CN111239318A - Method for determining urea content in biological sample based on combination of GC-MS and enzymatic chemical method - Google Patents

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

A method for the determination of urea content in biological samples based on the combination of GC-MS and enzymatic chemistry

technical field

The invention belongs to the field of biological material detection, in particular to a method for detecting urea content in biological samples based on GC-MS and an enzymatic chemical method.

Background technique

Urea alias carbonic diamide, carbonamide, urea. It is an organic compound composed of carbon, nitrogen, oxygen and hydrogen. Its chemical formula is CON ₂ H ₄ , CO(NH ₂ ) ₂ or CN ₂ H ₄ O, and the international generic drug name is Carbamide. Appearance is white crystal or powder. Urea is synthesized in the liver and is a nitrogen-containing metabolite excreted by mammals. This metabolic process is called the urea cycle. Urea is the first organic compound obtained by artificially synthesizing inorganic substances. Chemical formula: CO(NH ₂ ) ₂ , molecular weight 60.06, colorless or white needle-like or rod-like crystals, industrial or agricultural products are white slightly reddish solid particles, odorless and tasteless. The nitrogen content is about 46.67%, and the density is 1.335g/cm3. Soluble in water, alcohol, insoluble in ether, chloroform, weakly alkaline. CAS No.: 57-13-6, molecular weight: 60.05; melting point: 131-135 ℃; boiling point: 196.6, refractive index: n20/D 1.40; flash point: 72.7 ℃, density: 1.335; water solubility: 1080g/L ( 20°C). Chemical properties: It can react with acids to form salts. There is hydrolysis. The condensation reaction can be carried out at high temperature to generate biuret, triuret and cyanuric acid.

Urea was first synthesized by the German Wohler in 1828 and was widely used in the 1950s as a non-protein nitrogen source for ruminants. It is a low-toxic substance, with an oral LD ₅₀ of 15g/kg for rats and an oral LD ₅₀ of 11.5g/kg for mice. Ruminants can use urea to synthesize protein due to their unique digestive system structure. There are a large number of microorganisms with urease activity in the rumen of cattle. When cattle eat feed containing urea, urea can be decomposed into ammonia. These microorganisms can synthesize bacterial protein from ammonia and carbohydrates through ammoniation reaction or transamination reaction ( MCP), and then MCP is digested and absorbed in the bovine true stomach and small intestine. Although urea is a low-toxic substance, due to the fact that microorganisms in the rumen of cattle can decompose urea to produce ammonia, improper use will cause cattle poisoning and death. The main reasons are as follows: improper management of urea, causing urea to be stolen or ingested by cattle; excessive urea content in feed or urea and feed are not mixed evenly; feeding urease-containing feed at the same time feeding soybean cake or broad bean containing urease ; Cattle drink a lot of water after eating feed containing urea. All of the above can lead to the rapid decomposition of urea and the production of a large amount of ammonia, so that the microorganisms in the rumen of cattle cannot fully utilize the ammonia, so that the ammonia is absorbed into the blood through the rumen wall and causes poisoning. Cases of bovine urea poisoning are often encountered in forensic identification work, and such problems cannot be well resolved by the prior art reporting methods. Therefore, research and development of a rapid and accurate method for detecting urea in biological samples has always been a new topic to be solved.

SUMMARY OF THE INVENTION

Urea cannot be directly detected by gas chromatography-mass spectrometry (GC-MS) due to its high molecular polarity and difficulty in gasification. In view of the problems existing in the prior art, the object of the present invention is to provide an accurate and simple method for detecting urea in biological samples, after the urea is treated with urease and a derivatizing reagent, and a method for indirectly detecting urea using GC-MS, The urea content in the biological sample can be detected in a targeted manner, the interference of many external factors can be eliminated, the detection sensitivity is improved, the detection steps are greatly simplified, and time and cost are saved.

In order to achieve the above objects, the present invention adopts the following technical solutions.

A method for determining urea content in a biological sample based on the combination of GC-MS and enzymatic chemistry, specifically comprising the following steps.

(1) Detection conditions.

Gas chromatography-mass spectrometry: gas chromatography-mass spectrometry (GC-MS) was used.

Chromatographic column: HP-5MS capillary column, specification: 30m×0.25mm, 0.25μm.

The front inlet temperature, EI ion source temperature, quadrupole temperature and interface temperature were 280 °C, 150 °C, 230 °C, and 280 °C, respectively.

The carrier gas was high-purity helium (99.999%), the flow rate was 1.0 ml/min, and 1 μL was injected in a splitless manner.

Heating program: the initial column temperature was 60°C, held for 1 min, then increased to 280°C at a heating rate of 20°C/min, and held for 11 min.

(2) Sample processing.

Take 0.5mL of 2 blood samples or other biological samples to be tested and put them in 10mL centrifuge tubes respectively, add 10μL of 200μg/mL internal standard, add 1mL of PBS buffer, then add 0.5mL of 22U/mL urease, and add 0.5mL to the other part PBS buffer, sealed and placed in a water bath at 45°C for 30 min. At this point, urea can be completely decomposed into ammonia by urease. After the sample is cooled, add 1 mL of 3 mol/L NaOH solution, then add 50 μL of derivatization reagent heptafluorobutyryl chloride, and bathe it in a water bath at 40 °C for 10 min. Derivatization of the target. After the water bath, 2 mL of ethyl acetate: n-hexane = 9:1 was added, followed by shaking on a vortex for 5 min to extract, and centrifuged at 8000 rpm/min for 5 min. Take 1 mL of the organic solvent in the upper layer, blow dry nitrogen to near dryness, and then take 1 μL for injection and analysis by GC-MS.

(3) Atlas analysis.

Derivatized product heptafluorobutanamide, retention time 3.0min, characteristic mass numbers are m/z 214 (molecular ion peak), m/z194, m/z 166, m/z 146, m/z 100, m/z 69 and m/z 44 (base peak).

(4) Preparation of standard working curve sample solution.

Urease solution configuration: Dissolve urease in PH7.4 Phosphate Buffered Saline (PBS) and configure it into a 22U/ml urease solution (because the activity of urease will decrease with time, try to use it as needed).

Internal standard configuration: take an appropriate amount of 1,6-hexanediamine, dilute it with 0.1mol/L HCL to 200μg/ml, and store at 4°C.

Urea working solution configuration: Dilute urea into standard solution of urea with different concentrations according to the required linear range, sterilize by ultrasonic cleaning machine for 10min, and store in -20℃.

(5) Content calculation.

The standard working curve sample solution prepared in step (4) is processed according to the sample processing method in step (2), and then injected for analysis. Select the selected ion chromatogram of detection m/z 100, take the peak area of the derivatized product heptafluorobutanamide as the ordinate, and take the mass concentration of urea added in the blood as the abscissa, calculate the regression equation, when calculating the content, the The peak area of heptafluorobutanamide in the sample is substituted into the regression equation to obtain the content of the sample to be tested.

The biological samples are urine, gastric contents or plasma samples.

The minimum detection limit of this method was calculated with S/N=3, the minimum detection limit for blood urea was 0.05 μg/ml, and the quantification limit was 0.10 μg/ml.

Compared with the prior art, the beneficial effects of the present invention are as follows.

1. The detection principle of the method for determining the urea content in a biological sample based on the combination of GC-MS and an enzymatic chemical method provided by the present invention is to indirectly determine the urea in the biological sample.

2. The method for determining urea content in biological samples based on the combination of GC-MS and enzymatic chemistry provided by the present invention adopts derivatized mass spectrometry instead of traditional spectroscopy. It can effectively reduce some external factors such as blood coagulation state, hemolysis, and reagent interference.

3. The method for determining the urea content in a biological sample based on a combination of GC-MS and an enzymatic chemical method provided by the present invention uses fewer types of reagents, and the operation is simple and fast.

4. The method for determining urea content in a biological sample based on a combination of GC-MS and an enzymatic chemical method provided by the present invention can detect biological materials including urine, gastric contents or plasma samples.

5. The method for detecting urea content in biological samples based on the combination of GC-MS and enzymatic chemical method provided by the present invention is a method for detecting urea by GC-MS, the method utilizes the characteristic that urease can specifically decompose urea, and converts urea into Ammonia, and then use heptafluorobutyryl chloride to react with it relatively quickly and completely, so that urea, which cannot be detected by GC-MS, can be detected and quantified. The sensitivity of this method is higher than that of spectroscopy, and the linear relationship is better.

Description of drawings

Figure 1 is the chemical formula of urease decomposing urea.

Figure 2 is the derivatization reaction of derivatized ammonia and heptafluorobutyryl chloride (main reaction).

Figure 3 shows the effect of different alkaline systems on the derivatization effect.

Figure 4 is the effect of different temperatures on the derivatization effect.

Figure 5 is the effect of different reaction times on the derivatization effect.

Figure 6 is the determination of urea by gas chromatography-mass spectrometry (a typical chromatogram).

Figure 7 is the determination of urea by gas chromatography mass spectrometry (typical mass spectrum).

Fig. 8 is a linear relationship diagram of urea addition in blood.

Detailed ways

The specific embodiments of the present invention will be described in further detail below with reference to 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 embodiment adopts conventional experimental techniques, which are familiar to those skilled in the art, and can be performed according to the description provided by the material manufacturer according to this embodiment.

Example 1 Optimization of the method-related reaction process and conditions for the determination of urea content in biological samples based on the combination of GC-MS and enzymatic chemistry.

Urease is a nickel-containing oligomerase with an optimum pH of 7.4, with absolute specificity, which can specifically catalyze the decomposition of urea into ammonia and carbon dioxide. The reaction process is shown in Figure 1. Ammonia produced by the decomposition of urea by urease then reacts with the derivatizing reagent heptafluorobutyryl chloride to produce heptafluorobutanamide, which is detected by gas chromatography-mass spectrometry. The reaction equation is shown in Figure 2. In this reaction system, there are two steps of equilibrium that need to be controlled. First, the amount of PBS buffer solution in the reaction system, in the process of urease decomposing urea, one molecule of urea generates two molecules of ammonia, and combines with water molecules to generate ammonia water, so that the urease reaction environment becomes alkaline, and urease is in pH. Inactivated under conditions of about 9. When the urea content in the sample is too high, the urease will be inactivated before all the urea is decomposed. In this case, the volume of PBS buffer can be appropriately increased. A total of 2 mL of PBS buffer is added to the reaction system of the present invention, which can completely decompose a sample with a urea concentration of about 6 mg/mL; second, the amount of heptafluorobutyryl chloride and the amount of lye used, heptafluorobutyryl chloride reacts with ammonia and water to produce acid , the reaction product heptafluorobutanamide exists in the form of ions under acidic conditions and cannot be extracted by organic solvents. For this reason, an appropriate amount of lye should be added before the derivatization, so that the lower aqueous solution just becomes weakly alkaline after the reaction, so that the substance to be tested heptafluorobutyramide is freed, and the lye is added to neutralize the acid and alkali. to the effect of prompting the reaction to proceed in the positive direction. In the second derivatization step, we investigated the effects of different alkaline systems, different temperatures, and different reaction times on the entire detection process. The results are shown in Figures 3-5. Finally, when the pH was 14, 40 degrees Celsius was selected. React under such derivatization conditions for 5 min.

In addition, when using an organic solvent for extraction, since ethyl acetate and water have close densities and the reaction system is alkaline, if ethyl acetate alone is used for extraction, it is easy to cause emulsification. When using ethyl acetate: n-hexane = 9:1 as the extract Emulsification can be well avoided.

2. Chromatogram-mass spectrogram (minimum detection limit).

After enzymatic hydrolysis of urea, it reacts with heptafluorobutyryl chloride, and the mass spectrum of the typical product obtained is shown in Figure 6-7.

3. Standard curve (taking blood as an example).

Accurately draw an appropriate amount of urea standard working solution, add it to 0.5mL blank plasma, and configure the urea concentration as 0.1 μg/mL, 10 μg/mL, 200 μg/mL, 1 mg/mL, 3 mg/mL, 5 mg/mL. The blood samples were pretreated according to the above operation steps. After subtracting the total ammonia in the blank plasma added with urease, taking the concentration as the abscissa (x), the difference between the derivatized product of ammonia (heptafluorobutanamide) and the peak of the derivatized product of the internal standard The ratio is the ordinate (y), and the linear relationship is shown in Fig. 8, and the one-variable linear equation is obtained as y=0.0793x+1.9703 (R ² =0.9994, P<0.0001).

4. Methodological investigation.

4.1 Recovery and precision.

The intra-day and inter-day precisions were investigated for the three samples, respectively, and the ratio between the peak area of heptafluorobutyramide and the peak area of the internal standard was calculated. The intra-day precision RSD=3.7-6.2%; the inter-day precision RSD=4.0%-6.8%. 10μg/mL, 1mg/mL, and 5mg/mL of urea were added to the 0.5mL blank sample, and the urea originally present in the blank blood sample was subtracted after derivatization to calculate the recovery rate, and the measurements were performed in parallel three times. The average recovery rates of low, medium and high concentration groups were between 90.3% and 97.5%.

4.2 Stability test.

Based on the extremely active chemical properties of heptafluorobutyryl chloride, the covalent bond formed by the nucleophilic substitution reaction between ammonia and it is very stable and is not easy to break under normal external conditions. After comparing the peak area ratio of heptafluorobutyramide to the internal standard derivatized product in the samples after pretreatment at 0h, 12h, 24h, 48h and 96h, it was found that the ratio was relatively stable within 0-96h P=0.8621 ( P>0.05), RSD=1.65%.

Example 2 Practical case application.

A method for determining urea content in a biological sample based on the combination of GC-MS and enzymatic chemistry, specifically comprising the following steps.

(1) Detection conditions.

Gas chromatography-mass spectrometry: gas chromatography-mass spectrometry (GC-MS) was used.

Chromatographic column: HP-5ms capillary column, specification: 30m×0.25mm, 0.25μm.

The front inlet temperature, EI ion source temperature, quadrupole temperature and interface temperature were 280 °C, 150 °C, 230 °C, and 280 °C, respectively.

The carrier gas was high-purity helium (99.999%), the flow rate was 1.0 ml/min, and 1 μL was injected in a splitless manner.

Heating program: the initial column temperature was 60°C, held for 1 min, then increased to 280°C at a heating rate of 20°C/min, and held for 11 min.

(2) Sample processing.

Take 0.5 mL of the bovine blood sample to be tested and put it into a 10 mL centrifuge tube, add 10 μL of 200 μg/mL internal standard, add 1 mL of PBS buffer, and then add 0.5 mL of 22 U/mL urease (add 0.5 mL of PBS buffer for the other), After sealing, they were placed in a 45°C water bath and incubated for 30 min. At this point, urea can be completely decomposed into ammonia by urease. After the sample is cooled, add 1 mL of 3 mol/L NaOH solution, then add 50 μL of derivatization reagent heptafluorobutyryl chloride, and bathe it in a water bath at 40 °C for 10 min. Derivatization of the target. After the water bath, 2 mL of ethyl acetate: n-hexane = 9:1 was added, followed by shaking on a vortex for 5 min to extract, and centrifuged at 8000 rpm/min for 5 min. Take 1 mL of the organic solvent in the upper layer, blow dry nitrogen to near dryness, and then take 1 μL to inject and analyze by GC-MS.

(3) Atlas analysis.

Derivatized product heptafluorobutanamide, retention time 3.0min, characteristic mass number is m/z 214 (molecular ion peak), m/z 194, m/z 166, m/z 146, m/z 100, m/z 69 and m/z 44 (base peak).

(4) Preparation of standard working curve sample solution.

Urease solution configuration: Dissolve urease in PH7.4 Phosphate Buffered Saline (PBS) and configure it into a 22U/ml urease solution (because the activity of urease will decrease with time, try to use it as needed).

Internal standard configuration: take an appropriate amount of 1,6-hexanediamine, dilute it with 0.1mol/L HCL to 200μg/ml, and store at 4°C.

Urea working solution configuration: Dilute urea into urea standard solutions of different concentrations according to the required linear range, the specific concentrations are 0.1μg/ml, 10μg/ml, 200μg/ml, 2000μg/ml, 5000μg/ml, ultrasonic cleaning machine ultrasonic removal. Bacteria were stored at -20°C for 10 min.

(5) Content calculation.

The standard working curve sample solution prepared in step (4) is processed according to the sample processing method in step (2), and then injected for analysis. Select the selected ion chromatogram to detect m/z100, take the peak area of the derivatized product heptafluorobutanamide as the ordinate, and take the mass concentration of urea added in the blood as the abscissa, calculate the regression equation to obtain y=0.0793x+1.9703(R ² = 0.9994). When calculating the content, the peak area of heptafluorobutanamide in the sample to be tested is substituted into the regression equation to obtain the content of the sample to be tested.

The urea content in bovine blood in 7 cases was determined by the method based on the combination of GC-MS and enzymatic chemistry method provided above for the determination of urea content in biological samples. The specific urea content is shown in Table 1. The experimental results show that the determination method has strong applicability.

Table 1. The content of urea in bovine blood in 7 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.

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