A method for pre-treatment of polyethylene glycol derivative samples and determination of content

Abstract

The present invention relates to a method for pre-treatment of polyethylene glycol derivative samples and determination of content, and belongs to the technical field of chemical analysis. The method adopts polyethylene glycol derivative N3-PEG3-SPA to carry out derivatization reaction with benzylamine, and then carries out HPLC detection to obtain its content. N3-PEG3-SPA cannot be directly subjected to HPLC analysis to detect its purity because it contains an active ester group that is easily hydrolyzed. In the method of the present invention, the sample is subjected to derivatization reaction pre-treatment with aniline, and after the chromatographic conditions are optimized, including column temperature, flow rate, wavelength, mobile phase and chromatographic column, the HPLC content analysis method in the technical scheme of the present invention has simple steps, simple operation, high accuracy, good repeatability and high sensitivity.

Description

Technical Field

[0001] The invention relates to the field of chemical analysis, and in particular to a method for pre-treating a polyethylene glycol derivative sample and determining its content. Background Art

[0002] Polyethylene glycol derivatives are a new and widely used chemical intermediate. Azide (triethylene glycol) propionate succinimidyl ester is a short-chain monodisperse polyethylene glycol derivative, common name: N 3 -PEG 3 -SPA, CAS: 1245718-89-1, its molecular structure is as follows:

[0003]

[0004] N 3 -PEG 3 -SPA is a polyethylene glycol derivative with an azido group at one end and an active propionic acid ester at the other end, obtained by reacting triethylene glycol with tert-butyl acrylate and diphenylphosphoazide in sequence, followed by reduction, hydrolysis and condensation. The main function of the product is that the active propionic acid ester can couple with the amino or hydroxyl groups on protein molecules or small molecule drugs through amide condensation or ester condensation, thereby changing the structure of the drug molecule and optimizing its physical and chemical properties.

[0005] At present, there is no research on the detection of polyethylene glycol derivatives N 3 -PEG 3 -The main reason for the lack of reports on the analytical methods of SPA is that polyethylene glycol derivatives are usually complex in composition due to the particularity of their structure, synthesis method, purification method, and detection method, and many components are likely to interfere with the detection. Summary of the invention

[0006] The present invention provides a polyethylene glycol derivative N 3 -PEG 3 -SPA sample pretreatment method, which can reduce the interference of other impurities in the sample and fully extract the polyethylene glycol derivative N 3 -PEG 3 -SPA. On this basis, the present invention also provides a method for determining the content of polyethylene glycol derivative N in a sample. 3 -PEG 3 -SPA content method. This method has high accuracy, good repeatability and high sensitivity.

[0007] The present invention uses triethylene glycol as a starting material and synthesizes a monodisperse PEG derivative N having an azide at one end and an active ester at the other end by the process shown in the following figure. 3 -PEG 3-SPA, due to the easy hydrolysis of active esters, its purification is difficult, and the conventional process quality control standards, especially the impurity content, are difficult to meet the requirements for drug approval after modification. Therefore, this study aims to develop a production process and analytical method to optimize N 3 -PEG 3 - SPA purity and control of single impurity content:

[0008]

[0009] The above picture shows N 3 -PEG 3 -Synthesis route of SPA

[0010] The first aspect of the present invention relates to a polyethylene glycol derivative N 3 -PEG 3 -The method for pre-treatment of SPA samples comprises the following steps:

[0011] (1) N 3 -PEG 3 -SPA sample is subjected to derivatization reaction to obtain a reaction solution;

[0012] (2) Transfer the reaction solution obtained in step (1) into a volumetric flask, dilute to volume with acetonitrile, and shake well;

[0013] (3) Take the solution in step (2) and filter it through a membrane to obtain a sample solution.

[0014] In certain embodiments of the first aspect of the present invention, in step (1), the derivatization reaction is to convert the polyethylene glycol derivative N 3 -PEG 3 -SPA samples were reacted with benzylamine in dichloromethane.

[0015] In certain embodiments of the first aspect of the present invention, in step (1), the polyethylene glycol derivative N 3 -PEG 3 The molar ratio of SPA sample to benzylamine was 1:1 to 1:3.

[0016] In certain embodiments of the first aspect of the present invention, in step (1), the reaction temperature of the derivatization reaction is 20°C-40°C.

[0017] In certain embodiments of the first aspect of the present invention, in step (1), the reaction time of the derivatization reaction is 30 min to 3 hours.

[0018] In certain embodiments of the first aspect of the present invention, in step (2), if there is a precipitate in the reaction solution, the precipitate is also transferred to the volumetric flask.

[0019] In certain embodiments of the first aspect of the present invention, in the sample pretreatment method, N 3 -PEG 3 -SPA sample concentration is 2 mg / mL to 5 mg / mL, preferably 3 mg / mL to 4 mg / mL, for example 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL.

[0020] The second aspect of the present invention relates to a method for determining the content of polyethylene glycol derivative N in a sample. 3 -PEG 3 -SPA content method, comprising the following steps:

[0021] (1) a sample solution obtained by the pretreatment method according to the first aspect of the present invention;

[0022] (2) performing HPLC detection on the sample solution obtained in step (1);

[0023] (3) Calculate the polyethylene glycol derivative N in the sample according to the test results obtained in step (2) 3 -PEG 3 -SPA content.

[0024] In certain embodiments of the second aspect of the present invention, in step (2), the detection conditions of HPLC include one or more of the following a to f:

[0025] a. The chromatographic column is C18 chromatographic column;

[0026] Preferably, the chromatographic column is a Waters, Xbridge shield RP C18 chromatographic column; Extend-C18 chromatographic column;

[0027] Preferably, the chromatographic column specifications are 150 mm (length) × 4.6 mm (inner diameter), 3 μm (particle size);

[0028] b. The column temperature is 25°C to 35°C; preferably 30°C;

[0029] c. Column flow rate is 0.8 mL / min to 1.2 mL / min; preferably 1.0 mL / min;

[0030] d. The injection volume is 2 to 10 μL;

[0031] e. The detector is an ultraviolet detector;

[0032] Preferably, the detection wavelength is 190nm-210nm;

[0033] f. The mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous perchloric acid solution and the mobile phase B is acetonitrile;

[0034] Preferably, the concentration of the aqueous perchloric acid solution in the mobile phase A is 0.1‰ to 2.0‰; preferably 0.5‰;

[0035] Preferably, the elution program of the mobile phase is as shown in the following table:

[0036] Time (min) A(％) B(％) 0.00 90 10 4.00 90 10 10.00 50 50 15.00 40 60 17.00 10 90 20.00 10 90 20.10 90 10 24.00 90 10

[0037] In certain embodiments of the second aspect of the present invention, the ultraviolet detector is used to detect the absorbance at a wavelength of 210 nm.

[0038] In the technical solution of the present invention, the polyethylene glycol derivative N is creatively used 3 -PEG 3 -SPA is subjected to derivatization reaction with benzylamine and then tested by HPLC to obtain its content. 3 -PEG 3 -SPA cannot be directly subjected to HPLC analysis to detect its purity because it contains an active ester group that is easily hydrolyzed. In the method of the present invention, the sample is subjected to a derivatization reaction pretreatment with aniline, and after the chromatographic conditions are optimized, including the column temperature, flow rate, wavelength, mobile phase and chromatographic column, the HPLC content analysis method in the technical solution of the present invention has simple steps, simple operation, high accuracy, good repeatability and high sensitivity.

[0039] In summary, the present invention includes the following beneficial technical effects:

[0040] 1. The polyethylene glycol derivative N provided by the present invention 3 -PEG 3 -SPA sample pretreatment method can avoid hydrolysis of active ester groups in samples, produce impurities and interfere with content determination;

[0041] 2. The polyethylene glycol derivative N provided by the present invention 3 -PEG 3 -SPA content analysis method has simple steps, easy operation, high accuracy, good repeatability and high sensitivity.

[0042] In the description of the present invention, it should be understood that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0043] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is the NMR image in Example 4;

[0045] Figure 2 This is the chromatogram in Example 5. DETAILED DESCRIPTION

[0046] In order to enable those skilled in the art to better understand the technical solution of the present invention, some non-limiting embodiments are further disclosed below to further describe the present invention in detail.

[0047] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0048] In the present invention, min means minute; h means hour; g means gram; ml means milliliter;

[0049] In the present invention, HPLC means high performance liquid chromatography.

[0050] In the present invention, the concentration of the perchloric acid aqueous solution refers to the volume concentration, for example, a 5‰ perchloric acid aqueous solution means taking 0.5 mL of 70.0-72.0% perchloric acid solution and dissolving it in 1000 mL of water and mixing it evenly.

[0051] The materials used in the following examples and comparative examples are as follows:

[0052] Acetonitrile: chromatographic grade.

[0053] Benzylamine: analytical grade.

[0054] Perchloric acid: analytical grade.

[0055] Dichloromethane: analytical grade.

[0056] Water: Ultrapure water with conductivity ≥18.2MΩ·cm.

[0057] Polyethylene glycol derivatives N 3-PEG 3 -SPA: Buy or make your own.

[0058] Azide-PEG3-NHS standard: purchased.

[0059] Example 1 Synthesis of Compound IV

[0060] Compound N 3 -PEG 3 -tBu (23kg) was added to a 100L reactor, and 40L of tetrahydrofuran and 10L of hydrochloric acid (6M) were added. The reaction system was heated to 55°C and stirred for 14 hours. TLC showed that the reaction was complete. The tetrahydrofuran was removed under reduced pressure, and the pH was adjusted to 7-8 with cooled sodium hydroxide solution. The water system was washed 3 times with ethyl acetate (10L×3). The pH of the aqueous solution was adjusted to 5-6 with 4M hydrochloric acid. The aqueous phase was extracted 3 times with ethyl acetate (10L×3), the organic phases were combined and dried over anhydrous sodium sulfate. The organic phases were collected and concentrated to obtain a crude product, which was then chromatographed by column chromatography (silica gel, V (石油醚) :V (乙酸乙酯) =1:1) to obtain 9 kg (mmol) of light yellow liquid with a liquid purity of 95.3% N 3 -PEG 3 -COOH (TLC detection shows that there is an impurity point next to the product point). 3 -PEG 3 -COOH was added to a 50L spherical kettle, 1388g of sodium hydroxide was dissolved in 20L of water, ice was added to cool, and after cooling, it was added to the spherical kettle, and the cooling cycle was turned on to keep the reaction system below 5°C. Stir for 10 minutes to fully mix the reactants, add 5L of ethyl acetate to wash the aqueous phase, and at the same time, add 250ml of concentrated hydrochloric acid to the aqueous phase, stir for 5 minutes and then separate the liquids. 5L of ethyl acetate and 250ml of concentrated hydrochloric acid were added to the aqueous phase, and this extraction operation was repeated four times, and followed by TLC until there was no product in the aqueous phase. The organic phases were combined and dried with anhydrous sodium sulfate. After the organic phase was concentrated, about 8.2kg of light yellow liquid N with a liquid purity of 98% was obtained. 3 -PEG 3 -COOH product. The yield is 30.8%.

[0061] 1 H NMR (CDCl 3 , 400MHz), δ: 9.88 (s, 1H, COOH); 3.78 ~ 3.74 (t, 2H, J = 3.8Hz, CH 2 CH 2 COOH); 3.69~3.62(m,10H,CH 2 CH 2O); 3.40~3.38(t,2H,J=3.4Hz,CH 2 COOH); 2.65~2.62(t,2H,J=2.6Hz,CH 2 N 3 ).

[0062] Example 2 Synthesis of Compound V

[0063] In a 50L reactor, add N 3 -PEG 3 -COOH (3830g), add 20L solvent DCM, stir for 2min to mix evenly, cool the reaction system to about 0℃ under ice bath condition, add 3356g DCC solid in batches, add in about 30min. Stir for 5min and then add 1961g NHS in batches, let the reaction system naturally return to room temperature and keep reacting overnight. After the reaction is completed by TLC monitoring, there is a large amount of solid in the reaction liquid, freeze and filter the solid four times. After filtration, add the reaction liquid to a 50L reactor, add 10L water twice to wash, the solution is seriously emulsified, add industrial salt, stir for 2min and stand for separation. The organic phase is dried with anhydrous sodium sulfate, the organic phase is collected and concentrated to obtain about 5kg of crude product, the crude product is added to a 50L ball kettle, add 5L water and 8.5L methanol, stir vigorously for 5min, white solid appears on the liquid surface, and then the white solid is removed by suction, and the aqueous phase is added (PE / EA=2 / 1) solvent (15L×2) to wash twice. The aqueous phase was transferred to a kettle, 15L DCM was added for extraction, 10L DCM was added to the aqueous phase for extraction again, and the aqueous phase was extracted until TLC detected that there was no product. The organic phases were combined and dried with anhydrous sodium sulfate, and the organic phase was collected and concentrated to obtain 4.9kg of yellow liquid. About 5L of dichloromethane was added to the 4.9kg yellow crude product, and it was stirred for 1min to be fully mixed and dissolved. 15L of water was added once to wash five times, 12L of water was washed five times, and 10L of water was washed twice. The organic phase was dried with anhydrous sodium sulfate, and the solvent was concentrated to obtain 3.7kg of crude product. The crude product was pulped four times with methyl tert-butyl ether to obtain 2.5kg of product with a liquid phase of 97% and a single impurity content of less than 1%. It was purified by column chromatography (silica gel, V (dichloromethane): acetone) = 100 / 1-10 / 1) to obtain 2.3kg of light yellow liquid, with a liquid phase purity of 97.92% and a single impurity content of less than 1%. The quantitative nuclear magnetic content was 98.16%.

[0064] 1 H NMR (CDCl 3 , 400MHz), δ: 3.89~3.84 (t, 2H, J=3.9Hz, CH 2 CH 2 COOH); 3.72~3.64(m,10H,CH2 CH 2 O);3.42~3.38(t,2H,J=3.4Hz,CH 2 COOH); 2.90~2.88(t,2H,J=2.9Hz,CH 2 N 3 ); 2.82(s,4H,NHS).

[0065] Example 3 Analysis of N3-PEG3-SPA Product Content by NMR Internal Standard Method

[0066] The NMR analysis was performed using a 400MHz Oxford Zhongke high-resolution NMR spectrometer, with deuterated chloroform as the solvent and naphthalene (99%, w / w%; MACKLIN) as the internal standard. About 20 mg of the sample and internal standard were accurately weighed with a 1 / 10,000 balance, and a mixed solution was prepared with 1 ml of deuterated chloroform. The experimental conditions were as follows: measurement temperature: 22°C; observation frequency: 399.95MHz, spectrum width: 325.95Hz, pulse width: 15us, acquisition time: 5s; pulse delay time: 25s; number of scans: 16 times.

[0067] Calculation results:

[0068] Ws(%)=(As*Nr*Mr*Mws*Wr) / (Ar*Ns*Ms*Mwr);

[0069] As is the peak area of ​​the quantitative peak of the sample being tested;

[0070] Ar is the peak area of ​​the quantitative peak of the internal standard substance;

[0071] Nr is the number of protons contained in the quantitative peak of the internal standard substance;

[0072] Ns is the number of protons contained in the quantitative peak of the sample being tested;

[0073] Mr is the weighed mass of the internal standard;

[0074] Ms is the weight of the sample weighed;

[0075] Mws sample molecular weight;

[0076] Mwr internal standard molecular weight 128.17;

[0077] Wr is the mass percentage of the internal standard;

[0078] Sample weight: 23.9 mg; internal standard weight: 21.0 mg, 7.90 ppm and 7.53 ppm are internal standard peaks, the product content is calculated based on H at 3.84 ppm of the product, the internal standard results are as follows:

[0079] Ws(%)=(As*Nr*Mr*Mws*Wr) / (Ar*Ns*Ms*Mwr)

[0080] Example 4 Effect of condensing agent on the purity and content of N3-PEG3-SPA

[0081] Table 2 Condensation agent for compound N 3 -PEG 3 - Impact of SPA purity and yield

[0082]

[0083] In N 3 -PEG 3 -COOH(IV) and NHS (N-hydroxysuccinimide) reaction process, DCC, EDCI, DIC were used as condensing agents for comparative tests. After the three reactions were completed, solids were precipitated. We removed the solids by freezing and filtering four times, washed the filtrate twice with brine, dried the organic phase and removed the solvent under reduced pressure and sent it directly for inspection. The internal standard method NMR analysis was performed using 99% pure naphthalene as the internal standard. The results of the calculated NMR data are shown in Table 2. The product content obtained by using EDCI as the condensing agent is the lowest, only 82.45%, while DIC and DCC reached 92.10% and 98.16%, respectively. At the same time, according to Figure 1 The NMR images shown in the figure show that the characteristic peak integrals of NHS at 2.89ppm and 2.82 of the product using EDCI and DIC as condensation agents obviously exceed the theoretical value (theoretical integral is 6). After multiple treatments, the characteristic peak integrals at this location have not decreased. It is speculated that there may be an impurity containing an active ester group, and this main impurity is also the main reason for the low content of EDCI and DIC condensation reaction products (82.45% and 92.10%, respectively). The product batch using DCC as the condensation agent meets the theoretical value at 2.89ppm and 2.82, that is, DCC is the optimal condensation agent.

[0084] Example 5 Determination of the content of polyethylene glycol derivative N3-PEG3-SPA

[0085] Chromatographic conditions:

[0086]

[0087] Solution configuration:

[0088] Blank solution: acetonitrile.

[0089] Prepare blank: weigh 12 mg of benzylamine into a test tube containing 2 mL of dichloromethane, stir at room temperature for 1.5 hours, then transfer to a 100 mL volumetric flask, dilute to the mark with blank solution, and shake well.

[0090] System suitability solution: Weigh 32 mg of benzylamine and 69 mg of Azide-PEG3-NHS standard into a test tube containing 2 mL of dichloromethane, stir at room temperature for 1.5 hours, then transfer to a 100 mL volumetric flask, dilute to the mark with blank solution, and shake well.

[0091] Reporting limit solution: Take 0.1mL of system suitability solution into a 100mL volumetric flask, dilute to the mark with blank solution, and shake well.

[0092] Sample solution: weigh 32 mg of benzylamine and 69 mg of polyethylene glycol derivative N 3 -PEG 3 -SPA samples were added to test tubes containing 2 mL of dichloromethane, stirred at room temperature for 1.5 h, then transferred to a 100 mL volumetric flask, diluted to the mark with blank, and shaken well.

[0093] The above solution was subjected to HPLC detection under the above chromatographic conditions according to the following sequence table 1:

[0094]

[0095]

[0096] Calculation results:

[0097] The purity of the samples was calculated according to the following company:

[0098] Purity, %area:

[0099]

[0100] Acomponent = Azide-PEG3-NHS or peak area of ​​a single impurity

[0101] Atotal = the sum of all peak areas in the sample

[0102] N 3 -PEG 3 -SPA contains active ester groups that are easily hydrolyzed, so its purity cannot be directly tested by HPLC analysis. Therefore, the derivatization analysis method is a more reliable approach. 3 -PEG 3 -SPA was directly analyzed by HPLC after benzylamine derivatization according to the aforementioned standard operating procedures. Figure 2As shown. The retention time of the derivatized product is at 11.65min, while the retention time of the excess benzylamine is at 9.1min, and the remaining peaks are impurity peaks. We marked the main impurity peaks, which are distributed at 6.3min, 8.5min, 9.5min, 9.7min, 10.8min, 10.9min, 11.5min, 13.0min, 14.4min, 14.8min, and 16.0min. The UV detection control found that the EDCI batch and the DIC batch each had an impurity peak with a single impurity exceeding 1% at 11.5min and 16.0min, and the impurity peak at 11.5min was very close to the main peak position, especially the EDCI batch at 11.5min The impurity integrated area accounted for 13.09%, while the DCC batch derivatives did not detect impurities with a retention time of 11.5min, and all single impurity contents accounted for less than 1%. Due to N 3 -PEG 3 -SPA is easily hydrolyzed and cannot be exposed to air and water for a long time. We tried to analyze the N 3 -PEG 3 -SPA was used for rapid column chromatography purification, but it was found that the impurity of 11.5min still existed after column chromatography purification and then derivatization with benzylamine, and the integrated area ratio of the impurity was basically not reduced. This indicates that the nature of the impurity is very similar to that of the product and it is difficult to remove it by conventional purification methods.

[0103] The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A method for determining the content of N3-PEG3-SPA in a polyethylene glycol derivative sample, include: 23 kg of compound N3-PEG3-tBu was added to a 100 L reactor, 40 L of tetrahydrofuran and 10 L of 6 M hydrochloric acid were added, the reaction system was heated to 55 ° C, stirred for 14 hours, TLC showed that the reaction was complete, the tetrahydrofuran was removed under reduced pressure, and then the pH was adjusted to 7-8 with cooled sodium hydroxide solution, the water system was washed with ethyl acetate 3 times, 10 L each time, and then the pH of the aqueous solution was adjusted to 5-6 with 4 M hydrochloric acid, the aqueous phase was extracted with ethyl acetate 3 times, 10 L each time, the organic phases were combined and dried over anhydrous sodium sulfate, the organic phases were collected and concentrated to obtain a crude product, which was subjected to column chromatography. The column chromatography used silica gel, V (石油醚) :V (乙酸乙酯) =1:1 purification, 9kg of light yellow liquid with a liquid phase purity of 95.3% N3-PEG3-COOH was obtained, and TLC detection showed an impurity spot next to the product spot; Add N3-PEG3-COOH with a liquid purity of 95.3% to a 50L spherical kettle, dissolve 1388g of sodium hydroxide in 20L of water, add ice cubes to cool, add to the spherical kettle after cooling, open a cooling cycle to cool the reaction system to keep it below 5°C, stir for 10 minutes to fully mix the reactants, add 5L of ethyl acetate to wash the aqueous phase, and add 250ml of concentrated hydrochloric acid to the aqueous phase, stir for 5 minutes and then separate the liquids, continue to add 5L of ethyl acetate and 250ml of concentrated hydrochloric acid to the aqueous phase, repeat this extraction operation four times, track with TLC until there is no product in the aqueous phase, combine the organic phases and dry with anhydrous sodium sulfate, and concentrate the organic phase to obtain about 8.2kg of light yellow liquid N3-PEG3-COOH product with a liquid purity of 98%; In a 50L reactor, add 3830g N3-PEG3-COOH and 20L DCM solvent, stir for 2min to mix evenly, cool the reaction system to about 0°C under ice bath, add 3356g DCC solid in batches, add in about 30min, stir for 5min, and then add 1961g NHS, the reaction system was naturally warmed to room temperature and the reaction was maintained overnight. After the reaction was completed by TLC monitoring, there was a large amount of solid in the reaction solution. The solid was frozen and filtered four times. After filtration, the reaction solution was added to a 50L reactor and washed with 10L of water twice. The solution was severely emulsified. Industrial salt was added and stirred for 2 minutes and allowed to stand for separation. The organic phase was dried over anhydrous sodium sulfate. The organic phase was collected and concentrated to obtain about 5kg of crude product. The crude product was added to a 50L spherical kettle, 5L of water and 8.5L of methanol were added, and the mixture was stirred vigorously for 5min. A white solid appeared on the liquid surface, and the white solid was removed by filtration. The aqueous phase was washed twice with PE / EA=2 / 1 solvent, 15L each time, and the aqueous phase was transferred to the kettle. 15L of DCM was added for extraction, 10L of DCM was added to the aqueous phase for extraction again, and the aqueous phase was extracted until TLC detected that there was no product in the aqueous phase. The organic phases were combined and dried over anhydrous sodium sulfate. The organic phases were collected and concentrated to obtain 4.9kg of yellow liquid. About 5L of dichloromethane was added to the 4.9kg yellow crude product, and the mixture was stirred for 1min to fully mix and dissolve. 15L of water was added once for washing five times, 12L of water was added five times, and 10L of water was added twice. The organic phase was dried over anhydrous sodium sulfate, concentrated to remove the solvent, and 3.7kg of crude product was obtained. The crude product was slurried four times with methyl tert-butyl ether to obtain 2.5kg of product with a liquid phase content of 97% and a single impurity content of less than 1%. The product was subjected to column chromatography, and the column chromatography used silica gel, V (二氯甲烷) :V (丙酮) =10-100:1 purification, 2.3 kg of light yellow liquid was obtained, namely N3-PEG3-SPA sample; (1) subjecting the N3-PEG3-SPA sample to a derivatization reaction to obtain a reaction solution; (2) Transfer the reaction solution obtained in step (1) into a volumetric flask, dilute to volume with acetonitrile, and shake well; (3) taking the solution in step (2) and filtering it through a membrane to obtain a sample solution; (4) subjecting the sample solution obtained in step (3) to HPLC detection; (5) calculating the content of the polyethylene glycol derivative N3-PEG3-SPA in the sample according to the test results obtained in step (4); Wherein, in step (1), the derivatization reaction is to react the polyethylene glycol derivative N3-PEG3-SPA sample with benzylamine in dichloromethane; Wherein, in step (4), the detection conditions of HPLC include: a. The chromatographic column is C18 chromatographic column; b. Column temperature is 25℃~35℃; c. Column flow rate is 0.8 mL / min to 1.2 mL / min; d. The injection volume is 2μL~10μL; e. The detector is an ultraviolet detector with a detection wavelength of 190nm-210nm; f. The mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous perchloric acid solution and the mobile phase B is acetonitrile; Wherein, in step f, the elution procedure of the mobile phase is shown in the following table: 。 2. The method according to claim 1, wherein: In step (1), the reaction temperature of the derivatization reaction is 20°C-40°C.

3. The method according to claim 1, wherein: In step (1), the reaction time of the derivatization reaction is 30 minutes to 3 hours.

4. The method according to claim 1, wherein: In step (3), the N3-PEG3-SPA sample concentration in the sample solution is 2 mg / mL to 5 mg / mL.

5. The method according to claim 1, wherein: In step a, the chromatographic column is one of Waters, Xbridge shield RPC18 chromatographic column or Extend-C18 chromatographic column.

6. The method according to claim 1, wherein: In step f, the concentration of the perchloric acid aqueous solution in the mobile phase A is 0.1‰ to 2.0‰.

7. The method according to claim 1, wherein: In step (4), the chromatographic column specifications are 150 mm in length × 4.6 mm in inner diameter, and 3 μm in particle size.

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