Simple quantitative analysis using substrates preloaded with standards

Abstract

Dried blood spots (DBS) collected on substrate materials are used to quantify amino acids, acylcarnitines, organic acids, and numerous other small molecules. One of their main application areas is newborn screening. To properly quantify the target analyte small molecule in DBS, a stable isotope-labeled internal standard (typically a deuterium- or carbon-13-labeled version of the desired target analyte to be quantified) is preloaded onto the substrate before blood collection. For example, to quantify phenylalanine, phenylalanine, which has six carbon-13 atoms, is used as F.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a method and a microfluidic device for determining the amount of a target analyte, e.g., a metabolite including peptides, proteins, heavy metals, toxins, drugs and their metabolites, or other molecules, in a biological sample by using a labeled internal standard, in particular a stable isotope labeled internal standard pre-loaded onto a substrate to which the biological sample is to be loaded, for example, for the diagnosis of various metabolic disorders including newborn screening, cancer, diabetes, cardiovascular disease, drug monitoring, etc.

[0002] background Metabolomics Metabolomics offers a promising answer to this diagnostic challenge. The metabolome is the complete set of metabolites in a given system, whereas the lipidome is the complete set of lipid signatures; both are major molecular components of living systems. 1、2 Metabolites and lipids are both inputs and outputs of cellular and physiological processes, and therefore their levels are exquisitely sensitive to a wide range of perturbations linked to disease, genetic alterations, and environmental conditions. 3、4 Metabolomics is the precise and accurate measurement of metabolites in a given biological medium, and metabolomics and lipidomics have applications in many different areas; for example, cancer research. 5、6 , diabetes 7 , gut microbiome 8、9 , and newborn screening 10、11 In cancer, tumor cells primarily reprogram their metabolism to meet the demands of three main functions: bioenergetics (central carbon metabolism), biosynthesis (production of biomaterials such as nucleotides, amino acids, and lipids), and redox balance (the chemistry of the metabolic redox homeostasis state). 12、13Together, these processes support cancer cell maintenance and proliferation, tumor initiation, growth, and progression, and are considered the central dogma of cancer metabolism, allowing cancer cells to adapt to changing cellular and physiological conditions in the context of growth-related signals / stresses. Insights from genomics, epigenomics, proteomics, and transcriptomics have tremendously expanded our understanding of cancer and revealed novel, potentially targetable, tumor vulnerabilities. 14 Altered metabolism is recognized as a hallmark of cancer and offers valuable opportunities for cancer diagnosis, prognosis, and therapy. 15-18 Notably, there has been an ever-increasing clinical success of antimetabolites to treat cancer due to the increased metabolic demands of cancer cells on nucleotide biosynthesis, glycolysis, the TCA cycle, serine biosynthesis and the folate cycle, the methionine cycle, and fatty acid synthesis; however, their targets and associated pathways target only a few of the many possible metabolic dependencies altered to support cancer cell proliferation and tumor progression. 12、13、19 .

[0003] Inborn errors of metabolism Inborn errors of metabolism (IEM) are a group of diverse inherited metabolic disorders, each caused by an error in a single genetic code that results in insufficient or absent enzymatic activity required for intermediary metabolism.

[0004] Delays in the treatment of metabolic disorders in IEM can result in a variety of adverse events, including moderate to severe morbidity, such as neuropsychological dysfunction and mental retardation, and death (due to impaired metabolism and the resulting lack or accumulation of certain metabolic intermediates). Early diagnosis is therefore important for timely correction of symptoms through dietary or drug intervention before clinical symptoms become apparent in affected newborns. Each year, approximately 4 million infants in the United States are routinely screened for potential metabolic disorders through newborn screening.

[0005] Newborn screening is a set of tests for the earliest recognition and management of conditions that may affect a child's long-term health or survival in order to prevent associated severe clinical symptoms, disability, and death. Newborn screening for metabolic disorders of IEM is performed by collecting a few drops of the infant's blood on a special type of substrate, such as a cotton disc. The blood on the substrate is dried and then sent to the state health department. The dried blood spot (DBS) is extracted and analyzed by a laboratory for the quantification of numerous metabolites and small molecules, such as amino acids, free carnitine, acyl-carnitines, and organic acids, to identify infants at high risk for medical conditions.

[0006] For the quantification of metabolites and other small molecules in biological samples, which are target analytes for IEM diagnosis using newborn screening, internal standards (IS) are used as references. Internal standards are typically stable isotope-labeled versions of the desired target analyte, with the stable isotope typically being deuterium or carbon-13. For example, to quantify phenylalanine in a test sample such as blood, phenylalanine labeled with six carbon-13 atoms is used.

[0007] Quantitation of target analytes by adding an internal standard to a dried blood spot (DBS) sample can be complicated. Currently, the internal standard is added when an extraction solvent is added to a substrate loaded with the sample (blood). However, this use of an internal standard does not reflect the extraction efficiency of the target analyte from the substrate, nor does it reflect any potential degradation that may occur in the sample prior to analysis. In addition, loaded samples such as blood can vary in size, volume, and hematocrit, making accurate quantitation difficult.

[0008] Summary of the Invention It is an object of the present invention to provide an improved method and device for overcoming all or some of the above-mentioned drawbacks and problems associated with the state of the art.

[0009] In this disclosure, the feasibility of a new approach using a preloaded internal standard as a reference for quantifying a target molecule has been demonstrated. Based on the findings reported herein, one embodiment of the present invention relates to a method for determining the amount of at least one target molecule in a liquid test sample. The method involves delivering a predetermined volume of the liquid test sample to a substrate preloaded with a predetermined amount of an internal standard molecule, such that the predetermined volume of the liquid test sample contacts the internal standard molecule. The liquid test sample may be dried after delivery to the substrate. The substrate, or a portion thereof, is incubated with the liquid test sample in an extraction solvent to produce a supernatant containing at least one target molecule and the internal standard molecule. The method also involves detecting the at least one target molecule and the internal standard molecule in the supernatant; and quantifying the amount of the at least one target molecule in the liquid test sample based on the amount of the at least one target molecule and the amount of the internal standard molecule detected in the supernatant. In certain embodiments, and for the extraction experiments described below, the substrate (e.g., a cotton disk) is part of a volume-based microsampling device, such as a quantitative dried blood sample (qDBS) card.

[0010] Embodiments also relate to methods and / or microfluidic devices for determining the amount of at least one target molecule in a test sample by using a known amount of an internal standard molecule. The embodiments can be used to screen for or diagnose a variety of medical conditions, including diagnosing metabolic disorders. In exemplary embodiments, the internal standard is a stable isotope-labeled internal standard molecule corresponding to at least one stable isotope-labeled target molecule, and the stable isotope for labeling the internal standard molecule is: 2 H, 13 C. 15 N, 18 O. 34 S, or any combination thereof, and optionally, the stable isotope-labeled internal standard molecule may be in the amount range of 1 fmol to 5 mmol. Those skilled in the art will recognize that, depending on the target molecule, unlabeled internal standards may also be implemented. Unlabeled internal standards are believed to be particularly useful for analytical methods (e.g., ELISA, chromatography, etc.) other than mass spectrometry.

[0011] Additionally, test samples for which the present methods and microfluidic devices can be used are selected from biological fluids, such as excreted fluids (such as urine or sweat), secreted fluids (such as saliva, tears, milk or bile), fluids obtained from within a subject (such as blood, plasma, serum or cerebrospinal fluid), or fluids produced as a result of a pathological process (such as blister or cyst fluid), food samples, plant samples, environmental samples, and the like.

[0012] In certain embodiments, (i) loading a known amount of a solution containing at least one stable isotope-labeled internal standard molecule onto a substrate; (ii) drying the substrate for a predetermined period of time, e.g., 2 to 48 hours; (iii) adding a known volume of liquid test sample onto a substrate preloaded with at least one stable isotope-labeled internal standard molecule; (iv) thoroughly drying the substrate; (v) placing the substrate in a container; (vi) adding an extraction solvent and sonicating the vessel; (vii) incubating the tube containing the substrate and extraction solvent at room temperature for 30 minutes; and (viii) collecting the supernatant; and performing mass spectrometry with the supernatant, with or without chromatography, optionally derivatizing the target analytes before performing mass spectrometry. A method is disclosed that includes:

[0013] The method may involve delivering a predetermined volume of at least one liquid test sample to a first segment of a substrate, and the method may further include delivering a predetermined volume of at least one control sample of at least one target molecule to a second segment of the substrate; incubating the first segment or a portion thereof with the at least one liquid test sample in a first extraction solvent, and incubating the second segment or a portion thereof with the at least one control sample in a second extraction solvent, wherein the first extraction solvent and the second extraction solvent are the same or different.

[0014] In an alternative embodiment, the method may further include delivering a predetermined volume of at least one control sample of at least one target molecule to a second substrate pre-loaded with a predetermined amount of the internal standard molecule, such that the at least one control sample contacts the internal standard molecule, wherein the at least one control sample contains a known concentration of the at least one target molecule; optionally drying the at least one control sample on the second substrate; incubating the second substrate or a portion thereof with the at least one control sample in an extraction solvent to produce a supernatant for the at least one control sample containing the at least one target molecule and the internal standard molecule; detecting the at least one target molecule and the internal standard molecule in the supernatant of the control sample; creating a calibration curve based on the detection of the at least one target molecule and the internal standard molecule in the supernatant of the control sample; and quantifying the amount of the at least one target molecule in the test sample based on the amount of the at least one target molecule and the amount of the internal standard molecule detected in the supernatant of the test sample and correlation with the calibration curve.

[0015] Additionally, a screening kit is provided for determining the amount of at least one target molecule in a test sample by using an internal standard molecule, such as a stable isotope-labeled internal standard molecule of known amount, where the stable isotope-labeled internal standard molecule is a molecule corresponding to the at least one stable isotope-labeled target molecule. According to this embodiment, the kit comprises the following components: (i) at least one substrate; (ii) at least one stable isotope-labeled internal standard molecule, which may be in concentrated liquid or dry powder form; and / or (iii) at least one substrate preloaded with a known amount of at least one stable isotope-labeled internal standard molecule; and the kit optionally includes at least one extraction solvent.

[0016] In another embodiment, the kit comprises at least one substrate and at least one stable isotope-labeled internal standard molecule, and further comprises a loading means for loading the at least one stable isotope-labeled internal standard molecule onto the at least one substrate. Examples of the loading means include, but are not limited to, a syringe, a pipette, a container with a dropper nozzle, etc.

[0017] The screening kits are: - preparing a reconstituted solution comprising at least one stable isotope-labeled internal standard molecule; - preparing a working solution comprising at least one reconstituted stable isotope-labeled internal standard molecule together with an extraction solvent; and / or - the process of using the kit; (i) loading onto the substrate a known amount of working solution containing at least one stable isotope-labeled internal standard molecule; (ii) allowing the substrate to dry for a predetermined period of time; (iii) adding a known volume of the test sample onto a substrate preloaded with at least one stable isotope-labeled internal standard molecule; (iv) Allow the substrate to dry thoroughly; (v) placing the substrate into a container (e.g., a tube or other suitable container); (vi) adding an extraction solvent and sonicating the vessel; (vii) incubating the vessel containing the substrate and extraction solvent for a sufficient time and temperature (e.g., 30 minutes at room temperature); (viii) Collect the supernatant; and perform mass spectrometry using the supernatant. The instruction manual may further include:

[0018] According to another embodiment, a microfluidic device is provided that includes an inlet for application of a liquid test sample; a metering channel disposed in fluid communication with the inlet to receive at least a portion of the liquid test sample from the inlet, the metering channel having a predetermined volume; and a substrate configured to receive the metered volume of the liquid test sample from the metering channel, the substrate containing a predetermined amount of an internal standard molecule. In certain aspects, the substrate is composed of a liquid-absorbent material. In certain examples, the substrate is directed to a disk made of cotton, paper, fiber cloth, polymer resin, or a combination thereof. In other examples, the internal standard molecule optionally includes a label, and the label is optionally a stable isotope. Stable isotopes may include, but are not limited to, 2H, 13C, 15N, 18O, 34S, or any combination thereof. In certain examples, when a stable, labeled isotope is implemented as the internal standard molecule, it may be provided in an amount ranging from 0.1 to 5 mmol.

[0019] In certain embodiments, the stable isotope-labeled internal standard molecule is a stable isotope-labeled amino acid, free carnitine, acylcarnitine, and any combination thereof, such as 2H4-alanine, 13C6-,15N4-arginine, 13C4-,15N2-asparagine, 2H3-aspartic acid, 13C3-cysteine, 2H3-glutamic acid, 13C5-,15N2-glutamine, 13C-,15N-glycine, 13C6-,15N3-histidine, 13C6-,15N-isoleucine, 2H3-leucine, 13C6-,15N2-lysine, 2H3-methionine, 13C6-phenylalanine, 13C5-,15N-proline, 13C3-serine, 13C4 -threonine, 13C11-,15N2-tryptophan, 13C6-tyrosine, 2H8-valine, 2H2-citrulline, 2H6-ornithine, 2H9-carnitine, 2H3-acetylcarnitine, 2H3-propionylcarnitine, 2H3-butyrylcarnitine, 2H9-isovalerylcarnitine, 2H3-glutarylcarnitine, 2H3-hexanoylcarnitine, 2H3-octanoylcarnitine, 2H3-decanoylcarnitine, 2H3-lauroylcarnitine, 2H9-myristoylcarnitine, 2H3-palmitoylcarnitine, and / or 2H3-stearoylcarnitine, and / or any combination thereof. In a specific example, the stable isotope-labeled internal standard molecule is 13C6-phenylalanine.

[0020] In both the method and microfluidic device embodiments, the substrate may be made of any material capable of absorbing liquid, including cotton, paper, fiber cloth, polymer resins, etc.; extraction solvents include C1-3 straight or branched chain monoalcohols, particularly methanol, acetonitrile, acetone, chloroform, methyl-t-butyl ether, etc.

[0021] Furthermore, in certain embodiments, target molecules for detection and analysis according to the teachings and embodiments herein include free carnitine, acylcarnitines, and any combination thereof in a test sample, such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, citrulline, ornithine, free In particular, the target molecule is phenylalanine, which may include, but is not limited to, carnitine, acetylcarnitine, propionylcarnitine, butyrylcarnitine, isovalerylcarnitine, glutarylcarnitine, hexanoylcarnitine, octanoylcarnitine, decanoylcarnitine, lauroylcarnitine, myristoylcarnitine, palmitoylcarnitine, and / or stearoylcarnitine, and / or any combination thereof.

[0022] Furthermore, the stable isotope-labeled internal standard molecule may include a stable isotope-labeled amino acid, free carnitine, acylcarnitine, and any combination thereof, for example, 2 H4-alanine, 13 C6-, 15 N4-arginine, 13 C4-, 15 N2-asparagine, 2 H3-aspartic acid, 13 C3-cysteine, 2 H3-glutamic acid, 13 C5-, 15 N2-glutamine, 13 C-, 15 N-glycine, 13 C6-, 15 N3-histidine, 13 C6-, 15 N-isoleucine, 2 H3-leucine, 13 C6-, 15 N2-lysine, 2 H3-methionine, 13 C6-phenylalanine,13 C5-, 15 N-proline, 13 C3-serine, 13 C4-threonine, 13 C11-, 15 N2-tryptophan, 13 C6-tyrosine, 2 H8-valine, 2 H2-citrulline, 2 H6-ornithine, 2 H9-carnitine, 2 H3-acetylcarnitine, 2 H3-propionylcarnitine, 2 H3-butyrylcarnitine, 2 H9-Isovalerylcarnitine, 2 H3-glutarylcarnitine, 2 H3-hexanoylcarnitine, 2 H3-octanoylcarnitine, 2 H3-decanoylcarnitine, 2 H3-lauroylcarnitine, 2 H9-myristoylcarnitine, 2 H3-palmitoylcarnitine, and / or 2 In particular, stable isotope-labeled internal standard molecules may include, but are not limited to, H3-stearoylcarnitine, and / or any combination thereof. 13 C6-phenylalanine.

[0023] These and other embodiments are further described below. [Brief explanation of the drawings]

[0024] The detailed description of the preferred embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the drawings show certain, but not all, preferred embodiments. It should be understood that embodiments of the present invention are not limited to the precise arrangements and instrumentalities shown in the drawings. [Figure 1]Certain types of extraction methods are shown: Type A: aqueous reconstitution and dilution; Type B: extraction with organic solvent. Extraction of small molecules from dried blood spots (DBS) is performed following a Type B (bottom) step in which the DBS is extruded, transferred to a vial, and organic solvent is added. An internal standard is typically added just before the organic solvent is added. [Figure 2] Figure 1 shows the recovery of small molecules extracted from DBS cards. If there are no losses during extraction, 100% of the small molecule of interest is recovered from the spot (top workflow). However, there is always loss of analyte in the process, which results in lower recovery (middle workflow). By adding an internal standard before extraction, the improved method allows for the achievement of recoveries close to 100% (bottom workflow). The internal standard is represented as A*. [Figure 3] The stable isotope blood spot (SIBS) process for amino acid quantification is shown. A. The qDBS card is modified to include an internal standard (IS). A specific volume of blood can then be added to mix with the IS, allowing for easier and improved extraction and quantification. B. A representative amino acid profile is shown in the figure below. [Figure 4] Spectra (representing the change in response) for phenylalanine and phenylalanine 13C6 at various volumes added to the card are shown. [Figure 5] 1 shows a schematic diagram illustrating and explaining an embodiment of volume-based microsampling. [Figure 6] Schematic representations of various options for utilizing a control sample are shown. Figure 6A relates to a single substrate in which a liquid test sample is delivered to a first segment of the substrate and a control sample is delivered to a second segment of the substrate. Figures 6B and 6C illustrate a second option in which the liquid test sample is delivered to the first substrate (Figure 6B) and the control sample is delivered to the second substrate (Figure 6C). The substrate onto which the sample is delivered is cut, and the sample is then processed to produce a supernatant for analysis.

[0025] Table 1 shows the error observed when the internal standard was added after blood collection compared to when the internal standard was added before blood collection (Before).

[0026] Table 2 provides examples of volume-based metering or microsampling solutions.

[0027] Detailed Description Disclosed herein are processes involving preloading a substrate, typically used to collect and quantify components of a biological sample (e.g., a quantitative dried blood spot (qDBS) card), with a quantity of a desired internal standard for quantification. In one particular example, the substrate is a cotton disk to which a precise volume of sample is applied, and the substrate already contains a known amount of the desired internal standard for quantification. Preloading the internal standard onto the substrate facilitates immediate mixing of the internal standard with the blood sample prior to extraction, allowing for extraction recoveries approaching 100%. Providing a substrate preloaded with the internal standard described herein also reduces lab errors by eliminating the need to prepare the internal standard, providing population screening methods and devices with improved inter-laboratory reproducibility.

[0028] Mass spectrometry is an ideal standard for the quantification of many biochemicals due to its sensitivity, specificity, and selectivity. The use of stable isotope internal standards is essential in quantitative mass spectrometry. Most internal standards used are stable isotope versions of the target compound, such as tryptophan with three deuterium atoms as a standard for tryptophan. The use of stable isotope internal standards helps reduce errors associated with extraction and ionization efficiency in different individuals and different biological matrices. In a typical experiment, a volumetric aliquot of a biological fluid (e.g., plasma) is transferred to a tube, and an internal standard is added at a precise volume and concentration. The precise volume is important for accurate quantification across a set of samples. After mixing, the sample is extracted according to a desired process that optimizes the recovery of the target analyte. Because the internal standard was added at the beginning of the process, any testing errors that occur after this process are easily explained by the presence of an internal reference standard.

[0029] To this end, the present disclosure provides methods and microfluidic devices that use preloaded internal standards (to be used as more reliable standards) for the quantification of target molecules (e.g., amino acids, metabolites, lipids, peptides, proteins, heavy metals, toxins, drugs, or types of analytes) in obtained biological samples. Embodiments of the methods, microfluidic devices, and kits can be used to screen or diagnose a variety of medical conditions, particularly for metabolic disorder screening, such as newborn screening.

[0030] Additionally, the embodiments described herein can also be applied to new fields of use, including pharmaceutical quantification, illicit drug quantification, and home health testing. Furthermore, they can also be utilized in clinical trials conducted by pharmaceutical companies, which can help save on blood transportation costs and standardize blood collection procedures at multiple sites. While the implementation of the methods taught herein has been successfully demonstrated using small molecules, they can also be readily applied to peptide quantification, and potentially protein analysis.

[0031] As described in more detail in the Examples section below, quantification of amino acids using stable isotope-labeled analogs was demonstrated to demonstrate the feasibility of the new approach of using preloaded stable isotope-labeled internal standards as a reference for quantifying target molecules. In particular, phenylalanine 13C6 was used as a model compound. The observed errors were compared when the internal standard was added after blood collection versus before blood collection.

[0032] For these examples, a known volume of sample, such as blood, was collected on a substrate, such as cotton paper, in a qDBS card, preloaded with a known amount of internal standard, and allowed to dry to yield a dried blood spot (DBS). For extraction and quantification of amino acids from DBS, a cotton disk of the DBS sample was punched from the card and extracted with a solvent, such as methanol.

[0033] In certain embodiments, (ix) loading a known amount of a solution containing at least one stable isotope-labeled internal standard molecule onto the substrate; (x) drying the substrate for a predetermined period of time, for example, 2 to 48 hours; (xi) adding a known volume of a liquid test sample onto a substrate preloaded with at least one stable isotope-labeled internal standard molecule; (xii) thoroughly drying the substrate; (xiii) placing the substrate in a container; (xiv) adding an extraction solvent and sonicating the vessel; (xv) incubating the tube containing the substrate and extraction solvent at room temperature for 30 minutes; and (xvi) collecting the supernatant; and performing mass spectrometry with the supernatant, with or without chromatography, optionally derivatizing the target analytes before performing mass spectrometry. A method is disclosed that includes:

[0034] The method may involve delivering a predetermined volume of at least one liquid test sample to a first segment of a substrate, and the method may further include delivering a predetermined volume of at least one control sample of at least one target molecule to a second segment of the substrate; incubating the first segment, or a portion thereof, with the at least one liquid test sample in a first extraction solvent, and incubating the second segment, or a portion thereof, with the at least one control sample in a second extraction solvent, wherein the first extraction solvent and the second extraction solvent are the same or different. In a more specific embodiment, the substrate includes two or more segments to which two or more control samples of predetermined volumes are delivered. The two or more control samples may include a first volume of at least one target molecule at a first predetermined concentration and a second volume of at least one target molecule at a second predetermined concentration. The first and second volumes of at least one target molecule at the first and second predetermined concentrations may be the same or different.

[0035] In an alternative embodiment, the method may further include delivering a predetermined volume of at least one control sample of at least one target molecule to a second substrate pre-loaded with a predetermined amount of the internal standard molecule, such that the at least one control sample contacts the internal standard molecule, the at least one control sample comprising a known concentration of the at least one target molecule; optionally drying the at least one control sample on the second substrate; incubating the second substrate or a portion thereof with the at least one control sample in an extraction solvent to produce a supernatant for the at least one control sample comprising the at least one target molecule and the internal standard molecule; detecting the at least one target molecule and the internal standard molecule in the supernatant of the control sample; creating a calibration curve based on the detection of the at least one target molecule and the internal standard molecule in the supernatant of the control sample; and quantifying the amount of the at least one target molecule in the test sample based on the amount of the at least one target molecule and the amount of the internal standard molecule detected in the supernatant of the test sample and correlation with the calibration curve. In a more specific embodiment, the method may involve delivering at least one control sample to a third substrate, and the concentration of at least one target molecule in the at least one control sample delivered to the third substrate is different from the concentration of at least one target molecule in the at least one control sample delivered to the second substrate. As described above for the second substrate, the at least one control sample delivered to the third substrate can be incubated in an extraction solvent to produce a supernatant for the at least one control sample containing at least one target molecule and an internal standard molecule; the at least one target molecule and the internal standard molecule on the third substrate are detected in the supernatant of the control sample from the third substrate. Creating a calibration curve can be based on the detection of at least one target molecule and the internal standard molecule in the supernatant of the control sample from the second substrate and at least one target molecule and the internal standard molecule in the supernatant of the control sample from the third substrate.

[0036] FIG. 6 provides a schematic diagram of a method involving the running of a control sample. As shown in FIG. 6A, a substrate 600 has two distinct segments 601 and 602. A test sample 605 (e.g., blood) is delivered to segment 601, and a control sample 607 is delivered to segment 609. After delivery, the test sample 605 on the first segment 601 of the substrate 600 and the control sample 607 on the second segment 602 of the substrate are extracted as described herein. The entire sample-containing segment or a portion thereof can be used for extraction. FIG. 6B shows a substrate 610 onto which the test sample 605 is delivered, and FIG. 6C shows a substrate 611 onto which the control sample is delivered. The test sample 605 on substrate 610 and the control sample on substrate 611 are extracted as described herein. It should be noted that if more than one substrate is implemented, or if multiple segmented cards are implemented, the substrates can be housed in the same microfluidic device (e.g., a qDBS card) with separate channels delivering each sample to a different card or segment.

[0037] In one embodiment, a quantification process using stable isotope blood spots (SIBS) on a substrate was presented, and the resulting quantitative analysis spectra of the SIBS showed the presence of stable isotope-labeled amino acids along with their endogenous counterparts (Figure 3).

[0038] In another embodiment, phenylalanine 13 C6 was used to quantify the amino acid phenylalanine, an amino acid whose accumulation is associated with the development of the rare genetic metabolic disorder phenylketonuria (PKU). PKU is screened for by newborn screening tests and can be reliably diagnosed by detecting phenylalanine levels in the blood. Here, various volumes of phenylalanine were measured before adding blood to a cotton disc. 13 C6 was preloaded as an internal standard for phenylalanine. 13 When the volume and therefore the amount of C6 phenylalanine changes, 13The ratio of the relative abundance of phenylalanine to C6 is shown to have changed.

[0039] In another embodiment, the errors observed when the internal standard was added after blood collection and when the internal standard was added before blood collection are shown in Table 1. As a result, the standard deviation and relative standard deviation (RSD) of the ratios were smaller when the internal standard was added before blood collection than when the internal standard was added after blood collection.

[0040] The data provided herein demonstrate that the discovered approach of preloading with stable isotope-labeled internal standards is well suited for the quantification of a wide range of analytes.

[0041] definition Preferred materials and methods are described herein; any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In describing and claiming the present invention, the following terminology will be used. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0042] The articles "a," "an," "the," and the like are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article, unless specifically stated otherwise. By way of example, "an" means one element or more than one element. Unless otherwise indicated, "or" includes "and." By way of example, "A, B, or C" means A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, and A, B, and C in combination, unless otherwise specified.

[0043] It should be noted that the terms "first," "second," and the like, as used herein, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another unless otherwise specifically stated herein. The terms "a" and "an" do not denote a quantity limit, but rather indicate the presence of at least one of the referenced item. The modifier "about," when used in connection with a quantity, is inclusive of the stated value and has the meaning dictated by the context. The term "about" generally refers to a range of numbers that one of ordinary skill in the art would consider equivalent to the stated value (i.e., having the same function or result). In certain embodiments, the term "about" includes not only the stated numerical value but also values ​​that are ±30% of the stated numerical value. For example, about 40 degrees includes not only 40 degrees, but also 36 degrees and 44 degrees, and all values ​​therebetween. In further specific embodiments, the term "about" includes not only the stated numerical value but also values ​​that are ±25%. In many instances, the term "about" may include numbers that are rounded to the nearest significant figure. Furthermore, to the extent the terms "including," "including," "has," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to include in a manner similar to "comprising."

[0044] Any and all examples provided herein with respect to a particular embodiment, or the use of exemplary language (e.g., "such as"), are intended merely to better describe the disclosure and do not limit the scope of the otherwise claimed disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0045] As used herein, "amino acid" refers to an organic compound containing both an amino functional group and a carboxyl functional group. There are hundreds of amino acids in nature, which can be classified as alpha- (α-), beta- (β-), gamma- (γ-), or delta- (δ-) amino acids according to their structure based on the location of the core functional group. Alpha- (α-) amino acids are amino acids with the amino and carboxyl functional groups attached to the same carbon atom (α-carbon) and naturally occur in peptides and proteins, including the secondary amines proline and hydroxyproline. Due to this structure, all α-amino acids possess chirality, except for achiral glycine. In this disclosure, amino acids refer to all L / D isomers and S / R enantiomers. Beta- (β-), gamma- (γ-), or delta- (δ-) amino acids are non-proteinogenic amino acids in which the amino group is located on the carbon atom β, γ, or δ relative to the carboxyl group.

[0046] Proteinogenic α-amino acids, depending on the structure and ionization of their side chains, are classified as follows: nonpolar aliphatic amino acids, such as glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), and proline (Pro, P); polar neutral amino acids, such as serine (Ser, S) and threonine (Thr, T); sulfur-containing amino acids, such as cysteine ​​(Cys, C) and methionine (Met, M); aromatic amino acids, such as phenylalanine (Phenylalanine, M); Amino acids can be classified into the following groups: phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W); amide amino acids, such as asparagine (Asn, N) and glutamine (Gln, Q); anionic amino acids, such as aspartate ("aspartic acid," Asp, D) and glutamate ("glutamic acid," Glu, E); and cationic amino acids, such as histidine (His, H), lysine (Lys, K), and arginine (Arg, R). These 20 proteinogenic amino acids are standard / classical amino acids encoded by the universal genetic code. They are linked by peptide bonds, which are covalent amide bonds formed by joining the C1 group of the carboxyl group of one amino acid with the amino group of another amino acid, linking two consecutive alpha-amino acids to form linear, unbranched polymeric chains called peptides. Other amino acids are non-proteinogenic and are referred to as non-standard / non-classical, with the exception of two amino acids: selenocysteine ​​and pyrrolysine. Although these two non-standard / non-classical amino acids are rarely incorporated into peptides or proteins, they can be translationally incorporated by utilizing information not encoded in the universal genetic code. There are many known non-proteinogenic and non-standard / non-classical amino acids (e.g., carnitine, gamma-aminobutyric acid, levothyroxine, hydroxyproline, selenomethionine, etc.). Some non-proteinogenic amino acids are found in proteins, where they are formed by post-translational modifications that are often essential for protein function or regulation (e.g., carboxyglutamic acid / carboxyglutamate, hydroxyproline, hypusine, etc.).

[0047] Other non-proteinogenic amino acids are not found in proteins (e.g., gamma-aminobutyric acid neurotransmitter), and some of them are often present as intermediates in metabolic pathways for standard amino acids, as part of amino acid catabolism (e.g., ornithine and citrulline in the urea cycle). Amino acids in this disclosure include all of the above amino acids.

[0048] As used herein, "carnitine" refers to a quaternary ammonium compound that is involved in energy metabolism in most mammals.Generally, carnitine is a nutrient and is synthesized by the body as needed.The main role of carnitine is to transport long-chain fatty acids to mitochondria and serve as a carrier for producing energy through β-oxidation.In view of its metabolic role, carnitine is concentrated in tissues such as skeletal muscle and cardiac muscle that metabolize fatty acids as an energy source.

[0049] Although free carnitine and fatty acylcarnitines can freely diffuse from the cytosol through the porous outer mitochondrial membrane into the intermembrane space, they require carnitine-acylcarnitine translocase (CACT) to pass through the non-porous inner mitochondrial membrane and reach the mitochondrial matrix where β-oxidation occurs. CACT is a shuttle system for passive transport of carnitine and fatty acylcarnitines, functioning by transporting one molecule of free carnitine from the matrix to the intermembrane space, while transporting one molecule of fatty acylcarnitine from the intermembrane space into the matrix. Deficiency of this protein prevents the body from using fat for energy to some extent, especially during periods without food (fasting).

[0050] Acylcarnitine profiling is a diagnostic test for inherited disorders of fatty acid metabolism as well as branched-chain amino acid catabolism. Patients with this type of metabolic disorder accumulate disease-specific acylcarnitines that correlate with acyl-coenzyme A compounds in affected mitochondrial metabolic pathways. Newborns with high levels of these substances may have CACT deficiency, which may be caused by mutations in the SLC25A20 gene. Signs and symptoms of this disorder usually begin shortly after birth and may include respiratory problems, seizures, and irregular heartbeat (arrhythmia). Affected individuals typically have low blood sugar (hypoglycemia) and low levels of ketones, which are produced during fat breakdown and used for energy. Taken together, these symptoms are called hypoketotic hypoglycemia. People with CACT deficiency also usually have excess ammonia in the blood (hyperammonemia), an enlarged liver (hepatomegaly), and weakened heart muscle (cardiomyopathy). Many infants with CACT deficiency do not survive the neonatal period. Some affected individuals have a less severe form of the condition and do not develop signs and symptoms until early childhood. These individuals are at risk of liver failure, nervous system damage, coma, and sudden death.

[0051] Some infants lack sufficient carnitine due to carnitine uptake deficiency (CUD), also known as primary carnitine deficiency. CUD is an autosomal recessive disorder caused by mutations in the SLC22A5 gene, which encodes the high-affinity carnitine transporter OCTN2 in the plasma membrane. CUD results in unabsorbed carnitine being excreted in the urine and consequent systemic and intracellular carnitine deficiency, which ultimately leads to impaired beta-oxidation of fatty acids. CUD is a potentially fatal disease, and patients with CUD usually have premature cardiomyopathy (including poor contractility, ventricular wall thickening, or increased T waves on EKG), muscle weakness, recurrent hypoketotic hypoglycemic coma, or Reye-like syndrome. Laboratory evaluation reveals extremely low blood and tissue carnitine concentrations (<5% of normal), and most symptoms are reversible with early treatment.

[0052] As used herein, "metabolic disorder" refers to a disorder that negatively alters the ongoing biochemical reactions that maintain the balance of the body's metabolism, i.e., two processes: catabolism, a set of chemical reactions that break down larger molecules into smaller molecules to generate energy, e.g., the breakdown of carbohydrate molecules into glucose, and anabolism (i.e., biosynthesis), a set of chemical reactions that build molecules from smaller units by consuming energy. Metabolic disorders occur when abnormal chemical reactions in the body alter or disrupt these processes. The result of abnormal chemical reactions can be too much or too little of a substance, which can impair protein, fat, and carbohydrate metabolism and / or affect various organelle functions, leading to complex medical conditions.

[0053] There are various groups of metabolic disorders, among which inherited metabolic disorders, the majority of which are autosomal recessive genetic conditions. Most inherited metabolic disorders are caused by a single mutation in a gene, which results in a missing / defective enzyme, i.e., the enzyme is not produced at all by the body or is produced in a dysfunctional form, which can lead to the accumulation of toxic compounds or the lack of essential products (e.g., urea cycle defects, amino acid disorders such as phenylketonuria (PKU) and maple syrup urine disease (MSUD)), abnormal energy production or consumption (e.g., mitochondrial disorders, glycogen metabolism disorders, glycogen storage diseases, galactosemia, fatty acid oxidation disorders), and / or defective function of organelles (e.g., lysosomes, peroxisomes, Golgi, and endoplasmic reticulum). Hundreds of genetic / inherited metabolic disorders have been identified, and new ones are constantly being discovered.

[0054] Children and adolescents with inherited metabolic disorders have a wide spectrum of clinical manifestations, ranging from those who appear physically normal to those with distinctive dysmorphic physical features. The majority of them appear physically normal at birth, and many may present with significantly nonspecific signs and symptoms common to other serious medical conditions. Although each inherited metabolic disorder is extremely rare in the general population, when considered together, their cumulative incidence is relatively high, approximately 1 in 1,000 to 2,500 newborns.

[0055] As used herein, "newborn screening (NBS)" refers to a public health screening program in infants shortly after birth for conditions that are treatable but clinically silent in the neonatal period. The goal is to identify infants at risk for these conditions early enough to prevent or reverse clinical symptoms. Newborn screening is performed with a few drops of blood obtained by prick of the infant's heel. Target disorders for newborn screening include metabolic disorders such as amino acid disorders, urea cycle disorders, fatty acid oxidation disorders, organic acid plasma and lysosomal storage disorders, endocrine disorders, hemoglobinopathies, cystic fibrosis, congenital heart disease, severe combined immunodeficiency, hearing loss, and other conditions.

[0056] As used herein, "organic acid" refers to an organic compound with acidic properties. The most common organic acids are carboxylic acids, which have a carboxyl group -COOH and are relatively weak acids. Acidity is determined by the relative stability of the acid's conjugate base. For example, sulfonic acids, which have the group -SO2OH, are relatively strong acids. There are many other groups with weak acidity, including thiol groups -SH, enol groups, and phenolic groups. In biological systems, organic compounds containing these groups are commonly referred to as organic acids. A few common examples are lactic acid, acetic acid, formic acid, citric acid, oxalic acid, uric acid, malic acid, and tartaric acid.

[0057] As used herein, "phenylketonuria (PKU)" refers to an inherited autosomal recessive metabolic disorder conventionally caused by a deficiency of the enzyme phenylalanine hydroxylase (PAH), an enzyme that catalyzes the hydroxylation of the aromatic ring of phenylalanine to produce tyrosine, which is converted to a variety of important compounds, including thyroxine, norepinephrine, epinephrine, and melanin, or to p-hydroxyphenylpyruvate, which is ultimately degraded to acetoacetate and fumarate.

[0058] The hydroxylation of phenylalanine occurs primarily in the liver, but also in the kidney, and requires dihydropteridine reductase and tetrahydrobiopterin in addition to phenylalanine hydroxylase. Hydroxylation of phenylalanine occurs by a coupled oxidation / reduction reaction consisting of the oxidation of phenylalanine to tyrosine and tetrahydrobiopterin to quinonoid dihydrobiopterin with molecular oxygen as the electron donor, which is regenerated by reduction of quinonoid dihydrobiopterin by dihydropteridine reductase in the presence of NAD(P)H: Phenylalanine + O2 + tetrahydrobiopterin → → → Tyrosine + H2O + quinonoid dihydrobiopterin Phenylanine hydroxylase Quinonoid dihydrobiopterin + NAD(P)H + H + →→→NAD(P) + +Tetrahydrobiopterin Dihydropteridine reductase

[0059] When phenylalanine hydroxylase is deficient or absent, phenylalanine is not converted to tyrosine. Instead, it accumulates in the circulation and is converted by glutamine-phenylpyruvate transaminase to phenylpyruvate, a phenylketone that is ultimately excreted in the urine. Under normal conditions, phenylpyruvate levels are very low in the blood or urine; however, in PKU, levels of phenylpyruvate, as well as phenyllactate and phenylacetate, are very high in urine, sweat, and breath, producing a slight musty odor.

[0060] In addition to PAH deficiency, deficiencies in dihydropteridine reductase and tetrahydrobiopterin can also cause a substantial decrease in the rate of phenylalanine hydroxylation. The synthesis of tetrahydrobiopterin begins with GTP and requires reactions mediated by GTP-cyclohydrolase, 6-pyruvoyltetrahydrobiopterin synthase, and sepiapterin reductase. Deficiencies in these three enzymes also cause hyperphenylalaninemia (high levels of phenylalanine in the blood).

[0061] Phenylanine is a large neutral amino acid that crosses the blood-brain barrier (BBB) ​​via the large neutral amino acid transporter (LNAAT). Excess phenylalanine in the blood saturates the transporter, thereby significantly reducing the levels of other large neutral amino acids in the brain. However, because these amino acids are necessary for protein and neurotransmitter synthesis, phenylalanine accumulation disrupts brain development, resulting in microcephaly and mental retardation. If left untreated, abnormal irritability, hyperactivity, epileptic seizures, and skin lesions become evident by the age of one year. In addition to these symptoms, EEG abnormalities and severe learning disabilities are major clinical problems later in life.

[0062] As used herein, "qDBS card" refers to a quantitative dried blood spot (qDBS) card, such as the Capitainer® qDBS, which is a card for blood sample collection.

[0063] Volume-Based Weighing The accuracy of detecting metabolites using the methods and microfluidic devices described herein is directly related to the ability to apply a precise volume of sample to a substrate preloaded with an internal standard. In one embodiment, a CAPITAINER® B card microsampling system is implemented. Such a microfluidic device is described in International Publication No. 2015044454A2, the contents of which are incorporated herein by reference. The CAPITAINER® B card uses a combination of paper and polymer microfluidics to meter a fixed volume of 10 μl from an undetermined volume of finger-prick blood. The operation of this system is illustrated and explained in Figure 5. After applying a drop of blood to the inlet, the metering channel in the device automatically fills. A valve composed of a thin, dissolvable membrane then opens, removing excess blood at the inlet. A second membrane then opens at the outlet, transferring the metered blood volume onto the sample collection disk. The sample is dried, forming a high-quality DBS sample suitable for quantitative analysis.

[0064] Those skilled in the art will recognize that the embodiments described herein for preloading an internal standard can be applied to other volume-based microsampling systems known in the art. Although the majority of the microsampling market consists of solutions for non-quantitative sampling systems (traditional DBS) and semi-quantitative sampling systems, there are commercially available solutions for volume-based microsampling. Table 2 lists such commercially available solutions for sampling a fixed volume of whole blood.

[0065] [Table 1] TIFF2026503078000003.tif158165

[0066] Example Embodiments of the present invention are described in further detail by reference to the following experimental examples. These examples are provided for illustrative purposes only, and unless otherwise specified, they are not intended to be limiting. Therefore, the present invention should in no way be construed as limited to the following examples, but rather should be construed to encompass any and all variations that become apparent as a result of the teachings provided herein. It is believed that one of ordinary skill in the art will be able to make and utilize the compounds of the present invention and practice the claimed methods using the foregoing description and the following illustrative examples without further explanation. Thus, the following examples specifically point out preferred embodiments of the present invention, and should not be construed as limiting in any way the remainder of the disclosure.

[0067] Materials and Methods A standard qDBS card was provided by Capitainer. Due to the viscosity of blood, it is difficult to obtain the same volume of blood for spotting. To overcome this problem, a microfluidic quantitative DBS (qDBS) card was used to deliver precise sample volumes to a pre-cut DBS disk. In a typical version, the qDBS card precisely delivers 10 μL of whole blood to a cotton disk. The qDBS card was opened to expose the cotton disk. We added a mixture of stably labeled amino acids in various volumes (0.5 μL, 1.0 μL, and 2.0 μL). The card was then allowed to dry overnight. Blood from a finger prick was then added to the card. The card was then allowed to dry again. This replicates the process an individual would use. After sufficient drying, the card was punched out, placed in a tube, and extracted with an organic solvent. After a 30 minute incubation, the resulting supernatant was subjected to precursor ion scanning for amino acids (looking for the loss of 46) on a triple quadrupole mass spectrometer operating in positive electrospray ionization mode.

[0068] result Figure 3 provides a schematic diagram depicting the quantification process using stable isotope blood spots (SIBS) on a substrate and the results of quantitative analysis of amino acids in SIBS. A mixture of stably labeled amino acids was loaded onto the cotton disc of a qDBS card. After the cotton disc dried overnight, a drop of blood from a finger prick was added to the card. The cotton disc in the card was dried, punched out, and placed in a tube for extraction with organic solvent. The resulting spectrum indicated the presence of stable isotope-labeled amino acids along with their endogenous counterparts. The resulting spectrum indicated the presence of stably labeled amino acids along with their endogenous counterparts.

[0069] As shown in Figure 4, phenylalanine 13 Phenylanine in DBS was quantified using C6, where various volumes of phenylalanine were added before blood was added onto the cotton disc. 13 C6 (0.5 uL, 1.0 uL, and 2.0 uL) was preloaded onto the cotton disc of the qDBS card as an internal standard. 13 If the volume of the C6 phenylalanine internal standard and therefore its amount changes, 13 The ratio of the relative abundance of phenylalanine to C6 was changed.

[0070] Furthermore, the observed error when the internal standard was added after blood collection was compared with when the internal standard was added before blood collection. Table 2 shows the comparison of the standard deviation and relative standard deviation (RSD) of the ratio between the process in which the internal standard was added before blood collection and the process in which the internal standard was added after blood collection. In this comparison, the standard deviation was 0.08 with internal standard postloading and 0.06 with internal standard preloading; the relative standard deviation was 6.4% with internal standard postloading, improving to 4.5% with internal standard preloading, suggesting that internal standard preloading is a more reliable method for amino acid quantification.

[0071] In conclusion, these examples demonstrated that this approach of preloading with stable isotope-labeled internal standards is more suitable for the quantification of small molecules such as amino acids.

[0072] Metabolomics References [Table 2] TIFF2026503078000005.tif217165 TIFF2026503078000006.tif144165

[0073] Inborn errors of metabolism reference [Table 3]

Claims

1. 1. A method for determining the amount of at least one target molecule in a liquid test sample, comprising: delivering a predetermined volume of liquid test sample to a substrate preloaded with a predetermined amount of the internal standard molecule such that the predetermined volume of liquid test sample contacts the internal standard molecule; Optionally, drying the liquid test sample on the substrate; incubating the substrate or a portion thereof with the liquid test sample in an extraction solvent to produce a supernatant containing at least one target molecule and an internal standard molecule; Detecting at least one target molecule and an internal standard molecule in the supernatant; and quantitating the amount of the at least one target molecule in the liquid test sample based on the amount of the at least one target molecule and the amount of the internal standard molecule detected in the supernatant; A method comprising:

2. 2. The method of claim 1, wherein the internal standard molecule comprises a label, and the label is optionally a stable isotope label.

3. Stable isotope labeling ２ H. １３ C. １５ N. １８ O. ３４ S, or any combination thereof.

4. The method according to any one of claims 1 to 3, wherein the predetermined amount of the internal standard molecule is in the amount range of 0.1 to 5 mmol.

5. loading a predetermined amount of a solution containing an internal standard molecule onto a substrate; and Drying the substrate to produce a preloaded substrate The method of any one of claims 1 to 4, further comprising:

6. The method of any one of claims 1 to 5, wherein the substrate is made of a liquid-absorbing material.

7. 7. The method of claim 6, wherein the liquid absorbent material comprises cotton, paper, fiber cloth, polymer resin, or the like.

8. 8. The method of any one of claims 1 to 7, wherein the liquid test sample is delivered to the substrate using a microfluidic device including a metering channel having a predetermined volume, the substrate being positioned to receive a metered volume of the liquid test sample from the metering channel, and the method further comprising removing the substrate from the microfluidic device.

9. 9. The method of any one of claims 1 to 8, wherein a predetermined volume of at least one liquid test sample is delivered to a first segment of the substrate, and the method further comprises delivering a predetermined volume of at least one control sample of at least one target molecule to a second segment of the substrate; incubating the first segment or a portion thereof with the at least one liquid test sample in a first extraction solvent, and incubating the second segment or a portion thereof with the at least one control sample in a second extraction solvent, wherein the first extraction solvent and the second extraction solvent are the same or different.

10. delivering a predetermined volume of at least one control sample of the at least one target molecule to a second substrate preloaded with a predetermined amount of the internal standard molecule, such that the at least one control sample contacts the internal standard molecule, wherein the at least one control sample comprises a known concentration of the at least one target molecule; Optionally, drying at least one control sample on a second substrate; incubating the second substrate or a portion thereof with the at least one control sample in an extraction solvent to produce a supernatant for the at least one control sample comprising at least one target molecule and an internal standard molecule; detecting at least one target molecule and an internal standard molecule in the supernatant of the control sample; generating a calibration curve based on the detection of at least one target molecule and an internal standard molecule in the supernatant of the control sample; and quantitating the amount of at least one target molecule in the test sample based on the amount of the at least one target molecule and the amount of the internal standard molecule detected in the supernatant of the test sample and correlation with a calibration curve; The method of any one of claims 1 to 9, further comprising:

11. 11. The method of any one of claims 1 to 10, wherein the liquid test sample is selected from a biological fluid such as an excretory fluid (such as urine or sweat), a secretory fluid (such as saliva, tears, milk or bile), a fluid obtained from within a subject (such as blood, plasma, serum or cerebrospinal fluid), or a fluid produced as a result of a pathological process (such as blister or cyst fluid), a food sample, a plant sample, an environmental sample, or the like.

12. The method of any one of claims 1 to 11, wherein incubating comprises placing the substrate or a portion thereof in a container and sonicating the substrate in the presence of the extraction solvent.

13. 13. The method of any one of claims 1 to 12, wherein detecting comprises performing mass spectrometry and the extraction solvent optionally comprises a C1-3 straight or branched chain monoalcohol, in particular methanol.

14. The target molecule is selected from peptides, proteins, heavy metals, toxins, drugs and their metabolites, amino acids, free carnitine, acylcarnitines, and any combination thereof in the liquid test sample, or optionally the target molecule is selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, citrulline, 14. The method of any one of claims 1 to 13, wherein the carnitine is selected from the group consisting of ornithine, free carnitine, acetylcarnitine, propionylcarnitine, butyrylcarnitine, isovalerylcarnitine, glutarylcarnitine, hexanoylcarnitine, octanoylcarnitine, decanoylcarnitine, lauroylcarnitine, myristoylcarnitine, palmitoylcarnitine, and / or stearoylcarnitine, and / or any combination thereof.

15. 15. The method of claim 14, wherein the target molecule is phenylalanine.

16. The stable isotope-labeled internal standard molecule may be a stable isotope-labeled amino acid, free carnitine, acylcarnitine, or any combination thereof, for example, ２ H4-alanine, １３ C6-, １５ N4-arginine, １３ C4-, １５ N2-asparagine, ２ H3-aspartic acid, １３ C3-cysteine, ２ H3-glutamic acid, １３ C5-, １５ N2-glutamine, １３ C-, １５ N-glycine, １３ C6-, １５ N3-histidine, １３ C6-, １５ N-isoleucine, ２ H3-leucine, １３ C6-, １５ N2-lysine, ２ H3-methionine, １３ C6-phenylalanine, １３ C5-, １５ N-proline, １３ C3-serine, １３ C4-threonine, １３ C11-, １５ N2-tryptophan, １３ C6-tyrosine, ２ H8-valine, ２ H2-citrulline, ２ H6-ornithine, ２ H9-carnitine, ２ H3-acetylcarnitine, ２ H3-propionylcarnitine, ２ H3-butyrylcarnitine, ２ H9-isovalerylcarnitine, ２ H3-glutarylcarnitine, ２ H3-hexanoylcarnitine, ２ H3-octanoylcarnitine, ２ H3-decanoylcarnitine, ２ H3-lauroylcarnitine, ２ H9-myristoylcarnitine, ２ H3-palmitoylcarnitine, and / or ２ 16. The method of any one of claims 2 to 15, wherein the hydroxybenzoate is selected from the group consisting of hydroxybenzoates, ...

17. Stable isotope-labeled internal standard molecules １３ The method of claim 16, wherein the amino acid is C6-phenylalanine.

18. 18. The method of any one of claims 1 to 17, wherein at least one target molecule is associated with a metabolic disorder, and the metabolic disorder optionally comprises a neonatal metabolic disorder.

19. 19. The method of claim 18, wherein the metabolic disorder is phenylketonuria, CACT deficiency, or primary carnitine deficiency.

20. 20. The method of any one of claims 1 to 19, wherein the predetermined volume is about 1 to about 100 microliters, or about 5 to about 15 microliters, or about 35 to about 65 microliters, or about 10 microliters, or about 50 microliters.

21. an inlet for application of a liquid test sample; a metering channel disposed in fluid communication with the inlet for receiving at least a portion of the liquid test sample from the inlet, the metering channel having a predetermined volume; and a substrate positioned to receive a metered volume of the liquid test sample from the metering channel, the substrate containing a predetermined amount of an internal standard molecule; A microfluidic device comprising:

22. 22. The microfluidic device of claim 21, wherein the substrate is comprised of a liquid-absorbent material.

23. 23. The microfluidic device of claim 21 or 22, wherein the substrate comprises a disc made from cotton, paper, fiber cloth, polymer resin, or a combination thereof.

24. 24. The microfluidic device of claim 23, wherein the disc comprises a diameter of about 5-7 mm and a thickness of about 1-2 mm.

25. The microfluidic device of any one of claims 19 to 24, wherein the internal standard molecule optionally comprises a label, which label is optionally a stable isotope.

26. A stable isotope for labeling the internal standard molecule is ２ H. １３ C. １５ N. １８ O. ３４ 26. The microfluidic device of any one of claims 19 to 25, wherein the ionic liquid is selected from the group consisting of: , ...

27. The microfluidic device of any one of claims 19 to 26, wherein the internal standard molecule comprises a label, which label is optionally a stable isotope.

28. 27. The microfluidic device of claim 26, wherein the stable isotope-labeled internal standard molecule is in an amount within the range of 0.1 to 5 mmol.

29. The stable isotope-labeled internal standard molecule may be a stable isotope-labeled amino acid, free carnitine, acylcarnitine, or any combination thereof, for example, ２ H4-alanine, １３ C6-, １５ N4-arginine, １３ C4-, １５ N2-asparagine, ２ H3-aspartic acid, １３ C3-cysteine, ２ H3-glutamic acid, １３ C5-, １５ N2-glutamine, １３ C-, １５ N-glycine, １３ C6-, １５ N3-histidine, １３ C6-, １５ N-isoleucine, ２ H3-leucine, １３ C6-, １５ N2-lysine, ２ H3-methionine, １３ C6-phenylalanine, １３ C5-, １５ N-proline, １３ C3-serine, １３ C4-threonine, １３ C11-, １５ N2-tryptophan, １３ C6-tyrosine, ２ H8-valine, ２ H2-citrulline, ２ H6-ornithine, ２ H9-carnitine, ２ H3-acetylcarnitine, ２ H3-propionylcarnitine, ２ H3-butyrylcarnitine, ２ H9-isovalerylcarnitine, ２ H3-glutarylcarnitine, ２ H3-hexanoylcarnitine, ２ H3-octanoylcarnitine, ２ H3-decanoylcarnitine, ２ H3-lauroylcarnitine, ２ H9-myristoylcarnitine, ２ H3-palmitoylcarnitine, and / or ２ 29. The microfluidic device of claim 28, wherein the hydroxyl group is selected from the group consisting of H3-stearoylcarnitine, ... and / or any combination thereof.

30. Stable isotope-labeled internal standard molecules １３ The microfluidic device according to any one of claims 27 to 29, wherein the amino acid is C6-phenylalanine.

31. A screening kit for determining the amount of at least one target molecule in a test sample by using an internal standard molecule, comprising: at least one substrate; and At least one stable isotope-labeled internal standard molecule, which can be in the form of a concentrated liquid or a dry powder; or At least one substrate preloaded with at least one known amount of stable isotope-labeled internal standard molecule. Including, A screening kit, optionally comprising at least one extraction solvent.

32. 32. The screening kit of claim 31, comprising at least one substrate and at least one stable isotope-labeled internal standard molecule, and further comprising a loading means for loading at least one stable isotope-labeled internal standard molecule onto the at least one substrate.

33. 32. The screening kit of claim 31, wherein the loading means comprises a syringe, a pipette, or a container with a dropper nozzle.

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