CN111239318A - Method for determining urea content in biological sample based on combination of GC-MS and enzymatic chemical method - Google Patents
Method for determining urea content in biological sample based on combination of GC-MS and enzymatic chemical method Download PDFInfo
- Publication number
- CN111239318A CN111239318A CN202010250083.5A CN202010250083A CN111239318A CN 111239318 A CN111239318 A CN 111239318A CN 202010250083 A CN202010250083 A CN 202010250083A CN 111239318 A CN111239318 A CN 111239318A
- Authority
- CN
- China
- Prior art keywords
- urea
- sample
- content
- temperature
- urease
- 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
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 title claims abstract description 161
- 239000004202 carbamide Substances 0.000 title claims abstract description 86
- 238000000034 method Methods 0.000 title claims abstract description 41
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 title claims abstract description 26
- 239000012472 biological sample Substances 0.000 title claims abstract description 23
- 239000000126 substance Substances 0.000 title description 11
- 230000002255 enzymatic effect Effects 0.000 title description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims abstract description 44
- 239000000523 sample Substances 0.000 claims abstract description 37
- 108010046334 Urease Proteins 0.000 claims abstract description 30
- 229910021529 ammonia Inorganic materials 0.000 claims abstract description 22
- 238000001212 derivatisation Methods 0.000 claims abstract description 21
- 238000001514 detection method Methods 0.000 claims abstract description 15
- SGNZYJXNUURYCH-UHFFFAOYSA-N 5,6-dihydroxyindole Chemical compound C1=C(O)C(O)=CC2=C1NC=C2 SGNZYJXNUURYCH-UHFFFAOYSA-N 0.000 claims abstract description 13
- 108090000790 Enzymes Proteins 0.000 claims abstract description 8
- 102000004190 Enzymes Human genes 0.000 claims abstract description 8
- 239000012488 sample solution Substances 0.000 claims abstract description 7
- 238000012545 processing Methods 0.000 claims abstract description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 18
- 239000008280 blood Substances 0.000 claims description 18
- 210000004369 blood Anatomy 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- FOBJABJCODOMEO-UHFFFAOYSA-N 2,2,3,3,4,4,4-heptafluorobutanamide Chemical compound NC(=O)C(F)(F)C(F)(F)C(F)(F)F FOBJABJCODOMEO-UHFFFAOYSA-N 0.000 claims description 14
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 11
- 150000002500 ions Chemical class 0.000 claims description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000007853 buffer solution Substances 0.000 claims description 8
- 239000007789 gas Substances 0.000 claims description 8
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 8
- 239000003960 organic solvent Substances 0.000 claims description 8
- 238000004458 analytical method Methods 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 239000012224 working solution Substances 0.000 claims description 4
- 238000007664 blowing Methods 0.000 claims description 3
- 239000012159 carrier gas Substances 0.000 claims description 3
- 150000001793 charged compounds Chemical class 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 230000002496 gastric effect Effects 0.000 claims description 3
- 239000001307 helium Substances 0.000 claims description 3
- 229910052734 helium Inorganic materials 0.000 claims description 3
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 claims description 3
- NAQMVNRVTILPCV-UHFFFAOYSA-N hexane-1,6-diamine Chemical compound NCCCCCCN NAQMVNRVTILPCV-UHFFFAOYSA-N 0.000 claims description 3
- 230000014759 maintenance of location Effects 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000003672 processing method Methods 0.000 claims description 3
- 230000000630 rising effect Effects 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 238000010183 spectrum analysis Methods 0.000 claims description 3
- 239000012086 standard solution Substances 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 210000002700 urine Anatomy 0.000 claims description 3
- 238000004364 calculation method Methods 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 238000011002 quantification Methods 0.000 claims description 2
- 238000004506 ultrasonic cleaning Methods 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims 1
- 239000008363 phosphate buffer Substances 0.000 claims 1
- 238000001311 chemical methods and process Methods 0.000 abstract description 6
- 239000012620 biological material Substances 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000004611 spectroscopical analysis Methods 0.000 abstract description 3
- 235000013877 carbamide Nutrition 0.000 description 74
- 241000283690 Bos taurus Species 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 14
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 11
- 239000002953 phosphate buffered saline Substances 0.000 description 11
- 239000000047 product Substances 0.000 description 9
- 230000000694 effects Effects 0.000 description 7
- 239000003513 alkali Substances 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 210000004767 rumen Anatomy 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 238000000354 decomposition reaction Methods 0.000 description 3
- 231100000572 poisoning Toxicity 0.000 description 3
- 230000000607 poisoning effect Effects 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 241000282849 Ruminantia Species 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000004945 emulsification Methods 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 150000002894 organic compounds Chemical class 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241001391944 Commicarpus scandens Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 240000006677 Vicia faba Species 0.000 description 1
- 235000010749 Vicia faba Nutrition 0.000 description 1
- 235000002098 Vicia faba var. major Nutrition 0.000 description 1
- 210000003165 abomasum Anatomy 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- OHJMTUPIZMNBFR-UHFFFAOYSA-N biuret Chemical compound NC(=O)NC(N)=O OHJMTUPIZMNBFR-UHFFFAOYSA-N 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- -1 derivative of ammonia (heptafluorobutanamide) Chemical class 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000002050 international nonproprietary name Substances 0.000 description 1
- ZFSLODLOARCGLH-UHFFFAOYSA-N isocyanuric acid Chemical compound OC1=NC(O)=NC(O)=N1 ZFSLODLOARCGLH-UHFFFAOYSA-N 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 238000010534 nucleophilic substitution reaction Methods 0.000 description 1
- 230000009965 odorless effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000009967 tasteless effect Effects 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000005891 transamination reaction Methods 0.000 description 1
- WNVQBUHCOYRLPA-UHFFFAOYSA-N triuret Chemical compound NC(=O)NC(=O)NC(N)=O WNVQBUHCOYRLPA-UHFFFAOYSA-N 0.000 description 1
- 230000004143 urea cycle Effects 0.000 description 1
Images
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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
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)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the field of biological material detection, and particularly relates to a method for detecting urea content in a biological sample based on GC-MS and an enzyme chemistry method. The method comprises the following steps: (1) detecting conditions; (2) processing a sample; (3) analyzing a map; (4) preparing a standard working curve sample solution; (5) and (4) calculating the content. The method can specifically detect the urea content in the biological sample by a pre-column derivatization gas chromatography-mass spectrometry combined method, converts urea into ammonia by utilizing the characteristic that urease can specifically decompose urea, and then utilizes heptafluorobutyryl chloride to react with the ammonia rapidly and completely, so that the urea which cannot be detected by GC-MS can be detected and quantified. The method has higher sensitivity and better linear relation than a spectroscopic method.
Description
Technical Field
The invention belongs to the field of biological material detection, and particularly relates to a method for detecting urea content in a biological sample based on GC-MS and an enzyme chemistry method.
Background
Urea is also known as carbamide, urea. Is an organic compound consisting of carbon, nitrogen, oxygen and hydrogen. Its chemical formula is CON2H4、CO(NH2)2Or CN2H4O, international non-proprietary name, Carbamide. The appearance is white crystals or powder. Urea is synthesized in the liver and is the nitrogen-containing metabolism in the body of mammalsA compound (I) is provided. This metabolic process is called the urea cycle. Urea is the first organic compound obtained by artificially synthesizing inorganic substances. The chemical formula is as follows: CO (NH)2)2The molecular weight is 60.06, the crystal is colorless or white needle-shaped or rod-shaped crystal, and the industrial or agricultural product is white slightly reddish solid particles, and is odorless and tasteless. The nitrogen content was about 46.67%, and the density was 1.335g/cm 3. It is soluble in water and alcohol, insoluble in diethyl ether and chloroform, and weak alkaline. CAS No.: 57-13-6, molecular weight: 60.05; melting point: 131 ℃ and 135 ℃; boiling point: 196.6, refractive index: n 20/D1.40; flash point: 72.7 ℃, density: 1.335; water solubility: 1080g/L (20 ℃). The chemical properties are as follows: can react with acid to form salt. Has hydrolysis effect. Can carry out condensation reaction at high temperature to generate biuret, triuret and cyanuric acid.
Urea was first synthesized by Wohler, German in 1828 and was widely used in the 50 s of the 20 th century as a non-protein nitrogen source for ruminants. It is a low-toxic substance, and is orally administered to rats by LD5015g/kg, oral LD of mouse50It was 11.5 g/kg. Ruminants, due to their unique digestive system structure, enable them to utilize urea to synthesize proteins. In the rumen of cattle, a large number of microorganisms having urease activity exist, which decompose urea into ammonia when the cattle are fed with a feed containing urea, and these microorganisms synthesize Mycoprotein (MCP) from ammonia and carbohydrates by an ammoniation reaction or transamination reaction, and then the MCP is digested and absorbed in the abomasum and small intestine of the cattle. Although urea is a low-toxicity substance, improper use of urea due to the property that microorganisms in the rumen of cattle can decompose urea to produce ammonia can cause cattle poisoning and death. The main reasons are as follows: improper urea management causes the urea to be eaten by cattle or eaten by mistake; the content of urea in the feed is too high or the urea and the feed are not uniformly mixed; feeding the feed containing urea and simultaneously feeding the soybean cake or broad bean containing urease; after eating the feed containing urea, the cattle drink a large amount of water. The urea can be rapidly decomposed to generate a large amount of ammonia, and the ammonia cannot be fully utilized by microorganisms in the rumen of the cattle, so that the ammonia is absorbed into blood through the wall of the rumen to cause poisoning. Cases of bovine urea poisoning are frequently encountered in judicial identification work, but the prior art reports methods cannot be used for a long timeSolving such problems well. Therefore, the development of a method for rapidly and accurately detecting urea in a biological sample is a new problem to be solved urgently.
Disclosure of Invention
The urea has high molecular polarity and is difficult to gasify, so that the urea cannot be directly detected by a gas chromatography-mass spectrometry (GC-MS) combined technology. In view of the problems in the prior art, the invention aims to provide an accurate, simple and convenient method for detecting urea in a biological sample, which can be used for indirectly detecting urea by using GC-MS after urea is treated by urease and a derivatization reagent, can be used for pertinently detecting the urea content in the biological sample, can eliminate the interference of a plurality of external factors, improves the detection sensitivity, greatly simplifies the detection steps and saves time and cost.
In order to achieve the above object, the present invention adopts the following technical solutions.
A method for determining the urea content in a biological sample based on GC-MS and enzyme chemistry method specifically comprises the following steps.
(1) And (5) detecting the condition.
Gas chromatograph-mass spectrometer: gas chromatography-mass spectrometer (GC-MS) was used.
A chromatographic column: HP-5MS capillary column, specification: 30 m.times.0.25 mm, 0.25. mu.m.
The temperature of a front sample inlet, the temperature of an EI ion source, the temperature of a quadrupole rod and the temperature of an interface are respectively 280 ℃, 150 ℃, 230 ℃ and 280 ℃, a mass spectrometer adopts a single ion detection Scanning (SIM) mode, and the electron energy is 70 eV.
The carrier gas was high purity helium (99.999%) at a flow rate of 1.0ml/min, and 1 μ L was injected without splitting.
Temperature rising procedure: the initial column temperature was 60 ℃ and held for 1min, followed by a ramp up to 280 ℃ at a ramp rate of 20 ℃/min and held for 11 min.
(2) And (4) processing a sample.
Taking 2 samples of blood sample to be detected or other biological samples 0.5mL, respectively placing in 10mL centrifuge tubes, adding 200 μ g/mL internal standard 10 μ L, adding 1mL PBS buffer solution, then adding 22U/mL urease 0.5mL, adding 0.5mL PBS buffer solution in another sample, sealing, placing in 45 ℃ water bath, and incubating for 30 min. At the moment, urea can be completely decomposed into ammonia by urease, after the sample is cooled, 1mL of 3mol/L NaOH solution is added, 50 mu L of derivatization reagent, namely heptafluorobutyryl chloride, is added, and the mixture is subjected to water bath at 40 ℃ for 10min in a water bath kettle, so that the ammonia and the internal standard are subjected to derivatization. After the water bath was complete, 2mL of ethyl acetate were added: n-hexane ═ 9:1, followed by shaking on a vortex apparatus for 5min for extraction, and centrifugation at 8000rpm/min for 5 min. Taking 1mL of the upper organic solvent, blowing dry nitrogen to be nearly dry, taking 1 mu L of the upper organic solvent, and carrying out sample injection analysis on the obtained product by GC-MS.
(3) And (4) carrying out spectrum analysis.
The derivatization product, heptafluorobutanamide, has a retention time of 3.0min and characteristic mass numbers of m/z 214 (molecular ion peak), m/z194, m/z166, m/z 146, m/z100, m/z 69 and m/z 44 (base peak).
(4) Preparation of standard working curve sample solution.
Preparing a urease solution: urease was dissolved in Phosphate Buffered Saline (PBS) at pH7.4 to prepare a urease solution (prepared as soon as possible since urease activity decreased with time) at 22U/ml.
Internal standard configuration: taking an appropriate amount of 1, 6-hexanediamine, diluting to 200 mu g/ml with 0.1mol/L HCL, and refrigerating at 4 ℃.
Preparing a urea working solution: diluting urea into urea standard solutions with different concentrations according to the required linear range, performing ultrasonic sterilization by an ultrasonic cleaning machine for 10min, and freezing and storing at-20 ℃.
(5) And (4) calculating the content.
And (3) processing the standard working curve sample solution prepared in the step (4) according to the sample processing method in the step (2) and then carrying out sample injection analysis. Selecting a selective ion chromatogram for detecting m/z100, taking the peak area of a derivative product heptafluorobutanamide as a vertical coordinate, taking the mass concentration of urea added into blood as a horizontal coordinate, calculating a regression equation, and substituting the peak area of the heptafluorobutanamide in a sample to be detected into the regression equation during content calculation to obtain the content of the sample to be detected.
The biological sample is urine, gastric content or plasma sample.
The minimum detection limit of this method was calculated as S/N-3, and the minimum detection limit for urea in blood was 0.05 μ g/ml, and the quantification limit was 0.10 μ g/ml.
Compared with the prior art, the invention has the following beneficial effects.
1. The detection principle of the method for determining the urea content in the biological sample based on the combination of the GC-MS and the enzymatic chemical method is to indirectly determine the urea in the biological sample.
2. The method for determining the urea content in the biological sample based on the combination of the GC-MS and the enzymatic chemical method adopts a mass spectrometry method after derivatization, rather than the traditional spectrometry method. Can effectively reduce some external influence factors such as blood coagulation state, hemolysis, reagent interference and the like.
3. The method for determining the urea content in the biological sample based on the combination of the GC-MS and the enzymatic chemical method provided by the invention uses fewer types of reagents and is simple and quick to operate.
4. The biological material which can be detected by the method for determining the urea content in the biological sample based on the combination of the GC-MS and the enzyme chemistry method comprises urine, gastric content or plasma samples.
5. The method for detecting the urea content in the biological sample by using the GC-MS is based on the combination of the GC-MS and the enzyme chemistry method, the method utilizes the characteristic that urease can specifically decompose the urea, converts the urea into ammonia, and utilizes heptafluorobutyryl chloride to react with the heptafluorobutyryl chloride more quickly and completely, so that the urea which cannot be detected by the GC-MS can be detected and quantified. The method has higher sensitivity and better linear relation than a spectroscopic method.
Drawings
FIG. 1 shows the formula of urease decomposition of urea.
FIG. 2 shows derivatization reaction (main reaction) of the derivative with heptafluorobutyryl chloride.
FIG. 3 is a graph showing the effect of different basic systems on derivatization.
FIG. 4 is a graph of the effect of different temperatures on derivatization.
FIG. 5 is a graph showing the effect of different reaction times on the derivatization.
FIG. 6 is a typical chromatogram of urea as determined by gas chromatograph-mass spectrometer.
FIG. 7 shows the measurement of urea by gas chromatograph-mass spectrometer (typical mass spectrum).
FIG. 8 is a linear relationship diagram of urea addition to blood.
Detailed Description
The following detailed description of embodiments of the invention is provided in connection with the accompanying drawings and examples. The following examples illustrate the invention in detail, but are not intended to limit the scope of the invention. This example uses routine experimentation, which is well known to those skilled in the art, and can be performed according to the present example using the instructions provided by the materials manufacturer.
Example 1 optimization of reaction processes and conditions associated with methods for determining urea content in biological samples based on GC-MS in combination with enzymatic chemistry.
Urease is a nickeliferous oligoenzyme, has the optimum pH value of 7.4, has absolute specificity, can specifically catalyze the decomposition of urea into ammonia and carbon dioxide, and the reaction process is shown in figure 1. The ammonia generated by the decomposition of urea by urease is then reacted with a derivatizing agent, heptafluorobutyryl chloride, to produce heptafluorobutanamide, which is detected by gas chromatography-mass spectrometry. The reaction equation is shown in FIG. 2. In this reaction system, there are two steps of equilibrium to be controlled. Firstly, in the use amount of PBS buffer solution in the reaction system, in the process of decomposing urea by urease, one molecule of urea generates two molecules of ammonia, and the two molecules of ammonia are combined with water molecules to generate ammonia water, so that the reaction environment of the urease is changed into alkaline, and the urease is inactivated under the condition of pH of about 9. When the urea content in the sample is too high, the urease has not completely decomposed the urea and is inactivated, and in this case, the PBS buffer capacity can be increased appropriately. 2mL of PBS buffer solution is added into the reaction system of the invention, and the sample with urea concentration of about 6mg/mL can be decomposed completely to the maximum; secondly, the usage amount of the heptafluorobutyryl chloride and the usage amount of the alkali liquor, the reaction of the heptafluorobutyryl chloride with ammonia and water generates acid, and the reaction product of the heptafluorobutyrylamide exists in an ion form under an acidic condition and cannot be extracted by an organic solvent. For this reason, an appropriate amount of alkali liquor should be added before derivatization, so that the lower-layer aqueous solution becomes slightly alkalescent after the reaction is finished, the heptafluorobutanamide to be detected is dissociated, and the alkali liquor is added to neutralize acid and alkali, so that the reaction is promoted to be carried out in the positive direction. In the second derivatization step, we examined the influence of different alkaline systems, different temperatures and different reaction times on the whole detection process, and the results are shown in fig. 3-5, and finally, the derivatization conditions of reacting for 5 minutes at 40 ℃ and pH of 14 were selected.
In addition, when extraction is performed using an organic solvent, since ethyl acetate is close to water density and the reaction system is alkaline, if extraction with ethyl acetate alone is liable to cause emulsification, ethyl acetate: the emulsification phenomenon can be well avoided when n-hexane is 9:1 as the extraction liquid.
2. Chromatogram-mass spectrum (lowest detection limit).
The mass spectrogram of a typical product obtained by reacting urea with heptafluorobutyryl chloride after enzymolysis is shown in figures 6-7.
3. Standard curve (blood addition as an example).
Accurately sucking appropriate amount of standard working solution of urea, adding into 0.5mL of blank plasma respectively to obtain additive plasma samples with urea concentration of 0.1. mu.g/mL, 10. mu.g/mL, 200. mu.g/mL, 1mg/mL, 3mg/mL and 5mg/mL, pre-treating blood sample according to the above steps, subtracting total ammonia in blank plasma with urease, and taking concentration as abscissa (x), ratio of derivative of ammonia (heptafluorobutanamide) and internal standard derivative peak as ordinate (y), linear relation as shown in FIG. 8, to obtain unitary linear equation of y 0.0793x +1.9703(R is 0.0793x + 1.9703)2＝0.9994，P<0.0001)。
4. Methodology investigation.
4.1 recovery and precision.
And (3) respectively carrying out precision investigation in the day and in the daytime on the three samples, and calculating the ratio of the peak area of the heptafluorobutanamide to the peak area of the internal standard. Measuring the precision RSD in the day to be 3.7-6.2%; the daytime precision RSD is 4.0-6.8%. Urea was added at 10. mu.g/mL, 1mg/mL, 5mg/mL, respectively, to a 0.5mL blank sample, and urea originally present in the blank blood sample was subtracted after derivatization to calculate recovery, and was measured in triplicate. The average recovery rate of the low, medium and high concentration groups is between 90.3 and 97.5 percent.
4.2 stability test.
Based on the extremely active chemical property of heptafluorobutyryl chloride, a covalent bond formed by nucleophilic substitution reaction between ammonia and heptafluorobutyryl chloride is very stable and is not easy to break under common external conditions. After comparing the peak area ratios of heptafluorobutanamide to the internal standard derivatization product in the samples after standing for 0h, 12h, 24h, 48h and 96h for pretreatment, the ratio is more stable within 0-96h, namely P is 0.8621(P is more than 0.05), and RSD is 1.65%.
Example 2 practical case application.
A method for determining the urea content in a biological sample based on GC-MS and enzyme chemistry method specifically comprises the following steps.
(1) And (5) detecting the condition.
Gas chromatograph-mass spectrometer: gas chromatography-mass spectrometer (GC-MS) was used.
A chromatographic column: HP-5ms capillary column, specification: 30 m.times.0.25 mm, 0.25. mu.m.
The temperature of a front sample inlet, the temperature of an EI ion source, the temperature of a quadrupole rod and the temperature of an interface are respectively 280 ℃, 150 ℃, 230 ℃ and 280 ℃, a mass spectrometer adopts a single ion detection Scanning (SIM) mode, and the electron energy is 70 eV.
The carrier gas was high purity helium (99.999%) at a flow rate of 1.0ml/min, and 1 μ L was injected without splitting.
Temperature rising procedure: the initial column temperature was 60 ℃ and held for 1min, followed by a ramp up to 280 ℃ at a ramp rate of 20 ℃/min and held for 11 min.
(2) And (4) processing a sample.
Taking 0.5mL of bovine blood sample to be detected, respectively placing the bovine blood sample into a 10mL centrifuge tube, adding 10 μ L of 200 μ g/mL internal standard, adding 1mL of PBS buffer solution, then adding 0.5mL of 22U/mL urease (adding 0.5mL of PBS buffer solution into the other part), sealing, and then placing the sealed bovine blood sample into a 45 ℃ water bath kettle for incubation for 30 min. At the moment, urea can be completely decomposed into ammonia by urease, after the sample is cooled, 1mL of 3mol/L NaOH solution is added, 50 mu L of derivatization reagent, namely heptafluorobutyryl chloride, is added, and the mixture is subjected to water bath at 40 ℃ for 10min in a water bath kettle, so that the ammonia and the internal standard are subjected to derivatization. After the water bath was complete, 2mL of ethyl acetate were added: n-hexane ═ 9:1, followed by shaking on a vortex apparatus for 5min for extraction, and centrifugation at 8000rpm/min for 5 min. Taking 1mL of the upper organic solvent, blowing dry nitrogen to be nearly dry, taking 1 mu L of the upper organic solvent, and carrying out sample injection analysis on the obtained product by GC-MS.
(3) And (4) carrying out spectrum analysis.
The derivatization product, heptafluorobutanamide, has a retention time of 3.0min and characteristic mass numbers of m/z 214 (molecular ion peak), m/z194, m/z166, m/z 146, m/z100, m/z 69 and m/z 44 (base peak).
(4) Preparation of standard working curve sample solution.
Preparing a urease solution: urease was dissolved in Phosphate Buffered Saline (PBS) at pH7.4 to prepare a urease solution (prepared as soon as possible since urease activity decreased with time) at 22U/ml.
Internal standard configuration: taking an appropriate amount of 1, 6-hexanediamine, diluting to 200 mu g/ml with 0.1mol/L HCL, and refrigerating at 4 ℃.
Preparing a urea working solution: diluting urea into urea standard solutions with different concentrations of 0.1 μ g/ml, 10 μ g/ml, 200 μ g/ml, 2000 μ g/ml and 5000 μ g/ml respectively according to the required linear range, and performing ultrasonic sterilization with ultrasonic cleaner for 10min and freezing storage at-20 deg.C.
(5) And (4) calculating the content.
And (3) processing the standard working curve sample solution prepared in the step (4) according to the sample processing method in the step (2) and then carrying out sample injection analysis. Selecting a selective ion chromatogram for detecting m/z100, taking the peak area of a derivative product heptafluorobutanamide as an ordinate, taking the mass concentration of urea added into blood as an abscissa, and calculating a regression equation to obtain y of 0.0793x +1.9703 (R)20.9994). And when the content is calculated, substituting the peak area of heptafluorobutanamide in the sample to be detected into a regression equation to obtain the content of the sample to be detected.
The method for measuring the urea content in the biological sample based on GC-MS and enzyme chemistry provided above was used to measure the urea content in the bovine blood in 7 cases, and the specific urea content is shown in Table 1. The experimental result shows that the measuring method has strong applicability.
Table 1.7 urea content in bovine blood in the cases.
| Bovine blood | Urea content (mmol/L) | Ammonia content (mmol/L) |
| NO.1 | 14.44 | 7.54 |
| NO.2 | 6.05 | 9.7 |
| NO.3 | 2.63 | 15.61 |
| NO.4 | 6.08 | 6.08 |
| NO.5 | 2.934 | 15 |
| NO.6 | 16.41 | 3.86 |
| NO.7 | 9.24 | 7.43 |
Claims (3)
1. A method for determining the urea content in a biological sample based on GC-MS and enzyme chemistry is characterized by comprising the following steps:
detection conditions are as follows:
gas chromatograph-mass spectrometer: adopting a gas chromatography-mass spectrometer;
a chromatographic column: HP-5ms capillary column, specification: 30m × 0.25mm, 0.25 μm;
the temperature of a front sample inlet, the temperature of an EI ion source, the temperature of a quadrupole rod and the temperature of an interface are respectively 280 ℃, 150 ℃, 230 ℃ and 280 ℃, a mass spectrometer adopts a single ion detection scanning mode, and the electron energy is 70 eV;
the carrier gas is high-purity helium (99.999%), the flow rate is 1.0ml/min, and the sample introduction is 1 mu L without shunting;
temperature rising procedure: the initial column temperature is 60 ℃, the temperature is kept for 1min, then the temperature is increased to 280 ℃ at the heating rate of 20 ℃/min, and the temperature is kept for 11 min;
(2) sample treatment:
respectively placing 0.5mL of 2 blood samples to be detected or other biological samples into a 10mL centrifuge tube, adding 10 μ L of 200 μ g/mL internal standard, adding 1mL of PBS buffer solution, then adding 0.5mL of 22U/mL urease, adding 0.5mL of PBS buffer solution into the other part, sealing, and then placing into a 45 ℃ water bath kettle for incubation for 30 min; after the sample is cooled, adding 1mL of 3mol/L NaOH solution, adding 50 mu L of derivatization reagent, namely heptafluorobutyryl chloride, and carrying out water bath at 40 ℃ for 10min in a water bath kettle so as to perform derivatization on ammonia and the internal standard; after the water bath was complete, 2mL of ethyl acetate were added: n-hexane = 9:1, followed by shaking on a vortex instrument for 5min for extraction, and centrifuging at 8000rpm/min for 5 min;
taking 1mL of upper organic solvent, blowing dry nitrogen to be nearly dry, and taking 1 mu L of the upper organic solvent for sample injection analysis by GC-MS;
(3) and (3) spectrum analysis:
the derivatization product, namely heptafluorobutanamide, has the retention time of 3.0min and the characteristic mass numbers of m/z 213 (molecular ion peak), m/z194, m/z166, m/z 146, m/z100, m/z 69 and m/z 44 (base peak);
(4) preparation of standard working curve sample solution:
preparing a urease solution: dissolving urease in phosphate buffer PBS (pH 7.4) to prepare a urease solution with the concentration of 22U/ml, and preparing the urease solution as soon as possible;
internal standard configuration: taking a proper amount of 1, 6-hexanediamine, diluting to 200 mu g/ml with 0.1mol/L HCL, and refrigerating at 4 ℃;
preparing a urea working solution: diluting urea into urea standard solutions with different concentrations according to a required linear range, performing ultrasonic sterilization by an ultrasonic cleaning machine for 10min, and freezing and storing at-20 ℃;
(5) and (3) calculating the content:
processing the standard working curve sample solution prepared in the step (4) according to the sample processing method in the step (2) and then carrying out sample injection analysis; and (3) selecting a selective ion chromatogram for detecting m/z166, taking the peak area of a derivative product heptafluorobutanamide as a vertical coordinate, taking the mass concentration of urea added into blood as a horizontal coordinate, calculating a regression equation, and substituting the peak area of heptafluorobutanamide in the sample to be detected into the regression equation during content calculation to obtain the content of the sample to be detected.
2. The method of claim 1, wherein the biological sample is a urine, gastric content or plasma sample.
3. The method of claim 1, wherein the minimum detection limit of the method is S/N-3, the minimum detection limit for urea in blood is 0.05 μ g/ml, and the quantification limit is 0.10 μ g/ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010250083.5A CN111239318A (en) | 2020-04-01 | 2020-04-01 | Method for determining urea content in biological sample based on combination of GC-MS and enzymatic chemical method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010250083.5A CN111239318A (en) | 2020-04-01 | 2020-04-01 | Method for determining urea content in biological sample based on combination of GC-MS and enzymatic chemical method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111239318A true CN111239318A (en) | 2020-06-05 |
Family
ID=70873693
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010250083.5A Pending CN111239318A (en) | 2020-04-01 | 2020-04-01 | Method for determining urea content in biological sample based on combination of GC-MS and enzymatic chemical method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111239318A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115078564A (en) * | 2022-05-06 | 2022-09-20 | 沈阳药科大学 | Method for ultra-fast quantifying amine substances in urine |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104764850A (en) * | 2015-04-28 | 2015-07-08 | 江南大学 | Method for rapidly determining amount of urea in white spirit by means of gas chromatogram-mass spectrum |
| CN108414661A (en) * | 2018-03-13 | 2018-08-17 | 中国医科大学 | Derivative gas chromatography-mass spectrometry method of ammonia content in a kind of detection biological sample |
-
2020
- 2020-04-01 CN CN202010250083.5A patent/CN111239318A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104764850A (en) * | 2015-04-28 | 2015-07-08 | 江南大学 | Method for rapidly determining amount of urea in white spirit by means of gas chromatogram-mass spectrum |
| CN108414661A (en) * | 2018-03-13 | 2018-08-17 | 中国医科大学 | Derivative gas chromatography-mass spectrometry method of ammonia content in a kind of detection biological sample |
Non-Patent Citations (8)
| Title |
|---|
| DONALD G. SAUNDERS 等: "Derivatization and Gas Chromatographic Measurement of Some Thermally Unstable Ureas", 《ANALYTICAL CHEMISTRY》 * |
| SCOTT,S: "Determination of derivatized urea herbicides in water by solid-phase extraction, methylation and gas chromatography with a nitrogen–phosphorus detector", 《ANALYST》 * |
| 张天娇 等: "血清尿素同位素稀释气相色谱质谱法的建立和研究", 《中华检验医学杂志》 * |
| 张新春 等: "《临床检验技术与临床应用》", 30 June 2018, 上海交通大学出版社 * |
| 李国辉 等: "气相色谱-燃烧-同位素比值质谱法测定发酵液中尿素δ~(13)C和δ~(15)N", 《质谱学报》 * |
| 王晶 等: "适用于代谢组学研究的人体尿液保存与气相色谱-质谱联用分析方法", 《环境与健康杂志》 * |
| 申世刚 等: "高效液相色谱-荧光检测器法测定白酒中尿素含量方法研究", 《酿酒科技》 * |
| 袁慧雅 等: "GC-MS衍生化方法快速检测生物样品中的氨", 《法医学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115078564A (en) * | 2022-05-06 | 2022-09-20 | 沈阳药科大学 | Method for ultra-fast quantifying amine substances in urine |
| CN115078564B (en) * | 2022-05-06 | 2024-05-31 | 沈阳药科大学 | Method for quantifying amine substances in urine at ultra-fast speed |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113049699B (en) | Method for detecting biphenyl anhydride and related substances thereof and application | |
| CN110066655B (en) | Silver-doped carbon quantum dot and preparation method and application thereof | |
| CN101458238B (en) | Method for detecting Clenbuterol residual quantity in hair | |
| CN111239318A (en) | Method for determining urea content in biological sample based on combination of GC-MS and enzymatic chemical method | |
| CN108414661B (en) | Derivatization gas chromatography-mass spectrometry method for detecting ammonia content in biological sample | |
| CN110763535B (en) | Sample preparation method for measuring nitrite nitrogen 15 isotopic abundance | |
| CN111505152B (en) | Method for measuring residual amount of pentachlorophenol in pig hair | |
| CN114354800B (en) | Method for analyzing acetyl bromide content in cefuroxime axetil | |
| CN113311094B (en) | Method for simultaneously detecting androgen compounds in health food by liquid chromatography-tandem mass spectrometry | |
| CN103675145A (en) | Method for detecting residual quantity of cimaterol in pig hairs | |
| CN115902036A (en) | Method for determining urea content in allantoin aluminum | |
| CN111307992B (en) | Pre-column derivative liquid chromatography-mass spectrometry analysis method for quantitatively detecting organic acid in PM2.5 | |
| CN115326909A (en) | Method for detecting free fatty acid in human serum | |
| CN106770740A (en) | The detection method of N nitrosodimethylamines in a kind of malt or beer | |
| CN113138246A (en) | Detection method for targeted determination of short-chain fatty acid in biological sample | |
| CN117169386B (en) | Method for detecting content of laminaria oligosaccharide in health-care product | |
| CN108802244A (en) | A method of measuring methenamine content in rice and flour and product | |
| CN110174482A (en) | It is a kind of while measuring C14H25N4NaO11P2 and nine kinds of UPLC analysis methods in relation to substance | |
| CN112198254A (en) | Method for measuring existence form and content of semicarbazide in shellfish tissue | |
| CN117871750A (en) | Method for determining content of lysine | |
| CN114577943B (en) | Method for indirectly measuring aspartic acid and glutamic acid in infant formula milk powder and prepared milk powder | |
| CN116818957B (en) | Method for detecting content of sodium pentachlorophenolic acid and metabolite thereof in live pig sample | |
| CN113804785A (en) | Method for detecting urea by gas chromatography-mass spectrometry | |
| CN115219643A (en) | Detection method for confirming furacilin source and non-furacilin source semicarbazide in aquatic product | |
| Li et al. | Determination of phenobarbital in human urine and serum using flow injection chemiluminescence |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200605 |