Method for determining content of acetaminophen related substances by combining chemical derivatization with HPLC-ICP-MS (high performance liquid chromatography-inductively coupled plasma-mass spectrometry)

Abstract

The invention belongs to the field of drug detection, and particularly discloses a method for determining the content of related substances in acetaminophen by combining chemical derivatization with HPLC-ICP-MS. A to-be-detected sample is subjected to a derivatization reaction by adopting a compound # imgabs0 # as shown in a formula 1, and then HPLC-ICP-MS determination is performed. According to the method, a brand new thought for determining the content of the related substances in the acetaminophen based on ICP-MS is provided for the first time, the to-be-determined sample is innovatively derived by utilizing the formula 1, and the ICP-MS determination is further matched, so that the accurate determination of the related substances in the acetaminophen can be successfully realized on the premise of not needing an auxiliary standard substance.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a method for determining the contents of related substances o-acetaminophen, p-nitrophenol and p-aminophenol in acetaminophen. Technical Background

[0002] Drug impurities refer to other chemical substances in drugs besides the active ingredients, and are an important factor affecting the quality of drugs. According to the guidelines of the International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), drug impurities can be divided into organic impurities, inorganic impurities and residual solvents. Among them, organic impurities, also known as related substances, mostly have potential physiological activity, and some may even cause serious toxic side effects. The inspection of related substances in drug preparations is an important part of drug quality control, and the content of related substances is closely related to the safety and effectiveness of drugs. At present, the detection of related substances in drug preparations is mainly based on structure-dependent detection technologies such as liquid chromatography-ultraviolet detector (LC-UV), and its quantitative determination of related substances requires standard samples of the substances to be tested. However, standard samples of related substances are often difficult to obtain or lacking.

[0003] At present, the main component self-comparison method is mostly used in the pharmacopoeias of various countries to achieve indirect quantification of related substances, that is, the peak area of ​​the related substance is compared with the peak area of ​​the main component dilution solution with a specified concentration (generally 0.1% or 0.5%) to perform relative quantification of the related substances. The main means of determining the content of acetaminophen related substances is the non-derivatized HPLC-UV method. For example, the literature (Applied Ecology and Environmental Research, 2019, 17 (5): 10731-10742; Journal of Pharmaceutical Analysis, 2017, 37 (02): 237-242.) and pharmacopoeias have reported the HPLC-UV method. For example, the existing technologies with publication numbers CN106353428A, CN102147360A, CN101566608A, etc. have reported some similar methods.

[0004] However, the principal component self-control method can only achieve relatively accurate quantitative analysis of related substances when the structure of the related substances is extremely similar to that of the main drug (similar HPLC-UV response factors). When the analytical response difference between the related substance and the original drug is large (such as the response value of the related substance in HPLC-UV is much lower than that of the main component at the same concentration), the measured value of the related substance may be low, but its actual content is already higher than the safe range. Taking the α1 receptor blocker terazosin as an example, the HPLC-UV response of the main drug at the same concentration is more than 30 times that of its related substance 1-[(tetrahydrofuran-2-yl)formyl]piperazine. Therefore, it is extremely important to develop an analytical strategy that can accurately quantify drug-related substances without the need for standards to ensure drug quality and safety. Summary of the invention

[0005] In view of the problem that existing means for testing the content of related substances in acetaminophen are relatively scarce, the present invention provides a method for determining the content of related substances in acetaminophen by chemical derivatization combined with HPLC-ICP-MS, aiming to provide a new idea and means for testing related substances in acetaminophen without relying on standard samples.

[0006] A method for determining the content of related substances in acetaminophen by chemical derivatization combined with HPLC-ICP-MS, wherein the compound of formula 1 is used to carry out a derivatization reaction on a sample to be tested, followed by HPLC-ICP-MS determination;

[0007]

[0008] In Formula 1, X is a halogen.

[0009] The present invention proposes for the first time a new idea for determining the content of related substances in acetaminophen based on ICP-MS, which innovatively utilizes formula 1 to derive the sample to be tested, and further cooperates with ICP-MS determination, so that the accurate determination of related substances in acetaminophen can be successfully achieved without the need for auxiliary standard substances. The test method of the present invention has high detection sensitivity and the analysis response is not affected by the chemical structure of the test object, and can achieve quantitative analysis without the need for standard substances of the test object.

[0010] In the present invention, the related substance includes at least one of o-acetaminophen, p-nitrophenol and p-aminophenol.

[0011] In the present invention, the sample to be tested is a preparation containing acetaminophen for which related substances need to be identified, preferably a tablet.

[0012] In the present invention, the compound of formula 1 may preferably be a compound of formula 1-A;

[0013]

[0014] In the present invention, the derivatization reaction is carried out under the catalysis of an acid binding agent;

[0015] Preferably, the acid-binding agent comprises at least one of TEA, pyridine, ammonia water, and sodium hydroxide;

[0016] Preferably, the amount of the acid-binding agent is not less than 1 times the theoretical molar amount for completely acylation of hydroxyl groups and amino groups in the derivatization reaction system; considering the material cost, further, the acid-binding agent is more than 5 times the theoretical molar amount of acetaminophen and related substances in the sample to be tested, further 5 to 80 times; further 15 to 50 times;

[0017] Preferably, the amount of the compound of formula 1 is not less than 1 times the theoretical molar amount for completely acylation of the hydroxyl group and the amino group in the derivatization reaction system; considering the material cost, further, the compound of formula 1 is more than 5 times the theoretical molar amount of acetaminophen and its related substances in the sample to be tested, further 5 to 100 times; further 50 to 90 times.

[0018] In the present invention, the temperature of the derivatization reaction is 20 to 80°C; preferably 30 to 50°C;

[0019] Preferably, the derivatization reaction time is more than 5 minutes, and further preferably 5 to 60 minutes.

[0020] In the present invention, the HPLC conditions in the HPLC-ICP-MS determination process are: chromatographic column: Thermo AcclainTrinity P1 chromatographic column; eluent A: 8-12 mM ammonium acetate-water (pH=5); eluent B: acetonitrile;

[0021] The gradient elution program was as follows: 0-2 min: mobile phase A 80%→50%; 2-4 min: mobile phase A 50%; 4-8 min: mobile phase A 50%→15%; 8-10 min: mobile phase A 15%; 10-13 min: mobile phase A 15%→80%; 13-16 min: mobile phase A 80%;

[0022] Preferably, the size of the ThermoAcclain Trinity P1 chromatographic column is 3 μm, 2.1×50 mm;

[0023] Preferably, the column temperature is 34-36°C.

[0024] In the present invention, the ICP-MS conditions in the HPLC-ICP-MS determination process are: monitoring mode: No gas; measuring mass number: 127 I +; RF power: 1500-1600W; atomizing gas flow rate: 0.4-0.5L / min; reaction gas is O2;

[0025] Preferably, the temperature of the atomization chamber is -4 to -6°C; the sampling depth is 6 to 8 mm.

[0026] In the present invention, the related substances in the sample to be tested are determined based on the external standard method of element standards.

[0027] Beneficial Effects

[0028] The present invention innovatively combines chemical derivatization with HPLC separation technology and ICP-MS's non-chemical structure-dependent detection performance, innovatively pre-derivates the sample to be tested using Formula 1, and then uses HPLC-ICP-MS to determine the content of acetaminophen-related substances. The results of methodological validation show that the method has good accuracy and can achieve highly sensitive quantitative analysis of drug-related substances without the need for analytical standards, effectively solving the problem of quantitative analysis of related substances in drugs when there is a lack of analytical standards for related substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The structural diagram of acetaminophen and its related substances;

[0030] Figure 2 This is the derivatization reaction of acetaminophen and its related substances in Example 1;

[0031] Figure 3 is the ESI-MS spectrum of the paracetamol-related substance derivatization product in Example 1. a is the paracetamol-derivation product [M+H] + ESI-MS spectrum of a; b is the product of p-nitrophenol derivatization [MH] - ESI-MS spectrum of ; c is the product of p-aminophenol derivatization [M+H] + ESI-MS spectrum of .

[0032] Figure 4 The following are representative chromatograms of the relevant derivatization products of the HPLC-ICP-MS selective investigation of acetaminophen and its related substances in Example 1. a is a representative chromatogram of the derivatization products of the pure solution of the main drug acetaminophen (concentration of 2500 μM) in HPLC-ICP-MS; b is a representative chromatogram of the derivatization sample of the mixed solution containing acetaminophen (2500 μM) and its three related substances (all 2.5 μM) in HPLC-ICP-MS. DETAILED DESCRIPTION

[0033] The present invention is described in detail below in conjunction with embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some adjustments and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0034] Unless otherwise specified, the technical methods used in the examples are conventional methods known to those skilled in the art. In the examples, all raw materials added are commercially available conventional raw materials unless otherwise specified.

[0035] The testing method of the present invention may include the following specific steps:

[0036] Weigh appropriate amounts of acetaminophen and its related substances, o-acetaminophen, p-aminophenol and p-nitrophenol standards, and use acetonitrile as solvent to prepare stock solutions and working solutions of acetaminophen and its related substances. Mix the above working solutions in a certain proportion to prepare a mixed working solution. Take an appropriate amount of the mixed working solution of acetaminophen and its related substances for the development of the derivatization method.

[0037] Furthermore, the derivatization of acetaminophen and its related substances is carried out by using Formula 1 (such as Formula 1-A) as a derivatization reagent under the catalytic action of triethylamine. After optimizing the derivatization reaction conditions so that the related substances of acetaminophen are completely converted into corresponding derivatized products, the derivatized samples are used for the development of HPLC-ICP-MS methods.

[0038] Furthermore, the derivatized samples were directly injected into the HPLC-ICP-MS system to explore the optimal chromatographic separation conditions for each derivatized product. The derivatized samples were injected into the LC-MS / MS system to confirm the structure of each derivatized product.

[0039] Furthermore, the derivatized samples were injected into the HPLC-ICP-MS system for analysis. The selectivity, linearity, limit of quantification (LOQ), accuracy and precision of the established HPLC-ICP-MS analysis method were verified with reference to the ICH guideline "Q2(R1) Validation of Analytical Methods".

[0040] Example 1: Determination of the content of acetaminophen-related substances by chemical derivatization combined with HPLC-ICP-MS technology

[0041] The specific steps are:

[0042] 1. Derivatization reaction of acetaminophen and its related substances

[0043] 1. Preparation of derivatization reaction solution

[0044] Weigh 30.24 mg of acetaminophen standard in a 20 mL brown bottle, dissolve in 10 mL acetonitrile, vortex for 1 min, and prepare a 20 mM acetaminophen stock solution. Weigh 7.56 mg of o-acetaminophen, 6.99 mg of p-nitrophenol, and 5.46 mg of p-aminophenol standard in three 20 mL brown bottles, dissolve in 10 mL acetonitrile, vortex for 1 min, and prepare 5 mM stock solutions of the three related substances.

[0045] Weigh 12.65 mg of p-iodobenzoic acid standard in a 20 mL brown bottle, dissolve in 10 mL methanol, vortex for 1 min, and prepare a p-iodobenzoic acid stock solution with a concentration of 5 mM. Weigh 16.19 mg of triethylamine in a 20 mL brown bottle, dissolve in 10 mL acetonitrile, vortex for 1 min, and prepare a catalyst triethylamine stock solution with a concentration of 16 mM. Weigh 53.29 mg of formula 1-A standard in a 20 mL brown bottle, dissolve in 10 mL acetonitrile, vortex for 1 min, and prepare a derivatization reagent formula 1-A stock solution with a concentration of 20 mM. The above stock solutions are stored in a 4 ° C refrigerator.

[0046] Add 500 μL of catalyst triethylamine stock solution to a 1.5 mL EP tube containing 500 μL of acetonitrile to prepare a triethylamine working solution with a concentration of 8 mM. Add 20 μL of o-acetaminophen, p-nitrophenol and p-aminophenol stock solutions to three 1.5 mL EP tubes containing 980 μL of acetonitrile to prepare three related substance working solutions with a concentration of 100 μM. Add 10 μL of acetaminophen stock solution (20 μM), 50 μL of o-acetaminophen working solution (100 μM), 50 μL of p-nitrophenol working solution (100 μM) and 50 μL of p-aminophenol working solution (100 μM) to a 4 mL EP tube containing 1840 μL of acetonitrile, vortex for 30 seconds to prepare a mixed working solution containing 100 μM of acetaminophen and 5 μM of each of the three related substances.

[0047] 2. Optimization of derivatization reaction conditions

[0048] 1) Selection of Formula 1-A concentration: This experiment investigated the effect of Formula 1-A concentration on the reaction conversion rate. The working solution concentrations of Formula 1-A were 2, 4, 8, 12, 16, and 20 mM, respectively.

[0049] 2) Selection of catalyst triethylamine concentration: This experiment investigated the effect of triethylamine concentration on the reaction conversion rate. The concentrations of triethylamine working solution were 2, 4, 8, 12, and 16 mM, respectively.

[0050] 3) Selection of derivatization temperature: This experiment investigated the effect of derivatization temperature on the reaction conversion rate. The temperatures were room temperature (25°C), 40°C, 60°C, and 80°C.

[0051] 4) Selection of derivatization time: This experiment investigated the effect of derivatization time on the reaction conversion rate, with the times being 5, 15, 30, 45, and 60 min respectively.

[0052] 3. Structural confirmation of the corresponding derivatized products of related substances

[0053] The structures of the products were confirmed by LC-MS using an Agilent 1290 Infinity II ultra-high performance liquid chromatography coupled to an Agilent 6470 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source.

[0054] Conditions of the LC-MS system: chromatographic column: Waters Acquity UPLC HSS T3 chromatographic column (1.8 μm, 2.1×50 mm); column temperature: 40°C; injection volume: 1 μL; mobile phase A: 0.1% (v / v) formic acid-water; mobile phase B: acetonitrile. The gradient elution program is shown in Table 1. The mass spectrometry conditions are shown in Table 2.

[0055] Table 1 Gradient elution procedure for structural confirmation of derivatization reaction products

[0056]

[0057] Table 2 Mass spectrometry conditions for structural confirmation of derivatization reaction products

[0058]

[0059]

[0060] 2. Development of a method for the determination of related substances derived products in acetaminophen by HPLC-ICP-MS

[0061] 1. Preparation of acetaminophen and related substances working solution

[0062] Weigh 30.24 mg of acetaminophen standard in a 20 mL brown bottle, dissolve in 10 mL acetonitrile, vortex for 1 min, and prepare a 20 mM acetaminophen stock solution. Weigh 7.56 mg of o-acetaminophen, 6.99 mg of p-nitrophenol, and 5.46 mg of p-aminophenol standard in three 20 mL brown bottles, dissolve in 10 mL acetonitrile, vortex for 1 min, and prepare 5 mM stock solutions of the three related substances. All the above stock solutions are stored in a 4 ° C refrigerator.

[0063] 20 μL of acetaminophen, p-nitrophenol and p-aminophenol stock solutions were added to three 1.5 mL EP tubes containing 980 μL of acetonitrile to prepare three related substance working solutions with a concentration of 100 μM. 25 μL of acetaminophen stock solution (20 mM), 25 μL of acetaminophen working solution (100 μM), 25 μL of p-nitrophenol working solution (100 μM) and 25 μL of p-aminophenol working solution (100 μM) were added to a 4 mL EP tube containing 1900 μL of acetonitrile, and vortexed for 30 seconds to prepare a mixed working solution containing 250 μM of acetaminophen and 2.5 μM of each of the three related substances. After derivatization reaction under optimal conditions, it was used to test the separation effect of HPLC-ICP-MS (the concentration of the derivatized product of each related substance in the final reaction solution was 1 μM). A mixed solution containing 5000 μM acetaminophen and 5 μM three related substances was prepared according to similar steps and used as the system adaptability solution after derivatization (the concentration of the derivatized product of each related substance in the final reaction solution was 1 μM).

[0064] According to Table 3 below, the p-iodobenzoic acid stock solution was diluted stepwise with acetonitrile as solvent to obtain a series of working solutions with concentrations of 10, 8, 5, 4, 2, 1, and 0.5 μM, which were used to establish the standard curve of the I element for the quantitative determination of acetaminophen-related substances. According to Table 4 below, the QC-H, QC-M, and QC-L quality control samples of acetaminophen-related substances were prepared with similar steps and used for the validation of the HPLC-ICP-MS method after derivatization.

[0065] Table 3 HPLC-ICP-MS determination of acetaminophen related substances I standard curve sample concentration

[0066]

[0067]

[0068] Table 4 HPLC-ICP-MS quality control sample concentrations of acetaminophen-related substances

[0069]

[0070] 2. Selection of ICP-MS quantitative method

[0071] Choose the external standard method using elemental standards instead of related substance standards. A 7-point external standard method was used to establish a standard curve for a series of I (prepared in the form of p-iodobenzoic acid) standard solutions with a concentration range of 0.5-10 μM. Since gradient elution was used and the entire elution process was accompanied by a significant change in the proportion of the organic phase, which caused a change in sensitivity, the sensitivity needed to be corrected first. That is, first calculate the corresponding dead time through the dead volume of the instrument and the volume of the chromatographic column, and calculate the corresponding eluent composition at the peak time of the derivative product of each related substance based on the dead time and the peak time of each related substance. According to calculations, the eluent composition at the peak time of the o-acetaminophen derivatization product is: 47.1% aqueous phase-52.9% organic phase, the eluent composition at the peak time of the p-nitrophenol derivatization product is: 40.3% aqueous phase-59.7% organic phase, the eluent composition at the peak time of the external standard p-iodobenzoic acid is: 32.9% aqueous phase-67.1% organic phase; the peak time of the p-aminophenol derivatization product is: 24.2% aqueous phase-75.8% organic phase.

[0072] Under the above-mentioned eluent compositions, 6 parallel injections of 10 μM p-iodobenzoic acid were performed to obtain the response of I at different times, and the peak area correction factor f of the derivatized products of each related substance relative to the external standard p-iodobenzoic acid was calculated by the following formula.

[0073]

[0074] 3. HPLC-ICP-MS detection conditions

[0075] The samples were analyzed using an Agilent 1260infinity HPLC system equipped with an Agilent 1260infinity autosampler, an Agilent 1260infinity degasser, an Agilent 1260infinity binary pump and an Agilent 1260infinity thermostatic column chamber coupled to an Agilent 7900 triple quadrupole ICP-MS system.

[0076] High performance liquid chromatography conditions: chromatographic column: ThermoAcclain Trinity P1 chromatographic column (3 μm, 2.1×50 mm); column temperature: 35°C; injection volume: 2 μL; eluent A: 10 mM ammonium acetate-water (pH=5); eluent B: acetonitrile. The gradient elution program is shown in Table 5. The mass spectrometry conditions are shown in Table 6.

[0077] Table 5 HPLC-ICP-MS gradient elution program

[0078]

[0079] Table 6 Parameters of acetaminophen related substances determined by ICP-MS

[0080]

[0081]

[0082] 3. Results and Discussion

[0083] 1. Determination of derivatization reaction conditions

[0084] The optimal derivatization conditions after optimization are: add 100 μL of 8 mM triethylamine solution to 200 μL of mixed working solution, then add 100 μL of 20 mM solution of formula 1-A, vortex for 30 seconds, react in a constant temperature oscillator at 40°C for 30 minutes, and take out the reaction solution after the reaction is completed. After cooling to room temperature, add 100 μL of 0.5% (v / v) formic acid-water to the mixed solution to hydrolyze the excess derivatization reagent and acidify the sample.

[0085] 2. Structural confirmation of the corresponding derivatized products of related substances

[0086] The ESI-MS spectra of the corresponding derivatized products of the three related substances of acetaminophen are shown in Figure 3 As shown in the figure, the mass-to-charge ratios and isotope distributions observed when the peaks appeared in the mass spectra of the three related substance derivatization products were very consistent with expectations. The spectral peaks observed at the mass-to-charge ratios of m / z 381.9, 382.9 and 383.9 and the corresponding isotope distributions (100:17.2:2.0) of the acetaminophen derivatization products were consistent with their [M+H] + The molecular ion and its theoretical isotopic distribution (100:16.9:1.9) are very consistent. The spectral peaks observed at mass-to-charge ratios of m / z 368.2, 379.2, and 370.2 and the corresponding isotopic distribution (100:13.7:1.5) of the p-nitrophenol derivatized product are consistent with its [MH] - The molecular ion and theoretical isotope distribution (100:14.7:1.8) are consistent. The spectral peaks observed at the mass-to-charge ratios of m / z 569.9, 570.9, and 571.9 and the corresponding isotope distribution (100:24.5:3.5) of the p-aminophenol derivatized product are also consistent with its [M+H] + The molecular ion is consistent with its theoretical isotope distribution (100:22.3:2.9).

[0087] 3. Selection of ICP-MS quantitative method

[0088] Under the above-mentioned different eluent compositions, a 10 μM iodine-containing standard solution (p-iodobenzoic acid) was injected, and the relevant 127The I responses and calculated correction factors are shown in Table 7 below:

[0089] Table 7 Different eluent compositions 127 I response and correction factor calculation results

[0090]

[0091] 4. Validation of the HPLC-ICP-MS Method for Quantitative Derivatization of Paracetamol-Related Substances

[0092] (1) Selectivity: Figure 4 This is a representative chromatogram of the relevant samples in HPLC-ICP-MS. As can be seen from the figure, no interference peaks were observed at the elution time of the derivative products of acetaminophen and its related substances, and there was good separation between the main drug and its three related substances.

[0093] (2) Linearity: A standard curve was established for I standard solutions (prepared with p-iodobenzoic acid) in the concentration range of 0.5-10 μM. The equation of the I standard curve was: y = 34285x - 1336.2. The R 2 >0.999, and the back-calculated concentration deviations of the points taken from the calibration curve were all within ±10% (including LLOQ), so the calibration curve established had good linearity.

[0094] (3) LOQ: According to USP <621> The LOQs of o-acetaminophen, p-nitrophenol, and p-aminophenol determined by HPLC-ICP-MS were 0.089 μM, 0.097 μM, and 0.161 μM, respectively, based on the signal-to-noise ratio.

[0095] (4) Accuracy and precision: The accuracy and precision results of the three related substances are shown in Tables 8-10 below. The accuracy of HPLC-ICP-MS for the determination of o-acetaminophen is between 95.53% and 105.00%, and the precision is less than 4.03%. The accuracy of 4-nitrophenol is between 95.00% and 105.00%, and the precision is less than 4.79%. The accuracy of o-acetaminophen is between 95.12% and 104.80%, and the precision is less than 3.43%. The results show that the developed HPLC-ICP-MS method has good accuracy and precision.

[0096] Table 8 Accuracy and precision of HPLC-ICP-MS determination of acetaminophen derivatives

[0097]

[0098]

[0099] Table 9 Accuracy and precision results of HPLC-ICP-MS determination of p-nitrophenol derivatization products

[0100]

[0101] Table 10 Accuracy and precision results of HPLC-ICP-MS determination of p-aminophenol derivatization products

[0102]

[0103]

[0104] Due to the limitations of textual expression and the objective existence of other impurities with similar chemical structures, ordinary technicians in this technical field can make appropriate improvements or changes without departing from the principles of the present invention, and can also combine the above-mentioned technical features in an appropriate manner; these improvements, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be regarded as the scope of protection of the present invention.

Claims

1. A method for determining the content of related substances in acetaminophen by chemical derivatization combined with HPLC-ICP-MS, characterized in that: The compound of formula 1 is used to carry out a derivatization reaction on the sample to be tested, followed by HPLC-ICP-MS determination; In Formula 1, X is a halogen.

2. The method according to claim 1, characterized in that The related substances include at least one of o-acetaminophen, p-nitrophenol and p-aminophenol.

3. The method according to claim 2, characterized in that The sample to be tested is a preparation containing acetaminophen for which related substances need to be identified, preferably a tablet.

4. The method according to claim 1, characterized in that The compound of formula 1 is a compound of formula 1-A; 5. The method according to claim 1, characterized in that The derivatization reaction is carried out under the catalysis of an acid-binding agent; Preferably, the acid-binding agent comprises at least one of TEA, pyridine, ammonia water, and sodium hydroxide; Preferably, the amount of the acid-binding agent is not less than 1 times the theoretical molar amount for completely acylation of hydroxyl groups and amino groups in the derivatization reaction system; further, the amount of the acid-binding agent is more than 5 times, further 5 to 80 times, and further 15 to 50 times the theoretical molar amount of acetaminophen and related substances in the sample to be tested; Preferably, the amount of the compound of formula 1 is not less than 1 times the theoretical molar amount for completely acylation of the hydroxyl group and the amino group in the derivatization reaction system; further, the compound of formula 1 is more than 5 times, further 5 to 100 times, and further 50 to 90 times the theoretical molar amount of acetaminophen and its related substances in the sample to be tested.

6. The method according to claim 5, characterized in that The temperature of the derivatization reaction is 20 to 80°C; preferably 30 to 50°C; Preferably, the derivatization reaction time is more than 5 minutes, and further preferably 5 to 60 minutes.

7. The method according to claim 1, characterized in that The HPLC conditions during the HPLC-ICP-MS determination were as follows: chromatographic column: ThermoAcclain Trinity P1 chromatographic column; eluent A: 8-12 mM ammonium acetate-water (pH=5); eluent B: acetonitrile; The gradient elution program was as follows: 0-2 min: mobile phase A 80%→50%; 2-4 min: mobile phase A 50%; 4-8 min: mobile phase A 50%→15%; 8-10 min: mobile phase A 15%; 10-13 min: mobile phase A 15%→80%; 13-16 min: mobile phase A 80%; Preferably, the size of the ThermoAcclain Trinity P1 chromatographic column is 3 μm, 2.1×50 mm; Preferably, the column temperature is 34-36°C.

8. The method according to claim 1, characterized in that The ICP-MS conditions during the HPLC-ICP-MS determination were: monitoring mode: No gas; measured mass number: 127 I + ; RF power: 1500-1600W; atomizing gas flow rate: 0.4-0.5L / min; reaction gas is O2; Preferably, the temperature of the atomization chamber is -4 to -6°C; the sampling depth is 6 to 8 mm.

9. The method according to claim 1, characterized in that The relevant substances in the sample to be tested were determined based on the external standard method of elemental standards.

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