Detection and quantification of guanidinoacetic acid, creatine and creatinine by mass spectrometry

Abstract

The invention relates to detection and quantification of guanidinoacetic acid, creatine and creatinine by mass spectrometry. Methods of detecting or determining the amounts of guanidinoacetic acid (GAA), creatine, and creatinine by mass spectrometry are provided.

Description

[0001] This application is a divisional application. The application date of the original application is August 8, 2018, the application number is 2018800638894, and the name of the invention is "Detection and Quantification of Guanidine Acetic Acid, Creatine and Creatinine by Mass Spectrometry".

[0002] Cross-references to related patent applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 545,349, filed August 14, 2017, the entire contents of which are incorporated herein by reference. Background Art

[0004] Three human disorders characterized by defects in L-arginine:glycine amidinotransferase (AGAT), guanidinoacetate methyltransferase (GAMT), and the X-linked creatine transporter (SLC6A8) cause cerebral creatine deficiency syndrome (CCDS). Because the presenting clinical features (e.g., psychomotor retardation, growth retardation, intellectual disability, seizures, autistic behavior, spasticity, and movement abnormalities) are nonspecific features, they are underdiagnosed. Oral supplementation of creatine in AGAT and GAMT corrects brain creatine content. Diagnosis depends on the measurement of guanidinoacetate (GAA), creatine, and creatinine in urine and plasma. Elevated or decreased concentrations of these individual analytes in urine or plasma can distinguish the three disorders.

[0005] Accurate and sensitive determination of guanidinoacetic acid (GAA), creatine, and creatinine is required. Summary of the invention

[0006] Provided herein are methods for detecting or determining the amount of guanidinoacetic acid (GAA), creatine, and creatinine in a sample by mass spectrometry, including tandem mass spectrometry.

[0007] In certain embodiments, the methods provided herein are for detecting or determining the amount of guanidinoacetic acid (GAA), creatine, and creatinine, comprising: (a) purifying GAA, creatine, and creatinine in a sample; (b) ionizing GAA, creatine, and creatinine in the sample; (c) detecting or determining the amount of GAA, creatine, and creatinine ions (one or more) by mass spectrometry; wherein the amount of GAA, creatine, and creatinine ions (one or more) is related to the amount of GAA, creatine, and creatinine in the sample.

[0008] In certain embodiments, the methods provided herein are used to detect or determine the amount of guanidinoacetic acid (GAA), comprising: (a) purifying GAA in a sample; (b) ionizing GAA in the sample; (c) detecting or determining the amount of GAA ions (one or more) by mass spectrometry; wherein the amount of GAA ions (one or more) is related to the amount of GAA in the sample.

[0009] In certain embodiments, the methods provided herein are for detecting or determining the amount of creatine, comprising: (a) purifying GAA, creatine, and creatinine in a sample; (b) ionizing creatine in the sample; (c) detecting or determining the amount of creatine ion (s) by mass spectrometry; wherein the amount of creatine ion (s) is related to the amount of creatine in the sample.

[0010] In certain embodiments, the methods provided herein are used to detect or determine the amount of creatinine, comprising: (a) purifying creatinine in a sample; (b) ionizing creatinine in the sample; (c) detecting or determining the amount of creatinine ion (one or more) by mass spectrometry; wherein the amount of creatinine ion (one or more) is related to the amount of creatinine in the sample.

[0011] In some embodiments, guanidinoacetic acid (GAA), creatine, and creatinine are underivatized prior to mass spectrometry.

[0012] In some embodiments, the sample is urine or serum or plasma. In a preferred embodiment, the sample is urine. In some embodiments, the sample is whole blood. In some embodiments, the sample is saliva.

[0013] In some embodiments, the purification provided herein comprises liquid chromatography. In some embodiments, liquid chromatography comprises high performance liquid chromatography (HPLC).

[0014] In some embodiments, the purification provided herein comprises solid phase extraction (SPE).

[0015] In some embodiments, ionization comprises electrospray ionization (ESI). In some embodiments, ionization comprises ionization in a positive mode. In some embodiments, ionization comprises ionization in a negative mode.

[0016] In some embodiments, the methods provided herein further comprise adding an internal standard. In some embodiments, the internal standard is isotopically labeled.

[0017] In certain embodiments, the limit of quantitation of the method is less than or equal to 1 mg / L. In some embodiments, the limit of quantitation of the method is less than or equal to 0.9 mg / L. In some embodiments, the limit of quantitation of the method is less than or equal to 0.8 mg / L. In some embodiments, the limit of quantitation of the method is less than or equal to 0.7 mg / L. In some embodiments, the limit of quantitation of the method is less than or equal to 0.6 mg / L. In some embodiments, the limit of quantitation of the method is less than or equal to 0.5 mg / L. In some embodiments, the limit of quantitation of the method is less than or equal to 0.4 mg / L. In some embodiments, the limit of quantitation of the method is less than or equal to 0.3 mg / L.

[0018] In certain embodiments, the detection limit of the method is less than or equal to 1 mg / L. In some embodiments, the detection limit of the method is less than or equal to 0.9 mg / L. In some embodiments, the detection limit of the method is less than or equal to 0.8 mg / L. In some embodiments, the detection limit of the method is less than or equal to 0.7 mg / L. In some embodiments, the detection limit of the method is less than or equal to 0.6 mg / L. In some embodiments, the detection limit of the method is less than or equal to 0.5 mg / L. In some embodiments, the detection limit of the method is less than or equal to 0.4 mg / L. In some embodiments, the detection limit of the method is less than or equal to 0.3 mg / L. In some embodiments, the detection limit of the method is less than or equal to 0.2 mg / L. In some embodiments, the detection limit of the method is less than or equal to 0.1 mg / L.

[0019] In some embodiments, the ionization comprises generating a guanidinoacetic acid (GAA) precursor ion having a mass / charge ratio of 118.1 ± 0.5. In some embodiments, the method further comprises generating one or more fragment ions having a mass / charge ratio of 72.1 ± 0.5. In some embodiments, the ionization comprises generating a creatine precursor ion having a mass / charge ratio of 132.1 ± 0.5. In some embodiments, the method further comprises generating one or more fragment ions having a mass / charge ratio of 90.1 ± 0.5. In some embodiments, the ionization comprises generating a creatinine precursor ion having a mass / charge ratio of 114.1 ± 0.5. In some embodiments, the method further comprises generating one or more fragment ions having a mass / charge ratio of 44.1 ± 0.5.

[0020] In some embodiments, a method may include adding a reagent to a sample in an amount sufficient to deproteinize the sample.

[0021] As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise indicated. Thus, for example, reference to "a protein" includes a plurality of protein molecules.

[0022] As used herein, the term "purify" or "purify" does not refer to the removal of all substances other than the target analyte(s) from a sample. Rather, purification refers to the process of enriching the amount of one or more target analytes relative to other components in the sample that may interfere with the detection of the target analyte. Samples are purified herein by various means to allow the removal of one or more interfering substances, such as one or more substances that interfere with the detection of the selected analyte parent and daughter ions by mass spectrometry.

[0023] As used herein, the term "test sample" refers to any sample that can contain an analyte. As used herein, the term "body fluid" refers to any fluid that can be isolated from an individual's body. For example, "body fluid" can include blood, plasma, serum, bile, saliva, urine, tears, sweat, etc.

[0024] As used herein, the term "derivatization" refers to reacting two molecules to form a new molecule. The derivatizing agent may include an isothiocyanate group, a dinitro-fluorophenyl group, a nitrophenoxycarbonyl group, and / or a phthalaldehyde group, among others.

[0025] As used herein, the term "chromatography" refers to a process in which a chemical mixture carried by a liquid or gas is separated into components due to the differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase.

[0026] As used herein, the term "liquid chromatography" or "LC" refers to a method of selective delay of one or more components of a fluid solution as the fluid uniformly permeates through a column of subdivided material or through a capillary passage. The delay is due to the distribution of the mixture components between one or more stationary phases and the bulk fluid (i.e., mobile phase) as the fluid moves relative to the stationary phase(s). Examples of "liquid chromatography" include reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC), and high turbulence liquid chromatography (HTLC).

[0027] As used herein, the term "high performance liquid chromatography" or "HPLC" refers to liquid chromatography in which resolution is enhanced by forcing a mobile phase under pressure through a stationary phase, usually a densely packed column.

[0028] As used herein, the term "high turbulence liquid chromatography" or "HTLC" refers to a form of chromatography that uses the turbulent flow of the substance being measured through the column packing as the basis for making separations. HTLC has been used to prepare a sample containing two unnamed drugs before analysis by mass spectrometry. See, for example, Zimmer et al., J. Chromatogr. A 854: 23-35 (1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368, 5,795,469 and 5,772,874, which further explain HTLC. "Turbulent flow" is understood by one of ordinary skill in the art. When a fluid flows slowly and smoothly, the flow is called "laminar flow." For example, a fluid moving through an HPLC column at a low flow rate is laminar. In laminar flow, the motion of the fluid particles is orderly, with the particles moving generally in straight lines. At faster speeds, the inertia of the water overcomes the fluid friction and turbulence is generated. Fluid that is not in contact with an irregular boundary "outruns" fluid that is slowed by friction or deflected by an uneven surface. When a fluid flows turbulently, it flows in eddies and rotations (or vortices) that have a greater "drag force" than when the flow is laminar. Many references are available to help determine when a fluid flow is laminar or turbulent (e.g., Turbulent Flow Analysis: Measurement and Prediction, P.S. Bernard and J.M. Mallace, John Wiley & Sons, Inc., (2000); An Introduction to TurbulentFlow , Jean Mathieu and Julian Scott, Cambridge University Press (2001).

[0029] As used herein, the term "gas chromatography" or "GC" refers to a chromatography method in which a sample mixture is vaporized and injected into a carrier gas stream (as nitrogen or helium) which moves through a column containing a stationary phase composed of a liquid or particulate solid and separates into its component compounds based on their affinity for the stationary phase.

[0030] As used herein, the term "large particle column" or "extraction column" refers to a chromatography column comprising an average particle size greater than about 35 μm. As used herein, the term "about" refers to ±10%. In a preferred embodiment, the column comprises particles having a diameter of about 60 μm.

[0031] As used herein, the term "analytical column" refers to a chromatographic column having sufficient chromatographic plates to achieve separation of material in a sample, which material is eluted from the column sufficient to allow determination of the presence or amount of the analyte. Such columns are generally distinguished from "extraction columns" which have the general purpose of separating or extracting retained material from non-retained material to obtain a purified sample for further analysis. As used herein, the term "about" refers to ±10%. In a preferred embodiment, the analytical column comprises particles having a diameter of about 4 μm.

[0032] As used herein, the term "on-line" or "inline", such as used in "online automated manner" or "online extraction", refers to a process that is performed without operator intervention. In contrast, the term "off-line" as used herein refers to a process that requires manual intervention by an operator. Thus, if a sample is precipitated and the supernatant is then manually loaded into an autosampler, the precipitation and loading steps are off-line from subsequent steps. In various embodiments of the method, one or more steps may be performed in an online automated manner.

[0033] As used herein, the term "mass spectrometry" or "MS" refers to an analytical technique that identifies a compound by its mass. MS refers to a method of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m / z." MS techniques generally include (1) ionizing a compound to form a charged compound; and (2) detecting the molecular weight of the charged compound and calculating the mass-to-charge ratio. The compound can be ionized and detected by any suitable method. A "mass spectrometer" generally includes an ionizer and an ion detector. Generally, one or more target molecules are ionized and the ions are then introduced into a mass spectrometer where, due to a combination of magnetic and electric fields, the ions follow a path in space that depends on mass ("m") and charge ("z"). See, e.g., U.S. Pat. No. 6,204,500, entitled “Mass Spectrometry From Surfaces”; U.S. Pat. No. 6,107,623, entitled “Methods and Apparatus for Tandem Mass Spectrometry”; U.S. Pat. No. 6,268,144, entitled “DNA Diagnostics Based On Mass Spectrometry”; U.S. Pat. No. 6,124,137, entitled “Surface-Enhanced Photolabile Attachment And Release For Desorption And Detection Of Analytes”; Wright et al., Prostate Cancer and Prostatic Diseases 2:264-76 (1999); and Merchant and Weinberger, Electrophoresis 21:1164-67 (2000).

[0034] As used herein, the term "operating in negative ion mode" refers to those mass spectrometry methods in which negative ions are generated and detected. As used herein, the term "operating in positive ion mode" refers to those mass spectrometry methods in which positive ions are generated and detected.

[0035] As used herein, the term "ionization" or "ionization" refers to a process of producing analyte ions having a net charge equal to one or more electron units. Negative ions are those ions having a net negative charge of one or more electron units, while positive ions are those ions having a net positive charge of one or more electron units.

[0036] As used herein, the term "electron ionization" or "EI" refers to a process in which an analyte of interest in a gaseous or vapor phase interacts with a stream of electrons. The impact of the electrons with the analyte produces analyte ions, which can then be subjected to mass spectrometry techniques.

[0037] As used herein, the term "chemical ionization" or "CI" refers to a process in which a reactant gas (eg, ammonia) undergoes electron impact and analyte ions are formed by the interaction of the reactant gas ions with analyte molecules.

[0038] As used herein, the term "fast atom bombardment" or "FAB" refers to a method in which a beam of high energy atoms (usually Xe or Ar) strikes a non-volatile sample, which desorbs and ionizes the molecules contained in the sample. The test sample is dissolved in a viscous liquid matrix, such as glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether, 2-nitrophenyl octyl ether, sulfolane, diethanolamine, and triethanolamine. The selection of a suitable matrix for a compound or sample is an empirical process.

[0039] As used herein, the term "matrix-assisted laser desorption ionization" or "MALDI" refers to a method in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample through a variety of ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. For MALDI, the sample is mixed with an energy absorbing matrix, which facilitates the desorption of the analyte molecules.

[0040] As used herein, the term "surface enhanced laser desorption ionization" or "SELDI" refers to another method in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample through a variety of ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. For SELDI, the sample is generally combined with a surface that preferentially retains one or more target analytes. As in MALDI, this method can also use energy absorbing materials to promote ionization.

[0041] As used herein, the term "electrospray ionization" or "ESI" refers to a method in which a solution is passed along a short length of a capillary tube to which a high positive or negative potential is applied. The solution that reaches the end of the tube is vaporized (atomized) into a jet or spray of very small droplets of solution in a solvent vapor. The droplet mist flows through an evaporation chamber that is slightly heated to prevent condensation and evaporate the solvent. As the droplets become smaller, the electrical surface charge density increases until the natural repulsion between like polarity charges causes ions and neutral molecules to be released.

[0042] As used herein, the term "atmospheric pressure chemical ionization" or "APCI" refers to a mass spectrometry method similar to ESI; however, APCI produces ions by ion-molecule reactions that occur at atmospheric pressure in a plasma. The plasma is maintained by a discharge between a spray capillary and a counter electrode. The ions are then extracted into a mass analyzer, typically by using a set of differentially pumped skimmer stages. A countercurrent of dry and preheated N2 gas can be used to improve solvent removal. Gas phase ionization in APCI can be more efficient than in ESI when used to analyze less polar species.

[0043] As used herein, the term "atmospheric pressure photoionization" or "APPI" refers to a form of mass spectrometry in which the mechanism for the photoionization of a molecule M is photon absorption and electron ejection to form a molecular ion M+. Because the photon energy is generally just above the ionization potential, the molecular ion is not easily dissociated. In many cases, it is possible to analyze the sample without chromatography, thereby saving a lot of time and expense. In the presence of water vapor or a protic solvent, the molecular ion can extract H to form MH+. This situation tends to occur if M has a high proton affinity. This does not affect the quantitative accuracy because the sum of M+ and MH+ is constant. Drug compounds in protic solvents are usually observed as MH+, while non-polar compounds (such as naphthalene or testosterone) usually form M+. Robb, DB, Covey, TR and Bruins, AP (2000): See, e.g., Robb et al., Atmospheric pressure photoionization: An ionization method for liquid chromatography-Mass Spectrometry, Anal. Chem. 72(15): 3653-3659.

[0044] As used herein, the term "inductively coupled plasma" or "ICP" refers to a process in which a sample interacts with a partially ionized gas at a sufficiently high temperature so that most elements are atomized and ionized.

[0045] As used herein, the term "field desorption" refers to a method in which a non-volatile test sample is placed on an ionizing surface and a strong electric field is used to generate analyte ions.

[0046] As used herein, the term "desorption" refers to the removal of an analyte from a surface and / or the passage of an analyte into the gas phase.

[0047] As used herein, the term "limit of quantification," "limit of quantitation," or "LOQ" refers to the point at which measurements become quantitatively meaningful. The analyte response at this LOQ is identifiable, discrete, and reproducible with a precision of 20% and an accuracy of 80% to 120%.

[0048] As used herein, the term "limit of detection" or "LOD" is the point at which a measurement is greater than the uncertainty associated with it. The LOD is arbitrarily defined as 2 standard deviations (SD) from zero concentration.

[0049] As used herein, the "amount" of an analyte in a body fluid sample generally refers to an absolute value reflecting the amount of analyte detectable in a volume of body fluid. However, an amount also contemplates a relative amount compared to another amount of an analyte. For example, the amount of an analyte in a body fluid can be an amount that is greater or less than a control or normal level of the analyte normally present.

[0050] As used herein, the term "about" refers to the indicated value plus or minus 10% with reference to quantitative measurements that do not include measurement of the mass of the ion. Mass spectrometers can vary slightly in determining the mass of a given analyte. In the context of the mass of an ion or the mass / charge ratio of an ion, the term "about" refers to + / - 0.5 atomic mass units.

[0051] The above description of the invention is non-limiting and other features and advantages of the present invention will become apparent from the following detailed description of the invention and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 A typical chromatogram of a guanidine acetic acid standard / calibrator is shown.

[0053] Figure 2 A typical chromatogram of a creatine standard / calibrator is shown.

[0054] Figure 3 A typical chromatogram of a creatinine standard / calibrator is shown.

[0055] Figure 4 A guanidinoacetic acid calibration curve is shown, which includes 9 data points from 0.4 to 2500 mg / L.

[0056] Figure 5 A creatine calibration curve is shown, comprising 9 data points from 0.4 to 2500 mg / L.

[0057] Figure 6 A creatinine calibration curve is shown, comprising 9 data points from 0.8 to 5000 mg / L. DETAILED DESCRIPTION

[0058] Cerebral creatine deficiency syndrome (CCDS) - an inborn error of creatine metabolism - includes two disorders of creatine biosynthesis (guanidinoacetate methyltransferase [GAMT] deficiency and L-arginine:glycine amidinotransferase [AGAT or GATM] deficiency) as well as X-linked creatine transporter [SLC6A8] deficiency. Common to all three CCDS are intellectual disability and epilepsy. Most individuals with GAMT deficiency have behavioral impairments, which can include autistic behavior and self-mutilation; a significant proportion have pyramidal / extrapyramidal findings. Onset is between three months and three years of age. The phenotype of SLC6A8 deficiency in affected males ranges from mild intellectual disability and delayed speech development to severe intellectual disability, epilepsy, and behavioral disorders, with an age of diagnosis ranging from 2 to 66 years. Females heterozygous for SLC6A8 deficiency may have learning and behavioral problems. The biochemical diagnosis of CCDS relies on the measurement of guanidinoacetate (GAA), creatine, and creatinine in urine and plasma.

[0059] In certain embodiments, the methods provided herein are for detecting or determining the amount of guanidinoacetic acid (GAA), creatine, and creatinine, comprising: (a) purifying GAA, creatine, and creatinine in a sample; (b) ionizing GAA, creatine, and creatinine in the sample; (c) detecting or determining the amount of GAA, creatine, and creatinine ions (one or more) by mass spectrometry; wherein the amount of GAA, creatine, and creatinine ions (one or more) is related to the amount of GAA, creatine, and creatinine in the sample.

[0060] In certain embodiments, the method provided herein is for detecting or determining the amount of guanidinoacetic acid (GAA), comprising: (a) purifying GAA in a sample; (b) ionizing GAA in the sample; (c) detecting or determining the amount of GAA ions (one or more) by mass spectrometry; wherein the amount of GAA ions (one or more) is related to the amount of GAA in the sample.

[0061] In certain embodiments, the methods provided herein are for detecting or determining the amount of creatine, comprising: (a) purifying GAA, creatine, and creatinine in a sample; (b) ionizing creatine in the sample; (c) detecting or determining the amount of creatine ion (s) by mass spectrometry; wherein the amount of creatine ion (s) is related to the amount of creatine in the sample.

[0062] In certain embodiments, the methods provided herein are used to detect or determine the amount of creatinine, comprising: (a) purifying creatinine in a sample; (b) ionizing creatinine in the sample; (c) detecting or determining the amount of creatinine ion (one or more) by mass spectrometry; wherein the amount of creatinine ion (one or more) is related to the amount of creatinine in the sample.

[0063] In some embodiments, guanidinoacetic acid (GAA), creatine, and creatinine are underivatized prior to mass spectrometry.

[0064] In some embodiments, the sample is urine or serum or plasma. In a preferred embodiment, the sample is urine. In some embodiments, the sample is whole blood. In some embodiments, the sample is saliva.

[0065] In some embodiments, the purification provided herein comprises liquid chromatography. In some embodiments, liquid chromatography comprises high performance liquid chromatography (HPLC).

[0066] In some embodiments, the purification provided herein comprises solid phase extraction (SPE).

[0067] In some embodiments, ionization comprises electrospray ionization (ESI). In some embodiments, ionization comprises ionization in a positive mode. In some embodiments, ionization comprises ionization in a negative mode.

[0068] In some embodiments, the methods provided herein further comprise adding an internal standard. In some embodiments, the internal standard is isotopically labeled.

[0069] In certain embodiments, the limit of quantitation of the method is less than or equal to 1 mg / L. In some embodiments, the limit of quantitation of the method is less than or equal to 0.9 mg / L. In some embodiments, the limit of quantitation of the method is less than or equal to 0.8 mg / L. In some embodiments, the limit of quantitation of the method is less than or equal to 0.7 mg / L. In some embodiments, the limit of quantitation of the method is less than or equal to 0.6 mg / L. In some embodiments, the limit of quantitation of the method is less than or equal to 0.5 mg / L. In some embodiments, the limit of quantitation of the method is less than or equal to 0.4 mg / L. In some embodiments, the limit of quantitation of the method is less than or equal to 0.3 mg / L.

[0070] In certain embodiments, the detection limit of the method is less than or equal to 1 mg / L. In some embodiments, the detection limit of the method is less than or equal to 0.9 mg / L. In some embodiments, the detection limit of the method is less than or equal to 0.8 mg / L. In some embodiments, the detection limit of the method is less than or equal to 0.7 mg / L. In some embodiments, the detection limit of the method is less than or equal to 0.6 mg / L. In some embodiments, the detection limit of the method is less than or equal to 0.5 mg / L. In some embodiments, the detection limit of the method is less than or equal to 0.4 mg / L. In some embodiments, the detection limit of the method is less than or equal to 0.3 mg / L. In some embodiments, the detection limit of the method is less than or equal to 0.2 mg / L. In some embodiments, the detection limit of the method is less than or equal to 0.1 mg / L.

[0071] In some embodiments, a method may include adding a reagent to a sample in an amount sufficient to deproteinize the sample.

[0072] Suitable test samples include any test sample that may contain the target analyte. In some preferred embodiments, the sample is a biological sample; that is, a sample obtained from any biological source such as an animal, cell culture, organ culture, etc. In certain preferred embodiments, the sample is obtained from a mammal such as a dog, a cat, a horse, etc. Specific preferred mammals are primates, most preferably male or female humans. Specific preferred samples include blood, plasma, serum, hair, muscle, urine, saliva, tears, cerebrospinal fluid or other tissue samples. Such samples can be obtained, for example, from a patient, i.e., a living person (male or female), who presents himself in a clinical setting for diagnosis, prognosis or treatment of a disease or condition. The test sample is preferably obtained from a patient, such as serum.

[0073] Sample preparation for mass spectrometry

[0074] Methods that can be used to enrich the analyte relative to other components (e.g., proteins) in the sample include, for example, filtration, centrifugation, thin layer chromatography (TLC), electrophoresis including capillary electrophoresis, affinity separation including immunoaffinity separation, extraction methods including ethyl acetate extraction and methanol extraction, and the use of chaotropic agents, or any combination of the above.

[0075] Protein precipitation is a preferred method for preparing test samples. Such protein purification methods are well known in the art, for example, Polson et al., Journal of Chromatography B 785:263-275 (2003), describes protein precipitation techniques suitable for use in this method. Protein precipitation can be used to remove most of the protein from a sample while leaving the analyte in the supernatant. The sample can be centrifuged to separate the liquid supernatant from the precipitated protein. The resulting supernatant can then be applied to liquid chromatography and subsequent mass spectrometry analysis. In certain embodiments, the use of protein precipitation, such as acetonitrile protein precipitation, eliminates the need for high turbulence liquid chromatography (HTLC) or other on-line extraction prior to HPLC and mass spectrometry. Therefore, in such embodiments, the method involves (1) performing protein precipitation of a sample of interest; and (2) loading the supernatant directly onto an HPLC mass spectrometer without the use of on-line extraction or high turbulence liquid chromatography (HTLC).

[0076] In some preferred embodiments, HPLC can be used for analyte purification before mass spectrometry alone or in combination with one or more purification methods. In such an embodiment, the HPLC extraction column (extraction cartridge) of the captured analyte can be used to extract the sample, then eluted and chromatographic analysis on the second HPLC column or on an analytical HPLC column before ionization. Because the step involved in these chromatographic processes can be associated in an automatic manner, the demand for operator participation during analyte purification can be minimized. This feature can lead to the saving of time and cost, and eliminates the chance of operator error.

[0077] It is believed that turbulence, such as the turbulence provided by HTLC posts and methods, can improve mass transfer rate, which improves separation characteristics.HTLC posts are separated components by means of a high chromatographic flow rate through a packed column comprising hard particles. By adopting a high flow rate (such as 3-5mL / min), turbulence is produced in the post, which causes almost complete interaction between the stationary phase and the target analyte (one or more). The advantage of using HTLC posts is that the macromolecular accumulation associated with the biofluid matrix is ​​avoided, because high molecular weight species will not be retained under turbulent conditions. The HTLC method combined with multiple separations in a process reduces the demand for lengthy sample preparation, and runs at a significantly faster speed. Such methods have also achieved separation performance superior to laminar flow (HPLC) chromatography. HTLC allows direct injection of biological samples (blood plasma, urine, etc.). Because denatured proteins and other biological debris can rapidly block separation columns, it is difficult to achieve direct injection in the chromatography of traditional forms. HTLC also allows a very low sample volume less than 1mL, preferably less than 0.5mL, preferably less than 0.2mL, preferably 0.1mL.

[0078] Examples of HTLC applied to sample preparation prior to analysis by mass spectrometry have been described elsewhere. See, e.g., Zimmer et al., J. Chromatogr. A 854:23-35 (1999); see also, U.S. Pat. Nos. 5,968,367; 5,919,368; 5,795,469; and 5,772,874. In certain embodiments of the method, the sample is subjected to protein precipitation as described above prior to loading onto the HTLC column; in alternative preferred embodiments, the sample can be loaded directly onto the HTLC without protein precipitation. The HTLC extraction column is preferably a large particle column. In various embodiments, one of the multiple steps of the method can be performed in an online, automated manner. For example, in one embodiment, steps (i)-(v) are performed in an online, automated manner. In another aspect, the ionization and detection steps are performed online after steps (i)-(v).

[0079] Liquid chromatography (LC), including high performance liquid chromatography (HPLC), relies on relatively slow laminar flow techniques. Traditional HPLC analysis relies on column packing, in which the sample laminar flow through the column is the basis for separating the target analyte from the sample. Technicians will appreciate that separation in such a column is a diffusion process. HPLC has been successfully applied to the separation of compounds in biological samples, but before separation and subsequent analysis with a mass spectrometer (MS), a significant amount of sample preparation is required, which makes the technology labor-intensive. In addition, most HPLC systems do not fully utilize the full potential of mass spectrometers, and only allow an HPLC system to be connected to a single MS instrument, which results in tedious time requirements due to a large number of determinations.

[0080] Various methods for sample cleanup using HPLC prior to mass spectrometry analysis have been described. See, e.g., Taylor et al., Therapeutic Drug Monitoring 22:608-12 (2000); and Salm et al., Clin. Therapeutics 22 Supl. B:B71-B85 (2000).

[0081] Those skilled in the art can select HPLC instrument and column suitable for use with analyte.Chromatographic column usually includes medium (i.e. filling material) to promote the separation (i.e. fractionation) of chemical part. Medium can include tiny particles. Particles include binding surfaces that interact with various chemical parts to promote the separation of chemical parts. A suitable binding surface is a hydrophobic binding surface, such as an alkyl binding surface. The alkyl binding surface can include C-4, C-8, C-12 or C-18 binding alkyl groups, preferably C-18 binding groups.Chromatographic column includes an inlet for receiving a sample and an outlet for discharging an effluent (effluent) including the fractionation sample. In one embodiment, the sample (or pre-purified sample) is applied to the column at the inlet, eluted with a solvent or a solvent mixture, and discharged at the outlet. Different solvent modes can be selected to elute the target analyte (one or more). For example, liquid chromatography can be carried out using a gradient mode, an isocratic mode or a polytyptic (i.e. mixed) mode. During chromatography, separation of substances is achieved through variables such as choice of eluent (also called "mobile phase"), elution pattern, gradient conditions, temperature, and the like.

[0082] In some embodiments, analyte can be purified by applying sample to post under the following conditions, under which the target analyte is reversibly retained by the post filling material, while not retaining one or more other substances. In these embodiments, the first mobile phase condition can be used, wherein the target analyte is retained by the post, and once the non-retained material is washed through, the second mobile phase condition can be used subsequently to remove the retained material from the post. Alternatively, analyte can be purified by applying sample to post under mobile phase conditions, under which the target analyte is eluted at a different speed compared with one or more other substances. Such process can be relative to one or more other components of the sample, the amount of one or more target analytes enriched.

[0083] In a preferred embodiment, HTLC may be followed by HPLC on a hydrophobic column chromatography system. In certain preferred embodiments, the HPLC system based on TurboFlow Cyclone from Cohesive Technologies is used. The polymer column (particle size is 60 μm, column size is 50×1.0 mm, pore size is In a related preferred embodiment, Synergi from Phenomenex Inc. is used. Ether-linked phenyl analytical column with hydrophilic endcapping (particle size 4 μm, column size 150×2.0 mm, pore size In certain preferred embodiments, HTLC and HPLC are performed using HPLC grade ultrapure water and 100% methanol as mobile phases.

[0084] By carefully selecting valves and connector pipes, two or more chromatographic columns can be connected as needed so that substances are transferred from one chromatographic column to the next chromatographic column without any manual steps. In a preferred embodiment, the selection of valves and pipes is controlled by a computer preprogrammed to perform the necessary steps. Most preferably, the chromatographic system is also connected to a detector system, such as an MS system, in this online manner. Therefore, an operator can place a tray of samples in an autosampler and perform the remaining operations under computer control, which results in purification and analysis of all selected samples.

[0085] In certain preferred embodiments, the analyte or fragments thereof in the sample may be purified prior to ionization. In particularly preferred embodiments, the chromatography method is not gas chromatography.

[0086] Detection and quantification by mass spectrometry

[0087] In various embodiments, analyte or its fragment can be ionized by any method known to the technician. Mass spectrometry is performed using a mass spectrometer, which includes an ion source for ionizing fractionation samples and creating charged molecules for further analysis. For example, sample ionization can be performed by electron ionization, chemical ionization, electrospray ionization (ESI), photon ionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoionization (APPI), fast atom bombardment (FAB), liquid secondary ionization (LSI), matrix-assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray / plasma spray ionization, surface enhanced laser desorption ionization (SELDI), inductively coupled plasma (ICP) and particle beam ionization. The technician will understand that the selection of ionization method can be determined based on the type of analyte to be measured, the type of sample, the type of detector, the selection of positive and negative modes, etc. In a preferred embodiment, analyte or its fragment is ionized in positive or negative mode by heated electrospray ionization (HESI). In alternative embodiments, the analyte or fragments thereof are ionized by electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) in positive or negative mode.

[0088] After ionizing the sample, the positively or negatively charged ions thus produced can be analyzed to determine the mass-to-charge ratio. Suitable analyzers for determining the mass-to-charge ratio include quadrupole analyzers, ion trap analyzers, and time-of-flight analyzers. Several detection modes can be used to detect ions. For example, that is, the selected ions can be detected using a selective ion monitoring mode (SIM), or alternatively, the scanning mode of, for example, multiple reaction monitoring (MRM) or selected reaction monitoring (SRM) can be used to detect ions. Preferably, a quadrupole analyzer is used to determine the mass-to-charge ratio. For example, in a "quadrupole" or "quadrupole ion trap" instrument, the ions in the oscillating wireless radio frequency field are subjected to a force proportional to the DC potential, the amplitude of the RF signal, and the mass / charge ratio applied between the electrodes. Voltage and amplitude can be selected so that only ions with a specific mass / charge ratio travel the length of the quadrupole, and all other ions will be deflected. Therefore, a quadrupole instrument can serve as both a "mass filter" and a "mass detector" for the ions injected into the instrument.

[0089] The resolution of MS techniques is enhanced by using "tandem mass spectrometry" or "MS / MS". In this technique, precursor ions (also referred to as parent ions) generated from a target molecule can be filtered in an MS instrument, and the precursor ions are subsequently fragmented to produce one or more fragment ions (also referred to as daughter ions or product ions), which are then analyzed in a second MS process. By carefully selecting the precursor ions, only ions generated by certain analytes are transferred to the fragmentation chamber, where they collide with inert gas atoms to produce fragment ions. Because both precursor ions and fragment ions are produced in a reproducible manner under a given set of ionization / fragmentation conditions, the MS / MS technique can provide an extremely powerful analytical tool. For example, a combination of filtering / fragmentation can be used to eliminate interfering substances, and may be particularly useful in complex samples (e.g., biological samples).

[0090] Mass spectrometers usually provide users with ion scans; that is, the relative abundance of each ion with a specific mass / charge within a given range (e.g., 100 to 1000 amu). The result of the analyte assay by various methods known in the art, i.e., mass spectrum, can be related to the analyte amount in the original sample. For example, assuming that sampling and analysis parameters are carefully controlled, the relative abundance of a given ion can be compared with a table that converts the relative abundance to the absolute amount of the original molecule. Alternatively, molecular standards can be run with the sample, and a standard curve can be constructed based on the ions produced by those standards. Using such a standard curve, the relative abundance of a given ion can be converted to the absolute amount of the original molecule. In some preferred embodiments, an internal standard is used to generate a standard curve for calculating the number of analytes. The method for generating and using this standard curve is well known in the art, and a person of ordinary skill in the art can select an appropriate internal standard. For example, the isotope of the analyte can be used as an internal standard. For a person of ordinary skill in the art, many other methods for associating the amount of an ion with the amount of an original molecule are well known.

[0091] One or more steps of the method may be performed using automated machinery.In certain embodiments, one or more purification steps are performed online, and more preferably, all purification and mass spectrometry steps may be performed in an online manner.

[0092] In certain embodiments, such as MS / MS, where precursor ions are isolated for further fragmentation, collisionally activated dissociation is often used to generate fragment ions for further detection. In CAD, precursor ions gain energy by collision with an inert gas and are then fragmented by a process known as "unimolecular decomposition." Sufficient energy must be deposited in the precursor ions so that certain bonds in the ions are broken due to the increased vibrational energy.

[0093] In a specific preferred embodiment, MS / MS is used to detect and / or quantify analytes as follows. The sample is subjected to liquid chromatography, preferably HPLC, and the liquid solvent stream from the chromatographic column enters the heated nebulizer interface of the MS / MS analyzer, and the solvent / analyte mixture is converted into vapor in the heating tube of the interface. The analyte is ionized by the selected ion generator. Ions, such as precursor ions, pass through the hole of the instrument and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters that allow ions to be selected based on the mass-to-charge ratio (m / z) of ions (i.e., "precursor" and "fragment" ions). Quadrupole 2 (Q2) is a collision chamber in which ions are fragmented. The first quadrupole (Q1) of the mass spectrometer selects molecules with a mass / charge ratio of the analyte. Precursor ions with a mass / charge ratio of the correct analyte are allowed to be transferred to the collision chamber (Q2), while unwanted ions with any other mass / charge ratio collide with the side of the quadrupole and are eliminated. The precursor ions entering Q2 collide with neutral argon molecules and fragment. This method is called collision activated dissociation (CAD). The resulting fragment ions are passed to quadrupole 3 (Q3), where fragment ions of the analyte are selected, while other ions are eliminated.

[0094] The method may involve MS / MS performed in positive or negative ion mode. Using standard methods well known in the art, one of ordinary skill in the art can identify one or more fragment ions of a particular precursor ion of an analyte that can be used for selection in quadrupole 3 (Q3).

[0095] If the precursor ion of the analyte includes an alcohol or amine group, fragment ions are usually formed, which represent the dehydration or deamination of the precursor ion, respectively. In the case where the precursor ion includes an alcohol group, the fragment ions formed by dehydration are caused by the loss of one or more water molecules of the precursor ion (i.e., the difference in mass-to-charge ratio between the precursor ion and the fragment ion is about 18 for the loss of one water molecule; or about 36 for the loss of two water molecules, etc.). In the case where the precursor ion includes an amine group, the fragment ions formed by deamination are caused by the loss of one or more ammonia molecules (i.e., the difference in mass-to-charge ratio between the precursor ion and the fragment ion is about 17 for the loss of one ammonia molecule, or about 34 for the loss of two ammonia molecules, etc.). Similarly, precursor ions including one or more alcohol and amine groups usually form such fragment ions, which represent the loss of one or more water molecules and / or one or more ammonia molecules (i.e., the difference in mass-to-charge ratio between the precursor ion and the fragment ion is about 35 for the loss of one water molecule and the loss of one ammonia molecule). Generally, fragment ions representing the dehydration or deamination of the precursor ion are not specific fragment ions for specific analytes. Therefore, in a preferred embodiment of the invention, MS / MS is performed such that fragment ions of at least one analyte are detected which do not represent only the loss of one or more water molecules and / or the loss of one or more ammonia molecules from the precursor ion.

[0096] When ions collide with the detector, they generate electronic pulses, which are converted into digital signals. The acquired data are relayed to a computer, which plots the number of ions collected and the graph of time. The mass spectrum obtained is similar to the chromatogram generated in the traditional HPLC method. The area under the peak corresponding to the specific ion or the amplitude of such peak are measured, and the area or amplitude are associated with the amount of the target analyte. In some embodiments, the area under the curve or the peak amplitude of the fragment ion (one or more) and / or the precursor ion are measured to determine the amount of the analyte. As described above, the relative abundance of a given ion can be converted to the absolute amount of the original analyte using a calibration standard curve based on the peak of one or more ions of an internal molecular standard.

[0097] The following examples serve to illustrate the present invention. These examples are in no way intended to limit the scope of the method.

[0098] Example

[0099] Example 1 : Detection and Quantification of GAA, Creatine, and Creatinine by Mass Spectrometry

[0100] The samples were prepared by diluting urine 1:50 with ultrapure water. The minimum sample volume used was 100 μL. After dilution, the samples were spiked with an internal standard mixture (deuterium-labeled creatinine, C13-labeled guanidine acetic acid) and mixed. The diluted sample mixture was injected into an Agilent 1200 series HPLC system using a reverse phase column (reversed phase BDS250×4.6 mm). HPLC mobile phase: 0.1% formic acid / acetonitrile.

[0101] Analysis was performed using an Agilent 6410 triple quadrupole mass spectrometer by positive electrospray ionization. The run time was 10 minutes.

[0102] The calibration curves showed consistency in reproducibility and linearity. The method provided linear results in the range of 0.4-2500 mg / L for guanidinoacetic acid and creatine, and in the range of 0.8-5000 mg / L for creatinine. The lower limit of quantification was 0.3 mg / L for guanidinoacetic acid and creatinine, and 0.4 mg / L for creatine. The inter-assay coefficient of variation for guanidinoacetic acid at 25-500 mg / L was 8.1 to 4.7%, for creatine at 25-500 mg / L was 9.9 to 6.1%, and for creatinine at 120-2300 mg / L was 1.8 to 4.1%.

[0103] Table 1: Limit of quantitation and spike recovery studies

[0104]

[0105] Table 2: Intra-assay and inter-assay precision and accuracy of guanidine acetic acid

[0106]

[0107] The theoretical concentrations of spiked substances: low, medium and high are 25, 100 and 500 mg / L respectively.

[0108] Table 3: Creatine intra- and inter-assay precision and accuracy

[0109]

[0110] The theoretical concentrations of spiked substances: low, medium and high are 25, 100 and 500 mg / L respectively.

[0111] Table 4: Creatinine Intra-assay and Inter-assay Precision and Accuracy

[0112]

[0113] The theoretical concentrations of spiked substances were: low, medium and high were 125, 750 and 2500 mg / L respectively.

[0114] Precision / Sensitivity / Range

[0115]

[0116]

[0117] Measurement of these three analytes in urine allows for the biochemical diagnosis of CCDS. The ability to directly measure all three analytes in urine with very simple sample preparation methods is an improvement over previous methods involving derivatization and indirect calculations.

[0118] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein are incorporated herein by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all material and information from such articles, patents, patent applications, or other physical and electronic documents.

[0119] The methods illustratively described herein may be suitably practiced in the absence of any one or more elements, one or more limitations not expressly disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. should be understood broadly and non-restrictively. In addition, the terms and expressions used herein have been used as descriptive terms rather than restrictive terms, and there is no intention to use such terms and expressions that exclude any equivalent forms or portions thereof of the features shown and described. It should be recognized that various changes are possible within the scope of the claimed invention. Therefore, it should be understood that although the present invention is specifically disclosed by preferred embodiments and optional features, changes and variations of the present invention specifically implemented therein disclosed herein may be adopted by those of ordinary skill in the art, and such changes and variations are considered to be within the scope of the present invention.

[0120] The invention has been described broadly and generally herein. Each of the narrower classes and subclasses falling within the general disclosure also form part of the described methods. This includes the general description of the methods, the proviso or negative limitation being that any subject matter is removed from the class, whether or not the excluded material is expressly described herein.

[0121] Other embodiments are within the following claims.In addition, where features or aspects of the methods are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

1. A method for detecting or determining the amount of guanidinoacetic acid (GAA) in a sample, the method comprising: (a) purifying GAA in the sample; (b) ionizing GAA in the sample to produce one or more ions of GAA; (c) detecting the ion(s) from step (b) by mass spectrometry; The amount of the GAA ion(s) is related to the amount of GAA in the sample.

2. The method of claim 1, wherein the purification comprises liquid chromatography, optionally wherein the liquid chromatography comprises high performance liquid chromatography.

3. The method of claim 1, wherein the GAA is underivatized. The method of claim 1 , wherein the ionization comprises electrospray ionization (ESI). The method of claim 1 , wherein the ionizing comprises ionizing in a positive mode.

6. The method of claim 1, further comprising adding an internal standard, optionally wherein the internal standard is isotopically labeled.

7. The method of claim 1, wherein the sample is urine or serum.

8. The method of claim 1, wherein the limit of quantitation of the method is less than or equal to 0.3 mg / L.

9. The method of claim 1, wherein the ionizing comprises generating GAA precursor ions having a mass / charge ratio of 118.1±0.

5.

10. The method of claim 1, wherein the ionizing comprises generating one or more fragment ions having a mass / charge ratio of 72.1 ± 0.

5.

11. The method of claim 1, wherein the ionizing comprises generating a GAA precursor ion having a mass / charge ratio of 118.1±0.5 and one or more fragment ions having a mass / charge ratio of 72.1±0.5.

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