Method for detecting volatile substances in liquid biological sample

Abstract

The invention discloses a method for detecting volatile substances in a liquid biological sample, which comprises the following steps: acquiring the liquid biological sample, weighing a closed container for accommodating the sample before and after adding the sample, and determining the mass of the sample; transferring the blank liquid sample into a closed container, adding different amounts of standard volatile substances to be detected, and preparing a series of standard curve samples; preparing a series of quality control samples of which the concentrations are within a standard curve range by adopting a mode which is the same as a mode for preparing the standard curve sample; adding internal standard working gas into the standard curve sample, the quality control sample and the to-be-detected sample, and detecting the samples by using a gas phase analyzer; and substituting the ratio of the peak area of the to-be-detected substance to the peak area of the internal standard substance into the standard curve, and calculating the concentration of the to-be-detected substance in the liquid biological sample by combining the sample mass. The mass of the sample is obtained by using a precise weighing method, the sample can be directly transferred into the sample bottle at one time, other sample treatment steps are not needed, the sample is directly detected on a machine, and the error of sample detection is greatly reduced.

Description

Technical Field

[0001] The present invention relates to the research field of biological sample analysis and detection, and in particular to a method for detecting volatile substances in a liquid biological sample. Background Art

[0002] The detection of gaseous substances (such as ultrasound contrast agents perfluoropropane, perfluorobutane and sulfur hexafluoride, and other volatile organic compounds (VOCs)) in liquid biological samples (such as whole blood, urine, milk, etc.) is very necessary in drug development, disease diagnosis, environmental impact assessment, etc.

[0003] The conventional detection method for liquid biological samples is to precisely measure a certain volume of sample into a fixed container, and then remove the upper gas from the container through headspace sampling or manual injection needle and inject it into instruments such as gas chromatography (GC) or gas chromatography-mass spectrometry (GC-MS) for detection. The sample content unit is generally μg / mL or ng / mL, etc. Liquid biological samples may need to go through a certain amount of time and space from sample collection to analysis and detection, so that the gas in the sample can easily evaporate into the upper gas of the sample bottle. In order to prevent the detection concentration from being low due to gas volatilization, such samples cannot be packaged and removed before being tested on the machine. It is difficult to find equipment with precise measurement function for sampling, so the conventional calculation method for liquid samples is no longer applicable.

[0004] It is very necessary to find a method to accurately quantify the amount of sample to be tested after sampling and then use it for the detection of gas substances in liquid biological samples. Summary of the invention

[0005] The object of the present invention is to provide a method for detecting volatile substances in a liquid biological sample to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A method for detecting volatile substances in a liquid biological sample comprises the following steps:

[0008] Step S1, sample acquisition, obtaining a certain amount of liquid biological sample, and determining the sample mass by weighing the sealed container containing the sample before and after adding the sample, specifically: before obtaining the sample, weigh the sealed container without adding the sample as M0, obtain a certain amount of liquid biological sample and add it to the sealed container and weigh it again as M1, and obtain the sample mass by subtracting M0 from M1;

[0009] The sample quality calculation formula is as follows:

[0010] m 样本 =M1-M0

[0011] Among them, m 样本 represents the mass of the collected liquid biological sample, M0 represents the mass of the sealed container without adding the sample, and M1 represents the total mass of the sealed container and the sample after adding the liquid biological sample;

[0012] Step S2, standard curve preparation, pipetting a blank liquid sample into a sealed container, adding different amounts of standard volatile substances to be tested, and preparing a series of standard curve samples, wherein the mass of the blank liquid sample is M2, and M2 is obtained by accurately pipetting the same volume of blank liquid samples for more than 12 times and weighing the average value;

[0013] Step S3, quality control sample preparation, using the same method as the standard curve sample preparation to prepare a series of quality control samples with concentrations within the standard curve range;

[0014] Step S4, detection, adding internal standard working gas to the standard curve sample, quality control sample and the sample to be tested, and detecting the sample using a gas analyzer;

[0015] Step S5, result calculation, substituting the ratio of the peak area of ​​the analyte to the internal standard into the standard curve, and calculating the concentration of the analyte in the liquid biological sample in combination with the sample mass, the calculated concentration of the analyte in the liquid biological sample is in ng / g or μg / g;

[0016] The formula for calculating the concentration of the analyte in the liquid biological sample is as follows:

[0017]

[0018] Wherein, C represents the concentration of the analyte in the liquid biological sample, C1 represents the theoretical gas substance content calculated by substituting the ratio of the peak area of ​​the analyte to the internal standard into the standard curve, and M2 represents the mass of the blank liquid sample used to prepare the standard curve sample.

[0019] In the present invention, the sealed container is a sealed headspace bottle.

[0020] In the present invention, the certain amount of liquid biological sample is 2 mL, and the air having the same volume as the sample volume is extracted from the sealed headspace bottle in advance before adding the sample.

[0021] In the present invention, in step S1, when weighing the sealed headspace bottle, all contents of the sealed sample bottle except the sample during sampling need to be weighed.

[0022] In the present invention, in step S1, a liquid biological sample is obtained using a medical syringe and added into a sealed container.

[0023] In the present invention, in step S4, the gas analyzer is a gas chromatograph, a gas chromatography-mass spectrometer or a gas chromatography-triple quadrupole tandem mass spectrometer.

[0024] In the present invention, in step S4, the chromatographic column of the gas analyzer is selected according to the characteristics of the object to be analyzed, including GS-GASPRO, wherein the length of the chromatographic column is 60 meters and the inner diameter of the chromatographic column is 0.32 mm.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention uses a precise weighing method to obtain the mass of the sample, and can directly transfer the sample to a sample bottle at one time without other sample processing steps, and can be directly tested on a machine, thereby greatly reducing the error of sample testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A standard curve for a method for detecting volatile substances in a liquid biological sample of the present invention;

[0028] Figure 2 The chromatogram is a method for detecting volatile substances in a liquid biological sample according to the present invention. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The present invention provides a technical solution:

[0031] A method for detecting volatile substances in a liquid biological sample comprises the following steps:

[0032] Step S1, sample acquisition, obtaining a certain amount of liquid biological sample, the certain amount of liquid biological sample is 2 mL, using a medical syringe to obtain the liquid biological sample and add it to a sealed container, the sealed container is a sealed headspace bottle, the sealed headspace bottle is pre-drawn with air of the same volume as the sample before adding the sample, and the sealed container containing the sample is weighed before and after adding the sample to determine the sample mass. When weighing the sealed headspace bottle, it is necessary to weigh all the contents of the sealed sample bottle except the sample when injecting the sample, specifically: before obtaining the sample, the sealed container without the sample is weighed and recorded as M0, and after obtaining a certain amount of liquid biological sample and adding it to the sealed container, it is weighed again and recorded as M1, and the sample mass is obtained by subtracting M0 from M1;

[0033] The sample quality calculation formula is as follows:

[0034] m 样本=M1-M0

[0035] Among them, m 样本 represents the mass of the collected liquid biological sample, M0 represents the mass of the sealed container without adding the sample, and M1 represents the total mass of the sealed container and the sample after adding the liquid biological sample;

[0036] Step S2, standard curve preparation, pipetting a blank liquid sample into a sealed container, adding different amounts of standard volatile substances to be tested, and preparing a series of standard curve samples, wherein the mass of the blank liquid sample is M2, and M2 is obtained by accurately pipetting the same volume of blank liquid samples for more than 12 times and weighing the average value;

[0037] Step S3, quality control sample preparation, using the same method as the standard curve sample preparation to prepare a series of quality control samples with concentrations within the standard curve range;

[0038] Step S4, detection, adding internal standard working gas to the standard curve sample, the quality control sample and the sample to be tested, and detecting the sample using a gas analyzer, wherein the gas analyzer is a gas chromatograph, a gas chromatography-mass spectrometer or a gas chromatography-triple quadrupole tandem mass spectrometer, etc.; the chromatographic column of the gas analyzer is selected according to the characteristics of the analyte, including GS-GASPRO, wherein the length of the chromatographic column is 60 meters and the inner diameter of the chromatographic column is 0.32 mm;

[0039] Step S5, result calculation, substituting the ratio of the peak area of ​​the analyte to the internal standard into the standard curve, and calculating the concentration of the analyte in the liquid biological sample in combination with the sample mass, the calculated concentration of the analyte in the liquid biological sample is in ng / g or μg / g;

[0040] The formula for calculating the concentration of the analyte in the liquid biological sample is as follows:

[0041]

[0042] Wherein, C represents the concentration of the analyte in the liquid biological sample, C1 represents the theoretical gas substance content calculated by substituting the ratio of the peak area of ​​the analyte to the internal standard into the standard curve, and M2 represents the mass of the blank liquid sample used to prepare the standard curve sample.

[0043] In a specific embodiment, the steps for determining perfluoropropane in whole blood are as follows:

[0044] Step 1: Preparation of standard curve samples

[0045] Secondary standard gas: dilute the perfluoropropane standard gas with air to prepare the perfluoropropane secondary standard gas.

[0046] Standard curve sample: Add the mixed perfluoropropane secondary standard gas into the pre-capped headspace bottle in proportion (blank whole blood has been added in advance, with a mass of M2=2.11259 g, 2.11259 g is the average of 12 weighings of 2 mL of whole blood containing anticoagulant) to prepare perfluoropropane whole blood standard curve samples with concentrations of 0.094, 0.188, 0.471, 0.941, 3.765, 18.823, 30.117, and 37.646 ng / g, respectively.

[0047] Step 2: Preparation of quality control samples

[0048] Quality control samples: Use the same method as the preparation of perfluoropropane whole blood standard curve samples to prepare quality control samples with concentrations of 0.282, 11.294, and 28.234 ng / g.

[0049] Step 3: Sample processing

[0050] First, weigh the headspace bottle with anticoagulant but no sample added, recorded as M0. Transfer 2 mL of whole blood into a capped and sealed headspace bottle (2 mL of air is extracted in advance) and weigh it, recorded as M1. (M1-M0) is the mass of the sample. Add internal standard working gas to the standard curve samples, quality control samples, and samples to be tested, and perform detection on the machine.

[0051] Step 4: Testing instruments and conditions

[0052] The main instruments are as follows:

[0053]

[0054] Test conditions

[0055] Detection instrument: MSD (triple quadrupole)

[0056] Incubation temperature: 40°C

[0057] Quantitative loop temperature: 50°C

[0058] Equilibration time: 10 min

[0059] Injection volume: 1mL

[0060] Chromatographic column: GS-GASPRO (60m×0.32mm)

[0061] Flow rate: 1.2mL / min

[0062] Step 5: Test results

[0063] Standard curve

[0064] like Figure 1As shown, the standard curve is obtained with the response ratio of the analyte to the internal standard (x) as the horizontal coordinate, the concentration of the analyte (y) as the vertical coordinate, and the weight coefficient is 1 / x2. The concentration of the analyte is calculated according to the formula x=(yb) / a (where a is the slope of the standard curve after fitting, and b is the intercept of the standard curve after fitting).

[0065] Quality control sample test results: The average accuracy deviation of quality control samples of each concentration is within the range of ±15%, and the relative standard deviation (%CV) of quality control samples of each concentration is within 15%, which meets the requirements. The results are shown in the table below.

[0066]

[0067] In the above tables, “N / A” means not applicable.

[0068] Result calculation

[0069] Content C = C1*M2 / (M1-M0),

[0070] Among them, C1 is the concentration calculated by the standard curve, in ng / g;

[0071] M2 is the mass of the standard curve sample, in this experiment, M2 = 2.11259g;

[0072] M1 is the total weight of the sample to be tested and the headspace bottle;

[0073] M0 is the weight of the headspace bottle.

[0074] The test results of the two subjects at different time points before and after administration (perfluoropropane) are shown in the following table:

[0075]

[0076]

[0077] In the above table, BLLOQ means the concentration below the lower limit of quantification, and the lower limit of quantification of human whole blood is 0.094 ng / g.

[0078] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0079] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting volatile substances in a liquid biological sample, characterized in that: The following steps are involved: Step S1, sample acquisition, obtaining a certain amount of liquid biological sample, and determining the sample mass by weighing the sealed container containing the sample before and after adding the sample, specifically: before obtaining the sample, weigh the sealed container without adding the sample as M0, obtain a certain amount of liquid biological sample and add it to the sealed container and weigh it again as M1, and obtain the sample mass by subtracting M0 from M1; The sample quality calculation formula is as follows: m 样本 =M1-M0 Among them, m 样本 represents the mass of the collected liquid biological sample, M0 represents the mass of the sealed container without adding the sample, and M1 represents the total mass of the sealed container and the sample after adding the liquid biological sample; Step S2, standard curve preparation, pipetting a blank liquid sample into a sealed container, adding different amounts of standard volatile substances to be tested, and preparing a series of standard curve samples, wherein the mass of the blank liquid sample is M2, and M2 is obtained by accurately pipetting the same volume of blank liquid samples for more than 12 times and weighing the average value; Step S3, quality control sample preparation, using the same method as the standard curve sample preparation to prepare a series of quality control samples with concentrations within the standard curve range; Step S4, detection, adding internal standard working gas to the standard curve sample, quality control sample and the sample to be tested, and detecting the sample using a gas analyzer; Step S5, result calculation, substituting the ratio of the peak area of ​​the analyte to the internal standard into the standard curve, and calculating the concentration of the analyte in the liquid biological sample in combination with the sample mass, the calculated concentration of the analyte in the liquid biological sample is in ng / g or μg / g; The formula for calculating the concentration of the analyte in the liquid biological sample is as follows: Wherein, C represents the concentration of the analyte in the liquid biological sample, C1 represents the theoretical gas substance content calculated by substituting the ratio of the peak area of ​​the analyte to the internal standard into the standard curve, and M2 represents the mass of the blank liquid sample used to prepare the standard curve sample.

2. The method for detecting volatile substances in a liquid biological sample according to claim 1, characterized in that: The airtight container is a closed headspace bottle.

3. The method for detecting volatile substances in a liquid biological sample according to claim 2, characterized in that: The certain amount of liquid biological sample is 2 mL, and the air having the same volume as the sample volume is extracted from the sealed headspace bottle in advance before adding the sample.

4. The method for detecting volatile substances in a liquid biological sample according to claim 2, characterized in that: In the step S1, when the sealed headspace bottle is weighed, all the contents of the sealed sample bottle except the sample during sampling need to be weighed.

5. The method for detecting volatile substances in a liquid biological sample according to claim 1, characterized in that: In step S1, a liquid biological sample is obtained using a medical syringe and added into a sealed container.

6. The method for detecting volatile substances in a liquid biological sample according to claim 1, characterized in that: In step S4, the gas analyzer is a gas chromatograph, a gas chromatography-mass spectrometer or a gas chromatography-triple quadrupole tandem mass spectrometer.

7. The method for detecting volatile substances in a liquid biological sample according to claim 1, characterized in that: In step S4, the chromatographic column of the gas analyzer is selected according to the characteristics of the object to be analyzed, including GS-GASPRO, wherein the length of the chromatographic column is 60 meters and the inner diameter of the chromatographic column is 0.32 mm.

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