CN113075342B - Method for separating and detecting related substances of insulin diglucoside side chain - Google Patents
Method for separating and detecting related substances of insulin diglucoside side chain Download PDFInfo
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- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 title claims abstract description 94
- 229940125396 insulin Drugs 0.000 title claims abstract description 47
- 102000004877 Insulin Human genes 0.000 title claims abstract description 46
- 108090001061 Insulin Proteins 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 17
- 239000000126 substance Substances 0.000 title claims abstract description 17
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 93
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical group OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 claims abstract description 60
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims abstract description 40
- 239000007864 aqueous solution Substances 0.000 claims abstract description 29
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 6
- 150000001335 aliphatic alkanes Chemical class 0.000 claims abstract description 6
- 239000000741 silica gel Substances 0.000 claims abstract description 6
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 6
- 239000000945 filler Substances 0.000 claims abstract description 3
- 239000012535 impurity Substances 0.000 claims description 103
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 22
- 238000000926 separation method Methods 0.000 claims description 20
- 238000001514 detection method Methods 0.000 claims description 16
- -1 (2, 5-dioxopyrrolidin-1-yl) oxy Chemical group 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 11
- 239000002253 acid Substances 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 9
- 150000007524 organic acids Chemical class 0.000 claims description 8
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 6
- RTBFRGCFXZNCOE-UHFFFAOYSA-N 1-methylsulfonylpiperidin-4-one Chemical compound CS(=O)(=O)N1CCC(=O)CC1 RTBFRGCFXZNCOE-UHFFFAOYSA-N 0.000 claims description 2
- JFCQEDHGNNZCLN-UHFFFAOYSA-N anhydrous glutaric acid Natural products OC(=O)CCCC(O)=O JFCQEDHGNNZCLN-UHFFFAOYSA-N 0.000 claims description 2
- 238000004811 liquid chromatography Methods 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 238000004086 separation measurement method Methods 0.000 claims 1
- 239000000523 sample Substances 0.000 description 39
- 239000003085 diluting agent Substances 0.000 description 33
- 238000012360 testing method Methods 0.000 description 21
- 239000012488 sample solution Substances 0.000 description 16
- 238000002347 injection Methods 0.000 description 11
- 239000007924 injection Substances 0.000 description 11
- 238000003556 assay Methods 0.000 description 10
- 238000004128 high performance liquid chromatography Methods 0.000 description 10
- 230000014759 maintenance of location Effects 0.000 description 9
- 239000012490 blank solution Substances 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 239000011260 aqueous acid Substances 0.000 description 5
- 238000002372 labelling Methods 0.000 description 5
- 241000700124 Octodon degus Species 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- FYZPCMFQCNBYCY-WIWKJPBBSA-N Insulin degludec Chemical compound CC[C@H](C)[C@H](NC(=O)CN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H]1CSSC[C@@H]2NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CSSC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc3c[nH]cn3)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)Cc3ccccc3)C(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](Cc3c[nH]cn3)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](Cc3ccc(O)cc3)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](Cc3ccc(O)cc3)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](Cc3ccc(O)cc3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC2=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](Cc2ccccc2)C(=O)N[C@@H](Cc2ccccc2)C(=O)N[C@@H](Cc2ccc(O)cc2)C(=O)N[C@@H]([C@@H](C)O)C(=O)N2CCC[C@H]2C(=O)N[C@@H](CCCCNC(=O)CC[C@H](NC(=O)CCCCCCCCCCCCCCC(O)=O)C(O)=O)C(O)=O)NC1=O)[C@@H](C)O)[C@@H](C)CC FYZPCMFQCNBYCY-WIWKJPBBSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- YNPNZTXNASCQKK-UHFFFAOYSA-N phenanthrene Chemical compound C1=CC=C2C3=CC=CC=C3C=CC2=C1 YNPNZTXNASCQKK-UHFFFAOYSA-N 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 description 1
- AAEQXEDPVFIFDK-UHFFFAOYSA-N 3-(4-fluorobenzoyl)-2-(2-methylpropanoyl)-n,3-diphenyloxirane-2-carboxamide Chemical compound C=1C=CC=CC=1NC(=O)C1(C(=O)C(C)C)OC1(C=1C=CC=CC=1)C(=O)C1=CC=C(F)C=C1 AAEQXEDPVFIFDK-UHFFFAOYSA-N 0.000 description 1
- 101000610640 Homo sapiens U4/U6 small nuclear ribonucleoprotein Prp3 Proteins 0.000 description 1
- 101001110823 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 60S ribosomal protein L6-A Proteins 0.000 description 1
- 101000712176 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 60S ribosomal protein L6-B Proteins 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 102100040374 U4/U6 small nuclear ribonucleoprotein Prp3 Human genes 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009748 deglutition Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000003821 enantio-separation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229940026454 tresiba Drugs 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 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
- G01N30/60—Construction of the column
- G01N30/6052—Construction of the column body
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention relates to a method for separating and detecting related substances of a insulin diglucoside side chain, belonging to the field of analytical chemistry. In the method, a chromatographic column with polar inlaid C18 alkane silica gel as a filler is characterized in that a mobile phase is divided into an A phase and a B phase, wherein the A phase is perchloric acid aqueous solution or sulfuric acid aqueous solution, and the B phase is acetonitrile. The method is simple, quick and accurate.
Description
Technical Field
The invention relates to the field of analytical chemistry, in particular to a method for separating and detecting related substances of insulin diglucoside side chains.
Background
Insulin deglutition (Insulin degluec) was developed by novo nordisk corporation, marketed in japan 10 in 2012, and approved for the treatment of type 1 and type 2 diabetes. 25 months of 2015, the FDA approved a new Insulin deluge (instrument degluec) injection from Denmark and Norde, commercially available under the Tresiba name. Degu-insulin is a new generation of basic insulin analogues, and forms a polyhexamer after subcutaneous injection, thereby exerting 24h ultra-long-acting effect. Degluinsulin has CAS number 844439-96-9, molecular weight 6103.97204, and molecular formula C 274 H 411 N 65 O 81 S 6 。
Insulin deltoid side chain, CAS number: 943586-12-7, which is an important starting material in the process of synthesizing insulin digluconate, and the introduced impurities have important influence on the quality of insulin digluconate, so that strict quality control is required.
At present, related substance detection methods of Degu insulin side chains are not collected in U.S. pharmacopoeia USP, european pharmacopoeia EP, japanese pharmacopoeia JP and Chinese pharmacopoeia Ch.P, and in order to better and more accurately control related substances in products and ensure the quality of raw materials, the invention provides an analysis method suitable for the determination of related substances of Degu insulin side chains. The method can effectively separate the De-valley insulin side chain from other impurities, has good specificity, high sensitivity and reliable accuracy, and can be used for quality control of the De-valley insulin.
Disclosure of Invention
The invention aims to provide a method for separating and measuring substances related to a insulin diglucoside side chain by using a chiral chromatographic column, so as to realize separation and measurement of substances related to the insulin diglucoside side chain.
A method for separating and measuring a substance related to a insulin-binding side chain by liquid chromatography, comprising: the chromatographic column with polar inlaid C18 alkane silica gel stuffing has mobile phase comprising A phase of acid water solution and B phase of acetonitrile.
In some embodiments, the chromatographic column may be selected from BEH Shield RP18,4.6X100mm,2.5 μm, or polar-mosaic C18 alkane silica gel. In some embodiments, the chiral chromatography column is BEH Shield RP18,4.6x100mm,2.5 μm; the vendor is the phenanthrene door.
In some embodiments, the aqueous acid solution described in phase a comprises aqueous perchloric acid, aqueous sulfuric acid, or a combination thereof. In certain embodiments the aqueous acid solution described in phase a is an aqueous perchloric acid solution.
In some embodiments, the volume ratio of acid to water in the aqueous acid solution in phase a is 1:1000 to 50:1000. In some embodiments, the volume ratio of acid to water in the aqueous acid solution in phase a is 1:1000 to 3:1000. in certain embodiments the volume ratio of acid to water in the aqueous acid solution is 3:1000.
in some embodiments, the separation assay of the present invention can be implemented as follows:
1) Taking a proper amount of insulin diglucoside side chain or a sample containing the insulin diglucoside side chain, and dissolving the sample with a diluent or a blank solvent;
2) Setting instrument parameters: flow rate of mobile phase, detection wavelength, column box temperature of chromatographic column;
3) Taking a certain amount of the solution in the step 1), and injecting the solution into a high performance liquid chromatograph to finish the separation and determination of related substances of the insulin diglucoside side chain.
The insulin deltoid side chain described in step 1) may be of any purity.
The insulin deltoid side chain described in step 1) may be of any optical purity.
The diluent or the blank solvent in the step 1) is a mixed solution of acetonitrile and organic acid. The organic acid is selected from one or more of trifluoroacetic acid and acetic acid or a combination thereof. In some embodiments, the organic acid is trifluoroacetic acid.
In the diluent in the step 1), the volume ratio (V/V) of acetonitrile to organic acid is 40:60 to 90:10. In some embodiments, the volume ratio (V/V) of acetonitrile to organic acid in the diluent is 50:50.
The diluent contains 1 mg-5 mg of Degu insulin side chain sample per 1ml of diluent. In some embodiments, the diluent of step 1) contains 4mg of insulin diglucoside side chain sample per 1ml of diluent.
The flow rate of the mobile phase is 0.8 ml/min-1.2 ml/min. In some embodiments, the flow rate of the mobile phase is 0.8ml/min; in some embodiments, the flow rate of the mobile phase is 1.0ml/min.
The detection wavelength is 205nm to 215nm. In some embodiments, the detection wavelength is 205nm, facilitating detection.
The temperature of the chromatographic column box is 15-20 ℃. In some embodiments, the column box temperature is 15 ℃; in some embodiments, the column box temperature is 18 ℃; in some embodiments, the column box temperature is 20 ℃.
The sample injection amount of the sample solution is 1-10 mu l. In some embodiments, the sample solution is introduced in an amount of 2 μl. In some embodiments, the sample solution is introduced at 5 μl.
In some embodiments, the separation assay methods of the present invention can be implemented as follows:
1) Taking a proper amount of insulin diglucoside side chain or a sample containing insulin diglucoside side chain, dissolving the sample with a diluent or a blank solvent, and preparing 1-5 mg of sample solution containing insulin diglucoside side chain per 1 ml;
2) Setting the flow rate of the mobile phase to be 0.8 ml/min-1.2 ml/min, the detection wavelength to be 205nm-215 nm, and the temperature of a chromatographic column box to be 15-20 ℃;
3) Taking 1-10 mu l of the sample solution in the step 1), and injecting into a high performance liquid chromatograph to finish the separation and measurement of substances related to the insulin diglucoside side chain.
In the method provided by the invention, the high performance liquid chromatograph can be an Agilent 1200 type high performance liquid chromatograph system and a workstation in the United states.
In some embodiments, the separation assay described herein employs a chromatographic column of BEH Shield RP18,4.6X100mm,2.5 μm, phase A in the mobile phase being aqueous perchloric acid and phase B acetonitrile; the volume ratio of perchloric acid to water in the perchloric acid aqueous solution in the phase A is 1:1000 to 5:1000.
In some embodiments, the separation assay described herein employs a chromatographic column of BEH Shield RP18,4.6X100mm,2.5 μm, phase A in the mobile phase being aqueous perchloric acid and phase B acetonitrile; the volume ratio of perchloric acid to water in the perchloric acid aqueous solution in the phase A is 1:1000 to 3:1000.
In some embodiments, the separation assay described herein employs a chromatographic column of BEH Shield RP18,4.6X100mm,2.5 μm, phase A in the mobile phase being aqueous perchloric acid and phase B acetonitrile; the volume ratio of perchloric acid to water in the perchloric acid aqueous solution in the phase A is 1:1000 to 3:1000.
In some embodiments, the separation assay described herein employs a chromatographic column of BEH Shield RP18,4.6X100mm,2.5 μm, phase A in the mobile phase being aqueous perchloric acid and phase B acetonitrile; the volume ratio of perchloric acid to water in the perchloric acid aqueous solution in the phase A is 3:1000.
In some embodiments, the separation assay method of the present invention employs a chromatographic column of BEH Shield RP18,4.6X100mm,2.5 μm, phase A of the mobile phase being aqueous sulfuric acid and phase B of acetonitrile; the volume ratio of sulfuric acid to water in the sulfuric acid aqueous solution in the phase A is 10:990 to 100:900.
In some embodiments, the separation assay method of the present invention employs a chromatographic column of BEH Shield RP18,4.6X100mm,2.5 μm, phase A of the mobile phase being aqueous sulfuric acid and phase B of acetonitrile; the volume ratio of sulfuric acid to water in the sulfuric acid aqueous solution in the phase A is 10:990 to 50:950.
In some embodiments, the separation assay described herein employs a chromatographic column of BEH Shield RP18,4.6X100mm,2.5 μm, phase A in the mobile phase being aqueous sulfuric acid and phase B acetonitrile; the volume ratio of sulfuric acid to water in the sulfuric acid aqueous solution in the phase A is 50:950 to 100:900.
In some embodiments, the separation assay described herein employs a chromatographic column of BEH Shield RP18,4.6X100mm,2.5 μm, phase A in the mobile phase being aqueous sulfuric acid and phase B acetonitrile; the volume ratio of sulfuric acid to water in the sulfuric acid aqueous solution in the phase A is 50:950.
The separation method provided by the invention has the advantage that the time for separating and detecting related substances of the insulin diglucoside side chain is less than 40 minutes.
In the context of the foregoing or following, all numbers disclosed herein are approximations, whether or not the word "about" or "about" is used by the word "about". The numerical value of each number may vary by 1%, 2%, 5%, 7%, 8%, or 10%.
The invention adopts a chromatographic column with polar inlaid C18 alkane silica gel as a filler, takes perchloric acid aqueous solution or sulfuric acid aqueous solution as an A phase and acetonitrile as a mobile phase system of a B phase, can effectively separate the insulin deluge side chain from other impurities, has the separation degree of more than 1.5 or more than 2.5 or more than 5, and completely separates the base line.
Drawings
FIG. 1 is a high performance liquid chromatogram of a labeling sample of a test sample of insulin diglucoside side chains in example 1;
FIG. 2 is a high performance liquid chromatogram of a labeling sample of a test sample of insulin diglucoside side chains in example 2;
FIG. 3 is a high performance liquid chromatogram of a labeling sample of the insulin diglucoside side chain test sample of example 3;
FIG. 4 is a high performance liquid chromatogram of a labeling sample of the insulin diglucoside side chain test sample of example 4;
FIG. 5 is a high performance liquid chromatogram of a labeling sample of the insulin diglucoside side chain test sample of example 5;
FIG. 6 is a high performance liquid chromatogram of a test solution of insulin diglucoside side chains in example 3;
FIG. 7 is a high performance liquid chromatogram of a test solution of insulin diglucoside side chains in example 7;
FIG. 8 high performance liquid chromatography of the insulin diglucoside side chain test solution of example 8;
in the figure, the abscissa indicates retention time, minutes (min); the ordinate represents the electrical signal, mAU.
In the present invention, impurity 1 represents: (S) -12- ((1-carboxy-4- ((2, 5-dioxopyrrolidin-1-yl) oxy) -4-oxybutyl) amino) -12-oxodecanoic acid;
in the present invention, impurity 2 represents: n, N' -dicyclohexyl-carbonyl diamine;
in the present invention, impurity 3 represents: (S) -14- ((1-carboxy-4- ((2, 5-dioxopyrrolidin-1-yl) oxy) -4-oxybutyl) amino) -14-oxododecanoic acid;
in the present invention, impurity 4 represents: (2 s,2 'r) -2,2' - (hexadecyldiazadienyl) bis (5- ((2, 5-dioxapyrrolidin-1-yl) oxy) -5-oxopentanoic acid);
in the present invention, impurity 5 represents: (S) -2- (15-carboxypentadecyl) glutaric acid;
in the present invention, impurity 6 represents: (S) -2- (16- ((2, 5-dioxopyrrolidin-1-yl) oxy) -16-oxohexadecylamido) glutaric acid;
in the present invention, impurity 7 represents: (R) -1- (15-carboxypentadecyl) -5-oxopyrrolidine-2-carboxylic acid;
in the present invention, impurity 8 represents: (S) -20- ((1-carboxy-4- ((2, 5-dioxopyrrolidin-1-yl) oxy) -4-oxybutyl) amino) -20-oxooctadecanoic acid.
Detailed Description
The embodiment of the invention discloses a method for separating and detecting related substances of a insulin diglucoside side chain. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the method of the present invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the method described herein without departing from the spirit and scope of the invention.
The present invention will be described in detail with reference to examples.
Specification of instrument and chromatographic column: an Agilent 1200 high performance liquid chromatography system and workstation; automatic sample injection;
| chromatographic column | Specification of specification | Packing material |
| BEH Shield RP18 | 4.6x100mm，2.5μm | Polar inlaid C18 alkane silica gel |
Example 1
Instrument and conditions
Chromatographic column: BEH Shield RP18,4.6X100mm,2.5 μm
A detector: DAD (ultraviolet detector), detection wavelength 205nm;
flow rate: 1.0mL/min;
column temperature: 18 ℃;
sample injection amount: 2. Mu.L;
mobile phase a:0.3% aqueous perchloric acid;
mobile phase B: acetonitrile gradient elution:
| time (min) | Phase A (%) | Phase B (%) |
| 0 | 90 | 10 |
| 3 | 65 | 35 |
| 6 | 61 | 39 |
| 12 | 52 | 48 |
| 24 | 47 | 53 |
| 26 | 40 | 60 |
| 31 | 5 | 95 |
| 35 | 5 | 95 |
| 35.1 | 90 | 10 |
| 40 | 90 | 10 |
Run time: for 40min;
diluent/blank solution: acetonitrile: trifluoroacetic acid=50:50 (V: V);
experimental procedure
About 200mg of the insulin-in-Del side chain test sample is weighed into a 50mL brown volumetric flask, dissolved by using a diluent in an ultrasonic manner, and a proper amount (about 10mg each) of impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7 and impurity 8 are taken and added into the volumetric flask, diluted to a scale by using the diluent, and shaken uniformly to obtain a test standard sample solution.
Taking a sample to be tested and adding a standard, performing high performance liquid chromatography analysis according to the conditions, and recording a chromatogram, wherein the result is shown in figure 1.
As can be seen from fig. 1, the chromatographic peak with retention time of 15.55 minutes is that of GJD05, and the retention times corresponding to impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, and impurity 8 are respectively: 7.90min, 10.91min, 11.37min, 11.94min, 12.89min, 14.77min, 21.99min, 28.93min. The separation of the main peak from the adjacent peaks was 4.4.
Example 2
Instrument and conditions
Chromatographic column: BEH Shield RP18,4.6X100mm,2.5 μm
A detector: DAD (ultraviolet detector), detection wavelength 215nm;
flow rate: 1.0mL/min;
column temperature: 18 ℃;
sample injection amount: 2. Mu.L;
mobile phase a:0.3% perchloric acid aqueous solution
Mobile phase B: acetonitrile gradient elution:
| time (min) | Phase A (%) | Phase B (%) |
| 0 | 90 | 10 |
| 3 | 65 | 35 |
| 6 | 61 | 39 |
| 12 | 52 | 48 |
| 24 | 47 | 53 |
| 26 | 40 | 60 |
| 31 | 5 | 95 |
| 35 | 5 | 95 |
| 35.1 | 90 | 10 |
| 40 | 90 | 10 |
Run time: for 40min;
diluent/blank solution: acetonitrile: trifluoroacetic acid=50:50 (V: V);
experimental procedure
About 200mg of the insulin-in-Del side chain test sample is weighed into a 25mL brown volumetric flask, dissolved by using a diluent in an ultrasonic manner, and a proper amount (about 10mg each) of impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7 and impurity 8 are added into the volumetric flask, diluted to a scale by using the diluent, and shaken uniformly to obtain a test standard sample solution.
Taking a sample to be tested and adding a label, performing high performance liquid chromatography analysis according to the conditions, and recording a chromatogram, wherein the result is shown in figure 2.
As can be seen from fig. 2, when the wavelength is changed to 215nm, the absorption of each impurity and main peak is much weaker than that of 205 nm. Indicating that the individual impurities and main peaks should respond better at 205 nm.
Example 3
Instrument and conditions
Chromatographic column: BEH Shield RP18,4.6X100mm,2.5 μm
A detector: DAD (ultraviolet detector), detection wavelength 205nm;
flow rate: 1.0mL/min;
column temperature: 18 ℃;
sample injection amount: 2. Mu.L;
mobile phase a:0.5% perchloric acid aqueous solution
Mobile phase B: acetonitrile gradient elution:
| time (min) | Phase A (%) | Phase B (%) |
| 0 | 90 | 10 |
| 3 | 65 | 35 |
| 6 | 61 | 39 |
| 12 | 52 | 48 |
| 24 | 47 | 53 |
| 26 | 40 | 60 |
| 31 | 5 | 95 |
| 35 | 5 | 95 |
| 35.1 | 90 | 10 |
| 40 | 90 | 10 |
Run time: for 40min;
diluent/blank solution: acetonitrile: trifluoroacetic acid=50:50 (V: V);
experimental procedure
About 200mg of the insulin-in-Del side chain test sample is weighed into a 25mL brown volumetric flask, dissolved by using a diluent in an ultrasonic manner, and a proper amount (about 10mg each) of impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7 and impurity 8 are added into the volumetric flask, diluted to a scale by using the diluent, and shaken uniformly to obtain a test standard sample solution.
Taking a sample to be tested and adding a label, performing high performance liquid chromatography analysis according to the conditions, and recording a chromatogram, wherein the result is shown in figure 3.
As can be seen from fig. 3, when the concentration is changed from 0.3% perchloric acid aqueous solution to 0.5% perchloric acid aqueous solution, the chromatographic peak with retention time of 15.74 minutes is that of GJD05, and the retention times corresponding to impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, and impurity 8 are respectively: 7.96min, 10.99min, 11.49min, 12.11min, 13.05min, 14.94min, 22.35min, 29.09 min. The separation of the main peak from the adjacent peaks was 4.4.
Example 4
Instrument and conditions
Chromatographic column: BEH Shield RP18,4.6X100mm,2.5 μm
A detector: DAD (ultraviolet detector), detection wavelength 205nm;
flow rate: 1.0mL/min;
column temperature: 18 ℃;
sample injection amount: 5. Mu.L;
mobile phase a:0.1% perchloric acid aqueous solution
Mobile phase B: acetonitrile gradient elution:
| time (min) | Phase A (%) | Phase B (%) |
| 0 | 90 | 10 |
| 3 | 65 | 35 |
| 6 | 61 | 39 |
| 12 | 52 | 48 |
| 24 | 47 | 53 |
| 26 | 40 | 60 |
| 31 | 5 | 95 |
| 35 | 5 | 95 |
| 35.1 | 90 | 10 |
| 40 | 90 | 10 |
Run time: for 40min;
diluent/blank solution: acetonitrile: trifluoroacetic acid=50:50 (V: V);
experimental procedure
About 200mg of the insulin-in-Del side chain test sample is weighed into a 25mL brown volumetric flask, dissolved by using a diluent in an ultrasonic manner, and a proper amount (about 10mg each) of impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7 and impurity 8 are added into the volumetric flask, diluted to a scale by using the diluent, and shaken uniformly to obtain a test standard sample solution.
Taking a sample to be tested and adding a label, performing high performance liquid chromatography analysis according to the conditions, and recording a chromatogram, wherein the result is shown in figure 4.
As can be seen from fig. 4, when the concentration is changed from 0.3% perchloric acid aqueous solution to 0.1% perchloric acid aqueous solution, the chromatographic peak with retention time of 15.69 minutes is that of GJD05, and the retention times corresponding to impurity B, impurity K, impurity I, impurity L, impurity D, impurity C, impurity D02, impurity E, impurity F02, impurity N, impurity F01 and impurity G are respectively: 8.00min, 11.03min, 11.50min, 12.13min, 12.97min, 14.91min, 22.17min, 28.99min. The separation of the main peak from the adjacent peaks was 4.3.
Example 5
Instrument and conditions
Chromatographic column: BEH Shield RP18,4.6X100mm,2.5 μm
A detector: DAD (ultraviolet detector), detection wavelength 205nm;
flow rate: 1.0mL/min;
column temperature: 18 ℃;
sample injection amount: 2. Mu.L;
mobile phase a: (H) 2 SO 4 Water=50:950
Mobile phase B: acetonitrile gradient elution:
| time (min) | Phase A (%) | Phase B (%) |
| 0 | 90 | 10 |
| 3 | 65 | 35 |
| 6 | 61 | 39 |
| 12 | 52 | 48 |
| 24 | 47 | 53 |
| 26 | 40 | 60 |
| 31 | 5 | 95 |
| 35 | 5 | 95 |
| 35.1 | 90 | 10 |
| 40 | 90 | 10 |
Run time: for 40min;
diluent/blank solution: acetonitrile: trifluoroacetic acid=50:50 (V: V);
experimental procedure
About 200mg of the insulin-in-Del side chain test sample is weighed into a 25mL brown volumetric flask, dissolved by using a diluent in an ultrasonic manner, and a proper amount (about 10mg each) of impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7 and impurity 8 are added into the volumetric flask, diluted to a scale by using the diluent, and shaken uniformly to obtain a test standard sample solution.
Taking a sample to be tested and adding a label, performing high performance liquid chromatography analysis according to the conditions, and recording a chromatogram, wherein the result is shown in figure 5.
As can be seen from FIG. 5, when the aqueous solution of perchloric acid was changed from 0.3% to (1M H) 2 SO 4 : ultrapure water=50:950 (v: v)), the chromatographic peak with a retention time of 15.61 minutes is the chromatographic peak of GJD05, and the retention times corresponding to impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 are respectively: 8.00min, 10.91min, 11.48min, 12.13min, 12.94min, 14.87min, 21.99min, 28.89min. The separation of the main peak from the adjacent peaks was 4.2.
Example 6
Instrument and conditions
Chromatographic column: BEH Shield RP18,4.6X100mm,2.5 μm
A detector: DAD (ultraviolet detector), detection wavelength 205nm;
flow rate: 1.0mL/min;
column temperature: 18 ℃;
sample injection amount: 8. Mu.L;
mobile phase a:0.3% perchloric acid aqueous solution
Mobile phase B: acetonitrile gradient elution:
| time (min) | Phase A (%) | Phase B (%) |
| 0 | 90 | 10 |
| 3 | 65 | 35 |
| 6 | 61 | 39 |
| 12 | 52 | 48 |
| 24 | 47 | 53 |
| 26 | 40 | 60 |
| 31 | 5 | 95 |
| 35 | 5 | 95 |
| 35.1 | 90 | 10 |
| 40 | 90 | 10 |
Run time: for 40min;
diluent/blank solution: acetonitrile: trifluoroacetic acid=50:50 (V: V);
experimental procedure
About 10mg of the insulin diglucoside side chain test sample is taken and weighed into a 10mL brown volumetric flask, dissolved by the diluent in an ultrasonic manner, diluted to a scale by the diluent and shaken uniformly to obtain a test sample solution.
Taking the sample solution, performing high performance liquid chromatography under the above conditions, and recording the chromatogram, wherein the result is shown in FIG. 6.
Example 7
Instrument and conditions
Chromatographic column: BEH Shield RP18,4.6X100mm,2.5 μm
A detector: DAD (ultraviolet detector), detection wavelength 205nm;
flow rate: 1.0mL/min;
column temperature: 18 ℃;
sample injection amount: 2. Mu.L;
mobile phase a:0.3% perchloric acid aqueous solution
Mobile phase B: acetonitrile gradient elution:
run time: for 40min;
diluent/blank solution: acetone: trifluoroacetic acid=70:30 (V: V);
experimental procedure
About 40mg of the insulin diglucoside side chain test sample is taken and weighed into a 10mL brown volumetric flask, dissolved by the diluent in an ultrasonic manner, diluted to a scale by the diluent and shaken uniformly to obtain a test sample solution.
Taking the sample solution, performing high performance liquid chromatography under the above conditions, and recording the chromatogram, wherein the result is shown in FIG. 7.
Example 8
Instrument and conditions
Chromatographic column: BEH Shield RP18,4.6X100mm,2.5 μm
A detector: DAD (ultraviolet detector), detection wavelength 205nm;
flow rate: 1.0mL/min;
column temperature: 18 ℃;
sample injection amount: 2. Mu.L;
mobile phase a:0.3% perchloric acid aqueous solution
Mobile phase B: acetonitrile gradient elution:
| time (min) | Phase A (%) | Phase B (%) |
| 0 | 90 | 10 |
| 3 | 65 | 35 |
| 6 | 61 | 39 |
| 12 | 52 | 48 |
| 24 | 47 | 53 |
| 26 | 40 | 60 |
| 31 | 5 | 95 |
| 35 | 5 | 95 |
| 35.1 | 90 | 10 |
| 40 | 90 | 10 |
Run time: for 40min;
diluent/blank solution: acetic acid: trifluoroacetic acid=50:50 (V: V);
experimental procedure
About 40mg of the insulin diglucoside side chain test sample is taken and weighed into a 10mL brown volumetric flask, dissolved by the diluent in an ultrasonic manner, diluted to a scale by the diluent and shaken uniformly to obtain a test sample solution.
Taking the sample solution, performing high performance liquid chromatography under the above conditions, and recording the chromatogram, wherein the result is shown in FIG. 8.
Claims (2)
1. A method for separating and measuring related substances of a insulin diglucoside side chain by using a liquid chromatography is characterized in that a chromatographic column with polar inlaid C18 alkane silica gel as a filler is adopted, a mobile phase is divided into a phase A and a phase B, the phase A is an acid aqueous solution, and the phase B is acetonitrile;
wherein, the CAS number of the insulin deltoid side chain is 943586-12-7;
the chromatographic column is BEH Shield RP18,4.6X100mm,2.5 μm;
the acid aqueous solution is perchloric acid aqueous solution or sulfuric acid aqueous solution, wherein the mass concentration of the perchloric acid aqueous solution is 0.3-0.5%, the volume ratio of sulfuric acid to water in the sulfuric acid aqueous solution is 50:950, and the sulfuric acid is 1M sulfuric acid;
the gradient elution of mobile phase a and B phases is:
The method comprises the following steps:
1) Taking a proper amount of sample containing the insulin diglucoside side chain, and dissolving the sample by using a mixed solution of acetonitrile and organic acid;
2) Setting instrument parameters: the flow rate of the mobile phase, the detection wavelength and the temperature of a chromatographic column incubator;
3) Taking a certain amount of the solution in the step 1), and injecting the solution into a high performance liquid chromatograph to finish the separation and determination of related substances of the insulin diglucoside side chain;
wherein the volume ratio of acetonitrile to organic acid is 50:50, and the organic acid is selected from trifluoroacetic acid;
wherein the related substances are impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7 and impurity 8, wherein impurity 1 represents: (S) -12- ((1-carboxy-4- ((2, 5-dioxopyrrolidin-1-yl) oxy) -4-oxybutyl) amino) -12-oxodecanoic acid; impurity 2 represents: n, N' -dicyclohexyl-carbonyl diamine; impurity 3 represents: (S) -14- ((1-carboxy-4- ((2, 5-dioxopyrrolidin-1-yl) oxy) -4-oxybutyl) amino) -14-oxododecanoic acid; impurity 4 represents: (2 s,2 'r) -2,2' - (hexadecyldiazadienyl) bis (5- ((2, 5-dioxapyrrolidin-1-yl) oxy) -5-oxopentanoic acid); impurity 5 represents: (S) -2- (15-carboxypentadecyl) glutaric acid; impurity 6 represents: (S) -2- (16- ((2, 5-dioxopyrrolidin-1-yl) oxy) -16-oxohexadecylamido) glutaric acid; impurity 7 represents: (R) -1- (15-carboxypentadecyl) -5-oxopyrrolidine-2-carboxylic acid; impurity 8 represents: (S) -20- ((1-carboxy-4- ((2, 5-dioxopyrrolidin-1-yl) oxy) -4-oxybutyl) amino) -20-oxooctadecanoic acid.
2. The separation measurement method according to claim 1, wherein the flow rate of the mobile phase is 0.8ml/min to 1.2ml/min; the detection wavelength is 205nm-215m; the temperature of the chromatographic column box is 15-20 ℃.
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