CN114935609A - High-throughput target detection method for biological sample polyamine and related metabolites - Google Patents
High-throughput target detection method for biological sample polyamine and related metabolites Download PDFInfo
- Publication number
- CN114935609A CN114935609A CN202110671231.5A CN202110671231A CN114935609A CN 114935609 A CN114935609 A CN 114935609A CN 202110671231 A CN202110671231 A CN 202110671231A CN 114935609 A CN114935609 A CN 114935609A
- Authority
- CN
- China
- Prior art keywords
- related metabolites
- polyamines
- biological sample
- solution
- target detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to the technical field of biochemical detection, in particular to a high-flux target detection method for polyamines and related metabolites of a biological sample and application thereof. The method comprises the following specific steps: s1, extracting a biological sample to obtain an extracting solution; s2, performing derivatization treatment on the extracting solution to obtain a solution to be detected; and S3, determining the polyamine and the related metabolites in the solution to be determined by adopting a high performance liquid chromatography-mass spectrometer. The method can efficiently and accurately detect the levels of polyamines and related metabolites in the biological sample, and the detection result is accurate and reliable; the correlation coefficient between the chromatographic peak and the target compound is high, and the requirement of targeted metabonomics analysis can be met; the instrument has high response degree, can measure a low-content sample, has simple and convenient test steps, can complete the test in only 12min, and has high detection efficiency; the invention provides a new thought for the research of the fields of human metabolism and cancer prevention and treatment, and has important significance for the progress of life science.
Description
Technical Field
The invention relates to the technical field of biochemical detection, in particular to a high-flux target detection method for polyamines and related metabolites of a biological sample and application thereof.
Background
Polyamine is a compound molecule containing at least 2 amino groups, widely exists in biological cells, changes along with the proliferation and differentiation of the cells, and is an important factor for regulating the physiological activities and pathological processes of organisms; the research on the physiological polyamine level of organisms is an effective way for researching the DNA expression and the metabolic activity of human bodies. In recent years, the research finds that polyamine metabolism plays an important role in the formation mechanism of tumors, and the gene expression of ornithine decarboxylase and S-adenosylmethionine decarboxylase which are key enzymes for biosynthesis of polyamine is proved in vitro experimental research for treating tumors, so that a new idea is provided for tumor research. The study of polyamine metabolism in living bodies (particularly in the human body) is a hot topic in the field of life science.
In order to effectively acquire polyamine metabolism information in a living body, a method for measuring and analyzing polyamine is rapidly developed along with the progress of life science. The existing analysis methods of polyamines and related metabolites mainly comprise biological methods and chemical methods; biological methods are further classified into enzymatic assay, which performs quantification of an analyte by a specific enzyme catalyzing a substrate, and immunological assay, which combines polyamines with macromolecular proteins for radioimmunoassay. The chemical method is divided into a non-derivation method and a derivation method, wherein the non-derivation method comprises a thin layer chromatography and paper electrophoresis method, and the polyamine structure characteristic is not obvious enough, so that the sensitivity of the non-derivation method is low, and the derivation method is the mainstream method for the existing polyamine determination and analysis. The existing derivation method is divided into pre-column derivation and post-column derivation, the pre-column derivation is obtained by reacting a derivation reagent with a target substance to be detected before separation to obtain an ultraviolet or fluorescent derivative, for example, Chinese patent publication No. CN103575826 discloses a high performance liquid chromatography detection method for polyamine content in goose tissue, benzoyl chloride and sodium hydroxide are mixed, water bath derivation is carried out on goose tissue homogenate, then the derivation liquid is subjected to solid phase extraction through a C18 small column, and liquid chromatography detection is carried out after extraction; chinese published patent CN105424823 discloses a method for detecting polyamines in tobacco roots, stems and leaves, which adopts 3, 5-dinitrobenzoyl chloride to perform derivatization on tobacco samples, and the tobacco samples are detected by a liquid chromatogram-photodiode array detector. Derivatization operation in the prior art is more complicated, and the application of the derivatization operation in polyamine analysis is limited; the sensitivity of post-column derivatization is high, but a derivatization reagent access device and a reactor need to be introduced into the instrument, so that the requirement on the instrument is high. The mass spectrum is used as an analysis method with high sensitivity and strong specificity, and the combination of the mass spectrum and the liquid chromatogram is an effective means for improving the detection and analysis efficiency and the data quality. Under the background, the detection method of polyamines and metabolites in biological samples, which is accurate and reliable in measurement result, convenient for experimental operation, high in instrument responsiveness and strong in universality, is explored by applying the liquid chromatography-mass spectrometry to the biological samples, and becomes a problem to be solved in the field.
Disclosure of Invention
The invention provides a high-flux target detection method for polyamines and related metabolites in biological samples, overcomes the defects of poor precision of detection results of polyamines and related metabolites and complicated test method in the prior art, and realizes the high-flux target detection method for polyamines and related metabolites which has accurate and reliable measurement results, is convenient for experimental operation, has high instrument responsiveness and is suitable for wide biological samples.
The invention provides a method for detecting a high-flux target of polyamine and related metabolites of a biological sample, which comprises the following specific steps:
s1, extracting a biological sample to obtain an extracting solution;
s2, performing derivatization treatment on the extracting solution to obtain a solution to be detected;
and S3, determining the polyamine and the related metabolites in the solution to be determined by adopting a high performance liquid chromatography-mass spectrometer.
In a preferred embodiment, the polyamines and related metabolites include putrescine, cadaverine, herring spermine, spermidine, tyramine, histamine, phenethylamine, tryptamine, arginine, ornithine, S-adenosylmethionine, alanine, glutamic acid, glycine, tryptophan, threonine, methionine, valine, lysine, leucine, isoleucine and phenylalanine in combination with one or more of the foregoing.
In a preferred embodiment, when the biological sample is in a solid state, the step S1 is specifically: adding the biological sample into a centrifuge tube, adding the first extracting solution, uniformly mixing, grinding, performing ultrasonic treatment in an ice-water bath, centrifuging, and collecting supernatant to obtain the extracting solution.
In a preferred embodiment, when the biological sample is in a liquid state, the step S1 is specifically: and unfreezing the biological sample in an ice-water bath, transferring the biological sample into a centrifuge tube, adding the second extracting solution, uniformly mixing, performing ultrasonic treatment in the ice-water bath, centrifuging, and collecting supernatant to obtain the extracting solution.
In a preferred embodiment, the step S2 is specifically: and (3) taking the supernatant fluid into a centrifuge tube, adding a derivatization reagent, uniformly swirling, reacting for 0.5-1.5h at 35-45 ℃, adding 0.0.5-0.2% VOL organic acid aqueous solution, uniformly mixing, and centrifuging for 5-20min at 2-10 ℃, 11500-14000rpm to obtain the liquid to be detected.
In a preferred embodiment, the derivatizing agent comprises a combination of one or more of o-phthalaldehyde, dansyl chloride, 9-fluorenylmethylchloroformate, tosyl chloride, benzoyl chloride.
In a preferred embodiment, the hplc-ms is equipped with a chromatography column; the specification of the chromatographic column is 100 multiplied by 2.1mm and 1.8 mu m; the temperature of the chromatographic column is 30-40 ℃.
In a preferred embodiment, the sample introduction temperature of the high performance liquid chromatography-mass spectrometer is 2-10 ℃, and the sample introduction amount is 1-5 μ L.
In a preferred embodiment, the operating parameters of the mass spectrum in the hplc-ms are: capillary voltage: +4000/-3500V, nozzle voltage: +500/-500V, atomizing gas temperature: 200-350 ℃, atomizing airflow rate: 4-6.5L/min, sheath gas flow temperature: 200-350 ℃, the sheath flow air flow rate is 8-13L/min, and the pressure of the sprayer is 40-50 psi.
The invention provides an application of a high-flux target detection method of biological sample polyamine and related metabolites, and the detection method is applied to the research in the fields of human metabolism and cancer prevention and treatment.
Has the advantages that:
the high-throughput target detection method for the polyamines and the related metabolites in the biological sample has the following advantages:
(1) the invention can efficiently and accurately detect the levels of polyamine and related metabolites of biological samples (especially human liver and plasma), the detection result is accurate and reliable, and the dispersion of parallel samples is small;
(2) the method has the advantages that the feasibility of testing various target products is strong, each chromatographic peak can be effectively separated, the peak shape is good, the correlation coefficient between the chromatographic peak and the target compound is high, and the requirement of targeted metabonomics analysis can be met;
(3) the instrument has high response degree, can measure a low-content sample, has simple and convenient test steps, can finish the test in only 12min, has high detection efficiency, and is easy to popularize in the field of analysis and detection;
(4) the invention provides a new thought for the research of the fields of human metabolism and cancer prevention and treatment, and has important significance for the progress of life science.
Detailed Description
The disclosure may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the examples included therein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.
The term "prepared from …" as used herein is synonymous with "comprising". As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains" or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.
The conjunction "consisting of …" excludes any non-specified element, step, or component. If used in a claim, the phrase is intended to claim as closed, meaning that it does not contain materials other than those described, except for the conventional impurities associated therewith. When the phrase "consisting of …" appears in a clause of the claim body and not immediately after the subject matter, it defines only the elements described in the clause; other elements are not excluded from the claims as a whole.
When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.
The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. "optional" or "any" means that the subsequently described event or events may or may not occur, and that the description includes instances where the event occurs and instances where it does not.
Approximating language, as used herein in the specification and claims, is intended to modify a quantity, such that the invention is not limited to the specific quantity, but includes equivalent parts that are acceptable for use in a generic sense without departing from the spirit and scope of the invention. Accordingly, the use of "about" to modify a numerical value means that the invention is not limited to the precise value. In some instances, the approximating language may correspond to the precision of an instrument for measuring the value. In the present description and claims, range limitations may be combined and/or interchanged, including all sub-ranges contained therein if not otherwise stated.
In addition, the indefinite articles "a" and "an" preceding an element or component of the invention are not intended to limit the number requirement (i.e., the number of occurrences) of the element or component. Thus, "a" or "an" should be read to include one or at least one, and the singular form of an element or component also includes the plural unless the stated number clearly indicates that the singular form is intended.
In order to solve the above problems, the first aspect of the present invention provides a method for high-throughput target detection of polyamines and related metabolites in biological samples, comprising the following steps:
s1, extracting a biological sample to obtain an extracting solution;
s2, performing derivatization treatment on the extracting solution to obtain a solution to be detected;
and S3, determining the polyamine and the related metabolites in the solution to be determined by adopting a high performance liquid chromatography-mass spectrometer.
In some preferred embodiments, the polyamines and related metabolites include one or a combination of putrescine, cadaverine, herring spermine, spermidine, tyramine, histamine, phenylethylamine, tryptamine, arginine, ornithine, S-adenosylmethionine, alanine, glutamic acid, glycine, tryptophan, threonine, methionine, valine, lysine, leucine, isoleucine, and phenylalanine.
Further preferably, the polyamines and related metabolites include herring spermine, arginine, putrescine, cadaverine, ornithine, spermidine, spermine and S-adenosylmethionine.
In some preferred embodiments, the biological sample is stored in a-80 ℃ refrigerator protected from light prior to analysis.
In some preferred embodiments, when the biological sample is in a solid state, the step S1 is specifically: adding the biological sample into a centrifuge tube, adding the first extracting solution, uniformly mixing, grinding, performing ultrasonic treatment in an ice-water bath, centrifuging, and collecting supernatant to obtain the extracting solution.
Further preferably, when the biological sample is in a solid state, the step S1 is specifically: adding a biological sample into a centrifuge tube, adding a first extracting solution, uniformly mixing by swirling for 10-30s, grinding for 2-5min under the condition of 30-45Hz, carrying out ultrasonic treatment for 2-10min under an ice-water bath, standing for 0.5-2h at-20 to-80 ℃, centrifuging for 5-15min at-2 to-5 ℃ and at 11000 and 13000rpm, and collecting supernatant, namely the extracting solution.
More preferably, the first extract is 70-100% VOL acetonitrile aqueous solution.
More preferably, the solid-to-liquid ratio of the biological sample to the first extraction liquid is (3-20): 1.
in some preferred embodiments, when the biological sample is in a liquid state, the step S1 is specifically: and unfreezing the biological sample in an ice-water bath, transferring the biological sample into a centrifuge tube, adding the second extracting solution, uniformly mixing, performing ultrasonic treatment in the ice-water bath, centrifuging, and collecting supernatant to obtain the extracting solution.
Further preferably, when the biological sample is in a liquid state, the step S1 specifically includes: unfreezing the biological sample in an ice water bath at 4 ℃, transferring the biological sample into a centrifuge tube, adding a second extracting solution, uniformly mixing by vortex for 10-30s, carrying out ultrasonic treatment for 5-20min in the ice water bath, standing for 0.5-2h at-20 to-80 ℃, centrifuging for 5-30min at-2 to-5 ℃ and under the conditions of 11500 and 14000rpm, and collecting supernatant, namely the extracting solution.
Still more preferably, the volume ratio of the biological sample to the second extraction liquid is 1: (3-5).
The second extracting solution is one or the combination of more of methanol, acetonitrile, ethanol and acetone.
Polyamines and related metabolites in a biological sample have the characteristics of low content, easy degradation and the like, and need to be analyzed quickly and accurately in the actual detection process; the long time of pretreatment and computer detection often cannot obtain stable and effective detection results, while the short time of pretreatment often cannot effectively extract target compounds in biological samples. Meanwhile, different biological samples have the characteristics of dynamism and complexity, and the conventional polyamine analysis method is difficult to effectively and comprehensively carry out effective detection on polyamine levels of various biological samples. According to experimental exploration, the biological sample is extracted by acetonitrile, and the leaching of polyamine and related metabolites in the biological sample can be promoted by specific vortex-ultrasonic-standing-centrifugal operation; the operation temperature of ultrasonic treatment, standing treatment and centrifugation is set, the physiological degradation process of polyamine and related metabolites in the biological sample in the extraction process can be obviously inhibited, the lower limit of the detection quantification of target compounds in the biological sample is low, and the biological sample can be effectively detected in solid and liquid states. However, this extraction method is not only to dissolve metabolites such as polyamine into the extract solution, but also to carry out other impurity molecules in the living body while destroying the cell tissue, and thus has a problem that the endogenous interference is large and the data result is not stable enough.
In some preferred embodiments, the step S2 is specifically: and (3) taking the supernatant into a centrifuge tube, adding a derivatization reagent, uniformly swirling, reacting for 0.5-1.5h at 35-45 ℃, adding 0.0.5-0.2% VOL organic acid aqueous solution, uniformly mixing, and centrifuging for 5-20min at 2-10 ℃ and 14000rpm of 11500-.
In some preferred embodiments, the derivatizing agent comprises a combination of one or more of o-phthalaldehyde, dansyl chloride, 9-fluorenylmethylchloroformate, tosyl chloride, benzoyl chloride.
Further preferably, the derivatizing agent is dansyl chloride.
In some preferred embodiments, the volume ratio of the extraction solution to the derivatizing agent is (1-3): 1.
in some preferred embodiments, the hplc-ms is equipped with a Waters acquisition UPLC HSS T3 chromatography column; the specification of the chromatographic column is 100 multiplied by 2.1mm, 1.8 mu m; the temperature of the chromatographic column is 30-40 ℃.
In some preferred embodiments, the HPLC-MS is injected at a temperature of 2-10 ℃ and in an amount of 1-5. mu.L.
In some preferred embodiments, the mobile phase of the hplc-ms is divided into a mobile phase a and a mobile phase B, and the specific flow conditions are as follows:
0-0.5 min: mobile phase A is 75% → 75%, and mobile phase B is 25% → 25%;
0.5-5.9 min: mobile phase A is 75% → 2%, and mobile phase B is 25% → 98%;
5.9-9.4 min: mobile phase A phase is 2% → 2%, and mobile phase B phase is 98% → 98%;
9.4-9.5 min: mobile phase A phase is 2% → 75%, and mobile phase B phase is 98% → 25%;
9.5-12 min: mobile phase a was 75% → 75% and mobile phase B was 25% → 25%.
The flow rate of the mobile phase is 200-800 mu L/min.
In some preferred embodiments, the mobile phase a is an aqueous solution of formic acid and its derivatives; the mobile phase B is one or the combination of more of formic acid, methanol, acetonitrile, ethanol and acetone.
Further preferably, the mobile phase A is an 8-13mM aqueous formate solution; mobile phase B was acetonitrile.
Even more preferably, the mobile phase A also contains 0.05-0.15% VOL of formic acid.
The mobile phase A is 10mM ammonium formate aqueous solution; and mobile phase a also contained 0.1% VOL of formic acid.
The method for separating and measuring polyamine substances in biological samples is various, wherein the thin-layer chromatography has low sensitivity and is difficult to realize automation, the pretreatment step of samples of the gas chromatography is complex, the detection efficiency is low, and the capillary electrophoresis detection has high sensitivity, but the instrument maintenance cost is high and the popularization is difficult; the high performance liquid chromatography is an effective means for analyzing and detecting polyamine substances due to high sensitivity and convenient method. The polyamine does not have ultraviolet and fluorescent structural characteristics, and the on-machine detection result cannot be directly carried out; the dansyl chloride is used as a strong fluorescent marker, and the detection sensitivity of the dansyl chloride is 2-3 orders of magnitude higher than that of an ultraviolet marker; however, the derivatization time of dansyl chloride is long, the number of byproducts after derivatization is large, and effective separation of various target substances to be tested is difficult to realize when a plurality of polyamine substances are tested. The invention further researches show that after the biological sample extracting solution is subjected to derivatization treatment by dansyl chloride and the derivatization product is further separated by a Waters acquisition UPLC HSS T3 chromatographic column, effective separation of polyamine pathway target substances (especially herring spermine, arginine, putrescine, cadaverine, ornithine, spermidine, spermine and S-adenosylmethionine) can be realized by the specific elution procedure, the reproducibility of the test result is good, and the data quality is obviously improved.
In some preferred embodiments, the operating parameters of the mass spectrum in the hplc-ms are: capillary voltage: +4000/-3500V, nozzle voltage: +500/-500V, atomizing gas temperature: 200-350 ℃, atomizing airflow rate: 4-6.5L/min, sheath flow gas temperature: 200-350 ℃, the sheath flow air flow rate is 8-13L/min, and the pressure of the sprayer is 40-50 psi.
Further preferably, the working parameters of the mass spectrum plate are as follows: capillary voltage: +4000/-3500V, nozzle voltage: +500/-500V, atomizing gas temperature: 300 ℃, atomizing gas flow rate: 5L/min, sheath flow gas temperature: 250 ℃, sheath flow rate: 11L/min, nebulizer pressure: 45 psi.
Still further preferably, the atomizing gas and the sheath flow gas are both nitrogen.
The liquid phase in the high performance liquid chromatography-mass spectrometer adopted by the invention is Agilent 1290Infinity II series UHPLC System; the Mass spectrum is an Agilent6460 Triple Quadrupole Mass Spectrometer Triple tandem Quadrupole Mass Spectrometer; the mass spectrometer was configured with an AJS-ESI ion source. And (3) ionizing the substances to be detected through specific working parameter setting, and measuring the peak intensity of each ion spectrum to realize the analysis of the target substances. A large number of experiments are researched to find that when the temperature of atomizing gas is 300 ℃ and the flow rate of atomizing gas is 5L/min, target metabolite molecules in biological samples (particularly liver and plasma) are more easily broken to form fragment ions, so that substrate noise in the sample measuring process is avoided, the dispersion of polyamine target detection results of specific tissues of a human body is reduced, the accurate and reliable degree of the test results is improved, and the method has a positive promoting effect on the research of the fields of human metabolism and cancer prevention and treatment.
In some preferred embodiments, qualitative and quantitative analysis is performed on the biological sample by using an external standard method, that is, a standard solution with gradient concentration is prepared, the standard solution is subjected to derivatization treatment and is subjected to computer test, the relationship between the peak area and the concentration of polyamine and related metabolites in the standard solution is obtained, and a standard curve is drawn; and substituting the sample measurement result of the biological sample into the standard curve to obtain the high-flux target detection result of the polyamine and the related metabolites of the biological sample.
Further preferably, the preparation method of the standard solution comprises the following steps: accurately weighing corresponding amount of standard substances (herring spermine, arginine, putrescine, cadaverine, ornithine, spermidine, spermine and S-adenosylmethionine) in 8 10mL volumetric flasks, and making into 10mM single-standard stock solutions by using acetonitrile as solvent to perform constant volume. Respectively taking corresponding amounts of single-standard stock solutions in the same 10mL volumetric flask to prepare mixed-standard mother solution with a certain concentration; sequentially diluting the mixed standard mother solution to obtain a series of gradient standard solutions.
In some preferred embodiments, the concentration gradient of each standard in the gradient standard solution is 0, 10. mu.M, 20. mu.M, 50. mu.M, 100. mu.M.
Before high performance liquid chromatography-mass spectrometry analysis, a mixed standard solution containing target compounds is introduced into a mass spectrum, an ion pair with the best signal response condition is selected for each target compound, MRM parameters of the ion pair are screened and adjusted, the ion pair with the highest response strength is selected for quantitative analysis of polyamine and related metabolites, and other ion pairs are used for qualitative analysis of the polyamine and the related metabolites.
The invention provides an application of a high-flux target detection method of biological sample polyamine and related metabolites, and the detection method is applied to the research in the fields of human metabolism and cancer prevention and treatment.
Examples
In order to better understand the above technical solutions, the following detailed descriptions will be provided with reference to specific embodiments. It should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and that the insubstantial modifications and adaptations of the present invention by those skilled in the art based on the above disclosure are still within the scope of the present invention. In addition, the raw materials are commercially available and the extraction methods of the extract are all conventional extraction methods, if not otherwise specified.
Example 1.
The embodiment provides a high-throughput target detection method for polyamines and related metabolites in biological samples, which comprises the following specific steps:
s1, extracting a biological sample to obtain an extracting solution;
s2, performing derivatization treatment on the extracting solution to obtain a solution to be detected;
and S3, determining the polyamine and the related metabolites in the solution to be determined by adopting a high performance liquid chromatography-mass spectrometer.
The polyamines and related metabolites include herring spermine, arginine, putrescine, cadaverine, ornithine, spermidine, spermine and S-adenosylmethionine.
The biological sample is liver tissue and is from Shanghai Aqu Biotech limited.
The step of S1 is specifically as follows: adding a biological sample into a centrifuge tube, adding the first extracting solution, uniformly mixing by vortex for 15s, grinding for 4min under the condition of 40Hz, carrying out ultrasonic treatment for 5min in an ice-water bath, and repeating the grinding and ultrasonic treatment steps for 3 times; standing at-40 deg.C for 1h, centrifuging at-4 deg.C and 12000rpm for 15min, and collecting supernatant to obtain extractive solution.
The solid-liquid ratio of the biological sample to the first extracting solution is 10: 1.
the first extracting solution is 80% VOL acetonitrile water solution.
The step of S2 is specifically: 100 μ L of the supernatant was placed in a centrifuge tube and 50 μ L of LiNaHCO was added 3 Mixing saturated aqueous solution and 50 μ L derivatization reagent, vortexing, and keeping away from light at 40 deg.CReacting for 1h, taking out, adding 50 mu L of 0.1% VOL formic acid aqueous solution, mixing uniformly, centrifuging for 10min at 4 ℃ and 12000rpm, and taking 80 mu L of supernatant to a sample bottle to obtain the liquid to be detected.
The derivatization reagent is dansyl chloride aqueous solution with the concentration of 20 mg/mL.
The HPLC-MS is equipped with a Waters ACQUITY UPLC HSS T3 chromatographic column; the specification of the chromatographic column is 100 multiplied by 2.1mm and 1.8 mu m; the temperature of the column was 35 ℃.
The sample introduction temperature of the high performance liquid chromatography-mass spectrometer is 4 ℃, and the sample introduction amount is 2 mu L.
The mobile phase of the high performance liquid chromatography-mass spectrometer is divided into a mobile phase A and a mobile phase B, and the specific flow conditions are as follows:
0-0.5 min: mobile phase A is 75% → 75%, and mobile phase B is 25% → 25%;
0.5-5.9 min: mobile phase A is 75% → 2%, mobile phase B is 25% → 98%;
5.9-9.4 min: mobile phase A phase is 2% → 2%, and mobile phase B phase is 98% → 98%;
9.4-9.5 min: mobile phase A phase is 2% → 75%, and mobile phase B phase is 98% → 25%;
9.5-12 min: mobile phase a was 75% → 75% and mobile phase B was 25% → 25%.
The flow rate of the mobile phase was 400. mu.L/min.
The mobile phase A is 10mM ammonium formate aqueous solution; and mobile phase a also contained 0.1% VOL of formic acid. The mobile phase B is acetonitrile.
The working parameters of the mass spectrum plate are as follows: capillary voltage: +4000/-3500V, nozzle voltage: +500/-500V, atomizing gas temperature: 300 ℃, atomizing gas flow rate: 5L/min, sheath flow gas temperature: 250 ℃, sheath flow rate: 11L/min, nebulizer pressure: 45 psi.
The atomizing gas and the sheath flow gas are both nitrogen.
Performing qualitative and quantitative analysis on a biological sample by adopting an external standard method, namely preparing a standard solution with gradient concentration, performing derivatization treatment on the standard solution, performing on-machine test (the derivatization treatment and the on-machine test operation of the standard solution are consistent with those of the biological sample), obtaining the relation between the peak areas and the concentrations of polyamine and related metabolites in the standard solution, and drawing a standard curve; and substituting the sample measurement result of the biological sample into the standard curve to obtain the qualitative and quantitative results of the polyamine and the related metabolites of the biological sample.
The preparation method of the standard solution comprises the following steps: accurately weighing corresponding amount of standard substances (herring spermine, arginine, putrescine, cadaverine, ornithine, spermidine, spermine and S-adenosylmethionine) in 8 10mL volumetric flasks, and making into 10mM single-standard stock solutions by using acetonitrile as solvent to perform constant volume. Respectively putting corresponding amounts of single-label stock solutions into the same 10mL volumetric flask to prepare mixed-label mother solutions with certain concentrations; and sequentially diluting the mixed standard mother liquor to obtain a series of gradient standard solutions.
The concentration gradient of each standard substance in the gradient standard solution is 0, 10 mu M, 20 mu M, 50 mu M and 100 mu M.
Example 2.
The embodiment provides a high-throughput target detection method for polyamines and related metabolites in biological samples, which comprises the following specific steps:
s1, extracting a biological sample to obtain an extracting solution;
s2, performing derivatization treatment on the extracting solution to obtain a solution to be detected;
and S3, determining the polyamine and the related metabolites in the solution to be determined by adopting a high performance liquid chromatography-mass spectrometer.
The polyamines and related metabolites include herring spermine, arginine, putrescine, cadaverine, ornithine, spermidine, spermine and S-adenosylmethionine.
The biological sample is human plasma and is from Shanghai Aqu Biotech limited.
The step of S1 is specifically: thawing the biological sample in ice-water bath at 4 deg.C, transferring to a centrifuge tube, adding the second extractive solution, vortexing for 30s, mixing, performing ultrasonic treatment in ice-water bath for 15min, standing at-40 deg.C for 1h, centrifuging at-4 deg.C and 13000rpm for 15min, and collecting supernatant to obtain the extractive solution.
The volume ratio of the biological sample to the second extracting solution is 1: 4.
the second extracting solution is acetonitrile.
The step of S2 is specifically: 100 μ L of the supernatant was placed in a centrifuge tube and 50 μ L of NaHCO was added 3 And uniformly swirling the saturated aqueous solution and 50 mu L of derivatization reagent, carrying out a light-shielding reaction for 1h at 40 ℃, taking out, adding 50 mu L of 0.1% VOL formic acid aqueous solution, uniformly mixing, centrifuging for 10min at 4 ℃ and 12000rpm, and taking 80 mu L of supernatant into a sample bottle to obtain the liquid to be detected.
The derivatization reagent is dansyl chloride aqueous solution with the concentration of 20 mg/mL.
The HPLC-MS is equipped with a Waters ACQUITY UPLC HSS T3 chromatographic column; the specification of the chromatographic column is 100 multiplied by 2.1mm, 1.8 mu m; the temperature of the column was 35 ℃.
The sample introduction temperature of the high performance liquid chromatography-mass spectrometer is 4 ℃, and the sample introduction amount is 2 mu L.
The mobile phase of the high performance liquid chromatography-mass spectrometer is divided into a mobile phase A and a mobile phase B, and the specific flow conditions are as follows:
0-0.5 min: mobile phase A is 75% → 75%, and mobile phase B is 25% → 25%;
0.5-5.9 min: mobile phase A is 75% → 2%, mobile phase B is 25% → 98%;
5.9-9.4 min: mobile phase A is 2% → 2%, and mobile phase B is 98% → 98%;
9.4-9.5 min: mobile phase A is 2% → 75%, mobile phase B is 98% → 25%;
9.5-12 min: mobile phase a was 75% → 75% and mobile phase B was 25% → 25%.
The flow rate of the mobile phase was 400. mu.L/min.
The mobile phase A is 10mM ammonium formate aqueous solution; and mobile phase a also contained 0.1% VOL of formic acid. The mobile phase B is acetonitrile.
The working parameters of the mass spectrum plate are as follows: capillary voltage: +4000/-3500V, nozzle voltage: +500/-500V, atomizing gas temperature: 300 ℃, atomizing gas flow rate: 5L/min, sheath flow gas temperature: 250 ℃, sheath flow rate: 11L/min, nebulizer pressure: 45 psi.
The atomization gas and the sheath flow gas are both nitrogen.
Performing qualitative and quantitative analysis on a biological sample by adopting an external standard method, namely preparing a standard solution with gradient concentration, performing derivatization treatment on the standard solution, performing on-machine test (the derivatization treatment and the on-machine test operation of the standard solution are consistent with those of the biological sample), obtaining the relation between the peak areas and the concentrations of polyamine and related metabolites in the standard solution, and drawing a standard curve; and substituting the sample measurement result of the biological sample into the standard curve to obtain the qualitative and quantitative results of the polyamine and the related metabolites of the biological sample.
The preparation method of the standard solution comprises the following steps: accurately weighing corresponding amount of standard substances (herring spermine, arginine, putrescine, cadaverine, ornithine, spermidine, spermine and S-adenosylmethionine) in 8 10mL volumetric flasks, and making into 10mM single-standard stock solutions by using acetonitrile as solvent to perform constant volume. Respectively taking corresponding amounts of single-standard stock solutions in the same 10mL volumetric flask to prepare mixed-standard mother solution with a certain concentration; sequentially diluting the mixed standard mother solution to obtain a series of gradient standard solutions.
The concentration gradient of each standard substance in the gradient standard solution is 0, 10 mu M, 20 mu M, 50 mu M and 100 mu M.
Example 3.
The embodiment provides a method for detecting a high-throughput target of polyamine and related metabolites of a biological sample, and the specific implementation mode is the same as that of embodiment 1; the difference lies in that: the solid-liquid ratio of the biological sample to the first extracting solution is 15: 1.
example 4.
The embodiment provides a method for detecting polyamines and related metabolites in biological samples with high flux targets, which is the same as the embodiment 2; the difference lies in that: the volume ratio of the biological sample to the second extracting solution is 1: 7.
example 5.
The embodiment provides a method for detecting polyamines and related metabolites in biological samples with high flux targets, which is the same as the embodiment 1; the difference lies in that: the first extract is 90% VOL acetonitrile aqueous solution.
Example 6.
The embodiment provides a method for detecting polyamines and related metabolites in biological samples with high flux targets, which is the same as the embodiment 2; the difference lies in that: in the step S1, the centrifugation speed is 10000rpm, and the centrifugation time is 20 min.
Example 7.
The embodiment provides a method for detecting polyamines and related metabolites in biological samples with high flux targets, which is the same as the embodiment 1; the difference lies in that: the derivatization reagent is 30mg/mL dansyl chloride aqueous solution.
Example 8.
The embodiment provides a method for detecting polyamines and related metabolites in biological samples with high flux targets, which is the same as the embodiment 1; the difference lies in that: the amount of derivatizing agent added was 80. mu.L.
Example 9.
The embodiment provides a method for detecting polyamines and related metabolites in biological samples with high flux targets, which is the same as the embodiment 1; the difference lies in that: the temperature of the column was 40 ℃.
Example 10.
The embodiment provides a method for detecting polyamines and related metabolites in biological samples with high flux targets, which is the same as the embodiment 1; the difference lies in that: the flow rate of the mobile phase was 500. mu.L/min.
Performance test method
Coefficient of linear correlation R 2 ：
Based on the results of the gradient standard solutions measured in examples 1 to 10, a standard curve was plotted, and the correlation coefficient R of the concentration of herring spermine, arginine, putrescine, cadaverine, ornithine, spermidine, spermine and S-adenosylmethionine as the target test substance was calculated 2 Calculating the R of various herring spermine, arginine, putrescine, cadaverine, ornithine, spermidine, spermine and S-adenosylmethionine 2 Average value of (a).
And (3) recovery rate E:
using 50 μ M standard solution (the sum of the concentrations of herring spermine, arginine, putrescine, cadaverine, ornithine, spermidine, spermine and S-adenosylmethionine is 400 μ M) as sample to be tested, and detecting according to the analysis method of examples 1-10 to obtain test result C of polyamine and related metabolites in target compound total (C total Equal to the sum of the concentrations of herring spermine, arginine, putrescine, cadaverine, ornithine, spermidine, spermine and S-adenosylmethionine), the recovery E ═ 400-C x |/400*100％。
Lower limit of quantitation L:
milli Q water was used as a test sample, pretreated and tested on a machine according to the high-throughput target detection method of examples 1 to 10, 20-fold parallel determinations were made in each example analysis method, and the relative standard deviation RSD of the sum of the concentrations of the target test substances, i.e., herring spermine, arginine, putrescine, cadaverine, ornithine, spermidine, spermine and S-adenosylmethionine, was calculated m Calculating the lower limit of quantitation L-3 RSD m 。
Sample quality control RSD:
each group of examples was assayed 10 times in parallel, and the sum of the concentrations of the target test substances herring spermine, arginine, putrescine, cadaverine, ornithine, spermidine, spermine and S-adenosylmethionine was calculated, and the relative standard deviation RSD of the parallel test results was calculated.
Performance test data
TABLE 1 Performance test results
| R 2 | E(％) | L(nM) | RSD(％) | |
| Example 1 | 0.999 | 96.4 | 0.61 | 4.4 |
| Example 2 | 0.999 | 97.6 | 1.22 | 2.6 |
| Example 3 | 0.994 | 95.3 | 1.35 | 8.3 |
| Example 4 | 0.992 | 92.7 | 2.68 | 6.5 |
| Example 5 | 0.993 | 93.1 | 1.62 | 9.1 |
| Example 6 | 0.993 | 96.5 | 1.85 | 5.3 |
| Example 7 | 0.990 | 92.7 | 2.53 | 12.4 |
| Example 8 | 0.988 | 93.6 | 3.12 | 14.1 |
| Example 9 | 0.991 | 93.1 | 2.45 | 10.6 |
| Example 10 | 0.982 | 92.3 | 5.27 | 15.2 |
Finally, it is pointed out that the foregoing examples are merely illustrative and serve to explain some of the features of the method according to the invention. The appended claims are intended to claim as broad a scope as is contemplated, and the examples presented herein are merely illustrative of selected implementations in accordance with all possible combinations of examples. Accordingly, it is applicants' intention that the appended claims are not to be limited by the choice of examples illustrating features of the invention. Also, where numerical ranges are used in the claims, subranges therein are included, and variations in these ranges are also to be construed as possible being covered by the appended claims.
Claims (10)
1. A method for detecting polyamines and related metabolites in biological samples through high-flux targets is characterized by comprising the following specific steps:
s1, extracting a biological sample to obtain an extracting solution;
s2, performing derivatization treatment on the extracting solution to obtain a solution to be detected;
and S3, determining the polyamine and the related metabolites in the solution to be determined by adopting a high performance liquid chromatography-mass spectrometer.
2. The method for high throughput target detection of polyamines and related metabolites in biological samples according to claim 1, wherein the polyamines and related metabolites include putrescine, cadaverine, herring spermine, spermidine, tyramine, histamine, phenylethylamine, tryptamine, arginine, ornithine, S-adenosylmethionine, alanine, glutamic acid, glycine, tryptophan, threonine, methionine, valine, lysine, leucine, isoleucine and phenylalanine in combination with one or more of them.
3. The method for high throughput target detection of polyamines and related metabolites in biological samples according to claim 1 or 2, wherein the step of S1 comprises the steps of: adding the biological sample into a centrifuge tube, adding the first extracting solution, uniformly mixing, grinding, performing ultrasonic treatment in an ice-water bath, centrifuging, and collecting supernatant to obtain the extracting solution.
4. The method for high throughput target detection of polyamines and related metabolites in biological samples according to any one of claims 1-3, wherein when the biological sample is in liquid state, the step of S1 comprises: and unfreezing the biological sample in an ice-water bath, transferring the biological sample into a centrifuge tube, adding the second extracting solution, uniformly mixing, performing ultrasonic treatment in the ice-water bath, centrifuging, and collecting supernatant to obtain the extracting solution.
5. The method for high throughput target detection of polyamines and related metabolites in biological samples according to any one of claims 1 to 4, wherein the step S2 comprises the following steps: and (3) taking the supernatant fluid into a centrifuge tube, adding a derivatization reagent, uniformly swirling, reacting for 0.5-1.5h at 35-45 ℃, adding 0.0.5-0.2% VOL organic acid aqueous solution, uniformly mixing, and centrifuging for 5-20min at 2-10 ℃, 11500-14000rpm to obtain the liquid to be detected.
6. The method for high throughput target detection of polyamines and related metabolites in biological samples according to claim 5, wherein the derivatizing agent comprises one or more of o-phthalaldehyde, dansyl chloride, 9-fluorenylmethylchloroformate, tosyl chloride, and benzoyl chloride.
7. The method for high-throughput target detection of polyamines and related metabolites in biological samples according to any one of claims 1-6, wherein the HPLC-MS is equipped with a chromatographic column; the specification of the chromatographic column is 100 multiplied by 2.1mm and 1.8 mu m; the temperature of the chromatographic column is 30-40 ℃.
8. The method for detecting the high-throughput target of polyamines and related metabolites in biological samples according to any one of claims 1 to 7, wherein the sample injection temperature of the HPLC-MS is 2 to 10 ℃ and the sample injection amount is 1 to 5 μ L.
9. The method for detecting the high-throughput target of polyamines and related metabolites in biological samples according to any one of claims 1 to 8, wherein the working parameters of mass spectrometry in the HPLC-MS are as follows: capillary voltage: +4000/-3500V, nozzle voltage: +500/-500V, atomizing gas temperature: 200-350 ℃, atomizing airflow rate: 4-6.5L/min, sheath gas flow temperature: the temperature is 200-350 ℃, the sheath flow airflow speed is 8-13L/min, and the pressure of the sprayer is 40-50 psi.
10. Use of the method for high-throughput target detection of polyamines and related metabolites in biological samples according to any one of claims 1 to 9, wherein the method for high-throughput target detection is applied to the research in the fields of human metabolism and cancer prevention and treatment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110671231.5A CN114935609A (en) | 2021-06-17 | 2021-06-17 | High-throughput target detection method for biological sample polyamine and related metabolites |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110671231.5A CN114935609A (en) | 2021-06-17 | 2021-06-17 | High-throughput target detection method for biological sample polyamine and related metabolites |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114935609A true CN114935609A (en) | 2022-08-23 |
Family
ID=82861704
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110671231.5A Pending CN114935609A (en) | 2021-06-17 | 2021-06-17 | High-throughput target detection method for biological sample polyamine and related metabolites |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114935609A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000063683A1 (en) * | 1999-04-20 | 2000-10-26 | Target Discovery, Inc. | Polypeptide fingerprinting methods, metabolic profiling, and bioinformatics database |
| CN103822998A (en) * | 2012-11-19 | 2014-05-28 | 中国科学院大连化学物理研究所 | Method for analyzing amine substances in dansyl chloride derived-plasma based on liquid chromatography mass spectrometry |
| CN107300594A (en) * | 2017-07-31 | 2017-10-27 | 中国中医科学院医学实验中心 | The method of amino acid in liquid chromatography mass combination directly detection biological tissue Uniform Sample |
| CN110887910A (en) * | 2019-12-12 | 2020-03-17 | 上海阿趣生物科技有限公司 | Polyamine and detection method of synthetic pathway substance thereof |
-
2021
- 2021-06-17 CN CN202110671231.5A patent/CN114935609A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000063683A1 (en) * | 1999-04-20 | 2000-10-26 | Target Discovery, Inc. | Polypeptide fingerprinting methods, metabolic profiling, and bioinformatics database |
| CN103822998A (en) * | 2012-11-19 | 2014-05-28 | 中国科学院大连化学物理研究所 | Method for analyzing amine substances in dansyl chloride derived-plasma based on liquid chromatography mass spectrometry |
| CN107300594A (en) * | 2017-07-31 | 2017-10-27 | 中国中医科学院医学实验中心 | The method of amino acid in liquid chromatography mass combination directly detection biological tissue Uniform Sample |
| CN110887910A (en) * | 2019-12-12 | 2020-03-17 | 上海阿趣生物科技有限公司 | Polyamine and detection method of synthetic pathway substance thereof |
Non-Patent Citations (6)
| Title |
|---|
| IRIS SAMARRA ET AL: "Gender-Related Differences on Polyamine Metabolome in Liquid Biopsies by a Simple and Sensitive Two-Step Liquid-Liquid Extraction and LC-MS/MS", BIOMOLECULES * |
| JAE-HYUNG MAH ET AL: "Biogenic amines in Jeotkals, Korean salted and fermented fish products", FOOD CHEMISTRY * |
| LEO HAWEL ET AL: "A streamlined method for the isolation and quantitation of nanomole levels of exported polyamines in cell culture media", ANALYTICAL BIOCHEMISTRY * |
| XIN XIONG ET AL: "Rapid and Simultaneous Quantification of Polyamines in Human Plasma by LC–MS/MS After Pre‑column Derivatization with N‑(9‑Fluorenylmethoxycarbonyloxy)succinimide", CHROMATOGRAPHIA * |
| XIULI SU ET AL: "Simultaneous determination of methionine cycle metabolites, urea cycle intermediates and polyamines in serum, urine and intestinal tissue by using UHPLC-MS/MS", TALANTA * |
| 丁卓平 等: "高效液相色谱法同时测定水产品中10种生物胺的研究", 分析测试学报 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111175394B (en) | Method for detecting plasma catecholamine and metabolite thereof by liquid chromatography-tandem mass spectrometry | |
| Jia et al. | Simultaneous determination of 23 amino acids and 7 biogenic amines in fermented food samples by liquid chromatography/quadrupole time-of-flight mass spectrometry | |
| D'Alessandro et al. | A robust high resolution reversed-phase HPLC strategy to investigate various metabolic species in different biological models | |
| Liu et al. | Determination of polyamines in human urine by precolumn derivatization with benzoyl chloride and high-performance liquid chromatography coupled with Q-time-of-flight mass spectrometry | |
| Maráková et al. | Simultaneous determination of twelve biogenic amines in human urine as potential biomarkers of inflammatory bowel diseases by capillary electrophoresis–tandem mass spectrometry | |
| Wang et al. | Solid phase microextraction combined with thermal-desorption electrospray ionization mass spectrometry for high-throughput pharmacokinetics assays | |
| CN111638293A (en) | LC-MS/MS detection method for simultaneously determining amino acids and nucleotides in animal tissues | |
| CN113588804A (en) | Kit for detecting concentration of 5-hydroxytryptamine and melatonin in serum | |
| CN106959345A (en) | A kind of full Isotopic Internal Standard mass spectrum quantitative approach of neurotransmitter metabolite | |
| US8497471B2 (en) | Mass spectrometry assay for thiopurine-S-methyl transferase activity and products generated thereby | |
| Li et al. | Simultaneous determination of 24 free amino acids in MGC803 cells by hydrophilic interaction liquid chromatography with tandem mass spectrometry | |
| CN114428138A (en) | Liquid chromatography tandem mass spectrometry detection method of catecholamine and metabolites thereof based on magnetic solid phase extraction | |
| CN113533597A (en) | Method for determining esomeprazole in human plasma | |
| CN117030878A (en) | Stable free amino acid underivatization detection method and application thereof | |
| Swann et al. | Determination of amino acids and amines in mammalian decomposition fluid by direct injection liquid chromatography-electrospray ionisation-tandem mass spectrometry | |
| CN114935609A (en) | High-throughput target detection method for biological sample polyamine and related metabolites | |
| CN110806458A (en) | Method for simultaneously detecting leucine, isoleucine and valine contents in blood | |
| Sanwald et al. | A combined targeted/untargeted LC-MS/MS-based screening approach for mammalian cell lines treated with ionic liquids: Toxicity correlates with metabolic profile | |
| KR20090027896A (en) | Method for analysis of polyamines in urine or plasma using liquid chromatography/electronspray ionization-tandem mass spectrometry along with amine carbamylated derivatization | |
| CN104991027B (en) | The method for reducing fixedness buffer salt content in LC MS testers | |
| Wang et al. | A novel and sensitive screening method for β-agonists in porcine urine by using atmospheric solid analysis probe source coupled tandem mass spectrometry | |
| Han et al. | A novel reagent pre-column derivatization method for UHPLC-MS/MS to determine four α-dicarbonyl compounds in beer | |
| Tan et al. | Development and validation of the UPLC-MS method for simultaneous determination of six new psychoactive substances | |
| CN114624338A (en) | Method for quantitatively analyzing free amino acids in biological sample by using liquid chromatography-tandem mass spectrometry | |
| EP3359680B1 (en) | Methods for detecting or quantifying ctp and ctp synthase activity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |