Determination of antidepressants by mass spectrometry

Abstract

To provide methods for detecting or determining an amount of antidepressants and / or antidepressant metabolites in a sample.SOLUTION: A method for detecting or determining an amount of antidepressants and antidepressant metabolites in a sample, includes the steps of: subjecting the sample to ionization under conditions suitable to produce one or more ions detectable by mass spectrometry; determining an amount of one or more ions by mass spectrometry; and using the amount of one or more ions to determine the amount of antidepressants or antidepressant metabolites in the sample.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Cross-reference to related patent applications This application is filed on 2019, each of which is incorporated by reference in its entirety. This application claims the benefit of U.S. Provisional Application No. 62 / 855,863, filed May 13. [Background technology]

[0002] A baseline test was conducted to determine whether patients were using one or more antidepressants before treatment. It is useful for helping clinicians determine when to take medication. Monitoring patients prescribed antidepressants to avoid unwanted polypharmacy is difficult. Important: Some antidepressants, such as selective serotonin reuptake inhibitors (SSRIs), should not be mixed with drugs in the same class that may have adverse side effects. Accurate testing of antidepressants and metabolites is needed. Summary of the Invention [Means for solving the problem]

[0003] In one aspect, a method for detecting and quantifying antidepressants and antidepressant metabolites by mass spectrometry is provided. Provided herein.

[0004] Method for detecting the presence or amount of antidepressants and / or antidepressant metabolites in a sample by mass spectrometry A method is provided herein for detecting one or more ions that can be detected by mass spectrometry. subjecting the sample to ionization under conditions suitable for producing a single molecule by mass spectrometry; or determining the amount of a plurality of ions; and using the amount of one or more ions to measure the amount of the sample. and determining the presence or amount of antidepressant and / or antidepressant metabolites therein.

[0005] In some embodiments, the mass spectrometry comprises tandem mass spectrometry. The method comprises: a) ionizing a sample under conditions suitable for producing precursor ions; b) fragmenting the precursor ion to produce one or more fragment ions. and c) the amount of one or more ions produced in steps a) and b). and d) determining the amount of one or more ions using the amount of one or more ions determined in step c). and determining the presence or amount of the antidepressant and metabolites in the sample.

[0006] In some embodiments, a selective serotonin reuptake inhibitor, serotonin and norepinephrine and dopamine reuptake inhibitors, one or more antidepressants, including tricyclic antidepressants, sedatives, and / or antidepressant metabolites Methods for detecting or determining the amount of depressant and antidepressant metabolites are provided herein.

[0007] In some embodiments, fluoxetine, paroxetine, sertraline, citalopram, Pram, escitalopram, fluvoxamine, vilazodone, duloxetine, venrafa phenytoin, desmethylvenlafaxine, hydroxybupropion, imipramine, and nortol. Liptiline, amitriptyline, doxepin, trimipramine, desipramine, protriptyline Ptyline, amoxapine, clomipramine, maprotiline, trazodone, mirtazapine, Vortioxetine, desmethylcitalopram, desmethylclomipramine, desmethyld Xepin, norfluoxetine, norfluvoxamine, norsertraline, and 1,3- one or more antidepressants and antidepressants selected from the group consisting of chlorphenylpiperazine Methods for detecting or determining the amount of a drug metabolite are provided herein.

[0008] In some embodiments, one or more selective serotonin reuptake inhibitors (SREs) are Ibuprofen, paroxetine, sertraline, citalopram, escitalopram, fulvic acid oxamine, vilazodone), serotonin and norepinephrine reuptake inhibitors (duloxamine, vilazodone), cetin, venlafaxine, desmethylvenlafaxine), norepinephrine and doxorubicin amine reuptake inhibitors (hydroxybupropion), tricyclic antidepressants (imipramine, nortriptyline, amitriptyline, doxepin, trimipramine, desipramine, To detect or determine the amount of Other antidepressants used in this assay are sedatives and The metabolites tested are trazodone, mirtazapine, and vortioxetine. are desmethylcitalopram, desmethylclomipramine, desmethyldoxepin, and norethinib. Fluoxetine, norfluvoxamine, norsertraline, and 1,3-chlorophenyl It was piperazine.

[0009] In some embodiments, the amounts of 10 or more antidepressants and antidepressant metabolites simultaneously Methods for detecting or determining are provided herein.

[0010] In some embodiments, the amounts of 20 or more antidepressants and antidepressant metabolites simultaneously Methods for detecting or determining are provided herein.

[0011] In some embodiments, the amounts of 30 antidepressants and antidepressant metabolites are detected simultaneously. Methods for determining the above are provided herein.

[0012] In some embodiments, the methods provided herein comprise detecting one or more internal standards. In some embodiments, the one or more internal standards include: In some embodiments, the deuterated internal standard 1,3-chlorophenylpiperazine-D8, hydroxybupropion-D6, Descemet Tilvenlafaxine-D6, desmethylcitalopram-D3, trimipramine-D3 , Amitriptyline-D3, Nortriptyline-D3, Paroxetine-D6, Protriptyline Citalopram-D3, Citalopram-D6, Venlafaxine-D6, Imipramine-D3, selected from the group consisting of lazodone-D6, vilazodone-D4, and vortioxetine-D8 can be.

[0013] In some embodiments, the sample comprises a biological sample. In some embodiments, the sample is urine. In some embodiments, the sample is plasma or serum. In this embodiment, the sample is blood.

[0014] In some embodiments, the sample is subjected to liquid chromatography prior to ionization. In some embodiments, the liquid chromatography is high performance liquid chromatography. Includes:

[0015] In some embodiments, the method involves administering a concentration of about 4 ng / mL to about 5000 ng / mL (both ends). The present invention can detect levels of antidepressants and antidepressant metabolites in a range of concentrations (including values ​​of 0.01 to 0.1).

[0016] In some embodiments, the method comprises administering a dose of about 25 ng / mL to about 5000 ng / mL (both can detect levels of antidepressants and antidepressant metabolites within the range (including the end values) .

[0017] In some embodiments, the mass spectrometry is tandem mass spectrometry. In this state, tandem mass spectrometry is used for selected reaction monitoring, multiple reaction monitoring, precursor monitoring, and This is done by ion scanning or product ion scanning.

[0018] In a preferred embodiment, tandem mass spectrometry is performed by selected reaction monitoring. .

[0019] In some embodiments, ions having the following mass / charge ratios (m / z) are detected: Provided herein is a method for quantifying antidepressants and antidepressant metabolites, comprising the steps of: do.

[0020] [Table 1] JPEG2025170251000003.jpg254133JPEG2025170251000004.jpg154137

[0021] In some embodiments, the methods described herein provide a method for treating a patient with a pulmonary artery disease (PAGE) comprising administering a pulmonary artery disease (PAGE) to a patient in need thereof. Detect levels of antidepressants and antidepressant metabolites in the range of 0.1 mg / mL (inclusive). In some embodiments, the methods described herein can be used to detect 25 ng / mL to Antidepressants and antidepressant metabolites at levels within the range of 5000 ng / mL (inclusive) can be detected.

[0022] In some embodiments, the methods described herein provide an antidepressant with a lower limit of 10 ng / mL. In some embodiments, the present invention provides a method for quantifying the antidepressant and its metabolites. The method described here is capable of quantifying antidepressants and antidepressant metabolites down to a limit of 50 ng / mL. Cut.

[0023] In some embodiments, prior to ionization, the sample is extracted using a column such as a solid phase extraction (SPE) column. In some related embodiments, SPE and mass spectrometry are performed on the same column. This is done using line processing.

[0024] In some embodiments, the sample is subjected to high performance liquid chromatography (H In some related embodiments, the HPLC column is applied to an analytical column such as a HP LC and mass spectrometry are performed using online processing.

[0025] In some embodiments, the method comprises measuring an antidepressant in a biological sample, such as plasma or serum. and can be used to determine the presence or amount of antidepressant metabolites. In an embodiment, the biological sample is processed by one or more steps to obtain a processed A sample may be generated which may then be subjected to mass spectrometry. The one or more processing steps may include protein precipitation, filtration, liquid-liquid extraction, solid-phase extraction, liquid crystal extraction, or the like. chromatography, any immunopurification process, or the like, and any combination thereof. The method may include one or more purification steps, such as combining.

[0026] In certain preferred embodiments of the methods disclosed herein, mass spectrometry is performed using a positive Alternatively, mass spectrometry may be performed in negative ion mode. For example, atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI) A variety of ionization sources can be used in embodiments of the present invention, including: Therefore, antidepressants and antidepressant metabolites are measured using positive ion mode.

[0027] In a preferred embodiment, a singly detectable internal standard is added to the sample, the amount of which is also tested. In these embodiments, the analyte and endogenous components of interest present in the sample are determined. Both the solid and the standard are ionized in whole or in part, resulting in multiple ions that can be detected by the mass spectrometer. One or more ions generated from each are detected by mass spectrometry. In these embodiments, the presence or amount of ions generated from the analyte of interest is determined by the presence or amount of ions in the sample. It can be related to the amount of analyte present in the sample of interest.

[0028] In other embodiments, the amount of antidepressant and antidepressant metabolites in a sample is determined by one or more The determination can be made by comparison with an external reference standard. An exemplary external reference standard is an anti- Blank plasma or blood spiked with depressant and antidepressant metabolites or their isotope-labeled variants It is Qing.

[0029] As used herein, unless otherwise indicated, the singular forms "a," "an," and "the" Thus, for example, a reference to "a protein" includes multiple referents. It contains several protein molecules.

[0030] As used herein, the term "purification" or "to purify" refers to the purification of an analyte (single analyte) of interest. This does not mean that all materials other than the sample (or samples) are removed from the sample. The product is of interest relative to other components in the sample that may interfere with the detection of the analyte of interest. Refers to a procedure that increases the amount of one or more analytes. Purification of a sample by various means , one or more interfering substances, e.g., detection of selected parent or daughter ions by mass spectrometry. The relative reduction of one or more substances that may or may not interfere When using this term, the relative reduction refers to the amount of analyte of interest in the material to be purified. It is not necessary that all substances present with the product be completely removed by purification.

[0031] As used herein, the term "immunopurification" or "immunopurifying" refers to the process of immunopurifying one of the following: or antibodies, including polyclonal or monoclonal antibodies, for enrichment of multiple analytes. Immunopurification refers to a purification procedure utilizing any of the immunopurification methods known in the art. Immunopurification procedures can often be carried out using a solid support, e.g., a column, Conjugated particles bound to wells, tubes, gels, capsules, particles, or the like. Immunopurification, as used herein, is the use of antibodies attached or otherwise bound to the target protein. In this case, a procedure often referred to in the art as immunoprecipitation, as well as a procedure often referred to in the art as affinity chromatography, Examples of suitable chromatographic techniques include, without limitation, a procedure called affinity chromatography.

[0032] As used herein, the term "immunoparticle" refers to a particle that has a surface (on and / or within the particle) Capsules, beads having antibodies bound to, conjugated to, or otherwise attached to In certain embodiments utilizing immunopurification, immunoglobulins may be used. Immunospheres include sepharose or agarose beads. Alternative embodiments utilizing immunopurification In the method, the immunoparticles comprise glass, plastic or silica beads or silica gel. nothing.

[0033] As used herein, the term "sample" refers to a sample that may contain an analyte of interest. As used herein, the term "body fluid" refers to any fluid that can be separated from the body of an individual. For example, "body fluid" means blood, plasma, serum, bile, saliva, urine, tears, In some embodiments, the sample is a bodily fluid sample, preferably a sample of a bodily fluid, which may include sweat and the like. includes plasma or serum.

[0034] As used herein, the term "solid phase extraction" or "SPE" refers to the process by which a solution passes through or surrounds a sample. Dissolved or suspended in a solution (i.e., mobile phase) for a freely flowing solid (i.e., solid phase) SP means a method of separating a mixture of chemicals into components as a result of the affinity of the separated components. E, as used herein, is the affinity of the components in the mobile phase for the solid phase, in immunoaffinity. It differs from immunopurification in that it is not the result of a specific protein but rather the result of a chemical or physical interaction. As the mobile phase flows through or around the solid phase, undesirable components of the mobile phase are transported to the solid phase. The retention by the phase may result in the purification of the analyte in the mobile phase. In this method, the analyte is retained by the solid phase and undesired components of the mobile phase pass through or pass through the solid phase. In these cases, the next step may be to allow the material to flow around the The retained analytes are eluted from the solid phase using a second mobile phase. E can function by a single or mixed mode mechanism. A mixed mode mechanism is when multiple It utilizes ion exchange and hydrophobic retention in a mixed-mode SPE column. The phase may exhibit strong anion exchange and hydrophobic retention, or strong cation exchange and hydrophobic retention. It can show your strength.

[0035] As used herein, the term "chromatography" refers to the process of chromatography carried out by liquid or gas. The difference in the chemicals when a mixture of chemicals flows around or over a stationary liquid or solid phase. It refers to the process by which a substance is separated into its components as a result of differential partitioning.

[0036] As used herein, the term "liquid chromatography" or "LC" refers to a method for When the particles uniformly penetrate a column or capillary passage of fine material, one or more particles of the fluid solution This refers to a method by which components are selectively retarded. Retardation is achieved by separating one or more stationary phases from the bulk fluid ( the stationary phase(s) as this fluid moves relative to the stationary phase(s). It results from the partitioning of the components of a mixture. Examples of "liquid chromatography" include reversed-phase liquid chromatography. RPLC, High Performance Liquid Chromatography (HPLC) and Turbulent Flow Liquid Chromatography (TFLC) (sometimes called high turbulence liquid chromatography (HTLC) or known as high-throughput liquid chromatography).

[0037] As used herein, "high performance liquid chromatography" or "HPLC" (sometimes referred to as "high performance liquid chromatography" or "HPLC") The term "high pressure liquid chromatography" refers to the process of transferring a mobile phase under pressure to a stationary phase. A method of increasing the degree of separation by forcing a liquid through a column, typically a tightly packed column. It means chromatographic analysis.

[0038] As used herein, "turbulent flow liquid chromatography" or "TFLC" (sometimes referred to as "high turbulence liquid chromatography" or "TFLC") This is known as high-speed liquid chromatography or high-performance liquid chromatography. The term utilizes the turbulent flow of the substance being assayed through a column packing as the basis for separation. TFLC refers to a form of chromatography. TFLC is a method for separating two atomic fractions of a sample prior to analysis by mass spectrometry. This has been applied to the preparation of samples containing drugs. ogr, Vol. A854, pp. 23-35 (1999). For further explanation of TFLC, see U.S. Patent Nos. 5,968,367, 5,919,368, 5,795, See also U.S. Pat. Nos. 469 and 5,772,874. Those skilled in the art understand "turbulent flow." When a fluid flows slowly and smoothly, the flow is called "laminar flow." For example, Fluid moving at a low flow rate through an HPLC column is laminar. The motion of the particles is regular, with the particles moving generally in straight lines. At higher velocities, the water Turbulence occurs when inertia overcomes the frictional force of the fluid. It "outruns" that which is slowed by friction or deflected by an uneven surface. When a fluid is flowing turbulently, it is due to a greater "resistance" than when the flow is laminar. Flows in a swirling (or spiral) shape. Determine whether the fluid flow is laminar or turbulent. Many references are available to help in this regard (e.g., Turbule nt Flow Analysis: Measurement and Predict ion, PS Bernard & JMWallace, John Wiley & Sons, Inc. (2000), An Introduction to T. urbulent Flow, Jean Mathieu & Julian Scot t, Cambridge University Press (2001)).

[0039] As used herein, the term "gas chromatography" or "GC" refers to the process of analyzing a sample. The mixture is evaporated and the carrier moves through a column containing a stationary phase consisting of a liquid or particulate solid. Inject into a stream of gas (as nitrogen or helium) to measure the affinity of the compound to the stationary phase. This refers to chromatography, which separates a compound into its component compounds according to the following formula.

[0040] As used herein, the term "large particle column" or "extraction column" refers to a column having a particle size of approximately 50 μm. In this context, a chromatographic column containing particles with a mean particle diameter greater than m is referred to. When used herein, the term "about" means ±10%.

[0041] As used herein, the term "analytical column" refers to a column that is used to determine the presence or amount of an analyte. Enough chloroform to allow sufficient separation of the substances in the sample to be eluted from the column. means a chromatography column having a chromatographic plate. Such a column is Separating the retained material from the unretained material to obtain a purified sample for further analysis. This is often distinguished from "extraction columns," which have the general purpose of isolating or extracting. When used in context, the term "about" means ±10%. In the analytical column, particles of approximately 5 μm in diameter are included.

[0042] As used herein, the terms "online" and "inline" refer to, e.g., "online automated fashion" or "online automated fashion" As used in "online extraction," it refers to a procedure that is performed without the need for operator intervention. In contrast, the term "offline" as used herein refers to a process that is not manually performed by an operator. It refers to a procedure that requires intervention by industry. Thus, the sample is subjected to precipitation and then If the supernatant is manually loaded into the autosampler, the precipitation and loading steps are performed in the subsequent steps. In various embodiments of the method, one or more steps It can be carried out online automatically.

[0043] As used herein, the term "mass spectrometry" or "MS" refers to the analysis of compounds by their mass. MS refers to an analytical technique for identifying molecules by their mass-to-charge ratio or "m / z" of ions. MS technology is a method of filtering, detecting, and measuring ions based on the Generally, the method includes the steps of (1) ionizing a compound to produce a charged compound; and detecting the molecular weight of the compound and calculating the mass-to-charge ratio. The mass spectrometer generally refers to an ionization device that can ionize and detect the ions. and an ion detector. Generally, one or more molecules of interest are ionized and then The ions are introduced into a mass analyzer where a combination of magnetic and electric fields causes the ions to It follows a path in space that depends on mass ("m") and charge ("z"). For example, Mass Spectrometry From Surfaces No. 6,204,500, entitled "Method and Apparatus for Tandem Mass Spectrometry" hods and Apparatus for Tandem Mass Spect No. 6,107,623, entitled "Mass Spectrometry-Based DNA Diagnostics ( DNA Diagnostics Based On Mass Spectromet No. 6,268,144, entitled "Surface Enhancement for Analyte Desorption and Detection" Surface-Enhanced Photolabile Binding and Release Attachment And Release For Deoption And No. 6,124,137, entitled "Detection of Analytes"; Wright et al., Prostate Cancer and Prostatic Di seases, 1999, vol. 2, pp. 264-76 and Merchant and Wein Berger, Electrophoresis, 2000, 21, 1164-67 Please refer to page.

[0044] As used herein, the term "operating in negative ion mode" refers to the "Operating in positive ion mode" refers to a mass spectrometry method that generates and detects ions. The term "mass spectrometry" as used herein refers to a mass spectrometry method that generates and detects positive ions. do.

[0045] As used herein, the term "ionization" or "ionize" refers to the act of ionizing one or more electrons. Negative ions refer to a method for generating analyte ions with a net charge equal to the molecular unit. An ion is one that has a net negative charge of one or more electron units, while a positive ion is one that has a net negative charge of one or more electron units. has a net positive charge of electron units.

[0046] As used herein, the term "electron ionization" or "EI" refers to gas-phase or vapor-phase The term refers to the way in which the analyte of interest in a sample interacts with the flow of electrons. The collisions produce analyte ions that can then be subjected to mass spectrometry techniques.

[0047] As used herein, the term "chemical ionization" or "CI" refers to the process of ionizing a reagent gas (e.g., Ammonia (e.g., ammonia) is subjected to electron bombardment, resulting in the interaction of reagent gas ions with analyte molecules. This refers to the process by which analyte ions are generated.

[0048] As used herein, the term "fast atom bombardment" or "FAB" refers to high energy A beam of atoms (often Xe or Ar) bombards a non-volatile sample, dissolving the atoms contained in the sample. This refers to a method in which the test sample is dissolved in glycerol, thioglycerol, or Benzyl alcohol, m-nitrobenzyl alcohol, 18-crown-6 crown ether, 2-nitro Phenyloctyl ether, sulfolane, diethanolamine and triethanolamine The compound or sample is dissolved in a viscous liquid matrix such as Selection is an empirical process.

[0049] As used herein, "matrix-assisted laser desorption ionization" or "MALDI" The term "" refers to the process of photoionizing, protonating, deprotonating and clustering a non-volatile sample. A laser that desorbs and ionizes analytes in a sample through various ionization pathways, including decay For MALDI, the sample is exposed to radiation that promotes desorption of the analyte molecules. It is mixed with an energy absorbing matrix that accelerates the process.

[0050] As used herein, "surface-enhanced laser desorption ionization" or "SELDI" The term "" refers to the process of photoionizing, protonating, deprotonating and clustering a non-volatile sample. A laser that desorbs and ionizes analytes in a sample through various ionization pathways, including decay For SELDI, the sample is generally exposed to one of the following radiation sources: Alternatively, multiple analytes can be bound to a surface that preferentially retains them. Similar to MALDI, this method The method may also employ an energy absorbing material to facilitate ionization.

[0051] As used herein, the term "electrospray ionization" or "ESI" This refers to a method in which a solution is passed through a short capillary tube to the ends of which a high positive or negative potential is applied. The solution that reaches the target vaporizes (atoms) into a jet or stream of very small droplets of the solution in solvent vapor. This spray of droplets flows through the evaporation chamber. When the electrons are released, the natural repulsion between the like-charged molecules causes the release of ions and neutral molecules. The surface charge density increases until

[0052] As used herein, the term "atmospheric pressure chemical ionization" or "APCI" refers to ES APCI is a mass spectrometry technique similar to I, but APCI is an ion- Ions are generated by molecular reactions. The plasma is maintained by an electrical discharge between the atomizing capillary and the counter electrode. The ions are then pumped, typically using a pair of differentially pumped skimmer stages. The solvent is removed using countercurrent dry, preheated N2 gas. Gas phase ionization in APCI allows for the analysis of less polar species. It may be more effective than ESI to

[0053] The term "atmospheric pressure photoionization" or "APPI" as used herein refers to the photoionization of molecules M The photoionization mechanism of mass fraction M is the absorption of a photon and the emission of an electron to produce a molecular ion M+. This refers to the form of decomposition. Since the photon energy is generally just above the ionization potential, The ions are not susceptible to dissociation. In many cases, samples can be analyzed without the need for chromatography. This can save considerable time and money. In the presence of a ionic solvent, the molecular ion can abstract H to form MH+. This tends to happen if the sum of M+ and MH+ is constant. This does not affect the accuracy of the quantification. Drug compounds in protic solvents are generally Although it is usually observed as MH+, non-polar compounds such as naphthalene or testosterone are usually Usually, M+ is formed. For example, Robb et al., Anal. Chem., 2000, vol. 72 ( 15), pp. 3653-3659.

[0054] As used herein, the term "inductively coupled plasma" or "ICP" refers to most The sample is partially ionized at a temperature high enough that the elements are atomized and ionized. This refers to the way in which the gas interacts with the object.

[0055] As used herein, the term "field desorption" refers to the process of applying a non-volatile test sample to an ionizing surface. This refers to a method in which a sample is placed on a substrate and a strong electric field is used to generate analyte ions.

[0056] As used herein, the term "desorption" refers to the removal and / or cleavage of an analyte from a surface. Laser desorption / thermal desorption refers to the entry of the analyte into the gas phase. The laser is a technique for thermally desorbing atoms into the gas phase using a laser pulse. The backside of a specially designed 96-well plate is irradiated. The laser pulse heats the bottom, and the heat is transferred to the sample. The gas phase sample is then drawn into a mass spectrometer.

[0057] As used herein, the term "selected ion monitoring" refers to the monitoring of a relatively narrow mass range. detection mode of a mass spectrometer where only ions within a range of, typically about 1 mass unit, are detected It is Do.

[0058] As used herein, "multiple reaction monitoring," sometimes known as "selective reaction monitoring," refers to In the "reaction mode," precursor ions and one or more fragment ions are selectively detected. This is the detection mode of the mass spectrometer.

[0059] As used herein, the terms "lower limit of quantitation," "lower limit of quantitation," or "LLOQ" , the point at which the measurement becomes quantitatively meaningful. The responses of the substances are identifiable and individual, with relative standard deviations (RSD%) of less than 20%. It is reproducible with an accuracy of 85% to 115%.

[0060] As used herein, the term "limit of detection" or "LOD" refers to the amount of a measurement that is related to The LOD is the point at which a value is greater than the uncertainty associated with that measurement. The point above which the RSD is exceeded is defined as three times the RSD of the mean value at zero concentration.

[0061] As used herein, the "amount" of an analyte in a body fluid sample generally refers to the amount of analyte in a volume of sample. However, the amount is not necessarily the same as other analytes. Relative amounts are also contemplated, for example, the amount of an analyte in a sample compared to the amount normally present in the sample. The amount may be greater than a control or normal level of the analyte.

[0062] The term "about" is used herein to refer to quantitative measurements that do not include measurement of the mass of an ion. Where applicable, this means the stated value plus or minus 10%. Determining the mass of an ion can vary slightly depending on the mass or mass / charge ratio of the ion. The term "about" in this context means + / - 0.50 atomic mass units.

[0063] The above summary of the invention is not intended to be limiting, and other features and advantages of the invention may be found in the accompanying drawings, in which: These and other aspects of the present invention will become apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0064] [Figure 1] FIG. 1 shows the LC-MS / MS profiles of all analytes and metabolites. [Figure 2] 1 shows an example of baseline separation of (A) amitriptyline, (B) maprotiline, and (C) venlafaxine (analyte: left, internal standard (IS): right). [Figure 3] FIG. 1 shows the accuracy of citalopram compared to another laboratory, showing no bias greater than ±20% of the value. [Figure 4] FIG. 1 shows the accuracy of the metabolite desmethylcitalopram compared to another laboratory. No bias greater than ±20% of the value is shown. [Figure 5] Figure 1 shows cyclobenzaprine interference resolved. The figure shows 5 ng / mL maprotiline + 100x cyclobenzaprine. [Figure 6] FIG. 1 shows the mass spectral differences between desmethylvenlafaxine versus tramadol. [Figure 7] 1 shows tramadol interference isolated. The figure shows 5 ng / mL desmethylvenlafaxine + 100x tramadol. [Figure 8-1] FIG. 1 shows baseline separation of amitriptyline, maprotiline, and venlafaxine. [Figure 8-2] (Continuation of Figure 8-1) [Figure 9-1] FIG. 1 shows baseline separation of nortriptyline, protriptyline, and desmethylvenlafaxine. [Figure 9-2] (Continuation of Figure 9-1) [Figure 10] FIG. 1 shows baseline separation of desmethyldoxapine and mirtazapine. [Figure 11-1] FIG. 1 shows desipramine vs. mirtazapine identified by different transitions. [Figure 11-2] (Continued from Figure 11-1) [Figure 12-1] FIG. 1 shows that ion ratios and / or relative retention times (RRTs) are absent for desipramine in patients positive for mirtazapine. [Figure 12-2] (Continued from Figure 12-1) DETAILED DESCRIPTION OF THE INVENTION

[0065] In certain embodiments, the antidepressant panels described herein include a panel of drugs within this class. Along with compliance monitoring for patients with a history / risk of use and / or abuse Baseline testing before prescribing drugs in this class can help determine if Patients should be warned about the possibility of drug conflicts due to polypharmacy. Monitoring will identify the prescribed medications and non-prescribed medications that should be present for these patient populations. The absence of drugs that do not cause steroid use is necessary.

[0066] Certain brain chemicals, neurotransmitters, more specifically serotonin, norepinephrine and Dopamine is associated with depression. Most antidepressants affect these neurotransmitters. Different types / classes of antidepressants work in different ways to treat these neurotransmitters. These types include SSRIs, SNRIs, NDRIs, tricyclics, and non-steroidal antidepressants. Includes typical, MAOIs, etc. (see below).

[0067] Selective serotonin reuptake inhibitors (SSRIs). Doctors often prescribe SSRIs. These medications are safer than other types of antidepressants and generally less bothersome. SSRIs are less likely to cause serious side effects. (Selfemra), paroxetine (Paxil, Pexeva), Traline (Zoloft), citalopram (Celexa), escitalopram (Lexapro), Fluvoxamine (Faverin, Fevarin, Fluvoxamine) Floxyfral, Dumirox, Luvox, Includes vilazodone (Viibryd).

[0068] Serotonin and norepinephrine reuptake inhibitors (SNRIs) - Duloxetine Inbalta), venlafaxine (Effexor XR), desmethylvenlafaxine ( O-desmethylvenlafaxine, a synthetic form of the main metabolite of venlafaxine; Pristi levomilnacipran (Fetz) and levomilnacipran (Khedezla) SNRIs are unique drugs that increase both serotonin and norepinephrine levels. SNRIs have their own dual action, so they inhibit more than one cause of depression.

[0069] Norepinephrine and dopamine reuptake inhibitors (NDRIs). Bupropion (We Lubutrin, Aplenzin, Forfivo XL ) falls into this category. It is one of the few anti-inflammatory drugs that is rarely associated with sexual side effects. It is a type of depressant.

[0070] Tricyclic antidepressants (TCAs) tend to cause more side effects than newer antidepressants. Tricyclic antidepressants are generally recommended unless the patient first tries an SSRI and does not see improvement. Not prescribed. TCAs include imipramine (Tofranil), nortriptyline (Pamelo) Pamelor), amitriptyline (Elavil, Endep, Le Lentizol, Levate, Saroten ), Tryptanol, Tryptizol), Doxepin (Adapin) (Adapin), Curatin, Silenol, Sinequan quan), trimipramine (Surmontil), desipramine (Norpramine (No rpramin), protriptyline (Vivactil), Amoxa phenytoin (Asendin), clomipramine (Anafranil), and methadone Contains lotilin (Ludiomil).

[0071] Atypical antidepressants. These drugs do not fit neatly into any of the other antidepressant categories. These include trazodone (Oleptro), mirtazapine (Remeron) ) and vortioxetine (Brintellix). is sedating and is usually taken at night.

[0072] Monoamine oxidase inhibitors (MAOIs) are not included in this assay. The drug cannot be combined with an SSRI. A common MAOI is tranylcypromine. (Parnate), phenelzine (Nardil) and Includes isocarboxazid (Marplan).

[0073] A method for determining the amount of an analyte in a sample is described. More specifically, human plasma or serum. Abstract: A mass spectrometry method is described for detecting and / or quantifying an analyte in a biological sample, such as a nucleotide sequence. The method may utilize liquid chromatography, followed by a quantification of the analyte in the sample. This is followed by tandem mass spectrometry for identification.

[0074] Suitable test samples for use in the methods of the present invention are test samples that may contain the analyte of interest. In some preferred embodiments, the sample is a biological sample, i.e., an animal, a cell culture Samples obtained from biological sources such as cultures, organ cultures, etc. In certain preferred embodiments, In this case, the sample is obtained from a mammal such as a dog, cat, horse, etc. Particularly preferred mammals are The subject is a primate, most preferably a male or female human. Preferred samples are urine, blood, plasma, etc. Such samples include body fluid or tissue samples such as serum, saliva, cerebrospinal fluid, and preferably urine. is provided to, for example, a patient, i.e., a clinical facility, for diagnosis, prognosis, or treatment of a disease or condition. In some embodiments, the subject may be a living male or female individual appearing in court. A preferred sample may be obtained from a female human of childbearing potential. In embodiments comprising the steps of: To determine the amount of leflunomide metabolites (i.e., the amount of endogenous leflunomide metabolites in the sample) It can be used for.

[0075] The present invention also contemplates a kit for the quantitative assay of antidepressants. Kits for use in the treatment of cancer can include kits containing the compositions provided herein. For example, the kits can include , packaging materials, and a quantity of isotopically labeled internal standard sufficient for at least one assay. Generally, tangible information regarding the use of prepackaged reagents for quantitative assays of antidepressants is available. This also includes instructions recorded in writing (e.g., contained on paper or electronic media).

[0076] Calibration and QC pools used in embodiments of the present invention are preferably essentially free of analyte. Prepare using a matrix similar to the target sample matrix, provided that the matrix is ​​not present. do.

[0077] Preparation of samples for mass spectrometry In preparation for mass spectrometry, for example, liquid chromatography, filtration, centrifugation, Thin-layer chromatography (TLC), electrophoresis including capillary electrophoresis, immunoaffinity affinity separation, including cellulose-based separation; extraction methods, including ethyl acetate or methanol extraction; and including the use of chaotropic agents or any combination of the above or the like. Analytes can be isolated from a sample (e.g., a protein) by a variety of methods known in the art, including It may be concentrated relative to one or more other components.

[0078] Protein precipitation is used to prepare test samples, particularly biological test samples such as serum or plasma. Protein purification methods are well known in the art. See, for example, Polson et al. , Journal of Chromatography B 2003, Vol. 785: Pages 263-275 describe protein precipitation techniques suitable for use in the methods of the present invention. Proteins are removed from the sample in order to remove most of the protein, leaving the analyte in the supernatant. Protein precipitation can be used. The sample is centrifuged to separate the liquid from the precipitated proteins. The supernatant may be separated or the sample may be filtered to remove precipitated proteins. The resulting supernatant or filtrate may then be subjected directly to mass spectrometry or to liquid chromatography. The product may be subjected to additional purification methods, such as purification, and subsequent mass spectrometry. In this case, the mass can be reduced by using protein precipitation, e.g., acetonitrile protein precipitation. TFLC or prior to analysis or high performance liquid chromatography (HPLC) and mass spectrometry The need for other online extractions may be eliminated.

[0079] Another method of sample purification that can be used prior to mass spectrometry is liquid chromatography. (LC). Liquid chromatography, including high performance liquid chromatography (HPLC) Certain methods rely on relatively slow laminar flow techniques. Traditional HPLC analysis involves the use of Laminar flow of the sample through the column depends on the column packing, which is the basis for separating the analytes of interest from the sample. Those skilled in the art will understand that separation in such columns is a partitioning process, Select an LC, including HPLC, instrument, and column that is appropriate for use with the analyte Chromatographic columns are generally used for the separation of compound components (i.e., The medium may include fine particles. Particles generally interact with various compound components to facilitate their separation. One suitable binding surface is an alkyl bond, a cyano bond, or a biphenyl bond. The alkyl bonding surfaces are C-4, C-8, C- In a preferred embodiment, the column is The chromatographic column has an inlet for receiving the sample and a separator for separating the sample. The sample can be extracted directly into the inlet or by online extraction. It can be supplied from an SPE column or a TFLC column. In this embodiment, particles and phospholipids in the sample are removed before the sample reaches the HPLC column. To remove the ions, an online guard cartridge can be used before the HPLC column. In some embodiments, the guard cartridge is a biphenyl guard cartridge. It may be.

[0080] In one embodiment, the sample is applied to the LC column at the inlet port and a solvent or solvent mixture is added. The analyte(s) of interest can be eluted by the compound and discharged at the outlet. Various solvent modes for eluting the chromatographic components can be selected. The analysis can be performed using gradient, isocratic, or polymorphic (i.e., mixed) modes. During chromatography, separation of substances occurs via an eluent (known as the "mobile phase"). (also known), are affected by variables such as elution mode, gradient conditions, temperature selection, etc.

[0081] In certain embodiments, the analyte of interest is reversibly retained on the column packing, but The sample is applied to the column under conditions where one or more other substances are not retained. In these embodiments, the analyte of interest is purified by the column. A first mobile phase condition can be used in which the first retained material is retained and the unretained material is washed away. If so, a second mobile phase condition can then be used to remove the retained material from the column. Alternatively, the analyte of interest may dissolve at a different rate than one or more other substances. The analyte can be purified by applying the sample to the column under mobile phase conditions that favor Such procedures may involve the detection of one or more components of interest relative to one or more other components of the sample. The amount of the analyte may be increased.

[0082] In one preferred embodiment, the HPLC is a biphenyl column chromatographic system. In certain preferred embodiments, the method is carried out using a biphenyl analytical column (e.g., For example, a Pinnacle DB biphenyl analytical column (5 μm) manufactured by Restek Inc. In certain preferred embodiments, particles of 50x2.1 mm or equivalent are used. HPLC was performed using 0.1% aqueous formic acid for HPLC as solvent A and acetone as solvent B. The reaction is carried out using 0.1% formic acid in nitrile.

[0083] Careful selection of valves and fittings allows for one chromatic Two or more chromatography columns are used to pass material from one column to the next. In a preferred embodiment, the valves and piping are The selection is made by a computer preprogrammed to perform the necessary steps. Most preferably, the chromatography system is controlled in such an online manner. It is also connected to a detection system, e.g., an MS system. The tray can be attached to the autosampler and the rest of the operation is computer controlled. This is carried out under the conditions described above, resulting in the purification and analysis of all selected samples.

[0084] In some embodiments, TFLC is used for purification of analytes prior to mass spectrometry. In such an embodiment, a TFLC column can be used to capture the analyte. The sample can then be extracted using the HPLC. For example, the sample can be extracted using a TFLC extraction cartridge or a large particle column. This can be achieved using a packed column with a diameter of 50 μm. The sample is transferred online to an analytical HPLC column for further purification before mass spectrometry analysis. The steps involved in these chromatographic procedures can be linked in an automated fashion. This feature minimizes the need for operator intervention during the purification of the analyte. This can result in time and cost savings and eliminate opportunities for operator error .

[0085] Detection and quantification by mass spectrometry In various embodiments, the analyte can be ionized by methods known to those skilled in the art. Mass spectrometry ionizes the fractionated samples to produce charged molecules for further analysis. For example, ionization of a sample is performed using a mass spectrometer that includes an ion source for electron Ionization, Chemical Ionization, Electrospray Ionization (ESI), Photon Ionization, Atmospheric Pressure Chemical Ionization (APCI), Photoionization, Atmospheric Pressure Photoionization (APPI), Laser Diode Iode thermal desorption (LDTD), fast atom bombardment (FAB), liquid secondary ionization (LSI), Matrix-assisted laser desorption ionization (MALDI), field ionization, field desorption, Mospray / Plasmaspray Ionization, Surface Enhanced Laser Desorption Ionization (SE) This can be done by LDI), inductively coupled plasma (ICP) and particle beam ionization Those skilled in the art will appreciate that the choice of ionization method depends on the analyte being measured, the type of sample, the type of detector, and the potential It will be appreciated that the decision may be based on selection of positive vs. negative modes, etc.

[0086] Analytes may be ionized in positive mode or negative mode. In some embodiments, the analyte is ionized in positive mode. .

[0087] In mass spectrometry, the sample is generally ionized and then the resulting positive or negative ions are analyzed. The negatively charged ions can be analyzed to determine their mass-to-charge ratio (m / z). Suitable analyzers for determining γ are quadrupole analyzers, ion trap analyzers and in-flight analyzers. Exemplary ion trap methods include those described by Bartolucci et al., R apid Commun.Mass Spectrom., 2000, Vol. 14, 967 It is described on page 73.

[0088] According to some methods of the present invention, high resolution / high accuracy mass spectrometry is used to quantify analytes. That is, mass spectrometry can be performed at about 50 ppm or less of positive ion concentration for the ions of interest. A mass spectrometer capable of providing a resolving power (FWHM) of at least 10,000 was used. Preferably, the mass spectrometer has a resolving power (FWHM) of 20,000 or greater. M) and an accuracy of about 20 ppm or less, e.g., a resolution of 25,000 or more (FWHM) and accuracy of about 5 ppm or less, e.g., 25,000 or more The resolution (FWHM) is above 3 ppm and the accuracy is about 3 ppm or less. Three exemplary mass spectrometers that can demonstrate the required level of performance are -bitrap mass spectrometer, certain TOF mass spectrometers or Fourier transform ion cyclotrons The mass spectrometer includes a neutron resonance mass spectrometer.

[0089] Elements found in biologically active molecules, such as carbon, oxygen, and nitrogen, are naturally occurring in many different isotopes. For example, most carbon atoms exist in the 12 C, but not all naturally occurring About 1% of the carbon present is 13 C. Therefore, naturally occurring molecules containing The carbon fraction that 13 It contains a C atom. The inclusion of different elemental isotopes in a molecule results in multiple molecular isotopes. The difference in abundance is at least 1 atomic mass unit (amu). This means that the element isotopes are at least This is because the isotopes differ by one neutron (the mass of one neutron is approximately 1 amu). When ionized into a charged state, detection in mass spectrometry is based on mass-to-charge ratio (m / z). Therefore, the mass difference between isotopes can be difficult to distinguish. Two isotopes that differ in mass by 1 amu are ionized to the High resolution / high accuracy mass spectrometry is a highly versatile Charged ions (such as ions with ±4, ±5, ±6, ±7, ±8, ±9 or higher charges) It is possible to distinguish between isotopes of elements such as uranium and uranium.

[0090] Naturally occurring elemental isotopes allow for the determination of all molecular ions (with sufficiently sensitive mass spectrometers) (each of which may produce a separately detectable spectral peak when analyzed by an instrument) Multiple isotopes are commonly present in a molecule. The m / z ratios and relative abundances of multiple isotopes are In some embodiments, two or more molecules collectively comprise the isotopic signature of the ions. The m / z and relative abundance of the molecular isotopes are used to confirm the identity of the molecular ion under consideration. In some embodiments, the mass spectrometry peaks of one or more isotopes can be In some related embodiments, the molecular ion is quantified using a single isotope. In other related embodiments, a single mass spectrometric peak is used to quantify the molecular ion. The isotopic peak is used to quantify the molecular ion. The isotopic peaks can be subjected to appropriate mathematical treatments. These methods are well known in the art and include the sum of the areas under multiple peaks or the average of the responses from multiple peaks. , but not limited to these.

[0091] Generally in mass spectrometry techniques, ions can be detected using several detection modes. For example, the ions selected can be monitored in selected ion monitoring mode (SIM). Alternatively, or in place of, mass transitions due to collisionally activated dissociation (CAD) For example, multiple reaction monitoring (MRM) or selected reaction monitoring (SRM) CAD is often used to separate fragments for subsequent detection. In CAD, precursor ions are energized by collisions with an inert gas. They gain energy and then fragment in a process called "unimolecular decomposition." Enough energy is present in the ions so that the increased energy can break certain bonds within the ions. Alternatively, neutral loss can be monitored. can.

[0092] In some embodiments, the mass-to-charge ratio is determined using a quadrupole analyzer. In "quadrupole" or "quadrupole ion trap" instruments, ON is proportional to the DC potential applied between the electrodes, the amplitude of the RF signal, and the mass-to-charge ratio The voltage and amplitude are such that only ions with a specific mass / charge ratio traverse the quadrupole. However, all other ions can be selected to be deflected. acts as both a "mass filter" and a "mass detector" for the ions injected into the instrument possible.

[0093] The specificity of MS technology can be improved by "tandem mass spectrometry" or "MS / MS" In this technique, precursor ions (also called parent ions) are obtained from the target molecule. ) can be filtered by the MS instrument, and the precursor ions are then fragmented. and generate one or more fragment ions (daughter ions or Careful selection of precursor ions allows the generation of specific target ions. Only ions generated by the analyte are passed to the fragmentation chamber, where they are deactivated. Collisions with gas atoms produce fragment ions. Precursor and fragment ions Both amines are reproducibly generated under a set of defined ionization / fragmentation conditions, allowing for the S / MS technology can be an extremely powerful analytical tool. A combination of fragmentation can be used to remove interfering substances and This can be particularly useful for complex samples such as biological samples.

[0094] An alternative mode of operating a tandem mass spectrometer instrument is product ion scanning and pre- For a description of these modes of operation, see, e.g., E.Mi chael Thurman et al., Chromatographic-Mass Spec trometric Food Analysis for Trace Determinology ation of Pesticide Residues, Chapter 8( Edited by Amadeo R. Fernandez-Alba, Elsevier 2005)( 387).

[0095] The results of the analyte assay can be analyzed by a number of methods known in the art to determine the amount of analyte in the original sample. For example, if sampling and analytical parameters are carefully considered, the amount of precipitate can be related to the amount of If the relative abundance of a given ion is deeply controlled, the relative abundance can be calculated by first Alternatively, an external standard can be run with the sample. A standard curve can be generated based on the ions obtained from those standards. Using a standard curve, the relative abundance of a given ion can be converted to the absolute amount of the original molecule. In certain preferred embodiments, an internal standard can be used to calculate the amount of analyte. Methods for preparing and using such standard curves are well known in the art. and those skilled in the art can select an appropriate internal standard. For example, One or more forms of isotopically labeled molecules having / z can be used as internal standards. In some embodiments described herein, exemplary internal standards are isotopically labeled. Diazepam, but also many other compounds (isotopically labeled or otherwise) Many other methods for relating the amount of ion to the amount of the original molecule are known to those skilled in the art. It is well known.

[0096] As used herein, "isotopically labeled" refers to a compound that, when analyzed by mass spectrometry techniques, appears to be unlabeled. This results in a mass shift of the labeled molecule relative to the molecule. Examples of suitable labels include deuterium ( 2 H ), 13 C and 15 N. One or more isotope labels can be added to one or more molecules. One or more types of isotopic labels can be incorporated into the same isotopically labeled molecule. It can be used for.

[0097] One or more steps of the method may be performed using automated equipment. In an embodiment, one or more purification steps are performed online, more preferably All of the purification and mass spectrometry steps can be performed online.

[0098] In a particularly preferred embodiment, the analytes in a sample are determined using MS / MS as follows: The sample is preferably analyzed by liquid chromatography, preferably The liquid solvent flow from the chromatographic column is then analyzed by MS / MS. The solvent / analyte mixture enters the heated nebulizer interface of the analyzer. In these processes, the analyte (i.e., The ions, e.g., precursor ions, are analyzed through the aperture of the instrument. The ions pass through the first quadrupole and enter the second quadrupole. Selection of ions based on the ratio (m / z) (i.e., “precursor” in Q1 and Q3, respectively) The mass filter allows for the selection of " and "fragment" ions. Q2) is the collision cell where ions are fragmented. The quadrupole (Q1) selects molecules with the mass-to-charge ratio of the analyte. Precursor ions with other mass / charge ratios are passed into the collision chamber (Q2). Unwanted ions with π collides with the sides of the quadrupole and are removed. The fragment ions collide with neutral argon gas molecules and become fragments. is passed to quadrupole 3 (Q3), where fragment ions of the analyte are selected and other The ions are removed.

[0099] The method can be carried out in either positive or negative ion mode, preferably in positive ion mode. MS / MS performed in a holomorphic fashion may be performed by a skilled artisan using standard methods well known in the art. The authors identified one of the specific precursor ions of the analyte that could be used for selection in quadrupole 3 (Q3). One or more fragment ions can be identified.

[0100] When ions hit the detector, they emit a pulse of electrons that is converted into a digital signal. The acquired data is transferred to a computer, which then analyzes the collected information. The on counts are plotted against time. The resulting mass chromatogram is similar to the traditional HP Similar to a chromatogram obtained by LC-MS. Peaks corresponding to specific ions The area under or amplitude of such peaks can be measured and related to the amount of analyte of interest. In certain embodiments, the fragment ion(s) and / or precursor ion(s) can be The area under the curve or amplitude of the ON peak is measured to determine the amount of analyte. , the relative abundance of a given ion is determined by the peak of one or more ions of an internal or external molecular standard. The amount of the initial analyte can be converted to absolute amounts using a calibration standard curve based on

[0101] The following examples serve to illustrate the invention. These examples are provided to illustrate the scope of the present method. It is not a restriction. [Example]

[0102] Example 1: Sample preparation Simultaneous analysis of analytes and their metabolites for 23 prescription antidepressants as shown in Table 1 below. A validated LC-MS / MS method for the analysis is described.

[0103] [Table 2] JPEG2025170251000006.jpg223170

[0104] Quality Controls, Calibrators, and Internal Standards: Drug-Free Urine Control (UTAK) Calibration standards (4-5,000 ng / mL) were prepared by spiking a stock solution of the analyte into and quality controls (QCs) of 5, 12.5, and 4,000 ng / mL were prepared. The internal standards (IS) were 1,3-chlorophenylpiperazine-D8, hydroxybupropion, and thrombin-D6, desmethyl-venlafaxine-D6, desmethylcitalopram-D3, Rimipramine-D3, Amitriptyline-D3, Nortriptyline-D3, Paroxetine -D6, Protriptyline-D3, Citalopram-D6, Venlafaxine-D6, Imi pramin-D3, trazodone-D6, vilazodone-D4, and vortioxetine-D8 The mixture ranged from 25 to 100 ng / mL.

[0105] Sample preparation: Urine samples, calibrators, and QCs (25 μL each) were mixed in 1 mL 96-well plates. Mix with IS (25 µL) in a 10 mM ammonium formate in water (mobile phase A) in a 100 µL extraction plate. ) and diluted with 450 μL, then vortexed at 1,100 rpm for 2 minutes, and then injected and It was transferred to LC-MS / MS for analysis.

[0106] Example 2: Liquid Chromatography-Mass Spectrometry LC-MS / MS: Extracted samples (25 μL) were analyzed by Kinetex® fluorescein chromatography. A 2.6μ column (Phenomenex) was used. A / mobile phase B (25% methanol in acetonitrile) gradient was used for chromatography. Separation was performed using a 4-column LC multiplex with Prelude LX-4 Maximize throughput with MD™ (ThermoFisher Scientific) A Sciex 4500 Triple Quad™ mass spectrometer was used for the selected reaction model. Figure 1 shows a representative chromatogram for all analytes. , Figure 2 shows baseline separation of closely related analytes.

[0107] Table 2 shows the mass transitions used to detect each analyte in the mass spectrometry assay.

[0108] [Table 3] JPEG2025170251000008.jpg241155JPEG2025170251000009.jpg243157JPEG2025170251000010.jpg61169

[0109] Example 3: Verification and Results Validation: The following characteristics were determined by standard laboratory methods: limit of quantitation (LOQ), linearity (including upper linear limit [ULOL] by dilution), precision, accuracy, over 150 different drugs Interference from, stability, stability of extracted analytes, matrix effects, and carryover.

[0110] Linearity: Five- to nine-point calibration curves were obtained with a regression coefficient (r) >0.990 within a ±20% range of the target. Consistent linearity and reproducibility were demonstrated.

[0111] The CV was 7.5%-10%.

[0112] Analytical measurement range (AMR) for all antidepressant analytes and metabolites is 4-5.0 00ng / mL, LOQ was 10ng / ml (with one exception), and UL0 was 50,000n The exception was the metabolite norsertraline, whose AMR ranged from 25 to 5,000 nM. g / mL, and the LOQ was 50 ng / mL.

[0113] accuracy: Precision testing over 5 days showed a mean of >3 for low, medium and high level QCs. Sigma values ​​showed consistent results.

[0114] Accuracy: Sixty-five samples ranging in concentration from 4 to 20,000 ng / mL were collected from six other laboratories. Accuracy tests were performed by correlating the results of 5. Examples are shown in Figures 3 and 4.

[0115] Overall, Deming regression yields a correlation coefficient of 1.022 and an intercept of -0 without bias. .0681 was shown.

[0116] Disruption: (Over 150 multiple illicit and prescription drugs were tested, 100 times the cutoff. The study included both negative matrix controls and LoQ controls spiked with related substances. I went there.)

[0117] All interfering drugs tested showed a signal intensity of ≥ 20% of the LOQ panel drugs. Does not cause deviations.

[0118] Stability: The samples were stable for 7 days at room temperature, 14 days in the refrigerator, and 30 days in the freezer. was stable for 24 hours.

[0119] Matrix Effects: Samples were diluted with undiluted and diluted matrix at three different levels (0.5x, 2x). x, and 0.8xULOL).

[0120] No matrix effects were observed.

[0121] Carryover: Two samples were spiked consecutively at 4000 ng / mL, followed by four blank samples. The carryover effect was determined by repeating the experiment three times.

[0122] No carryover was observed.

[0123] All articles, patents and patent applications, and all other literature and electronic publications mentioned or cited herein. The content of publicly available information is subject to the exclusion of all representations, warranties, and conditions, unless each individual publication is specifically and individually incorporated by reference. No. 6,399,423, filed on Oct. 1, 2003, and incorporated herein by reference in its entirety to the same extent as if set forth herein. Applicants reserve the right to withdraw any and all copies of any such articles, patents, patent applications, or other physical and electronic documents. The right to physically incorporate into this application any and all materials and information from any and all sources is reserved.

[0124] The methods illustratively described herein may not include any element or components not specifically disclosed herein. may be suitably practiced in the absence of any element, limitation, or limitation. Thus, for example, the terms "comprising," "including," ) and "containing" are read broadly and without limitation. Further, the terms and phrases used herein are to be regarded as terms of description and not of limitation. and in the use of such terms and expressions This does not exclude any equivalents or portions of the features described herein that fall within the scope of the invention as claimed. It is recognized that various modifications are possible in the present invention. Although specifically disclosed by embodiments and optional features, the present disclosure and Variations and modifications of the invention embodied in the present invention may be used by those skilled in the art, and such variations may be made. It is to be understood that all such variations and modifications are considered to be within the scope of the present invention.

[0125] The invention has been described broadly and generically herein. Each of the narrower species and subspecies also forms part of the method. This includes the selection of any pair from a class. A general description of the method with conditions or negative limitations for removing material is provided to clarify whether the removed material is in accordance with the present invention. This includes whether or not it is specifically listed in the schedule.

[0126] Other embodiments are within the scope of the following claims. is described by a Markush group, one skilled in the art will understand that the present invention also It is understood that the term "group" may be described by any individual member or subgroup of members of the group. It will be.

Claims

1. Detecting the amount of one or more antidepressants and antidepressant metabolites in a sample by mass spectrometry is a method for determining a. under conditions suitable to produce one or more ions detectable by mass spectrometry; subjecting the sample to ionization; b. determining the abundance of one or more ions by mass spectrometry; c. Using the amount of one or more ions determined in step (b), determine the antidepressant in the sample. determining the amount of the drug or antidepressant metabolite; A method comprising:

2. the one or more antidepressants and antidepressant metabolites are selective serotonin reuptake inhibitors; agents, serotonin and norepinephrine reuptake inhibitors, norepinephrine and dopamine and antidepressant metabolites, including thiazol-3-one reuptake inhibitors, tricyclic antidepressants, sedatives, and antidepressant metabolites. The method according to claim 1.

3. The one or more antidepressants and antidepressant metabolites are selected from the group consisting of fluoxetine, paroxetine, Sertraline, citalopram, escitalopram, fluvoxamine, vilazodone, durozodone Xetine, venlafaxine, desmethylvenlafaxine, hydroxybupropion, Imipramine, nortriptyline, amitriptyline, doxepin, trimipramine, Cipramine, protriptyline, amoxapine, clomipramine, maprotiline, and torazolam Don, mirtazapine, vortioxetine, desmethylcitalopram, desmethylclomide Lamin, desmethyldoxepin, norfluoxetine, norfluvoxamine, norselt 1,3-chlorophenylpiperazine, and 1,3-chlorophenylpiperazine. The method described.

4. Simultaneously detecting or determining the amounts of 10 or more antidepressants and antidepressant metabolites. The method of claim 1 , comprising:

5. Simultaneously detecting or determining the amounts of 20 or more antidepressants and antidepressant metabolites. The method of claim 1 , comprising:

6. The method includes the step of simultaneously detecting or determining the amounts of 30 antidepressants and antidepressant metabolites. The method of claim 1 .

7. 10. The method of claim 1, wherein one or more internal standards are added.

8. 8. The method of claim 7, wherein the one or more internal standards comprise a deuterated internal standard.

9. The deuterated internal standard was 1,3-chlorophenylpiperazine-D8, hydroxybromine Propionibacterium-D6, desmethyl-venlafaxine-D6, desmethylcitalopram-D 3. Trimipramine-D3, Amitriptyline-D3, Nortriptyline-D3, Paroxetine Cetin-D6, Protriptyline-D3, Citalopram-D6, Venlafaxine-D6 , imipramine-D3, trazodone-D6, vilazodone-D4, and vortioxetine- 9. The method of claim 8, wherein the compound is selected from the group consisting of D8.

10. The method of claim 1 , wherein the sample comprises a biological sample.

11. The method of claim 1 , wherein the sample comprises urine.

12. 10. The method of claim 1, wherein the sample is subjected to liquid chromatography prior to ionization.

13. 13. The method of claim 12, wherein the liquid chromatography comprises high performance liquid chromatography. How to do it.

14. Antidepressants at levels ranging from about 4 ng / mL to about 5000 ng / mL, inclusive 10. The method of claim 1, wherein the method is capable of detecting antidepressant and antidepressant metabolites.

15. Antibodies at levels ranging from about 25 ng / mL to about 5000 ng / mL, inclusive The method of claim 1, capable of detecting neuroleptic and antidepressant metabolites.

16. The method of claim 1 , wherein the mass spectrometry is tandem mass spectrometry.

17. The tandem mass spectrometry may include selective reaction monitoring, multiple reaction monitoring, precursor ion 17. The method of claim 16, wherein the method is performed by scanning or product ion scanning. method.

18. 17. The method of claim 16, wherein the tandem mass spectrometry is performed by selected reaction monitoring. Law.

19. 2. The method of claim 1, wherein the lower limit of quantitation is 10 ng / mL.

20. 2. The method of claim 1, wherein the lower limit of quantitation is 50 ng / mL.

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