Determination of antidepressants by mass spectrometry

Abstract

Describes a method for detecting or determining the amount of antidepressants and / or antidepressant metabolites in a sample. More specifically, describes a mass spectrometry method for detecting and quantifying antidepressants and / or antidepressant metabolites in a sample.

Description

[0001] Cross - reference to related patent applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 855,863, filed on May 31, 2019, the entire content of which is incorporated herein by reference. Background Art

[0003] Baseline testing helps clinicians determine whether a patient uses or does not use one or more antidepressants before treatment. Monitoring patients taking antidepressants to ensure compliance and avoid accidental use of multiple medications is crucial. Some antidepressants, such as selective serotonin reuptake inhibitors (SSRI), may have harmful side effects and should not be used in combination with drugs of the same class. Precise testing of antidepressants and metabolites is needed. Summary of the Invention

[0004] In one aspect, provided herein are methods for detecting and quantifying antidepressants and antidepressant metabolites by mass spectrometry.

[0005] Provided herein are methods for detecting the presence or amount of an antidepressant and / or an antidepressant metabolite in a sample by mass spectrometry. The method includes ionizing the sample under conditions suitable for generating one or more ions detectable by mass spectrometry; determining the amount of one or more ions by mass spectrometry; and using the amount of one or more ions to determine the presence or amount of an antidepressant and / or an antidepressant metabolite in the sample.

[0006] In some embodiments, the mass spectrometry includes tandem mass spectrometry. In these embodiments, the method includes: a) ionizing the sample under conditions suitable for generating precursor ions; b) fragmenting the precursor ions to generate one or more fragment ions; c) determining the amount of one or more ions generated in steps a) and b); and d) using the amount of one or more ions determined in step c) to determine the presence or amount of an antidepressant and metabolite in the sample.

[0007] In some embodiments, provided herein are methods for detecting or determining the amount of one or more antidepressants and antidepressant metabolites, the antidepressants and antidepressant metabolites including selective serotonin reuptake inhibitors, serotonin and norepinephrine reuptake inhibitors, norepinephrine and dopamine reuptake inhibitors, tricyclic antidepressants, sedatives, and / or antidepressant metabolite metabolites.

[0008] In some embodiments, provided herein are methods for detecting or determining one or more antidepressants or antidepressant metabolites selected from fluoxetine, paroxetine, sertraline, citalopram, escitalopram, fluvoxamine, vilazodone, duloxetine, venlafaxine, desvenlafaxine, hydroxybupropion, imipramine, nortriptyline, amitriptyline, doxepin, trimipramine, desipramine, protriptyline, amoxapine, clomipramine, maprotiline, trazodone, mirtazapine, vortioxetine, norcitalopram, norclomipramine, nordoxepin, norfluoxetine, norfluvoxamine, norsertraline, and 1,3-chlorophenylpiperazine.

[0009] In some embodiments, provided herein are methods for detecting or determining the amount of one or more selective serotonin reuptake inhibitors (fluoxetine, paroxetine, sertraline, citalopram, escitalopram, fluvoxamine, vilazodone); serotonin and norepinephrine reuptake inhibitors (duloxetine, venlafaxine, desvenlafaxine); norepinephrine and dopamine reuptake inhibitor (hydroxybupropion); tricyclic antidepressants (imipramine, nortriptyline, amitriptyline, doxepin, trimipramine, desipramine, protriptyline, amoxapine, clomipramine, maprotiline). Other antidepressants used in the assay can also act as sedatives and are trazodone, mirtazapine, and vortioxetine. The metabolites tested are demethylcitalopram, norclomipramine, desmethyldoxepin, norfluoxetine, norfluvoxamine, norsertraline, and 1,3-chlorophenylpiperazine.

[0010] In some embodiments, provided herein are methods for simultaneously detecting or determining the amount of 10 or more antidepressants and antidepressant metabolites.

[0011] In some embodiments, provided herein are methods for simultaneously detecting or determining the amount of 20 or more antidepressants and antidepressant metabolites.

[0012] In some embodiments, provided herein are methods for simultaneously detecting or determining the amount of 30 antidepressants and antidepressant metabolites.

[0013] In some embodiments, the methods provided herein include adding one or more internal standards. In some embodiments, one or more internal standards include deuterated internal standards. In some embodiments, the deuterated internal standards are selected from 1,3-chlorophenylpiperazine-D8, hydroxybupropion-D6, desmethyl-venlafaxine-D6, demethylcitalopram-D3, trimipramine-D3, amitriptyline-D3, nortriptyline-D3, paroxetine-D6, protriptyline-D3, citalopram-D6, venlafaxine-D6, imipramine-D3, trazodone-D6, vilazodone-D4, and vortioxetine-D8.

[0014] In some embodiments, the sample comprises a biological sample. In a preferred embodiment, the sample is urine. In some embodiments, the sample is plasma or serum. In some embodiments, the sample is blood.

[0015] In some embodiments, the sample is subjected to liquid chromatography prior to ionization. In some embodiments, the liquid chromatography comprises high performance liquid chromatography.

[0016] In some embodiments, the method is capable of detecting levels of antidepressants and antidepressant metabolites in the range of about 4 ng / mL to about 5000 ng / mL, including the end values.

[0017] In some embodiments, the method is capable of detecting levels of antidepressants and antidepressant metabolites in the range of about 25 ng / mL to about 5000 ng / mL, including the end values.

[0018] In some embodiments, the mass spectrometry is tandem mass spectrometry. In some embodiments, the tandem mass spectrometry is performed by selected reaction monitoring, multiple reaction monitoring, precursor ion scanning or product ion scanning.

[0019] In a preferred embodiment, the tandem mass spectrometry is performed by selected reaction monitoring.

[0020] In some embodiments, provided herein is a method for determining antidepressants and antidepressant metabolites, which comprises detecting ions having the following mass-to-charge ratios (m / z).

[0021]

[0022]

[0023]

[0024] In some embodiments, the methods described herein are capable of detecting levels of antidepressants and antidepressant metabolites in the range of 4 ng / mL to 5000 ng / mL, including the end values. In some embodiments, the methods described herein are capable of detecting levels of antidepressants and antidepressant metabolites in the range of 25 ng / mL to 5000 ng / mL, including the end values.

[0025] In some embodiments, the methods described herein are capable of quantifying antidepressants and antidepressant metabolites at a lower limit of 10 ng / mL. In some embodiments, the methods described herein are capable of quantifying antidepressants and antidepressant metabolites at a lower limit of 50 ng / mL.

[0026] In some embodiments, the sample passes through an extraction column, such as a solid phase extraction (SPE) column, prior to ionization. In some related embodiments, SPE and mass spectrometry analysis are performed by on-line processing.

[0027] In some embodiments, the sample passes through an analytical column, such as a high performance liquid chromatography (HPLC) column, prior to ionization. In some related embodiments, HPLC and mass spectrometry are performed by on-line processing.

[0028] In some embodiments, the method can be used to determine the presence or amount of antidepressants and antidepressant metabolites in a biological sample, such as plasma or serum. In some related embodiments, the 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 embodiments, one or more processing steps include one or more purification steps, such as protein precipitation, filtration, liquid-liquid extraction, solid phase extraction, liquid chromatography, any immunopurification process, etc., and any combination thereof.

[0029] In certain preferred embodiments of the methods disclosed herein, mass spectrometry is performed in positive ion mode. Optionally, mass spectrometry is performed in negative ion mode. Various ionization sources can be used in the embodiments of the present invention, including, for example, atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI). In certain embodiments, positive ion mode is used to measure antidepressants and antidepressant metabolites.

[0030] In preferred embodiments, an internally detectable internal standard is provided in the sample, and its amount is also determined in the sample. In these embodiments, all or part of both the target analyte and the internal standard present in the sample are ionized to generate a variety of ions detectable in a mass spectrometer, and one or more ions generated by each are detected by mass spectrometry. In these embodiments, the presence or amount of ions generated by the target analyte can be related to the presence of the amount of the target analyte in the sample.

[0031] In other embodiments, the amount of antidepressants and antidepressant metabolites in the sample can be determined by comparison with one or more external reference standards. Exemplary external reference standards include blank plasma or serum spiked with antidepressants and antidepressant metabolites or isotopically labeled variants thereof.

[0032] As used herein, unless otherwise specified, the singular forms "a", "an" and "the" include plural forms. Thus, for example, reference to "a protein" includes a plurality of protein molecules.

[0033] As used herein, the terms "purification" or "purifying" do not refer to removing all materials from a sample other than the target analyte. Instead, purification refers to steps to enrich the amount of one or more target analytes relative to other components in the sample that may interfere with the detection of the target analyte. Purifying a sample in various ways can allow for a relative reduction of one or more interfering substances, e.g., one or more substances that may or may not interfere with the detection of selected parent or daughter ions by mass spectrometry. A relative reduction, as the term is used, does not require complete removal of any substances present with the target analyte in the material to be purified by purification.

[0034] As used herein, the terms "immunopurification" or "immunopurify" refer to purification steps that utilize antibodies (including polyclonal or monoclonal antibodies) to enrich one or more target analytes. Any immunopurification methods known in the art can be used for immunopurification. Typically, the immunopurification steps utilize antibodies that are bound, conjugated, or otherwise attached to a solid support (such as a column, well, tube, gel, capsule, particle, etc.). Immunopurification as used herein includes, but is not limited to, steps commonly referred to in the art as immunoprecipitation, and steps commonly referred to in the art as affinity chromatography.

[0035] As used herein, the term "immunoparticle" refers to capsules, beads, gel particles, etc. that have antibodies bound, conjugated, or otherwise attached to their surface (on and / or in the particle). In certain embodiments that utilize immunopurification, the immunoparticles include agarose gel or agarose beads. In alternative embodiments that utilize immunopurification, the immunoparticles include glass, plastic, or silica beads, or silica gel.

[0036] As used herein, the term "sample" refers to any sample that may include a target analyte. As used herein, the term "body fluid" refers to any fluid that can be separated from an individual's body. For example, "body fluid" can include blood, plasma, serum, bile, saliva, urine, tears, sweat, etc. In some embodiments, the sample includes a body fluid sample; preferably plasma or serum.

[0037] As used herein, the term "solid phase extraction" or "SPE" refers to the step of separating a chemical mixture into its components due to the affinity of the components dissolved or suspended in a solution (i.e., the mobile phase) for a solid (i.e., the solid phase) through or around which the solution passes. As used herein, SPE differs from immunopurification in that the affinity of the components in the mobile phase for the solid phase is the result of chemical or physical interactions rather than immunological affinity. In some cases, when the mobile phase passes through or around the solid phase, the undesired components of the mobile phase can be retained by the solid phase, resulting in the purification of the analyte in the mobile phase. In other cases, the analyte can be retained by the solid phase, allowing the undesired components of the mobile phase to pass through or around the solid phase. In these cases, a second mobile phase is then used to elute the retained analyte from the solid phase for further processing or analysis. SPE, including TFLC, can operate by single or mixed mode mechanisms. Mixed mode mechanisms utilize both ion exchange and hydrophobic retention in the same chromatographic column; for example, the solid phase of a mixed mode SPE column can exhibit strong anion exchange and hydrophobic retention; or it can be a column that exhibits strong cation exchange and hydrophobic retention.

[0038] As used herein, the term "chromatography" refers to the step of separating a chemical mixture into its components due to the different distribution of chemical entities when the chemical mixture carried by a liquid or gas flows around or through a stationary or solid phase.

[0039] As used herein, the term "liquid chromatography" or "LC" refers to the process in which one or more components of a fluid solution are selectively retarded as the fluid uniformly percolates through a column of finely divided material or through a capillary channel. The retardation is caused by the distribution of the components of the mixture between one or more stationary phases and the bulk fluid (i.e., the mobile phase) as the fluid moves relative to the stationary phase(s). Examples of "liquid chromatography" include reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC), and turbulent flow liquid chromatography (TFLC) (sometimes referred to as high turbulence liquid chromatography (HTLC) or high throughput liquid chromatography).

[0040] 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 separation is enhanced by forcing the mobile phase through the stationary phase (usually a densely packed column) under pressure.

[0041] As used herein, the term "turbulent flow liquid chromatography" or "TFLC" (sometimes referred to as high-turbulent flow liquid chromatography or high-throughput liquid chromatography) refers to a chromatographic format that utilizes the turbulent flow of an analyte through a column packing as the basis for separation. TFLC has been applied to the preparation of samples containing two unnamed drugs prior to analysis by mass spectrometry. See, 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 further explain TFLC. One of ordinary skill in the art understands "turbulence." When a fluid flows slowly and smoothly, this flow is referred to as "laminar flow." For example, a fluid moving through an HPLC column at a low flow rate is laminar. In laminar flow, the movement of fluid particles is orderly, where the particles generally move in a straight line. At faster speeds, the inertia of the water overcomes the fluid friction and creates turbulence. A fluid not in contact with an irregular boundary "overtakes" a fluid slowed by friction or deflected by an uneven surface. When a fluid flows turbulently, it flows in the form of eddies and vortices (or swirls), which have more "drag" than when in laminar flow. Many references are available to assist in determining 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 Mathieu & Julian Scott, Cambridge University Press (2001)).

[0042] As used herein, the term "gas chromatography" or "GC" refers to a chromatographic method in which a 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 material and is separated into its component compounds based on the affinity of the compounds for the stationary phase.

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

[0044] As used herein, the term "analytical column" refers to a chromatographic column having sufficient chromatographic plates to effect separation of materials in a sample eluted from the column to an extent sufficient to permit determination of the presence or amount of an analyte. Such a column is generally distinct from an "extraction column", the general purpose of which is to separate or extract a retained material from non-retained materials to obtain a purified sample for further analysis. As used in this context, the term "about" means ±10%. In a preferred embodiment, the analytical column comprises particles having a diameter of about 5 μm.

[0045] As used herein, the terms "on-line" and "inline", as used, for example, in "on-line automation mode" or "on-line extraction", refer to steps that are carried out without operator intervention. In contrast, the term "off-line" as used herein refers to steps that require manual operator intervention. Thus, if a sample is precipitated and then the supernatant is manually loaded into an autosampler, the precipitation and loading steps are off-line with respect to subsequent steps. In various embodiments of the method, one or more steps may be carried out in an on-line automation mode.

[0046] As used herein, the term "mass spectrometry" or "MS" refers to an analytical technique for identifying compounds by their mass. MS refers to a method of filtering, detecting, and measuring ions based on their mass-to-charge ratio or "m / z". MS techniques generally include (1) ionizing a compound to form a charged compound; and (2) detecting the molecular weight of the charged compound and calculating the mass-to-charge ratio. Ionization and detection of the compound can be performed by any suitable means. A "mass spectrometer" generally includes an ion generator and an ion detector. Generally, one or more target molecules are ionized and the ions are then introduced into the mass spectrometer where, due to the combination of magnetic and electric fields, the ions follow a path in space that depends on their mass ("m") and charge ("z"). See, e.g., U.S. Patent No. 6,204,500, entitled "Mass spectrometry From Surfaces"; 6,107,623, entitled "Methods and Apparatus for Tandem Mass spectrometry"; 6,268,144, entitled "DNA Diagnostics Based On Mass spectrometry"; 6,124,137, entitled "Surface-Enhanced Photolabile Attachment And Release For Desorption And Detection Of Analytes"; Wright et al., Prostate Cancer and Prostatic Diseases 1999, 2:264-76; and Merchant and Weinberger, Electrophoresis 2000, 21:1164-67.

[0047] As used herein, the term "operating in negative ion mode" refers to those mass spectrometry methods that produce and detect negative ions. As used herein, the term "operating in positive ion mode" refers to those mass spectrometry methods that produce and detect positive ions.

[0048] As used herein, the term "ionization" or "ionizing" refers to the process of generating analyte ions having a net charge equal to one or more electron units. Negative ions are those ions having a net negative charge of one or more electron units, while positive ions are those ions having a net positive charge of one or more electron units.

[0049] As used herein, the term "electron ionization" or "EI" refers to a method in which a target analyte in the gas phase or vapor phase interacts with an electron stream. The impact of the electrons with the analyte produces analyte ions, which can then be used in mass spectrometry techniques.

[0050] As used herein, the term "chemical ionization" or "CI" refers to a method in which a reaction gas (e.g., ammonia) is bombarded with electrons and analyte ions are formed by the interaction of reaction gas ions and analyte molecules.

[0051] As used herein, the term "fast atom bombardment" or "FAB" refers to a method in which a beam of high-energy atoms (usually Xe or Ar) bombards a non-volatile sample to desorb and ionize the molecules contained in the sample. The test sample is dissolved in a viscous liquid matrix such as glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether, 2-nitrophenyl octyl ether, sulfolane, diethanolamine, and triethanolamine. Selecting an appropriate matrix for a compound or sample is an empirical process.

[0052] As used herein, the term "matrix-assisted laser desorption ionization" or "MALDI" refers to a method in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample through various ionization pathways, including photo-ionization, protonation, deprotonation, and cluster decay. For MALDI, the sample is mixed with an energy-absorbing matrix, which aids in the desorption of analyte molecules.

[0053] 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 through various ionization pathways, including photo-ionization, protonation, deprotonation, and cluster decay. For SELDI, the sample is typically bound to a surface that preferentially retains one or more target analytes. As with MALDI, the process can also employ an energy-absorbing material to facilitate ionization.

[0054] As used herein, the term "electrospray ionization" or "ESI" refers to a method in which a solution passes along a short length of a capillary, where a high positive or negative electric potential is applied at the end of the capillary. The solution reaching the end of the tube is evaporated (atomized) into a jet or spray of very small solution droplets in a solvent vapor. This mist of droplets flows through an evaporation chamber. As the droplets become smaller, the surface charge density increases until the natural repulsion between like charges causes ions, as well as neutral molecules, to be released.

[0055] As used herein, the term "atmospheric pressure chemical ionization" or "APCI" refers to a mass spectrometry similar to ESI; however, APCI produces ions through ion-molecule reactions occurring in a plasma at atmospheric pressure. The plasma is sustained by a discharge between a spray capillary and a counter electrode. The ions are then typically extracted into a mass analyzer using a set of differential pumping skimmer stages. Dry and preheated N 2Countercurrent flow of the gas can be used to improve solvent removal. In the analysis of less polar substances, gas-phase ionization in APCI can be more effective than ESI.

[0056] As used herein, the term "atmospheric pressure photoionization" or "APPI" refers to a form of mass spectrometry in which the mechanism of photoionization of a molecule M is photon absorption and electron emission to form a molecular ion M + . Because the photon energy is typically just above the ionization potential, the molecular ions are less likely to dissociate. In many cases, samples can be analyzed without the need for chromatography, saving a significant amount of time and cost. In the presence of water vapor or a protonic solvent, the molecular ion can extract H to form MH+. This tends to occur if M has a high proton affinity. This does not affect the quantitative accuracy because the sum of M+ and MH+ is constant. Drug compounds in a protonic solvent are typically observed as MH+, while nonpolar compounds such as naphthalene or testosterone typically form M+. See, e.g., Robb et al., Anal. Chem. 2000, 72(15): 3653-3659.

[0057] As used herein, the term "inductively coupled plasma" or "ICP" refers to a method in which a sample interacts with a partially ionized gas at a sufficiently high temperature to atomize and ionize most elements.

[0058] As used herein, the term "field desorption" refers to a method in which a nonvolatile test sample is placed on an ionization surface and a strong electric field is used to generate analyte ions.

[0059] As used herein, the term "desorption" refers to the removal of an analyte from a surface and / or the entry of an analyte into the gas phase. Laser desorption / thermal desorption is a technique in which a sample containing an analyte is thermally desorbed into the gas phase by a laser pulse. The laser hits the back of a special 96-well plate with a metal base. The laser pulse heats the base, and the heat converts the sample into the gas phase. The gas-phase sample is then aspirated into a mass spectrometer.

[0060] As used herein, the term "selected ion monitoring" is a detection mode for a mass spectrometer in which only ions in a relatively narrow mass range, typically about one mass unit, are detected.

[0061] As used herein, "multiple reaction mode", sometimes called "selected reaction monitoring", is a detection mode for a mass spectrometer in which precursor ions and one or more fragment ions are selectively detected.

[0062] As used herein, the terms "lower limit of quantification", "limit of quantification", or "LLOQ" refer to the point at which a measurement becomes quantitatively meaningful. The analyte response at this LOQ is recognizable, discrete, and reproducible, with a relative standard deviation (RSD%) of less than 20% and an accuracy of 85% to 115%.

[0063] As used herein, the term "limit of detection" or "LOD" is the point at which the measured value is greater than the uncertainty associated with it. The LOD is the point at which a value exceeds the uncertainty associated with its measurement and is defined as three times the average RSD at zero concentration.

[0064] As used herein, the "amount" of an analyte in a body fluid sample generally refers to the absolute value reflecting the mass of the detectable analyte in the sample volume. However, the amount also takes into account the relative amount compared to the amount of another analyte. For example, the amount of an analyte in a sample can be an amount greater than the control or normal level of the analyte normally present in the sample.

[0065] As used herein, the term "about" in quantitative measurements involving measurements that do not include ion masses refers to plus or minus 10% of the indicated value. Mass spectrometers may vary slightly in determining the mass of a given analyte. In the context of ion mass or the mass-to-charge ratio of an ion, the term "about" refers to + / - 0.50 atomic mass units.

[0066] The above summary of the invention is non-limiting, and other features and advantages of the present invention will be apparent from the following detailed description of the invention and the claims. Brief Description of the Drawings

[0067] Figure 1 The LC-MS / MS curves of all analytes and metabolites are shown.

[0068] Figure 2 Examples of baseline separation of (A) amitriptyline, (B) maprotiline, and (C) venlafaxine (analyte: left panel, internal standard (IS): right panel) are shown.

[0069] Figure 3 The accuracy of citalopram compared to another experiment is shown. Deviations greater than ±20% values are not shown.

[0070] Figure 4 The accuracy of the metabolite demethylcitalopram compared to another experiment is shown. Deviations greater than ±20% values are not shown.

[0071] Figure 5 The isolated cyclobenzaprine interferent is shown. The figure shows 5 ng / mL maprotiline + cyclobenzaprine at 100×.

[0072] Figure 6Shows the mass spectrometry differences between desvenlafaxine vs. tramadol.

[0073] Figure 7 Shows the separated tramadol interferent. The figure shows 5 ng / mL desvenlafaxine + tramadol at 100×.

[0074] Figure 8 Shows the baseline separation of amitriptyline, maprotiline, and venlafaxine.

[0075] Figure 9 Shows the baseline separation of nortriptyline, protriptyline, and desvenlafaxine.

[0076] Figure 10 Shows the baseline separation of desmethyldoxepin and mirtazapine.

[0077] Figure 11 Shows desipramine vs mirtazapine identified by different transitions.

[0078] Figure 12 Shows that for desipramine in patients positive for mirtazapine, the ion ratio and / or relative retention time (RRT) will fail. Detailed Description

[0079] In certain embodiments, the group of antidepressants described herein can be used, where compliance monitoring is performed on patients with a history / risk of such drug use and / or abuse. Before prescribing such drugs, baseline testing alerts the provider to the possibility of polypharmacy drug conflicts. Compliance monitoring requires providing prescription drugs and no over-the-counter drugs for these patient groups.

[0080] Certain brain chemicals, neurotransmitters, are associated with depression, more specifically serotonin, norepinephrine, and dopamine. Most antidepressants treat depression by affecting these neurotransmitters. Different types / categories of antidepressants affect these neurotransmitters in different ways. These types include: SSRI, SNRI, NDRI, tricyclic, atypical, MAOI, and others (see below).

[0081] Selective serotonin reuptake inhibitors (SSRI). Doctors usually start by prescribing SSRI. Compared with other types of antidepressants, these drugs are safer and generally cause fewer troublesome side effects. SSRIs include fluoxetine (Prozac, Selfemra), paroxetine (Paxil, Pexeva), sertraline (Zoloft), citalopram (Celexa), escitalopram (Lexapro), fluvoxamine (Faverin, Fevarin, Floxyfral, Dumyrox, Luvox), vilazodone (Viibryd).

[0082] Serotonin and norepinephrine reuptake inhibitors (SNRIs) - duloxetine (Cymbalta), venlafaxine (Effexor XR), desvenlafaxine (synthetic form of the main metabolite of venlafaxine, O-desvenlafaxine; Pristiq, Khedezla), and levomilnacipran (Fetzima). SNRIs have a unique dual action in raising serotonin and norepinephrine levels; thus, SNRIs combat more than one cause of depression.

[0083] Norepinephrine and dopamine reuptake inhibitors (NDRIs). Bupropion (Wellbutrin, Aplenzin, Forfivo XL) belongs to this class. It is one of the few antidepressants not often associated with sexual side effects.

[0084] Tricyclic antidepressants (TCAs) tend to cause more side effects than newer antidepressants. TCAs are usually not prescribed unless a patient has first tried an SSRI without improvement. TCAs include imipramine (Tofranil), nortriptyline (Pamelor), amitriptyline (Elavil, Endep, Lentizol, Levate, Saroten, Tryptanol, Tryptizol), doxepin (Adapin, Curatin, Silenor, Sinequan), trimipramine (Surmontil), desipramine (Norpramin), protriptyline (Vivactil), amoxapine (Asendin), clomipramine (Anafranil), and maprotiline (Ludiomil).

[0085] Atypical antidepressants. These drugs do not fit neatly into any other antidepressant category. They include trazodone (Oleptro), mirtazapine (Remeron), and vortioxetine (Brintellix). These are sedatives and are usually taken at night.

[0086] The assay does not include monoamine oxidase inhibitors (MAOIs). These drugs cannot be used in combination with SSRIs. Common MAOIs include tranylcypromine (Parnate), phenelzine (Nardil), and isocarboxazid (Marplan).

[0087] A method for measuring the amount of an analyte in a sample is described. More specifically, a mass spectrometry method for detecting and / or quantifying an analyte in a biological sample (such as human plasma or serum) is described. The method can utilize liquid chromatography followed by tandem mass spectrometry to quantify the analyte in the sample.

[0088] Suitable test samples for the methods of the present invention include any test sample that may contain the target analyte. In some preferred embodiments, the sample is a biological sample; that is, a sample obtained from any biological source, such as an animal, cell culture, organ culture, etc. In certain preferred embodiments, the sample is obtained from a mammal, such as a dog, cat, horse, etc. A particularly preferred mammal is a primate, and most preferably a male or female human. Preferred samples include body fluids, such as urine, blood, plasma, serum, saliva, cerebrospinal fluid, or tissue samples; preferably urine. Such a sample can, for example, be obtained from a patient; that is, a living human, male or female, who presents in a clinical setting for the diagnosis, prognosis, or treatment of a disease or disorder. In some embodiments, the preferred sample can be obtained from a female human of childbearing potential. In embodiments where the sample comprises a biological sample, when the sample is obtained from a biological source, the method can be used to determine the amount of leflunomide metabolite in the sample (i.e., the amount of endogenous leflunomide metabolite in the sample).

[0089] The present invention also contemplates kits for the quantitative determination of antidepressants. A kit for the quantitative determination of antidepressants can include a kit containing the compositions provided herein. For example, the kit can include a packaging material and a measured amount of an isotopically labeled internal standard in an amount sufficient for at least one assay. Typically, the kit will also include instructions recorded in a tangible form (e.g., contained on paper or an electronic medium) for using the packaged reagents for the quantitative determination of antidepressants.

[0090] Calibration and QC pools for embodiments of the present invention are preferably prepared using a matrix similar to the expected sample matrix, provided that the analyte is substantially absent.

[0091] Sample Preparation for Mass Spectrometry

[0092] In preparation for mass spectrometry analysis, the analyte can be enriched relative to one or more other components in the sample (e.g., proteins) by various methods known in the art, including, for example, liquid chromatography, filtration, centrifugation, thin layer chromatography (TLC), electrophoresis (including capillary electrophoresis), affinity separation (including immunoaffinity separation), extraction methods (including extraction with ethyl acetate or methanol), and the use of chaotropic agents or any combination of the above.

[0093] Protein precipitation is a method for preparing test samples, especially biological test samples such as serum or plasma. Protein purification methods are well known in the art. For example, Polson et al., Journal of Chromatography B 2003, 785:263-275 describe protein precipitation techniques applicable to the methods of the present invention. Protein precipitation can be used to remove most of the proteins from a sample, leaving the analyte in the supernatant. The sample can be centrifuged to separate the liquid supernatant from the precipitated proteins; alternatively, the sample can be filtered to remove the precipitated proteins. The resulting supernatant or filtrate can then be used directly for mass spectrometry; or alternatively, for other purification methods such as liquid chromatography and subsequent mass spectrometry. In certain embodiments, the use of protein precipitation, such as, for example, acetonitrile protein precipitation, can obviate the need for TFLC or other on-line extraction prior to mass spectrometry or high performance liquid chromatography (HPLC) and mass spectrometry.

[0094] Another sample purification method that can be used prior to mass spectrometry is liquid chromatography (LC). Certain liquid chromatography methods, including high performance liquid chromatography (HPLC), rely on relatively slow laminar flow techniques. Conventional HPLC analysis relies on column packing where the laminar flow of the sample through the column is the basis for separating the target analyte from the sample. Those skilled in the art will understand that the separation in such columns is a partitioning process and the LC, including the HPLC, instrument and column applicable to the analyte, can be selected. The chromatographic column typically includes a medium (i.e., packing material) to facilitate the separation (i.e., fractionation) of chemical moieties. The medium can include fine particles. The particles typically include a bonded surface that interacts with the various chemical moieties to facilitate the separation of the chemical moieties. A suitable bonded surface is a hydrophobic bonded surface such as an alkyl-bonded, cyano-bonded or biphenyl-bonded surface. The alkyl-bonded surface can include C-4, C-8, C-12 or C-18 bonded alkyl groups. In a preferred embodiment, the column is a biphenyl column. The chromatographic column includes an inlet port for receiving the sample and an outlet port for discharging the effluent including the fractionated sample. The sample can be supplied directly to the inlet port, or from an SPE column such as an on-line extraction column or a TFLC column. In some embodiments, an on-line guard column can be used prior to the HPLC column to remove particulates and phospholipids from the sample before the sample reaches the HPLC column. In some examples, the guard column can be a biphenyl guard column.

[0095] In one embodiment, 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 target analyte(s). For example, liquid chromatography can be performed using a gradient mode, an isocratic mode or a multi-modal (i.e., mixed) mode. During chromatography, the separation of materials is affected by variables such as the choice of eluent (also referred to as the "mobile phase"), the elution mode, gradient conditions, temperature, etc.

[0096] In certain embodiments, an analyte can be purified by applying a sample to a column under conditions where the target analyte is reversibly retained by the column packing material while one or more other materials are not retained. In these embodiments, a first mobile phase condition can be employed where the target analyte is retained by the column, and once the non-retained materials are washed through, a second mobile phase condition can subsequently be employed to remove the retained material from the column. Optionally, an analyte can be purified by applying a sample to a column under mobile phase conditions where the target analyte elutes at a different rate compared to one or more other materials. Such a step can enrich the amount of one or more target analytes relative to one or more other components of the sample.

[0097] In a preferred embodiment, HPLC is performed using a biphenyl column chromatography system. In certain preferred embodiments, a biphenyl analytical column is used (e.g., Pinnacle DB biphenyl analytical column from Restek Inc. (5 μm particle size, 50×2.1 mm, or equivalent)). In certain preferred embodiments, HPLC is performed using an aqueous solution of 0.1% formic acid as solvent A and an acetonitrile solution of 0.1% formic acid as solvent B.

[0098] By carefully selecting valves and connector tubing, two or more chromatographic columns can be connected as needed such that materials can be transferred from one chromatographic column to another without any manual steps. In a preferred embodiment, the selection of valves and tubing is controlled by a pre-programmed computer to perform the necessary steps. Most preferably, the chromatographic system is also connected to a detector system, such as an MS system, in this on-line manner. Thus, an operator can place a tray of samples into an autosampler and the remaining operations are performed under computer control, thereby purifying and analyzing all selected samples.

[0099] In some embodiments, TFLC can be used to purify an analyte prior to mass spectrometry analysis. In such an embodiment, a TFLC column that captures the analyte can be used to extract the sample. The analyte is then eluted and transferred on-line to an analytical HPLC column. For example, sample extraction can be accomplished using a TFLC extraction column or a large particle size (50 μm) packed column. The sample eluted from this column is then transferred on-line to an HPLC analytical column for further purification prior to mass spectrometry. Since the steps involved in these chromatographic procedures can be connected in an automated manner, the requirement for operator involvement during analyte purification can be minimized. This feature can save time and cost and eliminate the opportunity for operator error.

[0100] Detection and Quantification by Mass Spectrometry

[0101] In various embodiments, an analyte can be ionized by any method known to those skilled in the art. Mass spectrometry is performed using a mass spectrometer that includes an ion source for ionizing a fractionated sample and generating charged molecules for further analysis. For example, ionization of the sample can be carried out by electron ionization, chemical ionization, electrospray ionization (ESI), photoionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoionization (APPI), laser diode thermal desorption (LDTD), fast atom bombardment (FAB), liquid secondary ionization (LSI), matrix-assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray / plasma spray ionization, surface-enhanced laser desorption ionization (SELDI), inductively coupled plasma (ICP), and particle beam ionization. Those skilled in the art will understand that the choice of ionization method can be determined based on the analyte to be measured, the sample type, the detector type, the choice of positive mode versus negative mode, and the like.

[0102] The analyte can be ionized in both positive and negative modes. In some embodiments, the analyte is ionized in the positive mode.

[0103] In mass spectrometry techniques, typically after the sample is ionized, the resulting positively or negatively charged ions can be analyzed to determine the 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 are described in Bartolucci, et al., Rapid Commun. Mass Spectrom. 2000, 14:967-73.

[0104] In some methods according to the present invention, high-resolution / high-accuracy mass spectrometry is used for quantification of the analyte. That is, mass spectrometry is performed using a mass spectrometer capable of exhibiting a resolving power (FWHM) of at least 10,000, which has an accuracy of about 50 ppm or less for the target ion; preferably, the mass spectrometer exhibits a resolving power (FWHM) of 20,000 or better and an accuracy of about 20 ppm or less; for example, a resolving power (FWHM) of 25,000 or better and an accuracy of about 5 ppm or less; for example, a resolving power (FWHM) of 25,000 or better and an accuracy of about 3 ppm or less. Three exemplary mass spectrometers capable of demonstrating the required performance levels for analyte ions are those that include an Orbitrap mass analyzer, certain TOF mass analyzers, or Fourier transform ion cyclotron resonance mass analyzers.

[0105] Elements found in bioactive molecules, such as carbon, oxygen, and nitrogen, naturally exist in a variety of different isotopic forms. For example, most carbon exists as 12 C, but about 1% of all naturally occurring carbon exists as 13exist in the form of C. Therefore, a portion of some naturally occurring carbon-containing molecules will contain at least one 13 C atom. The inclusion of naturally occurring elemental isotopes in a molecule gives rise to multiple molecular isotope forms. The mass difference between molecular isotope forms is at least 1 atomic mass unit (amu). This is because elemental isotopes differ by at least one neutron (the mass of one neutron ≈ 1 amu). When molecular isotope forms are ionized to multiply charged states, the mass differences between the isotope forms can become difficult to discern because mass spectrometry detection is based on the mass-to-charge ratio (m / z). For example, two isotope forms with a mass difference of 1 amu are both ionized to the 5+ state, and the difference in their m / z is only 0.2 (1 amu divided by the difference in charge states of 5). High-resolution / high-accuracy mass spectrometers are capable of distinguishing the isotope forms of highly multiply charged ions (e.g., ions with a charge of ±4, ±5, ±6, ±7, ±8, ±9, or higher).

[0106] Due to naturally occurring elemental isotopes, each molecular ion typically exists in multiple isotope forms (each of which may produce a separately detectable spectral peak if analyzed using a sufficiently sensitive mass spectrometer). The m / z ratios and relative abundances of the multiple isotope forms together constitute the isotope signature of the molecular ion. In some embodiments, the m / z and relative abundances of two or more molecular isotope forms can be used to confirm the identity of the molecular ion under study. In some embodiments, mass spectral peaks from one or more isotope forms are used to quantify the molecular ion. In some related embodiments, a single mass spectral peak from one isotope form is used to quantify the molecular ion. In other related embodiments, multiple isotope peaks are used to quantify the molecular ion. In these latter embodiments, the multiple isotope peaks can be subjected to any suitable mathematical treatment. Several mathematical treatments are known in the art and include, but are not limited to, summing the areas under multiple peaks or averaging the responses from multiple peaks.

[0107] In mass spectrometry techniques, several detection modes can generally be used to detect ions. For example, selected ions can be detected, i.e., using the selective ion monitoring mode (SIM), or alternatively, mass transitions generated by collision-activated dissociation (CAD), such as multiple reaction monitoring (MRM) or selected reaction monitoring (SRM). CAD is typically used to generate fragment ions for further detection. In CAD, precursor ions obtain energy by colliding with an inert gas and then fragment through a process called "unimolecular decomposition". Sufficient energy must be deposited in the precursor ion so that certain bonds within the ion can break due to an increase in vibrational energy. Optionally, neutral loss can be monitored.

[0108] In some embodiments, a quadrupole analyzer is used to determine the mass-to-charge ratio. For example, in a "quadrupole" or "quadrupole ion trap" instrument, the force on an ion in an oscillating radiofrequency field is proportional to the direct current potential applied between the electrodes, the amplitude of the RF signal, and the mass-to-charge ratio. The voltage and amplitude can be selected such that only ions with a specific mass-to-charge ratio travel the length of the quadrupole rod, while all other ions are deflected. Thus, a quadrupole rod instrument can act as a "mass filter" and a "mass detector" for the ions injected into the instrument.

[0109] The specificity of MS techniques can be enhanced by employing "tandem mass spectrometry" or "MS / MS". In this technique, precursor ions (also known as parent ions) generated from a target molecule can be filtered in an MS instrument, and then the precursor ions are subsequently fragmented to produce one or more fragment ions (also known as daughter ions or product ions), which are then analyzed in a second MS program. By carefully selecting the precursor ions, only the ions generated from certain analytes will enter the fragmentation chamber, where they collide with atoms of an inert gas to produce fragment ions. Since both the precursor ions and the fragment ions are generated in a reproducible manner under a given set of ionization / fragmentation conditions, the MS / MS technique can provide a very powerful analytical tool. For example, the combination of filtering / fragmenting can be used to eliminate interfering substances and may be particularly useful in complex samples such as biological samples.

[0110] Alternative modes of operating a tandem mass spectrometer include product ion scanning and precursor ion scanning. For descriptions of these modes of operation, see, for example, 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).

[0111] The results of analyte determination can be correlated with the amount of analyte in the original sample by a variety of methods known in the art. For example, if sampling and analysis parameters are carefully controlled, the relative abundance of a given ion can be compared to a table that converts relative abundance to the absolute amount of the original molecule. Optionally, an external standard can be run with the sample, and a standard curve can be constructed based on the ions generated from these standards. Using such a standard curve, the relative abundance of a given ion can be converted to the absolute amount of the original molecule. In certain preferred embodiments, an internal standard is used to generate a standard curve for calculating the amount of analyte. Methods for generating and using such standard curves are well known in the art, and one of ordinary skill in the art can select a suitable internal standard. For example, one or more forms of an isotopically labeled molecule having an m / z similar to the analyte can be used as an internal standard. In some of the embodiments described herein, an exemplary internal standard is isotopically labeled diazepam, but many other compounds (isotopically labeled or otherwise labeled) can be used. Many other methods for correlating the amount of ions with the amount of the original molecule will be known to one of ordinary skill in the art.

[0112] As used herein, when analyzed by mass spectrometry techniques, "isotopically labeled" results in a mass shift in the labeled molecule relative to the unlabeled molecule. Examples of suitable labels include deuterium ( 2 H), 13 C, and 15 N. One or more isotopic labels can be incorporated into one or more positions in the molecule, and one or more isotopic labels can be used on the same isotopically labeled molecule.

[0113] One or more steps of the method can be performed using an automated machine. In certain embodiments, one or more purification steps are performed online, and more preferably, all purification and mass spectrometry steps can be performed in an online manner.

[0114] In a particularly preferred embodiment, MS / MS is used to detect and / or quantify analytes in a sample as follows. The sample is preferably subjected to liquid chromatography, preferably HPLC; the liquid solvent stream from the chromatographic column enters the heated nebulizer interface of the MS / MS analyzer; and the solvent / analyte mixture is converted to vapor in the heated charged tube of the interface. During these processes, the analytes (i.e., antidepressants or metabolites) are analyzed. Ions, e.g., precursor ions, pass through the orifice of the instrument and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters that allow selection of ions based on their mass-to-charge ratio (m / z) (i.e., "precursor" and "fragment" ions in Q1 and Q3 are selected respectively). Quadrupole 2 (Q2) is a collision cell where ions are fragmented. The first quadrupole (Q1) of the mass spectrometer selects molecules having the mass-to-charge ratio of the analyte. Precursor ions having the correct mass-to-charge ratio are allowed to enter the collision cell (Q2), while unwanted ions having any other mass-to-charge ratio strike the sides of the quadrupole and are eliminated. The precursor ions entering Q2 collide with neutral argon molecules and are fragmented. The resulting fragment ions enter quadrupole 3 (Q3), where fragment ions of the analyte are selected while other ions are eliminated.

[0115] These methods may involve MS / MS performed in positive or negative ion mode; preferably positive ion mode. Using standard methods known in the art, one of ordinary skill in the art can identify one or more fragment ions of a particular precursor ion of the analyte that can be used for selection in quadrupole 3 (Q3).

[0116] When the ions strike the detector, they generate an electronic pulse that is converted into a digital signal. The data acquired is transferred to a computer, which plots a graph of the collected ion counts versus time. The resulting mass spectrum is similar to the chromatogram generated in traditional HPLC-MS methods. The area under the peak corresponding to a particular ion or the amplitude of such a peak can be measured and correlated with the amount of the target analyte. In certain embodiments, the area under the curve or peak amplitude of the fragment ion(s) and / or precursor ion is measured to determine the amount of the analyte. As described above, calibration standard curves of the peaks of one or more ions based on internal or external molecular standards can be used to convert the relative abundance of a given ion to the absolute amount of the original analyte.

[0117] The following examples are used to illustrate the present invention. These examples are in no way intended to limit the scope of the method.

[0118] Examples

[0119] Example 1: Sample Preparation

[0120] We describe a validated LC-MS / MS method for the simultaneous analysis of 23 prescription antidepressant analytes and their metabolites provided in Table 1 below.

[0121] Table 1. Antidepressants and metabolites determined by this assay

[0122]

[0123]

[0124] Quality control, calibrators, and internal standards: Calibration standards (4 - 5,000 ng / mL) at 5, 12.5, and 4,000 ng / mL and quality controls (QC’s) were prepared by spiking the analyte stock solutions into drug-free urine controls (UTAK). The internal standard (IS) was a 25 - 100 ng / mL mixture 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.

[0125] Sample preparation: Urine samples, calibrators, and QC (25 μL each) were mixed with IS (25 μL) in a 1 mL, 96-well extraction plate, diluted with 450 μL of 10 mM ammonium formate aqueous solution (mobile phase A), vortexed at 1,100 rpm for 2 minutes, and then transferred to LC-MS / MS for injection and analysis.

[0126] Example 2: Liquid Chromatography - Mass Spectrometry

[0127] LC-MS / MS: The extracted samples (25 μL) were chromatographically separated on a Phenyl-Hexyl 50x4.6 mm 2.6 μ column (Phenomenex) using a gradient of mobile phase A / mobile phase B (25% methanol in acetonitrile). On a Prelude LX-4MD TM (ThermoFisher Scientific), a 4-column LC multiplexer was used to maximize throughput. A Sciex 4500 Triple Quad TM mass spectrometer was used for selected reaction monitoring. Figure 1 is a representative chromatogram of all analytes and Figure 2 shows baseline separation of closely related analytes.

[0128] Table 2 provides the mass transitions for the detection of each analyte in the mass spectrometry assay

[0129] Table 2. Mass Spectral Transitions (m / z) for Detection of Antidepressants and Metabolites

[0130]

[0131]

[0132]

[0133]

[0134] Example 3: Verification and Results

[0135] Verification: The following characteristics were determined by standard laboratory methods: Limit of Quantitation (LOQ), linearity (including Upper Limit of Linearity of Dilution [ULOL]), precision, accuracy, interference from over 150 different drugs, stability, stability of extracted samples, matrix effect, and carryover.

[0136] Linearity:

[0137] The 5 - 9 - point calibration curves showed consistent linearity and reproducibility within ±20% of their targets, with a regression coefficient (r) > 0.990.

[0138] The CV was between 7.5% and 10%.

[0139] The Analytical Measurement Range (AMR) for all antidepressant analytes and metabolites was 4 to 5,000 ng / mL, the LOQ was 10 ng / ml (with 1 exception), and the ULOL was 50,000 ng / mL. The exception was the metabolite norsertraline, for which the AMR was 25 to 5,000 ng / mL and the LOQ was 50 ng / mL.

[0140] Precision:

[0141] A 5 - day precision study showed consistent results, with σ values for low, medium, and high levels of QC greater than 3.

[0142] Accuracy:

[0143] The accuracy study was conducted by correlating 65 samples in the concentration range of 4 to 20,000 ng / mL with another 65 results from another laboratory. Examples are Figure 3 and Figure 4 as presented.

[0144] On average, Deming regression showed a correlation coefficient of 1.022, an intercept of - 0.0681, and no bias.

[0145] Interference:

[0146] (Over 150 prohibited and prescription drugs were tested at 100-fold of the cut-off value. These tests were done in negative matrix controls and LOQ controls spiked with related substances)

[0147] None of the interfering drugs tested caused a signal intensity deviation of ≥20% for the group of drugs at the LOQ.

[0148] Stability:

[0149] The samples were stable for 7 days at room temperature, 14 days refrigerated, and 30 days frozen. After extraction, the samples were stable for 24 hours.

[0150] Matrix effect:

[0151] Samples were compared at 3 different levels (0.5×, 2×, and 0.8× ULOL) with neat matrix and diluted matrix.

[0152] No matrix effect was observed.

[0153] Cross-contamination:

[0154] Two samples were spiked back-to-back at a concentration of 4000 ng / mL, followed by 4 blank samples, to determine the cross-contamination effect. This was run in triplicate.

[0155] No cross-contamination was observed.

[0156] The contents of articles, patents, and patent applications, as well as all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication were specifically and individually indicated to be incorporated. The applicant reserves the right to actually incorporate any and all materials and information from any such article, patent, patent application, or other physical and electronic documents into this application.

[0157] The methods described illustratively herein can be suitably practiced in the absence of any element(s), limitation(s) not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. should be construed broadly and not restrictively. In addition, the terms and expressions employed herein are used as descriptive and not restrictive terms, and in using such terms and expressions, it is not intended to exclude any equivalents of the features shown and described or portions thereof. It should be recognized that various modifications can be made within the scope of the invention as claimed. Accordingly, it should be understood that although the invention has been specifically disclosed herein by way of preferred embodiments and optional features, modifications and variations of the invention disclosed herein can be resorted to by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention.

[0158] The present invention has been described herein in general and broadly. Each narrower genus and subgenus grouping within the general disclosure also forms part of the method. This includes the generic description of the method with the proviso or negative limitation of removing any subject matter from the genus, whether or not the excised material is specifically recited herein.

[0159] Other embodiments are within the appended claims. In addition, where features or aspects of the method are described in terms of a Markush group, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

1. A method for detecting or determining the amount of one or more selective serotonin reuptake inhibitors (SSRI) and SSRI metabolites in a sample by tandem mass spectrometry, wherein the SSRI and SSRI metabolites include fluoxetine and norfluoxetine, the method comprises: a. ionizing the sample under conditions suitable for generating fragment ions detectable by tandem mass spectrometry, wherein the fragment ions include a fluoxetine fragment ion with a mass-to-charge ratio of 148.1 ± 0.2 and a norfluoxetine fragment ion with a mass-to-charge ratio of 134.2 ± 0.2 or 30.1 ± 0.2; b. determining the amount of the fragment ions by tandem mass spectrometry; and c. using the amount of the fragment ions determined in step (b) to determine the amount of SSRI or SSRI metabolite in the sample.

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

3. The method according to claim 2, wherein the one or more internal standards include deuterated internal standards.

4. The method according to claim 3, wherein the deuterated internal standards are selected from norcitalopram-D3, paroxetine-D6, citalopram-D6, and vilazodone-D4.

5. The method according to claim 1, wherein the sample includes a biological sample.

6. The method according to claim 1, wherein the sample includes urine.

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

8. The method according to claim 7, wherein the liquid chromatography includes high performance liquid chromatography.

9. The method according to claim 1, wherein the method is capable of detecting levels of SSRI and SSRI metabolites in the range of 4 ng / mL to 5000 ng / mL, including the end values.

10. The method according to claim 1, wherein the method is capable of detecting levels of SSRI and SSRI metabolites in the range of 25 ng / mL to 5000 ng / mL, including the end values.

11. The method according to claim 1, wherein the tandem mass spectrometry is performed by selected reaction monitoring, multiple reaction monitoring, precursor ion scanning, or product ion scanning.

12. The method according to claim 1, wherein the tandem mass spectrometry is performed by selected reaction monitoring.

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

14. The method according to claim 1, wherein the lower limit of quantification is 50 ng / mL.

15. The method according to claim 1, wherein the sample includes serum.

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