A method for analyzing isomers in raw material fragment 1 for preparing idecalcitol

Abstract

The present invention belongs to the technical field of drug analysis and detection, and provides an analysis method for isomers in a fragment 1 of a raw material for preparing idecalcitol. In the analysis method provided by the present invention, the protective group tert-butyl dimethylsilyl ether in the fragment 1 of the raw material for preparing idecalcitol to be tested is first removed by a deprotection reaction, so that the polarity of the fragment 1, the enantiomer of the fragment 1, and the diastereomer of the fragment 1 becomes stronger, and the difference between the three also becomes larger. Then, a polysaccharide derivative chiral chromatographic column is used as a chromatographic column, so that the fragment 1 of the deprotected group, the enantiomer of the fragment 1, and the diastereomer of the fragment 1 show differences in physical characteristics, thereby achieving the purpose of separation and detection.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis and detection, and in particular to a method for analyzing isomers in a raw material fragment 1 for preparing idecalcitol. Background Art

[0002] Idecalcitol is a drug used to treat osteoporosis and is an active form of vitamin D. 3 Derivatives. Fragment 1 is a key material for the synthesis of idecalcitol. There is a chiral center in the structure of fragment 1, which will produce optical isomers. The optical impurity content in fragment 1 must be controlled, otherwise the impurities will participate in subsequent reactions and become impurities of idecalcitol, affecting product quality.

[0003] There are three chiral centers in the structure of fragment 1. The most likely optical isomers are enantiomers and diastereomers. The structures are as follows:

[0004]

[0005] From the chemical structures of fragment 1, fragment 1 enantiomers and fragment 1 diastereomers, we can see that they contain three protecting groups, tert-butyl dimethyl silyl ether, which protect the three hydroxyl groups in the structure, making the polarity of the three substances extremely small, and the polarity difference between the optical isomers is even smaller. Studies have found that, for example, supercritical CO 2 The analytical methods such as the chiral chromatography column analysis and the like cannot effectively separate the fragment 1, the fragment 1 enantiomer and the fragment 1 diastereomer, and therefore cannot detect the contents of the fragment 1 enantiomer and the fragment 1 diastereomer in the fragment 1. Summary of the invention

[0006] In view of this, the object of the present invention is to provide an analysis method for isomers in fragment 1, a raw material for preparing elcalcitol. The analysis method provided by the present invention realizes the determination of the content of fragment 1 enantiomers and fragment 1 diastereomers in fragment 1.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for analyzing isomers in a raw material fragment 1 for preparing idecalcitol, comprising the following steps:

[0009] The raw material fragment 1 for preparing idecalcitol to be tested, a solvent and a deprotection reagent are mixed to carry out a deprotection reaction to obtain the deprotected raw material fragment 1 for preparing idecalcitol;

[0010] The deprotected idecalcitol preparation raw material fragment 1 is subjected to liquid chromatography detection, and based on the area normalization method, the content of the fragment 1 enantiomer and the fragment 1 diastereomer in the idecalcitol preparation raw material fragment 1 to be tested is obtained;

[0011] The parameters of the liquid chromatography detection include:

[0012] The chromatographic column is a polysaccharide derivative chiral chromatographic column.

[0013] Preferably, the solvent comprises one or more of methanol, ethanol, acetone, tetrahydrofuran, toluene and methyl tert-butyl ether.

[0014] Preferably, the ratio of the raw material fragment 1 for preparing the idecalcitol to be tested to the solvent is 1 g: 1-20 mL.

[0015] Preferably, the deprotection agent comprises one or more of hydrogen chloride, hydrochloric acid, tetrabutylammonium fluoride, ammonium fluoride, pyridine hydrogen fluoride, triethylamine hydrogen fluoride, p-toluenesulfonic acid and camphorsulfonic acid.

[0016] Preferably, the deprotection reagent is calculated as an effective ingredient, and the usage ratio of the raw material fragment 1 for preparing idecalcitol to be tested and the deprotection reagent is 1 g: 0.03-0.1 mol.

[0017] Preferably, the temperature of the deprotection reaction is 10-40° C. and the time is 10-20 min.

[0018] Preferably, the parameters detected by liquid chromatography also include:

[0019] The mobile phase system is a mixture of n-hexane and isopropanol; the volume ratio of n-hexane to isopropanol in the mixture is 80:20 to 84:16;

[0020] The elution method is isocratic elution.

[0021] Preferably, the parameters of the liquid chromatography detection also include:

[0022] The detector is an ultraviolet detector;

[0023] The detection wavelength of the ultraviolet detector is 203-207 nm.

[0024] Preferably, the parameters of the liquid chromatography detection also include: the column temperature of the chromatographic column is 20-30°C.

[0025] Preferably, the parameters of the liquid chromatography detection also include:

[0026] The flow rate of the mobile phase system is 0.8-1.2 mL / min;

[0027] The injection volume was 10 ± 2 μL.

[0028] The invention provides an analysis method for isomers in a fragment 1 of a raw material for preparing idcalciferol, comprising the following steps: mixing the fragment 1 of a raw material for preparing idcalciferol to be tested, a solvent and a deprotection reagent, and performing a deprotection reaction to obtain the deprotected fragment 1 of the raw material for preparing idcalciferol; performing liquid chromatography detection on the deprotected fragment 1 of the raw material for preparing idcalciferol, and obtaining the contents of the enantiomer of the fragment 1 and the diastereomer of the fragment 1 in the fragment 1 of the raw material for preparing idcalciferol to be tested based on an area normalization method; and the parameters of the liquid chromatography detection include: the chromatographic column is a polysaccharide derivative chiral chromatographic column. In the analysis method provided by the present invention, the protective group tert-butyl dimethylsilyl ether in the raw material fragment 1 of the idecalcitol to be tested is firstly removed by a deprotection reaction, so that the polarity of the fragment 1, the enantiomer of the fragment 1, and the diastereomer of the fragment 1 becomes stronger, and the difference between the three also becomes larger. Then, a polysaccharide derivative chiral chromatographic column is used as a chromatographic column, so that the fragment 1, the enantiomer of the fragment 1, and the diastereomer of the fragment 1 without the protective group show differences in physical characteristics, thereby achieving the purpose of separation and detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 HPLC spectrum of the mixed solution in Example 1;

[0030] Figure 2 is the linear spectrum of the deprotected fragment 1 reference substance;

[0031] Figure 3 is the linear spectrum of the deprotected fragment 1 enantiomer reference substance;

[0032] Figure 4 This is the linear spectrum of the diastereoisomer reference substance of the deprotected fragment 1. DETAILED DESCRIPTION

[0033] The present invention provides a method for analyzing isomers in a raw material fragment 1 for preparing idecalcitol, comprising the following steps:

[0034] The raw material fragment 1 for preparing idecalcitol to be tested, a solvent and a deprotection reagent are mixed to carry out a deprotection reaction to obtain the deprotected raw material fragment 1 for preparing idecalcitol;

[0035] The deprotected idecalcitol preparation raw material fragment 1 is subjected to liquid chromatography detection, and the contents of fragment 1 enantiomer and fragment 1 diastereomer in the idecalcitol preparation raw material fragment 1 to be tested are obtained based on the area normalization method.

[0036] In the present invention, unless otherwise specified, the raw materials used in the present invention are preferably commercially available products.

[0037] The present invention mixes a raw material fragment 1 for preparing idecalcitol to be tested, a solvent and a deprotection reagent, and performs a deprotection reaction to obtain a deprotected raw material fragment 1 for preparing idecalcitol.

[0038] In the present invention, the solvent preferably includes one or more of methanol, ethanol, acetone, tetrahydrofuran, toluene and methyl tert-butyl ether, and more preferably ethanol. In the present invention, the amount ratio of the raw material fragment 1 to be tested for preparing idecalciferol and the solvent is preferably 1g: 1-20mL, and more preferably 1g: 2-10mL.

[0039] In the present invention, the deprotecting agent preferably includes one or more of hydrogen chloride, hydrochloric acid, tetrabutylammonium fluoride, ammonium fluoride, pyridine hydrogen fluoride, triethylamine hydrogen fluoride, p-toluenesulfonic acid and camphorsulfonic acid, and is more preferably hydrogen chloride. In the present invention, the hydrogen chloride is preferably used in the form of a hydrogen chloride solution; the solvent of the hydrogen chloride solution is preferably the same as the type of the solvent described above, which will not be repeated here. In the present invention, the deprotecting agent is calculated as an effective ingredient, and the amount ratio of the raw material fragment 1 to be tested for the preparation of idecalciferol and the deprotecting agent is preferably 1g: 0.03-0.1mol, and more preferably 1g: 0.04-0.06mol.

[0040] In the present invention, the mixing time is preferably 5 minutes; the mixing is preferably performed under shaking and sealing conditions.

[0041] In the present invention, the temperature of the deprotection reaction is preferably 10 to 40° C., more preferably 20 to 30° C.; the time is preferably 10 to 20 min, more preferably 15 min.

[0042] After the deprotection reaction, the present invention preferably further comprises nitrogen blowing and redissolution in sequence. In the present invention, the redissolution reagent preferably comprises one or more of methanol, anhydrous ethanol and acetonitrile, and more preferably anhydrous ethanol.

[0043] After obtaining the deprotected idecalcitol preparation raw material fragment 1, the present invention performs liquid chromatography detection on the deprotected idecalcitol preparation raw material fragment 1, and obtains the content of enantiomers and diastereomers in the idecalcitol preparation raw material fragment 1 to be detected based on the area normalization method.

[0044] In the present invention, the parameters of the liquid chromatography detection include:

[0045] The chromatographic column is a polysaccharide derivative chiral chromatographic column, and the polysaccharide derivative chiral chromatographic column is preferably DAICEL CHIRALPAKAD-H; the size of the chromatographic column is preferably 4.6×250 mm, 5 μm; the column temperature of the chromatographic column is preferably 20 to 30° C., and more preferably 25° C.;

[0046] The mobile phase system is preferably a mixture of n-hexane and isopropanol; the volume ratio of n-hexane to isopropanol in the mixture of n-hexane and isopropanol is preferably 80:20 to 84:16, and more preferably 82:18; the flow rate of the mobile phase system is preferably 0.8 to 1.2 mL / min, and more preferably 1.0 mL / min; the elution method is preferably isocratic elution;

[0047] The injection volume is preferably 10±2 μL, more preferably 10 μL;

[0048] The detector is preferably an ultraviolet detector, and the detection wavelength of the ultraviolet detector is preferably 203-207 nm, and more preferably 205 nm.

[0049] In the present invention, the liquid chromatography detection is preferably performed on a high performance liquid chromatograph (HPLC).

[0050] The present invention does not specifically limit the operation of the area normalization method, and a method well known to those skilled in the art may be used.

[0051] The following is a detailed description of the method for analyzing the isomers in the raw material fragment 1 for preparing idecalcitol provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0052] Example 1 Specificity

[0053] 1.1 Liquid chromatography detection conditions

[0054] Chromatographic column: CHIRALPAKAD-H (4.6×250mm, 5μm);

[0055] Column temperature: 25°C;

[0056] Mobile phase system: n-hexane-isopropanol volume ratio 82:18;

[0057] Mobile phase system flow rate: 1.0 mL / min;

[0058] The elution mode is: isocratic elution;

[0059] Injection volume: 10 μL;

[0060] Detector: UV detector;

[0061] Detection wavelength: 205nm;

[0062] 1.2 Solution preparation

[0063] Blank solvent: anhydrous ethanol.

[0064] Deprotected fragment 1 reference solution: Accurately weigh 4 mg of deprotected fragment 1 reference, place in a 10 mL volumetric flask, add anhydrous ethanol to dissolve and dilute to the scale, and shake well.

[0065] Deprotected fragment 1 enantiomer reference solution: Accurately weigh 4 mg of deprotected fragment 1 enantiomer reference, place in a 10 mL volumetric flask, add anhydrous ethanol to dissolve and dilute to the scale, and shake well.

[0066] Deprotected fragment 1 diastereoisomer reference solution: Accurately weigh 4 mg of deprotected fragment 1 diastereoisomer reference, place in a 10 mL volumetric flask, add anhydrous ethanol to dissolve and dilute to the scale, and shake well.

[0067] Mixed solution: Accurately weigh 4 mg of deprotected fragment 1 enantiomer reference substance, place it in a 10 mL volumetric flask, add 1 mL of deprotected fragment 1 diastereoisomer reference substance solution, dissolve and dilute to scale with anhydrous ethanol, shake well, and use as a stock solution. Accurately weigh 4 mg of deprotected fragment 1 reference substance, place it in a 1 mL volumetric flask, add the stock solution to dissolve and dilute to scale, shake well.

[0068] 1.3 Detection

[0069] The blank solvent, the deprotected fragment 1 reference solution, the deprotected fragment 1 enantiomer reference solution, the deprotected fragment 1 diastereoisomer reference solution and the mixed solution were respectively injected into the high performance liquid chromatograph to investigate the separation.

[0070] 1.4 Results

[0071] Figure 1 Table 1 is the HPLC spectrum of the mixed solution.

[0072] Table 1 Chromatographic information of mixed solution

[0073]

[0074] from Figure 1 It can be seen that: the peak with retention time 7.469min is the enantiomer peak of deprotected fragment 1; the peak with retention time 7.896min is the diastereomer peak of deprotected fragment 1; the peak with retention time 11.995min is the deprotected fragment 1 peak; it can be seen that: after deprotection treatment, the enantiomer of deprotected fragment 1, the diastereomer of deprotected fragment 1, and the deprotected fragment 1 can be well separated on the liquid chromatograph using a polysaccharide derivative chiral chromatographic column, and the analytical method provided by the present invention has good specificity.

[0075] Example 2 Linear

[0076] A deprotected fragment 1 reference substance, a deprotected fragment 1 enantiomer reference substance, and a deprotected fragment 1 diastereoisomer reference substance were taken to prepare a series of solutions, and the liquid chromatography detection conditions in 1.1 of Example 1 were used for detection to obtain chromatographic peaks of each series of solutions; a linear regression curve was made with the concentration of the series of solutions as the abscissa and the peak area as the ordinate, and the linear regression equation was calculated to examine the linear relationship. The results are shown in Tables 2 to 4 and Figures 2 to 4 shown.

[0077] Table 2 Linearity information of deprotected fragment 1 reference substance

[0078]

[0079]

[0080] Figure 2 This is the linear spectrum of the deprotected fragment 1 control substance.

[0081] Table 3 Linearity information of deprotected fragment 1 enantiomer reference substance

[0082]

[0083] Figure 3 This is the linear spectrum of the deprotected fragment 1 enantiomer reference substance.

[0084] Table 4 Linearity information of diastereoisomer reference substance of deprotected fragment 1

[0085]

[0086] Figure 4 This is the linear spectrum of the diastereoisomer reference substance of the deprotected fragment 1.

[0087] Example 3 Repeatability

[0088] Take 6 portions of fragment 1 test sample, add 2 mL of solvent ethanol to each, then add 8 mL of 5 mol / L deprotection reagent hydrogen chloride ethanol solution (0.04 mol hydrogen chloride), seal, shake for 5 min, and perform deprotection reaction at 25°C for 15 min; then blow dry with nitrogen, dissolve in 2 mL of anhydrous ethanol, and obtain 6 portions of test sample for the machine.

[0089] According to the liquid chromatography detection conditions in 1.1 of Example 1, 6 samples of the test product were tested, and the results are shown in Table 5. The closeness of the results of the enantiomer of fragment 1 and the diastereomer of fragment 1 was investigated.

[0090] Table 5 Repeatability test data

[0091]

[0092] It can be seen from Tables 1 to 5 that the analytical method provided by the present invention has good specificity, and its linearity and repeatability meet the requirements, and can be used for the detection of fragment 1 isomers.

[0093] Example 4 Accuracy

[0094] Take an appropriate amount of fragment 1 test solution, add high, medium and low concentrations of fragment 1 enantiomer reference substances and fragment 1 diastereoisomer reference substances, and prepare 3 copies in parallel to obtain a test sample spiked solution; the test sample spiked solution was tested according to the liquid chromatography detection conditions in 1.1 of Example 1. The results are shown in Table 6, and the degree of closeness of the results measured by the proposed analytical method to the true value was examined.

[0095] Table 6 Accuracy test data

[0096]

[0097]

[0098] Example 5 Solution Stability

[0099] Take the fragment 1 test sample, add 2 mL of solvent ethanol, then add 8 mL of 5 mol / L deprotection reagent hydrogen chloride ethanol solution (hydrogen chloride is 0.04 mol), seal, shake for 5 minutes, and perform deprotection reaction at 25°C for 15 minutes; then blow dry with nitrogen, dissolve in 2 mL of anhydrous ethanol to obtain the test sample solution, and place at room temperature for 12 hours to investigate the stability of the solution. The results are shown in Table 7.

[0100] Table 7 Solution stability test data

[0101]

[0102]

[0103] Example 6 Limit of Quantitation and Limit of Detection

[0104] A series of concentration solutions were prepared by taking the deprotected fragment 1 enantiomer reference substance and the deprotected fragment 1 diastereomer reference substance. When the S / N was close to but not less than 3, it was the detection limit concentration; when the S / N was close to but not less than 10, it was the quantification limit concentration. The results are shown in Table 8.

[0105] Table 8 Quantitation limit and detection limit test data

[0106]

[0107]

[0108] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for analyzing isomers in the raw material fragment 1 for preparing idecalcitol, characterized in that: The following steps are involved: The raw material fragment 1 for preparing idecalcitol to be tested, a solvent and a deprotection reagent are mixed to carry out a deprotection reaction to obtain the deprotected raw material fragment 1 for preparing idecalcitol; The deprotected idecalcitol preparation raw material fragment 1 is subjected to liquid chromatography detection, and based on the area normalization method, the content of the fragment 1 enantiomer and the fragment 1 diastereomer in the idecalcitol preparation raw material fragment 1 to be tested is obtained; The parameters of the liquid chromatography detection include: The chromatographic column is a polysaccharide derivative chiral chromatographic column; The specific structures of the fragment 1, the fragment 1 enantiomer and the fragment 1 diastereomer are as follows: The solvent includes one or more of methanol, ethanol, acetone, tetrahydrofuran, toluene and methyl tert-butyl ether; The deprotection reagent includes one or more of hydrogen chloride, tetrabutylammonium fluoride, ammonium fluoride, pyridine hydrogen fluoride, triethylamine hydrogen fluoride, p-toluenesulfonic acid and camphorsulfonic acid; The parameters of the liquid chromatography detection also include: The mobile phase system is a mixture of n-hexane and isopropanol; the volume ratio of n-hexane to isopropanol in the mixture is 80:20 to 84:16; the flow rate of the mobile phase system is 0.8 to 1.2 mL / min; The elution method is isocratic elution; The detector is an ultraviolet detector; the detection wavelength of the ultraviolet detector is 203-207nm; The column temperature of the chromatographic column is 20-30°C; The injection volume was 10 ± 2 μL.

2. The analysis method according to claim 1, characterized in that The dosage ratio of the raw material fragment 1 for preparing the idecalcitol to be tested and the solvent is 1 g: 1-20 mL.

3. The analysis method according to claim 1, characterized in that The deprotection reagent is calculated based on the effective ingredient, and the usage ratio of the raw material fragment 1 for preparing the idecalciferol to be tested and the deprotection reagent is 1g:0.03-0.1mol.

4. The analysis method according to claim 1, characterized in that The temperature of the deprotection reaction is 10-40° C. and the time is 10-20 min.

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