A method and kit for detecting fat-soluble vitamins

Abstract

The application discloses a method and a kit for detecting fat-soluble vitamins, and belongs to the technical field of in-vitro diagnosis. The kit comprises a sample extraction solution containing an internal standard, a calibrator and a quality control product; the sample extraction solution comprises acetonitrile, ethanol and isopropyl alcohol in a volume ratio of 7-8:1-2:1. The application selects a special combination of sample extraction solutions and further optimizes the ratio of the sample extraction solutions, thereby improving the responses of 25(OH)D2, 25(OH)D3 and VK1 and reducing the response of VE, compared with a conventional extraction solution composed of acetonitrile, methanol and isopropyl alcohol, and solving the problems of low responses of 25(OH)D2, 25(OH)D3 and VK1 and high concentration of VE in the protein precipitation method, which leads to high response. The pretreatment step of the detection method is simple, the cost is low, the sample detection time is short, the sensitivity is high, the components of the kit are stable and easy to store.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostics, and specifically to a method and kit for detecting fat-soluble vitamins. Background Technology

[0002] Fat-soluble vitamins include vitamin A (VA), 25-hydroxyvitamin D2 (25(OH)D2), 25-hydroxyvitamin D3 (25(OH)D3), vitamin E (VE), and vitamin K1 (VK1). Fat-soluble vitamins are essential for maintaining good health. Without sufficient vitamins, organs cannot function properly, skin ages rapidly, and vision deteriorates. Symptoms of insufficient fat-soluble antioxidant vitamins include immune dysfunction, recurrent infections, nervous system diseases, digestive and absorption disorders, cardiovascular diseases, chronic fatigue, and increased oxidative stress. A balanced and adequate concentration of antioxidant vitamins helps prevent free radical damage and the formation of chronic diseases. Vitamin A promotes growth and reproduction, maintains normal secretion of bones, epithelial tissues, vision, and mucous membranes, and has various physiological functions. Vitamin A and its analogues have a role in preventing precancerous lesions. Deficiency manifests as growth retardation and reduced dark adaptation, leading to night blindness. Vitamin D also promotes skin cell growth and differentiation and regulates immune function. Vitamin D deficiency can cause rickets in children and osteomalacia in adults. Vitamin E has antioxidant properties, protecting body cells from free radical damage, improving blood circulation, protecting tissues, lowering cholesterol, and preventing hypertension. Vitamin K1 is essential for the liver to synthesize factors II, VII, IX, and X, and is mainly used to treat various hemorrhagic diseases caused by vitamin K deficiency. It also has analgesic effects, relieves bronchospasm, and has significant effects on visceral smooth muscle colic, bile duct spasm, and colic caused by intestinal spasm. Fat-soluble vitamins have very important clinical significance and possess great diagnostic value.

[0003] Several commercial methods are currently available for the routine detection of fat-soluble vitamins, including high-performance liquid chromatography-ultraviolet (HPLC-UV), high-performance liquid chromatography-fluorescence (HPLC-FLD), high-performance liquid chromatography-diode array detector (LC-DAD), immunoassay, and liquid chromatography-tandem mass spectrometry (LC-MS / MS). Immunoassay systems, due to their poor specificity and low sensitivity, cannot accurately determine vitamin content. HPLC, with its limited detection limit, is mainly used for samples with high vitamin content and generally can only perform single-component or simultaneous determination of several components with high content. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) boasts extremely high specificity, detection sensitivity, and throughput, and is considered the gold standard for quantitative detection, widely used in various analytical industries. It can more accurately determine the content of fat-soluble vitamins in the human body, thus providing more accurate diagnostic information for clinical diagnosis. Currently, LC-MS / MS is internationally recognized as the most reliable method for detecting fat-soluble vitamins, especially 25(OH)D.

[0004] Existing Class II fat-soluble vitamin test kits, such as the fat-soluble vitamin detection kit (liquid chromatography-tandem mass spectrometry) offered by Mass Spectrometry Biotechnology Co., Ltd., can be used for the in vitro quantitative detection of vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and vitamin E in human serum samples. However, its calibrators and quality controls, after reconstitution, can only be stored for 5 days at ≤-20℃ in the dark; the detection targets are vitamins A, D, and E, and vitamin K cannot be detected simultaneously; and it must be stored at -20℃. For example, the fat-soluble vitamin detection kit (liquid chromatography-tandem mass spectrometry) offered by Hunan Kailai Spectrum Biotechnology Co., Ltd. is used for the in vitro quantitative detection of fat-soluble vitamins (vitamin A, 25-hydroxyvitamin D, vitamin E, and vitamin K1) in human serum samples, but this kit must be stored at -30 to -15℃.

[0005] Therefore, there is a need in the art for a method and kit for detecting fat-soluble vitamins that is quick, highly specific and selective, and stable in preservation. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method and kit for detecting fat-soluble vitamins. This invention utilizes the simplest pretreatment method, protein precipitation (PPT), for sample processing. By selecting a special combination of sample extraction solutions (i.e., protein precipitants) and further optimizing the ratio of the sample extraction solutions, compared to conventional extraction solutions composed of acetonitrile, methanol, and isopropanol, the responses of 25(OH)D2, 25(OH)D3, and VK1 are improved, while the response of VE is reduced. This solves the problems of low responses of 25(OH)D2, 25(OH)D3, and VK1, and excessively high responses due to high VE concentrations in the protein precipitation method. Furthermore, this invention effectively extends the product's stability by adding a combination of stabilizers and preservatives and selecting a suitable concentration of BSA as an alternative matrix.

[0007] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0008] In a first aspect, the present invention provides a kit for detecting fat-soluble vitamins, the kit comprising a sample extract containing an internal standard, a calibrator, and a quality control; the sample extract comprising acetonitrile, ethanol, and isopropanol in a volume ratio of 7-8:1-2:1.

[0009] As one embodiment of the present invention, the sample extract comprises acetonitrile, ethanol, and isopropanol in a volume ratio of 7:2:1.

[0010] As one embodiment of the present invention, the internal standard includes 25(OH)D2-d3, 25(OH)D3-d6, VA-d4, VE-d6, and VK1-d7.

[0011] As one embodiment of the present invention, the matrix of the calibrators and quality control samples comprises the following components by mass percentage:

[0012] Bovine serum albumin 1-5%,

[0013] PBS buffer 93-98%,

[0014] Preservative 0.1-2%,

[0015] Stabilizer 0.1-4%.

[0016] Furthermore, the preservative includes one or more of Proclin 300 and Proclin 950.

[0017] Furthermore, the stabilizer includes one or more of butylated hydroxyanisole (BHA), tert-butylhydroquinone (TBHQ), isoascorbic acid (D-VC), and citric acid (CA).

[0018] The preservatives and stabilizers in this invention are freeze-dried after preparation.

[0019] Secondly, the present invention provides a method for detecting fat-soluble vitamins using the aforementioned kit, comprising the following steps:

[0020] S1. Mix the test sample, sample extract, calibrator, and quality control sample by shaking, precipitate the protein, and centrifuge.

[0021] S2. Perform liquid chromatography-tandem mass spectrometry on the supernatant after centrifugation.

[0022] As one embodiment of the present invention, in step S1, the sample to be tested is accurately dissolved in ultrapure water according to the instructions, and after equilibration to room temperature, the next step can be carried out.

[0023] This invention utilizes a sample extraction buffer for simple protein precipitation to purify and extract the serum sample. The supernatant is then injected into the serum. This simple protein precipitation method allows for accurate quantification of fat-soluble vitamins in human serum; it is easy to operate and cost-effective.

[0024] As one embodiment of the present invention, in step S2, the chromatographic conditions for the liquid chromatography-tandem mass spectrometry detection include:

[0025] Chromatographic column: C18 column;

[0026] Gradient elution conditions: flow rate: 0.8 mL / min, column temperature: 40 ℃; autosampler: 10 ℃; injection volume: 40 μL;

[0027] Phase A: 0.1% FA aqueous solution;

[0028] Phase B: 0.1% FA (methanol);

[0029] The mobile phase gradient is as follows:

[0030]

[0031] As one embodiment of the present invention, in step S2, the mass spectrometry conditions for the liquid chromatography-tandem mass spectrometry detection include:

[0032] Ion source: Atmospheric pressure chemical ionization ion source; Acquisition mode: Positive ion; Scan mode: MRM; Curtain gas: 30 psi; Collision gas: 7 psi; Corona needle current: 4 uA; TEM: 450 o C; GS1: 60 psi;

[0033] Compound ion pair information includes:

[0034]

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1. The present invention selects the protein precipitation method (PPT) with the simplest pretreatment for sample processing, and solves the problems of low responses of 25(OH)D2, 25(OH)D3 and VK1 and too high response caused by high VE concentration by optimizing the composition ratio of the sample extraction solution. Compared with the liquid-liquid extraction method (LLE) with complex pretreatment steps adopted by most existing methods to ensure responses, the present invention compares the changes in responses when the protein precipitation method and the liquid-liquid extraction method are used to process the same serum sample through experiments. The results show that the responses of 25(OH)D2, 25(OH)D3 and VK1 by PPT > LLE, and the response of high-concentration VE by PPT < LLE, indicating that the optimized protein precipitation method is more superior to the complex liquid-liquid extraction method for the detection of fat-soluble vitamins. It has high sensitivity, can greatly reduce the time, manpower and material costs, improve the sample detection efficiency, and has obvious advantages in clinical detection. In addition, the extraction rates of VD and VK1 are higher than those of the complex liquid-liquid extraction pretreatment.

[0037] 2. The detection method of the present invention uses the protein precipitation method as the pretreatment method, and can accurately quantify 5 compounds including vitamin A (VA), 25-hydroxyvitamin D2 (25(OH)D2), 25-hydroxyvitamin D3 (25(OH)D3), vitamin E (VE) and vitamin K1 (VK1) in human serum within 5 minutes. A liquid chromatography method and a mass spectrometry method are determined. Under this method, it can ensure the accurate and efficient simultaneous determination of the contents of 5 fat-soluble vitamins in serum in a short time, providing possibilities and new ideas for clinical detection. This method has a short sample measurement time, stable method, good specificity and high selectivity.

[0038] 3. The present invention effectively extends the stability of the product by optimizing the product composition components (adding a combination of stabilizers and preservatives, screening the content of a suitable alternative matrix BSA, and in the form of freeze-drying), and determining a suitable storage method. It can be stored in the dark at 2-8 °C, and the validity period is 12 months. After the reagent is unsealed and reconstituted, it is stored sealed at -20±5 °C, and the validity period is 28 days. The kit of the present invention has a low cost, stable kit components and is easy to store. Description of the Drawings

[0039] By reading the detailed description of the non-restrictive embodiments with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:

[0040] Figure 1 It is the detection flow chart of the present invention;

[0041] Figure 2The following is a stability data graph for Example 3; where (a) is the accuracy result of 5 compounds after 7 days of acceleration at the low concentration point S1; (b) is the accuracy result of 5 compounds after 7 days of acceleration at the high concentration point S6; (c) is the accuracy result of 5 compounds after 7 days of opening at the low concentration point S1; and (d) is the accuracy result of 5 compounds after 7 days of opening at the high concentration point S6.

[0042] Figure 3 The left image shows the chromatogram of VA in Example 5; the left image is the external standard chromatogram, and the right image is the internal standard chromatogram.

[0043] Figure 4 The left chromatogram is the VD2 chromatogram from Example 5; the right chromatogram is the external standard chromatogram.

[0044] Figure 5 The left image shows the chromatogram of VD3 in Example 5; the left image is the external standard chromatogram, and the right image is the internal standard chromatogram.

[0045] Figure 6 The left image shows the chromatogram of vitamin E in Example 5; the left image is the external standard chromatogram, and the right image is the internal standard chromatogram.

[0046] Figure 7 The image shows the chromatogram of VK1 in Example 5; the left image is the external standard chromatogram, and the right image is the internal standard chromatogram. Detailed Implementation

[0047] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, providing detailed implementation methods and specific operating procedures, which will help those skilled in the art to further understand the present invention. It should be noted that the scope of protection of the present invention is not limited to the following embodiments; any adjustments and improvements made under the concept of the present invention are all within the scope of protection of the present invention.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0049] Reagent preparation:

[0050] The calibrators are divided into S1, S2, S3, S4, S5, and S6, with the following contents: vitamin A content of 50, 100, 200, 400, 1000, and 1600 ng / mL, 25-hydroxyvitamin D2 content of 2, 4, 8, 16, 40, and 64 ng / mL, 25-hydroxyvitamin D3 content of 5, 10, 20, 40, 100, and 160 ng / mL, vitamin E content of 781.25, 1562.5, 3125, 6250, 15625, and 25000 ng / mL, and vitamin K1 content of 0.15, 0.3, 0.6, 1.2, 3, and 4.8 ng / mL.

[0051] The quality control materials are divided into LQC, MQC, HQC, and blank quality control material H0. Among them, the vitamin A content of LQC, MQC, and HQC is 150, 450, and 1280 ng / mL, respectively; the content of 25-hydroxyvitamin D2 is 6, 18, and 51.2 ng / mL, respectively; the content of 25-hydroxyvitamin D3 is 15, 45, and 128 ng / mL, respectively; the content of vitamin E is 2343.75, 7031.25, and 20000 ng / mL, respectively; and the content of vitamin K1 is 0.45, 1.35, and 3.84 ng / mL, respectively. The blank quality control material H0 does not contain any of the compounds.

[0052] The parameters for detection by liquid chromatography-tandem mass spectrometry are as follows:

[0053] Chromatographic column: C18 column;

[0054] Gradient elution conditions: flow rate: 0.8 mL / min, column temperature: 40 ℃; autosampler: 10 ℃; injection volume: 40 μL;

[0055] Phase A: 0.1% FA aqueous solution;

[0056] Phase B: 0.1% FA (methanol);

[0057] The mobile phase gradient is shown in Table 1 below:

[0058] Table 1

[0059]

[0060] Mass spectrometry conditions include:

[0061] Ion source: Atmospheric pressure chemical ionization ion source; Acquisition mode: Positive ion; Scan mode: MRM; Curtain gas: 30 psi; Collision gas: 7 psi; Corona needle current: 4 uA; TEM: 450 o C; GS1: 60 psi;

[0062] The compound ion pair information is shown in Table 2 below:

[0063] Table 2

[0064]

[0065] Example 1

[0066] Optimize and screen suitable sample extraction solutions:

[0067] In this embodiment, six sample extraction solutions containing internal standards were prepared using the six sample extraction solution systems listed in Table 3 below. Serum samples of the same specific concentration were pretreated using the protein precipitation method, and then analyzed by liquid chromatography-tandem mass spectrometry. The peak areas of the lipid-soluble vitamin ADEK response were measured as shown in Table 3 below:

[0068] Table 3

[0069]

[0070] The table above shows that Group 1, using acetonitrile alone as the sample extraction solvent, showed a low response to fat-soluble vitamins. The concentrations of VD2 and VK1 in human serum were very low. Therefore, improving the response of VD2 and VK1 is a key challenge to be addressed in establishing the method for this kit, as the extraction efficiency of acetonitrile as the extraction solvent was clearly insufficient. Group 2, by introducing isopropanol into the acetonitrile system to form an acetonitrile:isopropanol ratio of 9:1, treated the same sample and found that the VK1 response increased by more than 5 times, while the VD2 response also improved. Further, Group 3, by introducing ethanol to form an acetonitrile:ethanol:isopropanol ratio of 8:1:1, further improved the compound response. Group 4, by adjusting the ratio of the three components to obtain an acetonitrile:ethanol:isopropanol ratio of 7:2:1, treated this sample and increased the VD2 response by nearly 2 times (from 996 to 1639) and the VK1 response by 7.6 times (from 1120 to 8590). In addition, control groups 5 (acetonitrile:ethanol:isopropanol = 17:2:1) and 6 (acetonitrile:methanol:isopropanol = 7:2:1) were set up to explore the effects of more combinations on the response of fat-soluble vitamins. The experimental results showed that the acetonitrile:ethanol:isopropanol = 7:2:1 system had a higher response than control groups 5 and 6, especially for VD2 and VK1. Therefore, the optimal ratio can be determined to be acetonitrile:ethanol:isopropanol = 7:2:1.

[0071] By screening and optimizing a suitable sample extraction solution system, the low extraction rate of protein precipitation as a pretreatment method for fat-soluble vitamins was resolved, improving the response at low levels of VD2 and VK1 and ensuring high sensitivity and specificity for the detection of the five fat-soluble vitamin compounds. Furthermore, the VE response was reduced in the acetonitrile:ethanol:isopropanol = 7:2:1 system compared to the acetonitrile system, helping to address the issue of excessively high VE response.

[0072] Example 2

[0073] Samples were processed using an optimized protein precipitation method compared to liquid-liquid extraction:

[0074] The specific procedure involves taking a serum sample, using method one: (refer to...) Figure 1 The procedure shown is as follows: Method 1: Protein precipitation (sample extraction solution system: acetonitrile:ethanol:isopropanol = 7:2:1) for simple protein precipitation, centrifugation, and supernatant collection for instrument detection; Method 2: Liquid-liquid extraction (acetonitrile precipitation of protein, hexane extraction of sample, nitrogen blowing enrichment, reconstitution, and loading). The response (peak area) of the two pretreatment methods to the same serum sample is compared (as shown in Table 4 below).

[0075] Table 4

[0076]

[0077] Currently, most commercially available reagent kits use liquid-liquid extraction (LLPE) with a more complex pretreatment process to ensure the response of VD and VK1. This involves adding a larger proportion of extractant to achieve a high extraction rate, followed by concentration and enrichment using methods such as nitrogen blowing, and finally reconstitution with a rehydration solution before detection by liquid chromatography-tandem mass spectrometry (LC-MS / MS). While LLPE can improve the response of target compounds, it requires more pretreatment steps, is more complex, and takes longer, resulting in low efficiency in clinical use. As shown in Table 4, protein precipitation (using an acetonitrile:ethanol:isopropanol = 7:2:1 system as the extraction solution) achieves higher extraction rates for VD2, VD3, and VK1 than LLPE. Since the levels of VD and VK1 in human serum are relatively low, the optimized protein precipitation method has significant advantages, enabling efficient extraction and accurate quantification in actual sample assays.

[0078] Example 3

[0079] Stability of a matrix-based fat-soluble vitamin assay kit with different proportions of bovine serum albumin (BSA) as a substitute:

[0080] Stability tests were conducted at low concentration points (S1) and high concentration points (S6) under the same stabilizer formulation, using 1% BSA, 3% BSA, and 5% BSA as alternative matrices (Table 5).

[0081] Table 5

[0082]

[0083] The results are as follows Figure 2As shown, 1% and 5% BSA showed stability results exceeding the accuracy by 100±15% in accelerated and open-bottle tests at both low and high concentration points, while 3% BSA remained stable. This invention screened out the product alternative matrix with the best stability performance for fat-soluble vitamins.

[0084] Example 4

[0085] Accuracy experiment:

[0086] The product calibrator was used to test the standard substance three times. VA, VE, and VK1 were tested using NIST 968f, and 25(OH)D2 and 25(OH)D3 were tested using NIST 2970. The product standard curve values ​​were compared with the standard substance target values. The test results and relative deviations are shown in Table 6 below.

[0087] Table 6

[0088]

[0089] The product calibrators were tested three times with the standard reference material. NIST 968f was used for VA, VE, and VK1, and NIST 2970 was used for 25(OH)D2 and 25(OH)D3. The relative deviations between the test results and the target values ​​of the standard reference material were all less than 15%, which meets the technical requirements.

[0090] Precision experiment:

[0091] The product calibrators were used to test low-concentration and high-concentration precision samples 10 times each, and the CV was calculated from the results. The results are shown in Table 7 below:

[0092] Table 7

[0093]

[0094] The low-concentration and high-concentration samples were tested 10 times each, and the repeatability CV of the quality control samples was ≤15%, which met the technical requirements.

[0095] Stability test:

[0096] 1. Stability after opening:

[0097] After opening, the product was stored at -20±5 ℃, and the standard substance was measured after 31 days. The relative deviation between the test results and the target value of the standard substance was less than 15%, indicating that the product was stable at the test time point.

[0098] Table 8

[0099]

[0100] Storage stability: Table 9

[0101]

[0102] The present invention stores the standard substance at 2-8℃ for 13 months and measures the standard substance. The relative deviation between the test results and the target value of the standard substance is less than 15%, indicating that the product is stable at the test time point.

[0103] Stability results were obtained after 31 days of opening, and the result was satisfactory. The calibration period for open-bottle storage was set at 28 days. Long-term storage stability was determined at 13 months, and the result was satisfactory. The calibration period for long-term stability was set at 12 months. The above are the accurate results from laboratory monitoring. Actual stability can be further investigated and extended.

[0104] Example 5

[0105] Using the method selected in this invention, VA, 25(OH)D2, 25(OH)D3, VE, VK1 and their corresponding internal standard diagrams are respectively as follows: Figure 3-7 As shown, the external and internal standard peak diagrams of the detected target analytes are clean and interference-free, indicating that this method has good specificity.

[0106] In summary, based on the above demonstrations, this invention solves the existing technical problems in the detection of fat-soluble vitamins, such as complex pretreatment, long detection time, and unstable methods.

[0107] In summary, the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A kit for detecting fat-soluble vitamins, characterized in that, The kit includes a sample extraction solution containing an internal standard, calibrators, and quality control materials; the sample extraction solution includes acetonitrile, ethanol, and isopropanol in a volume ratio of 7:2:

1. The internal standards include 25(OH)D2-d3, 25(OH)D3-d6, VA-d4, VE-d6, and VK1-d7; The matrix of the calibrators and quality control samples comprises the following components by mass percentage: Bovine serum albumin 1-5%, PBS buffer 93-98%, Preservative 0.1-2%, Stabilizer 0.1-4%; The preservative includes one or more of Proclin 300 and Proclin 950; The stabilizer includes one or more of butylated hydroxyanisole, tert-butylhydroquinone, isoascorbic acid, and citric acid.

2. The use of the kit as described in claim 1 in the detection of fat-soluble vitamins.

3. A method for detecting fat-soluble vitamins using the kit as described in claim 1, characterized in that, Includes the following steps: S1. Mix the test sample, sample extract, calibrator, and quality control sample by shaking, precipitate the protein, and centrifuge. S2. Perform liquid chromatography-tandem mass spectrometry on the supernatant after centrifugation.

4. The method according to claim 3, characterized in that, In step S2, the chromatographic conditions for the liquid chromatography-tandem mass spectrometry detection include: Chromatographic column: C18 column; Gradient elution conditions: flow rate: 0.8 mL / min, column temperature: 40 ℃; autosampler: 10 ℃; injection volume: 40 μL; Phase A: 0.1% FA aqueous solution; Phase B: 0.1% FA (methanol); The mobile phase gradient is as follows: 。 5. The method according to claim 3, characterized in that, In step S2, the mass spectrometry conditions for the liquid chromatography-tandem mass spectrometry detection include: Ion source: Atmospheric pressure chemical ionization ion source; Acquisition mode: Positive ion; Scan mode: MRM; Curtain gas: 30 psi; Collision gas: 7 psi; Corona needle current: 4 uA; TEM: 450 o C; GS1: 60 psi; Compound ion pair information includes: 。

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