to the volume of body fluid analyzed, allowing for the acquisition of the original amino acid concentration in the body fluid. Quantitative analysis is preferably performed using internal standards.

[0012] In one embodiment, the body fluid may be processed to obtain a fraction containing high concentrations of amino acids, after which further analysis may be performed. In another method, a low molecular weight fraction of the body fluid may be obtained (for example, by passing the body fluid through a molecular weight filter).

[0013] In one embodiment, this method may be used to detect at least 20 distinct individual amino acids. In another embodiment, this method may be used to detect at least 25, 30, 35, or 40 distinct amino acids.

[0014] For example, the method of the present invention may be useful for detecting and / or quantifying individual amino acids in any combination, including, but not limited to, the following: phosphoserine, sulfocysteine, arginosuccinate, hydroxyproline, aspartic acid, asparagine, glutamic acid, serine, phosphoethanolamine (PEA), glutamine, glycine, histidine Sarcosine, taurine, carnosine, citrulline, arginine, anserine, 1-methylhistidine, 3-methylhistidine, alpha-aminoadipic acid (AAD), threonine, alanine, beta-alanine (BALA), proline, ethanolamine, gamma-aminobutyric acid (GABA), beta- Minoisobutyric acid (BAIA), α-aminobutyric acid (AAB), cysteine, tyrosine, valine, methionine, L-allocystathionine (cystathionine A), L-cystathionine (cystathionine B), cystine, isoleucine, allisoleucine, leucine, DL-hydroxylysine (Hydro Roxylysine (1), DL-Allohydroxylysine (Hydroxylysine (2)), Phenylal Nin, ornithine, tryptophan, homocystine, arginosuccinate (ASA), lysine , and Hawkinsin (2-L-cysteine-S-yl-1,4-dihydroxycyclo Hexy-5-en-1-yl)acetic acid. Furthermore, any individual detected in body fluids using this method. A disease state may be diagnosed based on amino acid levels.

[0015] In some embodiments, the method of the present invention may be useful for detecting and / or quantifying individual amino acids in any combination, including, but not limited to, the following: aspartic acid, ASA, S-cysteine, glutamic acid, OH-proline, serine, asparagine, PEA, AAD, glycine, glutamine, sarcosine, histidine, β-alanine, taurine, citrulline, and ka. Lunosine, threonine, arginine, anserine, 1-methylhistidine, 3-methylhistidine, alanine, GABA, BAIB, proline, ethanolamine, AAB, tyrosine, valine, Methionine, cystathionine A, cystathionine B, cystine, isoleucine, allisoleucine, leucine, OH-lysine-1, OH-lysine-2, homocystine, phenylalanine, tryptophan, ornithine, and lysine.

[0016] In another embodiment, the method of the present invention may be useful for detecting and / or quantifying individual amino acids in any combination, including, but not limited to, the following: hydroxyproline, aspartic acid, asparagine, glutamic acid, serine, glutamine, glycine, histidine, sarcosine, taurine, citrulline, arginine, 1,3-methylhistidine, α- Aminoadipic acid, threonine, alanine, β-alanine, proline, ethanolamine γ-aminobutyric acid, β-aminoisobutyric acid, α-aminobutyric acid, tyrosine, valine, methionine L-cystathionine, isoleucine, leucine, phenylalanine, ornithine, tryptophan, homocystine, and lysine.

[0017] In yet another embodiment, the method of the present invention may be useful for detecting and / or quantifying individual amino acids in any combination, including, but not limited to, the following: hydroxyproline, aspartic acid, asparagine, glutamic acid, serine, glutamine, glycine, histidine, sarcosine, taurine, citrulline, arginine, 1,3-methylhistidine, α-aminoadipic acid, threonine, alanine, β-alanine, proline, ethanolamine, γ-aminobutyric acid, β-aminoisobutyric acid, α-aminobutyric acid, tyrosine, valine, methionine L-cystathionine, isoleucine, leucine, phenylalanine, ornithine, tryptophan, homocystine, lysine, cystine, and hydroxylysine.

[0018] In yet another aspect, the method of the present invention may be useful for detecting and / or quantifying individual amino acids including, but not limited to, the following in any combination: hydroxyproline, aspartic acid, asparagine, glutamic acid, serine, glutamine, glycine, histidine, sarcosine, taurine, citrulline, arginine, α-aminoadipic acid, threonine, alanine, β-alanine, proline, γ-aminobutyric acid, β-aminoisobutyric acid, α-aminobutyric acid, tyrosine, valine, methionine, isoleucine, leucine, phenyl alanine, ornithine, tryptophan, homocystine, and lysine.

[0019] In one aspect, the method of the present invention may be useful for detecting and / or quantifying individual amino acids including, but not limited to, the following in any combination: phosphoserine, sulfocysteine, arginosuccinic acid, hydroxyproline, aspartic acid, phosphoethanolamine, sarcosine, carnosine, anserine, 1,3-methylhistidine, α-amino adipic acid, β-alanine, proline, ethanolamine, γ-aminobutyric acid, β-aminoi sobutyric acid, cysteine, L-allocystathionine A, L-cystathionine, cystine, alloiso leucine, DL-hydroxylysine, DL-allohydroxylysine, and homocystine.

[0020] In another aspect, the method of the present invention may be useful for detecting and / or quantifying individual amino acids including, but not limited to, the following in any combination: phosphoserine, sulfocysteine, arginosuccinic acid, hydroxyproline, aspartic acid, phosphoethanolamine, sarcosine, carnosine, anserine, 1,3-methylhistidine, α-amino adipic acid, β-alanine, proline, ethanolamine, γ-aminobutyric acid, β-aminoi Butyrate, cysteine, L-allocystathionine A, L-cystathionine, cystine, DL-hydroxylysine, DL-allohydroxylysine, and homocystine. In yet another embodiment, this The method can be used to identify either cysteine, phosphoserine, or arginosuccinate.

[0021] In yet another aspect, the method of the present invention may be useful for detecting and / or quantifying individual amino acids in any combination, including, but not limited to, the following: phosphoserine, sulfocysteine, arginosuccinate, hydroxyproline, phosphoethanolamine, sarcosine, carnosine, anserine, 1,3-methylhistidine, α-aminoadipic acid, β-alanine, proline, ethanolamine, γ-aminobutyric acid, β-aminoisobutyric acid, cis Thein, L-allocystathionine A, L-cystathionine, cystine, alloisoleucine, DL-hydroxylysine, DL-allohydroxylysine, and homocystine.

[0022] In further embodiments, the methods of the present invention may be useful for detecting and / or quantifying individual amino acids in any combination, including, but not limited to, the following: phosphoserine, sulfocysteine, arginosuccinate, hydroxyproline, phosphoethanolamine, sarcosine, carnosine, anserine, 1,3-methylhistidine, α-aminoadipic acid, etc. Tanolamine, γ-aminobutyric acid, β-aminoisobutyric acid, L-alosisthionine A, L-cis Tathionine, cystine, alloisoleucine, DL-hydroxylysine, DL-allohydroxylysine, and homocystine.

[0023] In further embodiments, the methods of the present invention may be useful for detecting and / or quantifying individual amino acids in any combination, including, but not limited to, the following: phosphoserine, cysteine, arginosuccinate, hydroxyproline, phosphoethanolamine, sarcosine, carnosine, anserine, 1,3-methylhistidine, α-aminoadipic acid, ethanol Luamine, γ-aminobutyric acid, β-aminoisobutyric acid, L-alosystathionine A, L-cystathio Nin, cystine, DL-hydroxylysine, DL-allohydroxylysine, and homocystine.

[0024] In other embodiments, the method of the present invention quantifies at least two, at least three, at least four, at least five, at least seven, at least ten, at least fifteen, at least twenty, at least twenty-five, or at least thirty or more individual amino acids.

[0025] The disclosed methods can be used as criteria for diagnosing or monitoring the effectiveness of treatment for various diseases known to be associated with abnormal levels of individual amino acids (i.e., single amino acids isolated from dipeptides and polypeptides). For example, levels of leucine, isoleucine, valine, lysine, and / or histidine may be used to diagnose and / or monitor renal failure; levels of cystine and / or glutamine may be used to diagnose and / or monitor Crohn's disease, ulcerative colitis, and / or chronic fatigue syndrome; levels of tryptophan and / or histidine may be used to diagnose and / or monitor Wilson's disease; levels of alanine may be used to diagnose and / or monitor Cushing's disease or gout; levels of valine, leucine, and / or isoleucine may be used to diagnose and / or monitor diabetes mellitus and / or maple syrup urine disease; levels of tyrosine and / or phenylalanine may be used to diagnose and / or monitor attention deficit hyperactivity disorder; and levels of hawkinsin may be used to diagnose and / or monitor hawkinsinuria.

[0026] LC separation of derivatized amino acids can be performed using any type of commercially available LC system. Suitable LC columns are those that can handle fine particles (e.g., approximately 2-5 μm in diameter, preferably approximately 3 μm). It contains a filler (silica particles). The particles generally have fine pores of about 50 to 300 angstroms, preferably about 150 angstroms. The particles generally have pores of about 50-600 m 2 / g Preferably about 100m 2 It has a surface area of ​​ / g.

[0027] The particles may have a hydrophobic stationary phase bonded to their surface. In one embodiment, the hydrophobic stationary phase may be an alkyl phase containing C-4, C-8, and C-18 (preferably C-18).

[0028] The column can have suitable dimensions. Preferably, the column has a diameter of about 0.5 mm to about 5 mm and a length of about 15 mm to about 300 mm, most preferably a diameter of about 2 mm and a length of about 50 mm.

[0029] LC separation of derivatized amino acids can be performed by using a hydrophobic solvent or a solvent mixture containing a hydrophobic solvent as a mobile phase in a gradient and eluting the amino acids. In one embodiment, the derivatized fraction may be applied to a column in an aqueous buffer (i.e., a hydrophilic solvent), and the amino acids may be eluted by applying a mobile phase containing an increasing amount of organic solvent (i.e., a hydrophobic solvent) to the column. For example, the aqueous buffer may contain (95% H2O, 5% acetonitrile). Often, the acetonitrile concentration in the mobile phase is gradually increased to approximately 100% to dissolve the amino acids from the column. It may be discharged. If necessary, heat the mobile phase to a temperature of about 40-60°C, preferably about 50°C. Alternatively, the mobile phase may contain one or more additional reagents useful during LC and / or MS (e.g., ammonium acetate or acetic acid), if necessary.

[0030] MS analysis of derivatized amino acids can be performed by ionizing the sample. Suitable ionization methods include electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). Photoionization, electron ionization, fast electron bombardment (FAB) / liquid secondary ionization (LSIMS), matrix-assisted laser desorption / ionization (MALDI), field ionization, field These include desorption methods, thermal spray / plasma spray ionization methods, and particle beam ionization methods. Preferably, MS is performed using ESI. Furthermore, MS is performed in negative or positive ion modes (preferably Alternatively, this may be done using negative ion mode.

[0031] MS analysis of derivatized amino acids may be performed using any of several types of ion analyzers (quadrupole analyzers, ion trap analyzers, and time-of-flight analyzers). Preferably, MS is performed using a quadrupole analyzer. The ions generated during MS may be detected using several detection methods (such as selected ion monitoring (SIM) and scanning methods). In addition, ions are detected using SIM. A MS other than tandem MS is preferred.

[0032] Furthermore, a method is provided for diagnosing the presence of metabolic disorders involved in amino acid metabolism in an individual. This method includes determining whether body fluids contain one or more amino acids at abnormal levels by: (a) derivatization of the amino acids in the body fluids; (b) separation of the derivatized amino acids by liquid chromatography (LC); (c) mass spectrometry (MS) of the separated derivatized amino acids; and (d) quantification of the derivatized amino acids by MS analysis by comparison with structurally similar amino acids from a group of amino acid standards. Various aspects of this method are the same as those already described. [Brief explanation of the drawing]

[0033] [Figure 1] Figure 1 is a table showing various amino acids detected and quantified by the methods described herein. The third column shows the MW "molecular weight"; the fourth column shows the molecular weight of the PITC; the fifth column shows the molecular weight of each PITC-derivative amino acid; the sixth column shows the LC retention time; and the seventh column shows the ions observed by mass spectrometry. [Figure 2] Figure 2 shows the MS analysis results after LC of a single sample containing the indicated amino acid. [Modes for carrying out the invention]

[0034] Detailed description of preferred embodiments As used herein, “amino acid” means any molecule containing an α-carbon covalently bonded to an amino group and an acidic group. The acidic group may be a carboxyl group. The “acidic group” may be a molecule having one of the following formulas: [ka] In the formula, R is a side chain and Z contains at least three carbon atoms. The term "amino acid" includes, but is not limited to, 20 human endogenous amino acids and their derivatives, such as: lysine, asparagine, threonine, serine, isoleucine, methionine, proline, histidine, glutamine, arginine, glycine, aspartic acid, glutamic acid, arani , valine, phenylalanine, leucine, tyrosine, cysteine, tryptophan, phosphoserine (PSER), sulfocysteine, arginosuccinate (ASA), hydroxypropyl Phosphoethanolamine (PEA), sarcosine (SARC), taurine (TAU), carno Syn (CARN), Citrulline (CIT), Anserine (ANS), 1,3-Methylhistidine (ME-HIS), α-Aminoadipic Acid (AAA), β-Alanine (BALA), Ethanolamine (ETN), γ-Aminobutyric Acid (GABA), β-Aminoisobutyric Acid (BAIA), α-Aminobutyric Acid (BABA), L-Alloth Stathonine (cystathionine A; CYSTA-A), L-cystathionine (cystathionine B; CYSTA-B), cystine, alloisoleucine (alloILE), DL-hydroxylysine (hydroxylysine(1)), DL-allohydroxylysine (hydroxylysine(2)), ornithine (ORN), homocystine (HCY), and their derivatives. "Amino acids" include D-amino acids and L-amino acids. This also includes stereoisomers such as the anoic acid form. Unless otherwise specified, the term “amino acid” refers to individual (i.e., free) amino acid molecules separated from the amino acids present in dipeptides, polypeptides, and proteins.

[0035] As used herein, “body fluid” means any liquid that can be isolated from the body of an individual. For example, “body fluid” includes blood, plasma, serum, bile, saliva, urine, tears, sweat, cerebrospinal fluid (CSF), etc. This includes body fluids. Preferably, the body fluids are plasma, serum, cerebrospinal fluid, urine, or saliva, and most preferably plasma.

[0036] As used herein, “derivativeization” refers to the process of reacting two molecules to produce a new molecule. This means that... For example, amino acid derivatization may be carried out by reacting an amino acid with a derivatizing reagent to produce a derivatized amino acid. Derivatization may also involve reacting the α-amino group of an amino acid with an electrophilic atom of a derivatizing reagent to form a covalent bond. The derivatizing reagent may be an isothiocyanate group, a dinitrofluorophenyl group, a nitrophenoxycarbonyl group, and / or a phthalaldehyde group.

[0037] As used herein, “liquid chromatography (LC)” refers to the selective delay process of one or more body fluid components as a body fluid passes uniformly through a column or capillary pathway of fine matter. The delay is due to the distribution of mixture components between one or more stationary phases and bulk fluid (i.e., mobile phase), which occurs when the liquid flows counterflowing relative to the stationary phase. This process is used for the analysis and separation of mixtures of two or more substances. “Liquid chromatography” includes reversed-phase liquid chromatography (RPLC) and high-pressure liquid chromatography (HPLC).

[0038] As used herein, “mass spectrometry” or “mass spectrometry” (MS analysis) means an analytical technique for identifying unknown compounds, including: (1) ionization of the compound and generation of a charged compound by electrochemical fractionation of the compound; and (2) detection of the molecular weight of the charged compound and calculation of the mass-to-charge ratio (m / z). The compound may be ionized and detected by any suitable method. The "quantitative analyzer" includes means for the ionization of compounds and the detection of charged compounds.

[0039] As used herein, “electrospray ionization” is a technique used in mass spectrometry to ionize polymers and overcome their tendency to fragment. In electrospray ionization, a liquid is pushed out from a micro-metallic capillary charged by a carrier gas. The liquid contains the substance of study (analyte) and a large amount of solvent (usually much more volatile than the analyte). The charge contained in the capillary is transferred to the liquid, which charges the analyte molecules. Since like charges repel each other, the liquid is pushed out of the capillary column, forming a mist or aerosol of tiny droplets approximately 10 μm in diameter. The distance between molecules with similar charges increases. A neutral carrier gas is used to volatilize the neutral solvent in the microdroplet, causing the charged analyte molecules to move closer together. However, this proximity of molecules becomes unstable, and when molecules with similar charges approach each other, the droplet explodes again. This process is repeated until the analyte no longer contains solvent and isolated ions are generated. The isolated ions are then transported to the mass spectrometer.

[0040] As used herein, the term "quadrupole analyzer" refers to a quadrupole (i.e., two pairs of parallel poles) A mass spectrometer consisting of aligned metal rods, where one pair of rods has a positive potential and the other pair has a negative potential. For detection, ions must pass through the center of parallel orbits along the aligned rods. When the quadrupole is operated with a predetermined DC amplitude and high-frequency voltage, only ions with a predetermined m / z resonate and enter a stable orbit in the quadrupole. It passes through and is detected. "Cation mode" means a mode in which ions with a positive potential are detected by the mass spectrometer. "Anion mode" means a mode in which ions with a negative potential are detected by the mass spectrometer. In "single-ion monitoring" or "selected monitoring" (i.e., SIM), only a specific mass is observed. As you might guess, the DC amplitude and high-frequency voltage are adjusted.

[0041] As used herein, “low molecular weight fraction” refers to a fraction containing one or more low molecular weight molecules at a high concentration. The molecular weight of low molecular weight molecules is generally less than approximately 1000 daltons, and more generally less than approximately 500 daltons.

[0042] As used herein, "hydrophobic" means insoluble or sparingly soluble in water. Examples of hydrophobic compounds include long-chain alkanes. A hydrophobic solvent is a solvent capable of dissolving hydrophobic compounds.

[0043] In this specification, the term “approximately” in relation to quantities means plus or minus 10% of that quantity.

[0044] A method for identifying and / or quantifying amino acids in body fluids is disclosed. Body fluids may include blood, plasma, serum, bile, saliva, urine, cerebrospinal fluid, etc. Preferred body fluids are plasma, serum, and CSF. This can be urine or saliva, with plasma being the most preferred.

[0045] Preferably, a group of individual amino acid standards corresponding to the types of amino acids that may be present in a particular body fluid is added to the body fluid sample, after which any step is performed. Preferably, the group of amino acid standards contains a known amount of each individual amino acid contained therein. The group of amino acid standards may contain one or more amino acids from the following group: lysine, asparagine, threonine, serine, isoleucine, methionine, proline, histidine, glutamine, arginine, glycine, aspartic acid, glutamic acid, alanine, valine, phenylalanine, leucine, tyrosine, cysteine, tryptophan, phosphoserine, sulfocysteine, arginosuccinic acid, hydroxyproline, phosphoethanolamine, Sarcosine, taurine, carnosine, citrulline, anserine, 1,3-methylhistidine, α-aminoadipic acid, β-alanine, ethanolamine, γ-aminobutyric acid, β-aminoisobutyric acid, α-aminobutyric acid, L-alocystathionine (cystathionine A), L-cystathionine (cystathionine B), cystine, alloisoleucine, leucine, DL-hydroxylysine (hydroxylysine (1)), DL-allohydroxylysine (hydroxylysine (2)), ornithine , tryptophan, homocystine, and their isomers (e.g., stereoisomers).

[0046] The amino acids in the amino acid standard material group may be modified so that they can be easily distinguished from similar amino acids found in body fluids. Internal standard amino acids preferably exhibit behavior closest to amino acids selected to be chemically and physically equivalent, but they fragment into ions of different masses during mass spectrometry. Therefore, preferred amino acid standard materials are deuterated.

[0047] Body fluids may be treated before derivatization to obtain high-concentration amino acid preparations. Various operations may be used for this purpose depending on the type of body fluid. These include filtration, precipitation, centrifugation, and combinations thereof. Separation of low molecular weight fractions is a preferred method. Size separation of small samples is preferably performed by filtration using a low molecular weight cutoff filter. The filtered body fluid sample (i.e., permeate) contains free amino acids, and the retained components (i.e., residue) contain high molecular weight components (e.g., proteins). Suitable filters for obtaining filtrate are 45 micron, 22 micron, and 100,000, 50,000, and 10,000 denier filters. A Luton cutoff filter is available. Furthermore, alcohol (e.g., methanol) or acid may be added to the sample to precipitate high molecular weight components from the plasma sample. Alternatively, high molecular weight components may be removed from the sample by high-speed centrifugation.

[0048] Amino acid derivatization may be performed after any necessary processing of the body fluid sample. Generally, amino acids in the sample are derivatized to obtain free amino acids in the sample during LC-MS. This facilitates the separation and / or detection of the derivatives (e.g., pre-column derivatization followed by LC). The derivatization reagent may contain substituents (e.g., phosphors or chromophores) that facilitate the detection of the derivatized amino acids during or after chromatography. Furthermore, the derivatization reagent may contain substituents that facilitate the ionization of the derivatized amino acids during mass spectrometry. Common derivatization reagents include isothiocyanates (e.g., phenyl isothiocyanate (PITC)), o-phthalidaldehyde (OPA), 2,4-dinitrofluorobenzene (DNFB), and Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA). In a preferred embodiment, the derivatizing reagent is PITC.

[0049] After derivatizing the amino acids in the sample, chromatographic separation of the sample (preferably) Alternatively, high-pressure liquid chromatography (LC-MS) and mass spectrometry (i.e., LC-MS) are performed.

[0050] Liquid chromatography and mass spectrometry can be performed by placing the derivatized sample in an apparatus that includes a chromatography column in communication with a mass spectrometer. Generally, chromatography columns contain a medium (i.e., packing material) to facilitate the separation (i.e., fractionation) of derivatized amino acids. The medium may contain fine particles with a diameter of about 2-6 μm, preferably about 3 μm. For example, the particles may be silica particles. The particles may have fine pores with a diameter of about 50-300 angstroms, preferably 150 angstroms. Furthermore, the particles may have pores of about 50-600 μm. 2 / g, preferably 100m 2 It may have a surface area of ​​ / g.

[0051] The particles may have a binding surface that interacts with the derivatized amino acid to facilitate the separation of the amino acid. One preferred binding surface is a hydrophobic binding surface, such as an alkyl binding surface. The alkyl binding surface may contain C-4, C-8, or C-18 alkyl binding groups, preferably C-18 binding groups.

[0052] The column can have suitable dimensions. In particular, the column has a diameter of approximately 0.5-5 mm and a length The length may be approximately 15-300 mm. Preferably, the column has a diameter of approximately 2 mm and a length of approximately 50 mm.

[0053] Suitable media and / or pre-prepared columns for the preparation of chromatography columns are available commercially. In particular, suitable columns are available from Thermo Electron. (For example, 250 x 2.1 mm, 5 μm, BetaBasic C18 column).

[0054] A chromatography column includes an inlet port for receiving the sample and an outlet port for releasing the eluent containing the fractionated sample. In this method, the derivatized sample is applied to the column at the inlet port, eluted with a solvent or solvent mixture, and discharged at the outlet port. Various solvent modes may be selected for amino acid elution. For example, liquid chromatography can use gradient mode, isochromatic... Liquid chromatography may be performed using a gradient mode or a polytypic (i.e., mixed) mode. Preferably, liquid chromatography is performed using a gradient mode. In gradient mode, the derivatized sample is applied to the column and a mixture of two solvents (i.e., mobile phase) is used. The amino acids are eluted by passing the solvent through a column. Generally, as is well known in the art, one solvent tends to be relatively hydrophilic, and the other solvent tends to be relatively hydrophobic. As a specific example of a solvent composition that has been shown to be suitable for carrying out the method of the present invention, even if the hydrophilic solvent is 95% H2O and 5% acetonitrile and the hydrophobic solvent is 100% acetonitrile, Good. If necessary, the compounding solvent may contain one or more reagents (e.g., 20 mM ammonium acetate) to facilitate the separation and / or detection of the derivatized amino acids. Several reagents may be added to the mobile phase to improve the peak shape of the chromatography and / or to provide an ion source for LC-MS.

[0055] In most cases, liquid chromatography with a gradient solvent involves mixing two solvents using two pumps. First, when mixing the solvents, the solvent mixture passing through the column (i.e., the mobile phase) contains the most hydrophilic solvent. The solvent gradient is then constructed by gradually decreasing the amount of hydrophilic solvent in the mixture and increasing the amount of hydrophobic solvent. Finally, the solvent mixture passing through the column contains the most hydrophobic solvent. In this way, hydrophilic amino acids are eluted before hydrophobic amino acids.

[0056] A mass spectrometer includes an inlet port for receiving fractionated samples, which communicates with the outlet port for the chromatography column. The mass spectrometer can produce one or more mass spectrometry datasets for identifying one or more amino acids in the sample. Suitable instruments for performing LC-MS are commercially available. In particular... Suitable instruments for performing LC-MS are available from Agilent Technologies (e.g., Agilent 1100 series LC / MSD).

[0057] The mass spectrometer includes an ion source for ionizing the fractionated sample for further analysis and generating charged molecules. Sample ionization may be carried out by: electrospray ionization (ESI), atmospheric pressure chemical ionization (ACPI), photoionization, Electron ionization, fast atomic collision (FAB) / liquid secondary ionization (LSIMS), matrix-assisted laser desorption / ionization (MALDI), field ionization, electrolytic desorption, thermal spray / Plasma spray ionization and particle beam ionization. Electrospray ionization is preferred.

[0058] After ionizing the sample, the resulting positively or negatively charged ions can be analyzed, and the mass-to-charge ratio (i.e., m / z) can be measured. Preferably, the negatively charged ions are separated. Analysis is performed. Suitable analyzers for measuring the mass-to-charge ratio include quadrupole analyzers, ion trap analyzers, and time-of-flight analyzers. Preferably, the mass-to-charge ratio is measured using a quadrupole. Ion detection may be performed using several detection methods. For example, selected ions may be detected (i.e., using a selected ion monitoring mode (SIM)), or skim ions. Ions may be detected using a cyan mode. Preferably, ions are detected using a SIM. . [Examples]

[0059] Example 1: Analytical Method Urine, plasma, and cerebrospinal fluid (CSF) samples were obtained from healthy individuals. Patients were fasted overnight. Heparinized plasma samples were collected from the subjects. Non-fasted samples were used from pediatric patients. Amino acids in urine, plasma, and CSF were quantified using the following procedure.

[0060] Add the deuterated internal standard to approximately 100 μl of the test material (i.e., plasma, urine, or CSF). A test mixture was prepared. The test mixture was then passed through a filter with a molecular weight cutoff of 10,000 to obtain a low molecular weight filtrate fraction as a test sample, which was then dried under a nitrogen atmosphere at 40-75°C. The dried sample was dissolved in approximately 25 μl of re-dried solution (equal volumes of methanol, 1 M sodium acetate, and triethylamine) and dried at 40°C under a nitrogen atmosphere. The sample was then divided into 50 μl of derivatives. Embodied solution (1.12 μl of 100% methanol, 1.60 μl of water, 1.60 μl of 100% triethylamine, It was then dissolved in 1.60 μl of 100% phenylisothiocyanate (PITC). The prepared sample was heated at approximately 40°C for approximately 15-20 minutes, and then dried under a nitrogen atmosphere at 50-60°C. The dried sample was dissolved in 100 μl of reconstitution solution (95% H2O, 5% acetonitrile) and then volt-coated. The sample was squeegeeed and transferred to an LC / MS vial.

[0061] LC / MS analysis of amino acid samples was performed using an Agilent 1100 series LC / MSD with a Thermo Beta-Basic C-18 (250 x 2.1 mm) HPLC column. The mobile phase composition was (A) 20 mM acetate The solutions were mononium and (B) 100% acetonitrile (heated to 50°C). The amino acid samples were Elution was performed from the column using the following step gradient:

[0062] [Table 1]

[0063] The separated amino acids were eluted from the HPLC column and introduced into a spray chamber, where the eluate was sprayed and desolvated by an electrospray ion source. The PITC derivatives were negatively charged in the electrospray step and then further separated using a quadrupole mass filter. Amino acid ions were detected and their abundance was measured. The area abundance of each amino acid ion and its ratio to the internal standard were plotted against a six-point calibration curve. Typical measurement results are shown in Figures 1 and 2.

[0064] Example 2: Determination of amino acid content in plasma Amino acid analysis was performed on heparinized plasma samples collected from neonates (under 30 days), infants (1–23.9 months), children (2–17.9 years), and adults (18 years and older). All individuals were clinically diagnosed as normal or obtained from samples submitted for infectious disease assessment. All subjects were fully amblyable, community-dwelling, healthy, and not taking any medications. The demographics of the study groups are as follows:

[0065] [Table 2]

[0066] Based on the analysis of these plasma samples, normal reference ranges were constructed using standard parametric and nonparametric statistical methods. If the data showed a Gaussian distribution, a suitable mean ± 2SD range was selected. If the data did not show a Gaussian distribution, the 95th percentile range or the observed range was selected using nonparametric methods. Table 1 shows the normal reference ranges for each assayed amino acid, along with the limit of quantification (LOQ). Reference ranges are expressed in μM (micromoles / liter). For undetectable amino acids, the normal range is either less than the LOQ or... They should be treated as equivalent.

[0067] [Table 3]

[0068] [Table 4]

[0069] Example 3: Determination of amino acid content in urine Amino acid analysis was performed on urine samples collected from neonates (under 30 days), infants (1–23.9 months), children (2–17 years), and adults (17 years and older). All individuals were clinically diagnosed as normal or obtained from samples submitted for infectious disease assessment. All subjects Participants were fully able to walk, lived in the community, were healthy, and were not taking any medications. The demographics of the test group were as follows:

[0070] [Table 5]

[0071] First, the amino acid content in urine was measured based on a concentration standard (amino acid μmol / liter of urine; μM). Then, the amino acid concentrations were standardized based on urinary creatinine levels, and normal reference ranges were constructed using standard parametric and nonparametric statistical methods. If the data showed a Gaussian distribution, a suitable mean ± 2SD range was selected. If the data did not show a Gaussian distribution, the 95th percentile range or the observed range was selected using a nonparametric method. Table 2 shows the normal reference range (mmol / mol creatinine) for each amino acid assayed. The limit of quantification (LOQ) was determined based on the urinary amino acid measurement, supplemented by creatinine. The test was not performed correctly. The normal range for undetectable amino acids is less than or equal to LOQ. It should be.

[0072] [Table 6]

[0073] [Table 7]

[0074] Example 4 Determination of amino acid content in cerebrospinal fluid (CSF) Newborns (under 3 months), infants (3-23.9 months), children (2-10 years), and adults (10 years and older) Amino acid analysis was performed on CSF samples collected from the individuals. All individuals were clinically normal. The samples were obtained from individuals diagnosed with or submitted for infectious disease assessment. All subjects were fully mobile, community-dwelling, healthy, and not taking any medications. The demographics of the study group were as follows:

[0075] [Table 8]

[0076] Based on the analysis of these CSF samples, standard parametric and nonparametric methods Normal reference ranges were constructed using statistical methods. If the data showed a Gaussian distribution, a suitable range of mean ± 2SD was selected. If the data did not show a Gaussian distribution, the 95th percentile range or the observed range was selected using a non-parametric method. Table 3 shows the normal reference ranges for each assayed amino acid, along with the limit of quantification (LOQ). Reference ranges are expressed in μM (micromoles / liter). For undetectable amino acids, the normal range is either less than the LOQ or... They should be treated as equivalent.

[0077] [Table 9]

[0078] [Table 10]

[0079] Example 5: Determination of amino acid content in saliva Amino acid analysis was performed on saliva samples from nine adults (3 men and 6 women). All subjects were diagnosed as clinically normal, fully ambly, community-dwelling, healthy, and not taking any medications. Table 4 shows the range of measured amino acid levels. For diagnostic purposes, these ranges may be considered the "normal" range. The normal range for undetectable amino acids is It should be less than or equal to LOQ.

[0080] [Table 11]

[0081] [Table 12]

[0082] All patents and other references cited herein represent the level of skill of the articulate person in the field to which the present invention pertains, and all of them, including tables and drawings, are incorporated herein by reference to the same extent as each reference is incorporated by reference as an individual reference.

[0083] As will be readily apparent to those skilled in the art, the present invention is well suited to achieving the purposes and benefits described herein, as well as those inherently incidental thereto. The methods, modifications, and compositions described herein as typical of currently preferred embodiments are representative examples and are not intended to limit the scope of the present invention. Modifications and other uses made by those skilled in the art are also permitted. This is included in the intent of the present invention, but is defined in the claims.

[0084] Various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and intent of the invention, as will be readily apparent to those skilled in the art. Accordingly, further embodiments thereof are included within the scope of the invention and the appended claims.

[0085] The present invention, as suitably illustrated herein, may be carried out without any element(s) or limitations(s), but these are not specifically disclosed herein. Therefore, for example, in each of the cases described herein, the terms "including," "essentially..." are used. The terms “consisting of” and “consisting of” may be substituted for each other by any of the other two. The terms and expressions used are for illustrative purposes only, not limitations, and there is no intention to exclude any or any part of the features presented and described in the use of those terms and expressions, and various modifications may be made within the scope of the claimed invention as recognized. Accordingly, although the invention has been specifically disclosed by preferred embodiments and features as necessary, it should be understood that those skilled in the art may modify and change the concepts disclosed herein, and such modifications and changes will be considered to fall within the scope of the invention as defined in the appended claims.

[0086] Furthermore, when describing the features or aspects of the present invention in the Markush group or other group of options, the present invention may also be described in terms of individual members or subgroups of members of the Markush group or other group, as will be apparent to those skilled in the art.

[0087] Furthermore, unless otherwise stated, when various numerical values ​​are described in relation to the embodiments, further embodiments are described by setting any two different numerical values ​​as the endpoints of the range. Those ranges are also included within the scope of the invention described.

[0088] Accordingly, further embodiments are included within the scope of the present invention and the appended claims.

Claims

1. A method for determining the amounts of at least four amino acids in a sample by simultaneous analysis using mass spectrometry, (a) Purify the at least four amino acids, including arginosuccinic acid, phosphoserine, taurine, and sulfocysteine, by liquid chromatography. (b) Ionizing the at least four amino acids under conditions suitable for generating ions detectable by mass spectrometry, (c) The amount of ions of at least four amino acids is detected by mass spectrometry, and (d) Determine the amount of the at least four amino acids in the sample from the amount of ions determined in step (c), Methods that include...

2. The method according to claim 1, wherein the at least four amino acids further comprise alloisoleucine or β-alanine.

3. The method according to claim 1, wherein the at least four amino acids further comprise aspartic acid, threonine, serine, asparagine, glutamic acid, glutamine, glycine, alanine, citrulline, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, gamma-aminobutyric acid, lysine, and histidine.

4. The method according to claim 1, wherein the at least four amino acids further comprise aspartic acid, hydroxyproline, threonine, serine, asparagine, glutamic acid, glutamine, sarcosine, α-aminoadipic acid, proline, glycine, alanine, α-aminobutyric acid, valine, cysteine, methionine, homocitrulline, cystathionine, alloisoleucine, isoleucine, leucine, tyrosine, phenylalanine, β-aminoisobutyric acid, homocystine, γ-aminobutyric acid, tryptophan, hydroxylysine, lysine, and histidine.

5. The method according to claim 1, wherein the sample is urine.

6. The method according to claim 1, wherein the sample is plasma.

7. The method according to claim 1, wherein the amount of the at least four amino acids is used for the diagnosis or monitoring of one or more diseases selected from the group consisting of maple syrup urine disease, renal failure, Crohn's disease, ulcerative colitis, chronic fatigue syndrome, Wilson's disease, Cushing's disease, gout, and attention deficit hyperactivity disorder.

8. The method according to claim 1, comprising using one or more internal standards.

9. The method according to claim 1, wherein the mass spectrometry method is tandem mass spectrometry.

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