All-isotope internal standard mass spectrometry quantitation method for carnitine-based compounds

A technology of isotope labeling and compounds, which is applied in measuring devices, instruments, and material analysis through electromagnetic means, can solve the problems of inaccurate quantification and achieve the effects of accurate quantification, low cost and important application value

Inactive Publication Date: 2017-12-05
上海谱领生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a kind of carnitine compound full isotope internal standard mass spectrometry quantitative method, solve the quantitative

Method used

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  • All-isotope internal standard mass spectrometry quantitation method for carnitine-based compounds
  • All-isotope internal standard mass spectrometry quantitation method for carnitine-based compounds
  • All-isotope internal standard mass spectrometry quantitation method for carnitine-based compounds

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Embodiment 1

[0033] The purpose of this embodiment is to establish the UPLC-MS / MS quantitative method of carnitine compound isotope reagent derivatization method, such as Figure 1-3 shown.

[0034] Prepare the methanol solution (mixed standard mother solution) of carnitine and acylcarnitine mixed standard, dilute, get 100 microliters of dilutions in two parts, blow dry with nitrogen, add 70 microliters (range is 50-100 microliters, The following range values ​​are marked by this method) acetyl chloride and n-butanol were heat treated at 80°C for 30 minutes, and the volume ratio (v / v) of acetyl chloride and n-butanol was 7:1; another part was added with 70 microliters (50- 100 microliters) D9-acetyl chloride and n-butanol (v / v is 7:1) were heat-treated at 80°C for 20 minutes, and after drying with nitrogen, 500 microliters (200-1000 microliters) of 80% acetonitrile aqueous solution (volume ratio ) were redissolved to obtain esterification derivatives of carnitine compounds and n-butanol o...

Embodiment 2

[0038] The purpose of this example is to use the established UPLC- / MS / MS quantitative method for carnitine compounds to semi-quantitatively analyze mouse islet β-cell meat, so as to provide important research data for scientific research.

[0039] Take the methanol solution of the mixed standard substance of carnitine and acylcarnitine, dilute it, take 50 microliters, dry it with nitrogen, add 100 microliters (50-100 microliters) of 3N hydrochloric acid D7-n-butanol, seal it, and heat it at 90°C After 10 minutes, blow dry with nitrogen, add 1000 microliters of 80% acetonitrile aqueous solution (volume ratio) to redissolve, and further dilute 10 times to obtain the internal standard mother solution of the esterification derivative product of carnitine compound and D7-n-butanol.

[0040] Take cryopreserved islet β cells (cell number 4×10 5 ), add 300 microliters (range 200-500 microliters) of HPLC grade methanol (pre-cooled), store at -80°C overnight, take out the low temperatur...

Embodiment 3

[0043] The purpose of this example is to use the established UPLC- / MS / MS quantitative method for carnitine compounds to quantitatively analyze human plasma samples, so as to provide important data for early prediction studies of metabolic diseases such as diabetes.

[0044] Prepare methanol solution of carnitine compound mixed standard, dilute, take 100 microliters, blow dry with nitrogen, add 50 microliters of acetyl chloride / D5-n-butanol (14:1, v / v), seal, and heat at 40°C Treat for 50 minutes, blow dry with nitrogen, add 1000 microliters of 80% acetonitrile aqueous solution (volume ratio) to redissolve, and further dilute 10 times to obtain carnitine compounds and D5-n-butanol esterification derivatives (internal standard solution).

[0045] Take cryopreserved human plasma samples, thaw, shake and mix for a short time, take 20 microliters (5-50 microliters) of thawed plasma, add 100 microliters (50-450 microliters) of HPLC grade methanol (pre-cooled), set- Keep at 20°C for ...

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Abstract

The present invention discloses a new all-isotope mass spectrometry quantitation method for carnitine-based compounds in biological samples, wherein the method is the all-isotope internal standard quantitation method for carnitine-based compounds, uses the esterification derivative of a stable isotope labeled n-butanol reagent and a carnitine-based compound standard substance as a quantitative internal standard, and uses liquid chromatography-tandem quadrupole mass spectrometry as a detection method. According to the present invention, the method can simultaneously perform the quantitative analysis on all the carnitine-based compounds, has advantages of accurate qualitation, accurate quantitation, high throughput and low cost, can overcome the disadvantages of high interference, inaccurate quantitation, requirement of a large amount of expensive deuterium-labeled carnitine-based compound standard substances and the like of the current carnitine-based compound test, can significantly improve the accuracy of neonatal screening, clinical diagnosis of suspected metabolic diseases and scientific research, and other detections, and has important application value.

Description

technical field [0001] The invention relates to a mass spectrometry quantitative detection and analysis method for carnitine compounds in biological samples, and further to a mass spectrometric quantitative method for all isotope internal standard mass spectrometry of carnitine compounds in biological samples. Background technique [0002] L-carnitine (3-hydroxy-4-trimethylamine butyric acid internal salt) is a biosynthesis product from lysine and methionine widely distributed in various organs of the human body, of which about 25% are in the liver , kidney and brain synthesis, and the rest comes from red meat and dairy products. Complete oxidation of fatty acids must take place in the mitochondria, but cannot enter the mitochondria by itself. The main function of L-carnitine is to combine with the deamination products of fatty acids or branched-chain amino acids to form acylcarnitine, transport these metabolites from the cytoplasm to the mitochondria, and completely degrad...

Claims

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Application Information

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IPC IPC(8): G01N30/89G01N27/62
CPCG01N30/89G01N27/62
Inventor 占同文
Owner 上海谱领生物科技有限公司
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