CN115792031A

CN115792031A - Method for quantitative detection of organic acid

Info

Publication number: CN115792031A
Application number: CN202211643340.7A
Authority: CN
Inventors: 雷焕娜; 赵晓雯; 余文祥; 赵亚丽
Original assignee: Beijing Novogene Technology Co ltd
Current assignee: Beijing Novogene Technology Co ltd
Priority date: 2022-12-20
Filing date: 2022-12-20
Publication date: 2023-03-14

Abstract

The invention provides a quantitative detection method of organic acid, and relates to the technical field of detection. The method for quantitatively detecting an organic acid provided by the invention comprises the following steps: and (3) carrying out derivatization treatment on the sample to be detected, adding an internal standard solution, and carrying out quantitative analysis on the organic acid in the sample by adopting a liquid chromatography-tandem mass spectrometry method. Wherein the derivatization treatment comprises: performing derivatization reaction on organic acid in a sample by using 3-nitrophenylhydrazine; the internal standard solution comprises organic acid which is subjected to derivatization treatment by adopting C13-3-nitrophenylhydrazine; the organic acids include: itaconic acid, citraconic acid, mesaconic acid, L-lactic acid, succinic acid, malic acid, citric acid, isocitric acid, fumaric acid, aconitic acid, ketoglutaric acid, pyruvic acid, and oxaloacetic acid. The quantitative detection method for the organic acid has the advantages of high sensitivity, low detection limit, high accuracy and good stability, and can realize the quantitative detection of 13 organic acids in the tricarboxylic acid cycle.

Description

Method for quantitative detection of organic acid

Technical Field

The invention relates to the technical field of detection, in particular to a quantitative detection method of organic acid.

Background

The tricarboxylic acid cycle (TCAcycle) is a ubiquitous metabolic pathway in aerobic organisms. Prokaryotes are distributed in the cytoplasm, and eukaryotes are distributed in mitochondria. The tricarboxylic acid cycle is the final metabolic pathway of three major nutrients (carbohydrate, lipid, amino acid) and is also the pivotal part of the metabolic connection of carbohydrate, lipid, amino acid. The tricarboxylic acid cycle is also the most efficient way for the body to oxidize sugars or other substances to obtain energy. In sugar metabolism, the most energy is produced by oxidation of sugars via this pathway. The tricarboxylic acid cycle plays an important role in all living bodies and is increasingly emphasized. Therefore, the detection demand of organic acids in the tricarboxylic acid cycle is increased, and in the detection of organic acids, it is found that such substances contain isomers, and the isomers are poor in separation degree or even difficult to separate in the detection, and some substances have low detection sensitivity and cannot meet the detection requirements.

The separation degree of isomers is poor or even isomers can not be separated by a common mass spectrum detection means, the sensitivity of individual substances is low, the quantitative limit cannot meet the detection requirement, the price of an isotope internal standard is high, isotopes can not be bought from each standard, and the internal standard endogenous substance matrix effect is difficult to test.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The present invention is directed to a method for quantitatively detecting an organic acid, which solves at least one of the above problems.

In a first aspect, the present invention provides a method for quantitatively detecting an organic acid, comprising the steps of: performing derivatization treatment on a sample to be detected, adding an internal standard solution, and performing quantitative analysis on organic acid in the sample by adopting a high performance liquid chromatography-tandem mass spectrometry method;

the derivatization treatment comprises: performing derivatization reaction on organic acid in a sample by using 3-nitrophenylhydrazine;

the internal standard solution comprises organic acid subjected to derivatization treatment by adopting C13-3-nitrophenylhydrazine;

the organic acid includes: itaconic acid, citraconic acid, mesaconic acid, L-lactic acid, succinic acid, malic acid, citric acid, isocitric acid, fumaric acid, aconitic acid, ketoglutaric acid, pyruvic acid, and oxaloacetic acid;

the conditions of the high performance liquid chromatography comprise:

and (3) chromatographic column: waters HSS T3.1 × 150mm, waters BEH C18.1 × 100mm, or Waters HSS T3.1 × 100mm;

the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is formic acid aqueous solution, and the mobile phase B is acetonitrile;

elution procedure: 0-1min 40% of mobile phase B,1-3.5min of mobile phase B gradually rises to 60%,3.5-8.5min of mobile phase B gradually rises to 75%,8.5-11.5min of mobile phase B gradually rises to 100%, 11.5-13.5% of mobile phase B gradually falls to 40%, and 14-1695in 40% of mobile phase B gradually falls to 13.5-14min of mobile phase B;

the conditions of the mass spectrum include: the electrospray ionization source has the ion source temperature of 350300-400 ℃, the ion source voltage negative/positive mode of-2500V/3500V, the sheath gas 35 (-2500 to-3000V)/(3500 to 4000V), the sheath gas 30-40psi, the auxiliary gas 105-15psi and the collision gas 1.5-2psi, and multiple reaction monitoring is adopted for scanning.

As a further technical scheme, the derivatization treatment is that derivatization reaction is carried out in the presence of a catalyst and an activating agent;

the catalyst comprises pyridine, 4-dimethylaminopyridine or acetic acid;

the activator comprises N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide;

the medium of the derivatization reaction comprises aqueous methanol;

in the derivatization reaction system, the concentration of 3-nitrophenylhydrazine or C13-3-nitrophenylhydrazine is 2.5-50mmol, the volume concentration of the catalyst is 0.1% -5%, and the concentration of the activating agent is 5-50mmol.

As a further technical scheme, the catalyst is pyridine.

As a further technical scheme, the temperature of the derivatization reaction is 4-50 ℃, and preferably 40 ℃;

the time of the derivatization reaction is 10-60min, and preferably 40min.

As a further technical scheme, the concentration of the formic acid aqueous solution is 0.01-0.2%.

As a further technical scheme, the concentration of the formic acid aqueous solution is 0.1%.

As a further technical solution, the conditions of the high performance liquid chromatography further include:

the column temperature of the chromatographic column is 35-55 ℃, and preferably 40 ℃;

the amount of the sample is 2 to 5. Mu.L, preferably 2. Mu.L.

As a further technical scheme, the method also comprises a step of extracting the sample before the sample is subjected to derivatization treatment.

As a further technical solution, the extracting step includes: dissolving a sample in a solvent, and taking clear liquid after solid-liquid separation;

the solvent comprises a methanol solution;

the concentration of the methanol solution is 70-95%, preferably 80%.

As a further technical scheme, the mass spectrum parameters of the organic acid after derivatization are as follows:

fumaric acid:

positive and negative modes: negative, parent ion: 384.988, daughter ion: 231.97, collision energy: 17.89eV, radio frequency voltage: 88V; or, positive and negative mode: negative, parent ion: 384.988, daughter ion: 234.042, collision energy: 14.69eV, radio frequency voltage: 88V;

citric acid:

positive and negative modes: negative, parent ion: 596, daughter ion: 222.03, collision energy: 27.7eV, radio frequency voltage: 97V; or, positive and negative mode: negative, parent ion: 596, daughter ion: 401.054, collision energy: 18.1eV, radio frequency voltage: 97V;

citraconic acid:

positive and negative modes: negative, parent ion: 399.05, daughter ion: 246.071, collision energy: 17.38eV, radio frequency voltage: 76V; or, positive and negative mode: negative, parent ion: 399.05, daughter ion: 248.095, collision energy: 14.73eV, radio frequency voltage: 76V;

malic acid:

positive and negative modes: negative, parent ion: 403.088, daughter ion: 207.97, collision energy: 17.72eV, radio frequency voltage: 74V; or, positive and negative mode: negative, parent ion: 403.088, daughter ion: 250.071, collision energy: 13.47eV, radio frequency voltage: 74V;

l lactic acid:

positive and negative modes: negative, parent ion: 224, daughter ion: 152.042, collision energy: 14.77eV, radio frequency voltage: 50V; or, positive and negative mode: negative, parent ion: 224, daughter ion: 137.042, collision energy: 18.27eV, radio frequency voltage: 50V;

aconitic acid:

positive and negative modes: negative, parent ion: 577.95, daughter ion: 425.083, collision energy: 15.45eV, radio frequency voltage: 101V; or, positive and negative mode: negative, parent ion: 577.95, daughter ion: 178.054, collision energy: 20.88eV, radio frequency voltage: 101V;

succinic acid:

positive and negative modes: negative, parent ion: 387.088, daughter ion: 234.042, collision energy: 17.59eV, radio frequency voltage: 85V; or, positive and negative mode: negative, parent ion: 387.088, daughter ion: 98.071, collision energy: 34.82eV, radio frequency voltage: 85V;

mesaconic acid:

positive and negative modes: negative, parent ion: 399.05, daughter ion: 246.024, collision energy: 16.29eV, radio frequency voltage: 80V; or, positive and negative mode: negative, parent ion: 399.05, daughter ion: 248.042, collision energy: 14.56eV, radio frequency voltage: 80V;

isocitric acid:

positive and negative modes: negative, parent ion: 595.95, product ion: 387.083, collision energy: 18.94eV, radio frequency voltage: 89V; or, positive and negative mode: positive, parent ion: 598.038, daughter ion: 445.125, collision energy: 13.64eV, radio frequency voltage: 83V;

pyruvic acid:

positive and negative modes: negative, parent ion: 357.038, daughter ion: 150.042, collision energy: 19.41eV, radio frequency voltage: 79V; or, positive and negative mode: negative, parent ion: 357.038, daughter ion: 137.042, collision energy: 22.02eV, radio frequency voltage: 79V;

ketoglutaric acid:

positive and negative modes: negative, parent ion: 550.088, daughter ion: 371.125, collision energy: 21.93eV, radio frequency voltage: 117V; or, positive and negative mode: negative, parent ion: 550.088, daughter ion: 233.042, collision energy: 27.75eV, radio frequency voltage: 117V;

itaconic acid:

positive and negative modes: negative, parent ion: 398.975, daughter ion: 246.042, collision energy: 17.85eV, radio frequency voltage: 77V; or, positive and negative mode: negative, parent ion: 398.975, daughter ion: 234.042, collision energy: 16.79eV, radio frequency voltage: 77V;

oxaloacetic acid:

positive and negative modes: negative, parent ion: 536.062, daughter ion: 357.054, collision energy: 21.55eV, radio frequency voltage: 95V; or, positive and negative mode: negative, parent ion: 536.062, daughter ion: 247.042, collision energy: 24.33eV, radio frequency voltage: 95V;

c13-itaconic acid:

positive and negative modes: negative, parent ion: 411.132, daughter ion: 252.054, collision energy: 17.51eV, radio frequency voltage: 86V; or, positive and negative mode: negative, parent ion: 411.132, daughter ion: 240.042, collision energy: 17.3eV, radio frequency voltage: 86V;

c13-fumaric acid:

positive and negative modes: negative, parent ion: 397.082, daughter ion: 238.042, collision energy: 18.9eV, radio frequency voltage: 100V; or, positive and negative mode: negative, parent ion: 397.082, daughter ion: 240.113, collision energy: 15.95eV, radio frequency voltage: 100V;

c13-citric acid:

positive and negative modes: negative, parent ion: 614.162, product ion: 437.137, collision energy: 19.36eV, radio frequency voltage: 104V; or, positive and negative mode: negative, parent ion: 614.162, product ion: 228.071, collision energy: 29.6eV, radio frequency voltage: 104V;

c13-citraconic acid:

positive and negative modes: negative, parent ion: 411.132, daughter ion: 252.054, collision energy: 17.59eV, radio frequency voltage: 97V;

c13-malic acid:

positive and negative modes: negative, parent ion: 415.142, daughter ion: 214.042, collision energy: 18.9eV, radio frequency voltage: 80V; or, positive and negative mode: negative, parent ion: 415.142, daughter ion: 143.071, collision energy: 36.93eV, radio frequency voltage: 80V;

C13-L lactic acid:

positive and negative modes: negative, parent ion: 230.012, daughter ion: 158.054, collision energy: 15.87eV, radio frequency voltage: 53V; or, positive and negative mode: negative, parent ion: 230.012, daughter ion: 143.071, collision energy: 19.66eV, radio frequency voltage: 53V;

c13-succinic acid:

positive and negative modes: negative, parent ion: 399.11, daughter ion: 240.054, collision energy: 18.61eV, radio frequency voltage: 90V; or, positive and negative mode: negative, parent ion: 399.11, daughter ion: 158.054, collision energy: 22.9eV, radio frequency voltage: 90V;

c13-aconitic acid:

positive and negative modes: negative, parent ion: 596.18, daughter ion: 252.054, collision energy: 26.52eV, radio frequency voltage: 101V; or, positive and negative mode: negative, parent ion: 596.18, daughter ion: 184.113, collision energy: 21.6eV, radio frequency voltage: 101V;

c13-isocitric acid:

positive and negative modes: negative, parent ion: 614.21, daughter ion: 437.137, collision energy: 19.28eV, radio frequency voltage: 96V; or, positive and negative mode: positive, parent ion: 616.21, product ion: 457.208, collision energy: 13.8eV, radio frequency voltage: 82V;

c13-mesaconic acid:

positive and negative modes: negative, parent ion: 411.132, daughter ion: 252.054, collision energy: 17.55eV, radio frequency voltage: 88V; or, positive and negative mode: negative, parent ion: 411.132, daughter ion: 226.125, collision energy: 23.32eV, radio frequency voltage: 88V;

c13-pyruvic acid:

positive and negative modes: negative, parent ion: 369.092, daughter ion: 143.042, collision energy: 24.25eV, radio frequency voltage: 89V; or, positive and negative mode: negative, parent ion: 369.092, daughter ion: 156.024, collision energy: 21.3eV, radio frequency voltage: 89V;

c13-ketoglutaric acid:

positive and negative modes: negative, parent ion: 568.152, product ion: 383.137, collision energy: 23.45eV, radio frequency voltage: 142V; or, positive and negative mode: negative, parent ion: 568.152, product ion: 239.125, collision energy: 29.22eV, radio frequency voltage: 142V;

c13-oxaloacetic acid:

positive and negative modes: negative, parent ion: 554.062, daughter ion: 253.03, collision energy: 23.7eV, radio frequency voltage: 89V; or, positive and negative mode: negative, parent ion: 554.062, daughter ion: 395.196, collision energy: 21.39eV, radio frequency voltage: 89V.

Compared with the prior art, the invention has the following beneficial effects:

the quantitative detection method of the organic acid provided by the invention has the advantages of high sensitivity, low detection limit, high accuracy and good stability, and can realize the quantitative detection of 13 organic acids in the tricarboxylic acid cycle.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a XIC diagram of the derivatized standard;

FIG. 2 is a XIC diagram of an underivatized standard;

FIG. 3 is the XIC chart provided in comparative example 2;

FIG. 4 is the XIC chart provided in comparative example 3;

FIG. 5 is the XIC chart provided in comparative example 4;

FIG. 6 is the XIC chart provided in comparative example 5;

FIG. 7 is the XIC chart provided in comparative example 6;

FIG. 8 is the XIC chart provided in comparative example 7;

fig. 9 is the XIC chart provided for comparative example 8.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.

the conditions of the high performance liquid chromatography comprise:

a chromatographic column: waters HSS T3.1 × 150mm, waters BEH C18.1 × 100mm, or Waters HSS T3.1 × 100mm;

The research of the inventor finds that the separation degree of isomers can be improved by adopting 3-nitrophenylhydrazine to perform derivatization on the organic acid and then adopting a liquid chromatography-tandem mass spectrometry to perform quantitative analysis on the organic acid. For example, mesaconic acid, itaconic acid and citraconic acid have uniform nonderivative retention times of 1.04 and are indistinguishable, and citric acid and isocitric acid have retention times of 0.95 and are likewise indistinguishable; after derivatization, itaconic RT =2.78, citraconic RT =2.97, mesaconic RT-3.44, completely separable, isocitric RT =3.53, citric RT =3.90, and the two isomers are also completely separable. In addition, each substance is realized by derivatization with a corresponding standard. In addition, the Waters HSS T3.1 × 150mm, waters BEH C18.1 × 100mm and Waters HSS T3.1 × 100mm columns RT have good stability, but separation of isomers is difficult to achieve with conventional methods, whereas the use of specific derivatization methods in the present invention in combination with the columns described above retains the advantage of good RT stability and simultaneously achieves separation of isomers such as mesaconic acid, itaconic acid, citraconic acid, etc.

In some preferred embodiments, the derivatizing treatment is a derivatizing reaction in the presence of a catalyst and an activating agent;

the catalyst comprises pyridine, 4-dimethylamino pyridine or acetic acid, wherein the pyridine is used as the catalyst with the best effect, so the catalyst is preferably pyridine;

the activator comprises N- (3-dimethylaminopropyl) -N' -Ethylcarbodiimide (EDC);

preferably, the medium of the derivatization reaction comprises aqueous methanol.

In some preferred embodiments, the concentration of the derivatizing reagent (3-NPH) of the derivatization reaction may be, for example, but not limited to, 2.5mmol, 10mmol, 25mmol, 40mmol, 50mmol, preferably 40mmol.

In some preferred embodiments, the concentration of the activating agent (EDC) for the derivatization reaction may be, for example, but not limited to, 5mmol, 10mmol, 20mmol, 30mmol, 50mmol, preferably 30mmol.

In some preferred embodiments, the catalyst (pyridine) volume concentration of the derivatization reaction may be, for example, but not limited to, 0.1%, 0.5%, 1%, 2%, 5%, preferably 2%.

In some preferred embodiments, the temperature of the derivatization reaction may be, for example, but not limited to, 4 ℃,20 ℃, 30 ℃,40 ℃,50 ℃, preferably 40 ℃;

preferably, the time of the derivatization reaction may be, but is not limited to, 10min, 20min, 30min, 40min, 60min, preferably 40min.

In some preferred embodiments, the concentration of the aqueous formic acid solution may be, for example, but not limited to, 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, or 0.2%, preferably 0.1%.

In some preferred embodiments, the conditions of the high performance liquid chromatography further comprise:

the column temperature of the column may be, for example, but not limited to, 35 ℃,40 ℃, 45 ℃,50 ℃, 55 ℃, preferably 40 ℃;

the amount of sample may be, for example, but not limited to, 2. Mu.L, 3. Mu.L, 4. Mu.L or 5. Mu.L, preferably 2. Mu.L.

In some preferred embodiments, the sample further comprises a sample extraction step before the derivatization treatment.

The extraction step comprises: dissolving a sample in a solvent, and taking a clear solution after solid-liquid separation;

preferably, the solvent comprises a methanol solution;

preferably, the concentration of the methanol solution in the final system may be, for example, but not limited to, 70%, 75%, 85%, 90%, 95%, preferably 90%.

In some preferred embodiments, the mass spectrometric parameters of the derivatized organic acids are shown in table 1.

TABLE 1

Continuing with Table 1:

note: in the table, Q1 is a parent ion, Q3 is a daughter ion, RF is a radio frequency voltage, CE is collision energy, and Polarity is a positive-negative mode.

The invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for purposes of illustration only and are not to be construed as limiting the invention in any way.

Example 1

A quantitative detection method of organic acid comprises the following steps:

and (3) carrying out derivatization treatment on the sample to be detected, adding an internal standard solution, and carrying out quantitative analysis on the organic acid in the sample by adopting a liquid chromatography-tandem mass spectrometry method.

Mixing and preparing a standard: transferring 13 organic acid mother solutions by a liquid transfer device respectively, and mixing uniformly to prepare mixed standard mother solution 1 (the concentration of each organic acid is 200 ug/ml) of 10 standard products (itaconic acid, citraconic acid, mesaconic acid, L-lactic acid, succinic acid, malic acid, citric acid, isocitric acid, fumaric acid and aconitic acid) and mixed standard mother solution 2 (the concentration of each organic acid is 200 ug/ml) of 3 standard products (ketoglutaric acid, pyruvic acid and oxaloacetic acid). The organic acid names and CAS numbers are shown in Table 2.

TABLE 2

Serial number	Name(s)	CAS number
			1	Itaconic acid	97-65-4
2	Fumaric acid	110-17-8
			3	Oxaloacetic acid	328-42-7
4	Citric acid	77-92-9
			5	Citraconic acid	498-23-7
6	Malic acid	6915-15-7
			7	L lactic acid	79-33-4
8	Succinic Acid (SA)	110-15-6
			9	Aconitic acid	585-84-2
10	Ketoglutaric acid	328-50-7
			11	Isocitric acid	1637-73-6
12	Mesaconic acid	498-24-8
			13	Pyruvic acid	127-17-3

。

Preparing an internal standard: transferring 100 mul of mixed

standard mother liquor

1 and 2 by a pipettor respectively, adding 500 mul of 80% methanol aqueous solution respectively to obtain 600 mul of mixed liquor, mixing uniformly, centrifuging, taking 50 mul of supernatant respectively, adding 50u1160mM C13-3-NPH (C13-3-nitrophenylhydrazine) (80% methanol water), 50u1120mM EDC (N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide) (methanol), 50u18% pyridine (methanol), deriving for 40min at 40 ℃ to obtain internal standard mother liquor 1 and internal standard mother liquor 2, transferring 100u1 of each of the internal standard mother liquor 1 and the internal standard mother liquor 2 respectively to obtain internal standard mother liquor, transferring 20 mul of the internal standard mother liquor by the pipettor, adding 80% methanol aqueous solution of 80u1, mixing uniformly to obtain mixed internal standard solution (20 ug/ml), and performing derivatization treatment on samples to be detected by the same method, so that calculation is convenient, the dilution of the solutions is not considered, and the following concentration is the same.

Linear preparation: mu.1 of mixed

standard mother liquor

1 and 2 were pipetted separately, and then 500. Mu.l of 80% aqueous methanol was added to the pipettes to obtain 600. Mu.l of a mixed solution, which was mixed and centrifuged, and 50. Mu.l of supernatant was pipetted separately, and 50u1160mM 3-NPH (3-nitrophenylhydrazine) (80% aqueous methanol), 50ul120mM EDC (methanol), 50ul8% pyridine (methanol) was added to the supernatant separately, and derivatization was carried out at 40 ℃ for 40min to obtain linear mother liquor 1 and

linear mother liquor

2, and 100. Mu.l of each of linear mother liquor 1 and linear mother liquor 2 was pipetted separately to obtain 100ug/ml linear mother liquor. After dilution to linear form, 100ul of each spot was taken and 1ul of mixed internal standard solution (20 ug/ml) was added.

The extraction method comprises the following steps: 100mg or 100ul of the sample was extracted with 500ul 80% methanol, centrifuged, and the supernatant was collected.

The derivation method comprises the following steps: with 50ul of the above extraction reagent, 50ul 160mM 3-NPH (80% methanol solution, derivatization reagent), 50ul8% pyridine (methanol solvent, catalyst), 50ul 160mM EDC (methanol solution, activator) were added and reacted at 40 ℃ for 40min, supernatant fluid 100ul,1ul mixed internal standard solution (20 ug/ml) were taken and mixed well to obtain a liquid on-machine.

Chromatographic conditions are as follows:

a chromatographic column: waters HSS T32.1 × 150mm;

mobile phase: phase A: 0.1% formic acid water; phase B: acetonitrile;

column temperature: 40 ℃ C:

sample introduction amount: 2 μ l.

The chromatographic gradient is shown in table 3 below.

TABLE 3

Time min	Flow rate ml/min	A％	B％
					0	0.4	60	40
1	0.4	60	40
				3.5	0.4	40	60
8.5	0.4	25	75
				11.5	0.4	0	100
13.5	0.4	0	100
				14	0.4	60	40
16	0.4	60	40

。

Mass spectrum conditions:

electrospray ionization (ESI) source, ion source temperature 350 deg.C, ion source voltage negative/positive mode-2500V/3500V, sheath gas 35psi, auxiliary gas 10psi, and collision gas 1.5psi, scanning using Multiple Reaction Monitoring (MRM). The mass spectrometric detection parameters for the 26 standards and internal standards are shown in table 1.

Comparative example 1

A method for detecting an organic acid, which is different from example 1 in that a sample is not subjected to a derivatization treatment.

Comparative example 2

A method for detecting an organic acid, which is different from example i in that the mobile phase: phase A: 10mM ammonium acetate; phase B: methanol; the chromatographic gradients are shown in table 8 below (mobile phase a and mobile phase B add up to 100% in tables 8-11).

TABLE 8

Time min	Flow rate ml/min	B％
				0	0.4	2
2	0.4	2
			9	0.4	60
13	0.4	75
			17	0.4	100
19	0.4	100
			19.1	0.4	2
21	0.4	2

。

Comparative example 3

A method for detecting an organic acid, which is different from example 1 in that a mobile phase: phase A: 10mM ammonium acetate; phase B: methanol; the chromatographic gradient is shown in table 9 below.

TABLE 9

Time min	Flow rate ml/min	B％
				0	0.4	2
l	0.4	2
			9	0.4	100
12	0.4	100
			13	0.4	2
16	0.4	2

。

Comparative example 4

A method for detecting an organic acid, which is different from example 1 in that a chromatographic gradient is shown in table 10 below.

Watch 10

。

Comparative example 5

A method for detecting an organic acid, which is different from example 1 in that a chromatographic gradient is shown in table 11 below.

TABLE 11

Time min	Flow rate ml/min	B％
				0	0.4	2
1	0.4	2
			9	0.4	100
12	0.4	100
			13	0.4	2
16	0.4	2

。

Comparative example 6

A method for detecting an organic acid, which is different from example 1 in that liquid chromatography uses isocratic elution: mobile phase a 80%, mobile phase B20%.

Comparative example 7

A method for detecting an organic acid, which is different from example 1 in that NN-dimethylethylenediamine is used as a derivatization reagent. The ion pair is selected according to the substance to be detected.

Comparative example 8

An organic acid detection method, which is different from example 1 in that AETE (2-aminoethyl) trimethylammonium chloride was used as a derivatization reagent. The ion pair is selected according to the substance to be detected.

Test example 1 detection of sensitivity

The linear mother liquor provided in example 1 was diluted to 500ng/mL and examined by the methods provided in example 1 and comparative examples 1 to 8, respectively, and the results are shown in Table 4 and FIGS. 1 to 9.

TABLE 4

As can be seen from table 4, the peak area of the organic acid after the derivatization treatment is significantly increased, which indicates that the method provided in example 1 has higher sensitivity. The XIC of example 1 is shown in fig. 1, and it can be seen from the graph that the RT times of the respective organic acids are different, and the XIC of comparative example 1 is shown in fig. 2, and the organic acid peaks at about 1 min. The XIC diagram of comparative example 2 is shown in fig. 3, oxaloacetate does not peak, itaconic RT =10.64, and citraconic RT =10.68 are hardly separable. The XIC diagram of comparative example 3 is shown in fig. 4, oxaloacetate does not peak, citraconic RT =7.8, itaconic RT =7.76, mesaconic RT =8.05, poor resolution, isocitric RT =8.33, citric RT =8.4, and hardly separable. The XIC diagram of comparative example 4 is shown in fig. 5, oxaloacetate, ketoglutarate do not peak, citraconic RT =9.2, and itaconic RT =9.16 are hardly separable. The XIC diagram of comparative example 5 is shown in figure 6, oxaloacetate does not peak, isocitrate RT =7.01, citrate RT =7.18, and is poorly separated, itaconate RT =7.71, citraconate RT =7.8, mesaconate RT =6.99, and is poorly separated. The XIC pattern of comparative example 6 is shown in FIG. 7, where only L-lactic acid and malic acid appeared; the XIC diagram of comparative example 7 is shown in figure 8, with itaconic, mesaconic and citraconic RT all at 1.08, citric and isocitric RT all at 0.65, others, fumaric RT =0.75, oxaloacetic RT =0.62, malic RT =1.22, l-lactic RT =1.08, succinic RT =0.72, aconitic RT =0.66, ketoglutaric RT =0.6, pyruvic RT =0.71, RT times are all earlier; the XIC diagram of comparative example 8 is shown in fig. 9, where itaconic acid, mesaconic acid, citraconic acid RT are all 1.09, citric acid RT =0.72, isocitric acid RT =0.76, other materials, fumaric acid RT =1.09, oxaloacetic acid RT =0.6, malic acid RT =0.73, l-lactic acid RT =0.71, succinic acid RT =1.09, aconitic acid RT =1.05, ketoglutaric acid RT =0.68, pyruvic acid RT =0.57, and RT times are all earlier.

Test example 2 quantitative lower limit, linear and Linear Range detection

The linear mother liquor provided in example 1 was used as a sample, and was diluted in a gradient manner to 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000ng/mL, 100ul of each point was taken, and 1ul of mixed internal standard solution was added to each point, and the results were measured by the method provided in example 1, and are shown in table 5.

TABLE 5

Test example 3 detection of matrix Effect, accuracy and precision

1) Matrix effect test method:

preparation of a linear solution: the internal standard mother liquor provided in example 1 was diluted to 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000ng/mL and subjected to on-machine detection.

1. Using water as matrix

An internal standard solution 1 of 10ug/ml was prepared from the internal standard mother liquor provided in example 1.

Mu.l of mass-spectrometric water was pipetted by a pipette, and then 500. Mu.l of 80% aqueous methanol was added to obtain 600. Mu.l of a mixed solution, followed by mixing and centrifugation, 50. Mu.l of the supernatant was collected, 50ul 160mM 3-NPH (80% aqueous methanol), 50ul120mM EDC (methanol), 50ul8% pyridine (methanol) was added thereto, derivatization was carried out at 40 ℃ for 40min, 100ul of the supernatant was collected, 1ul of 1 (10 ug/ml) of an internal standard solution was added thereto, and the mixture was mixed. And obtaining a low-point standard water matrix sample.

Mu.l of mass spectrometric water was pipetted separately by a pipette, and 500. Mu.l of 80% aqueous methanol was added to obtain 600. Mu.l of a mixed solution, mixed well, centrifuged, 50. Mu.l of the supernatant was taken, 50ul 160mM 3-NPH (80% aqueous methanol), 50ul120mM EDC (methanol), 50ul8% pyridine (methanol) was added separately, derivatization was carried out at 40 ℃ for 40min, 100ul of the supernatant was taken, 1ul of an internal standard solution (20 ug/ml) was added thereto, and mixed well. A midpoint spiked water matrix sample was obtained.

Mu.l of mass-spectrometric water was pipetted by a pipette, and 500. Mu.l of 80% aqueous methanol was added to obtain 600. Mu.l of a mixed solution, followed by mixing, centrifugation, and 50. Mu.l of the supernatant was added thereto, 50ul 160mM 3-NPH (80% aqueous methanol), 50ul120mM EDC (methanol), 50ul8% pyridine (methanol), derivatization was carried out at 40 ℃ for 40min, 100ul of the supernatant was taken, 1ul of an internal standard mother liquor (100 ug/ml) was added thereto, and the mixture was mixed. And obtaining a high-point standard water matrix sample.

2 using the sample as a matrix

Mu.l/100 mg of each sample was pipetted and 500. Mu.l of 80% aqueous methanol was added to obtain 600. Mu.l of the mixture, which was mixed and centrifuged, 50. Mu.l of the supernatant was collected, 50ul of 160mM 3-NPH (80% aqueous methanol), 50ul of 120mM EDC (methanol), 50ul of 8% pyridine (methanol) was added to the supernatant, 40 ℃ derivatization was carried out for 40min, 100ul of the supernatant was collected, 1ul of 1 (10 ug/ml) of the internal standard solution was added and mixed. And obtaining a low-point labeled sample matrix sample.

Mu.l/100 mg of each sample was pipetted and 500. Mu.l of 80% aqueous methanol was added to obtain 600. Mu.l of the mixture, which was mixed and centrifuged, 50. Mu.l of the supernatant was collected and 50ul of 160mM 3-NPH (80% aqueous methanol), 50ul of 120mM EDC (methanol), 50ul of 8% pyridine (methanol), derivatization was carried out at 40 ℃ for 40min, 100ul of the supernatant was collected and 1ul of the internal standard solution (20 ug/ml) was added and mixed. And obtaining a middle point labeled sample matrix sample.

Mu.l/100 mg of sample was pipetted and 500. Mu.l of 80% aqueous methanol was added to obtain 600. Mu.l of the mixture, mixed, centrifuged, 50. Mu.l of the supernatant was collected, 50ul of 160mM 3-NPH (80% aqueous methanol), 50ul of 120mM EDC (methanol), 50ul of 8% pyridine (methanol) was added to the supernatant, derivatized at 40 ℃ for 40min, 100ul of the supernatant was collected, 1ul of the internal standard mother liquor (100 ug/ml) was added thereto, and mixed. And obtaining a high-point labeled sample matrix sample.

3 groups were prepared in parallel per spot above.

Blank matrix sample treatment:

transfer 100ul of mass-spectrometric water and 500ul of 80% aqueous methanol solution to each other with a pipette to obtain 600 ul of mixed solution, mix well, centrifuge, collect 50ul of supernatant, add 50ul 160mM 3-NPH (80% aqueous methanol), 50ul120mM EDC (methanol), 50ul8% pyridine (methanol), derivatize at 40 ℃ for 40min, collect 100ul of supernatant, add 1ul of 80% aqueous methanol, mix well. A blank sample was obtained. Groups 3 were prepared in parallel.

The matrix effect was calculated by measuring the samples and blanks by the external standard method and using the following formula.

ME% = { (QC matrix-B blank)/QC mass water-1 } × 100%;

note: ME: matrix effect, QC matrix: adding a quality control internal standard sample into a sample matrix, and carrying out QC mass spectrometry: mass spectrometry water additivity.

Samples were run from the above samples (3 spiked water matrix samples, 3 spiked sample matrix samples, blank samples) and linear solution samples, with 3 replicates per sample set, and the matrix effect results are shown in table 6.

2) Stability experiment quality control sample:

the linear mother liquor provided in example 1 was used as a sample, and was diluted in a gradient manner to 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000ng/mL, and 100ul of each point was taken and 1ul of mixed internal standard solution (20 ug/mL) was added thereto.

Transfer 100. Mu.l of mixed standard mother liquor 1 and mixed standard mother liquor 2 by a pipette, and then add 500. Mu.l of 80% aqueous methanol solution to obtain 600. Mu.l of mixed liquor, mix well, centrifuge, take 50. Mu.l of supernatant, add 50ul 160mM 3-NPH (80% aqueous methanol), 50ul120mM EDC (methanol), 50ul8% pyridine (methanol), derivatize for 40min at 40 ℃ to obtain linear mother liquor 1 and linear mother liquor 2, transfer 100ul of linear mother liquor 1 and linear mother liquor 2 respectively, and mix to obtain 100ug/ml of linear mother liquor. Diluting to 100ng/ml,200ng/ml,1000ng/ml, transferring 100ul of each point in the low, medium and high points, adding 1ul of mixed internal standard solution, and mixing.

Blank matrix

Transfer 100ul of mass water separately with a pipette, add 500ul of 80% aqueous methanol to obtain 600 ul of mixed solution, mix well, centrifuge, take 50ul of supernatant, add 50ul 160mM 3-NPH (80% aqueous methanol), 50ul120mM EDC (methanol), 50ul8% pyridine (methanol), derivatize for 40min at 40 deg.C, take 100ul of supernatant, add 1ul of mixed internal standard solution, mix well.

Sample introduction is carried out on the stability experiment quality control samples, each sample is set to be 3 times, sample introduction is carried out for 1 time every day, three days are repeated, an internal standard method is adopted to determine samples and blank samples, stability and precision are achieved, the recovery rate result is shown in table 6, sample introduction is carried out for 24 hours repeatedly on the last day (namely, the stability experiment quality control samples are advanced, and then 12 gradient samples are circularly introduced), and the stability result is shown in table 7.

TABLE 6

TABLE 6, continuation:

13 organic acid linear blood matrix effect results-17.53% < ME% < 19.92%, cell matrix effect results-16.27% < ME% < 13.18%, leaf matrix effect results-18.78% < ME% < 17.55%, root matrix effect results-19.21% < ME% < 15.83%, liver matrix effect results-17.68% < ME% < 17.21%, kidney matrix effect results-15.53% < ME% < 17.92%, lung matrix effect results-16.86% < ME% < 15.95%, between-20% and 20% indicate that 7 types of matrix effects have less influence; the precision result of the first day in the day is more than 0.89% and less than 10.72% CV%, the precision result of the second day in the day is more than 1.01% and less than 10.94% CV%, the precision result of the third day in the day is more than 0.52% and less than 10.51% CV%, and the precision results are all less than 15%, and the acceptance standard is met; the precision result of three days in the day is more than 0.15 percent and less than 11.15 percent of CV percent and less than 15 percent, and the precision result meets the acceptance standard; the recovery results ranged from 86.57% < R% < 107.43%, and ranged from 85% to 115%, meeting the acceptance criteria.

TABLE 7

QCL results of 24-hour stability of 13 organic acid samples in a sample injector after treatment are 2.33% < CV% < 7.75%, QCM results are 3.12% < CV% < 12.06%, QCH results are 3.05% < CV% < 8.33%, and the results all fall within the range of CV < 15%, and the results meet the acceptance standards.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for quantitatively detecting an organic acid, comprising the steps of: performing derivatization treatment on a sample to be detected, adding an internal standard solution, and performing quantitative analysis on organic acid in the sample by adopting a high performance liquid chromatography-tandem mass spectrometry method;

the conditions of the high performance liquid chromatography comprise:

a chromatographic column: waters HSS T3.1 × 150mm, waters BEH C18.1 × 100mm or Waters HSS T3.1 × 100mm;

the conditions of the mass spectrum include: the electrospray ionization source has the ion source temperature of 300-400 ℃, the ion source voltage negative/positive mode (-2500 to-3000V)/(3500 to 4000V), the sheath gas of 30-40psi, the auxiliary gas of 5-15psi and the collision gas of 1-2psi, and multiple reaction monitoring is adopted for scanning.

2. The method for quantitatively detecting an organic acid according to claim 1, wherein the derivatization treatment is a derivatization reaction in the presence of a catalyst and an activating agent;

the catalyst comprises pyridine, 4-dimethylaminopyridine or acetic acid;

the activator comprises N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide;

the medium of the derivatization reaction comprises aqueous methanol;

3. The method for quantitatively detecting an organic acid according to claim 2, wherein the catalyst is pyridine.

4. The method for quantitatively detecting an organic acid according to claim 2, wherein the temperature of the derivatization reaction is 4 to 50 ℃;

the time of the derivatization reaction is 10-60min.

5. The method for quantitatively detecting an organic acid according to claim 1, wherein the concentration of the aqueous formic acid solution is 0.01 to 0.2%.

6. The method for quantitatively detecting an organic acid according to claim 5, wherein the concentration of the aqueous formic acid solution is 0.1%.

7. The method for the quantitative detection of an organic acid according to claim 1, wherein the conditions of the high performance liquid chromatography further comprise:

the temperature of the chromatographic column is 35-55 ℃;

the sample amount is 2-5 mu L.

8. The method for quantitatively detecting an organic acid according to claim 1, wherein the sample further comprises a sample extraction step before the derivatization treatment.

9. The method for the quantitative detection of organic acids according to claim 8, wherein the extraction step comprises: dissolving a sample in a solvent, and taking clear liquid after solid-liquid separation;

the solvent comprises a methanol solution;

the concentration of the methanol solution is 70-95%.

10. The method for the quantitative detection of an organic acid according to any one of claims 1 to 9, wherein mass spectrometric parameters of the derivatized organic acid are as follows:

fumaric acid:

citric acid:

citraconic acid:

malic acid:

l lactic acid:

aconitic acid:

succinic acid:

mesaconic acid:

isocitric acid:

pyruvic acid:

ketoglutaric acid:

itaconic acid:

oxaloacetic acid:

c13-itaconic acid:

c13-fumaric acid:

c13-citric acid:

c13-citraconic acid:

c13-malic acid:

C13-L lactic acid:

c13-succinic acid:

positive and negative modes: negative, parent ion: 399.11, daughter ions: 240.054, collision energy: 18.61eV, radio frequency voltage: 90V; or, positive and negative mode: negative, parent ion: 399.11, daughter ion: 158.054, collision energy: 22.9eV, radio frequency voltage: 90V;

c13-aconitic acid:

c13-isocitric acid:

c13-mesaconic acid:

c13-pyruvic acid:

c13-ketoglutaric acid:

c13-oxaloacetic acid:

positive and negative modes: negative, parent ion: 554.062, daughter ion: 253.03, collision energy: 23.7eV, radio frequency voltage: 89V;

or, positive and negative mode: negative, parent ion: 554.062, daughter ion: 395.196, collision energy: 21.39eV, radio frequency voltage: 89V.