TW202229867A - Quantitative analysis method of adenosine phosphate capable of reliably calculating the content ratios of adenosine monophosphate, adenosine diphosphate, and adenosine triphosphate in an adenosine phosphate component - Google Patents

Abstract

A quantitative analysis method of adenosine phosphate includes: subjecting a mixture comprising an adenosine phosphate component and an ionic liquid matrix to the ultraviolet laser desorption ionization treatment to form an ionized sample, wherein the adenosine phosphate component includes adenosine monophosphate, adenosine diphosphate, and adenosine triphosphate. The ionic liquid matrix includes at least one of an ionic liquid formed by a dihydroxybenzoic acid anion and a pyridinium cation, and an ionic liquid formed by an [alpha]-cyano-4-hydroxycinnamate anion and a tripropylammonium onium ion; and analyzing the ionized sample to obtain the signal intensities of adenosine monophosphate parent ion, adenosine diphosphate parent ion, and adenosine triphosphate parent ion, and calculating the content ratios of adenosine monophosphate, adenosine diphosphate, and adenosine triphosphate.

Description

Quantitative Analysis of Adenosine Phosphate

The present invention relates to a quantitative analysis method, particularly a quantitative analysis method of adenosine phosphate.

US Patent Publication No. 2005158863A discloses a matrix for use in UV matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and is an ionic liquid at room temperature, and the matrix comprises Organic substances and amine salts. The amine salt is selected from 3-aminoquinoline, pyridine, primary amine, secondary amine, tertiary amine, or imidazole. The organic substance is selected from 2,5-dihydroxybenzoic acid, 2-hydroxy-5-methoxybenzoic acid, picolinic acid, 3-hydroxypicolinic acid, nicotinic acid, 5-chloro-2-mercaptobenzothiazole, 6 -Aza-2-thio-thymine, 2',4',6'-trihydroxyacetophenone hydrate, 2',6'-dihydroxy-acetophenone, 9H-pyrido[3,4 -b] Indole, 1,8,9-Anthratriol, trans-3-indoleacrylic acid, phosphonium, ferulic acid, 2,5-dihydroxyacetophenone, 1-nitroazole, 7-amine Ethyl-4-methylcoumarin, 2-(p-hydroxybenzazepine)-benzoic acid, 8-aminopyrene-2,3,4-trisulfonic acid, 2[2E-3-(4-th tributylphenyl)-2-methylprop-2-alkenyl]malononitrile, 4-methoxy-3-hydroxycinnamic acid, or 3,4-dihydroxycinnamic acid. The matrix of this US patent can be used in a MALDI mass spectrometry method for analyzing analytes such as polymers, proteins or nucleotides.

Although the matrix of this U.S. patent case can be used in the MALDI mass spectrometry method for analyzing nucleotides, since the nucleotides are labile molecules, during the MALDI mass spectrometry analysis, the phosphate groups of the nucleotides are not affected. It is easily detached from this nucleotide and, therefore, when analytes comprising adenosine monophosphate, adenosine diphosphate and adenosine triphosphate are analyzed by MALDI mass spectrometry, the adenosine triphosphate is due to phosphoric acid. The root is detached and converted into adenosine monophosphate or adenosine diphosphate, and the adenosine diphosphate is converted into adenosine monophosphate. At this time, the adenosine diphosphate parent ion cannot be confirmed in the mass spectrum. The signal peak is from adenosine diphosphate or adenosine triphosphate, and it cannot be confirmed that the adenosine monophosphate parent ion signal peak is from adenosine monophosphate, adenosine diphosphate or adenosine triphosphate, As a result, it is impossible to quantify the ratio between adenosine monophosphate, adenosine diphosphate and adenosine triphosphate, and even lead to distortion of the quantitative results. The ratio of glycoside triphosphates in biological cells affects the metabolism and energy transfer of biological cells, so how to effectively quantify analytes containing adenosine monophosphate, adenosine diphosphate and adenosine triphosphate Analytics is critical and urgently needed to be developed.

Therefore, the object of the present invention is to provide a quantitative analysis method of adenosine phosphate.

Therefore, the quantitative analysis method of adenosine phosphate of the present invention comprises the following steps: The mixture is subjected to ultraviolet laser desorption ionization treatment to form an ionized sample, wherein the mixture contains an adenosine phosphate component and an ionic liquid matrix, and the adenosine phosphate component includes adenosine monophosphate (Adenosine monophosphate, AMP for short), adenosine diphosphate (ADP for short) and adenosine triphosphate (ATP for short), and the ionic liquid matrix includes at least one ionic liquid, and the ionic liquid is selected from dihydroxybenzene Ionic liquid formed by formate anion and pyridinium cation or ionic liquid formed by α-cyano-4-hydroxycinnamate anion and tripropylammonium onium ion; The ionized sample is analyzed with a mass analyzer and a mass detector to obtain the ion signal intensity of the signal peak derived from the adenosine monophosphate parent ion of the adenosine monophosphate, the ion signal intensity derived from the adenosine diphosphate the ion signal intensity of the signal peak of the adenosine diphosphate parent ion, and the ion signal intensity of the signal peak of the adenosine triphosphate parent ion derived from the adenosine triphosphate; and According to the intensity of the plasma signal, the content ratio of the adenosine monophosphate, the adenosine diphosphate, and the adenosine triphosphate is calculated.

The effect of the present invention lies in: reducing the phosphate radicals in the adenosine monophosphate, adenosine diphosphate and adenosine triphosphate of the adenosine phosphate component through the ionic liquid matrix from the adenosine monophosphate, Adenosine diphosphate and adenosine triphosphate are detached. In the quantitative analysis method of adenosine phosphate of the present invention, the adenosine monophosphate parent ion is derived from adenosine monophosphate and adenosine diphosphate. The parent ion is derived from adenosine diphosphate, and the adenosine triphosphate parent ion is derived from adenosine triphosphate, enabling the adenosine monophosphate, the adenosine monophosphate in the adenosine phosphate component The content ratio between the diphosphate and the adenosine triphosphate was quantitatively analyzed.

The content of the present invention will be described in detail below.

The quantitative analysis method of adenosine phosphate of the present invention comprises the following steps: The mixture is subjected to ultraviolet laser desorption ionization treatment to form an ionized sample, wherein the mixture comprises an adenosine phosphate component and an ionic liquid matrix, and the adenosine phosphate component includes adenosine monophosphate, adenosine diphosphate Phosphate ester and adenosine triphosphate, and the ionic liquid matrix includes at least one ionic liquid, and the ionic liquid is selected from the ionic liquid formed by dihydroxybenzoate anion and pyridinium cation or by α-cyano-4-hydroxyl The ionic liquid formed by cinnamate anion and tripropylammonium onium ion; The ionized sample is analyzed with a mass analyzer and a mass detector to obtain the ion signal intensity of the signal peak derived from the adenosine monophosphate parent ion of the adenosine monophosphate, the ion signal intensity derived from the adenosine diphosphate the ion signal intensity of the signal peak of the adenosine diphosphate parent ion, and the ion signal intensity of the signal peak of the adenosine triphosphate parent ion derived from the adenosine triphosphate; and According to the intensity of the plasma signal, the content ratio of the adenosine monophosphate, the adenosine diphosphate, and the adenosine triphosphate is calculated.

＜Ultraviolet laser desorption ionization treatment＞

In a specific example of the present invention, the wavelength of the ultraviolet laser is 355 nm. The repetition rate of the ultraviolet laser is not particularly limited, and the repetition rate commonly used in the conventional MALDI mass spectrometry method can be used. In a specific example of the present invention, the repetition frequency of the ultraviolet laser is 100 Hz.

In order to enable the quantitative analysis method of adenosine phosphate of the present invention to more accurately quantify the content ratio between the adenosine monophosphate, the adenosine diphosphate, and the adenosine triphosphate, preferably, the ionic liquid It is an ionic liquid formed by dihydroxybenzoate anion and pyridinium cation.

<Mass Analyzer>

The mass analyzer is, for example, a time-of-flight (TOF for short) mass analyzer. In a specific example of the present invention, the mass analyzer is a time-of-flight mass analyzer, and the flight mode is a reflection mode.

＜Quality Detector＞

The mass detector is, for example, an electron multiplier tube type ion detector or a microchannel plate (MCP for short) type ion detector.

The present invention will be further described with respect to the following examples, but it should be understood that the examples are only used for illustration and should not be construed as a limitation of the implementation of the present invention.

Preparation Example 1 Ionic liquid matrix

0.2 mmole of dihydroxybenzoic acid was dissolved in methanol, then, 0.2 mmole of aniline was added, then, a test tube shaker was used to shake for 2 to 3 minutes, so that the dihydroxybenzoic acid and the aniline were mixed uniformly, then, The treatment was carried out for 24 hours in a vacuum oven set at room temperature and pressure set at 0.1 torr to evaporate methanol and residual aniline. Next, ethanol (solvent) is added to obtain a matrix component comprising an ionic liquid matrix and ethanol, wherein, in the matrix component, the concentration of the ionic liquid matrix is 0.2M, and the ionic liquid matrix comprises a mixture of dihydroxybenzoic acid An ionic liquid formed by an anion and a phenylammonium cation.

Preparation Examples 2 to 11

The preparation steps of Preparation Examples 2 to 11 are roughly the same as those of Preparation Example 1. The main difference is that the sources of carboxylate anions and onium cations are changed, see Table 1.

Preparation Example 12

The dihydroxybenzoic acid was dissolved in a mixed solvent of water and acetonitrile with a volume ratio of 1:3 to form a solution, wherein, in the solution, the concentration of the dihydroxybenzoic acid was 1M.

Preparation Example 13

Dissolve α-cyano-4-hydroxycinnamic acid in a mixed solvent of water and acetonitrile with a volume ratio of 1:3 to form a solution, wherein, in the solution, the concentration of the α-cyano-4-hydroxycinnamic acid is 0.1M.

Table 1 Preparation example source of carboxylate anion Onium Cation Source In the matrix component, the concentration (M) of the ionic liquid matrix 1 Dihydroxybenzoic acid (DHB) Aniline (A) 0.2 2 Dihydroxybenzoic acid (DHB) Pyridine (P) 0.2 3 Dihydroxybenzoic acid (DHB) Butylamine (B) 1 4 Dihydroxybenzoic acid (DHB) 1-Methazole (MI) 1 5 Dihydroxybenzoic acid (DHB) Tri-n-butylamine (TBA) 1 6 Dihydroxybenzoic acid (DHB) Tri-n-propylamine (TPA) 0.2 7 Alpha-cyano-4-hydroxycinnamic acid (CHCA) Aniline (A) 0.2 8 Alpha-cyano-4-hydroxycinnamic acid (CHCA) Butylamine (B) 0.2 9 Alpha-cyano-4-hydroxycinnamic acid (CHCA) 1-Methazole (MI) 1 10 Alpha-cyano-4-hydroxycinnamic acid (CHCA) Tri-n-butylamine (TBA) 1 11 Alpha-cyano-4-hydroxycinnamic acid (CHCA) Tri-n-propylamine (TPA) 1

Evaluation item

Measurement of detection efficiency of the ATP precursor ion signal peak: The matrix components of Preparation Examples 1 to 11 were combined with 1 nmol of adenosine monophosphate disodium salt (purchased from Sigma-Aldrich, USA, with a purity of 99%) Mixing to obtain a mixture, wherein the molar ratio of the adenosine monophosphate disodium salt to the ionic liquid matrix in the matrix component is 1:500. Then, 1 μL of the mixture was spotted in a UV laser desorption ionization time-of-flight tandem mass spectrometer (brand: Bruker Daltonics, Germany; model: Ultraflex III) stainless steel using the Dried droplet method. On the plate, and after the ethanol volatilizes, a test sample is formed. The detection sample is placed in the UV laser desorption ionization time-of-flight tandem mass spectrometer, and mass spectrometry analysis is performed in negative mode. The ultraviolet laser is produced from neodymium-doped yttrium aluminum garnet (Nd-YAG) material through frequency triple technology, and the wavelength of the ultraviolet laser is 355nm, and the single-shot repetition frequency is 100Hz and the strike The number of times is 1000 times, wherein, the laser energy (laser energy) of Preparation Examples 1 to 6 is 95% of the total energy of the ultraviolet laser, and the laser energy of Preparation Examples 7 to 11 is the ultraviolet laser energy. 75% of the total energy, and the number of blows is distributed in 20 different locations for each test sample. The mass analyzer of the ultraviolet laser desorption ionization time-of-flight tandem mass spectrometer is a time-of-flight mass analyzer and the flight mode adopts a reflection mode. The mass detector of the ultraviolet laser desorption ionization time-of-flight tandem mass spectrometer is a microchannel chip ion detector. The analysis results are shown in Figures 1 and 2.

Measurement of detection efficiency of ADP precursor ion signal peaks: Mix the matrix components of Preparation Examples 1 to 11 with 1 nmol of adenosine diphosphate disodium salt (purchased from Sigma-Aldrich, USA, with a purity of more than 95%) to obtain The mixture, wherein the molar ratio of the adenosine diphosphate disodium salt to the ionic liquid matrix in the matrix component is 1:500. Then, according to the above-mentioned method for measuring the detection efficiency of the ATP precursor ion signal peak. The analysis results are shown in Figures 3 and 4.

Measurement of detection efficiency of AMP precursor ion signal peaks: The matrix components of Preparation Examples 1-2, Preparation Examples 5-6, and Preparation Examples 8-11 were combined with 1 nmol of adenosine triphosphate disodium salt (purchased from Sigma in the United States). -Aldrich, 99% pure) to obtain a mixture in which the molar ratio of the adenosine triphosphate disodium salt to the ionic liquid matrix in the matrix component is 1:500. Then, according to the above-mentioned method for measuring the detection efficiency of the ATP precursor ion signal peak. The analysis results are shown in Figures 5 and 6.

Referring to FIG. 1, in the mass spectra of Preparation Examples 1-2 and Preparation Examples 5-6, there is a precursor ion signal peak of [ATP-H] ^- at the mass-to-charge ratio (m/z) of 506.5, while Preparation Example 3 The mass spectrum of ~4 does not have the precursor ion signal peak, which means that the ionic liquid matrix of Preparation Examples 3~4 is not suitable for the analysis of ATP. Referring to FIG. 2, in the mass spectra of Preparation Examples 7-11, there is a signal peak of the parent ion of [ATP-H] ^- at the mass-to-charge ratio (m/z) of 506.5, which indicates the ionic liquids of Preparation Examples 7-11 All matrices are suitable for the analysis of ATP.

Referring to FIG. 3, in the mass spectra of Preparation Examples 1-6, there is a signal peak of [ADP-H] ^- parent ion at the mass-to-charge ratio (m/z) of 426.8, which indicates the ionic liquids of Preparation Examples 1-6 All matrices are suitable for the analysis of ADP. Referring to FIG. 4, in the mass spectra of Preparation Examples 7-11, there is a signal peak of [ADP-H] ^- parent ion at the mass-to-charge ratio (m/z) of 426.8, which indicates the ionic liquids of Preparation Examples 7-11 All matrices are suitable for the analysis of ADP.

Referring to Fig. 5, in the mass spectra of Preparation Examples 1-2 and Preparation Example 5, there is a signal peak of [AMP-H] ^- parent ion at the mass-to-charge ratio (m/z) of 346.2, but the parent ion signal peak of Preparation Example 5 The signal intensity of the ion signal peak is very small, and the mass spectrum of Preparation Example 6 does not have the parent ion signal peak, which means that the ionic liquid matrix of Preparation Examples 5~6 has a poor dissociation efficiency for AMP, and there are There is a problem of low detection efficiency, so the ionic liquid matrix of Preparation Examples 5-6 is not suitable for analyzing AMP. Referring to FIG. 6 , in the mass spectra of Preparation Examples 8 to 11, there is a precursor ion signal peak of [AMP-H] ^- at a mass-to-charge ratio (m/z) of 346.2, however, the signal of the precursor ion signal peak The intensity is very small, which means that the ionic liquid matrix of Preparation Examples 8 to 10 has a poor freeing efficiency for AMP, and there is a problem of low detection efficiency.

It can be seen from the above that the ionic liquid matrices of Preparation Examples 1 to 2 and Preparation Example 11 can be used for the analysis of ATP, ADP and AMP in matrix-assisted laser desorption ionization mass spectrometry analysis. However, referring to Figure 1, Figure 3 and Figure 5, In Preparation Example 1, the difference between the ion signal intensity of the signal peak of the adenosine triphosphate parent ion and the ion signal intensity of the signal peak of the adenosine diphosphate parent ion is too large, which is not conducive to quantitative analysis. Based on the above, the ionic liquid matrix that can simultaneously analyze ATP, ADP and AMP is suitable for use in matrix-assisted laser desorption ionization mass spectrometry as Preparation Example 2 and Preparation Example 11.

Example 1

Mix 0.33nmol of adenosine monophosphate disodium salt, 0.33nmol of adenosine diphosphate disodium salt, 0.33nmol of adenosine triphosphate disodium salt with deionized water to obtain a component containing adenosine phosphate ester A composition with deionized water, wherein the concentration of the adenosine phosphate component in the composition is 2 mM.

The composition was mixed with the matrix component of Preparation Example 2 to obtain a mixture, wherein the molar ratio of the adenosine phosphate ester component in the composition to the ionic liquid matrix in the matrix component was 1:500. Then, according to the above-mentioned method for measuring the detection efficiency of the ATP precursor ion signal peak.

Examples 2 to 10

The preparation steps of Example 2 are roughly the same as those of Example 1, and the main difference lies in: changing the type of ionic liquid matrix, or the content of each component in the adenosine phosphate component, see Table 2.

Table 2 matrix Adenosine Phosphate Component Preparation example AMP:ADP:ATP (molar ratio) Example 1 2 1:1:1 2 2 1:1:2 3 2 1:1:3 4 2 1:1:4 5 2 1:1:5 6 11 1:1:1 7 11 1:1:2 8 11 1:1:3 9 11 1:1:4 10 11 1:1:5 Comparative example 1 12 1:1:1 2 12 1:1:2 3 12 1:1:3 4 12 1:1:4 5 12 1:1:5 6 13 1:1:1 7 13 1:1:2 8 13 1:1:3 9 13 1:1:4 10 13 1:1:5

，獲得離子訊號強度比。以該等離子訊號強度比為縱座標，而AMP：ADP：ATP的莫耳比例為橫坐標，繪製一張曲線圖，如圖7所示。值得說明的是，在圖7中，因對實施例1、實施例6、比較例1、及比較例6的實驗數據進行歸一化處理，故該等離子訊號強度比皆為1。 The ion signal intensity (abbreviated as I _1-ATP ) of the signal peaks of the adenosine triphosphate parent ions in Example 1, Example 6, Comparative Example 1 and Comparative Example 6 obtained by the detection efficiency measurement method was compared with that of adenosine triphosphate. The ratio A (i.e., I _1-ATP /I _1-ADP ) of the ion signal intensity (abbreviated as I _1-ADP ) of the signal peak of the diphosphate precursor ion is normalized to obtain the ion intensity ratio and is 1. The ion signal intensity (abbreviated as I _ATP ) of the signal peak of the adenosine triphosphate parent ion in Examples 2 to 5, Examples 7 to 10, Comparative Examples 2 to 5 and Comparative Examples 7 to 10 was compared with that of adenosine diphosphate. The ratio of the ion signal intensity (I _ADP for short) of the signal peak of the precursor ion (referred to as I _ATP /I _ADP ) is multiplied by

, to obtain the ion signal intensity ratio. Taking the plasma signal intensity ratio as the ordinate and the molar ratio of AMP:ADP:ATP as the abscissa, a graph is drawn, as shown in FIG. 7 . It should be noted that, in FIG. 7 , since the experimental data of Example 1, Example 6, Comparative Example 1, and Comparative Example 6 are normalized, the plasma signal intensity is all 1.

，獲得離子訊號強度比。以該等離子訊號強度比為縱座標，而AMP：ADP：ATP的莫耳比例為橫坐標，繪製一張曲線圖，如圖8所示。值得說明的是，在圖7中，因對實施例1、實施例6、比較例1、及比較例6的實驗數據進行歸一化處理，故該等離子訊號強度比皆為1。 The ion signal intensity (abbreviated as I _1-ADP ) of the signal peaks of the adenosine diphosphate parent ions in Example 1, Example 6, Comparative Example 1 and Comparative Example 6 obtained by the detection efficiency measurement method was compared with that of adenosine diphosphate. The ratio B (ie, I _1-ADP /I _1-AMP ) of the signal peaks of the signal peaks of the monophosphate precursor ions is normalized to obtain the ion intensity ratio (I ₁ -AMP ) and is 1. The ion signal intensities (abbreviated as I _ADP ) of the signal peaks of the adenosine diphosphate parent ions of Examples 2 to 5, Examples 7 to 10, Comparative Examples 2 to 5 and Comparative Examples 7 to 10 were compared with adenosine monophosphate. The ratio of the ion signal intensity (I _AMP for short) of the signal peak of the precursor ion (I _ADP /I _AMP for short) is multiplied by

, to obtain the ion signal intensity ratio. Taking the plasma signal intensity ratio as the ordinate and the molar ratio of AMP:ADP:ATP as the abscissa, a graph is drawn, as shown in FIG. 8 . It should be noted that, in FIG. 7 , since the experimental data of Example 1, Example 6, Comparative Example 1, and Comparative Example 6 are normalized, the plasma signal intensity is all 1.

，獲得離子訊號強度比。以該等離子訊號強度比為縱座標，而AMP：ADP：ATP的莫耳比例為橫坐標，繪製一張曲線圖，如圖9所示。值得說明的是，在圖7中，因對實施例1、實施例6、比較例1、及比較例6的實驗數據進行歸一化處理，故該等離子訊號強度比皆為1。 The ion signal intensity (abbreviated as I _1-ATP ) of the signal peaks of the adenosine triphosphate parent ions in Example 1, Example 6, Comparative Example 1 and Comparative Example 6 obtained by the detection efficiency measurement method was compared with that of adenosine triphosphate. The ratio C (ie, I _1-ATP /I _1-AMP ) of the signal peaks of the signal peaks of the monophosphate precursor ions is normalized to obtain the ion intensity ratio (I ₁ -AMP ) and is 1. The ion signal intensities (abbreviated as I _ATP ) of the signal peaks of the adenosine triphosphate parent ions in Examples 2 to 5, Examples 7 to 10, Comparative Examples 2 to 5, and Comparative Examples 7 to 10 were compared with adenosine monophosphate. The ratio of the ion signal intensity of the signal peak of the precursor ion (abbreviated as I _AMP ) (abbreviated as I _ATP /I _AMP ) is multiplied by

, to obtain the ion signal intensity ratio. Taking the plasma signal intensity ratio as the ordinate and the molar ratio of AMP:ADP:ATP as the abscissa, a graph is drawn, as shown in FIG. 9 . It should be noted that, in FIG. 7 , since the experimental data of Example 1, Example 6, Comparative Example 1, and Comparative Example 6 are normalized, the plasma signal intensity is all 1.

In Figures 7, 8 and 9, the theoretical ion signal intensity ratio is also obtained as described above, and theoretically, the theoretical ion signal intensity ratio is the molar ratio of AMP:ADP:ATP.

Referring to Figures 7 to 9, the ionic liquid matrices of Preparation Example 2 and Preparation Example 11 were used in MALDI mass spectrometry to analyze the adenosine phosphate ester component. The actual ion signal intensity ratio obtained is almost the theoretical ion signal intensity ratio. Consistent, but the matrix of Preparation Example 12 and Preparation Example 13 was used in MALDI mass spectrometry to analyze the adenosine phosphate ester component and the deviation of the actual ion signal intensity ratio and the theoretical ion signal intensity ratio was too large, especially ADP and The ion signal intensity ratio of AMP (see Figure 8) and the ion signal intensity ratio of ATP and AMP (see Figure 9), which indicate that the ionic liquid matrix of Preparation Example 2 and Preparation Example 11 can indeed effectively reduce the adenosine phosphate group. The probability that the phosphate radicals in the divided adenosine monophosphate, adenosine diphosphate and adenosine triphosphate are separated from the adenosine monophosphate, adenosine diphosphate and adenosine triphosphate, respectively, so that it is possible to The content ratio among the adenosine monophosphate, the adenosine diphosphate, and the adenosine triphosphate in the adenosine phosphate fraction is quantitatively analyzed.

To sum up, reducing the phosphate radicals in the adenosine monophosphate, adenosine diphosphate and adenosine triphosphate of the adenosine phosphate component through the ionic liquid matrix are derived from the adenosine monophosphate, adenosine monophosphate, and adenosine triphosphate, respectively. In the quantitative analysis method of adenosine phosphate of the present invention, the adenosine monophosphate parent ion is derived from adenosine monophosphate, the adenosine diphosphate parent ion The ion is derived from adenosine diphosphate, and the adenosine triphosphate parent ion is derived from adenosine triphosphate, enabling the adenosine monophosphate, the adenosine diphosphate in the adenosine phosphate component The phosphate ester and the content ratio of the adenosine triphosphate can be quantitatively analyzed, so the object of the present invention can be achieved.

However, the above are only examples of the present invention, and should not limit the scope of implementation of the present invention. Any simple equivalent changes and modifications made according to the scope of the patent application of the present invention and the contents of the patent specification are still included in the scope of the present invention. within the scope of the invention patent.

Other features and effects of the present invention will be clearly presented in the embodiments with reference to the drawings, wherein: Fig. 1 is a mass spectrogram for illustrating the detection efficiency of the ionic liquid matrix of Preparation Examples 1-6 to the ATP precursor ion signal peak; Fig. 2 is a mass spectrogram for illustrating the detection efficiency of the ionic liquid matrix of Preparation Examples 7-11 to the ATP precursor ion signal peak; Fig. 3 is a mass spectrogram for illustrating the detection efficiency of the ionic liquid matrix of Preparation Examples 1-6 to the ADP parent ion signal peak; Fig. 4 is a mass spectrogram, used to illustrate the detection efficiency of the ionic liquid matrix of Preparation Examples 7-11 to the ADP parent ion signal peak; Fig. 5 is a mass spectrum, used to illustrate the detection efficiency of the ionic liquid matrix of Preparation Examples 1-2 and Preparation Examples 5-6 to the AMP parent ion signal peak; Fig. 6 is a mass spectrum for illustrating the detection efficiency of the AMP precursor ion signal peak using the ionic liquid matrix of Preparation Examples 8-11; Fig. 7 is a graph for illustrating that the quantitative analysis method of adenosine phosphate of the present invention can quantitatively analyze ATP, ADP and AMP in the adenosine phosphate fraction; 8 is a graph illustrating that the quantitative analysis method of adenosine phosphate of the present invention can quantitatively analyze ATP, ADP and AMP in the adenosine phosphate fraction; and Fig. 9 is a graph illustrating that the quantitative analysis method of adenosine phosphate of the present invention can quantitatively analyze ATP, ADP and AMP in the adenosine phosphate fraction.

Claims (3)

A method for quantitative analysis of adenosine phosphate, comprising the following steps: The mixture is subjected to ultraviolet laser desorption ionization treatment to form an ionized sample, wherein the mixture comprises an adenosine phosphate component and an ionic liquid matrix, and the adenosine phosphate component includes adenosine monophosphate, adenosine diphosphate Phosphate ester and adenosine triphosphate, and the ionic liquid matrix includes at least one ionic liquid, and the ionic liquid is selected from the ionic liquid formed by dihydroxybenzoate anion and pyridinium cation or by α-cyano-4-hydroxyl The ionic liquid formed by cinnamate anion and tripropylammonium onium ion; The ionized sample is analyzed with a mass analyzer and a mass detector to obtain the ion signal intensity of the signal peak derived from the adenosine monophosphate parent ion of the adenosine monophosphate, the ion signal intensity derived from the adenosine diphosphate the ion signal intensity of the signal peak of the adenosine diphosphate parent ion, and the ion signal intensity of the signal peak of the adenosine triphosphate parent ion derived from the adenosine triphosphate; and According to the intensity of the plasma signal, the content ratio of the adenosine monophosphate, the adenosine diphosphate, and the adenosine triphosphate is calculated. The quantitative analysis method of adenosine phosphate according to claim 1, wherein the mass analyzer is a time-of-flight mass analyzer. The method for quantitative analysis of adenosine phosphate according to claim 1, wherein the ionic liquid is an ionic liquid formed of a dihydroxybenzoic acid anion and a pyridinium cation.

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