CN118033001A - High performance liquid chromatography separation assay CALO-B1And method for impurity thereof - Google Patents
High performance liquid chromatography separation assay CALO-B1And method for impurity thereof Download PDFInfo
- Publication number
- CN118033001A CN118033001A CN202410359526.2A CN202410359526A CN118033001A CN 118033001 A CN118033001 A CN 118033001A CN 202410359526 A CN202410359526 A CN 202410359526A CN 118033001 A CN118033001 A CN 118033001A
- Authority
- CN
- China
- Prior art keywords
- impurity
- mobile phase
- calcitriol
- starting material
- solution
- 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
- 239000012535 impurity Substances 0.000 title claims abstract description 99
- 238000000034 method Methods 0.000 title claims abstract description 81
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 38
- 238000000926 separation method Methods 0.000 title claims description 23
- 238000003556 assay Methods 0.000 title 1
- 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 claims abstract description 252
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 132
- GMRQFYUYWCNGIN-NKMMMXOESA-N calcitriol Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@@H](CCCC(C)(C)O)C)=C\C=C1\C[C@@H](O)C[C@H](O)C1=C GMRQFYUYWCNGIN-NKMMMXOESA-N 0.000 claims abstract description 79
- 229960005084 calcitriol Drugs 0.000 claims abstract description 79
- 235000020964 calcitriol Nutrition 0.000 claims abstract description 79
- 239000011612 calcitriol Substances 0.000 claims abstract description 79
- 239000007858 starting material Substances 0.000 claims abstract description 65
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 44
- FIQMHBFVRAXMOP-UHFFFAOYSA-N triphenylphosphane oxide Chemical compound C=1C=CC=CC=1P(C=1C=CC=CC=1)(=O)C1=CC=CC=C1 FIQMHBFVRAXMOP-UHFFFAOYSA-N 0.000 claims abstract description 44
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 22
- 239000011259 mixed solution Substances 0.000 claims abstract description 22
- 239000007864 aqueous solution Substances 0.000 claims abstract description 15
- 238000012937 correction Methods 0.000 claims abstract description 15
- 238000010828 elution Methods 0.000 claims abstract description 11
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 9
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 5
- FPLYNRPOIZEADP-UHFFFAOYSA-N octylsilane Chemical group CCCCCCCC[SiH3] FPLYNRPOIZEADP-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000000741 silica gel Substances 0.000 claims abstract description 5
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 5
- 239000012071 phase Substances 0.000 claims description 98
- 239000000243 solution Substances 0.000 claims description 81
- 238000001514 detection method Methods 0.000 claims description 61
- 230000014759 maintenance of location Effects 0.000 claims description 30
- 239000000523 sample Substances 0.000 claims description 27
- 239000003085 diluting agent Substances 0.000 claims description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 239000012085 test solution Substances 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 238000005259 measurement Methods 0.000 claims description 3
- 230000006399 behavior Effects 0.000 claims description 2
- 239000013068 control sample Substances 0.000 claims description 2
- 239000000126 substance Substances 0.000 abstract description 20
- 238000002360 preparation method Methods 0.000 abstract description 10
- 238000004458 analytical method Methods 0.000 abstract description 8
- 238000003908 quality control method Methods 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 239000012488 sample solution Substances 0.000 description 26
- 238000007865 diluting Methods 0.000 description 19
- 239000000047 product Substances 0.000 description 18
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 15
- 239000011550 stock solution Substances 0.000 description 15
- 238000005303 weighing Methods 0.000 description 14
- 238000012360 testing method Methods 0.000 description 11
- 238000011084 recovery Methods 0.000 description 10
- 239000013558 reference substance Substances 0.000 description 9
- 239000012467 final product Substances 0.000 description 8
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 239000000463 material Substances 0.000 description 5
- -1 (tert-butyldimethylsilyl) oxy Chemical group 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000010354 integration Effects 0.000 description 4
- 238000004090 dissolution Methods 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- FGWRMMTYIZKYMA-UHFFFAOYSA-N tert-butyl-hydroxy-dimethylsilane Chemical compound CC(C)(C)[Si](C)(C)O FGWRMMTYIZKYMA-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Natural products CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000010812 external standard method Methods 0.000 description 2
- 125000001981 tert-butyldimethylsilyl group Chemical group [H]C([H])([H])[Si]([H])(C([H])([H])[H])[*]C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- PGUIOHNOYADLMU-UHFFFAOYSA-N 1,1,1,3,3,3-hexafluoro-2-[3-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]propan-2-ol Chemical compound FC(F)(F)C(C(F)(F)F)(O)C1=CC=CC(C(O)(C(F)(F)F)C(F)(F)F)=C1 PGUIOHNOYADLMU-UHFFFAOYSA-N 0.000 description 1
- IMROMDMJAWUWLK-UHFFFAOYSA-N Ethenol Chemical group OC=C IMROMDMJAWUWLK-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- NXTYDKDYJFSZSO-UHFFFAOYSA-N butyl-hydroxy-dimethylsilane Chemical group CCCC[Si](C)(C)O NXTYDKDYJFSZSO-UHFFFAOYSA-N 0.000 description 1
- 239000002302 calcium metabolism regulator Substances 0.000 description 1
- WMKGGPCROCCUDY-PHEQNACWSA-N dibenzylideneacetone Chemical compound C=1C=CC=CC=1\C=C\C(=O)\C=C\C1=CC=CC=C1 WMKGGPCROCCUDY-PHEQNACWSA-N 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- BDAGIHXWWSANSR-UHFFFAOYSA-N formic acid Substances OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 238000012946 outsourcing Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 239000013074 reference sample Substances 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- FDDKCJBJJYNRPN-UHFFFAOYSA-N tert-butyl(silyloxy)silane Chemical group CC(C)(C)[SiH2]O[SiH3] FDDKCJBJJYNRPN-UHFFFAOYSA-N 0.000 description 1
- WBLNEDUTMUGDSW-UHFFFAOYSA-N tert-butyl-cyclohexyloxy-dimethylsilane Chemical compound CC(C)(C)[Si](C)(C)OC1CCCCC1 WBLNEDUTMUGDSW-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention belongs to the technical field of chemical analysis, and particularly relates to a method for separating and measuring CALO-B 1 and impurities thereof by using a high performance liquid chromatography. Impurities include impurity B 1e, impurity B 1f, impurity B 1g, impurity B 1h, impurity B 1j, and/or triphenylphosphine oxide; the method comprises the following steps: adopting octyl silane bonded silica gel as a chromatographic column stationary phase, wherein a mobile phase is a mixed solution of phosphoric acid aqueous solution and acetonitrile, and separating CALO-B 1 and 6 impurities through gradient elution; then detecting by an ultraviolet detector, and calculating the content of the impurity B 1g according to a main component self-comparison method added with correction factors; the contents of impurity B 1j, triphenylphosphine oxide, impurity B 1h, impurity B 1f and impurity B 1e were calculated by limit method. The method has the advantages of strong specificity, high sensitivity, high accuracy and good durability, and has important significance for quality control of calcitriol starting material B 1 and calcitriol and quality improvement of preparation products.
Description
Technical Field
The invention belongs to the technical field of chemical analysis, and particularly relates to a method for separating and measuring CALO-B 1 and impurities thereof by using a high performance liquid chromatography.
Background
Calcitriol is a calcium metabolism regulator, and has chemical name of (5Z, 7E) -9, 10-open loop cholest-5, 7, 10 (19) -triene-1 alpha, 3 beta, 25-triol monohydrate, molecular formula of C 27H44O3·H2 O, molecular weight of 434.65, and structural formula shown in formula I. B 1 is an important segment for synthesizing calcitriol, is a key starting material of calcitriol, is named as calcitriol starting material B 1 (CALO-B 1).CALO-B1 for short, has the chemical name of (1R, 3S, Z) -5- (2-hydroxy-ethylene) -4-methylene-1, 3-di (tert-butyldimethylsilyloxy) cyclohexane, has a molecular formula of C 21H42O3Si2 and a molecular weight of 398.73, and has a key effect on quality and impurity tracing of calcitriol by separation and impurity control of the structural formula of CALO-B 1 as shown in formula II.
It was found that CALO-B 1 introduces its specific process impurities during the synthesis: impurity B 1e, impurity B 1f, impurity B 1g, impurity B 1h, impurity B 1j and triphenylphosphine oxide. Impurity B 1e has the chemical name of 2- ((1R, 4R, 6S) -4- ((tert-butyldimethylsilyl) oxy) -1-methyl-7-oxabicyclo [4.1.0] heptane-2-subunit) acetate, the molecular formula of C 17H30O4 Si, the molecular weight of 326.50, and the structural formula of the impurity B is shown as formula III. Impurity B 1f has the chemical name of ethyl 2- [ (1E, 3S, 5R) -5- [ (tert-butyldimethylsilyl) oxy ] -3-hydroxy-2-methylenecyclohexylidene ] acetate, and has the molecular formula of C 17H30O4 Si, the molecular weight of 326.50, and the structural formula shown in formula IV. Impurity B 1g has the chemical name of (E) -2- ((3S, 5R) -3, 5-bis ((tert-butyldimethylsilyl) oxy) -2-methylenecyclohexylidene) ethyl acetate, and has the molecular formula of C 23H44O4Si2 and the molecular weight of 440.76, and the structural formula is shown in formula V. The chemical name of the impurity B 1h is dibenzylidene acetone, the molecular formula is C 17H14 O, the molecular weight is 234.29, and the structural formula is shown in a formula VI. The chemical name of the impurity B 1j is 9-fluorenone-4-formic acid, the molecular formula is C 14H8O3, the molecular weight is 224.21, and the structural formula is shown as formula VII. Triphenylphosphine oxide alias triphenylphosphine oxide has a molecular formula of C 18H15 OP, a molecular weight of 278.28, and a structural formula shown in formula VIII.
From the analysis of the synthetic process of CALO-B 1, the possibility that the impurities exist in CALO-B 1 is high, and if detection control is not performed in CALO-B 1, the purity and quality of the calcitriol of the final product can be affected, the safety risk of a patient in using the drug can be increased, and the following intermediate and the difficulty in tracking the impurities of the final product can be increased. However, there is no legal standard at present, and no literature or patent reports on the detection of calcitriol starting material B 1 and the above 6 impurities in the same analysis method, and only a part of patent literature reports on the detection method of calcitriol or related substances thereof. For example, the invention patent publication No. CN115109802A discloses a high performance liquid chromatography detection method of related impurity PZA of calcitriol, which adopts octyl silane bonded silica gel as a chromatographic column filling agent and adopts a mixed solution of 1.0g/L of tris (hydroxymethyl) aminomethane solution and acetonitrile as a mobile phase; the invention patent with publication number CN109752471A discloses a calcitriol detection method, calcitriol is detected by an HPLC method, a chromatographic column is a C18 column, a mobile phase A is methanol, and a mobile phase B is methanol and acetonitrile. However, none of these patents disclose the foregoing CALO-B 1 and its 6 process impurities, and do not allow for qualitative and quantitative detection of CALO-B 1 and its 6 process impurities.
Therefore, in order to avoid the above 6 impurities affecting the purity and quality of the product, it is necessary to establish a new method for separating and measuring calcitriol starting material B 1 and 6 impurities thereof to control the quality of calcitriol starting material B 1 and calcitriol.
Disclosure of Invention
Accordingly, one of the purposes of the present invention is to provide a method for separating process impurities in calcitriol starting material B 1 based on high performance liquid chromatography, which can achieve simultaneous separation of calcitriol starting material B 1 and 6 impurities thereof within 50 minutes or more, and provides support for qualitative and quantitative detection of each impurity in calcitriol starting material B 1.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
A method of separating process impurities in calcitriol starting material B 1 based on high performance liquid chromatography, the process impurities comprising any one or more of impurity B 1e, impurity B 1f, impurity B 1g, impurity B 1h, impurity B 1j and triphenylphosphine oxide; the high performance liquid chromatography comprises the step of adopting octyl silane bonded silica gel as a chromatographic column stationary phase, wherein a mobile phase is a mixed solution of phosphoric acid aqueous solution and acetonitrile, the mobile phase comprises a mobile phase A and a mobile phase B, and the volume ratio of the phosphoric acid aqueous solution to the acetonitrile in the mobile phase A is 40-50:60-50; in the mobile phase B, the volume ratio of the phosphoric acid aqueous solution to the acetonitrile is 3-7:97-93; sequentially separating the impurity B 1j, triphenylphosphine oxide, the impurity B 1h, the impurity B 1f, the impurity B 1e, the calcitriol starting material B 1 and the impurity B 1g by gradient elution;
The calcitriol starting material B 1 has a structural formula shown in a formula II, the impurity B 1e has a structural formula shown in a formula III, the impurity B 1f has a structural formula shown in a formula IV, the impurity B 1g has a structural formula shown in a formula V, the impurity B 1h has a structural formula shown in a formula VI, the impurity B 1j has a structural formula shown in a formula VII, and the triphenylphosphine oxide has a structural formula shown in a formula VIII;
Further, the components may be characterized according to the elution order.
Further, the gradient elution procedure was:
Setting the volume ratio of the mobile phase A to the mobile phase B to be 90-100 after 0 minutes: 10-0;
Setting the volume ratio of the mobile phase A to the mobile phase B to be 90-100 after 2 minutes: 10-0;
Setting the volume ratio of the mobile phase A to the mobile phase B to be 0-10 after 22 minutes: 100-90;
setting the volume ratio of the mobile phase A to the mobile phase B to be 0-10 after 40 minutes: 100-90;
41 minutes, setting the volume ratio of the mobile phase A to the mobile phase B to be 90-100:10-0;
setting the volume ratio of the mobile phase A to the mobile phase B to be 90-100 after 50 minutes: 10-0.
Still further, the gradient elution procedure was:
0 minutes, the volume ratio of mobile phase A to mobile phase B is set to be 100:0;
2 minutes, the volume ratio of mobile phase A to mobile phase B is set as 100:0;
22 minutes, the volume ratio of mobile phase A to mobile phase B is set to 0:100;
40 minutes, the volume ratio of mobile phase A to mobile phase B is set to 0:100;
41 minutes, the volume ratio of mobile phase A and mobile phase B was set to 100:0;
50 minutes, the volume ratio of mobile phase A to mobile phase B is set to be 100:0.
Further, the concentration of the phosphoric acid aqueous solution is 0.05% to 0.15%, preferably 0.098% to 0.102%, more preferably 0.1%.
Further, the column temperature is 20℃to 40℃and preferably 29℃to 31℃and more preferably 30 ℃.
Further, the flow rate is 0.5ml/min to 1.5ml/min, preferably 0.9ml/min to 1.1ml/min, more preferably 1.0ml/min. Preferably, in the mobile phase a, the volume ratio of the phosphoric acid aqueous solution to the acetonitrile is 43-47:57-53, more preferably 45:55; in the mobile phase B, the volume ratio of the phosphoric acid aqueous solution to the acetonitrile is 4-6:96-94, more preferably 5:95.
Preferably, the chromatographic column is Agilent ZORBAX Eclipse XDB-C8.
Preferably, the column size is 4.6mm.times.150mm, 5. Mu.m.
Preferably, the sample volume is 20. Mu.l.
Preferably, the separation time is 50 minutes.
Furthermore, the method can realize separation and detection of 6 process impurities in calcitriol starting material B 1 and related substances thereof; the related substances comprise an impurity B 1a, an impurity B 1b, an impurity B 1c, an impurity B 1i, an impurity tert-butyl dimethyl silanol and an impurity imidazole; the structural formula of the impurity B 1a is shown in a formula IX; the structural formula of the impurity B 1b is shown in a formula X; the structural formula of the impurity B 1c is shown in a formula XI; the structural formula of the impurity B 1i is shown in a formula XII; the structural formula of the impurity tertiary butyl dimethyl silanol is shown in a formula XIII; the structural formula of the impurity imidazole is shown in a formula XIV;
the second object of the present invention is to provide a method for identifying whether calcitriol starting material B 1 contains process impurities, which can effectively detect impurities with concentration lower than 0.02% in a sample, and has a lower detection limit.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
A method for identifying whether calcitriol starting material B 1 contains process impurities comprising the steps of:
(1) Separating any one or more of calcitriol starting material B 1 and process impurities thereof by adopting the method;
(2) Detecting by an ultraviolet detector with detection wavelength of 220nm-250nm after separation, and obtaining a chromatogram;
(3) Judging whether calcitriol starting material B 1 contains any one or more of impurity B 1j, triphenylphosphine oxide, impurity B 1h, impurity B 1f, impurity B 1e and impurity B 1g according to the consistency of the retention behaviors of the detection sample and the control sample.
Further, the retention time of each component is sequentially from short to long as impurity B 1j, triphenylphosphine oxide, impurity B 1h, impurity B 1f, impurity B 1e, calcitriol starting material B 1 and impurity B 1g.
The components may be characterized according to the length of the retention time.
Preferably, the detection wavelength is 240 nm.+ -. 2nm, more preferably 240nm.
Further, a mixed solution of 0.1% phosphoric acid aqueous solution and acetonitrile is taken as a mobile phase, and in the mobile phase A, the volume ratio of the 0.1% phosphoric acid aqueous solution to the acetonitrile is 45:55, in the mobile phase B, the volume ratio of the 0.1% phosphoric acid aqueous solution to the acetonitrile is 5:95; the flow rate of the mobile phase is 1.0ml/min, the specification of a chromatographic column is 4.6mm multiplied by 150mm, the column temperature is 5 mu m, and the temperature of the column is 30 ℃; the detection wavelength is 240nm; gradient elution was performed according to the following gradient elution procedure to obtain a chromatogram:
0 minutes, the volume ratio of mobile phase A to mobile phase B is set to be 100:0;
2 minutes, the volume ratio of mobile phase A to mobile phase B is set as 100:0;
22 minutes, the volume ratio of mobile phase A to mobile phase B is set to 0:100;
40 minutes, the volume ratio of mobile phase A to mobile phase B is set to 0:100;
41 minutes, the volume ratio of mobile phase A and mobile phase B was set to 100:0;
50 minutes, the volume ratio of mobile phase A to mobile phase B is set to be 100:0.
Further, when the relative retention time was 0.11±0.02 with calcitriol starting material B 1 as a reference peak, impurity B 1j was judged; when the relative retention time is 0.14+/-0.02, judging that the triphenylphosphine oxide is obtained; when the relative retention time is 0.33.+ -. 0.02, it is determined as impurity B 1h; when the relative retention time is 0.50.+ -. 0.02, impurity B 1f is judged; when the relative retention time is 0.70.+ -. 0.02, impurity B 1e is judged; when the relative retention time is 1.30.+ -. 0.02, impurity B 1g is judged; the calcitriol starting material B 1 has a retention time of 23.4±0.5min.
Further, impurity B 1j is present for a retention time of 2.6.+ -. 0.5 min; the retention time is 3.3+/-0.5 min, which is triphenylphosphine oxide; impurity B 1h with retention time of 7.8+ -0.5 min; impurity B 1f with retention time of 11.6+ -0.5 min; impurity B 1e with retention time of 16.4+ -0.5 min; impurity B 1g was present at a retention time of 30.4.+ -. 0.5 min.
It is a further object of the present invention to provide a method for determining the process impurity content of calcitriol starting material B 1 which allows for the quantification of impurities in a sample having a concentration of less than 0.05% with a low quantification limit.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for determining the process impurity content in calcitriol starting material B 1 comprising the steps of:
(1) Separation and detection: separating and detecting calcitriol starting material B 1 and process impurities by adopting the identification method to obtain a chromatogram;
(2) And (3) content measurement: calculating the content of impurity B 1g by adopting a main component self-comparison method added with correction factors according to the chromatogram measured in the step (1); the contents of impurity B 1j, triphenylphosphine oxide, impurity B 1h, impurity B 1f and impurity B 1e in calcitriol starting material B 1 are calculated respectively by adopting a limiting method.
From the above-mentioned structural formulae of each impurity, it can be seen that: the polarity difference between each impurity and the main component (CALO-B 1) is very large, the ultraviolet absorption difference is also very large, the impurities with strong polarity are reserved in the same analysis method, the impurities with weak polarity can be smoothly eluted, and the quantitative detection by selecting proper wavelength is difficult, so that the method is one of the main innovations of the method.
Further, preparing a solution to be detected by adopting a diluent before separation; the diluent is any one or more of the mobile phase, acetonitrile, ethanol and methanol.
Further, the solution to be tested comprises any one or more of a test solution, a control solution and a control solution.
Preferably, the concentration of the sample solution is 1.5mg/ml, the concentration of the control solution is 1.5 mug/ml, and the concentration of the control solution is 1.5 mug/ml each containing impurity B 1e, impurity B 1f, impurity B 1h, impurity B 1j and triphenylphosphine oxide.
Preferably, the diluent is acetonitrile.
Preferably, the method specifically comprises the following steps:
1. preparing a test sample solution: precisely weighing a sample, adding acetonitrile for dissolution to prepare a sample solution with the concentration of 1.5 mg/ml;
2. preparing a control solution: precisely measuring a proper amount of sample solution, and diluting with acetonitrile to prepare a control solution with the concentration of 1.5 mug/ml;
3. Control solution: accurately weighing impurity B 1e, impurity B 1f, impurity B 1h, impurity B 1j and triphenylphosphine oxide reference substances, adding acetonitrile to dissolve and quantitatively diluting to obtain a solution containing about 1.5 μg of each impurity in 1 ml.
4. Taking the sample solution prepared in the step 1, the control solution prepared in the step 2 and the control solution prepared in the step 3, carrying out high performance liquid chromatography analysis, recording a chromatogram, calculating the content of the impurity B 1g in the calcitriol starting material B 1 according to a main component self-contrast method added with a correction factor, and calculating the contents of the impurity B 1j, triphenylphosphine oxide, the impurity B 1h, the impurity B 1f and the impurity B 1e according to a limit method respectively.
Further, the correction factor of the impurity B 1g is 0.23.
Further, the impurity B 1g is in the range of concentration 0.5981 μg/ml to 9.6480 μg/ml, y= 0.4736X-0.0065, r=1.0000; the calcitriol starting material B 1 has a concentration ranging from 0.6211 mu g/ml to 10.2600 mu g/ml, Y=0.1095X+0.0006, r=1.0000; wherein Y is Y axis and represents peak area; x is X axis, represents concentration, R represents correlation coefficient.
Further, the impurity control (product) solution is continuously sampled for 6 times, CALO-B 1, impurity B 1j, triphenylphosphine oxide, impurity B 1h, impurity B 1f and impurity B 1e, the peak areas RSD of the impurities are all smaller than 5.0%, tailing factors are all smaller than 1.8, and theoretical plates are all larger than 2000.
Further, if chromatographic peaks consistent with retention time of the impurity B 1e, the impurity B 1f, the impurity B 1h, the impurity B 1j and/or the triphenylphosphine oxide exist in the chromatogram of the detection sample, and the peak area of each impurity is larger than the peak area of the corresponding impurity in the reference sample solution, the content of each process impurity in the calcitriol initial material B 1 sample is unqualified; if the chromatogram of the detection product does not have chromatographic peaks consistent with the retention time of the impurity B 1e, the impurity B 1f, the impurity B 1h, the impurity B 1j and/or the triphenylphosphine oxide, or the chromatogram of the detection product has chromatographic peaks consistent with the retention time of the impurity B 1e, the impurity B 1f, the impurity B 1h, the impurity B 1j and/or the triphenylphosphine oxide, but the peak area of each impurity is smaller than or equal to the peak area of the corresponding impurity in the reference substance solution, the process impurity content in the calcitriol starting material B 1 sample is qualified.
Further, preparing a standard substance to obtain a standard sample chromatogram; and (3) testing the chromatograms obtained by the samples, and calculating the impurity content according to the peak area by a main component self-comparison method and a limit method added with correction factors.
The standard substance is the known component content, and the component content of the test sample is unknown.
The obtained standard sample chromatograms can be stored in a database and used as quality control map materials in an intelligent pharmaceutical factory, and the quality control map materials are used for helping a robot to judge whether products produced by the robot are qualified or not, and quantitatively improved analysis suggestions are provided for unqualified products.
The invention has the beneficial effects that:
1. The method utilizes the high performance liquid chromatography to separate and measure the calcitriol starting material B 1 and 6 impurities, realizes the effective control of the impurities, fundamentally determines the quality of the product, and has the advantages of specificity, sensitivity, simplicity, convenience, rapidness, high accuracy, good precision, good durability and the like. The invention provides technical support for quality control of calcitriol starting material B 1 and calcitriol and quality improvement of preparation products.
2. The detection method provided by the invention can realize simultaneous determination of calcitriol starting material B 1 and 6 impurities thereof within 50 minutes, and has the characteristic of short analysis time.
3. According to the detection method provided by the invention, the quantitative limit concentration of the calcitriol starting material B 1 is 0.6211 mug/ml, the quantitative limit concentration of the impurity B 1g is 0.5981 mug/ml, the quantitative limit concentration of the impurity B 1j is 0.1658 mug/ml, the quantitative limit concentration of the impurity triphenylphosphine oxide is 0.6364 mug/ml, the quantitative limit concentration of the impurity B 1h is 0.3194 mug/ml, the quantitative limit concentration of the impurity B 1f is 0.5457 mug/ml, and the quantitative limit concentration of the impurity B 1e is 0.5821 mug/ml; calcitriol starting material B 1 has a detection limit concentration of 0.2174 μg/ml, impurity B 1g has a detection limit concentration of 0.2093 μg/ml, impurity B 1j has a detection limit concentration of 0.0580 μg/ml, impurity triphenylphosphine oxide has a detection limit concentration of 0.2227 μg/ml, impurity B 1h has a detection limit concentration of 0.1118 μg/ml, impurity B 1f has a detection limit concentration of 0.1910 μg/ml, and impurity B 1e has a detection limit concentration of 0.2037 μg/ml. The detection method has high sensitivity, strong practicability and accurate and reliable detection result, and has important significance for realizing the quality control of calcitriol starting material CALO-B 1.
Drawings
FIG. 1 is an HPLC plot of diluent (acetonitrile);
FIG. 2 is an HPLC diagram of a system applicability solution;
FIG. 3 is an HPLC plot of a control solution;
FIG. 4 is an HPLC diagram of a sample solution;
FIG. 5 is an HPLC plot of the mixed solution;
FIG. 6 is an HPLC plot of a quantitative limiting solution;
FIG. 7 is an HPLC plot of the detection limit solution;
FIG. 8 is an HPLC plot of control solution in precision;
FIG. 9 is an HPLC plot of the sample solution for precision;
FIG. 10 is an HPLC plot of a standard addition solution;
FIG. 11 is an HPLC plot of a control solution in accuracy;
FIG. 12 is an HPLC plot of a control solution in accuracy;
FIG. 13 is an HPLC plot of a sample solution without a standard in accuracy;
FIG. 14 is an HPLC plot of a 50% solution with added accuracy;
FIG. 15 is an HPLC plot of a 100% solution with added accuracy;
FIG. 16 is an HPLC plot of a 150% solution with accuracy added;
FIG. 17 is an HPLC plot of the mixed solution at a durability-flow rate of 0.9 ml/min;
FIG. 18 is an HPLC plot of the mixed solution at a durability-flow rate of 1.1 ml/min;
FIG. 19 is an HPLC plot of the mixed solution at a durability-column temperature of 29 ℃;
FIG. 20 is an HPLC plot of the mixed solution at a durability-column temperature of 31 ℃;
FIG. 21 is an HPLC plot of the mixed solution under conditions of durability-mobile phase ratio 1 (mobile phase A (43:57), mobile phase B (4:96));
FIG. 22 is an HPLC plot of the mixed solution under conditions of durability-mobile phase ratio 1 (mobile phase A (47:53), mobile phase B (6:94));
FIG. 23 is an HPLC plot of the mixed solution with a durability versus phosphoric acid concentration of 0.098% in the mobile phase;
FIG. 24 is an HPLC plot of the mixed solution at a phosphoric acid concentration of 0.102% in the durable-mobile phase;
FIG. 25 is a chromatogram of acetonitrile-water as mobile phase in example 2;
FIG. 26 is a chromatogram of example 2 with acetonitrile-0.1% phosphoric acid in water as the mobile phase;
FIG. 27 is a chromatogram of the column stationary phase of example 2 being octadecylsilane chemically bonded silica;
FIG. 28 is a chromatogram of example 2 wherein the column stationary phase is octyl silane bonded silica gel;
FIG. 29 is a 3D plot of the test wavelength selection experiment of example 2;
FIG. 30 is a chromatogram of example 2 for a detection wavelength of 220 nm;
FIG. 31 is a chromatogram of example 2 for a detection wavelength of 230 nm;
FIG. 32 is a chromatogram of example 2 for a detection wavelength of 240 nm.
Detailed Description
The technical scheme of the present invention will be further clearly and completely described in connection with specific embodiments. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. Therefore, all other embodiments obtained by those skilled in the art without undue burden are within the scope of the invention based on the embodiments of the present invention.
In the embodiment of the invention, the reference substance is derived from Chongqing Huabang pharmaceutical Co., ltd, chongqing Hua Bangsheng Kai pharmaceutical Co., ltd or outsourcing.
In the embodiment of the invention, the contents of the impurities B 1e, B 1f, B 1h, B 1j and triphenylphosphine oxide are calculated: the chromatographic peak with the retention time consistent with the impurity is not larger than the peak areas of impurity B 1e, impurity B 1f, impurity B 1h, impurity B 1j and triphenylphosphine oxide (0.10%) in the control solution.
In the embodiment of the invention, the preparation method of the solution comprises the following steps:
System applicability solution: a suitable amount of CALO-B 1 system applicability control I (about impurity B 1g 1.5.5. Mu.g and calcitriol starting material B 1 1.5.5 mg per 1.5 mg) was taken, dissolved and diluted with acetonitrile to prepare a solution containing about 1.5mg per 1 ml.
Test solution: the product is taken to be proper, precisely weighed, dissolved by acetonitrile and quantitatively diluted to prepare a solution containing about 1.5mg of the product in each 1 ml.
Control (product) solution: taking appropriate amounts of impurity B 1e, impurity B 1f, impurity B 1h, impurity B 1j and triphenylphosphine oxide reference substance, precisely weighing, precisely measuring an appropriate amount of sample solution, placing into a same measuring flask, adding acetonitrile for dissolving and diluting to obtain a solution containing about CALO-B 1 1.5 mug and 1.5 mug of each impurity in each 1 ml.
In the embodiment of the present invention, the isomer impurities possibly existing in calcitriol starting material B 1 include: impurity B 1a, chemical name of (E) -2- ((3S, 5R) -3, 5-bis ((tert-butyldimethylsilyl) oxo) -2-methylalkylenecyclohexylene), molecular formula of C 21H42O3Si2, molecular weight of 398.73, and structural formula of IX; impurity B 1b, chemical name is (Z) -2- ((3R, 5R) -3, 5-bis ((tert-butyldisilyl) oxy) -2-methylenedicyclohexyl) ethane-1-ol, molecular formula is C 21H42O3Si2, molecular weight is 398.73, and structural formula is shown in formula X; impurity B 1c, chemical name of (Z) -2- ((3S, 5S) -3, 5-bis ((tert-butyldimethylsilyl) oxy) -2-methylenedicyclohexyl) ethane-1-ol, molecular formula of C 21H42O3Si2, molecular weight of 398.73, and structural formula of XI; impurity B 1i, 1, 3-bis (hexafluoro-hydroxyisopropyl) benzene with molecular formula of C 12H6F12O2 and molecular weight of 410.16, and structural formula of XII; the impurity tert-butyl dimethyl silanol has a molecular formula of C 6H16 OSi, a molecular weight of 132.28 and a structural formula shown in a formula XIII; the molecular formula of the impurity imidazole is C 3H4N2, the molecular weight is 68.08, and the structural formula is shown in formula XIV;
example 1 method for isolation determination of calcitriol starting Material B 1 and 6 impurities thereof
(1) Instrument and chromatographic conditions
The high performance liquid chromatograph is Shimadzu SPD-20A; the chromatographic column is Agilent ZORBAX Eclipse XDB-C8, the specification is 4.6mm×150mm,5 μm; the mobile phase comprises a mobile phase A and a mobile phase B; mobile phase A is prepared from phosphoric acid aqueous solution with concentration of 0.1% and acetonitrile according to volume ratio of 45:55, the mobile phase B is composed of phosphoric acid aqueous solution with concentration of 0.1% and acetonitrile according to the volume ratio of 5: 95. Gradient elution was performed as in table 1; the flow rate is 1.0ml/min; the column temperature is 30 ℃; the detection wavelength is 240nm; the sample volume was 20. Mu.l.
TABLE 1 gradient elution Table
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 2 | 100 | 0 |
| 22 | 0 | 100 |
| 40 | 0 | 100 |
| 41 | 100 | 0 |
| 50 | 100 | 0 |
(2) Preparing a solution to be tested
A diluent: acetonitrile.
System applicability solution: weighing CALO-B 1 system applicability reference substance I2.928 mg, placing into a 2ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking to obtain 1.4640mg/ml solution.
Control (product) solution: taking appropriate amounts of impurity B 1e, impurity B 1f, impurity B 1h, impurity B 1j and triphenylphosphine oxide reference substance, precisely weighing, precisely measuring an appropriate amount of sample solution, placing into a same measuring flask, adding acetonitrile for dissolving and diluting to prepare a solution containing CALO-B 1 1.5470 mug, impurity B 1e 1.4552 mug, impurity B 1f 1.3644 mug, impurity B 1h 1.5972 mug and impurity B 1j 1.6578 mug in each 1 ml.
Test solution: taking a proper amount of the product (calcitriol starting material B 1), precisely weighing, adding acetonitrile for dissolving and quantitatively diluting to prepare a solution containing 1.4996mg in each 1 ml.
(3) Detection of
Respectively taking a diluent, a system applicability solution, a control (product) solution and a test sample solution, performing high performance liquid chromatography according to the chromatographic conditions, and recording a chromatogram; and the impurity contents were calculated.
The impurity B 1g content calculation formula is:
Wherein: peak areas of respective impurity peaks in the Ar-sample solution chromatogram;
A S -the main peak area in the control (product) solution chromatogram;
0.23-correction factor for impurity B 1g.
Impurity B 1e, impurity B 1f, impurity B 1h, impurity B 1j and triphenylphosphine oxide content calculation: the chromatographic peak with the retention time consistent with the impurity is not larger than the peak areas of impurity B 1e, impurity B 1f, impurity B 1h, impurity B 1j and triphenylphosphine oxide (0.10%) in the control solution.
Results: as shown in fig. 1 to 4, the integration results corresponding to fig. 2 to 4 are shown in tables 2 to 4. From the results, the diluent did not interfere with the detection of the sample. In the system applicability solution chromatogram, the peak outlet sequence is CALO-B 1 and impurity B 1g in sequence, so that the separation can be effectively carried out, and the separation degree is more than 1.5; and calculating the content of impurity B 1g in the solution of the test sample according to the self-comparison method of the main component added with the correction factors.
TABLE 2 integral results Table of FIG. 2
TABLE 3 integral results Table of FIG. 3
TABLE 4 integral results Table of FIG. 4
EXAMPLE 2 selection of mobile phase, chromatographic column and detection wavelength
The present example examined the choice of mobile phase, chromatographic column and detection wavelength, respectively. Other experimental conditions were the same as in example 1 except for the examination item. The results are shown in Table 5.
TABLE 5 selection results of mobile phases, chromatographic columns and detection wavelengths
EXAMPLE 3 specificity experiments
The present example performed a proprietary verification of the detection scheme of example 1, including a blank solvent interference experiment, and performed a separation verification of the chromatographic system for impurities that may be present, including impurity B 1a, impurity B 1b, impurity B 1c, impurity B 1i, impurity t-butyldimethylsilyl alcohol and impurity imidazole. The method specifically comprises the following steps: weighing a proper amount of each impurity, adding acetonitrile, and diluting to prepare a solution with the concentration of 3-9 mug/ml serving as a single needle positioning solution. Then, a mixed solution containing calcitriol starting material B 1, impurity B 1g, impurity B 1e, impurity B 1f, impurity B 1h, impurity B 1j, triphenylphosphine oxide and the above impurities (about 1.5 mg/1 ml of calcitriol starting material B 1, about 1.5 to 4.5. Mu.g of each impurity) was prepared. The single needle positioning solution and the mixed solution of each impurity were sampled respectively, and detection was performed according to the experimental conditions of example 1, and a chromatogram was obtained.
Results: the pattern of the blank solvent is shown in fig. 1, the pattern of the mixed solution is shown in fig. 5, and the specific detection results are shown in table 6, wherein the results show that the blank solvent (acetonitrile) and the known impurities do not interfere with the detection of calcitriol starting material B 1 and 6 impurities (impurity B 1g, impurity B 1e, impurity B 1f, impurity B 1h, impurity B 1j and triphenylphosphine oxide). Imidazole, tert-butyl dimethyl silanol and impurity B 1i do not show peaks at the wavelength, and do not interfere with detection; impurity B 1a, impurity B 1b and impurity B 1c are overlapped with CALO-B 1 peaks, and detection of impurities in the system is not interfered. The separation degree between the main peak and the adjacent impurity peak in the color spectrum of the mixed solution is more than 1.5, and the separation degree between the known impurity peaks is more than 1.5. The result shows that other impurities, the main component and 6 impurities have better separation degree, and the method has good specificity.
TABLE 6 specificity results Table
Example 4 sensitivity experiment
(1) Preparing a solution to be tested
Quantitative limiting solution: the calcitriol starting material B 1 and 6 impurities thereof are weighed, respectively added with acetonitrile for dissolution and dilution to prepare a solution containing the calcitriol starting material B 1 0.6211 mug, the impurity B 1j 0.1658 mug, the triphenylphosphine oxide 0.6364 mug, the impurity B 1h 0.3194 mug, the impurity B 1f 0.5457 mug, the impurity B 1e 0.5821 mug and the impurity B 1g 0.5981 mug which are contained in 1ml of the solution as a quantitative limiting solution.
Detection limit solution: acetonitrile was added to prepare a solution containing 1 0.2174. Mu.g of calcitriol starting material B, 0.0580. Mu.g of impurity B 1j, 0.2227. Mu.g of triphenylphosphine oxide, 1h 0.1118. Mu.g of impurity B 1f 0.1910. Mu.g of impurity B 1e 0.2037. Mu.g of impurity B and 1g 0.2093. Mu.g of impurity B as a detection limit solution per 1 ml.
(2) Detection of
The quantitative limiting solution and the detection limiting solution are taken and respectively injected, and detection is carried out according to the experimental conditions of the example 1, and a chromatogram is obtained.
Results: the quantitative limit map and the detection limit map are shown in fig. 6 and 7, respectively, and the integration results are shown in tables 7 and 8, respectively. The quantitative limit concentration of calcitriol starting material B 1 is 0.6211 mug/ml, the quantitative limit concentration of impurity B 1g is 0.5981 mug/ml, the quantitative limit concentration of impurity B 1j is 0.1658 mug/ml, the quantitative limit concentration of impurity triphenylphosphine oxide is 0.6364 mug/ml, the quantitative limit concentration of impurity B 1h is 0.3194 mug/ml, the quantitative limit concentration of impurity B 1f is 0.5457 mug/ml, the quantitative limit concentration of impurity B 1e is 0.5821 mug/ml, the concentration of each component in a sample is less than 0.05%, the RSD of peak area is less than 5.0%, and the signal to noise ratio is greater than 10, thus indicating that each component can be accurately quantified at the concentration. The detection limit concentration of calcitriol starting material B 1 is 0.2174 mug/ml, the detection limit concentration of impurity B 1g is 0.2093 mug/ml, the detection limit concentration of impurity B 1j is 0.0580 mug/ml, the detection limit concentration of impurity triphenylphosphine oxide is 0.2227 mug/ml, the detection limit concentration of impurity B 1h is 0.1118 mug/ml, the detection limit concentration of impurity B 1f is 0.1910 mug/ml, the detection limit concentration of impurity B 1e is 0.2037 mug/ml, the concentration of each component in a sample is less than 0.02%, and the signal to noise ratio is greater than 10, so that each component can be effectively detected at the concentration. The method has high sensitivity.
TABLE 7 integral results Table of FIG. 6
TABLE 8 integral results Table of FIG. 7
Example 5 linearity and correction factor
And weighing appropriate amounts of calcitriol starting material B 1 and impurity B 1g, and preparing a series of linear solutions containing calcitriol starting material B 1 and impurity B 1g simultaneously by adopting a progressive dilution method, wherein the concentration of the solutions is quantitatively limited to 0.7% of the concentration of the solution to be tested. The dilution of each 1 ml contains calcitriol starting material B 1 and impurity B 1g, the concentration gradients of which are used to establish a standard curve, the linear range of the evaluation method and the lower limit of quantification. Sampling is carried out according to the order of the concentration from low to high, a chromatogram is recorded, the peak area Y is linearly regressed by a least square method according to the concentration X (mug/ml), and a regression equation and a correlation coefficient are calculated.
Results: the regression equation of the impurity B 1g is Y= 0.4736X-0.0065, and the correlation coefficient R is 1.0000, which shows that the linear relation of the impurity B 1g is good within the concentration range of 0.5981-9.6480 mug/ml (LOQ-0.7 percent); the regression equation of calcitriol starting material B 1 is Y=0.1095X+0.0006, and the correlation coefficient R is 1.0000, which shows that calcitriol starting material B 1 has good linear relationship in the range of the concentration of 0.6211 mug/ml to 10.2600 mug/ml (LOQ to 0.7%).
According to the linear slope, the correction factor of the obtained impurity B 1g is calculated to be 0.23, so that the quantitative mode of the impurity B 1g in the calcitriol starting material B 1 is selected as 'calculated according to the principal component self-comparison method added with the correction factor'.
EXAMPLE 6 precision
(1) Preparing a solution to be tested
Impurity B 1g stock solution a: accurately weighing 3.115mg of impurity B 1g reference substance, placing into a 10ml measuring flask, adding acetonitrile for dissolving and diluting to scale, shaking uniformly, and taking as impurity B 1g stock solution; and precisely transferring 1.0ml, placing into a 50ml measuring flask, adding acetonitrile to dilute to a scale, and shaking uniformly to obtain the final product.
Test solution: taking about 15mg of calcitriol initial material B 1, precisely weighing, placing into a 10ml measuring flask, precisely transferring 2.5ml of impurity B 1g stock solution A, placing into the same 10ml measuring flask, adding acetonitrile, dissolving, diluting to scale, and shaking to obtain the final product. (parallel preparation of 6 parts)
Control solution: precisely measuring 1.0ml of the sample solution, placing into a 100ml measuring flask, adding acetonitrile for dilution to the scale, shaking, precisely measuring 1.0ml, placing into a 10ml measuring flask, adding acetonitrile for dilution to the scale, and shaking to obtain the final product.
(2) Detection of
20. Mu.l of each of the sample solution and the control solution was sampled, and the samples were examined under the experimental conditions of example 1, and a chromatogram was recorded. The content of the related substances in 6 parts of test sample solution and the RSD thereof are calculated according to the main component self-comparison method added with the correction factors.
To examine the effect of random variation factors on precision, tests were performed by different analysts at different times. 6 parts of sample solutions to be tested and control solutions are prepared in parallel by the same method, a chromatogram is recorded, the content of related substances and RSD in the 6 parts of sample solutions are calculated according to the main component self-comparison method added with correction factors, and RSD of the content of the related substances in 12 parts of samples detected by different analysts is calculated.
Results: as shown in fig. 8 and 9, the integration results are shown in tables 9 to 10, the average value of the content of detected impurity B 1g in 6 sample solutions of different persons is about 0.09%, the RSD is 1.2% and 2.2%, respectively, and the RSD of the content of detected impurity J in 12 total samples of both persons is 3.1% and less than 5.0%. The technical scheme is proved to have good precision.
TABLE 9 integral results Table of FIG. 8
TABLE 10 integral results Table of FIG. 9
EXAMPLE 7 Standard addition
This example was run against the potential impurities "impurity B 1e, impurity B 1f, impurity B 1h, impurity B 1j and triphenylphosphine oxide" and is specifically as follows:
(1) Solution preparation
Impurity mixed solution a: impurity mixed solution a: accurately weighing appropriate amounts of impurity B 1e, impurity B 1f, impurity B 1h, impurity B 1i, impurity B 1j and triphenylphosphine oxide stock solution, adding acetonitrile, dissolving, and diluting to obtain solution containing 3 μg of each impurity per 1 ml.
Control solution: the control (product) solution was the same as in example 1.
Unlabeled test solution: the sample solution was the same as in example 1.
Standard addition of solution: and (3) taking about 15mg of a sample to be tested, precisely weighing, placing the sample into a 10ml measuring flask, precisely transferring 5.0ml of the impurity mixed solution A, placing the sample into the same 10ml measuring flask, adding a diluent for dissolution, diluting to a scale, and shaking uniformly to obtain the product. (parallel preparation of 2 parts)
(2) Measurement
The standard addition solution was sampled, tested according to the experimental conditions in example 1 and the chromatogram recorded. The accuracy of each impurity was calculated based on the average value of the peak areas of each impurity in the control (product) solution in example 1.
Results: as shown in fig. 10, the integrated results are shown in table 11, and the average recovery of impurity B 1j was 100.2%, the average recovery of triphenylphosphine oxide was 99.9%, the average recovery of impurity B 1h was 92.8%, the average recovery of impurity B 1f was 100.4%, and the average recovery of impurity B 1e was 95.2%. The recovery rate of each impurity is in the range of 90% -108%, which indicates that the technical scheme has good accuracy.
TABLE 11 integral results Table of FIG. 10
Example 8 accuracy
(1) Solution preparation
Impurity B 1g accuracy stock solution: accurately weighing a proper amount of impurity B 1g, adding acetonitrile for dissolving and diluting to prepare a solution containing 7.5 mug of impurity B 1g per 1 ml.
Control solution: accurately removing 2.0ml of impurity B 1g accurate stock solution, placing into a 10ml measuring flask, diluting to scale with diluent, and shaking.
Sample stock solution: accurately weighing calcitriol initial material B 1 249.93mg, placing into 25ml measuring flask, adding acetonitrile, dissolving, diluting to scale, and shaking.
Test solution (no standard solution): precisely measuring 2.0ml of the stock solution of the sample, placing into a 10ml measuring flask, adding acetonitrile to dilute to scale, and shaking to obtain the final product.
Control solution: precisely measuring 1ml of the sample solution, placing in a 100ml measuring flask, adding acetonitrile for dilution to the scale, shaking up, precisely measuring 1.5ml, placing in a 10ml measuring flask, adding acetonitrile for dilution to the scale, and shaking up to obtain the final product.
Unlabeled test solution: precisely transferring 1.5ml of sample stock solution, placing into a 10ml measuring flask, diluting to scale with diluent, and shaking.
Control solution: precisely transferring 1.0ml of the sample solution, placing into a 100ml measuring flask, diluting to scale with diluent, and shaking; and precisely transferring 1.0ml of the solution, placing into a 10ml measuring flask, diluting to scale with a diluent, and shaking uniformly to obtain the final product.
50% Of the labeled test sample solution: accurately removing 1.0ml of impurity B 1g accurate stock solution and 1.5ml of sample stock solution, placing into the same 10ml measuring flask, diluting to scale with diluent, and shaking. (parallel preparation of 3 parts)
100% Of standard test solution: accurately removing 2.0ml of impurity B 1g accurate stock solution and 1.5ml of sample stock solution, placing into the same 10ml measuring flask, diluting to scale with diluent, and shaking. (parallel preparation of 3 parts)
150% Of labeled test sample solution: accurately removing 3.0ml of impurity B 1g accurate stock solution and 1.5ml of sample stock solution, placing into the same 10ml measuring flask, diluting to scale with diluent, and shaking. (parallel preparation of 3 parts)
(2) Measurement method
Taking the sample solution, the reference substance solution, the reference solution, the sample solutions with the standard and the sample solution without the standard, respectively, carrying out sample injection, detecting according to the experimental conditions in the embodiment 1, recording a chromatogram, and calculating the recovery rate of the impurity B 1g according to an impurity reference substance external standard method and a main component self-comparison method with the correction factors.
Results: as shown in fig. 11 to 16, the integration results are shown in table 12, and the average recovery rate of impurity B 1g calculated by the impurity reference external standard method is 98.0% and RSD is 1.3%; the average recovery rate of impurity B 1g calculated by the main component self-comparison method added with correction factors is 99.3%, RSD is 1.3%, and the recovery rates are all in the range of 90% -108%, which indicates that the technical scheme has good accuracy.
TABLE 12 integral result tables of FIGS. 11-16
Example 9 durability
Taking the mixed solution in the embodiment 2, respectively adjusting the column flow rate, the column temperature, the mobile phase proportion and the phosphoric acid concentration in the mobile phase on the basis of the chromatographic conditions in the embodiment 1, respectively testing after the instrument system is stable, and recording the separation degree between the peaks.
Results: as shown in FIGS. 17 to 24 and Table 13, the column flow rate was 0.9ml/min and 1.1ml/min, and the degree of separation between the peaks was more than 1.5. When the mobile phase proportion 1 and the mobile phase proportion 2 are adopted, the separation degree between peaks is more than 1.5; the separation degree between peaks is more than 1.5 when the column temperature is 29 ℃ and 31 ℃; the phosphoric acid concentration in the mobile phase was 0.098% and 0.102%, and the separation degree between peaks was greater than 1.5. Namely, when the flow rate of the column, the proportion of the mobile phase, the concentration of phosphoric acid in the mobile phase and the temperature of the column fluctuate, the separation degree between the calcitriol starting material B 1 and 6 impurities is more than 1.5, which proves that the method has good durability.
TABLE 13 durability results Table
/>
Claims (10)
1. A method for separating process impurities in calcitriol starting material B 1 based on high performance liquid chromatography, characterized in that the process impurities comprise any one or more of impurity B 1e, impurity B 1f, impurity B 1g, impurity B 1h, impurity B 1j and triphenylphosphine oxide; the high performance liquid chromatography comprises the step of adopting octyl silane bonded silica gel as a chromatographic column stationary phase, wherein a mobile phase is a mixed solution of phosphoric acid aqueous solution and acetonitrile, the mobile phase comprises a mobile phase A and a mobile phase B, and the volume ratio of the phosphoric acid aqueous solution to the acetonitrile in the mobile phase A is 40-50:60-50; in the mobile phase B, the volume ratio of the phosphoric acid aqueous solution to the acetonitrile is 3-7:97-93; sequentially separating the impurity B 1j, triphenylphosphine oxide, the impurity B 1h, the impurity B 1f, the impurity B 1e, the calcitriol starting material B 1 and the impurity B 1g by gradient elution;
The calcitriol starting material B 1 has a structural formula shown in a formula II, the impurity B 1e has a structural formula shown in a formula III, the impurity B 1f has a structural formula shown in a formula IV, the impurity B 1g has a structural formula shown in a formula V, the impurity B 1h has a structural formula shown in a formula VI, the impurity B 1j has a structural formula shown in a formula VII, and the triphenylphosphine oxide has a structural formula shown in a formula VIII;
2. the method of claim 1, wherein the gradient elution procedure is:
Setting the volume ratio of the mobile phase A to the mobile phase B to be 90-100 after 0 minutes: 10-0;
Setting the volume ratio of the mobile phase A to the mobile phase B to be 90-100 after 2 minutes: 10-0;
Setting the volume ratio of the mobile phase A to the mobile phase B to be 0-10 after 22 minutes: 100-90;
setting the volume ratio of the mobile phase A to the mobile phase B to be 0-10 after 40 minutes: 100-90;
41 minutes, setting the volume ratio of the mobile phase A to the mobile phase B to be 90-100:10-0;
setting the volume ratio of the mobile phase A to the mobile phase B to be 90-100 after 50 minutes: 10-0.
3. The method of claim 1, wherein the aqueous phosphoric acid solution has a concentration of 0.05% to 0.15%.
4. The method according to claim 1, wherein the column temperature is 20-40 ℃ and the flow rate is 0.5-1.5 ml/min.
5. The method for identifying whether calcitriol starting material B 1 contains process impurities is characterized by comprising the following steps:
(1) Separating calcitriol starting material B 1 and any one or more of its process impurities by the method of any one of claims 1-4;
(2) Detecting by an ultraviolet detector with the detection wavelength of 220nm-240nm after separation, and obtaining a chromatogram;
(3) Judging whether calcitriol starting material B 1 contains any one or more of impurity B 1j, triphenylphosphine oxide, impurity B 1h, impurity B 1f, impurity B 1e and impurity B 1g according to the consistency of the retention behaviors of the detection sample and the control sample.
6. The method of claim 5, wherein the retention time of each component is from short to long, impurity B 1j, triphenylphosphine oxide, impurity B 1h, impurity B 1f, impurity B 1e, calcitriol starting material B 1, and impurity B 1g.
7. The method according to claim 5, wherein the calcitriol starting material B 1 is used as a reference peak, and the impurity B 1j is determined when the relative retention time is 0.11±0.02; when the relative retention time is 0.14+/-0.02, judging that the triphenylphosphine oxide is obtained; when the relative retention time is 0.33.+ -. 0.02, it is determined as impurity B 1h; when the relative retention time is 0.50.+ -. 0.02, impurity B 1f is judged; when the relative retention time is 0.70.+ -. 0.02, impurity B 1e is judged; when the relative retention time is 1.30.+ -. 0.02, impurity B 1g is judged; the calcitriol starting material B 1 has a retention time of 23.4±0.5min.
8. A method for determining the process impurity content in calcitriol starting material B 1 comprising the steps of:
(1) Separation and detection: separating and detecting calcitriol starting material B 1 and its technical impurities by the method according to any one of claims 5-7 to obtain a chromatogram;
(2) And (3) content measurement: calculating the content of impurity B 1g by adopting a main component self-comparison method added with correction factors according to the chromatogram measured in the step (1); the contents of impurity B 1j, triphenylphosphine oxide, impurity B 1h, impurity B 1f and impurity B 1e in calcitriol starting material B 1 are calculated respectively by adopting a limiting method.
9. The method of claim 8, wherein prior to separation, a diluent is used to prepare the test solution; the diluent is any one or more of the mobile phase, acetonitrile, ethanol and methanol.
10. The method of claim 8, wherein impurity B 1g is in the range of concentration 0.5981 μg/ml to 9.6480 μg/ml, Y = 0.4736X-0.0065, r = 1.0000; the calcitriol starting material B 1 has a concentration ranging from 0.6211 mu g/ml to 10.2600 mu g/ml, Y=0.1095X+0.0006, r=1.0000; wherein Y is Y axis and represents peak area; x is X axis, represents concentration, R represents correlation coefficient.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410359526.2A CN118033001A (en) | 2024-03-27 | 2024-03-27 | High performance liquid chromatography separation assay CALO-B1And method for impurity thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410359526.2A CN118033001A (en) | 2024-03-27 | 2024-03-27 | High performance liquid chromatography separation assay CALO-B1And method for impurity thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118033001A true CN118033001A (en) | 2024-05-14 |
Family
ID=90987926
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410359526.2A Pending CN118033001A (en) | 2024-03-27 | 2024-03-27 | High performance liquid chromatography separation assay CALO-B1And method for impurity thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118033001A (en) |
-
2024
- 2024-03-27 CN CN202410359526.2A patent/CN118033001A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sripalakit et al. | High-performance liquid chromatographic method for the determination of pioglitazone in human plasma using ultraviolet detection and its application to a pharmacokinetic study | |
| CN115453012B (en) | Reversed-phase HPLC method for simultaneously measuring multiple positional isomers in voathixetine hydrobromide | |
| CN113237988B (en) | Method for detecting content of degraded impurity aldol dimer in oxycodone liquid preparation | |
| CN107870209B (en) | Method for determining impurity content in linagliptin bulk drug | |
| CN111208215B (en) | Method for detecting impurity 2-mercaptobenzothiazole in ceftriaxone sodium | |
| CN116297978A (en) | HPLC method for separating and measuring posaconazole Z 3 And methods for impurity and solvent thereof | |
| CN114689737B (en) | Analysis method of S-o-chlorophenylglycine methyl tartrate related substances | |
| CN118033001A (en) | High performance liquid chromatography separation assay CALO-B1And method for impurity thereof | |
| CN114354789B (en) | Method for simultaneously measuring cabozantinib analogue and related substances thereof | |
| CN114264765B (en) | Analytical method for determining related substances in glimepiride intermediate by utilizing HPLC | |
| CN114646701A (en) | HPLC (high Performance liquid chromatography) test method for related substances in L-prolinamide | |
| CN108760937B (en) | Determination of residual ethylenediamine in caspofungin acetate and application thereof | |
| CN114660183A (en) | High performance liquid chromatography analysis method for separating and measuring L-alanine isopropyl ester hydrochloride enantiomer | |
| CN114594168A (en) | Method for detecting indobufen impurity | |
| CN114910596B (en) | Analysis method for determining related substances of pyridone compounds | |
| CN118033002A (en) | Separation and determination of calcitriol starting material SM by GC method1And method for impurity thereof | |
| CN111323493A (en) | Method for detecting enantiomer of posaconazole starting material | |
| CN114113402B (en) | Method for measuring pinanediol content in bortezomib by adopting high performance liquid chromatography | |
| CN114324673B (en) | Analysis method for simultaneously determining clemastine fumarate and isomer thereof | |
| CN114295750B (en) | Method for detecting related substances in ursodeoxycholic acid synthesis | |
| CN114088842B (en) | Rocuronium bromide intermediate and impurity detection method | |
| CN114200067B (en) | High performance liquid chromatography analysis method for 6-bromo-3-hydroxy pyrazine-2-carboxamide and impurities | |
| CN117191969A (en) | Method for detecting intermediate V in buprenorphine intermediate VI | |
| CN117705987A (en) | Detection method for related substances in Wupattinib intermediate | |
| CN117825547A (en) | Isolation assay of clotrimazole intermediate Z 1 High performance liquid chromatography of related substances in (B) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination |