A rapid method for determining the concentration of ethanol in blood

Abstract

The application relates to a rapid determination method of ethanol concentration in blood, in particular to a high-speed centrifugal purification-continuous flow analyzer rapid determination method of ethanol content in blood, which is characterized in that: after a sample is diluted by a saturated sodium chloride solution and high-speed centrifugation, interfering substances such as hemoglobin in the sample are removed, and the upper clear liquid is used to rapidly determine the ethanol content in the extraction solution by using a self-designed continuous flow analysis module. The application has the advantages that: after high-speed centrifugation treatment by salting-out, the colored interfering substances in the sample can be effectively removed, so that the sample is fully purified; then based on the principle that ethanol and diazo-p-aminobenzoic acid produce orange-yellow compounds under alkaline conditions, a continuous flow analyzer rapid detection module is designed, so that automatic detection of the sample is realized, the efficiency is high, the method has good repeatability and recovery rate, and the method is suitable for analysis of a large number of samples.

Description

Technical Field

[0001] The present invention relates to a method for measuring the ethanol concentration in blood, in particular to a method for quickly measuring the ethanol content in blood using a high-speed centrifugal purification-continuous flow analyzer. Background Art

[0002] Currently, traffic police often rely on both on-site identification using handheld rapid-detection devices and blood sample testing to maintain public road safety. On-site testing is based on the "Blood and Breath Alcohol Content Thresholds and Test Methods for Drunk Driving" (GB19522-2004), but accurate identification often requires laboratory testing of blood samples based on the "Headspace-Gas Chromatographic Method for the Determination of Ethanol, Methanol, n-Propanol, Acetaldehyde, Acetone, Isopropanol, and n-Butanol in Blood and Urine" (GA / T 1073-2013). Due to the high concentration of water in the sample, the ethanol is difficult to completely vaporize and distribute, resulting in biased test results. Furthermore, the sample preparation process involves negative pressure in the headspace vial, which is cumbersome and can lead to inconsistent results across batches. With the increasing number of alcohol testing cases, analytical methods with shorter test cycles and minimal human intervention are needed. Developing a rapid, standardized, and automated alcohol testing system has become a key focus of modern forensic science research.

[0003] The present invention aims at the current demand for rapid detection of ethanol in blood. Since ethanol detection in blood is often performed on samples screened according to the GB19522-2004 method, which belongs to constant analysis, it is very necessary to establish a fast and simple sample pretreatment and efficient continuous flow analysis method. Summary of the Invention

[0004] The main purpose of the present invention is to determine the ethanol content in blood. A method for quickly determining the ethanol content in blood using a high-speed centrifugal purification-continuous flow analyzer is established. The method effectively removes colored interfering substances in the sample through salting-out high-speed centrifugation treatment, thereby fully purifying the sample. Then, based on the principle that ethanol and diazo-p-aminobenzoic acid produce an orange-yellow compound through coupling reaction under alkaline conditions, a rapid detection module of the continuous flow analyzer is designed, thereby achieving automated sample detection with high efficiency, good method repeatability and recovery rate, and being suitable for analyzing large quantities of samples.

[0005] The object of the present invention is to be achieved by following steps:

[0006] A method for determining the concentration of ethanol in blood, which uses a saturated sodium chloride solution to dilute the sample and then centrifuge it at high speed to remove interfering substances (such as hemoglobin). ， The supernatant was taken and the ethanol content in the extraction solution was rapidly determined using a self-designed continuous flow analysis module.

[0007] The method of the present invention comprises the following steps:

[0008] (1) Sample dilution and purification: Weigh 0.5-1.0 mL of the sample to be tested and dilute it to 5 mL with saturated sodium chloride solution. Then centrifuge at a speed of 2000-50000 rps to remove interfering substances in the sample, such as hemoglobin and other macromolecules.

[0009] (2) Determination of ethanol content: First, the reaction module pipeline of the analyzer was modified so that the inlet a of the glass four-way 11 on the analyzer was introduced into the NaOH solution through a pump tube with a flow rate of 1.20 ml / min, the inlet b was introduced into the air through a pump tube with a flow rate of 0.32 ml / min, and the liquid inlet c was introduced into the sample solution to be tested through a pump tube with a flow rate of 0.23 ml / min. The liquid outlet d of the glass four-way was connected to the 5-turn mixing ring; the outlet of the 5-turn mixing ring was connected to the inlet of the T-shaped glass tee 12, and the color developer was introduced into the branch end of the T-shaped glass tee. The color developer was introduced into the sample solution by a pump tube with a flow rate of 0.23 ml / min. A sodium nitrite solution is introduced into an ice-water bath reaction cell I through a pump tube with a flow rate of 0.1 ml / min, and a p-aminobenzoic acid solution is introduced into a pump tube with a flow rate of 0.23 ml / min, so as to online generate a diazo p-aminobenzoic acid complex in an ice-water bath reaction cell I. The outlet of a T-shaped glass tee is connected to an ice-water bath reaction cell II, and the outlet end of the ice-water bath reaction cell II is connected to a 440 nm colorimetric cell through a 20-turn mixing ring. A sample solution, a sodium hydroxide solution, and a color developer solution are introduced into the ice-water bath reaction cell II for a color reaction to perform a measurement. During the measurement process, standard solution and sample analysis are performed in sequence to ultimately obtain the ethanol content in the solution to be measured, and then the ethanol concentration in the blood is calculated.

[0010] In the present invention, the ice-water bath reaction tank II is an ice-water bath reaction tank provided with 20 turns of mixing rings, and the ice-water bath reaction tank I is an ice-water bath reaction tank provided with 40 turns of mixing rings.

[0011] The reagents used were prepared in the following manner:

[0012] Saturated sodium chloride solution: Weigh 36.5 g of sodium chloride and add it to 100 g of water to dissolve.

[0013] Para-aminobenzoic acid solution: Weigh 12.5 g of para-aminobenzoic acid and add it into 100 mL of 5 mol / L hydrochloric acid solution to dissolve it.

[0014] The sodium hydroxide solution: weigh 10g of sodium hydroxide and add it into 100g of water to dissolve.

[0015] Described sodium nitrite solution: Weigh 7.5 grams of sodium nitrite and add it to 92.5 grams of water to dissolve it.

[0016] In the present invention, a reaction detection module assembled using AAⅢ type (Seal Company, UK) (the original instrument was not designed with an ethanol detection module) can realize the principle of producing an orange-yellow compound through the coupling reaction of ethanol and diazo-p-aminobenzoic acid under alkaline conditions. The module is designed as an online color development reaction, which can significantly reduce the amount of reagents used, make the operation simpler, and reduce the detection cost.

[0017] In the present invention, an online reactor for the color developer diazo-p-aminobenzoic acid complex, ice-water bath reaction tank I, is designed, which can achieve both temperature control (0-4°C) and the generation of the color developer, thereby avoiding the disadvantage of the color developer being difficult to preserve.

[0018] In the present invention, a color reaction reaction tank, ice-water bath reaction tank II, is designed, which can achieve temperature control (0-4°C) and realize the coupling reaction of ethanol and diazo-p-aminobenzoic acid complex under alkaline conditions to produce an orange-yellow compound.

[0019] In the present invention, the orange-yellow compound produced by the coupling reaction is subjected to 20 turns of mixing so that the reaction solution can be returned to room temperature for detection (usually the colorimetric method is performed at room temperature) to avoid endothermic phenomenon caused by excessive cooling of the reaction solution.

[0020] Compared with the analytical method of GA / T 1073-2013 in the prior art, the sample purification operation is simple, and the detection process is fully automatic, fast and efficient. The invented method has good precision and recovery rate, and is suitable for the determination of ethanol content in large quantities of blood samples after screening by the GB19522-2004 method. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of the module configuration (i.e., test pipeline) connection of the present invention.

[0022] The serial numbers in the figure are: 1 is air, 2 is 10% sodium hydroxide solution, 3 is sample solution, 4 is p-aminobenzoic acid solution, 5 is sodium nitrite solution, 6 is pump tube with flow rate 0.32mL / min, 7 is pump tube with flow rate 1.20mL / min, 8 is pump tube with flow rate 0.23mL / min, 9 and 10 are pump tubes with flow rate 0.23mL / min, 11 is glass four-way pipe, 12 is glass three-way pipe, 13 is detector (with 440nm filter and 1cm flow cell), 14 is ice-water bath reaction cell I, and 15 is ice-water bath reaction cell II. DETAILED DESCRIPTION

[0023] The following combination Figure 1 The determination method is described as follows: a blood sample is diluted with a saturated sodium chloride solution, and after high-speed centrifugation, the supernatant is taken and the ethanol content in the extraction solution is rapidly determined using a self-designed continuous flow analysis module.

[0024] The method of the present invention comprises the following steps:

[0025] (1) Sample dilution and purification: Weigh 0.5-1.0 mL of the sample to be tested and dilute it to 5 mL with saturated sodium chloride solution. Then centrifuge at a speed of 2000-50000 rps to remove interfering substances in the sample, such as hemoglobin and other macromolecules.

[0026] (2) Determination of ethanol content: First, the reaction module pipeline of the analyzer was modified so that the inlet a of the glass four-way 11 on the analyzer was introduced into the NaOH solution through a pump tube with a flow rate of 1.20 ml / min, the inlet b was introduced into the air through a pump tube with a flow rate of 0.32 ml / min, and the liquid inlet c was introduced into the sample solution through a pump tube with a flow rate of 0.23 ml / min. The liquid outlet d of the glass four-way 11 was connected to the 5-turn mixing ring; the outlet of the 5-turn mixing ring was connected to the inlet of the T-shaped glass tee 12, and the color developer was introduced into the branch end of the T-shaped glass tee. The color developer was introduced into the T-shaped glass tee by a A pump tube with a flow rate of 0.23 ml / min is used to introduce a sodium nitrite solution, and a pump tube with a flow rate of 0.23 ml / min is used to introduce a p-aminobenzoic acid solution, and a diazo p-aminobenzoic acid complex is generated online in an ice-water bath reaction tank I 14 (with a 40-turn mixing ring); the outlet of the T-shaped glass tee 12 is connected to an ice-water bath reaction tank II 15 (with a 20-turn mixing ring), and the outlet end of the ice-water bath reaction tank II is connected to a 440 nm colorimetric cell 13 through a 20-turn mixing ring, and the sample solution, sodium hydroxide solution and color developer solution are introduced into the ice-water bath reaction tank II for color reaction to perform determination. ， During the detection process, the standard solution and sample analysis are carried out in sequence, and finally the ethanol content in the test solution is obtained, and then the ethanol concentration in the blood is obtained by calculation.

[0027] The present invention is further described below with reference to specific examples:

[0028] Example 1

[0029] According to the operating steps of the inventive method, 0.3 mL of 10 portions of 0.1 mg / mL minimum concentration standard solution were taken, and 4.7 mL of saturated sodium chloride solution were added to 10 mL centrifuge tubes and mixed evenly. The centrifuge tubes were placed on a high-speed centrifuge and centrifuged at 2000 rps for 5 minutes. Then the supernatant was taken into a sample cup and tested using the designed continuous flow analyzer module. The standard deviation of the test results was calculated 10 times, and 3 times the standard deviation was used as the detection limit and 10 times the standard deviation was used as the quantitative limit. The method detection limit was 0.024 mg / mL and the quantitative limit was 0.080 mg / mL, indicating that the method had a lower detection limit.

[0030] Example 2

[0031] According to the operating steps of the inventive method, 0.5 mL of blood sample is taken, 4.5 mL of saturated sodium chloride solution is added to a 10 mL centrifuge tube and mixed evenly, the centrifuge tube is placed on a high-speed centrifuge, and centrifuged at a speed of 2500 rps for 5 minutes; then the supernatant is taken to a sample cup, and the designed continuous flow analyzer module is used for detection, and the ethanol concentration in the sample solution to be tested is obtained to be 12.5 mg / mL. The result is converted into an alcohol content of 125 mg / mL in the blood sample, and the degree of drunkenness can be judged based on this result.

[0032] Example 3

[0033] Sample number The present invention (mg / mL) GA / T 1073—2013 (mg / mL) 1 125.0 129.1 2 67.5 64.8 3 88.6 90.1 4 35.6 34.2

[0034] Example 4

[0035] A blood sample was taken and the precision and spiked recovery of the method were investigated in turn according to the operating steps of the invented method. The results showed that the precision of the method was 2.3%, and the spiked recovery was between 97.2% and 1019%. Both evaluation results were better than those of the GA / T 1073-2013 method (precision 3.9% and spiked recovery between 94.7% and 104.3%), indicating that the invented method can be used for the rapid determination of ethanol in blood.

Claims

1. A method for rapidly determining the concentration of ethanol in blood, characterized in that: Dilute the sample with saturated sodium chloride solution and centrifuge at high speed to remove interfering substances in the sample ， The supernatant was taken and the ethanol content in the extraction solution was rapidly determined using a self-designed continuous flow analysis module suitable for an AAⅢ continuous flow analyzer produced by Seal Company, UK. The rapid assay method comprises the following steps: (1) Sample dilution and purification: Weigh 0.5-1.0 mL of the sample to be tested and dilute it to 5 mL with saturated sodium chloride solution. Then centrifuge at a speed of 2000-50000 rps to remove interfering substances in the sample. (2) Determination of ethanol content: First, the reaction module pipeline of the analyzer was modified so that the glass four-way inlet a on the analyzer was introduced into the NaOH solution through a pump tube with a flow rate of 1.20 ml / min, the inlet b was introduced into the air through a pump tube with a flow rate of 0.32 ml / min, and the liquid inlet c was introduced into the sample solution through a pump tube with a flow rate of 0.23 ml / min. The liquid outlet d of the glass four-way was connected to the 5-turn mixing ring; the outlet of the 5-turn mixing ring was connected to the inlet of the T-type glass tee, and the color developer was introduced into the branch end of the T-type glass tee. The color developer was introduced into the sample solution by a pump tube with a flow rate of 0.23 ml / min. A sodium nitrite solution is introduced through a pump tube with a flow rate of 0.23 ml / min, and a p-aminobenzoic acid solution is introduced through a pump tube with a flow rate of 0.23 ml / min, so as to online generate a diazo p-aminobenzoic acid complex in an ice-water bath reaction tank I; the outlet of the T-shaped glass tee is connected to the ice-water bath reaction tank II, and the outlet end of the ice-water bath reaction tank II is connected to a 440 nm colorimetric cell through a 20-turn mixing ring. The sample solution, sodium hydroxide solution and color developer solution are introduced into the ice-water bath reaction tank II for color development reaction and thus determination is performed. During the detection process, the standard solution and the sample are analyzed in sequence, and finally the ethanol content in the extraction solution is obtained, and then the ethanol concentration in the blood is obtained by calculation.

2. The method for rapidly measuring blood ethanol concentration according to claim 1, wherein: The ice-water bath reaction tank II is an ice-water bath reaction tank with 20 turns of mixing rings.

3. The method for rapid determination of blood ethanol concentration according to claim 1, wherein: The ice-water bath reaction tank I is an ice-water bath reaction tank with 40 turns of mixing rings.

4. The method for rapid determination of blood ethanol concentration according to claim 1, wherein: The saturated sodium chloride solution was prepared by weighing 36.5 g of sodium chloride and adding the solution to 100 g of water to dissolve the solution.

5. The method for rapid determination of blood ethanol concentration according to claim 1, wherein: The preparation method of the p-aminobenzoic acid solution is as follows: 12.5 g of p-aminobenzoic acid is weighed and added into 100 mL of 5 mol / L hydrochloric acid solution to dissolve the mixture.

6. The method for rapid determination of blood ethanol concentration according to claim 1, wherein: The sodium hydroxide solution is prepared by weighing 10 g of sodium hydroxide and adding it into 100 g of water to dissolve it.

7. The method for rapidly measuring blood ethanol concentration according to claim 1, wherein: The sodium nitrite solution is prepared by weighing 7.5 grams of sodium nitrite and adding it to 92.5 grams of water to dissolve it.

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