Method for detecting propylene glycol and glycerol in urine

Abstract

The invention belongs to the technical field of chemical detection, and particularly relates to a method for detecting propylene glycol and glycerol in urine. The method for detecting propylene glycol and glycerol in urine comprises the following steps: mixing urine with benzoyl chloride in an alkaline environment, carrying out derivatization reaction through vortex at the temperature of 50-70 DEG C, drying after derivatization reaction, and redissolving with an organic solvent to obtain a solution to be detected, and respectively carrying out LC-MS-MS test analysis on the solution to be detected and the solution to be detected diluted by 50-80 times so as to detect propylene glycol and glycerol. According to the present invention, the heating vortex is adopted to perform the derivatization reaction, the secondary sample introduction is adopted, and under the specific quantitative ion pair parameter condition, the accurate quantitative analysis can be simultaneously performed on the propylene glycol content and the glycerol content in the urine, and the detection method has characteristics of simpleness, high sensitivity and high accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical detection, and particularly relates to a method for detecting propylene glycol and glycerol in urine. Background Art

[0002] It is very necessary to find specific biomarkers for vaping e-cigarettes or heated tobacco products and further conduct exposure and risk assessments. In addition to nicotine, e-cigarette liquids contain propylene glycol and glycerol, which are aerosolizing agents in heated tobacco and e-cigarette products and account for 80-95% in e-cigarette liquids; vaporized propylene glycol and glycerol generate aerosols, and after being inhaled by the smoker, 45% of the propylene glycol and glycerol prototypes are excreted from the body through urine and become potential biomarkers for vaping e-cigarettes in vitro. By analyzing the contents of propylene glycol and glycerol in urine, the vaping situation and risks of e-cigarettes can be continuously tracked and monitored.

[0003] On October 9, 2021, Burkhardt et al. disclosed a method for quantitatively determining the main components 1,2-propylene glycol (PG) and glycerol (G) in e-liquid by high performance liquid chromatography-mass spectrometry (LC-MS-MS) in the article "1,2-Propylene Glycol: A Biomarker of Exposure Specific to e-Cigarette Consumption". Sodium hydroxide and benzoyl chloride were added to a sample containing an internal standard and urine, and then n-pentane was added and stirred in a multi-tube vortex mixer for 15 min for derivatization reaction. Then, an aqueous glycine solution was added and mixed for another 15 min to quench the excessive derivatization reagent benzoyl chloride. The supernatant was taken by centrifugation and vacuum concentrated and evaporated to dryness, and then re-dissolved in acetonitrile and analyzed by LC-MS-MS. The multiple reaction monitoring mode (MRM) was adopted, and the quantitative ion pairs Q1 / Q3 of propylene glycol and glycerol were 285 / 163 and 405 / 283 respectively to evaluate the contents of propylene glycol and glycerol in the plasma and urine of users of different types of tobacco products.

[0004] However, the above detection method still has problems of low detection efficiency, low sensitivity and low accuracy. Summary of the Invention

[0005] The present invention provides a method for detecting propylene glycol and glycerol in urine, which solves the problems of low detection efficiency, low sensitivity and low accuracy of the existing detection methods.

[0006] To solve the above technical problems, the technical solution of the method for detecting propylene glycol and glycerol in urine of the present invention is as follows:

[0007] A method for detecting propylene glycol and glycerol in urine, comprising the following steps: mixing urine with benzoyl chloride in an alkaline environment and performing a derivatization reaction by vortexing, where the temperature of the vortexing is 50-70°C. After the derivatization reaction is completed, it is dried and redissolved in an organic solvent to obtain a test solution. The test solution and the test solution diluted 50-80 times are respectively subjected to LC-MS-MS test analysis to detect propylene glycol and glycerol.

[0008] The present invention improves the existing technology and provides a method for detecting propylene glycol and glycerol in urine. By using heating vortex for derivatization reaction, the target analytes are more fully mixed with the derivatization reagent. Under heating conditions, the derivatization speed is faster, and the time required to complete the reaction is short, only 3-5 minutes are needed to complete the derivatization. The derivatization reaction completely and directionally converts small molecule substances into macromolecular derivatization products that are easy to analyze, ensuring the accuracy of the analysis results. And a secondary injection method is adopted for detection. By performing LC-MS-MS analysis on the diluted test solution, the derivatization products of glycerol change linearly with the concentration, enabling accurate quantitative analysis of the contents of propylene glycol and glycerol in urine at the same time. It realizes the simultaneous detection and analysis of two target substances, propylene glycol and glycerol, with large differences in response. The instrument analysis time is short, and only 8 minutes are needed for each sample analysis, greatly improving the work efficiency.

[0009] The present invention establishes a general and efficient pretreatment method that is simple to operate, has strong versatility, low cost, is environmentally friendly, and can meet the simultaneous derivatization and extraction of propylene glycol and glycerol in human urine. Combined with LC-MS-MS coupling technology, it simultaneously analyzes propylene glycol and glycerol in urine. The detection method provided by the present invention is easy to operate, simple, has high sensitivity and high accuracy, can simultaneously detect and analyze propylene glycol and glycerol in urine, and has a good linear relationship between the response peak area and its concentration in a relatively large concentration range. The R values of linear fitting for propylene glycol and glycerol 2 are 0.9997 and 0.9996 respectively. The linear ranges of propylene glycol and glycerol are 8-200 ng / mL and 0.16-4 ng / mL respectively, and the quantitative limits are 6.5 ng / mL and 0.08 ng / mL respectively, which can provide technical support for the risk assessment of e-cigarette exposure.

[0010] To further improve the derivatization efficiency, preferably, the rotation speed of the vortexing is 150-200 r / min, and the time of the vortexing is 3-5 minutes.

[0011] To further make the target substances in urine derivatize more completely, preferably, 100-120 μL of benzoyl chloride is added for every 100 μL of urine.

[0012] To further improve the derivatization efficiency, preferably, n-hexane is added during vortexing, and 1 - 1.5 mL of n-hexane is added per 100 μL of urine.

[0013] To further quench the excessive derivatization reagent, preferably, a glycine solution is added for vortexing before drying, and 500 - 600 μL of a glycine solution with a mass fraction of 10 - 15% is added per 100 μL of benzoyl chloride. More preferably, the time for vortexing is 15 - 20 min.

[0014] To further improve the separation effect of the target substance, preferably, the chromatographic column used in LC-MS-MS test analysis is an Agilent Poroshell 120SB-C18 chromatographic column.

[0015] To further improve the separation effect of the target substance, preferably, the chromatographic conditions in LC-MS-MS test analysis are as follows: column temperature: 40 - 50 °C; injection volume: 5 - 10 μL; flow rate: 0.50 - 0.60 mL / min; mobile phase: A: aqueous formic acid solution with a concentration of 0.1 - 0.2%, B: acetonitrile, gradient elution is adopted, and the gradient elution conditions are: 50% B from 0 to 0.5 min, then changing uniformly to 95% B at 4 min, then changing uniformly to 50% B at 6.1 min, and 50% B from 6.1 to 8 min.

[0016] To further improve the detection accuracy, preferably, the mass spectrometry conditions in LC-MS-MS test analysis are: ion source: ESI(+); CAD: 8 psi; GS1: 60 psi; GS2: 50 psi; CUR: 35 psi; electrospray voltage: 5500 - 6000 V; drying temperature: 450 - 500 °C; scan time: 40 - 50 ms; detection mode: multiple reaction monitoring mode.

[0017] To further improve the detection accuracy, preferably, when using the multiple reaction monitoring mode, the quantitative ion pairs Q1 / Q3 of propylene glycol and glycerol are 307 / 163 and 427 / 283 respectively; the qualitative ion pairs Q1 / Q3 of propylene glycol and glycerol are 307 / 105 and 427 / 105 respectively.

[0018] To further improve the detection accuracy and stability of a large number of samples, preferably, after testing and analyzing 200 to 250 samples by LC-MS-MS, the detection system and the chromatographic column are rinsed with a mixed solution of isopropanol and methanol with a volume ratio of (7 to 8):(2 to 3) for 1 to 2 hours, and the chromatographic column is re-equilibrated with the mobile phase for 1 to 2 hours. After continuously injecting about 200 to 250 samples, the chromatographic peaks gradually bifurcate and the response intensity also decreases. By rinsing the detection system and the chromatographic column and re-equilibrating the chromatographic column, the chromatographic system reaches the initial analysis state, and the cleanliness of the analysis system and the stability of the analysis results of a large number of samples can be maintained. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a flowchart of the method for detecting propylene glycol and glycerol in urine according to Embodiment 1 of the present invention;

[0020] Figure 2 It is a column chart of the response intensity of propylene glycol and glycerol derivatives under different vortex conditions;

[0021] Figure 3 It is an LC-MS-MS spectrum of propylene glycol and glycerol in a standard product and a urine sample. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] The technical concept of the method for detecting propylene glycol and glycerol in urine of the present invention is as follows:

[0023] When the prior art detects propylene glycol and glycerol in urine, only vortex is used for derivatization, the derivatization time is long (15 minutes), the derivatization efficiency is low, which is not conducive to the accuracy of LC-MS-MS analysis and detection; and the method of injecting samples once is used to detect propylene glycol and glycerol at the same time. The response peak area of glycerol has a power exponential relationship with its concentration, and the linear relationship is poor. It is impossible to accurately quantify glycerol, and the accuracy of quantitative analysis of propylene glycol and glycerol is poor. The existing detection methods have problems of low detection efficiency, low sensitivity and low accuracy when detecting propylene glycol and glycerol.

[0024] However, the present invention improves the derivatization efficiency by using heating vortex, and tests propylene glycol by directly injecting the test solution, and tests glycerol by injecting the test solution after dilution, so that the response peak area of glycerol shows a good linear relationship with its concentration, greatly improving the detection sensitivity and accuracy of propylene glycol and glycerol.

[0025] The method for detecting propylene glycol and glycerol in urine of the present invention includes the following steps:

[0026] (1) Mix urine with benzoyl chloride, internal standard solution, and n-hexane in an alkaline environment, and carry out a derivatization reaction by vortexing at 50-70 °C. For every 100 μL of urine, add 100-120 μL of benzoyl chloride, 1-1.5 mL of n-hexane. The rotation speed of vortexing is 150-200 r / min, and the time of vortexing is 3-5 min. Then add glycine solution and vortex for 15-20 min. For every 100 μL of benzoyl chloride, add 500-600 μL of glycine solution. After that, dry and re-dissolve with an organic solvent to obtain a solution to be tested.

[0027] (2) Inject the solution to be tested directly for LC-MS-MS test analysis to detect propylene glycol, and then dilute the solution to be tested by 50-80 times and inject it for LC-MS-MS test analysis to detect glycerol. When performing LC-MS-MS test analysis, the chromatographic conditions are as follows: chromatographic column: Agilent Poroshell 120SB-C18 chromatographic column; column temperature: 40-50 °C; injection volume: 5-10 μL; flow rate: 0.50-0.60 mL / min; mobile phase: A: 0.1-0.2% formic acid aqueous solution, B: acetonitrile, gradient elution is adopted, and the gradient elution conditions are: 50% B for 0-0.5 min, 95% B for 4-6 min, 50% B for 6.1-8 min. When performing LC-MS-MS test analysis, the mass spectrometry conditions are as follows: ion source: ESI(+); CAD: 8 psi; GS1: 60 psi; GS2: 50 psi; CUR: 35 psi; electrospray voltage: 5500-6000 V; drying temperature: 450-500 °C; scanning time: 40-50 ms; detection mode: multiple reaction monitoring mode. The quantitative ion pairs Q1 / Q3 of propylene glycol and glycerol are 307 / 163 and 427 / 283 respectively, and the qualitative ion pairs Q1 / Q3 of propylene glycol and glycerol are 307 / 105 and 427 / 105 respectively. After testing and analyzing 200-250 samples, rinse the detection system and chromatographic column with a mixed solution of isopropanol and methanol with a volume ratio of (7-8):(2-3) for 1-2 h, and re-equilibrate the chromatographic column with the mobile phase for 1-2 h.

[0028] In the specific implementation manner, the alkaline environment in step (1) is obtained by adding sodium hydroxide solution. For every 100 μL of urine, add 500-600 μL of sodium hydroxide solution, and the concentration of the sodium hydroxide solution is 4-5 mol / L.

[0029] In the specific implementation manner, after the derivatization reaction in step (1) is completed, let it stand and take the supernatant for drying, and the drying is carried out by nitrogen blowing to dryness.

[0030] In the specific embodiment, the organic solvent in step (1) is acetonitrile, and 1 - 2 mL of acetonitrile is added corresponding to 400 - 500 μL of the supernatant; before the LC-MS-MS test analysis, it is filtered through a 0.22 - 0.25 μm organic phase filter membrane to obtain the solution to be tested.

[0031] In the specific embodiment, the internal standard solution includes PG- 13 C 2 and G-D 5 ,and the concentrations of PG- 13 C 2 and G-D 5 are 25 - 30 μg / mL and 12.5 - 15 μg / mL respectively.

[0032] The following further illustrates the embodiments of the present invention in conjunction with specific examples. The chemical reagents involved in the following examples are all commercially available conventional products without special instructions. Among them, an ExionLCTM AD liquid chromatograph (SCIEX Company, USA) is selected; an AB SCIEX liquid chromatography tandem mass spectrometry (HPLC-MS-MS) combined instrument.

[0033] I. Specific examples of the method for detecting propylene glycol and glycerol in urine of the present invention

[0034] Example 1

[0035] The flow chart of the method for detecting propylene glycol and glycerol in urine in this example is as Figure 1 shown, and the specific detection method is as follows:

[0036] (1) Thaw the pre-collected urine sample at room temperature. Add 500 μL of 4 mol / L sodium hydroxide solution, 100 μL of urine, and 50 μL of the mixed internal standard working solution (the concentration of PG- 13 C 2 is 25 μg / mL, and the concentration of G-D 5 is 12.5 μg / mL) into the reaction flask in sequence. After mixing, add 100 μL of benzoyl chloride and 1 mL of n-hexane in sequence, cover the lid, place the above sample on a heating vortex instrument, and vortex at 50 °C at a speed of 150 r / min for 5 min; then add 500 μL of 10% glycine solution by mass fraction, vortex for another 15 min, let it stand, take 400 μL of the supernatant, blow it to dry with nitrogen, re-dissolve it with 1 mL of acetonitrile, and filter it through a 0.22 μm organic phase filter membrane to obtain the solution to be tested into the chromatographic vial.

[0037] (2) Inject the solution to be tested directly for LC-MS-MS test analysis to quantitatively analyze propylene glycol in the urine sample; dilute the solution to be tested by 50 times and then inject it for LC-MS-MS test analysis to quantitatively analyze glycerol in the urine sample.

[0038] When performing LC-MS-MS test and analysis, the chromatographic conditions are as follows: chromatographic column: Agilent Poroshell 120SB-C18 chromatographic column (3.0×100mm, 2.7μm); column temperature: 40°C; injection volume: 5μL; flow rate: 0.50mL / min; mobile phase: A: 0.1 - 0.2% formic acid aqueous solution, B: acetonitrile, gradient elution is adopted, and the gradient elution conditions are shown in Table 1.

[0039] Table 1 High-performance liquid chromatography gradient elution conditions

[0040] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0 50 50 0.5 50 50 4.00 5 95 6.00 5 95 6.10 50 50 8.00 50 50

[0041] Note: The specific preparation process of mobile phase A is as follows: accurately add 1mL of formic acid to 1 liter of deionized water.

[0042] When performing LC-MS-MS test and analysis, the mass spectrometry conditions are as follows: ion source: ESI(+); CAD: 8psi; GS1: 60psi; GS2: 50psi; CUR: 35psi; electrospray voltage: 5500V; drying temperature: 450°C; scan time: 40ms; detection mode: multiple reaction monitoring (MRM) mode, and the MRM parameters of propylene glycol and glycerol are shown in Table 2.

[0043] Table 2 MRM parameters of propylene glycol and glycerol

[0044]

[0045] II. Experimental examples

[0046] (1) Optimization of pretreatment conditions

[0047] Take 20 portions of the same urine sample, with the other conditions being the same. Perform derivatization reactions under room temperature vortex and heated vortex conditions respectively. Take samples with vortex times of 2min, 5min, 10min, 20min, and 30min for LC-MS-MS determination, and the test results are as Figure 2 shown. Figure 2 In (a), it shows the change in the response intensity (intensity) of the propylene glycol derivative product under different vortex conditions, Figure 2 and in (b), it shows the change in the response intensity of the glycerol derivative product. As can be seen from Figure 2 , when vortexing at room temperature, as the vortex time extends, the area response of the derivatization reaction products of propylene glycol and glycerol continuously increases and reaches stability at 20 minutes. However, after heating, only 5 minutes of vortexing is required, and the absolute amount of the derivatization reaction products of propylene glycol and glycerol basically reaches stability, indicating that heated vortex can significantly improve the efficiency of the derivatization reaction.

[0048] (2) Determination of Working Curve and Quantitation Limit

[0049] Using deionized water as the solvent, a series of standard solutions containing propylene glycol and glycerol were prepared respectively. The internal standard PG- 13 C 2 had a concentration of 25 μg / mL, and G-D 5 had a concentration of 12.5 μg / mL. After the calibration curve was prepared, direct injection was performed for LC-MS-MS analysis. The injection concentrations of the 6-level calibration curve of propylene glycol derivatives were 4.0, 8.0, 20, 40, 80, and 200 ng / mL respectively. The prepared calibration curve was diluted 50 times, and injection was performed for LC-MS-MS analysis. The injection concentrations of the 6-level calibration curve of glycerol were 0.08, 0.16, 0.4, 0.8, 1.6, and 4.0 ng / mL respectively. Linear regression analysis was performed on the ratio of the peak area of each target substance to the peak area of the internal standard and the ratio of the concentration of each target substance to the concentration of the internal standard to obtain the standard working curve. Within the linear concentration range, the working curves of propylene glycol and glycerol had good linearity and were suitable for quantitative analysis. The standard working solution with the lowest concentration of each target substance was determined in parallel 10 times, and the standard deviation was calculated. The quantitation limit of the method was 10 times the standard deviation. The correlation coefficients, linear ranges, and quantitation limits of the standard curves are shown in Table 3.

[0050] Table 3 Linear Equations, Linear Ranges, Correlation Coefficients, and Quantitation Limits of Each Analyte

[0051]

[0052] *Note: Y—the ratio of the peak area of the target compound to the peak area of the internal standard; X—the ratio of the concentration of the target compound to the concentration of the internal standard

[0053] (3) Recovery and Precision Tests

[0054] The same urine sample was determined in parallel 5 times within the day and between days to investigate the repeatability of the method; the recoveries were determined at three concentration levels of high, medium, and low to investigate the accuracy of the method. The test results are shown in Table 4. The results show that the precision (RSD) within the day and between days was between 2.46% and 5.65%, and the recoveries of the analytes at the three levels of high, medium, and low were between 94.4% and 106.0%. Therefore, the method had good precision and high recovery.

[0055] Table 4 Test Results of Precision and Recovery

[0056]

[0057] (4) Determination of Target Analytes in Urine Samples

[0058] 51 urine samples from smokers of different tobacco products were analyzed. The propylene glycol content ranged from 0.3 to 33 μg / mL, and the glycerol content ranged from 0.4 to 9 μg / mL. The total amount of propylene glycol in urine is the product of the propylene glycol concentration in urine and the urine volume, and the total metabolic amount of glycerol in urine is the product of the glycerol concentration in urine and the urine volume. The specific test results are shown in Table 5. The LC-MS-MS spectra of propylene glycol and glycerol in the standard and urine sample No. D5002 are as Figure 3 shown, where Figure 3 (a) in Figure 3 and (b) in

[0059] are the spectra of the standard and sample D5002, respectively.

[0059] Table 5 Test results of propylene glycol and glycerol in urine samples from a certain city

[0060]

[0061] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for detecting propylene glycol and glycerol in urine, characterized in that: The method comprises the following steps: mixing urine with benzoyl chloride in an alkaline environment and then performing a derivatization reaction by vortexing, the vortexing temperature being 50-70°C; after the derivatization reaction is completed, drying and re-dissolving with an organic solvent to obtain a test solution; and performing LC-MS-MS test analysis on the test solution and the test solution diluted 50-80 times to detect propylene glycol and glycerol.

2. The method for detecting propylene glycol and glycerol in urine according to claim 1, characterized in that: The rotation speed of the vortex is 150-200 r / min, and the vortex time is 3-5 min.

3. The method for detecting propylene glycol and glycerol in urine according to claim 1, characterized in that: For every 100 μL of urine, add 100-120 μL of benzoyl chloride.

4. The method for detecting propylene glycol and glycerol in urine according to claim 1, characterized in that: During the vortexing, n-hexane was added, with 1-1.5 mL of n-hexane added for every 100 μL of urine.

5. The method for detecting propylene glycol and glycerol in urine according to claim 1, characterized in that: Before the drying, a glycine solution is added and vortexed, and 500-600 μL of a glycine solution with a mass fraction of 10-15% is added for every 100 μL of benzoyl chloride.

6. The method for detecting propylene glycol and glycerol in urine according to claim 1, characterized in that: The chromatographic column used in the LC-MS-MS test analysis was an Agilent Poroshell 120 SB-C18 column.

7. The method for detecting propylene glycol and glycerol in urine according to claim 1, characterized in that: The chromatographic conditions for LC-MS-MS test analysis were: column temperature: 40~50°C; injection volume: 5~10μL; flow rate: 0.50~0.60 mL / min; Mobile Phase: A: 0.1~0.2% formic acid aqueous solution, B: acetonitrile, gradient elution, the gradient elution conditions are: 50% B from 0 to 0.5 min, then change at a constant speed to 4 min, 95% B from 4 to 6 min, then change at a constant speed to 6.1 min, 50% B from 6.1 to 8 min.

8. The method for detecting propylene glycol and glycerol in urine according to claim 1, characterized in that: The mass spectrometry conditions for LC-MS-MS test analysis were as follows: ion source: ESI (+); CAD: 8 psi; GS1: 60 psi; GS2: 50 psi; CUR: 35 psi; electrospray voltage: 5500~6000 V; drying temperature: 450~500 ºC; scanning time: 40~50 ms; detection mode: multiple reaction monitoring mode.

9. The method for detecting propylene glycol and glycerol in urine according to claim 8, characterized in that: In the multiple reaction monitoring mode, the quantitative ion pairs Q1 / Q3 of propylene glycol and glycerol were 307 / 163 and 427 / 283, respectively; the qualitative ion pairs Q1 / Q3 of propylene glycol and glycerol were 307 / 105 and 427 / 105, respectively.

10. The method for detecting propylene glycol and glycerol in urine according to claim 1, characterized in that: After 200 to 250 samples were analyzed by LC-MS-MS, the detection system and chromatographic column were flushed with a mixed solution of isopropanol and methanol in a volume ratio of (7 to 8): (2 to 3) for 1 to 2 hours, and the chromatographic column was re-equilibrated with the mobile phase for 1 to 2 hours.

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