CN110274973B - Detection method of sulfur-containing amino acid in serum and enrichment material thereof - Google Patents
Detection method of sulfur-containing amino acid in serum and enrichment material thereof Download PDFInfo
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Abstract
The invention relates to a method for detecting sulfur-containing amino acid in serum and an enrichment material thereof, which comprises the following steps: reducing a serum sample by adding a reducing agent, pretreating the reduced serum sample solution by a specific purifying material to obtain a purified sample eluting solution, carrying out liquid chromatography-tandem mass spectrometry detection and analysis, and determining sulfur-containing amino acids such as methionine, cysteine, homocysteine and the like. The specific material is a metal affinity material taking silica gel as a matrix, and serum is purified by the specific material, so that the interference of the matrix in the serum can be removed, the sensitivity of sulfur-containing amino acid is improved, and the accuracy of sulfur-containing amino acid detection is better ensured. The purification of the specific material is to realize the selective enrichment of the sulfur-containing amino acid by utilizing the specific affinity action between the sulfydryl of the sulfur-containing amino acid and metal, and solve the problem that the sulfur-containing amino acid can not be effectively enriched in a reversed phase chromatography.
Description
Technical Field
The invention belongs to the technical field of medical inspection and analysis, and particularly relates to a liquid chromatography-tandem mass spectrometry detection method for detecting sulfur-containing amino acids such as methionine, cysteine and homocysteine in a serum sample.
Technical Field
Sulfur-containing amino acids in human body are closely related to metabolism of various sulfur-containing compounds in human body, and lack of methionine can cause appetite decrease, growth retardation, kidney enlargement, etc., and can be used for preventing acute or chronic hepatitis, liver cirrhosis, etc. The sulfydryl brought by the cysteine can relieve the poisoning degree of toxic substances and prevent and treat radioactive rays and the like. Homocysteine, an intermediate product of methionine metabolism in humans, is also present in humans, and elevated homocysteine is a risk factor for atherosclerotic vascular disease [ Jung YS, y.et al, biochimie.2013; 95(8):1605-10]. The clinical detection of sulfur-containing amino acid in human body has important significance for the auxiliary diagnosis and treatment of clinical patient-related diseases. The method for clinically detecting the sulfur-containing amino acid comprises a radioactive enzyme immunoassay method, a fluorescence polarization immunoassay method, a high performance liquid chromatography method and the like, wherein the enzyme immunoassay method has high sensitivity and strong specificity, but has poor precision and long operation time and is not suitable for single samples and emergency samples. Fluorescence polarization immunoassay methods are costly to use and require specialized instrumentation. The high performance liquid chromatography has good accuracy and stability, but is not suitable for trace analysis of small molecular substances in vivo. The liquid chromatography-tandem mass spectrometry has the advantages of extremely wide application range, high sensitivity, strong specificity, capability of providing relative molecular mass and structural information, high flux and short quantitative time on the premise of higher accuracy and precision. When common liquid chromatography-tandem mass spectrometry is used for detecting sulfur-containing amino acid in serum, matrix interference in a serum sample is large, ion chromatogram base line interference is large, and pollution to a chromatographic column for liquid phase separation and mass spectrometry is serious, so that an enrichment purification material specific to sulfur-containing amino acid in serum is designed, and a separation analysis method specific to specific detection of sulfur-containing amino acid in serum is developed. [ Ubbink JB, Y.et al, Semin Thromb Hemost.2000; 26(3):233-41].
In recent years, amino acids in complex biological samples are quantitatively analyzed by a simple and sensitive high performance liquid chromatography-tandem mass spectrometry method, wherein the amino acids are separated by ion exchange solid phase extraction according to the structural properties of the amino acids. Due to the characteristics of mass spectrometry, the influence of other interference peaks caused by matrix effect cannot be observed on a chromatogram, but co-effluent components of solid phase extraction still enter a chromatography-mass spectrometry system to pollute the chromatography-mass spectrometry separation and analysis system, and the sensitivity of mass spectrometry detection is reduced. For example, CN201710129265 discloses a liquid chromatography tandem mass spectrometry method for detecting sulfur-containing amino acids in plasma by a non-derivatization method, wherein an isotope internal standard solution is added into a plasma sample, then a reducing agent is added for reduction, and after protein precipitation treatment, the supernatant is analyzed and collected by LC-MS/MS, but the obtained precision is not high. For another example, CN201610314061 discloses a plasma sample pretreatment method for endogenous amino acid detection, which uses silica-bonded C1-30 alkyl particles as solid adsorbent to adsorb amino acid components, but has low specificity for sulfur-containing amino acids. Aiming at the structural characteristics of sulfydryl of sulfur-containing amino acid, a solid phase extraction material for enriching and purifying the sulfur-containing amino acid specificity in serum is designed and developed, silicon dioxide is used as a matrix, and metal ions are introduced on the basis of proper coordination. Realizing the exclusive specificity of the sulfur-containing amino acid. [ XINXING T, Y et al, Se Pu.2012,30(7):696-
Disclosure of Invention
The invention aims to provide a method for detecting sulfur-containing amino acid in serum and a specific purifying material used for the detection method. The specific purification material is a metal affinity material taking silica gel as a matrix, and a specific solid phase extraction material is adopted to effectively enrich and purify sulfur-containing amino acid in serum and remove the interference of the matrix in the serum, so that simple, efficient and feasible pretreatment process and high-sensitivity detection of liquid chromatography-tandem mass spectrometry are realized.
In order to solve the technical problem, the invention provides a method for detecting sulfur-containing amino acid in serum, which comprises the following steps:
1) reduction of serum samples: adding a deuterated internal standard into a serum sample, carrying out vortex mixing, adding a reducing agent for reduction, and carrying out vortex mixing reaction for a period of time to obtain a serum sample solution for later use;
2) filling a solid phase extraction column: and (3) filling specification: 50-500mg of solid phase extraction material, and 1-6mL of column tube volume;
3) solid phase extraction and separation: activating, balancing, loading, leaching and eluting the extraction column, and finally collecting all elution fractions;
4) analysis of the samples: drying the elution fraction collected in the step 3) by using nitrogen, redissolving by using an initial mobile phase, performing component separation by using a high performance liquid based on a hydrophilic reverse phase chromatography principle, detecting by using a triple quadrupole tandem mass spectrometry to obtain a sample spectrogram, and detecting to obtain the positive ion of the sulfur-containing amino acid.
In a preferred embodiment of the present invention, the sulfur-containing amino acids are methionine, cysteine and homocysteine.
In a preferred embodiment of the invention, the serum sample is sampled in a volume of 5 μ L to 500 μ L; 0.1-10 muL of deuterated internal standard containing sulfur amino acid is added into each 1 muL of serum sample, and the concentration of the deuterated internal standard containing sulfur amino acid is 1 muM-50 muM.
In a preferred embodiment of the invention, the reducing agent is dithiothreitol, and the concentration of the reducing agent is in the range of 0.5g/L (w/v) to 10g/L (w/v).
In a preferred embodiment of the present invention, the solid phase extraction material is a specific purification material for sulfur-containing amino acid, the specific purification material uses silica gel as a matrix, and the bonding phases are ligand L and metal ion M, and the structure is as follows:
wherein M is Fe3+、Cu2+、Co2+、Ni2+L is one of histidine, glutamic acid, aspartic acid and lysine, and n is equal to 8-30; the particle size of the silica gel of the solid phase extraction material is 15-100 mu m, and the pore diameter is
Specific surface area 150m2/g-400m2(ii)/g; the specific purifying material is prepared by the conventional bonding reaction in the field, and the structure of the specific purifying material is verified by a general identification method.
In a preferred embodiment of the present invention, the structure of the specific purification material is such that M is Co2+L is lysine, n is 18, the particle size of silica gel of the solid phase extraction material is 50 μm, and the pore diameter is
Specific surface area 300m2/g。
In a preferred embodiment of the present invention, the solid phase extraction column first employs a volume ratio of organic solvent: 70 parts of water: 30 washing the first mixed solution; activating an organic solvent; adding organic solvent in volume ratio: water 5: 95 balancing the second mixed solution; after the serum sample solution is loaded; adding the second mixed solution for leaching; finally, using organic solvent with volume ratio: water: base 50: 49.9: and 0.1, eluting with the third mixed solution, and collecting all elution fractions.
In a more preferred embodiment of the present invention, the organic solvent and the alkali solution in the first mixed solution, the second mixed solution and the third mixed solution are corresponding, the organic solvent is one or more of methanol, acetonitrile, isopropanol, ethanol and acetone, the buffer salt solution is ammonia water or ethylenediamine solution, and the alkali concentration is 0.1-5%.
In a preferred embodiment of the invention, the high performance liquid chromatography conditions in step 4) are:
a chromatographic column: CORTECS 2.7 μm 2.1 μm × 100 mm; the flow rate of the mobile phase is 0.3 mL/min; gradient elution; the column temperature is 30-45 ℃; sample introduction amount: 5 uL;
mass spectrometry conditions:
ionization mode: ESI+(ii) a Spraying voltage: 4.0 KV; desolventizing gas temperature: at 450 ℃; atomizing: 50 psi; assisting atomization gas: 50 psi; air curtain air: 30, of a nitrogen-containing gas; the scanning mode is as follows: multiple Reaction Monitoring (MRM).
According to the detection method of the sulfur-containing amino acid in the serum and the enrichment material thereof, the detection limits of the methionine, cysteine and homocysteine containing the sulfur-containing amino acid are respectively 3 mug/mL, 6 mug/mL and 1.2 mug/mL;
the RSD of methionine, cysteine and homocysteine is 3.72%, 3.22% and 4.07% respectively. The invention can accurately determine the content of the sulfur-containing amino acid in the serum.
The invention has the beneficial effects that:
(1) the structure is novel. The invention firstly proposes that silica gel is taken as a substrate, a bonding phase is taken as a ligand and metal ions, the filler metal ion structure has specific affinity effect on sulfur-containing amino acid, and the filler metal ion structure can be specifically enriched and purified on the sulfur-containing amino acid;
(2) the application range is wide. The metal affinity material provided by the invention has specificity to a sulfhydryl group;
(3) simple operation and high flux. The pretreatment method provided by the invention is simple and reliable to operate, and is beneficial to realizing high flux of clinical serum samples;
(4) the qualitative and quantitative determination is accurate. The method adopts high performance liquid chromatography-tandem mass spectrometry for detection, the serum sample is corrected by adding an isotope internal standard, the standard curve is quantitative, the result accuracy is high and stable, and the method can be used for quantitative quantification of the methionine, cysteine and homocysteine which are contained in clinical blood samples.
Drawings
FIG. 1 is an MRM chromatogram of an ion channel of a methionine standard solution of example 1;
FIG. 2 is an MRM chromatogram of methionine in serum of example 1;
FIG. 3 is an MRM chromatogram of the ion channel of the cysteine standard solution of example 2;
FIG. 4 is an MRM chromatogram of cysteine in serum of example 2;
FIG. 5 is an MRM chromatogram of an ion channel of a homocysteine standard solution in example 3;
FIG. 6 is an MRM chromatogram of homocysteine in serum of example 3;
Detailed Description
The invention will now be further described with reference to the following examples, which are intended to be illustrative of the invention and are not to be construed as limiting the invention.
Example 1 detection of methionine containing sulfur amino acids in serum:
1. materials and reagents
A chromatographic column: (CORTECS, 2.7 μm 2.1. mu. m.times.100 mm) (Vout. Co., USA)
The methionine standard substance and the methionine isotope internal standard are purchased from Shanghai Hui Shi medicine science and technology Limited; dithiothreitol is available from Shanghai Brilliant Biotech, Inc.; methanol, acetonitrile, isopropanol, formic acid, ammonium formate (chromatographically pure), ammonia (chromatographically pure); ultrapure water: and preparing a Mili-Q ultrapure water machine.
2. Apparatus and device
A high performance liquid chromatography-tandem mass spectrometer equipped with an electrospray ionization (ESI) ionization source (4500MD, AB, USA), wherein the liquid chromatography separation mode is hydrophilic reversed phase chromatography separation, and the detector is triple quadrupole tandem mass spectrometry; one-ten-thousandth electronic analytical balance; SPE solid phase extraction device.
3. The method for detecting methionine containing sulfur amino acid by using high performance liquid chromatography-tandem mass spectrometry comprises the following steps:
1) preparing a standard working solution: accurately preparing a methionine standard substance stock solution (1mg/mL), diluting with pure water to obtain a methionine standard solution with a concentration of 300 mug/mL, and respectively diluting with pure water to six concentration levels of 150 mug/mL, 75 mug/mL, 15 mug/mL, 6 mug/mL and 3 mug/mL for later use;
2) preparing an internal standard solution: accurately preparing standard stock solution (1mg/mL) of methionine isotope internal standard, and diluting with pure water to obtain the standard stock solution with the concentration of 6 mug/mL for later use;
3) preparation of a standard solution: respectively taking 10 mu L of standard solutions with different concentrations of 300 mu g/mL, 150 mu g/mL, 75 mu g/mL, 15 mu g/mL, 6 mu g/mL and 3 mu g/mL in the step 1), adding 90 mu L of blank matrix solution without methionine, adding 30 mu L of mixed isotope internal standard (d3-Met 6 mu g/mL), uniformly mixing by vortex, adding 300 mu L of dithiothreitol (1.5g/L, w/v), mixing by vortex, and standing for 15min to obtain a serum sample solution for later use.
4) And (3) extracting a sample: adding 30 μ L mixed isotope internal standard (d3-Met 6 μ g/mL) into 100 μ L serum, vortex mixing, adding 300uL dithiothreitol (1.5g/L, w/v), vortex mixing, standing for 15min to obtain serum sample solution for use.
5) Filling a solid phase extraction column: filling 100mg of filler into a 1mL column tube (in the structure of the specific purification filler, M is Co2+L is lysine, n is 18, the particle size of silica gel of the solid phase extraction material is 50 μm, and the pore diameter is
Specific surface area 300m2Per gram), filling 200mg of filler into a 1mL column tube for later use;
6) solid phase extraction and separation: the solid phase extraction column is firstly washed by 1mL of 70% methanol water; activating by 1mL of methanol; then adding 5% methanol water for balancing; loading 400uL of each serum sample solution obtained in the step 3) and the step 4); adding 200uL of 5% methanol water for leaching; finally, using 400uL of methanol: water: ammonia water 50: 49.9: 0.1 elution, collecting all elution fractions, drying by nitrogen, redissolving the initial mobile phase, and waiting for sample loading analysis.
7) Analysis of the samples: loading the complex solution of the sample to be detected in the step 6) for detection, separating the component to be detected by using a hydrophilic reverse chromatogram, detecting by using a triple quadrupole tandem mass spectrometry to obtain a sample spectrogram, and detecting methionine positive ions: high performance liquid chromatography conditions and mass spectrometry conditions
i) High performance liquid chromatography conditions
A chromatographic column: CORTECS 2.7 μm 2.1 μm × 100 mm; mobile phase: acetonitrile (a), 0.2% formic acid (B), gradient elution is shown in table 1; column temperature 35 ℃, sample injection volume: 5 μ L.
TABLE 1 ultra high performance liquid chromatography gradient conditions
| Time/min | Flow rate/mL/min | A/％ | B/％ |
| 0 | 0.6 | 10 | 90 |
| 1.5 | 0.6 | 10 | 90 |
| 1.8 | 0.6 | 90 | 10 |
| 2.7 | 0.6 | 90 | 10 |
| 3 | 0.6 | 10 | 90 |
| 3.8 | 0.6 | 10 | 90 |
ii) Mass Spectrometry conditions
Ionization mode: ESI+(ii) a Spraying voltage: 4.0 KV; desolventizing gas temperature: at 450 ℃; atomizing GAS (GAS 1): 50 psi; assisting atomising GAS (GAS 2): 50 psi; air curtain air: 30 psi; the scanning mode is as follows: the qualitative and quantitative ion pairs, residence time collision energies, etc. of the Multiple Reaction Monitoring (MRM) methionine are shown in Table 2.
TABLE 2 Mass Spectrometry parameters of methionine
| Name of Compound | Parent ion | Daughter ions | Dwell time | DP(V) | CE(V) |
| Met | 150.100 | 104.000 | 200 | 45 | 15 |
| Met | 150.100 | 56.000 | 200 | 45 | 15 |
| D3-Met | 153.100 | 136.000 | 200 | 45 | 15 |
4. And (3) analyzing a quantitative calculation result: and (3) according to the ratio of the peak area of the methionine ion chromatographic peak in the sample chromatogram to the peak area of the methionine deuterated internal standard ion chromatographic peak as a response, making a linear regression equation (shown in table 3) with the corresponding concentration, and calculating the concentration of methionine in the serum sample by using an external standard method to obtain the concentration of methionine in the serum sample.
TABLE 3 analysis of quantitative calculation results
| Compound (I) | Linear regression equation | Detection limit | Coefficient of correlation r |
| Met | y＝0.278294x-0.05961 | 3μg/mL | 0.997909 |
The concentration of methionine containing amino acid for quantitative analysis in the same serum sample is detected to be 4.23 mug/mL, 4.49 mug/mL and 4.53 mug/mL, the RSD of the methionine in the serum sample is 3.72 percent, and the stability of the target compound of the sample is good.
Example 2 detection of the sulfur-containing amino acid cysteine in serum:
1. materials and reagents
The difference from example 1 is that the internal standard of cysteine and cysteine isotopes is purchased from Shanghai Hui Shi pharmaceutical science and technology, Inc.;
2. apparatus and device
The difference from example 1 is that a high performance liquid chromatography-tandem mass spectrometer was equipped with an Electrospray (ESI) ionization source (TQD, wawter, usa);
3. the method for detecting the sulfur-containing amino acid cysteine by using the high performance liquid chromatography-tandem mass spectrometry comprises the following steps:
1) preparing a standard working solution: accurately preparing a cysteine standard stock solution (1mg/mL), diluting with pure water to obtain a cysteine standard solution with a concentration of 600 mug/mL, and respectively diluting with pure water to six concentration levels of 300 mug/mL, 120 mug/mL, 30 mug/mL, 12 mug/mL and 6 mug/mL for later use;
2) preparing an internal standard solution: accurately preparing standard stock solution (1mg/mL) of cysteine isotope internal standard, and diluting with pure water to obtain the standard stock solution with the concentration of 12 mug/mL for later use;
3) preparation of a standard solution: respectively taking 10 mu L of standard solutions with different concentrations of 600 mu g/mL, 300 mu g/mL, 120 mu g/mL, 30 mu g/mL, 12 mu g/mL and 6 mu g/mL in the step 1), adding 90 mu L of blank matrix solution without cysteine, adding 30 mu L of mixed isotope internal standard (d2-Cys 12 mu g/mL), uniformly mixing by vortex, adding 200 mu L of dithiothreitol (2.0g/L, w/v), mixing by vortex, and standing for 15min to obtain a serum sample solution for later use;
4) and (3) extracting a sample: adding 30 μ L of mixed isotope internal standard (d2-Cys 12 μ g/mL) into 100 μ L of serum, mixing by vortex, adding 200 μ L of dithiothreitol (2.0g/L, w/v), mixing by vortex, and standing for 15min to obtain serum sample solution for use;
5) filling a solid phase extraction column: the filling specification and procedure were the same as in example 1;
6) solid phase extraction and separation: the difference from example 1 is that 300. mu.L of serum sample solution was loaded;
7) analysis of the samples: loading the sample to be detected in the step 6) into a complex solution for detection, and performing hydrophilic reverse chromatography on the component to be detected
Separating, detecting triple quadrupole tandem mass spectrometry to obtain a sample spectrogram, and detecting cysteine positive ions:
the difference from example 1 is that the capillary voltage under mass spectrometry conditions: 2.0 KV; taper hole voltage: 50V; desolventizing gas temperature:
450 ℃; desolventizing air flow rate: 800L/Hr; taper hole air flow rate: 150L/Hr;
the qualitative and quantitative ion pairs, residence time collision energies, etc. for cysteine are shown in table 4.
TABLE 4 Mass Spectrometry parameters for cysteine
| Name of Compound | Parent ion | Daughter ions | Dwell time | DP(V) | CE(V) |
| Cys | 122.000 | 76.000 | 200 | 35 | 19 |
| Cys | 122.000 | 86.900 | 200 | 35 | 18 |
| D3-Cys | 124.000 | 61.200 | 200 | 35 | 19 |
5. And (3) analyzing a quantitative calculation result: according to the peak area of a cysteine ion chromatographic peak and cysteine deuteration in a sample chromatogram
The peak area ratio of the standard ion chromatographic peak as the response and the corresponding concentration are used as the linear regression equation (as shown in Table 5)
Shown), calculating the concentration of cysteine in the serum sample by an external standard method to obtain the concentration of cysteine in the serum sample.
TABLE 5 quantitative calculation results best analysis
| Compound (I) | Linear regression equation | Detection limit | Coefficient of correlation r |
| Cys | y＝0.0135x+0.0334 | 6μg/mL | 0.9970 |
The concentration of the cysteine containing the sulfur amino acid is detected to be 38.57 mu g/mL, 37.18 mu g/mL and 37.04 mu g/mL in the same serum sample through quantitative analysis, the RSD of the cysteine in the serum sample is 3.22 percent, and the stability of the target compound in the sample is good.
Example 3 detection of sulfur-containing amino acid homocysteine in serum:
1. materials and reagents
The difference from example 1 is that homocysteine and homocysteine isotope are internally labeled from Shanghai Hui Shiyao
Science and technology limited;
2. apparatus and device
Same as in example 1
3. The method for detecting sulfur-containing amino acid homocysteine by high performance liquid chromatography-tandem mass spectrometry comprises the following steps:
1) preparing a standard working solution: accurately preparing a homocysteine standard stock solution (1mg/mL), diluting with pure water to obtain a homocysteine standard solution with the concentration of 120 mug/mL, and respectively diluting with pure water to six concentration levels of 60 mug/mL, 24 mug/mL, 6 mug/mL, 2.4 mug/mL and 1.2 mug/mL for later use;
2) preparing an internal standard solution: accurately preparing standard stock solution (1mg/mL) of homocysteine isotope internal standard, and diluting with pure water to obtain a concentration of 2 mug/mL for later use;
3) preparation of a standard solution: respectively taking 5 mu L of standard solutions with different concentrations of 120 mu g/mL, 60 mu g/mL, 24 mu g/mL, 6 mu g/mL, 2.4 mu g/mL and 1.2 mu g/mL in the step 1), adding 45 mu L of blank matrix solution without homocysteine, adding 50 mu L of mixed isotope internal standard (d2-Cys 2 mu g/mL), mixing uniformly by vortex, adding 200 mu L of dithiothreitol (2.0g/L, w/v) for mixing uniformly by vortex, and mixing uniformly for 15min by oscillation to obtain a serum sample solution for later use;
4) and (3) extracting a sample: adding 50 μ L of serum into 50 μ L of mixed isotope internal standard (d2-Cys 2 μ g/mL), mixing by vortex, adding 200 μ L of dithiothreitol (2.0g/L, w/v), mixing by vortex, and shaking for 15min to obtain serum sample solution;
5) filling a solid phase extraction column: the filling specification and procedure were the same as in example 1;
6) solid phase extraction and separation: the difference from example 1 is that 300. mu.L of serum sample solution was loaded;
7) analysis of the samples: loading the complex solution of the sample to be detected in the step 6) for detection, separating the components to be detected by using a hydrophilic reverse chromatogram, detecting by using a triple quadrupole tandem mass spectrometry to obtain a sample spectrogram, and detecting cysteine positive ions: ionization mode: ESI+(ii) a Spraying voltage: 4.5 KV; desolventizing gas temperature: 500 ℃; atomizing GAS (GAS 1): 50 psi; assisting atomising GAS (GAS 2): 50 psi; air curtain air: 35 psi; the scanning mode is as follows: multiple Reaction Monitoring (MRM)
Qualitative and quantitative ion pairs, residence time collision energies, etc. for homocysteine are shown in table 6.
TABLE 6 Mass Spectrometry parameters of homocysteine
| Name of Compound | Parent ion | Daughter ions | Dwell time | DP(V) | CE(V) |
| Hcy | 136.000 | 56.000 | 200 | 40 | 24 |
| Hcy | 136.000 | 90.000 | 200 | 40 | 16 |
| D4-Hcy | 140.100 | 94.100 | 200 | 40 | 16 |
6. And (3) analyzing a quantitative calculation result: and (3) according to the ratio of the peak area of the homocysteine ion chromatographic peak in the sample chromatogram to the peak area of the homocysteine deuterated internal standard ion chromatographic peak as a response, making a linear regression equation (shown in table 7) with the corresponding concentration, and calculating the concentration of homocysteine in the serum sample by using an external standard method to obtain the concentration of homocysteine in the serum sample.
TABLE 7 analysis of quantitative calculation results
| Compound (I) | Linear regression equation | Detection limit | Coefficient of correlation r |
| Cys | y＝0.119394x-0.03225 | 1.2μg/mL | 0.99806 |
The concentration of sulfur-containing amino acid homocysteine in the same serum sample is detected to be 25.13 mug/mL, 27.77 mug/mL and 31.89 mug/mL in quantitative analysis, the RSD of the methionine in the serum sample is 4.07 percent, and the stability of the target compound of the sample is good.
Example 4: the pretreatment processes of the solid phase extraction column PEP-2 and the X amide and the specific solid phase extraction column are compared:
1. materials and reagents
Homocysteine, cysteine and methionine were purchased from Shanghai Hui Shi pharmaceutical science and technology Limited;
2. apparatus and device
Same as example 1;
3. three pretreatment processes of a solid phase extraction column X amide, PEP and a specific solid phase extraction column comprise the following steps:
1) preparing and filling a specific solid-phase extraction column: acidifying and bonding silica gel to obtain specific filler (in the structure of the specific purifying filler, M is Co)2+L is lysine, n is 18, the particle size of silica gel of the solid phase extraction material is 50 μm, and the pore diameter is
Specific surface area 300m2Per gram), filling 200mg of filler into a 1mL column tube for later use;
2) preparing a standard working solution: respectively and accurately preparing 1mg/mL of homocysteine standard stock solution, cysteine standard stock solution and methionine standard stock solution, and diluting with pure water to obtain standard work solution with methionine concentration of 75 mug/mL, cysteine concentration of 120 mug/mL and homocysteine concentration of 6 mug/mL;
3) and (3) extracting a sample: taking 100 mu L of blank calf serum (without target compounds), adding 200uL of dithiothreitol (3.0g/L, w/v) for vortex mixing, and uniformly mixing for 20min by oscillation to obtain a serum sample solution for later use;
4) solid phase extraction column X amide: activating by using 1mL of methanol; balancing acetonitrile and pure water (60: 40); loading 300uL of the serum sample solution obtained in the step 3); adding 0.5mL of n-hexane for leaching and drying; eluting with acetonitrile and pure water (30: 70), collecting all the eluate fractions, and sampling for analysis;
5) the PEP treatment process of the solid phase extraction column comprises the following steps: activating by using 1mL of methanol; balancing 1mL of pure water; loading 300uL of the serum sample solution obtained in the step 3); adding 0.6mL of pure water for leaching; finally, eluting with 1mL of methanol, collecting all elution fractions, blowing the nitrogen for drying, redissolving the initial mobile phase, and carrying out sample loading analysis;
6) the treatment process of the specific solid phase extraction column comprises the following steps: the material in the specific solid phase extraction column was identical to that in example 1. Washing with 1mL of 70% methanol water; activating by 1mL of methanol; then adding 5% methanol water for balancing; loading 300uL of the serum sample solution obtained in the step 3); adding 300uL of 50% methanol water for leaching; finally, eluting with 1mL of methanol, water and ammonia water at the ratio of 50: 49.8: 0.2, collecting all elution fractions, drying with nitrogen, redissolving the initial mobile phase, and carrying out sample loading analysis;
7) and (3) sample analysis: loading the complex solution of the sample to be detected in the steps 4), 5) and 6) to detect, wherein the difference of the analysis method and the embodiment 1 is mass spectrum ion pair parameters;
| name of Compound | Parent ion | Daughter ions | Dwell time | DP(V) | CE(V) |
| Hcy | 136.000 | 56.000 | 80 | 40 | 24 |
| Hcy | 136.000 | 90.000 | 80 | 40 | 16 |
| D4-Hcy | 140.100 | 94.100 | 80 | 40 | 16 |
| Cys | 122.000 | 76.000 | 80 | 35 | 19 |
| Cys | 122.000 | 86.900 | 80 | 35 | 18 |
| D3-Cys | 124.000 | 61.200 | 80 | 35 | 19 |
| Met | 150.100 | 104.000 | 80 | 45 | 15 |
| Met | 150.100 | 56.000 | 80 | 45 | 15 |
| D3-Met | 153.100 | 136.000 | 80 | 45 | 15 |
8) And (3) analyzing a quantitative calculation result:
TABLE 8 solid phase extraction column treatment with three different packings-Cys results
TABLE 9 solid-phase extraction column treatment with three different fillers-Hcy results
TABLE 10 solid phase extraction column treatment of three different packing-Met results
TABLE 11 Signal to noise ratio (S/N) of three different target compounds measured after treatment with different fillers
The results show that the response and RSD% of the same serum sample treated by the specific solid extraction column are obviously higher than those of an X amide and PEP solid phase extraction column, and the signal-to-noise ratio of a detection spectrogram is also obviously different.
The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.
Claims (2)
1. A detection method of sulfur-containing amino acid in serum is characterized in that the sulfur-containing amino acid is methionine, cysteine and homocysteine; the method comprises the following steps:
1) reduction of serum samples: adding a deuterated internal standard into a serum sample, carrying out vortex mixing, adding a reducing agent for reduction, and carrying out vortex mixing reaction for a period of time to obtain a serum sample solution for later use; wherein the volume of the serum sample is 5-500 μ L; adding 0.1-10 muL of deuterated internal standard containing sulfur-containing amino acid into each 1 muL of serum sample, wherein the concentration of the deuterated internal standard containing sulfur-containing amino acid is 1-50 muM; the reducing agent is dithiothreitol, and the concentration of the reducing agent is 0.5g/L-10 g/L;
2) filling a solid phase extraction column: and (3) filling specification: 50-500mg of solid phase extraction material, 1-6mL of column tube volume, wherein the solid phase extraction material is a specific purification material for sulfur-containing amino acid, the specific purification material takes silica gel as a matrix, and a bonding phase is a ligand L and a metal ion M, and the structure is as follows:
m is Co2+, L is lysine, n is equal to 18, the particle size of the solid phase extraction material silica gel is 50 mu M, the pore diameter is 80A, and the specific surface area is 300M 2/g;
3) solid phase extraction and separation: washing the solid phase extraction column with 70% methanol water; activating by 1mL of methanol; then adding 5% methanol water for balancing; loading the serum sample solution; adding 5% methanol water for leaching; finally, using methanol: water: ammonia = 50: 49.9: 0.1 elution, collecting all elution fractions;
4) analysis of the samples: drying the elution fraction collected in the step 3) by using nitrogen, redissolving by using an initial mobile phase, performing component separation by using ultra-high performance liquid chromatography based on a hydrophilic interaction chromatography principle, detecting by using triple quadrupole tandem mass spectrometry to obtain a sample spectrogram, and detecting to obtain a positive ion containing sulfur-containing amino acid, wherein the conditions of the ultra-high performance liquid chromatography and the mass spectrometry are as follows:
i) ultra-high performance liquid chromatography conditions:
a chromatographic column: CORTECS 2.7 μm, 2.1mm × 100 mm; mobile phase: acetonitrile A and 0.2% formic acid water B, and gradient elution is shown in the following table; column temperature 35 ℃, sample injection volume: 5 muL;
ultra high performance liquid chromatography gradient conditions
ii) mass spectrometry conditions:
for methionine: ionization mode: ESI +; spraying voltage: 4.0 KV; desolventizing gas temperature: at 450 ℃; atomizing GAS GAS 1: 50 psi; assisting atomizing GAS 2: 50 psi; air curtain air: 30 psi; the scanning mode is as follows: monitoring multiple reactions;
for cysteine: capillary voltage: 2.0 KV; taper hole voltage: 50V; desolventizing gas temperature: 450 ℃; desolventizing air flow rate: 800L/Hr; taper hole air flow rate: 150L/Hr;
for homocysteine: ionization mode: ESI +; spraying voltage: 4.5 KV; desolventizing gas temperature: 500 ℃; atomizing GAS GAS 1: 50 psi; assisting atomizing GAS 2: 50 psi; air curtain air: 35 psi; the scanning mode is as follows: and (5) monitoring multiple reactions.
2. The detection method according to claim 1, wherein the detection limit of methionine, cysteine and homocysteine is 1.2 μ g/mL to 6 μ g/mL.
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