CN106908559B - Separation and determination method of calcipotriol intermediate L and related impurities - Google Patents
Separation and determination method of calcipotriol intermediate L and related impurities Download PDFInfo
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- 239000012535 impurity Substances 0.000 title claims abstract description 75
- LWQQLNNNIPYSNX-UROSTWAQSA-N calcipotriol Chemical compound C1([C@H](O)/C=C/[C@@H](C)[C@@H]2[C@]3(CCCC(/[C@@H]3CC2)=C\C=C\2C([C@@H](O)C[C@H](O)C/2)=C)C)CC1 LWQQLNNNIPYSNX-UROSTWAQSA-N 0.000 title claims abstract description 38
- 229960002882 calcipotriol Drugs 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 33
- 238000000926 separation method Methods 0.000 title abstract description 13
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 33
- 239000013558 reference substance Substances 0.000 claims abstract description 15
- 238000004458 analytical method Methods 0.000 claims abstract description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 6
- OAYLNYINCPYISS-UHFFFAOYSA-N ethyl acetate;hexane Chemical compound CCCCCC.CCOC(C)=O OAYLNYINCPYISS-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000000741 silica gel Substances 0.000 claims abstract description 5
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 29
- 239000003085 diluting agent Substances 0.000 claims description 19
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 15
- 239000000523 sample Substances 0.000 claims description 13
- 230000014759 maintenance of location Effects 0.000 claims description 11
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 10
- 239000012488 sample solution Substances 0.000 claims description 7
- 238000001514 detection method Methods 0.000 claims description 5
- 239000000945 filler Substances 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
- 239000003960 organic solvent Substances 0.000 claims description 2
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 claims 1
- 229910052796 boron Inorganic materials 0.000 claims 1
- 239000002245 particle Substances 0.000 claims 1
- 239000000126 substance Substances 0.000 abstract description 6
- 230000005526 G1 to G0 transition Effects 0.000 abstract description 2
- 239000011259 mixed solution Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000006227 byproduct Substances 0.000 description 3
- 230000000711 cancerogenic effect Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 231100000219 mutagenic Toxicity 0.000 description 3
- 230000003505 mutagenic effect Effects 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- 125000003118 aryl group Chemical group 0.000 description 2
- 230000001747 exhibiting effect Effects 0.000 description 2
- 230000001738 genotoxic effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- -1 Alkyl hydrazine Chemical compound 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- NOWKCMXCCJGMRR-UHFFFAOYSA-N Aziridine Chemical compound C1CN1 NOWKCMXCCJGMRR-UHFFFAOYSA-N 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- QDHHCQZDFGDHMP-UHFFFAOYSA-N Chloramine Chemical compound ClN QDHHCQZDFGDHMP-UHFFFAOYSA-N 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 201000004681 Psoriasis Diseases 0.000 description 1
- XSTXAVWGXDQKEL-UHFFFAOYSA-N Trichloroethylene Chemical compound ClC=C(Cl)Cl XSTXAVWGXDQKEL-UHFFFAOYSA-N 0.000 description 1
- 125000005354 acylalkyl group Chemical group 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 150000001350 alkyl halides Chemical class 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- 150000004982 aromatic amines Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000003180 beta-lactone group Chemical group 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 108091092356 cellular DNA Proteins 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000012039 electrophile Substances 0.000 description 1
- 125000003700 epoxy group Chemical group 0.000 description 1
- 231100000025 genetic toxicology Toxicity 0.000 description 1
- 231100000024 genotoxic Toxicity 0.000 description 1
- OAKJQQAXSVQMHS-UHFFFAOYSA-N hydrazine Substances NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 1
- BLNWTAHYTCHDJH-UHFFFAOYSA-O hydroxy(oxo)azanium Chemical compound O[NH+]=O BLNWTAHYTCHDJH-UHFFFAOYSA-O 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229960004961 mechlorethamine Drugs 0.000 description 1
- 231100000299 mutagenicity Toxicity 0.000 description 1
- 230000007886 mutagenicity Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000000269 nucleophilic effect Effects 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000013074 reference sample Substances 0.000 description 1
- 238000007142 ring opening reaction Methods 0.000 description 1
- 150000003335 secondary amines Chemical class 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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Abstract
The invention belongs to the field of analytical chemistry, and particularly relates to a separation and determination method of a calcipotriol intermediate L and related impurities. The method for separating the calcipotriol intermediate L and the related impurities adopts silica gel as a stationary phase and ethyl acetate-n-hexane as a mobile phase for separation, and the method can realize effective separation of the intermediate L and the related impurities; the determination method comprises the steps of adopting high performance liquid chromatography, taking the intermediate L and a related impurity reference substance to prepare a reference substance solution and a test substance solution respectively, carrying out high performance liquid chromatography analysis, recording a chromatogram, and comparing peak areas of impurities corresponding to peak-out time in the test substance solution and the reference substance solution, so that the intermediate L and the related impurities are effectively separated and detected, and the method is simple to operate and high in accuracy.
Description
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to a separation and determination method of a calcipotriol intermediate L and related impurities.
Background
The chemical name of the calcipotriol is 9, 10-ring-opening cholest-24-cyclopropyl-5, 7, 10(19), 22-tetraene-1, 3, 24 triol, and the molecular formula is C27H40O3Molecular weight: 412.61. calcipotriol is used primarily for the topical treatment of psoriasis vulgaris. Compound L is an important intermediate in the synthesis of calcipotriol and has the following structural formula:
during the synthesis of intermediate L, various impurities such as M are generatedbBy-product Mb20S-isomer of (A), McAnd Mc20S-isomer of (a), and the like, and the structural formulae of these impurities are as follows:
among the above impurities, MbAnd its by-product MbThe 20S-isomer of (A) has some groups exhibiting toxic, mutagenic or carcinogenic effects, thus alerting it to possible genotoxicity, i.e., the ability to directly or indirectly damage cellular DNA, resulting in mutagenic and carcinogenic effects. The genotoxic groups generally have the property of electrophiles, and these groups undergo substitution reactions with nucleophilic centers in nucleic acids, proteins or other important components in vivo under physiological conditions, causing irreversible damage to these components, manifesting as toxicity, mutagenicity or carcinogenesis. Groups exhibiting toxic, mutagenic or carcinogenic effects are exemplified by:
| a. anxiety acyl alkyl ester | b. Aromatic nitro radical | c. Aromatic azo compounds |
| d. Aromatic nitrogen oxide | e. Aromatic primary or secondary amines | f. Alkyl hydrazine |
| g. Ester aldehyde group | h. N-carbinol radical | i. Halogenated olefins |
| j. Nitrogen mustard radical | k. Chloramine | l, β -lactone |
| m, ethylene imine | n, alkyl halides | o, Uratan |
| p, N-nitrosamines | q, aromatic amines or phenols | r, epoxy group |
The impurities, in particular M, produced during the synthesis of calcipotriol intermediate LbAnd its by-product MbIncomplete removal of the 20S-isomer ultimately affects the purity and quality of the drug. Therefore, in order to control the quality of intermediate L, it is necessary to separate and measure intermediate L and its related impurities. However, there is currently no HPLC method for separating and determining intermediate L and its related impurities.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a method for separating a calcipotriol intermediate L and related impurities, which can achieve effective separation of the intermediate L and related impurities; the second purpose of the invention is to provide a method for separating and determining the calcipotriol intermediate L and the related impurities by using the high performance liquid chromatography, which can realize effective separation and determination of the intermediate L and the related impurities.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the method for separating the calcipotriol intermediate L and the related impurities adopts silica gel as a stationary phase and ethyl acetate-n-hexane as a mobile phase for separation.
The method is suitable for separating the calcipotriol intermediate L and the related impurities, can be used for separating a certain substance independently, and can also be used for separating the calcipotriol intermediate L and the related impurities simultaneously.
In the method for separating the calcipotriol intermediate L and the related impurities, preferably, the related impurities are Mb、Mb20S isomer of (1), McAnd McOne or more of the 20S isomers of (a).
A method for separating and measuring calcipotriol intermediate L and related impurities by high performance liquid chromatography comprises the following specific steps: dissolving the intermediate L and related impurity reference substances with a diluent to prepare a reference substance solution with a known concentration, dissolving a test sample with the diluent to prepare a test sample solution, respectively sampling the reference substance solution and the test sample solution, performing high performance liquid chromatography, recording a chromatogram, comparing peak areas of impurities corresponding to peak emergence times in the test sample solution and the reference sample solution, and calculating the contents of the calcipotriol intermediate L and the related impurities in the test sample; the high performance liquid chromatography adopts a chromatographic column with silica gel as a filler, and ethyl acetate-n-hexane as a mobile phase for analysis.
The method for separating and determining the calcipotriol intermediate L and the related impurities by using the high performance liquid chromatography is suitable for separating and determining the calcipotriol intermediate L and the related impurities, can be used for independently detecting a certain substance, and can also be used for simultaneously separating and detecting the calcipotriol intermediate L and the related impurities.
Further, the method comprises the following steps: respectively taking the intermediate L and the related impurity reference substance, respectively dissolving the intermediate L and the related impurity reference substance by using a diluent to prepare an intermediate L positioning solution and a positioning solution of each related impurity, respectively taking the intermediate L positioning solution and the positioning solution of each related impurity for sample injection, carrying out high performance liquid chromatography analysis, and determining the retention time of the intermediate L and each related impurity.
Further, the method for separating and determining the calcipotriol intermediate L and the related impurities by using the high performance liquid chromatography is characterized in that the related impurities are Mb、Mb20S isomer of (1), McAnd McOne or more of the 20S isomers of (a).
Further, the method for separating and determining the calcipotriol intermediate L and related impurities by using the high performance liquid chromatography comprises the step of using a diluent, namely any one of a mobile phase ethyl acetate-n-hexane or a mobile phase organic solvent such as ethyl acetate or n-hexane.
Further, the method for separating and determining the calcipotriol intermediate L and the related impurities by using the high performance liquid chromatography has the mobile phase flow rate of 0.5-1.5ml/min, and preferably 1 ml/min.
Further, according to the method for separating and determining the calcipotriol intermediate L and related impurities by using the high performance liquid chromatography, the volume ratio of ethyl acetate to n-hexane in the mobile phase is 1-4: 96-99.
Further, the method for separating and determining the calcipotriol intermediate L and the related impurities by using the high performance liquid chromatography adopts an ultraviolet detector for detection, and the detection wavelength is 272 +/-2 nm.
Further, the method for separating and determining the calcipotriol intermediate L and related impurities by using the high performance liquid chromatography is characterized in that the specification of a chromatographic column is 4.6 x 250mm and 5 microns.
The invention has the beneficial effects that: (1) according to the method for separating the calcipotriol intermediate L and the related impurities, the calcipotriol intermediate L and the related impurities can be effectively separated, the impurities are effectively controlled, the product quality is fundamentally determined, and the method has the advantages of simplicity, convenience, rapidness, high accuracy and the like; (2) the method for separating and determining the calcipotriol intermediate L and the related impurities by using the high performance liquid chromatography can realize effective separation and determination of the calcipotriol intermediate L and the related impurities, and is simple to operate and high in accuracy by using the high performance liquid chromatography; (3) the method is suitable for separating and/or determining the calcipotriol intermediate L and the related impurities, can be used for separating and/or determining a certain substance independently, and can also be used for separating and/or determining the calcipotriol intermediate L and the related impurities simultaneously.
Drawings
FIG. 1 is an HPLC chart of the diluent in example 1, and the chromatographic peak in the chart is the chromatographic peak of the diluent.
FIG. 2 shows M in example 1bHPLC of 20S isomer of (5), wherein the chromatographic peak is MbThe 20S isomer of (1) with a retention time of 9.2 min.
FIG. 3 shows M in example 1bThe peak of the chromatogram in the HPLC chart is MbRetention time of the chromatographic peak of (1) is 9.8 min.
FIG. 4 shows M in example 1cHPLC of 20S isomer of (5), wherein the chromatographic peak is McThe 20S isomer of (1) with a retention time of 17.2 min.
FIG. 5 shows M in example 1cThe peak of the chromatogram in the HPLC chart is McRetention time of the chromatographic peak of (1) is 19.3 min.
FIG. 6 is an HPLC chart of the mixed solution of example 2, in which the chromatographic peaks are sequentially Mb20S isomer of (1), Mb、Mc20S isomer of (1), McAnd the retention time of chromatographic peaks of the intermediate L are as follows in sequence: 9.4min, 9.9min, 17.4min, 19.8min and 82.8 min.
FIG. 7 is an HPLC chart of the sample in example 3, in which the chromatographic peak is the chromatographic peak of the sample and the retention time is 81.9 min.
Detailed Description
The examples are provided for better illustration of the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
Sources of controls referred to in the examples:
l: chongqing Huabang pharmaceutical Co., Ltd, batch number: 99.0% of Cal-L-140903;
Mb: chongqing Huabang pharmaceutical Co., Ltd, batch number: Cal-Mb-140809，96.4％；
Mb20S-isomer of (a): chongqing Huabang pharmaceutical Co., Ltd, batch number: 140701, 97.8%;
Mc: chongqing Huabang pharmaceutical Co., Ltd, batch number: Cal-Mc-140903，96.3％；
Mc20S-isomer of (a): chongqing Huabang pharmaceutical Co., Ltd, batch number: 140803-2, 92.9%;
in the following examples, the apparatus and chromatographic conditions used in the process of the invention were as follows:
high performance liquid chromatograph: SHIMADZU LC-2010 AHT;
a chromatographic column: ZORBAX SIL (4.6X 250mm, 5 μm);
mobile phase: ethyl acetate: n-hexane (2: 98);
wavelength: 272 nm;
column temperature: 25 ℃;
flow rate: 1.0 ml/min;
sample introduction volume: 20 mu l of the mixture;
diluent agent: mobile phase (ethyl acetate: n-hexane (2: 98)).
Example 1 related impurity Mb、Mb20S isomer of (1), Mc、McLocalization of the 20S isomer of
(1)Mb、Mb20S isomer of (1), Mc、McPreparation of a positioning solution of the 20S isomer
MbPositioning solution: weighing impurity Mb15.11mg, placing in a 50ml measuring flask, adding a diluent to dissolve and dilute to the scale, and shaking up to obtain the product.
McPositioning solution: weighing impurity Mc15.26mg, placing in a 50ml measuring flask, adding a diluent to dissolve and dilute to the scale, and shaking up to obtain the product.
Mb20S isomer positioning solution: weighing impurity Mb15.28mg of the 20S isomer is put into a 50ml measuring flask, the diluent is added to dissolve and dilute the 20S isomer to the scale mark, and the mixture is shaken up to obtain the compound.
Mc20S isomer positioning solution: weighing impurity Mc15.22mg of the 20S isomer is put into a 50ml measuring flask, the diluent is added to dissolve and dilute the 20S isomer to the scale mark, and the mixture is shaken up to obtain the compound.
(2)Mb、Mb20S isomer of (1), Mc、McLocalization of the 20S isomer of
Respectively taking diluent and MbPositioning solution, McPositioning solution, Mb20S isomer of (5) McPerforming high performance liquid chromatography analysis according to the above chromatographic conditions, recording chromatogram, and obtaining results shown in FIGS. 1-5, wherein the diluent does not interfere with the detection of the sample, MbHas a retention time of 9.2min for the 20S isomer of (5)bRetention time 9.8min, McHas a retention time of 17.0min for the 20S isomer of (5)cThe retention time was 19.3 min.
Example 2 calcipotriol intermediate L and related impurity Mb、Mb20S isomer of (1), Mc、McSeparation of the 20S isomer of
(1) Intermediate L and related impurity Mb、Mb20S isomer of (1), Mc、McPreparation of a mixed solution of 20S isomer of (1)
M prepared in example 1 was separately precision-transferredb、Mc、Mb20S isomer of (1) and Mc3.3ml of each 20S isomer positioning solution is placed in the same 100ml measuring flask, a diluent is added to dilute the solution to a scale, the solution is shaken up to obtain a mixed positioning stock solution, then 10.26mg of an intermediate L reference substance is precisely weighed and placed in a 10ml measuring flask, 1.0ml of the mixed positioning stock solution is added, the diluent is added to dissolve and dilute the solution to the scale, and the solution is shaken up to obtain a mixed solution.
(2) Sampling 20 μ L of mixed solution, performing high performance liquid chromatography according to the above chromatographic conditions, recording chromatogram, and finding the result shown in FIG. 6, wherein the retention time of intermediate L is 81.9min, under the separation condition of the invention, the intermediate L is completely separated from each related impurity, and M isbThe separation degree of the 20S isomer from other impurities is satisfactory. Intermediate L reference product with no detectable impurity Mb20S isomer of (1).
EXAMPLE 3 isolation test of test article
(1) Preparing a test solution: taking about 10mg of the sample, placing the sample in a 10ml measuring flask, adding a diluent to dissolve and dilute the sample to a scale, and shaking up the sample to obtain the reagent.
(2) Respectively sampling 20 μ l sample of the test solution and mixed solution, performing high performance liquid chromatography according to the above chromatography conditions, recording chromatogram, which is shown in FIG. 7, and detecting no related impurity M in the test solutionb、Mb20S isomer of (1), McAnd Mc20S isomer of (1).
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Claims (4)
1. A method for separating and determining calcipotriol intermediate L and related impurities through high performance liquid chromatography is characterized by comprising the following specific steps of taking the intermediate L and related impurity reference substances, adding a diluent to dissolve the intermediate L and related impurities to prepare reference substance solutions with known concentrations, taking sample solutions of the reference substance solutions and the sample solutions to prepare sample solutions, respectively injecting samples, performing high performance liquid chromatography analysis, recording color products, calculating a test spectrogram, comparing the contents of the calcipotriol intermediate L and related impurities contained in peak surface products of the impurities corresponding to peak times in the sample solutions and the reference substance solutions, performing analysis by using a chromatographic column with silica gel as a filling agent and ethyl acetate-n-hexane as a mobile phase, wherein the specific chromatographic conditions are that the chromatographic column is ZOAX RBSIL (cubic boron silica gel) with a diameter of 4.6 × 0mm and a particle size of 5 mu M, the volume ratio of the ethyl acetate to the n-hexane is 2:98, the column temperature is 25 ℃, the flow rate is 1.0ml/min, and the related impurities are Mb、Mb20S isomer of (1), Mc20S isomer of (1) and Mc(ii) a The M isb20S isomer of (1), Mb、Mc20S isomer of (1), McAnd the retention time of chromatographic peaks of the intermediate L are as follows in sequence: 9.4min, 9.9min, 17.4min, 19.8min and 82.8 min.
2. The method for separating and determining calcipotriol intermediate L and related impurities by using high performance liquid chromatography as claimed in claim 1, wherein the method further comprises the following steps: respectively taking the intermediate L and the related impurity reference substance, respectively dissolving the intermediate L and the related impurity reference substance by using a diluent to prepare an intermediate L positioning solution and a positioning solution of each related impurity, respectively taking the intermediate L positioning solution and the positioning solution of each related impurity for sample injection, carrying out high performance liquid chromatography analysis, and determining the retention time of the intermediate L and each related impurity.
3. The method for separating and determining calcipotriol intermediate L and related impurities by high performance liquid chromatography as claimed in claim 1, wherein said diluent is any one of mobile phase ethyl acetate-n-hexane or mobile phase organic solvent.
4. The method for separating and determining calcipotriol intermediate L and related impurities according to claim 1, wherein the high performance liquid chromatography is used for detection by an ultraviolet detector, and the detection wavelength is 272 +/-2 nm.
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| EP1692123A1 (en) * | 2003-11-20 | 2006-08-23 | Eli Lilly And Company | Phenyl-furan compounds as vitamin d receptor modulators |
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