Vitamin K1 and isomer detection and content determination method thereof

Abstract

The invention belongs to the technical field of detection, and particularly discloses a method for detecting vitamin K1 and isomers thereof and determining the content of the vitamin K1, and the method for detecting vitamin K1 and isomers thereof and determining the content of the vitamin K1 adopts liquid chromatography to qualitatively and / or quantitatively detect vitamin K1 and isomers thereof in a to-be-detected sample. Chromatographic conditions of the liquid chromatography are as follows: a chromatographic column is a triacontylsilane bonded silica gel chromatographic column; the column temperature is 25-40 DEG C; the detector is an ultraviolet detector or a diode array detector; performing isocratic elution by taking methanol as a mobile phase A and water as a mobile phase B; according to the method, methanol is taken as a mobile phase A, and water is taken as a mobile phase B, so that the complexity of mobile phase preparation is reduced, rapid separation of VK1 and cis-VK1 can be realized only by isocratic elution of methanol and water in the test, the test efficiency can be improved, damage of the mobile phase containing a strong-solubility organic reagent to an instrument pipeline and a chromatographic column is reduced, and the method is suitable for industrial production. Wide application prospects are realized.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to a method for detecting and determining the content of vitamin K1 and its isomers. Background Art

[0002] Vitamin K1 is a derivative of 2-methyl-1,4-naphthoquinone, with the chemical formula C 31 H 46 O2, which is a fat-soluble vitamin, is a yellow to orange transparent viscous liquid, odorless, easily soluble in chloroform, ether and vegetable oil, slightly soluble in ethanol, and insoluble in water. It will not decompose until heated to 120°C, but it is easily destroyed by ultraviolet rays, so it should be stored away from light. Vitamin K1 is mainly found in vegetables, such as green leafy vegetables, spinach, kale, broccoli and cabbage. After ingestion, vitamin K1 can increase intestinal peristalsis and secretion function. At the same time, it can also affect the metabolism of some hormones, such as delaying the decomposition of glucocorticoids in the liver. Vitamin K1 is mainly used as a food fortifier, mostly used in infant food, with a usage of 420-475μg / kg. It is also used as a vitamin drug for the prevention and treatment of vitamin K1 deficiency, low thrombin, and spontaneous hemorrhage of the newborn. Vitamin K1 is dissolved in the lipids of the mitochondrial membrane, plays a role in electron transfer, increases intestinal motility and secretory function, and affects the metabolism of some hormones, such as delaying the decomposition of glucocorticoids in the liver. It also has an effect similar to hydrocortisone, and long-term injection can increase the endocrine activity of the thyroid gland.

[0003] The existing national standard liquid chromatography testing technology is to dissolve in methanol, use a mobile phase containing methanol, tetrahydrofuran, glacial acetic acid, zinc chloride, and sodium acetate, pass through a C18 column and a zinc reduction column, and determine the content of vitamin K1 under a fluorescence detector with an excitation wavelength of 243nm and an emission wavelength of 430nm. The retention time of the chromatographic peak is used for qualitative analysis and the external standard method is used for quantitative analysis. However, this analysis method uses tetrahydrofuran, which can easily damage the instrument pipeline, reduce the service life of instrument accessories, and cause instrument failure. Summary of the invention

[0004] The invention provides a method for detecting and determining the content of vitamin K1 and its isomers. The invention controls the chromatographic conditions of a liquid chromatography method, adopts methanol as a mobile phase A and water as a mobile phase B, abandons the step of using a zinc reduction column in a test process, thereby reducing the complexity of mobile phase preparation, so that the test can achieve rapid separation of VK1 and cis-VK1 by only relying on isocratic elution of methanol water, is conducive to improving the test efficiency, reduces the damage of the mobile phase containing a strongly soluble organic reagent to the instrument pipeline and the chromatographic column, and has broad application prospects.

[0005] The present invention solves the technical problem by adopting the following technical solutions: A method for detecting and determining the content of vitamin K1 and its isomers, which uses liquid chromatography to qualitatively and / or quantitatively detect vitamin K1 and its isomers in a sample to be tested, wherein the chromatographic conditions of the liquid chromatography are as follows: the chromatographic column is a triacontylsilane bonded silica gel chromatographic column; the column temperature is 25-40° C.; the detector is an ultraviolet detector or a diode array detector; methanol is used as mobile phase A, water is used as mobile phase B for isocratic elution, and the flow rate is 0.5 mL / min-1.0 mL / min; The isomer of vitamin K1 is the cis-isomer of vitamin K1.

[0006] The present invention creatively controls the chromatographic conditions of the liquid chromatography method, adopts methanol as the mobile phase A and water as the mobile phase B, abandons the step of using a zinc reduction column in the test process, thereby reducing the complexity of mobile phase preparation, so that the test can achieve rapid separation of VK1 and cis-VK1 by relying only on isocratic elution of methanol and water, which is beneficial to improving test efficiency and reducing the damage of the mobile phase containing highly soluble organic reagents to instrument pipelines and chromatographic columns, and has broad application prospects.

[0007] As a preferred embodiment of the present invention, the specifications of the chromatographic column are 150 mm×3.0 mm×2.7 μm, and the particle size is 2-3 μm.

[0008] As a preferred embodiment of the present invention, during the isocratic elution, the volume ratio of methanol to water is (90-99): (1-10).

[0009] As a preferred embodiment of the present invention, the chromatographic conditions further include: an injection volume of 5 to 20 μL.

[0010] As a preferred embodiment of the present invention, the chromatographic conditions also include: a detection wavelength of 220-280 nm.

[0011] As a preferred embodiment of the present invention, the sample to be tested is Biostime milk powder.

[0012] As a preferred embodiment of the present invention, the liquid chromatography qualitative and / or quantitative detection is specifically: (1) Vitamin K1 and the cis-isomer of vitamin K1 are prepared into a reference solution using a solvent, and the reference solution is measured using a liquid chromatograph to obtain a chromatogram; (2) preparing the sample to be tested into a test solution, measuring the test solution by liquid chromatography, and obtaining a comparative chromatogram; (3) Compare the chromatograms with the chromatograms for qualitative analysis; (4) Based on the peak area, the area normalization method was used to quantitatively calculate the content of vitamin K1 and vitamin K1 cis isomer.

[0013] The specific calculation method of area normalization method is:

[0014] Where: ω is the mass fraction of VK1 or cis-VK1 in the sample, unit is %; Ai is the peak area of ​​each component of the sample.

[0015] As a preferred embodiment of the present invention, the concentration of vitamin K1 in the reference solution is 0.05-5 μg / mL, and the concentration of the cis isomer of vitamin K1 in the reference solution is 0.05-5 μg / mL.

[0016] As a preferred embodiment of the present invention, the preparation method of the test solution is as follows: (1) accurately weigh 1 g to 5 g of the homogenized sample into a 50 mL centrifuge tube, add 0.25 mL of isotope internal standard solution (1 μg / mL), add 5 mL of warm water to dissolve (5 mL of liquid sample is directly absorbed, and vegetable oil does not need to be diluted with water), add 5 mL of phosphate buffer (pH 8.0), mix well, add 0.2 g of lipase and 0.2 g of amylase (amylase may not be added to samples without starch), cover, vortex for 2 min to 3 min, mix well, and place in a constant temperature water bath oscillator at 35 to 39 ° C for more than 2 h to allow it to be fully enzymatically hydrolyzed; (2) Take out the sample after enzymatic hydrolysis, add 10 mL of ethanol and 1 g of potassium carbonate respectively, mix well, add 10 mL of n-hexane, vortex extract for 10 min, centrifuge at 6000 r / min for 3 min, transfer the supernatant to another 50 mL centrifuge tube, add 10 mL of n-hexane to the lower layer, vortex for 5 min, centrifuge at 6000 r / min for 3 min, combine the supernatants, make up to 25 mL with n-hexane, add 20 mL of water to the above extract, shake for 0.5 min, let stand for stratification, take 5 mL of supernatant into a 10 mL glass test tube, blow nitrogen to dryness, add 1 mL of methanol to dissolve, filter with a 0.22 μm filter membrane to obtain the test solution.

[0017] As a preferred embodiment of the present invention, the solvent is at least one of methanol and ethanol.

[0018] The beneficial effects of the present invention are as follows: the present invention controls the chromatographic conditions of the liquid chromatography method, adopts methanol as the mobile phase A and water as the mobile phase B, abandons the use of the zinc reduction column as a chromatographic column, thereby reducing the complexity of the mobile phase preparation, so that the test can achieve rapid separation of VK1 and cis-VK1 by only relying on isocratic elution of methanol and water, which is beneficial to improving the test efficiency, reducing the damage of the mobile phase containing a strongly soluble organic reagent to the instrument pipeline and the chromatographic column, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the corresponding spectrum of the mixed standard double peaks.

[0020] Figure 2~Figure 3 This is the linear relationship diagram of the reference solution.

[0021] Figure 4~Figure 5 This is the repeatability test chart of low concentration reference solution.

[0022] Figure 6~Figure 7 This is the repeatability test chart of high concentration reference solution.

[0023] Figure 8~Figure 10 This is the detection limit test diagram.

[0024] Fig.11 The graph is the test solution.

[0025] Figures 12-13 Repeatability of the test solution. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0028] In the present invention, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0029] In the present invention, there is no particular limitation on the specific dispersion and stirring treatment methods.

[0030] The reagents or instruments used in the present invention without indicating the manufacturer are all conventional products that can be obtained through commercial purchase.

[0031] Example 1 1. A method for detecting and determining the content of vitamin K1 and its isomers, using liquid chromatography to qualitatively and quantitatively detect vitamin K1 and its isomers in a sample to be tested, specifically: (1) Vitamin K1 and the cis-isomer of vitamin K1 are prepared into a reference solution using a solvent, and the reference solution is measured using a liquid chromatograph to obtain a chromatogram; (2) Prepare the sample to be tested (Biostime milk powder) into a test solution, measure the test solution by liquid chromatography, and obtain a comparative chromatogram; (3) Compare the chromatograms with the chromatograms for qualitative analysis; (4) Based on the peak area, the area normalization method was used to quantitatively calculate the content of vitamin K1 and vitamin K1 cis isomer.

[0032] 1.1 Chromatographic conditions: Detection instrument: Thermo Fisher U3000 high performance liquid chromatograph; Chromatographic column: C30 chromatographic column 150mm×3.0mm×2.7μm; Flow rate: 0.8mL / min; Elution mode: isocratic elution (96:4); Detector: UV detector or diode array detector; Detection wavelength: 254nm Column temperature: 35℃ Injection volume: 10μL Mobile phase A: Chromatographic grade methanol; Mobile phase B: Ultrapure water.

[0033] 1.3 Preparation of reference solution: Weigh 100 mg of cis-VK1 (CAS: 16033-41-3, brand: SHIMADZU, purity: 99%) into a 100 mL volumetric flask, make up to volume with ethanol, and sonicate until the solution becomes clear and transparent without obvious fat sediment to obtain a standard stock solution of vitamin K1 isomers (1 mg / mL); weigh 100 mg of VK1 (CAS: 84-80-0, brand: macklin, purity: 98%) into a 100 mL volumetric flask, make up to volume with ethanol, and sonicate until the solution becomes clear and transparent without obvious fat sediment to obtain a standard stock solution of vitamin K1 (1 mg / mL); then weigh 1 mL of the standard stock solution of vitamin K1 (1 mg / mL) and 1 mL of the standard stock solution of vitamin K1 and its isomers into 100 mL volumetric flasks, make up to volume with ethanol, and sonicate to obtain a mixed standard intermediate solution of vitamin K1 and its isomers (10 μg / mL).

[0034] Take an appropriate amount of vitamin K1 and its isomer mixed standard intermediate solution, and use ethanol to dilute the samples to 10 ng / mL, 40 ng / mL, 100 ng / mL, 200 ng / mL, and 400 ng / mL, respectively, to obtain the reference solution to be tested.

[0035] Take the vitamin K1 isomer standard stock solution and dilute the samples to 40 ng / mL with ethanol to obtain the cis-VK1 standard solution.

[0036] 1.4 Spectrum of the correspondence between the mixed standard double peaks Test samples: cis-VK1 standard solution, 40ng / mL reference solution. The spectrum is shown in Figure 1.

[0037] 1.5 Linearity test Chromatographic conditions: Same as 1.1 Chromatographic conditions.

[0038] Test samples: 10ng / mL, 40ng / mL, 100ng / mL, 200ng / mL, 400ng / mL The test results are as follows Figure 2~3 As shown: the correlation of VK1 is 0.9999 and the correlation of cis-VK1 is 0.9995.

[0039] 1.6 Repeatability test Chromatographic conditions: Same as 1.1 Chromatographic conditions.

[0040] Test samples: 40ng / mL, 400ng / mL Low concentration 40ng / mL: Low concentration test results such as Figure 4 , Figure 5 As shown: VK1: The injection was repeated three times continuously, and the relative standard deviation of retention time was 0.02%, the relative standard deviation of peak area was 1.25%, and the relative standard deviation of plate number was 0.88%.

[0041] cis-VK1: After three consecutive injections, the relative standard deviation of retention time was 0.04%, the relative standard deviation of peak area was 0.48%, and the relative standard deviation of plate number was 1.91%.

[0042] High concentration test results such as Figure 6 , Figure 7 As shown: High concentration 400ng / mL: VK1: After three consecutive injections, the relative standard deviation of retention time was 0.02%, the relative standard deviation of peak area was 0.59%, and the relative standard deviation of plate number was 0.63%.

[0043] cis-VK1: After three consecutive injections, the relative standard deviation of retention time was 0.02%, the relative standard deviation of peak area was 0.65%, and the relative standard deviation of plate number was 0.94%.

[0044] VK1 and cis-VK1 were tested using a dedicated chromatographic column that specifically separates VK1 and its isomers, with stable performance and good reproducibility.

[0045] 1.7 Detection limit test Chromatographic conditions: Same as 1.1 Chromatographic conditions.

[0046] Test sample: 10ng / mL, test results are as follows Figure 8 , 9 , as shown in 10 According to the formula: Detection limit = 3 × standard deviation × (1 + 1 / signal-to-noise ratio) VK1: When tested at a concentration of 10 ng / mL, the average signal-to-noise ratio was 55.93, which means that the detection limit of this substance is 3.05 ng / mL.

[0047] cis-VK1: When tested at a concentration of 10 ng / mL, the average signal-to-noise ratio was 6.6, which means that the detection limit of this substance was 3.45 ng / mL.

[0048] 2. Pretreatment of test samples: 1. Enzymatic hydrolysis: Accurately weigh 1g~5g (accurate to 0.01g, vitamin K1 content not less than 0.01μg) of the homogenized sample into a 50mL centrifuge tube, add 0.25mL of isotope internal standard solution (1μg / mL), add 5mL of warm water to dissolve (5mL of liquid sample is directly absorbed, and vegetable oil does not need to be diluted with water), add 5mL of phosphate buffer (pH8.0), mix well, add 0.2g of lipase and 0.2g of amylase (amylase is not required for samples without starch), cover, vortex for 2min~3min, mix well, and place in a constant temperature water bath oscillator at 37℃±2℃ for more than 2h to allow it to be fully hydrolyzed.

[0049] 2. Extraction: Take out the enzymatically hydrolyzed sample, add 10 mL of ethanol and 1 g of potassium carbonate respectively, mix well, add 10 mL of n-hexane, vortex extraction for 10 minutes, centrifuge at 6000 r / min for 3 minutes, transfer the supernatant to another 50 mL centrifuge tube, add 10 mL of n-hexane to the lower layer, vortex for 5 minutes, centrifuge at 6000 r / min for 3 minutes, combine the supernatants, make up to 25 mL with n-hexane, and wait for purification.

[0050] 3. Purification: Add 20 mL of water to the above extract, shake for 0.5 min, let stand for stratification, take 5 mL of supernatant into a 10 mL glass test tube, blow nitrogen to dryness, add 1 mL of methanol to dissolve, filter with a 0.22 μm filter membrane, and prepare the filtrate for injection. Do a blank test without adding a sample according to the same operation method.

[0051] 2. Preparation of test sample: accurately weigh 1g~5g of the homogenized sample into a 50mL centrifuge tube, add 0.25mL of isotope internal standard solution (1μg / mL), add 5mL of warm water to dissolve (5mL of liquid sample is directly absorbed, and vegetable oil does not need to be diluted with water), add 5mL of phosphate buffer (pH8.0), mix well, add 0.2g of lipase and 0.2g of amylase (amylase can be omitted for samples without starch), cover, vortex for 2min~3min, mix well, and place in a constant temperature water bath oscillator at 35~39℃ for more than 2h to allow it to be fully enzymatically hydrolyzed; (2) Take out the sample after enzymatic hydrolysis, add 10 mL of ethanol and 1 g of potassium carbonate respectively, mix well, add 10 mL of n-hexane, vortex extract for 10 min, centrifuge at 6000 r / min for 3 min, transfer the supernatant to another 50 mL centrifuge tube, add 10 mL of n-hexane to the lower layer, vortex for 5 min, centrifuge at 6000 r / min for 3 min, combine the supernatants, make up to 25 mL with n-hexane, add 20 mL of water to the above extract, shake for 0.5 min, let stand for stratification, take 5 mL of supernatant into a 10 mL glass test tube, blow nitrogen to dryness, add 1 mL of methanol to dissolve, filter with a 0.22 μm filter membrane to obtain the test solution.

[0052] The test spectrum of the test product is as follows Fig.11 Shown 2.1 Repeatability test Chromatographic conditions: Same as 1.1.

[0053] The test results are as follows Figures 12-13 As shown: VK1: The injection was repeated three times continuously, with the relative standard deviation of retention time being 0.15%, the relative standard deviation of sample amount being 0.96%, and the relative standard deviation of plate number being 1.63%.

[0054] cis-VK1: After three consecutive injections, the relative standard deviation of retention time was 0.19%, the relative standard deviation of sample volume was 0.34%, and the relative standard deviation of plate number was 1.60%.

[0055] With the above-mentioned ideal embodiments of the present invention as inspiration, through the above-mentioned description, relevant personnel can make various changes and modifications without departing from the technical concept of the present invention. The technical scope of the present invention is not limited to the contents of the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A method for detecting and determining the content of vitamin K1 and its isomers, characterized in that: The method comprises the following steps: qualitatively and / or quantitatively detecting vitamin K1 and its isomers in a sample to be tested by liquid chromatography, wherein the chromatographic conditions of the liquid chromatography are as follows: the chromatographic column is a triacontylsilane bonded silica gel chromatographic column; the column temperature is 25-40° C.; the detector is an ultraviolet detector or a diode array detector; methanol is used as mobile phase A, water is used as mobile phase B for isocratic elution, and the flow rate is 0.5 mL / min-1.0 mL / min; The isomer of vitamin K1 is the cis-isomer of vitamin K1.

2. The method for detecting and determining the content of vitamin K1 and its isomers according to claim 1, characterized in that: The specifications of the chromatographic column are 150 mm×3.0 mm×2.7 μm, and the particle size is 2-3 μm.

3. The method for detecting and determining the content of vitamin K1 and its isomers according to claim 1, characterized in that: During the isocratic elution, the volume ratio of methanol to water is (90-99): (1-10).

4. The method for detecting and determining the content of vitamin K1 and its isomers according to claim 1, characterized in that: The chromatographic conditions also include: an injection volume of 5 to 20 μL.

5. The method for detecting and determining the content of vitamin K1 and its isomers according to claim 1, characterized in that: The chromatographic conditions also include: a detection wavelength of 220-280 nm.

6. The method for detecting and determining the content of vitamin K1 and its isomers according to claim 1, characterized in that: The sample to be tested is Biostime milk powder.

7. The method for detecting and determining the content of vitamin K1 and its isomers according to claim 1, characterized in that: The liquid chromatography qualitative and / or quantitative detection is specifically: (1) Vitamin K1 and the cis-isomer of vitamin K1 are prepared into a reference solution using a solvent, and the reference solution is measured using a liquid chromatograph to obtain a chromatogram; (2) preparing the sample to be tested into a test solution, measuring the test solution by liquid chromatography, and obtaining a comparative chromatogram; (3) Compare the chromatograms with the chromatograms for qualitative analysis; (4) Based on the peak area, the area normalization method was used to quantitatively calculate the content of vitamin K1 and vitamin K1 cis isomer.

8. The method for detecting and determining the content of vitamin K1 and its isomers according to claim 1, characterized in that: The concentration of vitamin K1 in the reference solution is 0.05-5 μg / mL, and the concentration of the cis isomer of vitamin K1 in the reference solution is 0.05-5 μg / mL.

9. The method for detecting and determining the content of vitamin K1 and its isomers according to claim 1, characterized in that: The preparation method of the test solution is: (1) Accurately weigh 1g~5g of the homogenized sample into a 50mL centrifuge tube, add 0.25mL of isotope internal standard solution (1μg / mL), add 5mL of warm water to dissolve (5mL of liquid sample is directly absorbed, and vegetable oil does not need to be diluted with water), add 5mL of phosphate buffer (pH8.0), mix well, add 0.2g of lipase and 0.2g of amylase (amylase can be omitted for samples without starch), cover, vortex for 2min~3min, mix well, and place in a constant temperature water bath oscillator at 35~39℃ for more than 2h to allow for sufficient enzymatic hydrolysis; (2) Take out the sample after enzymatic hydrolysis, add 10 mL of ethanol and 1 g of potassium carbonate respectively, mix well, add 10 mL of n-hexane, vortex extract for 10 min, centrifuge at 6000 r / min for 3 min, transfer the supernatant to another 50 mL centrifuge tube, add 10 mL of n-hexane to the lower layer, vortex for 5 min, centrifuge at 6000 r / min for 3 min, combine the supernatants, make up to 25 mL with n-hexane, add 20 mL of water to the above extract, shake for 0.5 min, let stand for stratification, take 5 mL of supernatant into a 10 mL glass test tube, blow nitrogen to dryness, add 1 mL of methanol to dissolve, filter with a 0.22 μm filter membrane to obtain the test solution.

10. The method for detecting and determining the content of vitamin K1 and its isomers according to any one of claims 7 to 9, characterized in that: The solvent is at least one of methanol and ethanol.

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