Determination of antidepressants by mass spectrometry

Abstract

The invention relates to the determination of antidepressants by mass spectrometry. Methods for detecting or determining the amount of an antidepressant and / or an antidepressant metabolite in a sample are described. More specifically, mass spectrometry is described for detecting and quantifying antidepressants and / or antidepressant metabolites in a sample.

Description

[0001] This application is a divisional application. The application date of the original application is May 29, 2020, the application number is 2020800512037, and the name of the invention is "Determination of antidepressants by mass spectrometry".

[0002] Cross-references to Related Patent Applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 855,863, filed May 31, 2019, which is incorporated herein by reference in its entirety. Background Art

[0004] Baseline testing helps clinicians determine whether a patient is taking or not taking one or more antidepressants before treatment. Monitoring patients taking antidepressants to ensure adherence and avoid inadvertent polypharmacy is crucial. Some antidepressants, such as selective serotonin reuptake inhibitors (SSRIs), can have harmful side effects and should not be combined with other drugs in the same class. Accurate testing of antidepressants and their metabolites is necessary. Summary of the Invention

[0005] In one aspect, provided herein are methods for detecting and quantifying antidepressants and antidepressant metabolites by mass spectrometry.

[0006] 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 comprises ionizing the sample under conditions suitable for producing one or more ions detectable by mass spectrometry; determining the amount of the one or more ions by mass spectrometry; and using the amount of the one or more ions to determine the presence or amount of the antidepressant and / or antidepressant metabolite in the sample.

[0007] In some embodiments, the mass spectrometry method comprises tandem mass spectrometry. In these embodiments, the method comprises: a) ionizing the sample under conditions suitable for producing a precursor ion; b) fragmenting the precursor ion to produce one or more fragment ions; c) determining the amount of the one or more ions produced in steps a) and b); and d) using the amount of the one or more ions determined in step c) to determine the presence or amount of the antidepressant and metabolite in the sample.

[0008] In some embodiments, provided herein are methods for detecting or determining the amount of one or more 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 metabolites.

[0009] In some embodiments, provided herein are methods for detecting or determining one or more antidepressants or antidepressant metabolites selected from the group consisting of 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.

[0010] 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 inhibitors (hydroxybupropion); tricyclic antidepressants (imipramine, nortriptyline, amitriptyline, doxepin, trimipramine, desipramine, protriptyline, amoxapine, clomipramine, maprotiline). Other antidepressants used in this test also act as sedatives and are trazodone, mirtazapine, and vortioxetine. The metabolites tested were desmethylcitalopram, desmethylclomipramine, desmethyldoxepin, desmethylfluoxetine, desmethylfluvoxamine, desmethylsertraline, and 1,3-chlorophenylpiperazine.

[0011] In some embodiments, provided herein are methods for simultaneously detecting or determining the amounts of 10 or more antidepressant drugs and antidepressant drug metabolites.

[0012] In some embodiments, provided herein are methods for simultaneously detecting or determining the amounts of 20 or more antidepressant drugs and antidepressant drug metabolites.

[0013] In some embodiments, provided herein are methods for simultaneously detecting or determining the amounts of 30 antidepressant drugs and antidepressant drug metabolites.

[0014] In some embodiments, the methods provided herein include adding one or more internal standards. In some embodiments, the one or more internal standards include a deuterated internal standard. In some embodiments, the deuterated internal standard is selected from 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.

[0015] 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.

[0016] In some embodiments, the sample is subjected to liquid chromatography prior to ionization. In some embodiments, liquid chromatography comprises high performance liquid chromatography.

[0017] In some embodiments, the method is capable of detecting antidepressants and antidepressant metabolites at levels ranging from about 4 ng / mL to about 5000 ng / mL, inclusive.

[0018] In some embodiments, the method is capable of detecting antidepressants and antidepressant metabolites at levels ranging from about 25 ng / mL to about 5000 ng / mL, inclusive.

[0019] 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.

[0020] In a preferred embodiment, tandem mass spectrometry is performed by selected reaction monitoring.

[0021] In some embodiments, provided herein are methods for identifying antidepressants and antidepressant metabolites comprising detecting ions comprising the following mass-to-charge ratios (m / z).

[0022]

[0023]

[0024]

[0025] In some embodiments, the methods described herein are capable of detecting antidepressants and antidepressant metabolites at levels ranging from 4 ng / mL to 5000 ng / mL, inclusive. In some embodiments, the methods described herein are capable of detecting antidepressants and antidepressant metabolites at levels ranging from 25 ng / mL to 5000 ng / mL, inclusive.

[0026] 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.

[0027] In some embodiments, the sample is passed 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 online processing.

[0028] In some embodiments, the sample is passed 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 online processing.

[0029] In some embodiments, the methods can be used to determine the presence or amount of antidepressants and antidepressant metabolites in a biological sample (e.g., 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, the 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, and the like, and any combination thereof.

[0030] In certain preferred embodiments of the methods disclosed herein, mass spectrometry is performed in positive ion mode. Alternatively, mass spectrometry is performed in negative ion mode. Various ionization sources can be used in embodiments of the present invention, including, for example, atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI). In certain embodiments, antidepressants and antidepressant metabolites are measured using positive ion mode.

[0031] In preferred embodiments, a separately 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 produce a plurality of ions detectable in a mass spectrometer, and the one or more ions produced by each are detected by mass spectrometry. In these embodiments, the presence or amount of ions produced by the target analyte can be correlated with the presence of the amount of the target analyte in the sample.

[0032] In other embodiments, the amount of the antidepressant 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 the antidepressant and antidepressant metabolites or isotopically labeled variants thereof.

[0033] As used herein, the singular forms "a," "an," and "the" include plural forms unless otherwise indicated. Thus, for example, reference to "a protein" includes a plurality of protein molecules.

[0034] As used herein, the terms "purification" or "purifying" do not refer to the removal of all materials other than the target analyte from a sample. Rather, purification refers to the step of enriching 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 by various means can allow for a relative reduction in one or more interfering substances, for example, one or more substances that may or may not interfere with the detection of a selected parent ion or daughter ion by mass spectrometry. Relative reduction, as the term is used, does not require the complete removal by purification of any substance present with the target analyte in the material to be purified.

[0035] As used herein, the term "immunopurification" or "immunopurify" refers to a purification step that utilizes antibodies (including polyclonal or monoclonal antibodies) to enrich for one or more target analytes. Immunopurification can be performed using any immunopurification method known in the art. Typically, immunopurification steps utilize antibodies that are bound, conjugated, or otherwise attached to a solid support (e.g., 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.

[0036] As used herein, the term "immunoparticle" refers to a capsule, bead, gel particle, or the like having an antibody bound, conjugated, or otherwise attached to its surface (on and / or in the particle). In certain embodiments utilizing immunopurification, the immunoparticle comprises agarose gel or agarose beads. In alternative embodiments utilizing immunopurification, the immunoparticle comprises glass, plastic, or silica beads, or silica gel.

[0037] As used herein, the term "sample" refers to any sample that may include an analyte of interest. As used herein, the term "body fluid" refers to any fluid that can be isolated 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 comprises a body fluid sample; preferably, plasma or serum.

[0038] 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., mobile phase) for the solid (i.e., solid phase) through which the solution passes or around it. 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 immunoaffinity. In some cases, as the mobile phase passes through or around the solid phase, undesirable components of the mobile phase can be retained by the solid phase, resulting in purification of the analyte in the mobile phase. In other cases, the analyte can be retained by the solid phase, thereby allowing undesirable 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 be operated via single or mixed mode mechanisms. Mixed-mode mechanisms utilize 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 be a column that exhibits strong cation exchange and hydrophobic retention.

[0039] As used herein, the term "chromatography" refers to a procedure in which a chemical mixture carried by a liquid or gas is separated into its components due to the different distributions of the chemical entities as the mixture flows around or through a stationary or solid phase.

[0040] 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 delayed as the fluid uniformly permeates through a column of finely divided material or through a capillary channel. The delay is caused by the distribution of the components of the mixture between the one or more stationary phases and the bulk 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 referred to as high turbulence liquid chromatography (HTLC) or high throughput liquid chromatography).

[0041] As used herein, the term "high performance liquid chromatography" or "HPLC" (sometimes called "high pressure liquid chromatography") refers to liquid chromatography in which resolution is enhanced by forcing a mobile phase under pressure through a stationary phase, usually a densely packed column.

[0042] 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 utilizes the turbulent flow of an analyte through a column packing as the basis for separation. TFLC has been applied to the preparation of a sample containing two unnamed drugs prior to analysis by mass spectrometry. See, for example, Zimmer et al., J Chromatogr A 854:23-35 (1999); see also U.S. Patent Nos. 5,968,367, 5,919,368, 5,795,469, and 5,772,874, which further explain TFLC. Those of ordinary skill in the art understand "turbulent flow." When a fluid flows slowly and smoothly, the flow is called "laminar flow." For example, a fluid moving through an HPLC column at a low flow rate is laminar. In laminar flow, the motion of fluid particles is orderly, with the particles generally moving in straight lines. At faster speeds, the inertia of the water overcomes fluid friction and produces turbulence. A fluid that is not in contact with an irregular boundary "outpaces" a fluid that is slowed by friction or deflected by an uneven surface. When a fluid flows turbulently, it moves in the form of eddies and vortices (or vortices), which have more "drag" than when it flows laminarly. Many references are available to help determine whether a 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)).

[0043] As used herein, the term "gas chromatography" or "GC" refers to a chromatography method in which a sample mixture is vaporized and injected into a stream of carrier gas (such as nitrogen or helium) moving through a column containing a stationary phase composed of a liquid or particulate material and separated into its component compounds according to their affinity for the stationary phase.

[0044] As used herein, the term "large particle column" or "extraction column" refers to a chromatography column containing an average particle size greater than about 50 μm. As used in this context, the term "about" refers to ±10%.

[0045] As used herein, the term "analytical column" refers to a chromatographic column having sufficient chromatographic plates to achieve separation of the material in the sample eluted from the column sufficient to allow determination of the presence or amount of the analyte. Such a column is generally distinguished from an "extraction column," the general purpose of which is to separate or extract retained material from non-retained material to obtain a purified sample for further analysis. As used in this context, the term "about" refers to ±10%. In a preferred embodiment, the analytical column comprises particles having a diameter of approximately 5 μm.

[0046] As used herein, the terms "online" and "inline," for example, as used in "online automated format" or "online extraction," refer to steps that are performed without operator intervention. In contrast, the term "offline" as used herein refers to steps that require manual operator intervention. Thus, if a sample is precipitated and the supernatant is then manually loaded into an autosampler, the precipitation and loading steps are offline from subsequent steps. In various embodiments of the method, one or more steps can be performed in an online automated format.

[0047] As used herein, the term "mass spectrometry" or "MS" refers to an analytical technique that identifies compounds by their mass. MS refers to a method for filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m / z." MS techniques generally involve (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. Compounds can be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector. Generally, one or more target molecules are ionized and the ions are then introduced into a mass spectrometer 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, for example, U.S. Patent Nos. 6,204,500, entitled “Mass spectrometry From Surfaces”; 6,107,623, entitled “Methods and Apparatus for Tandem Mass spectrometry”; 6,268,144, entitled “DNA Diagnostics Based On Mass spectrometry”; 6,124,137, entitled “Surface-Enhanced Photolabile Attachment And Release For Desorption And Detection Of Analytes”; Wright et al., Prostate Cancer and Prostatic Diseases 1999, 2:264-76; and Merchant and Weinberger, Electrophoresis 2000, 21:1164-67.

[0048] As used herein, the term "operating in negative ion mode" refers to those mass spectrometry methods that generate and detect negative ions. As used herein, the term "operating in positive ion mode" refers to those mass spectrometry methods that generate and detect positive ions.

[0049] As used herein, the term "ionization" or "ionizing" refers to the process of producing 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.

[0050] As used herein, the term "electron ionization" or "E1" refers to a process in which an analyte of interest in the gas or vapor phase interacts with a stream of electrons. The collision of the electrons with the analyte produces analyte ions, which can then be used in mass spectrometry techniques.

[0051] As used herein, the term "chemical ionization" or "CI" refers to a process in which a reagent gas (eg, ammonia) is bombarded with electrons, and analyte ions are formed by the interaction of the reagent gas ions and the analyte molecules.

[0052] 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) strikes a nonvolatile sample, desorbing and ionizing 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-nitrophenyloctyl ether, sulfolane, diethanolamine, and triethanolamine. Selecting the appropriate matrix for a compound or sample is an empirical process.

[0053] As used herein, the term "matrix-assisted laser desorption ionization" or "MALDI" refers to 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 photoionization, protonation, deprotonation, and cluster decay. For MALDI, the sample is mixed with an energy-absorbing matrix, which facilitates the desorption of the analyte molecules.

[0054] As used herein, the term "surface-enhanced laser desorption ionization" or "SELDI" refers to another method in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample through various ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. For SELDI, the sample is typically bound to a surface that preferentially retains one or more target analytes. As with MALDI, the process can also employ energy-absorbing materials to facilitate ionization.

[0055] As used herein, the term "electrospray ionization" or "ESI" refers to a method in which a solution is passed along a short length of a capillary tube with a high positive or negative potential applied at the end of the capillary tube. 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 droplet flows through an evaporation chamber. As the droplets become smaller, the surface charge density increases until the natural repulsion between like charges causes ions and neutral molecules to be released.

[0056] As used herein, the term "atmospheric pressure chemical ionization" or "APCI" refers to a mass spectrometry method similar to ESI; however, APCI produces ions by ion-molecule reactions occurring within a plasma at atmospheric pressure. The plasma is maintained by an electrical discharge between a spray capillary and a counter electrode. The ions are then typically extracted into a mass analyzer using a set of differentially pumped skimmer stages. A countercurrent of dry and preheated N2 gas can be used to improve solvent removal. Gas-phase ionization in APCI can be more efficient than ESI when analyzing less polar substances.

[0057] As used herein, the term "atmospheric pressure photoionization" or "APPI" refers to a process in which the mechanism of photoionization of a molecule M is photon absorption and electron emission to form a molecular ion M + A form of mass spectrometry. Because the photon energy is usually 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 lot of time and money. In the presence of water vapor or protic solvents, the molecular ions can extract H to form MH+. This often happens if M has a high proton affinity. This does not affect the quantitative accuracy because the sum of M+ and MH+ is constant. Pharmaceutical compounds in protic solvents are usually observed as MH+, while non-polar compounds such as naphthalene or testosterone usually form M+. See, for example, Robb et al., Anal. Chem. 2000, 72(15): 3653-3659.

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

[0059] As used herein, the term "field desorption" refers to a method in which a non-volatile test sample is placed on an ionizing surface and a strong electric field is used to generate analyte ions.

[0060] As used herein, the term "desorption" refers to the removal of an analyte from a surface and / or the passage of the analyte into the gas phase. Laser desorption and thermal desorption are techniques in which a sample containing an analyte is thermally desorbed into the gas phase by a laser pulse. Laser light strikes the back of a custom-made 96-well plate with a metal base. The laser pulse heats the base, and the heat causes the sample to transition to the gas phase. The gas phase sample is then drawn into a mass spectrometer.

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

[0062] As used herein, "multiple reaction mode," sometimes referred to as "selected reaction monitoring," is a detection mode for a mass spectrometer in which a precursor ion and one or more fragment ions are selectively detected.

[0063] As used herein, the term "lower limit of quantification," "lower limit of quantification," or "LLOQ" refers to the point at which measurements become quantitatively meaningful. The analyte response at this LOQ is identifiable, discrete, and reproducible with a relative standard deviation (RSD%) of less than 20% and an accuracy of 85% to 115%.

[0064] As used herein, the term "limit of detection" or "LOD" is the point at which a 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, defined as three times the RSD of the mean at zero concentration.

[0065] As used herein, the "amount" of an analyte in a bodily fluid sample generally refers to an absolute value reflecting the amount of analyte detectable in the sample volume. However, the amount also contemplates relative amounts compared to the amount of another analyte. For example, the amount of an analyte in a sample can be an amount that is greater than a control or normal level of the analyte normally present in the sample.

[0066] As used herein, the term "about" in relation to quantitative measurements that do not include measurement of the mass of an ion refers to the indicated value plus or minus 10%. 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.

[0067] The foregoing summary of the invention is non-limiting, and other features and advantages of the invention will be apparent from the following detailed description of the invention and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 LC-MS / MS profiles for all analytes and metabolites are shown.

[0069] Figure 2 Examples of baseline separation are shown for (A) amitriptyline, (B) maprotiline, and (C) venlafaxine (analytes: left, internal standard (IS): right).

[0070] Figure 3 The precision of Citalopram compared to another experiment is shown. No deviation greater than ±20% of the value was shown.

[0071] Figure 4 The precision of the metabolite desmethylcitalopram compared to another experiment is shown. No deviation greater than ±20% of the value was shown.

[0072] Figure 5The separated cyclobenzaprine interferent is shown. The graph shows 5 ng / mL maprotiline + cyclobenzaprine at 100×.

[0073] Figure 6 Shown are the differences in mass spectra of desmethylvenlafaxine vs. tramadol.

[0074] Figure 7 The separated tramadol interferent is shown. The graph shows 5 ng / mL desmethylvenlafaxine + tramadol at 100×.

[0075] Figure 8 Baseline separation is shown for amitriptyline, maprotiline, and venlafaxine.

[0076] Figure 9 Baseline separation of nortriptyline, protriptyline, and desvenlafaxine is shown.

[0077] Figure 10 Baseline separation between nordoxepin and mirtazapine is shown.

[0078] Figure 11 Desipramine vs. mirtazapine identified by different transitions are shown.

[0079] Figure 12 It was shown that ion ratios and / or relative retention times (RRT) would fail for desipramine in mirtazapine-positive patients. DETAILED DESCRIPTION

[0080] In certain embodiments, the antidepressant panels described herein can be used with adherence monitoring for patients with a history / risk of use and / or abuse of such medications. Before prescribing such medications, baseline testing can alert providers to the potential for polypharmacy conflicts. Adherence monitoring requires that these patient populations be provided with prescription medications and without over-the-counter medications.

[0081] Certain brain chemicals, called neurotransmitters, are associated with depression, more 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 include: SSRIs, SNRIs, NDRIs, tricyclics, atypicals, MAOIs, and others (see below).

[0082] Selective serotonin reuptake inhibitors (SSRIs). Doctors usually start by prescribing an SSRI. These medications are safer and generally cause fewer troublesome side effects than other types of antidepressants. SSRIs include fluoxetine (Prozac, Selfemra), paroxetine (Paxil, Pexeva), sertraline (Zoloft), citalopram (Celexa), escitalopram (Lexapro), fluvoxamine (Faverin, Fevarin, Floxyfral, Dumyrox, Luvox), and vilazodone (Viibryd).

[0083] Serotonin and norepinephrine reuptake inhibitors (SNRIs)—duloxetine (Cymbalta), venlafaxine (Effexor XR), desmethylvenlafaxine (the synthetic form of venlafaxine's main metabolite, O-desmethylvenlafaxine; Pristiq, Khedezla), and levomilnacipran (Fetzima). SNRIs have a unique dual effect of raising both serotonin and norepinephrine levels; thus, SNRIs fight more than one cause of depression.

[0084] Norepinephrine and dopamine reuptake inhibitors (NDRIs). Bupropion (Wellbutrin, Aplenzin, Forfivo XL) belongs to this class. It is one of the few antidepressants not frequently associated with sexual side effects.

[0085] Tricyclic antidepressants (TCAs) tend to cause more side effects than newer antidepressants. Tricyclic antidepressants are not usually prescribed unless the 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).

[0086] Atypical antidepressants. These medications don't fit neatly into any other antidepressant class. They include trazodone (Oleptro), mirtazapine (Remeron), and vortioxetine (Brintellix). These are sedatives and are usually taken at night.

[0087] This test does not include monoamine oxidase inhibitors (MAOIs). These medications should not be used in combination with SSRIs. Common MAOIs include tranylcypromine (Parnate), phenelzine (Nardil), and isocarboxazid (Marplan).

[0088] Methods for measuring the amount of an analyte in a sample are described. More specifically, mass spectrometry methods for detecting and / or quantifying an analyte in a biological sample (such as human plasma or serum) are described. The methods can utilize liquid chromatography followed by tandem mass spectrometry to quantify the analyte in the sample.

[0089] 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. Particularly preferred mammals are primates, most preferably male or female humans. Preferred samples include body fluids, such as urine, blood, plasma, serum, saliva, cerebrospinal fluid, or tissue samples; preferably urine. Such samples can, for example, be obtained from a patient; that is, a living person, male or female, who appears in a clinical setting for the diagnosis, prognosis, or treatment of a disease or condition. In some embodiments, preferred samples can be obtained from female humans of reproductive 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 metabolites in the sample (i.e., the amount of endogenous leflunomide metabolites in the sample).

[0090] The present invention also contemplates kits for quantitative determination of antidepressants. Kits for quantitative determination of antidepressants may include kits comprising the compositions provided herein. For example, the kit may include packaging materials and a measured amount of an isotopically labeled internal standard sufficient for at least one assay. Typically, the kit will also include instructions recorded in a tangible form (e.g., contained on paper or electronic media) for using the packaged reagents for quantitative determination of antidepressants.

[0091] Calibration and QC pools for use with embodiments of the present invention are preferably prepared using a matrix similar to the expected sample matrix, provided that the analyte is substantially absent.

[0092] Sample preparation for mass spectrometry analysis

[0093] In preparation for mass spectrometry analysis, the analyte can be enriched relative to one or more other components (e.g., proteins) in the sample 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 (including ethyl acetate or methanol extraction), and use of chaotropic agents, or any combination of the foregoing.

[0094] Protein precipitation is a method for preparing a test sample, particularly a biological test sample, 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 describes a protein precipitation technique suitable for use in the methods of the present invention. Protein precipitation can be used to remove most of the protein from a sample, leaving the analyte in the supernatant. The sample can be centrifuged to separate the liquid supernatant from the precipitated protein; alternatively, the sample can be filtered to remove the precipitated protein. The resulting supernatant or filtrate can then be used directly for mass spectrometry analysis; or alternatively, for other purification methods, such as liquid chromatography and subsequent mass spectrometry analysis. In certain embodiments, the use of protein precipitation, such as, for example, acetonitrile protein precipitation, can avoid the need for TFLC or other online extraction prior to mass spectrometry or high performance liquid chromatography (HPLC) and mass spectrometry.

[0095] Another sample purification method that can be used before mass spectrometry is liquid chromatography (LC). Certain liquid chromatography, including high performance liquid chromatography (HPLC), relies on relatively slow laminar flow technology. Traditional HPLC analysis relies on column packing, wherein the laminar flow of the sample through the column is the basis for separating the target analyte from the sample. Technicians will understand that separation in such a column is a distribution process and LC can be selected, including HPLC, instruments and columns suitable for analytes. The chromatographic column generally includes a medium (i.e., a packing material) to promote the separation (i.e., fractionation) of the chemical part. The medium may include microparticles. The particles generally include a bonded surface that interacts with various chemical parts to promote the separation of the chemical parts. 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. 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 comprising the fractionated sample. The sample can be supplied directly to the inlet port, or from an SPE column, such as an online extraction column or a TFLC column. In some embodiments, an online guard column can be used before the HPLC column to remove particulates and phospholipids in the sample before the sample reaches the HPLC column. In some embodiments, the guard column can be a biphenyl guard column.

[0096] In one embodiment, the sample can be applied to an 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 polymorphic (i.e., mixed) mode. During chromatography, the separation of materials is affected by variables such as the selection of an eluent (also referred to as "mobile phase"), an elution mode, a gradient condition, a temperature, etc.

[0097] In certain embodiments, the analyte can be purified by applying the sample to the post under the condition that the target analyte is reversibly retained by the column packing material and one or more other materials are not retained. In these embodiments, a first mobile phase condition can be adopted, wherein the target analyte is retained by the post, and once the non-retained material is washed through, a second mobile phase condition can be adopted to remove the retained material from the post subsequently. Alternatively, the analyte can be purified by applying the sample to the post under the mobile phase condition in which the target analyte is eluted at a different rate than one or more other materials. This step can enrich the amount of one or more target analytes relative to one or more other components of the sample.

[0098] In a preferred embodiment, HPLC is performed using a biphenyl column chromatography system. In certain preferred embodiments, a biphenyl analytical column (e.g., a Pinnacle DB biphenyl analytical column (5 μm particle size, 50×2.1 mm, or equivalent) from Restek Inc.) is used. In certain preferred embodiments, HPLC is performed using HPLC grade 0.1% formic acid in water as solvent A and 0.1% formic acid in acetonitrile as solvent B.

[0099] By carefully selecting valves and connector tubing, two or more chromatography columns can be connected as needed, allowing material to be transferred from one column to another without any manual steps. In a preferred embodiment, the selection of valves and tubing is controlled by a preprogrammed computer to perform the necessary steps. Most preferably, the chromatography system is also connected to a detector system, such as an MS system, in this online manner. Thus, an operator can place a tray of samples into the autosampler, and the remaining operations are carried out under computer control, allowing all selected samples to be purified and analyzed.

[0100] In some embodiments, TFLC can be used to purify analytes prior to mass spectrometry analysis. In this embodiment, a TFLC column that captures the analyte can be used to extract the sample. The analyte is then eluted and transferred online 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 the column is then transferred online to an HPLC analytical column for further purification prior to mass spectrometry. Because 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 saves time and cost and eliminates the opportunity for operator error.

[0101] Detection and quantification by mass spectrometry

[0102] In various embodiments, analyte can be ionized by any method known to the skilled person. Mass spectrometry is performed using a mass spectrometer comprising an ion source for ionizing the fractionated sample and producing charged molecules for further analysis. For example, the ionization of the sample can be performed by electron ionization, chemical ionization, electrospray ionization (ESI), photon ionization, 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. It will be understood by those skilled in the art that the selection of ionization method can be determined based on the analyte to be measured, sample type, detector type, positive mode to negative mode selection etc.

[0103] The analyte can be ionized in positive and negative modes. In some embodiments, the analyte is ionized in positive mode.

[0104] In mass spectrometry, typically after a 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. An exemplary ion trap method is described in Bartolucci, et al., Rapid Commun. Mass Spectrom. 2000, 14:967-73.

[0105] According to some methods of the present invention, high resolution / high precision mass spectrometry is used for quantification of analytes. 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; such as a resolving power (FWHM) of 25,000 or better and an accuracy of about 5 ppm or less; such as a resolving power (FWHM) of 25,000 or better and an accuracy of about 3 ppm or less. Three exemplary mass spectrometers capable of exhibiting the required performance levels for analyte ions are those comprising an orbital trap mass analyzer, certain TOF mass analyzers, or a Fourier transform ion cyclotron resonance mass analyzer.

[0106] Elements found in biologically active molecules, such as carbon, oxygen, and nitrogen, naturally exist in many different isotopic forms. For example, most carbon exists in 12 C, but about 1% of all naturally occurring carbon is in the form 13 C forms. Therefore, some naturally occurring carbon-containing molecules will contain at least one 13 C atoms. Including naturally occurring isotopes of elements in molecules will produce a variety of molecular isotopic forms. The mass difference between molecular isotopic forms is at least 1 atomic mass unit (amu). This is because the isotopes of elements differ by at least one neutron (the mass of a neutron ≈ 1 amu). When molecular isotopic forms are ionized into multiple charge states, the mass difference between the isotopic forms may become difficult to distinguish because mass spectrometry detection is based on the mass-to-charge ratio (m / z). For example, two isotopic 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 (the difference in 1 amu / 5 charge states). High resolution / high precision mass spectrometers are able to distinguish isotopic forms of highly multiply charged ions (such as ions with a charge of ±4, ±5, ±6, ±7, ±8, ±9 or more).

[0107] Due to naturally occurring element isotopes, there are usually multiple isotopic forms for each molecular ion (if analyzed using a sufficiently sensitive mass spectrometer, each of which may produce a separately detectable spectral peak). The m / z ratio and relative abundance of multiple isotopic forms together constitute the isotopic signature of the molecular ion. In some embodiments, the m / z and relative abundance of two or more molecular isotopic forms can be utilized to confirm the identity of the molecular ion under study. In some embodiments, mass spectrometry peaks from one or more isotopic forms are used for quantitative molecular ions. In some related embodiments, a single mass spectrometry peak from a kind of isotopic form is used for quantitative molecular ions. In other related embodiments, multiple isotopic peaks are used to quantitative molecular ions. In these latter embodiments, multiple isotopic peaks can be subjected to any appropriate 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.

[0108] In mass spectrometry, several detection modes can generally be used to detect ions. For example, selected ions can be detected, i.e., using selective ion monitoring mode (SIM), or alternatively, mass conversion produced by impact-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 gain energy by colliding with an inert gas and are then fragmented by a process known as "unimolecular decomposition." Sufficient energy must be deposited in the precursor ions so that certain bonds within the ions can be broken due to the increase in vibrational energy. Alternatively, neutral loss can be monitored.

[0109] In some embodiments, a quadrupole analyzer is used to determine mass-to-charge ratio. For example, in a "quadrupole" or "quadrupole ion trap" instrument, the force experienced by ions in an oscillating radio frequency field is proportional to the DC potential applied between the electrodes, the amplitude of the RF signal, and the mass-to-charge ratio. The voltage and amplitude can be selected so that only ions with a specific mass-to-charge ratio travel through the length of the quadrupole rods, while all other ions are deflected. Thus, a quadrupole instrument can act as both a "mass filter" and a "mass detector" for ions injected into the instrument.

[0110] The specificity of the MS technique can be enhanced by employing "tandem mass spectrometry" or "MS / MS." In this technique, precursor ions (also called parent ions) generated by 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 called daughter ions or product ions), which are then analyzed in a second MS procedure. By carefully selecting the precursor ions, only ions generated by certain analytes enter the fragmentation chamber, where they collide with atoms of an inert gas to produce the fragment ions. Because both the precursor ions and the fragment ions are produced 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 filtration / fragmentation combination can be used to eliminate interfering substances and can be particularly useful in complex samples such as biological samples.

[0111] Alternative modes of operating a tandem mass spectrometer include product ion scanning and precursor ion scanning. For a description 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).

[0112] By various methods known in the art, the result of the analyte determination can be associated with the amount of the analyte in the original sample. For example, if the sampling and analysis parameters are carefully controlled, the relative abundance of a given ion can be compared with a table that converts the relative abundance into the absolute amount of the original molecule. Alternatively, an external standard can be run together with the sample, and a standard curve can be constructed based on the ions generated by these standards. Using such a standard curve, the relative abundance of a given ion can be converted into the absolute amount of the original molecule. In certain preferred embodiments, an internal standard is used to generate a standard curve for calculating the number of analytes. The method for generating and using such a standard curve is well known in the art, and those of ordinary skill can select a suitable internal standard. For example, one or more forms of isotope-labeled molecules with m / z similar to the analyte can be used as an internal standard. In some embodiments described herein, an exemplary internal standard is isotope-labeled diazepam, but many other compounds (isotope-labeled or otherwise labeled) can be used. Many other methods of associating the amount of ions with the amount of the original molecule will be known to those of ordinary skill in the art.

[0113] As used herein, an "isotopic label" produces a mass shift in the labeled molecule relative to the unlabeled molecule when analyzed by mass spectrometry techniques. Examples of suitable labels include deuterium ( 2 H), 13 C and 15 N. One or more isotopic labels may be incorporated into one or more positions in a molecule, and one or more isotopic labels may be used on the same isotopically labeled molecule.

[0114] One or more steps of the method can be performed using automated machinery. 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.

[0115] 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 into vapor in the heated charged tube of the interface. During these processes, the analyte (i.e., antidepressant or metabolite) is 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 ions to be selected based on their mass-to-charge ratio (m / z) (i.e., "precursor" and "fragment" ions in Q1 and Q3, respectively). Quadrupole 2 (Q2) is a collision cell in which ions are fragmented. The first quadrupole (Q1) of the mass spectrometer selects molecules with an analyte mass-to-charge ratio. Precursor ions with the correct mass-to-charge ratio are allowed to enter the collision chamber (Q2), while undesirable ions with any other mass-to-charge ratio collide with the sides of the quadrupole and are eliminated. The precursor ions entering Q2 collide with neutral argon molecules and fragment. The resulting fragment ions enter quadrupole 3 (Q3), where they select the fragment ions of the analyte while eliminating other ions.

[0116] 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 can identify one or more fragment ions of a specific precursor ion of a selected analyte that can be used in quadrupole 3 (Q3).

[0117] When ions collide with the detector, they produce an electronic pulse, which is converted into a digital signal. The data obtained are transferred to a computer, which plots the relationship between the ion counts collected and time. The mass spectrum generated is similar to the chromatogram generated in the traditional HPLC-MS method. The area under the peak corresponding to a specific ion or the amplitude of this peak can be measured and associated with the amount of the target analyte. In some embodiments, the area under the curve or the peak amplitude of fragment ions (one or more) and / or precursor ions are measured to determine the amount of the analyte. As mentioned above, the relative abundance of a given ion can be converted into the absolute amount of the original analyte using a calibration standard curve based on the peak of one or more ions of an internal or external molecular standard.

[0118] The following examples serve to illustrate the present invention. These examples are in no way intended to limit the scope of the method.

[0119] Example

[0120] Example 1: Sample preparation

[0121] 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.

[0122] Table 1. Antidepressants and metabolites identified by this assay

[0123]

[0124]

[0125] Quality Control, Calibrators, and Internal Standards: Calibration standards (4-5000 ng / mL) at 5, 12.5, and 4000 ng / mL and quality controls (QCs) were prepared by spiking analyte stock solutions into a drug-free urine control (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.

[0126] Sample preparation: Urine samples, calibrators, and QCs (25 μL each) were mixed with IS (25 μL) in a 1 mL, 96-well extraction plate, diluted with 450 μL of 10 mM aqueous ammonium formate (mobile phase A), and vortexed at 1,100 rpm for 2 minutes before being transferred to LC-MS / MS for injection and analysis.

[0127] Example 2: Liquid chromatography-mass spectrometry

[0128] LC-MS / MS: The extracted sample (25 μL) was Chromatographic separation was performed on a Phenyl-Hexyl 50x4.6mm 2.6μ column (Phenomenex) using a mobile phase A / mobile phase B (25% methanol in acetonitrile) gradient. TM (ThermoFisher Scientific), a 4-column LC multiplexer was used to maximize throughput. TM Mass spectrometry was used for selected reaction monitoring. Figure 1 is a representative chromatogram for all analytes, and Figure 2 Baseline separation of closely related analytes is demonstrated.

[0129] Table 2 provides the mass transitions used to detect each analyte in the mass spectrometry assay.

[0130] Table 2. Mass spectral transitions (m / z) used to detect antidepressants and metabolites

[0131]

[0132]

[0133]

[0134]

[0135] Example 3: Verification and Results

[0136] Validation: The following properties were determined by standard laboratory methods: limit of quantitation (LOQ), linearity (including upper limit of linearity [ULOL] of dilution), precision, accuracy, interferences from over 150 different drugs, stability, extracted sample stability, matrix effects, and carryover.

[0137] Linearity:

[0138] The 5-9 point calibration curves showed consistent linearity and reproducibility within ±20% of their targets, with regression coefficients (r) >0.990.

[0139] The CV is between 7.5% and 10%.

[0140] 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 one exception) and a ULOL of 50,000 ng / mL. The exception was the metabolite norsertraline, which had an AMR of 25 to 5,000 ng / mL and a LOQ of 50 ng / mL.

[0141] Precision:

[0142] A 5-day precision study showed consistent results with σ values greater than 3 for low-, medium-, and high-level QCs.

[0143] Accuracy:

[0144] The precision study was conducted by correlating 65 samples ranging in concentration from 4 to 20,000 ng / mL with another 65 results from another laboratory. Figure 3 and Figure 4 Presented.

[0145] On average, the Deming regression showed a correlation coefficient of 1.022 and an intercept of -0.0681, indicating no bias.

[0146] interference:

[0147] (Over 150 illicit and prescription drugs were tested at 100x the cut-off value. These tests were performed using negative matrix controls and controls spiked with the substances of interest at LOQ.)

[0148] None of the interfering drugs tested caused a signal intensity deviation of ≥20% of the group drugs at LOQ.

[0149] stability:

[0150] Samples are stable for 7 days at room temperature, 14 days in the refrigerator, and 30 days in the freezer. After extraction, samples are stable for 24 hours.

[0151] Matrix Effects:

[0152] Samples were compared to neat and diluted matrix at 3 different levels (0.5x, 2x and 0.8x ULOL).

[0153] No matrix effects were observed.

[0154] Cross contamination:

[0155] Two samples were spiked back-to-back at a concentration of 4000 ng / mL, followed by four blank samples, to determine the effect of carryover. This was run in triplicate.

[0156] No cross contamination was observed.

[0157] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are incorporated herein by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such article, patent, patent application, or other physical and electronic file.

[0158] The methods illustratively described herein may be suitably implemented in the absence of any element(s), limitation(s) not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like should be interpreted broadly and not restrictively. Furthermore, the terms and expressions employed herein are used as descriptive and not restrictive terms, and the use of such terms and expressions is not intended to exclude any equivalents of the features shown and described, or portions thereof. It should be recognized that various modifications may be made within the scope of the claimed invention. Therefore, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the invention disclosed herein may be adopted by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention.

[0159] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also forms part of the method. This includes the general method description with a proviso or negative limitation removing any subject matter from the genus, regardless of whether the removed material is specifically recited herein.

[0160] Other embodiments are within the following claims.In addition, where features or aspects of the methods are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

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

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

3. The method of claim 1 , wherein the one or more antidepressants and antidepressant metabolites are selected from the group consisting of 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.

4. The method of claim 1, wherein the method comprises simultaneously detecting or determining the amounts of 10 or more antidepressant drugs and antidepressant drug metabolites.

5. The method of claim 1, wherein the method comprises simultaneously detecting or determining the amounts of 20 or more antidepressant drugs and antidepressant metabolites.

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

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

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

9. The method according to claim 8, wherein the deuterated internal standard is selected from the group consisting of 1,3-chlorophenylpiperazine-D8, hydroxybupropion-D6, desmethyl-venlafaxine-D6, norcitalopram-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 of claim 1, wherein the sample comprises a biological sample.

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