CN114184720A - Method for measuring urea content in culture medium - Google Patents
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- CN114184720A CN114184720A CN202111518720.3A CN202111518720A CN114184720A CN 114184720 A CN114184720 A CN 114184720A CN 202111518720 A CN202111518720 A CN 202111518720A CN 114184720 A CN114184720 A CN 114184720A
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- 238000000034 method Methods 0.000 title claims abstract description 52
- 239000001963 growth medium Substances 0.000 title claims abstract description 35
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000004949 mass spectrometry Methods 0.000 claims abstract description 15
- 238000001514 detection method Methods 0.000 claims abstract description 11
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims abstract description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000005055 methyl trichlorosilane Substances 0.000 claims description 9
- JLUFWMXJHAVVNN-UHFFFAOYSA-N methyltrichlorosilane Chemical compound C[Si](Cl)(Cl)Cl JLUFWMXJHAVVNN-UHFFFAOYSA-N 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 9
- VIYXXANHGYSBLY-UHFFFAOYSA-N trimethylsilyl 2,2,2-trifluoroacetate Chemical compound C[Si](C)(C)OC(=O)C(F)(F)F VIYXXANHGYSBLY-UHFFFAOYSA-N 0.000 claims description 7
- 239000001307 helium Substances 0.000 claims description 6
- 229910052734 helium Inorganic materials 0.000 claims description 6
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 239000012086 standard solution Substances 0.000 claims description 5
- 150000003672 ureas Chemical class 0.000 claims description 5
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- 241000195649 Chlorella <Chlorellales> Species 0.000 claims description 2
- 241000195619 Euglena gracilis Species 0.000 claims description 2
- 238000000861 blow drying Methods 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
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- 230000035945 sensitivity Effects 0.000 abstract description 8
- 238000001212 derivatisation Methods 0.000 abstract description 7
- 238000006243 chemical reaction Methods 0.000 abstract description 5
- IJOOHPMOJXWVHK-UHFFFAOYSA-N chlorotrimethylsilane Chemical compound C[Si](C)(C)Cl IJOOHPMOJXWVHK-UHFFFAOYSA-N 0.000 abstract description 4
- DCERHCFNWRGHLK-UHFFFAOYSA-N C[Si](C)C Chemical compound C[Si](C)C DCERHCFNWRGHLK-UHFFFAOYSA-N 0.000 abstract description 3
- 230000009471 action Effects 0.000 abstract description 3
- 125000003277 amino group Chemical group 0.000 abstract description 3
- 125000004435 hydrogen atom Chemical group [H]* 0.000 abstract description 3
- 239000002253 acid Substances 0.000 abstract description 2
- 239000011230 binding agent Substances 0.000 abstract description 2
- XCOBLONWWXQEBS-KPKJPENVSA-N N,O-bis(trimethylsilyl)trifluoroacetamide Chemical compound C[Si](C)(C)O\C(C(F)(F)F)=N\[Si](C)(C)C XCOBLONWWXQEBS-KPKJPENVSA-N 0.000 abstract 1
- 238000004737 colorimetric analysis Methods 0.000 description 7
- 239000000126 substance Substances 0.000 description 6
- 230000014759 maintenance of location Effects 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
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- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000004885 tandem mass spectrometry Methods 0.000 description 2
- BGNGWHSBYQYVRX-UHFFFAOYSA-N 4-(dimethylamino)benzaldehyde Chemical compound CN(C)C1=CC=C(C=O)C=C1 BGNGWHSBYQYVRX-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- XSQUKJJJFZCRTK-VMIGTVKRSA-N bis(azanyl)methanone Chemical compound [15NH2][13C]([15NH2])=O XSQUKJJJFZCRTK-VMIGTVKRSA-N 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- OBRMNDMBJQTZHV-UHFFFAOYSA-N cresol red Chemical compound C1=C(O)C(C)=CC(C2(C3=CC=CC=C3S(=O)(=O)O2)C=2C=C(C)C(O)=CC=2)=C1 OBRMNDMBJQTZHV-UHFFFAOYSA-N 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
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- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- FSEUPUDHEBLWJY-HWKANZROSA-N diacetylmonoxime Chemical compound CC(=O)C(\C)=N\O FSEUPUDHEBLWJY-HWKANZROSA-N 0.000 description 1
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- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- CUILPNURFADTPE-UHFFFAOYSA-N hypobromous acid Chemical compound BrO CUILPNURFADTPE-UHFFFAOYSA-N 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001948 isotopic labelling Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
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- 238000004476 mid-IR spectroscopy Methods 0.000 description 1
- 238000002552 multiple reaction monitoring Methods 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229940054441 o-phthalaldehyde Drugs 0.000 description 1
- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical compound O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
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Images
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- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- 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)
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Abstract
The invention relates to the technical field of biological detection, in particular to a method for determining urea content in a culture medium. Under the action of pyridine as an acid binding agent, urea and BSTFA (containing 1% TMCS) are subjected to derivatization reaction, hydrogen groups in two amino groups in the urea are respectively replaced by trimethyl silicon, the original urea with the molecular weight of 60.06 is derivatized to form a derivative with the molecular weight of 204.11, a mass spectrogram detected by gas-mass spectrometry is formed, the urea in a culture medium can be qualitatively analyzed according to the mass spectrogram, and the urea in the culture medium can be quantitatively analyzed according to a standard curve of a standard sample and a gas-mass spectrometry detection result. The method has the advantages of easy operation, short time, less interference and high sensitivity.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a method for determining urea content in a culture medium.
Background
Microalgae are used as lower plants and widely distributed in oceans, lakes, lands and the like. The microalgae have extremely high carbon sequestration efficiency and have extremely high potential contribution value for realizing the goals of carbon peak arrival and carbon neutralization. The urea is usually used as a nitrogen source of the microalgae, and the growth of the microalgae is influenced by the content of the urea, so that the accurate qualitative and quantitative determination of the urea change in a microalgae culture medium is particularly critical to the growth of the microalgae. However, microalgae can secrete some metabolites into water, and the concentration change of urea cannot be accurately detected by using a conventional method.
At present, there are many methods for measuring urea at home and abroad, and the methods are summarized as follows: direct colorimetry, indirect colorimetry, chromatography, mid-infrared spectroscopy, and the like. 1) The direct colorimetric method is that some special reagent is directly acted with urea to produce color reaction, and the absorbance is measured in spectrophotometer at specific wavelength and converted into urea content. The more common direct colorimetric methods include 3 methods of diacetyl monoxime method, o-phthalaldehyde method and p-dimethylaminobenzaldehyde method, while the nitrite-griss reagent method can only be qualitative and not quantitative at the present stage. 2) The indirect colorimetric method is to detect the product of urea after enzymolysis and then convert the product into urea content. But with much interference. Currently, indirect colorimetry methods such as urease-bosch colorimetry, enzyme coupling method, cresol red method, enzyme electrode method and the like are commonly used. 3) The chromatographic analysis method has the characteristics of high speed, high efficiency, high sensitivity and the like, is suitable for separating complex mixtures, realizes simultaneous determination of multiple components, and is widely applied to the fields of environmental monitoring, food, agriculture and the like. The chromatographic determination of urea is based, for a review of the literature, mainly on liquid chromatographic correlation techniques.
Chromatography: the chromatographic analysis method has the characteristics of high speed, high efficiency, high sensitivity and the like, is suitable for separating complex mixtures, realizes simultaneous determination of multiple components, and is widely applied to the fields of environmental monitoring, food, agriculture and the like. The chromatographic determination of urea is based, for a review of the literature, mainly on liquid chromatographic correlation techniques. 1) The method is mainly applied clinically in recent years by measuring a luminophore generated by the reaction of urea and hypobromous acid under a certain condition and detecting the concentration of urea by high performance liquid chromatography combined with a fluorescence detector, but the method has general accuracy at present, a linear correlation coefficient r only reaches 0.93, and interference exists, so the method needs further improvement. 2) The liquid chromatography isotope dilution tandem mass spectrometry adopts Urea isotope labeling substance (Urea-13C, 15N2) as an internal standard, ZORBAXRX-SIL as a chromatographic separation column, acetonitrile solution as a mobile phase, and uses a multiple reaction monitoring mode measurement of electrospray triple quadrupole tandem mass spectrometry and a bracket method of isotope dilution for quantification. In the serum urea detection, the pretreatment operation of the method is simple and quick, complex derivatization reaction is not needed, the quantification is accurate, and the repetition precision is high. The method is verified by adopting the NIST serum standard substance in the United states, and the relative deviation of the determination result and the standard value is less than 0.5 percent, thereby proving that the method has higher accuracy.
However, the above methods still have the defects of long time consumption, complex operation, low sensitivity, much interference, incapability of simultaneous quantification and characterization and the like. Therefore, the invention develops a method which is easy to operate, short in operation time, less in interference, high in sensitivity, qualitative and quantitative in urea based on gas phase-mass spectrometry combined detection, and provides a technical support for detecting urea for scientific research and application in various fields, particularly microalgae culture.
Disclosure of Invention
In view of the above, the present invention provides a method for determining urea, which can quantitatively detect urea and has the characteristics of easy operation, short operation time, less interference and high sensitivity.
In order to achieve the above object, the present invention provides the following technical solutions:
a method for determining the urea content of a culture medium, comprising the steps of:
1) taking a culture medium sample to be detected, sequentially adding pyridine and N, O-bis (trimethylsilyl) trifluoroacetic acid solution containing methyltrichlorosilane after membrane filtration and helium blow drying, and performing water bath after oscillation to obtain a liquid to be detected;
2) detecting the urea derivative with the formula I structure in the liquid to be detected by adopting gas-mass spectrometry, and calculating the urea content in the culture medium;
in some embodiments, the culture medium is a microalgae culture medium, and may be a culture medium in which microalgae are not cultured or a culture medium in which microalgae are cultured; the microalgae comprise Euglena gracilis and/or Chlorella.
In some embodiments, the volume ratio of the pyridine to the methyltrichlorosilane-containing N, O-bis (trimethylsilyl) trifluoroacetic acid solution in step 1) is 4: 1.
In some embodiments, the time of the shaking in step 1) is 30 seconds.
In some embodiments, the water bath in step 1) is a 50 ℃ water bath for 5 min.
In some embodiments, the membrane filtration in step 1) has a filter membrane pore size of 0.22 μm.
In some embodiments, the mass fraction of methyltrichlorosilane in the methyltrichlorosilane-containing N, O-bis (trimethylsilyl) trifluoroacetic acid solution in step 1) is 1%.
In some embodiments, the conditions for gas-mass spectrometric detection are:
the temperatures of a sample inlet, a quadrupole rod, an ion source and a transmission line of the gas-mass spectrometry are respectively 150 ℃, 280 ℃ and 320 ℃;
the temperature rising procedure is as follows: maintaining the temperature at 60 ℃ for 1-3 minutes, heating to 180 ℃ for 1-3 minutes, and operating for 12-15 minutes in total;
the split ratio is 100: 1;
the sample injection volume is 1 mul;
the flow rate of pure helium is 1.2mL/min
The scanning mode is set, 50-500 m/z.
The method also comprises the following steps before the step 2): preparing a standard solution with gradient concentration, detecting the standard solution by adopting a gas-mass spectrometry, and establishing a standard curve by taking a peak area as a vertical coordinate and a urea concentration as a horizontal coordinate.
In some embodiments, the gradient concentration standard solution consists of water and urea, wherein the concentration of the urea is 0.05-1 mg/ml; preferably, the concentration of urea may be specifically 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.1mg/ml and 0.05 mg/ml.
Under the action of pyridine, urea and BSTFA (containing 1% TMCS) are subjected to derivatization reaction, hydrogen groups in two amino groups in the urea are respectively replaced by trimethyl silicon, the original urea with the molecular weight of 60.06 is derivatized to form a derivative with the molecular weight of 204.11, a mass spectrogram detected by gas-mass spectrometry is formed, the urea in a culture medium can be qualitatively analyzed according to the mass spectrogram, the urea in the culture medium can be quantitatively analyzed according to a standard curve of a standard sample and a gas-mass spectrometry detection result, and the method is easy to operate, short in operation time, less in interference and high in sensitivity.
Drawings
FIG. 1 shows the detection principle of the present invention and the mass spectrum after urea derivatization;
FIG. 2 shows the retention time of a standard;
FIG. 3 is the time of the urea peak in the microalgal culture medium;
FIG. 4 is a standard curve and R correlation coefficient established from the concentration gradient and peak area of the standard;
FIG. 5 is a graph showing the calculation of urea concentration in the microalgal culture medium based on the standard curve and the peak area of the sample.
Detailed Description
The invention provides a method for measuring urea content in a culture medium. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1
Preparing a sample
1. Filtering 1mL of microalgae culture medium by using a 0.22-micron membrane, and transferring 100 mu l of microalgae culture medium into an Agilent flask;
2. drying the supernatant by using common helium gas for about 20 minutes;
3. add 400. mu.l pyridine to nitrogen blown bottles, add 100. mu.l N, O-bis (trimethylsilyl) trifluoroacetic acid (BSTFA) (containing 1% methyltrichlorosilane, TMCS), shake for 30 seconds;
4. water bath is carried out for 5 minutes at the temperature of 50 ℃ in a water bath kettle;
5. and (4) performing machine detection on the sample.
(II) establishing a standard sample curve
1. Weighing 10mg of urea into an Agilent bottle, adding 1ml of deionized water, and fully dissolving;
2. preparing urea with concentration gradient of 1,0.5,0.25,0.1,0.05mg/ml and volume of 500 μ l; take 100. mu.l and proceed according to the above 2-5 steps.
(III) gas-Mass Spectrometry conditions
The temperatures of the gas-mass spectrum sample inlet, the four-stage rod, the ion source and the transmission line are respectively 150 ℃, 280 ℃ and 320 ℃. The temperature of the case is maintained for 1 to 3 minutes at 60 ℃, the temperature is increased to 180 ℃ and maintained for 1 to 3 minutes, and the operation is carried out for 12 to 15 minutes in total. The split ratio is as follows: 100:1. The flow rate of pure helium is 1.2 mL/min. The injection volume was 1. mu.l. The scanning mode is set, 50-500 m/z.
Analysis of results
1. The principle of detecting the concentration of urea and a mass spectrogram after derivatization thereof (figure 1) are disclosed; urea is derivatized with BSTFA (containing 1% of TMCS) under the action of acid-binding agent pyridine, and hydrogen groups in two amino groups of the urea are respectively substituted by trimethyl silicon. Derivative of urea with original molecular weight of 60.06 forms derivative with molecular weight of 204.11, and finally forms mass spectrum detected by gas-mass spectrometry. According to the mass spectrogram, the qualitative analysis of urea can be carried out.
2. Determining the retention time of the standard sample (fig. 2); under the set gas-mass spectrometry conditions, the retention time of the substance after urea derivatization is 8.671, the peak pattern is normal, and the peak pattern is not mixed with other peaks. And quantifying the concentration of the urea derivative in the solution to be detected according to the retention time and the peak area, and further calculating to obtain the concentration of urea in the culture medium.
3. Determining the time to peak of urea derivatives in microalgal culture medium (figure 3); the retention time of the urea-derived substances in the microalgal culture medium is 8.671, which is the same as that of the standard sample in fig. 2 and can be well separated from other peaks, which shows that under the set gas-mass spectrometry conditions, the urea in the microalgal culture medium can be not only derivatized but also not interfered by other substances. The result provides a basis for the quantification of urea in the microalgae culture medium.
4. Preparing and analyzing a linear regression curve equation and an R correlation coefficient according to the concentration gradient and the peak area of the urea in the standard sample (figure 4); the result shows that substances subjected to derivatization with the concentration of urea within the range of 0.1-1 mg/mL have positive correlation with peak area, wherein R2The correlation coefficient reached 0.9956, meeting the quantitative standard.
5. The concentration of urea in the microalgal culture medium was calculated from the regression curve equation and the peak area of the sample (fig. 5). The results show that the concentration of urea in the culture medium was 0.98g/L at day 0 and decreased by 0.74g/L (24.49%) by day 3 when the microalgae were cultured by the method, indicating that the microalgae absorbed the urea in the culture medium. By the later culture period (day 6), the concentration of urea in the culture medium is reduced to 0.32g/L (67.34%), which indicates that the urea absorption speed of the microalgae is increased by the later culture period. The result provides a research basis for optimizing the microalgae culture method. Meanwhile, the invention explains the qualitative and quantitative application of the urea in the microalgae culture successfully.
The results show that the urea concentration is in the range of 0.1-1 mg/mL and has positive correlation with the peak area, wherein R2The correlation coefficient reached 0.9956, meeting the quantitative standard. The concentration of urea in the culture medium can still be detected when the concentration of urea is 0.1mg/mL, which shows that the method for detecting urea has extremely high sensitivity, and in addition, as can be seen from FIG. 3, a target peak (urea derivative) and other miscellaneous peaks have good separation effect, which shows that the method has the characteristic of less detection interference.
Claims (10)
1. A method for determining the urea content in a culture medium is characterized by comprising the following steps:
1) taking a culture medium sample to be detected, sequentially adding pyridine and N, O-bis (trimethylsilyl) trifluoroacetic acid solution containing methyltrichlorosilane after membrane filtration and helium blow drying, and performing water bath after oscillation to obtain a liquid to be detected;
2) detecting the urea derivative with the formula I structure in the liquid to be detected by adopting gas-mass spectrometry, and calculating the urea content in the culture medium;
2. the method of claim 1, wherein the culture medium is a microalgae medium; the microalgae comprise Euglena gracilis and/or Chlorella.
3. The process of claim 1, wherein the volume ratio of the pyridine to the methyltrichlorosilane-containing N, O-bis (trimethylsilyl) trifluoroacetic acid solution in step 1) is 4: 1.
4. The method as claimed in claim 1, wherein the oscillation time in step 1) is 30 seconds.
5. The method according to claim 1, wherein the water bath in step 1) is a 50 ℃ water bath for 5 min.
6. The method as claimed in claim 1, wherein the membrane filtration in step 1) has a membrane pore size of 0.22 μm.
7. The method according to claim 1, wherein the mass fraction of methyltrichlorosilane in the methyltrichlorosilane-containing N, O-bis (trimethylsilyl) trifluoroacetic acid solution in step 1) is 1%.
8. The method of claim 1, wherein the gas-mass spectrometric detection conditions are:
the temperatures of a sample inlet, a quadrupole rod, an ion source and a transmission line of the gas-mass spectrometry are respectively 150 ℃, 280 ℃ and 320 ℃;
the temperature rising procedure is as follows: maintaining the temperature at 60 ℃ for 1-3 minutes, heating to 180 ℃ for 1-3 minutes, and operating for 12-15 minutes in total;
the split ratio is 100: 1;
the sample injection volume is 1 mul;
the flow rate of pure helium is 1.2mL/min
The scanning mode is set, 50-500 m/z.
9. The method of claim 1, wherein step 2) is preceded by: preparing a standard sample solution with gradient concentration, detecting the standard solution by adopting a gas-mass spectrometry, and establishing a standard curve by taking a peak area as a vertical coordinate and a urea concentration as a horizontal coordinate.
10. The method according to claim 9, wherein the gradient concentration standard solution is composed of water and urea, and the concentration of the urea is 0.05-1 mg/ml.
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| 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 |
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| 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 |
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| Title |
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| 王宇成主编: "《最新色谱分析检测方法及应用技术实用手册 第4卷》", 银声音像出版社 * |
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