Detection kit for water-soluble vitamins in serum and its preparation and detection method

Abstract

The present invention relates to a kind of water-soluble vitamin detection kit in serum and its preparation and detection method, prepare water-soluble vitamin calibrators and quality control products and corresponding supporting reagents to form a water-soluble vitamin detection kit, utilize sulfosalicylic acid ammonium acetate system to carry out protein precipitation, assisted by protein precipitation tube by vacuum negative pressure device to remove protein precipitation, obtain clean filtrate, and then use mass spectrometry detection. The present invention not only avoids the cumbersome steps that organic solvent protein precipitation method needs centrifugation and concentration, simplifies water-soluble vitamin detection pre-treatment process, and effectively solves the problem that spike recovery rate is high, improves the efficiency of water-soluble vitamin detection;Also provides new automation ideas for the detection of other indicators.

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] Currently, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) is the predominant method for the detection of water-soluble vitamins. This method boasts high sensitivity and specificity, making it the "gold standard" for vitamin detection. However, its analytical performance is significantly affected by the sample, placing high demands on sample preparation. Inadequate sample pretreatment or cleanup can compromise the accuracy and reproducibility of quantitative analysis results, contaminate the instrument, and incur high maintenance costs. Long-term operation can even lead to instrument failure. Currently, the main methods for serum sample preparation include magnetic bead extraction, solid-phase extraction, and protein precipitation (PPT). While the magnetic bead method is easily automated, it requires specificity, cannot simultaneously detect multiple parameters, and may even require conjugated antibodies, resulting in poor reproducibility and high costs. Protein precipitation, on the other hand, often uses organic solvents as protein precipitants, but requires a complex series of steps, including centrifugation, nitrogen purging, and reconstitution, which is time-consuming and labor-intensive, making it difficult to automate. Furthermore, the large amounts of volatile organic solvents present health risks to laboratory operators.

[0004] Chinese patent CN106324142A discloses a method for determining water-soluble vitamins using semi-automated sample processing liquid chromatography. The method uses methanol or acetonitrile to precipitate proteins, centrifuges, and extracts the supernatant using a semi-automated solid-phase extractor. The extract is then eluted with a methanol-water solution, purged with nitrogen, and reconstituted for analysis. This method is complex and time-consuming, making it unsuitable for rapid clinical testing. Chinese patent CN110412175A discloses a method for detecting water-soluble vitamins in blood. The method uses sulfosalicylic acid as a protein precipitant, centrifuges, and extracts the supernatant for analysis. Although simple to operate, the method suffers from significant interference in the detection of 5-methyltetrahydrofolate (5-MTHF), resulting in spike recovery rates as high as approximately 150%, leading to quantitative inaccuracies and failing to meet clinical quantitative testing requirements. Summary of the Invention

[0005] In order to overcome the shortcomings of the existing technology, the present invention provides a detection kit for water-soluble vitamins in serum and its preparation and detection method. The present invention not only avoids the tedious steps of centrifugation and concentration required by the organic solvent protein precipitation method, simplifies the pretreatment process of water-soluble vitamin detection, but also effectively solves the problem of high spike recovery rate, improves the efficiency of water-soluble vitamin detection; and also provides a new automation idea for the detection of other indicators.

[0006] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0007] A kit for detecting water-soluble vitamins in serum comprises a calibrator, a quality control product, an isotope internal standard solution, a protein precipitant, a calibrator diluent, and a mobile phase concentrate. The calibrator comprises a water-soluble vitamin at one concentration; the quality control product comprises a water-soluble vitamin 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 mobile phase concentrate is an ammonium acetate methanol aqueous solution; the water-soluble vitamins comprise vitamin B1 (VB1), vitamin B2 (VB2), vitamin B3 (VB3-NM), vitamin B5 (VB5), vitamin B6 (VB6-PA), and 5-methyltetrahydrofolate (5-MTHF).

[0008] Preferably, the concentration of the calibrator is 30-220 ng / mL, the concentration of the quality control is 10-220 ng / mL, the concentration of the isotope internal standard solution is 30-600 ng / mL, the protein precipitant composition is 4% (mass / volume) sulfosalicylic acid and 150 mM ammonium acetate; the mobile phase concentrate composition is 2 M (mol / L) ammonium acetate and 50% (volume / volume) methanol.

[0009] The present invention also provides a method for preparing the above-mentioned serum water-soluble vitamin detection kit, comprising the following steps:

[0010] (1) Prepare the calibrant diluent: adsorb bovine albumin (BSA) using activated carbon; filter through a water filter membrane; dilute the BSA with phosphate buffered saline (PBS), and then add ProClin 300 and ascorbic acid;

[0011] (2) Prepare calibrators and quality control products: Prepare calibrators and quality control products using phosphate buffer containing bovine albumin;

[0012] (3) Prepare isotope internal standard solution: add water-soluble vitamin isotope internal standard stock solution into phosphate buffer;

[0013] (4) Prepare protein precipitant: add sulfosalicylic acid and ammonium acetate to ultrapure water, mix well, aliquot and store at 2-8°C;

[0014] (5) Prepare mobile phase concentrate: dissolve ammonium acetate in 50% methanol, dilute to volume with 50% methanol after complete dissolution, mix well, aliquot and store at -20±5℃;

[0015] (6) Freeze drying: freeze drying of calibrators, quality control products and isotope internal standards;

[0016] (7) Place the calibrator, quality control, isotope internal standard solution, protein precipitant, calibrator diluent and mobile phase concentrate into the kit and store at -20±5℃.

[0017] Preferably, in step (1), the bovine albumin is 5% (mass / volume) bovine albumin, and the filtration process is sequentially filtered through 0.45 μm and 0.22 μm water filter membranes, 0.45 μm water membrane filtration once, and 0.22 μm water membrane filtration twice; the phosphate buffer concentration is 0.1 M; the bovine albumin is diluted 25 times; the volume fraction of ProClin300 is 0.05%, and the ascorbic acid is 0.1% (mass / volume) ascorbic acid;

[0018] In step (2), the bovine albumin is 1.25% (mass / volume) bovine albumin, and the phosphate buffer further contains 0.5% (mass / volume) ascorbic acid and 0.05% (volume / volume) ProClin300;

[0019] In step (3), the concentration of the phosphate buffer used is 0.02 M, and the phosphate buffer contains 20% (mass / volume) VC and 1% (volume / volume) acetic acid.

[0020] The present invention also provides a method for detecting water-soluble vitamins in serum for non-diagnostic purposes, using the above-mentioned kit, comprising the following steps:

[0021] (i) Reagent preparation;

[0022] (ii) Pretreatment: Place the sample in a protein precipitation tube, add isotope internal standard solution, add protein precipitant, mix well, and use vacuum negative pressure to transfer the extract in the protein precipitation tube to the well plate, and then detect it on the mass spectrometer.

[0023] Preferably, step (i) specifically comprises:

[0024] (i-1) Prepare calibrators;

[0025] (i-2) Prepare a standard curve;

[0026] (i-3) Prepare isotope internal standard solution;

[0027] (i-4) Prepare mobile phase: Dilute the mobile phase concentrate with ultrapure water or methanol to prepare mobile phase A or mobile phase B.

[0028] Preferably, in step (ii), the chromatographic parameters are controlled as follows:

[0029] Chromatographic column: Agilent ZORBAX Eclipse Plus-C18, size 4.6*50 mm, particle size 1.7 μm;

[0030] Precolumn: Agilent ZORBAX Eclipse Plus-C18, size 4.6*12.5 mm, particle size 1.7 μm;

[0031] Flow rate: 0.5 mL / min; column temperature: 35°C; injection: 10 μL;

[0032] LC elution gradient:

[0033] 0-1.0 min, the proportion of mobile phase A is 95%, and the proportion of mobile phase B is 5%;

[0034] 1.0-1.5 min, mobile phase A ratio is 60-95%, mobile phase B ratio is 5-40%;

[0035] 1.5-2.5 min, mobile phase A ratio is 50-60%, mobile phase B ratio is 40-50%;

[0036] 2.5-3.0 min, mobile phase A ratio is 5-50%, mobile phase B ratio is 50-95%;

[0037] 3.0-3.8 min, mobile phase A ratio is 5%, mobile phase B ratio is 95%;

[0038] 3.8-4.2 min, mobile phase A ratio is 5-95%, mobile phase B ratio is 5-95%;

[0039] 4.2-5.0 min, the proportion of mobile phase A is 95%, and the proportion of mobile phase B is 5%.

[0040] Preferably, in step (ii), the mass spectrometry parameters are controlled as follows:

[0041] Ionization mode: electrospray positive ion mode; capillary voltage: 0.5 kV; cone voltage: 30 V; desolvation temperature: 500 °C; desolvation gas flow rate: 1000 L / Hr; cone gas flow rate: 20 L / Hr;

[0042] Scan mode: multiple reaction monitoring; scan interval 15-200 ms, cone voltage 20-50 V, collision energy 20-30 eV.

[0043] The beneficial effects of the present invention are:

[0044] The present invention does not require nitrogen blowing, redissolution and other complex, time-consuming and difficult to automate processing steps, greatly improving the pre-treatment efficiency, and is conducive to meeting clinical rapid, efficient and simplified needs. 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, and ensures the accuracy of detection; the present invention effectively solves the problem of high spike recovery during water-soluble vitamin detection, and can more accurately quantify the water-soluble vitamins in serum. In addition, the present invention also combines a vacuum negative pressure device to carry out sample pretreatment, realizes semi-automatic detection of water-soluble vitamins, and provides a practical solution for the automated research and development of water-soluble vitamin detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the pre-processing flow chart;

[0046] Figure 2 is the linear fitting regression equation and scatter plot. DETAILED DESCRIPTION

[0047] The technical solutions of the present invention are further specifically described below through examples. These examples are provided for the purpose of illustrating the present invention and are not intended to limit the present invention. All other examples obtained by persons of ordinary skill in the art based on the examples in this application without creative work are intended to fall within the scope of protection of this application.

[0048] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0049] 1. Kit Preparation

[0050] 1. Kit components are shown in Table 1:

[0051] Table 1 Components of the water-soluble vitamin detection kit

[0052]

[0053] 2. Preparation of each component of the kit

[0054] (1) Calibrator dilution: 5% BSA was adsorbed on activated carbon for 1 hour. The solution was filtered through 0.45 μm and 0.22 μm water filter membranes, once through a 0.45 μm filter membrane and twice through a 0.22 μm filter membrane. The 5% BSA was diluted 25-fold with 0.1 M PBS, and 0.05% (v / v) ProClin 300 and 0.1% ascorbic acid were added.

[0055] (2) Calibrators and quality control products: Use 1.25% BSA-PBS solution (0.5% ascorbic acid, 0.05% ProClin300) to prepare calibrators and quality control products according to the concentrations shown in Table 2 (unit: ng / mL). Calibrators should be packaged in 600 μL bottles and quality control products in 500 μL bottles. Store at -20±5℃.

[0056] Table 2 Concentrations of water-soluble vitamin calibrators and quality control products

[0057]

[0058] (3) Isotope internal standard solution: Take the water-soluble vitamin isotope internal standard stock solution and add it to 0.02M PBS (containing 20% ​​VC and 1% acetic acid). According to the concentration shown in Table 3, the unit is ng / mL. Aliquot 750μL into each bottle and store at -20±5℃.

[0059] Table 3 Isotope internal standard concentrations of water-soluble vitamins

[0060]

[0061] (4) Protein precipitant: The ingredients are 4% sulfosalicylic acid and 150 mM ammonium acetate. Add 1.7 g sulfosalicylic acid and 0.5 g ammonium acetate to 42 mL ultrapure water, mix well, and store at 2-8 °C after aliquoting.

[0062] (5) Mobile phase concentrate: The composition is 2M ammonium acetate-50% methanol. Dissolve 15.4 g ammonium acetate in 50% methanol, dilute to 100 mL, mix well, and store at -20±5°C after aliquoting.

[0063] (6) Freeze drying: Calibrators, quality control products and isotope internal standards need to be freeze dried. The freeze drying process of the freeze dryer is shown in Table 4 below;

[0064] Table 4 Lyophilization procedures for calibrators, quality control products, and isotope internal standards

[0065]

[0066] During the pre-freezing process, the inlet temperature is less than -45℃ for ≥300min;

[0067] During sublimation drying, the inlet temperature must be less than -15°C for ≥660 min, and the vacuum must be ≤50 Pa;

[0068] During analytical drying, the inlet temperature is greater than 15°C for ≥300 minutes.

[0069] (7) The above-mentioned calibrators, quality control products, isotope internal standard solution, protein precipitant, calibrator diluent and mobile phase concentrate are placed in the kit and stored at -20±5℃.

[0070] 2. Detection Method

[0071] 1. Reagent preparation:

[0072] (1) Calibrator J6 and quality control products (Z1, Z2): Let stand at room temperature for more than 10 min, add 1.2 mL of ultrapure water to reconstitute the calibrator and 1 mL of ultrapure water to reconstitute the quality control, mix well, and set aside.

[0073] (2) Preparation of standard curve: Take the reconstituted calibrator J6 and prepare the calibration curve according to the method in Table 5;

[0074] Table 5 Preparation of standard curve

[0075]

[0076] (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 to dissolve it, mix well, and set aside.

[0077] (4) Mobile phase: Dilute the mobile phase concentrate 200 times with ultrapure water or methanol to prepare mobile phase A or mobile phase B, sonicate for 10 minutes, and set aside.

[0078] 2. Pre-processing steps:

[0079] Take 50µL of sample and place it in a protein precipitation tube. Add 10µL of internal standard working solution and 150µL of protein precipitation agent. Mix for 1 minute. Use vacuum negative pressure to transfer the extract in the protein precipitation tube to a 96-well plate. Then, detect it on a Waters TQD mass spectrometer. Figure 1 shown.

[0080] 3. Chromatographic parameters

[0081] (1) Chromatographic column: Agilent ZORBAX Eclipse Plus-C18 (4.6*50 mm, 1.7 μm);

[0082] (2) Pre-column: Agilent ZORBAX Eclipse Plus-C18 (4.6*12.5 mm, 1.7 μm) + High performance ZORBAX guard fittings;

[0083] (3) Flow rate: 0.5 mL / min; column temperature: 35°C; injection: 10 μL;

[0084] (4) Liquid chromatography elution gradient: see Table 6:

[0085] Table 6 Chromatographic gradient elution program for water-soluble vitamins

[0086]

[0087] 4. Mass spectrometry parameters

[0088] (1) Ion source: electrospray ionization (ESI). Specific parameters are shown in Table 7:

[0089] Table 7 Ion source parameters

[0090]

[0091] (2) Scanning mode: Multiple reaction monitoring (MRM). Specific parameters are shown in Table 8:

[0092] Table 8 Mass spectrometry conditions for water-soluble vitamins

[0093]

[0094] 3. Optimization of sulfosalicylic acid-ammonium acetate system

[0095] (1) Preparation of different concentrations of sulfosalicylic acid:

[0096] ① 2% sulfosalicylic acid: Weigh 2 g of sulfosalicylic acid and dissolve it in 100 mL of ultrapure water. Ultrasonicate for 10 min to obtain 2% sulfosalicylic acid, which is recorded as Group 1 and set aside.

[0097] ②4% sulfosalicylic acid: Weigh 4 g of sulfosalicylic acid and dissolve it in 100 mL of ultrapure water. Ultrasonicate for 10 min to obtain 4% sulfosalicylic acid, which is recorded as Group 2 and set aside.

[0098] ③ 6% sulfosalicylic acid: Weigh 6 g of sulfosalicylic acid and dissolve it in 100 mL of ultrapure water. Ultrasonicate for 10 min to obtain 6% sulfosalicylic acid, which is recorded as Group 3 and set aside.

[0099] ④8% sulfosalicylic acid: Weigh 8 g of sulfosalicylic acid and dissolve it in 100 mL of ultrapure water. Ultrasonicate for 10 min to obtain 8% sulfosalicylic acid, which is recorded as Group 4 and set aside.

[0100] (2) Preparation of different concentrations of sulfosalicylic acid-ammonium acetate

[0101] ①4% sulfosalicylic acid-50 mM ammonium acetate: Weigh 4 g sulfosalicylic acid and 385 mg ammonium acetate and dissolve them in 100 mL ultrapure water. Ultrasonicate for 10 min to obtain 4% sulfosalicylic acid-50 mM ammonium acetate, which is designated as Group 5 and reserved for future use.

[0102] ②4% sulfosalicylic acid-100 mM ammonium acetate: Weigh 4 g of sulfosalicylic acid and dissolve 770 mg of ammonium acetate in 100 mL of ultrapure water. Ultrasonicate for 10 min to obtain 4% sulfosalicylic acid-100 mM ammonium acetate, which is designated as Group 6 and reserved for future use.

[0103] ③4% sulfosalicylic acid-150 mM ammonium acetate: Weigh 4 g sulfosalicylic acid and dissolve 1155 mg ammonium acetate in 100 mL ultrapure water. Ultrasonicate for 10 min to obtain 4% sulfosalicylic acid-150 mM ammonium acetate, which is designated as Group 7 and reserved for future use.

[0104] ④4% sulfosalicylic acid-200 mM ammonium acetate: Weigh 4 g of sulfosalicylic acid and dissolve 1540 mg of ammonium acetate in 100 mL of ultrapure water. Ultrasonicate for 10 min to obtain 4% sulfosalicylic acid-200 mM ammonium acetate, which is designated as Group 8 and reserved for future use.

[0105] Actual human serum was selected as the base sample for the recovery experiments. Recovery experiments were performed on experimental groups 1-8. Three replicates of the base sample and spiked sample were run in each group. The samples were treated with the aforementioned protein precipitants. The experimental results were recorded and statistically analyzed. The spiked recoveries of each group were compared, as shown in Table 9. The low concentration was 5 times the lower limit of the linear range; the medium concentration was the middle concentration of the linear range; and the high concentration was 75% of the upper limit of the linear range.

[0106] Table 9 Spiked recovery of various substances with different protein precipitants

[0107]

[0108] The results showed that VB1 had the highest recovery rate when 4% sulfosalicylic acid was used as the protein precipitant, while the recoveries of all other substances were greater than 85%. The addition of ammonium acetate significantly increased VB1 recovery. Overall, the 4% sulfosalicylic acid-150 mM ammonium acetate solution provided the best recoveries for all substances.

[0109] Detection data processing:

[0110] 1. Calibration curve linearity test results

[0111] Within the linear range of each index of water-soluble vitamins, the correlation coefficients of the calibration curves all meet the requirements (R 2 ≥0.99), the results are shown in Table 10:

[0112] Table 10 Linearity and retention time of water-soluble vitamins

[0113]

[0114] 2. Precision

[0115] The concentrations of samples at low and high concentration levels were tested separately, with 3 repetitions per day for 5 consecutive days. The intra-batch and inter-batch precision were calculated, with the requirements: intra-batch CV value < 6.25% and inter-batch CV value < 8.33%.

[0116] The 5-day precision experiment showed that the method of the present invention has good intra-batch and inter-batch precision. The results are shown in Table 11-1 and Table 11-2. Both intra-batch and inter-batch precision meet the requirements.

[0117] Table 11-1 VB1, VB2, VB3-NM 5-day precision

[0118]

[0119] Table 11-2 5-day precision of VB5, VB6-PA, and 5-MTHF

[0120]

[0121] 3. Correctness Verification

[0122] Actual human serum was used as the base sample for the recovery experiment. 50 μL of low, medium, and high standard samples were mixed with 950 μL of the base sample, respectively. As shown in Table 12, the low concentration was 5 times the lower limit of the linear range; the medium concentration was the middle of the linear range; and the high concentration was 75% of the upper limit of the linear range. The "base sample" and "experimental sample" were measured in parallel six times. The experimental results were recorded and statistically analyzed for the spiked recoveries at the low, medium, and high concentration levels. Recovery = (test value - base sample value) / theoretical value × 100%, with a requirement of 85% < recovery < 115%.

[0123] Table 12 Spike recovery preparation table

[0124]

[0125] The accuracy of the spike recovery verification method was verified. The results are shown in Table 13. The average recovery rates of various indicators of water-soluble vitamins all met the requirements.

[0126] Table 13 Verification results of water-soluble vitamin accuracy

[0127]

[0128] 4. Lower limit of quantification

[0129] The lower limit of quantification (LLQ) is the lowest value of the target analyte that can be detected by the LC-MS / MS method while meeting the laboratory's accuracy and precision requirements. Five samples with concentrations close to the detection limit were selected for the LLQ. Each concentration sample was divided into five portions for processing, and each sample was measured three times. Three batches were measured consecutively, and the overall precision (CV) of each concentration sample and the deviation of its mean concentration from the theoretical concentration were evaluated. The mean value of the lowest concentration sample with a CV ≤ 20% and a bias < 15% was used as the LLQ of the method. The results of the LLQ for water-soluble vitamins are shown in Tables 14-1 and 14-2:

[0130] Table 14-1 Results of the lower limit of quantification of VB1, VB2, and VB3-NM

[0131]

[0132] Table 14-2 Results of the lower limit of quantification of VB5, VB6-PA and 5-MTHF

[0133]

[0134] 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 of the present invention can still ensure the reliability of the results when detecting low-concentration samples.

[0135] 5. Dilution Reliability

[0136] Human serum near the upper limit of the linear range (AMR) was collected and diluted 2, 4, and 8 times with ultrapure water, respectively. The dilution was considered to pass the validation if 80% ≤ the diluted test value / theoretical value ≤ 120%. The dilution reliability results for water-soluble vitamins are shown in Tables 15-1 and 15-2:

[0137] Table 15-1 VB1, VB2, VB3-NM dilution reliability results

[0138]

[0139] Table 15-2 Dilution reliability results of VB5, VB6-PA, and 5-MTHF

[0140]

[0141] It can be seen from Table 15-1 and Table 15-2 that the maximum dilution factor of each indicator of water-soluble vitamins is 8, indicating that the method of the present invention can still ensure the reliability of the results when diluting 8 times when detecting high-concentration samples.

[0142] 6. Linear Verification

[0143] Referencing the requirements of the "Guidelines for Linearity Evaluation of Clinical Chemistry Equipment" (WS / T 480-2012), the Health Industry Standard of the People's Republic of China, for linear range verification, samples at five concentration levels were selected for measurement, with each sample measured four times. All samples were pre-processed and quantified on the instrument as quickly as possible. The linearity verification results for water-soluble vitamins are shown in Table 16:

[0144] Table 16 Linearity verification results of water-soluble vitamins

[0145]

[0146] The obtained data were fitted with a regression equation and a scatter plot was drawn. The linear fitting regression equation and scatter plot were as follows: Figure 2 As shown, VB1 linear validation regression equation: y=1.0015x+0.0701, R 2 =0.9999; VB2 linear verification regression equation: y=1.017x-0.478, R 2 =0.9995; VB3-NM linear validation regression equation: y=0.9926x+0.0543, R 2 =0.9999; VB5 linear verification regression equation: y=0.9953x+0.2753, R 2 =0.9998; VB6-PA linear verification regression equation: y=0.998x-0.1515, R 2 =0.9999; 5-MTHF linear validation regression equation: y=1.0035x-0.4351, R 2 =0.9998.

[0147] When the correlation coefficient R 2 ≥0.95, and the regression coefficient b is in the range of 0.97 to 1.03, indicating that it is qualified within the linear range. Figure 2 It can be seen that the regression coefficient b and correlation coefficient R are obtained by performing regression statistical analysis on the expected values ​​and measured values ​​of various indicators of water-soluble vitamins. 2 All of them meet the requirements of the "Guidelines for Linearity Evaluation of Clinical Chemistry Equipment", indicating that the linearity of this method meets the validation requirements.

[0148] 7. Matrix Effects

[0149] The experiment selected pure solutions of the target analytes, biological matrix samples, and a 1:1 mixture of the two, each of which was processed and analyzed. If the response of the 1:1 mixture sample differed by less than a certain percentage (20%) from the mean response of the biological matrix sample and the pure solution sample, it confirmed that the matrix effect existed or did not affect the accurate quantification of the target analyte. The matrix effect results for different concentrations of water-soluble vitamins are shown in Table 17:

[0150] Table 17 Matrix effect results of different concentrations of water-soluble vitamins

[0151]

[0152] By investigating and analyzing the matrix effects of various indicators of water-soluble vitamins at different concentrations, the results showed that the deviations at 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 effectively solves the problem of high spike recovery and reduces interference during detection. The method of the present invention is less susceptible to interference and does not affect the quantification of the target compound.

[0153] 8. Extract stability test

[0154] Ten human serum samples were selected and treated with the sulfosalicylic acid-ammonium acetate system. After being placed at 2-8°C, the samples were tested at 0h, 24h, 48h, and 72h. The test results were recorded and analyzed. The extract stability results are shown in Tables 18-1, 18-2, and 18-3:

[0155] Table 18-1 Stability results of VB1 and VB2 extracts

[0156]

[0157] Table 18-2 Stability results of VB3-NM and VB5 extracts

[0158]

[0159] Table 18-3 Stability results of VB6-PA and 5-MTHF extracts

[0160]

[0161] It can be seen from Table 18-1, Table 18-2, and Table 18-3 that 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.

[0162] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples and is subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention are considered to be within the scope of protection of the present invention.

Claims

1. A kit for detecting water-soluble vitamins 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 a concentration of a water-soluble vitamin; The quality control product includes two or more concentrations of water-soluble vitamins; 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 ammonium acetate methanol aqueous solution; The water-soluble vitamins include vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6 and 5-methyltetrahydrofolate; The protein precipitant comprises 4% sulfosalicylic acid and 150 mM ammonium acetate.

2. The test kit for detecting water-soluble vitamins in serum according to claim 1, wherein: The concentration of the calibrator is 30-220 ng / mL; the concentration of the quality control is 10-220 ng / mL; the concentration of the isotope internal standard solution is 30-600 ng / mL; and the mobile phase concentrate consists of 2 M ammonium acetate and 50% methanol.

3. A method for preparing a test kit for detecting water-soluble vitamins in serum according to claim 1, characterized in that The steps include: (1) Prepare the calibrant diluent: adsorb bovine albumin using activated carbon; filter through a water filter membrane; dilute the bovine albumin using phosphate buffer, and then add ProClin 300 and ascorbic acid; (2) Prepare calibrators and quality control products: Prepare calibrators and quality control products using phosphate buffer containing bovine albumin; (3) Prepare isotope internal standard solution: add water-soluble vitamin isotope internal standard stock solution into phosphate buffer; (4) Prepare protein precipitant: add sulfosalicylic acid and ammonium acetate to ultrapure water, mix well, aliquot and store at 2-8°C; (5) Prepare mobile phase concentrate: dissolve ammonium acetate in 50% methanol, dilute to volume with 50% methanol after complete dissolution, mix well, aliquot and store at -20±5℃; (6) Freeze drying: freeze drying of calibrators, quality control products and isotope internal standards; (7) Place the calibrator, quality control, isotope internal standard solution, protein precipitant, calibrator diluent and mobile phase concentrate into the kit and store at -20±5℃.

4. The preparation method according to claim 3, wherein: In step (1), the bovine albumin is 5% bovine albumin, and the filtration process is sequentially filtered through 0.45 μm and 0.22 μm water filter membranes, 0.45 μm water membrane filtration once, and 0.22 μm water membrane filtration twice; the concentration of phosphate buffer is 0.1 M; the bovine albumin is diluted 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 further 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: The kit according to claim 1 comprises the following steps: (i) Reagent preparation; (ii) Pretreatment: Place the sample in a protein precipitation tube, add isotope internal standard solution, add protein precipitant, mix well, and use vacuum negative pressure to transfer the extract in the protein precipitation tube to the well plate, and then detect it on the mass spectrometer.

6. The detection method according to claim 5, characterized in that Step (i) specifically includes: (i-1) Prepare calibrators; (i-2) Prepare a standard curve; (i-3) Prepare isotope internal standard solution; (i-4) Prepare mobile phase: Dilute the mobile phase concentrate with ultrapure water or methanol to prepare mobile phase A or mobile phase B.

7. The detection method according to claim 6, characterized in that In step (ii), the chromatographic parameters are controlled as follows: Chromatographic column: Agilent ZORBAX Eclipse Plus-C18, size 4.6*50 mm, particle size 1.7 μm; precolumn: Agilent ZORBAX Eclipse Plus-C18, size 4.6*12.5 mm, particle size 1.7 μm; Flow rate: 0.5 mL / min; column temperature: 35°C; injection: 10 μL; LC 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, mobile phase A ratio is 60-95%, mobile phase B ratio is 5-40%; 1.5-2.5 min, mobile phase A ratio is 50-60%, mobile phase B ratio is 40-50%; 2.5-3.0 min, mobile phase A ratio is 5-50%, mobile phase B ratio is 50-95%; 3.0-3.8 min, mobile phase A ratio is 5%, mobile phase B ratio is 95%; 3.8-4.2 min, mobile phase A ratio is 5-95%, mobile phase B ratio 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, characterized in that 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 temperature: 500 °C; desolvation gas flow rate: 1000 L / Hr; cone gas flow rate: 20 L / Hr; Scan mode: multiple reaction monitoring; scan interval 15-200 ms, cone voltage 20-50 V, collision energy 20-30 eV.

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