WO2022262245A1 - Method for analyzing and detecting trace aflatoxin in water-soluble fermented medicament - Google Patents

Abstract

A method for analyzing and detecting trace aflatoxin in a water-soluble fermented medicament. The method comprises: (1) extracting an aqueous solution of a sample with dichloromethane, blow-drying same with nitrogen, and then dissolving same with a 30% aqueous solution (V/V) of methanol to obtain a test solution; and (2) performing detection by means of ultra-high performance liquid chromatography-mass spectrometry. The problems of a matrix interfering with the determination of a target peak and residual samples in an instrument causing the contamination of the instrument are solved by means of extraction. Aflatoxin and a sample can be fully and effectively separated within 7 min, the linearly dependent coefficient r of aflatoxin in respective linear ranges is more than 0.99, the lowest limit of quantification is 3 ng/L, the lowest limit of detection is 1 ng/L, and the average spiked recovery rate is 80%-120%. The detection method has the characteristics of high speed, high efficiency, high sensitivity, no matrix interference in sample detection, good durability, etc., and can be suitable for quantitative and qualitative detection of aflatoxin in a water-soluble medicament.

Description

A method for the analysis and detection of trace aflatoxins in water-soluble fermented medicines technical field

The invention relates to the technical field of medicine, more specifically, to a method for analyzing and detecting trace amounts of aflatoxins in water-soluble fermented medicines

Background technique

Aflatoxin (AFLATOXIN, referred to as AFT) is a product of the metabolism of Aspergillus flavus and Aspergillus parasiticus, and is recognized as the strongest natural carcinogen. These strains mainly parasitize in edible oil, corn, rice, peanuts, dried fruits, condiments, feed and various foods, and produce toxins, which can cause poisoning of humans and livestock. The chemical structure of aflatoxin has been determined, including more than 10 kinds of aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), among which AFB1, AFB2 , AFG1, AFG2 structures are as follows:

The grain raw materials used in fermented drugs contain Aspergillus flavus or Aspergillus parasitica, which may produce aflatoxin, and the pharmacopoeia of various countries requires the limit to be controlled below 2 μg/kg. Aflatoxin detection methods include thin-layer chromatography (TIC), liquid chromatography (HPLC), enzyme-linked immunoassay (ELISA), fluorescence spectrometry (IAC/SFB), ultra-performance liquid chromatography-mass spectrometry (UPLC-MS ), etc., these methods have high detection sensitivity (mg/kg level), sample pretreatment methods are cumbersome, and the matrix in the sample interferes with the aflatoxin determination and other shortcomings. This method adopts ultra-high performance liquid chromatography for separation, high selectivity and high sensitivity mass spectrometry for detection, and the detection sensitivity is higher (μg/kg level); the sample is dissolved and extracted, the pretreatment is simple, and the sample matrix does not interfere with the determination of the target peak ; The method has good durability, and can detect the content of trace aflatoxin in the sample very well, so as to ensure the product quality of downstream products.

Contents of the invention

The inventors of the present application found that the grain raw materials used in fermented medicines may be contaminated by aflatoxin. In order to realize the quality control of trace aflatoxins in fermented medicines, it is necessary to detect the traces of aflatoxins in fermented medicines.

The purpose of the present invention is to provide a method for detecting trace amounts of aflatoxin in water-soluble fermented medicines. The method has the advantages of high selectivity and high sensitivity, simple sample pretreatment, no interference of the sample matrix to the determination of the target peak, and good durability of the method. , the specific scheme is as follows:

A method for detecting trace amounts of aflatoxin in water-soluble fermented medicines, said aflatoxin comprising aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, characterized in that said method comprises:

Step 1: After dissolving the sample in water, extract it with dichloromethane to obtain the extract, dry the extract with nitrogen, and then dissolve it with 30% aqueous methanol (V/V) to obtain the test solution;

Step 2: using ultra-high performance liquid chromatography-mass spectrometry to detect the test solution in step 1;

Wherein, the chromatographic condition of the ultra-high performance liquid chromatography described in step 2 is:

Chromatographic column: octadecylsilane bonded silica gel column;

Mobile phase: Mobile phase A is 10mM ammonium formate aqueous solution, and mobile phase B is methanol; The gradient elution mode of mobile phase is gradient elution, and the program of described gradient elution is as follows:

time (min)	Mobile phase A%	Mobile phase B%
0.0	70	30
3.0	20	80
5.0	20	80
5.1	70	30

7.0

70

30

The ultra-high performance liquid chromatography-mass spectrometer described in step 2 is: ultra-high performance liquid chromatography-triple quadrupole mass spectrometry;

The water-soluble fermented medicine is selected from acarbose, 1,4-butanedisulfonic acid ademetionine, L-lysine and glutathione.

In step 1, after the sample is dissolved in water, it is prepared to dissolve 1-1000 mg, preferably 1-800 mg, more preferably 1-700 mg, most preferably 10-500 mg of sample solution in 1 mL; add 2 mL of dichloromethane, mix well, After standing, the upper layer solution was discarded, and 1 mL of the lower layer solution was pipetted, blown dry with nitrogen, and directly dissolved with 30% methanol aqueous solution (V/V) to obtain the test solution.

The octadecylsilane-bonded silica gel column described in step 2 has a column diameter of 2.1 mm to 4.6 mm, a column length of 50 mm to 150 mm, and a particle diameter of 1.6 μm to 3.5 μm; preferably octadecylsilane bonded silica gel The column diameter of the chromatographic column is 2.1 mm to 3.5 mm, the column length is 50 mm to 100 mm, and the particle size is 1.6 μm to 2.5 μm; more preferably, the column diameter of the octadecylsilane bonded silica gel column is 2.1 mm to 3.0 mm, The column length is 50mm ~ 100mm, the particle size is 1.6μm ~ 1.8μm; the most preferred Waters

C18 chromatographic column, the column diameter is 2.1mm, the column length is 100mm, and the particle size is 1.6μm.

The column temperature of the chromatographic column in step 2 is 25°C-45°C, preferably 30°C-45°C, more preferably 35°C-40°C, most preferably 40°C.

The flow rate of the mobile phase in step 2 is 0.2mL/min-0.6mL/min, preferably 0.3mL/min-0.5mL/min, more preferably 0.3mL/min-0.4mL/min, most preferably 0.3mL/min.

The parameters of mass spectrometry described in step 2 are:

Scan mode: mass spectrometry multiple reaction monitoring;

Ion source: electrospray ion source;

Ion source mode: positive mode;

Capillary voltage: 0.3-1.0KV, preferably 0.4-0.8KV, more preferably 0.5KV;

Drying gas temperature: 300-650°C, preferably 450-600°C, more preferably 550°C;

Drying gas flow rate: 1000L/Hr;

Cone voltage: 15-40V, preferably 20-30V, more preferably 35V;

Source temperature: 150°C.

The ultra-high performance liquid chromatography-mass spectrometer described in step 2 is Waters ACQUITY I

PLUS\XEVO TQ-XS, Waters ACQUITY I UPLC Class-TQ-S micro, Agilent 1290-6470, preferably Waters ACQUITY I

PLUS\XEVO TQ-XS.

In the aforementioned ultra-high performance liquid phase-mass spectrometry method for detecting aflatoxins of the present invention, the ultra-high performance liquid phase-mass spectrometry chromatographic conditions are as follows:

Column: Waters

C18, 2.1mmx100mm, 1.6μm

Column temperature: 40°C

Flow rate: 0.3mL/min

Detectors were selected from triple quadrupole mass detectors.

The ultra-high performance liquid phase-mass spectrometry method for detecting trace aflatoxins provided by the present invention has the following advantages: the sample pretreatment is simple, and the method of sample dissolution and extraction is used to solve the problem of sample matrix interference target peak determination and sample residue in the instrument. Contamination; good separation, all effective separation of aflatoxin and sample in 7 minutes; good linear correlation coefficient, linear correlation coefficient r>0.99 of aflatoxin in their respective linear ranges; high sensitivity, the lowest quantitative limit of instrument detection is 3ng /L, the minimum detection limit is 1ng/L; the recovery rate is high, and the recovery rate of the standardization accuracy of aflatoxin samples is 80% to 120%. Compared with the existing aflatoxin determination method, the method has the characteristics of rapidity, high efficiency, high sensitivity, no matrix interference in sample detection, good durability, etc., and can be applied to the quantitative and qualitative detection of aflatoxin in water-soluble drugs.

Description of drawings:

Fig. 1 is an ultra-high performance liquid chromatography-mass spectrometry spectrum of aflatoxin (AFB1, AFB2, AFG1, AFG2) standard solution in Example 1 of the method of the present invention.

Fig. 2 is an ultra-high performance liquid chromatography-mass spectrometry spectrum of aflatoxin (AFB1, AFB2, AFG1, AFG2) quantitative limit solution in Example 1 of the method of the present invention.

3 is an ultra-high performance liquid chromatography-mass spectrometry spectrum of detection limits of aflatoxins (AFB1, AFB2, AFG1, AFG2) in Example 1 of the method of the present invention.

detailed description

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and examples.

Using aflatoxin as a reference substance, the trace aflatoxin content in the water-soluble fermented medicine is detected as follows:

Example 1:

Chromatographic conditions:

Instrument: Waters ACQUITY I UPLC Class-TQ-S micro

Column: Waters

C18 2.1mm*100mm*1.6μm

Flow rate: 0.3mL/min

Column temperature: 40°C

Injection volume: 10.0μL

Mobile phase A: 10mM ammonium formate aqueous solution (weigh 0.32g of mass spectrometry grade ammonium formate, dissolve with 500mL ultrapure water, and mix well)

Mobile Phase B: Methanol

Diluent: 30% aqueous methanol (V/V)

Needle washing solution: 80% methanol water solution (V/V)

Blank solvent: same as diluent

gradient:

time (min)	Mobile phase A%	Mobile phase B%
0.0	70	30
3.0	20	80
5.0	20	80
5.1	70	30
7.0	70	30

Mass Spectrometry Conditions:

Preparation of aflatoxin reference standard solution: Precisely pipette an appropriate amount of aflatoxin reference stock solution, and dilute it step by step to prepare aflatoxin (AFB1, AFB2, AFG1, AFG2) concentrations of 30ng/L, 60ng/L, and 120ng respectively /L, 150ng/L, 250ng/L solutions, as the standard curve measurement solution.

The linear regression equation and the linear correlation coefficient r of the four aflatoxins in Example 1 are shown in Table 1. In the equation, y is the peak area of the corresponding aflatoxin, and x is the mass concentration of the corresponding aflatoxin.

Table 1 Linear regression equation, linear correlation coefficient r, detection limit and quantification limit of aflatoxin in Example 1.

Analyte

Linear range

linear equation

Correlation coefficient r

detection limit

The limit of quantitation

the	(ng/L)	the	the	(ng/L)	(ng/L)
AFB1	30～250	y=36364.7536x-1102.8058	0.9997	9	30
AFB2	30～250	y=28317.6766x-238.3089	0.9995	9	30
AFG1	30～250	y=33927.5089x+552.9885	0.9925	9	30
AFG2	30～250	y=18460.1754x+7.3658	0.9967	9	30

Note: detection limit and quantitative limit = corresponding reference substance solution concentration/sample concentration

Detection of Aflatoxin in Acarbose API:

Acarbose sample solution: Weigh 400mg of acarbose sample into a 15mL centrifuge tube, accurately add 1.0mL ultrapure water for ultrasonic dissolution for 5min, then precisely add 2.0mL of dichloromethane, mix well, let stand for 2min, discard For the upper layer solution, 1.0 mL of the lower layer solution was precisely pipetted into a sample injection vial, blown dry with nitrogen, then added 1.0 mL of diluent to dissolve, shake well, and serve as the test solution. The above-mentioned LC-MS method was used for detection, and the standard curve method was used to calculate the content of aflatoxin in the sample. Aflatoxin was not detected in the acarbose sample solution, which was less than the detection limit (0.045 μg/kg).

Accuracy of table 2 aflatoxin in embodiment 1

Example 2:

Instrument: Waters ultra-high performance liquid chromatography-triple quadrupole mass spectrometer (Waters ACQUITY I

PLUS\XEVO TQ-XS)

Column: Waters

C18 2.1mm*100mm*1.6μm

Flow rate: 0.3mL/min

Column temperature: 40°C

Injection volume: 10.0μL

Mobile phase A: 10mM ammonium formate aqueous solution (weigh 0.32g of mass spectrometry grade ammonium formate and dissolve in 500mL ultrapure water)

Mobile Phase B: Methanol

Diluent: 30% aqueous methanol (V/V)

Needle washing solution: 80% methanol water solution (V/V)

Blank solvent: same as diluent

gradient:

time (min)	Mobile phase A%	Mobile phase B%
0.0	70	30
3.0	20	80
5.0	20	80
5.1	70	30
7.0	70	30

Mass spectrometry conditions:

Preparation of aflatoxin reference standard solution: Precisely pipette an appropriate amount of aflatoxin reference solution, and dilute it step by step to prepare aflatoxin concentrations of 3ng/L, 6ng/L, 12ng/L, 15ng/L, and 25ng/L The solution was used as the standard curve measurement solution.

The linear regression equation and the linear correlation coefficient r of the four aflatoxins in Example 2 are shown in Table 3. In the equation, y is the peak area of the corresponding aflatoxin, and x is the mass concentration of the corresponding aflatoxin.

Table 3 Linear regression equation, linear correlation coefficient r, detection limit and quantification limit of aflatoxin in Example 2.

Note: detection limit and quantitative limit = corresponding reference substance solution concentration/sample concentration

Detection of aflatoxin in 1,4-butanedisulfonic acid adenosylmethionine API:

1,4-butanedisulfonic acid adenosylmethionine sample solution: Weigh 48mg of 1,4-butanedisulfonic acid adenosylmethionine sample into a 15mL centrifuge tube, add 1.0mL water for ultrasonic dissolution for 5min, then add 2.0mL dichloromethane , mix well, let it stand for 2min, discard the upper layer solution, pipette 1.0mL of the lower layer solution into the injection vial, blow dry with nitrogen, then add 1.0mL diluent to dissolve, shake well, and use it as the test solution. The above-mentioned LC-MS method was used for detection, and the standard curve method was used to calculate the content of aflatoxin in the sample. Aflatoxin was not detected in the 1,4-butanedisulfonic acid adenosylmethionine sample solution, which was less than the detection limit (0.0375 μg/kg).

Table 4 Aflatoxin Accuracy in Example 2 Sample 1,4-Ademetionine Butanedisulfonate

Detection of aflatoxins in L-lysine raw materials:

L-lysine sample solution: Weigh 30mg of L-lysine sample into a 15mL centrifuge tube, add 1.0mL water to ultrasonically dissolve for 5min, then add 2.0mL dichloromethane, mix well, let stand for 2min, discard the upper solution , Pipette 1.0mL of the lower layer solution into the sample injection vial, dry it with nitrogen, then add 1.0mL of diluent to dissolve, shake well, and use it as the test solution. The above-mentioned LC-MS method was used for detection, and the standard curve method was used to calculate the content of aflatoxin in the sample. Aflatoxins were not detected in the L-lysine sample solution, which was less than the detection limit (0.06 μg/kg).

Table 5 aflatoxin accuracy in embodiment 2 sample L-lysine

Detection of aflatoxins in glutathione raw materials:

Glutathione sample solution: Weigh 60 mg of L-lysine sample into a 15 mL centrifuge tube, add 1.0 mL of water for ultrasonic dissolution for 5 min, then add 2.0 mL of dichloromethane, mix well, let stand for 2 min, discard the upper layer solution, Pipette 1.0mL of the lower layer solution into the sample injection vial, dry it with nitrogen, then add 1.0mL diluent to dissolve, shake well, and use it as the test solution. The above-mentioned LC-MS method was used for detection, and the standard curve method was used to calculate the content of aflatoxin in the sample. Aflatoxins were not detected in the glutathione sample solution, which was less than the detection limit (0.03 μg/kg).

Table 6 aflatoxin accuracy in embodiment 2 sample glutathione

Example 3:

Instrument: Agilent UPLC-triple quadrupole mass spectrometer (Agilent 1290-6470)

Column: Waters

C18 2.1mm*100mm*1.6μm

Flow rate: 0.3mL/min

Column temperature: 40°C

Injection volume: 10.0μL

Mobile phase A: 10mM ammonium formate aqueous solution (weigh 0.32g of mass spectrometry grade ammonium formate and dissolve in 500mL ultrapure water)

Mobile Phase B: Methanol

Diluent: 30% aqueous methanol (V/V)

Needle washing solution: 80% methanol water solution (V/V)

Blank solvent: same as diluent

gradient:

time (min)	Mobile phase A%	Mobile phase B%
0.0	70	30
3.0	20	80
5.0	20	80
5.1	70	30

7.0

70

30

Mass Spectrometry Conditions:

Preparation of aflatoxin reference standard solution: Precisely pipette an appropriate amount of aflatoxin reference solution, and dilute it step by step to prepare aflatoxin concentrations of 30ng/L, 60ng/L, 120ng/L, 150ng/L, and 250ng/L The solution was used as the standard curve measurement solution.

The linear regression equations and linear correlation coefficients r of the four aflatoxins in Example 3 are shown in Table 7. In the equation, y is the peak area of the corresponding aflatoxin, and x is the mass concentration of the corresponding aflatoxin.

Table 7 Linear regression equation, linear correlation coefficient r, detection limit and quantification limit of aflatoxin in Example 3.

Note: detection limit and quantitative limit = corresponding reference substance solution concentration/sample concentration

Detection of aflatoxin in 1,4-butanedisulfonic acid adenosylmethionine API:

1,4-butanedisulfonic acid adenosylmethionine sample solution: Weigh 600mg of 1,4-butanedisulfonic acid adenosylmethionine sample into a 15mL centrifuge tube, add 1.0mL water for ultrasonic dissolution for 5min, then add 2.0mL dichloromethane , mix well, let it stand for 2min, discard the upper layer solution, pipette 1.0mL of the lower layer solution into the injection vial, blow dry with nitrogen, then add 1.0mL diluent to dissolve, shake well, and use it as the test solution. The above-mentioned LC-MS method was used for detection, and the standard curve method was used to calculate the content of aflatoxin in the sample. Aflatoxin was not detected in the 1,4-butanedisulfonic acid adenosylmethionine sample solution, which was less than the detection limit (0.03 μg/kg).

Table 8 Aflatoxin Accuracy in Example 3 Sample 1,4-Ademetionine Butanedisulfonate

Detection of aflatoxins in L-lysine raw materials:

L-lysine sample solution: Weigh 300mg of L-lysine sample into a 15mL centrifuge tube, add 1.0mL water for ultrasonic dissolution for 5min, then add 2.0mL dichloromethane, mix well, let stand for 2min, discard the upper solution , Pipette 1.0mL of the lower layer solution into the sample injection vial, dry it with nitrogen, then add 1.0mL of diluent to dissolve, shake well, and use it as the test solution. The above-mentioned LC-MS method was used for detection, and the standard curve method was used to calculate the content of aflatoxin in the sample. Aflatoxins were not detected in the L-lysine sample solution, which was less than the detection limit (0.06 μg/kg).

Accuracy of table 9 aflatoxin in embodiment 3 sample L-lysine

Detection of aflatoxins in glutathione raw materials:

Glutathione sample solution: Weigh 1000mg of L-lysine sample into a 15mL centrifuge tube, add 5.0mL water for ultrasonic dissolution for 5min, then add 2.0mL dichloromethane, mix well, let stand for 2min, discard the upper layer solution, Pipette 1.0mL of the lower layer solution into the sample injection vial, dry it with nitrogen, then add 1.0mL diluent to dissolve, shake well, and use it as the test solution. The above-mentioned LC-MS method was used for detection, and the standard curve method was used to calculate the content of aflatoxin in the sample. Aflatoxins were not detected in the glutathione sample solution, which was less than the detection limit (0.018 μg/kg).

Accuracy of table 10 aflatoxin in embodiment 3 sample glutathione

As can be seen from the above-mentioned embodiments, the detection method provided by the present invention has simple sample pretreatment, and adopts the sample dissolution and extraction method to solve the problem of sample matrix interference target peak measurement and sample residue in the instrument causing instrument pollution; the separation is good, and Aspergillus flavus The toxin and the sample can be effectively separated within 7 minutes; the linear correlation coefficient is good, and the linear correlation coefficient r of aflatoxin in the linear range of 3ng/L-25ng/L or 30ng/L-250ng/L is greater than 0.99; the sensitivity is high, and the aflatoxin The minimum quantification limit for toxin instrument detection is 3ng/L, the minimum limit is 0.1μg/kg, the minimum detection limit is 1ng/L, and the minimum limit is 0.03μg/kg; Between 80% and 120%. Compared with the existing aflatoxin determination method, the method has the characteristics of rapidity, high efficiency, high sensitivity, no matrix interference in sample detection, good durability, etc., and can be applied to the quantitative and qualitative detection of aflatoxin in water-soluble drugs.

Claims (7)

1. A method for detecting trace amounts of aflatoxin in water-soluble fermented medicines, said aflatoxin comprising aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, characterized in that said method comprises:

   Step 1: After dissolving the sample in water, extract it with dichloromethane to obtain the extract, dry the extract with nitrogen, and then dissolve it with 30% aqueous methanol (V/V) to obtain the test solution;

   Step 2: using ultra-high performance liquid chromatography-mass spectrometry to detect the test solution in step 1;

   Wherein, the chromatographic condition of the ultra-high performance liquid chromatography described in step 2 is:

   Chromatographic column: octadecylsilane bonded silica gel column;

   Mobile phase: Mobile phase A is 10mM ammonium formate aqueous solution, and mobile phase B is methanol; The gradient elution mode of mobile phase is gradient elution, and the program of described gradient elution is as follows:

   time (min) Mobile phase A% Mobile phase B% 0.0 70 30 3.0 20 80 5.0 20 80 5.1 70 30 7.0 70 30

   The ultra-high performance liquid chromatography-mass spectrometer described in step 2 is: ultra-high performance liquid chromatography-triple quadrupole mass spectrometry;

   The water-soluble fermented medicine is selected from acarbose, 1,4-butanedisulfonic acid ademetionine, L-lysine and glutathione.
2. The method according to claim 1, characterized in that in step 1, after the sample is dissolved in water, it is configured to dissolve 1-1000 mg, preferably 1-800 mg, more preferably 1-700 mg, most preferably 10-500 mg in 1 mL. Add 2 mL of dichloromethane, mix well, discard the upper layer solution after standing, pipette the lower layer solution 1 mL, blow dry with nitrogen, and directly dissolve with 30% aqueous methanol (V/V) to obtain the test solution.
3. The method according to any one of claims 1-2, wherein the column diameter of the octadecylsilane-bonded silica gel chromatographic column described in step 2 is 2.1 mm to 4.6 mm, and the column length is 50 mm to 150 mm. The particle size is 1.6 μm to 3.5 μm; preferably, the octadecylsilane bonded silica gel column has a column diameter of 2.1 mm to 3.5 mm, a column length of 50 mm to 100 mm, and a particle size of 1.6 μm to 2.5 μm; more preferably octadecylsilane The column diameter of the alkylsilane bonded silica gel column is 2.1mm-3.0mm, the column length is 50mm-100mm, and the particle size is 1.6μm-1.8μm; the most preferred Waters

   

   C18 chromatographic column, the column diameter is 2.1mm, the column length is 100mm, and the particle size is 1.6μm.
4. The method according to any one of claims 1-3, characterized in that the column temperature of the chromatographic column in step 2 is 25°C to 45°C, preferably 30°C to 45°C, more preferably 35°C to 40°C, 40°C is most preferred.
5. The method according to any one of claims 1-4, wherein the flow rate of the mobile phase in step 2 is 0.2mL/min～0.6mL/min, preferably 0.3mL/min～0.5mL/min, more preferably Preferably 0.3mL/min～0.4mL/min, most preferably 0.3mL/min.
6. The method according to any one of claims 1-5, wherein the parameters of the mass spectrum in step 2 are:

   Scan mode: mass spectrometry multiple reaction monitoring;

   Ion source: electrospray ion source;

   Ion source mode: positive mode;

   Capillary voltage: 0.3-1.0KV, preferably 0.4-0.8KV, more preferably 0.5KV;

   Drying gas temperature: 300-650°C, preferably 450-600°C, more preferably 550°C;

   Drying gas flow rate: 1000L/Hr;

   Cone voltage: 15-40V, preferably 20-30V, more preferably 35V;

   Source temperature: 150°C.
7. The method according to any one of claims 1-6, wherein the ultra-high performance liquid chromatography-mass spectrometer described in step 2 is Waters ACQUITY I

   

   PLUS\XEVO TQ-XS, Waters ACQUITY I UPLC Class-TQ-S micro, Agilent 1290-6470, preferably Waters ACQUITY I

   

   PLUS\XEVO TQ-XS.

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