Quantitation of antidepressants by mass spectrometry

Abstract

Methods for detecting or determining the amount of an antidepressant and / or antidepressant metabolite in a sample are described. More particularly, mass spectrometry methods for detecting and quantifying an antidepressant and / or antidepressant metabolite in a sample are described.

Description

[Technical Field]

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

[0002] Baseline testing is useful in helping clinicians determine whether a patient is using one or more antidepressants prior to treatment. Monitoring patients prescribed antidepressants is very important to ensure compliance and avoid unintended polypharmacy. Some antidepressants, such as selective serotonin reuptake inhibitors (SSRIs), can have adverse side effects and should not be mixed with other drugs in the same class. Accurate testing of antidepressants and metabolites is needed. Summary of the Invention [Means for solving the problem]

[0003] In one aspect, provided herein is a method for the detection and quantification of antidepressants and antidepressant metabolites by mass spectrometry.

[0004] Provided herein are methods for detecting the presence or amount of an antidepressant and / or antidepressant metabolites in a sample by mass spectrometry, the methods comprising subjecting the sample to ionization under conditions suitable to produce 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 metabolites in the sample.

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

[0006] 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.

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

[0008] 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, desmethylvenlafaxine), norepinephrine and dopamine reuptake inhibitors (hydroxybupropion), tricyclic antidepressants (imipramine, nortriptyline, amitriptyline, doxepin, trimipramine, desipramine, protriptyline, amoxapine, clomipramine, maprotiline). Other antidepressants used in this assay also function as sedatives and are trazodone, mirtazapine, and vortioxetine. The metabolites tested were desmethylcitalopram, desmethylclomipramine, desmethyldoxepin, norfluoxetine, norfluvoxamine, norsertraline, and 1,3-chlorphenylpiperazine.

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

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

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

[0012] 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 the group consisting of 1,3-chlorophenylpiperazine-D8, hydroxybupropion-D6, desmethyl-venlafaxine-D6, desmethylcitalopram-D3, trimipramine-D3, amitriptyline-D3, nortriptyline-D3, paroxetine-D6, protriptyline-D3, citalopram-D6, venlafaxine-D6, imipramine-D3, trazodone-D6, vilazodone-D4, and vortioxetine-D8.

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

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

[0015] In some 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, inclusive.

[0016] 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, inclusive.

[0017] In some embodiments, the mass analysis is tandem mass analysis. In some embodiments, the tandem mass analysis is performed by selected reaction monitoring, multiple reaction monitoring, precursor ion scanning, or product ion scanning.

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

[0019] In some embodiments, provided herein is a step of quantifying antidepressants and antidepressant metabolites, comprising detecting ions comprising the following mass / charge ratios (m / z):

[0020] [Table 1] JPEG0007742207000002.jpg254133JPEG0007742207000003.jpg154137

[0021] 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, inclusive. 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, inclusive.

[0022] In some embodiments, the methods described herein are capable of quantifying antidepressants and antidepressant metabolites down to a limit of 10 ng / mL, hi some embodiments, the methods described herein are capable of quantifying antidepressants and antidepressant metabolites down to a limit of 50 ng / mL.

[0023] In some embodiments, prior to ionization, the sample is subjected to an extraction column, such as a solid phase extraction (SPE) column. In some related embodiments, SPE and mass spectrometry are performed using online processing.

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

[0025] In some embodiments, the methods 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 can be processed by one or more steps to generate a processed sample, which can then be subjected to mass spectrometry. 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, or the like, and any combination thereof.

[0026] 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.

[0027] In preferred embodiments, a separately detectable internal standard is added to the sample, and its amount is also determined in the sample. In these embodiments, all or a portion of both the analyte of interest and the internal standard present in the sample are ionized to produce multiple ions that can be detected by a mass spectrometer, and one or more ions produced from each are detected by mass spectrometry. In these embodiments, the presence or amount of ions produced from the analyte of interest can be related to the amount of analyte of interest present in the sample.

[0028] In other embodiments, the amount of antidepressant and antidepressant metabolites in a sample can be determined by comparison to one or more external reference standards, exemplary of which are blank plasma or serum spiked with antidepressant and antidepressant metabolites or isotopically labeled variants thereof.

[0029] As used herein, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated. Thus, for example, reference to "a protein" includes a plurality of protein molecules.

[0030] As used herein, the terms "purification" or "purifying" do not refer to the removal of all substances other than the analyte(s) of interest from a sample. Instead, purification refers to a procedure that enhances the amount of one or more analytes of interest relative to other components in the sample that may interfere with the detection of the analytes of interest. Purification of a sample by various means allows for the relative reduction of one or more interfering substances, e.g., one or more substances that may or may not interfere with the detection of selected parent or daughter ions by mass spectrometry. Relative reduction, as used in this term, does not require that the purification completely removes substances that are present along with the analytes of interest in the material to be purified.

[0031] As used herein, the terms "immunopurification" or "immunopurifying" refer to a purification procedure that utilizes antibodies, including polyclonal or monoclonal antibodies, to enrich for one or more analytes of interest. Immunopurification can be performed using any of the immunopurification methods known in the art. Often, immunopurification procedures utilize antibodies bound, conjugated, or otherwise attached to a solid support, such as a column, well, tube, gel, capsule, particle, or the like. Immunopurification, as used herein, includes, without limitation, procedures often referred to in the art as immunoprecipitation, as well as procedures often referred to in the art as affinity chromatography.

[0032] 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 within the particle). In certain embodiments utilizing immunopurification, the immunoparticle comprises sepharose or agarose beads. In alternative embodiments utilizing immunopurification, the immunoparticle comprises glass, plastic, or silica beads, or silica gel.

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

[0034] As used herein, the term "solid-phase extraction" or "SPE" refers to a method for separating a mixture of chemicals into components as a result of the affinity of the components dissolved or suspended in a solution (i.e., a mobile phase) for a solid (i.e., a solid phase) through which the solution flows. SPE, as used herein, 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 flows through or around the solid phase, undesired components of the mobile phase may be retained by the solid phase, resulting in the purification of the analyte in the mobile phase. In other cases, the analyte may be retained by the solid phase, allowing the undesired components of the mobile phase to flow 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 function by a single- or mixed-mode mechanism. A mixed-mode mechanism utilizes 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 may exhibit strong cation exchange and hydrophobic retention.

[0035] As used herein, the term "chromatography" means a process by which a mixture of chemicals carried by a liquid or gas is separated into components as a result of differential partitioning of the chemicals as they flow around or over a stationary liquid or solid phase.

[0036] As used herein, the term "liquid chromatography" or "LC" refers to a method in which one or more components of a fluid solution are selectively retarded as the fluid uniformly permeates a column or capillary passage of fine material. The retardation results from the partitioning of the components of the mixture between 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 known as high-turbulence liquid chromatography (HTLC) or high-throughput liquid chromatography).

[0037] As used herein, the term "high performance liquid chromatography" or "HPLC" (sometimes known as "high pressure liquid chromatography") refers to liquid chromatography that increases the degree of separation by forcing a mobile phase under pressure through a stationary phase, typically a tightly packed column.

[0038] As used herein, the term "turbulent flow liquid chromatography" or "TFLC" (sometimes known as high-turbulence liquid chromatography or high-throughput liquid chromatography) refers to a form of chromatography that utilizes the turbulent flow of assayed substances through a column packing as the basis for separation. TFLC was applied to the preparation of samples containing two unknown drugs prior to analysis by mass spectrometry. See, e.g., Zimmer et al., J Chromatogr A854, 23-35 (1999). See also U.S. Pat. Nos. 5,968,367, 5,919,368, 5,795,469, and 5,772,874, which further describe TFLC. Those skilled in the art understand "turbulent flow." When a fluid flows slowly and smoothly, the flow is called "laminar flow." For example, a fluid moving at a low flow rate through an HPLC column is laminar. In laminar flow, the motion of fluid particles is orderly, with particles generally moving in straight lines. At higher velocities, the inertia of the water overcomes the frictional forces of the fluid, and turbulent flow results. Fluid not in contact with an irregular boundary will "overtake" it, slowed by friction or deflected by an uneven surface. When a fluid is flowing turbulently, it will swirl (or spiral) in eddies with greater "drag" than if the flow were laminar. Many references are available to help determine when 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)).

[0039] As used herein, the term "gas chromatography" or "GC" refers to chromatography in which a sample mixture is vaporized and injected into a stream of carrier gas (such as nitrogen or helium) that moves through a column containing a stationary phase consisting of a liquid or particulate solid, separating its component compounds according to their affinity for the stationary phase.

[0040] As used herein, the term "large particle column" or "extraction column" refers to a chromatography column containing an average particle diameter greater than about 50 μm. As used in this context, the term "about" means ±10%.

[0041] As used herein, the term "analytical column" refers to a chromatography column having a chromatographic plate sufficient to achieve separation of materials in a sample eluting from the column sufficient to allow for the determination of the presence or amount of an analyte. Such columns are often distinguished from "extraction columns," which have the general purpose of separating or extracting retained materials from unretained 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 contains particles about 5 μm in diameter.

[0042] As used herein, the terms "online" and "in-line" refer to a procedure that is performed without the need for operator intervention, as used, for example, in "online automated fashion" or "online extraction." In contrast, the term "offline," as used herein, refers to a procedure that requires manual operator intervention. Thus, if a sample is subjected to precipitation and then the supernatant is manually loaded into an autosampler, the precipitation and loading steps are offline from the subsequent steps. In various embodiments of the method, one or more steps can be performed in an online automated fashion.

[0043] 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 for filtering, detecting, and measuring ions based on the ions' mass-to-charge ratio, or "m / z." MS techniques generally include the steps of (1) ionizing compounds to produce charged compounds, and (2) detecting the molecular weight of the charged compounds and calculating their 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 molecules of interest are ionized, and the ions are then introduced into a mass analyzer, where a combination of magnetic and electric fields causes the ions to follow a path in space that depends on their mass ("m") and charge ("z"). See, for example, U.S. Patent No. 6,204,500, entitled "Mass Spectrometry From Surfaces," U.S. Patent No. 6,107,623, entitled "Methods and Apparatus for Tandem Mass Spectrometry," U.S. Patent No. 6,268,144, entitled "DNA Diagnostics Based On Mass Spectrometry," U.S. Patent No. 6,124,137, entitled "Surface-Enhanced Photolabile Attachment And Release For Desoption 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.

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

[0045] As used herein, the terms "ionization" or "ionize" refer to a process that produces analyte ions having a net charge equal to one or more electron units. Negative ions are those that have a net negative charge of one or more electron units, while positive ions are those that have a net positive charge of one or more electron units.

[0046] As used herein, the term "electron ionization" or "EI" refers to a method in which an analyte of interest in the gas or vapor phase interacts with a stream of electrons. Collisions between the electrons and the analyte produce analyte ions that can then be subjected to mass spectrometry techniques.

[0047] As used herein, the term "chemical ionization" or "CI" refers to a method in which a reagent gas (e.g., ammonia) is subjected to electron bombardment to produce analyte ions through interaction of the reagent gas ions with analyte molecules.

[0048] As used herein, the term "fast atom bombardment" or "FAB" refers to a method in which a beam of high-energy atoms (often Xe or Ar) bombards a nonvolatile sample, desorbing and ionizing the molecules contained within. 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. The selection of an appropriate matrix for a compound or sample is an empirical process.

[0049] 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 radiation that desorbs and ionizes analytes in the sample by various ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. For MALDI, the sample is mixed with an energy-absorbing matrix that facilitates desorption of analyte molecules.

[0050] As used herein, the term "surface-enhanced laser desorption ionization" or "SELDI" refers to another method in which a nonvolatile sample is exposed to laser radiation that desorbs and ionizes analytes in the sample by 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 analytes of interest. Like MALDI, this method can also use energy-absorbing materials to facilitate ionization.

[0051] As used herein, the term "electrospray ionization" or "ESI" refers to a method in which a solution is passed through a short capillary tube, to the end of which a high positive or negative potential is applied. The solution reaches the end of the tube and is evaporated (atomized) into a jet or spray of very small droplets of the solution in solvent vapor. This spray of droplets flows through an evaporation chamber. As the droplets become smaller, the surface charge density increases to the point where natural repulsion between like-sign charges causes the release of ions as well as neutral molecules.

[0052] As used herein, the term "atmospheric pressure chemical ionization" or "APCI" refers to a mass spectrometry technique similar to ESI, but APCI generates ions through ion-molecule reactions that occur in a plasma at atmospheric pressure. The plasma is sustained by an electrical discharge between a nebulizing capillary and a counter electrode. Ions are then extracted into a mass spectrometer, typically using a pair of differentially pumped skimmer stages. Counterflow dry, preheated N2 gas can be used to improve solvent removal. Gas-phase ionization in APCI can be more effective than ESI for analyzing less polar species.

[0053] The term "atmospheric pressure photoionization" or "APPI," as used herein, refers to a form of mass spectrometry in which the mechanism of photoionization of a molecule M is the absorption of a photon and the ejection of an electron to produce a molecular ion, M+. Because the photon energy is typically just above the ionization potential, the molecular ion is not susceptible to dissociation. In many cases, it is possible to analyze samples without the need for chromatography, which can save considerable time and money. In the presence of water vapor or a protic solvent, the molecular ion can abstract H to form MH+. This tends to occur when M has a high proton affinity. This does not affect the accuracy of quantification, since the sum of M+ and MH+ is constant. Drug compounds in protic solvents 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.

[0054] 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 temperature high enough that most elements are atomized and ionized.

[0055] 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.

[0056] As used herein, the term "desorption" refers to the removal of an analyte from a surface and / or the entry of the 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 illuminates the backside of a custom-made 96-well plate with a metal base. The laser pulse heats the bottom, and the heat transfers the sample into the gas phase. The gas phase sample is then drawn into a mass spectrometer.

[0057] As used herein, the term "selected ion monitoring" refers to a detection mode in a mass spectrometer instrument in which only ions within a relatively narrow mass range, typically within about one mass unit, are detected.

[0058] As used herein, "multiple reaction mode," sometimes known as "selective reaction monitoring," is a detection mode of a mass spectrometer instrument in which a precursor ion and one or more fragment ions are selectively detected.

[0059] As used herein, the terms "lower limit of quantitation," "lower limit of quantitation," or "LLOQ" refer to the point at which a measurement becomes quantitatively meaningful. The analyte response at this LOQ is identifiable, distinct, and reproducible with a relative standard deviation (RSD%) of less than 20% and an accuracy of 85% to 115%.

[0060] As used herein, the term "limit of detection" or "LOD" is the point at which a measurement is greater than its associated uncertainty. The LOD is the point at which a value exceeds the uncertainty associated with its measurement and is defined as three times the RSD of the mean at zero concentration.

[0061] As used herein, the "amount" of an analyte in a bodily fluid sample generally refers to an absolute value reflecting the mass of analyte that can be detected in a volume of sample. However, amount also contemplates a relative amount compared to the amount of other analytes. 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 that is normally present in the sample.

[0062] The term "about," as used herein with respect to quantitative measurements that do not involve measurement of the mass of an ion, means plus or minus 10% of the stated value. Mass spectrometry instruments may vary slightly in determining the mass of a given analyte. The term "about," with respect to the mass or mass-to-charge ratio of an ion, means + / - 0.50 atomic mass units.

[0063] The above summary of the invention is non-limiting; other features and advantages of the invention will be apparent from the following detailed description of the invention and from the claims. [Brief explanation of the drawings]

[0064] [Figure 1] FIG. 1 shows the LC-MS / MS profiles of all analytes and metabolites. [Figure 2] 1 shows an example of baseline separation of (A) amitriptyline, (B) maprotiline, and (C) venlafaxine (analyte: left, internal standard (IS): right). [Figure 3] FIG. 1 shows the accuracy of citalopram compared to another laboratory, showing no bias greater than ±20% of the value. [Figure 4] FIG. 1 shows the accuracy of the metabolite desmethylcitalopram compared to another laboratory. No bias greater than ±20% of the value is shown. [Figure 5] Figure 1 shows cyclobenzaprine interference resolved. The figure shows 5 ng / mL maprotiline + 100x cyclobenzaprine. [Figure 6] FIG. 1 shows the mass spectral differences between desmethylvenlafaxine versus tramadol. [Figure 7] 1 shows tramadol interference isolated. The figure shows 5 ng / mL desmethylvenlafaxine + 100x tramadol. [Figure 8-1] FIG. 1 shows baseline separation of amitriptyline, maprotiline, and venlafaxine. [Figure 8-2] (Continued from Figure 8-1) [Figure 9-1] FIG. 1 shows baseline separation of nortriptyline, protriptyline, and desmethylvenlafaxine. [Figure 9-2] (Continuation of Figure 9-1) [Figure 10] FIG. 1 shows baseline separation of desmethyldoxapine and mirtazapine. [Figure 11-1]FIG. 1 shows desipramine vs. mirtazapine identified by different transitions. [Figure 11-2] (Continued from Figure 11-1) [Figure 12-1] FIG. 1 shows that ion ratios and / or relative retention times (RRTs) are not seen for desipramine in patients positive for mirtazapine. [Figure 12-2] (Continued from Figure 12-1) DETAILED DESCRIPTION OF THE INVENTION

[0065] In certain embodiments, the antidepressant panels described herein can be used in conjunction with medication compliance monitoring for patients with a history / risk of use and / or abuse of drugs within this class. Baseline testing prior to prescribing drugs in this class alerts providers to potential drug conflicts due to polypharmacy. Medication compliance monitoring requires the absence of prescribed and unprescribed medications for these patient populations.

[0066] Certain brain chemicals, neurotransmitters, more specifically serotonin, norepinephrine, and dopamine, are associated with depression. Most antidepressants treat depression by affecting these neurotransmitters. Different types / classes of antidepressants affect these neurotransmitters in different ways. These types include SSRIs, SNRIs, NDRIs, tricyclics, atypicals, MAOIs, etc. (see below).

[0067] Selective serotonin reuptake inhibitors (SSRIs). Doctors often start by prescribing an SSRI. These medications are safer than other types of antidepressants 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, Dumirox, Luvox), and vilazodone (Viibryd).

[0068] Serotonin and norepinephrine reuptake inhibitors (SNRIs) - duloxetine (Cymbalta), venlafaxine (Effexor XR), desmethylvenlafaxine (synthetic form of venlafaxine's major metabolite, O-desmethylvenlafaxine; Pristiq, Khedezla), and levomilnacipran (Fetzima). SNRIs have a unique dual action of increasing both serotonin and norepinephrine levels, thus inhibiting more than one cause of depression.

[0069] Norepinephrine and dopamine reuptake inhibitors (NDRIs). Bupropion (Wellbutrin, Aplenzin, Forfivo XL) falls into this category. It is one of the few antidepressants associated with few sexual side effects.

[0070] Tricyclic antidepressants (TCAs) tend to cause more side effects than newer antidepressants, and are generally not prescribed unless the patient has first tried an SSRI and seen improvement. TCAs include imipramine (Tofranil), nortriptyline (Pamelor), amitriptyline (Elavil, Endep, Lentizolinol, Levate, Saroten, tryptanol, tryptizol), doxepin (Adapin, Curatin, Silenol, Sinequan), trimipramine (Surmontil), desipramine (Norpramin), protriptyline (Vivactil), amoxapine (Asendin), clomipramine (Anafranil), and maprotiline (Ludiomil).

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

[0072] Monoamine oxidase inhibitors (MAOIs) are not included in this assay. These drugs cannot be combined with SSRIs. Common MAOIs include tranylcypromine (Parnate), phenelzine (Nardil), and isocarboxazid (Marplan).

[0073] Methods for determining 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 may utilize liquid chromatography followed by tandem mass spectrometry to quantify the analyte in the sample.

[0074] Suitable test samples for use in the methods of the present invention are those that may contain the analyte of interest. In some preferred embodiments, the sample is a biological sample, i.e., a sample obtained from a 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 bodily fluid or tissue samples, such as urine, blood, plasma, serum, saliva, cerebrospinal fluid, etc., preferably urine. Such samples can be obtained, for example, from patients, i.e., living male or female individuals presenting to a clinical facility for diagnosis, prognosis, or treatment of a disease or condition. In some embodiments, preferred samples can be obtained from female humans of childbearing potential. In embodiments in which the sample comprises a biological sample, 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) when the sample is obtained from a biological source.

[0075] The present invention also contemplates a kit for the quantitative assay of antidepressants.The kit for the quantitative assay of antidepressants can include a kit containing the composition provided herein.For example, the kit can include packaging material and a certain amount of isotope-labeled internal standard in an amount sufficient for at least one assay.Generally, it also includes a tangible recorded instruction manual (for example, contained on paper or electronic media) for the use of the packaged reagent for the quantitative assay of antidepressants.

[0076] Calibration and QC pools used in embodiments of the present invention are preferably prepared using a matrix similar to the sample matrix of interest, provided that the analyte is essentially absent.

[0077] Preparation of samples for mass spectrometry In preparation for mass spectrometry, the analyte can be enriched relative to one or more other components in the sample (e.g., proteins) by a variety of 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 ethyl acetate or methanol extraction, and the use of chaotropic agents, or any combination of the above or the like.

[0078] Protein precipitation is one method for preparing test samples, particularly 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, describes protein precipitation techniques suitable for use in the methods of the present invention. Protein precipitation can be used to remove the majority of proteins from a sample, leaving the analyte in the supernatant. The sample can be centrifuged to separate the liquid supernatant from the precipitated proteins, or the sample can be filtered to remove the precipitated proteins. The resulting supernatant or filtrate can then be subjected directly to mass spectrometry analysis or to additional purification methods, such as liquid chromatography, followed by mass spectrometry analysis. In certain embodiments, the use of protein precipitation, such as acetonitrile protein precipitation, can eliminate the need for TFLC or other online extraction prior to mass spectrometry analysis, or high-performance liquid chromatography (HPLC) and mass spectrometry analysis.

[0079] Another method of sample purification that can be used prior to mass spectrometry analysis is liquid chromatography (LC). Certain methods of liquid chromatography, including high-performance liquid chromatography (HPLC), rely on relatively slow laminar flow techniques. Traditional HPLC analysis relies on column packing, where laminar flow of the sample through the column is the basis for separating the analytes of interest from the sample. Those skilled in the art will understand that separation in such columns is a partitioning process and will be able to select an appropriate LC, including HPLC, instrument, and column for use with the analyte. Chromatographic columns generally contain a medium (i.e., packing material) to facilitate separation (i.e., fractionation) of compound components. The medium may include fine particles. The particles generally contain binding surfaces that interact with various compound components to facilitate separation of the compound components. One suitable binding surface is a hydrophobic binding surface, such as an alkyl-, cyano-, or biphenyl-bound surface. The alkyl-bound surface may include C-4, C-8, C-12, or C-18-bound alkyl groups. In a preferred embodiment, the column is a biphenyl column. The chromatographic column includes an inlet port for receiving a sample and an outlet port for discharging an eluate containing the fractionated sample. The sample can be supplied to the inlet port directly or from an SPE column, such as an online extraction column, or a TFLC column. In some embodiments, an online guard cartridge can be used before the HPLC column to remove particles and phospholipids in the sample before it reaches the HPLC column. In some embodiments, the guard cartridge can be a biphenyl guard cartridge.

[0080] In one embodiment, a sample can be applied to an LC column at an inlet port, eluted with a solvent or solvent mixture, and discharged at an outlet port. Various solvent modes can be selected to elute the analyte(s) of interest. For example, liquid chromatography can be performed using gradient, isocratic, or polymorphic (i.e., mixed) modes. During chromatography, separation of substances is affected by variables such as the choice of eluent (also known as the "mobile phase"), elution mode, gradient conditions, temperature, etc.

[0081] In certain embodiments, analytes can be purified by applying a sample to a column under conditions in which the analyte of interest is reversibly retained by the column packing, while one or more other substances are not. In these embodiments, first mobile phase conditions can be used in which the analyte of interest is retained by the column, and second mobile phase conditions can be subsequently used to remove the retained substances from the column once the unretained substances have been washed away. Alternatively, analytes can be purified by applying a sample to a column under mobile phase conditions in which the analyte of interest elutes at a different rate than one or more other substances. Such a procedure can enhance the amount of one or more analytes of interest relative to one or more other components of the sample.

[0082] In one preferred embodiment, HPLC is performed using a biphenyl column chromatographic 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) manufactured by Restek Inc., or equivalent) is used. In certain preferred embodiments, HPLC is performed using 0.1% aqueous formic acid for HPLC as solvent A and 0.1% formic acid in acetonitrile as solvent B.

[0083] By careful selection of valves and connecting piping, two or more chromatography columns can be connected as needed to allow material to pass from one chromatography column to the next without the need for manual steps. In a preferred embodiment, the selection of valves and piping is controlled by a computer preprogrammed to perform the necessary steps. Most preferably, the chromatography system is also connected to a detection system, e.g., an MS system, in such an on-line manner. Thus, an operator can load a tray of samples into the autosampler, and the remaining operations are performed under computer control, resulting in the purification and analysis of all selected samples.

[0084] In some embodiments, TFLC can be used for analyte purification prior to mass spectrometry analysis. In such embodiments, samples can be extracted using a TFLC column that captures the analyte. The analyte is then eluted and transferred online to an analytical HPLC column. For example, sample extraction can be achieved using a TFLC extraction cartridge or a large particle size (50 μm) packed column. The sample eluted from this column is transferred online to an analytical HPLC column for further purification prior to mass spectrometry analysis. The steps involved in these chromatographic procedures can be linked in an automated manner, minimizing the need for operator involvement during analyte purification. This feature potentially saves time and money and eliminates the opportunity for operator error.

[0085] Detection and quantification by mass spectrometry In various embodiments, analytes can be ionized by methods known to those skilled in the art. Mass analysis is performed using a mass spectrometer that includes an ion source for ionizing fractionated samples and generating charged molecules for further analysis. For example, sample ionization can be achieved 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. Those skilled in the art will appreciate that the choice of ionization method can be determined based on the analyte to be measured, the type of sample, the type of detector, the choice of positive versus negative mode, and the like.

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

[0087] In mass spectrometry techniques, a sample is generally ionized and the resulting positively or negatively charged ions can then 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.

[0088] According to some methods of the present invention, high-resolution / high-accuracy mass spectrometry is used for quantitation of analytes. That is, mass spectrometry is performed using a mass spectrometer capable of exhibiting a resolving power (FWHM) of at least 10,000 with an accuracy of about 50 ppm or less for ions of interest. Preferably, the mass spectrometer exhibits a resolving power (FWHM) of 20,000 or more and an accuracy of about 20 ppm or less, such as a resolving power (FWHM) of 25,000 or more and an accuracy of about 5 ppm or less, such as a resolving power (FWHM) of 25,000 or more and an accuracy of about 3 ppm or less. Three exemplary mass spectrometers capable of exhibiting the required level of performance for analyte ions include orbitrap mass spectrometers, certain time-of-flight mass spectrometers, or Fourier transform ion cyclotron resonance mass spectrometers.

[0089] Elements found in biologically active molecules, such as carbon, oxygen, and nitrogen, occur naturally in many different isotopes. For example, most carbon is 12 C, but about 1% of all naturally occurring carbon is 13 C. Therefore, a portion of naturally occurring carbon containing molecules contains at least one 13The inclusion of naturally occurring elemental isotopes in molecules results in multiple molecular isotopes. The difference in mass between molecular isotopes is at least 1 atomic mass unit (amu). This is because elemental isotopes differ by at least one neutron (the mass of one neutron is approximately 1 amu). When molecular isotopes are ionized to multiple charge states, the mass difference between isotopes can be difficult to distinguish because detection in mass spectrometry is based on mass-to-charge ratio (m / z). For example, two isotopes that differ in mass by 1 amu, both ionized to the 5+ state, exhibit an m / z difference of only 0.2 (1 amu difference / 5 charge states). High-resolution / high-accuracy mass spectrometry can distinguish isotopes of highly multiply charged ions (such as ions with ±4, ±5, ±6, ±7, ±8, ±9, or higher charges).

[0090] Due to naturally occurring elemental isotopes, multiple isotopes typically exist for every molecular ion (each of which may produce a separately detectable spectral peak when analyzed by a sufficiently sensitive mass spectrometer). The m / z ratios and relative abundances of the multiple isotopes collectively comprise the isotopic signature of the molecular ion. In some embodiments, the m / z and relative abundances of two or more molecular isotopes can be utilized to confirm the identity of the molecular ion under consideration. In some embodiments, one or more isotopic mass spectrometric peaks are used to quantify the molecular ion. In some related embodiments, a single mass spectrometric peak of one isotope is used to quantify the molecular ion. In other related embodiments, multiple isotopic peaks are used to quantify the molecular ion. In these latter embodiments, the multiple isotopic peaks can be subjected to appropriate mathematical treatments. Some mathematical treatments are known in the art and include, but are not limited to, summing the areas under multiple peaks or averaging the responses due to multiple peaks.

[0091] In mass spectrometry, ions can generally be detected using several detection modes. For example, selected ions can be detected using selected ion monitoring (SIM), or alternatively, using mass transitions resulting from collisionally activated dissociation (CAD), such as multiple reaction monitoring (MRM) or selected reaction monitoring (SRM). CAD is often used to generate fragment ions for subsequent detection. In CAD, precursor ions gain energy through collisions with an inert gas and then fragment through a process called "unimolecular decomposition." Sufficient energy must be deposited in the precursor ion so that the increased vibrational energy can break specific bonds within the ion. Alternatively, neutral loss can be monitored.

[0092] In some embodiments, mass-to-charge ratios are determined using a quadrupole analyzer. For example, in a "quadrupole" or "quadrupole ion trap" instrument, ions in an oscillating radio frequency field experience a force proportional to the DC potential applied between 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 particular mass-to-charge ratio traverse the quadrupole, while all other ions are deflected. Thus, a quadrupole instrument can function as both a "mass filter" and a "mass detector" for ions injected into the instrument.

[0093] The specificity of MS techniques can be enhanced by "tandem mass spectrometry" or "MS / MS." In this technique, precursor ions (also called parent ions) derived from molecules of interest can be filtered by an MS instrument, which then fragments them to produce one or more fragment ions (also called daughter ions or product ions) that are analyzed in a second MS step. By careful selection of precursor ions, only ions produced by a specific analyte are passed to a fragmentation chamber, where the fragment ions are generated by collision with atoms of an inert gas. Because both precursor and fragment ions are reproducibly generated under a set of defined ionization / fragmentation conditions, MS / MS techniques can be an extremely powerful analytical tool. For example, a combination of filtration / fragmentation can be used to remove interfering substances, which can be particularly useful for complex samples such as biological samples.

[0094] Alternative modes of operating tandem mass spectrometry instruments 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).

[0095] The results of an analyte assay can be related to the amount of analyte in the original sample by many methods known in the art. For example, if sampling and analytical parameters are carefully controlled, the relative abundance of a given ion can be compared to a table that converts the relative abundance to the absolute amount of the original molecule. Alternatively, external standards can be run with the sample, and a standard curve is generated based on the ions from those standards. Such a standard curve can be used to convert the relative abundance of a given ion 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 an analyte. Methods for generating and using such standard curves are well known in the art, and one of skill in the art can select an appropriate internal standard. For example, one or more forms of isotopically labeled molecules with a similar m / z to the analyte can be used as the internal standard. In some embodiments described herein, an exemplary internal standard is isotopically labeled diazepam, although many other compounds (isotopically labeled or otherwise) can be used. Many other methods for relating the amount of an ion to the amount of the original molecule will be known to those of skill in the art.

[0096] As used herein, an "isotopic label" results in a mass shift of the labeled molecule compared 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 can be incorporated at one or more positions in a molecule, and one or more types of isotopic labels can be used in the same isotopically labeled molecule.

[0097] One or more steps of the method can be performed using automated equipment. In certain embodiments, one or more purification steps can be performed online, and more preferably, all of the purification and mass spectrometry steps can be performed online.

[0098] In a particularly preferred embodiment, analytes in a sample are detected and / or quantified using MS / MS as follows: The sample is preferably subjected to liquid chromatography, preferably HPLC, where the liquid solvent flow 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 charging tube of the interface. During these processes, the analyte (i.e., antidepressant or metabolite) is analyzed. Ions, e.g., precursor ions, pass through the instrument's orifice and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters that allow ion selection based on the ion's mass-to-charge ratio (m / z) (i.e., selection of "precursor" and "fragment" ions in Q1 and Q3, respectively). Quadrupole 2 (Q2) is a collision cell where ions are fragmented. The first quadrupole (Q1) of the mass spectrometer selects molecules with the mass-to-charge ratio of the analyte. Precursor ions with the correct mass-to-charge ratio are passed into the collision chamber (Q2), while unwanted ions with other mass-to-charge ratios collide with the sides of the quadrupole and are removed. Precursor ions entering Q2 collide with neutral argon gas molecules and are fragmented. The resulting fragment ions are passed into quadrupole 3 (Q3), where the analyte fragment ions are selected and other ions are removed.

[0099] The method may involve MS / MS performed in positive or negative ion mode, preferably positive ion mode. Using standard methods well known in the art, one skilled in the art can identify one or more fragment ions of a particular precursor ion of an analyte that can be used for selection in quadrupole 3 (Q3).

[0100] When ions strike the detector, they produce pulses of electrons that are converted into digital signals. The acquired data is transferred to a computer, which plots the counts of collected ions versus time. The resulting mass chromatogram is similar to that obtained with traditional HPLC-MS methods. The area under the peaks corresponding to specific ions or the amplitude of such peaks can be measured and correlated with the amount of the analyte of interest. In certain embodiments, the area under the curve or amplitude of the fragment ion(s) and / or precursor ion peaks is measured to determine the amount of the analyte. As described above, the relative abundance of a given ion can be converted to the absolute amount of the original analyte using a calibration standard curve based on one or more ion peaks of an internal or external molecular standard.

[0101] The following examples serve to illustrate the invention, but are not intended to limit the scope of the method. [Example]

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

[0103] [Table 2] JPEG0007742207000005.jpg223170

[0104] Quality Controls, Calibrators, and Internal Standards: Calibration standards (4–5,000 ng / mL) and quality controls (QCs) at 5, 12.5, and 4,000 ng / mL were prepared by spiking drug-free urine controls (UTAK) with stock solutions of the analytes. The internal standards (IS) were mixtures of 1,3-chlorphenylpiperazine-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 at concentrations ranging from 25 to 100 ng / mL.

[0105] 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 ammonium formate in water (mobile phase A), and then vortexed at 1,100 rpm for 2 min before being transferred to the LC-MS / MS for injection and analysis.

[0106] Example 2: Liquid Chromatography-Mass Spectrometry LC-MS / MS: Extracted samples (25 μL) were chromatographically separated on a Kinetex® Phenyl-Hexyl 50x4.6 mm 2.6 μ column (Phenomenex) using a mobile phase A / mobile phase B (25% methanol in acetonitrile) gradient. A four-column LC multiplex was used to maximize throughput on a Prelude LX-4 MD™ (ThermoFisher Scientific). A Sciex 4500 Triple Quad™ mass spectrometer was used for selected reaction monitoring. Figure 1 shows a representative chromatogram for all analytes, and Figure 2 shows baseline separation of closely related analytes.

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

[0108] [Table 3] JPEG0007742207000007.jpg241155JPEG0007742207000008.jpg243157JPEG0007742207000009.jpg61169

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

[0110] Linearity: Five- to nine-point calibration curves showed consistent linearity and reproducibility within ±20% of the target, with regression coefficients (r) >0.990.

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

[0112] The analytical measurement range (AMR) for all antidepressant analytes and metabolites was 4–5,000 ng / mL, with an LOQ of 10 ng / mL (with one exception) and an ULOL of 50,000 ng / mL, except for the metabolite norsertraline, which had an AMR of 25–5,000 ng / mL and an LOQ of 50 ng / mL.

[0113] accuracy: Precision testing over 5 days showed consistent results with sigma values ​​greater than 3 for low, medium and high levels of QC.

[0114] Accuracy: Accuracy testing was performed by correlating 65 samples spanning the concentration range 4-20,000 ng / mL with 65 other results from different laboratories. Examples are shown in Figures 3 and 4.

[0115] Overall, the Deming regression showed a correlation coefficient of 1.022 and an intercept of −0.0681, without bias.

[0116] Disruption: (Over 150 multiple illicit and prescription drugs were tested at 100x the cutoff. These tests were performed with both negative matrix controls and LoQ controls spiked with related substances.)

[0117] None of the interfering drugs tested causes a deviation of ≧20% in the signal intensity of the panel drugs at the LOQ.

[0118] Stability: The samples were stable at room temperature for 7 days, refrigerated for 14 days, and frozen for 30 days. After extraction, the samples were stable for 24 hours.

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

[0120] No matrix effects were observed.

[0121] Carryover: Two samples were spiked consecutively at 4000 ng / mL followed by four blank samples to determine carryover effects, which were performed in triplicate.

[0122] No carryover was observed.

[0123] The contents of the articles, patents, and patent applications, and all other literature and electronically available information mentioned or cited herein are incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic literature.

[0124] The methods illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc., should be read expansively and without limitation. Moreover, the terms and expressions used herein are used for purposes of description and not limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the shown and described features or portions thereof. It is recognized that various modifications are possible within the scope of the invention as claimed. Thus, while the invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and variations of the invention disclosed and embodied therein may be employed by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention.

[0125] The invention has been described broadly and generically herein. Each of the narrower species and subspecies falling within the generic disclosure also forms part of the method. This includes a general description of the method with a condition or negative limitation that removes any subject matter from the genus, whether or not the removed material is specifically set forth herein.

[0126] Other embodiments are within the scope of the following claims. Furthermore, where features or aspects of a method are described in terms of a Markush group, one of ordinary skill in the art will understand that the invention may also be described in terms of any individual component or subgroup of components of the Markush group.

Claims

1. 1. A method for simultaneously detecting or determining the amounts of 20 or more antidepressants and antidepressant metabolites in a sample by mass spectrometry, comprising: Over 20 antidepressants and antidepressant metabolites, including selective serotonin reuptake inhibitors (SSRIs) and SSRI metabolites, including fluoxetine and norfluoxetine; a. subjecting the sample to a multi-channel high performance liquid chromatography (HPLC) system prior to ionization to produce a second sample, the multi-channel HPLC system comprising multiple analytical columns; b. subjecting said second sample to ionization under conditions suitable to produce a plurality of ions detectable by mass spectrometry; c. determining the amount of a plurality of ions by mass spectrometry, wherein the plurality of ions comprises a fluoxetine fragment having a mass / charge ratio of 148.1±0.50 and a norfluoxetine fragment having a mass / charge ratio of 134.2±0.50 or 30.1±0.50; d. Using the amounts of the plurality of ions determined in step (c), simultaneously detecting or determining the amounts of 20 or more antidepressants and / or antidepressant metabolites in the sample; A method comprising:

2. 2. The method of claim 1, wherein the 20 or more antidepressants and antidepressant metabolites include serotonin and norepinephrine reuptake inhibitors, norepinephrine and dopamine reuptake inhibitors, tricyclic antidepressants, sedatives, and antidepressant metabolites.

3. 2. The method of claim 1, wherein the 20 or more antidepressants and antidepressant metabolites are selected from the group consisting of paroxetine, sertraline, citalopram, escitalopram, fluvoxamine, vilazodone, duloxetine, venlafaxine, desmethylvenlafaxine, hydroxybupropion, imipramine, nortriptyline, amitriptyline, doxepin, trimipramine, desipramine, protriptyline, amoxapine, clomipramine, maprotiline, trazodone, mirtazapine, vortioxetine, desmethylcitalopram, desmethylclomipramine, desmethyldoxepin, norfluvoxamine, norsertraline, and 1,3-chlorphenylpiperazine.

4. 10. The method of claim 1, comprising simultaneously detecting or determining the amounts of 30 antidepressants and antidepressant metabolites.

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

6. 6. The method of claim 5, wherein the one or more internal standards comprises a deuterated internal standard.

7. 7. The method of claim 6, wherein the deuterated internal standard is selected from the group consisting of 1,3-chlorphenylpiperazine-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.

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

9. The method of claim 8 , wherein the sample comprises urine.

10. The method of claim 1, capable of detecting levels of antidepressants and antidepressant metabolites in the range of 4 ng / mL to 5000 ng / mL (inclusive).

11. The method of claim 10, capable of detecting levels of antidepressants and antidepressant metabolites in the range of 25 ng / mL to 5000 ng / mL (inclusive).

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

13. The method of claim 12 , wherein the tandem mass spectrometry is performed by selected reaction monitoring, multiple reaction monitoring, precursor ion scanning, or product ion scanning.

14. The method of claim 12 , wherein the tandem mass spectrometry is performed by selected reaction monitoring.

15. 2. The method of claim 1, wherein the lower limit of quantitation of the antidepressant and antidepressant metabolites is 10 ng / mL.

16. 2. The method of claim 1, wherein the lower limit of quantitation of the antidepressant and antidepressant metabolites is 50 ng / mL.

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