Carryover-free method for semaglutide quantification

Abstract

This invention provides a method and composition for separating, purifying, detecting, measuring, or analyzing a sample using liquid chromatography. [Solution] The sampling means of the liquid chromatography system is rinsed with a first rinsing solution. The first rinsing solution contains two or more solvents selected from the group consisting of trifluoroacetic acid, ammonia, formic acid, acetonitrile, methanol, water, and 2-propanol. Furthermore, the sampling means is rinsed with a second rinsing solution. The second rinsing solution contains one or more solvents selected from the group consisting of N,N-dimethyl sulfoxide (DMSO), acetonitrile, methanol, trifluoroacetic acid, formic acid, ammonium, and water.

Description

Detailed Description of the Invention

[0001] Background Analysis of synthetic peptides such as semaglutide using high-performance liquid chromatography (HPLC) followed by mass spectrometry (MS) is extremely difficult, among other reasons, due to nonspecific adsorption observed in the parts of the HPLC autosampler and the analytical column. This results in a large carryover of material analysis and limits the analytical method from achieving the desired limit of quantitation (LOQ). Conventional approaches involve the use of highly affinity solvents such as water, organic solvents, harsh solvents or harsh aqueous solutions having a specific pH, mixtures of solvents, etc. to remove contamination from the previous injection, but it is an approach one by one at a time.

[0002] In addition, when analyzing higher concentrations of such molecules, even more carryover occurs. To avoid this situation, many conventional LC-MS / MS methods cover a limited quantitative calibration range. Methods with a small calibration range have limited applicability to formulations with a wide range of label claims or dose concentrations, in terms of the study of its bioavailability and bioequivalence (BABE) in drug development. Therefore, there is a need for a new sensitive, accurate and robust analytical method for quantifying synthetic peptides such as semaglutide using LC-MS / MS without carryover of contaminants from the previous injection and with a wide dynamic range, which is applicable to formulations with a wide range of dose concentrations.

[0003] Summary In one embodiment, the disclosure provides a method for detecting, measuring, or analyzing a sample using liquid chromatography, the method comprising rinsing the sampling means of a liquid chromatography system with a first rinse solution. In some embodiments, the method further comprises rinsing the sampling means with a second rinse solution. In one embodiment, the disclosure provides a composition for detecting, measuring, or analyzing a sample using liquid chromatography as described herein.

[0004] Brief explanation of the drawing The accompanying drawings, incorporated into and constituting part of this disclosure, illustrate exemplary embodiments and, together with the description, serve to illustrate the principles disclosed. [Brief explanation of the drawing]

[0005] [Figure 1] Figure 1 shows the internal rinsing of the needle using rinsing solutions (R0, R1, and R2). [Figure 2] Figure 2 shows diagrams of the internal and external rinse processes. [Figure 3] Figure 3 shows the structure of semaglutide. [Figure 4] Figure 4 shows the analysis conditions. [Figure 5] Figure 5 shows the chromatograms of the blank, LLOQ, ULOQ, and calibration curves. [Figure 6] Figure 6 shows a diagram of the sample analysis. [Figure 7] Figure 7 shows the system accuracy and specificity. [Figure 8] Figure 8 shows the linearity range. [Figure 9] Figure 9 shows the results for accuracy and precision. [Figure 10] Figure 10 shows chromatographic overlays of blank, LLOQ, and ULOQ to demonstrate that there is no carryover after the ULOQ sample. [Figure 11] Figure 11 shows the results of the blank analysis. [Figure 12-1] Figure 12 shows the methods report. [Figure 12-2] Figure 12 shows the methods report. [Figure 12-3] Figure 12 shows the methods report. [Figure 12-4] Figure 12 shows the methods report. [Figure 12-5] Figure 12 shows the methods report. [Figure 12-6] Figure 12 shows the methods report. [Figure 13] Figure 13 shows the chromatography results for semaglutide. [Figure 14-1] Figure 14 shows the chromatography results. [Figure 14-2] Figure 14 shows the chromatography results. [Figure 14-3] Figure 14 shows the chromatography results. [Figure 14-4] Figure 14 shows the chromatography results. [Figure 14-5] Figure 14 shows the chromatography results. [Figure 14-6] Figure 14 shows the chromatography results. [Figure 14-7] Figure 14 shows the chromatography results.

[0006] Detailed explanation The following provides a more detailed explanation of this disclosure to aid in understanding it.

[0007] Unless otherwise specified, "%v / v" as used herein represents a volume percentage based on the total volume of the standard, unless otherwise stated.

[0008] When the term "about" is used, it is used to mean a particular effect or result obtained within a specified tolerance, and the method of obtaining that tolerance is well known to those skilled in the art. When the term "about" is used in describing a range of values or endpoints, the present disclosure must be understood as including the particular values or endpoints shown. In one aspect, the term "about" means plus or minus 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the value of the number to which the term is applied.

[0009] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", or any other variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises the recited elements is not necessarily limited to only those elements, and may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0010] As used herein, the phrase "sampling means" can include any hardware, component, part, or apparatus that can be used to collect a sample for analysis using liquid chromatography, examples of which include needles, pipettes, syringes, tubes, and their equivalents.

[0011] The transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim and prohibits the claim from including materials other than those recited, except for the usual accompanying impurities. When the phrase "consist of" is in the body of the claim rather than immediately following the preamble, it limits only the elements recited in that clause, and other elements are not excluded from the claim as a whole.

[0012] The transitional phrase "consisting essentially of" limits the scope of the claim to the recited materials or steps and those that do not substantially affect the basic and novel characteristics (s) of the claimed invention. A "consisting essentially of" claim lies midway between a closed claim written in the "consisting of" format and a fully open claim drafted in the "comprising" format. Any suitable level of additives, and trace impurities, as specified herein, are not excluded from the composition by the term "consisting essentially of".

[0013] Further, "or" and "and / or" mean inclusive rather than exclusive, unless otherwise clearly stated. For example, the condition A or B, or A and / or B is satisfied by any one of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

[0014] The use of "a" or "an" to describe various elements and components herein is for convenience only and is meant to convey the general sense of the disclosure. This description must be construed to include one or at least one, and the singular form includes the plural form unless it is obvious otherwise.

[0015] As used herein, the term "chromatography" means a process in which a mixture of chemical substances carried by a liquid or gas is separated into components as a result of the specific distribution of the chemical substances as they flow around or over a stationary liquid or solid phase.

[0016] As used herein, the terms “liquid chromatography” or “LC” refer to the process of selectively delaying one or more components of a fluid solution as it uniformly permeates through a column of finely divided substances or through capillary channels.

[0017] This delay arises from the distribution of the components of a mixture between one or more stationary phases and a bulk fluid (i.e., mobile phase) as the fluid moves relative to the stationary phases. Examples of "liquid chromatography" include reverse-phase liquid chromatography (RPLC), high-performance liquid chromatography (HPLC), and turbulent flow liquid chromatography (TFLC) (also known as high-turbulence liquid chromatography (HTLC) or high-throughput liquid chromatography).

[0018] As used herein, the terms “mass spectrometry” or “MS” mean an analytical technique for identifying compounds by their mass. MS means a method of filtering, detecting, and measuring ions based on their mass-to-charge ratio, i.e., “m / z”. MS techniques generally include (1) ionizing a compound to form a charged compound, and (2) detecting the molecular weight of the charged compound and calculating its mass-to-charge ratio. Ionization and detection of compounds can be carried out by any suitable means. A “mass spectrometer” generally includes an ionizer, a mass spectrometer, and an ion detector. Generally, one or more target molecules are ionized, the ions are then introduced into a mass spectrometer, where, by a combination of magnetic and electric fields, the ions follow a path in space that depends on their mass ("m") and charge ("z").

[0019] In one embodiment, the disclosure provides a method for separating, purifying, detecting, measuring, or analyzing a sample. In some embodiments, the method uses liquid chromatography. In some embodiments, the method includes rinsing the sampling means of the liquid chromatography with a first rinse solution.

[0020] In some embodiments, this method involves separating or purifying a sample, or components of the sample, such as proteins. In some embodiments, the sample or protein may be semaglutide.

[0021] In some embodiments, the method includes detecting, measuring, or analyzing the presence or absence, quantity, concentration, or mass of a sample or a component therein, such as a protein. In some embodiments, the sample or protein may be a semaglutide.

[0022] In some embodiments, detection may involve using a detector to identify and / or quantify the compounds separated in the column. In some embodiments, the compounds include a sample or components of the sample, such as proteins. In some embodiments, the sample or protein may be semaglutide.

[0023] "Sample" means a quantity of material from a biological, environmental, medical, or patient source that is the subject of separation, purification, detection, measurement, or analysis. On the one hand, it means including specimens or cultures (e.g., microbiological cultures). On the other hand, it means including both biological and environmental specimens. A specimen may include specimens of synthetic origin. Environmental specimens include environmental substances such as surface materials, soil, water, and industrial specimens, as well as specimens obtained from food and dairy processing equipment, apparatus, instruments, tools, disposable and non-disposable items. A specimen may be obtained from sources including, but not limited to, whole blood, serum, plasma, urine, saliva, sweat, feces, tears, intestinal fluid, mucosal specimens, lung tissue, tumors, transplanted organs, fetuses, and / or other sources. A specimen may originate from animals, including humans, fluids, solids (e.g., feces), or tissues. The sample may include, but is not limited to, cultures, blood, saliva, cerebrospinal fluid, pleural fluid, lactation, lymph, sputum, semen, needle aspirates, and other substances collected from a patient. In some embodiments, the sample contains semaglutide contained in the above-mentioned biological sample. In some embodiments, the sample consists of semaglutide contained in the biological sample. In some embodiments, the sample is semaglutide contained in the biological sample.

[0024] In some embodiments, the first rinse solution contains one, two or more, three or more, or four or more solvents from the group consisting of trifluoroacetic acid, ammonia, formic acid, acetonitrile, methanol, water, and 2-propanol. In some embodiments, the first rinse solution contains trifluoroacetic acid. In some embodiments, the first rinse solution contains ammonia. In some embodiments, the first rinse solution contains formic acid. In some embodiments, the first rinse solution contains acetonitrile. In some embodiments, the first rinse solution contains methanol. In some embodiments, the first rinse solution contains water. In some embodiments, the first rinse solution contains 2-propanol. In some embodiments, the first rinse solution further contains at least one selected from the group consisting of acetonitrile, methanol, water, and 2-propanol. In some embodiments, the first rinse solution further contains acetonitrile. In some embodiments, the first rinse solution further contains methanol. In some embodiments, the first rinse solution further contains water. In some embodiments, the first rinse solution further comprises 2-propanol. In some embodiments, the first rinse solution comprises an aqueous solvent and an organic solvent.

[0025] In some embodiments, the first rinse solution contains trifluoroacetic acid. In some embodiments, the first rinse solution contains 1% v / v trifluoroacetic acid. In some embodiments, the first rinse solution contains at least 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, or 50% v / v trifluoroacetic acid. In some embodiments, the first rinse solution contains up to 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1%, or 0.5% v / v trifluoroacetic acid. In some embodiments, the first rinse solution is approximately 0.1% to 50%, approximately 0.1% to 40%, approximately 0.1% to 30%, approximately 0.1% to 20%, approximately 0.1% to 10%, approximately 0.1% to 5%, approximately 0.1% to 3%, approximately 0.1% to 1%, approximately 0.5% to 50%, approximately 0.5% to 40%, approximately 0.5% to 30%, approximately 0.5% to 20% Contains trifluoroacetic acid in %, approximately 0.5% to 10%, approximately 0.5% to 5%, approximately 0.5% to 3%, approximately 0.5% to 1.5%, approximately 0.5% to 1%, approximately 1% to 50%, approximately 1% to 40%, approximately 1% to 30%, approximately 1% to 20%, approximately 1% to 10%, approximately 1% to 5%, approximately 1% to 4%, approximately 1% to 3%, or approximately 1% to 2% v / v.

[0026] In some embodiments, the first rinse solution contains 1% v / v of a solvent from the group consisting of trifluoroacetic acid, ammonia, formic acid, acetonitrile, methanol, water, and 2-propanol. In some embodiments, the first rinse solution contains at least 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, or 50% v / v of a solvent from the group consisting of trifluoroacetic acid, ammonia, formic acid, acetonitrile, methanol, water, and 2-propanol. In some embodiments, the first rinse solution contains a solvent from the group consisting of trifluoroacetic acid, ammonia, formic acid, acetonitrile, methanol, water, and 2-propanol in amounts of up to 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1%, or 0.5% v / v. In some embodiments, the first rinse solution contains a solvent from the group consisting of trifluoroacetic acid, ammonia, formic acid, acetonitrile, methanol, water, and 2-propanol in concentrations of approximately 0.1% to 50%, approximately 0.1% to 40%, approximately 0.1% to 30%, approximately 0.1% to 20%, approximately 0.1% to 10%, approximately 0.1% to 5%, approximately 0.1% to 3%, approximately 0.1% to 1%, and approximately 0.5% to 5%. Includes 0%, approximately 0.5% to 40%, approximately 0.5% to 30%, approximately 0.5% to 20%, approximately 0.5% to 10%, approximately 0.5% to 5%, approximately 0.5% to 3%, approximately 0.5% to 1.5%, approximately 0.5% to 1%, approximately 1% to 50%, approximately 1% to 40%, approximately 1% to 30%, approximately 1% to 20%, approximately 1% to 10%, approximately 1% to 5%, approximately 1% to 4%, approximately 1% to 3%, or approximately 1% to 2% v / v.

[0027] In some embodiments, the first rinse solution further comprises at least one selected from the group consisting of acetonitrile, methanol, water, and 2-propanol. In some embodiments, the first rinse solution further comprises acetonitrile, methanol, water, and 2-propanol. In some embodiments, the first rinse solution comprises equivolutes of acetonitrile, methanol, water, and 2-propanol. In some embodiments, the first rinse solution comprises unequal volumes of acetonitrile, methanol, water, and 2-propanol.

[0028] In some embodiments, rinsing the sampling means with the first rinse solution includes purging the first rinse solution from the sampling means. In some embodiments, rinsing the sampling means with the first rinse solution further includes rinsing the injection port.

[0029] In some embodiments, the first rinse fluid can be used as an internal rinse. In some embodiments, the first rinse fluid can be used as an external rinse. In some embodiments, the first rinse fluid can rinse the autosampler flow path.

[0030] In some embodiments, the method further includes rinsing the sampling means with a second rinse solution. In some embodiments, the second rinse solution comprises one, two or more, three or more, or four or more solvents selected from the group consisting of N,N-dimethyl sulfoxide (DMSO), acetonitrile, methanol, trifluoroacetic acid, formic acid, and ammonium, and water. In some embodiments, the second rinse solution comprises DMSO. In some embodiments, the second rinse solution comprises acetonitrile. In some embodiments, the second rinse solution comprises methanol. In some embodiments, the second rinse solution comprises trifluoroacetic acid. In some embodiments, the second rinse solution comprises formic acid and ammonium. In some embodiments, the second rinse solution comprises formic acid. In some embodiments, the second rinse solution comprises ammonium. In some embodiments, the second rinse solution comprises water. In some embodiments, the second rinse solution can be used to clean the outside of the needle.

[0031] In some embodiments, the second rinse solution contains about 5% v / v DMSO. In some embodiments, the second rinse solution contains at least 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, or 50% v / v DMSO. In some embodiments, the second rinse solution contains up to 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1%, or 0.5% v / v DMSO. In some embodiments, the second rinse solution is approximately 0.1% to 50%, approximately 0.1% to 40%, approximately 0.1% to 30%, approximately 0.1% to 20%, approximately 0.1% to 10%, approximately 0.1% to 5%, approximately 0.1% to 3%, approximately 0.1% to 1%, approximately 0.5% to 50%, approximately 0.5% to 40%, approximately 0.5% to 30%, approximately 0.5% to 20%, approximately 0.5% to 10%, approximately 0.5% to 5%, approximately 0.5% to Contains 3%, approximately 0.5% to 1.5%, approximately 0.5% to 1%, approximately 1% to 50%, approximately 1% to 40%, approximately 1% to 30%, approximately 1% to 20%, approximately 1% to 10%, approximately 1% to 5%, approximately 1% to 4%, approximately 1% to 3%, or approximately 1% to 2%, approximately 3% to 50%, approximately 3% to 40%, approximately 3% to 30%, approximately 3% to 20%, approximately 3% to 10%, approximately 3% to 5%, or approximately 3% to 4% v / v DMSO.

[0032] In some embodiments, rinsing the sampling means with a second rinse solution includes immersing a portion of the sampling means in the second rinse solution. In some embodiments, rinsing the sampling means with a second rinse solution further includes agitating or pumping the second rinse solution while a portion of the sampling means is immersed in it.

[0033] In some embodiments, the method further includes introducing a sample into a liquid chromatography column via a sampling means and / or performing liquid chromatography using the liquid chromatography column.

[0034] In some embodiments, the method further includes performing mass spectrometry on the eluent from liquid chromatography. The eluent from liquid chromatography can be detected, measured, or analyzed by other methods, such as UV detection. In some embodiments, the eluent from liquid chromatography can be detected, measured, or analyzed by other methods, such as a UV-Vis spectrophotometer using a photodiode array detector ("UV detector").

[0035] In some embodiments, the sample contains a peptide or protein. In some embodiments, the sample contains a peptide or protein contained in a biological sample. In some embodiments, the sample consists of a peptide or protein. In some embodiments, the sample is a peptide contained in a biological sample. In some embodiments, the sample contains a synthetic peptide or protein. In some embodiments, the sample contains a synthetic peptide or protein contained in a biological sample. In some embodiments, the sample consists of a synthetic peptide or protein contained in a biological sample. In some embodiments, the sample is a synthetic peptide or protein contained in a biological sample. In some embodiments, the sample contains a non-synthetic, naturally occurring peptide or protein. In some embodiments, the sample contains a naturally occurring peptide or protein contained in a biological sample. In some embodiments, the sample consists of a naturally occurring peptide or protein contained in a biological sample. In some embodiments, the sample is a naturally occurring peptide or protein contained in a biological sample. In some embodiments, the sample contains semaglutide.

[0036] In some embodiments, liquid chromatography includes high-performance liquid chromatography (HPLC). In some embodiments, liquid chromatography includes fast protein liquid chromatography (FPLC). In some embodiments, liquid chromatography includes liquid-liquid chromatography.

[0037] In some embodiments, the column conditioning solution comprises one or more solvents selected from the group consisting of DMSO, acetonitrile, methanol, and formic acid. In some embodiments, the column conditioning solution comprises DMSO. In some embodiments, the column conditioning solution comprises acetonitrile. In some embodiments, the column conditioning solution comprises methanol. In some embodiments, the column conditioning solution comprises formic acid.

[0038] In some embodiments, the column conditioning solution contains 0.5% v / v formic acid. In some embodiments, the column conditioning solution contains 1% v / v formic acid in water. In some embodiments, the column conditioning solution contains 1% v / v formic acid in methanol. In some embodiments, the column conditioning solution contains at least 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, or 50% v / v formic acid. In some embodiments, the column conditioning solution contains up to 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1%, or 0.5% v / v formic acid. In some embodiments, the column conditioning solution is approximately 0.1% to 50%, approximately 0.1% to 40%, approximately 0.1% to 30%, approximately 0.1% to 20%, approximately 0.1% to 10%, approximately 0.1% to 5%, approximately 0.1% to 3%, approximately 0.1% to 1%, approximately 0.5% to 50%, approximately 0.5% to 40%, approximately 0.5% to 30%, approximately 0.5% to 20%, approximately 0.5% to 10%, approximately 0.5% to 5%, approximately 0 Contains 0.5% to approximately 3%, approximately 0.5% to approximately 1.5%, approximately 0.5% to approximately 1%, approximately 1% to approximately 50%, approximately 1% to approximately 40%, approximately 1% to approximately 30%, approximately 1% to approximately 20%, approximately 1% to approximately 10%, approximately 1% to approximately 5%, approximately 1% to approximately 4%, approximately 1% to approximately 3%, or approximately 1% to approximately 2%, approximately 3% to approximately 50%, approximately 3% to approximately 40%, approximately 3% to approximately 30%, approximately 3% to approximately 20%, approximately 3% to approximately 10%, approximately 3% to approximately 5%, or approximately 3% to approximately 4% v / v formic acid.

[0039] In some embodiments, the column conditioning solution contains 1.0% v / v DMSO. In some embodiments, the column conditioning solution contains at least 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, or 50% v / v DMSO. In some embodiments, the column conditioning solution contains up to 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1%, or 0.5% v / v DMSO. In some embodiments, the column conditioning solution is available in concentrations of approximately 0.1% to 50%, 0.1% to 40%, 0.1% to 30%, 0.1% to 20%, 0.1% to 10%, 0.1% to 5%, 0.1% to 3%, 0.1% to 1%, 0.5% to 50%, 0.5% to 40%, 0.5% to 30%, 0.5% to 20%, 0.5% to 10%, 0.5% to 5%, and 0. Contains DMSO in amounts of 5% to approximately 3%, approximately 0.5% to approximately 1.5%, approximately 0.5% to approximately 1%, approximately 1% to approximately 50%, approximately 1% to approximately 40%, approximately 1% to approximately 30%, approximately 1% to approximately 20%, approximately 1% to approximately 10%, approximately 1% to approximately 5%, approximately 1% to approximately 4%, approximately 1% to approximately 3%, or approximately 1% to approximately 2%, approximately 3% to approximately 50%, approximately 3% to approximately 40%, approximately 3% to approximately 30%, approximately 3% to approximately 20%, approximately 3% to approximately 10%, approximately 3% to approximately 5%, or approximately 3% to approximately 4% v / v.

[0040] In some embodiments, the sample exhibits a considerable amount of nonspecific adsorption on the liquid chromatography channel. In some embodiments, the sample is soluble in DMSO. In some embodiments, the sample is soluble in a second rinse solution.

[0041] In some embodiments, the compositions described herein exclude at least one solvent selected from the group consisting of benzene, carbon disulfide, carbon tetrachloride, dichloromethane (DCM), methylene chloride, toluene, xylene, white spirit, acetone, and ethyl acetate. In some embodiments, the compositions described herein contain water, organic solvents, or at least one solvent selected from the group consisting of benzene, carbon disulfide, carbon tetrachloride, dichloromethane (DCM), methylene chloride, toluene, xylene, white spirit, acetone, and ethyl acetate in concentrations of 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1% v / v.

[0042] In some embodiments, this method measures samples of at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 ng / mL. In some embodiments, this method measures samples of up to 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 ng / mL.In some embodiments, this method is used for approximately 0.01 to approximately 1500 ng / mL, approximately 0.01 to approximately 1000 ng / mL, approximately 0.01 to approximately 900 ng / mL, approximately 0.01 to approximately 800 ng / mL, approximately 0.01 to approximately 700 ng / mL, approximately 0.01 to approximately 600 ng / mL, approximately 0.01 to approximately 500 ng / mL, approximately 0.01 to approximately 400 ng / mL, approximately 0.01 to approximately 300 ng / mL, approximately 0.01 to approximately 200 ng / mL, approximately 0.01 to approximately 100 ng / mL, approximately 0.01 to approximately 50 ng / mL, approximately 0.1 to approximately 1500 ng / mL, and approximately 0. .1 to about 1000ng / mL, about 0.1 to about 900ng / mL, about 0.1 to about 800ng / mL, about 0.1 to about 700ng / mL, about 0.1 to about 600ng / mL, about 0.1 to about 500ng / mL, about 0.1 to about 400ng / mL, about 0.1 to about 300ng / mL, about 0.1 to about 200ng / mL, about 0.1 to about 100ng / mL, about 0.1 to about 50ng / mL, about 0.2 to about 1500ng / mL, about 0.2 to about 1000ng / mL, about 0.2 to about 900ng / mL, about 0.2 to about 800ng / mL, about 0.2 to about 700ng / mL, about 0.2 to about 600ng / mL, about 0.2 to about 500ng / mL, about 0.2 to about 400ng / mL, about 0.2 to about 300ng / mL, about 0.2 to about 200ng / mL, about 0.2 to about 100ng / mL, about 0.2 to about 50ng / mL, about 0 .5 to about 1500ng / mL, about 0.5 to about 1000ng / mL, about 0.5 to about 900ng / mL, about 0.5 to about 800ng / mL, about 0.5 to about 700ng / mL, about 0.5 to about 600ng / mL, about 0.5 to about 500ng / mL, about 0.5 to about 400ng Measure samples with concentrations of approximately 0.5 to 300 ng / mL, 0.5 to 200 ng / mL, 0.5 to 100 ng / mL, 0.5 to 50 ng / mL, 1 to 1500 ng / mL, 1 to 1000 ng / mL, 1 to 900 ng / mL, 1 to 800 ng / mL, 1 to 700 ng / mL, 1 to 600 ng / mL, 1 to 500 ng / mL, 1 to 400 ng / mL, 1 to 300 ng / mL, 1 to 200 ng / mL, 1 to 100 ng / mL, or 1 to 50 ng / mL.

[0043] In one embodiment, the Disclosure provides a composition for detecting, measuring, or analyzing a sample using liquid chromatography as described herein, the composition comprising at least one solvent from the group consisting of trifluoroacetic acid, ammonia, formic acid, acetonitrile, methanol, water, and 2-propanol. In some embodiments, the composition comprises trifluoroacetic acid. In some embodiments, the composition comprises ammonia. In some embodiments, the composition comprises formic acid. In some embodiments, the composition comprises acetonitrile. In some embodiments, the composition comprises methanol. In some embodiments, the composition comprises water. In some embodiments, the composition comprises 2-propanol.

[0044] In some embodiments, the composition contains trifluoroacetic acid in the range of about 0.5% to about 5% v / v. In some embodiments, the composition further comprises acetonitrile, methanol, water, and 2-propanol. In some embodiments, the composition comprises equivolutes of acetonitrile, methanol, water, and 2-propanol.

[0045] In one embodiment, the present disclosure provides a composition for detecting, measuring, or analyzing a sample using liquid chromatography as described herein, the composition comprising at least one solvent from the group consisting of N,N-dimethyl sulfoxide (DMSO), acetonitrile, methanol, trifluoroacetic acid, formic acid, ammonium, and water. In some embodiments, the composition comprises N,N-dimethyl sulfoxide (DMSO). In some embodiments, the composition comprises acetonitrile. In some embodiments, the composition comprises methanol. In some embodiments, the composition comprises trifluoroacetic acid. In some embodiments, the composition comprises formic acid. In some embodiments, the composition comprises ammonium. In some embodiments, the composition comprises water.

[0046] In some embodiments, the composition contains DMSO in the range of about 1% to about 10% v / v. In some embodiments, the composition cleans liquid chromatography autosampler components, including at least one selected from the group consisting of needles, high-pressure valves, injection ports, and stainless steel tubing used for connections.

[0047] In some embodiments, the lower limit quality control (LLQC) according to the method herein includes at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 ng / mL. In some embodiments, the method achieves lower limit quality control (LLQC) levels of up to 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 ng / mL.In some embodiments, this method is used for approximately 0.01 to approximately 1500 ng / mL, approximately 0.01 to approximately 1000 ng / mL, approximately 0.01 to approximately 900 ng / mL, approximately 0.01 to approximately 800 ng / mL, approximately 0.01 to approximately 700 ng / mL, approximately 0.01 to approximately 600 ng / mL, approximately 0.01 to approximately 500 ng / mL, approximately 0.01 to approximately 400 ng / mL, approximately 0.01 to approximately 300 ng / mL, approximately 0.01 to approximately 200 ng / mL, approximately 0.01 to approximately 100 ng / mL, approximately 0.01 to approximately 50 ng / mL, approximately 0.1 to approximately 1500 ng / mL, and approximately 0.1 to Approximately 1000ng / mL, approximately 0.1 to approximately 900ng / mL, approximately 0.1 to approximately 800ng / mL, approximately 0.1 to approximately 700ng / mL, approximately 0.1 to approximately 600ng / mL, approximately 0.1 to approximately 500ng / mL, approximately 0.1 to approximately 400ng / mL, approximately 0.1 to approximately 300ng / mL, Approximately 0.1 to approximately 200ng / mL, approximately 0.1 to approximately 100ng / mL, approximately 0.1 to approximately 50ng / mL, approximately 0.2 to approximately 1500ng / mL, approximately 0.2 to approximately 1000ng / mL, approximately 0.2 to approximately 900ng / mL, approximately 0.2 to approximately 800ng / mL, approximately 0.2 to approximately 700ng / mL, about 0.2 to about 600ng / mL, about 0.2 to about 500ng / mL, about 0.2 to about 400ng / mL, about 0.2 to about 300ng / mL, about 0.2 to about 200ng / mL, about 0.2 to about 100ng / mL, about 0.2 to about 50ng / mL, about 0.5 to about 15 00ng / mL, about 0.5 to about 1000ng / mL, about 0.5 to about 900ng / mL, about 0.5 to about 800ng / mL, about 0.5 to about 700ng / mL, about 0.5 to about 600ng / mL, about 0.5 to about 500ng / mL, about 0.5 to about 400ng / mL, about 0. Achieve lower limit quality control (LLQC) of 5 to approximately 300 ng / mL, approximately 0.5 to approximately 200 ng / mL, approximately 0.5 to approximately 100 ng / mL, approximately 0.5 to approximately 50 ng / mL, approximately 1 to approximately 1500 ng / mL, approximately 1 to approximately 1000 ng / mL, approximately 1 to approximately 900 ng / mL, approximately 1 to approximately 800 ng / mL, approximately 1 to approximately 700 ng / mL, approximately 1 to approximately 600 ng / mL, approximately 1 to approximately 500 ng / mL, approximately 1 to approximately 400 ng / mL, approximately 1 to approximately 300 ng / mL, approximately 1 to approximately 200 ng / mL, approximately 1 to approximately 100 ng / mL, or approximately 1 to approximately 50 ng / mL.

[0048] In some embodiments, the upper limit quality control (ULQC) of the method described herein includes at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 ng / mL. In some embodiments, the method achieves an upper limit of quality control (ULQC) of up to 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 ng / mL.In some embodiments, this method is used for approximately 1 to approximately 1500 ng / mL, approximately 1 to approximately 1000 ng / mL, approximately 1 to approximately 900 ng / mL, approximately 1 to approximately 800 ng / mL, approximately 1 to approximately 700 ng / mL, approximately 1 to approximately 600 ng / mL, approximately 1 to approximately 500 ng / mL, approximately 1 to approximately 400 ng / mL, approximately 1 to approximately 300 ng / mL, approximately 1 to approximately 200 ng / mL, approximately 1 to approximately 100 ng / mL, and approximately 1 to approximately 50 ng / mL, about 2 to about 1500ng / mL, about 2 to about 1000ng / mL, about 2 to about 900ng / mL, about 2 to about 800ng / mL, about 2 to about 700ng / mL, about 2 to about 600ng / mL , about 2 to about 500ng / mL, about 2 to about 400ng / mL, about 2 to about 300ng / mL, about 2 to about 200ng / mL, about 2 to about 100ng / mL, about 2 to about 50ng / mL, about 5 to about 150 0ng / mL, about 5 to about 1000ng / mL, about 5 to about 900ng / mL, about 5 to about 800ng / mL, about 5 to about 700ng / mL, about 5 to about 600ng / mL, about 5 to about 500ng / mL , about 5 to about 400ng / mL, about 5 to about 300ng / mL, about 5 to about 200ng / mL, about 5 to about 100ng / mL, about 5 to about 50ng / mL, about 10 to about 1500ng / mL, about 10 to about Achieve an upper limit of quality control (ULQC) of 1000 ng / mL, approximately 10-900 ng / mL, approximately 10-800 ng / mL, approximately 10-700 ng / mL, approximately 10-600 ng / mL, approximately 10-500 ng / mL, approximately 10-400 ng / mL, approximately 10-300 ng / mL, approximately 10-200 ng / mL, approximately 10-100 ng / mL, or approximately 10-50 ng / mL.

[0049] In some embodiments, the flow rate used in the method described herein may be 0.3 mL / min. In some embodiments, the flow rate may be at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mL / min. In some embodiments, the flow rate may be up to 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or 0.05 mL / min.In some embodiments, the flow rates are approximately 0.05 to 100, 0.05 to 90, 0.05 to 80, 0.05 to 70, 0.05 to 60, 0.05 to 50, 0.05 to 40, 0.05 to 30, 0.05 to 20, 0.05 to 10, 0.1 to 100, 0.1 to 90, 0.1 to 80, 0.1 to 70, 0.1 to 60, 0.1 to 50, 0.1 to 40, 0.1 to 30, 0.1 to 20, 0.1 to 10, 0.1 to 5, and 0.1 ~approximately 4, approximately 0.1~approximately 3, approximately 0.1~approximately 2, approximately 0.1~approximately 1, approximately 0.1~approximately 0.5, approximately 0.1~approximately 0.4, approximately 0.1~approximately 0.3, approximately 0.1~approximately 0.2, approximately 0.2~approximately 100, approximately 0.2~approximately 90, approximately 0.2~approximately 80, approximately 0.2~approximately 70, approximately 0.2~approximately 60, approximately 0.2~approximately 50, approximately 0.2~approximately 40, approximately 0.2~approximately 30, approximately 0.2~approximately 20, approximately 0.2~approximately 10, approximately 0.2~approximately 5, approximately 0.2~approximately 4, approximately 0.2~approximately 3, approximately 0.2~approximately 2, approximately 0.2~approximately 1, approximately 0.2~approximately 0.5, approximately 0.2~approximately 0.4, approximately 0. 2 to approximately 0.3, approximately 0.3 to approximately 100, approximately 0.3 to approximately 90, approximately 0.3 to approximately 80, approximately 0.3 to approximately 70, approximately 0.3 to approximately 60, approximately 0.3 to approximately 50, approximately 0.3 to approximately 40, approximately 0.3 to approximately 30, approximately 0.3 to approximately 20, approximately 0.3 to approximately 10, approximately 0.3 to approximately 5, approximately 0.3 to approximately 4, approximately 0.3 to approximately 3, approximately 0.3 to approximately 2, approximately 0.3 to approximately 1, approximately 0.3 to approximately 0.5, approximately 0.3 to approximately 0.4, approximately 0.4 to approximately 100, approximately 0.4 to approximately 90, approximately 0.4 to approximately 80, approximately 0.4 to approximately 70, approximately 0.4 to approximately 60, approximately 0.4 to approximately 50, approximately 0.4 to approximately 40, The flow rates may range from approximately 0.4 to 30, 0.4 to 20, 0.4 to 10, 0.4 to 5, 0.4 to 4, 0.4 to 3, 0.4 to 2, 0.4 to 1, 0.4 to 0.5, 0.5 to 100, 0.5 to 90, 0.5 to 80, 0.5 to 70, 0.5 to 60, 0.5 to 50, 0.5 to 40, 0.5 to 30, 0.5 to 20, 0.5 to 10, 0.5 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, or 0.5 to 1 mL / min.

[0050] In some embodiments, the volume of sample injected into the liquid chromatography described herein may be about 25 μL. In some embodiments, the injection volume of the analytical conditions may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μL. In some embodiments, the injection volume of the analytical conditions includes up to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4500, or 5000 μL. In the application, the injection volume is approximately 1 to 5000, approximately 1 to 4000, approximately 1 to 3000, approximately 1 to 2000, approximately 1 to 1000, approximately 1 to 500, approximately 1 to 400, approximately 1 to 300, approximately 1 to 200, approximately 1 to 100, approximately 1 to 50, approximately 1 to 30, approximately 5 to 5000, approximately 1 to 4000, approximately 5 to 3000, approximately 5 to Approximately 2000, approximately 5-1000, approximately 5-500, approximately 5-400, approximately 5-300, approximately 5-200, approximately 5-100, approximately 5-50, approximately 5-30, approximately 10-5000, approximately 10-4000, approximately 10-3000, approximately 10-2000, approximately 10-1000, approximately 10-500, approximately 10-400, approximately 10-300, approximately 10 ~200, 10~100, 10~50, 10~40, 10~30, 10~20, 20~5000, 20~4000, 20~3000, 20~2000, 20~1000, 20~500, 20~400, 20~300, 20~2000, 20~1000, 20~500, 20~400, 20~300, 20~200, 20~100, 20~50 The range may be approximately 20-40, 20-30, 25-5000, 25-4000, 25-3000, 25-2000, 25-1000, 25-500, 25-400, 25-300, 25-200, 25-100, 25-50, 25-40, or 25-30 μL.

[0051] In some embodiments, the accuracy of the methods described herein may be at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120%. In some embodiments, the accuracy may be up to 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150%. In some embodiments, the accuracy is approximately 75-150, 75-140, 75-130, 75-120, 75-115, 75-110, 75-105, 75-100, 75-95, 80-150, 80-140, 80-130, 80-120, 80-115, 80-110, 80-105, 80-100, 80-90, 80-95, 85-140, 85-130, 85- It may fall within the range of approximately 120, 85-115, 85-110, 85-105, 85-100, 85-95, 85-90, 90-150, 90-140, 90-130, 90-120, 90-115, 90-110, 90-105, 90-100, 90-95, 95-140, 95-130, 95-120, 95-115, 95-110, 95-105, and 95-100%.

[0052] In some embodiments, the accuracy (relative standard deviation (RSD)) of the methods described herein is within about 15%. In some embodiments, the accuracy of the methods described herein may be at least about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 20, 25, or 30%. In some embodiments, the accuracy (relative standard deviation (RSD)) may be at most about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50%. In several implementations, the precision (relative standard deviation (RSD)) values ​​are approximately 1 to 20, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 20, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, and 2 to 10. 9, approximately 2-8, approximately 2-7, approximately 2-6, approximately 2-5, approximately 2-4, approximately 2-3, approximately 4-15, approximately 4-14, approximately 4-13, approximately 4-12, approximately 4-11, approximately 4-10, approximately 4-9, approximately 4-8, approximately 4-7, approximately 4-6, approximately 4-5, approximately 6-15, approximately 6-14, approximately 6-13, approximately 6-12, approximately 6-11, approximately 6-10, approximately 6-9, approximately 6-8, or within the range of approximately 6-7%. [Examples]

[0053] The present disclosure will be described in more detail below with reference to the following embodiments. However, the following embodiments are intended to illustrate these embodiments, and the scope of the embodiments is not limited thereto.

[0054] Examples In this example, two solvent mixtures are prepared and used for LC-MS / MS analysis, each containing multiple solvents in optimized proportions, specifically designed to remove contamination from HPLC autosampler components. These two solvent mixtures are used sequentially according to pre- and post-treatment programs utilizing the autosampler multi-rinse function. These solvent mixtures are used to clean HPLC autosampler components, such as needles, high-pressure valves, injection ports, and stainless steel tubing used for connections. In addition, attention is paid to potential nonspecific adsorption by utilizing a completely bioinert HPLC column with a unique bioinert coating on the inside of the ss column body.

[0055] Figure 1 shows a diagram of the internal rinsing of the needle with rinsing solution, and Figure 2 shows diagrams of both internal and external rinsing.

[0056] Semaglutide is a peptide used as an antidiabetic drug for the treatment of type 2 diabetes. It is also used as an anti-obesity drug to reduce weight. In recent years, studies have shown that semaglutide also acts on the brain, suggesting its potential usefulness for various diseases, including Parkinson's disease and Alzheimer's disease. To overcome these challenges, there have been attempts to develop an MRM-based LC-MS / MS method that is ideal for pharmacokinetic studies of semaglutide in human plasma, with low limit of quantification (LLOQ) levels, no carryover, and a wide dynamic range. Low levels of semaglutide in plasma were determined using Shimadzu's LCMS-8060NX.

[0057] Human plasma was obtained from local suppliers to prepare calibration standards and quality control (QC) samples. Precursor ion selection and MRM optimization at different collision energies and voltages were performed using Shimadzu's "Optimization for method" tool. Optimized MRMs were developed for two product ions at optimized voltages and collision energies (CE).

[0058] An LC method (Figure 4) was created using a UHPLC column (Shim-pack Claris), and semaglutide was eluted without carryover. Using the created LC method and optimized MRM, an LLOQ of 0.2 ng / ml and an upper limit of quantification (ULOQ) of 600 ng / ml were achieved without carryover.

[0059] For quantification, wide linearity batches ranging from 0.2 to 600 ng / ml were processed in human plasma. For QC checks, lower quality control (LLQC), low quality control (LQC), medium quality control (MQC), and high quality control (HQC) samples were repeatedly processed and linearity was quantified. The accuracy of the calibration standard and QC samples was found to be within acceptable limits (Figure 5).

[0060] A schematic diagram of the sample analysis is shown in Figure 6. 0.3 mL of pre-spiked plasma was vortexed for 2 minutes with 0.7 mL of precipitation solvent and 0.1 mL of 2% ammonia solution, and then centrifuged at 7000 rpm for 5 minutes. Next, the sample was introduced into a pre-prepared cartridge under positive pressure. The evaporated eluent was reconstituted with 0.3 mL of diluent.

[0061] In accordance with the ICH M10 guidelines, validation parameters such as specificity, linearity, accuracy, precision, and carryover were studied.

[0062] System accuracy: System accuracy was evaluated by calculating the variation in peak area and retention time (RT) of six replicas of a 300 ng / ml treated semaglutide standard. The %RSD was found to be less than 5 for peak area, and the difference in RT for the six replica injections was found to be within +0.1 minutes. The specificity of this method was determined by comparing the response of blank samples (reagent and matrix) to the reported level. The response in reagent / matrix blank samples was good, within <20% of the reporting limit, and met the acceptance criteria (Figure 7).

[0063] Linearity Study: Processed calibration standards were used for the linearity study. All calibration standards were found within the range of 85–115% accuracy (Figure 8). Linearity is shown in Figure 8.

[0064] Accuracy and Precision Studies: QC samples were repeatedly processed at four different levels—LLQC, LQC, MQC, and HQC—and quantified for accuracy and precision studies. The observed results were within the acceptable limit of %RSD ± 15% (Figure 9).

[0065] Carryover: Carryover was evaluated by analyzing blank samples after injecting the highest calibration standard. The area response in semaglutide retention time of blank samples analyzed after the highest calibration standard was found to be less than 20.0% of the area response of the LLOQ standard (Figure 10).

[0066] These experimental data demonstrate a highly sensitive and accurate method for quantifying GLP-1 peptide in human plasma using Shimadzu's LCMS-8060NX system. This method addresses common challenges, including achieving low LLOQ, a wide dynamic range, and carryover-free detection. Furthermore, the results meet the accuracy and precision criteria of the ICH M10 guidelines, confirming the reliability of this method.

[0067] Figure 11 shows the results of a blank analysis performed before the analysis of semaglutide. A blank analysis (BLK) was performed without injecting a sample. Normally, the signal intensity should be 0 in a blank analysis, but due to noise, a very small signal was generated as shown in Figure 11 (BLK002).

[0068] Figure 12 shows the detailed method used in the example.

[0069] These examples demonstrate that, even after analysis of high-concentration quality-checked human plasma samples, semaglutide can be quantitatively analyzed with excellent accuracy due to the absence of carryover. These examples allow for the quantitative analysis of semaglutide in biological matrices such as human plasma in the range of 0.2 ng / mL to 600 ng / mL. Multi-rinse autosampler options / settings, programmably optimized rinse solutions, and proprietary bioinert columns are the minimum features required for highly sensitive and accurate quantitative analysis of synthetic peptides (Figures 13 and 14). Exemplary Embodiments Embodiment 1. A method for separating, purifying, detecting, measuring, or analyzing a sample using liquid chromatography, comprising rinsing the sampling means of a liquid chromatography system with a first rinse solution. Embodiment 2. The method according to Embodiment 1, wherein the first rinse solution comprises two or more solvents from the group consisting of trifluoroacetic acid, ammonia, formic acid, acetonitrile, methanol, water, and 2-propanol. Embodiment 3. The method according to Embodiment 1 or Embodiment 2, wherein the first rinse solution contains trifluoroacetic acid. Embodiment 4. The method according to any one of the preceding embodiments, wherein the first rinse solution contains trifluoroacetic acid in a range of about 0.5% to about 5% v / v. Embodiment 5. The first rinse solution is the method according to any one of the preceding embodiments, comprising 1% v / v trifluoroacetic acid. Embodiment 6. The method according to any one of the preceding embodiments, wherein the first rinse solution further comprises at least one selected from the group consisting of acetonitrile, methanol, water, and 2-propanol. Embodiment 7. The method according to any one of the preceding embodiments, wherein the first rinse solution further comprises acetonitrile, methanol, water, and 2-propanol. Embodiment 8. The method according to any one of the preceding embodiments, wherein the first rinse solution comprises equivolutes of acetonitrile, methanol, water, and 2-propanol. Embodiment 9. The method according to any one of the prior embodiments, wherein rinsing the sampling means with the first rinse solution includes purging the first rinse solution from the sampling means. Embodiment 10. The method according to any one of the prior embodiments, further comprising rinsing the sampling means with a first rinse solution, and rinsing the injection port. Embodiment 11. The method according to any one of the prior embodiments, further comprising rinsing the sampling means with a second rinsing solution. Embodiment 12. The method according to any one of the preceding embodiments, wherein the second rinse solution comprises one or more solvents selected from the group consisting of N,N-dimethyl sulfoxide (DMSO), acetonitrile, methanol, trifluoroacetic acid, formic acid, ammonium, and water. Embodiment 13. The method according to any one of the prior embodiments, wherein the second rinse solution contains DMSO in the range of about 1% to about 10% v / v. Embodiment 14. The method according to any one of the preceding embodiments, wherein the second rinse solution contains DMSO in the range of about 4% to about 6% v / v. Embodiment 15. The method according to any one of the prior embodiments, wherein the second rinse solution comprises 5% v / v DMSO. Embodiment 16. The method according to any one of the preceding embodiments, wherein rinsing the sampling means with a second rinse solution includes immersing a portion of the sampling means in the second rinse solution. Embodiment 17. The method according to any one of the preceding embodiments, wherein rinsing the sampling means with a second rinse solution further comprises stirring or pumping the second rinse solution while a portion of the sampling means is immersed in it. Embodiment 18. The method according to any one of the prior embodiments, wherein rinsing the sampling means includes washing the components of the liquid chromatography autosampler. Embodiment 19. The method according to Embodiment 18, wherein the autosampler component includes at least one selected from the group consisting of a needle, a high-pressure valve, an injection port, and a stainless steel tube used for connection. Embodiment 20. The method according to any one of the prior embodiments, further comprising introducing a sample into a liquid chromatography column via a sampling means and / or performing liquid chromatography using the liquid chromatography column. Embodiment 21. The method according to any one of the prior embodiments, further comprising performing mass spectrometry on the eluate from liquid chromatography. Embodiment 22. The method according to any one of the prior embodiments, wherein the sample comprises a peptide. Embodiment 23. The method according to any one of the prior embodiments, wherein the sample comprises a synthetic peptide. Embodiment 24. The method according to any one of the prior embodiments, wherein the sample comprises semaglutide. Embodiment 25. Liquid chromatography is a method according to any one of the prior embodiments, comprising high-performance liquid chromatography (HPLC). Embodiment 26. A method according to any one of the prior embodiments, comprising high-performance protein liquid chromatography (FPLC). Embodiment 27. Liquid chromatography is a method according to any one of the prior embodiments, including liquid-liquid chromatography. Embodiment 28. The method according to any one of the preceding embodiments, wherein the column conditioning solution comprises one or more solvents selected from the group consisting of N,N-dimethyl sulfoxide (DMSO), acetonitrile, methanol, and formic acid. Embodiment 29. The column conditioning solution is the method according to any one of the prior embodiments, wherein the column conditioning solution contains about 0.1% to about 5% v / v of formic acid. Embodiment 30. The column conditioning solution is the method according to any one of the prior embodiments, comprising 0.5% v / v formic acid. Embodiment 31. The method according to any one of the prior embodiments, wherein the column conditioning solution comprises about 0.1% to about 10% v / v of N,N-dimethyl sulfoxide (DMSO). Embodiment 32. The column conditioning solution comprises 1.0% v / v N,N-dimethyl sulfoxide (DMSO) as described in any one of the preceding embodiments. Embodiment 33. The present method, comprising separating, purifying, detecting, measuring, or analyzing proteins from a sample, according to any one of the prior embodiments. Embodiment 34. The present method is a method according to any one of the prior embodiments, comprising separation or purification. Embodiment 35. The present method is a method according to any one of the prior embodiments, comprising detection, measurement, or analysis. Embodiment 36. The method according to any one of the prior embodiments, comprising detecting, measuring, or analyzing the amount, concentration, and / or mass of protein in a sample. Embodiment 37. The method according to any one of the prior embodiments, wherein the protein is semaglutide. Embodiment 38. A composition for detecting, measuring, or analyzing a sample using liquid chromatography as described in any one of the prior embodiments, wherein the composition comprises at least one solvent from the group consisting of trifluoroacetic acid, ammonia, formic acid, acetonitrile, methanol, water, and 2-propanol. Embodiment 39. The composition according to Embodiment 38, wherein the composition contains trifluoroacetic acid in the range of about 0.5% to about 5% v / v. Embodiment 40. The composition according to Embodiment 38 or 39, further comprising acetonitrile, methanol, water, and 2-propanol. Embodiment 41. The composition according to any one of Embodiments 38 to 40, comprising equivolutes of acetonitrile, methanol, water, and 2-propanol. Embodiment 42. A composition for detecting, measuring, or analyzing a sample using liquid chromatography as described in any one of Embodiments 1 to 37, wherein the composition comprises at least one solvent from the group consisting of N,N-dimethyl sulfoxide (DMSO), acetonitrile, methanol, trifluoroacetic acid, formic acid, ammonium, and water. Embodiment 43. The composition is the same as in Embodiment 42, wherein the composition contains DMSO in the range of about 1% to about 10% v / v. Embodiment 44. The composition according to either one of Embodiments 42 or 43, wherein the composition cleans a liquid chromatography autosampler component comprising at least one selected from the group consisting of a needle, a high-pressure valve, an injection port, and a stainless steel tube used for connection.

Claims

1. A method for detecting, measuring, or analyzing a sample using liquid chromatography, the method comprising rinsing the sampling means of a liquid chromatography system with a first rinse solution.

2. The method according to claim 1, wherein the first rinse solution comprises two or more solvents selected from the group consisting of trifluoroacetic acid, ammonia, formic acid, acetonitrile, methanol, water, and 2-propanol.

3. The method according to claim 1 or 2, wherein the first rinse solution contains trifluoroacetic acid.

4. The method according to any one of the preceding claims, wherein the first rinse solution further comprises at least one selected from the group consisting of acetonitrile, methanol, water, and 2-propanol.

5. The method according to any one of the preceding claims, wherein rinsing the sampling means with a first rinse solution includes purging the first rinse solution from the sampling means.

6. The method according to any one of the preceding claims, further comprising rinsing the sampling means with the first rinsing solution to rinsing the injection port.

7. The method according to any one of the preceding claims, further comprising rinsing the sampling means with a second rinse solution.

8. The method according to any one of the preceding claims, wherein the second rinse solution comprises one or more solvents selected from the group consisting of N,N-dimethyl sulfoxide (DMSO), acetonitrile, methanol, trifluoroacetic acid, formic acid, ammonium, and water.

9. The method according to any one of the preceding claims, wherein the second rinse solution contains DMSO in the range of about 1% to about 10% v / v.

10. The method according to any one of the preceding claims, wherein rinsing the sampling means with the second rinse solution includes immersing a portion of the sampling means in the second rinse solution.

11. The method according to any one of the preceding claims, wherein rinsing the sampling means with the second rinse solution further comprises stirring or pumping the second rinse solution while the portion of the sampling means is immersed in it.

12. The method according to any one of the preceding claims, wherein rinsing the sampling means includes washing the components of the liquid chromatography autosampler.

13. The method according to claim 12, wherein the autosampler component includes at least one selected from the group consisting of a needle, a high-pressure valve, an injection port, and a stainless steel tube used for connection.

14. The aforementioned method, Introducing the sample into a liquid chromatography column via the sampling means, and / or The method according to any one of the preceding claims, further comprising performing liquid chromatography using the liquid chromatography column.

15. The method according to any one of the preceding claims, further comprising performing mass spectrometry on the eluate from the liquid chromatography.

16. The method according to any one of the preceding claims, wherein the sample comprises a peptide.

17. The method according to any one of the preceding claims, wherein the sample comprises semaglutide.

18. The method according to any one of the preceding claims, wherein the liquid chromatography includes high-performance liquid chromatography (HPLC).

19. The method according to any one of the preceding claims, wherein the column conditioning solution comprises one or more solvents selected from the group consisting of N,N-dimethyl sulfoxide (DMSO), acetonitrile, methanol, and formic acid.

20. The method according to any one of the preceding claims, wherein the column conditioning solution contains about 0.1% to about 5% v / v of formic acid.

21. The method according to any one of the preceding claims, wherein the column conditioning solution comprises about 0.1% to about 10% v / v of N,N-dimethyl sulfoxide (DMSO).

22. A composition for detecting, measuring, or analyzing a sample using liquid chromatography as described in any one of the above methods, wherein the composition comprises at least one solvent from the group consisting of trifluoroacetic acid, ammonia, formic acid, acetonitrile, methanol, water, and 2-propanol.

23. A composition for detecting, measuring, or analyzing a sample using liquid chromatography as described in any one of the above methods, wherein the composition comprises at least one solvent from the group consisting of N,N-dimethyl sulfoxide (DMSO), acetonitrile, methanol, trifluoroacetic acid, formic acid, ammonium, and water.

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