Method for measuring content of pentafluorophenol

Abstract

The invention relates to a method for determining the content of pentafluorophenol, which adopts HPLC (High Performance Liquid Chromatography) for detection, and the chromatographic conditions of the HPLC are as follows: a chromatographic column is a C8 chromatographic column or a C18 chromatographic column; the mobile phase A is a trifluoroacetic acid-water solution, and the mobile phase B is a trifluoroacetic acid-acetonitrile solution; the gradient elution procedure is 0 min to 5 min, wherein the volume percent of the mobile phase A and the volume percent of the mobile phase B are 70%-80% and 30%-20% respectively; 5-26 minutes: the volume percentages of the mobile phase A and the mobile phase B are 50%-45% and 50%-55% respectively; 26-33min: the volume percentages of the mobile phase A and the mobile phase B are 0% and 100% respectively; 33 min to 40 min: the volume percentages of the mobile phase A and the mobile phase B are 70% to 80% and 30% to 20% respectively. The method is good in specificity, high in sensitivity and strong in universality, and can be used as a platform method.

Description

A method for determining pentafluorophenol content Technical Field

[0001] The invention belongs to the field of analytical chemistry, and specifically relates to a method for determining the content of pentafluorophenol, and in particular to a method for determining the content of pentafluorophenol in a medicine by adopting high performance liquid chromatography. Background Art

[0002] The development of new chemistries and conjugation technologies is of great significance for the construction of proteins with enhanced functionality, such as antibody-drug conjugates (ADCs), which have been successfully used as cancer therapeutics. One of the most versatile methods for protein modification is through lysine conjugation, which is achieved by forming an amide bond between a lysine residue and an activated ester such as N-hydroxysuccinimide (NHS), due to solvent accessibility and the high abundance of these residues on the protein surface.

[0003] In peptide synthesis reactions, alkyl esters of amino acid protecting groups are often used in amide bond formation reactions, but the reaction rate of this type of reaction is very slow. The goal of peptide bond formation reactions is to have high reaction efficiency and no side reactions. Pentafluorophenol ester is a potential active ester for protein coupling due to its high reactivity and stability, as well as high coupling efficiency. It has been widely used in peptide synthesis. Due to its stability in the body circulation, it is often used as a linker for ADC drugs. It is of the non-cleavable type and relies on lysosomal degradation in the cell to release the effective small molecule payload in the ADC. Pentafluorophenol (PFP) is a common raw material for the preparation of pentafluorophenyl active esters in peptide synthesis.

[0004] Quality control of ADC final products is crucial for the drug's in vivo efficacy. However, reactions using pentafluorophenol ester linkers can produce free PFP impurities. For example, during small molecule synthesis or ADC-drug conjugation, the PFP impurities generated can be teratogenic, depending on the reaction conditions or the stability of the ADC drug linker and toxin linker. Premature release of the cytotoxic payload can also lead to off-target toxicity, posing a safety risk and requiring strict control. The detection of PFP residues in ADC drugs is limited by the low residual amount and the presence of large proteins. Existing reported methods are not suitable for the detection of PFP residues in ADC drugs.

[0005] Currently, domestic companies use gas chromatography to analyze the purity of PFP, but there are few reports on the quantitative analysis of PFP as a pharmaceutical impurity, especially the quantitative analysis of PFP as an impurity in recombinant antibody-drug conjugates. Research and development of a PFP residual detection method is particularly necessary for quality control of small molecules and ADC drugs.

[0006] Summary of the Invention

[0007] The purpose of this application is to establish a reverse-phase high-performance liquid chromatography (RP-HPLC) method for determining residual PFP. Validation of the method demonstrates its simplicity, ease of operation, and robustness, along with its high sensitivity and ability to accurately and quantitatively determine even very low levels of PFP impurities. This method is widely applicable to the quality control of small and large molecule pharmaceuticals, achieving efficient, platform-based, and environmentally friendly quality control, and facilitating industrial application.

[0008] This application provides a method for determining pentafluorophenol content using HPLC. By selecting and adjusting chromatographic conditions such as the chromatographic column, mobile phase, and elution procedure, pentafluorophenol can be accurately and effectively determined. Specifically for ADC macromolecule drugs, the method provided in this application exhibits very high detection sensitivity, enabling accurate quantitative determination of pentafluorophenol even at very low levels.

[0009] The present application provides a method for determining the content of pentafluorophenol, characterized in that HPLC is used for detection, and the chromatographic conditions of the HPLC include:

[0010] The chromatographic column is a C8 chromatographic column or a C18 chromatographic column;

[0011] Mobile phases A and B are selected from trifluoroacetic acid-water solution or trifluoroacetic acid-acetonitrile solution respectively; and

[0012] The gradient elution program was:

[0013] 0-5 min: the volume percentages of mobile phase A and mobile phase B are 70%-80% and 30%-20%, respectively;

[0014] 5-26 min: The volume percentages of mobile phase A and mobile phase B are 50%-45% and 50%-55%, respectively;

[0015] 26-33 min: the volume percentages of mobile phase A and mobile phase B were 0% and 100%, respectively;

[0016] 33-40 min: The volume percentages of mobile phase A and mobile phase B are 70%-80% and 30%-20%, respectively.

[0017] Detailed Description of the Invention

[0018] The present application provides a method for determining the content of pentafluorophenol, characterized in that HPLC is used for detection, and the chromatographic conditions of the HPLC include:

[0019] The chromatographic column is a C8 chromatographic column or a C18 chromatographic column;

[0020] Mobile phases A and B are selected from trifluoroacetic acid-water solution or trifluoroacetic acid-acetonitrile solution respectively; and

[0021] The gradient elution program was:

[0022] 0-5 min: the volume percentages of mobile phase A and mobile phase B are 70%-80% and 30%-20%, respectively;

[0023] >5-26 min: The volume percentages of mobile phase A and mobile phase B are 50%-45% and 50%-55%, respectively;

[0024] >26-33 min: The volume percentages of mobile phase A and mobile phase B are 0% and 100%, respectively;

[0025] >33-40 min: The volume percentages of mobile phase A and mobile phase B are 70%-80% and 30%-20%, respectively.

[0026] In some embodiments, mobile phase A is a trifluoroacetic acid-water solution, and mobile phase B is a trifluoroacetic acid-acetonitrile solution; preferably, mobile phase A is a trifluoroacetic acid-water solution with a volume percentage of 0.08%-0.12%, and mobile phase B is a trifluoroacetic acid-acetonitrile solution with a volume percentage of 0.08%-0.12%; more preferably, mobile phase A is a trifluoroacetic acid-water solution with a volume percentage of 0.1%, and mobile phase B is a trifluoroacetic acid-acetonitrile solution with a volume percentage of 0.1%.

[0027] In some embodiments, HPLC is used for detection, and the chromatographic conditions include: a C8 chromatographic column, mobile phase A is a 0.1% by volume trifluoroacetic acid-water solution, mobile phase B is a 0.1% by volume trifluoroacetic acid-acetonitrile solution, and the gradient elution program is selected from any one of the following:

[0028] (1)

[0029] (2)

[0030] (3)

[0031] (4)

[0032] In some embodiments, in the method for determining the content of pentafluorophenol, the chromatographic column is an SB-C8 chromatographic column or a CSH C18 chromatographic column; preferably, the chromatographic column is an SB-C8, 5μm 4.6×250mm chromatographic column or a CSH C18, 3.5μm 4.6×150mm chromatographic column; preferably, the chromatographic column is an Agilent Zorbax 300 SB-C8, 5μm 4.6×250mm chromatographic column or an Xselect CSH C18, 3.5μm 4.6×150mm chromatographic column; more preferably, the chromatographic column is an Agilent Zorbax 300 SB-C8, 5μm 4.6×250mm chromatographic column.

[0033] In some embodiments, in the method for determining the content of pentafluorophenol, a chromatographic column with similar performance to the Agilent Zorbax300SB-C8 column can also be selected.

[0034] In some embodiments, in the method for determining the content of pentafluorophenol, the injection volume is 50-100 μl; preferably, the injection volume is 100 μl.

[0035] In some embodiments, in the method for determining the content of pentafluorophenol, the limit of quantification of pentafluorophenol is 0.1 μg / ml.

[0036] In some embodiments, in the method for determining the content of pentafluorophenol, the chromatographic conditions also include: a detection wavelength of 254-265 nm, a flow rate of 1-1.5 ml / min, and a column temperature of 55-65°C; preferably, the chromatographic conditions also include: a detection wavelength of 264 nm, a flow rate of 1-1.5 ml / min, and a column temperature of 57-63°C.

[0037] In some embodiments, in the method for determining the content of pentafluorophenol, the chromatographic conditions further include: a detection wavelength of 264 nm, a flow rate of 1 ml / min, and a column temperature of 60°C.

[0038] In some embodiments, in the method for determining the content of pentafluorophenol, the preparation method of mobile phase A is: measure 1000 ml of ultrapure water, add 1 ml of trifluoroacetic acid, mix evenly, and filter through a 0.22 μm filter membrane to obtain; the preparation method of mobile phase B is: measure 1000 ml of acetonitrile, add 1 ml of trifluoroacetic acid, mix evenly, and filter through a 0.22 μm filter membrane to obtain.

[0039] In some embodiments, the method for determining the content of pentafluorophenol comprises the following steps:

[0040] HPLC was used for detection, and the chromatographic conditions of the HPLC included:

[0041] The chromatographic column was an Agilent Zorbax 300 SB-C8, 5 μm 4.6 × 250 mm column;

[0042] Mobile phase A was a 0.1% by volume trifluoroacetic acid-water solution, and mobile phase B was a 0.1% by volume trifluoroacetic acid-acetonitrile solution;

[0043] The injection volume was 100 μl;

[0044] The detection wavelength is 264 nm;

[0045] The flow rate is 1 ml / min;

[0046] 'The column temperature is 60°C; and

[0047] The gradient elution program was

[0048] In some embodiments, the method for determining the content of pentafluorophenol comprises the following steps:

[0049] (1) Prepare pentafluorophenol standard solutions, test solutions, and diluents of different concentrations;

[0050] (2) Setting the above HPLC conditions;

[0051] (3) Inject each solution in step (1) into a liquid chromatograph according to the above injection volume, and record the chromatogram using an ultraviolet detector.

[0052] In some embodiments, the concentration of the pentafluorophenol standard solution is selected from 0.1 μg / ml, 0.2 μg / ml, 0.5 μg / ml, 1.0 μg / ml, 2.0 μg / ml and 5.0 μg / ml.

[0053] In some embodiments, the pentafluorophenol standard solution is obtained by dissolving the pentafluorophenol reference substance in a diluent; preferably, the diluent is an acetonitrile-water solution with a volume concentration of 30%.

[0054] In some embodiments, the test solution is a drug solution. Preferably, the drug is an ADC or a small molecule compound; preferably, the ADC has a molecular weight of 150±50 kD, and the ADC may produce pentafluorophenol during its preparation; preferably, the toxin in the ADC drug is an auristatin compound, or the small molecule compound is a small molecule compound that is prone to producing pentafluorophenol during the production process; preferably, the small molecule compound is an auristatin compound.

[0055] The determination method provided in the present application can be used to test the pentafluorophenol content in a drug; preferably, the drug is an ADC-conjugated drug or a small molecule drug; preferably, the molecular weight of the ADC-conjugated drug is 150±50KD; preferably, the ADC-conjugated drug is a recombinant antibody-conjugated drug that produces free pentafluorophenol molecules in the conjugated product.

[0056] The present application provides a method for determining the content of pentafluorophenol in drugs, especially ADC macromolecular drugs, by high performance liquid chromatography. The method has simple chromatographic conditions, convenient operation, high sensitivity, and good durability. It can effectively separate the components and perform quantitative calculations, quickly and accurately achieving quality control of pentafluorophenol impurities, and ensuring product quality and safety.

[0057] The method provided in this application is highly versatile, efficient, and rapid. Since the pentafluorophenol detected is unrelated to the structure of the test sample, this detection method can be used as a platform method to detect ADC drugs and their small molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIG1 shows the HPLC spectrum of Test Example 1 of Example 1;

[0059] FIG2 shows the HPLC spectrum of Test Example 2 of Example 1;

[0060] FIG3 shows the HPLC spectrum of Test Example 3 of Example 1;

[0061] FIG4 shows the HPLC profile of elution procedure 3 of Example 2;

[0062] FIG5 shows the HPLC profile of elution procedure 4 of Example 2;

[0063] FIG6 shows the HPLC profile of Example 4. Example

[0064] The present invention is further illustrated below by describing in detail embodiments, but this is not intended to limit the present invention. Those skilled in the art may make various modifications or improvements based on the basic idea of ​​the invention. However, as long as they do not depart from the basic idea of ​​the invention, they are all within the scope of the present invention.

[0065] According to the ICH guidelines (Q3C (R8) impurities: residual solvents guidelines), the rat subcutaneous LD 50 The calculated maximum tolerated daily dose (PDE) of pentafluorophenol in the injection is 1.932 μg / day, and the maximum tolerated concentration (PFP) is 0.134 μg / ml. Through exploratory research, the applicant found that the limit of quantification (LOQ) of PFP in the drug to be measured should be no higher than 0.1 μg / ml to meet the requirements of quantitative detection.

[0066] Example 1-1. Screening of mobile phase and chromatographic column

[0067] 1) Solution preparation:

[0068] Diluent: 30% acetonitrile aqueous solution, used for solution preparation.

[0069] PFP reference substance mother solution: Weigh pentafluorophenol standard, dissolve it in acetonitrile, and prepare a solution with a concentration of about 1 mg / ml as the PFP reference substance mother solution.

[0070] PFP standard working solution: Dilute the PFP reference substance stock solution with 30% acetonitrile in water to 0.1 μg / ml, 0.2 μg / ml, 0.5 μg / ml, 1.0 μg / ml, 2.0 μg / ml, and 5.0 μg / ml, respectively, to prepare the PFP standard working solutions. Use the 1.0 μg / ml solution as the system suitability solution.

[0071] Preparation of the test solution: Take an appropriate amount of the ADC drug prepared according to CN108697809A into an injection vial. The molecular weight of the ADC drug is 150±50 kD, and prepare the test solution.

[0072] Spiked solution: Take the test solution and add a certain amount of PFP standard to it by spiking to prepare spiked solutions with PFP concentrations of 0.1μg / ml, 1.0μg / ml and 5.0μg / ml, respectively. Prepare 3 copies of each concentration.

[0073] 2) Mobile phase:

[0074] Mobile phase 1:

[0075] Mobile phase A: 0.1% trifluoroacetic acid-water solution (v / v). Preparation method: Measure 1000 ml of ultrapure water, add 1 ml of trifluoroacetic acid, and mix well.

[0076] Mobile phase B: 0.1% trifluoroacetic acid-acetonitrile solution (v / v). Preparation method: Measure 1000 ml of acetonitrile, add 1 ml of trifluoroacetic acid, and mix well.

[0077] Mobile phase 2:

[0078] Mobile phase A: phosphate buffer. Preparation: weigh 2.5 g potassium dihydrogen phosphate and 2.5 g dipotassium hydrogen phosphate, dissolve in 1000 ml ultrapure water, adjust pH to 7.5, mix well, and filter through a 0.22 μm filter membrane.

[0079] Mobile phase B: methanol.

[0080] Column 1: Agilent Zorbax 300 SB-C8, (5 μm 4.6 × 250 mm); Column 2: Xselect CSH C18, (3.5 μm 4.6 × 150 mm);

[0081] Flow rate: 1.0 ml / min (mobile phase 1) or 1.5 ml / min (mobile phase 2), detection wavelength: 264 nm, column temperature: 60 °C;

[0082] Elution gradient 1:

[0083] Elution gradient 2:

[0084] 3) Test methods

[0085] Accurately measure 100 μl of the system suitability solution in 1) and inject it into the liquid chromatograph to observe the detection results.

[0086] Example 1. Screening of mobile phase and chromatographic column

[0087] 1) Solution preparation:

[0088] Diluent: 30% acetonitrile aqueous solution, used for solution preparation.

[0089] PFP reference substance mother solution: Weigh pentafluorophenol standard, dissolve it in acetonitrile, and prepare a solution with a concentration of about 1 mg / ml as the PFP reference substance mother solution.

[0090] PFP standard working solution: Dilute the PFP reference substance stock solution with 30% acetonitrile in water to 0.1 μg / ml, 0.2 μg / ml, 0.5 μg / ml, 1.0 μg / ml, 2.0 μg / ml, and 5.0 μg / ml, respectively, to prepare the PFP standard working solutions. Use the 1.0 μg / ml solution as the system suitability solution.

[0091] Preparation of the test solution: Take an appropriate amount of ADC drug I-1 prepared according to CN106729743A into an injection vial. The molecular weight of the ADC drug is 150±50 kD, and prepare the test solution.

[0092] Spiked solution: Take the test solution and add a certain amount of PFP standard to it by spiking to prepare spiked solutions with PFP concentrations of 0.1μg / ml, 1.0μg / ml and 5.0μg / ml, respectively. Prepare 3 copies of each concentration.

[0093] 2) Mobile phase:

[0094] Mobile phase 1:

[0095] Mobile phase A: 0.1% trifluoroacetic acid-water solution (v / v). Preparation method: Measure 1000 ml of ultrapure water, add 1 ml of trifluoroacetic acid, and mix well.

[0096] Mobile phase B: 0.1% trifluoroacetic acid-acetonitrile solution (v / v). Preparation method: Measure 1000 ml of acetonitrile, add 1 ml of trifluoroacetic acid, and mix well.

[0097] Mobile phase 2:

[0098] Mobile phase A: phosphate buffer. Preparation: weigh 2.5 g potassium dihydrogen phosphate and 2.5 g dipotassium hydrogen phosphate, dissolve in 1000 ml ultrapure water, adjust pH to 7.5, mix well, and filter through a 0.22 μm filter membrane.

[0099] Mobile phase B: methanol.

[0100] Column 1: Agilent Zorbax 300 SB-C8, (5 μm 4.6 × 250 mm); Column 2: Xselect CSH C18, (3.5 μm 4.6 × 150 mm);

[0101] Flow rate: 1.0 ml / min (mobile phase 1) or 1.5 ml / min (mobile phase 2), detection wavelength: 264 nm, column temperature: 60 °C;

[0102] Elution gradient 1:

[0103] Elution gradient 2:

[0104] 3) Test methods

[0105] Accurately measure 100 μl of the system suitability solution in 1) and inject it into the liquid chromatograph to observe the detection results.

[0106] 4) Test results

[0107] Table 1 Mobile phase types and chromatographic columns

[0108] Test Examples 1-2 used mobile phase 1 and columns 1 and 2. The target peaks all elute normally, with good peak shape and adequate resolution from other peaks. Test Example 3 used mobile phase 2. The chromatographic peaks of the system suitability solution and the diluent were essentially identical, indicating that PFP did not elute under these conditions. Therefore, mobile phase A: 0.1% trifluoroacetic acid in water and mobile phase B: 0.1% trifluoroacetic acid in acetonitrile were used as the mobile phases for subsequent investigations. The Xselect CSH C18 column has a smaller pore size, requiring sample pretreatment (ACN precipitation). Therefore, an Agilent Zorbax 300 SB-C8 was used for subsequent investigations.

[0109] Example 2. Elution program screening

[0110] 100 μl of a spiked solution containing 1.0 μg / ml PFP was precisely measured and injected into a liquid chromatograph. Mobile phase 1 and column 1, selected in Example 1, were used at a flow rate of 1.0 ml / min. The following elution procedures were used to assess sample detection. The experimental protocols and results are shown in the table below.

[0111] Elution procedure 1

[0112] Elution procedure 2

[0113] Elution procedure 3

[0114] Elution procedure 4

[0115] Table 2 Elution procedure investigation

[0116] When elution procedure 3 was used, the PFP peak had a high absorption and a sensitive response, and the PFP peak was well separated from other impurity peaks, and the protein was completely eluted. Shortening the elution time resulted in a poorer separation between the target peak and other impurity peaks, and the protein could not be completely eluted. Therefore, elution procedure 3 was selected for subsequent experiments.

[0117] Example 3. Investigation of injection volume

[0118] The mobile phase 1 and chromatographic column 1 of Example 1 and the elution procedure 3 of Example 2 were used as chromatographic conditions. The following injection volumes of the system suitability solution were used to investigate the sample detection performance. The experimental scheme and results are shown in the table below.

[0119] Table 3. Sample injection volume investigation

[0120] According to the requirements of the Chinese Pharmacopoeia, the baseline signal-to-noise ratio at the quantitative limit concentration needs to be greater than or equal to 10. When using a 100μl injection volume, the signal-to-noise ratio is around 15-30, which can meet the quantitative detection requirements. Therefore, a 100μl injection volume is selected.

[0121] Example 4. Specificity Investigation

[0122] Using mobile phase 1 and chromatographic column 1 of Example 1, and elution procedure 3 of Example 2 as chromatographic conditions, 100 μl of the process intermediate solution was precisely measured and injected into the liquid chromatograph to examine the peak elution of each component. The process intermediate solution is an intermediate product in the preparation of an ADC drug and contains PFP, small toxin molecules, and related impurities.

[0123] The results are shown in Figure 6. Under this chromatographic condition, the peaks of PFP, toxin small molecules and impurities in the process intermediate solution can be effectively separated, with good peak shapes, meeting the specificity requirements.

[0124] Example 5. Investigation of limit of quantification

[0125] The mobile phase 1, chromatographic column 1 of Example 1 and the elution procedure 3 of Example 2 were used as chromatographic conditions. Three spiked solutions with a PFP concentration of 0.1 μg / ml prepared in Example 1 were taken, and 100 μl of each solution was accurately measured and injected into the liquid chromatograph to investigate the ratio of the measured concentration to the theoretical concentration (recovery rate), the recovery rate RSD%, and the signal-to-noise ratio.

[0126] Table 4. Limit of quantification investigation

[0127] The results showed that the signal-to-noise ratios for the spiked solution of the test sample at a concentration of 0.1 μg / ml were 27.9, 28.0, and 28.5, respectively; the recoveries were 98%, 99%, and 102%, respectively, with an RSD of 2.1%, meeting the standard requirements for method development. This indicates that PFP in the test sample can be accurately and quantitatively detected at this concentration, so 0.1 μg / ml was selected as the limit of quantification for this method.

[0128] Example 6. Linearity Investigation

[0129] The PFP standard working solutions prepared in Example 1 at different concentrations of 0.1 μg / ml, 0.2 μg / ml, 0.5 μg / ml, 1.0 μg / ml, 2.0 μg / ml, and 5.0 μg / ml were used. The mobile phase 1 and chromatographic column 1 of Example 1, along with the elution procedure 3 of Example 2, were used as chromatographic conditions. 100 μg of each of the six different concentrations of PFP standard solutions was precisely measured and injected into the liquid chromatograph. The chromatograms were recorded to examine the linear relationship between PFP concentration and peak area. The results showed that within the PFP concentration range of 0.1 μg / ml to 5.0 μg / ml, the concentration and peak area showed a linear relationship. The standard curve had good linearity, with the linear function being y = 19,707.52x - 185.03, and R = 0. 2 is 0.999974, and the residual sum of squares is 174110, which satisfies R 2 The requirement of ≥0.99 indicates that the linearity of this method is good.

[0130] Example 7. Accuracy Investigation

[0131] Three aliquots of the PFP spiked solutions prepared in Example 1, each containing 0.1 μg / ml, 1.0 μg / ml, and 5.0 μg / ml, were injected into the sample vial. The mobile phase 1 and chromatographic column 1 of Example 1, along with the elution procedure 3 of Example 2, were used as chromatographic conditions. The ratio of the measured PFP concentration to the theoretical concentration (% recovery) and the RSD of the recovery were examined to determine the accuracy of the method.

[0132] Table 5. Accuracy test

[0133] The recoveries of PFP in the spiked solutions at the three concentration levels of low, medium, and high were low (98%, 99%, and 102%), medium (90%, 91%, and 90%), and high (86%, 85%, and 84%), respectively. The RSDs of the recoveries were 2.1%, 0.7%, and 1.2%, respectively, meeting the accuracy verification requirements, indicating that this method can accurately and quantitatively determine PFP within the above concentration range.

[0134] Example 8. Durability Study (Column Temperature)

[0135] The mobile phase 1 and chromatographic column 1 of Example 1 and the elution procedure 3 of Example 2 were used as chromatographic conditions, and the peak elution of the sample to be tested was investigated under different chromatographic column temperature conditions (57° C., 60° C., and 63° C.).

[0136] Accurately measure 100 μl of the system suitability solution of Example 1 and inject it into the liquid chromatograph. Record the chromatogram. The test data and results are shown in the table below.

[0137] Table 6 Column temperature investigation

[0138] The percentage differences in the detection values ​​of pentafluorophenol at different column temperatures (57°C, 63°C) and the method-set column temperature (60°C) were 1.4% and 3.1%, respectively, which met the durability verification requirements.

Claims

1. A method for determining the content of pentafluorophenol, characterized in that: HPLC was used for detection, and the chromatographic conditions of the HPLC included: The chromatographic column is a C8 chromatographic column or a C18 chromatographic column; Mobile phases A and B are selected from trifluoroacetic acid-water solution or trifluoroacetic acid-acetonitrile solution respectively; and The gradient elution program was: 0-5 min: the volume percentages of the mobile phase A and the mobile phase B are 70%-80% and 30%-20% respectively; 5-26 min: the volume percentages of the mobile phase A and the mobile phase B are 50%-45% and 50%-55% respectively; 26-33 min: the volume percentages of the mobile phase A and the mobile phase B are 0% and 100% respectively; 33-40 min: the volume percentages of the mobile phase A and the mobile phase B are 70%-80% and 30%-20% respectively.

2. The measuring method according to claim 1, wherein The chromatographic column is an SB-C8 chromatographic column or a CSH C18 chromatographic column; preferably, the chromatographic column is an SB-C8, 5μm 4.6×250mm chromatographic column or a CSH C18, 3.5μm 4.6×150mm chromatographic column; preferably, the chromatographic column is an Agilent Zorbax 300 SB-C8, 5μm 4.6×250mm chromatographic column or an Xselect CSH C18, 3.5μm 4.6×150mm chromatographic column; more preferably, the chromatographic column is an Agilent Zorbax 300 SB-C8, 5μm 4.6×250mm chromatographic column.

3. The measuring method according to claim 1 or 2, characterized in that The injection volume is 50-100 μl; preferably, the injection volume is 100 μl.

4. The assay method according to any one of claims 1 to 3, wherein The limit of quantification of pentafluorophenol was 0.1 μg / ml.

5. The assay method according to any one of claims 1 to 4, characterized in that The chromatographic conditions also include: a detection wavelength of 254-265 nm, a flow rate of 1-1.5 ml / min, and a column temperature of 55-65°C; preferably, the chromatographic conditions also include: a detection wavelength of 264 nm, a flow rate of 1-1.5 ml / min, and a column temperature of 57-63°C; preferably, the chromatographic conditions also include: a detection wavelength of 264 nm, a flow rate of 1 ml / min, and a column temperature of 60°C.

6. The assay method according to any one of claims 1 to 5, characterized in that The mobile phase A is a trifluoroacetic acid-water solution, and the mobile phase B is a trifluoroacetic acid-acetonitrile solution; preferably, the mobile phase A is a trifluoroacetic acid-water solution with a volume percentage of 0.08%-0.12%, and the mobile phase B is a trifluoroacetic acid-acetonitrile solution with a volume percentage of 0.08%-0.12%; more preferably, the mobile phase A is a trifluoroacetic acid-water solution with a volume percentage of 0.1%, and the mobile phase B is a trifluoroacetic acid-acetonitrile solution with a volume percentage of 0.1%.

7. A method for determining the content of pentafluorophenol, characterized in that: HPLC was used for detection, and the chromatographic conditions of the HPLC included: The chromatographic column is a C8 chromatographic column or a C18 chromatographic column; Mobile phases A and B are selected from trifluoroacetic acid-water solution or trifluoroacetic acid-acetonitrile solution respectively; and The gradient elution program was selected from any of the following: (1) (2) (3) (4) 8. The assay method according to any one of claims 1 to 7, comprising the following steps: (1) Prepare pentafluorophenol standard solutions, test solutions, and diluents of different concentrations; (2) Setting HPLC conditions according to the assay method according to any one of claims 1 to 7; (3) Inject each solution in step (1) into a liquid chromatograph according to the injection volume described in claim 3, and record the chromatogram using an ultraviolet detector.

9. The assay method according to any one of claims 1 to 8, wherein the concentration of the pentafluorophenol standard solution is selected from 0.1 μg / ml, 0.2 μg / ml, 0.5 μg / ml, 1.0 μg / ml, 2.0 μg / ml and 5.0 μg / ml; Preferably, the pentafluorophenol standard solution is obtained by dissolving the pentafluorophenol reference substance in a diluent; Preferably, the diluent is an acetonitrile solution with a volume concentration of 30%.

10. The assay method according to any one of claims 1 to 9, which is used to determine the content of pentafluorophenol in a compound; preferably, the compound is an ADC or a small molecule compound; preferably, the molecular weight of the ADC is 150±50 kD; preferably, the ADC is an ADC that produces pentafluorophenol during the preparation process; and the small molecule is a small molecule compound that may produce pentafluorophenol during the preparation process.

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