DETERMINATION OF ANTIDEPRESSANTS BY MEANS OF MASS SPECTROMETRY.

Abstract

The present invention relates to a method for simultaneously detecting or determining the amount of 20 or more antidepressants and antidepressant metabolites in a sample by means of mass spectrometry, wherein the 20 or more antidepressants and antidepressant metabolites include selective serotonin reuptake inhibitors (SSRIs) and SSRI metabolites comprising fluoxetine and norfluoxetine, said method comprising: a. subjecting the sample to ionization under suitable conditions to produce multiple ions detectable by means of mass spectrometry; b. determining the amount of multiple ions by means of mass spectrometry, wherein the multiple ions comprise a fluoxetine fragment ion with a mass / charge ratio of 148.1 ± 0.50 and a norfluoxetine fragment ion with a mass / charge ratio of 134.2 ± 0.50 or 30.1 ± 0.50; and c.Use the quantity of multiple ions determined in step (b) to determine the quantity of 20 or more antidepressants or antidepressant metabolites in the sample.

Description

DETERMINATION OF ANTIDEPRESSANTS BY MEANS OF MASS SPECTROMETRY FIELD OF INVENTION

[0001] The invention relates to described methods for detecting or determining the amount of antidepressants and / or antidepressant metabolites in a sample. More specifically, mass spectrometric methods are described for detecting and quantifying antidepressants and / or antidepressant metabolites in a sample. BACKGROUND OF THE INVENTION

[0002] Baseline testing is useful in helping a clinician determine whether or not a patient is using antidepressant drugs for treatment. It is crucial to monitor patients prescribed antidepressants to ensure adherence and avoid unintended polypharmacy. Some antidepressants, such as selective serotonin reuptake inhibitors (SSRIs), can have side effects and should not be mixed with other drugs in the same class. Accurate testing of the antidepressant and its metabolites is necessary. SUMMARY OF THE INVENTION

[0003] In one aspect, methods for detecting and quantifying antidepressants and antidepressant metabolites by mass spectrometry are provided herein.

[0004] This document provides methods for detecting the presence or quantity of antidepressants and / or antidepressant metabolites in a sample by mass spectrometry. The methods include subjecting the sample to ionization under suitable conditions to produce one or more ions detectable by mass spectrometry; determining the quantity of one or more ions by mass spectrometry; and using the quantity of one or more ions to determine the presence or quantity of antidepressants and / or antidepressant metabolites in the sample.

[0005] In some modalities, mass spectrometry comprises tandem mass spectrometry. In such modalities, the methods include: a) ionizing the sample under suitable conditions to produce a precursor ion; b) fragmenting a precursor ion to produce one or more fragment ions; c) determining the amount of one or more ions produced in steps a) and b); and d) using the amount of one or more ions in step c) to determine the presence or amount of antidepressants and metabolites in the sample.

[0006] In some embodiments, methods are provided herein for detecting or determining the amount of one or more antidepressants and antidepressant metabolites comprising selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, norepinephrine-dopamine reuptake inhibitors, tricyclic antidepressants, sedatives, and antidepressant metabolites.

[0007] In some embodiments, methods are provided herein for detecting or determining the amount of one or more antidepressants and antidepressant metabolites selected from the group consisting of fluoxetine, paroxetine, sertraline, citalopram, escitalopram, fluvoxamine, vilazodone, duloxetine, venlafaxine, desmethylvenlafaxine, hydroxybupropion, imipramine, notriptyline, amitriptyline, doxepin, trimipramine, desipramine, protriptyline, amoxapine, clomipramine, maprotiline, trazodone, mirtazapine, vortioxetine, desmethylcitalopram, desmethylclomipramine, desmethyldoxepin, norfluoxetine, norfluvoxamine, norsertraline, and 1,3-chlorphenylpiperazine.

[0008] In some modalities, methods are provided herein for detecting or determining the amount of one or more selective serotonin reuptake inhibitors (fluoxetine, paroxetine, sertraline, citalopram, escitalopram, fluvoxamine, vilazodone); selective serotonin reuptake inhibitors (duloxetine, venlafaxine, desmethylvenlafaxine); norepinafrine and dopamine reuptake inhibitors (hydroxybupropion); tricyclic antidepressants (imipramine, nortriptyline, amitriptyline, doxepin, trimipramine, desipramine, protriptyline, amoxapine, clomipramine, maprotiline). Other antidepressants used in this assay may also act as sedatives and include trazodone, mirtazapine, and vortioxetine. The metabolites tested were desmethylcitalopram, desmethylclomipramine, desmethyldoxepin, norfluoxetine, norfluvoxamine, norsertraline, and 1,3-chlorphenylpiperazine.

[0009] In some forms, methods are provided herein for simultaneously detecting or determining the amount of 10 or more antidepressants and antidepressant metabolites.

[0010] In some forms, methods are provided herein for simultaneously detecting or determining the amount of 20 or more antidepressants and antidepressant metabolites.

[0011] In some forms, methods are provided herein for simultaneously detecting or determining the amount of 30 antidepressants and antidepressant metabolites.

[0012] In some embodiments, the methods provided herein comprise adding one or more internal standards. In some embodiments, one or more standards comprises deuterated internal standards. In some embodiments, the deuterated internal standards are selected from the group consisting of 1,3-chlorophenylpiperazine-D8, hydroxybupropion-D6, desmethyl-venlafaxine-D6, desmethylcitalopram-D3, trimipramine-D3, amitriptyline-D3, nortriptyline-D3, paroxetine-D6, protriptyline-D3, citalopram-D6, venlafaxine-D6, amipramine-D3, trazodone-D6, vilazodone-D4, and vortioxetine-D8.

[0013] In some modalities, the sample comprises a biological sample. In a preferred modality, the sample is urine. In some modalities, the sample is plasma or serum. In some modalities, the sample is blood.

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

[0015] In some modalities, the method is able to detect antidepressants and antidepressant metabolites at levels within the range of approximately 4 ng / ml to approximately 5000 ng / ml, global.

[0016] In some modalities, the method is able to detect antidepressants and antidepressant metabolites at levels within the range of approximately 25 ng / ml to approximately 5000 ng / ml, global.

[0017] In some modalities, mass spectrometry is tandem mass spectrometry. In some modalities, tandem mass spectrometry is conducted by selected reaction monitoring, multiple reaction monitoring, precursor ion scanning, or product ion scanning.

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

[0019] In some modalities, the present provides the step of determining antidepressants and antidepressant metabolites which comprises detecting ions comprising the following mass / charge (m / z) ratios. Precursor Q1 (m / z) Fragment Q3 (m / z) Analit 1 196.993 118 1,3-Chlorphenylpiperazine 1 2 196.993 119.1 1,3-Chlorphenylpiperazine 2 3 205.065 158.1 1,3-Chlorphenylpiperazine D8 4 278.096 105 Amitriptyline 1 5 278.096 115 Amitriptyline 2 6 281 202.1 Amitriptyline-D3 7 314.006 271.1 Amoxapina 1 8 314.006 193.1 Amoxapina 2 9 256.02 130 Hydroxibupropion 1 10 .....11............ 256.02 103 Hydroxibupropion 2 262.061 130.1 Hidroxybupropion-D6 12 325.07 109 Citalopram 1 13 325.07 262.1 Citalopram 2 14 331.103 109 Citalopram-D6 15 264.083 91 Nortriptilina 1 16 264.083 105 Nortriptilina 2 17 267.095 105 Notriptilina-D3 18 311.043 109 Desmetilcitalopram 1 Precursor Q1 (m / z) Fragment Q3 (m / z) Analyte 19 311.043 262.1 Desmethylcitalopram 2 20 314.072 108.9 Desmethylcitalopram-D3 21 315.054 86.1 Clomipramine 1 22 315.054 58 Clomipramine 2 23 301,037 72 Desmethylclomipramine 1 24 301,037 227.1 Desmethylclomipramine 2 25 267,091 72 Desipramine 1 26 267,091 193.1 Desipramine 2 27 280,096 107 Doxepin 1 28 280,096 165.1 Doxepin 2 29 266.074 107 Desmethyldoxapine 1 30 31............ 266.074 77 Desmethyldoxapine 2 298.03 154.1 Duloxetine 1 32 298.03 44.1 Duloxetine 2 33 310.07 148.1 Fluoxetine 1 34 310.07 44.1 Fluoxetine 2 35 296.066 134.2 Norfluoxetine 1 36 37.......... 296.066 30.1 Norfluoxetine 2 319.057 71 Fluvoxamine 1 38 39.......... 319.057 200.1 Fluvoxamine 2 305.025 229.1 Norfluvoxamine 1 40 305.025 188.1 Norfluvoxamine 2 41 281.098 86 Imipramine 1 42 281.098 58 Imipramine 2 43 284.013 89 Imipramine-D3 44 278.094 191.2 Maprotiline 1 45 278.094 189 Maprotiline 2 46 266.081 195.1 Mirtazapine 1 47 266.081 194.1 Mirtazapine 2 48 330.033 192.1 Paroxetina 1 49 330.033 70 Paroxetina 2 50 336.092 198.2 Paroxetina-D6 51 264.096 191 Protriptilina 1. Precursor Q1 (m / z) Fragmento Q3 (m / z) Analito 52 264.096 189 Protriptilina 2 53 267.095 191.1 Protriptilina-D3 54 306 159 Sertralina 1 55 306 275 Sertralina 2 56 292.005 159 Desmetilsertralina 1 57 292.005 123 Desmetilsertralina 2 58 372.096 176.1 T razodona 1 59 372.096 148 Trazodona 2 60 378.114 182.1 Trazodona-D6 61 295.128 100.1 Trimipramine 1 62 295.128 58.1 Trimipramine 2 63 298.138 103.1 Trimipramine-D3 64 278.126 58 Venlafaxine 1 65 278.126 121 Venlafaxine 2 66 284.139 64.1 Venalfaxine-D6 67 264.106 58 Desmethylvenlafaxine 1 68 264.106 107 Desmethylvenlafaxine 2 69 270.134 64.1 Desmethylvenlafaxine-D6 70 442.133 155.1 Vilazodone 1 71 442.133 197.2 Vilazodone 2 72 446.163 155.1 Vilazodone-D4 73 299.059 150 Vortioxetina 1 74 299.059 109 Vortioxetina 2 75 307.082 153.1 Vortioxetina-D8

[0020] In some modalities, the methods described herein are capable of detecting antidepressants and antidepressant metabolites at levels within the range of 4 ng / ml to 5000 ng / ml, global. In some modalities, the methods described herein are capable of detecting antidepressants and antidepressant metabolites at levels within the range of 25 ng / ml to 5000 ng / ml, global.

[0021] In some modalities, the methods described herein are capable of quantifying antidepressants and antidepressant metabolites to a lower limit of 10 ng / ml. In some modalities, the methods described herein are capable of quantifying antidepressants and antidepressant metabolites to a lower limit of 50 ng / ml.

[0022] In some modes, the sample is subjected to an extraction column, such as a solid-phase extraction (SPE) column, prior to ionization. In some related modes, SPE and mass spectrometry are conducted with online processing.

[0023] In some modalities, the sample is subjected to an analytical column, such as a high-performance liquid chromatography (HPLC) column, prior to ionization. In some related modalities, HPLC and mass spectrometry are conducted with online processing.

[0024] In some modalities, the methods can be used to determine the presence or quantity of antidepressants and antidepressant metabolites in a biological sample, such as plasma or serum. In some related modalities, a biological sample is processed through one or more steps to generate a processed sample, which can then be subjected to mass spectrometry analysis. In some modalities, one or more processing steps comprise one or more purification steps, such as protein precipitation, filtration, liquid-liquid extraction, solid-phase extraction, liquid chromatography, any immunopurification process, and any combination thereof.

[0025] In some preferred embodiments of the methods disclosed herein, mass spectrometry is performed in a positive ion mode. Alternatively, mass spectrometry is performed in a negative ion mode. Various ionization sources, including, for example, atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI), may be used in embodiments of the present invention. In some embodiments, antidepressants and antidepressant metabolites are measured using a positive ion mode.

[0026] In preferred embodiments, a separately detectable internal standard is provided in the sample, the amount of which is also determined in the sample. In such embodiments, all or a portion of both the analyte of interest and the internal standard present in the sample is ionized to produce a plurality of ions detectable by mass spectrometry, and one or more ions produced from each are detected by mass spectrometry. In such embodiments, the presence or amount of ions generated from the analyte of interest can be related to the presence of the amount of analyte of interest in the sample.

[0027] In other modalities, the amount of antidepressants and antidepressant metabolites in a sample can be determined by purchasing one or more external reference standards. External reference standards include plasma or white serum augmented with antidepressants and antidepressant metabolites or an isotopically labeled variant thereof.

[0028] As used herein, unless otherwise stated, the singular forms “a”, “one”, “an” and “the” include their plural references. Thus, for example, a reference to “a protein” includes a plurality of protein molecules.

[0029] As used herein, the term “purification” or “purify” does not refer to the removal of 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 the detection of the analyte of interest. Purification of the sample by various means may allow for the relative reduction of one or more interfering substances, e.g., one or more substances that may or may not interfere with the detection of ESA selected mother or daughter ions by mass spectrometry. Relative reduction, as used herein, does not require that any substance present with the analyte of interest in the material to be purified be completely removed by means of purification.

[0030] As used herein, the term “immunopurification” or “immunopurify” refers to a purification procedure that uses antibodies, including polyclonal or monoclonal antibodies, to enrich one or more analytes of interest. Immunopurification may be performed using any of the immunopurification methods known in the prior art. Often, the immunopurification procedure uses the binding of antibodies, conjugates, or other forms attached to a solid support; for example, a column, well, tube, gel, capsule, particle, or other similar medium. Immunopurification, as used herein, includes, among others, procedures often referred to in the prior art as immunoprecipitation and procedures often referred to in the prior art as affinity chromatography.

[0031] As used herein, the term “immunoparticle” refers to a capsule, bead, gel particle, or other similar object having antibodies, conjugates, or other antibodies attached to its surface (either on and / or within the particle). In some modalities using immunopurification, the immunoparticles comprise sepharose or agarose beads. In alternative modalities using immunopurification, the immunoparticles comprise glass, plastic, or silica beads, or silica gel.

[0032] As used herein, the term “sample” refers to any sample that may contain an analyte of interest. As used herein, the term “body fluid” means any fluid that can be isolated from an individual’s body. For example, “body fluid” may include blood, plasma, serum, bile, saliva, urine, tears, perspiration, and others. In some modalities, the sample comprises a sample of body fluid; preferably plasma or serum.

[0033] 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 components dissolved or suspended in a solution (i.e., mobile phase) for a solid through or around which the solution is passed (i.e., solid phase). SPE, as used herein, differs from immunopurification in that the affinity of the components in the mobile phase to the solid phase results from a chemical or physical interaction, rather than immunoaffinity. Occasionally, as the mobile phase passes through or around the solid phase, unwanted components of the mobile phase may be retained by the solid phase, resulting in purification of the analyte in the mobile phase.In other cases, the analyte may be retained by the solid phase, allowing unwanted components of the mobile phase to pass through or around it. In such cases, a second mobile phase is subsequently used to elute the retained analyte from the solid phase for further processing or analysis. SPE, including TFLC, can operate via a unit-mode or mixed-mode mechanism. Mixed-mode mechanisms utilize both ion exchange and hydrophobic retention in the same column; for example, the solid phase of a mixed-mode SPE column may exhibit strong anion exchange and hydrophobic retention, or it may exhibit a column that displays strong cation exchange and hydrophobic retention.

[0034] As used herein, the term “chromatography” refers to a process in which a chemical mixture carried out by a liquid or gas is separated into components as a result of differential distribution of the chemical entities as it flows around or along a stationary liquid or solid phase.

[0035] As used herein, the term “liquid chromatography” or “LC” means a process of selectively retarding one or more components of a fluid solution as the fluid is uniformly filtered through a column of a substance divided very finely, or through capillary passages. The retardation results in the distribution of the mixture components between one or more stationary phases and the bulk volume of fluid (i.e., mobile phase) as the fluid moves relative to the stationary phase(s). Examples of “liquid chromatography” include reversed-phase liquid chromatography (RPLC), high-performance liquid chromatography (HPLC), and turbulent flow liquid chromatography (TFLC) (sometimes known as high-turbulence liquid chromatography (HTLC) or high-throughput liquid chromatography).

[0036] As used herein, the term “high-performance liquid chromatography” or “HPLC” (sometimes referred to as “high-pressure liquid chromatography”) refers to liquid chromatography in which the degree of separation is increased by forcing the mobile phase under pressure through a stationary phase, usually a densely packed column.

[0037] As used herein, the term “turbulent flow liquid chromatography” or “TFLC” (sometimes referred to as high turbulence liquid chromatography or high-throughput liquid chromatography) refers to a form of chromatography that uses turbulent flow of the assay material 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. Patent Nos. 5,968,367, 5,919,368, 5,795,469, and 5,772,874, which explain TFLC in more detail. Those skilled in the art understand what is meant by “turbulent flow.” When the fluid flows slowly and subtly, 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 fluid particles is uniform, with particles generally moving in straight lines. At faster speeds, the inertia of the water overcomes fluid friction forces, resulting in turbulent flow. Fluid not in contact with the irregular boundary "overtakes" fluid that is slowed by friction or deflected by an irregular surface. When a fluid flows turbulently, it flows in eddies and vortices, with more "resistance" compared to laminar flow. Many references are available to help determine whether fluid flow is laminar or turbulent (e.g., Turbulent Flow Analysis: Measurement and Prediction, P.S. Bernard & J.M. Wallace, John Wiley & Sons, Inc.)., (2000); An Introduction to Turbulent Flow, Jean Mathleu & Julián Scott, Cambridge University Press (2001)).

[0038] As used herein, the term “gas chromatography” or “GC” refers to a chromatography in which the sample mixture is vaporized and injected into a carrier gas stream (such as nitrogen or helium) that moves through a column containing a stationary phase composed of a liquid or particulate solid and is separated into its component compounds according to the affinity of the stationary phase compounds.

[0039] As used herein, the term “large particle column” or “extraction column” refers to the chromatography column containing an average particle diameter greater than approximately 50 pm. As used in this context, the term “approximately” means ± 10%.

[0040] As used herein, the term “analytical column” refers to a chromatography column having sufficient chromatographic plates to effect a separation of the materials in a sample eluted from the column in sufficient quantity to permit 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 unretained material in order to obtain a purified sample for further analysis. As used in this context, the term “approximately” means ±10%. In a preferred embodiment, the analytical column contains particles approximately 5 pm in diameter.

[0041] As used herein, the term “online,” for example, as used in “automated online” or “online extraction,” refers to a procedure performed without the need for operator intervention. Conversely, the term “offline,” as used herein, refers to a procedure that requires manual operator intervention. Thus, if samples are subjected to precipitation, and the supernatants are subsequently loaded manually into an automated sample processor, the precipitation and loading steps are offline from subsequent steps. In several embodiments of the methods, one or more steps may be performed in an automated online manner.

[0042] As used herein, the term “mass spectrometry” or “MS” refers to a technique for identifying compounds by means of 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 compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating a mass-to-charge ratio. Compounds can be ionized and detected by any suitable means. A “mass spectrometry” 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 spectrometry instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that depends on their mass (“m”) and charge (“z”). See, e.g., US Patent Numbers 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 Desorption Detection of Analytes”; Wright et al., Prostate Cancer and Prostatic Diseases 1999, 2: 264-76: and Merchand and Weinberger, Electrophoresis 2000, 21: 1164-67.

[0043] As used herein, the term “negative ion mode operation” refers to those mass spectrometry methods where negative ions are generated and detected. The term “positive ion mode operation,” as used herein, refers to those mass spectrometry methods where positive ions are generated and detected.

[0044] As used herein, the term “ionization” refers to the process of generating an analyte ion that has a net electrical charge equal to one or more electron units. Negative ions are those that have a net negative charge of one or more electron units, while positive ions are those that have a net positive charge of one or more electron units.

[0045] As used herein, the term “electron ionization” or “El” refers to methods in which the analyte of interest, in a gas or vapor phase, interacts with a flow of electrons. The impact of the electrons with the analyte produces analyte ions, which can then be subjected to a mass spectrometry technique.

[0046] As used herein, the term “chemical ionization” or “Cl” refers to methods in which a reactant gas (e.g., ammonia) is subjected to electron impact, and analyte ions are formed by means of the interaction of reactant gas ions and analyte molecules.

[0047] 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 a non-volatile sample, desorbing and ionizing molecules contained in the sample. Test samples are dissolved in a viscous liquid matrix, such as, for example, glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6-crown ether, 2-nitrophenylethyl ether, sulfolane, diethanolamine, and triethanolamine. The selection of a suitable matrix for a compound or sample is an empirical process.

[0048] 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 means of ionization pathways, including photoionization, protonation, deprotonation, and heavy-ion decay. For MALDI, the sample is mixed with an energy-absorbing matrix, which facilitates the desorption of the analyte molecules.

[0049] 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 means of ionization pathways, including photoionization, protonation, deprotonation, and heavy-ion decay. For SELDI, the sample is typically bonded 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.

[0050] As used herein, the term “electrospray ionization” or “ESI” refers to methods in which a solution is passed along a short length of capillary tubing, at the end of which a high negative and positive electrical potential is applied. The solution reaching the end of the tubing is vaporized (nebulized) into a stream or spray of very small droplets of the solution in solvent vapor. This mist of droplets flows through an evaporation chamber. As the droplets become smaller, the surface electrical charge density decreases to the point where the natural repulsion between like charges causes ions, as well as neutral molecules, to be released.

[0051] 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 means of ion-molecule reactions that occur within a plasma at atmospheric pressure. The plasma is maintained by an electrical discharge between the spray capillary and a counter electrode. The ions are then typically extracted to the mass analyzer by means of a set of differentially pumped frother stages. A counterflow of preheated, dry N2 gas may be used to enhance solvent removal. Gas-phase ionization in APCI can be more effective than ESI for analyzing less polar species.

[0052] The term “atmospheric pressure photoionization” or “APPI,” as used herein, refers to the form of mass spectrometry where the photoionization mechanism of molecule M is photon absorption and electron ejection to form the molecular ion M+. Because the photon energy is usually just above the ionization potential, the molecular ion is less susceptible to dissociation. In many cases, it may be possible to analyze the samples without chromatography, thereby saving considerable time and expense. In the presence of water vapor or a protic solvent, the molecular ion can abstract H+ to form MH+. This tends to occur if M has a high proton affinity. This does not affect the accuracy of quantification because the sum of M+ and MH+ is constant.Drug compounds in protic solvents are often observed as MH+, whereas nonpolar compounds, such as naphthalene or testosterone, often form M+. See, e.g., Robb et al., Anal. Chem. 2000, 72(15): 3653-3659.

[0053] 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, so that most of the elements are atomized or ionized.

[0054] As used herein, the term “field desorption” refers to methods in which a non-volatile test sample is placed on an ionization surface, and an intense electric field is used to generate analyte ions.

[0055] 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 gas phase. Thermal desorption / laser desorption is a technique in which a sample containing the analyte is thermally desorbed into the gas phase by means of a laser pulse. The laser strikes 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 to the gas phase. The gas-phase sample is then taken to mass spectrometry.

[0056] As used herein, the term “ion-selective monitoring” is a detection mode of a mass spectrometry instrument in which only ions within a relatively narrow mass range, usually one mass unit, are detected.

[0057] As used herein, “multiple reaction mode”, sometimes referred to as “selected reaction monitoring”, is a detection mode for a mass spectrometry instrument in which a precursor ion and one or more fragment ions are selectively detected.

[0058] As used herein, the term “lower limit of quantification” or “LLOQ” refers to the point at which measurements become quantitatively significant. The analyte response at such LOQ is identifiable, discrete, and reproducible with a relative standard deviation (RSD %) of less than 20% and an accuracy of 85% to 115%.

[0059] As used herein, the term “limitation of detection” or “LOD” is the point at which the measurement value is greater than the uncertainty associated with it. The LOD is the point at which the value exceeds the uncertainty associated with its measurement and is defined as three times the RSD of the mean at zero concentration.

[0060] As used herein, an “amount” of analyte in a body fluid sample generally refers to an absolute value that reflects the mass of the detectable analyte in the sample volume. However, an amount also encompasses a relative amount compared to another amount of analyte. For example, an amount of an analyte in a sample may be an amount that is greater than a control or a level of the analyte normally present in the sample.

[0061] The term “approximately,” as used herein in reference to quantitative measurements excluding the measurement of the mass of an ion, refers to the stated value of plus or minus 10%. Mass spectrometry instruments may vary slightly in determining the mass of a given analyte. The term “approximately” in the context of the mass of an ion or the mass-to-charge ratio of an ion refers to + / - 0.50 atomic mass units.

[0062] The summary of the invention described above is non-limiting and other features and advantages of the invention will be evident from the description of the invention and the claims. BRIEF DESCRIPTION OF THE FIGURES

[0063] Figure 1 shows an LC-MS / MS profile of all analytes and metabolites.

[0064] Figure 2 shows an example of reference separation of (A) amitriptyline, (B) marprotiline, and (C) venlafaxine (analytes: left, internal standard (IS): right).

[0065] Figure 3 shows the accuracy of citalopram compared to another laboratory. It shows no bias greater than ±20% of the values.

[0066] Figure 4 shows the accuracy of the desmethylcitalopram metabolite compared to another laboratory. It shows no bias greater than ±20% of the values.

[0067] Figure 5 shows interference from separate Cyclobenzaprine. The figure shows 5 ng / ml of Maprotiline + Cyclobenzaprine at 100x.

[0068] Figure 6 shows mass spectral distinction of Desmethylvenlafaxine vs. Trelling.

[0069] Figure 7 shows separate Tramadol interference. The figure shows 5 ng / ml of Desmethylvenlafaxine + Tramadol at 100x.

[0070] Figure 8 shows a reference separation of Amitriptyline, Maprotiline, and Venlafaxine.

[0071] Figure 9 shows a reference separation of Notriptyline, Protritiline, Desmethylvenlafaxine.

[0072] Figure 10 shows a reference separation of Desmethyldoxapine and Mirtazapine.

[0073] Figure 11 shows Desipramine vs Mirtazapine identified by means of different transitions.

[0074] Figure 12 shows a proportion of ion and / or relative retention time (RRT) will fail for Desipramine in Mirtazapine-positive patients. DETAILED DESCRIPTION OF THE INVENTION

[0075] In some modalities, the antidepressant panels described herein may be used in conjunction with compliance monitoring for patients with a history of / risk for the use and / or abuse of drugs within that class. Prescribing baseline testing for this class of drugs alerts the provider to the potential for polypharmacy drug conflicts. Compliance monitoring requires the presence of prescribed drugs and the absence of non-prescribed drugs for these patient populations.

[0076] Certain brain chemicals, neurotransmitters, are associated with depression, specifically serotonin, norepinephrine, and dopamine. Most antidepressants treat depression by affecting these neurotransmitters. Different types / classes of antidepressants affect these neurotransmitters in different ways. These types include: SSRIs, SNRIs, NDRIs, tricyclics, atypical antidepressants, MAOIs, and others (see below).

[0077] Selective serotonin reuptake inhibitors (SSRIs). Doctors often start by prescribing an SSRI. These medications are safe and generally cause fewer troublesome side effects compared to other types of antidepressants. SSRIs include fluoxetine (Prozac, Selfemra), paroxetine (Paxil, Pexeva), sertraline (Zoloft), citalopram (Celexa), escitalopram (Lexapro), fluvoxamine (Faverina, Fevarina, Floxifral, Dumyrox, Luvox), and vilazodone (Viirbryd).

[0078] Serotonin-norepinephrine reuptake inhibitors (SNRIs) – dulexetine (Cymbalta), vanlafaxine (Effexor XR), desmethyl venlafaxine (a synthetic form of the major metabolite of venlafaxine, O-desmethylvenlafaxine; Pristiq, Khedezla), and levomilnacipran (Fetzima). SNRIs have a unique dual action in increasing levels of both serotonin and norepinephrine; therefore, SNRIs combat more than one cause of depression.

[0079] Norepinephrine and dopamine reuptake inhibitors (NDRIs). Bupropion (Wellbutrin, Aplenzin, Forfivo XL) falls into this category. It is one of the few antidepressants that are not frequently associated with sexual side effects.

[0080] Tricyclic antidepressants (TCAs) tend to cause more side effects compared to newer antidepressants. Tricyclic antidepressants are generally not prescribed unless the patient has tried an SSRI first, but without any improvement. TCAs include imipramine (Tofranil), notriptyline (Pamelor), amitriptyline (Elavil, Endep, Lentizol, Levate, Saroten, Triptanol, Triptizol), doxepin (Adapin, Curatin, Silenor, Sinequan), trimipramine (Surmontil), desipramine (Nopramin), protriptyline (Vivactil), amoxapine (Asendin), clomipramine (Anafranil), and maprotiline (Ludiomil).

[0081] Atypical Antidepressants. These medications do not fit into any of the antidepressant categories. They include trazodone (Oleptro), mirtazapine (Remeron), and vortioxetine (Brintellix). These are sedatives and are usually taken in the evening / night.

[0082] Monoamine oxidase inhibitors (MAOIs) are not included in this trial. These drugs may be combined with SSRIs. Common MAOIs include tranylcypromine (Parnato), phenelzine (Nardil), and isocarboxazid (Marplan).

[0083] Methods are described for measuring the amount of analyte in a sample. More specifically, mass spectrometry methods are described for detecting and / or quantifying analyte in a biological sample, such as human plasma or serum. The methods may use liquid chromatography followed by tandem mass spectrometry to quantify the analyte in the sample. IVIA / a / ZUZ I / υΊ

[0084] Test samples suitable for use in methods of the present invention include any test sample that may contain the analyte of interest. In some preferred embodiments, a sample is a biological sample; that is, a sample obtained from any biological source, such as, for example, an animal, a cell culture, an organ culture, etc. In some preferred embodiments, the samples are obtained from a mammalian animal, such as, for example, a dog, cat, horse, etc. In particular, the preferred mammalian animals are primates, with human males and females being the most preferred. The preferred samples comprise body fluids, such as, for example, urine, blood, plasma, serum, saliva, cerebrospinal fluid, or tissue samples; preferably urine.These samples may be obtained, for example, from a patient; that is, a living person, male or female, who presents voluntarily in a clinical setting for diagnosis, prognosis, or treatment of a disease or condition. In some modalities, preferred samples may be obtained from female humans of childbearing potential. In modalities where the sample comprises a biological sample, methods may be used to determine the amount of leflunomide metabolite in the sample when the sample is obtained from the biological source (i.e., the amount of endogenous leflunomide metabolite in the sample).

[0085] The present invention also contemplates equipment for an antidepressant quantification assay. An antidepressant quantification assay kit may include equipment comprising the compositions provided herein. For example, such equipment may include packaging material and measured quantities of an isotopically labeled internal standard, in sufficient quantity for at least one assay. Typically, the kits will also include instructions recorded in a tangible form (e.g., paper content or an electronic medium) for using the packaged reagents in an antidepressant quantification assay.

[0086] The calibration and QC mixture for use in modalities of the present invention are preferably prepared using a matrix similar to the intended sample matrix, provided that the analyte is essentially absent. Sample Preparation for Mass Spectrometry Analysis

[0087] In preparation for mass spectrometry analysis, analytes may be enriched relative to one or more other components in the sample (e.g., protein) 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, extraction methods including ethyl acetate and methanol extraction, and the use of chaotropic agents or any combination of the foregoing or similar methods.

[0088] Protein precipitation is a method of preparing a test sample, especially a biological test sample, such as serum or plasma. Protein purification methods are known in the prior art. For example, Polson et al., Journal of Chromatography B 2003, 785,263-275, describes protein precipitation techniques suitable for use in the methods of the present invention. Protein precipitation can be used to remove most of the protein from the sample, leaving analyte in the supernatant. Samples can be centrifuged to separate the liquid supernatant from the precipitated proteins; alternatively, samples can be filtered to remove the precipitated proteins. The resulting supernatant or filtrate can then be applied directly to mass spectrometry analysis; or alternatively, to further purification methods, such as liquid chromatography, and subsequent mass spectrometry analysis.In some modalities, the use of protein precipitation, such as acetonitrile protein precipitation, can eliminate the need for TFLC or other online extraction prior to mass spectrometry or high-performance liquid chromatography (HPLC) and mass spectrometry.

[0089] Another method of sample purification that can be used prior to mass spectrometry is liquid chromatography (LC). Some liquid chromatography methods, including high-performance liquid chromatography (HPLC), rely on relatively slow laminar flow technology. Traditional HPLC analysis depends on a column packing in which the laminar flow of the sample through the column is the basis for separating the analyte of interest from the sample. Those skilled in the art will understand that separation in such columns is a partitioning process and can select LC, including HPLC, instruments and columns that are suitable for use with the analytes. The chromatographic column usually includes a medium (i.e., packing material) to facilitate the separation of the chemical halves (i.e., fractionation). The medium may include minute particles.The particles typically include a bonded surface that interacts with the various chemical halves to facilitate their separation. A suitable bonded surface is a hydrophobic one, such as an alkyl, cyanide, or biphenyl bonded surface. Alkyl bonded surfaces may include C4, C8, C12, or C18 alkyl groups. In preferred embodiments, the column is a biphenyl column. The chromatographic column includes an inlet port for receiving a sample and an outlet port for discharging an effluent containing the fractionated sample. The sample may be supplied directly to the inlet port or from a sample separation (SPE) column, such as an online extraction (OEE) column or a fractionated liquid chromatography (TFLC) column.In some embodiments, an inline guard cartridge can be used upstream of the HPLC column to remove particulates and phospholipids from the sample before they reach the HPLC column. In some embodiments, the guard cartridge may be a biphenyl guard cartridge.

[0090] In one mode, the sample can 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 can be selected to elute the analyte(s) of interest. For example, liquid chromatography can be performed using a gradient mode, an isocratic mode, or a polytypical (i.e., mixed) mode. During chromatography, the separation of materials is achieved through variables such as the choice of eluent (also known as a “mobile phase”), elution mode, gradient conditions, temperature, etc.

[0091] In some embodiments, an analyte can be purified by applying a sample to a column under conditions where the analyte of interest is retained in reverse by the column packing material, while one or more other materials are not retained. In such embodiments, a first mobile-phase condition can be employed where the column retains the analyte of interest, and a second mobile-phase condition can be subsequently employed to remove the retained material from the column, once the unretained materials have been thoroughly washed. Alternatively, an analyte can be purified by applying a sample to a column under mobile-phase conditions where the analyte of interest elutes at a differential rate compared to one or more of the other materials. Such procedures can enrich the amount of one or more analytes of interest relative to one or more other components of the sample.

[0092] In a preferred embodiment, HPLC is carried out using a biphenyl column chromatographic system. In some preferred embodiments, an analytical biphenyl column was used (e.g., the Pinnacle DB Byphenyl analytical column from Restek Inc. (5 pm particle size, 50 x 2.1 mm), or equivalent). In some preferred embodiments, HPLC is performed using an HPLC grade of 0.1% aqueous formic acid as solvent A, and 0.1% formic acid in acetonitiridion as solvent B.

[0093] Through careful selection of valves and connecting tubing, two or more chromatography columns can be connected as required, so that material is passed from one to the next without any manual steps. In preferred embodiments, the selection of valves and tubing is controlled by a computer pre-programmed to perform the necessary steps. More preferably, the chromatography system is also connected online to the detector system, e.g., an MS system. Therefore, an operator can place a sample tray in an automated sample processor, and the remaining operations are performed under computer control, resulting in the purification and analysis of all selected samples.

[0094] In some modalities, TFLC can be used for analyte purification prior to mass spectrometry. In such modalities, samples can be extracted using a TFLC column, which captures the analyte. The analyte is then eluted and transferred online to an analytical HPLC column. For example, sample extraction can be achieved with a TFLC extraction cartridge, which can be obtained with a packed column of large particle size (50 pm). The sample is eluted from this column and then transferred online to an analytical HPLC column for further purification prior to mass spectrometry. Because the steps involved in such chromatography procedures can be linked in an automated manner, the requirement for operator involvement during analyte purification can be minimized.This feature can result in time and cost savings, and eliminate the possibility of operator error. Detection and Quantification by Mass Spectrometry

[0095] In several modalities, analytes can be ionized by any method known to a person skilled in the art. 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, sample ionization can be performed by electron ionization, chemical ionization, electrospray ionization (ESI), photon ionization, atmospheric pressure chemical ionization (APCI), photoionization, photoionization, atmospheric pressure photoionization (APPI), laser thermal desorption diode (LDTD), 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 (SELDI), inductively coupled plasma (ICP), and particle beam ionization. Those skilled in the field will understand that the choice of ionization method can be determined based on the analyte being measured, the sample type, the detector type, and the choice of positive versus negative mode. negative, etc.

[0096] The analytes can be ionized in either a positive or negative mode. In some modalities, the analytes were ionized in a positive mode.

[0097] In general, mass spectrometry techniques, after the sample has been ionized, allow the positively or negatively charged ions created in this way to be analyzed to determine a mass-to-charge ratio (m / z). Suitable analyzers for determining m / z include quadrupole analyzers, ion trap analyzers, and time-of-flight analyzers. Exemplary ion trap methods were described in Bartolucci, et al., Rapid Commun. Mass Spectrom. 2000, 14:967-73.

[0098] According to some methods of the present invention, high-resolution / high-precision mass spectrometry is used for the quantification of the analytes. That is, the mass spectrometry is conducted with a mass spectrometer capable of exhibiting a resolving power (FWHM) of at least 10,000 with an accuracy of approximately 50 ppm or less for the ions of interest; preferably, the mass spectrometry exhibits a resolving power (FWHM) of 20,000 or better and an accuracy of approximately 20 ppm or less; such as, for example, a resolving power (FWHM) of 25,000 or better and an accuracy of approximately 5 ppm or less; such as, for example, a resolving power (FWHM) of 25,000 or better and an accuracy of approximately 3 ppm or less.Three exemplary mass spectrometers capable of exhibiting the required performance level for analyte ions are those that include orbitrap mass analyzers, some TOF mass analyzers, or Fourier transform ion cyclotron resonance mass analyzers. wiA / ai¿v¿i iv 144^0

[0099] Elements found in biologically active molecules, such as carbon, oxygen, and nitrogen, exist naturally in a number of different isotopic forms. For example, most carbon is present as 12C, but approximately 1% of all naturally occurring carbon is present as 13C. Therefore, some fraction of naturally occurring carbon-containing molecules will contain at least one atom of 13C. The inclusion of naturally occurring elemental isotopes in molecules gives rise to multiple molecular isotopic forms. The difference in masses of molecular isotopic forms is at least 1 atomic mass unit (amu). This is because elemental isotopes differ by at least one neutron (mass of one neutron = 1 amu).When molecular isotopes are ionized to multiply charged states, the mass distinction between the isotopes can become difficult to discern because mass spectrometry detection is based on the mass-to-charge ratio (m / z). For example, two isotopes that differ in mass by 1 amu and are both ionized to a 5+ state will exhibit an m / z difference of only 0.2 (1 amu difference / charge state of 5). High-resolution / high-precision mass spectrometers are capable of discerning between isotopes of highly multiplied charged ions (such as ions with charges of ±4, ±5, ±6, ±7, ±8, ±9, or greater).

[00100] Due to naturally occurring elemental isotopes, multiple isotopic forms typically exist for each molecular ion (each of which gives rise to a separate spectrometric peak detectable by a sufficiently sensitive mass spectrometer). The m / zy ratios, or relative abundances, of multiple isotopic forms collectively comprise an isotopic signature for a molecular ion. In some embodiments, the m / zy relative abundances of two or more molecular isotopic forms can be used to confirm the identity of a molecular ion under investigation. In some embodiments, the mass spectrometric peak of one or more isotopic forms is used to quantify a molecular ion. In some related embodiments, a single mass spectrometric peak of one isotopic form is used to quantify a molecular ion.In other related modalities, a plurality of isotopic peaks was used to quantify a molecular ion. In these later modalities, the plurality of isotopic peaks can be subjected to any suitable mathematical treatment. Several mathematical treatments are known in the prior art and include, among others, summarizing the area under multiple peaks or averaging the response of multiple peaks.

[00101] In mass spectrometry techniques, ions can typically be detected using various detection modes. For example, selected ions can be detected using a selective ion monitoring (SIM) mode, or alternatively, mass transitions resulting from collision-activated dissociation (CAD) can be detected, e.g., multiple reaction monitoring (MRM) or selected reaction monitoring (SRM). CAD is often used to generate fragment ions for further detection. In CAD, precursor ions gain energy through collisions with an inert gas and are subsequently fragmented 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 break due to the increased vibrational energy. Alternatively, neutral loss can be monitored.

[00102] In some configurations, the mass-to-charge ratio is determined using a quadrupole analyzer. For example, in a quadrupole or quadrupole ion trap instrument, ions in a radio frequency oscillator field experience a force proportional to the DC potential applied between electrodes, the RF signal amplitude, and the mass-to-charge ratio. The voltage and amplitude can be selected so that only ions with a particular mass-to-charge ratio travel the length of the quadrupole, while the remaining ions are deflected. Therefore, quadrupole instruments can act as both a mass filter and a mass detector for ions injected into the instrument.

[00103] One can improve the specificity 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 into an MS instrument, and the precursor ion is subsequently fragmented to produce one or more fragmented ions (also called daughter ions or product ions) that are then analyzed in a second MS procedure. Through careful selection of precursor ions, the ions produced by certain analytes are passed into the fragmentation chamber, where collisions with atoms of an inert gas produce the fragment ions. Because both the precursor and fragment ions are produced reproducibly under a given set of ionization / fragmentation conditions, the MS / MS technique can provide an extremely powerful analytical tool.For example, the combination of filtration / fragmentation can be used to remove interfering substances, and can be particularly useful in complex samples, such as biological samples.

[00104] Alternative operating modes of a tandem mass spectrometry instrument include a product ion scan and a precursor ion scan. For a description of such operating modes, see, e.g., E. Michael Thurman, et al., Chromatographic-Mass Spectrometric Food Analysis for Trace Determination of Pesticide Residues, Chapter 8 (Amadeo R. Fernandez-Alba, ed., Elsevier 2005) (387).

[00105] The results of an analyte assay can be related to the amount of analyte in the original sample by means of numerous methods known in the prior art. For example, given that the sampling and analysis parameters are carefully controlled, the relative abundance of a given ion can be compared to a table that converts this relative abundance to an absolute amount of the original molecule. Alternatively, external standards can be run on the samples, and a standard curve constructed based on ions generated from these standards. By using this standard curve, the relative abundance of a given ion can be converted to an absolute amount of the original molecule. In some preferred embodiments, an internal standard is used to generate a standard curve for calculating the amount of analytes.The methods for generating and using such standard curves are well known in the prior art, and a person skilled in the art is able to select a suitable internal standard. For example, one or more forms of an isotopically labeled molecule with a similar m / z ratio as analytes can be used as internal standards. In some embodiments described herein, an exemplary internal standard is isotopically labeled diazepam, although numerous other compounds (isotopically labeled or not) can be used. Numerous other methods for relating the amount of an ion to the amount of the original molecule will be known to those skilled in the art.

[00106] As used herein, an “isotopic tag” produces a mass change in the tagged molecule relative to the untagged molecule when analyzed by mass spectrometry techniques. Examples of suitable tags include deuterium (2H), 13C, and Y15N. One or more isotopic tags may be incorporated at one or more positions on the molecule, and one or more types of isotopic tags may be used on the same isotopically tagged molecule.

[00107] One or more stages of the methods may be carried out using automated machines. In some embodiments, one or more purification stages are carried out online, and more preferably all purification and mass spectrometry stages may be performed online.

[00108] In particular, in the preferred modalities, the analytes in a sample are detected and / or quantified using MS / MS as follows. The preferred samples are subjected to liquid chromatography, preferably HPLC. The liquid solvent flow from a chromatographic column enters the heated nebulizer interface of an MS / MS analyzer, and the solvent / analyte mixture is vaporized in the heated, charged tubing of the interface. During these processes, the analyte (i.e., antidepressants or metabolites) is analyzed. Ions, e.g., precursor ions, pass through the instrument orifice and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters, enabling the selection of ions (i.e., selection of “precursor” and “fragment” ions in Q1 and Q3, respectively) based on their mass-to-charge ratio (m / z). Quadrupole 2 (Q2) is the collision cell, where the ions are fragmented.The first quadrupole of the mass spectrometer (Q1) selects molecules with the mass to charge ratios of the analytes. Precursor ions with the correct mass-to-charge ratios are allowed to pass into the collision chamber (Q2), while unwanted ions with any other mass-to-charge ratio collide with the sides of the quadrupole and are removed. Precursor ions entering Q2 collide with neutral argon gas molecules and fragments. The generated fragment ions are passed to quadrupole 3 (Q3), where the analyte fragment ions are selected while other ions are removed.

[00109] The methods may involve MS / MS performed in either a positive or negative ion mode; preferably a positive ion mode. Using standard methods known in the prior art, a person skilled in the art is able to identify one or more fragment ions of a particular analyte precursor ion that can be used for quadrupole selection 3 (Q3).

[00110] As ions collide with the detector, they can produce an electron pulse that is converted into a digital signal. The acquired data is transmitted to a computer, which plots the counts of collected ions versus time. The mass chromatograms are similar to chromatograms generated in traditional HPLC-MS methods. The areas under the peaks corresponding to particular ions, or the amplitude of those peaks, can be measured and correlated to the amount of the analyte of interest. In some modalities, the area under the curves, or peak amplitude, for fragment ion(s) and / or precursor ion(s) is measured to determine the amount of analytes. As described above, the relative abundance of a given ion can be converted to an absolute amount of the original analyte using calibration standard curves based on peaks of one or more ions from an internal or external molecular standard.

[00111] The following Examples serve to illustrate the invention. These Examples are in no way intended to limit the scope of the methods. EXAMPLES EXAMPLE 1: Sample Preparation

[00112] We describe a validated LC-MS / MS method for simultaneous analysis for 23 prescribed antidepressant analytes and their metabolites provided in Table 1 below.

[00113] Table 1. Antidepressants and metabolites determined by the assay Name Companies and Brand Names Amitriptyline TCA Elavil, Endep, Lentizol, Levate, Saroten, Tryptanol, Tryptizol Amoxapine TOA Asendin Citalopram / Escitalopram SSR 1 Celexa, Lexapro Desmethylcitalopram - METABOLITE Clomipramine TCA Anafranil Desmethylclomipramine - METABOLITE Desipramine TCA Norpramin Doxepin TCA Adapin, Curatin, Silenor, Sinequan Desmethyldoxepin - METABOLITE Duloxetine SNR 1 Cymbalta Fluoxetine SSR 1 Prozac, Selfemra Norfluoxetine - METABOLITE Fluvoxamine SSR I Faverin, Fevarin, Floxyfral, Dumyrox, Luvox Norfluvoxamine - METABOLITE Hydroxybupropion NDR I Wellbutrin, Aplenzin, Forfivo XL Imipramine TOA Tofranil Maprotiline TOA Ludiomil Mirtazapine Atypical Remeron Nortriptyline - METABOLITE Amitriptyline & Prescribed Drug TCA Pamelor Paroxetine SSR I Paxil, Pexeva Protriptyline TCA Vivactil Sertraline SSR I Zoloft Norsertraline - METABOLITE Trazodone Atypical Oleptro 1,3-chlorphenylpiperazine (metaCPP) - METABOLITE Trimipramine TCA Surmontil Venalfaxine SNR I Effexor XR O-Desmethylvenlafaxine - METABOLITE & Prescription Drug SNR I Pristiq, Khedezla Vilazodone SSR I Viibryd Vortioxetine Atypical Brintellix

[00114] Quality Controls, Calibrators and Internal Standards: The calibration standards (4-5,000 ng / ml) and quality controls (QC) at 5, 12.5 and 4,000 ng / ml are prepared by adding analyte stock solutions to drug-free urine controls (UTAK). The internal standard (IS) was a 25-100 ng / ml mixture of 1,3-chlorophenylpiperazine-D8, Hydroxybupropion-D6, desmethyl-venlafaxine-D6, desmethylcitalopram-D3, trimipramine-D3, amitriptyline-D3, nortriptyline-D3, paroxetine-D6, protriptyline-D3, citalopram-D6, venlafaxine-D6, imipramine-D3, trazodone-D6, vilazodone-D4, and vortioxetine-D8.

[00115] Sample Preparation: Urine samples, calibrators and QC (25 μI each) were mixed with IS (25 μI) in a 1 μI, 96-well extraction plate, diluted with 450 μI of 10 mM ammonium formate in water (mobile phase A), and subsequently vortexed at 1,100 rpm for 2 minutes before moving to the LC-MS / MS for injection and analysis. Example 2: Liquid Chromatography-Mass Spectrometry

[00116] LC-MS / MS: Extracted samples (25 μL) were chromatographically reduced on a Kinetex® Phenyl-Hexyl 50 x 4.6 mm 2.6 μ column (Phenomenex) using mobile phase A / mobile phase B gradients (25% methanol in acetonitrile). A column LC multiplexer of 4 was employed to maximize throughput on a Prelude LX-4 MD™ (ThermoFisher Scientific). A Sciex 4500 Triple Quad™ Mass Spectrometer was used for selected reaction monitoring. Figure 1 is a representative chromatogram for all analytes, and Figure 2 demonstrates a reference separation of closely related analytes.

[00117] Table 2 provides the mass transitions used to detect each analyte in the mass spectrometry assay. ινΐΛ / a / zuz i / u I 44^0

[00118] Table 2. Mass spectrometry transitions (m / z) used to detect antidepressants and metabolites Q1 (m / z) Q3 (m / z) ID DP EP j CE CXP 1 196,993 118 1,3-Chlorphenylpiperazine 1 76 5 60 8 2 196,993 119.1 1,3-Chlorphenylpiperazine 2 76 5 45 10 3 205,065 158.1 1,3-Chlorphenylpiperazine D8 56 10 29 12 4 278,096 105 Amitriptyline 1 81 10 50 10 5 278,096 115 Amitriptyline 2 81 10 95 10 6 281 202.1 Amitriptyline-D3 81 10 79 14 7 314.006 271.1 Amoxapine 1 101 10 48 10 8 9.............. 314.006 193.1 Amoxapine 2 101 10 78 14 256.02 130 Hydroxibupropion 1 56 10 85 12 10 .....11............ 256.02 103 Hydroxibupropion 2 56 10 53 10 262.061 130.1 Hydroxibupropion-D6 1 10 65 10 12 325.07 109 Citalopram 1 81 10 90 10 13 325.07 262.1 Citalopram 2 81 10 39 10 14 331,103 109 Citalopram-D6 76 10 33 10 15 264,083 91 Nortriptyline 1 76 10 70 8 16 264,083 105 Nortriptyline 2 76 10 45 10 Q1 (m / z) Q3 (m / z) ID DP EP CE CXP 17 267.095 105 Notriptyline-D3 71 10 27 10 18 311.043 109 Desmethylcitalopram 1 81 10 85 10 19 311.043 262.1 Desmethylcitalopram 2 81 10 36 10 20 314.072 108.9 Desmethylcitalopram-D3 81 10 31 10 21 315.054 86.1 Clomipramine 1 81 10 55 8 22 315.054 58 Clomipramine 2 81 10 30 16 23 301.037 72 Desmethylclomipramine 1 76 10 60 8 24 301.037 227.1 Desmethylclomipramine 2 76 10 51 16 25 267.091 72 Desipramine 1 71 5 55 10 26 267.091 193.1 Desipramine 2 71 5 60 14 27 280.096 107 Doxepin 1 76 10 55 10 28 280.096 165.1 Doxepin 2 76 5 95 14 29 266.074 107 Desmethyldoxapin 1 66 10 52 10 30 266.074 77 Desmethyldoxapin 2 66 10 90 8 31 298.03 154.1 Duloxetine 1 11 5 8 8 32 298.03 44.1 Duloxetine 2 11 5 90 9 33 310.07 148.1 Fluoxetine 1 16 5 13 10 34 310.07 44.1 Fluoxetine 2 16 5 13 9 35 296.066 134.2 Norfluoxetine 1 17 5 13 10 36 296.066 30.1 Norfluoxetine 2 10 5 35 8 37 319.057 71 Fluvoxamine 1 16 5 33 8 38 319.057 200.1 Fluvoxamine 2 16 5 31 8 39 305.025 229.1 Norfluvoxamine 1 1 5 23 10 40 305.025 188.1 Norfluvoxamine 2 1 5 27 8 41 281.098 86 Imipramine 1 66 10 60 8 42 281.098 58 Imipramine 2 66 10 115 6 43 284.013 89 Imipramine-D3 66 10 21 8 44 278.094 191.2 Maprotiline 1 81 10 65 8 45 278.094 189 Maprotiline 2 81 10 105 14 46 266.081 195.1 Mirtazapine 1 86 10 60 14 47 266.081 194.1 Mirtazapine 2 135 10 67 7 48 330.033 192.1 Paroxetine 1 106 10 29 8 49 330.033 70 Paroxetine 2 106 10 70 8 50 336.092 198.2 Paroxetine-D6 71 10 29 8. Q1 (m / z) Q3 (m / z) ID DP EP CE CXP 51 264.096 191 Protriptyline 1 81 10 55 16 52 264.096 189 Protriptyline 2 81 10 95 14 53 267.095 191.1 Protriptyline-D3 86 10 39 14 54 306 159 Sertraline 1 66 5 39 10 55 306 275 Sertraline 2 66 5 17 12 56 292.005 159 Desmethylsertraline 1 6 5 35 8 57 292.005 123 Desmethylsertraline 2 6 5 67 10 58 372.096 176.1 T razodone 1 111 5 45 12 59 372.096 148 Trazodone 2 111 5 70 12 60 378.114 182.1 Trazodone-D6 116 10 33 14 61 295.128 100.1 Trimipramine 1 1 10 55 10 62 295.128 58.1 Trimipramine 2 1 10 115 6 63 298.138 103.1 Trimipramine-D3 31 10 23 10 64 278.126 58 Venlafaxine 1 1 10 90 16 65 278.126 121 Venlafaxine 2 1 10 60 12 66 284.139 64.1 Venlafaxine-D6 1 10 57 6 67 264.106 58 Desmethylvenlafaxine 1 1 10 75 6 68 264.106 107 Desmethylvenlafaxine 2 1 10 65 10 69 270.134 64.1 Desmethylvenlafaxine-D6 1 10 49 6 70 442.133 155.1 Vilazodone 1 151 5 95 12 71 442.133 197.2 Vilazodone 2 151 5 45 8 72 446.163 155.1 Vilazodona-D4 151 5 69 12 73 299.059 150 Vortioxetine 1 126 10 44 7 74 299,059 109 Vortioxetine 2 126 10 44 7 75 307,082 153.1 Vortioxetine-D8 101 5 38 8 278.2 202.1 Amitriptyline 3 50 10 35 5 310.1 117.1 Fluoxetine 3 50 10 35 5 310.1 91.1 Fluoxetine 4 50 10 35 5 310.1 259.1 Fluoxetine 5 50 10 35 5 319.2 145.1 Fluvoxamine 3 50 10 35 5 319.2 130.1 Fluvoxamine 4 50 10 35 5 266.2 209.2 Mirtazapine 3 50 10 35 5 330.1 135.1 Paroxetine 3 50 10 35 5 330.1 109 Paroxetine 4 50 10 35 5. Q1 (m / z) Q3 (m / z) ID DP EP CE CXP 264.2 155.2 Protriptyline 3 50 10 35 5 264.2 178.2 Protriptyline 4 50 10 35 5 306 129.1 Sertraline 3 50 10 35 5 295.2 193.1 Trimipramine 3 50 10 35 5 295.2 208.2 Trimipramine 4 50 10 35 5 278.2 147.1 Venlafaxine 3 50 10 35 5 278.2 91.1 Venlafaxine 4 50 10 35 5 278.2 191.1 Amitriptyline 4 50 10 35 5 Example 3: Validation and Results

[00119] Validation: The following characteristics were determined by standard laboratory methods: limit of quantification (LOQ), linearity (including upper limit of linearity [ULOL] with dilution), precision, interference by over 150 different drugs, stability, stability of extracted specimen, matrix effect and remnant.

[00120] Linearity:

[00121] A 5-9 point calibration curve exhibited consistent linearity and reproducibility in the ±20$ range of its target with a regression coefficient (r) > 0.990.

[00122] The CVs were between 7.5% and 10%.

[00123] The analytical measurement range (AMR) for all antidepressant analytes and metabolites was 4 to 5,000 ng / ml, with a LOQ of 10 ng / ml (with 1 exception), and a ULOL of 50,000 ng / ml. The exception was the metabolite, norsertraline, with an AMR of 25 to 5,000 ng / ml and a LOQ of 50 ng / ml.

[00124] Accuracy:

[00125] A precision study over a 5-day period showed results consistent with a sigma value greater than 3 for low, medium, and high level QC.

[00126] Accuracy:

[00127] The accuracy study was carried out by correlating 65 samples across the concentration range of 4 to 20,000 ng / ml with another 65 results from another laboratory. Examples are presented in Figures 3 and 4.

[00128] On average, Deming regression showed a correlation coefficient of 1.022 and an intercept of -0.0681 with no bias.

[00129] Interference:

[00130] Tested (over 150 multiple legal drugs and prescription drugs at 100 times the cutoff. Such testing was performed using both a negative matrix control and a LoQ control added with the relevant substances).

[00131] None of the tested interference drugs caused a >20% deviation in panel drug signal intensities at LOQ.

[00132] Stability:

[00133] The specimens are stable for 7 days at room temperature, 14 days refrigerated, and 30 days frozen. Upon subsequent extraction, the samples were stable for 24 hours.

[00134] Matrix Effects:

[00135] The samples were compared at 3 different levels (0.5 x, 2x, and 0.8 x ULOL) with sorted and dilute matrices.

[00136] No matrix effects were observed.

[00137] Remaining:

[00138] Two samples at 4000 ng / ml were added back-to-back followed by 4 blank samples to determine remnant effects. The above was performed in triplicate.

[00139] No remnant was observed.

[00140] The contents of the articles, patents, and patent applications, as well as all other electronically available documents and information mentioned or cited herein, are incorporated herein by reference in their entirety to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any other material or information from any of these articles, patents, patent applications, or other physical or electronic documents.

[00141] The methods illustratively described herein can be properly implemented 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 broadly and without limitation. In addition, the terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions to exclude any equivalent features shown and described or portions thereof. It is acknowledged that various modifications are possible within the scope of the claimed invention.Therefore, it should be understood that, although the present invention has been specifically disclosed by means of preferred embodiments and features, optional modifications and variations of the invention embodied and disclosed herein may be restored by those skilled in the art, and that such modifications and variations are considered to be within the scope of the invention.

[00142] The invention has been broadly and generically described herein. Each of the more restricted species and subgeneric groupings falling within the generic disclosure also forms part of the methods. The foregoing includes the generic description of the methods with a positive or negative limitation that excludes any subject matter from the genus, regardless of whether or not the excluded material is specifically cited herein.

[00143] Other embodiments are found within the following claims. Furthermore, 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 described in this way in terms of any individual member or subgroup of members of the Markush group.

Claims

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

2. The method according to claim 1, wherein said one or more antidepressants and antidepressant metabolites comprise selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, norepinephrine-dopamine reuptake inhibitors, tricyclic antidepressants, sedatives, and antidepressant metabolites.

3. The method according to claim 1, wherein said one or more antidepressants and antidepressant metabolites are selected from the group consisting of fluoxetine, paroxetine, sertraline, citalopram, escitalopram, fluvoxamine, vilazodone, duloxetine, venlafaxine, desmethylvenlafaxine, hydroxybupropion, imipramine, notriptyline, amitriptyline, doxepin, trimipramine, desipramine, protriptyline, amoxapine, clomipramine, maprotiline, trazodone, mirtazapine, vortioxetine, desmethylcitalopram, desmethylclomipramine, desmethyldoxepin, norfluoxetine, norfluvoxamine, norsertraline, and 1,3-chlorphenylpiperazine.

4. The method according to claim 1, wherein the method comprises simultaneously detecting or determining the quantity of 10 or more antidepressants and antidepressant metabolites.

5. The method according to claim 1, wherein the method comprises simultaneously detecting or determining the quantity of 20 or more antidepressants and antidepressant metabolites.

6. The method according to claim 1, wherein the method comprises simultaneously detecting or determining the quantity of 30 antidepressants and antidepressant metabolites.

7. The method according to claim 1, wherein one or more internal standards are added.

8. The method according to claim 7, wherein one or more standards comprises deuterated internal standards.

9. The method according to claim 8, wherein the deuterated internal standards are selected from the group consisting of 1,3-chlorophenylpiperazine-D8, hydroxybupropion-D6, desmethylvenlafaxine-D6, desmethylcitalopram-D3, trimipramine-D3, amitriptyline-D3, nortriptyline-D3, paroxetine-D6, protriptyline-D3, citalopram-D6, venlafaxine-D6, imipramine-D3, trazodone-D6, vilazodone-D4, and vortioxetine-D8 10. The method according to claim 1, wherein the sample comprises a biological sample.

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

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

13. The method according to claim 12, wherein said liquid chromatography comprises high-performance liquid chromatography.

14. The method according to claim 1, wherein the method is capable of detecting antidepressants and antidepressant metabolites at levels within the range of approximately 4 ng / ml to approximately 5000 ng / ml, overall.

15. The method according to claim 1, wherein the method is capable of detecting antidepressants and antidepressant metabolites at levels within the range of approximately 25 ng / ml to approximately 5000 ng / ml, overall.

16. The method according to claim 1, wherein said mass spectrometry is tandem mass spectrometry.

17. The method according to claim 16, wherein said tandem mass spectrometry is conducted by selected reaction monitoring, multiple reaction monitoring, precursor ion scanning, or product ion scanning.

18. The method according to claim 16, wherein said tandem mass spectrometry is conducted by selected reaction monitoring.

19. The method according to claim 1, wherein the lower limit of quantification is 10 ng / ml.

20. The method according to claim 1, wherein the lower limit of quantification is 10 ng / ml.