Composition for preservation of vitamin D and vitamin K, substitute matrix and application

Abstract

The invention provides a composition for preservation of vitamin D and vitamin K, an alternative matrix and application, and relates to the technical field of detection reagents. The composition comprises 0.5-3 mol / L of sodium pyrosulfite, 0.5-3 mol / L of sodium hydrogen sulfite and 0.5-3 wt% of lipoprotein lipase, and the technical problems that vitamin D and vitamin K are unstable and prone to failure are solved.

Description

Technical Field

[0001] The invention relates to the technical field of detection reagents, and in particular to a composition, a replacement matrix and application for preserving vitamin D and vitamin K. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Vitamins are important compounds involved in regulating the body's metabolic processes and physiological functions. Vitamin D can promote the absorption of calcium and phosphorus. Vitamin D deficiency can lead to deformities in growing bone cells and induce rickets. Vitamin K participates in blood coagulation, anticoagulation, bone metabolism regulation, cell growth and proliferation, and reduces the probability of physiological and pathological events such as oxidative stress response and inflammatory response.

[0004] The stability of vitamin D and vitamin K has always been the key to accurate detection. Vitamin D and vitamin K are easily affected by factors such as light, temperature, humidity, and oxidation, which can reduce their activity and cause loss, which has become a difficult problem in clinical detection. The self-related properties that affect vitamin D and vitamin K include: (1) Antioxidant property: Vitamin D is sensitive to oxidants, while vitamin K is not. After adding oxidants, vitamin K loses more than 60% after being stored at room temperature for one week. (2) Anti-reduction property: Vitamin K is sensitive to reducing agents, while vitamin D is not. After adding reducing agents, vitamin D loses more than 30% after being stored at room temperature for one week. (3) Acid-base environment: Neutral and weakly alkaline environments are conducive to the stability of vitamin D, while neutral environments are conducive to the stability of vitamin K.

[0005] Environmental factors that affect the stability of vitamin D and vitamin K include: (1) trace elements: trace elements in serum have a considerable impact on vitamin D and vitamin K, especially certain trace element sulfates with high crystal water, such as ferrous sulfate heptahydrate and zinc sulfate heptahydrate. Some trace element additives are both oxidants or reductants, and promoters of certain oxidation reactions (such as copper). In the presence of trace elements, some unstable vitamins are prone to failure. The higher the concentration of trace elements, the longer the time, the more failures. For example, it has been determined that serum with extremely low trace element content only loses 17% of vitamin K after being stored at room temperature for one week, while if the serum contains trace elements, the loss of vitamin K is more than 70%. (2) Enzymes: Various enzymes in serum also have a certain impact on the stability of vitamins. Vitamins are coenzymes that play a physiological role in enzymes. If the ambient temperature and pH value are suitable, the enzymes will consume vitamins. (3) Adsorption: Vitamin K has a strong adsorption to ordinary carriers and endogenous lipids. It is reported that the recovery rate of vitamin K in ordinary EP tubes is only 30% after being stored for one week.

[0006] Accurate test results are crucial for diagnosing vitamin D deficiency (such as rickets, osteoporosis, etc.) and vitamin K deficiency (such as coagulation dysfunction). The instability of vitamin D and vitamin K during the testing process will lead to deviations in test results, resulting in false positive and false negative results, and affecting the accurate judgment of the vitamin levels in patients. Therefore, how to improve the stability of vitamin D and vitamin K is an issue to be solved.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The object of the present invention is to provide a composition for preserving vitamin D and vitamin K, so as to alleviate the technical problem that vitamin D and vitamin K are unstable and easy to lose efficacy.

[0009] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0010] In a first aspect, a composition for preserving vitamin D and vitamin K is provided, the composition comprising 0.5-3 mol / L sodium pyrosulfite, 0.5-3 mol / L sodium bisulfite and 0.5-3 wt % lipoprotein lipase.

[0011] In a second aspect, a use of the composition of the first aspect for preserving vitamin D and vitamin K is provided.

[0012] In a third aspect, a method for preserving vitamin D and vitamin K is provided, the method comprising adding the composition described in the first aspect to a system for preserving vitamin D and vitamin K.

[0013] In a fourth aspect, a replacement matrix for vitamin D and vitamin K is provided, wherein the replacement matrix comprises the composition described in the first aspect and a diluent.

[0014] In a fifth aspect, a method for preparing a replacement matrix for vitamin D and vitamin K is provided, the preparation method comprising mixing the composition and a diluent.

[0015] In a sixth aspect, a reagent containing vitamin D and vitamin K is provided, wherein the reagent comprises predetermined amounts of vitamin D and vitamin K, and the alternative matrix for vitamin D and vitamin K described in the fourth aspect.

[0016] In the seventh aspect, a detection kit for vitamin D and vitamin K is provided, the detection kit comprising one or more of the composition described in the first aspect, the alternative matrix described in the fourth aspect, and the reagent described in the sixth aspect.

[0017] In an eighth aspect, a method for detecting vitamin D and vitamin K is provided, the method comprising using the composition described in the first aspect to reduce the loss of vitamin D and vitamin K in the sample during the detection process.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The composition for preserving vitamin D and vitamin K provided by the present invention comprises sodium pyrosulfite, sodium bisulfite and lipoprotein lipase, and the degradation of vitamin D and vitamin K can be alleviated through the synergistic effect of the three components, and vitamin D and vitamin K can be prevented from binding to proteins and / or fats in the environment. The composition is applied to the detection of vitamin D and vitamin K, and the degradation and binding of vitamin D and vitamin K to proteins and / or fats in the sample to be tested and the alternative matrix can be prevented, thereby improving the detection accuracy. The composition provided by the present invention is of great significance for maintaining the stability of vitamin D and vitamin K during the detection process, improving the accuracy of the detection results, ensuring the evaluation of the therapeutic effect, improving the quality of laboratory testing, and promoting the reliability of clinical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is the vitamin D2 chromatogram of experimental group 3 of Example 3;

[0022] Figure 2 This is the vitamin D3 chromatogram of experimental group 3 of Example 3;

[0023] Figure 3 This is the chromatogram of vitamin K (MK-4) in experimental group 3 of Example 3;

[0024] Figure 4 This is the chromatogram of vitamin K (MK-7) in experimental group 3 of Example 3;

[0025] Figure 5 This is the vitamin D2 chromatogram of experimental group 7 in Example 3;

[0026] Figure 6 This is the vitamin D3 chromatogram of experimental group 7 in Example 3;

[0027] Figure 7 This is the chromatogram of vitamin K (MK-4) in experimental group 7 of Example 3;

[0028] Figure 8 This is the chromatogram of vitamin K (MK-7) in experimental group 7 of Example 3;

[0029] Fig. 9 This is the vitamin D2 chromatogram of experimental group 8 in Example 3;

[0030] Fig.10 This is the vitamin D3 chromatogram of experimental group 8 in Example 3;

[0031] Fig.11 This is the chromatogram of vitamin K (MK-4) in experimental group 8 of Example 3;

[0032] Fig.12 This is the chromatogram of vitamin K (MK-7) in experimental group 8 of Example 3;

[0033] Fig.13 This is the vitamin D2 chromatogram of experimental group 12 of Example 3;

[0034] Fig.14 This is the vitamin D3 chromatogram of experimental group 12 of Example 3;

[0035] Fig.15 This is the chromatogram of vitamin K (MK-4) in experimental group 12 of Example 3;

[0036] Fig.16 This is the chromatogram of vitamin K (MK-7) in experimental group 12 of Example 3;

[0037] Fig.17 This is the vitamin D2 chromatogram of experimental group 16 in Example 3;

[0038] Fig.18 This is the vitamin D3 chromatogram of experimental group 16 in Example 3;

[0039] Fig.19 This is the chromatogram of vitamin K (MK-4) in experimental group 16 of Example 3;

[0040] Fig. 20 This is the chromatogram of vitamin K (MK-7) in experimental group 16 of Example 3. DETAILED DESCRIPTION

[0041] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than 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.

[0042] In the first aspect, a composition for preserving vitamin D and vitamin K is provided, the composition comprising sodium pyrosulfite, sodium bisulfite and lipoprotein lipase. Sodium pyrosulfite and sodium bisulfite as reducing agents can alleviate the degradation of vitamin D and vitamin K; lipoprotein lipase can prevent vitamin D and vitamin K from binding to proteins and / or fats in the environment. The concentration of sodium pyrosulfite in the composition is 0.5 to 3 mol / L, for example, it can be but not limited to 0.5, 1, 1.5, 2, 2.5 or 3 mol / L; the concentration of sodium bisulfite is 0.5 to 3 mol / L, for example, it can be but not limited to 0.5, 1, 1.5, 2, 2.5 or 3 mol / L; the concentration of lipoprotein lipase is 0.5 to 3wt%, for example, it can be but not limited to 0.5, 1, 1.5, 2, 2.5 or 3wt%.

[0043] In an optional embodiment, the composition comprises 0.5-2 mol / L sodium pyrosulfite, 0.5-2 mol / L sodium bisulfite and 0.5-2 wt % lipoprotein lipase.

[0044] In an optional embodiment, the composition comprises 1 mol / L sodium pyrosulfite, 1 mol / L sodium bisulfite and 1 wt % lipoprotein lipase.

[0045] In a second aspect, a use of the composition of the first aspect for preserving vitamin D and vitamin K is provided.

[0046] In an optional embodiment, the vitamin D includes vitamin D2 and / or vitamin D3.

[0047] In an optional embodiment, the vitamin K includes vitamin K2.

[0048] In an optional embodiment, the vitamin K2 includes tetraenyl menaquinone (MK-4) and / or heptenyl menaquinone (MK-7).

[0049] In an optional embodiment, the composition is used to preserve vitamin D and vitamin K in a sample to be tested, for example, to preserve vitamin D and vitamin K in serum after blood collection.

[0050] In an optional embodiment, the composition is used to preserve vitamin D and vitamin K in a reagent preparation, such as vitamin D and vitamin K in a standard substance used to construct a standard curve in a test or a quality control substance used for quality control.

[0051] In a third aspect, a method for preserving vitamin D and vitamin K is provided, the method comprising adding the composition described in the first aspect to a system for storing vitamin D and vitamin K.

[0052] In an optional embodiment, the storage method further comprises storing vitamin D and vitamin K in a dark environment and at a temperature not exceeding 4°C.

[0053] In an alternative embodiment, vitamin D and vitamin K are stored at -20°C.

[0054] In an optional embodiment, the system for preserving vitamin D and vitamin K includes buffer, blood, plasma or serum, preferably serum.

[0055] In a fourth aspect, a replacement matrix for vitamin D and vitamin K is provided, wherein the replacement matrix comprises the composition described in the first aspect and a diluent.

[0056] In an optional embodiment, the diluent comprises a buffer, blood, plasma or serum, preferably serum.

[0057] In an optional embodiment, the diluent is blood, plasma or serum from which vitamin D and vitamin K have been removed. The "removal of vitamin D and vitamin K" refers to reducing the content of vitamin D and vitamin K inherent in the diluent, so as to reduce the effect of the original vitamin D and vitamin K in the diluent on the quantitative determination of vitamin D and vitamin K in the reagent. It is understood that an acceptable amount of vitamin D and vitamin K residues are allowed.

[0058] In an optional embodiment, the diluent is serum from which vitamin D and vitamin K have been removed.

[0059] In an optional embodiment, the serum is subjected to solid phase extraction, adsorption by an adsorbent and ultraviolet light irradiation to remove the original vitamin D and vitamin K in the serum.

[0060] In an optional embodiment, the serum is sequentially subjected to solid phase extraction, adsorption by an adsorbent and ultraviolet light irradiation to remove the original vitamin D and vitamin K in the serum.

[0061] In an optional embodiment, the solid phase extraction uses a Strata C8 solid phase extraction column.

[0062] In an optional embodiment, the adsorbent comprises dextran-coated activated carbon.

[0063] In a fifth aspect, a method for preparing a replacement matrix for vitamin D and vitamin K is provided, the preparation method comprising mixing the composition described in the first aspect and a diluent.

[0064] In an optional embodiment, the diluent is serum from which vitamin D and vitamin K have been removed; the serum is subjected to solid phase extraction, adsorbent adsorption and ultraviolet light irradiation to remove the original vitamin D and vitamin K in the serum.

[0065] In an optional embodiment, the serum is sequentially subjected to solid phase extraction, adsorbent adsorption and ultraviolet light irradiation to remove the original vitamin D and vitamin K in the serum.

[0066] In an optional embodiment, the solid phase extraction uses a Strata C8 solid phase extraction column.

[0067] In an optional embodiment, the adsorbent comprises dextran-coated activated carbon.

[0068] In a sixth aspect, a reagent containing vitamin D and vitamin K is provided, wherein the reagent containing vitamin D and vitamin K comprises a predetermined amount of vitamin D and vitamin K, and the alternative matrix for vitamin D and vitamin K described in the fourth aspect. The reagent includes but is not limited to standard products and quality control products with different concentrations of vitamin D and vitamin K.

[0069] In the seventh aspect, a detection kit for vitamin D and vitamin K is provided, the detection kit comprising one or more of the composition described in the first aspect, the alternative matrix described in the fourth aspect, and the reagent containing vitamin D and vitamin K described in the sixth aspect.

[0070] In an eighth aspect, a method for detecting vitamin D and vitamin K is provided, the method comprising using the composition of the first aspect to reduce the loss of vitamin D and vitamin K in a sample during the detection process. The method is a detection method for non-diagnostic and non-therapeutic purposes.

[0071] The loss includes degradation of vitamin D and vitamin K; and / or, the loss includes binding of vitamin D and vitamin K to substances in the environment making them undetectable.

[0072] In an optional embodiment, the detection method includes constructing a standard curve using the reagent containing vitamin D and vitamin K described in the sixth aspect.

[0073] In an optional embodiment, the detection method includes using chromatography to separate vitamin D and vitamin K in the sample to be tested.

[0074] In an optional embodiment, the chromatographic conditions include:

[0075] Chromatographic column: ACQUITY UPLC BEH Phenyl column (2.1×50mm, 1.7μm);

[0076] Mobile phase: Mobile phase A contained 0.5 mM NH 4 Mobile phase F and 0.1% v / v formic acid in methanol; mobile phase B was an aqueous solution containing 0.1% v / v formic acid.

[0077] The elution gradient is:

[0078] At 0 to 0.5 min, the volume ratio of the mobile phase A to the mobile phase B is maintained at 30:70;

[0079] At 0.5 to 2 minutes, the volume ratio of the mobile phase A to the mobile phase B changes from 30:70 to 5:95;

[0080] At 2 to 4.5 min, the volume ratio of the mobile phase A to the mobile phase B is maintained at 5:95;

[0081] At 4.5-4.6 min, the volume ratio of the mobile phase A to the mobile phase B was changed from 5:95 to 30:70;

[0082] At 4.6 to 5 min, the volume ratio of the mobile phase A to the mobile phase B was maintained at 30:70.

[0083] In an alternative embodiment, vitamin D and vitamin K are detected using mass spectrometry.

[0084] In an optional embodiment, the mass spectrometry conditions include: ionization mode: ESI (+); detection mode: MRM (detection mode: MRM); ion source temperature (TEM): 500°C; nebulizer gas (Gas1): 25.0psi; voltage (NC): 5.0V; curtain gas (GurtainGas): 25.0psi.

[0085] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0086] Example 1

[0087] Embodiment 1 provides a method for detecting vitamin D and vitamin K, comprising:

[0088] 1. Pre-treatment process of vitamin D and vitamin K:

[0089] 200 μL serum + 20 μL internal standard (vitamin D2-deuterium 6, vitamin D3-deuterium 5, vitamin MK4-deuterium 7 and vitamin MK7-deuterium 7) + 800 μL ethanol-acetonitrile (1:1), vortex for 10 min, centrifuge at 14000 rpm for 10 min, take 700 μL of supernatant + 1300 μL n-hexane: isooctane = 1:1, extract for 10 min, centrifuge at 14000 rpm for 5 min, take 1000 μL of supernatant, blow with nitrogen, reconstitute with 80 μL 80% ethanol and shake for 2 min.

[0090] 2. Chromatographic conditions:

[0091] Chromatographic column: ACQUITY UPLC BEH Phenyl column (2.1×50 mm, 1.7 μm); injection volume: 5 μL; mobile phase: mobile phase A-methanol (0.5 mM NH 4 F+0.1% v / v formic acid), mobile phase B-water (0.1% v / v formic acid), flow rate: 0.4 mL / min. The gradient elution program is shown in Table 1:

[0092] Table 1 Elution gradient

[0093] Time (min) A(%v / v) B (%v / v) 0 30 70 0.5 30 70 2 5 95 4.5 5 95 4.6 30 70 5 30 70

[0094] 3. Mass spectrometry conditions:

[0095] Ionization mode: ESI (+); detection mode: MRM (detection mode: MRM); ion source temperature (TEM): 500°C; nebulizer gas (Gas1): 25.0psi; voltage (NC): 5.0V; curtain gas (Gurtain Gas): 25.0psi; corresponding MRM channels and parameters are shown in Table 2.

[0096] Table 2 Mass spectrometry parameters of the components to be tested and internal standard substances

[0097]

[0098]

[0099] The following Examples 2 to 4 all adopt the method of Example 1 for the detection of vitamins in samples.

[0100] Example 2

[0101] This example investigates methods for removing vitamin D and vitamin K from serum matrix, including using ion exchangers to remove trace elements in serum and thereby reduce the effects of trace elements on vitamins, using ultrafiltration tubes to remove large molecular weight enzyme proteins and thereby reduce the effects of enzymes on vitamins, using adsorbents to remove vitamins from serum, using ultraviolet light to treat serum to degrade vitamins, using solid phase extraction to remove vitamins from serum, and the like. The experimental groupings are shown in Table 3:

[0102] Table 3 Experimental groups for removing vitamin D and vitamin K from serum matrix

[0103] Experimental Grouping Pre-treatment method Experimental Group 1 Ion exchangers Experimental Group 2 Ultrafiltration tube + ion exchanger Experimental Group 3 Adsorbent + ion exchanger Experimental Group 4 UV irradiation + ion exchanger Experimental Group 5 Solid Phase Extraction - Strata C8 Experimental Group 6 Solid Phase Extraction-Strata Silica Experimental Group 7 Sequential solid phase extraction-Strata C8+adsorbent+UV irradiation

[0104] The steps for each treatment method are as follows:

[0105] (1) Ion exchangers remove trace elements from serum:

[0106] Weak cation exchange column Oasis WCXμElution Plate 30μm was used;

[0107] Activation: 200 μL 85% ACN / 1% Fa (aqueous solution containing 85% acetonitrile / 1% formic acid);

[0108] Balance: 200 μL HO 2 O;

[0109] Sample loading: 600 μL sample;

[0110] Wash 1: 200 μL HO 2 O;

[0111] Elution 2: 200 μL ACN (acetonitrile);

[0112] Elution: 60 μL 85% ACN / 1% Fa (85% acetonitrile / 1% formic acid);

[0113] Reconstitution: 50 μL 0.01% Vc (0.01% vitamin C aqueous solution).

[0114] (2) Use ultrafiltration tubes to remove large molecular weight enzyme proteins:

[0115] Serum samples were treated for 30 minutes using ultrafiltration centrifuge tubes (15 ml 50KD), UFC905096, Millipore.

[0116] (3) Using adsorbents to remove vitamins from serum:

[0117] Add 4 mL of ultrapure water, 0.1 g of activated carbon, and 0.01 g of dextran to a centrifuge tube, shake, and centrifuge at 18,000 rpm for 5 min. Discard the supernatant and leave the dextran-coated activated carbon. Take 5 mL of serum, add it to the drained dextran-coated activated carbon, shake, and centrifuge (18,000 rpm, 5 min). Take the supernatant and add it to the drained dextran-coated activated carbon. Shake, centrifuge (18,000 rpm, 5 min), and collect the supernatant.

[0118] (4) Using ultraviolet light to treat serum to degrade vitamins:

[0119] Take a serum sample and place it under ultraviolet light for 20 minutes.

[0120] (5) Use solid phase extraction (Strata C8 solid phase extraction column) to remove vitamins from serum:

[0121] Solid phase extraction column: Strata C8, 25 mg, 8E-S005-CGB;

[0122] Activation: MeOH (methanol);

[0123] Balance: H 2 O;

[0124] Loading: After centrifugation of the serum, take the supernatant and load it onto the SPE plate to receive the loading liquid.

[0125] (6) Use solid phase extraction (Strata Silica solid phase extraction column) to remove vitamins from serum:

[0126] Solid phase extraction column: Strata Silica, 50 mg, 8E-S012-DGB;

[0127] Activation: n-hexane;

[0128] Loading: After centrifugation of the serum, take the supernatant and load it onto the SPE plate to receive the loading liquid.

[0129] According to the experimental grouping in Table 3, vitamin D and vitamin K in the serum matrix to which the standard substances were added were removed, and the recoveries of serum vitamin D2, vitamin D3, and vitamin K2 (MK-4 (menaquinone) and MK-7 (menaquinone)) after treatment were tested. The results are shown in Table 4.

[0130] Table 4 Vitamin D and vitamin K content in serum matrix after treatment

[0131]

[0132] The results are shown in Table 4. Ion exchangers and ultrafiltration tubes cannot significantly remove vitamin D and vitamin K from serum. C8 solid phase extraction based on reversed phase retention mode has a significant removal effect, and the best effect is achieved after combining adsorbent and ultraviolet irradiation. The combination of solid phase extraction-Strata C8+adsorbent+ultraviolet irradiation can remove vitamin D and vitamin K from serum.

[0133] Example 3

[0134] This example investigates the composition of the composition for preserving vitamin D and vitamin K, and the components investigated include oxidants, reductants, anti-adsorbents, hydrolases and antibacterial agents, specifically including: ProClin TM 300. Sodium metabisulfite (NaHSO 3 ), sodium bisulfite, sodium azide, mannitol and lipoprotein lipase.

[0135] The composition prepared by using the above 6 substances was added to the serum, and the recovery rate was measured after being placed in the light at room temperature for one day after the addition of the spike. The results are shown in Table 6. The chromatograms of vitamin D2, vitamin D3, vitamin K (MK-4) and vitamin K (MK-7) in experimental groups 3, 7, 8, 12 and 16 are shown in Figures 1 to 20 shown.

[0136] Table 5 Composition

[0137] Experimental Grouping Composition and final concentration of each component Experimental Group 1 No components to be investigated Experimental Group 2 <![CDATA[0.1%ProClin TM 300]]> Experimental Group 3 1M Sodium metabisulfite Experimental Group 4 1M Sodium bisulfite Experimental Group 5 1M Sodium Azide Experimental Group 6 0.2wt% Mannitol Experimental Group 7 1wt% lipoprotein lipase Experimental Group 8 1M sodium pyrosulfite + 1M sodium bisulfite + 1wt% lipoprotein lipase Experimental Group 9 0.5M sodium pyrosulfite + 2M sodium bisulfite + 0.5wt% lipoprotein lipase Experimental Group 10 2M sodium metabisulfite + 0.5M sodium bisulfite + 2wt% lipoprotein lipase Experimental Group 11 0.5M sodium metabisulfite + 3M sodium bisulfite + 0.5wt% lipoprotein lipase Experimental Group 12 3M sodium metabisulfite + 0.5M sodium bisulfite + 3wt% lipoprotein lipase Experimental Group 13 1M sodium metabisulfite + 1M sodium bisulfite Experimental Group 14 1M sodium bisulfite + 1wt% lipoprotein lipase Experimental Group 15 1M sodium pyrosulfite + 1wt% lipoprotein lipase Experimental Group 16 1M sodium pyrosulfite + 1M sodium bisulfite + 0.2wt% mannitol Experimental Group 17 1M sodium metabisulfite + 1M sodium bisulfite + 1M sodium azide

[0138] Table 6 Recovery of each vitamin after adding components of each experimental group

[0139]

[0140]

[0141] It can be seen from the above experimental results that the combination of sodium pyrosulfite, sodium bisulfite and lipoprotein lipase has the best protective effect on vitamin D and vitamin K, which is better than using only two combinations of sodium pyrosulfite, sodium bisulfite and lipoprotein lipase and using them alone, and replacing lipoprotein lipase with mannitol or sodium azide has the best protective effect on vitamin D and vitamin K than the combination of sodium pyrosulfite, sodium bisulfite and lipoprotein lipase. Among them, the effect is best when the concentration of sodium pyrosulfite is 1M, sodium bisulfite is 1M and lipoprotein lipase is 1wt%. And from the experimental results in Table 4, it can be seen that the combination of sodium pyrosulfite, sodium bisulfite and lipoprotein lipase is more suitable for alleviating the degradation of vitamin D and vitamin K, but has no significant advantage over other formula compositions in terms of the protective effect on vitamin A and vitamin B1.

[0142] Example 4

[0143] This example investigates the effects of light and storage temperature on the degradation of vitamin D and vitamin K in serum. The experimental method is as follows:

[0144] 1. Prepare low-value quality control (QCL), mid-value quality control (QCM) and high-value quality control (QCH), add vitamin D, vitamin D3, MK-4 and MK-7 to the target concentration, and then group them according to the following conditions: experimental group 1: light-proof frozen (-20℃); experimental group 2: light-proof refrigerated (-4℃); experimental group 3: light-proof room temperature (25℃); experimental group 4: light-proof room temperature (25℃). The recovery rates of vitamin D, vitamin D3, MK-4 and MK-7 in the samples were measured after 1 day, 2 days and 7 days of storage, and the results are shown in Tables 7 to 10.

[0145] Table 7 Experimental results of experimental group 1

[0146]

[0147] Table 8 Experimental results of experimental group 2

[0148]

[0149] Table 9 Experimental results of experimental group 3

[0150]

[0151] Table 10 Experimental results of experimental group 4

[0152]

[0153] It can be seen from Tables 7 to 10 that the storage temperature significantly affects the stability of MK4, and light significantly affects the stability of MK7. Serum samples need to be protected from light and frozen after blood collection.

[0154] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition for preserving vitamin D and vitamin K, characterized in that: The invention comprises 0.5-3 mol / L of sodium pyrosulfite, 0.5-3 mol / L of sodium bisulfite and 0.5-3 wt % of lipoprotein lipase.

2. The composition according to claim 1, characterized in that Containing 0.5-2 mol / L sodium pyrosulfite, 0.5-2 mol / L sodium bisulfite and 0.5-2 wt% lipoprotein lipase; Optionally, the composition comprises 1 mol / L sodium pyrosulfite, 1 mol / L sodium bisulfite and 1 wt % lipoprotein lipase.

3. Use of the composition according to claim 1 or 2 in preserving vitamin D and vitamin K; Optionally, the vitamin D includes vitamin D2 and / or vitamin D3; Optionally, the vitamin K includes vitamin K2; Optionally, the vitamin K2 comprises menatetrenone and / or heptenequinone.

4. A method for preserving vitamin D and vitamin K, characterized in that: Comprising: adding the composition of claim 1 or 2 to a system for preserving vitamin D and vitamin K; Optionally, the storage method further comprises storing vitamin D and vitamin K in a dark environment and at a temperature not exceeding 4°C; Optionally, the system for preserving vitamin D and vitamin K includes buffer, blood, plasma or serum, preferably serum.

5. A substitute matrix for vitamin D and vitamin K, characterized in that Comprising the composition according to claim 1 or 2 and a diluent; Optionally, the diluent comprises a buffer, blood, plasma or serum, preferably serum; Optionally, the diluent is blood, plasma or serum from which vitamin D and vitamin K have been removed.

6. The method for preparing a substitute matrix for vitamin D and vitamin K according to claim 5, characterized in that: comprising mixing the composition and a diluent; Optionally, the diluent is serum from which vitamin D and vitamin K have been removed; the serum is subjected to solid phase extraction, adsorption by an adsorbent and ultraviolet irradiation to remove vitamin D and vitamin K in the serum; Optionally, the solid phase extraction uses a Strata C8 solid phase extraction column; Optionally, the adsorbent comprises dextran-coated activated carbon.

7. A reagent containing vitamin D and vitamin K, characterized in that: Contains predetermined amounts of vitamin D and vitamin K, and the replacement matrix for vitamin D and vitamin K according to claim 5.

8. A test kit for vitamin D and vitamin K, characterized in that: Comprising one or more of the composition of claim 1 or 2, the alternative matrix for vitamin D and vitamin K of claim 5, and the reagent of claim 7.

9. A method for detecting vitamin D and vitamin K, characterized in that: The method comprises using the composition of claim 1 or 2 to reduce the loss of vitamin D and vitamin K in a sample during the detection process; Optionally, a standard curve is constructed using the reagent described in claim 7.

10. The detection method according to claim 9, characterized in that: The detection method comprises separating vitamin D and vitamin K in a sample to be tested by chromatography.

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