CN118033002A - Separation and determination of calcitriol starting material SM by GC method1And method for impurity thereof - Google Patents
Separation and determination of calcitriol starting material SM by GC method1And method for impurity thereof Download PDFInfo
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- 239000007858 starting material Substances 0.000 title claims abstract description 304
- 239000012535 impurity Substances 0.000 title claims abstract description 230
- 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 title claims abstract description 76
- 229960005084 calcitriol Drugs 0.000 title claims abstract description 76
- 235000020964 calcitriol Nutrition 0.000 title claims abstract description 76
- 239000011612 calcitriol Substances 0.000 title claims abstract description 76
- 238000000034 method Methods 0.000 title claims abstract description 56
- 238000000926 separation method Methods 0.000 title claims abstract description 19
- 238000004817 gas chromatography Methods 0.000 claims abstract description 12
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 9
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 6
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 6
- 238000001514 detection method Methods 0.000 claims description 37
- 230000014759 maintenance of location Effects 0.000 claims description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 4
- 239000012159 carrier gas Substances 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 230000006399 behavior Effects 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 239000000243 solution Substances 0.000 description 38
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- 238000005303 weighing Methods 0.000 description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 238000007865 diluting Methods 0.000 description 6
- 239000013557 residual solvent Substances 0.000 description 6
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- 239000007789 gas Substances 0.000 description 5
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- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 4
- 238000003908 quality control method Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- -1 (tert-butyldimethylsilyl) oxy Chemical group 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 238000009835 boiling Methods 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
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- 238000010521 absorption reaction Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
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- 230000000694 effects Effects 0.000 description 2
- SHZIWNPUGXLXDT-UHFFFAOYSA-N ethyl hexanoate Chemical compound CCCCCC(=O)OCC SHZIWNPUGXLXDT-UHFFFAOYSA-N 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
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- FGWRMMTYIZKYMA-UHFFFAOYSA-N tert-butyl-hydroxy-dimethylsilane Chemical compound CC(C)(C)[Si](C)(C)O FGWRMMTYIZKYMA-UHFFFAOYSA-N 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 description 1
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- NXTYDKDYJFSZSO-UHFFFAOYSA-N butyl-hydroxy-dimethylsilane Chemical group CCCC[Si](C)(C)O NXTYDKDYJFSZSO-UHFFFAOYSA-N 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 239000002302 calcium metabolism regulator Substances 0.000 description 1
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- 239000004205 dimethyl polysiloxane Substances 0.000 description 1
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- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 description 1
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- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for separating and measuring calcitriol starting material SM 1 and impurities thereof by a GC method. The gas chromatography adopts a capillary column as a chromatographic column, and a chromatographic column stationary phase is polyethylene glycol; sequentially separating impurities SM 1e, SM 1d, SM 1b, SM 1a, calcitriol starting material SM 1 and SM 1c by adjusting column temperature and using temperature programming; the column temperature is 100-260 ℃; detecting by using an FID detector after separation; according to the measured chromatograms, calculating the contents of impurities SM 1e, SM 1d, SM 1b and SM 1a according to a main component self-comparison method; the content of impurity SM 1c was calculated by limit method. The method of the invention realizes the effective control of 5 impurities in the calcitriol starting material SM 1, and has the advantages of intuitiveness, specialization, sensitivity, simplicity and convenience and high accuracy.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for separating and measuring calcitriol starting material SM 1 and impurities thereof by a GC method.
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. Calcitriol can be used for treating osteoporosis and rickets. The oral calcitriol can restore normal absorption of calcium in intestinal tract, correct hypocalcemia, relieve musculoskeletal pain, and simultaneously help to reduce excessive serum alkaline phosphatase and thyroxine levels.
SM 1 is an important fragment of synthesized calcitriol, is a key starting material of calcitriol, is named as calcitriol starting material SM 1 (CAT-SM 1).CAT-SM1 for short, separation and impurity control have a key effect on quality and impurity tracing of calcitriol), and the chemical name of CAT-SM 1 is (1R, 3aR,7 aR) -1- ((R) -6-hydroxy-6-methylhept-2-yl) -7-methyl octahydro-1 d-inden-4-ol, and has a molecular formula of C 18H34O2 and a molecular weight of 282.46.CAT-SM 1 as shown in a structural formula II.
Research shows that CAT-SM 1 can introduce specific process impurities in the synthesis process: impurity SM 1a, impurity SM 1b, impurity SM 1c, impurity SM 1d, and impurity SM 1e. The chemical name of the impurity SM 1a is (1R, 3aS,7 aR) -1- ((R) -6-hydroxy-6-methylhept-2-yl) -7-methyl octahydro-1H-inden-4-ol, the molecular formula is C 18H34O2, the molecular weight is 282.46, and the structural formula is shown as formula III. The chemical name of the impurity SM 1b is (1R, 3aR,7 aR) -1- ((S) -6-hydroxy-6-methylhept-2-yl) -7-methyl octahydro-1H-inden-4-ol, the molecular formula is C 18H34O2, the molecular weight is 282.46, and the structural formula is shown as formula IV. The chemical name of the impurity SM 1c is (1R, 3aR,7 aR) -1- ((R) -7-hydroxy-7-methyloctan-2-yl) -7-methyl octahydro-1H-indene-4-alcohol, the molecular formula is C 19H36O2, the molecular weight is 296.50, and the structural formula is shown as formula V. Impurity SM 1d has the chemical name of (6R) -6- ((1R, 3aR,7 aR) -4- ((tert-butyldimethylsilyl) oxy) -7 a-methyl octahydro-1H-inden-1-yl) -2-methyl hept-2-ol, the molecular formula is C 24H48O2 Si, the molecular weight is 396.73, and the structural formula is shown in a formula VI. The chemical name of the impurity SM 1e is (5R) -5- ((1R, 3aR,7 aR) -4- ((tert-butyldimethylsilyl) oxy) -7 a-methyl octahydro-1H-inden-1-yl) ethyl caproate, the molecular formula is C 24H46O3 Si, the molecular weight is 410.71, and the structural formula is shown as a formula VII.
From the analysis of the synthesis process of CAT-SM 1, the possibility that the impurities exist in CAT-SM 1 is high, if the detection control is not performed in CAT-SM 1, the purity and quality of the final product calcitriol can be affected, the safety risk of patients using the medicine is increased, and the difficulty of tracking the impurities of subsequent intermediates and final products is increased. However, no legal standard exists at present, and no literature or patent reports on the detection of calcitriol starting material SM 1 and the 5 impurities in the same analysis method. Only a part of the prior art reports a calcitriol and a detection method of related substances thereof, for example, a patent with publication number of CN116466016B discloses a method for measuring the content of related substances in calcitriol oral liquid, which adopts high performance liquid chromatography to detect calcitriol precursors and degradation impurities in the calcitriol oral liquid. The patent with publication number CN109752471A discloses a calcitriol detection method, which detects calcitriol by an HPLC method, wherein a chromatographic column is a C18 column, a mobile phase A is methanol, and a mobile phase B is methanol and acetonitrile. However, these methods have all been difficult to achieve effective determination of impurities SM 1a, SM 1b, SM 1c, SM 1d and SM 1e in calcitriol.
Therefore, in order to avoid the influence of 5 impurities, namely, impurity SM 1a, impurity SM 1b, impurity SM 1c, impurity SM 1d and impurity SM 1e, on the purity and quality of the product, a new method needs to be established to separate and detect calcitriol starting material SM 1 and 5 impurities thereof so as to control the quality of calcitriol starting materials SM 1 and calcitriol.
Disclosure of Invention
Accordingly, one of the purposes of the present invention is to provide a method for separating calcitriol starting material SM 1 and its impurities by gas chromatography, which can simultaneously achieve effective separation of calcitriol starting material SM 1 and its 5 impurities within 110 minutes or more than 110 minutes, and provides support for qualitative and quantitative detection of calcitriol starting material SM 1 and its 5 impurities.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
A method of separating calcitriol starting material SM 1 and its impurities by gas chromatography, the impurities comprising any one or more of impurities SM 1a, SM 1b, SM 1c, SM 1d, SM 1e; the structural formula of the calcitriol starting material SM 1 is shown in a formula II, the structural formula of the impurity SM 1a is shown in a formula III, the structural formula of the impurity SM 1b is shown in a formula IV, the structural formula of the impurity SM 1c is shown in a formula V, the structural formula of the impurity SM 1d is shown in a formula VI, and the structural formula of the impurity SM 1e is shown in a formula VII;
The gas chromatography adopts a capillary column as a chromatographic column, polyethylene glycol as a chromatographic column stationary phase and nitrogen as carrier gas; sequentially separating impurities SM 1e, SM 1d, SM 1b, SM 1a, calcitriol starting material SM 1 and SM 1c by adjusting column temperature and using temperature programming; the column temperature is 100-260 ℃;
From the above structural formulae, it is apparent that: the ultraviolet absorption of each impurity and the main component (CAT-SM 1) is very weak, and a liquid chromatography-reverse phase system is almost not reserved, and the gas chromatography method is adopted to separate and measure the impurities by considering that the boiling points of CAT-SM 1, impurities SM 1a, impurities SM 1b and impurities SM 1c can meet the gas detection requirement. However, CAT-SM 1, impurities SM 1a and SM 1b are isomers, the boiling points of the impurities SM 1d and SM 1e are much higher than those of other components, and the detection of impurities with large boiling points difference is considered in a gas phase analysis method, so that the better separation and detection of the isomer impurities is a difficult point of the method and is an innovation of the method.
Preferably, the elution time is 110 minutes.
Further, the column box temperature-increasing program is: the initial temperature is 100-150 ℃, and the temperature is maintained for 2-3 minutes; heating to 210-230deg.C at a rate of 1-2deg.C/min, and maintaining for 8-15 min; raising the temperature to 240-260 ℃ at the speed of 4-6 ℃/min, and maintaining for 20-25 minutes.
Still further, the column box temperature program is: the initial temperature is 150 ℃ and is maintained for 2 minutes; raising the temperature to 220 ℃ at a speed of 1 ℃/min, and maintaining for 10 minutes; the temperature was raised to 250℃at a rate of 5℃per minute and maintained for 22 minutes.
Further, the temperature of the sample inlet is 180-230 ℃, preferably 200 ℃.
Further, the flow rate is 0.5 to 3ml/min, preferably 0.9 to 1.1ml/min, more preferably 1.0ml/min.
Preferably, the chromatographic column stationary phase is 100% polyethylene glycol.
More preferably, the column is an Agilent DB-WAX, having a specification of 30m by 0.32mm, 0.25. Mu.m.
Preferably, the sample volume is 2. Mu.l.
The second object of the present invention is to provide a method for qualitatively identifying calcitriol starting material SM 1 and its impurities, which can be used for qualitatively identifying impurities SM 1a, SM 1b, SM 1c, SM 1d and SM 1e, which are less than 0.02% in a sample, and has a low detection limit.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
The method for qualitatively identifying calcitriol starting material SM 1 and impurities thereof comprises the following steps:
(1) Separating calcitriol starting material SM 1, impurity SM 1a, impurity SM 1b, impurity SM 1c, impurity SM 1d, impurity SM 1e by using the method for separating calcitriol starting material SM 1 and impurities thereof by gas chromatography;
(2) Detecting by adopting an FID detector to obtain a chromatogram;
(3) And comparing the chromatographic behaviors of the detection product and the control product, and judging whether the sample contains any one or more components of calcitriol starting material SM 1, impurity SM 1a, impurity SM 1b, impurity SM 1c, impurity SM 1d and impurity SM 1e.
Further, the detector temperature is 200-300 ℃, preferably 250 ℃.
Further, the retention time is sequentially from short to long as the impurities SM 1e, SM 1d, SM 1b, SM 1a, calcitriol starting material SM 1 and SM 1c, and the components can be characterized according to the length of the retention time.
Further, as a preferable technical scheme:
Setting a column box temperature-rising program as follows: the initial temperature is 150 ℃ and is maintained for 2 minutes; raising the temperature to 220 ℃ at a speed of 1 ℃/min, and maintaining for 10 minutes; raising the temperature to 250 ℃ at a rate of 5 ℃/min, and maintaining for 22 minutes; the flow rate is 1.0ml/min, the chromatographic column is Agilent DB-WAX, and the specification is 30m multiplied by 0.32mm,0.25 mu m; the temperature of the sample inlet is 200 ℃; the detector temperature was 250 ℃; any one or more of calcitriol starting material SM 1, impurity SM 1a, impurity SM 1b, impurity SM 1c, impurity SM 1d, impurity SM 1e was isolated and detected as described above and a chromatogram was obtained.
Further, with calcitriol starting material SM 1 as a reference peak, impurity SM 1e was found to have a relative retention time of 0.79±0.02, impurity SM 1d was found to have a relative retention time of 0.81±0.02, impurity SM 1b was found to have a relative retention time of 0.95±0.02, impurity SM 1a was found to have a relative retention time of 0.96±0.02, and impurity SM 1c was found to have a relative retention time of 1.10±0.02.
Further, when the retention time is 61.4±0.5min, it is determined that the impurity SM 1e; when the retention time is 62.9+ -0.5 min, the impurity SM 1d is judged; when the retention time is 73.3+ -0.5 min, the impurity SM 1b is judged; when the retention time is 74.0.+ -. 0.5min, the impurity SM 1a is judged; when the retention time is 77.3+/-0.5 min, judging that the calcitriol starting material SM 1 is used; when the retention time was 85.3.+ -. 0.5min, the impurity SM 1c was judged.
The third objective of the present invention is to provide a method for quantitatively detecting calcitriol starting material SM 1 and its impurities, which can quantitatively detect impurities SM 1a, SM 1b, SM 1c, SM 1d and SM 1e with a content of less than 0.05% in a sample, and has a low quantitative limit.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
The method for quantitatively detecting calcitriol starting material SM 1 and impurities thereof comprises the following steps:
(1) Separating and detecting any one or more of calcitriol starting materials SM 1, impurities SM 1a, impurities SM 1b, impurities SM 1c, impurities SM 1d and impurities SM 1e by adopting the method for qualitatively identifying calcitriol starting materials SM 1 and impurities thereof, so as to obtain a chromatogram;
(2) According to the chromatograms measured in the step (1), calculating the contents of impurities SM 1e, SM 1d, SM 1b and SM 1a according to a main component self-comparison method; the content of impurity SM 1c was calculated by limit method.
Further, before separation, preparing a sample to be tested by taking any one or more of absolute ethyl alcohol, methyl alcohol and methylene dichloride as a diluent.
Preferably, the diluent is absolute ethanol.
Preferably, the sample to be tested comprises a sample solution, a control solution and a control solution.
More preferably, the concentration of the sample solution is 7mg/ml, the concentration of the control solution is 35. Mu.g/ml, and the concentration of the control solution is 1c. Mu.g/ml containing the impurity SM 1c.
Preferably, the method specifically comprises the following steps:
Step 1, precisely weighing a proper amount of a test sample, adding absolute ethyl alcohol for dissolution, and preparing a solution with the concentration of 7mg/ml as a test sample solution;
step 2, precisely measuring a proper amount of sample solution, and diluting the sample solution with absolute ethyl alcohol to prepare a solution with the concentration of 35 mug/ml serving as a control solution;
step 3, precisely weighing a proper amount of an impurity SM 1c reference substance, adding absolute ethyl alcohol for dissolution and quantitatively diluting to prepare a solution with the concentration of 7 mug/ml, and taking the solution as a reference substance solution;
And 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, respectively injecting 2 μl, performing gas chromatographic analysis, and recording a chromatogram. According to the peak area, the contents of impurities SM 1e, SM 1d, SM 1b and SM 1a are respectively calculated by adopting a main component self-comparison method; the content of impurity SM 1c was calculated using the limit method.
Further, the calculation formula of the main component by the self-contrast method is as follows:
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.
Further, whether the content of the impurity SM 1c in the sample to be detected is qualified or not is judged by using a limit method. Specifically, when chromatographic peaks consistent with the retention time of the impurity SM 1c exist in the chromatogram of the detection product, if the peak area of the chromatogram of the detection product is less than or equal to the peak area of the corresponding chromatographic peak in the reference product, the sample is qualified; if the peak area of the chromatogram of the detection product is larger than the peak area of the corresponding chromatographic peak in the reference product, the sample is unqualified.
Further, the impurity SM 1e is in the range of concentration 2.683 μg/ml to 46.953 μg/ml, y= 0.0672X-0.0112, r=1.0000; the impurity SM 1d is in the concentration range of 1.978-34.619 mug/ml, Y=0.0712X-0.0081, R=1.0000; the impurity SM 1b is in the range of 1.925-33.685 mug/ml, Y=0.715X-0.0056, R=1.0000; the impurity SM 1a is in the range of 2.058-36.012 mug/ml, Y=0.0677X-0.0043, R=1.0000; the calcitriol starting material SM 1 is in the concentration range of 3.192 μg/ml to 106.416 μg/ml, y=0.0733x+0.0297, r=1.0000; wherein Y is Y axis and represents peak area; x is X axis, represents concentration, R represents correlation coefficient.
Further, preparing a standard substance to obtain a standard sample chromatogram; the chromatograms obtained from the samples were tested and the content of each component was calculated as peak area according to the principal component self-control method and the limiting method.
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. According to the invention, the calcitriol starting material SM 1 and 5 impurities thereof are separated and measured by utilizing a gas chromatography, so that effective control of impurities SM 1a, SM 1b, SM 1c, SM 1d and SM 1e in the calcitriol starting material SM 1 is realized, the product quality is fundamentally determined, and the method has the advantages of intuitiveness, specificity, sensitivity, simplicity, convenience, high accuracy and the like. The invention provides technical support for quality control of calcitriol starting materials SM 1 and calcitriol and quality improvement of preparation products.
2. According to the detection method disclosed by the invention, the calcitriol starting material SM 1 and 5 impurities thereof can be detected in one set of analysis method, the quantitative limit concentration of the calcitriol starting material SM 1 is 3.1925 mug/ml, the quantitative limit concentration of the impurity SM 1a is 2.0578 mug/ml, the quantitative limit concentration of the impurity SM 1b is 1.9249 mug/ml, the quantitative limit concentration of the impurity SM 1c is 2.2495 mug/ml, the quantitative limit concentration of the impurity SM 1d is 1.9782 mug/ml, and the quantitative limit concentration of the impurity SM 1e is 2.6830 mug/ml. Calcitriol starting material SM 1 had a detection limit concentration of 1.5962 μg/ml, impurity SM 1a had a detection limit concentration of 1.0289 μg/ml, impurity SM 1b had a detection limit concentration of 0.9624 μg/ml, impurity SM 1c had a detection limit concentration of 1.1247 μg/ml, impurity SM 1d had a detection limit concentration of 0.9891 μg/ml, and impurity SM 1e had a detection limit concentration of 1.3415 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 SM 1.
3. The invention can simultaneously realize the effective determination of calcitriol starting material SM 1 and 5 impurities thereof within 110 minutes, and has the characteristics of short separation time and high separation efficiency.
4. The method for separating and measuring calcitriol starting material SM 1 and 5 impurities thereof provided by the invention is not interfered by residual solvents such as blank solvent, tert-butyldimethylsilanol, tetrahydrofuran, ethyl acetate, n-hexane and the like, and has the characteristics of strong specificity and good separation degree.
Drawings
FIG. 1 is a GC diagram of a diluent (absolute ethanol);
FIG. 2 is a GC diagram of a system applicability solution;
FIG. 3 is a GC diagram of a control (control) solution;
FIG. 4 is a GC diagram of a sample solution;
FIG. 5 is a GC diagram of the mixed solution;
fig. 6 and 7 are GC diagrams of the same needle metering limiting solution, wherein fig. 6 is a GC diagram printed on full scale, and fig. 7 is a GC diagram printed on more distinct peaks by adjusting the scale;
fig. 8 and 9 are GC diagrams of the same needle detection limit solution, wherein fig. 8 is a GC diagram printed on full scale, and fig. 9 is a GC diagram printed on more distinct peaks by adjusting the scale;
FIG. 10 is a GC diagram of example 2, column model HP-5;
FIG. 11 is a GC diagram of example 2, column model DB-624;
FIG. 12 is a GC diagram for example 2, column model DB-WAX.
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 molecular formula of the impurity tertiary butyl dimethyl silanol is C 6H16 OSi, the molecular weight is 132.28, and the structural formula is shown in formula VIII;
In the embodiment of the invention, the diluent is absolute ethyl alcohol.
In the embodiment of the invention, the reference substance is derived from Chongqing Hua Bangsheng Kai pharmaceutical Co-Ltd or outsourcing.
In the embodiment of the invention, the preparation method of the solution comprises the following steps:
System applicability solution: a suitable amount of CAT-SM 1 system applicability control I (each 7mg contains about impurity SM 1a, impurity SM 1b, impurity SM 1d, impurity SM 1e each 3.5-105 mug and calcitriol starting material SM 1 mg) is taken, and absolute ethyl alcohol is added to dissolve and dilute the mixture to prepare a solution containing about 7mg of each 1 ml.
Test solution: the product is taken to be proper, precisely weighed, dissolved by absolute ethyl alcohol and quantitatively diluted to prepare a solution with the concentration of about 7mg in each 1 ml.
Control (product) solution: taking appropriate amounts of CAT-SM 1 and impurity SM 1c reference substances, precisely weighing, placing into a same measuring flask, adding absolute ethyl alcohol for dissolving and diluting to prepare a solution containing about CAT-SM 1 mug and impurity SM 1c mug in each 1 ml.
In the embodiment of the invention, the contents of impurities SM 1a, SM 1b, SM 1d and SM 1e are calculated according to a main component self-comparison method, and the calculation formula is as follows:
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.
In the embodiment of the invention, the content of the impurity SM 1c is calculated: the chromatographic peak of the sample solution chromatogram, which is consistent with the retention time of the impurity SM 1c, has a peak area which is not more than that of the impurity SM 1c in the reference solution (0.10%). Impurities SM 1a, SM 1b, SM 1d and SM 1e are not more than 0.15%.
Example 1
(1) Instrument and chromatographic conditions
The gas chromatograph is Agilent 7890B; the chromatographic column is Agilent DB-WAX with the specification of 30m multiplied by 0.32mm and 0.25 mu m; the carrier gas is nitrogen; the column temperature was 150 ℃ at the initial temperature, maintained for 2 minutes, raised to 220 ℃ at a rate of 1 ℃ per minute, maintained for 10 minutes, and raised to 250 ℃ at a rate of 5 ℃ per minute, maintained for 22 minutes; the flow rate is 1.0ml per minute; the temperature of the sample inlet is 200 ℃, the detector is FID, and the temperature of the detector is 250 ℃; the sample volume was 2. Mu.l.
(2) Preparing a solution:
A diluent: absolute ethyl alcohol.
System applicability solution: 7mg of CAT-SM 1 system applicability reference substance (each 7mg contains about impurity SM 1a, impurity SM 1b, impurity SM 1d, impurity SM 1e each 3.5-105 mug and calcitriol starting material SM 1 mg) is taken, placed into a 1ml measuring flask, added with a diluent to be dissolved and diluted to scale, and shaken uniformly to obtain the product.
Test solution: accurately weighing 14.34mg of sample, accurately weighing, placing into 2ml measuring flask, adding diluent, dissolving, diluting to scale, and shaking.
Impurity SM 1c stock solution: accurately weighing impurity SM 1c 13.92.92 mg, placing into a 10ml measuring flask, adding diluent, dissolving, diluting to scale, and shaking.
Control (product) solution: and precisely transferring the sample solution and the impurity SM 1c stock solution respectively by 0.5ml, placing into a 100ml measuring flask, adding a diluent, diluting to a scale, and shaking uniformly to obtain the final product.
(3) Measurement
Respectively taking diluent (absolute ethyl alcohol), system applicability solution, control (product) solution and test sample solution, performing gas chromatographic analysis according to the chromatographic conditions, and recording a chromatogram.
The results are shown in FIGS. 1-4, and the integration results corresponding to FIGS. 2-4 are shown in tables 1-3. From the results, the diluent (absolute ethanol) did not interfere with the detection of the sample. In the system applicability solution chromatogram, the peak-out sequence is sequentially an impurity SM 1e, an impurity SM 1d, an impurity SM 1b, an impurity SM 1a and a CAT-SM 1, the separation degree between the impurity SM 1a and the impurity SM 1b is 1.47, the effective separation can be realized, and the separation degree is more than 1.2; the content of the impurity SM 1a in the sample is 0.11%, the impurities SM 1b, SM 1d and SM 1e are not detected, and the content of other unknown impurities is 0.53% according to the calculation of the main component by a self-comparison method. The impurity SM 1c was not detected as calculated by the limit method.
TABLE 1 integral results Table of FIG. 2
TABLE 2 integral results Table of FIG. 3
TABLE 3 integral results Table of FIG. 4
Example 2 selection of chromatographic column
The detection effect of chromatographic columns of different types/different polarities/different stationary phases is examined in the embodiment, and the detection method comprises the following steps: 1) Model HP-5, stationary phase (5% -phenyl) -methyl polysiloxane, nonpolar chromatographic column; 2) Model DB-624, stationary phase 6% cyanopropylphenyl-94% dimethylpolysiloxane, medium polarity chromatographic column; 3) Model DB-WAX, stationary phase is 100% polyethylene glycol, strong polarity chromatographic column.
As shown in FIGS. 10 to 12 and Table 4, when separation was performed using a column having a model DB-WAX, a stationary phase of 100% polyethylene glycol, and a strong polarity, the column was preferable because each peak type was good and each peak-to-peak separation was good.
TABLE 4 selection results of chromatographic columns
EXAMPLE 3 specificity experiments
The present study performed a proprietary verification of the assay protocol of example 1, including a blank solvent interference experiment, and performed a separation verification of the chromatography system for the impurities that may be present (isomer impurities: impurity SM 1a, impurity SM 1b, impurity SM 1c, impurity SM 1d, impurity SM 1c, impurity t-butyldimethylsilanol, and residual solvents that may be present: tetrahydrofuran, ethyl acetate, n-hexane, may also be present in calcitriol starting material SM 1). The verification method comprises the following steps: weighing a proper amount of each impurity, weighing a proper amount of each residual solvent, adding absolute ethyl alcohol to dilute the solution to prepare a solution of about 20 mug/ml of each impurity and a limiting solution of each residual solvent as a single needle positioning solution, preparing a mixed solution containing calcitriol starting material SM 1, impurity SM 1a, impurity SM 1b, impurity SM 1c, impurity SM 1d, impurity SM 1e and the residual solvents (about 7mg of calcitriol starting material SM 1 containing about 10 mug of each impurity and limiting concentration of each residual solvent in each 1 ml), and respectively injecting samples.
Results: the pattern of the blank solvent (absolute ethyl alcohol) is shown in fig. 1, the pattern of the mixed solution is shown in fig. 5, the specific detection result is shown in table 5, and the result shows that the detection of the sample is not interfered; the separation degree between the main peak and the adjacent impurity peak in the mixed solution is more than 1.5; the separation degree between the impurities SM 1a and SM 1b is more than 1.2; the separation degree of other impurities is more than 1.5. The method has good specificity.
TABLE 5 specificity results Table
Example 4 sensitivity experiment
Weighing calcitriol starting material SM 1 and proper amounts of 5 impurities thereof, respectively adding absolute ethyl alcohol to dissolve and dilute the calcitriol starting material SM 1 and the 5 impurities to prepare a solution containing calcitriol starting material SM 1 3.1893 mug, impurity SM 1a 2.0578 mug, impurity SM 1b 1.9249 mug, impurity SM 1c 2.2495 mug, impurity SM 1d 1.9782 mug and impurity SM 1e 2.6830 mug which are per 1ml as quantitative limiting solution; adding absolute ethyl alcohol to dilute to prepare a solution which is used as a detection limit solution and contains 1ml of calcitriol starting material SM 1 1.5946 mug, impurity SM 1a 1.0289 mug, impurity SM 1b 0.9624 mug, impurity SM 1c 1.1247 mug, impurity SM 1d 01.0289 mug and impurity SM 1e 1.3415 mug.
Results: the quantitative limit patterns are shown in fig. 6 and 7, the detection limit patterns are shown in fig. 8 and 9, and the integration results are shown in tables 6 and 7, respectively. The calcitriol starting material SM 1 has a quantitative limit concentration of 3.1893 mug/ml, the impurity SM 1a has a quantitative limit concentration of 2.0578 mug/ml, the impurity SM 1b has a quantitative limit concentration of 1.9249 mug/ml, the impurity SM 1c has a quantitative limit concentration of 2.2495 mug/ml, the impurity SM 1d has a quantitative limit concentration of 1.9782 mug/ml, the impurity SM 1e has a quantitative limit concentration of 2.6830 mug/ml, the concentration of each component in the sample is less than 0.05%, the RSD of the peak area is less than 10.0%, and the signal to noise ratio is greater than 10, which indicates that each component can be accurately quantified at the concentration. The detection limit concentration of calcitriol starting material SM 1 is 1.5946 mug/ml, the detection limit concentration of impurity SM 1a is 1.0289 mug/ml, the detection limit concentration of impurity SM 1b is 0.9624 mug/ml, the detection limit concentration of impurity SM 1c is 1.1247 mug/ml, the detection limit concentration of impurity SM 1d is 0.9891 mug/ml, the detection limit concentration of impurity SM 1e is 1.3415 mug/ml, the concentration of each component in the sample is less than 0.02%, and the signal to noise ratio is greater than 10, thus indicating that each component can be effectively detected at the concentration. The method has high sensitivity.
TABLE 6 integral result tables of FIGS. 6 and 7
Table 7. Integral result tables of fig. 8 and 9
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Claims (10)
1. A method for separating calcitriol starting material SM 1 and its impurities by gas chromatography, characterized in that the impurities comprise any one or more of impurities SM 1a, SM 1b, SM 1c, SM 1d, SM 1e; the structural formula of the calcitriol starting material SM 1 is shown in a formula II, the structural formula of the impurity SM 1a is shown in a formula III, the structural formula of the impurity SM 1b is shown in a formula IV, the structural formula of the impurity SM 1c is shown in a formula V, the structural formula of the impurity SM 1d is shown in a formula VI, and the structural formula of the impurity SM 1e is shown in a formula VII;
The gas chromatography adopts a capillary column as a chromatographic column, polyethylene glycol as a chromatographic column stationary phase and nitrogen as carrier gas; sequentially separating impurities SM 1e, SM 1d, SM 1b, SM 1a, calcitriol starting material SM 1 and SM 1c by adjusting column temperature and using temperature programming; the column temperature is 100-260 ℃;
2. The method of claim 1, wherein the column box temperature increase program is: the initial temperature is 100-150 ℃, and the temperature is maintained for 2-3 minutes; heating to 210-230deg.C at a rate of 1-2deg.C/min, and maintaining for 8-15 min; raising the temperature to 240-260 ℃ at the speed of 4-6 ℃/min, and maintaining for 20-25 minutes.
3. The method of claim 1, wherein the sample inlet temperature is 180 ℃ to 230 ℃; the flow rate is 0.5-3ml/min.
4. The method for qualitatively identifying calcitriol starting material SM 1 and impurities thereof is characterized by comprising the following steps:
(1) Isolating any one or more of calcitriol starting material SM 1, impurity SM 1a, impurity SM 1b, impurity SM 1c, impurity SM 1d, impurity SM 1e using the method of claim 1;
(2) Detecting by adopting an FID detector to obtain a chromatogram;
(3) And comparing the chromatographic behaviors of the detection product and the control product, and judging whether the sample contains any one or more components of calcitriol starting material SM 1, impurity SM 1a, impurity SM 1b, impurity SM 1c, impurity SM 1d and impurity SM 1e.
5. The method of claim 4, wherein the detector temperature is 200-300 ℃.
6. The method according to claim 4, wherein the impurity SM 1e is determined when a retention time is 61.4±0.5 min; when the retention time is 62.9+ -0.5 min, the impurity SM 1d is judged; when the retention time is 73.3+ -0.5 min, the impurity SM 1b is judged; when the retention time is 74.0.+ -. 0.5min, the impurity SM 1a is judged; when the retention time is 77.3+/-0.5 min, judging that the calcitriol starting material SM 1 is used; when the retention time was 85.3.+ -. 0.5min, the impurity SM 1c was judged.
7. The method for quantitatively detecting calcitriol starting material SM 1 and impurities thereof is characterized by comprising the following steps:
(1) Separating and detecting any one or more of calcitriol starting material SM 1, impurity SM 1a, impurity SM 1b, impurity SM 1c, impurity SM 1d, and impurity SM 1e by the method of any one of claims 4-6 to obtain a chromatogram;
(2) According to the chromatograms measured in the step (1), calculating the contents of impurities SM 1e, SM 1d, SM 1b and SM 1a according to a main component self-comparison method; the content of impurity SM 1c was calculated by limit method.
8. The method of claim 7, wherein the sample to be tested is formulated with any one or more of absolute ethanol, methanol, methylene chloride as a solvent prior to separation.
9. The method of claim 7, wherein when a chromatographic peak with a retention time consistent with that of the impurity SM 1c is present in the detected chromatogram, if the peak area of the detected chromatogram is less than or equal to the peak area of the corresponding chromatographic peak in the control, the sample is qualified; if the peak area of the chromatogram of the detection product is larger than the peak area of the corresponding chromatographic peak in the reference product, the sample is unqualified.
10. The method of claim 7, wherein the impurity SM 1e is in the range of concentration 2.683 μg/ml to 46.953 μg/ml, Y = 0.0672X-0.0112, r = 1.0000; the impurity SM 1d is in the concentration range of 1.978-34.619 mug/ml, Y=0.0712X-0.0081, R=1.0000; the impurity SM 1b is in the range of 1.925-33.685 mug/ml, Y=0.715X-0.0056, R=1.0000; the impurity SM 1a is in the range of 2.058-36.012 mug/ml, Y=0.0677X-0.0043, R=1.0000; the calcitriol starting material SM 1 is in the concentration range of 3.192 μg/ml to 106.416 μg/ml, y=0.0733x+0.0297, r=1.0000; wherein Y is Y axis and represents peak area; x is X axis, represents concentration, R represents correlation coefficient.
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