Sample pretreatment method generally suitable for screening organophosphate in marine organisms

Abstract

The invention relates to a sample pretreatment method generally suitable for screening organophosphate in marine organisms. The sample pretreatment method comprises the following steps: purifying and freezing, drying and grinding, extracting to remove water, degreasing to remove water for the second time, and analyzing and detecting; the method comprises the following steps: purifying fish with seawater, dissecting the fish, cutting the fish into 1-2cm broken sections, and freezing the broken sections to obtain a frozen fish sample; carrying out vacuum drying and ball milling on the frozen fish sample, adding an extraction agent, a water removal agent and a salting-out agent after ball milling for extraction and water removal, and adding a degreasing agent and a water removal agent into the fish sample subjected to extraction and water removal to obtain a degreased fish sample subjected to secondary water removal; and concentrating the degreased and secondarily dewatered fish sample to be nearly dry, and compounding and analyzing the concentrated fish sample by using an organic solvent to obtain the content of organophosphate in the fish sample. According to the sample pretreatment method, two different instrument detections can be realized through one-time sample feeding.

Description

Technical Field

[0001] The present invention relates to the technical field of sample pretreatment, and in particular to a sample pretreatment method generally applicable to screening organophosphates in marine organisms. Background Art

[0002] Currently, organophosphates in marine organisms primarily originate from flame retardant products, plastic waste, and industrial wastewater. Due to their lipophilic nature, organophosphate esters (OPEs) tend to accumulate in adipose tissue, particularly at higher concentrations in organisms at higher trophic levels (such as fish, seals, and whales). Some OPEs, such as chlorinated OPEs, exhibit biomagnification, potentially increasing in concentration as they ascend the food chain. Human ingestion of OPEs through seafood (such as shellfish and fish) can affect the liver and immune system. Therefore, a sample pretreatment method for detecting organophosphates in marine organisms is urgently needed.

[0003] Existing sample pretreatment methods for organophosphates generally use acetonitrile as an extraction solvent, which has a poor effect in extracting organophosphates from samples. In response to this, the present invention proposes a sample pretreatment method that is generally applicable to screening organophosphates in marine organisms. Summary of the Invention

[0004] To address the above-mentioned issues, the present invention proposes a sample pretreatment method generally applicable to screening for organophosphates in marine organisms. The method enables a single sample to be tested in two different instruments. Equal volume ratios of ethyl acetate / dichloromethane and methanol are used as the solution for extracting organophosphates from biological samples, greatly increasing the extraction efficiency.

[0005] According to one aspect of the present invention, a sample pretreatment method generally applicable to screening for organophosphates in marine organisms is provided, wherein the sample pretreatment comprises purification and freezing, drying and grinding, extraction and water removal, defatting and secondary water removal, and analysis and detection; Step 1, purification and freezing: the fish is purified with seawater, dissected, cut into 1-2 cm pieces, and frozen to obtain frozen fish samples; Step 2, drying and grinding: vacuum drying and ball milling the frozen fish sample obtained in step 1 to obtain a ground fish sample; Step 3, extraction and dehydration: adding an extractant, a dehydrating agent, and a salting-out agent to the ground fish sample obtained in step 2, vortexing, ultrasonicating, and centrifuging to obtain a fish sample after extraction and dehydration; Step 4, defatting and secondary water removal: adding a degreasing agent and a water remover to the fish sample after extraction and water removal obtained in step 3, vortexing, ultrasonicating, centrifuging, and filtering to obtain a defatted and secondary water-removed fish sample; Step 5, analysis and detection: the defatted and secondary dewatered fish sample obtained in step 4 is concentrated to near dryness, the concentrated fish sample is compounded with an organic solvent, and the organic phosphate content in the fish sample is analyzed.

[0006] Furthermore, the purification time in step 1 is 12 to 24 hours; The freezing temperature in step 1 is -20~-18°C; The freezing time in step 1 is 10 to 12 hours.

[0007] Furthermore, the vacuum drying conditions in step 2 are as follows: The vacuum drying temperature is -50~-60°C; The vacuum drying time is 48 to 56 hours; The conditions for ball milling in step 2 are as follows: The ball mill has a rotation speed of 200-400 rpm; The ball milling time is 2 to 3 minutes.

[0008] Furthermore, the extractant in step 3 is selected from one of ethyl acetate / n-hexane with a volume ratio of 1:1, ethyl acetate / cyclohexane with a volume ratio of 1:1, ethyl acetate / dichloromethane with a volume ratio of 1:1, ethyl acetate / dichloromethane with a volume ratio of 1:2, and acetonitrile.

[0009] Furthermore, the extractant is ethyl acetate / dichloromethane with a volume ratio of 1:1; The dehydrating agent in step 3 and step 4 is selected from at least one of MgSO4 and Na2SO4.

[0010] The salting-out agent in step 3 is NaCl; Furthermore, the mass ratio of the ground fish sample, extractant, dehydrating agent and salting-out agent in step 3 is 1:7~12:4:1.

[0011] Furthermore, the vortexing time in step 3 and step 4 is 1 to 2 minutes; The ultrasonic time in step 3 and step 4 is 20 to 30 minutes; The centrifugal conditions in step 3 and step 4 are as follows: The centrifugal speed is 5000-6000 rpm; The centrifugation time is 5 to 10 min.

[0012] Furthermore, the degreasing agent in step 4 is N-propylethylenediamine and reverse phase silica gel C 18 ; The mass ratio of the fish sample after extraction and dehydration in step 4, the degreasing agent, and the water removing agent is 500-1000:150-300:475-950.

[0013] Furthermore, the concentration to near dryness in step 5 is carried out under a nitrogen atmosphere; The flow rate of the nitrogen is 0.5-1.0 ml / min; The concentration time is 40 to 60 minutes. The organic solvent in step 5 is selected from one of isooctane and methanol. When the organic solvent is isooctane, the reconstituted fish sample is subjected to gas phase mass spectrometry analysis; when the organic solvent is methanol, the reconstituted fish sample is subjected to liquid phase mass spectrometry analysis; The analysis conditions of the gas phase mass spectrometry are as follows: The analysis conditions of the gas phase mass spectrometry are as follows: The injection volume was 1–2 μL; The injection port temperature is 260~300℃; The column temperature program is controlled as follows: Hold at 50-60 °C for 1-2 min, then increase the temperature to 110-120 °C at a rate of 30-40 °C / min, and then increase the temperature to 260-300 °C at a rate of 4-5 °C / min and hold for 6-7.5 min; The ion source temperature was 260–300°C; the quenching gas helium flow rate was 2.0–2.25 mL / min, the collision gas nitrogen flow rate was 1.2–1.5 mL / min, the gas flow rate was 0.5–1 mL / min, and the nebulizer pressure was 30–40 psi. The analysis conditions of the liquid phase mass spectrometry are as follows The injection volume was 1–2 μL; Column temperature is 30~40℃; The mobile phase consisted of 0.1% formic acid solution (A) and 1 mmol / L ammonium acetate in methanol (B), with a flow rate of 0.2–0.30 mL / min. The mobile phase gradient was set as follows: 0 ~ 10 min, 50 ~ 62% B; 10 ~ 12 min, 62 ~ 70% B; 12 ~ 22 min, 70 ~ 75% B; 22 - ~ 27 min, 75 ~ 100% B; 27 ~ 32 min, 100% B; Electrospray ionization was performed in positive ion source mode with multiple reaction monitoring as the scan type. The ion source temperature was set between 220 and 250 °C. The ionization voltage was between -4000 and -4500 V, and the gas flow rate was between 5 and 6 L / min. The sheath gas temperature was set between 300 and 350 °C, and the sheath gas flow rate was between 10 and 11 L / min. The capillary voltage was between +2500 and +3000 V, and the nebulizer pressure was set between 20 and 30 psi.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) The technical solution adopted by the present invention is to place the collected sea crucian carp in fresh seawater and culture it for 24 hours to remove the remaining impurities in the organism.

[0015] (2) The technical solution adopted by the present invention is to crush the fish sample before placing it in a vacuum freeze dryer and then freeze it, which improves the freezing efficiency and shortens the sample processing time.

[0016] (3) The technical solution adopted by the present invention uses ethyl acetate / dichloromethane in an equal volume ratio as a solution for extracting organophosphates from biological samples, which greatly increases the extraction efficiency.

[0017] (4) The technical solution adopted by the present invention is to divide the sample into two parts when concentrating and compounding, and exchange the solvent into isooctane and methanol respectively, so that one sample can be tested by two different instruments, which increases the comparability of the data. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The content of organophosphate in the fish sample after extraction and dehydration as described in Examples 1 and 4 to 7 of the present invention; Figure 2 The content of organophosphate in the fish sample after extraction and dehydration as described in Examples 8 to 10 of the present invention. DETAILED DESCRIPTION

[0019] The present invention will be further described below with reference to specific examples, but the present invention is not limited thereto in any way.

[0020] The sea crucian carp used in Examples 1 to 10 were all caught from the Bohai Sea; The fish meat, viscera and gills used in the present invention are obtained by dissecting a sea crucian carp that has been purified for 24 hours and then caught. The fish meat, viscera and gills are cut into 1-2 cm pieces using scissors, evenly placed in a culture dish, marked, and placed in a refrigerator below -20°C for 12 hours until all frozen, thereby obtaining frozen fish meat samples, viscera samples and gill samples.

[0021] In the present invention, ultra-performance liquid chromatography (Ultimate 1290, Thermo Fisher Scientific, USA) combined with triple quadrupole tandem mass spectrometry (triple-quad 6470, AB SCIEX, USA) (UPLC-MS / MS) was used to detect organophosphates in the extracted samples using an Inertsil ODS-4 column (150 mm × 2.1 mm × 1.8 μm, LB-AQ, Japan).

[0022] The extractants n-hexane, ethyl acetate, acetonitrile and cyclohexane used in the examples of the present invention were purchased from Shanghai Anpu Company.

[0023] Example 1 Step 1, purification and freezing: The caught crucian carp was placed in seawater for purification for 24 hours. The purified crucian carp was dissected to obtain the fish meat. The fish meat was cut into 1-2 cm pieces using scissors and evenly placed in a Petri dish. The pieces were marked and placed in a refrigerator below -20°C for 12 hours until completely frozen, thereby obtaining the frozen fish meat sample. Step 2, drying and grinding: Open the top cover of the vacuum freeze dryer, place the fish meat sample frozen in step 1 in the vacuum dryer, and dry it at -60°C for 50 h. Place the dried fish meat sample in a ball mill and grind it at 300 rpm for 2 min to obtain a ground fish meat sample; Step 3, extraction and dehydration: weigh 1 g of the ground fish sample obtained in step 2 into a 50 mL glass centrifuge tube, add 10 mL of ethyl acetate / dichloromethane with a volume ratio of 1:1 as an extractant, add 4 g of MgSO4 as a dehydrating agent, add 1 g of NaCl as a salting-out agent, vortex for 2 min, sonicate for 30 min, and centrifuge at 5000 rpm for 5 min. Transfer the supernatant to a 15 mL glass centrifuge tube to obtain the extracted and dehydrated fish sample; Step 4, defatting and secondary water removal: 900 mg of MgSO4 as a water removal agent, 150 mg of N-propylethylenediamine (PSA) and 150 mg of reversed-phase silica gel C were added to the fish meat sample after extraction and water removal. 18 (octadecyl bonded silica gel) was used as a degreasing agent, vortexed for 1 minute, centrifuged at 5000 rpm for 5 minutes, and the supernatant was collected and filtered using a 0.22 μm organic polypropylene membrane syringe filter to obtain the defatted and secondary dehydrated fish meat sample; Step 5, analysis and detection: 1 ml of the defatted and twice dehydrated fish meat sample obtained in step 4 was taken and divided into sample A and sample B, and concentrated to near dryness under a nitrogen atmosphere with a nitrogen flow rate of 0.5 mL / min and concentrated for 60 min; the concentrated sample A was reconstituted with 1 ml of isooctane, and then subjected to gas phase mass spectrometry analysis (analysis conditions: injection volume of 1 μL; injection port temperature of 300 °C; column program temperature control as follows: 60 °C for 1 min, 40 °C / min to 120 °C, and finally to 300 °C at 5 °C / min and maintained for 7.5 min. The ion source temperature was set to 300 °C. The quenching gas helium flow rate was 2.25 mL / min, the collision gas nitrogen flow rate was 1.5 mL / min, and the gas flow rate was 1 mL / min. The nebulizer pressure was 40 psi); the concentrated sample B was reconstituted with 1 mL of methanol, and then subjected to liquid phase mass spectrometry detection (analysis conditions: injection volume of 2 μL; the column temperature was set at 40 °C. The mobile phase consisted of a solution containing 0.1% formic acid (A) and a methanol solution containing 1 mmol / L ammonium acetate (B), with a flow rate of 0.30 mL / min. The mobile phase gradient was set as follows: 0 ~ 10 min, 50 ~ 62% B; 10 ~ 12 min, 62 ~ 70% B; 12 ~ 22 min, 70 ~ 75% B; 22 ~ 27 min, 75 ~ 100% B; 27 ~ 32 min, 100% B. Electrospray ionization (ESI) positive ion source mode was used, and the scan type was multiple reaction monitoring (MRM). The ion source temperature was set at 250 °C. The ionization voltage was -4500 V, and the gas flow rate was 6 L / min. The sheath gas temperature was set at 350 °C, and the sheath gas flow rate was 11 L / min. The capillary voltage was +3000 V, and the nebulizer pressure was set at 30 psi. The nozzle voltage was 0 V), the liquid chromatography mass spectrometry test results are as follows Figure 1 shown.

[0024] Example 2 Step 1, purification and freezing: Place the caught crucian carp in seawater for 24 hours, dissect the purified crucian carp, remove the internal organs, cut the internal organs into 1-2 cm pieces with scissors, place them evenly in a petri dish, mark them, and freeze them in a refrigerator below -20°C for 12 hours until they are completely frozen, to obtain the frozen internal organs sample; Step 2, drying and grinding: Open the top cover of the vacuum freeze dryer, place the viscera sample frozen in step 1 in the vacuum dryer, and dry it at -60°C for 48 h. Place the dried viscera sample in a ball mill and grind it at 300 rpm for 2 min to obtain the ground viscera sample; Step 3, extraction and dehydration: weigh 1 g of the ground viscera sample obtained in step 2 into a 50 mL glass centrifuge tube, add 10 mL of ethyl acetate / dichloromethane with a volume ratio of 1:1 as an extractant, add 4 g of MgSO4 as a dehydrating agent, add 1 g of NaCl as a salting-out agent, vortex for 2 min, sonicate for 30 min, and centrifuge at 5000 rpm for 5 min. Transfer the supernatant to a 15 mL glass centrifuge tube to obtain the viscera sample after extraction and dehydration; Step 4, defatting and secondary water removal: add 900 mg of MgSO4 as a water removal agent, 150 mg of N-propylethylenediamine (PSA) and 150 mg of reversed-phase silica gel C to the viscera sample after extraction and water removal. 18 (octadecyl bonded silica gel) was used as a degreasing agent, vortexed for 1 minute, centrifuged at 5000 rpm for 5 minutes, and the supernatant was collected and filtered using a 0.22 μm organic polypropylene membrane syringe filter to obtain the viscera sample after secondary degreasing and water removal; Step 5, analysis and detection: Take 1 ml of the defatted and secondary dehydrated viscera sample obtained in step 4 and divide it into sample A and sample B, and concentrate them to near dryness under a nitrogen atmosphere with a nitrogen flow rate of 0.5 mL / min for 60 min; reconstitute the concentrated sample A with 1 ml of isooctane, and then perform gas chromatography-mass spectrometry analysis (the analysis conditions are consistent with those in implementation 1); reconstitute the concentrated sample B with 1 mL of methanol, and then perform liquid chromatography-mass spectrometry detection (the analysis conditions are consistent with those in implementation 1).

[0025] Example 3 Step 1, purification and freezing: The captured crucian carp was placed in seawater for purification for 24 hours. The purified crucian carp was dissected to obtain the gills. The gills were cut into 1-2 cm pieces using scissors and evenly placed in a Petri dish. The pieces were marked and placed in a refrigerator below -20°C for 12 hours until completely frozen. The frozen gill samples were obtained. Step 2, drying and grinding: open the top cover of the vacuum freeze dryer, place the fish gill sample frozen in step 1 in the vacuum dryer, dry at -60 ° C for 48 h, place the dried fish gill sample in a ball mill, and grind it at a speed of 300 rpm for 2 min to obtain a ground fish gill sample; Step 3, extraction and dehydration: weigh 1 g of the ground fish gill sample obtained in step 2 into a 50 mL glass centrifuge tube, add 10 mL of ethyl acetate / dichloromethane with a volume ratio of 1:1 as an extractant, add 4 g MgSO4 as a dehydrating agent, add 1 g NaCl as a salting-out agent, vortex for 2 minutes, ultrasonicate for 30 minutes, and centrifuge at 5000 rpm for 5 minutes. Transfer the supernatant to a 15 mL glass centrifuge tube to obtain the extracted and dehydrated fish gill sample; Step 4, defatting and secondary water removal: 900 mg MgSO4, 150 mg N-propylethylenediamine (PSA) and 150 mg reverse phase silica gel C were added to the fish gill sample after extraction and water removal. 18 (octadecyl bonded silica gel), vortex for 1 min, centrifuge at 5000 rpm for 5 min, take the supernatant, and filter the supernatant using a 0.22 μm organic polypropylene membrane syringe filter to obtain the defatted and secondary dehydrated fish gill sample; Step 5, analysis and detection: 1 ml of the defatted and secondary dehydrated fish gill sample obtained in step 4 was taken and divided into sample A and sample B, and concentrated to near dryness under a nitrogen atmosphere with a nitrogen flow rate of 0.5 mL / min for 60 min; the concentrated sample A was reconstituted with 1 ml of isooctane, and then subjected to gas chromatography-mass spectrometry analysis (the analysis conditions were consistent with those in Example 1); the concentrated sample B was reconstituted with 1 mL of methanol, and then subjected to liquid chromatography-mass spectrometry detection (the analysis conditions were consistent with those in Example 1).

[0026] Example 4 The difference from Example 1 is that the extractant in step 3 is ethyl acetate / n-hexane (ie, EA / HEX) with a volume ratio of 1:1, and the remaining steps are consistent with Example 1. The detected organophosphates are as follows Figure 1 and as shown in Table 1.

[0027] Example 5 The difference from Example 1 is that the extractant in step 3 is ethyl acetate / cyclohexane (ie, EA / CHX) with a volume ratio of 1:1, and the remaining steps are consistent with Example 1. The detected organophosphate is as follows Figure 1 and as shown in Table 1.

[0028] Example 6 The difference from Example 1 is that the extractant in step 3 is ethyl acetate / dichloromethane (ie, EA / DCM) with a volume ratio of 1:1, and the remaining steps are consistent with Example 1. The detected organophosphate is as follows Figure 1 and as shown in Table 1.

[0029] Example 7 The difference from Example 1 is that the extractant in step 3 is acetonitrile, and the remaining steps are consistent with Example 1. The detected organophosphate is as follows Figure 1 and as shown in Table 1.

[0030] Example 8 The difference from Example 1 is that the extractant in step 3 is ethyl acetate / dichloromethane (1:2 in volume ratio), i.e., EA / DCM (1:2). Figure 2 As shown, the detected organophosphates are Figure 2and as shown in Table 1.

[0031] Example 9 The difference from Example 1 is that the extractant in step 3 is acetonitrile, i.e. ACN. The test results are as follows: Figure 2 As shown, the detected organophosphates are Figure 2 and as shown in Table 1.

[0032] Example 10 The difference from Example 1 is that the extractant in step 3 is ethyl acetate / dichloromethane (1:1 by volume), i.e., EA / DCM (1:1). The test results are as follows: Figure 2 As shown in the results, the extraction effect was the best when ethyl acetate / dichloromethane with a volume ratio of 1:1 was used as the extraction solvent. The detected organophosphates such as Figure 2 and as shown in Table 1.

[0033] Table 1 shows the organic phosphates extracted and detected in Examples 1 to 8 of the present invention.

[0034] The above descriptions are merely several embodiments of the present invention and do not constitute any form of limitation to the present invention. Although the present invention is disclosed as above in terms of preferred embodiments, they are not intended to limit the present invention. Any technician familiar with the present profession who, without departing from the scope of the technical solution of the present invention, makes slight changes or modifications using the technical contents disclosed above are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A sample pretreatment method generally applicable to screening for organophosphates in marine organisms, characterized in that: The sample pretreatment includes purification and freezing, drying and grinding, extraction and water removal, defatting and secondary water removal, and analysis and detection; Step 1, purification and freezing: the fish is purified with seawater, dissected, cut into 1-2 cm pieces, and frozen to obtain frozen fish samples; Step 2, drying and grinding: vacuum drying and ball milling the frozen fish sample obtained in step 1 to obtain a ground fish sample; Step 3, extraction and dehydration: adding an extractant, a dehydrating agent, and a salting-out agent to the ground fish sample obtained in step 2, vortexing, ultrasonicating, and centrifuging to obtain a fish sample after extraction and dehydration; Step 4, defatting and secondary water removal: adding a degreasing agent and a water remover to the fish sample after extraction and water removal obtained in step 3, vortexing, ultrasonicating, centrifuging, and filtering to obtain a defatted and secondary water-removed fish sample; Step 5, analysis and detection: the defatted and secondary dewatered fish sample obtained in step 4 is concentrated to near dryness, the concentrated fish sample is compounded with an organic solvent, and the organic phosphate content in the fish sample is analyzed.

2. The sample pretreatment method according to claim 1, characterized in that The purification time in step 1 is 12 to 24 hours; The freezing temperature in step 1 is -20~-18°C; The freezing time in step 1 is 10 to 12 hours.

3. The sample pretreatment method according to claim 1, characterized in that The conditions for vacuum drying described in step 2 are as follows: The vacuum drying temperature is -50~-60°C; The vacuum drying time is 48 to 56 hours; The conditions for ball milling in step 2 are as follows: The ball mill has a rotation speed of 200-400 rpm; The ball milling time is 2 to 3 minutes.

4. The sample pretreatment method according to claim 1, wherein The extractant in step 3 is selected from one of ethyl acetate / n-hexane with a volume ratio of 1:1, ethyl acetate / cyclohexane with a volume ratio of 1:1, ethyl acetate / dichloromethane with a volume ratio of 1:1, ethyl acetate / dichloromethane with a volume ratio of 1:2, and acetonitrile; Preferably, the extractant is ethyl acetate / dichloromethane in a volume ratio of 1:1; The dehydrating agent in step 3 and step 4 is selected from at least one of MgSO4 and Na2SO4; The salting-out agent in step 3 is NaCl; Preferably, the mass ratio of the ground fish sample, extractant, dehydrating agent and salting-out agent in step 3 is 1:7 to 12:4:

1.

5. The sample pretreatment method according to claim 1, characterized in that: The vortexing time in step 3 and step 4 is 1 to 2 minutes; The ultrasonic time in step 3 and step 4 is 20 to 30 minutes; The centrifugal conditions in step 3 and step 4 are as follows: The centrifugal speed is 5000-6000 rpm; The centrifugation time is 5 to 10 min.

6. The sample pretreatment method according to claim 1, characterized in that: The degreasing agent in step 4 is N-propylethylenediamine and reverse phase silica gel C 18 ; The mass ratio of the fish sample after extraction and dehydration in step 4, the degreasing agent, and the water removing agent is 500-1000:150-300:475-950.

7. The sample pretreatment method according to claim 1, characterized in that: The concentration to near dryness in step 5 is carried out under a nitrogen atmosphere; The flow rate of the nitrogen is 0.5-1.0 ml / min; The concentration time is 40 to 60 minutes. The organic solvent in step 5 is selected from one of isooctane and methanol. When the organic solvent is isooctane, the reconstituted fish sample is subjected to gas phase mass spectrometry analysis; when the organic solvent is methanol, the reconstituted fish sample is subjected to liquid phase mass spectrometry analysis; The analysis conditions of the gas phase mass spectrometry are as follows: The injection volume was 1–2 μL; The injection port temperature is 260~300℃; The column temperature program is controlled as follows: Hold at 50-60 °C for 1-2 min, then increase the temperature to 110-120 °C at a rate of 30-40 °C / min, and then increase the temperature to 260-300 °C at a rate of 4-5 °C / min and hold for 6-7.5 min; The ion source temperature was 260–300°C; the quenching gas helium flow rate was 2.0–2.25 mL / min, the collision gas nitrogen flow rate was 1.2–1.5 mL / min, the gas flow rate was 0.5–1 mL / min, and the nebulizer pressure was 30–40 psi. The analysis conditions of the liquid phase mass spectrometry are as follows The injection volume was 1–2 μL; Column temperature is 30~40℃; The mobile phase consisted of 0.1% formic acid solution (A) and 1 mmol / L ammonium acetate in methanol solution (B), with a flow rate of 0.2–0.3 mL / min. The mobile phase gradient was set as follows: 0 ~ 10 min, 50 ~ 62% B; 10 ~ 12 min, 62 ~ 70% B; 12 ~ 22 min, 70 ~ 75% B; 22 - ~ 27 min, 75 ~ 100% B; 27 ~ 32 min, 100% B; Electrospray ionization was performed in positive ion source mode with multiple reaction monitoring as the scan type. The ion source temperature was set between 220 and 250°C. The ionization voltage was between -4000 and -4500 V, and the gas flow rate was between 5 and 6 L / min. The sheath gas temperature was set between 300 and 350°C, and the sheath gas flow rate was between 10 and 11 L / min. The capillary voltage was between +2500 and +3000 V, and the nebulizer pressure was set between 20 and 30 psi.

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