Method and device for determining blood folic acid deficiency boundary value by liquid chromatography-mass spectrometry

Abstract

The invention discloses a method and a device for judging a blood folic acid deficiency boundary value by liquid chromatography-mass spectrometry in the technical field of medical detection. The method comprises the following steps: step 1, pre-treating a sample; step 2, UPLC-MS / MS detection is carried out; step 3, data calculation and verification; and 4, performing boundary value conversion and judgment. According to the method, the regression model is constructed by fitting experimental data, the boundary value of folic acid deficiency and edge deficiency detected by the liquid chromatography-mass spectrometry is defined, serum pretreatment and liquid chromatography are integrated through the developed micro-fluidic chip, the speed and accuracy of serum folic acid detection by the liquid chromatography-mass spectrometry are further improved, and the method is suitable for large-scale popularization and application. The clinical detection requirements are met, and the efficiency and accuracy of judging the folic acid nutritional status are improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical detection, and specifically relates to a blood folate deficiency cutoff determination method and device of liquid chromatography-mass spectrometry. BACKGROUND

[0002] Serum folate detection began in the 1950s, and the initial microbial method (Lactobacillus casei) was the "gold standard", which was then replaced by radioimmunoassay (RIA). However, RIA was gradually eliminated due to radioactive contamination and complicated procedures. Subsequently, the protein binding method was developed, which is fast and easy to automate, and has become a routine clinical method, but it can only report total folate and the difference between kits is large. In the past decade, liquid chromatography-tandem mass spectrometry (LC-MS / MS) has emerged, which can simultaneously quantify multiple folate monomers, is sensitive and specific, and is considered a new reference method that can be standardized, but it has not yet been popularized.

[0003] The World Health Organization has established the microbial improvement method as the gold standard for evaluating the folate nutritional status of the population, and has developed folate nutritional status cutoffs based on the microbial improvement method. Folate deficiency is defined as folate concentration < 6.8 nmol / L, and marginal deficiency is defined as 6.8 ≤ folate concentration < 13.5 nmol / L. Although the microbial improvement method is the gold standard for folate detection, it has problems such as complex operation and long time-consuming. The microbial improvement method first requires dilution of serum, and then adds the diluted serum, folate culture medium and strain into a 96-well enzyme plate, seals it and incubates it at 37°C in the dark for 42 hours. From sample processing to result reading, it takes more than 42 hours. Therefore, it is difficult for clinics to use the microbial improvement method to detect serum folate, and mass spectrometry is often used instead. However, mass spectrometry for folate nutritional status detection still lacks clear cutoffs, and it is difficult to convert the results with the microbial method.

[0004] Liquid chromatography-tandem mass spectrometry can output different folate subtype detection levels with high throughput, mainly 5-methyltetrahydrofolate, which accounts for more than 90% of total circulating folate. Therefore, the results of liquid chromatography-tandem mass spectrometry and the microbial improvement method cannot be directly corresponded, and need to be converted. Currently, there is no cutoff determination standard for the results of liquid chromatography-tandem mass spectrometry.

[0005] Therefore, it is necessary to propose a liquid chromatography-mass spectrometry blood folate deficiency cutoff determination method and device that can convert the results of liquid chromatography-tandem mass spectrometry and set the 5-methyltetrahydrofolate deficiency and marginal deficiency cutoffs of liquid chromatography-tandem mass spectrometry. SUMMARY

[0006] In order to solve the above problems, the purpose of the present application is to provide a blood folate deficiency threshold determination method and device for liquid chromatography tandem mass spectrometry, a regression model is constructed by fitting experimental data, the deficiency and marginal deficiency threshold of liquid chromatography tandem mass spectrometry for detecting folate are defined, the serum pretreatment and liquid chromatography are integrated through the developed microfluidic chip, the speed and accuracy of liquid chromatography tandem mass spectrometry for detecting serum folate are further improved, the clinical detection demand is adapted, and the efficiency and accuracy of folate nutritional status determination are improved.

[0007] In order to achieve the above purpose, the technical scheme of the present application is as follows: a blood folate deficiency threshold determination method and device for liquid chromatography tandem mass spectrometry, comprising the following steps:

[0008] Step one, sample pretreatment, collecting blood samples and separating serum, taking quantitative serum samples for dilution, then purifying through a micro-extraction column to remove matrix interference, and then mixing with a mixed internal standard solution containing 5-methyltetrahydrofolate isotope internal standard and pteroyl glutamic acid isotope internal standard;

[0009] Step two, UPLC-MS / MS detection, chromatographically separating the pretreated sample, and synchronously detecting the peak areas of 5-methyltetrahydrofolate and free pteroyl glutamic acid in a multiple reaction monitoring mode by mass spectrometry;

[0010] Step three, data calculation and verification, according to the detected peak areas, the internal standard method is used to calculate the concentration C1 of 5-methyltetrahydrofolate and the concentration C2 of free pteroyl glutamic acid in serum respectively, and the ratio R=C1 / C2 is calculated; when the ratio R is within the preset threshold range, the detection result is determined to be valid;

[0011] Step four, threshold conversion and determination, the valid 5-methyltetrahydrofolate concentration C1 is substituted into the preset regression equation to convert into the equivalent microbial method total folate concentration X, and then the folate nutritional status of the individual is determined according to the preset threshold standard.

[0012] Further, the preset threshold range in the data calculation and verification step is R∈[8.0, 12.0]; if the R value exceeds the range, a re-detection process is automatically triggered and reported.

[0013] Further, the preset regression equation in the threshold conversion and determination step is X= (C1-b) / k, wherein the initial coefficient k=0.656, b=2.309; the preset threshold standard is: C1<6.77 nmol / L is determined as deficiency, 6.77≤C1<11.17 nmol / L is determined as marginal deficiency, and C1≥11.17 nmol / L is determined as sufficiency.

[0014] The principle of the basic scheme is: the scheme takes high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) technology as the core, combines internal standard method quantification and data verification logic, constructs the correlation between mass spectrometry detection results and microbiological method, and realizes the accurate determination of blood folate deficiency cutoff. The core principle includes three aspects: first, through sample pretreatment, remove serum matrix interference and offset the loss of components in the sample processing process, provide pure and stable sample basis for subsequent detection; second, use the high separation and high specificity of UPLC-MS / MS to capture the peak area of 5-methyltetrahydrofolate and free pteroyl glutamic acid in the multiple reaction monitoring mode, ensure the accuracy of key component quantification; third, exclude errors through data verification, and then convert the 5-methyltetrahydrofolate concentration detected by mass spectrometry into the equivalent total folate concentration of microbiological method based on the preset regression equation (C1=0.656X+2.309), and finally complete the nutritional status determination according to the C1 classification cutoff (<6.77nmol / L for deficiency, 6.77≤C1<11.17nmol / L for marginal deficiency, and ≥11.17nmol / L for sufficiency), solving the core problem of difficult conversion of mass spectrometry and gold standard results and no clear cutoff.

[0015] The beneficial effects of the basic scheme are: 1. The sample pretreatment link removes serum matrix interference through microextraction column purification, avoiding the influence of impurities on the detection results. At the same time, double isotope internal standard is introduced to effectively offset the loss of target components during sample processing, reducing quantitative errors. In the data calculation and verification link, valid detection data is selected through the ratio R∈[8.0, 12.0], if the R value exceeds this range, a re-detection process is automatically triggered and reported, further excluding invalid data caused by operation or instrument failure, ensuring the accuracy of the detection results from multiple links and reducing the risk of misjudgment.

[0016] 2. Through the preset regression equation C1=0.656X+2.309, the 5-methyltetrahydrofolate concentration detected by mass spectrometry is converted into the equivalent total folate concentration of microbiological method, establishing the correlation between the results of the two detection methods. At the same time, the folate nutritional status classification cutoff based on C1 is clear, solving the problem of no clear cutoff for blood folate deficiency detected by liquid chromatography tandem mass spectrometry and the difficulty in converting the detection results to microbiological method.

[0017] 3. Compared with the microbiological improvement method which takes more than 42 hours from sample processing to reading results, the scheme uses UPLC-MS / MS detection combined with standardized sample pretreatment process, greatly shortening the detection period and better meeting the rapid demand of blood folate detection in clinical practice. At the same time, the whole detection process is clear and easy to operate without complex manual intervention, which is convenient for clinical routine and suitable for large-scale population folate nutritional status screening and evaluation.

[0018] A blood folate deficiency threshold determination device of liquid chromatography-mass spectrometry, comprising a microfluidic chip, the microfluidic chip is internally provided with a sample pretreatment assembly for realizing sample dilution, micro-extraction purification and mixing with double internal standards, and a detection adapter assembly for connecting an external chromatography mass spectrometer;

[0019] A control and data processing system for controlling the operation of the microfluidic chip and performing data calculation, verification, threshold conversion and determination logic.

[0020] Further, the sample pretreatment assembly comprises a sample addition hole opened in the top wall of the microfluidic chip, the sample addition hole is provided with a sealing cover, the sample addition hole is communicated with a conical buffer cavity, the top wall of the microfluidic chip is further provided with a liquid injection hole, the liquid injection hole is provided with a first micro-flow pump, the liquid injection hole is communicated with a liquid injection pipe, the liquid injection pipe is communicated with the buffer cavity, the side of the buffer cavity opposite to the liquid injection pipe is communicated with a dilution pipe, the dilution pipe is communicated with a dilution cavity, a plurality of mixing columns are fixedly connected to the inner bottom wall of the dilution cavity, the side of the dilution cavity opposite to the dilution pipe is communicated with a mixing pipe, a plurality of mixing islands are fixedly connected in the mixing pipe, a control valve is arranged at the communication position between the dilution cavity and the mixing pipe, the mixing pipe is communicated with an extraction column, and the first micro-flow pump and the control valve are signal-connected with the control and data processing system.

[0021] Further, the extraction column is arranged in an inclined manner, filter membranes are arranged at both ends of the extraction column, an adsorbent is filled in the extraction column, the bottom end of the extraction column is communicated with a reversing valve, the output end of the reversing valve is communicated with a liquid discharge hole opened in the bottom wall of the microfluidic chip, the other output end of the reversing valve is communicated with a sample outlet pipe, the sample outlet pipe is communicated with an internal standard cavity, an internal standard hole is opened in the top wall of the internal standard cavity, the internal standard hole is provided with a second micro-flow pump, the internal standard cavity is communicated with a serpentine pipe at one side, mixing platforms are fixedly connected to the curved positions of the pipe wall of the serpentine pipe, and the reversing valve and the second micro-flow pump are signal-connected with the control and data processing system.

[0022] Further, the detection adapter assembly comprises a sample loading cavity, the sample loading cavity is communicated with the end of the serpentine pipe, a sample loading valve is arranged at the communication position between the sample loading cavity and the serpentine pipe, the sample loading cavity is communicated with liquid phase pipes at both sides, the liquid phase pipes are communicated with liquid phase holes opened in the top wall of the microfluidic chip at the ends, the liquid phase holes are provided with third micro-flow pumps, the other side of the sample loading cavity is communicated with a chromatographic column, filter plates are arranged at both ends of the chromatographic column, a stationary phase is filled in the chromatographic column, an electrothermal film for maintaining column temperature is arranged on the inner wall of the chromatographic column, the end of the chromatographic column is communicated with a mass spectrometer sample loading cavity, the mass spectrometer sample loading cavity is communicated with a mass spectrometer, and the sample loading valve, the third micro-flow pumps, the electrothermal film and the mass spectrometer are signal-connected with the control and data processing system.

[0023] Further, the control and data processing system is configured to:

[0024] receive peak area data of 5-methyltetrahydrofolate and free pteroylglutamic acid from the mass spectrometer;

[0025] The concentrations C1, C2 and the ratio R thereof are calculated, and the detection validity is verified according to the threshold range R∈[8.0, 12.0];

[0026] The effective C1 value is substituted into the regression equation to convert into the X value, and the final folate nutritional status determination conclusion is output.

[0027] Further, the control and data processing system is also integrated with a dynamic calibration algorithm, when the cumulative effective sample amount reaches a set threshold, the external statistical software package is automatically called to update the coefficients of the regression equation.

[0028] The beneficial effects of the basic scheme are: 1. The sample pretreatment component of the device can automatically complete the whole process of sample dilution, micro-extraction purification and mixing with double internal standards through the coordinated design of structures such as sample hole, buffer cavity, dilution cavity, mixing tube, extraction column, combined with the signal linkage of the first micro-pump, control valve, reversing valve, etc. and the control and data processing system. Without a large amount of manual operation, both manual operation errors are avoided and the sample pretreatment steps are unified and standardized, providing pure, stable and high-consistency samples for subsequent detection, thus ensuring the detection accuracy from the upstream.

[0029] 2. In the detection adaptation component, the design of the sample cavity, liquid phase tube and third micro-pump can realize the accurate control and transportation of the sample and mobile phase; the electrothermal film on the inner wall of the chromatographic column can stably maintain the column temperature, avoiding the influence of column temperature fluctuation on the chromatographic separation effect, and the stationary phase in the chromatographic column and the two end screens can ensure efficient separation of the target component; the stable connection between the mass spectrometry sampling cavity and the external mass spectrometer ensures the effective transmission of the detection signal. At the same time, the control and data processing system receives the peak area data of the mass spectrometer in real time, calculates the concentration and ratio through standardized logic, verifies the detection validity according to R∈[8.0, 12.0], further excludes invalid data, and multiple links jointly improve the accuracy of the detection result.

[0030] 3. The control and data processing system not only can control the orderly operation of the components of the microfluidic chip, but also can automatically execute data calculation (C1, C2 and R value), validity verification, limit conversion (substitute the effective C1 into the regression equation to convert into the X value) and folate nutritional status determination, and directly output the conclusion, without manual calculation and judgment, greatly shortening the time from detection to result. In addition, the dynamic calibration algorithm integrated in the system can automatically call the external statistical software package to update the regression equation coefficients when the cumulative effective sample amount reaches the set threshold, so that the detection standard can be dynamically optimized without manual intervention, ensuring the accuracy and timeliness of the long-term detection result, and better meeting the needs of clinical rapid detection and large-scale sample screening.

[0031] 4、The device integrates the sample pretreatment component and the detection adaptation component in the microfluidic chip, and each component is compactly arranged and orderly connected, so that a complex decentralized experimental device does not need to be additionally built, and the laboratory operation space is greatly saved. Meanwhile, the integrated design reduces the loss and pollution risk of the sample in the transfer process, reduces the waste of reagents and samples, indirectly controls the detection cost, and the overall device structure is clear and convenient to maintain, so that the device is convenient for clinical routine use. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is a linear correlation fitting graph of the microbiological method and the liquid chromatography-mass spectrometry method.

[0033] Figure 2 is a nonlinear correlation fitting graph of the microbiological method and the liquid chromatography-mass spectrometry method.

[0034] Figure 3 is an isometric view of a blood folate deficiency boundary determination device of the liquid chromatography-mass spectrometry method in the embodiment.

[0035] Figure 4 is a front view of a blood folate deficiency boundary determination device of the liquid chromatography-mass spectrometry method in the embodiment.

[0036] Figure 5 is a side view of a blood folate deficiency boundary determination device of the liquid chromatography-mass spectrometry method in the embodiment.

[0037] The reference signs in the drawings of the specification include: 1, microfluidic chip; 2, liquid phase hole; 3, internal standard hole; 4, sealing cover; 5, liquid injection hole; 6, liquid phase tube; 7, sample loading cavity; 8, sample loading valve; 9, serpentine tube; 10, mixing table; 11, internal standard cavity; 12, filter membrane; 13, mixing tube; 14, mixing island; 15, control valve; 16, mixing column; 17, dilution tube; 18, liquid injection tube; 19, sieve plate; 20, reversing valve; 21, liquid discharge hole; 22, buffer cavity. DETAILED DESCRIPTION

[0038] The following is further described in detail through specific embodiments:

[0039] Embodiment 1

[0040] Basically as shown in the drawings: Figure 1 and Figure 2 A blood folate deficiency boundary determination method of a liquid chromatography-mass spectrometry method includes the following steps:

[0041] Step one, sample pretreatment, collecting a blood sample and separating serum, taking a quantitative serum sample for dilution, then purifying through a micro-extraction column to remove matrix interference, and then mixing with a mixed internal standard solution containing 5-methyltetrahydrofolate isotope internal standard and pteroyl glutamic acid isotope internal standard.

[0042] Step two, UPLC-MS / MS detection, the pretreated sample is chromatographically separated, and the peak areas of 5-methyltetrahydrofolate and free pteroylglutamic acid are simultaneously detected by mass spectrometry in multiple reaction monitoring mode.

[0043] Step three, data calculation and verification, according to the detected peak area, the internal standard method is used to calculate the concentration C1 of 5-methyltetrahydrofolate and the concentration C2 of free pteroylglutamic acid in serum respectively, and the ratio R = C1 / C2 of the two is calculated; when the ratio R is within the preset threshold range, it is determined that the detection result is valid, and the preset threshold range is R ∈ [8.0, 12.0]; if the R value exceeds the range, the re-detection process is automatically triggered.

[0044] Step four, threshold conversion and determination, the valid 5-methyltetrahydrofolate concentration C1 is substituted into the preset regression equation to convert into the equivalent microbial method total folate concentration X, and then the folate nutritional status of the individual is determined according to the preset threshold standard. The preset regression equation is X = (C1-b) / k, wherein the initial coefficients k = 0.656 and b = 2.309; the preset threshold standard is: C1 < 6.77 nmol / L is determined as deficiency, 6.77 ≤ C1 < 11.17 nmol / L is determined as marginal deficiency, and C1 ≥ 11.17 nmol / L is determined as sufficiency.

[0045] It also includes a dynamic calibration step: when the cumulative number of valid detection samples reaches a set threshold, the coefficients k and b of the regression equation are updated based on new valid sample data through a nonlinear regression model.

[0046] The specific implementation process is as follows: the microbial improvement method is the gold standard for folate detection, but it has problems such as complex operation and long time consumption. The microbial improvement method first needs to dilute the serum, then add the diluted serum, folate culture medium and strain into a 96-well enzyme-labeled plate, seal it and incubate it at 37°C in the dark for 42 hours. From sample processing to result reading, it takes more than 42 hours.

[0047] The same batch of serum samples (n = 573) were detected for folate levels using the improved microbial method and liquid chromatography tandem mass spectrometry, respectively. Then the correlation between the detection results of the two methods was evaluated using Pearson correlation and multivariate fractional polynomial, the correlation curve was fitted, and the regression model was constructed. Statistical analysis was based on R software (version 4.4.3). The "cor" function in the "stats package" was used to calculate the Pearson correlation coefficient, and the "lm" function was used to construct the linear regression model. The "mfp" function in the "mfp package" was used to construct the nonlinear regression model, and the linear correlation graph Figure 1 and the nonlinear correlation graph Figure 2 .

[0048] The results of microbial method and mass spectrometry were linearly correlated when the concentration of folic acid was low. Based on the improved microbial method for folic acid nutritional status established by the World Health Organization, the folic acid nutritional status was divided into sufficient (≥13.5 nmol / L), marginal deficiency (6.8-<13.5 nmol / L), and deficiency (<6.8 nmol / L). The linear regression equation was used to calculate the deficiency and marginal deficiency cutoff values of five-methyl tetrahydrofolic acid detected by mass spectrometry.

[0049] The linear regression equation was: mass spectrometry result=0.656*microbial method result+2.309.

[0050] According to the calculation of the equation, the folic acid nutritional status cutoff values of mass spectrometry were: sufficient (≥11.17 nmol / L), marginal deficiency (6.77-<11.17 nmol / L), and deficiency (<6.77 nmol / L).

[0051] The blood folic acid deficiency cutoff value determination method of liquid chromatography-mass spectrometry obtained from the above study was used for implementation detection.

[0052] Before starting the detection, reagent preparation and instrument parameter setting were completed: 100 ng / mL mixed internal standard solution (containing 5-methyl tetrahydrofolic acid isotope internal standard and folic acid isotope internal standard) was prepared; 0.1% formic acid aqueous solution was prepared as diluent and equilibration liquid, and methanol was prepared as activation liquid and eluent. At the same time, the high performance liquid chromatography-mass spectrometry system was initialized, the column temperature was set to 35℃, the injection volume was 5 μL, the flow rate of mobile phase was 0.3 mL / min, the mobile phase A was 0.1% formic acid aqueous solution, the mobile phase B was acetonitrile, and the preset gradient elution program was set; the mass spectrometer used electrospray ion source and multiple reaction monitoring mode, the ion source temperature was set to 550℃, the atomization gas pressure was 50 psi, the auxiliary gas pressure was 50 psi, the curtain gas pressure was 35 psi, the collision gas pressure was Medium, and the detection parameters of 5-methyl tetrahydrofolic acid (ion pair m / z 460.2→m / z 314.1, collision energy 28eV) and folic acid (ion pair m / z 442.2→m / z 296.1, collision energy 26eV) were determined. In addition, the extraction column was activated (methanol was flushed at a flow rate of 1 mL / min for 3 min) and equilibrated (0.1% formic acid aqueous solution was flushed at a flow rate of 1 mL / min for 3 min), and the system self-checking and preparation were completed.

[0053] 5mL fasting venous blood of the subject is collected, and after centrifugation (3500r / min, 15min) to separate the serum, the serum is diluted with 0.1% formic acid aqueous solution in the above-mentioned liquid chromatography-mass spectrometry method blood folate deficiency cutoff determination device, and the diluted sample liquid is passed through the activated and equilibrated extraction column at a flow rate of 1mL / min, followed by elution of impurities with 5% methanol aqueous solution, and then elution of the target component with methanol. A quantitative mixed internal standard solution is added to the eluate as the sample to be detected, and the pretreatment is completed.

[0054] The sample to be detected is injected into the high performance liquid chromatography-mass spectrometry system, and the detection program is started. In the chromatographic separation stage, the preset gradient elution program is run: 0-1min maintains 5% B mobile phase, 1-3min raises B phase proportion from 5% to 30%, 3-5min continues to raise to 90%, 5-7min maintains 90% B phase, 7-7.1min quickly falls to 5% B phase, 7.1-10min maintains 5% B phase to balance the chromatographic column. The separated 5-methyltetrahydrofolate and free pteroyl glutamic acid components enter the mass spectrometer, which detects the peak areas of the two components and their corresponding internal standards in multiple reaction monitoring mode, and records the peak area data in real time.

[0055] After receiving the peak area data detected by the mass spectrometer, the concentration is calculated by the internal standard method: according to the standard curve (linear correlation coefficient R²≥0.999) and the formula C=(sample peak area / internal standard peak area)×internal standard concentration×dilution multiple / standard curve slope, the concentrations C1 of 5-methyltetrahydrofolate and C2 of free pteroyl glutamic acid in the serum are calculated respectively, and then the ratio R=C1 / C2 is calculated.

[0056] The R value is compared with the preset threshold range [8.0, 12.0], if the R value is within this range (such as a sample C1=19.2nmol / L, C2=1.98nmol / L, R≈9.7), the detection result is determined to be valid; if the R value is out of range (such as R=7.2 or 13.5), re-detection is triggered, and the sample pretreatment and detection process is repeated once and reported.

[0057] For the effective detection results, C1 is substituted into the regression equation X= (C1-b) / k (initial coefficient k=0.656, b=2.309), and the total folate concentration X in the equivalent microbial method is converted through X= (C1-b) / k (for example, when C1=19.2 nmol / L, X≈25.7 nmol / L). According to the cutoff standard (C1<6.77 nmol / L is deficiency, 6.77≤C1<11.17 nmol / L is marginal deficiency, and C1≥11.17 nmol / L is sufficient), the folate nutritional status of the subjects (the sample C1≥11.17 nmol / L, determined as sufficient) is determined and a report is generated.

[0058] The system automatically records the effective sample data. When the cumulative effective sample amount reaches the set threshold (such as 100 cases), the external statistical software is called, the regression equation is refitted based on the C1 data of these samples and the total folate concentration data detected by the corresponding microbial method through a nonlinear regression model (least squares method), and the coefficients k and b are updated (such as k=0.662, b=2.285). The updated coefficients are used for the cutoff conversion of subsequent samples to ensure the accuracy of the detection.

[0059] Embodiment 2

[0060] Different from the above embodiment, as shown in Figures 3 to 5 A blood folate deficiency cutoff determination device for liquid chromatography-mass spectrometry includes a microfluidic chip 1, a sample pretreatment assembly inside the microfluidic chip 1 for realizing sample dilution, micro-extraction purification, and mixing with double internal standards, and a detection adapter assembly for connecting an external chromatograph-mass spectrometer.

[0061] The sample pretreatment assembly includes a sample addition hole opened in the top wall of the microfluidic chip 1, the sample addition hole is hinged with a sealing cover 4, the sample addition hole is communicated with a conical buffer cavity 22, the top wall of the microfluidic chip 1 is also provided with a liquid injection hole 5, the liquid injection hole 5 is installed with a first microflow pump, the liquid injection hole 5 is communicated with a liquid injection tube 18, the liquid injection tube 18 is communicated with the buffer cavity 22, the side of the buffer cavity 22 opposite to the liquid injection tube 18 is communicated with a dilution tube 17, the dilution tube 17 is communicated with a dilution cavity, a plurality of mixing columns 16 are integrally formed on the inner bottom wall of the dilution cavity, the side of the dilution cavity opposite to the dilution tube 17 is communicated with a mixing tube 13, a plurality of mixing islands 14 are integrally formed in the mixing tube 13, a control valve 15 is arranged at the communication position between the dilution cavity and the mixing tube 13, the mixing tube 13 is communicated with an extraction column, and the first microflow pump and the control valve 15 are both signal-connected with a control and data processing system.

[0062] The extraction column is arranged obliquely, filter membranes 12 are arranged at both ends of the extraction column, adsorbents are filled in the extraction column, a reversing valve 20 is communicated with the bottom end of the extraction column, a liquid discharge hole 21 opened in the bottom wall of the microfluidic chip 1 is communicated with the output end of the reversing valve 20, another output end of the reversing valve 20 is communicated with a sample outlet tube, the sample outlet tube is communicated with an internal standard cavity 11, an internal standard hole 3 is opened in the top wall of the internal standard cavity 11, a second micro flow pump is installed on the internal standard hole 3, a serpentine tube 9 is communicated with one side of the internal standard cavity 11, mixing tables 10 are integrally formed at the curved positions of the tube wall of the serpentine tube 9, and the reversing valve 20 and the second micro flow pump are signal connected with a control and data processing system.

[0063] The detection adaptation assembly comprises a sample loading cavity 7, the sample loading cavity 7 is communicated with the end of the serpentine tube 9, a sample loading valve 8 is arranged at the communication position between the sample loading cavity 7 and the serpentine tube 9, liquid phase tubes 6 are communicated with both sides of the sample loading cavity 7, liquid phase holes 2 opened in the top wall of the microfluidic chip 1 are communicated with the ends of the liquid phase tubes 6, third micro flow pumps are installed on the liquid phase holes 2, a chromatographic column is communicated with the other side of the sample loading cavity 7, sieve plates 19 are arranged at both ends of the chromatographic column, stationary phases are filled in the chromatographic column, electrothermal films for maintaining column temperature are arranged on the inner wall of the chromatographic column, a mass spectrometry sample cavity is communicated with the end of the chromatographic column, a mass spectrometer is communicated with the mass spectrometry sample cavity, and the sample loading valve 8, the third micro flow pumps, the electrothermal films and the mass spectrometer are signal connected with the control and data processing system.

[0064] The control and data processing system is used for controlling the operation of the microfluidic chip 1 and performing data calculation, verification, limit conversion and determination logic. The control and data processing system is configured to receive peak area data of 5-methyltetrahydrofolate and folic acid from the mass spectrometer, calculate the concentrations C1, C2 and the ratio R of the two, verify the detection effectiveness according to the threshold range R ∈ [8.0, 12.0], substitute the effective C1 value into the regression equation to convert into an X value, and output the final folic acid nutrition status determination conclusion. The control and data processing system is also integrated with a dynamic calibration algorithm, when the cumulative effective sample amount reaches a set threshold, an external statistical software package is automatically called to update the coefficients of the regression equation.

[0065] The specific implementation process is as follows: before detection starts, the whole liquid chromatography-mass spectrometry blood folate deficiency boundary determination device is initialized. The control and data processing system starts the self-checking program, respectively establishes signal connection with the first micro flow pump, the second micro flow pump, the third micro flow pump, the control valve 15, the reversing valve 20, the sample valve 8, the electric heating film and the mass spectrometer, and confirms that the running state of each component is normal. Subsequently, a predetermined volume of mixed internal standard solution (containing 5-methyltetrahydrofolate isotope internal standard and folate isotope internal standard, concentration 100 ng / mL) is injected into the internal standard cavity 11 through the internal standard hole 3, the internal standard solution reserve is completed; the extraction column in the microfluidic chip 1 is activated and balanced, the first micro flow pump transports methanol to the liquid injection pipe 18 through the liquid injection hole 5, the methanol enters the dilution cavity through the buffer cavity 22 and the dilution pipe 17, then flows into the mixing pipe 13 through the opened control valve 15, and finally enters the extraction column, with a flow rate of 1 mL / min for 3 min to realize activation, and then switches to transport 0.1% formic acid solution, also with a flow rate of 1 mL / min for 3 min to complete the balance, the waste liquid generated in the activation and balance process is switched to the liquid discharge hole 21 by the reversing valve 20 for discharge. At the same time, control the electric heating film to start working, heat the column temperature to 35℃ and maintain constant temperature, and prepare for subsequent chromatographic separation.

[0066] 5mL fasting venous blood of the subject is collected, after centrifugal separation of serum, the sealing cover 4 of the top wall of the microfluidic chip 1 is opened, 0.5mL serum sample is injected into the sample hole, and the sample enters the conical buffer cavity 22 along the sample hole. The control and data processing system sends a command to start the first microfluidic pump, and 1.5mL of 0.1% formic acid aqueous solution (diluent) is transported to the injection tube 18 through the injection hole 5, and the diluent flows into the buffer cavity 22 through the injection tube 18 and mixes with the serum sample, and after preliminary dilution, the mixed solution enters the dilution cavity through the dilution tube 17 on one side of the buffer cavity 22. A plurality of mixing columns 16 fixed on the bottom wall of the dilution cavity form a barrier and a flow to the mixed solution, so as to promote the mixed solution to be fully stirred in the cavity, so as to realize further dilution (the final dilution ratio is 1:3). After dilution is completed, the control and data processing system controls the control valve 15 to be opened, and the diluted sample liquid flows to the extraction column through the mixing tube 13. A plurality of mixing islands 14 fixed in the mixing tube 13 further strengthen the uniformity of the sample liquid. The sample liquid flows through the extraction column at a flow rate of 1mL / min, and the filter membrane 12 at both ends of the extraction column prevents the loss of the adsorbent filled inside. The adsorbent specifically adsorbs 5-methyltetrahydrofolic acid and folic acid in the sample liquid, while removing matrix interference impurities. After the impurities are adsorbed, the control valve 20 is switched to stop discharging the liquid from the discharge hole 21, and the second microfluidic pump is started to quantitatively transport the mixed internal standard liquid in the internal standard cavity 11 to the sample outlet tube. The internal standard liquid and the target component purified by the extraction column are flowed into the internal standard cavity 11, and the mixing of the target component and the double internal standard is completed. The mixed liquid then enters the serpentine tube 9 connected to one side of the internal standard cavity 11, and the mixing platform 10 fixed on the curved part of the tube wall of the serpentine tube 9 makes the mixed liquid produce vortex during the flow process, further improving the mixing uniformity of the target component and the internal standard. Finally, the uniformly mixed sample liquid to be detected flows into the sample cavity 7 of the detection adapter assembly.

[0067] In combination Figure 3 , Figure 4 and Figure 5As shown, after the sample liquid to be detected enters the sample chamber 7, the control and data processing system controls the sample valve 8 to open, and at the same time, the third micro-pump is started to transport the mobile phase (the mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is acetonitrile) to the liquid phase tube 6 through the liquid phase hole 2. The mobile phase pushes the sample liquid to be detected in the sample chamber 7 into the chromatographic column at a flow rate of 0.3 mL / min. The screen plate 19 at both ends of the chromatographic column prevents the internal filling of the stationary phase from leaking. The sample liquid starts chromatographic separation under the action of the stationary phase, and the components are separated according to the preset gradient elution program: 0-1 min, 5% B mobile phase is maintained, 1-3 min, the proportion of B phase is increased from 5% to 30%, 3-5 min, it is continuously increased to 90%, 5-7 min, 90% B phase is maintained, 7-7.1 min, it is quickly reduced to 5% B phase, and 7.1-10 min, 5% B phase is maintained to balance the chromatographic column. The separated 5-methyl tetrahydrofolic acid and folic acid components enter the mass spectrometry sample chamber in turn, and then enter the mass spectrometer connected thereto. The mass spectrometer uses an electrospray ion source and a multiple reaction monitoring mode for detection, with an ion source temperature of 550°C, an atomization gas pressure of 50psi, an auxiliary gas pressure of 50psi, a curtain gas pressure of 35psi, and a collision gas pressure of Medium. The peak areas of 5-methyl tetrahydrofolic acid (ion pair m / z 460.2→m / z 314.1, collision energy 28eV) and folic acid (ion pair m / z 442.2→m / z 296.1, collision energy 26eV) and their corresponding internal standards are monitored, and the peak area data is transmitted to the control and data processing system in real time.

[0068] After the control and data processing system receives the peak area data transmitted by the mass spectrometer, the internal standard method concentration calculation program is called, and the standard curve (linear correlation coefficient R 2 ≥0.999) and the formula C=(sample peak area / internal standard peak area) x internal standard concentration x dilution multiple / standard curve slope are used to calculate the concentration C1 of 5-methyl tetrahydrofolic acid and the concentration C2 of free folate glutamate in the serum, respectively, and automatically calculate the ratio R=C1 / C2 of the two. The system compares the R value calculated with the preset threshold range [8.0, 12.0], if the R value is in the range (such as a sample calculated C1=19.2 nmol / L, C2=1.98 nmol / L, R≈9.7), the detection result is valid, and the subsequent threshold conversion link is entered; if the R value is out of the range, the system immediately issues a re-detection instruction, and the control device automatically empties the flow path and re-executes the whole process of sample pretreatment and detection.

[0069] For effective detection results, the system substitutes C1 value into the preset regression equation X=(C1-b) / k (initial coefficient k=0.656, b=2.309), converts into equivalent microbial method total folate concentration X (for example, when C1=19.2 nmol / L, X≈25.7 nmol / L) by the formula X=(C1-b) / k, and then compares with the preset threshold standard (C1<6.77 nmol / L is deficiency, 6.77≤C1<11.17 nmol / L is marginal deficiency, and C1≥11.17 nmol / L is sufficiency) to determine the folate nutritional status of the subject (the sample C1≥11.17 nmol / L is determined to be sufficient), and generate a determination report. At the same time, the system automatically records the effective detection sample data, and when the cumulative effective detection sample amount reaches the set threshold (for example, 100 cases), the integrated dynamic calibration algorithm is triggered, the external statistical software package is automatically called, the regression equation is refitted by a nonlinear regression model (least squares method) based on the C1 data of the first 100 samples and the corresponding microbial method total folate concentration data, the coefficients k and b are updated, and the updated coefficients are stored in the system for subsequent sample threshold conversion, ensuring the long-term accuracy of the detection results.

[0070] Specific experimental process:

[0071] I. Experimental purpose

[0072] 1. Verify the repeatability and accuracy of the liquid chromatography-mass spectrometry (UPLC-MS / MS) method in blood folate detection.

[0073] 2. Confirm the effectiveness of the preset regression equation C1=0.656X+2.309.

[0074] 3. Evaluate the clinical applicability of the C1-based threshold standard (deficiency: <6.77 nmol / L; marginal: 6.77-11.17 nmol / L; sufficiency: ≥11.17 nmol / L).

[0075] II. Experimental samples

[0076] Total sample size: 600 cases

[0077] Sample source: hospital health examination center, nutrition department clinic

[0078] Sample type: serum separated from fasting venous blood

[0079] Sample grouping: healthy control group (n=300); suspected folate deficiency group (n=300)

[0080] III. Experimental equipment and reagents

[0081] Equipment: The microfluidic chip integrated device, UPLC-MS / MS system (Agilent 1290 Infinity II + Sciex Triple Quad 5500 +)

[0082] Reagent: Mixed internal standard solution (containing 5-methyl tetrahydrofolic acid and folic acid isotope internal standard, 100 ng / mL); 0.1% formic acid aqueous solution, methanol, acetonitrile (chromatographic pure)

[0083] Micro-extraction column filler: C18 adsorbent

[0084] Four, experimental steps

[0085] 1. Sample pretreatment

[0086] Collect 5 mL of fasting venous blood, centrifuge at 3500 r / min for 15 min, and separate the serum.

[0087] Take 0.5 mL of serum, add 1.5 mL of 0.1% formic acid aqueous solution for dilution (1:3).

[0088] Pass the diluted sample through the activated (methanol) and balanced (0.1% formic acid) micro-extraction column at a flow rate of 1 mL / min.

[0089] Wash with 5% methanol water, and elute the target components with methanol.

[0090] Add 100 μL of mixed internal standard solution to the eluent and mix well for standby.

[0091] 2. UPLC-MS / MS detection

[0092] Chromatographic conditions:

[0093] Chromatographic column: C18 column; column temperature: 35℃; mobile phase: A: 0.1% formic acid water; B: acetonitrile

[0094] Gradient program: 0-1 min 5% B → 3 min 30% B → 5 min 90% B → 7 min 90% B → 7.1 min 5% B → 10 min 5% B

[0095] Flow rate: 0.3 mL / min; injection volume: 5 μL

[0096] Mass spectrometry conditions:

[0097] Ion source: ESI+; monitoring mode: MRM

[0098] 5-methyltetrahydrofolate: m / z 460.2→ 314.1 (CE 28 eV); folic acid: m / z 442.2→ 296.1 (CE 26 eV)

[0099] 3. Data calculation and validation

[0100] 1. Calculate C1 (5-methyltetrahydrofolate) and C2 (folic acid) concentrations using internal standard method.

[0101] 2. Calculate the ratio R = C1 / C2.

[0102] 3. If R ∈ [8.0, 12.0], determine as valid data; otherwise, re-detect once and report.

[0103] 4. Cut-off conversion and determination

[0104] Convert valid C1 into equivalent microbiological concentration X using the regression equation X = (C1-2.309) / 0.656.

[0105] Determine folic acid nutritional status based on C1 value: Deficiency: C1 < 6.77 nmol / L; Marginal deficiency: 6.77 ≤ C1 < 11.17 nmol / L; Adequate: C1 ≥ 11.17 nmol / L.

[0106] When the cumulative number of valid samples reaches 100, automatically call the R language mfp package to update the regression equation coefficients.

[0107] Five, experimental data

[0108] As shown in the following table:

[0109] Table 1. Detection data example (first 10 cases)

[0110]

[0111] Table 2. Data statistical analysis

[0112]

[0113] Six, experimental conclusion

[0114] Among the 600 samples, 98% of the data were verified by the ratio R (R ∈ [8.0, 12.0]), indicating that the pretreatment and detection process was stable.

[0115] The determination results based on C1 have high consistency with clinical evaluation, and the detection rates of deficiency and marginal deficiency meet the expectations.

[0116] The initial regression equation C1 = 0.656X + 2.309 performs well in most samples, and can be further optimized after dynamic calibration.

[0117] Strong clinical applicability: the method has fast detection speed (<15 minutes per sample), high degree of automation, and is suitable for large-scale screening.

[0118] It should be noted that, in this text, relational terms such as first and second are used only to distinguish one entity or action from another, and do not necessarily require or imply that there is any such actual relationship or order between these entities or actions. Moreover, the terms "include", "contain" or any other variants thereof are intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such process, method, article or equipment.

[0119] The above is only an embodiment of the present application, and the common knowledge of the scheme and the characteristics of the prior art are not described in detail. The person skilled in the art knows all the common technical knowledge in the field of the present application before the filing date or the priority date, can obtain all the prior art in the field, and has the ability to apply conventional experimental means before that date. The person skilled in the art can improve and implement the present scheme based on the disclosure given in this application, and some typical known structures or known methods should not be an obstacle for the person skilled in the art to implement the present application. It should be noted that, for those skilled in the art, without departing from the structure of the present application, a number of modifications and improvements can be made, which should also be considered as the protection scope of the present application, and these will not affect the effect and practicality of the present application. The protection scope of the present application should be subject to the content of its claims, and the specific implementation mode and the like in the specification can be used to explain the content of the claims.

Claims

1. A method and apparatus for determining the blood folate deficiency threshold using liquid chromatography-mass spectrometry, characterized in that: Includes the following steps: Step 1: Sample pretreatment. Collect blood samples and separate serum. Take a quantitative amount of serum sample and dilute it. Then, purify it by microextraction column to remove matrix interference. Then mix it with a mixed internal standard solution containing 5-methyltetrahydrofolate isotope internal standard and pteroylglutamate isotope internal standard. Step 2: UPLC-MS / MS detection. The pretreated sample was separated by chromatography, and the peak areas of 5-methyltetrahydrofolate and free pteroylglutamic acid were simultaneously detected by mass spectrometry in multiple reaction monitoring mode. Step 3: Data calculation and verification. Based on the detected peak area, the concentrations of 5-methyltetrahydrofolate (C1) and free pteroylglutamate (C2) in serum are calculated using the internal standard method, and the ratio R = C1 / C2 is calculated. When the ratio R is within the preset threshold range, the test result is considered valid. Step 4: Threshold conversion and judgment. Substitute the effective 5-methyltetrahydrofolate concentration C1 into the preset regression equation to convert it into the equivalent total folate concentration X by microbial method. Then, determine the individual's folate nutritional status according to the preset threshold standard.

2. The method for determining the blood folate deficiency threshold by liquid chromatography-mass spectrometry according to claim 1, characterized in that: The preset threshold range in the data calculation and verification steps is R∈[8.0, 12.0]; if the R value exceeds this range, a re-detection process will be automatically triggered and a report will be issued.

3. The method for determining the blood folate deficiency threshold by liquid chromatography-mass spectrometry according to claim 2, characterized in that: The preset regression equation in the threshold conversion and judgment step is X=(C1-b) / k, where the initial coefficients k=0.656 and b=2.309; the preset threshold criteria are: C1<6.77 nmol / L is judged as deficient, 6.77≤C1<11.17 nmol / L is judged as marginally deficient, and C1≥11.17 nmol / L is judged as sufficient.

4. The method for determining the blood folate deficiency threshold by liquid chromatography-mass spectrometry according to claim 3, characterized in that: It also includes a dynamic calibration step: when the cumulative number of valid detection samples reaches a set threshold, the coefficients k and b of the regression equation are updated using a nonlinear regression model based on the new valid sample data.

5. An apparatus for implementing the method for determining the blood folate deficiency threshold using liquid chromatography-mass spectrometry as described in any one of claims 1-4, characterized in that: The microfluidic chip (1) is provided with a sample pretreatment component for sample dilution, microextraction purification and mixing with dual internal standards, and a detection adapter component for connecting to an external chromatography-mass spectrometer. The control and data processing system is used to control the operation of the microfluidic chip (1) and to perform data calculation, verification, threshold conversion and judgment logic.

6. The apparatus for determining the blood folate deficiency threshold by liquid chromatography-mass spectrometry according to claim 5, characterized in that: The sample pretreatment component includes a sample loading port on the top wall of the microfluidic chip (1), the sample loading port is equipped with a sealing cap (4), the sample loading port is connected to a conical buffer chamber (22), the top wall of the microfluidic chip (1) is also provided with an injection port (5), the injection port (5) is equipped with a first microfluidic pump, the injection port (5) is connected to an injection tube (18), the injection tube (18) is connected to the buffer chamber (22), and a dilution tube is connected to one side of the buffer chamber (22) opposite to the injection tube (18). 17) The dilution tube (17) is connected to the dilution chamber. Several mixing columns (16) are fixedly connected to the bottom wall of the dilution chamber. A mixing tube (13) is connected to the side of the dilution chamber opposite to the dilution tube (17). Several mixing islands (14) are fixedly connected inside the mixing tube (13). A control valve (15) is provided at the connection between the dilution chamber and the mixing tube (13). An extraction column is connected to the mixing tube (13). The first microfluidic pump and the control valve (15) are both connected to the control and data processing system.

7. The apparatus for determining the blood folate deficiency threshold by liquid chromatography-mass spectrometry according to claim 6, characterized in that: The extraction column is inclined and has filter membranes (12) laid at both ends. The extraction column is filled with adsorbent. The bottom end of the extraction column is connected to a reversing valve (20). The output end of the reversing valve (20) is connected to a drain hole (21) opened on the bottom wall of the microfluidic chip (1). The other output end of the reversing valve (20) is connected to a sample tube. The sample tube is connected to an internal standard cavity (11). An internal standard hole (3) is opened on the top wall of the internal standard cavity (11). A second microfluidic pump is provided on the internal standard hole (3). A serpentine tube (9) is connected to one side of the internal standard cavity (11). A mixing platform (10) is fixedly connected to the bend of the serpentine tube (9). The reversing valve (20) and the second microfluidic pump are both connected to the control and data processing system.

8. The apparatus for determining the blood folate deficiency threshold by liquid chromatography-mass spectrometry according to claim 5, characterized in that: The detection adapter component includes a sample loading chamber (7), which is connected to the end of a serpentine tube (9). A sample loading valve (8) is provided at the connection between the sample loading chamber (7) and the serpentine tube (9). Liquid phase tubes (6) are connected to both sides of the sample loading chamber (7). A liquid phase hole (2) is opened on the top wall of the microfluidic chip (1) at the end of the liquid phase tube (6). A third microfluidic pump is provided in each of the liquid phase holes (2). A chromatographic column is connected to the other side of the sample loading chamber (7). A sieve plate (19) is laid at both ends of the chromatographic column. The chromatographic column is filled with a stationary phase. An electric heating film for maintaining the column temperature is laid on the inner wall of the chromatographic column. A mass spectrometry injection chamber is connected to the end of the chromatographic column. The mass spectrometry injection chamber is connected to a mass spectrometer. The sample loading valve (8), the third microfluidic pump, the electric heating film and the mass spectrometer are all connected to the control and data processing system.

9. The apparatus for determining the blood folate deficiency threshold by liquid chromatography-mass spectrometry according to claim 5, characterized in that: The control and data processing system is configured as follows: Receive peak area data for 5-methyltetrahydrofolate and free pteroylglutamic acid from the mass spectrometer; Calculate the concentrations C1 and C2 of both and their ratio R, and verify the detection effectiveness based on the threshold range R∈[8.0, 12.0]. Substitute the valid C1 value into the regression equation to convert it into an X value, and output the final conclusion on folic acid nutritional status.

10. The apparatus for determining the blood folate deficiency threshold by liquid chromatography-mass spectrometry according to claim 9, characterized in that: The control and data processing system also integrates a dynamic calibration algorithm, which automatically calls an external statistical software package to update the coefficients of the regression equation when the cumulative effective sample size reaches a set threshold.

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