阅读说明：本技术 用于检测脂溶性维生素的试剂盒 (Kit for detecting fat-soluble vitamins ) 是由 韦弢 倪君君 张同兰 谭琛 彭孝枝 于 2020-12-30 设计创作，主要内容包括：本发明提供了用于检测脂溶性维生素的试剂盒,包括：至少三瓶脂溶性维生素的校准品、至少三瓶脂溶性维生素的质控品、混合内标蛋白沉淀剂、流动相添加剂和提取液,其中,提取液用于提取待处理样品中的脂溶性维生素；至少三瓶脂溶性维生素的校准品和至少三瓶脂溶性维生素的质控品均为脂溶性维生素的冻干品,其中,脂溶性维生素包括维生素A和维生素E；混合内标蛋白沉淀剂包括：含有蛋白沉淀剂、维生素A-d3内标物和维生素E-d6内标物的混合溶液；流动相添加剂包括：含有甲酸的甲醇溶液。本发明能够提高试剂盒中脂溶性维生素的稳定性。(The invention provides a kit for detecting fat-soluble vitamins, comprising: the method comprises the following steps of (1) calibrating at least three bottles of fat-soluble vitamins, quality control products of at least three bottles of fat-soluble vitamins, a mixed internal standard protein precipitator, a mobile phase additive and an extracting solution, wherein the extracting solution is used for extracting the fat-soluble vitamins in a sample to be treated; at least three bottles of calibrator of fat-soluble vitamins and at least three bottles of quality control products of the fat-soluble vitamins are freeze-dried products of the fat-soluble vitamins, wherein the fat-soluble vitamins comprise vitamin A and vitamin E; the mixed internal standard protein precipitant comprises: a mixed solution containing a protein precipitant, a vitamin A-d3 internal standard and a vitamin E-d6 internal standard; the mobile phase additive comprises: a methanol solution containing formic acid. The invention can improve the stability of fat-soluble vitamins in the kit.)

1. A kit for detecting a fat-soluble vitamin, comprising:

the method comprises the following steps of (1) calibrating at least three bottles of fat-soluble vitamins, quality control products of at least three bottles of fat-soluble vitamins, a mixed internal standard protein precipitator, a mobile phase additive and an extracting solution, wherein the extracting solution is used for extracting the fat-soluble vitamins in a sample to be treated;

the calibrator of the at least three bottles of fat-soluble vitamins and the quality control product of the at least three bottles of fat-soluble vitamins are freeze-dried products of the fat-soluble vitamins, wherein the fat-soluble vitamins comprise vitamin A and vitamin E;

the mixed internal standard protein precipitant comprises: a mixed solution containing a protein precipitant, a vitamin A-d3 internal standard and a vitamin E-d6 internal standard;

the mobile phase additive comprises: a methanol solution containing formic acid.

2. The kit according to claim 1,

the protein precipitating agent comprises: ethanol solution and/or methanol solution.

3. The kit according to claim 1,

the mobile phase additive comprises: mobile phase additive a and mobile phase additive B;

when the elution mobile phase A used for detecting the sample to be treated comprises a methanol solution, the mobile phase additive A comprises a methanol solution containing 2% -4% of formic acid;

when the eluting mobile phase B for detecting the sample to be treated comprises a methanol solution containing acetonitrile, the mobile phase additive B comprises a methanol solution containing 3% -5% formic acid.

4. The kit according to claim 1,

the extracting solution comprises: a solution of n-hexane containing 0.5% -3% ethanol or epoxyethyl ether containing 0.5% -3% ethanol.

5. The kit according to claim 1,

the freeze-dried product of the fat-soluble vitamin comprises: bovine serum albumin, excipient, antioxidant, cosolvent and fat-soluble vitamin.

6. The kit according to claim 5,

the freeze-dried product of the fat-soluble vitamin is obtained by the following operations:

uniformly mixing the bovine serum albumin, the excipient, the antioxidant, the cosolvent and the purified water under the conditions of light shielding and sealing to obtain a mixture;

adding the fat-soluble vitamins into the mixture and uniformly mixing under the conditions of light protection and sealing to obtain a semi-finished product;

and (3) freeze-drying the semi-finished product under the light condition with the wavelength of more than 500nm and the preset freeze-drying condition to obtain a freeze-dried product of the fat-soluble vitamin.

7. The kit according to claim 6,

the lyophilization conditions include:

freezing at-50 deg.C to-30 deg.C for 2-5h, sublimating at-35 deg.C to-20 deg.C for 6-12h, desorbing and drying at 0-10 deg.C for 3-5h, and intensively drying at 15-25 deg.C for 3-10 h.

8. The kit according to claim 5,

when the excipient comprises mannitol, wherein the mass ratio of the mannitol to the bovine serum albumin comprises 14:100-24: 100;

and/or the presence of a gas in the gas,

when the antioxidant comprises dibutyl hydroxy toluene and butyl hydroxy anisole, the mass ratio of the dibutyl hydroxy toluene to the bovine serum albumin comprises 9:100-12:100, and the mass ratio of the butyl hydroxy anisole to the bovine serum albumin comprises 9:100-12: 100.

9. The kit according to claim 5,

when the cosolvent comprises lecithin and polyoxyethylene sorbitol fatty acid ester, the mass ratio of the lecithin to the bovine serum albumin is 0.2:100-0.5:100, and the mass ratio of the polyoxyethylene sorbitol fatty acid ester to the bovine serum albumin is 0.16:100-0.26: 100.

10. The kit of any one of claims 1 to 9, wherein the kit further comprises: the composite solution is used for pretreating a sample to be treated, wherein the composite solution comprises a methanol solution containing 0-20% of water.

Technical Field

The invention relates to the technical field of biological detection, in particular to a kit for detecting fat-soluble vitamins.

Background

Fat-soluble vitamins are naturally occurring micronutrients in food products that can provide and maintain essential and broad physiological and biochemical functions. The fat-soluble vitamin A is an enzyme prosthetic group, has continuous enzyme composition, plays a role in regulating the metabolism of organisms, and is one of essential important nutrient elements for growth and development. The fat-soluble vitamin E is a general term of tocopherol and tocotrienol, participates in fat metabolism, and maintains the normal function of endocrine.

At present, the stability of fat-soluble vitamins in a kit for detecting the fat-soluble vitamins is generally poor, and therefore, low-temperature cryopreservation, ultra-low-temperature storage and even nitrogen-filled storage are required. However, the degradation rate of the fat-soluble vitamins in the kit is still high when the fat-soluble vitamins are stored under the above conditions, so that the effect of improving the stability of the fat-soluble vitamins in the kit is poor.

Disclosure of Invention

The invention provides a kit for detecting fat-soluble vitamins, which can improve the stability of the fat-soluble vitamins in the kit.

The embodiment of the invention provides a kit for detecting fat-soluble vitamins, which comprises:

the method comprises the following steps of (1) calibrating at least three bottles of fat-soluble vitamins, quality control products of at least three bottles of fat-soluble vitamins, a mixed internal standard protein precipitator, a mobile phase additive and an extracting solution, wherein the extracting solution is used for extracting the fat-soluble vitamins in a sample to be treated;

the calibrator of the at least three bottles of fat-soluble vitamins and the quality control product of the at least three bottles of fat-soluble vitamins are freeze-dried products of the fat-soluble vitamins, wherein the fat-soluble vitamins comprise vitamin A and vitamin E;

the mixed internal standard protein precipitant comprises: a mixed solution containing a protein precipitant, a vitamin A-d3 internal standard and a vitamin E-d6 internal standard;

the mobile phase additive comprises: a methanol solution containing formic acid.

The calibrator of the fat-soluble vitamin and the quality control product of the fat-soluble vitamin in the kit provided by the invention are freeze-dried products, can be placed in a brown ampoule, and can be redissolved by adding an aqueous solution, and the kit has the advantages of instant use, convenience in use, difficulty in degradation and convenience in long-distance transportation.

Specifically, the content of the same fat-soluble vitamin in each bottle of calibration product of the fat-soluble vitamin is different, and the content of the same fat-soluble vitamin in each bottle of quality control product of the fat-soluble vitamin is also different.

Specifically, the kit is stored in a light-proof and sealed manner at the temperature of 2-8 ℃ so as to be beneficial to improving the stability of the fat-soluble vitamins in the kit.

Specifically, in order to more accurately detect the concentration of the fat-soluble vitamin in the sample to be detected, the internal standard substances in the mixed internal standard protein precipitator are all isotopes of each fat-soluble vitamin, so that the influence on the detection of the target substance caused by the reaction between the internal standard substances and the target substance during the detection of the target substance can be avoided.

Preferably, the protein precipitating agent comprises: ethanol solution and/or methanol solution.

Specifically, the protein precipitant in the mixed internal standard protein precipitant can be used for dissolving the vitamin A-d3 internal standard substance and the vitamin E-d6 internal standard substance, and can better remove impurities and purify a target substance. The protein precipitator comprises a single ethanol solution, a single methanol solution or a solution formed by mixing the ethanol solution and the methanol solution in any proportion.

Preferably, the mobile phase additive comprises: mobile phase additive a and mobile phase additive B;

when the elution mobile phase A used for detecting the sample to be treated comprises a methanol solution, the mobile phase additive A comprises a methanol solution containing 2% -4% of formic acid;

when the eluting mobile phase B for detecting the sample to be treated comprises a methanol solution containing acetonitrile, the mobile phase additive B comprises a methanol solution containing 3% -5% formic acid.

For formic acid in the mobile phase additive a, 2% -4% refers to any ratio in the range of 2% to 4%, for example, 2%, 2.5%, 3%, 3.5%, and 4% formic acid.

For formic acid in the mobile phase additive B, 3% -5% refers to any ratio in the range of 2% to 4%, for example, 3%, 3.5%, 4%, 4.5%, and 5% formic acid.

In particular, when fat-soluble vitamins are detected under mass spectrometric conditions, it is generally necessary to add volatile acids, such as trifluoroacetic acid, acetic acid and formic acid, to the elution mobile phase in order to ensure better protonation. Due to long-term use of trifluoroacetic acid, the device is easy to be polluted and not easy to be washed, and even the response of the device under the condition of negative source is influenced. And the additive in the selective elution mobile phase is formic acid because acetic acid is easy to crystallize at the temperature lower than 20 ℃. Meanwhile, in order to ensure that the chromatogram of the fat-soluble vitamin with a perfect peak shape is obtained, the mobile phase additive A is selected to comprise a methanol solution containing 2% -4% formic acid, and the mobile phase additive B is selected to comprise a methanol solution containing 3% -5% formic acid.

Preferably, in order to ensure the concentration of the fat-soluble vitamins extracted from the sample to be tested, the extracting solution comprises: a solution of n-hexane containing 0.5% -3% ethanol or epoxyethyl ether containing 0.5% -3% ethanol.

0.5% -3% of ethanol in the extract refers to any ratio in the range of 0.5% to 3%, for example, 0.5%, 1%, 1.5%, 2%, 2.5% and 3% ethanol.

Preferably, the lyophilizate of fat-soluble vitamins comprises: bovine serum albumin, excipient, antioxidant, cosolvent and fat-soluble vitamin.

Specifically, in order to reduce the matrix effect and improve the accuracy of the detection result, bovine serum albumin is selected as the substitute matrix. In order to solve the problem that vitamin A and vitamin E are easy to oxidize, an antioxidant is added into a freeze-dried product of fat-soluble vitamin. In order to ensure that the fat-soluble vitamin in the freeze-dried product of the fat-soluble vitamin can be well redissolved, a cosolvent is added into the freeze-dried product of the fat-soluble vitamin.

Preferably, the freeze-dried product of the fat-soluble vitamin is obtained by the following operations:

uniformly mixing the bovine serum albumin, the excipient, the antioxidant, the cosolvent and the purified water under the conditions of light shielding and sealing to obtain a mixture;

adding the fat-soluble vitamins into the mixture and uniformly mixing under the conditions of light protection and sealing to obtain a semi-finished product;

and (3) freeze-drying the semi-finished product under the light condition with the wavelength of more than 500nm and the preset freeze-drying condition to obtain a freeze-dried product of the fat-soluble vitamin.

Specifically, the bovine serum albumin, the excipient, the antioxidant, the cosolvent and the purified water are stirred and mixed uniformly in a light-proof container (such as a brown round-bottom flask and a brown mobile phase bottle) which is sealed at normal temperature (20-25 ℃) and normal pressure, and can be stirred for 15-20 min at the rotating speed of 1500-2000 rpm to obtain a mixture; then adding fat-soluble vitamins, stirring and mixing uniformly under the same condition, and stirring at the rotating speed of 1000-1500 rpm for 12-20min to obtain a semi-finished product.

Specifically, the pH value of the mixture needs to be adjusted to be within a range of pH 6.8-pH 7.6 so as to be used for simulating the pH environment of blood to be detected, so that the accuracy of detecting the fat-soluble vitamins in the blood to be detected by using the kit is further improved.

Specifically, the bovine serum albumin, the excipient, the antioxidant, the cosolvent and the purified water are uniformly mixed and then added with the fat-soluble vitamin for uniform mixing, so that the uniform mixing time of the fat-soluble vitamin can be effectively reduced on the premise of ensuring uniform mixing, the exposure time of the fat-soluble vitamin is shortened, the degradation of the fat-soluble vitamin is reduced, and the stability of the fat-soluble vitamin in the freeze-dried product of the fat-soluble vitamin is further improved.

Specifically, the semi-finished product may be dispensed into a bottle (for example, a light-shielding sealable bottle such as a brown ampoule bottle) under a light condition with a wavelength greater than 500nm, and then a half stopper is added, and freeze-drying is performed under a preset freeze-drying condition, so as to directly obtain a calibrator for fat-soluble vitamins and a quality control product for fat-soluble vitamins in the kit. Wherein, the half plug is added to ensure that the moisture in the semi-finished product can be smoothly removed under the preset freeze-drying condition. Or lyophilizing the semi-finished product under the light condition with wavelength of more than 500nm and preset lyophilization condition to obtain lyophilized product of fat-soluble vitamin, and bottling to obtain calibrator of fat-soluble vitamin and quality control product of fat-soluble vitamin.

Particularly, as vitamin A and vitamin E are sensitive to light and easy to oxidize and degrade, the degradation of the vitamin A and the vitamin E can be effectively reduced under the condition of light with the wavelength of more than 500nm, namely, ultraviolet light and blue-violet light below 500nm are eliminated. For example, a safety ultraviolet-protected yellow light may be used to achieve the above-described light conditions.

Preferably, the lyophilization conditions comprise:

freezing at-50 deg.C to-30 deg.C for 2-5h, sublimating at-35 deg.C to-20 deg.C for 6-12h, desorbing and drying at 0-10 deg.C for 3-5h, and intensively drying at 15-25 deg.C for 3-10 h.

Specifically, the semi-finished product can be freeze-dried by using a vacuum freeze dryer, and the vacuum freeze dryer is pre-cooled for 1-3 hours at-30 ℃ to-50 ℃ to provide a stable low-temperature environment for the semi-finished product; then the semi-finished product is put in a freeze dryer, frozen for 2-5h at-50 to-30 ℃, sublimated for 6-12h at-35 to-20 ℃, then analyzed and dried for 3-5h at 0 to 10 ℃, and enhanced dried for 3-10h at 15 to 25 ℃, thus the semi-finished product is dried under vacuum, and is in a high anoxic state, so that the fat-soluble vitamin easy to oxidize is fully protected.

Specifically, the sublimation can directly remove about 90% of water from the frozen solid semi-finished product, and meanwhile, the original solid skeleton structure is kept, the product is spongy, and the rehydration of the obtained product is excellent. In order to improve the storage stability of a freeze-dried product of a fat-soluble vitamin and to prolong the protective period thereof, it is necessary to remove bound water by resolution drying. To further ensure adequate removal of the bound water, enhanced drying is used to more thoroughly remove the remaining bound water.

Preferably, when the excipient comprises mannitol, the mass ratio of mannitol to bovine serum albumin comprises 14:100-24: 100.

When the excipient comprises mannitol, the mass ratio of mannitol to bovine serum albumin includes 14:100-24:100, and 14:100-24:100 refers to any ratio in the range of 14:100 to 24:100, such as 14:100, 16:100, 18:100, 20:100, 22:100, and 24:100.

Preferably, when the antioxidant comprises dibutyl hydroxy toluene and butyl hydroxy anisole, the mass ratio of the dibutyl hydroxy toluene to the bovine serum albumin comprises 9:100-12:100, and the mass ratio of the butyl hydroxy anisole to the bovine serum albumin comprises 9:100-12: 100.

When the antioxidant comprises dibutyl hydroxy toluene and butyl hydroxy anisole, the mass ratio of the dibutyl hydroxy toluene to the bovine serum albumin comprises 9:100-12:100, and the mass ratio of the butyl hydroxy anisole to the bovine serum albumin comprises 9:100-12:100, wherein 9:100-12:100 refers to any ratio in the range of 9:100 to 12:100, such as 9:100, 10:100, 11:100 and 12: 100.

Specifically, the antioxidant is selected from dibutyl hydroxy toluene (BHT) and Butyl Hydroxy Anisol (BHA), and as BHA is stable to heat, BHA is not easily damaged under weak base condition, and does not color under the action of metal ions; moreover, the BHT has high stability and strong oxidation resistance, and the BHT and the BHA can further protect vitamin A from being oxidized and improve the stability of fat-soluble vitamins in the kit.

Preferably, when the cosolvent comprises lecithin and polyoxyethylene sorbitol fatty acid ester, the mass ratio of the lecithin to the bovine serum albumin comprises 0.2:100-0.5:100, and the mass ratio of the polyoxyethylene sorbitol fatty acid ester to the bovine serum albumin comprises 0.16:100-0.26: 100.

When the cosolvent comprises lecithin and polyoxyethylene sorbitol fatty acid ester, the mass ratio of the lecithin to the bovine serum albumin is 0.2:100-0.5:100, and the mass ratio of the polyoxyethylene sorbitol fatty acid ester to the bovine serum albumin is 0.16:100-0.26:100, wherein 0.2:100-0.5:100 refers to any ratio in the range of 0.2:100 to 0.5:100, such as 0.2:100, 0.3:100, 0.4:100 and 0.5: 100; 0.16:100-0.26:100 refers to any ratio in the range of 0.16:100 to 0.26:100, such as 0.16:100, 0.18:100, 0.20:100, 0.22:100, 0.24:100, and 0.26: 100.

Specifically, when the cosolvent comprises lecithin and polyoxyethylene sorbitol fatty acid ester, the quick redissolution of the freeze-dried product of the fat-soluble vitamin can be realized, and the stability of the fat-soluble vitamin in the freeze-dried product of the fat-soluble vitamin is improved.

Preferably, the kit further comprises: the compound solution is used for pretreating a sample to be treated, wherein the compound solution comprises a methanol solution containing 0-20% of water.

For a reconstituted solution, 0% -20% refers to any proportion in the range of 0% to 20%, for example, containing 0%, 5%, 10% and 15% and 20% water.

The kit for detecting fat-soluble vitamins provided by the invention comprises: at least three bottles of calibrator of fat-soluble vitamins, at least three bottles of quality control products of fat-soluble vitamins, mixed internal standard protein precipitator, mobile phase additive and extracting solution for extracting the fat-soluble vitamins in a sample to be treated, wherein the calibrator of the fat-soluble vitamins and the quality control products of the fat-soluble vitamins are freeze-dried products of the fat-soluble vitamins, and the fat-soluble vitamins comprise vitamin A and vitamin E. The fat-soluble vitamins in the kit provided by the invention have higher stability.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a schematic diagram of a kit for detecting fat-soluble vitamins according to an embodiment of the present invention;

FIG. 2 is a flow chart of a method for preparing a lyophilized product of a fat-soluble vitamin according to an embodiment of the present invention;

FIG. 3 is a flow chart of a method for detecting fat-soluble vitamins based on a kit according to an embodiment of the present invention;

FIG. 4 is a graph showing the linear relationship of vitamin A detection based on the kit according to one embodiment of the present invention;

FIG. 5 is a graph showing the linear relationship of vitamin E detection based on the kit according to one embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer and more complete, the technical solutions in the embodiments of the present invention will be described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention, and based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative efforts belong to the scope of the present invention.

At present, because vitamin A and vitamin E are easy to oxidize and are easy to be influenced by light, oxygen and metal ions, the fat-soluble vitamins in the existing kit for detecting the fat-soluble vitamins are usually in a liquid state, and in order to improve the stability of the fat-soluble vitamins in the existing kit, low-temperature cryopreservation, ultralow-temperature preservation and even nitrogen-filled preservation are required, but the stability of the liquid fat-soluble vitamins in the kit preserved by adopting the method is still poor.

One embodiment of the present invention provides a kit for detecting a fat-soluble vitamin, comprising:

the method comprises the following steps of (1) calibrating at least three bottles of fat-soluble vitamins, quality control products of at least three bottles of fat-soluble vitamins, a mixed internal standard protein precipitator, a mobile phase additive and an extracting solution, wherein the extracting solution is used for extracting the fat-soluble vitamins in a sample to be treated;

at least three bottles of calibrator of fat-soluble vitamins and at least three bottles of quality control products of the fat-soluble vitamins are freeze-dried products of the fat-soluble vitamins, wherein the fat-soluble vitamins comprise vitamin A and vitamin E;

the mixed internal standard protein precipitant comprises: a mixed solution containing a protein precipitant, a vitamin A-d3 internal standard and a vitamin E-d6 internal standard;

the mobile phase additive comprises: a methanol solution containing formic acid.

The invention provides a kit for detecting fat-soluble vitamins, which comprises the following components in part by weight: the kit comprises at least three bottles of calibrator of fat-soluble vitamins, at least three bottles of quality control products of the fat-soluble vitamins, a mixed internal standard protein precipitator, a mobile phase additive and an extracting solution for extracting the fat-soluble vitamins in a sample to be treated, wherein the calibrator of the fat-soluble vitamins and the quality control products of the fat-soluble vitamins are freeze-dried products, and are dissolved immediately, convenient to use, not easy to degrade and convenient for long-distance transportation, and the fat-soluble vitamins in the kit have high stability.

The kit for detecting fat-soluble vitamins provided by the present invention is described in detail below with several examples.

Example 1: kit for detecting fat-soluble vitamins

The main components of the kit are shown in table 1:

TABLE 1

Composition of matter	Component identification	Dosage forms	Specification of
				Fat-soluble vitamin calibrator	C1、C2、C3、C4、C5、C6	Solid body	6 bottle
Fat-soluble vitamin quality control product	QC(L)、QC(M)、QC(H)	Solid body	3 bottle
				Mixed internal standard protein precipitant	P14	Liquid, method for producing the same and use thereof	24mL × 1 bottle
Mobile phase additive A	A10	Liquid, method for producing the same and use thereof	8mL × 1 bottle
				Flow additive B	B11	Liquid, method for producing the same and use thereof	8mL × 1 bottle
Extract liquid	T12	Liquid, method for producing the same and use thereof	24mL × 1 bottle
				Compound solution	F13	Liquid, method for producing the same and use thereof	12mL × 1 bottle

Specifically, after the fat-soluble vitamin calibrator and the fat-soluble vitamin quality control product are respectively added with 500 μ L of water for redissolving, the concentration of each fat-soluble vitamin is as follows:

calibrator for fat-soluble vitamins C1: the vitamin A is 0.0281 +/-0.005 mu g/mL, and the vitamin E is 1.125 +/-0.23 mu g/mL;

calibrator for fat-soluble vitamins C2: vitamin A is 0.1125 +/-0.02 mu g/mL, and vitamin E is 2.25 +/-0.45 mu g/mL;

calibrator for fat-soluble vitamins C3: vitamin A is 0.225 +/-0.02 mu g/mL, and vitamin E is 4.5 +/-0.5 mu g/mL;

calibrator for fat-soluble vitamins C4: vitamin A is 0.45 +/-0.05 mu g/mL, and vitamin E is 9.0 +/-0.9 mu g/mL;

calibrator for fat-soluble vitamins C5: vitamin A is 0.9 +/-0.1 mu g/mL, and vitamin E is 18 +/-1.8 mu g/mL;

calibrator for fat-soluble vitamins C6: vitamin A is 1.8 +/-0.2 mu g/mL, and vitamin E is 36 +/-3.6 mu g/mL;

quality control of fat-soluble vitamins QC (L): vitamin A is 0.2 +/-0.02 mu g/mL, and vitamin E is 5 +/-0.5 mu g/mL;

fat-soluble vitamin quality control QC (M): vitamin A is 0.6 +/-0.06 mu g/mL, and vitamin E is 15 +/-1.5 mu g/mL;

fat-soluble vitamin quality control QC (H): vitamin A is 1.2 +/-0.12 mu g/mL, and vitamin E is 30 +/-3 mu g/mL;

mixed internal standard protein precipitant P14: an ethanol mixed solution containing 0.2 μ g/mL vitamin A-d3 internal standard and 0.5 μ g/mL vitamin E-d6 internal standard;

mobile phase additive a (a10) was a methanol solution containing 2.5% formic acid;

mobile phase additive B (B11) was a methanol solution containing 4% formic acid;

the extractive solution T12 is n-hexane solution containing 1% ethanol;

the composite solution F13 is a methanol solution containing 2% of water;

the kit further comprises: instructions for use, certification, lining, packaging box, EP tube, 96-well plate, heat-sealable film, instructions relating to the use, storage, transport process of the kit.

Specifically, the schematic diagram of the detection kit for fat-soluble vitamins is shown in fig. 1, wherein the fat-soluble vitamin calibrator and the fat-soluble vitamin quality control product are lyophilized products, and when in use, the fat-soluble vitamin calibrator and the fat-soluble vitamin quality control product are respectively added with aqueous solutions according to the operation of an instruction for redissolution, and the volume is 0.5 mL/bottle; the components of the kit are stored in a dark and sealed manner at the temperature of 2-8 ℃.

The specification describes the detection method in example 3; the preparation method in example 2 can be used to prepare a calibrator for fat-soluble vitamins and a quality control product for fat-soluble vitamins.

Example 2: freeze-dried product of fat-soluble vitamin and its preparation method

The embodiment of the present invention provides a method for preparing a lyophilized product of fat-soluble vitamins based on the kit of example 1, as shown in fig. 2, including:

step 201: uniformly mixing bovine serum albumin, an excipient, an antioxidant, a cosolvent and purified water under the conditions of light protection and sealing to obtain a mixture;

step 202: adding fat-soluble vitamins into the mixture, and uniformly mixing under the conditions of light protection and sealing to obtain a semi-finished product;

step 203: and (3) freeze-drying the semi-finished product under the light condition with the wavelength of more than 500nm and the preset freeze-drying condition to obtain a freeze-dried product of the fat-soluble vitamin.

Specifically, the excipient comprises mannitol, and the mass ratio of the mannitol to the bovine serum albumin comprises 20: 100; the antioxidant comprises dibutyl hydroxy toluene and butyl hydroxy anisole, the mass ratio of the dibutyl hydroxy toluene to the bovine serum albumin comprises 10:100, and the mass ratio of the butyl hydroxy anisole to the bovine serum albumin comprises 10: 100; the cosolvent comprises lecithin and polyoxyethylene sorbitol fatty acid ester, the mass ratio of the lecithin to the bovine serum albumin is 0.4:100, and the mass ratio of the polyoxyethylene sorbitol fatty acid ester to the bovine serum albumin is 0.2: 100.

Specifically, 50g of bovine serum albumin, 10g of mannitol, 5g of dibutylhydroxytoluene, 5g of butylhydroxyanisole, 0.2g of lecithin, 0.1g of polyoxyethylene sorbitol fatty acid ester and 1000g of purified water are placed in a brown round-bottom flask, the pH of the mixture in the brown round-bottom flask is adjusted to be in a range of pH 6.8-pH 7.6, and then the mixture is stirred for 15min at a rotation speed of 1200rpm under a closed condition at 20 ℃ in the brown round-bottom flask by using magnetic stirring to obtain a mixture; then, according to the content of the vitamins in the calibrator and the quality control product in the embodiment 1, adding corresponding mass of fat-soluble vitamins, and stirring for 5min at the rotating speed of 1200rpm by adopting magnetic stirring under the closed condition of 20 ℃ to obtain a semi-finished product;

sub-packaging the semi-finished product into brown ampoule under the environment of using an ultraviolet-proof yellow safety lamp and clean dust-free, adding a semi-stopper, then placing the bottled semi-finished product into a vacuum freeze-drying machine which is pre-cooled for 2h at-50 ℃, freezing for 3h at-50 ℃, subliming for 7h at-25 ℃, then performing resolution drying for 3h at 10 ℃, performing enhanced drying for 5h at 20 ℃, and tightly sealing the brown ampoule to obtain a calibrator of the fat-soluble vitamins and a quality control product of the fat-soluble vitamins in the kit.

Example 3: method for detecting fat-soluble vitamins

The embodiment of the present invention provides a method for detecting fat-soluble vitamins based on the kit in embodiment 1, as shown in fig. 3, including:

step 301: respectively adding aqueous solution into at least three bottles of calibrator of fat-soluble vitamins in the kit for redissolution to obtain standard working solutions with at least three concentrations;

step 302: respectively adding the mixed internal standard protein precipitant in the kit into the standard working solution with at least three concentrations to obtain standard solutions with at least three concentrations, wherein the amounts of the mixed internal standard protein precipitant in the standard solutions with at least three concentrations are the same;

step 303: preparing a mobile phase by using a mobile phase additive in the kit;

step 304: respectively detecting each standard solution by using a liquid chromatograph-mass spectrometer and a mobile phase under a preset detection condition to obtain a first detection result corresponding to each standard solution;

step 305: fitting a standard curve equation corresponding to each fat-soluble vitamin according to each first detection result, the concentration of each fat-soluble vitamin in the standard solution and the concentration of an internal standard substance of each fat-soluble vitamin in the mixed internal standard protein precipitator;

step 306: determining whether the slope and intercept of a standard curve equation are respectively in a preset range by using at least three bottles of quality control products of fat-soluble vitamins in the kit;

step 307: if so, carrying out centrifugal treatment on the sample to be treated, and taking a first supernatant after centrifugation;

step 308: adding an extracting agent, an extracting solution in the kit and a mixed internal standard protein precipitator into the first supernatant, uniformly mixing by vortex, centrifuging, and taking the centrifuged upper-layer solution as a sample to be detected;

step 309: detecting the sample to be detected by using a liquid chromatograph-mass spectrometer under the detection condition to obtain a second detection result of the sample to be detected;

step 310: and obtaining the concentration of each fat-soluble vitamin in the sample to be detected based on the standard curve equation and the second detection result.

In the embodiment of the invention, the kit for detecting the fat-soluble vitamin can be used for obtaining the standard solutions of the fat-soluble vitamin with at least three concentrations, the standard solutions containing the fat-soluble vitamin with different concentrations are detected by the LC-MS, the first detection result corresponding to the standard solution with each concentration can be obtained, and the standard solution contains the internal standard substance of the fat-soluble vitamin, so that the standard curve equation of each fat-soluble vitamin is obtained by fitting based on the concentrations of the fat-soluble vitamin in the standard solutions with various concentrations, the concentrations of the internal standard substance and a plurality of detection results. Preliminary purification can be performed by performing centrifugal treatment on a sample to be treated, so that the interference of impurities on sample detection is reduced. And adding an extracting agent, an extracting solution in the kit and a mixed internal standard protein precipitator into the centrifuged first supernatant, performing vortex mixing, further purifying, and reducing the interference of impurities on sample detection. And detecting by using a liquid chromatograph-mass spectrometer under the same detection condition as the standard solution to obtain a second detection result, and obtaining the concentration of each fat-soluble vitamin in the sample to be detected based on the standard curve equation and the second detection result. Therefore, the kit can be used for simultaneously and rapidly detecting fat-soluble vitamins.

It is noted that the first supernatant comprises serum or plasma.

Specifically, the detection conditions include:

the liquid phase condition among the detection conditions includes:

the column includes, but is not limited to, a C18 column, packing having a particle size of 2.7 μm, an inner diameter of 3.0mm, and a length of 50 mm;

the column temperature is 25-35 ℃; the flow rate comprises 0.3-0.6mL/min, and the sample amount is 1-10 μ L.

Mass spectrometry detection conditions among detection conditions, comprising:

the LC-MS adopts an ESI ion source, a positive ion scanning mode, a multi-reaction monitoring mode, an ion spray voltage: 3000V-6000V; heating gas temperature: 250 ℃ and 550 ℃; spray voltage: 30-70 psi; air curtain pressure: 20-50 psi.

Specifically, whether the slope and intercept of a standard curve equation are respectively in a preset range is determined by using at least three bottles of fat-soluble vitamin quality control products in the kit, the at least three bottles of fat-soluble vitamin quality control products are detected by using the standard curve equation, whether the concentration of the fat-soluble vitamin quality control products obtained by detection is in the range of the true concentration (target value) plus or minus 1.96SD of the fat-soluble vitamin quality control products is judged, and if yes, the concentration of the fat-soluble vitamin in a sample to be detected by using the standard curve is determined; otherwise, the detection result is inaccurate, and the slope and intercept of the standard curve need to be corrected according to the concentration of the fat-soluble vitamin quality control product and the real concentration of the fat-soluble vitamin quality control product obtained by detection.

The method for detecting fat-soluble vitamins based on the kit of example 1 will be described in detail below with reference to several examples.

3.1 preparation of Standard solutions of Serial concentrations

Respectively adding 0.5mL of aqueous solution into six bottles of fat-soluble vitamin calibrator in example 1 for redissolution to obtain six standard working solutions with different concentrations, respectively transferring 200 mu L of the six standard working solutions with different concentrations and 210 mu L of mixed internal standard protein precipitant in the kit into each hole of a 96-hole plate by using a liquid transfer machine, mixing to prepare six mixed solutions with different concentrations, wherein the amounts of internal standard substances in the six standard solutions are the same, adding an extracting agent and an extracting solution in the kit into the mixed solution, performing vortex mixing, and centrifuging to obtain a centrifuged upper layer solution, namely the standard solution.

It should be noted that the standard solutions with different concentrations can be obtained according to the pretreatment operation when the sample to be treated is treated, that is, the extraction agent, the extraction solution, the vortex time and rotation speed and the centrifugal rotation speed and time after the extraction agent is added, the complex solution, the vortex time and rotation speed and the centrifugal rotation speed and time after the complex solution is added are all consistent with the pretreatment in example 3.3, so as to eliminate the system error and improve the accuracy of the detection result.

3.2 fitting the equation of the standard curve

The six standard solutions in example 3.1 were respectively detected by a liquid chromatograph-mass spectrometer to obtain chromatograms of the six standard solutions of fat-soluble vitamins with different concentrations.

Respectively obtaining peak areas corresponding to each fat-soluble vitamin in six standard solutions and an internal standard substance of each fat-soluble vitamin in a mixed internal standard protein precipitator from the chromatogram of the standard solution of the fat-soluble vitamins, respectively, taking the ratio of the peak area of each fat-soluble vitamin obtained from the chromatogram of the standard solution with each concentration to the chromatographic peak area of the internal standard substance of each fat-soluble vitamin as the ordinate y of a standard curve equation, taking the ratio of the concentration of each fat-soluble vitamin in the standard solution to the concentration of the internal standard substance of each fat-soluble vitamin as the abscissa x of the standard curve equation, performing linear regression on the detected data with different concentrations, fitting to obtain a standard curve equation of y a x + b, and obtaining weight coefficients a and b, wherein the weight coefficient a is the slope of the standard curve equation, the weight coefficient b is the intercept of the standard curve equation.

Specifically, a first standard curve equation for vitamin a and a second standard curve equation for vitamin E can be obtained separately.

The detection conditions include:

a high performance liquid chromatography-tandem mass spectrometry detection system, such as the AB SCIEX Triple quad 4500MD system, is used.

Liquid phase conditions among the detection conditions:

a chromatographic column: agilent Poroshell 120EC-C18, filler particle size 2.7 μm, inner diameter 3.0mm, length 50 mm;

the mobile phase A is an aqueous solution containing 0.1 percent of formic acid;

mobile phase B was methanol solution containing 0.1% formic acid.

Volume ratio of mobile phase a to mobile phase B:

0.00min：10％:90％；0.30min：10％:90％；

0.31min：2％:98％；4.00min：2％:98％；

4.01min：10％:90％；5.00min：10％:90％；

the column temperature was 28 ℃; the flow rate was 0.4mL/min and the amount of sample was 5. mu.L.

Specifically, 8mL of mobile phase additive a (methanol solution containing 2.5% formic acid) in the kit was added to 192mL of purified water to obtain mobile phase a; mobile phase B was obtained by adding 8mL of mobile phase additive B (4% formic acid in acetonitrile) in the kit to 292mL of 25% acetonitrile in methanol.

Mass spectrometric detection conditions of detection conditions:

the LC-MS adopts an ESI ion source, a positive ion scanning mode and a multi-reaction monitoring mode; ion spray voltage: 5500V; heating gas temperature: at 450 ℃; spray voltage: 40 psi; air curtain pressure: 30 psi.

3.3 pretreatment of samples to be tested

3.3.1: taking at least 500 mu L of blood to be processed, centrifuging for 10min at the centrifugation speed of 3000-3500rpm, taking supernatant serum or plasma as a first supernatant, and storing the serum or plasma under the refrigeration of-20 ℃ for standby before analysis.

3.3.2: transferring 500 μ L of the serum or plasma obtained in step 3.3.1 to each well of a 96-well plate by using a pipette gun, adding 100 μ L of purified water, 200 μ L of n-hexane, 210 μ L of the mixed internal standard protein precipitant obtained in example 1 and 200 μ L of the extract obtained in example 1, sealing the 96-well plate with a sealing membrane (for example, sealing with a sealing membrane), vortex-mixing at 2000rpm for 8min, then centrifuging at 4000rpm for 15min, collecting 350 μ L of the centrifuged supernatant (upper organic phase), blow-drying the transferred supernatant with nitrogen, sequentially adding 100 μ L of the reconstituted solution obtained in example 1 (a methanol solution containing 2% water), vortex-mixing at 2000rpm for 4min, and centrifuging at 4000rpm for 10min, and collecting 20 μ L of the centrifuged second supernatant as a sample to be tested.

In the embodiment of the invention, the 96-well plate is adopted to realize the batch pretreatment process of the samples to be detected, a plurality of samples to be detected are treated simultaneously, and the 96-well plate is transposed for automatic sample injection, thereby further simplifying the detection process of the fat-soluble vitamins.

3.4 detection of sample to be detected

And (3) detecting the sample to be detected by using a liquid chromatograph-mass spectrometer under the detection conditions in the embodiment 3.2 to obtain a chromatogram of the sample to be detected.

The chromatographic peak area of each fat-soluble vitamin in the sample to be detected and the chromatographic peak area of the internal standard substance of each fat-soluble vitamin in the sample to be detected can be obtained from the chromatogram of the sample to be detected, the chromatographic peak area of each fat-soluble vitamin in the sample to be detected and the chromatographic peak area of the internal standard substance of each fat-soluble vitamin are taken as the ordinate y and are substituted into the standard curve equation y a x + b in the embodiment 3.2, and the concentration of each fat-soluble vitamin in the sample to be detected can be obtained because the weight coefficients a and b are known. Specifically, the concentration of vitamin A in the sample to be detected can be obtained according to the first standard curve equation, and the concentration of vitamin E in the sample to be detected can be obtained according to the second standard curve equation.

Example 4: description of the stability of fat-soluble vitamins in the intermediate product

The semi-finished product of the calibrator for the fat-soluble vitamin and the semi-finished product of the quality control product for the fat-soluble vitamin in the kit of example 1, which are obtained in example 2, are respectively packaged and then stored at 2-8 ℃, three samples are respectively selected at each concentration point in the semi-finished product at different times to be detected according to the method in example 3, and the percentage of the actual concentration of the fat-soluble vitamin and the theoretical input fat-soluble vitamin concentration (target concentration) obtained by detection is calculated by using a mean value, as shown in tables 2 and 3, wherein table 2 is the ratio of the actual concentration of vitamin a in the semi-finished product to the target concentration, and table 3 is the ratio of the actual concentration of vitamin E in the semi-finished product to the target concentration.

TABLE 2

Number of days

C1

C2

C3

C4

C5

C6

QC(L)

QC(M)

QC(H)

0

100.5％

99.8％

100.2％

98.6％

97.5％

100.1％

99.2％

99.6％

97.9％

1

99.6％

100.1％

98.6％

99.1％

98.2％

100％

100.5％

97.2％

99.1％

3

100.2％

99.5％

99.2％

98.2％

99.3％

100.3％

99.1％

99％

98.7％

5

99.1％

98.6％

98％

97.3％

97.8％

99.2％

98.6％

97.9％

97.1％

7

98.1％

97.6％

96.8％

96.3％

95.8％

98.1％

96.4％

95.9％

96％

TABLE 3

Number of days

C1

C2

C3

C4

C5

C6

QC(L)

QC(M)

QC(H)

0

100.1％

99.8％

99.2％

100.6％

98.5％

100％

99.8％

98.6％

98.9％

1

100％

100.3％

99.3％

99.5％

99.2％

100.4％

99.7％

98.2％

99.1％

3

99.9％

100％

99.7％

98.4％

97.9％

98.2％

98.1％

97.9％

98.7％

5

97.3％

97.6％

98.8％

99％

98.8％

98.1％

97.6％

97.1％

97.3％

7

97.1％

96.4％

95.8％

95.8％

96.1％

96％

95.4％

96.7％

97％

As can be seen from tables 2 and 3, the relative deviations of the actual concentrations of vitamin a and vitamin E from the target values did not exceed 3% when the semi-finished product was left to stand at 2 to 8 ℃ for 5 days, nor did it exceed 5% when it was left to stand for 7 days, and therefore the fat-soluble vitamins in the semi-finished product prepared in example 2 had better stability.

Example 5: description of the moisture content of fat-soluble vitamins in lyophilized products

The 4 kits of example 1 were randomly collected and stored in the dark at room temperature, and the moisture content of the freeze-dried products of the fat-soluble vitamins (including the calibrator of the fat-soluble vitamins and the quality control products of the fat-soluble vitamins) in the randomly-collected kits was measured by volumetric titration using a Karl Fischer moisture meter. As shown in tables 4 and 5, wherein table 4 is the moisture content in the freeze-dried product, and table 5 is the appearance of the freeze-dried product, wherein the appearance comprises appearance and appearance of the packaged freeze-dried product at different times and the state after reconstitution.

TABLE 4

Time

C1

C2

C3

C4

C5

C6

QC(L)

QC(M)

QC(H)

0 month

1.35％

1.56％

1.32％

1.48％

1.62％

1.56％

1.45％

1.38％

1.52％

1 month

1.35％

1.52％

1.36％

1.26％

1.54％

1.52％

1.29％

1.35％

1.49％

3 month

1.64％

1.68％

1.57％

1.48％

1.58％

1.78％

1.61％

1.58％

1.62％

Month 5

1.68％

1.65％

1.72％

1.68％

1.69％

1.72％

1.98％

1.62％

1.64％

6 month

1.7％

1.72％

1.69％

1.72％

1.68％

1.86％

1.74％

1.71％

1.87％

TABLE 5

As can be seen from tables 4 and 5, when the freeze-dried product of the fat-soluble vitamin is stored at room temperature for 6 months in the dark, the moisture content in the freeze-dried product of the fat-soluble vitamin does not exceed 3%, and the freeze-dried product of the fat-soluble vitamin is a white solid and is a transparent liquid after redissolution, so that the stability of the fat-soluble vitamin in the kit is good.

Example 6: description of the stability of fat-soluble vitamins in lyophilisates

The freeze-dried product of the fat-soluble vitamin (including the calibrator of the fat-soluble vitamin and the quality control product of the fat-soluble vitamin) obtained in example 2 in the kit of example 1 is stored at room temperature in the dark, three samples are selected for each concentration point in the freeze-dried product at different times respectively, and the detection is performed according to the method in example 3, and the percentage of the actual concentration of the fat-soluble vitamin obtained by the detection and the theoretical input concentration (target concentration) of the fat-soluble vitamin is calculated by using a mean value, as shown in table 6 and table 7, where table 6 is the ratio of the actual concentration of vitamin a to the target concentration in the freeze-dried product, and table 7 is the ratio of the actual concentration of vitamin E to the target concentration in the freeze-dried product.

TABLE 6

Time

C1

C2

C3

C4

C5

C6

QC(L)

QC(M)

QC(H)

0 month

100％

99.9％

100.1％

99.2％

98.9％

100.1％

99.9％

100％

99.5％

1 month

99.8％

100％

996％

99.3％

98.3％

99.8％

99.5％

100％

99.1％

3 month

100.2％

99.6％

99.2％

99.1％

98.4％

99.5％

99.4％

99.6％

98.7％

Month 5

99.6％

98.9％

98.5％

98.5％

98.2％

99.2％

98.6％

99.4％

98.6％

6 month

98.9％

98.2％

98.6％

97.9％

97.8％

98.1％

97.6％

98.9％

98.1％

TABLE 7

Time

C1

C2

C3

C4

C5

C6

Q7

Q8

Q9

0 month

100.2％

100％

99.5％

100％

99.5％

99.5％

100％

99.9％

99％

1 month

99.9％

99.8％

99.3％

99.8％

99.2％

99.4％

99.1％

99.2％

99.1％

3 month

99.7％

99.6％

99.1％

99.6％

98.9％

98.9％

98.9％

99％

98.6％

Month 5

98.9％

99.1％

98.6％

99.1％

98.8％

98.4％

98.4％

98.4％

98.1％

6 month

98.1％

98.4％

98.4％

98.4％

97.9％

98.5％

97.9％

97.9％

97.6％

As can be seen from tables 6 and 7, when the freeze-dried product of the fat-soluble vitamin was left at room temperature for 6 months in the dark, the relative deviations between the actual concentrations of vitamin a and vitamin E and the target concentrations did not exceed 3%, and therefore, the fat-soluble vitamin in the freeze-dried product of the fat-soluble vitamin prepared in example 2 had better stability.

Example 7: instructions for Linear investigation of kits

The kit described in example 1 was processed according to the detection method described in example 3 using the instructions and fitted with a standard curve equation according to example 3.2 to examine the linearity of the calibrator.

Specifically, for vitamin a, six standard solutions prepared from six calibrators of fat-soluble vitamins are obtained, the ratio of the concentration of vitamin a in each concentration of standard solution to the concentration of a standard substance in vitamin a-d3 is used as the abscissa x1 of a standard curve equation, the ratio of the peak area of vitamin a obtained in the chromatogram of each concentration of standard solution to the chromatographic peak area of a standard substance in vitamin a-d3 is used as the ordinate y1 of the standard curve equation, linear regression is performed on the data of different concentrations obtained by the above detection, the standard curve equation is fitted to y1 a1 x1+ b1, and weight coefficients a1 and b1 are obtained, the weight coefficient a1 is the slope of the standard curve equation, the weight coefficient b1 is the intercept of the standard curve equation, and the linear relationship diagram of vitamin a is shown in fig. 4, wherein the linear equation 1 in fig. 4 is y 3.9259 x1+ 1060.7, the correlation coefficient was 0.99947.

Specifically, the target value concentration, actual concentration, and recovery rate of vitamin a in each calibrator for vitamin a in the calibrator are shown in table 8.

TABLE 8

Specifically, for vitamin E, six standard solutions prepared from six calibrators of fat-soluble vitamins are obtained, the ratio of the concentration of vitamin E in each concentration of standard solution to the concentration of a standard substance in vitamin E-d6 is used as the abscissa x2 of a standard curve equation, the ratio of the peak area of vitamin E obtained in the chromatogram of each concentration of standard solution to the chromatographic peak area of the standard substance in vitamin E-d6 is used as the ordinate y2 of the standard curve equation, linear regression is performed on the data of different concentrations obtained by the above detection, the standard curve equation is obtained by fitting y2 a2 x2+ b2, and weight coefficients a2 and b2 are obtained, the weight coefficient a2 is the slope of the standard curve equation, the weight coefficient b2 is the intercept of the standard curve equation, and the linear relationship diagram 5 of vitamin E is obtained, wherein the linear equation 2 x 2.3767-85 0.2821 in the diagram 5, the correlation coefficient was 0.99875.

Specifically, the target concentration, actual concentration, and recovery rate of vitamin E in each calibrator for vitamin E in the calibrator are shown in table 9.

TABLE 9

It can be known from the verification tests and tables 8 and 9 that the recovery rate of the present embodiment meets the requirements, and the method for detecting each fat-soluble vitamin in blood has good reproducibility and good recovery rate, thereby improving the accuracy of the detection result and eliminating the system error. In this embodiment, the correlation coefficient corresponding to the linear relationship data of each fat-soluble vitamin is greater than 0.9900, and the linear relationship is good.

Example 8: instructions for in-batch and inter-batch precision of the kit

Randomly drawing 1 batch of the kit described in example 1, repeatedly detecting the quality control products (QC (L), QC (M) and QC (H)) of the three fat-soluble vitamins in the kit according to the pretreatment and detection conditions of the sample to be detected in example 3, and determining the concentration of each fat-soluble vitamin in the quality control products of the fat-soluble vitamins, wherein the concentration of each fat-soluble vitamin in the quality control products of the three fat-soluble vitamins is shown in table 10, and VA is vitamin A and VE is vitamin E.

Watch 10

As can be seen from Table 10, the detection concentration of each fat-soluble vitamin in the quality control product of the fat-soluble vitamins is within the given target value range, and the batch precision range is 0.69-4.80%, which meets the standard of a kit enterprise, namely the standard batch precision required by the precision of the finished kit: CV < 10%.

Randomly extracting 3 batches of the kit described in example 1, repeatedly detecting the quality control products (qc (l), qc (m), and qc (h)) of the three fat-soluble vitamins in the extracted kit according to the pretreatment and detection conditions of the sample to be detected in example 3, and determining the concentration of each fat-soluble vitamin in the quality control products of the fat-soluble vitamins, wherein the concentration of each fat-soluble vitamin in the quality control products of the three fat-soluble vitamins is shown in table 11, in which VA is vitamin a and VE is vitamin E.

TABLE 11

As can be seen from Table 11, the detection concentration of each fat-soluble vitamin in the quality control product of the fat-soluble vitamins is within the given target value range, the batch-to-batch precision range is 1.47-5.76%, and the relative range is less than 8%, which meets the requirements of the related batch-to-batch precision.

It should be noted that the missing graphs in fig. 4 and 5 do not affect the technical content of the present solution.

Furthermore, it should be understood that various changes or modifications, such as addition or subtraction of one or more raw materials in the lyophilized product and adjustment of the ratio of the raw materials, can be made by those skilled in the art, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a" does not exclude the presence of other similar elements in a process, method, article, or apparatus that comprises the element.

Finally, it is to be noted that: the above description is only a preferred embodiment of the present invention, and is only used to illustrate the technical solutions of the present invention, and not to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.