An LC-MS / MS method for detecting neurotransmitter metabolites in a biological sample

Abstract

The application relates to an LC-MS / MS method for detecting neurotransmitter metabolites in a biological sample, comprising the following steps: S1, constructing a mass spectrum database containing neurotransmitter metabolites and isotope internal standards; S2, preparing a mixed standard solution containing neurotransmitter standards and drawing standard curves of each neurotransmitter metabolite standard; S3, extracting a biological sample by a protein precipitation method to obtain a sample solution to be detected; S4, separating the sample solution to be detected by using liquid chromatography, wherein a mobile phase A of the liquid chromatography is an aqueous solution containing ammonium fluoride and formic acid, and a mobile phase B is an organic phase containing formic acid; and S5, performing mass spectrum detection on the sample separated by the liquid chromatography. The method can realize baseline separation of 57 metabolites within 9 minutes.

Description

Technical Field

[0001] This application relates to the field of analytical chemistry, and in particular to an LC-MS / MS method for detecting neurotransmitter metabolites in biological samples. Background Technology

[0002] Neurotransmitters (NTs) are chemical substances that transmit information between nerve cells. They are key molecules in the transmission of chemical signals in the nervous system and play a central role in the regulation of physiological functions and the development of diseases. Studies have shown that imbalances in neurotransmitters and their metabolites are closely related to various pathological processes, including neurodegenerative diseases, mental disorders, and sleep-wake cycle disorders. For example, dopaminergic system abnormalities are associated with Parkinson's disease, while serotonin metabolism disorders are common in patients with depression. This correlation makes neurotransmitter testing an important tool for clinical diagnosis and drug development.

[0003] At the technical level, neurotransmitter detection faces numerous challenges. Due to the wide variety of neurotransmitters (including monoamines, amino acids, choline derivatives, etc.) and their diverse concentration range in the central nervous system (from pg to μg), traditional methods (microdialysis, electrochemical sensor technology, and fluorescent probe technology) often struggle to achieve simultaneous detection of multiple substances. In recent years, liquid chromatography-tandem mass spectrometry (LC-MS / MS) has become the mainstream method for neurotransmitter detection due to its high sensitivity, high specificity, and wide coverage. However, neurotransmitter metabolites exhibit a wide range of polarities, are present in low concentrations in biological samples, and experience significant matrix interference. Traditional reversed C18 separation methods or hydrophilic chromatographic (HILIC) column methods still have limitations. For example, the former either fails to retain highly polar neurotransmitters, leading to severe matrix interference and affecting the accuracy of quantitative results, or requires derivatization to promote retention, but the resulting compounds are complex and the pretreatment is very time-consuming. The latter, while retaining most highly polar substances, suffers from poor stability, making the detection results difficult to reproduce, and it does not significantly improve sensitivity for metabolites in low concentrations. Furthermore, neurotransmitters are easily degraded in vitro (e.g., acetylcholine is easily hydrolyzed by cholinesterase at pH > 7), placing extremely high demands on biological sample pretreatment methods. Summary of the Invention

[0004] This invention provides an LC-MS / MS method for detecting neurotransmitter metabolites in biological samples. Addressing the shortcomings of existing technologies, this method uses LC-MS / MS to quantitatively detect 57 neurotransmitters (including monoamines, amino acids, choline, and organic acids) in common biological samples such as human serum, rat brain tissue, and human cells. This method optimizes key chromatographic parameters to solve the retention problem of the 57 neurotransmitter metabolites, and achieves baseline separation and improved sensitivity for these metabolites. It also incorporates 45 isotope internal standards for quantitative correction of actual samples. This method offers advantages such as simple pretreatment, detection of multiple neurotransmitter types, comprehensive coverage of biological sample types, good reproducibility, low sample volume, short analysis time, high sensitivity, and more accurate quantification, making it suitable for rapid analysis of large batches of samples.

[0005] This application provides an LC-MS / MS method for detecting neurotransmitter metabolites in biological samples, comprising the following steps: S1: constructing a mass spectrometry database containing neurotransmitter metabolites and their isotope internal standards; S2: preparing a mixed standard solution containing neurotransmitter standards and plotting standard curves for each neurotransmitter metabolite standard; S3: extracting the biological sample by protein precipitation to obtain a sample solution to be tested; S4: separating the sample solution to be tested by liquid chromatography, wherein the mobile phase A of the liquid chromatography is an aqueous solution containing ammonium fluoride and formic acid, and the mobile phase B is an organic phase containing formic acid; S5: performing mass spectrometry detection on the sample separated by liquid chromatography.

[0006] Optionally, in step S1, the neurotransmitter metabolites include 57 neurotransmitter metabolites, and the 57 neurotransmitter metabolites include at least one of choline, 4-aminobutyric acid, 5-hydroxytryptamine, dopamine, and thyroxine.

[0007] Optionally, the 57 neurotransmitter metabolites include pyridinecarboxylic acid, dopamine, 3-methoxytyramine, 5-hydroxyindoleacetic acid, 5-hydroxytryptophan, 5-methoxyindole-3-acetic acid, 5-methoxytryptamine, acetylcholine, choline, dopa (L-DOPA), adrenaline, choline, 4-aminobutyric acid, histamine, indole-3-carboxaldehyde, kynurenine, melatonin, DL-3-methoxyadrenaline hydrochloride, N-acetyl-5-hydroxytryptamine, norepinephrine, DL-noradrenaline hydrochloride, phenylalanine, phenylethylamine, 5-hydroxytryptamine, and thyroid hormones. At least one of the following: tryptophan, tryptophan, tyramine, tyrosine, dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, 3-hydroxy-an-aminobenzoic acid, (R)-3-hydroxybutyric acid, homovanillic acid, homovanillic acid, kynurenic acid, cinnamic acid, xanthuric acid, arginine, aspartic acid, betaine, betaine aldehyde chloride, glutamic acid, glutamine, glycine, histidine, L-leucine, L-lysine, L-methionine, ornithine, methylglycine (sarcosine), L-serine, threonine, phenylpyruvic acid, p-hydroxyphenylpyruvic acid, glutathione, and succinic acid.

[0008] Optionally, in step S2, the mixed standard solution of neurotransmitter standards contains 45 isotope internal standards.

[0009] Optionally, in step S3, the biological sample includes at least one of human serum, brain tissue, and human cells.

[0010] Optionally, in step S3, the protein precipitation extraction of the biological sample includes the following steps: a: Human serum samples were extracted using a mixed solvent of acetonitrile and methanol in a volume ratio of 3:(9-17); b: Brain tissue samples were extracted using a methanol-water solution with a methanol volume fraction of 65-75%; c: Add a methanol aqueous solution with a methanol volume fraction of 75-85% to the human cell sample and extract it by freezing and thawing in liquid nitrogen 2-4 times.

[0011] Optionally, in step S4, mobile phase A is an aqueous solution containing 0.4-0.6 mM ammonium fluoride and 0.08-0.12% (v / v) formic acid, and mobile phase B is an organic phase containing 0.08-0.12% (v / v) formic acid, wherein the volume ratio of methanol to acetonitrile in the organic phase is (45-55):(55-45).

[0012] Optionally, in step S4, the liquid chromatography column used for liquid chromatography detection is an ACQUITY UPLC HSSPFP column.

[0013] Optionally, in step S4, gradient elution is used for the mobile phase and gradient elution is used for liquid chromatography separation. The gradient elution conditions are 0 min-7 min 95% A / 5% B (V / V), 7-7.6 min 5% A / 95% B (V / V), and 7.6-9 min 95% A / 5% B (V / V).

[0014] Optionally, in step S4, the flow rate of the liquid chromatography column is 0.28-0.32 mL / min, and the column temperature is 39-41℃.

[0015] Optionally, in step S5, the mass spectrometry detection uses an electrospray ionization source with multi-channel reaction monitoring. The positive ion spray voltage is 5500V, the negative ion spray voltage is -4500V, and the ion source temperature, collision gas, curtain gas, nebulizer, and auxiliary gas positive and negative modes are all 550℃, 7psi, 35psi, 50psi, and 60psi, respectively.

[0016] In summary, the present invention has at least one of the following beneficial technical effects: 1. This application provides an LC-MS / MS method for detecting neurotransmitter metabolites in biological samples. It can be applied to the detection of human serum, rat brain tissue, human cells and other sample types. It has the advantages of covering a wide range of biological sample types and has a wide range of applications. It is of great significance for the screening, diagnosis and identification of neurological diseases.

[0017] 2. This application provides an LC-MS / MS method for detecting neurotransmitter metabolites in biological samples. By adjusting the chromatographic column, mobile phase additives, and mobile phase composition, the retention enhancement, baseline separation, and sensitivity improvement of 57 metabolites can be achieved. At the same time, 45 isotope internal standards are added to reduce the matrix interference of metabolites in biological samples. The entire analysis time is only 9 minutes.

[0018] 3. This application provides an LC-MS / MS method for detecting neurotransmitter metabolites in biological samples, which has the advantages of simple pretreatment, use of protein precipitation method, only one pretreatment required for a single sample type, small sample volume, non-degradable metabolites, low cost, high efficiency, and easy automation. Attached Figure Description

[0019] Figure 1 The total ion chromatogram shows the separation, sensitivity, and response of 57 neurotransmitter metabolite standards using the chromatographic column and mobile phase system of Example 1. Figure 2 Extraction ion chromatograms showing the separation, sensitivity, and response of 57 neurotransmitter metabolite standards using the chromatographic column and mobile phase system described in Example 1; Figure 3The total ion chromatogram shows the separation, sensitivity, and response of 57 neurotransmitter metabolites in human serum using the chromatographic column and mobile phase system of Example 1. Figure 4 The total ion chromatogram shows the separation, sensitivity, and response of 57 neurotransmitter metabolites in rat brain tissue using the chromatographic column and mobile phase system of Example 1. Figure 5 The total ion chromatogram shows the separation, sensitivity, and response of 57 neurotransmitter metabolites in human cells using the chromatographic column and mobile phase system of Example 1. Figure 6 The total ion chromatogram shows the separation, sensitivity, and response of the column used in Comparative Example 1 for 57 neurotransmitter metabolite standards. Figure 7 The total ion chromatogram shows the separation, sensitivity, and response of the column used in Comparative Example 2 for 57 neurotransmitter metabolite standards. Figure 8 Extraction ion chromatograms showing the separation, sensitivity, and response of 57 neurotransmitter metabolite standards using the mobile phase system in Comparative Example 3; Figure 9 The extraction ion chromatograms show the separation, sensitivity, and response of the mobile phase systems in Example 1 and Comparative Example 3 to the cholineamine standard. in, Figure 9 In the figure, (a) is the extraction ion chromatogram of the mobile phase system in Example 1 for the separation, sensitivity and response of choline standard; Figure 9 In the figure, (b) is the extraction ion chromatogram of the mobile phase system in Comparative Example 3 for the separation, sensitivity and response of choline standard; Figure 10 The extraction ion chromatograms show the separation, sensitivity, and response of the mobile phase systems in Example 1 and Comparative Example 3 to the 4-aminobutyric acid standard. in, Figure 10 In the figure, (a) is the extraction ion chromatogram of the mobile phase system in Example 1 for the separation, sensitivity and response of 4-aminobutyric acid standard; Figure 10 In the figure, (b) is the extraction ion chromatogram of the mobile phase system in Comparative Example 3 for the separation, sensitivity and response of 4-aminobutyric acid standard; Figure 11 The extraction ion chromatograms show the separation, sensitivity, and response of the mobile phase systems in Example 1 and Comparative Example 3 to the 5-hydroxytryptamine standard. in, Figure 11 In the figure, (a) is the extraction ion chromatogram of the mobile phase system in Example 1 for the separation, sensitivity and response of 5-hydroxytryptamine standard; Figure 11 In the figure, (b) is the extraction ion chromatogram of the mobile phase system in Comparative Example 3 for the separation, sensitivity and response of 5-hydroxytryptamine standard; Figure 12 The extraction ion chromatograms show the separation, sensitivity, and response of the mobile phase systems in Example 1 and Comparative Example 3 to dopamine standards. in, Figure 12 In the figure, (a) is the extraction ion chromatogram of the mobile phase system in Example 1 for the separation, sensitivity and response of dopamine standard; Figure 12 In the figure, (b) is the extraction ion chromatogram of the mobile phase system in Comparative Example 3 for the separation, sensitivity and response of dopamine standard; Figure 13 The extraction ion chromatograms show the separation, sensitivity, and response of the mobile phase systems in Example 1 and Comparative Example 3 to the thyroxine standard. in, Figure 13 In the figure, (a) is the extraction ion chromatogram of the mobile phase system in Example 1 for the separation, sensitivity and response of thyroxine standard; Figure 13 In the figure, (b) is the extraction ion chromatogram of the mobile phase system in Comparative Example 3 for the separation, sensitivity and response of thyroxine standard. Detailed Implementation

[0020] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0021] Example 1

[0022] An LC-MS / MS method for detecting neurotransmitter metabolites in biological samples includes the following steps: 1. Database Construction The establishment of the database of 57 neurotransmitter metabolites and 45 isotopes in this invention includes the following methods: Fifty-seven commercially available neurotransmitter standards and 45 isotope standards were purchased. 1 mg of each of the 57 neurotransmitter standards was weighed and dissolved in 1 mL of 70% (v / v) methanol aqueous solution to obtain single-standard stock solutions of the 57 neurotransmitter standards. Similarly, 1 mg of each of the 45 isotope standards was weighed and dissolved in 1 mL of 70% (v / v) methanol aqueous solution to obtain single-standard stock solutions of the 45 isotope standards. Both the single-standard stock solutions of the 57 neurotransmitter standards and the 45 isotope standards were collectively referred to as neurotransmitter metabolites. The mass spectrometry parameters (including optimal Q1, Q3, etc.) of 102 neurotransmitter metabolites were then precisely optimized using a flow injection method with a syringe pump to construct a neurotransmitter metabolite database. The results are shown in Table 1.

[0023] Table 1 Qualitative information on neurotransmitter metabolites

[0024]

[0025]

[0026]

[0027] Note: IS indicates an isotope internal standard. 2. Construction of Standard Curve 15 μL of each of the 57 neurotransmitter standard stock solutions (1 mg / mL) were mixed and diluted to obtain a mixed standard solution of 57 neurotransmitter metabolites. The concentration of each neurotransmitter standard in the mixed standard solution was 15 μg / mL, and the solvent used for dilution was 70% (v / v) methanol aqueous solution. The mixed standard solution of 57 neurotransmitter metabolites was then diluted with 70% (v / v) methanol aqueous solution to obtain a series of standard curve concentrations of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, and 10000 ng / mL for each neurotransmitter standard. Each standard curve concentration solution contained 45 isotope internal standards, and the concentration of each isotope internal standard was 200 ng / mL.

[0028] A mixed standard solution of 57 neurotransmitter metabolites with a concentration of 100 ng / mL was prepared for liquid chromatography separation and detection (preparation method: 15 μL of each of the 57 neurotransmitter standard stock solutions of 1 mg / mL was mixed and diluted to obtain a mixed standard solution of 57 neurotransmitter metabolites. The concentration of each neurotransmitter standard in the mixed standard solution was 100 ng / mL. The solvent used for dilution was 70% (v / v) methanol aqueous solution, which contained 45 isotope internal standards, and the concentration of each isotope internal standard was 100 ng / mL).

[0029] 3. Extraction of biological samples (1) Add 45 isotopic internal standards to a mixed solvent of acetonitrile and methanol at a volume ratio of 1:4, with a concentration of 250 ng / mL for each isotopic internal standard, to obtain a 20% (v / v) acetonitrile-methanol internal standard extract; take 50 μL of human serum sample (human serum sample C used in precision and accuracy tests). 加标 Human serum sample C 本底 Add 200 μL of 20% (v / v) acetonitrile methanol internal standard extraction solution, vortex at 2500 r / min for 5 min, let stand at -20℃ for 30 min, and then centrifuge at 4℃ and 12000 r / min for 15 min. After centrifugation, take the supernatant for analysis (liquid chromatography and mass spectrometry). (2) Add 45 isotopic internal standards to a 70% (v / v) methanol aqueous solution, with each isotopic internal standard having a concentration of 200 ng / mL, to obtain a 70% (v / v) methanol aqueous internal standard extract; take 50 mg of rat brain tissue sample (rat brain tissue sample C used in precision and accuracy tests). 加标 Rat brain tissue sample C 本底 After adding one steel ball and grinding evenly, add 500 μL of 70% (v / v) methanol-water internal standard extraction solution, vortex at 2500 r / min for 5 min, let stand at -20℃ for 30 min, and then centrifuge at 4℃ and 12000 r / min for 15 min. After centrifugation, take the supernatant for analysis (liquid chromatography and mass spectrometry). (3) Add 45 isotopic internal standards to an 80% (v / v) methanol aqueous solution, with each isotopic internal standard having a concentration of 200 ng / mL, to obtain an 80% (v / v) methanol aqueous internal standard extract; take 1×10 6 Personal cell samples (human cell samples used in precision and accuracy testing C) 加标 Human cell sample C 本底Add 500 μL of 80% (v / v) methanol-water internal standard extraction solution, freeze and thaw repeatedly with liquid nitrogen 3 times, vortex at 2500 r / min for 5 min, let stand at -20℃ for 30 min, and then centrifuge at 4℃ and 12000 r / min for 15 min. After centrifugation, take the supernatant for analysis (liquid chromatography and mass spectrometry).

[0030] 4. Liquid Chromatography and Mass Spectrometry Conditions The methods for standard curve preparation, liquid chromatography separation and detection of 57 neurotransmitter metabolites using mixed standard solutions, and mass spectrometry analysis, as well as the analysis of extracted biological samples, are all as follows: Instrument parameters: The data acquisition instrument system used for LC-MS / MS detection in this invention mainly includes ultra-high performance liquid chromatography (Waters HClass) and tandem mass spectrometry MS / MS (Applied Biosystems 6500 QuadrupoleTrap).

[0031] Chromatographic conditions: The column was an ACQUITY UPLC HSS PFP column, id 2.1×100mm, 1.8μm; mobile phase A was ultrapure water (containing 0.5mM ammonium fluoride and 0.1% (v / v) formic acid); mobile phase B was methanol and acetonitrile (containing 0.1% (v / v) formic acid) in a 1:1 volume ratio; column temperature was 40℃; flow rate was 0.3mL / min; injection volume was 2μL; gradient elution program: 0 min A / B 95:5 (v / v), 7 min A / B 5:95 (v / v), 7.5 min 5:95 (v / v), 7.6 min A / B 5:95 (v / v), 9 min 95:5 (v / v).

[0032] Mass spectrometry conditions: The ion source was an electrospray ionization (ESI) source, with simultaneous detection in both positive and negative ion modes, and the scanning mode was multiple reaction monitoring (MRM). The spray voltage for positive mode was 5500V, and the spray voltage for negative mode was -4500V. The ion source temperature, collision gas, curtain gas, nebulizer, and auxiliary gas were 550℃, 7psi, 35psi, 50psi, and 60psi for both positive and negative modes.

[0033] The total ion chromatogram of the separation, sensitivity, and response of the 57 neurotransmitter metabolites in a mixed standard solution of 57 neurotransmitter metabolites at a concentration of 100 ng / mL is shown below. Figure 1 As shown, the extraction ion chromatograms of 57 neurotransmitter metabolite standards at a concentration of 100 ng / mL are as follows: Figure 2As shown, the separation, sensitivity, and extraction ion chromatograms of cholineamine, 4-aminobutyric acid, 5-hydroxytryptamine, dopamine, and thyroxine standards in a mixed standard solution of 57 neurotransmitter metabolites at a concentration of 100 ng / mL are respectively shown in the figures. Figure 9 (a)-13(a) is shown.

[0034] To verify the reliability of the results obtained by the method of the present invention in detecting human serum samples, rat brain tissue samples, and human cell samples, methodological investigation experiments (including limit of quantitation, linearity, accuracy, precision, etc.) were designed to verify the reliability of the detection method.

[0035] (1) Linear range and linear equation The solutions prepared above at a series of standard curve concentration points were tested according to the measurement conditions of this embodiment, and the linear equations, linear ranges and limits of quantitation (LLOQ) of 57 neurotransmitter metabolites were obtained as shown in Table 2.

[0036] Table 2. Quantitative standard curves and related limits of quantification for neurotransmitter metabolites.

[0037]

[0038]

[0039] Experimental results on the correlation coefficient of linear equations show that R 2 All values ​​are greater than 99%, indicating that the linear relationship meets the test requirements.

[0040] (2) Precision and accuracy Sufficient amounts of human serum, rat brain tissue, and human cell samples were selected and thoroughly vortexed for homogenization. Each sample was divided into 36 aliquots, with each aliquot containing 50 μL of human serum, 50 mg of rat brain tissue, and 1 × 10⁻⁶ human cells. 6 Personal cell samples were stored at -80°C. Twelve samples of each sample type were removed on the first day, and six of these were added to a mixed standard solution of 57 neurotransmitter metabolites (preparation method: 15 μL of each of the 57 neurotransmitter standard stock solutions at 1 mg / mL was mixed and diluted to obtain the mixed standard solution of 57 neurotransmitter metabolites; the concentration of each neurotransmitter standard in the mixed standard solution was 500 ng / mL; the solvent used for dilution was 70% (v / v) methanol aqueous solution). This was designated as human serum sample C. 加标 Rat brain tissue sample C 加标 Human cell sample C 加标 The other six samples were used as background and did not contain a mixed standard solution of 57 neurotransmitter metabolites; these were designated as human serum sample C.本底 Rat brain tissue sample C 本底 Human cell sample C 本底 After processing using the above-described extraction and processing methods for biological samples, analysis was performed using liquid chromatography and mass spectrometry (LC-MS) according to the aforementioned methods to determine accuracy and intra-day precision. The total ion chromatogram of the separation, sensitivity, and response of 57 neurotransmitter metabolites in the background human serum sample is shown below. Figure 3 As shown, the total ion chromatograms of the separation, sensitivity, and response of 57 neurotransmitter metabolites in rat brain tissue samples are as follows. Figure 4 As shown, the total ion current chromatograms of the separation, sensitivity, and response of 57 neurotransmitter metabolites in human cell samples are as follows. Figure 5 As shown in Table 3, the steps of the first day were repeated on the second and third days, and the daytime precision was obtained after three consecutive days of measurement. The accuracy calculation formula is as follows: (C 加标 -C 本底 ) / C 理论 ×100%; Among them, C 加标 This indicates the concentration measured after extraction from a sample containing a mixed standard solution of 57 neurotransmitter metabolites; C 本底 This indicates the concentration measured after extraction of the sample background; C 理论 This indicates the theoretical concentration of a mixed standard solution of 57 neurotransmitter metabolites added to the sample.

[0041] Table 3. Accuracy and Intra-day and Inter-day Precision in Biological Samples

[0042]

[0043] As shown in Table 3, the precision and accuracy of the method of this invention vary in different biological samples. Precision refers to the stability of continuous detection. The intra-day precision and inter-day precision for three consecutive days of substances with a precision of less than 20% can reach over 93%, indicating that the method of this invention has good stability. In addition, the accuracy of this method is relatively good in human serum and human cell biological samples. The proportion of substances with an accuracy of 70-130% in human serum and human cells is 91% (52 / 57, i.e., 52 out of 57 metabolites can be accurately detected) and 81% (46 / 57, i.e., 46 out of 57 metabolites can be accurately detected) and 81% (46 out of 57 metabolites can be accurately detected) and 81% (46 out of 57 metabolites can be accurately detected) and 81% (46 out of 57 metabolites can be accurately detected) and 81% (46 out of 57 metabolites can be accurately detected) and 81% (46 out of 57 metabolites can be accurately detected) and 81% (46 out of 57 metabolites can be accurately detected) and 81% (46 out of 57 metabolites can be accurately detected) respectively. In rat brain tissue, the proportion of substances with an accuracy of 70-130% is 56% (32 / 57, i.e., 32 out of 57 metabolites can be accurately detected). Compared with human serum and human cell samples, the accuracy of metabolite detection in rat brain tissue samples is lower. This may be because the high content of neurotransmitter metabolites and the complex matrix in rat samples lead to poor accuracy of some substances.

[0044] Combining Example 1 and Figure 3-5 It is evident that the detection method described in this application exhibits good separation efficiency, high sensitivity, and high response value for neurotransmitter metabolites in human serum, rat brain tissue, and human cell samples.

[0045] At the same time, by Figure 1 and Figure 2 It is evident that the chromatographic column used in this application, when combined with flow chromatography, exhibits good separation performance, high sensitivity, and high response value for 57 neurotransmitter metabolite standards.

[0046] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that, in the liquid chromatography detection, the ACQUITY UPLC HSS PFP column was replaced with a reverse-flow T3 column. The liquid chromatography detected a mixed standard solution of 57 neurotransmitter metabolites at a concentration of 100 ng / mL.

[0047] The total ion chromatogram of the separation, sensitivity, and response of 57 neurotransmitter metabolite standards in a mixed standard solution of 57 neurotransmitter metabolites at a concentration of 100 ng / mL on a reverse-phase T3 column is shown below. Figure 6 As shown.

[0048] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that, for liquid chromatography detection, the ACQUITY UPLC HSS PFP column was a hydrophilic column (HILIC). The liquid chromatography detected a mixed standard solution of 57 neurotransmitter metabolites at a concentration of 100 ng / mL.

[0049] The total ion chromatogram of the separation, sensitivity, and response of the hydrophilic chromatographic column for the 57 neurotransmitter metabolite standards in a mixed standard solution of 57 neurotransmitter metabolites at a concentration of 100 ng / mL is shown in the figure below. Figure 7 As shown.

[0050] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that, in the liquid chromatography detection, the mobile phase of this application was replaced with a conventional mobile phase system. Specifically, mobile phase A was ultrapure water (containing 0.1% (v / v) formic acid); mobile phase B was acetonitrile (containing 0.1% (v / v) formic acid). The liquid chromatography detected a mixed standard solution of 57 neurotransmitter metabolites at a concentration of 100 ng / mL.

[0051] The separation, sensitivity, and response of the 57 neurotransmitter metabolite standards in a mixed standard solution of 57 neurotransmitter metabolites at a concentration of 100 ng / mL using a conventional mobile phase system are shown in the extraction ion chromatogram. Figure 8 As shown, the separation, sensitivity, and extraction ion chromatograms of cholineamine, 4-aminobutyric acid, 5-hydroxytryptamine, dopamine, and thyroxine standards in a mixed standard solution of 57 neurotransmitter metabolites at a concentration of 100 ng / mL are respectively shown in the figures. Figure 9 As shown in (b)-13(b).

[0052] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that, for liquid chromatography detection, mobile phase B of this application was replaced with a methanol-formic acid system. Specifically, mobile phase A was ultrapure water (containing 0.5 mM ammonium fluoride and 0.1% (v / v) formic acid); mobile phase B was methanol (containing 0.1% (v / v) formic acid). The liquid chromatography detected a mixed standard solution of 57 neurotransmitter metabolites at a concentration of 100 ng / mL.

[0053] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that, in the liquid chromatography detection, mobile phase A of this application was replaced with mobile phase A without added ammonium fluoride. Specifically, mobile phase A was ultrapure water (containing 0.1% (v / v) formic acid); mobile phase B was methanol and acetonitrile (containing 0.1% (v / v) formic acid) in a volume ratio of 1:1. The liquid chromatography detected a mixed standard solution of 57 neurotransmitter metabolites at a concentration of 100 ng / mL.

[0054] Conclusion Analysis and Summary In conjunction with Example 1 and Comparative Example 1, Figure 1 and Figure 6It can be seen that the ACQUITY UPLC HSS PFP column used in Example 1 has good separation effect, high sensitivity and high response value for 57 neurotransmitter metabolite standards; in Comparative Example 1, the ACQUITY UPLC HSS PFP column was replaced with a reverse T3 column, and the separation effect and response value were worse than those in Example 1.

[0055] In conjunction with Example 1 and Comparative Example 2, Figure 1 and Figure 7 It can be seen that in Comparative Example 2, after replacing the ACQUITY UPLC HSS PFP column with a hydrophilic column, the separation effect of 57 neurotransmitter metabolite standards was significantly worse, and the response value was also worse than that in Example 1.

[0056] Combined with Example 1, Comparative Example 3 and Figure 9-13 As can be seen, in Comparative Example 3, after replacing the mobile phase of this application with a conventional mobile phase, the sensitivity and response value of the choline standard decreased; the response value of the 4-aminobutyric acid standard decreased; the separation effect, sensitivity, and response value of the 5-hydroxytryptamine standard decreased; the sensitivity of the dopamine standard decreased; and the sensitivity of the thyroxine standard decreased.

[0057] Based on Example 1 and Comparative Example 4, it can be seen that in Comparative Example 4, after replacing the mobile phase B with a methanol system (containing 1% formic acid (v / v)), the overall liquid phase pressure was higher, resulting in a poorer separation effect, reduced sensitivity, and prolonged retention time for 57 neurotransmitter metabolites.

[0058] Based on Example 1 and Comparative Example 5, it can be seen that in Comparative Example 5, when mobile phase A was replaced with ultrapure water (containing 0.1% (v / v) formic acid), the separation effect of 57 neurotransmitter metabolites deteriorated and the sensitivity decreased.

[0059] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. An LC-MS / MS method for detecting neurotransmitter metabolites in biological samples, characterized in that, Includes the following steps: S1: Construct a mass spectrometry database containing neurotransmitter metabolites and their isotope internal standards; S2: Prepare mixed standard solutions containing neurotransmitter standards and plot standard curves for each neurotransmitter metabolite standard; S3: Extract the biological sample using the protein precipitation method to obtain the sample solution to be tested; S4: The sample solution to be tested is separated by liquid chromatography. The mobile phase A of the liquid chromatography is an aqueous solution containing ammonium fluoride and formic acid, and the mobile phase B is an organic phase containing formic acid. S5: Perform mass spectrometry analysis on the sample separated by liquid chromatography.

2. The method according to claim 1, characterized in that, In step S1, the neurotransmitter metabolites include 57 neurotransmitter metabolites, which include at least one of choline, 4-aminobutyric acid, 5-hydroxytryptamine, dopamine, and thyroxine.

3. The method according to claim 1, characterized in that, In step S2, the mixed standard solution of neurotransmitter standards contains 45 isotope internal standards.

4. The method according to claim 1, characterized in that, In step S3, the biological sample includes at least one of human serum, brain tissue, and human cells.

5. The method according to claim 4, characterized in that, In step S3, protein precipitation and extraction of the biological sample are performed. Includes the following steps: a: Human serum samples were extracted using a mixed solvent of acetonitrile and methanol in a volume ratio of 3:(9-17); b: Brain tissue samples were extracted using a methanol-water solution with a methanol volume fraction of 65-75%; c: Add a methanol aqueous solution with a methanol volume fraction of 75-85% to the human cell sample and extract it by freezing and thawing in liquid nitrogen 2-4 times.

6. The method according to claim 1, characterized in that, In step S4, mobile phase A is an aqueous solution containing 0.4-0.6 mM ammonium fluoride and 0.08-0.12% (v / v) formic acid, and mobile phase B is an organic phase containing 0.08-0.12% (v / v) formic acid. In the organic phase, the volume ratio of methanol to acetonitrile is (45-55):(55-45).

7. The method according to claim 1, characterized in that, In step S4, the liquid chromatography column used for liquid chromatography detection is an ACQUITY UPLC HSS PFP column.

8. The method according to claim 1, characterized in that, In step S4, the liquid chromatography separation uses gradient elution, and the gradient elution conditions are 0 min-7 min 95% A / 5% B (V / V), 7-7.6 min 5% A / 95% B (V / V), and 7.6-9 min 95% A / 5% B (V / V).

9. The method according to claim 1, characterized in that, In step S4, the flow rate of the liquid chromatography column is 0.28-0.32 mL / min, and the column temperature is 39-41℃.

10. The method according to claim 1, characterized in that, In step S5, mass spectrometry detection uses an electrospray ionization source with multi-channel reaction monitoring. The positive ion spray voltage is 5500V, the negative ion spray voltage is -4500V, and the ion source temperature, collision gas, curtain gas, nebulizer, and auxiliary gas positive and negative modes are 550℃, 7psi, 35psi, 50psi, and 60psi, respectively.

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