Method and kit for detecting methylmalonic acid in blood plasma through high-performance liquid chromatography-tandem mass spectrometry

A high-performance liquid chromatography, methylmalonic acid technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., to achieve the effect of good reproducibility, high accuracy and saving analysis costs

Inactive Publication Date: 2017-04-19
郭嘉亮
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve some of the problems existing in the detection method of methylmalonic acid in plas...

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  • Method and kit for detecting methylmalonic acid in blood plasma through high-performance liquid chromatography-tandem mass spectrometry
  • Method and kit for detecting methylmalonic acid in blood plasma through high-performance liquid chromatography-tandem mass spectrometry
  • Method and kit for detecting methylmalonic acid in blood plasma through high-performance liquid chromatography-tandem mass spectrometry

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Experimental program
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Embodiment 1

[0067] Embodiment 1: instrument, material and reagent

[0068] Tandem mass spectrometer (AB Sciex API 5500), high performance liquid phase (Shimadzu LC-20AD XR system). Methylmalonic acid, succinic acid, ammonium acetate and acetic acid were all products from Sigma. Methylmalonate-d3 was purchased from Cambridge Isotope Laboratories. Charcoal-adsorbed serum was purchased from Equitech-Bio, Inc. (USA). HPLC grade ethanol was purchased from J.T. Baker Company, mass spectrometry grade acetonitrile was purchased from Merck Company, 15-mL centrifuge tubes were purchased from Kirgen Company, and volumetric flasks were purchased from BRAND GMBH+CO KG (Germany). Pure water was purified by Millipore Simplicity UA water purifier.

Embodiment 2

[0069] Embodiment 2: the preparation method of calibrator and internal standard solution, comprises the following steps:

[0070] (1) Preparation of standard mother liquor

[0071] Accurately weigh 12.5mg of methylmalonic acid standard substance, dissolve it with pure water and make it to 50mL to obtain a stock solution of 0.25mg / mL (2.1mmol / L), and dilute it successively with pure water to obtain the standard substance mother solution I: 21μmol / L L and standard stock solution II: 100 μmol / L.

[0072] (2) Preparation of standard products

[0073] Preferably, the mother liquor of the standard is diluted with a diluent to obtain 6 different concentrations of the calibrator: 0.05, 0.10, 0.25, 0.50, 1.0, 2.0 μmol / L methylmalonic acid. Each 1 mL of the calibrator with different concentrations was stored at -20°C.

[0074] (3) Preparation of internal standard solution methylmalonic acid-d3 aqueous solution

[0075] Weigh methylmalonic acid-d3, dissolve it in ultrapure water, and...

Embodiment 3

[0076] Embodiment 3: Determination of the plasma sample pretreatment process, including the following steps:

[0077] Take 200 μL plasma into a 2 mL centrifuge tube, add urease, incubate at 37°C for 30 minutes to remove urea, add 20 μL internal standard and 800 μL absolute ethanol, centrifuge at 10,000 rpm for 5 minutes to remove protein, take supernatant, 50 Blow dry with high-purity nitrogen at ℃, redissolve in 500 μL 80% acetonitrile water, vortex for 3 minutes; centrifuge at 13000 r / min for 3 minutes, and inject for detection.

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Abstract

The invention relates to a method and kit for detecting methylmalonic acid in blood plasma through high-performance liquid chromatography-tandem mass spectrometry. The method realizes detection of methylmalonic acid in blood plasma, especially separation of methylmalonic acid in blood plasma from a butanedioic acid detection baseline, through pretreatment and optimized selection of a chromatographic column, chromatographic conditions, and the types and parameters of mass spectrometry. The method is simple and fast, has good precision, accuracy and stability and can provide a more accurate and reliable detection method for clinical practice.

Description

technical field [0001] The invention belongs to the field of in vitro detection of biological markers, in particular to a high performance liquid chromatography-tandem mass spectrometry detection method and a kit for methylmalonic acid, in particular to a special method for detecting methylmalonic acid in plasma samples Assay methods and kits. Background technique [0002] Methylmalonic acidemia (MMA) is a common organic acidemia, an autosomal recessive genetic disease, mainly due to methylmalonyl-CoA mutase (methylmalonyl-CoA mutase, MCM) or its coenzyme cobalamin (VitB12) metabolic defects. Methylmalonic acidemia can start shortly after birth or at any age and deteriorate rapidly, prone to repeated metabolic ketoacidosis, refusal to eat, lethargy, coma, recurrent vomiting, dehydration, dyspnea, muscle tension Symptoms such as depression and growth retardation lead to early death or various neurological dysfunctions. [0003] The biochemical diagnosis of genetic metaboli...

Claims

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Application Information

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IPC IPC(8): G01N30/72G01N30/02
CPCG01N30/7266G01N30/02
Inventor 郭嘉亮张乔轩孙平华江正瑾龚道原林航
Owner 郭嘉亮
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