Method for detecting bromo-phytol in vitamin K1

Abstract

The invention provides a UPLC (Ultra Performance Liquid Chromatography) detection method for bromo-phytol in vitamin K1, and belongs to the technical field of pharmaceutical analysis. According to the method, an octadecyl silane bonded silica gel chromatographic column is adopted, a mobile phase A is a methanol-ethanol-water volume ratio (68-72): (18-22): 10, a mobile phase B is a methanol-ethanol volume ratio of 50: 50, and gradient elution is carried out; the method is used for detecting the bromo-phytol in the vitamin K1. The method has excellent system applicability, specificity, sensitivity, repeatability, accuracy and durability, has good linearity in a wide detection range, can effectively separate and detect bromo-phytol in vitamin K1, and provides safety guarantee for clinical application of vitamin K1.

Description

Technical Field

[0001] The present invention relates to the field of drug detection and analysis, and in particular to a method for detecting brominated phytol in vitamin K1. Background Art

[0002] Vitamin K1 injection is clinically used for bleeding caused by vitamin K deficiency, such as bleeding caused by obstructive jaundice, bile fistula, chronic diarrhea, etc., hypoprothrombinemia caused by coumarins, sodium salicylate, etc., neonatal bleeding, and vitamin K deficiency in the body caused by long-term use of broad-spectrum antibiotics. During the research process, the inventors found that in addition to the known impurities disclosed, vitamin K1 may also contain potential impurities such as bromophytol, whose structure is shown below.

[0003]

[0004] Brominated plant alcohol is a potential mutagenic impurity, and its residual amount in vitamin K1 needs to be controlled to ensure the safety of clinical use of vitamin K1. At present, there is no relevant literature reporting the detection method of brominated plant alcohol, nor is there any pharmacological and toxicological data related to brominated plant alcohol. It is known that the maximum daily dose of vitamin K1 injection is 10 mg. The limit of brominated plant alcohol is 0.015% calculated by the TTC method (toxicological concern threshold). Since the boiling point of brominated plant alcohol is 390±11℃ (source: ChemicalBook), it is difficult to detect by GC or GC-MS. The inventors tried to detect brominated plant alcohol by LC-MS and found that its structure is difficult to ionize on ESI or APCI sources, that is, it is difficult to form charged ions to be detected by MS. Summary of the invention

[0005] The purpose of the present invention is to provide a method for detecting brominated phytol in vitamin K1 based on the problems existing in the prior art. The method can effectively detect brominated phytol in vitamin K1 and provide safety guarantee for the clinical application of vitamin K1.

[0006] The technical solution adopted by the present invention is as follows:

[0007] A method for detecting brominated phytol in vitamin K1 adopts an octadecylsilane bonded silica gel chromatographic column, uses a methanol-ethanol-water volume ratio of (68-72):(18-22):10 as a mobile phase A, and a methanol-ethanol volume ratio of 50:50 as a mobile phase B, and performs gradient elution to detect brominated phytol in vitamin K1.

[0008] In a preferred embodiment of the present invention, the volume ratio of methanol-ethanol-water in mobile phase A is 70:20:10.

[0009] In a preferred embodiment of the present invention, the gradient elution program is as shown in the following table.

[0010] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 21 100 0 23 30 70 32 30 70 33 100 0 42 100 0

[0011] In a preferred embodiment of the present invention, the flow rate is 0.1-0.5 ml / min; preferably, the flow rate is 0.2±0.05 ml / min.

[0012] In a preferred embodiment of the present invention, the column temperature is 20-40°C; preferably, the column temperature is 30±2°C.

[0013] In a preferred embodiment of the present invention, the detection wavelength is 223±2 nm.

[0014] In a preferred embodiment of the present invention, the specifications of the chromatographic column are: particle size 1.5 μm to 3 μm, inner diameter 1.0 mm to 3.0 mm, and column length 30 mm to 150 mm.

[0015] The beneficial effects of the present invention are:

[0016] The present invention provides a UPLC detection method for brominated phytol in vitamin K1. The method has excellent system applicability and specificity, sensitivity, repeatability, accuracy and durability, and good linearity within a wide detection range. The method can effectively separate and detect brominated phytol in vitamin K1, and provides safety guarantee for the clinical application of vitamin K1. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the detection chromatogram of Comparative Example 1;

[0018] Figure 2 It is the detection chromatogram of Comparative Example 2;

[0019] Figure 3 It is the detection chromatogram of Comparative Example 3;

[0020] Figure 4 It is the detection chromatogram of Comparative Example 4;

[0021] Figure 5 It is the detection chromatogram of Example 1;

[0022] Figure 6 Chromatogram for verification of specificity and system suitability in Example 2;

[0023] Figure 7 It is the linear spectrum in Example 4. DETAILED DESCRIPTION

[0024] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0025] Comparative Example 1

[0026] Test solution: Take an appropriate amount of vitamin K1 raw material (source: Sichuan Ren'an Pharmaceutical Co., Ltd., the same below), dissolve it in ethanol and dilute it to make a solution containing approximately 5 mg of vitamin K1 per 1 ml.

[0027] Mixed solution: Take an appropriate amount of vitamin K1 raw material and an appropriate amount of bromophytol reference substance, place them in the same volumetric flask, add ethanol to dissolve and dilute to make a solution containing approximately 5 mg of vitamin K1 and 2 μg of bromophytol per 1 ml.

[0028] The detection wavelength was 223 nm; the flow rate was 0.2 ml / min; the column temperature was 30 °C, and the injection volume was 5 μl. Different mobile phase compositions were used for isocratic elution. The different mobile phase compositions are as follows:

[0029] Mobile phase ①: acetonitrile-water volume ratio 98:2;

[0030] Mobile phase ②: ethanol-water volume ratio is 90:10;

[0031] Mobile phase ③: methanol-water volume ratio 98:2;

[0032] Mobile phase ④: acetonitrile-ethanol-methanol volume ratio is 20:15:65;

[0033] Mobile phase ⑤: ethanol-methanol-water volume ratio is 10:85:5.

[0034] Test results such as Figure 1 shown. Figure 1 In the figure, AE are the detection chromatograms of mobile phases ①-⑤ respectively. Figure 1 -A and 1-B, using mobile phases ① and ②, the elution time of brominated plant alcohol in the mixed solution was 9.402min and 5.388min, respectively, and the separation degree with the adjacent peak was 0.4 and 0.7, respectively, which was far from meeting the separation requirement (≥1.5), and when using mobile phase ①, the unknown impurity peak with an elution time of 9.732min in the test solution would interfere with the detection of brominated plant alcohol; when using mobile phase ②, the unknown impurity peaks with elution times of 5.270min and 5.823min would interfere with the detection of brominated plant alcohol; Figure 1-C, using mobile phase ③, the elution time of brominated plant alcohol in the mixed solution is 5.655min, and the separation degree from the adjacent unknown peak is 1.3, which does not meet the requirement (≥1.5), and the unknown peak at 5.730min in the test solution partially overlaps with the elution position of brominated plant alcohol; Figure 1 -D, using mobile phase ④, the peak time of brominated plant alcohol in the mixed solution is 3.683min, the unknown peak at 3.595min in the test sample overlaps with the peak position of brominated plant alcohol, and the baseline is abnormally convex between 6min and 10min. Figure 1 -E, using mobile phase ⑤, the peak time of brominated plant alcohol in the mixed solution is 7.827min, and the peaks adjacent to the unknown peaks are 2.0 and 1.5 respectively, both ≥1.5, and the separation degree meets the requirements, but the unknown peak of 7.835min in the test solution may overlap with the peak position of brominated plant alcohol. Based on the above analysis of the mobile phase compositions and their separation conditions, compared with other mobile phase compositions, the mobile phase compositions of ethanol, methanol and water have more prospects for effectively separating brominated plant alcohol.

[0035] Comparative Example 2

[0036] Test solution 1: Take an appropriate amount of vitamin K1 raw material, dissolve it in ethanol and dilute it into a solution containing approximately 20 mg of vitamin K1 per 1 ml.

[0037] Test solution 2: Take an appropriate amount of vitamin K1 raw material, dissolve it in ethanol and dilute it to make a solution containing approximately 5 mg of vitamin K1 per 1 ml.

[0038] Bromophytol solution 1: Take an appropriate amount of bromophytol reference substance, weigh it accurately, dissolve it in ethanol and dilute it to make a solution containing about 10 μg of bromophytol per 1 ml.

[0039] Bromophytol solution 2: Take an appropriate amount of bromophytol solution (about 10 μg / ml), add ethanol to dissolve and dilute to make a solution containing about 0.2 μg of bromophytol per 1 ml.

[0040] The detection wavelength was 223 nm; the column temperature was 30°C; the mobile phase A was methanol; the mobile phase B was ethanol; the mobile phase C was water; and the elution procedure was shown in Table 1.

[0041] Table 1 Elution procedure in comparative example 2

[0042] Time (min) Mobile phase A (%) Mobile phase B (%) Mobile phase C (%) 0 75 18 7 23 75 18 7 28 65 32 3 41 65 32 3 43 75 18 7 50 75 18 7

[0043] The following chromatographic columns were used for detection:

[0044] Column 1: GL Sciences Inertsil C8-3 3 μm, 4.6 mm × 150 mm, flow rate 0.5 ml / min, injection volume 20 μl;

[0045] Column 2: Thermo Hypersil GOLD C8 3 μm, 4.6 mm × 150 mm, flow rate 0.5 ml / min, injection volume 20 μl;

[0046] Column 3: Agilent ZORBAX SB-C8 5 μm, 4.6 mm × 150 mm, flow rate 0.5 ml / min, injection volume 20 μl;

[0047] Column 4: Waters Acquity BEH C18 1.7μm, 2.1mm×100mm, flow rate 0.2ml / min, injection volume 5μl.

[0048] The samples injected into chromatographic columns 1-3 are test solution 1 and brominated plant alcohol solution 1, and the samples injected into chromatographic columns are test solution 2 and brominated plant alcohol solution 2.

[0049] Test results such as Figure 2 As shown, Figure 2 In the figure, AD are the detection chromatograms using chromatographic columns 1-4, respectively. Figure 2 -A, the retention time of brominated plant alcohol is 20.920min, and the unknown peak at 20.867min in the test solution overlaps with the elution position of brominated plant alcohol, which will interfere with the detection of brominated plant alcohol. Figure 2 -B, the retention time of brominated plant alcohol is 10.440min, the elution point of brominated plant alcohol is obviously covered by another peak, and the unknown peak at 10.923min in the test solution partially overlaps with the elution point of brominated plant alcohol, which affects the detection of brominated plant alcohol. Figure 2 -C, the retention time of brominated plant alcohol is 13.770min, the unknown peak at 14.023min in the test solution completely overlaps with the elution position of brominated plant alcohol, and the impurities in the test solution are concentrated at the elution position of brominated plant alcohol, resulting in poor separation effect. Figure 2-D, the retention time of brominated plant alcohol is 11.620min. Although there is an unknown impurity peak with an elution time of 11.657min in the test solution, which partially overlaps with the elution position of brominated plant alcohol, the S / N of the brominated plant alcohol solution (about 0.2μg / ml) is 13.5, which meets the range of 10 to 30, and this concentration is lower than 30% of the limit concentration, which can be used as the quantitative limit and can meet the detection sensitivity requirements. Based on the above analysis, compared with chromatographic columns ① to ③, chromatographic column ④ has a better prospect of meeting the detection sensitivity requirements and effectively separating brominated plant alcohol.

[0050] Comparative Example 3

[0051] Test solution: Take an appropriate amount of vitamin K1 raw material, dissolve it in ethanol and dilute it to make a solution containing approximately 5 mg of vitamin K1 per 1 ml.

[0052] Mixed solution: Take an appropriate amount of vitamin K1 raw material and an appropriate amount of bromophytol reference substance, place them in the same volumetric flask, add ethanol to dissolve and dilute to make a solution containing approximately 5 mg of vitamin K1 and 2 μg of bromophytol per 1 ml.

[0053] The detection wavelength was 223 nm; the flow rate was 0.2 ml / min; the column temperature was 30°C, and the injection volume was 5 μl.

[0054] Mobile phase A was methanol-ethanol-water with a volume ratio of 74:18:8; mobile phase B was methanol-ethanol with a volume ratio of 50:50. The elution program is shown in Table 2.

[0055] Table 2 Elution procedure in comparative example 3

[0056] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 16 100 0 18 30 70 26 30 70 27 100 0 35 100 0

[0057] Test results such as Figure 3 The retention time of brominated phytol is 12.968min, and there are multiple unknown peaks in the test solution (peak times are 12.492min and 12.963min) that overlap with the peak positions of brominated phytol, interfering with the detection of brominated phytol.

[0058] Comparative Example 4

[0059] Mixed solution: Take an appropriate amount of vitamin K1 raw material and an appropriate amount of bromophytol reference substance, place them in the same volumetric flask, add ethanol to dissolve and dilute to make a solution containing approximately 5 mg of vitamin K1 and 2 μg of bromophytol per 1 ml.

[0060] The detection wavelength was 223 nm; the flow rate was 0.2 ml / min; the column temperature was 30°C, and the injection volume was 5 μl.

[0061] Mobile phase A was methanol-ethanol-water with a volume ratio of 70:20:10, and mobile phase B was methanol-ethanol with a volume ratio of 50:50. Two elution processes shown in Tables 3 and 4 were used for elution.

[0062] Table 3 Elution procedure 1 in comparative example 4

[0063] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 18 90 10 22 25 75 32 25 75 33 90 10 42 90 10

[0064] Table 4 Elution procedure 2 in comparative example 4

[0065] Time (min) Mobile phase A (%) Mobile phase B (%) 0 95 5 20 95 5 22 35 65 32 35 65 33 95 5 42 95 5

[0066] Test results such as Figure 4 As shown in Figure A and Figure B are the detection chromatograms of elution program 1 and elution program 2, respectively. Figure 4 -A, using elution procedure 1, the retention time of brominated plant alcohol is 13.782min, and the separation degree with the previous adjacent peak (retention time is 12.993min) and the next adjacent peak (retention time is 15.025min) is 1.2 and 1.4 (both <1.5), which does not meet the separation requirements. Using elution procedure 2, the retention time of brominated plant alcohol is 15.597min, and the separation degree with the previous adjacent peak (retention time is 14.830min) is 1.3, which does not meet the separation requirements.

[0067] Example 1

[0068] Test solution: Take an appropriate amount of vitamin K1 raw material, dissolve it in ethanol and dilute it to make a solution containing approximately 5 mg of vitamin K1 per 1 ml.

[0069] Mixed solution: Take an appropriate amount of vitamin K1 raw material and an appropriate amount of bromophytol reference substance, place them in the same volumetric flask, add ethanol to dissolve and dilute to make a solution containing approximately 5 mg of vitamin K1 and 2 μg of bromophytol per 1 ml.

[0070] Bromophytol solution (about 0.2 μg / ml): Take an appropriate amount of bromophytol reference substance, accurately weigh it, add ethanol to dissolve it and dilute it to make a solution containing about 0.2 μg of bromophytol per 1 ml.

[0071] The detection wavelength was 223 nm; the flow rate was 0.2 ml / min; the column temperature was 30°C, and the injection volume was 5 μl.

[0072] Mobile phase A was methanol-ethanol-water with a volume ratio of 70:20:10; mobile phase B was methanol-ethanol with a volume ratio of 50:50. The elution program shown in Table 5 was used for elution:

[0073] Table 5 Elution procedure in Example 1

[0074] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 21 100 0 23 30 70 32 30 70 33 100 0 42 100 0

[0075] Test results such as Figure 5 shown. Figure 5 It is shown in the results that the retention time of bromophytol is 18.532min, there is no unknown peak interference detection at the peak position, and the separation degree of bromophytol from the front adjacent impurity (retention time is 17.582min) and the rear adjacent impurity (retention time is 20.065min) is 1.6 and 1.5, respectively, both ≥1.5, meeting the separation requirements. In addition, the S / N of bromophytol solution (about 0.2μg / ml) is 14.6, between 10 and 30. It can be seen that the method provided in this embodiment can effectively separate and detect bromophytol in vitamin K1.

[0076] Example 2

[0077] Blank solution: ethanol.

[0078] Stock solution of brominated plant alcohol reference substance (about 150μg / ml): take about 30mg of brominated plant alcohol reference substance, accurately weigh it, put it into a 20ml volumetric flask, add ethanol to dissolve and dilute to the scale, shake well, and use it as stock solution①; then measure 1ml of stock solution①, put it into a 10ml volumetric flask, dilute to the scale with ethanol, shake well, and obtain.

[0079] System suitability solution: Take appropriate amounts of vitamin K1 API and brominated phytol alcohol reference stock solutions, dilute with ethanol to prepare a solution containing approximately 5 mg of vitamin K1 and 2.2 μg of brominated phytol alcohol per 1 ml.

[0080] Bromophytol reference solution (about 0.75 μg / ml): Accurately measure an appropriate amount of the reference stock solution and dilute it with ethanol to make a solution containing about 0.75 μg of brominated phytol per 1 ml.

[0081] The detection method in Example 1 was used to test and verify its specificity and system applicability. The results are shown in Table 6 and Figure 6 shown. Figure 6 In the chromatographic elution curves ①′-④′ respectively represent the blank solution, the system suitability solution, the brominated plant alcohol reference solution and the test solution. The retention time of plant brominated alcohol in the brominated plant alcohol reference solution is 17.820min. In the system suitability solution, the retention time of plant brominated alcohol is 17.783min, and the retention times of the front adjacent and rear adjacent peaks are 16.873min and 19.300min, respectively. The test results show that the blank solution and various impurities have no interference with the detection of brominated plant alcohol; the minimum separation between brominated plant alcohol and the front and rear adjacent peaks is 1.6 (>1.5), which meets the separation requirements. It can be seen that the method has good specificity and system applicability.

[0082] Table 6 Specificity and system suitability verification results

[0083]

[0084] Example 3

[0085] This example verifies the sensitivity of the detection method provided in Example 1.

[0086] (1) Solution preparation

[0087] Take the brominated plant alcohol reference solution and gradually dilute it until the detection limit S / N is in the range of 3 to 10 and the quantification limit S / N is in the range of 10 to 30.

[0088] (2) Results

[0089] The test results are shown in Table 7. The detection limit S / N is in the range of 3 to 10, the quantification limit S / N is in the range of 10 to 30, and the quantification limit concentration is equivalent to the limit concentration of 20.4%, that is, it does not exceed 30% of the limit concentration of brominated phytol, indicating that the detection sensitivity of this method is good.

[0090] Table 7 Detection limit and quantification limit results

[0091] name Concentration μg / ml Peak area S / N Equivalent to limit concentration% Limit of Quantitation 0.1529 0.0477 19.2 20.4 Detection limit 0.0459 0.0195 7.2 6.1

[0092] Example 4

[0093] This example examines the linearity and range of the detection method provided in Example 1.

[0094] (1) Solution preparation

[0095] Take an appropriate amount of brominated plant alcohol reference solution, and take quantitative limit concentration solution, 40%, 100%, 200% and 500% limit concentration solutions as a series of concentration solutions.

[0096] (2) Results

[0097] The test results are shown in Table 8 and Figure 7 As shown. The results show that brominated plant alcohols show good linearity in the range of quantification limit to 500% of the limit concentration, y = 0.2661x + 0.0147 (r = 0.9998), the reported Y-intercept is 0.0147, the slope is 0.2661, the residual square sum is 0.0002, and the Y-intercept does not exceed ± 25% of the 100% limit concentration response value. It can be seen that the method has good linearity.

[0098] Table 8 Linearity and range results

[0099]

[0100] Example 5

[0101] Blank solution, system suitability solution, and brominated plant alcohol reference solution: the same as in Example 2.

[0102] Test solution: Accurately measure an appropriate amount of vitamin K1 raw material, dissolve it in ethanol and dilute it to make a solution containing approximately 5 mg of vitamin K1 per 1 ml.

[0103] The method provided in Example 1 was used for testing, and the test results showed that no brominated plant alcohol was detected in the 6 test solutions. It can be seen that the repeatability of this method is good.

[0104] Example 6

[0105] Accuracy determination sample: Take about 100 mg of the test sample and add an appropriate amount of about 150 μg / ml or 7.5 μg / ml brominated plant alcohol reference substance stock solution to prepare accuracy determination samples with 200%, 100%, and 20% limit concentrations.

[0106] The method provided in Example 1 was used for testing to verify its accuracy. The test results are shown in Table 9. The recovery rates of brominated plant alcohols at three concentration levels were in the range of 95.33% to 104.35%, all between 90.0% and 108.0%, and the maximum RSD of the recovery rates at each concentration level and all concentration levels was 4.6% (≤10.0%). It can be seen that the accuracy of the method meets the requirements.

[0107] Table 9 Accuracy results

[0108]

[0109] Example 7

[0110] Blank solution: ethanol.

[0111] System suitability solution, bromophytol reference solution and test solution: prepare the same as in Example 2.

[0112] Spiked test sample solution: Take about 100 mg of the test sample and add an appropriate amount of about 150 μg / ml brominated plant alcohol reference stock solution to prepare a 100% and limiting concentration spiked test sample solution.

[0113] The test conditions were fine-tuned based on the method provided in Example 1 to verify its durability. The specific test conditions are shown in Table 10.

[0114] Table 10 Durability test conditions

[0115] serial number name Initial conditions Range of variation Inspection conditions 1 Column flow rate 0.2ml / min ±0.05ml / min 0.15ml / min、0.25ml / min 2 Column temperature 30℃ ±2℃ 28℃、32℃ 3 wavelength 223nm ±2nm 221nm、225nm 4 Initial ratio of mobile phase 70:20:10 Methanol and ethanol change ±2 68:22:10、72:18:10

[0116] The test results are shown in Table 11-12. When the column temperature is 28°C and 32°C, the flow rate is 0.15ml / min and 0.25ml / min, the wavelength is 221nm and 225nm, and the initial ratio of the mobile phase is changed to (methanol-ethanol-water) 68:22:10 and 72:18:10:

[0117] Blank solution: no interference at the peak position of brominated plant alcohol;

[0118] System suitability solution: The minimum resolution between brominated plant alcohol and the adjacent peaks is 1.5 (≥1.5);

[0119] Spiked test solution: When the above chromatographic conditions were changed, the recovery of brominated plant alcohols in the spiked test solution ranged from 90.16% to 104.31%, all between 90.0% and 108.0%, and the RSD of the recovery under all conditions was 1.9% (≤10.0%).

[0120] It can be seen that the durability is good under the conditions of column temperature of 28°C to 32°C, flow rate of 0.15ml / min to 0.25ml / min, wavelength of 221nm to 225nm, and initial ratio of mobile phase of 68:22:10 to 72:18:10.

[0121] Table 11 Durability-System Suitability Results

[0122]

[0123] Table 12 Durability - Results of spiked test solution

[0124]

[0125] Note: The original conditions are for sample 2-1 in the accuracy recovery.

Claims

1. A method for detecting brominated phytol in vitamin K1, characterized in that: An octadecylsilane bonded silica gel column was used, with a methanol-ethanol-water volume ratio of (68-72):(18-22):10 as mobile phase A and a methanol-ethanol volume ratio of 50:50 as mobile phase B for gradient elution to detect brominated phytol in vitamin K1.

2. The detection method according to claim 1, characterized in that: The volume ratio of methanol-ethanol-water in mobile phase A was 70:20:

10.

3. The detection method according to claim 1 or 2, characterized in that: The gradient elution program was: 。 4. The detection method according to claim 3, characterized in that: The flow rate is 0.1-0.5 ml / min; preferably, the flow rate is 0.2±0.05 ml / min.

5. The detection method according to claim 3, characterized in that: The column temperature is 20-40°C; preferably, the column temperature is 30±2°C.

6. The detection method according to claim 3, characterized in that: The detection wavelength is 223±2nm.

7. The detection method according to any one of claims 4 to 6, characterized in that: The specifications of the chromatographic column are: particle size 1.5μm~3μm, inner diameter 1.0mm~3.0mm, column length 30mm~150mm.

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