CN113607829B - Method for detecting concentration of 5-hydroxytryptamine and melatonin in serum - Google Patents

Abstract

The invention provides a method for detecting the concentration of 5-hydroxytryptamine and melatonin in serum, which adopts sodium carbonate solution as a pH regulator and methyl tertiary butyl ether as an extraction liquid in the pretreatment process of a serum sample, can enhance the extraction efficiency of the 5-hydroxytryptamine, screen out the optimal chromatographic conditions such as mobile phase, gradient and chromatographic column, can detect the 5-hydroxytryptamine and the melatonin in the serum at one time, has high sensitivity, strong specificity and simple pretreatment process, can separate and detect the 5-hydroxytryptamine and the melatonin in the serum within 5.5 minutes, basically meets the requirements on accuracy and precision, and can be used for quantitative analysis of the 5-hydroxytryptamine and the melatonin in the serum clinically and provide a simple and quick detection method for monitoring the concentration of the 5-hydroxytryptamine and the melatonin in clinic.

Description

Method for detecting concentration of 5-hydroxytryptamine and melatonin in serum

Technical Field

The invention belongs to the technical field of blood detection, and particularly relates to a method for detecting the concentration of 5-Hydroxytryptamine (5-HT) and Melatonin (MLT) in serum.

Background

5-hydroxytryptamine and melatonin are used as important indoleamine neurotransmitters in human bodies, have various biological activities of adjusting biological rhythms, emotion, cognition, scavenging free radicals, promoting inflammatory signal transduction and the like, and have important influence on bone metabolism. Melatonin is a metabolite of 5-hydroxytryptamine, which can be converted to melatonin via 5-hydroxytryptamine N-acetyltransferase and 5-hydroxytryptamine N-acetylmethoxytransferase. Recent domestic and foreign studies have found that 5-hydroxytryptamine participates in the processes of bone formation and bone resorption, including the osteogenesis promoting effect of central 5-hydroxytryptamine and the negative regulation effect of peripheral 5-hydroxytryptamine on bone formation; melatonin also has the functions of promoting osteoblast proliferation and differentiation and bone differentiation marker expression, and can inhibit osteoclast differentiation by promoting OPG secretion and eliminating osteoclast free radical, and 5-hydroxytryptamine and melatonin play a critical role in maintaining bone metabolism dynamic balance, thereby providing a new target and direction for bone metabolism diseases such as osteoporosis. The concentration of 5-hydroxytryptamine and melatonin in serum is detected simultaneously by utilizing an ultra-high performance liquid chromatography tandem mass spectrometry technology, and the changes of the bone mass and the bone structure of a human body can be reflected by circulating the levels of 5-hydroxytryptamine and melatonin, so that the method is used for evaluating the bone shortness condition of the crowds such as the aged, postmenopausal women, type 2 diabetes patients and the like, and provides scientific basis for clinical diagnosis and treatment.

The invention discloses a method and a kit for detecting melatonin in saliva by using a high performance liquid chromatography tandem mass spectrometry technology, which provides a method for detecting melatonin in saliva by using a high performance liquid chromatography tandem mass spectrometry method, has single detection component and simple matrix, and is not suitable for pretreatment of serum samples due to the fact that pretreatment conditions are very simple, the toxicity of an extract is very high, the nitrogen blowing time is long, and even if chromatographic conditions and pretreatment methods in the patent are adopted, 5-hydroxytryptamine and melatonin cannot be detected simultaneously, so that the clinical detection of melatonin in serum is not very high in reference value. Another patent (application No. 201910058794. X) discloses "a method for simultaneously detecting the contents of various biogenic amines in soy sauce by HPLC", in which the sample to be detected is not a biological sample although it contains 5-hydroxytryptamine, the substrate is simple, and the pretreatment is very complicated by a derivatization method.

Because 5-hydroxytryptamine and melatonin belong to alkaline and neutral substances respectively, the detection is difficult, the detection of 5-hydroxytryptamine and melatonin in serum together is not reported at present, and Chau RM (Chau RM, patel BA. Determination of serotin, melatonin and metabolites in gastrointestinal tissue using high-performance liquid chromatography with electrochemical detection. Biomed chromatogrI 2009,23 (2): 175-81), and the like detect 5-hydroxytryptamine and melatonin in the gastrointestinal tract together by adopting an electrochemical analysis technology, and the detection sensitivity is low (the detection limit of melatonin is 70 ng/ml), cannot meet the detection requirement of clinical blood samples, the analysis time is long (30 min), and the clinical application is also unfavorable.

Disclosure of Invention

The invention aims to provide a method for detecting the concentration of 5-hydroxytryptamine and melatonin in serum based on the prior art.

The technical scheme of the invention is as follows:

a method for detecting the concentration of 5-hydroxytryptamine and melatonin in serum,

wherein, the internal standard substances corresponding to the 5-hydroxytryptamine and the melatonin are respectively as follows: 5-hydroxytryptamine-d 4 (5-HT-d 4), melatonin-d 4 (MLT-d 4);

after pretreatment, oscillating and centrifuging a serum sample, taking supernatant and sampling, detecting 5-hydroxytryptamine and melatonin in the pretreated serum by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology, separating an object to be detected from a serum matrix by utilizing an ultra-high performance liquid chromatography, and then establishing a calibration curve by utilizing a mass spectrometry isotope internal calibration method with the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, wherein the specific chromatographic conditions are as follows:

(1) Ultra-high performance liquid chromatography conditions:

mobile phase a:0.01 to 0.2 percent of formic acid-1 to 5mM ammonium acetate aqueous solution; mobile phase B: methanol;

chromatographic column model: waters BEH C18 (2.1X100 mm,1.7 μm);

adopting a mode that a mobile phase A and a mobile phase B are mixed mobile phases for gradient elution, wherein the initial proportion of the mobile phase A to the mobile phase B is 85-100:25-0; the gradient elution process is as follows: the volume ratio of the mobile phase A to the mobile phase B is gradually changed from the initial proportion to 2:98 at a constant speed within 0-1.5 minutes; the volume ratio of mobile phase A to mobile phase B is kept constant at 2:98 within 1.5-3.5 minutes; in 3.5-5.5 minutes, the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 2:98 to the initial ratio at a constant speed; each sample was collected for 5.5 minutes;

(2) Mass spectrometry conditions:

in an electrospray ionization positive ion detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, and the capillary voltage is 0.5kV (ESI+); the temperature of the ion source is 150 ℃; the desolventizing temperature is 400 ℃; the flow rate of the desolventizing gas is 800L/hr; the flow rate of the taper hole air is 150L/hr; and simultaneously monitoring each target object and the corresponding isotope internal standard.

In order to improve the chromatographic selectivity, it may be considered to adjust the polarity of the mobile phase. According to the invention, formic acid and ammonium acetate are added into the mobile phase A, so that the ionization efficiency of a target compound can be effectively improved, and under the cooperation of other conditions, compared with the detection of 5-hydroxytryptamine and melatonin in serum by adopting an LC-MS/MS method in the prior art, the method has the advantages of higher sensitivity, simple pretreatment process, low cost, high sensitivity and strong specificity, and the separation and detection of the 5-hydroxytryptamine and the melatonin can be completed within 5.5 minutes. In a preferred embodiment, mobile phase A is a 0.1% to 0.15% formic acid-1.5 to 2.5mM ammonium acetate aqueous solution, preferably mobile phase A is a 0.1% formic acid-2 mM ammonium acetate aqueous solution without affecting the effectiveness of the present invention.

In chromatography, the choice of the chromatographic column is important, and the requirements for the chromatographic column are: high column efficiency, good selectivity, high analysis speed, etc. The invention adopts methanol and 0.01 to 0.2 percent formic acid-1 to 5mM ammonium acetate aqueous solution as mobile phase, the model of chromatographic column is Waters BEH C18 (2.1 multiplied by 100mM,1.7 mu m), under the cooperation of other conditions, endogenous substances do not interfere with the measurement of the sample, the sensitivity is high, the specificity is strong, and the accuracy and precision basically meet the requirements.

The selection of the internal standard is a very important task when using the internal standard method. The ideal internal standard should be capable of being added to the sample in accurate, known amounts and have substantially the same or as consistent as possible physicochemical properties, chromatographic behavior and response characteristics as the sample being analyzed; the internal standard must be sufficiently separated from the components of the sample under chromatographic conditions. The invention adopts deuterated isotope as an internal standard, the internal standard and an object to be detected have the same chemical property and matrix effect, and the repeatability and the accuracy are good when 5-hydroxytryptamine and melatonin in serum are measured.

In a preferred embodiment, the initial ratio of mobile phase A to mobile phase B is 90-95:10-5. Further preferably, the initial ratio of mobile phase a to mobile phase B is 90:10.

In a preferred embodiment, the flow rate is from 0.2 to 0.4ml/min, preferably 0.3ml/min.

Further, the column temperature is 30 to 50 ℃, preferably 40 ℃.

In one embodiment, the sample volume is 1 to 10. Mu.L, for example, 1. Mu.L, 5. Mu.L, or 10. Mu.L.

In a preferred embodiment, when the ultra performance liquid chromatography tandem mass spectrometry technology is used for detecting 5-hydroxytryptamine and melatonin in the pretreated serum, specific chromatographic conditions are as follows:

(1) Ultra-high performance liquid chromatography conditions:

mobile phase a:0.1% formic acid-2 mM ammonium acetate aqueous solution; mobile phase B: methanol;

chromatographic column model: waters BEH C18 (2.1X100 mm,1.7 μm);

adopting a mode that a mobile phase A and a mobile phase B are mixed mobile phases for gradient elution, wherein the initial ratio of the mobile phase A to the mobile phase B is 90:10; the gradient elution process is as follows: the volume ratio of the mobile phase A to the mobile phase B gradually changes from 90:10 to 2:98 at a constant speed within 0-1.5 minutes; the volume ratio of mobile phase A to mobile phase B is kept constant at 2:98 within 1.5-3.5 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 2:98 to 90:10 at a constant speed within 3.5-5.5 minutes; each sample was collected for 5.5 minutes; the gradient elution mode is specifically shown in table 1; the flow rate was 0.3ml/min, the column temperature was 40℃and the sample volume was 5. Mu.L.

TABLE 1 gradient elution parameters for mobile phases

Time (min)	Flow rate (ml/min)	％A	％B	Curve
					0.0	0.3	90	10	-
1.5	0.3	2	98	6
					3.5	0.3	2	98	6
5.5	0.3	90	10	1

(2) Mass spectrometry conditions:

in an electrospray ionization positive ion detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, and the capillary voltage is 0.5kV (ESI+); the temperature of the ion source is 150 ℃; the desolventizing temperature is 400 ℃; the flow rate of the desolventizing gas is 800L/hr; the flow rate of the taper hole air is 150L/hr; the mass spectrum source parameters are shown in table 2, and the mass spectrum parameters of each target and the corresponding isotope internal standard are also monitored, and the mass spectrum parameters of each target are shown in table 3.

Table 2 mass spectrometry source parameters

Table 3 5-hydroxytryptamine and melatonin detection mass spectrometry parameters

The serum mentioned in the present invention is human or animal serum.

The pretreated serum mentioned in the present invention is prepared as follows: adding an internal standard working solution, a pH regulator and an extraction solution into serum for extraction, oscillating, taking supernatant after centrifugal treatment, drying by nitrogen flow, mixing with a complex solution, and taking supernatant for sample injection after centrifugal treatment again; wherein the pH regulator is 0.1M-0.5M sodium carbonate solution, preferably 0.15M-0.25M sodium carbonate solution, more preferably 0.2M sodium carbonate solution. The extract was methyl tert-butyl ether. The compound solution is 10-80% methanol water solution, preferably 50% methanol water solution. The internal standard working solution is diluted 100 times as the mixed internal standard solution.

Because 5-hydroxytryptamine and melatonin belong to alkaline substances and neutral substances respectively, the detection is difficult, and the detection of 5-hydroxytryptamine and melatonin in serum is not reported at present. Based on the characteristics of 5-hydroxytryptamine and melatonin, the invention discovers that the pH regulator (sodium carbonate solution) is adopted in the pretreatment process of serum samples, and methyl tertiary butyl ether is used as an extraction liquid, so that the extraction efficiency of the 5-hydroxytryptamine can be enhanced, the purpose of detecting the 5-hydroxytryptamine together with the melatonin is achieved, the extraction mode is simple, the pretreatment is rapid, and the method is easy to popularize and use in clinic. When a specific pH regulator is selected, similar alkaline solutions, such as sodium hydroxide solution and potassium hydroxide solution, are used as the pH regulator, and similar extracting solutions, such as n-hexane, ethyl acetate or a mixed solution of the two are used as the extracting solutions for extracting the serum sample, the extraction efficiency of the 5-hydroxytryptamine is very low, the 5-hydroxytryptamine cannot be extracted well, and the quantitative analysis of the 5-hydroxytryptamine in the serum sample cannot be satisfied.

In a preferred embodiment, the pretreated serum of the present invention is prepared as follows: 200 mu l of serum is taken in a 1.5ml centrifuge tube, 20 mu l of internal standard working solution, 20 mu l of 0.2M sodium carbonate solution and 900 mu l of methyl tertiary butyl ether are added into the centrifuge tube, after shaking for 3-10 min, the centrifuge tube is centrifuged for 4-10 min at the temperature of between 12000 and 15000r/min and the temperature of between 4 and 20 ℃; and then taking 850 mu l of supernatant, drying by nitrogen flow, mixing with 80 mu l of 50% methanol aqueous solution, oscillating for 1-5 min, centrifuging for 1-5 min at the temperature of 12000-15000 r/min and the temperature of 4-20 ℃, and taking the supernatant to be sampled.

In a more preferred embodiment, the pretreated serum of the present invention is prepared as follows: 200. Mu.l of serum was taken in a 1.5ml centrifuge tube, to which 20. Mu.l of an internal standard working solution, 20. Mu.l of a 0.2M sodium carbonate solution and 900. Mu.l of methyl tert-butyl ether were added, and after shaking for 5min, the mixture was centrifuged at 14800r/min at 10℃for 5min; and then taking 850 μl of supernatant, drying by nitrogen flow, mixing with 80 μl of 50% methanol water solution, oscillating for 3min, centrifuging at 14800r/min and 10deg.C for 3min, and taking supernatant to sample.

In one embodiment, the internal standard working fluid of the present invention is prepared as follows:

the following internal standard mother liquor is prepared by methanol aqueous solution: 5-hydroxytryptamine-d 4 1mg/ml, melatonin-d 4 0.01mg/ml;

and respectively transferring each internal standard mother solution: 5-hydroxytryptamine-d 4. Mu.l, melatonin-d 4 4. Mu.l, was added to 986. Mu.l aqueous methanol to give 1ml of a mixed internal standard solution comprising: 5-hydroxytryptamine-d 4 10. Mu.g/ml, melatonin-d 4 0.04. Mu.g/ml.

10uL of the mixed internal standard solution is added into 0.99ml of pure methanol, and the internal standard working solution is obtained, wherein the internal standard working solution comprises the following components: 5-hydroxytryptamine-d 4 100ng/ml, melatonin-d 4 0.4ng/ml.

In a preferred embodiment, the aqueous methanol solution is selected to be 10% to 80% aqueous methanol, for example 50% aqueous methanol, when the internal standard solution is formulated and mixed.

In a more preferred embodiment, the internal standard working fluid mentioned in the present invention is prepared as follows:

the 5-hydroxytryptamine-d 4 and melatonin-d 4 internal standard mother solutions are respectively prepared by 50% methanol aqueous solution, then added into 986 mu l of 50% methanol aqueous solution, uniformly mixed to obtain 1ml of mixed internal standard solution, 10 mu l of the mixed internal standard solution is added into 0.99ml of pure methanol, and the internal standard working solution is obtained, and the concentration of the internal standard working solution is shown in the table 4 below. It is recommended to be packaged and frozen in a refrigerator at-80 ℃ to be taken and used.

Table 4 internal standard working solution formulation

In one embodiment, the standard of the invention is prepared as follows:

5-hydroxytryptamine and melatonin were formulated as standard stock solutions at the following concentrations: 5-hydroxytryptamine 1mg/ml, melatonin 0.01mg/ml;

and respectively transferring mother solutions of all standard substances: 20 μl of 5-hydroxytryptamine and 2 μl of melatonin; then adding the mixture into 978 mu l of methanol aqueous solution to obtain 1ml of mixed standard stock solution; the stock solution of the mixed standard comprises: 5-hydroxytryptamine 20. Mu.g/ml, melatonin 0.02. Mu.g/ml.

Preparing the mixed standard stock solution into a calibrator solution with seven different concentration points by using a blank serum matrix, wherein the seven concentration points of the calibrator solution are as follows:

the seven concentration points of 5-hydroxytryptamine are in turn: 1ng/ml, 2.5ng/ml, 5ng/ml, 25ng/ml, 50ng/ml, 250ng/ml, 500ng/ml;

the seven concentration points of melatonin are in turn: 0.001ng/ml, 0.0025ng/ml, 0.005ng/ml, 0.025ng/ml, 0.05ng/ml, 0.25ng/ml, 0.5ng/ml.

In a preferred embodiment, the aqueous methanol solution selected in the preparation of the mixed standard stock solution is 10% to 80% aqueous methanol, for example 50% aqueous methanol.

In a preferred embodiment, the blank serum matrix is a 5% to 15% aqueous methanol solution, and more preferably, the blank serum matrix is a 10% aqueous methanol solution.

In a more preferred embodiment, the standard solution is prepared as follows:

standard stock solutions of 5-hydroxytryptamine and melatonin were removed separately and added to 978 μl of 50% aqueous methanol to give 1ml of mixed standard stock solution, with concentrations as given in table 5 below.

TABLE 5 Standard stock solutions

The invention prepares the mixed standard stock solution into seven calibration material solutions with different concentration points by using a blank serum matrix (10% methanol aqueous solution), and the preparation process is as follows:

10 μl of the mixed standard solution was added to 390 μl of 10% aqueous methanol solution as the first high-value concentration point; taking 130 mu l of the first high-value concentration point, and diluting the first high-value concentration point with 130 mu l of 10% methanol aqueous solution to obtain a second high-value concentration point; taking the first high-value concentration point, and diluting the first high-value concentration point with a 10% methanol aqueous solution with the volume of 9 times to obtain a third high-value concentration point; taking the second high-value concentration point and diluting the second high-value concentration point with a 10% methanol aqueous solution with the volume of 9 times to obtain a fourth high-value concentration point; taking the third high-value concentration point, and diluting the third high-value concentration point with a 10% methanol aqueous solution with the volume of 9 times to obtain a fifth high-value concentration point; taking a fourth high-value concentration point, and diluting the fourth high-value concentration point with a 10% methanol aqueous solution with the volume of 9 times to obtain a sixth high-value concentration point; and diluting the fifth high-value concentration point with 4 times of 10% methanol water solution to obtain a seventh high-value concentration point.

The invention adopts a gradient dilution method to prepare standard yeast, takes out standard solution from a refrigerator at-20 ℃, and then vortex for 10s, uses standard solution to prepare the highest concentration point of the standard yeast within 2min, and stores the standard yeast at-80 ℃ after preparation, wherein the specific process is shown in the following table 6 (unit: ng/ml).

Table 6 standard curve preparation

Standard curve	Remove solution (μL)	Blank serum matrix (mu L)	5-hydroxytryptamine	Melatonin
					S7	Mixed standard stock solution 10	390	500	0.5
S6	S7 130	130	250	0.25
					S5	S7 30	270	50	0.05
S4	S6 30	270	25	0.025
					S3	S5 30	270	5	0.005
S2	S4 30	270	2.5	0.0025
					S1	S3 50	200	1	0.001

200 μl of each concentration point of seven different calibrator samples is taken in a 1.5ml centrifuge tube, 20 μl of internal standard working solution, 20 μl of 0.2M sodium carbonate solution and 900 μl of methyl tert-butyl ether are added into the centrifuge tube, after shaking for 5min, the mixture is centrifuged at 14800r/min at 10deg.C for 5min; then, 850. Mu.l of the supernatant was dried with a nitrogen stream and mixed with 80. Mu.l of a 50% aqueous methanol solution, followed by shaking for 3min, centrifuging at 14800r/min at 10℃for 3min, and then, 65. Mu.l of the supernatant was obtained and 5. Mu.l of the sample was introduced.

The invention also comprises the preparation of a quality control product, wherein the quality control product is blank serum containing 5-hydroxytryptamine and melatonin, and the three concentrations of the quality control product are QC (L), QC (M) and QC (H) respectively;

QC (L) was diluted 10000-fold with the above mixed standard stock solution as a blank serum matrix.

QC (M) was diluted 1000-fold with the blank serum matrix for the mixed standard stock solution described above.

QC (H) was diluted 50-fold with blank serum matrix for the above mixed standard stock solution.

For the purposes of the present invention, the blank serum matrix is a blank serum that does not contain the compound of interest. In a preferred embodiment, the blank serum matrix is a 5% to 15% aqueous methanol solution, for example, the blank serum matrix is a 10% aqueous methanol solution.

In a preferred embodiment, the quality control is prepared as follows: the above mixed standard stock solution was prepared as a blank serum matrix (10% aqueous methanol) to three different concentrations of QC (L), QC (M), QC (H), as shown in table 7.

TABLE 7 quality control product concentration (Unit: ng/ml)

QC (L) contains: 5-hydroxytryptamine 2ng/ml and melatonin 0.002ng/ml.

The QC (M) includes: 5-hydroxytryptamine 20ng/ml and melatonin 0.02ng/ml.

The QC (H) includes: 5-hydroxytryptamine 400ng/ml and melatonin 0.4ng/ml.

By adopting the technical scheme of the invention, the advantages are as follows:

the invention provides a method for detecting the concentration of 5-hydroxytryptamine and melatonin in serum, which adopts sodium carbonate solution as a pH regulator and methyl tertiary butyl ether as an extraction liquid in the pretreatment process of a serum sample, can enhance the extraction efficiency of the 5-hydroxytryptamine, screen out the optimal chromatographic conditions such as mobile phase, gradient and chromatographic column, can detect the 5-hydroxytryptamine and the melatonin in the serum at one time, has high sensitivity, strong specificity and simple pretreatment process, can separate and detect the 5-hydroxytryptamine and the melatonin in the serum within 5.5 minutes, basically meets the requirements on accuracy and precision, and can be used for quantitative analysis of the 5-hydroxytryptamine and the melatonin in the serum clinically and provide a simple and quick detection method for monitoring the concentration of the 5-hydroxytryptamine and the melatonin in clinic.

Drawings

FIG. 1 is an extract ion flow chart of 5-hydroxytryptamine and melatonin standards;

FIG. 2 is a graph of the extracted ion flow of 5-hydroxytryptamine and melatonin in the serum;

FIG. 3 is a graph comparing the lower limit of 5-hydroxytryptamine quantification and the response of serum samples with different pretreatment;

FIG. 4 is a graph comparing melatonin lower limit on quantification and response of serum samples with different pretreatment;

FIG. 5 is a graph of the lower limit of 5-hydroxytryptamine quantification versus the response of serum samples using different mobile phases;

fig. 6 is a graph comparing melatonin lower limit on quantification and response of serum samples with different mobile phases.

Detailed Description

The invention will be better understood from the following examples. However, it will be readily appreciated by those skilled in the art that the description of the embodiments is provided for illustration only and should not limit the invention as described in detail in the claims.

Example 1:

1. experimental materials and instruments

1. Material

The samples were from serum samples collected in 12 month ward of the first hospital affiliated to the university of chinese medical science, 2020.

(1) Instrument: a Xex TQ-S triple quadrupole mass spectrometer (Waters Corporation); UPLC I-Class ultra-high performance liquid chromatography system (auto sampler, waters Corporation); SCILOGEX D2012 high speed table centrifuge (usa); ultrapure water meter (ELGA LabWater, uk); multitube Vortex mixer (Vortex genie2, usa); an adjustable pipette (Eppendorf 0.5-10. Mu.l, 10-100. Mu.l, 100-1000. Mu.l); glassware, measuring cylinders, etc.

(2) Reagent consumable: MS grade methanol (Fisher, USA); MS grade formic acid (Fisher, USA); MS grade ammonium acetate (Sigma, usa); HPLC grade methyl tert-butyl ether (Fisher, usa); sodium carbonate (national medicine); chromatographic column model: waters BEH C18 (2.1X100 mm,1.7 μm).

(3) Standard substance: standards and their corresponding internal standards are shown in table 8 below.

Table 8 standards and internal standards

Sequence number	Chinese name	Manufacturer' s
			1	5-hydroxytryptamine	TRC
2	Melatonin	TRC
			3	5-hydroxytryptamine-d 4	TRC
4	Melatonin-d 4	TRC

(4) Quality control product: the blank serum containing 5-hydroxytryptamine and melatonin was divided into three concentrations of QC (L), QC (M) and QC (H), and the concentrations are shown in Table 7.

2. Liquid condition

(1) Chromatographic conditions: mobile phase a:0.1% formic acid-2 mM ammonium acetate aqueous solution; mobile phase B: methanol. Chromatographic column model: waters BEH C18 (2.1X100 mm,1.7 μm) was eluted by gradient, as detailed in Table 1. The flow rate was 0.3ml/min, the column temperature was 40℃and the sample volume was 5. Mu.L.

(2) In an electrospray ionization positive ion detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, and the capillary voltage is 0.5kV (ESI+); the temperature of the ion source is 150 ℃; the desolventizing temperature is 400 ℃; the flow rate of the desolventizing gas is 800L/hr; the flow rate of the taper hole air is 150L/hr; the mass spectrum source parameters are shown in table 2, and the mass spectrum parameters of each target and the corresponding isotope internal standard are also monitored, and the mass spectrum parameters of each target are shown in table 3.

3. Experimental procedure

(1) Preparing a standard substance:

5-hydroxytryptamine and melatonin were formulated as standard stock solutions at the following concentrations: 5-hydroxytryptamine 1mg/ml, melatonin 0.01mg/ml;

and respectively transferring mother solutions of all standard substances: 20 μl of 5-hydroxytryptamine and 2 μl of melatonin; then added to 978. Mu.l of 50% aqueous methanol to give 1ml of a mixed standard stock solution. The stock solution of the mixed standard comprises: the details of 5-hydroxytryptamine 20. Mu.g/ml and melatonin 0.02. Mu.g/ml are given in Table 5.

The mixed standard stock solution is prepared into seven calibration material solutions with different concentration points by a blank serum matrix (10% methanol aqueous solution), and the details are shown in table 6, wherein the seven concentration points of the calibration material solutions are as follows:

the seven concentration points of 5-hydroxytryptamine are in turn: 1ng/ml, 2.5ng/ml, 5ng/ml, 25ng/ml, 50ng/ml, 250ng/ml, 500ng/ml;

the seven concentration points of melatonin are in turn: 0.001ng/ml, 0.0025ng/ml, 0.005ng/ml, 0.025ng/ml, 0.05ng/ml, 0.25ng/ml, 0.5ng/ml.

(2) Internal standard working fluid

The following internal standard mother liquor was prepared with 50% aqueous methanol: 5-hydroxytryptamine-d 4 1mg/ml, melatonin-d 4 0.01mg/ml;

respectively transferring internal standard mother liquor: 5-hydroxytryptamine-d 4. Mu.l, melatonin-d 4 4. Mu.l; then, the mixture was added to 986. Mu.l of 50% aqueous methanol to obtain 1ml of a mixed internal standard solution comprising: 5-hydroxytryptamine-d 4 10. Mu.g/ml, melatonin-d 4 0.04. Mu.g/ml.

Adding 10 μl of the mixed internal standard solution into 0.99ml of pure methanol to obtain internal standard working solution, wherein the internal standard working solution comprises: 5-hydroxytryptamine-d 4 100ng/ml, melatonin-d 4 0.4ng/ml.

(3) Preparing a quality control product:

the standard stock solutions were prepared as three different concentrations of QC (L), QC (M), QC (H) in a blank serum matrix (10% aqueous methanol) as shown in table 7.

QC (L) contains: 5-hydroxytryptamine 2ng/ml and melatonin 0.002ng/ml.

The QC (M) includes: 5-hydroxytryptamine 20ng/ml and melatonin 0.02ng/ml.

The QC (H) includes: 5-hydroxytryptamine 400ng/ml and melatonin 0.4ng/ml.

(4) Sample processing

1) Standard substance treatment: 200 μl of each concentration point of seven different calibrator samples is taken in a 1.5ml centrifuge tube, 20 μl of internal standard working solution, 20 μl of 0.2M sodium carbonate solution and 900 μl of methyl tert-butyl ether are added into the centrifuge tube, after shaking for 5min, the mixture is centrifuged at 14800r/min at 10deg.C for 5min; then, 850. Mu.l of the supernatant was dried with a nitrogen stream and mixed with 80. Mu.l of a 50% aqueous methanol solution, followed by shaking for 3min, centrifuging at 14800r/min at 10℃for 3min, and then, 65. Mu.l of the supernatant was obtained and 5. Mu.l of the sample was introduced.

2) Pretreatment of serum samples: 200. Mu.l of serum was taken in a 1.5ml centrifuge tube, to which 20. Mu.l of an internal standard working solution, 20. Mu.l of a 0.2M sodium carbonate solution and 900. Mu.l of methyl tert-butyl ether were added, and after shaking for 5min, the mixture was centrifuged at 14800r/min at 10℃for 5min; then, 850. Mu.l of the supernatant was dried with a nitrogen stream and mixed with 80. Mu.l of a 50% aqueous methanol solution, followed by shaking for 3min, centrifuging at 14800r/min at 10℃for 3min, and then, 65. Mu.l of the supernatant was obtained and 5. Mu.l of the sample was introduced.

3) Pretreatment of quality control products: 200 μl of each of the quality control solutions QC (L), QC (M) and QC (H) was taken in a 1.5ml centrifuge tube, and then the samples were pretreated in accordance with the serum samples, and the details thereof were omitted.

4. Method verification

1. Extracting an ion flow chromatogram: the peak shapes of the standard products of 5-hydroxytryptamine and melatonin and the serum sample are symmetrical, and the peak shapes are free from interference of miscellaneous peaks, which indicates that the sample can be well detected under the condition, wherein the figure 1 is a flow chart of the extracted ions of the standard products of 5-hydroxytryptamine and melatonin, and the figure 2 is a flow chart of the extracted ions of the 5-hydroxytryptamine and melatonin in serum.

2. Calibration curve: and (3) using an isotope internal calibration method, using TargetLynx software to establish a calibration curve by taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the concentration of an object to be detected in serum. The linear fitting equation of the 5-hydroxytryptamine and the melatonin in the respective concentration ranges has good linearity, and the correlation coefficient is more than 0.99, so that the quantitative requirement is met, and the table 9 is shown.

Table 95 Linear regression equation and Linear correlation coefficient of hydroxytryptamine and melatonin

3. Accuracy investigation: and (5) evaluating the accuracy of the method by adopting a standard adding recovery rate test. A mixed blank serum sample is prepared, mixed standard substances with low, medium and high concentrations are respectively added, the treatment and measurement are repeated for 5 times by the same steps, and the result shows that the standard recovery rate of 5-hydroxytryptamine and melatonin is between 97.45% and 108.06%, the RSD of 5 repeated tests is between 0.75% and 1.97%, and the statistical result is shown in Table 10.

TABLE 10 results of recovery of 5-hydroxytryptamine and melatonin addition

4. Precision test: taking a non-interference blank serum sample, adding 5-hydroxytryptamine with different concentrations and melatonin standard substances to obtain serum samples with low, medium and high concentrations, repeatedly processing 6 batches in one day, continuously processing for three days, quantitatively measuring the concentrations of the 5-hydroxytryptamine and the melatonin by an internal standard method, processing 3 batches in three days, and calculating the precision between batches to obtain the results shown in Table 11, wherein the precision between batches is 0.75% -3.00%.

TABLE 11 results of within-and inter-lot precision tests

5. Discussion of the invention

The invention adopts an ID-UPLC-MS/MS method to measure the concentration of 5-hydroxytryptamine and melatonin in human serum. Meanwhile, the detection is carried out on the peak-out time and the ion pair of the target object, the sensitivity is high, meanwhile, the substrate interference can be greatly eliminated by adopting the isotope internal standard method for quantification, and the accurate quantification can be achieved without being influenced by the pretreatment process, the sample loading volume, the flow and other conditions.

The accuracy of the method is evaluated by a labeling recovery rate test, and the result shows that the labeling recovery rate of the 5-hydroxytryptamine and the melatonin is 97.45-108.06 percent, the RSD of 5 repeated tests is 0.75-1.97 percent, and the accuracy is good.

The repeatability result of the method shows that the precision of 5-hydroxytryptamine and melatonin in batches is 0.75% -3.00%, 3 batches are processed in three days, and the precision between batches is calculated to be 1.57% -2.86%. And the pretreatment process of the established serum sample is very simple, and the serum dosage is only 200 mu L.

Comparative example 1:

the method for detecting 5-hydroxytryptamine and melatonin in serum provided in the comparative example comprises the following steps:

1. liquid condition

(1) Mobile phase a:0.1% formic acid-2 mM ammonium acetate aqueous solution; mobile phase B: methanol. Chromatographic column model: waters BEH C18 (2.1X100 mm,1.7 μm) was eluted by gradient, as detailed in Table 1. The flow rate was 0.3ml/min, the column temperature was 40℃and the sample volume was 5. Mu.L.

(2) Mass spectrometry conditions: in an electrospray ionization positive ion detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, and the capillary voltage is 0.5kV (ESI+); the temperature of the ion source is 150 ℃; the desolventizing temperature is 400 ℃; the flow rate of the desolventizing gas is 800L/hr; the flow rate of the taper hole air is 150L/hr; the mass spectrum source parameters are shown in table 2, and the mass spectrum parameters of each target and the corresponding isotope internal standard are also monitored, and the mass spectrum parameters of each target are shown in table 3.

2. Experimental procedure

(1) Preparing a standard substance:

5-hydroxytryptamine and melatonin were formulated as standard stock solutions at the following concentrations: 5-hydroxytryptamine 1mg/ml, melatonin 0.01mg/ml;

and respectively transferring mother solutions of all standard substances: 20 μl of 5-hydroxytryptamine and 2 μl of melatonin; then added to 978. Mu.l of 50% aqueous methanol to give 1ml of a mixed standard stock solution. The stock solution of the mixed standard comprises: 5-hydroxytryptamine 20. Mu.g/ml, melatonin 0.02. Mu.g/ml.

The above mixed standard stock solution was formulated as a blank serum base (10% aqueous methanol) into 1ng/ml 5-hydroxytryptamine and 0.001ng/ml melatonin (lower limit sample).

(2) Internal standard working fluid

The following internal standard mother liquor was prepared with 50% aqueous methanol: 5-hydroxytryptamine-d 4 1mg/ml, melatonin-d 4 0.01mg/ml;

respectively transferring internal standard mother liquor: 5-hydroxytryptamine-d 4. Mu.l, melatonin-d 4 4. Mu.l; then, the mixture was added to 986. Mu.l of 50% aqueous methanol to obtain 1ml of a mixed internal standard solution comprising: 5-hydroxytryptamine-d 4 10. Mu.g/ml, melatonin-d 4 0.04. Mu.g/ml.

Adding 10 μl of the mixed internal standard solution into 0.99ml of pure methanol to obtain internal standard working solution, wherein the internal standard working solution comprises: 5-hydroxytryptamine-d 4 100ng/ml, melatonin-d 4 0.4ng/ml.

(3) Sample processing

1) Standard pretreatment 1: preparing 500 mu l of standard curve quantitative lower limit sample, adding 10 mu l of internal standard working solution into the sample, mixing the sample for 60s, adding 100 mu l of NaOH solution (1 mol/l) and 1ml of extraction solution (the volume ratio of n-hexane to ethyl acetate is 1:1), oscillating the sample for 5min, separating the sample from the sample for 10min at 13000 revolutions per minute, and taking 800 mu l of supernatant; drying with nitrogen, re-dissolving 100 μl of 10% methanol water solution, and oscillating for 5min; separating the core at 13000 rpm for 3min, and collecting supernatant.

2) Serum sample pretreatment 1: taking 500 μl of serum sample, adding 10 μl of internal standard working solution, mixing for 60s, adding 100 μl of NaOH solution (1 mol/l) and 1ml of extract (volume ratio of n-hexane and ethyl acetate is 1:1), oscillating for 5min, separating heart for 10min at 13000 rpm, and collecting 800 μl of supernatant; drying with nitrogen, re-dissolving 100 μl of 10% methanol water solution, and oscillating for 5min; separating the core at 13000 rpm for 3min, and collecting supernatant.

3) Standard pretreatment 2: 200 μl of standard curve quantitative lower limit sample is prepared, 20 μl of internal standard working solution, 20 μl of 0.2M sodium carbonate solution and 900 μl of methyl tert-butyl ether are added thereto, shaking is carried out for 5min, the heart is separated for 10min at 13000 rpm, and 800 μl of supernatant is taken; drying with nitrogen, re-dissolving 100 μl of 50% methanol water solution, and oscillating for 5min; separating the core at 13000 rpm for 3min, and collecting supernatant.

4) Serum sample pretreatment 2: 200 μl of serum sample was taken, 20 μl of internal standard working solution, 20 μl of 0.2M sodium carbonate solution and 900 μl of methyl tert-butyl ether were added thereto, shaking was performed for 5min, and the heart was separated for 10min at 13000 rpm, and 800 μl of supernatant was taken; drying with nitrogen, re-dissolving 100 μl of 50% methanol water solution, and oscillating for 5min; separating the core at 13000 rpm for 3min, and collecting supernatant.

The standard and serum samples in pretreatment 1 and pretreatment 2 were taken and tested according to the above chromatographic conditions, and the experimental results are shown in fig. 3 and 4.

Pretreatment is carried out on the standard substance and the serum sample by adopting a pretreatment mode 1 and a pretreatment mode 2 respectively, and the difference of melatonin and 5-hydroxytryptamine in response intensity is examined. As can be seen from fig. 3 and 4, melatonin has little difference in response intensity, but the pretreatment 2 is slightly better in treatment mode; however, in terms of the response intensity, the treatment efficiency of the pretreatment 2 is about 50 times higher than that of the pretreatment 1, and a remarkable effect is obtained, whereas the pretreatment 1 cannot satisfy the quantitative analysis of 5-hydroxytryptamine in the serum sample due to the lower treatment efficiency, that is, the pretreatment 1 is adopted to treat the serum sample, so that the 5-hydroxytryptamine and melatonin in the serum cannot be simultaneously detected at one time.

Comparative example 2

The procedure for detecting 5-hydroxytryptamine and melatonin in serum provided in this comparative example is the same as that of comparative example 1, and the pretreatment method uses pretreatment 2, except that mobile phase A is different. Wherein, mobile phase A1:0.1% formic acid-2 mM ammonium acetate aqueous solution; mobile phase A2:0.05% formic acid in water.

The standard and serum samples of pretreatment 2 were taken and tested with reference to the chromatographic conditions of comparative example 1, respectively, and the experimental results are shown in fig. 5 and 6.

Pretreatment was performed on the standard and serum samples by the pretreatment 2, and the difference in response intensity between melatonin and 5-hydroxytryptamine was examined by referring to the chromatographic conditions in comparative example 1. As can be seen from fig. 5 and 6, in the same sample pretreatment mode, when the mobile phase A1 and the mobile phase A2 are used for chromatographic analysis, the ionization efficiency of 5-hydroxytryptamine is not greatly different, and the ionization efficiency is slightly better when the mobile phase A1 is used; however, the difference in ionization efficiency is evident for 5-hydroxytryptamine, and the ionization efficiency when using mobile phase A1 is about 6 times that when using mobile phase A2, that is, 0.1% formic acid-2 mM ammonium acetate aqueous solution-methanol is significantly better than 0.05% formic acid aqueous solution-methanol as mixed mobile phase under the same pretreatment mode and substantially the same chromatographic conditions.

The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments may be modified or some technical features may be replaced equivalently; such modifications and substitutions do not depart from the spirit of the invention.

Claims (7)

1. A method for detecting the concentration of 5-hydroxytryptamine and melatonin in blood serum, which is characterized in that,

the internal standard substances corresponding to the 5-hydroxytryptamine and the melatonin are respectively as follows: 5-hydroxytryptamine-d 4 and melatonin-d 4;

detecting 5-hydroxytryptamine and melatonin in pretreated serum by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology, separating an object to be detected from a serum matrix by utilizing the ultra-high performance liquid chromatography, and then establishing a calibration curve by taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis by utilizing a mass spectrometry isotope internal calibration method, wherein the specific chromatographic conditions are as follows:

(1) Ultra-high performance liquid chromatography conditions:

mobile phase a:0.1% formic acid-2 mM ammonium acetate aqueous solution; mobile phase B: methanol;

chromatographic column model: waters BEH C18;

adopting a mode that a mobile phase A and a mobile phase B are mixed mobile phases for gradient elution, wherein the initial ratio of the mobile phase A to the mobile phase B is 90:10; the gradient elution process is as follows: the volume ratio of the mobile phase A to the mobile phase B is gradually changed from the initial proportion to 2:98 at a constant speed within 0-1.5 minutes; the volume ratio of mobile phase A to mobile phase B is kept constant at 2:98 within 1.5-3.5 minutes; in 3.5-5.5 minutes, the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 2:98 to the initial ratio at a constant speed; each sample was collected for 5.5 minutes;

the pretreated serum was prepared as follows: 200 μl of serum is taken in a 1.5ml centrifuge tube, 20 μl of internal standard working solution, 20 μl of 0.2M sodium carbonate solution and 900 μl of methyl tert-butyl ether are added into the centrifuge tube, and after shaking for 3-10 min, the centrifuge tube is centrifuged for 4-10 min at the temperature of 12000-15000 r/min and 4-20 ℃; taking 850 μl of supernatant, drying by nitrogen flow, mixing with 80 μl of 50% methanol aqueous solution, oscillating for 1-5 min, centrifuging for 1-5 min at 12000-15000 r/min and 4-20deg.C, and taking supernatant to be sampled;

(2) Mass spectrometry conditions:

in the electrospray ionization positive ion detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, and the capillary voltage is 0.5 kV; the temperature of the ion source is 150 ℃; the desolventizing temperature is 400 ℃; the flow rate of the desolventizing gas is 800L/hr; the flow rate of the taper hole air is 150L/hr; and simultaneously monitoring each target object and the corresponding isotope internal standard.

2. The method according to claim 1, wherein the flow rate is 0.2-0.4 ml/min; the column temperature is 30-50 ℃; the sample injection volume is 1-10 mu L.

3. The method according to claim 2, wherein the flow rate is 0.3ml/min; column temperature is 40 ℃; the sample injection volume is 5 mu L.

4. The method of claim 1, wherein the serum is human or animal serum.

5. The method of claim 1, wherein the step of determining the position of the substrate comprises,

the internal standard working solution is prepared according to the following method:

the following internal standard mother liquor is prepared by methanol aqueous solution: 5-hydroxytryptamine-d 4 1mg/ml, melatonin-d 4 0.01mg/ml;

and respectively transferring each internal standard mother solution: 5-hydroxytryptamine-d 4. Mu.l, melatonin-d 4 4. Mu.l; then adding the mixture into 986 mu l of 50% methanol aqueous solution to obtain 1ml of mixed internal standard solution;

10uL of the mixed internal standard solution is added into 0.99ml of pure methanol, and then the internal standard working solution is obtained.

6. The method of claim 1, wherein the step of determining the position of the substrate comprises,

the standard is prepared according to the following method:

5-hydroxytryptamine and melatonin were formulated as standard stock solutions at the following concentrations: 5-hydroxytryptamine 1mg/ml, melatonin 0.01mg/ml;

and respectively transferring mother solutions of all standard substances: 20 μl of 5-hydroxytryptamine and 2 μl of melatonin; then adding the mixture into 978 mu l of 50% methanol aqueous solution to obtain 1ml of mixed standard stock solution;

preparing the mixed standard stock solution into seven calibrator solutions with different concentration points by using a blank serum matrix, wherein the blank serum matrix is 5% -15% methanol water solution, and the seven concentration points of the calibrator solutions are as follows:

the seven concentration points of 5-hydroxytryptamine are in turn: 1ng/ml, 2.5ng/ml, 5ng/ml, 25ng/ml, 50ng/ml, 250ng/ml, 500ng/ml;

the seven concentration points of melatonin are in turn: 0.001ng/ml, 0.0025ng/ml, 0.005ng/ml, 0.025ng/ml, 0.05ng/ml, 0.25ng/ml, 0.5ng/ml.

7. The method of claim 6, wherein the blank serum matrix is a 10% aqueous methanol solution.

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