Method for detecting polysorbate 80 based on MALDI-MS and UHPLC-Q-TOF / MS combined technology

Abstract

The invention discloses a method for detecting polysorbate 80 based on an MALDI-MS and UHPLC-Q-TOF / MS. The method comprises the following steps: taking a polysorbate 80 sample, preparing a polysorbate 80 solution by using acetonitrile, respectively carrying out MALDI-MS detection and UHPLC-Q-TOF / MS detection on the polysorbate 80 solution, and determining the content of the polysorbate 80 in the polysorbate 80 sample according to the MALDI-MS and UHPLC-Q-TOF / MS detection results. The method comprises the following steps: acquiring PS80 principal component distribution according to an MALDI-MS (Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry) map: a polyoxyethylene chain delta m / z 44.0 + / -0.2 and an esterified derivative sodium adduct ion gradient peak cluster, and carrying out qualitative analysis on components of the polysorbate 80 in combination with a characteristic interval of m / z 44.0 + / -0.2 in the MALDI map; according to the retention time in the UHPLC-Q-TOF / MS detection result, identifying the monoester, diester and trace unesterified sorbitan in the polysorbate 80. According to the invention, molecular weight distribution analysis and component separation and identification of PS80 are realized.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical excipient analysis, and relates to a method for detecting polysorbate 80 (PS80) based on matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) coupled with ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF / MS). Specifically, it relates to a method for determining the molecular weight distribution and analyzing the component separation efficiency of polysorbate 80 based on MALDI-MS and UHPLC-Q-TOF / MS. Background Technology

[0002] Polysorbate 80 is a nonionic surfactant widely used in biopharmaceutical formulations, often for the stability protection of protein drugs. However, polysorbate 80 has a complex structure, mainly composed of sorbitol, polyoxyethylene chains, and fatty acids (such as oleic acid) linked by ester bonds. It exists as isomers with various degrees of polymerization and esterification forms, and the composition varies significantly between different batches, posing challenges to quality control.

[0003] Currently, common analytical methods for polysorbate 80 include HPLC-UV and ESI-MS. However, these methods suffer from insufficient resolution, significant component overlap, and an inability to comprehensively characterize complex components. Therefore, there is an urgent need for a novel analytical method that is highly sensitive, has high resolution, is easy to operate, and is suitable for complex sample matrices to achieve comprehensive quality evaluation of polysorbate 80. Summary of the Invention

[0004] The purpose of this invention is to solve the problems of low resolution, difficulty in component identification, and long analysis cycle in the prior art, and to provide a method for detecting polysorbate 80 based on the coupling technology of MALDI-MS and UHPLC-Q-TOF / MS. This method can realize molecular weight distribution analysis and component separation efficiency evaluation of polysorbate 80.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for detecting polysorbate 80 based on MALDI-MS and UHPLC-Q-TOF / MS coupling technology includes: taking a polysorbate 80 sample, preparing a polysorbate 80 solution using acetonitrile, and performing MALDI-MS and UHPLC-Q-TOF / MS detection on the polysorbate 80 solution respectively; obtaining the main component distribution of PS80 based on the MALDI-MS spectrum: polyoxyethylene chain Δm / z 44.0±0.2, and sodium adduct ion gradient peak cluster of esterified derivatives; and performing qualitative analysis of the components of polysorbate 80 based on the characteristic interval of m / z 44.0±0.2 in the MALDI spectrum; and matching the retention time in the UHPLC-Q-TOF / MS detection results with the theoretical component database of polysorbate 80 to identify the monoesters, diesters, and trace amounts of unesterified sorbitan in polysorbate 80.

[0007] Preparation of the polysorbate 80 solution: Dissolve the polysorbate 80 sample in acetonitrile to prepare a solution with a concentration of 0.2-1 mg / mL, and filter it through a 0.22 μm organic filter membrane to obtain the polysorbate 80 solution.

[0008] Preferably, the concentration of polysorbate 80 solution is 1 mg / mL when performing MALDI-MS detection; and the concentration of polysorbate 80 solution is 0.2 mg / mL when performing UHPLC-Q-TOF / MS detection.

[0009] The conditions for MALDI-MS detection are as follows:

[0010] Matrix: 2,5-Dihydroxybenzoic acid (DHB);

[0011] Cation reagent: Na⁺ solution, promotes the formation of [M+Na]⁺ ions;

[0012] Spotting method: The mixed spotting method is adopted. The matrix solution, cationic reagent and polysorbate 80 solution are mixed and the mixture is precisely spotted into the center of the well of the 48-well stainless steel target plate. It is dried at room temperature in the dark until the solvent evaporates completely to form a homogeneous co-crystallization.

[0013] Mass spectrometry conditions: reflectance positive ion mode, scanning range m / z 500~2500, laser energy 90~100, frequency 50Hz.

[0014] The matrix solution is a DHB concentration of 5–15 mg / mL prepared using 50% v / v acetonitrile aqueous solution as solvent.

[0015] Preferably, the concentration of DHB in the matrix solution is 10 mg / mL.

[0016] Preferably, the cationic reagent is an aqueous solution of sodium chloride with a concentration of 3 to 6 mg / mL.

[0017] Specifically, the cationic reagent is an aqueous solution of sodium chloride with a concentration of 5 mg / mL.

[0018] The volume ratio of the matrix solution, cationic reagent, and polysorbate 80 solution is 10:5:10.

[0019] Specifically, the spotting method is as follows: using a mixed spotting method, add 10 μL of matrix solution, 5.0 μL of cationic reagent and 10 μL of polysorbate 80 solution to a 0.6 mL centrifuge tube, vortex to mix, use a micropipette to take 1 μL of the mixture and accurately spot it onto the center of the well of a 48-well stainless steel target plate, and dry at room temperature in the dark for about 10 min - 40 min until the solvent has completely evaporated.

[0020] The conditions for UHPLC-Q-TOF / MS detection are as follows:

[0021] Column: Agilent ZORBAX RRHD SB-C8 (100 mm × 2.1 mm, 1.8 μm) or equivalent column;

[0022] Mobile phase: Mobile phase A is a 0.05%-0.2% (v / v) formic acid aqueous solution, preferably a 0.1% (v / v) formic acid aqueous solution, and mobile phase B is a 0.1% (v / v) formic acid acetonitrile;

[0023] Gradient elution program: 0–30 min, 5% B → 100% B; 30–33 min, 100% B; 33–36 min, 100% B → 5% B; 36–46 min, 5% B;

[0024] Flow rate: 0.4-0.6 mL / min;

[0025] Column temperature 45-55°C;

[0026] Injection volume 20 μL;

[0027] Mass spectrometry conditions: positive ion mode, scan range m / z 100–3000. In secondary mass spectrometry (MS / MS) analysis, collision-induced dissociation (CID) mode was used, with collision energies set to 80 eV and 120 eV for dicharged ions and monocharged ions, respectively.

[0028] The aforementioned polysorbate 80 theoretical component database covers 31 categories and a total of 952 compounds.

[0029] The beneficial effects of this invention are:

[0030] This invention uses liquid chromatography (separating 12 peaks in 46 min) coupled with high-resolution mass spectrometry to accurately identify monoesters, diesters, and trace impurities (unesterified sorbitan). The two complement each other to form a dual mode of "high-throughput primary screening - in-depth component tracing", realizing the molecular weight distribution analysis and component separation and identification of PS80.

[0031] The technologies are highly complementary: MALDI technology, with its high surface sensitivity and salt resistance, can quickly obtain the spatial distribution characteristics and relative abundance information of the main components of PS80, making it suitable for preliminary screening of large batches of samples; UHPLC-Q-TOF / MS system, relying on the excellent separation performance of ultra-high pressure liquid chromatography and the high resolution of quadrupole-time-of-flight mass spectrometry, can systematically elucidate the degree of esterification and fatty acid composition of PS80. The combination of the two can provide multidimensional data support, comprehensively covering the molecular weight distribution and component analysis needs of PS80.

[0032] High analytical precision: By optimizing the matrix (DHB was selected as the optimal matrix), cation (sodium ion was selected as the optimal charge control reagent), and target application method (the mixed method was selected as the standardized target application scheme) of the MALDI method, the accuracy of PS80 molecular weight distribution analysis is improved; UHPLC-Q-TOF / MS combined with the PS80 theoretical component database can accurately identify unesterified polyols, monoesters, diesters, and polyesters in PS80, and clarify the degree of polymerization range of each component;

[0033] Highly practical: The MALDI method requires no complex pretreatment and has a fast analysis speed, which can meet the high-throughput initial screening needs of the production site; UHPLC-Q-TOF / MS can achieve fine characterization of complex components of PS80, providing data support for PS80 quality control, process optimization and application risk assessment (such as avoiding toxic components), and is suitable for biopharmaceutical R&D and pharmaceutical excipient quality testing scenarios.

[0034] Addressing technical challenges: Addressing the issue that existing reversed-phase chromatography cannot completely separate PS80 monoesters, diesters, and polyesters, UHPLC-Q-TOF / MS high-resolution mass spectrometry, matched with a database, enables accurate identification of ester components even if they are not completely separated, filling the technical gap in the systematic characterization of complex PS80 components. Attached Figure Description

[0035] Figure 1 The effect of different matrices (DHB, CHCA, SA, DI) on the MALDI signal of polysorbate 80.

[0036] Figure 2This study compares the ionic addition efficiencies of cations Na⁺, K⁺, and Ag⁺ to polysorbate 80.

[0037] Figure 3 A comparison of the signal stability of polysorbate 80 using the mixing method, sandwich method, and coating method.

[0038] Figure 4 The MALDI superimposed spectra of polysorbate 20 / 40 / 60 / 80 show the effect of fatty acid chain length and double bond on molecular weight distribution.

[0039] Figure 5 The optimized typical chromatogram of polysorbate 80 showed 11 major peak groups separated within 46 min: a. polyoxyethylene sorbitan, b. polyoxyethylene isosorbide and polyoxyethylene glycol, c. polyoxyethylene sorbitan monooleate, d. polyoxyethylene isosorbide monooleate, e. polyoxyethylene sorbitan dioleate, f. polyoxyethylene isosorbide dioleate, g. polyoxyethylene sorbitan trioleate, and h. polyoxyethylene sorbitan tetraoleate.

[0040] Figure 6Representative MS / MS spectra represent a series of fragments from polyoxyethylene sorbitan monooleate to tetraoleate; a. polyoxyethylene sorbitan, b. polyoxyethylene isosorbide and polyoxyethylene glycol, c. polyoxyethylene sorbitan monooleate, d. polyoxyethylene isosorbide monooleate, e. polyoxyethylene sorbitan dioleate, f. polyoxyethylene isosorbide dioleate, g. polyoxyethylene sorbitan trioleate, h. polyoxyethylene sorbitan tetraoleate.

[0041] Figure 7 The total ion chromatograms and polarity distribution patterns of different ester components are shown.

[0042] Figure 8 The MALDI spectrum of polysorbate 80 shows a characteristic interval of m / z 44.0±0.2. Detailed Implementation

[0043] Example 1

[0044] 1.1 Instruments and Reagents

[0045] Instrument: Shimadzu AXIMA Performance MALDI-TOF / MS, linear / reflection dual mode;

[0046] Reagents: Polysorbate 20 (Batch No.: 20210502-1, Nanjing Well Pharmaceutical Co., Ltd.); Polysorbate 40 (Shanghai Aladdin Biochemical Technology Co., Ltd.); Polysorbate 60 (Shanghai Aladdin Biochemical Technology Co., Ltd.); Polysorbate 80 (Batch No. 20210401, Nanjing Well Pharmaceutical Co., Ltd.); CHCA (α-cyano-4-hydroxycinnamic acid, LaserBio Labs, France); DI (anthratriol, LaserBio Labs, France); SA (sinapic acid, LaserBio Labs, France); DHB (2,5-dihydroxybenzoic acid, LaserBio Labs, France); TOf mix (standard polypeptide mixture) standard (LaserBio Labs, France); NaCl (Sinopharm Group, analytical grade); KCl (Sinopharm Group, analytical grade); AgTFA (Sinopharm Group, analytical grade); Acetonitrile (Merck, LC-MS grade); Formic acid (Merck, LC-MS grade).

[0047] 1.2 Matrix Screening

[0048] α-Cyano-4-hydroxycinnamic acid (CHCA) solution (5 mg / mL): Accurately weigh 5 mg of α-cyano-4-hydroxycinnamic acid (CHCA) into a 2 mL centrifuge tube, add 1 mL of matrix diluent (50% v / v acetonitrile aqueous solution) to dissolve, and vortex thoroughly until no precipitate is formed to obtain a CHCA solution with a concentration of 5 mg / mL; using 50% v / v acetonitrile aqueous solution, prepare anthraquinone (DI) solution with a concentration of 5 mg / mL, an SA solution with a concentration of 20 mg / mL, and a 2,5-dihydroxybenzoic acid (DHB) solution with a concentration of 10 mg / mL using the same method.

[0049] 1.3 Sodium ion solution (5 mg / mL): Accurately weigh 5 mg of sodium chloride into a 2 mL centrifuge tube, add 1 mL of purified water, and vortex at low speed to mix well to obtain the sodium ion solution.

[0050] 1.4 Polysorbate 80 solution: Accurately weigh 25 mg of polysorbate 80 sample into a 25 mL volumetric flask, dilute with acetonitrile and bring to the mark, mix thoroughly, and filter through a 0.22 μm organic filter membrane to obtain a polysorbate 80 solution with a concentration of 1 mg / mL.

[0051] The performance of four typical matrices—DI, CHCA, SA, and DHB—was systematically evaluated. Parallel tests were conducted using a 1 mg / mL polysorbate 80 solution under identical instrument parameters via a homogeneous mixing spotting method, focusing on the signal response characteristics in the m / z range of 500–2000. Mixing spotting method: 10 μL of matrix solution, 5.0 μL of sodium chloride solution, and 10 μL of polysorbate 80 solution were added to a 0.6 mL centrifuge tube, vortexed, and 1 μL of the mixture was precisely spotted onto the center of each well in a 48-well stainless steel target plate using a micropipette. The mixture was dried at room temperature in the dark. Mass spectrometry conditions: reflectance positive ion mode, scan range m / z 500–2500, laser energy 90–100 kW, frequency 50 Hz.

[0052] Depend on Figure 1 It is evident that using DI as the matrix results in baseline fluctuations and signal heterogeneity; using SA as the matrix only produces effective ionization in the m / z range of 1250–2000; and using CHCA as the matrix exhibits significant baseline drift. In contrast, using DHB as the matrix demonstrates excellent broadband response, maintaining stable signal intensity across the entire detection range, and exhibiting a uniform Gaussian peak distribution. Considering ionization efficiency, signal stability, and spectral quality parameters, DHB is selected as the optimal matrix.

[0053] 1.3 Screening of cations

[0054] Sodium ion solution (5 mg / mL): Accurately weigh 5 mg of sodium chloride into a 2 mL centrifuge tube, add 1 mL of purified water, and vortex at low speed to mix well to obtain a sodium ion solution with a sodium chloride concentration of 5 mg / mL; accurately weigh potassium chloride (KCl) and silver trifluoroacetate (AgTFA) respectively, and prepare potassium ion solution (potassium chloride concentration of 5 mg / mL) and silver ion solution (silver trifluoroacetate concentration of 5 mg / mL) in the same way.

[0055] Using DHB as a matrix, cationic environments were constructed using sodium chloride, potassium chloride, and silver trifluoroacetate, respectively. Parallel tests were conducted using a mixed spotting method to systematically evaluate the charge regulation mechanisms of Na⁺, K⁺, and Ag⁺.

[0056] Depend on Figure 2 It is evident that in the low m / z range of 500–1000, the signal intensity using Na⁺ is significantly superior to that of K⁺ and Ag⁺, with lower background interference. The Na⁺-DHB complex exhibits a regular layered structure, and its cation-π interaction can promote directional energy transfer, thereby improving laser absorption efficiency and making it suitable for high-throughput analysis of polymers. Based on ionization efficiency and spectral quality, sodium ions are selected as the optimal charge-modifying reagent.

[0057] Example 2

[0058] Establishment of a rapid screening method using MALDI-TOF / MS

[0059] 1.1 Instruments and Reagents

[0060] Same as Example 1.

[0061] 1.2 Solution Preparation

[0062] (1) Matrix solution:

[0063] 2,5-Dihydroxybenzoic acid (DHB) solution (5 mg / mL): Accurately weigh 5 mg of 2,5-dihydroxybenzoic acid into a 2 mL centrifuge tube, add 1 mL of matrix diluent (50% v / v acetonitrile aqueous solution) to dissolve, and vortex thoroughly until no precipitate is formed, thus obtaining a DHB solution with a concentration of 5 mg / mL.

[0064] (2) Cation solution:

[0065] Sodium ion solution (5 mg / mL): Accurately weigh 5 mg of sodium chloride into a 2 mL centrifuge tube, add 1 mL of purified water, and vortex at low speed to mix well to obtain the sodium ion solution.

[0066] (3) Sample stock solution:

[0067] Polysorbate 80 stock solution (1 mg / mL): Accurately weigh 25 mg of polysorbate 80 sample into a 25 mL volumetric flask, dilute with acetonitrile and bring to the mark, mix thoroughly, and filter through a 0.22 μm organic filter membrane to obtain polysorbate 80 stock solution.

[0068] Accurately weigh samples of polysorbate 60, polysorbate 40, and polysorbate 20, and prepare stock solutions of polysorbate 60 (1 mg / mL), polysorbate 40 (1 mg / mL), and polysorbate 20 (1 mg / mL) using the same method.

[0069] (4) Spotting solution:

[0070] Mixing method: Take 10.0 μL of each of the stock solutions of polysorbate 20, 40, 60 and 80 with a concentration of 1 mg / mL, add them to 0.6 mL centrifuge tubes with 10.0 μL of matrix solution and 5.0 μL of cation solution respectively, and vortex to mix well to obtain polysorbate 20, 40, 60 and 80 spotting solutions respectively.

[0071] Sandwich method: Add matrix solution to the target plate, remove the matrix after 5-10 seconds to form a matrix film, add sample solution to the film, let it dry, cover it with an equal volume of matrix solution, and let it dry.

[0072] Covering method: First, drop the sample solution onto the target plate and let it dry. Then, cover it with an equal volume of matrix solution to form a matrix film and let it dry.

[0073] 1.3 Sampling and Detection

[0074] 1 μL of sample solution was spotted onto a 48-well stainless steel target plate and dried at room temperature in the dark for 10 min to form a uniform eutectic layer. A linear positive ion mode was used with a laser energy of 95 Hz, a frequency of 50 Hz, and a mass range of m / z 500–2500. Each sample spot was bombarded with lasers a total of 500 times to obtain the average spectrum.

[0075] Using DHB as a matrix, 1 mg / mL polysorbate 20, 40, 60, and 80 sample solutions were prepared by mixing, sandwiching, and covering methods. The crystallization regulation mechanism of the three methods was systematically evaluated.

[0076] Depend on Figure 3 It is evident that the mixing method exhibits optimal performance in the m / z range of 500–2000, with low baseline variability, making it suitable for precise mass spectrometry analysis of complex polysorbate systems. The mixing method achieves molecular-level dispersion of the sample and DHB matrix through vortex mixing, forming homogeneous microcrystals; the sandwich method results in increased lattice defect density due to solvent extraction; and the covering method generates a concentration gradient due to differences in droplet diffusion kinetics. Based on crystallization quality and detection sensitivity, the mixing method is established as the standardized target-targeting scheme.

[0077] 1.4 Data Processing

[0078] Baseline correction, spectral smoothing, and alignment were performed using MALDI Solution software. The filter width minus the baseline was set to 30, Gaussian smoothing was used, the smoothing filter was set to 30, and the peak width was set to 1. Through system optimization of MALDI detection parameters, leveraging its high surface sensitivity and rapid analysis characteristics, the core structural commonality of the polysorbate series was successfully captured: all samples exhibited characteristic signals of sorbitan anhydride and polyoxyethylene chains (Δm / z 44.0±0.2), and were further characterized by sodium adduct ions derived from fatty acid esterification ([M+Na)). + The distribution of gradient peak clusters reflects the regularity of the esterification reaction.

[0079] Different polysorbates show differences in fatty acid structure in their MALDI spectra (mixed method). Figure 4 From the molecular weight distribution, PS20 (lauric acid, C12) has the lowest molecular weight ([M+Na)). +≈1200-1600 Da); the main peaks of PS40 (palmitic acid, C16) and PS60 (stearic acid, C18) shift sequentially towards the high-quality region ([M+Na)). + ≈1400-1800 Da), among which PS60 has a broader peak distribution due to its long-chain saturated fatty acids; the main peak of PS80 (oleic acid, C18:1) continues to shift towards the high-quality region ([M+Na)). + (≈1600-2000 Da). Furthermore, although the molecular weight of PS80 is similar to that of PS60, the presence of the C9-C10 double bond results in peaks of oxidative degradation byproducts (such as fragment peaks in the m / z < 1000 region) in the spectrum. Regarding peak complexity, the straight-chain saturated fatty acid structure of PS20 / 40 / 60 concentrates the main peak signal with less background interference, while PS80, due to the oxidation sensitivity of unsaturated fatty acids, often shows additional peaks of epoxides or peroxide derivatives in the low-mass region. In addition, ionization efficiency exhibits a hydrophobicity-dependent characteristic: PS80, due to its strong hydrophobicity, co-crystallizes with the matrix, and the main component signal intensity is generally higher than the other three, but its oxidation byproducts may mask low-abundance components. In contrast, PS20, due to its short-chain hydrophilicity, is affected by matrix inhibition in high-salt systems, requiring desalting pretreatment to improve detection sensitivity.

[0080] The preferred conditions for MALDI-TOF / MS were selected based on Examples 1 and 2:

[0081] Matrix: 2,5-Dihydroxybenzoic acid (DHB); a matrix solution with a DHB concentration of 10 mg / mL was prepared using 50% v / v acetonitrile aqueous solution;

[0082] Cation reagent: Sodium chloride solution (NaCl); Accurately weigh 5 mg of sodium chloride into a 2 mL centrifuge tube, add 1 mL of purified water, and vortex at low speed to mix well to obtain a sodium chloride solution with a NaCl concentration of 5 mg / mL;

[0083] Sample solution: Polysorbate 80 (PS80); Accurately weigh 25 mg of polysorbate 80 sample into a 25 mL volumetric flask, dilute with acetonitrile and bring to the mark, mix thoroughly, and filter through a 0.22 μm organic filter membrane to obtain a polysorbate 80 solution with a concentration of 1 mg / mL;

[0084] Spotting method: The mixed spotting method was adopted. 10 μL of matrix solution, 5.0 μL of sodium chloride solution and 10 μL of polysorbate 80 solution were added to a 0.6 mL centrifuge tube and vortexed to mix. 1 μL of the mixture was accurately spotted into the center of the well of a 48-well stainless steel target plate using a micropipette. The mixture was dried at room temperature in the dark to form a homogeneous co-crystallization.

[0085] Mass spectrometry conditions: reflectance positive ion mode, scanning range m / z 500~2500, laser energy 90~100, frequency 50Hz.

[0086] Example 3

[0087] Establishment of a precise verification method using UHPLC-Q-TOF / MS

[0088] 2.1 Instruments and Reagents

[0089] Instrument: Agilent 1290 Infinity II UHPLC coupled with 6545 Q-TOF / MS, AJS ESI source.

[0090] 2.2 Sample solution preparation

[0091] Accurately weigh 25 mg of polysorbate 80 sample into a 25 mL volumetric flask, dilute with acetonitrile and bring to the mark, mix thoroughly to obtain a polysorbate 80 solution with a concentration of 1 mg / mL.

[0092] Accurately measure 2.0 mL of 1 mg / mL polysorbate 80 solution and place it in a 10 mL volumetric flask. Dilute with acetonitrile and bring the volume to the mark. Mix thoroughly to obtain a polysorbate 80 solution with a concentration of 0.2 mg / mL. Filter the solution through a 0.22 μm organic microporous membrane and collect the filtrate for UHPLC-Q-TOF / MS analysis.

[0093] 2.3 Chromatographic conditions

[0094] Chromatographic columns: Agilent ZORBAX RRHD SB-C8 (length 100 mm × inner diameter 2.1 mm, particle size 1.8 μm), Agilent Eclipse XDB C18 (length 150 mm × inner diameter 4.6 mm, particle size 3.5 μm), Waters Xbridge C18 (length 250 mm × inner diameter 4.6 mm, particle size 5 μm).

[0095] Table 1. Detailed information on different chromatographic columns

[0096]

[0097] Column selection: Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: 0.1% formic acid acetonitrile solution; Gradient elution: 0–30 min, 5% B → 100% B; 30–33 min, 100% B; 33–36 min, 100% B → 5% B; 36–46 min, equilibration with 5% B; Flow rate: 0.5 mL / min-1 The column temperature was 50℃, and the injection volume was 20 μL. The Agilent ZORBAX RRHD SB-C8 (100 mm × 2.1 mm, 1.8 μm) column effectively separated polysorbate 80, yielding more chromatographic peaks with high resolution and good peak shape. The other two columns exhibited poor resolution, numerous shoulder peaks, and noticeable tailing. Therefore, the Agilent ZORBAX RRHD SB-C8 (100 mm × 2.1 mm, 1.8 μm) column was selected for subsequent chromatographic condition exploration experiments.

[0098] Mobile phase selection: An Agilent ZORBAX RRHD SB-C8 (100 mm × 2.1 mm, 1.8 μm) column was used. Formic acid was added as a modifier to the mobile phase. The 0.1% formic acid aqueous solution in mobile phase A (as described in "Column Selection") was replaced with 0.05% and 0.2% formic acid aqueous solutions, while all other parameters remained the same as in "Column Selection." The effects of 0.05%, 0.1%, and 0.2% formic acid on the chromatographic results were explored. Baseline fluctuations were larger under the 0.05% and 0.2% formic acid conditions. In contrast, 0.1% formic acid had a greater effect on peak shape adjustment and a more stable baseline.

[0099] Elution gradient screening: An Agilent ZORBAX RRHD SB-C8 (100 mm × 2.1 mm, 1.8 μm) column was used. Mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: 0.1% formic acid acetonitrile solution. Elution gradient 1: 0–30 min, 5%B → 100%B; 30–33 min, 100% B; 33–36 min, 100%B → 5%B; 36–46 min, equilibration with 5%B. Elution gradient 2: 0–10 min, 5%B → 100%B; 10–12 min, 100% B; 12–15 min, 100%B → 5%B; 15–20 min, equilibration with 5%B. Elution gradient 3: 0–40 min, 5%B → 100%B; 40–45 min, 100% B; 45–50 min, 100%B → 5%B. %B; 50–60 min, equilibrate with 5%B; flow rate 0.5 mL / min -1The column temperature was 50℃, and the injection volume was 20 μL. The effect of injection time (20, 46, and 60 min) was investigated. The results showed that the separation effect was poor within 20 min, with most components failing to achieve complete separation and elution. Within 46 min, polysorbate 80 was completely detected with good separation. Gradient elution for 60 min was inefficient and time-consuming. Therefore, gradient elution 1 was used as the final elution gradient condition.

[0100] Determining UHPLC-Q-TOF / MS conditions

[0101] (1) UHPLC conditions

[0102] Column: Agilent ZORBAX RRHD SB-C8 (100 mm × 2.1 mm, 1.8 μm); Mobile phase A: aqueous solution containing 0.1% formic acid; Mobile phase B: acetonitrile containing 0.1% formic acid; Flow rate: 0.5 mL / min; Gradient elution: 0–30 min, 5%B → 100%B; 30–33 min, 100%B; 33–36 min, 100%B → 5%B; 36–46 min, equilibration with 5%B; Injection volume: 20 μL.

[0103] (2) Mass spectrometry conditions

[0104] AJS ESI ion source, positive ion detection mode, dry gas flow rate: 8 L / min; dry gas temperature: 320 ℃; nebulizer pressure: 0.18 MPa; sheath gas flow rate: 11 L / min; sheath gas temperature: 325 ℃; capillary voltage: 3500 V; nozzle voltage: 1500 V; scan mode: Auto MS / MS; scan range: m / z 100~3000; resolution: 10 GHz; fragmenter voltage: 175 V; in the secondary mass spectrometry (MS / MS) analysis, collision-induced dissociation (CID) mode was used, and the collision energies were set to 80 eV and 120 eV for doubly charged ions (M+2H²⁺) and monocharged ions (M+H⁺), respectively.

[0105] 2.4 Data Processing Agilent Mass Hunter qualitative software (version B.10.0) was used for data acquisition and analysis of polysorbate 80. The components were analyzed and identified by retention time, primary and secondary high-resolution mass spectrometry data.

[0106] 2.5 Database Establishment

[0107] The Mass Hunter PCDL Manager polysorbate 80 extended database (Agilent database) provided by Agilent Technologies was used. The construction principle of this database is consistent with the polysorbate 80 library in the Excipient Profiler pharmaceutical excipient intelligent analysis system, both based on mathematical models established according to the correlation between the degree of polymerization and retention time of each component of polysorbate 80 reported in the literature. Based on this, the components contained in the sample were identified by matching the mass spectrometry detection results with the theoretical values ​​in the database and then filtering according to the matching results. This extended database not only covers more components bound to fatty acids but also has a higher degree of esterification, enabling the identification of components with added Na+. + or NH4 + The component peaks.

[0108] Using a chromatographic elution gradient, PS80 successfully separated 11 main chromatographic peaks within 46 minutes (see...). Figure 5 ). Figure 6 Typical mass spectra further revealed the structural characteristics of each component. As can be seen from the mass spectra, due to the different degrees of polymerization of the same component, the difference in m / z between components with single-charged addition ions is 44 Da, and the difference in m / z between components with double-charged addition ions is 22 Da. The mass spectra of the same component exhibit a normal distribution. For example, Figure 6 The m / z of the single-charge parent ion of polyoxyethylene sorbitan (PS) in group a ranges from 1045.6037 (n=20) to 1485.8602 (n=30), with a difference of 44 Da between adjacent degrees of polymerization, forming a continuous cluster of normally distributed peaks (see...). Figure 8 ).

[0109] Taking sample polysorbate 80 (number: 20210401) as an example, the obtained mass-to-nucleus ratio was compared with the mass-to-nucleus ratio data in the database to infer the component structure and obtain the ester classification of each peak component and the polymerization degree range of each component, as shown in Table 2.

[0110] Table 2. Summary of component names and degrees of polymerization in polysorbate 80

[0111]

[0112] Based on the mass spectrometry peak assignments of polysorbate 80 samples, it was found that in the early stage of elution (t... R =0.50-13.06 min, peaks 1-3) mainly consist of unesterified polyols with strong hydrophilicity, including polyoxyethylene, polyoxyethylene isosorbide, and polyoxyethylene sorbitan. This is followed by monoester components, at t RThe two eluting peaks (peaks 4-5) at approximately 18.00 min were identified as polyoxyethylene sorbitan monooleate and polyoxyethylene isosorbitan monooleate, respectively. Diester components (peaks 6-8, PSD and PID) were eluted at approximately 25.00 min. The triester and tetraester components were the most hydrophobic, eluting at approximately 18.00 min. R Separation was achieved in ≥27.00 min (see...) Figure 7 It can be seen that the monoesters and diesters, and the diesters and polyesters, have similar polarities. While reversed-phase chromatography cannot completely separate different esters, it can determine their approximate ranges without affecting the mass spectrometric resolution of the components. UHPLC-Q-TOF / MS, through optimization of liquid chromatography coupled with high-resolution mass spectrometry, systematically resolved the complex composition of polysorbate 80. Based on the constructed theoretical database, this method accurately identified unesterified polyols, monoesters, diesters, and polyesters, and clarified their degree of polymerization ranges, highlighting its ability to resolve trace components.

[0113] In summary, the two technologies can complement each other in practical applications. MALDI rapidly provides data on molecular weight distribution and principal component abundance, suitable for high-throughput initial screening and stability assessment in production environments; while UHPLC-Q-TOF / MS achieves fine characterization through chromatographic retention time and high-resolution mass spectrometry, serving component tracing in process optimization, oxidative degradation mechanism research, and toxic impurity identification in safety evaluation. In the application of polysorbate 80, combined use can comprehensively guide the formulation of polysorbate 80 quality standards (principal component thresholds), stabilizer formulation optimization (based on esterification degree control), and risk control in biopharmaceutical development (avoiding toxic components).

[0114] The MALDI and UHPLC-Q-TOF / MS methods used in this invention to analyze polysorbate 80 not only provide a methodological basis for its component analysis, but also lay a technical foundation for subsequent research on its degradation behavior and mechanism in different buffer systems.

Claims

1. A method for detecting polysorbate 80 based on MALDI-MS coupled with UHPLC-Q-TOF / MS, characterized in that: include: Polysorbate 80 (PS80) samples were taken, and PS80 solutions were prepared using acetonitrile. The PS80 solutions were analyzed by MALDI-MS and UHPLC-Q-TOF / MS. Based on the MALDI-MS chromatogram, the main component distribution of PS80 was obtained: polyoxyethylene chain Δm / z 44.0±0.2, and the gradient peak cluster of sodium adduct ions of esterified derivatives. The components of PS80 were qualitatively analyzed using the characteristic interval of m / z 44.0±0.2 in the MALDI chromatogram. Based on the retention times in the UHPLC-Q-TOF / MS results, the results were matched with the theoretical component database of PS80 to identify the monoesters, diesters, and trace amounts of unesterified sorbitan in PS80.

2. The method for detecting polysorbate 80 according to claim 1, characterized in that: Preparation of the polysorbate 80 solution: Dissolve the polysorbate 80 sample in acetonitrile to prepare a solution with a concentration of 0.2-1 mg / mL, and filter it through a 0.22 μm organic filter membrane to obtain the polysorbate 80 solution.

3. The method for detecting polysorbate 80 according to claim 1, characterized in that: The conditions for MALDI-MS detection are as follows: Matrix: 2,5-Dihydroxybenzoic acid; Cation reagent: Na⁺ solution; Spotting method: The mixed spotting method is adopted. The matrix solution, cationic reagent and polysorbate 80 solution are mixed and the mixture is precisely spotted into the center of the well of the 48-well stainless steel target plate. It is dried at room temperature in the dark until the solvent evaporates completely to form a homogeneous co-crystallization. Mass spectrometry conditions: reflectance positive ion mode, scanning range m / z 500–2500, laser energy 90–100, frequency 50 Hz.

4. The method for detecting polysorbate 80 according to claim 3, characterized in that: The matrix solution is a DHB concentration of 5–15 mg / mL prepared using 50% v / v acetonitrile aqueous solution as solvent; the cation reagent is a sodium chloride aqueous solution with a concentration of 3–6 mg / mL.

5. The method for detecting polysorbate 80 according to claim 4, characterized in that: The matrix solution is a DHB solution with a concentration of 10 mg / mL prepared using 50% v / v acetonitrile aqueous solution as solvent; the cation reagent is a sodium chloride aqueous solution with a concentration of 5 mg / mL.

6. The method for detecting polysorbate 80 according to claim 3, characterized in that: The volume ratio of the matrix solution, cationic reagent, and polysorbate 80 solution is 10:5:

10.

7. The method for detecting polysorbate 80 according to any one of claims 3-6, characterized in that: The spotting method is as follows: A mixed spotting method is used. 10 μL of matrix solution, 5.0 μL of cationic reagent and 10 μL of polysorbate 80 solution are added to a 0.6 mL centrifuge tube, vortexed and mixed. 1 μL of the mixture is then precisely spotted onto the center of the well of a 48-well stainless steel target plate using a micropipette. The plate is then dried at room temperature in the dark for about 10 min to 40 min until the solvent has completely evaporated.

8. The method for detecting polysorbate 80 according to claim 1, characterized in that: The conditions for UHPLC-Q-TOF / MS detection are as follows: Column: Agilent ZORBAX RRHD SB-C8 (100 mm × 2.1 mm, 1.8 μm) or equivalent column; Mobile phase: Mobile phase A is a 0.05-0.2% v / v formic acid aqueous solution, preferably a 0.1% v / v formic acid aqueous solution, and mobile phase B is 0.1% (v / v) formic acid acetonitrile; Gradient elution program: 0–30 min, 5% B → 100% B; 30–33 min, 100% B; 33–36 min, 100% B → 5% B; 36–46 min, 5% B; Flow rate: 0.4-0.6 mL / min; Column temperature 45-55°C; Injection volume 20 μL; Mass spectrometry conditions: positive ion mode, scan range m / z 100~3000. In secondary mass spectrometry analysis, collision-induced dissociation mode was used, and the collision energies were set to 80 eV and 120 eV for dicharged ions and monocharged ions, respectively.

9. The method for detecting polysorbate 80 according to claim 8, characterized in that: Column: Agilent ZORBAX RRHD SB-C8 (100 mm × 2.1 mm, 1.8 μm).

10. The method for detecting polysorbate 80 according to claim 8, characterized in that: Mobile phase A is a 0.1% v / v formic acid aqueous solution.

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