CN114354795A - Detection method and application of related substances in hydroxypropyl betacyclodextrin - Google Patents
Detection method and application of related substances in hydroxypropyl betacyclodextrin Download PDFInfo
- Publication number
- CN114354795A CN114354795A CN202111646543.7A CN202111646543A CN114354795A CN 114354795 A CN114354795 A CN 114354795A CN 202111646543 A CN202111646543 A CN 202111646543A CN 114354795 A CN114354795 A CN 114354795A
- Authority
- CN
- China
- Prior art keywords
- mobile phase
- elution
- related substances
- solution
- betacyclodextrin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 39
- ODLHGICHYURWBS-LKONHMLTSA-N trappsol cyclo Chemical compound CC(O)COC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)COCC(O)C)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1COCC(C)O ODLHGICHYURWBS-LKONHMLTSA-N 0.000 title claims abstract description 36
- 239000000126 substance Substances 0.000 title claims abstract description 25
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims abstract description 91
- 238000000034 method Methods 0.000 claims abstract description 77
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims abstract description 63
- 229960004853 betadex Drugs 0.000 claims abstract description 63
- 229960004063 propylene glycol Drugs 0.000 claims abstract description 56
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 54
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 45
- 238000010828 elution Methods 0.000 claims abstract description 33
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 4
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims abstract description 4
- 239000000741 silica gel Substances 0.000 claims abstract description 4
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 4
- -1 Phenyl Chemical group 0.000 claims description 3
- 239000003643 water by type Substances 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 238000003786 synthesis reaction Methods 0.000 claims description 2
- 229920000858 Cyclodextrin Polymers 0.000 abstract description 61
- 239000001116 FEMA 4028 Substances 0.000 abstract description 60
- 235000011175 beta-cyclodextrine Nutrition 0.000 abstract description 60
- 238000002347 injection Methods 0.000 abstract description 26
- 239000007924 injection Substances 0.000 abstract description 26
- 238000007796 conventional method Methods 0.000 abstract description 3
- 239000000523 sample Substances 0.000 description 48
- 239000000243 solution Substances 0.000 description 33
- 230000014759 maintenance of location Effects 0.000 description 32
- 239000012488 sample solution Substances 0.000 description 20
- DNIAPMSPPWPWGF-GSVOUGTGSA-N (R)-(-)-Propylene glycol Chemical compound C[C@@H](O)CO DNIAPMSPPWPWGF-GSVOUGTGSA-N 0.000 description 19
- 235000013772 propylene glycol Nutrition 0.000 description 19
- 239000012490 blank solution Substances 0.000 description 16
- 239000012088 reference solution Substances 0.000 description 16
- 238000011084 recovery Methods 0.000 description 15
- 238000000926 separation method Methods 0.000 description 14
- 239000012535 impurity Substances 0.000 description 13
- 238000003908 quality control method Methods 0.000 description 13
- 238000012360 testing method Methods 0.000 description 13
- 239000013558 reference substance Substances 0.000 description 8
- 239000011550 stock solution Substances 0.000 description 8
- 239000012085 test solution Substances 0.000 description 8
- 238000005303 weighing Methods 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 5
- 238000007865 diluting Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000010812 external standard method Methods 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- GOOHAUXETOMSMM-UHFFFAOYSA-N Propylene oxide Chemical compound CC1CO1 GOOHAUXETOMSMM-UHFFFAOYSA-N 0.000 description 1
- QJJXYPPXXYFBGM-LFZNUXCKSA-N Tacrolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1\C=C(/C)[C@@H]1[C@H](C)[C@@H](O)CC(=O)[C@H](CC=C)/C=C(C)/C[C@H](C)C[C@H](OC)[C@H]([C@H](C[C@H]2C)OC)O[C@@]2(O)C(=O)C(=O)N2CCCC[C@H]2C(=O)O1 QJJXYPPXXYFBGM-LFZNUXCKSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000003255 drug test Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000011514 reflex Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229960001967 tacrolimus Drugs 0.000 description 1
- QJJXYPPXXYFBGM-SHYZHZOCSA-N tacrolimus Natural products CO[C@H]1C[C@H](CC[C@@H]1O)C=C(C)[C@H]2OC(=O)[C@H]3CCCCN3C(=O)C(=O)[C@@]4(O)O[C@@H]([C@H](C[C@H]4C)OC)[C@@H](C[C@H](C)CC(=C[C@@H](CC=C)C(=O)C[C@H](O)[C@H]2C)C)OC QJJXYPPXXYFBGM-SHYZHZOCSA-N 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Landscapes
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention provides a detection method and application of related substances in hydroxypropyl betacyclodextrin, wherein the related substances comprise 1, 2-propylene glycol and betacyclodextrin, and the detection is carried out by adopting liquid chromatography under the following conditions: phenyl bonded silica gel chromatographic column; mobile phase A: water; mobile phase B: methanol or acetonitrile; elution procedure: eluting with mobile phase A for at least 15 min; switching the mobile phase to reduce the volume fraction of the mobile phase A in the mobile phase A and the mobile phase B from 100% to 0-60%; maintaining the elution of the switched mobile phase for a period of time; switching the mobile phase to increase the volume fraction of the mobile phase A in the mobile phase A and the mobile phase B from 0-60% to 100%; mobile phase a elution was maintained for a period of time. The method has the advantages of high specificity, applicability, accuracy, precision, durability and repeatability, and solves the problem that the related substances in hydroxypropyl betacyclodextrin are difficult to accurately detect under the condition of continuous sample injection in the conventional method.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, and particularly relates to a detection method and application of related substances in hydroxypropyl betacyclodextrin.
Background
Hydroxypropyl betacyclodextrin (i.e., hydroxypropyl-beta-cyclodextrin, hydroxypropyl-p-cyclodextrin for short, HP-B-CD) is a hydroxyalkylated hydrophilic derivative formed by condensing beta-cyclodextrin and propylene oxide, and is a common pharmaceutical adjuvant. Various impurities may be introduced during the synthesis of hydroxypropyl betacyclodextrin, including 1, 2-propanediol and betacyclodextrin. The impurity residue is directly related to the medication safety, so the impurity content of hydroxypropyl betacyclodextrin needs to be strictly controlled.
The chromatography is a common method for detecting 1, 2-propylene glycol and betacyclodextrin in hydroxypropyl betacyclodextrin, and in order to realize accurate quantification of the two substances, the two substances are generally required to be detected under different chromatographic conditions, so that the detection cost is increased, and the detection efficiency is influenced. Therefore, in the four pharmaceutic adjuvants in the '2020 edition of Chinese pharmacopoeia', a method for simultaneously detecting 1, 2-propylene glycol and betacyclodextrin in hydroxypropyl betacyclodextrin is provided, and the chromatographic detection conditions are as follows: precisely weighing about 2.5g of a sample, placing in a 25ml measuring flask, adding 15ml of water with the temperature of 60 ℃, shaking to dissolve, cooling to room temperature, diluting with water to a scale, and shaking up to obtain a sample solution; and precisely weighing 50mg of a betacyclodextrin reference substance and 50mg of a1, 2-propylene glycol reference substance, placing the two reference substances into a 100ml measuring flask, adding water to dissolve and dilute the two reference substances to a scale, and shaking up the two reference substances to obtain a reference substance solution. Measuring by high performance liquid chromatography (general rule 0512), and using phenyl bonded silica gel as filler; water is used as a mobile phase, and the flow rate is 1.5 ml/min; a differential refractive detector; the column temperature was 40 ℃; the temperature of the detector is 40 ℃; the amount of sample was 20. mu.l. According to an external standard method, the content of beta-cyclodextrin is not more than 0.5 percent and the content of 1, 2-propylene glycol is not more than 0.5 percent by peak area quantification; the single impurities except for betacyclodextrin and 1, 2-propanediol should not exceed 0.1% (calculated as 1, 2-propanediol) and the total of the impurities except for betacyclodextrin and 1, 2-propanediol should not exceed 1.0% (calculated as 1, 2-propanediol, only the peak between betacyclodextrin and 1, 2-propanediol).
However, the above analysis method has poor reproducibility and cannot ensure the quantitative accuracy of the sample in the continuous sample injection detection process.
Disclosure of Invention
The present invention is directed to solving at least one of the above problems in the prior art. Therefore, the invention provides a method for detecting related substances in hydroxypropyl betadex, which can realize quantitative detection of 1, 2-propylene glycol and betadex in hydroxypropyl betadex and can be used for detecting unknown impurities between chromatographic peaks of 1, 2-propylene glycol and betadex. The method has the advantages of high specificity, applicability, accuracy, precision, durability and repeatability.
The invention also provides application of the detection method.
The invention provides a method for detecting related substances in hydroxypropyl betacyclodextrin, which adopts liquid chromatography to detect, wherein the related substances comprise 1, 2-propylene glycol and betacyclodextrin, and the conditions of the liquid chromatography are as follows:
a chromatographic column: phenyl bonded silica gel chromatographic column;
mobile phase A: water;
mobile phase B: methanol or acetonitrile;
the elution procedure was as follows:
eluting with the mobile phase A for at least 15 min;
switching the mobile phase to reduce the volume fraction of the mobile phase A in the mobile phase A and the mobile phase B from 100% to 0-60%;
maintaining the elution of the switched mobile phase for a period of time;
switching the mobile phase to enable the volume fraction of the mobile phase A in the mobile phase A and the mobile phase B to be increased from 0-60% to 100%;
the mobile phase a elution was maintained for a period of time.
The method for detecting related substances in hydroxypropyl betadex has at least the following beneficial effects:
the specificity is good: the blank solution has no interference to the chromatographic peak of the substance to be detected in the reference solution and the sample solution.
The system has good applicability: after 10 needles of accurate continuous sample injection, the chromatographic peak types of the 1, 2-propylene glycol and the beta-cyclodextrin of the quality control needle are good, and the peak area and the retention time are stable, while in the existing method, the beta-cyclodextrin chromatographic peak of the quality control needle is combined with other chromatographic peaks, the integral of the chromatographic peak is interfered, and the peak area RSD of the applicability of the quality control needle and a system is 23 percent, so that the applicability requirement of the system is not met.
The accuracy is good: in the process of accurate continuous sample injection of 10 needles, the reproducibility of the retention time of the 1, 2-propylene glycol and the beta-cyclodextrin in the mixed sample solution is good, wherein the stability of the retention time of the beta-cyclodextrin is obviously superior to that of the existing method. For the recovery, the RSD of 1, 2-propanediol at different concentrations was less than 2% at concentrations of 0.05% (limit 10%), 0.50% (limit 100%) and 0.75% (limit 150%). The recovery rate RSD of betacyclodextrin is significantly lower than that of the prior art methods, wherein the recovery rate RSD of betacyclodextrin is lower than 1% at the concentrations of 0.50% (limit 100%) and 0.75% (limit 150%). In the process of accurate continuous sample injection of 10 needles, the separation degrees of 1, 2-propylene glycol and beta-cyclodextrin in the solution of the test sample are both greater than 4.4, while only the first 4 needles of the existing method can meet the requirement that the separation degree is greater than 4.
The precision is good: in the process of accurately and continuously feeding samples into 6 needles, the reproducibility of the retention time of the 1, 2-propylene glycol and the beta-cyclodextrin in the sample solution is good, wherein the difference value between the maximum retention time and the minimum retention time of the beta-cyclodextrin is 0.776min, while the difference value of the maximum retention time and the minimum retention time of the beta-cyclodextrin is 2.43 min. In the aspect of precision repeatability, after 6 needles of accurate continuous sample injection, the actually measured RSD of the 1, 2-propylene glycol content is 0.7%, the actually measured RSD of the betacyclodextrin content is 1.4%, wherein the actually measured RSD of the betacyclodextrin content is obviously superior to the level of 5.1% which can be realized by the prior art. In the process of continuous sample injection of 6 needles with precision, the separation degrees of 1, 2-propylene glycol and beta-cyclodextrin in the solution of the test sample are both greater than 4.5, while only the first 2 needles in the existing method can meet the requirement that the separation degree is greater than 4.
The durability is good: in the process of adding the impurity sample solution and continuously feeding 9 needles, the retention time of 1, 2-propylene glycol and beta-cyclodextrin and the RSD of the peak area are both less than 2 percent. And changing the elution program, wherein when the mobile phase switched by the second section of elution program is changed into the mobile phase A with the volume fraction of 80%, in the process of continuously feeding the sample solution into 9 needles, the retention time of beta-cyclodextrin is reduced along with the increase of the feeding times, the peak area is also reduced along with the increase of the feeding times, the RSD of the retention time is 7.13%, the RSD of the peak area is 2.41%, and the retention time and the RSD of the peak area are both more than 2.0%.
Based on the verification in various aspects such as specificity, system applicability, accuracy, precision, durability and the like, the result shows that the detection method has good applicability and can be used for quantitative detection of 1, 2-propylene glycol and betacyclodextrin in hydroxypropyl betacyclodextrin.
The detection method can accurately detect the 1, 2-propylene glycol and the betacyclodextrin in the hydroxypropyl betacyclodextrin under the condition of continuous sample injection, wherein the repeatability and the accuracy of the detection of the betacyclodextrin are obviously superior to those of the existing method.
The detection method provided by the invention can be used for detecting unknown impurities between chromatographic peaks of the 1, 2-propylene glycol and the beta-cyclodextrin besides the 1, 2-propylene glycol and the beta-cyclodextrin in the hydroxypropyl beta-cyclodextrin.
According to some embodiments of the invention, the maintaining of the switched mobile phase for elution for a period of time is: the mobile phase elution after switching was maintained for at least 5 min.
According to some embodiments of the invention, said maintaining of said mobile phase a elution for a period of time is in particular: the mobile phase a elution was maintained for at least 30min to fully equilibrate the system. Further, the elution time of the mobile phase A is maintained to be 30-80 min.
According to some embodiments of the invention, the detector of the liquid chromatography detection is a refractive index detector. Further, the temperature of the detector was 40 ℃ ± 2 ℃.
According to some embodiments of the invention, the chromatographic column is WATERS
Phenyl group. And optionally, a gauge of 10 μm,
according to some embodiments of the invention, the column temperature of the chromatography column is 40 ℃ ± 2 ℃. Too high a column temperature may affect the separation of 1, 2-propanediol from betacyclodextrin, and too low a column temperature may affect the peak profile of betacyclodextrin.
According to some embodiments of the invention, the total flow rate of the mobile phase during the liquid chromatography detection is constant at 1.5 ± 0.3 ml/min. The flow rate decreases and may affect the peak profile of betacyclodextrin, and too high a flow rate may affect the degree of separation of 1, 2-propanediol and betacyclodextrin.
It should be noted that, the present invention is not particularly limited to the column temperature, the detector temperature, the mobile phase flow rate, and other parameters, and those skilled in the art can easily and reasonably determine the column temperature, the detector temperature, the mobile phase flow rate, and other parameters according to the actual detection requirements.
According to some embodiments of the invention, the elution procedure is as follows:
0 min-15 min, wherein the mobile phase A is obtained;
15-16 min, wherein the volume fraction of the mobile phase A in the mobile phase A and the mobile phase B is reduced from 100% to 0-60%;
16-21 min, wherein the volume fraction of the mobile phase A in the mobile phase A and the mobile phase B is maintained to be 0-60%;
the volume fraction of the mobile phase A in the mobile phase A and the mobile phase B is increased from 0-60% to 100% within 21-22 min;
after 22min, the mobile phase a elution was maintained for at least 30 min.
According to some embodiments of the invention, the elution of mobile phase a is maintained for a period of 30-80min during the last phase of the elution procedure.
The second aspect of the invention provides the application of the detection method of related substances in hydroxypropyl betacyclodextrin in synthesizing hydroxypropyl betacyclodextrin, and particularly can be used for purity inspection of hydroxypropyl betacyclodextrin, thereby being used for quality control of products or optimizing reaction conditions to obtain products with higher purity. The detection method has obvious application advantages due to the detection effect under the continuous sample feeding condition, and the detection efficiency can be improved.
The content in the present invention means the mass percentage content unless otherwise specified.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a chromatogram of a prior art method of interest specificity;
FIG. 2 is a chromatogram for specificity in accordance with example 1 of the present invention;
FIG. 3 is a chromatogram of a prior art method regarding system applicability;
FIG. 4 is a chromatogram relating to the applicability of the system in example 1 of the present invention;
FIG. 5 is a chromatogram of prior art methods with respect to accuracy;
FIG. 6 is a chromatogram for accuracy of example 1 of the present invention;
FIG. 7 is a chromatogram relating to precision of a prior art method;
FIG. 8 is a chromatogram for precision according to example 1 of the present invention;
FIG. 9 is a chromatogram for durability according to example 2 of the present invention;
FIG. 10 is a chromatogram for comparative example 1 of the present invention regarding durability.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
1. Related assay devices, components and materials
Liquid chromatograph, Thermo U3000; differential refractive detector, Thermo reflex Max 520; phenyl-bonded silica gel chromatography columns, Waters
Phenyl 10μm，
Electronic balance, mettleltoduo, model MS205 DU;
hydroxypropyl betacyclodextrin, manufactured by rogowatt, france, lot No. E0023; 1, 2-propanediol (lot 190001-201905), beta-cyclodextrin (lot 100317-202005), produced by the institute of food and drug assay in China; methanol, manufactured by Supelco, batch No. I11465071116; acetonitrile, produced by Honeywell, run No. U9OA 1H.
2. Solution preparation
Blank solution: and (3) water.
Control solution: taking 50mg of 1, 2-propylene glycol, precisely weighing, placing in a 100ml measuring flask, adding water for dissolving, diluting to a scale, and shaking up to obtain a1, 2-propylene glycol positioning solution; taking 50mg of beta-cyclodextrin, precisely weighing, placing in a 100ml measuring flask, adding water to dissolve and dilute to a scale, and shaking up to obtain a beta-cyclodextrin positioning solution.
Mixing the reference solution: taking 50mg of beta-cyclodextrin and 50mg of 1, 2-propylene glycol, accurately weighing, placing into the same 100ml measuring flask, adding water to dissolve and dilute to scale, shaking up to obtain a mixed reference solution.
Mix control stock solution: taking 250mg of beta-cyclodextrin and 250mg of 1, 2-propylene glycol, precisely weighing, placing into the same 20ml measuring flask, adding water to dissolve and dilute to scale, shaking up, and using as a mixed reference stock solution.
Test solution: taking about 2.5g of hydroxypropyl betacyclodextrin, precisely weighing, placing in a 25ml measuring flask, adding 15ml of water with the temperature of 60 ℃, shaking to dissolve, cooling to room temperature, diluting with water to scale, and shaking up to obtain a sample solution.
Adding a test sample solution: taking about 2.5g of hydroxypropyl betacyclodextrin, precisely weighing, placing in a 25ml measuring flask, adding 15ml of water with the temperature of 60 ℃, shaking to dissolve, cooling to room temperature, respectively adding 0.1ml of mixed reference stock solution (the relative content of single impurity is 0.05 percent (limit 10 percent), 3 parts of mixed reference stock solution are prepared in parallel and are respectively numbered from 1# to 3#), 1ml of mixed reference stock solution (the relative content of single impurity is 0.5 percent (limit 100 percent), 6 parts of mixed reference stock solution are prepared in parallel and are respectively numbered from 4# to 9#), and 1.5ml of mixed reference stock solution (the relative content of single impurity is 0.75 percent (limit 150 percent), 3 parts of mixed reference stock solution are prepared in parallel and are respectively numbered from 10# to 12), diluting to scale with water, shaking uniformly, and using as a mixed sample solution.
10% methanol solution: 100ml of methanol is weighed and placed in a reagent bottle, water is added to 1000ml, and the mixture is shaken up and treated by ultrasound to be used as a 10 percent methanol solution.
10% acetonitrile solution: 100ml of acetonitrile is weighed and placed in a reagent bottle, water is added to 1000ml, and the mixture is shaken up and treated by ultrasound to be used as 10 percent acetonitrile solution.
3. Chromatographic conditions
Example 1:
0 min-15 min, mobile phase A;
the volume fraction of the mobile phase A in the mobile phase A and the mobile phase B is reduced from 100% to 30% within 15-16 min;
16 min-21 min, and the volume fraction of the mobile phase A in the mobile phase A and the mobile phase B is maintained to be 30%;
the volume fraction of the mobile phase A in the mobile phase A and the mobile phase B is increased from 30% to 100% within 21-22 min;
22 min-100 min, mobile phase A.
Existing methods
The method of the '2020 version of Chinese pharmacopoeia' is that the column temperature is 40 ℃, the detector temperature is 40 ℃, water is a mobile phase, the flow rate is 1.5ml/min, and the sample volume is 20 mul.
System applicability requirements: the separation of the betacyclodextrin peak and the 1, 2-propanediol peak should be no less than 4.
4. Calculation of results
Calculated by peak area according to an external standard method.
5. Test results
5.1 specificity
The sample introduction sequence is as follows:
1. in the existing method, samples are sequentially injected according to the following sequence: blank solution 2 needles, 1, 2-propylene glycol positioning solution (reference solution), betacyclodextrin positioning solution (reference solution), mixed reference solution, contaminated test solution (content 0.50% (limit 100%)), blank solution, 10% methanol solution, 10% acetonitrile solution;
2. example 1, samples were injected sequentially in the following order: blank solution 3 needles, 1, 2-propylene glycol positioning solution, betacyclodextrin positioning solution, mixed reference solution, mixed test solution (content 0.50% (limit 100%)), blank solution, 10% methanol solution and 10% acetonitrile solution.
The chromatograms of the prior art method and example 1 are shown in fig. 1 and fig. 2, and in fig. 1 and fig. 2, the blank solution, the 1, 2-propylene glycol positioning solution, the betacyclodextrin positioning solution, the mixed reference solution, the impurity sample solution (with a content of 0.50% (limit of 100%)), the 10% methanol solution and the 10% acetonitrile solution are sequentially mapped from top to bottom. In fig. 2, the peak after 15min is due to the difference in refractive index of the mobile phase during the gradient elution procedure, which does not affect the quantification of 1, 2-propanediol and betacyclodextrin.
As can be seen from fig. 1 and 2, the blank solvent was not interfered in both the conventional method and example 1.
10% methanol peaked at 2.23min under both chromatographic conditions, with a retention time close to that of 1, 2-propanediol (about 2.26min), and the 10% methanol peak area was about 83 times the peak area of the 1, 2-propanediol limit concentration, indicating that the presence of low concentrations of methanol in the system would interfere with the detection of 1, 2-propanediol.
10% acetonitrile peaks at 2.55min under both chromatographic conditions, the retention time is also very close to that of 1, 2-propanediol (about 2.26min), and the peak area of 10% acetonitrile is about 103 times the peak area of the limit concentration of 1, 2-propanediol, indicating that the presence of low concentrations of acetonitrile in the system also interferes with the detection of 1, 2-propanediol.
5.2 suitability and accuracy
The sample introduction sequence is as follows:
1. in the existing method, samples are sequentially injected according to the following sequence: blank solution 2 needles, mixed reference substance solution 5 needles, test sample solution, mixed test sample solution 9 needles (serial numbers 1# to 6#, 10# to 12#), and mixed reference substance solution (quality control needle);
2. example 1, samples were injected sequentially in the following order: blank solution 3 needles, mixed reference solution 5 needles, test solution, mixed test solution 9 needles (serial numbers 1# to 6#, 10# to 12#), and mixed reference solution (quality control needle).
The detection requirements are as follows:
the measured value is less than or equal to 0.0001%, and the recovery rate is 70-125%;
0.0001% < measured value less than or equal to 0.001%, and recovery rate of 75-120%;
the measured value is more than 0.001% and less than or equal to 0.01%, and the recovery rate is 80-115%;
the measured value is more than 0.01 percent and less than or equal to 0.1 percent, and the recovery rate is 85 to 110 percent;
the measured value is less than or equal to 1 percent when the ratio is more than 0.1 percent, and the recovery rate is 90-108 percent;
1 percent < the measured value is less than or equal to 10 percent, and the recovery rate is 92 to 105 percent;
the measured value is less than or equal to 100 percent and the recovery rate is 95 to 102 percent.
5.2.1 System applicability
The chromatograms of the prior art method and example 1 related to the applicability of the system are respectively shown in fig. 3 and fig. 4, and in fig. 3 and fig. 4, the chromatograms of the blank solution, the mixed reference solution 5 needle, and the mixed reference solution (quality control needle) are sequentially corresponding from top to bottom.
The results of the applicability of the prior art process and the system of example 1 are shown in tables 1 and 2, respectively.
It can be seen that, after accurate continuous sample injection of 10 needles is carried out under the two chromatographic conditions, the peak area and retention time of the 1, 2-propylene glycol of the quality control needle have no significant change.
In the existing method, after 10 needles of sample are accurately and continuously fed, a betacyclodextrin chromatographic peak of a quality control needle is combined with other chromatographic peaks, the integral of the chromatographic peak is interfered (the last chromatogram of a graph in figure 3), and the peak area RSD of the applicability of the quality control needle and a system is 23 percent, so that the requirement of the applicability of the system is not met.
Example 1 after 10 needles of sample were injected continuously with accuracy, the peak pattern of the quality control needle for the betacyclodextrin chromatography was good (fig. 4), and the peak area and retention time were stable. The results show that the chromatographic conditions of example 1 are systematically reproducible.
TABLE 1
TABLE 2
5.2.2 Retention time and degree of separation
The chromatograms of the conventional method and example 1 are shown in fig. 5 and 6, respectively, and the chromatograms of the test solution, the miscellaneous test solution 1# to 6# and the miscellaneous test solution 10# to 12# are sequentially corresponding from top to bottom in fig. 5 and 6. The retention time and resolution results are shown in Table 3.
As can be seen, the retention time of the 1, 2-propylene glycol in the solution of the test sample is not significantly changed in the process of accurate continuous sample injection of 10 needles under the two chromatographic conditions.
In the existing method, in the process of accurate continuous sample injection of 10 needles, the retention time of the beta-cyclodextrin in the solution of the test sample is in a decreasing trend in the continuous sample injection process, and the retention time difference value of the 1 st needle and the 10 th needle is 3.46min (see fig. 5 and table 3); the separation degree of the 1, 2-propylene glycol and the beta-cyclodextrin in the mixed sample solution is reduced, and the separation degree of the 5 th needle is 3.54 (< 4).
Example 1 in the process of accurate continuous sample injection for 10 needles, the retention time of the betacyclodextrin in the chromatogram of the solution of the test sample is kept stable under the same limit concentration, and as the concentration of the sample increases, the retention time only shows a slight forward trend as a whole, and the difference between the maximum value and the minimum value is 0.776min, which shows that the chromatographic reproducibility of example 1 is obviously better.
TABLE 3
5.2.3 recovery
The recovery results for the prior art process and example 1 are shown in tables 4 and 5, respectively.
TABLE 4
TABLE 5
It can be seen that, after accurate continuous sample injection of 10 needles under the two chromatographic conditions, the recovery rate of the 1, 2-propylene glycol can be kept at a higher level. Example 1-fold recovery of the tacrolimus was higher than the existing process and the RSD recovery at concentrations of 0.05% (limit 10%), 0.50% (limit 100%) and 0.75% (limit 150%) was significantly lower than the existing process. The results show that the accuracy of example 1 is superior to the existing methods and the repeatability is good.
5.3 precision
The sample introduction sequence is as follows:
1. in the existing method, samples are sequentially injected according to the following sequence: blank solution 2 needles, mixed reference solution 5 needles, mixed sample solution 6 needles (serial numbers 4# to 9#, content 0.50% (limit 100%)), mixed reference solution (quality control needle);
2. example 1, samples were injected sequentially in the following order: blank solution 3 needles, mixed reference solution 5 needles, mixed sample solution 6 needles (serial numbers 4# to 9#, content 0.50% (limit 100%)), mixed reference solution (quality control needle);
the detection requirements are as follows:
the measured value is less than or equal to 0.0001 percent, and the RSD is not more than 15 percent;
0.0001 percent is less than or equal to 0.001 percent in measured value, and RSD is not more than 8 percent;
the measured value is less than or equal to 0.01 percent when the ratio of 0.001 percent to 0.01 percent, and the RSD is not more than 6 percent;
the measured value is less than or equal to 0.1 percent when the ratio is more than 0.01 percent, and the RSD is not more than 4 percent;
the measured value is less than or equal to 1 percent when the ratio is more than 0.1 percent, and the RSD is not more than 3 percent;
1 percent is more than the actual measurement value and less than or equal to 10 percent, and the RSD is not more than 2 percent;
the measured value is less than or equal to 100 percent when the ratio is more than 10 percent, and the RSD is not more than 1.5 percent.
The detection results are as follows:
FIG. 7 and FIG. 8 are chromatograms of a prior art method and a sample solution of example 1 with 6 needles mixed in continuous sample injection. The retention times and the separation contrast of the two methods are shown in table 6.
It can be seen that, in the process of continuous sample injection with precision and 6 needles under two chromatographic conditions, the retention time of the 1, 2-propylene glycol in the mixed sample solution has no significant change. In the prior art, in the process of continuous sample injection of 6 needles with precision, the retention time of the beta-cyclodextrin in the solution of the test sample is reduced in the continuous sample injection process, and the difference value of the retention time of the 1 st needle and the 6 th needle is 2.43min (see fig. 7 and table 6); the separation degree of the 1, 2-propylene glycol and the beta-cyclodextrin in the mixed sample solution tends to be small, the separation degree of the 3 rd needle is 3.98 (less than 4), and the repeatability is poor.
Example 1 in the process of accurate continuous sample injection of 6 needles, the retention time of the betacyclodextrin in the chromatogram of the solution of the test sample is stable, basically has no difference (see fig. 8 and table 6), and the separation degrees are all more than 4.5, which shows that the chromatogram reproducibility of example 1 is good.
TABLE 6
TABLE 7
TABLE 8
Table 7 and table 8 show the precision repeatability of the prior art method and example 1, respectively. It can be seen that, after 6 needles of precision repetitive continuous sample injection are carried out under two chromatographic conditions, the actually measured RSD of the 1, 2-propylene glycol content is lower, but the actually measured RSD of the 1-fold content of the beta-cyclodextrin in the example is obviously lower than that of the existing method, and the precision is higher.
5.4 durability
The effect of elution program variation was examined.
Example 2
The elution procedure was as follows, and the chromatographic conditions were the same as in example 1 except for the elution procedure:
0 min-15 min, mobile phase A;
the volume fraction of the mobile phase A in the mobile phase A and the mobile phase B is reduced to 60 percent from 100 percent in 15-16 min;
16 min-21 min, and the volume fraction of the mobile phase A in the mobile phase A and the mobile phase B is maintained to be 60%;
the volume fraction of the mobile phase A in the mobile phase A and the mobile phase B is increased from 60% to 100% within 21-22 min;
22 min-100 min, mobile phase A.
Comparative example 1
The elution procedure was as follows, and the chromatographic conditions were the same as in example 1 except for the elution procedure:
0 min-15 min, mobile phase A;
the volume fraction of the mobile phase A in the mobile phase A and the mobile phase B is reduced to 80 percent from 100 percent for 15 to 16 min;
16 min-21 min, and the volume fraction of the mobile phase A in the mobile phase A and the mobile phase B is maintained to be 80%;
the volume fraction of the mobile phase A in the mobile phase A and the mobile phase B is increased from 80% to 100% within 21-22 min;
22 min-100 min, mobile phase A.
The sample injection sequence of example 2 was: blank solution 2 needles, contaminated test sample solution (content 0.50% (limit 100%)) 9 needles. Example 2 after the completion of the detection, the elution procedure of example 1 was used to sample 2 needles of the blank solution, and the sample injection detection of comparative example 1 was performed in the following sample injection order: blank solution, solution with test sample (content 0.50% (limit 100%)) 9 needles.
FIGS. 9 and 10 are chromatograms of example 2 and comparative example 1, respectively, and in FIGS. 8 and 9, a blank solution and a solution to be tested are chromatograms of a continuous 9-pin probe in this order from top to bottom. The retention times and peak areas for the spiked samples for both elution procedures are shown in Table 9.
TABLE 9
From the above results, when the methanol content in the elution procedure is reduced to 40%, the retention time and peak area RSD of 1, 2-propanediol and betacyclodextrin in the chromatogram of the sample solution continuously injected into the 9-pin sample are both less than 2.0%. In contrast, in comparative example 1, the methanol content is reduced to 20%, the retention time of beta-cyclodextrin is reduced with the increase of the number of sample injections, the peak area is also reduced with the increase of the number of sample injections, and both the retention time and the peak area RSD are greater than 2.0%.
It should be noted that, in the above examples, only the chromatographic detection effect using methanol as the mobile phase B was exemplified, and in addition, methanol may be replaced by acetonitrile, and other detection parameters are not changed, and similar effects (results regarding acetonitrile are omitted) may be similarly achieved.
The results are combined to show that the chromatographic detection method has good reproducibility, good specificity, applicability, accuracy, precision and durability, and can accurately detect the 1, 2-propylene glycol and the beta-cyclodextrin in the hydroxypropyl beta-cyclodextrin under the condition of continuous sample injection, wherein the reproducibility and the accuracy of the detection of the beta-cyclodextrin are obviously superior to those of the existing method. Besides 1, 2-propylene glycol and beta-cyclodextrin, the method can also be used for detecting unknown impurities between chromatographic peaks of the 1, 2-propylene glycol and the beta-cyclodextrin.
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the foregoing and various other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention.
Claims (10)
1. The method for detecting related substances in hydroxypropyl betadex adopts liquid chromatography for detection, wherein the related substances comprise 1, 2-propylene glycol and betadex, and is characterized in that the conditions of the liquid chromatography for detection are as follows:
a chromatographic column: phenyl bonded silica gel chromatographic column;
mobile phase A: water;
mobile phase B: methanol or acetonitrile;
the elution procedure was as follows:
eluting with the mobile phase A for at least 15 min;
switching the mobile phase to reduce the volume fraction of the mobile phase A in the mobile phase A and the mobile phase B from 100% to 0-60%;
maintaining the elution of the switched mobile phase for a period of time;
switching the mobile phase to enable the volume fraction of the mobile phase A in the mobile phase A and the mobile phase B to be increased from 0-60% to 100%;
the mobile phase a elution was maintained for a period of time.
2. The method for detecting related substances in hydroxypropyl betadex according to claim 1, wherein the mobile phase elution after the maintaining of the switching is a period of time, specifically: the mobile phase elution after switching was maintained for at least 5 min.
3. The method for detecting related substances in hydroxypropyl betadex according to claim 1, wherein the elution of the mobile phase A is maintained for a period of time, specifically: the mobile phase a elution was maintained for at least 30 min.
4. The method for detecting related substances in hydroxypropyl betadex according to claim 1, wherein the detector for liquid chromatography detection is a differential refraction detector.
5. The method for detecting related substances in hydroxypropyl betadex according to claim 4, wherein the temperature of the detector is 40 ℃ ± 2 ℃.
6. The method for detecting related substances in hydroxypropyl betacyclodextrin as claimed in claim 1, wherein the chromatographic column is WATERS
Phenyl。
7. The method for detecting related substances in hydroxypropyl betadex according to claim 1, wherein the column temperature of the chromatographic column is 40 ℃ ± 2 ℃.
8. The method for detecting related substances in hydroxypropyl betadex according to claim 1, wherein the total flow rate of the mobile phase is constant at 1.5 ± 0.3ml/min during the liquid chromatography detection.
9. The method for detecting related substances in hydroxypropyl betacyclodextrin according to claim 1, wherein the elution procedure is as follows:
0 min-15 min, wherein the mobile phase A is obtained;
15-16 min, wherein the volume fraction of the mobile phase A in the mobile phase A and the mobile phase B is reduced from 100% to 0-60%;
16-21 min, wherein the volume fraction of the mobile phase A in the mobile phase A and the mobile phase B is maintained to be 0-60%;
the volume fraction of the mobile phase A in the mobile phase A and the mobile phase B is increased from 0-60% to 100% within 21-22 min;
after 22min, the mobile phase a elution was maintained for at least 30 min.
10. Use of the method according to any one of claims 1 to 9 for the detection of substances involved in hydroxypropyl betacyclodextrin in the synthesis of hydroxypropyl betacyclodextrin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111646543.7A CN114354795A (en) | 2021-12-29 | 2021-12-29 | Detection method and application of related substances in hydroxypropyl betacyclodextrin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111646543.7A CN114354795A (en) | 2021-12-29 | 2021-12-29 | Detection method and application of related substances in hydroxypropyl betacyclodextrin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114354795A true CN114354795A (en) | 2022-04-15 |
Family
ID=81103958
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111646543.7A Pending CN114354795A (en) | 2021-12-29 | 2021-12-29 | Detection method and application of related substances in hydroxypropyl betacyclodextrin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114354795A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102375032A (en) * | 2010-08-10 | 2012-03-14 | 石药集团中奇制药技术（石家庄）有限公司 | Quality control method of hydroxypropyl-beta-cyclodextrin |
| CN105116075A (en) * | 2015-09-15 | 2015-12-02 | 昆药集团股份有限公司 | Quantitative detection method for hydroxypropyl-beta-cyclodextrin |
-
2021
- 2021-12-29 CN CN202111646543.7A patent/CN114354795A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102375032A (en) * | 2010-08-10 | 2012-03-14 | 石药集团中奇制药技术（石家庄）有限公司 | Quality control method of hydroxypropyl-beta-cyclodextrin |
| CN105116075A (en) * | 2015-09-15 | 2015-12-02 | 昆药集团股份有限公司 | Quantitative detection method for hydroxypropyl-beta-cyclodextrin |
Non-Patent Citations (1)
| Title |
|---|
| 宋更申;冯丽;裴丽娟;王茉莉;: "药用辅料羟丙基-β-环糊精的研究概况", 中国药房, no. 45, 1 December 2011 (2011-12-01) * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112394127B (en) | Method for determining content of 3-amino-1-adamantanol and L-prolinamide in vildagliptin | |
| CN114113405B (en) | High performance liquid chromatography analysis method for glycerophosphorylcholine and isomer thereof | |
| CN112630365B (en) | Method for determining content of dimyristoyl phosphatidylcholine by high performance liquid chromatography | |
| CN114354795A (en) | Detection method and application of related substances in hydroxypropyl betacyclodextrin | |
| CN109425666B (en) | LC-MS analysis method of acyl chloride derivative | |
| CN111426760B (en) | Method for determining genotoxic impurities in doxofylline raw material medicine | |
| CN109374791B (en) | Method for determining acid residue in remifentanil hydrochloride raw material by using high performance liquid chromatography | |
| CN114280191A (en) | Method for detecting related substances in bis-cysteine and preparation thereof | |
| CN110095554B (en) | Method for analyzing milrinone related substances by high performance liquid chromatography | |
| CN109646426B (en) | Composition and method for detecting purity and related substances of 1-amino-2-propanol | |
| CN116930368B (en) | Detection method of settop alcohol isomer | |
| CN107884496B (en) | Method for determining content of succinic acid in trelagliptin succinate | |
| CN112710762A (en) | Method for measuring residual quantity of dimethyl sulfate | |
| CN114264765A (en) | Analysis method for determining related substances in glimepiride intermediate by using HPLC | |
| CN114324663B (en) | Method for detecting lornoxicam-related substances | |
| CN117030871B (en) | Method for detecting palmitic acid in paliperidone palmitate | |
| CN117368390B (en) | Method for determining impurity substances of loteprednol etabonate bulk drug by high performance liquid chromatography | |
| CN115236255B (en) | Method for detecting related substances of loxoprofen sodium | |
| CN112305100B (en) | Method for detecting content of genotoxic impurity benzyl bromide in medicine | |
| CN112595793B (en) | Earthworm injection detection method based on phenol determination | |
| CN113176371B (en) | Method for determining free polyethylene glycol in fatty alcohol-polyoxyethylene ether | |
| CN115420840A (en) | Method for detecting chloral hydrate related substances | |
| CN114609277A (en) | Method for measuring content of compound acetylsalicylic acid tablet | |
| CN114184721A (en) | Method for detecting residual posaconazole solvent | |
| CN117538457A (en) | Method for detecting 1, 4-butyrolactone in succinic anhydride |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |