Quantitation of tamoxifen and metabolites thereof 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]

[0001] CROSS-REFERENCE TO RELATED PATENT APPLICATIONS This application was filed on May 12, 2014, and is incorporated herein by reference in its entirety. This application claims priority to U.S. Provisional Application No. 61 / 992,214. [Background technology]

[0002] The following background of the invention is provided solely as an aid in understanding the invention. and are described as prior art to the present invention or admitted to constitute prior art to the present invention. I can't.

[0003] Tamoxifen is the standard of care for patients with hormone receptor-positive breast cancer after first-line treatment. Tamoxifen therapy for 5 to 10 years reduces recurrence and death in these patients. Nevertheless, many patients are still concerned about the often unpleasant side effects of the drug. Due to the effects, they do not complete their full course of treatment.

[0004] Tamoxifen is converted to highly active endoxifen for full efficacy Tamoxifen is a prodrug. The conversion of tamoxifen to endoxifen is catalyzed by CYP2D6 ( Due to metabolic pathways that depend on genetic mutations, such as the genetic mutation in the 2D6 gene. Typing has been promoted to predict response and toxicity to tamoxifen, There is some debate as to the direct relevance to the individual level. Summary of the Invention [Problem to be solved by the invention]

[0005] There is a need for an effective method of predicting response to tamoxifen. [Means for solving the problem]

[0006] The present invention relates to the detection of tamoxifen and its analogs in a sample by mass spectrometry, including tandem mass spectrometry. Methods for quantifying metabolites are provided.

[0007] In one embodiment, the method is for determining the amount of norendoxife in a sample by mass spectrometry. (a) ionizing said norenoxifen to produce one or more compounds detectable by mass spectrometry; (b) generating a norendoxifene ion or ions from the process by mass spectrometry; detecting the amount of said norenoxifen ions (ion(s)) from said mixture; The method further comprises providing a method for determining whether the amount of ion(s) is related to the amount of norendooxifen in the sample. will be done.

[0008] In one embodiment, tamoxifen and its metabolism in a sample are analyzed in a single mass spectrometry assay. 1. A method for determining the amount of a product comprising: (a) ionizing said tamoxifen and metabolites; (b) generating one or more ions detectable by mass spectrometry; and detecting the amount of said ions (s) from the process by analysis, The amount of ion(s) is related to the amount of tamoxifen and metabolites, respectively, in the sample. A method is provided.

[0009] In some embodiments, the metabolite comprises norenoxifen. In embodiments, the metabolite is endoxifen or N-desmethyl-4-hydroxybenzoate. In some embodiments, the metabolite comprises 4'-hydroxybutyric acid. In some embodiments, the metabolite comprises 4-hydroxytamoxifen. In some embodiments, the metabolite comprises N-desmethyl-4'- In some embodiments, the metabolite comprises N-hydroxytamoxifen. In some embodiments, the metabolite comprises norendoxin. Tamoxifen, endoxifen, 4'-hydroxytamoxifen, 4-hydroxytamoxifen Tamoxifen, N-desmethyl-4'-hydroxytamoxifen, and N-desmethyl- 4'-hydroxytamoxifen. In embodiments, the metabolite is norenoxifen, endoxifen, 4'-hydro Roxitamoxifen, 4-hydroxytamoxifen, N-desmethyl-4'-hydroxy Tamoxifen, N-desmethyltamoxifen, and any combination thereof. In some embodiments, the metabolite is norenoxifen, endoxifen, 4' -Hydroxytamoxifen, 4-hydroxytamoxifen, N-desmethyl-4'- Hydroxytamoxifen, and N-desmethyl-4'-hydroxytamoxifen In some embodiments, the metabolite is norendoxifen, endoxifen 4'-Hydroxytamoxifen, 4-Hydroxytamoxifen, N-Desmethionine any combination of N-4'-hydroxytamoxifen, N-desmethyltamoxifen, Includes combinations.

[0010] In one aspect, the methods provided herein comprise protein precipitation. In some embodiments, the methods provided herein include purification. In some embodiments, the purification comprises liquid chromatography. In some embodiments, the liquid chromatography is high pressure 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 norenoxifen. do.

[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, the mass spectrometry is tandem mass spectrometry.

[0016] In one embodiment, the amount of tamoxifen or one or more tamoxifen metabolites is Provided herein are methods for predicting tamoxifen response in a patient by determining In some embodiments, a large amount of one or more of tamoxifen or tamoxifen is provided. Xifene metabolites are responsible for the positive response to tamoxifen in patients. In some embodiments, the metabolite comprises norenoxifene. The metabolite is endoxifen or N-desmethyl-4-hydroxytamoxifen. In some embodiments, the metabolite includes 4'-hydroxytamoxifen. In some embodiments, the metabolite includes 4-hydroxytamoxifen. In some embodiments, the metabolite comprises N-desmethyl-4'-hydroxy In some embodiments, the metabolite comprises N-desmethyltamoxifen. In some embodiments, the metabolites include norendoxifene, Endoxifen, 4'-hydroxytamoxifen, 4-hydroxytamoxifen, N-desmethyl-4'-hydroxytamoxifen, and N-desmethyl-4'-hydroxy Contains roxtamoxifen.

[0017] In some embodiments, the methods provided herein are measured by limit of quantitation (LOQ). In some embodiments, the method for quantifying tamoxifen has a sensitivity of 5n In some embodiments, the quantification method for tamoxifen has a limit of quantification of 0.1 mg / mL or less. The method has a limit of quantitation of 4 ng / mL or less. The quantification method has a limit of quantification of 3 ng / mL or less. The method for quantification of xifene has a limit of quantification of 3 ng / mL or less. In some cases, tamoxifen quantification methods have a limit of quantification of 2 ng / mL or less. In this embodiment, the method for quantifying tamoxifen has a limit of quantification of 1.5 ng / mL or less. do.

[0018] In some embodiments, the method of quantification of N-desmethyl tamoxifen is 5 ng / m In some embodiments, the N-desmethyl tamoxifen has a limit of quantitation of 0.1 μg / mL or less. The quantification method has a limit of quantification of 4 ng / mL or less. The quantification method for smethyltamoxifen has a limit of quantification of 3 ng / mL or less. In some embodiments, the method for quantifying N-desmethyl tamoxifen comprises quantifying N-desmethyl tamoxifen at levels below 3 ng / mL. In some embodiments, the method for quantifying N-desmethyl tamoxifen has a limit of quantification. The method has a limit of quantitation of 2 ng / mL or less. The tamoxifen quantification method has a limit of quantification of ≦1.5 ng / mL.

[0019] In some embodiments, the method for quantifying 4'-hydroxy tamoxifen is 5 ng / In some embodiments, 4'-hydroxytamoxifen has a quantitation limit of 0.1 mL or less. The quantification method for 4ng / mL or less has a limit of quantification of 4ng / mL or less. The method for quantification of '-hydroxy tamoxifen has a limit of quantification of 3 ng / mL or less. In some embodiments, the method of quantification of 4'-hydroxy tamoxifen is 3 ng / ml In some embodiments, 4'-hydroxy tamoxifen has a limit of quantitation of 0.1 to 1.0 L. The quantification method of has a limit of quantification of 2 ng / mL or less. -The quantification method for hydroxytamoxifen has a limit of quantification of 1.5 ng / mL or less. In some embodiments, the method for quantifying 4'-hydroxy tamoxifen is 1 ng / In some embodiments, 4'-hydroxytamoxifen has a quantitation limit of 0.1 mL or less. In some embodiments, the quantification method for the agonist has a limit of quantification of 0.5 ng / mL or less. The quantification method for 4'-hydroxytamoxifen has a limit of quantification of 0.4 ng / mL or less. In some embodiments, the method for quantifying 4'-hydroxy tamoxifen comprises: It has a limit of quantification of less than 0.2ng / mL.

[0020] In some embodiments, the method of quantification of 4-hydroxy tamoxifen is 5 ng / m In some embodiments, the quantitation limit of 4-hydroxytamoxifen is 1. The quantification method has a limit of quantification of 4 ng / mL or less. The quantification method for droxitamoxifen has a limit of quantification of 3 ng / mL or less. In some embodiments, the method for quantifying 4-hydroxy tamoxifen comprises quantifying 4-hydroxy tamoxifen at levels below 3 ng / mL. In some embodiments, the method for quantifying 4-hydroxy tamoxifen has a limit of quantification. The method has a limit of quantitation of 2 ng / mL or less. Tamoxifen quantification methods have limits of quantification below 1.5 ng / mL. In embodiments, the method for quantifying 4-hydroxy tamoxifen provides a quantitative determination of 1 ng / mL or less. In some embodiments, the method for quantifying 4-hydroxy tamoxifen has limitations. In some embodiments, the 4-hydroxytryptamine has a limit of quantitation of 0.5 ng / mL or less. Tamoxifen quantification methods have limits of quantification below 0.4 ng / mL. In an embodiment, the method for quantifying 4-hydroxy tamoxifen comprises quantifying 4-hydroxy tamoxifen at 0.2 ng / mL or less. It has a limit of quantification.

[0021] In some embodiments, N-desmethyl-4-hydroxytamoxifen (endoxyl) The quantification method for sifen has a limit of quantification of 5 ng / mL or less. So how do we quantify N-desmethyl-4-hydroxytamoxifen (endoxifen)? The method has a limit of quantitation of 4 ng / mL or less. The quantification method for 4-hydroxytamoxifen (endoxifen) is below 3 ng / mL. In some embodiments, N-desmethyl-4-hydroxytamol has a lower limit of quantification. The quantification method for xifen (endoxifen) has a limit of quantification of 3 ng / mL or less. In some embodiments, N-desmethyl-4-hydroxytamoxifen (endokis The method for quantification of schizophrenia (Schizophrenia) has a limit of quantification of 2 ng / mL or less. So how do we quantify N-desmethyl-4-hydroxytamoxifen (endoxifen)? The method has a limit of quantification of 1.5 ng / mL or less. The quantification method for ethyl-4-hydroxytamoxifen (endoxifen) is 1 ng / ml In some embodiments, the N-desmethyl-4-hydroxybenzoate has a limit of quantitation of 0.1 to 1.0 L. The quantification method for tamoxifen (endoxifen) has a quantification limit of 0.5 ng / mL or less. In some embodiments, N-desmethyl-4-hydroxy tamoxifen ( The quantification method for endoxifen) has a limit of quantification of 0.4 ng / mL or less.

[0022] In some embodiments, quantification of N-desmethyl-4'-hydroxy tamoxifen The method has a limit of quantitation of 5 ng / mL or less. The quantification method for 4'-hydroxy-tamoxifen has a limit of quantification of 4 ng / mL or less. In some embodiments, the definition of N-desmethyl-4'-hydroxy tamoxifen is The quantification method has a limit of quantification of 3 ng / mL or less. The quantification method for methyl-4'-hydroxytamoxifen has a quantification limit of 3ng / mL or less. In some embodiments, N-desmethyl-4'-hydroxy tamoxifen The quantification method for N- The quantification method for desmethyl-4'-hydroxytamoxifen was designed to detect concentrations below 1.5 ng / mL. In some embodiments, N-desmethyl-4'-hydroxytamoxifen is The method for quantifying sifen has a limit of quantification of 1 ng / mL or less. The quantification method for N-desmethyl-4'-hydroxytamoxifen is 0.5 ng / ml. In some embodiments, the N-desmethyl-4'-hydroxybutyric acid The quantification method for tamoxifen has a limit of quantification of 0.4 ng / mL or less.

[0023] In some embodiments, the method for quantifying norendipine includes quantifying norendipine at or below 5 ng / mL. In some embodiments, the method for quantifying norendipine has a limit of quantification of 4 In some embodiments, the norendoxifen has a quantification limit of ng / mL or less. The quantification method has a limit of quantification of 3 ng / mL or less. The method for quantification of indoxifene has a limit of quantification of 3 ng / mL or less. In its formulation, the quantification method for norendiphene has a limit of quantification of 2 ng / mL or less. In some embodiments, the method for quantifying norendoxifenin includes determining whether the concentration is 1.5 ng / mL or less. In some embodiments, the method for quantifying norenoxifene has a lower limit of quantification. In some embodiments, the norendoxylanase has a limit of quantification of 1.2 ng / mL or less. The quantification method for phene has a limit of quantification of 1 ng / mL or less. The method for quantification of norenoxifene has a limit of quantification of 0.5 ng / mL or less.

[0024] In some embodiments, the methods provided herein are measured by limit of detection (LOD). In some embodiments, the method for detecting tamoxifen has a sensitivity of 5 ng. In some embodiments, the method for detecting tamoxifen has a detection limit of 0.1 ml / mL or less. In some embodiments, the detection limit for tamoxifen is 4 ng / mL or less. The detection method has a detection limit of 3 ng / mL or less. The method for detecting the phenotype has a detection limit of 3 ng / mL or less. The method for detecting moxifen has a detection limit of 2 ng / mL or less. In this study, the detection method for tamoxifen has a detection limit of 1.5 ng / mL or less. In some embodiments, the method for detecting tamoxifen has a detection limit of 1 ng / mL or less. In some embodiments, the method for detecting tamoxifen comprises detecting 0.6 ng / mL or less. It has limitations.

[0025] In some embodiments, the method for detecting N-desmethyl tamoxifen is 5 ng / mL In some embodiments, the detection limit for N-desmethyl tamoxifen is The extraction method has a detection limit of 4 ng / mL or less. The detection method for tiltamoxifen has a detection limit of 3 ng / mL or less. In an embodiment, the method for detecting N-desmethyl tamoxifen has a detection limit of 3 ng / mL or less. In some embodiments, the method for detecting N-desmethyl tamoxifen comprises the steps of: In some embodiments, N-desmethyltamoxifen has a detection limit of 0.1 mg / mL or less. The method for detecting the enzyme has a detection limit of 1.5 ng / mL or less. The method for detecting N-desmethyltamoxifen has a detection limit of 1 ng / mL or less. In some embodiments, the method for detecting N-desmethyl tamoxifen comprises detecting 0.6 ng / ml It has a detection limit of less than L.

[0026] In some embodiments, the method for detecting 4'-hydroxy tamoxifen comprises detecting 5 ng / ml In some embodiments, 4'-hydroxy tamoxifen has a detection limit of 0.1 L or less. The method for detecting 4'- The detection method for hydroxytamoxifen has a detection limit of 3 ng / mL or less. In some embodiments, the method for detecting 4'-hydroxy tamoxifen comprises detecting a concentration of 3 ng / mL or less. In some embodiments, the method for detecting 4'-hydroxy tamoxifen has a detection limit. The method has a detection limit of 2 ng / mL or less. The detection method for tamoxifen has a detection limit of 1.5 ng / mL or less. In an embodiment, the method for detecting 4'-hydroxy tamoxifen comprises detecting less than 1 ng / mL. In some embodiments, the method for detecting 4'-hydroxy tamoxifen has limitations. In some embodiments, the 4'-hydroxylase has a detection limit of 0.5 ng / mL or less. The detection method for tamoxifen has a detection limit of 0.4 ng / mL or less. In an embodiment, the method for detecting 4'-hydroxy tamoxifen comprises detecting a concentration of 0.2 ng / mL or less. In some embodiments, the method for detecting 4'-hydroxy tamoxifen has a detection limit. The method has a detection limit of 0.1 ng / mL or less.

[0027] In some embodiments, the method for detecting 4-hydroxy tamoxifen is 5 ng / mL In some embodiments, the detection limit for 4-hydroxy tamoxifen is The extraction method has a detection limit of 4 ng / mL or less. The detection method for tamoxifen has a detection limit of 3 ng / mL or less. In an embodiment, the method for detecting 4-hydroxy tamoxifen has a detection limit of 3 ng / mL or less. In some embodiments, the method for detecting 4-hydroxy tamoxifen comprises the steps of: In some embodiments, 4-hydroxytamoxifen has a detection limit of 0.1 mg / mL or less. The method for detecting the enzyme has a detection limit of 1.5 ng / mL or less. The method for detecting 4-hydroxy tamoxifen has a detection limit of 1 ng / mL or less. In some embodiments, the method for detecting 4-hydroxy tamoxifen comprises detecting 0.5 ng / ml In some embodiments, the detection limit of 4-hydroxytamoxifen is 1. The detection method has a detection limit of 0.4 ng / mL or less. The detection method for hydroxytamoxifen has a detection limit of 0.2 ng / mL or less. In some embodiments, the method for detecting 4-hydroxy tamoxifen comprises detecting a concentration of 0.1 ng / mL It has the following detection limits:

[0028] In some embodiments, N-desmethyl-4-hydroxytamoxifen (endoxyl) In some embodiments, the detection method for .sigma.fen has a detection limit of 5 ng / mL or less. How can I detect N-desmethyl-4-hydroxytamoxifen (endoxifen)? In some embodiments, N-desmethyl-4 -Hydroxytamoxifen (endoxifen) detection method is 3ng / mL or less In some embodiments, N-desmethyl-4-hydroxytamoxifen is The detection method for endoxifen has a detection limit of 3 ng / mL or less. In some embodiments, N-desmethyl-4-hydroxytamoxifen (endoxifen) In some embodiments, the detection method for N- The detection method for desmethyl-4-hydroxytamoxifen (endoxifen) is 1.5 In some embodiments, N-desmethyl-4-hydroxyphenylalanine has a detection limit of ng / mL or less. The detection method for droxitamoxifen (endoxifen) has a detection limit of 1 ng / mL or less. In some embodiments, N-desmethyl-4-hydroxytamoxifen The detection method for (endoxifen) has a detection limit of 0.5 ng / mL or less. In some embodiments, N-desmethyl-4-hydroxytamoxifen (endoxifen) ) has a detection limit of 0.4 ng / mL or less. In some embodiments, The detection method for N-desmethyl-4-hydroxytamoxifen (endoxifen) is In some embodiments, the N-desmethyl-4 -Hydroxytamoxifen (endoxifen) detection method is 0.15ng / mL or less has a lower detection limit.

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

[0030] In some embodiments, the method for detecting norenoxifen comprises detecting a concentration of 5 ng / mL or less. In some embodiments, the detection method for norendoxife has an extinction limit of 4 ng. In some embodiments, the detection of norenoxifen has a limit of detection of 0.1 ml / mL or less. The method has a detection limit of 3 ng / mL or less. The method for detecting Shifen has a detection limit of 3 ng / mL or less. , several methods for detecting norenoxifen have detection limits of 2 ng / mL or less. In one embodiment, the method for detecting norenoxifen has a detection limit of 1.5 ng / mL or less. In some embodiments, the method for detecting norendoxifenin has a concentration of 1.2 ng / mL. In some embodiments, the method for detecting norenoxifen has a detection limit of less than 1 mL. The method has a detection limit of 1 ng / mL or less. The detection method for phene has a detection limit of 0.5 ng / mL or less.

[0031] As used herein, the term "purification" or "to purify" refers to the purification of a desired component. It does not refer to the removal of all material from a sample other than the analyte(s). The preparation comprises the determination of one or more analytes of interest relative to one or more other constituents of the sample. Purification, as used herein, refers to the procedure of enriching a substance in an amount from all others. No isolation of the analyte is required. In a preferred embodiment, a purification step or procedure is used to One or more interfering substances, e.g., one or more substances that interfere with the operation of the equipment used in the method. or removing a plurality of substances or substances that may interfere with the detection of analyte ions by mass spectrometry. This can be done.

[0032] As used herein, "about" refers to a quantitative measurement that does not involve measuring the mass of an ion. " refers to the stated value plus or minus 10%.

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

[0034] As used herein, the term "sample" refers to any sample that may contain an analyte of interest. As used herein, the term "body fluid or tissue" refers to a sample from an individual's body. "A" refers to any fluid or tissue that can be isolated from a body fluid or tissue. " may include blood, plasma, serum, bile, saliva, urine, tears, sweat, etc. If solid tissue is analyzed If the tissue can be processed to release a liquid fraction that may contain any analytes present in the tissue, The liquid fraction may then be subjected to the methods described herein.

[0035] As used herein, the term "size separation technique" refers to a technique that separates a sample by measuring the weight and shape of the molecule. and allowing for the separation of at least one species from a test sample based on any one or more of: "This refers to any technique (physical or chemical) that can be used to separate or remove all or part of a substance. Examples of such techniques are filtration, chromatographic Certain aspects of the present invention include, but are not limited to, fluorometric and mass spectrometry. .

[0036] As used herein, the term "chromatography" refers to the process of separating liquids or gases. Chemical mixtures transported by the and / or separation into constituent components as a result of differential partitioning of chemical components as they flow through it. This refers to the process of

[0037] As used herein, the term "liquid chromatography" or "LC" , a fluid solution as it permeates a column of fine material or a capillary passage uniformly This refers to the process of selective retardation of one or more components of the As the ase(s) move relative to the stationary phase(s), the retardation occurs due to the interaction between the stationary phase(s) and the bulk fluid (i.e., The term "liquid chromatography" refers to the partitioning of the components of a mixture between the liquid phase (the moving phase) and the sample. Reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC) ) and high turbulence liquid chromatography (HTLC).

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

[0039] As used herein, the term "mass spectrometry" or "MS" refers to a method for analyzing MS refers to an analytical technique for identifying compounds by their m / z. MS techniques generally involve (1) filtering, detecting, and measuring compounds (2) determining the molecular weight of the ions by electrochemically ionizing the ions to form charged species (e.g., ions); The compounds can be ionized by any suitable means and their m / z calculated. The term "mass spectrometer" generally refers to a device that includes an ionizer and an ion detector. Generally, one or more molecules of interest are ionized and the ions are then mass-dispersed. The ions are introduced into a spectrometer where, due to a combination of magnetic and electric fields, the ions are split into smaller groups based on mass ("m") ) and charge ("z"). For example, "Mass Spectrometry" U.S. Patent No. 6,204,500 entitled "Methods From Surfaces" No. 0; “Methods and Apparatus for Tandem Mas No. 6,107,623, entitled "DNA D Spectrometry"; "Diagnostics Based on Mass Spectrometry" No. 6,268,144 entitled "Surface-Enhanced Photo labile Attachment And Release For Desorp The sixth issue of the same publication, entitled "Measures and Detection of Analytes" ,124,137;Wright et al.,Prostate Cancer and Prostatic Diseases 2:264-76(1999); and Merchant and Weinberger,Electrophoresis 21:1164-67(2000).

[0040] As used herein, the term "operating in positive ion mode" means "Negative ion mode" refers to a mass spectrometry method in which positive ions are detected. The term "detecting" refers to mass spectrometry in which negative ions are detected.

[0041] As used herein, the term "ionization" or "ionizing" means A process that generates analyte ions having a net charge equal to one or more electron units. A cation is an ion that has a net positive charge of one or more electron units. An anion is one that has a net negative charge of one or more electron units. .

[0042] As used herein, the term "electron ionization" or "EI" refers to gas-phase or refers to the way in which an analyte of interest in the vapor phase interacts with the electron stream. Collisions between electrons and the analyte produces analyte ions, which can then be subjected to mass spectrometric techniques.

[0043] As used herein, the term "chemical ionization" or "CT" refers to the reaction of a reagent gas with a A gas (e.g., ammonia) is subjected to electron bombardment to induce interactions between reagent gas ions and analyte molecules. This refers to the manner in which analyte ions are formed by reaction.

[0044] As used herein, the term "fast atom bombardment" or "FAB" refers to a high-energy A beam of energy atoms (often Xe or Ar) bombards a non-volatile sample, The method refers to a method for desorbing and ionizing molecules contained in a sample. The test sample is glycerol, Thioglycerol, m-nitrobenzyl alcohol, 18-crown-6-crown ace ether, 2-nitrophenyl octyl ether, sulfolane, diethanolamine, and 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 radiation, which results in photoionization. in the sample through various ionization pathways including protonation, deprotonation and cluster decay. MALDI refers to a method of desorbing and ionizing analytes in a sample. - mixed with an absorbent matrix, which promotes desorption of the analyte molecules.

[0046] As used herein, "surface-enhanced laser desorption ionization" or "SELDI" The term refers to the process in which a non-volatile sample is exposed to laser radiation, which causes photoionization, protonation, and Analytes in a sample are isolated by various ionization pathways including protonation, deprotonation, and cluster decay. For SELDI, the sample is typically a single molecule of interest. The analyte or analytes are bound to a surface that preferentially retains them. As with MALDI, this The process may also use energy absorbing materials to facilitate ionization.

[0047] As used herein, the term "electrospray ionization" or "ESI" The term refers to passing a solution along a short length of capillary tube and producing a highly positive or negative charge at the end. This refers to the method by which a negative potential is applied. The solution that reaches the end of the tube is very much of the solution in the solvent vapor. It is vaporized (atomized) into a jet or spray of tiny droplets. This mist of droplets is vaporized. It flows through an evaporation chamber where it is slightly heated to prevent condensation and evaporate the solvent. As the droplets get smaller, the natural repulsion between like charges causes the ions as well as The electronic surface charge density increases to such a point that it causes neutral molecules to be released.

[0048] As used herein, the term "atmospheric pressure chemical ionization" or "APCI" APCI is a mass spectrometry technique similar to ESI, but uses ionization that occurs in a plasma at atmospheric pressure. The plasma is generated between the spray capillary and the counter electrode. The ions are then typically pumped through a set of differentially pumped skimmer stages (d The mixture is then extracted into a mass spectrometer using commercially available pumped skimmer stages. Preheat N 2 Countercurrent flow of gas may be used to improve solvent removal. Ionization can be more effective than ESI for analyzing less polar species.

[0049] The term "atmospheric pressure photoionization" or "APPI" as used herein means The mechanism for photoionization of molecule M is photon absorption and electron emission to form molecule M+. This refers to a form of mass spectrometry that uses photon energy that is usually just above the ionization potential. In many cases, the molecular ion is less susceptible to dissociation. It may be possible to analyze the variances in the number of ..., thus saving significant time and money. In the presence of water vapor or protic solvents, the molecular ion can extract H to form MH+ This tends to occur when M has a high proton affinity. The sum of MH+ and MH+ is constant, so there is no effect on the quantitative accuracy. Compounds are usually observed as MH+, whereas compounds such as naphthalene or testosterone Any non-polar compound will usually form an 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 Please refer to 3-3659.

[0050] As used herein, the term "inductively coupled plasma" or "ICP" refers to most The sample is then immersed in a partially ionized gas at a temperature high enough to atomize and ionize most of the elements. This refers to the way in which a device interacts with another device.

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

[0052] As used herein, the term "desorption" refers to the removal and / or cleavage of an analyte from a surface. or the transfer of an analyte into the gas phase.

[0053] As used herein, the term "limit of quantitation" or "LOQ" refers to the degree to which a measurement is quantitative. The analyte response at this LOQ is within 20% precision and accuracy. It is identifiable, discrete, and reproducible to a degree of 80% to 120%.

[0054] In certain preferred embodiments of the methods disclosed herein, mass spectrometry is Certain particularly preferred implementations of the methods disclosed herein are In one embodiment, mass spectrometry is performed using ESI.

[0055] In other preferred embodiments, a separately detectable internal standard is provided in the sample.

[0056] In one embodiment, the method involves a combination of LC and mass spectrometry. In some embodiments, the mass spectrometry is tandem mass spectrometry (MS / MS).

[0057] The summary of the invention described above is not limiting, and other features and advantages of the invention are described in the following text. This is apparent from the detailed description of the invention and the claims. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] Methods for quantitatively measuring tamoxifen and / or its metabolites in patient samples. This quantitative measurement is achieved by using LC-MS / MS techniques. Prior to use in C-MS / MS, samples were prepared by any of the following techniques: The first purification of tamoxifen and / or its metabolites in a sample can be carried out by protein purification. This may be accomplished by the use of catalytic purification, filtration or chromatography.

[0059] Any suitable size separation technique may be utilized, but in the example below, a first and second size Both separation techniques are filtration through molecular weight cut-off filters. As discussed in the examples, the same filter is used for both the first and second size separations. The molecular weight cutoff filter has an appropriate molecular weight cutoff so that it can be used for both It is possible to select a filter.

[0060] LC, most preferably HPLC, can be used alone or in combination with other purification methods of choice. This purification can be used in combination with MS / MS to The method provides an assay system for quantifying a selected analyte in a test sample. The amount of selected analyte in the sample is used to determine the amount of tamoxifen in the original test sample. The quantification method provided herein has increased specificity and overcomes methodological issues (antibody interference). etc.) are less susceptible to

[0061] Suitable samples 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, a cell culture, an organ culture, etc. In certain preferred embodiments, the sample is obtained from any biological source, such as The sample may be obtained from a mammal such as a dog, cat, horse, etc. Particularly preferred mammals are , primates, and most preferably humans. Particularly preferred samples include blood, plasma, serum, and urine. Examples of such samples include saliva, tears, cerebrospinal fluid, or other bodily fluid or tissue samples. For example, a patient, i.e., a living individual in a clinical context relevant to the diagnosis, prognosis, or treatment of a disease or condition. The test sample may be obtained from a human. The test sample is preferably obtained from a patient, e.g., 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. Such purification typically involves chromatography, such as liquid chromatography. and often includes further purification steps carried out prior to chromatography. Various procedures may be used depending on the type of sample or type of chromatography. Examples include filtration, centrifugation, combinations thereof, and the like.

[0063] Filtration is used to separate test samples for chromatography, particularly biological test samples such as serum or plasma. This is one preferred method of preparing the sample. The species having a larger molecular weight are separated from those having a molecular weight less than the cutoff of the filter. This is performed by filtering the test sample through a molecular weight cut-off filter to separate The test sample remaining on the filter after complete (or near complete) filtration is considered to be filtrate. Substantially free of potentially interfering species having a molecular weight below the filter cutoff .

[0064] Various methods have been described, including the use of HPLC for sample cleanup prior to mass spectrometry analysis. For example, Taylor et al., Therapeutic Drug Mon sedimentation 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, Continued) Manual C18 solid phase extraction, HPLC for chromatography on C18 analytical columns and MS / MS analysis). Those skilled in the art will recognize suitable The HPLC instrument and column may be selected based on the specifications of the column. , containing a medium (i.e., packing material) to facilitate separation (i.e., fractionation) of chemical moieties. The matrix may include microparticles. The particles may be arranged to accommodate various chemical moieties for facilitating separation of the chemical moieties. One suitable binding surface is a loosely bonded surface, such as an alkyl binding surface. The alkyl bonded surface is a C-4, C-8, or C-18 bonded alkyl surface. The chromatography column may include a cyclohexyl group, preferably a C-8 bond. and an outlet port for discharging the eluate containing the fractionated sample. Includes

[0065] In certain embodiments, the analyte of interest is reversibly retained by the column packing material. By applying the sample to the column under conditions that do not retain one or more other materials, In these embodiments, the analyte of interest is retained by the column, and the analyte can be purified. A first mobile phase condition can be used that is maintained, followed by a second mobile phase condition that is not maintained. It can be used to remove retained material from the column once the unretained material has been washed away. Alternatively, the analyte of interest may be differently expressed as compared to one or more other materials. Analytes can be purified by applying the sample to the column under mobile phase conditions that result in rapid elution. Such procedures may involve the detection of one or more components of interest relative to one or more other components of the sample. The amount of a number of analytes may be enriched.

[0066] In one embodiment, the sample to be analyzed is applied to the column at the inlet port and a solvent or The analytes of interest are eluted with a solvent mixture and discharged at the outlet port. For example, liquid chromatography can be performed in a gradient mode. Use isotropic, isocratic, or polytyptic (i.e., mixed) modes. In a preferred embodiment, the HPLC is carried out using HPLC gray water as the mobile phase. C8 was prepared using 0.2% formic acid in ultrapure water and 0.2% formic acid in 100% methanol. This is carried out on an analytical HPLC system using a solid phase.

[0067] A large number of column packings are available for the chromatographic separation of samples, allowing for suitable separation plans. The choice of protocol depends on the characteristics of the sample, the analyte of interest, the presence of interfering substances and their characteristics, etc. Commercially available HPLC columns include polar, ion exchange (cationic) both ionic and anionic), hydrophobic interactions, phenyl, C-2, C-8, C-18 and polar coatings on porous polymer columns. .

[0068] In one embodiment, the HPLC column has a median particle size of 5 μm (nominal) and In a preferred embodiment, the column dimensions are: The diameter is 1.0 mm and the length is 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, separation of materials is determined by the choice of eluent (also known as the "mobile phase"); This is achieved by the choice of gradient elution and variables such as gradient conditions, temperature, etc.

[0070] Detection and quantification by mass spectrometry In various embodiments, the analyte can be ionized by any method known to one of skill in the art. Mass spectrometry is performed using a mass spectrometer, which ionizes the fractionated sample and Contains an ion source to create charged molecules for further analysis; used in a variety of MS techniques The ion sources used are electron ionization, chemical ionization, and electrospray ionization (E SI), photon ionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoionization ionization (APPI), fast atom bombardment (FAB) / liquid secondary ionization (LSIMS), matrix Microcatalyst-assisted laser desorption / ionization (MALDI), field ionization, field desorption, thermospray - / Plasma spray ionization, Surface-enhanced laser desorption ionization (SELDI), Inductively coupled These include, but are not limited to, inductively coupled plasma (ICP) and particle beam ionization. The choice of ionization method depends on the analyte to be measured, the type of sample, the type of detector, and the It will be understood by those skilled in the art that the determination may be made based on the selection of active vs. negative modes, etc. cormorant.

[0071] In a preferred embodiment, the analyte is electrosprayed to create analyte precursor ions. In a related preferred embodiment, the analyte precursor is ionized by electrospray ionization (ESI). The collision ions are present in a gaseous state, and the inert collision gas is argon.

[0072] After the sample is ionized, the positively charged ions created thereby are analyzed to determine m / Suitable analyzers for determining m / z include quadrupole analyzers, ion traps, and the like. The ions are detected in several detection modes: For example, only selected ions can be detected using selective ion monitoring. The ions may be detected using a scanning inversion mode (SIM) or multiple ions may be detected using a scanning inversion mode (SIM). modes, such as multiple reaction monitoring (MRM) or selected reaction monitoring (SRM) In a preferred embodiment, the ions are detected using SRM. can be.

[0073] Preferably, m / z is determined using a quadrupole instrument. In an "ion trap" device, ions in an oscillating radio frequency electromagnetic field are trapped by a DC voltage applied between electrodes. The probe receives a force proportional to the position, amplitude, and m / z of the RF signal. The voltage and amplitude are Only ions with m / z of one m / z may be selected to travel the length of the quadrupole, while others Therefore, quadrupole instruments have a high probability of deflecting all ions injected into the instrument. Thus, it can act as both a "mass filter" and a "mass detector."

[0074] By using "tandem sample analysis" or "MS / MS", the resolution of the MS technique is increased. In this technique, precursor ions (also called parent ions) are generated from the molecule of interest. The precursor ions are then fragmented into a single ion. or multiple fragment ions (also called daughter ions or product ions). The precursor ion is then analyzed in a second MS step. By careful selection of the ions produced by a particular analyte, only the ions produced by that particular analyte are directed into the fragmentation chamber. The precursor ions travel to the nucleus where they are generated by collision with atoms of an inert gas. Both complex and fragment ions are produced in a reproducible manner under a given set of ionization / fragmentation conditions. Because of the large number of molecules produced, MS / MS techniques can provide a very powerful analytical tool. A combination of purification / fragmentation may be used to eliminate interfering substances and to improve the quality of complex samples such as biological samples. This may be particularly useful for

[0075] In addition, a matrix-assisted laser desorption / ionization (MAD) system coupled to a time-of-flight analyzer was developed. Recent advances in techniques such as LDI-TOF (Lapse-Doppler Time of Flight) allow for very short ion pulses. This allows for the analysis of analytes at the femtomole level. Mass spectrometers that combine They may also be combined in a manner known as "MS / MS". MS / MS / TOF, MALD SELDI / MS / MS / TOF, or SELDI / MS / MS / TOF mass spectrometry Other combinations may be used.

[0076] Mass spectrometers typically allow the user to perform an ion scan, i.e., a series of measurements over a given range (e.g., 400–1 Provides the relative abundance of each ion with a specific m / z across a range of 600 amu. The results of the assay, i.e., the mass spectrum, can be analyzed to determine the amount of analyte in the original sample by a number of methods known to those skilled in the art. For example, if sampling and analysis parameters are carefully Since the relative abundance of a given ion is controlled by the relative abundance of the original molecule, A comparison can be made to a table for conversion to logarithmic values. Alternatively, molecular standards can be run along with the samples. and a standard curve can be constructed based on the ions generated by those standards. Such a standard curve can be used to convert the relative abundance of a given ion into the absolute amount of the original molecule. In certain preferred embodiments, an internal standard is used to calculate the amount of tamoxifen. Methods for preparing and using such standard curves are known to those skilled in the art. It is well known in the art and those skilled in the art can select an appropriate internal standard. Numerous other methods of relating quantities to the amount of the original molecule are known to those of skill in the art.

[0077] One or more steps of the method may be performed using automated machinery. In certain embodiments, one or more purification steps are performed online, more preferably Alternatively, the LC purification and mass spectrometry steps can all be performed in an on-line fashion.

[0078] In certain embodiments, techniques such as MS / MS may be used to identify chromatographic sequences for further fragmentation. Precursor ions are isolated. In these embodiments, collisionally activated dissociation (CAD) is used to , which may generate fragment ions for further detection. In CAD, the precursor ions are They gain energy by colliding with an inert gas, followed by a process called "unimolecular decomposition." Sufficient energy must be deposited in the precursor ion. 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. In ETD, radical anions are used to transfer electrons to highly charged peptides. Transfer of the peptide to a cation or protein to induce random cleavage along the peptide backbone. Drop.

[0079] In a particularly preferred embodiment, the analytes are detected using LC-MS / MS as follows: The analyte-enriched sample prepared as described above is subjected to LC. The liquid solvent stream from the chromatography column is passed through the heated nebulizer of the LC-MS / MS analyzer. The solvent / analyte mixture proceeds to the riser interface, where it is converted to vapor in a heated tube at the interface. The analyte contained in the spray solvent is detected by applying a large voltage to the spray solvent / analyte mixture. The ions are ionized by a corona discharge needle on the surface of the instrument. The ions pass through an opening in the instrument and are absorbed by the first four Quadrupoles 1 and 3 (Q1 and Q3) are mass filters; Allows for the selection of ions (i.e., "precursor" and "fragment" ions) based on m / z Quadrupole 2 (Q2) is the collision cell where ions undergo fragmentation. Q1 is The m / z of the precursor ion is used to select the ion. The selected precursor ion is then passed through the collision chamber. ions with any other m / z are allowed to pass to the second member (Q2). The precursor ions that proceed to Q2 are ejected by collision with the side of Q1. It may undergo fragmentation by collision with a proton via collision-activated dissociation (CAD). Alternatively, Q2 If the precursor ions that proceed to the electron transfer solution are multiple charged cations, they The resulting fragment ions are passed to Q3, where they are In, selected fragment ions are collected while other ions are rejected.

[0080] Using standard methods well known in the art, one of skill in the art can easily identify the nucleotide sequence used for selection in Q3. It is possible to identify one or more fragment ions of a particular precursor ion that may be produced. Specific fragment ions must be formed in significant amounts by other molecules with similar molecular structures. In contrast, non-specific fragment ions are formed by molecules other than the desired analyte. Suitable specific fragment ions are those fragment ions formed by the selected analyte. Determine whether the ions are also formed by other molecules with similar structures or characteristics. The analyte can be identified by testing various molecular standards. At least one fragment ion specific to an ion having an m / z corresponding to the m / z of the on be identified.

[0081] As the ions collide with the detector, they release beams of electrons that are converted into a digital signal. The acquired data is relayed to a computer, which calculates the time The ion counts per unit area are plotted. The area under the peak corresponding to a particular ion, or The amplitude of such peaks is measured and the area or amplitude is correlated with the amount of the analyte of interest. In certain embodiments, the under-curve concentration for the fragment ions (ion(s)) and / or precursor ions is The area of ​​the peak, or the amplitude of the peak, is measured to determine the amount of analyte using m / z. Thus, the relative abundance of a given ion is determined by the peak abundance of one or more ions of an internal molecular standard. Using a calibration standard curve based on the LC-MS / MS data, the amount of the original analyte can be converted to an absolute amount. - The absolute amount of the analyte detected by MS / MS is calculated based on the absolute amount of the analyte present in the original test sample. It can be converted to logarithmic value.

[0082] In some embodiments, the methods provided herein are measured by limit of quantitation (LOQ). In some embodiments, the method for quantifying tamoxifen has a sensitivity of 5n In some embodiments, the quantification method for tamoxifen has a limit of quantification of 0.1 mg / mL or less. The method has a limit of quantitation of 4 ng / mL or less. The quantification method has a limit of quantification of 3 ng / mL or less. The method for quantification of xifene has a limit of quantification of 3 ng / mL or less. In , the quantification method for tamoxifen has a limit of quantification of 2 ng / mL or less. In this embodiment, the method for quantifying tamoxifen has a limit of quantification of 1.5 ng / mL or less. do.

[0083] In some embodiments, the method of quantification of N-desmethyl tamoxifen is 5 ng / m In some embodiments, the N-desmethyl tamoxifen has a limit of quantitation of 0.1 μg / mL or less. The quantification method has a limit of quantification of 4 ng / mL or less. The quantification method for smethyltamoxifen has a limit of quantification of 3 ng / mL or less. In some embodiments, the method for quantifying N-desmethyl tamoxifen comprises quantifying N-desmethyl tamoxifen at levels below 3 ng / mL. In some embodiments, the method for quantifying N-desmethyl tamoxifen has a limit of quantification. The method has a limit of quantitation of 2 ng / mL or less. The tamoxifen quantification method has a limit of quantification of ≦1.5 ng / mL.

[0084] In some embodiments, the method for quantifying 4'-hydroxy tamoxifen is 5 ng / In some embodiments, 4'-hydroxytamoxifen has a quantitation limit of 0.1 mL or less. The quantification method for 4ng / mL or less has a limit of quantification of 4ng / mL or less. The method for quantification of '-hydroxy tamoxifen has a limit of quantification of 3 ng / mL or less. In some embodiments, the method of quantification of 4'-hydroxy tamoxifen is 3 ng / ml In some embodiments, 4'-hydroxy tamoxifen has a limit of quantitation of 0.1 to 1.0 L. The quantification method of has a limit of quantification of 2 ng / mL or less. -The quantification method for hydroxytamoxifen has a limit of quantification of 1.5 ng / mL or less. In some embodiments, the method for quantifying 4'-hydroxy tamoxifen is 1 ng / In some embodiments, 4'-hydroxytamoxifen has a quantitation limit of 0.1 mL or less. In some embodiments, the quantification method for the agonist has a limit of quantification of 0.5 ng / mL or less. The quantification method for 4'-hydroxytamoxifen has a limit of quantification of 0.4 ng / mL or less. In some embodiments, the method for quantifying 4'-hydroxy tamoxifen comprises: It has a limit of quantification of less than 0.2ng / mL.

[0085] In some embodiments, the method of quantification of 4-hydroxy tamoxifen is 5 ng / m In some embodiments, the quantitation limit of 4-hydroxytamoxifen is 1. The quantification method has a limit of quantification of 4 ng / mL or less. The quantification method for droxitamoxifen has a limit of quantification of 3 ng / mL or less. In some embodiments, the method for quantifying 4-hydroxy tamoxifen comprises quantifying 4-hydroxy tamoxifen at levels below 3 ng / mL. In some embodiments, the method for quantifying 4-hydroxy tamoxifen has a limit of quantification. The method has a limit of quantitation of 2 ng / mL or less. Tamoxifen quantification methods have limits of quantification below 1.5 ng / mL. In embodiments, the method for quantifying 4-hydroxy tamoxifen provides a quantitative determination of 1 ng / mL or less. In some embodiments, the method for quantifying 4-hydroxy tamoxifen has limitations. In some embodiments, the 4-hydroxytryptamine has a limit of quantitation of 0.5 ng / mL or less. Tamoxifen quantification methods have limits of quantification below 0.4 ng / mL. In an embodiment, the method for quantifying 4-hydroxy tamoxifen comprises quantifying 4-hydroxy tamoxifen at 0.2 ng / mL or less. It has a limit of quantification.

[0086] In some embodiments, N-desmethyl-4-hydroxytamoxifen (endoxyl) The quantification method for sifen has a limit of quantification of 5 ng / mL or less. So how do we quantify N-desmethyl-4-hydroxytamoxifen (endoxifen)? The method has a limit of quantitation of 4 ng / mL or less. The quantification method for 4-hydroxytamoxifen (endoxifen) is below 3 ng / mL. In some embodiments, N-desmethyl-4-hydroxytamol has a lower limit of quantification. The quantification method for xifen (endoxifen) has a limit of quantification of 3 ng / mL or less. In some embodiments, N-desmethyl-4-hydroxytamoxifen (endokis The method for quantification of schizophrenia (Schizophrenia) has a limit of quantification of 2 ng / mL or less. So how do we quantify N-desmethyl-4-hydroxytamoxifen (endoxifen)? The method has a limit of quantification of 1.5 ng / mL or less. The quantification method for ethyl-4-hydroxytamoxifen (endoxifen) is 1 ng / ml In some embodiments, the N-desmethyl-4-hydroxybenzoate has a limit of quantitation of 0.1 to 1.0 L. The quantification method for tamoxifen (endoxifen) has a quantification limit of 0.5 ng / mL or less. In some embodiments, N-desmethyl-4-hydroxy tamoxifen ( The quantification method for endoxifen) has a limit of quantification of 0.4 ng / mL or less.

[0087] In some embodiments, quantification of N-desmethyl-4'-hydroxy tamoxifen The method has a limit of quantitation of 5 ng / mL or less. The quantification method for 4'-hydroxy-tamoxifen has a limit of quantification of 4 ng / mL or less. In some embodiments, the definition of N-desmethyl-4'-hydroxy tamoxifen is The quantification method has a limit of quantification of 3 ng / mL or less. The quantification method for methyl-4'-hydroxytamoxifen has a quantification limit of 3ng / mL or less. In some embodiments, N-desmethyl-4'-hydroxy tamoxifen The quantification method for N- The quantification method for desmethyl-4'-hydroxytamoxifen was designed to detect concentrations below 1.5 ng / mL. In some embodiments, N-desmethyl-4'-hydroxytamoxifen is The method for quantifying sifen has a limit of quantification of 1 ng / mL or less. The quantification method for N-desmethyl-4'-hydroxytamoxifen is 0.5 ng / ml. In some embodiments, the N-desmethyl-4'-hydroxybutyric acid The quantification method for tamoxifen has a limit of quantification of 0.4 ng / mL or less.

[0088] In some embodiments, the method for quantifying norendipine includes quantifying norendipine at or below 5 ng / mL. In some embodiments, the method for quantifying norendipine has a limit of quantification of 4 In some embodiments, the norendoxifen has a quantification limit of ng / mL or less. The quantification method has a limit of quantification of 3 ng / mL or less. The method for quantification of indoxifene has a limit of quantification of 3 ng / mL or less. In its formulation, the quantification method for norendiphene has a limit of quantification of 2 ng / mL or less. In some embodiments, the method for quantifying norendoxifenin includes determining whether the concentration is 1.5 ng / mL or less. In some embodiments, the method for quantifying norenoxifene has a limit of quantification below In some embodiments, the norendoxylanase has a limit of quantification of 1.2 ng / mL or less. The phene quantification method has a limit of quantification of 1 ng / mL or less. The method for quantification of norenoxifene has a limit of quantification of 0.5 ng / mL or less.

[0089] In some embodiments, the methods provided herein are measured by limit of detection (LOD). In some embodiments, the method for detecting tamoxifen has a sensitivity of 5 ng. In some embodiments, the method for detecting tamoxifen has a detection limit of 0.1 ml / mL or less. In some embodiments, the detection limit for tamoxifen is 4 ng / mL or less. The detection method has a detection limit of 3 ng / mL or less. The method for detecting the phenotype has a detection limit of 3 ng / mL or less. The method for detecting moxifen has a detection limit of 2 ng / mL or less. In this study, the detection method for tamoxifen has a detection limit of 1.5 ng / mL or less. In some embodiments, the method for detecting tamoxifen has a detection limit of 1 ng / mL or less. In some embodiments, the method for detecting tamoxifen comprises detecting 0.6 ng / mL or less. It has limitations.

[0090] In some embodiments, the method for detecting N-desmethyl tamoxifen is 5 ng / mL In some embodiments, the detection limit for N-desmethyl tamoxifen is The extraction method has a detection limit of 4 ng / mL or less. The detection method for tiltamoxifen has a detection limit of 3 ng / mL or less. In an embodiment, the method for detecting N-desmethyl tamoxifen has a detection limit of 3 ng / mL or less. In some embodiments, the method for detecting N-desmethyl tamoxifen comprises the steps of: In some embodiments, N-desmethyltamoxifen has a detection limit of 0.1 mg / mL or less. The method for detecting the enzyme has a detection limit of 1.5 ng / mL or less. The method for detecting N-desmethyltamoxifen has a detection limit of 1 ng / mL or less. In some embodiments, the method for detecting N-desmethyl tamoxifen comprises detecting 0.6 ng / ml It has a detection limit of less than L.

[0091] In some embodiments, the method for detecting 4'-hydroxy tamoxifen comprises detecting 5 ng / ml In some embodiments, 4'-hydroxy tamoxifen has a detection limit of 0.1 L or less. The method for detecting 4'- The detection method for hydroxytamoxifen has a detection limit of 3 ng / mL or less. In some embodiments, the method for detecting 4'-hydroxy tamoxifen comprises detecting a concentration of 3 ng / mL or less. In some embodiments, the method for detecting 4'-hydroxy tamoxifen has a detection limit. The method has a detection limit of 2 ng / mL or less. The detection method for tamoxifen has a detection limit of 1.5 ng / mL or less. In an embodiment, the method for detecting 4'-hydroxy tamoxifen comprises detecting less than 1 ng / mL. In some embodiments, the method for detecting 4'-hydroxy tamoxifen has limitations. In some embodiments, the 4'-hydroxylase has a detection limit of 0.5 ng / mL or less. The detection method for tamoxifen has a detection limit of 0.4 ng / mL or less. In an embodiment, the method for detecting 4'-hydroxy tamoxifen comprises detecting a concentration of 0.2 ng / mL or less. In some embodiments, the method for detecting 4'-hydroxy tamoxifen has a detection limit. The method has a detection limit of 0.1 ng / mL or less.

[0092] In some embodiments, the method for detecting 4-hydroxy tamoxifen is 5 ng / mL In some embodiments, the detection limit for 4-hydroxytamoxifen is The detection method has a detection limit of 4 ng / mL or less. The detection method for tamoxifen has a detection limit of 3 ng / mL or less. In an embodiment, the method for detecting 4-hydroxy tamoxifen has a detection limit of 3 ng / mL or less. In some embodiments, the method for detecting 4-hydroxy tamoxifen comprises the steps of: In some embodiments, 4-hydroxytamoxifen has a detection limit of 0.1 mg / mL or less. The method for detecting the enzyme has a detection limit of 1.5 ng / mL or less. The method for detecting 4-hydroxy tamoxifen has a detection limit of 1 ng / mL or less. In some embodiments, the method for detecting 4-hydroxy tamoxifen comprises detecting 0.5 ng / ml In some embodiments, the detection limit of 4-hydroxytamoxifen is 1. The detection method has a detection limit of 0.4 ng / mL or less. The detection method for hydroxytamoxifen has a detection limit of 0.2 ng / mL or less. In some embodiments, the method for detecting 4-hydroxy tamoxifen comprises detecting a concentration of 0.1 ng / mL It has the following detection limits:

[0093] In some embodiments, N-desmethyl-4-hydroxytamoxifen (endoxyl) In some embodiments, the detection method for .sigma.fen has a detection limit of 5 ng / mL or less. How can I detect N-desmethyl-4-hydroxytamoxifen (endoxifen)? In some embodiments, N-desmethyl-4 -Hydroxytamoxifen (endoxifen) detection method is 3ng / mL or less In some embodiments, N-desmethyl-4-hydroxytamoxifen is The detection method for endoxifen has a detection limit of 3 ng / mL or less. In some embodiments, N-desmethyl-4-hydroxytamoxifen (endoxifen) In some embodiments, the detection method for N- The detection method for desmethyl-4-hydroxytamoxifen (endoxifen) is 1.5 In some embodiments, N-desmethyl-4-hydroxyphenylalanine has a detection limit of ng / mL or less. The detection method for droxitamoxifen (endoxifen) has a detection limit of 1 ng / mL or less. In some embodiments, N-desmethyl-4-hydroxytamoxifen The detection method for (endoxifen) has a detection limit of 0.5 ng / mL or less. In some embodiments, N-desmethyl-4-hydroxytamoxifen (endoxifen) ) has a detection limit of 0.4 ng / mL or less. In some embodiments, The detection method for N-desmethyl-4-hydroxytamoxifen (endoxifen) is In some embodiments, the N-desmethyl-4 -Hydroxytamoxifen (endoxifen) detection method is 0.15ng / mL or less has a lower detection limit.

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

[0095] In some embodiments, the method for detecting norenoxifen comprises detecting a concentration of 5 ng / mL or less. In some embodiments, the detection method for norendoxife has an extinction limit of 4 ng. In some embodiments, the detection of norenoxifen has a limit of detection of 0.1 ml / mL or less. The method has a detection limit of 3 ng / mL or less. The method for detecting Shifen has a detection limit of 3 ng / mL or less. , several methods for detecting norenoxifen have detection limits of 2 ng / mL or less. In one embodiment, the method for detecting norenoxifen has a detection limit of 1.5 ng / mL or less. In some embodiments, the method for detecting norendoxifenin has a concentration of 1.2 ng / mL. In some embodiments, the method for detecting norenoxifen has a detection limit of less than 1 mL. The method has a detection limit of 1 ng / mL or less. The detection method for phene has a detection limit of 0.5 ng / mL or less.

[0096] The following examples serve to illustrate the invention. These examples are not to be construed as limiting the scope of the invention in any way. It is not intended to limit the scope of the methods. EXAMPLES

[0097] Determination of tamoxifen and its metabolites In the following procedure, tamoxifen and its metabolites are extracted from serum. After addition to a filter plate and subsequent mixing with acetonitrile / IS, a precipitate was formed. The mixture is then placed on a positive pressure manifold to force the organic fraction through to a collection plate. After the plate was covered, it was inserted into the Cohesive system for injection onto the MS / MS. ) and the MS was in APCI positive mode. Quantification was performed using specific parent- Analytes with similar migration were separated chromatographically.

[0098] [Table 1]

[0099] [Table 2]

[0100] [Table 3]

[0101] Calibrators / standards used A 12-point calibration is used for each analyte. Initially, only one standard is made ( Std-12), and serial dilutions are performed to generate the remaining standards. 2) should be removed from the -70°C freezer and allowed to thaw. While thawing, Label four 12 x 75 mm tubes.

[0102] Add 3.0 mL of std-12 to tube 12. From this standard, the following table shows A standard curve is generated accordingly. Standards should be generated with each assay. Place the original standards back into the -60 to -90°C freezer.

[0103] [Table 4]

[0104] Once these analytes have been added, a sufficient amount of Biocell serum is used to Mix and then dispense into a 15 mL centrifuge tube. 12) After that, place the tube in a freezer at -60 to -90°C for storage. It is stable for one year. .

[0105] Target concentration of reference material

[0106] [Table 5]

[0107] [Table 6]

[0108] [Table 7]

[0109] [Table 8]

[0110] Equipment and Supplies

[0111] Assay Platform This assay was performed using a Thermo LC / MS / MS system containing the following modules: Used:

[0112] [Table 9]

[0113] [Table 10]

[0114] [Table 11]

[0115] [Table 12]

[0116] Prediction

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

[0118] Analytical Measurement Range (AMR) Tamoxifen: 1.47 to 1500 ng / mL N-desmethyltamoxifen: 1.47 to 1500 ng / mL 4'-Hydroxy tamoxifen: 0.2 to 200 ng / mL 4-Hydroxytamoxifen: 0.2 to 200 ng / mL N-desmethyl-4-hydroxytamoxifen (endoxifen): 0.39-4 00ng / mL N-desmethyl-4'-hydroxytamoxifen: 0.39 to 400 ng / mL

[0119] Precision: Inter-assay (time period of assay) and intra-assay (day-to-day) precision studies were performed versus low, medium and high All analytes showed less than 12% change over the validation period. The dynamic coefficients are shown.

[0120] Interfering substances: Mild or moderate icteric and lipemic samples are acceptable. Hemolytic samples are , are unacceptable as they clog filters during sample preparation. Icteric and lipemic samples are unacceptable.

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

[0122] Validation of Tamoxifen and Its Metabolites Assays This report includes the LC / MS / MS analysis of tamoxifen and its five key Phase 1 A detailed overview of validation for metabolites is included. Assays are laboratory developed and This is a test.

[0123] Methods: Tamoxifen and its five major phase 1 metabolites (N-desmethyltamoxifen) were Tamoxifen, N-desmethyl-4-hydroxytamoxifen, N-desmethyl-4'-hydroxy Roxitamoxifen, 4-hydroxytamoxifen, and 4'-hydroxytamoxifen The serum is extracted with sucrose (Sifen). The extraction is by protein precipitation followed by filtration. Then, the analysis and quantification is performed by LC / MS / MS.

[0124] [Table 13]

[0125] Precision studies on laboratory developed tests (LDTs) Within-run precision: Low, medium, and high controls were analyzed within one run (10). All QCs were The results were within the acceptable range (coefficient of variation less than 20%).

[0126] [Table 14]

[0127] [Table 15]

[0128] [Table 16]

[0129] Total Precision: Total precision is based on all QC runs for all assays during the validation process. Acceptance was based on a coefficient of variation of less than 20%. All QC was within this criterion. It's settled.

[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 time to take the low pool (containing all of the analyte) and This was performed by extracting 1:2 of 100% ... Assuming that linearity continues below the limit of quantitation (LOQ) level, the following values ​​are obtained: This would be the lowest quantifiable concentration. The experiment was carried out over 5 days. Tamoxifen: 0.59ng / mL N-desmethyltamoxifen: 0.59 ng / mL 4'-hydroxytamoxifen: 0.1ng / mL N-desmethyl-4'-hydroxytamoxifen: 0.5ng / mL 4-hydroxytamoxifen: 0.1ng / mL N-desmethyl-4-hydroxytamoxifen (endoxifen): 0.15ng / mL

[0134] Limit of Quantitation (LOQ): The acceptance criterion for LOQ is the lowest concentration with a coefficient of variation of less than 20%. To determine the LOQ, the mid-level standard was diluted 1:2. I explained. Tamoxifen: 1.5ng / mL N-desmethyltamoxifen: 1.5 ng / mL 4'-hydroxytamoxifen: 0.4ng / mL N-desmethyl-4'-hydroxytamoxifen: 0.4ng / mL 4-hydroxytamoxifen: 0.2ng / mL N-desmethyl-4-hydroxytamoxifen (endoxifen): 0.4ng / mL

[0135] Accuracy Recoveries of known standards: Serum was spiked with all analytes to the specified concentrations, extracted, and then run in triplicate. All mixes were spiked to cover the linear and therapeutic range of each analyte. It was done.

[0136] [Table 20]

[0137] Interference studies Acceptance Criteria: Differences due to potential interfering substances must be greater than TEa / It should be 4 or less.

[0138] Hemolysis interference: Low and high pools were spiked with hemolyzed RBCs at low, medium and high concentrations. Samples were extracted in quadruplicate. Hemolysis indicated interference with tamoxifen or the metabolites measured. However, due to the difficulty of filtering from moderately and highly hemolyzed samples, Only hemolyzed samples should be acceptable.

[0139] [Table 21]

[0140] Lipidemia interference: The low and high pools were spiked with lipidemia samples at low, medium and high concentrations. Samples were extracted in quadruplicate. Lipidemia samples were diluted with tamoxifen or the metabolites being measured. showed no interference.

[0141] [Table 22]

[0142] Bilirubin interference: To the low and high pools, bilirubin was added at low, medium and high concentrations. Samples were extracted in quadruplicate. Samples spiked with bilirubin were treated with either tamoxifen or No interference from metabolites was observed.

[0143] [Table 23] EXAMPLES

[0144] Validation of the Norendoxifen Assay This report includes a validation study of norendoxifen by LC / MS / MS. Contains a detailed overview of: Assays are laboratory developed tests.

[0145] Norendoxifene is extracted from the serum using protein precipitation followed by filtration. , analysis and quantification is performed by LC / MS / MS.

[0146] [Table 24]

[0147] Precision studies on laboratory developed tests (LDTs) Intra-run precision: Low, medium, and high controls (ng / mL) were analyzed within a single run (10). All QCs fell within the acceptance criteria (coefficient of variation less than 20%).

[0148] [Table 25]

[0149] Total Precision: Total precision is based on all QC runs for all assays during the validation process. Acceptance was based on a coefficient of variation of less than 20%. All QC was within this criterion. It's settled.

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

[0151] Limit of quantification (LOQ): Norendoxifen = 1.2 ng / mL

[0152] Accuracy Recovery of known standards: Serum was spiked with all analytes to the specified concentrations, extracted, and then analyzed in quadruplicate. All mixes were spiked to cover the linear and therapeutic range of each analyte. It was done.

[0153] [Table 26]

[0154] Interference studies Acceptance Criteria: Differences due to potential interfering substances must be greater than TEa / It should be 4 or less.

[0155] Hemolysis interference: Low and high pools were spiked with hemolyzed RBCs at low, medium and high concentrations. Samples were extracted in quadruplicate. Hemolysis showed no interference with norendoxifen. However, due to the difficulty of filtering from moderately and highly hemolyzed samples, only mildly hemolyzed samples were filtered. It should be tolerated.

[0156] [Table 27]

[0157] Lipidemia interference: The low and high pools were spiked with lipidemia samples at low, medium and high concentrations. Samples were extracted in quadruplicate. Lipidemia samples showed no interference with norendoxifen. Recoveries of known standards: Serum was spiked with all analytes to a specific concentration and extracted. Quadruplicates were analyzed. All mixes were run to cover the linear and therapeutic range of each analyte. Added.

[0158] [Table 28]

[0159] Bilirubin interference: To the low and high pools, bilirubin was added at low, medium and high concentrations. Samples were extracted in quadruplicate. Samples spiked with bilirubin were used to reduce interference with norenoxifen. did not show any.

[0160] [Table 29] EXAMPLES

[0161] Clinical quantification and response studies Tamoxifen in patient samples was detected using the standard operating protocols of Examples 1 to 3. The phenotype and its metabolites were quantified and correlated with tamoxifen response.

[0162] [Table 30] JPEG2025081382000031.jpg242106

[0163] [Table 31] JPEG2025081382000033.jpg203161

[0164] [Table 32] JPEG2025081382000035.jpg242106

[0165] [Table 33] JPEG2025081382000037.jpg242109

[0166] [Table 34] JPEG2025081382000039.jpg241118

[0167] [Table 35] JPEG2025081382000041.jpg241108

[0168] [Table 36] JPEG2025081382000043.jpg202150

[0169] [Table 37] JPEG2025081382000045.jpg242116

[0170] [Table 38] JPEG2025081382000047.jpg203153

[0171] [Table 39] JPEG2025081382000049.jpg241110

[0172] [Table 40] JPEG2025081382000051.jpg202143

[0173] [Table 41] EXAMPLES

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

[0175] [Table 42] JPEG2025081382000054.jpg230156JPEG2025081382000055.jpg224156

[0176] [Table 43] JPEG2025081382000057.jpg227158JPEG2025081382000058.jpg199157

[0177] [Table 44] JPEG2025081382000060.jpg229163JPEG2025081382000061.jpg209164

[0178] [Table 45] JPEG2025081382000063.jpg230153

[0179] The articles, patents, patent applications and all other documents and electronic documents mentioned or cited herein are The contents of the publicly available information are detailed below with each publication incorporated by reference. Each of the above-mentioned references is incorporated by reference in its entirety to the same extent as if each of the above-mentioned references was individually and explicitly indicated. Applicants hereby disclaim all liability in connection with any such articles, patents, patent applications, or other physical and We reserve the right to physically incorporate any and all materials and information from electronic documents.

[0180] The methods illustratively described herein may be used in combination with any element not specifically disclosed herein. The present invention may be suitably carried out in the absence of the limitation(s). For example, terms such as "comprises," "includes," and "contains" are used broadly and without limitations. Moreover, the terms and expressions used in this specification are intended to be used for explanatory purposes only. It is used as a general term and not as a term of limitation, shown and described. The intention of the use of such terms and expressions to the exclusion of any equivalents or parts thereof of the features It will be appreciated that various modifications are possible within the scope of the invention as claimed. Thus, the present invention has been specifically disclosed by preferred embodiments and optional features. However, modifications and variations of the invention embodied herein disclosed may occur to those skilled in the art. and such modifications and variations are deemed to be within the scope of the present invention. should be understood.

[0181] The invention has been described broadly and comprehensively herein. Each narrower species and subgeneric classification also forms part of the method. Removal of any subject matter from the genus, whether or not the material is specifically recited herein, is not permitted. The present invention includes a comprehensive description of the method, subject to any proviso or limitation.

[0182] Other embodiments are within the scope of the following claims. has been described in terms of Markush groups, and the present invention also provides It will be understood by those of skill in the art that the present invention is described in terms of any individual member or subgroup of members of .

Claims

1. Tamoxifen and N-desmethyltamoxifen in human samples in a single mass spectrometric assay 1. A method for determining an amount of moxifen, comprising: (a) purifying the sample by liquid chromatography; (b) ionizing the tamoxifen and N-desmethyl tamoxifen to obtain a mass spectrometry result; analyzing to produce one or more detectable ions; (c) detecting the amount of said ions from step (b) by mass spectrometry, The amount of the ions detected represents the amount of tamoxifen and N-desmethyltamoxifen in the sample. Related to each amount of Shifen, Tamoxifen and N-desmethyltamoxifen at 1.5 ng / mL or less A method having a limit of quantification.

2. 10. The method of claim 1, further comprising protein precipitation prior to step (a).

3. The method of claim 1 further comprising filtering prior to step (a).

4. The liquid chromatography is high pressure liquid chromatography (HPLC). The method according to claim 1.

5. The liquid chromatography is high turbulence liquid chromatography (HTLC); The method of claim 1.

6. The method of claim 1 , further comprising detecting the amount of an internal standard.

7. The method of claim 1 , wherein the ionization is in positive ion mode.

8. The method of claim 1 , wherein the sample is a serum or plasma sample.

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

10. Norendoxifen, endoxifen, 4'-hydroxytamoxifen, 4-hydroxy From droxytamoxifen, and N-desmethyl-4'-hydroxytamoxifen 10. The method of claim 1, further comprising determining the amount of a metabolite selected from the group consisting of: 。

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