CN103822998A - Method for analyzing amine substances in dansyl chloride derived-plasma based on liquid chromatography mass spectrometry - Google Patents
Method for analyzing amine substances in dansyl chloride derived-plasma based on liquid chromatography mass spectrometry Download PDFInfo
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- 238000004895 liquid chromatography mass spectrometry Methods 0.000 title claims abstract description 6
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Abstract
本发明公开了一种基于液质联用的丹磺酰氯衍生血浆中胺类物质的分析方法，其特征在于：采用丹磺酰氯作为衍生化试剂对含伯胺、仲胺的代谢物进行衍生化，从而增加胺类物质在反相色谱中的保留能力，并提高在质谱检测中的离子化效率，达到提高胺类物质定量准确性和检测灵敏度的目的。本发明对胺类代谢物覆盖面广（包括氨基酸及其甲基化、乙酰化产物，二肽等），可以利用100μL血浆样品检测到113个胺类物质，提供胺类物质代谢组的轮廓信息，并且具有特异性好、精确、重复性高、试剂消耗少等特点。The invention discloses a method for analyzing amines in blood plasma derived from dansyl chloride based on liquid chromatography-mass spectrometry, which is characterized in that: using dansyl chloride as a derivatization reagent to derivatize metabolites containing primary and secondary amines , so as to increase the retention capacity of amine substances in reversed-phase chromatography, and improve the ionization efficiency in mass spectrometry detection, so as to achieve the purpose of improving the quantitative accuracy and detection sensitivity of amine substances. The present invention covers a wide range of amine metabolites (including amino acids and their methylated and acetylated products, dipeptides, etc.), and can detect 113 amines in 100 μL of plasma samples, providing profile information of amine metabolites, And it has the characteristics of good specificity, precision, high repeatability, and low reagent consumption.
Description
Technical field
The present invention relates to analytical chemistry and medical domain, is that a kind of dansyl Cl based on liquid chromatograph mass spectrography is derivative to improve the analytical approach of amine substance metabolic profile in blood plasma.
Background technology
Amine substance is the basis of body metabolism, and its main represented amino acid forms the base unit of protein especially, has important biochemical activity.The protein fragments that the amido link that dipeptides is formed by two amino acid dehydrating condensations forms or material, with nutrition, hormone, ferment suppress, regulate immune, antibacterial, antiviral, the anti-oxidant very close relation that has.In addition, research shows amino acid whosely to methylate, acetylate and human health status closely related, be the key character of tumor development.Therefore realize amine substance and carry out metabolic profiling analysis, to researching human body physiological status, disease process and therapeutic evaluation etc. all has important Research Significance.
Amine substance polarity is strong, is difficult to retain in reversed-phase liquid chromatography, and it is serious that ion suppresses, quantitative analysis difficult.At present, conventional quantitative analysis method is by many reaction detection of liquid chromatography-tandem mass spectrometry method (LC-MRM).This method speed is fast, and sample pretreatment is simple.But because different amine substance polarity differences is larger, a little less than many amine substance chromatogram reserve capabilities, and differing greatly in ionization process, the Ionization Efficiency of many amines in electric spray ion source is very low, causes its detection sensitivity poor.Therefore in reality detects, generally can only detect and quantitative test for the amino acid of high-load at present.
Derivatization method is to improve a kind of more conventional method of Ionization Efficiency.Utilize the chemical characteristic of amine substance, its reactivity is conventionally different with alkaline power, and substituent size is larger on the impact of reactivity, and the amine reactivity that steric hindrance is larger reduces.In the present invention, dansyl Cl can specificity derive moderate primary amine and the secondary amine metabolin of steric hindrance, strengthens the reservation of amine substance in reverse-phase chromatography, reaches higher detection sensitivity thereby improve Ionization Efficiency.Document and the patent of report are measured several seed amino acids in blood plasma with dansyl Cl derivatization method at present, but coverage rate is little, detect amine substance few, there is no a kind of amine metabolic profiling analysis method in blood plasma.For above-mentioned situation, we have proposed a kind of new detection technique and quilitative method that is expected to cover amine metabolism group.
Summary of the invention
The present invention is directed to prior art deficiency, the analytical approach of the full spectrum of amine substance in the derivative blood plasma of a kind of dansyl Cl based on liquid chromatograph mass spectrography is provided, make it for detection of amine substance metabolic profile in human plasma, for understand body physiology course, disease generation development mechanism, body to pathology and medicine reply and personalized therapy program provides effective information and foundation.The method has the features such as specificity is good, accurate, repeatability is high, reagent consumption is few.The mark spectrum that examination goes out according to the method can provide basis for the further investigation of the diseases such as cancer.
For achieving the above object, the technical solution used in the present invention is as follows:
Concrete steps are as follows,
The first step, collects blood plasma with the heparin tube containing ethylenediamine tetraacetic acid (EDTA) anti-coagulants, and-80 ℃ of low temperature are preserved.Before analysis, blood plasma unfreezing, to normal temperature, is quantitatively pipetted to 100 μ L in centrifuge tube and adds containing interior mark Glycine-d5 DL-Alanine-3,3,3-d3, L-Leucine-5,5,5-d3, Thymine-d3, the acetonitrile solution 400 μ L of Phenylalanine-d5 and Tryptophan-d5, the concentration being inside marked in acetonitrile solution is 400ng/mL.
Second step, the sample that the first step is obtained is at 4 ° of C, centrifugal 10min under rotating speed 15000rmp/min.Get supernatant volume 450 μ L to centrifuge tube freeze-drying on freeze dryer with liquid-transfering gun.
The 3rd step adds 40 μ L concentration 5mg/mL dansyl Cl acetonitrile solutions and 40 μ L concentration to be the Na of 0.15mol/L in blood plasma extract after the freeze-drying obtaining in second step
2cO
3/ NaHCO
3aqueous solution (PH=9.4), vortex time 1min, seals airtight rear 60 ℃ of water-bath 50min by centrifuge tube.
The 4th step adds the butylamine aqueous solution that 20 μ L concentration are 0.5mol/L in centrifuge tube, and vortex time is 1min.By centrifuge tube seal airtight after 60 ℃ of water-bath 40min again.
The 5th step, by the sample having derived, at 4 ℃, under rotating speed 15000rmp/min, centrifugal 10min, gets supernatant, and sample introduction 10 μ L carry out liquid chromatograph mass spectrography detection.
3) the derivative plasma sample of dansyl Cl is carried out to liquid chromatography matter combination analysis successively;
The instrument that described liquid chromatograph mass spectrography detects is LTQ-Orbitrap(Thermo Fisher Scientific, USA).
Liquid-phase condition is: ACQUITY HSS T3 post (10cm × 2.1mm, 1.8 μ m, Waters, Ireland), and 60 ℃ of column temperatures, sample size 10 μ L, mobile phase A is volumetric concentration 0.1% formic acid/aqueous solution, B is 0.1% formic acid/acetonitrile; Condition of gradient elution: maintain 98/2(A/B when 0～2min, V/V), change gradually gradient and become 20/80(A/B to 20min, V/V), become 0/100(A/B, V/V to 28min) and be maintained to 29min, 29.0～29.1min becomes initial gradient 98/2(A/B, and be maintained to 30min V/V), flow velocity 0.35mL/min, after post, efflux directly imports mass spectrometer system detection without shunting.
Mass spectrum condition is: electric spray ion source (ESI), adopts positive ion mode to detect; Use high-purity N
2assistant spray ionization and desolventizing, Qiao Qi35Ge unit, 5 units of assisted gas; 325 ℃ of ion source temperatures, spray voltage is 4500V, Centroid type collection mass spectrometric data; Tube lens voltage 90V, mass scanning scope m/z 80～1100, Orbitrap resolution 30000, obtains the total ion current figure of analyzed sample; Finally obtain the total ion current figure of plasma sample amine substance, it is amine substance metabolic profile spectrum.
Amine substance is one of most important metabolic product in human body, and the variation of its content can not only be reacted the physiological situation in human normal situation, also can react the variation that external environment stimulates and pathology causes simultaneously.The profile analysis of amine substance metabolism group is a kind of method that characterizes human body comprehensive situation.This method adopts the research platform of the metabolism group based on Ultra Performance Liquid Chromatography-high resolution mass spectrum coupling (UHPLC-LTQ Orbitrap), still can keep good degree of separation under higher linear velocity, has shortened analysis time, has improved analysis throughput.The mass spectrographic resolution of LTQ Orbitrap is high, and sweep velocity is fast, once analyzes and can obtain macrometabolic element information, and the quality precision of metabolin is high, for the evaluation of mark provides reliable foundation, saves qualification time, reduces and identifies difficulty.Detect the amine substance metabolism group in blood plasma in conjunction with derivatization method, contrast characteristic ion 234.05887+M and 307.1480+M carry out qualitative (accompanying drawing 1), once analyze the concentration information that can obtain 113 important amine substances, thereby obtain the profile information of amine substance metabolism group, make to understand from metabolism angle amine substance variation and the response of entirety in human body.In sample preprocessing process, add amine Isotopic Internal Standard, can proofread and correct in preprocessing process and instrument operational process and respond the error that small drift brings by interior target response.In sample sequence analytic process, insert the potpourri of plasma sample as quality control (QC) sample.By the monitoring to QC sample total ion current figure, can detect the response of analytical sequence Instrumental and have or not drift, guarantee the reliability of experimental data.
Blood plasma, as the body fluid storehouse of the most directly reflecting body metabolism situation, is widely used in the medicine and pharmacology such as medical diagnosis on disease and drug evaluation.And blood examination is as the most frequently used clinical health check-up index determining method, is suitable for by the research of metabolism group and clinical health check-up examination combination, for clinical health check-up index provides new thinking and foundation.By detecting amine metabolism group in conjunction with statistical method, can be for the discovery of the early stage biomarker of disease, thereby for the diagnosis of disease provide the treatment of new thinking and disease provide may be new target.The present invention has the features such as specificity is good, accurate, repeatability is high, reagent consumption is few.
Accompanying drawing explanation
Fig. 1 is technology path and the mass spectrometry rule of the derivative amine substance of dansyl Cl of the present invention.
Fig. 2 is that as derivatization reagent, contrast derives and non-derivative blood plasma and chromatogram derivative and non-17 kinds of derivative amine substances dansyl Cl of the present invention.(a) plasma sample of non-derivative; (b) plasma sample of derivatization; (c) 17 of non-derivative kinds of content are the mixed mark of 50ng/mL amine substance; (d) 17 of derivatization kinds of content are the mixed mark of 50ng/mL amine substance.The full name that abbreviation is corresponding: H(Histidine), R(Arginine), N(Asparagine), Q(Glutamine), S(Serine), D(Aspartate), E(Glutamic acid), G(Glycine), β-A(β-Alanine), A(Alanine), GABA(Gamma-Aminobutyric acid), P(Proline), M(Methionine), W(Tryptophan), I(Isoleucine), L(Leucine).
Embodiment
Embodiment 1
By the blood plasma unfreezing in-80 ℃ of refrigerators to normal temperature, quantitatively pipette 100 μ L blood plasma to centrifuge tube and add containing Isotopic Internal Standard Glycine-d5, DL-Alanine-3,3,3-d3, L-Leucine-5,5,5-d3, Thymine-d3, Phenylalanine-d5 and Tryptophan-d5(concentration are 400ng/mL) acetonitrile solution 400 μ L.17 kinds of amine substances are made into the mixed mark of 50ng/mL, the samely add the Isotopic Internal Standard that 6 kinds of concentration are 400ng/mL.By after two kinds of potpourris difference vortex 1min, sample is centrifugal 10min under 4 ℃ of rotating speed 15000rmp/min.Get supernatant volume 450 μ L to centrifuge tube freeze-drying on freeze dryer with liquid-transfering gun.In blood plasma extract after freeze-drying, add 40 μ L concentration 5mg/mL dansyl Cl acetonitrile solutions and 40 μ L concentration to be the Na of 0.15mol/L
2cO
3/ NaHCO
3aqueous solution (PH=9.4), vortex time 1min, seals airtight rear 60 ℃ of water-bath 50min by centrifuge tube.Add the butylamine aqueous solution that 20 μ L concentration are 0.5mol/L, vortex time is 1min, by centrifuge tube seal airtight after 60 ℃ of water-bath 40min again.The sample having derived is at 4 ℃, and centrifugal 10min under rotating speed 15000rmp/min, gets supernatant sample introduction 10 μ L and carry out liquid chromatograph mass spectrography detection.
Liquid-phase condition is: ACQUITY HSS T3 post (10cm × 2.1mm, 1.8 μ m, Waters, Ireland), and 60 ℃ of column temperatures, sample size 10 μ L, mobile phase A is volumetric concentration 0.1% formic acid/aqueous solution, B is 0.1% formic acid/acetonitrile; Condition of gradient elution: maintain 98/2(A/B when 0～2min, V/V), change gradually gradient and become 20/80(A/B to 20min, V/V), become 0/100(A/B, V/V to 28min) and be maintained to 29min, 29.0～29.1min becomes initial gradient 98/2(A/B, and be maintained to 30min V/V), flow velocity 0.35mL/min, after post, efflux directly imports mass spectrometer system detection without shunting.
Mass spectrum condition is: electric spray ion source (ESI), adopts positive ion mode to detect; Use high-purity N 2 assistant spray ionization and desolventizings, Qiao Qi35Ge unit, 5 units of assisted gas; 325 ℃ of ion source temperatures, spray voltage is 4500V, Centroid type collection mass spectrometric data; Tube lens voltage 90V, mass scanning scope m/z 80～1100, Orbitrap resolution 30000, obtains the total ion current figure of analyzed sample; Finally obtain the total ion current figure of plasma sample and mixed sample amine substance, it is amine substance metabolic profile spectrum.
The method of the derivative blood plasma of dansyl Cl for this experiment, can be in 100 μ L blood plasma by Internal standard correction methods to realizing the metabolic profiling analysis of amine substance.As shown in Figure 2, originally in 1min, go out the amine substance at peak, because appearance time is near the dead time, peak shape is poor, and it is serious that ion suppresses, and reduced detection sensitivity.In the blood plasma and standard model spectrogram of underivatized, amine substance is suppressed by other peaks, is almost difficult to see.Amine substance after derivative retains enhancing, and peak shape is improved obviously, on reverse-phase chromatography, well separates, and sensitivity improves 1～3 order of magnitude, and analysis result more accurately and reliably.
Embodiment 2
Before sampled plasma, puncture and carry out pathological diagnosis by multiple spot, guarantee the accuracy of sampling.As a child, gather morning on an empty stomach in patient's fasting 12.Sample comprises that prostate specific antigen (PSA) concentration is all at benign prostate cancer hyperplasia patient and malignant prostate carninomatosis people (being benign prostate cancer hyperplasia patient and the malignant prostate carninomatosis people that PSA cannot the distinguish) blood plasma of 4～10ng/mL.After collection, put into rapidly-80 ℃ of Refrigerator stores.
Before analysis, by blood plasma unfreezing to normal temperature, quantitatively pipette 100 μ L blood plasma to centrifuge tube and add containing interior mark Glycine-d5 DL-Alanine-3,3,3-d3, L-Leucine-5,5,5-d3, Thymine-d3, Phenylalanine-d5 and Tryptophan-d5(concentration are 400ng/mL) acetonitrile solution 400 μ L.After vortex 1min, sample is centrifugal 10min under 4 ℃ of rotating speed 15000rmp/min.Get supernatant volume 450 μ L to centrifuge tube freeze-drying on freeze dryer with liquid-transfering gun.In blood plasma extract after freeze-drying, add 40 μ L concentration 5mg/mL dansyl Cl acetonitrile solutions and 40 μ L concentration to be the Na of 0.15mol/L
2cO
3/ NaHCO
3aqueous solution (PH=9.4), vortex time 1min, seals airtight rear 60 ℃ of water-bath 50min by centrifuge tube.Add the butylamine aqueous solution that 20 μ L concentration are 0.5mol/L, vortex time is 1min, by centrifuge tube seal airtight after 60 ℃ of water-bath 40min again.The sample having derived is at 4 ℃, and centrifugal 10min under rotating speed 1 5000rmp/min, gets supernatant sample introduction 10 μ L and carry out liquid chromatograph mass spectrography detection.Liquid-phase condition is: ACQUITYHSS T3 post (10cm × 2.1mm, 1.8 μ m, Waters, Ireland), and 60 ℃ of column temperatures, sample size 10 μ L, mobile phase A is volumetric concentration 0.1% formic acid/aqueous solution, B is 0.1% formic acid/acetonitrile; Condition of gradient elution: maintain 98/2(A/B when 0～2min, V/V), change gradually gradient and become 20/80(A/B to 20min, V/V), become 0/100(A/B, V/V to 28min) and be maintained to 29min, 29.0～29.1min becomes initial gradient 98/2(A/B, and be maintained to 30min V/V), flow velocity 0.35mL/min, after post, efflux directly imports mass spectrometer system detection without shunting.Mass spectrum condition is: electric spray ion source (ESI), adopts positive ion mode to detect; Use high-purity N 2 assistant spray ionization and desolventizings, 35 units of sheath gas nitrogen, 5 units of assisted gas nitrogen; 325 ℃ of ion source temperatures, spray voltage is 4500V, Centroid type collection mass spectrometric data; Tube lens voltage 90V, mass scanning scope m/z 80～1100, Orbitrap resolution 30000, obtains the total ion current figure of analyzed sample; Finally obtain the total ion current figure of plasma sample amine substance, it is amine substance metabolic profile spectrum.
Raw data utilizes Thermo Scientific SIEVE 1.2 softwares to extract compound information, and carries out obtaining parent mass peak table after peak match.Parent mass peak table is carried out to missing values processing according to 80% rule (retaining at least 80% sample peak area in each class sample is not 0 value), and Isotopic Internal Standard is proofreaied and correct and is reduced error.By the peak table obtaining, shown in accompanying drawing 1, the law of regularity of contrast characteristic ion 234.05887+M and 307.1480+M calculates the accurate molecular weight of amine substance.Carry out accurate mass contrast by HMDB database and Metlin database, more further confirm with MS/MS second order ms, wherein part of compounds is finally verified with standard specimen, qualitative go out amine substance (subordinate list 1) in 113 kinds of blood plasma.
Then utilize self-programmed software MSACS to carry out T check analysis, there were significant differences (p < 0.05) in benign prostatic hyperplasis and malignant prostate cancer blood plasma to find in 113 kinds of amine substances 31 kinds (subordinate list 1).The Difference of Metabolism of amine substance can be good at distinguishing benign prostatic hyperplasis and the malignant prostate carninomatosis people that PSA value is difficult to diagnosis, and finds the metabolic pathway wherein getting muddled.
Amine substance qualitative results and the difference in benign prostatic hyperplasis and prostate cancer thereof in subordinate list 1. blood plasma
The present invention is the analytical approach based on the full spectrum of amine substance in the derivative blood plasma of the dansyl Cl of liquid chromatograph mass spectrography, in conjunction with Chemical Measurement data analysis screening mark.In the blood plasma that the present invention sets up, amine metabolic profiling analysis method adds interior mark in preprocessing process, is farthest reduced in the error causing in experimentation; In addition, realize the monitoring to retention time and corresponding signal in instrument operational process by quality control (QC), guaranteed the reliability of data.The present invention filters out multiple distinguishing benign hyperplasia of prostates and malignant prostate carninomatosis people's potential mark, makes up the shortcoming of PSA poor specificity, for prostatic cancer early diagnosis and study of incident mechanism provide foundation.
Claims (9)
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