CN113884609A - Method for detecting urea in white spirit based on ultra-high performance liquid chromatography fluorescence detector - Google Patents
Method for detecting urea in white spirit based on ultra-high performance liquid chromatography fluorescence detector Download PDFInfo
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Abstract
Provides a method for quantifying urea in white spirit based on an ultra-high performance liquid chromatography fluorescence detector. Optimizing the optimal derivative dosage, derivative time, elution gradient and sample amount through a single-factor test, and finally determining the detection method as follows: sucking 0.8mL of sample, and placing the sample in a brown liquid storage bottle; adding 0.6mL of 9-hydroxy xanthene alcohol solution of 0.02mol/L, 0.2mL of hydrochloric acid solution of 1.5mol/L, and uniformly mixing by oscillation; deriving for 40min in dark at room temperature; filtering with 0.22 μm organic filter membrane; taking the filtrate and using the filtrate for the measurement of the ultra-high performance liquid chromatograph. The average recovery rate of the invention is 102.46%; the precision of sample introduction is 1.44%, and the repeatability of the sample is 1.90%; LOD and LOQ are respectively 0.030mg/L and 0.093 mg/L; compared with the traditional method, the method provided by the invention is simple and rapid to operate, high in accuracy and good in stability, and provides a better choice for the quantification of urea in the white spirit.
Description
Technical Field
The invention belongs to the technical field of physical measurement and testing, and particularly relates to a method for detecting urea in white spirit based on an ultra-high performance liquid chromatography fluorescence detector.
Background
White spirit is one of the six distilled spirits in the world and is the traditional distilled spirit in China. It is naturally inoculated by various microorganisms, and subjected to long-time open solid fermentation and other unique processes, so that the unique characteristics, fragrance and taste of the fermented product are created. Is popular with consumers and is unique in distilled liquor in the world.
Ethyl Carbamate (Ethyl Carbamate, EC, H)2NCOOC2H5) Also called urethane, has genotoxicity and carcinogenicity, is a 2A class carcinogen, is commonly present in white spirit and other fermented foods, and is mainly formed by related precursor substances in the processes of food fermentation and storage. Research has shown that urea is an important precursor for the formation of EC in white spirit. In view of the wide existence of EC, genotoxicity and carcinogenicity, in order to actively deal with the potential safety hazard problem of EC to the white spirit industry and maintain the legal rights and interests of wide consumers, the risk assessment and content control of EC are made from the source, so that the generation of EC is blocked, the content control research of urea which is an important precursor substance is required, and the establishment of a rapid and accurate quantitative method of urea in white spirit is urgent.
At present, common methods for detecting urea in quantitative white spirit mainly comprise spectrophotometry, high performance liquid chromatography, a biological enzyme method and an online dialysis-flow injection photometry. The current disadvantages of the method are as follows: low efficiency, complex operation, limited accuracy and poor stability; especially, the derivation dosage and the derivation time directly influence the accuracy of the urea content detection. In view of this, the following improvement is proposed.
Disclosure of Invention
The technical problems solved by the invention are as follows: the method for detecting urea in white spirit based on the ultra-high performance liquid chromatography fluorescence detector is provided, and accurate detection of urea is realized in the shortest time through control of derivative dosage and derivative time; the quick, accurate and high-precision detection of the excellent separation degree of the white spirit urea is realized.
The technical scheme adopted by the invention is as follows: the method for detecting urea in white spirit based on the ultra-high performance liquid chromatography fluorescence detector is characterized by comprising the following steps:
s001, sucking a 0.8mL sample, and placing the sample in a brown liquid storage bottle;
s002, adding 0.6mL of 9-hydroxy xanthene alcohol solution of 0.02mol/L, 0.2mL of hydrochloric acid solution of 1.5mol/L, and uniformly mixing by oscillation;
s003, deriving for 40min in a dark place at room temperature;
s004, filtering by using a 0.22 mu m organic filter membrane;
and S005, taking the filtrate and using the filtrate for the measurement of the ultra-high performance liquid chromatograph.
In the above technical solution, further: the method comprises the following steps of:
preparing an acetic acid solution with a volume fraction of 1.0 percent: sucking 1.0mL of glacial acetic acid into a 100mL volumetric flask, adding water to a constant volume to a scale, and uniformly mixing;
preparing 0.02mol/L sodium acetate solution: weighing 1.64g of anhydrous sodium acetate, dissolving with ultrapure water, diluting to a constant volume of 1000mL, adjusting the pH to 7.2 with 1.0% of acetic acid solution, filtering, and ultrasonically degassing for 15min for later use;
preparing 1.5mol/L hydrochloric acid solution: sucking 6.2mL of concentrated hydrochloric acid into a 50mL volumetric flask, diluting to 50mL of volume with water, oscillating for 1min by a vortex mixer, and mixing uniformly;
preparing 0.02 mol/L9-hydroxyl xanthene alcohol solution: weighing 0.198g of 9-hydroxyl xanthene, adding n-propanol solution into a 50mL volumetric flask to fix the volume to 50mL scale mark, uniformly mixing, and performing ultrasonic treatment for 15min for later use;
preparing 1.0mg/mL urea standard stock solution: weighing 0.100g of urea standard substance, dissolving with absolute ethyl alcohol, fixing the volume to the scale mark of a 100mL volumetric flask, oscillating for 1min by a vortex mixer, and mixing uniformly for later use;
preparing 100mg/L urea intermediate solution: sucking 1mL of urea standard stock solution into a 10mL volumetric flask, metering the volume to 10mL scale mark with absolute ethyl alcohol, oscillating for 1min by a vortex mixer, and uniformly mixing for later use.
The technical proposal is thatFurther: the liquid chromatographic conditions of the ultra-high performance liquid chromatograph are as follows: a chromatographic column: 2.1x100mm, 2 μm C18A chromatographic column; column temperature: 35 ℃; detection wavelength: λ ex213nm, λ em308 nm; flow rate: 0.2 mL/min; sample introduction volume: 10 mu L of the solution; the mobile phase A is: pH 7.2, 0.02mol/L sodium acetate solution; the mobile phase B is as follows: and (3) acetonitrile.
In the above technical solution, further: the elution gradient of the mobile phase B is 0-4.5 min, and the elution gradient of the mobile phase B is 80-65%; 4.5-12 min, and 65-70% of a mobile phase B; 12-18 min, and 70-0% of mobile phase B; 18-21 min, and 0-80% of mobile phase B; and (5) 80% of mobile phase B for 21-25 min.
In the above technical solution, further: the method comprises the following experimental steps of sample injection quantity selection: after the standard working solution with the concentration of 5.0mg/L is derived, 2 muL, 5 muL and 10 muL are respectively set for sample injection analysis, and when the sample injection amount is 10 muL, the separation effect is best.
In the above technical solution, further: the method comprises the following steps of drawing a standard curve of urea: sucking 100mg/L urea intermediate solution with a certain volume into a 10mL volumetric flask, and preparing 0.05mg/L, 0.1mg/L, 0.4mg/L, 1mg/L, 5mg/L, 10mg/L and 20mg/L urea standard curve working solution by using absolute ethyl alcohol in sequence; oscillating for 1min by vortex mixer, mixing, and performing liquid phase analysis on the mixture at the present location; drawing the standard curve working solution by the area of the target chromatographic peak to obtain a regression equation and a correlation coefficient; the regression equation is that Y is 1.0 multiplied by 107X+3×106，R20.9994; the recovery rate of the added standard is 102.46%; the precision of sample injection is 1.44%, and the repeatability of the sample is 1.90%.
In the above technical solution, further: the method comprises the following steps of precision testing: taking a standard working solution with the concentration of 6.0mg/L, deriving and filtering the standard working solution, continuously feeding samples for 6 times, and calculating the RSD of the target peak concentration; the average concentration of the derivative was 6.15mg/L, RSD and was 1.44%.
In the above technical solution, further: comprises the following steps of repeated research: after multiple Feng-flavor liquor samples are derived, carrying out sample injection analysis according to chromatographic conditions: the average concentration of the samples was 1.50mg/L with a relative standard deviation of 1.90%.
In the above technical solution, further: the method comprises the following accuracy test steps: taking 6 parts of phoenix-flavor liquor samples which are numbered as 1#, 2#, 3#, 4#, 5# and 6# respectively; adding 4 μ L of standard working solution with concentration of 1.0mg/L into No. 1 and No. 2; adding 5 μ L of standard working solution with concentration of 1.0mg/L into No. 3 and No. 4; adding 6 μ L of standard solution standard working solution with concentration of 1.0mg/L into No. 5 and No. 6; performing liquid phase analysis after derivatization and filtration to obtain the actually measured urea content, and calculating the standard recovery rate of each sample: the average recovery rate of the added standard is 102.46%, and each recovery rate is 80-120%.
In the above technical solution, further: the LOD of the detection limit of the method is 0.030 mg/L; the method has a limit of quantitation LOQ of 0.093 mg/L.
Compared with the prior art, the invention has the advantages that:
1. the invention establishes a method for quickly and accurately quantifying urea in white spirit based on an ultra-performance liquid chromatography fluorescence detector (UPLC-FLD); in the method, a white spirit sample is derived by 9-hydroxyl xanthene alcohol under acidic and dark conditions, and C is adopted18Separating the target substance by using a reversed phase chromatographic column, wherein the mobile phase comprises acetonitrile and a 0.02mol/L sodium acetate solution with the pH value of 7.2, and then detecting and analyzing by using FLD (flash chromatography) with the wavelength of a detector being lambda ex-213 nm and lambda em-308 nm, and completing the quantitative detection of urea in the white spirit in 25 min; the test shows that the standard curve equation of urea is Y ═ 1.0X 107X+3×106，R20.9994, the linear relation is good; the average recovery rate of the added standard is 102.46%; the sample injection precision is 1.35%, and the repeatability of the sample is 1.5%; LOD and LOQ are respectively 0.030mg/L and 0.093 mg/L; compared with the traditional method, the method is simple and rapid to operate, high in accuracy and good in stability, and provides a better choice for quantifying urea in the white spirit.
2. The optimal selection of the derivatization dose of 800 mu L and the derivatization time of 40min effectively ensures the accuracy of the urea content and achieves the aim of accurate quantification.
3. The selection of the optimal elution gradient ensures that the peak shape and the separation degree of the urea target peak are more reliable, and a better urea target object peak spectrogram can be obtained.
4. Because the sample amount can affect various factors such as the target peak shape and the separation degree of urine, the invention can achieve better separation effect when the sample amount is determined to be 10 mu L through experiments.
5. According to the precision test step, the average concentration of the obtained derivative is 6.15mg/L, RSD and is 1.44%, and the sample injection precision of the method is high; in addition, the average concentration of the sample is 1.50mg/L, the relative standard deviation is 1.90 percent and is less than 5 percent of the requirement, so the repeatability of the method meets the requirement and the effect is better.
6. The accuracy test steps prove that the average standard addition recovery rate of the method is 102.46%, the test recovery rate is 80-120%, the test recovery rate meets the requirements, and the result is ideal.
7. The invention provides a method for quickly and accurately quantifying urea in white spirit by using UPLC-FLD; the standard curve equation is that Y is 1.0 multiplied by 107X+3×106，R20.9994; the recovery rate of the added standard is 102.46%; the precision of sample injection is 1.44%, and the repeatability of the sample is 1.90%. Compared with the traditional method, the method is a quick and accurate detection method with high precision and good separation degree suitable for urea in white spirit; the establishment of the method for quickly quantifying urea in the white spirit can greatly promote the formation mechanism and control research of ethyl carbamate in the white spirit.
Drawings
FIG. 1 is a graph of peak area of a target at the same derivatization dose;
FIG. 2 is a graph of peak area of a target at the same time as the derivatization;
FIG. 3 is a peak spectrum of a target compound of urea under different elution gradients;
FIG. 4 is a chromatogram of urea at different sample injection volumes;
FIG. 5 is a standard curve of urea.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to fig. 1 to 5 in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The materials used were: phoenix-flavor liquor.
Reagents used: anhydrous sodium acetate (AR, komeo), anhydrous ethanol (GR, komeo), n-propanol (AR, komeo); 9-hydroxy occupies xanthenol (not less than 98%, Sigma), urea standard (purity not less than 99%, Sigma); hydrochloric acid (AR, group of national drugs), acetonitrile (GR, MREDA), glacial acetic acid (AR, Shang Yang Gong).
The apparatus used was: ultra-high performance liquid chromatography (LC-40B, Shimadzu), vortex mixer (XW-80A, Shanghai Chitang electronics Co., Ltd.), ten-thousandth balance (AE240, Mettler-Torlo), pH meter (FE20, Mettler-Torlo instruments Co., Ltd.), ultrasonic cleaner (SB5200DT, Ningbo New Techiba), and spectrofluorometer (RF-20AXS, Shimadzu).
The method for detecting urea in white spirit based on the ultra-high performance liquid chromatography fluorescence detector comprises the following steps:
s001, sucking a 0.8mL sample, and placing the sample in a brown liquid storage bottle;
s002, adding 0.6mL of 9-hydroxy xanthene alcohol solution of 0.02mol/L, 0.2mL of hydrochloric acid solution of 1.5mol/L, and uniformly mixing by oscillation;
step S003, performing light-shielding derivatization for 40min at room temperature;
step S004, filtering by using a 0.22 mu m organic filter membrane;
and step S005, taking the filtrate and using the filtrate for the measurement of the ultra high performance liquid chromatograph.
In the above embodiment, further: the method comprises the following steps of:
preparing an acetic acid solution with a volume fraction of 1.0 percent: and (3) sucking 1.0mL of glacial acetic acid into a 100mL volumetric flask, adding water to a constant volume to a scale, and uniformly mixing.
Preparing 0.02mol/L sodium acetate solution: weighing 1.64g of anhydrous sodium acetate, dissolving with ultrapure water, diluting to 1000mL, adjusting pH to 7.2 with 1.0% acetic acid solution, filtering, and ultrasonic degassing for 15 min.
Preparing 1.5mol/L hydrochloric acid solution: sucking 6.2mL of concentrated hydrochloric acid into a 50mL volumetric flask, diluting to 50mL of volume with water, oscillating for 1min by a vortex mixer, and mixing uniformly.
Preparing 0.02 mol/L9-hydroxyl xanthene alcohol solution: weighing 0.198g of 9-hydroxy xanthene, adding n-propanol solution into a 50mL volumetric flask to reach a constant volume of 50mL, uniformly mixing, and performing ultrasonic treatment for 15min for later use.
Preparing 1.0mg/mL urea standard stock solution: weighing 0.100g of urea standard substance, dissolving with absolute ethyl alcohol, fixing the volume to the scale mark of a 100mL volumetric flask, oscillating for 1min by a vortex mixer, and mixing uniformly for later use.
Preparing 100mg/L urea intermediate solution: sucking 1mL of urea standard stock solution into a 10mL volumetric flask, metering the volume to 10mL scale mark with absolute ethyl alcohol, oscillating for 1min by a vortex mixer, and uniformly mixing for later use.
In the above embodiment, further: the liquid chromatographic conditions of the ultra-high performance liquid chromatograph are as follows: a chromatographic column: 2.1X100mm, 2 μm C18A chromatographic column; column temperature: 35 ℃; detection wavelength: λ ex213nm, λ em308 nm; flow rate: 0.2 mL/min; sample introduction volume: 10 mu L of the solution; the mobile phase A is: pH 7.2, 0.02mol/L sodium acetate solution; the mobile phase B is as follows: and (3) acetonitrile.
Mobile phase a, mobile phase B elution gradient: see table 1:
TABLE 2 mobile phase elution gradient
Selection of the conditions for derivatization:
the derivative dosage and the derivative time directly influence the accuracy of the urea content, so that the derivative dosage and the derivative time need to be optimally selected, the sample can be fully derived in the shortest time, and the purpose of accurate quantification is further achieved.
As can be seen from fig. 1 and 2, the urea peak area shows a trend of substantially stabilizing after a significant increase with the increase of the derivative dosage, i.e., the derivative further reacts with the increase of the derivative dosage until the reaction is complete at the derivative dosage of 800 μ L. Therefore, the optimal derivatization dose is 800. mu.L. Meanwhile, with the extension of the derivation time, the peak area of the derivative continuously rises and finally keeps flat, and the derivation time is finally selected to be 40min in consideration of the time cost and the sufficiency of the derivation process.
Optimization of elution gradient with respect to the invention:
the elution gradient can directly influence the peak shape and the separation degree of the urea target peak, even whether the urea target peak appears or not, so that the optimization of the elution gradient is important in the establishment of the method. In order to obtain a better peak spectrum of the urea target compound, a plurality of elution gradients are tried in the experiment, and finally, the elution gradient in the peak spectrum No. 1 in the figure 3 is selected through comparison.
That is, in the above embodiment, further: the elution gradient of the mobile phase B is 0-4.5 min, and the elution gradient of the mobile phase B is 80-65%; 4.5-12 min, and 65-70% of a mobile phase B; 12-18 min, and 70-0% of mobile phase B; 18-21 min, and 0-80% of mobile phase B; and (3) the optimal elution gradient is 80 percent of the mobile phase B in 21-25 min.
Regarding the selection of the sample size:
the sample amount can affect the peak shape, the separation degree and other aspects of the urea target peak. Therefore, after the standard working solution with the concentration of 5.0mg/L is derived according to the optimized conditions, 2 muL, 5 muL and 10 muL are respectively set for sample injection analysis, and as can be seen from figure 4, when the sample injection amount is 10 muL, a better separation effect can be achieved.
Therefore, in the above embodiment, further: the method comprises the following experimental steps of sample injection quantity selection: after the standard working solution with the concentration of 5.0mg/L is derived, 2 muL, 5 muL and 10 muL are respectively set for sample injection analysis, and when the sample injection amount is 10 muL, the separation effect is best.
Standard curve for urea:
in the above embodiment, further: the method comprises the following steps of drawing a standard curve of urea: sucking 100mg/L urea intermediate solution with a certain volume into a 10mL volumetric flask, and preparing 0.05mg/L, 0.1mg/L, 0.4mg/L, 1mg/L, 5mg/L, 10mg/L and 20mg/L urea standard curve working solution by using absolute ethyl alcohol in sequence; the mixture was shaken for 1min using a vortex mixer and the liquid phase analysis was performed as-is. Drawing the standard curve working solution by the area of the target chromatographic peak to obtain a regression equation and a correlation coefficient; the standard curve for urea is shown in FIG. 4.
The regression equation is that Y is 1.0 multiplied by 107X+3×106，R20.9994; the recovery rate of the added standard is 102.46%; the precision of sample injection is 1.44%, and the repeatability of the sample is 1.90%.
For the precision test:
in order to verify the precision, a sample injection precision test and a repeatability test are carried out. In order to perform precision tests to verify the precision, in the above examples, further: the method comprises the following steps of precision testing: taking a standard working solution with the concentration of 6.0mg/L, deriving and filtering the standard working solution, continuously feeding samples for 6 times, and calculating the RSD of the target peak concentration; as can be seen from Table 2, the average concentration of the derivative is 6.15mg/L, RSD and is 1.44%, and the injection precision of the method is high.
TABLE 3 sample introduction precision test results
To study reproducibility:
6 parts of Feng-flavor wine sample is derived and then subjected to sample injection analysis according to the optimized chromatographic conditions, and the average concentration of the sample is 1.50mg/L, the relative standard deviation is 1.90 percent and is less than 5 percent of the requirement, so that the repeatability of the method meets the requirement and the effect is good.
TABLE 4 results of the repeatability tests
That is, in the above embodiment, further: comprises the following steps of repeated research: after multiple Feng-flavor liquor samples are derived, carrying out sample injection analysis according to chromatographic conditions: the average concentration of the samples was 1.50mg/L with a relative standard deviation of 1.90%.
For the accuracy test:
in order to research the standard recovery rate of the method, 6 parts of Feng-flavor liquor samples are accurately sucked, the numbers of the Feng-flavor liquor samples are respectively 1#, 2#, 3#, 4#, 5# and 6#, and 4 mu L of standard solution with the concentration of 1.0mg/L is added into the 1# and 2 #; adding 5 μ L of standard solution with concentration of 1.0mg/L into No. 3 and No. 4; 6. mu.L of a 1.0mg/L standard solution was added to 5# and 6 #. Liquid phase analysis was performed after derivatization and filtration to obtain the actual measured urea content, and the spiked recovery for each sample was calculated, as shown in table 4.
TABLE 5 results of the spiked recovery test
As can be seen from the above table, the average recovery rate of the method is 102.46%, and each recovery rate is between 80-120%, which meets the requirements and results are ideal.
With respect to method limit of detection (LOD) and limit of quantitation (LOQ):
the detection Limit (LOD) and quantification Limit (LOQ) are determined by the following three methods: visual methods, signal-to-noise methods, and methods based on deviation and slope. Deviation and slope based methods are selected herein to determine detection limits and quantitation limits.
The specific determination method comprises the steps of filtering the derived standard solution, carrying out liquid phase analysis, observing a peak spectrogram of a liquid phase, selecting the standard solution when the peak area response value is minimum, carrying out continuous sample introduction 10 times on the standard solution, calculating the Standard Deviation (SD) of the peak area, marking as the blank standard deviation, and calculating the detection limit and the quantification limit according to the following formula.
Limit of detection (LOD) ═ 3.3 sigma/S (1)
Limit of quantitation (LOQ) ═ 10 sigma/S (2)
In the formula, σ: standard deviation of blanks; s: the slope of the calibration curve.
Experiments show that in the above examples, further: the LOD of the detection limit of the method is 0.030 mg/L; the method has a limit of quantitation LOQ of 0.093 mg/L.
From the above description it can be found that: the invention establishes a method for quickly and accurately quantifying urea in white spirit based on an ultra-performance liquid chromatography fluorescence detector (UPLC-FLD); in the method, a white spirit sample is derived by 9-hydroxyl xanthene alcohol under acidic and dark conditions, and C is adopted18Separating the target substance by using a reversed phase chromatographic column, wherein the mobile phase comprises acetonitrile and a 0.02mol/L sodium acetate solution with the pH value of 7.2, and then detecting and analyzing by using FLD (flash chromatography) with the wavelength of a detector being lambda ex-213 nm and lambda em-308 nm, and completing the quantitative detection of urea in the white spirit in 25 min; the test shows that the standard curve equation of urea is Y ═ 1.0X 107X+3×106，R20.9994, the linear relation is good; the average recovery rate of the added standard is 102.46%; the sample injection precision is 1.35%, and the repeatability of the sample is 1.5%; LOD and LOQ are respectively 0.030mg/L and 0.093 mg/L; compared with the traditional method, the method is simple and rapid to operate, high in accuracy and good in stability, and provides a better choice for quantifying urea in the white spirit.
The optimal selection of the derivatization dose of 800 mu L and the derivatization time of 40min effectively ensures the accuracy of the urea content and achieves the aim of accurate quantification.
The selection of the optimal elution gradient ensures that the peak shape and the separation degree of the urea target peak are more reliable, and a better urea target object peak spectrogram can be obtained.
Because the sample amount can affect various factors such as the target peak shape and the separation degree of urine, the invention can achieve better separation effect when the sample amount is determined to be 10 mu L through experiments.
According to the precision test step, the average concentration of the obtained derivative is 6.15mg/L, RSD and is 1.44%, and the sample injection precision of the method is high; in addition, the average concentration of the sample is 1.50mg/L, the relative standard deviation is 1.90 percent and is less than 5 percent of the requirement, so the repeatability of the method meets the requirement and the effect is better.
The accuracy test steps prove that the average standard addition recovery rate of the method is 102.46%, the test recovery rate is 80-120%, the test recovery rate meets the requirements, and the result is ideal.
In summary, the following steps: the invention provides a method for quickly and accurately quantifying urea in white spirit by using UPLC-FLD; the standard curve equation is that Y is 1.0 multiplied by 107X+3×106，R20.9994; the recovery rate of the added standard is 102.46%; the precision of sample injection is 1.44%, and the repeatability of the sample is 1.90%. Compared with the traditional method, the method is a quick and accurate detection method with high precision and good separation degree suitable for urea in white spirit; the establishment of the method for quickly quantifying urea in the white spirit can greatly promote the formation mechanism and control research of ethyl carbamate in the white spirit.
In summary, the embodiments in the present specification are described in a related manner, and the same and similar parts among the embodiments are referred to each other, and each embodiment focuses on the differences from the other embodiments.
The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Claims (10)
1. The method for detecting urea in white spirit based on the ultra-high performance liquid chromatography fluorescence detector is characterized by comprising the following steps:
s001, sucking a 0.8mL sample, and placing the sample in a brown liquid storage bottle;
s002, adding 0.6mL of 9-hydroxy xanthene alcohol solution of 0.02mol/L, 0.2mL of hydrochloric acid solution of 1.5mol/L, and uniformly mixing by oscillation;
s003, deriving for 40min in a dark place at room temperature;
s004, filtering by using a 0.22 mu m organic filter membrane;
and S005, taking the filtrate and using the filtrate for the measurement of the ultra-high performance liquid chromatograph.
2. The method for detecting urea in white spirit based on the ultra-high performance liquid chromatography fluorescence detector according to claim 1, which is characterized by comprising the steps of:
preparing an acetic acid solution with a volume fraction of 1.0 percent: sucking 1.0mL of glacial acetic acid into a 100mL volumetric flask, adding water to a constant volume to a scale, and uniformly mixing;
preparing 0.02mol/L sodium acetate solution: weighing 1.64g of anhydrous sodium acetate, dissolving with ultrapure water, diluting to a constant volume of 1000mL, adjusting the pH to 7.2 with 1.0% of acetic acid solution, filtering, and ultrasonically degassing for 15min for later use;
preparing 1.5mol/L hydrochloric acid solution: sucking 6.2mL of concentrated hydrochloric acid into a 50mL volumetric flask, diluting to 50mL of volume with water, oscillating for 1min by a vortex mixer, and mixing uniformly;
preparing 0.02 mol/L9-hydroxyl xanthene alcohol solution: weighing 0.198g of 9-hydroxyl xanthene, adding n-propanol solution into a 50mL volumetric flask to fix the volume to 50mL scale mark, uniformly mixing, and performing ultrasonic treatment for 15min for later use;
preparing 1.0mg/mL urea standard stock solution: weighing 0.100g of urea standard substance, dissolving with absolute ethyl alcohol, fixing the volume to the scale mark of a 100mL volumetric flask, oscillating for 1min by a vortex mixer, and mixing uniformly for later use;
preparing 100mg/L urea intermediate solution: sucking 1mL of urea standard stock solution into a 10mL volumetric flask, metering the volume to 10mL scale mark with absolute ethyl alcohol, oscillating for 1min by a vortex mixer, and uniformly mixing for later use.
3. The method for detecting urea in white spirit based on the ultra-high performance liquid chromatography fluorescence detector according to claim 1, wherein the ultra-high performance liquid chromatography is performed under the following liquid chromatography conditions:
a chromatographic column: 2.1X100mm, 2 μm C18A chromatographic column; column temperature: 35 ℃; detection wavelength: λ ex213nm, λ em308 nm; flow rate: 0.2 mL/min; sample introduction volume: 10 mu L of the solution; the mobile phase A is: pH 7.2, 0.02mol/L sodium acetate solution; the mobile phase B is as follows: and (3) acetonitrile.
4. The method for detecting urea in white spirit based on the ultra-high performance liquid chromatography fluorescence detector according to claim 3, wherein the elution gradient of the mobile phase B is 0-4.5 min, and the mobile phase B is 80-65%; 4.5-12 min, and 65-70% of a mobile phase B; 12-18 min, and 70-0% of mobile phase B; 18-21 min, and 0-80% of mobile phase B; and (5) 80% of mobile phase B for 21-25 min.
5. The method for detecting urea in white spirit based on the ultra-high performance liquid chromatography fluorescence detector according to claim 1, which is characterized by comprising the sample size selection experiment step of: after the standard working solution with the concentration of 5.0mg/L is derived, 2 muL, 5 muL and 10 muL are respectively set for sample injection analysis, and when the sample injection amount is 10 muL, the separation effect is best.
6. The method for detecting urea in white spirit based on the ultra-high performance liquid chromatography fluorescence detector according to claim 1, which comprises the step of drawing a standard curve of urea: sucking 100mg/L urea intermediate solution with a certain volume into a 10mL volumetric flask, and preparing 0.05mg/L, 0.1mg/L, 0.4mg/L, 1mg/L, 5mg/L, 10mg/L and 20mg/L urea standard curve working solution by using absolute ethyl alcohol in sequence; oscillating for 1min by vortex mixer, mixing, and performing liquid phase analysis on the mixture at the present location; drawing the standard curve working solution by the area of the target chromatographic peak to obtain a regression equation and a correlation coefficient; the regression equation is that Y is 1.0 multiplied by 107X+3×106，R20.9994; the recovery rate of the added standard is 102.46%; the precision of sample injection is 1.44%, and the repeatability of the sample is 1.90%.
7. The method for detecting urea in white spirit based on the ultra-high performance liquid chromatography fluorescence detector according to claim 1, characterized by comprising the following precision testing steps: taking a standard working solution with the concentration of 6.0mg/L, deriving and filtering the standard working solution, continuously feeding samples for 6 times, and calculating the RSD of the target peak concentration; the average concentration of the derivative was 6.15mg/L, RSD and was 1.44%.
8. The method for detecting urea in white spirit based on the ultra-high performance liquid chromatography fluorescence detector according to claim 1, which is characterized by comprising the following repeated research steps: after multiple Feng-flavor liquor samples are derived, carrying out sample injection analysis according to chromatographic conditions: the average concentration of the samples was 1.50mg/L with a relative standard deviation of 1.90%.
9. The method for detecting urea in white spirit based on the ultra-high performance liquid chromatography fluorescence detector according to claim 1, which is characterized by comprising the accuracy test steps of: taking 6 parts of phoenix-flavor liquor samples which are numbered as 1#, 2#, 3#, 4#, 5# and 6# respectively; adding 4 μ L of standard working solution with concentration of 1.0mg/L into No. 1 and No. 2; adding 5 μ L of standard working solution with concentration of 1.0mg/L into No. 3 and No. 4; adding 6 μ L of standard solution standard working solution with concentration of 1.0mg/L into No. 5 and No. 6; performing liquid phase analysis after derivatization and filtration to obtain the actually measured urea content, and calculating the standard recovery rate of each sample: the average recovery rate of the added standard is 102.46%, and each recovery rate is 80-120%.
10. The method for detecting urea in white spirit based on the ultra-high performance liquid chromatography fluorescence detector according to claim 1, wherein the method has a detection limit LOD of 0.030 mg/L; the method has a limit of quantitation LOQ of 0.093 mg/L.
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