阅读说明：本技术 一种透析液中神经递质的检测方法 (Method for detecting neurotransmitter in dialysate ) 是由 潘凌云 姜珊 贾益群 王宇 刘新华 王雨 于 2021-09-24 设计创作，主要内容包括：本发明属于分析化学技术领域,提供了一种透析液中神经递质的检测方法。本发明以乙腈为流动相B、甲酸铵水溶液作为流动相A,正、负离子同时扫描的质谱检测模式,首次完成了4类共13个神经递质的同时定性和定量检测,分析时间短,定性和定量检测结果准确、可靠,无需将待测透析液分别采用不同的检测仪器或不同的流动相条件进行分析,节省了样本量、样本的检测时间,也为在线检测提供了必要的前提,具有可期的临床应用前景。本发明在进行超高效液相色谱-质谱检测前,对待测透析液进行富集,实现了对低含量神经递质的透析液的检测。(The invention belongs to the technical field of analytical chemistry, and provides a method for detecting neurotransmitter in dialysate. The invention uses acetonitrile as a mobile phase B, uses ammonium formate aqueous solution as a mobile phase A, adopts a mass spectrum detection mode of simultaneous scanning of positive ions and negative ions, completes simultaneous qualitative and quantitative detection of 13 neurotransmitters of 4 types for the first time, has short analysis time and accurate and reliable qualitative and quantitative detection results, does not need to analyze dialysate to be detected by different detection instruments or different mobile phase conditions, saves the sample amount and the detection time of the sample, provides necessary premise for on-line detection, and has promising clinical application prospect. Before the ultra-performance liquid chromatography-mass spectrometry detection, the method enriches the dialysate to be detected, and realizes the detection of the dialysate with low neurotransmitter content.)

1. A method for detecting a neurotransmitter in a dialysate, comprising the steps of:

enriching the dialysate to be detected to obtain an enriched sample;

performing ultra-high performance liquid chromatography-mass spectrometry detection on the enriched sample;

the ultra-high performance liquid chromatography-mass spectrometry detection comprises ultra-high performance liquid chromatography detection and mass spectrometry detection;

the parameters of the ultra-high performance liquid chromatography detection comprise:

a chromatographic column: waters ACQUITY UPLC BEH C₁₈Chromatography column, 2.1mm × 100mm, 1.7 μm;

mobile phase A: an aqueous ammonium formate solution;

mobile phase B: acetonitrile;

gradient elution procedure:

(1) isocratic elution is carried out for 11.5-12.5 min by using acetonitrile with the volume ratio of 2-5%;

(2) the volume ratio of acetonitrile is increased to 65-75% at a constant speed, and gradient elution is carried out for 2.5-3.5 min;

(3) isocratic elution is carried out for 1.0-2.0 min by using acetonitrile with the volume ratio of 65-75%;

(4) instantaneously switching acetonitrile with the volume ratio of 65-75% into acetonitrile with the volume ratio of 2-5%, and then isocratically eluting for 1.0-2.0 min;

flow rate of mobile phase: 0.2-0.3 mL/min;

column temperature: 30-45 ℃;

the parameters of the mass spectrometric detection include:

the ion source is an electrospray ion source, and the scanning mode is a positive or negative ion mode; ion source spray voltage: 4.0-4.5 kV; ion source gas curtain gas: 193.0 to 275.8 kPa; collision gas: 41.4-55.1 kPa; ion source temperature: 400-600 ℃; atomizing: 344.7 to 413.7 kPa; auxiliary gas: 344.7 to 413.7 kPa; analysis time: 8.0-10 min; multiple reaction monitoring mode.

2. The detection method according to claim 1, wherein the enrichment is quantitative loop enrichment; the specification of the quantitative ring is 5-20 mu L.

3. The detection method according to claim 2, wherein the quantification ring is a quantification ring having a ten-way valve.

4. The detection method according to claim 1, 2 or 3, wherein the enrichment time is 2.5min to 20 min.

5. The detection method according to claim 1, wherein when the neurotransmitter is glutamic acid, the monitor ion pair is 148.1/84.1; when the neurotransmitter is aspartic acid, the monitoring ion pair is 134.1/74.0; when the neurotransmitter is 5-hydroxytryptamine, the monitoring ion pair is 177.2/160.3; when the neurotransmitter is histamine, the monitoring ion pair is 112.0/95.1; when the neurotransmitter is acetylcholine, the monitoring ion pair is 146.3/87.0; when the neurotransmitter is glycine, the monitoring ion pair is 76.2/30.0; when the neurotransmitter is 5-oxindole acetic acid, the monitoring ion pair is 192.1/146.1; when the neurotransmitter is glutamine, the monitoring ion pair is 147.1/130.1; when the neurotransmitter is gamma-aminobutyric acid, the monitoring ion pair is 104.1/87.2; when the neurotransmitter is adenosine, the monitoring ion pair is 268.4/136.5; the above 10 neurotransmitters were monitored in positive ion mode;

when the neurotransmitter is adenosine monophosphate, the monitoring ion pair is 268.4/136.5; when the neurotransmitter is adenosine diphosphate, the monitored ion pair is 426.2/133.8; when the neurotransmitter is adenosine triphosphate, the monitoring ion pair is 505.9/158.9; the above 3 neurotransmitters were monitored using the negative ion mode.

6. The detection method according to claim 1, wherein the diluent of the dialysate to be detected is ringer's solution.

7. The detection method of claim 1, wherein the ultra performance liquid chromatography-mass spectrometry detection comprises quantitative detection; the quantitative detection adopts a standard curve method; the standard curve in the standard curve method is a standard curve of neurotransmitter concentration and chromatographic peak area.

Technical Field

The invention relates to the technical field of analytical chemistry, in particular to a method for detecting neurotransmitter in dialysate.

Background

Neurotransmitters are chemical substances that are synthesized by presynaptic neurons and released at the terminals, specifically act on receptors on postsynaptic neurons or effector cells, and perform a role in information transmission. The neurotransmitter mainly comprises amino acids such as gamma-aminobutyric acid, glutamic acid, glycine and the like, monoamines such as 5-hydroxytryptamine, epinephrine, norepinephrine and the like, cholines such as acetylcholine, choline and the like, purines and peptides such as adenosine and derivatives thereof, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate and the like, and the like.

Amino acid neurotransmitters are classified into Excitatory (EAAs) and Inhibitory (IAAs), which are important substances for regulating physiological activities of the body. The excitatory amino acids include glutamic acid, aspartic acid and the like, which play an important role in maintaining normal signal transmission of neurons, and the inhibitory amino acids include gamma-aminobutyric acid, glycine and the like, which are mainly used as inhibitory neurotransmitters to mediate fast inhibitory synaptic transmission of spinal cords and brainstems. Monoamine neurotransmitters include catecholamines (dopamine, norepinephrine and epinephrine) and indoles (5-hydroxytryptamine), which are stored in presynaptic membrane vesicles and play important roles in regulating nervous and cardiovascular systems of human bodies. Choline neurotransmitter mainly refers to acetylcholine, which is a neurotransmitter of many peripheral nerves, and changes of choline are related to various diseases such as muscular diseases of nervous system, epilepsy, brain trauma and encephalitis. Purine neurotransmitters mainly comprise adenine, adenosine and derivatives thereof, and purine has an important position in body metabolism, can protect nervous system, modulate pain sensation, and play an important role in cerebrovascular and gastrointestinal tract activities, nervous system immune response, canceration and nerve regeneration.

There are a large number of neurotransmitters in the peripheral nervous system of muscles, and changes in the content thereof have an important influence on organisms and are closely related to various diseases including muscular stiffness, rheumatoid arthritis, osteoarthritis, and the like. Studies have shown that targeting glutamatergic signals in peripheral nerves is an important pathway to promote regeneration and repair and pain control; 5-hydroxytryptamine participates in various physiological regulation processes such as muscle contraction, blood pressure regulation and control, platelet aggregation and the like; myotonic patients have increased levels of acetylcholine in their muscle tissues (very low levels in normal muscle). Therefore, assaying biological samples such as tissue homogenates and monitoring changes in neurotransmitter levels in muscle tissue are of great interest for neurological studies. However, most transmitters are low in biological samples, the matrix is complex, and the interference of endogenous components is large. Therefore, the measurement of the content thereof is highly required for pretreatment of a sample, sensitivity of an instrument, and the like.

Currently, there are many reports on the detection method of neurotransmitters in biological samples. Among them, the monoamine neurotransmitters are commonly used in electrochemical, fluorescence, capillary electrophoresis and liquid chromatography-mass spectrometry (LC-MS/MS); amino acid neurotransmitters are detected by a fluorescence method after pre-column derivatization and a liquid chromatography-mass spectrometry method; choline is mainly detected by electrochemistry; adenosine is detected by capillary electrophoresis and liquid chromatography-mass spectrometry. The detection of different types of neurotransmitters has mostly been separated by high performance liquid systems, with only some differences in the choice of detectors. The problems faced in the detection of neurotransmitters in a sample mainly include the following aspects: 1. the content of neurotransmitter is very low, and the neurotransmitter belongs to endogenous substances, and other substances have great interference on the neurotransmitter; 2. the chemical property of the monoamine component is unstable, and the monoamine component is easy to be oxidized and decomposed under the illumination and alkaline conditions; 3. the HPLC-fluorescence method adopted by the amino acid transmitter needs to perform derivatization on a detection sample to enable the detection sample to generate fluorescence, and difficulty in analysis and detection and establishment of methodology is increased.

Compared with other detection means, the LC-MS/MS method does not need derivatization, is simple to operate, high in sensitivity, short in analysis time and high in selectivity, is a powerful tool for analyzing trace components in a complex biological sample, and can meet the analysis requirements of the biological sample by properly optimizing the pretreatment conditions and the chromatographic and mass spectrum conditions of the sample. When the mass spectrometry is used for measurement, monoamines, amino acids and choline mostly adopt a mass spectrometry method in a positive mode to obtain higher instrument response, adenosine monophosphate, adenosine diphosphate and adenosine triphosphate in purines are more suitable for detection in a negative ion mode under the condition of an alkaline mobile phase because of the existence of phosphate radicals with negative charges, and although the detection in the positive ion mode can be carried out after ion pair reagents (N, N-dimethylhexylamine, dibutylamine acetic acid and the like) are adopted as the mobile phase in literature reports, the repeatability is poor in earlier research. Therefore, it is necessary to find more suitable chromatographic conditions and mass spectrum conditions to simultaneously detect adenosine monophosphate neurotransmitter and other neurotransmitters, thereby achieving the purpose of reducing the detection cost and time.

Disclosure of Invention

In view of the above, the present invention provides a method for detecting a neurotransmitter in a dialysate. The detection method provided by the invention can realize the simultaneous detection of the adenosine monophosphate neurotransmitter, the choline neurotransmitter, the amino acid neurotransmitter and the monoamine neurotransmitter in the purine neurotransmitters.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for detecting neurotransmitter in dialysate, which comprises the following steps:

enriching the dialysate to be detected to obtain an enriched sample;

performing ultra-high performance liquid chromatography-mass spectrometry detection on the enriched sample;

the ultra-high performance liquid chromatography-mass spectrometry detection comprises ultra-high performance liquid chromatography detection and mass spectrometry detection;

the parameters of the ultra-high performance liquid chromatography detection comprise:

a chromatographic column: waters ACQUITY UPLC BEH C₁₈Chromatography column, 2.1mm × 100mm, 1.7 μm;

mobile phase A: an aqueous ammonium formate solution;

mobile phase B: acetonitrile;

gradient elution procedure:

(1) isocratic elution is carried out for 11.5-12.5 min by using acetonitrile with the volume ratio of 2-5%;

(2) the volume ratio of acetonitrile is increased to 65-75% at a constant speed, and gradient elution is carried out for 2.5-3.5 min;

(3) isocratic elution is carried out for 1.0-2.0 min by using acetonitrile with the volume ratio of 65-75%;

(4) instantaneously switching acetonitrile with the volume ratio of 65-75% into acetonitrile with the volume ratio of 2-5%, and then isocratically eluting for 1.0-2.0 min;

flow rate of mobile phase: 0.2-0.3 mL/min;

column temperature: 30-45 ℃;

the parameters of the mass spectrometric detection include:

the ion source is an electrospray ion source, and the scanning mode is a positive or negative ion mode; ion source spray voltage: 4.0-4.5 kV; ion source gas curtain gas: 193.0 to 275.8 kPa; collision gas: 41.4-55.1 kPa; ion source temperature: 400-600 ℃; atomizing: 344.7 to 413.7 kPa; auxiliary gas: 344.7 to 413.7 kPa; analysis time: 8.0-10 min; multiple reaction monitoring mode.

Preferably, the enrichment is quantitative loop enrichment; the specification of the quantitative ring is 5-20 mu L.

Preferably, the dosing ring is a dosing ring with a ten way valve.

Preferably, the enrichment time is 2.5min to 20 min.

Preferably, when the neurotransmitter is glutamate, the monitor ion pair is 148.1/84.1; when the neurotransmitter is aspartic acid, the monitoring ion pair is 134.1/74.0; when the neurotransmitter is 5-hydroxytryptamine, the monitoring ion pair is 177.2/160.3; when the neurotransmitter is histamine, the monitoring ion pair is 112.0/95.1; when the neurotransmitter is acetylcholine, the monitoring ion pair is 146.3/87.0; when the neurotransmitter is glycine, the monitoring ion pair is 76.2/30.0; when the neurotransmitter is 5-oxindole acetic acid, the monitoring ion pair is 192.1/146.1; when the neurotransmitter is glutamine, the monitoring ion pair is 147.1/130.1; when the neurotransmitter is gamma-aminobutyric acid, the monitoring ion pair is 104.1/87.2; when the neurotransmitter is adenosine, the monitoring ion pair is 268.4/136.5; the above 10 neurotransmitters were monitored in positive ion mode;

when the neurotransmitter is adenosine monophosphate, the monitoring ion pair is 268.4/136.5; when the neurotransmitter is adenosine diphosphate, the monitored ion pair is 426.2/133.8; when the neurotransmitter is adenosine triphosphate, the monitoring ion pair is 505.9/158.9; the above 3 neurotransmitters were monitored using the negative ion mode.

Preferably, the diluent of the dialysate to be tested is ringer's solution.

Preferably, the ultra performance liquid chromatography-mass spectrometry detection comprises quantitative detection; the quantitative detection adopts a standard curve method; the standard curve in the standard curve method is a standard curve of neurotransmitter concentration and chromatographic peak area.

The invention provides a method for detecting neurotransmitter in dialysate, which comprises the following steps: enriching the dialysate to be detected to obtain an enriched sample; performing ultra-high performance liquid chromatography-mass spectrometry detection on the enriched sample; the ultra-high performance liquid chromatography-mass spectrometry detection comprises ultra-high performance liquid chromatography detection and mass spectrometry detection; the parameters of the ultra-high performance liquid chromatography detection comprise: a chromatographic column: WatersACQUITY UPLC BEH C₁₈Chromatography column, 2.1mm × 100mm, 1.7 μm; mobile phase A: an aqueous ammonium formate solution; mobile phase B: acetonitrile; gradient elution procedure: (1) isocratic elution is carried out for 11.5-12.5 min by using acetonitrile with the volume ratio of 2-5%; (2) the volume ratio of acetonitrile is increased to 65-75% at a constant speed, and gradient elution is carried out for 2.5-3.5 min; (3) isocratic elution is carried out for 1.0-2.0 min by using acetonitrile with the volume ratio of 65-75%; (4) instantaneously switching acetonitrile with the volume ratio of 65-75% into acetonitrile with the volume ratio of 2-5%, and then isocratically eluting for 1.0-2.0 min; flow rate of mobile phase: 0.2-0.3 mL/min; column temperature: 30-45 ℃; the parameters of the mass spectrometric detection include: the ion source is an electrospray ion source, and the scanning mode is a positive ion mode and a negative ion mode; ion source spray voltage: 4.0-4.5 kV; ion source gas curtain gas: 193.0 to 275.8 kPa; collision gas: 41.4-55.1 kPa; ion source temperature: 400-600 ℃; atomizing: 344.7 to 413.7 kPa; auxiliary gas: 344.7 to 413.7 kPa; analysis time: 8.0-10 min; multiple reaction monitoring mode.

The invention takes acetonitrile as a mobile phase B and ammonium formate aqueous solution as a mobile phase A, and a mass spectrum detection mode of positive and negative ion simultaneous scanning completes simultaneous qualitative and quantitative detection of 13 neurotransmitters (acetylcholine, glycine, glutamic acid, glutamine, gamma-aminobutyric acid, aspartic acid, histamine, 5-hydroxytryptamine, 5-oxindole acetic acid, adenosine monophosphate, adenosine diphosphate and adenosine triphosphate) of 4 classes for the first time, has short analysis time and accurate and reliable qualitative and quantitative detection results, does not need to analyze a dialysate to be detected by different detection instruments or different mobile phase conditions respectively, saves the sample amount and the detection time of the sample, provides necessary preconditions for on-line detection, and has a promising clinical application prospect. Before the ultra-performance liquid chromatography-mass spectrometry detection, the method enriches the dialysate to be detected, and realizes the detection of the dialysate with low neurotransmitter content.

Furthermore, the dialysate is directly detected through the ultra-high performance liquid chromatography-mass spectrometry after being enriched by the quantitative ring, so that the sample loss caused by complex pretreatment is avoided.

Furthermore, the switchable ten-way valve on the quantitative ring can directly sample and analyze the enriched dialysate, so that the loss and pollution of the sample in the transferring and storing processes are avoided.

Further, the dialysate is diluted by the ringer's solution to obtain the dialysate to be measured, which can improve the peak shape and reduce the matrix effect.

Drawings

FIG. 1 is a schematic diagram of the operation of a dosing ring having a ten way valve;

FIG. 2 is a schematic flow diagram of an on-line microdialysis-ultra high performance liquid chromatography-mass spectrometry detection and analysis platform;

FIG. 3 is a multiple reactive ion monitoring chromatogram of a Glu (glutamic acid) standard in example 1;

FIG. 4 is a multiple reactive ion monitoring chromatogram of an Asp (aspartic acid) standard in example 1;

FIG. 5 is a multiple reactive ion monitoring chromatogram of the 5-HT (5-hydroxytryptamine) standard of example 1;

FIG. 6 is a multiple reactive ion monitoring chromatogram of the HA (histamine) standard in example 1;

FIG. 7 is a multiple reactive ion monitoring chromatogram of the ACh (acetylcholine) standard of example 1;

FIG. 8 is a multiple reactive ion monitoring chromatogram of a Gly (glycine) standard in example 1;

FIG. 9 is a multiple reaction ion monitoring chromatogram of the 5-HIAA (5-oxindole acetic acid) standard of example 1;

FIG. 10 is a multiple reactive ion monitoring chromatogram of the Gln (glutamine) standard in example 1;

FIG. 11 is a multi-reactive ion monitoring chromatogram of the gamma-GABA (gamma-aminobutyric acid) standard in example 1;

FIG. 12 is a multiple reactive ion monitoring chromatogram of the ADO (adenosine) standard in example 1;

FIG. 13 is a multiple reaction ion monitoring chromatogram of the AMP (adenosine monophosphate) standard in example 1;

FIG. 14 is a multi-reactive ion monitoring chromatogram of the ADP (adenosine diphosphate) standard in example 1;

FIG. 15 is a multiple reactive ion monitoring chromatogram of the ATP (adenosine triphosphate) standard in example 1;

FIG. 16 is a graph of in vitro recovery vs. concentration trends for 13 neurotransmitters in example 2;

FIGS. 17-18 are bar graphs of the levels of 13 neurotransmitter concentrations in rat muscle dialysate of example 3.

Detailed Description

The invention provides a method for detecting neurotransmitter in dialysate, which comprises the following steps:

enriching the dialysate to be detected to obtain an enriched sample;

and carrying out ultra-performance liquid chromatography-mass spectrometry detection on the enriched sample.

In the present invention, the starting materials used in the present invention are preferably commercially available products unless otherwise specified.

In the present invention, the dialysate to be tested is preferably purchased commercially or made by oneself.

In the present invention, when the detection method is directed to real-time detection, the preparation method of the dialysate to be detected preferably comprises the following steps:

after the animal body to be detected in real time is perfused, the dialysate containing the neurotransmitter is taken out of the body, and the dialysate to be detected is obtained.

In the present invention, the agent for perfusion preferably comprises Ringer's Solution.

In the present invention, the perfusion apparatus preferably comprises a microdialysis pump.

In the present invention, the perfusion preferably comprises the steps of:

after an animal body to be detected in real time is anesthetized, firstly embedding a linear probe into the tissue of the animal body to be detected in real time, pumping Ringer's Solution into one end, into which the probe is inserted, through a microdialysis pump for perfusion, and then enabling dialysate to flow out from the other end of the probe; and after perfusion is balanced for 30min, collecting the effluent liquid at the other end of the probe as dialysate to be detected.

In the present invention, the flow rate of the perfusion is preferably 1. mu.L/min to 2. mu.L/min, and more preferably 1. mu.L/min.

In the present invention, the enrichment is preferably quantitative ring enrichment; the standard of the quantitative determination loop is preferably 5. mu.L to 20. mu.L, and more preferably 10. mu.L. The dialysate to be detected is directly detected by ultra-high performance liquid chromatography-mass spectrometry after being enriched by the quantitative ring, so that the sample loss caused by complex pretreatment is avoided.

In the present invention, the dosing ring is preferably a dosing ring having a ten-way valve. The switchable ten-way valve on the quantitative ring can directly sample and analyze the enriched dialysate, thereby avoiding the loss and pollution caused by the transfer and storage processes of the sample.

Fig. 1 is a schematic diagram of the working principle of a dosing ring with a ten-way valve.

In the present invention, the time for the enrichment is preferably 2.5min to 20min, and more preferably 10 min.

In the invention, the dialysis containing 13 neurotransmitters is enriched before the ultra performance liquid chromatography-mass spectrometry detection, so that the detection of the dialysate with low content of the neurotransmitters is realized.

After an enriched sample is obtained, the invention carries out ultra performance liquid chromatography-mass spectrometry detection on the enriched sample.

In the invention, the ultra performance liquid chromatography-mass spectrometry detection comprises ultra performance liquid chromatography detection and mass spectrometry detection.

In the invention, the parameters of the ultra-high performance liquid chromatography detection comprise:

a chromatographic column: waters ACQUITY UPLC BEH C₁₈Chromatography column, 2.1mm × 100mm, 1.7 μm;

mobile phase A: an aqueous ammonium formate solution; the concentration of the ammonium formate aqueous solution is preferably 1 mmol/L-5 mmol/L, and more preferably 2 mmol/L; the pH value of the ammonium formate aqueous solution is preferably 9.8-10.0, and particularly preferably 9.8;

mobile phase B: acetonitrile;

gradient elution procedure:

(1) isocratic elution is carried out for 11.5-12.5 min by using acetonitrile with the volume ratio of 2-5%;

(2) the volume ratio of acetonitrile is increased to 65-75% at a constant speed, and gradient elution is carried out for 2.5-3.5 min;

(3) isocratic elution is carried out for 1.0-2.0 min by using acetonitrile with the volume ratio of 65-75%;

(4) instantaneously switching acetonitrile with the volume ratio of 65-75% into acetonitrile with the volume ratio of 2-5%, and then isocratically eluting for 1.0-2.0 min;

the gradient elution procedure is preferably:

0 → 12.0 min: 98% mobile phase a;

12.0 → 15.0 min: 98% → 30% mobile phase a;

15.0 → 16.0 min: 30% mobile phase a;

16.0 → 18.0 min: 98% mobile phase a;

flow rate of mobile phase: 0.2-0.3 mL/min, preferably 0.25 mL/min;

column temperature: 30-45 ℃ and preferably 35 ℃.

In the present invention, the parameters of the mass spectrometric detection include:

the ion source is an electrospray ion source, and the scanning mode is a positive or negative ion mode;

ion source spray voltage: 4.0-4.5 kV, and particularly preferably 4.5 kV;

ion source gas curtain gas: 193.0 to 275.8kPa, preferably 241.3 kPa;

collision gas: 41.4 to 55.1kPa, preferably 48.3 kPa;

ion source temperature: 400-600 ℃, preferably 500 ℃;

atomizing: 344.7 to 413.7kPa, preferably 413.7 kPa;

auxiliary gas: 344.7 to 413.7kPa, preferably 413.7 kPa;

analysis time: 8.0-10 min, preferably 8.0 min;

multiple reaction monitoring mode (MRM).

In the present invention, when the neurotransmitter is glutamate, the monitored ion pair is 148.1/84.1; when the neurotransmitter is aspartic acid, the monitoring ion pair is 134.1/74.0; when the neurotransmitter is 5-hydroxytryptamine, the monitoring ion pair is 177.2/160.3; when the neurotransmitter is histamine, the monitoring ion pair is 112.0/95.1; when the neurotransmitter is acetylcholine, the monitoring ion pair is 146.3/87.0; when the neurotransmitter is glycine, the monitoring ion pair is 76.2/30.0; when the neurotransmitter is 5-oxindole acetic acid, the monitoring ion pair is 192.1/146.1; when the neurotransmitter is glutamine, the monitoring ion pair is 147.1/130.1; when the neurotransmitter is gamma-aminobutyric acid, the monitoring ion pair is 104.1/87.2; when the neurotransmitter is adenosine, the monitoring ion pair is 268.4/136.5; the above 10 neurotransmitters were monitored using positive ion mode.

In the present invention, when the neurotransmitter is adenosine monophosphate, the monitoring ion pair is 268.4/136.5; when the neurotransmitter is adenosine diphosphate, the monitored ion pair is 426.2/133.8; when the neurotransmitter is adenosine triphosphate, the monitoring ion pair is 505.9/158.9; the above 3 neurotransmitters were monitored using the negative ion mode.

In the present invention, the ultra performance liquid chromatography-mass spectrometry detection preferably comprises quantitative detection; the quantitative determination is preferably performed using a standard curve method. In the present invention, the standard curve in the standard curve method is a standard curve of neurotransmitter concentration and peak area.

In the present invention, the standard curve method preferably comprises the steps of:

preparing a mixed standard solution dissolved with a neurotransmitter standard;

performing ultra-high performance liquid chromatography-mass spectrometry detection on the mixed standard solution dissolved with the neurotransmitter standard substance to obtain the chromatographic peak area of the neurotransmitter standard substance;

drawing a chromatographic peak area-concentration standard curve of each neurotransmitter based on the chromatographic peak area and the concentration of the neurotransmitter standard;

and substituting the measured chromatographic peak area of each neurotransmitter of the dialysate to be measured into the corresponding chromatographic peak area-concentration standard curve of the neurotransmitter to obtain the concentration of each neurotransmitter in the dialysate to be measured.

The invention prepares a mixed standard solution dissolved with neurotransmitter standard.

In the present invention, the method for preparing a mixed standard solution in which a neurotransmitter standard is dissolved preferably includes the steps of:

preparing a single standard solution of neurotransmitter standard or preparing a mixed standard solution dissolved with a plurality of neurotransmitter standards;

diluting with ringer's solution to obtain mixed standard solution containing neurotransmitter standard.

In the present invention, the solvent for preparing a single standard solution of neurotransmitter standard or preparing a mixed standard solution in which a plurality of neurotransmitter standards are dissolved preferably includes methanol, acetonitrile or water; the method specifically comprises the following steps: dissolving glutamic acid, aspartic acid, adenosine monophosphate, adenosine diphosphate and adenosine triphosphate by using ultrapure water; the remaining neurotransmitters were dissolved with methanol.

After the mixed standard solution dissolved with the neurotransmitter standard is obtained, the method carries out ultra-performance liquid chromatography-mass spectrometry detection on the mixed standard solution dissolved with the neurotransmitter standard to obtain the chromatographic peak area of the neurotransmitter standard.

In the invention, the parameters of the ultra performance liquid chromatography-mass spectrometry detection are preferably consistent with the technical scheme, and are not described herein again.

After the chromatographic peak area of the neurotransmitter standard is obtained, the invention draws a chromatographic peak area-concentration standard curve of each neurotransmitter based on the chromatographic peak area and the concentration of the neurotransmitter standard.

The drawing method is not particularly limited in the present invention, and the drawing operation known to those skilled in the art may be adopted.

After a chromatographic peak-concentration standard curve of each neurotransmitter is obtained, the measured chromatographic peak area of each neurotransmitter of the dialysate to be measured is substituted into the corresponding chromatographic peak area-concentration standard curve of the neurotransmitter to obtain the concentration of each neurotransmitter in the dialysate to be measured.

The substitution operation is not particularly limited in the present invention, and a substitution operation known to those skilled in the art may be employed.

FIG. 2 is a schematic flow diagram of an on-line microdialysis-ultra high performance liquid chromatography-mass spectrometry detection and analysis platform.

The following examples are provided to describe the method for detecting a neurotransmitter in a dialysate of the present invention in detail, but they should not be construed as limiting the scope of the present invention.

Example 1

The purpose of this example is to establish a test method for the determination of 13 neurotransmitters in a muscle microdialysis sample based on an on-line microdialysis-LC-MS combination. 13 neurotransmitters include: choline neurotransmitters include acetylcholine; amino acids include: glycine, glutamic acid, glutamine, gamma-aminobutyric acid, aspartic acid, histamine; monoamines include: 5-hydroxytryptamine, 5-oxindole acetic acid; the purines include: adenosine, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate.

Preparing a standard solution: precisely weighing 13 reference substances, namely glutamic acid, aspartic acid, adenosine monophosphate, adenosine diphosphate and adenosine triphosphate, dissolving the 13 reference substances in ultrapure water, dissolving the rest neurotransmitter reference substances in methanol, respectively preparing 5.0mg/mL single-standard stock solutions, and storing the single-standard stock solutions at-20 ℃. Methanol is adopted to prepare the mixed standard mother liquor of the 13 reference substances, and the concentration is 200 mug/mL. Diluting the mixed standard solution mother solution with ringer's solution before use to prepare a series of mixed standard solutions with different concentration distributions of each neurotransmitter in the mixed standard solution.

Collecting samples: after a rat is anesthetized by isoflurane, a linear probe is embedded into forelimb (peripheral) muscle tissue of the rat, dialysis liquid is immediately collected after the ringer's solution perfusion is balanced for 30min at the rate of 1 mu L/min, the dialysis liquid is enriched in a quantitative ring with the volume of 10 mu L, the detection time of each dialysis liquid is set to be 18min, wherein the enrichment time is 10min, and the mass spectrometry time is 8 min. Enrichment and analysis of the next dialysate continued after 18 min. The sample injection analysis device runs in a reciprocating mode, and therefore the purpose of real-time sample injection analysis is achieved. The working schematic diagram of the quantitative ring with the ten-way valve and the flow schematic diagram of the online microdialysis-ultra high performance liquid chromatography-mass spectrometry detection and analysis platform are respectively shown in fig. 1 and fig. 2.

Liquid chromatography conditions: waters accuracy ultra performance liquid chromatograph (Waters corporation, USA), Waters accuracy UPLC BEH C₁₈Chromatography column (2.1 mm. times.100 mm, 1.7 μm); mobile phase B: acetonitrile; mobile phase A: 2mmol/L ammonium formate in water at pH 9.8;

gradient elution procedure:

0 → 12.0 min: 98% mobile phase a; 12.0 → 15.0 min: 98% → 30% mobile phase a; 15.0 → 16.0 min: 30% mobile phase a; 16.0 → 18.0 min: 98% mobile phase a;

the column temperature was 35 ℃ and the flow rate of the mobile phase was 0.25 mL/min.

Mass spectrum conditions: API6500+ triple quadrupole tandem mass spectrometer (AB Sciex, usa); electrospray ion source (ESI), positive, negative ion scan mode; ion source spray voltage: 4.5 kV; ion source gas curtain gas: 241.3 kPa; collision gas: 48.3 kPa; ion source temperature: 500 ℃; atomizing: 413.7 kPa; auxiliary gas: 413.7 kPa; analysis time (duration): 8.0 min; multiple reaction monitoring mode (MRM), instrument parameters and monitoring ion pairs are shown in table 1. The obtained multi-reactive ion monitoring chromatogram of the neurotransmitter standard product is shown in figures 3 to 15.

TABLE 1 Instrument parameters and monitoring ion pairs

Drawing a standard curve: taking a series of standard working solutions under the item of 'standard solution preparation', quantifying by adopting an external standard method, taking the peak area of an object to be detected as a vertical coordinate (Y value), taking the concentration of the object to be detected as a horizontal coordinate (X value), and performing regression calculation by using a weighted (W ═ 1/X) least square method to obtain a corresponding regression equation shown in Table 2. S/N ≧ 3 determines the detection limit, S/N ≧ 10 determines the quantitation limit, where S represents the signal value and N represents the noise value.

TABLE 2 Standard Curve for neurotransmitters

As can be seen from Table 2, the standard curve of each neurotransmitter has a good linear relationship between the peak area of the analyte and the concentration of the analyte in the corresponding linear range.

Precision and accuracy: taking a series of standard working solutions with the concentration of 5, 50 and 400ng/mL under the item of 'standard solution preparation', distributing the working solutions in low, medium and high concentrations, preparing 5 parts of each concentration in parallel, and carrying out sample injection analysis; the measurement is continuously carried out for 3 days, and the standard curve equation of the current day is substituted, so that the precision and the accuracy between batches and in batches are calculated, and the results are shown in Table 3.

TABLE 3 inter-and intra-batch precision and accuracy test results

As can be seen from Table 3, the in-batch and inter-batch precision and accuracy of each neurotransmitter component in the biological sample are good, the in-batch and inter-batch precision range of the sample to be tested is 0.06% -9.25%, and the accuracy range of the sample to be tested is-8.80% -9.60%, which all meet the requirements of the biological analysis method.

Matrix effect: taking solvent standard samples with the concentration levels of low, medium and high of 5, 50 and 400ng/mL respectively under the item of 'standard solution preparation', carrying out sample injection analysis, and recording the peak area A.

And (3) diluting the mixed standard solution mother liquor by using a proper amount of the blank muscle dialysate of the healthy rat, preparing a matrix standard sample containing three concentration levels of low, medium and high of 5, 50 and 400ng/mL, carrying out sample injection analysis, and recording the peak area B.

Directly feeding the collected blank muscle dialysate of the healthy rats for analysis, and recording the peak area C.

The matrix effect of 13 neurotransmitters was calculated by the above 3 preparation methods, each concentration being 5 parts in parallel. The formula for the calculation of the Matrix Effect (ME) is as follows:

ME(％)＝(B-C)/A×100％；

in the formula, B is the peak area of a matrix standard sample, C is the peak area of a blank sample, and A is the peak area of a solvent standard sample.

The matrix effect results obtained are shown in table 4.

TABLE 4 matrix Effect

As can be seen from table 4: the stroma effect range of 13 neurotransmitters is 75.62% -112.5%, and the RSD range is less than 15%, which indicates that the dialysate has no obvious interference on the substance to be detected.

Example 2

The purpose of this example was to study the relationship between in vitro probe recovery and neurotransmitter concentration levels for 13 neurotransmitters using established detection methods.

Using ringer's solution as the perfusion fluid, a perfusion rate of 1.0 μ L/min was selected to examine the in vitro probe recovery rate (n ═ 3) at concentration levels of 5 μ g/mL, 10 μ g/mL, and 20 μ g/mL, and one sample was collected every 30 min. The solution samples with the three concentration levels are obtained by directly diluting the mixed standard solution with the ringer's solution. According to the formula R ═ C_s/C_m100 calculation of Probe recovery, C_mRepresenting a mixed control solution of known concentration, C_sRepresentative of the concentrations measured for the microdialysis samples, the results are shown in table 5 and fig. 16.

In vitro recovery of 513 neurotransmitters

From table 5 and fig. 16, it can be found that: the in-vitro probe recovery rates of the neurotransmitters with different concentrations are basically consistent, and the probe recovery rate has no obvious correlation with the transmitter concentration; the recovery rate of the except purine neurotransmitters is lower, ADO is (7.39 +/-0.97)%, AMP is (4.24 +/-0.66)%, ADP is (5.50 +/-2.20)%, ATP is (5.97 +/-0.88)%, and the recovery rate of different concentrations of the other neurotransmitters is higher than 8.5%.

Example 3

The purpose of this example 3 was to study the effect of acupuncture on the levels of 13 neurotransmitters in the peripheral muscles of rats using established assays.

Needle puncture and muscle tissue dialysis sampling: healthy male rats 5 were dialyzed, one each, through the hole-most point, and then acupuncture was performed on Tianfu and Chize points. Perfusing ringer's solution at a flow rate of 1.0 μ L/min, balancing perfusion for 60min, and collecting muscle tissue dialysate during and after acupuncture.

The results of the content of rat muscle tissue dialysate between the two groups are shown in FIG. 17 and FIG. 18. As can be seen from fig. 17 and 18: the levels of Glu, Asp, HA, ACh, Gly, γ -GABA, ADO, ADP, AMP and ATP were reduced in the post-acupuncture group compared to the time-acupuncture group, but there was no statistical difference; the content of 5-HIAA and Gln is increased, and the Gln has statistical difference (P < 0.05). The results show that the concentration level of neurotransmitters such as Gln, AMP and ADP in the peripheral nervous system of the muscle is higher than that of other neurotransmitters; most of the neurotransmitters in the muscle tissue were changed during and after the rat acupuncture.

Example 4

Preparing a dialysate containing 13 neurotransmitters by using ringer's solution, wherein the concentration of glutamic acid in the dialysate is 14.71 mug/mL; the concentration of the aspartic acid is 13.31 mu g/mL; the concentration of the 5-hydroxytryptamine is 17.62 mug/mL; the concentration of histamine was 11.11. mu.g/mL; the concentration of the acetylcholine is 16.32 mug/mL; the concentration of the glycine is 15.0 mu g/mL; the concentration of the 5-oxindole acetic acid is 19.12 mu g/mL; the concentration of glutamine is 14.61 mug/mL; the concentration of the gamma-aminobutyric acid is 10.31 mu g/mL; the concentration of adenosine is 26.72 mug/mL; the concentration of adenosine monophosphate is 34.72 mug/mL; the concentration of adenosine diphosphate is 42.72 mu g/mL; the concentration of adenosine triphosphate was 50.72. mu.g/mL.

And (3) enriching the dialysate samples in a quantitative loop with the volume of 10 mu L, wherein the detection time of each sample is set to be 18min, the enrichment time is 10min, and the mass spectrometry time is 8 min.

The parameters of the ultra-high liquid chromatography and the mass spectrometry are the same as those of the embodiment 1.

Measuring the concentration of glutamic acid in the dialysate to be 15.12 mug/mL, the concentration of aspartic acid to be 12.75 mug/mL and the concentration of 5-hydroxytryptamine to be 18.01 mug/mL; the concentration of histamine was 10.94. mu.g/mL; the concentration of the acetylcholine is 15.18 mug/mL; the concentration of glycine is 15.78 mug/mL; the concentration of the 5-oxindole acetic acid is 19.96 mu g/mL; the concentration of glutamine is 13.70 mug/mL; the concentration of the gamma-aminobutyric acid is 9.91 mu g/mL; the concentration of adenosine is 28.88 mug/mL; the concentration of adenosine monophosphate is 35.97 mu g/mL; the concentration of adenosine diphosphate is 43.87 mu g/mL; the concentration of adenosine triphosphate was 48.79. mu.g/mL. The results show that: the detection method provided by the invention has accurate and reliable results.

It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be construed as the protection scope of the present invention.