A method for detecting the content of VK2 cis-isomer by UPLC-CAD-DAD

Abstract

This invention relates to the field of VK2 detection technology and discloses a method for detecting the content of VK2 cis isomers using UPLC-CAD-DAD, comprising the following steps: analyzing a reference solution using an ultra-high performance liquid chromatograph equipped with a CAD detector and a DAD detector to obtain a standard chromatogram; preparing a test sample solution and analyzing the test sample solution using an ultra-high performance liquid chromatograph equipped with a CAD detector and a DAD detector to obtain a chromatogram of the test sample; comparing the chromatogram of the test sample with the standard chromatogram for qualitative analysis of components; and step 3, analyzing the component content using the external standard method. This invention achieves efficient separation of VK2 and the cis isomer by optimizing the ratio of the chromatographic column to the mobile phase, with a resolution R≥1.5, meeting the requirements for quantitative analysis; moreover, the mobile phase composition is stable, ensuring separation effect without complex adjustments, and the operation is convenient.

Description

Technical Field

[0001] This invention relates to the field of VK2 detection technology, and in particular to a method for detecting the content of VK2 cis isomers using the UPLC-CAD-DAD method. Background Technology

[0002] Vitamin K2 is a collective term for a series of compounds containing a 2-methyl-1,4-naphthoquinone core and a varying number of isoprene structural units at the C3 position. Among them, MK-7 (menaquinone-7) is an important form of vitamin K2, with a side chain containing seven isoprene units. Its chemical name is vitamin K2(35), and its chemical formula is C... 46 H 64 O2. Vitamin K2 (using MK-7 as an example) is a yellow crystal at room temperature. It is a fat-soluble vitamin, insoluble in water, but readily soluble in organic solvents (such as chloroform, ether, petroleum ether, acetone, and isopropanol) and vegetable oils. Vitamin K2 is heat-stable, but its structure is easily destroyed by ultraviolet radiation, therefore it needs to be stored away from light. Vitamin K2 is mainly synthesized by microorganisms and is commonly found in foods such as natto, animal liver, egg yolks, and meat. Certain microorganisms in the human gut can also automatically synthesize trace amounts of vitamin K2.

[0003] Vitamin K2 is the only biologically active form of vitamin K and is one of the essential vitamins for the human body. Supplementing with vitamin K2 can promote blood clotting, ensure bone health, reduce cardiovascular disease, improve immunity, and effectively prevent and treat diabetes. In addition, vitamin K2 may help prevent and treat liver disease, rheumatoid arthritis, chronic kidney disease, and neurological disorders. However, excessive intake can also affect metabolism and the absorption of other medications; therefore, it should be used strictly according to prescribed instructions.

[0004] Current national standards use ion-pairing reagents as the mobile phase, combined with a C18 detector and a fluorescence detector, to separate MK-4, MK-7, and MK-9. For existing vitamin K2 assays, the preparation of the mobile phase is quite complex. Both the ion-pairing reagent and the mixture of the four solvents are difficult to prepare and prone to contamination. Furthermore, laboratory verification shows that the water ratio in this assay affects the retention time and resolution between vitamin K2 and its cis isomers for determining the relative content of vitamin K2 and its cis isomers. Currently, the assay methods in national standards primarily target DAD or FLD detectors. CAD, as an emerging detector type, offers high sensitivity and accuracy for the detection of targets such as sugar alcohols. Expanding the detection pathway for VK2 in CAD can effectively broaden the possibility of co-testing VK2 with targets such as sugar alcohols, laying the foundation for expanding the testing range. Therefore, finding a VK2 assay method that can simultaneously function in both CAD and DAD detectors is of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting the content of VK2 cis isomers using UPLC-CAD-DAD. This method can achieve efficient separation of VK2 and its cis isomers, while having low detection limits (0.6 μg / mL for VK2 and 10 μg / mL for VK2 isomers). It also exhibits good repeatability and excellent stability, and can accurately determine the relative content of VK2 and its cis isomers, providing reliable technical support for the quality control of VK2 products.

[0006] To achieve the above objectives, the present invention provides a method for detecting the content of VK2 cis isomers using the UPLC-CAD-DAD method, comprising the following steps: Step 1: Analyze the reference solution using an ultra-high performance liquid chromatograph equipped with a CAD detector and a DAD detector to obtain a standard chromatogram; Step 2: Prepare the test sample into a test solution, analyze the test solution using an ultra-high performance liquid chromatograph equipped with a CAD detector and a DAD detector to obtain the spectrum of the test sample, compare the spectrum of the test sample with the standard color spectrum, and perform qualitative analysis of the components. Step 3: Analyze the component content using the external standard method; The analytical conditions were as follows: the chromatographic column was a C30 column, the column temperature was 28~32℃, the flow rate was 0.35~0.45mL / min, and an anhydrous ethanol solution containing 60~80mM ammonium acetate was used as the mobile phase. The volume ratio of anhydrous ethanol to water in the anhydrous ethanol solution was (90~98):(2~10).

[0007] Furthermore, in steps 1 and 2, the wavelength of the DAD detector is 250~260nm; the temperature of the CAD detector is 38~42℃.

[0008] Furthermore, the wavelength of the DAD detector is 254 nm; the temperature of the CAD detector is 40 °C.

[0009] Furthermore, the CAD detector is VH-D20-A; the DAD detector is VF-D11-A.

[0010] Further, in step 1, the preparation method of the reference solution is as follows: 35-45 mg of vitamin K2 standard and 1.5-2.5 mL of tetrahydrofuran are mixed evenly, and anhydrous ethanol is added to prepare a 100 mL stock solution; the stock solution is then diluted with ethanol to 20 μg / mL, 40 μg / mL, 80 μg / mL, 100 μg / mL, and 160 μg / mL, respectively, to obtain the reference solution.

[0011] Further, in step 2, the preparation method of the test solution is as follows: 12-16 mL of the sample to be tested, 1.5-2.5 mL of tetrahydrofuran, and 48-52 mL of anhydrous ethanol are mixed evenly, and the volume is adjusted to 100 mL with anhydrous ethanol. The mixture is dried to obtain a dried product. The dried product is reconstituted with 1.5-2.5 mL of anhydrous ethanol, and the volume is adjusted to 100 mL with anhydrous ethanol. The mixture is mixed evenly, filtered through a membrane, and the test solution is obtained.

[0012] Furthermore, in step 3, the column temperature is 30℃ and the flow rate is 0.4mL / min.

[0013] Furthermore, in step 3, an anhydrous ethanol solution containing 70 mM ammonium acetate is used as the mobile phase, wherein the volume ratio of anhydrous ethanol to water in the anhydrous ethanol solution is 95:5.

[0014] Furthermore, in step 3, the injection volume of the reference solution and the test solution is 18~22 μL during the analysis.

[0015] Furthermore, in step 3, the chromatographic column has dimensions of 150 mm × 3.0 mm × 2.7 μm.

[0016] The advantages and positive effects of the UPLC-CAD-DAD method for detecting the content of VK2 cis isomers described in this invention are as follows: 1. This invention uses a mixed solution of ethanol aqueous solution and ammonium acetate as the mobile phase (mobile phase A and mobile phase B are not separated, only a mixed solution is used as the mobile phase because CAD baseline is sensitive, so the mobile phase cannot be mixed online on the instrument). It contains only two solvents and one additive, which is simple to prepare, has a low risk of contamination, avoids the corrosiveness of tetrahydrofuran and the damage to the chromatographic column by ion-pair reagents in the prior art, extends the service life of the instrument and consumables, and reduces the detection cost.

[0017] 2. This invention achieves efficient separation of VK2 and its cis isomer by optimizing the ratio of the chromatographic column to the mobile phase, with a resolution R≥1.5, meeting the requirements for quantitative analysis; moreover, the mobile phase composition is stable, and the separation effect can be guaranteed without complex adjustments, making the operation convenient.

[0018] 3. This invention is compatible with both CAD and DAD detectors. The DAD detector detects compounds by their absorption of ultraviolet light at a specific wavelength, responding only to compounds containing chromophores (structures that absorb ultraviolet light). The CAD detector atomizes the eluent and measures the charge carried by the dried particles; its response is independent of the compound structure and can detect most non-volatile and semi-volatile organic compounds. When used in combination, DAD detects hydrophobic drugs with ultraviolet absorption, while CAD detects substances without ultraviolet absorption. A single injection provides a comprehensive understanding of the drug and excipient content in the formulation. Specifically, in this invention, the CAD detector overcomes the DAD detector's insufficient response to substances without ultraviolet absorption, broadening the possibility of co-testing VK2 and sugar alcohols, and expanding the application scenarios. Both detectors exhibit a linear correlation of 0.9990, demonstrating high detection accuracy.

[0019] 4. The detection method of this invention has a low detection limit, with a detection limit of 0.6 μg / mL for VK2 and 10 μg / mL for VK2 isomers. It has good repeatability and excellent stability, and can accurately determine the relative content of VK2 and cis isomers, providing reliable technical support for the quality control of VK2 products.

[0020] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0021] Figure 1 This is a correlation detection diagram for CAD detection in an embodiment of the present invention; Figure 2 This is a repeatability detection diagram for CAD detection in an embodiment of the present invention; Figure 3 This is a CAD detection sample spectrum in an embodiment of the present invention, where A is the test result of the sample and B is the VK2 sensitivity test result; Figure 4 This is a correlation detection graph for DAD detection in an embodiment of the present invention; Figure 5 This is a repeatability detection diagram for DAD detection in an embodiment of the present invention; Figure 6 This is a DAD detection spectrum of the test sample in an embodiment of the present invention, where A is the test result of the test sample and B is the VK2 sensitivity test result; Figure 7 This is a spectrum of vitamin K2, the test sample, in an embodiment of the present invention. Figure 8 This is a spectral diagram of vitamin K2, a standard product, in an embodiment of the present invention. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0024] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0025] In this invention, there are no particular limitations on the specific dispersion and stirring methods.

[0026] Unless otherwise specified, all reagents or instruments used in this invention are commercially available conventional products. Unless otherwise specified, the raw materials used in each comparative example and the parallel experiments of each embodiment are the same commercially available products.

[0027] This invention provides a method for detecting the content of VK2 cis isomers using UPLC-CAD-DAD, comprising the following steps: (1) The reference solution was analyzed by an ultra-high performance liquid chromatograph equipped with CAD and DAD to obtain a standard chromatogram; (2) Prepare the test sample into a test solution, analyze the test solution using an ultra-high performance liquid chromatograph equipped with a CAD detector and a DAD detector, obtain the spectrum of the test sample, compare the spectrum of the test sample with the standard color spectrum, and perform qualitative analysis of the components. (3) Analyze the component content using the external standard method; The analytical conditions were as follows: the chromatographic column was a C30 column, the column temperature was 28~32℃, the flow rate was 0.35~0.45mL / min, and an anhydrous ethanol solution containing 60~80mM ammonium acetate was used as the mobile phase, with the volume ratio of anhydrous ethanol to water in the anhydrous ethanol solution being (90~98):(2~10).

[0028] As a preferred embodiment of this application, when analyzing the component content by external standard method: the chromatographic column is a C30 column, the column temperature is 30℃, and the flow rate is 0.4mL / min.

[0029] As a preferred embodiment of this application, the analysis uses an anhydrous ethanol solution containing 70 mM ammonium acetate as the mobile phase, and further, the volume ratio of anhydrous ethanol to water in the anhydrous ethanol solution is 95:5.

[0030] As a preferred embodiment of this application, the injection volume of the analytical reference solution and the test solution is 18~22 μL.

[0031] As a preferred embodiment of this application, the chromatographic column has dimensions of 150 mm × 3.0 mm × 2.7 μm.

[0032] In a preferred embodiment of this application, the wavelength of the DAD detector is 250~260nm; the temperature of the CAD detector is 38~42℃.

[0033] In a preferred embodiment of this application, the wavelength of the DAD detector is 254 nm; the temperature of the CAD detector is 40 °C.

[0034] In a preferred embodiment of this application, the CAD detector is VH-D20-A; the DAD detector is VF-D11-A.

[0035] As a preferred embodiment of this application, the preparation method of the reference solution is as follows: 35-45 mg of vitamin K2 standard and 1.5-2.5 mL of tetrahydrofuran are mixed evenly, and anhydrous ethanol is added to prepare a 100 mL stock solution; the stock solution is then diluted with ethanol to 20 μg / mL, 40 μg / mL, 80 μg / mL, 100 μg / mL, and 160 μg / mL, respectively, to obtain the reference solution.

[0036] As a preferred embodiment of this application, the preparation method of the test solution is as follows: 12-16 mL of the sample to be tested, 1.5-2.5 mL of tetrahydrofuran, and 48-52 mL of anhydrous ethanol are mixed evenly, and the volume is adjusted to 100 mL with anhydrous ethanol. The mixture is dried to obtain a dried product. The dried product is reconstituted with 1.5-2.5 mL of anhydrous ethanol, and the volume is adjusted to 100 mL with anhydrous ethanol. The mixture is mixed evenly and filtered through a membrane to obtain the test solution.

[0037] The following examples will provide further details.

[0038] Example I. Instruments, Reagents and Methods: 1. Instruments: A Vanquish Horizon ultra-high performance liquid chromatograph equipped with a VH-D20-A CAD and a VF-D11-A DAD.

[0039] The component content was calculated using the external standard method.

[0040] 2. Vitamin K2 stock solution: Weigh 40 mg of vitamin K2 standard (brand: BePure, CAS No.: 2124-57-4) into a 100 mL brown volumetric flask, add 2 mL of tetrahydrofuran and shake to dissolve, dilute to the mark with anhydrous ethanol, mix well, filter through a membrane, and use as a stock solution. Store at 0℃~4℃ protected from light. It is recommended that the storage period not exceed 1 month.

[0041] 3. Vitamin K2 standard solution: Take an appropriate amount of vitamin K2 stock solution and dilute the sample with ethanol to 20 μg / mL, 40 μg / mL, 80 μg / mL, 100 μg / mL and 160 μg / mL respectively to obtain the test reference solution.

[0042] 4. Vitamin K2 test solution: Weigh 15 mL of calcium, vitamin D, and vitamin K oral solution into a 100 mL amber volumetric flask, add 2 mL of tetrahydrofuran and 50 mL of anhydrous ethanol, shake to dissolve, and dilute to the mark with anhydrous ethanol. Pipette 1.0 mL into a centrifuge tube, purge with nitrogen to dryness, redissolve with 2 mL of anhydrous ethanol, transfer to a 10 mL amber volumetric flask, dilute to the mark with anhydrous ethanol, mix well, and filter through a membrane (protect from light; inject immediately after preparation).

[0043] II. Test conditions: Testing instrument: Vanquish high-performance liquid chromatograph; Chromatographic column: Core-shell reversed-phase C30 column, 150 mm × 3.0 mm × 2.7 μm; Flow rate: 0.4 mL / min; Mobile phase: prepared from chromatographic grade anhydrous ethanol-ultrapure water (95:5 v / v) and 70 mM ammonium acetate: isocratic elution; Column temperature: 30℃; Injection volume: 20 μL; DAD detector wavelength: 254nm; CAD detector temperature: 40℃.

[0044] III. CAD Test Results: 3.1 Linearity: Test samples: Vitamin K2 standard solution: 20 μg / mL, 40 μg / mL, 80 μg / mL, 100 μg / mL, 160 μg / mL; like Figure 1 As shown, the correlation of VK2 is 0.9990.

[0045] 3.2 Limit of Detection: Test sample: Vitamin K2 standard solution: 20 μg / mL; According to the formula: Detection limit = Sample concentration ÷ (Signal-to-noise ratio / 3).

[0046] As shown in Table 1, the mean signal-to-noise ratio of VK2 at a concentration of 20 μg / mL was 96.3, indicating that the detection limit of this substance was 0.6 μg / mL. VK2 isomer: tested at a concentration of 20 μg / mL, the average signal-to-noise ratio was 6.0, indicating that the detection limit of this substance was 10 μg / mL.

[0047] Table 1

[0048] 3.3 Repeatability: Test sample: Vitamin K2 standard solution: 40 μg / mL; Six consecutive injections, such as Figure 2 As shown, the relative standard deviation (RSD) of the peak area response value of VK2 is 2.62%, which is less than 3.0%, and the separation degree (R) between VK2 and the cis isomer of VK2 is greater than 1.5.

[0049] 3.4 Spectrum of the test sample: Test sample: Vitamin K2 test solution; The sample was injected three times consecutively, and the chromatogram is shown below. Figure 3 As shown, the relative standard deviation (RSD) of the peak area response value of VK2 is 2.84%, which is less than 3.0%, and the separation degree (R) between VK2 and the VK2 cis isomer is 2.12, which is greater than 1.5.

[0050] IV. DAD Test Results: 4.1 Linearity: Test samples: Vitamin K2 standard solutions: 20 μg / mL, 40 μg / mL, 80 μg / mL, 100 μg / mL, 160 μg / mL.

[0051] like Figure 4 As shown, the correlation of VK2 is 0.9990.

[0052] 4.2 Limit of Detection: Test sample: Vitamin K2 standard solution: 20 μg / mL; According to the formula: Detection limit = Sample concentration ÷ (Signal-to-noise ratio / 3); As shown in Table 2, the mean signal-to-noise ratio of VK2 at a concentration of 20 μg / mL was 96.3, indicating that the detection limit of this substance was 0.6 μg / mL.

[0053] VK2 isomer: tested at a concentration of 20 μg / mL, the average signal-to-noise ratio was 6.0, indicating that the detection limit of this substance was 10 μg / mL.

[0054] Table 2

[0055] 4.3 Repeatability: Test sample: Vitamin K2 standard solution: 40 μg / mL; Six consecutive injections, such as Figure 5 As shown, the relative standard deviation (RSD) of the peak area response value of VK2 is 0.06%, which is less than 3.0%, and the separation degree (R) between VK2 and the cis isomer of VK2 is greater than 1.5.

[0056] 4.4 Spectrum of the test sample: Test sample: Vitamin K2 test solution; The test sample was injected continuously for 6 injections, such as... Figure 6 As shown, the relative standard deviation (RSD) of the peak area response value of VK2 is 1.02%, which is less than 3.0%, and the separation degree (R) between VK2 and the VK2 cis isomer is 2.12, which is greater than 1.5.

[0057] 4.5 Spectrum: Test samples: Vitamin K2 test solution and Vitamin K2 standard solution: 40 μg / mL; The spectra of the test sample and the standard vitamin K2 are as follows: Figure 7 , 8 As shown, the spectra of the test sample and the standard vitamin K2 have similar ripples, with both wavelengths greater than 990 nm, indicating that the purity is qualified.

[0058] This invention is compatible with both CAD and DAD detectors. The DAD detector detects the absorption of ultraviolet light at a specific wavelength by a compound, responding only to compounds containing a chromophore (a structure that absorbs ultraviolet light). The CAD detector atomizes the eluent and measures the charge carried by the dried particles; its response is independent of the compound structure and can detect most non-volatile and semi-volatile organic compounds. When used in combination, DAD detects hydrophobic drugs with ultraviolet absorption, while CAD detects substances without ultraviolet absorption. A single injection provides a comprehensive understanding of the drug and excipient content in the formulation. Specifically, in this invention, the CAD detector overcomes the DAD detector's insufficient response to substances without ultraviolet absorption, broadening the possibility of co-testing VK2 and sugar alcohols, and expanding the application scenarios. Both detectors exhibit a linear correlation of 0.9990, demonstrating high detection accuracy.

[0059] Therefore, the present invention employs the above-mentioned UPLC-CAD-DAD method for detecting the content of VK2 cis isomer. By optimizing the ratio of the chromatographic column to the mobile phase, the efficient separation of VK2 and the cis isomer is achieved, with a resolution R≥1.5, which meets the requirements for quantitative analysis. Furthermore, the mobile phase composition is stable, and the separation effect can be guaranteed without complex adjustments, making the operation convenient.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for determining the content of VK2 cis isomers using UPLC-CAD-DAD, characterized in that, Includes the following steps: Step 1: Analyze the reference solution using an ultra-high performance liquid chromatograph equipped with a CAD detector and a DAD detector to obtain a standard chromatogram; Step 2: Prepare the test sample into a test solution, analyze the test solution using an ultra-high performance liquid chromatograph equipped with a CAD detector and a DAD detector to obtain the chromatogram of the test sample, compare the chromatogram of the test sample with the standard chromatogram, and perform qualitative analysis of the components. Step 3: Analyze the component content using the external standard method; The analytical conditions were as follows: the chromatographic column was a C30 column, the column temperature was 28~32℃, the flow rate was 0.35~0.45mL / min, and an anhydrous ethanol solution containing 60~80mM ammonium acetate was used as the mobile phase. The volume ratio of anhydrous ethanol to water in the anhydrous ethanol solution was (90~98):(2~10).

2. The method for detecting the content of VK2 cis isomers by UPLC-CAD-DAD method according to claim 1, characterized in that, In steps 1 and 2, the wavelength of the DAD detector is 250~260nm; the temperature of the CAD detector is 38~42℃.

3. The method for detecting the content of VK2 cis isomers by UPLC-CAD-DAD method according to claim 2, characterized in that, The wavelength of the DAD detector is 254 nm; the temperature of the CAD detector is 40 °C.

4. The method for detecting the content of VK2 cis isomers by UPLC-CAD-DAD method according to claim 1, characterized in that, The CAD detector is VH-D20-A; the DAD detector is VF-D11-A.

5. The method for detecting the content of VK2 cis isomers by UPLC-CAD-DAD method according to claim 1, characterized in that, In step 1, the preparation method of the reference solution is as follows: 35-45 mg of vitamin K2 standard and 1.5-2.5 mL of tetrahydrofuran are mixed evenly, and anhydrous ethanol is added to prepare a 100 mL stock solution; the stock solution is then diluted with ethanol to 20 μg / mL, 40 μg / mL, 80 μg / mL, 100 μg / mL, and 160 μg / mL, respectively, to obtain the reference solution.

6. The method for detecting the content of VK2 cis isomers by UPLC-CAD-DAD method according to claim 1, characterized in that, In step 2, the preparation method of the test solution is as follows: 12-16 mL of the sample to be tested, 1.5-2.5 mL of tetrahydrofuran, and 48-52 mL of anhydrous ethanol are mixed evenly, and the volume is adjusted to 100 mL with anhydrous ethanol. The mixture is dried to obtain a dried product. The dried product is reconstituted with 1.5-2.5 mL of anhydrous ethanol, and the volume is adjusted to 100 mL with anhydrous ethanol. The mixture is mixed evenly, filtered through a membrane, and the test solution is obtained.

7. The method for detecting the content of VK2 cis isomers by UPLC-CAD-DAD according to claim 1, characterized in that, In step 3, the column temperature was 30℃ and the flow rate was 0.4 mL / min.

8. The method for detecting the content of VK2 cis isomers by UPLC-CAD-DAD according to claim 1, characterized in that, In step 3, an anhydrous ethanol solution containing 70 mM ammonium acetate is used as the mobile phase, and the volume ratio of anhydrous ethanol to water in the anhydrous ethanol solution is 95:

5.

9. The method for detecting the content of VK2 cis isomers by UPLC-CAD-DAD according to claim 1, characterized in that, In step 3, the injection volume of the reference solution and the test solution is 18~22 μL during the analysis.

10. The method for detecting the content of VK2 cis isomers by UPLC-CAD-DAD according to claim 1, characterized in that, In step 3, the chromatographic column has dimensions of 150 mm × 3.0 mm × 2.7 μm.

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