Quantification of tamoxifen and its metabolites by mass spectrometry

Abstract

To provide methods for quantitation of tamoxifen and its metabolites in a sample by mass spectrometry, including tandem mass spectrometry.SOLUTION: In one aspect, methods are provided for determining the amount of tamoxifen and metabolites thereof in a sample in a single mass spectrometry assay, the methods comprising: (a) ionizing the tamoxifen and metabolites to produce one or more ions detectable by mass spectrometry; and (b) detecting the amount of the ions from the step by mass spectrometry; where the amount of the ions detected is related to the amount of each of tamoxifen and metabolites in the sample.SELECTED DRAWING: None

Description

Technical Field

[0005] , , , ,

[0001] Cross - reference to related patent applications This application claims priority to U.S. Provisional Application No. 61 / 992,214, filed on May 12, 2014, which is hereby incorporated by reference in its entirety.

Background Art

[0002] The following description of the background of the present invention is provided merely as an aid in understanding the present invention and is not admitted to describe or constitute prior art of the present invention.

[0003] Tamoxifen is the standard of care for hormone receptor - positive breast cancer patients after primary treatment. Tamoxifen therapy for 5 - 10 years reduces the risk of recurrence and death in these patients. Nevertheless, many patients do not complete their full treatment course, often due to unpleasant side effects of the drug. <000…21> [[ID=…]]

[0004] Tamoxifen is a prodrug that is converted to highly active endoxifen for full efficacy. The conversion of tamoxifen to endoxifen is via a metabolic pathway that depends on genetic mutations such as genetic mutations in CYP2D6 (2D6). 2D6 gene typing has been promoted to predict response and toxicity to tamoxifen, but there is debate about its direct relevance at the individual level. <…]]

Summary of the Invention

Problems to be Solved by the Invention

[0005] An effective method for predicting response to tamoxifen is needed.​​​​​​​​​ [Means for solving the problem]

[0006] The present invention relates to tamoxifen and the inclusion of tamoxifen in a sample obtained by mass spectrometry, including tandem mass spectrometry. This provides a method for quantifying metabolites.

[0007] In one embodiment, the amount of norendoxiphene in a sample was determined by mass spectrometry. (a) Ionize the norendoxiphene and obtain one detectable by mass spectrometry. (b) generating multiple norendoxphene ions, and (b) mass spectrometry to determine the process The method includes detecting the amount of norendoxphene ions (ion(s)) detected A method is provided in which the amount of ions (ion(s)) relates to the amount of norendoxifene in the sample. It will be done.

[0008] In one embodiment, tamoxifen and its metabolism in a sample are observed in a single mass spectrometry assay. A method for determining the amount of product, comprising (a) ionizing the tamoxifen and its metabolites (b) Mass The analysis includes detecting the amount of the ions (ion(s)) from the process, and the detected ions The amount of ions is related to the amounts of tamoxifen and its metabolites in the sample. A method is provided.

[0009] In some embodiments, the metabolite includes norendoxiphene. In the embodiment, the metabolite is endoxifen or N-desmethyl-4-hydrox Contains cytamoxifen. In some embodiments, the metabolite is 4'-hydroxy Contains tamoxifen. In some embodiments, the metabolite is 4-hydroxytamoxifen. Contains xifene. In some embodiments, the metabolite is N-desmethyl-4'- Contains hydroxytamoxifen. In some embodiments, the metabolite is N-des Contains methyltamoxifen. In some embodiments, the metabolite is norendoxy Cyfen, Endoxifen, 4'-Hydroxytamoxifen, 4-Hydroxytamoxifen Cyphen, N-desmethyl-4'-hydroxytamoxifen, and N-desmethyl- It contains metabolites selected from the group consisting of 4'-hydroxytamoxifen. In the embodiment, the metabolites are norendoxiphene, endoxiphene, and 4'-Hyd Roxitamoxifen, 4-hydroxytamoxifen, N-desmethyl-4'-hydroxy This includes any combination of xitamoxifen and N-desmethyltamoxifen. In several embodiments, the metabolites are norendoxiphene, endoxiphene, and 4' -Hydroxytamoxifen, 4-Hydroxytamoxifen, N-Desmethyl-4'- Hydroxytamoxifen and N-desmethyl-4'-hydroxytamoxifen Includes. In some embodiments, the metabolites are norendoxiphen, endoxif 4'-hydroxytamoxifen, 4'-hydroxytamoxifen, N-desmethicone Any combination of ru-4'-hydroxytamoxifen and N-desmethyltamoxifen Includes combinations.

[0010] In one embodiment, the method provided herein includes protein precipitation. Several embodiments In some embodiments, the method provided herein includes purification. includes filtration. In some embodiments, the purification includes liquid chromatography . In some embodiments, the liquid chromatography is high performance liquid chromatography - (HPLC).

[0011] In some embodiments, the methods provided herein include detecting the amount of an internal standard . In some embodiments, the internal standard is deuterated norendoxifen .

[0012] In some embodiments, ionization is by atmospheric pressure chemical ionization (APCI) . In some embodiments, the ionization is in positive ion mode

[0013] In some embodiments, ionization is by electrospray ionization (ESI) . In some embodiments, the ionization is in positive ion mode .

[0014] In some embodiments, the sample is a serum sample

[0015] In some embodiments, mass spectrometry is tandem mass spectrometry

[0016] In one aspect, a method for predicting tamoxifen response in a patient by determining the amount of tamoxifen or one or more tamoxifen metabolites is provided herein . In some embodiments, high amounts of one or more tamoxifen or tamoxifen metabolites indicate a positive response to tamoxifen in the patient . In some embodiments, the metabolites include norendoxifen . In some embodiments ​The metabolites are endoxifen or N-desmethyl-4-hydroxytamoxy Contains fen. In some embodiments, the metabolite is 4'-hydroxytamoxif Contains 4-hydroxytamoxifen. In some embodiments, the metabolite is 4-hydroxytamoxifen. Includes. In some embodiments, the metabolite is N-desmethyl-4'-hydroxy Contains tamoxifen. In some embodiments, the metabolite is N-desmethyltamoxifen. Contains xifene. In some embodiments, the metabolite is noendoxiphen. Endoxifen, 4'-hydroxytamoxifen, 4'-hydroxytamoxifen, N-desmethyl-4'-hydroxytamoxifen, and N-desmethyl-4'-Hyd Contains roxitamoxifen.

[0017] In some embodiments, the methods provided herein are measured by the limit of quantification (LOQ). It has a defined sensitivity. In some embodiments, the quantification method for tamoxifen is 5n It has a quantitative limit of g / mL or less. In some embodiments, the quantitative method of tamoxifen The method has a quantitative limit of 4 ng / mL or less. In some embodiments, tamoxifen The quantification method has a quantification limit of 3 ng / mL or less. In some embodiments, Tamo The method for quantifying xifene has a quantification limit of 3 ng / mL or less. Several embodiments Therefore, the quantitative analysis method for tamoxifen has a quantitative limit of 2 ng / mL or less. In this embodiment, the method for quantifying tamoxifen has a quantification limit of 1.5 ng / mL or less. do.

[0018] In some embodiments, the method for quantifying N-desmethyltamoxifen is 5 ng / m³ It has a limit of quantification of L or less. In some embodiments, N-desmethyltamoxifen The quantification method has a quantification limit of 4 ng / mL or less. In some embodiments, N-de The quantitative method for methyltamoxifen has a quantification limit of 3 ng / mL or less. In that embodiment, the method for quantifying N-desmethyltamoxifen is 3 ng / mL or less. It has a limit of quantification. In some embodiments, methods for quantifying N-desmethyltamoxifen The method has a quantitative limit of 2 ng / mL or less. In some embodiments, N-desmethyl The quantification method for tamoxifen has a quantification limit of 1.5 ng / mL or less.

[0019] In some embodiments, the method for quantifying 4'-hydroxytamoxifen is 5 ng / It has a limit of quantification of less than mL. In some embodiments, 4'-hydroxytamoxyfe The method for quantifying ng has a quantification limit of 4 ng / mL or less. In some embodiments, 4 The method for quantifying hydroxytamoxifen has a quantification limit of 3 ng / mL or less. In some embodiments, the method for quantifying 4'-hydroxytamoxifen is 3 ng / m³. It has a limit of quantification of L or less. In some embodiments, 4'-hydroxytamoxifen The quantification method has a quantification limit of 2 ng / mL or less. In some embodiments, 4' - The quantification method for hydroxytamoxifen has a quantification limit of 1.5 ng / mL or less. In some embodiments, the method for quantifying 4'-hydroxytamoxifen is 1 ng / It has a limit of quantification of less than mL. In some embodiments, 4'-hydroxytamoxyfe The quantification method has a quantification limit of 0.5 ng / mL or less. In some embodiments, The quantification method for 4'-hydroxytamoxifen has a quantification limit of 0.4 ng / mL or less. It has. In some embodiments, the method for quantifying 4'-hydroxytamoxifen is 0 It has a quantitative limit of 0.2 ng / mL or less.

[0020] In some embodiments, the method for quantifying 4-hydroxytamoxifen is 5 ng / m³. It has a limit of quantification of L or less. In some embodiments, 4-hydroxytamoxifen The quantification method has a quantification limit of 4 ng / mL or less. In some embodiments, 4-H The quantification method for droxitamoxifen has a quantification limit of 3 ng / mL or less. In that embodiment, the method for quantifying 4-hydroxytamoxifen is 3 ng / mL or less. It has a limit of quantification. In some embodiments, the quantification method of 4-hydroxytamoxifen The method has a quantitative limit of 2 ng / mL or less. In some embodiments, 4-hydroxy The quantitative method for tamoxifen has a limit of quantification of 1.5 ng / mL or less. In this embodiment, the method for quantifying 4-hydroxytamoxifen is to quantify it to a level of 1 ng / mL or less. It has limitations. In some embodiments, the method for quantifying 4-hydroxytamoxifen is It has a quantitative limit of 0.5 ng / mL or less. In some embodiments, 4-hydroxy The quantitative method for tamoxifen has a quantification limit of 0.4 ng / mL or less. In this embodiment, the method for quantifying 4-hydroxytamoxifen is to obtain a concentration of 0.2 ng / mL or less. The limit of quantification is reached.

[0021] In some embodiments, N-desmethyl-4-hydroxytamoxifen (Endo The quantification method for cyphen has a quantification limit of 5 ng / mL or less. Several embodiments So, how to quantify N-desmethyl-4-hydroxytamoxifen (endoxifen)? The method has a quantitative limit of 4 ng / mL or less. In some embodiments, N-desmethyl The method for quantifying -4-hydroxytamoxifen (endoxifen) is 3 ng / mL or less. It has the following limits of quantification. In some embodiments, N-desmethyl-4-hydroxytamol The quantitative method for xifene (endoxifene) has a quantitative limit of 3 ng / mL or less. In some embodiments, N-desmethyl-4-hydroxytamoxifen (Endo) The quantification method for cyphen has a quantification limit of 2 ng / mL or less. Several embodiments So, how to quantify N-desmethyl-4-hydroxytamoxifen (endoxifen)? The method has a quantitative limit of 1.5 ng / mL or less. In some embodiments, N-Desmet The quantitative method for til-4-hydroxytamoxifen (endoxifen) is 1 ng / m³. It has a limit of quantification of L or less. In some embodiments, N-desmethyl-4-hydroxy The quantitative method for tamoxifen (endoxifen) is a limit of quantification of 0.5 ng / mL or less. It has N-desmethyl-4-hydroxytamoxifen ( The quantification method for endoxifen has a quantification limit of 0.4 ng / mL or less.

[0022] In some embodiments, the quantification of N-desmethyl-4'-hydroxytamoxifen The method has a quantitative limit of 5 ng / mL or less. In some embodiments, N-desmethic The quantitative method for ru-4'-hydroxytamoxifen has a limit of quantification of 4 ng / mL or less. In some embodiments, the determination of N-desmethyl-4'-hydroxytamoxifen The quantification method has a quantitative limit of 3 ng / mL or less. In some embodiments, N-DES is used. The quantitative method for methyl-4'-hydroxytamoxifen has a quantification limit of 3 ng / mL or less. It has N-desmethyl-4'-hydroxytamoxifen. In some embodiments, it has N-desmethyl-4'-hydroxytamoxifen. The quantification method has a quantification limit of 2 ng / mL or less. In some embodiments, N- The quantification method for desmethyl-4'-hydroxytamoxifen is 1.5 ng / mL or less. It has a limit of quantification. In some embodiments, N-desmethyl-4'-hydroxytamoxy The method for quantifying cyphen has a quantification limit of 1 ng / mL or less. In some embodiments, The quantitative method for N-desmethyl-4'-hydroxytamoxifen is 0.5 ng / m³. It has a limit of quantification of L or less. In some embodiments, N-desmethyl-4'-hydrox The quantification method for cytamoxifen has a quantification limit of 0.4 ng / mL or less.

[0023] In some embodiments, the method for quantifying norendoxiphen is such that the result is 5 ng / mL or less. It has a limit of quantification. In some embodiments, the method for quantifying norendoxiphene is 4 It has a limit of quantification of ng / mL or less. In some embodiments, norendoxiphen The quantification method has a quantification limit of 3 ng / mL or less. In some embodiments, Norwegian The quantification method for doxifen has a quantification limit of 3 ng / mL or less. Several methods are available. In terms of morphology, the method for quantifying norendoxiphen has a quantitative limit of 2 ng / mL or less. In some embodiments, the method for quantifying norendoxiphene is such that the concentration is 1.5 ng / mL or less. It has the following limits of quantification. In some embodiments, the method for quantifying norendoxiphene is It has a quantitative limit of 1.2 ng / mL or less. In some embodiments, norendoxy The method for quantifying fen has a quantification limit of 1 ng / mL or less. In some embodiments, The method for quantifying norendoxiphen has a quantification limit of 0.5 ng / mL or less.

[0024] In some embodiments, the methods provided herein are measured by the limit of detection (LOD). It has a defined sensitivity. In some embodiments, the method for detecting tamoxifen is 5 ng It has a detection limit of less than / mL. In some embodiments, the method for detecting tamoxifen is It has a detection limit of 4 ng / mL or less. In some embodiments, tamoxifen testing The extraction method has a detection limit of 3 ng / mL or less. In some embodiments, tamoxif The detection method for fertilizer has a detection limit of 3 ng / mL or less. In some embodiments, The method for detecting moxifen has a detection limit of 2 ng / mL or less. Several embodiments Therefore, the detection method for tamoxifen has a detection limit of 1.5 ng / mL or less. In that embodiment, the method for detecting tamoxifen has a detection limit of 1 ng / mL or less. In some embodiments, the method for detecting tamoxifen is to detect levels of 0.6 ng / mL or less. Reach the limit.

[0025] In some embodiments, the method for detecting N-desmethyltamoxifen is 5 ng / mL. The following detection limits are observed. In some embodiments, the detection of N-desmethyltamoxifen The extraction method has a detection limit of 4 ng / mL or less. In some embodiments, N-desme The detection method for tiltamoxifen has a detection limit of 3 ng / mL or less. In terms of application methods, the detection limit for N-desmethyltamoxifen is 3 ng / mL or less. It has. In some embodiments, the method for detecting N-desmethyltamoxifen is 2n It has a detection limit of g / mL or less. In some embodiments, N-desmethyltamoxif The detection method for ethanol has a detection limit of 1.5 ng / mL or less. In some embodiments, The detection method for N-desmethyltamoxifen has a detection limit of 1 ng / mL or less. In some embodiments, the detection method for N-desmethyltamoxifen is 0.6 ng / m³ It has a detection limit of L or less.

[0026] In some embodiments, the method for detecting 4'-hydroxytamoxifen is 5 ng / m³ It has a detection limit of L or less. In some embodiments, 4'-hydroxytamoxifen The detection method has a detection limit of 4 ng / mL or less. In some embodiments, 4'- The detection method for hydroxytamoxifen has a detection limit of 3 ng / mL or less. In that embodiment, the method for detecting 4'-hydroxytamoxifen is 3 ng / mL or less. It has a detection limit. In some embodiments, the detection method for 4'-hydroxytamoxifen The method has a detection limit of 2 ng / mL or less. In some embodiments, 4'-hydroxy The detection method for cytamoxifen has a detection limit of 1.5 ng / mL or less. In this embodiment, the method for detecting 4'-hydroxytamoxifen is to detect levels of 1 ng / mL or less. It has limitations. In some embodiments, the detection method for 4'-hydroxytamoxifen is It has a detection limit of 0.5 ng / mL or less. In some embodiments, 4'-hydroxy The detection method for cytamoxifen has a detection limit of 0.4 ng / mL or less. In this embodiment, the method for detecting 4'-hydroxytamoxifen is 0.2 ng / mL or less. It has a detection limit. In some embodiments, the detection method for 4'-hydroxytamoxifen The method has a detection limit of 0.1 ng / mL or less.

[0027] In some embodiments, the method for detecting 4-hydroxytamoxifen is 5 ng / mL. The following detection limits are observed. In some embodiments, the detection of 4-hydroxytamoxifen The extraction method has a detection limit of 4 ng / mL or less. In some embodiments, 4-hydro The detection method for xitamoxifen has a detection limit of 3 ng / mL or less. In terms of application methods, the detection limit for 4-hydroxytamoxifen is 3 ng / mL or less. It has. In some embodiments, the method for detecting 4-hydroxytamoxifen is 2n It has a detection limit of g / mL or less. In some embodiments, 4-hydroxytamoxif The detection method for ethanol has a detection limit of 1.5 ng / mL or less. In some embodiments, The detection method for 4-hydroxytamoxifen has a detection limit of 1 ng / mL or less. In some embodiments, the method for detecting 4-hydroxytamoxifen is 0.5 ng / m³ It has a detection limit of L or less. In some embodiments, 4-hydroxytamoxifen The detection method has a detection limit of 0.4 ng / mL or less. In some embodiments, 4- The detection method for hydroxytamoxifen has a detection limit of 0.2 ng / mL or less. In some embodiments, the method for detecting 4-hydroxytamoxifen is 0.1 ng / mL It has the following detection limits.

[0028] In some embodiments, N-desmethyl-4-hydroxytamoxifen (Endo The detection method for cyphen has a detection limit of 5 ng / mL or less. In some embodiments, The detection method for N-desmethyl-4-hydroxytamoxifen (endoxifen) is... It has a detection limit of 4 ng / mL or less. In some embodiments, N-desmethyl-4 - The detection method for hydroxytamoxifen (endoxifen) is a test for levels of 3 ng / mL or less. It has an output limit. In some embodiments, N-desmethyl-4-hydroxytamoxif The detection method for endoxifen has a detection limit of 3 ng / mL or less. In that embodiment, N-desmethyl-4-hydroxytamoxifen (endoxifen) The detection method of ) has a detection limit of 2 ng / mL or less. In some embodiments, N- The detection method for desmethyl-4-hydroxytamoxifen (endoxifen) is 1.5 It has a detection limit of ng / mL or less. In some embodiments, N-desmethyl-4-H The detection method for droxitamoxifen (endoxifen) has a detection limit of 1 ng / mL or less. It has a field. In some embodiments, N-desmethyl-4-hydroxytamoxifen The detection method for (endoxifen) has a detection limit of 0.5 ng / mL or less. In that embodiment, N-desmethyl-4-hydroxytamoxifen (endoxifen) The detection method of ) has a detection limit of 0.4 ng / mL or less. In some embodiments, The detection method for N-desmethyl-4-hydroxytamoxifen (endoxifen) is: It has a detection limit of 0.2 ng / mL or less. In some embodiments, N-desmethyl-4 - The detection method for hydroxytamoxifen (endoxifen) is 0.15 ng / mL or less. It has the following detection limits.

[0029] In some embodiments, the detection method for N-desmethyl-4'-hydroxytamoxifen The method has a detection limit of 5 ng / mL or less. In some embodiments, N-desmethyl The detection method for -4'-hydroxytamoxifen has a detection limit of 4 ng / mL or less. In some embodiments, methods for detecting N-desmethyl-4'-hydroxytamoxifen The method has a detection limit of 3 ng / mL or less. In some embodiments, N-desmethyl The detection method for -4'-hydroxytamoxifen has a detection limit of 3 ng / mL or less. In some embodiments, methods for detecting N-desmethyl-4'-hydroxytamoxifen The method has a detection limit of 2 ng / mL or less. In some embodiments, N-desmethyl The detection method for -4'-hydroxytamoxifen has a detection limit of 1.5 ng / mL or less. In some embodiments, the test of N-desmethyl-4'-hydroxytamoxifen The extraction method has a detection limit of 1 ng / mL or less. In some embodiments, N-desme The detection method for til-4'-hydroxytamoxifen has a detection limit of 0.5 ng / mL or less. It has N-desmethyl-4'-hydroxytamoxifen. In some embodiments, it has N-desmethyl-4'-hydroxytamoxifen. The detection method has a detection limit of 0.4 ng / mL or less.

[0030] In some embodiments, the method for detecting norendoxiphen is such that the detection level is 5 ng / mL or less. It has an output limit. In some embodiments, the method for detecting norendoxiphene is 4ng It has a detection limit of less than / mL. In some embodiments, the detection of norendoxiphen is performed. The method has a detection limit of 3 ng / mL or less. In some embodiments, norendokey The cyphen detection method has a detection limit of 3 ng / mL or less. In some embodiments, The detection method for norendoxiphen has a detection limit of 2 ng / mL or less. In this embodiment, the method for detecting norendoxiphen has a detection limit of 1.5 ng / mL or less. It has. In some embodiments, the method for detecting norendoxiphene is 1.2 ng / It has a detection limit of less than mL. In some embodiments, the detection method of norendoxiphen The method has a detection limit of 1 ng / mL or less. In some embodiments, norendoxy The fen detection method has a detection limit of 0.5 ng / mL or less.

[0031] As used herein, the terms “purify” or “to purify” refer to the intended portion. This does not mean removing all material from the sample other than the analytes. Instead, The preparation involves applying the target analyte to one or more components of the sample. This refers to a procedure for enriching the quantity. Purification, as used herein, means removing from all other substances. Isolation of the analyte is not required. In a preferred embodiment, a purification step or procedure is used. One or more interfering substances, for example, one that interferes with the operation of the equipment used in the method Alternatively, remove multiple substances or substances that may interfere with the detection of analyte ions by mass spectrometry. It is possible.

[0032] When used in this specification, "about" refers to quantitative measurements that do not involve the measurement of ion mass. The term "plus or minus 10%" refers to a value that is either plus or minus 10% of the stated value.

[0033] As used herein, the term “substantially all” means more than 50% Preferably above 60%, more preferably above 70%, more preferably above 80% It refers to any percentage that is rotating, and more preferably, any percentage greater than 90%.

[0034] As used herein, the term “sample” means any sample that may contain the analyte of interest. Refers to a sample. As used herein, the term “body fluid or tissue” refers to the body of an individual. It means any liquid or tissue that can be isolated from body fluids or tissue. For example, "body fluids or tissue." "This may include blood, plasma, serum, bile, saliva, urine, tears, sweat, etc. Solid tissue is analyzed. If possible, the process is designed to release a liquid fraction that may contain any analytes present in the tissue. Processing may be carried out. Subsequently, liquid fractionation may be performed by the method described herein.

[0035] As used herein, the term “size separation technique” refers to the weight and shape of molecules. Based on one or more of the following, it is possible to isolate at least one species from the test sample. This refers to any technique (physical or chemical). Examples of such techniques include filtration and chromatography. Examples include, but are not limited to, certain aspects of tography and mass spectrometry. .

[0036] As used herein, the term "chromatography" refers to the process of transforming a liquid or gas into a liquid or gas. More than transported chemical mixtures, they are near the fixed liquid or solid phase, on top of it, / or, as it flows through it, it separates into its constituent components as a result of differential distribution of chemical components. It refers to the process of doing something.

[0037] As used herein, the term "liquid chromatography" or "LC" is used in this specification. , when a fluid permeates uniformly into a column of fine material or into a capillary passage, the fluid solution This refers to the process of selective delay of one or more components of the fluid. Because it moves relative to ase(s), the delay is between one or more stationary phases and bulk fluid (i.e., This is caused by the distribution of the components of the mixture between the mobile phase and the surrounding environment. Reverse-phase liquid chromatography (RPLC), high-performance liquid chromatography (HPLC) Examples include ) and highly turbulent liquid chromatography (HTLC).

[0038] As used herein, "high-performance liquid chromatography" or "HPLC" refers to The term refers to the process of moving the mobile phase under pressure through a stationary phase, usually a densely packed column. This refers to liquid chromatography that increases the degree of separation.

[0039] As used herein, the terms “mass spectrometry” or “MS” refer to the mass of those masses. MS refers to an analytical technique for identifying compounds. MS uses ions at their m / z to filter them. MS techniques generally refer to methods of detection and measurement of compounds. (1) to convert the material into a charged species (e.g., an ion), and (2) to determine the molecular weight of the ion. This includes extracting the compounds and calculating their m / z. The compounds are then subjected to any appropriate means. It can be ionized and detected. A "mass spectrometer" generally consists of an ionizer and an ion detector. Includes. Generally, one or more molecules of interest are ionized, and then the ions are divided by mass. When introduced into the analytical instrument, the ions, due to the combination of magnetic and electric fields, have a mass ("m"). ) and trace the space which depends on the charge ("z"). For example, "Mass Spectr U.S. publication titled "Methodology From Surfaces" 6,204,50 No. 0; “Methods and Apparatus for Tandem Mas Patent Nos. 6,107,623, titled "s Spectrometry"; "DNA D "Iagnostics Based on Mass Spectrometry" The same publication as the title, No. 6,268,144; "Surface-Enhanced Photo labile Attachment And Release For Desorp The sixth volume of the same publication is titled "Synopsis and Detection of Analytes". ,124,137;Wright et al.,Prostate Cancer and Prostatic Diseases 2:264-76(1999); and Merchant and Weinberger,Electrophoresis See 21:1164-67(2000).

[0040] As used herein, the term "operating in positive ion mode" means This refers to a mass spectrometry method in which positive ions are detected. Similarly, it refers to a method that operates in negative ion mode. The term "detecting anions" refers to mass spectrometry, a method of detecting anions.

[0041] As used herein, the terms "ionization" or "to ionize" are defined as follows: A pruning process that generates analyte ions with a net charge equal to one or more electron units. This refers to a ion. A positive ion is one that has a net positive charge from one or more electron units. An anion is a substance that has a net negative charge from one or more electron units. .

[0042] As used herein, the terms “electron ionization” or “EI” refer to the gas phase or This refers to the way the target analyte in the vapor phase interacts with the electron flow. This involves collisions between electrons and the analyte. This generates analyte ions, which can then be subjected to mass spectrometry.

[0043] As used herein, the terms "chemical ionization" or "CT" refer to reagent gas When a substance (for example, ammonia) is subjected to electron collisions, the interaction between the reagent gas ions and the analyte molecules occurs. This refers to a method by which analyte ions are formed through a reaction.

[0044] As used herein, the terms “fast atomic impact” or “FAB” refer to high-energy atomic impacts. A beam of energy atoms (often Xe or Ar) collides with a non-volatile sample, and the sample This refers to a method of desorption and ionization of molecules contained within. The test sample is glycerol. Thioglycerol, m-nitrobenzyl alcohol, 18-crown-6-crown A Tel, 2-nitrophenyloctyl ether, sulfolane, diethanolamine, and It is dissolved in a viscous liquid matrix such as triethanolamine. The selection of a suitable matrix is ​​an empirical process.

[0045] As used herein, "matrix-assisted laser desorption / ionization" or "MA" The term "LDI" refers to the process of exposing a non-volatile sample to laser irradiation, which then photoionizes it. Various ionization pathways, including protonation, deprotonation, and cluster decay, are used to ionize the sample. This refers to a method of desorption and ionization of the analyte. In relation to MALDI, the sample is energy —When mixed with the absorption matrix, it promotes the detachment of analyte molecules.

[0046] As used herein, "surface-enhanced laser desorption / ionization" or "SELDI" The term refers to the process of exposing a non-volatile sample to laser irradiation, which then leads to photoionization, prototyping. Various ionization pathways, including deprotonation, deprotonation, and cluster decay, can be used to analyze the analyte in a sample. This refers to another method of desorption and ionization. With respect to SELDI, the sample is usually one of the desired components. It is bonded to a surface that preferentially holds one or more analytes. Similar to MALDI, this The process may also use energy-absorbing materials to promote ionization.

[0047] As used herein, "electrospray ionization" or "ESI" is used. The term refers to passing a solution along a short capillary tube to a high positive or negative terminal. This refers to a method in which a negative potential is applied. The solution that reaches the end of the tube is very different from the solution in the solvent vapor. It is vaporized (sprayed) into a jet or spray of tiny droplets. This mist of droplets is vapor It flows through the heating chamber, is slightly heated, prevents condensation, and evaporates the solvent. As the droplets become smaller, the natural repulsion between similar charges causes ions and The electron surface charge density increases until it reaches a point where neutral molecules are released.

[0048] As used herein, the term "atmospheric pressure chemical ionization" or "APCI" is used in this specification. While it refers to a mass spectrometry method similar to ESI, APCI refers to the ion formation that occurs in the plasma at atmospheric pressure. Ions are generated by a molecular reaction. The plasma is between the spray capillary and the counter electrode. It is maintained by discharge. Next, the ions are typically supplied by a set of differential pump skimmer stages (di Extracted into a mass spectrometer using (specially pumped skimmer stages). Drying and The removal of the solvent may be improved by using a counterflow of preheated N2 gas. (Gas phase in APCI) Ionization may be more effective than ESI for analyzing less polar species.

[0049] The terms "atmospheric pressure photoionization" or "APPI" are used herein, The mechanism for the photoionization of molecule M is that it is a photon absorption and electron emission molecule, forming molecule M+ This refers to a form of mass spectrometry. The photon energy is usually slightly above the ionization potential. Molecular ions are less susceptible to dissociation. In many cases, the sample can be processed without chromatography. It may be possible to analyze this, thus saving significant time and money. In the presence of water vapor or a protic solvent, molecular ions can extract H to form MH+. This tends to occur when M has a high proton affinity. This is because M+ and Since the sum of MH+ is constant, it does not affect quantitative accuracy. Drugs in protic solvents The compound is usually observed as MH+, whereas naphthalene or testosterone... Most nonpolar compounds typically form M+. Robb, DB, Covey, TR and Bruins, AP (2000): For example, Robb et al., At mospheric pressure photoionization: An io nization method for liquid chromatograph y-mass spectrometry.Anal.Chem.72(15):365 See 3-3659.

[0050] As used herein, the terms "inductively coupled plasma" or "ICP" are mostly used in this context. At a temperature high enough to atomize and ionize most elements, the sample is partially ionized into a gas. This refers to a method of causing interaction between two things.

[0051] As used herein, the term "field desorption" refers to the process by which a non-volatile test sample is ionized. This refers to a method of generating analyte ions by placing them on a surface and using a strong electric field.

[0052] As used herein, the term “desorption” means the removal of analytes from a surface and / Alternatively, it refers to the transfer of the analyte into the gas phase.

[0053] As used herein, the term “Limit of Quantitative Quotient” or “LOQ” means that the measurement is quantitative. This refers to the point that is meaningful in terms of accuracy. The analyte response in this LOQ is accurate with a 20% precision and accurate It is identifiable with a degree of accuracy of 80% to 120%, is discrete, and is reproducible.

[0054] In certain preferred embodiments of the methods disclosed herein, mass spectrometry is used to obtain positive It is carried out in ion mode. A particular particularly preferred implementation of the method disclosed herein Morphologically, mass spectrometry is performed using ESI.

[0055] In another preferred embodiment, a separately detectable internal standard is supplied to the sample.

[0056] In one embodiment, the method encompasses a combination of LC and mass spectrometry. Another preferred embodiment In terms of form, the mass spectrometry is tandem mass spectrometry (MS / MS).

[0057] The above summary of the present invention is not limiting, and other features and advantages of the present invention are described below. This is evident from the detailed description of the invention and the claims. [Modes for carrying out the invention]

[0058] Regarding a method for quantitatively measuring tamoxifen and / or its metabolites in patient samples This will be described. This quantitative measurement is achieved using the LC-MS / MS technique. Prior to using C-MS / MS, the sample is prepared using one or more of the following techniques. It is possible. The first purification of tamoxifen and / or its metabolites in the sample is protein This can be carried out by filtration, chromatography, or other methods.

[0059] Any suitable size separation technique can be used, but in the example below, the first and second sizes Both separation techniques involve filtration through a molecular weight cutoff filter. Similarly, the following procedures are performed. As will be discussed in the example, the same filter can be applied to both the first and second size separations. A molecular weight cutoff that has an appropriate molecular weight cutoff so that it can be used by It is possible to select a filter.

[0060] LC, most preferably HPLC, can be used alone or in combination with other purification methods. This can be used to purify the analyte. This purification can be combined with MS / MS. This provides an assay system for quantifying selected analyses in test samples. Subsequently, The amount of tamoxifen in the original test sample is determined using the amount of the selected analyte in the sample. The quantification methods provided herein offer enhanced specificity and address methodological issues (antibody interference). It is less susceptible to (such as)

[0061] A suitable sample may include any test sample that may contain the analyte of interest. In a preferred embodiment, the sample is a biological sample, i.e., an animal, cell culture, organ culture, etc. The sample is obtained from any biological source such as the above. In a particular preferred embodiment, The samples are obtained from mammals such as dogs, cats, and horses. Particularly preferred mammals are Primates, most preferably humans. Particularly preferred samples include blood, plasma, serum, and urine. Examples include saliva, tears, cerebrospinal fluid, or other bodily fluid or tissue samples. Such samples are, for example, For example, the patient, that is, the living person with a clinical background regarding the diagnosis, prognosis, or treatment of a disease or condition. It can be obtained from humans. The test sample is preferably obtained from a patient, for example, serum or plasma. .

[0062] Sample preparation for mass spectrometry The sample is processed or purified to obtain a preparation suitable for analysis by mass spectrometry. This may also involve chromatography, such as liquid chromatography. Furthermore, it often includes additional purification steps performed prior to chromatography. However, this is also acceptable. The various procedures depend on the type of sample or the type of chromatography. It can be used for various purposes. Examples include filtration, centrifugation, and combinations thereof.

[0063] Filtration is used for filtration of test samples for chromatography, especially biological test samples such as serum or plasma. This is one preferred method for preparing the material. Such filtration is more effective than the filter's cutoff. Species with a large molecular weight are separated from species with a molecular weight smaller than the filter's cutoff. This is performed by filtering the test sample through a molecular weight cutoff filter to separate the molecules. The test sample remaining on the filter after complete (or nearly complete) filtration is then processed. It is substantially free of potentially interfering species with molecular weights smaller than Luther's cutoff. .

[0064] Various methods are described, including the use of HPLC for sample purification prior to mass spectrometry analysis. For example, Taylor et al., Therapeutic Drug Mon itoring 22:608-12(2000)(Manual sedimentation of blood samples, followed by manual C1 8. Solid-phase extraction, injection into HPLC for chromatography on a C18 analytical column. and MS / MS analysis; and Salm et al., Clin. Therapeu tics 22 Supl.B:B71-B85(2000) (Manual sedimentation of blood samples, continuation) Manual C18 solid-phase extraction, HPLC for chromatography on a C18 analysis column. See injection into and MS / MS analysis). Those skilled in the art will know how to use in this method. You may choose the HPLC instrument and column that you are using. Chromatography columns are typically The medium (i.e., packing material) is included to facilitate the separation (i.e., fractionation) of the chemical parts. The material may contain fine particles. The particles may contain various chemical components to facilitate the separation of the chemical parts. It includes a bonding surface that interacts with the molecules. One suitable bonding surface is a sparse bonding surface such as an alkyl bonding surface. It is an aqueous bonded surface. The alkyl bonded surface is a C-4, C-8, or C-18 bonded alkyl The chromatography column may contain a C-8 bond group, preferably a C-8 bond group. An inlet port for collecting and an outlet port for discharging eluted material, including the fractionated sample. Includes "t".

[0065] In a particular embodiment, the target analyte is reversibly held by the column packing material. Alternatively, the sample can be subjected to column spectroscopy under conditions where one or more other materials are not retained. The analyte can be further purified. In these embodiments, the analyte of interest is retained by the column. The first mobile phase condition can be used, and then the second mobile phase condition is not maintained. Once the material has been washed away, the remaining material is used to remove it from the column. This can be done. Alternatively, the target analyte may be different from one or more other materials. By running the sample through a column under mobile phase conditions that allow for rapid elution, the analyte can be purified. The procedure involves applying one or more of the following treatments to one or more components of the sample: The quantity of the numerical analytes may be enriched.

[0066] In one embodiment, the sample to be analyzed is placed on the column at the inlet port and then subjected to a solvent or The analyte is eluted in the solvent mixture and discharged through the outlet port. Various solvent modes are available for the target analyte. Regarding elution, one option may be chosen. For example, liquid chromatography can be used with a gradient motion. Using either constant composition mode or polytyptic (i.e., mixed) mode It may be carried out in this manner. In a preferred embodiment, HPLC is performed using HPLC Gray as the mobile phase. Using 0.2% formic acid in ultrapure water and 0.2% formic acid in 100% methanol, C8 This is performed using a solid-phase analytical HPLC system.

[0067] Numerous column packing options are available for chromatographic separation of samples, and suitable separation options are available. The selection of a rotol depends on the characteristics of the sample, the target analyte, the presence and characteristics of interfering substances, etc. It is an empirical process that depends on polarity and ion exchange (positive). (Both ions and anions), hydrophobic interactions, phenyl, C-2, C-8, C-18 Examples include, but are not limited to, polar coatings of porous polymer columns. .

[0068] In one embodiment, the HPLC column has a median particle size of 5 μm (nominal) and median It has a C8 solid phase with a particle pore size of 100 Å. In a preferred embodiment, the column dimensions It has a diameter of 1.0 mm and a length of 50 mm (Phenomenex Corp. Luna 5μ C8(2) 100Å New Column 50x1.0mm, Pheno (menex Cat.No.00B-4249-A0 or equivalent).

[0069] During chromatography, the separation of materials involves the selection of the eluent (also known as the "mobile phase"). This is achieved through the selection of gradient elution, gradient conditions, temperature, and other variables.

[0070] Detection and quantification by mass spectrometry In various embodiments, the analyte can be ionized by any method known to those skilled in the art. Mass spectrometry is performed using a mass spectrometer, which ionizes the fractionated sample. Includes an ion source for generating charged molecules for further analysis. Used in various MS techniques. As ion sources, electron ionization, chemical ionization, and electrospray ionization (E SI), photon ionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoion Chemical ionization (APPI), fast atomic bombardment (FAB) / liquid secondary ionization (LSIMS), matrix Laser-assisted desorption / ionization (MALDI), electrolytic ionization, electrolytic desorption, thermospress - Plasma spray ionization, surface-enhanced laser desorption ionization (SELDI), induction ionization Examples include, but are not limited to, integrated plasma (ICP) and particle beam ionization. The selection of the ionization method depends on the analyte to be measured, the type of sample, the type of detector, and the positive Those skilled in the art will understand that this can be determined based on the selection of positive versus negative modes, etc. cormorant.

[0071] In a preferred embodiment, the analyte is electrosprayed to create analyte precursor ions. It is ionized by ionization (ESI). In related preferred embodiments, the analyte precursor Body ions exist in a gaseous state, and the inert collisional gas is argon.

[0072] After the sample is ionized, the positively charged ions created are analyzed, and m / The z-axis can be determined. Suitable analyzers for determining m / z include quadrupole analyzers and ion transistors. Examples include hop analyzers and time-of-flight analyzers. Ions are detected in several detection modes. One of them can be used for detection. For example, only selected ions can be detected using selective ion monitoring. Detection may be performed using scanning mode (SIM), or multiple ions may be detected using scanning mode For example, multiple reaction monitoring (MRM) or selective reaction monitoring (SRM) It may be detected using a method. In a preferred embodiment, the ions are detected using an SRM. It can be done.

[0073] Preferably, the m / z is determined using a quadrupole instrument. In an "ion trap" device, ions in a vibrating radio frequency electromagnetic field are trapped by a DC current applied between electrodes. It receives a force proportional to the position, the amplitude of the RF signal, and m / z. The voltage and amplitude are specific Only ions with m / z can be selected to propagate along the length of the quadrupole, while others All of the ions are deflected. Therefore, the quadrupole instrument is deflected with respect to the ions injected into the instrument. It can function as both a "mass filter" and a "mass detector".

[0074] By using "tandem sample analysis" or "MS / MS," the resolution of MS techniques can be improved. It may be increased. In this technique, the precursor ion (also called the parent ion) generated from the target molecule The precursor ions are then filtered by an MS instrument, and then fragmented into one. Alternatively, it can produce multiple fragment ions (also called daughter ions or product ions). Next, one or more fragment ions are analyzed in a second MS procedure. Precursor ions Through careful selection, only ions produced by a specific analyte are filtered into the fragmentation chamber. It moves to the next location, where fragment ions are generated through collisions with atoms of the inert gas. Both whole and fragment ions are produced in a reproducible manner under a given set of ionization / fragmentation conditions. Therefore, MS / MS techniques can provide an extremely powerful analytical tool. For example, filtration / A combination of fragmentation may be used to eliminate interfering substances, such as in complex samples like biological samples. This may be particularly useful.

[0075] Furthermore, matrix-assisted laser desorption and ionization ("MA") linked to a time-of-flight analyzer. Recent advances in technology, such as LDI-TOF, allow for very short ion pulses. This enables analysis of analytes at the femtomole level. This is achieved using a time-of-flight analyzer and tandem MS. Mass spectrometers that combine these are also well known to those skilled in the art. Furthermore, the multiple mass spectrometry process is They may be combined in a way known as "MS / MS," such as MS / MS / TOF or MALD. Various methods such as I / MS / MS / TOF, or SELDI / MS / MS / TOF mass spectrometry Other combinations may also be used.

[0076] Mass spectrometers typically allow the user to perform an ion scan, i.e., within a predetermined range (e.g., 400-1000). Provides the relative abundance of each ion using a specific m / z over a range of 600 amu. The assay results, i.e., the mass spectrum, can be obtained in the original sample by numerous methods known to those skilled in the art. It can be related to the amount of analyte. For example, if the sampling and analytical parameters are carefully considered Because it is controlled by gravity, the relative abundance of a given ion is such that its relative abundance is an absolute value of the original molecule. It can be compared with a table for converting to logarithmic scales. Alternatively, molecular standards can be used together with the sample. Furthermore, standard curves can be constructed based on the ions generated from these standard materials. Using such standard curves, the relative abundance of a given ion is converted to the absolute amount of the original molecule. Obtain. In a particular preferred embodiment, an internal standard is used to calculate the amount of tamoxifen. Create a standard curve for outputting. The method for creating and using such a standard curve is the technique. It is well known in the field, and those skilled in the art can select an appropriate internal standard. Numerous other methods for relating a quantity to the quantity of the original molecule are well known to those skilled in the art.

[0077] One or more steps of the method may be carried out using automated machinery. In certain embodiments, one or more purification steps are carried out online, which is preferable. Furthermore, the LC purification and mass spectrometry processes can all be performed online.

[0078] In certain embodiments, techniques such as MS / MS are used for further fragmentation. The precursor ions are isolated. In these embodiments, collision-activated dissociation (CAD) is used. Fragment ions may be generated for further detection. In CAD, the precursor ions are Energy is gained through collisions with inert gases, followed by a process called "single-molecule decomposition." Fragmentation occurs due to the ions. Sufficient energy must be deposited on the precursor ions. As a result, certain bonds within the ion are broken due to the increase in vibrational energy. In an alternative embodiment, electron transfer dissociation (ETD) is used to generate fragment ions. It may be done. In ETD, radical anions are used to transfer electrons to a large number of stacked charged peptides. Transfer to cytoplasm or protein cations, and random cleavage along the peptide backbone. To drip.

[0079] In a particularly preferred embodiment, the analyte is detected using LC-MS / MS as described below. and / or quantified. The analyte-rich sample prepared as described above is subjected to LC. The liquid solvent flow from the chromatography column is heated by the LC-MS / MS analyzer. The solvent / analyte mixture proceeds to the riser interface, where it is converted into vapor by a heating tube at the interface. The analyte contained in the spray solvent is subjected to a high voltage applied to the spray solvent / analyte mixture. The surface is ionized by a corona discharge needle. The ions pass through the opening of the instrument and into the first four We move on to the quadrupoles. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters, and they Based on the m / z, ion selection (i.e., "precursor" and "fragment" ions) is possible. Yes. Quadrupole 2 (Q2) is a collision cell where ions undergo fragmentation. Q1 is, The precursor ion is selected using its m / z. The selected precursor ion is then subjected to collisions. While it becomes possible to pass through the numbar (Q2), any other ion with any other m / z, It collides with the side of Q1 and is eliminated. The precursor ion that proceeds to Q2 is neutral argon gas. It may undergo fragmentation via collision-activated dissociation (CAD) due to collisions with its offspring. Alternatively, Q2 If the precursor ions that proceed to are multiple stacked charged cations, then they are electron transfer solutions. It may undergo fragmentation during ETD. The resulting fragment ions pass through to Q3, where Then, the selected fragment ions are collected, while the other ions are excluded.

[0080] Using standard methods well known in the art, a person skilled in the art can use the following to make the selection in Q3. It is possible to identify one or more fragment ions of a specific precursor ion. Specific fragment ions are formed in considerable quantities by other molecules with similar molecular structures. In contrast, nonspecific fragment ions are formed by molecules other than the desired analyte. Appropriate specific fragment ions are formed by the selected analyte. It also determines whether the ON is formed by other molecules with similar structures or characteristics. This can be identified by testing various molecular standards. Preferably, the analyte At least one fragment ion specific to an ion having an m / z corresponding to the m / z of ON is It is identified.

[0081] As ions collide with the detector, they are converted into digital signals. This generates a delay. The acquired data is relayed to the computer, and the computer processes it over a unit of time. Plot the ion count per unit area. The area under the peak corresponding to a specific ion, or The amplitude of a peak is measured, and the area or amplitude is correlated with the amount of the target analyte. In certain embodiments, the curve relating to fragment ions (ion(s)) and / or precursor ions is shown below The area or peak amplitude is measured, and the amount of analyte is determined using m / z. Thus, the relative abundance of a given ion is the P of one or more ions in the internal molecular standard. Using a calibration standard curve based on the LC, it can be converted to the absolute amount of the original analyte. Subsequently, LC - The absolute amount of analytes detected by MS / MS is the absolute amount of analytes present in the original test sample. It can be converted to logarithmic scale.

[0082] In some embodiments, the methods provided herein are measured by the limit of quantification (LOQ). It has a defined sensitivity. In some embodiments, the quantification method for tamoxifen is 5n It has a quantitative limit of g / mL or less. In some embodiments, the quantitative method of tamoxifen The method has a quantitative limit of 4 ng / mL or less. In some embodiments, tamoxifen The quantification method has a quantification limit of 3 ng / mL or less. In some embodiments, Tamo The method for quantifying xifene has a quantification limit of 3 ng / mL or less. Several embodiments Therefore, the quantitative analysis method for tamoxifen has a quantitative limit of 2 ng / mL or less. In this embodiment, the method for quantifying tamoxifen has a quantification limit of 1.5 ng / mL or less. do.

[0083] In some embodiments, the method for quantifying N-desmethyltamoxifen is 5 ng / m³ It has a limit of quantification of L or less. In some embodiments, N-desmethyltamoxifen The quantification method has a quantification limit of 4 ng / mL or less. In some embodiments, N-de The quantitative method for methyltamoxifen has a quantification limit of 3 ng / mL or less. In that embodiment, the method for quantifying N-desmethyltamoxifen is 3 ng / mL or less. It has a limit of quantification. In some embodiments, methods for quantifying N-desmethyltamoxifen The method has a quantitative limit of 2 ng / mL or less. In some embodiments, N-desmethyl The quantification method for tamoxifen has a quantification limit of 1.5 ng / mL or less.

[0084] In some embodiments, the method for quantifying 4'-hydroxytamoxifen is 5 ng / It has a limit of quantification of less than mL. In some embodiments, 4'-hydroxytamoxyfe The method for quantifying ng has a quantification limit of 4 ng / mL or less. In some embodiments, 4 The method for quantifying hydroxytamoxifen has a quantification limit of 3 ng / mL or less. In some embodiments, the method for quantifying 4'-hydroxytamoxifen is 3 ng / m³. It has a limit of quantification of L or less. In some embodiments, 4'-hydroxytamoxifen The quantification method has a quantification limit of 2 ng / mL or less. In some embodiments, 4' - The quantification method for hydroxytamoxifen has a quantification limit of 1.5 ng / mL or less. In some embodiments, the method for quantifying 4'-hydroxytamoxifen is 1 ng / It has a limit of quantification of less than mL. In some embodiments, 4'-hydroxytamoxyfe The quantification method has a quantification limit of 0.5 ng / mL or less. In some embodiments, The quantification method for 4'-hydroxytamoxifen has a quantification limit of 0.4 ng / mL or less. It has. In some embodiments, the method for quantifying 4'-hydroxytamoxifen is 0 It has a quantitative limit of 0.2 ng / mL or less.

[0085] In some embodiments, the method for quantifying 4-hydroxytamoxifen is 5 ng / m³. It has a limit of quantification of L or less. In some embodiments, 4-hydroxytamoxifen The quantification method has a quantification limit of 4 ng / mL or less. In some embodiments, 4-H The quantification method for droxitamoxifen has a quantification limit of 3 ng / mL or less. In that embodiment, the method for quantifying 4-hydroxytamoxifen is 3 ng / mL or less. It has a limit of quantification. In some embodiments, the quantification method of 4-hydroxytamoxifen The method has a quantitative limit of 2 ng / mL or less. In some embodiments, 4-hydroxy The quantitative method for tamoxifen has a limit of quantification of 1.5 ng / mL or less. In this embodiment, the method for quantifying 4-hydroxytamoxifen is to quantify it to a level of 1 ng / mL or less. It has limitations. In some embodiments, the method for quantifying 4-hydroxytamoxifen is It has a quantitative limit of 0.5 ng / mL or less. In some embodiments, 4-hydroxy The quantitative method for tamoxifen has a quantification limit of 0.4 ng / mL or less. In this embodiment, the method for quantifying 4-hydroxytamoxifen is to obtain a concentration of 0.2 ng / mL or less. The limit of quantification is reached.

[0086] In some embodiments, N-desmethyl-4-hydroxytamoxifen (Endo The quantification method for cyphen has a quantification limit of 5 ng / mL or less. Several embodiments So, how to quantify N-desmethyl-4-hydroxytamoxifen (endoxifen)? The method has a quantitative limit of 4 ng / mL or less. In some embodiments, N-desmethyl The method for quantifying -4-hydroxytamoxifen (endoxifen) is 3 ng / mL or less. It has the following limits of quantification. In some embodiments, N-desmethyl-4-hydroxytamol The quantitative method for xifene (endoxifene) has a quantitative limit of 3 ng / mL or less. In some embodiments, N-desmethyl-4-hydroxytamoxifen (Endo) The quantification method for cyphen has a quantification limit of 2 ng / mL or less. Several embodiments So, how to quantify N-desmethyl-4-hydroxytamoxifen (endoxifen)? The method has a quantitative limit of 1.5 ng / mL or less. In some embodiments, N-Desmet The quantitative method for til-4-hydroxytamoxifen (endoxifen) is 1 ng / m³. It has a limit of quantification of L or less. In some embodiments, N-desmethyl-4-hydroxy The quantitative method for tamoxifen (endoxifen) is a limit of quantification of 0.5 ng / mL or less. It has N-desmethyl-4-hydroxytamoxifen ( The quantification method for endoxifen has a quantification limit of 0.4 ng / mL or less.

[0087] In some embodiments, the quantification of N-desmethyl-4'-hydroxytamoxifen The method has a quantitative limit of 5 ng / mL or less. In some embodiments, N-desmethic The quantitative method for ru-4'-hydroxytamoxifen has a limit of quantification of 4 ng / mL or less. In some embodiments, the determination of N-desmethyl-4'-hydroxytamoxifen The quantification method has a quantitative limit of 3 ng / mL or less. In some embodiments, N-DES is used. The quantitative method for methyl-4'-hydroxytamoxifen has a quantification limit of 3 ng / mL or less. It has N-desmethyl-4'-hydroxytamoxifen. In some embodiments, it has N-desmethyl-4'-hydroxytamoxifen. The quantification method has a quantification limit of 2 ng / mL or less. In some embodiments, N- The quantification method for desmethyl-4'-hydroxytamoxifen is 1.5 ng / mL or less. It has a limit of quantification. In some embodiments, N-desmethyl-4'-hydroxytamoxy The method for quantifying cyphen has a quantification limit of 1 ng / mL or less. In some embodiments, The quantitative method for N-desmethyl-4'-hydroxytamoxifen is 0.5 ng / m³. It has a limit of quantification of L or less. In some embodiments, N-desmethyl-4'-hydrox The quantification method for cytamoxifen has a quantification limit of 0.4 ng / mL or less.

[0088] In some embodiments, the method for quantifying norendoxiphen is such that the result is 5 ng / mL or less. It has a limit of quantification. In some embodiments, the method for quantifying norendoxiphene is 4 It has a limit of quantification of ng / mL or less. In some embodiments, norendoxiphen The quantification method has a quantification limit of 3 ng / mL or less. In some embodiments, Norwegian The quantification method for doxifen has a quantification limit of 3 ng / mL or less. Several methods are available. In terms of morphology, the method for quantifying norendoxiphen has a quantitative limit of 2 ng / mL or less. In some embodiments, the method for quantifying norendoxiphene is such that the concentration is 1.5 ng / mL or less. It has the following limits of quantification. In some embodiments, the method for quantifying norendoxiphene is It has a quantitative limit of 1.2 ng / mL or less. In some embodiments, norendoxy The method for quantifying fen has a quantification limit of 1 ng / mL or less. In some embodiments, The method for quantifying norendoxiphen has a quantification limit of 0.5 ng / mL or less.

[0089] In some embodiments, the methods provided herein are measured by the limit of detection (LOD). It has a defined sensitivity. In some embodiments, the method for detecting tamoxifen is 5 ng It has a detection limit of less than / mL. In some embodiments, the method for detecting tamoxifen is It has a detection limit of 4 ng / mL or less. In some embodiments, tamoxifen testing The extraction method has a detection limit of 3 ng / mL or less. In some embodiments, tamoxif The detection method for fertilizer has a detection limit of 3 ng / mL or less. In some embodiments, The method for detecting moxifen has a detection limit of 2 ng / mL or less. Several embodiments Therefore, the detection method for tamoxifen has a detection limit of 1.5 ng / mL or less. In that embodiment, the method for detecting tamoxifen has a detection limit of 1 ng / mL or less. In some embodiments, the method for detecting tamoxifen is to detect levels of 0.6 ng / mL or less. Reach the limit.

[0090] In some embodiments, the method for detecting N-desmethyltamoxifen is 5 ng / mL. The following detection limits are observed. In some embodiments, the detection of N-desmethyltamoxifen The extraction method has a detection limit of 4 ng / mL or less. In some embodiments, N-desme The detection method for tiltamoxifen has a detection limit of 3 ng / mL or less. In terms of application methods, the detection limit for N-desmethyltamoxifen is 3 ng / mL or less. has. In some embodiments, the detection method of N-desmethyltamoxifen has a detection limit of 2n g / mL or less. In some embodiments, the detection method of N-desmethyltamox ifen has a detection limit of 1.5 ng / mL or less. In some embodiments , the detection method of N-desmethyltamoxifen has a detection limit of 1 ng / mL or less. In some embodiments, the detection method of N-desmethyltamoxifen has a detection limit of 0.6 ng / m L or less.

[0091] In some embodiments, the detection method of 4'-hydroxytamoxifen has a detection limit of 5 ng / m L or less. In some embodiments, the detection method of 4'-hydroxytamoxifen has a detection limit of 4 ng / mL or less. In some embodiments, 4'- hydroxytamoxifen detection method has a detection limit of 3 ng / mL or less. Some embodiments, the detection method of 4'-hydroxytamoxifen has a detection limit of 3 ng / mL or less . In some embodiments, the detection method of 4'-hydroxytamoxifen has a detection limit of 2 ng / mL or less. In some embodiments, 4'- hydroxytamoxifen detection method has a detection limit of 1.5 ng / mL or less. Some [[ID= 28]]embodiments, the detection method of 4'-hydroxytamoxifen has a detection limit of 1 ng / mL or less . In some embodiments, the detection method of 4'-hydroxytamoxifen has a detection limit of 0.5 ng / mL or less. In some embodiments, 4'- hydroxytamoxifen detection method has a detection limit of 0.4 ng / mL or less. Some embodiments, the detection method of 4'-hydroxytamoxifen has a detection limit of 0.2 ng / mL or less . In some embodiments, the detection method of 4'-hydroxytamoxifen has a detection limit of 0.4 ng / mL or less. Some embodiments, the detection method of 4'-hydroxytamoxifen has a detection limit of 0.2 ng / mL or less It has a detection limit. In some embodiments, the detection method of 4'-hydroxytamoxifen has a detection limit of 0.1 ng / mL or less.

[0092] In some embodiments, the detection method of 4-hydroxytamoxifen has a detection limit of 5 ng / mL or less. In some embodiments, the detection method of 4-hydroxytamoxifen has a detection limit of 4 ng / mL or less. In some embodiments, the detection method of 4-hydroxy tamoxifen has a detection limit of 3 ng / mL or less. In some embodiments, the detection method of 4-hydroxy tamoxifen has a detection limit of 3 ng / mL or less. In some embodiments, the detection method of 4-hydroxytamoxifen has a detection limit of 2 n g / mL or less. In some embodiments, the detection method of 4-hydroxytamoxifen has a detection limit of 1.5 ng / mL or less. In some embodiments the detection method of 4-hydroxytamoxifen has a detection limit of 1 ng / mL or less. In some embodiments, the detection method of 4-hydroxytamoxifen has a detection limit of 0.5 ng / m [[ID=_{26}]] L or less. In some embodiments, the detection method of 4-hydroxytamoxifen has a detection limit of 0.4 ng / mL or less. In some embodiments, 4- hydroxytamoxifen detection method has a detection limit of 0.2 ng / mL or less. In some embodiments, the detection method of 4-hydroxytamoxifen has a detection limit of 0.1 ng / mL or less.

[0093] In some embodiments, N-desmethyl-4-hydroxytamoxifen (endoxyl) The detection method for cyphen has a detection limit of 5 ng / mL or less. In some embodiments, The detection method for N-desmethyl-4-hydroxytamoxifen (endoxifen) is... It has a detection limit of 4 ng / mL or less. In some embodiments, N-desmethyl-4 - The detection method for hydroxytamoxifen (endoxifen) is a test for levels of 3 ng / mL or less. It has an output limit. In some embodiments, N-desmethyl-4-hydroxytamoxif The detection method for endoxifen has a detection limit of 3 ng / mL or less. In that embodiment, N-desmethyl-4-hydroxytamoxifen (endoxifen) The detection method of ) has a detection limit of 2 ng / mL or less. In some embodiments, N- The detection method for desmethyl-4-hydroxytamoxifen (endoxifen) is 1.5 It has a detection limit of ng / mL or less. In some embodiments, N-desmethyl-4-H The detection method for droxitamoxifen (endoxifen) has a detection limit of 1 ng / mL or less. It has a field. In some embodiments, N-desmethyl-4-hydroxytamoxifen The detection method for (endoxifen) has a detection limit of 0.5 ng / mL or less. In that embodiment, N-desmethyl-4-hydroxytamoxifen (endoxifen) The detection method of ) has a detection limit of 0.4 ng / mL or less. In some embodiments, The detection method for N-desmethyl-4-hydroxytamoxifen (endoxifen) is: It has a detection limit of 0.2 ng / mL or less. In some embodiments, N-desmethyl-4 - The detection method for hydroxytamoxifen (endoxifen) is 0.15 ng / mL or less. It has the following detection limits.

[0094] In some embodiments, the detection method for N-desmethyl-4'-hydroxytamoxifen The method has a detection limit of 5 ng / mL or less. In some embodiments, N-desmethyl The detection method for -4'-hydroxytamoxifen has a detection limit of 4 ng / mL or less. In some embodiments, methods for detecting N-desmethyl-4'-hydroxytamoxifen The method has a detection limit of 3 ng / mL or less. In some embodiments, N-desmethyl The detection method for -4'-hydroxytamoxifen has a detection limit of 3 ng / mL or less. In some embodiments, methods for detecting N-desmethyl-4'-hydroxytamoxifen The method has a detection limit of 2 ng / mL or less. In some embodiments, N-desmethyl The detection method for -4'-hydroxytamoxifen has a detection limit of 1.5 ng / mL or less. In some embodiments, the test of N-desmethyl-4'-hydroxytamoxifen The extraction method has a detection limit of 1 ng / mL or less. In some embodiments, N-desme The detection method for til-4'-hydroxytamoxifen has a detection limit of 0.5 ng / mL or less. It has N-desmethyl-4'-hydroxytamoxifen. In some embodiments, it has N-desmethyl-4'-hydroxytamoxifen. The detection method has a detection limit of 0.4 ng / mL or less.

[0095] In some embodiments, the method for detecting norendoxiphen is such that the detection level is 5 ng / mL or less. It has an output limit. In some embodiments, the method for detecting norendoxiphene is 4ng It has a detection limit of less than / mL. In some embodiments, the detection of norendoxiphen is performed. The method has a detection limit of 3 ng / mL or less. In some embodiments, norendokey The tamoxifen detection method has a detection limit of 3 ng / mL or less. In some embodiments , the norendoxifen detection method has a detection limit of 2 ng / mL or less. In some embodiments, the norendoxifen detection method has a detection limit of 1.5 ng / mL or less . In some embodiments, the norendoxifen detection method has a detection limit of 1.2 ng / mL or less. In some embodiments, the norendoxifen detection method has a detection limit of 1 ng / mL or less. In some embodiments, the norendoxifen detection method has a detection limit of 0.5 ng / mL or less.

[0096] The following examples are useful for explaining the present invention. These examples are not intended to limit the scope of the method in any way.

Example

[0100] [Table 3]

[0101] Calibration materials / standard materials to use A 12-point calibration is used for each analyte. Initially, only one standard material is prepared ( Std-12), a series of dilutions are carried out to produce the remaining standard substance. Standard substance (#1 2) It should be removed from the -70°C freezer and allowed to thaw. While thawing, 12 Label each 12 x 75 mm tube.

[0102] Add 3.0 mL of std-12 to tube 12. From this standard substance, the following table is obtained. Following this pattern, a standard curve is created. Standard materials should be prepared using each assay. Return the original standard substance to a freezer at -60 to -90°C.

[0103] [Table 4]

[0104] Once these analytes are added, use a sufficient amount of Biocell serum to 200°C. Prepare to mL. After mixing, transfer to a 15 mL centrifuge tube. Label (Std- 12) Afterwards, place the tubes in a freezer at -60 to -90°C for storage. They will remain stable for one year. .

[0105] Standard concentration of standard substance

[0106] [Table 5]

[0107] [Table 6]

[0108] [Table 7]

[0109] [Table 8]

[0110] Equipment and supplies

[0111] Assay platform A Thermo LC / MS / MS system containing the following modules can be used for this assay. Used:

[0112] [Table 9]

[0113] [Table 10]

[0114] [Table 11]

[0115] [Table 12]

[0116] Predicted value

[0117] Reference range: Tamoxifen: 12.54~233.07 ng / mL N-Desmethyltamoxifen: 2.59~373.96 ng / mL 4'-Hydroxytamoxifen: 0.4~6.33 ng / mL 4-Hydroxytamoxifen: 0.24~5.05 ng / mL N-desmethyl-4-hydroxytamoxifen (endoxifen): 0.93~4 3.19 ng / mL N-desmethyl-4'-hydroxytamoxifen: 1.17~19.95 ng / mL

[0118] Analytical measurement range (AMR) Tamoxifen: 1.47-1500 ng / mL N-Desmethyltamoxifen: 1.47-1500 ng / mL 4'-Hydroxytamoxifen: 0.2-200 ng / mL 4-Hydroxytamoxifen: 0.2-200 ng / mL N-desmethyl-4-hydroxytamoxifen (endoxifen): 0.39~4 00 ng / mL N-desmethyl-4'-hydroxytamoxifen: 0.39~400 ng / mL

[0119] Precision: Inter-assay (assay duration) and intra-assay (1 day) precision studies, low, medium, and high pair The analysis was performed using light. All analytes showed less than 12% change over the validation period. The dynamic coefficient was shown.

[0120] Interfering substances: Samples with mild or moderate jaundice and dyslipidemia are acceptable. Hemolytic samples are... These clog the filter during sample preparation, which is unacceptable. They are extremely hemolytic. Samples exhibiting jaundice and dyslipidemia are unacceptable.

[0121] Clinical sensitivity (LOQ): Tamoxifen: 1.47 ng / mL N-Desmethyltamoxifen: 1.46 ng / mL 4'-Hydroxytamoxifen: 0.2 ng / mL 4-Hydroxytamoxifen: 0.2 ng / mL N-desmethyl-4-hydroxytamoxifen (endoxifen): 0.39 ng / mL N-desmethyl-4'-hydroxytamoxifen: 0.39 ng / mL [Examples]

[0122] Validation of Tamoxifen and its Metabolite Assays This report includes tamoxifen and its five major Phase 1 studies using LC / MS / MS. It includes a detailed overview of the validation of metabolites. The assay was developed in the laboratory. This was the test.

[0123] Method: Tamoxifen and its five major Phase 1 metabolites (N-desmethyltamoxifen) Fen, N-desmethyl-4-hydroxytamoxifen, N-desmethyl-4'-Hyd Roxitamoxifen, 4-hydroxytamoxifen, and 4'-hydroxytamoxi Cyphen is extracted from serum. The extraction method involves protein precipitation followed by filtration. Next, analysis is performed. Quantification is performed using LC / MS / MS.

[0124] [Table 13]

[0125] Accuracy study of the test (LDT) developed in the laboratory. In-run precision: Low, medium, and high controls were analyzed in a single run (10). QC was complete for all tests. It fell within the criteria (coefficient of variation less than 20%).

[0126] [Table 14]

[0127] [Table 15]

[0128] [Table 16]

[0129] Overall accuracy: Overall accuracy is based on all QC executions for all assays during the validation process. The pass / fail criteria were based on a coefficient of variation of less than 20%. All QC was within this judgment criterion. It's settled down.

[0130] [Table 17]

[0131] [Table 18]

[0132] [Table 19]

[0133] Analytical sensitivity (detection limit) Limit of Detection (LOD): The limit of detection (LOD) is the low pool (containing all of the analyte). This is done by first dispensing the solution, and then gradually diluting it (1:2) to the lowest observable level. Assuming that linearity continues below the limit of quantification (LOQ), the following values ​​are obtained: This is likely the lowest concentration that can be quantified. This experiment was conducted over five days. Tamoxifen: 0.59 ng / mL N-Desmethyltamoxifen: 0.59 ng / mL 4'-Hydroxytamoxifen: 0.1 ng / mL N-desmethyl-4'-hydroxytamoxifen: 0.5 ng / mL 4-Hydroxytamoxifen: 0.1 ng / mL N-desmethyl-4-hydroxytamoxifen (endoxifen): 0.15 ng / mL

[0134] Limit of Quantitative Quality (LOQ): The passing criterion for LOQ is the lowest concentration with a coefficient of variation of less than 20%. It is defined as follows: To determine the LOQ, intermediate level standards are diluted in a 1:2 ratio. He explained. Tamoxifen: 1.5 ng / mL N-desmethyltamoxifen: 1.5 ng / mL 4'-Hydroxytamoxifen: 0.4 ng / mL N-desmethyl-4'-hydroxytamoxifen: 0.4 ng / mL 4-Hydroxytamoxifen: 0.2 ng / mL N-desmethyl-4-hydroxytamoxifen (endoxifen): 0.4 ng / mL

[0135] Accuracy Recovery rate of known standard substances: All analytes are added to serum to a specific concentration, extracted, and then triple-shot. The analysis was performed in iterative form. All mixes were added to cover the linear and therapeutic range of each analyte. It was done.

[0136] [Table 20]

[0137] Interference research Acceptance Criteria: Differences due to potential interfering substances are considered acceptable if TEa / It should be 4 or less.

[0138] Hemolysis interference: Hemolyzed RBCs were added to low and high pools at low, medium, and high concentrations. The sample was extracted using quadruple repeats. Hemolysis indicates interference from tamoxifen or the metabolite being measured. However, due to the difficulty of filtering moderately and highly hemolytic samples, mild cases were observed. Only hemolyzed samples should be acceptable.

[0139] [Table 21]

[0140] Lipid-lowering interference: Lipid-lowering samples were added to low and high pools at low, medium, and high concentrations, respectively. Samples were extracted using quadruple repeat extraction. Lipidemia samples were extracted using tamoxifen or the metabolite to be measured. It did not show any interference.

[0141] [Table 22]

[0142] Bilirubin interference: Bilirubin was added to low and high pools at low, medium, and high concentrations. Samples were extracted using quadruple repeat extraction. Samples to which bilirubin was added were then tested for tamoxifen or measured. No interference from metabolites was observed.

[0143] [Table 23] [Examples]

[0144] Validation of noendoxiphen assay This report includes validation of norendoxiphen by LC / MS / MS. This includes a detailed overview. The assay is a laboratory-developed test.

[0145] Norendoxiphene is extracted from serum using protein precipitation followed by filtration. Analysis and quantification will be performed using LC / MS / MS.

[0146] [Table 24]

[0147] Accuracy study of the test (LDT) developed in the laboratory. In-run precision: Low, medium, and high control (ng / mL) were analyzed in a single run (10) All QC (Quality Control) tests met the passing criteria (coefficient of variation less than 20%).

[0148] [Table 25]

[0149] Overall accuracy: Overall accuracy is based on all QC executions for all assays during the validation process. The pass / fail criteria were based on a coefficient of variation of less than 20%. All QC was within this judgment criterion. It's settled down.

[0150] Limit of detection (LOD): Norendoxiphen = 1.2 ng / mL

[0151] Limit of Quantitative

[0152] Accuracy Recovery rate of known standard substances: All analytes are added to serum to a specific concentration, extracted, and then quadrupled. The analysis was performed in iterative form. All mixes were added to cover the linear and therapeutic range of each analyte. It was done.

[0153] [Table 26]

[0154] Interference research Acceptance Criteria: Differences due to potential interfering substances are considered acceptable if TEa / It should be 4 or less.

[0155] Hemolysis interference: Hemolyzed RBCs were added to low and high pools at low, medium, and high concentrations. The sample was extracted using quadruple repeats. Hemolysis did not show interference with norendoxiphen. However, However, due to the difficulty of filtering moderately and highly hemolytic samples, only mildly hemolytic samples are filtered. It should be permissible.

[0156] [Table 27]

[0157] Lipid-lowering interference: Lipid-lowering samples were added to low and high pools at low, medium, and high concentrations, respectively. The samples were extracted using quadruple repeats. Lipid-lowering samples did not show interference with noendoxiphen. Recovery rate of known standard substances: After adding all analytes to serum to a specific concentration and extracting, The analysis was performed using quadruple replicates. All mixes were designed to cover the linear and therapeutic range of each analyte. It was added.

[0158] [Table 28]

[0159] Bilirubin interference: Bilirubin was added to low and high pools at low, medium, and high concentrations. Samples were extracted using quadruple repeats. Samples with added bilirubin showed interference from noendoxiphen. It did not show that.

[0160] [Table 29] [Examples]

[0161] Clinical Quantification and Response Studies Using the standard operating protocols of Examples 1 to 3, tamoxifen in patient samples was investigated. Tamoxifen and its metabolites were quantified and correlated with the tamoxifen response.

[0162] [Table 30] JPEG0007884626000031.jpg242106

[0163] [Table 31] JPEG0007884626000033.jpg203161

[0164] [Table 32] JPEG0007884626000035.jpg242106

[0165] [Table 33] JPEG0007884626000037.jpg242109

[0166] [Table 34] JPEG0007884626000039.jpg241118

[0167] [Table 35] JPEG0007884626000041.jpg241108

[0168] [Table 36] JPEG0007884626000043.jpg202150

[0169] [Table 37] JPEG0007884626000045.jpg242116

[0170] [Table 38] JPEG0007884626000047.jpg203153

[0171] [Table 39] JPEG0007884626000049.jpg241110

[0172] [Table 40] JPEG0007884626000051.jpg202143

[0173] [Table 41] [Examples]

[0174] Further clinical quantification and response studies Using the standard operating protocols of Examples 1 to 3, tamoxifen in patient samples was investigated. Tamoxifen and its metabolites were quantified and correlated with the tamoxifen response.

[0175] [Table 42] JPEG0007884626000054.jpg230156JPEG0007884626000055.jpg224156

[0176] [Table 43] JPEG0007884626000057.jpg227158JPEG0007884626000058.jpg199157

[0177] [Table 44] JPEG0007884626000060.jpg229163JPEG0007884626000061.jpg209164

[0178] [Table 45] JPEG0007884626000063.jpg230153

[0179] The papers, patents, patent applications and all other documents and electronic documents mentioned or cited herein The content of the information available to children is designed so that each publication incorporates it by reference. The whole is incorporated by reference to the same extent as if it were shown physically and individually. The applicants shall not include in this application any such papers, patents, patent applications, or other physical and We retain the right to physically incorporate any material and information from electronic documents.

[0180] The methods described herein as illustrative are any elements not specifically disclosed herein. It can be properly implemented in the absence of (singular or plural) or limitation (singular or plural). However For example, terms such as "including," "contains," and "contains" are broad and limited in scope. It is to be interpreted without limitation. Furthermore, the terms and expressions used herein are for illustrative purposes only. It is used as a word, not as a restrictive term, and is shown and described. The intent behind using such terms and expressions is to exclude any equivalents or parts thereof of the features. It is recognized that various modifications are possible within the scope of the claimed invention. Therefore, the present invention has been more specifically disclosed by preferred embodiments and optional features. However, modifications and variations of the invention as disclosed herein can be performed by those skilled in the art. It is possible that such changes and variations may be deemed to be within the scope of the present invention. This should be understood.

[0181] The present invention has been described extensively and comprehensively in this specification. Each of the narrower species and subgenus classifications also constitutes part of this method. This is deleted. Whether or not materials are specifically listed in this specification, any subject may be removed from the genus. The comprehensive description of the method is to include a condition or negative limitation.

[0182] Other embodiments are found within the scope of the following claims. Furthermore, features and aspects of the method However, it is described in terms of the Marcush group, and the present invention also thereby relates to the Marcush group It will be understood by those skilled in the art that the terms used are those of any individual member or subgroup of members. .

Claims

1. A method for determining the amount of norendoxiphene in a sample obtained from a human by mass spectrometry, The aforementioned sample is purified by liquid chromatography. Add an internal standard containing deuterated norendoxifene. Ionizing the aforementioned norendoxifene to produce one or more norendoxifene ions detectable by mass spectrometry, and ionizing the aforementioned deuterated norendoxifene to produce one or more deuterated norendoxifene ions detectable by mass spectrometry, The amount of the internal standard is detected by detecting the amount of one or more deuterated norendoxphene ions by mass spectrometry, and The process includes detecting the amount of one or more norendoxiphene ions by mass spectrometry, wherein the amount of one or more norendoxiphene ions detected is related to the amount of norendoxiphene in the sample. A method for determining whether orendoxiphene has a quantitative limit of 5 ng / mL or less.

2. The method according to claim 1, wherein the liquid chromatography is high-pressure liquid chromatography.

3. The method according to claim 1, wherein the ionization is by atmospheric pressure chemical ionization.

4. The method according to claim 1, wherein the ionization is in a positive ion mode.

5. The method according to claim 1, wherein the sample is a serum sample.

6. The method according to claim 1, wherein the mass spectrometry is tandem mass spectrometry.

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