LC-MS/MS high-throughput detection method of saxagliptin and 5-hydroxysaxagliptin in human blood plasma

A technology of hydroxysaxagliptin and detection method, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of increasing blood concentration, shortening running time, and low sensitivity, so as to improve detection accuracy and chromatographic running time Short, the effect of solving the residual problem

Active Publication Date: 2017-08-15
苏州海科医药技术有限公司
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Problems solved by technology

[0002] In recent years, the prevalence of diabetes in China has a clear upward trend, and type 2 diabetes is the main type. Saxagliptin is a competitive inhibitor of dipeptidyl peptidase 4 (DPP4), which can reduce the secretion of incretin hormone. The inactivation rate can increase its blood concentration, thereby reducing the fasting and postprandial blood glucose concentration of patients with type 2 diabetes in a glucose-dependent manner; from the current domestic and foreign related literature, the analytical method for the determination of gliptins in biological samples There are mainly high performance liquid phase ultraviolet detection (HPLC-UV) and liquid chromatography tandem mass spectrometry (LC-MS / MS), among which HPLC-UV has the characteristics of simple operation and low test cost, but the sensitivity of this type of method is low. It cannot be used in low-dose pharmacokinetic and bioequivalence studies; LC-MS / MS is a chromatographic technique using mass spectrometry as the detection method, which combines the separation ability of high-performance liquid chromatography with the detection and structural analysis functions of mass spectrometers Ingeniously combined, it has high sensitivity and high selectivity unmatched by other instruments. It is the best when running multi-level reaction monitoring (MRM) mode, and the determination of drug concentration can reach pg level. One of the most powerful analytical tools for efficacy research
[0003] At present, several literatures use LC-MS / MS technology to detect the concentration of saxagliptin and 5-hydroxy saxagliptin in plasma. The detection method is mainly used in clinical and non-clinical pharmacokinetic studies. The sample extraction method covers the protein precipitation method , solid-phase extraction and liquid-liquid extraction, the sensitivity can be as low as 50pg / mL; in addition, it has been reported in the literature that saxagliptin binds to dipeptidyl peptidase 4 (DPP4) in plasma to produce specificity Adsorption, this adsorption is more obvious in plasma samples with low drug concentration, which may easily lead to the low concentration of saxagliptin measured in human plasma. -MS / MS Chromatography for high throughput while shortening run times

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  • LC-MS/MS high-throughput detection method of saxagliptin and 5-hydroxysaxagliptin in human blood plasma
  • LC-MS/MS high-throughput detection method of saxagliptin and 5-hydroxysaxagliptin in human blood plasma
  • LC-MS/MS high-throughput detection method of saxagliptin and 5-hydroxysaxagliptin in human blood plasma

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Embodiment 1

[0029] Example 1 Establishment of LC-MS / MS determination method for saxagliptin and its metabolite 5-hydroxy saxagliptin in human plasma

[0030] 1. Solution and sample preparation

[0031] 1.1 Standard series samples: Accurately weigh the appropriate amount of each reference substance, dissolve saxagliptin in acetonitrile and constant volume, and dissolve 5-hydroxy saxagliptin in acetonitrile:water (50:50, v / v) and constant volume, and prepare Prepare a stock solution with a concentration of about 1.00 mg / mL, accurately absorb the appropriate amount of each stock solution, and dilute step by step with acetonitrile: water (3:1, v / v) to obtain a mixed standard series of working solutions, and dilute the working solution with human blank plasma , the concentration ranges of saxagliptin and 5-hydroxy saxagliptin in the mixed standard series samples were 0.200-50.0 and 0.298-74.4ng / mL respectively, which were used to draw the standard curve;

[0032] 1.2 Quality control samples: ...

Embodiment 2

[0067] Embodiment two Determination of saxagliptin and its metabolite 5-hydroxyl saxagliptin in human plasma

[0068] Apply the established LC-MS / MS assay method for the content of saxagliptin and its metabolite 5-hydroxy saxagliptin in human plasma to determine the concentration of saxagliptin and 5-hydroxy saxagliptin in plasma for evaluation Bioequivalence study of saxagliptin tablets.

[0069] 48 healthy subjects were enrolled, half male and half male, among them, 24 were administered on an empty stomach, and 24 were administered after meals. The drug group took the test or reference preparations on an empty stomach in a standing position 30 minutes after the start of meal timing, before administration and after administration 0.25h, 0.5h, 0.75h, 1.0h, 1.5h, 2.0h, 3.0h, Collect about 4mL of venous blood at 4.0h, 6.0h, 8.0h, 12.0h, 16.0h, 24.0h, 36.0h, and 48.0h. After the blood sample is collected, it is placed in a test tube that has been labeled in advance, and the plas...

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Abstract

The invention relates to an LC-MS / MS high-throughput detection method of saxagliptin and 5-hydroxysaxagliptin in human blood plasma. The method comprises the following steps: taking a blood plasma sample to be detected, adding an internal standard solution, adding an aqueous solution of CHAPS, carrying out eddy current heating, adding acetonitrile, centrifuging the obtained solution, taking the obtained supernatant, blowing the supernatant with nitrogen until the supernatant is dry, adding a redissolving solution to redissolve the dried supernatant in order to obtain a sample to be detected, and carrying out LC-MS / MS analysis, wherein the internal standard solution is an acetonitrile and water (50:50, v / v) mixed solution with the isotopic label <13>CD2-saxagliptin concentration being 5.00 ng / mL and the <13>CD2-hydroxysaxagliptin concentration being 10.0 ng / mL, and the redissolving solution is an acetonitrile: water: formic acid mixed solution with the volume ratio being 95:5:0.1. The method satisfies the demands of clinical large-scale sample analysis; and compared with the prior art, the method has the advantages of simplicity in operation, short extraction time, and suitableness for high-throughput sample pretreatment.

Description

technical field [0001] The invention relates to an analysis and detection method of gliptins in biological samples, in particular to a LC-MS / MS high-throughput detection method of saxagliptin and 5-hydroxy saxagliptin in human plasma. Background technique [0002] In recent years, the prevalence of diabetes in China has a clear upward trend, and type 2 diabetes is the main type. Saxagliptin is a competitive inhibitor of dipeptidyl peptidase 4 (DPP4), which can reduce the secretion of incretin hormone. The inactivation rate can increase its blood concentration, thereby reducing the fasting and postprandial blood glucose concentration of patients with type 2 diabetes in a glucose-dependent manner; from the current domestic and foreign related literature, the analytical method for the determination of gliptins in biological samples There are mainly high performance liquid phase ultraviolet detection (HPLC-UV) and liquid chromatography tandem mass spectrometry (LC-MS / MS), among ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/02
CPCG01N30/02G01N30/06
Inventor 钮小英杨勇钟勘
Owner 苏州海科医药技术有限公司
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