1. A kit for simultaneously detecting tryptophan and metabolites thereof in a biological sample based on ultra performance liquid chromatography-tandem mass spectrometry is characterized in that the kit comprises a standard substance of tryptophan and metabolites thereof, wherein the tryptophan and the metabolites thereof comprise: 3-hydroxykynurenine, 5-hydroxytryptamine, kynurenine, xanthurenic acid, tryptophan, kynurenic acid, 3-hydroxyanthranilic acid, 5-oxindole acetic acid, 3-indoleacetic acid and 3-indolepropionic acid.

2. The kit of claim 1, wherein the standard is a mixed standard of the standards for tryptophan and metabolites thereof.

3. The kit of claim 1, further comprising an internal standard.

4. The kit of claim 1, wherein the internal standard comprises tryptophan-d 5 and carbamazepine.

5. The method according to any one of claims 1 to 4, wherein the kit further comprises eluent A and eluent B, wherein eluent A is 0.1% aqueous formic acid (v/v); eluent B is 100% acetonitrile.

6. A method for simultaneously detecting tryptophan and metabolites thereof in a biological sample based on ultra high performance liquid chromatography tandem mass spectrometry is characterized by comprising the following steps:

eluting and separating a biological sample by using a mobile phase and a chromatographic column of an ultra-high performance liquid chromatograph, scanning a parent ion-ion pair of tryptophan and metabolites thereof by using a mass spectrometer to obtain peak areas of the analytes, establishing a standard curve by using the concentration of each analyte as an X axis and the ratio of the response peak area of the analytes to the response peak area of an internal standard as a Y axis, and calculating the content of the tryptophan and the metabolites thereof, wherein the tryptophan and the metabolites thereof comprise 3-hydroxykynurenine, 5-hydroxytryptamine, kynurenine, xanthurenic acid, tryptophan, kynurenic acid, 3-hydroxyanthranilic acid, 5-hydroxyindoleacetic acid, 3-indoleacetic acid and 3-indolepropionic acid.

7. The method according to claim 6, characterized in that the method comprises the following specific steps:

s1, obtaining a biological sample to be detected;

s2, chromatographic separation: subjecting the biological sample obtained in step S2 to gradient elution with a chromatographic column, wherein eluent A is 0.1% formic acid aqueous solution (v/v), and eluent B is 100% acetonitrile;

s3, mass spectrometric detection: selecting a positive ion electrospray ionization mode, wherein the ion source parameters are respectively as follows: the capillary voltage is 2.24 kV; the temperature of the solvent gas is 450 ℃; the flow rate of the cone/desolvated gas was 150/900L/h.

8. The method of claim 7, wherein in step S1, the method further comprises the step of preprocessing the sample: the biological sample was deproteinized using acetonitrile and the supernatant was taken for the next step.

9. The method according to claim 7, wherein the specific procedure of the gradient elution is as shown in the following table:

10. the method according to claim 7, wherein the mass spectrometric parameters of tryptophan and metabolites thereof are as shown in the following table:

Background

Tryptophan (TRP) is one of essential amino acids of the human body, and plays an important role in protein biosynthesis and in the in vivo metabolic regulation network as a precursor of various important bioactive molecules. Cancer, coronary heart disease, Alzheimer's disease, irritable bowel syndrome and other diseases are related to metabolic disorder of tryptophan through research. Ingested tryptophan is metabolized in the body mainly through three pathways: kynurenine (Kynurenine), Serotonin (Serotonin) and bacterial degradation pathways.

Approximately 95% of tryptophan is metabolized via the kynurenine pathway and converted to a series of downstream metabolites, including kynurenine, Kynurenic acid (Kynurenic acid), 3-Hydroxykynurenine (3-Hydroxykynurenine, 3-HK), 3-Hydroxyanthranilic acid (3-Hydroxyanthranilic acid, 3-HAA), and Xanthurenic Acid (XA). The Kynurenine pathway can be divided into a liver pathway and a non-liver pathway, wherein 90% of Tryptophan is catalyzed and metabolized by Tryptophan-2, 3-dioxygenase (TDO) in the liver, the enzymes which metabolize Tryptophan in the non-liver pathway are mainly Indoleamine-2,3-dioxygenase (IDO-2, 3-dioxygenase, IDO), both TDO and IDO are key rate-limiting enzymes for converting TRP into N-formyl Kynurenine (N-forryneurine, NFK), and the NFK can be further hydrolyzed by Kynurenine carboxamide to generate Kynurenine (KYN). Kynurenine is metabolized mainly through two pathways, one pathway is that 3-hydroxykynurenine is generated under the catalysis of Kynurenine-3-monooxygenase (Kynurenine3-monooxygenase, KMO), then 3-Hydroxyanthranilic acid (3-Hydroxyanthranilic acids, 3-HAA) and Xanthurenic Acid (XA) are generated under the catalysis of Kynurenine enzyme (Kynurenine aminotransferase, KAT) and 3-Hydroxyanthranilic acid (3-Hydroxyanthranilic acids, 3-HAA) and Xanthurenic Acid (XA) respectively, and finally active molecules such as Quinolinic acid (Quinolinic acid, QA), pyridinecarboxylic acids and Nicotinamide Adenine Dinucleotide (NAD) are generated through multi-stage enzymatic reaction. The other pathway is the production of kynurenine under the action of kynurenine aminotransferase. Metabolites of the kynurenine metabolic pathway play important roles in the physiological functions of immunomodulation, neural excitability and vasodilation. It has been reported that increased pathological levels of kynurenine and kynurolinic acid in plasma are associated with increased risk of death and cardiovascular events in patients with coronary heart disease. In addition, kynurenine, an endogenous ligand that promotes tumor proliferation, binds to the Aromatic Hydrocarbon Receptor (AHR) and activates the AHR pathway, thereby promoting the growth and migration of tumor cells.

Approximately 1% -2% of tryptophan is catalyzed by tryptophan hydroxylase (TPH) and decarboxylase to produce the neurotransmitter 5-hydroxytryptamine (5-HT), and a portion of 5-hydroxytryptamine is catalyzed by hepatic monoamine oxidase (MAO) to produce 5-hydroxyindoleacetic acid (HIAA). As monoamine hormones and neurotransmitters, 5-hydroxytryptamine is involved in a variety of physiological processes, and plays an important role in, inter alia, regulating neurotransmission, blood pressure regulation and growth stimulating functions. Abnormal serotonin release can trigger pathophysiological processes such as depression, irritable bowel syndrome and thrombosis.

Tryptophan is metabolized by the intestinal flora to form 3-indolopropionic acid (IPA) and 3-Indoleacetic acid (IAA). The 3-indole propionic acid is a powerful antioxidant and has neuroprotective effect. It activates PXR, eliminates hydroxyl radicals, reduces DNA damage and lipid peroxidation of neuronal cells, and inhibits β amyloid production, thus having a protective effect on neuronal damage caused by ischemia, and is considered to be an indoleamine that effectively scavenges free radicals.

Based on the key roles of tryptophan and its metabolites in the development of diseases, energy metabolism and maintaining the steady state of the body, it is very important to develop a detection method for simultaneously quantifying the above 10 metabolites in human plasma. Tryptophan and its metabolites, however, are difficult to determine using conventional analytical methods because: (1) the organism content is low, and the requirement on the sensitivity of the method is high; (2) the composition in biological samples is complex and interfering components interfere with quantifying the metabolite concentration of the sample.

At present, genetic metabolism micromolecule diagnosis based on a liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupling technology is an effective method for separating and detecting components in a complex system, has the characteristics of high sensitivity, good selectivity, wide linear range and the like, and has very obvious advantages for mixture analysis with small sample amount and complex matrix. Liquid chromatography tandem mass spectrometry detection methods for simultaneously measuring tryptophan and metabolites thereof in human plasma have been reported at home and abroad, but the reported methods mainly have the following problems: (1) considering that the analyte is an endogenous substance and therefore there is no analyte-free biological matrix, the pure solution or artificial matrix used to construct the standard curve may not ensure the same mass spectral response to an equivalent concentration of analyte present in the standard and study samples; (2) background subtraction methods used to determine stromal effects may not be applicable to endogenous metabolites with higher concentrations in human plasma; (3) sample preparation methods that employ solid phase extraction or vacuum blow drying redissolution are complex and time consuming for high throughput assays; (4) the reported analysis methods mostly require higher plasma consumption or longer analysis time to realize quantitative analysis of tryptophan metabolites, and simultaneously can accurately quantify the tryptophan metabolites less, so that the correlation between a plurality of metabolites in a tryptophan metabolic pathway and the health condition of the organism cannot be better considered. The methods not only have low working efficiency, but also increase the demand of samples, and are not suitable for clinical large-scale sample detection.

Therefore, there is a need in the art to develop a method and a detection kit based on a liquid chromatography tandem mass spectrometry technology with high sensitivity, accuracy and stability, which are used for simultaneously detecting human plasma tryptophan and the concentration of downstream metabolites thereof, are helpful for the development of the work of diagnosis and treatment, disease detection and the like of related diseases, and provide an analysis method and a kit for the future research of the action mechanism of drugs on related diseases based on tryptophan metabolic pathway association.

Disclosure of Invention

The invention aims to provide a kit and a method capable of accurately, conveniently and high-flux detecting human plasma tryptophan and metabolites thereof aiming at the defects of few metabolic species, long time consumption, high blood consumption, complex pretreatment and the like in the existing tryptophan metabolite detection method.

The purpose of the invention is realized by the following technical scheme:

a kit for simultaneously detecting tryptophan and metabolites thereof in a biological sample based on ultra performance liquid chromatography-tandem mass spectrometry comprises a standard product of tryptophan and metabolites thereof, wherein the tryptophan and the metabolites thereof comprise: 3-hydroxykynurenine (3-HK), 5-hydroxytryptamine (5-HT), Kynurenine (KYN), Xanthurenic Acid (XA), Tryptophan (TRP), kynurenic acid (KYNA), 3-hydroxyanthranilic acid (3-HAA), 5-hydroxyindoleacetic acid (5-HIAA), 3-indoleacetic acid (IAA) and 3-indolepropionic acid (IPA).

In some embodiments of the invention, the standard is a mixed standard of standards for the tryptophan and metabolites thereof.

In some embodiments of the invention, the standard for tryptophan and metabolites thereof utilizes methanol (chromatographic grade), dimethyl sulfoxide (DMSO, analytical grade) or ultrapure water as a formulation solution. In some embodiments of the invention, the standard of tryptophan and metabolites thereof is formulated as follows: the above 10 standards were weighed out precisely, and methanol was added to prepare stock solutions of 5-HT (0.1mg/mL), KYN (1mg/mL), TRP (2mg/mL), 3-HAA (0.1mg/mL), 5-HIAA (1mg/mL), IPA (1mg/mL) and IAA (1 mg/mL). Stock solutions of KYNA (0.1mg/mL) were prepared with DMSO/methanol (1: 9, v/v). Stock solutions of 3-HK (0.1mg/mL) and XA (0.1mg/mL) are preferably prepared with water/methanol (9: 1, v/v) and stored at-80 ℃.

Preferably, aliquots of each stock solution are mixed and serially diluted with acetonitrile into 8 standard solutions of different concentrations.

3-HK：15、30、75、150、300、750、1500、3000ng/mL；

5-HT：7.5、15、37.5、75、150、375、750、1500ng/mL；

KYN：125、250、625、1250、2500、6250、12500、25000ng/mL；

XA：5、10、25、50、100、250、500、1000ng/mL；

TRP：500、1000、2500、5000、10000、25000、50000、100000ng/mL；

KYNA：25、50、125、250、500、1250、2500、5000ng/mL；

3-HAA：10、20、50、100、200、500、1000、2000ng/mL；

5-HIAA：12.5、25、62.5、125、250、625、1250、2500ng/mL；

IPA and IAA: 50. 100, 250, 500, 1000, 2500, 5000, 10000 ng/mL.

In some embodiments of the invention, the kit further comprises an internal standard. In some embodiments of the invention, the internal standard comprises tryptophan-d 5 and carbamazepine. In some preferred embodiments of the present invention, the internal standard is prepared by the following method: the tryptophan-d 5 and the carbamazepine are precisely weighed, stock solutions of 200 mu g/mL and 100 mu g/mL are respectively prepared by adding methanol to serve as internal standard substances, the two stock solutions of the internal standard substances are finally diluted by using methanol to obtain a mixed internal standard solution containing 20 mu g/mL of the tryptophan-d 5 and 1 mu g/mL of the carbamazepine, and the mixed internal standard solution is stored at the temperature of 80 ℃ below zero.

In some embodiments of the invention, the kit further comprises eluent a and eluent B, wherein eluent a is 0.1% aqueous formic acid (v/v); eluent B is 100% acetonitrile.

In the present invention, the biological sample is selected from a plasma sample, a serum sample and/or a urine sample.

In some embodiments of the invention, the biological sample is a plasma sample. Further, the kit also comprises a protein precipitation solution, and preferably, the protein precipitation solution is acetonitrile.

Still further, the kit further comprises a quality control product. The preparation method of the quality control product comprises the following steps: adding the standard substance solution of tryptophan and metabolites thereof into blank plasma matrix solution, preferably preparing LLOQ, LQC, MQC and HQC with quantitative lower limit, low, medium and high concentration.

LLOQ: accurately transferring 10 mu L of mixed standard substance working solution containing the tryptophan and the metabolites thereof with the lowest quantitative lower limit, and diluting the mixed standard substance working solution to 50 mu L by using a diluent;

and (3) LQC: accurately transferring 10 mu L of mixed standard substance working solution containing the tryptophan and the metabolites thereof with the second standard concentration, and diluting the mixed standard substance working solution to 50 mu L by using a diluent;

MQC: accurately transferring 10 mu L of mixed standard substance working solution containing the tryptophan and the metabolites thereof, which corresponds to 50% of the standard range of the analyte, and diluting the mixed standard substance working solution to 50 mu L by using a diluent;

HQC: accurately transferring 10 μ L of mixed standard working solution containing tryptophan and metabolites thereof and corresponding to the highest standard concentration of 75%, and diluting to 50 μ L with diluent.

The preparation method of the blank plasma matrix solution comprises the following steps: 1g of activated carbon was added to 10mL of plasma of a healthy person, and the mixture was rotated at 4 ℃ for 6 hours by using a test tube tumbler. Centrifuging at 16,128 Xg for 20min at 4 deg.C, collecting supernatant, and filtering with 0.22 μm microporous membrane for 2 times to obtain supernatant as blank plasma matrix solution, which is used as diluent for diluting and preparing the quality control product.

The second aspect of the invention provides a method for simultaneously detecting tryptophan and metabolites thereof in a biological sample based on ultra high performance liquid chromatography tandem mass spectrometry, which comprises the following steps:

eluting and separating a biological sample by using a mobile phase and a chromatographic column of an ultra-high performance liquid chromatograph, scanning a parent ion-daughter ion pair of tryptophan and metabolites thereof by using a mass spectrometer to obtain peak areas of the analytes, establishing a standard curve by using the concentration of each analyte as an X axis and the ratio of the response peak area of the analytes to the response peak area of an internal standard as a Y axis, and calculating the content of the tryptophan and the metabolites thereof, wherein the tryptophan and the metabolites thereof comprise 3-hydroxykynurenine, 5-hydroxytryptamine, kynurenine, xanthurenic acid, tryptophan, kynurenic acid, 3-hydroxyanthranilic acid, 5-hydroxyindoleacetic acid, 3-indoleacetic acid and 3-indolepropionic acid.

In some embodiments of the invention, the method comprises the specific steps of:

s1, obtaining a biological sample to be detected;

s2, chromatographic separation: subjecting the biological sample obtained in step S2 to gradient elution with a chromatographic column, wherein eluent A is 0.1% formic acid aqueous solution (v/v), and eluent B is 100% acetonitrile;

s3, mass spectrometric detection: selecting a positive ion electrospray ionization mode, wherein the ion source parameters are respectively as follows: the capillary voltage is 2.24 kV; the temperature of the solvent gas is 450 ℃; the flow rate of the cone/desolvated gas was 150/900L/h.

In some embodiments of the present invention, in step S1, the method further comprises the step of preprocessing the sample: the biological sample is deproteinized with acetonitrile, and the supernatant is taken for the next step. In a specific embodiment of the present invention, the step of pre-treating comprises: placing a biological sample in an EP tube, adding a mixed internal standard solution, adding ice-cold acetonitrile to precipitate protein, mixing by vortex, and placing at-20 ℃ to completely precipitate the protein. Centrifuging at 16,128 Xg at 4 deg.C, collecting supernatant, loading into 96-well plate, and detecting by sample injection.

In some embodiments of the invention, in step S1, the chromatography column is of the type: waters Acquity UPLC HSS T3 reverse phase chromatography column (3.0 mm. times.100 mm, 1.8 μm).

In some embodiments of the invention, in step S1, the flow rate: 0.3 mL/min; column temperature: 30 ℃; sample introduction amount: 2 μ L.

In some embodiments of the invention, the specific procedure for the gradient elution is shown in the following table:

in some embodiments of the invention, the mass spectrometric detection is performed using a triple quadrupole mass spectrometer.

In some embodiments of the invention, the mass spectrometry conditions are as follows:

(1) an ion source: electrospray ionization

(2) Ion source parameters: capillary voltage, 2.24 kV; the temperature of the desolvation gas is 450 ℃; cone/desolvation gas (nitrogen) flow rate, 150/900L/h.

In the present invention, the ion source parameters may vary according to the actual instrument model.

(3) Scanning mode: positive ion multiple reaction mode monitoring (MRM) analysis, the parent ion → daughter ion channels were selected as:

3-HK：225.07→110.06；

5-HT：177.00→160.00；

KYN：209.10→94.07；

XA：206.00→159.99；

TRP-d5：210.08→192.20；

TRP：205.13→145.97；

KYNA：190.00→143.99；

3-HAA：154.00→108.00；

5-HIAA：192.04→146.02；

IAA：176.04→130.02；

IPA：190.04→130.03；

CAR：237.20→194.20。

in some embodiments of the invention, the mass spectrometry parameters of tryptophan and metabolites thereof are as shown in the following table:

the invention has the advantages of

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

(1) by utilizing the method, the selectivity, linearity, precision, accuracy and matrix effect, and the recovery rate and stability all meet the requirements of quantitative analysis through a specific gradient elution program and a double internal standard quantitative method.

(2) Compared with the reported method set forth in the background technology, the method of the invention uses an ultra-high performance liquid chromatography tandem mass spectrometer for detection, the pretreatment operation is simple and convenient, the plasma consumption is low, and the quantitative analysis of tryptophan and 9 metabolites thereof in plasma can be realized within 5min, so that the clinical detection is simpler, more convenient and faster, and the method has the advantage of high flux, and is beneficial to the determination of large sample size.

(3) The diluent provided by the kit provided by the invention obtains plasma without an analyte by an activated carbon adsorption method to remove endogenous interference in a biological matrix, so that the mass spectrum response of the analyte in the diluent is closer to that of the original biological matrix, the accuracy of a quantitative analysis result is improved, and the quantitative analysis result is used for constructing a standard curve, verifying a method and clinically applying the quantitative analysis result.

Drawings

Figure 1 shows chromatograms of tryptophan and its 9 metabolites, 2 internal standards in plasma.

Figure 2 shows the mass spectra of tryptophan and its 9 metabolites, 2 internal standards.

Fig. 3 shows the plasma histogram of tryptophan and its 9 metabolites in 100 healthy people.

Detailed Description

Unless otherwise indicated, implied from the context, or customary in the art, all parts and percentages herein are by weight and the testing and characterization methods used are synchronized with the filing date of the present application. Where applicable, the contents of any patent, patent application, or publication referred to in this application are incorporated herein by reference in their entirety and their equivalent family patents are also incorporated by reference, especially as they disclose definitions relating to synthetic techniques, products and process designs, polymers, comonomers, initiators or catalysts, and the like, in the art. To the extent that a definition of a particular term disclosed in the prior art is inconsistent with any definitions provided herein, the definition of the term provided herein controls.

The numerical ranges in this application are approximations, and thus may include values outside of the ranges unless otherwise specified. A numerical range includes all numbers from the lower value to the upper value, in increments of 1 unit, provided that there is a separation of at least 2 units between any lower value and any higher value. For example, if a compositional, physical, or other property (e.g., molecular weight, melt index, etc.) is recited as 100 to 1000, it is intended that all individual values, e.g., 100, 101, 102, etc., and all subranges, e.g., 100 to 166, 155 to 170, 198 to 200, etc., are explicitly recited. For ranges containing a numerical value less than 1 or containing a fraction greater than 1 (e.g., 1.1, 1.5, etc.), then 1 unit is considered appropriate to be 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digit numbers less than 10 (e.g., 1 to 5), 1 unit is typically considered 0.1. These are merely specific examples of what is intended to be expressed and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.

The terms "comprising," "including," "having," and derivatives thereof do not exclude the presence of any other component, step or procedure, and are not intended to exclude the presence of other elements, steps or procedures not expressly disclosed herein. To the extent that any doubt is eliminated, all compositions herein containing, including, or having the term "comprise" may contain any additional additive, adjuvant, or compound, unless expressly stated otherwise. Rather, the term "consisting essentially of … …" excludes any other components, steps or processes from the scope of any of the terms hereinafter recited, insofar as such terms are necessary for performance. The term "consisting of … …" does not include any components, steps or processes not specifically described or listed. Unless explicitly stated otherwise, the term "or" refers to the listed individual members or any combination thereof.

In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.

Examples

The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

The experimental methods not specifically described in the following examples are conventional methods unless otherwise specified. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Example method for detecting tryptophan and metabolite thereof in plasma simultaneously by ultra high performance liquid chromatography-tandem mass spectrometry and operation method of kit

Instrument, reagent, standard and internal standard

(1) The instrument comprises the following steps: ACQUILY UPLC ultra high performance liquid chromatograph (Waters, USA), Xevo TQ-S triple quadrupole mass spectrometer (Waters, USA), high speed refrigerated centrifuge (Allegra X-30R, Beckman Coulter, Germany), ultra pure water spectrometer (EMD Millipore, Billerica, USA), vortex apparatus (Lenberger instruments manufacturing, China), and the like.

(2) Reagent: activated carbon (100 mesh size) was purchased from Sigma-Aldrich. Chromatographic grade methanol and acetonitrile were purchased from Thermo Fisher Scientific (Waltham, MA, USA), chromatographic grade formic acid was purchased from Sigma-Aldrich (st. louis, MO, USA), and ultrapure water was self-made.

(3) And (3) standard substance: l-tryptophan (99%, purity), 3-indolylpropanic acid (98%, purity), 3-indolylacetic acid (99%, purity) and 5-hydroxytryptamine (97.5%, purity) were purchased from J & K Scientific (Beijing, China). L-kynurenine (> 98% purity) and xanthurenic acid (96% purity) were purchased from Aladdin (shanghai, china). Kynurenine (99%, purity) and 3-hydroxykynurenine (99%, purity) were purchased from Sigma-Aldrich (st. louis, MO, USA). 5-Hydroxyindoleacetic acid (> 98.0% purity) was purchased from Tokyo Chemical Industry (Tokyo, Japan). 3-hydroxy anthranilic acid (> 98.0% purity) was purchased from Macklin (Shanghai, China).

(4) Internal standard: L-Tryptophan-d 5 (98%, purity) and carbamazepine (99.7%, purity) were purchased from Toronto Research Chemicals (Toronto, Canada) and Chinese drug laboratory (Beijing, China).

Preparation of stock solution, mixed standard solution, quality control product and mixed internal standard solution

(1) Preparation of stock solutions

Precisely weighing 3-hydroxykynurenine (3-HK), 5-hydroxytryptamine (5-HT), Kynurenine (KYN), Xanthurenic Acid (XA), Tryptophan (TRP), kynurenic acid (KYNA), 3-hydroxyanthranilic acid (3-HAA), 5-hydroxyindoleacetic acid (5-HIAA), 3-indoleacetic acid (IAA) and 3-indolepropionic acid (IPA) as reference substances, and adding methanol to prepare stock solutions of 5-HT (0.1mg/mL), KYN (1mg/mL), TRP (2mg/mL), 3-HAA (0.1mg/mL), 5-HIAA (1mg/mL), IPA (1mg/mL) and IAA (1 mg/mL); DMSO/methanol (1: 9, v/v) is added to prepare stock solution of KYNA (0.1 mg/mL); stock solutions of 3-HK (0.1mg/mL) and XA (0.1mg/mL) were prepared with water/methanol (9: 1, v/v) and stored at-80 ℃.

(2) Preparation of Mixed Standard solutions

Aliquots of each stock solution were mixed and serially diluted with acetonitrile into 8 concentration-gradient mixed standard solutions. 3-HK: 15. 30, 75, 150, 300, 750, 1500, 3000 ng/mL; 5-HT: 7.5, 15, 37.5, 75, 150, 375, 750, 1500 ng/mL; KYN: 125. 250, 625, 1250, 2500, 6250, 12500, 25000 ng/mL; XA: 5. 10, 25, 50, 100, 250, 500, 1000 ng/mL; TRP: 500. 1000, 2500, 5000, 10000, 25000, 50000, 100000 ng/mL; KYNA: 25. 50, 125, 250, 500, 1250, 2500, 5000 ng/mL; 3-HAA: 10. 20, 50, 100, 200, 500, 1000, 2000 ng/mL; 5-HIAA: 12.5, 25, 62.5, 125, 250, 625, 1250, 2500 ng/mL; IPA and IAA: 50. 100, 250, 500, 1000, 2500, 5000, 10000 ng/mL. Storing at-80 deg.C.

(3) Preparation of quality control product

Adding the above 10 mixed standard solutions into blank plasma matrix solution to obtain LLOQ, LQC, MQC, and HQC with lowest quantitative limit, low, medium, and high concentrations.

LLOQ: accurately transferring 10 mu L of mixed standard substance working solution containing 10 analytes at the lowest quantitative lower limit, and diluting the mixed standard substance working solution to 50 mu L by using a diluent;

and (3) LQC: accurately transferring 10 mu L of mixed standard substance working solution with a second standard concentration containing 10 analytes, and diluting the mixed standard substance working solution to 50 mu L by using a diluent;

MQC: accurately transferring 10 mu L of mixed standard working solution containing 10 analytes and corresponding to the standard range of 50% of the analytes, and diluting the mixed standard working solution to 50 mu L by using a diluent;

HQC: the 10 μ L of the mixed standard working solution containing 10 analytes corresponding to the highest standard concentration of 75% was accurately removed and diluted to 50 μ L with a diluent.

(4) Preparation of a Mixed internal Standard solution

Tryptophan-d 5 and carbamazepine were precisely weighed and added with methanol to prepare stock solutions of 200. mu.g/mL and 100. mu.g/mL, respectively.

Both internal standard stocks were finally diluted with methanol to a mixed internal standard solution containing 20. mu.g/mL tryptophan-d 5 and 1. mu.g/mL carbamazepine and stored at-80 ℃.

Preparation of blank plasma matrix

1g of activated carbon was added to 10mL of plasma of a healthy person, and the mixture was rotated at 4 ℃ for 6 hours by using a test tube tumbler. Centrifuging at 16,128 Xg for 20min at 4 deg.C, collecting supernatant, and filtering with 0.22 μm microporous membrane for 2 times to obtain supernatant as diluent.

Fourth, pretreatment of plasma sample

50 mu L of plasma is placed in a 1.5mL EP tube, 10 mu L of mixed internal standard solution is added, 150 mu L of ice-cold acetonitrile is added for precipitating protein, and after vortex mixing is carried out for 4min, the mixture is placed at-20 ℃ for 10min so as to completely precipitate the protein. Centrifuging at 16,128 Xg for 20min at 4 deg.C, collecting supernatant 100 μ L, loading into 96-well plate, and detecting by sample injection.

Fifth, chromatographic conditions

(1) Mobile phase A: 0.1% formic acid aqueous solution, v/v, formic acid purity is chromatographic purity;

(2) mobile phase B: 100% acetonitrile, the purity is chromatographic purity;

(3) the type of the chromatographic column: waters Acquity UPLC HSS T3 reverse phase chromatography column (3.0 mm. times.100 mm, 1.8 μm);

(4) flow rate: 0.3 mL/min;

(5) column temperature: 30 ℃;

(6) sample introduction amount: 2 mu L of the solution;

(7) and (3) an elution mode: gradient elution, the specific procedure is shown in table 1.

TABLE 1 gradient elution procedure

The chromatogram obtained by detection is shown in figure 1, and the result shows the chromatogram of tryptophan and 9 metabolites thereof in plasma and 2 internal standard substances in the chromatographic conditions of the method, and all the compounds have obtained the best symmetric peak and separation degree. .

Sixthly, mass spectrum conditions

(1) An ion source: electrospray ionization

(2) Ion source parameters: capillary voltage, 2.24 kV; the temperature of the desolvation gas is 450 ℃; cone/desolvation gas (nitrogen) flow rate, 150/900L/h.

(3) Scanning mode: positive ion multiple reaction mode monitoring (MRM) analysis, with the parent → daughter ion channels selected to be 3-HK: 225.07 → 110.06; 5-HT: 177.00 → 160.00; KYN: 209.10 → 94.07; XA: 206.00 → 159.99; TRP-d 5: 210.08 → 192.20; TRP: 205.13 → 145.97; KYNA: 190.00 → 143.99; 3-HAA: 154.00 → 108.00; 5-HIAA: 192.04 → 146.02; IAA: 176.04 → 130.02; IPA: 190.04 → 130.03; CAR: 237.20 → 194.20.

(4) Mass spectrum parameters: the mass spectral parameters for the 12 compounds are shown in table 2.

TABLE 2 Tryptophan and its metabolites and 2 internal standards for the collection of ion pairs and Mass Spectrometry parameters

The mass spectrum obtained by detection is shown in FIG. 2. The results show that mass spectrometric detection of 12 compounds was performed in positive ion mode, and the optimal mass spectrometric response of each compound was obtained by optimizing the parent ion → daughter ion pairs of 12 compounds.

Example 2 Selective verification of the method established in example 1

The selectivity of the method established in example 1 was examined by analyzing the responses at the peaks of blank plasma from 6 different individual sources with the lowest limit of quantitation of the analyte and the internal standard.

The results show that the peak area of the blank plasma analyte does not exceed 20.0% of the lowest limit of quantitation and the internal standard does not exceed 5.0%, indicating that no interfering components are present and the selectivity of the method of the invention is good.

Example 3 Linear verification of the method established in example 1

Taking the mass concentration of 8 analytes as the abscissa x and the ratio of the peak area of the analyte to the peak area of the internal standard substance as the ordinate y, performing linear regression analysis by using a weighted least square method (the weight coefficient is 1/x)²) A mixed internal standard solution containing tryptophan-d 5 and carbamazepine was selected. According to the chromatographic behavior and the electric potential of two internal standards and the analyte in the method of the inventionThe separation efficiency, tryptophan-d 5 as an internal standard for kynurenine and tryptophan and carbamazepine as an internal standard for other analytes, and the concentration of tryptophan and its metabolites in plasma was calculated using a dual internal standard method.

The results show that 10 analytes are better linear with a correlation coefficient (r) in the following range²)>0.99, meeting the quantitative requirement. 3-HK: 3-600 ng/mL; 5-HT: 1.5-300 ng/mL; KYN: 25-5000 ng/mL; XA: 1-200 ng/mL; TRP: 100-20,000 ng/mL; KYNA: 5-1000 ng/mL; 3-HAA: 2-400 ng/mL; 5-HIAA: 2.5-500ng/mL, IAA and IPA: 10-2000 ng/mL.

Example 4 precision and accuracy testing of the method established in example 1

Quality control substances containing 10 analytes with the lowest quantitative limit, low, medium and high concentrations of 4 are prepared according to the method of example 1, and the operation is carried out according to the item of 'pretreatment of plasma samples'. 6 samples were prepared in parallel for each mass concentration, each batch followed by a standard curve and the assay was continued for 3 days. The measured concentration of the quality control sample was calculated from the standard curve of the day, and the precision and accuracy were determined by calculating the relative standard deviation (RSD%) and the relative error (RE%) of the measured values. The results show that: the within-day and the day-to-day precision (% RSD) and accuracy (% RE) of LLOQ are within 20%, and the other three QC levels are within 15%, and the determination results are shown in Table 3.

TABLE 3 Intra-day precision, Interday precision and accuracy of Tryptophan and its metabolites

Example 5 matrix Effect and recovery test

Preparing a quality control product with low, medium and high concentrations of 3 containing 10 analytes according to the method of the embodiment 1, and obtaining a peak area A according to the operation of a 'plasma sample pretreatment'; pretreating blank plasma according to 'plasma sample pretreatment', taking supernate, respectively adding mixed standard substance solutions with low, medium and high concentrations containing 10 analytes, adding mixed internal standard solution, vortex mixing uniformly, and carrying out sample injection analysis to obtain a peak area B; preparing a mixed standard solution containing 10 analytes and having low, medium and high concentrations, wherein a solvent is a acetonitrile solution containing 40%, adding a mixed internal standard working solution, and carrying out sample injection analysis to obtain a peak area C.

Matrix effect ═ B/Cx 100%

The absolute extraction recovery rate is C/B × 100%

The relative extraction recovery rate is A/B multiplied by 100%

The results show that: the inventors first used a blank plasma prepared by activated carbon adsorption as a substitute matrix for simulating actual biological samples, observed matrix effects of 109.86% and 108.44% of two internal standards TRP-d5 and CAR in plasma from 6 plasma samples from different sources, respectively, and matrix effects of analytes in the range of 86.57% -109.95%, except that slight ion suppression (matrix effect of 83.60%) was observed in HQC of 3-HK, and ion enhancement (matrix effect of 128.17%) was observed in LQC of 3-HAA. However, as in previous studies, the normal 3-HK concentration in serum of healthy humans is close to 4.79 ng/mL. Moreover, the ion enhancement observed in 3-HAA in the method of the invention was constant (relative standard deviation of 4.02% in 6 different blank plasma sources). However, the ion inhibition or enhancement phenomena do not affect the accuracy and precision of the method, and the matrix effect of the method does not affect the quantification of the two analytes in the actual plasma sample, which indicates that the blank plasma matrix solution can replace human plasma as a matrix to prepare a quality control product and construct a standard curve.

The relative recovery rates of 10 analytes in the plasma are 68.65% -100.72%, the absolute recovery rate is 57.39% -106.84%, the relative recovery rates of two internal standards TRP-d5 and CAR in the plasma are 76.76% + -3.41% and 75.70% + -5.61%, and the absolute recovery rates are 84.32% + -3.86% and 82.11% + -6.76%, respectively, which indicates that the pretreatment method of the invention is suitable for the determination of biological samples. The results of the matrix effect and recovery measurements are shown in Table 4.

TABLE 4 substrate Effect, Absolute recovery and relative recovery of Tryptophan and its metabolites

Example 6 stability test

Preparing quality control samples with low, medium and high concentrations, processing the samples under the item of 'plasma sample processing', observing the stability of standing for 3.5h at room temperature, standing for 12h in a sample injector, repeatedly freezing and thawing for three times within 24h, and paralleling each quality control sample for 6 times. The results show that 10 analytes deviate in concentration by less than 15% under different conditions. Indicating stability under assay and storage conditions, the assay results are shown in table 5.

TABLE 5 stability of Tryptophan and its metabolites

Example 7 plasma sample testing

FIG. 3 is a histogram obtained by measuring the concentrations of tryptophan and its metabolites in 100 normal human plasma, the measured concentrations being in agreement with the literature report (Henykova, E., et al, Stable isotope dilution ultra-high performance chromatography method and standard chromatography method) and the measured concentrations being in accordance with the method of measuring the accuracy of the measurement of the present invention, i.e., the method of measuring the accuracy of the measurement of the present invention, which is described in detail in specimen chromatography, J.Chromatogramator A,2016.1437: p.145-157.; Amirkhani A., quantitative analysis, kynurenic acid and kynurenic acid in specimen chromatography method, and specimen chromatography method, 2002.780.7.7. Meanwhile, the method is not only suitable for detecting the tryptophan and the metabolites thereof in the blood plasma, but also suitable for detecting the metabolites such as the tryptophan in other body fluids such as blood serum and urine, and has universality.

Example 2: kit composition, formulation and specification

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.