Method for detecting lactate in whole blood

Abstract

The application discloses a method for detecting lactic acid in whole blood. Specifically, the method comprises a lactic acid extraction method, an ion mobility spectrometer sampling and detection analysis process and method. The lactic acid solution obtained by pretreating the whole blood sample is combined with the negative pressure sampling ion mobility tube technology, the sample is directly introduced into the photoionization center point of the ion mobility tube reaction zone, and then high-sensitivity direct detection of lactic acid is realized. The lactic acid measurement can realize quantitative analysis of lactic acid in whole blood in the concentration range of 100-1000 μg / ml. The analysis time of a single sample is less than 0.5 min, and the software can automatically output the lactic acid blood concentration detection analysis result. The detection method is simple, efficient, accurate and reliable, and has very important significance for clinical diagnosis of diseases through lactic acid blood concentration analysis.

Description

Technical Field

[0001] This invention relates to the field of separation, analysis and detection technology, and specifically to a method for detecting lactate in whole blood. Background Technology

[0002] Lactic acid (C3H6O3), M=90.078, melting point 18℃, boiling point 122℃, density 1.21g / mL. It is a product of anaerobic respiration, produced in skeletal muscle and brain tissue. D-lactic acid cannot be absorbed by the human body and can lead to increased blood urinary acidity, causing metabolic disorders. The WHO recommends a daily intake of less than 100mg per kilogram of body weight. L-lactic acid can be completely digested and absorbed by the human body.

[0003] Clinical blood lactate concentration range: 0.5–5.5 mmol / L (45–495 mg / L). In patients with asphyxia, blood lactate levels will rise, pH will decrease, BE value will decrease, partial pressure of oxygen (PaO2) will decrease, and the 1-min Apgar score will be lower. The ratio of oxygen concentration (FiO2) is calculated using the following formula: FiO2 = (21 + oxygen flow rate (L / min) × 4) / 100. Therefore, an oxygenation index PaO2 / FiO2 ≤ 100 mmHg is one of the criteria for severe acute respiratory distress syndrome. In a clinical study of 52 cases, the mortality rate was 0% for blood lactate levels below 1.4 mmol / L (126 ppm), 22% for levels below 4.4 mmol / L (400 ppm), 78% for levels below 8.7 mmol / L (800 ppm), and 100% for levels above 13 mmol / L (1170 ppm). Lactate levels are closely related to patient prognosis, and changes in lactate concentration are a good indicator of treatment effectiveness.

[0004] Zhang Ming et al. invented a blood gas analyzer for medical testing (patent number ZL201821788414.5). This utility model discloses a blood gas analyzer for medical testing, including a blood gas analyzer body. A display screen is installed on the upper front side of the blood gas analyzer body. A test port and a test result output port are installed on the lower front side of the blood gas analyzer body. A protective cover assembly is installed on the outer wall of the blood gas analyzer body near the test port. A support plate is fixed at the bottom of the blood gas analyzer body. A support column and a moving mechanism are installed at the lower end of the support plate.

[0005] Barbara Lynn EATON et al. invented (patent number US2018 / 0291430A1), a laboratory method for diagnosing bacterial vaginosis in subjects. In a single sample, the invention also discloses methods and compositions for detecting nucleic acids of *Lactobacillus*, *Gardnerella vaginalis*, and / or *Vitalis* in a sample.

[0006] Gan Yiwu et al. invented a highly stable lactate detection reagent (patent number ZL201410697752.8), belonging to the field of clinical in vitro detection technology. The reagent comprises two components: reagent R1 and reagent R2. The rational combination of these components ensures the stability of the entire reagent system, demonstrating excellent stability.

[0007] Blood lactate analyzers use an enzyme-current detection method to rapidly measure blood lactate concentration, evaluating the training intensity and functional status of professional athletes and testing the gradually changing lactate content in the blood. However, due to limitations in instruments, consumables, resources, and cost, widespread clinical use is not feasible. Summary of the Invention

[0008] The technical problem this invention aims to solve is: purification of lactate from whole blood, and addressing issues such as low lactate transfer efficiency, low detection sensitivity, long analysis time, and memory effect in traditional lactate injectors. Specific details include:

[0009] A method for detecting lactate in whole blood, characterized in that:

[0010] 1) Dissolve lactic acid standard in the mixed blood of more than 10 healthy people to prepare whole blood solutions with at least 5 different lactic acid contents in the range of 50-1000 μg / ml;

[0011] 2) The lactic acid-containing solution obtained in step 1) is detected and analyzed using a negative ion mode photoionization ion mobility spectrometer to obtain the detection signal; the peak migration time of lactic acid in negative ion mode and the signal tracking change trend curve of lactic acid peak within 0-30 seconds are recorded; the signal intensity data summed value, i.e., the peak area corresponding to different lactic acid concentrations, can be obtained by retrieving the lactic acid peak tracking change trend curve in the software.

[0012] 3) Fit the peak area values ​​of the tracking signal corresponding to different lactic acid concentrations in 1) and 2) to obtain the standard curve equation. Substitute the peak area of ​​the lactic acid sample with unknown concentration into the standard curve equation to calculate the lactic acid concentration, thereby achieving quantitative analysis.

[0013] 4) Extract lactic acid from the blood sample to be tested to obtain a solution containing lactic acid;

[0014] 5) The lactic acid-containing solution obtained in step 4) is detected and analyzed using a negative ion mode photoionization ion mobility spectrometer; the detection signal is obtained; based on the comparison between the obtained sample peak migration time and the lactic acid signal peak migration time in the spectrum of step 2), the software retrieves the peak area of ​​the target peak and calculates the blood lactic acid concentration according to the method in 3).

[0015] For steps 1) and 4), the extraction process is as follows: the blood sample extraction solvent is one or both of ethyl acetate or methyl tert-butyl ether; the extraction solution is subjected to sonication (2-5 min, power 500-600w), centrifugation (2-5 min, speed 10000-12000 rpm), and the supernatant is filtered through a membrane (pore size 0.22μm) to obtain a solution containing lactic acid. 0.5-1 mL of extraction solvent is used for every 0.5-1 mL of blood (preferably, volume ratio 1:1).

[0016] Specific process,

[0017] 1) Lactic acid standard sample analysis test: Dissolve lactic acid standard in the mixed blood of more than 10 healthy people, and prepare whole blood solutions of 100, 200, 400, 600, 800, and 1000 μg / ml using a stepwise dilution method; use 0.5-1 mL of extraction solvent (volume ratio, 1:1) for each 0.5-1 mL of blood according to the extraction process in claim 2, measure 2-5 μL of the lactic acid-containing solution, and then use a negative ion mode photoionization ion mobility spectrometer for detection and analysis to obtain the detection signal; record the peak migration time of lactic acid in negative ion mode and the signal tracking trend curve of lactic acid peak within 30 seconds.

[0018] 2) Whole blood sample analysis test: Accurately measure a certain amount of blood sample to be tested, perform lactic acid extraction pretreatment according to the extraction process of claim 2 above, and then measure 2-5 μL of lactic acid-containing solution for analysis; perform detection and analysis in negative ion mode of ion mobility spectrometry to obtain detection signal; compare the obtained sample peak migration time with the lactic acid signal peak migration time of the above spectrum, and select the target peak tracking signal intensity data for quantitative analysis.

[0019] The ion migration tube used includes four through holes as gas interfaces, an ion migration tube outlet port (4) for connecting to the gas inlet of the pump in the ionization region (2), an interface (5) for connecting to the chemical dopant source, and an ion migration tube sample inlet tube (6); a drift gas inlet (8) is provided in the migration region (3); the airflow circulation field inside the ion migration tube is such that, except for the outlet port (4), which is the outlet, the rest are inlets.

[0020] The chemical dopant interface (5) is located between the vias (4) and (6), and the chemical dopant is one or both of acetone or butanone.

[0021] The ion migration tube sample inlet tube (6) is made of metal, with a variable-diameter insulating inner sleeve (7) nested inside. The micro-syringe (9) contains the sample, and its tip needle is inserted into the variable-diameter insulating inner sleeve. The inner sleeve releases the sample at the end of the migration tube to achieve the best ionization effect.

[0022] The variable-diameter insulating inner sleeve (7) is a hollow tube formed by sealing the open end of one of two hollow tubes with different inner diameters to the open end of the other. The annular surface formed at the connection of the two hollow tubes inside the hollow tube is the stepped platform, which is perpendicular to the axis of the hollow tube. The hollow tube with the larger inner diameter is set at one end outside the ion migration tube, and the hollow tube with the smaller inner diameter is set at one end of the ionization region. The sample front end is placed on one side of the hollow tube with the smaller inner diameter, and the sample front end is ionized in the ionization region in sequence.

[0023] After 2-5 μl of the sample extract of lactic acid in whole blood is injected into the variable diameter insulating inner sleeve (7), one end of the lower end of the inner sleeve is located in the core photoionization region of the ion migration tube. The sample is then slowly pyrolyzed and vaporized by a small airflow (100-200 ml / min) from the vacuum pump connected to the through hole 4.

[0024] The data acquisition time in the method is 0-30 seconds, and the quantitative analysis is calculated based on the cumulative peak intensity of the tracked signal within 0-30 seconds.

[0025] The migration tube used in the experiment is a vacuum injection method; the drift gas is 500-600 ml / min; the dopant gas is 50-100 ml / min; the carrier gas carrying the sample is 100-200 ml / min; the pump flow rate in the experiment is the sum of the drift gas, dopant gas and carrier gas, and the flow rate is 650-900 ml / min; the migration tube in the experiment is a cylindrical structure. If the lamp window radius is set to "R" (usually 0.5-1 cm), the ionization region radius is (1-8)R, the migration region radius is greater than the ionization region radius and less than 10R; the axial distance of the ionization region is set to L (usually designed to be 2-3 cm), and the migration region length is designed to be 3-4L.

[0026] Advantages of the present invention

[0027] 1. Ion mobility spectrometry is used as an analytical method for lactate in whole blood. The pretreatment process involves reagent screening and deep purification of the extraction process, which is conducive to direct injection of the extract into the center point.

[0028] 2. This measurement method does not require an external sample injector, thus avoiding sample transfer losses associated with traditional sample injectors and improving the sensitivity of whole blood lactate analysis; the sample is directly injected at the photoionization center point, improving ionization efficiency.

[0029] 3. In this invention, the sample is slowly and gradually pyrolyzed and vaporized by a small airflow carried by an external air pump, which helps to solve the adsorption of lactic acid by the material in the ionization region and avoids the memory effect. Attached Figure Description

[0030] Figure 1 is a schematic diagram of the structure and principle of the ion mobility spectrometer for detecting lactic acid;

[0031] Figure 1-1 In the diagram, 1 is the VUV lamp ionization source; 2 is the ionization region of the ion migration tube; 3 is the migration region of the ion migration tube; 4 is the ion migration tube outlet port connected to the vacuum pump; 5 is the interface for connecting the chemical dopant; 6 is the ion migration tube sample inlet tube; 7 is the variable diameter insulating inner sleeve; 8 is the drift gas inlet; 9 is the micro-injector; 10 is the amplifier; 11 is the A / D converter; 12 is the data processing system; 13 is the ion gate; and 14 is the pump.

[0032] Figure 1-2 This is a schematic diagram of the stepped structure of the variable-diameter inner sleeve. 7-1 shows the upper frustum structure of the inner sleeve; 7-2 shows the first-step frustum.

[0033] Figure 1-3 This is a schematic diagram of the dopant cylinder device. In the diagram: 51 is the dopant cylinder (left, diagram C), 52 is the dopant gas air source inlet, 53 is the dopant gas outlet, 54 is the PTFE tube, 55 is the sample vial (right, diagram D), and 56 is the chemical dopant acetone.

[0034] Figure 2 Ion mobility spectra for qualitative analysis of lactic acid blood samples using ion mobility spectrometry.

[0035] Figure 3 The standard curve fitting diagram for quantitative analysis of lactic acid in blood samples using ion mobility spectrometry.

[0036] Figure 4 The ion mobility spectrum is used to detect the tracking signal in lactic acid blood samples using an ion mobility spectrometer. Detailed Implementation

[0037] In the following embodiments, all ion mobility spectrometry operations were performed using the ion mobility spectrometer apparatus shown in Figure 1.

[0038] The ion migration tube is a VUV lamp ionization source with a circular lamp window cross-section radius of 6 mm. It includes a coaxial cylindrical ionization region and a cylindrical migration region. The ionization region is 25 mm long and has an inner cylinder diameter of 20 mm; the migration region is 90 mm long and has an inner cylinder diameter of 25 mm. An ion gate is provided between the ionization region and the migration region. A photoionization source is located on the side of the ionization region away from the ion gate. An ion migration tube sample inlet 6 is provided on the outer wall of the ion migration tube in the ionization region.

[0039] A variable diameter insulating inner sleeve 7 is inserted into the sample inlet 6 of the ion migration tube. One end of the variable diameter insulating inner sleeve 7 is located in the ionization region of the ion migration tube, and the other end is located outside the ion migration tube.

[0040] The variable-diameter insulating inner sleeve 7 is hollow with a stepped inner diameter structure; the inner diameter of the other end located outside the ion migration tube is larger than the inner diameter of the end located in the ionization region.

[0041] The stepped variable diameter structure has one step or step, and is a hollow tube with two open ends formed by sealing one open end of one hollow tube with the other open end of two hollow tubes with different inner diameters. The two hollow tubes are coaxial, and the annular surface formed at the connection between the two hollow tubes inside the hollow tube is the stepped platform, which is perpendicular to the axis of the hollow tube. The hollow tube with the larger inner diameter is set at the other end outside the ion migration tube, and the hollow tube with the smaller inner diameter is set at one end of the ionization region.

[0042] The inner diameter of the variable-diameter insulating inner sleeve 7 at one end in the ionization region is 0.8 mm, the radial width of the stepped platform is 0.35 mm, and the inner diameter at the other end located outside the ion migration tube is 1.5 mm.

[0043] Both the ionization region and the migration region are coaxial cylindrical, with their axial centerlines placed parallel to the horizontal plane. The injection port 6 is located in the upper middle part of the ion migration tube.

[0044] Insert a variable diameter insulating inner sleeve 7 into the sample inlet 6 of the ion migration tube. The stepped platform with variable diameter inside the variable diameter insulating inner sleeve can be used as a storage platform for all or part of the liquid sample. When the liquid sample is injected into the ion migration tube, the liquid sample can be dropped onto the stepped platform. Then the inlet 6 can be connected to the carrier gas source. The inlet 6 does not need to be connected to the injector separately.

[0045] The inlet 6 is a circular through hole, and the variable diameter insulating inner sleeve 7 is a circular tube. The outer diameter "r1" of the variable diameter insulating inner sleeve 7 is less than or equal to the inner diameter "r2" of the ion migration tube sample inlet 6, that is, r1≤r2. r1 and r2 satisfy r2=5mm, and the difference between r2 and r1 is 0.05mm. The gap between the two can realize the function of evacuating and replenishing gas into the migration tube, reducing the sample airflow.

[0046] A hollow, annular, inverted frustum-shaped protrusion is provided radially on the outer wall of the other end of the variable-diameter insulating inner sleeve 7, which is located outside the ion migration tube. The cross-section of the protrusion along the axis of the variable-diameter insulating inner sleeve is two symmetrical right-angled triangles with the axis of the variable-diameter insulating inner sleeve as the axis of symmetry. One right-angled side A in the radial direction of the triangle is located away from the ion migration tube, and the vertex A corresponding to right-angled side A is located closer to the ion migration tube. The other right-angled side of the triangle is parallel to the axis of the variable-diameter insulating inner sleeve. The length of right-angled side A is greater than the difference between r2 and r1.

[0047] One groove is provided on the outer wall of the annular inverted truncated cone-shaped protrusion along the axial direction (from the lower bottom edge of the cone to the upper bottom edge). The radial cross-sectional area of ​​the groove gradually decreases from the right angle side A to the apex A (i.e. from the lower bottom edge of the cone to the upper bottom edge). The width of the groove on the radial cross-section is 0.15mm and the groove depth is 0.10mm. When the air pump is working, the groove can also realize the function of air replenishment.

[0048] The ion migration tube includes a photoionization source (e.g., a VUV lamp ionization source) and a Faraday disk arranged opposite to each other at the left and right ends, and an ion gate located between the photoionization source and the Faraday disk. The region between the photoionization source and the ion gate is the ionization region, and the region between the ion gate and the Faraday disk is the migration region.

[0049] Three through holes are sequentially provided on the outer wall of the ion migration tube, from the side of the ionization region near the photoionization source towards the ion gate. These holes are the ion migration tube outlet 4 for connecting to the vacuum pump, the interface 5 for connecting to the chemical dopant source, and the ion migration tube sample inlet 6. These three through holes, together with the drift gas inlet 8 provided on the outer wall of the ion migration tube near the ion detector, constitute the external interface of the gas circulation system of the ion migration tube. The ion migration tube outlet 4 is connected to the vacuum pump inlet as the gas outlet of the ion migration tube, and the rest are gas inlets.

[0050] The material of the variable diameter insulating inner sleeve 7 is polytetrafluoroethylene.

[0051] The variable-diameter insulating inner sleeve 7 is 6cm long. One end, located outside the ion migration tube, serves as the sample inlet port. The other end, located in the ionization region, is inserted into the area between the side of the cylindrical light path (light irradiation area) emitted from the circular light window of the photoionization source and the central axis. The radius of the cylindrical light path is equal to the radius of the circular light window of the photoionization source (e.g., VUV lamp), and the radius of the cylindrical ionization region is 1.5 times the radius of the light window.

[0052] The axial distance between the photoionization source lamp window and the ion gate is set to "L". The axial distance between the other end of the variable-diameter insulating inner sleeve 7 in the ionization region and the photoionization source lamp window is 18 / 25L, and its radial position is located on the central axis of the light path emitted by the lamp window. In this embodiment, the axial distance "L" between the lamp window and the ion gate is 25mm, and the center point sample injection position is 18mm.

[0053] Liquid samples, or liquid phases extracted after dilution with organic solvents, or liquid phases extracted after dissolving with organic solvents, are used as liquid injection samples. The liquid injection samples are loaded into a syringe barrel equipped with a syringe needle. After the syringe needle outlet is inserted into the variable diameter insulating inner sleeve, the sample is ejected from the syringe barrel. Subsequently, the sample is enriched on the stepped surface inside the variable diameter insulating inner sleeve and continuously vaporized in the ionization region under the action of the carrier gas, resulting in efficient thermal ionization.

[0054] Before being loaded into the microsyringe, the liquid sample must undergo a pretreatment process by filtering it through a filter membrane. The microporous filter membrane has a pore size of 0.22 μm, and the amount of sample injected into the variable-diameter insulating inner sleeve by the microsyringe is 2 μl.

[0055] The photoionization ion mobility spectrometer has three through-hole tubes near the VUV lamp in the ionization region: ion mobility tube outlet 4 (connected to the vacuum pump); dopant outlet 5; and sample inlet 6. In this embodiment, the drift gas (air) flow rate is 600 sccm, the vacuum pump flow rate is 850 sccm, the dopant gas (air as carrier gas) flow rate is 50 sccm, and the sample carrier gas (air) flow rate is 200 sccm. The ion mobility tube temperature is set to 100°C, and the ion gate opening time is set to 50 μs.

[0056] The ion migration tube is connected to the chemical dopant interface 5 via a conduit to a dopant cartridge device (such as...). Figure 1-3 The dopant cartridge assembly includes a cylindrical dopant cartridge (e.g., open at the top) for connection. Figure 1-3 (Left, Figure C) The outer wall of the dopant cylinder 51 has external threads at the open end. A cylindrical sealing cap with internal threads is screwed onto the open end of the dopant cylinder, sealing the open end of the dopant cylinder. A lower through hole is provided on the lower side wall of the cylindrical dopant cylinder as a dopant gas inlet 52 connected to an air source. An upper through hole is provided on the upper side wall of the cylindrical dopant cylinder as a dopant gas outlet 53, which is connected to the chemical dopant (dopant) interface 5 via a conduit. A hollow, sealed sample vial (e.g., ...) is placed inside the dopant cylinder. Figure 1-3 (Right, Figure D) Acetone 56, a chemical dopant, is placed inside the sample vial. A rod (outer diameter) is inserted into the top of the sample vial 55. The PTFE tube 54 has its upper open end inside the dopant cartridge and its lower open end above the liquid surface of acetone, the chemical dopant, inside the sample vial. The sample dopant gas (air) purging flow rate is 50 sccm. Acetone vapors through the PTFE tube opening on the sample vial cap. Inside the dopant cartridge, the acetone vapors are diluted by air and then purged into the ion migration tube.

[0057] In the following examples, the experimental conditions for ion mobility spectrometry were as follows: ion mobility spectrometry using a photoionization source, and direct injection of lactic acid samples at the center point. During the experiment, the migration tube temperature was maintained at 100°C, and the flow rates of the sample carrier gas (air), drift gas (air), and dopant gas (acetone) were 200, 600, and 50 mL / min, respectively.

[0058] The ion mobility spectrometer in the invention uses a VUV lamp ionization source. The ion mobility tube detector, from left to right, consists of a VUV lamp (1), an ionization region structure (2), and a migration region structure (3). An ion gate (13) is located between the ionization region (2) and the migration region (3). The sample is ionized in the ionization region by the VUV lamp ionization source and sequentially passes through the ion gate into the migration region. Due to different migration speeds, the sample is finally received and processed by the ion receiver. In the experiment, the ion mobility tube lamp window has a cross-sectional radius of 6 mm, an ionization region length of 25 mm, and an inner cylinder diameter of 20 mm; the migration region length is 90 mm, and the inner cylinder diameter is 25 mm. The axial distance "L" between the lamp window and the ion gate is 25 mm, and the center injection point is 18 mm from the ion gate.

[0059] To achieve accurate, rapid, and sensitive detection of lactate in whole blood, this invention uses ion mobility spectrometry as the analytical method. Lactic acid standards are dissolved in whole blood (prepared into lactate solutions with concentrations of 100, 200, 400, 600, 800, and 1000 μg / mL) as standard samples. The ion mobility spectrometer in negative ion mode is used as the detection instrument to obtain detection signals. The software automatically identifies relevant information to obtain a standard curve equation. Samples with unknown lactate concentrations are substituted into the standard curve equation to directly calculate the lactate content in whole blood.

[0060] Example 1

[0061] Initial experiments explored methanol, acetonitrile, n-hexane, carbon tetrachloride, ethyl acetate, and methyl tert-butyl ether as solvents for extracting lactic acid from whole blood. A 1 mL sample of whole blood (mixed blood from 10 healthy individuals aged 18-60 years) with a lactic acid concentration of 1000 μg / mL was placed in a sample vial. 1 mL of each of the above-mentioned solvents was added, followed by sonication for 2 min (500 W), centrifugation for 2 min (12000 rpm), and then the supernatant was filtered through an organic membrane (0.22 μm pore size). 2 μL of the supernatant was injected centrally into the ionization zone of an ion migration tube. Methanol and acetonitrile were unsuitable due to their miscibility with blood; n-hexane and carbon tetrachloride extracted almost no lactic acid and were therefore unsuitable as well. By comparing the signal intensity of lactic acid extraction with ethyl acetate and methyl tert-butyl ether, at the same lactic acid concentration, ethyl acetate showed the strongest lactic acid detection signal; therefore, ethyl acetate was selected for subsequent experiments.

[0062] Example 2

[0063] Qualitative analysis and detection of lactic acid samples:

[0064] A microsyringe accurately dispenses 1 mL of a 400 μg / mL lactic acid standard sample (using mixed blood from 10 healthy individuals aged 18-60 as the solvent) into a sample vial. Add 1 mL of ethyl acetate, sonicate for 2 min (500 W), centrifuge for 2 min (12000 rpm), and then pass the supernatant through an organic membrane (0.22 μm pore size). Inject 2 μL of the extract into the ionization region of an ion migration tube. Sample molecules are directly ionized into positive and negative ions in the ionization region. The hot carrier gas passes through periodically opening ion gates and enters the drift region formed by a uniform electric field, where separation and detection are achieved to obtain a detection signal. Figure 2 The ion mobility spectrum of lactic acid detected by the negative ion mode ion mobility spectrometry of the device of the present invention is given. It can be seen from the figure that the target peak of lactic acid detection in negative ion mode is 4.555ms.

[0065] Example 3

[0066] Pretreatment method for lactic acid standard samples:

[0067] Accurately measure 1 mL of lactic acid standard samples (using mixed blood from 10 healthy individuals aged 18-60 years as solvent) at a concentration of 100, 200, 400, 600, 800, and 1000 μg / mL into a sample vial using a microsyringe. Add 1 mL of ethyl acetate, sonicate for 2 min (500 W), centrifuge for 2 min (12000 rpm), and after centrifugation, pass the supernatant through an organic membrane (0.22 μm pore size). Inject 2 μL of the extract into the ionization region of an ion migration tube.

[0068] Quantitative analysis and detection of lactic acid standard samples:

[0069] The lactic acid-containing solution obtained from the above pretreatment was detected and analyzed using a negative ion mode photoionization ion mobility spectrometer to obtain the detection signal; the peak migration time of lactic acid in negative ion mode and the signal tracking change trend curve of lactic acid peak within 0-30 seconds were recorded; the signal intensity data summed, i.e., the peak area corresponding to different lactic acid concentrations, can be obtained by retrieving the lactic acid peak tracking change trend curve using software.

[0070] Experiments at concentrations of 100, 200, 400, 600, 800, and 1000 μg / ml revealed that the lactic acid peak (4.555 ms) exhibited a linear relationship within the concentration range of 100-1000 μg / ml. Figure 3 The equation is: Y = 4646.44 + 2.28 * X, with a correlation coefficient of 0.9865.

[0071] Example 4

[0072] Detection of lactate in whole blood samples:

[0073] Accurately measure 1 mL of whole blood sample with known lactate concentration (e.g., 200 μg / mL, prepared by mixing blood from 10 healthy individuals aged 18-60 years as solvent) into a sample bottle, add 1 mL of ethyl acetate, sonicate for 2 min (500 W power), centrifuge for 2 min (12000 rpm), and after centrifugation, take the supernatant and pass it through a membrane (organic membrane, 0.22 μm pore size);

[0074] Using the device of this invention, a 2 μL sample is injected at the center point into the ionization region of the ion migration tube and ionized into positive and negative ions. These ions then pass through periodically opening ion gates and enter a drift region formed by a uniform electric field. Separation and detection are achieved in the drift region, obtaining the instrument's background detection signal. The detected ion migration spectrum is obtained by continuously acquiring a 4.555 ms signal peak for 0.5 min. Figure 4 As shown.

[0075] The cumulative sum of the recorded lactic acid peak signals were 4812 mV, 5017 mV, and 5528 mV, respectively. Substituting these signals into the standard curve equation, Y = 4646.44 + 2.28 * X, the calculated lactic acid concentration was 207 μg / mL, which is close to the true value of 200 μg / mL.

Claims

1. A method for detecting lactate in whole blood, characterized in that: 1) Dissolve lactic acid standard in the mixed blood of more than 10 healthy people to prepare whole blood solutions with at least 5 different lactic acid contents in the range of 50-1000 μg / mL; 2) The lactic acid-containing solution obtained in step 1) was detected and analyzed using a negative ion mode photoionization ion mobility spectrometer to obtain the detection signal; the peak migration time of lactic acid in negative ion mode and the signal tracking change trend curve of lactic acid peak within 30 seconds were recorded; the signal intensity data summed value, i.e., the peak area corresponding to different lactic acid concentrations, can be obtained by retrieving the lactic acid peak tracking change trend curve through the instrument software. 3) Fit the peak area values ​​of the tracking signal corresponding to different lactic acid concentrations in steps 1) and 2) as the horizontal and vertical axes respectively to obtain the standard curve equation; substitute the peak area of ​​the lactic acid sample with unknown concentration into the standard curve equation to calculate the lactic acid concentration and thus achieve quantitative analysis. 4) Extract lactic acid from the blood sample to be tested to obtain a solution containing lactic acid; 5) The lactic acid-containing solution obtained in step 4) is detected and analyzed using a negative ion mode photoionization ion mobility spectrometer; the detection signal is obtained; the peak migration time of the sample obtained is compared with the peak migration time of the lactic acid signal in the spectrum of step 2) above, and the peak area of ​​the target peak is retrieved by the instrument software and the corresponding blood lactic acid concentration is calculated according to the method in 3). The ion migration tube used includes a photoionization source and a Faraday disk arranged opposite to each other at the left and right ends, and an ion gate located between the photoionization source and the Faraday disk. The region between the photoionization source and the ion gate is the ionization region, and the region between the ion gate and the Faraday disk is the migration region. Three through holes are provided sequentially on the ionization region of the outer wall of the ion migration tube from the side near the photoionization source to the ion gate. These holes are the ion migration tube outlet (4) for connecting the gas pump, the chemical dopant interface (5) for connecting the chemical dopant, and the ion migration tube sample inlet (6). These three through holes, together with the drift gas inlet (8) provided on the migration region of the outer wall of the ion migration tube near the ion detector, form the external interface of the gas circulation system of the ion migration tube. The ion migration tube outlet (4) is connected to the gas pump inlet as the gas outlet of the ion migration tube, and the rest are gas inlets. A variable diameter insulating inner sleeve (7) is inserted into the sample inlet (6) of the ion migration tube. One end of the variable diameter insulating inner sleeve (7) is located in the ionization region of the ion migration tube, and the other end is located outside the ion migration tube. The variable diameter insulating inner sleeve (7) is hollow and has a stepped inner diameter structure. The inner diameter of the other end located outside the ion migration tube is larger than the inner diameter of the end located in the ionization region. The number of steps in the inner diameter stepped variable diameter structure is 1. The variable diameter insulating inner sleeve (7) is a hollow tube with two open ends, composed of two hollow tubes with different inner diameters. The open end of one of them is sealed to the open end of the other. The annular surface formed at the connection of the two hollow tubes inside the hollow tube is the stepped platform. The stepped platform is perpendicular to the axis of the hollow tube. The hollow tube with the larger inner diameter is set at the other end outside the ion migration tube, and the hollow tube with the smaller inner diameter is set at one end of the ionization region.

2. The method according to claim 1, characterized in that: For steps 1) and 4), the extraction process is as follows: the blood sample extraction solvent is one or both of ethyl acetate or methyl tert-butyl ether; the extraction solution is sonicated, centrifuged, and the supernatant is filtered through a membrane to obtain a solution containing lactic acid. 0.5-1 mL of extraction solvent is used for every 0.5-1 mL of blood. The sonication time is 2-5 min, the sonication power is 500-600 W, the centrifugation time is 2-5 min, the rotation speed is 10000-12000 rpm, and the filter membrane pore size is 0.22 μm.

3. The method according to claim 2, characterized in that: the specific process, 1) Lactic acid standard sample analysis test: Dissolve lactic acid standard in the mixed blood of more than 10 healthy people, and prepare whole blood solutions of 100, 200, 400, 600, 800, and 1000 μg / mL using a stepwise dilution method; extract 0.5-1 mL of blood with 0.5-1 mL of extraction reagent according to the extraction process in claim 2, measure 2-5 μL of the lactic acid-containing solution, and then use a negative ion mode photoionization ion mobility spectrometer for detection and analysis to obtain the detection signal; record the peak migration time of lactic acid in negative ion mode and the signal tracking trend curve of lactic acid peak within 30 seconds. 2) Whole blood sample analysis test: Accurately measure a certain amount of blood sample to be tested, perform lactic acid extraction pretreatment according to the extraction process of claim 2 above, and then measure 2-5 μL of lactic acid-containing solution for analysis; perform detection and analysis in negative ion mode of ion mobility spectrometry to obtain detection signal; compare the obtained sample peak migration time with the lactic acid signal peak migration time of the above spectrum, and select the target peak tracking signal intensity data for quantitative analysis.

4. The method according to claim 1, characterized in that: The inner diameter of the variable diameter insulating inner sleeve (7) at one end of the ionization zone is 0.5-1.0 mm, and the radial width of the stepped platform is 0.25-1.0 mm. The ionization zone and the migration zone are both coaxial cylindrical, with their axial center lines placed parallel to the horizontal plane. The ion migration tube inlet (6) is located in the upper middle part of the ion migration tube. Insert a variable diameter insulating inner sleeve (7) into the sample inlet (6) of the ion migration tube. The stepped platform with variable diameter inside the variable diameter insulating inner sleeve can be used as a storage platform for all or part of the liquid sample. When the liquid sample is injected into the ion migration tube, the liquid sample can be dripped onto the stepped platform. Then the ion migration tube inlet (6) can be connected to the carrier gas source. The ion migration tube inlet (6) does not need to be connected to the injector separately. The ion migration tube inlet (6) is a circular through hole, and the variable diameter insulating inner sleeve (7) is a circular tube. The outer diameter r1 of the variable diameter insulating inner sleeve (7) is less than or equal to the inner diameter r2 of the ion migration tube inlet (6), that is, r1≤r2. r1 and r2 satisfy 4mm≤r2≤6mm, and the difference between r2 and r1 is less than 0.1mm. When r1<r2, the gap between the two can realize the function of evacuating and replenishing gas into the migration tube, reducing the sample gas flow.

5. The method according to claim 4, characterized in that: A hollow, annular, inverted frustum-shaped protrusion is provided radially on the outer wall of the other end of the variable diameter insulating inner sleeve (7) located outside the ion migration tube. The cross-section of the protrusion along the axis of the variable diameter insulating inner sleeve is two symmetrical right triangles with the axis of the variable diameter insulating inner sleeve as the axis of symmetry. One right-angled side A in the radial direction of the triangle is located on the side away from the ion migration tube, and the vertex corresponding to right-angled side A is located on the side closer to the ion migration tube. The other right-angled side of the triangle is parallel to the axis of the variable diameter insulating inner sleeve. The length of right-angled side A is greater than the difference between r2 and r1. One or two grooves are provided on the outer wall of the annular inverted truncated cone-shaped protrusion along the axial direction, i.e. from the lower base to the upper base of the cone. The radial cross-sectional area of ​​the grooves gradually decreases from the right-angle side A to the apex A, i.e. from the lower base to the upper base of the cone. The width of the grooves on the radial cross-section is less than 0.2 mm, and the groove depth is less than 0.2 mm. When the air pump is working, the grooves can also realize the function of air replenishment. The material of the variable diameter insulating inner sleeve (7) is one or two of polytetrafluoroethylene or high temperature resistant rubber. The high temperature resistant rubber can withstand high temperatures of 120-150℃ without any change in physical or chemical properties. The variable-diameter insulating inner sleeve (7) is 4-6cm long. One end outside the ion migration tube serves as the sample inlet port, and the other end of the port in the ionization region is inserted at the cylindrical light path emitted from the circular light window of the photoionization source, i.e., the area between the side of the variable-diameter insulating inner sleeve and the central axis of the light irradiation region. The radius of the cylindrical light path is equal to the radius of the circular light window of the photoionization source, and the radius of the cylindrical ionization region is between 1 and 2 times the radius of the circular light window. The axial distance between the photoionization source window and the ion gate, i.e. the axial distance between the ionization region and the ionization source window is set to L. The axial position of the variable diameter insulating inner sleeve (7) at the other end of the ionization region is within the ion migration tube and the photoionization source window is within the range of (1 / 2-5 / 6)L. The radial position is within the range of the central axis of the light path emitted by the light window to a radius R less than or equal to that of the circular light window.

6. The method according to any one of claims 1-5, characterized in that: Liquid samples, or liquid phases extracted after dilution with organic solvents, or liquid phases extracted after dissolving with organic solvents, are used as liquid injection samples. The liquid injection samples are loaded into a microsyringe. After the outlet of the microsyringe is inserted into the variable diameter insulating inner sleeve, the sample in the microsyringe is pushed out. Subsequently, the sample is enriched on the stepped surface inside the variable diameter insulating inner sleeve and continuously vaporized in the ionization region under the action of the carrier gas, resulting in efficient thermal ionization. Before being loaded into the microsyringe, the liquid sample is pretreated by filtering through a filter membrane. The microporous filter membrane has a pore size of 0.22 μm. The amount of sample injected into the variable-diameter insulating inner sleeve by the microsyringe is between 2-5 μL. The chemical dopant interface (5) is located between the ion migration tube outlet interface (4) and the ion migration tube sample inlet (6). The chemical dopant is one or two of acetone or butanone. The ion migration tube sample inlet (6) is nested inside a variable diameter insulating inner sleeve (7) that is coaxial with it; the micro-injector (9) contains the sample, and the front outlet, i.e. the needle, is inserted into the variable diameter insulating inner sleeve. The inner sleeve enters the outlet end of the migration tube to release the sample, so as to achieve the best ionization effect.

7. The method according to claim 6, characterized in that: After 2-5 μL of the sample extract of lactic acid in whole blood is injected into the variable diameter insulating inner sleeve (7), one end of the lower end of the inner sleeve is located in the core photoionization region of the ion migration tube. The sample is then slowly pyrolyzed and vaporized by a pump connected to the gas outlet (4) of the ion migration tube with a small gas flow of 100-200 mL / min.

8. The method according to any one of claims 1-3, characterized in that: The data acquisition time in the method is 30 seconds, and the quantitative analysis is calculated based on the cumulative peak intensity, i.e., peak area, of the tracked signal within 30 seconds. The migration tube used in the experiment was a vacuum injection method; the drift gas was 500-600 mL / min; the chemical dopant gas was 50-100 mL / min; the carrier gas carrying the sample was 100-200 mL / min; the pump flow rate in the experiment was the sum of the drift gas, chemical dopant gas and carrier gas, and the flow rate was 650-900 mL / min; the migration tube in the experiment was a cylindrical structure, the radius R of the circular light window was set to 0.5-1 cm, the radius of the ionization region was (1-8)R, the radius of the migration region was greater than the radius of the ionization region but less than 10R; the axial distance L of the ionization region was 2-3 cm, and the length of the migration region was designed to be (3-4)L.

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