Methods for detecting catecholamines by mass spectrometry

Abstract

Provided are methods for determining the amount of one or more of one or more of norepinephrine (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 the ions to determine the amount of one or more of NE, E, and D in the sample.

Description

METHODS FOR DETECTING CATECHOLAMINES BY MASS SPECTROMETRYCROSS-REFERENCE TO RELATED APPLICATIONS[00011 This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 517,410, filed on August 3, 2023, the contents of which are incorporated herein by reference in their entirety.FIELD

[0002] The present technology relates to the detection of catecholamines. In a particular aspect, the technology relates to methods for detecting epinephrine, norepinephrine, and / or dopamine by mass spectrometry.BACKGROUND

[0003] Catecholamines include the hormones norepinephrine (NE), epinephrine (E), and dopamine (DA), which are the principal secretory products of neural tissue. In particular, catecholamines can be produced in chromaffin cell tumors, pheochromocytoma, ganglioneuroma, and neuroblastoma. Accordingly, the measurement of circulating catecholamines is valuable in the diagnosis of catecholamine-secreting tumors associated with hypertension (e.g., pheochromocytomas, neuroblastomas, and / or gangliomas) and with the evaluation of orthostatic hypotension. Since early diagnosis and treatment of these diseases is critical, fast and accurate detection of these hormones is desirable.

[0004] Existing methods using high-performance liquid chromatography (HPLC) coupled with an electrochemical detector (ECD) have limitations including, specificity, long run times, and outdated unsupported instrument components. In contrast, liquid chromatography with tandem mass spectrometry (LC-MS / MS) offers several advantages such as increased sensitivity and specificity, shorter run times with more advanced pump systems, and multiplexing capabilities for faster sample throughput. Provided herein are LC-MS / MS methods for detecting catecholamines in a biological sample (e.g., a urine or plasma sample).

[0005] Urine is a complex matrix for LC-MS / MS and usually requires enrichment of catecholamines to be a viable alternative to ECD. In the present disclosure, the LC-MS / MS methods described herein can detect catecholamines after their specific extraction from urine.|0006] Plasma catecholamine levels reflect the balance between biosynthesis, release, uptake, catabolism, and excretion of the catecholamines at the time of sampling. The concentration of catecholamines and their metabolites in urine represent an overall estimate of sympathetic activity during relatively long periods of time and may not reflect the influence of transient changes in plasma levels. Therefore, in some cases of pheochromocytoma when paroxysmal attacks are very short, urine values may not be raised above the normal limits. In such patients, the measurement of plasma catecholamines levels during a spontaneous paroxysm or during an attack provoked by histamine or glucagon is an effective way of confirming the diagnosis. Measurements of plasma norepinephrine are useful in diagnosing patients with orthostatic hypotension. Failure to increase the supine norepinephrine concentration on standing suggests a sympathetic nervous system disorder. Plasma norepinephrine is an independent risk factor in patients with chronic congestive heart failure that relates to subsequent mortality.Norepinephrine is useful in evaluating patients with hypertension. Under conditions of relatively stable sympathetic activity or when the induced changes are prolonged, the urinary levels of catecholamines may be directly proportional to those in the blood.

[0007] Accordingly, it is important to detect these hormones with sufficient accuracy and efficiency. Thus, improved methods for measuring the concentrations of catecholamines are desirable.SUMMARY

[0008] Disclosed herein are methods for determining an amount of an analyte in a sample, the method comprising: purifying the sample by liquid chromatography; subjecting the sample to a derivatizing agent to generate a derivatized sample; subjecting the derivatized sample to electrospray ionization (ESI) under conditions suitable to produce one or more ions detectable by mass spectrometry; andquantifying the amount of the analyte by tandem mass spectrometry, wherein the quantifying comprises quantifying an amount of the ion(s), and the amount of the ion(s) detected is related to the amount of the analyte in the sample; wherein: the limit of quantitation 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 agent comprises phenylisothiocyanate (PITC).10009] In some embodiments, the one of the one or more analytes is norepinephrine. In some embodiments, the quantifying comprises quantifying the amount of precursor ion having a mass-to-charge ratio of 305.2 ± 0.5. In some embodiments, the quantifying comprises quantifying the amount of one or more fragment ion(s) having a mass-to-charge ratio selected from the group consisting of 107.0 ± 0.5 and 152.2 ± 0.5.

[0010] In some embodiments, the one of the one or more analytes is epinephrine. In some embodiments, the quantifying comprises quantifying the amount of precursor ion having a mass-to-charge ratio of 319.1±0.5. In some embodiments, the quantifying comprises quantifying the amount of one or more fragment ion(s) having a mass-to-charge ratio selected from the group consisting of 166.1± 0.5 and 107.0 ± 0.5.

[0011] In some embodiments, the one of the one or more analytes is dopamine. In some embodiments, the quantifying comprises quantifying the amount of precursor ion having a mass- to-charge ratio of 289.1 ± 0.5. In some embodiments, the quantifying comprises quantifying the amount of one or more fragment ion(s) having a mass-to-charge ratio selected from the group consisting of 137.2 ± 0.5 and 153.2 ± 0.5.]0012] In some embodiments, the sample is a plasma sample.[0013J In some embodiments, the limit of quantitation of the methods is less than or equal to 10 pg / mL.

[0014] In some embodiments, the analytes are purified by high performance liquid chromatography (HPLC) prior to ionization. In some embodiments, the analytes are purified bysolid phase extraction (SPE) prior to ionization. In some embodiments, the analytes are purified by immobilized boronic acid extraction prior to ionization.

[0015] In some embodiments, the amounts of two or more of the analytes from the group consisting of epinephrine, norepinephrine, and dopamine are determined in the same sample injection.

[0016] In some embodiments, the amounts of epinephrine, norepinephrine, and dopamine are determined in the same sample injection.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIGS. 1A-1C show the Limit of Detection and Limit of Quantitation for Norepinephrine (FIG. 1A), Epinephrine (FIG. IB), and Dopamine (FIG. 1C), respectively.

[0018] FIGS. 2A-2C show the calibration curve for Norepinephrine (FIG. 2A), Epinephrine (FIG. 2B), and Dopamine (FIG. 2C), respectively.

[0019] FIGS. 3A-3C show the analytical measurement range validation by dilution (ClinCal® Standards) for Norepinephrine (FIG. 3A), Epinephrine (FIG. 3B), and Dopamine (FIG. 3C), respectively.

[0020] FIG. 4A shows Norepinephrine recovery of known standards. FIG. 4B shows Epinephrine recovery of known standards. FIG. 4C shows Dopamine recovery of known standards.

[0021] FIG. 5A shows norepinephrine split sample comparison (all samples included). FIG. 5B shows norepinephrine split sample comparison (samples above linearity excluded). FIG. 5C shows epinephrine split sample comparison. FIG. 5D shows dopamine split sample comparison.

[0022] FIGS. 6A-6C show the interference study for Norepinephrine (FIG. 6A), Epinephrine (FIG. 6B), and Dopamine (FIG. 6C), respectively.

[0023] FIG. 7A shows the sample type study of norepinephrine split sample comparison. FIG. 7B shows the sample type study of epinephrine split sample comparison. FIG. 7C shows the sample type study of dopamine split sample comparison.

[0024] FIGS. 8A-8C show the Limit of Detection, Limit of Quantitation and Limit ofQuantitation for Norepinephrine (FIG. 8A), Epinephrine (FIG. 8B), and Dopamine (FIG. 8C), respectively.

[0025] FIG. 9A shows Norepinephrine recovery of known standards. FIG. 9B shows Epinephrine recovery of known standards. FIG. 9C shows Dopamine recovery of known standards.

[0026] FIG. 10A shows norepinephrine split sample comparison (bottom right: corrected to pg / 24hr or pg / g CREAT). FIG. 10B shows epinephrine split sample comparison (bottom right: corrected to pg / 24hr or pg / g CREAT). FIG. 10C shows dopamine split sample comparison (bottom right: corrected to pg / 24hr or pg / g CREAT).

[0027] FIGS. 11A-11C show the Limit of Blank, Limit of Detection, and Limit of Quantitation for Norepinephrine (FIG. 11 A), Epinephrine (FIG. 11B), and Dopamine (FIG. 11C), respectively.

[0028] FIGS. 12A-12C show the calibration curve for Norepinephrine (FIG. 12A), Epinephrine (FIG. 12B), and Dopamine (FIG. 12C), respectively.

[0029] FIG. 13A shows norepinephrine split sample comparison. Note: the 7 outlier points (shown in the box) that are skewing at a notably higher bias towards Ops and greatly lowering the observed r-value, are due to the expected higher interference found in the ECD- HPLC compared to the newer more precise LC-MSMS utilized in R&D. FIG. 13B shows epinephrine split sample comparison. FIG. 13C shows dopamine split sample comparison.

[0030] FIGS. 14A-14C show the hemolysis interference study for Norepinephrine (FIG. 14 A), Epinephrine (FIG. 14B), and Dopamine (FIG. 14C), respectively.

[0031] FIGS. 15A-15C show the bilirubin interference study for Norepinephrine (FIG. 15 A), Epinephrine (FIG. 15B), and Dopamine (FIG. 15C), respectively.

[0032] FIGS. 16A-16C show the lipemia interference study for Norepinephrine (FIG. 16 A), Epinephrine (FIG. 16B), and Dopamine (FIG. 16C), respectively.DETAILED DESCRIPTIONDefinitions

[0033] In order for the present technology to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the technology and to provide additional detail regarding its practice are hereby incorporated by reference.

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

[0035] As used herein, the term “purification” or “purifying” does not refer to removing all materials from the sample other than the analyte(s) of interest. Instead, purification refers to a procedure that enriches the amount of one or more analytes of interest relative to other components in the sample that may interfere with detection of the analyte of interest.Purification of the sample by various means may allow relative reduction of one or more interfering substances, e.g., one or more substances that may or may not interfere with the detection of selected precursor or fragment ions by mass spectrometry. Relative reduction as this term is used does not require that any substance, present with the analyte of interest in the material to be purified, is entirely removed by 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” means any fluid that can be isolated from the body of an individual. For example, “body fluid” may include blood, plasma, serum, bile, saliva, urine, tears, perspiration, and the like.

[0037] As used herein, the term “solid phase extraction” or “SPE” refers to a process in which a chemical mixture is separated into components as a result of the affinity of componentsdissolved or suspended in a solution (i.e., mobile phase) for a solid through or around which the solution is passed solid phase). In some instances, as the mobile phase passes through or around the solid phase, undesired components of the mobile phase may be retained by the solid phase resulting in a purification of the analyte in the mobile phase. In other instances, the analyte may be retained by the solid phase, allowing undesired components of the mobile phase to pass through or around the solid phase. In these instances, a second mobile phase is then used to elute the retained analyte off of the solid phase for further processing or analysis.

[0038] As used herein, the term “chromatography” refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase.

[0039] As used herein, the term “liquid chromatography” or “LC” means a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this fluid moves relative to the stationary phase(s). Examples of “liquid chromatography” include reverse phase liquid chromatography (RPLC), high-performance liquid chromatography (HPLC), and turbulent flow liquid chromatography (sometimes known as 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 in which the degree of separation is increased by forcing the mobile phase under pressure through a stationary phase, typically a densely packed column.

[0041] As used herein, the term “turbulent flow liquid chromatography” or “TFLC” (sometimes known as high turbulence liquid chromatography (HTLC) or high throughput liquid chromatography) refers to a form of chromatography that utilizes turbulent flow of the material being assayed through the column packing as the basis for performing the separation. TFLC has been applied in the preparation of samples containing two unnamed drugs prior to analysis by mass spectrometry. See, e.g., Zimmer et al., J Chromatogr A 854: 23-35 (1999); see also, U.S.Pat. Nos. 5,968,367, 5,919,368, 5,795,469, and 5,772,874, which further explain TFLC. Persons of ordinary skill in the art understand “turbulent flow”. When fluid flows slowly and smoothly, the 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 the particles of fluid is orderly with particles moving generally in straight lines. At faster velocities, the inertia of the water overcomes fluid frictional forces and turbulent flow results. Fluid not in contact with the irregular boundary “outruns” that which is slowed by friction or deflected by an uneven surface. When a fluid is flowing turbulently, it flows in eddies and whirls (or vortices), with more “drag” than when the flow is laminar. Many references are available for assisting in determining when fluid flow is laminar or turbulent (e.g., Turbulent Flow Analysis: Measurement and Prediction, P. S. Bernard and J. M. Wallace, John Wiley and Sons, Inc., (2000); An Introduction to Turbulent Flow, jean Mathieu and Julian Scott, Cambridge University Press (2001)).

[0042] As used herein, the term “gas chromatography” or “GC” refers to chromatography in which the sample mixture is vaporized and injected into a stream of carrier gas (such as nitrogen or helium) moving through a column containing a stationary phase composed of a liquid or a particulate solid and is separated into its component compounds according to the affinity of the compounds for the stationary phase.

[0043] As used herein, the term “large particle column” or “extraction column” refers to a chromatography column containing an average particle diameter greater than about 50 pm. As used in this context, the term “about” means±10%.

[0044] As used herein, the term “analytical column” refers to a chromatography column having sufficient chromatographic plates to affect a separation of materials in a sample that elute from the column sufficient to allow a determination of the presence or amount of an analyte. Such columns are often distinguished from “extraction columns”, which have the general purpose of separating or extracting retained material from non-retained materials in order to obtain a purified sample for further analysis. As used in this context, the term “about” means 10%. In some embodiments, the analytical column contains particles of about 4 pm in diameter.

[0045] As used herein, the term “on-line” or “inline”, for example as used in “on-line automated fashion” or “on-line extraction” refers to a procedure performed without the need foroperator intervention. In contrast, the term “off-line” as used herein refers to a procedure requiring manual intervention of an operator. Thus, if samples are subjected to precipitation, and the supernatants are then manually loaded into an autosampler, the precipitation and loading steps are off-line from the subsequent steps. In various embodiments of the methods, one or more steps may be performed in an on-line automated fashion.

[0046] As used herein, the term “sample injection” refers to introducing an aliquot of a single sample into an analytical instrument, for example a mass spectrometer. This introduction may occur directly or indirectly. An indirect sample injection may be accomplished, for example, by injecting an aliquot of a sample into a HPLC column that is connected to a mass spectrometer in an on-line fashion.

[0047] As used herein, the term “same sample injection” with respect to multiple analyte analysis by mass spectrometry means that the ions for two or more different analytes are determined essentially simultaneously by measuring ions for the different analytes from the same (i.e. identical) sample injection.

[0048] As used herein, the term “mass spectrometry” or “MS” refers to an analytical technique to identify compounds by their mass. MS refers to methods of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or “m / z”. MS technology generally includes (1) ionizing the compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating a mass-to-charge ratio. The compounds may be ionized and detected by any suitable means. A “mass spectrometer” generally includes an ionizer and an ion detector. In general, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrographic instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent upon mass (“m”) and charge (“z”). See, e.g., U.S. Pat. Nos. 6,204, 500, entitled “Mass Spectrometry From Surfaces,” 6,107, 623, entitled “Methods and Apparatus for Tandem Mass Spectrometry,” 6,268,144, entitled “DNA Diagnostics Based On Mass Spectrometry,” 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 those mass spectrometry methods where negative ions are generated and detected. The term “operating in positive ion mode” as used herein, refers to those mass spectrometry methods where positive ions are generated and detected. As used herein, the term “ionization” or “ionizing” refers to the process of generating an analyte ion having a net electrical charge equal to one or more electron units. Negative ions are those having a net negative charge of one or more electron units, while positive ions are those having a net positive charge of one or more electron units.

[0050] As used herein, the term “electron ionization” or “El” refers to methods in which an analyte of interest in a gaseous or vapor phase interacts with a flow of electrons. Impact of the electrons with the analyte produces analyte ions, which may then be subjected to a mass spectrometry technique.(0051 ] As used herein, the term “chemical ionization” or “CI” refers to methods in which a reagent gas (e.g. ammonia) is subjected to electron impact, and analyte ions are formed by the interaction of reagent gas ions and analyte molecules.

[0052] As used herein, the term “fast atom bombardment” or “FAB” refers to methods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in the sample. Test samples are dissolved in a viscous liquid matrix such as glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether, 2- nitrophenyl octyl ether, sulfolane, diethanolamine, and triethanolamine. The choice of an appropriate matrix for a compound or sample is an empirical process.|0053] As used herein, the term “matrix-assisted laser desorption ionization” or “MALDI” refers to methods in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample by various ionization pathways, including photo-ionization, protonation, deprotonation, and cluster decay. For MALDI, the sample is mixed with an energy-absorbing matrix, which facilitates 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 analytes in the sample by various ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. For SELDI the sample istypically bound to a surface that preferentially retains one or more analytes of interest. As in MALDI, this process may also employ an energy-absorbing material to facilitate ionization.

[0055] As used herein, the term “electrospray ionization” or “ESI,” refers to methods in which a solution is passed along a short length of capillary tube, to the end of which is applied a high positive or negative electric potential. Solution reaching the end of the tube is vaporized (nebulized) into a jet or spray of very small droplets of solution in solvent vapor. This mist of droplets flows through an evaporation chamber, which may be heated to prevent condensation and to facilitate solvent evaporation. As the droplets get smaller the electrical surface charge density increases until such time that the natural repulsion between like charges causes ions as well as neutral molecules to be released.

[0056] As used herein, the term “atmospheric pressure chemical ionization” or “APCI,” refers to mass spectrometry methods that are similar to ESI; however, APCI produces ions by ion-molecule reactions that occur within a plasma at atmospheric pressure. The plasma is maintained by an electric discharge between the spray capillary and a counter electrode. Then ions are typically extracted into the mass analyzer by use of a set of differentially pumped skimmer stages. A counterflow of dry and preheated N2 gas may be used to improve removal of solvent. The gas-phase ionization in APCI can be more effective than ESI for analyzing less- polar species.

[0057] The term “atmospheric pressure photoionization” or “APPI” as used herein refers to the form of mass spectrometry where the mechanism for the photoionization of molecule M is photon absorption and electron ejection to form the molecular ion M+. Because the photon energy typically is just above the ionization potential, the molecular ion is less susceptible to dissociation. In many cases it may be possible to analyze samples without the need for chromatography, thus saving significant time and expense. In the presence of water vapor or protic solvents, the molecular ion can extract H to form MH+. This tends to occur if M has a high proton affinity. This does not affect quantitation accuracy because the sum of M+ and MH+ is constant. Drug compounds in protic solvents are usually observed as MH+, whereas nonpolar compounds such as naphthalene or testosterone usually form M+, See, e.g., Robb et al., Anal. Chem. 2000, 72(15): 3653-3659.(0058] As used herein, the term “inductively coupled plasma” or “ICP” refers to methods in which a sample interacts with a partially ionized gas at a sufficiently high temperature such that most elements are atomized and ionized.

[0059] As used herein, the term “field desorption” refers to methods in which a nonvolatile test sample is placed on an ionization surface, and an intense electric field is used to generate analyte ions.

[0060] As used herein, the term “desorption” refers to the removal of an analyte from a surface and / or the entry of an analyte into a gaseous phase. Laser desorption thermal desorption is a technique wherein a sample containing the analyte is thermally desorbed into the gas phase by a laser pulse. The laser hits the back of a specially made 96-well plate with a metal base. The laser pulse heats the base and the heat causes the sample to transfer into the gas phase. The gas phase sample is then drawn into the mass spectrometer.[00611 As used herein, the term “selective ion monitoring” is a detection mode for a mass spectrometric instrument in which only ions within a relatively narrow mass range, typically about one mass unit, are detected.

[0062] As used herein, “multiple reaction mode,” sometimes known as “selected reaction monitoring,” is a detection mode for a mass spectrometric instrument in which a precursor ion and one or more fragment ions are selectively detected.

[0063] As used herein, the term “limit of quantification”, “limit of quantitation” or “LOQ” refers to the point where measurements become quantitatively meaningful. The analyte response at this LOQ is identifiable, discrete and reproducible 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” is the point at which the measured value is larger than the uncertainty associated with it. The LOD is the point at which a value is beyond the uncertainty associated with its measurement and is defined as two times the RSD of the mean at the zero concentration.|0065] As used herein, an “amount” of an analyte in a body fluid sample refers generally to an absolute value reflecting the mass of the analyte detectable in volume of body fluid. However, an amount also contemplates a relative amount in comparison to another analyte amount. For example, an amount of analyte in a body fluid can be an amount which is greater than a control or normal level of analyte normally present.

[0066] The term “about” as used herein in reference to quantitative measurements not including the measurement of the mass of an ion, refers to the indicated value plus or minus 10%. Mass spectrometry instruments can vary slightly in determining the mass of a given analyte. The term “about” in the context of the mass of an ion or the mass / charge ratio of an ion refers to 0.50 atomic mass unit.

[0067] The summary above is non-limiting and other features and advantages of the technology will be apparent from the following detailed description, and from the claims.

[0068] Disclosed herein are methods for measuring the amount of one or more catecholamines from the group consisting of norepinephrine, epinephrine, and dopamine in a sample. In particular, mass spectrometric methods are described for detecting in a sample (e.g., urine or plasma sample) one or more catecholamines from the group consisting of norepinephrine, epinephrine, and dopamine. For example, any one of the catecholamines NE, E, and D in a sample may be detected by mass spectrometry; or any two of NE, E, and D in a sample may be detected by mass spectrometry; or all three of NE, E, and D in a sample may be detected by mass spectrometry. The methods may utilize high-performance liquid chromatography (HPLC), to perform purification of selected analytes, and combine this purification with methods of mass spectrometry (MS), thereby providing a high-throughput assay system for detecting and quantifying one or more catecholamines from the group consisting of NE, E, and D in a sample. The preferred embodiments are particularly well suited for application in large clinical laboratories for automated catecholamine assay.

[0069] Suitable samples for use in methods include any sample that may contain the analyte of interest. In some embodiments, a sample is a biological sample; that is, an aqueous sample obtained from any biological source, such as an animal, a cell culture, an organ culture, etc. In certain preferred embodiments, samples are obtained from a mammalian animal, such asa dog, cat, horse, etc. Particularly preferred mammalian animals are primates, most preferably male or female humans. In some embodiments, a sample includes bodily fluids such as urine, blood, plasma, serum, saliva, cerebrospinal fluid, or tissue samples. In some embodiments, a sample is a urine sample. In some embodiments, a sample is a plasma sample.

[0070] Samples may be obtained, for example, from a patient; that is, a living person, male or female, presenting oneself in a clinical setting for diagnosis, prognosis, or treatment of a disease or condition, such as pheochromocytoma, ganglioneuroma, neuroblastoma, gangliomas, 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). A preservative (i.e., an agent that maintains a pH below about 3, such as 6N HC1) should be added to urine specimen that is not immediately analyzed because the stability of catecholamines declines as the pH rises, with destruction becoming extremely rapid in an alkaline medium.

[0071] Also presented are kits for an NE and / or E and / or D quantitation assay. Such kits comprise one or more internal standards, in amounts sufficient for at least one assay. Typically, the kits will also include instructions recorded in a tangible form (e.g., contained on paper or an electronic medium) for using the packaged reagents for use in a measurement assay for determining the amount of NE and / or E and / or D.

[0072] Calibration and QC pools for use in various embodiments are preferably prepared using a matrix similar to the intended sample matrix.Sample Preparation

[0073] Samples may be prepared for mass spectrometry by enriching NE and / or E and / or D in the sample by any appropriate methods. Enrichment of NE and / or E and / or D relative to other components in the sample (e.g. protein) prior to mass spectrometry may be accomplished by various methods known in the art, including for example, liquid chromatography, filtration, centrifugation, thin layer chromatography (TLC), electrophoresis including capillary electrophoresis, affinity separations including immunoaffinity separations, liquid-liquid or solid phase extraction methods including ethyl acetate or methanol extraction or pretreatment withimmobilized boronic acid (via a gel or otherwise), the use of chaotropic agents, or any combination of the above or the like.

[0074] Protein precipitation is another method of preparing a test sample, especially a biological test sample, such as serum, plasma, or urine. Such protein purification methods are well known in the art, for example, Poison et al., Journal of Chromatography B 2003, 785:263- 275, describes protein precipitation techniques suitable for use in methods described herein. Protein precipitation may be used to remove most of the protein from the sample leaving NE and / or E and / or D in the supernatant. The samples may be centrifuged to separate the liquid supernatant from the precipitated proteins; alternatively the samples may be filtered to remove precipitated proteins. The resultant supernatant or filtrate may then be applied directly to mass spectrometry analysis; or alternatively to liquid chromatography and subsequent mass spectrometry analysis. In certain embodiments, the use of protein precipitation such as for example, formic acid protein precipitation, may obviate the need for HTLC or other on-line extraction prior to mass spectrometry or HPLC and mass spectrometry.10075 [ One means of sample purification that may be used prior to mass spectrometry is liquid chromatography (LC). Certain methods of liquid chromatography, including HPLC, rely on relatively slow, laminar flow technology. Traditional HPLC analysis relies on column packing in which laminar flow of the sample through the column is the basis for separation of the analyte of interest from the sample. The skilled artisan will understand that separation in such columns is a diffusional process and may select HPLC instruments and columns that are suitable for use with NE (or derivatized NE), E (or derivatized E), and / or D (or derivatized D). The chromatographic column typically includes a medium i.e., a packing material) to facilitate separation of chemical moieties (i.e., fractionation). The medium may include minute particles. The particles include a bonded surface that interacts with the various chemical moieties to facilitate separation of the chemical moieties. One suitable bonded surface is a hydrophobic bonded surface such as an alkyl bonded, a cyano-bonded, or a pentafluorophenyl propyl (F5) surface. Alkyl bonded surfaces may include C-4, C-8, C-12, or C-18 bonded alkyl groups. In preferred embodiments, the column is an F5 column. The chromatographic column includes an inlet port for receiving a sample directly or indirectly from a solid-phase extraction or HTLC column and an outlet port for discharging an effluent that includes the fractionated sample.

[0076] In one embodiment, the sample may be applied to the LC column at the inlet port, eluted with a solvent or solvent mixture, and discharged at the outlet port. Different solvent modes may be selected for eluting the analyte(s) of interest. For example, liquid chromatography may be performed using a gradient mode, an isocratic mode, or a polytyptic (i.e., mixed) mode. During chromatography, the separation of materials is affected by variables such as choice of eluent (also known as a “mobile phase”), elution mode, gradient conditions, temperature, etc.

[0077] In certain embodiments, an analyte may be enriched in a sample by applying a sample to a column under conditions where the analyte of interest is reversibly retained by the column packing material, while one or more other materials are not retained. In these embodiments, a first mobile phase condition can be employed where the analyte of interest is retained by the column, and a second mobile phase condition can subsequently be employed to remove retained material from the column, once the non-retained materials are washed through. Alternatively, an analyte may be enriched in a sample by applying a sample to a column under mobile phase conditions where the analyte of interest elutes at a differential rate in comparison to one or more other materials. Such procedures may enrich the amount of one or more analytes of interest relative to one or more other components of the sample.

[0078] In one preferred embodiment, HPLC is conducted with a hydrophobic column chromatographic system. In certain preferred embodiments, a F5 analytical column (e.g., a Discovery HS F5 analytical column from Sigma-Aldrich, Inc. (5 pm particle size, 50x4.6 mm), or equivalent) is used. In certain preferred embodiments, HTLC and / or HPLC are performed using HPLC, Grade 0.1% aqueous formic acid and 1% formic acid in acetonitrile as the mobile phases.

[0079] By careful selection of valves and connector plumbing, two or more chromatography columns may be connected as needed such that material is passed from one to the next without the need for any manual steps. In preferred embodiments, the selection of valves and plumbing is controlled by a computer pre-programmed to perform the necessary steps. Most preferably, the chromatography system is also connected in such an on-line fashion to the detector system, e.g., an MS system. Thus, an operator may place a tray of samples in anautosampler, and the remaining operations are performed under computer control, resulting in purification and analysis of all samples selected.

[0080] In some embodiments, HTLC may be used for enrichment of NE (or derivatized NE), E (or derivatized E), and / or D (or derivatized D) prior to mass spectrometry. In such embodiments, samples may be extracted using an extraction cartridge which captures the analyte, then eluted and chromatographed on a second HTLC column or onto an analytical HPLC column prior to ionization. For example, sample extraction with an HTLC extraction cartridge may be accomplished with a large particle size (50 pm) packed column. Sample eluted off of this column may then be transferred to an HPLC analytical column, such as a F5 analytical column, for further purification prior to mass spectrometry. Because the steps involved in these chromatography procedures may be linked in an automated fashion, the requirement for operator involvement during the purification of the analyte can be minimized. This feature may result in savings of time and costs, and eliminate the opportunity for operator error, Detection and Quantitation by Mass Spectrometry.Detection and Quantitation by Mass Spectrometry[00811 In various embodiments, NE (or derivatized NE), E (or derivatized E), and / or D (or derivatized D) present in a test sample may be ionized by any methods known to a skilled artisan. Mass spectrometry is performed using a mass spectrometer, which includes an ion source for ionizing the fractionated sample and creating charged molecules for further analysis. For example ionization of the sample may be performed by electron ionization, chemical ionization, electrospray ionization (ESI), photon ionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoionization (APPI), fast atom bombardment (FAB), liquid secondary ionization (LSI), matrix assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray / plasmaspray ionization, surface enhanced laser desorption ionization (SEMI), inductively coupled plasma (ICP) and particle beam ionization. The skilled artisan will understand that the choice of an ionization method may be determined based on the analyte to be measured, type of sample, the type of detector, the choice of positive versus negative mode, etc. In some embodiments, NE (or derivatized NE), E (or derivatized E), and / or D (or derivatized D) present in a test sample may be ionized by ESI.(0082] E (or derivatized E), NE (or derivatized NE), and / or D (or derivatized D) may be ionized in positive or negative mode. In preferred embodiments, E (or derivatized E), NE (or derivatized NE), and / or D (or derivatized D) is ionized by heated ESI in positive mode. In related preferred embodiments, 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 techniques generally, after the sample has been ionized, the positively charged or negatively charged ions thereby created may be analyzed to determine a mass-to-charge ratio, Suitable analyzers for determining mass-to-charge ratios include quadrupole analyzers, ion trap analyzers, and time-of-flight analyzers. Exemplary ion trap methods are described in Bartolucci, et al., Rapid Commun. Mass Spectrom. 2000, 14:967-73.(0084] The ions may be detected using several detection modes. For example, selected ions may be detected, i.e., using a selective ion monitoring mode (SIM), or alternatively, ions may be detected using a scanning mode, e.g., multiple reaction monitoring (MRM) or selected reaction monitoring (SRM). Preferably, the mass-to-charge ratio is determined using a quadrupole analyzer. For example, in a “quadrupole” or “quadrupole ion trap” instrument, ions in an oscillating radio frequency field experience a force proportional to the DC potential applied between electrodes, the amplitude of the RF signal, and the mass / charge ratio. The voltage and amplitude may be selected so that only ions having a particular mass / charge ratio travel the length of the quadrupole, while all other ions are deflected. Thus, quadrupole instruments may act as both a “mass filter” and as a “mass detector” for the ions injected into the instrument.(0085] One may enhance the resolution of the MS technique by employing “tandem mass spectrometry,” or “MS / MS”. In this technique, a precursor ion (also called a parent ion) generated from a molecule of interest can be filtered in an MS instrument, and the precursor ion is subsequently fragmented to yield one or more fragment ions (also called daughter ions or product ions) that are then analyzed in a second MS procedure. By careful selection of precursor ions, only ions produced by certain analytes are passed to the fragmentation chamber, where collisions with atoms of an inert gas produce the fragment ions. Because both the precursor and fragment ions are produced in a reproducible fashion under a given set ofionization / fragmentation conditions, the MS / MS technique may provide an extremely powerful analytical tool. For example, the combination of filtration / fragmentation may be used to eliminate interfering substances, and may be particularly useful in complex samples, such as biological samples.

[0086] The mass spectrometer typically provides the user with an ion scan; that is, the relative abundance of each ion with a particular mass / charge over a given range (e.g., 90 to 1000 amu). The results of an analyte assay, that is, a mass spectrum, may be related to the amount of the analyte in the original sample by numerous methods known in the art. For example, given that sampling and analysis parameters are carefully controlled, the relative abundance of a given ion may be compared to a table that converts that relative abundance to an absolute amount of the original molecule. Alternatively, molecular standards may be run with the samples, and a standard curve constructed based on ions generated from those standards. Using such a standard curve, the relative abundance of a given ion may be converted into an absolute amount of the original molecule. In certain preferred embodiments, one or more internal standards may be used to generate standard curves for calculating the quantity of E (or derivatized E), NE (or derivatized NE), and / or D (or derivatized D). Methods of generating and using such standard curves are well known in the art and one of ordinary skill is capable of selecting appropriate internal standards. For example, an isotopically labeled catecholamine may be used as an internal standard; in certain preferred embodiments, norpeinephrine-d6 (de-NE), epinephrine-d6 (de-E), dopamine-d4 (d4-D), may be used as internal standards. Numerous other methods for relating the amount of an ion to the amount of the original molecule will be well known to those of ordinary skill in the art.

[0087] One or more steps of the presented methods may be performed using automated machines. In certain embodiments, one or more purification steps are performed on-line, and more preferably all of the purification and mass spectrometry steps may be performed on-line.

[0088] In certain embodiments, such as MS / MS, where precursor ions are isolated for further fragmentation, collision activation dissociation is often used to generate the fragment ions for further detection. In collision activated dissociation (CAD), precursor ions in energy through collisions with an inert gas, and subsequently fragment by a process referred to as“unimolecular decomposition.” Sufficient energy must be deposited in the precursor ion so that certain bonds within the ion can be broken due to increased vibrational energy.(0089] In some embodiments, the methods described herein comprise derivatizing the catecholamines (e.g., NE, E, and / or D) in a sample with a derivatizing agent such as PITC prior to mass spectrometry analysis. As used herein, “derivatizing” or “derivatization” means reacting two molecules to form a new molecule. Thus, a derivatizing agent is an agent that is reacted with another substance to derivatize the substance. For example, phenylisothiocyanate (PITC) is a derivatizing reagent that may be reacted with catecholamine (e.g., norepinephrine, epinephrine, and / or dopamine) to form a PITC-derivatized catecholamine (e.g., PITC-derivatized norepinephrine, PITC-derivatized epinephrine, and / or PITC-derivatized dopamine). For example, exemplary Scheme 1 shows derivatizing dopamine with PITC. Accordingly, the catecholamines (e.g., NE, E, and / or D) in a sample may be detected and / or quantified using MS / MS as follows. The samples may be filtered through and eluted from immobilized boronic acid gel. Then, the catecholamines (e.g., NE, E, and / or D) in the purified sample are derivatized with PITC or an isotopic variant thereof. The derivatized and purified samples may then be subjected to liquid chromatography, preferably HPLC. The flow of liquid solvent from the chromatographic column enters the heated ESI probe of an MS / MS analyzer and the analytes ionized.Scheme 1 - Phenylisothiocyanate (PITC) derivitization

[0090] Alternatively, in some embodiments, the methods described herein do not comprise derivatizing the catecholamines (e.g., NE, E, and / or D) in a sample. Accordingly, the catecholamines (e.g., NE, E, and / or D) in a sample may be detected and / or quantified using MS / MS as follows. The samples may be filtered through and eluted from immobilized boronic acid gel. The purified samples (e.g., the resulting eluent) may then be subjected to liquidchromatography, preferably HPLC. The flow of liquid solvent from the chromatographic column enters the heated ESI probe of an MS / MS analyzer and the analytes ionized.

[0091] The ions, e.g., precursor ions, pass through the orifice of the instrument and enter the first quadrupole. Quadrupoles 1 and 3 (QI and Q3) are mass filters, allowing selection of ions (i.e., selection of “precursor” and “fragment” ions in QI and Q3, respectively) based on their mass-to-charge ratio (m / z). Quadrupole 2 (Q2) is the collision cell, where ions are fragmented. The first quadrupole of the mass spectrometer (QI) selects for molecules with the mass-to-charge ratios of NE (or derivatized NE), E (or derivatized E), or D (or derivatized D). Precursor ions with the correct mass / charge ratios are allowed to pass into the collision chamber (Q2), while unwanted ions with any other mass / charge ratio collide with the sides of the quadrupole and are eliminated. Precursor ions entering Q2 collide with neutral argon gas molecules and fragment. This process is called collision activated dissociation (CAD). The fragment ions generated are passed into quadrupole 3 (Q3), where the fragment ions are selected while other ions are eliminated.[ 00921 During analysis of a single sample, QI and / or Q3 may be adjusted such that mass / charge ratios of one or more precursor ion / fragment ion pairs specific to one catecholamine is first selected, followed at some later time by the selection of mass / charge ratios of one or more precursor ion / fragment ion pairs specific to a second catecholamine, optionally followed at some later time by the selection of mass / charge ratios of one or more precursor ion / fragment ion pairs specific to a third catecholamine. In particularly preferred embodiments, mass-to-charge ratios of precursor / fragment ion pairs specific to NE (or derivatized NE), mass- to-charge ratios of precursor / fragment ion pairs specific to E (or derivatized E), and mass-to- charge ratios of precursor / fragments ion pairs specific to D (or derivatized D) are detected during analysis of a single sample, although the sequence of detection may occur in any order.10093] The methods may involve MS / MS performed in either positive or negative ion mode, preferably positive ion mode. Using standard methods well known in the art, one of ordinary skill is capable of identifying one or more fragment ions of a particular precursor ion of NE (or derivatized NE), E (or derivatized E), and / or D (or derivatized D) that may be used for selection in quadrupole 3 (Q3).|0094] As ions collide with the detector, they produce a pulse of electrons that are converted to a digital signal. The acquired data is relayed to a computer, which plots counts of the ions collected versus time. The resulting mass chromatograms are similar to chromatograms generated in traditional HPLC methods. The areas under the peaks corresponding to particular ions, or the amplitude of such peaks, are measured and the area or amplitude is correlated to the amount of the analyte of interest. In certain embodiments, the area under the curves, or amplitude of the peaks, for fragment ion(s) and / or precursor ions are measured to determine the amount of analyte or analytes detected. As described above, the relative abundance of a given ion or parent / fragment ion pair may be converted into an absolute amount of an original analyte, e.g., NE (or derivatized NE), E (or derivatized E), or D (or derivatized D), using calibration standard curves based on peaks of one or more ions of an internal molecular standard, such as norpeinephrine-d6 (de-NE), epinephrine-d6 (de-E), and / or dopamine-d4 (d4-D).

[0095] Thus, in accordance with some aspects, the present disclosure provides methods for determining an amount of an analyte in a sample, the methods comprise one or more of: purifying the sample by liquid chromatography; subjecting the sample to a derivatizing agent to generate a derivatized sample; subjecting the sample (e.g., the purified sample or the derivatized sample) to electrospray ionization (ESI) under conditions suitable to produce one or more ions detectable by mass spectrometry; and quantifying the amount of the analyte by tandem mass spectrometry, wherein the quantifying comprises quantifying an amount of the ion(s), and the amount of the ion(s) detected is related to the amount of the analyte in the sample.

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

[0097] In some embodiments, the analyte comprises epinephrine, norepinephrine, dopamine, or a mixture of any two or more thereof.

[0098] In some embodiments, the methods described herein comprise subjecting the sample to a derivatizing agent to generate a derivatized sample. In some embodiments, the derivatization agent comprises phenylisothiocyanate (PITC). In some embodiments, the one ofthe one or more analytes is norepinephrine, and the quantifying comprises quantifying the amount of precursor ion having a mass-to-charge ratio of 305.2 ± 0.5; and / or quantifying the amount of one or more fragment ion(s) having a mass-to-charge ratio selected from the group consisting of 107.0 ± 0.5 and 152.2 ± 0.5. In some embodiments, the one of the one or more analytes is epinephrine, and the quantifying comprises quantifying the amount of precursor ion having a mass-to-charge ratio of 319. l±0.5; and / or quantifying the amount of one or more fragment ion(s) having a mass-to-charge ratio selected from the group consisting of 166.1± 0.5 and 107.0 ± 0.5. In some embodiments, the one of the one or more analytes is dopamine, and the quantifying comprises quantifying the amount of precursor ion having a mass-to-charge ratio of 289.1 ± 0.5; and / or quantifying the amount of one or more fragment ion(s) having a mass-to- charge ratio selected from the group consisting of 137.2 ± 0.5 and 153.2 ± 0.5.

[0099] In some embodiments, the methods described herein do not comprise subjecting the sample to a derivatizing agent to generate a derivatized sample. In some embodiments, the one of the one or more analytes is norepinephrine, and the quantifying comprises quantifying the amount of precursor ion having a mass-to-charge ratio of 152.1 ± 0.5; and / or quantifying the amount of one or more fragment ion(s) having a mass-to-charge ratio of 76.9 ± 0.5. In some embodiments, the one of the one or more analytes is epinephrine, and the quantifying comprises quantifying the amount of precursor ion having a mass-to-charge ratio of 166.1 ± 0.5; and / or quantifying the amount of one or more fragment ion(s) having a mass-to-charge ratio selected from the group consisting of 51.0 ± 0.5 and 107.0 ± 0.5. In some embodiments, the one of the one or more analytes is dopamine, and the quantifying comprises quantifying the amount of precursor ion having a mass-to-charge ratio of 137.1 ± 0.5; and / or quantifying the amount of one or more fragment ion(s) having a mass-to-charge ratio of 91.1 ± 0.5.10.1.00 [ In some embodiments, the sample is a plasma sample. In some embodiments, the sample is a urine sample.[01011 In some embodiments, the analytes are purified by high performance liquid chromatography (HPLC) prior to ionization. In some embodiments, the analytes are purified by solid phase extraction (SPE) prior to ionization. In some embodiments, the analytes are purified by immobilized boronic acid extraction prior to ionization.

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

[0103] The following examples serve to illustrate various aspects of this disclosure.These examples are in no way intended to limit the scope of the methods.EXAMPLESExample 1 - Detection of Catecholamines in Human Plasma (PITC derivatization method)

[0104] An illustrative flowchart showing the preparation of samples and detection of plasma catecholamines is provided in Scheme 2.Scheme 2.5.6 hours / 2 hours1.5 hours 30 minutes piste

[0105] Patient plasma samples were prepared by combining heparinized plasma with internal standards (ISTD), stabilizers, and ammonium phosphate monobasic buffer. These steps were automated using a Hamilton liquid handler. A solid phase extraction (SPE) was conducted using an Agilent PBA plate on a SPEware ALD. The solid phase extraction (SPE) plate containing phenylboronic acid (PBA) packing can capture the catecholamines. Sample eluent was collected and dried down under a nitrogen stream on a Porvair.

[0106] A “Redry” solution was added to each well and thoroughly mixed on anEppendorf plate mixer prior to the second “dry down” step. PITC derivatization solution wasthen added to each sample, mixed, and allowed time to incubate. The PITC adds 135 to the molecular mass. The collection plate was again dried down under nitrogen heated at 35°C.

[0107] Samples were reconstituted with acetonitrile / water, mixed, and filtered through an Agilent Captiva filter plate. The samples were injected on a StreamSelect multiplexing HPLC system coupled to an Agilent 6495C Triple Quadrupole mass spectrometer in positive ion mode with a heated electrospray source. Chromatographic separation was achieved by using a Zorbax SB-Aq column (Agilent). Agilent MassHunter quantitative software was used to fit the calibration curve based on peak area ratios of the analyte to internal standard.

[0108] Quality Control (QC) Material. The QC pools were prepared by spiking stripped serum with known concentrations of norepinephrine, epinephrine and dopamine. The pools were split into three aliquots that were spiked with stock standards at concentrations which covered the presumptive reportable range of the assay. The QC pools were separated into 2.5 mL aliquots and stored in an ultralow freezer at -90°C to -60°C to be used for precision data.The analyte QC materials were spiked with the following concentrations:

[0109] The MS uses HESI-positive mode for detection. The quantitation is based upon unique precursor-product transitions. The following precursor and fragment ions were used:

[0110] A brief summary of results obtained in Example 1 are provided in the table below.

[0111] Precision.

[0112] Within Run Precision (Intra-Assay). Ten samples of each quality control level (Low, Mid, and High) were extracted within one run. The intra-assay coefficients of variation were <8.3% for all analytes, which is within the acceptable criteria. Acceptable criteria: CV <8.3% or TEa / 3.

[0113] Total Precision. 5 replicates of each spiked pool level (Low, Mid, and High) were extracted over 5 different days for a total of 25 injections. The inter-assay coefficients of variation were <8.3% for all analytes, which is within the acceptable criteria. Acceptable criteria: SD<TEa / 3 or CV <8.3%, unacceptable if Total SD > l / 2Tea or Total SD must be less than a defined maximum SD or CV.

[0114] Inter-Operator Precision. Comparison of similar extracted plates, both containing equal amounts of quality controls, spiked pools, and standards, were assayed by different lab personnel. Comparison of these results yielded inter-operator coefficients of variation <8.3% for all analytes, which is within the acceptable criteria. Acceptable criteria: CV <8.3% or TEa / 310115] Analytical Sensitivity (Detection Limits)

[0116] Limit of Blank (LOB). 5 stripped serum blanks were extracted over 5 different days for a total of 25 injections. Calculation: LOB= mean of blank + 2SD. The LOB for the analytes are as follows:|0117| Limit of Detection (LOD). 5 stripped serum blanks were extracted over 5 different days for a total of 25 injections. Calculation: LOD= mean of blank + 4SD. The LOB for the analytes is as follows (FIGS. 1A-1C).

[0118] Limit of Quantitation (LOQ). Stripped serum was spiked with known concentrations of each analyte. 5 replicates of 3 levels were extracted and analyzed across 5 days.Acceptability criteria: The lowest concentration at which SD < TEa / 3. TEa for all three analytes is 25%. The LOQ for the analytes are as follows (FIGS. 1A-1C). All analytes are acceptable for CV<8.3%.

[0119] Analyte Measurement Range (AMR)

[0120] Calibration Verification. 19 sets of standards were analyzed in 19 separate extractions. A linear regression from these runs yielded correlation coefficients of 0.9900 or greater for all analytes, and average recoveries of 92.1% - 108.7%, revealing a quantifiable range for all analytes (FIGS. 2A-2C). Acceptability criteria: The average of the observed values should deviate from the expected range by no more than TEa / 4. TEa / 4 and AMR range met validation criteria and are acceptable.[0121 | AMR Validation by Dilution. A lyophilized calibrator (ClinCal®), containing norepinephrine, epinephrine, and dopamine, was reconstituted with 5mL of HPLC grade water. The reconstituted solution was then mixed for 15 minutes before extraction. Dilutions of 1 / 2, 1 / 4, 1 / 10, 1 / 15, and 1 / 30 were prepared from the reconstituted calibrator and extracted as unknowns (FIGS. 3A-3C). Calculated values were then compared to target values to obtain % recovery. Acceptability criteria: The difference between values based on the regression line and the observed values should be < TEa / 4. TEa / 4 and recovery range met validation criteria and are acceptable.

[0122] Reportable Range (RR)|0123| Accuracy

[0124] Recovery Studies

[0125] Recovery of Known Standards. Two sets of known standards were tested in separate extractions as a means of quantitating results. These standards were spiked to a level of 1,200 pg / mL. Target dilution levels of 600 pg / mL, 300 pg / mL, 120 pg / mL, 60 pg / mL, 20 pg / mL, and 10 pg / mL were obtained by diluting the 1,200 pg / mL spike level with stripped serum (FIGS. 4A-4C). Acceptability criteria: the error due to lack of perfect recovery (amount recovered MINUS amount added) should be < TEa / 4. The recoveries for all analytes are within the acceptable criteria.

[0126] Recovery by Dilution. Recovery by dilution of 1 / 2, 1 / 4, 1 / 10 / , 1 / 15, and 1 / 30 dilutions were performed on a purchased ClinCal® calibrator standard (Refer to section 7.2 for preparation). The diluted samples were injected in duplicate and the mean calculated values were compared to neat values to obtain % recovery. Acceptability criteria: The average of the observed values should deviate from the expected range by no more than TEa / 3 (which is 8.3%). The recoveries for all analytes are within the acceptable criteria.

[0127] Split-Sample Comparison Study. A comparison study was performed using up to 51 samples (FIGS. 5A-5D). Samples were analyzed on both the ECD-HPLC platform and LCMSMS method. The LCMSMS method monitors only specific derivatized target molecules resulting in enhanced specificity when compared to the ECD-HPLC methodology. 16 samples were below detectable levels for epinephrine and 17 for dopamine and were excluded. Two plots were calculated for norepinephrine, the first plot containing all analyzed samples and the second plot excluding results above 1,000 pg. This was done to display an alternative sample comparison lower concentration levels.

[0128] Measurement Uncertainty (MU)101291 Measurement Uncertainty. Definition: Degree to which one is certain of a result of a particular measurement. Expressed as the 95th percentile confidence interval around a given number determined by the mean + / - 1.96*SD of multiple determinations. MUConfidence Intervals are calculated using the Precision sheet of the Assay Validation Calculatortemplate. Analyze data by calculating the mean and standard deviation, the MU is then calculated by multiplying the SD by 1.96 and adding or subtracting this to the mean. Each QC level was analyzed as 3 separate levels with 5 replicates in 5 different days.

[0130] Specimen Stability

[0131] Pooled patient plasma was divided into 5 groups and spiked with known concentrations of catecholamines across the existing reference range. Each sample was extracted in duplicate for all stability studies. Acceptability criteria: A sample is considered stable as long as the average difference between the baseline value and the time / temperature sample value is < TEa / 4 for that analyte.

[0132] Interference Study.101331 Acceptability criteria: The difference due to a potential interfering substance should be <TEa / 4 to be considered acceptable (FIGS. 6A-6C).

[0134] Hemolysis Interference. 3 separate patient plasma pools were spiked with catecholamine stock standards and analyzed in triplicate for baseline results. 250uL of hemolzyed RBC’s was added to 4,750uL of lOmM PBS solution, this new solution was used as grossly hemolyzed interference. The grossly hemolzyed solution was further diluted with lOmM PBS at 1 : 10 and 1 :20 for moderately and slightly hemolyzed interference. 90uL of eachinterfering solution was mixed with 810uL of each spiked plasma level. All samples were extracted in triplicate. Plasma catecholamines were acceptable for slight, moderately, and grossly hemolyzed samples.10135] Lipemia Interference. 3 separate patient plasma pools were spiked with catecholamine stock standards and analyzed in triplicate for baseline results. ImL of intralipid was added to 4mL of lOmM PBS solution, this new solution was used as the grossly lipemic interference. The additional interference levels were prepared by diluting the intralipid stock with lOmM PBS solution to 1 : 10, for moderately lipemic, and 1 :20, for slightly lipemic. 90uL of each interfering solution was mixed with 810uL of each spiked plasma level. All samples were extracted in triplicate. Plasma catecholamines were acceptable for slight and moderately lipemic samples.

[0136] Bilirubin Interference. 3 separate patient plasma pools were spiked with catecholamine stock standards and analyzed in triplicate for baseline results. A Img / mL bilirubin sample was prepared with lOmM PBS solution, this new solution was used as grossly icteric interference. The grossly icteric solution was further diluted with lOmM PBS at 1 : 10 and 1 :20 for moderately and slightly icteric interference. 90uL of each interfering solution was mixed with 810uL of each spiked plasma level. All samples were extracted in triplicate. Plasma catecholamines were acceptable for slight, moderately, and grossly icteric samples.

[0137] Drug Interference. Drug interference study was conducted on carbidopa and levodopa due to their similar molecular structures. Samples were prepared by spiking stripped serum with carbidopa and levodopa to a final concentration of 10,000 ng / mL. The spiked samples were extracted in duplicate and analyzed. Acceptability criteria: The difference due to a potential interfering substance should be < TEa / 4 to be considered acceptable. Interfering drugs were below LOQ and were determined not to cause interference. Results are as follows.

[0138] Ion Suppression. The method of post-column infusion was utilized to detect ion suppression. A constant flow (20 uL / min) of extracted catecholamine standard (1,000 pg / mL) was infused, followed by the injection of six extracted samples (1 blank, 5 patients). Variation in signal response of the infused analyte at the corresponding retention time would indicate that ionization suppression exists. No ion suppression was observed.. Acceptability criteria: No more than ±25% change in signal intensity of post-column infusion.

[0139] Carryover.

[0140] Both injection carryover and liquid handler (Hamilton) carryover were evaluated for this study. For samples, stripped serum was spiked to achieve low (50 pg / mL) and high (l,000pg / mL) levels of catecholamines. Assay sequence was 2 low replicates, followed up by 2 high replicates then followed by 3 low replicates. This sequence was repeated 2 additional times within the same assay. Results of the low levels spikes were compared to the low levels spikes after the high levels spikes in order to test for carryover. Acceptability criteria: The difference due to carryover should be < TEa / 4 to be considered acceptable. Results were acceptable and it was determined that no carryover was observed in this assay.[0.141] Specimen Type (Split Sample Comparison)101421 Acceptable sample type for the described assay on ECD / HPLC platform is plasma drawn from a vacutainer tube with sodium heparin as a preservative. The study was performed by testing blood samples from 34 donors collected in 2 types of vacutainer tubes, sodium heparin and EDTA as preservatives. All samples were extracted and analyzed. The plasma results from EDTA were compared against the results from sodium heparin. Results for norepinephrine, epinephrine, and dopamine are acceptable (FIGS. 7A-7C).Example 2 - Detection of Catecholamines in Human Urine without Derivatization|0143| Catecholamines in a human urine sample can be detected in accordance with methods summarized in the table below.10.1441 Patient urine samples were prepared by combining samples with internal standards (ISTD), stabilizers, and ammonium phosphate monobasic buffer. A solid phaseextraction (SPE) plate containing phenylboronic acid (PBA) packing captures the catecholamines through their cis-diols. The eluent was collected and dried down under nitrogen. Samples were reconstituted with HPLC-grade water.

[0145] The samples were injected on a StreamSelect multiplexing HPLC system coupled to an Agilent 6495C Triple Quadrupole mass spectrometer in positive ion mode with a heated electrospray source. Chromatographic separation was achieved by using a Kinetex PFP column (Phenomenex). Agilent Masshunter quantitative software was used to fit the calibration curve based on peak area ratios of the analyte ions to internal standard.

[0146] The following precursor and fragment ions are used:[01471 Quality Control (QC) Material

[0148] Precision101491 Intra-Assay Precision. Acceptance Criteria: The %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). 5 replicates of each level for Recipe and Sigma Cal QC were extracted over 5 different days for a total of 25 injections for each level. Results are shown in the table below. Conclusion: Intra-Assay Precision data meet the acceptable reproducibility requirements of SD or %CV <TEa / 4.

[0150] Inter- Assay Precision. Acceptance Criteria: The %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). 2 replicates of Bio-Rad QC at each level (I, II, III) were extracted over 20 different extraction for a total of 40 injections at each level. Results are shown in the table below. Conclusion: Inter-Assay Precision data meet the acceptable reproducibility requirements of SD or %CV <TEa / 4.

[0151] Analytical Sensitivity (Detection Limits)

[0152] Limit of Blank (LoB). Acceptance Criteria: LoB = mean of blank + 2SD. Water blanks were extracted over 3 different days for a total of 25 injections. Results: No quantifiable peaks of catecholamines were seen, therefore a mean and standard deviation could not be determined (FIGS. 8A-8C). Conclusion: Limit of Blank (LoB) could not be determined for urine catecholamines by LC / MS / MS.

[0153] Limit of Detection (LoD). Acceptance Criteria: LoD = mean of blank + 4SD. Water blanks were extracted over 3 different days for a total of 25 injections. Results: No quantifiable peaks of catecholamines were seen, therefore a mean and standard deviation could not be determined (FIGS. 8A-8C). Conclusion: Limit of Detection (LoD) could not be determined for urine catecholamines by LC / MS / MS.

[0154] Limit of Quantitation (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 standard) spiked urine with known concentrations of each analyte were diluted down as various levels. 5 replicates of 5 levels were extracted and analyzed across 5 days (FIGS. 8A-8C).

[0155] Conclusion: The Limit of Quantitation (LoQ) was determined for each catecholamine and for practical purposes was then rounded up to the nearest whole number. Results are summarized in the table below.The LOQ for the analytes are as follows:

[0156] Reportable Range (RR)

[0157] Analyte Measurement Range (AMR). Definition for Quantitative and selectSemi-Quantitative assays: The reportable range is the entire span of result values for which theaccuracy of the instrument or test system was established or verified. Expressed as the lower limit of the AMR (usually LoQ) and extending to or past the upper limit of the AMR.

[0158] Calibration Verification. Acceptance Criteria: The %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). 20 sets of the BioRad standard were analyzed in 20 separate extractions as well as 21 sets of the Sigma standard were analyzed in 21 separate extractions. Results are summarized in the table below.Conclusion: A linear regression from these runs yielded correlation coefficients of 0.9900 or greater for all analytes and standards met requirements of SD or %C V <TEa / 3.

[0159] AMR Validation by Dilution. Acceptance Criteria: The %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). Commercially available lyophilized standards were used to generate 3 separate calibration curves across theAMR at 6 different levels. Results were correlated against their target concentrations. Results are summarized in the tables below.[0160| Conclusion: Calibration linearity was verified across the AMR and met the acceptable criteria. The analytical measurement range (AMR) is as follows:[01611 Reportable Range (RR)

[0162] Accuracy

[0163] Recovery Study

[0164] Recovery of Known Standards. Acceptance Criteria: The % 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 separate extractions as a means of quantitating results. The first standard is a commercially available lyophilized standard (BioRad) and the second standard was spiked diluent with a sigma stock standard prepared in-house. 20 sets of the BioRad standard were analyzed in 20 separate extractions as well as 21 sets of the Sigma standard were analyzed in 21 separate extractions (FIGS. 9A-9C). Results are summarized in the table below. Conclusion: The difference between the calculated concentrations and the expected concentrations met the acceptable criteria of TEa / 3 for either percent accuracy or standard deviation.

[0165] Recovery by Dilution. Acceptance Criteria: The %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 (Img / mL) for each standard, norepinephrine, epinephrine, and dopamine were spiked into human urine at high concentrations above the AMR. Recovery by dilution of 1 / 2, 1 / 5, 1 / 10 / , 1 / 25, 1 / 50, and 1 / 100 dilutions were performed on the spiked human urine in triplicate. The diluted samples were injected and the mean calculated values were compared to spiked urine values to obtain % recovery. Results are summarized in the table below. Conclusion: The difference between the calculated concentrations and the expected concentrations met the acceptable criteria of TEa / 3 for either percent accuracy or standard deviation.

[0166] Recovery of Previously Tested (Known) Proficiency Test Samples.Acceptance Criteria: The %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 Test samples, containing known amounts of urine catecholamines, were extracted in 5 replicates and injected. Proficiency samples are also surveyed across external laboratories on LCMSMS. Results were correlated against known amounts. Results are summarized in the table below. Conclusion: Thedifference between the calculated concentrations and the expected concentrations met the acceptable criteria of TEa / 3 for either percent accuracy or standard deviation.10167] Method Comparison Study.

[0168] Acceptance Criteria: The 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 on both the ECD-HPLC platform and the LCMSMS method on the Agilent 6495 StreamSelect system (FIGS. 10A-10C). Results are summarized in the table below. Conclusion: The LCMSMS method on the Agilent 6495 StreamSelect system is acceptable for norepinephrine and dopamine. Epinephrine acceptable at low concentration due to low sample volume size at higher concentrations.

[0169] Measurement Uncertainty (MU)

[0170] Definition: Degree to which one is certain of a result of a particular measurement. Expressed as the 95th percentile confidence interval around a given number determined by the mean + / - 1.96*SD of multiple determinations. For modified or unmodified FDA Cleared orApproved tests, measurement uncertainty is determined by measuring each QC level 5 times perday for 5 separate days. MU Confidence Intervals are calculated using the Precision sheet of the Assay Validation Calculator template. Analyze data by calculating the mean and standard deviation, the MU is then calculated by multiplying the SD by 1.96 and adding or subtracting this to the mean. Confidence intervals for each QC level of each catecholamine was established and summarized in the table below.1’0.1.71 J Specimen Stability

[0172] Patient urine was collected for 24hr and Random sample types. All samples were extracted for background measurement and divided into groups ensuring concentrations over theexisting reference range. A total of 5 24hr urine samples and 6 random urine samples were extracted in triplicate for all stability studies. Results are summarized in the table below.

[0173] Analytical Specificity

[0174] Interfering Substances. Acceptance Criteria: The difference due to a potential interfering substance should be < TEa / 4 to be considered acceptable. Drug interference study was conducted on carbidopa and levodopa due to their similar molecular structures. Samples were prepared by spiking assay diluent with carbidopa and levodopa to a final concentration of 2,500 ng / mL. The spiked samples were extracted in triplicate and analyzed. Conclusion: Interfering drugs are below LOQ and are determined not to cause interference.

[0175] Ion Suppression. Acceptance Criteria: No more than a 25% change in signal intensity of post column infusion. Examine the total ion chromatogram (TIC) to determine if there is a change in signal intensity when the analyte is eluted. The method of post column infusion was utilized to detect ion suppression. A constant flow (20 uL / min) of diluted catecholamine standard mix (2 ug / mL for norepinephrine, 1 ug / mL for epinephrine, and 3 ug / mL for dopamine) was infused, followed by the injection of 10 extracted blank samples containing internal standard mix. Variation in signal response of the infused analyte at the corresponding retention time would indicate that ionization suppression exists. Conclusion: Ion suppression was not observed.

[0176] Carryover. Both injection carryover and liquid handler (Hamilton) carryover were evaluated for this study. For samples, a commercially available spike lyophilized standard was reconstituted and used for the high sample and then diluted 1 :50 for the low sample. Acceptance Criteria: The difference due to a potential interfering substance should be < TEa / 4 to be considered acceptable. Assay sequence was 2 low replicates, followed up by 2 high replicates then followed by 3 low replicates. This sequence was repeated 2 additional times on separateassay extractions. Results of the low levels spikes were compared to the low levels spikes after the high levels spikes in order to determine carryover. Conclusion: Results were acceptable, as < TEa / 4, and it was determined that no carryover was observed in this assay.[01.77] Reference Interval (RI).

[0178] Split-patient comparison between ECD-HPLC and LCMSMS for urine catecholamines indicates good correlation between the two methods. All catecholamine analytes, corrected and raw values, yield acceptable R2values of 0.94 to 1 and results within 95% confidence.(0179] Specimen TypesExample 3 - Detection of Catecholamines in Human Plasma without Per ivatization

[0180] Catecholamines in a human plasma sample can be detected in accordance with methods summarized in the table below.(0181 ] Patient plasma samples. Patient plasma samples were prepared by combining heparinized plasma with internal standards (IS), stabilizers, and ammonium phosphate monobasic buffer. A solid phase extraction plate (SPE) containing phenylboronic acid (PBA) packing captures the catecholamines through their cis-diols. The eluent was collected, dried down and reconstituted.

[0182] Catecholamine-Free Serum Samples. These samples were prepared by combining 70% catecholamine-free filtered serum diluted with 30% HPLC-grade water with internal standards (ISTD), stabilizers, and ammonium phosphate monobasic buffer. These steps were automated through the Hamilton liquid handler. A solid-phase extraction (SPE) was done through an Agilent PBA plate on a Tecan A200. Sample eluent was collected and dried down under nitrogen gas and then reconstituted prior to injection.

[0183] The extracted samples were injected at 45 pL on Transcend TLX-4 multiplexing LC system with Vanquish pumps (ThermoFisher Scientific) coupled to Sciex Triple Quad 7500 mass spectrometer in positive ion mode with a heated electrospray source. Chromatographic separation was achieved by using a PFP column (Phenomenex). SciexOS quantitative software was used to fit the calibration curve based on peak area ratios of the analyte ions to internal standard.

[0184] The following precursor and fragment ions were used:10185] Total Error Allowable (TEa) for each analyte is as follows:

[0186] Quality Control (QC) Material. QC materials were prepared by spiking the diluted Catecholamine-Free Serum with 25 pg / mL, 100 pg / mL, and 500 pg / mL of Norepinephrine, Epinephrine, and Dopamine. The spiked pools were split into 3 groups at concentrations spanning the expected reportable range of the assay. The QC pools are then separated into 1.8mL aliquots, to be stored in an ultra-low freezer at -90°C to -60°C to be used for precision data.

[0187] Precision

[0188] Precision Studies.

[0189] Intra- Assay Precision. Acceptance Criteria: Within run CV < TEa / 4 which is <6% or SD < 3pg / mL for Epinephrine, < 6% or SD < 6 pg / mL for Dopamine, and < 5% or SD < 19 pg / mL for Norepinephrine. Five Replicates at three control levels (Low, Mid, and High) were tested within a single run, in repeating sequential ascending order of concentration. Control level replicates were stored in duplicates per tube; each tube contained 1.8 mL of control sample which was used for two separately extracted replicates. Results are summarized in the table below. The intra-assay CV are as follows: < 6% for all 3 QC levels of Epinephrine; < 5% for QC 1 & 3, < 7% for QC 2 but SD < 19 pg / mL for Norepinephrine; and 6% for Dopamine.Conclusion: The results for all analytes are within the acceptance criteria.101901 Inter-Assay Precision. Acceptance Criteria: Within run CV < TEa / 3 which is < 8% or SD < 4 pg / mL for Epinephrine, < 8% or SD < 7pg / mL for Dopamine and < 7% or SD < 25 pg / mL for Norepinephrine with Sigma > 3. Each control level was analyzed in replicates of five over five separate setups. Results are summarized in the table below. The inter-assay CV are as follows: < 7% for QC levels of Epinephrine except for level 1 which is 11%, but SD < 4 pg / mL; < 7% QC levels of Norepinephrine except for level 1 which is 14%, but SD < 25 pg / mL, and < 8% for all QC levels of Dopamine. Conclusion: The results are acceptable for all analytes, with CV or concentrations < TEa / 3 and overall Sigma > 3.

[0191] Inter-Operator Precision. Acceptance Criteria: SE < TEa / 4. Comparison of the spiked standards and patient samples, and QC replicates, and standards. The samples and standards were extracted by two different lab personnel. Results are summarized in the table below. All analytes are acceptable for SE < TEa / 4. Conclusion: The results meet the acceptable reproducibility requirements for all three analytes.

[0192] Analytical Sensitivity (detection limits)

[0193] Limit of Blank (LoB). LoB = mean of blank + 2SD. Twenty (20) blank diluent samples were analyzed in one assay. No quantifiable peak of catecholamine were observed (epinephrine LoB: 0.46 pg / mL; norepinephrine LoB: 0.76 pg / mL; dopamine LoB: 0.06 pg / mL) (FIGS. 11A-11C)

[0194] Limit of Detection (LoD). LoD = mean of blank + 4SD. Twenty (20) blank diluent samples were analyzed across two assays. No quantifiable peak of catecholamine (Epinephrine LoD: 0.80 pg / mL; Norepinephrine LoD: 1.35 pg / mL; Dopamine LoD: 0.12 pg / mL) (FIGS. 11A-11C).

[0195] Limit of Quantitation (LoQ). Acceptance Criteria: The lowest detectable concentration at which SD < TEa / 3 and > LOD. To assess the LoQ, three pools were prepared using standard material spiked in diluted serum at levels close to the or below the expected reportable. Epinephrine and Dopamine Each pool was measured 5 times per run, across 5 runs total. 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 is greater than the corresponding LoD and SD < TEa / 3 (FIGS. 11A-11C).

[0196] Reportable Range (RR).

[0197] Analyte Measurement Range (AMR). Definition for Quantitative and select Semi-Quantitative assays: The reportable range is the entire span of result values for which the accuracy of the instrument or test system was established or verified. Expressed as the lower limit of the AMR and extending to or past the upper limit of the AMR. Based on the studies, the AMR are as follows: Epinephrine: 5 to 1,200 pg / mL; Norepinephrine: 8 to 1,200 pg / mL; Dopamine: 5 to 1,200 pg / mL.

[0018] Calibration Verification. Acceptance Criteria: The average of the observed values at each level should deviate from the expected by < TEa / 3. A set of calibrators was prepared and analyzed over the course of 3 different extraction dates, with approximately a month between each. All injections were done in single replicates. The difference between the target concentration and observed concentrations were analyzed (FIGS. 12A-12C). Results are summarized in the table below. The linear regression from the five runs all yield a correlation coefficient of 0.990 or greater for all analytes, and the average observed values CV < TEa / 3. The statistics performed on the data indicate an acceptable calibration performance. Conclusion: The calibration linearity was verified across the AMR, meeting the acceptable accuracy criteria.

[0199] AMR Validation by Dilution. Acceptance Criteria: The difference between values based on the regression line should be < TEa / 3. A dilution study was done using 5 samples with known concentrations of Norepinephrine, Epinephrine, and Dopamine. Results are summarized in the table below. Conclusion: All analytes are found to have an acceptable accuracy up to dilution factor 2.

[0200] Accuracy[02011 Recovery Study(0202] Recovery of Known Standards. Acceptance Criteria: Spike recovery average SE < TEa / 4. A spike recovery study was performed to evaluate if there is any apparent matrix effect within the assay. Base recovery serum samples (5 in total) were spiked with 50uL of 1200 pg / mL stock solution containing Epinephrine, Norepinephrine, and Dopamine to 950uL of each sample. Results are summarized in the table below. The average SE% of each analyte is < TEa / 4. Conclusion: Recovery ranges for all analytes are deemed acceptable by the predefined criteria.

[0203] Split-Sample Comparison. Acceptance Criteria: The absolute value of difference in averages should be less than TEa / 4. Patient samples were collected, and each separated into two aliquots; from which one aliquot was assayed using the original methodology in single replicates and the other was assayed with the proposed new methodology in duplicates (FIGS. 13A-13C). Results are summarized in the table below. At least 102 samples were analyzed on both the current platform (ECD-HPLC) and the proposed LCMSMS platform. When viewing the acceptability of the split sample comparison study, consideration should be taken since the new study being compared is of a different extraction method and different detector. Conclusion: There is a negative bias across all three analytes, trending at higher detected values from data provided by Operations relative to the R&D observed values. The proposed method is more specific than the current method platform.

[0204] Measurement Uncertainty (MU).

[0205] For modified or unmodified FDA Cleared or Approved tests, measurement uncertainty is determined by measuring each QC level 5 times per day for 5 separate days. If it is necessary to complete the MU study over a shorter time frame than 5 separate days, each set of 5 QC values must be in separate runs and an acknowledgement of the shortened timeframe documented in the final precision study. MU Confidence Intervals are calculated using the Precision sheet of the Assay Validation Calculator template. Analyze data by calculating the mean and standard deviation, the MU is then calculated by multiplying the SD by 1.96 and adding or subtracting this to the mean. Results are summarized in the tables below.|0206| Specimen Stability.10207 [ The stability of the specimen is shown in the table below.

[0208] Analytical Specificity.

[0209] Hemolysis Interference. Acceptance Criteria: the difference due to potential interfering substances should be <TEa / 4 to be considered acceptable. The effects of hemolysis in the assay was evaluated by spiking three patient samples with hemolyzed RBCs with a concentration of approximately 150 g / L at low (1 :20), medium (1 : 10), and high (1 :5) concentrations, to simulate slight, moderate, and gross hemolysis These spiked samples were assayed with a set of the same samples without the interfering substance spike and instead an equivalent volume of lOmM PBS, as a control comparison (FIGS. 14A-14C). Results: The difference due to a potential interfering substance for all analytes are <TEa / 4 up to moderate Hemolysis levels. Conclusion: Plasma samples exhibiting slight to moderate hemolysis are acceptable. Gross hemolysis is unacceptable.

[0210] Bilirubin Interference. Acceptance Criteria: The difference due to potential interfering substances should be <TEa / 4 to be considered acceptable. A 1 mg / mL bilirubin sample was prepared in 10 mM PBS solution, further diluted to 1 : 10 and 1 :20 to simulate slight and moderate levels of icteria. The effects of bilirubin in the assay were evaluated by spiking three patient samples with bilirubin at low, medium, and high concentrations, to simulate slight, moderate, and gross icteric interference. These spiked samples were assayed with a set of the same samples without the interfering substance spike and instead an equivalent volume of lOmM PBS, as a control comparison (FIGS. 15A-15C). Results: The difference due to a potential interfering substance for all analytes are <TEa / 4 at all Bilirubin levels. Bilirubin doesnot interfere with quantitation of human plasma catecholamines. Conclusion: Plasma samples exhibiting slight to gross icteria are acceptable.

[0211] Lipemia Interference. Acceptance Criteria: the difference due to potential interfering substances should be <TEa / 4 to be considered acceptable. The effects of Lipemia in the assay was evaluated by spiking three patient samples with intralipid at low, medium, and high concentrations, to simulate slight, moderate, and gross lipemia. 1 mL of intralipid was diluted in 4 mL of lOmM PBS. This was further diluted into 1 : 10 and 1 :20, to simulate slight, moderate, and gross lipemia. These spiked samples were assayed with a set of the same samples without the interfering substance spike and instead an equivalent volume of lOmM PBS, as a control comparison (FIGS. 16A-16C). Results: The difference due to a potential interfering substance for all analytes are < TEa / 4. Lipemia does not interfere with quantitation of human plasma catecholamines. Conclusion: Plasma samples exhibiting slight to gross lipemia are acceptable.

[0212] Drug Interference. Acceptance Criteria: The difference due to potential interfering substances should be < TEa / 4 to be considered acceptable. The drug interference study was conducted using Levodopa, which is one of the known to increase Dopamine levels in plasma and urine (Grouzmann 2013), but with a molecular different than that of the three analytes. Samples were prepared by spiking stripped serum with a known concentration of catecholamines with Levodopa The samples were analyzed in duplicates on LCMSMS. These spiked samples were assayed with a set of the same samples without the interfering substance spike and instead an equivalent volume of lOmM PBS, as a control comparison. Results: Product peaks associated with Levodopa have a retention time is consistently separated from the expected retention times for all analytes, supporting that there is no interference due to Levodopa. Conclusion: There is no interference detected in response to Levodopa spike.1021 [ Ion Suppression. Acceptance Criteria: No more than a ± 25% change in signal intensity of post-column infusion. 100,000pg / mL of catecholamine standard infused postcolumn at 9 uL / min while injecting 1 blank and 5 patient samples. The chromatogram was observed and then it was determined whether a change in signal intensity occurred during sample elution. The percent ion suppression was calculated as the percent difference between the approximate average TIC height before the analyte RT and directly around the analyte RT.Results: Minimal ion suppression was observed. Conclusion: There is no detectable interference due to ion suppression, greater then 25%.

[0214] Carryover.

[0215] Acceptance Criteria: The difference between sample types should be < TEa / 2. The carry over test were performed across 3 separate assays. During each extraction, samples were injected in the specific order of 2 low samples, then 2 high samples, and then 3 low samples. Results: The data shows that the average % difference for Epinephrine, Norepinephrine, and Dopamine are all less then TEa / 4. Conclusion: There is no carry over for detection of Epinephrine, Norepinephrine, and Dopamine.

[0216] Reference Interval (RI).

[0217] Reference range interval is as follows:

[0218] Specimen types.

[0219] Acceptance Criteria: the difference due to difference in storage container and / or sample type should be <TEa / 4 to be considered acceptable. 5 venous blood samples were drawn from each subject using standard venipuncture technique from up to 10 random patients. The plasma and serum results were compared against the results from plasma drawn from the current acceptable tube type for ECD-HPLC platform, sodium heparin. Results: The data shows that the average carryover for Epinephrine, Norepinephrine, and Dopamine are all less then TEa / 4.Conclusion: Plasma samples collected from tubes containing EDTA and Lithium Heparin Tubes are acceptable.

[0220] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0221] The methods illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof. It is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the invention embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0222] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the methods. This includes the generic description of the methods with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0223] Other embodiments are within the following claims. In addition, where features or aspects of the methods are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

WHAT IS CLAIMED IS:

1. A method for determining an amount of an analyte in a sample, the method comprising: purifying the sample by liquid chromatography; subjecting the sample to a derivatizing agent to generate a derivatized sample; subjecting the derivatized sample to electrospray ionization (ESI) under conditions suitable to produce one or more ions detectable by mass spectrometry; and quantifying the amount of the analyte by tandem mass spectrometry, wherein the quantifying comprises quantifying an amount of the ion(s), and the amount of the ion(s) detected is related to the amount of the analyte in the sample; wherein: the limit of quantitation 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 agent 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 precursor ion having a mass-to-charge ratio of 305.2 ± 0.5.

4. The method of claim 2 or claim 3, wherein the quantifying comprises quantifying the amount of one or more fragment ion(s) having a mass-to-charge ratio selected from the group consisting of 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 precursor ion having a mass-to-charge ratio of 319. l±0.5.

7. The method of claim 5 or 6, wherein the quantifying comprises quantifying the amount of one or more fragment ion(s) having a mass-to-charge ratio selected from the group consisting of 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 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 ion(s) having a mass-to-charge ratio selected from the group consisting of 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 quantitation of the methods is less than or equal to 10 pg / mL.

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

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

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

16. The method of any one of claims 1-15, wherein the amounts of two or more of the analytes from 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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