A UPLC-MS analytical method for the simultaneous determination of 14 water-soluble vitamins and metabolites in human serum

Abstract

This invention belongs to the field of in vitro diagnostic medical testing technology, and discloses an analytical method for the simultaneous determination of 14 water-soluble vitamins and metabolites in human serum using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS). This method uses organic acid precipitation of proteins in human serum samples, eliminating the need for nitrogen blowing; matrix purification is achieved solely through mixing and centrifugation. Detection is then performed using UPLC-MS, with quantification using an internal standard method. Verification showed good linearity for the 14 water-soluble vitamins and metabolites within their respective concentration ranges, with correlation coefficients (R0 and R1). 2 The values ​​were all greater than 0.99. The method detection limit was 0.03 μg / L to 1.50 μg / L, and the quantitation limit was 0.10 μg / L to 5.0 μg / L. The recoveries of the three levels of spiking ranged from 88.21% to 110.63%, with RSD values ​​ranging from 1.72% to 10.98% (n=6). This method achieves a unified experimental protocol for the detection of multiple water-soluble vitamins, and features high sensitivity, good recovery, good stability, and simple and convenient operation. It is suitable for the simultaneous determination of multiple water-soluble vitamins and metabolites in real human serum.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic medical testing technology, specifically relating to an UPLC-MS analytical method for simultaneously determining 14 water-soluble vitamins and metabolites in human serum. Background Technology

[0002] There are many types of water-soluble vitamins and their metabolites, including numerous B vitamins alone, such as: vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin, niacinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxal, pyridoxic acid), vitamin B7 (biotin), vitamin B9 (folic acid), and vitamin B2. 12 (Cobalamin), etc., among which pyridoxal phosphate is the metabolically active form of vitamin B6, 5-methyltetrahydrofolate is the metabolically active form of vitamin B9, and methylmalonic acid is the metabolically active form of vitamin B1. 12 The lack of water-soluble vitamins and their metabolites can lead to metabolic disorders and various adverse clinical symptoms when there are abnormalities in the intake, transport, or biotransformation of water-soluble vitamins. Therefore, accurate analysis of the levels of water-soluble vitamins and their metabolites in the blood is of great significance for studying human physiological and pathological states.

[0003] Among existing detection technologies, commonly used methods for detecting water-soluble vitamins include microbiological methods, radioimmunoassay, protein binding assays, spectrophotometry, fluorescence assays, chemiluminescence assays, capillary electrophoresis, high-performance liquid chromatography (HPLC), and HPLC-tandem mass spectrometry (HPLC-MS). However, microbiological methods, spectrophotometry, chemiluminescence, capillary electrophoresis, and HPLC all have certain drawbacks, such as long detection times, low throughput, complex operation, large variability, and low sensitivity. HPLC-MS, on the other hand, offers high throughput, high sensitivity, and high selectivity, enabling the simultaneous analysis of multiple water-soluble vitamins at low detection levels.

[0004] Currently, there are many published methods for detecting water-soluble vitamins in blood samples using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). For example, Chinese patent application number CN202111628441.2, "A method for simultaneous detection of multiple water-soluble vitamins in serum based on liquid chromatography-tandem mass spectrometry," proposes to extract water-soluble vitamins by combining ultrafiltration tube treatment of blood samples. This method is costly, cumbersome, and time-consuming. Chinese patent application number CN202111534070.1, "A method for detecting multiple water-soluble vitamins," proposes to extract water-soluble vitamins by using an anion exchange solid-phase extraction column combined with nitrogen blowing. This pretreatment process requires the use of an anion exchange solid-phase extraction column, is costly, cumbersome, and time-consuming, and can only detect five water-soluble vitamins. Chinese patent application CN202211319154.8, entitled "Pretreatment Kit and Pretreatment and Detection Method for Water-Soluble Vitamins in Serum," proposes a method of extracting five water-soluble vitamins using protein precipitation combined with nitrogen blowing. However, this method detects only a limited number of target analytes and does not detect metabolically active substances that are difficult to detect (pyridoxal phosphate, methylmalonic acid, and 5-methyltetrahydrofolate). Furthermore, the pretreatment requires nitrogen drying, making the operation complex and increasing detection costs. It does not address multiple issues simultaneously and cannot achieve a comprehensive assessment of nutritional health. Therefore, it is necessary to provide a method for the simultaneous determination of multiple water-soluble vitamins and metabolites in human serum based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS). Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for simultaneously determining 14 water-soluble vitamins and metabolites in human serum using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS), the method comprising the following steps: Step S1: Add 20 μL of internal standard solution to 100 μL of serum sample containing 14 water-soluble vitamins and metabolites, then add 50 μL~150 μL of acid protein precipitant, vortex to mix, centrifuge, and take the supernatant as the test solution. Step S2: Load the test solution onto ultra-high performance liquid chromatography-tandem mass spectrometry for detection, and obtain the peak area values ​​of each water-soluble vitamin and metabolite and its internal standard; Step S3: Calculate the concentration of each water-soluble vitamin based on the standard curve of each water-soluble vitamin; The acid protein precipitant is one or more of the following: 2%~15% sulfosalicylic acid, 10%~30% citric acid, and 5%~20% perchloric acid by volume. The serum sample contained 14 water-soluble vitamins and metabolites, namely vitamin B1, vitamin B2, vitamin B3-niacin (niacin), vitamin B3-nicotinamide (nicotinamide), vitamin B5 (pantothenic acid), vitamin B6-pyridoxine (pyridoxine), vitamin B6-pyridoxal (pyridoxal), vitamin B6-pyridoxic acid (pyridoxic acid), pyridoxal phosphate, vitamin B7 (biotin), vitamin B9-folic acid (folic acid), 5-methyltetrahydrofolate, and vitamin B2. 12 (Cobalamin), methylmalonic acid; The standard curve is obtained by the following steps: Step a1: Prepare 14 intermediate standard solutions of 10 μg / mL water-soluble vitamins and metabolites from stock solutions of 14 water-soluble vitamins and metabolites with a concentration of 1 mg / mL; Step a2: Take the 14 intermediate standard solutions from step a1, mix them, and dilute them with human serum to different mass concentrations to obtain mixed standard working solutions; prepare a total of 9 mixed standard working solutions, with each water-soluble vitamin and metabolite having a different concentration in the different mixed standard working solutions; Step a3: Perform the same treatment as step S1 on the mixed standard working solution prepared in step a2 to obtain the standard test solution; The standard test solution was loaded onto ultra-high performance liquid chromatography-tandem mass spectrometry for detection under the same detection conditions as in step S2. Step a4: For each water-soluble vitamin and metabolite, plot the standard curve with the ratio of the peak area of ​​the nine target substances to the peak area of ​​the internal standard as the ordinate and the concentration of each target substance as the abscissa.

[0006] The internal standards for the 14 water-soluble vitamins and their metabolites mentioned above are, in order: Vitamin B1- 13 C4; Vitamin B2- 13 C4, 15 N2; Vitamin B3-Niacin-D4; Vitamin B3-Niacinamide-D4; Vitamin B5- 13 C3, 15 N; Vitamin B6-pyridoxine- 13 C4; Vitamin B6-pyridoxal-D3; Vitamin B6-pyridoxic acid-D3; Pyridoxal phosphate-D3; Vitamin B7-D4; Vitamin B9-folic acid-D4; 5-methyltetrahydrofolate- 13 C, D3; Vitamin B 12 - 13 C7; Methylmalonic acid-D3.

[0007] Preferably, the acid protein precipitant is sulfosalicylic acid with a volume percentage concentration of 2% to 15%. More preferably, the acid protein precipitant is sulfosalicylic acid with a volume percentage concentration of 3% and an addition amount of 100 μL.

[0008] Preferably, in step S2, the chromatographic column used is an E-Gen water-soluble vitamin-specific chromatographic column, the inner diameter of which is 2.1 mm, the length is 100 mm, and the particle size is 3 μm.

[0009] Preferably, in step S2, the mobile phase A is an ammonium formate-formic acid aqueous solution, and the mobile phase B is an ammonium formate-formic acid methanol solution; the molar concentration of ammonium formate in the mobile phase A is 1 mM~10 mM, and the volume percentage concentration of formic acid is 0.01%~2%; the molar concentration of ammonium formate in the mobile phase B is 1 mM~10 mM, and the volume percentage concentration of formic acid is 0.01%~2%.

[0010] Preferably, in step S2, the flow rate of the mobile phase is 0.4 mL / min, and the elution program of mobile phase A is: 0 min, 100%; 1.3 min, 100%; 3.0 min, 5%; 4.5 min, 5%; 4.51 min, 100%; 6.0 min, 100%; and the elution program of mobile phase B is: 0 min, 0; 1.3 min, 0; 3.0 min, 95%; 4.5 min, 95%; 4.51 min, 0; 6.0 min, 0.

[0011] Preferably, in step S2, the mass spectrometry conditions are: electrospray ionization source, positive and negative ion multiple reaction monitoring mode scanning analysis; ion source parameters: positive mode voltage 3000V, negative mode voltage 3000V, sheath gas: 40Arb, auxiliary gas: 10Arb, purge gas: 0Arb, ion transmission tube temperature: 350℃, ion source temperature: 350℃.

[0012] Preferably, in step S1, 20 μL of internal standard solution is added to 100 μL of serum sample, followed by 50 μL to 150 μL of acid protein precipitant. After vortexing for 1 min, the mixture is centrifuged at 11000 r / min for 15 min, and the supernatant is taken as the test solution.

[0013] Preferably, in step S1, the serum sample is one or more of human serum and / or animal serum. More preferably, the serum is human serum.

[0014] Preferably, this method can simultaneously detect 14 water-soluble vitamins and metabolites, with a detection limit of 0.03~1.50 μg / L and a quantitation limit of 0.10~5.0 μg / L.

[0015] This invention discloses a method for the simultaneous determination of 14 water-soluble vitamins and metabolites in human serum using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The method involves acid precipitation of proteins in the serum sample, followed by purification of the sample matrix through centrifugation. Detection and analysis are performed using UPLC-MS / MS with internal standard quantification, enabling simultaneous quantitative analysis of 14 water-soluble vitamins and metabolites. This invention features a simple sample processing method, short processing time, and low serum sample consumption. The detection method can simultaneously quantify 14 water-soluble vitamins and metabolites within 6 minutes, exhibiting high specificity, high throughput, high sensitivity, low cost, and reliable results. It effectively monitors the levels of multiple water-soluble vitamins and metabolites in the human body, making it particularly suitable for widespread clinical application.

[0016] Compared with the prior art, the present invention has at least the following advantages: (1) The present invention optimizes the sample pretreatment method and ultra-high performance liquid chromatography-mass spectrometry conditions, and establishes an analytical method for simultaneously determining 14 water-soluble vitamins and metabolites in human serum by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS), and performs accurate qualitative and quantitative analysis. It can save sample processing and detection time, and reduce the detection cost of multiple vitamins. Overall, it is a detection method with simple sample processing, high throughput, strong specificity, high sensitivity and reliable results.

[0017] (2) This invention is the first to realize the simultaneous detection of 14 water-soluble vitamins and metabolites in a serum sample using UPLC-MS technology. The use of two pairs of ions for quantitative and qualitative analysis ensures the specificity of the analytes and reduces the influence of interfering substances. This method is simple and fast to operate, with an analysis time of only 6 minutes. It has high throughput and low cost, effectively monitors the levels of water-soluble vitamins and metabolites in the human body, and is easy to promote and popularize in clinical practice. Attached Figure Description

[0018] Figure 1a This is a chromatogram of 14 water-soluble vitamins and metabolites in a human serum sample from Embodiment 2 of the present invention; Figure 1b This is a chromatogram of another portion of 14 water-soluble vitamins and metabolites in a human serum sample from Embodiment 2 of the present invention; Figure 2 This is a standard curve diagram of VB1 in Embodiment 3 of the present invention; Figure 3 This is the standard curve diagram of VB2 in Embodiment 3 of the present invention.

[0019] Figure 4 This is the standard curve diagram of VB3 in Embodiment 3 of the present invention; Figure 5 This is a standard curve diagram of VB3-NM in Embodiment 3 of the present invention; Figure 6 This is a standard curve diagram of VB5 in Embodiment 3 of the present invention; Figure 7 This is a standard curve diagram of VB6-PA in Embodiment 3 of the present invention; Figure 8 This is a standard curve diagram of VB6-PL in Embodiment 3 of the present invention; Figure 9 This is a standard curve diagram of VB6-PLP in Embodiment 3 of the present invention; Figure 10 This is the standard curve diagram of VB6-PN in Embodiment 3 of the present invention; Figure 11 This is the standard curve diagram of VB7 in Embodiment 3 of the present invention; Figure 12 This is the standard curve diagram of VB9-FA in Embodiment 3 of the present invention; Figure 13 This is a standard curve diagram of 5-MTHF in Embodiment 3 of the present invention; Figure 14 This is a standard curve diagram of MMA in Embodiment 3 of the present invention; Figure 15 This is a standard curve diagram of VB12 in Embodiment 3 of the present invention. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0021] Unless otherwise specified, the raw materials and ingredients used in the preparation examples and embodiments of the present invention were purchased through general commercial channels.

[0022] Vitamin B1 (CAS No.: 59-43-8), Vitamin B2 (CAS No.: 83-88-5), Vitamin B3-Niacin (CAS No.: 59-67-6), Vitamin B3-Niacinamide (CAS No.: 98-92-0), Vitamin B5 (CAS No.: 79-83-4), Vitamin B6-Pyridoxine (CAS No.: 58-56-0), Vitamin B 6- Pyridoxal (CAS No.: 65-22-5), Vitamin B6-pyridoxic acid (CAS No.: 82-82-6), Pyridoxal phosphate (CAS No.: 54-47-7), Vitamin B7 (CAS No.: 58-85-5), Vitamin B9-folic acid (CAS No.: 59-30-3), 5-methyltetrahydrofolate (CAS No.: 31690-09-2), Vitamin B 12(CAS No.: 68-19-9), Methylmalonic acid (CAS No.: 516-05-2), the above vitamin standards are all from Shanghai Anpu Experimental Technology Co., Ltd.; Vitamin B1- 13 C4 (CAS No.: 1257525-77-1), Vitamin B2- 13 C4, 15 N2 (CAS No.: 1217461-14-7), Vitamin B3-Niacin-D4 (CAS No.: 66148-15-0), Vitamin B3-Niacinamide-D4 (CAS No.: 347841-88-7), Vitamin B5- 13 C3, 15 N (CAS No.: 356786-94-2), Vitamin B6-pyridoxine- 13 C4 (CAS No.: [58-56-0]_13C4), Vitamin B6-pyridoxal-D3 (CAS No.: 1354560-58-9), Vitamin B6-pyridoxic acid-D3 (CAS No.: 1435934-03-4), Pyridoxal phosphate-D3 (1354560-58-9), Vitamin B7-D4 (CAS No.: 1217850-77-5), Vitamin B9-folic acid-D4 (CAS No.: 171777-72-3), 5-methyltetrahydrofolate- 13 C, D3 (CAS No.: 1356019-94-7), Vitamin B 12 - 13 C7 (CAS No.: 1978303-01-3), Methylmalonic acid-D3 (CAS No.: 42522-59-8), the above internal standards are all from Shanghai Anpu Laboratory Technology Co., Ltd.; Human serum, sourced from Shanghai Anpu Laboratory Technology Co., Ltd.; Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS), model RZ-500, Shanghai Ruikang Biotechnology Co., Ltd.

[0023] <Preparation Example 1> Preparation of standard stock solution (1 mg / mL): Accurately weigh 10.00 mg of each of the following standards: vitamin B2, pyridoxine, biotin, and 5-methyltetrahydrofolate. Dissolve each standard in 2 mL of DMSO, then dilute with 70% methanol aqueous solution and bring the volume to 10 mL in a brown volumetric flask. After shaking well, transfer the solutions to brown sample bottles and store them at -20°C in the dark to obtain standard stock solutions with a concentration of 1 mg / mL for each of the following standards: vitamin B2, pyridoxine, biotin, and 5-methyltetrahydrofolate.

[0024] Accurately weigh 10.00 mg of each of the following standards: vitamin B1, nicotinic acid, nicotinamide, pantothenic acid, pyridoxine, pyridoxal, pyridoxal phosphate, folic acid, cobalamin, and methylmalonic acid. Dilute each standard with 70% (v / v) methanol aqueous solution and bring the volume to 10 mL in a brown volumetric flask. After shaking well, transfer the solutions to brown sample bottles and store them at -20°C in the dark to obtain standard stock solutions with a concentration of 1 mg / mL for the aforementioned vitamin B1, nicotinic acid, nicotinamide, pantothenic acid, pyridoxine, pyridoxal, pyridoxal phosphate, folic acid, cobalamin, and methylmalonic acid.

[0025] Preparation of standard intermediate solution (10 μg / mL): Accurately pipette 0.1 mL of the above standard stock solution, dilute with 70% methanol aqueous solution (v / v) and bring to a final volume of 10 mL in a brown volumetric flask. Shake well and transfer to a brown sample bottle. Store at -20°C protected from light.

[0026] Preparation of mixed standard working solution (1 μg / mL): Accurately pipette 1 mL of the above standard intermediate solution, dilute with 70% methanol aqueous solution (v / v) and bring to a final volume of 10 mL in a brown volumetric flask. Shake well and transfer to a brown sample bottle. Prepare fresh for each use.

[0027] Preparation of mixed standard working solution: According to experimental requirements, accurately pipette the above-mentioned intermediate standard solutions and dilute them with human serum to the appropriate mass concentration to prepare mixed standard working solutions. Prepare solutions immediately before use. Specific mass concentrations are shown in Table 1. In VB3-NM, the suffix NM indicates that VB3 exists in the form of nicotinamide; in VB6-PA, the suffix PA indicates that VB6 exists in the form of pyridoxine; in VB6-PL, the suffix PL indicates that VB6 exists in the form of pyridoxal; in VB6-PLP, the suffix PLP indicates that VB6 exists in the form of pyridoxal phosphate; in VB6-PN, the suffix PN indicates that VB6 exists in the form of pyridoxine; and in VB9-FA, the suffix FA indicates that VB9 exists in the form of folic acid. In Table 1, C1 to C9 represent nine standards used for standard working curves, with concentrations ranging from low to high.

[0028] Table 1. Concentration points of standard working curve

[0029] Preparation of quality control samples: Accurately pipette the above-mentioned mixed standard working solution and dilute it with human serum to three different concentration levels of quality control samples. Prepare the samples fresh for each use. The specific mass concentrations are shown in Table 2. In VB3-NM, the suffix NM indicates that VB3 is present in the form of nicotinamide; in VB6-PA, the suffix PA indicates that VB6 is present in the form of pyridoxine; in VB6-PL, the suffix PL indicates that VB6 is present in the form of pyridoxal; in VB6-PLP, the suffix PLP indicates that VB6 is present in the form of pyridoxal phosphate; in VB6-PN, the suffix PN indicates that VB6 is present in the form of pyridoxine; and in VB9-FA, the suffix FA indicates that VB9 is present in the form of folic acid. In Table 2, LQC to HQC represent the three quality control samples with concentrations from low to high, and the concentrations of the 14 water-soluble vitamins and metabolites are all ng / mL.

[0030] Table 2. Concentration points of quality control samples

[0031] Preparation of internal standard stock solution (200 μg / mL): Accurately weigh out vitamin B1- 13 C4, Vitamin B2 13 C4, 15 N2, Vitamin B3-Niacin-D4, Vitamin B3-Niacinamide-D4, Vitamin B5- 13 C3, 15 N, Vitamin B6-pyridoxine- 13 C4, Vitamin B6-pyridoxal-D3, Vitamin B6-pyridoxine-D3, Vitamin B7-D4, 5-methyltetrahydrofolate- 13 C, D3, Vitamin B9-Folic Acid-D4, Vitamin B 12 - 13 C7 and methylmalonic acid-D3 standards, 2.00 mg each, were first dissolved in 5 mL DMSO, then diluted with pure methanol and brought to a final volume of 10 mL in a brown volumetric flask. After shaking well, the solutions were transferred to brown sample vials to obtain internal standard stock solutions with a concentration of 200 μg / mL for each of the aforementioned internal standards. The solutions were then aliquoted and stored at -20℃ protected from light.

[0032] Preparation of mixed internal standard intermediate solution (10 μg / mL): Accurately pipette 0.5 mL of the above internal standard stock solution, dilute with 70% methanol aqueous solution (v / v) and bring to a final volume of 10 mL in a brown volumetric flask. Shake well and transfer to a brown sample bottle. Store at -20°C in the dark.

[0033] Preparation of mixed internal standard working solution (0.1 μg / mL): Accurately pipette 0.1 mL of the above mixed internal standard intermediate solution, dilute with 70% methanol aqueous solution (v / v) and bring to a final volume in a 10 mL brown volumetric flask, shake well and transfer to a brown sample bottle for use immediately.

[0034] <Example 1> In this embodiment, sample processing is performed to obtain a test solution. Three parallel tests are conducted for each sample.

[0035] Pipette 100 μL of human serum into a 1.5 mL centrifuge tube, add 20 μL of the mixed internal standard working solution from Preparation Example 1, then add 100 μL of 3% sulfosalicylic acid solution, mix well for 1 min, centrifuge at 11000 r / min for 15 min, and take the supernatant as the test solution for UPLC-MS determination.

[0036] <Example 2> In this embodiment, the supernatant obtained in Example 1 is used as the test solution for detection.

[0037] 1. Chromatographic conditions a) Chromatographic column: E-Gen water-soluble vitamin column, 2.1×100 mm, 3μm; b) Mobile phase: A is a 0.2% (v / v) aqueous solution of formic acid (containing 2 mM ammonium formate), and B is a 0.2% (v / v) formic acid-methanol solution (containing 2 mM ammonium formate). The volume ratio of formic acid to methanol is 1:499. Gradient elution is used, and the elution program is shown in Table 3.

[0038] Table 3. Gradient elution procedure

[0039] c) Flow rate: 0.4 mL / min; d) Column temperature: 45 ℃; e) Injection volume: 5 µL; 2. Mass spectrometry conditions Electrospray ionization (ESI) source, scanning analysis in positive and negative ion multiple reaction monitoring (MRM) modes; ion source parameters: positive mode voltage 3000V, negative mode voltage 3000V, sheath gas: 40Arb, auxiliary gas: 10Arb, purge gas: 0Arb, ion transfer tube temperature: 350℃, ion source temperature: 350℃. Ion pair information is shown in Table 4, where * indicates quantitative ions.

[0040] Table 4. Monitoring ion pairs of 14 water-soluble vitamins and metabolites with internal standards

[0041]

[0042] The water-soluble vitamins and metabolites in the serum sample to be tested (the supernatant obtained in Example 1) were determined, and the results are as follows: Figures 1a-1b The measurement time shown is 6 minutes. Figures 1a-1b The A-N regions correspond to methylmalonic acid, nicotinamide, nicotinic acid, pyridoxal, pyridoxine, pyridoxine, vitamin B5, vitamin B7, pyridoxal phosphate, vitamin B1, vitamin B2, folic acid, 5-methyltetrahydrofolate, and vitamin B1, respectively. 12 The figure shows the retention time (RT) of each analyte.

[0043] from Figures 1a-1b The results show that the detection baselines for various vitamins and metabolites are stable and free from matrix interference.

[0044] <Example 3> This embodiment uses a mixed standard working solution to plot a standard curve.

[0045] 100 μL of each of the nine concentrations of the mixed standard working solution from the preparation example was precisely measured as a sample (target analyte), and processed according to the method in Example 1. The resulting supernatant was used as a standard test solution for UPLC-MS analysis.

[0046] Quantitative detection was performed according to the method in Example 2. A standard curve was plotted with the ratio of the peak area of ​​the target analyte at each concentration to the peak area of ​​the internal standard on the ordinate and each concentration (theoretical concentration) on the abscissa. Measurements were performed within the same analytical batch, and three batches were repeated. Results are shown below. Figures 2-15 And Table 5.

[0047] like Figure 2-15 As shown, in Figure 2-15 In the diagram, a~h correspond to vitamin B1, vitamin B2, niacin, nicotinamide, vitamin B5, pyridoxine, pyridoxal, and pyridoxal phosphate, respectively; i~n correspond to pyridoxine, vitamin B7, folic acid, 5-methyltetrahydrofolate, and vitamin B1, respectively. 12 Methylmalonic acid.

[0048] As shown in Table 5, this method exhibits good linearity for 14 water-soluble vitamins and metabolites within their respective concentration ranges, with correlation coefficients (R0 and R0). 2 The values ​​were all greater than 0.99, the method detection limit was 0.03 μg / L to 1.50 μg / L, and the quantitation limit was 0.10 μg / L to 5.0 μg / L.

[0049] Table 5. Linear equations and correlation coefficients for 14 water-soluble vitamins and their metabolites

[0050] <Example 4> Method accuracy and precision testing 100 μL of each of the low-concentration (LQC), medium-concentration (MQC), and high-concentration (HQC) quality control samples from Table 2 of the preparation examples were precisely measured. The quality control samples were processed according to the method in Example 1 and quantitatively detected according to the method in Example 2. Three batches of quality control samples at three different concentration levels were tested at different times. The ratio of the corresponding vitamin peak area to the internal standard was substituted into the standard curve equation to obtain the detection concentrations of 14 water-soluble vitamins and metabolites in the serum samples. The recovery rates of the 14 water-soluble vitamins and metabolites in the serum samples were then calculated using the following formula: X = C / A * 100%. Where X is the recovery rate, C is the detection concentration obtained from the standard curve by UPLC-MS testing, and A is the theoretical concentration to be added. The precision and accuracy within batches (6 parallel samples) and between batches (3 batches, 18 parallel samples) were obtained through the above calculations, and the results are shown in Table 6. In Table 6, the concentrations of the quality control samples are shown in Table 2 of the preparation examples.

[0051] Table 6. Intra-batch and inter-batch accuracy and precision of 14 water-soluble vitamins and metabolites

[0052]

[0053] As shown in Table 6, the recoveries of the three concentration levels of this test method ranged from 88.21% to 110.63%, with RSD values ​​ranging from 1.72% to 10.98% (n=6). This indicates that the detection method has high accuracy and good precision.

[0054] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A UPLC-MS analytical method for simultaneously determining 14 water-soluble vitamins and metabolites in human serum, characterized in that, Includes the following steps: Step S1: Add 20 μL of internal standard solution to 100 μL of serum sample containing 14 water-soluble vitamins and metabolites, then add 50 μL~150 μL of acid protein precipitant, vortex to mix, centrifuge, and take the supernatant as the test solution. Step S2: The test solution is loaded onto an ultra-high performance liquid chromatography-tandem mass spectrometry instrument for detection to obtain the peak area values ​​of each water-soluble vitamin, metabolite and internal standard; Step S3: Calculate the concentration of each water-soluble vitamin and metabolite based on the standard curves of each water-soluble vitamin and metabolite; The acid protein precipitant is one or more of the following: sulfosalicylic acid (volume percentage concentration of 2%~15%), citric acid (volume percentage concentration of 10%~30%), and perchloric acid (volume percentage concentration of 5%~20%). The serum sample contained 14 water-soluble vitamins and metabolites, including vitamin B1, vitamin B2, vitamin B3-niacin, vitamin B3-nicotinamide, vitamin B5, vitamin B6-pyridoxine, vitamin B6-pyridoxal, vitamin B6-pyridoxic acid, pyridoxal phosphate, vitamin B7, vitamin B9-folic acid, 5-methyltetrahydrofolate, and vitamin B1. 12 Methylmalonic acid; The standard curve is obtained by the following steps: Step a1: Prepare 14 intermediate standard solutions of 10 μg / mL water-soluble vitamins and metabolites from stock solutions of 14 water-soluble vitamins and metabolites with a concentration of 1 mg / mL; Step a2: Take the 14 intermediate standard solutions from step a1, mix them, and dilute them with human serum to different mass concentrations to obtain mixed standard working solutions; prepare a total of 9 mixed standard working solutions, with each water-soluble vitamin and metabolite having a different concentration in the different mixed standard working solutions; Step a3: Perform the same treatment as step S1 on the mixed standard working solution prepared in step a2 to obtain the standard test solution; The standard test solution was loaded onto ultra-high performance liquid chromatography-tandem mass spectrometry for detection under the same detection conditions as in step S2. Step a4: For each water-soluble vitamin and metabolite, the standard curve is plotted with the ratio of the peak area of ​​the nine target substances to the peak area of ​​the internal standard as the ordinate and the concentration of each target substance as the abscissa. In step S2, the chromatographic column used is an E-Gen water-soluble vitamin-specific chromatographic column with an inner diameter of 2.1 mm, a length of 100 mm, and a particle size of 3 μm. In step S2, mobile phase A is an ammonium formate-formic acid aqueous solution, and mobile phase B is an ammonium formate-formic acid-methanol solution; the molar concentration of ammonium formate in mobile phase A is 1 mM~10 mM, and the volume percentage concentration of formic acid is 0.01%~2%; the molar concentration of ammonium formate in mobile phase B is 1 mM~10 mM, and the volume percentage concentration of formic acid is 0.01%~2%, and the volume ratio of formic acid to methanol is 1:499; In step S2, the flow rate of the mobile phase is 0.4 mL / min. The elution program for mobile phase A is: 0 min, 100%; 1.3 min, 100%; 3.0 min, 5%; 4.5 min, 5%; 4.51 min, 100%; 6.0 min, 100%. The elution program for mobile phase B is: 0 min, 0%; 1.3 min, 0%; 3.0 min, 95%; 4.5 min, 95%; 4.51 min, 0%; 6.0 min, 0%. In step S2, the mass spectrometry conditions are as follows: electrospray ionization source, positive and negative ion multiple reaction monitoring mode scanning analysis; ion source parameters: positive mode voltage 3000V, negative mode voltage 3000V, sheath gas: 40Arb, auxiliary gas: 10Arb, purge gas: 0Arb, ion transmission tube temperature: 350℃, ion source temperature: 350℃.

2. The UPLC-MS analytical method for simultaneously determining 14 water-soluble vitamins and metabolites in human serum as described in claim 1, characterized in that, In step S1, the acid protein precipitant is sulfosalicylic acid with a volume percentage concentration of 3% and an addition amount of 100 μL.

3. The UPLC-MS analytical method for simultaneously determining 14 water-soluble vitamins and metabolites in human serum as described in claim 1, characterized in that, In step S1, the internal standard is vitamin B1- 13 C4, Vitamin B2 13 C4, 15 N2, Vitamin B3-Niacin-D4, Vitamin B3-Niacinamide-D4, Vitamin B5- 13 C3, 15 N, Vitamin B6-pyridoxine- 13 C4, Vitamin B6-pyridoxal-D3, Vitamin B6-pyridoxic acid-D3, Pyridoxal phosphate-D3, Vitamin B7-D4, Vitamin B9-folic acid-D4, 5-methyltetrahydrofolate- 13 C, D3, Vitamin B 12 - 13 C7, Methylmalonic acid-D3.

4. The UPLC-MS analytical method for simultaneously determining 14 water-soluble vitamins and metabolites in human serum as described in claim 1, characterized in that, The method can simultaneously detect 14 water-soluble vitamins and metabolites, with a detection limit of 0.03–1.50 μg / L and a quantitation limit of 0.10–5.0 μg / L.

5. The UPLC-MS analytical method for simultaneously determining 14 water-soluble vitamins and metabolites in human serum as described in claim 1, characterized in that, In step S1, 20 μL of internal standard solution is added to 100 μL of serum sample, followed by 50 μL to 150 μL of acid protein precipitant. After vortexing for 1 min, the mixture is centrifuged at 11000 r / min for 15 min, and the supernatant is taken as the test solution.

6. The UPLC-MS analytical method for simultaneously determining 14 water-soluble vitamins and metabolites in human serum as described in claim 1, characterized in that, In step S1, the serum sample is one or more of human serum and / or animal serum.

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