CN116297915B - Analysis and identification method for polysorbate auxiliary materials - Google Patents
Analysis and identification method for polysorbate auxiliary materials Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 56
- 229920000136 polysorbate Polymers 0.000 title claims abstract description 31
- 229950008882 polysorbate Drugs 0.000 title claims abstract description 30
- 238000004458 analytical method Methods 0.000 title claims abstract description 26
- 239000000463 material Substances 0.000 title claims abstract description 20
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims abstract description 44
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 claims abstract description 43
- 229920000053 polysorbate 80 Polymers 0.000 claims abstract description 43
- 229940068968 polysorbate 80 Drugs 0.000 claims abstract description 43
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims abstract description 18
- 229920001213 Polysorbate 20 Polymers 0.000 claims abstract description 17
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims abstract description 17
- 229940068977 polysorbate 20 Drugs 0.000 claims abstract description 17
- 229920001214 Polysorbate 60 Polymers 0.000 claims abstract description 16
- 239000001818 polyoxyethylene sorbitan monostearate Substances 0.000 claims abstract description 15
- 235000010989 polyoxyethylene sorbitan monostearate Nutrition 0.000 claims abstract description 15
- 229940113124 polysorbate 60 Drugs 0.000 claims abstract description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 48
- 239000000523 sample Substances 0.000 claims description 18
- 238000002347 injection Methods 0.000 claims description 14
- 239000007924 injection Substances 0.000 claims description 14
- 238000004949 mass spectrometry Methods 0.000 claims description 13
- 239000002904 solvent Substances 0.000 claims description 11
- 238000005259 measurement Methods 0.000 claims description 10
- 238000001514 detection method Methods 0.000 claims description 8
- 239000013558 reference substance Substances 0.000 claims description 8
- 239000012488 sample solution Substances 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 7
- 238000000889 atomisation Methods 0.000 claims description 7
- 238000012937 correction Methods 0.000 claims description 6
- 150000002009 diols Chemical class 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 238000003908 quality control method Methods 0.000 claims description 6
- 238000004808 supercritical fluid chromatography Methods 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 6
- 238000011156 evaluation Methods 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- 238000004090 dissolution Methods 0.000 claims description 2
- 238000001228 spectrum Methods 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 abstract description 9
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 abstract description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 4
- 238000012544 monitoring process Methods 0.000 abstract description 4
- 239000012530 fluid Substances 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 3
- 239000001569 carbon dioxide Substances 0.000 abstract description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 abstract description 2
- 239000000203 mixture Substances 0.000 description 16
- 239000012071 phase Substances 0.000 description 10
- 150000001875 compounds Chemical class 0.000 description 5
- 239000000546 pharmaceutical excipient Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 150000002148 esters Chemical class 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 229940079593 drug Drugs 0.000 description 3
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 229940124531 pharmaceutical excipient Drugs 0.000 description 3
- KLDXJTOLSGUMSJ-JGWLITMVSA-N Isosorbide Chemical compound O[C@@H]1CO[C@@H]2[C@@H](O)CO[C@@H]21 KLDXJTOLSGUMSJ-JGWLITMVSA-N 0.000 description 2
- 229920006192 POE isosorbide Polymers 0.000 description 2
- 229920001219 Polysorbate 40 Polymers 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000010829 isocratic elution Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000249 polyoxyethylene sorbitan monopalmitate Substances 0.000 description 2
- 235000010483 polyoxyethylene sorbitan monopalmitate Nutrition 0.000 description 2
- 229940101027 polysorbate 40 Drugs 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- -1 sorbitol anhydride Chemical class 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- WSGYTJNNHPZFKR-UHFFFAOYSA-N 3-hydroxypropanenitrile Chemical compound OCCC#N WSGYTJNNHPZFKR-UHFFFAOYSA-N 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 150000005690 diesters Chemical class 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 238000004896 high resolution mass spectrometry Methods 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229960002479 isosorbide Drugs 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 238000013441 quality evaluation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 150000005691 triesters Chemical class 0.000 description 1
- 238000001195 ultra high performance liquid chromatography Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
Abstract
The invention discloses an analysis and identification method of polysorbate auxiliary materials. In the analysis method established by adopting the supercritical fluid phase-combining chromatography, carbon dioxide and acetonitrile/ethanol are used as mobile phases, and the components of polysorbate 20, polysorbate 60 and polysorbate 80 are separated to obtain better separation degree, so that a referenceable method is provided for monitoring the consistency and stability quality of polysorbate products among batches.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and particularly relates to an analysis and identification method of polysorbate auxiliary materials.
Background
Polysorbate series auxiliary materials are important surfactant auxiliary materials, and quality standards of polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80 are collected in four parts of Chinese pharmacopoeia. The composition of polysorbate is quite complex and component characterization presents challenges in terms of quality control. Currently, there are reports of rapid analysis of polysorbate 80 using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), however, due to lack of sufficient mass resolution, it is difficult to identify near-mass oligomers with MALDI-TOF-MS alone; there are also reports of rapid analysis of polysorbate-series excipients using ultra-high performance liquid chromatography/mass spectrometry (UPLC-MS) (Wang Z, wang Y, cai T, et al journal of Chromatography A, 2019:460450.) but the database of compounds used for analysis and identification contains only 853 compounds, far less than the total number of compounds that may be present in polysorbate-series excipients, and the analysis process requires a large amount of organic solvents.
The polysorbate auxiliary materials represented by polysorbate 80 and polysorbate 20 have very important roles in pharmaceutical preparations, are mainly used as solubilizers of insoluble drugs or stabilizers of biological macromolecular drugs, and belong to critical auxiliary materials in the formulation. Its compositional variation will directly affect the solubilization and stabilization functions of the drug. Therefore, the high-performance analysis method has important significance for precisely identifying the polysorbate composition and realizing quality control.
Disclosure of Invention
The invention aims to provide a method for analyzing and identifying polysorbate auxiliary materials by adopting supercritical fluid chromatography and mass spectrometry.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the analytical identification method of the polysorbate auxiliary materials comprises the following steps:
step 1, taking a test sample, adding methanol for dissolution, and preparing a solution with the concentration of 0.2mg/mL as a test sample solution;
step 2, detecting the sample solution by adopting the combination of supercritical fluid chromatography and mass spectrometry to obtain a determination result;
the supercritical fluid chromatography conditions were as follows:
ACQUITY UPC 2 torus Diol chromatography column with a specification of 3.0mm×100mm,1.7 μm,130A
Column temperature: 50 DEG C
Flow rate: 1.0mL/min
Sample injection volume: 1 mu L
Back pressure: 2500psi
Mobile phase: a is CO 2 B is methanol and acetonitrile according to the volume ratio of 30:70, and the gradient elution procedure is as follows: 0-0.2 min,8% B;0.2 to 12.0min,8 to 15 percent of B;12.0 to 18.0min,15 to 20 percent of B;18.0 to 31.0min,20 to 35 percent of B;31.0 to 34.0min,35 to 50 percent of B;34.0 to 34.2min,50 to 8 percent of B;34.2 to 37.0min,8 percent of B; the ionizing solvent is a mixed solution of methanol and 5mmol/L ammonium formate, and the flow rate is 0.2mL/min;
the mass spectrometry conditions were as follows:
ionization mode: ESI (electronic service provider interface) + The method comprises the steps of carrying out a first treatment on the surface of the Atomization gas temperature: 500 ℃; atomizing gas flow rate: 800L/h; source temperature: 120 ℃; capillary voltage: 2.5KV; taper hole voltage: 40V; acquisition mode: MS (MS) E The method comprises the steps of carrying out a first treatment on the surface of the Scanning range: m/z is 50-3000;
step 3, comparing the measurement result with the established polysorbate 80 fingerprint to realize analysis and identification of polysorbate auxiliary materials;
the polysorbate auxiliary materials are polysorbate 20, polysorbate 60 and polysorbate 80.
Further, the method for establishing the polysorbate 80 fingerprint comprises the following steps: respectively preparing polysorbate 80 sample solution and reference substance solution, sequentially sampling and detecting, integrating all the peaks of the basic peak chromatograms of all the sampled samples, introducing the obtained basic peak chromatograms and integral data into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, taking the reference substance map as a correction reference, automatically matching the peaks by using the median, and completing multi-point correction to obtain the polysorbate 80 fingerprint.
In step 3, the measurement result is compared with the established polysorbate 80 fingerprint, the fingerprint similarity of the measurement result is evaluated by adopting an included angle cosine method, and the polysorbate 20, the polysorbate 60 and the polysorbate 80 are distinguished according to the similarity result.
Furthermore, the measurement result is compared with the established polysorbate 80 fingerprint, the fingerprint similarity is evaluated by adopting an included angle cosine method, and the quality control of polysorbate 80 can be realized according to the similarity result.
Compared with the prior art, the invention has the following beneficial effects:
1. the analysis method established by the supercritical fluid phase-combining chromatography is adopted to separate the components of the polysorbate 20, the polysorbate 60 and the polysorbate 80, 50-70 chromatographic peaks can be successfully detected within the analysis time length of 37 minutes, and the organic solvent used by single-needle sample injection is less than 10mL. Compared with the traditional high performance liquid chromatography, the analysis efficiency is greatly improved.
2. The UPCC-MS polysorbate 80 fingerprint established after the UPCC and the QTOF-MS are combined realizes the similarity evaluation of the total component distribution of the polysorbate 80 products from different sources, and can be effectively distinguished from polysorbate 20 and polysorbate 60.
The polysorbate 80UPCC-TOF-MS fingerprint analysis method established by the invention has good precision and repeatability, simple sample preparation and high analysis speed, can be used as a polysorbate product stability monitoring or quality consistency evaluation method, can provide a theoretical basis for further researching the quality standard and spectral efficiency relation of polysorbate auxiliary materials, and can provide a borrowable technical method and valuable data reference for the popularization of a 'green inspection' standard system.
Drawings
Fig. 1 to 4 are spectrograms of the procedure of the UPCC-MS method established in example 1.
FIG. 5 is a UPCC-MS fingerprint of 12 batches of polysorbate 80 in example 2.
FIG. 6 shows the base peak chromatograms of polysorbate 20 (PS 20), polysorbate 60 (PS 60), and polysorbate 80 (PS 80) in example 2.
Detailed Description
Polysorbate is a commonly used class of nonionic surfactant adjuvants. Polysorbate 20, polysorbate 40, polysorbate 60 and Polysorbate 80 are all collected in the United states Pharmacopeia 2020, european Pharmacopeia, and Polysorbate 20 and Polysorbate 80 are most widely used, and particularly in high risk formulations, they are also generally used in higher amounts for prescription. However, polysorbate adjuvant composition is extremely complex, which arises from the variety and various uncertainties in the raw materials and synthesis processes, including: (1) there may be two types of parent cores (i.e. sorbitol anhydride and isosorbide), (2) polydispersity of the side chains (i.e. different numbers of polyoxyethylene repeat units), (3) type of fatty acid (derived from different fatty acid constitution of the raw material), (4) uncertainty of degree of esterification (i.e. unesterified, monoester, diester, triester). Table 1 lists the theoretical structural formulas and the classification of the components of the actual composition of the polysorbate and the chemical structural formulas thereof. The theoretical composition deduces that the compound types of the polyoxyethylene sorbitan and the esters thereof, the polyoxyethylene isosorbide and the esters thereof, and the polyethylene glycol and the esters thereof can be obtained, and the polyoxyethylene isosorbide and the esters thereof totally contain more than 1 ten thousand theoretical compounds.
TABLE 1 classification of components of theoretical structural formula and actual composition of Polysorbate and general chemical structural formula thereof
Fingerprint is mostly used for identification and quality evaluation of traditional Chinese medicinal materials, and few applications of identification and analysis in complex pharmaceutical excipients are reported. In fact, many of the pharmaceutical excipients have complex mixture systems such as tween, span surfactants, triglycerides and oils of natural origin, which all face more quality control problems. Due to the extremely complex composition, it is often difficult to find differences in composition or complicated steps of the method employed by conventional analytical detection means, and the detection efficiency is low. Therefore, in recent years, the qualitative or quantitative research of such auxiliary materials based on the liquid phase/high resolution mass spectrometry has been carried out. Generally, the creation of a custom database is a common strategy for the compositional analysis and identification of such pharmaceutical excipients. However, this approach is time consuming and labor intensive and is not an optimal choice for certain quality control scenarios (e.g., for product batch-to-batch stability monitoring, etc.). In addition, although the conventional high performance liquid chromatography can effectively separate the multiple components, the problem that the elution time has to be increased due to the limitations of the liquid chromatography, so that a large amount of organic solvents are consumed is avoided, and the high quality development of the green test is not met.
In the analysis method established by adopting the supercritical fluid phase-combining chromatography, carbon dioxide and acetonitrile/ethanol are used as mobile phases, and the components of polysorbate 20, polysorbate 60 and polysorbate 80 are separated to obtain better separation degree, so that a referenceable method is provided for monitoring the consistency and stability quality of polysorbate products among batches.
The invention will now be described in further detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The experimental procedures and reagents not shown in the formulation of the examples were all in accordance with the conventional conditions in the art.
The following examples employ the following instruments and materials:
ACQUITY UPCC, xex G2-XS Q-TOF-MS (WATERS Co., USA); WATERS MassLynx V4.1.1 mass spectrometry workstation; water UNIFI 1.9.3 analysis software; chromatographic column: ACQUITYUPC2 Torrs Diol chromatography column (3.0 mm. Times.100 mm,1.7 μm, WATERS).
Polysorbate 20, polysorbate 60 and Polysorbate 80 are available from He Dachio Chemie, nanjing Wil pharmaceutical Co., ltd; the 10 batches of polysorbate 80 samples were from j.t. baker chemicals, jiangsu easily pharmaceutical limited, croda chemicals, uk, and south tokyo wil pharmaceutical limited, respectively. Polysorbate 20USP standard (lot number: R079C 0), polysorbate 60USP standard (lot number: R09110) and Polysorbate 80USP standard (lot number: R072G 0) were used as controls.
The invention will now be described in further detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The experimental procedures and reagents not shown in the formulation of the examples were all in accordance with the conventional conditions in the art.
Example 1
Establishment of UPCC-MS method and method reproducibility investigation
The UPCC chromatographic conditions were searched using different ratios of mobile phases, different chromatographic columns and protonated solvents.
Condition 1: ACQUITY UPC 2 Torus Diol chromatography column (3.0 mm. Times.100 mm,1.7 μm, 130A);
column temperature: 50 ℃; flow rate: 1.0mL/min; sample injection volume: 1 μl; back pressure: 2500psi;
mobile phase: a is CO 2 B is methanol and acetonitrile according to the volume ratio of 30:70, and isocratic elution: 60% A,40% B; the ionizing solvent is a mixed solution of methanol and 5mmol/L ammonium formate, and the flow rate is 0.2mL/min;
mass spectrometry conditions: ionization mode: ESI (electronic service provider interface) + The method comprises the steps of carrying out a first treatment on the surface of the Atomization gas temperature: 500 ℃; atomizing gas flow rate: 800L/h; source temperature: 120 ℃; capillary voltage: 2.5KV; taper hole voltage: 40V; acquisition mode: MS (MS) E The method comprises the steps of carrying out a first treatment on the surface of the Scanning range: m/z is 50-3000.
Condition 2: ACQUITY UPC 2 Torus Diol chromatography column (3.0 mm. Times.100 mm,1.7 μm, 130A);
column temperature: 50 ℃; flow rate: 1.0mL/min; sample injection volume: 1 μl; back pressure: 2500psi;
mobile phase: a is CO 2 B is methanol and acetonitrile according to the volume ratio of 30:70, and isocratic elution: 80% A,20% B; the ionizing solvent is a mixed solution of methanol and 5mmol/L ammonium formate, and the flow rate is 0.2mL/min;
mass spectrometry conditions: ionization mode: ESI (electronic service provider interface) + The method comprises the steps of carrying out a first treatment on the surface of the Atomization gas temperature: 500 ℃; atomizing gas flow rate: 800L/h; source temperature: 120 ℃; capillary voltage: 2.5KV; taper hole voltage: 40V; acquisition mode: MS (MS) E The method comprises the steps of carrying out a first treatment on the surface of the Scanning range: m/z is 50-3000.
Condition 3: vinsis HSS C 18 SB Column chromatography Column (100A, 1.8 μm,3 mm. Times.150mm);
column temperature: 50 ℃; flow rate: 1.0mL/min; sample injection volume: 1 μl; back pressure: 2500psi;
mobile phase: a is CO 2 B is methanol and acetonitrile according to the volume ratio of 30:70, and the gradient elution procedure is as follows: 0-0.2 min,8% B;0.2 to 12.0min,8 to 15 percent of B;12.0 to 18.0min,15 to 20 percent of B;18.0 to 31.0min,20 to 35 percent of B;31.0 to 34.0min,35 to 50 percent of B;34.0 to 34.2min,50 percentAbout 8% B;34.2 to 37.0min,8 percent of B; flow rate: 1.0mL/min; sample injection volume: 1 μl; back pressure: 2500psi;
the ionizing solvent is a mixed solution of methanol and 5mmol/L ammonium formate, and the flow rate is 0.2mL/min;
mass spectrometry conditions: ionization mode: ESI (electronic service provider interface) + The method comprises the steps of carrying out a first treatment on the surface of the Atomization gas temperature: 500 ℃; atomizing gas flow rate: 800L/h; source temperature: 120 ℃; capillary voltage: 2.5KV; taper hole voltage: 40V; acquisition mode: MS (MS) E The method comprises the steps of carrying out a first treatment on the surface of the Scanning range: m/z is 50-3000.
Condition 4: vinsis HSS C 18 SB Column chromatography Column (100A, 1.8 μm,3 mm. Times.150mm);
column temperature: 50 ℃; flow rate: 1.0mL/min; sample injection volume: 1 μl; back pressure: 2500psi;
mobile phase: a is CO 2 B is methanol and acetonitrile according to the volume ratio of 30:70, and the gradient elution procedure is as follows: 0-0.2 min,8% B;0.2 to 12.0min,8 to 15 percent of B;12.0 to 18.0min,15 to 20 percent of B;18.0 to 31.0min,20 to 35 percent of B;31.0 to 34.0min,35 to 50 percent of B;34.0 to 34.2min,50 to 8 percent of B;34.2 to 37.0min,8 percent of B; flow rate: 1.0mL/min; sample injection volume: 1 μl; back pressure: 2500psi;
the ionizing solvent is methanol with the flow rate of 0.2mL/min;
mass spectrometry conditions: ionization mode: ESI (electronic service provider interface) + The method comprises the steps of carrying out a first treatment on the surface of the Atomization gas temperature: 500 ℃; atomizing gas flow rate: 800L/h; source temperature: 120 ℃; capillary voltage: 2.5KV; taper hole voltage: 40V; acquisition mode: MS (MS) E The method comprises the steps of carrying out a first treatment on the surface of the Scanning range: m/z is 50-3000.
The detection method of the invention increases CO in the mobile phase by different proportions of mobile phase, different chromatographic columns and column temperature conditions and protonated solvent when establishing, as shown in figures 1 and 2 2 As the ratio of (2), the number of separated chromatographic peaks increases; as shown in FIG. 3, when a less polar column is used, the number of eluted peaks is smaller than that of a more polar column, and the separation efficiency is poor, as shown in FIG. 4, when ammonium formate is not added to the ionizing solvent, the response is poor due to the low ionization efficiency.
Finally, the following detection conditions are obtained:
1. solution preparation
Proper amount of sample is precisely weighed, placed in a 100ml volumetric flask, dissolved and diluted to a scale by adding methanol, and uniformly shaken to prepare a solution with the concentration of about 0.2mg/ml as a sample solution.
2. UPCC phase-combining chromatographic conditions
ACQUITY UPC 2 Torus Diol chromatography column (3.0 mm. Times.100 mm,1.7 μm, 130A); column temperature: 50 ℃; flow rate: 1.0mL/min; sample injection volume: 1 μl; back pressure: 2500psi; mobile phase:
A＝CO 2 b=methanol-acetonitrile (30/70, V/V); ionizing solvent: methanol+5 mmol/L ammonium formate, 0.2mL/min; the gradient elution procedure was: 0-0.2 min,8% B;0.2 to 12.0min,8 to 15 percent of B;12.0 to 18.0min,15 to 20 percent of B;18.0 to 31.0min,20 to 35 percent of B;31.0 to 34.0min,35 to 50 percent of B;34.0 to 34.2min,50 to 8 percent of B; 34.2-37.0 min,8% B.
3. Mass spectrometry conditions
Ionization mode: esi+; atomization gas temperature: 500 ℃; atomizing gas flow rate: 800L/h; source temperature: 120 ℃; capillary voltage: 2.5KV; taper hole voltage: 40V; acquisition mode: MSE; scanning range: m/z is 50-3000.
The control solution of polysorbate 80 was prepared according to the above method and sequentially subjected to sample injection detection, and 6-needle parallel experiments were performed to investigate the reproducibility of the method. The chromatogram is recorded, and chromatographic peaks which are continuously stabilized to form peaks and have relative peak areas larger than 5% are taken as main chromatographic peaks, wherein the RSD of the retention time is smaller than 0.3%, and the RSD of the relative peak areas is mostly smaller than 10%. The result shows that the method has good repeatability and can meet the index requirement of fingerprint establishment.
Example 2
Establishment and analysis of polysorbate fingerprint
And respectively carrying out infrared spectrum tests on the polysorbate 20, the polysorbate 60, the polysorbate 80 and the respective reference substances before establishing the fingerprint spectrum, and obtaining infrared spectrograms which are basically consistent with the reference substances by the samples.
Preparing a polysorbate 80 sample solution and a reference substance solution respectively according to the method of example 1, sequentially injecting samples for detection, integrating all peaks of all injected base peak chromatograms (Base Peak Chromatogram, BPC), introducing all obtained UPCC-MS base peak chromatograms and integrated data into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (version number: 2012.130723), using the reference substance map as a correction reference, automatically matching peaks by using a median, and completing multipoint correction to generate a UPCC-QTOF-MS fingerprint of polysorbate 80.
12 batches of polysorbate 80 from different sources and their USP controls were analyzed by the established UPCC-QTOF-MS method to determine 22 common peaks, the chromatograms and common patterns are shown in FIG. 5. The similarity of fingerprints is evaluated on 12 batches of samples from different sources by adopting an included angle cosine method, and the results are shown in table 2, wherein the closer the similarity is 1, the more similar the fingerprints are, and the closer the overall chemical composition distribution is.
TABLE 2 similarity analysis of Polysorbate 80
| R | S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | S9 | S10 | S11 | S12 | |
| R | 1 | 0.955 | 0.906 | 0.963 | 0.957 | 0.955 | 0.926 | 0.944 | 0.948 | 0.884 | 0.966 | 0.925 | 0.963 |
| S1 | 0.955 | 1 | 0.789 | 0.992 | 0.980 | 0.944 | 0.903 | 0.925 | 0.921 | 0.758 | 0.995 | 0.886 | 0.983 |
| S2 | 0.906 | 0.789 | 1 | 0.794 | 0.805 | 0.900 | 0.904 | 0.812 | 0.833 | 0.990 | 0.806 | 0.880 | 0.854 |
| S3 | 0.963 | 0.992 | 0.794 | 1 | 0.982 | 0.942 | 0.900 | 0.948 | 0.947 | 0.770 | 0.996 | 0.876 | 0.969 |
| S4 | 0.957 | 0.980 | 0.805 | 0.982 | 1 | 0.931 | 0.883 | 0.958 | 0.960 | 0.785 | 0.990 | 0.915 | 0.960 |
| S5 | 0.955 | 0.944 | 0.900 | 0.942 | 0.931 | 1 | 0.983 | 0.932 | 0.935 | 0.884 | 0.948 | 0.897 | 0.976 |
| S6 | 0.926 | 0.903 | 0.904 | 0.900 | 0.883 | 0.983 | 1 | 0.908 | 0.897 | 0.873 | 0.901 | 0.902 | 0.956 |
| S7 | 0.944 | 0.925 | 0.812 | 0.948 | 0.958 | 0.932 | 0.908 | 1 | 0.993 | 0.793 | 0.945 | 0.910 | 0.928 |
| S8 | 0.948 | 0.921 | 0.833 | 0.947 | 0.960 | 0.935 | 0.897 | 0.993 | 1 | 0.826 | 0.946 | 0.893 | 0.920 |
| S9 | 0.884 | 0.758 | 0.990 | 0.770 | 0.785 | 0.884 | 0.873 | 0.793 | 0.826 | 1 | 0.783 | 0.831 | 0.818 |
| S10 | 0.966 | 0.995 | 0.806 | 0.996 | 0.990 | 0.948 | 0.901 | 0.945 | 0.946 | 0.783 | 1 | 0.894 | 0.977 |
| S11 | 0.925 | 0.886 | 0.880 | 0.876 | 0.915 | 0.897 | 0.902 | 0.910 | 0.893 | 0.831 | 0.894 | 1 | 0.924 |
| S12 | 0.963 | 0.983 | 0.854 | 0.969 | 0.960 | 0.976 | 0.956 | 0.928 | 0.920 | 0.818 | 0.977 | 0.924 | 1 |
The results show that the similarity between 12 samples and the control map is 0.884-0.966, and the similarity degree in composition distribution among products can be effectively distinguished by adopting the method of the invention.
The sample injection analysis is carried out on the polysorbate 20 and the polysorbate 60 by adopting the same experimental method as the polysorbate 80, the similarity of the polysorbate 20, the polysorbate 60 and the polysorbate 80 is respectively calculated by adopting an included angle cosine method according to the measurement result by taking a comparison fingerprint of the polysorbate 80 as a reference, and the results are respectively 0.408 and 0.450, which shows that the UPCC-MS fingerprint established by the method can distinguish different varieties of polysorbate, as shown in fig. 6.
The results obtained with the method of the invention allow the establishment of a theoretical mass spectrum database of polysorbate 80 for the identification of the composition of polysorbate 80 on the market.
Claims (2)
1. The analysis and identification method of the polysorbate auxiliary materials is characterized in that the polysorbate auxiliary materials are selected from polysorbate 20, polysorbate 60 and polysorbate 80;
the method comprises the following steps:
step 1, taking a test sample, adding methanol for dissolution, and preparing a solution with the concentration of 0.2mg/mL as a test sample solution;
step 2, detecting the sample solution by adopting the combination of supercritical fluid chromatography and mass spectrometry to obtain a determination result;
the supercritical fluid chromatography conditions were as follows:
ACQUITY UPC 2 torus Diol chromatographic column, specification is: 3.0mm×100mm,1.7 μm,130A
Column temperature: 50. DEG C
Flow rate: 1.0mL/min
Sample injection volume: 1. mu L (mu L)
Back pressure: 2500psi of
Mobile phase: a is CO 2 B is methanol and acetonitrile according to the volume ratio of 30:70, and the gradient elution procedure is as follows: 0 to 0.2min,8 percent of B;0.2 to 12.0min,8 to 15 percent of B;12.0 to 18.0min,15 to 20 percent of B;18.0 to 31.0min,20 to 35 percent of B;31.0 to 34.0min,35 to 50 percent of B;34.0 to 34.2min,50 to 8 percent of B; 34.2-37.0 min,8% B; the ionizing solvent is a mixed solution of methanol and 5mmol/L ammonium formate, and the flow rate is 0.2mL/min;
the mass spectrometry conditions were as follows:
ionization mode: ESI (electronic service provider interface) + The method comprises the steps of carrying out a first treatment on the surface of the Atomization gas temperature: 500. the temperature is lower than the temperature; atomizing gas flow rate: 800L/h; source temperature: 120. the temperature is lower than the temperature; capillary voltage: 2.5KV; taper hole voltage: 40V, V; acquisition mode: MS (MS) E The method comprises the steps of carrying out a first treatment on the surface of the Scanning range: m/z is 50-3000;
step 3, comparing the measurement result with the established polysorbate 80 fingerprint to realize analysis and identification of polysorbate auxiliary materials;
the analysis and identification of the polysorbate auxiliary materials are selected from the following (a) or (b):
(a) Comparing the measurement result with the established polysorbate 80 fingerprint, evaluating the similarity of the fingerprint of the measurement result by adopting an included angle cosine method, and distinguishing the polysorbate 20, the polysorbate 60 and the polysorbate 80 according to the similarity result;
(b) Comparing the measurement result with the established polysorbate 80 fingerprint, evaluating the similarity of the fingerprint of the measurement result by adopting an included angle cosine method, and realizing the quality control of the polysorbate 80 according to the similarity result.
2. The analytical identification method of claim 1 wherein: the method for establishing the polysorbate 80 fingerprint comprises the following steps:
respectively preparing polysorbate 80 sample solution and reference substance solution, sequentially carrying out sample injection detection by adopting supercritical fluid chromatography and mass spectrometry, integrating each peak of all the sample injection base peak chromatograms, introducing the obtained base peak chromatograms and integrated data into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, taking the reference substance spectrum as a correction reference, automatically matching the peaks by using a median, and completing multi-point correction to obtain the polysorbate 80 fingerprint.
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