Kit for detecting water-soluble vitamins in serum and preparation and detection method thereof

Abstract

The invention relates to a detection kit for water-soluble vitamins in serum and a preparation and detection method of the detection kit, a water-soluble vitamin calibration product, a quality control product and corresponding matched reagents are prepared to form the detection kit for the water-soluble vitamins, and a sulfosalicylic acid-ammonium acetate system is used for carrying out protein precipitation; and removing protein precipitate by a protein precipitation tube through a vacuum negative pressure device to obtain a clean filtrate, and then detecting by using mass spectrometry. The complex steps of centrifugation and concentration required by an organic solvent protein precipitation method are avoided, the pretreatment process of water-soluble vitamin detection is simplified, the problem that the adding standard recovery rate is high is effectively solved, and the water-soluble vitamin detection efficiency is improved; and meanwhile, a new automation thought is provided for detection of other indexes.

Description

Technical Field

[0001] The invention belongs to the technical field of vitamin detection, and in particular relates to a detection kit for water-soluble vitamins in serum and a preparation and detection method thereof. Background Art

[0002] Water-soluble vitamins (such as vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, and 5-methyltetrahydrofolate) are a group of organic substances necessary for humans to prevent metabolic disorders. Their content in the human body is extremely small, but they play an important role in the body's growth, development, metabolism, and other processes.

[0003] At present, high performance liquid chromatography tandem mass spectrometry is mainly used to detect water-soluble vitamins. This method has high sensitivity and strong specificity and is the "gold standard" for detecting vitamins. However, the analytical performance of this method is greatly affected by the sample, and it has high requirements for sample processing. If the sample pretreatment or purification effect is not good, it will affect the accuracy and repeatability of the quantitative analysis results, and it will also contaminate the instrument, resulting in high maintenance costs. Long-term operation may even lead to the risk of instrument scrapping. At present, the main serum sample processing methods include magnetic bead method, solid phase extraction method, protein precipitation method (PPT), etc. Although the magnetic bead method is easy to automate, it requires specificity, cannot meet the requirements of multi-index detection at the same time, and even requires coupled antibodies, which has the disadvantages of poor repeatability and high cost. The protein precipitation method mostly uses organic solvents as protein precipitants, but it requires a series of complex operations such as centrifugation, nitrogen blowing, and re-dissolution, which is time-consuming and labor-intensive, and is not conducive to automated operation. Moreover, a large amount of easily volatile organic solvents is also detrimental to the health of experimental operators.

[0004] Chinese patent CN106324142A discloses a method for determining water-soluble vitamins by semi-automatic sample processing liquid chromatography technology, which uses methanol or acetonitrile to precipitate proteins, takes the supernatant after centrifugation, extracts it with a semi-automatic solid phase extractor, then elutes it with methanol aqueous solution, blows it with nitrogen, and then dissolves it for detection on the machine. This method has a complicated operation process and a long processing time, which is not conducive to rapid clinical detection. Chinese patent CN110412175A discloses a method for detecting water-soluble vitamins in blood, which uses sulfosalicylic acid as a protein precipitant, centrifuges, and takes the supernatant for detection on the machine. Although the method is simple to operate, there is obvious interference in the detection of 5-methyltetrahydrofolate (5-MTHF), the spike recovery rate is as high as about 150%, and there is a problem of inaccurate quantification, which cannot meet the requirements of clinical quantitative detection. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, the present invention provides a kit for detecting water-soluble vitamins in serum, as well as a preparation and detection method thereof. The present invention not only avoids the cumbersome steps of centrifugation and concentration required by the organic solvent protein precipitation method, simplifies the pretreatment process for detecting water-soluble vitamins, but also effectively solves the problem of high spike recovery rate, improving the detection efficiency of water-soluble vitamins; at the same time, it also provides a new automation idea for the detection of other indicators.

[0006] To achieve the above-mentioned invention objectives, the technical solution adopted by the present invention is as follows: A kit for detecting water-soluble vitamins in serum, the kit includes calibrators, quality control products, isotope internal standard solutions, protein precipitants, calibrator diluents, mobile phase concentrates. The calibrators include water-soluble vitamins at one concentration; the quality control products include water-soluble vitamins at two or more concentrations; the isotope internal standard solutions are water-soluble vitamin isotope internal standards; the protein precipitants are a combination of sulfosalicylic acid and ammonium acetate; the mobile phase concentrates are ammonium acetate methanol aqueous solutions; the water-soluble vitamins include vitamin B1 (VB1), vitamin B2 (VB2), vitamin B3 (VB3-NM), vitamin B5 (VB5), vitamin B6 (VB6-PA), 5-methyltetrahydrofolic acid (5-MTHF).

[0007] Preferably, the concentration of the calibrators is 30-220 ng / mL, the concentration of the quality control products is 10-220 ng / mL, the concentration of the isotope internal standard solutions is 30-600 ng / mL, the components of the protein precipitants are 4% (mass / volume) sulfosalicylic acid and 150 mM ammonium acetate; the components of the mobile phase concentrates are 2 M (mol / L) ammonium acetate and 50% (volume / volume) methanol.

[0008] The present invention also provides a preparation method for the above-mentioned kit for detecting water-soluble vitamins in serum, including the following steps: (1) Prepare the calibrator diluent: Adsorb bovine albumin (BSA) with activated carbon; filter through a water-based filter membrane; dilute the above bovine albumin with phosphate buffer solution (PBS), and then add ProClin300 and ascorbic acid. (2) Prepare the calibrators and quality control products: Prepare the calibrators and quality control products with phosphate buffer solution containing bovine albumin. (3) Prepare the isotope internal standard solution: Take the water-soluble vitamin isotope internal standard stock solution and add it to the phosphate buffer solution. (4) Prepare the protein precipitant: Add sulfosalicylic acid and ammonium acetate to ultrapure water, mix well, dispense, and store at 2-8 °C. (5) Prepare the mobile phase concentrate: Dissolve ammonium acetate in 50% methanol, and after complete dissolution, make up the volume with 50% methanol, mix well, dispense, and store at -20 ± 5 °C. (6)Lyophilization: The calibrator, quality control product, and isotope internal standard are lyophilized. (7)Load the calibrator, quality control product, isotope internal standard solution, protein precipitant, calibrator diluent, and mobile phase concentrate into the kit and store it at -20 ± 5°C.

[0009] Preferably, in step (1), the bovine albumin is 5% (mass / volume) bovine albumin. The filtration process is carried out by filtering successively through 0.45 μm and 0.22 μm aqueous filtration membranes, filtering once through the 0.45 μm water membrane and twice through the 0.22 μm water membrane; the concentration of the phosphate buffer solution is 0.1 M; the dilution factor of the bovine albumin is 25 times; the volume fraction of ProClin300 is 0.05%, and the ascorbic acid is 0.1% (mass / volume) ascorbic acid. In step (2), the bovine albumin is 1.25% (mass / volume) bovine albumin, and the phosphate buffer solution also contains 0.5% (mass / volume) ascorbic acid and 0.05% (volume / volume) ProClin300. In step (3), the concentration of the phosphate buffer solution used is 0.02 M, and the phosphate buffer solution contains 20% (mass / volume) VC and 1% (volume / volume) acetic acid.

[0010] The present invention also provides a method for detecting water-soluble vitamins in serum for non-diagnostic purposes. Using the above-mentioned kit, it includes the following steps: (i) Reagent preparation; (ii) Pretreatment: Take the sample in a protein precipitation tube, add the isotope internal standard solution, add the protein precipitant, mix well, transfer the extract in the protein precipitation tube to the well plate using vacuum negative pressure, and then detect it with a mass spectrometer.

[0011] Preferably, step (i) specifically includes: (i-1) Prepare the calibrator; (i-2) Prepare the standard curve; (i-3) Prepare the isotope internal standard solution; (i-4) Prepare the mobile phase: Dilute the mobile phase concentrate with ultrapure water or methanol to prepare mobile phase A or mobile phase B.

[0012] Preferably, in step (ii), the chromatographic parameters are controlled as follows: Chromatographic column: Agilent ZORBAX Eclipse Plus-C18, specification 4.6 * 50 mm, particle size 1.7 μm; Pre-column: Agilent ZORBAX Eclipse Plus-C18, specification 4.6 * 12.5 mm, particle size 1.7 μm; Flow rate: 0.5 mL / min; Column temperature: 35 °C; Injection volume: 10 μL; Liquid chromatography elution gradient: From 0 to 1.0 min, the proportion of mobile phase A is 95% and the proportion of mobile phase B is 5%; From 1.0 to 1.5 min, the proportion of mobile phase A is 60 - 95% and the proportion of mobile phase B is 5 - 40%; From 1.5 to 2.5 min, the proportion of mobile phase A is 50 - 60% and the proportion of mobile phase B is 40 - 50%; From 2.5 to 3.0 min, the proportion of mobile phase A is 5 - 50% and the proportion of mobile phase B is 50 - 95%; From 3.0 to 3.8 min, the proportion of mobile phase A is 5% and the proportion of mobile phase B is 95%; From 3.8 to 4.2 min, the proportion of mobile phase A is 5 - 95% and the proportion of mobile phase B is 5 - 95%; From 4.2 to 5.0 min, the proportion of mobile phase A is 95% and the proportion of mobile phase B is 5%.

[0013] Preferably, in step (ii), the mass spectrometry parameters are controlled as follows: Ionization mode: electrospray positive ion mode; Capillary voltage: 0.5 kV; Cone voltage: 30 V; Desolvation gas temperature: 500 °C; Desolvation gas flow rate: 1000 L / Hr; Cone gas flow rate: 20 L / Hr; Scanning mode: multiple reaction monitoring; Scanning interval time 15 - 200 ms, cone voltage 20 - 50 V, collision energy 20 - 30 eV.

[0014] The beneficial effects of the present invention are as follows: The present invention does not require complex and time-consuming processing steps such as nitrogen blowing and reconstitution, which are not easy to automate, greatly improving the pretreatment efficiency and facilitating meeting the clinical requirements of rapidity, high efficiency, and simplicity. At the same time, the sulfosalicylic acid - ammonium acetate system also provides an acidic environment for water-soluble vitamins, which is more conducive to the stability of vitamins such as 5-MTHF, ensuring the accuracy of detection; the present invention effectively solves the problem of high spike recovery rate during the detection of water-soluble vitamins and can more accurately quantify water-soluble vitamins in serum. In addition, the present invention also combines a vacuum negative pressure device for sample pretreatment to achieve semi-automation of water-soluble vitamin detection, providing a practical solution for the automated research and development of water-soluble vitamin detection. Description of the Drawings

[0015] Figure 1 is the pretreatment flow chart; Figure 2 is the linear fitting regression equation and scatter plot. Detailed implementation mode

[0016] The following is a further specific description of the technical solution of the present invention through embodiments. These embodiments are for the purpose of explaining the present invention and not for limiting it. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of this application.

[0017] In the experimental methods described in the embodiments, unless otherwise specified, they are all conventional methods; the reagents and materials, unless otherwise specified, can all be obtained from commercial channels.

[0018] I. Preparation of the kit 1. The components of the kit are shown in Table 1: Table 1 Components of the water-soluble vitamin detection kit

[0019] 2. Preparation of each component of the kit (1) Calibrator diluent: Adsorb 5% BSA with activated carbon for 1 h; filter successively through 0.45 μm and 0.22 μm aqueous filter membranes, filter once with the 0.45 μm water membrane and twice with the 0.22 μm water membrane. Dilute the above 5% BSA 25-fold with 0.1 M PBS, and then add 0.05% (volume ratio) ProClin300 and 0.1% ascorbic acid.

[0020] (2) Calibrators and quality control products: Prepare calibrators and quality control products with 1.25% BSA-PBS solution (0.5% ascorbic acid, 0.05% ProClin300). According to the concentrations shown in Table 2, in ng / mL, each bottle of calibrator is encapsulated with 600 μL, and each bottle of quality control product is encapsulated with 500 μL, and stored at -20 ± 5 °C.

[0021] Table 2 Concentrations of water-soluble vitamin calibrators and quality control products

[0022] (3) Isotope internal standard solution: Take the water-soluble vitamin isotope internal standard mother liquor and add it to 0.02 M PBS (containing 20% VC and 1% acetic acid). According to the concentrations shown in Table 3, in ng / mL, each bottle is aliquoted with 750 μL and stored at -20 ± 5 °C.

[0023] Table 3 Concentrations of water-soluble vitamin isotope internal standards

[0024] (4) Protein precipitant: Composed of 4% sulfosalicylic acid and 150 mM ammonium acetate. 1.7 g of sulfosalicylic acid and 0.5 g of ammonium acetate are added to 42 mL of ultrapure water, mixed well, aliquoted, and stored at 2 - 8 °C.

[0025] (5) Mobile phase concentrate: Composed of 2 M ammonium acetate - 50% methanol. 15.4 g of ammonium acetate is dissolved in 50% methanol, made up to 100 mL, mixed well, aliquoted, and stored at -20 ± 5 °C.

[0026] (6) Freeze-drying: Calibrators, quality control samples, and isotope internal standards need to be freeze-dried. The freeze-drying process of the freeze-dryer is shown in Table 4 below; Table 4 Freeze-drying procedures for calibrators, quality control samples, and isotope internal standards

[0027] During the pre-freezing process, the inlet temperature is less than -45 °C for ≥ 300 min; During the sublimation drying, the inlet temperature is less than -15 °C for ≥ 660 min, and the vacuum must be ≤ 50 Pa; During the desorption drying, the inlet temperature is greater than 15 °C for ≥ 300 min.

[0028] (7) The above calibrators, quality control samples, isotope internal standard solutions, protein precipitants, calibrator diluents, and mobile phase concentrates are loaded into the kit and stored at -20 ± 5 °C.

[0029] II. Detection method 1. Reagent preparation: (1) Calibrator J6 and quality control samples (Z1, Z2): Let stand at room temperature for more than 10 min. Add 1.2 mL of ultrapure water to the calibrator for reconstitution, and add 1 mL of ultrapure water to the quality control samples for reconstitution. Mix well and set aside.

[0030] (2) Preparation of the standard curve: Take the reconstituted calibrator J6 and prepare the calibration curve according to the method in Table 5; Table 5 Preparation of the standard curve

[0031] (3) Internal standard working solution: Let the isotope internal standard stand at room temperature for more than 10 min. Add 1.5 mL of ultrapure water for reconstitution, mix well, and set aside.

[0032] (4) Mobile phase: Dilute the mobile phase concentrate 200-fold with ultrapure water or methanol to prepare mobile phase A or mobile phase B. Sonicate for 10 min and set aside.

[0033] 2. Pretreatment operation steps: Take 50 μL of the sample in a protein precipitation tube, add 10 μL of the internal standard working solution, add 150 μL of the protein precipitant, mix well for 1 min, transfer the extract in the protein precipitation tube to a 96-well plate using vacuum negative pressure, and then detect it on a Waters TQD mass spectrometer. The pretreatment operation process is as Figure 1 shown.

[0034] 3. Chromatographic parameters (1) Chromatographic column: Agilent ZORBAX Eclipse Plus-C18 (4.6 * 50 mm, 1.7 μm); (2) Pre-column: Agilent ZORBAX Eclipse Plus-C18 (4.6 * 12.5 mm, 1.7 μm) + High performance ZORBAX guard fittings; (3) Flow rate: 0.5 mL / min; Column temperature: 35 °C; Injection volume: 10 μL; (4) Liquid chromatography elution gradient: See Table 6: Table 6 Chromatographic gradient elution program for water-soluble vitamins

[0035] 4. Mass spectrometry parameters (1) Ion source: Electrospray ionization source (ESI), specific parameters are shown in Table 7: Table 7 Ion source parameters

[0036] (2) Scanning mode: Multiple reaction monitoring (MRM), specific parameters are shown in Table 8: Table 8 Mass spectrometry conditions for water-soluble vitamins

[0037] III. Optimization of the sulfosalicylic acid-ammonium acetate system (1) Preparation of sulfosalicylic acid solutions with different concentrations: ① 2% sulfosalicylic acid: Weigh 2 g of sulfosalicylic acid and dissolve it in 100 mL of ultrapure water, sonicate for 10 min to obtain 2% sulfosalicylic acid, denoted as group 1, and reserve it; ② 4% sulfosalicylic acid: Weigh 4 g of sulfosalicylic acid and dissolve it in 100 mL of ultrapure water, sonicate for 10 min to obtain 4% sulfosalicylic acid, denoted as group 2, and reserve it; ③ 6% sulfosalicylic acid: Weigh 6 g of sulfosalicylic acid and dissolve it in 100 mL of ultrapure water, sonicate for 10 min to obtain 6% sulfosalicylic acid, denoted as group 3, and reserve it; ④ 8% Sulfosalicylic Acid: Weigh 8 g of sulfosalicylic acid and dissolve it in 100 mL of ultrapure water. Sonicate for 10 min to obtain 8% sulfosalicylic acid, denoted as Group 4, and set aside for use. (2) Preparation of Sulfosalicylic Acid - Ammonium Acetate with Different Concentrations ① 4% Sulfosalicylic Acid - 50 mM Ammonium Acetate: Weigh 4 g of sulfosalicylic acid and 385 mg of ammonium acetate, dissolve them in 100 mL of ultrapure water, sonicate for 10 min to obtain 4% sulfosalicylic acid - 50 mM ammonium acetate, denoted as Group 5, and set aside for use. ② 4% Sulfosalicylic Acid - 100 mM Ammonium Acetate: Weigh 4 g of sulfosalicylic acid and 770 mg of ammonium acetate, dissolve them in 100 mL of ultrapure water, sonicate for 10 min to obtain 4% sulfosalicylic acid - 100 mM ammonium acetate, denoted as Group 6, and set aside for use. ③ 4% Sulfosalicylic Acid - 150 mM Ammonium Acetate: Weigh 4 g of sulfosalicylic acid and 1155 mg of ammonium acetate, dissolve them in 100 mL of ultrapure water, sonicate for 10 min to obtain 4% sulfosalicylic acid - 150 mM ammonium acetate, denoted as Group 7, and set aside for use. ④ 4% Sulfosalicylic Acid - 200 mM Ammonium Acetate: Weigh 4 g of sulfosalicylic acid and 1540 mg of ammonium acetate, dissolve them in 100 mL of ultrapure water, sonicate for 10 min to obtain 4% sulfosalicylic acid - 200 mM ammonium acetate, denoted as Group 8, and set aside for use. Select actual human serum as the basic sample for the recovery experiment. Conduct the recovery experiment on experimental groups 1 - 8. For each group, the basic sample and the spiked sample are prepared in triplicate. Treat the samples with the above protein precipitants respectively, record the experimental results and conduct statistical analysis, and compare the spiked recoveries of each group of experiments. The results are shown in Table 9. Among them, the low concentration is 5 times the lower limit of the linear range; the medium concentration is the middle concentration of the linear range; the high concentration is 75% of the upper limit of the linear range.

[0038] Table 9 Spiked Recoveries of Each Substance with Different Protein Precipitants

[0039] The results show that when 4% sulfosalicylic acid is used as the protein precipitant, the recovery rate of VB1 is the highest, and the recovery rates of other substances are all greater than 85%. After adding ammonium acetate, the recovery of VB1 increases significantly. Considering comprehensively, when 4% sulfosalicylic acid - 150 mM ammonium acetate is used as the protein precipitant, the recovery rates of each substance are the best.

[0040] Detection Data Processing: I. Results of the Linear Test of the Calibration Curve Within the linear range of each index of water - soluble vitamins, the correlation coefficients of their calibration curves all meet the requirements (the requirement is R 2 ≥ 0.99), and the results are shown in Table 10: Table 10 Linearity and Retention Time of Water - Soluble Vitamins

[0041] II. Precision Detect the concentrations of samples at two concentration levels, low and high, respectively. Conduct 3 replicates each day for 5 consecutive days, and statistically analyze the within-batch and between-batch precision. Requirements: within-batch CV value < 6.25%, between-batch CV value < 8.33%.

[0042] The 5-day precision experiment shows that the method described in the present invention has good within-batch and between-batch precision. The results are shown in Table 11-1 and Table 11-2, and both the within-batch and between-batch precision meet the requirements.

[0043] Table 11-1 5-day precision of VB1, VB2, VB3-NM

[0044] Table 11-2 5-day precision of VB5, VB6-PA, 5-MTHF

[0045] III. Accuracy Verification Select actual human serum as the basic sample for the recovery experiment. Mix 50 μL of low, medium, and high standard samples with 950 μL of the basic sample respectively, as shown in Table 12. The low concentration is 5 times the lower limit of the linear range; the medium concentration is the middle concentration of the linear range; the high concentration is 75% of the upper limit of the linear range. Repeat the parallel determination of the above "basic sample" and "experimental sample" 6 times, record the experimental results and conduct statistical analysis of the spiked recovery at 3 concentration levels of low, medium, and high. Recovery rate = (detected value - basic sample value) / theoretical value × 100%. Requirements: 85% < recovery rate < 115%.

[0046] Table 12 Preparation table for spiked recovery

[0047] Verify the accuracy of the spiked recovery method. The results are shown in Table 13, and the average recovery rates of all indexes of water-soluble vitamins meet the requirements.

[0048] Table 13 Results of accuracy verification of water-soluble vitamins

[0049] IV. Lower Limit of Quantification The lower limit of quantification is the lowest value of the target analyte detected by the LC-MS / MS method while meeting the laboratory's requirements for accuracy and precision. For the lower limit of quantification, 5 samples with concentrations close to the detection limit were selected. Each concentration sample was divided into 5 portions for processing, and each portion was measured 3 times. Three batches were measured continuously. The total precision (CV) of each concentration sample and the deviation between the mean value of the concentration measurement and the theoretical concentration were evaluated respectively. The mean value of the lowest concentration sample with CV ≤ 20% and bias < 15% was used as the lower limit of quantification of the method. The results of the lower limit of quantification of water-soluble vitamins are shown in Tables 14-1 and 14-2: Table 14-1 Results of the lower limit of quantification of VB1, VB2, and VB3-NM

[0050] Table 14-2 Results of the lower limit of quantification of VB5, VB6-PA, and 5-MTHF

[0051] As can be seen from Tables 14-1 and 14-2, the lower limit of quantification of VB1 is 0.47 ng / mL, the lower limit of quantification of VB2 is 1.25 ng / mL, the lower limit of quantification of VB3-NM is 1.875 ng / mL, the lower limit of quantification of VB5 is 5.63 ng / mL, the lower limit of quantification of VB6-PA is 1.25 ng / mL, and the lower limit of quantification of 5-MTHF is 0.94 ng / mL, indicating that the method described in the present invention can still ensure the reliability of the results when detecting low-concentration samples.

[0052] V. Dilution reliability Human serum near the upper limit of the linear range (AMR) was collected and diluted 2, 4, and 8 times with ultrapure water respectively. It was required that 80% ≤ diluted test value / theoretical value ≤ 120% to determine that the dilution passed the verification. The results of the dilution reliability of water-soluble vitamins are shown in Tables 15-1 and 15-2: Table 15-1 Results of the dilution reliability of VB1, VB2, and VB3-NM

[0053] Table 15-2 Results of the dilution reliability of VB5, VB6-PA, and 5-MTHF

[0054] As can be seen from Tables 15-1 and 15-2, the maximum dilution factor for each index of water-soluble vitamins is 8, indicating that the method described in the present invention can still ensure the reliability of the results when diluting high-concentration samples 8 times.

[0055] VI. Linear verification According to the requirements of the "Guidelines for the Linear Evaluation of Clinical Chemistry Equipment" (WS / T 480-2012) of the People's Republic of China, when verifying the linear range, samples at 5 concentration levels are selected for determination, and each sample is measured 4 times. All samples should be pretreated and analyzed quantitatively on the instrument within the shortest time. The linear verification results of water-soluble vitamins are shown in Table 16: Table 16 Linear verification results of water-soluble vitamins

[0056] Perform a first-order fitting regression equation and scatter plot for the obtained data. The linear fitting regression equation and scatter plot are as Figure 2 shown. The regression equation for the linear verification of VB1: y = 1.0015x + 0.0701, R 2 = 0.9999; The regression equation for the linear verification of VB2: y = 1.017x - 0.478, R 2 = 0.9995; The regression equation for the linear verification of VB3-NM: y = 0.9926x + 0.0543, R 2 = 0.9999; The regression equation for the linear verification of VB5: y = 0.9953x + 0.2753, R 2 = 0.9998; The regression equation for the linear verification of VB6-PA: y = 0.998x - 0.1515, R 2 = 0.9999; The regression equation for the linear verification of 5-MTHF: y = 1.0035x - 0.4351, R 2 = 0.9998.

[0057] When the correlation coefficient R 2 ≥ 0.95 and the regression coefficient b is within the range of 0.97 - 1.03, it indicates that it is qualified within this linear range. From Figure 2 it can be seen that by performing regression statistical analysis on the expected values and measured values of various indicators of water-soluble vitamins, the regression coefficient b and the correlation coefficient R 2 both meet the requirements of the "Guidelines for the Linear Evaluation of Clinical Chemistry Equipment", indicating that the linearity of this method meets the verification requirements.

[0058] VII. Matrix effect In the experiment, pure solutions of the target analyte, biological matrix samples, and a 1:1 mixture of the two are selected, pretreated respectively and then injected for analysis. If the response value of the 1:1 mixture sample is lower than a certain proportion (20%) compared with the mean of the response values of the biological matrix sample and the pure solution sample, it proves whether the matrix effect exists or does not affect the accurate quantification of the target analyte. The matrix effect results of water-soluble vitamins at different concentrations are shown in Table 17: Table 17 Matrix effect results of water-soluble vitamins at different concentrations

[0059] By investigating and analyzing the matrix effects of various indicators of water-soluble vitamins at different concentrations, the results showed that the deviations of the low, medium and high levels were all less than 15%, indicating that the use of the sulfosalicylic acid-ammonium acetate system in the present invention has well solved the problem of high spike recovery rate and reduced interference during detection. The method of the present invention is less susceptible to interference and does not affect the quantification of the target compound.

[0060] 8. Extraction stability test 10 human serum samples were selected, treated with sulfosalicylic acid-ammonium acetate system and placed in 2-8℃ environment, and tested at 0h, 24h, 48h, and 72h, respectively, and the test results were recorded and analyzed. The stability results of the extract are shown in Table 18-1, Table 18-2, and Table 18-3: Table 18-1 Stability results of VB1 and VB2 extracts

[0061] Table 18-2 Stability results of VB3-NM and VB5 extracts

[0062] Table 18-3 Stability results of VB6-PA and 5-MTHF extracts

[0063] It can be seen from Table 18-1, Table 18-2 and Table 18-3 that the water-soluble vitamins in serum can be stable at 2-8°C for 72 hours after being treated with sulfosalicylic acid-ammonium acetate protein precipitant.

[0064] Finally, it should be noted that the above are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A water-soluble vitamin detection kit in serum, characterized in that: The kit includes a calibrator, a quality control product, an isotope internal standard solution, a protein precipitant, a calibrator diluent, and a mobile phase concentrate; The calibrator includes water-soluble vitamins at one concentration; The quality control product includes water-soluble vitamins at two or more concentrations; The isotope internal standard solution is a water-soluble vitamin isotope internal standard; The protein precipitant is a combination of sulfosalicylic acid and ammonium acetate; The calibrator diluent includes bovine albumin, ProClin300, and ascorbic acid; The mobile phase concentrate is an aqueous solution of ammonium acetate in methanol; The water-soluble vitamins include vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, and 5-methyltetrahydrofolic acid.

2. The water-soluble vitamin detection kit in serum according to claim 1, wherein: The concentration of the calibrator is 30 - 220 ng / mL; the concentration of the quality control product is 10 - 220 ng / mL; the concentration of the isotope internal standard solution is 30 - 600 ng / mL; the components of the protein precipitant are 4% sulfosalicylic acid and 150 mM ammonium acetate; the components of the mobile phase concentrate are 2 M ammonium acetate and 50% methanol.

3. A method for preparing the water-soluble vitamin detection kit for the serum as described in claim 1, characterized in that It includes the following steps: (1) Prepare the calibrator diluent: Adsorb bovine albumin with activated carbon; filter through a water-based filter membrane; after diluting the above bovine albumin with phosphate buffer, then add ProClin300 and ascorbic acid; (2) Prepare the calibrator and the quality control product: Prepare the calibrator and the quality control product with phosphate buffer containing bovine albumin; (3) Prepare the isotope internal standard solution: Take the mother liquor of the water-soluble vitamin isotope internal standard and add it to the phosphate buffer; (4) Prepare the protein precipitant: Add sulfosalicylic acid and ammonium acetate to ultrapure water, mix well, aliquot, and store at 2 - 8 °C; (5) Prepare the mobile phase concentrate: Dissolve ammonium acetate in 50% methanol, after complete dissolution, make up the volume with 50% methanol, mix well, aliquot, and store at -20 ± 5 °C; (6) Lyophilize: Lyophilize the calibrator, the quality control product, and the isotope internal standard; (7) Load the calibrator, the quality control product, the isotope internal standard solution, the protein precipitant, the calibrator diluent, and the mobile phase concentrate into the kit, and store at -20 ± 5 °C.

4. The preparation method according to claim 3, characterized in that: In step (1), the bovine albumin is 5% bovine albumin, and the filtration process is to filter successively through 0.45 μm and 0.22 μm water-based filter membranes, filter once with the 0.45 μm water membrane and twice with the 0.22 μm water membrane; the concentration of the phosphate buffer is 0.1 M; the dilution multiple of the bovine albumin is 25 times; the volume fraction of ProClin300 is 0.05%, and the ascorbic acid is 0.1% ascorbic acid; In step (2), the bovine albumin is 1.25% bovine albumin, and the phosphate buffer also contains 0.5% ascorbic acid and 0.05% ProClin300; In step (3), the concentration of the phosphate buffer used is 0.02 M, and the phosphate buffer contains 20% VC and 1% acetic acid.

5. A method for detecting water-soluble vitamins in serum for non-diagnostic purposes, characterized in that: Using the kit described in claim 1, it includes the following steps: (i) Reagent preparation; (ii) Pretreatment: Take the sample in a protein precipitation tube, add the isotope internal standard solution, add the protein precipitant, mix well, transfer the extract in the protein precipitation tube to a well plate using vacuum negative pressure, and then detect it on a mass spectrometer.

6. The detection method according to claim 5, wherein Step (i) specifically includes: (i-1) Prepare calibration standards; (i-2) Prepare a standard curve; (i-3) Prepare an isotopic internal standard solution; (i-4) Prepare the mobile phase: The mobile phase concentrate is diluted with ultrapure water or methanol to prepare mobile phase A or mobile phase B.

7. The detection method according to claim 6, wherein In step (ii), the chromatographic parameters are controlled as follows: Chromatographic column: Agilent ZORBAX Eclipse Plus-C18, specification 4.6*50 mm, particle size 1.7 μm; Pre-column: Agilent ZORBAX Eclipse Plus-C18, specification 4.6*12.5 mm, particle size 1.7 μm; Flow rate: 0.5 mL / min; Column temperature: 35°C; Injection volume: 10 μL; Liquid chromatography elution gradient: 0 - 1.0 min, the proportion of mobile phase A is 95%, and the proportion of mobile phase B is 5%; 1.0 - 1.5 min, the proportion of mobile phase A is 60 - 95%, and the proportion of mobile phase B is 5 - 40%; 1.5 - 2.5 min, the proportion of mobile phase A is 50 - 60%, and the proportion of mobile phase B is 40 - 50%; 2.5 - 3.0 min, the proportion of mobile phase A is 5 - 50%, and the proportion of mobile phase B is 50 - 95%; 3.0 - 3.8 min, the proportion of mobile phase A is 5%, and the proportion of mobile phase B is 95%; 3.8 - 4.2 min, the proportion of mobile phase A is 5 - 95%, and the proportion of mobile phase B is 5 - 95%; 4.2 - 5.0 min, the proportion of mobile phase A is 95%, and the proportion of mobile phase B is 5%.

8. The detection method according to claim 6, wherein In step (ii), the mass spectrometry parameters are controlled as follows: Ionization mode: Electrospray positive ion mode; Capillary voltage: 0.5 kV; Cone voltage: 30 V; Desolvation gas temperature: 500°C; Desolvation gas flow rate: 1000 L / Hr; Cone gas flow rate: 20 L / Hr; Scanning mode: Multiple reaction monitoring; Scanning interval time 15 - 200 ms, cone voltage 20 - 50 V, collision energy 20 - 30 eV.

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