Method for detecting catecholamines by mass spectrometry

Abstract

The present invention provides a method for determining the amount of one or more of one or more noradrenaline (NE), epinephrine (E), and dopamine (D) in a sample using mass spectrometry. The methods generally involve ionizing one or more of NE (or derivatized NE), E (or derivatized E), and D (or derivatized D) in a sample, and detecting and quantifying the amount of ions to determine the amount of one or more of NE, E, and D in the sample.

Description

[0001] Cross-reference to related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 517,410, filed August 3, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This technology relates to the detection of catecholamines. Specifically, it relates to a method for detecting adrenaline, noradrenaline, and / or dopamine by mass spectrometry. Background Technology

[0003] Catecholamines, including the hormones norepinephrine (NE), epinephrine (E), and dopamine (DA), are major secretory products of nerve tissue. In particular, catecholamines can be produced in chromaffin cell tumors, pheochromocytomas, gangliocytomas, and neuroblastomas. Therefore, measuring circulating catecholamines is of significant value in diagnosing catecholamine-secreting tumors associated with hypertension (e.g., pheochromocytomas, neuroblastomas, and / or gangliocytomas) and in assessing orthostatic hypotension. Since early diagnosis and treatment of these diseases are crucial, rapid and accurate detection of these hormones is desirable.

[0004] Existing methods using high-performance liquid chromatography (HPLC) coupled with electrochemical detectors (ECD) have limitations, including specificity, long run times, and outdated and poorly supported instrument components. In contrast, liquid chromatography-tandem mass spectrometry (LC-MS / MS) offers several advantages: improved sensitivity and specificity, shorter run times with more advanced pump systems, and faster sample throughput through multiplexing capabilities. This article presents an LC-MS / MS method for the detection of catecholamines in biological samples, such as urine or plasma.

[0005] Urine is a complex matrix for LC-MS / MS and often requires enrichment of catecholamines to be a viable alternative to ECD. In this disclosure, the LC-MS / MS method described herein enables the detection of catecholamines after specific extraction from urine.

[0006] Plasma catecholamine levels reflect the balance between catecholamine biosynthesis, release, uptake, metabolism, and excretion at the sampling time point. Urinary catecholamine and metabolite concentrations represent a comprehensive assessment of sympathetic activity over a longer period and may not reflect the effects of transient changes in plasma levels. Therefore, in some pheochromocytomas, urinary values ​​may not rise beyond normal limits during very short paroxysmal attacks. In such patients, measuring plasma catecholamine levels during spontaneous attacks or histamine- or glucagon-induced attacks is an effective way to confirm the diagnosis. Measuring plasma norepinephrine can be used to diagnose orthostatic hypotension. Failure to elevate norepinephrine concentration in the supine position while standing suggests sympathetic nervous system dysfunction. Plasma norepinephrine is an independent risk factor for patients with chronic congestive heart failure and is associated with subsequent mortality. Norepinephrine can be used to assess hypertensive patients. Under conditions of relatively stable sympathetic activity or when induced changes are prolonged, urinary catecholamine levels may be proportional to blood catecholamine levels.

[0007] Therefore, detecting these hormones with sufficient accuracy and efficiency is crucial. Thus, improved methods for measuring catecholamine concentrations are desired. Summary of the Invention

[0008] This article discloses a method for determining the amount of an analyte in a sample, the method comprising: The sample was purified by liquid chromatography. The sample is subjected to a derivatization reagent to generate a derivatized sample; Under conditions suitable for generating one or more ions detectable by mass spectrometry, the derivatized sample is subjected to electrospray ionization (ESI); and The amount of analyte is quantified by tandem mass spectrometry, where quantification includes the amount of ions, and the amount of ions detected is correlated with the amount of analyte in the sample. in: The limit of quantitation for this method is less than 15 pg / mL; The analytes include adrenaline, noradrenaline, dopamine, or any mixture of two or more thereof; and Derivatizing reagents include phenyl isothiocyanate (PITC).

[0009] In some embodiments, one of the analytes is norepinephrine. In some embodiments, quantification includes quantifying the amount of a precursor ion with a mass-to-charge ratio of 305.2 ± 0.5. In some embodiments, quantification includes quantifying the amount of one or more fragment ions with mass-to-charge ratios selected from 107.0 ± 0.5 and 152.2 ± 0.5.

[0010] In some embodiments, one of the analytes is adrenaline. In some embodiments, quantification includes quantifying the amount of a precursor ion with a mass-to-charge ratio of 319.1 ± 0.5. In some embodiments, quantification includes quantifying the amount of one or more fragment ions with mass-to-charge ratios selected from 166.1 ± 0.5 and 107.0 ± 0.5.

[0011] In some embodiments, one of the analytes is dopamine. In some embodiments, quantification includes quantifying the amount of a precursor ion with a mass-to-charge ratio of 289.1 ± 0.5. In some embodiments, quantification includes quantifying the amount of one or more fragment ions with mass-to-charge ratios selected from 137.2 ± 0.5 and 153.2 ± 0.5.

[0012] In some implementations, the sample is a plasma sample.

[0013] In some implementations, the limit of quantitation of this method is less than or equal to 10 pg / mL.

[0014] In some embodiments, the analyte is purified by high-performance liquid chromatography (HPLC) before ionization. In some embodiments, the analyte is purified by solid-phase extraction (SPE) before ionization. In some embodiments, the analyte is purified by immobilized boric acid extraction before ionization.

[0015] In some implementations, the amounts of two or more analytes in a group consisting of adrenaline, noradrenaline, and dopamine are determined in the same sample injection.

[0016] In some implementations, the amounts of adrenaline, noradrenaline, and dopamine are determined in the same sample injection. Attached Figure Description

[0017] Figure 1A-1C They showed norepinephrine ( Figure 1A ), adrenaline ( Figure 1B ) and dopamine Figure 1C The limits of detection and limits of quantitation of )

[0018] Figure 2A-2C They showed norepinephrine ( Figure 2A ), adrenaline ( Figure 2B ) and dopamine Figure 2C The calibration curve.

[0019] Figures 3A-3C The effects of dilution (ClinCal® standard) on norepinephrine ( Figure 3A ), adrenaline ( Figure 3B ) and dopamine Figure 3C The analytical measurement range was verified.

[0020] Figure 4A The recovery rate of norepinephrine from known standards is shown. Figure 4B The adrenaline recovery rate of known standards is shown. Figure 4C The dopamine recovery rate of known standards is shown.

[0021] Figure 5A The comparison of norepinephrine samples (including all samples) is shown. Figure 5B The norepinephrine sample comparison is shown (excluding samples outside the linear range). Figure 5C The comparison of adrenaline samples is shown. Figure 5D The dopamine sample comparison is shown.

[0022] Figures 6A-6C They showed norepinephrine ( Figure 6A ), adrenaline ( Figure 6B ) and dopamine Figure 6C Interference research.

[0023] Figure 7A This study shows a comparison of sample types for norepinephrine fractionation. Figure 7B This study shows a comparison of sample types for adrenaline fractionation. Figure 7C This study shows a comparison of sample types for dopamine fractionation.

[0024] Figures 8A-8C They showed norepinephrine ( Figure 8A ), adrenaline ( Figure 8B ) and dopamine Figure 8C The limits of detection, limits of quantitation, and limits of quantitative analysis of ) are specified.

[0025] Figure 9A The recovery rate of norepinephrine from known standards is shown. Figure 9B The adrenaline recovery rate of known standards is shown. Figure 9C The dopamine recovery rate of known standards is shown.

[0026] Figure 10A The comparison of norepinephrine samples is shown (bottom right: corrected to μg / 24h or μg / g CREAT). Figure 10B The comparison of epinephrine samples is shown (bottom right: corrected to μg / 24h or μg / g CREAT). Figure 10C The dopamine sample comparison is shown (bottom right: corrected to μg / 24h or μg / g CREAT).

[0027] Figure 11A-11C They showed norepinephrine ( Figure 11A ), adrenaline ( Figure 11B ) and dopamine Figure 11CThe blank limit, detection limit and quantitation limit of )

[0028] Figures 12A-12C They showed norepinephrine ( Figure 12A ), adrenaline ( Figure 12B ) and dopamine Figure 12C The calibration curve.

[0029] Figure 13A The comparison of norepinephrine samples is shown. Note: The seven outliers (significantly larger shifts towards Ops and a substantial decrease in observed r values) are due to the expected higher interference from ECD-HPLC compared to the newer, more precise LC-MS used in R&D. Figure 13B The comparison of adrenaline samples was shown. Figure 13C The dopamine sample comparison is shown.

[0030] Figures 14A-14C They showed norepinephrine ( Figure 14A ), adrenaline ( Figure 14B ) and dopamine Figure 14C Research on hemolytic interference.

[0031] Figures 15A-15C They showed norepinephrine ( Figure 15A ), adrenaline ( Figure 15B ) and dopamine Figure 15C ) bilirubin interference study.

[0032] Figures 16A-16C They showed norepinephrine ( Figure 16A ), adrenaline ( Figure 16B ) and dopamine Figure 16C A study on lipid interference. Detailed Implementation

[0033] definition To facilitate understanding of this technology, certain terms are defined below. Further definitions of the following terms, as well as others, will be provided throughout this specification. All literature and other references cited to illustrate the technical background and provide practical details are incorporated herein by reference.

[0034] As used herein, unless otherwise stated, the singular forms “a,” “an,” and “the” include plural references. Thus, for example, a reference to “a protein” includes multiple protein molecules.

[0035] As used herein, the terms "purification" or "purifying" do not refer to the removal of all substances other than the target analyte from a sample. Rather, purification refers to the process of enriching one or more target analytes in an amount relative to other components in the sample that may interfere with the detection of the target analyte. Purifying a sample in various ways allows for a relative reduction of one or more interfering substances, such as those that may or may not interfere with the detection of selected precursor or fragment ions by mass spectrometry. This relative reduction when using the term does not require the complete removal of any substances present in the material to be purified along with the target analyte through purification.

[0036] As used herein, the term "test sample" refers to any sample that may contain NE, E, or D. As used herein, the term "body fluid" refers to any fluid that can be separated from an individual's body. For example, "body fluid" may include blood, plasma, serum, bile, saliva, urine, tears, sweat, and so on.

[0037] As used herein, the term "solid phase extraction" or "SPE" refers to a process in which a chemical mixture is separated into components by the affinity of the components dissolved or suspended in a solution (i.e., the mobile phase) for the solid (the solid phase) through which the solution passes or bypasses. In some cases, unwanted components of the mobile phase may be retained by the solid phase as it passes through or bypasses it, resulting in the purification of the analyte in the mobile phase. In other cases, the analyte may be retained by the solid phase, allowing unwanted components of the mobile phase to pass through or bypass it. In these cases, a second mobile phase is then used to elute the retained analyte from the solid phase for further processing or analysis.

[0038] As used herein, the term “chromatography” refers to the process by which a chemical mixture carried by a liquid or gas flows around or above a stationary liquid or solid phase, and separates into components due to the differential distribution of the chemical entities.

[0039] As used herein, the term "liquid chromatography" or "LC" refers to the process of selectively delaying the distribution of one or more components of a fluid solution when a fluid is uniformly permeated through a column of finely broken material or through a capillary channel. The delay is due to the distribution of the components of the mixture between one or more stationary phases and the bulk fluid (i.e., the mobile phase), as the fluid moves relative to the stationary phase. Examples of "liquid chromatography" include reversed-phase liquid chromatography (RPLC), high-performance liquid chromatography (HPLC), and turbulent liquid chromatography (sometimes called high-turbulence liquid chromatography (HTLC) or high-throughput liquid chromatography).

[0040] As used herein, the term "high-performance liquid chromatography" or "HPLC" refers to liquid chromatography that improves resolution by forcing the mobile phase through a stationary phase (usually a densely packed column) under pressure.

[0041] As used herein, the term "turbulent flow liquid chromatography" or "TFLC" (sometimes referred to as high turbulent flow liquid chromatography (HTLC) or high-throughput liquid chromatography) refers to a form of chromatography that utilizes the turbulent flow of the analyte through a packed column as the basis for separation. TFLC has been applied to the preparation of samples containing two unnamed drugs prior to analysis by mass spectrometry. See, for example, Zimmer et al. J Chromatogr A 854:23-35 (1999); see also U.S. Patents 5,968,367, 5,919,368, 5,795,469, and 5,772,874, which further explain TFLC. Those skilled in the art understand “turbulence.” When a fluid flows slowly and smoothly, this flow is called “laminar flow.” For example, fluid moving through an HPLC column at low flow rates is laminar. In laminar flow, the motion of fluid particles is ordered, and the particles generally move in a straight line. At higher speeds, the inertia of water overcomes fluid friction, resulting in turbulence. Fluid that does not come into contact with irregular boundaries “overtakes” fluid that is slowed by friction or deflected by uneven surfaces. When a fluid flows turbulently, it flows in the form of eddies and vortices (or eddies), with greater “drag” than when the flow is laminar. Numerous references can be used to help determine when a fluid flow is laminar or turbulent (e.g., Turbulent Flow Analysis: Measurement and Prediction , PSBernard & JMWallace, John Wiley &Sons, Inc., (2000); An Introduction to Turbulent FlowJean Mathieu & Julian Scott, Cambridge University Press (2001).

[0042] As used herein, the term “gas chromatography” or “GC” refers to a chromatographic method in which a mixture of samples is vaporized and injected into a carrier gas stream (such as nitrogen or helium) that moves through a column containing a stationary phase consisting of liquid or particulate solids, and is separated into its component compounds based on the affinity of the compounds for the stationary phase.

[0043] As used herein, the terms "large particle column" or "extraction column" refer to a chromatographic column with an average particle size greater than about 50 μm. As used in this context, the term "about" means ±10%.

[0044] As used herein, the term "analytical column" refers to a chromatographic column with sufficient chromatographic plates to influence the separation of materials in a sample, eluting from the column in a manner sufficient to allow for the determination of the presence or amount of an analyte. Such columns are generally distinct from "extraction columns," whose primary purpose is to separate from non-retaining materials or extract retained materials to obtain a purified sample for further analysis. As used herein, the term "about" means 10%. In some embodiments, the analytical column contains particles with a diameter of about 4 μm.

[0045] As used herein, the terms "on-line" or "inline," such as those used in "online automated method" or "online extraction," refer to procedures that do not require operator intervention. In contrast, the term "off-line," as used herein, refers to procedures that require manual operator intervention. Therefore, if the sample is precipitated and then the supernatant is manually loaded into the autosampler, the precipitation and loading steps are offline from subsequent steps. In various embodiments of this method, one or more steps can be performed in an online automated manner.

[0046] As used herein, the term "sample injection" refers to the process of introducing an aliquot of a single sample into an analytical instrument (e.g., a mass spectrometer). This introduction can be achieved directly or indirectly. Indirect sample injection can be accomplished by injecting an aliquot of the sample into a high-performance liquid chromatography column connected in-line to the mass spectrometer.

[0047] As used in this article, the term "same sample injection" for the detection of multiple analytes by mass spectrometry means that by measuring the ions of different analytes from the same (i.e. completely identical) sample injection, the ions of two or more different analytes are essentially determined simultaneously.

[0048] As used herein, the term “mass spectrometry” or “MS” refers to an analytical technique for identifying compounds by their mass. MS refers to the method of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or “m / z”. MS techniques typically involve (1) ionizing the compound to form a charged compound; and (2) detecting the molecular weight of the charged compound and calculating its mass-to-charge ratio. These compounds can be ionized and detected by any suitable means. A “mass spectrometer” typically includes an ionizer and an ion detector. Generally, one or more target molecules are ionized, and the ions are then introduced into the mass spectrometer, where, due to the combination of magnetic and electric fields, the ions follow a path in space that depends on their mass (“m”) and charge (“z”). See, for example, U.S. Patent No. 6,204,500 entitled "Mass Spectrometry From Surfaces"; U.S. Patent No. 6,107,623 entitled "Methods and Apparatus for Tandem Mass Spectrometry"; U.S. Patent No. 6,268,144 entitled "DNA Diagnostics Based On Mass Spectrometry"; U.S. Patent 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 1999, 2:264-76; and Merchant and Weinberger, Electrophoresis 2000, 21:1164-67.

[0049] As used herein, the term "operating in negative ion mode" refers to a mass spectrometry method for generating and detecting negative ions. The term "operating in positive ion mode" refers to a mass spectrometry method for generating and detecting positive ions. As used herein, the term "ionization" or "ionizing" refers to the process of generating analyte ions with a net charge equal to one or more electron units. A negative ion is an ion with a net negative charge of one or more electron units, while a positive ion is an ion with a net positive charge of one or more electron units.

[0050] As used herein, the term "electron ionization" or "EI" refers to a method in which a target analyte interacts with a stream of electrons in a gaseous or vapor phase. Collisions between electrons and the analyte produce analyte ions, which can then be analyzed using mass spectrometry.

[0051] As used herein, the term "chemical ionization" or "CI" refers to a method in which a reagent gas (e.g., ammonia) is subjected to electron collisions, and analyte ions are formed through the interaction between the reagent gas ions and the analyte molecules.

[0052] As used herein, the term "fast atom bombardment" or "FAB" refers to the method of bombarding non-volatile samples with a high-energy atomic beam (typically Xe or Ar), desorbing and ionizing molecules contained within 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-nitrophenyloctyl ether, sulfolane, diethanolamine, and triethanolamine. Selecting a suitable matrix for the compound or sample is an empirical process.

[0053] As used herein, the terms “matrix-assisted laser desorption / ionization” or “MALDI” refer to a method in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes the analytes in the sample through various 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 analyte molecules.

[0054] 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 the analytes in the sample through various ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. For SELDI, the sample is typically bound to a surface that preferentially retains one or more target analytes. Similar to MALDI, this process can also utilize energy-absorbing materials to facilitate ionization.

[0055] As used herein, the term "electrospray ionization" or "ESI" refers to a method of passing a solution through a short-length capillary tube, at which a high positive or negative potential is applied. The solution reaching the end of the tube is evaporated (atomized) into a jet or spray of very small solution droplets in solvent vapor. This droplet mist flows through an evaporation chamber, which can be heated to prevent condensation and promote solvent evaporation. As the droplets become smaller, the surface charge density increases until the natural repulsion between similar charges leads to the release of ions and neutral molecules.

[0056] As used herein, the terms "atmospheric pressure chemical ionization" or "APCI" refer to a mass spectrometry method similar to ESI; however, APCI generates ions through ionic-molecular reactions occurring within a plasma at atmospheric pressure. The plasma is maintained by a discharge between a spray capillary and a counter electrode. The ions are then typically extracted to the mass analyzer using a set of differentially pumped skimmer stages. Countercurrent flow of dried and preheated N2 gas can be used to improve solvent removal. For analyzing species with lower polarity, gas-phase ionization in APCI may be more efficient than ESI.

[0057] As used herein, the term "atmospheric pressure photoionization" or "APPI" refers to a form of mass spectrometry in which the photoionization mechanism of molecule M is photon absorption and electron ejection to form molecule M+. Because the photon energy is typically slightly above the ionization potential, the molecular ion does not readily dissociate. In many cases, samples can be analyzed without chromatography, saving significant time and costs. In the presence of water vapor or proton solvents, the molecular ion can be extracted with H+ to form MH+. + This often occurs if M has a high proton affinity. This does not affect quantitative accuracy because M... + and MH + The sum is constant. Drug compounds in proton solvents are typically observed to have MH.+ Nonpolar compounds such as naphthalene or testosterone typically form M... + See, for example, Robb et al. Anal.Chem. 2000, 72(15):3653-3659.

[0058] As used herein, the term "inductively coupled plasma" or "ICP" refers to a method in which a sample interacts with a partially ionized gas at a sufficiently high temperature, causing most elements to be atomized and ionized.

[0059] As used in this article, the term "field desorption" refers to a method of placing a non-volatile test sample on an ionizing surface and using a strong electric field to generate analyte ions.

[0060] As used herein, the term "desorption" refers to the removal of an analyte from a surface and / or the transfer of an analyte into the gas phase. Laser desorption / thermal desorption is a technique in which a sample containing an analyte is thermally desorbed into the gas phase by a laser pulse. The laser strikes the back of a specially designed 96-well plate with a metal substrate. The laser pulse heats the substrate, and the heat transfers the sample into the gas phase. The gas phase sample is then drawn into a mass spectrometer.

[0061] As used in this article, the term “selective ion monitoring” is a detection mode of a mass spectrometer in which only ions within a relatively narrow mass range are detected, typically about one mass unit.

[0062] As used in this article, “multiple reaction mode,” sometimes also called “selective reaction monitoring,” is a detection mode of a mass spectrometer in which precursor ions and one or more fragment ions are selectively detected.

[0063] As used herein, the terms “limit of quantification,” “limit of quantitation,” or “LOQ” refer to the point at which a measurement is quantitatively significant. The analyte response at this LOQ is identifiable, discrete, and repeatable, with a relative standard deviation (RSD%) of 20% and an accuracy of 80% to 120%.

[0064] As used herein, the term "limit of detection" or "LOD" refers to the point at which a measured value exceeds the uncertainty associated with it. LOD is defined as twice the RSD of the mean value at zero concentration.

[0065] As used herein, the “amount” of an analyte in a body fluid sample typically refers to the absolute amount of analyte that is detectable within the body fluid volume. However, amount also takes into account the relative amount compared to the amount of another analyte. For example, the amount of an analyte in a body fluid can be greater than that of a control or a normally present level of the analyte.

[0066] As used herein, the term "about" when referring to quantitative measurements that do not include ion mass means ±10% of the indicated value. Mass spectrometry instruments may vary slightly in determining the mass of a given analyte. In the context of ion mass or ion mass-to-charge ratio, the term "about" means 0.50 atomic mass units.

[0067] The above overview is not a limiting statement, and other features and advantages of the technology will be apparent from the following detailed description and the appended claims.

[0068] This document discloses a method for measuring the amount of one or more catecholamines in the group consisting of norepinephrine, epinephrine, and dopamine from a sample. Specifically, a mass spectrometry method is described for detecting one or more catecholamines in the group consisting of norepinephrine, epinephrine, and dopamine in a sample (e.g., urine or plasma samples). For example, any one of the catecholamines NE, E, or D in a sample can be detected by mass spectrometry; or any two of NE, E, and D in a sample can be detected by mass spectrometry; or all three of NE, E, and D in a sample can be detected by mass spectrometry. This method can employ high-performance liquid chromatography (HPLC) to purify the selected analyte and combine this purification with mass spectrometry (MS) to provide a high-throughput assay system for detecting and quantifying one or more catecholamines in the group consisting of NE, E, and D in a sample. Preferred embodiments are particularly suitable for applications in automated catecholamine assays in large clinical laboratories.

[0069] Samples suitable for this method include any sample that may contain the target analyte. In some embodiments, the sample is a biological sample, i.e., an aqueous sample obtained from any biological source (e.g., animal, cell culture, organ culture, etc.). In some preferred embodiments, the sample is obtained from mammals such as dogs, cats, horses, etc. Particularly preferred mammals are primates, and most preferably male or female humans. In some embodiments, the sample includes bodily fluids, such as urine, blood, plasma, serum, saliva, cerebrospinal fluid, or tissue samples. In some embodiments, the sample is a urine sample. In some embodiments, the sample is a plasma sample.

[0070] Samples may be obtained from, for example, a patient, meaning a living human individual (male or female) presenting in a clinical setting for the diagnosis, prognosis, or treatment of a disease or condition (such as pheochromocytoma, gangliocytoma, neuroblastoma, ganglioma, or paraganglioma). In some embodiments, the sample is a urine specimen (e.g., a urine specimen obtained from a patient), preferably a 24-hour urine specimen. In some embodiments, the sample is a plasma specimen (e.g., a plasma specimen obtained from a patient). For urine samples that cannot be analyzed immediately, a preservative (i.e., a reagent that maintains the pH below approximately 3, such as 6NHCl) should be added, as the stability of catecholamines decreases with increasing pH and deteriorates extremely rapidly in alkaline media.

[0071] The present invention also provides kits for quantitative assays of NE and / or E and / or D. Such kits include one or more internal standards in sufficient quantities to perform at least one assay. Typically, the kit also includes instructions in tangible form (e.g., contained in paper or electronic media) for performing a measurement assay using the packaged reagents to determine the amounts of NE and / or E and / or D.

[0072] The calibration and QC pools used in various implementations are preferably prepared using a matrix similar to that of the intended sample matrix.

[0073] Sample preparation NE and / or E and / or D in a sample can be enriched by any suitable method to prepare a sample for mass spectrometry. Prior to mass spectrometry, enrichment of NE and / or E and / or D relative to other components (e.g., proteins) in the sample can be accomplished by a variety of methods known in the art, including but not limited to liquid chromatography, filtration, centrifugation, thin-layer chromatography (TLC), electrophoresis (including capillary electrophoresis), affinity separation (including immunoaffinity separation), liquid-liquid or solid-phase extraction (including extraction with ethyl acetate or methanol or pretreatment with immobilized boric acid (by gel or other means)), the use of a dissociative agent, or any combination thereof, etc.

[0074] Protein precipitation is another method for preparing test samples (especially biological test samples such as serum, plasma, or urine). Such protein purification methods are well-known in the field, for example, by Poison et al. Journal of Chromatography B2003, 785:263-275, describes a protein precipitation technique suitable for the methods described herein. Protein precipitation can be used to remove most proteins from a sample, leaving NE and / or E and / or D in the supernatant. The sample can be centrifuged to separate the supernatant from the precipitated proteins; optionally, the sample can be filtered to remove the precipitated proteins. The resulting supernatant or filtrate can be used directly for mass spectrometry analysis; or optionally for liquid chromatography and subsequent mass spectrometry analysis. In some embodiments, using protein precipitation (such as formic acid protein precipitation) can eliminate the need for HTLC or other online extraction steps prior to mass spectrometry or HPLC-mass spectrometry.

[0075] Prior to mass spectrometry, a sample purification method that could be used was liquid chromatography (LC). Some liquid chromatography methods (including HPLC) rely on relatively slow laminar flow techniques. Traditional HPLC analysis relies on column packing, in which the laminar flow of the sample through the column is the basis for separating the target analyte from the sample. Those skilled in the art will understand that such separation in the column is a diffusion process, and suitable HPLC instruments and columns can be selected for use with NE (or derivatized NE) and / or E (or derivatized E) and / or D (or derivatized D). Chromatographic columns typically include a medium (i.e., packing material) to facilitate the separation (i.e., fractionation) of chemical fractions. The medium may include microparticles. These particles include binding surfaces that interact with various chemical fractions to facilitate their separation. A suitable binding surface is a hydrophobic binding surface, such as an alkyl-bound, cyano-bound, or pentafluorophenylpropyl (F5) surface. Alkyl-bound surfaces may include C-4, C-8, C-12, or C-18 bound alkyl groups. In a preferred embodiment, the column is an F5 column. A chromatographic column includes an inlet for receiving samples directly or indirectly from a solid-phase extraction or HTLC column and an outlet for discharging an effluent containing fractionated samples.

[0076] In one implementation, the sample can be applied to the LC column at the inlet, eluted with a solvent or solvent mixture, and discharged at the outlet. Different solvent modes can be selected to elute the target analyte. For example, liquid chromatography can be performed using gradient mode, isocratic mode, or polymorphic (i.e., mixed) mode. During chromatography, the separation of materials is affected by variables such as the choice of eluent (also known as the "mobile phase"), elution mode, gradient conditions, and temperature.

[0077] In some embodiments, analytes in a sample can be enriched by applying the sample to a column under conditions where the target analyte is reversibly retained by the column packing material, while one or more other substances are not retained. In these embodiments, a first mobile phase condition may be used, in which the target analyte is retained by the column, and once the unretained substances have been washed through, a second mobile phase condition may then be used to remove the retained substances from the column. Alternatively, the sample can be enriched by applying the sample to a column under mobile phase conditions where the target analyte elutes at a differential rate relative to one or more other substances. Such procedures allow for the enrichment of one or more target analytes relative to one or more other components in the sample.

[0078] In a preferred embodiment, HPLC analysis is performed using a hydrophobic column system. In some preferred embodiments, an F5 analytical column (e.g., the Discovery HS F5 analytical column (5 μm particle size, 50 × 4.6 mm) from Sigma-Aldrich, Inc., or an equivalent) is used. In some preferred embodiments, HTLC and / or HPLC are performed using HPLC with a mobile phase of HPLC-grade 0.1% formic acid aqueous solution and 1% formic acid acetonitrile solution.

[0079] By carefully selecting valves and connecting lines, two or more chromatographic columns can be connected as needed, allowing substances to be transferred from one to the next without any manual steps. In a preferred embodiment, the selection of valves and lines is controlled by a pre-programmed computer to execute the necessary steps. Most preferably, the chromatographic system is also connected online to a detection system (e.g., an MS system). Therefore, the operator can place the sample tray into the autosampler, and the remaining operations are performed under computer control, resulting in the purification and analysis of all selected samples.

[0080] In some embodiments, HTLC can be used to enrich NE (or derivatized NE), E (or derivatized E), and / or D (or derivatized D) prior to mass spectrometry. In such embodiments, the sample can be extracted using an extraction column that captures the analyte, followed by elution and chromatographic separation on a second HTLC column or analytical HPLC column, and then ionized. For example, sample extraction using an HTLC extraction column can be performed using a large-particle-size (50 μm) packed column. The sample eluted from this column can then be transferred to an HPLC analytical column (such as an F5 analytical column) for further purification, followed by mass spectrometry. Because the steps involved in these chromatographic processes can be linked in an automated manner, the operator intervention required during analyte purification can be minimized. This feature can lead to time and cost savings and eliminate the chance of operator error in mass spectrometry detection and quantification.

[0081] Detection and quantification by mass spectrometry In various embodiments, NE (or derivatized NE), E (or derivatized E), and / or D (or derivatized D) in the sample can be ionized using any method known to those skilled in the art. Mass spectrometry is performed using a mass spectrometer, which includes an ion source used to ionize the fractionated sample and generate 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 atomic bombardment (FAB), liquid secondary ionization (LSI), matrix-assisted laser desorption / ionization (MALDI), field ionization, field desorption, thermal spray / plasma spray ionization, surface-enhanced laser desorption / ionization (SEMI), inductively coupled plasma (ICP), and particle beam ionization. Those skilled in the art will understand that the choice of ionization method can be determined based on the analyte, sample type, detector type, positive / negative mode selection, etc. In some implementations, NE (or derivatized NE), E (or derivatized E) and / or D (or derivatized D) in the test sample can be ionized by ESI.

[0082] E (or derivatized E), NE (or derivatized NE), and / or D (or derivatized D) can be ionized in either positive or negative ion mode. In a preferred embodiment, E (or derivatized E), NE (or derivatized NE), and / or D (or derivatized D) are ionized in positive ion mode by heating ESI. In a related preferred embodiment, the E (or derivatized E), NE (or derivatized NE), and / or D (or derivatized D) ions are in a gaseous state, and the inert collision gas is argon or nitrogen; preferably argon.

[0083] In mass spectrometry, after sample ionization, the resulting positively or negatively charged ions are 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. Bartolucci et al. […]. Rapid Commun. Mass Spectrom. An exemplary ion trap method is described in 2000, 14:967-73.

[0084] Several detection modes can be used to detect ions. For example, selected ions can be detected using selective ion monitoring (SIM) mode, or alternatively, ions can be detected using scanning modes, such as multiple reaction monitoring (MRM) or selected reaction monitoring (SRM). Preferably, a quadrupole analyzer is used to determine the mass-to-charge ratio. For example, in a quadrupole or quadrupole ion trap instrument, the force on ions in an oscillating radio frequency field is proportional to the DC potential applied between the electrodes, the amplitude of the RF signal, and the mass-to-charge ratio. The voltage and amplitude can be selected such that only ions with a specific mass / charge ratio travel along the length of the quadrupole, while all other ions are deflected. Therefore, a quadrupole instrument can function as both a "mass filter" and a "mass detector" for ions injected into the instrument.

[0085] The resolution of MS techniques can be enhanced by employing tandem mass spectrometry, or MS / MS. In this technique, precursor ions (also called parent ions) generated by the target molecule are filtered in the MS instrument, then fragmented to produce one or more fragment ions (also called daughter ions or product ions), which are then analyzed in a second MS program. By carefully selecting the precursor ions, only ions generated by certain analytes are passed to the fragmentation chamber, where they collide with atoms in an inert gas to produce fragment ions. Because both the precursor and fragment ions are generated in a reproducible manner under given ionization / fragmentation conditions, MS / MS can provide an extremely powerful analytical tool. For example, the combination of filtration / fragmentation can be used to eliminate interfering substances and can be particularly useful in complex samples, such as biological samples.

[0086] Mass spectrometers typically provide users with ion scan spectra, which are the relative abundances of each ion with a specific mass-to-charge ratio within a given range (e.g., 90 to 1000 amu). The results of analyte determination (i.e., mass spectra) can be correlated with the amount of analyte in the original sample using various methods known in the art. For example, given careful control of sampling and analytical parameters, the relative abundance of a given ion can be compared to a table converting that relative abundance into the absolute amount of the original molecule. Optionally, molecular standards can be run with the sample, and a standard curve can be constructed based on the ions generated from these standards. Using such a standard curve, the relative abundance of a given ion can be converted into the absolute amount of the original molecule. In some preferred embodiments, one or more internal standards can be used to generate the standard curve for calculating the amounts of E (or derivatized E), NE (or derivatized NE), and / or D (or derivatized D). Methods for generating and using such standard curves are well known in the art, and those skilled in the art can select appropriate internal standards. For example, isotopically labeled catecholamines can be used as internal standards; in some preferred embodiments, norepinephrine-d6 (d6-NE), epinephrine-d6 (d6-E), or dopamine-d4 (d4-D) can be used as internal standards. Many other methods for relating the amount of ions to the amount of the original molecules are well known to those skilled in the art.

[0087] One or more steps of the presented method can be performed using automated machines. In some embodiments, one or more purification steps are performed online, and more preferably, all purification and mass spectrometry steps can be performed online.

[0088] In some implementations, such as MS / MS, where precursor ions are separated for further fragmentation, collision-activated dissociation (CAD) is typically used to generate fragment ions for further detection. In CAD, precursor ions gain energy through collisions with an inert gas and are subsequently broken up through a process called "unimolecular decomposition." Sufficient energy must be deposited in the precursor ions so that some bonds within the ions can break due to increased vibrational energy.

[0089] In some embodiments, the methods described herein include derivatizing catecholamines (e.g., NE, E, and / or D) in a sample using a derivatizing reagent (such as PITC) prior to mass spectrometry analysis. As used herein, “derivatizing” or “derivatization” refers to the reaction of two molecules to form a new molecule. Thus, a derivatizing reagent is an agent that reacts with another substance to derivatize that substance. For example, phenyl isothiocyanate (PITC) is a derivatizing reagent that can react with catecholamines (such as norepinephrine, epinephrine, and / or dopamine) to generate PITC-derived catecholamines (such as PITC-derived norepinephrine, PITC-derived epinephrine, and / or PITC-derived dopamine). For example, exemplary embodiment 1 shows the derivatization of dopamine using PITC. Therefore, catecholamines (such as NE, E, and / or D) in a sample can be detected and / or quantified using MS / MS as follows. The sample can be filtered through and eluted from an immobilized boric acid gel. Subsequently, the catecholamines (such as NE, E, and / or D) in the purified sample are derivatized using PITC or its isotopic variants. The derivatized and purified sample can then undergo liquid chromatography (preferably HPLC). A liquid solvent flows from the column into a heated ESI probe of the MS / MS analyzer, ionizing the analyte.

[0090] Scheme 1—Phenyl isothiocyanate (PITC) derivatization Optionally, in some embodiments, the methods described herein do not include derivatization of catecholamines (e.g., NE, E, and / or D) in the sample. Therefore, MS / MS can be used to detect and / or quantify catecholamines (e.g., NE, E, and / or D) in the sample as follows: The sample can be filtered through and eluted from an immobilized boric acid gel. The purified sample (e.g., the resulting eluent) can then undergo liquid chromatography (preferably HPLC). A liquid solvent flows from the column into a heated ESI probe of the MS / MS analyzer and ionizes the analyte.

[0091] Ions (e.g., precursor ions) pass through the instrument's nozzle and enter the first quadrupole. Quadrupole 1 (Q1) and quadrupole 3 (Q3) act as mass filters, allowing ion selection based on their mass-to-charge ratio (m / z) (i.e., selecting "precursor" ions and "fragment" ions in Q1 and Q3, respectively). Quadrupole 2 (Q2) is the collision chamber where ion fragmentation occurs. The mass spectrometer's first quadrupole (Q1) selects molecules with a mass-to-charge ratio of NE (or derived NE), E (or derived E), or D (or derived D). Precursor ions with the correct mass / charge ratio are allowed to enter the collision chamber (Q2), while unwanted ions with any other mass / charge ratio collide with the sides of the quadrupole and are eliminated. The precursor ions entering Q2 collide with neutral argon molecules and undergo fragmentation. This process is called collision-activated dissociation (CAD). The resulting fragment ions enter quadrupole 3 (Q3), where fragment ions are selected while other ions are eliminated.

[0092] During single-sample analysis, Q1 and / or Q3 can be adjusted such that the mass-to-charge ratio of one or more precursor ion / fragment ion pairs specific to one catecholamine is selected first, followed by the selection of the mass-to-charge ratio of one or more precursor ion / fragment ion pairs specific to a second catecholamine at a later time, and optionally, the selection of the mass-to-charge ratio of one or more precursor ion / fragment ion pairs specific to a third catecholamine at a later stage. In a particularly preferred embodiment, the mass-to-charge ratio of precursor ion / fragment ion pairs specific to NE (or derivatized NE), the mass-to-charge ratio of precursor ion / fragment ion pairs specific to E (or derivatized E), and the mass-to-charge ratio of precursor ion / fragment ion pairs specific to D (or derivatized D) are detected during single-sample analysis, although the detection sequence can occur in any order.

[0093] This method can be performed using MS / MS in either positive or negative ion mode (preferably positive ion mode). Using standard methods known in the art, those skilled in the art can identify one or more fragment ions of specific precursor ions of NE (or derivatized NE), E (or derivatized E), and / or D (or derivatized D), which can be used for selection in a quadrupole 3 (Q3).

[0094] When ions collide with the detector, they generate electronic pulses, which are converted into digital signals. The acquired data is transmitted to a computer, which plots a curve showing the relative change in the number of collected ions over time. The resulting mass spectrometry chromatogram is similar to that generated in conventional HPLC methods. The area under the curve (AUC) or amplitude of the peak corresponding to a specific ion is measured, and this AUC or amplitude is correlated with the amount of the target analyte. In some embodiments, the amount of one or more analytes detected is determined by measuring the AUC or amplitude of the peaks of fragment ions and / or precursor ions. As described above, a calibration standard curve based on the peaks of one or more ions, such as norepinephrine-d6 (d6-NE), epinephrine-d6 (d6-E), and / or dopamine-d4 (d4-D), can be used to convert the relative abundance of a given ion or precursor ion / fragment ion pair into the absolute amount of the original analyte, such as NE (or derivatized NE), E (or derivatized E), or D (or derivatized D).

[0095] Therefore, according to some aspects, this disclosure provides a method for determining the amount of an analyte in a sample, the method comprising one or more of the following steps: purifying the sample by liquid chromatography; subjecting the sample to a derivatizing reagent to generate a derivatized sample; subjecting the sample (e.g., the purified sample or the derivatized sample) to electrospray ionization (ESI) under conditions suitable for generating one or more ions detectable by mass spectrometry; and quantifying the amount of the analyte by tandem mass spectrometry, wherein quantification includes quantifying the amount of ions, and the amount of ions detected is correlated with the amount of the analyte in the sample.

[0096] In some embodiments, the limit of quantitation of the method described herein is less than or equal to 15 ng / mL, such as less than or equal to 10 ng / mL, less than or equal to 5 ng / mL, or less than or equal to 3 ng / mL. In some embodiments, the limit of quantitation of the method described herein is less than or equal to 15 pg / mL, such as less than or equal to 10 pg / mL.

[0097] In some embodiments, the analyte includes adrenaline, noradrenaline, dopamine, or any two or more of them.

[0098] In some embodiments, the methods described herein include subjecting a sample to a derivatizing reagent to generate a derivatized sample. In some embodiments, the derivatizing reagent comprises phenyl isothiocyanate (PITC). In some embodiments, one or more analytes is norepinephrine, and quantification includes the amount of a precursor ion with a mass-to-charge ratio of 305.2 ± 0.5; and / or the amount of one or more fragment ions with mass-to-charge ratios selected from 107.0 ± 0.5 and 152.2 ± 0.5. In some embodiments, one or more analytes is epinephrine, and quantification includes the amount of a precursor ion with a mass-to-charge ratio of 319.1 ± 0.5; and / or the amount of one or more fragment ions with mass-to-charge ratios selected from 166.1 ± 0.5 and 107.0 ± 0.5. In some embodiments, one of the analytes is dopamine, and quantification includes the amount of a precursor ion with a mass-to-charge ratio of 289.1 ± 0.5; and / or the amount of one or more fragment ions with mass-to-charge ratios selected from 137.2 ± 0.5 and 153.2 ± 0.5.

[0099] In some embodiments, the methods described herein do not involve subjecting the sample to a derivatizing reagent to generate a derivatized sample. In some embodiments, one of the analytes is norepinephrine, and quantification includes the amount of a precursor ion with a mass-to-charge ratio of 152.1 ± 0.5; and / or the amount of one or more fragment ions with a mass-to-charge ratio of 76.9 ± 0.5. In some embodiments, one of the analytes is adrenaline, and quantification includes the amount of a precursor ion with a mass-to-charge ratio of 166.1 ± 0.5; and / or the amount of one or more fragment ions with mass-to-charge ratios selected from 51.0 ± 0.5 and 107.0 ± 0.5. In some embodiments, one of the analytes is dopamine, and quantification includes the amount of a precursor ion with a mass-to-charge ratio of 137.1 ± 0.5; and / or the amount of one or more fragment ions with a mass-to-charge ratio of 91.1 ± 0.5.

[0100] In some embodiments, the sample is a plasma sample. In some embodiments, the sample is a urine sample.

[0101] In some embodiments, the analyte is purified by high-performance liquid chromatography (HPLC) before ionization. In some embodiments, the analyte is purified by solid-phase extraction (SPE) before ionization. In some embodiments, the analyte is purified by immobilized boric acid extraction before ionization.

[0102] In some embodiments, the amounts of two or more analytes from the group consisting of adrenaline, noradrenaline, and dopamine are determined in the same sample injection. In some embodiments, the amounts of adrenaline, noradrenaline, and dopamine are determined in the same sample injection.

[0103] The following examples are intended to illustrate various aspects of this disclosure. These examples are in no way intended to limit the scope of the method.

[0104] Example Example 1 - Detection of catecholamines in human plasma (PITC derivatization method) Scheme 2 provides a schematic flowchart illustrating sample preparation and plasma catecholamine detection.

[0105] Option 2 Patient plasma samples were prepared by mixing heparinized plasma with an internal standard (ISTD), stabilizer, and ammonium dihydrogen phosphate buffer. These steps were automated using a Hamilton Liquid Processor. Solid-phase extraction (SPE) was performed on an Agilent PBA plate on a SPEware ALD system. The PBA-filled SPE plate captures catecholamines. Sample eluent was collected and dried under a nitrogen stream on a Porvair.

[0106] Before the second "dry down" step, "redry" solution was added to each well and thoroughly mixed on an Eppendorf plate mixer. Then, PITC derivatization solution was added to each sample, mixed, and incubated. PITC increases the molecular weight by 135. The collected plates were then dried again at 35°C under nitrogen.

[0107] Samples were reconstituted with acetonitrile / water, mixed, and filtered through an Agilent Captiva filter. Samples were injected into a StreamSelect multiplexed HPLC system coupled to an Agilent 6495C triple quadrupole mass spectrometer in positive ion mode with a heated electrospray ionization source. Chromatographic separation was achieved using a Zorbax SB-Aq column (Agilent). Calibration curves were fitted using Agilent MassHunter quantitative software based on the peak area ratio of the analyte to the internal standard.

[0108] Quality Control (QC) Materials. A QC mixture sample was prepared by adding known concentrations of norepinephrine, epinephrine, and dopamine to stripped serum. This mixture sample was aliquoted into three equal aliquots, each containing a stock standard at a presumed reportable range concentration for coverage testing. The QC mixture sample was aliquoted into 2.5 mL aliquots and stored in an ultra-low temperature freezer at -90°C to -60°C for accuracy data. The analyte QC materials were prepared at the following concentrations: The MS was detected using the HESI positive ion mode. Quantitative analysis was based on unique precursor-product transitions. The following precursor ions and fragment ions were used: In the following table, a simple summary of the results obtained in Example 1 is provided. Accuracy.

[0109] Run accuracy (within batch). Ten samples at each quality control level (low, medium, and high) were extracted within the same run. The within-batch coefficient of variation for all analytes was less than 8.3%, meeting the acceptable criteria. Acceptable criteria: CV ≤ 8.3% or TEa / 3. Total accuracy. Five replicate samples at each spiked matrix sample level (low, medium, and high) were extracted on 5 different working days, for a total of 25 injections. The between-batch coefficient of variation for all analytes was ≤ 8.3%, meeting the acceptable criteria. Acceptable criteria: SD < TEa / 3 or CV ≤ 8.3%; Unacceptable: If the total SD ≥ 1 / 2Tea, or the total SD must be less than the defined maximum SD or CV. Inter-operator accuracy. The comparison of similar extraction plates containing equal amounts of quality control samples, spiked matrix samples, and standards was determined by different laboratory personnel. The result comparison showed that the inter-operator coefficient of variation for all analytes was ≤ 8.3%, meeting the acceptable criteria. Acceptable criteria: CV ≤ 8.3% or TEa / 3 Analytical sensitivity (limit of detection) Limit of blank (LOB). Five stripped serum blank samples were extracted on 5 different dates, for a total of 25 injections. Calculation: LOB = blank mean + 2SD. The LOB for the analytes is as follows: Limit of detection (LOD). Five stripped serum blank samples were extracted on 5 different dates, for a total of 25 injections. Calculation: LOD = blank mean + 4SD. The LOD for the analytes is as follows ( Figure 1A-1C ). Limit of quantitation (LOQ). Each analyte at a known concentration was added to the stripped serum. Five replicate samples at 3 levels were extracted and analyzed over 5 days. Acceptability criteria: Lowest concentration, SD < TEa / 3. The TEa for all three analytes is 25%. The LOQ for the analytes is as follows ( Figure 1A-1C All analytes are acceptable for a CV ≤ 8.3%. Analyte Measurement Range (AMR) Calibration validation. Nineteen standards were analyzed in 19 independent extractions. Linear regression analysis of these runs showed correlation coefficients of 0.9900 or greater for all analytes, with average recoveries ranging from 92.1% to 108.7%, indicating that all analytes were within the quantifiable range. Figure 2A-2C Acceptability criterion: The deviation of the mean of the observed values ​​from the expected range should not exceed TEa / 4. Both TEa / 4 and the AMR range meet the validation criteria and are acceptable. AMR was validated by dilution. A lyophilized calibrator (ClinCal®) containing norepinephrine, epinephrine, and dopamine was reconstituted with 5 mL of HPLC-grade water. The reconstituted solution was then mixed for 15 minutes before extraction. Dilutes of 1 / 2, 1 / 4, 1 / 10, 1 / 15, and 1 / 30 were prepared from the reconstituted calibrator and extracted as unknown samples. Figures 3A-3C Then, the calculated values ​​are compared with the target values ​​to obtain the recovery rate (%). Acceptability criterion: The difference between the regression line value and the observed value should be ≤ TEa / 4. Both TEa / 4 and the recovery rate range meet the validation criteria and are acceptable. Reportable Scope (RR) Accuracy Recycling Research Recovery of known standards. Two sets of known standards were tested in independent extractions to quantify the results. These standards were added to a level of 1200 pg / mL. Target dilution levels were obtained by diluting the 1200 pg / mL level with stripped serum: 600 pg / mL, 300 pg / mL, 120 pg / mL, 60 pg / mL, 20 pg / mL, and 10 pg / mL. Figures 4A-4C Acceptability criteria: The error due to imperfect recovery (recovery amount minus spiking amount) should be ≤ TEa / 4. The recovery rates of all analytes met the acceptable criteria.

[0110] Recovery by dilution. Purchased ClinCal® calibration standards (preparation see Section 7.2) were recovered by dilutions of 1 / 2, 1 / 4, 1 / 10, 1 / 15, and 1 / 30. The diluted samples were injected repeatedly, and the calculated mean was compared to the net value to obtain the recovery rate (%). Acceptability criteria: The deviation of the mean observed values ​​from the expected range should not exceed TEa / 3 (i.e., 8.3%). Recovery rates for all analytes met the acceptable criteria. Comparative analysis using a maximum of 51 samples. Figures 5A-5D Samples were analyzed simultaneously using both ECD-HPLC and LCMS-MS methods. Compared to ECD-HPLC, LCMS-MS monitors only specific derivatized target molecules, thus improving specificity. Sixteen samples of epinephrine and 17 samples of dopamine were below detectable levels and were excluded. For norepinephrine, two plots were calculated: the first plot includes all analyzed samples, and the second plot excludes results exceeding 1000 pg. This was done to provide comparisons with alternative samples at lower concentration levels. Measurement uncertainty (MU) Measurement uncertainty. Definition: The degree of confidence in a particular measurement result. Expressed as a 95% confidence interval around a given number, determined by the mean of multiple measurements ± 1.96 × SD. The MU confidence interval is calculated using the "Precision" worksheet in the Detection Validation Calculator template. MU is calculated by analyzing the data using the mean and standard deviation, then multiplying the SD by 1.96 and adding or subtracting from the mean. Each QC level is analyzed as three independent levels, with five replicates per level on five different dates. Specimen stability The mixed patient plasma was divided into 5 groups and catecholamines of known concentration (covering existing reference ranges) were added. For all stability studies, each sample was extracted repeatedly. Acceptability criterion: a sample was considered stable as long as the mean difference between baseline values ​​and time / temperature sample values ​​was ≤ TEa / 4 of the analyte.

[0111] Interference research Acceptability criterion: Differences caused by potential interfering substances should be ≤TEa / 4 to be considered acceptable. Figures 6A-6C ).

[0112] Hemolytic interference. Three independent patient plasma samples were mixed and supplemented with catecholamine stock standards, and analyzed in triplicate to obtain baseline results. 250 μL of hemolyzed RBCs were added to 4750 μL of 10 mM PBS solution; this fresh solution was used as severe hemolytic interference. This severe hemolytic solution was further diluted with 10 mM PBS at 1:10 and 1:20 to obtain moderate and mild hemolytic interference. 90 μL of each interfering solution was mixed with 810 μL of each spiked plasma level. All samples were extracted in triplicate. Plasma catecholamines were acceptable for mild, moderate, and severe hemolytic samples.

[0113] Lipid interference. Three independent patient plasma samples were mixed and supplemented with catecholamine stock standards, and triple-replicated analyses were performed to obtain baseline results. 1 mL of intralipid was added to 4 mL of 10 mM PBS solution; this fresh solution was used for severe lipemia interference. Other interference levels were prepared by diluting the intralipid stock solution to 1:10 (moderate lipemia interference) and 1:20 (mild lipemia interference) with 10 mM PBS solution. 90 μL of each interference solution was mixed with 810 μL of each spiked plasma level. All samples were extracted tripletly. Plasma catecholamines were acceptable for both mild and moderate lipemia samples.

[0114] Bilirubin interference. Three independent patient plasma aliquots were added to catecholamine stock standards and analyzed in triplicate to obtain baseline results. A 1 mg / mL bilirubin sample was prepared using 10 mM PBS; this fresh solution was used for severe jaundice interference. This severe jaundice solution was further diluted with 10 mM PBS at 1:10 and 1:20 to obtain moderate and mild jaundice interference. 90 μL of each interference solution was mixed with 810 μL of each spiked plasma level. All samples were extracted in triplicate. Plasma catecholamines were acceptable for mild, moderate, and severe jaundice samples.

[0115] Drug interference. Due to the similarity in molecular structure between carbidopa and levodopa, drug interference was investigated. Samples were prepared by adding carbidopa and levodopa to stripped serum to a final concentration of 10,000 ng / mL. Spiked samples were repeatedly extracted and analyzed. Acceptability criteria: Differences caused by potential interfering substances should be ≤TEa / 4 to be considered acceptable. Interfering drugs below the LOQ were determined not to cause interference. Results are as follows: Ion inhibition. Ion inhibition was detected using a post-column infusion method. Extracted catecholamine standards (1000 pg / mL) were infused at a constant flow rate (20 μL / min), followed by injection of six extract samples (1 blank sample and 5 patient samples). Changes in the signal response of the infused analyte at corresponding retention times indicated the presence of ionization inhibition. No ion inhibition was observed in this experiment. Acceptability criterion: Post-column infusion signal intensity change not exceeding ±25%.

[0116] Carryover.

[0117] For this study, both injection residue and Hamiltonian HPLC residue were evaluated. Stripped serum was added to achieve low (50 pg / mL) and high (1000 pg / mL) catecholamine levels. The assay sequence was: two low-concentration replicates, then two high-concentration replicates, followed by three low-concentration replicates. This sequence was repeated twice within the same assay. The results of the low-level spikes were compared with the low-level spikes after the high-level spikes to test for residue. Acceptability criteria: a difference due to residue ≤ TEa / 4 was considered acceptable. The results were acceptable, and no residue was observed in the assay.

[0118] Specimen type (sampling comparison) For the experiments described on the ECD / HPLC platform, acceptable sample types were plasma collected using vacuum blood collection tubes containing sodium heparin as a preservative. This study tested blood samples collected from 34 donors in two types of vacuum blood collection tubes (sodium heparin and EDTA as preservatives). All samples were extracted and analyzed. Results from plasma containing EDTA were compared with those from plasma containing sodium heparin. Results for norepinephrine, epinephrine, and dopamine were all acceptable. Figures 7A-7C ).

[0119] Example 2 - Detection of catecholamines in human urine without derivatization Catecholamines in human urine samples can be detected using the methods summarized in the table below. Patient urine samples were prepared by combining the sample with an internal standard (ISTD), a stabilizer, and ammonium dihydrogen phosphate buffer. Solid-phase extraction (SPE) plates filled with phenylboronic acid (PBA) captured catecholamines via their cis-diol content. The eluent was collected and dried under nitrogen. The sample was reconstituted with HPLC-grade water.

[0120] Samples were injected into a StreamSelect multiplexed HPLC system coupled to an Agilent 6495C triple quadrupole mass spectrometer, using positive ion mode and a heated electrospray ionization source. Chromatographic separation was achieved using a Kinetex PFP column (Phenomenex). Calibration curves were fitted using Agilent Masshunter quantitative software based on the peak area ratio of analyte ions to internal standards.

[0121] Use the following precursor ions and fragment ions: Quality Control (QC) Materials accuracy Intra-batch precision. Acceptance criteria: %CV or SD should be ≤ TEa / 4 (norepinephrine and epinephrine: %CV ≤ 5% or 1.25 ng / mL, where TEa = 20% or 5 ng / mL; dopamine: %CV ≤ 5% or 2.50 ng / mL, where TEa = 30% or 10 ng / mL). Five replicates were extracted at each level according to the formulation and Sigma Cal QC on five different dates, for a total of 25 injections per level. Results are shown in the table below. Conclusion: Intra-batch precision data meet acceptable reproducibility requirements (SD or %CV ≤ TEa / 4). Batch-to-batch accuracy. Acceptance criteria: %CV or SD should be ≤ TEa / 3 (norepinephrine and epinephrine: %CV ≤ 6.67% or 1.67 ng / mL, where TEa = 20% or 5 ng / mL; dopamine: %CV ≤ 10% or 5 ng / mL, where TEa = 30% or 10 ng / mL). Two replicates of Bio-Rad QC samples for each level (I, II, III) were extracted using 20 different extractions, for a total of 40 injections per level. Results are shown in the table below. Conclusion: Batch-to-batch accuracy data meet acceptable reproducibility requirements (SD or %CV ≤ TEa / 4). Analytical sensitivity (limit of detection) Blank limit (LoB). Acceptance criteria: LoB = blank mean + 2SD. Water blank samples were extracted over three different dates, with a total of 25 injections. Results: No quantifiable catecholamine peaks were observed, therefore the mean and standard deviation could not be determined. Figures 8A-8C Conclusion: The blank limit (LoB) of urinary catecholamines cannot be determined by LC / MS / MS.

[0122] Limit of Detection (LoD). Acceptance criteria: LoD = blank mean + 4SD. Water blank samples were extracted over three different dates, with a total of 25 injections. Results: No quantifiable catecholamine peaks were observed, therefore the mean and standard deviation could not be determined. Figures 8A-8C Conclusion: The limit of detection (LoD) for urinary catecholamines cannot be determined by LC / MS / MS.

[0123] Limit of Quantification (LoQ). Acceptance criteria: Lowest concentration at which SD ≤ TEa / 3 (norepinephrine and epinephrine: %CV ≤ 6.67%, where TEa = 20%; dopamine: %CV ≤ 10%, where TEa = 30%). Commercially available (BioRad standards) were added to urine samples (each analyte at known concentrations) and diluted to various levels. Extraction and analysis were performed on five replicates of five levels over five days. Figures 8A-8C ). Conclusion: A limit of quantitation (LoQ) was determined for each catecholamine and rounded to the nearest integer for practical purposes. The results are summarized in the table below.

[0124] The LOQ of the analyte is as follows: Reportable Scope (RR) Analyte Measurement Range (AMR). Quantitative and Selected Semi-Quantitative Test Definition: The reportable range is the entire range of results for which the accuracy of the instrument or detection system has been established or validated. It is expressed as the lower limit of AMR (usually LoQ) and extends to or beyond the upper limit of AMR.

[0125] Calibration Validation. Acceptance Criteria: CV or SD should be ≤ TEa / 3 (Norepinephrine and epinephrine: CV ≤ 6.67% or 1.67 ng / mL, where TEa = 20% or 5 ng / mL; Dopamine: CV ≤ 10% or 5 ng / mL, where TEa = 30% or 10 ng / mL). Twenty Bio-Rad standards were analyzed in 20 independent extractions, and 21 Sigma standards were analyzed in 21 independent extractions. Results are summarized in the table below. Conclusion: For all analytes and standards, the correlation coefficients of the linear regressions from these runs were 0.9900 or greater, meeting the requirement of SD or %CV ≤ TEa / 3. Validate the AMR by dilution. Acceptance criteria: %CV or SD should be ≤ TEa / 4 (Norepinephrine and epinephrine: %CV ≤ 5% or 1.25 ng / mL, where TEa = 20% or 5 ng / mL; Dopamine: %CV ≤ 5% or 2.50 ng / mL, where TEa = 30% or 10 ng / mL). Generate three independent calibration curves at six different levels within the AMR range using commercially available lyophilized standards. Correlate the results with the target concentration. Summarize the results in the table below. Conclusion: Calibration linearity was validated within the AMR and met acceptable criteria. The Analytical Measurement Range (AMR) is as follows: Reportable Scope (RR) Accuracy Recycling Research Recovery of known standards. Acceptance criteria: Accuracy % or SD should be ≤ TEa / 3 (norepinephrine and epinephrine: accuracy % ≤ 6.67% or 1.67 ng / mL, where TEa = 20% or 5 ng / mL; dopamine: accuracy % ≤ 10% or 5 ng / mL, where TEa = 30% or 10 ng / mL). Two sets of known standards were tested in independent extractions to quantify the results. The first set was commercially available lyophilized standards (Bio-Rad), and the second set was a self-made diluent containing Sigma stock standards. Twenty sets of BioRad standards were analyzed in 20 independent extractions, while 21 sets of Sigma standards were analyzed in 21 independent extractions. Figures 9A-9C The results are summarized in the table below. Conclusion: The difference between the calculated concentration and the expected concentration meets the acceptable standard of TEa / 3 in terms of both percentage of accuracy and standard deviation. Recovery by dilution. Acceptance criteria: Accuracy % or SD should be ≤ TEa / 3 (norepinephrine and epinephrine: accuracy % ≤ 6.67% or 1.67 ng / mL, where TEa = 20% or 5 ng / mL; dopamine: accuracy % ≤ 10% or 5 ng / mL, where TEa = 30% or 10 ng / mL). Stock solutions (1 mg / mL) of each standard (norepinephrine, epinephrine, dopamine) were added to human urine at concentrations higher than the AMR. Spiked human urine was recovered in triplicate through dilutions of 1 / 2, 1 / 5, 1 / 10, 1 / 25, 1 / 50, and 1 / 100. The diluted samples were injected, and the calculated average was compared with the spiked urine value to obtain the recovery % (%). Results are summarized in the table below. Conclusion: The difference between the calculated concentration and the expected concentration met the acceptable TEa / 3 criterion in terms of both percentage of accuracy and standard deviation. Recovery of previously tested (known) proficiency testing samples. Acceptance criteria: Accuracy % or SD should be ≤ TEa / 3 (norepinephrine and epinephrine: accuracy % ≤ 6.67% or 1.67 ng / mL, where TEa = 20% or 5 ng / mL; dopamine: accuracy % ≤ 10% or 5 ng / mL, where TEa = 30% or 10 ng / mL). Proficiency testing samples containing known amounts of urinary catecholamines were extracted and injected in 5 replicates. Proficiency testing samples were also investigated in an external laboratory on LC-MS / MS. Results were correlated with known amounts. Results are summarized in the table below. Conclusion: The difference between calculated and expected concentrations met the acceptable TEa / 3 criterion in terms of both percentage of accuracy and standard deviation. Comparative study of methods.

[0126] Acceptance criteria: Standard error (SE) should be less than TEa / 3 (norepinephrine and epinephrine: SE ≤ 6.67, where TEa = 20%; dopamine: SE ≤ 10, where TEa = 30%). Up to 112 samples were analyzed using the LC-MS / MS method on an ECD-HPLC platform and an Agilent 6495 StreamSelect system. Figures 10A-10C The results are summarized in the table below. Conclusion: The LCMSMS method on the Agilent 6495 StreamSelect system is acceptable for norepinephrine and dopamine. Epinephrine is acceptable at low concentrations because the sample volume is too small at high concentrations. Measurement uncertainty (MU) Definition: The degree of confidence in a specific measurement result. Expressed as a 95% confidence interval around a given number, it is determined by the mean of multiple measurements ± 1.96 × SD. For FDA-approved modified or unmodified tests, measurement uncertainty is determined by performing five measurements per day for five consecutive days at each QC level. The MU confidence interval is calculated using the precision worksheet in the Detection Validation Calculator template. MU is calculated by analyzing the data using the mean and standard deviation, then multiplying the SD by 1.96 and adding or subtracting from the mean. Confidence intervals for each QC level for each catecholamine are established and summarized in the table below. Specimen stability 24-hour urine samples and randomized samples were collected from patients. All samples were extracted for background measurements and grouped to ensure concentrations exceeded existing reference ranges. For all stability studies, a total of 5 24-hour urine samples and 6 randomized urine samples were extracted, with each sample being tested in triplicate. Results are summarized in the table below. Analytical specificity Interfering substances. Acceptance criteria: Differences caused by potential interfering substances should be ≤TEa / 4 to be considered acceptable. Due to the structural similarity between carbidopa and levodopa, a drug interference study was conducted. Samples were prepared by adding carbidopa and levodopa to the assay diluent to a final concentration of 2500 ng / mL. The spiked samples were extracted and analyzed three times. Conclusion: The interfering drug was below the limit of quantitation and determined not to cause interference.

[0127] Ion inhibition. Acceptance criteria: Signal intensity change after column infusion not exceeding 25%. Examine the total ion chromatogram (TIC) to determine if the signal intensity changes during analyte elution. Ion inhibition was detected using a post-column infusion method. A diluted catecholamine standard mixture (norepinephrine 2 μg / mL, epinephrine 1 μg / mL, dopamine 3 μg / mL) was infused at a constant flow rate (20 μL / min), followed by injection of 10 extraction blank samples containing the internal standard mixture. Changes in the signal response at corresponding retention times after analyte infusion indicated ionization inhibition. Conclusion: No ion inhibition was observed.

[0128] Residue. For this study, both injection residue and liquid processor (Hamilton) residue were assessed. For samples, commercially available spiked lyophilized standards were reconstituted and used for high-concentration samples, then diluted 1:50 for low-concentration samples. Acceptance criteria: Differences due to potential interfering substances ≤TEa / 4 were considered acceptable. The test sequence was: 2 low-concentration replicates, then 2 high-concentration replicates, followed by 3 low-concentration replicates. For independent test extractions, this sequence was repeated twice. Low-level spiked results were compared with low-level spiked results following high-level spiked results to determine residue. Conclusion: The results were acceptable, ≤TEa / 4, and no residue was observed in this test.

[0129] Reference interval (RI).

[0130] Patient-group comparisons between ECD-HPLC and LCMS-MS for urinary catecholamines indicated good correlation between the two methods. Acceptable R values ​​(R0.94 to 1) were obtained for all catecholamine analytes (corrected vs. raw). 2 The value is within the 95% confidence interval.

[0131] Specimen type Example 3 - Detection of catecholamines in human plasma without derivatization Catecholamines in human plasma samples can be detected using the methods summarized in the table below. Patient plasma samples. Patient plasma samples were prepared by combining heparinized plasma with an internal standard (IS), a stabilizer, and ammonium dihydrogen phosphate buffer. A solid-phase extraction plate (SPE) filled with phenylboronic acid (PBA) captured catecholamines via its cis-diol. The eluent was collected, dried, and reconstituted.

[0132] Catecholamine-free serum samples. These samples were prepared by combining 70% filtered catecholamine-free serum (diluted with 30% HPLC-grade water) with an internal standard (ISTD), stabilizer, and ammonium dihydrogen phosphate buffer. These steps were automated using a Hamilton Liquid Processor. Solid-phase extraction (SPE) was performed using an Agilent PBA plate on a Tecan A200. The eluent was collected, dried under nitrogen, reconstituted, and then injected.

[0133] Extracted samples were injected in 45 µL volumes into a Transcend TLX-4 multiplexed LC system (equipped with a Vanquish pump, ThermoFisher Scientific), coupled to a Sciex Triple Quad 7500 mass spectrometer in positive ion mode with a heated electrospray ionization source. Chromatographic separation was achieved using a PFP column (Phenomenex). Calibration curves were fitted using SciexOS quantitative software based on the peak area ratio of analyte ions to internal standards.

[0134] Use the following precursor ions and fragment ions: The total permissible error (TEa) for each analyte is as follows: Quality control (QC) materials were prepared by adding 25 pg / mL, 100 pg / mL, and 500 pg / mL of norepinephrine, epinephrine, and dopamine to diluted catecholamine-free serum. The spiked mixtures were divided into three groups at concentrations covering the expected reportable range of the assay. The QC mixtures were then aliquoted into 1.8 mL aliquots and stored in an ultra-low temperature freezer at -90°C to -60°C for accuracy data.

[0135] accuracy Accuracy study.

[0136] Intra-batch precision. Acceptance criteria: Within the run, CV ≤ TEa / 4 (≤ 6%) or SD ≤ 3 pg / mL (epinephrine), ≤ 6% or SD ≤ 6 pg / mL (dopamine), ≤ 5% or SD ≤ 19 pg / mL (norepinephrine). Five replicates of three control levels (low, medium, and high) were tested in a single run, in ascending order of replicate concentration. Control level replicates were stored in duplicate per tube; each tube contained 1.8 mL of control sample, which was used for two independently extracted replicates. Results are summarized in the table below. Intra-batch CVs are as follows: all three QC levels for epinephrine < 6%; norepinephrine QC 1 and 3 < 5%, QC 2 < 7% but SD < 19 pg / mL; dopamine 6%. Conclusion: Results for all analytes met the acceptance criteria. Precision between batches. Acceptance criteria: within run, CV ≤ TEa / 3 (≤ 8%) or SD < 4 pg / mL (epinephrine), ≤ 8% or SD < 7 pg / mL (dopamine), ≤ 7% or SD < 25 pg / mL (norepinephrine), Sigma ≥ 3. Five replicate sample analyses were performed for each control level in five independent settings. The results are summarized in the table below. The between-batch CVs are as follows: for the epinephrine QC levels, ≤ 7% (except 11% for level 1, but SD ≤ 4 pg / mL); for the norepinephrine QC levels, ≤ 7% (except 14% for level 1, but SD ≤ 25 pg / mL); for all dopamine QC levels, < 8%. Conclusion: For all analytes, the results are acceptable, with CV or concentration ≤ TEa / 3 and overall Sigma ≥ 3. Precision between operators. Acceptance criteria: SE < TEa / 4. Spiked standards, patient samples, QC replicate samples, and standards were compared. Samples and standards were extracted by two different laboratory personnel. The results are summarized in the table below. All analytes are acceptable, with SE < TEa / 4. Conclusion: For all three analytes, the results meet the acceptable reproducibility requirements. Analytical sensitivity (limit of detection) Limit of blank (LoB). LoB = blank mean + 2SD. Twenty (20) blank dilution samples were analyzed in one trial. No quantifiable catecholamine peaks were observed (epinephrine LoB: 0.46 pg / mL; norepinephrine LoB: 0.76 pg / mL; dopamine LoB: 0.06 pg / mL)( Figure 11A-11C ).

[0137] Limit of detection (LoD). LoD = blank mean + 4SD. Twenty (20) blank dilution samples were analyzed in two trials. No quantifiable catecholamine peaks were observed (epinephrine LoD: 0.80 pg / mL; norepinephrine LoD: 1.35 pg / mL; dopamine LoD: 0.12 pg / mL)( Figure 11A-11C )

[0138] Limit of Quantitation (LoQ). Acceptance criteria: The lowest detectable concentration at which SD < TEa / 3 and > LoD. To assess LoQ, three spiked samples were prepared using reference materials and added to diluted serum at levels close to or below the expected reportable levels. For epinephrine and dopamine: Five measurements were made for each spiked sample per run, for a total of five runs. The LoQ was determined by the lowest concentration at which SD < TEa / 3. Epinephrine LoQ: 4.84 or 5 pg / mL; norepinephrine LoQ: 7.81 or 8 pg / mL; dopamine LoQ: 5.11 or 5 pg / mL. Conclusion: The mean LoQ for all analytes was greater than the corresponding LoD, and SD < TEa / 3( Figure 11A-11C ).

[0139] Reportable Range (RR).

[0140] Analytical Measurement Range (AMR). Definition of quantitative and selected semi - quantitative tests: The reportable range is the result values of the entire range for which the accuracy of the instrument or testing system is established or verified. Expressed as the lower limit of the AMR and extending to or beyond the upper limit of the AMR. Based on the study, the AMR is as follows: Epinephrine: 5 to 1200 pg / mL; norepinephrine: 8 to 1200 pg / mL; dopamine: 5 to 1200 pg / mL.

[0141] Calibration Verification. Acceptance criteria: The deviation of the mean of the observed values from the expected values for each level should be < TEa / 3. A set of calibration standards was prepared and analyzed over three different extraction dates (approximately one month apart). All injections were made as single replicates. The differences between the target and observed concentrations were analyzed( Figures 12A-12C ). The results are summarized in the table below. For all analytes, the correlation coefficient of the linear regression for five runs was 0.990 or greater, and the mean observed CV < TEa / 3. Statistical analysis of the data indicated acceptable calibration performance. Conclusion: Calibration linearity was verified across the entire AMR range, meeting the acceptable accuracy criteria. Verification of AMR by dilution. Acceptance criteria: The differences between the values based on the regression line should be ≤ TEa / 3. A dilution study was performed using five samples of norepinephrine, epinephrine, and dopamine with known concentrations. The results are summarized in the table below. Conclusion: All analytes had acceptable accuracy up to a dilution factor of 2. Accuracy Recovery Study Recovery of known standards. Acceptance criterion: mean SD of spiked recovery ≤ TEa / 4. A spiked recovery study was conducted to assess the presence of a significant matrix effect in the assay. Baseline recovered serum samples (5 samples in total) were supplemented with 50 μL of 1200 pg / mL stock solution containing epinephrine, noradrenaline, and dopamine to a final volume of 950 μL per sample. Results are summarized in the table below. The mean SE% for each analyte was ≤ TEa / 4. Conclusion: The recovery ranges for all analytes were considered acceptable according to the pre-specified criteria. Sampling comparison. Acceptance criteria: The absolute value of the difference between the means should be less than TEa / 4. Patient samples were collected and each sample was divided into two equal aliquots; one aliquot was tested using the original method in a single replicate sample, and the other aliquot was tested using the proposed new method in a double replicate sample. Figures 13A-13C The results are summarized in the table below. At least 102 samples were analyzed on both the current platform (ECD-HPLC) and the proposed LCMS / MS platform. When assessing the acceptability of the fractional comparative study, care should be taken to note that the newer comparative study used different extraction methods and detectors. Conclusion: Negative bias was observed for all three analytes, with detection values ​​from the operations data generally higher than R&D observations. The proposed method has higher specificity than the existing platform. Measurement uncertainty (MU).

[0142] For modified or unmodified tests approved by the FDA, measurement uncertainty is determined by performing five measurements per day for five consecutive days at each QC level. If the MU study must be completed within fewer than five independent working days, each of the five QC values ​​must be performed in a separate run, and this reduction in time must be noted in the final accuracy study. The MU confidence interval is calculated using the accuracy worksheet in the Detection Validation Calculator template. The MU is calculated by analyzing the data using the mean and standard deviation, then multiplying the SD by 1.96 and adding or subtracting from the mean. The results are summarized in the table below: Specimen stability.

[0143] Specimen stability is shown in the table below. Analyze specificity.

[0144] Hemolytic interference. Acceptance criteria: Differences caused by potential interfering substances should be ≤TEa / 4 to be considered acceptable. The effect of hemolysis in the assay was assessed by adding hemolyzed RBCs at concentrations of approximately 150 g / L to three patient samples (low concentration 1:20, medium concentration 1:5, and high concentration 1:10 to simulate mild, moderate, and severe hemolysis). These spiked samples were measured together with a control group of identical samples (without interfering substances spiked, but with an equal volume of 10 mM PBS). Figures 14A-14C Results: Differences due to potential interfering substances in all analytes were ≤TEa / 4, ranging from upper to moderate hemolysis levels. Conclusion: Mild to moderate hemolysis in plasma samples is acceptable. Severe hemolysis is unacceptable.

[0145] Bilirubin interference. Acceptance criteria: Differences caused by potential interfering substances should be ≤TEa / 4 to be considered acceptable. 1 mg / mL bilirubin samples were prepared in 10 mM PBS solution and further diluted to 1:10 and 1:20 to simulate mild and moderate jaundice levels. The effect of bilirubin on the detection was assessed by adding low, medium, and high concentrations of bilirubin to three patient samples to simulate mild, moderate, and severe jaundice interference. These spiked samples were measured together with a control group of identical samples (without interfering substances, but with an equal volume of 10 mM PBS). Figures 15A-15C Results: At all bilirubin levels, the differences in potential interfering substances for all analytes were ≤TEa / 4. Bilirubin did not interfere with the quantification of human plasma catecholamines. Conclusion: Plasma samples presenting with mild to severe jaundice are acceptable.

[0146] Lipid interference. Acceptance criteria: Differences caused by potential interfering substances ≤ TEa / 4 are considered acceptable. The effect of lipemia on the test was assessed by adding low, medium, and high concentrations of fat emulsion to three patient samples to simulate mild, moderate, and severe lipemia. 1 mL of fat emulsion was diluted in 4 mL of 10 mM PBS. This was further diluted to 1:10 and 1:20 to simulate mild, moderate, and severe lipemia. These spiked samples were measured together with a control group of identical samples (without interfering substances spiked, but with an equal volume of 10 mM PBS). Figures 16A-16C Results: Differences in all analytes due to potential interfering substances were ≤TEa / 4. Lipidemia did not interfere with the quantification of human plasma catecholamines. Conclusion: Plasma samples with mild to severe lipemia are acceptable.

[0147] Drug interference. Acceptance criteria: The difference caused by potential interfering substances should be ≤ TEa / 4 and is considered acceptable. The drug interference study was conducted using levodopa, which is known to increase dopamine levels in plasma and urine (Grouzmann 2013), but its molecular structure is different from that of the three analytes. Samples were prepared by adding known concentrations of catecholamines and levodopa to stripped serum. All samples were analyzed in duplicate on LCMSMS. These spiked samples were assayed together with a set of identical samples (without interfering substance spikes, but with an equal volume of 10 mM PBS) as controls. Results: The retention times of the product peaks related to levodopa were always separated from the expected retention times of all analytes, confirming that levodopa did not cause interference. Conclusion: No interference in response to levodopa spikes was detected.

[0148] Ion suppression. Acceptance criteria: The change in signal intensity upon post-column infusion should not exceed ±25%. A catecholamine standard of 100000 pg / mL was infused post-column at 9 μL / min, and 1 blank sample and 5 patient samples were injected simultaneously. The chromatograms were observed, and then it was determined whether a change in signal intensity occurred during the elution of the samples. The percentage of ion suppression was calculated as the percentage difference between the approximate average TIC heights before the analyte RT and around the direct analyte RT. Results: Minimal ion suppression was observed. Conclusion: No interference exceeding 25% due to ion suppression was detected.

[0149] Carryover.

[0150] Acceptance criteria: The difference between sample types should be < TEa / 2. The carryover test was conducted in 3 independent trials. During each extraction, the samples were injected in a specific order: 2 low-concentration samples, then 2 high-concentration samples, and then 3 low-concentration samples. Results: The data showed that the average percentage differences for epinephrine, norepinephrine, and dopamine were all less than TEa / 4. Conclusion: No carryover was detected for the detection of epinephrine, norepinephrine, and dopamine.

[0151] Reference interval (RI).

[0152] The reference range intervals are as follows: Specimen type.

[0153] Acceptance criteria: Differences due to variations in storage containers and / or specimen type should be ≤TEa / 4 to be considered acceptable. Five venous blood samples were collected from each of up to 10 randomized patients using standard venipuncture techniques. Plasma and serum results were compared with plasma results obtained from currently acceptable tubing types (heparin sodium) using the ECD-HPLC platform. Results: Data showed that the mean residues of adrenaline, noradrenaline, and dopamine were all below TEa / 4. Conclusion: Plasma samples collected from EDTA-containing tubes and lithium heparin tubes are acceptable. All articles, patents, and patent applications mentioned or cited herein, as well as all other documents and electronically available information, are incorporated herein by reference in their entirety to the same extent that each individual publication is specifically and individually indicated to be incorporated herein by reference. The applicant reserves the right to materialize and incorporate any and all material and information in any such articles, patents, patent applications, or other tangible and electronic documents into this application.

[0154] The methods described herein can be suitably practiced without any one or more elements or limitations not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be interpreted broadly and without limitation. Furthermore, the terms and expressions used herein have been used as descriptive rather than restrictive terms, and their use is not intended to exclude any equivalents of the shown and described features or portions thereof. It should be recognized that various modifications can be made within the scope of the invention as described in the claims. Therefore, it should be understood that although the invention has been specifically disclosed by way of preferred embodiments and optional features, modifications and variations can be made to the invention disclosed herein by those skilled in the art, and such modifications and variations are considered to fall within the scope of protection of this invention.

[0155] This invention has been described broadly and generally. Narrower species and subgenus groups falling within this general disclosure constitute part of the method. This includes a general description of the method, wherein additional limiting conditions or negative restrictions may exclude any subject matter of the general description, whether or not the excluded content is expressly listed in this specification.

[0156] Other embodiments are all included in the claims. Furthermore, when features or aspects of the method are described in Markush group form, those skilled in the art will understand that the present invention also covers any single member or subgroup of members within the Markush group.

Claims

1. A method of determining the amount of an analyte in a sample, the method comprising: purifying the sample by liquid chromatography; subjecting the sample to a derivatization reagent to generate a derivatized sample; subjecting the derivatized sample to electrospray ionization (ESI) under conditions suitable to produce one or more ions capable of detection by mass spectrometry; and quantifying the amount of the analyte by tandem mass spectrometry, wherein the quantifying comprises quantifying the amount of the ions, and the amount of the ions detected is correlated to the amount of the analyte in the sample; wherein: the limit of quantification of the method is less than 15 pg / mL; the analyte comprises epinephrine, norepinephrine, dopamine, or a mixture of any two or more thereof; and the derivatization reagent comprises phenylisothiocyanate (PITC).

2. The method of claim 1, wherein one of the one or more analytes is norepinephrine.

3. The method of claim 2, wherein the quantifying comprises quantifying the amount of a precursor ion having a mass-to-charge ratio of 305.2 ± 0.

5.

4. The method of claim 2 or 3, wherein the quantifying comprises quantifying the amount of one or more fragment ions having a mass-to-charge ratio selected from 107.0 ± 0.5 and 152.2 ± 0.

5.

5. The method of claim 1, wherein one of the one or more analytes is epinephrine.

6. The method of claim 5, wherein the quantifying comprises quantifying the amount of a precursor ion having a mass-to-charge ratio of 319.1 ± 0.

5.

7. The method of claim 5 or 6, wherein the quantifying comprises quantifying the amount of one or more fragment ions having a mass-to-charge ratio selected from 166.1 ± 0.5 and 107.0 ± 0.

5.

8. The method of claim 1, wherein one of the one or more analytes is dopamine.

9. The method of claim 8, wherein the quantifying comprises quantifying the amount of a precursor ion having a mass-to-charge ratio of 289.1 ± 0.

5.

10. The method of claim 8 or 9, wherein the quantifying comprises quantifying the amount of one or more fragment ions having a mass-to-charge ratio selected from 137.2 ± 0.5 and 153.2 ± 0.

5.

11. The method of any one of claims 1-10, wherein the sample is a plasma sample.

12. The method of any one of claims 1-11, wherein the limit of quantification of the method is less than or equal to 10 pg / mL.

13. The method of any one of claims 1-12, wherein the analyte is purified by high performance liquid chromatography (HPLC) prior to ionization.

14. The method of any one of claims 1-13, wherein the analyte is purified by solid phase extraction (SPE) prior to ionization.

15. The method of any one of claims 1-14, wherein the analyte is purified by immobilized boronic acid extraction prior to ionization.

16. The method of any one of claims 1-15, wherein the amount of two or more analytes in the group consisting of epinephrine, norepinephrine, and dopamine are determined in the same sample injection.

17. The method of any one of claims 1-16, wherein the amounts of epinephrine, norepinephrine, and dopamine are determined in the same sample injection.

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