Liquid chromatography-mass spectrometry detection kit and detection method for serum seleno-amino acid based on mass spectrometry probe labeling

Abstract

The invention belongs to the technical field of health, clinical and medical related detection, and relates to a liquid chromatography-mass spectrometry detection kit and a detection method for serum seleno-amino acid labeled on the basis of a mass spectrometry probe. The detection kit comprises a mass spectrum probe solution, a protein precipitant solution and a buffering agent solution, the mass spectrum probe is 6-aminoquinolyl-N-hydroxysuccinimido carbamic acid ester, and the mass spectrum probe is 2-aminoquinolyl-N- The buffer solution contains a seleno-amino acid isotope internal standard. According to the kit, a mass spectrum probe is used for labeling, a serum sample is subjected to protein precipitation and then mixed with the mass spectrum probe at room temperature, and simultaneous high-flux accurate quantification of multiple seleno-amino acids can be directly achieved through liquid chromatography-tandem mass spectrometry in a conventional reversed-phase chromatography mode. The 6-aminoquinolyl-N-hydroxysuccinimido carbamate mass spectrum probe adopted in the kit disclosed by the invention has the advantages of high reaction activity, convenience in operation, high sensitivity and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of health, clinical and medical related detection, and in particular relates to a liquid chromatography-mass spectrometry detection kit and detection method for serum selenoamino acids based on mass spectrometry probe labeling. BACKGROUND

[0002] Selenium (Se) is an important trace element essential for human and animal life activities. The content limit between beneficial and harmful in the human body is very small: studies have shown that daily intake of less than 40 μg will lead to selenium deficiency, and further lead to diseases such as Kashin-Beck disease and Keshan disease; more than 400 μg will cause symptoms of selenium poisoning such as gastrointestinal discomfort, nerve damage, and hair loss. The recommended daily intake of the U.S. Institute of Medicine is 55 μg / day, and the World Health Organization is 40 μg for men and 30 μg for women. In 2017, the Chinese Nutrition Society recommended that the selenium intake of adults be 60-400 μg / day.

[0003] As a component of the earth's crust, selenium is ubiquitous in soil, rock, air, water, and plants and animals. However, the distribution of selenium content in natural soil is very uneven, leading to selenium deficiency, which has become a global health problem that needs to be solved urgently. Only the United States, Venezuela, Canada, Switzerland, Japan, New Zealand, and Australia are countries with adequate selenium intake. The main way for the human body to intake selenium is still through diet, especially the selenium in plants, which is the main source of selenium intake for the human body. The main form of selenium in selenium-rich plants is selenium-containing organic small molecule compounds in the form of selenoamino acids, which have higher safety and bioavailability compared to inorganic selenium such as selenate or selenite. More and more studies have shown that selenium has an important influence on cancer development, drug resistance, and immune escape processes, and selenoamino acids significantly improve immune response by regulating the cross-linking of tumor cells and immune cells and restructuring the tumor microenvironment, thus also showing great potential in tumor immunotherapy. Therefore, quantitative detection of selenoamino acids in serum, a circulating medium in the human body, has important health, clinical, and medical significance.

[0004] Current research generally believes that the selenoamino acids present in the mammalian body are mainly selenocysteine and selenomethionine, and the representative selenoamino acids in plants and yeast also include methylselenocysteine. Although it has been more than 60 years since the first selenoamino acid, methylselenocysteine, was discovered in 1960, and the health effects of selenium have also attracted more and more attention, there are still many challenges in the quantitative detection of selenoamino acids.

[0005] The circulating level of selenoamino acids in organisms is low (ppb, μg / kg), and the currently developed analysis methods usually use traditional elemental analysis methods such as atomic fluorescence spectrometry or inductively coupled plasma mass spectrometry in combination with solid-phase extraction and other pre-enrichment methods. Since atomic fluorescence spectrometry and inductively coupled plasma mass spectrometry can only detect selenium elements, they cannot distinguish selenoamino acids with different chemical structures, and therefore must rely on chromatographic separation. However, selenoamino acids, as amino acid substances, generally have a low LogP, and they have almost no retention in conventional reversed-phase chromatography mode, making it difficult to achieve effective chromatographic separation using conventional reversed-phase chromatography. The currently available technologies all use cation exchange chromatography or hydrophilic interaction chromatography to achieve a certain degree of separation, but the separation degree of cation exchange chromatography and hydrophilic interaction chromatography is small and the stability is poor, and it is still difficult to meet the requirements of high selectivity, high sensitivity and high throughput simultaneous detection of selenoamino acids under the extremely low circulating level of selenoamino acids in serum clinical samples. At present, there is no method that can achieve high-throughput simultaneous detection of multiple selenoamino acids in reversed-phase chromatography mode, and it is difficult to meet the demand for high-throughput simultaneous quantitative detection of selenoamino acids in serum biological samples in health, clinical and medical research. SUMMARY

[0006] In view of the problems of complicated sample processing and poor stability of quantitative results in the prior art, the application provides a liquid chromatography-mass spectrometry detection kit and a detection method for serum selenoamino acids based on mass spectrometry probe labeling. The kit developed by the application uses mass spectrometry probe labeling, does not need to rely on time-consuming, labor-intensive and high-cost solid-phase extraction column pre-enrichment, and can directly achieve simultaneous high-throughput accurate quantification of multiple selenoamino acids in conventional reversed-phase chromatography mode after mixing the serum sample after protein precipitation with the mass spectrometry probe at room temperature. Further, the mass spectrometry probe labeling realizes effective retention in conventional reversed-phase chromatography mode by enhancing the original hydrophobicity of selenoamino acids, and solves the significant influence of sample pH difference on the stability of quantitative results when using cation exchange chromatography mode. The 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate mass spectrometry probe used in the kit has the advantages of high reactivity, convenient operation and high sensitivity.

[0007] The detection kit provided by the application can realize high-throughput analysis without professional personnel, only by conventional use training, greatly reducing the application threshold. The labeled products of multiple selenoamino acids all have good separation degree, sensitivity and accuracy, and can meet the quantitative detection requirements of selenoamino acids in clinical and related medical research.

[0008] In order to achieve the above-mentioned purposes, the first aspect of the application provides a liquid chromatography-mass spectrometry detection kit for serum selenoamino acids based on mass spectrometry probe labeling, which comprises a mass spectrometry probe solution, a protein precipitant solution and a buffer solution;

[0009] The mass spectrometry probe is 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate;

[0010] The buffer solution contains a selenoamino acid isotope internal standard.

[0011] The second aspect of the present application provides the use of a detection kit, wherein the selenoamino acids detected by the detection kit include selenocystine, methylselenocysteine, selenomethionine and selenoethionine.

[0012] The third aspect of the present application provides a detection method for serum selenoamino acids based on mass spectrometry probe labeling and liquid chromatography-mass spectrometry, wherein the detection method is performed by using the detection kit, and comprises the following steps:

[0013] S1, adding a protein precipitant solution to a serum sample to be detected, mixing uniformly and incubating, and after the incubation is completed, separating to obtain a supernatant;

[0014] S2, adding a mass spectrometry probe solution and a buffer solution to the supernatant obtained in step S1 to mix uniformly, and performing a labeling reaction;

[0015] S3, after the labeling reaction is completed, performing quantitative analysis to obtain a selenoamino acid detection result.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] In the liquid chromatography-mass spectrometry detection kit and simultaneous detection method for serum selenoamino acids based on mass spectrometry probe labeling, the serum sample to be detected can be labeled by a simple and convenient mass spectrometry probe, without the need for pre-enrichment treatment. In addition, the four kinds of selenoamino acids can be simultaneously detected in liquid chromatography-mass spectrometry by directly using a reversed-phase chromatography mode, thereby avoiding the significant influence of the pH difference of the sample on the stability of the quantitative result when ion exchange chromatography is used, and the selenoamino acids all have good chromatographic retention and separation degree. The mass spectrometry probe labeling enhances the analysis sensitivity by changing the chemical structure of the selenoamino acid, and can meet the high-throughput and high-sensitivity quantitative detection requirements of selenoamino acids in clinical and related medical research.

[0018] Other features and advantages of the present application will be described in detail in the following specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0019] The above and other objects, features and advantages of the present application will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings in which like reference characters refer to like parts throughout the several views, and wherein the exemplary embodiments of the present application are shown.

[0020] Figure 1 The chemical structure of the mass spectrometry probe of the present application is shown.

[0021] Figure 2 The schematic diagram of the labeling reaction of the mass spectrometry probe of the present application is shown.

[0022] Figure 3 The comparison diagram of the secondary mass spectrometry of the selenoamino acid before and after the labeling by the mass spectrometry probe of the present application is shown, wherein, Figure 3 A- Figure 3 D respectively show the comparison diagram of the secondary mass spectrometry of selenocystine, methylseleno cysteine, selenomethionine and selenoethionine before and after the labeling by the mass spectrometry probe.

[0023] Figure 4 The fragmentation behavior of the selenoamino acid mass spectrometry probe labeling product in the tandem mass spectrometry is shown.

[0024] Figure 5 The reverse phase chromatogram of the selenoamino acid before and after the labeling by the mass spectrometry probe and the extracted ion current chromatogram of the selenoamino acid labeling product are shown, wherein, Figure 5 A- Figure 5 B respectively show the reverse phase chromatogram of selenocystine and methylseleno cysteine before and after the labeling by the mass spectrometry probe, Figure 5 C shows the extracted ion current chromatogram of the labeling product of selenocystine and methylseleno cysteine.

[0025] Figure 6 The stability of the selenoamino acid labeling product under the continuous sample injection analysis for 24 hours at 4℃ is shown. DETAILED DESCRIPTION

[0026] The preferred embodiments of the present application will be described in more detail below. Although the preferred embodiments of the present application are described below, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein.

[0027] The first aspect of the present application provides a liquid chromatography-mass spectrometry detection kit for the serum selenoamino acid based on the mass spectrometry probe labeling, which comprises a mass spectrometry probe solution, a protein precipitant solution and a buffer solution;

[0028] The mass spectrometry probe is 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.

[0029] The buffer solution contains a selenoamino acid isotope internal standard.

[0030] In the present application, the chemical structure of the mass spectrometry probe is as follows: Figure 1As shown, the chemical structure of the mass spectrometric probe adopted is composed of a "6-aminoquinoline" chromatographic retention-enhancing group with a 171.1 Da reporter ion, an "urethane" specific reactive group and an "N-hydroxysuccinimide" protecting group. The "quinoline" structure in the chromatographic retention-enhancing group has strong hydrophobicity and thus functions as a chromatographic retention-enhancing group. In the present application, by designing the chemical structure of the mass spectrometric probe, on the basis of ensuring that the mass spectrometric probe can be labeled with selenoamino acids efficiently and rapidly, the 171.1 Da reporter ion can effectively improve the selectivity of the mass spectrometric quantitative channel, the protecting group can effectively avoid the loss of the mass spectrometric probe during storage, and the chromatographic retention-enhancing group can significantly enhance the hydrophobicity of the selenoamino acid to achieve effective retention in the reversed-phase chromatographic mode.

[0031] According to the present application, preferably, the solvent of the mass spectrometric probe solution is at least one of methanol, acetonitrile and isopropanol; and the concentration of the mass spectrometric probe in the mass spectrometric probe solution is 0.5-5 mg / mL.

[0032] In the present application, by controlling the concentration of the mass spectrometric probe, the loss of excess mass spectrometric probe during the analysis process can be reduced while ensuring the sensitivity of the analysis method, and the analysis cost can be reduced.

[0033] According to the present application, preferably, the protein precipitant is at least one of acetonitrile, methanol and isopropanol containing 0.5%-1.0% formic acid.

[0034] According to the present application, preferably, the buffer solution is at least one of a borate aqueous solution, a tris-hydroxymethyl aminomethane-hydrochloric acid buffer solution and a phosphate buffer solution; the concentration of the buffer in the buffer solution is 0.1-0.5 M; and the amount of the buffer solution added is such that the pH of the labeling reaction solution environment is 7.5-9.

[0035] In the present application, by controlling the concentration of the buffer and the pH of the labeling reaction solution environment, the smooth occurrence of the mass spectrometric probe labeling reaction can be effectively controlled, the occurrence of side reactions can be avoided, the mass spectrometric probe can be consumed and by-products can be produced, and the negative influence on the quantitative analysis result can be avoided.

[0036] According to the present application, preferably, the selenoamino acid isotopic internal standard is at least one of 13 C1-methyl-selenomethionine, L-cysteine-2,3,3-d3 and L-cystine-1,1'-d2. 13 C2; and the concentration of the selenoamino acid isotopic internal standard in the buffer solution is 1-5 ng / mL.

[0037] The second aspect of the present application provides the use of a detection kit, and the selenoamino acids detected by the detection kit include selenocystine, methylselenocysteine, selenomethionine and selenoethionine.

[0038] The third aspect of the present application provides a detection method based on mass spectrometry probe labeling of serum selenoamino acids liquid chromatography-mass spectrometry, which is carried out by using the detection kit, and comprises the following steps:

[0039] S1, adding a protein precipitant solution to the serum sample to be detected, mixing uniformly and incubating, and after the incubation is completed, separating to obtain a supernatant;

[0040] S2, adding a mass spectrometry probe solution and a buffer solution to the supernatant obtained in step S1 to mix uniformly, and performing a labeling reaction;

[0041] S3, after the labeling reaction is completed, performing quantitative analysis to obtain a selenoamino acid detection result.

[0042] In the present application, the mass spectrometry probe labeling reaction is as shown in Figure 2 The mechanism of the mass spectrometry probe labeling process is an amidation reaction between the primary amino group in the selenoamino acid and the carboxyl group on the carbamic acid in the mass spectrometry probe. Specifically, the mass spectrometry labeling process utilizes the nucleophilicity of amine. The mass spectrometry probe is first hydrolyzed under the action of the alkaline buffer solution as an acid scavenger, and the protecting group N-hydroxysuccinimide is removed to expose the carbamic acid reaction group. The removed N-hydroxysuccinimide also plays the role of a carboxylic acid activator in the reaction solution, promoting the rapid occurrence of the amidation reaction of the mass spectrometry probe labeling process.

[0043] The comparison chart of the secondary mass spectrometry graphs of the four selenoamino acids before and after the mass spectrometry probe labeling is as shown in Figure 3 After the mass spectrometry probe labeling, the selenoamino acid substances are converted into asymmetric urea composed of 6-aminoquinoline and selenoamino acid. The presence of the 6-aminoquinoline group significantly improves the hydrophobicity and ionization efficiency of different selenoamino acid labeling products, thereby realizing sufficient separation in the conventional reversed-phase chromatography mode and enhancing the subsequent mass spectrometry response. At the same time, the target analyte of the mass spectrometry detection is changed from small molecular weight selenoamino acid to larger molecular weight asymmetric urea labeling product, avoiding the background interference of small molecular weight regions in the mass spectrometry of biological samples, and specifically improving the analysis resolution and method sensitivity of selenoamino acid in the mass spectrometry.

[0044] According to the present application, preferably, in step S1, the volume ratio of the serum sample to be detected to the protein precipitant is 1:3-5;

[0045] The temperature of the incubation is -30 to -10℃, and the time is 10-60 min;

[0046] The separation is centrifugal separation, and the conditions of the centrifugal separation include a rotation speed of 10000-20000 rpm and a time of 5-20 min.

[0047] Preferably, step S1 further comprises: thawing the frozen serum to be tested at -5-10 ℃, and after thawing, vortexing and mixing at 10-40 ℃ to obtain the serum sample to be tested.

[0048] According to the present application, preferably, in step S2, the volume ratio of the supernatant, the mass spectrometry probe solution and the buffer solution is 1:0.2-2:0.2-2.

[0049] The labeling reaction is vortexed at 10-40 ℃ for 10-40 s.

[0050] According to the present application, preferably, in step S3, further comprising adding a constant volume agent solution to the reaction solution after the labeling reaction is completed to obtain a solution to be tested, and then performing quantitative analysis on the solution to be tested.

[0051] The constant volume agent solution is selected from at least one of a formic acid aqueous solution, a formic acid methanol solution and a formic acid acetonitrile solution.

[0052] The concentration of formic acid in the constant volume agent solution is 0.1%-1%.

[0053] The quantitative analysis uses ultra-high performance liquid chromatography-tandem mass spectrometry in a reversed-phase chromatography mode.

[0054] In the present application, in the high performance liquid chromatography-tandem mass spectrometry in the conventional reversed-phase chromatography mode, the chromatographic column stationary phase is a silica gel matrix particle bonded with C8, C18 or C30 ligand, the mobile phase water phase and organic phase are water and methanol or acetonitrile added with formic acid, the flow rate is 0.2 to 1.0 mL / min, the column temperature is 30 to 50 ℃, and the elution program is a gradient elution program.

[0055] Further, in the high performance liquid chromatography-tandem mass spectrometry in the conventional reversed-phase chromatography mode, the chromatographic column stationary phase is a silica gel matrix particle bonded with C18 ligand, the mobile phase water phase and organic phase are water and acetonitrile added with formic acid, the flow rate is 0.2 to 0.6 mL / min, the column temperature is 30 to 40 ℃, and the elution program is a gradient elution program.

[0056] In the present application, by controlling the chromatographic separation conditions, the analysis time can be effectively reduced while effectively separating a plurality of seleno-amino acid labeling products, thereby improving the analysis throughput.

[0057] In the present application, the fragmentation behavior characteristics of the seleno-amino acid mass spectrometry probe labeling product in tandem mass spectrometry are as shown in the figure. Figure 4 The seleno-amino acid mass spectrometry probe labeling product releases a characteristic fragment ion (m / z 171 Da) containing a quinoline structure during fragmentation, which can be used as a reporter ion for liquid chromatography-mass spectrometry quantitative detection

[0058] In the present application, the contrast chart of reversed-phase chromatography retention behavior of selenomethionine (A) and selenocysteine (B) before and after labeling, and the reversed-phase chromatography retention extraction ion flow chart (C) of four characteristic selenoamino acids are shown as follows Figure 5 As shown in the figure, the chromatographic retention of the original unlabeled selenoamino acids is less than 1 minute, and the retention time of the mass spectrometry probe labeled product is extended to 3 minutes and 5 minutes, respectively. All selenoamino acid mass spectrometry probe labeled products exhibit good reversed-phase chromatography retention and separation degree.

[0059] In the present application, the stability of the mass spectrometry probe labeled product at 4℃ for 24h is shown as follows Figure 6 As shown in the figure, the relative intensity change of the mass spectrometry response of the selenoamino acid labeled product within 24h at 4℃ is maintained within the range of 91% to 108%, maintaining good stability and meeting the requirements of high-throughput analysis of large batches of samples.

[0060] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0061] Unless otherwise specified, the raw materials used in the embodiments of the present application are commercially available, the instrument equipment used is conventional instrument equipment in the art, and the operation method used is a conventional method in the art.

[0062] Unless otherwise specified, the percentage content used in the embodiments of the present application refers to the mass fraction percentage content.

[0063] Example 1

[0064] The method for simultaneously detecting a plurality of selenoamino acids in serum provided in the present embodiment comprises the following steps:

[0065] S1, freeze-preserved serum samples are naturally thawed at 4℃, and after complete thawing, vortex mixing is performed at room temperature to obtain the serum samples to be tested.

[0066] S2, 50uL of the serum sample to be tested is accurately taken into a 1.5mL centrifuge tube, and 150uL of acetonitrile solution containing 0.1% formic acid pre-cooled at -20℃ for 30min is added, and vortex mixing is performed.

[0067] S3, the mixed solution containing the protein precipitant is incubated at -20℃ for 30min, and after the incubation is completed, the mixed solution is centrifuged at 13,000rpm for 10min, and 50uL of the supernatant is accurately taken for subsequent processing.

[0068] S4, add borate buffer solution (25uL, pH 9.0, 0.1M, 0.1% formic acid) to the supernatant, vortex for 20s at room temperature. 13 The concentration of C1-methyl-selenomethionine is 1ng / mL, and the mass spectrometry probe (50uL, 2.5mg / mL) is vortexed for 20s at room temperature.

[0069] S5, add 375uL of 0.1% formic acid aqueous solution to the reaction mixture to make up the volume, centrifuge to obtain the supernatant for determination.

[0070] S6, use ultra-high performance liquid chromatography-tandem mass spectrometry in reverse phase chromatography mode to quantitatively analyze the test solution.

[0071] S7, according to the mass spectrometry response obtained for each selenoamino acid and its standard working curve, the original concentration of each selenoamino acid in the serum sample is calculated.

[0072] In this embodiment, the standard working curve is obtained by the internal standard method, that is, a stable isotope internal standard is added to each standard concentration point in the standard working curve, and the standard working curve is calculated.

[0073] In this embodiment, the standard concentration points cover 0.1 to 100ng / mL (ppb), and the actual concentrations of each standard concentration point are shown in Table 1.

[0074] Table 1. Standard concentration points in the standard working curve of various selenoamino acids

[0075] analyte Std1 Std2 Std3 Std4 Std5 Std6 Std7 Std8 Std 9 selenocysteine 0.1 0.5 1 2.5 5 10 25 50 100 methylselenocysteine 0.1 0.5 1 2.5 5 10 25 50 100 selenomethionine 0.1 0.5 1 2.5 5 10 25 50 100 selenoethionine 0.1 0.5 1 2.5 5 10 25 50 100

[0076] Specifically, the standard concentration points are obtained by stepwise dilution of the standard sample stock solution. The standard sample stock solution is obtained by accurately weighing 5.0mg of the analysis standard powder and dissolving it in 5mL of deionized water.

[0077] In this embodiment, the standard working curve should be automatically calculated by the analysis instrument according to each standard concentration point, and should be reconfigured, produced and calculated every fixed period during the sample analysis process.

[0078] In this embodiment, the labeling efficiency is used to evaluate the effectiveness of the mass spectrometry probe labeling.

[0079] Specifically, after the standard solution is labeled by the mass spectrometry probe, the remaining content of the original selenoamino acid analyte is quantitatively detected, and the labeling efficiency is calculated.

[0080] Specifically, the labeling efficiency (%) is calculated by the formula: labeling efficiency (%) = (1-c' / c0) x 100%, wherein c' is the residual content of the original seleno-amino acid analyte after labeling, and c0 is the initial content.

[0081] In this embodiment, the labeling efficiency of each seleno-amino acid after mass spectrometry probe labeling is shown in Table 2.

[0082] Table 2. Labeling efficiency of each seleno-amino acid

[0083] analyte labeling efficiency (%) selenocysteine >99 methylselenocysteine >99 selenomethionine >99 selenoethionine >99

[0084] In this embodiment, the limit of detection (LOD) and the limit of quantification (LOQ) are used to evaluate the sensitivity of the method.

[0085] Specifically, the analyte concentrations at response signal to baseline noise ratios (S / N) of 3 and 10 are obtained, which are the limit of detection (LOD) and the limit of quantification (LOQ), respectively.

[0086] The limit of detection (LOD) and the limit of quantification (LOQ) in this embodiment are shown in Table 3.

[0087] Table 3. Limit of detection (LOD) and limit of quantification (LOQ) of each seleno-amino acid

[0088] analyte limit of detection (ppb) limit of quantitation (ppb) selenocysteine 0.02 0.05 methylselenocysteine 0.01 0.04 selenomethionine 0.03 0.10 selenoethionine 0.01 0.03

[0089] In this embodiment, the standard working curve accuracy is evaluated using the method of standard addition recovery, and the standard addition recovery rate should be within the acceptable range of ±20%.

[0090] Specifically, serum samples are randomly selected, and standard solutions at low, medium and high concentrations are added to the samples, respectively, to obtain spiked samples. The actual concentrations of each concentration point are shown in Table 4. Each concentration of spiked sample is 5 parallel samples, and each parallel sample is analyzed repeatedly for 3 times. The standard addition recovery rate at each spiked concentration point is calculated.

[0091] Table 4. Spiked recovery concentration points in accuracy evaluation

[0092] spike recovery concentration point theoretical concentration / ppb LQC 0.5 MQC 10 HQC 50

[0093] Specifically, the spiked samples at each spiked concentration point are obtained by the same sample pretreatment method to obtain the test solution, and the same analysis conditions are used for quantitative analysis. The quantitative results of 5 parallel samples at each concentration point are averaged to calculate the spiked recovery rate.

[0094] Specifically, the recovery rate is calculated by the formula: recovery rate (%) = (|cmean-c0| / c) x 100%, wherein cmean is the average of the quantitative results of the spiked samples, c0 is the initial concentration of the original unspiked sample, and c is the theoretical spiked concentration.

[0095] In this embodiment, the recovery rates of each selenoamino acid at different spiked recovery concentration points are shown in Table 5.

[0096] Table 5. Recovery rates of each selenoamino acid at different spiked concentration points

[0097]

[0098] The foregoing description of the various embodiments of the application has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the application to the precise form disclosed. Many modifications and variations are possible in light of the above teaching. It is intended that the scope of the application be limited not with this detailed description, but rather by the claims appended hereto.

Claims

1. A liquid chromatography-mass spectrometry based probe-labeled serum selenoamino acid detection kit, characterized in that, The detection kit comprises a mass spectrometry probe solution, a protein precipitant solution and a buffer solution; The mass spectrometry probe is 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; The buffer solution contains a selenoamino acid isotope internal standard.

2. The test kit according to claim 1, wherein, The solvent of the mass spectrometry probe solution is at least one of methanol, acetonitrile and isopropyl alcohol; The concentration of the mass spectrometry probe in the mass spectrometry probe solution is 0.5-5 mg / mL.

3. The test kit according to claim 1, wherein, The protein precipitant is at least one of acetonitrile, methanol and isopropyl alcohol containing 0.5%-1.0% formic acid.

4. The test kit according to claim 1, wherein, The buffer solution is at least one of a borate buffer solution, a tris-hydroxymethyl aminomethane-hydrochloric acid buffer solution and a phosphate buffer solution; The concentration of the buffer in the buffer solution is 0.1-0.5 M; and the amount of the buffer solution added is such that the pH of the labeling reaction solution environment is 7.5-9.

5. The test kit according to claim 1, wherein, The selenoamino acid isotopic internal standard is 13 C1-methyl-selenomethionine, L-cysteine-2,3,3-d3 and L-cystine-1,1'-d2 13 at least one of C2; The concentration of the selenoamino acid isotope internal standard in the buffer solution is 1-5 ng / mL.

6. Use of a test kit according to any one of claims 1 to 5, characterized in that, The selenoamino acids detected by the detection kit include selenocystine, methyl seleno cysteine, selenomethionine and selenoethionine.

7. A method for detecting serum selenoamino acids by mass spectrometry probe labeling based liquid chromatography tandem mass spectrometry, characterized in that, The detection method is performed by using the detection kit of any one of claims 1-6, and comprises the following steps: S1, adding the protein precipitant solution to the serum sample to be detected, mixing uniformly and incubating, and after the incubation is completed, separating to obtain supernatant; S2, adding the mass spectrometry probe solution and the buffer solution to the supernatant obtained in step S1 to mix uniformly, and performing labeling reaction; S3, after the labeling reaction is completed, performing quantitative analysis to obtain the detection result of the selenoamino acid.

8. The detection method according to claim 7, wherein, In step S1, the volume ratio of the serum sample to be detected to the protein precipitant is 1:3-5; The incubation temperature is -30 to -10℃, and the incubation time is 10-60 min; The separation is centrifugal separation, and the centrifugal separation conditions include a rotation speed of 10000-20000 rpm and a time of 5-20 min; Preferably, step S1 further comprises: thawing the frozen serum to be detected at -5 to 10℃, and after the thawing is completed, vortexing uniformly at 10-40℃ to obtain the serum sample to be detected.

9. The detection method according to claim 7, wherein, In step S2, the volume ratio of the supernatant to the mass spectrometry probe solution to the buffer solution is 1:0.2-2:0.2-2; The labeling reaction is vortexing at 10-40℃ for 10-40 s.

10. The detection method according to claim 7, wherein, In step S3, further comprising adding a constant volume agent solution to the reaction solution after the labeling reaction is completed to constant volume, obtaining a solution to be detected, and then performing quantitative analysis on the solution to be detected; The constant volume agent solution is at least one of a formic acid aqueous solution, a formic acid methanol solution and a formic acid acetonitrile solution; The concentration of formic acid in the constant volume agent solution is 0.1%-1%; The concentration of formic acid in the constant volume agent solution is 0.1%-1%. The quantitative analysis uses ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry in a reversed phase chromatographic mode, preferably, the conditions of the ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry in a reversed phase chromatographic mode include: the stationary phase is a silica gel matrix particle bonded with C8, C18 or C30 ligand, preferably a silica gel matrix particle bonded with C18 ligand, the mobile phase is an aqueous phase and an organic phase, the aqueous phase is water added with formic acid, and the organic phase is methanol and / or acetonitrile, preferably acetonitrile, the flow rate is 0.2-1.0 mL / min, preferably 0.2-0.6 mL / min, the column temperature used is 30-50℃, preferably 30-40℃, and the elution procedure used is a gradient elution procedure.

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